Graduate Thesis Or Dissertation
 

The significance of glutathione conjugation for aflatoxin B₁ metabolism in rainbow trout and coho salmon

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  • In rodent models as well as in fish models there are significant species differences in the susceptibility toward aflatoxin B₁ (AFB₁) carcinogenesis. Mouse is less susceptible toward AFB₁ carcinogenesis than rat. Researchers have come to the conclusion that the lower susceptibility of mice is not a result of less effective activation of AFB₁, but rather of more effective inactivation of the toxic intermediate AFB₁-2,3-epoxide, and especially inactivation through the glutathione (GSH) conjugation of the epoxide. Rainbow trout fed Oregon Test Diet are more sensitive toward AFB₁ hepatocarcinogenesis than coho salmon, or, than trout fed the inhibitors β-naphthoflavone (BNF), 1ndole-3-carbinol (I3C), or Aroclor 1254 (PCB). This study examined the role of AFB1-glutathione (AFB₁-SG) conjugation In these differences. A tritiated AFB₁-glutathione conjugate standard (³H-AFB₁-S6) was produced in vitro using mouse liver S-9 fraction as a source of GSH transferase. It was purified by reverse phase HPLC, and its structure verified by amino acid analysis and mass spectrometry. Coho salmon and rainbow trout fed the various diets were injected i.p. with ³H-AFB₁ (49μCi, 50 μg/ kg fish); bile, liver and kidney were collected at 24 h. Recovery of total aflatoxin radioactivity was determined for all three tissues, and the hepatic AFB₁-DNA binding was also determined. Bile metabolites were quantitated by reverse phase HPLC using the mouse ³H-AFB₁-SG as a standard. The resistance of coho salmon toward AFB₁ carcinogenicity was supported by a 20-fold lower hepatic AFB₁-DNA binding compared to control trout. AFB₁-SG was detected in bile only in control, BNF, and I3C fed trout, at < 1% of total recovered metabolites, and at < 0.2% of the original dose, being highest in control trout. The major conjugates were glucuronides of aflatoxicol (AFL) and aflatoxicol-M₁ (AFL-M₁) (80-902 of the total recovered metabolites). In vitro metabolism studies using isolated liver cell fractions supported the in vivo metabolism results. Less than 0.531 of the original AFB₁ dose was converted to AFB₁-SG conjugate in salmon and trout samples. In contrast, with isolated mouse liver cell fractions approximately 25% of the original AFB₁ dose was conjugated with GSH. The GSH concentration of control trout liver was 2.9 mmol/ kg. Coho salmon had GSH concentration 70% of that found in control trout. In BNF pre-fed trout liver GSH concentration was enhanced by 25% compared to controls. Liver GSH transferase activity using l-chloro-2,4- dlnitrobenzene as substrate was 1.15 μmol/min/mg protein in control trout. This enzyme activity In salmon was only 26% of that found in control trout. A 62% elevation In GSH transferase activity compared to controls was detected in trout fed BNF diet. There is no apparent correlation between liver GSH or GSH transferase activities among the various groups, and their relative sensitivities to AFB₁ carcinogenesis. This study indicates that AFB₁-SG conjugation is not a significant pathway in salmon and trout fed control diets, or trout fed various inhibitors, and cannot account for the variation In AFB₁ sensitivity.
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