Epidemiological evidence suggests that aflatoxin B1 (AFB1), a potent animal hepatotoxin and hepatocarcinogen may be an etiologic agent in the induction of primary liver cancer in humans. It undergoes activation via epoxidation and the interaction of its epoxide with DNA is believed to responsible for its initiation of carcinogenesis. There is a wide difference in carcinogenicity of AFB1 between rats and hamsters and AFB1-DNA binding in vivo and in vitro. Recent work with hepatosubcellular fractions of these two species from my laboratory has shown that the binding of DNA with AFB1 is strongly modulated by a competive detoxication reaction with reduced glutathione (GSH), catalyzed by one or more of cytosolic GSH S-transferases. The purpose of this study is to explore and delinate further a possible role of GSH and its S-transferases in AFB1 corcinogenesis.
The specific aims of this project are to extend and amplify our current preliminary studies by (1) investigating the kinetics of AFB1-DNA binding and how it is affected by AFB1-GSH conjugation and excretion. Comparative studies will be conducted in livers of rats and hamsters, using as experimental models isolated hepatocytes, the perfused liver and whole animals. Pretreatment of animals with buthionine sulfoximine (BSO) will lower hepatic GSH levels, pretreatment with 2-oxothiazolidine-4-carboxylate will increase hepatic GSH levels and administration of phenobarbital (PB) will increase GSH S-transferases, activities. Manipulation of the levels of GSH and its S-transferases by these means should provide effective data to assess the role of conjugation in AFB1 hepatocarcinogenesis. (2) Investigate the biological significance of GSH and its S-transferases in modulation of hepatic AFB1-DNA binding and hepatacarcinogenesis by examining production of preneoplastic enzyme altered foci of hepatocytes as well as ultimate hepatocarcinomas in rats and hamsters in which GSH is reduced by pretreatment with BSO before the administration of AFB1. In similar experiments the effect of increased GSH S-transferases will be examined by administration of PB with and without BSO treatment. (3) Finally, to investigate whether one or more of the known specific cytosolic GSH S-transferases are responsible for conversion of AFB1-epoxide to the AFB1-GSH conjugate. These studies would entail purification of various GSH S-transferases from rat and hamster livers, and examining their catalytic and kinetic properties in a reconstituted system. The long term objectives of this program are aimed at understanding these metabolic mechanisms in AFB1 carcinogenesis in other sensitive and resistant species.
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