A comparison of the effects of pretreatment with phenobarbitone and 3-methylcholanthrene on the metabolism of aflatoxin B1 by rat liver microsomes and isolated hepatocytes in vitro

Abstract

In agreement with previous reports (Neal and Colley, Biochem. J., 174 (1978) 839; Gurtoo and Dave, Cancer Res., 35 (1975) 382) the capacity of isolated hepatic microsomes to metabolise aflatoxin B1 (AFB1) is enhanced by pretreatment in vivo with phenobarbitone. Microsomes isolated from rats pretreated with 3-methylcholanthrene also exhibit increased metabolism of AFB1. The levels of metabolism are increased in proportion to the increased levels of cytochromes P-450 and P-448 indicating similar overall efficiencies of the two cytochromes to metabolise AFB1. However, the qualitative aspects of the metabolism induced by the two compounds are very different. In agreement with the previous data (Neal and Colley, Biochem J., 174 (1978) 839) the formation of aflatoxins Q1, M1 and B1-dihydrodiol are enhanced by pretreatment with phenobarbitone whereas pretreatment with 3-methylcholanthrene, while not affecting the production of aflatoxin Q1 and slightly lowering AFB1-dihydrodiol formation, increases the production of aflatoxin M1 greater than 10-fold. Covalent binding of AFB1 to microsomal protein in vitro is increased by pretreatment in vivo with phenobarbitone, but decreased by pretreatment with 3-methylcholanthrene. The changes in the level of protein binding in vitro brought about by these pretreatments are quantitatively similar to the relative changes in AFB1-dihydrodiol tris complex production induced by the two compounds indicating a role for this metabolite in protein binding. Covalent binding of AFB1 to DNA in vitro is also increased by pretreatment in vivo with phenobarbitone, but not by 3-methylcholanthrene and the total level of binding of AFB1 to DNA in the in vitro system is greater than binding to microsomal protein. The possible mechanisms for binding AFB1 to microsomal protein and DNA are discussed. In contrast to the microsomal incubations, only low levels of aflatoxins Q1 and M1 are present after 2 h incubation AFB1 with intact hepatocytes isolated from control, methylcholanthrene or phenobarbitone pretreated rats. Pretreatment with either phenobarbitone or 3-methylcholanthrene induces the formation of polar metabolites, presumably conjugates, emphasising the predominant role of phase 2 metabolism in intact cellular systems.