Tumors in fish from the Detroit River

A fish tumor survey was conducted in the Lower Detroit River during 1985 to 1987. Five species of fish were collected from sediment deposition zones and were examined for neoplasia. Neoplasms and related lesions were found in bullhead (Ictalurus nebulosus), walleye (Stizostedion vitreum), redhorse sucker (Moxostoma, sp), white sucker (Catostomus commersoni), and bowfin (Amia calva). Overall, 8.2 percent of the fish examined had oral/dermal lesions and 10.1 percent had liver lesions. Liver and skin tumors were found to be age-related in bullheads and size-related in walleye. Based on the fish surveyed, the incidence of tumors in fish from the Detroit River was similar to that observed in other chemically contaminated waterways.     

The use of tumors in wild populations of fish to assess ecosystem health

Evidence has linked toxicants in aquatic systems with cancer in fish and population level effects on species. Thus some types of tumors may be useful monitors of ecosystem health, at least as affected by genotoxins and promoters. However, tumors caused by purely genetic mechanisms or by virus would not be good indicators. Only neoplasms which have chemicals as a portion of their etiology (either as initiators or promoters) would be useful in assessing ecosystem health. Lesions which may fit these criteria include liver neoplasms (both biliary and hepatic) and skin lesions in a variety of primarily benthic fishes, and neural lesions in various drum species and in butterfly fish species. Two studies purporting to demonstrate a lack of tumors in fish from polluted areas have been reexamined and found either to have insufficient data on vulnerable species or to actually support a tumor-pollution linkage. Thus certain lesions in vulnerable species or species groups may serve as a mechanism to assess one facet of ecosystem health.     

Metalloproteinases 2 and -9 activity during promotion and progression stages of rat liver carcinogenesis

Activity of metalloproteinases 2 and 9 (MMP-2 and 9) during promotion and progression of rat liver carcinogenesis was investigated in a modified resistant hepatocyte model. Development of preneoplastic liver lesions positive for glutathione S-transferase 7-7-(GST-P 7-7-positive PNL) and tumors besides hepatocytes positive for proliferating cell nuclear antigen (PCNA) were quantified and compared to MMP-2 and-9 activity using gelatin zymography. Marked increases in GST-P 7-7-positive PNL development, PCNA labeling indices, MMP-2 (pro, intermediate and active forms) and pro-MMP-9 activity were observed after proliferative stimulus induced by 2-acetylaminofluorene (2-AAF) exposure cycles. After 2-AAF withdrawal, increase in MMP-2 activity was detected only in neoplastic mixed lesions, whereas active MMP-9 was increased in both PLN and neoplastic tissues. Our findings suggest that MMP-2 may be associated with proliferative events induced by 2-AAF rather than with selective growth of PNL and that MMP-9 could be associated with progression of PNL and neoplastic mixed lesions.     

Establishing the Causal Relationship between Polycyclic Aromatic Hydrocarbon (PAH) Exposure and Hepatic Neoplasms and Neoplasia-Related Liver Lesions in English Sole (Pleuronectes vetulus)

For almost 25 years our laboratory has studied the impact of PAHs and related industrial contaminants on benthic fish, following an interdisciplinary approach involving chemical exposure assessment linked to synoptic detection of various effects at several levels of biological organization. These data demonstrate a cause-and-effect relationship between neoplastic and neoplasia-related liver lesions in English sole, and exposure to PAHs, and to a lesser degree, chlorinated hydrocarbons such as PCBs. In statistical analyses of data from multiple field studies conducted since 1978, exposure to PAHs measured in various compartments has consistently been identified as a highly significant, major risk factor for neoplasms and related lesions in this species, with PCB exposure shown to be a significant, but less consistent and less strong risk factor for these lesions. A cause-and-effect relationship between PAHs and toxicopathic liver lesions in this species is further supported by the experimental induction of toxicopathic lesions identical to those observed in field-collected fish, in sole exposed in the laboratory to model carcinogenic PAHs such as BaP or to PAH-rich extracts of sediments from Eagle Harbor, a severely PAH-contaminated site in Puget Sound. More recent field studies have identified significant associations between hepatic cytochrome P4501A (CYP1A) induction and xenobiotic-DNA adduct formation, and hepatic lesion prevalences in wild subadult English sole. Field studies in Eagle Harbor subsequent to capping of the most PAH-contaminated region of this harbor with clean dredge spoils have shown a decline in exposure to PAHs as assessed by biliary fluorescent aromatic compounds (FACs) and hepatic xenobiotic-DNA adducts. This decline in PAH exposure has been accompanied by a dramatic decline in risk of occurrence of toxicopathic hepatic lesions in English sole from Eagle Harbor. Further, laboratory studies have induced lesions in English sole by injections of extracts from PAHcontaminated sediments. Overall, these findings relating to exposure to PAHs and chlorinated hydrocarbons and the occurrence of hepatic neoplasms and neoplasiarelated lesions in English sole fulfill the classic criteria for causality in epizootiological or ecological risk assessment studies, including: (1) strength of association, (2) consistency of association, (3) specificity of association, (4) toxicological and biological plausibility, (5) temporal sequence/timing (i.e., exposure precedes disease, effect decreases when the cause is decreased or removed), (6) dose-response or biological gradient, and (7) supportive experimental evidence.     

Analysis of fish bile with HPLC — fluorescence to determine environmental exposure to benzo(a)pyrene

Brown bullhead from the Black River, Ohio, have a high incidence of liver neoplasia which is associated with elevated concentrations of polynuclear aromatic hydrocarbons (PAHs) in the sediment. We evaluated the use of biliary concentrations of benzo(a)pyrene [B(a)P] equivalents as a means for determining PAH exposure. Bile was collected from 16 brown bullheads and 8 common carp taken from each of two Lake Erie tributaries in Ohio, the industrialized Black River and the non-industrialized Old Woman Creek. Hatchery bullhead (n = 8) were used to determine base levels of PAHs. A high performance liquid chromatography (HPLC) — fluorescence technique was used to determine the concentration of B(a)P equivalents in the bile samples. The area of all peaks fluorescing at 380/430 nm was summed to give a single value for B(a)P equivalents in each sample. Concentrations of B(a)P equivalents generally reflected concentrations of PAH in sediment where fish were collected. Bile taken from Black River carp contained the highest concentration of B(a)P equivalents and was significantly different from all other groups. The value obtained for Black River bullhead was also high and was found to be significantly different from hatchery bullhead. B(a)P equivalents varied between carp and bullhead from the same habitat possibly because of differing food habits or metabolic pathways. However, our results indicate that relative levels of B(a)P equivalents in the bile of fish correspond well to B(a)P levels in sediment and may offer a means of determining environmental exposure of fish to the parent compound.     

Altered expression of the xenobiotic transporter P-glycoprotein in liver and liver tumours of mummichog Fundulus heteroclitus from a creosote-contaminated environment

P-glycoproteins (Pgps) are involved in efflux of xenobiotics from drug-resistant cell lines and tumours, and in excretion of toxicants from normal tissues. Recently, investigators have proposed that Pgp activity contributes to resistance or tolerance of certain aquatic species to pollutants. In the present study using immunoblot and immunohistochemical techniques, we found elevation of Pgp in liver and liver tumours of creosote-resistant mummichog from a contaminated site in the Elizabeth River, Virginia. Immunoblots of mummichog liver extracts showed an immunoreactive band at 170 kDa and indicated two- to three-fold elevation of Pgp in livers of resistant fish relative to those from a reference site. Laboratory exposures of reference site fish to a model PAH (3-methylcholanthrene), however, produced no increase in liver Pgp levels as measured by immunoblot. Normal mummichog liver sections showed specific immunohistochemical staining for Pgp on the canalicular surface of hepatocytes. In the majority of hepatic neoplasms we observed a high level of over-expression and altered patterns of Pgp expression. However we did not observe Pgp over-expression in early proliferative lesions. Elevation of Pgp in livers and liver tumoursof these resistant mummichog may contribute to their survival in a heavily contaminated environment.     

Altered expression of the xenobiotic transporter P-glycoprotein in liver and liver tumours of mummichog Fundulus heteroclitus from a creosote-contaminated environment

P-glycoproteins (Pgps) are involved in efflux of xenobiotics from drug-resistant cell lines and tumours, and in excretion of toxicants from normal tissues. Recently, investigators have proposed that Pgp activity contributes to resistance or tolerance of certain aquatic species to pollutants. In the present study using immunoblot and immunohistochemical techniques, we found elevation of Pgp in liver and liver tumours of creosote-resistant mummichog from a contaminated site in the Elizabeth River, Virginia. Immunoblots of mummichog liver extracts showed an immunoreactive band at 170 kDa and indicated two- to three-fold elevation of Pgp in livers of resistant fish relative to those from a reference site. Laboratory exposures of reference site fish to a model PAH (3-methylcholanthrene), however, produced no increase in liver Pgp levels as measured by immunoblot. Normal mummichog liver sections showed specific immunohistochemical staining for Pgp on the canalicular surface of hepatocytes. In the majority of hepatic neoplasms we observed a high level of over-expression and altered patterns of Pgp expression. However we did not observe Pgp over-expression in early proliferative lesions. Elevation of Pgp in livers and liver tumoursof these resistant mummichog may contribute to their survival in a heavily contaminated environment.     

Histomoniasis and reticuloendotheliosis in a wild turkey (Meleagris gallopavo) in North Carolina

A moribund wild turkey (Meleagris gallopavo) died shortly after it was discovered in Martin County, North Carolina (USA). The 4.3-kg female turkey appeared in good condition with no visible external lesions or evidence of injury. There were 2- to 5-mm yellow-white plaques on the mucosal surfaces of the oral cavity and mid-esophagus. The liver had large, multifocal, irregular pale areas on cut and uncut surfaces. The spleen contained multifocal, pale, hard, nodules. Microscopic changes in the liver consisted of large multifocal coalescing areas of necrosis. Occasional spherical 10 to 15 microns in diameter organisms consistent with Histomonas meleagridis were present in the necrotic areas. Viable hepatic parenchyma contained multifocal infiltrations of numerous mononuclear cells interpreted as neoplastic cells resembling lymphoblasts and plasma cells. Similar neoplastic cell infiltrates, consistent with the lymphoproliferative disease reticuloendotheliosis, were present in spleen, lung, and esophageal and oral mucosa. Reticuloendotheliosis virus, subtype 2, was isolated from samples of liver and spleen.     

Histopathological study on six cases of calf leukosis.

A pathological study was carried out on six calves 4 to 10 months of age affected with lymphoid tumors. Most of the lymph nodes enlarged in consequence of leukotic changes in all the calves. These changes occurred also in other various organs. When the leukotic lesions were investigated to clarify the distribution and histological manifestation, three pathological patterns were discriminated among them. One of the patterns was seen in four cases, in which leukotic lesions were present constantly in bone marrow, thymus, liver and kidney. In the lymph nodes, tonsils, and intestinal lymphatic apparatus, neoplastic cellular proliferation took place in paracortical or interfollicular areas and medullary cords obliterating the lymph follicles. Leukotic involvement was observed in the interstitial and the vascular connective tissue in the thymus, as well as in liver and kidney. A second pattern of lesion was revealed in one of the other two cases. Besides lymph nodes in which neoplastic proliferation was seen in the paracortical area, only the thymus manifested intralobular neoplastic involvement in this case. In the remaining case, the leukotic lesion was characterized by the presence of neoplastic cellular masses resembling large lymph follicles in appearance in the lymphatic tissues. It was manifested distinctly in the spleen. Severe thymic atrophy and granulocytic hyperplasia in bone marrow were present in this case.     

Biochemical and toxicopathic biomarkers assessed in smallmouth bass recovered from a polychlorinated biphenyl-contaminated river

Smallmouth bass (Micropterus dolomieu) were collected to quantify the nature and prevalence of biomarker responses, including biochemical indices, toxicopathic lesions and general health indices, among fish collected from polychlorinated biphenyl (PCB)-contaminated and nearby uncontaminated reaches of the Kalamazoo River, Michigan, USA. Blood and tissue samples (gill, liver, spleen, head kidney, trunk kidney, thyroid and gonads) were collected and preserved at necropsy for biochemical and histological analyses. The body condition factor and liver somatic index were significantly lower in fish collected from the downstream, contaminated site. Plasma vitellogenin was not detected in male fish collected from either site. Liver ethoxyresorufin-O-deethylase activity and liver and spleen superoxide dismutase activity were significantly depressed in fish collected from the downstream site. Significant toxicopathic lesions such as glycogen depletion, enhanced macrophage aggregates, hepatic foci of cellular alteration (i.e. preneoplastic lesions) and neoplasia were also detected in the liver of fish collected from the downstream site. This study indicates that many of the biochemical and histopathological biomarker responses were associated with liver and body tissue PCB concentrations. Taken together, the biomarkers of exposure and effect strongly suggest that fish within the downstream site are adversely affected by PCBs and other chemical stressors.     

Biochemical and toxicopathic biomarkers assessed in smallmouth bass recovered from a polychlorinated biphenyl-contaminated river

Smallmouth bass (Micropterus dolomieu) were collected to quantify the nature and prevalence of biomarker responses, including biochemical indices, toxicopathic lesions and general health indices, among fish collected from polychlorinated biphenyl (PCB)-contaminated and nearby uncontaminated reaches of the Kalamazoo River, Michigan, USA. Blood and tissue samples (gill, liver, spleen, head kidney, trunk kidney, thyroid and gonads) were collected and preserved at necropsy for biochemical and histological analyses. The body condition factor and liver somatic index were significantly lower in fish collected from the downstream, contaminated site. Plasma vitellogenin was not detected in male fish collected from either site. Liver ethoxyresorufin-O-deethylase activity and liver and spleen superoxide dismutase activity were significantly depressed in fish collected from the downstream site. Significant toxicopathic lesions such as glycogen depletion, enhanced macrophage aggregates, hepatic foci of cellular alteration (i.e. preneoplastic lesions) and neoplasia were also detected in the liver of fish collected from the downstream site. This study indicates that many of the biochemical and histopathological biomarker responses were associated with liver and body tissue PCB concentrations. Taken together, the biomarkers of exposure and effect strongly suggest that fish within the downstream site are adversely affected by PCBs and other chemical stressors.     

The buffering power of plasma in brown bullhead (Ameiurus nebulosus)

Brown bullhead (Ameiurus nebulosus) blood plasma was found to exhibit an unusually high non-bicarbonate buffer capacity (beta) in relation to that of other teleost fish. In brown bullhead, the non-bicarbonate buffer capacity of plasma (beta(plasma)), at -5.72 +/- 0.34 mmol l(-1) pH unit(-1) (mean +/- S.E.M., N=30), constituted 37% of whole blood beta and was 2.5 times higher than beta(plasma) in rainbow trout (-2.33 +/- 0.42 mmol l(+/-1) pH unit(-1); N=7). The strong buffering power of bullhead plasma was not the result of unusually high plasma protein levels. Size separation chromatography in conjunction with a spectrophotometric assay for buffering capacity were used to isolate a plasma fraction of high buffering power. SDS-polyacrylamide gel electrophoresis revealed that this fraction contained four proteins, but was dominated by a protein of approximately 68-70 kDa molecular mass. On the basis of the amino acid composition of this fraction, the dominant protein was identified as albumin. In comparison to other fish albumins, bullhead albumin appears to be histidine-rich (6.7%). Thus, the unusually high non-bicarbonate buffer capacity of bullhead plasma appears to stem from the presence in the plasma of a histidine-rich albumin.     

Comparative metabolism of benzo[f]quinoline by liver microsomes from brown bullheads and rats

1. Liver microsomes from rats were considerably more active in metabolizing benzo[f]quinoline (B f Q) than those from brown bullheads (Ictalurus nebulosus). 2. The main B f Q metabolites formed by both rat and brown bullhead liver microsomes were qualitatively similar and included B f Q-7,8-dihydrodiol, B f Q-9,10-dihydrodiol, B f Q-N-oxide, 7-hydroxy B f Q, and 9-hydroxy B f Q. 3. The liver microsomes from control brown bullheads and rats metabolized B f Q primarily at the 7,8-and 9,10-positions, respectively, whereas in the case of microsomes from 3-methylcholanthrene (3-MC)-treated rats or brown bullheads, the major site of metabolic attack was the 7,8-position. 4. A 3-MC-type of cytochrome P-450 appears to be primarily responsible for the oxidation of B f Q by control brown bullhead liver microsomes, whereas a phenobarbital-inducible type of cytochrome P-450 seems to be involved in the metabolism of B f Q by control rat liver microsomes.     

Fine needle aspiration biopsy of metastatic melanoma. A morphologic analysis of 174 cases

The prognostic and therapeutic decisions in cases of metastatic melanoma depend upon the morphologic documentation of metastatic disease, which may rapidly and accurately be done by fine needle aspiration (FNA) biopsy of clinically suspicious lesions. The tumor cells derived from malignant melanomas demonstrate a wide range of appearances, however, and other neoplasms may be mimicked. Furthermore, additional neoplasms of other types are more frequent in melanoma patients: the possibility of a new primary tumor must be considered if the morphology of the tumor cells is uncharacteristic. Therefore, a study was undertaken to analyze the morphologic changes seen in FNA biopsy specimens from metastatic malignant melanoma and to determine which features could be the most useful in establishing a definitive diagnosis. A total of 174 consecutive cases, comprising 151 malignant aspirates and 23 inconclusive aspirates, were reviewed. The most significant features for identification of melanoma over other tumor types were the cell shape and nuclear position, the presence of numerous isolated neoplastic cells and occasional binucleated or multinucleated cells. Intracellular melanin in neoplastic cells was diagnostic when present, but it was absent in 60% of the cases. Macronucleoli and/or intranuclear cytoplasmic invaginations were characteristic but variable features. Morphology was also found to vary by site and cell type. Lung aspirates were less cellular and more likely to contain melanin. Aspirates of subcutaneous nodules were more often composed of spindle-shaped cells or of other variant cell types. Lymph node aspirates more often yielded epithelioid cells with macronucleoli and/or intranuclear invaginations. Spindle-cell melanomas usually demonstrated inconspicuous nuclei and rarely showed enlarged nucleoli. Epithelioid-cell tumors contained multinucleated cells and areas of cell wrapping more frequently than did spindle-cell tumors. The findings in this study emphasize that a full awareness of the spectrum of morphologic presentations of metastatic melanoma as well as of the clinical history are needed for greater precision in its diagnosis and for avoidance of the pitfall of misdiagnosing nonmelanomas with similar appearances.     

Fine needle aspiration of the liver and pancreas with ultrasound guidance

The experience of the Institut Gustave-Roussy in the diagnosis of hepatic and pancreatic lesions by fine needle aspiration (FNA) is reported. Totals of 116 consecutive percutaneous ultrasound-guided FNAs of the liver and 27 of the pancreas were performed without complication in patients with ultrasonically suspected neoplastic lesions. In 12 cases, the material was not suitable for diagnosis. In the liver, 97 cases were correctly diagnosed and confirmed by follow-up. Immunohistologic studies were helpful in distinguishing primary liver tumors from other malignancies. One false-positive result was reported. In the pancreas, malignancy was detected in 17 cases. Cytology alone provided the correct tumor diagnosis in 15 cases: 10 primary carcinomas, 2 endocrine tumors and 3 metastases. The sensitivities of FNA in this study were 87.6% for the liver and 85% for the pancreas, similar to those reported in other series.     

Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study

Fish play host to viral, bacterial, and parasitic diseases in addition to non-infectious conditions such as cancer. The National Marine Monitoring Programme (NMMP) provides information to the U.K. Government on the health status of marine fish stocks. An aspect of this work relates to the presence of tumors and other pathologies in the liver of the offshore sentinel flatfish species, dab (Limanda limanda). Using internationally agreed quality assurance criteria, tumors and pre-tumors are diagnosed using histopathology. The current study has expanded upon this work by integrating these traditional diagnostic approaches with ones utilizing modern technologies for analysis of proteomic and metabolomic profiles of selected lesions. We have applied SELDI and FT-ICR technologies (for proteomic and metabolomic analyses, respectively) to tumor and non-tumor samples resected from the liver of dab. This combined approach has demonstrated how these technologies are able to identify protein and metabolite profiles that are specific to liver tumors. Using histopathology to classify "analysis groups" is key to the success of such an approach since it allows for elimination of spurious samples (e.g., those containing parasite infections) that may confuse interpretation of "omic" data. As such, the pathology laboratory plays a central role in collating information relating to particular specimens and in establishing sampling groups relative to specific diagnostic questions. In this study, we present pilot data, which illustrates that proteomics and metabolomics can be used to discriminate fish liver tumors and suggest future directions for work of this type.     

Involvement of hepatocyte growth factor on hepatocarcinogenesis induced by peroxisome proliferators

Hepatocyte growth factor (HGF) has been known to enhance the growth of normal hepatocytes, but also to inhibit the growth of neoplastic cells. This article examines the involvement of HGF in the hepatocarcinogenesis caused by peroxisome proliferators (PPs). Up to 78 wk after male F-344 rats were orally given (4-chloro-6-[2,3-xylidino]-2-pyrimidinylthio) acetic acid (Wy-14,643), the hepatocarcinomas and (pre)neoplastic nodules in the livers were observed. At that time, the content of HGF and the expression of HGF mRNA in the liver tumors were significantly decreased. These changes were observed also in the liver of rats treated with other PPs, such as dehydroepiandrosterone and di(2-ethylhexyl)phthalate, but were not observed in tumors induced by genotoxic carcinogens (diethylnitrosamine-phenobarbital). In in vivo experiments, the formation of preneoplastic lesions and the tumors caused by Wy-14,643 administration were markedly suppressed by iv-injection of HGF in a dose-dependent manner. In the colony assay using (pre)neoplastic cells from livers of Wy-14,643-treated rats, HGF inhibited the colony formation of (pre)neoplastic cells in a dose-dependent manner. These findings may indicate that decreases in hepatic HGF levels are common and specific events induced by PPs, but not by genotoxic carcinogens, and that those changes play an important role in the promotion of neoplastic or preneoplastic cell growth induced by PPs.     

Seasonal changes in territoriality and frequency of agonistic behavior in two densities of juvenile brown bullhead,Ictalurus nebulosus

Synopsis Incidence of territoriality and frequency of six agonistic action patterns of juvenile brown bullhead, Ictalurus nebulosus, were observed in the laboratory over a two year period at two fish densities under conditions of constant light and temperature. High density fish showed little territoriality or agonistic behavior while low density fish displayed an annual cycle of agonistic behavior and territoriality. Agonistic behavior frequency and incidence of territoriality peaked in February and March and were higher the second year when compared with the first.     

Histopathological definition of the adult and calf types of bovine leukosis

Histopathological distinctions were examined on 36 cattle 2 months to 12 years of age affected with leukosis. The distribution of tumors pointed out previously as the characteristic of the adult type of leukosis was observed in 27 adult cases, which, except two, were positive for the immunodiffusion test of bovine leukemia virus (BLV). The noticeable development of massive and lumpy tumors in serosa, loose connective tissue, and lymph nodes seemed to suggest a neoplastic accumulation in areas related to the lymphatic way. Splenic involvement and intrasinusoidal leukotic immigration in the liver were present in cases with the leukemic manifestation of peripheral blood. The neoplastic involvement of bone marrow, however, was difficult to find. On the other hand, in 5 calves and 4 adolescent or adult cattle, the neoplastic proliferation was observed throughout such hematopoietic organs as lymph nodes, bone marrow, liver, spleen, thymus and tonsil. The hepatic lesion was characterized by the neoplastic proliferation in the portal triads No BLV antibody could be detected from these cattle, except one adult. As the conclusion of the present study, the tumor of adult type regarded as the pathologic manifestation of enzootic bovine leukosis is defined as malignant lymphoma (non-Hodgkin's lymphoma or lymphosarcoma) of undifferentiated-, histiocytic-, or poorly- differentiated lymphocytic- cell type. And lymphocytic leukemia seems to be desirable term to distinguish the lymphoid malignancy regarded as the characteristic of the calf type from that of the adult type.