Immunochemical study on the contributions of two molecular species of microsomal cytochrome P-450 to the metabolism of 2-acetylaminofluorene by rat liver microsomes.

The roles of two species of cytochrome P-450, the major cytochrome P-450 components of liver microsomes of phenobarbital-treated rats (PB-P-450) and of 3-methylcholanthrenetreated rats (MC-P-448), in the metabolism of 2-acetylaminofluorene were studied in rat liver microsomes in vitro. 2-Acetylaminofluorene was incubated with the microsomes of rats treated with three different inducers of monooxygenase, and then the metabolites were separated into ethyl acetatesoluble, water-soluble, and covalently protein-bound fractions. High-pressure liquid Chromatographie analysis of the ethyl acetate-soluble metabolites revealed that the total amounts of hydroxylated metabolites were 1.4, 7.6, and 13.3 times higher than normal in the microsomes of rats treated with phénobar bital, 3-methylcholanthrene, and polychlorinated biphenyls, re spectively. The water-soluble metabolites in the microsomes of these respective groups were also increased 1.8, 7.5, and 21.3 times, and the covalent binding of 2-acetylaminofluorene metabolites to protein was increased 1.8, 6.0, and 17.7 times. The effects of the antibodies against PB-P-450 and MC-P448 on the metabolism of 2-acetylaminofluorene were investi gated in polychlorinated biphenyl-treated rat liver microsomes in which these two species are the major cytochrome P-450 components and are present in almost equal amounts. Anti bodies against MC-P-448 caused 90% inhibition of the forma tion of 1and 3-, 5-, 7-, and /V-hydroxy-2-acetylaminofluorene. On the other hand, antibodies against PB-P-450 caused 10 to 20% inhibition of the formation of hydroxylated metabolites. Antibodies against MC-P-448 also caused 90% inhibition of the formation of water-soluble metabolites and 80% inhibition of covalent binding of 2-acetylaminofluorene metabolites to protein, while antibodies against PB-P-450 caused 25 and 5% inhibition, respectively. These results show that MC-P-448 has about 10 times higher activity than PB-P-450 in the metabolism of 2-acetylaminoflu