Concentration of lipocalin region of collagen XXVII alpha 1 in the serum of dogs with hemangiosarcoma.

BACKGROUND: Hemangiosarcoma (HSA) is a common malignancy of dogs with characteristic early, aggressive metastasis. Diagnosis of HSA is challenging because of lack of sensitive and specific diagnostic tests. HYPOTHESIS: Specific proteins that are increased in serum of dogs with HSA might represent useful biomarkers of the disease. ANIMALS: Thirty-four dogs with HSA and 42 healthy dogs from the Ontario Veterinary College Teaching Hospital. METHODS: This case-control study compared serum proteins in dogs with HSA and healthy dogs. Proteins were separated by 2-dimensional difference gel electrophoresis and identified by liquid chromatography and tandem mass spectrometry. RESULTS: Western blot analysis showed that serum collagen XXVII peptide concentration in serum of dogs with large metastatic HSA burdens (1,488, 231-3,754 DU; median, minimum-maximum); was, on average, 9.5-fold higher than in healthy dogs (156; 46-2,101 DU). While concentrations for dogs with osteosarcomas (678; 124-3,251 DU), lymphomas (423; 92-2,777 DU), carcinomas (1,022; 177-3,448 DU), and inflammatory disease were also increased, values were consistently lower than those for HSA. Receiver operating characteristic curves revealed an estimated area under the curve of 83% for HSA cases whereas areas for other neoplastic and nonneoplastic diseases were nondiscriminatory. Serum collagen XXVII peptide concentration before splenectomy (1,350; 1,156-1,929 DU) was reduced after tumor removal (529; 452-562 DU) and chemotherapy but increased in 2 dogs with tumor recurrence (511-945 DU; 493-650 DU). CONCLUSIONS AND CLINICAL IMPORTANCE: Collagen XXVII peptide might be useful for diagnosis and monitoring of advanced HSA.

Proteomic analysis of ovomucoid hypersensitivity in mice by two-dimensional difference gel electrophoresis (2D-DIGE).

There is a need to develop reliable methods to assess the safety of genetically modified and other novel foods. The aim of this study was to identify protein biomarkers of food allergy in mice exposed to ovomucoid (OVM), a major food allergen found in chicken egg white. BALB/c mice were repeatedly sensitized by gavage with OVM and cholera toxin (CT) and control mice were exposed to a mixture of amino acids with CT. At the endpoint, all mice were challenged intraperitoneally with OVM and alum. Type-1 hypersensitivity was confirmed in OVM-sensitized mice by observation of clinical signs of anaphylaxis and elevated levels of plasma histamine, OVM-specific IgE and OVM-specific IgG by ELISA. Differential protein expression was assessed in albumin-depleted plasma as well as in mesenteric lymph node, liver, spleen, and ileum by two-dimensional difference gel electrophoresis (2D-DIGE). Differentially expressed proteins were identified by liquid chromatography with tandem mass spectrometry. Plasma proteins overexpressed in OVM-sensitized mice included haptoglobin (41-fold), serum amyloid A (19-fold) and peroxiredoxin-2 (1.9-fold). Further validation of these plasma proteins in other animal models of food allergy with different food allergens is required to assess their potential as candidate biomarkers for use in evaluating the allergenicity of novel foods.

Gender-dependent reductions in vertebrae length, bone mineral density and content by doxorubicin are not reduced by dexrazoxane in young rats: effect on growth plate and intervertebral discs.

Doxorubicin (DOX) is widely used in anti-cancer cocktails. Dexrazoxane (DXR) is a cardioprotectant approved for use with DOX. The effect of DOX, with or without DXR, on bone in children is not well understood. The aim of this study was to examine the effect of DOX on vertebrae and femur length and bone density acquisition in young rats, as well as to test the hypothesis that young females are more susceptible to DOX-induced tissue damage than young males. The results of this study suggest that a single injection of DOX in young female and not male rats is associated with low bone turnover resulting in vertebrae and femur bone growth deficits. DOX selectively decreased BMD and BMC accrual in the lumbar vertebrae that was not prevented by DXR. DOX-treated rats also exhibited growth plate and intervertebral disc defects. This information will be useful in the design of interventions to promote bone growth or retard bone loss during DOX treatment.

Identification of a 43-kDa protein in human liver cytosol that binds to the 3'-untranslated region of CYP2A6 mRNA.

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.

Diminished molecular response to doxorubicin and loss of cardioprotective effect of dexrazoxane in Egr-1 deficient female mice.

Doxorubicin (DOX) and VP16 are DNA topoisomerase II inhibitors yet only DOX induces an irreversible cardiotoxicity, likely through DOX-induced oxidative stress. Egr-1 is overexpressed after many stimuli that increase oxidative stress in vitro and after DOX-injection into adult mice in vivo. To investigate Egr-1 function in the heart, we compared the molecular and histological responses of wild type (+/+) and Egr-1 deficient (-/-) female mice to saline, DOX, VP16, the cardioprotectant dexrazoxane (DZR), or DOX+DZR injection. DOX, and to a lesser extent VP16, induced characteristic increases in cardiac muscle and non-muscle genes typical of cardiac damage in +/+ mice, whereas only beta-MHC and Sp1 were increased in -/- mice. DZR-alone treated +/+ mice showed increased cardiomyocyte transnuclear width without a change to the heart to body weight (HW/BW) ratio. However, DZR-alone treated -/- mice had an increased HW/BW, increased cardiomyocyte transnuclear width, and gene expression changes similar to DOX-injected +/+ mice. DZR pre-injection alleviated DOX-induced gene changes in +/+ mice; in DZR+DOX injected -/- mice the increases in cardiac and non-muscle gene expression were equal to, or exceeded that, detected after DOX-alone or DZR-alone injections. We conclude that Egr-1 is required for DOX-induced molecular changes and for DZR-mediated cardioprotection.

Allele-specific PCR analysis of p53 codon 249 AGT transversion in liver tissues from patients with viral hepatitis.

AGG to AGT mutations in codon 249 of the p53 tumor-suppressor gene are frequently observed in hepatocellular carcinomas (HCC) from areas where exposure to aflatoxin B1 (AFB) occurs. We developed a sensitive allele-specific polymerase chain reaction (AS-PCR) assay to detect this point mutation in non-neoplastic human liver tissues. Three oligonucleotide primers, 1 specific for the mutant allele and 2 specific for the wild-type allele were used. The mutant allele primer differed from the wild-type allele due to a G-to-T transversion in its terminal 3' nucleotide. The first stage involved amplification of exon 7 of p53 followed by a selective amplification of mutant codon 249 sequences. This method allowed for the detection of a mutant codon 249 allele in the presence of as many as 105 copies of the wild-type allele and was 100-fold more sensitive than the restriction fragment length polymorphism-PCR technique. We have applied this AS-PCR protocol to examine codon 249 AGT transversion in tumor and matched non-tumor liver samples from North American patients with hepatitis and from Mozambiquan patients exposed to AFB. Mutations were detected in 5 of 6 samples of non-neoplastic liver from Mozambiquan patients, all of whom were HBsAg- or HBcAg-positive and AFB-exposed. In contrast, no mutations were detected in non-neoplastic liver from North American patients with either HBV- or HCV-derived hepatitis and cirrhosis. This procedure is a simple and powerful approach for screening p53 codon 249 AGT mutation in heterogeneous non-neoplastic hepatocyte populations.

Overexpression of cytochrome P-450 isoforms involved in aflatoxin B1 bioactivation in human liver with cirrhosis and hepatitis.

Studies were carried out to test the hypothesis that inflammatory liver disease increases the expression of specific cytochrome P-450 isoenzymes involved in aflatoxin B1 (AFB) activation. The immunohistochemical expression and localization of various human cytochrome P-450 isoforms, including CYP2A6, CYP1A2, CYP3A4, and CYP2B1, were examined in normal human liver and liver with hepatitis and cirrhosis. The constitutive expression of CYP3A4 in normal liver showed a characteristic pattern of distribution in centrilobular hepatocytes, whereas CYP1A2, CYP2A6, and CYP2B1 were expressed uniformly throughout the liver acinus. In sections of liver infected with hepatitis B virus (HBV) or hepatitis C virus (HCV), the expression of CYP2A6 was markedly increased in hepatocytes immediately adjacent to areas of fibrosis and inflammation. CYP3A4 and CYP2B1 were induced to a lesser degree, and expression of CYP1A2 was unaffected. In HBV-infected liver, double immunostaining revealed that overexpression of CYP2A6 occurred in hepatocytes expressing the HBV core antigen. In HCV-infected liver, CYP2A6, CYP3A4, and CYP2B1 were overexpressed in hepatocytes with hemosiderin pigmentation. These results suggest that alterations in phenotypic expression of specific P-450 isoenzymes in hepatocytes associated with hepatic inflammation and cirrhosis might increase susceptibility to AFB genotoxicity.

Influences of chronic cholangiohepatitis and cholestasis on hepatic metabolism of benzo[a]pyrene in white suckers (Catostomus commersoni) from industrially polluted areas of Lake Ontario.

White suckers from polluted regions of western Lake Ontario have an increased prevalence of cholangiocellular and hepatocellular and hepatocellular neoplasms associated with an idiopathic chronic cholangiohepatitis. We examined the hypothesis that bile duct obstructions and cholestasis in these fish might increase the susceptibility of liver to administered benzo[a]pyrene (B[a]P). Cytosolic glutathione S-transferase (GST) activity (CDNB) was reduced in obstructed liver to 45% of activity in adjacent unobstructed liver. At micromolar concentrations, chenodeoxycholic acid, deoxycholic acid, bilirubin and haematin each inhibited GST activity of hepatic cytosolic and S-hexylglutatione-affinity-purified GST preparations from unobstructed liver. Liver cytosol and affinity-purified hepatic GSTs from normal white sucker liver reduced DNA binding of 3H-benzo[a]-pyrene-7,8-diol-9,10-epoxide (3H-BPDE) after preincubation in vitro in the presence of 5 mM GSH. Under these conditions, cytosol from adjacent unobstructed liver had a moderately stronger protective activity against DNA binding by BPDE (16.4 +/- 1.3 pmol BPDE/mg DNA) than did cytosol from obstructed liver (20.6 +/- 1.6 pmol BPDE/mg DNA). Suckers with obstructed livers identified by laparotomy were orally administered 3H-benzo[a]pyrene (3H-B[a]P) (0.2 mmol/kg) or unlabelled B[a]P (2.0 mg/kg) and the level of B[a]P macromolecular binding was analyzed in liver tissue by liquid scintillation counting and by immunohistochemistry with antibodies to BPDE-DNA adducts. Covalent binding of 3H-B[a]P to hepatic protein was 30% less in adjacent unobstructed liver compared to obstructed liver; however, there was no significant difference in the levels of 3H-B[a]P bound to DNA in the obstructed lobes compared with non-obstructed adjacent liver. These studies demonstrate that some endogenous non-substrate ligands that accumulate during cholestasis can reduce hepatic GST activity in white suckers. While these changes are insufficient to influence total 3H-B[a]P-DNA adducts in obstructed liver, the preferential localization of BPDE-DNA adducts in GST-deficient hyperplastic biliary tracts suggests that cholangiohepatitis might increase susceptibility to cholangiolar neoplasia in fish exposed to genotoxic polycyclic aromatic hydrocarbons.

Enhanced hexachloro-1:3-butadiene nephrotoxicity in rats with a preexisting adriamycin-induced nephrotic syndrome.

Renal damage was assessed by histopathology and urinalysis in male Wistar rats treated with either hexachloro-1:3-butadiene (HCBD; a single 170-mg/kg ip dose that caused proximal tubule necrosis), adriamycin (ADR; a single 5-mg/kg ip dose that caused minimal glomerular changes up to 35 days), or HCBD given 2 wk after ADR and compared with age-matched control rats for 21 days. Urinalysis values in ADR-treated rats showed minimal renal changes. HCBD significantly elevated urine volume (10-fold), protein (5-fold), glucose (175-fold), and brush border enzymes (10-600-fold), indicating severe proximal tubular damage, but most parameters returned to pretreatment levels 6 days after treatment. In ADR-pretreated rats subsequently given HCBD, both the urinary alkaline phosphatase and the ratio of kidney: body weight were significantly higher for longer periods. Histopathology demonstrated that the HCBD-induced proximal tubular lesion was confined to the outer stripe of the outer medulla. Advanced regeneration and repair was evident 21 days after HCBD treatment. In the ADR-pretreated rats the HCBD-induced lesion was more severe and affected the entire cortex and was characterized by marked tubular epithelial calcification, with little evidence of repair and tubular restitution 21 days after treatment. Enzyme histochemistry showed gamma-glutamyltranspeptidase localized to the proximal tubules. After HCBD treatment the enzyme staining was lost and subsequently returned in parallel with histological recovery up to 21 days. The distribution and intensity of gamma-glutamyltranspeptidase was unchanged in ADR-treated rats. The distribution and intensity of gamma-glutamyltranspeptidase in kidneys of ADR-pretreated rats given HCBD had not returned to normal by day 21. The results of this study indicate that pretreatment with ADR increases HCBD-induced nephrotoxic damage and decreases renal cortical repair capacity.

Metabolism of nitrosamines and aflatoxin B1 by hamster liver CYP2A enzymes.

Using a specific antibody against mouse CYP2A5 and coumarin as inhibitors, and pretreatments known to affect the activities of CYP2A-related enzymes, we studied the contribution of hamster liver CYP2As in the dealkylation of N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and hydroxylation of aflatoxin B1 (AFB1). CYP2A5 antibody inhibited AFB1 and NDEA metabolism by 40-60% in untreated hamsters and after treatment with phenobarbital (PB) or 3-methylcholanthrene (MC). After pyrazole (PYR) treatment, there was no inhibition, although PYR increased the metabolism above the controls. NDMA metabolism was inhibited only weakly in all groups. This suggests that CYP2As may contribute significantly to NDEA and AFB1 metabolism and less to NDMA metabolism in untreated hamster liver and after MC or PB treatments, but that PYR treatment may change the expression pattern of CYPs so that the metabolism is mainly catalysed by CYPs not recognized by the antibody. Coumarin inhibited NDEA and NDMA metabolism in all groups of hamsters, including pyrazole-pretreated animals, suggesting that it interacts not only with CYP2As but also with other CYPs that are important in nitrosamine metabolism. On the other hand, coumarin inhibited AFB1 metabolism significantly only in control and PB-treated animals but not at all after PYR or MC treatment. This suggests that enzymes involved in AFB1 metabolism may be different depending on the pretreatment. It is concluded that CYP2As may contribute significantly to nitrosamine and aflatoxin metabolism in hamster liver. However, in view of previous results showing essential differences in their regulation between mouse and hamster, interspecies comparisons may be difficult.

Induction of specific cytochrome P450s involved in aflatoxin B1 metabolism in hepatitis B virus transgenic mice.

The relative roles of hepatitis B virus (HBV) and aflatoxin and their possible mechanism of interaction in the etiopathogenesis of hepatocellular carcinoma (HCC) are not understood. One hypothesis is that viral infection and associated liver injury alter expression of carcinogen-metabolizing enzymes. We tested this hypothesis in an HBV-transgenic mouse model in which a synergistic interaction occurs between aflatoxin B1 (AFB1) and HBV in the induction of HCC (Sell et al., Cancer Res 51:1278-1285, 1991). In this transgenic mouse lineage, overproduction of the HBV large envelope protein results in progressive liver cell injury, inflammation, and regenerative hyperplasia. Initially, two cytochrome P450s of importance in AFB1 metabolism in the mice were identified, namely Cyp2a-5 and Cyp3a, using specific antibodies and chemical inhibitors. The expression of these P450 isoenzymes and an alpha-class glutathione S-transferase (GST) isoenzyme, YaYa, were examined. Increased expression and altered distribution of Cyp2a-5 were demonstrated, by immunohistochemical analysis, to be associated with the development of liver injury in mice and to increase with age between 1 and 12 months. Cyp3a expression was also increased in HBV-transgenic mice, but the increase was not as clearly related to age. GST YaYa levels were the same in HBV-transgenic mice and their nontransgenic littermates of all ages. These results show that expression of specific cytochrome P450s is altered in association with overexpression of HBV large envelope protein and liver injury in this model. This may have general relevance to human HCC, the etiology of which is associated with a diverse range of liver-damaging agents.

Association of liver fluke (Opisthorchis viverrini) infestation with increased expression of cytochrome P450 and carcinogen metabolism in male hamster liver.

Synergy between exposure to chemical carcinogens (nitrosamines) and infestation with the liver fluke Opisthorchis viverrini has been demonstrated in a hamster model of hepatocarcinogenesis (Flavell et al., Carcinogenesis 4:927-930, 1983; Thamavit et al., Carcinogenesis 8:1351-1353, 1987). To elucidate the mechanisms of this interaction we tested the hypothesis that liver parasitism might influence the expression and activity of carcinogen metabolizing enzymes. We found that one, and perhaps more, hamster liver cytochrome P450 (CYP) isozymes immunorelated to mouse CYP2A5 contributed up to 50 or 60% of the hepatic aflatoxin B1 (AFB) and N-nitrosodiethylamine (NDEA) metabolism, respectively. As inferred from average enzyme activities and from western blot, immunoinhibition, and substrate (coumarin) inhibition analyses, O. viverrini infestation increased the expression of enzymes detectable by anti-CYP2A5 antibody as well as NDEA metabolism in male but not in female hamsters. Immunohistochemical analysis of CYP2A expression by anti-mouse CYP2A5 antibody demonstrated that the O. viverrini-associated increase was not uniformly distributed throughout the liver but occurred in hepatocytes immediately adjacent to areas of inflammation. Immunohistochemical analysis of AFB-DNA adducts in the livers of O. viverrini-infested hamsters treated with AFB showed that the highest levels of adducts were found in the regions of liver where hepatocellular expression of enzymes detectable by anti-CYP2A5 antibody is induced. These results suggest that a high local expression of CYP isozymes in O. viverrini-infested livers could be a contributing risk factor in the development of liver cancers associated with parasitic hepatitis.

In vitro metabolism of aflatoxin B1 by normal and tumorous liver tissue from Thailand.

Liver tissues were obtained from 20 liver cancer patients from Thailand, an area where the incidence of this tumour is high and where exposure to aflatoxin occurs. The expression of hepatic cytochrome P450s (P450) and glutathione S-transferase (GST) was examined and this expression was compared to the in vitro metabolism of aflatoxin B1 (AFB1). There was a > 10-fold inter-individual variation in expression of the various P450s including CYP3A4 (57-fold), CYP2B6 (56-fold) and CYP2A6 (120-fold). Microsomal metabolism of AFB1 to AFB1 8,9-epoxide (as measured by AFB1 tris-diol formation) and aflatoxin Q1 (AFQ1), the major metabolite produced, was significantly correlated with CYP3A3/4 expression (P < 0.001) and, to a lesser extent, with CYP2B6 expression (P < 0.01). There was a significantly reduced expression of major P450 proteins in microsomes from liver tumours compared to microsomes from the paired normal liver when analysed by Western immunoblot analysis. The production of AFQ1 and AFB1 tris-diol was almost uniformly reduced in tumours, but interestingly, the production of AFP1 was significantly increased. The immunoreactive expression of the major human classes of cytosolic GSTs (alpha, mu and pi) was also analyzed in normal and tumorous liver tissue. The expression of GSTA (alpha) and GSTM (mu) class proteins was markedly decreased and GSTP (pi) increased in the majority of tumour cytosols compared to normal liver. The cytosolic GST activity (1-chloro-2,4-dinitrobenzene conjugation) was significantly lower in liver tumours compared to normal liver (193 +/- 149 versus 875 +/- 299 nmol/min/mg, P < 0.0001), as was glutathione peroxidase (GPx) activity (cumene hydroperoxide) (26 +/- 23 versus 70 +/- 26 nmol/min/mg respectively, P < 0.005). Ten out of 14 individuals (71%) were homozygous null when genotyped for GSTM1. There was no detectable conjugation of AFB1 8,9-epoxide to glutathione by cytosol either from tumorous or normal liver. Thus, capacity of human cytosols to conjugate reactive AFB1 metabolites to GSH resembled AFB1-sensitive species such as rat, trout and duck rather than resistant species such as mouse and hamster. These data indicate a strong capacity of multiple forms of human hepatic P450s to metabolize AFB1 to both the reactive intermediate AFB1 8,9-epoxide and the detoxification product AFQ1. These results suggest that in view of the lack of significant GST-mediated protection against AFB1 in human liver, variations in expression of hepatic P450, due either to genetic polymorphisms or to modulation by environmental factors, may be important determinants in the risk of liver cancer development in AFB1-exposed populations.

Loss of glutathione S-transferases in pollution-associated liver neoplasms in white suckers (Catostomus commersoni) from Lake Ontario.

White suckers (Catostomus commersoni) are one of two species of bottom-feeding fish in which various liver neoplasms are more prevalent in urban/industrial sites in western Lake Ontario than in less polluted sites in the Great Lakes. Previous studies indicate that white suckers excrete metabolites of various polycyclic aromatic hydrocarbons (PAHs) in bile, and that glutathione transferase (GST)-mediated conjugation is a major detoxification pathway for the PAH benzo[alpha]pyrene. To determine whether hepatocarcinogenesis in these wild fish is associated with induced GST-dependent resistance to carcinogens, we examined the expression of immunoreactive GSTs in liver neoplasms and putatively preneoplastic altered hepatocellular foci from white suckers collected from several polluted sites in western Lake Ontario. Histological sections of liver with altered hepatocellular foci, hepatocellular adenomas, hepatocellular carcinomas, bile duct adenomas and bile duct carcinomas were examined for GST immunoreactivity by the peroxidase-antiperoxidase (PAP) technique with polyclonal antiserum specific for all major GST isoenzyme subunits found in normal liver of white suckers. All bile duct adenomas, bile duct carcinomas and hepatocellular carcinomas were markedly or completely deficient in immunoreactive GST in comparison with surrounding normal hepatocytes. The majority of the hepatocellular adenomas were also deficient. Most altered hepatocellular foci had normal GST staining, but several GST-deficient altered hepatocellular foci were observed. However, none of the preneoplastic or advanced liver neoplasms expressed induced GST, suggesting that carcinogenesis is not associated with selection for GST-dependent resistance. Loss of hepatocellular GSTs may be incidental to neoplastic progression in these fish, or might be important in increasing susceptibility of some preneoplastic populations of hepatocytes to further DNA damage by environmental or endogenous chemicals that are normally detoxified by GSTs.

Expression of immunoreactive glutathione S-transferases in hepatic neoplasms induced by aflatoxin B1 or 1,2-dimethylbenzanthracene in rainbow trout (Oncorhynchus mykiss).

The expression of immunoreactive glutathione S-transferase (GST) was examined in hepatic neoplasms induced in rainbow trout by aflatoxin B1 (AFB) or 1,2-dimethylbenzanthracene (DMBA). Tumors were induced in adult trout by continuous dietary exposure to 8 p.p.b. AFB1 for 12 months or embryo bath exposure to DMBA (5 p.p.m. for 24 h, 3 times with 12 h intervals between exposures). Polyclonal antiserum specific for the two major trout hepatic GST subunits in trout liver was produced by immunizing rabbits with affinity-purified trout GST. Hepatocellular, cholangiolar and mixed neoplasms as well as foci of hepatocellular alteration were examined for GST immunoreactivity by the PAP technique. The majority of lesions were GST-deficient (AFB treated, 67%; DMBA treated, 54%), whereas GST expression was induced in 21% (AFB treated) and 31% (DMBA treated) of altered hepatic foci. The GST-induced foci were consistently small (AFB treated, 0.07 +/- 0.05 mm2; DMBA treated, 0.02 +/- 0.01 mm2) and none had progressed beyond the altered focus stage. The majority of larger advanced lesions (adenomas and carcinomas) were GST deficient (AFB treated, 2.33 +/- 0.35 mm2; DMBA treated, 2.95 +/- 0.59 mm2). These studies demonstrate that induced GST expression occurs in some small populations of hepatocytes, but not in larger advanced stages of malignant progression of aflatoxin- or PAH-induced hepatic neoplasms in rainbow trout.

The role of glutathione S-transferases in the hepatic metabolism of benzo[a]pyrene in white suckers (Catostomus commersoni) from polluted and reference sites in the Great Lakes.

1. Orally administered 3H-benzo[a]pyrene (3H-BaP) was excreted in the bile of White Suckers predominantly as water soluble metabolites some of which were hydrolyzed by arylsulfatase or beta-glucuronidase. 2. Non-hydrolysible polar metabolites comprised a substantial proportion of biliary metabolites. 3. HPLC analysis revealed fluorescent and 3H-labelled peaks which co-eluted with standards of the glucuronide and sulfate conjugates of BaP. 4. The most polar peak co-chromatographed with a double-radiolabelled metabolite produced in vitro with 3H-BaP and 35S-glutathione. 5. Inhibition of epoxide hydrolase in vitro reduced all water soluble metabolites except the glutathione conjugate of BaP. 6. Glutathione conjugation represents a major hepatic detoxication pathway of BaP in White Suckers.