Peroxisomen proliferazioaren markatzaileak diren geneen klonazioa eta espresio ikerketak

Transcription

1 Jakintza-arloa: Biologia Peroxisomen proliferazioaren markatzaileak diren geneen klonazioa eta espresio ikerketak Xenobiotiko organikoen eragina Egilea: EIDER BILBAO CASTELLANOS Urtea: 2007 Zuzendaria: Unibertsitatea: IBON CANCIO URIARTE UPV/EHU ISBN:

2 Hitzaurrea Itsasoa eta ozeanoak, sortzen ditugun milaka tona kutsatzaileren azken geralekua dira eta beraz, bertan bizi direnengan eragin ditzaketen kalteak identifikatu, baloratu eta zuzenduko dituzten metodologiak garatzea eta aplikatzea ezinbestekoa da. Urteetan zehar, ekosistemetako gune ezberdinetan dagoen toxiko partikularren berri izateko, analisi kimikoak erabili izan badira ere, azken urteotan kutsatzaileek biotaren gainean eragindako aldaketak aztertzen dituzten neurketen, hots neurketa biologikoen, garrantziaz jabeturik, biomarkatzaileen gaineko ikerketa-proiektuak ugaritu dira. Horrela, xenobiotiko organikoek organismo urtarretan peroxisomen proliferazioa eragiten dutela aski ezaguna da eta beraz, egitura aldetik erlaziorik ez duten konposatu hauen eraginpean edo/eta baldintza metaboliko aldakorren pean peroxisomek erakusten duten plastikotasunak, interes handia sortzen du ikuspegi ekotoxikologikotik. Izan ere, egun, peroxisomen proliferazioa aukeratutako organismo zentinelak bizi direneko ingurunearen egoeraren kalitatea aztertzea helburu duten biojarraipen-programetan esposiziobiomarkatzaile gisa erabiltzen da, esate baterako Prestige ontziak isuritako fuelolioak organismoen gain eragindako kalteak ebaluatzerako orduan. Hala ere, ur ingurunearen zentinela gisa erabiltzen diren arrain eta moluskuen peroxisomek ugaztunenekin antzekotasunak erakutsi arren, hainbat dira itsas organismoen peroxisomen molekula mailako erregulazioaren gainean erantzun beharreko galderak. Hori dela eta, tesi-lan honetan egoera fisiologiko ezberdinetan zein kutsatzaile ezberdinen pean molekula mailan eta bereziki, transkripto mailan ematen diren aldaketak aztertu nahi izan dira. Aukeratutako espezieen genomaren gaineko informazio ezak ordea, toxikologikoki garrantzitsuak diren hainbat itu-generen gaineko sekuentzia, (bereziki peroxisometako -oxidazio induzigarrirako kodetzen duten geneena, palmitoil- CoA oxidasa, proteina multifuntzionala eta 3-ketoazil-CoA tiolasa) lortu eta deskribatzera behartu du ikertzailea. Ondoren, baldintza fisiologiko ezberdinetan eta epe laburrean xenobiotiko organikoen pean modu akutuan mantendutako legatz, lazun eta muskuiluetan sekuentziok eta bereziki, peroxisometako β-oxidazio induzigarria transkripzionalki erregulatu egiten direla frogatu da, nahiz eta legatz eta muskuiluen kasuan ikerketa sakonagoak beharko diren zelaian aplikatu aurretik. Lazunetan berriz, ugaztunetan gertatzen denaren kontrara, peroxisomen proliferazioak ez du eragiten peroxisomen mintzeko 70 proteinaren erregulaziorik, agian itsas espezieek duten proliferatzeko gaitasuna ugaztun sentikorrena baino apalagoa delako. Azkenik eta in silico bada ere, arrain eta moluskuen peroxisometako - oxidazioko entzimak kodetzen dituzten geneek PPRE sekuentziak agertzen dituztela ikusi da, hau da, ugaztunetan ematen den erregulazio-mekanismoa posible litzatekeela. Hortaz, peroxisometako oxidazioaren karakterizazioaren ondoren, hemendik aurrera, peroxisomen proliferazioa itsas-inguruneko xenobiotiko organikoen aurreko esposizio-biomarkatzaile sentikor eta egoki gisa erabili ahal izateko, besteak beste, inguruneko baldintzetan eta egoera fisiologiko normalen pean peroxisomen biogenesia gobernatzen duten mekanismo molekularrak ezagutu beharko genituzke, peroxisometan eman daitezkeen fluktuazio posibleak

3 detektatzeko aukera eduki eta inguruneko kutsatzaileen eraginpean dauden organismo urtarretan ematen diren aldaketen interpretazioan urratsak eman ahal izateko. Ildo honetan, tesi-lanean in silico aurkitutako PPRE sekuentzia, klonatutako geneen eta bereziki, palmitoil-coa oxidasaren genearen gune sustatzailean agertzen den frogatu beharko litzateke, eta in vitro espresaturiko hartzaile nuklearrek (PPAR eta RXR) PPRE-ak ezagutu, lotu eta erregulatzeko gaitasuna dutenentz zehaztu; galdera hauek erantzun nahian, gene walking eta gel shift assay bezalako teknikak garatzen dihardugu egun. Gainera, orain arte, peroxisomen proliferatzaile gisa jo diren konposatuek erregulazio mota hau gauzatzeko gai diren frogatzeko gene reporter assay bat diseinatzeko asmoa dago lazunetan. Azkenik, palmitoil-coa oxidasaren RNAzundak prestatu dira lazun eta muskuiluetan, gene honek ehun ezberdinetan izan dezakeen espresio-patroia determinatzeko asmoz.

4 ZOOLOGIA ETA ANIMALI BIOLOGIA ZELULARRA SAILA PEROXISOMEN PROLIFERAZIOAREN MARKATZAILEAK DIREN GENEEN KLONAZIOA ETA ESPRESIO-IKERKETAK: XENOBIOTIKO ORGANIKOEN ERAGINA CLONING AND EXPRESSION ANALYSIS OF PEROXISOME PROLIFERATION MARKER GENES IN AQUATIC ORGANISMS: EFFECTS OF ORGANIC XENOBIOTICS PhD THESIS EIDER BILBAO CASTELLANOS Leioa, August, 2007.eko abuztua

5 I. SARRERA Sarreraren zenbait atal zientzia-gaien artikulu dibulgatibo gisa argitaratu dira: - I. Cancio eta E. Bilbao. "Arrainak, geneak eta txipak: kutsaduraren aurrean adierazteko askatasuna". Elhuyar Zientzia eta Teknologia Aldizkaria (2006). 3. saria euskaraz idatzitako zientzia-gaien artikulu dibulgatiboetan. CAF-Elhuyar Sariak XII. edizioa. 219,

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7 Aurkibidea I. SARRERA 1 1. POLUITZAILEEK ORGANISMO ITSASTARRETAN ERAGINDAKO EFEKTU BIOLOGIKOAK Inguruneko kutsaduraren azterketa Biomarkatzaileak Jarraipen-programak Kalitate-bermea biomarkatzaileen ezarpenean 8 2. TOXIKOEN ERAGINPEKO GENEEN MOLDATZE-ERANTZUNAK Molekula mailako moldatze defentsa-mekanismoak, defentsoma kimikoa Proteina garraiatzaileak (0 faseko bioeraldaketa-metabolismoa) Bioeraldaketa oxidatiboa (I faseko metabolismoa) Erredukzio- eta konjugazio-bioeraldaketa (II. faseko metabolismoa) Proteina antioxidatzaileak Hartzailea eta seinale-transdukzioa Metalen detoxifikazioa Bero-talka proteinak EKOIZPEN ALTUKO TRANSKRIPTOMA-IKERKETAK ARRAIN ETA MOLUSKUETAN Arrain eta moluskuen gene eta genomen gaineko informazioa Ekoizpen altuko sekuentziazio- eta transkriptoma-teknikak Homologia bidezko klonazioa edo geneen ehiza Ezabatze-hibridazio subtraktiboa Differential display RT-PCRa (ddrt-pcr) Gene-espresioaren analisi seriatua Mikro eta makrotxipak QA ekoizpen maila altuko mikrotxip bidezko ikerketetan Geneen espresio-profilak espezie itsastarretan Patogenoen aurreko ostalariaren erantzunak Inguruneko baldintza fisikoen aurreko moldatze-erantzunak edo baldintza fisiologikoen aldaketaren ondoriozko geneen espresio-profilak Konposatu kimiko toxikoen eraginpean ZERGAITIK ZENTRATU BEHAR DUGU PEROXISOMEN PROLIFERAZIOAREN IKERKETAN EKOIZPEN ALTUKO TOXIKOGENOMIKAREN AROAN? Peroxisomak Peroxisomak arrain eta moluskuetan Peroxisomen biogenesia eta proliferazioa Peroxisomen proliferazioaren mekanismoa: peroxisomen proliferatzaileek aktibaturiko 47 hartzaileak (PPAR) 4.5. Peroxisomen erantzunak itsas-organismoetan AZKEN OHARRAK 51 II GAIAREN EGOERA, HIPOTESIA ETA HELBURUAK 137 III. EMAITZAK ETA EZTABAIDA Cloning and expression pattern of a polyamine oxidase, xanthine oxidoreductase and catalase in mussel Mytilus galloprovincialis and thicklip grey mullet Chelon labrosus Molecular characterisation of the peroxisomal β-oxidation in aquatic organisms: cloning and expression pattern of palmitoyl-coa oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase Differential expression of genes involved in peroxisome proliferation in thicklip grey mullets Chelon labrosus injected with benzo(a)pyrene Transcriptional regulation of peroxisome proliferation and biotransformation metabolism in thicklip grey mullets Chelon labrosus exposed to perfluorooctane sulfonate and to Prestige-like heavy fuel oil 233 3

8 Aurkibidea 3.5. Xenobiotiko organikoen peko Mytilus galloprovincialis muskuiluen liseri-guruineko geneen espresioa: zelai eta laborategiko saioak Toxikologikoki garrantzitsuak diren geneen klonazioa eta palmitoil-coa oxidasaren (AOX1) espresio-patroia Merluccius merluccius legatzean 293 IV. ONDORIOAK ETA TESIA 317 V. ERANSKINA I 321 VI. ERANSKINA II 327 4

9 Sarrera 1. Poluitzaileek organismo itsastarretan eragindako efektu biologikoak Organismo bizidunak bizi direneko ingurunea, aldakorra da gehientsuenetan, beraz, baldintza biotiko eta abiotikoek eskainitako fluktuazioak gainditzen ikasi behar dute. Izan ere, espezie baten historia ebolutiboa ingurunean ematen diren aldakortasunetara moldatzeko duen gaitasunaren menpe dago. Hau, bizi-estrategiak, fisiologia eta jarrera moldatuz edo/eta aldaketok erresistentzia-mugen barruan aurkituz gero, hauen aurrean eutsiz lor daiteke. Ia edozein organismok inguruneko fluktuazioetara moldatu behar da, horien artean, tenperatura, oxigenoa, ura, elikagaiak, edo/eta argi eskuragarritasuna, irradiazio ultramorea, ph, gazitasuna, patogeno eta harraparien presentzian. Ingurunearen konposizio kimikoaren aldaketa, bizidunek gainditu beharreko beste faktore abiotiko bat da. Horrela, gene espresioaren erregulazioa da inguruneko baldintzen aurrean zein biziraupena bermatzeko organismoek darabilten giltzarria (Cossins et al. 2006; Gibson 2006). Organismo batek har dezakeen egoera transkripzionalen kantitatea harrigarria da eta eboluzioa gidatzen duten indarretako bat izan daiteke (Gibson 2006). Geneen espresioan ematen diren aldaketen garrantzia King eta Wilsonen adibide batek argitzen ditu hobeto (1975). Gizaki eta txinpanzeen arteko distantzia genetikoa eta ezberdintasun biokimikoak txikiegiak dira organismo bien ezberdintasun fenotipikoak azaldu ahal izateko, beraz beraien ustetan, "anatomian eta bizitzeko eran emandako aldaketak, geneen espresioa kontrolatzen duten mekanismoetan gauzatu behar izan da, proteinen sekuentzia-aldaketan baino gehiago". Zentzu honetan, XIX. mendeko iraultza industriala eta 1950.eko "kimikaren aroak", Lurrak ordura arte ezagutu ez zuen abiadura arinean eta bide ezberdin gehiagoren bitartez ingurunea eraldatzen hasi ziren milaka substantzia kimiko berrien ekoizpena eta askatzea ekarriz. Substantziok gai biologikoentzat arrotzak dira (xenobiotikoak) (1. Irudia). Gaur egun, Europako merkatuak konposatu kimiko inguru eskaintzen ditu eta horietatik ia herena tona bat baino kantitate handiagotan (1-100 tona) ekoizten da urtero (Hengstler et al. 2006). Honek, itsas-organismoak transkripzio mailan neur daitezkeen estrategia adaptatiboak garatzera behartzen ditu. 1. Irudia. DDT pestizida organoklorinatua, Munduko Bigarren Gerratearen bukaeran emandako "Aro kimikoa" ondoen islatzen duen konposatu kimikoa da. Intsektizida gisa oso erabilia izan zen bere ekoizpena debekatu zuten arte, izan ere konposatu hau iraunkorra baita ingurunean eta konposatu xenoestrogeno eta kartzinogenikoa da. Argazkian New York-eko Beach State Park-ean eltxoen aurkako DDTaren zabaltzea ikusten da urtean. Argazkia National Geographic-etik hartua. 3 S A R R E R A

10 Sarrera 1.1. Inguruneko kutsaduraren azterketa Lan hau itsasoko kutsaduran oinarritzen da, izan ere, itsasoa baita ingurunera bide ezberdinetatik heltzen diren eta jatorri ezberdina duten konposatu kimiko nahasketen azken geralekua (ICES 2006). Egitura aldetik ezberdinak izan arren, xenobiotiko asko hidrofoboak dira eta ezaugarri horrek, mintzak zeharkatuz zeluletan sartzea errazten die eta beraz, itsas-organismoen ehunetan meta daitezke eta bertan kalteak sortarazi. Edozein kasutan, kutsatzaileen pean egondako animaliak erantzuten saiatuko dira, beraien ingurune fisikoan edo bizitzeko behar dituzten baldintza naturaletan emandako aldaketen aurrean egiten duten moduan. Organismoek bideraturiko konpentsaziorako mekanismoa ez bada gai erantzun beharreko aldaketa gainditzeko, animaliak efektu toxikoak pairatuko ditu, homeostasi egoeratik urrunduz. Horrela, konposatu kimikoek ugalketan edo/ta garapenean eragin dezakete, animaliaren jarrera alda dezakete edo molekula mailan eritasuna eragin dezakete; azken batean, itsasoko komunitateetan egitura eta funtzioak aldatuz (GESAMP 2001). Itsaso eta ozeanoak gizakiaren jardueraren ondorioz kaltetzen ari direnez, itsas-ingurunera heltzen diren kutsatzaileek eragin ditzaketen kalteak identifikatu, baloratu eta zuzenduko lituzketen metodologiak garatzeko beharra dago (Cajaraville et al. 2000; Broeg et al. 2005; Ankley et al. 2006; Denslow et al. 2007). Analisi kimikoak beharrezkoak dira inguruneko kutsadura aztertzean, ekosistemetako gune ezberdinetan, ura, sedimentua eta biota, dagoen toxiko partikularren berri ematen baitu. Baina neurtu beharreko konposatu kimiko ezberdinen kantitatea handia da. Eskuragarri dauden kutsatzaileen kontzentrazio erlatiboa denboran eta espazioan organismo biziek emandako erantzunek integratzen dituztenez, biota da inguruneko osasun arriskuak determinatzean euskarri esanguratsuena (McCarthy eta Shugart, 1990). Horrela, urtean, "The Mussel Watch" kontzeptua proposatu zen itsasoko uretan metatzen ziren konposatu kimikoen jarraipena egiteko, muskuilu eta ostrak organismo zentinela legez erabiliz (Goldberg 1975). Inguruneko kutsaduraren biojarraipena egiteko, moluskuen erabilpenak, bereziki bibalboenak, gainontzeko taxonetako organismoen erabilpena aise gainditu du, organismo zurgatzaile hauek inguruneko konpartimentu guztietatik kutsatzaileak inguruneko mailen gainetik gora metatzeko gaitasuna baitute (Rainbow 1997). Gainera, ekonomikoki garrantzitsuak diren organismo sesilak dira, urte osoan zehar eskuragarri, lagintzeko errazak, banaketa zabala dutenak eta ekosisteman gune kritikoan kokatuta daudenak. Ezaugarri guzti hauek, Mytilus spp muskuiluek ere betetzen dituzte. Hala ere, ingurune-kutsaduraren azterketa ezin da oinarritu soilik analisi kimikoetan, hauek, biotan eragindako kalteen berri ez dute ematen eta (Cajaraville et al. 2000; Allan et al. 2006). Gainera, egunez egun ekoitzi, merkaturatze eta ingurunera askatzen den kutsatzaileen kopurua eta motak (bifenil polibrominatuak, konposatu perfluoronatuak, droga terapeutikoak, nanopartikulak..) hazten daudenez, kutsaduraren ebaluaziorako estrategia berrien garapena beharrezkoa da. Ondorioz, inguruneko-kutsadura aztertzerakoan neurketa kimiko eta biologiko ezberdinak bateratzearen aldeko adostasun orokor bat badago (Cajaraville et al. 2000; Goldberg eta Bertine, 2000; JAMP 2003; Lam eta Gray 2003; Allan et al. 2006). Zentzu honetan, muskuiluekin batera, arrainak ikertu dira organismo zentinela legez, kutsatzaileek eragindako kalteak maila biologikoan aztertzeko egokiak baitira. Arrainen genomaren gaineko ikerketak gero eta gehiago erabiltzen dira ingurunean ematen diren aldaketen gaineko ezagutzan sakontzeko asmoz; horien artean, hipoxia, zoldurak edo kutsatzaile ezberdinen zein konposatu terapeutikoen esposiziopean emandako aldaketak aztertzen dira. Arrainek, espezie kopuruari dagokionez ornodunen talderik handiena osatzen dute, izan ere espezie katalogatu dira ( ornodun guztien baturak ematen duen espezie kopurua baino gehiago. Ia edozein ur ingurune betetzeko gaitasuna dute, bizitzeko estrategia dibertsitate aberatsa garatuz. Hortaz, erantzun metabolikoak, gorputzeko-formak, ugaltzeko estrategiak edo/eta 4

11 Sarrera beste edozein jarrera garatu dituzten arrain espezieak aurki daitezke. Arrainak uretan murgilduta bizi eta ugaltzen dira. Ura, beraz, arrainen zakatz eta liseri traktuko molekula, zelula eta ehunekin harreman zuzenean dago. Beraz, arrainak organismo zentinela onak dira ingurunean ematen diren aldaketak eta gorabehera biotiko eta abiotikoek eragindako moldatze biologikoak aztertzeko (Cossins eta Crawford, 2005). Arrainak, muskuiluak bezalako ornogabeak ez bezala, ornodunak dira, eta beraz, ugaztunekin garapen-programa, mekanismo eta moldatze fisiologikoak, organoak eta sistemak konpartitzen dituzte. Azken ezaugarri hau, ez da nolanahikoa, arrainek muskuiluek ez bezala, gibela baitute, eta ugaztunetan bezala, xenobiotikoen metabolismoan organo nagusiena da (Hahn et al. 2006; Incardona et al. 2006). Arrainen genoma oso interesgarria da agertzen duen plastikotasuna dela eta, poliploidiak, intronen banaketa eta gene, kromosoma edo genoma osoko duplikazioak, gainontzeko ornodun taldeetan baino maizago gertatzen dira (Maglich et al. 2003; Cossins eta Crawford, 2005; Volff 2005; Annilo et al. 2006; Hahn et al. 2006; Kasahara et al. 2007; Metpally et al. 2007; Semon eta Wolfe, 2007). Konposatu kimikoen pean sistema biologikoen erantzuteko gaitasuna ikertzeko helburuz, zein osasun-egoera kuantifikatzeko (organismo zentineletan), antolakuntza biologikoko maila ezberdinetan emandako efektu biologikoak bateratzea proposatu dute hainbat autorek (Attrill eta Depledge 1997, Allen eta Moore 2004, Broeg et al. 2005; Ankley et al. 2006). Honek, beharrezko egiten du biomarkatzaile eta bioentsaioetan burututako molekula, zelula eta ehun mailako azterketen datuak, organismo mailan burututako bioentsaioak eta komunitate eta populazio mailako azterketa ekologikoak bateratzea (ICES 2005, 2006). Konposatu kimiko berezien aurreko biomarkatzaile espezifikoak bideratzen dituen geneen espresio mailako perfilak ikertzea denez lan honen xedea, biomarkatzaileen nondik norakoak kokatzeari helduko zaio ondoren Biomarkatzaileak McCarthy eta Shugart-ek (1990) honela definitu zituzten biomarkatzaileak: "Kutsatzaileen presentzia (esposizio-biomarkatzaileak) edo ostalariak emandako erantzunaren maila (efektubiomarkatzaileak) adierazten duten gorputzeko fluido, zelula edo ehunetan maila biokimiko edo zelularrean burututako neurketak. Gaur egun, biomarkatzaile molekularrak dira ikerketa ekotoxikologikoen ate nagusia eta biomarkatzaileen garapenaren parte handi bat, xenobiotikoen peko ikerketak burutzeko tresna molekularren aplikaziotik datoz (Williams et al. 2003; Tom eta Auslander 2005; Dondero et al. 2006a; Moens et al. 2006; Venier et al. 2006; Roberts et al. 2006; Denslow et al. 2007; Mortensen eta Arukwe, 2007). Erantzun arinak ematen dituztenez, molekula eta zelula mailako biomarkatzaileak, inguruneko kutsatzaileek sortarazitako efektu biologikoen seinale goiztiar legez erabiltzen dira, antolakuntza biologiko maila altuagoetan, populazioa, komunitatea edo ekosistema mailetan hain zuzen ere, eman daitezkeen eta atzeraezinak izan daitezkeen aldaketak aurreikusteko (Cajaraville et al. 1998; Ankley et al. 2006; Denslow et al. 2007). Orokorrean, maila biologiko baxuetan emandako erantzunak, espezifikoagoak, sentikorragoak, errepikakorragoak eta zehazteko errazagoak dira. Aldaketa ekologikoekin erlazionatzeko aldiz, zailagoak dira (Van der Oost et al. 2003; Denslow et al. 2007). Bestalde, maila biologiko altuetan emandako erantzunak, ekosistemaren osasunaren isla zuzena dira eta hortaz, ingurunearen kudeaketarako eraginkorragoak. Hala ere, zehazteko zailagoak dira, ez dira hain espezifikoak, eta ingurunean kaltea dagoeneko eman denean bakarrik gertatzen dira, eta hortaz, kudeatzeko edo zuzentzeko neurri eskasak har daitezke ordurako (Connell et al. 1999; Van der Oost et al. 2003; Au 2004). Azken urteotako ikerketek Europan "biomarkatzaileen hurbilketa" aplikatu dute itsasoko sistema biologikoetan kutsatzaileek eragindako ekintza kaltegarrien ebaluaziorako (Handy et al. 2003; Lam eta Gray 2003; Galloway et al. 2004; Schiedek et al. 2006; Bilbao et al. 2006a; 2006b; Zorita et al. 2007a; Dondero et al. 2006a). Hala ere, oraindik ere, eztabaida zabala dago biomarkatzaileen definizio eta bereziki inguruneko 5 S A R R E R A

12 Sarrera arriskuen ebaluaziorako beraien erabilpenari dagokionean (Forbes et al. 2006; Ankley et al. 2006), eta badirudi nazioarteko zein administrazio lokalak biomarkatzaileen hurbilketa, ingurunea aztertzeko kalitate-programetan aplikatzearen aurrean mesfidati agertzen direla. Inguruneko azterketetan, biomarkatzaileen mugetako bat, biomarkatzaile bakar baten erabilpenean dago, izan ere biomarkatzaile bakarrak ez baitu islatzen organismoaren erantzun osoa eta beraz, efektu biologikoen biojarraipenak, biomarkatzaile ezberdinak erabiliz burutu beharko lirateke (Viarengo eta Canesi 1991; JAMP 1997; Ringwood et al. 1999; UNEP/RAMOGE 1999; Cajaraville et al. 2000; JAMP 2003; Galloway et al. 2004; Broeg et al. 2005). Are gehiago, biomarkatzaile bakoitzarentzat, espezifizitatea, denboraren erlazioa, inter eta intra-indibiduoen aldakortasuna, oinarrizko balioak eta errakuntzara eraman dezaketen faktoreen presentzia, hala nola, faktore biotiko zein abiotikoak, beraien aplikazioa baino lehen definitu beharko lirateke (Schulte eta Talaska 1995; Fossi et al. 2002; Lam eta Gray 2003). Esaterako, hainbat biomarkatzaile gorputzaren pisuaren arabera sexua, ugalketa-fasea, tamaina, sasoia, gazitasuna, pha eta uraren tenperaturaren arabera aldatzen direla ikusi da, (Roesijadi 1994; Bordin et al. 1997; Cancio et al. 1999; Serra et al. 1999; Hylland et al. 1992; Olsson et al. 1995; Serafim et al. 2002; Vidal et al. 2002; Cajaraville et al. 2003; Leiniö eta Lehtonen 2005; Pfeifer et al. 2005). Honek esan nahi du, efektu biologikoen jarraipena kontu handiarekin planifikatu behar dela, neurtu beharreko parametroetan, sasoiaren araberako aldaketak nola ematen diren kontutan hartuz. Faktore biotiko eta abiotikoekin batera, itsasingurunean nekez izango da konposatu kimiko bat kutsaduraren iturri bakarra eta hortaz kutsatutako guneetan, konposatu kimikoen nahasteak agertzen dira efektu gehigarriak, sinergiko edo antagonikoak gertatuz. Beraz, biomarkatzaile jakin batek konposatu espezifiko baten aurrean eman dezakeen erantzuna, bestelako kutsatzaileek estali dezakete, emaitza faltsuak ondorioztatuz. Esaterako, P450 1A1 zitokromoa (CYP1A1), xenobiotikoak metabolizatzen dituzten entzimak kodetzen dituzten multigene familiako partaide da eta inguruneko kutsatzaile organiko (PAHak) mordoak induzi dezake. Horrela, biojarraipen ikerketa ugaritan erabilia da (Aas et al. 2000; George et al. 2004; Pacheco et al. 2005). CYP1A1ren erregulazio positiboa ornodunetan, geneon promotoreetan agertzen den xenobiotikoen peko erantzun elementuek (XRE) bideratzen dute (Lewis et al. 2004). Adibidez, Platichthys flesusen CYP1A1en promotoreko 7 XRE guneekin batera, urgoran, metalen peko erantzun elementu bat ere badago (MRE). Frogatu da CYP1A1en indukzioa eragiten duten PAHen pean, platuxek dosiaren menpeko erantzuna ematen dutela CYP1A1en promotorean, kadmiorekin tratatzean berriz (1.0 µm), transkripzio gutxi eraginez promotore berberetan (Lewis et al. 2006). Kadmio eta PAHen bidezko kotratamentuak berriz, indukzioa esangarriki murriztu zuen, PAHekin bakarrik tratatzean lortutako emaitzekin alderatuz gero. MRE guneko mutazioek, kadmioari lotutako promotorearen jardueraren gutxitzea ezabatu egin zuten. Emaitza hauek CYP1A1en indukzioaren gaineko interpretazioen konplexutasuna zein mailakoa den, erakusten dute konposatu kimikoen nahasteen jarraipen-ikerketak burutu behar direnean (Lewis et al. 2006). Ingurune-kutsadura ebaluatzeko helburuz, molekula, zelula eta ehun mailako biomarkatzaile erabiltzea gomendatzen da, hortaz Jarraipen-programak Oraintsu sortutako European Marine Strategy (EMS) deritzon Europa mailako programa, ekosisteman oinarritutako hurbilpena erabiliz (Ecosysten Based Approach, EBA), gauzatu beharko litzateke hau da, lur, ura eta bizi-iturriak bateratzen dituen kudeaketarako estrategia bat, kontserbazioa eta iraunkortasuna modu zuzenean sustatzen dituztenak (CBD 2000). Ebaluazio honen oinarrian, inguruneko itu-talde neurgarria eta asoziaturiko indikatzaileak zehaztu beharko lirateke Europako uretarako, beharrezkoak diren jarraipen-programekin batera, ekosistemak kalitate-egoera onean ote dauden aztertzeko (Borja 2006). EMS arteztaraua, urtean sartu zen indarrean eta jarraipen-programen ezarpena urterako garatu beharko litzateke Europako itsasoen 6

13 Sarrera ingurunearen egoera ona urterako lortzeko (COM2005; Borja 2006). Orain, ekotoxikologisten erronka, itsasoko ekoeskualde ezberdinetan ezarri daitezkeen jarraipen-sareak eta metodologia egokiak garatzea da (Borja 2006; Lehtonen eta Schiedek 2006). Biomarkatzaileen hurbilketak, itsas-ingurunearen bizi-irauterako jarraipen-programetan ezartzeko erabilpen zabala agintzen bazuen ere, efektu biologikoen ebaluazioa oso modu mugatuan bakarrik sartu da arteztarau berrietan, analisi kimikoen diagnostikoa osatuz (JAMP 2003; Allan et al. 2006; Lehtonen eta Schiedek 2006). 2 hamarkada baino gehiagotan, zientzialariek biomarkatzaileek inguruneko kutsaduraren ebaluazioan aurkezten dituzten abantailak erakutsi dituzte, baina ekotoxikologian ez dago aurrekaririk non biomarkatzaileen datuak erabaki arautzailerik hartzeko erabili izanaz (Ankley et al. 2006). Efektu biologikoen jarraipena, aurretik ezagutzen ez diren konposatu baten edo konposatuen nahasteen presentzia adierazteko erabil daiteke, kutsatzaile eta erantzun ekologikoen arteko loturak erakutsiz eta inguruneko kalitatea kaltetutako eskualdeak identifikatzeko erabiliz (Lehtonen eta Schiedek, 2006). Efektu biologikoen neurketarako metodo ezberdinak ebaluatu dira International Council for the Exploration of the Sea-ak antolaturiko (ICES) lan-talde ezberdinetan, zenbait biomarkatzaile kutsadura ebaluatzen duten jarraipen-programetan eransteko egokiak zirela ondorioztatuz (Stebbing eta Dethlefsen 1992; ICES 2005; UNEP/MAP 2005). Gaur egun, urteko urtarriletik, organoestainozko konposatuen pean molusku gatropodoetan ematen den imposexa (emeen gaineko berezitazun anatomiko arren inposaketa) da OSPAR CEMPen derrigorrezkoa den biomarkatzaile bakarra. Hazten ari diren biomarkatzaile berriak egun ebaluazio pean dauden bitartean, markatzaile arrunt batzuk balidazio-fasean daude eta itsas-ingurunearen kalitatearen azterpenerako tresna gisa erabili dira (Bilbao et al. 2006a; 2006b; Zorita et al. 2007a). Beste biomarkatzaile batzuk, munduan zehar dauden ikertzaile eta laborategi ezberdinentzako intereseko ikerketa-proiektu espezifikoen pean garatzen ari dira eta borondatezko aplikazioa dute CEMPn (ICES 2006). Biomarkatzaile "konbentzionalez" gain, "omic" hurbilketen erabilpena, transkriptomika, proteomika eta metabolomika, inguruneko kutsadura aztertzeko, etorkizuneko tresna legez ikusten dira (Hogstrand et al. 2002; Neumann eta Galvez 2002; Larkin et al. 2003a; 2003b; Snape et al. 2004; Dondero et al. 2006b; Ankley et al. 2006; Denslow et al. 2007). Ikerketa ekotoxikogenomiko berriek geneen transkriptoen, proteinen eta metabolitoen profiletan ematen diren patroi-aldaketak ikertzen ditu kutsatzaileen pean egon ondorengo, zelula, ehun edo organismoetan (Snape et al. 2004; Miracle eta Ankley, 2005; Ankley et al. 2006). Geneen txipek (3.2.5 atala) ehunka-milaka geneen espresioa era berean ikertzeko abantaila eskaintzen dute, zenbait espezie modelotan genoma osoa izanik aztergai (Bartosiewicz et al. 2001; Williams et al. 2003; 2006; Ankley et al. 2006; Denslow et al. 2007), eta kutsatzaileek eragindako gene ezberdinen sareen elkarrekintzak adieraziz (Larkin et al. 2003b; Williams et al. 2006). Euskarri toxikogenomikoen aplikazioak milaka generen analisia baimentzen du era batera, hala beharrez konposatu kimiko bakoitzaren aurrean ehunka biomarkatzaile posible aztertuz. Izan ere, geneen txipak azken urteotan biomarkatzaile konbentzionalen kasuan proposatu den multibiomarkatzaileen hurbilpenerako laburbide bat dira (Viarengo eta Canesi, 1991, JAMP 1997; UNEP/RAMOGE 1999; Cajaraville et al. 2000; JAMP 2003; Broeg et al. 2005; Lewis et al. 2006). Zenbat eta genomaren gaineko analisi zabalagoa burutu, hots, toxikogenomikak baimentzen duen indibiduo baten aukera fenotipiko guztien edo gehienen espektroa aztertzea suposatuko lukeena, biomarkatzaile soilen hurbilketak ondorioztatzen duen bidezidor espezifiko jakinen azterketaren gabezia saihesten du (Ankley et al. 2006). Ekotoxikologistak, sarritan saiatu dira zelaian hartutako laginetan biomarkatzaileak aplikatuz, esposatutako animaliek erakusten dituzten osasun aldaketei sentsua aurkitzen, baina horretarako emaitzak ezagunak diren toxikotasun-mekanismoetara lotu behar dira. 7 S A R R E R A

14 Sarrera Hala ere, "omic" teknologiek atea irekitzen dio ekintza-mekanismo berriak ulertzeari eta hauek kutsatzaileekin izan ditzaketen elkarrekintzak ezagutzeari (Miracle eta Ankley, 2005). Mikrotxipek konposatu kimiko ezberdinen ekintzarako mekanismo eta jarduteko moduen inguruko informazioa eta konposatu kimikoen toxikotasun-sinadurak emateko gaitasuna dute (2. Irudia, Miracle eta Ankley, 2005; Ankley et al. 2006; Williams et al. 2006; Denslow et al. 2007). Bidezidor biokimiko ugariren aldi bereko analisi paraleloen bidez, posible da esposizioa eman deneko agertokiaren eta bere toxikotasunaren ulermen zabala lortzea. Antzera, baina geneen txipak baino gutxiago garatuta, proteina mailako sinadurak daude, ikerketa proteomikoen barruan xenobiotiko sinpleek zein beraien nahasteek eragindako toxikotasun-mekanismoak errazago ulertzeko informazioa eskaintzen dute (Bandara eta Kennedy 2002; Mi et al. 2005; Apraiz et al. 2006) eta inguruneko kutsaduraren ebaluazioan erabilgarriak diren biomarkatzaile nobelen garapena ondoriozta dezakete Kalitate-bermea (QA) biomarkatzaileen ezarpenean Konfiantzazko datu esangarrien lorpena, edozein ikerketa edo/eta jarraipen-programatan ezinbesteko osagaia da. Datuok lortzeko, eta hurbilketa hauek ekotoxikologian ezartzeko gomendatzen diren teknika analitiko guztiak, kalitate bermea (QA) daramaten programa egokien pean garatu behar dira (ICES 2004; JAMP 2003; Rees 2004; Allan et al. 2006; Ankley et al. 2006). QAren helburua, datuek erakusten duten aldakortasunaren tamaina eta beronen iturri posibleak identifikatzea da, errore analitikoak gutxitzeko, emaitzek kalitate onargarria izateko, ikertzaileok zein administrazioaek aurkezten zaizkien emaitzetaz fidatu ahal izateko eta laborategi ezberdinek eskuratzen dituzten emaitzak konparagarriak direla bermatzeko. Horrela, talde ezberdinen arteko neurketa-frogetan parte hartzea ezinbesteko kontsideratu beharko litzateke. Zelai zein laborategiko jarduera analitikoak beteko dituzten lan-prozedura estandarren prestaketa (SOP) hobetzea garrantzitsua da edozein etekin zientifiko lortu nahi bada (JAMP 2003). SOP ezberdinen laborategi arteko konparaketek akatsak identifika ditzakete eta beraz metodologiaren armonizazioa bultzatu. Orain arte, Technics in the Marine Environmental Sciences-en (Times Series), itsas-ingurunean ematen diren efektu biologikoen neurketarako 16 metodo eta prozeduren deskribapen zehaztua argitaratu da ( QUASIMEMEren (Quality Assurance of Information for Marine Environmental Monitoring) barruan, analisi kimikoetarako QA etengabeko hazkundea bultzatu da eta ahalegin honek prozedura horiek azterketa biologikoetan ezartzeko markoa bidera dezakete etorkizunean. Azken hamarkadetan geografikoki mugatutako interkalibrazio ariketak burutu dira Mediterranean Pollution Biomonitoring Programme (MED POL) Mediterraneoan, Biological Effects Quality Assurance in Monitoring Programmes (BEQUALM) Ipar Itsasoan eta esparru mugatuagokoak laborategi ezberdinen artean. 1995ean, MED POLek biomarkatzaileen erabilpena bideratu zuen Mediterraneoan zehar burututako eskala handiko biojarraipen-programa batean, non QA protokoloak garatu ziren, laborategien arteko biomarkatzaileen gaineko datu konparagarriak lortu asmoz (Gabrielides 1997). BEQUALM proiektua, urtean ezarri zen OSPARen eskakizunen aurreko erantzun zuzen legez, nazio zein nazioarteko itsasoko jarraipen-programetan parte hartzen duten laborategiek, OSPAR JAMP eta CEMP esaterako, efektu biologikoen kontrol-kalitatea lortzeko beharrezkoa den infraestruktura europarra ezartzeko asmoz. Itsas-organismoetan neurturiko biomarkatzaileen erantzunen orekarako QA programa Norwegian Institute for Water Research-ak (NIVA) ( zuzentzen du. Programa honetan, interkalibrazio ariketak eta lanketasaioak antolatu dira metodo biologiko ezberdinen kasuan. Emaitzek, tekniketako asko jarraipen- 8

15 Sarrera programetan erabiltzeko nahiko sendoak direla adierazten ari dira ( 2. Irudia. Bioinformatikak lagunduta, "omic" izenaz ezagutzen diren teknologien bidez, toxikotasun-mekanismoak ezagutu daitezke, esposizio mota bakoitzaren aurrean sinadura espezifikoak emanez eta ekosistemen osasun-egoeraren inguruko aurreikuspenak baimenduz. Horrela, inguruneko arriskuen azterketa burutzea posible egiten dute. K. Chipmanek luzatutako irudia. QA erronka sendoa da ingurunearen kalitatea aztertuko duten etorkizuneko teknika molekularrentzako, bereziki teknika hauen ekoizpen maila altua dela eta. Konposatu kimikoen pean modu ezberdinean erregulatutako geneen kuantifikazioan erabiltzen den RT-PCR teknikan, ez da QArik ezartzen aplikatzen kutsaduraren jarraipen-programetan. Esate baterako, ingurunekutsaduraren azterketan gehien neurtzen den espresioa, besteak beste, honako geena da: CYP1A1, konposatu aromatiko lauen peko biomarkatzaile legez (George et al. 2004; Pacheco et al. 2005), bitelogenina eta zona erradiatako proteinak, konposatu estrogenikoen peko biomarkatzaile legez (Rotchell eta Ostrander, 2003), metalotioneinak metalen peko biomarkatzaile legez (Lemoine eta Laulier 2003; Tom et al. 2004; Zorita et al. 2007b) eta hsp proteinak proteinen desnaturalizazioak eragindako estresa adierazteko biomarkatzaile legez (Franzelliti eta Fabbri, 2005; Cellura et al. 2007). RT-PCR (Q-PCR) teknikak, zehaztasun maila baxuagoa erakusten duten teknika semikuatitatiboen gainetik ezartzeko adostasunera heltzeko beharra ikusten ari da. Bestalde, Q-PCRn erabilitako kimika antzekoa da ikerketa ekotoxikologiko gehienetan (SYBR-Green) (Dondero et al. 2005; Mortensen eta Arukwe 2007). Adostasun maila hor amaitzen da, RNA erauzteko eta cdna ekoizteko erabiltzen diren metodoak ezberdinak baitira. Izan ere, kuantifikaziorako zein datuen tratamendurako modu ezberdinak daude (Cellura et al. 2006; Dondero et al. 2006; Mortensen eta Arukwe, 2007). Zelaian lan egiten duten ekotoxikologista gehienek behatzen duten arazoa, housekeepingekiko burutu beharreko intereseko genearen normalizazioaren beharra da. Konposatu kimikoen pean mantendutako organismoetan ez dago espresioa aldatzen ez duen generik, izan ere β- aktina edo 18S rrna bezalako housekeeping klasikoek esposizio-egoera ezberdinen pean erregulatzen direla plazaratzen ari da behin eta berriro (Arukwe 2006; Filby eta Tyler, 2007). Modu honetan, housekeeping ugari erabiltzea proposatzen dituzten zientzialariak daude (Filby eta Tyler, 2007) eta baita housekeepingik gabeko datu gordinak erabiltzea proposatzen dutenak ere (Arukwe 2006). 9 S A R R E R A

16 Sarrera Mikrotxipak darabiltzaten ekotoxikogenomikako ekoizpen altuko tekniketan QA ezartzetik urrun samar gaude oraindik. Sakonago aztertu beharreko gaia denez, ekoizpen altuko tekniken ondorengo atalean eztabaidatuko dugu (3.3. atala). 2. Toxikoen eraginpeko geneen moldatze-erantzunak Ikerketa ekotoxikogenomiko gehienek animalia esposatuen geneen espresioa animalia kontrolen transkriptomarekin konparatzen dute. Hala ere, transkripzio-profila alda dezaketen berezko iturri ugari daude, horien artean, organismoen egoera fisiologikoa, beraien garapen-fasea, adina, analisia burutzen deneko sasoia, generoa eta polimorfismo genetikoak. Esate baterako, geneen espresioan eragiten duten DNAren eskualdeak oso aldakorrak dira, %0.6 gune polimorfiko agertuz (Oleksiak et al. 2002). Oleksiak eta laguntzaileek (2002) Fundulus heteroclitus populazio berean, ikertutako 907 geneen %18an gutxi gora behera espresio mailako ezberdintasun esangarriak aurkitu zituzten. Espresio-ezberdintasuna gehienetan 1.5 faktore ingurukoa zen eta sarritan 2.0koa. Populazioen arteko ezberdintasunek aldagarritasuna areagotzen zuten (Oleksiak et al. 2002). Laborategian baldintza bertsuetan aklimataturiko F. heteroclitus arrainen hiru ar populazio ezberdinetatik isolatutako bihotzeko bentrikuluetan burututako gene metabolikoen espresio mailako ikerketen arabera, geneen %94aren espresioa ezberdina zen populazio bereko indibiduoen artean. Ezberdintasun hauek, maila genetiko zein bestelako iturri biologikoek ondorioztatutako esperotako aldagarritasuna osatzen dute populazio naturalen barruan (Oleksiak et al. 2005). Geneen espresio mailako aldagarritasun normala ezartzea urrats kritikoa da beraz, espresio mailetan emandako aldaketa, estresatzaile jakin batekiko erantzuna den ezberdindu nahi denean. Aldagarritasuna ondorioztatzen duen beste faktore bat generoa da. Tyne itsasadar kutsatuko Platichthys flesus platuxak dentsitate baxuko mikrotxip batean (160 gene) Alde itsasadar garbiarekin konparatzean, 11 gene kutsaduraren markatzaileak zirela ondorioztatu zuten Williams eta lagunztaileek (2003). Hala ere, gene hauek ar helduetan bakarrik ziren esangarriki ezberdinak (>2 geneen espresio aldagarritasuna), emeen populazioan, espresioaren aldakortasuna oso altua izatean, populazioen arteko ezberdintasunak estalita geratu ziren. Orduz geroztik, ikertzaile-talde hau zelaian jasotako organismoetan burututako mikrotxip mailako ikerketak bideratzean, platuxa arren populazioetan zentratu dira (Chipman, komunikazio pertsonala). Antzera, M. edulis muskuiluaren kasuan, konposatu kimikoen eraginpeko transkripzio mailako erantzuna emeetan arretan baino altuagoa dela deskribatu da (Brown et al. 2006), beraz, organismo emeak erabiltzea proposatzen da biomarkatzaile berrien bilaketan. Aldakortasun honen jakitun izatea eta indar estatistiko nahikoa izango duten esperimentuak planifikatzea beharrezkoa da emaitza erabilgarriak lortu ahal izateko. Laginak taldeetan elkartzea estatistikaren indarra areagotzeko modua izan daiteke, populazio barneko aldakortasuna estalduz. Bestalde, indibiduoen arteko espresio-profilen aldakortasuna neurtu nahiko bagenu, lagin kopurua handitu beharko genuke (Denslow et al. 2007). Garrantzitsua da, geneen espresio mailaren gainean denbora eta dosiak duten menpekotasuna finkatzea, (eko)toxikologian burutzen diren transkripzio mailako ikerketetan, ekintza-mekanismoa zehazterako orduan horiek baitira kontutan hartu beharreko puntuak. Kutsatzaile baten eraginpean egon eta ordu gutxiren buruan, geneen espresio-patroiak zeharo aldatzen dira. Epe laburrean edo dosi baxuetan, induzitzen diren geneak, toxikotasunaren aurrean organismoa babesten duten moldatzegeneak izaten dira oro har (3. Irudia). Dosia emendatu ahala, organismoa, estresatzailearen eraginpean egotearen ondorioz galdutako oinarrizko bidezidor biokimikoak konpentsatzen saiatzen da eta ordezko bidezidor ezberdinak aktibatzen dira gene multzo berrien espresioa eraginez. 10

17 Sarrera Konpentsazio-aldaketak, esposizioak eragindako homeostasiaren kontrako efektuekin daude lotuta beraz. Dosia edo esposizio-denbora oraindik gehiago areagotuz gero, organismoak ezingo du konpentsatzen jarraitu eta efektu kaltegarri larriak emango dira. Egoera honen pean, geneen espresioaldaketek jarraituko dute, baina ezingo da homeostasirik lortu. Maila honetako esposizio etengabeak gaixotasuna ondoriozta dezake, hazkuntzaren zein ugalketaren etetea edo/eta heriotza. Moldatzeak, konpentsazioak eta toxikotasunak erantzun etengabeak emango dituzte eta erantzunaren fase bakoitzarekin asoziaturiko gene multzoak aldatzen joango dira (Denslow et al. 2007). Konposatu kimiko jakinen eragiteko modua aztertu nahi bada, garrantzitsua da espresio mailako ikerketak erantzunaren moldatze-fase edo/eta konpentsazio-fasean burutzea (3. Irudia). Kaltea eragiten duten geneak aktibatzen badira berriz, organismoak emandako erantzuna ezingo da erlazionatu kutsatzaileak eragindako toxikotasun-mekanismo espezifikoarekin (Denslow et al. 2007). 3. Irudia. Toxikotasunaren urratsak. Kutsatzailearen kontzentrazio baxu pean eta epe laburrean, organismoek moldatze-bidezidor biokimikoen bidez erantzungo dute. Kutsatzailearen kontzentrazioa (edo denbora) areagotu ahala, konpentzazio-mekanismoak aktibatuko dira homeostasia mantentzeko asmoz. Kutsatzaileen maila toxikoen eraginpean, organismoek nekea agertzen dute eta ezingo diote luzaroan erantzun kutsatzaileari, toxikotasun terminalean sartuz. Ikerketa toxikogenomikoek lehenengo bi faseetan ematen diren aldaketak aztertzen zentratu beharko lirateke, eragin kaltegarriak agertu aurretiko aurreikuspena egiteko gai izateko helburuz. Denslow et al. (2007)-tik moldatutako irudia Molekula mailako moldatze defentsa-mekanismoak, "defentsoma kimikoa" Baldintza fisiko, kimiko eta biologikoei aurre egin beharrak, eboluzioan zehar moldatze bidezidorretan parte hartuko duen gene-sarea garatzera behartu du animalietan; honek, kaltearen aurrean babesa eskaintzen du eta hainbat kasutan kalteen konponketa bideratu ere. Aipaturiko babes hau gauzatzeko gai den gene eta proteina sarea, konposatu kimikoen aurreko molekula mailako moldatze-defentsa-mekanismoaren frontea da eta "dentsoma-kimiko" izenarekin ezagutzen da (Goldstone et al. 2006). Bidezidor horien erregulazio egokiaren bidez lortutako zelularen homeostasiak, ez da bakarrik konposatu xenobiotikoen inaktibazio/eliminazioan arituko. Bestelako konposatuen eliminazioan ere arituko da, hala nola: a.- Esteroideak, oxigeno espezie erreaktiboak, ROS, lipido-peroxidoak, eta hemo taldeen anderatze-produktuen bezalako molekula endogenoena. 11 S A R R E R A

18 Sarrera b.- mikrobioen produktuak, fitotoxinak, eta bestelako inguruneko konposatu naturalena (ez bakarrik gizakiaren jardueren ondorioz agertzen direnak), horien artean naturalki esistitzen diren metal eta PAHena. Defentsoma kimiko honek, lotugai bidez aktibatzen diren transkripzio-faktoreak biltzen ditu nagusiki; zeluletako toxikoen aurrean, toxikotasun gutxiagoko konposatu hidrofiliko eta iraizteko errezagoak diren konposatuak, oxidazio, erredukzio edo konjugazioz eraldatzen dituzten bioeraldaketa-entzimak, kutsatzaileak edo/eta beraien metabolitoak zitosoletik aktiboki ponpatuz diharduten garraiatzaileak eta ROSen aurrean zein bestelako makromolekulen erradikalen aurkako babesean diharduten entzima antioxidatzaileak (Nakata et al. 2006) Proteina garraiatzaileak (0 faseko bioeraldaketa-metabolismoa) Konposatu toxikoen aurkako defentsa, zelula barrura sartu beharreko konposatu anfipatiko edo arinki lipofilikoen barneraketa murriztuz hasten da. Hau lortzeko, konposatu hauen kanporaketa aktiboa gauzatzen da ATP Binding Cassette (ABC) edo droga anitzen kanpora garraio-proteina izenarekin ezagunak diren proteina garraiatzaileen bitartez (Annilo et al. 2006). Gizakiaren genoman 48 ABC gene garraiatzaile identifikatu dira. 7 azpifamilitan daude antolatuta, ABC A-G bitartean izendatzen direlarik (Dean eta Annilo, 2005). ABCB, C eta G familien artean (p-glikoproteinak) konposatu toxikoak kanporatzen dituzten eta eta droga anitzen edo xenobiotiko anitzen garraiatzaile (multidrug or multixenobiotic transporters) izenarekin ezagunak diren proteinak daude (Cole eta Deeley, 1998; Sarkadi et al. 2004; Annilo et al. 2006). Genoma eukarioten artean, ABC gene kopuruan aldakortasun esangarria badago ere, (Annilo et al. 2006; 1 Taula), familia bakoitzaren proportzio erlatiboak konstante matentzeko joera agertu du. Gizakian aurkitutako ABC proteinen azpifamilia guztiek homologoak dituzte Ciona instestinalis eta zebraarrainaren genometan, zebra-arrainek ABCH gene bakarrak osatutako familia berri bat agertzen duelarik (Annilo et al. 2006). Itsas-trikuek 65 ABC gene dituzte 8 azpifamiliatan antolatuta, tartean droga anitzen garraiatzaileak diren 3 azpifamiliak (Goldstone et al. 2006). Itsas-trikuen kasuan ABCC familia bestelako deuterostomoen genometan baino %25 handiagoa da, gutxienez 30 gene agertuz, ABC garraiatzaile guztien ia-ia erdia betez. Moluskuetan, ABC garraiatzaile ezberdinen cdna zatiak klonatu dira. M. galloprovincialis eta M. edulis muskuiluan, 2 ABC garraiatzailearen zatiak klonatu dira droga anitzen erresistentziarekin erlazionaturiko proteina bat (ABCC azpifamiliakoa) eta p-glikoproteina bat (ABCB azpifamiliakoen antzekotasun handiagoa) (Luedeking eta Koehler, 2004; Franzellitti eta Fabbri, 2006). 1. Taula. Phyla ezberdinetako animalien genoma-sekuentzien homologiaren analisiaren bitartez aurkitutako defentsomako 4 gene-talde kanonikoen kopuru. Arrain teleosteoen datuak Fugu eta zebra-arrainetik lortu dira, tunikatuenak C. intestinalisetik, ekinodermoenak S. purpuratusetik, intsektuenak D. melanogasteretik (CYP geneak dira: 46 Apis mellifera, 81 Bombix mori, 85 D. melanogaster, 103 Anopheles gambiae, 132 Aedes aegypti eta 143 Tribolium castaneum) eta nematodoenak C. elegansetik, hainbat artikulutan argitaratu bezala (Enmark eta Gustafsson, 2001; Maglich et al. 2003; Yagi et al. 2003; Sheps et al. 2004; Menzel et al. 2005; van Gilst et al. 2005; Annilo et al. 2006; Feyereisen 2006; Goldstone et al. 2006; Velarde et al. 2006). bhlh-pas Nuclear Receptors CYP genes ABC transporters Human Teleost? 68, /56 Tunicate /31 Echinoderm Insect 15 18/19 +3/2 pseudogens 46 to Nematode pseudogens >

19 Sarrera Konposatu kimiko ezberdinek, tartean ugaztunen LXR eta FXR-ren (ikus aurrerago) lotugaiak, ugaztunen ABCC geneak gainerregulatzen dituzte (Kast et al. 2002; Kullak-Ublick eta Becker, 2003) eta itsas-trikuaren ABCC9A ere induzi dezakete (Goldstone et al. 2006). Zenbait konposatu kimikok ere, p-glikoproteinaren espresioa gainerregulatzen du arrain ezberdinen gibel eta zakatzetan (Bard et al. 2002a; 2002b Zaja et al. 2007). Moluskuen ABC garraiatzaileen espresioa ere metalen (Franzellitti eta Fabbri, 2006) eta gazitasun baxu zein anaerobiosiaren eraginpean (Luedeking and Koehler, 2004) erregulatu egiten dela ikusi da. Modu berean, p-glikoproteina garraiatzailearen jarduera Diesel-2 olioaren eraginpean mantendutako muskuiluetan emendatu egiten dela ikusi da (Smital et al. 2003). ABC garraiatzaileekin batera, birikako erresistentzia-proteinak ere, III. faseko sistemako partaideak dira eta konjugaturiko metabolitoen iraizketan dihardute (Luedeking eta Koehler, 2004). Familia honetako partaide induzigarri bat klonatu da muskuiluan (Luedeking eta Koehler, 2004; Franzellitti eta Fabbri, 2006) eta 3 gene ezberdin sekuentziatu dira Aplysia californicaren neuronetako transkriptoman (Moroz et al. 2006) Bioeraldaketa oxidatiboa (I faseko metabolismoa) Oro har, toxikoak zitoplasman barneratu ondoren, hauek eraldatu beharra dago konposatuon eliminazio edo inaktibazioa bideratzeko. Konposatu kimikoen eraldaketa oxidatiboa (I fasea) metabolito hidrofilikoagoak eratzeko ematen da eta sarritan, iraizketarako lehenengo urratsa da (Rewitz et al. 2006). Oxidazioa P450 zitokromoek (CYP), flabin monooxigenasek (FMO) eta aldehido dehidrogenasa (ALDH) entzimek burutzen dute. CYP superfamilian, CYP1, CYP2, CYP3 eta CYP4 familiak xenobiotikoen bioeraldaketan espezializatu dira ornodunetan (Lewis et al. 2002; Rewitz et al. 2006). Itsas-trikuak 120 CYP gene ditu eta 1-4 gene-familiei dagozkienek kopuru totalaren zati handi bat betetzen dute (%80), genefamilia hauetan funtzionalitatearen hedatzea emateko presioaren isla. Zuhaitz filogenetikoan zehar klonaturiko CYP gene familia berriek eta CYP genearen duplikazio anitzen arrasto argien agerpenaren ondorioz (tunikatu, ekinodermo eta intsektuen genoma kasu, 1. Taula), CYP nomenklaturan oinarritutako izendegia konplikatu egin da eta eta nomenklatura sinpleago bat bideratu da non, CYP geneak familiarteko erlazioak islatzen dituzten klan ezberdinetan antolatzen diren (Nelson et al. 2004; Rewitz et al. 2006). Zentzu honetan, 2. klanak CYP2, CYP1 eta beste enztima batzuk biltzen ditu eta 3. klanak CYP3 eta estukien erlazionaturiko intsektuen CYP6 eta CYP9 entzimak (Nelson et al. 2004). Ornodunen CYPak, xenobiotikoak metabolizatzen dituzten entzima kritikoak dira tartean, hainbat PAH kartzinogenikoren aktibazioa katalizatzen dutenak. Hidrokarburo aromatiko polizikliko eta halogenatu lauek zein hainbat konposatu naturalek CYP gene asko induzitzen dituzte aril hidrokarburoen hartzailearen bidez (AHR, 4. Irudia) (George et al. 2004; Godard et al. 2005; Hahn, et al. 2006; Lewis et al. 2006). CYP1aren antzeko 11 gene daude itsas-trikuaren genoman, familia honetako ordezkariak ugariagoak izanik C. intestinalis (5 ordezkari) edo ornodun gehienetan (3-4 ordezkari) baino (Godard et al. 2005; Goldstone et al. 2006). CYP1A azpifamilia ornodunetara mugatuta dagoela ematen duen arren (Rewitz et al. 2006), CYP1A-aren antzeko proteinak ornogabeetan, tartean Pazifikoko ostrak (Boutet et al. 2004a), klonatu izana azaldu da, sekuentzien arteko konparaketak gauza bera esaten ez badu ere. Esangarriki induzigarriak diren bi CYP1A daude ugaztunetan eta 1 arrainetan, ornodunetan CYP1B1 bat dago, Superfund estuarioetan bizi diren Fundulus heteroclitus arrainetan esangarriki azpiespresatzen dela deskribatu dena (Fisher eta Oleksiak, 2007) eta bi CYP1C gene ezberdin esistitzen dira arrainetan (Godard et al. 2005). 13 S A R R E R A

20 Sarrera Ugaztunetan, CYP2 entzima dibertsoak substratu ugariren oxidazioaren arduradunak dira (Lewis et al. 2004). Itsas-triku eta intsektuetan (bereziki zenbait diptero eta koleoptero) CYP2ren antzeko gene kopurua beste genomekiko areagotu da, eta itsas-trikuan aurresandako 73 daude, 76 Aedes aegyptin eta 70 Tribolium castaneum. Honek itsas-trikuen CYP totalen %60 baino gehiago eta koleopteroen %57a ordezkatzen du, ornodunen genomako %30-49arekin kontrajarriz (Feyereisen 2006; Goldstone et al. 2006). Azpifamilia honetako partaideak krustazeotan ere deskribatu dira, hainbat konposatu organikoen eraginpean induzigarriak izanik (Rewitz et al. 2006). 4. Irudia. Ornodunetan hainbat gene, CYP geneak besteak beste, aril hidrokarbonoen hartzailearen (AHR) bitartez aktibatzen dira. Hainbat konposatu natural eta hidrokarburo aromatiko polizikliko (dioxinak, TCDD) zein halogenatu lau AHR-ren agonista dira. Agonisten lotura eman ondoren, AHRek xenobiotikoen erantzun elementuen gainean dihardute (XRE edo DRE) bioeraldaketa metabolismoaren gainespresioa eraginez. Biocarta-tik hartutako irudia. Gizakiaren gibeleko CYP3A entzimek klinikoki erabiltzen diren drogen %40-60 metabolizatzen dute (Guengerich 1999), hormona endogeno, onddo eta landareen produktu eta inguruneko kutsatzaileekin batera (Guengerich, 1999; Thummel eta Wilkinson, 1998). Tunikatuek lau gene dituzte filogenetikoki ornodunen CYP3aren antzekoak direnak (Verslycke et al. 2006). CYP3aren antzeko 10 gene oso eta guztiz osatu gabeko 7 identifikatu dira itsas-trikuaren genoman (Goldstone et al. 2006). Analisi filogenetikoek adierazten dute gene hauek 3. klaneko gainontzeko geneekin bat datozela, ornodun ta tunikatuen CYP3 geneekin, intsektuen CYP6 eta CYP9 geneekin eta ornodunen CYP5 geneekin batera. CYP6 genea esate baterako, fenobarbital eta pestizidek induzitzen dute eta CYP6G1 geneari intsektuek intsektiziden aurrean duten erresistentziaren erantzule izatea egozten zaio. Ikusi denez, zenbait polimorfismok geneen espresioa areagotu egiten dute, honek zenbait intsektiziden iraizpena ondorioztatzen duelarik (ffrench-constant 2007; Feyereisen 2006). Moluskuetan, CYP3 genearen klaneko geneekin homologia altuena agertzen duen CYP gene bat klonatu da Mercenaria mercenariaren gonadan (Rewitz et al. 2006). CYP4 familia, animalietan ezagutzen diren CYP familietatik zaharrena da, knidario, anelido poliketo eta moluskuetan agertuz (Rewitz et al. 2006). Peroxisomen proliferatzaileak CYP4ren substratuak dira eta ugaztunetan CYP4ren espresioa peroxisomen proliferatzaileek aktibaturiko hartzailearen (PPAR) erregulazioaren pean dago (Waxman 1999; Mandard et al. 2004). Antzera, peroxisomen proliferatzaileak diren di(2-etilhexil)phtalato eta 2,4-diklorofenoxi azido azetikoaren,, ziztada intraperitonealak, CYP4T2 genearen espresioa eragiten du Dicentrarchus labraxen 14

21 giltzurrunean (Sabourault et al. 1998). Itsas-trikuaren genomak 10 gene dauzka CYP4aren antzekoak direnak, intsektuek CYP4ren klaneko gene dituzten bitartean (Feyereisen 2006; Goldstone et al, 2006). Interesgarria da, CYP4 entzima bat, CYP4BB1 alegia, benzo(a)antrazeno, olio gordin eta klofibratoaren (peroxisomen proliferatzailea da) eraginpean induzigarriak direla Nereis diversicolor poliketoan (Rewitz et al. 2006). FMO entzimak NADPH-menpeko nukleofilo ahulen N- edo S-oxidazioa katalizatzen du (Cashman 2005; Ziegler 2002). Gizakiaren genomak 6 FMO funtzional ditu eta 5 pseudogene (Hernandez et al. 2004). Itsas-trikuaren genomak 16 FMO ditu (Goldstone et al. 2006). Flabina duen 2-monooxigenasa (FMO-2) eta monoamino oxidasa A (MAO A) kodetzen duten cdna osoak klonatu dira Crassostrea gigasen hidrokarburoen eta bi pestiziden eraginpean mantendu ostean (Boutet et al. 2004a). Hidrokarburo eta pestiziden tratamendua, glifosato eta atrazina, diuron eta isoproturonak osatutako nahastea, gene bien espresio diferentzial bortitzaren erantzule da, gainespresio hau ehuna, denbora eta tratamenduarekiko espezifikoa izanik (Boutet et al. 2004a; 2004b). Aldehido dehidrogenasa entzimek (ALDH), oso erreaktiboak diren aldehido elektrofilikoak oxidatzen dituzte; konposatu hauen artean, amino azidoen metablismoan sortutako aldehido endogenoak, karbohidratoak, aminak, esteroideak eta inguruneko konposatu kimiko ugari daude. ALDH entzimek zelulako homeostasia mantentzen jardun dezakete ere, zelulako erredox oreka mantenduz, ROSen eliminazioa dela bide, eta NADPH edo NADH bezalako baliokide erreduzitzaileak sortuz. Gizakiak 19 ALDH gene ditu eta itsas-trikuak Errudukzio- eta konjugazio-bioeraldaketa (II. faseko metabolismoa) Sarrera Oxidazioaren ondoren erredukzio edo konjugazioen bidezko eraldaketak ematen dira glutation-stransferasa (GST), sulfotransferasa (SULT), UDP-glukuronosil transferasa (UGT), N-azetil transferasa (NAT), aldo-keto erreduktasa (AKR), epoxido hidrolasa (EPHX), eta NAD(P)H-kinona oxidoerreduktasa (NQO) entzimen bidez. Entzima hauek (GST, SULT, UGT, NAT) lehenengo edo bigarren mailako konjugazio erreakzioak burutzen dituzte substantzia toxikoen gainean edo toxikoak erreduzitu edo hidrolizatu egiten dituzte (NQO, AKR, EPHX), guztien artean II. faseko bioeraldaketa metabolismoa osatuz. Lortzen diren metabolito konjugatuak, zeluletako mintzen zehar kanporatzen dira ABC-garraiatzaileen bitartez. Hori dela eta, ABC-garraiatzaileak III. faseko metabolismoan ere dihardutela kontsideratzen da (Luedeking eta Koehler, 2004). Kontutan eduki behar dugu, prozesu hauek guztiak eragin kolateralak izan ditzaketela, bioeraldaketa metabolismoak orokorrean detoxifikazioa gauzatzen badu ere, oxidazio eta N-azetilazioa eta sulfato edo glutationaren konjugazioan metabolito toxiko eta mutagenikoak sor daitezke, benzo(a)pirenoaren metabolismoaren bidezko aktibazioa horren adibide izanik (Gamage et al. 2003; 2006). II. faseko bioeraldaketa metabolismoan diharduten geneen kopurua gizaki eta itsas-trikuen genometan baino ez da katalogatu (Goldstone et al. 2006). Itsas-trikuek xenobiotikoak konjugatzen dituzten hainbat entzima dituzte, horien artean, 36 SULT gene zitosoliko, 49 UGT gene eta 38 GST gene. Honek, gene hauen dibertsifikazio handia eman dela suposatzen du, gizakiarekin konparatuz gero, non soilik 13 SULT, 13 UGT eta 17 GST gene dauden (Gamage et al. 2006; Mackenzie et al. 2005; Nebert eta Vasiliou, 2004; Pearson, 2005). Hala ere, intsektuen genomak gene-familia hauetako partaide gehiago izan ditzake, esate baterako, D. melanogasteraren genomak 33 UGT gene ditu (Luque eta O'Reilly, 2002). UGTri homologia ezartzea zaila da, baina dbest datu baseetan burututako bilaketa arinak 20 UGT ezberdinekin homologia erakusten duten EST sekuentziak azaltzen ditu (1A1, 2 eta 5, 1B1 eta 2, 2A1 eta 3, 2B2, 3, 4, 7, 9, 10, 13, 15, 17, 20 eta 34, 2C1 eta 15 S A R R E R A

22 Sarrera 3) arrain espezie ezberdinetan. Konposatu kimikoen eraginpean, hainbat UGT ezberdin gainespresatzen dira (Williams et al. 2003). Itsas-trikuaren 38 GSTek, GSTen arteko 6 mota nagusiak ordezkatzen dituzte (9 alfa, 1 pi, 17 sigma, 6 teta, 1 zeta, 4 omega). Ugaztunetan, alfa, pi, eta mu GSTak, PAH-metabolitoen konjugazioaz arduratzen dira (Sundberg et al. 1997). Omega motako GST bat gainespresatu egiten da hidrokarburoen eraginpean mantedutako Pazifikoko ostretan (Boutet et al. 2004b). GST mikrosomalak (MGST) bestalde, substratu espezifizitate zabala duten mintzari lotutako transferasak dira, horietako batzuek ugaztunen hantura erantzunetan parte-hartze zuzena izanik (Trebino et al. 2003). MGST motako hamaika gene oso eta partzialki lortutako bat aurkitu dira itsas-trikuaren genoman, gizakiaren genoman 3 besterik ez dauden bitartean (Pearson, 2005). SULT zitosolikoak xenobiotikoen metabolismoaren eta substratu endogeno txikien erantzuleak dira (SULT1 eta SULT2), mintzari lotutako SULTak peptido, lipido eta glikosaminoglikano endogenoen sulfonazioan ari diren bitartean (Gamage et al. 2006). 36 SULT zitosoliko daude itsastrikuaren genoman. 10 SULT zitosoliko klonatu dira zebra-arrainean gaur egun arte, 6 SULT1 gene, 3 SULT2, beste batek ezagutzen diren SULT gene-familia guztietatik kanpo dagoela ematen duelarik(yasuda et al. 2006). II. faseko entzima erreduzitzaile eta hidrolizatzaileak ere, itsastrikuaren genoman badaude ordezkatuta (Goldstone et al. 2006). Mintzetako bi EPHX gene daudela ematen du, hauek, ugaztunetako xenobiotikoen bidez induzigarria den EPHX1en antzekoak izanik, eta 3 EPHX solugarri, ugaztunetako EPHX2 geneekin erlazionatuta daudenak (Goldstone et al. 2006) Proteina antioxidatzaileak Babes antioxidatzaileak defentsa-sistemaren ezinbesteko osagaiak dira ingurune aerobikoetan bizi diren organismoetan. ROSak metabolismo normalean zehar ekoizten dira baina beraien ekoizpena areagotu egiten da organismoak toxikoen zein izpi ultramoreen eraginpean egotean, makromolekulak kaltetu eta toxikotasuna sortuz. Proteina antioxidatzaileen artean, superoxide dismutasa, katalasa, eta peroxidasek, horien arten, glutation peroxidasa, tioredoxinak eta aurretik aipatutako GSTak, jarduten dute ROSen detoxifikazioan glutation-glutation disulfuro (GSH-GSSG) erredox zikloaren bitartez. Gene guzti hauek, metazooen genometan daude ordezkatuta eta zenbait konposatu kimikoren eraginpean aipaturiko entzimen jarduera areagotu egiten dela ikusi da arrain eta muskuiluetan (Boutet et al. 2004b; Mimeault et al. 2006) Hartzaileak eta seinale-transdukzioa Zenbait proteina-familia, bhlh-pas proteinak, hormonen hartzaile nuklearrak eta estres oxidatiboari erantzuten dioten transkripzio-faktoreak konposatu kimikoak detektatzen eta bioeraldaketa metabolismoan parte hartzen duten proteinen espresioaren erregulazioan parte hartzen dute (0-tik III. fasera), gutxienez ornodunetan (Ulrich et al. 2003; Hahn et al. 2006). The bhlh-pas receptor family bhlh-pas proteina-familiaren barruan, garapen-fasean zeharreko seinaleztapenean, erritmo zirkadianoak ezarri eta mantentzen eta oxigenoa, hormonak eta xenobiotikoak bezalako inguruneko aldagaiak detektatzen parte hartzen duten transkripzio-faktoreak aurkitzen ditugu (Gu et al. 2000; Kewley et al. 2004). Gene-familia hau hedatu egin da ornodunetan, soilik intsektu, nematodo, tunikatu eta ekinodermoen genoman aurkitzen den gene bakoitzeko bi edo hiru paralogo erakutsiz (Hahn, 2002; Hahn et al. 2006; Goldstone et al. 2006). Familia honetan ikertuen dagoen partaidea 16

23 sentsore kimiko gisa betetzen duen funtzioa dela eta, AHR eta berarekin erlazionaturiko hartzaileak dira, aril hidrokarburuaren hartzailearen translokatzaile nuklearra (ARNT) eta aril hidrokarburuaren hartzailearen errepresorea (AHRR) (Hahn et al. 2006; Mortensen eta Arukwe, 2006). AHRa interesgarria da hidrokarburo aromatiko lauek, dioxina edo hainbat PAHek, eta bestelako zenbait konposatu kimikok hartzaile hau aktibatzeko ahalmena dutelako (Denison eta Nagy, 2003). Ondoren, AHRak zenbait CYP generen eta II. faseko bioeraldaketa metabolismoko entzimak kodetzen dituzten geneen erregulazioa gauzatzen du (Nebert et al. 2004; Hahn et al. 2006; Mortensen eta Arukwe, 2007). Hala ere, ornodunen AHRak ez bezala, ornogabeetan ezagutzen diren AHR ortologoek, Mya arenaria moluskuarenak edo intsektu eta nematodoenak adibidez, ez dituzte lotzen ornodunen AHRren lotugai tipikoak diren xenobiotikoak (Butler et al. 2001; 2004; MacMillan eta Bradfield, 2007), aril hidrobarburoen metabolismoa ornodunen moldatze goiztiarra izan daitekeela iradokiz. AHR eta HIF1α (biek erabiltzen dute ARNT heterodimerisaziorako bikote legez), duplikatuta agertzen dira itsas-trikuaren genoman, Ciona eta protostomoek gene bakarra agertzen baitute (Goldstone et al. 2006). Teleosteotan ere bi AHR gene esistitzen dira (Hahn et al. 2006). Itsastrikuaren bi HIF geneek ornodunen paralogoak dirudite. HIF proteinek, oxigeno maila baxuaren aurreko erantzuna, eboluzioan zehar kontserbatuta dagoen eta garapenerako metazoo gehienetan ezinbestekoa den seinalizazio-mekanismo baten bidez bideratzen dute (Lavista-Llanos et al. 2002; Shen et al. 2005; Nikinmaa eta Rees, 2005). Ornodunetan, hipoxiaren aurreko moldatze-fisiologikoa, basodilatazioa eta erantzun angiogenikoak, metabolismoaren beharrizanen murrizketa eta energia lortzeko bidezidor anaerobioen piztea erregulatzen dituzten seinale konplexuen bidez lortzen da. Oxigenoaren eskari ez bada asetzen zelula kopurua murriztu egin behar da ere (Ton et al. 2003; Papandreou et al. 2005). Prozesu hauek, HIFren kontrolpean daude espezie ornodunetan (Gracey et al. 2001, Ton et al. 2003; Papandreou et al. 2005). Bestalde, ezaguna da marearteko organismoek beraien bidezidor metabolikoak moldatzen dituztela hipoxia epe labur edo/eta luzeei aurre egin ahal izateko, egunean birritan airepean egotean esaterako. Zentzu honetan, Crassostrea virginica (CD648099) marearteko moluskuan, HIF-1 motako proteina bat identifikatu den arren, Crassostrea gigasen oraintsu burututako ikerketa batean, hipoxia gogorraren eraginpean gene talde bat gain- eta azpierregulatu egiten dela ikusi da (David et al. 2005), baina ez da islatzen ornodunetan ikusten den zelulen prozesu proliferatzaileen ezta erantzun apoptotikoen emendioaren gaineko erregulazioan. Hartzaile nuklearren superfamilia Sarrera Hartzaile nuklearren (NR) superfamiliak konposatu kimikoen aurreko sentsore legez jokatzen duen talde zabala osatzen du. Pregnano-X-hartzailea (PXR), androstano-hartzaile konstitutiboa (CAR), BXR (anfibioetan) eta CXR (txorietan) PXR-ren homologo ez ugaztundarrak, peroxisomen proliferatzaileek aktibaturiko hartzaileak (PPAR), erritinoide-x-hartzailea (RXR), estrogeno hartzaileak (ER), androgeno hartzaileak (AR), glukokortikoideen hartzaileak, tiroideko hormonen hartzailea (THR), gibeleko-x-hartzailea (LXR) eta farnesoide-x-hartzailea (FXR) hainbat xenobiotikok eta konposatu fisiologikok (nagusiki konposatu lipidiko aktiboak) aktibatu eta erantzun koordinatu bat bideratzen dute lipidoen homeostasia, eta I. eta II. faseko hainbat entzima eta garraiatzaileren bidezidorrak eraenduz (Handschin eta Meyer 2003; Ulrich et al. 2003; Handschin et al. 2004; Reschly eta Krasowski, 2006). PXR, CAR, eta PPARek xenobiotikoen aurreko CYP2, CYP3 eta CYP4ren erantzun espezifikoa induzitzen dute (Ulrich et al. 2003). Sei NR identifikatu dira A. californicaren transkriptoma neuronalean (Moroz et al. 2006). Ustezko 33 NR identifikatu dira S. purpuratusen genoman (Goldstone et al. 2006) eta ematen duenez, NR hauetako 4, NRen 1H azpifamiliarekin erlazionatuta daude. Gene hauetako batek, LXR-ren klusterrean agertzen den lotugaia lotzeko domeinua du, DNA lotzeko domeinua FXRen klusterrean agertzen den bitartean. Beste 3 NR1H motako geneak dira eta, NR1 barneko azpifamilia berria 17 S A R R E R A

24 Sarrera osatzen dute (Goldstone et al. 2006). Ez da detektatu NR1I familiako geneen ortologorik (PXR, CAR, BXR, CXR, VDR) genoma bildumetan ezta EST liburutegietan ere (Goldstone et al. 2006). Ornodunetan, xenobiotikoen sentsore gisa jokatzen duten geneon itxurazko gabezi hau, deuterostomo talde ezberdinen artean dagoen sentsore-banaketa ezberdintasunaren isla izan daiteke. Hala ere, NR1Hrekin erlazionaturiko gene nobelen esistentziak, xenobiotikoen sentsore gisa jokatzen izan dezaketen nolabaiteko aukera zabaltzen du (Goldstone et al. 2006). Zentzu honetan, artropodoen ekdisona-hartzailea (NR1H1) eta C. elegansen DAF-12 (NR1J) NRak ugalketa eta garapenerako garrantzitsua den esteroideen seinalizazioa bideratzen du, CYPak inplikatuz (Motola et al. 2006). Edozein kasutan ere, NR1I gene-familiaren ortologoak ere ezagutzen dira ekdisozoetan. C. elegansen NHR-8 eta D. melanogasterren DHR96, xenobiotikoen aurreko erantzunak eraentzen dituztela ezaguna da, tartean, fenobarbitala edo DDTarekiko fruta-eulian (King-Jones et al. 2006) eta koltxizina edo klorokinonarekikoa nematodoan (Lindblom et al. 2001). CARek ugaztunen espezifikoa ematen du, PXR eta CARek hegaztien CXR genearen duplikazioz agertuz (Handschin et al. 2004). PXR arrain-espezie ezberdinetan klonatu da eta arrainek erakusten dute, ornodun guztien NRen artean, aldagarritasun handiena lotugaia lotzeko domeinuan (Krasowski et al. 2005). PXR-ren aktibaziorako, C27dun behazuneko alkohol sulfatoen espezifizitate estua izatetik (D. rerio), espezifizitate zabalagoa izatera pasatu da hegazti eta ugaztunetan (Krasowski et al. 2005; Reschly eta Krasowski, 2006). Edozein kasutan ere, zebra-arrainen eta arrankarien PXR hartzailea xenobiotikoen bidez aktibatzen dela ikusi da, normalean ugaztunetan PXRek zuzentzen dituen CYP3A eta droga anitzen jasankortasun geneen indukzioa arrainetan ere ondorioztatzen duelarik (Bresolin et al. 2005; Celander et al. 2007). S. purpuratusek RXR (NR2B) eta HNF4 (NR2A)-ren homologoak ditu. RXRek ornodunetan, eta ornogabeetan agertzen den USP homologoak, NR1H eta NR1I proteinen dimerisazio-bikote legez dihardute transkripzioa erregulatzeko. RXRen ortologoak klonatu dira molusku-espezie ezberdinetan, eta imposexa (arren ezaugarri genitalen agerpena emeetan) eragiten duten mekanismo molekularrekin erlazionatu dira konposatu organometalikoen eraginpean mantendutako gastropodoetan (Nishikawa et al. 2004). Ikusi denez, RXRek espezifikoki lotzen ditu organoestainuak afinitate handiarekin, 9-cis azido erretinoikoa berriz, ornodunetan bezala, moluskuen RXR-ren lotugai naturala da eta Thais clavigeran imposexa eragin dezake (Nishikawa et al. 2004). PPARek (NR1C) ornodunetan, peroxisomen proliferatzaileei erantzuten diete, beraien itu-geneek lipidoen metabolismoan, energiaren homeostasian eta zelularen desberdintzaketan parte hartzen dutelarik (Mandard et al. 2004). Ornodunek ehun-banaketa eta funtzio ezberdina duten 3 PPAR isoforma (PPARα, PPARβ eta PPARγ) dituzte; berezitasuna, arrainen genomak PPARα ren bi paralogo dituela da (Maglich et al. 2003; Metapally et al. 2007). Peroxisomen proliferazioa (ikus beranduago) izenaz ezaguna den erantzun zelular pleiotropikoa, PPARα isoformak eraentzen du ornodunetan (Mandard et al. 2004). S. purpuratusen genomak bi PPAR paralogo ditu, ornogabeetan deskribatu diren lehenengoak (Goldstone et al. 2006). Hartzaile nuklearren nematodoetako sare handiak (1 Taula) ez du PPAR-ren homologorik, baina NHR-49 hartzaileak, estrukturalki ornodunen HNF4rekin erlazionatuta dagoena, PPAR-ren funtzioarekin antz handien izan dezakeen jarduera biologikoa betetzen du lipidoen kontsumo eta erregulazioan (van Gilst et al. 2005). Ornodunek, inguruneko inpaktuaren azterketan garrantzitsuak diren estrogeno eta konposatu estrogenikoen erantzunean parte hartzen duten estrogeno hartzaileak dituzte (ER, NR3A) ematen duenez, tunikatu, ekinodermo eta ekdisozoetan agertzen ez direnak ez dauden (Thornton et al. 2003; Goldstone et al. 2006). Hala ere, estrogeno hartzailearekin erlazionaturiko hartzaileak (ERR, NR3B) identifikatu dira intsektu eta ekiodermoetan (Thornton et al. 2006; Goldstone, et al. 2006). ERRak disrupzio-endokrinoa eragiten duten konposatuen ituak dira (Yang eta Chen, 1999) eta gainera, itsas- 18

25 trikuaren hartzaile batek, Sp-Shr2, arraultzetan estradiola lotzen duela jakin da oraintsu; hau, amarengandik hartutako estradiol tratamantuaren aurrean gainespresatu egiten da (Roepke et al. 2006). ER oraintsu klonatu da molusku espezie ezberdinetan: A. californica opistobrankioan (Thronton et al. 2003), Octopus vulgaris zefalopodoan (Keay et al. 2006) edo M. edulis eta Crassostrea gigas bibalbioetan (Puinean et al. 2006; Matsumoto et al. 2007), baina hala ere, moluskuetan ikusi da ez dutela estrogenorik lotzen eta beraz, transkripzioaren aktibatzaile konstitutiboak direla (Thronton et al. 2003; Keay et al. 2006). Era berean, estradiolarekin burututako tratamenduak ez du efekturik ER-ren espresio mailetan, ezta ERak erregulatutako bitelogenina (arrainetan) bezalako geneen espresioan ere (Puinean et al. 2006). Estres oxidatiboari erantzuten dioten transkripzio-faktoreak Ornodunetan, ingurunearen sentsore gisa jokatzen duten bestelako transkripzio-faktoreen artean (CNC-bZIP familia) eritroide-deribatu 2 faktore nuklearra (NFE2) eta NFE2rekin erlazionaturiko (NFE2 motakoak) 1, 2 eta 3 (Nguyen et al. 2003) gehi erlazionaturiko "cap'n'collar" (CNC)-basicleucine zipper (bzip) familiako proteinak daude; oxidatzaile eta elektrofiloek aktibatzen dituzte eta Maf proteina txikiekin heterodimeroak eratzen dituzte entzima antioxidatzaile ezberdinak kodetzen dituzten geneen transkripzioa eragiteko (Nguyen et al. 2003). Drosophilak gene bakarra du CNC (Veraksa et al. 2000). S. purpuratusen genomak, ustezko CNC-bZIP gene bakarra du, Sp-Cnc, kordatuetan emandako CNC-bZIP genearen duplikazio eta dibertsifikazioa ekinodermoak deuterostomoetatik banatu ondoren eman zela isalatuz (Goldstone et al. 2006). Ez du ematen BTBbZIP proteinak eta Maf proteina txikia ornogabeetan daudenik (Goldstone et al. 2006) Metalen detoxifikazioa Sarrera Gorputzean barneratutako metalek, metal eskuragarrien iraizketa eta detoxifikazioaren arteko ratioa gainditzen badute, metalek toxikotasuna sortzen dute (Rainbow 2002). Metalen tolerantzia, zelulako lotugaiek, metalotioneinek (MT) esate baterako, metalak bahitzeko duten gaitasunean oinarritzen da. MTak pisu molekular baxuko, zisteina kopuru altuko eta beroaren aurreko egonkortasun handia erakusten duten proteina ez-entzimatikoak dira (Kägi eta Schaffer 1988). Zisteina erresiduoen tiol taldeek baimendu egiten diete MTei metalen katioi jakinak afinitate handiz lot ditzaten (Viarengo 1989), metala-tiolato taldeak eratuz (Kägi eta Kojima 1987). MT motako proteinak organismo askotan aurkitu dira, horien artean ornodun urtarrak, arrainak nabarmenki (Olsson et al. 1998; Roeva et al. 1999), moluskuak (Bebianno eta Langston 1992; Zorita et al. 2007a, 2007b), krustazeoak (Roesijadi 1992, Engel eta Brouwer 1993) eta itsas-trikuak bezalako ornogabe urtarrak (Nemer et al. 1991). MTei beste funtzio batzuk hala nola, erradiazio ionizatzaileen aurkako babesean (Cai et al. 1999) eta oxierradikalekiko defentsa antioxidatzaile orokorran egotzi zaizkie MTei (Thornalley eta Vasak 1985; Andrews 2000; Cavalletto et al. 2002; Colangelo et al. 2004). Neurgarria den MTen lehenengo efektua mrnaren sintesia denez gero, metalek eragindako MT geneen induzigarritasuna biomarkatzaile gisa proposatu da ingurune toxikologiaren azterketan (Lemoine eta Laulier 2003; Rebelo et al. 2003; Tom et al. 2004; Zorita et al. 2007b). Ornodunetan, MT geneen eraenketa modu sakonean ikertu da (Coyle et al. 2002; Haq et al lanak ikusi), baina ornogabeetan berriz, MTen eraenketaren erantzule diren sekuentziak oso gutxi ikertu dira (Langston et al. 1998). Ornogabeetatik ornodunetara amankomunean ematen den MTen eraenketa-motiboa, MT geneen promotoreetan hainbat kopiatan agertzen den metalen aurreko erantzun-elementua da (MRE) (Samson eta Gedamu 1998). MTen biosintesiaren eraenketa, transkripzio mailakoa da nagusiki (Hamer 1986, Palmiter 1987). Estres edo metalen eraginaren ondoren, metalen transkripzio-faktorea (MTF-1) zitoplasmatik nukleora lekuz aldatzea da (Smirnova et al. 2000) eta MTen promotoreetako MRE guneetan lotu ondoren. Horrela, MT genearen transkripzio basala zein metalek bideraturiko 19 S A R R E R A

26 Sarrera transkripzioa eraentzen dute (Heuchel et al. 1994). MTF-1en homologo bat aurkitu da itsas-trikuaren genoman (Goldstone et al. 2006). Metal-ioiez, aparte, zenbait hormonek, zitokinek, hazkuntzafaktoreek, bitaminek, oxierradikalek, tumoreen sustatzaileek eta beste hainbat konposatu kimikok ere MTen biosintesia eragiten dute in vivo zein kultibatutako zeluletan Metalak lotzen dituzten bestelako geneen artean, Cu eta Fe lotzen duten zeruplasmina, Fe lotzen duen transferrina eta Fe gordetzen duen ferritina proteinaren katea astuna zein arina daude, eta moluskuetan hemolinfako Fe, baina baita Cu, Ni, Co eta Zn metalak lotzen dituzten kabortina eta pernina proteina bereziak ere aurki ditzakegu (Huvet et al. 2004; Scotti et al. 2001). Fitokelatinak nagusiki GSH taldeek osatzen dituzten eta metalak lotzen dituzten peptidoak dira; onddo eta landareetan metal detoxifikatzaile garrantzitsuak dira. Fitokelatina sintasa animalia lerro askotan aurkitu da (Clemens 2006; Goldstone et al. 2006), nahiz eta ornodunetan ez den aurkitu. Bestalde, fitokelatinak, anelido oligoketoetan klonatu dira eta beraien espresioa metal astunen eraginpean gainespresatu egiten dela ikusi da (Asensio, komunikazio pertsonala). Metal toxikoen garraio aktiboa detoxifikaziorako beste bide bat da. Zentzu honetan, ABC-garraiatzaileek metalak zuzenean edo glutationarekin konjugatuta kanpora ditzakete (Leslie et al. 2001; Sweet, 2005) Bero-talka proteinak Bero-talka proteinak (hsp) konposatu toxiko eta erradikal aske ezberdinen aurkako erantzunean parte hartzen dute animalietan (Feder eta Hofmann, 1999; Luckenbach et al. 2003; Iwama et al. 2004; Franzellitti eta Fabbri, 2006). hsp-ren mrna eta proteinen indukzioa zelularen estres orokorraren erantzun gisa agertzen dira bero-talka faktorearen (HSF) bitartez. hsp90ak toxikoen itu diren proteinak egonkortzen dituztela ikusi da, hsp90ak proteinak egonkortuz, drogen aurreko erresistentzia eskuratzen dela iradokiz (Cowen eta Lindquist, 2005). Gainera, hsp90ak esteroideen hormona-hartzaileak eusten ditu. Ezaguna da, hsp70ak, katea polipeptidikoen tolespenean laguntzen duela, txaperona gisa jokatuz eta honela eraldatu edo desnaturalizatutako proteinen konponketa zein degradazioa bideratzen lagunduz. Zenbait bero-talka gene-familia aurki daitezke animalien genoman, tartean, hsp110, 100, 90, 70, 69, 40 eta hsp α-kristalino txiki bat (Feder eta Hofmann, 1999). hsp70 da bero-talka proteinen arteko familiarik nagusiena eta itsas-trikuetan 17 gene ditu gutxienez (Goldstone et al. 2006). Zuhaitz filogenetikoetan zehar organismoek agertzen duten defentsomari erreparatuz gero, lanean diharduen gene talde zabala dagoela ikusten dugu. Esate baterako, molusku eta arrainetan MT edo hsp70, inguruneko hamaikatxo faktorek induzitzen dituzte (Iwama et al. 2004). Espezie batzuen gene kopuruetan areagotze nabarmenak eman dira, nematodoen hartzaile nuklearren kasua esate baterako, edo ekinodermo eta intsektuen CYP geneak (1 Taula). Beraz, bidezidor batzuk osagarriak izan daitezke edo/eta zenbait kasutan errepikatuta ager daitezke, baina edozein kasutan, bidezidor hauek guztiak modu koordinatuan eraendu behar dira. Toxikoen aurrean moldatze-erantzuna ematen duten geneen eraenketa koordinatua, CYP eta UGTren gaineko AHR-ren eraenketa edo ugaztunetan CYP3A4, UGT1A, eta ABCC2ren gainean PXRak bideratzen duen koeraenketa, xenobiotikoen aurreko erantzuna azkartzeko modu bat izan daiteke. Seinale-transdukzioaren erredundantzia, erantzun arinak emateko mekanismoa izan daiteke, zelularen estres orokorreko erantzuna zuzenduko duen estres-seinale mota ezberdin ugari baimenduz (Goldstone et al. 2006). Honek guztiak, moldatze-bidezidorrak ikertu eta kuantifikatzea baimentzen duten metodo analitikoak eskatzen ditu, bidezidorretako gene bakanen determinazio soila ordezkatuz. Horrela, inguruneko toxikologiaren helburu nagusi bilakatu da toxikoen aurreko erantzunean parte hartzen duten gene sareen ikerketa, horretarako ekoizpen altuko teknologiak aplikatuz. 20

27 Sarrera 3. Ekoizpen altuko transkriptoma-ikerketak arrain eta moluskuetan 3.1. Arrain eta moluskuen gene eta genomen gaineko informazioa Arrain espezieen gaineko informazioa genomikoari dagokionez, asko dago egiteke oraindik. Espezie urtarren artean, arrainen taldea da gehien ikertu dena gaur egun arte. Gizakiaren genomaren ezagutza osotu ostean, ezagutu ziren hurrengo ornodunen genomak Takifugu rubripes (Aparicio et al. 2002) eta Tetraodon nigroviridis arrainenak izan ziren (Jaillon et al. 2004) (5. Irudia). Arrain espezie biek dute ornodunen artean ezagutzen diren genomarik laburrenetakoak, gizakiarena baino 7 aldiz laburragoak. Espezie bietan falta da ornodunen genoman ohikoak diren DNA-gune errepikakorrak eta honek, gene-sekuentzia eta gene-eraentzaileen detekzio eta analisia erraztu egiten du. Bestalde, genoma biak publikoki argitaratu dira Ensembl-en ( EMBL-European Bioinformatics Institute (EBI) eta Wellcome Trust Sanger Institute-ak (WTSI) elkarrekin burututako proiektuan (Hubbard et al. 2007) urtetik, arrain handiago baten genoma, medaka arrainarena (Oryzias latipes) hain zuzen ere, (700 Mb, Tetraodonen 343 Mb-ekin alderatuz) dago eskuragarri Ensembl-en (Kasahara et al. 2007). Argitaratutako sekuentzia hauekin batera, datu-base berak WTSIn sekuentziatzen diharduten Danio rerioren genoma eta Broad institutua ( sekuentziatzen ari den Gasterosteus aculeatusena daude eskuragarri. Gainera, ornodunen arteko talde primitiboenaren (Agnathanak) ordezkarian, Petromyzon marinus, genomako 5,9X lortu dira behin-behinean, Washington University-ko Genome Sequencing Centre-ak urteko ekainean Ensembl-en argitaratu bezala. Asko geratzen da sekuentziatzeko oraindik, kontutan hartuz, ia teleosteo espezien artetik, 5en genoma baino ez dagoela eskuragarri. Egun, Salmo salar (Genome British Columbia) eta Oreochromis niloticus (University of New Hampshire) teleosteoen genoma ezagutzeko proiektuak garatzen ari dira eta Leucoraja erinacea eta Squalus acanthias (NIG Intramural Sequencing Center) kondriktioen genoma partzialaren gaineko proiektuak hasi dira. 5. Irudia. Gizakiaren genoma sekuentziatu ondoren, Takifugu rubripes (Aparicio et al. 2002) eta Tetraodon nigroviridisen (Jaillon et al. 2004) genoma txikiak izan dira ondoren argitaratu ziren ornodunen genomak. Moluskuei dagokienean, urtean zehar osatu da lehenengo moluskuaren genoma, izan ere, Lottia gigantearen 0.43 pg-tako genoma txikia sekuentziatu da Californiako Unibertsitatean, 21 S A R R E R A

28 Sarrera Berkeley (Chapman et al. 2007). Molusku ezberdinen genoma ezagutzeko proiektu ezberdinak daude martxan, ikuspuntu ekotoxikologikotik garrantztsuenetakoa, Stanford-eko Unibertsitatean Andrew Gracey eta George Somero burutzen ari diren Mytilus californianus muskuiluarena izanik. Martxan dauden bestelako genoma-proiektuen artean, Biomphalaria glabrata gastropodoa (Washington University) eta Aplysia californica opistobrankioarena (Broad Institute) dira. Inguruneko genomika-konparatiboan garrantzitsua izan da zuhaitz filogenetikoko taxa ezberdinetako espezie adierazgarrien genomak ezagutzea. Baten bat aipatzearren, ekdisozoa taldeko Caenorabditis elegans nematodoa eta Drosophila melanogaster fruta-euliaren (ondoren, bestelako intsektuena etorriko zen tartean, eltxoa edo erlearena, alegia) genomak publikatu dira. Ciona intestinalis urokordatuarena eta azkenik, ekinodermatuen artean, Strongylocentrotus purpuratus itsas-trikuarena (The Sea urchin Genome Sequencing Consortium, 2006) ere argitaratu dira. Protostomioen artean hutsune handia dago, molusko edo anelidoen genomarik ez baitago publikoki eskuragarri oraindik. Transkripzio-sekuentziei dagokienez, hainbat espresio itu-sekuentzia (EST) dago eskuragarri datubaseetan, ESTak sekuentziatzeko programa handien ondorioz edo munduan zeharreko hainbat ikertzaile beraien intereseko espezie eta sekuentzietan kontzentratzearen ondorioz (2. Taula). Oraintsu, A. californicaren transkriptoma neuronalaren %50-70 sekuentziatu da eta neuronetako gene-produktu identifikatu dira (Moroz et al. 2006). Euprymna scolopesen 11 cdna liburutegitik lortutako sekuentzia ez-errepikakor argitaratu dira ere (Chun et al. 2006). Haliotis asininearen mantuan espresatzen diren geneen %25a, "sekretoma" izenarekin ezagutzera eman dena, sekuentziatu dira (Jackson et al. 2006). Spisula solidissima molusku bibalboaren transkriptoma Marine Biological Laboratory-an sekuentziatzen ari dira Hersko Nobel saridunak (molusku berean proteinen anderaketarako beharrezkoa den ubikitina aurkitu zuen) proposatutako programaren barruan. Crassostrea virginican EST programa martxan dago (Auburn University) eta Mytilus galloprovicialis muskuiluan 1700 EST-sekuentzia baino gehiago argitaratu dira dagoeneko Italian burututako sekuentziazio-programa batean (Venier et al. 2006). Era berean, arrainetan hainbat EST sekuentziazio-programa dago martxan. Baten bat aipatzearren, Pleurogene proiektuaren barruan Solea senegalensis mihi-arrainean EST-proiektu garrantzitsu bat burutzen ari dira Espainian eta Hippoglossus hippoglossusen Canadan (Douglas et al. 2007) ( 2 zeluletako fasean, egun 1eko larba edo halibut heldugabeen ESTen gaineko EST-informazio gehigarria dago eta beraz, amarengandik datorren zein garapen fasearen araberako mrna-transkriptoen inguruko informazioa eskuragarri utzi dute ere Norbegian (Bai et al. 2007). Akuakulturan edo/eta arrantzan esanguratsuak diren Sparus aurata urraburua (Birdgemap project, Dicentrarchus labrax lupia (BassMap project, Chini et al. 2006), Gadus morhua makailaua (Codgene project, Ictalurus punctatus katu-arraina (Ju et al. 2000), Paralichthys olivaceus (Kurobe et al. 2005) eta Scophthalmus maximus erreboiloa (Martínez, komunikazio pertsonala) espezietan ehunaren araberako zein garapen-fasearen araberako ESTen sekuentziazio-programa ezberdinak daude martxan. Ikerketa hauetako askoren helburua, akuakulturaren munduan horren garrantzitsua den bakterio eta birusek eragindako zolduren aurrean organismoek erakusten duten babes immunea ikertzea da. Honek esan nahi du, proiektu hauetako asko giltzurrun, gibel, bare edo/eta odoleko transkriptoman zentratzen direla. Informazio hau ikuspuntu ekotoxikologikotik oso garrantzitsua da, konposatu kimiko asko immunoeraentzaileak baitira, esaterako PAHak immunoezabatzaileak izanik (Carlson et al. 2004a). Ingurunean adierazgarriak diren espezieen gainean toxikoek eragiten dituzten kalte eta estreserantzunen transkriptoma ezagutzeko garatu diren EST-proiektuek interes berezia dute. Ikerketa 22

29 Sarrera hauetako askok geneen espresio diferentziala edo subtraktiboa erabiltzen dute populazio batean espresatzen den mrna molekulak aberasteko (ikus beranduago). Ikerketa proiektu handiagoak gauzatu dira konposatu kimiko ezberdinen eraginpean mantendutako organismoen liburutegiak normalizatzeko. Europako Genipol proiektuan, Platichthys flesus platuxak intraperitonealki ziztatu ziren 3-metilkolantreno, benzo(a)pireno, aroklor 1254, azido perfluorootanoikoa, t- butilhidroxiproxidoa, 17-β estradiola, 17-α metiltestosterona, kadmio kloruroa, pregnelona16-αkarbonitriloa eta lindanoarekin eta ondoren, gibeleko RNA erauzi zitzaien liburutegi normalizatu bat eraikitzeko asmoz (Williams et al. 2006). Ezabatze-hibridazio subtraktiboko (SSH) eta cdna liburutegiko klonak konbinatuz, 5211 nukleotido-sekuentzia lortu ziren eta horietako 2232 gene 2. Taula eko ekainean NCBIn egondako dbest sarrera publikoen kopurua Fish Number of public ESTs Molluscs Danio rerio Aplysia californica Salmo salar Euprymna scolopes Oryzias latipes Mytilus californianus Gasterosteus Biomphalaria aculeatus glabrata Oncorhynchus Crassostrea mykiss virginica Pimephales Argopecten promelas irradians Petromyzon Crassostrea marinus gigas Fundulus Mytilus heteroclitus galloprovinicalis Number of public ESTs Others (tunicates, echinoderms, crustacea) Number of public ESTs Ciona intestinalis Hydra magnipapillata Strongylocentrotus purpuratus Paracentrotus lividus Carcinus maenas Homarus americanus Daphnia magna Callinectes sapidus Ictalurus punctatus Chlamys farreri 3375 Fenneropenaeus chinensis Gadus morhua Litopenaeus vannamei Squalus acanthias Takifugu rubripes Misgurnus anguillicaudatus Paralabidichromis chilotes Cyprinus carpio bakanei zegozkien (Williams et al. 2006). Antzeko hurbilketaren bitartez, 4700 klonetako cdnaliburutegia eratu zen Lithognathus mormyrus arrain esparidoan (Auslander et al. 2005). Genipol proiektutik beste proiektu berri bat garatu zen etinil estradiol, dibentzo(a)antrazeno eta kuprearen eraginpean mantendutako Gasterosteus acculeatusen cdna liburutegi normalizatu eta subtraktiboa eratzeko asmoz. Kasu honetan, 979 EST-sekuentzia lortu ziren (Williams et al. 2007). Stressgene proiektuan, metal, PCB eta PAHen eraginpean mantendutako makailauen gibel, giltzurrun, bare eta odoleko cdna liburutegian EST baino gehiago lortu ziren eta horietatik, 746 garrantzitsuak direnez metabolismoan eta erantzun immunean, zein estres mailako erantzunean, eskala txikiko mikrotxip bat egiteko erabili dira ( 23 S A R R E R A

30 Sarrera 3.2. Ekoizpen altuko sekuentziazio- eta transkriptoma-teknikak Homologia bidezko klonazioa edo "geneen ehiza" Phyla ezberdinetako sekuentzia nukleotidikoak Genbank datu-baseetan batzen ari direnez, ClustalW bidez homologia bilaketak egin eta gene ortologoen artean kontserbazio maila altuko guneak ezagutzea errezagoa da. Gene baten sekuentzia ebolutiboki nahiko kontserbatua badago, hasle anderatuak diseina daitezke eskuz zein programa egokiak erabiliz eta RT-PCR bidez anplifika daiteke intereseko espeziean. Arrain teleosteo ugarirentzat nukleotido-sekuentzia mailako informazio ugari dagoenez datu-baseetan, nahiko erraza suertatzen da arrainen cdna edo DNA sekuentziak anplifikatzeko moduko hasleak diseinatzea. Kordatuak ez diren organismoentzat berriz, informazio gutxiago dago, bereziki molusku eta anelidoen kasuan, beraz, sarritan hasleak ez dira diseinatzen konfidantza askorekin. Askotan, aztertu nahi dugun espezietik ebolutiboki oso aldenduta dauden taxetako organismoen sekuentziak lerrokatuz diseinatu behar dira hasleak, sekuentziaren kontserbazio mailak oso ezberdinak izan daitezkeelarik. Homologian oinarrituriko gene bakanen klonazioa oso erabilia izan da azken urteotan. Esate baterako, Raingeard eta laguntzaileek Chelon labrosus lazunaren PPARα-ren zati bat klonatu zuten hurbilketa hau erabiliz. Song eta laguntzaileek (2006) Argopecten irradiansen hsp70 klonatu zuten eta Franzilletti eta Fabbrik (2006) p-glikoproteina, major vault protein eta multidrug resistance related protein klonatu zituzten Mytilus galloprovinicialisen modu bertsuan. Williams eta laguntzaileek (2003) metodologia bera erabili zuten ekoizpen altuagoa lortzeko, estresarekin erlazionaturiko zein bestelako 110 gene anplifikatu zituztenean platuxaren cdna eta DNAn hasle anderatuak erabiliz. Azkenik, 110 horietatik 89 anplikoi bakarrik zeuden hasieran bilatzen zituzten horien artean eta platuxan ezagunak ziren beste sekuentzia batzuekin batera dentsitate baxuko mikrotxip batean sartu zituzten, Tyne eta Alde estuarioetako platuxen geneen espresio diferentziala ikertzeko (Williams et al. 2003). Hurbilketa honen bidez, genearen irakurketa-markoa (ORF) osoaren portzentaje ezberdina estaltzen duten ESTak lortzen dira. Modu ezberdinak daude orduan, 3' eta 5' muturretako sekuentziak lortzeko. Esate baterako, Kullman eta laguntzaileek (2000) Oryzias latipesen CYP3A klonatu zuten hasle anderatuak erabiliz eta PCR produktu indibidualak sekuentziatu eta P450 zitokromoaren familian identifikatu ondoren, DNA-liburutegi osagarri bat arakatzeko zunda espezifikoak ekoiztu diseinatu ziren, ORF osoa lortuz. Beste aukera bat, 3' eta 5' muturretan anplifikazio arina burutzea (RACE) da, falta diren muturretako cdna zatiak eskuratzeko (Song et al. 2006) Ezabatze-hibridazio subtraktiboa (SSH) Ezabatze-hibridazio subtraktiboa (SSH) Diatchenko eta laguntzaileek urtean garatutako teknika da modu ezberdinean espresatutako geneak identifikatzeko. Bi iturri ezberdinetako RNA totaletik cdna sintetizatzen da alderantzizko transkripzioz eta transkriptometako bat, lagin bietan komunean berdintsu agertzen diren transkriptoak beste transkriptomatik ezabatzeko erabiltzen da. Modu honetan, kontrol-populazio batean agertzen diren transkriptoak, toxiko baten eraginpean mantendutako laginaren transkriptoetatik ezabatzeko erabili daitezke, geratzen den transkriptomak, egoera jakin horretan gainespresatutako transkriptoetan aberastutako liburutegi subtraktiboa osatzen duelarik. Teknika honekin PCRa ere konbina daiteke, ezabatze PCR (suppression PCR) delakoan (6. Irudia). Ezabaketa optimizatzeko, cdna errestrikzio-entzimen bidez mozten da (RsaI) DNA-zati txikiak lortzeko (~ nukleotidotako zatiak). Diseinaturiko bi DNA-adaptadore ezberdin eta 24

31 Sarrera berezi ligatzen dira cdna laginetako batean (orokorrean, kutsatzailearen eraginpean egondako organismoena), cdna-tester izenarekin ezaguna dena, eta bi hibridazio-ziklo burutzen dira. Lehenengorako, adaptadore ezberdinarekin ligaturiko bi azpipopulazioetan banatutako tester bakoitza, kontrol edo driverarekin ligatzen da bakoitza bere aldetik. Bigarrenean, lehenengo hibridazioko populazioak jartzen dira kontaktuan. Lortutako nahastea eta hibridatutako azido nukleikoen populazioa RT-PCR bidez anplifikatzen da adaptadoreetako batekiko espezifikoa den Fw eta bigarren adaptorearekiko espezifikoa den Rv hasleak erabiliz. Metodo hau, PCRan oinarrituta dagoenez, tester-ean gainespresatuta dauden geneak anplifikatzen dira. Modu honetan, forwardliburutegi bat eratzen da. Reverse-liburutegia, azpiespresatutako geneen produktuetan aberastutakoa, kontrola tester gisa erabiliz lor daiteke. 6. Irudia. Ezabatze-hibridazio subtraktiboaren oinarria modu ezberdinean espresatutako transkriptoetan aberastutako liburutegien sorpenerako. Clontech-etik hartutako eskema. 25 S A R R E R A

32 Sarrera Klonen sekuentziazioak, liburutegi ezberdinetan baldintza jakin batzuen aurrean, modu ezberdinean adierazi den gene taldea kualitatiboki identifikatzea baimentzen du. Emaitza hauek, teknika kuantitatiboen bidez balidatu behar dira Differential display RT-PCRa (ddrt-pcr) Differential display RT-PCR (ddrt-pcr) PCRan oinarrituriko metodoa da, non egoera jakinen aurrean, ehun edo zeluletan gainespresatuta dauden transkriptoen isolaketa ematen den. 7. Irudian erakusten dira jarraitu beharreko urratsak. Bi populazio edo egoera esperimental ezberdinen eraginpeko (kontrola eta esposatua) RNA isolatu ondoren, (totala zein mrna): 1.- Alderantzizko transkripzioa burutzen da hasle jakin batzuk erabiliz (berdinak RNA-populazio bien kasuan). 2.- cdna anplifikatu egiten da hasle arbitrario seriatuak erabiliz 3.- Populazio bakoitzeko anplikonak elektroforetikoki banatzen dira akrilamida geletan 4.- Baldintza esperimental bietan modu ezberdintzatuan espresatzen diren anplikonak berriro anplifikatzen dira, klonatu eta sekuentziatu. Modu ezberdintzatuan espresaturiko ahalik eta gene gehien aztertu ahal izateko, RT-hasleek ahalik eta cdna-polupalzio ugariena eratzea baimendu behar dute, ondorengo PCR anplifikazioetarako ituhasleekin hornituz. Berez, T11MN motako 12 oligo-dt hasle erabiltzea gomendatzen da (Liang and Pardee 1992); bertan, M, A, G edo C izan daitekeelarik eta N edozein nukleotido izan daitekeelarik. Hasle hauek 12 cdna azpifrakzio ezberdin eratzea baimentzen dute eta bakoitzak transkriptoma jakin batean (RNA erauzi deneko organismoaren zelula edo ehunekoa) espresatzen diren geneen hamabirena ordezkatzen du. Urrengo urratsa, ahalik eta cdna gehien anplifikatzea da PCR bidez. Urrats honetan, itu motako hasleek reverse hasle gisa jokatzen dute. Forward hasleak ausazko 7. Irudia. Differential display delakoaren oinarria. Kontrol eta aztertu beharreko laginen mrna alderantzizko transkripzioz cdna bihurtzen dira, bakoitza bere aldetik N eta M nukleotidoak konbinazio ezberdinetan agertzen dituzten oligo(dt) itu-hasleen presentzian. Hasle bera eta ausazko dekameroak erabiltzen dira ondoren PCR bat burutzeko hasle gisa. Lortutako produktuak elektroforesian migratu eta kontrol eta tratatutako laginetan modu ezberdinean agertzen diren bandak (geziek adierazi bezala) aztertu egiten dira. 26

33 Sarrera sekuentzia duten 10-mero oligonukleotidoak dira (zenbait murrizpenekin). Esistitzen den mrna bakoitza anplifikatzeko, hasle ezberdin inguru beharko lirateke, bakoitza reverse motako hasle guztiekin erabiliz, banakako PCRetan (Bauer et al. 1994). PCR-erreakzio bakoitzeko anplikonak elektroforetikoki banatzen dira bi mrna populazio ezberdinetako zatiak, hasle bereekin anplifikatutakoak, bata bestearen alboan migratuz beti (8. Irudia). Nukleotido erradiaktiboak nahiago izaten dira, kimioluminiszentzia eta fluoreszentzia bitarteko markaketak ere eskuragarri badaude ere. Kontrol eta esposatuetan ezberdintzatzen diren intereseko bandak geletik moztu, beranplifikatu eta zatiak sekuentziaziorako bektore egokietan klonatzen dira. Banda bakoitzean cdna espezie bat baino gehiago ager daitezkeenez, kolonien isolatze eta karakterizazioa gomendatzen da. 8. Irudia. ddrt-pcrren aplikazioa paper-fabrikek isuritako konposatuen eraginpeko Micropterus salmoides arrainean. Geziek hasle arbitrarioen bidez (AP-1, AP-21 eta AP-10, hurrenez hurren) arrain kontrol eta tratatuetan modu ezberdinean anplifikatu diren mrna zatiak adierazten dituzte. Denslow et al Teknika hau oso neketsua da, garestia eta teknikoki eskakizun handiduna. Modu ezberdintzatuan lortutako transkriptoen %50a baino gehiago positibo faltsuak izaten dira eta hasleak 3' muturreko 500 bp-rekin lotu behar direnez, RNAren zati bat baino ez da anplifikatzen. Beste arazo bat, bukaerako produktuko PCRan oinarritzen den hurbilketa teknikoa denez, ezberdinki espresaturiko zenbait transkripto ezin dira harrapatu beraien espresio ezberdintasuna oso handia ez delako Gene-espresioaren analisi seriatua (SAGE) SAGE bi irizpide nagusi aplikatzen dituen teknika da. 20 bp-tako oligonukleotido sekuentzia labur batek, errestrikzio endonukleasa espezifiko batek (itu-entzima, AE) polya isatsetik distantzia finko batetara moztuz definituta, mrna transkripto espezifikoak identifika ditzake. Teorikoki, 10- bp-etako ESTak 4 10 sekuentzia konbinazio ezberdin eman ditzake, gizakiaren genomatik ondorioztatutako traskripto guztien artean diskriminatzeko bestekoa (Tuteja eta Tuteja, 2004). 27 S A R R E R A

34 Sarrera Protokoloak mrnaren purifikazioa eskatzen du, ondoren fase solidoan dauden oligo(dt) bola magnetikoetara lotzeko. cdna zuzenean boletan sintetisatzen da eta ondoren NlaIII (AE) ituentzimarekin liseritzen da, 3' muturra oligo(dt)-boletan utzita (9. Irudia). SAGE motako esperimentu gehienek 256 bp-ero eta beraz, mrna transkripto bakoitzean, 4 bp ezagutzen dituen NlaIII ituentzima erabiltzen dute. Bigarren SAGE liburutegi bat, itu-entzima ezberdin batez eratutakoa, NlaIII gune bako transkriptoak detektatzeko erabilgarria izan daiteke. Lagina bi hoditan banatzen da eta lotugai (linker) ezberdin birekin lotzen da, A edo B. Lotugai biek dute BsmFI-ek, itu-entzimaren ezagutza-gunetik 10-bp-tara mozten duen IIS motako errestrikzio-entzima ezagutzen duen gunea. BsmFI etiketa(tag)-entzima (TE) legez ezagutzen da, SAGE-etiketa (SAGE-tag) izeneko oligonukleotido bakarrak eratzen dituelako. Ondoren, SAGEetiketak oligo(dt)-boletatik askatu, eta beraien artean lotzen dira dietiketak (ditagak) eratuz. Dietiketak PCR bidez anplifikatzen dira, jarraian lotugaietatik banatu, geletik purifikatu, modu seriatuan ligatu, klonatu eta sekuentziatuak izateko sekuentziadore automatizatuan. 9. Irudia. Gene-espresioaren analisi seriatuak (SAGE) oligo(dt)-boletan burututako cdnaren sintesi zuzena eta ituentzima ezberdinen bidezko ondorengo liseriketa eskatzen ditu. Banatutako laginak etiketa-entzimak (TE) ezagutuko dituen errestrikzio-guneak dituzten lotugai ezberdin birekin lotzen dira. SAGE-etiketak boletatik banatu, ligatu eta PCR bidez anplifikatu ondoren, sekuentziatu egiten dira. 10 bp-tako etiketak ezin ditu genomako gene guztiak identifikatu. SAGEluzeak hala ere, (LongSAGE) IIS errestrikzio-endonukleasa ezberdin bat erabiltzen du, MmeI esaterako, 3'-ko itugunetik 17 bp-tara mozten duena. Sekuentziadoreak emandako sekuntzien artxiboak SAGE2000 sofware-a erabiliz aztertzen dira ( Geneen espresio diferentzialari dagokion zerrenda ondoren azaldu bezala lortzen da: (1) SAGE2000 software-a erabiliz, SAGE-etiketak ezberdindu, dietiketak erauziz eta duplikaturiko dietiketak konprobatuz (2) Sekuentzia erreferentzialen datubase bat deskargatu (gizaki eta organismo modeloentzako bakarrik eskuragarri) NCBIren Web orrialdetik (SAGEmap, 28

35 (3) Etiketak espresaturiko geneen datu-basearekin erlazionatu (Lash et al. 2000). Transkriptoen ugaritasun erlatiboa kalkula daiteke ondoren, etiketa bakarren kontaketa zati sekuentziatutako etiketa kopuru guztia eginez Mikro eta makrotxipak Sarrera Mikrotxipen teknologia azido nukleikoen arteko hibridazioan oinarritzen da, bietako bat euskarri solido batean egonkortuta egonik. Euskarri honetan egonkortutako zunden sareak osatzen du mikrotxiparen euskarria (10. Irudia). Zunda bakarra edo/eta zunda gutxi batzuk (esate baterako, transkripto baten 5'-3' luzeran zeharreko sekuentzia ezberdinak) eratzen dira PCR bidez anplifikatutako DNA osagarria (cdna) sortuz edo intereseko cdna-sekuentzien gune espezifikoetan eratutako DNA-oligonukleotido sintetikoak lortuz. Oligonukleotidoak ordenagailu bidez diseinatzen dira eta hortaz, hibridatzeko baldintza optimo berak zein antzeko (bestelako) sekuentziekin ez hibridatzea bermatzen da. cdna edo anplikoiz eratutako mikrotxipekin alderatzean, abantaila esangarri bat dago, antzeko sekuentzia duten transkriptoen arteko hibridazio gurutzatua, esate baterako, gene paralogoen arteko hibridazio gurutzatua, ekidin daitekeela. Gainera, behar diren oligonukleotidoak errazago eratzen dira modu automatizatu batean eta sekuentziako akatsak edo euskarrian atxikitzerako orduan burututako erroreak ez dira horren maiz ematen. Bestalde, ez dago cdna liburutegirik eratzeko beharrizanik. Beraz, oligonukleotidoz eratutako txipek egungo teknologia osatzen dute, bereziki espezie modeloetan txip handiak garatzerako orduan, genoma mailako informazioa guztiz eskuragarri baitago datubaseetan. Konpainia bik dute oligonukleotidoz eratutako txipen ekoizpenaren lidergoa, Affymetrix eta Agilent Technologies-ek, hain zuzen ere. Biek imprimatzen dituzte oligonkleotidoak in situ, Affymetrix-en kasuan fotolitografia erabiliz eta Agilent-en kasuan Hewlett-Packard inprimagailuen bidezko inpresio kimikoa erabiliz. Affymetrix-ek 25-meroak eratzen ditu Agilent-ek 60-meroak eratzen dituen bitartean. Intereseko genearen sekuentziak dituen cdna liburutegi bateko klon indibidualetan burututako PCR-erreakzioetan lortutako anplikoiak euskarrian gehituz lortzen dira cdna-txipak. Bi txip mota ezberdin ditzakegu: makro eta mikrotxipak. Makrotxipetan, cdna nilonezko mintzean lotzen da eskuz pipeteatu ondoren. Lan neketsu, intentsu eta akats ugari izaten dituena, hortaz, oso dentsitate txikiko cdna txipekin baino ez da erabiltzen, 200 sekuentzia ingurutakoak (Williams et al. 2003; Meyer et al. 2005; Brown et al. 2006). Salbuespena, EcoArray-k konposatu estrogenikoek Micropterus salmoidesen duten eragina aztertzeko komertzializaturiko makrotxipa izan daiteke ( 500 cdna sekuentzia inguru dituena (Larkin et al. 2003a; 2003b). Txipak lortzeko hainbat aukera daude: (1) prestatutako txipak (Affymetrix, Agilent, Amersham, ABI, eta beste hainbat), (2) bezeroaren araberako txipak (Affymetrix, Agilent), (3) PCR bidez anplifikaturiko klonen bilduma erabiliz nork berak lortutako txipak (nork berak egindakoak, zein komertzialak) Aztertu beharreko gene kopurua interes handikoa da. Ikerketa mekanistikoak bideratzeko zein toxikotasun-patroiak edo konposatu toxikoen eragina aztertzean gene-kopurua ahalik eta handiena izaten da, horrek esan nahi du, gaur egun, genomen sekuentziazioren aroan, birtualki izaki baten gene guztiak erabiltzen direla. Estatistika bidezko geneen selekzioa burutuz eta konposatu jakin batzuen aurreko geneen espresioa-profila ezagututa edo gaisotasunen fenotipoak ezagututa, konposatu toxikoen sinaturak 29 S A R R E R A

36 Sarrera (peroxisomen proliferatzaileena, konposatu estrogenikoena edo AHR-ren agonistak) edo gaisotasunak (minbizia edo hantura) espezifikoki azter daitezke dentsitate baxuagoko txip zuzendu batean (ikus Superarray-k argitaratutako minbizia, apoptosia, hanturaren diagnosirako bidezidorrekiko espezifikoak diren txipen zerrenda Mikrotxipen azterketak kalitate altuko RNA purifikatua eskatzen duenez, RNAren kalitatea aztertzeko makinaria garatu egin da, Bioanalyser-a esate baterako (Agilent). RNA cdnaren sintesirako erabiltzen da, ondoren crna sintetisatzeko. Sintesian zehar edo sintesia burutu ondoren, cdna edo crna biotina (Affymetrix), fluoreszentzia bidez (beste txip batzuk) edo erradiaktiboki (makrotxipak) markatzen da. Kalitatearen kontrola oso lagin txikietan burutu behar denez Nanodrop espektrofotometroa (NanoDrop technologies) bezalako teknologia garatu da mikrotxipetan erabili beharreko cdna kuantifikatzeko. Markaturiko azido nukleikoak txiparekin hibridatzen dira ondoren. Affymetrix euskarria eta makrotxipak markaturiko crna lagin bakarra erabiltzen dute hibridaziorako (kanal sinpleko txipak), euskarri gehienek eta Agilent-eko mikrotxipek, Cy3 fluoreszentzia berdez eta Cy5 gorriz markaturiko bi lagin erabiltzen dituzte, bata kontrola, bestea tratatua (gainespresatutako geneak gorriz agertuko dira, azpiespresatutakoak berdez ta antzera espresatzen direnak horiz). Nahiago izanez gero, ikerketa kuantitatibo absolutuen kasuan hibridaziorako kolore bakarra erabili daiteke, kasu honetan berdea erabiliz, gorria (Cy5) sentikorragoa baita, bereziki ozonizazio prozesuekiko. 10. Irudia. Mikrotxipen prestaketa. Modu ezberdinean markaturiko kontrolaren eta tratatuaren cdna edo crnak antzeko kantitateetan nahasten dira, ondoren txiparekin hibridatzeko. Txipa eskaneatzean, gainespresatutako, azpiespresatutako zein antzeko mailan espresatutako kolore ezberdineko puntuak lortzen dira. 30

37 Hibridatu eta garbitu ondoren, eskaner baten bidez irakurri egiten da txipa (edo autoerradiografiaz errebelatu edo fosforo bidez irudia harrapatzen da) eta fluoreszentzia edo kimioluminiszentziaren (Affymetrix) intentsitatea neurtzen da. Eskaneatutako irudiak, irudi-analisi bidezko programen bidez (ArrayVision, GenePix, Imagene) prozesatzen dira, puntuko datu bilketa eginez. Esperimentuan zehar zein esperimentuaren ondoren, lortutako emaitzak fidagarriak direla ziurtatuko diguten kontrolak burutu behar dira. Hibridaziorako kontrol orokorren artean, antzeko tindaketa intentsitatea txip osoan zehar, zaborrik ez agertzea daude besteak beste. Cy3/Cy5 motako hibridazioetan, crna populazio biak markatu behar dira kolore biekin, gorri berde, eta berde gorri, fluorokromo biak erabiltzean, markaketako efizientzian eman daitezkeen ezberdintasunak normalizatzeko. Mikrotxipen datuen interpretazio egokia burutu ahal izateko, garrantzitsuena emaitzak fenomeno biologikoekin lotzea da agian. Metodo orokor bat, tratamenduak aberastutako bidezidor edo funtzioei erreparatzea da. Geneen ontologian (GO) oinarritutako mota honetako analisiak, ( Web-etan oinarritutako hainbat iturri erabiliz burutu daitezke. Maila honetako analisiek gene asko egokiro anotatuta ez egoteari egin behar diete aurre. Bereziki, modeloak ez diren organismoetan, horietako batzuk ondo anotatutako espezietatik urrun samar daudenak, informazio gehiena ESTen sekuentziazio-programen bidez lortu da, askotan 3' muturrak sekuentziatzera mugatuz (homologia bidez sekuentzion anotazioa zaila da). Beraz, bibliografian inguruneko baldintza ezberdinen aurrean ezberdinki espresatzen diren EST-sekuentzia asko daude, baina sarritan oraindik funtzio ezezaguna dutenak (Brown et al. 2005; Venier et al. 2006; Williams et al. 2006) QA ekoizpen maila altuko mikrotxip bidezko ikerketetan Sarrera Maila altuko ekoizpena duten teknikek hedatze zabala izan dute azken urteotan eta datu kantitate handia sortzen ari da baina mikrotxipek estandarizazioa behar dute. Gaur egun, ikertzaile gehienek, datuen bilketarako, analisirako eta aurkezpenerako tekniken estandarizazioaren beharra azpimarratzen dute (Mattes et al. 2004; Benson and DiGiulio, 2005; Ankley et al. 2006; Denslow et al. 2007). Espezie modeloetan esaterako, erabili beharreko euskarri fisikoen gisako gauzak eztabaidatu beharko lirateke. Zebra-arrainetan, euskarri ezberdin ugari esistitzen da, komertzialak zein irakaskuntzan oinarritutako zentroenak (Agilent, Affymetrix, Compugen/Sigma-Aldrich, MWG-Biotech, Quiagen-Operon, Norwegian School of Veterinary Science; University of Antwerp) zunda mota ezberdindunak, ehunekiko espezifikoak eta genomaren kopuru aldakorrak estaltzen dituztenak, ehunka gene gutxi batzutatik genoma osorartean (Meijer et al. 2005; Van der Ven et al. 2005, 2006). Hurbilketa ezberdinak erabiltzen dira toxikogenomikako datuak aztertzeko eta gero eta behar handiagoa dago oinarrizko gauzak finkatzerako orduan, tartean, esperimentu jakin batean, gene bat gainespresatu edo azpiespresatu den erabakitzeko (Ankley et al. 2006; Baken et al. 2007). Zentzu honetan, iraganean nahiko hedatuta zegoen 2x gainespresatu edo azpiespresatutako balioak mozten zitueneko zuzenak ezartzea (Williams et al. 2003), maila baxuagoetan, baina hala ere, esangarriki erregulatutako geneak baztertuz. Egun, tresna estatistiko zehatzagoak erabiltzen dira bioinformatikan, mikrotxipak erabilitako esperimentuetan geneak gainespresatu edo azpiespresatu ote diren erabakitzeko. Zentzu honetan, Health and Environmental Science Institute (HESI)-ak, 30 erakunde biltzen zituen laborategiarteko lankidetzan oinarritutako ikerketa-programa jarri zuen martxan, toxikogenomikako protokolo esperimentaletan ematen diren aldaketa tekniko eta biologikoen iturriak identifikatzeko (Pennie et al. 2004). Laborategiek, geneen espresioak, esposizio-toxikoei erantzuten dieten aldaketa-fenotipiko eta bidezidor biologikoekin lotu zituzten sendoki. 31 S A R R E R A

38 Sarrera Toxicogenomics Research Consortiumak (TRC) ( ere, era ezberdinean erregulatutako geneen espresio profilak euskarri ezberdinen artean korrelazionatzen zirela ondorioztatu zuen eta aldakortasun iturririk nagusiena laborategi arteko aldaketak ematen zuen (Mattes et al. 2004). Mikrotxipen datuen sorrera eta aurkezpena formalizatzeko saiakerak egin dira Microarray Gene Expression Data (MGED) Society-ren pean, ildo nagusienak ezarriz (Minimum Information About a Microarray Experiment, MIAME). MIAME ( mikrotxipen emaitzekin batera aurkeztu beharreko informazioaren berri ematen duen dokumentua da, gainontzeko ikerlariek emaitzok interpreta eta konproba ahal ditzaten (Brazma et al. 2001). Jarraitu beharreko ildoak MIAME/TOXera ere hedatu dira, toxikogenomikak zehazki eskatzen dituen ildoak ere zehazteko ( Modu berean, mikrotxip baten inguruko informazioa konpartitu ahal izateko, emaitzak eredu estandarrak jarraituz depositatu behar dira espresio mailen inguruko datu-base berezietan. Mota horretako 3 datu-base ezberdin esistitzen dira, EEBBetan Europan eta Japonen eskuragarri dauden Genbank-i lotuta: Gene Expression Omnibus-a (GEO) ( ArrayExpress EBIn ( eta Japoniako Center for Information Biology Gene Expression Database (CIBEX) ( Transkripzio mailako ikerketak ekotoxikologian guztiz erabilgarriak izan daitezen, asko dago egiteko oraindik arlo honetan, eta hainat urrats aurreratu dira dagoeneko Organisation for economic Cooperation and development (OECD) delakoaren bidez, erregulazioan genomikaren erabilerak duen erabilpena aztertzeko talde bat bideratu baitute (OECD, 2005; Ankley et al. 2006). Teknika hauek beste erronka bati egin behar diete aurre, izan ere, beraien emaitzak sinplifikatu beharra dago konposatu kimikoen osasunerako arriskua aztertzen dutenei azaltzeko. Hori dela eta, gene ontology delakoa (hiztegi arrunta, gene bakoitzak zelularen ze konpartimentutan diharduen, bere funtzio molekularra zein den eta ze prozesu biologikotan diharduen azaltzen duena) garrantzitsua da jargoia murriztu eta terminologia estandarizatzeko ( Bestalde, lortutako emaitza multzoa kondentsatzeko beharra dago. Badaude hainbat ekimen, toxikogenomikako datu-base publikoak eratzeko, geneen espresio mailako datuak gaineratzeaz gain, jopuntu toxikologikoen inguruko informazioa gaineratzen dutenak, horrela, ezagutza osoa bermatzen duten baseak eraikiz. Honela, geneen espresio-patroietan emandako erantzun toxiko adaptatiboen interpretazio sakonagoetan aurreratzea baimenduko da. Zenbait adibide aurki daitezke, Chemical Effects in Biological Systems (CEBS)-en ezagutza-basean ( adibidez, non inguruneko esposizioek giza-osasunean zelan eragiten duten, nola indibiduoek esposizio horien aurrean sentikortasun ezberdina agertzen duten eta nola sentikortasun horiek denboraren arabera aldatzen doazen definitzeko saioak egiten ari diren, eta ArrayExpress datu-baseari loturiko Tox- MIAMExpressen ( adibidez, faktore biologikoak (behaketa klinikoak, patologia klinikoa, patologia eta histopatologia) geneen espresioaren inguruko datuekin lotzea baimentzen da Geneen espresio-profilak espezie itsastarretan Itsas-organismoen geneen espresio-profilak, inguruneko baldintzetan emandako aldaketen erantzun adaptatiboen ikerketara bideratu da gehienbat edo/eta garapen-fase ezberdinetan dauden indibiduoen arteko ezberdintasunen ikerketara (Cossins eta Crawford, 2005; Douglas 2006; Denslow et al. 2007; Gracey 2007). Zehaztasun handiagoz, transkriptomen inguruko 3 ikerketa mota ezberdin daitezke itsas-organismoetan: 32

39 Sarrera 1.- Zoldura edo hanturari erantzuten dioten bidezidor molekularrak ezagutarazten saiatzen direnak 2-. Konposatu kimiko jakin baten gehienetan, ingurunean agertzeko joera duena, ekintza-bidea ikertzen ahalegintzen direnak 3-. Tenperatura, hipoxia, superpopulaketak eragindako estresa edo garapen-fase jakin bati edo gaixotasun egoera jakin bati erantzuten dieten populazio barneko ezberdintasunak, inguruneko aldaketa fisikoen aurreko aklimatazioa baimentzen duten bidezidor adaptatiboak topatu nahian Ikerketa gehienetan, inguruneko aldaketen aurrean esposizioa edo esposizioaren efektua adierazten duten biomarkatzaileak bilatzea izan zen hasierako asmoa (11. Irudia). Beraz, hasieran banakako geneen erregulazioan jarri zen jomuga. Lehenengo egun horietatik, genomika mailako ezagutza handitu egin da, GO gaiak gene ezberdinentzako ezarri direnez gero, geneen bidezidorren eraenketaren analisiak, banakako geneen eraenketak baino garrantzi handiagoa hartu du. Bidezidor geniko eta metabolikoen ulermenak, genomaren ikuspuntu zabal batetik, organismoaren egoera fisiologikoa ulertzea ekar lezakeela uste da. 11. Irudia. Konposatu toxiko modeloen eraginpean (disruptore-endokrinoak, peroxisomen proliferatzaileak eta metal astunak) gene espezifiko ezberdinen espresio-patroi jakinak lortzen dira. Konposatu ezezagunen eraginpean ere, espresio-patroi zehatz bat lortzen da, kasu honetan disruptore-endokrinoek eragindakoaren parekoa. Hortaz, konposatu ezezagunak disruptore-endokrinoak badituela jakin daiteke. Horrez gain, bitelogenina disruptore-endokrinoen eraginpeko esposizio-biomarkatzaile egokia dela ondoriozta daiteke. Irudia: K. Chipman Patogenoen aurreko ostalariaren erantzunak Itsas-organismoetan burututako ikerketa transkriptomikoak, akuakulturan erabiltzen diren arrainespezieetan eta krustazeoetan oinarritu dira batik bat, zoldura eta hantura prozesuetan inplikatuta dauden gene eta bidezidor genikoen gaineko ezagutza handitzeko helburuarekin. Zoldura prozesuetan parte hartzen duten erresistentzia ezaugarriak ezberdintzeak manipulazio genetikorako eta ugaltzaileen kudeaketa estrategiak baimenduko lituzke akuakultura-enpresetan, zoldurek diru-galera handienak ondorioztatzen dituzten arazoa baitira. Modu berean, ekoizpen handiko ikerketa hauek, kultiboko ale guztiak kaltetu aurretik edo ondorio kaltegarriak ekiditeko 33 S A R R E R A

40 Sarrera moduko fasean zoldura posibleak aurreikus ditzaketen seinale goiztiar legez funtzionatzen duten biomarkatzaileak azalerazten dituzte. Ekoizpen altuko zenbait metodo, erantzun immunerako transkriptoetan aberastutako ESTliburutegien sorrera (Nam et al. 2000; Kono eta Sakai, 2001; Savan eta Sakai, 2002; Kocabas et al. 2002; Kono et al. 2004) eta EST-liburutegi subtraktiboak (Bayne et al. 2001, Fujiki et al. 2001; 2003; Tsoi et al. 2004; Dios et al. 2007; Atlantic Cod Genomics and Broodstock Development Project) barne arrain teleosteoen erantzun immuneko gene berriak eta ostalari-patogeno elkarrekintzak identifikatzeko saiakeretan erabili dira. Piscirickettsia salmonalis (Rise et al. 2004), Mycobacterium marinum (Meijer et al. 2005) eta Aeromonas salmonicida (furunkulosia eragiten duen agentea, Ewart et al. 2005) patogeno intrazelularrekin zein zakatzetako Neoparamoeba spp (Morrison et al. 2006) ektoparasitoekin, Saprolegnia (Roberge et al. 2007) onddo-moduko protistoarekin edo mota ezberdinetako birusekin, tartean, septizemia hemorragikoaren birusa (Byon et al. 2005; 2006), hirame errabdobirusa (Kurobe et al. 2005; Yasuike et al. 2006) edo nekrosi infekziosohematopoietikoa eragiten duen moduko birusek (Purcell et al. 2006) zoldutako zebra-arrainean, Atlantiar izokinean eta Japoneko platuxan mikrotxipen bidezko ikerketak burutu dira. LPS bezalako PAParen injekzioaren transkripzio mailako eraginak aztertzeko ikerketak burutu dira ere arrain espezie hauetan (Kurobe et al. 2005; MacKenzie et al. 2006). Interes berezia dute, birusek eragindako gaixotasun ezberdinen aurkako txertoek eragindako transkripzio mailako efektuek, modu ezberdinean erregulatzen diren geneek birusek eragindako zolduraren aurrean organismoei abantaila eskaintzeko aukera handiena duten geneak baitira (Byon et al. 2005; 2006; Yasuike et al. 2006). Organismo ornogabeei dagokienez, gutxiago egin da, nahiz eta molusku eta krustazeoen gainean zeozer egin den. Esate baterako, bakterioek zoldutako Crassostrea gigas hemozitoen (Gueguen et al. 2003) edo puntu-zuridun sindromearen birusak zoldutako (WSSV) Penaeus monodon postlarben EST-liburutegiak eratu dira (Leu et al. 2007). Subtrakzio bidez, liburutegiak eratu dira ere elkarrekintza ezberdinetan parte hartzen duten geneak identifikatzeko: Biomphalaria glabrata eta Schistosoma mansoni (Lockyer, et al. 2007) edo Echinostoma caproniren (Guillou et al. 2007) artean, C. gigas edo virginica eta Perkinsus marinus parasitoaren artean (Tanguy et al. 2004) eta Penaeus monodonen hemozitoak eta WSSVren artean (Genbank). Mikrotxipen erabilpena krustazeotara mugatu da, non WSSVren, munduan zeharreko izkira talde ugariren patogeno garrantzitsuenetakoa, efektuak ikertu diren, izkira espezie ezberdinetan Penaeus stylirostris (Dhar et al. 2003), Fenneropenaeus chinensis (Wang et al. 2006) eta Litopenaeus vannamei (Robalinho et al. 2007). Ehuneko trauma edo patogeno zein parasitoen inbasioek aldaketak eragiten dituzte animalien gorputzeko fluidoetako hainbat makromolekulen kantitateetan (Bayne eta Gerwick, 2001). Aldaketa hauek, fase akutuko erantzunaren (APR) alde bat osatzen dute, organismo osoaren organo sistema ezberdinetan aldaketa metabolikoak eragiten dituena, alegia. Ornodunetan ematen den erantzunaren seinale bat, gibelean ematen den eta ondoren jariatzen diren plasmako proteinen emendioan dago, era berean, beste zenbait proteinen gutxitzea emanik. Fase akutuko proteina hauek defentsarekin lotutako jardueretan ari dira, agente infekziosoen dispertsioa mugatuz, kaltetutako ehuna konponduz, proteasak inaktibatuz mikrobioak eta bestelako patogenoak hilez eta osasun egoera berreskuratuz (Bayne eta Gerwick, 2001). Proteina hauetako batzuek bakardadean egiten dute lan, beste batzuek ordea proteina-kateetan parte hartzen dute. Batzuk mikrobioentzako zuzenean dira kaltegarriak, beste batzuek bitartean, ituak eraldatzen dituzte, erantzun zelularrerako prestatuz. Esate baterako, transferrinak bakterioen hazkuntzaren inhibitzaile gisa jokatzen du, bakterioaren mantenurako 34

41 Sarrera beharrezkoa den burdina bahituz. Interferona hazkuntzaren beste inhibitzaile bat da, Mx edo/eta hepzidina bezalako birusen aurkako peptidoen espresioa eragiten duena. α2-makroglobulina bezalako zenbait proteasa inhibitzaile, arrainen gorputzeko fluidoetan agertzen dira eta patogenoen aurkako defentsan parte hartzen dute. Hainbat entzima litiko, lisozima, kitinasa, katepsinak konplementu sistemaren bidezidor litikoa eta bestelako entzima bakteriolitiko/hemolitiko, bakterioen aurkako defentsarako elementu garrantzitsuak dira. Mukosa edo gazurreko aglutinina eta prezipitinek, C motako lektinak eta pentraxinek, zenbait karbohidrato lotzen dituzte espezifikoki, opsonizazioa, fagozitosia eta konplementuko sistemaren aktibazioa eraginez (oso aberatsa arrainetan) (Bayne eta Gerwick, 2001; Secombes et al. 2001). Jaiotzetiko sistema immuneak mikrobioen molekula adierazgarriak identifikatzen ditu. Patogenoekin asoziaturiko patroi molekularra (PAMP) delako terminoa erabiltzen da orokorrean izaki zelulanitzetan espresatzen ez diren eta oso kontserbatuta dauden molekula hauek izendatzeko (Mackenzie et al. 2006; Magnadóttir 2006). Molekula hauen artean, polisakaridoak, lipopolisakaridoak (LPS), peptidoglikanoak, bakterioen DNA, birusen dsrna eta izaki zelulanitzen gainazalean aurkitzen ez diren bestelako molekulak daude (Magnadóttir 2006). Behin aktibatuta, ezagutzarako molekulek opsonizazioa eta patogenoaren fagozitosia eragin dezakete, zelula zitotoxiko naturalak estimulatu edo seinalizazio prozesu ezberdinak aktiba ditzakete, tartean, konplementu-sistema eta bidezidor litikoa edo fase akutuko erantzuna (Magnadóttir 2006). Erantzun hau guztia, plasmarekin zerikusia duten seinaleek, inflamazioaren aldeko zitokinek, induzitzen dute, aurretik aipatutako geneen trankripzioa eraginez (Bayne eta Gerwick, 2001). Zitokinak zelularen mintzeko hartzaile espezifikoekin lotzen dira nukleoko gene ugariren indukzioa, areagotzea edo eta inhibizioa eragingo duten erantzun-kateak piztuz (Secombes et al. 2001; Mulder et al. 2007). Zitokina garrantzitsu asko identifikatu dira arrain teleosteoetan, mota ezberdinetako interleukinak, nekrosi tumoraleko α faktorea (TNF-α), interferona, hazkuntzaren β transformazio-faktorea (TGFβ) eta zenbait kemokina; azken hauek, zoldura gunera neutrofilo eta makrofagoen migrazioa eman dadin beharrezkoak dira. Azkenik, garrantzitsua da azpimarratzea, erantzun immunearen azterketa espezie urtarretan ere garrantzitsua dela ikuspegi toxikologikotik, konposatu xenobiotiko asko immunoezabatzaileak direla deskribatu baita; PAHak esaterako arrainen immunoezabatzaile bortitzak dira (Carlson et al. 2004; Reynaud eta Deschaux, 2006). Interesgarria da aipatzea, kronikoki kutsatutako estuarioetan bizi diren arrainetan erantzun immunearekin zerikusia duten geneak zeharo erregulatuta daudela (Williams et al. 2003; Fisher eta Oleksiak, 2007; Roling et al. 2007). Ez dugu ahaztu behar gainera, sistema immunearekin erlazionaturiko geneak direla espresio maila aldakorrenak erakusten dituztenak arrain populazioen artean (Bayne et al. 2006). Arrain-populazioak zoldurak sortzen dituzten agenteen eraginpean egotean, arrunta izaten da superbibentzia ezberdina eta ugalketarako arrakasta ezberdina erakustea, agentearen aurrean jaiotzezko erresistentzia altuagoa duen populazio baterantz bideratuz (Bayne et al. 2006). Ondoren, hilkortasun ezberdina dela eta, populazio batek patogenoaren aurreko jaiotzetiko erresistentzia altuagoarekin iraun beharko luke; fenotipo jakin horien erantzule diren eta konstitutiboki espresatutako proteinak kodetzen dituzten gene edo aleloak maiztasun handiagoz mantentzen dira populazioan eta ondorioz, ikerketa transkriptomiko konparatiboz aurki zitezkeen (Bayne et al. 2006). Argazki hau zeharo nahastu daiteke xenobiotikoen esposizio kronikopean. 35 S A R R E R A

42 Sarrera Inguruneko baldintza fisikoen aurreko moldatze-erantzunak edo baldintza fisiologikoen aldaketaren ondoriozko geneen espresio-profilak Hipoxia Organismo urtarrak sarritan, oxigeno mailen gora-beherak dituzten inguruneetan bizi dira, bai sasoiaren zein egunaren arabera, askotan baldintza extremoetan edo/eta tenperatura-aldaketa leunen menpe ere egonik (Gracey et al. 2007). Ornodunetan hipoxiaren aurreko moldatze fisiologikoa basodilatazioa eta erantzun angiogenikoak, beharrizan metabolikoen murrizpena eta bidezidor anaerobikoen bidezko energiaren lorpena erregulatzen duten seinaleen bidez gauzatzen da. Behar den oxigeno eskaria ez bada betetzen, behar den zelula kopurua ere murriztu egin behar da (Ton et al. 2003; Papandreou et al. 2005). Prozesu hauek, hipoxiak induzituriko faktoreen kontrolpean daude ornodunetan (Gracey et al. 2001; Ton et al. 2003; Papandreou et al. 2005). Arrainetan garatu zen lehenengo mikrotxipak Gillichthys mirabilis arrain eurioxikoaren 5376 anplikon zituen (arrainak baldintza hipoxikoetan mantenduz lortu ziren sekuentzia horiek) eta baldintza hipoxikoetan (%10 oxigenoa) 6 egunez mantendu ondoren, ehun ezberdinetan ematen diren aldaketa transkripzionalak aztertzea zuen helburutzat (Gracey et al. 2004). Hipoxiaren arabera erregulatutako 126 gene deskubritu ziren, 56 gibelaren espezifikoak, 34 muskuluarenak eta 36 ehun bietan espresatzen zirenak. Energia beharrizan handiagoa suposatzen duten prozesuetan, hazkuntza, lokomozioa eta proteinen sintesia, inplikatutako geneak azpiespresatu egin ziren, glukoneogenesiko geneak gainespresatu egin ziren bitartean (Gracey et al. 2004). Ondoren, antzeko ikerketak burutu dira euskarri ezberdinekin eta hipoxia erregimen ezberdinen pean, zebra-arrainaren ehun eta garapen-fase ezberdinetan nagusiki (Ton et al. 2002; 2003; van der Mer et al. 2005) eta baita Oryzias latipesen burmuin, zakatz eta gibelean (Ju et al. 2006). Cyprinus carpioren 13K-ko beste mikrotxip bat erabiliz, muskulua ez den bestelako ehunetan mioglobinak esposizio-biomarkatzaile gisa aurkitzea baimendu zuen (Fraser et al. 2006; Gracey et al. 2007). Mioglobinak muskulu-ehunetan betetzen duen funtzioa oxigenoaren garraioa erraztea da eta gibelean agertu zuen ezusteko gainespresioak, oxigenoaren banaketan betetzen duen funtzioa iradoki dezake, horrela, karpek hipoxia baldintzetan duten tolerantzia altua azalduz (Gracey et al. 2007). Ekoizpen altuko hurbilketa transkriptomikoen bitartezko hipoxiaren efektuaren azterketa ez da oso zabala ornogabe urtarretan. Kontsideratu beharra dago, marearteko guneetan bizi diren molusku bibalbioek airepean egoteari aurre egin behar diotela egunean bi aldiz. SSH hurbilketa erabili zen Crassostrea gigas ostrak 7-10 eta 24 egun baldintza hipoxikoetan mantendu ostean, zakatz, mantu eta liseri-guruinean azpi eta gainespresatutako geneak espezifikoki identifikatzeko (David et al. 2005). Metodo honek modu ezberdinean erregulatutako 616 sekuentzia azaldu zituen, hauek, arnasketa, karbohidratoen metabolismoa, lipidoen metabolismoa, metabolismo oxidatiboa, sistema immunea, proteina eta azido nukleikoen eraenketa, metalen bahiketa eta estres erantzuna bezalako 12 funtzio fisiologiko nagusiri zegozkielarik. Crassostrea virginica ostren hemozitoetatik abiatuta, forward-eta reverse-liburutegi subtraktiboak eratu dira ere eta lortutako ESTak Genbank-ean utzi dira CV CV bitarteko sarrera-kodeak erabiliz. Metodologia bera erabili zen Callinectes sapidusen hepatoarean hipoxiari erantzuten dioten geneak aztertzeko (Brouwer et al. 2004). Tenperatura Tenperaturan ematen diren aldaketak, arrainetan ematen den transkripzio mailako epe laburreko hotzaren aurreko aklimatazioa bereziki, Ictalurus punctatus arrainaren burmuinean (Ju et al. 2002), C. carpio (Gracey et al. 2004) eta Gillichthys mirabilisen ehun ezberdinetan (Buckley et al. 2006) 36

43 edo zebra-arrainen muskuluan (Malek et al. 2004) ikertu dira dentsitate altuko mikrotxipen bitartez (3. Taula). C. carpiok hotzaren aurrean ematen duen erantzun akutuan parte hartzen duten metabolismoarekin lotutako prozesu biologikoak ikertzean, burmuina da ehunik aktiboena gainespresatzen diren prozesu metabolikoei dagokienez (Gracey et al. 2004). Kolesterol, gantz-azido eta esterolaren metabolismoan ematen diren aldaketak argiak dira burmuinean, lipidoek nerbio sistema zentralean duten garrantziaren adierazle. Zitratoaren zikloan (TCA zikloa) parte hartzen duten geneak ikertutako sei-zazpi ehunetan emendatzen dira, hotzari egokitutako arrain populazioen entzima mitokondrialen jarduera emendatuak erakusten dituzten ikerketekin bat etorriz (Gracey et al. 2004; 2007). Hotzaren aurreko aklimatazioa, proteinen espresioa erregulatzen duten, ubikitinaren menpeko proteinen katabolismoan diharduten edo/eta proteinen tolespenean diharduten geneen espresio diferentzialarekin ere erlazionatuta dago. Tenperaturak eragindako estres kronikoa edo epe-luzeko tenperatura-aldaketen aurreko moldapenak ere, cdna mikrotxipen bidez ikertu dira Austrofundulus limnaeus arrainen gibelean (Podrabsky eta Somero, 2004) eta amuarrainen bihotzean (Vornanen et al. 2005). EEBBetako Atlantiar kostaldean bizi diren Fundulus heteroclitus arrainen bihotzeko 363 gene metabolikoren espresio-profiletan ere ezberdintasunak aurkitu dira (Paschall et al. 2004). Gene hauen analisi estatistikoaren arabera, 62 genetako talde bat identifikatu zen zeinen espresioa Atlantiar kostaldean zeharreko tenperaturarekin erregresio esangarria erakusten zuen (Paschall et al. 2004). Hau da, termoklinan zehar gune ezberdinetan jasotako animaliak baldintza fisiologiko berdinetara 9 hilabetez aklimatatu eta aztertu ondoren, gene metabolikoen %17ak, erlazio lineala agertu zuen mrna kantitatea eta animaliak bizi ziren inguruneko tenperaturaren artean. Geneen hibridazio heterologoa zebra-arrainen oligonukleotido-txip batean ere aplikatu da Pomacentrus moluccensis koraletako arrainetan beroak eragindako estresaren aurreko gene goiztiarren erantzuna identifikatzeko, izan ere, arrain hauek klima-aldaketak eragindako tenperaturaaldaketen menpe egon daitezke (Kassahn et al. 2007). Ornogabe urtarrei dagokienez, Crassostrea gigasen zakatz eta mantuko tenperaturaren menpeko liburutegi subtraktiboak baino ez dira sortu eta EST-sekuentziak Genbank-ean utzi ditu Meistertzheim A-L-ek. Bestalde, estres termiko akutu pean mantendutako Gadus morhua makailauaren muskulu eskeletikoa, gibela eta giltzurrun-bihotzeko SSH liburutegiak eta amuarrainean epeltasunak, hotzak eta hipoxiak eragindako estresaren SSH liburutegia jarri dira eskuragarri Genbank-ean azken hilabeteotan. Estresa Sarrera Kortisolaren (estresarekin erlazionatutako hormona nagusia arrainetan) transkripzio mailako efektuaren ikerketak garrantzi handia hartu du. Zentzu honetan, odoleko kortisol mailak emendatzen dituen arrainak esku tartean erabiltzearen estresa transkripzio mailan testatu da amuarrainen burmuin eta giltzurrunetan testatu dira (Krasnov et al. 2005). Giltzurruneko transkripzio-profilak cdnamikrotxipen bidez ikertu dira kortisol inplanteak jasotako Sparus aurata arrain heldugabeetan (Sarroupoulou et al. 2005), non karbohidratoen metabolismoan, poliaminen biosintesian, burdinaren homeostasian eta ioien garraioan diharduten geneak baitziren afektatuenak. McKenzie eta laguntzaileen lana (2006) oso interesgarria da zentzu honetan, hauek, kortisolaren jarduera immunoezabatzailea aztertu zuten arrankarien monozito/makrofagoetan. LPS bidezko makrofagoen estimulazioak makrofagoen aktibazioaren (zelulen diferentziazioa, apoptosiaren erregulazioa, zelulen atxekidura) gainerregulazioa ondorioztatzen du, kortisolarekin batera 37 S A R R E R A

44 Sarrera inkubatuta berriz, LPSk gain edo azpierregulatutako geneen %53 edo %78ko inhibizioa ondorioztatzen du, hurrenez hurren (12. Irudia). Figure 12. LPSek eragindako monozito/makrofagoen aktibazioaren analisi transkripzionala O. mykiss arrainean. Kortosolarekin ko-inkubatzean, LPSak gain- edo azpiespresatutako geneen%53 eta %78, hurrenez hurren, inhibitu egiten da. GOren bidez, LPS eta LPS + kortisol tratamenduak bereiz daitezke (Mackenzie et al. 2006). Gazitasunaren eraginpeko Anguilla anguillla aingiraren ehun osmoerregulatzaile nagusienen geneen espresio-profila aztertu da ere, 6144ko cdna-mikrotxip bat erabiliz (Kalujnaia et al. 2007) Konposatu kimiko toxikoen eraginpean Aurreko atalean (2.1.) ikusi dugu zeintzuk diren konposatu toxikoen eraginpean erregulatzen diren gene eta bidezidorrak bai ornodunetan eta baita ornogabeetan ere (ornodunen geneen homologoak). Geneon produktuen ekintza bateratuak esposizioaren aurreko moldatzea ekar dezake. Urteetan zehar, gene hauetako askoren eraenketa banaka aztertu da espezie urtar ezberdinetan, gehienak arrainak, eta urtetik aurrera ekoizpen altuko ikerketak aplikatzen ari dira, konposatu ezberdinek arrainen geneen espresioan duten jokatzeko mekanismoa aztertzeko asmoz (3. Taula). Arrainetatik kanpo, interesa erakutsi da propikonazola eta fenarimola (Soetaert et al. 2006; 2007), ibuprofenoa (Heckmann et al. 2006), kadmioa, kuprea eta zinka (Poynton et al. 2007) bezalako konposatuek Daphnia magnan ondorioztatzen duten erantzun toxikogenomikoa ikertzeko. Arrainen artean, zebra-arraina da konposatu kimikoen jarduteko modua aztertzeko erabiltzen den animalia modeloa, bere genoma teleosteo guztien artetik ondoen ezagutzen dena baita eta ornodunetan burutzen diren garapen mailako ikerketetan onartuen dagoen modeloa baita. Zebraarrainen mikrotxip ezberdinak erabili dira ehun ezberdinen edo/eta garapen-faseko moldatzeerantzunak aztertzeko klorpromazina edo mianserina bezalako neurofarmazeutiko (Van der Ven et al. 2005; 2006), zein dioxina (Hanley-Goldstone et al. 2005; Andreasen et al. 2006), 17α-etinilestradiol (Hoffmann et al. 2006; Martyniuk et al. 2007; Santos et al. 2007), Aroklor 1254 (Kreiling et al. 2007), artsenikoa (Lam et al. 2006), 3, 4 dikloroanilinaren (Voelker et al. 2007) aurrean eta DMBAk eragindako kartzinomen eraginpean (Lam et al. 2006). 38

45 Sarrera Konposatu kimikoei dagokienez, disruptore-endokrinoek eragindako transkripzio mailako aldaketen azterketan interes handia jarri da, bereziki estradiol eta konposatu estrogenikoei dagokienez. Zebra-arrainetik kanpo, 17β-estradiolaren eragina Micropterus salmoidesen (Larkin et al. 2003), medaka japoniarrean (Kishi et al. 2006), Platichthys flesusen (Williams et al. 2006) eta Cyprinodon variegatesen (Knoebl et al. 2006) gibelean aztertu da. 17α-etinilestradiolak sortutako eraginak amuarrainean ikusi dira (Hook et al. 2006; Finne et al. 2007), Cyprinus carpio heldugabeen gibelean (Moens et al. 2006) eta Carasius auratus ar helduen burmuinean (Martyniuk et al. 2006). DDE, dihidrotestosterona, 11-ketotestosterona eta 4-nonilfenola bezalako beste konposatu kimiko batzuen eragina arrain-espezie ezberdinetan ikertu da ere euskarri ezberdinak erabiliz (Larkin et al. 2003; Blum et al. 2004; Kishi et al. 2006; Moens et al. 2006). Moens eta laguntzaileen lana (2006) azpimarratzekoa da, izan ere 14 disruptore endokrino ezberdinek eragindako transkripzio-profilak aztertu dituzte (17β-estradiol, 17α-etinilestradiol, 4-nonilfenol, bisfenol A, tamoxifenoa, 17αmetiltestosterona, 11-ketotestosterona, dibutil phtalatoa, flutamida, binclozolina, hidrokortisona, CuCl2, propiltiouraziloa, eta L-triiodotironinaren gei L-tiroxinaren nahastea) Cyprinus carpioren cdna-mikrotxip batean. Konposatu ezberdin bakoitzari asoziaturiko transkripzio-profil espezifikoa diskriminatzeko gai izan ziren. Espresioan antzekotasunak aurkitu ziren modu analogoan jokatzen duten konposatuen artean (konposatu estrogenikoak, androgenikoak, kortisolaren modulatzaileak eta hormona tiroideoak) baina konposatu bakoitzak bere espresio-patroi propioa sortu zuen txipean (13. Irudia). Gainera, konposatu ezberdinen artean diskriminatzeko gai ziren geneak identifikatu zituzten. 13. Irudia. 14 disruptore-endokrinok C. carpion eragiten duten espresio-profila. Konposatu bakoitzak espresio-patroi zehatza eragiten du txipean. Taldekatzeanalisiek, konposatu estrogenikoen artean eta kortisolaren modulatzaileen artean ekintza-mekanismo berdintsuak identifikatu dituzte, horiek hormona tiroideo eta androgenoen ezberdinak izanik. Konposatu ezberdinen artean ezberdintzea lortzen duten 12 gene identifikatu dira. (Moens et al. 2006). Gehien ikertu den konposatu kimikoa kadmioa da, berak eragindako trankripzio-profila amuarrainean (Koskinen et al. 2004), platuxan (Sheader et al. 2006; Williams et al. 2006a), karpan (Reynders et al. 2006) eta Mediterraneoko muskuiluan (Venier et al. 2006) ikertu baita. Konposatu kimikoek eragindako transkripzio-profil diferentziala, espezie bakarraren gene-euskarrietan ere burutu da. Modu honetan, β-naftoflabona, Cd, CCl 4 eta pirenoaren (Koskinen et al. 2004; Krasnov et al. 2005b) eta 17α-etinilestradiol, benzo(a)pireno, Cr-VI, dikuat, trenbolona eta 2,2,4,4'- 39 S A R R E R A

46 Sarrera tetrabromodifenil eterraren jarduteko modua ikertu da amuarrainaren ehun ezberdinetan (Hook et al. 2006). Cd, CCl 4 eta pirenoaren eraginpeko Salmo trutta lacustrisen gibeleko transkriptoma ere ikertu da (Krasnov et al. 2007). Koskinen eta laguntzaileen ikerketaren (2004) arabera, epe labur eta ertaineko esposizioek bakarrik eragiten dituzte gene funtzionaletan espresio diferentzialak. Dosi altuetan, toxikotasunari erantzuten dion erantzun inespezifikoak erantzun espezifikoak estali egiten ditu. Venier eta laguntzaileak (2006) Mediterraneoko muskuiluek, metalen aurrean konposatu organikoekin konparatuz, ematen dituzten erantzun diferentzialak karakterizatzen saiatu dira. Helburu honekin, Cd, Cu eta Hg nahastearen pean mantendu zituzten muskuilu batzuk eta benzo(a)pireno, fluoranteno, 1-nitopireno, 1-aminopirene eta dioxina nahastearen pean beste zenbait. Beraien arabera, mikrotxiparen ikerketatik lortutako transkriptoma ezberdinak Veneziako urmaelako kutsadura ezberdineko guneetan hartutako organismoen kutsadura iturriak diskriminatzeko lagungarri izan daitezke (Venier et al. 2006). Zelaian hartutako organismoetan burututako ekoizpen altuko teknika mota hauen gaineko saiakerak azpimarratu nahiko genituzke, konposatu toxikoen esposizio akutu peko moldatzeerantzun asko (defentsoma eratzen dutenak) transitorioak izateko joera dute eta esposizio kroniko pean konpentsazio-geneek ordezka baititzakete. Straub eta laguntzaileek (2004) Pseudopleuronectes americanus neguko platuxen gibeleko liburutegi subtraktiboak eratu zituzten New Jersey-ko bi itsasadarretan (oso inpaktaturiko Raritan- Hudson itsasadarrean versus industrializazio maila baxuagoko itsasadarrak). Transkriptoen profil ezberdinak lortu ziren arrainen habitat ezberdinetan. Gainerregulatutako liburutegian erantzun immuneko transkriptoen artean, C3, C7 konplementoa, H faktorea, Bf/C2 faktorea, modu diferentzialean erregulatutako amuarrainaren 1 proteina eta mikrobioen aurkako hepzidinak agertu ziren, kutsatutako guneko arrainak birus eta bakterioen eragin garrantzitsuaren eraginpean zeudela aditzera emanez. Lortutako transkriptoen artean, I eta II. faseko xenobiotikoen metabolismoko CYP1A, CYP3A eta GST ere aurkitu ziren. VTG I eta II eta arraultza inguratzen duten proteina (zp) azpieranduta agertu ziren ere. Beraz, ematen duenez xenobiotikoen esposizioaren adierazle gisa erabiltzen diren gene klasikoak kronikoki kutsatutako estuarioetan ere desberdinki eraendu egiten dira. Williams eta laguntzaileek (2003) antzeko SSH ikerketak burutu dituzte Tyne itsasadar kutsatuko eta Alde itsasadar garbiko platuxen transkriptomak erabiliz, eta lortutako EST sekuentziak, "geneen ehiza" bidez lortutako estresarekin lotutako beste zenbait sekuentziarekin batera Europako platuxan egindako 160 genetako lehenengo makrotxipa osotu zen. Mikrotxipak adierazi zuen espresio mailak oso aldakorrak zirela emeen populazioetan, beraz, ezin izan zen estuario bien artean ezberdintasunik ezarri. Bestalde, platuxa arretan 11 gene espresatu ziren modu ezberdinean Tyne itsasadar kutsatuan, CYP1A1, UGT, aldehido dehidrogenasa, paraoxanasa eta Cu/Zn SOD bereziki aipagarriak izanik, bitartean, C3 konplementua azpieraendu egin zelarik (Williams et al. 2003). Bestalde, Fundulus heteroclitus itsasadarreko arrainak laborategian pirenoaren eraginpean mantendu ostean pirenoaren eraginari erantzuten dioten biomarkatzaile ugari lortu ziren SSH bidez. Gene-biomarkatzaile hauek kreosatoz kutsatutako Hego Carolinako Superfund eskualdean ikertu ziren Q-PCR bidez. Pirenoaren eraginez modu diferentzialean espresatutako geneetako asko ez ziren aldatu kreosatoz kutsaturikoetan. Hala ere, CYP1A1 eta EST bat bai induzitu zirela (Roling et al. 2004). 40

47 Sarrera Era berean, kromoz kutsatutako Hego Carolinako Superfund eskualdeko erremediazioak aukera aproposa eskaini zuen 270 EST sekuentzia dituen Fundulus makrotxiparen erabilera ikertzeko. Cr(VI), Cr(III), As(III) eta pireno pean mantendutako arrainetan txipa lortzeko SSH eta ddrt-pcr erabili ziren, horrela erremediazioaren eraginkortasunaren jarraipena egiteko (Roling et al. 2007). Fundulusak urteko erremediazioaren aurretik eta erremediazioa hasi ondorengo 2003 eta urteetan hartu ziren. Era berean, arrainak erreferentzia-gune batean ere hartu ziren. Shipyard Creekeko eta gune garbi bateko urteko Fundulusen gibeleko geneen espresioa zeharo ezberdina zen. Erremediazioak gune bietan hartutako Fundulusen geneen espresio-patroiak homogenizatu zituen, modu ezberdinean espresatutako gene kopurua urtean 22 izatetik urtean 4 izatera jaitsiz (Roling et al. 2007). Ikerketa hau oso aproposa da mikrotxipak arriskuen ebaluazioan izan dezaketen erabilgarritasuna islatzeko. Bestalde, Fisher eta Oleksiak (2007)-ek Fundulusen cdna-mikrotxipak erabili dituzte 384 gene metabolikoren burmuineko espresio-patroiak alderatzeko bederatzi populazio desberdinetan: kutsatutako 3 Superfund populazio independenteak eta Superfund populazio bakoitzarentzat genetikoki antzekoak diren bi, erreferentzia-populazioak. Kutsatutako populazioen geneen %17ak moldatze-aldaketak erakutsi zituen geneen espresioan. Gene hauen artean, bi (CYP1B1 eta NADHubikinona oxidoerreduktasa AGGG azpiunitatearen aitzindaria) erantzun kontserbatua erakutsi zuten hiru Superfund estuarioetan, inguruneko kutsaduraren aurreko moldatze-mekanismo independiente eta komunak iradokiz populazio hauetan. Azkenik, zelaiko M. galloprovincialisen 2K-ko cdna-mikrotxipa erabiliz (Venier et al. 2006), Veneziako urmaeleko petrokimika inguruko muskuiluak oso erraz bereiz zitezkeen itsaso zabalaren alboan bizi ziren muskuiluetatik beraien espresio patroia zela eta. Lortutako gene-markatzaileetako batzuk, kutsatzaile organikoak adierazten zituzten metalikoak baino gehiago, gune horretako emaitza kimikoekin bat eginez (Venier et al. 2006). 4. Zergaitik zentratu behar dugu peroxisomen proliferazioaren ikerketan ekoizpen altuko toxikogenomikaren aroan? Toxikogenomikaren garapenean eta ezarpenean aurreratu arren, hainbat oztopok geneen espresioaren gaineko datuen interpretazioa eta beraiengandik esangarria eta erabilgarria den informazioaren erauzketa egokia trabatu dute. Esate baterako, konposatuen ekintza-mekanismoa, dosia, denbora eta esposizio-luzeraren eta zelularen fenotipoaren menpe egon daitezke (Hanley- Goldstone et al. 2005; Krasnov et al. 2005a; 2005b; van der Ven et al. 2005; Reynders et al. 2006). Gainera, geneen espresio-erantzunak dinamikoak eta itzulgarriak dira, beste emaitz toxikologiko zenbaiten kontrara (Volz et al. 2005). Honek dosi-erantzun erlazioak ezartzea eta sistema modeloekin estrapolatzera garamatza. Arriskuen karakterizazio zehatza burutzeko, ikerkuntz genomikoetan oinarrituriko emaitzak osasun egoeraren nondik-norako zehatzekin erlazionatu behar da. Ezin da ondorioztatu geneen espresioan emaniko aldaketa esangarri batek efektu kaltegarri bat ondorioztatuko duenik (edo aldaketa txiki batek, bere eza) emaitzak kontestu egokian jarri eta geneen espresioaren aldagarritasun fisiologikoak baldintza naturaletan zeintzuk diren ezagutu arte (Ankley et al. 2006; Denslow et al. 2007). Genotipo jakin bakoitzari fenotipo bat ezarri behar zaio ikerketa toxikogenomikoetan (Moggs 2005). Peroxisomen proliferazioari dagokionez, kontu bi sortzen dira prozesu hau ekoizpen altuko ikerketa toxikogenomikoetan ikertzean: 1.- Konposatu xenobiotiko eta endobiotikoen eraginpean, espezie urtarretan prozesua nola kontrolatzen den ulertzea, horrela geneen erregulazio-patroi jakin bat ezarri dakioke konposatu baten esposizioaren ekintzari. Konposatu kimikoen eraginpean, peroxisometako geneen erregulazioa 41 S A R R E R A

48 Sarrera badago espezie urtarretan, eta zer? Horrek esan nahi du peroxisomen proliferazioa ematen dela maila zelularrean? Horrek esan nahi du badagoela aukerarik kartzinogenizitate ez-mutagenikorako (ugaztun-espezie sentikorretan gertatzen den legez)? Peroxisomen proliferazioa espezie urtarretan ulertu behar dugu, bere esanahia genomaren erantzun zabalen kontestuan, interpretatzeko gai izan aurretik 2.- Oraindik arrain eta ornogabe urtarren mikrotxipek (eta Genbank datu-baseak) peroxisomen proliferazioan adierazgarriak diren gene-sekuentziarik ez dute agertzen. Beraz, prozesu zelular honi dagokion informazio gutxi lortzea espero da esisititzen diren DNA-euskarrietan. Arrainen artean, zebra-arrainen mikrotxipek bakarrik betetzen dute organismoaren ia genoma osoa, beraz, sekuentzia peroxisomikoak barneratzen dituzte, baina agian zebra-arraina ez da espezie erantzuleena peroxisomen proliferatzaileen aurrean. Arrain-espezie guztiek ez dute erantzuten beste arrain batzuetan peroxisomen proliferazioa eragiten duten kinaden aurrean (Bilbao et al. 2006a; 2006b). Toxikologian erabiltzen diren beste arrain espezie batzuen mikrotxipei dagokienez, arrankariaren 1380 sekuentziatako cdna-mikrotxipa, pirenoak gibel eta burmuinean eragiten duen transkripzio-profila aztertzeko erabili da (Koskinen et al. 2004; Krasnov et al. 2005b), baina gene hauen artean PTS2 hartzailearen sekuentzia, azil-coa tioester 1 hidrolasarena, katea oso luzeko azil- CoA sintetasa (VLCAS), aldehido oxidasa, serina-pirubato aminotransferasa, katalasa, proteina multifuntzionala (MFP) eta azil-coa lotzen duen proteina aurki daitezke gene peroxisomiko gisa (Krasnov, komunikazio pertsonala). Peroxisomen proliferazioaren ikuspuntutik VLCAS eta MFP baino ez dira esangarriak. Ugaztunetan peroxisomen proliferatzaile tipikotzat jokatzen duten konposatu tipikoei dagokienez, dibutil phtalatoaren efektu endokrinoak bakarrik ikertu dira karpa arruntaren tamaina erdiko mikrotxip batean (960 gene zati) (Moens et al. 2006), baina bertan ez dago gene peroxisomiko bat bera ere. Beraz, ingurune toxikologirako bidezidor adierazgarria izanik, peroxisomen gaineko ikerketan zentratzeko beharrizana dago oraindik Peroxisomak Peroxisomak, saguen giltzurruneko tubuluetan behatu zituen lehen aldiz Rhodinek (1954), transmisiozko mikroskopio elektronikoa erabiliz. "Mikrogorputz" terminoa erabili zuen mintzez inguratutako organulu elektrodentsook definitzeko. Mikrorgorputz terminoa gaur egun ere, zelula eukariotikoetan agertzen diren mota honetako organuloak definitzeko erabiltzen da. De Duve eta laguntzaileen hasierako ikerketa lanetan izendatu zen organulu hau lehen aldiz "peroxisoma" (de Duve 1965; de Duve eta Baudhuin, 1966). De Duve eta Baudhuinek (1966) deskribatu zuten, peroxisomak gutxienez H 2 O 2 produzitzen duen flabin oxidasa bat eta berau anderatzen duen katalasa dituen organulua dela. Peroxisomak oso organulu heterogeneoak dira, beraien tamaina, forma eta proteinen konposizioa espezie, ehuna, zelula, egoera metaboliko eta garapen-fasearen arabera aldatuz. Orokorrean, peroxisomak pikor fineko matrix elektrodentsoa duten eta mintz bakar batez inguratutako organuluak dira nm-ko lodiera duen mintzarekin (Fujiki et al. 1982; Zaar 1992) 140/200 nmol/mg-ko fosfolipido/proteina ratioa dute (Fujiki et al. 1982; Lazarow eta Fujiki, 1985). 0.1 eta 1.5 µm bitarteko diametroa dute nahiz eta kopurua, tamaina eta forma ikertzen den organu bakoitzeko aldatu egiten diren; hala ere, orokorrean lipidoen metabolismo aktiboa duten ehunetan ugariagoak dira (Gorgas 1987). Karraskarien gibelean peroxisomen gaineko ikerketa ugari egin da; bertan peroxisomak, esferikoak dira eta kristalizaturiko urato oxidasak (UOX) eratzen duen barne kristaloidea agertzen dute (Völkl et al 1988). Xantina oxidasa (XOD) ere karraskarien gibeleko 42

49 peroxisomen nukleoidearekin asoziatu da (Angermüller et al. 1987). Gizakia bezalako beste zenbait espezieren gibelean ez da kristaloiderik aurkitu, ez baita UOXik agertzen. Oro har, animalien peroxisomek, laborategietan ohikoak diren ugaztun edo gizakiez eta legamiez gain, ez dute aparteko atentziorik jaso. Arrainen eta moluskuen peroxisomen morfologia eta proteinen konposizioaren inguruan eskuragarri dauden datu gehienak Fahimi eta Cajaravillek (1995), Cancio eta Cajaravillek (2000) eta Cajaraville eta laguntzaileek (2003) bildutakoak dira. Peroxisomen funtzio metabolikoak Sarrera Peroxisomek animalien zeluletako hainbat prozesu metabolikotan parte hartzen dute, nagusiki, lipidoen homeostasiarekin erlazionaturikoak: gantz-azidoen β-oxidazioa, kolesterolaren sintesia, behazun-azidoen sintesia, xenobiotikoen metabolismoa, ROSen metabolismo eta sorrera, poliamina, purina, aminoazido eta glioxilatoaren katabolismoa, plasmalogenoaren biosintesia, azido fitanikoaren α-oxidazioa eta gantz-azidoen elongazioa (Van den Bosch et al. 1992; Mannaerts eta Van Veldhoven, 1993; Singh 1997). Peroxisomen β-oxidazioa ikerketa honen gai garrantzitsuena den heinean, prozesu hau sakonago deskribatuko da. Animalia zeluletan, β-oxidazioa mitokondrio eta peroxisometan ematen da (Mannaerts eta Van Veldhoven, 1993). Arau orokor gisa, peroxisomek katea luzeko gantz-azidoen β-oxidazioa betetzen dute, mitokondrioek bitartean, 18C-arteko gantz-azidoena (Chance eta McIntosch, 1994; Reddy eta Mannaerts, 1994; Fan et al. 1996). Peroxisomek katea luzeko gantz-azidoen oxidazioaren %5-20 bitartean parte hartzen dute (Singh 1997), gibel, burmuin eta kultibatutako azaleko fibrobalastoetan katea oso luzeko gantz-azidoen oxidazioa (>C22) esklusiboki peroxisometan ematen den bitartean (Mannaerts eta Van Veldhoven, 1993; Chance eta McIntosch, 1994; Reddy eta Mannaerts, 1994; Singh 1997). Peroxisomen β-oxidazioa beste zenbait bidezidorretan ere ezinbestekoa, tartean, kolesterolaren alboko katearen mozketa behazunaren sintesia gauzatzeko, katea luzeko azido dikarboxilikoen katabolismoa, azil adarkadura duten xenobiotikoak, prostaglandinak eta zenbait eikosanoide, asegabeko gantz-azidoak eta azido pristanikoa, azido pipekolikoa eta azido glutarikoa (Mannaerts eta Van Veldhoven, 1993; Devchand et al. 1996; Singh 1997). Mitokondrio zein peroxisometako β-oxidazioak, aktibaturiko gantz-azidoen gainean bakarrik jarduten du, hau da, beraien azil-coa deribatuen gainean dihardu. Aktibazio hau gauza dadin, peroxisomek azil-coa ligasak edo sintetasak dituzte beraien mintzean (Singh 1992). Katearen luzera ezberdinen gainean espezifizitatea duten ligasak daude zelulan. C12-C22 gantz-azidoen gainean diharduen palmitoil-coa ligasa mitokondrioetan, mikrosometan eta peroxisometan dago (Singh 1992). Mitokondioen barne-mintzean zeharreko translokazioa burutzeko, azil-coa-ren deribatuak azil-karnitinaren deribatu bihurtu behar dira, peroxisometan beharrezko ez den urratsa. Ligasa berezi bat, lignozeroil-coa ligasa beharrezkoa da peroxisometan 22 C-tik gorako gantz-azidoen aktibaziorako eta entzima hau mikrosoma eta peroxisometan agertzen da (Singh 1992). Behin azil-coa deribatuak aktibatuta β-oxidazioan sartzen dira hiru entzimek lau urratsetan, dehidrogenazioa, hidratazioa, dehidrogenazioa eta mozketa tiolitikoa, burutzen duten bidezidorrean (Tolbert 1981; Schepers et al. 1990; Van den Bosch et al. 1992; Mannaerts eta Van Veldhoven, 1993; Reddy eta Mannaerts, 1994; Baumgart et al. 1996; Dieauaide-Noubhani, et al. 1996; Singh 1997; Baes et al. 2000). Azil-CoA oxidasa (AOX), proteina multifuntzionala (MFP) eta 3-keto azil-coa 43 S A R R E R A

50 Sarrera tiolasa peroxisomen matrizean daude eta egitura aldetik mitokondrioetan funtzio bera betetzen duten entzimen ezberdinak dira. AOX entzima mugatzailea da bidezidorrean eta sustratu ezberdinak hartzen dituzten hiru forma deskribatu dira arratoietan (Schepers et al. 1990). Induzigarria den palmitoil-coa oxidasak (AOX1) aseturiko katea luze eta oso luzeko gantz azidoen gainean dihardu, azido dikarboxiliko eta prostaglandinen gainean ere bai. Pristanoil-CoA oxidasak (AOX3) adarkaturiko gantz-azidoen gainean dihardu eta gibelean espezifikoki agertzen den trihidroxikoprostanoil-coa oxidasak (AOX2) azido biliarren bitartekoetan (Baumgart et al. 1996). AOX2 orainarte, ugaztun, hegazti eta anfibioetan baino ez da deskribatu (Morais et al. 2007). MFPk β-oxidazioko bigarren eta hirugarren urratsak katalizatzen ditu, gantz-azido asegabeak katabolizatzeko beharrezkoa den isomerizazio urratsarekin batera (Tolbert 1981; Mannaerts eta Van Velhoven, 1993; Reddy eta Mannaerts, 1994; Dieauaide-Noubhani, et al. 1996; Singh 1997). MFPren bi forma karakterizatu dira arratoien gibeleko peroxisometan, batak gantz-azidoen β-oxidazioan parte hartzen du eta besteak behazun azidoenarenean (Dieauaide-Noubhani et al. 1996). Bidezidorreko azken erreakzioa 3-ketoazil-CoA tiolasak katalizatzen du eta jatorrizko molekula baino bi karbono laburragoa den azil-coa deribatua eta azetil-coa molekula bat lortzen dira. Prozesu honetan bi entzimek hartzen dute parte gutxienez, 3-ketoazil-CoA tiolasa (THIO) eta sterol carrier protein-x-ek (ScpX). Arratoietan bi THIO gene daude, THIOA eta THIOB (Hijikata et al. 1990; Osumi 1993; Hansmannel et al. 2003). Azil-CoAk ez dira guztiz oxidatzen peroxisometan, 6- tik 12 karbono bitarteko gantz-azidoak lortzen dira eta mitokondrioetara garraiatzen dira oxidazio osoa eman dadin. Peroxisomek katea ertaineko karnitina tranferasa dute eta beharbada substratuen transferentzian dihardu (Singh 1997). Azil-xenobiotiko konposatuetako batzuk edo ω-oxidazioren bidez alboko kate karboxiliko bat hartzen duten xenobiotikoak, β-oxidazioan sar daitezke, laburrago, polarrago eta iraizteko errazagoak diren molekulak emateko (Mannaerts eta Van Velhoven, 1993). Azil-xenobiotikoen metabolitoak konposatu beraren eraginpean mantendutako arratoien gernuan aurkitu dira (Yamada et al. 1986). Prozesua karraskarien hepatozitoetan estimulatu egiten da peroxisomen proliferazioa eragiten duten konposatuen eraginpean (Yamada et al. 1986; 1987). Peroxisometako β-oxidazioak mitokondrioetako β-oxidazioak baino nabarmenago oxidatzen ditu xenobiotikoak (Yamada et al. 1987) Peroxisomak arrain eta moluskuetan Arrainen peroxisomak µm bitartekoak dira baina Chelon labrosus lazunean peroxisoma handiagoak deskribatu dira, µm (Orbea et al. 1999a). Espezie honetan peroxisomak handiagoak dira udan eta animalia zaharretan (Orbea et al. 1999a). Lazunean, peroxisomak taldekatuta agertzen dira eta peroxisomen dentsitate bolumetrikoa altuagoa da hepatozito periportaletan perizentraletan baino, ezberdintasun hauek neguan nabermenagoak izanik (Orbea et al. 1999a). Arrainetako peroxisomek lipidoen β-oxidazioa eta purinen katabolismoa betetzen dute. AOX, MFP eta karnitina azetiltransferasa identifikatu dira arrain espezie ezberdinetan (Cancio eta Cajaraville, 2000). Western-blot analisien bidez, C. labrosus eta Danio rerio arrainen gibeleko AOXek arratoien AOXen aurkako antigorputzarekin erreakzionatzen duela ikusi da, ugaztunetan deskribatutako hiru AOX azpiunitateak azalduz (Orbea et al. 1998; Ortiz-Zarragoitia et al. 2006). Entzimaren kokapena lazunen peroxisometan immunozitokimikoki ziurtatu da (Orbea et al. 1999; Cajaraville et al. 2003). Karnitina azetiltransferasa mitokondrioetako β-oxidaziorako oinarrizko entzima da eta Raja erinacea eta Carassius auratusen peroxisometan ere aurkitu da peroxisometan 44

51 Sarrera laburtu diren gantz-azidoen mitokondrioranzko garraioa errazten (Böck et al. 1980; Stewart et al. 1994). Lipidoak arrain espezie askoren osagai nagusiena dira eta bertan hazkuntza, ugalketa zein mugimendurako beharrezkoa den energia lortzeko funtzio garrantzitsua betetzen dute (Nevejan et al. 2003; Tocher et al. 2003). Organismo urtarrak eta bereziki itsasoko organismoak ugaztunen metabolismo peroxisomikoko substratuak diren katea luze eta oso luzeko ω3 gantz-azidoetan aberatsak dira, nabarmenki, azido dokosahexaenoikoa eta azido eikopentaenoikoa (EPA) (Nevejan et al. 2003; Tocher et al. 2003). Zentzu honetan, jatorri itsastarreko gantz-azido poliasegabeek (PUFA) ugaztunen β-oxidazioa induzi dezakete eta dietako zenbait gantz-azidok gibeleko β-oxidazioko jarduera erregulatzen du izokin espezieetan (Torstensen eta Stubhaug 2004; Leaver et al. 2006) baina ez ase arte elikatzean gibel lipidiko handiak garatzen dituzten gadidoetan (Nanton et al. 2003). Ematen duenez gantz-azidoetan aberatsa den dieta pean gibeleko β-oxidazioa erregulatzen ez duten espezieetan lipidoak metatu egiten dira gibelan. Lipido eskuragarri asko dauden sasoietan, uda, gadidoen gibel koipetsuak beha daitezke (Nanton et al. 2003). Peroxisomen β-oxidazioaren bidezko gantz-azidoen oxidazioaren gaitasuna eta bere ekarpena, mitokondrioetako β-oxidazioarekin alderaketa, espeziearen araberakoa da. Zenbait arrainen gibel eta muskuluan peroxisometako β- oxidazioa garrantzisua dela dirudi, non gantz-azido ugari oxidatzeko gai den (Crockett eta Sidell, 1993a; 1993b). Modu honetan, Melanogrammus aeglefinus eta Salmo salarren gibeleko β-oxidazioa %100 peroxisomiko da (Nanton et al. 2003; Froyland et al. 2000), muskulu gorri (91%) eta zurian (97%) β-oxidazioa nagusiki mitokondriala den bitartean (Nanton et al. 2003). Kontrara, beste zenbait arrain teleosteoren gibeleko β-oxidazioa %30-50 peroxisomikoa da (Crockett eta Sidell, 1993a; 1993b). Arratoi normalaren hepatozitoetan, peroxisometako β-oxidazioak palmitatoaren oxidazio zelularrean %10-30 bitartean parte hartzen duela estimatu da (Mannaerts et al. 1979; Kondrup eta Lazarow, 1985). Espezie batzuetan peroxisometako β-oxidazioak mitokondrioetakoak baino nagusitasun handiagoa izatearen esangura fisiologikoa ez da ikertu oraindik eta ikerketa gehiago eskatzen du, aurkezten duten katea luzeko gantz-azidoen (LCFA) kantitate handiaren aurreko moldaketa izan baitaiteke. Peroxisomak muskuiluen liseri-epitelioan ikertu dira (Cancio eta Cajaraville 2000). Mytilus galloprovincialis muskuiluaren liseri-guruineko peroxisometan nukleoideak nekez aurki daitezke eta soilik liseri-zeluletan (Cajaraville 1991; Fahimi eta Cajaraville, 1995), giltzurruneko peroxisometan nukleoide amorfoak aurkitzen diren bitartean (Cancio eta Cajaraville, 2000). Gastropodo lurtarretan, nukleoideak Ariolimax columbianusen (Dannen eta Beard, 1977) giltzurrunean maiz agertzen direla ikusi da baina ez Arion ateren (Dannen eta Beard, 1977). β-oxidazioko entzimak zein katalasa moluskuen peroxisometan aurkitu dira. AOX jarduera A. ater barearen peroxisometan aberastutako frakzioetan eta liseri-guruinean (Malik et al. 1987), M. galloprovincialisen liseri-guruinean (Cajaraville et al. 1992b; Cancio, 1998) eta Placopecten magellanicusen liseri-guruin eta bihotzean (Stewart et al. 1994). Arratoiaren AOX eta MFPren kontrako antigorputz poliklonalekin immunoerreaktiboak diren eta ugaztunen proteinen antzeko mugikortasun elektroforetikoa agertzen duten bi polipeptido aurkitu dira M. galloprovincialisen liseri-guruineko frakzio peroxisomikoetan (Cancio 1998; Cajaraville et al. 2003). 70 kda-etako peroxisomen mintzetako proteina (PMP70) ere muskuiluen liseri-guruineko frakzio peroxisomikoetan immunolokalizatu da arratoiaren aurkako antigorputza erabiliz (Cancio 1998). Moluskuetan peroxisometako β-oxidazioak duen garrantziaz ezer gutxi ezagutzen da, liseriguruinean nagusitzen dela jakin arren (Cancio eta Cajaraville, 2000). Moluskuen lipido-konposizioa arrain teleosteoen oso antzekoa denez, arrainen bidezidorrak duen garrantzi bera dueneko hipotesia bota daiteke. 45 S A R R E R A

52 Sarrera 4.3. Peroxisomen Biogenesia eta proliferazioa Peroxisomak organulu plastikoak dira eta zenbait baldintzaren pean tamaina, forma eta zenbakiari dagozkion erabateko aldaketak eta proteina edukiari dagozkion aldaketa arinak jasan ditzakete zelula mota ezberdinetan. Peroxisomak aurretik esisititzen diren peroxisomen fusio eta fisioz eratzen dira (Lazarow eta Fujiki, 1985; Lüers et al. 1990; Fahimi et al. 1993a; 1993b; Stefanini et al. 1994). Peroxisometako proteinak genoma nuklearrak kodetuta, zitosoleko polisoma askeetan sintetizatzen dira, gehienetan beraien bukaerako tamainuan (Lazarow eta Fujiki, 1985). Peroxisometara garraiatzeko ez da behar ez glikosilaziorik ezta itzulpen-ondorengo bestelako aldaketarik ere. Peroxisometarako garraioa bideratzeko bi itu-seinale peroxisomiko (PTS) nagusi identifikatu zaizkie matrizeko proteinei. Peroxisomako matrizeko proteina gehienek PTS1 izeneko motibo tripeptido kontserbatua darabilte; ondorioz, serina-lisina-leuzina (SKL) aminoazido-eskuentzia edo bere aldagaiak (S/A/C) (K/R/H)(L) (Gould et al. 1987; Subramani, 1996a; 1996b; Nöhammer et al. 2000; Emmanuelsson et al. 2003). SKLren aurkako antigorputzek animalia, landare eta legamien peroxisometako, hazien glioxisometako eta Tripanosomen glikosometako proteinak ezagutzen dituzte (Gould et al. 1990; Keller et al. 1991; Gietl 1996). Karboxilo terminalean kokatutako seinale hau ez da mozten garraioa bideratu ondoren. Bigarren seinalea amino terminalean kokatutako PTS2 da eta entzima gutxiagoren seinale gisa funtzionatzen duen nonapetido kontserbatua da (R/K)(L/V/I)(X 5 )(H/Q)(L/A) (Swinkles et al. 1991; Subramani, 1996a; 1996b; Waterham eta Cregg, 1997; Kunau 2001). THIOk esate baterako, PTS2 seinalea dauka (Kunau 2001) eta ugaztunetan 44 kda-eko aintzindari gisa sintetizatzen da. Peroxisometara garraiatu ondoren, proteinak itu-sekuntzia galtzen du eta behin heldu ondoren, tamaina 41 kda da (Swinkles et al. 1991; van Roermund et al. 1995). Peroxisomek proliferazio handia jasan dezakete, sarritan entzimen jardueraren indukzioarekin. Peroxisomen proliferazioarekin batera, erretikulo endoplasmatiko (Grasso 1993) eta mitokondrioetan ere aldaketak behatzen dira (Meijer eta Afzelius, 1989; Cherkaoui Malki et al. 1991). Peroxisomen proliferazioa droga hipolipidemikoen, klofibratoa, eraginpean mantendutako arratoien gibelean deskribatu zen lehenengo aldiz (Hess et al. 1965). Ondoren, interes handiagoa hartu zuen peroxisomen proliferazioa kartzinogenesiarekin erlazionatu zenean arratoietan (Reddy et al. 1980). Peroxisomen proliferazioa eragiten duten konposatuen kopurua ikaragarria da eta itxuraz ez dute antzik egiturari dagokionez, talde azidiko komun baten edukia, normalean talde karboxilikoa, ez bada (Bentley et al. 1993; Lake, 1995). Dieta koipetsuak, hotzaren aurreko moldatzea, E bitaminaren gabezia, riboflabina gabezia, obesitate genetikoa, diabetesa eta jan ezak ere peroxisomen proliferazioa eragin ditzakete karraskarietan (Bentley et al. 1993). Ugaztunen zeluletako peroxisomen proliferazioa lipidoen homeostasian ematen diren aldaketekin asoziatuta dago, gantz-azidoen mantenuan diharduten entzimen jardueren aldaketen emaitza gisa; horien artetik, aipagarriena peroxisometako β-oxidazioa eta mikrosometako ω-oxidazio dira (Meijer eta Afzelius, 1989; Keller et al. 1993; Reddy eta Mannaerts, 1994; Lemberger et al. 1996a; Mandard et al. 2004). Peroxisometako β-oxidazioko hiru entzimak induzitu egiten dira bai proteina zein mrna mailan, 30 aldiz jarduera altuagoak neurtuz (Nemali et al. 1988; Osumi et al. 1993; Schad et al. 1996; Mandard et al. 2004). Hau prozesu ez-paraleloa da animalien zeluletan, bidezidor metaboliko berean parte hartu eta jarraian dauden bi entzimaren jarduera baino gehiago ez baita induzitzen (Osumi et al. 1993). Jardueraren emendioa izaten duten beste entzima peroxisomiko batzuk 2, 2 -dienoil-coa erreduktasa 2 (DECR), karnitina azetiltransferasa eta karnitina oktanoiltransferasa (Mandard et al. 2004) dira. Peroxisomen matrizeko entzimen indukzioa 46

53 Sarrera peroxisomen mintzen ekoizpenarekin bateratu behar da, gibelean gainontzeko ehunetan baino nagusitasun handiagoz ematen den prozesu batean (Nemali et al. 1989). Peroxisomen proliferazioa eragiten duten baldintzek katalasa eta beste hainbat oxidasa bezalako entzima peroxisomikoetan eragiten dute. Peroxisometatik kanpoko hainbat entzimaren jarduera ere areagotu egiten da, bereziki gantz-azidoen ω-oxidazioan parte hartzen duten mikrosometako CYP4A familiakoena (4A1, 4A2, eta 4A3) bi-bost aldiz induzitzen da (Bell et al. 1992; Muerhoff et al. 1992; Mandard et al. 2004). Ugaztunek modu ezberdinean erantzuten diete proliferazioa eragiten duten konposatuei. Esate baterako, arratoi eta saguak oso erantzule onak dira, marmoset eta kaputxin tximinoek ahulki erantzuten dute eta akuri eta gizakiek ez dute erantzuten (Fahimi et al. 1993a). Kontutan hartu beharreko beste parametro batzuk, proliferazioa eragiten duten agenteen potentzia eta dosia eta organismoen generoa dira, karraskari arrek emeek baino indartsuago erantzuten baitute (Bentley et al. 1993; Rao et al. 1994). Peroxisomen proliferatzaile gisa erabili diren konposatu klasikoek "konposatu hipolipidemiko" izen generikoa jaso dute eta horietako batzuk, arteriosklerosi eta hiperkolesterolemiadun gaixoetan agente terapeutiko legez erabili dira. Beste konposatu talde baten jarduera proliferatzailea ere oso modu zabalean ikertu da, plastifikatzaileena alegia, bereziki phtalato-esterrena (Bentley et al. 1993; Lake 1995; Cancio et al. 1998). Peroxisomen proliferatzaileak diren beste konposatu batzuk, esteroideak, pestizidak, elikagaiei zaporea ematen dieten konposatuak, industriako hainbat konposatu kimiko, hainbat droga terapeutiko, hidrokarburo eta protuktu natural (Bentley et al. 1993; Lake 1995). Egitura aldetik loturarik ez badute ere, konposatu mota gehienek funtzio azidikoa dute, gehienetan talde karboxilikoa jatorrizko konposatuan edo beraien metabolitoren batean (Lake 1995). Ugaztunetan peroxisomen proliferazioa ikusgarrien gibelean bada ere, gibelaz kanpoko ehunetako peroxisomek ere erregulazioa jasotzen dute peroxisomen proliferatzaileen eragin pean (Hinton eta Price, 1993). Karraskarien gibeletik kanpo, peroxisoma gehien kortexean aurkitzen da (Zaar, 1992). Entzima eta mrnaren indukzio maila ez da gibelean ematen denaren parekoa eta entzima gutxiago induzitzen dira (Nemali et al. 1988; Bell et al. 1992; Hinton eta Price, 1993). AOXen jarduera entzimatikoa 28 aldiz areagotzen da gibelean eta 9 aldiz bakarrik zipofibratoz trataturiko arratoien giltzurrunetan (Nemali et al. 1988; Bell et al. 1992). Giltzurrunak, baina ez beste edozein ehun, gibeleko mikrosomako CYP4A1en induzigarritasuna parekatzen du (Bell et al. 1992; Hinton eta Price, 1993). Beste karraskari batzuen ehunetan, heste, barrabil, ehun adipotsu arre eta adrenalean, muskulu eskeletiko eta kardiakoan eta birikan erantzun mugatuagoak aurkitzen dira (Baumgart, komunikazio pertsonala). Peroxisomen proliferatzaileen eraginpeko sagu eta arratoiek gibeleko tumoreen intzidentzia altuagoa dute, erabilitako agentearen arabera (Reddy eta Lalwani, 1983; Lake 1995). Proliferatzaileek espeziearekiko espezifikoa den erantzun hau eragiten dute konposatu genotoxikoak ez badira ere (Kraupp-Grasl et al. 1991; Bayly et al. 1994; Roberts et al. 1995). Peroxisomen proliferatzaileak diren konposatuek eragindako hepatokartzinogenizitatea azaltzeko hainbat mekanismo proposatu dira karraskarietan: a) estres oxidatiboaren indukzioa, b) zelulen erreplikazio edo mitosien areagotzea, c) gibeleko kalteen eragitea eta d) apoptosiaren ezabatzea Peroxisomen proliferazioaren mekanismoa: peroxisomen proliferatzaileek aktibaturiko hartzaileak (PPARak) Peroxisomen proliferatzaileek aktibaturiko hartzailea (PPAR), hartzaile nuklearraren superfamiliako kide da eta Isseman eta Green-ek (1990) saguetan klonatu eta peroxisomen proliferazioaren erantzunaren arduradun gisa identifikatu zuten (Mangelsdorf eta Evans, 1995; 47 S A R R E R A

54 Sarrera Mangelsdorf et al. 1995). Ondoren, PPAR berriak klonatu dira ornodunetan (Dreyer et al. 1992; Göttlicher et al. 1992; Jow and Mukherjee, 1995) zenbait arrain espezie barne (Ruyter et al. 1997; Maglich et al. 2003; Boukuvala et al. 2004; Raingeard et al. 2006; Kondo et al. 2007). PPARek, hartzaile nuklearren superfamiliako gainontzeko kideak bezala, funtzio ezberdinak bideratzen dituzten sei domeinu funtzional dituzte. Domeinu hauetako batzuei funtzio jakina egotzi zaie dagoeneko: a) A/B domeinuak transaktibazio funtzioa betetzen du b) C domeinuak bi Zn-hatz agertzen ditu eta DNA lotzen du, c) D domeinuak C eta E/F domeinuen arteko zubi gisa funtzionatzen du eta d) lotugaia lotzen dueneko E domeinuak lotugaiaren menpeko transaktibazioa bideratzen du eta dimerizazio nagusia hornitzen du (Lemberger et al. 1996; Robinson-Rechavi et al. 2003; Raingeard et al. 2006). 14. Irudia. Peroxisomen proliferatzaileek PPARα bidez eragiten duten mekanismoa. PPARαaren lotugaiak kutxa berdez adierazi dira eta erregulatutako geneak kutxa gorriz. Biocarta-tik hartutako irudia. Hiru PPAR mota ezberdin identifikatu dira espezie ezberdinetan, PPARα, PPARβ eta PPARγ, arrainen genoman 2 PPARα isoforma aurresan diren bitartean (Maglich et al. 2003; Metpally et al. 2007; Kondo et al. 2007). α isoforma kodetzen duen genearen itu-disrupzioak erakutsi du peroxisomen proliferatzaileen efektuen arduraduna dela karraskarietan (Lee et al. 1995). Nukleoko hartzaile hormonalek DNA batzen dute itu-sekuentziak ezagutzen dituztenean hormonen erantzun-elementuetan, PPRE (peroxisomen proliferatzailen erantzun-elementuak) PPARen kasuan. PPARek AGGTCA kontsentsu-sekuentzia daraman motibo-gunea ezagutzen dute (Lemberger et al. 1996a; 1996b; Boukouvala et al. 2004). Mota honetako hartzaileak dimerizatu egin 48

55 behar dira eta dimeroa da DNAn lotzen dena; hortaz, bi erantzun elementu behar dira dimeroa lotzeko. Loturarako, PPARek RXR-rekin eratzen du heterodimeroa (Kliewer, et al. 1992; Keller et al. 1993). RXRak ligandoaren menpeko aktibazio sinergistikoa burutzen du PPRE duten sustatzaileetan (Gearing et al. 1993; Henry et al. 1995; Boukouvala et al. 2004). PPAR-RXR, arrainen PPAR-RXR barne (Boukouvala et al. 2004; Kondo et al. 2007), DR-1 elementuetan lotzen da, hau da, base pare bakarrak banatzen dituen PPREren erreplika zuzenetan (AGGTCA-X- AGGTCA) (Dreyer et al. 1992; Keller eta Wahli, 1993). PPREak karraskarien entzima ugaritan deskribatu dira, beraien artean peroxisometako β- oxidazioan parte hartzen duten entzimetan eta CYP4A azpifamiliaren partaideetan (Mandard et al. 2004). PPAR, hartzailea aktibatzeko gaitasunarekin korrelazionatzen den indar proliferatzaile ona agertzen duten peroxisomen proliferatzaileek akibatzen dute (Isseman eta Green, 1990; Dreyer et al. 1992). Peroxisomen proliferatzaile bat PPAR rekin lotzen deneko lehenengo arrastoa, Devchand eta laguntzaileek aurkitu zuten (1996), izan ere Wy Xenopusen PPAR ren lotugai gisa identifikatu zuten. Ondoren, Kim eta laguntzaileek (2005) demostratu zuten inguruneko zenbait kutsatzailek, PAHak esaterako, ugaztunen PPARα aktiba dezaketela. Oraintsuago, Wy k Fuguren PPARα isoformen jarduera transkripzionala aktibatzen duela ikusi da (Kondo et al. 2007) Peroxisomen erantzunak itsas-organismoetan Sarrera Zipofribatoa eta gemfibrozila bezalako ugaztunen peroxisomen proliferatzaile tipikoak Onchorynchus mykiss arrankariari intraperitonealki ziztatzeak AOX, MFP eta katalasaren jardueraren emendioa ondorioztatzen du, horrekin batera, gibelaren gorputzarekiko pisuaren erratioa emendatzen delarik (Yang et al. 1990; Scarano et al. 1994). Antzera, Japoniako medaka arrainak, Oryzias latipes, bi asteren buruan gemfibrozilaren eraginpean mantendu ondoren, AOX eta MFPren jarduerak emendatzen direla ikusi da (Scarano et al. 1994). Modu paraleloan, ikerketa morfometrikoek erakusten dute zipofibratoa ziztatutako amuarrainetan peroxisomen dentsitate bolumetrikoaren emendioa ematen dela, dentsitate numerikoa aldatu barik (Yang et al. 1990). Salmo salar Atlantikoko izokinaren hepatozitoak kultibatu eta klofibrato eta bezafibrato pean mantendu ostean AOX jardueraren indukzioa neurtu da (Ruyter et al. 1997). Arrainak normalean, peroxisomen proliferazioa eragin lezaketen plastifikatzaile, herbizida, pestizida edo industriako konposatuen pean daude (Fahimi eta Cajaraville, 1995). Inguruneko kutsatzaileen menpe mantendutako arrainen katalasak arreta handia piztu du. Katalasa eta beste entzima antioxidatzaile batzuk, arrain zein beste espezie batzutan induzitzen dira xenobiotikoek eragindako oxigeno erradikalen gainekoizpena dela eta. Ictalarus punctatus arrainak enpresek gaiak zuritzean sortzen duten isurkariaren eraginpean egotean (Mather-Mihaich eta Di Giulio, 1991), dinitro-o-kresolaren pean egondako Anguilla anguilla aingiran (Braunbeck eta Völkl, 1991) eta dieldrina ziztatu ondorengo Sparus auratan (Pedrajas et al. 1996) katalasaren indukzioa behatu da. Dicentrarchus labrax eta Limanda limandari intraperitonealki 3-metilkolantrenoa ziztatzean katalasa eta superoxido dismutasaren jarduera, transitorioki eta ez modu esangarrian, eragiten dute (Lemaire et al. 1996). Droga hipolipidemikoak ez diren xenobiotiko organikoek peroxisometako β-oxidazioan eragiten dituzten efektuak ikertzen dituzten lan gutxi daude. Pikloramak eta azido 2,4- diklorofenoxiazetikoak I. punctatusen AOXen jardueran aldaketarik sortzen ez duen bitartean (Gallagher eta Di Giulio, 1991), enpresek gaiak zuritzean sortzen duten isurkari batek espezie berean, AOXen jarduera zazpi aldiz areagotu zuen (Mather-Mihaich eta Di Giulio, 1991). S. auratari zizitatutako dieldrinak 9.3 aldiz areagotu zuen AOXen jarduera (Pedrajas et al. 1996). 49 S A R R E R A

56 Sarrera Peroxisomen proliferazioa endosulfan eta disulfotonen eraginpean mantendutako arrankarietan ere deskribatu da (Arnold et al. 1995). Arrankarietan, peroxisomek betetzen duten bolumen totalaren handipen absolutua ikusi da (Arnold et al. 1995). Peroxisomen proliferazioa deskribatu da ere, baina ez kuantifikatu, arrankarien giltzurrunetako tubulo proximaletan, arrainak, atrazinaren kontzentrazio ezberdinen eta 30 µg/l linuronen pean mantendu ondoren (Oulmi et al. 1995a; 1995b). Etinilestradiolak ere peroxisomen proliferazioa eragiten du zebra-arrainen fase goiztiarretan baina ez eme helduetan (Ortiz-Zarragoitia et al. 2006). Ostera, xenoestrogeno ezberdinen eraginpean, etinilestradiol, 17β-estradiol, dibutilphtalato, metoxiklor eta 4-tert-oktilfenol, 15 egunez mantendutako zebra-arrain arrek peroxisomen azalera-dentsitatean eta dentsitate numerikoan eta AOX jardueran emendioa erakutsi zuten (Ortiz-Zarragoitia eta Cajaraville, 2005). Peroxisomen proliferazioa moluskuetan ere deskribatu da (Cancio eta Cajaraville, 2000; Cajaraville et al. 2003; Cajaraville eta Ortiz-Zarragoitia, 2006). Hiru olio ezberdinen uretan egokitutako frakzioak (WAF) katalasaren jardueraren emendio esangarria eta liseri-guruineko peroxisomen proliferazioa eragin zituen (Cajaraville et al. 1990b; 1992a; 1997). Horrela, Ural eta Maya motako olio gordin eta olio lubrifikatzaile komertzialaren kontzentrazio ezberdinen uretango esposizioak, 21 egunen buruan, peroxisomen bolumen- eta azalera-dentsitatea eta dentsitate numerikoa emendatu zituen muskuiluen liseri-guruineko epitelioan. Olio lubrifikatzaile komertzialak erakutsi zuen peroxisomen proliferazio bortitzena eragiteko gaitasuna. Esposizioak 49 edo 91 egunetan zehar jarraitu zuenean, erantzunaren garrantzia txikiagotu edo guztiz desagertu zen (Cajaraville et al. 1997). Ondoriozta daiteke, petroliotik deribatutako hidrikarburuek peroxisomen proliferatzaile gisa jokatzen dutela muskuiluen liseri-guruinean. Era berean, magurioak epelaburrean naftaleno hidrokarburo diaromatikoarekin tratatu ondoren, Littorina littorearen giltzurruneko mikrogorputz kopurua, (ustezko peroxisomak) areagotu egiten da (Cajaraville et al. 1990a). Deroceras reticulatum barean emaitza kualitatibo bera behatu eta deskribatu dute % 4 karbamatoren eraginpean (Triebskorn 1989). Beranduago, olio lubrifikatzaile komertzial baten WAFarekin, benzo(a)pirenoarekin (B(a)P), klofibrato eta dietilhexil phtalatoarekin burututako ikerketetan ez zen peroxisomen proliferaziorik behatu muskuiluetan, 21 egunez B(a)P eta WAFaren eraginaren pean mantendutako muskuiluen peroxisomen oxidasen jardueran emendio arinak neurtu baziren ere (Cancio et al. 1998; Cajaraville eta Ortiz-Zarragoitia 2006). Antzera, B(a)P, phtalato eta klofibratoarekin ziztatutako muskuiluek AOX jardueraren emendioa erakutsi zuten 7. egunean. Gainera, peroxisomen dentsitate numerikoaren eta dentsitate bolumetrikoaren emendioa aurkitu zen WAF eta klofibratoz ziztatutako muskuiluetan (7 egunetan) (Cancio et al. 1998). Ipar Itsasoko olioaren (NSO) eta NSO eta alkilfenolen nahaste baten pean mantendutako Mytilus edulis muskuiluen AOX jarduera eta peroxisomen dentsitate bolumetrikoa esangarriki emendatu ziren (Cajaraville eta Ortiz-Zarragoitia 2006). Diallilphtalato eta bisfenol A-ren eraginpean mantendutako muskuiluetan ere detsitate bolumetriko altagoak neurtu ziren (Cajaraville eta Ortiz- Zarragoitia 2006). Kutsatzaile organikoen kontzentrazio maila ezberdinak daudeneko zelaiko gune ezberdinetan jasotako Mytilus sp. muskuiluek peroxisomen kopuru eta tamainan ezberdintasunak erakutsi dituzte (Krishnakumar et al. 1995; Orbea et al. 1999b; Porte et al. 2001; Orbea eta Cajaraville 2006; Zorita et al. 2007a). Krishnakumar eta laguntzaileek (1995) erlazio positiboa aurkitu zuten Puget Sound-en, Washington, jasotako muskuiluen liseri-guruinean metatutako, PAH, PCB eta DDTen kontzentrazio eta peroxisomen proliferazioaren agerpenaren artean. Bizkaiko golkoko bi estuario ezberdinetan jasotako muskuiluek peroxisomen dentsitate bolumetriko eta numeriko eta katalasa jarduera altuagoa erakutsi zuten PAH kontzentrazio altuena azaldu zuen estuarioko organismoetan (Orbea et al. 50

57 1999b). 1996ko martxoan Bartzelonan lagindutako muskuiluek peroxisomen dentsitate numeriko altuagoa erakutsi zuten Espainiako kostalde Mediterraneoko gainontzeko laginketa-puntuetan jasotakoekin alderatuta, azken hauek PAH maila baxuagoak zituzten (Porte et al. 2001). Antzera, Zorita eta laguntzaileek (2007a) AOX jarduera altuenak, peroxisomen proliferatzaileak izan daitezkeen konposatuen kontzentrazio altuena agertzen zeneko laginketa-puntuan eskuratu zituzten, Mediterranear Itsasoan bi urtetan zehar burututako biojarraipen-programan. Ikusi denez, peroxisomen proliferazioa muskuiluek kutsatzaileen aurrean emaniko erantzun arin eta itzulgarria da (Cajaraville et al. 2003, Orbea eta Cajaraville 2006). Horrela, peroxisomen proliferazioa, peroxisomen proliferazioa eragiten duten konposatu organikoen esposizioaren aurreko biomarkatzaile aproposa kontsideratzen da muskuiluetan (Cajaraville eta Ortiz-Zarragoitia 2006) eta hortaz, arrainetan oso erabilia den eta kutsatzaile organikoen aurreko esposizio-biomarkatzaile den CYP1A1 genearen gainespresioaren (edo asoziaturiko EROD jarduerarena) neurketaren alternatiba gisa proposatu da (Aas et al. 2000; Cajaraville et al. 2000; George et al. 2004; Lewis et al. 2006). Testu-inguru honetan, garrantzitsua da baldintza fisiko eta fisiologiko normaletan peroxisomen proliferatzaileen maila basalak ezagutzea. Iberiar Penintsulako M. galloprovincialis muskuiluen liseri-guruinean burututako sasoiaren araberako ikerketetan, peroxisometako entzimen jarduera eta egituran aldaketa nabarmenak ematen direla ikusi da (Cancio et al. 1999; Orbea et al. 2007), aldaketok peroxisomen proliferatzaileekin trataturiko ugaztun-espezie erantzuleen antzekoak izanik (Cancio et al. 1999). Modu honetan, apirilean jasotako animaliek AOX jarduera altuena agertu zuten, palmitoil-coa oxidatzeko duten gaitasuna neguko hilabete inaktiboena baino 25 aldiz altuagoa izanik. Berdintsu, peroxisomen dentsitate numerikoa zortzikoiztu egin zen udaberri hasieran (Cancio et al. 1999). AOX jardueran eta peroxisomen dentsitate bolumetrikoan neurtutako aldaketak positiboki eta esangarriki korrelazionatu ziren urteko zikloan zehar, katalasaren jarduera entzimatikoan gauza bera gertatuz. Aldeketa hauek, lipido neutroen liseri-tubuloen epiteliotik duktuen epitelioranzko mobilizazioarekin bat zetozen, sasoiaren araberako elikagaien barneraketa eta ugalketa-zikloak, peroxisomen proliferazio tipikoarekin konpara daitezkeen parametro peroxisomikoen aldaketak eragiten dituztela adieraziz. Orduan, peroxisomek molusku hauen lipidoen homeostasian eta ugalketa-zikloan funtzio garrantzisua bete lezakete (Cancio et al. 1999). 5. AZKEN OHARRAK Sarrera Peroxisomek funtzio bitalak betetzen dituzten entzima talde heterogeneoa dute, funtzioon artean, gantz-azidoen β-oxidazioa, kolesterol eta behazun-azidoen sintesia, xenobiotikoen metabolismoa, ROSen metabolismo eta sorrera egonik. Organismo eta ehun ezberdinek ezaugarri ezberdinak dituzten eta bidezidor metaboliko ezberdinetan parte hartzen duten peroxisomak dituzte. Arrain eta moluskuen datu-bilketak adierazten du organismo mota bien peroxisomek ezaugarri funtzional komun ugari dituztela. Hala ere, peroxisomek organismo urtarretan betetzen dituzten funtzioen gaineko datu gutxi ditugu oraindik (lipidoen homeostasia esate baterako) eta beraz, ikerketa sakonagoak behar dira arlo honetan. Bereziki, peroxisometako erreakzioen garrantzia organismo urtarren metabolismo orokorrean zein den eta peroxisometako metabolismo lipidikoa eta esteroidegenesia eta ugalketa-arrakastaren arteko lotura ikertu beharko lirateke, inguruneko faktoreek (ugalketa-sasoi, elikadura, sasoia, generoa) eta faktore-fisiologikoek peroxisomen funtzioetan duten garrantziarekin batera. Peroxisomen biogenesiko hainbat arlo beraien kasuan bakarrik ematen da eta galdera asko daude oraindik erantzuteke, peroxisomen sorrera nola ematen denetik hasita. Ugaztun eta legamietan peroxisomen sorreran parte hartzen duten mekanismo molekularren eta proteinen inguruan ezagutza zabalagoa lortu da, baina oraindik ez da ezer ezagutzen beste phyla batzuetako organismoetan. 51 S A R R E R A

58 Sarrera Interes handia sortzen du organulu hauen plastikotasunak eta baldintza metaboliko aldakor zein egitura aldetik erlaziorik ez duten konposatuen eraginpean duten proliferatzeko gaitasunak. Proliferatzeko duten gaitasun honek interesgarri bihurtzen ditu peroxisomak ikuspegi ekotoxikologikotik, ingurunean kontzentrazio altutan agertzen diren hainbat konposatuk peroxisomen proliferazioa eragin baitezakete animalietan. Organismo urtarrak luzaroan erabili dira bizi direneko ingurunearen egoeraren indikatzaile gisa eta peroxisomen proliferazioa zenbait konposaturen aurreko esposizio-biomarkatzaile gisa erabili daiteke, tartean, PAHak, PCBak, enpresek zuritze-tratamenduetatik isuritzen dituzten konposatuak etab, denak baitira ezagunak organismo urtar ezberdinetan peroxisomen proliferazioa eragiteagaitik. Organismo urtarretan peroxisomen proliferazioa erregulatzen duen mekanismo molekularrak ikerketa sakonagoak behar ditu. Inguruneko baldintzetan eta egoera fisiologiko normalen pean peroxisomen biogenesia gobernatzen duten mekanismo molekularrak ikertzeko beharrizana dago, peroxisometan eman daitezkeen fluktuazio posibleak detektatzeko aukera eduki eta inguruneko kutsatzaileen eraginpean dauden organismo urtarretan ematen diren aldaketen interpretazioan lagungarri izan daitezen. 52

59 4000 hepatic amplicons cdna Saxitoxin effects Aeromonas salmonicida (liver, spleen) Bard et al Ewart et al genes from 175 EST GRASP3.5K, cdna Sneaker male vs immature male Aubin-Horth et al salmonid libraries genes from 175 EST salmonid libraries GRASP16K, cdna Heterologous gene hybridisation Von Schalburg et al Lithognathus mormyrus 4700 hepatic clones cdna Multichemical exposure analysis Auslander et al. PRIMO 2005 unique sequences, obtained after injecting lindane, Aroclor-1254, estradiol, Cd, PFOA, B(a)P, TBT, MetHg, nonylphenol. Platichthys flesus 601 EST sequences (350 ESTs cdna Environmental stress Marchand et al. SETAC 2004 after pesticide treatment; 251 from P. americanus) hepatic clones, 3600 cdna Cd, estradiol treatment Williams et al. 2006, 2007 unique gene sequences Paralichthys olivaceus genes cdna Immune response, concanavalin-a and LPS injection, DNA vaccination Yasuike et al. 2004, Byon et al. 2005a, 2005b, Emmadi et al 2005 Pseudopleuronectes 192 clones from a SSH library cdna Cr(VI) tretament Chapman et al americanus Ictalurus punctatus 660 brain genes cdna Cold acclimation Ju et al Micropterus salmoides unique gene sequences 24mer NimbleGen Systems Inc. LPS injection Li & Waldbieser, 2006 GSE cdnas, mostly responsive cdna (Nylon) Pulp and paper mill effluent effects in liver Denslow et al to estrogenic compounds ECOARRAY 2000 genes 60mer Agilent Unpublished ECOARRAY Sparus aurata clones, cdna library of cdna Cortisol implants Sarropoulou et al mixed embryonic & larval stages Brain specific Nylon macroarray Viral infection Dios et al Gillichthys mirabilis 5376 cdna clones (half cdna Hypoxia Gracey et al

60 generated by SSH hypoxia), 3840 clones from liver, 1152 skeletal muscle, 384 from brain) 9207 cdna clones cdna Heat stress Buckley et al Cyprinus carpio 643 Cd-related genes obtained cdna Cd induced expression profile in liver Reynders et al by SSH 960 hormone-responding and cdna Liver expression profiles of 14 different Moens et al gender-related genes by SSH endocrine disrupting compounds cdna probes cdna Effects of increasing levels of cold Gracey et al Carassius auratus 1200 brain specific clones cdna EE2 effects in brain Martyniuk et al Microgadus tomcod 4416 unidentified heart cdnas cdna PCB mixture treatment (tomcod populations from 3 sites with different PCB & TCDD burdens) Carlson et al. SETAC 2004; 2005 Pimephales promelas 200 endocrine related genes Nylon macroarray MeHg treatment Klaper et al. 2006, ECOARRAY 1000 genes 60mers Agilent Estradiol and fadrozole treatment Villeneuve et al. SETAC genes 60mers Agilent Unpublished ECOARRAY Rutilus rutilus cdna gonad?? Estrogenic chemicals Tyler et al. SETAC 2005 Sebastes schlegeli clones, obtained by SSH?? B(a)P treatment Lee et al. SETAC 2005 after B(a)P treatment Gadus morhua 746 ESTs, metabolism, immune cdna Cold shock STRESSGEN and stress related genes Scophthalmus maximus 3482 singletons, from EST-s of 60mer Agilent Unpublished P. Martinez (personal bacterially challenged head communication) kidney, liver and spleen Hippoglossus 9150 ESTs 50mer Unpublished Pleurogene project hippoglossus Solea senegalensis 5230 singletons, 4550 ESTs + 60mer Unpublished Pleurogene project 320 mirna Danio ESTs halibut Cyprinodon variegatus???? EE2 Knoebl et al

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89 II. GAIAREN EGOERA, HIPOTESIA ETA HELBURUAK II. STATE OF THE ART, HYPOTHESIS AND OBJECTIVES 137

90 2

91 Hypothesis and Ojectives STATE OF THE ART Peroxisome proliferation is a cellular process known in mammalian species to be associated with the induction of peroxisomal β-oxidation pathway, all three enzymes of the peroxisomal β-oxidation being induced, both at the protein and at the mrna level. Genes coding the enzymes in this inducible pathway, contain proliferation response elements (PPREs) in their promoter regions that the heterodimer formed by the peroxisome proliferator activated receptor α (PPARα) and retinoid X receptor is able to bind in the presence of PPARα agonists (peroxicome proliferator compounds) for upregulation of gene expression. Interestingly, it has been demonstrated that apart from natural ligands and typical mammalian peroxisome proliferators such as hypolipidemic drugs, mammalian PPARα can also be activated by some environmental pollutants such as some polycyclic aromatic hydrocarbons (PAHs), perfluorinated compounds and phthalate ester plasticizers. Aquatic environments are continuously exposed to xenobiotics that may cause peroxisome proliferation in aquatic species, fish and molluscs. In this context, peroxisome proliferation is being measured as a specific biomarker of exposure to such compounds, both in laboratory exposure experiments and in pollution biomonitoring programmes. However, the transcriptional regulation of peroxisome proliferation has never been studied in fish and/or molluscs. Additionally, although different PPARs have been identified in different fish species and more recently in the sea-urchin Stronglylocentrotus purpuratus genome, little is known about the molecular mechanisms that govern this process in aquatic species. HYPOTHESIS The molecular mechanism leading to peroxisome proliferation in aquatic organisms is similar to that in mammalian responsive species and it is regulated at the transcriptional level by exposure to organic xenobiotics. Thus, the measurement of peroxisomal gene expression levels might be useful as biomarker of exposure to peroxisome proliferator compounds in pollution monitoring programmes. OBJECTIVES In order to proof this hypothesis true, the present work attempts to address the following general objectives: 1.- To characterise peroxisomes, specially the peroxisomal b-oxidation pathway, at the molecular level in aquatic organisms by cloning genes coding for peroxisomal proteins. 2.- To study alterations in peroxisomal gene expression levels under exposure to different organic toxic compounds in different laboratory and field conditions. These general objectives have been subdivided in a series of partial objectives that are shown bellow and are addressed in the different chapters of the Results and Discussion section: - To clone genes coding for enzymes involved in the most relevant peroxisomal functions, specially those involved in the peroxisome proliferation process, in different aquatic organisms 139

92 Cloning and expression of peroxisome proliferation marker genes in aquatic organisms (Mediterranean mussel Mytilus galloprovincialis, thicklip grey-mullet Chelon labrosus and European hake Merluccius merluccius). - To characterise molecularly the inducible peroxisomal β-oxidation pathway in fish and mussels. - To study the tissue expression pattern of the cloned peroxisomal genes depending on different physiological and physical conditions (gender, developmental stage and season). - To study the expression levels of the cloned peroxisomal genes in the thicklip grey mullet Chelon labrosus after a single intraperitoneal injection with the carcinogenic model PAH benzo(a)pyrene. - To study the expression levels of the cloned peroxisomal genes in Chelon labrosus and Mytilus galloprovincialis after exposure to Prestige-like heavy fuel oil and to the typical mammalian peroxisome proliferator compound, perfluorooctane sulfonate. - To compare peroxisomal gene expression profiles under exposure to organic xenobiotics, with the cellular process of peroxisome proliferation and with the expression pattern of genes responsive to the xenobiotics studied (phase I and II biotransformation metabolism genes in fish and hsp70 in mussels). - To study the usefulness of quantifying peroxisomal gene expression levels as biomarker of fuel oil exposure in Mytilus galloprovincialis and Merluccius merluccius, captured in two monitoring campaigns for the evaluation of the biological effects of the Prestige oil spill in the North Iberian Peninsula in the years 2004 and

93 III. EMAITZAK ETA EZTABAIDA III. RESULTS AND DISCUSSION 141

94 1.- Cloning and expression pattern of a polyamine oxidase, xanthine oxidoreductase and catalase in mussel Mytilus galloprovincialis and thicklip grey mullet Chelon labrosus Parts of this chapter have been published at: BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I (2006). "Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: generation of new tools to study peroxisome proliferation". Marine Environmental Research 62S, Parts of this chapter have been presented at: 13 th International Symposium on Pollutant Responses in Marine Organisms (PRIMO), Alessandria, Italy. June 19-22, BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I. "Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: generation of new tools to study peroxisome proliferation". 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry (SETAC)-Europe, The Hague, Netherlands, May 7-11, CANCIO I, BILBAO E, RAINGEARD D, SAEZ-MORQUECHO C, DIAZ DE CERIO O, CAJARAVILLE MP. "Cloning and expression pattern of peroxisomal genes in the thicklip grey mullet Chelon labrosus: a differential gene expression analysis to study peroxisome proliferation". 143

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96 ABSTRACT Cloning and expression pattern of a POX, XOR, and CAT Thicklip grey mullet Chelon labrosus is an abundant species in Eastern-Atlantic estuaries where it is able to endure highly polluted environments displaying several of the characteristics required in an estuarine sentinel species. Mussels on the other hand, are worldwide used as model indicator organisms to monitor the effects of marine pollution. Although many studies determine biomarkers of oxidative stress in fish and mussels, reactive oxygen species (ROS) producing and eliminating enzymes are known to be differentially regulated depending on gender and season. Thus, the aim of the present study was to clone and determine expression patterns of peroxisomal oxidases such as xanthine oxidoreductase (XOR) and polyamine oxidase (POX) that produce ROS, and catalase (CAT) that is involved in the elimination of H 2 O 2, in different tissues of mussels and mullets. In addition, expression levels in mullet liver, as main detoxifying tissue in fish, and tissues of mussels collected in winter and in late spring were compared. First, fragments of XOR, POX and CAT were cloned in both species by using degenerate primers and then their expression pattern was studied by semi-quantitative RT-PCR. In mussels, XOR, POX and CAT were expressed in digestive gland, mantle and gills. In mullets, POX and CAT were expressed in liver, spleen, brain, heart, muscle and gills while XOR was mainly expressed in liver and heart. XOR, POX and CAT were similarly expressed in liver of mullets captured both in winter and in spring but significant seasonal differences were found in mussel tissues. CAT expression levels were at their highest in gills of mussels collected in winter while POX showed the highest expression levels in spring. In situ hybridisation studies in mussels allowed specific localisation of CAT expression in digestive and gill epithelial cells and in ovocytes. In conclusion, XOR, POX, and CAT showed a widespread tissue distribution in their expression both in mussels and in mullets. Mullets did not show season related expression alterations in the liver while mussels showed a clear season related regulation of POX and CAT expression. Developmental stage and gender related differences in mullet XOR tissue expression levels were detected. This physiological regulation in antioxidant and pro-oxidant enzyme transcription should be taken into account in monitoring programs using these enzyme activities as biomarkers of pollution effects. 145

97 Results and Discussion RESUMEN El muble Chelon labrosus es una especie abundante en los estuarios del Atlántico Este donde vive en ambientes altamente contaminados mostrando algunas de las características de especies centinela de estuarios. Por otro lado, el mejillón se utiliza en todo el mundo como organismo modelo indicador en los seguimientos de contaminación marina. A pesar de que hay estudios que determinan biomarcadores de estrés oxidativo en peces y mejillones, se sabe que los enzimas que producen y eliminan las especies reactivas de oxigeno (ROS) se regulan diferencialmente en función del sexo y la estación del año. Así, el objetivo de este estudio fue clonar y determinar los patrones de expresión de oxidasas peroxisómicas tales como xantina oxidoreductasa (XOR) y poliamino oxidasa (POX) que producen ROS y catalasa (CAT) que se encarga de eliminar H 2 O 2 en diferentes tejidos, tanto de peces como de mejillones. Además, se compararon los niveles de expresión en hígado de muble, como principal órgano encargado de la detoxificación en peces, y en los diferentes tejidos de mejillones recogidos en invierno y primavera. Para ello, en primer lugar se clonaron fragmentos de XOR, POX y CAT en ambas especies mediante cebadores degenerados y después se estudió el patrón de expresión mediante RT-PCR semicuantitativa. En mejillones, XOR, POX y CAT se expresaron en branquias, manto y glándula digestiva. En mubles, POX y CAT se expresaron en hígado, bazo, cerebro, corazón, músculo y branquias mientras que XOR se expresó principalmente en hígado y corazón. La expresión de XOR, POX y CAT fue similar en el hígado de mubles capturados tanto en invierno como en primavera, pero se encontraron diferencias significativas en los tejidos de mejillón. El nivel de expresión de CAT en branquia de mejillón, alcanzó el máximo en invierno, mientras que POX mostró los mayores niveles de expresión en primavera. Los estudios de hibridación in situ, permitieron localizar la expresión de CAT en ovocitos y células epiteliales de la branquia y de la glándula digestiva. Por lo tanto, XOR, POX y CAT mostraron una amplia distribución tisular en su expresión en mejillones y mubles. Además, mientras que los mubles no presentaron expresión diferenciada de los genes estudiados dependiente de la estación, los mejillones mostraron clara regulación de POX y CAT. Se observaron también diferencias en el nivel de expresión de XOR dependientes del grado de desarrollo y el género de los mubles. Esta regulación fisiológica en la transcripción de enzimas antioxidantes y prooxidantes debería tenerse en cuenta en programas de seguimiento que midan como biomarcadores de efecto de la contaminación las actividades enzimáticas estudiadas. 146

98 Cloning and expression pattern of a POX, XOR, and CAT Introduction During normal metabolism, oxidases in aerobic organisms use oxygen as an electron acceptor to catalyse the formation of an oxidised product together with reactive oxygen species (ROS) as by-products such as hydrogen peroxide (H 2 O 2 ) and superoxide anion radical (O - 2 ), which in the presence of iron ions can give rise to highly reactive hydroxyl radicals. ROS can be removed by the antioxidant defence system, however, excess ROS generation above cellular antioxidant capacity, may result in damage to DNA, proteins, and lipids, leading to impaired cell function and pathological alterations (Gutteridge and Halliwell 2000; Mate's 2000; Schrader and Fahimi 2006; Alves de Almeida et al. 2007). Exposure to xenobiotics induces an increased generation of ROS (Livingstone et al. 1990; Livingstone 2001; Gorbi and Regoli 2003), so, in environmental toxicology it is of great interest to know how ROS-generating oxidases and antioxidant enzymes are regulated depending on biotic and abiotic parameters. In rat liver, peroxisomes are responsible for 20% of total oxygen consumption, resulting in the production of 35% of all H 2 O 2, mainly through peroxisomal β-oxidation (Schrader and Fahimi 2006). In aquatic organisms, involvement of peroxisomes in β-oxidation seems to be species specific. In this way, hepatic β-oxidation is 100% peroxisomal in the salmon Salmo salar (Nanton et al. 2003; Froyland et al. 2000), whereas peroxisomes accounted for 30-50% of the total hepatic β-oxidation in other marine teleost fishes (Crockett and Sidell 1993a; 1993b). In either case, peroxisomal acyl-coa oxidases should play an important role in ROS generation in aquatic organisms. However, although to a lower extent, other peroxisomal enzymes such as xanthine oxidoreductase (XOR) and (poly)amine oxidase could play an important role in ROS formation, as well (Cancio and Cajaraville 1997; Strolin Benedetti et al. 2006; Liu et al. 2006; Schrader and Fahimi 2006). XOR was identified by Angermüller and coworkers (1987) as a peroxisomal enzyme, however, posterior approaches have considered that XOR is located in the cytosol (Kooij et al. 1991; Cancio and Cajaraville 1997; Frederiks and Vreeling-Sindelarova 2002), on cell membranes (Rouquette et al. 1998) and in various organelles of Kupffer and sinusoidal cells including peroxisomes, rough endoplasmic reticulum, lysosomes and endocytic vesicles (Frederiks and Vreeling-Sindelarova 2002; Harrison 2004). XOR is involved in purine metabolism acting either as oxidase or dehydrogenase. In addition, XOR is also implicated in a multitude of physiological processes such as cell proliferation and differentiation, defence against micro-organisms and metabolism of xenobiotics (Kooij et al. 1994; Pritsos 2000; Berry and Hare 2004; Harrison 2004; Martin et al. 2004). Both forms are able to reduce oxygen in the presence of hypoxanthine, but xanthine oxidase (XOX) is more efficient than xanthine dehydrogenase (XDH) (Saito and Nishino 1989). On the contrary, XDH is more efficient in NADH oxidase activity than XOX (Sanders et al. 1997). The dehydrogenase activity of this enzyme has been biochemically and histochemically detected in the digestive gland of mussels (Cancio and Cajaraville 1997; 1999) while XOX activity has only been described in stenoxic bivalves (Dykens and Shick 1988). On the other hand, both XOX and XDH activities have been measured in different fish species (Cleare et al. 1976; Liu et al. 2006; Giuffrida et al. 2007). In addition, it has been observed that XOR can be regulated transcriptionally, its activity being induced in aquatic organisms after exposure to different pollutants (Basha and Rani 2003; Pandey et al. 2003; Liu et al. 2006). On the other hand, mammalian amine oxidases form a heterogeneous family of enzymes that metabolise various endobiotic, dietary or xenobiotic monoamines, diamines and polyamines (Benedetti 2001; Strolin Benedetti et al. 2006; Gong and Boor 2006). Polyamines (i.e., putrescine, spermidine and spermine) are found in every living cell and are believed to participate in cellular proliferation and differenciation (Heby 1981; Wallace et al. 2003; Jäne et al. 2004), being essential for normal cell growth (Wallace et al. 2003; Jäne et al. 2004; Igarashi 2006). The mobilisation and catabolism 147

99 Results and Discussion of polyamines and N-acetylated polyamines is primarily carried out by the flavin oxidases polyamine oxidases (POX), a group of enzymes that in the last few years have been found to be involved in maintaining the balance of amine oxidase and antioxidant enzyme activities and in the control of cancer inhibition or progression (Seiler, 2003; Wallace 2003; Seiler and Raul 2005; Toninello et al. 2006). The metabolism of polyamines via spermidine/spermine N1- acetyltransferase (SSAT) and POX produces H 2 O 2 that may induce both SSAT and cell death, thus causing a positive cell-death signalgenerating cycle (Wallace et al. 2003; Seiler and Raul 2005). In addition, the products of oxidative deamination of polyamines, form important cytotoxic agents (Seiler amd Raul 2005). Changes in POX activities have been demonstrated in tissues during postnatal development or aging, after infectious agents and mediators of inflammation, after treatment with certain drugs, hormones or hyperplastic factors, (Babbar et al. 2007) or even after exposure to peroxisome proliferators (Hayashi et al, 1989; Hayashi and Miwa 1989), regulation occurring at several levels, including transcription and translation (Wallace et al. 2003). Nothing is known about their regulation or function in aquatic species. Besides hydrogen peroxide-generating oxidases, peroxisomes also contain the hydrogen peroxide-catabolising enzyme catalase (CAT). It is a heme-containing enzyme that catalyses the conversion of H 2 O 2 to water and molecular oxygen without consuming cellular reducing equivalents. Hence, CAT provides the cell with a very energy efficient mechanism to remove hydrogen peroxide (Scandalios 2005). CAT also uses H 2 O 2 to oxidise xenobiotics such as phenols, formic acid, formaldehyde and alcohols (Singh 1997; Scandalios 2005). Changes in the levels of antioxidant enzymes such as CAT have been proposed as biomarkers of contaminantmediated prooxidant challenge in a variety of marine organisms, including mussels (Regoli and Principato 1995; Livingstone 2001; Regoli et al. 2004), and fish (Orbea et al. 2002; Regoli et al. 2002). However, the relationships between response and contaminant exposure are still less well established, and more research is required to elucidate the mechanism behind these effects on selected sentinel species (Van der Oost et al. 2003). Since biological responses can be modulated seasonally and depending on biological factors such as gender and developmental stage, the application of biomarkers of ROS production and detoxification, requires assessing the effect of such parameters on the normal pattern of ROS producing and detoxifying enzymes. The main aim of this study was to assess whether these physiological variations in enzyme activities are regulated at the transcriptional level. Material and Methods Reactives All chemicals were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless specified otherwise. Animals and experimental procedure Five mussels Mytilus galloprovincialis and 6 thicklip grey mullets Chelon labrosus were collected from Arriluze, Biscay Bay (43º20 N, 3º01 W) in June 2004 and January Animals were immediately processed after collection but mullets were classified as juveniles and mature male or females (2 individuals per group) prior to dissection. Fish were anesthetised by immersion in a saturated solution of 3-aminobenzoic acid ethyl ester before being sacrificed. Liver, gills, brain, muscle, heart and gonad of mullets and digestive gland, gills and mantle of mussels were dissected and frozen, embedded in RNA later, in liquid nitrogen for gene expression studies. All the samples were stored at -80ºC until processing. Cloning of target genes 50 to 100 mg of tissue were homogenised in Trizol (Invitrogen, Carlsbad, California, USA) using an Hybaid Ribolyser TM (Hybaid, Ashford, UK) at 4 m/s for 20 s (mullet brain, heart, spleen, gonad and liver and mussel mantle) and 40 seconds (gills and mussel digestive gland). RNA was extracted following manufacturer`s 148

100 Cloning and expression pattern of a POX, XOR, and CAT recommendations. Then, 2 µg of mussel and 1 µg of mullet total RNA were used for cdna synthesis (Invitrogen) using random hexamers and according to manufacturer s instructions in the thermal cycler (icycler TM, Bio-Rad, San Diego, California, USA). Degenerate primers were generated aligning known sequences belonging to different species. POX of Mus musculus (NM_153783), Homo sapiens (NM_152911) and Danio rerio (CF924898) were aligned and Fw 5'- GACAGYRCKGATGAYCCTCT-3' and Rv 5'- TDCYGGAAGAACTCCTGRGTC-3' primers were synthesised. Similarly, XOR was amplified using Fw 5'-GGDGGMTGYGGBGCBTG-3' and Rv 5'- TADCCHGTRCARCGRCAMAGRTT-3' which were obtained aligning Poecilia reticulata (AY034103), Homo sapiens (HSU39487), Ceratitis capitata (AY014961) and Caenorhabditis elegans (CAB05902) XOR sequences. Finally, CAT was amplified in both species using Fw 5'- TTCATHCAYACRCAGAAGMG-3' and Rv 5'- CTGYTCHACYTCMGCRAA-3' degenerate primers designed by the alignment of Mytilus edulis (AY580271), Mytilus californianus (AY580259) and Litopeneaus vannamei (AY518322) CAT. PCR conditions were: 2' at 94ºC, 35 cycles of 30'' at 94ºC, 30'' at 56ºC and 30'' at 72ºC and a final extension step of 8' at 72ºC. PCR products were visualised with an ethidium bromide agarose gel (1.5%) electrophoresis. After purification of PCR amplicons (Qiagen, Hilden, Germany), fragments were cloned using a TOPO-TA cloning kit (Invitrogen). Plasmids were extracted from bacteria using the SNAP miniprep kit (Invitrogen) and digested with BamHI and NotI for 2 hours. PCR products for each gene were sequenced using the specific degenerate Fw primers described above, while cloned inserts were sequenced using the universal M13 Fw/Rv primers. Sequencing was carried out in the Sequencing Service of the Department of Genetics, Physical Anthropology and Animal Physiology (University of the Basque Country). Semiquantification of gene expression Monoplex-PCR conditions were optimised for the amplification of CAT, POX and XOR fragments bp long using Taq polymerase (Invitrogen) in a convencional icycler termocycler (Bio-Rad) (Bilbao et al. 2006). PCR products were visualised in an agarose gel (1.5%) electrophoresis stained with ethidium bromide and analysed using a computer-aided gel analyser (Gel-Doc-2000, Bio-Rad). β-actin, and 18S rrna were measured as housekeeping genes following PCR conditions described by Raingeard et al. (2006). Results have been expressed in arbitrary semiquantitative units. In situ hybridisation for the localisation of CAT mrna Preparation of riboprobes For generation of digoxigenin-labeled crna probes, specific CAT PCR amplicons (273 bp of mussel CAT and 240 bp of mullet CAT) were inserted in a plasmid vector (pgem -T Easy kit; Promega, Wisconsin, USA) containing T7 and SP6 RNA polymerase promoters on each side of the multicloning site. E. coli JM109 highefficiency competent cells were transformed according to manufacturer s instructions. White colonies were selected from X-Gal/IPTG ampicillin agar plates and grown in LB/ampicillin liquid media. Plasmids were purified using the QIAprep spin miniprep kit (Qiagen). Cloned PCR fragments were sequenced to verify that inserts belonged to CAT and to assess the direction of insertion. Plasmids were linearised with SacII and SpeI restriction enzymes and purified by phenol/chloroform extraction method prior to perform the in vitro transcription for the synthesis of both antisense and sense probes using DIG-RNA labelling kit (Roche Diagnostics, Switzerland). Briefly, 1 µg of linearised template DNA was incubated for 2 h at 37ºC in 20 µl of a mixture containing 40 U of the appropriate polymerase, NTP labelling mixture (1 mm each ATP, CTP, GTP, 0.65 mm UTP, and 0.35 mm digoxigenin-labeled UTP), transcription buffer, and 20 U RNAse inhibitor (RNAsin). The reaction was stopped by adding 2 µl of 0.2 M EDTA (ph 8.0) and the transcripts 149

101 Results and Discussion were precipitated with 2.5 µl 4 M LiCl and 75 µl ethanol at -20ºC overnight. After pelleting and washing with cold 70% ethanol, transcripts were resuspended in 50 µl diethyl-pyrocarbonate (DEPC)-treated water. In addition, sense probes were also generated as negative controls using the opposite RNA polymerase promoter depending on cloning direction. In situ hybridisation procedure After deparaffinisation and rehydration, 1 µm thick mussel and mullet tissue sections were pretreated with 0.1 M HCl and digested for 30 min at 37ºC with 5 µg/ml proteinase K in TE buffer (100 mm Tris, 50 mm EDTA, ph 8.0), followed by 5 min postfixation in freshly prepared 4% paraformaldehyde in PBS (ph 7.4). Subsequently, sections were acetylated with 0.25% (v/v) acetic anhydre in 0.1 M triethanolamine at ph 8.0, followed by dehydration in ethanol and air-drying. Sections were prehybridised for 2 h at 45ºC in a mixture consisting of 50% (v/v) formamide, 50 mm Tris- HCl (ph 7.5), 25 mm EDTA, 20 mm NaCl, 250 µg/ml yeast trna, and 2.5 x Denhardt s solution (50 x Denhardt s consists of 1%, w/v, each of Ficoll 400, polyvinyl-pyrolidane and bovine serum albumin (BSA)). The hybridisation mixture contained 5 ng/ µl riboprobe, 50% formamide, 20 mm Tris-HCl (ph 7.5), 1 mm EDTA, 0.33 M NaCl and 10% dextran sulphate in a total volume of 20 µl per section. Each section was covered by a siliconised (Sigmacote; Sigma-Aldrich) coverslip, sealed with rubber cement, and hybridised overnight at 45ºC. To remove the excess probe, sections were washed at 45ºC with 2 x SSC (standard saline citrate buffer, 300 mm Na Cl, 30 mm sodium citrate, ph 7.2) for 30 min and 1 x SSC with 50% (v/v) formamide at 45ºC followed by 0.5 x SSC twice for 10 min at room temperature. Before digoxigenin detection, nonspecific binding sites were blocked with 1% (w/v) blocking medium, 0.5% (w/v) BSA in Tris/saline (TBS) buffer (100 mm Tris, 150 mm NaCl, ph 7.5) for 30 min. Sections were incubated overnight at 4ºC with alkaline phosphatase-labeled anti-digoxigenin Fab fragments (Roche Diagnostics, Basel, Switzerland) diluted 1:500 in blocking buffer. After removal of unbound Fab fragments by several TBS washes followed by a 2 min incubation in tetranitromethano (TNM) buffer (100 mm Tris, 50 mm MgCl2, 100 mm NaCl, ph 9.5), the staining reaction for alkaline phosphatase was performed at 37ºC in TNM buffer containing nitroblue tetrazolium salt/5- bromo-4-chloro-3-indolyl phosphate color reaction (Promega GmbH, Germany). Sections were then counterstained with hematoxylin, mounted in glycerol gelatine and observed under a Leitz Laborlux S microscope (Wetzlar, Germany). Results Cloning of target genes Fragments of 266 bp and 290 bp encoding XOR were cloned in C. labrosus (AY876387) and Mytilus galloprovincialis (AY876388), respectively. Mullet XOR showed 85% amino acid identity with xanthine dehydrogenase of Poecilia reticulata (AAK59699) while mussel XOR showed 42% amino acid identity with Strongylocentrotus purpuratus xanthine dehydrogenase/oxidase (XP_ ) (Table 1). On the other hand, a fragment of 385 bp belonging to a putative POX (AY876389) was cloned in Chelon labrosus (Table 1). The fragment showed 90% amino acid identity with Danio rerio spermine oxidase (BC066413) and 82% amino acid identity with human polyamine oxidase (NM_175840). In mussels, the 326 bp fragment cloned (AY876390) showed 91% of amino acid identity with D. rerio spermine oxidase (BC066413) and 85% with human polyamine oxidase (NM_175840) (Table 1). Finally, 273 bp of CAT were cloned in C. labrosus (AY743715), showing 94% amino acid identity with Oplegnathus fasciatus CAT (AAU44617). Similarly, a 552 bp fragment was amplified in M. galloprovincialis (AY743716). This fragment showed 95% amino acid identity with M. edulis CAT (AAT06168) (Table 1). Gene expression pattern in Mytilus galloprovincialis tissues Since 18S rrna expression showed the 150

102 Cloning and expression pattern of a POX, POX and CAT Table 1. Putative identity of PCR amplification products belonging to genes coding for peroxisomal enzymes in Chelon labrosus and Mytilus galloprovincialis. Gene accession number Size of RT-PCR product amino acid identity with known sequences Chelon labrosus AY bp 85% Poecilia reticulata XOR AY NM_ bp 90% Danio rerio Spermine oxidase 82% Homo sapiens POX AY bp 94% Oplegnathus fasciatus CAT M. galloprovincialis AY bp 55% Strongylocentrotus purpuratus XOR AY NM_ bp 91% Danio rerio Spermine oxidase 85% Homo sapiens POX AY bp 95% Mytilus edulis CAT lowest intertissue and seasonal expression variability among the studied housekeeping genes, expression of target genes was normalised against this gene. XOR, POX and CAT were expressed in gills, mantle and digestive gland of mussels; however, the low XOR expression levels did not allow semiquantification neither in winter nor in spring. In all tissues POX expression was higher in spring than in winter, such difference being significant in mantle and digestive gland (Figure 1). CAT expression did not change seasonally in mantle and digestive gland, but in gills, CAT expression was significantly higher in winter than in spring (Figure 1). Gene expression pattern in Chelon labrosus tissues Since 18S rrna expression showed the lowest intertissue and seasonal expression variability among the studied housekeeping genes, expression of target genes was normalised against this gene. CAT and POX were expressed in all studied tissues in juvenile and mature (female and male) mullets both in winter and spring (Figure 2). XOR expression was not detected in gills of mature mullets in winter, while in juveniles, XOR expression was exclusively hepatic (Figure 2). In spring, XOR expression was found in all tissues of mature mullets, including a very weak expression in gills. In juveniles, spring expression was limited to high expression levels in liver and spleen and weak expression levels in muscle (Figure 2). Both in winter and in spring mature tissues POX expression was specially high in brain and spleen while in juveniles higher expression levels were measured in brain, spleen and gills than in liver, heart and muscle (Figure 2). Female gonad showed a very high expression level in spring (Figure 2). In winter mature individuals, the highest CAT expression levels were observed in liver and spleen while in juveniles, expression was mainly observed in spleen, heart, gill, and muscle (Figure 2). In spring juveniles, expression was mainly restricted to brain and liver while in mature individuals was high in all tissues but specially in female gonad (Figure 2). Comparing expression levels in the livers of all the mussels captured in winter to those in the animals captured in spring, no seasonal variations in gene expression levels were detected, although the variability among 151

103 Results and Discussion Figure 1. (a) Expression of POX, CAT and 18S rrna in gills, mantle and digestive gland of mussels collected in winter 2004 and spring (b) Mean expression values of genes coding for POX and CAT in mussel tissues after normalisation against 18S rrna (n = 5). Vertical bars indicate standard deviation and asterisks statistically significant differences between winter and spring after U-test (p<0.05). individuals increased in spring (Figure 3). In situ hybridisation for the localisation of CAT mrna Staining control using sense probe was completely negative confirming that the staining found using the antisense probe was specific. CAT mrna was found to be ubiquitous being localised in both digestive gland epithelial cells (digestive and basophilic cells), ovocytes and gill epithelial cells of mussels (Figure 4). Probes designed to localise mullet CAT were not able to hybridise with our tissue sections. Discussion In the present study, XOR, POX and CAT cdnas were partially cloned in mussels Mytilus galloprovincialis and thicklip grey mullets Chelon labrosus. Mullets and mussels have been reported to suffer seasonal metabolic and enzyme activity variations related both to physical changes in the environment, such as changes in temperature, food availability and oxygen levels and to physiological factors such as gonadal development (Widdows, 1978; Orbea et al; 2002; Ferreira et al. 2005), so the expression pattern of cloned genes was studied with the aim of assessing the effect of these variables in their expression regulation. XOR is considered the rate-limiting enzyme in purine metabolism acting either as oxidase or as dehydrogenase (Berry and Hare 2004; Benedetti et al. 2006). However, in recent years, additional functions have been revealed such as its involvement in the regulation of adipogenesis and PPARγ activity (Cheung et al. 2007), its antimicrobial properties (Hancock et al. 2002) or its participation in the metabolism of xenobiotics 152

104 Cloning and expression pattern of a POX XOR, and CAT Figure 2. Expression pattern of XOR, POX and CAT in different tissues (B = brain, L = liver, S = spleen, H = heart, G = gill, M = muscle and Gn = gonad) of juvenile and mature male and female thicklip grey mullets Chelon labrosus, collected in winter (2004) and spring (2005) in the Bay of Biscay. N.a. = gonad not available. that have been reported to act via the aryl hydrocarbon receptor such as tetrachlorodibenzo-p-dioxin (Sugihara et al. 2001). In the present study, XOR amplification in mullet liver resulted in the cloning and sequencing of a 266 bp long fragment which showed 85% amino acid identity with other fish XORs while a fragment of 290 bp was obtained in mussels. This fragment showed its highest homology with the sea urchin Strongylocentrotus purpuratus XOR (Table 1). Since low XOR expression levels were measured in most of the studied tissues, quantification of its expression level by semiquantitative-rt-pcr was impossible in mussel tissues and very difficult in mullets. In agreement to previous data in the literature (Resende et al. 2005), the highest XOR expression levels were measured in liver and no significant differences were found when comparing winter and spring expression levels. However, in winter no XOR expression was detected in gills at all, while a very weak expression could be detected in spring. XOR expression was more widely distributed in mature individuals than in juveniles where expression was restricted to liver in winter and spleen in spring. Thus, it seems that XOR expression depends on the developmental stage and season. However, since XOR in mussel and mullet seems to be expressed at very basal levels, alterations in the expression level should be measured using a more sensitive technique such as QPCR. In mammals, liver and intestine present the highest XOR activity levels (Pritsos 2000; Harrison 2004) XOR expression being mainly regulated by cytokines, steroid hormones and 153

105 Results and Discussion 1,2 0,8 XOR significantly induced in summer months coinciding with an increase in the metabolic demand during this season crucial in the reproductive cycle. 0,4 0 1,5 1 0,5 0 2,5 W W POX CAT S S In addition, a fragment of 385 bp and 326 bp long POX showing high deduced amino acid identity with N-acetylspermine/Nacetylspermidine oxidase and specially with spermine oxidase of different fish species was cloned in C. labrosus and M. galloprovincialis, respectively. Spermine oxidase has been identified as polyamine oxidase able to convert spermine back into spermidine without the need of an acetylation step (Wang et al. 2001). Therefore, while spermine oxidase is only able to oxidise spermine, N-acetylspermine/Nacetylspermidine oxidase oxidises N- acetylspermine and N-acetylspermidine (Vujcic et al. 2002; 2003). However, both contribute to the homeostasis of polyamines. 2 1,5 1 0,5 0 W Figure 3. Mean expression values of genes coding for XOR, POX and CAT in liver of 6 mullets C. labrosus captured in winter (W) and spring (S). Vertical bars indicate standard deviation. No statistically significant differences were detected by the U-test. oxygen tension (Levinson and Chalker 1980; Lanzillo et al. 1996; Terada et al. 1997; Berry and Hare 2004). Androgens are supposed to behave as XOR activity inducers while estrogens, on the contrary, could be inhibitory. Therefore, since steroid hormones control the reproductive cycle, gender related XOR expression could be expected. In female Salmo trutta liver and kidney XOR activity was highest in May and it decreased during vitellogenesis, male livers showing a similar variation pattern (Resendde et al. 2005). Seasonality in XOR activity was also reported by Cancio and Cajaraville (1999) in the digestive gland of mussels. Intense and season dependent variations in XDH activity were detected, being S As XOR, POXs are also flavin-containing enzymes. When they oxidise their substrates, FAD is reduced, being reoxidised by O 2 to generate H 2 O 2. Another product of the oxidation of N-acetylated polyamines is 3- acetamidopropanal that after deacetylation may cause tissue damage. Oxidation of spermine, on the other hand, produces aminopropanal (Vujcic et al. 2002; 2003; Wallace et al. 2003). Polyamines (putrescine, spermine and spermidine) participate in important cellular functions such as cell growth and proliferation and can be involved in tumour development. In this sense, it has been suggested that there may be a system of polyamine reutilisation after their acetylation in fish too (Kumazawa and Suzuki 1987), but up to now, little is known on POX functions in aquatic organisms. In catfish POX activity was found in brain, intestine, kidney and liver being at its highest in the intestine and liver and at its lowest in brain (Kumazawa and Suzuki 1987). In mice, liver and stomach present the highest POX expression levels followed by heart, spleen, thymus, small intestine, muscle, pancreas, uterus and breast at various developmental stages (Wu et al. 2003). In agreement, the highest POX activity has been measured in rat liver (Pavlov et al. 1991) while 154

106 Cloning and expression pattern of a POX, XOR, and CAT Figure 4. Localisation of catalase mrna by in situ hybridisation in the cytosol of oocytes (a), gill epithelial cells (b) and both digestive gland epithelial cells (digestive and basophilic cells) (c, d) of mussels Mytilus galloprovincialis. Scale bars: (a) = 100 µm, (b and d) = 20 µm and (c) = 200 µm. in human the highest activity has also been reported in liver followed by testis, kidney, spleen and small intestine (Suzuki et al. 1984). We also detected high expression levels in mullet liver but the highest POX expression levels were found in brain and spleen. Regarding mussels, POX, expression levels were high in all studied tissues, suggesting POXs carry out important metabolic functions in molluscs. In rats, females showed lower POX activity levels in all the studied tissues than males (Ferioli et al. 1999). In our case, no obvious differences were observed between male and female mullets. Additionally, no seasonal dependent POX expression alterations were observed in liver. On the contrary, POX expression levels in mussel tissues were highest in spring, differences with winter samples being significant in mantle and digestive gland. These differences could be related to reproduction, since an active polyamine metabolism may be expected to be needed during that period in order to regulate cell proliferation. CAT is the main peroxisomal protein and constitutes an important barrier against oxygen radicals, degrading H 2 O 2 produced by the activity of peroxisomal flavin-oxidases. In this sense, it has been proposed to be inducible under ROS generating conditions (Livingstone et al. 1990; Solé et al. 1995; Scandalios 2005; Schrader and Fahimi 2006). In the present study, CAT expression was localised in oocytes, gill and digestive gland of mussels by in situ hybridisation, being expressed in similar levels in mantle and digestive gland of organisms collected both in winter and in spring. However, significant higher CAT expression was measured in gills of mussels collected in winter than in gills of mussels collected in spring. Similarly, in M. edulis, antioxidant gluthation S-transferase activity measured in gill peaked during the winter months and showed little seasonal variability in digestive gland (Power and Sheehan 1996). In addition, it has been widely reported that digestive gland of mussels Mytilus sp and Perna perna collected in spring show higher activity levels than in winter (Cancio and Cajaraville 1999; Sceehan and Power 1999; Orbea et al. 2002; Petrovic et al. 2004; Leiniö and Lehtonen 2005; Bocchetti and Regoli 2006; 155

107 Results and Discussion Alves de Almeida et al. 2007). Most of these authors report that elevated temperature in late spring and higher bioavailability of nutrients would induce oxygen consumption and cellular oxyradical generation, which could be compensated by the increase of antioxidant defences. However, present results indicate that this increase in activity is not regulated at the gene expression level. Alternatively, it might be speculated that as organisms were collected in a chronic polluted harbour where other factors may have modulated CAT expression all along the year. In this sense, organisms in this location do not show a clear reproductive seasonality, the gonad of most mussels being in spawning or advanced gametogenetic phases all along the year (Porte et al. 2006). Similarly, CAT expression level in mullet liver did not change when comparing expression levels in winter and spring. On the contrary, mullets Mugil cephalus showed significantly higher CAT activity in spring than in winter (Orbea et al. 2002; Ferreira et al. 2005). On the contrary, seasonal variations of antioxidants in Mediterranean M. cephalus did not appear strictly related to the summer period (Gorbi et al. 2005), so it was defended that biological intrinsic variables such as reproductive cycle could have a greater effect in modulating these responses in mullets. Similarly, changes in CAT activity fluctuated randomly and were not seasonally dependent in the estuarine fish Pomatoschistus minutes or in Geophafus brasiliensis and Prochilodus lineatus (Filho et al. 2001; Camargo and Martínez 2006; Solé et al. 2006). Anyway, Significant CAT expression levels were detected in all the studied tissues, both in mature and in juvenile mullets, indicating the importance of this antioxidant enzyme in most of the tissues. In conclusion, XOR, POX and CAT showed a widespread tissue distribution, being differently expressed under the studied phyiological and physical conditions. This physiological regulation in antioxidant and prooxidant enzyme transcription should be taken into account when these enzyme activities are measured as biomarkers of pollution effects. Acknowledgements: This work was supported by the Spanish MEC (PRESTEPSE, VEM C06), Basque Government (ETORTEK-IMPRES) and University of the Basque Country (predoctoral grant to E. Bilbao and grant for consolidated research groups). Authors would like to thank Dr. Arno Schad (University of Heidelberg) and Dr. Izaskun Zorita (University of the Basque Country) for their contribution to in situ hybridisation studies. References Alves de Almeida E, Celso Dias Bainy A, Paula de Melo Loureiro A, Regina Martinez G, Miyamoto S, Onuki J, Fujita Barbosa L, Carrião Machado Garcia C, Manso Prado F, Eliza Ronsein G, Alexandre Sigolo C, Barbosa Brochini C, Maria Gracioso Martins A, Helena Gennari de Medeiros M, Di Mascio P (2007). "Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: Antioxidants, lipid peroxidation and DNA damage". Comparative Biochemistry and Physiology Part A 146, Angermuller S, Bruder G, Volkl A, Wesch H, Fahimi HD (1987). "Localization of xanthine oxidase in crystalline cores of peroxisomes. A cytochemical and biochemical study". European Journal of Cell Biology 45, Babbar N, Murray-Stewart T, Casero RA (2007). Inflammation and polyamine catabolism: the good, the bad and the ugly. Biochemical Society Transactions 35, Basha PS, Rani AU (2003). "Cadmium-induced antioxidant defense mechanism in freshwater teleost Oreochromis mossambicus (Tilapia)". Ecotoxicology and Environmental Safety 56, Benedetti MS (2001). "Biotransformation of xenobiotics by amine oxidases". Fundamental and Clinical Pharmacology 15, Berry CE Hare JM (2004). "Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications". The Journal of Physiology 555,

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112 Results and Discussion 55

113 POX, XOR eta CATen klonazio eta espresio-patroia 1.- Poliamino oxidasa, xantina oxidoerreduktasa eta katalasaren klonazio eta espresio-patroia Mytilus galloprovincialis muskuiluan eta Chelon labrosus lazunean Atal honetako emaitzak ondorengo artikuluan argitaratu dira: BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I (2006). "Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: generation of new tools to study peroxisome proliferation". Marine Environmental Research 62S, Atal honetako emaitzak aipatzen diren kongresuetan aurkeztu dira: 13 th International Symposium on Pollutant Responses in Marine Organisms (PRIMO), Alessandria, Italia. Ekainak 19-22, BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I. "Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: generation of new tools to study peroxisome proliferation" 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, The Hague, Herbeherak, Maiatzak 7-11, CANCIO I, BILBAO E, RAINGEARD D, SAEZ-MORQUECHO C, DIAZ DE CERIO O, CAJARAVILLE MP. "Cloning and expression pattern of peroxisomal genes in the thicklip grey mullet Chelon labrosus: a differential gene expression analysis to study peroxisome proliferation". 161

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115 POX, XOR eta CATen klonazio eta espresio-patroia SARRERA Chelon labrosus lazuna, Atlantiar Ekialdeko estuarioetan kutsadura maila altuko guneetan bizi den espezie ugaria da. Espezie honek, estuarioetako espezie zentinelek betetzen dituzten hainbat ezaugarri betetzen ditu. Muskuilua bestalde, mundu osoan zehar erabiltzen da itsasoko kutsaduraren jarraipenak burutzeko organismo indikatzaile legez. Arrain eta muskuiluetan estres oxidatiboa determinatzen duten biomarkatzaileak neurtzen dituzten ikerketak badaude ere, jakina da oxigeno espezie erreaktiboak (ROS) ekoitzi eta eliminatzen dituzten entzimak sexu eta sasoiaren araberako erregulazio diferentziala dutela. Beraz, lan honen helburua, ROSak ekoizten dituzten xantina oxidoerreduktasa (XOR) eta poliamino oxidasa (POX) oxidasa peroxisomikoak eta H 2 O 2 -a ezabatzen duen CAT kodetzen dituzten geneen zatiak klonatzea eta beraien espresio-patroiak arrain zein muskuiluen ehun ezberdinetan aztertzea izan zen. Gainera, arrainetan detoxifikazioaz arduratzen den organo nagusian, alegia gibela, eta muskuiluen ehun ezberdinetan espresio mailak konparatu ziren, neguan eta udaberrian batutako organismoetan. Horretarako, lehenik, espezie bietan XOR, POX eta CAT kodetzen dituzten geneen zatiak klonatu ziren hasle anderatuak erabiliz, ondoren, RT-PCR semikuantitatiboz beraien espresio-patroia aztertu zelarik. Muskuiluetan, XOR, POX eta CAT zakatz, mantu eta liseri-guruinean espresatu ziren. Lazunetan, POX eta CAT gibel, bare, burmuin, bihotz, muskulu, zakatz eta gonadan espresatu ziren bitartean, XOR gibel eta zakatzetan espresatu zen nagusiki. Garapen maila eta jeneroaren araberako XORen espresio maila ezberdina aurkitu zen. XOR, POX eta CAT geneen espresioa antzekoa izan zen udaberrian eta neguan arrantzatutako lazunetan, muskuiluetan aldiz, ezberdintasun nabarmenak aurkitu ziren. Muskuiluen zakatzetako CAT geneak neguan azaldu zuen espresio maila gorena, POXek berriz, udaberrian agertu zituen maila altuenak. Bestalde in situ hibridazioaren bitartez CAT genearen espresioa zakatz eta liseri-guruineko zelula epitelialetan zein obozitoetan aurkitu zen. Beraz, XOR, POX eta CATek lazun zein muskuiluen ehunetan espresio-banaketa zabala erakutsi zuten. Hala ere, lazunek ez zuten agertu sasoiaren araberako espresio ezberdintasun esangarririk; muskuiluen kasuan, sasoiaren araberako POX eta CATen transkripzio mailako erregulazio argia behatu zen bitartean. Hortaz, entzima antioxidatzaile eta prooxidatzaileen transkripzio mailako erregulazio fisiologikoa kontutan hartu beharrekoa da kutsaduraren efektuak ikertzeko helburuz, azterturiko geneen produktu entzimatikoak biomarkatzaile legez neurtzen dituzten jarraipenprogrametan. 163 L A B U R P E N A

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117 POX, XOR eta CATen klonazio eta espresio-patroia Emaitzak Itu-geneen klonazioa XOR kodetzen duen genearen 266 bp eta 290 bp klonatu ziren C. labrosus lazunean (AY876387) eta Mytilus galloprovincialis muskuiluan (AY876388), hurrenez hurren. Lazunaren XORak %85eko amino azido identitatea agertu zuen Poecilia reticulataren xantina deshidrogenasarekin (AAK59699) eta muskuiluarenak berriz, %42koa Strongylocentrotus purpuratusen xantina deshidrogenasa/oxidasarekin (XP_ ) (1 Taula). Bestalde, ustezko POXen 385 bp klonatu ziren Chelon labrosus lazunean (AY876389) (1 Taula). Anplifikatutako zatiak, %90eko amino azido identitatea agertu zuen Danio rerioren spermina oxidasarekin (BC066413) eta %82koa gizakiaren poliamino oxidasarekin (NM_175840). Muskuiluetan klonatutako 326 bp-tako zatiak (AY876390), %91ko amino azido identitatea erakutsi zuen D. rerioren espermina oxidasarekin (BC066413) eta %91ko amino azido identitatea berriz, gizakiaren poliamino oxidasarekin (NM_175840) (1 Taula). Azkenik, Oplegnathus fasciatusen CATekin (AAU44617) %94ko amino azido identitatea agertu zuen 273 bp-tako zatia klonatu zen C. labrosus lazunean (AY743715). Berdintsu, M. edulisen CATekin (AAT06168) %95eko amino azido identitatea agertu zuen 552 bp-tako zatia klonatu zen M. galloprovincialis muskuiluan (AY743716) Geneen espresio-patroia Mytilus galloprovincialisen ehunetan 18S rrna-ak erakutsi zuen, ehun ezberdinen eta sasoiaren araberako espresio-aldaketarik baxuena, eta beraz, neurtutako geneen espresio mailak, housekeeping horrekiko normalizatu ziren. XOR, POX eta CAT muskuiluen zakatz, mantu eta liseri-guruinean espresatu ziren, hala ere, XORak erakutsitako espresio maila baxua zela eta, gene horren espresioa ezin izan zen egokiro semikuantifikatu ez neguan ezta udaberrian ere. Aztertutako ehun guztietan, POXen espresioa altuagoa izan zen udaberrian neguan baino, ezberdintasun hori mantuan eta liseri-guruinean esangarria izanik (1 Irudia). Liseri-guruin eta mantuan ez zen sasoiaren araberako aldaketarik behatu CATen espresioan, zakatzetan ordea, CATen espresio altuagoa neurtu zen neguan udaberrian baino (1 Irudia). Geneen espresioa Chelon labrosusen 18S rrna-ak erakutsi zuen, ehun ezberdinen eta sasoiaren araberako espresio-aldaketarik baxuena, eta beraz, neurtutako geneen espresio mailak, housekeeping horrekiko normalizatu ziren. POX eta CAT, aztertutako lazun heldu eta heldugabeen ehun guztietan espresatu ziren, bai neguan zein udaberrian (2. Irudia). Lazun helduen zakatzetan ez zen XORen espresiorik behatu neguan, heldugabeetan berriz, XORen espresioa gibelean aurkitu zen bereziki (2. Irudia). Udaberrian, lazun helduen ehun guztietan aurkitu zen XORen espresioa, zakatzetan aurkitutako XOR espresio arina barne. Udaberriko heldugabeetan, XOR gibel eta barean espresatu zen nabarmenki eta arinki muskuluan (2. Irudia). Negu zein udaberriko lazun helduen ehunetako POX espresio maila bereziki altua izan zen burmuin eta barean, heldugabeetan berriz, burmuin, bare eta zakatzetan, gibel, bihotz eta muskuluan baino espresio maila altuagoak neurtu ziren (2. Irudia). Emeen gonadak POX genearen espresio maila altua agertu zuen udaberrian (2. Irudia). Neguko organismo helduetan, CAT genearen espresio mailarik altuenak gibel eta barean neurtu ziren, heldugabeetan berriz, espresioa bare, bihotz, zakatz eta muskuluan aurkitu zen nagusiki (2. Irudia). Udaberriko heldugabeetan berriz, CATen espresioa burmuin eta gibelera mugatu zen nagusiki eta helduetan berriz, espresio maila altuak aurkitu ziren aztertutako ehun guztietan baina bereziki gonadan (2. Irudia). Neguan arrantzatutako lazunen gibeleko 165 E M A I T Z A K

118 Emaitzak eta Eztabaida espresio mailak udaberrikoekin alderatzean, ez zen behatu sasoiaren araberako espresio mailaren aldaketa esangarririk, udaberrian indibiduoen arteko aldakortasuna emendatu egin bazen ere (3. Irudia). In situ hibridazioaren bidezko CATen mrnaren kokapena zela aurkitu zen, hau liseri-guruineko zelula epitelialetan (liseri-zelulak eta zelula basofilikoak), zakatzetako zelula epitelialetan eta obozitoetan lokalizatuz (4. Irudia). Lazunen CAT lokalizatzeko diseinatutako zundek ez zuten hibridatu erabilitako ebakinetan. Tindaketaren kontrol gisa erabilitako sense zundak ez zuen tindaketarik eman, horrela, antisense zundak emandako seinalea espezifikoa zela ziurtatu zen. CAT genearen mrna ubikuoa 166

119 Ondorioak POX, XOR eta CATen klonazio eta espresio-patroia 1.- Oxigeno espezie erreaktiboen eraketan diharduten xantina oxidoerreduktasa (XOR) eta poliamino oxidasa (POX) eta H 2 O 2 -ren degradazioan aritzen den katalasa (CAT) entzimak kodetzen dituzten geneen zati bana klonatu eta sekuentziatu da Chelon labrosus lazunean eta Mytilus galloprovincialis muskuiluan. 2.- XOR, POX eta CATek espresio-banaketa zabala agertzen dute aztertutako C. labrosus eta M. galloprovincialisen ehunetan. CATren espresio zabala in situ hibridazioaren bidez ere ikusi ahal izan da, muskuiluaren liseri-guruin eta zakatzetako zelula epitelialetan eta baita obozitoetan ere. 3.- XORek garapen fase eta sexuaren araberako espresio diferentziala erakusten du lazunetan. Bestalde, lazunek ez dute agertu sasoiaren araberako espresio-aldaketarik gibelean, muskuiluen ehun ezberdinetan, POX eta CATek sasoiaren araberako aldaketak erakutsi dituzten bitartean. Geneon espresioan ematen den egoera fisiologiko zein inguru-fisikoaren kutsaduraren araberako erregulazioaren efektua aztertzeko, biomarkatzaile legez geneok kodetzen dituzten entzima antioxidatzaile edo prooxidatzaileen jarduera entzimatikoak neurtzen dituzten jarraipen-programetan kontutan hartu beharreko faktoreak dira. 167 O N D O R I O A K

120 2.- Molecular characterisation of the peroxisomal b-oxidation pathway in aquatic organisms: cloning and expression pattern of palmitoyl-coa oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase This chapter has been sent for publication to: BILBAO E, CAJARAVILLE MP, CANCIO I (submitted). "Molecular characterisation of the peroxisomal β-oxidation pathway in aquatic organisms: cloning and expression pattern of palmitoyl-coa oxidase, multifunctional protein and 3- ketoacyl-coa thiolase". Experimental Cell Research. Parts of this chapter have been presented at: 14 th International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14), Florianópolis, Brazil, May 6-9, BILBAO E, CAJARAVILLE MP, CANCIO I. "Cloning and regulation of the peroxisomal β-oxidation genes (acyl-coa oxidase, multifunctional protein, thiolase) in thicklip grey mullets and mussels after treatment with organic xenobiotics". 17 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, Porto, Portugal, May 20-24, BILBAO E, CAJARAVILLE MP, CANCIO I. "A field study of changes in peroxisomal gene expression in mussel Mytilus galloprovincialis". 169

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122 ABSTRACT Molecular characterisation of β-oxidation in aquatic organisms Due to the ability to respond after exposure to organic toxic compounds, peroxisome proliferation is an endpoint that has been proposed as a biomarker of exposure to organic pollutants in mussels and in fish. Mussels are worldwide studied as sentinels of pollution in marine environments while mullets such as the thicklip grey mullet Chelon labrosus have been proposed as ideal sentinel species since they inhabit highly polluted environments. In order to molecularly characterise the peroxisomal β- oxidation pathway in mussels Mytilus galloprovincialis and in C. labrosus, genes coding for the enzymes participating in all three steps in the pathway, palmitoyl-coa oxidase (AOX1), multifunctional protein (MFP1 in mullet and MFP2 in mussels), and 3-ketoacyl-CoA thiolase (THIO), were cloned. Additionally, a fragment of the peroxisomal 2, 4 dienoyl-coa reductase 2 (DECR) necessary for the β-oxidation of unsaturated fatty acids was cloned in mullets. All four genes were found to be expressed in all studied tissues in both species. The whole open reading frame of AOX1 was cloned and sequenced in both mussels and mullets revealing high homology with known AOX1 sequences, with highly conserved important domains such as the FAD binding motif or the typical peroxisomal targeting signal (PTS1). The presence of PTS1 in mullet peroxisomal proteins was also confirmed by immunoelectron microscopy. A thorough in silico analysis of the gene and genome databases allowed to identify in fish and molluscs sequence homologs of all the enzymes necessary for 2 of the 3 different paralog peroxisomal β-oxidation pathways described in metazoans (AOX1, AOX3, MFP1, MFP2, THIO and sterol carrier protein X). Only the enzyme necessary for the oxidation of branched chain fatty acids, AOX2, described in mammalian, avian and amphibian species seems to be lacking from the genomes of fish and molluscs. Expression of cloned genes was studied after exposure of mullets and mussels to two compounds reported to act as peroxisome proliferators, perfluorooctanoic sulfonic acid and benzo(a)pyrene. Induction of both AOX1 and MFP1 expression was determined. In silico studies of the promoter regions in the piscine genomes available in the Ensembl genome repository, allowed the identification of putative peroxisome proliferator response elements that would explain the possible cellular and molecular mechanisms leading to peroxisome proliferation in fish. Further gene expression and morphological studies will reveal the usefulness of studying peroxisomal alterations in aquatic organisms as a biomarker of exposure to organic chemicals. 171

123 Results and Discussion RESUMEN Dada la capacidad para responder ante la exposición a contaminantes orgánicos tóxicos, la proliferación de peroxisomas se ha propuesto como biomarcador de exposición a contaminantes orgánicos tanto en mejillones como en peces. Los mejillones se utilizan ampliamente como centinelas de la contaminación en medios marinos mientras que los mubles, como Chelon labrosus, han sido propuestos como especie centinela ideal debido a que habitan ambientes altamente contaminados. Con el fin de caracterizar molecularmente la β-oxidación peroxisómica en el mejillón Mytilus galloprovincialis y en C. labrosus, se clonaron los genes que codifican los tres enzimas implicados, palmitoil-coa oxidasa (AOX1), proteína multifuncional (MFP1 en muble y MFP2 en mejillón) y 3-ketoacil-CoA tiolasa (THIO). Además en muble, se clonó un fragmento del gen que codifica 2, 4 dienoil-coa reductasa 2 (DECR) peroxisómico, necesario para la oxidación de ácidos grasos poliinsaturados. Se clonó el open reading frame (ORF) completo de AOX1 tanto en mejillones como en mubles y éste reveló alta homología con secuencias de AOX1 conocidas, presentando una conservación alta de dominios importantes como son el motivo de unión a FAD o la señal típica de entrada a peroxisomas (PTS1). La presencia de PTS1 en proteínas peroxisómicas de muble se confirmó además mediante microscopía immunoelectrónica. El análisis in silico de bases de datos de genes y genomas permitieron identificar secuencias homólogas en peces y moluscos de todos los enzimas necesarios en 2 de las 3 vías parálogas de la β-oxidación peroxisómica descrita en metazoos (AOX1, AOX3, MFP1, MFP2, THIO, sterol carrier protein X). Así, en peces y moluscos sólo parece faltar el enzima necesario para la oxidación de ácidos grasos ramificados, AOX2, descrito en mamíferos, aves, y anfibios. La expresión de los genes clonados se estudió tras la exposición de peces y mejillones a dos proliferadores de peroxisómas, perfluorooctano sulfonato y benzo(a)pireno, donde se observó una sobreexpresión de AOX1 y MFP1. Los estudios in silico de los promotores en genomas de especies de peces disponibles en la base de datos Ensembl, permitieron la identificación de posibles elementos de respuesta a proliferadores de peroxisomas que explicarían los posibles mecanismos celulares y moleculares que dirigen la proliferación de peroxisomas en peces. Además, los estudios de expresión génica y los estudios morfológicos revelarán la utilidad de estudiar alteraciones peroxisómicas en organismos acuáticos como biomarcador de exposición a contaminantes orgánicos. 172

124 Molecular characterisation of β-oxidation in aquatic organisms Introduction Lipids are the principal constituents of many fish species where they play major roles for the generation of the energy necessary for growth, reproduction and movement (Nevejan et al. 2003; Tocher et al. 2003). In bivalve molluscs more than half of the energy requirements for embryogenesis is met by lipids (mainly polyunsaturated fatty acids, PUFAs) the rest being met by proteins (Whyte 1991; 1992). Aquatic organisms, and particularly marine organisms are rich in ω3 long and very long chain fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (Nevejan et al. 2003; Tocher et al. 2003), that are substrates of the peroxisomal metabolism at least in mammals (Sampath and Ntambi 2005; Inoue 2005). In this sense, peroxisomal β-oxidation seems to be very important in the liver and muscle of different fish species where it is capable of oxidising a wide range of fatty acid substrates (Crockett and Sidell 1993a; 1993b). The physiological significance for the dominance of peroxisomal over mitochondrial β-oxidation in the liver of some fish (Nanton et al. 2003; Froyland et al. 2000) has not been studied and deserves further investigation since it may be an adaptation to the high amount of long-chain fatty acids (LCFA) present in marine lipids. Regarding molluscs nothing is known on the relative importance of the peroxisomal β- oxidation that is mainly concentrated in the digestive gland (Cancio and Cajaraville 2000), but as the lipid composition is highly similar to that in teleost species it could be hypothesised that the relative importance of the pathway would match that reported for fish. Peroxisomal β-oxidation is responsible for the chain-shortening of a variety of acylcoenzyme A (CoA) derivatives of different lipid substrates such as, LCFAs and very-long-chain fatty acids (VLCFA), PUFAs, long-chain dicarboxylyc acids, eicosanoids, 2-methylbranched fatty acids, bile acid intermediates and the carboxyl side chain of some xenobiotics (Mannaerts and Van Veldhoven 1993; Mannaerts et al. 2000; Reddy and Hashimoto, 2001). The activated forms of these lipid compounds enter the β-oxidation where a first oxidation step produces a double bond between carbon atoms 2 and 3, followed by a hydratation and an oxidation of that trans double bond. The last step is a thiolytic cleavage. In this cycling process acyl-chains are shortened liberating a fatty acyl- CoA shortened in two carbons and acetyl-coa (Kim and Battaile 2002). Each of these steps can be catalysed in peroxisomes by at least two different proteins while shortening of unsaturated fatty acids requires auxiliary enzymes: trans- 3 -cis- 2 - trans-enoyl-coa isomerase and 2, 4 dienoyl- CoA reductase 2 (DECR). DECR reduces 2- trans, 4-cis-dienoyl-CoA generated from fatty acids with a cis-double bond in an even numbered position to trans-enoyl-coa, which after conversion into 2-trans-enoyl-CoA by the isomerase can reenter the oxidation spiral (Schulz, 1994; Kunau et al. 1995). This, at least doubled paralogue enzymatic system is generally accepted of consisting on an enzymatic machinery which allows efficient metabolism of unmodified and modified acyl-coas (Baes et al. 2000; Qi et al. 2000) but where substrate capture seems to occur depending on the specificity or/and affinity of the individual enzymes (Van Veldhoven et al. 2001). In the metazoans studied there are two distinct fatty acid oxidation pathways one based on the activity of palmitoyl- CoA oxidase (AOX1), L-specific multifunctional protein (MFP1) and 3-ketoacyl- CoA thiolase (THIO) and the other one based on the activity of pristanoyl-coa oxidase (AOX3) or trihydroxycoprostanoyl-coa oxidase (AOX2), D-specific multifunctional protein (MFP2) and sterol carrier protein X (SCPX) (Hashimoto 1999; Baes et al. 2000; Mannaerts et al. 2000; Reddy and Hashimoto, 2001). The first of these pathways has been reported to be inducible at the transcriptional level in certain responsive species upon exposure to peroxisome proliferator compounds such as hypolipidemic drugs, phthalate ester plasticisers or perfluorinated compounds (Beier and Fahimi 1991; Masters et al. 1998; Cancio and Cajaraville, 2000). Due to the environmental relevance of the inducible pathway as a biomarker of exposure to xenobiotics that might act as peroxisome proliferators, the present study is focused on AOX1, MFP1 and THIO. 173

125 Results and Discussion AOX1 is the first and rate-limiting enzyme of the inducible peroxisomal β-oxidation pathway and increased AOX1 activity is usually measured as peroxisome proliferation marker (Cancio and Cajaraville 2000; Cajaraville and Ortiz- Zarragoitia 2006). MFP1 which is also inducible under exposure to peroxisome proliferators catalyses the second and third steps of the pathway together with an isomerisation step, that is necessary to catabolise unsaturated substrates (Hiltunen and Qin 2000; Mannaerts et al. 2000; Reddy & Hashimoto 2001). The last reaction in the pathway depends on a THIO, which in rodents is coded by two genes, A and B. THIO- A is constitutively expressed whereas expression of THIO-B is induced by peroxisome proliferators (Hijikata et al. 1990, Hansmannel et al. 2003). In rodents, AOX1, MFP1 and THIO are target genes of the peroxisome proliferation activated receptor α (PPARα). After binding some of the lipids and peroxisome proliferators mentioned above, PPARα is able to heterodimerise with retinoid X receptor (RXR) and induce transcription of these target genes (Lemberger et al. 1996; Keller et al. 2000) by acting on the peroxisome proliferator response elements (PPRE) in their promotor regions. Although functional PPREs have been described in mammalian genes responsive to peroxisome proliferators (Latruffe et al. 2001; Mandard et al. 2004), no PPREs, which could elucidate the mechanism of β-oxidation induction have been reported in genes of aquatic organisms. However, PPREs have been described in CYP19A1 and glutathione-s transferase A1 (GST-A1) genes of different fish species (Leaver et al. 1997; Gardner et al. 2005). On the other hand, three PPAR subtypes PPARα (2 genes), β and γ have been described in several fish species (Leaver et al. 2005; Liu et al. 2005), among them in thicklip grey mullet Chelon labrosus (Ibabe et al. 2004; Raingeard et al. 2006). No PPARs have been described yet in invertebrates, although 2 PPAR paralogs have been described on the first assembly of the Strongilocentrotus purpuratus genome (Goldstone et al. 2006). The presence of some nuclear receptors belonging to the same superfamily as PPARs such as the estrogen receptor or RXR have been described in molluscs (Thornton et al. 2003; Nishikawa et al. 2004; Bouton et al. 2005). In aquatic organisms, peroxisome proliferation measured as a significant induction of AOX1 activity has been observed in mussels in different laboratory studies after exposure to a water accomodated fraction of lubricant oil (Cancio et al. 1998), North Sea Oil (Cajaraville and Ortiz-Zarragoitia 2006), clofibrate (Cancio et al. 1998), di(2-ethylhexyl)phathalate (Orbea et al. 2002) and after benzo(a)pyrene (B(a)P) injection (Cancio et al. 1998; Orbea et al. 2002). In fish, peroxisome proliferation has been measured in different fish species inhabiting environments with high burdens of organic toxic compounds (Bilbao et al. 2006a; Marigómez et al. 2006). The aim of this work was to characterise molecularly the inducible β-oxidation pathway in two aquatic organisms, mussel M. galloprovincialis and thicklip grey mullet C. labrosus. Since peroxisome proliferation has been proposed as a novel biomarker of exposure to organic pollutants in aquatic organisms (Cajaraville et al. 2003) and in order to evaluate the possible usefulness of cloned sequences as tools to study such proliferation, we measured their transcriptional inducibility in fish injected with B(a)P and in mussels exposed to perfluorooctanoic sulfonate. Materials and methods Materials All chemicals were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless specified otherwise. Cloning of Target Sequences: Mullets Chelon labrosus and mussels Mytilus galloprovincialis were sampled in Arriluze, Bay of Biscay (43º20 48 N, 3º01 42 W). Liver of mullets and digestive gland of mussels were frozen in RNA later and stored at -80 until processing mg of tissue were homogenised in Trizol (Invitrogen, Carlsbad, California, USA) using a Hybaid Ryboliser TM (Hybaid, Ashford, UK) cell disruptor at a shaking speed of 4 m/s for 20 seconds for liver and 40 seconds for digestive gland. Total RNA 174

126 Molecular characterisation of β-oxidation in aquatic organisms was isolated following the manufacturer s protocol. Then 3 µg of total RNA were used as template for cdna synthesis by reverse transcriptase PCR (Invitrogen) using random hexamers as primers and following the manufacturer's recommendations. Degenerate primers designed aligning known sequences deposited in the nucleotide repositories from different phyla were used to amplify fragments of genes coding for mullet and mussel AOX1, MFPs, THIO and DECR. Danio rerio (BC083524) and Phascolarctos cinereus (AF63453) AOX1 were aligned and Fw1: 5'-GMACYTATGCCCARACRG-3', Fw2: 5'-GARACYACAGCHACYTAYGA-3' and Fw3: 5'-ACDTAYGGHACCATGGTGTT-3' were designed as forward primers and Rv1: 5'- AGCATCATVACVGTRTTYTC-3', Rv2: 5'- GARTATCCATGYCCACCAC-3', Rv3: GTTTTCDCCYTCAWAKGTGC-3', Rv4: 5'- CCTKTARAAAGTCCCCAGAAT-3' and Rv5: 5'-AYTTCTTGGCCCAYTCAAACA-3' as reverse primers for amplification of AOX1 in mussels and mullets. Primers were coupled and a unique PCR was cycled: 2' at 94ºC, 35 cycles of (30'' at 94ºC, 30'' at 52ºC and 30'' at 72ºC) and 8' of extension at 72ºC. Amplification of MFP1 was carried out using the primers generated by aligning MFP1 of D. rerio (NM207068), Oncorhynchus mykiss (CA383240) and Salmo salar (CX355439). Fw: 5'- GAGGKGCDGACATCMGKG-3' and Rv: 5'- GAGTCTGGGYAGACGYTGTG-3' were used as primers in a PCR where cdna samples were denatured for 2' at 94ºC, amplified by 35 cycles at 94º for 30'', 58ºC for 30'' and 72ºC for 30'' and extended for 8' at 72ºC. D. rerio (NP956430), Carcinus maenas (CX994266) and Hydra magnipapillata (DN253014) were aligned and Fw: 5'-GCTGTRGCMAACTATGATTCWGT-3' and Rv: 5'-TTKGCCTGHCCRAAGTTVCCA- 3' were used as primers for amplification of MFP in mussels cycled as before but at a melting temperature of 57ºC. THIO was amplified in C. labrosus as published by our group (Bilbao et al. 2006b). For that purpose, Ictalurus punctatus (BM496229), D. rerio (NP ), Fundulus heteroclitus (CN981172) and Homo sapiens (NP001598) were aligned and Fw: 5'- GCTGCTGAGYGCHGTGAT-3' and Rv: 5'- CTCYGAVGTKATGCCCATSG-3' were designed as primers. PCR conditions were 2' at 94ºC, 35 cycles of 94º for 15'', 56ºC for 15'' and 72ºC for 15'' with a final elongation step at 72ºC for 8'. In mussels THIO was amplified using an EST sequence without assigned homology obtained from the Genbank (AJ624743). Fw was 5'-AGCAGTTATTAAACAGTCTGGTAT-3' and Rv: 5'-TCATCTGTTGTTACAGTTATTTGT-3'. PCR was cycled at a melting temperature of 54ºC using the same conditions as for mullet THIO. Finally, Takifugu rubripes (Ensembl accession number: SINFRUT ), Tetraodon nigroviridis (CR638586), Gasterosteus aculeatus (DW647728) and Platichtys flessus (DV568015) were aligned and DECR was amplified using Fw: 5'- ATACACACACATCTACAGYCC-3' and Rv: 5'-CRTCRTTGSCTGARGTMAG-3' as primers in a PCR cycled at the same conditions as mussel THIO. PCR products were visualised in a 1.5 % agarose gel, stained with ethidium bromide and purified using a PCR purification kit (Qiagen, Hilden, Germany). Single bands were directly sequenced but whenever more than one band was amplified, purified products were cloned using the TOPO-TA cloning kit (Invitrogen). Plasmids were extracted from bacteria using the S.N.A.P miniprep kit (Invitrogen) and digested with BamHI and NotI for 2 hours. PCR products for each gene were sequenced using the specific degenerate Fw primers described above while cloned inserts were sequenced in the Sequencing Service of the Department of Genetics, Physical Anthropology and Animal Physiology (University of the Basque Country) using the universal M13 Fw/Rv primers. RACE-PCR of AOX1 In order to obtain the whole open reading frame (ORF) of AOX1, 5 and 3 RACE-PCRs were carried out on the cdna of both mullets and mussels. 3 RACE-PCR was performed using the BD SMART RACE cdna Amplification kit (BD Biosciences, Clontech, 175

127 Results and Discussion California, USA) where 5 - ACCTGGCTCTGCTCTACGCCCTGA-3 and 5 -GCCAAGCTTGCCTATGGTTCTATGGTG- 3 were used as gene specific outer primers in mullets and mussels, respectively. Each nested PCR was cycled using 5 - TGTGGCGCTAGTGGATGCCTTTGACG-3 and 5 - CTACCCAGGGTGGAGAAGAACCCCAGAT -3 as gene specific inner primers. 5 RACE-PCR was carried out using the FirstChoice RLM- RACE kit (Ambion, Applied Biosystems, Texas, USA) where 5 - CCACCTTGGCGTATTTCATCAGC-3 and 5 - CACTTCAATCCCTGGCAAGACA-3 were used as gene specific outer primers in mullets and mussels, respectively. 5 - GTTGGAGGTCTTTCCAAGTCCTC-3 and 5 -TGAGTGTACAGTTGCGCCATCA-3 were used as gene specific inner primers. Both RACE- PCRs were cycled as suggested by the manufacturers. A second nested primer 5'- CCACCATTTGATGGAGCTGACC-3' was used as gene specific inner Rv primer in order to specifically obtain the complete sequence of the 5' extreme of mullet AOX1 Semiquantification of gene expression Specific primers amplifying fragments between bp were designed according to the new sequences obtained in mussels and mullets. PCR conditions for the amplification of AOX1, MFP1 and THIO were optimised (Table 1) using Taq polymerase (Invitrogen) and specific primers. Obtained PCR products were visualised by ethidium bromide stained agarose gel (1.5%) electrophoresis and analysed using a computer-aided gel analyser (Gel Doc 2000, Bio-Rad, San Diego, California, USA). Expression of target genes was measured in different tissues (digestive gland, mantle and gills of mussels and liver, brain, heart, muscle, gonad and gills of mullets) of 5 organisms collected in winter and 5 collected in spring. Then, 5 different mussels were exposed to 2 ppm of perfluorooctane sulfonate (waterborne) and 5 different mullets were intraperitoneally injected with 5 ppm of B(a)P using corn oil as vehicle. Results were expressed in arbitrary semiquantitative units, normalised against β-actin (AY836368, mullet and AF157491, mussel) and 18S rrna (AY825252, mullet and L33452, mussel) (Raingeard et al 2006; in prep). Statistical differences were calculated (p<0.05) by the Mann-Whitney U-test. Immunocytochemistry Small tissue blocks (<1 mm 3 ) of mullet liver were processed as previously described (Orbea et al. 1999). Briefly, tissue was fixed by immersion in 0.1 M phosphate buffer, ph 7.4, containing 2% paraformaldehyde, 0.05% glutaraldehyde and 2.5% NaCl for 1 h at room temperature. After washing, samples were dehydrated in ethanol by progressive lowering of temperature (Robertson et al. 1992) in a Rechert AFS (Leica Instruments GmbH, Germany) embedded in Lowicryl HM20 and polymerised under ultraviolet light. Ultrathin sections were cut in and picked up onto nickel grids. For the immunolabeling, sections were blocked in Tris-buffred saline (TBS) containing 4% BSA for 30 min at room temperature. Incubation with the specific polyclonal anti-skl antibody was carried out overnight in a humid chamber at room temperature. Antibody was raised in rabbits and immunised with a synthetic SKL peptide coupled to keyhole limpet hemocyanin as described by Usuda et al. (1999) (kindly provided by Prof. S. Subramani). After rinsing in several changes of TBS, sections were incubated in a protein-a-gold (10 nm) solution for 1 h at room temperature, rinsed in distilled water and counterstained in uranyl acetate and lead citrate. Electron micrographs were obtained with a JEOL JEM-100SX electron microscope (Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 60 kev. Results Cloning of AOX1, MFP, THIO and DECR Although specific bands were obtained in mullets using all the primer couples designed for AOX1 the longest fragments were obtained employing the couple Fw2-Rv5. Fw2-Rv5 amplified 3 bands of around 2000, 1500 and

128 Molecular characterisation of β-oxidation in aquatic organisms Table 1: Optimal PCR conditions for the specific amplification AOX1, MFP1 and THIO in thicklip grey mullets Chelon labrosus and mussels Mytilus galloprovincialis. *, Not measured. AOX1 Fw and Rv (5'-3') PCR conditions Tm (ºC) n Cycles Chelon labrosus CGCTACAGTGCTGTTCGTCACCA 94ºC CGGCACACTTCGATCACATGACAT 94ºC-15 MFP1 GTCTGGGCACCATGGGAAGAG CTTATCCAGTGCAGGAGCCAACC THIO TGCACCTGGAAGCCGGCG TTCCCATGGGGATGATGCAGTCTC Mytilus galloprovincialis AOX1 TACGATACAGTGCTGTTCGTC CCACAGCATATTCTACATACTTCTAT MFP2 * * THIO AGCAGTTATTAAACAGTCTGGTAT TCATCTGTTGTTACAGTTATTTGT Tm-15 n ºC-30 72ºC ºC ºC-25 Tm-25 n 72ºC-30 72ºC bps. After cloning the PCR products and digesting with BamHI and NotI, a specific band of 1450 bp that matched the expected amplicon size was obtained. The sequence of this band revealed a deduced 86% amino acid identity with Gallus gallus AOX1 (E value 0.0), so after cleaning primer sequences, a 1408 bp AOX1 fragment was obtained in mullets. In mussels, Fw1-Rv1, Fw1-Rv2, Fw2-Rv1 and Fw2-Rv2 amplified specific bands of 870, 790, 820 and 740 bp respectively. A final fragment of 845 bp was sequenced showing a deduced 64% amino acid identity with G. gallus AOX1 (E value: 2e ). Degenerate primers generated to amplify MFPs amplified fragments of bp in both species. Therefore, 213 bp identified as MFP1 through nucleotide homology were obtained in C. labrosus (EF407560) and 182 bp identified as MFP2 in M. galloprovincialis (EF525543). Mullet MFP1 showed a deduced amino acid identity of 66% with Tetraodon nigroviridis MFP1 (CAG09300) while mussel MFP2 showed 86% amino acid identity with Strongylocentrotus purpuratus MFP2 (XP ) (Figure 1). Using the same methodology, 327 bp of THIO (DQ021958) were amplified in mullets, showing 78% amino acid identity with Danio rerio THIO (AAH72706) while using specific primers, 498 bp of mussel THIO were amplified. Mussel THIO showed 56% amino acid identity with G. gallus THIO (XP ) (Figure 2). Finally, 283 bp of DECR were amplified from mullet liver (EF407559). The deduced protein sequence showed 88% amino acid identity with Tetraodon nigroviridis DECR (CAG04352) (Figure 3). RACE-PCR of AOX1 3'RACE PCR resulted in amplification of long products in both species. Sequencing of amplified products in mullets, determined a new region of 97 bp which corresponded with the last approximately 32 amino acids of the 3' terminal end of AOX1 cdna. In this way, 1505 bp of the AOX1 ORF (76%) were obtained in C. labrosus. In mussels, a fragment coding 257 amino acids (772 bp) was obtained. A total of 1616 bp of the whole AOX1 ORF were obtained in M. galloprovincialis by 3'RACE-PCR. On the other hand, 5'RACE-PCR amplified bands of bp. In mussels, this fragment contained 394 new nucleotides that code for the first 131 amino acids of AOX1. Similarly, 478 new nucleotides of the mullet AOX1 sequence were obtained, completing the ORF of mullet AOX1. Thus, the complete cdna sequence of mussel AOX1 consists of 2010 nucleotides coding for a deduced amino acid sequence of 670 residues (EF525542) showing a 51% amino acid identity with Xenopus tropicalis AOX1. In mullets, AOX1 contains 1983 bp that code for 661 amino acids (EF525541). Mullet AOX1 showed 79% amino acid identity with D. rerio AOX1. Although AOX1 in mussels differed from known AOX1 sequences in some regions several similarities were found when aligning 177

129 Results and Discussion (a) TnMFP1 VIGLGTMGRGITVALAQAGLSVVAVETQAKQLTEARKAVAGMLKRAAVRR---GTTPEPG 216 ClMFP1 --GLGTMGRGITVALAQAGLSVVAVETHAKQLMEAKQAVSGMLERGAKRR---GLAPALD 55 DrMFP1 VIGLGTMGRGIVVSLARVGISVIAVESEKKLLETGRQMVIGMLERDAKRR---GVSASLN 356 RnMFP1 VLGLGTMGRGIAISFARVGISVVAVESDPKQLDAAKKIITFTLEKEASRAHQNGQASAKP 360 *********.:::*:.*:**:***:. * *.:: : *:: * * * :. TnMFP1 SITYSDQLQAAARADLVIEAVFEDMEVKETLFRQLSAVCRPDALLCTNTSSLDVDHLASQ 276 ClMFP1 KISYMQSIKSTSDADL DrMFP1 LLKFSLSLQDLKDVDLVIEAVFEDMALKKQIFRELSRVCRPATLLCSNTSGLDVDALADV 416//718 RnMFP1 KLRFSSSTKELSTVDLVVEAVFEDMNLKKKVFAELSALCKPGAFLCTNTSALNVDDIASS 420 : :. :.** (b) DrMFP2 EIRAAGGKAVANYDSVEDGEKLIQTALDAFGRIDVVVNNAGILRDRSFARTSDVDWDLIQ 120 RnMFP2 EIRRRGGKAVANYDSVEAGEKLVKTALDTFGRIDVVVNNAGILRDRSFSRISDEDWDIIQ 120 MgMFP EDRSFASISDQDWDLIH 17 SpMFP2 EIRSKGGNAVANYDSVEDGDKLVQTALDNFGRIDIVINNAGILRDRSFARISDMDWDLVH 120.****: ** ***::: DrMFP2 RVHLRGSFLVTRAAWNHMKQQKFGRIIMTSSAAGIYGNFGQANYSAAKLGLLGLANTLAI 180//725 RnMFP2 RVHLRGSFQVTRAAWDHMKKQNYGRIIMTASASGIYGNFGQANYSAAKLGLLGLANTLVI 180 MgMFP2 RVHLRGSFQVTRAAWPHMKKNKYGRIIMVTSAAGIYGNFGQAK SpMFP2 RVHLRGSFMVTRAAWPHMKKQKFGRIIMTSSAAGLYGNFGQTNYSAAKLGLVGMSNTLSR 180 ******** ****** ***::::*****.:**:*:******:: Figure 1: (a) Alignment of multifunctional protein 1 (MFP1) amino acid sequences of Tetraodon nigroviridis (TnMFP1), Danio rerio (DrMFP1), Rattus norvegicus (RnMFP1) and deduced Chelon labrosus (ClMFP1) MFP1 amino acid sequence. (b) Alignment of multifunctional protein 2 (MFP2) sequences of Stronglylocentrotus purpuratus (SpMFP2), Danio rerio (DrMFP2), Rattus norvegicus (RnMFP2) and deduced MFP2 amino acid sequence of Mytilus galloprovincialis (Mg MFP2). Whole length of Danio rerio MFP1 and MFP2 amino acid sequences are indicated (718 and 725). different AOX1 sequences (Figure 4). In this way, both AOX1 sequences contained residues Gly101 and Trp123, considered markers of the Exon3I variant. Additionally, both showed a typical peroxisomal targeting sequence in the C- terminus, SKL in mullets and AKL in mussels. The conserved unique fatty acyl CoA oxidase sequence KWWPGG was found at residues in mullets while in mussels was slightly different (KYWPGC, residues ). The FAD binding motif CGGHGY is conserved in both species and localised at residues in mussels and in mullets. However, the proteolytic cleavage site between Val468 and Ala469 and the consensus sequence for a Ser/Thr dehydratase pyridoxal-phosphate attachment site (VVDINDLTSLVEVYKLRAAILVDL) were only present in mullet AOX1. Gene expression studies AOX1 and THIO were expressed in mantle, gills and digestive gland of mussels and in brain, 178 heart, spleen, muscle, gonad, gills and liver of mullets. Mullet liver and mussel digestive gland showed the the highest AOX1 and THIO expression levels. Similar AOX1 and THIO expression levels were measured in liver of mullets captured in winter and spring, although both genes showed slightly lower expression levels in spring than in winter (Figure 5). On the contrary, higher AOX1 and THIO expression levels were detected in the digestive gland of mussels collected in spring (Figure 5). On the other hand, 2 days after exposure to PFOS, AOX1 gene expression was significantly induced in the digestive gland of mussels (Figure 6) and 1 day after a single intraperitoneal B(a)P injection, MFP1 expression was significantly induced in the liver of mullets (Figure 6). Immuncytochemistry The tripeptide sequence SKL was exclusively localised in peroxisomes of mullet hepatocytes, as shown by gold labelling randomly distributed

130 Molecular characterisation of β-oxidation in aquatic organisms DrTHIOA AKRGSFKDTTPDELLSAVMSAVIKDVGLKPSLLGDVCVGNVLQPGAGALMARVAHFFSGF 104 ClTHIO VPXXLGXVCVGYVXHLEAGALVAXVAXFLSGF 32 RnTHIOA AGRGGFKDTTPDELLSAVLTAVLQDVKLKPECLGDISVGNVLEPGAGAVMARIAQFLSGI 120 RnTHIOB AGRGGFKDTTPDELLSAVLTAVLQDVKLKPECLGDISVGNVLQPGAGAAMARIAQFLSGI 110 SpTHIO DTRPDDLLAAAFKGVLDETKISPDRLGDIVVGNVLLPGGGAIHARFAQFYSGI 53 MgTHIO LEILLWTVGD-GRGAVXLRTGQFMAGI 26!! : : * **. * :*: DrTHIOA PESVPVYTVNRQCSSGLQALFNIAGGIRGGSYDLGLACGVESMSL-RSPNNPGDISPRLM 163 ClTHIO XEHVPVYTVNRQCSSGLQALFNIAGAIRSRSIDLGLACGVESMSL-RSVGNPGDLSSRLT 91 RnTHIOA PETVPLSAVNRQCSSGLQAVANIAGGIRNGSYDIGMACGVESMSL-SNRGNPGNISSRLL 179 RnTHIOB PETVPLSAVNRQCSSGLQAVANIAGGIRNGSYDIGMACGVESMTL-SERGNPGNISSRLL 169 SpTHIO PESVPINTVNRQCSSGLQAFMNAAGGIKSGVYDVAMASGVESMSR-SPMYQGEDLNPKVM 112 MgTHIO PDSVPSSAVNRQCSSGLQAVMNVAGSIRMGQCNMGIGAGVESMSFHRGENNSAPLNPKIF 86 : ** :***********. * **.*: ::.:..*****: : :..:: DrTHIOA DNEKARDCIIPMGITSENVAERFGITREKQDRFALSSQQKAAMAQKNGIFDQEITPVTTK 223 ClTHIO DNDKARDCIIPMGITSE RnTHIOA ESDKARDCLIPMGITSENVAERFGISRQKQDAFALASQQKAASAQSKGCFRAEIVPVTTT 239 RnTHIOB ENEKARDCLIPMGITSENVAERFGISRQKQDAFALASQQKAASAQSKGCFRAEIVPVTTT 229 SpTHIO LDQTTKDCLIPMGITSENVAEKFGVTRQQQDQMALESQLKAAAAQTKGYFDREIIPVTTV 172 MgTHIO EHEMARKCLIPMGITSENVAEKYGITREQQDNMALASQQKALKATKDGLFKDEIIPTTVT 146 : ::.*:********^^^^;;^;;^;;^^ ;^^ ^^ ^^ ^..^ ^ ^^ ^.^. DrTHIOA FVEENGTERTITVTKDDGIRPGTTLEGLAKLRPAFKDTGSTTAGNASQVSDGAAAVLIGR 283//418 ClTHIO RnTHIOA VLDDKGDRKTITVSQDEGVRPSTTMEGLAKLKPAFKDGGSTTAGNSSQVSDGAAAVLLAR 299 RnTHIOB VLDDKGDRKTITVSQDEGVRPSTTMEGLAKLKPAFKDGGSTTAGNSSQVSDGAAAVLLAR 289 SPTHIO IIDEAGQERPITVTKDDGVRGNTTIEGLAKLKPAFSPEGTTTAGNSSQVSDGAAAVLVAS 232 MgTHIO IKDQDXNERQITVTNR ;;.; ^^^;; Figure 2: Alignment of thiolase (THIO) A sequences of Stronglylocentrotus purpuratus (SpTHIO), Danio rerio (DrTHIOA), Rattus norvegicus (RnTHIOA), Rattus norvegicus THIOB (RnTHIOB) and deduced THIO amino acid sequence of Mytilus galloprovincialis (MgTHIO) and Chelon labrosus (ClTHIO). Same amino acids are indicated by *,! and ^ depending on the sequences that have been aligned. Total length of DrTHIOA has been indicated (418). throughout the peroxisomal matrix (Figure 7). Discussion In the present work, genes coding for AOX1, MFP (1 in mullets and 2 in mussels), THIO and DECR have been cloned and their expression studied after exposure to two different organic xenobiotics. In addition, putative peroxisome proliferation response elements in the promoter region of those genes as well as in genes coding all the enzymes necessary for both different paralog peroxisomal β-oxidation pathways described in teleosts (AOX1, AOX3, MFP1, MFP2, THIO and SCPX) have been identified by in silico studies. The enzyme that catalyses the first and ratelimiting step of the peroxisomal β-oxidation pathway, AOX1, is usually measured as marker of peroxisome proliferation (Mandard et al. 2004) since its activity is transcriptionally induced after exposure to some organic compounds. AOX1 in mullets contains 1983 bp coding for 661 amino acids and shows high amino acid identity (79%) with Danio rerio AOX1. In mussels AOX1 gene contains a coding domain sequence of 2010 nucleotides coding for 670 amino acids, 9 amino acids longer than in vertebrate species, as it is shown in Figure 4. Nothing had been published on the genes that code the peroxisomal β-oxidation enzymes in aquatic species until Morais et al. (2007) published their work on the cloning of AOX1 in D. rerio during the preparation of this manuscript. This first published piscine cdna shows a high degree of sequence homology with homologous mammalian genes as it has been also shown in the present study with mussel and mullet AOX1. Morais et al. (2007) reveal that 179

131 Results and Discussion ClDECR VQDHGGNIVNISATLGYRGQG 21 TnDECR AAGNFLCPASSLSFNAFKTVMEIDTMGTFNTSKVVYEKWFQNHGGNIVNISATLGYRGQG 179 DrDECR AAGNFLCPATSLSFNAFKTVMEIDTMGTFNTSKVIYDKWFKDHGGSIVNISATLGYRGQA 180 HsDECR AAGNFLCPAGALSFNAFKTVMDIDTSGTFNVSRVLYEKFFRDHGGVIVNITATLGNRGQA 161.::*** ****:**** ***. ClDECR LQVHAGSAKAANDAMTKHLAVEWGPSGVRVNTLAPGPVSGTEGYRRLGGPQGEAAGVFCS 81 TnDECR LQVHAGSAKAANDAMTRHLAVEWGPSGVRVNAMAPGPISGTEGFRRLGGTRGEAAGLFQS 239 DrDECR LQVHAGSAKAANDAMTRHLAVEWGPSGVRVNTVAPGPISGTEGYRRLGGSHAETAGAFHS 240 HsDECR LQVHAGSAKAAVDAMTRHLAVEWGPQNIRVNSLAPGPISGTEGLRRLGGPQASLSTKVTA 221 *********** ****:********..:***::****:***** *****.:.. :. : ClDECR IPLQRAGNXXEM TnDECR IPLQRAGNKTEMAHCALFLASRSSSYVTGATLVADGGSWLTSANDVSMLLGTASSKSAKL 299 DrDECR IPLQRAGNKTEMAHAVLFLASRASSYVTGSVLVADGGAWLTSANDVERLLGIVSSRSAKL 300 HsDECR SPLQRLGNKTEIAHSVLYLASPLASYVTGAVLVADGGAWLTFPNGVKGLPDFAS-FSAKL 280 **** ** *: Figure 3: 2, 4 dienoyl-coa reductase 2 (DECR) sequence alignment of Homo sapiens (hsdecr), Danio rerio (DrDECR), Tetraodon nigroviridis (TnDECR) and deduced amino acid sequence of Chelon labrosus (ClDECR). PTS1 sequence is shown in bold. through alternative splicing of the zebrafish AOX1 gene, 2 transcripts differing only in the 3 rd exon are produced. This characteristic has also been reported in mammalian AOX1, where both transcripts have been proved to show different substrate specificity (Setoyama et al. 1995). In fish, both transcripts show tissue specific expression differentially regulated after feeding (Morais et al. 2007). Indeed, an analysis of the available teleost genomes reveals that alternative 3 rd exon utilisation is a general feature among teleosts, as demonstrated by the Gasterosteus aculeatus predicted transcript sequences ENSGAC and in the Ensembl annotation project (Birney et al. 2006). Blast analysis of the EST sequences in the Genbank with the nucleotide sequences of the two alternative 3 rd exons of stickleback and zebrafish allows to find two different Takifugu rubripes AOX1 transcripts differing exclusively in the 160 nucleotides stretch matching the 3 rd gene exon (CA33313 and BU805260) proving that these alternative two transcripts are transcribed in vivo. Regarding aquatic invertebrates, the recently sequenced Strongylocentrotus purpuratus genome allows to predict an AOX1 sequence. Blast search of this genomic sequence only revealed one possible 3 rd exon. Accordingly, AOX1 in mussels and mullets contained Gly101 and Trp123 used as markers of Exon 3I (Morais et al. 2007) although existence of a possible Exon 3II can not be disregarded. When comparing mussel AOX1 sequence with known AOX1 sequences it is noted that it specially differs in the 5' and 3' ends and that shows a short deletion around nucleotide 470. Sequencing of AOX1 in both aquatic organisms revealed conservation of main AOX enzyme features. Among these similarities, the conserved unique fatty acyl CoA oxidase sequence KWWPGG (Hooks et al 1999; Ngo et al. 2003; Morais et al. 2007) is localised at residues in mussels and at residues in mullets. This sequence is slightly divergent in mussels (KKYPGC), although substitutions are conservative. The FAD binding motif CGGHGY localised in mullets and mussels, as well as the Glu421 catalytic active site, are completely conserved both in vertebrates and as it has been described by Morais et al. (2007), comparing vertebrates and arthropods. Another characteristic of AOX1 is the presence of a proteolytic cleavage site between Val468 and Ala469, where the A subunit (72 kda) may be cleaved into B and C subunits (51 and 21 kda, respectively) detected through western blotting in mammalians (Miyazawa et al. 1987; Nöhammer et al. 2000). However, in zebrafish only Val467 has been described while in C. labrosus and in Gasterosteus aculeatus both residues are conserved. Accordingly, Orbea et al. (1999) immunodetected the 3 subunits in mullet liver showing that AOX1 is cleaved as in rat liver. Differently, since a small fragment of 9 180

132 Molecular characterisation of β-oxidation in aquatic organisms ClAOX MNPDITKERENATXXVEKLTNILDGGPEKTKRRRQIESLVFSDPDFK-EEDPNFL 54 GaAOX MNPDITRERQNATFDVEKLTHILDGGYEKTKRRREIEAMVFSDPDFK-EEDPNFL 54 DrAOX MNPDISRERENASFNLEILTNVLDGGAEKTNRRREIESLVIGDPDFQ-HEDLNFL 54 HsAOX MNPDLRRERDSASFNPELLTHILDGSPEKTRRRREIENMILNDPDFQ-HEDLNFL 54 MgAOX1 MSQGNVTPDLARERSKATFDVENLAEFMIGGRDRLKRKRFLQNIAIQDPFFKKQKSWSFC 60 :.**: :**..*: * *:..: *. ::.*:* :: : : ** *:.:..* ClAOX1 SRSERYDQAVRKSAQMILKLREYGIADPEEIYYYKNMVKGNQQEAMGLHFAMFLPTLYSQ 114 GaAOX1 SRSERYDQAVKKSAQMILKLREYGIADPEEIYCYKNMVLGHHHEAMGLHFVMFLPTLYSQ 114 DrAOX1 SRSERYDAAVRKSAQMILKLREYGISDPEEIYSYKTVVRGVFQEPLGVHNVMFIPTLKSQ 114 HsAOX1 TRSQRYEVAVRKSAIMVKKMREFGIADPDEIMWFKKLHLVNFVEPVGLNYSMFIPTLLNQ 114 MgAOX1 SVEEQYDMGLSKSIYIQKKLQEMGITDMQEAFYYRECAAAHENSPLGLHYAMFLPTINKQ 120 :.::*:.: ** : *::* **:* :* ::..:*:: **:**:.* ClAOX1 CDPEQSKKWLPLAESFQAIGTYAQTELGHGTHLRGLETTATYDPATQEFVLNSPTVSSIK 174 GaAOX1 CDPQQARKWLPLAESLQAVGTYAQTEMGHGTHLRGLETTATYDPATQEFVLNSPTVSSIK 174 DrAOX1 CTAEQRKKWIPLAESFHMLGTYAQTELGHGTHIRALETTATYDPSTQEFVLNSSTISSIK 174 HsAOX1 GTTAEKEKWLLSSKGLQIIGTYAQTEMGHGTHLRGLETTATYDPETQEFILNSPTVTSIK 174 MgAOX1 ATPEQKKKWLPLAENYKMIGTYAQTELGHGTFIRGLETTATYDPKTKEFILDSPTLTSKK 180. :.**: ::. : :*******:****.:*.********* *:**:*:*.*::* * ClAOX1 WWPGGLGKTSNHAIVLAQLYSLGNCHGLHAFIVPIRDMNTHEPLPGIVVGDIGPKFGFNE 234 GaAOX1 WWPGGLGKTSNHAIVLAQLYTQGNCHGLHAFIVPIRDLDTHVPLPGIVVGDIGPKFGFSE 234 DrAOX1 WWPGGLGKTSNHAIVLAQLYTQGKCHGLHAFITPIRCMKTHMPLPGVVVGDIGPKFGFDE 234 HsAOX1 WWPGGLGKTSNHAIVLAQLITKGKCYGLHAFIVPIREIGTHKPLPGITVGDIGPKFGYDE 234 MgAOX1 YWPGCLGKTSNHCVVMAQLYTQGKSHGPHMFMVQIRSMXDHSVLPGIEVGVIGNKFGYGT 240 :*** *******.:*:*** : *:.:* * *:. ** : * ***: ** ** ***:. ClAOX1 VDNGFLKLENVRIPRENMLMKYAKVDPDGTYMKPPSAKLTYGTMVFIRSMIVGESARALV 294 GaAOX1 VDNGFLKLENVRIPRENMLMKFAKVEADGTYLKPPSTKLTYGTMVFIRSLLVAESARALS 294 DrAOX1 VDNGYLKLENVRIPRENMLMKYAQVEPDGTYVKPPSDKLTYGTMVFIRSMIVGESARALS 294 HsAOX1 IDNGYLKMDNHRIPRENMLMKYAQVKPDGTYVKPLSNKLTYGTMVFVRSFLVGEAARALS 294 MgAOX1 NDNGFLSFDKLRIPRENMLMRYSQVHEDGTYVKPQNAKLAYGSMVLIRSSIVSDAARGXA 300 ***:*.::: *********::::*. ****:**. **:**:**::** :*.::**. ClAOX1 KSCTIAIRYSAVRHQSEIRPGEPEPQILDYQTQQYKLFPLLATAYAFTFVGQYMQQTYQR 354 GaAOX1 KSCTIAIRYSSVRHQSEIRPGHPEPQILDYQTQQYKLLPLLAMAYAFTFVGQYMRQTYHR 354 DrAOX1 KSCTIAIRYSAVRHQSELRPGEPEPQILDYQTQQYKLFPLLATAYAFHFVGQYMNKTYHR 354 HsAOX1 KACTIAIRYSAVRHQSEMKPGEPEPQILDFQTQQYKLFPLLATAYAFQFVGAYMKETYHR 354 MgAOX1 QXCVISIRYSAVRXQTEAYPGGEEPQILDYQTQQYRLFPLLASAYSLQLAGKYVFNTYLE 360 : *.*:****:** *:* ** ******:*****:*:**** **:: :.* *: :**. ClAOX1 ITGDINQGDFSELPELHALSAGLKAFTTWETNSAIEVCRMSCGGHGYSRSSALPDIYVEF 414 GaAOX1 ISGDINEGDFSELPELHALSAGLKAFTTWATNSAIEVCRMSCGGHGYSRSSGLPDIYVEF 414 DrAOX1 ISGDISLGDFSELPELHALSAGLKAFTTWAANTGIEVCRMSCGGHGYSRCSSLPDIYVTF 414 HsAOX1 INEGIGQGDLSELPELHALTAGLKAFTSWTANTGIEACRMACGGHGYSHCSGLPNIYVNF 414 MgAOX1 VNDQIEKGNLESMPELHALSAGLKAFSTYEASAGIEVCRICCGGHGYSQASGLPKIYVDV 420 :. * *::..:******:******::: :.:.**.**:.*******:.*.**.***. ClAOX1 TPTCTYEGENTVMMLQTARYLVKSYRQAKAGQQLSGIVSYLNEAGDRRLQPQPVAARPTV 474 GaAOX1 TPTCTYEGENTVMMLQTARYLVKSYRQAQAGQQLSGIVSYLNDLKHQRVQPQPVAGRPTV 474 DrAOX1 TPTCTYEGENTVMMLQTARYLVKSYKQARAGQQLTGIVSYLNES-QSRIQPHSVSSRPTV 473 HsAOX1 TPSCTFEGENTVMMLQTARFLMKSYDQVHSGKLVCGMVSYLNDLPSQRIQPQQVAVWPTM 474 MgAOX1 VPGCTYEGENTVMMLKTARYLMKCYSRAKQGQKLPGFVQYIGQNLNKKS CM 471.* **:*********:***:*:*.* :.: *: : *:*.*:.: : : ClAOX1 VDINDLTSLVEVYKLRAAILVDLAAKSIQQELQRRKSQEDAWNNSAIDLVRASDAHCHYV 534 GaAOX1 VDINDLASLVEVYKQRAAILVELAAKSIQQELHHSKSQEDAWNNSAIDLVRASDAHCHYV 534 DrAOX1 VNINDLVSLVEAYKFRAAKLVEVAAKNLQLELQHSKSNEDAWNNTSIDLVRASDAHCHYV 533 HsAOX1 VDINSPESLTEAYKLRAARLVEIAAKNLQKEVIHRKSKEVAWNLTSVDLVRASEAHCHYV 534 MgAOX1 SEEVALGCLVMAYEHRAARLVEAAAMRMQSLIQGGKKQHEAWNGSSVQLFWAAKAHCHLF 531 :.*..*: *** **: ** :* : *.:. *** ::::*. *:.****. 181

133 Results and Discussion ClAOX1 VVKLFTDKLGEVG-DTAIHSVLSNLALLYALNGITKNSGDFLLAGLLSVPQVXQSSVRIK 593 GaAOX1 VVKLFTDKLGEVS-DTAIHSVMSTLALLYALHGITKNSGDFLQAGLLNVPQVLQISNRIK 593 DrAOX1 VVKLFAAKLSEIG-DKAVHSVLSTLALLYALHGVAQNSGDFLKAGLLSVSQLDQISQRLK 592 HsAOX1 VVKLFSEKLLKIQ-DKAIQAVLRSLCLLYSLYGISQNAGDFLQGSIMTEPQITQVNQRVK 593 MgAOX1 CVKNFVDAIQAKSLSLQTTTVMKSVCQLYXVHGILENLGEFMQDGFFSGKQVGYLQRKML 591 ** * :. :*:.:. ** : *: :* *:*:.::. *:. :: ClAOX1 ELLSKLRPNAVALVDAFDVHDKKLNSVLGRYDGNVYENMFEWARRSPLNKTQVHESYHKY 653 GaAOX1 QLLSQLRPNAVALVDAFDFHDNRLKSVLGRYDGNVYENLFEWARRSPLNSTEVHESFHKY 653 DrAOX1 GLLLEIRPNAVALVDAFDYRDEMLNSSLGRYDGNVYEHMFEWAKKSPLNHTEVHESHNKY 652 HsAOX1 ELLTLIRSDAVALVDAFDFQDVTLGSVLGRYDGNVYENLFEWAKNSPLNKAEVHESYHKH 653 MgAOX1 ALFEHIRPNAVALVDAFDYPDMVLGSCLGRYDGQVYQALYDYAKSAPMNQTEIHSTYYKH 651 *: :*.:********* * * * ******:**: ::::*: :*:* :::*.:. *: ClAOX1 LKPLR SKL 661 GaAOX1 LKPLR AKL 661 DrAOX1 LKPLR SKL 660 HsAOX1 LKSLQ SKL 661 MgAOX1 LKPLINPETTSDAMQFAKL 670 **.* :** Figure 4: Alignment of palmitoyl-coa oxidase (AOX1) sequences of Gasterosteus aculeatus (GaAOX1), Danio rerio (DrAOX1), Homo sapiens (HsAOX1) and deduced AOX1 amino acid sequences of Chelon labrosus (ClAOX1) and Mytilus galloprovincialis (MgAOX1) where conserved residues are showed as: G and W, Gly101 and Trp123, markers of the Exon 3I; KWWPGG, conserved fatty acyl-coa oxidase sequence; CGGHGY, FAD binding domain; E, Glu421 catalytic active site; VA,Val468 and Ala469 proteolytic cleavage site; VDINDLTSLVEVYKLRAAILE, consensus sequence for a Ser/Thr dehydratase pyridoxal-phosphate attachment site; S (A)KL, typical peroxisomal targeting signals 1 (PTS1). residues is depleted in the same region of mussel AOX1, the proteolytic cleavage site is absent. Accordingly, Cancio et al (2000) described only two immnoreactive polypeptides of around 72 and 43 kda in the digestive gland of mussels and suggested that a different cleavage may occur in mussel AOX1. Interestingly, the consensus sequence for a Ser/Thr dehydratase pyridoxal-phosphate attachment site found in mammals (Nöhammer (a) ,5 1 0 (b) 0 W S W S Figure 5: Mean expression values of AOX1 measured in mullet liver (a) and mussel digestive gland (b) collected in W = winter and S = spring. AOX1 expression was normalised against 18S rrna. Vertical segments show standard deviation. No statistical differences were detected by U-test (p<0.05). et al. 2000) and zebrafish (Morais et al. 2007) was found in mullets but not in mussel AOX1. Finally, both mussel and mullet AOX1 sequences contain a potential peroxisomal targeting signal in their C-terminus. The majority of peroxisomal matrix proteins contain a typical peroxisomal targeting signal 1 (PTS1) for their import from the cytosol to peroxisomes (Nöhammer et al. 2000; Emanuelsson et al. 2003) something that has been observed to be conserved from yeast to mammals (Van der Klei and Veenhuis 2006), PTS1 consists of a tripeptide located at the C-terminus characterised by the sequence SKL or conservative variants (S/A/C)(K/R/H)(L/M). In the present work sequencing of the AOX1 ORF revealed the typical peroxisomal targeting signals SKL in the C-terminus of mullet AOX1 and AKL in mussels. Furthermore, the presence of SKL has been determined immunocytochemically in mullet liver peroxisomes as previously reviewed by Cajaraville et al. (2003). Regarding the different AOX genes, AOX2 has only been found in mammalian, avian and amphibian species (Morais et al. 2007) although

134 Molecular characterisation of β-oxidation in aquatic organisms AOX1 Mussel MFP1 Mullet Figure 6: Expression of palmitoyl-coa oxidase 1 (AOX1) in the digestive gland of 5 mussels exposed to PFOS for 2 days (mean value = 1.93 ± 0.4) and of multifunctional protein 1 (MFP1) measured in liver of 3 mullets after 1 day of a single intraperitoneal injection with 5 ppm of (B(a)P) in corn oil (mean value = 2.22 ± 0.2). Cn indicates control organisms. Expression was normalised against β-actin in the case of mussels (mean value = 1.26 ± 0.3) and against 18S rrna in the case of mullets (mean value = 1.5 ± 0.4). one of the Ciona intestinalis predicted AOX homologs shows highest structural identity with this isoform. Clearly, teleost fish seem to be an exception among vertebrates since their genomes do not show AOX2 related genes (Figure 8). On the other hand, AOX3 has been found in all the vertebrate genomes analysed to date. We have been able to find AOX3 sequences in the five teleost fish genomes available in Ensembl, and sequence homologs can also be found in the genomes of urochordates (C. intestinalis) and ecdysozoa (insects and nematodes), where all the AOX-like sequences, present in many copies, are most similar to AOX3. Additionally, a possible AOX3 ortholog sequence has been predicted in the S. purpuratus genome. The Aplysia genome project that has studied the neuronal transcriptome of this opistobranch mollusc (Moroz et al. 2006) has resulted in the cloning of an EST with high deduced amino acid identity with known AOX3 sequences ( The 2E-enoyl-CoAs generated by peroxisomal AOX, are hydrated to either 3S- or 3R-hydroxyacyl-CoAs, also referred to as L-3- and D-3hydroxyacyl-CoA, which are subsequently dehydrogenated to 3-ketoacyl- CoAs. These reactions are catalysed in a substrate specific way by MFP1 and MFP2. A fragment of 213 bp of MFP1 have been cloned from mullet liver which are aligned with the C domain of rat MFP1 (Figure 1). Five functional domains (A, B, C, D and E) have been described in mammalian MFP1, C domain and D domain constituting the (3S)-dehydrogenase region (Kiema et al. 2002). MFP2 plays a central role in peroxisomal β-oxidation as it handles most peroxisomal β-oxidation substrates (Huyghe et al. 2006), those derived from AOX2 and AOX3 oxidation steps and the enoyl-coas of very long chain fatty acids oxidised by AOX1. Mammalian MFP2 can be subdivided into three domains: the N-terminal domain similar to the short-chain alcohol dehydrogenase which displays 3Rhydroxyacyl-CoA dehydrogenase activity, a central domain displaying 2-enoyl-CoA hydratase activity and a C-terminal domain that is similar to sterol carrier protein 2 (Huyghe et al. 2006). The fragment of 182 bp amplified in mussels is aligned with the N-terminal domain (Figure 1). Although we were not able to clone neither MFP1 in mussels nor MFP2 in mullets, an analysis of gene and genome databases allows detecting MFP2 genes in teleost fish (Table 2). In the same way, in the Aplysia neuronal transcriptome two ESTs can be found, one similar to described MFP2 genes, as the mussel sequence reported hereby, an another one similar to MFP1 (see additional information in annex 1). This allows to predict that in other molluscs, such as bivalves, an MFP1 ortholog could be present as it is also clearly suggested by western blot studies carried out using a polyclonal antibody against rat MFP1 (Cancio et al. 1998). Our laboratory will continue trying to clone this gene in the future in order to complete the inducible peroxisomal pathway in mussels. The third step in the peroxisomal β-oxidation 183

135 Results and Discussion Figure 7: Electron micrographs of mullet (Chelon labrosus) liver. Ultrathin sections were fixed with glutaraldehyde and formaldehyde, Lowicryl-embedded and incubated with anti-skl antibody and protein-a-gold. Colloidal gold particles (10 nm) show the presence of the peroxisomal targeting sequence SKL in peroxisomal matrix proteins. P = peroxisome, M = mitochondria, L = lysosome. Scale bars = 3.5 µm. pathway is catalysed by two different enzymes in mammals, birds and amphibians, THIO and SCPx, which in the case of mice and rats becomes 3 enzymes with the appearance of two different THIO genes: thiolase A and thiolase B (Hijikata et al. 1990; Bodnar and Rachubinski 1990; Chevillard et al. 2004). Both rodent enzymes have the same specificities regarding ketoacyl-coa substrates (Antonenkov et al. 1999; Chevillard et al. 2004), but they are differently regulated: thiolase A is constitutively expressed whereas the expression of thiolase B is induced by peroxisome proliferators (Hijikata et al. 1990; Antonenkov et al. 2000). The main sequence difference between thiolase A and thiolase B has been described in the early portion of exon I in both genes (Osumi 1993). Fish seem to show the same enzymatic content as humans or chicken, and thus THIO and SCPx orthologs have been found in the 5 teleost genomes available in Ensembl (Table 3). The same applies to nematodes, insects, ascidians, echinoderms and also molluscs, as observed with the mussel THIO presented here and the putative THIO and SCPx sequences found in the Aplysia neuronal transcriptome (Moroz et al. 2006) (see additional information in annex 1). Since reductases in combination with isomerases appear to play an important role in the oxidation of virtually all unsaturated fatty acids (Kunau et al. 1995) which are so important in marine organisms, 283 bp of mullet peroxisomal DECR were cloned. This sequence showed 86% amino acid identity with Tetraodon nigroviridis DECR and it is located between nucleotides (Figure 3). Induction on DECR expression has been reported in rodent liver after exposure to different peroxisome proliferators such as clofibrate (Lei et al. 2003) and it was cloned in a Platichtys flesus liver induced cdna library after exposure to a cocktail of pollutants (Williams et al. 2006). In invertebrates a putative DECR S. purpuratus (CD305935) EST sequence was identified. Additionally, DECR was found to be up-regulated by subtractive hybridisation in Crassostrea gigas (CB617391) after exposure to hydrocarbons (Boutet et al. 2004). The transcriptional inducibility of different genes in the presence of peroxisome proliferators has been reported to be linked to the presence of peroxisome proliferator responsive elements in their promoter regions (Mandard et al. 2004). There is no information related to the presence of PPREs in peroxisomal β-oxidation genes outside mammals, birds or amphibians. Regarding fish, in the last years it has become clear that the promoter regions of aromatase genes among other regulatory elements possess PPREs. In this sense, CYP19A1 of Dicentrarchus labrax (Galay-Burgos et al. 2006), Lates calcarifer and Cromopolites altivelis (Gardner et al. 2005), and CYP19A2 of Danio rerio (Kazeto et al. 2001), L. calcarifer, Gobiodon histrio (Gardner et al. 2005) and Mugil cephalus (Nocillado et al. 2007) have been reported to show putative PPAR/RXR- 184

136 Molecular characterisation of β-oxidation in aquatic organisms AOX1 S. purpuratus AOX M. galloprovincialis AOX1 D. rerio AOX1 C. labrosus AOX1 G. aculeatus AOX1 X. tropicalis Palmitoyl-CoA oxidase AOX1 AOX1 G. gallus AOX1 R. norvegicus AOX1a H. sapiens AOXCG9527-PA D. melanogaster AOXCG17544-PA D. melanogaster AOX3 X. tropicalis AOX3 G. aculeatus AOX3 D. rerio AOX3 G. gallus Pristanoyl-CoA oxidase AOX3 AOX3 R. norvegicus AOX3 H. sapiens AOX3 S. purpuratus Euprymna scolopes EST AOX2 G. gallus AOX2 X. tropicalis AOX2 R. norvegicus Trihydroxycoprostanoyl-CoA oxidase AOX2 AOX2 H. sapiens AOXCG5009-PA D. melanogaster AOX57D-d D. melanogaster AOX57D-p-RA D. melanogaster AOX C. elegans F08A8 Ecdysozoa specific acyl-coa oxidases AOX C. elegans F25C8 0.1 AOX C. elegans F59F4 Figure 8: Phylogenetic tree was produced by ClustalX and TreeView using available amino acid sequences (or amino acid sequences deduced from existing nucleotide sequences) corresponding to the three acyl-coa oxidases, palmitoyl- CoA oxidase (AOX1), trihydroxicopristanoyl-coa oxidase (AOX2) and pristanoyl-coa oxidase (AOX3). The scale bar indicates the distance corresponding to 10% amino acid differences. Fish and molluscan sequences are marked in bold. Notice that E. scolopes sequence represented in the tree is only a partial sequence covering 19% of the whole ORF, including the most 3 end of the AOX3 sequence with its SKL peroxisomal targeting sequence and showing 55% sequence homology with D. rerio AOX3 (e value 6 e -35 ). 185

137 Results and Discussion Table 2. Scores found searching for PPARα/RXR-binding motifs in the 3000 nucleotides immediately upstream of the start codon of all the peroxisomal β-oxidation and CYP4 genes localised in piscine genomes (Ensembl). The Ensembl codes for each gene are given in bold letters and numbers and scores above 69 have been considered as putative PPREs (in some promoter regions more than one putative PPRE has been localised). N.a. = no promoter region sequence available; 0 = no putative PPRE present. GENE D. rerio ENSDARG AOX , 72 AOX MFP MFP THIO , 72 SCPX DECR CYP , 70 G. aculeatus ENSGACG , , O. latipes ENSORLG , , N.a , , 70, , 70 T. rubripes NEWSINFRUG , binding sites. Three PPREs have also been reported in the promoter region of Pleuronectes platessa GSTA1 gene (Leaver et al. 1997), two of which have been shown to bind piscine PPARs heterodimerised with RXR in electrophoretic mobility shift assays (Boukouvala et al. 2004). These two PPREs when searched for in the Transfac database using the Motif search software ( show 79 and 71% sequence identity with the consensus CWRAWCT AGGNCA A AGGTCA PPARα/RXR-binding motif. Similar levels of homology where checked in the 3000 nucleotides immediately upstream of the initiation codons of the β-oxidation genes that were identified in the D. rerio, G. aculeatus, Oryzias latipes and T. rubripes genomes available in Ensembl. Wintz et al. (2006) made a similar study with the 1500 nucleotides upstream of different apolipoproteins and of fatty-acidbinding protein in the Ensembl D. rerio genome browser, failing to find any PPREs in these genes. In the present study, the analysis was carried out with the 3000 nucleotides immediately upstream of initiation codons, as for instance the PPRE of the highly regulated rat MFP1 gene has been detected at position from the transcription start site (Zhang et al. 1992). We also checked the CYP4 family related genes in these fish, as CYP4A6 shows typical PPREs in rodents where its transcription is highly up-regulated under exposure to peroxisome proliferators (Boukouvala et al. 2004). In this sense, putative PPREs were found in the promoter regions of many of the genes studied (Table 2) and although this is not a clear proof of the inducibility of these genes under regulation of piscine PPARα, it clearly suggests that this possibility exists. In order to study the expression and regulation capacity of genes coding for peroxisomal β-oxidation, expression of AOX1 and THIO was measured in mussels and mullet tissues. As it was expected, AOX1 and THIO were mainly expressed in liver of mullets and digestive gland of mussels (Bilbao et al. 2006b) as it occurs in mammals (Nemali et al. 1988). In liver, similar expression levels were measured in mullets captured in winter or spring, contrary to seasonal related peroxisomal size changes reported in female Salmo salar (Rocha et al. 1999) and mullet (Orbea et al. 1999). On the other hand, in the present study, higher expression levels of genes coding for AOX1 and 186

138 Molecular characterisation of β-oxidation in aquatic organisms THIO were measured in spring than in winter in mussels. Accordingly, Cancio et al. (1999) detected the annual highest AOX1 activities in digestive glands of mussels collected in April and May. Therefore, these results in mussels could be related to an increase in β-oxidation substrates, such as eicosanoids (prostaglandins and leukotrienes) which show a marked seasonal variability linked to the reproductive cycle and food availability (Cancio et al. 1999). Since peroxisomal volume, surface and numerical density, peroxisomal enzyme activities and the transcription of genes coding for peroxisomal enzymes of mussels are increased in spring, it may be suggested that the molecular mechanism necessary to elicit peroxisome proliferation is present in mussels. In order to assess whether the peroxisomal β- oxidation pathway is transcriptionally regulated after exposure to peroxisome proliferator compounds we carried out a preliminary study semiquantifying the expression of AOX1 in mussels exposed to PFOS and of MFP1 in fish injected with B(a)P. PFOS is a persistent, bioaccumulative fluorinated compound whose biological effects have mainly been studied in rodents where it has been described to be an important inducer of peroxisomal β-oxidation (Ikeda et al. 1987; Sohlenius et al. 1993; Guruge et al. 2006). The action of this compound could be explained by the structural similarity between PFOS and endogenous fatty acids (Hu et al. 2005). Fluorinated compounds have demonstrated to date to cause a similar response in aquatic species. Exposure to very high perfluorooctanoic acid doses provokes significant increases in AOX1 activity in Pimephales promelas (Oakes et al. 2004) and accordingly a slight but significant AOX1 overexpression has been observed in mussels exposed to PFOS in the present study. Additionally, 5 ppm of B(a)P provoked a significant induction of MFP1 expression in mullets. As it has been previously reported, the expression of MFP1 is strongly induced by peroxisome proliferators and other PPARα biological ligands (Reddy and Hashimoto, 2001). Therefore, induction of the MFP activity has been observed for instance in Oryzias latipes after exposure to gemfibrozil (Scarano et al. 1994). In Onchorynchus mykiss a relatively mild induction of MFP has also been reported after exposure to ciprofibrate (Yang et al. 1990). Thus, both experiments demonstrate that β-oxidation genes can be transcriptionally induced by peroxisome proliferators in the digestive gland of mussels and in the liver of mullets, and further experiments are being conducted to evaluate the extent of the response of the peroxisomal β- oxidation genes to the exposure to different organic compounds. In conclusion, sequencing and expression of genes coding for enzymes participating in the peroxisomal β-oxidation pathway in mussels and mullets, as well as in silico studies of the piscine available promoter regions, allow a deeper understanding of this pathway, so important for lipid homeostasis, and its regulation in aquatic organisms. Further gene expression and morphological studies will reveal the usefulness of studying peroxisomal alterations in aquatic organisms as a biomarker of exposure to organic chemicals. Acknowledgements This work was supported by the Spanish MEC (BIOMTOOLS, REN: /MAR, PRESTEPSE, VEM C06), Basque Government (ETORTEK-IMPRES) and University of the Basque Country (predoctoral grant to E. Bilbao and grant for consolidated research groups). References Antonenkov VD, Croes K, Waelkens E, Van Veldhoven PP, Mannaerts GP (2000). "Identification, purification and characterization of an acetoacyl-coa thiolase from rat liver peroxisomes". European Journal of Biochemistry 267, Antonenkov VD, Van Veldhoven PP, Waelkens E, Mannaerts GP (1999). "Comparison of the stability and substrate specificity of purified peroxisomal 3-oxoacyl- CoA thiolases A and B from rat liver". Biochimica et Biophysica Acta 1437, Baes M, Huyghe S, Carmeliet P, Declercq PE, Collen D, Mannaerts GP, Van Veldhoven PP (2000). "Inactivation 187

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144 Molecular characterisation of β-oxidation in aquatic organisms ANNEX 1 193

145

146 PEROXISOMAL MULTIFUNCTIONAL PROTEINS MFP2 H. sapiens MFP2 R. norvegicus MFPb C. intestinalis MFP2 G. gallus MFP1 G. gallus MFP1 R. norvegicus MFP1 H. sapiens MFP1 D. rerio MFP1 G. aculeatus MFP1 D. melanogaster 0.1 MFP2 D. rerio MFP2 G. aculeatus MFP2 C. intestinalis MFP2 A. californica MFP2 D. melanogaster MFP2b C. elegans MFP2 C. elegans Phylogenetic tree was produced by ClustalX and TreeView using available amino acid sequences (or amino acid sequences deduced from existing nucleotide sequences) corresponding to multifunctional protein 1 (MFP1) and multifunctional protein 2 (MFP2). The scale bar indicates the distance corresponding to 10% amino acid differences. Fish and molluscan sequences are marked in bold. Molluscan sequences are partial. Molecular characterisation of β-oxidation in aquatic organisms

147 196 PT S. purpuratus PEROXISOMAL THIOLASES Scpx G. gallus Scpx C. ferrari Results and Discussion PT M. californianus PT C. intestinalis Scpx C. elegans PT C. elegans PT2 D. melanogaster Scpx C. intestinalis PTb C. intestinalis Scp S. purpuratus PT D. melanogaster PT G. gallus Scpx D. melanogaster PT H. sapiens PTa R. norvegicus PTb R. norvegicus Scpx G. aculeatus Scpx D. rerio 0.1 PT D. rerio PT G. aculeatus Scpx X. tropicalis Scpx H. sapiens Scpx R. norvegicus 0.1 Phylogenetic tree was produced by ClustalX and TreeView using available amino acid sequences (or amino acid sequences deduced from existing nucleotide sequences) corresponding to 3-ketoacyl-CoA thiolase (THIO) and sterol carrier protein X (SCPX). The scale bar indicates the distance corresponding to 10% amino acid differences. Fish and molluscan sequences are marked in bold. Moluuscan sequences are partial.

148 β-oxidazioaren molekula mailako karakterizazioa itsas organismoetan 2.- Peroxisometako b-oxidazioaren molekula mailako karakterizazioa itsas organismoetan: palmitoil-coa oxidasa, proteina multifuntzionala eta 3-ketoazil-CoA tiolasaren klonazioa eta espresio-patroia Atal honetako emaitzak argitaratzeko bidali dira: BILBAO E, CAJARAVILLE MP, CANCIO I. "Molecular characterisation of the peroxisomal β-oxidation pathway in aquatic organisms: cloning and expression pattern of palmitoyl-coa oxidase, multifunctional protein and 3-ketoacyl- CoA thiolase". Experimental Cell Research. Atal honetako emaitzak kongresuetan aurkeztu dira: 14 th International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14), Florianópolis, Brazil, Maiatzak 6-9, BILBAO E, CAJARAVILLE MP, CANCIO I. "Cloning and regulation of the peroxisomal β- oxidation genes (acyl-coa oxidase, multifunctional protein, thiolase) in thicklip grey mullets and mussels after treatment with organic xenobiotics". 17 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, Porto, Portugal, Maiatzak 20-24, BILBAO E, CAJARAVILLE MP, CANCIO I. "A field study of changes in peroxisomal gene expression in mussel Mytilus galloprovincialis". 197

149

150 LABURPENA β-oxidazioaren molekula mailako karakterizazioa itsas organismoetan Peroxisomen proliferazioa, kutsadura organikoaren aurrean proposatutako esposizio-biomarkatzailea da muskuilu zein arrainetan, kutsatzaile organiko toxikoen aurrean duen erantzuteko gaitasuna dela eta. Muskuilua kutsaduraren zentinela gisa erabiltzen da munduan zehar, eta lazuna, Chelon labrosus espeziea esaterako, kutsadura altua dagoeneko guneetan bizitzeko duen gaitasunari lotuta, espezie zentinela egoki legez proposatu da. Peroxisometako β-oxidazioa molekularki karakterizatzeko helburuarekin, bidezidor horretan parte hartzen duten 3 entzimak kodetzen dituzten geneak klonatu ziren, palmitoil-coa oxidasa (AOX1), proteina multifuntzionala (MFP1) eta 3-ketoazil-CoA tiolasa (THIO). Gainera, gantz-azido polisaturatugabeen oxidaziorako beharrezkoa den 2, 4 dienoil-coa erreduktasa 2 peroxisomikoa kodetzen duen genearen zati bat ere klonatu zen lazunean. AOX1 kodetzen duen genearen open reading frame osoaren klonazioak muskuilu eta lazunean, ezagutzen diren espezieen AOX1 genearen sekuentziekin homologia altua erakutsi zuen, FAD lotzeko motiboa edo peroxisometarako itu-seinale tipikoa den PTS1 bezalako domeinu garrantzitsuak oso kontserbatuta egonik. Lazunaren peroxisometan PTS1dun proteinen presentzia mikroskopia immunoelektronikoz ere konfirmatu zen. Datu-baseetako gene eta genomen gainean burututako in silico azterketak burutu ziren. Bertan, metazooen peroxisometako β-oxidazioan deskribatutako 3 bidezidor paralogoetako 2-tan beharrezkoak diren entzima guztiak kodetzen dituzten geneen homologoak (AOX1, AOX3, MFP1, MFP2, THIO, sterol carrier protein X) identifikatzea lortu zen arrain eta moluskuetan. Horrela, ematen du, arrain eta moluskuetan, ugaztun, hegazti eta anfibioetan deskribatu den eta gantz-azido adarkatuak oxidatzeko beharrezkoa den AOX2 entzima bakarrik falta dela. Arrain eta muskuiluak peroxisomen proliferatzaile biren pean, perkluorooktano sulfonatoa eta benzo(a)pirenoa, mantendu ondoren, AOX1 eta MFP1en gainespresioa neurtu zen. Ensembl datubaseetan eskuragarri dauden arrainen gene eta genomen promotoreetan in silico azterketak burutzeak bestalde, peroxisomen proliferazioa zuzentzen duten mekanismo molekular eta zelularrak azalduko lituzketen peroxisomen proliferatzaileen erantzun elementuak identifikatzea baimendu zuen arrainetan. Gainera, geneen espresio mailako azterketa zein azterketa morfologiko sakonek, kutsatzaile organikoen aurrean aldaketa peroxisomikoek organismo itsastarretan esposiziobiomarkatzaile legez izan dezaketen erabilgarritasuna argituko dute. 199 L A B U R P E N A

151

152 β-oxidazioaren molekula mailako karakterizazioa itsas organismoetan Emaitzak gibelean (EF407559). Deduzitutako amino azido sekuentziak %88ko amino azido identitatea AOX1, MFP, THIO eta DECR-ren azaldu zuen Tetraodon nigroviridisen DECRrekin klonazioa (CAG04352) (3. Irudia). AOX1 lazunetan anplifikatzeko erabilitako hasle bikote guztiekin luzera ezberdinetako bandak lortu baziren ere, Fw2-Rv5 izan zen anplikon luzeenak eman zituena. Fw2-Rv5 hasleen bidez 2000, 1500 eta 600 bp ingurutako 3 banda lortu ziren. PCR produktuak klonatu eta BamHI eta NotI errestrikzio-entzimekin moztu ondoren, esperotako anplikonaren tamainarekin bat zetorren 1450 bp ingurutako banda bat lortu zen. Banda honen sekuentziak, Gallus gallusen AOX1en sekuentziarekin %86ko amino azido identitatea zuen sekuentzia eman zuen (E balioa 0.0). Horrela, hasleen sekuentziak ezabatu ondoren, 1408 bp-tako AOX1 zatia lortu zen lazunean. Muskuiluan, Fw1-Rv1, Fw1-Rv2, Fw2-Rv1 eta Fw2-Rv2 hasle-bikoteekin 870, 790, 820, eta 740 bp ingurutako bandak lortu ziren, hurrenez hurren. 845 bp-tako zati bat sekuentziatu zen azkenean, eta horrek %64ko amino azido identitatea azaldu zuen G. gallusen AOX1ekin (E balioa 2e -103 ). MFP anplifikatzeko diseinatutako hasle anderatuek bp ingurutako zatiak anplifikatu zituzten espezie bietan. Horrela, sekuentzia nukleotidikoak erakutsitako homologiaren bidez, 213 bp, MFP1 gisa identifikatu ziren C. labrosusen (EF407560), eta 182 bp, MFP2 gisa identifikatu ziren M. galloprovincialis muskuiluan (EF525543). Lazunaren MFP1ek %66ko amino azido identitatea azaldu zuen Tetraodon nigroviridisen MFP1ekin (CAG09300) eta muskuiluaren MFP2k Strongylocentrotus purpuratusen MFP2rekin %86ko amino azido identitatea erakutsi zuen (XP ) (1 Irudia). Metodologia bera erabiliz, Danio rerioren THIOrekin (AAH72706) %78ko amino azido identitatea agertu zuten 327 bp (DQ021958) anplifikatu ziren lazunetan. Hasle espezifikoak erabiliz berriz, muskuiluaren THIOren 498 bp anplifikatu ziren. Muskuiluaren THIOk %56ko amino azido identitatea azaldu zuen G. gallusen THIOrekin (XP ) (2. Irudia). Azkenik, DECR-ren 283 bp anplifikatu ziren lazunaren AOX1en RACE-PCR 3 RACE-PCR bidez produktu luzeak anplifikatzea lortu zen espezie bietan. Lazunetan, produktuon anplifikazioak, AOX1en 3' muturreko azken 32 amino azidoei zegozkien 97 bp-tako sekuentzia nukleotidiko berria eman zuen. Horrela, AOX1en open reading frame (ORF) osoaren 1505 bp eskuratu ziren C. labrosusen (sekuentzia osoaren %76a). Muskuiluetan, 257 amino azido kodetzen dituen 772 bp-tako AOX1 zatia lortu zen. Guztira, 3' RACE-PCR erabiliz, M. galloprovincialisen 1616 bp lortu ziren. Bestalde, 5 RACE-PCR-ren bidez, bp ingurutako bandak anplifikatu ziren. Muskuiluetan, AOX1en 131 amino azido kodetzen dituen 394 nukleotido berri lortu ziren. Antzera, lazunaren AOX1 sekuentziaren 478 nukleotido berri lortu ziren, lazunaren AOX1en ORF osoa eskuratuz. Hortaz, muskuiluaren AOX1en ORF osoak 2010 nukleotido ditu (EF525542), eta honek, %51ko amino azido identitatea erakutsi zuen Xenopus tropicalisen AOX1ekin. Lazunetan berriz, AOX1ek 661 amino azido kodetzen dituen 1983 bp ditu (EF525541). Lazunaren AOX1ek D. rerioren AOX1ekin %79 amino azido identitatea agertu zuen. Muskuiluaren AOX1 sekuentzia zenbait gunetan ezagunak diren AOX1 sekuentzien ezberdina bazen ere, AOX1 sekuentzia ezberdinen lerrokatzeak kontserbaturiko gune nabariak azaldu zituen (4. Irudia). Modu honetan, organismo bien AOX1 sekuentziek Gly101 eta Trp123 erresiduoak dituzte, Exon 3I bariantearen markatzaile kontsideratzen direnak. Gainera, biek agertzen dute C-terminusean peroxisometan sartzeko itu-sekuentzia (PTS1), SKL lazunetan eta AKL muskuiluetan. Kontserbatutako gantzen azil-coa oxidasaren KWWPGG sekuentzia kontserbatua, erresiduoetan kokatzen da lazunetan, eta muskuiluetan apur bat ezberdina da (KYPGC, n kokatuta). FAD lotzeko CGGHGY motiboa espezie bietan kontserbatu egiten da eta erresiduetan dago kokatuta arrainetan, 201 E M A I T Z A K

153 Emaitzak eta Eztabaida muskuiluetan n dagoen bitartean. Hala ere, Val468 eta Ala469 guneen artean kokatzen den gune proteolitikoa eta Ser/Thr dehidratasa piridoxal-fosfatoaren lotura-gunea (VVDINDLTSLVEVYKLRAAILVDL), lazunen AOX1 sekuentzian bakarrik ageri dira. Geneen espresioa AOX1 eta THIO muskuiluaren liseri-guruin, mantu eta zakatzetan espresatu ziren, baita arrainen garun, bihotz, bare, muskulu, gonada, zakatz eta gibelean ere. Denetatik, lazunen gibelak eta muskuiluen liseri-guruinak agertu zuten espresio mailarik altuena, hortaz, etorkizunean egin beharreko espresio mailako azterketak bi ehun hauek erabiliz burutuko ziren nagusiki. Neguan eta udaberrian arrantzatutako lazunen gibelean, antzeko espresio mailak neurtu ziren, nahiz eta gene biek espresio maila baxuagoa erakutsi zuten udaberrian (5. Irudia). Kontrara, AOX1 eta THIO maila altuagoak neurtu ziren udaberrian jasotako muskuiluetan (5. Irudia). Bestalde, 2 egunez PFOS pean mantendutako muskuiluek kontrolek baino AOX1 espresio esangarriki altuagoa erakutsi zuten liseri-guruinean (6. Irudia). Lazunen gibelean berriz, AOX1 espresio maila gainespresatu egin zen arrainei intraperitonealki B(a)P ziztatu ondoren (6. Irudia). Immunozitokimika SKL tripeptido-sekuentzia lazunen hepatozitoetako peroxisometan lokalizatu zen, ausaz banatutako urre partikulek erakutsi zuten moduan. 203

154 Ondorioak β-oxidazioaren molekula mailako karakterizazioa itsas organismoetan 1.-Peroxisometako β-oxidazioko 3 urrats nagusietan beharrezkoak diren entzimak kodetzen dituzten geneak klonatu dira, palmitoil-coa oxidasa (AOX1) eta 3-ketoazil-CoA tiolasa (THIO) M. galloprovincialis muskuilu eta C. labrosus lazunean, proteina multifuntzionala 1 (MFP1) eta 2, 4 dienoil-coa erreduktasa 2 (DECR) lazunean eta proteina multifuntzionala 2 (MFP2) muskuiluan. 2.-Transkripzionalki induzigarria den AOX1 genearen open reading frame osoa klonatu da M. galloprovincialis muskuilu eta C. labrosus lazunean. Sekuentziek homologia altua agertu dute kontserbatutako guneetan, horien artean, FAD lotzeko motiboa, gantzen azil-coa oxidasen sekuentzia eta C-muturreko peroxisometarako itu-sekuentzia. Gainera, PTS1 proteinak gibeleko hepatozitoetan kokatu dira immunozitokimika bidez. 3.-In silico analisien bidez ugaztun, hegazti eta narrastien peroxisometako β-oxidazioko 3 bidezidor paralogoetako bitan diharduten entzima guztien sekuentzia homologoak aurkitu dira arrain eta moluskuetan, gantz-azido adarkatuen oxidaziorako beharrezkoa den AOX2 salbu. 4.-Datu baseetan eskuragarri dauden arrainen sekuentzien promotoreetan burututako in silico azterketen bidez, konposatu kimiko organikoen pean mantendutako arrainetan peroxisomen proliferazioa eragiten duten mekanismo molekularrak azal ditzaketen peroxisomen proliferatzailekiko erantzun elementuak identifikatu dira. 5.-Ohiko peroxisomen proliferatzaileei erantzuten dieten espezieetan bezala, peroxisomen proliferazioa eragiten duten konposatu organikoen pean AOX1 eta MFP1 transkripzionalki erregulatzen direla frogatu da muskuilu eta lazunean; hori dela eta, RT-PCR tresna egokia izan daiteke peroxisomen proliferazioa aztertzeko itsas organismoetan, kutsaduraren eragina ikertzeko biojarraipen programen barruan O N D O R I O A K

155 3.- Differential expression of genes involved in peroxisome proliferation in thicklip grey mullets Chelon labrosus injected with benzo(a)pyrene This chapter has been sent for publication to: BILBAO E, CAJARAVILLE MP, CANCIO I (submitted). Differential expression of genes involved in peroxisome proliferation in thicklip grey mullets Chelon labrosus injected with benzo(a)pyrene. Toxicology and Applied Pharmacology. Parts of this chapter have been presented at: 6 th Iberian and 3 rd Iberoamerican Congress of Environmental Contamination and Toxicology (CICTA), Cadiz, Spain. September 25-28, BILBAO E, CAJARAVILLE MP, CANCIO I. "Cloning of peroxisomal and phase I xenobiotic metabolism genes in the thicklip grey mullet Chelon labrosus: Induction of expression after exposure to benzo(a)pyrene". 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, The Hague, Netherlands, May 7-11, BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I. "Cloning and expression pattern of peroxisomal genes in the thicklip grey mullet Chelon labrosus: different gene expression analysis to study peroxisome proliferation". VII th International Congress on the Biology of Fish. St. John s, Newfoundland, Canada, July 18-22, BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I. "Differential gene expression in mullets injected with B(a)P: phase I metabolism and peroxisome proliferation". Awarded: 1.- Student and Posdoctoral Travel award to participate in the VII th International Congress on the Biology of Fish ICBF 2006, Canada. 2.- Best oral presentation award in Biomarkers session in same congress. 205

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157 ABSTRACT Differential gene expression after B(a)P injection Benzo(a)pyrene (B(a)P) is a model mutagenic polycyclic aromatic hydrocarbon (PAH) commonly released into the environment, whose toxicological mechanisms have been widely studied in aquatic organisms. B(a)P induces phase I biotransformation metabolism and it may also induce peroxisome proliferation which is characterised by increased peroxisomal volume density, accompanied in rodents by the transcriptional induction of peroxisomal enzymes, mainly those involved in peroxisomal β-oxidation. Since the study of biological effects caused by xenobiotics in aquatic environments requires selection of proper species and effective biomarkers, the aim of the present work was to study peroxisome proliferation as a novel biomarker at the gene and protein level, in comparison to the expression of the well-characterised cytochrome P450 1A1 gene (CYP1A1) in a fish species able to endure high concentrations of pollutants. For that purpose, juvenile thicklip grey mullets Chelon labrosus were single injected with B(a)P (5 mg/kg) and expression of palmitoyl-coa oxidase (AOX1), multifunctional protein (MFP1), 3-ketoacyl-CoA thiolase (THIO), 2, 4 dienoyl- CoA reductase 2, PMP70 and catalase (CAT) was compared to CYP1A1 expression in liver and gills. For this purpose, the major peroxisomal membrane protein PMP70 and CYP1A1 were previously cloned using degenerate primers. AOX1 and CYP1A1 expression was induced in gills 1 day after B(a)P injection. CYP1A1 expression was also induced in mullet liver one day after injection, indicating that B(a)P was available for the liver. This was further proved by the significant accumulation of B(a)P-like metabolites in bile after 7 days of injection. In liver, AOX1, MFP1 and THIO expression was induced at day 1 followed by the induction of CAT expression at day 7 that may reflect a response to an oxidative stress caused by B(a)P itself or its induction through the biotransformation metabolism and the peroxisomal β-oxidation. However, these hepatic responses were not reflected at AOX1 protein or activity level. Thus, it has been proved for the first time that the three enzymes in the fish peroxisomal β-oxidation pathway are transcriptionally inducible after B(a)P injection. It remains to be tested whether this overexpression is reflected in an increase in the number of peroxisomes, although PMP70, marker of peroxisome increased numbers, was not transcriptionally induced, nor AOX1 protein levels showed any variation. Further studies will help to determine the usefulness of studying this cellular process at the molecular level as a biomarker of exposure to PAHs. 207

158 Results and Discussion RESUMEN El benzo(a)pireno (B(a)P) es un hidrocarburo aromático policíclico (PAH) mutagénico modelo, liberado comúnmente al medio y cuyos mecanismos toxicológicos han sido ampliamente estudiados en organismos acuáticos. El B(a)P induce el metabolismo de biotransformación de fase I y puede a su vez, inducir proliferación de peroxisomas, que se caracteriza por el aumento de la densidad volumétrica de los peroxisomas, acompañado en roedores, por la inducción transcripcional de enzimas peroxisómicos, principalmente los que participan en la β-oxidación. Debido a que el estudio de los efectos biológicos causados por xenobióticos en ambientes marinos requiere la selección de especies apropiadas y biomarcadores efectivos, el objetivo del presente trabajo fue estudiar la proliferación de peroxisomas como biomarcador novel a nivel génico y proteico en una especie de pez capaz de vivir en altas concentraciones de contaminantes, y compararlo con la expresión del gen citocromo P450 1A1 (CYP1A1) ampliamente caracterizado. Con ese fin, se inyectó intraperitonealmente B(a)P (5 mg/kg) a mubles inmaduros Chelon labrosus y se comparó la expresión de los genes que codifican para palmitoil-coa oxidasa (AOX1), proteína multifuncional (MFP1), 3-ketoacil-CoA tiolasa (THIO), 2, 4 dienoil-coa reductasa 2 (DECR), PMP70 y catalasa (CAT) junto con la expresión de CYP1A1 tanto en hígado como en branquias. Para llevar a cabo este objetivo, previamente se clonaron el gen que codifica la principal proteína de membrana de los peroxisomas PMP70 y CYP1A1. La expresión de AOX1 y CYP1A1 en branquias se indujo 1 día después de la inyección. La expresión de CYP1A1 también se indujo en el hígado de los mubles 1 día después de la inyección, indicando que el B(a)P era disponible para el hígado. Ésto se confirmó mediante la acumulación significativa de metabolitos tipo B(a)P en bilis 7 días después de la inyección. Además, el día 1 se indujo la expresión de AOX1, MFP1 y THIO en hígado, seguido de la inducción de CAT 7 días después de la inyección, lo cual podría reflejar la respuesta al estrés oxidativo causado por el B(a)P, o el causado a través de la inducción del metabolismo de biotransformación y la β-oxidación peroxisómica. Sin embargo, estas respuestas hepáticas no se reflejaron ni en el nivel de proteína ni en la actividad de AOX1. Por lo tanto, se ha probado por primera vez, que los tres enzimas de la β-oxidación peroxisómica son transcripcionalmente inducibles tras una inyección de B(a)P. Sin embargo, queda por probar si esta sobreexpresión se refleja en el aumento numérico de peroxisomas, a pesar de que en el presente estudio, la expresión de PMP70, marcador del aumento peroxisómico, no varió transcripcionalmente y los niveles proteicos de AOX1 tampoco presentaron variaciones. Futuros estudios determinarán la utilidad de estudiar este proceso celular a nivel molecular como biomarcador de exposición a PAHs. 208

159 Differential gene expression after B(a)P injection Introduction Organic toxic pollutants such as the model mutagenic polyclyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (B(a)P) (Michel et al. 1992) are widespread in aquatic environments and upon accumulation into biota exert a wide variety of biological effects. Induction of biotransformation metabolism and peroxisome proliferation are two examples of such effects that may be employed in sentinel fish as biomarkers of exposure to these compounds in pollution biomonitoring programmes. In this sense, it can be considered that mullets, such as thicklip grey mullets Chelon labrosus, may be good sentinel species for such monitoring programs (Orbea et al. 1999; 2002b; Bilbao et al. 2006a; Ferreira et al. 2006) since they are abundant in coastal and estuarine habitats, being able to live in highly polluted environments where they have been described to accumulate high concentrations of PAHs and organochlorine contaminants (Ferreira et al. 2004). Peroxisome proliferation is defined as a pleiotropic cellular response characterised by increased peroxisomal volume density (Cancio and Cajaraville 2000; Cajaraville et al. 2003; Cajaraville and Ortiz-Zarragoitia 2006) which in rodents is usually accompanied by the transcriptional induction of some peroxisomal enzymes, mainly those involved in lipid homeostasis such as the three enzymes participating in the peroxisomal β-oxidation pathway; palmitoyl-coa oxidase (AOX1), multifunctional protein (MFP) and 3-ketoacyl- CoA thiolase (THIO) (Qi et al. 2000). Peroxisome proliferation has also been reported in terms of AOX1 induction in laboratory experiments in mussels exposed to different chemical organic compounds (Cancio et al. 1998; Cancio and Cajaraville 2000; Cajaraville and Ortiz-Zarragotia 2006) and different fish species such as Sparus aurata (Pedrajas et al. 1996), Ictalurus puntactus (Mather-Mihaich and Di Giulio 1991), Onchorynchus mykiss (Yang et al. 1990; Scarano et al. 1994; Oakes et al. 2005), Danio rerio (Ortiz-Zarragoitia and Cajaraville 2005) and Pimephales promelas, Semotilus atromaculatus, Notropis hudsonius or Catastomus commersoni (Oakes et al. 2005). However, other studies have reported low or no sensitivity towards peroxisome proliferators in Cyprinus carpio and Oryzias latipes (Scarano et al. 1994; Hoff et al. 2003). In vertebrate responsive species, peroxisome proliferation is under the regulation of the peroxisome proliferator activated receptor α (PPARα), a transcription factor that belongs to the nuclear receptor superfamily (Isseman and Green 1990), whose expression has been already reported in liver and gills of mullets (Ibabe et al. 2004; Raingeard et al. 2006). However, the study of peroxisome proliferation after treatment with any toxic organic compound has not been studied in mullets yet. Moreover, peroxisome proliferation has never been studied at the transcriptional level in any fish species. On the other hand, PAHs can be biotransformed effectively in fish liver by the phase I biotransformation metabolism, where cytochrome P450 1A1 (CYP1A1) participates converting the parent compounds to easily excretable metabolites. CYP1A1 gene expression is regulated in aquatic organisms by the aryl hydrocarbon receptor (AhR) whose agonists are xenobiotics such as planar PAHs and polychlorinated biphenyls (PCBs) and dioxins. Transcriptional induction of CYP1A1 is a sensitive and specific adaptive response in fish exposed to xenobiotics such as B(a)P (Hahn and Stegeman 1994; Woodin et al. 1997; Jönsson et al. 2006), which is considered a potent AhR inducer (Hahn and Stegeman 1994). Thus, regulation of gene expression by AhR plays a crucial role in the metabolism and clearance of xenobiotics that reach the organism from the environment (Wang and LeCluyse, 2003). Therefore, the transcriptional activation of CYP1A1 in fish exposed to organic compounds is commonly used as biomarker of exposure to planar organic compounds (Hahn and Stegeman 1994). In addition, since some PAHs are excreted as polar metabolites in bile, the levels of biliary PAH metabolites are also sensitive biomarkers to assess recent exposure to PAHs (Van der Oost et al. 2003). However, by catalysing the oxidative metabolism of lipophilic compounds through the phase I biotransformation metabolism, many 209

160 Results and Discussion compounds such as B(a)P and other PAHs, are transformed to mutagenic/carcinogenic intermediates (Buters et al. 1999). In addition, highly reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide and hydroxyl radicals are produced in this process as by-products (Jifa et al. 2006) causing oxidative stress. The same applies to the peroxisome proliferation process where an increased β- oxidation activity results in increased H 2 O 2 production (Schrader and Fahimi 2006). Different enzymatic and non-enzymatic mechanisms within the cell are involved in avoiding oxidative damage and among them, the main peroxisomal enzyme catalase (CAT) participates in the elimination of H 2 O 2. Since peroxisome proliferation in fish could be regulated at the transcriptional level as in well-characterised responsive species, the aim of this work was to test the transcriptional regulation of β-oxidation genes in thicklip grey mullets C. labrosus in order to understand the peroxisome proliferation process in fish and explore the possibility of studying peroxisomal gene expression profiling as a sensitive biomarker of exposure to organic xenobiotics. For that purpose, the expression levels of several peroxisomal genes (AOX1, MFP1, THIO, 2, 4 -dienoyl-coa reductase 2 (DECR), peroxisomal membrane protein 70 (PMP70) and CAT) was determined in liver and gills of thicklip grey mullets injected with B(a)P, in comparison with the expression regulation of the well-characterised and B(a)P-responsive CYP1A1 gene. Materials and methods Materials All chemicals were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless specified otherwise. Animals and experimental procedure 45 immature thicklip grey mullets Chelon labrosus (12 ± 5 cm) were collected from Plentzia (43º24 N, 2º56 W), situated in an unpolluted estuary (Orbea and Cajaraville 2006) in the Biscay Bay in April Organisms were acclimatised to laboratory conditions during 1 week. Water used during the experiment, which came from a clean place and is used to keep shellfish for human consumption, had the same conductivity (48.7 ms) and ph (7.84) as water from Plentzia and was previously sterilised with UV light and filtered through active charcoal. Water was renewed and specimens were fed with dried bread daily during the experiment. Animals were maintained at a constant temperature of 20ºC. Mullets were randomly divided into 3 groups prior to the beginning of the experiment where fish in the first group were intraperitoneally single injected with 5 mg of B(a)P / kg, using corn oil as vehicle. Fish in the second group were injected corn oil (control) and animals in the third group were not treated. One and seven days after the injection fish were anesthetised by immersion in a saturated solution of 3- aminobenzoic acid ethyl ester before being sacrificed. Liver and gill samples were dissected, embedded in RNA later and frozen in liquid nitrogen for gene expression studies. Liver and bile samples were directly frozen in liquid nitrogen for biochemical and immunochemical studies as well as for the measurement of PAH metabolites. All the samples were stored at -80ºC until processing. Fixed wavelength fluorescence (FF) analysis At day 7, bile samples of 5 control and 5 B(a)P injected organisms were diluted 1:1000 in 50% ethanol. FF was then measured at the excitation/emission wavelength of 380/430 nm (FF 380/430 ) where B(a)P type of metabolites are best detected (Krahn et al. 1987). Measurements were performed in quartz cuvettes on a SLM2 Aminco luminescence spectrometer (Spectronic Instruments, Rochester, New York, USA) and slit widths were set at 4 nm for both excitation and emission wavelengths. The FF values were recorded in arbitrary fluorescence and the signal levels of the solvent were subtracted and represented as fold induction over control values. 210

161 Differential gene expression after B(a)P injection Palmitoyl-CoA oxidase (AOX1) activity measurement: AOX1 activity was quantified determining the H 2 O 2 dependent oxidation of dichlorofluorescein diacetate (Molecular Probes, Eugene, Oregon, USA) catalysed by an exogenous peroxidase using 30 µm palmitoyl- CoA as substrate according to Small et al. (1985). Each liver sample, 5 animals per experimental group, was homogenised individually in TVBE buffer (1 mm sodium bicarbonate, 1 mm EDTA, 0.1 % ethanol and 0.01 % Triton X-100), ph 7.6 (4 ml / g tissue) in a Hybaid Ribolyser TM (Hybaid, Ashford, UK). After centrifugation at 500 g at 4ºC for 15 min in a Jouan CR-312 centrifuge (Jouan, Saint- Herblain, France), supernatants were diluted 1:40 in TVBE buffer and assayed for AOX1 activity at a wavelength of 502 nm using a Shimadzu UV-1603 spectrophotometer (Shimadzu, Duisburg, Germany). Total protein concentration was measured using the DC protein assay (BioRad, San Diego, California, USA) based on the method of Lowry and measuring γ-globulin as standard. AOX1 activity is given as mu AOX mg -1 total protein equivalent to nmol H 2 O 2 min -1 mg -1 protein. SDS-PAGE and Western blotting: 15 µg of total protein was loaded for each organism (3 organisms per experimental group, 1 day after injection) and separated by their molecular weight in 12.5% sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred onto Hybond-ECL nitrocellulose membranes (Amersham Biosciences, Bucks, UK) by wet-transference. Membranes were blocked for 1 h at room temperature in phosphate-buffered saline plus 0.1% Tween 20 (PBST) plus 5% non-fatty dried milk. Incubations with the specific rabbit anti-rat AOX1 polyclonal antibody (supplied by Prof. HD. Fahimi, Universität Heidelberg, Germany) diluted 1:1000 were carried out overnight at 4ºC (Orbea et al. 1999). Membranes were washed for 3x15 min in PBST and incubated with horseradish peroxidase labelled secondary antirabbit IgG antibody for 1 h at room temperature. After washing for 3x15 min in PBST, the peroxidase activity was visualised with an enhanced chemiluminescence ECL kit (Amersham Biosciences, Buckinghamshire, UK). RNA extraction and cdna synthesis 50 to 100 mg of liver or gill tissue were homogenised in Trizol (Invitrogen, Carlsbad, California, USA) using an Hybaid Ryboliser at 4 m/s and for 20 and 40 seconds, respectively. 4 µg of total RNA were used for cdna synthesis by RT-PCR (Invitrogen) using random hexamers and according to manufacturer s instructions in the icycler TM thermal cycler (icycler TM, Bio- Rad). Cloning of target genes: CYP1A1 and PMP70 Degenerate primers Fw-5'- ACMATCTCYACTGCCCTGTC-3', Rv-5'- TTCGTGCAATGACCTCRCC-3' and Fw-5'- ATGATACTGCCATTCATTGGA-3 ' and Rv-5'- TGTGGCTCTCAGWTGGC-3', designed for the amplification of CYP1A1, were obtained aligning known CYP1A1 sequences belonging to different mugilid fish family species (Liza aurata AF022433, Liza saliens AF072899, and Mugil curema AY827103). On the other hand, forward Fw-5'- CTBTACTCCAACCTCAGCAAG-3' and Fw- 5'-AACAGTGARGAGATYGCMTTCTA-3', and reverse Rv-5'- TAGAAKGCRATCTCYTCACTGTT-3' and Rv-5'-GRCTGTAGATGWARTCCTCCA-3' degenerate primers were designed for the amplification of the peroxisomal membrane protein of 70 kda (PMP70), aligning PMP70 sequences of Homo sapiens (M81182), Xenopus laevis (EF070607) and Danio rerio (NM_213482) available in the Genbank. PCR products 1100 and 230 bp long were expected. After purification of all specific PCR amplicons using a Minielute PCR purification kit (Qiagen, Hilden, Germany) fragments were sequenced in the Sequencing service of the Department of Genetics, Physical Anthropology 211

162 Results and Discussion and Animal Physiology (University of the Basque Country). Semiquantification of gene expression Monoplex-PCR conditions for the amplification of CYP1A1 and PMP70 fragments around bp in length were optimised using Taq polymerase (Invitrogen), following PCR conditions shown in Table 1. PCR conditions described in Bilbao et al. (2006a; submitted) were applied for the amplification of AOX1 (EF525541), MFP1 (EF407560), THIO (DQ021958), CAT (AY743715) and DECR (EF883076) in 5 organisms per group. β-actin (AY836368) and 18S rrna (AY825252) were analysed as housekeeping genes following PCR conditions described in Raingeard et al. (2006). PCR products were visualised by electrophoresis in ethidium bromide stained agarose gels (1.5%) and analysed using a computer-aided gel analyser (Gel-Doc-2000, Bio-Rad). Results were expressed in arbitrary semi-quantitative units using 18S rrna as housekeeping gene for normalisation of the expression. Statistical analysis: Statistical analysis was carried out using SPSS 13 where the Mann-Whitney U test was applied to establish significant differences between groups. A 95% significance level was established. Results No mortality was observed neither along the acclimatisation period to laboratory conditions nor during the experiment. Cloning of target genes: CYP1A1 and PMP70 Nearly the complete CYP1A1 (DQ438983) mrna sequence (% 91 of the coding domain sequence) was amplified from C. labrosus liver. The amplification product contained 1408 bps coding for 470 residues that show high homology with known vertebrate CYP1A1 sequences. As it was expected, the highest amino acid identity was obtained with sequences of other mugilid species, Liza saliens and Liza aurata, where 98% amino acid identity was observed. After sequencing and cleaning primer sequences, 1002 bp of PMP70 (~51% of cds) were obtained (EF407558). The deduced protein sequence presented 85% amino acid identity with Danio rerio, 82% with Xenopus laevis and 81% with Homo sapiens PMP70 sequences (Figure 1). The last 178 residues of the deduced mullet PMP70 corresponded to the typical ATPbinding cassette transporter motif that has been reported to be involved in the import of very long-chain fatty acids into peroxisomes in mammals. The amplified fragment was not long enough to determine whether predicted pex19p binding site, or peroxisomal targeting and insertion information sites, were present or not, but comparison of the Danio rerio and Gasterosteus aculeatus sequences available in the Ensembl genome repository allow to identify such regions in fish PMP70. PAH metabolites Fixed-wavelength fluorescence measurements at the excitation/emission Table 1. PCR conditions for the specific amplification and expression semiquantification of thicklip grey mullet CYP1A1 and PMP70 are shown. Before cycling conditions, a denaturising step was added for 2 minutes in both cases. A final elongation step was added for 8 minutes. Optimised number of cycles for CYP1A1 was 30 and for PMP CYP1A1 PMP70 Primers 5-3 Fw: GGAGAGGCTTTATCAAGAACT Rv: CGTGCAATGACCTCACCAA Fw: GACCCTACATGACGCTTGGT Rv: GCTTTTCCCCTCCACTAAGG PCR conditions 15 at 94ºC, 15 at 52ºC, 1 at 72ºC 30 at 94ºC, 30 at 57ºC, 45 at 72ºC 212

163 Differential gene expression after B(a)P injection DrPMP70 -MAAVSKYLTAKNSAVAGGVLLVLYILKQRRRAAALNRKKGSANEL-NSEKDGKKERAAV 58 ClPMP HsPMP70 -MAAFSKYLTARNSSLAGAAFLLLCLLHKRRRALGLHGKKSGKPPLQNNEKEGKKERAVV 59 XlPMP70 MAPVYSKYLNARNTSVAGAALFLLYLISKRR---GASGKKNGKSSLQNNEKDGKKEKAVV 57 DrPMP70 DKLFFIRISRILKIMVPKFFSKETWYLLLIAVMLVTRTYCDVWMIQNGTMIESAIIGRST 118 ClPMP HsPMP70 DKVFFSRLIQILKIMVPRTFCKETGYLVLIAVMLVSRTYCDVWMIQNGTLIESGIIGRSR 119 XlPMP70 DRVFFGRIGQILKILVPKTFCKESGYLLFIAVMLIARTYCDVWMIQNGTFIESAIIGRSR 117 DrPMP70 KGFKKYLFNFMTAMPISALVNNFLKLGLNELKLCFRVRLTKHLYDEYLKGYTYYKMGNLD 178 ClPMP HsPMP70 KDFKRYLLNFIAAMPLISLVNNFLKYGLNELKLCFRVRLTKYLYEEYLQAFTYYKKGNLD 179 XlPMP70 KDFKRYLFNFIAAMPAISLVNNFLKFGLNELKLCFRERLTKYLYDEYLKSFTYYKMGNLD 177 DrPMP70 NRIANADQLLTQDVERFCNSVVDLYSNLSKPLLDIGLYIFKLTTAIGAQGPATMMAYLLI 238 ClPMP HsPMP70 NRIANPDQLLTQDVEKFCNSVVDLYSNLSKPFLDIVLYIFKLTSAIGAQGPASMMAYLVV 239 XlPMP70 NRIANADQLLTQDVERFCNSVVDLYSNLSKPFLDIVLYIFKLTSAIGAQGPASMMAYLLV 237 DrPMP70 SGLFLTRLRRPIGKMTVTEQKYEGEYRYVNSRLITNSEEIAFYNGNTREKQTIHSTFKKL 298 ClPMP GNVRETESIYATFKKL 16 HsPMP70 SGLFLTRLRRPIGKMTITEQKYEGEYRYVNSRLITNSEEIAFYNGNKREKQTVHSVFRKL 299 XlPMP70 SGLFLTRLRRPIGKMTVAEQRYEGEYRYVNSRLITNSEEIAFYNGNKREKLTIYSSFQKL 297 ** **. :::: *:** DrPMP70 VDHLHNFIFFRFSMGMVDSIIAKYFATVVGYLVVSRPFLNLSHSRHLNSSHAELLEDYYQ 358 ClPMP70 VDHLHNFIFFRFSMGFVDSIIAKYIATVVGYLVVSRPFLNPSDHRHLHSTHSELLEDYYQ 76 HsPMP70 VEHLHNFILFRFSMGFIDSIIAKYLATVVGYLVVSRPFLDLSHPRHLKSTHSELLEDYYQ 359 XlPMP70 VEHLHNFILFRFSMGFVDSIIAKYLATVVGYLVVSRPFLAINHPRHLNSSHAELLEDYYQ 357 *:******:******::*******:**************.. ***:*:*:******** DrPMP70 SGRMLLRMSQALGRIVLAGREMTRLSGFTARITELMRVLKELNSGKYERTMVSQSE--KD 416 ClPMP70 SGRMLLRMSQALGRIVLAGREMTRLSGFTAXITELIKVLKELNAGKYERTMVSQQNG-AD 135 HsPMP70 SGRMLLRMSQALGRIVLAGREMTRLAGFTARITELMQVLKDLNHGKYERTMVSQQEKGIE 419 XlPMP70 SGRMLLRMSQALGRIVLAGREMTRLSGFTARITELMQVLKDLNQGKYERTMVSQQDKDVE 417 *************************:**** ****::***:** **********.: : DrPMP70 ASEKLTLVPGSGRIINIDNIIKFDHTPLATPNGDVLIRDLCFEVKSGTNVLVCGPNGCGK 476 ClPMP70 TAGEPKLVPGRGQVTNRDNIIKFDHTPLSTPNGDVLIRDXTXEVRSGTNVLVCGPNGCGK 195 HsPMP70 GVQVIPLIPGAGEIIIADNIIKFDHVPLATPNGDVLIRDLNFEVRSGANVLICGPNGCGK 479 XlPMP70 AVPSIPLIPGSGKVINADKIIKFDHVPLATPNGDLLIRDLNFEVRSGTNVLVCGPNGCGK 477 *:** *.: *:******.**:*****:**** **:**:***:******** DrPMP70 SSLFRVLGELWPLFGGNLTKPERGKLFYVPQRPYMTLGSLRDQVIYPDTHESQKKKGISD 536 ClPMP70 SSLFRVLGELWPLFGGHLTKPERGKLFYVPQRPYMTLGSLRDQVIYPDTFEDQRKKGISD 255 HsPMP70 SSLFRVLGELWPLFGGRLTKPERRKLFYVPQRPYMTLGTLRDQVIYPDGREDQKRKGISD 539 XlPMP70 SSLFRVLGELWPLFGGSLTKPERGKLFYVPQRPYMTLGTLRDQVIYPDTQEDQKRKGISD 537 **************** ****** **************:********* *.*::***** DrPMP70 LVLKEYLDNVQLGHILDREGSWDTVQDWMDVLSGGEKQRMAMARLFYHKPQFAILDECTS 596 ClPMP70 QVLKEYLDNVQLGHILDREGSWDSVQDWMDVLSGGEKQRMAMARLFYHKPQFAILDECTS 315 HsPMP70 LVQKEYLDNVQLGHILEREGGWDSVQDWMDVLSGGEKQRMAMARLFYHKPQFAILDECTS 599 XlPMP70 KVLKEYLDNVQLGHILDREGGWDSVQDWMDVLSGGEKQRMAMARLFYHKPQFAILDECTS 597 * *************:***.**:************************************ DrPMP70 AVSVDVEDYIYSHCRKVGITLFTVSHRKSLWKHHEYYLHMDGRGNYEFKPITPETVEFGS 656 ClPMP70 AVSVDVEDFIYSQTPGTG HsPMP70 AVSVDVEGYIYSHCRKVGITLFTVSHRKSLWKHHEYYLHMDGRGNYEFKQITEDTVEFGS 659 XlPMP70 AVSVDVEGYIYNHCRKVGITLFTVSHRKSLWKHHEYYLHMDGRGNYDFKQITEDTDAFGS 657 *******.:**.:.* Figure 1. Alignment of the deduced amino acid sequence of Chelon labrosus peroxisomal membrane protein 70 (ClPMP70) and the predicted Danio rerio PMP70 sequence (DrPMP70, NM_213482), and Homo sapiens (HsPMP70, M81182) and Xenopus laevis (XlPMP70, EF070607) PMP70 sequences. Residues 56 to 337 in D. rerio correspond to the predicted transmembrane region. Predicted transmembrane segments in human PMP70 are underlined, predicted Pex19p-binding sites are in white and clusters of positively charged amino acid residues are indicated in bold letters (Van Ael and Fransen 2006). This shows the high degree of conservation of these structures within piscine PMP70. Last 178 residues shaded in grey, correspond to the typical ATP-binding cassette transporter motif. 213

164 Results and Discussion wavelengths to measure B(a)P-like metabolites recorded 10-fold increased fluorescence intensities in B(a)P injected organisms 7 days after the injection. Thus, uptake of B(a)P resulted in bile accumulation of B(a)P Cn B(a)P Control B(a)P Figure 2. Fluorescence intensity measurements of B(a)P type metabolites in bile at the excitation emission wavelengths 380/430 nm 7 days after B(a)P injection. Results are given as fold fluorescence intensity increase taking control values to 1. Mean values were obtained from 5 different bile samples. Vertical segments indicate standard deviations. metabolites in injected mullets (Figure 2). Control individuals (vehicle injected) showed background levels of fluorescence intensity at the wavelengths utilised to measure B(a)P metabolites. In the same way, no accumulation of pyrene-like or naphthalene-like metabolites was observed in the fish biles analysed when a) 10 FF 341/383 and FF 290/335 were measured (data not shown). AOX1 enzyme activity levels and immunochemically detected protein levels AOX1 enzyme activity showed no significant variations along the experiment in the 3 treatment groups (Only results from vehicle and B(a)P+ vehicle injected animals are represented, Figure 3a). In fact, similar AOX1 protein levels were also detected by western-blot at day 1 when comparing oil and B(a)Pinjected mullets (Figure 3b) where three reactive bands of 72, 52 and 21 kda, corresponding to the native (A band) and the proteolytically cleaved protein (B and C bands) were detected. Gene expression studies B(a)P injection inhibited β-actin expression, so semiquantitative results were normalised using 18S rrna as housekeeping gene. The vehicle (corn-oil) significantly repressed AOX1 expression in liver at day 1, while the rest of the genes were similarly expressed in oil injected and in uninjected control organisms at both sampling days (data not shown). All the graphs presented here compare B(a)P and corn-oil injected groups (Figures 4-5). B(a)P injection significantly induced CYP1A1 gene expression at day 1 in liver and b) 72 kda 52 kda 6 22 kda Cn B(a)P 2 0 DAY1 Control DAY7 B(a)P Figure 3. (a) Spectrophotometric measurement of AOX1 enzyme activity (mu/mg protein) in liver of mullets 1 and 7 days after corn-oil (control) and B(a)P in corn-oil injection. Values are given as means ± standard deviations (n = 5). (b) Results of the immunoblot analysis showing the crossreactivity of the polyclonal antirat AOX1 antibody. Cn = cornoil injected group; B(a)P = B(a)P in corn-oil injected group. 214

165 Differential gene expression after B(a)P injection * AOX * MFP DAY1 DAY7 0 DAY1 DAY * THIO DECR DAY1 DAY7 0 DAY1 DAY7 5 CAT 1.5 PMP * DAY1 DAY7 DAY1 DAY7 CYP1A1 4 Control B(a)P * DAY1 DAY7 Figure 4. Semiquantification of AOX1, MFP1, THIO, DECR, CAT, PMP70 and CYP1A1 gene expression levels normalised using 18S rrna as housekeeping gene is shown in liver of mullets after 1 and 7 days of corn-oil (control) and B(a)P in corn-oil injection. Values are given as means ± standard deviations (n = 5) taking control mean values to 1 in each case. Asterisks indicate statistically significant differences between controls and B(a)Pinjected mullets at the same day (p<0.05). 215

166 Results and Discussion gills (Figures 4-5), confirming that injection was effective and that animals were responding to the treatment. Gene expression patterns returned to control levels at day 7. In a similar way, AOX1, MFP1 and THIO expression levels were significantly increased in liver at day 1 (Figure 4). In gills, a significant upregulation of AOX1 gene expression was also observed one day after B(a)P injection (Figure 5). Expression in all cases returned to control levels after 7 days of injection. Following induction of CYP1A1 and genes coding for enzymes of the peroxisomal β- oxidation pathway at day one, hepatic CAT expression was observed to be upregulated 7 days after B(a)Pinjection (Figure 4). No changes were observed in CAT gene expression at day 1 in liver or in any of the sampling days in gills (Figures 4-5). Finally, no alterations were observed in liver PMP70 and DECR expression * AOX1 levels among control, vehicle and B(a)P injected organisms (Figure 4). Discussion In order to determine whether exposure to a heavy PAH in thicklip grey mullets Chelon labrosus, elicits peroxisome proliferation and whether this response is regulated at the transcriptional level, different peroxisomal parameters have been studied, 1 and 7 days after a single intraperitoneal injection of 5 mg / kg B(a)P. All gene expression results have been only normalised against 18S rrna gene expression, due to the inhibition caused by B(a)P injection on β-actin expression. 18S rrna gene expression did not show significant variations between experimental groups and among individuals, however, alterations on the expression of the so-called housekeeping genes THIO DAY 1 DAY 7 0 DAY 1 DAY CAT 4 3 * CYP1A DAY 1 DAY 7 0 DAY 1 DAY Control B(a)P Figure 5. Semiquantification of AOX1, THIO, CAT and CYP1A1 gene expression levels normalised using 18S rrna as housekeeping gene is shown in gills of mullets after 1 and 7 days of corn-oil (control) and B(a)P in corn-oil injection. Values are given as means ± standard deviations (n = 5) taking control mean values to 1 in each case. Asterisks indicate statistically significant differences between controls and B(a)P-injected mullets at the same day (p<0.05).

167 Differential gene expression after B(a)P injection have been widely described (Arukwe 2006). In fact, it is considered that in toxicology, housekeeping genes vary considerably under exposure to different chemical compounds, as it is the case of β-actin in laboratory chemical exposure experiments, and thereby may lead to an erroneous interpretation of the expression profile of target genes (Arukwe 2006). However, and contrary to our observations, Hoffmann et al. (2006) did not observe alterations on the expression of β-actin in liver of Danio rerio after waterborne exposure to B(a)P. With the aim of confirming the effect of the B(a)P injection, B(a)P metabolites were measured in biles 7 days after the injection. Fish bile metabolites have been shown to be sensitive biomarkers for detection of PAH exposure (Van der Oost et al. 2003) and in fact, a 10 fold higher fluorescence intensity due to B(a)P metabolites was observed in the B(a)P injected group in comparison with oil injected mullets. In addition, the well-characterised modulation of CYP1A1 gene expression upon B(a)P exposure was semiquantified in liver and gills. CYP1A1 is a monooxigenase involved in the phase I biotransformation metabolism of a wide range of planar aromatic compounds. Induction of its enzymatic activity (EROD activity), protein or transcript levels have been widely reported as biomarkers of fish exposure to organic toxic compounds in laboratory and field studies (Hahn and Stegeman, 1994; Craft et al. 2001; Williams et al. 2003). It has been shown that CYP1A1 induction by PAHs and PCBs is regulated via the aryl hydrocarbon receptor (Hahn 1998; Van der Oost et al. 2003). Therefore, CYP1A1 induction has been described after treatment with B(a)P in many fish species such as Fundulus heteroclitus (Van Veld et al. 1997; Patel et al. 2006), Sparus aurata (Ortiz-Delgado et al. 2005), Oncorhynchus mykiss (Levine and Oris 1999; Jönsson et al. 2006), Trematomus bernacchii (Regoli et al. 2005), Anguilla rostrata (Schlezinger and Stegeman 2000) and the mugilid fish Mugil cephalus (Ferreira et al. 2004). In this study, 1408 bp of thicklip grey mullet CYP1A1 were cloned and semiquantitative studies revealed an induction of the biotransformation metabolism, measured as a significant induction of CYP1A1 gene expression, both in gills and in liver, 1 day after B(a)P injection. This induction confirmed that animals were responding specifically to B(a)P uptake. 7 days after injection, CYP1A1 expression was similar in treated and untreated organisms, suggesting that B(a)P was already removed from the studied tissues, although B(a)P metabolites were still present in the bile. Similarly, Ferreira et al. (2006) showed a significant decrease of EROD activity in grey mullets coming from a polluted site in the Douro estuary and allowed to depurate for 1 month while recovery of bile metabolite levels required further 3 months of depuration. Apart from the induction of phase I biotransformation metabolism, B(a)P elicits many other cytological and biochemical responses and alterations in fish. In Solea ovata for instance, 5 mg/kg of B(a)P provoked clear cytological and biochemical alterations such as increased abundance of lipofuscin granules, peroxisomes, mitochondria, lipid droplets and lysosomes, slight proliferation of the endoplasmic reticulum and glycogen depletion (Au et al. 1999). Some PAHs such as B(a)P have been reported to activate PPARα in rodents and to alter expression of target genes such as CYP4 (Kim et al. 2005). Additionally, the hypolipidemic drug and typical mammalian peroxisome proliferator WY induced CYP1A1 gene expression and EROD activity in humans, and interestingly 2 peroxisome proliferation response elements (PPREs) have been found in the promoter of human CYP1A1 (Sérée et al. 2004) suggesting that the AhR and the PPAR pathways could be interconnected. In liver of thicklip grey mullets we have measured induction of AOX1, MFP1 and THIO gene expression at day 1. In gills, only AOX1 gene expression was upregulated, indicating that the uptake of B(a)P by intraperitoneal injection, results in a mainly hepatic uptake of B(a)P, not suggested by the CYP1A1 data, or that peroxisome proliferation is mainly an hepatic event as in rodents. Therefore, expression of the three genes coding all the enzymes participating in the peroxisomal β-oxidation was induced in liver of mullets after 1 day of B(a)P injection. 217

168 Results and Discussion In summary, B(a)P seems to act as a typical peroxisome proliferator and mullets respond transcriptionally in a similar way to species responsive to peroxisome proliferators. However, no expression alterations were observed in genes coding for peroxisomal PMP70 and DECR. DECR plays an important role in the oxidation of virtually all unsaturated fatty acids (Kunau et al. 1995). Polyunsaturated fatty acids (PUFAs) are very important components of cellular structures in marine fish species (Tocher 2003) and are typical mammalian peroxisome proliferators (Price et al. 2000; Nakamura et al. 2004). The typical piscine PUFAs docosahexaenoic and eicosapentaenoic acids have been described to be agonists of piscine PPARs in Dicentrarchus labrax (Boukouvala et al. 2004). In addition, induction of DECR expression has been reported in rodents after exposure to different peroxisome proliferators such as clofibrate (Lei et al. 2003) and it has been cloned from a normalised library obtained from livers of Aroclor 1254 exposed Platichthys flesus (Williams et al. 2006). On the other hand, PMP70, major component of peroxisomal membranes (Imanaka et al. 2000), is markedly inducible in rodents by the administration of peroxisome proliferators, in parallel with increased peroxisome numbers (Imanaka et al. 1999). Also in the liver of Fundulus heteroclitus a significant induction of PMP70 was observed with a polyclonal antibody against the mammalian protein after waterborne exposure to the model peroxisome proliferator 2,4-dichlorophenoxyacetic acid (Ackers et al. 2000). In the present work, 1002 bp of the gene encoding PMP70 have been cloned in mullets. The cloned fragment showed high homology with known vertebrate PMP70s containing a well conserved peroxisomal ATP-binding cassette transporter motif. Fish PMP70s show also a well conserved ABC transporter transmembrane region that allows interaction to Pex19p and import into peroxisomes as predicted in human PMP70 by Van Ael and Fransen (2006). However, the cloned fragment was not long enough to determine whether predicted pex19p binding site or peroxisomal targeting and insertion information sites are present in mullets. Anyway, expression studies did not reveal any significant alteration in PMP70 expression levels after B(a)P injection. AOX1 protein levels in liver of oil and B(a)P injected mullets were similar at day 1 where three reactive bands of 72, 52 and 21 kda corresponding to the native protein and its proteolytic cleavage peptides described in the mammalian protein were visualised, as reported by Orbea et al. (1999). Similarly, no alterations were detected in AOX1 activity in liver so, levels of gene transcription and protein products did not seem to be related. In Danio rerio, 17βestradiol and the xenoestrogens dibutylphathalate, methoxychlor, 4-tertoctylphenol and 17α-ethynylestradiol caused significant induction of AOX1 activity together with increased peroxisomal surface and numerical densities 7 and 15 days after exposure (Ortiz-Zarragoitia and Cajaraville 2005). In mullets exposed to a heavy fuel oil for 2 days (Bilbao et al. 2006b), AOX1 gene transcription and enzyme activity induction were found, suggesting the possibility that 1 day was not enough to elicit a response at the protein level and that the protein level increase might have occurred in the lapse in between both samplings. On the other hand, peroxisome proliferation and CYP1A1 induction can result in an increased production of reactive oxygen species (ROS) that can cause oxidative stress (Jifa et al. 2006). For instance, Oakes et al. (2005) observed AOX1 activity induction followed by increased oxidative damage in Pimephales promelas, O. mykiss, Catastomus commersoni, Notropis hudsonius and Semotilus atromaculatus after exposure to another typical peroxisome proliferator, perfluorooctane sulfonate. CAT is the main peroxisomal enzyme and it is involved in the elimination of H 2 O 2, main by-product of peroxisomal β-oxidation pathway. Increased CAT activity and gene expression have been reported in fish after xenobiotic exposure (Au et al. 1999), but however, CAT activity has been disregarded as a valid biomarker for environmental risk assessment since both induction and inhibition are observed after exposure to environmental pollutants (Van der Oost et al. 2003). In this way, in the present study, we report a significant induction of CAT 218

169 Differential gene expression after B(a)P injection expression in liver 7 days after B(a)P injection following induced peroxisomal gene and CYP1A1 expression at day 1, while in the Antarctic fish Trematomus bernachii exposure to B(a)P inhibited liver catalase activity (Regoli et al. 2005). In Lateolabrax japonicus no alterations in CAT activity have been observed, showing that hydrogen peroxide produced during B(a)P metabolism was mainly catalised by glutathione peroxidase but barely by CAT (Jifa et al. 2006). Similarly, in O. mykiss B(a)P does not cause statistically significant effects on CAT activity (Sturve et al. 2005) while, in isolated trout hepatocytes exposed to a complex of 20 environmentally relevant contaminants (PAHs, PCBs and pesticides), increased CAT activity has been described (Strmac and Braunbeck 2002). In Lepidorhombus boscii from a fuel-oil impacted area (Martínez-Gomez et al. 2006) CAT induction was determined. In the same way, in Carassius auratus the typical fibrate peroxisome proliferator gemfibrozil caused a significant induction of the antioxidant defence (Mimeault et al. 2006). In conclusion, we have shown that expression of all 3 genes of the peroxisomal β-oxidation pathway is modulated in liver and gills of mullets injected with B(a)P. However, further studies are needed to conclude whether this induction in the transcriptional activities of peroxisomal genes results in peroxisome proliferation, specially since no alterations in the transcription of PMP70 and AOX1 protein levels have been detected, and to determine the usefulness of studying this cellular process at the molecular level as a biomarker of exposure to organic pollutants. Acknowledgements This work was supported by the Spanish MEC (BIOMTOOLS, REN: /MAR, PRESTEPSE, VEM C06), Basque Government (ETORTEK-IMPRES) and University of the Basque Country (predoctoral grant to E. Bilbao and grant for consolidated research groups). Thanks are due to Urtzi Izagirre for his help in the collection of fish for this experiment and to Dr. Gorka Basañez (UPV/EHU) for his contribution in FF measurements. References Arukwe A (2006). "Toxicological housekeeping genes: do they really keep the house?". Environmental Science and Technology 40, Au DWT, Wu RSS, Zhou BS, Lam PKS (1999). "Relatioship between ultraestructural changes and EROD activities in liver of fish exposed to benzo(a)pyrene". Environmental Pollution 104, Bilbao, E, Cajaraville MP, Cancio I (submitted). "Molecular characterisation of the peroxisomal β- oxidation pathway in aquatic organisms: cloning and expression pattern of palmitoyl-coa oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase". Experimental Cell Research. Bilbao E, Cajaraville MP, Cancio I (2006b). "Induction of peroxisomal gene expression in mullets exposed to PFOS and a heavy fuel oil similar to the Prestige oil". VII th International Congress on the Biology of Fish, Canada. Bilbao E, Diaz de Cerio O, Cajaraville MP and Cancio I (2006a). "Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: Generation of new tools to study peroxisome proliferation". Marine Environmental Research 62, S Boukouvala E, Antonopolou E, Favre-Krey L, Diez A, Bautista JM, Leaver MJ, Tocher DR, Krey G (2004). "Molecular characterization of three peroxisome-activated receptors from the sea bass (Dicentrarchus labrax)". Lipids 39, Buters JTM, Doehmer J, Gonzalez FJ (1999). "Cytochrome P450-null mice". Drug Metabolism Reviews 31, Cajaraville MP, Cancio I, Ibabe A, Orbea A (2003). "Peroxisome proliferation as a biomarker in environmental pollution assessment". Microscopy Research and Technique 61, Cajaraville MP, Ortiz-Zarragoitia M (2006). "Specificity of the peroxisome proliferation response in 219

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175 Geneen espresio diferentziala B(a)P-a ziztatu ondoren 3.- Peroxisomen proliferazioan diharduten geneen espresio diferentziala benzo(a)pirenoz ziztaturiko Chelon labrosus lazunetan Atal hau argitaratzeko bidali da: BILBAO E, CAJARAVILLE MP, CANCIO I. Differential expression of genes involved in peroxisome proliferation in thicklip grey mullets Chelon labrosus injected with benzo(a)pyrene. Toxicology and Applied Pharmacology. Atal honetako emaitzak kongresuetan aurkeztu dira: 6 th Iberian and 3 rd Iberoamerican Congress of Environmental Contamination and Toxicology (CICTA), Cadiz, Espainia. Irailak 25-28, BILBAO E, CAJARAVILLE MP, CANCIO I. "Cloning of peroxisomal and phase I xenobiotic metabolism genes in the thicklip grey mullet Chelon labrosus: Induction of expression after exposure to benzo(a)pyrene". 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, Haya, Herbeherak, Maiatzak 7-11, BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I. "Cloning and expression pattern of peroxisomal genes in the thicklip grey mullet Chelon labrosus: different gene expression analysis to study peroxisome proliferation". VII th International Congress on the Biology of Fish. St. John s, Newfoundland, Canada, Uztailak 18-22, BILBAO E, DIAZ DE CERIO O, CAJARAVILLE MP, CANCIO I. "Differential gene expression in mullets injected with B(a)P: phase I metabolism and peroxisome proliferation". Saritua: 1.- VII th International Congress on the Biology of Fish ICBF 2006, Canada, kongresuan parte hartzeko Student and Posdoctoral Travel award. 2.- Kongresu bereko Biomarkers sesioan Best oral presentation award. 225

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177 Geneen espresio diferentziala B(a)Pa ziztatu ondoren LABURPENA Benzo(a)pirenoa (B(a)P), mutazioak eragiten dituen, ingurunera modu zabalean askatzen den eta organismo itsastarretan mekanismo toxikologiko aski ezaguna duen hidrokarburo aromatiko polizikliko (PAH) modeloa da. B(a)Pak fase I-eko bioeraldaketa metabolismoa emendatu egiten du eta peroxisomen proliferazioa ere sortarazi dezake; azken hau, peroxisomen dentsitate bolumetrikoaren emendioak karakterizatzen du, karraskarietan, peroxisometako entzimen, batez ere β-oxidazioko entzimen, transkripzio mailako emendioa ere ematen delarik. Ingurune itsastarretan bizi diren organismoetan xenobiotikoek eragiten dituzten efektuen azterketak, espezie egokien zein biomarkatzaile eraginkorren aukeraketa beharrezko egiten ditu. Horrela, lan honen helburua, maila geniko zein proteikoan peroxisomen proliferazioa biomarkatzaile nobel gisa ikertzea eta aski ezaguna den P450 1A1 zitokromoa (CYP1A1) kodetzen duen genearen espresioarekin konparatzea izan zen, kutsatzaileen kontzentrazio altuen pean bizitzeko gai den arrain espezie batean. Helburu honekin, Chelon labrosus lazun heldugabei B(a)Pa (5 mg/kg) ziztatu zitzaien intraperitonealki eta palmitoil-coa oxidasa (AOX1), proteina multifuntzionala (MFP1), 3-ketoazil-CoA tiolasa (THIO), 2, 4 dienoil-coa erreduktasa 2 (DECR), PMP70 eta katalasa (CAT) geneen espresioa, CYP1A1 genearen espresioarekin konparatu zen gibel eta zakatzetan. Horretarako, peroxisomen mintzetako proteina nagusia den PMP70 kodetzen duen genea eta CYP1A1 klonatu ziren lehenengo. Zakatzetan, AOX1 eta CYP1A1en espresioa emendatu egin zen ziztadaren hurrengo egunean. CYP1A1en gibeleko espresioa ere, esangarriki igo zen B(a)Pa ziztatu ondorengo egunean. Igoera hau gainera, behazuneko PAHen metaketa esangarriak ere baieztatu zuen 7. egunean, B(a)Pa gibelean eskuragarria zenaren adierazle. Gainera, gibeleko AOX1, MFP1 eta THIOren espresioa ziztadaren hurrengo egunean emendatu egin bazen ere, CAT genearen emendioa ziztada eman eta 7 egunetara eman zen. CAT genearen gainespresioak, B(a)Pak zuzenean eragindako estres oxidatiboari erantzun diezaioke edo eta bioeraldaketa metabolismoak zein peroxisometako β-oxidazioak eragindakoari. Hala ere, gibeleko erantzun hauek ez ziren AOX1en jarduera edo proteina mailan islatu. Beraz, organismo itsastarren peroxisometako β-oxidazioko entzimak, B(a)Paren pean, transkripzionalki induzigarriak direla frogatu dugu lehenengo aldiz. Hala ere, ikerketa sakonagoak beharko dira gene peroxisomiko hauen gainespresioak, peroxisomen emendio numerikoan gauzatzen diren jakiteko, nahiz eta lan honetan, peroxisomen emendio numerikoaren adierazle den PMP70 genearen espresioa ez zen transkripzionalki aldatu, ezta AOX1 proteinaren maila ere. Etorkizunean burutu beharreko ikerketek argituko dute zelula mailako prozesu hau, PAHen peko biomarkatzaile gisa aplikatzeko molekula mailan burututako ikerketak erabilgarriak direnentz. 227 L A B U R P E N A

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179 Geneen espresio diferentziala B(a)Pa ziztatu ondoren Emaitzak Aklimatazioan zehar eta esperimentuan zehar ez zen lazunik hil. Itu-geneen klonazioa: CYP1A1 eta PMP70 CYP1A1en ia open reading frame osoa (%91) anplifikatu zen C. labrosus lazunaren gibelean (DQ438983). Anplifikatutako 1408 bp-tako produktuak, ornodunen CYP1A1 sekuentziekin homologia altua agertu zuen 470 amino azidotako zati kodetzen du. Espero bezala, amino azido identitate altuena mugilidoen espezieekin lortu zen, Liza saliens eta Liza auratarekin %98ko amino azido identitatea aurkitu zen. Sekuentziatu eta hasleen sekuentziak ezabatu ondoren, PMP70en 1002 bp (%51) lortu ziren (EF407558). Deduzituriko proteina-sekuentziak %85eko amino azido identitatea agertu zuen Danio reriorekin, %82koa Xenopus laevisekin eta %81ekoa Homo sapiensen PMP70arekin (1 Irudia). Deduzituriko lazunaren PMP70 sekuentziaren azken 178 amino azidoak, ugaztunen peroxisometan katea luzeko gantzazidoak peroxisometan barneratzeko beharrezkoa den ATP lotzeko motiboarekin lerrokatu ziren. Anplifikaturiko zatiak ez zuen luzera nahikoa Pex19p lotzeko aurreikusi den edo peroxisometan lotzeko zein barneraketa burutzeko beharrezkoak diren informazioguneak zeuden edo ez argitzeko. Hala ere, Ensemblen eskuragarri dauden Danio rerio eta Gasterosteus aculeatusen sekuentzien alderatzeak, arrainen PMP70en gune horiek identifikatzea baimendu zuen. PAHen metabolitoak B(a)P motako PAHak neurtzean, finkatutako uhin-luzeretan burututako exzitazio- eta emisiofluoreszentzia neurketetan, B(a)Pa ziztatu eta 7 egunetara, 10 aldiz fluoreszentzia maila altuagoak neurtu ziren kontroletan baino. Hori dela eta, ziztatutako arrainetan, B(a)Paren barneraketa eta B(a)Paren behazuneko metaketa eman ziren (2. Irudia). Kontrolek (olioa ziztaturikoak), B(a)P motako metabolitoak neurtzeko erabilitako uhin-luzeran background mailako fluoreszentzia intentsitatea azaldu zuten. Era berean, ez zen naftaleno edo pireno motako metabolitoen metaketarik eman arrainen behazunean FF 341/383 eta FF 290/335 neurketak burutu ondoren (ez dira datuak erakusten). AOX1 entzimaren jarduera maila eta immunozitokimikoki neurtutako proteina maila Esperimentuan zehar, AOX1 entzimaren jarduerak ez zuen aldaketa esangarririk erakutsi talde batean ere ez (3a. Irudian, olioarekin eta olio + B(a)Parekin ziztatutako arrainak aurkeztu dira soilik). Izan ere, antzeko AOX1 proteina mailak aurkitu ziren western-blott bidez lehenengo eguneko olio eta B(a)P + olioarekin ziztatutako arrainak konparatzean (3b Irudia), non 3 banda erreaktibo ikus zitezkeen, 72, 52 eta 21 kda-ei zegozkienak, hain zuzen ere. Banda hauek proteina natiboa (A banda) eta proteolitikoki moztutako proteinarekin bat zetozen (B eta C bandak). Geneen espresio maila B(a)Pak β-aktinaren espresioa inhibitu egin zuen, beraz, geneen espresio mailako azterketa semikuantitatiboak 18S rrna housekeeping gisa erabiliz normalizatu ziren. Olioak, AOX1 espresio maila erreprimitu egin zuen lehenengo egunean disekzionaturiko lazunen gibeletan, gainontzeko geneen espresio maila, egun bietan (1 eta 7), tratatu bakoen parekoa izan zen bitartean (datuok ez dira erakusten). Hemen aurkeztutako grafiko guztiek, olioarekin eta olioa + B(a)Parekin ziztatutako arrainen espresio mailak azaltzen dituzte (4. eta 5. irudiak). B(a)P-injekzioak CYP1A1 genearen gainespresio esangarria sortarazi zuen lehenengo eguneko arrainen gibel eta zakatzetan (4. eta 5. Irudia), injekzioa efektiboa zela eta animaliek tratamenduari erantzun egiten ziotela adieraziz. Geneen espresio-patroiak kontrolen mailetara jaitsi ziren 7. egunean. Antzera, AOX1, MFP1 eta THIO geneen espresio mailak esangarriki gainespresatu ziren lehenengo egunean (4. Irudia). Zakatzetan, AOX1 genearen gainespresio esangarria behatu zen ere, B(a)Pa ziztatu eta hurrengo egunean (5. Irudia). Kasu 229 E M A I T Z A K

180 Emaitzak eta Eztabaida ziztatu eta hurrengo egunean (5. Irudia). Kasu guztietan espresio mailak kontrolen mailetara jaitsi ziren ziztada eman eta 7 egunetara. CYP1A1 eta peroxisometako β-oxidaziorako kodetzen duten geneen lehenengo eguneko gainespresioaren ondoren, gibeleko CAT espresioaren gainerregulazioa behatu zen B(a)Pa ziztatu eta 7. egunean (4. Irudia). Gibelean, lehenengo egunean edo zakatzetan, 1 zein 7. egunean, ez zen CATren espresioan aldaketarik behatu (4-5. Irudiak). Azkenik, olioarekin ziztatutako organismoak eta B(a)Prekin ziztatutakoak konparatzean, ez zen espresio mailen aldaketarik behatu gibeleko PMP70 eta DECR geneen kasuan (4. Irudia). 230

181 Ondorioak Geneen espresio diferentziala B(a)Pa ziztatu ondoren 1.-I Faseko bioeraldaketa metabolismoan diharduen CYP1A1en sekuentzia kodetzailearen %91 eta peroxisomen mintzetako proteina nagusia den PMP70en sekuentzia kodetzailearen %51 klonatu ditugu Chelon labrosus lazunean. 2.-B(a)Pa ziztatu ondoren, Chelon labrosus lazunen gibel eta zakatzetan behatutako CYP1A1 genearen indukzioak eta behazunean metatutako B(a)P metabolitoek, I faseko bioeraldaketa metabolismoa gainespresatu egin zela adierazi zuten. 3.-Peroxisometako β-oxidazioko entzima guztiak (AOX1, MFP1 eta THIO) maila transkripzionalean erregulatuta daude Chelon labrosus lazunen gibel eta zakatzetan, B(a)Pa ziztatu ondoren. Hala ere, ez zen behatu AOX1 proteina edo jarduera mailako aldaketarik organismo beretan. 4.-I faseko bioeraldaketa metabolismoko eta peroxisometako β-oxidazioako proteinak kodetzen dituzten geneen transkripzio mailako indukzioaren ondoren CAT genearen transkripzio mailako gainespresioa eman zen. Azken honek, oxigeno erreaktibo espezieen metabolismoan dihardu, eta beraz, zelularen oxigeno erreaktibo espezieen homeostasian aldaketak eman zirela adierazi zuen. 5.-PMP70 genearen espresio mailan ez zen aldaketa esangarririk eman, β-oxidazioaren indukzio transkripzionala beraz, ez zen peroxisoma kopurua emendatzeko nahikoa izan. Ikerketa sakonagoak beharko dira transkripzio mailan emandako aldaketok peroxisomen proliferazioa eragin dezaketenentz argitzeko. 231 O N D O R I O A K

182 4.- Transcriptional regulation of peroxisome proliferation and biotransformation metabolism in thicklip grey mullets Chelon labrosus exposed to perfluorooctane sulfonate and to Prestige-like heavy fuel oil This chapter has been sent for publication to: BILBAO E, RAINGEARD D, DIAZ DE CERIO O, ORTIZ-ZARRAGOITIA M, RUIZ P, IZAGIRRE U, ORBEA A, MARIGÓMEZ I, CAJARAVILLE MP, CANCIO I. "Effects of exposure to Prestige-like heavy fuel oil on convencional biomarkers and target gene expression of the thicklip grey mullet Chelon labrosus". Toxicology and Applied Pharmacology. Parts of this chapter have been presented at: 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, The Hague, The Netherlands, May 7-11, CANCIO I, BILBAO E, RAINGEARD D, SAEZ-MORQUECHO C, DIAZ DE CERIO O, CAJARAVILLE MP. "Differential gene expression in sentinel marine species under exposure to organic xenobiotics". Open European Peroxisome Meeting 2006, Leuven, Belgium. September 18-19, BILBAO E, CAJARAVILLE MP, CANCIO I. "Cloning and expression analysis of peroxisome proliferation marker genes in aquatic organisms: effects of organic xenobiotics". VII th International Congress on the Biology of Fish. St. John s, Newfounland, Canada, July 18-22, BILBAO E, CAJARAVILLE MP, CANCIO I. "Induction of peroxisomal gene expression in mullets exposed to PFOS and to a heavy fuel oil similar to the Prestige oil". SYMPOSIUM on Marine Accidental Oil spills (VERTIMAR), Vigo, Spain, June 5-8, BILBAO E, RAINGEARD D, DIAZ DE CERIO O, ORTIZ-ZARRAGOITIA M, RUIZ P, IZAGIRRE U, ORBEA A, MARIGÓMEZ I, CAJARAVILLE MP, CANCIO I. "Effects of exposure to Prestige-like heavy fuel oil on convencional biomarkers and target gene expression of the thicklip grey mullet Chelon labrosus". 233

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184 ABSTRACT Transcriptional regulation of peroxisome proliferation and biotransformation metabolism Thicklip grey mullets Chelon labrosus inhabit coastal and estuarine areas where they can bioaccumulate pollutants that may alter their transcriptional profiles. Peroxisome proliferation which in rodents is usually manifested as a transcriptional induction of enzymes involved in lipid homeostasis such as palmitoyl-coa oxidase (AOX1), L-specific multifunctional protein (MFP1), 3- ketoacyl-coa thiolase (THIO) and 2, 4 -dienoyl-coa reductase-2 (DECR), may be one of such effects. In this sense, in order to study transcriptional alterations after exposure to environmentally relevant pollutants, mullets C. labrosus were exposed for 2 and 16 days to a typical mammalian peroxisome proliferator perfluorooctane sulfonate (PFOS), and to fresh (F) and weathered (WF) heavy fuel oil, similar to that spilled by the Prestige tanker in the Northern Iberian Peninsula in November However, as polyclyclic aromatic hydrocarbons (PAHs) such as the ones present in the fuel oil induce phase I and phase II biotransformation metabolism, glutathione-s-transferase (GST) and UDP-glucuronosyltransferase (UGT) were partially cloned and their expression was also studied together with expression of cytochrome P450 1A1 (CYP1A1), involved in phase I biotransformation metabolism. In addition, since some PAHs are excreted as polar metabolites in bile, PAH metabolites were also measured through fixed wavelength fluorescence. It was demonstrated that exposed animals accumulated PAH metabolites in their bile, WF exposed animals being enriched in B(a)P-like metabolites and F exposed ones in naphthalene-like metabolites. AOX1 and CYP1A1 gene expressions and AOX1 enzyme activity were significantly induced in liver after 2 days of F and WF exposure. At day 16, CYP1A1 overexpression was still significant in respect to controls while only F kept AOX1 expression and activity levels significantly above control levels. Only a slightly increased expression of the major peroxisomal membrane protein PMP70 was measured 16 days after WF exposure. Although not significantly, PFOS slightly induced AOX1 gene expression while AOX1 activity was significantly induced after 2 days of PFOS exposure. On the other hand, F inhibited DECR expression 2 days after exposure and WF induced GST expression at day 16. Catalase (CAT) expression in liver was significantly induced after 16 days in all exposure groups, reflecting a possible oxidative stress situation that could be linked to the induced biotransformation and β-oxidation activity recorded at day 2. However, in gills F induced CAT expression already 2 days after exposure together with CYP1A1 overexpression. Expression returned to control levels before day 16 in both cases. In conclusion, exposure to a fresh and weathered heavy fuel oil similar to that spilled by the Prestige tanker, altered hepatic expression and activity levels of the peroxisomal β-oxidation ratelimiting enzyme AOX1 and the biotransformation metabolism followed at later stages by an overexpression of the antioxidant enzyme CAT. These results show that AOX1 is regulated at the transcriptional level in fish and that the process of peroxisome proliferation could follow the same molecular mechanism as in responsive mammalian species. 235

185 Results and Discussion RESUMEN Los mubles Chelon labrosus habitan zonas costeras y estuarios donde pueden bioacumular contaminantes que pueden llegar a alterar sus perfiles transcripcionales. La proliferación de peroxisomas, que en roedores se manifiesta habitualmente como inducción transcripcional de enzimas implicados en la homeostasis lipídica, tales como palmitoil-coa oxidasa (AOX1), proteína multifuncional (MFP1), 3-ketoacil-CoA tiolasa (THIO) y 2, 4 -dienoil-coa reductasa 2 (DECR) puede ser uno de los efectos producidos por los contaminantes. En este sentido, con el fin de estudiar las alteraciones transcripcionales tras la exposición a contaminantes relevantes medioambientalmente, se expusieron mubles C. labrosus durante 2 y 16 días a perfluorooctano sulfonato (PFOS) y fuel-oil pesado, tanto fresco (F) como envejecido (WF), similar al vertido por el Prestige en el Norte de la Peninsula Ibérica en noviembre de Sin embargo, como los hidrocarburos policíclicos aromáticos (PAHs) entre ellos, los presentes en el fuel-oil procedente del Prestige, inducen las fases I y II del metabolismo de biotransformación, se clonaron parcialmente los genes que codifican para glutation-s-transferasa (GST) y UDP-glucuronosiltransferasa (UGT) y se estudió su expresión génica, junto con la expresión del gen que codifica para citocromo P450 1A1 (CYP1A1), implicado en el metabolismo de fase I. Además, como algunos PAHs se excretan como metabolitos polares en bilis, se midieron los metabolitos de PAHs mediante fluorescencia a longitud de onda fija. Así, se demostró que los animales expuestos a fuel oil acumularon metabolitos de PAHs en la bilis; los expuestos a WF acumularon principalmente metabolitos de tipo benzo(a)pireno, mientras que los expuestos a F acumularon metabolitos de tipo naftaleno. La expresión de AOX1 y CYP1A1 así como la actividad de AOX1 se sobreexpresaron en el hígado 2 días después de la exposición tanto a F como a WF. El día 16, la sobreexpresión de CYP1A1 todavía se mantenía por encima de los controles mientras que sólo F era capaz mantener la actividad y la expresión de AOX1 significativamente por encima de los controles. En el caso del principal componente proteico de las membranas peroxisómicas, PMP70, sólo se observó una ligera sobreexpresión tras 16 días de exposición a WF. Aunque no significativamente, PFOS indujo la expresión de AOX1 ligeramente mientras que su actividad se indujo significativamente 2 días después de la exposición. Por otro lado, F inhibió la expresión de DECR tras 2 días de exposición y WF indujo la expresión de GST el día 16. La expresión de catalasa (CAT) en hígado se indujo significativamente tras 16 días de exposición, reflejando una posible situación de estrés oxidativo que podría estar relacionada con la inducción del metabolismo de biotransformación y la β-oxidación determinadas el día 2. Sin embargo, en branquias, F indujo la exposición de CAT tras 2 días de exposición junto con la sobreexpresión de CYP1A1. La expresión volvió a niveles similares obtenidos en los grupos control antes del día 16 en todos los casos. Por lo tanto, la exposición a fuel oil pesado, fresco y envejecido, similar al vertido por el Prestige en el norte de la Península Ibérica, alteró el nivel de expresión y actividad del primer enzima de la β-oxidación peroxisómica AOX1, así como el metabolismo de biotransformación; en fases posteriores, se dio la sobreexpresión del enzima antioxidante CAT. Estos resultados demostraron que la regulación diferencial de AOX1 podría emplearse como biomarcador temprano de exposición a fuel oil pesado. 236

186 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism Introduction Coastal and marine areas are important pollutant recipients via tanker spills, urban runoffs, industrial and sewage effluents or atmospheric deposition. In November 2002 the tanker Prestige sunk offshore the Galician coast affecting more than 2600 km of the North Iberian coastline with more than tones of heavy fuel oil, being one of the 20 major oil spills since 1967 (The International Tanker Owners Pollution Federation Ltd, In subsequent months, heavy fuel oil enriched in heavy polycyclic aromatic hydrocarbons (PAHs) and metals such as Se, Mo, Ni and V, arrived at beaches and cliffs all along the North coast of the Iberian Peninsula. In order to study the biological effects of such pollutants, different exposure and effect biomarkers can be used and among them, peroxisome proliferation has been proposed in aquatic organisms as a promising biomarker of exposure to organic compounds such as PAHs (Cancio and Cajaraville 2000; Cajaraville et al. 2000; 2003; Cajaraville and Ortiz-Zarragoitia 2006). In this way, peroxisome proliferation has been observed in mussels and in different fish species exposed to crude and lubricant oils, PAHs, polychlorinated biphenyls (PCBs) and phthalate ester plasticisers (Cancio and Cajaraville 2000; Cajaraville et al. 2003; Cajaraville and Ortiz-Zarragoitia 2006). Peroxisome proliferation is characterised by increased hepatic peroxisomal volume density, which in rodents is usually accompanied by the transcriptional upregulation of enzymes involved in lipid homeostasis, such as those participating in peroxisomal β-oxidation: palmitoyl-coa oxidase (AOX1), L-specific multifunctional protein (MFP1) and 3-ketoacyl- CoA thiolase (THIO) (Qi et al. 2000). In addition, peroxisomal reductase, in combination with an isomerase, play an important role in the peroxisomal oxidation of virtually all unsaturated fatty acids (Kunau et al. 1995) and induction on 2, 4 dienoyl-coa reductase 2 (DECR) has been reported after exposure to different peroxisome proliferators such as clofibrate (Lei et al. 2003). In vertebrate responsive species, peroxisome proliferation is under the regulation of the peroxisome proliferator activated receptor α (PPARα), a transcription factor that belongs to the nuclear receptor superfamily (Isseman and Green 1990) whose expression has been already described in different tissues of fish (Boukouvala et al. 2004; Ibabe et al. 2004; Raingeard et al. 2006). Considering that piscine genes coding for peroxisomal β-oxidation enzymes contain putative PPARα/RXR binding motifs in their promoter regions (Bilbao et al. submitted a), induction of peroxisomal genes in aquatic organisms might be regulated by a mechanism similar to that in rodents. However, up to day, most of the studies reporting peroxisome proliferation in aquatic species have only measured the process in terms of increased peroxisomal volume density or increased AOX1 activity (Braunbeck and Völkl 1991; Oulmi and Braunbeck 1996; Au et al 1999; Cancio and Cajaraville 2000; Oakes et al. 2003; 2004; Porte et al. 2001; Cajaraville et al. 2003; Bilbao et al 2006; Zorita et al. 2007). Toxicity of some pollutants depends among other factors, on the rate in which they are biotransformed (Ferreira et al. 2006). In this way, the biotransformation metabolism transforms PAHs into mutagenic/carcinogenic intermediates (Buters et al. 1999). Two processes can be distinguished in the biotransformation metabolism. Phase I metabolism consists in oxidative reactions where cytochrome P450 (CYP) monooxygenases play the central role as catalysts, resulting in hydroxylation of parent compounds and leading to a suitable functionalised substrate for phase II metabolism. As a consequence, lipophilic compounds are excreted as polar metabolites in bile, the levels of biliary metabolites constituting a sensitive biomarker to assess recent exposure to organic toxic compounds (Van der Oost et al. 2003). Among the different CYP families, in vertebrates, organic contaminants such as PCBs or PAHs are specifically oxidised by cytochrome P450 1A coded by the CYP1A gene. This gene in turn, is regulated by the substrates that its 237

187 Results and Discussion product enzyme helps to metabolise, so its expression is upregulated in the presence of PCBs or PAHs (Hahn and Stegeman 1994; Jönsson et al. 2006). The induction of CYP1A expression is widely used as a biomarker of exposure to these compounds (Hahn and Stegeman 1994; Arinç et al 2000; Van der Oost et al. 2003). Phase II metabolism involves the conjugation of the functionalised compound with an endogenous substrate, thus facilitating the excretion of lipophilic chemicals by the addition of a polar group (Van der Oost et al. 2003). Addition of glutathion is one of such steps catalysed by glutathione-s-transferases (GST) which are quantitatively the most important phase II detoxification enzymes (Fitzpatrick et al. 1997). Glucuronidation is another important conjugation reaction for the excretion of lipophilic chemicals (George and Leaver 2002) adding UDP-glucuronic acid to xenobiotics in a process catalysed by UDPglucuronosyltransferases (UGT) (George and Taylor 2002). Significant effects of PAHs and halogenated xenobiotics on fish GST and UGT enzyme activities have been reported in field and laboratory studies (Clarke et al. 1992; Vigano et al. 1998; Van der Oost et al. 2003; Gaworecki et al. 2004). Interestingly, human CYP1A1 contains 2 peroxisome proliferation response elements (PPRE) in the promoter region and several peroxisome proliferators are able to enhance CYP1A1 activity by a mechanism that still remains to be elucidated (Sérée et al. 2004). Similarly, Barbier et al. (2003) identified UGT as a PPARα and PPARγ target gene in mice while GSTA1 in the flatfish Pleuronectes platessa contains functional PPREs in its promoter region (Leaver and George 1996; Boukouvala et al 2004). Induction of biotransformation metabolism and peroxisomal β-oxidation produce highly reactive oxygen species (ROS) as by-products (Jifa et al. 2006) leading to oxidative stress, increasing the possibilities for appearance of mutagenic or carcinogenic lesions (Reid and MacFarlane 2003). In this way, the antioxidant defence may be induced in cells as response to ROS generated after chemical exposure, so measurement of components of the antioxidant defence such as catalase activity may be also helpful to determine organism exposure to lipophilic organic compounds (Cheung et al. 2001; Livingstone 2001). Therefore, a laboratory experiment was designed where thicklip grey mullets Chelon labrosus, common in estuarine environments with high pollutant burdens, were exposed to different pollutants: fresh Prestige-like heavy fuel oil, trying to mimic first instants after the spill and weathered fuel oil in order to mimic the situation in the coastline that received the fueloil after long periods in the water. Mullets were additionally exposed to a perfluorinated compound, perfluorooctane sulfonate (PFOS) as a typical mammalian peroxisome proliferator. Fluorinated alkyl substances constitute a diverse class of chemicals that occur in a wide range of products. PFOS is used as surfactant and surface protector in carpets, leather and paper as well as in chemicals such as floor-polishes or firefighting foams (Giesy and Kannan 2002). Perfluorinated compounds are resistant to hydrolysis, photolysis, biodegradation and metabolism in the environment, resulting in a high degree of environmental persistence, PFOS being biomagnified along the marine food chain (Schultz et al. 2003; Bossi et al. 2005). Effects caused by fluorinated compounds have been studied in mammalian model species in which they have been shown to be peroxisome proliferators (Ikeda et al. 1987; Sohlenius et al. 1993) and hypolipidemic agents (Seacat et al. 2003). Information regarding toxic effects caused by fluorinated compounds in aquatic organisms is scarce but Oakes et al. (2004) observed AOX1 activity induction in liver of the fathead minnow Pimephales promelas. Thus, in the present study, peroxisome proliferation and biotransformation metabolism were studied at the gene (AOX1, MFP, THIO, DECR, PMP70, CAT, CYP1A, GST and UGT) expression level in gills which are in direct contact with the water, and in liver, which in marine species is the main site of biotransformation and peroxisomal β-oxidation metabolism. 238

188 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism Material and Methods Materials All chemicals were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) unless specified otherwise. Animals and experimental procedure Thicklip grey mullets Chelon labrosus (11-19 cm in length) were fished in Plentzia, Bay of Biscay (43º N; 2º W), in September This locality is considered to be relative clean of toxic chemical compounds (Orbea and Cajaraville 2006). Organisms were acclimatised to laboratory conditions in continuously aerated sea water for two weeks. No mortalities were observed during the second acclimatisation week. Water was from a non-polluted site in Getaria (43º17'22''N; 2º08'48''W) that is supplied to local restaurants to keep shellfish for human consumption. After acclimatisation, organisms were divided into 4 different polyethylene tanks containing 300 l of sea water and a sediment bed composed of 5 kg of gravel and 6 kg of sand. During the experiment, water was constantly aerated (dissolved oxygen mg/l; ph ; salinity 35 ; temperature 20ºC) and it was not replaced. Animals were kept under a constant 12 h light/dark cycle and were fed daily with dried bread. Organisms were exposed for 2 and 16 days to 2 ppm of PFOS dissolved in sea water, fresh heavy fuel oil (F) and weathered heavy fuel oil (WF). Animals in the forth group were unexposed. Fresh fuel oil was prepared mixing 150 ml of heavy fuel oil similar to the one spilled by the Prestige tanker (IFO 380, marine fuel RMG 35-ISO 8217) with the sediment substrate just before the beginning of the experiment. Weathered fuel oil was prepared mixing the same amount of fuel oil with the substrate but the tanks were prepared 2 ½ months before the beginning of the experiment. During the weathering process, constant aeration was maintained in the aquarium. For all studies, fish were anesthetised by immersion in a saturated solution of 3- aminobenzoic acid ethyl ester before being sacrificed. Liver and gill samples of 5 mullets per experimental group were embedded in RNA later and frozen in liquid nitrogen (whole gills, 100 mg liver) for genomic studies. Additionally, liver samples of the same 5 organisms were frozen in liquid nitrogen for the spectrophotometrical determination of palmitoyl-coa oxidase activity. Bile samples of 10 mullets belonging to each control and F and WF exposed tanks were also frozen in liquid nitrogen for the measurement of naphthalene, pyrene and benzo(a)pyrene like metabolites. All collected samples were stored at -80ºC until further required for downstream procedures. Fixed wavelength fluorescence analysis Bile samples were diluted 1:1000 in 50% ethanol. Fixed wavelength fluorescence (FF) was then measured at the excitation/emission wavelength pairs 290/335, 341/383 and 380/430 nm, denoted FF 290/335, FF 341/383 and FF 380/430, respectively. At FF 290/335, mainly naphthalene type of low molecular weight PAH metabolites, typically associated with petroleum products, are detected. Benzo(a)pyrene (B(a)P) type of heavy PAH metabolites are best detected at FF 380/430 (Krahn et al. 1987). By FF 341/383, mainly pyrene-derived metabolites are detected (Aas et al. 2000). Measurements were performed in quartz cuvettes in a SLM2 Aminco luminescence spectrometer (Spectronic Instruments, Rochester, New York, USA) and slit widths were set at 4 nm for both excitation and emission wavelengths. The FF values were expressed as arbitrary fluorescence and the signal levels of the solvent were subtracted. PAH and PFOS concentration in water PAH and PFOS concentration in water were measured in the Department of Analytical Chemistry of the University of the Basque Country. PAHs were measured as described by Navarro et al. (2006) while methodology for the measurement of PFOS was slightly modified from Taniyasu et al. (2005). Palmitoyl-CoA oxidase (AOX1) activity measurement: AOX1 activity was determined 239

189 Results and Discussion spectrophotometrically measuring the H 2 O 2 dependent oxidation of dichlorofluorescein diacetate (Molecular Probes, Eugene, Oregon, USA) catalysed by an exogenous peroxidase, using 30 µm palmitoyl-coa as substrate according to Small et al. (1985). Liver samples of 5 animals were individually homogenised in TVBE buffer (1 mm sodium bicarbonate, 1 mm EDTA, 0.1 % ethanol and 0.01 % Triton X-100), ph 7.6 (4 ml / g tissue) in a Hybaid Ribolyser TM (Hybaid, Ashford, UK). After centrifugation at 500g at 4ºC for 15 min in a Jouan CR-312 centrifuge (Jouan, Saint-Herblain, France), supernatants were diluted 1:40 in TVBE buffer and assayed for AOX1 activity using a Shimadzu UV-1603 spectrophotometer (Shimadzu, Duisburg, Germany). Total protein concentration was measured using the DC protein assay (BioRad, San Diego, California, USA) based on the Lowry method and measuring γ-globulin as standard. AOX1 activity is given as mu AOX mg -1 total protein equivalent to nmol H 2 O 2 min - 1 mg -1 protein. Cloning of target sequences mg of liver or gills were homogenised in Trizol (Invitrogen, Carlsbad, California, USA) using a Hybaid Ryboliser TM homogeniser for 20 (liver) or 40 seconds (gill), at 4 m/s. 3 µg of total RNA were used for cdna synthesis by RT-PCR (Invitrogen) using random hexamers according to manufacturer's instructions. UGT, GST and PMP70 cdna fragments were amplified by PCR using degenerate primers. By aligning known UGT sequences of Gasterosteus aculeatus (ENSGACT ), Pleuronectes platessa (X74116), Pleuronectes yokohamae (AB120133), Epinephelus coioides (AY735003) and Tetraodon nigroviridis (CAAE ), Fw 5'- CTGGTGCCYGAAASYAGCCTG-3' and Rv 5'-TCTYYGCACAGTTGATVCCTCC-3' were designed and used as primers for the amplification of a UGT fragment of 660 bp. Fw- 5'-AGGACATGACTCTGCTGTGGG-3' and Rv-5'-CCCTCAAACATGCGYTGGTACAT-3' designed by aligning GST sequences of Sparus aurata (AY362762), Pagrus major (AB158412), Micropterus salmoides (AY335905), Platichthys flesus (AJ310428) and Pleuronectes platessa (X95199) were used as degenerate primers for the amplification of a mullet GST fragment of 290 bp. Fragments were purified using a PCR purification kit (Qiagen, Hilden, Germany) and cloned using a TOPO-TA cloning kit (Invitrogen). Products were sequenced in the Sequencing Service of the Department of Genetics, Physical Anthropology and Animal Physiology (University of the Basque Country) using M13 Fw and Rv primers. Semiquantification of gene expression Monoplex-PCR conditions were optimised for the amplification of bp fragments of UGT and GST (Table 1) using Taq polymerase (Invitrogen) in a conventional icycler termocycler (BioRad). PCR products were visualised through an agarose gel (1.5%) electrophoresis where amplicons were visualised using ethidium bromide and analysed using a computer-aided gel analyser (Gel-Doc-2000, BioRad). Specific sequences of mullet catalase (CAT, AY743715), palmitoyl-coa oxidase (AOX1, EF525541), L-specific multifunctional protein (MFP1, EF407560), 3-ketoacyl-CoA thiolase (THIO, DQ021958), 2, 4 -dienoyl- CoA reductase 2 (DECR, EF407559), 70 kda peroxisomal membrane protein (PMP70, EF407558) and CYP1A1 (DQ438983) were obtained from previous studies by our group Table 1: PCR conditions for the specific amplification of GST and UGT cdnas in liver of thicklip grey mullets Chelon labrosus. All protocols began with a 2 minute denaturising step at 94ºC. At the end a final extension step of 8 minutes at 72ºC was added. Number of cycles for GST, 30 and for UGT, 27. GST UGT Fw and Rv 5-3 Fw-CCTGCTGGAGGGTTATGATTGC Rv-GCTTGGTTCCCTGGGACTTGTA Fw-CCAGCCTGTAATGAGTCGAC Rv-CGCACAGTTGATGCCTCC PCR conditions 94º C for 25, 58º C for 25, 72º C for 45 94º C for 30, 56º C for 30, 72º C for

190 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism (Bilbao et al. submitted a; b). β-actin (AY836368) and 18S rrna (AY825252) were measured as housekeeping genes following PCR conditions described by Raingeard et al. (2006). Results have been expressed against the less variable housekeeping gene. Statistical analysis Statistical analysis was performed using SPSS 13 where the Mann Whitney U test was applied to establish significant differences between groups in gene expression determinations. Student t test was applied to establish significant differences between groups in AOX1 activity measurements. Significant differences between means were established at p<0.05. Results Cloning of target sequences A 556 bp long cdna fragment of the coding domain sequence (cds) of UGT was amplified and sequenced from mullet liver (EF407557). The deduced amino acid sequence showed 75% amino acid identity with the UGT sequence of E. coioides (AAW29020), 71% with a Gasterosteus aculeatus EST (DN699857), and 57% with P. yokohamae UG1B2 (AB120133). The same methodology was applied in the cloning of a mullet 282 bp GST fragment corresponding to its N-terminal domain (EF407556), which showed a deduced 80% amino acid identity with P. platessa GSTA (GST class-theta, CAA64495) and M. salmoides GST (AY335905). Experimental behaviour No mortality was observed during the experiment. However, mullets exposed to PFOS did not eat or ate less than organisms in the rest of the experimental groups. In addition, mullets seemed to be suffering of some kind of narcotic effect and limited to swim in the surface of the tank from the first day of exposure. Chemical analysis of water dissolved compounds PFOS concentration in water was similar after 2 and 16 days of experiment, 0.46 and 0.44 ppm respectively. In fuel exposed tanks, the highest PAH concentration (16 Environmental Protection Agency priority PAHs) was measured at day 2 in the F exposed tank (97.18 ppm vs ppm in control and 18.2 ppm in WF). However PAH concentrations decreased considerably at day 16, all experimental groups showing similar concentrations (3.79 ppm control, 3.5 ppm F and 6.28 ppm WF), probably due to evaporation, adsorption to tank walls, and bioaccumulation. PAH metabolites The measurement of PAH bile metabolites revealed that animals exposed to F and WF accumulated and, at least partially, metabolised PAHs. B(a)P-, pyrene- and naphthalene-like metabolites were determined in biles of all measured individuals (Figure 1). Results are given as fluorescence intensity fold induction in comparison with fluorescence intensity in control biles. High fluorescence intensity at the emission wavelength of the heavy PAHs (B(a)Plike) was detected in the WF exposed samples at day 2 (Figure 1), the fluorescence intensity being 14 times higher than in control samples. F exposed individuals showed also high fluorescence intensity at this wavelength but just 10 times above control levels. Pyrene-like metabolites showed similar fluorescence intensities in both exposure groups (10 times more than in controls) while naphthalene-like metabolites in F exposed samples were enriched (8 fold increase over controls) above WF levels (5 fold increase). In all cases, fluorescence intensities significantly decreased at day 16 although they kept above control levels (Figure 1). However, at day 16 B(a)P- and pyrene-like metabolites showed slightly higher fluorescence intensities in WF than in F, while F showed higher fluorescence intensities than WF when naphthalene-like metabolites were measured. 241

191 Results and Discussion DAY2 DAY B(a)P-like metabolites Pyr-like metabolites Naph-like metabolites 0 B(a)P-like metabolites Pyr-like metabolites Naph-like metabolites Figure 1. Fluorescence intensity measured at 380/430, 341/383 and 290/335 nm excitation/emission wavelengths for benzo(a)pyrene-like, pyrene-like and naphthalene-like metabolites represented as fluorescence emission fold increase in biles of mullets exposed to fresh (grey bars) and weathered (dark grey bars) heavy fuel oil for 2 and 16 days, considering that emission intensity in control samples was 1 (white bars). Vertical bars represent standard deviations. Target gene expression levels Among the housekeeping genes measured in gills and liver, 18S rrna showed the lowest coefficient of variability in gills, while β-actin was less variable in liver. Thus, results in liver have been normalised against β-actin and against 18S rrna in gills. CYP1A1 expression was significantly induced in liver 2 and 16 days after exposure to F and WF oil while in gills, CYP1A1 induction was only observed at day 2 (Figure 2). When looking at the expression levels of genes coding for phase II biotransformation enzymes, induction of GST was observed after 16 days of WF exposure (Figure 2). However, no changes were observed in UGT expression levels. On the other hand, PFOS had no effect on any of the biotransformation genes studied (Figure 2) Regarding the expression of peroxisomal genes, expression of AOX1 was induced in liver 2 and 16 days after F exposure (Figure 3) while WF only induced AOX1 expression at day 2. In gills AOX1 expression showed high variability in organisms within each experimental group though a trend indicating expression induction was observed, mainly in the F exposed group (data not shown). AOX1 expression was only slightly induced in liver and gills after PFOS exposure (Figure 3) and MFP1 and THIO expression levels showed no variations along the experiment (Figure 3) as it was the case with PMP70 which only showed a slight increasing trend after 16 days of WF exposure (Figure 4). DECR was slightly induced 16 days after WF exposure while F inhibited DECR expression at day 2 (Figure 3). Finally, induction of CAT expression was observed after 16 days of exposure to PFOS and to both fuel oils (Figure 5). In gills, a significant CAT overexpression was observed 2 days after F exposure, while no CAT expression could be detected in the 16 days WF exposed animals (Figure 5). Palmitoyl-CoA oxidase activity AOX1 activity was induced after 2 and 16 days of F exposure while WF induced AOX1 activity only at day 16 (Figure 6). In addition, AOX1 activity was also induced by PFOS exposure, induction being significant only after 2 days of exposure. Discussion A short exposure to fresh and weathered heavy fuel oil similar to that spilled by the Prestige tanker and to the perfluorinated compound PFOS, altered the activity of the β- 242

192 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism 3 DAY2 * * CYP1A1 in gills 3 DAY Cn PFOS F WF Cn PFOS F WF CYP1A1 in liver * * * * Cn PFOS F WF 0 Cn PFOS F WF 2 UGT in liver Cn PFOS F WF 0 Cn PFOS F WF GST in liver * Cn PFOS F WF 0 Cn PFOS F WF Figure 2. Mean expression values of cytochrome P450 1A1 (CYP1A1) in gills and liver. Mean expression values of glutathione-s-transferase (GST) and UDP-glucuronyltransferase (UGT) measured in liver of 5 mullets, 2 and 16 days after exposure to perfluorooctanesulfonate (PFOS), fresh (F) and weathered (WF) fuel oil. All control mean values have been taken to 1, so fold-relative variations in expression can be best observed. Vertical segments show standard deviations. Statistically significant differences between treatment and control (Cn) means are indicated by asterisks. 243

193 Results and Discussion DAY2 AOX1 DAY * * * Cn PFOS F WF 0 Cn PFOS F WF MFP Cn PFOS F WF 0 Cn PFOS F WF THIO Cn PFOS F WF 0 Cn PFOS F WF 4 DECR * Cn PFOS F WF 0 Cn PFOS F WF Figure 3. Mean expression values of palmitoyl-coa oxidase (AOX1), multifunctional protein 1 (MFP1), thiolase (THIO) and 2, 4 -dienoyl-coa reductase 2 (DECR) in liver of 5 mullets after 2 and 16 days of exposure to perfluorooctanesulfonate (PFOS), fresh (F) and weathered (WF) fuel oil. Control means have been taken to 1, so foldrelative variations in expression can be best observed. Vertical segments show standard deviations. Statistically significant differences between treatment and control (Cn) means are indicated by asterisks. Statistical significance was established according to the U-test (p<0.05). 244

194 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism DAY2 DAY16 PMP Cn PFOS F WF 0 Cn PFOS F WF Figure 4. Mean expression values of peroxisomal membrane protein of 70 kda (PMP70) measured in liver of 5 mullets after 2 and 16 days of exposure to perfluorooctanesulfonate (PFOS), fresh (F) and weathered (WF) fuel oil. Control means have been taken to 1, so fold-relative variations in expression can be best observed. Vertical segments show standard deviations. Statistical significance was established according to the U-test (p<0.05). oxidation rate limiting enzyme AOX1 at the transcriptional level in mullets Chelon labrosus. Similarly, biotransformation metabolism was induced by oil exposure but not by PFOS. Both processes were accompanied by an overexpression of CAT. Low PAH concentrations were measured in WF water at day 2, probably due to the high grade of accumulation of medium or high molecular weight PAHs in fish as indicated by the high levels of metabolites detected in their biles, in addition to adsorption to the tank walls or evaporation of low molecular weight PAHs. Additionally, PAH concentration in water decreased significantly at day 16. In fish, some PAHs are excreted after accumulation in the liver as polar metabolites so the levels of PAH metabolites in bile, are sensitive biomarkers to assess recent exposure to PAHs (Van der Oost et al. 2003). In the present study, fluorescence emission intensities determined in biles of organisms exposed to Prestige-like fuel oil showed that fish readily accumulated and metabolised PAHs to polar compounds for elimination above control levels. These results are in agreement with Ferreira et al. (2006) who reported that mullets have the ability to metabolise and eliminate PAHs in this way. Measured PAH metabolites also reflected that the PAH composition in both fuel exposure groups was different, in this sense, naphthalenelike compounds predominated in F while high molecular weight ones (B(a)P-like) predominated in WF. Thus, we believe that we were able to mimic the situation in the field after the Prestige oil spill. Low molecular weight PAHs such as naphthalene, are associated with acute toxicity whereas some of the high molecular weight PAHs such as B(a)P, form carcinogenically active metabolites after being biotransformed (Neff 2002). Among the effects that such compounds may cause on biota, peroxisome proliferation is defined as a pleiotropic cellular response characterised by increased volume density of peroxisomes usually accompanied in rodents by the transcriptional induction of enzymes involved in lipid homeostasis, being specially relevant those involved in peroxisomal β- oxidation (Qi et al. 2000). Several laboratory and field studies have shown that hypolipidemic drugs, phthalate ester plasticisers, PAHs and oil derivatives, PCBs, certain pesticides, bleached kraft pulp and paper mill effluents (BKME), alkylphenols and estrogens may provoke peroxisome proliferation (Beier and Fahimi 1991; Cancio and Cajaraville 2000; Cajaraville et al. 2000; Dzhekova-Stojkova et al. 2001; Nakayama et al. 2005; Oakes et al. 2005; Cajaraville and Ortiz-Zarragoitia 2006). However, peroxisome proliferation has been 245

195 Results and Discussion DAY2 DAY16 CAT in gills 3 * * 0 Cn PFOS F WF 0 Cn PFOS F WF CAT in liver 2 2 * * * Cn PFOS 0 F WF Cn PFOS F WF Figure 5. Mean expression values of catalase (CAT) measured in gills and liver of 5 mullets after 2 and 16 days of exposure to perfluorooctanesulfonate (PFOS), fresh (F) and weathered (WF) fuel oil. Control means have been taken to 1, so fold-relative variations in expression can be best observed. Vertical segments show standard deviations. Statistically significant differences between treatment and control (Cn) means are indicated by asterisks. Statistical significance was established according to the U-test (p<0.05). mainly measured in terms of increased peroxisomal volume density or activity inductions of AOX1, the first and rate limiting enzyme in peroxisomal β-oxidation. Peroxisome proliferation has been measured in different fish species such as Sparus aurata (Pedrajas et al. 1996), Ictalurus puctatus (Mather-Mihaich and Giulio 1991), Onchorynchus mykiss (Yang et al. 1990; Scarano et al. 1994; Oakes et al. 2005), Danio rerio (Ortiz-Zarragoitia and Cajaraville 2005) and Pimephales promelas, Semotilus atromaculatus, Notropis hudsonius or Catastomus commersoni (Oakes et al. 2005), Clupea harengus and Pollachius virens exposed to different compounds (Bilbao et al. 2006). In addition, induction of AOX1 expression was observed in mullets after a single intraperitoneal injection with B(a)P (Bilbao et al. submitted b). However, other studies have reported low or no sensitivity towards peroxisome proliferators in Cyprinus carpio and Oryzias latipes (Scarano et al. 1994; Hoff et al. 2003). In the present work, 2 and 16 days of F exposure induced AOX1 gene expression and enzyme activity level in liver. However, since WF only induced AOX1 expression after 2 days of exposure, followed by an induction of AOX1 activity at day 16, it could be suggested that WF needs longer for altering peroxisomal β-oxidation at the protein level. In the same way, the fact that intraperitoneal B(a)P injection induced the 3 genes coding for peroxisomal β-oxidation enzymes in mullet liver, but it did not induce AOX1 activity (Bilbao et al. submitted b), could indicate that protein translation needs some time to follow gene transcription. In addition, in the present experiment, Bilbao et al. (2007) also reported an induction of PPARα expression 2 days after exposure to both heavy fuel oils. This expression co-regulation of both AOX1 and PPARα, 246

196 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism suggests that peroxisome proliferation may occur through a PPARα driven mechanism, acting on a possible peroxisome proliferation response element (PPRE) in mullet AOX1 gene promoter region. Presence of putative PPREs in piscine peroxisomal genes has been proved to be feasible by in silico analysis of the available piscine genome sequences (Bilbao et al. submitted a). In gills AOX1 expression showed high variability within organisms of the same group though an increasing trend was observed, mainly in the F group, suggesting that probably since liver is the tissue where the peroxisomal β- oxidation is most relevant (Nanton et al. 2003), it is more sensitive to the effect of peroxisome proliferators than gills. On the other hand, no alterations in the hepatic expression levels of the other two β-oxidation enzymes coding genes was detected although a slight induction of THIO expression was observed after 16 days of exposure. On the contrary, the model PAH B(a)P induced expression of both genes in liver of mullets 1 day after a single intraperitoneal injection (Bilbao et al. submitted b). DECR plays an important role in the oxidation of virtually all unsaturated fatty acids (Kunau et al. 1995) and induction of its expression has been reported in rodents after exposure to different peroxisome proliferators such as clofibrate (Lei et al. 2003). Similarly, DECR was cloned from a normalised library obtained from livers of Aroclor 1254 exposed Platichthys flesus (Williams et al. 2006) and from a suppression subtractive library obtained from oysters Crassostrea gigas exposed to a mixture of hydrocarbons (Boutet et al. 2004). On the contrary, in the present work down-regulation of DECR expression was observed in F exposed organisms at day 2 while in a previous study, B(a)P injection did not alter its expression (Bilbao et al. submitted b). The 70 kda peroxisomal membrane protein (PMP70) major component of peroxisomal membranes, is markedly inducible by the administration of peroxisome proliferating compounds in parallel with the induction of peroxisomal fatty acid β-oxidation enzymes in responsive species (Imanaka et al. 1999; 2000). In the liver of the mummichog Fundulus heteroclitus for instance, a significant increase in PMP70 protein was observed immunochemically using polyclonal antibodies against the mammalian protein after 7, 14 and 21 days waterborne exposure to the model peroxisome proliferator 2, 4- dichlorophenoxyacetic acid (Ackers et al. 2000). Presently, expression studies have not revealed significant alterations after exposure, although there was an increasing trend after exposure to fuel oils for 16 days. When measuring peroxisomal volume density stereologically through catalase histochemistry, a significant increase was detected in animals exposed to F for 16 days (Bilbao et al. 2007). Thus, it seems that heavy fuel oil provokes a moderate increase in peroxisomal volume densities that does not require any significant transcriptional upregulation of PMP70. Hereby only a significant but moderate induction of AOX DAY 2 DAY 16 * AOX activity * Cn PFOS F WF * * Cn PFOS F WF Figure 6. Spectrophotometric measurement of AOX1 enzyme activity (mu/mg protein) in liver of mullets after 2 and 16 days of exposure to perfluorooctanesulfonate (PFOS), fresh (F) and weathered (WF) fuel oil. Values are given as means ± standard deviations (n=5) and asterisks indicate statistically significant differences between controls and exposed mullets. Statistical significance was established according to the t-test (p<0.05). 247

197 Results and Discussion gene expression and enzyme activity, in comparison with the 25-fold induction detected in rodent species (Cancio and Cajaraville, 2000), has been detected without any transcriptional alteration in the rest of the enzymes participating in the peroxisomal β-oxidation pathway. This could indicate that a moderate increase in peroxisomal enzymes does not require any de novo synthesis of new peroxisomes. In an injection experiment using B(a)P all the three genes in the peroxisomal pathway were significantly up-regulated, but similarly to the observations presented here no regulation of PMP70 expression was detected (Bilbao et al. submitted). The cytochrome P450 1A (CYP1A) subfamily of monooxygenases is involved in phase I biotransformation metabolism and can oxidise PAHs effectively in the liver of mullets (Wang et al. 2003; Ferreira et al. 2006). Transcriptional induction of CYP1A is a sensitive and specific adaptive response in fish exposed to PAHs, PCBs or BKMEs (Hahn and Stegeman 1994; Woodin et al. 1997; Mimura and Fujii-Kuriyama 2003; Van der Oost et al. 2003; Jönsson et al. 2006) and thus, it is commonly used as biomarker of exposure to such compounds (Hahn and Stegeman 1994). In the present study, CYP1A1 expression was induced in liver after 2 and 16 days of heavy fuel oil exposure while in gills, expression was only induced after 2 days of exposure. Liver is the main detoxifying organ in vertebrates however, gills are in direct and continuous contact with the surrounding water, so gills might respond to low concentrations of pollutants. Moreover, it has been suggested that the response might disappear more rapidly in gills than in liver after termination of exposure (Jönsson et al. 2003). In this sense, CYP1A expression under exposure to rapidly metabolisable substances should preferably be measured in tissues in direct contact with the environment such as intestine or gills (Levine and Oris 1999). Anyway, according to our results, both tissues showed similar inducibility under the exposure conditions applied. Phase II biotransformation metabolism, measured in terms of GST expression levels in liver of mullets, was only induced after 16 days exposure to WF while UGT expression was unaltered. Hepatic GST has been reported to increase in several studies where fish were exposed to PAHs, PCBs and polychlorinated dibenzo-p-dioxins, however, most studies did not demonstrate any significant alterations (Van der Oost et al. 2003). Β-naphthoflavone upregulated a GSTA mrna in plaice tissues, and expression of 2 different GSTA mrnas was increased in plaice liver and gill by pretreatment with the peroxisome proliferating agent perfluorooctanoic acid (Leaver et al. 1997) A time-dependent hepatic GST induction was observed, after exposure to a water-soluble fraction of diesel oil in Prochilodus lineatus (Simonato et al. 2007). Similarly, GST activity has been correlated with PAH tissue content in several bivalve species (Cheung et al. 2001; Gowland et al. 2002; Rocher et al. 2006). However, significant decreases in GST activity have been reported in Dicentrarchus labrax and Lepomis macrochirus exposed to PAHs (Lemaire et al. 1992), and in fish species from polluted environments (Otto et al. 1996; Tuvikene et al. 1999). Similarly, GST liver activity did not clearly correlate with accumulated PCB and DDT levels or PAH bile metabolites in mullets from the Douro river (Ferreira et al. 2004; 2006). In this sense, in agreement to present results, Sturve et al. (2006) observed significant increases in CYP1 protein expression but a lower responsiveness in GST catalytic activity in PAH exposed cod Gadus morhua. Therefore, they suggested that hepatic total GST activity might not be a reliable biomarker of PAH exposure in fish, unless specific GST isoenzymes that exhibit selective responses to pollutants are investigated (Leaver et al., 1997; Schreiber et al. 2006). Similarly, Ferreira et al. (2006) and Van der Oost et al. (2003) concluded that this enzyme does not seem to be very useful as an exposure biomarker for environmental risk assessment. Regarding UGT expression it is well documented that it is under regulation of the aryl hydrocarbon receptor (Zhou et al. 2005), so exposure to chemicals that induce CYP1A expression may also increase UGT expression as it was the case in flounder from the contaminated Tyne estuary (Williams et al. 2003). UGT 248

198 Transcriptional regulation of peroxisome proliferation and biotransformation metabolism activity has also been reported to increase in fish exposed in laboratory conditions to PAHs and PCBs, as reviewed by Van der Oost et al. (2003), or in fish collected from sites with high sediment PAH burdens in correlation with EROD activity levels (Schreiber et al. 2006). On the contrary, significant decreases in UGT activities were observed in BKME, which is also an AhR agonist, exposed Coregonus lavaretus and in trouts caged in a BKME-polluted environment (Oikari and Nakari 1982). In our study, exposure to Prestige-like heavy fuel oil did not alter UGT hepatic expression in mullets. A perfluorinated compound, PFOS, was also studied in the present report as a typical peroxisome proliferating compound in mammals (Hu et al. 2005; Guruge et al. 2006) trying to compare the response of mullet peroxisomes in the present exposure scenario. The perfluorinated compound perfluorooctanic acid (PFOA) did induce AOX1 activity in the fathead minnow Pimephales promelas (Oakes et al. 2004) while PFOS consistently increased hepatic AOX1 activity and increased oxidative damage in P. promelas, Oncorhynchus mykiss, Semotilus atromaculatus, Notropis hudsonius and Catastomus commersoni (Oakes et al. 2005). Perfluorinated compounds are utilised in many industrial and commercial processes and due to their persistence, bioaccumulation capacity and low to no degradation possibility (Key et al. 1998) are globally accumulated in living organisms (Bossi et al. 2005). After 2 and 16 days of exposure to 2 ppm of PFOS in water, PFOS concentration in water was quite low (around 0.45 ppm). Among the studied effects of exposure to perfluorinated compounds, changes in genes that code for peroxisomal β-oxidation enzymes and GST have been observed in rats (Hu et al. 2005; Guruge et al. 2006). Under the applied exposure conditions mullets suffered some kind of narcotic effect and they were limited to swim in the surface of the tank from the first day of exposure. Mullets did not eat or ate less than controls. This was suggested also in rats exposed to PFOA where suppression of G- protein associated signalling genes was observed, possibly leading to a disregulation of food intake and energy homeostasis (Guruge et al. 2006). In addition, significant AOX1 expression was observed 16 days after exposure in gills, while in liver only a slight induction at AOX1 expression levels was observed. AOX1 activity was significantly induced at day 2 in liver and was slightly increased at day 16. Similar to observations in heavy fuel oil exposed animals, AOX1 activity induction was not paralleled by any regulation of PMP70 or any of the other β-oxidation genes. As expected, no effects were detected at all on the expression of the biotransformation enzymes as PFOS is not acting through the AhR pathway. Finally, following alterations in studied metabolisms after exposure to different pollutants, significant induction of CAT expression was observed in liver at day 16 in the exposed groups, in agreement with results obtained after injection of B(a)P in mullets (Bilbao et al. submitted b). This is interpreted as a response to the oxidative stress resulting from an increased oxidative metabolism produced by CYP1A1 and AOX1 enzyme activities. In this sense, mullets exposed in the field to a mixture of contaminants showed high levels of antioxidant defences which after 1, 4 and 8 months of depuration were significantly decreased, CAT activity showing the most significant decrease in correlation with a lowered EROD activity (Ferreira et al. 2005; 2007). In isolated rainbow trout hepatocytes exposed to a complex of 20 environmentally relevant contaminants (PAHs, PCBs and pesticides) increased CAT activity was detected (Strmac and Braunbeck 2002). In Lepidorhombus boscii from a fuel-oil impacted area (Martínez-Gomez et al. 2006) CAT induction was determined and in fathead minnow, rainbow trout, Catastomus commersoni, Notropis hudsonius and Semotilus atromaculatus exposed to PFOS, increased oxidative damage was reported following AOX1 induction (Oakes et al. 2005). However, CAT activity has been disregarded as a valid biomarker since both induction and inhibition are observed after exposure to environmental pollutants (Van der Oost et al. 2003). In this sense, although F induced CAT expression in gills 2 days after exposure, WF completely depleted CAT expression at day 16. Thus, CAT response in gills seems to be more variable than 249

199 Results and Discussion in liver. In addition, one of the lines of defence against lipid peroxidation is provided by the peroxidase activity of GSTs towards lipid hydroperoxides. However, hydroperoxides may also be decomposed to unsaturated alkenals, hydroxylalkenals and malondialdehyde which are extremely toxic to cells and can bind to DNA and proteins unless detoxified by GST-catalysed conjugation to glutathione (Martínez-Lara et al. 2002). Thus, ROS production could also have induced the GST induction detected in WF after 16 days of exposure. In conclusion, the perfluorinated compound PFOS slightly induced expression of AOX1. F and WF exposure conditions, that tried to mimic the situation in the North Iberian coast after the Prestige oil spill, induced alterations in the expression of genes coding for enzymes in the biotransformation metabolism and in genes coding for peroxisomal proteins. However many responses may have been masked by the presence of different kinds of chemical compounds such as metals mixed with PAHs. In addition, different rates of response were observed in liver and gills after exposure to both fuel oil conditions which may be related with the different chemical composition of both fuel oils and with the different role of each organ in metabolising such compounds. It has been observed that peroxisomes respond moderately to exposure to the chemical compounds tested. The up-regulation of AOX1 transcription was not accompanied by the typical alterations in the rest of peroxisomal parameters (induction of MFP1, THIO and PMP70). Deciphering whether peroxisome proliferation in mullets follows the same molecular mechanisms as in typical mammalian responsive species, should await studies of exposure to potent piscine peroxisome proliferators where significant increases in peroxisomal volume densities can be determined at the transcriptional level. Acknowledgements This work was supported by the Spanish MEC (PRESTEPSE, VEM C06), Basque Government (ETORTEK-IMPRES) and University of the Basque Country (predoctoral grant to E. Bilbao and grant for consolidated research groups). Thanks are also due to Dr. Gorka Basañez (UPV/EHU) for his contribution in FF measurements. References Aas E, Beyer J, Goksøyr A (2000). "Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: An evaluation of compound specificity, inner filter effect and signal interpretation". Biomarkers 5, Ackers JT, Johnston MF, Haasch ML (2000). "Immunodetection of hepatic peroxisomal PMP70 as an indicator of peroxisomal proliferation in the mummichog, Fundulus heteroclitus". Marine Environmental Research 50, Au DWT, Wu RSS, Zhou BS, Lam PKS (1999). "Relatioship between ultraestructural changes and EROD activities in liver of fish exposed to benzo(a)pyrene". Environmental Pollution 104, Arinç E, sen A, Bozcaarmutlu (2000). "Cytochrome P450 and associated mixed-function oxidase induction in fish as a biomarker for toxic carcinogenic pollutants in the aquatic environment". Pure and Applied Chemistry 72, Barbier O, Villeneuve L, Bocher V, Fontaine C, Pineda Torra I, Duherm C, Kosykh V, Fruchart JC, Guillermette C, Staels B (2003). "UDP-glucuronosyltransferase 1A9 enzyme is a peroxisome proliferator-activated receptor α and γ target gene". Journal of Biological Chemistry 278, Beier K, Fahimi HD (1991). "Environmental pollution by common chemicals and peroxisome proliferation: efficient detection by cytochemistry and automatic image analysis". Progress in Histochemistry and Cytochemistry, 23, Bilbao E, Cajaraville MP, Cancio I (submitted a). "Molecular characterisation of the peroxisomal β- oxidation pathway in aquatic organisms: cloning and expression pattern of palmitoyl-coa oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase". Experimental Cell Research. Bilbao E, Cajaraville MP, Cancio I (submitted b). "Differential expression of genes involved in peroxisome 250

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203 Results and Discussion Oakes KD, McMaster ME, Pryce AC, Munkittrick KR, Portt CB, Hewitt LM, MacLean DD, Van Der Kraak GJ (2003). "Oxidative stress and bioindicators of reproductive function in pulp and paper mill effluent exposed white sucker". Toxicological Sciences 74, Oakes KD, Sibley PK, Martin JW, McLean DD, Solomon KR, Mabury SA, Van der Kraak GJ (2005). "Short term exposures of fish to perfluoroocane sulfonate: acute effects on fatty acyl-coa oxidase activity, oxidative stress, and circulating sex steroids". Environmental Toxicology and Chemistry 24, Oakes KD, Sibley PK, Solomon KR, Mabury SA, Van der Kraak GJ (2004). "Impact of perfluorooctanoic acid on fathead minnow (Pimephales promelas) fatty acyl-coa oxidase activity, circulating steroids, and reproduction in outdoor microcosms". Environmental Toxicology and Chemistry 23, Oikari AOJ, Nakari T (1982). "Kraft pulp mill effluent components cause liver dysfunction in trout". Bulletin of Environmental Contamination and Toxicology 28, Orbea A, Cajaraville MP (2006). "Peroxisome proliferation and antioxidant enzymes in transplanted mussels of four basque estuaries with different levels of polycyclic aromatic hydrocarbon and polychlorinated biphenyl pollution". Environmental Toxicology and Chemistry 25, Ortiz-Zarragoitia M, Cajaraville MP (2005). "Effects of selected xenoestrogens on liver peroxisomes, vitellogenin levels and spermatogenic cell proliferation in male zebrafish". Comparative Biochemistry and Physiology Part C 141, Otto DME, Moon TW (1996). "Phase I and II enzymes and antioxidant responses in different tissues of brown bullheads from relatively polluted and non-polluted systems". Archives of Environmental Contamination and Toxicology 31, Oulmi Y, Braunbeck T (1996). "Toxicity of 4- chloroaniline in early life-stages of zebrafish (Brachydanio rerio): I. Cytopathology of liver and kidney after microinjection". Archives of Environmental Contamination and Toxicology 30, Pedrajas JR, López-Barea J, Peinado J (1996). "Dieldrin induces peroxisomal enzymes in fish (Sparus aurata) liver". Comparative Biochemistry and Physiology Part C 115, Porte C, Solé M, Borghi V, Martínez M, Chamorro J, Torreblanca A, Ortiz M, Orbea A, Soto M, Cajaraville MP (2001). "Chemical, biochemical and cellular responses in the digestive gland of the mussel Mytilus galloprovincialis from the Spanish Mediterranean coast". Biomarkers 6, Qi C, Zhu Y, Reddy JK (2000). "Peroxisome proliferator-activated receptors, coactivators, and downstream targets". Cell Biochemistry and Biophysics 32, Raingeard D, Cancio I, Cajaraville MP (2006). "Cloning and expression pattern of peroxisome proliferators-activated receptor alpha in the thicklip grey mullet Chelon labrosus". Marine Envirnomental Research 62S, Reid DJ, MacFarlene GR (2003). "Potencial biomarkers of crude oil exposure in the gastropod mollusc, Austrocochlea porcata: laboratory and manipulative field studies". Environmental Pollution 126, Rocher B, Le Goff J, Peluchet L, Briand M, Manduzio H, Gallois J, Devier MH, Geffard O, Gricourt L, Augagneur S, Budzinski H, Pottier D, André V, Lebailly P, Cachot J (2006). "Genotoxicant accumulation and cellular defence activation in bivalves chronically exposed to waterborne contaminants from the Seine River". Aquatic Toxicology 79, Scarano SC, Calebrese EJ, Kostecki PT, Baldwin LA, Leonard DA (1994). "Evaluation of a rodent peroxisome proliferator in two species of freshwater fish: rainbow trout (Oncorhynchus mykiss) and Japaneses medada (Oryzias latipes)". Ecotoxicology and Environmental Safety 29, Schlenius AK, Eriksson AM, Hongdtrom C, Kimland M, DePierre JW (1993). "Perfluorooctane sulfonic acid is a potent inducer of peroxisomal fatty acid beta-oxidation and other activities known to be affected by peroxisome proliferators in mouse liver". Pharmacology and Toxicology 72,

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205 Results and Discussion as sentinel organisms". Environmental Pollution 148,

206 Peroxisomen proliferazioa eta bioeraldaketa metabolismoaren erregulazio transkripzionala 4.- Peroxisomen proliferazio eta bioeraldaketa metabolismoaren transkripzio mailako erregulazioa perfluorooktano sulfonato eta Prestige ontziak isuritako fuelaren antzeko fuel-olio astunaren pean mantendutako Chelon labrosus lazunetan Atal honetako emaitzak argiataratzeko prestatu dira: BILBAO E, RAINGEARD D, DIAZ DE CERIO O, ORTIZ-ZARRAGOITIA M, RUIZ P, IZAGIRRE U, ORBEA A, MARIGÓMEZ I, CAJARAVILLE MP, CANCIO I. "Effects of exposure to Prestige-like heavy fuel oil on convencional biomarkers and target gene expression of the thicklip grey mullet Chelon labrosus". Toxicology and Applied Pharmacology. Atal honetako emaitzak kongresuetan aurkeztu dira: 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, Haya, Herbeherak, Maiatzak 7-11, CANCIO I, BILBAO E, RAINGEARD D, SAEZ-MORQUECHO C, DIAZ DE CERIO O, CAJARAVILLE MP. "Differential gene expression in sentinel marine species under exposure to organic xenobiotics". Open European Peroxisome Meeting 2006, Leuven, Belgika. Irailak 18-19, BILBAO E, CAJARAVILLE MP, CANCIO I. "Cloning and expression analysis of peroxisome proliferation marker genes in aquatic organisms: effects of organic xenobiotics". VII th International Congress on the Biology of Fish. St. John s, Newfounland, Canada, Uztailak 18-22, BILBAO E, CAJARAVILLE MP, CANCIO I. "Induction of peroxisomal gene expression in mullets exposed to PFOS and to a heavy fuel oil similar to the Prestige oil". SYMPOSIUM on Marine Accidental Oil spills (VERTIMAR), Vigo, Espainia, Ekainak 5-8, BILBAO E, RAINGEARD D, DIAZ DE CERIO O, ORTIZ-ZARRAGOITIA M, RUIZ P, IZAGIRRE U, ORBEA A, MARIGÓMEZ I, CAJARAVILLE MP, CANCIO I. "Effects of exposure to Prestige-like heavy fuel oil on convencional biomarkers and target gene expression of the thicklip grey mullet Chelon labrosus". 257

207 Results and Discussion 258

208 Peroxisomen proliferazioa eta bioeraldaketa metabolismoaren erregulazio transkripzionala Laburpena Chelon labrosus lazunak kostako guneetan zein estuarioetan bizi dira, eta bertan, beraien perfil transkripzionalak alda ditzaketen kutsatzaieleen kontzentrazio altuak metatzeko gaitasuna dute. Efektu horietako bat, peroxisomen proliferazioa da, karraskarietan lipidoen homeostasian diharduten geneek maila transkripzionalean duten indukzioa erakutsiz agertzen dena, hala nola, palmitoil-coa oxidasa (AOX1), proteina multifuntzionala (MFP1), 3-ketoazil-CoA tiolasa (THIO) eta 2, 4 dienoil-coa erreduktasa 2 (DECR) kodetzen dituzten geneak. Zentzu honetan, kutsatzaile organikoen pean egotean ematen diren maila transkripzionaleko aldaketak ikertu asmoz, C. labrosus lazunak 2 eta 16 egunen buruan perfluorooktano sulfonato (PFOS) eta Prestige ontziak Iberiar Penintsulako iparraldean 2002.eko azaroan isuritako fuel olioaren antzeko olio astun fresko (F) eta zaharkituaren (WF) pean mantendu ziren. Hidrokarburo aromatiko poliziklikoek (PAH), Prestige ontziak isuritako fuelak zituenak esaterako, fase I eta fase II-ko bioeraldaketa metabolismoa induzitzen dituzte, haatik, glutation-s transferasa (GST) eta UDP-glukuronosiltransferasa (UGT) klonatu eta beraien espresioa neurtu zen, fase I-eko bioeraldaketa metabolismoan diharduen CYP1A1 genearen espresioarekin batera. Gainera, PAH batzuk metabolito polar gisa behazunean kanporatzen direnez, PAHen metabolitoak neurtu ziren uhin-luzera finkoko fluoreszentzia erabiliz. Horrela, PAH pean mantendutako organismoek, behazunean PAHen metabolitoak metatzen zituztela frogatu zen; WF pean mantendutakoek, B(a)Pmotako metabolitoak metatu zituzten gehienbat, F-ren pean mantendutakoek berriz, nafataleno motakoak. AOX1 eta CYP1A1 geneen espresioa zein AOX1 entzimaren jarduera induzitu egin ziren gibelean F eta WF pean bi egunetan mantendu ondoren. 16. egunean, CYP1A1en gainespresioa kontrolen mailen gainetik mantendu zen, eta Fk bakarrik mantendu zituen AOX1en espresioa eta jarduera kontrolen gainetik. Peroxisometako proteina nagusia den PMP70en kasuan, 16 egunetan WF pean mantendu ostean soilik behatu zen gainespresio arina. Bestalde, esangarriki ez bazen ere, PFOSek AOX1 espresioa emendatu zuen 2. egunean, bere jarduera esangarriki emendatu zuen bitartean. Bestalde, Fk DECR espresioa inhibitu egin zuen 2. egunean eta WFk 16. eguneko GST induzitu. Katalasaren (CAT) espresioa gibelean 16. egunean induzitu egin zen, zelulak zuen estres oxidatibo egoera posiblea azaleraziz; hau, 2. egunean behatutako bioeraldaketa metabolismo eta β-oxidazioaren indukzioarekin egon daiteke lotuta. Hala ere, zakatzetan, Fk 2. egunean eragin zuen CATen gainespresioa, CYP1A1enarekin batera. Espresioa 16. egunean kontrolen maila berdintsuetara bueltatu zen kasu bietan. Prestige ontziak isuritako fuel-olio astunaren antzeko fuel-olio fresko eta zaharkituaren pean mantendutako lazunetan beraz, peroxisometako β-oxidazioko lehenengo entzima eta era berean, mugatzailea den AOX1en espresio zein jarduera mailako indukzioa eragin zen, horrekin batera, I faseko bioeraldaketa metabolismoan ere aldaketak eraginik; aldaketa hauekin batera, ondorengo faseetan, CAT entzima antioxidatzailearen gainespresioa eman zen. Emaitza hauen bitartez, AOX1 genearen erregulazio diferentziala, fuel-olio astunaren eraginak aztertzeko esposizio biomarkatzaile gisa erabili daitekeela frogatu zen. 259 L A B U R P E N A

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210 Emaitzak Peroxisomen proliferazioa eta bioeraldaketa metabolismoaren erregulazio transkripzionala PAH-metabolitoak Esperimentuan zehar ez zen arrainik hil. Hala ere, PFOS pean mantendutako lazunek gainontzeko arrainontzietakoak baino gutxiago edo/eta apenas jaten zuten. Gainera, PFOS peko arrainak hasieratik narkotikoen efektupean zeudela zirudien, arrainontziaren gainazalean igeri egitera mugatuz. Klonatutako itu-sekuentziak UGTren domeinu kodetzailearen sekuentziaren 556 bp-tako cdna zati bat anplifikatu eta sekuentziatu zen lazunaren gibelean (EF407557). Deduzituriko amino azido sekuentziak %75eko amino azido identitatea erakutsi zuen E. coioidesen UGT sekuentziarekin (AAW29020), %71koa Gasterosteus aculeatusen ESTarekin (DN699857) eta %57koa P. yokohammaenaren UGT1B2rekin (AB ). Metodologia bera erabiliz, lazunaren GSTaren N-terminalari zegozkion 282 bp klonatu ziren (EF407556). Lortutako sekuentzia zati horrek, P. platessaren GSTArekin (CAA64495) eta M. salmoidesen GSTrekin (AY335905) %80ko amino azido identitatea erakutsi zuen. Esposizio-esperimentua Uretan disolbatutako konposatuen analisi kimikoa PFOS pean mantendutako arrainontziko uretan neurtu zen PFOS kontzentrazioa antzekoa izan zen esperimentuko 2. eta 16. egunetan, 0.46 eta 0.44 ppm, hurrenez hurren. Fuelaren pean mantendutakoetan, PAH kontzentrazio altuena (Environmental Protection Agengy-ak lehenetsitako 16 PAHak) 2. egunean neurtu zen F pean mantendutako uretan (97.18 ppm vs ppm kontrolean eta 18.2 ppm WF taldean). Hala ere, PAH kontzentrazioa nabarmenki jaitsi zen 16. egunean lurrunketa, hormetan emandako adsortzio edo/eta biometaketa zirela eta, talde esperimental guztiek antzeko kontzentrazioak erakutsiz (3.79 ppm kontroletan, 3.5 ppm F taldean eta 6.28 ppm WF taldean). Behazunean neurtutako PAH-metabolitoek erakutsi zutenez, F eta WF pean mantendutako animaliek PAHak metatu eta partzialki bazen ere, metabolizatu egin zituzten. Horrela, B(a)P, pireno eta naftaleno moduko metabolitoak, neurtutako arrain guztietan determinatu ziren (1 Irudia). Emaitzak, organismo kontroletan neurtutako fluoreszentzia intentsitatearekin konparatuz eman dira, tratatuak zenbat aldiz induzitu ziren azalduz. PAH astunen uhinluzeran (B(a)P motako PAHak) fluoreszentziaintentsitate altuak neurtu ziren WF pean 2 egunez mantendutako arrainen behazunean (1 Irudia), fluoreszentzia-intentsitatea kontroletan baino 14 aldiz altuagoa izanik. F pean mantendutako arrainek ere, fluoreszentziaintentsitate handia erakutsi zuten uhin-luzera honetan, baina kasu honetan, kontrolek baino 10 aldiz altuagoa izan zen. Pireno motako metabolitoek antzeko fluoreszentziaintentsitatea erakutsi zuten trataturiko talde bietan (kontroletan baino 10 aldiz altuagoa), naftaleno motako metabolitoak berriz, F pean mantendutako laginetan (kontrolek baino 8 aldiz gehiago), WF pean mantendutakoen gainetik zeuden (kontrolak baino 5 aldiz gehiago). Kasu guztietan, fluoreszentzia-intentsitatea kontrolen gainetik mantendu arren, 16. egunean esangarriki jaitsi zen (1 Irudia). Hala eta guztiz ere, 16. egunean, B(a)P eta pireno motako metabolitoek, WF pean mantendutako organismoetan F pean mantendutakoetan baino fluoreszentzia-intentsitate arinki altuagoak azaldu zituzten, naftaleno motako metabolitoek berriz, fluoreszentzia-intentsitate altuagoak erakutsi zituzten F pean mantendutako organismoetan WF pean mantendutakoetan baino. Itu-geneen espresio mailak Zakatz eta gibelean neurtutako housekeeping geneen artetik, 18S rrnak agertu zuen aldakortasun mailarik baxuena zakatzetan eta β- aktinak gibelean. Beraz, gibelean neurtutako espresio mailak β-aktinarekiko normalizatu ziren eta zakatzean 18S rrnarekiko. CYP1A1 genearen espresioa esangarriki 261 E M A I T Z A K

211 Emaitzak eta Eztabaida induzitu zen gibelean, arrainak F eta WF pean 2 eta 16 egun egon ondoren. Zakatzetan berriz, CYP1A1en indukzioa 2. egunean bakarrik behatu zen (2. Irudia). Fase II-ko bioeraldaketa metabolismoan diharduten entzimak kodetzen dituzten geneei zegokienez, GSTren gainsepresioa behatu zen 16 egunetan WF pean mantendutako arrainetan. Hala ere, UGTren espresio mailetan ez zen aldaketarik neurtu. Bestalde, PFOS konposatuak ez zuen izan bioeraldaketa metabolismoko geneen gaineko efekturik (2. Irudia). Peroxisometako proteinak kodetzen dituzten geneei zegokienez berriz, AOX1 gibelean gainespresatu egin zen F pean 2 eta 16 egunez mantendutako animalietan (Figure 3), WFk, AOX1, 2. egunean bakarrik gainespresatu zuen bitartean. Zakatzetan, AOX1en espresioak aldakortasun handia erakutsi zuen talde bereko organismoetan, nahiz eta gainespresioa adierazten zuen joera orokorra behatu zen, batez ere, F pean mantendutako organismoetan (ez dira datuak erakusten). PFOS pean mantendutako organismoen gibel zein zakatzetan, AOX1 arinki bakarrik gainespresatu zen eta MFP1 eta THIOk ez zuten inongo aldaketarik erakutsi esperimentuan zehar (3. Irudia). Modu berean, PMP70ak gainespresio arin bat besterik ez zuen erakutsi WF pean 16 egunetan mantendutako arrainen gibelean (4. Irudia). 16 egunetan zehar WF pean mantendutako arrainek, DECR arinki gainespresatu zuten gibelean, 2 egunez F pean mantendutakoetan berriz, DECR-ren espresioa inhibitu egin zen (3. Irudia). Azkenik, CAT genearen gainespresioa behatu zen PFOS pean zein fuel mota bien pean 16 egun egondako arrainetan (5. Irudia). Zakatzetan, CATen gainespresio esangarria behatu zen F pean 2 egunez mantendutako arrainetan. WF pean 16 egunez mantendutako animalietan berriz, ez zen behatu CAT espresioaren aldaketarik (5. Irudia). Palmitoil-CoA oxidasaren jarduera F pean mantendutako arrainetan, AOX1 entzimaren jardueraren indukzioa behatu zen 2 eta 16. egunean, WFk berriz, 16. eguneko AOX1 jarduera bakarrik induzitu zuen (6. Irudia). Gainera, AOX1en jarduera PFOS pean ere induzitu egin zen, indukzio hau esposizioa hasi eta 2 egunetara esangarria izanik. 262

212 Peroxisomen proliferazioa eta bioeraldaketa metabolismoaren erregulazio transkripzionala Ondorioak 1.- Chelon labrosus lazunek, Prestige ontziak isuritako fuelaren antzeko fuel-olio astunaren pean (freskoa eta zaharkitua), PAH-metabolitoak metatu zituzten behazunean. WF pean mantendutako arrainen behazuna B(a)P motako PAHetan aberastu zen bitartean, F pean mantendutako arrainena, naftaleno motakoetan aberastu zen. 2.- Fuel-olio astunak, 2 egunen buruan CYP1A1 genearen gainespresio esangarria eragin zuen gibel eta zakatzetan, PFOSek ordea, ez zuen eragin bioeraldaketa metabolismoko espresio mailetan. Gibeleko espresio mailek 16. egunean maila berdintsuan mantendu baziren ere, zakatzetan, espresio mailak kontrolen parera jaitsi ziren. 3.- AOX1 genearen espresio maila eta entzimaren jarduera induzitu egin ziren, bai PFOS zein fuel olioen pean, baina ez zen gauza bera gertatu β-oxidazioko MFP1 eta THIO-rekin. Gainera, peroxisomen mintzetako proteina nagusian, PMP70, ez zen espresio mailako aldaketarik behatu, peroxisomen de novoko sintesia beharrezkoa ez zela islatuz. 4.- Fuel-olio astunen pean, peroxisometako β-oxidazioko AOX1-en zein fase I-eko bioeraldaketa metabolismoko CYP1A1-en gainespresioen ondotik, CAT eta GST geneen gainespresioa eman zen, erabilitako kutsatzaile organikoek estres oxidatiboa eragin zutela islatuz. 5.- AOX1-ek erakutsi zuen erregulazio diferentziala, fuel-olio astunaren peko organismoetan erabil daiteke esposizio-biomarkatzaile goiztiar legez. 263 O N D O R I O A K

213 5.- Xenobiotiko organikoen peko Mytilus galloprovincialis muskuiluen liseri-guruineko geneen espresioa: zelai eta laborategiko saioak Atal honetako emaitzen argitaratzea prestatzen ari da: ORBEA A, RUIZ P, BILBAO E, CANCIO I, CAJARAVILLE MP. "Peroxisomal acyl-coa oxidase activity and expression of peroxisomal genes in mussel, Mytilus galloprovincialis: long-term effects of the Prestige oil spill and seasonal variations". Marine Pollution Bulletin. RAINGEARD D, BILBAO E, GARMENDIA L, ORTIZ-ZARRAGOITIA M, ORBEA A, MARIGOMEZ I, CAJARAVILLE MP, CANCIO I. "Biomarker and gene expression responses in mussels Mytilus galloprovincialis experimentally exposed to perfluorooctanoic sulfonic acid and to Prestige-like heavy fuel-oil. Toxicological Sciences. Atal honetako emaitzak aipatzen diren kongresuetan aurkeztu dira: 16 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, Haya, Herbeherak, Maiatzak 7-11, CANCIO I, BILBAO E, RAINGEARD D, SAEZ-MORQUECHO C, DIAZ DE CERIO O, CAJARAVILLE MP. "Differential gene expression in sentinel marine species under exposure to organic xenobiotics". 17 th Annual Meeting of the Society of Environmental Toxicology and Chemistry-Europe, Oporto, Portugal, Maiatzak 20-24, BILBAO E, CAJARAVILLE MP, CANCIO I. "A field study of changes in peroxisomal gene expression in mussel Mytilus galloprovincialis". VERTIMAR 2007, SYMPOSIUM on Marine Accidental Oil Spills, Vigo, Espainia, Ekainak 5-8, ORBEAA, RUIZ P, BILBAO E, CANCIO I, CAJARAVILLE MP. "Peroxisomal acyl-coa oxidase activity and expression of peroxisomal genes in mussel, Mytilus galloprovincialis: monitoring effects of the Prestige oil spill and seasonal variations". 265

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215 LABURPENA Geneen espresio diferentziala M. galloprovincialis muskuiluan Prestige bezalako petrolio-ontziek sortarazitako isurketak direla eta, uraren kalitatearen jarraipen zuzena zein uretan bizi diren organismoek bizi dituzten eraginen azterketaren beharra agerian geratzen dira. Mytilus galloprovincialis muskuilua eragin horien lekuko eta zentinela egokia kontsideratzen denez, organismo hauetan esposizio biomarkatzaile egokiak aztertzea garrantzitsua da. Horrela, hidrokarburo aromatiko poliziklikoen (PAH) aurrean erantzun dezakeen biomarkatzaileetako bat, peroxisomen proliferazioa da. PAHak bezalako xenobiotiko organikoen aurrean, transkripzio mailako geneen azterketa peroxisomen proliferazioa neurtzeko hurbilpen egokia izan daiteke. Lan honetan, kutsatzaile organiko ezberdinen pean zelaian hartutako zein laborategian mantendutako muskuiluen liseri-guruineko zenbait gene adierazgarriren espresioa neurtu zen PCR kuantitatibo zein semikuantitatiboz. Horrela, palmitoil-coa oxidasa (AOX1) eta katalasa geneen espresio-aldaketak neurtu ziren, muskuiluak laborategian North Sea Oil (NSO), Prestige-ak isuritako fuelaren antzeko olio fresko (F) eta zaharkitua (WF) eta perfluorooktano sulfonatoaren (PFOS) pean mantendu ostean. Gauza bera egin zen Prestige ontzia hondoratu ondoren, Iberiar Penintsulako iparraldeko 5 laginketa-puntutan jasotako muskuiluetan. Muskuilu hauen estres egoera orokorra neurtzeko helburuz, hsp70 genearen espresioa ere neurtu zen. Laborategian mantendutako muskuiluetan NSO, F eta WFak ez zuten gene peroxisomikoen espresioaren aldaketarik eragin, nahiz eta, WFak hsp70 genearen gainespresioa eragin zuen 2. egunean. PFOSek ordea, AOX1 genearen gainespresioa eragin zuen 2. egunean, jardueraren aldaketarik eman ez bazen ere. Zelaian, Prestige ontzia hondoratu eta urte bete eta erdira hartutako muskuiluen ehunetan neurtutako PAH kontzentrazioarekin bat ez zetozen AOX1 eta CAT geneen espresioaren aldaketa arinak neurtu ziren. Espresio mailako aldaketa hauek, bi gune ezberdindu zituzten geografikoki, ekialderen zeuden laginketa-estazioetan espresio maila arinki altuagoak erakutsiz. Ikerketa sakonagoak beharko dira, denbora tarte ezberdinetan eta esposizio-eszenario ezberdinetan mantendutako muskuiluek erakusten duten peroxisomen proliferazioa (peroxisomen dentsitate bolumetrikoaren emendioa eta AOX1 jardueraren indukzioa) maila transkripzionalean erregulatuta dagoen determinatzeko. 267 L A B U R P E N A

216 Emaitzak eta Eztabaida RESUMEN Los efectos causados por vertidos como el del Prestige, entre otros, manifiestan la necesidad de llevar a cabo seguimientos de la calidad del agua, así como la necesidad de estudiar los efectos causados en los organismos que viven en el medio marino. El mejillón Mytilus galloprovincialis se considera testigo directo de esos efectos y centinela apropiado; por lo tanto, se considera importante el estudio de biomarcadores de exposición en estos organismos. Así, uno de los biomarcadores que responde ante la exposición a hidrocarburo aromáticos policíclicos (PAHs) es la proliferación de peroxisomas. Como la medición transcripcional de genes diana podría ser una aproximación adecuada para medir la proliferación de peroxisomas tras la exposición a xenobióticos orgánicos como los PAHs, en este estudio se ha medido mediante PCR semicuantitativa y cuantitativa la expresión de ciertos genes diana en la glándula digestiva de mejillones expuestos a contaminantes orgánicos, tanto en el campo como en el laboratorio. Se midió la expresión de palmitoil-coa oxidasa (AOX1) y catalasa tras exponer los mejillones en el laboratorio a North Sea Oil (NSO), fuel fresco (F) y envejecido (WF) similar al vertido por el Prestige y perfluorooctano sulfonato (PFOS). Del mismo modo, se midió la expresión génica en mejillones recogidos en 5 puntos de muestreo del norte de la Península Ibérica tras el vertido del Prestige. Con el fin de determinar la situación de estrés general de los mejillones se midieron también los niveles de expresión del gen que codifica para hsp70. Los mejillones expuestos en el laboratorio a NSO, F y WF, no presentaron variaciones en el patrón transcripcional, a pesar de que hsp70 se sobreexpresó tras la exposición a WF durante 2 días. Sin embargo, PFOS provocó la sobreexpresión de AOX1 2 días después del comienzo de la exposición, sin que su actividad aumentara. Los mejillones recogidos en el campo un año y medio después del vertido del Prestige, presentaron variaciones transcripcionales en la expresión de AOX1 y CAT que no correspondían con las concentraciones de PAHs medidas en tejidos blandos de mejillón; sin embargo eran capaces de diferenciar dos zonas, por un lado, las estaciones situadas al oeste donde el impacto del vertido fue directo y las estaciones del este donde el fuel llegó meses después de haber estado flotando en el mar. Por lo tanto, se puede decir, que el tiempo transcurrido tras la exposición, el periodo de estabulación y/o el efecto producido por la posible presencia de contaminantes que pueden presentarse junto con los PAHs, son factores ha tener en cuenta ante el estudio de la proliferación de peroxisomas. Además, se necesitarán estudios más profundos en mejillones que presenten proliferación de peroxisomas (aumento de densidad volumétrica junto con inducción de AOX1) con el fin de determinar si esa regulación se da a nivel transcripcional. 268

217 Geneen espresio diferentziala M. galloprovincialis muskuiluan Sarrera Industriaren garapen arina eta gizakiaren etengabeko jardueren ondorioz besteak beste, itsas ekosistemak aldatzen ari dira. Horrela, kostaldeko uretan bizi diren organismoek, inguruneko baldintza fisiko-kimiko naturalek zein kutsatzaileek eragindako baldintza estresagarriei egin behar diete aurre. Itsas ingurunea aztertzea helburu duten biojarraipen programetan, organismoetan eragindako aldaketak aztertzerako orduan, Mytilus galloprovincialis muskuiluak berebiziko garrantzia du organismo zentinela legez. Izan ere, azterketak muskuiluetan burutzeak abantaila ugari baititu: besteak beste, muskuiluak sesilak dira, laginketak erraztuz eta tokian tokiko informazioa gordez, banaketa zabala erakusten dute munduko kostalde gehienetan, honek, mundu mailako gune ezberdinetan espezie bera erabiltzea baimentzen duelarik. Muskuiluak, filtrazioz elikatzen dira ur kopuru handiak barneratuz eta kutsatzaile organiko zein ezorganikoak biometatzeko gaitasun handia dute, kutsatzaileok biotransformatzeko gaitasun baxua agertzen dutelarik (Cajaraville et al., 2000; Smolders et al. 2003). Horrela, 70. hamarkadan muskuiluak erabiltzen hasi ziren biojarraipenetarako organismo legez "Mussel watch" jarraipen-programaren barruan, estuario eta itsasertzeko inguruetan kutsadura kimikoak denbora zein espazioaren arabera agertzen zituen aldaketak aztertzeko helburuz (Goldberg, 1975). Orduz geroztik, muskuiluak gero eta gehiago erabili dira poluzioak eragindako efektu biologikoen ikerketan (Bayne, 1989; Cajaraville et al. 2000; Livingstone et al. 2000; Roméo et al. 2003; Petrovic et al. 2004). Zentzu honetan, itsas inguruneko poluzioa aztertzeko muskuiluek duten garrantzia ikusirik, ezinbestekoa bilakatzen da erantzun sentikorrak emango dituzten biomarkatzaile egokiak garatzea. Biomarkatzaileak, maila biokimiko zein zelularrean kutsatzaileen presentzia edo hauen aurrean ostalariak duen erantzuteko gaitasuna adierazten duten gorputzeko fluido, zelula edo ehun mailan egindako neurketak dira (Livingstone et al. 2000). Horrela, organismo bat kutsatzaileen pean egon denentz edo/eta kutsatzailearen aurrean organismoak erantzuten duenentz adieraz dezakete. Alta, biomarkatzaileen ezaugarri nagusienetako bat, epe luzera organismoan, populazioan edo/eta ekosisteman gerta daitezkeen efektu biologikoen epe-laburreko adierazleak izatea da (Cajaraville et al. 2000; Cajaraville et al. 2003). Esposiziobiomarkatzaileen artean, muskuiluetan neurturiko peroxisomen proliferazioa kutsatzaile organikoen aurrean ematen den erantzun gisa proposatu da, honek, arrainetan neurtzen den eta ornogabeetan oso gutxi garaturiko bioeraldaketa metabolismoaren neurketa ordezka dezakeelakoan (Cajaraville et al. 2000). Peroxisomen proliferazioa ematean, peroxisomen dentsitate bolumetrikoaren handipenarekin batera, entzima peroxisomiko batzuen jardueraren emendioa, batik bat β- oxidazioko entzimena, behatzen da (Qi et al. 2000). Karraskarietan, jardueraren emendio hori, peroxisomen β-oxidazioko entzimen transkripzio mailako indukzioaren ondorioz ematen da. Muskuiluetan ere, laborategiesperimentu ezberdinek peroxisometako β- oxidazioko lehenengo entzima den palmitoil- CoA oxidasaren (AOX1) jardueran indukzioa deskribatu dute peroxisomen dentsitate bolumetrikoaren emendioarekin batera, olio lubrifikatzailearen urari egokitutako frakzioaren pean (WAF), mikroenkapsulatutako hidrokarburo polizikliko aromatikoen (PAH) zein North Sea Oil (NSO) olioaren pean (Cancio et al. 1998; Krishnakumar et al. 1997; Cajaraville eta Ortiz-Zarragoitia 2006). Konposatu horiez gain, muskuiluen peroxisometan aldaketak eragiten dituzten beste zenbait xenobiotiko organiko deskribatu dira ere (Cancio eta Cajaraville 2000; Cajaraville eta Ortiz-Zarragoitia 2006). Zelaian, muskuiluen liseri-guruineko peroxisomen proliferazioaren gaineko zenbait ikerketa burutu da, besteak beste, Ipar Itsasoko petrolio plataformek ekoiztutako uretan mantendutako Mytilus edulisetan (Bilbao et al. 2006) zein Mediterraneo eta Kantauri itsasoko Mytilus galloprovincialisetan egindako biojarraipen programen barruan (Bocchetti eta Regoli 2006; Cajaraville et al. 2006; Marigómez et al. 2006; Orbea et al. 2006; Zorita et al. 2007). Hala ere, zelaian egindako ikerketetan, inguruneko faktore naturalek, biomarkatzaileek ematen dituzten erantzunetan eragina dutela 269 S A R R E R A

218 Emaitzak eta Eztabaida ikusi da eta beraz, biomarkatzaileak aldagai naturalen arabera nola aldatzen diren ezagutzea garrantzitsua da. Horrela, peroxisomen proliferazioa determinatzeko sarritan neurtzen den AOX1 entzimaren jarduerak urte sasoiaren arabera Kantauri itsasoko zein Adriatikoko muskuiluetan izaten dituen aldaketak aztertu dira (Cancio et al. 1999, Bocchetti eta Regoli 2006; Orbea et al. 2007). Gainera, kutsadura maila ezberdinak dituzten zelaiko guneen artean muskuiluekin burututako transplante esperimentuetan, peroxisomen proliferazioa prozesu induzigarria eta era berean, itzulgarria dela ikusi da, eta hortaz, inguruneko kutsadura aztertzen duten jarraipen programetan biomarkatzaile egokia izan litekeela berretsi da (Orbea eta Cajaraville 2006). Era berean, estres egoeretan, olio gordinetan dauden PAHen pean esaterako, zelula barneko oxigeno espezie erreaktiboen kontzentrazioa (ROS) emendatu egiten da, PAHek zuzenean eraginda edota ROS ekoizleak diren entzima ezberdinen indukzioak eraginda, hala nola, AOX1 (Van der Oost et al. 2003; Orbea eta Cajaraville 2006; Schrader eta Fahimi 2006; Valavanidis et al. 2006). Kutsatzaileekiko esposizioak beraz, zelularen ROS kontzentrazioan desorekak ondoriozta ditzake, ROS kontzentrazioaren neurrigabeko emendioak berriz, zelularen DNA, lipido zein proteinetan kalteak eta ondorioz, patologia eta toxikotasun ezberdinak gara daitezke (Livingstone et al. 1990; Van der Oost et al. 2003; Valavanidis et al. 2006). Egoera hori ekidin asmoz, entzima antioxidatzaileen jardueraren emendioa espero daiteke, zelulan ematen den kalte oxidatzailearen aurkako babes mekanismo gisa (Niyogi et al. 2001; Manduzio et al. 2005; Bocchetti eta Regoli 2006; Orbea eta Cajaraville 2006). Horrela, erantzun antioxidatzailean ematen diren indukzio eta inhibizioek zein kalte oxidatiboak berak, kutsatzaile organikoen aurrean emandako erantzunak islatzen dituzte, eta hortaz, esposizio eta efektu biomarkatzaile legez proposatu dira (Cheung et al. 2001; Valavanidis et al. 2006), tartean peroxisometako entzima nagusia egonik, katalasa (CAT), alegia. Gainera, estres egoeren aurrean, estresproteina ezberdinek ere, berebiziko garrantzia hartzen dute organismo itsastarren homeostasi metabolikoan. Bero-talka proteinek (heat shock protein, hsp) familia zabala osatzen dute eta orohar, zelulako proteinen biosintesia ematen denean, proteinen heltze prozesuan, tolespen, destolespen eta mintzen zeharreko translokazioan dihardute (Fink, 1999; Hartl and Hayer-Hartl 2002). Estres egoeretan, hsp70 taldeko proteinak induzitzen direla deskribatu da, ziur aski estresak kaltetutako proteinak birtolespen egokian lagundu asmoz. Hori dela eta, hsp70 estresaren aurreko markatzaile orokor gisa erabiltzen da (Radlowska eta Pempkowiak 2002; David et al. 2005). Horrela, hainbat lanek hsp70 genearen espresioa inguruneko estresatzaile ezberdinen arabera nola aldatzen den aztertzen du moluskuetan, erantzun zitobabesleak ikertzeko orduan. Aztertutako estresatzaileak, hala nola, sasoiaren araberako aldaketak (Hoffman eta Somero 1995; Minier et al. 2000; Lesser eta Kruse 2004), gazitasuna (Werner 2004), tenperatura (Luedeking eta Koehler 2004; Cellura et al. 2006), hipoxia (David et al. 2005), patogenoak (Cellura et al. 2006), bifenilo poliklorinatuak (PCB) (Cruz- Rodriguez et al. 2000), PAHak (Cruz-Rodriguez et al. 2002) eta metal astunen metaketa (Radlowska eta Pempkowiak 2002; Franzelliti eta Fabbri 2006) dira. Lan honen xede nagusia, kutsatzaile organikoen aurrean muskuiluen liseri-guruineko peroxisomen AOX1 eta CAT proteinak kodetzen dituzten geneetan gertatzen diren transkripzio mailako aldaketak aztertzea izan zen. Horretarako, muskuiluak uretan disolbatutako NSO, perfluorooktano sulfonatoa (PFOS), zein Prestige ontziak isuritako olioaren antzeko ezaugarriak dituen olio astunaren pean mantendu ziren laborategiko esperimentu desberdinetan. Gainera, hsp70 genearen espresioa estres zelularraren biomarkatzaile orokor gisa erabili zen. Horrez gain, geneen espresioa aztertzerako orduan, PCR kuantitatiboa eta semikuantitatiboa konparatu ziren. Azkenik, aztertutako geneen transkripzio mailako ikerketek zelaian izan dezaketen erabilera aztertu nahirik, ingurunearen kalitatea aztertzeko biojarraipen programa batean aplikatu ziren. Kasu honetan, Prestige ontziak zeraman fuelaren isurketak eragindako kalteak aztertzeko burutzen ari diren lanen 270

219 Geneen espresio diferentziala M. galloprovincialis muskuiluan barruan, Iberiar Penintsulako iparraldeko 5 laginketa-puntu aukeratu ziren eta isurketaren ondorengo urteetan, 2004 eta 2005.eko uztailan, geneen espresioan eman zitezkeen aldaketak aztertu ziren. Material eta Metodoak Erreaktiboak Bestelako azalpenik ematen ez den bitartean, erabilitako erreaktibo guztiak Sigma-Aldrich (St Louis, Missouri, EEBB) etxekoak dira. Ipar Itsasoko olioaren pean mantendutako muskuiluak Mytilus edulis muskuilu helduak Norvegiako Førlandsfjorden hartu ziren eta RF-Akvamiljø Zentruan (Randaberg, Norvegia) BEEP proiektuaren baitan burututako ikerketa lanen barruan, Statfjord B NSO olioaren pean mantendu ziren 3 astez (Cajaraville eta Ortiz- Zarragoitia 2006). Esperimentua eko azaroa-abendua bitartean burutu zen. Esperimentuan zehar, muskuiluak etengabeko fluxu bidezko ur aireztatuan mantendu ziren, Isochrysis eta Rhodomonasekin elikatuz. Erabilitako olio kontzentrazioa 0.5 ppm-koa zen, erauzketa plataformetan lortzen den ur ekoizkinak duenaren parekoa, alegia. Erreferentzia gisa, iragazitako itsasoko ura (10-12ºC, 34) erabili zen. Olio-dispertsioa, presio altuko balbula nahasle bat erabiliz burutu zen (Sanni et al. 1998). Sakabanatzea olio partikulak 10 µm-takoak izan zitezen burutu zen, ekoitzitako uretan bezala. Diluitutako ura itsasoko urarekin nahastu ondoren, esperimentua burutu zeneko akuariora ponpatu zen, esandako kontzentrazio nominal egokia lortuz. Fuel-olio astuna eta PFOSaren pean mantendutako muskuiluen esperimentua cm-ko luzeradun Mytilus galloprovincialis muskuiluak Mundakan (43º 12'58'' N; 2º 42'14'' W) hartu ziren eko irailean. Laborategiko baldintzetara moldatzeko itsasoko ur naturaletan mantendu ziren 15 egunetan zehar, etengabe aireztatuz. Mantenurako, kutsadurarik gabeko ura hartu zen Getarian (43º18 15, 1º20 ), jatetxeek gizakontsumorako saltzen dituzten itsaskiak mantentzeko erabiltzen duten ura, alegia. Organismoak laborategiko baldintzetara moldatu ondoren, 4 akuariotan banatu ziren. Akuarioen oinaldea 5 kg harri eta 6 kg harea nahastuz prestatu zen eta bertara 300 litro itsasoko ur gehitu zen. Esperimentuan zehar, ura ez zen aldatu, etengabe aireztatuz mantendu zen (disolbatutako oxigenoa: mg/l; ph-a: ; gazitasuna: 0 / 00 35; tenperatura: 20ºC) eta 12 ordutako argi/iluntasun ziklo konstantea ezarri zen gelan. Animaliak egunero elikatu ziren, ornogabeak elikatzeko erabiltzen den JBL Korall Fluid-a (JBL, Alemania) akuarioetara isuriz. Organismoak 2 eta 16 egun mantendu ziren 2 mg/l-ko PFOS kontzentrazioaren pean eta fuel-olio fresko eta zaharkitutako fuelolioaren pean. Fuel-olio freskoa prestatzeko, Prestige ontziak isuritako fuelaren antzekoa zen fuel astunaren (IFO 380, RMG 35-ISO 8217 itsasoko fuela) 150 ml nahastu ziren substratuarekin esperimentua hasi aurretik; zaharkitua prestatzeko berriz, nahasketa, esperimentua hasi baino 10 aste lehenago prestatu zen eta etengabeko aireztapenarekin mantendu zen esperimentua amaitu arte. Laugarren akuarioko organismoak tratatugabeak ziren eta kontrol gisa erabili ziren. Zelaiko ikerketa: Prestige biojarraipena cm bitarteko M. galloprovincialis muskuiluak marea baxueneko egun eta orduetan jaso ziren marearteko gunean, eta 2005.eko uztailean. Prestige ontzia hondoratu zenean, askatutako olioak Galiziako kostan eragin zuen batik bat lehenengo eta Iberiar Penintsulako iparraldeko kostan zehar sakabanatu zen ondorengo hilabeteetan; hori dela eta, laginketa Iberiar Penintsulako iparraldeko 5 puntutan egin zen (1 Irudia), São Bartolomeu do Mar (41º34'50''N; 8º47'82''W), Camelle (43º18'N; 9º08'W), Arrigunaga (43º20'50''; 0º40'40''), Gorliz (43º43'16''N; 2.9º40'44''W) eta Mundakan (43º 12'58'' N; 2º 42'14'' W), hain zuzen ere. São Bartolomeu do Mar laginketa-puntua erreferentzia gisa erabiltzeko helburuz aukeratu zen, Cairrão eta laguntzaileek lortutako emaitzen arabera (2004). 271 M E T O D O L O G I A

220 Emaitzak eta Eztabaida N 1. Irudia. Iberiar Pernintsulako Iparraldean burututako biojarraipenean, maila transkripzionalean burutu beharreko ikerketetak, 2004 eta urteetako uztailean S = São Bartolomeu do Mar, C = Camelle, A = Arrigunaga, G = Gorliz eta M = Mundakan hartutako muskuiluetan egin ziren. Izarrak Prestige ontzia hondoratu zeneko gunea adierazten du. Laginen prestaketa Kasu guztietan, talde esperimentaleko 5 muskuiluren liseri-guruina disekzionatu zen geneen espresioa aztertzeko eta RNAlaterdun hodi banatan izoztu ziren nitrogeno likidotan. PFOS eta fuel-olio astunaren pean mantendutako muskuiluen AOX1en jarduera neurtzeko, 5 muskuiluren liseri-guruina disekzionatu eta nitrogeno likidotan izoztu ziren banaka. 25 muskuiluren ehun bigun guztiak analisi kimikoak burutzeko izoztu ziren. Laginak -80ºCtan gorde ziren prozesatu arte. AOX1 jardueraren neurketa AOX1en jarduera H 2 O 2 -aren menpeko diklorofluoreszeina diazetatoaren (Molecular Probes, Eugene, Oregon, EEBB) oxidazioan oinarrituz neurtu zen. Aipatutako erreakzioa peroxidasa exogeno batek katalizatzen du 30 µm palmitoil-coa substratu gisa erabiliz (Small et al. 1985). Bost organismoren liseri-guruinak indibidualki homogenizatu ziren 7.6 ph-dun TVBE indargetzailetan (1mM sodio bikarbonatoa, 1 mm EDTA, % 0.1 etanol eta % 0.01 Triton X-100), Hybaid Ribolyser TM -a (Hybaid, Ashford, Britainia Handia) erabiliz. Ondoren, 500g-tan zentrifugatu ziren 15 minutuz, 4ºC-tan, Jouan CR-312 zentrifuga batean (Jouan, Saint-Herblain, Frantzia). Gainjalkinak TVBE indargetzailetan diluitu eta AOX1 jarduera Shimadzu UV-1603 espektrofotometro batean (Shimadzu, Duisberg, Alemania) neurtu zen. Proteina totalaren kontzentrazioa Lowry-ren metodoan oinarritutako DC protein assaya (Bio-Rad, San Diego, California, EEBB) erabiliz neurtu zen, γ- globulina proteina estandar gisa erabiliz. AOX1 jarduera mu AOX mg -1 proteina total gisa adierazi da (nmol H 2 O 2 min -1 mg -1 proteina). PAH eta PFOS kontzentrazioa PFOS, F eta WF-aren pean mantendutako muskuiluen PAH eta PFOS kontzentrazioak Euskal Herriko Unibertsitateko Kimika Analitika Sailean neurtu ziren. PAH-ak Navarro et al. (2006)-en deskribatutako metodologia jarraituz eta PFOS-a Taniyasu et al. (2005)-en metodologia arinki moldatuz. Gene-espresioaren semikuantifikazioa Liseri-guruinak ( mg) Trizoletan (Invitrogen, Carlsbad, California, EEBB) homogenizatu ziren Hybaid Ryboliser homogenizatzailea erabiliz, 20 s eta 4 m/s-ko baldintzen pean. Ondoren, RT-PCR (Invitrogen) bidezko cdnaren sintesia, RNA totalaren 3 µg eta ausazko hexameroak erabiliz burutu zen. PCR bidezko AOX1 (EF525542), CAT (AY743716) eta hsp70 (AY861684) geneen base pare inguruko anplifikazioa, baldintzak Taq polimerasa (Invitrogen) entzimarekin optimizatu ondoren burutu zen. AOX1 genearen anplifikazio espezifikoa burutzeko beharrezkoak ziren baldintzak, aurretik burututako lanetan deskribatutako berberak izan ziren (Tesi honetako bigarren kapitulua). CAT eta hsp70 berriz, 1. Taulan zehaztu bezala anplifikatu ziren. hsp70-en anplifikaziorako erabilitako hasleak Cellura et al. (2006)-tik lortu ziren. PCR 272