Toxicology in Vitro 26 (2012) Contents lists available at SciVerse ScienceDirect. Toxicology in Vitro

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1 Toxicology in Vitro 26 (212) Contents lists vilble t SciVerse ScienceDirect Toxicology in Vitro journl homepge: Crbonyl stress nd combintion of stxnthin/vitmin C induce biochemicl chnges in humn neutrophils B.A. Guerr, A.P. Bolin, R. Otton Postgrdute Progrm, Helth Sciences, CBS, Universidde Cruzeiro do Sul, 3342 São Pulo, SP, Brzil rticle info bstrct Article history: Received 3 April 212 Accepted 22 June 212 Avilble online 3 June 212 Keywords: Antioxidnt Free rdicl High glucose Leukocyte Glyction Methylglyoxl Crotenoid The purpose of the present study ws to find out whether co-tretment of humn neutrophils with high glucose nd methylglyoxl (MGO) cn lter the biochemicl prmeters of humn neutrophils. We lso exmined if stxnthin ssocited with vitmin C cn improve those biochemicl prmeters. Neutrophils from helthy subjects were treted with 2 mm of glucose nd 3 lm MGO followed or not by the ddition of the ntioxidnts stxnthin (2 lm) nd vitmin C (1 lm). MGO/high glucose tretment reduced the phgocytic cpcity nd the G6PDH, totl/sod nd GR ctivities. Additionlly, there ws n increse in the ctivity of myeloperoxidse (MPO) with consequent increse in the hypochlorous cid production, CAT ctivity nd in the relese of IL-6 cytokine without chnges in intrcellulr clcium mobiliztion. Our study lso shows tht the ssocition of stxnthin with vitmin C gretly improved neutrophil phgocytic cpcity, decresing ll rective oxygen species mesured, pro-inflmmtory IL-1b nd TNF- relese, MPO ctivity nd HClO production. The combintion of stxnthin with vitmin C lone hs more ntioxidnt nd nti-inflmmtory effects thn when they were in the presence of MGO/high glucose. Injury to the function of neutrophils due to high glucose nd methylglyoxl ppers not to involve oxidtive stress or clcium relese. The ssocition of ntioxidnts stxnthin nd vitmin C promoted significnt improvement in the function of neutrophils nd in the redox sttus. Ó 212 Elsevier Ltd. Open ccess under the Elsevier OA license. 1. Introduction Methylglyoxl (MGO), highly rective dicrbonyl metbolite produced during glucose metbolism, is mjor precursor of the dvnced glyction end products (AGEs). AGEs re the result of the non-enzymtic glyction of proteins/lipids which ccumulte during nturl ging. In generl, they re lso gretly ugmented in disorders such s dibetes, renl filure nd Alzheimer s disese (Brownlee, 1995; Schmidt et l., 1994; Tkedo et l., 1996). MGO clinicl significnce is bsed on the fct tht there is strong ssocition between the pthophysiology of type 2 dibetes long with ssocited vsculr nd neuronl complictions, nd incresed plsm MGO nd AGEs concentrtions (Turk, 21). Dhr et l. (28) showed tht vsculr smooth muscle cells treted with high glucose (25 mm) incresed intrcellulr MGO concentrtion ccompnied by incresed oxidtive stress. Both MGO nd high glucose my ctivte different pthwys, incresing rective species of oxygen nd nitrogen production (ROS/RNS) which in turn, leds to oxidtive stress (Wng et l., 29). AGEs formed from Corresponding uthor. Address: Av. Regente Feijó, 1295, 3342 São Pulo, SP, Brzil. Tel./fx: E-mil ddresses: rosemri.otton@cruzeirodosul.edu.br, rosemriotton@ hotmil.com (R. Otton). high glucose nd/or MGO cn lso link to specific AGE-receptor (RAGE) present in the plsm membrne of different cell types, including immune cells, nd trigger inflmmtory response by incresing ctivtion of NFjB signling pthwy (Klpos, 1999). Immune cell dysfunction is common feture involved in the pthogenesis nd/or lte complictions of severl chronic diseses. Phgocytosis nd killing of the pthogens re the primry functions of neutrophils in the innte immune response in order to contin nd kill invding microbil pthogens. This process is chieved through series of rpid nd coordinted responses (Filkow et l., 27). Neutrophils exhibit potent ntimicrobil rsenl tht includes oxidnts, proteinses, nd ntimicrobil peptides. Neutrophils lso produce prodigious quntities of ROS nd RNS such s superoxide nd nitric oxide through the ctivity of oxidntgenerting systems such s the phgocyte NADPH oxidse (Shepprd et l., 25) nd nitric oxide synthse (NOS), respectively (Filkow et l., 27; Gebsk et l., 25; Kleinert et l., 24). Astxnthin (ASTA) is n ornge-reddish crotenoid pigment found in living orgnisms prticulrly in the mrine environment where it is present in microlge, plnkton, krill nd sefood. It gives slmon, trout, nd crustcens such s shrimp nd lobster their distinctive pinkish colortion (Fssett nd Coombes, 211). ASTA belongs to the xnthophyll clss of crotenoids nd is closely relted to b-crotene, lutein, lycopene, nd zexnthin, shring Ó 212 Elsevier Ltd. Open ccess under the Elsevier OA license.

2 1182 B.A. Guerr et l. / Toxicology in Vitro 26 (212) with them mny of helth-promoting effects ttributed to crotenoids (Yun et l., 211). Severl studies hve demonstrted tht stxnthin exhibits wide vriety of biologicl ctivities, including the prevention nd tretment of vrious diseses, such s cncers, chronic inflmmtory diseses, metbolic syndrome, dibetes, dibetic nephropthy, crdiovsculr diseses, gstrointestinl diseses, liver diseses, nd neurodegenertive diseses (Chew et l., 1999; Jyonouchi et l., 2; Kishimoto et l., 21; Mrin et l., 211; Nguib, 2; Otton et l., 211). The presence of the hydroxyl nd keto moieties on ech ionone ring (Fig. 1) explins some of its unique fetures such s the bility to be esterified, higher ntioxidnt ctivity, nd more polr nture thn other crotenoids (Hussein et l., 26). Astxnthin my ct s strong ntioxidnt by donting the electrons nd recting with free rdicls to convert them into more stble products nd terminte free rdicl chin rection in wide vriety of living orgnisms. The nonpolr middle segment of the stxnthin molecule is series of crbon-crbon double bonds, which lternte with crbon-crbon single bonds, termed conjugted. This polrnonpolr-polr lyout lso llows the stxnthin molecule to tke trnsmembrne orienttion, mking precise fit into the polrnonpolr-polr spn of the cell membrne (Kidd, 211). As mentioned by mny uthors, the ntioxidnt ctivity of stxnthin ppers to be greter thn tht of bet-crotene nd lph-tocopherol (Fukuzw et l., 1998; Nguib, 2). However, studies from our group which evluted the ntioxidnt effect of stxnthin on leukocytes in humn nd niml models, showed modest ntioxidnt ction (Bolin et l., 21; Guerr nd Otton, 211; Mcedo et l., 21; Mttei et l., 211; Otton et l., 21, 211), minly observed in the reduction of superoxide nd hydrogen peroxide production. Vitmin C is n essentil micronutrient, which hs been implicted in vriety of biologicl processes, including immune response (Meng et l., 29). Vitmin C or L-scorbic cid is the body s most importnt intrcellulr nd extrcellulr queousphse ntioxidnt. This ntioxidnt esily scvengers peroxyl rdicls, superoxide nion, singlet oxygen nd hypochlorite (Sies nd Sthl, 1995). The oxidtion of vitmin C by recting with ROS genertes the scorbyl rdicl tht hs little rectivity, crucil to the ntioxidnt effect of vitmin C. Ascorbic cid is considered physiologicl substrte for myeloperoxidse (MPO) nd its effect on myeloperoxidse-dependent processes is widely ttributed to scvenger or quencher ctions on hypochlorous cid (Myzk nd Crr, 22; Svenkov et l., 1994). Polymorphonucler cells such s neutrophils present millimolr concentrtions of scorbic cid in their cytosol which cn be understood s n importnt protective role ginst the ction of rective species produced by neutrophils (Guiquil et l., 21; Wng et l., 1997). Deficiency of this vitmin is ssocited with impired function of this cell type, including the reduction of its ntimicrobil ctivity (Goldschmidt, 1991) nd decresed spontneous poptosis (Vissers nd Wilkie, 27). Becuse both ntioxidnts re present in specific microenvironment in cells comprtments, we believe tht combintion of stxnthin with vitmin C cn improve the ntioxidnt effect of both. The purpose of the present study ws to find out whether cotretment of humn neutrophils with high glucose (2 mm) nd MGO cn lter the biochemicl prmeters of these immune cells. High glucose ws used s physiologicl intrcellulr source of MGO s previously described (Dhr et l., 28). We lso exmined if stxnthin ssocited with vitmin C cn improve those biochemicl prmeters. In ddition, we evluted the mechnism underlying this modultion. 2. Mterils nd methods 2.1. Regents Methylglyoxl, D-glucose, stxnthin, dihydroethidium, vitmin C, propidium iodide nd most of the other chemicls were purchsed from Sigm-Aldrich Chemicl Compny (St. Louis, MO, USA), except RPMI-164 culture medium, lucigenin nd pluronic cid, nd cetoxymethylester (Fur-2AM), which cme from Invitrogen (CA, USA). Common regents for buffers (e.g. PBS) nd regulr lbortory solutions were obtined from Lbsynth (Didem, SP, Brzil) Subjects The Ethicl Committee of the Universidde Cruzeiro do Sul pproved the experimentl procedure of this study. Around 3 helthy dult women nd men (men ge 21. ± 4.) were included in the present study. The subjects recruited did not present ny systemic or topicl therpeutic regimen, smoking history, lcohol hbits, obesity or ny other systemic diseses t lest for the lst 2 months (bsed on n nmnesis protocol) Cell isoltion nd culture condition Neutrophils were obtined through the collection of humn peripherl blood by venipuncture procedure in vcuum/siliconized tubes contining.1 mm EDTA. Peripherl blood neutrophils were isolted under sterile conditions by using density grdient present in the regent Histopque 177 (Sigm Aldrich), ccording to the mnufcturer s instruction. After obtined, neutrophils were counted in Neubuer chmber using Trypn blue (1%). Neutrophils (1 1 6 /ml) from ech volunteer were cultured in 1 ml of RPMI- 164 medium supplemented with 1% fetl bovine serum, 2 mm Hepes, 2 mm glutmine, nd ntibiotics (streptomycin 1 units/ ml nd penicillin 2 units/ml) or ressuspended in Tyrode s solution (137 mm NCl, 2.68 mm KCl,.49 mm MgCl 2, 12 mm NHCO 3,.36 mm NH 2 PO 4, 5.6 mm D-glucose, nd 5 mm cid HEPES, ph 7.4) for cute ssys. Before strting our experiments we evluted the toxicity of incresing concentrtions of MGO on neutrophils. For this purpose, cells ( ) were treted for 18 h with MGO in concentrtions rnging from 1 to 5 lm. After this period the cells were collected nd nlyzed by Trypn blue exclusion. In the literture, the Fig. 1. Chemicl structure of stxnthin.

3 B.A. Guerr et l. / Toxicology in Vitro 26 (212) physiologicl concentrtion of MGO in plsm is bout 5 lm, but levels cn be 5 6 times higher in ptients with dibetes types 1 nd 2 (Dutr et l., 25). Bsed on those dt, the concentrtion of MGO selected to be used in the present study ws 3 lm MGO (nontoxic, dt not shown) in Tyrode s solution. Glucose concentrtion ws used t 2 mm, lso confirmed s nontoxic concentrtion (Trypn blue exclusion, dt not shown). Astxnthin t 2 lm ws solubilized in DMSO, wheres vitmin C t 1 lm ws solubilized in Tyrode s solution. The following experimentl groups were creted: control (without tretment), AV (stxnthin + vitmin C), GM (glucose + methylglyoxl) nd AVGM (stxnthin + vitmin C + glucose + methylglyoxl). Cells were cultured t 5% CO 2 for 18 h t 37 C nd then were collected, centrifuged nd stored t 8 C to ssy glutthione content nd ntioxidnt enzyme ctivity. To mesure cytokines relese, cells were cultured for 18 h nd the superntnt ws collected nd stored under the sme condition. ROS production nd phgocytic cpcity were ssyed in neutrophils fter cute tretment of cells Cell membrne integrity To ssess whether the concentrtion of MGO, glucose nd both ntioxidnts stxnthin nd vitmin C selected for the experiments cused toxicity in neutrophils, we ssyed cell vibility by using flow cytometer nlysis. Immeditely fter being obtined nd t the end of the culture period (24 h), cells (5 1 5 ) were treted s previously described nd then used to test the membrne integrity. This ssy ws crried out in FACSclibur flow cytometer (Becton Dickinson, Mountin View, CA) using propidium iodide (PI) (5 lg/ml) dissolved in phosphte buffered sline (.137 M NCl, 2.7 mm KCl, 8. mm N 2 HPO 4, ph 7.4). PI is highly wter-soluble fluorescent compound tht cnnot pss through intct membrnes nd is generlly excluded from vible cells. When cells lose membrne integrity it psses through membrne nd binds to DNA. Therefore, n increse in fluorescence to PI indictes decrese in the proportion of vible cells. Fluorescence of PI ws determined in FL2 chnnel (ornge-red fluorescence-585/42 nm). The results were expressed s percentge of the control group Neutrophil functionl prmeters Phgocytic cpcity Neutrophils (5 1 5 cell/well) were treted nd incubted for 6 min t 37 C in 1 ml RPMI 164 medium with opsonised zymosn prticles. Zymosn prticles (5 1 6 /well) were opsonized by incubtion in the presence of control serum for 6 min. Afterwrds cells were hrvested, citocentrifuged, stined nd counted in n opticl microscope. The score of phgocytosis ws expressed by the number of cells tht hd one, two, three, four or more phgocyted zymosn prticles (Smpio et l., 21) Mesurement of hypochlorous cid (HOCl) production Production of HOCl by neutrophils ws evluted ccording to the method described by Dypbukt et l. (Dypbukt et l., 25). In short, fter tretment neutrophils (6 1 5 /well) were stimulted with phorbol myristte cette (PMA) (6 ng/well) for 6 minutes. The rection ws performed in modified PBS (NCl 14 mm, KCl 1 mm, MgCl 2.5 mm, CCl 2 1 mm, glucose 1 mg/ml nd turine 5 mm), ph 7.4. Rections were stopped by the ddition of 26.8 units/ml of ctlse. Cells were then centrifuged, the superntnt (2 ll) ws collected nd dded with 5 ll of solution contining 2 mm of 3,3,5,5-tetrmethylbenzidine (TMB), 1 lm sodium iodide, nd 1% dimethylformmide in 4 mm cette buffer. After 5 min, bsorbnce ws recorded t 65 nm in microplte reder nd stndrd curve (1 4 lm of HOCl) ws used to determine the concentrtion of hypochlorous cid Mesurement of myeloperoxidse (MPO) relese from neutrophils The mesurement of MPO enzyme ctivity ws performed by oxidtion of luminol in the presence of H 2 O 2 nd PMA ccording to Htnk et l. (26). Neutrophils (2 1 6 cells/well) were exposed for 3 min, t 37 C, with or without 2 lm of stxnthin; 1 lm of vitmin C nd/or 2 mm of glucose, nd 3 lm ofmgo in the presence or bsence of PMA. After incubtion, the medium ws immersed into ice nd centrifuged t 5g for 1 min, t 4 C, to seprte the superntnt from the cells. The superntnt ws used to mesure MPO ctivity. The rection ws run in PBS, H 2 O 2 (.1 mm) nd luminol (1 mm), t 37 C, in finl volume of 3 ll. Chemiluminescence ws determined in microplte reder. Results re expressed s reltive luminescence unit (RLU) of degrnultion Glucose-6-phosphte dehydrogense (G6PDH) ctivity Glucose-6-phosphte dehydrogense (G6PDH), EC 1.1.l.49, is key regultory enzyme of the oxidtive segment of the pentosephosphte pthwy. It produces equivlent reducing gents in the form of NADPH to meet some cellulr needs for reductive biosynthesis nd s contribution to the mintennce of the cellulr redox stte (Cost Ros et l., 1995). The mximum ctivity of this enzyme ws previously described (Guerr nd Otton, 211). The extrction buffer consisted of Tris-HCl (5 mm), EDTA (1 mm) t ph 8.. The rection buffer used contined Tris-HCl (86 mm), MgCl 2 (6.9 mm), NADP+(.4 mm), glucose-6-phosphte (1.2 mm) nd Triton X-1.5% (v/v) t ph 7.6. The totl volume of the smple ws 374 ll. The rection ws strted by dding glucose- 6-phosphte to the medium. The bsorbnce t 34 nm ws nlyzed in microplte reder (Tecn, Slzburg, Austri), nd the results re expressed s nmol/min/mg of protein Relese of pro-inflmmtory cytokines Cytokines IL-6, IL-1b nd TNF- were ssyed in cell culture superntnt with ELISA kits ccording to the mnufcturer s instructions (Quntikine, R&D System, Minnepolis, MN, USA). Neutrophils (1 1 6 /ml) were cultured for 18 h in the presence or bsence of LPS s stimulus (1 lg/ml). Afterwrds, cells were centrifuged (1g, 4 C, 1 min) nd the superntnt ws collected nd stored t 8 C until they re used for cytokines determintion. The lower limits of detection for the ELISA nlyses were s follows: 1.17 pg/ml for IL-6 nd 1.95 pg/ml for IL1-b nd TNF Oxidtive prmeters Dihydroethidium ssy Dihydroethidium (DHE) is fluorescent probe used to mesure intrcellulr superoxide nion production. Once inside the cell, DHE is rpidly oxidized to ethidium ( red fluorescent compound) by superoxide nd/or H 2 O 2 (in the presence of peroxidse). Neutrophils (5 1 5 /well) were incubted with 5 lm DHE for 15 min t room temperture in the drk. Afterwrds, the cells were treted nd stimulted with PMA (2 ng/well). As internl control, cells were treted with either 1 lm DPI or 5 lm rotenone ( complex 1 electron trnsport chin inhibitor), nd.4 mm sodium zide (SA), complex III electron trnsport chin inhibitor for 3 min prior to tretment. Also, to ensure the specificity of DHE to superoxide nion, hydrogen peroxide (5 lm) ws dded to control- PMA stimulted cells. The fluorescence ws nlyzed in microplte reder (Tecn, Slzburg, Austri) (396 nm wvelength excittion nd 59 nm wvelength emission). The results were expressed s percentge of the control group Lucigenin The lucigenin chemiluminescent probe ws utilized to mesure the extrcellulr superoxide nion content minly produced

4 1184 B.A. Guerr et l. / Toxicology in Vitro 26 (212) through NADPH-oxidse ctivtion. Lucigenin releses energy in the form of light fter excittion by superoxide nion. The chemiluminescence produced ws monitored by luminometer for 6 min (Tecn, Slzburg, Austri). Lucigenin (5 lm) ws dded to cells (5 1 5 / well) treted with or without 2 mm of glucose nd 3 lm of MGO, in the presence or bsence of 2 lm of stxnthin, 1 lm of vitmin C in Tyrode s buffer supplemented with fetl bovine serum 1%. The experiments were crried out in triplicte in the presence nd bsence of opsonized zymosn prticles (1 1 6 /well) used s ROS-inducer. As internl control, 1 lm diphenyleneiodonium (DPI), NADPH-oxidse inhibitor, or.4 mm sodium zide (SA), complex III electron trnsport chin inhibitor, were dded to control cells 3 min prior to the lucigenin evlution. Results re expressed s chemiluminescence reltive units. The sttisticl nlysis ws performed by AUC clcultion (re under the curve) of t lest three different experiments performed in triplicte Hydrogen peroxide production by phenol red ssy Hydrogen peroxide (H 2 O 2 ) production ws mesured ccording to Pick nd Mizel (1981), bsed on horserdish peroxidse, which ctlyzes the phenol red oxidtion by H 2 O 2.Neutrophils(5 1 5 /well) were incubted with or without 2 lmofstxnthin,1lm of vitmin C nd 2 mm of glucose, nd 3 lm of MGO in Tyrode s buffer, mixed with.28 mm phenol red nd horserdish peroxidse (1, units/mg) t 37 C for 1 h. The production of H 2 O 2 ws mesured in the bsence nd presence of PMA (2 ng/well). The rection ws terminted by lkliniztion (ddition of 1 ll of NOH 1 M solution) nd bsorbnce t 62 nm ws mesured to evlute H 2 O 2 concentrtion (compred to stndrd curve). The results were expressed s percentge of the control group Generl ROS production ssyed by DCFH-DA The probe DCFH-DA is primrily used s n indictor of the production of H 2 O 2 (Brndt nd Keston, 1965) but it is lso described s being oxidized by other ROS such s HO., ROO., NO nd peroxynitrite (Crow, 1997). The cells (5 1 5 /well) were preloded with DCFH-DA (5 lm) by incubtion in culture medium for 3 minutes. DCFH-DA is cleved inside the cells by non specific esterse nd turns to high fluorescent 2,7-dichlorofluoroscein (DCF) upon oxidtion by ROS. After the loding period, cells were treted with or without 2 lm of stxnthin, 1 lm of vitmin C nd 2 mm of glucose, nd 3 lm of MGO in Tyrode s buffer for 6 minutes. The experiments were conducted in the presence or bsence of PMA (2 ng/well). Afterwrds, cells were centrifuged nd resuspended in 3 ll of Tyrodés buffer, nd the fluorescence ws monitored in spectrofluorimeter Tecn (Slzburg, Austri) with excittion t 485 nm nd emission t 53 nm. As n internl control 5 lm of H 2 O 2 ws dded to control cells under PMAstimultion to ensure the specificity of DCFH-DA. The results were expressed s percentge of the control group Nitric oxide production (NO ) NO production ws performed ccording to Ding et l. (1988) through nitrite determintion. Nitric oxide is rpidly converted into nitrite in queous solutions nd, therefore, the totl nitrite cn be used s n indictor of nitric oxide concentrtion. The spectrophotometric nlysis of the totl nitrite content ws performed by using the Griess regent (1% sulfnilic cid,.1% N-1-nphthylethylenedimine dihydrochloride) in superntnts. Neutrophils (5 1 5 /1 ll) in RPMI 164 medium were treted with or without 2 lm of stxnthin, 1 lm of vitmin C nd 2 mm of glucose nd 3 lm of MGO nd stimulted with lipopolyscchride (LPS) t 1 lg/well for 4 h. Then, the sme volume of Griess (187 ll) ws dded to cells nd the bsorbnce ws mesured in 55 nm. The nitrite concentrtion ws determined using sodium nitrite s stndrd ( 6 lm). The results were expressed s percentge of the control group Intrcellulr C 2+ concentrtion Chnges in cytosolic C 2+ levels were monitored by fluorescence using the clcium-sensitive probe Fur 2-AM (Otton et l., 27). Neutrophils (1 1 6 /well) were treted with or without 2 lm of stxnthin, 1 lm of vitmin C nd 3 lm of MGO in the presence of opsonized zymosn prticles (1 1 6 /well). Totl intrcellulr relese of C 2+ ws monitored for 6 min in microplte reder (Tecn, Slzburg, Austri). Trnsformtion of the fluorescent signl to C 2+ (in nmol C 2+ per minute) ws performed by clibrtion with ionomycin (1 lm, mximum concentrtion) followed by EGTA ddition (6 lm, minimum concentrtion) ccording to the Grynkiewicz eqution (Grynkiewicz et l., 1985) Antioxidnt profile of neutrophils Antioxidnt enzyme ctivity To evlute ntioxidnt enzyme ctivities s well s GSH nd GSSG content, we performed these specific ssys fter 24 h of culture s previously described. After this period, cells (5 1 6 ) were hrvested, centrifuged nd the pellet ws dded with specific extrction buffer. Cells were then ruptured by ultrsoniction in Vibr Cell pprtus (Connecticut, USA), centrifuged for 1 min, 1,g t 4 C nd the superntnt ws used for nlysis. Superoxide dismutse (SOD), Ctlse (CAT), glutthione peroxidse (GPx) nd glutthione reductse (GR) ctivities were determined in neutrophils using microplte reder (Tecn, Slzburg, Austri). CAT ctivity ws mesured s described by Aebi (1984) bsed on the direct decomposition of hydrogen peroxide (H 2 O 2 ). SOD ctivity ws mesured using the method described by Ewing nd Jnero (1995) which involves the reduction of O 2 rdicls by nitroblue tetrzolium (NBT) for 3 min. Glutthione peroxidse (Mnnervik, 1985) nd glutthione reductse (Crlberg nd Mnnervik, 1985) ctivities were mesured bsed on the oxidtion of b-nadph in the presence of tert-butyl hydroperoxide, used s substrte GSH nd GSSG content Reduced (GSH) nd oxidized (GSSG) glutthione content in neutrophils were mesured s described by Rhmn et l. (26). The method is bsed on the rection between reduced thiol groups (such s in GSH) with 5,5 -dithiobis-2-nitrobenzoic cid (DTNB) to form 5-thio-2-nitrobenzoic cid (TNB), which is stoichiometriclly detected by bsorbnce t 412 nm. Purified GSH nd GSSG (Sigm- Aldrich) were used s stndrds Protein mesurement The totl protein content of cells ws mesured by the method of Brdford, using BSA s stndrd (Brdford, 1976) Sttisticl nlyses All dt points re presented s the men vlues with stndrd errors of t lest three independent experiments, ech one performed in triplicte. The dt were nlyzed by one-wy ANOVA followed by the Tukey s post-test. The softwre employed for sttisticl nlysis ws GrphPd Prism (version4; GrphPd Softwre, Sn Diego, CA, USA). 3. Results 3.1. Cell membrne integrity Cell membrne integrity ws tested by using flow cytometer nd propidium iodide s probe. After 24 h of culture, none of

5 B.A. Guerr et l. / Toxicology in Vitro 26 (212) the groups showed ny significnt loss of cell membrne integrity. These results indicte tht the concentrtions of MGO, glucose, stxnthin nd vitmin C selected to evlute the functionl prmeters of neutrophils did not cuse cell deth (Fig. 2). Additionlly, MGO, high glucose, stxnthin nd vitmin C lone did not promote chnges in cell vibility (dt not shown) Phgocytic cpcity In order to determine the potentil of MGO nd glucose to modulte the phgocytic cpcity of humn neutrophils, we mesured the incorportion of opsonized zymosn prticles in the cells (Tble 1). There ws significnt decrese of 3% in the phgocytic cpcity of neutrophils fter tretment with glucose + methylglyoxl (GM group), wheres there ws n increse of 22% in the phgocytic cpcity fter AV-tretment s compred to the control group. When GM-treted cells were dded with ntioxidnts (AVGM group) we observed complete restortion in the phgocytic cpcity. Neither glucose nor MGO lone promoted the sme effect observed when those compounds were combined (dt not shown). Vitmin C lone promoted improvement in the phgocytic cpcity (dt not shown) Myeloperoxidse (MPO) ctivity MPO ctivity ws evluted in neutrophils fter induction of neutrophil-degrnultion by ddition of PMA for 3 min (Tble 1). As compred with the control group, MPO ctivity ws incresed by 4% in the GM-group nd reduced 86% nd 94% in the AV nd AVGM groups, respectively Hypochlorous cid production Neutrophils were stimulted to produce hypochlorous cid by the ddition of PMA (6 ng/well). Hypochlorous cid concentrtion ws significntly reduced by 25% in the AV group nd incresed by 135% nd 99% in the GM nd AVGM groups, respectively, when compred with the control group (Tble 1) Glucose-6-phosphte dehydrogense ctivity The mximum G6PDH ctivity ws ssessed by the reduction of the co-fctor NADP + into NADPH in humn neutrophils (Tble 1). GM promoted significnt reduction of 37% in G6PDH ctivity % of cells 15 Vible cells Non-vible cells 1 5 nd stxnthin + vitmin C ddition (AVGM group) incresed the G6PDH ctivity by 52% when compred to the GM group Cytokines Relese TNF-, IL-1b, nd IL-6 re inflmmtory cytokines which ply importnt roles in immune responses to vriety of inflmmtory stimuli. Therefore, we evluted the effects of GM on TNF-, IL-1b, nd IL-6 fter 18 h of LPS-stimultion. The levels of these cytokines in the culture superntnts were mesured using ELISA kits. Control neutrophils treted with LPS showed significnt increse in cytokine production when compred with the bsl condition (1 ± 1 pg/ml, dt not shown). The production of proinflmmtory cytokines IL-6, IL-1b nd TNF- by humn neutrophils in the AVGM group ws significntly decresed by 46%, 36% nd 77%, respectively, when compred with the GM-group. IL-1b nd TNF- were lso reduced in the AV-group by 42% nd 89%, respectively, when compred with the control group Mesurement of superoxide nion production The production of rective oxygen species is mong the key wepons used by neutrophils to exterminte pthogens. In order to evlute some possible modultion of MGO + glucose nd stxnthin nd vitmin C in few of these species we used different probes. Superoxide nion production ws mesured by using two different probes, DHE nd lucigenin. As ssyed by the DHE probe, when GM-treted cells were stimulted with PMA there ws n increse of 41% in the superoxide nion production compred with the PMA-control cells. Cells treted with stxnthin plus vitmin C decresed production of superoxide nion by 54% s compred with the control-stimulted group. Addition of ntioxidnts to cells treted with GM (AVGM group) promoted reduction of 66% in superoxide s compred with the GM group in stimulted conditions. Rotenone + Sodium Azide nd DPI were dded to neutrophils under PMA-stimultion. Both inhibitors significntly reduced superoxide nion production to bsl levels. SOD enzyme ddition ws used to evlute the specificity of DHE probe to superoxide nion (Fig. 3A), nd s expected there ws no significnt fluorescence in this group. As n internl control, we lso crried out the ddition of 5 lm ofh 2 O 2 to PMAtreted cells. As expected, there ws no increse in the fluorescence produced, thus ensuring the specificity of DHE for superoxide nion (dt not shown). The lucigenin probe (Fig. 3B) ws used to mesure the ctivtion of NADPH-oxidse nd then extrcellulr superoxide nion production. For this purpose neutrophils were chllenged with opsonized zymosn prticles. All tretments promoted reduction in the superoxide nion production s compred with controlzymosn group. DPI (1 lm) ddition 3 min before the tretment with zymosn prticles promoted totl inhibition in the lucigenin signl, indicting tht, indeed, superoxide nion production occurred vi NADPH-oxidse ctivtion. Sodium zide (SA) did not promote significnt reduction in the lucigenin light emission, indicting the specificity of lucigenin probe to superoxide nion present in the extrcellulr comprtment Hydrogen peroxide production Fig. 2. Vibility of humn neutrophils (5 1 5 /well) fter 24 h of culture. Cells were treted with glucose (2 mm) nd MGO (3 lm) with or without the ntioxidnts stxnthin (2 lm) nd vitmin C (1 lm), nd cultured s previously described. Afterwrds, cells were hrvested nd then nlyzed by flow cytometry using propidium iodide s probe. Results re presented s men ± SEM of t lest three independent experiments, ech one performed in triplicte. Hydrogen peroxide production ws evluted by the method of phenol red oxidtion (Fig. 3C) nd DCFH-DA probe (Fig. 3D). MGO + glucose did not promote ny modifiction in the H 2 O 2 production. However, in both ssys when the neutrophils were treted with the ntioxidnts in the AV nd AVGM groups, there ws

6 1186 B.A. Guerr et l. / Toxicology in Vitro 26 (212) Tble 1 Functionl prmeters of humn neutrophils. Phgocytosis (Score of phgocitic cpcity) 263 ± ± ± ± 9 b MPO ctivity (reltive luminescence unit) 1516 ± ± ± ± 16,b HOCl production (lm of HOCl/6 1 5 cells) 18.8 ± ± ± ± 1.52,b G6PDH ctivity (nmol/min/mg/protein) 48.6 ± ± ± ± 3.34 b IL-6 (pgml 1 ) 428 ± ± ± ± 56 b IL-1b (pgml 1 ) 121 ± ± 5 13 ± 8 66 ± 5,b TNF- (pgml 1 ) 69±6 8±1 53 ± 4 11 ± 1,b The results re presented s men ± SEM of t lest three experiments performed in triplicte. In the ssy of cytokines production, ll groups were LPS-stimulted nd in the myeloperoxidse ssy nd hypochlorous cid production, both experiments were stimulted with PMA. Compred to control group (p <.1). b Compred to GM group (p <.1). A % of the control B Arbitrry unit of chemiluminescence Unstimulted cells PMA-stimulted cells RT+SA SOD DPI Control SA+Zym DPI+Zym Time (minutes) DPI+SA+Zym,b Control+Zym AV+Zym GM+Zym AVGM+Zym,b C % of the control D % of the control 11 Unstimulted-cells PMA-Stimulted cells 1 9 8,b Unstimulted cells PMA-stimulted cells ,b H 2 O 2 E 4 Unstimulted cells LPS-stimulted cells % of the control c c,b Fig. 3. (A) Intrcellulr superoxide nion production ssyed by DHE probe, (B) extrcellulr superoxide nion production ssyed by lucigenin probe, (C) hydrogen peroxide production ssyed by phenol red oxidtion, nd by using DCFH-DA probe (D), NO production ssyed by Griess regent (E). Neutrophils were stimulted s described in the Mteril nd Methods section. The results re presented s men ± SEM of t lest three independent experiments, ech one performed in triplicte. () compred to controlstimulted group (p <.1); (b) compred to GM-stimulted group (p <.1); (c) compred to control non-stimulted group (p <.1). significnt reduction in the production of hydrogen peroxide fter PMA-stimultion. As positive control for the DCFH-DA probe we dded 5 lmofh 2 O 2. Our dt show tht the DCFH-DA probe hs high specificity to hydrogen peroxide Nitric oxide production The NO production ws evluted in cells t bsl nd LPS-stimulted conditions (Fig. 3E). In bsl conditions there ws

7 B.A. Guerr et l. / Toxicology in Vitro 26 (212) Intrcellulr clcium concentrtion (nm) n increse of 115% nd 88% in the AV nd AVGM groups when compred with the control group. After LPS-stimultion there ws n increse in NO production of 52% nd 37% in the AV nd AVGM groups respectively, s compred with the control group Intrcellulr clcium concentrtion Intrcellulr clcium mobiliztion ws monitored for 2 minutes by using Fur 2-AM probe in neutrophils chllenged with opsonized zymosn prticles (Fig. 4). There ws no significnt difference in clcium relese mong ll groups Activity of ntioxidnt enzymes Totl SOD ctivity ws decresed in the GM nd AV groups by 28% nd 23%, respectively, s compred to the control group (Tble 2). In the AVGM group there ws n increse of 35% in the totl SOD ctivity in comprison with the GM group. Mximum ctivity of ctlse (CAT) incresed in 43% in the GM group, wheres there ws reduction of 32% nd 17% in the AV nd AVGM groups, respectively, both compred with the control group. In the AVGM treted cells we observed reduction of 42% when compred with the GM group. However, there ws 3-fold increse in the GPx ctivity in the AVGM group compred with the control. GM nd AV reduced the GR ctivity in 82% nd 25%, respectively, compred to the control group, wheres in the AVGM group there ws n 11- fold increse in GR ctivity compred to the GM group (Tble 2) GSH nd GSSG content nd GSH/GSSG rtio The content of GSH incresed 93% fter ddition of ntioxidnts in the AV group when compred with the control group. As consequence, GSH/GSSG rtio ws incresed in the AV group when compred with the control group (Tble 2). 4. Discussion Fig. 4. Totl intrcellulr clcium [C 2+ ]i (nm) mobiliztion monitored during 2 min. Cells (1 1 6 /well) were previously loded with 5 lm Fur 2-AM for 1 h nd then incubted with glucose + MGO nd/or stxnthin + vitmin C nd stimulted with opsonized zymosn prticles (5 1 6 /well). The results re presented s men ± SEM of t lest three independent experiments, ech one performed in triplicte. Dibetic ptients suffer from mny common infections whose cuses remin unknown. One hypothesis suggests tht the dmge would be cused by immune glyction which cn be responsible for incresed susceptibility to infections (Price et l., 21). Among the glyction gents we cll ttention to methylglyoxl, which is dicrbonyl rective tht origintes from the brekdown of glucose (Desi nd Wu, 27). The results of this study showed tht co-tretment of humn neutrophils with MGO/high glucose promoted importnt modifictions in the neutrophil function in vitro. Tretment of neutrophils with MGO/high glucose did not promote citotoxicity; however, it reduced the phgocytic cpcity nd the G6PDH, totl/sod nd GR ctivities. Additionlly, there ws n increse in the ctivity of myeloperoxidse (MPO) with consequent increse in the hypochlorous cid production, CAT ctivity nd in the relese of IL-6 cytokine without chnges in intrcellulr clcium mobiliztion. Contrsting with other studies (Dhr et l., 28), MGO/high glucose did not show strong prooxidnt effect, s demonstrted by the rtings in the production of superoxide nion, hydrogen peroxide nd nitric oxide. These results indicte which MGO/high glucose effects did not involve oxidtive stress or clcium relese. In ddition, our study shows tht the ssocition of stxnthin with vitmin C gretly improved neutrophil phgocytic cpcity, decresing ll rective oxygen species mesured, pro-inflmmtory IL-1b nd TNF- relese, MPO ctivity nd HClO production. The combintion of stxnthin with vitmin C lone hs more ntioxidnt nd nti-inflmmtory thn when they were in the presence of MGO/high glucose. The bnorml glucose homeostsis in dibetes due to the formtion of the highly rective metbolite MGO (Fleming et l., 211; Tjim et l., 22; Thornlley, 25) my be the key step in triggering the neutrophil dysfunction. Neutrophils re the first immune cells to enter the site of infection or injury nd there neutrophils kill microorgnisms by ingesting them into phgocytic vcuoles (phgosomes). Therefore, phgocytosis is undoubtedly one of the most importnt roles of neutrophils. During phgocytosis, grnules in the cytoplsm of neutrophils merge with the newly formed phgosome, forming the phgolysosome (Kuijpers et l., 21). The cytoplsmic grnules of neutrophils hve s one of their min constituent myeloperoxidse, the enzyme tht ctlyzes the rection of hydrogen peroxide in the presence of hlide ions such s chloride, bromide nd iodide hipohlosos cids, in prticulr hypochlorous cid (Hmpton et l., 1998; Kettle et l., 1997). Hypochlorous cid is considered one of the most importnt ntimicrobil gents produced by neutrophils. During phgocytosis there is ctivtion of the NADPH oxidse, n enzyme complex tht ssembles in the phgosoml membrne nd converts oxygen into the superoxide rdicl nion (O 2 ). Superoxide nion is generted in the externl surfce (i.e. inside the phgosome) with reducing equivlents supplied by intrcellulr NADPH. During grnules secretion, phgocytosis nd killing of pthogens, levels of clcium in the cytosol re usully incresed (Lee et l., 23). In cells co-treted with MGO/high glucose (GM group) there ws n increse in MPO enzyme ctivity nd consequently in the production of hypochlorous cid (Tble 1), wheres neither high glucose nor MGO lone yielded the sme effect (dt not shown). Myeloperoxidse is n enzyme stored in zurophilic grnules of polymorphonucler neutrophils nd relesed into extrcellulr fluid during inflmmtory processes. Severl studies hve shown its involvement in oxidtive stress nd inflmmtion. Recently, MPO hs been considered s possible mrker of plque instbility nd useful tool for the prognostic evlution of ptients with coronry rtery disese (Gustpne et l., 211). Possibly, incresed relese nd ctivity of MPO in neutrophils promoted by co-tretment of neutrophils with MGO/high glucose could contribute to the development of the micro- nd mcro-vsculr complictions observed in dibetic condition. In contrst, cells treted with ntioxidnts promoted mrked reduction in the MPO nd HClO production long with drstic reduction in ll rective oxygen species. This effect ws not observed when cells were treted with either stxnthin or vitmin C lone. Superoxide is physiologicl substrte for MPO nd their interctions re centrl to n importnt host defense mechnism. When relesed by neutrophils, MPO enzyme opertes in the

8 1188 B.A. Guerr et l. / Toxicology in Vitro 26 (212) Tble 2 Antioxidnt enzyme ctivities nd glutthione content. Totl/SOD (U/mg protein) ± ± ± ±.33 b,c CAT (lmol/min/mg protein) 4.51 ± ± ± 1.4,b ±.98,b GPx (mu/mg protein) 4.54 ± ± ± ± 1.9 GR (mu/mg protein) 31.3 ± ± ± ± 6.17,b GSH (lm of GSH/mg of protein) 2.78 ± ± ± ±.34 c GSSG (lm of GSH/mg of protein).37 ±.4.43 ±.3.24 ±.5.32 ±.7 GSH/GSSG (lm of GSH/mg of protein) 6.37 ± ± ± ± 1.34 c The results re presented s men ± SEM of t lest three experiments performed in triplicte. Compred to the control group (p <.1). b Compred to the MG group (p <.1). c Compred to the AV group (p <.1). presence of flux of superoxide. Winterbourn nd Kettle (24) showed tht superoxide hs profound influence on oxidtive rections ctlysed by MPO. It rects directly with the enzyme to modulte production of hypochlorous cid. Within neutrophil phgosomes, where MPO cts by killing micro-orgnisms, it my be the preferred substrte for the enzyme. Superoxide lso rects rpidly with rdicls generted by MPO forming different species. These species re likely to be toxic nd contribute to the pthophysiologicl ctions of MPO (Winterbourn nd Kettle, 24). Therefore, reduced superoxide nion nd hydrogen peroxide production promoted by stxnthin nd vitmin C cn be involved in the reduced MPO nd HClO production s well s direct scvenger effect promoted by ntioxidnts. In ddition, the phgocytic cpcity of neutrophils nd G6PDH ctivity, key enzyme of pentose pthwy involved in NADPH formtion, were decresed in cells fter tretment with MGO/high glucose. A decrese in the phgocytic cpcity ccompnied by decrese in NADPH vilbility could men minor neutrophil effectiveness to destroy pthogens. This fct hs been ssocited with the impirment in neutrophil function observed in dibetes (Lecube et l., 211). Decresed phgocytic cpcity by induced by MGO/ high glucose ws prevented by tretment of cells with the combintion of ntioxidnts stxnthin nd vitmin C. Previous studies from our group lso showed tht ddition of stxnthin lter the cpcity of neutrophils to phgocytose opsonized zymosn prticles under different conditions (Guerr nd Otton, 211; Mcedo et l., 21; Mrin et l., 211). The mechnism by which the ntioxidnt stxnthin improves phgocytic cpcity of neutrophils remins to be elucidted in future studies. Although it is well known tht phgocytosis in neutrophil cells is process which involves intrcellulr clcium mobiliztion, in the present study we did not observe ny chnges in intrcellulr clcium concentrtion mong ll groups. By mens of Millrd rection, MGO is ble to cross-link with cellulr proteins on trgeted mino cids (rginine, lysine), leding to the formtion of dvnced glyction end-products (AGEs), nd thus contributing to ging nd complictions in chronic diseses (Fleming et l., 211; Thornlley, 25). Similrly to our results, some uthors showed which MGO inctivte the enzyme glutthione reductse (Pget et l., 1998; Prk et l., 23; Wu nd Juurlink, 22). Glutthione reductse recycles GSSG using NADPH s cofctor, reestblishing the intrcellulr content of reduced glutthione (GSH) (Juurlink, 1999; Wu nd Juurlink, 22). Other studies hve shown tht MGO reduced GSH content mking cells more sensitive to oxidtive stress (Kikuchi et l., 1999; Meister, 1988; Shinpo et l., 2). The inctivtion of MGO is process ctlyzed by the glyoxlse system tht uses glutthione (GSH) s cofctor. MGO inctivted bovine glutthione peroxidse in time nd dosedependent mnner, forming connection with glutthione to sites of rginine 184 nd 185 (Prk et l., 23). High concentrtion of MGO in plsm nd ort re ssocited with incresed levels of superoxide, significntly reduced levels of GSH, decresed ctivity of glutthione peroxidse nd glutthione reductse in SHR rts with high blood pressure (Wng et l., 25). Contrsting with these studies, we did not observe ny chnge in the content of GSH, GSSG nd in the rte GSH/GSSG (Tble 2). Studies by Chng nd collegues (Chng et l., 25) demonstrted tht MGO cused mitochondril oxidtive stress by incresing the mitochondril production of superoxide, nitric oxide nd peroxynitrite. MGO cn inhibit complex III nd thereby disrupt the electron trnsport chin, leding to lekge of electrons to form superoxide nion (Wng et l., 29). The direct effect of MGO on mitochondri ws investigted by Desi nd collegues (Desi nd Wu, 27) using MitoSOX, mitochondril specific probe used to detect mitochondril superoxide production. Incubtion of vessel smooth muscle cells with MGO 3 lmol/l significntly induced mitochondril superoxide production s compred with the group of untreted cells. Another study showed tht incubtion of vsculr smooth muscle cells (VSMCs) of rt ort with MGO significntly incresed production of superoxide in dose-dependent mnner, which ws prevented by the ddition of enzyme superoxide dismutse (SOD) or inhibition of NADPH oxidse with DPI (Chng et l., 25). MGO lso incresed the genertion of hydrogen peroxide in VSMCs nd incresed formtion of peroxynitrite (ONOO ) through the induction of inducible NOS (inos) (Chng et l., 25). Similr results were found by Wrd nd McLeish, who dded MGO in neutrophils nd found tht there ws significnt increse in bsl production of hydrogen peroxide nd superoxide nion in dose-dependent mnner of the MGO concentrtion, indicting incresed respirtory burst ctivity (Wrd nd McLeish, 24). The effect of MGO ws significntly higher in pltelets pretreted with n gent tht depletes GSH nd glutthione peroxidse (Leoncini nd Poggi, 1996). Contrsting with these results, our dt show tht MGO/high glucose did not cuse ny mjor chnge in the production of rective oxygen/nitrogen species in neutrophils (Fig. 3). One cceptble reson for the wek pro-oxidnt effect of MGO/ high glucose could be the MGO concentrtion used in the present study. Mny uthors demonstrte modultion of MGO on different cell types using high MGO concentrtions rnging from 1 lm to 1 mm (Chng et l., 25; Desi et l., 21; Wng et l., 29). We used MGO t 3 lm, which is considered by some uthors high concentrtion usully found in the dibetic plsm (Dutr et l., 25). In ddition, the incubtion time of neutrophils which MGO/high glucose could be short to promote ny permnent modifiction in the neutrophil function. Severl uthors hve shown tht, to be effective s glyction gent, MGO needs to be incubted for long periods, which ws not observed in this work, due to the short hlf-life of neutrophils in culture. On the other hnd, ssocition of stxnthin with vitmin C promoted cler ntioxidnt effect (Fig. 3) s observed by the mrked reduction in the production of superoxide nion nd hydrogen peroxide production. Compred with previous study

9 B.A. Guerr et l. / Toxicology in Vitro 26 (212) from our group tht showed wek stxnthin ntioxidnt-effect (Bolin et l., 21; Cmpoio et l., 211; Guerr nd Otton, 211; Mcedo et l., 21), the ssocition of both ntioxidnts llowed gret ntioxidnt ction. Mny uthors hve reported the effective ntioxidnt ction of either stxnthin or vitmin C lone, but not in combintion. In our model, the stxnthin/vitmin C system mimics the recycling system of vitmin C/vitmin E. Astxnthin provides cell membrnes with potent protection ginst free rdicls or other oxidtive ttck. Experimentl studies confirm tht this nutrient hs lrge cpcity to neutrlize free rdicls or other oxidnt ctivity in the nonpolr ( hydrophobic ) zones of phospholipid ggregtes, s well s long their polr (hydrophilic) boundry zones (Fssett nd Coombes, 211). Vitmin C, in turn, promotes ntioxidnt effects minly in wterphse microenvironment. Neutrophils re cpble of expressing vriety of proteins involved in inflmmtion nd immune responses, including cytokines such s IL-1b, IL-1r (interleukin receptor ntgonist), IL-8, IL-12, TNF-, TGF -b, MIP-1, MIP-1b, GM-CSF nd IFN- (Scpini et l., 2). Although cytokines induce pthology when expressed inppropritely, they ply importnt roles in vriety of physiologicl processes. Wng nd collegues demonstrted tht 3 lmol/l MGO for 12 h significntly incresed the secretion of pro-inflmmtory cytokines such s interleukin-6 (IL-6), IL-8 nd tumor necrosis fctor (TNF-), nd induced poptosis in neutrophils (Wng et l., 27). In this study, MGO/high glucose incresed IL-6 production in cells stimulted with LPS. When ntioxidnts were dded to MGO/high glucose treted cells (AVGM group), there ws n importnt reduction in ll pro-inflmmtory cytokines when compred to the GM group. In summry, our results show tht tretment of neutrophils with high glucose nd MGO promotes n injury to the function of neutrophils, nd this process ppers not to involve oxidtive stress or clcium relese. In ddition, when cells were treted with the ssocition of ntioxidnts stxnthin nd vitmin C, we observed significnt improvement in the function of neutrophils nd in the redox sttus. The use of ntioxidnts to prevent or reverse dibetic complictions seems to be necessry; however, single substnce cnnot chieve this effect. Therefore, we re proposing combintion of two substnces tht ct differently in cell microenvironment, working in collbortive wy. The collbortive wy in which the ntioxidnts work ws evidenced in lmost ll experiments performed s compred with cells treted with ntioxidnts lone. In the ner future, the combintion of ntioxidnts stxnthin nd vitmin C might ct s n djuvnt therpy for the tretment of vriety of diseses, including dibetes mellitus. The nswer to the question of whether the in vitro neutrophils protection chieved by this combintion of therpy cn be trnslted to subjects with dibetes will hve to wit until completion of the ongoing clinicl tril. Conflict of interest sttement All the uthors of the present mnuscript declre tht there is no ny ctul or potentil conflict of interest including ny finncil, personl or other reltionships with other people or orgniztions tht could inppropritely influence, or be perceived to influence our work. Acknowledgements The uthors re grteful to the technicl ssistnce of T.R. Cmpoio, Mrinovic MP nd A.C. Morndi. This reserch ws supported by Fundção de Ampro Pesquis do Estdo de São Pulo (FAPESP 9/ nd 9/ ), Conselho Ncionl de Desenvolvimento Científico e Tecnológico (Bols Produtividde em Pesquis, Nivel 2, #31219/29-3, CNPq, Brzil) nd Universidde Cruzeiro do Sul. References Aebi, H., Ctlse in vitro. Methods Enzymol. 15, Bolin, A.P., Mcedo, R.C., Mrin, D.P., Brros, M.P., Otton, R., 21. Astxnthin prevents in vitro uto-oxidtive injury in humn lymphocytes. Cell Biol. Toxicol. 26, Brdford, M.M., A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye binding. Anl. Biochem. 72, Brndt, R., Keston, A.S., Synthesis of dicetyldichlorofluorescin: stble regent for fluorometric nlysis. Anl. Biochem. 11, 6 9. Brownlee, M., Advnced protein glycosyltion in dibetes nd ging. Annu. Rev. Med. 46, Cmpoio, T.R., Oliveir, F.A., Otton, R., 211. Oxidtive stress in humn lymphocytes treted with ftty cid mixture: role of crotenoid stxnthin. Toxicol. in Vitro 25, Crlberg, I., Mnnervik, B., Glutthione reductse. Methods Enzymol. 113, Chng, T., Wng, R., Wu, L., 25. Methylglyoxl-induced nitric oxide nd peroxynitrite production in vsculr smooth muscle cells. Free Rdic. Biol. Med. 38, Chew, B.P., Prk, J.S., Wong, M.W., Wong, T.S., A comprison of the nticncer ctivities of dietry bet-crotene, cnthxnthin nd stxnthin in mice in vivo. Anticncer Res. 19, Cost Ros, L.F., Curi, R., Murphy, C., Newsholme, P., Effect of drenline nd phorbol myristte cette or bcteril lipopolyscchride on stimultion of pthwys of mcrophge glucose, glutmine nd O2 metbolism. Evidence for cyclic AMP-dependent protein kinse medited inhibition of glucose-6- phosphte dehydrogense nd ctivtion of NADP+-dependent mlic enzyme. Biochem. J. 31 (Pt 2), Crow, J.P., Dichlorodihydrofluorescein nd dihydrorhodmine 123 re sensitive indictors of peroxynitrite in vitro: implictions for intrcellulr mesurement of rective nitrogen nd oxygen species. Nitric Oxide 1, Desi, K., Wu, L., 27. Methylglyoxl nd dvnced glyction endproducts: new therpeutic horizons? Recent Pt Crdiovsc Drug Discov 2, Desi, K.M., Chng, T., Wng, H., Bnigesh, A., Dhr, A., Liu, J., Untereiner, A., Wu, L., 21. Oxidtive stress nd ging: is methylglyoxl the hidden enemy? Cn. J. Physiol. Phrmcol. 88, Dhr, A., Desi, K., Kzchmov, M., Yu, P., Wu, L., 28. Methylglyoxl production in vsculr smooth muscle cells from different metbolic precursors. Metbolism 57, Ding, A.H., Nthn, C.F., Stuehr, D.J., Relese of rective nitrogen intermedites nd rective oxygen intermedites from mouse peritonel mcrophges. Comprison of ctivting cytokines nd evidence for independent production. J. Immunol. 141, Dutr, F., Ciriolo, M.R., Clbrese, L., Bechr, E.J., 25. Aminocetone induces oxidtive modifiction to humn plsm ceruloplsmin. Chem. Res. Toxicol. 18, Dypbukt, J.M., Bishop, C., Brooks, W.M., Thong, B., Eriksson, H., Kettle, A.J., 25. A sensitive nd selective ssy for chlormine production by myeloperoxidse. Free Rdic. Biol. Med. 39, Ewing, J.F., Jnero, D.R., Microplte superoxide dismutse ssy employing nonenzymtic superoxide genertor. Anl. Biochem. 232, Fssett, R.G., Coombes, J.S., 211. Astxnthin: potentil therpeutic gent in crdiovsculr disese. Mr Drugs 9, Filkow, L., Wng, Y., Downey, G.P., 27. Rective oxygen nd nitrogen species s signling molecules regulting neutrophil function. Free Rdic. Biol. Med. 42, Fleming, S.T., Love, M.M., Bennett, K., 211. Dibetes nd cncer screening rtes mong Applchin nd non-applchin residents of Kentucky. J. Am. Bord Fm. Med. 24, Fukuzw, K., Inokmi, Y., Tokumur, A., Tero, J., Suzuki, A., Rte constnts for quenching singlet oxygen nd ctivities for inhibiting lipid peroxidtion of crotenoids nd lph-tocopherol in liposomes. Lipids 33, Gebsk, A., Olsznecki, R., Korbut, R., 25. Endotoxemi in rts: role of leukocyte sequestrtion in rpid pulmonry nitric oxide synthse-2 expression. J. Physiol. Phrmcol. 56, Goldschmidt, M.C., Reduced bctericidl ctivity in neutrophils from scorbutic nimls nd the effect of scorbic cid on these trget bcteri in vivo nd in vitro. Am. J. Clin. Nutr. 54, 1214S 122S. Grynkiewicz, G., Poenie, M., Tsien, R.Y., A new genertion of C2+ indictors with gretly improved fluorescence properties. J. Biol. Chem. 26, Guiquil, V.H., Ver, J.C., Golde, D.W., 21. Mechnism of vitmin C inhibition of cell deth induced by oxidtive stress in glutthione-depleted HL-6 cells. J. Biol. Chem. 276, Guerr, B.A., Otton, R., 211. Impct of the crotenoid stxnthin on phgocytic cpcity nd ROS/RNS production of humn neutrophils treted with free ftty cids nd high glucose. Int. Immunophrmcol. 11, Gustpne, M., Crdillo, M.T., Bisillo, G., Bisucci, L.M., 211. Myeloperoxidse s possible dignostic nd prognostic mrker of cute coronry syndrome. Recenti Prog. Med. 12,