Abstract

A reconstituted purified mixed-function oxidase (MFO) system converts ³H(-)t-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [³H(-)t-7,8-dihydrodiol BP] to r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide I) and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide II). The addition of DNA to the incubation mixture results in covalent binding of metabolites to DNA. The binding of metabolites to DNA is a linear function of cytochrome P-450 LM₄ content. The addition of epoxide hydratase reduces the binding of metabolites to DNA by 35%-43%. Human monocytes, lymphocytes, and Fisher rat liver cells (TRL-2) also catalyze the binding of ³H(-)t-7,8-dihydrodiol BP to added DNA. Cells that have been induced by benzanthracene (BA) exhibit greater levels of DNA binding. The addition of epoxide hydratase to the medium reduces the amount of reactive metabolites binding to DNA by 54% and 31% in control and induced monocytes, 45% and 26% in control and induced lymphocytes, and by 18%-28% in control and 13%-24% in BA-preinduced rat liver TRL-2 cells. Thus, with both purified reconstituted MFO systems or with whole cell systems of either human monocytes, lymphocytes, or rat cells, the addition of purified epoxide hydratase reduces the binding of ³H(-)t-7,8-diol metabolites to DNA. These results indicate that epoxide hydratase functions in the hydrolytic inactivation of the diol epoxides. When heat-inactivated epoxide hydratase was used, no alteration in DNA binding was detected. The MFO system autocatalyzes the binding of ³H(-)t-7,8-dihydrodiol BP to the two protein components of the MFO enzyme system. The metabolites are bound to cytochrome P-450 LM₄ to the greatest extent both with respect to specific activity and total binding and to a lesser extent to cytochrome c (P-450) reductase. DNA can compete for reactive metabolite since the addition of DNA considerably reduces the binding of metabolites to the protein components. Added bovine serum albumin is also covalently bound to metabolites but does not reduce the binding to either DNA or the purified proteins of the enzyme system. Added epoxide hydratase is also covalently bound and reduces the binding to the other proteins. Diol epoxide I and II hydrolysis was measured by high-pressure liquid chromatography of the four tetrol products. The addition of DNA reduced the formation of tetrols I-2 (7/8,8,10), II-1 (7,9/8,10), and II-2 (7,9,19/8) and had no effect on the major I-1 (7,10/8,9) tetrol, indicating a stereoselective effect of DNA on diol epoxide hydrolysis. The addition of epoxide hydratase reduced the formation of three of the four tetrols by 11%-44% and increased tetrol II-1 formation, indicating a hydratase-induced alteration in the ratio of hydrolysis products. The latter results and the inhibition of DNA binding by hydratase indicate that the hydratase has a stereospecific interaction with diol epoxides, binding them covalently, altering their mode of hydrolysis, and apparently reducing their activity as carcinogenic intermediates that bind to DNA. Thus, epoxide hydratase may play am important role in diol epoxide detoxification.