Temporal and spatial patterns in tumour prevalence in brown bullhead Ameiurus nebulosus (Lesueur) in the tidal Potomac River watershed (USA).

For two decades, fish tumour surveys have been used to monitor habitat quality in the Chesapeake Bay (USA) watershed. Tributaries with sediments contaminated with polynuclear aromatic hydrocarbons (PAHs), known to cause liver neoplasia, were frequently targeted. Here, we compare surveys in brown bullhead Ameiurus nebulosus conducted in 2009-2011 in the tidal Potomac River watershed (including the Anacostia River) with previous surveys. Using logistic regression, we identified length and sex as covariates for liver and skin tumours. We reported a statistically significant decrease in liver tumour probabilities for standardized 280 mm Anacostia bullheads between the 1996 and 2001 samplings (merged collections: female-77.5%, male-43.0%) and 2009-2011 (female-42.2%, male-13.6%). However, liver tumour prevalence in bullheads from the Anacostia, Potomac River (Washington, DC) and Piscataway Creek (17 km downriver) was significantly higher than that for Chesapeake Bay watershed reference locations. The causes of skin tumours in bullheads are uncertain, requiring further research. The similar liver tumour prevalence in these three locations suggests that the problem is regional rather than restricted to the Anacostia. To monitor habitat quality and the success of pollution control actions, we recommend conducting tumour surveys on a 5-year cycle coordinated with sediment chemistry analyses.

Neoplasms of the urinary tract in fish.

The veterinary literature contains scattered reports of primary tumors of the urinary tract of fish, dating back to 1906. Many of the more recent reports have been described in association with the Registry of Tumors in Lower Animals, and most of the spontaneous neoplasms of the kidney and urinary bladder are single case reports. In rare instances, such as described in nephroblastomas of Japanese eels and tubular adenomas/adenocarcinomas of Oscars, there is suggestion of a genetic predisposition of certain populations to specific renal neoplasms, environmental carcinogenesis, or potentially an unknown infectious etiology acting as a promoter. Hematopoeitic neoplasms have been infrequently described as primary to the kidney of a variety of fish species, and therefore those case reports of renal lymphoma and plasmacytic leukemia are addressed within the context of this review.

Tumor prevalence and biomarkers of exposure in brown bullhead (Ameiurus nebulosus) from Back River, Furnace Creek, and Tuckahoe River, Maryland.

Brown bullheads (Ameiurus nebulosus) were collected from 2 locations near Baltimore, Maryland, Back River and Furnace Creek, and 1 (reference) location, Tuckahoe River, to compare the prevalence of tumors (liver and skin) and visible skin lesions (fin erosion and abnormal barbels). Cytochrome P450 activity measured as ethoxyresorufin-O-deethylase, biliary PAH-like fluorescent metabolites, and fillet contaminant concentrations were determined as indicators of exposure in a randomly selected subset of the fish. There were no significant differences in liver tumor prevalence: Back River = 8% (4/50), Furnace Creek = 0% (0/50), and Tuckahoe River = 2.6% (1/39; p = 0.20, extension of Fisher's exact test). Skin tumor prevalence was as follows: Furnace Creek = 12% (6/50), Back River = 8% (4/50), and Tuckahoe River = 0% (0/39; p = 0.063). In the Back River fish, there was a 40% (20/50) prevalence of fin erosion and a 28% (14/50) prevalence of abnormal (shortened, clubbed, or missing) barbels. Fin erosion was not observed in the other collections, and only 10% (5/50) of the Furnace Creek fish had abnormal barbels (p < 0.001 for both lesions). There were statistically significant differences in mean EROD activity, with levels in Furnace Creek and Back River fish approximately twice that found in Tuckahoe River fish. There were also significant differences in mean benzo(a)pyrene-like bile metabolite concentrations: the lowest mean was in the Tuckahoe River fish, 8 times higher in Furnace Creek fish, and 13 times higher in Back River fish. Of the 3 groups, the Back River bullheads appear to be most adversely affected by contaminant exposure because they had the highest prevalence of liver tumors, fin erosion, and abnormal barbels.

Hepatic cholangiocarcinoma and testicular mesothelioma in a wild-caught blue shark, Prionace glauca (L.).

An adult male blue shark, Prionace glauca (L.), caught in July 2000 by a recreational fisherman off Long Island, New York, USA, had a retained fishing hook from a previous capture. The hook penetrated the gastric wall and lacerated the right liver lobe. Macroscopic lesions consisted of transmural gastritis and peritonitis. Alteromonas sp. and Vibrio alginolyticus were isolated from the peritoneal fluid. In addition, a well delineated, sessile mass was found on the otherwise normal serosa of the right testis. Histopathological findings included mesothelial hyperplasia and hypertrophy involving diffusely the gastric, hepatic and parietal serosae, and forming a discrete testicular capsular mass compatible with mesothelioma. In the liver an intrahepatic cholangiocarcinoma, chronic hepatitis, biliary hyperplasia and increased numbers of melanomacrophages were found. In addition organisms compatible with histozoic and coelozoic myxosporeans were found within the skeletal muscle of the abdomen and intrahepatic bile ducts, respectively. This is the third literature report of a liver tumour and the first report of a coelomic mesothelioma from a shark.

MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

Tumor prevalence and biomarkers of exposure in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River, USA, watershed.

Associations between contaminant exposure and liver and skin tumor prevalence were evaluated in brown bullheads (Ameiurus nebulosus) from the tidal Potomac River, USA, watershed. Thirty bullheads (> or = age 3) were collected from Quantico embayment, near a Superfund site that released organochlorine contaminants; Neabsco Creek, a tributary with petroleum inputs from runoff and marinas; and Anacostia River (spring and fall), an urban tributary designated as a Chesapeake Bay region of concern, that was contaminated with polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and organochlorine pesticides. Fish were collected from the Tuckahoe River, as a reference. Cytochrome P450 activity, bile PAH metabolites, and muscle organochlorine pesticide and PCB concentrations were measured in randomly selected individuals and sediment contaminants were analyzed. We found statistically significant differences in liver tumor prevalences: Anacostia (spring), 50%; Anacostia (fall), 60%; Neabsco, 17%; Quantico, 7%; and Tuckahoe, 10%. Skin tumor prevalences were significantly different: Anacostia (spring), 37%; Anacostia (fall), 10%; Neabsco, 3%; Quantico, 3%; and Tuckahoe, 0%. Tumor prevalence in Anacostia bullheads warrants concern and was similar to those at highly contaminated sites in the Great Lakes. Evidence was found of higher PAH exposure in Anacostia fish but a cause-effect linkage could not be established. Fish tumor surveys, with histopathologic examination of internal and external organs, are recommended for monitoring the status of regions of concern.

A proposed classification scheme for Xiphophorus melanomas based on histopathologic analyses.

We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous-macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.

Lesser known aquarium fish tumor models.

The repeated use of particular species for experimental oncology in fish increases their future value by accumulating background information for these models and justifies the establishment of genetic stock centers. However, the wide diversity that exists within the class Osteichthyes and Chondrichthyes suggests that the ideal surrogate models for studying some types of neoplasms might be found among lesser known species. To help assess cultured fish as surrogates for some other types of human neoplasia, we examined cases in the archives of the Registry of Tumors in Lower Animals and reviewed reports in the literature. Spontaneous and induced neoplasms originating from a spectrum of cell types were seen in more than 215 fish species commonly raised in aquaria or cultured for study among 69 families. Prominent families include the Poeciliidae (livebearers), Cyprinidae (carps and minnows), Cichlidae (cichlids), Cyprinodontidae (killifish), Characidae (tetras), Adrianichthyidae (medakas), Aplocheilidae (rivulins), and Salmonidae (salmon and trout). The following are examples of potential fish tumor models that have received less consideration than some others: papilloma and carcinoma of the urinary bladder in oscar (Astronotus ocellatus); osteogenic neoplasms, peripheral nerve sheath tumors, and ependymoblastoma in coho salmon fingerlings (Oncorhynchus kisutch); and nephroblastoma resembling Wilms' tumor in Japanese eels (Anguilla japonica).

Suprasellar germinoma in three lake whitefish (Coregonus clupeaformis).

Suprasellar germinomas were identified in three wild-caught lake whitefish (Coregonus clupeaformis) from the St. Lawrence River, Quebec, Canada. Histologically, the three tumors expanded the subarachnoid space of the ventral surface of the brain immediately adjacent to the pituitary gland and, in one case, infiltrated the adjacent neuropil. These tumors were characterized by nests and sheets of round cells with a high mitotic rate, separated by a scant amount of loose fibrovascular stroma. The stroma was infiltrated by a moderate number of small mononuclear cells, including rare CD3-immunoreactive lymphocytes. This is the first report of intracranial germinoma in a fish species.

Ichthyophonus-like infection in wild amphibians from Québec, Canada.

Myositis associated with infection by Ichthyophonus-like organisms was diagnosed in 35 of 260 (13%) wild amphibians collected in Quebec, Canada, from 1959 to 1964 (n = 30), and 1992 to 1999 (n = 230). Infection was diagnosed in 17 green frogs Rana clamitans, 9 wood frogs R. sylvatica, 4 red-spotted newts Notophthalmus viridescens, 3 bullfrogs R. catesbeiana, 1 spring peeper Pseudacris crucifer, and 1 pickerel frog R. palustris. The spring peeper and one of the bullfrogs were collected in 1964 from the Mont Saint-Hilaire Biosphere Reserve, indicating long-term presence of the organism. Spores of the organisms invaded striated muscle fibers and were associated with variable degrees of granulomatous and eosinophilic inflammation. Infection was considered fatal in 2 green frogs, 1 wood frog, and 1 red-spotted newt. It was considered potentially significant in 3 additional green frogs in which up to 100% of the fibers of some muscles were replaced by spores associated with a severe granulomatous reaction. Ultrastructural features of Ichthyophonus-like spores included a thick trilaminated wall, a paramural cytoplasm, multiple nuclei, oval mitochondria with short tubulo-vesicular cristae and numerous ribosomes. This report represents 4 new host records and shows that ichthyophonosis is enzootic in amphibians from Quebec.

Ethylnitrosourea induces neoplasia in zebrafish (Danio rerio).

The zebrafish (Danio rerio) has been successfully used to discover hundreds of genes involved in development and organogenesis. To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors. Germ line mutations are most commonly induced for zebrafish mutant screens by exposing adult male zebrafish to the alkylating agent, ethylnitrosourea (ENU). To determine whether ENU induces tumors, we compared the incidence of tumors in ENU-treated fish with untreated controls. Interestingly, 18 of 18 (100%) fish mutagenized with either 2.5 or 3.0 mM ENU developed epidermal papillomas, which numbered 1 to 22 per fish, within 1 year of treatment. The induced epidermal lesions included epidermal hyperplasia, flat papillomas (0.2 to 1.2 mm), and pedunculated papillomas (1.2 to 8 mm in greatest dimension), but no skin cancers. Angiogenesis was evident in papillomas larger than approximately 1 mm. All but two papillomas contained the three cell types (keratinocytes, club, and mucous cells) of normal zebrafish epidermis; histologic variants lacked either club cells or mucous cells. Two cavernous hemangiomas and a single malignant peripheral nerve sheath tumor were also found in the treated fish. None of five untreated controls developed tumors. These studies establish the feasibility of the zebrafish as an experimental model for the study of skin tumors.

Intersexes in Mississippi River shovelnose sturgeon sampled below Saint Louis, Missouri, USA.

The Missouri Department of Conservation detected high organochlorine levels in tissues of fish from the Mississippi River south of Saint Louis. Chlordane, which is both a carcinogen and an endocrine disrupter, reached 2960 micrograms/kg in fillets of shovelnose sturgeon, Scaphirhynchus platyorynchus, and 1926 micrograms/kg in their roe, wet weight basis. Polychlorinated biphenyls (PCBs) and p,p'-dichlorodiphenyldichloroethylene (DDE) in roe have reached 5810 and 780 micrograms/kg, respectively. The purpose of this study was to determine if endocrine disrupters and carcinogens are a potential cause of the significantly diminished reproduction capacity and population declines in the endangered pallid sturgeon, Scaphirhynchus albus. Twenty-four surrogate shovelnose sturgeons were sampled north of Saint Louis where contaminant levels were lower compared with 17 sampled south of Saint Louis where contaminant levels were high. Liver and gonad tissues were formalin-fixed, histologically processed and examined. Neoplasms were not found. The principal finding was that the testes of two of the seven downstream male fish contained ovigerous lamellae, in addition to mature sperm. Since sturgeons are gonochoristic and intersexes are rare and unusual, the occurrence of intersex in 29% of the male Mississippi River shovelnose sturgeon examined from a population contaminated with chlordane and other chemicals raise concern for human health, species biodiversity and preservation of waterway quality. These findings, however, need to be corroborated by expanded studies with a higher sample size.

Cutaneous mastocytomas in the neotenic caudate amphibians Ambystoma mexicanum (axolotl) and Ambystoma tigrinum (tiger salamander).

Spontaneous mastocytomas studied in 18 axolotls (Ambystoma mexicanum) and six tiger salamanders (Ambystoma tigrinum) were gray-white, uni- to multilobular cutaneous protrusions from 2 mm to 2 cm in diameter. Tumors were moderately cellular unencapsulated masses that usually infiltrated the dermis and hypodermis with the destruction of intervening tissues. Some tumors were invading superficial bundles of the underlying skeletal muscle. Tumors consisted of mitotically active cells derived from a single lineage but showing a range of differentiation. Immature cells had nearly smooth to lightly cleft or folded basophilic nuclei bordered by a band of cytoplasm with few cytoplasmic processes and containing a few small uniform eccentric granules. Mature cells had basophilic nuclei with deep clefts or folds and abundant eosinophilic cytoplasm with multiple long intertwining cytoplasmic extensions packed with metachromatic granules. The axolotls were old individuals from an inbred laboratory colony. The tiger salamanders were wild animals from a single polluted pond. They could have been old and inbred. Both groups were neotenic. These are the first mastocytomas discovered in cold-blooded animals.

Expression of growth arrest and DNA damage genes and DNA mismatch repair genes in snake melanomas.

Snake melanomas are rare tumours and their pathogenesis is still unclear. In this study the expression of DNA mismatch repair genes and Growth Arrest DNA Damage Genes was investigated. The expression only of 3 of them GADD34, MLH1 and MSH2 was found. The role of these genes in animal tumorigenesis is discussed.

Diagnostic criteria for degenerative, inflammatory, proliferative nonneoplastic and neoplastic liver lesions in medaka (Oryzias latipes): consensus of a National Toxicology Program Pathology Working Group.

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from Medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

Immunohistochemical and photometric analysis of snake-melanomas.

Snake melanomas are very rare tumours in the class of Reptilia. This study demonstrates the ploidy status and S100 and HMB45 expression in snake melanomas in connection to their morphology and clinical history.

Prevalence and histopathology of shell disease in turtles from Lake Blackshear, Georgia.

Turtles in Lake Blackshear, Crisp County, Georgia (USA) were evaluated for shell disease during intensive trapping efforts on 8 and 9 May 1990. The disease was most prevalent in Pseudemys concinna (74%) and Trachemys scripta (35%). The degree of necrosis on the carapace was significantly positively correlated with the degree of necrosis on the plastron in T. scripta (rs = 0.50), but not in P. concinna (rs = 0.06). Female T. scripta with lesions were significantly larger than females without lesions. Lesions were not detected on six other species of turtles. Some areas contained multinucleate osteoclasts that were destroying bone. No tumors were detected in soft tissue samples.

Association of prokaryotes with symptomatic appearance of withering syndrome in black abalone Haliotis cracherodii.

Withering syndrome (WS) is an epizootic fatal wasting disease that is devastating California Channel Island populations of black abalone Haliotis cracherodii. Our studies suggest a strong pathogen-disease association. The pathogen is an intracellular prokaryote that infects epithelial cells lining the gut and enzyme secreting cells of the digestive diverticula. It multiplies by binary fission in round to oval, basophilic, membrane-bound colonies teeming in the cytoplasm. Infection of the digestive diverticula is accompanied by a complete loss of digestive enzyme granules and metaplasia of enzyme secretory cells to a morphology similar to epithelium lining the gut. Extensive infection of digestive diverticular cells and the resultant deficiency in digestive enzymes correlates to the degree of pedal muscle atrophy and the severity of signs associated with WS. Electron microscopically the intracellular pathogen is a rod-shaped, ribosome-rich, gram-negative, prokaryote with a trilaminar cell wall consistent with the order Rickettsiales. Microbiological and protozoological methods produced no patterns that implicated other types of microbes. Chemical analysis of tissue from animals from a population with WS did not support an association between WS and environmental pollutant exposure to polycyclic aromatic hydrocarbons, polychlorinated biphenyls, or chlorinated pesticides.