Benzo(a)pyrene metabolism by murine spleen microsomes

The immunosuppressive actions of benzo(a)pyrene have been proposed to be mediated by reactive metabolites rather than the parent compound. Reactive metabolites which suppress splenic humoral immune responses are thought to be generated within the spleen rather than in distant tissues. Although the spleen has been shown to be capable of metabolizing benzo(a)pyrene, the relative amounts and types of metabolites generated have not been determined. In this study, high-pressure liquid chromatography was used to separate benzo(a)pyrene metabolites generated by splenic microsomes. The major metabolites generated by the splenic microsomal preparations of untreated female B6C3F1 mice were found to be the 9,10- and 7,8-dihydrodiols and 9-, 7-, and 3-hydroxy benzo(a)pyrene. The 1,3-, 3,6-, and 6,12-diones and 4,5-dihydrodiol constituted only a small fraction of the metabolites generated. The generation of all metabolites were inhibited by alpha-naphthoflavone and antiserum to NADPH-cytochrome P-450 reductase, whereas SKF 525-A had only a minimal effect. Dihydrodiol production was completely inhibited by the epoxide hydrolase inhibitor, trichloropropylene oxide. Benzo(a)pyrene pretreatment of mice produced a dramatic increase in the amount of metabolites formed; however, the pattern of metabolites remained similar to that generated by splenic microsomes of untreated mice. The role of prostaglandin synthetase in generating these metabolites was also examined. The addition of arachidonic acid in place of NADPH resulted in the formation of only quinones. Dihydrodiols and phenols were undetectable. The results of this study indicate that splenocytes may be capable of generating the 7,8-dihydrodiol, the precursor to the highly reactive 7,8-dihydrodiol-9,10-epoxide. Furthermore, the addition of the 7,8-dihydrodiol-9,10-epoxide to splenocyte cultures resulted in a decreased in vitro antibody forming cell response to sheep red blood cells. Thus, benzo(a)pyrene-induced immunosuppression may be mediated by the dihydrodiol-epoxide generated within the spleen. Since benzo(a)pyrene exposure was found to increase its own metabolism, immunosuppression produced by the administration of benzo(a)pyrene over several days may be the result of an increased production of immunosuppressive metabolites. The pattern of metabolites generated and the effects of the two types of cytochrome P-450 inhibitors suggests that the major isozyme of cytochrome P-450 that mediates the metabolism of benzo(a)pyrene within the spleen of untreated mice may be similar to the isozyme induced in the liver upon pretreatment with polycyclic aromatic hydrocarbons.