Epizootic neoplasia of the lateral line system of lake trout (Salvelinus namaycush) in New York's Finger Lakes.

This article documents an epizootic of inflammation and neoplasia selectively affecting the lateral line system of lake trout (Salvelinus namaycush) in 4 Finger Lakes in New York from 1985 to 1994. We studied more than 100 cases of this disease. Tumors occurred in 8% (5/64) of mature and 21% (3/14) of immature lake trout in the most severely affected lake. Lesions consisted of 1 or more neoplasm(s) in association with lymphocytic inflammation, multifocal erosions, and ulcerations of the epidermis along the lateral line. Lesions progressed from inflammatory to neoplastic, with 2-year-old lake trout showing locally extensive, intense lymphocytic infiltrates; 2- to 3-year-old fish having multiple, variably sized white masses up to 3 mm in diameter; and fish over 5 years old exhibiting 1 or more white, cerebriform masses greater than 1 cm in diameter. Histologic diagnoses of the tumors were predominantly spindle cell sarcomas or benign or malignant peripheral nerve sheath neoplasms, with fewer epitheliomas and carcinomas. Prevalence estimates did not vary significantly between sexes or season. The cause of this epizootic remains unclear. Tumor transmission trials, virus isolation procedures, and ultrastructural study of lesions failed to reveal evidence of a viral etiology. The Finger Lakes in which the disease occurred did not receive substantially more chemical pollution than unaffected lakes in the same chain during the epizootic, making an environmental carcinogen an unlikely primary cause of the epizootic. A hereditary component, however, may have contributed to this syndrome since only fish of the Seneca Lake strain were affected.

Epizootic ameloblastomas in Chinook salmon (Oncorhynchus tshawytscha) of the northwestern United States.

Abnormal growths were observed on the lips and in the oral cavities of 2- and 3-year-old Chinook salmon (Oncorhynchus tshawytscha) maintained in one freshwater and one saltwater captive fish-rearing facility in the Columbia River (Pacific Northwest). Initially presenting as bilaterally symmetrical, red, irregular plaques on oral mucosal surfaces, the lesions developed progressively into large, disfiguring masses. Of the 502 natural parr collected for captive broodstock, 432 (86%) displayed these tumors, whereas cohort salmon (i.e., same year classes) in these same facilities remained unaffected. Morphologically similar neoplasms were collected occasionally from adult Chinook salmon that had returned to their natal streams. Histologic features of the tumors suggested that they were derived from the portion of dental lamina destined to form tooth root sulci; therefore, these neoplasms were diagnosed as ameloblastomas. The lesions also resembled archived specimens of Chinook salmon oral tumors, which had been described decades earlier. Etiologic investigations performed during the current outbreak included bacteriologic, virologic, genetic, ultrastructural analyses, and cohabitation exposure studies. Results of these efforts did not indicate an obvious genetic basis for this syndrome, attempts to isolate potentially causative viruses or bacteria were negative, and disease transmission to naïve fish was unsuccessful. A few intracytoplasmic hexagonal structures, possibly consistent with viral particles (approximately 100 nm), were observed ultrastructurally in a tumor cell from 1 of 6 specimens submitted for transmission electron microscopy. Although the presence of these particles does not constitute sufficient evidence for causality, an infectious or multifactorial etiology seems plausible.

An in vivo method for using 5-bromo-2'-deoxyuridine (BrdU) as a marker of chemically-induced hepatocellular proliferation in the Japanese medaka (Oryzias latipes).

Japanese medaka fish (Oryzias latipes) were used to develop an in vivo method to assess hepatocellular proliferation in a nonmammalian model. Proliferative responses were assessed in medaka at 7, 17, 24, and 94 days after a 48-hour exposure to 10 or 100 mg/L diethylnitrosamine (DEN). Subgroups of medaka were exposed to 50 or 75 mg/L of 5-bromo-2'-deoxyuridine (BrdU) in water for 72 hours, sacrificed, and then processed for immunohistochemical staining. Proliferative indices of BrdU-labeled hepatocytes were quantified and compared using both count and area measurements. There was a significant increase (p < 0.05) in hepatocellular proliferation in the 100 mg/L DEN-treated fish as compared to controls and 10 mg/L DEN-treated fish for the first 3 time points. Hepatocarcinogenicity was evaluated 26 weeks post-DEN exposure. There was a significant increase (p < 0.0001) in hepatocellular neoplasms in 100 mg/L DEN-treated fish compared to other fish. Effective BrdU-labeling of S-phase hepatocytes in medaka was achieved by adding BrdU to the aquarium water, and an increase in hepatocellular proliferation using this method was detected 7 days after exposure to a carcinogenic concentration of DEN. Additionally, the new method of area measurement indices of proliferation were as precise as count indices (R2 > or = 0.92).

Chronic toxicity of chloroform to Japanese medaka fish.

Japanese medaka (Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured chloroform concentrations of 0.017, 0.151, or 1.463 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic chloroform concentration. Histopathology was evaluated at 6 and 9 months. Chloroform was not hepatocarcinogenic to the medaka at the concentrations tested. Chronic toxicity was evidenced at these time points by statistically significant ([alpha] = 0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.463 mg/L chloroform. We assessed hepatocellular proliferation by exposing test fish to 5-bromo-2'-deoxyuridine in the aquarium water for 72 hr after 4 and 20 days of chloroform exposure; we then quantified area-labeling indices of the livers using computer-assisted image analysis. We observed no treatment-related increases in cellular proliferation. We analyzed cells in circulating blood in medaka after 6 months of chloroform exposure. Hematocrit, leukocrit, cell viability, and cell counts of treated fish were not significantly different from those of control fish. Using gas chromatography (GC), we evaluated intrahepatic concentrations of chloroform in fish after 9 months of exposure. Livers from the 0.151 and 1.463 mg/L chloroform-treated fish had detectable amounts of chloroform, but these levels were always lower than the aquaria concentrations of chloroform. Thus, it appeared that chloroform did not bioaccumulate in the liver. Unidentified presumptive metabolite peaks were found in the GC tracings of these fish livers.

Chronic toxicity of bromodichloromethane to the Japanese medaka (Oryzias latipes).

Japanese medaka (Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured bromodichloromethane (BDCM) concentrations of 0.018, 0.143, or 1.424 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic BDCM concentration. BDCM was not hepatocarcinogenic to medaka at the concentrations tested. Chronic toxicity was evidenced at 6 and 9 months by statistically significant (alpha = 0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.424 mg/L BDCM. Hepatocellular proliferation was assessed after 1, 4, and 20 days of BDCM exposure. Treatment-related increases or decreases in cellular proliferation were not observed at any time point. Hematocrit, leukocrit, cell viability, and cell counts of treated fish after 9 months of BDCM exposure were not significantly different from control fish. Intrahepatic concentrations were evaluated by gas chromatography after 9 months of BDCM exposure. Fish livers from all three BDCM treatments had detectable amounts of BDCM, with median intrahepatic concentrations of 1.02, 2.89, and 21.25 mg BDCM/kg fish liver in the low, middle, and high concentrations, respectively. Medaka chronic toxicity effects of statistically significant gallbladder and bile duct abnormalities occurred at 1.424 mg/L BDCM, well above median drinking water levels.

Histopathology of Japanese medaka (Oryzias latipes) chronically exposed to a complex environmental mixture.

Japanese medaka (Oryzias latipes) were used to evaluate the carcinogenicity of a complex groundwater that contained 5 U.S. Environmental Protection Agency priority pollutant heavy metals and 13 chlorinated aliphatic hydrocarbons. A test protocol that used 10 mg/L diethylnitrosamine (DEN) prior to groundwater exposure was designed to assess both initiation and promotion. The fish were exposed continuously for 9 mo with 0, 1, 5, or 25% groundwater, by volume, with either West Branch of Canal Creek water (Aberdeen Proving Ground-Edgewood Area, Aberdeen Proving Ground, MD) or dechlorinated tap water as the diluent, while concurrent controls were run in the laboratory. Incidental findings included various neoplasms in the nares, ovary, skeletal muscle, skin, swim bladder, testis, thymus, and thyroid. Factors evaluated during statistical analyses of fish neoplasm prevalence included diluent type, groundwater percentage, fish gender, and DEN initiation. Liver neoplasm prevalence was higher in DEN-initiated fish and was frequently higher in males. Concentrations of up to 25% groundwater, by volume, showed no evidence of being a complete carcinogen and showed no consistent, conclusive evidence of being a promoter.

Environmental complex mixture toxicity assessment.

Trichloroethylene (TCE) was found as a contaminant in the well supplying water to an aquatic testing laboratory. The groundwater was routinely screened by a commercial laboratory for volatile and semivolatile compounds, metals, herbicides, pesticides, and polychlorinated biphenyls using U.S. Environmental Protection Agency methods. Although TCE was the only reportable peak on the gas chromatograph, with average concentrations of 0.200 mg/l, other small peaks were also present, indicating the possibility that the contamination was not limited to TCE alone. A chronic 6-month carcinogenicity assay was conducted on-site in a biomonitoring trailer, using the Japanese medaka fish (Oryzias latipes) in an initiation-promotion protocol, with diethylnitrosamine (DEN) as the initiator and the TCE-contaminated groundwater as a promoter. Study results indicated no evidence of carcinogenic potential of the groundwater without initiation. There was, however, a tumor-promotional effect of the groundwater after DEN initiation. A follow-up laboratory study was conducted using reagent grade TCE added to carbon-filtered groundwater to simulate TCE concentrations comparable to those found in the contaminated groundwater. Study results indicated no promotional effects of TCE. These studies emphasize the necessity for on-site bioassays to assess potential environmental hazards. In this instance, chemical analysis of the groundwater identified TCE as the only reportable contaminant, but other compounds present below reportable limits were noted and may have had a synergistic effect on tumor promotion observed with the groundwater exposure. Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species but cannot take into account the possible toxic effects of mixtures of compounds.

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.

Reactivity of tissue-specific antigens in N-methyl-N'-nitro-N-nitrosoguanidine-induced neoplasms and normal tissues from medaka (Oryzias latipes).

To further characterize the distribution of tissue-specific antigens in fish neoplasms, juvenile medaka were exposed to 30 mg/L of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 1 hr and allowed to grow out for up to 16 mo. Using a streptavidin peroxidase technique, keratin, vimentin, and neurofilament intermediate filament proteins, and actin and S-100 proteins were labeled in MNNG-induced neoplasms and normal medaka tissues using specific monoclonal or polyclonal antibodies. In vascular tumors, rhabdomyosarcoma, and teratoma, muscle tissues were positive for actin. Other sarcomas including hemangiopericytoma, fascial sarcoma, and undifferentiated sarcoma were negative for all antibodies tested. An unusual scale-associated neoplasm, composed of clusters of scale-forming cells surrounding spicules of scale, had keratin-positive stroma. The epithelial neoplasms were also positive for keratin, except for pancreatic acinar carcinoma, which had limited positivity. Both teratoma and olfactory carcinoma had S-100-positive intraepithelial cells morphologically reminiscent of neurosensory epithelial cells, which were S-100 positive in normal tissues. Although positive reactivity in fish tissues correlated with mammalian data, the antibodies used were raised against mammalian antigens. Therefore, a negative reaction may be indicative of lack of antibody sensitivity to specific fish antigens rather than absence of the antigen in the tissues. However, these data show that tissue-specific antigen detection may assist in elucidating the biology of neoplasia in fish.

N-methyl-N'-nitro-N-nitrosoguanidine-induced neoplasms in medaka (Oryzias latipes).

To test the sensitivity of the small fish species Oryzias latipes to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), medaka were exposed at 15 days of age to 30 mg/L for 1 hr and followed for up to 16 mo. One hundred neoplasms were diagnosed in 84 of 213 exposed fish, with approximately equal percentages in males and females. Many neoplasms (62%) were of mesenchymal origin and were categorized as blood vascular neoplasms (hemangioma and hemangiosarcoma), invasive sarcomas, and scale-associated neoplasms. Invasive sarcomas included rhabdomyosarcoma, fascial sarcoma, hemangiopericytoma, and undifferentiated sarcoma. A scale-associated neoplasm, termed lepidocytoma, was an unusual neoplasm of scale anlage. Thyroid follicular neoplasms, with a 100% incidence in males, and pancreatic acinar carcinoma were the most common epithelial tumors. Neoplasms of the gills, swim bladder, and olfactory epithelium were also seen as well as teratoma with mixed epithelial and mesenchymal components. The study showed a broad range of neoplasms induced by MNNG in medaka, with a tissue distribution that might support direct action on exposed tissues.

Hepatocyte clusters in the spleen: a normal feature of some populations of brown bullheads in New York State.

Since 1985, fish pathologists at Cornell University have studied lesions in freshwater fish inhabiting natural waters throughout New York State in order to clarify possible adverse effects of contaminants on fish health. In the course of these studies, we have conducted complete necropsies on over 400 brown bullheads and histologic examinations of the major organs of over 370 of these fish. In the course of our histologic studies, we observed islands of well-differentiated hepatocytes in the spleens of neoplasm-free brown bullheads from 2 of the 37 diverse lakes, rivers, and ponds that we sampled. The splenic hepatocyte island occurred in 11-100% of fish from the 2 affected sites, with 1-50 hepatocyte clusters per section of spleen. Hepatocyte islands measured up to 1 mm in diameter. Unlike the metastases of hepatocellular carcinomas, which we have described elsewhere in this issue (9), these islands of hepatocytes in the spleens of neoplasm-free fish were not evident grossly. These hepatocyte clusters in the spleen of certain populations of brown bullheads may lead to confusion in studies of neoplasia in brown bullheads. Caution is advised in interpretation of metastasis of liver neoplasms to the spleen of brown bullheads.

The riddle of hepatic neoplasia in brown bullheads from relatively unpolluted waters in New York State.

Since 1985, pathologists at Cornell University have investigated the causes of lesions in freshwater fish throughout New York waters in order to clarify possible impairment of fish health by environmental contaminants. Fishermen and biologists alerted us to several relatively protected reservoirs and ponds in which we have found no evidence of elevated levels of anthropogenic environmental contaminants but in which up to 100% of brown bullheads exhibited skin neoplasia. Complete necropsies and histologic study revealed that over 30% of mature brown bullheads from some of these sites had benign or malignant hepatocellular or biliary liver neoplasia. Up to 50% of brown bullheads had benign or malignant liver neoplasia in other relatively unpolluted waters with no evidence of skin neoplasia in bullheads. Multiple samplings of brown bullheads from several of these sites have revealed puzzling variability in the prevalence of skin, liver, and other neoplasia in these fish populations. The cause of these striking epizootics of neoplasia in brown bullheads in unpolluted waters in New York State remains unclear. We hypothesize that natural carcinogens such as N-nitroso compounds formed in aquatic sediments or radon from geologic formations may contribute to epizootic fish neoplasia in New York waters.

Naturally occurring orocutaneous papillomas and carcinomas of brown bullheads (Ictalurus nebulosus) in New York State.

Ninety-four brown bullheads (Ictalurus nebulosus) with spontaneous orocutaneous neoplasms (papillomas and carcinomas) were studied grossly and by light microscopy. Of these 94 fish, 71 were selected from 505 fish examined macroscopically during field surveys and 23 were selected from those submitted for diagnostic study. Fish with neoplasms came from 17 locations throughout New York State: Buffalo River, Canaan Lake, Cazenovia Lake, Delta Lake, Fort Pond, Greenwood Lake, Hudson River, Lake George, Lake Tiorati, Lincoln Hall Pond. Lincoln Pond, Oneida Lake, Onondaga Lake, Rutland Pond, Salmon River, Silver Stream Reservoir and Swan Lake. The prevalence varied from 0 to 100%. Multiple neoplasms were more common (84/94, 89%) than solitary ones (10/94, 11%). In order of decreasing frequency, neoplasms arose on the head, especially the ventral aspect, the lower dental plate, the upper dental plate, the trunk, the barbels, the fins, the tongue, and the tail. Of the 38/94 brown bullheads (40%) with tumors in both upper and lower lips and dental plates, 24/38 (63%) had the tumors in opposition. Macroscopically, soft, pink or yellowish papillary masses protruded above the normal epithelial surfaces. Histologically, cutaneous and oral neoplasms originated from the morphologically similar Malpighian epithelial cells of the surface epithelia and from the outer cells of the enamel organ. Based on histopathologic criteria, cutaneous and oral neoplasms were considered the same disease in different anatomic locations. No apparent difference in biologic behavior was noted between cutaneous and oral neoplasms. The lesions apparently progressed from benign papilloma to locally invasive carcinoma (28/94, 30%). Neoplastic emboli were seen in one case, and no metastases were detected. There was no statistically significant difference between the susceptibility of males (20/57, 35%) and females (6/26, 23%) to carcinomas. An apparent correlation was noted between a higher frequency of malignant tumors and longer body size (3/13 fish [23%] < 30 cm, 17/54 fish [31%] 30-34 cm, and 7/18 fish [39%] > 34 cm).

DNA alterations and enzyme activities in Japanese medaka (Oryzias latipes) exposed to diethylnitrosamine.

Several molecular and biochemical markers of genotoxicity were adapted for measurement in the medaka, and were used to describe the effects of treatment of the organism with diethylnitrosamine (DEN). DEN treatment inhibited the activity of a detoxication enzyme activity (ethoxyresorufin-O-deethylase) and increased the activity of glutathione-S-transferase. This pattern of response has been described in preneoplastic rodent cells. No O6-ethyl guanine adducts were detected, and a slight, but statistically significant, increase in DNA strand breaks was observed. These results are consistent with the hypothesis that prolonged exposure to high levels of DEN induced alkyltransferase activity which enzymatically removes any O6-ethyl guanine adducts but does not result in strand breaks or hypomethylation of the DNA such as might be expected from excision repair of chemically modified DNA.

Seasonal prevalence of skin tumors from walleye (Stizostedion vitreum) from Oneida Lake, New York.

A seasonal survey of skin tumor prevalence in walleyes (Stizostedion vitreum) was conducted during the ice-free period on Oneida Lake, New York in 1986. During the survey, 1,028 walleyes were collected and examined for the presence of lymphocystis disease, dermal sarcoma, discrete epidermal hyperplasia and diffuse epidermal hyperplasia. Skin growths were high in prevalence in early spring, low in prevalence during the summer, and again high in prevalence in the fall. Lymphocystis disease and dermal sarcoma were more frequently observed than either discrete or diffuse epidermal hyperplasia. Histologically, a moderate to severe inflammatory response was associated with dermal sarcoma in the early spring and late spring but not in the fall. Regardless of the time of year, varying degrees of inflammatory response were seen associated with lymphocystis disease. Samples were inadequate to assess seasonal trends in incidence of discrete and diffuse epidermal hyperplasia.

A lymphosarcoma in an Atlantic salmon (Salmo salar).

A lymphosarcoma that appeared to be of thymic origin and of lymphoblastic type was found in a 3.5-yr-old Atlantic salmon (Salmo salar). The fish was from a population of 60 broodfish maintained at a research fish laboratory. A large tumor mass was found under the left operculum. Small tumor nodules were found on the swim bladder and in the abdominal adipose tissue. The location of this neoplasm differed from those of previously described tumors in this fish species.

The comparative pathology of non-viral bullous skin diseases in domestic animals.

In a review of non-viral bullous skin diseases of domestic animals and a 4-year study of cases presented to the New York State College of Veterinary Medicine, we found 15 diseases: pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus, pemphigus erythematosus, bullous pemphigoid, systemic lupus erythematosus, dermatitis herpetiformis, toxic epidermal necrolysis, drug eruption, epidermolysis bullosa, epidermolysis bullosa simplex, familial acantholysis, bovine congenital porphyria, impetigo and subcorneal pustular dermatosis. The 15 diseases were placed in five categories: autoimmune, immune-mediated, hereditary, bacterial and idiopathic. A histologic classification of these disorders based on the site of blister formation and other important clinicopathologic, histologic and immunopathologic findings was developed.