Manual for the Microscopic Diagnosis of Proliferative Liver and Skin Lesions in the Brown Bullhead (Ameiurus nebulosus)

Transcription

1 Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed (meiurus neulosus) Viki S. lzer 1, John W. Fournie 2, Jeffrey. Wolf 3, nd Mrilyn J. Wolfe 3 1 Ntionl Fish Helth Reserh Lortory, U.S. Geologil Survey, Leetown Rod, Kerneysville, WV U.S. Environmentl Protetion geny, Ntionl Helth nd Environmentl Effets Reserh Lortory, Gulf Eology Division, 1 Sine Islnd Drive, Gulf reeze, FL The Registry of Tumors in Lower nimls, Experimentl Pthology Lortories, In., Shw Rod, Sterling, V 20166

2 Pennsylvni Se Grnt, prt of the Ntionl Se Grnt Progrm, is prtnership of Penn Stte, the ommonwelth of Pennsylvni, nd the Ntionl Oeni nd tmospheri dministrtion. This pulition is ville in lterntive medi upon request. Penn Stte is ommitted to ffirmtive tion, equl opportunity, nd the diversity of its workfore. Pulished in Ferury 2007.

3 TLE OF ONTENTS 1.0 Introdution kground Informtion Purpose of the Mnul Speimen Prepertion Field olletion Speimen Proessing Liver Norml Liver Histology Non-neoplsti Hepti Lesions Prolifertion of Mrophge ggregtes Prolifertive Response to Prsites Puttively Preneoplsti Hepti Lesions - Foi of ellulr ltertion sophili Fous Eosinophili Fous Vuolted nd ler ell Foi Neoplsti Hepti Lesions Heptoellulr denom Heptoellulr rinom Non-Neoplsti iliry Lesions ile Dut Prolifertion nd Firosis ile Dut Prsitism Neoplsti iliry Lesions holngiom holngiorinom Metstti Neoplsi Skin Norml Skin, rel, nd Orl vity Mirosopi pperne lk Pigmented Skin Lesions Non-Rised Melnisti Spots Rised lk Skin Lesions Prsite-ssoited lk, Rised Lesions Melnoti Hyperplsi Melnom Melnoti Ppillom Ple res of Disolortion Non-Rised res of Disolortion Rised Ple res Ppillom Squmous ell rinom Misellneous Rised Growths knowledgements Referenes

4 FIGURES Figure 1. Illustrtion of liver piees to e smpled nd pled in fixtive in the field... Figure 2. Mirosopi pperne of norml ullhed liver... Figure 3. Mrophge ggregtes in the liver setions of rown ullhed... Figure 4. Grossly visile lesions in rown ullhed liver... Figure 5. Helminth prsites nd ompnying inflmmtory response in ullhed liver Figure 6. sophili fous in the liver of rown ullhed... Figure 7. Eosinophili fous in rown ullhed liver... Figure 8. Vuolted ell fous in rown ullhed liver... Figure 9. ler or mixed ell fous in rown ullhed liver... Figure 10. Heptoellulr denom in rown ullhed liver... Figure 11. Heptoellulr rinom in rown ullhed liver... Figure 12. Norml ile dutule nd dut mirosopi pperne in rown ullhed liver Figure 13. ile dut prolifertion nd firosis in rown ullhed liver... Figure 14. ile dut myxozon prsite of rown ullhed liver... Figure 15. holngiom within the liver of rown ullhed liver... Figure 16. holngiorinom within the liver of rown ullhed liver... Figure 17. Mirosopi pperne of norml skin of rown ullhed... Figure 18. Mirosopi pperne of norml rown ullhed rels... Figure 19. Mirosopi pperne of norml orl vity... Figure 20. Melnisti spots on the skin of rown ullhed... Figure 21. Smll rised, lk res on the rels of rown ullhed... Figure 22. Slightly rised melnisti lesion on the ody surfe of rown ullhed... Figure 23. Melnom on the rel of rown ullhed... Figure 24. Melnom on the ody surfe of rown ullhed... Figure 25. Lrge melnom on the ody surfe of rown ullhed... Figure 26. Sessile ppillom on the hed of rown ullhed... Figure 27. Non-rised yellowish re of disolortion of the ventrl surfe of ullhed Figure 28. Rised growths on the mouth of rown ullhed... Figure 29. Mirosopi pperne of the ppilloms illustrted in Figure Figure 30. Squmous ell rinom in rown ullhed... Figure 31. Osteom nd osteosrom in rown ullhed

5 1.0 INTRODUTION 1.1 kground Informtion The Gret Lkes Wter Qulity greement, in whih the United Sttes nd nd greed to restore nd preserve the iologil, physil nd hemil integrity of the Gret Lkes sin Eosystem, ws first signed in In 1987, Protool ws signed y oth governments whih defined Gret Lkes res of onern (O) s "geogrphi res tht fil to meet the generl or speifi ojetives of the greement where suh filure hs used or is likely to use impirment of enefiil use of the re's ility to support quti life." The U.S. nd ndin governments identified 43 suh res; 26 in the U.S., 12 in nd nd five shred etween the U.S. nd nd on onneting river systems. The Gret Lkes Wter Qulity greement, s mended vi the 1987 protool, direts the two federl governments to ooperte with stte nd provinil governments to develop nd implement Remedil tion Plns (RPs) for eh O. The Protool lso lled for reports on restortive progress nd for the Interntionl Joint ommission (IJ) to review RPs proposed y the 43 res of onern (IJ, 1987). Three Os (ollingwood Hror, Severn Sound nd Oswego River) hve een delisted nd Presque Isle y hs een designted n re of Reovery. Fourteen enefiil use impirments were identified nd the vrious Os hve different omintions of these impirments. One impirment, listed s fish tumors or other deformities is defined s ourring when the inidene rte of fish tumors or other deformities exeeds rtes t unimpted or ontrol sites or when survey dt onfirm the presene of neoplsti or preneoplsti liver tumors in ullhed or sukers (IJ, 1989; lkeerie/ui/reports). urrently, 16 of the 40 Os hve the presene of tumors or other deformities listed s one of their enefiil use impirments. It is importnt to reognize tht lthough neoplsi hs een linked with speifi ontminnts very little use nd effet evidene, prtiulrly well designed exposure studies, exists for rown ullhed. 1.2 Purpose of the Mnul Over the yers numerous surveys hve een undertken to ssess the inidene of skin nd liver tumors, s well s other ontminnt-ssoited mirosopi lesions t Os. However, mjor prolem is lk of onsistent riteri for evluting mirosopi (histologil) hnges in ullhed liver nd skin. For instne, in some studies there is no distintion etween preneoplsti (ltered foi) nd neoplsti lesions, or no good desriptions of individul lesions (lk 1983; Pyron et l. 2001). Some studies inluded only rinoms s neoplsi, while in others denoms, rinoms, nd holngioms were inluded (umnn et l. 2000) nd some inluded rinoms, denoms nd ltered foi (Muin nd Ersing 1991). Skin tumors re often dignosed visully without the supporting histologil verifition. Hene, stndrdiztion of the riteri eing used to evlute the impirment is neessry, so tht dt from different yers, referene sites nd res of onern n e dequtely ompred. Pennsylvni Deprtment of Environmentl Protetion, U.S. Environmentl Protetion geny Gret Lkes Ntionl Progrm Offie nd Pennsylvni Se Grnt sponsored numer of onferenes relted to Fish Tumors in Gret Lkes res of onern. t the initil meeting, ommittees were formed to work on 1) stndrdized monitoring protools nd 2) riteri for stndrdized ssessment of skin nd liver lesions. This mnul is the produt of the Histopthology Suommittee nd the purpose is to desrie neoplsti nd non-neoplsti prolifertive lesions of the liver nd skin of the rown ullhed nd suggest terminology tht n e onsistently used t Os throughout the Gret Lkes nd other res. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 1

6 2.0 SPEIMEN PREPRTION 2.1 Field olletion ompnion mnul Field Mnul for ssessing Internl nd Externl nomlies in rown ullhed (meiurus neulosus) y Rfferty nd Grzio (2007) provides guidne for the identifition of lesions nd deformities oserved during gross exmintion nd reommends stndrd operting proedures for the olletion, neropsy, nd preservtion of tissue olleted in the field. riefly, externl lesions, primrily rised lesions of ny olor nd onsisteny should e smpled. It is est to remove the whole lesion plus some norml surrounding tissue, if possile. If the lesion is lrge, susmple should e removed. For the identifition of preneoplsti or neoplsti liver lesions it is neessry to smple ll livers s most of the lesions will not e oserved grossly. It is suggested tht t lest five piees of liver e pled in fixtive in the field (Figure 1). Piees of skin nd liver re generlly fixed in 10% uffered formlin, Dvidson s (Lun 1992), or Z-Fix (nteh LTD, ttle reek, MI). 2.2 Speimen Proessing There hve een no studies to sttistilly vlidte the numer of setions tht need to e exmined from n individul ullhed liver to hve given onfidene of dignosing prtiulr lesion if present. To our knowledge the only morphometri study of liver neoplsms nd ltered foi urrently ville ws onduted on mummihogs Fundulus heterolitus, muh smller fish speies (Stine et l. 2004). In tht study six livers (five of whih hd visile lesions) were serilly setioned. It ws determined tht from 3-15 (men of 7) setions were required to dignose neoplsi or ltered foi from eh liver with 95% ury. It is likely tht for fish with lrger livers more setions will e required. Hene, it is suggested tht from the five or more piees olleted in the field, six to ten piees should e pled in ssettes nd routinely proessed for emedding in prffin, setioned t thikness of 4-6 μm nd stined with hemtoxylin nd eosin (Lun 1992; Profet et l. 1992). Figure 1. Illustrtion of liver piees to e smpled nd pled in fixtive in the field. 3.0 LIVER Prt of the definition of the enefiil use impirment is when survey dt onfirm the presene of neoplsti or preneoplsti liver tumors in ullhed or sukers. Liver neoplsms nd other mirosopi liver lesions hve een ssoited with PH nd other ontminnt exposure in rown ullheds (umnn 1992; umnn nd Hrshrger 1995; umnn nd Hrshrger 1998; Pinkney et l. 2001, 2004) nd other fish speies, inluding English sole Prophyrs vetulus (Myers et l. 1990), mummihog Fundulus heterolitus (Vogelein et l.1990), nd winter flounder Pseudopleuronetes merinus (Grdner et l. 1989). lthough it is esy to define nd desrie neoplsti lesions, those to e onsidered preneoplsti re more diffiult s there hve een no good exposure studies to doument the progression of liver neoplsi in ullhed. While oth ile dut prolifertion nd foi of ellulr ltertion my e ssoited with hemil exposures nd re onsidered preneoplsti in some fish speies, ile dut prolifertion my lso e ssoited with myxozon prsites nd is often retive in ullhed. Hene, lthough ile dut prolifertion should e doumented, we do not suggest it e onsidered hemillyindued hnge t Os. In ddition, there is insuffiient evidene to indite tht it is preneoplsti lesion. onversely, foi of ellulr ltertion re lmost lwys ssoited with toxiopthi effets. In wild fish surveys their presene hs een highly orrelted with exposure to PHs, Ps, DDTs, hlordnes, dieldrin, merury, pulp mill effluent nd romti hydroron metolites in English sole, white roker Genyonemus linetus, Pifi stghorn sulpin Leptoottus rmtus, strry flounder Pltihthys stelltus, d Limnd limnd, flounder Pltihthys Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 2

7 flesus, rok sole Lepidopsett ilinet nd Dover sole Mirostomus pifius (reviewed y u 2004; Feist et l. 2004; Koehler 2004) nd re reommended for ssessing hroni toxiity (Hinton 1994). Lortory exposures of numer of fish speies hve indited ltered foi to e preneoplsti, prtiulrly sophili foi (Hendrik et l. 1984; Grizzle nd Thiygrjh 1988; Hinton et l. 1988; Hwkins et l. 1990; Lw et l. 1994). Hene, we reommend tht these foi of ellulr ltertion e onsidered preneoplsti. 3.1 Norml Liver Histology ullhed livers, s in most other teleosts, re omposed of hepti tuules. In liver setions from ullheds olleted t referene sites oservle ile duts re often sprse nd not prominent in setions exmined y light mirosopy (Figure 2 nd ). Portl veins re ommonly surrounded y pnreti tissue nd ile duts my e oserved within this tissue (Figure 2). Norml heptoyte pperne n vry from hving little to no oservle vuoliztion, lipid or glyogen storge (Figure 2 nd ), to hving extensive vuoliztion (Figure 2 nd D). Mrophge ggregtes re lso oserved within pnreti nd hepti tissue (Figure 2 nd ). More detiled desriptions on norml pisine hepti histology nd heptotoxiity n e found in (oormn et l. 1997; Metlfe 1998; Hinton et l. 2001; Wolf nd Wolfe 2005). D Figure 2. Mirosopi pperne of norml ullhed liver.. Setion of norml liver illustrting the tuulr struture, ile dut () nd mrophge ggregte (rrow) within the exorine pnreti tissue. r = 150 μm.. Norml liver with highly vuolted heptoytes. lood vessels re often surrounded y pnreti tissue () whih osionlly ontin ile duts (rrow). r = 150 μm.. Higher mgnifition of illustrting the tuulr struture nd mrophge ggregte (rrow). r = 50 μm. D. Higher mgnifition of illustrting highly vuolted heptoytes, pnreti tissue () nd ile dut (rrow). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 3

8 3.2 Non-Neoplsti Hepti Lesions Prolifertion of Mrophge ggregtes Mrophge ggregtes re strutures found in the spleen, kidney nd liver of fish (reviewed y guis nd Roerts 2003; Wolke 1992). They hve een shown to inrese with ge (lzer et l. 1987) nd in response to environmentl stress (Fournie et l. 2001). Hene, in young fish, nd/or fish from referene sites, mrophge ggregtes my not e oservle (Figure 2) or they my e present t low density within the liver or hepti pnreti tissue (Figure 2 nd 3), while t impted sites higher density my e oserved (Figure 3). Three pigments - melnin, hemosiderin nd the lipopigments, lipofusin/eroid, n e oserved within these strutures using the Perl s Prussin lue method whih stins hemosiderin lue (Lun 1992). In ullheds the lipopigments nd hemosiderin re most ommonly oserved. Some hve higher proportion of hemosiderin, n iron-ontining pigment, whih stins lue (Figure 3 nd D), while others ontin more of the lipofusin/eroid pigments, formed y oxidtion of lipid nd retining yellowish-rown pperne (Figure 3 nd ). D Figure 3. Mrophge ggregtes in liver setions of rown ullhed stined y the Perl s Prussin lue method.. Liver setion from ullhed olleted t referene site illustrting n ggregte in the heptopnreti tissue () nd nother within hepti tissue (). r = 100 μm.. liver setion from ullhed olleted t n O illustrting numerous ggregtes within heptopnreti () nd hepti () tissue. r = 100 μm.. Mrophge ggregte () with moderte mount of hemosiderin (lue) nd eroid/lipofusin (rrows). r = 20 μm. D. Mrophge ggregte () with high levels of hemosiderin (lue). r = 20 μm. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 4

9 3.2.2 Prolifertive Responses to Prsites Helminth prsites re ommonly oserved within liver tissue of ullhed olleted in some res. They n inlude tremtodes, nemtodes, nthoephlns nd estodes. These my e the most ommonly oserved, grossly visile lesions (Figure 4). They my pper s irregulrly-shped, elongte or rounded, ple, rised res. However, rised ple res my lso e tumors (Figure 4). Figure 4. Grossly visile lesions in rown ullhed liver.. Numerous ple irregulrly-shped res, some on the surfe of the liver nd some within the prenhym (yellow rrows).. Ple disolored res of vrile size nd shpe on the surfe nd within the prenhym of the liver. These inluded prsites nd the prolifertive inflmmtory response to them (yellow rrows), illustrted in Figure 5, nd holngiorinom (lk rrow), illustrted in Figure 16. Gllldder is present (white rrow). Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 5

10 D Figure 5. Helminth prsites nd the ompnying inflmmtory responses in rown ullhed liver. nd. estode prsite () on the surfe nd extending into the prenhym of the liver. r = 150 μm.. Remnnts of helminth prsite () nd extensive inflmmtory response () within the liver. r = 150 μm. D. hroni grnulomtous inflmmtory response () nd gint ells (rrows) within the liver of rown ullhed. r = 50 μm. H&E stin. 3.3 Puttively Preneoplsti Lesions - Foi of ellulr ltertion Four tegories of foi of ellulr ltertion re reognized in fish livers stined with hemtoxylin nd eosin, sed on the tintoril hrteristis of the heptoyte ytoplsm. In rts, it hs een doumented tht these differenes in stining re due to umultions of speifi omponents within the ells. ells within eosinophili foi re rih in smooth endoplsmi retiulum, while ells within sophili foi re rih in RN. ler ell foi ontin undnt glyogen. Foi of vuolted ells ontin lipid in some fish speies nd mixture of lipid nd glyogen in others (reviewed y unton 1996). The mrgins of these foi re generlly distint ut the hepti tuules re rrnged in reltively norml pttern, merge impereptily with the surrounding prenhym, nd little to no ompression is oserved. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 6

11 3.3.1 sophili Foi sophili foi re round to irregulr lusters of heptoytes rih in rough endoplsmi retiulum nd inresed sophili stining ompred to djent ells (Figure 6). The ells my e smller thn djent heptoytes, ut ellulr typi nd mitoti figures re generlly not present (Figure 6). Figure 6. sophili fous in the liver of rown ullhed.. Disrete fous of heptoytes () stining more sophili thn surrounding () heptoytes. r = 150 μm.. Higher mgnifition of sophili fous (), illustrting the mrgin of the fous (rrow), whih merges into the djent heptoytes (). r = 100 μm. H&E stin Eosinophili Fous Eosinophili foi re round to irregulr res of heptoytes with inresed eosinophili ompred to djent ells (Figure 7). The ells my e slightly enlrged with grnulr eosinophili ytoplsm (Figure 7). Figure 7. Eosinophili fous in rown ullhed liver.. re of heptoytes with inresed eosinophili () when ompred to surrounding heptoytes (). rrows indite the order of the lesion. r = 150 μm.. Heptoytes within the eosinophili fous () hve finely grnulr ytoplsm nd re slightly lrger thn surrounding heptoytes (). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 7

12 3.3.3 Vuolted nd ler ell Foi Vuolted foi re desried s round to irregulr, ontining heptoytes with medium to lrge, ler, lipidontining, ytoplsmi vuoles. The nulei re often displed to the periphery of these ells (Figure 8). ler ell foi, s desried in other fish speies, re hrterized y enlrged heptoytes with ground glss pperne to the ytoplsm nd the nulei re generlly entrlly loted within the ells. The ler ytoplsm is the result of glyogen umultion whih is dissolved out in queous fixtives. ler ell foi re less ommonly oserved thn vuolted foi (oormn et l. 1997; Feist et l. 2004). In ullhed, these foi often pper to e mixed foi with ells hrteristi of oth ler ell nd vuolted foi (Figure 9). ells within the fous my hve oth entrlly-loted nd eentri nulei nd the ground glss pperne is not s evident. Figure 8. Vuolted ell Foi.. Fous () with order lending into surrounding heptoytes (). r = 150 μm.. Higher mgnifition illustrting the lk of ompression or enpsultion etween the order (rrow) of the fous () nd surrounding heptoytes (). r = 100 μm.. ells within the fous hve vrily-sized ler vuoles nd eentri nulei (rrows). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 8

13 Figure 9. ler or Mixed ell Fous.. Fous () with order lending into surrounding heptoytes (). r = 150 μm.. Higher mgnifition illustrting the lk of ompression or enpsultion etween the order (rrow) of the fous () nd surrounding heptoytes (). r = 100 μm.. Some ells within the fous hve entrlly-loted nulei (rrows), while others re eentri. Some ells hve ytoplsm with ground glss pperne (yellow rrows). r = 50 μm. H&E stin. 3.4 Neoplsti Heptoellulr Lesions Heptoellulr denom denoms re disrete lesions whih generlly hve distint order. The ells my exhiit ltered stining properties nd hene pper more eosinophili, sophili or vuolted ompred to the surrounding tissue. ells re usully not rrnged in norml growth ptterns nd usully there is very little pleomorphism. Mitoti figures re rrely oserved. Mrophge ggregtes, pnreti tissue nd other strutures re often missing or sprse within the neoplsti lesion. denoms my e demrted y ompression of the djent prenhyml ells (Figure 10), however in some ses this is not s ovious (Figure 10). Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 9

14 D Figure 10. Heptoellulr denom in the liver of rown ullhed.. Neoplsti ells within the denom () stin eosinophili ompred to surrounding heptoytes (). r = 150 μm.. denom () showing ovious ompression (rrows) of surrounding tissue. r = 150 μm. nd D. Higher mgnifition of the periphery of two denoms () whih hve some ellulr typi nd moderte ompression (rrows) of surrounding tissue. r = 50 μm. H&E stin Heptoellulr rinom Heptoellulr rinoms, mlignnt hepti neoplsms, re often diffusely spred throughout the hepti prenhym (Figure 11 nd ) in rown ullhed, ut my e distint foi with irregulr orders (Figure 11 nd E). Neoplsti ells invde the djent prenhym (Figure 11D). ellulr pleomorphism nd nuler typi re key fetures (Figure 11F) nd there is often n inrese in the numer of mitoti figures. There n e some tumor gint ell formtion nd these my e smll or lrge lesions. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 10

15 d e D e E F e Figure 11. Heptoellulr rinom in the liver of rown ullhed.. Foi of neoplsti ells () re oserved throughout the norml hepti prenhym (). Inflmmtion (), proly in response to helminth prsites (d) is lso oserved. r = 150 μm.. Higher mgnifition of, inditing fous of neoplsti ells (), norml liver () nd mrophge ggregte (e). r = 50 μm.. Lrger, more invsive rinom () infiltrting norml liver (). r = 150 μm. D. Higher mgnifition of illustrting the invsiveness of the neoplsti ells (). r = 50 μm. E. Well-differentited heptoellulr rinom (). r = 100 μm. F. Higher mgnifition of E illustrting neoplsti ells whih vry in size nd shpe nd my hve multiple nuleoli within the nuleus (rrow). mrophge ggregte is evident (e). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 11

16 3.5 Non-Neoplsti iliry Lesions ile Dut Prolifertion nd Firosis Norml smll nd intermedite ile dutules/duts hve olumnr epithelium nd thin nd of onnetive tissue (Figure 12). Lrge ile duts re surrounded y onnetive tissue nd re prominent ner the ommon ile dut (Figure 12). ile dut hyperplsi onsists of n inresed numer of vrily-shped ile duts, when ompred to the norml pperne (Figure 2 nd ). ile duts re often sttered throughout the liver nd do not form disrete msses (Figure 13), lthough osionlly smll lusters re oserved (Figure 13). The iliry epithelium is lwys well-differentited (norml-ppering), however there my e moderte firosis ssoited with proliferting ile duts (Figure 13). holngiofirosis onsists of ile duts or dutules with extensive peridutl firosis (Figure 13D), however the iliry epithelium is still norml. Figure 12. ullhed liver showing norml ile dutule nd dut.. Within the hepti tissue () ile dutules (rrow) re ommonly loted within the pnreti tissue () nd re ounded y thin nd of onnetive tissue. r = 50 μm.. Lrger duts hve thiker lyer of onnetive tissue (rrow) nd olumnr epithelium. (). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 12

17 D Figure 13. ile dut prolifertion nd firosis.. Prolifertion of individul ile dutules nd duts (rrows) throughout the liver. r = 150 μm.. Fol prolifertion of ile duts () with moderte mount of firosis round the duts. r = 150 μm.. Higher mgnifition illustrting inresed firous tissue () nd norml epithelium (). r = 100 μm. D. Extensive firosis () round fol re of ile dut prolifertion. r = 50 μm. H&E stin ile Dut Prsitism Sporoplsms of myxozon prsite hve een oserved within the ile duts of rown ullhed from numerous res. The ffeted ile duts re often thikened nd in some res there is evidene of hyperplsi of the epithelium or nerosis (Figure 14). It is urrently not known wht ssoition this prsite my hve on ile dut prolifertion or iliry neoplsi. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 13

18 D Figure 14. ile dut myxozon prsites of rown ullhed.. Sporoplsms () of myxozon prsite within the ile duts (rrows). r = 150 μm.. Higher mgnifition of the sporoplsm () ontining mture spores (rrows) s well s immture stges. Spores re elongte with polr psules on either end. r = 20 μm.. ffeted ile duts my show signs of epithelil hyperplsi (rrow) ssoited with the presene of the sporoplsm (). r = 50 μm. D. Some ffeted ile duts show signs of epithelil nerosis (rrow) ssoited with the prsite (). r = 50 μm. H&E stin. 3.6 Neoplsti iliry Lesions holngiom holngioms re enign tumors of ile duts within the liver. These re lusters of ile duts whih re welldifferentited nd often hve disrete order etween the nodule nd surrounding hepti prenhym (Figure 15). Mny of the ile duts my e irregulrly-shped nd dilted. Figure 15. holngiom within the liver of rown ullhed.. Nodule of neoplsti ile duts () is seprted from the norml hepti tissue () y well-defined order (rrows). r = 50 μm.. Higher mgnifition illustrting the well-defined order (rrows) nd neoplsti ile duts () whih re irregulrly shped nd dilted.. r = 25 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 14

19 3.6.2 holngiorinom holngiorinoms re mlignnt tumors of ile duts. Some tumors ontin modertely to well-differentited ile duts, whih vry gretly in size nd shpe nd invde into the surrounding prenhym (Figure 16 nd ). The proliferting ile duts my e ssoited with proliferting strom nd inflmmtion (Figure 16 nd D). In some tumors, there is very little norml dut formtion nd the neoplsti epithelil ells re pleomorphi with mitoti figures nd re surrounded y proliferting spindle ell strom (Figure 16E nd F). D d d E F Figure 16. holngiorinom in rown ullhed liver.. Proliferting neoplsti ile duts () invde into the surrounding hepti prenhym (). r = 150 μm.. Higher mgnifition of invding neoplsti ile duts (). r = 100 μm.. holngiorinom in whih the proliferting neoplsti ile duts surround pnreti tissue (). r = 150 μm. D. Higher mgnifition illustrting inflmmtion (d) round the proliferting ile duts (). r = 50 μm. E. Smll nodule of undifferentited ile duts () seprted from norml liver () y inflmmtion. r = 100 μm. F. Higher mgnifition illustrting pleomorphi ells with mitoti figure (rrow). r = 20 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 15

20 3.7 Metstti Neoplsi Metstsis of mlignnt neoplsms to the liver is rre in fish. However, metstses of derml melnoms to the liver hve een oserved in rown ullhed (M. Wolfe, personl oservtion). 4.0 SKIN The presene of externl neoplsms, in ullhed nd white suker tostomus ommersoni in the Gret Lkes region nd elsewhere, hs een used s n inditor of environmentl helth (umnn 1992). Skin tumors reported in rown ullhed inlude epiderml ppilloms, squmous ell rinoms nd melnoms (Sonstegrd 1977; Kim et l. 1989; owser et l. 1991). Henry Dvid Thoreu my hve een the first to doument rised growth in ullhed. study written y Thoreu nd reported y Osurn (1925) of ullheds in pond on pe od, desried lesions vrying from melnisti spots to lrge rised tumors, ttriuted to lk ooid teri, whih were proly melnosomes (orgnelles responsile for melnin prodution). Elevted prevlenes of rised skin lesions hve een reported t ontminted nd/or industrilized sites in numerous fish speies (Smith et l. 1989; Hyes et l. 1990; Kine et l. 1990; Okihiro et l. 1993; Mikelin et l. 2000), lthough, the ssoition with hemil exposure in ullheds is less onlusive thn with liver neoplsi (Poulet et l. 1994; Pinkney et l. 2001). Ppilloms re the most frequently reported utneous neoplsms nd ffet wide vriety of freshwter nd sltwter fishes. Some lortory studies hve indited hemil etiology for ullhed ppilloms (lk 1983; Grizzle et l. 1984), while others hve suggested virl etiology sed on the oservtion of virl prtiles (Edwrds nd Smsonoff 1977). numer of studies hve looked for, ut not onfirmed, the presene of virl prtiles (owser et l. 1991; Poulet et l. 1996) nd ttempts to trnsmit the oroutneous neoplsms hve not een suessful, suggesting multiftoril etiology (Poulet et l. 1993). Viruses hve een implited in the indution of tumors or tumor-like growths in numer of other fish speies (Ymmoto et l. 1985; Lee nd Whitfield 1992; nders nd Yoshimizu 1994; Qukenush et l. 2001). It hs een suggested higher prevlene of skin tumors in fish olleted t ontminted res my e explined y lower resistne or immunosuppression (u et l. 2004). These rised growths re most ommonly oserved on the lips nd rels, however n e found nywhere on the ody surfe nd fins. 4.1 Norml Skin, rel, nd Orl vity Mirosopi pperne The histologi pperne of ullhed skin nd other tissues is similr to tht desried for hnnel tfish Itlurus punttus (Grizzle nd Rogers 1976). ullhed skin, like tht of other tfishes, does not ontin sles. The skin is omposed of the epidermis nd underlying dermis nd hypodermis nd vries in thikness in different res of the ody. The epidermis is omposed of olumnr sl ells whih eome squmous in the outer lyer. Golet or muous ells re present in ll res ut vry in undne. lrm sustne, lu or fright ells re lso ommon, while melnophores nd tste uds re found in some res (Figure 17 nd ). The dermis is omposed of ompt lyer of firous onnetive tissue elow the epidermis. In most res there is thin lyer of melnophores immeditely eneth the epidermis. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 16

21 Figure 17. Mirosopi pperne of norml rown ullhed skin.. Skin from the lterl surfe with numerous lrm sustne ells () in the epidermis (etween doule-heded rrow). The dermis () immeditely elow the epidermis ontins thin line of melnophores (rrow). r = 150 μm.. n re of skin with fewer lrm sustne ells () nd more muous or golet ells (). The epithelil ells t the se of the epidermis (yellow rrow) re olumnr ells, eoming more squmous (lk rrow) t the surfe. r = 50 μm. H&E stin. The rels re omposed of entrl ore of pseudortilge nd nervous tissue, overed with dermis nd epidermis, oth generlly ontining melnophores. Tste uds re very numerous in the epidermis (Figure 18 nd ) of the rels. e f e f d Figure 18. Mirosopi pperne of rown ullhed rels.. rels onsist of n epidermis (), dermis () with lyer of melnophores (rrow), entrl portion of nervous tissue () nd pseudortilge (d). r = 150 μm.. The epidermis of the rel hs undnt tste uds (e), some lrm sustne ells (f) nd melnoytes (yellow rrows). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 17

22 The orl vity hs n epithelium similr to the outer skin, however it hs more folded pperne (Figure 19). Most res hve fewer lrm sustne ells, while tste uds re undnt. Teeth re nkylosed to the premxill nd dentry ones of the mouth (Figure 19). The teeth re overed with thin enmel-like mteril nd formed of dentine-like mteril whih surrounds pulp vity tht ontins nerves, lood vessels nd firous onnetive tissue. Modified epithelil ells surround the teeth. d Figure 19. Mirosopi pperne of the norml orl vity.. Muosl epithelium (etween doule heded rrow) ontins tste uds () nd lrm sustne ells (). r = 150 μm.. Teeth () extend from the one through the propi muos nd muos, nd re surrounded y modified epithelil ells (rrows). The suderml re (d) onsists of highly vsulrized loose onnetive tissue. r = 150 μm. H&E stin. 4.2 lk Pigmented Skin Lesions lk pigmented lesions in rown ullhed rnge from non-rised melnisti res to lrge rised lk res. In ullheds, there hve een no studies tht ddress the progression of pigmented lesions. It is unknown whether the non-rised melnisti res (melnoti hyperpigmenttion) re prenerous lesions tht progress to melnoms. We hve oserved exmples of ll in wild-ught ullheds (Figures 20-26). The Xiphophorus model hs een used extensively to study moleulr nd geneti mehnisms involved in melnom indution (Setlow et l. 1989; Shrtl 1995; Nirn et l. 1996). In this fish speies, lssifition sheme for spontneous tumors nd neoplsti lesions indued y UV rdition nd exposure to the rinogen N-methyl-N-nitrosoure hs een desried. These lesions inlude nonneoplsti melnoti hyperpigmenttion, preneoplsti melnosis nd melnoms (Gimenez- onti et l. 2001). orreltion etween exposure to environmentl hemils nd pigment ell neoplsi hs een suggested in some fish speies (Kimur et l. 1984; Kine et l. 1990; Okihiro et l. 1993) Non-Rised Melnisti Spots Melnisti res re non-rised, lk pigmented spots on the hed, ody surfe or fins. They n vry gretly in size nd shpe (Figure 20 nd ). Histologilly, these melnisti lesions onsist of inresed numers of melnoytes primrily within the epidermis, ut sometimes the dermis s well. The melnoyte my hve extensive ytoplsmi extensions or e in lrge groups (Figure 20 nd D). Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 18

23 D Figure 20. Melnisti spots.. Multiple, nonrised, lk spots on the lterl ody surfe.. lrge, irregulr, non-rised lk re on the hed nd ody surfe.. Mirosopi pperne of melnisti spot. Epidermis (etween doule-heded rrow) ontins groups of melnoytes (lk rrow) s well s individul ells with extensive ytoplsmi extensions (yellow rrows). r = 150 μm. D. Higher mgnifition illustrting groups of melnoytes of vrying sizes (lk rrow) nd individul melnoytes with extensive ytoplsmi extensions (yellow rrows). r = 50 μm. H&E stin Rised lk Skin Lesions These res n vry from smll rised res to lrger rised res on fins, ody surfe, lips nd rels. It is neessry to exmine these lesions histologilly to determine whether they re prsite-ssoited rised lesions (Figure 21), melnoti hyperplsi (Figure 22), melnoms (Figures 23-25), or ppilloms with inresed melnoytes within the proliferting epidermis (Figure 26). Grossly, these my ll look very similr Prsite-ssoited lk, Rised Lesions Some of the rised lk res oserved on rels nd the hed region re found to ontin tremtode meterrie. These prsites pper primrily round nd within the nerves of the rels, nd re ssoited with prolifertion of dermis, underlying onnetive tissue, derml melnoytes nd in some ses prolifertion of the overlying epidermis (Figure 21). Similr rised lk lesions re known to our in lue tfish (Itlurus furtus), ssoited with Ihthyophonus infetions (Fournie, personl oservtions). Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 19

24 Figure 21.. Smll rised, lk res (white rrows) on the rels of rown ullhed.. Mirosopi pperne of the rels illustrting prsiti ysts () within the nerves () nd dense onnetive tissue. In some res this response is ssoited with prolifertion of the epidermis (). r = 150 μm. H&E stin Melnoti Hyperplsi Slightly rised nd irregulr pigmented res hve een noted in some ullhed, whih mirosopilly exhiited epiderml hyperplsi nd norml-ppering melnin umultions, nd re possily preneoplsti (Figure 22). D Figure 22.. Slightly rised melnisti re on the ody surfe of rown ullhed.. Mirosopi pperne of setion of the re. The epidermis (etween the doule-heded rrows) is of vrying thikness nd ontins groups of norml, spindleshped ells with melnin (). Norml melnoytes re present in the epidermis nd dermis (rrows). r = 150 μm.. Higher mgnifition showing norml melnoytes in dermis nd epidermis (rrows) nd groups of norml melnoytes in epidermis (). r = 100 μm. D. norml melnoytes re spindle-shped nd vuolted (). r = 50 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 20

25 Melnom Lesions dignosed s melnoms rnge from very smll rised res on the skin or rels (Figure 23 nd 24) to very lrge rised res (Figure 25). numer of types of melnoms similr to those desried y Gimenez-onti et l. (2001) hve een oserved in rown ullhed nd re illustrted in the following figures. These inlude the melnoyti melnoms (Figure 23) whih re hrterized y prolifertion of melnoytes tht re dendriti in shpe nd ontin vrile mounts of pigment. D Figure 23. Melnom on the rel of rown ullhed.. Smll rised, lk re on the rel. - D. Histologi pperne of rel melnom.. Epidermis (rrow) is intt over most of the melnom (). r = 300 μm.. The proliferting neoplsti ells extend into the dense onnetive tissue of the dermis (). There re numerous lood vessels throughout the tumor (rrows). r = 50 μm. D. In res there re foi of neoplsti ells () within the epidermis (). The neoplsti ells surround lood vessels (), re dendriti nd ontin vrying mounts of pigment. r = 10 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 21

26 The melnom on the ody surfe (Figure 24) is usully polymorphi melnom. These tumors re heterogenous with oth hevily nd lightly pigmented res. ell types within the tumor inlude melnoytes, epithelioid ells nd mromelnophores. D Figure 24. Melnom on the ody surfe of rown ullhed.. Grossly visile, rised, lk lesion (rrow) on the lterl surfe of rown ullhed whih lso hs lrge disolored lesion (), overed y fungl growth.. Mirosopi pperne of the melnom. In some res the epidermis overing the tumor is thikened () nd ontins pegs of the neoplsti ells (rrow). The tumor ontins some res tht re less densely stined () thn others (). r = 300 μm.. Higher mgnifition of the tumor illustrting the vrily sized nd shped ells nd izrre growth pttern. r = 50 μm. D. Neoplsti ells inlude very lrge mromelnophore ells () nd ells of reltively norml size (). r = 20 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 22

27 The lrge, lk rised lesion on the ody surfe (Figure 25) resemles spindle ell melnom histologilly, nd is very similr to derml melnom previously desried from rown ullhed s hving shwnnom-like differentition (Skmoto nd White 2002). D Figure 25. Lrge melnom on the ody surfe of rown ullhed.. Irregulr rised lk re on the skin.. Mirosopi pperne illustrting herringone pttern with onnetive tissue strom (). r = 150 μm.. Osionl lusters of rounded, densely pigmented melnophores () were oserved. r = 100 μm. D. The mjority of neoplsti ells were spindle-shped with vrying mounts of melnin. r = 20 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 23

28 Melnoti Ppillom Some rised lk res re ppilloms (see ) tht re hrterized y prolifertion of melnoytes within the epidermis nd long the onnetive tissue treule (Figure 26). D Figure 26.. Sessile ppillom (rrow) on the hed of rown ullhed.. Mirosopilly, the lesion ws omposed of proliferting epithelil ells with melnoytes within these proliferting res (lk rrows). r = 150 μm.. These proliferting epithelil ells form the typil ppillomtous folds supported y the onnetive tissue strom lined y melnoytes (white rrows). r = 150 μm. D. Higher mgnifition illustrting umultions of melnoytes in the strom (white rrow) nd individul melninontining ells in the proliferting epidermis (lk rrow). r = 100 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 24

29 4.3 Ple res of Disolortion res of disolortion, generlly white to yellow, rnge from smll, non-rised to lrge, rised, multiolored nd multiloed lesions. Mirosopilly, these res show epiderml hyperplsi of one or more ell types, ppilloms or squmous ell rinoms. numer of studies hve suggested progression from epiderml hyperplsi to ppillom nd osionlly to rinom in rown ullhed (Luké nd Shlumerger 1941; Smith et l. 1989; Poulet et l. 1994; unton 2000) Non-Rised res of Disolortion These res on ullhed re generlly gry or yellow res suh s illustrted in Figure 27. Mirosopilly, they pper to e due to prolifertion of ertin ell types. The ple gry res ontin inresed epithelil nd muous ells, while the yellow res ontin inresed muous nd lrm sustne ells. D Figure 27.. Non-rised yellow spot on the ventrl surfe of rown ullhed.. Mirosopi pperne t the periphery of the disolored re showing the differene etween the norml epithelium () nd the disolored re (). r = 150 μm.. Mirosopilly, the yellow lesions ontin res with inresed muous ells (rrows) nd lrm sustne ells (). r = 150 μm. D. Higher mgnifition of the ffeted re in, illustrting the prolifertion of muous ells (rrows) nd lrm sustne ells (). r = 100 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 25

30 4.3.2 Rised Ple res Rised, ple growths n e oserved on the lips, fins nd ody surfe nd n vry from smll, slightly rised, plque-like res to very lrge multiple, olesing nodules. In ullheds they re most ommon in the orl vity nd on the lips. Most often these growths re ppilloms or squmous ell rinoms Ppillom Ppilloms re onsidered enign growths nd n rnge from slightly rised, homogeneous-ppering res (Figure 28) to smll rised, slightly eroded nd reddened plques (Figure 28) to lrger, often multiple nodules (Figure 28 nd D) whih my vry in olor from ple to lk (Figure 28). D Figure 28. Rised growth on the mouth of rown ullhed. ll were determined to e ppilloms y mirosopi evlution.. slightly rised, plque-like, ple growth on the lower lip.. smll, slightly rised, reddened nd eroded growth on the upper lip.. Lrger, multiple rised growths on the lips, some drk (lk rrows) nd some very ple (white rrows). D. Multiple, rised growths on the lower lip. Histologilly, the epithelil prolifertion uses ppillry folds to form whih re supported y onnetive tissue ords. These folds extend ove the norml skin surfe nd extend into the underlying dermis. Prolifertion is primrily of the epiderml mlpighin ells. However, in some res there my e prolifertion of muous or lrm sustne ells using the re to pper ple, while in others there my e prolifertion of melnoytes giving the re drk pperne. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 26

31 d e e D e E f F g Figure 29. Mirosopi pperne of the ppilloms illustrted in Fig Proliferting epiderml (mlpighin ) ells () form folds or ppille supported y onnetive tissue (). Neoplsti ells do not infiltrte elow the sement memrne (rrow). r = 150 μm.. Rised growths tht pper reddened nd/or rded (Figure 28) ontin res of hemorrhge or ongestion () nd the outer surfes my e neroti (d). r = 150 μm.. Individul growths my pper very ple nd trnsluent (Figure 28) due to prolifertion of muous ells (e). r = 100 μm. D. Higher mgnifition illustrting the well-defined folds of the epidermis () within the onnetive tissue support (). r = 100 μm. E. The proliferting ells re squmous (f) in the outer regions nd olumnr to uoidl (g) towrd the dermis. r = 50 μm. F. Proliferting ells vry in size nd shpe, nulei re enlrged nd my ontin multiple nuleoli (rrow). r = 20 μm. H&E stin Squmous ell rinom Squmous ell rinoms hve een desried in numer of fish speies (Fournie et l. 1987; Hrshrger nd lrk 1990; Hnjvnit nd Mulhy 2004) inluding rown ullhed (unton 2000; Pinkney et l. 2001). In study of ullhed from the k River, MD, n re with elevted PH levels, progression from hyperplsi to Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 27

32 ppillom to rinom ws suggested. Lol invsion through the sl lmin ours s the lesion progresses to rinom (unton 2000). Some res re msses of neoplsti ells in whih smll islnds of supportive tissue, muous or lrm sustne ells my eome entrpped (Figure 30 ). Other res my ontin ords or nests of neoplsti ells interspersed with firous onnetive tissue (Figure 30 nd D). rinoms re hrterized y pleomorphism of the epithelil ells. Lrge, often polygonl ells, with enlrged nulei, re oserved with loss of norml orienttion (Figure 30 E nd F). They re lso hrterized y extension through the sement memrne (Figure 30 F). D E F d e Figure 30. Squmous ell rinom in rown ullhed.. Lrge growth on the lower lip nd jw of rown ullhed.. Mirosopi pperne of the neoplsti growth illustrting the lk of well-defined folds () nd islnds of supporting tissue (). r = 150 μm.. In some res, ords () or nests (rrows) of neoplsti ells re found within firous tissue (). r = 100 μm. D. Higher mgnifition of neoplsti ells () tht hve extended through the sl lmin nd smll nests of neoplsti ells (rrows) in the dermis nd hypodermis. r = 50 μm. E. re of the rinom illustrting the lk of norml orienttion of the lrge, often polygonl ells. Neroti res (), muous ells (d) nd mitoti figure (rrow) my e oserved. r = 50 μm. F. Invsion of the neoplsti ells (e) through the sement memrne (doule rrow) nd into the underlying tissues (). r = 20 μm. H&E stin. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 28

33 Misellneous Rised Growths Osionlly, other tumor types re found to e responsile for the rised growths oserved on ullhed fins, ody surfe or mouth. n osteom nd osteosrom were oth dignosed from the sme ullhed. These lesions were desried grossly s firm, rised mss on the petorl fin nd hyperplsti lesion on the upper lip, respetively. The fin lesion ws dignosed mirosopilly s n osteom (Figure 31 nd ) while the jw lesion ws n osteosrom (Figure 31 nd D). To our knowledge, there re no other reports of ony tumors in ullhed, however individul ses hve een desried in few other fish speies (Grizzle et l. 1995; Mner nd ivti 1999; Pereir nd Peleteiro 2002). The osteom is omprised of treule of mture one, generlly with well-defined order (Figure 31 nd ). The osteosrom ontined less mture one nd the periphery ws omposed of irregulrly-shped extensions of osteoid surrounded y pleomorphi, sophili osteolsts (Figure 31 nd D). D Figure 31. Rised ony tumors on the fin nd jw of rown ullhed.. Mirosopi pperne of the osteom on the fin. Treule of mture one () with well-defined order (rrow) in most res. r = 150 μm.. In some res of the tumor () there is osteolsti tivity (rrow) on the periphery. r = 50 μm.. Mirosopi pperne of n osteosrom on the jw of rown ullhed. Periphery is omposed of irregulrly-shped extensions of osteoid (rrows). r = 150 μm. D. Higher mgnifition illustrting the pleomorphi, sophili osteolsts (rrows) surrounding the osteoid rnhes. r = 50 μm. H&E stin. 5.0 KNOWLEDGEMENTS We thnk ll those within Pennsylvni Deprtment of Environmentl Protetion nd Pennsylvni Se Grnt involved in the olletion of fish nd other logistil ssistne. We ppreite the tehnil ssistne of Kthy Spring nd Drlene owling for preprtion of histologil slides. Funding ws provided y the U.S. Geologil Survey, the U.S. Environmentl Protetion geny, Pennsylvni Se Grnt nd the projet ws performed, in prt y using the servies provided y the Ntionl ner Institute s Registry of Tumors in Lower nimls, operted under ontrt y Experimentl Pthology Lortories, In., N We ppreite the ritil reviews provided y Pul owser, Mrk Myers, Willim Hwkins, Wolfgng Vogelein nd John Hrshrger. Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 29

34 6.0 REFERENES guis,. nd R.J. Roerts Melno-mrophge entres nd their role in fish pthology. J. Fish Dis. 26: nders, K. nd M. Yoshimizu Role of viruses in the indution of skin tumours nd tumour-like prolifertions of fish. Dis. qut. Org. 19: u, D.W.T The pplition of histo-ytopthologil iomrkers in mrine pollution monitoring: review. Mr. Pollut. ull. 48: umnn, P The use of tumors in wild fish to ssess eosystem helth. qut. Eosystem Helth 1: umnn P.., V. irns, W. Kurey, L. Lmert, I. Smith nd R. Thom Fish tumors or other deformities. Lke Erie Lkewide Mngement Pln (LMP) Tehnil Report Series, Report No. 6, 59 pp. umnn, P.. nd J.. Hrshrger Deline in liver neoplsms in wild rown ullhed tfish fter oking plnt loses nd environmentl PHs plummet. Environ. Helth Perspet. 2: umnn, P.. nd J.. Hrshrger Long term trends in liver neoplsm epizootis of rown ullhed in the lk River, Ohio. Environ. Monitor. ssess. 53: lk, J.J Epiderml hyperplsi nd neoplsi in rown ullheds (Itlurus neulosus) in response to repeted pplitions of PH ontining extrt of polluted river sediment. In: Polynuler romti hydrorons: Seventh Interntionl Symposium on Formtion, Metolism nd Mesurment. M.W. ooke nd.j. Dennis (editors). ttelle Press, olumus, OH, pp lzer, V.S., R.E. Wolke, J. rown nd.. Powell Pisine mrophge ggregte prmeters s helth monitors: effet of ge, sex, reltive weight, seson nd site qulity in lrgemouth ss (Miropterus slmoides). qut. Toxiol. 10: oormn, G.., S. otts, T.E. unton, J.W. Fournie, J.. Hrshrger, W.E. Hwkins, D.E. Hinton, M.P. Jokinen, M.S. Okihiro nd M.J. Wolfe Dignosti riteri for degenertive, inflmmtory, prolifertive nonneoplsti nd neoplsti liver lesions in medk (Oryzis ltipes): onsensus of Ntionl Toxiology Progrm Pthology Working Group. Toxiol. Pthol. 25: owser, P.R., M.J. Wolfe, J. Reimer nd.s. Shne Epizooti ppilloms in rown ullheds, Itlurus neulosus from Silver Strem reservoir, New York. Dis. qut. Org. 11: unton, T.E Experimentl hemil rinogenesis in fish. Toxiol. Pthol. 24: Edwrds, M.R. nd W.. Smsonoff Eletron mirosopi oservtions on virus-like prtiles of tfish ppillom. Pro. nnul Eletron Miro. So. mer. 35: Feist, S.W., T. Lng, G.D. Stentiford nd. Köhler iologil effets of ontminnts: Use of liver pthology of the Europen fltfish d (Limnd limnd L.) nd flounder (Pltihthys flesus L.) for monitoring. IES Tehniques in Mrine Environmentl Sienes No.38. Fournie, J.W., J.K. Summers, L.. ourtney, V.D. Engle nd V.S. lzer Utility of spleni mrophge ggregtes s n inditor of fish exposure to degrded environments. J. qut. niml Helth 13: Field Mnul for the Mirosopi Dignosis of Prolifertive Liver nd Skin Lesions in the rown ullhed 30