Neoplastic and Nonneoplastic Hepatic Changes in Lake Whitefish(Coregonus clupeaformis) from the St. Lawrence River, Quebec, CanadaIgor Mikaelian,1 Yves de Lafontaine,2 Chantal M6nard,2 Pierre Tellier,3 John C. Harshbarger,4 and DanielMartineaul1Canadian Cooperative Wildlife Health Centre and Centre Qu6b6cois sur la Sant6 des Animaux Sauvages, D6partement de Pathologieet de Microbiologie, Facult6 de M6decine V6terinaire, Universite de Montr6al, Saint-Hyacinthe, Quebec, Canada; 2St. Lawrence Centre,Environment Canada, Montreal, Quebec, Canada; 3Bio-Research Laboratories Ltd, Senneville, Quebec, Canada; 4Registry of Tumors in

Lower Animals, George Washington University Medical Center, Department of Pathology, Washington, DC 20037 USA

As part of a survey of fish diseass, lake whitefish (Coregons clupeaformis) were coliected in fail1995 from the St. Lawrence River 15 km upstream of Quebec City, Quebec, Canada, to assessthe prevalence of liver lesions. A total of 141 fish were captured and necropsied, and three stan-dard s ons of liver were taken for histological exmination. Prvlences of altered hepatocytefoci, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma were 0.7%, 2.1%, 0.7%,and 2.1%, respectively. Thus, the overall prevalence of liver neoplasia was 4.9% (71141).Hepatic tumors were only observed in fish 7 years old or older. Fish age was siganificantdy andpositively correlated with the index assessing the number and size of macrophage aregtes(pcO.001; r = 0.16). Hepatocyte vacuolatin anisokaryosis, lymphocytic infilon, and bileduct hyperplasia were also observed but were not related to the age, length, sex, or condition fac-tor of the fish. These results represent the first report on a series of hepatic tumors in a wildsalmonid species. Key words: cancer, environmental carcinogenesis, fish, liver, pathology.Environ Heal Perspect 106:179-183 (1998). [Online 26 February 1998]bhp://ehpntl.nieh.ni.gov/docsI9.98/1 06p179-183mikclian/abstract.html

The occurrence of hepatic neoplastic andcertain associated nonneoplastic lesions over20 fish species has been attributed to thepresence of chemical carcinogens in aquaticecosystems (1-8). Consequently, monitor-ing hepatic lesions in target fish species mayprovide an indicator of pollution (9-12).

In 1994 and 1995, a general assessment ofthe health of the St. Lawrence River was spon-sored by Environment Canada, and fish dis-eases were monitored as part of this program.In 1994, 16 of 197 lake whitefish (Coregonusclupeaformis) from the St. Lawrence Riverwere selected for necropsy on the basis ofexternal abnormalities during a survey atSaint-Nicolas, Quebec (13). Two of these 16fish were diagnosed with cholangiocarcinomas[Registry of Tumors in Lower Animals(RTLA), #6185 and #6186], but no livermass was found upon macroscopic examina-tion of 1,291 randomly selected fish belong-ing to 15 species, including 297 channel cat-fish (Ictalurus punctatus), 200 longnose suck-ers (Catostomus catostomus), and 184 whitesuckers (Catostomus commersonO). The presentstudy was undertaken in 1995 to determinemore precisely the prevalence of liver neo-plasms and other hepatic lesions in lake white-fish from the St. Lawrence River and to exam-ine the effects of fish age and size on theoccurrence of these pathological conditions.

Materials and MethodsLake whitefish were collected using fixedfishing gear installed at Saint-Nicolas(46°43' N, 71O19' W), Quebec, Canada, 15

km upstream of Quebec City, from 30August 1995 to 30 October 1995. All lakewhitefish caught during this period werekept for examination.

Upon collection, fish were measuredfor total length to the nearest millimeterand weighed to the nearest gram. Fish werehumanely euthanized with an overdose oftricaine methane sulfonate (MS 222), anda complete postmortem examination wasperformed. The weight of liver, gonads,and gastric content and the evisceratedweight were recorded.

Samples of liver and of any internallesion were collected from every fish. Threestandard sections of liver were preserved in10% buffered formalin for histologicalanalyses. Samples were routinely embed-ded in paraffin wax, sectioned at 5 pm,and stained with hematoxylin-phloxin-saf-fron. Scales were removed from the ventro-lateral side of the body and fish age wasdetermined by scale reading with a stere-omicroscope. Due to the difficulty of pre-cisely determining the age of older fishwith scale reading, all fish 9 years old orolder were grouped as 9+ year fish.

All sections were examined by the samepathologist; lesions were further examinedby a second pathologist, and tumors wereconfirmed by the RTLA. Foci of alteredhepatocytes and liver neoplasms were clas-sified according to criteria outlined byBoorman et al. (14). Other hepatic lesionswere classified and graded as describedbelow.

Hepatocyte vacuolation was graded 0(no vacuolation), 1 (<5% hepatocytes wereaffected), 2 (5-40% were affected), and 3(>40% were affected). The macrophageaggregates (MAs) were scored 0 (no MAs),1 (presence of a few MAs scattered withinthe parenchyma), 2 (presence of numerousMAs), and 3 (more than 20% of the sectionconsisted of MAs). Lymphocytic infiltrationwas graded 0 (no lymphocytes), 1 (a fewlymphocytes and/or plasma cells around afew bile tracts), 2 (some bile tracts sur-rounded by 1-3 layers of lymphocytesand/or plasma cells), and 3 (some bile tractssurrounded by >3 layers of lymphocytesand/or plasma cells). Bile duct hyperplasiawas graded 0 (no increase), 1 (slightincrease in the number of bile ducts withina few randomly distributed portobiliarytracts), 2 (several markedly convoluted bileducts within most portobiliary tracts or ran-domly scattered in the hepatic parenchyma;occasional loss of polarity or luminal infold-ing of the epithelium), and 3 (numerousmarkedly convoluted bile ducts in thehepatic parenchyma and/or in portobiliarytracts; prominent loss of polarity andanisokaryosis). Hepatocyte anisokaryosiswas graded 0 (absence) and 1 (presence).Individual fish were scored according to thehighest grade diagnosed on three micro-scopic sections.

Fish condition factor (CF), used as anoverall physiological index, was calculatedas follows:

Address correspondence to D. Martineau, CanadianCooperative Wildlife Health Centre and CentreQudbdcois sur la Santd des Animaux Sauvages,Ddpartement de Pathologie et de Microbiologie,Facultd de Mddecine Vdtdrinaire, Universitd deMontrral, Saint-Hyacinthe, Quebec, J2S 7C6, Canada.We are grateful to Fernand Gingras for his commit-ment to the collection of specimens; to DenisLabontd, Sylvie Legendre, and David Routier forfield work; and to Jacinthe Cardin, Jules Deslandes,Lyne Pdpin, and Bibiane Pdpin-Faille for skillful lab-oratory assistance. Funded by St. Lawrence Vision2000, Environment Canada, Montreal, Quebec; theCanadian Cooperative Wildlife Health Centre,Saskatoon, Saskatchewan; and the Centre Qudbecoissur la Santd des Animaux Sauvages, Saint-Hyacinthe,Quebec, Canada.Received 5 August 1997; accepted 5 December 1997.

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Articles * Mikaelian et al.

CF= 100 x (total weight - gonad weight)(length)3

where weight is expressed in grams andlength in centimeters.

The difference between tumor preva-lence in categories of length, age, and sexwas statistically evaluated by a %2 test. CFof fish with and without hepatic lesionswas compared using a Wilcoxon-rank-sumtest. The relationship between the preva-lence of each kind of lesion and fish ageand CF was tested using Spearman rankcorrelation.

ResultsThe lake whitefish liver is composed of twolarge lobes suspended over the digestive tractfrom a ligament. These features facilitateeasy removal and examination of the liver.

The MAs score increased with age (Fig.1; Spearman correlation, r, = 0.16; p<0.001;n = 141). Various degrees of hepatocyte vac-uolation, anisokaryosis, nuclear pleomor-phism, bile duct hyperplasia, lymphocyticproliferation, and foci of coagulative necrosiswere found, irrespective of the length, sex,and CF of the fish or the presence of neo-plastic lesions (Table 1).

Eosinophilic foci of altered hepatocyteswere found in one fish (RTLA # 6354;Table 2). They consisted of two round andsharply demarcated areas that were 300 pm

2.5

2

@ .5

U ||I||SI |*IX__El _| 1lI_

and 1 mm in diameter and composed ofslightly enlarged hepatocytes with an abun-dant eosinophilic and finely granular cyto-plasm (Fig. 2).

A total of seven fish were found withhepatic tumors, which yields an overallprevalence of 4.9% (7/141). Fish withhepatic tumors were 7 years of age and olderand were longer than 450 mm (Table 2).The prevalence of hepatic tumors is 8.4%(7/83) for fish aged 7 years or older and11.4% (4/35) for fish aged 9 years or older.

Hepatocellular carcinomas were found inthree females. They consisted of poorlydemarcated, translucent (RTLA #6264 and#6265) or pale brown (RTLA #6265), fleshynodules located within the hepatic parenchy-ma. Microscopically, these tumors werenodular, single to multiple, poorly demarcat-ed, mildly compressive, and unencapsulated(Fig. 3). They were composed of two- tothree-cell thick irregularly convoluted trabec-ulae that were generally perpendicular to themargins of the mass. Neoplastic cells wereabout 50% larger than normal hepatocytesand their cytoplasm was slightly morebasophilic. Their nuclei, 50% larger thannormal, showed moderate hypochromasiaand minimal anisokaryosis and each had asingle large nudeolus that was eccentricallylocated and amphophilic. The nudeus/cyto-plasm ratio was normal, and there were fewmitoses (0-2/high power field). MAs andbile ducts were not present in tumors. In onespecimen (RTLA #6260), neoplastic trabec-ulae were focally separated by dilated sinu-soids measuring up to 100 plm in width.

A cholangioma was found histologicallyin one fish (Fig. 4). It consisted of a single,well-delimited and unencapsulated clusterof well-differentiated to slightly irregularbile duct-like structures that were separatedby a small amount of fibrovascular stroma.

Cholangiocarcinomas were found macro-scopically in one fish (RTLA #6258) and

histologically in two additional fish (RTLA#6261 and #6266; Table 2). Macroscopicallyvisualized tumors consisted ofwell-demarcat-ed white solid nodules located within theparenchyma. Microscopically, these tumorswere unencapsulated nodules, 0.3-3 mm indiameter, that were single or multiple, well-defined, locally infiltrative, and consisting ofirregularly shaped bile duct-like structuresseparated by variable amounts of well-vascu-larized stroma (Fig. 5). Bile duct-like struc-tures located at the center of the tumors wereoften dilated, some being cystic, with occa-sional infolding of the flattened epithelium.Peripheral bile duct-like structures were linedby a cuboidal epithelium, and their smallerdiameter was less variable than that of theperipheral structures. Tumor cells had indis-tinct borders; variable amounts of pale aci-dophilic, finely granular cytoplasm; and anelongated, often monstrous, vesicular nude-us with a small basophiic nucleolus. A fewneoplastic cells were binudeated. Anisokar-yosis was more apparent at the periphery oftumors, and the nudeus/cytoplasm ratio waselevated (1/2 to 2/1). Mitoses were rare (lessthan 1/x400 power field).

Changes in other organs with grosslesions were not neoplastic and are notreported here.

DiscussionThe lake whitefish population in the St.Lawrence River, 15 km upstream fromQuebec City, seems to be affected by a highprevalence of hepatic neoplasia. Prior to thisstudy and the previous report from ourgroup (13), a single hepatic neoplasm (acholangiocarcinoma) had been diagnosed ina fish from the genus Coregonus (lake her-ring, Coregonus artedi; Essex County, NY;RTLA #1426). To our knowledge, the pre-sent report is the first description of a seriesof hepatic neoplasia in a wild population ofsalmonid fish, although the domesticated

Age (year)

Figure 1. Mean index (± 1 standard deviation) ofthe number and size of macrophage aggregates(MAs) as a function of fish age. The numbersabove or in the bars represent the sample size foreach age class.

Table 1. Severity of nonneoplastic hepatic lesionsin lake whitefish (Coregonus clupeaformis)caught in 1995 in the St. Lawrence River at Saint-Nicolas, Quebec, Canada (n= 141)

LesionHepatocyte vacuolationMacrophage aggregatesLymphocytic infiltrationBile duct hyperplasiaFoci of coagulatve necrosisNuclear pleomorphism

Histopathologic index0 1 2 3

77 22 28 1496 20 21 440 93 8 069 66 5 1137 4139 2

Table 2. Preneoplastic and neoplastic hepatic lesions in lake whitefish (Coregonus clupeaformis) caughtin 1995 in the St. Lawrence river at Saint-Nicolas, Quebec, Canada

Number LesionRTLA Length of diameternumber (mm) Age Sex lesions (mm)

Foci of altered hepatocytes 6354 489 6 M 2 1Hepatocellular carcinoma 6264 523 9 F ±15 2-6

6265 521 9+ F 3 1-36260 547 9+ F 1 14

Cholangioma 6262 478 7 M 1 0.3Cholangiocarcinoma 6258 532 9+ F 2 0.4-2

6261 506 8 M 2 16266 458 7 M 1 3

Fish without neoplasia - - - 69 F - -60M - -4U - -

Abbreviations: RTLA, Registry of Tumors in Lower Animals; F, female; M, male; U, unknown.

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Figure 2. Eosinophilic foci of altered hepatocytes in lake whitefish (Coregonusclupeaformis). Altered hepatocytes form anastomosing trabeculae (two cellsthick). Their cytoplasm is slightly more acidophilic and more abundant thannormal hepatocytes. Bar = 200 pm; hematoxylin-phloxin-saffron.

Figure 3. Hepatocellular carcinoma in lake whitefish (Coregonusclupeaformis). The tumor is composed of cords 2-4 cells thick. The tumorslightly compresses the adjacent parenchyma and is devoided ofmelanomacrophage aggregates and bile duct tracts. Bar = 300 pm; hema-toxylin-phloxin-saffron.

rainbow trout is susceptible to a variety ofcarcinogenic compounds that cause hepato-cellular carcinomas (15-18).

In humans and in other mammals,common causes of hepatic neoplasia arechemical carcinogens and viruses (19-21).Helicobacter infections (22,23) and parasiticliver flukes (24,25) have been associatedwith liver cancer. In feral fish, high preva-lences of liver neoplasms have generallybeen attributed to anthropogenic (2,3,6,7,9,26-28) and occasionally to natural (29)chemical contamination of the environ-ment. Hepatic neoplasms were not detectedin lake whitefish exposed to bleached kraftmill effluent subject to primary treatment(30) or in those inhabiting unpollutedOntarian (2,30) and Quebec lakes (31).However, these studies are not comparablebecause histology was not done (2,31) orwas done on a limited number of fish (30).

All previous series of hepatic neoplasmsreported in wild fish occurred in bottom-feeding nonsalmonid species (5,27). Becausebenthic invertebrates are known to concen-trate contaminants including polychlorinat-ed biphenyls and powerful carcinogens such

Figure 4. Cholangioma in lake whitefish (Coregonus clupeaformis). The tumor forms a small unencapsulat-ed cluster of well-differentiated bile duct-like structures. Bar = 75 pm; hematoxylin-phloxin-saffron.

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Articles * Mikaelian et al.

Figure 5. Cholangiocarcinoma in lake whitefish (Coregonus clupeaformis). Thetumor is composed of closely packed, well-differentiated bile duct-like struc-tures that infiltrate the adjacent hepatic parenchyma. Bar = 150 pm; hema-toxylin-phloxin-saffron.

as polycyclic aromatic hydrocarbons (PAHs)(32-35), fish feeding on benthos areexposed to higher doses of carcinogenic con-taminants. The gastric content of our lakewhitefish consisted of gastropods admixedwith varying numbers of amphipods(Gammarusspp.) and small amounts of sedi-ments (de Lafontaine, unpublished observa-tion), which indicates that lake whitefishfeed on benthic organisms (30,36). Such adiet may play an etiological role in this seriesof tumors.

The MA score was statistically associatedwith age. In other studies, the number andsize of hepatic and splenic MAs have beencorrelated with levels of environmental cont-aminants (37-40) as well as with aging (41).A control population from an area with dif-ferent contamination levels would be neededto determine whether the increased MAscore in older lake whitefish is the result oflonger exposure to toxic substances.

We found hepato-cyte vacuolation andanisokaryosis. Thesechanges have alsobeen associated withexposure to contami-nants in fish (12,42).However, these non-neoplastic changeswere not associatedwith the presence oftumors, sex, age, orCF, and their signifi-cance remains unde-termined.

Lake whitefishpopulations inhabitinglarge rivers in northernQuebec are anadro-mous (43). In the St.Lawrence River, rep-orts by local fishermenand our observations(unpublished data) areconsistent with ananadromous reproduc-tive pattern. Conse-quently, the occur-rence of hepatic neo-plasms in lake white-fish, as opposed totheir absence in theother fish species thatwe examined from thesame area, may reflectthe exposure of lakewhitefish to differentchemical compounds.It may also reflect dif-ferences in the abilityto generate carcino-genic metabolites

from xenobiotic compounds such as PAHs(44). Further studies assessing the relation-ship of contaminant levels in lake whitefishtissues to their diet are required to supportthe hypothetical link between contaminantsand neoplasia in this species and to docu-ment the spatial movement and distributionof lake whitefish in the St. Lawrence River.

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