Glutathione S-transferase expression in pollution-associated hepatic lesions of brown bullheads (Ameiurus nebulosus) from the Cuyahoga River, Cleveland, Ohio.

In rodents, overexpression of glutathione S-transferase pi is a characteristic feature of foci of cellular alteration (FCA) and neoplastic liver lesions induced by genotoxic chemicals. Alterations of glutathione S-transferase (GST) expression in hepatic lesions have also been reported in fish exposed to environmental carcinogens, and cellular GST expression may be an important determinant of growth and progression of chemical-associated liver tumors in certain fish species. In the present study, GST expression was examined in hepatic lesions of brown bullheads (n = 44) from the Cuyahoga River, a highly industrialized site located in Cleveland, Ohio. GST proteins were detected by immunohistochemistry and polyclonal antibodies that recognize either two major bullhead GST proteins or a pi-like GST isoform. Hepatic lesions were present in 70% of the fish and included biliary hyperplasia and biliary fibrosis; eosinophilic, basophilic, clear cell, and vacuolated FCA; and biliary neoplasms. Eosinophilic FCA and biliary tumors were the most prevalent preneoplastic and neoplastic lesions. GST expression in hyperplastic biliary tissue, FCA and tumors did not markedly differ from that of surrounding normal hepatocytes or biliary epithelium. Some hepatocytes within eosinophilic FCA had decreased GST expression. A complete absence of GST immunoreactive protein was not observed in any lesion, and there were no marked differences when comparing GST pi to overall GST expression. Our results indicate that GST expression in hepatic lesions of brown bullhead exposed to environmental carcinogens does not significantly differ from that in surrounding normal cells and is therefore not a useful predictor of environmental carcinogenesis in this species. Furthermore, the regulation and expression of GST pi in bullhead hepatocarcinogenesis appears to differ markedly from that during hepatocarcinogenesis in rats and some other fish species.

Induction of glutathione S-transferase activity and protein expression in brown bullhead (Ameiurus nebulosus) liver by ethoxyquin.

The inducibility of hepatic cytosolic glutathione S-transferases (GSTs) was examined in brown bullheads, a freshwater fish that is highly susceptible to hepatic neoplasia following exposure to carcinogen-contaminated sediments. Juvenile bullheads were fed a semi-purified antioxidant-free diet supplemented with ethoxyquin (0.5% w/w dissolved in 3% corn oil), a prototypical rodent GST-inducing agent, twice daily for 14 days. Control bullheads received the antioxidant-free diet supplemented with corn oil (3% w/w). A significant increase (1.6-fold, p < or = 0.01) in hepatic cytosolic GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) was observed in the ethoxyquin-treated bullheads relative to control fish. A trend toward increased GST-NBC activity was observed in the ethoxyquin-treated fish (1.2-fold, p = 0.06), whereas no treatment-related effects were observed on GST activities toward ethacrynic acid (ECA). In contrast, GST activity toward (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) was repressed in affinity-purified cytosolic fractions prepared from ethoxyquin-treated bullheads relative to control bullheads. Silver staining and densitometric analysis of isoelectric-focused, affinity-purified GST proteins revealed increased expression of two basic GST-like isoforms in ethoxyquin-treated fish. In summary, exposure to ethoxyquin increases brown bullhead GST-CDNB catalytic activity and hepatic cationic GST protein expression. However, the increase in overall GST-CDNB activity by ethoxyquin is associated with repression of GST-BPDE activity, suggesting differential effects on hepatic bullhead GST isoforms by ethoxyquin. The potential repression of bullhead GST isoforms that conjugate the carcinogenic metabolites of PAH metabolism under conditions of environmental chemical exposure could be a contributing factor in the sensitivity of bullheads to pollutant-associated neoplasia.

Conservation of a glutathione S-transferase in marine and freshwater fish.

We have previously reported the isolation and cloning of glutathione S-transferase (GST) cDNAs from two marine fish, English sole (Pleuronectes vetulus) and starry flounder (Platichthys stellatus), that exhibited > 95% identity to plaice (Pleuronectes platessa) GST-A Aquatic Toxicol., 44, 171-182]. In the present study, we have used reverse transcription-polymerase chain reaction (RT-PCR) analysis to isolate a 471 nucleotide GST-like cDNA from largemouth bass (Micropterus salmoides) liver. Sequence identity of the largemouth bass partial cDNA to plaice GST-A was approximately 90%. Northern blotting analysis using the partial GST cDNA from English sole as a probe detected a single band of approximately 1 kb in English sole liver and a slightly larger GST-like band that was highly expressed in largemouth bass liver. In addition, a faint band of similar size was recognized in brown bullhead (Ameriurus nebulosus) liver, but not in channel catfish (Ictalurus punctatus) liver. In conclusion, we have extended our studies of GST expression in flatfish and have isolated an additional GST-A-like cDNA from a largemouth bass. Conservation of a GST-A like cDNA among certain marine flatfish and freshwater species suggests an important function for this gene.

Regression of subcutaneous lymphoma following removal of an ovarian granulosatheca cell tumor in a horse.

A 9-year-old Arabian mare was admitted for evaluation of multiple subcutaneous nodules and infertility. Fine-needle aspiration of one of the subcutaneous nodules resulted in a cytologic diagnosis of histiolymphocytic lymphoma. Palpation per rectum and transrectal ultrasonography revealed a mass associated with the left ovary. Excision of the ovarian tumor was performed, and a histopathologic diagnosis of granulosa-theca cell tumor was made. After removal of the granulosa-theca cell tumor, subcutaneous nodules regressed. The referring veterinarian reported that the nodules had also disappeared and then recurred after administration of a synthetic progestin. To further characterize the lymphoma and investigate this possible hormonal relationship, immunophenotyping and estrogen and progesterone receptor assays were performed. The subcutaneous lymphoma was classified as a T-cell rich B-cell lymphoma, results of estrogen receptor assays were negative, and results of progesterone receptor assays were positive. Clinical observations of subcutaneous lymphoma in horses indicate that the waxing and waning nature of these tumors may be associated with the estrous cycle, pregnancy, foaling, and lactation. Clinical observations and identification of progesterone receptors suggest that a relationship between serum steroid hormone concentrations, such as estrogen and progesterone, and subcutaneous lymphoma may exists.