Presence Of Microsomal Activation System Research Articles

Examination of microsomal cytochrome P450-catalyzed in vitro activation of o-phenylphenol to DNA binding metabolite(s) by 32P-postlabeling technique.

It has been previously reported that the reactive metabolites phenylsemiquinone and phenylbenzoquinone are generated during microsomal cytochrome P450-catalyzed redox cycling of o-phenylphenol (OPP). However, covalent modification of DNA by OPP-reactive metabolites has yet not been demonstrated. In the present study we have investigated the covalent binding in DNA by OPP-reactive metabolites using 32P-postlabeling. Analysis of adducts by 32P-postlabeling in products of chemical reaction of DNA with phenylbenzoquinone revealed four major and several minor adducts. The chemical reaction of deoxyguanosine 3'-phosphate with phenylbenzoquinone also showed four major adducts. The chromatographic mobility of major adducts of deoxyguanosine 3'-phosphate-phenylbenzoquinone was identical to that of major adducts of DNA-phenylbenzoquinone. The major adducts are demonstrated to be stable. The total covalent binding in deoxyguanosine 3'-phosphate by phenylbenzoquinone (686,000-687,000 amol/nmol nucleotide) was higher than that observed in DNA (26,500-28,000 amol/nmol nucleotides). Reaction of DNA with OPP or a hydroxylated metabolite of OPP, phenylhydroquinone, in the presence of microsomes and NADPH or cumene hydroperoxide showed four major adducts. Adduct formation in DNA by OPP or phenylhydroquinone in the presence of the microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. The chromatographic mobility of major adducts in DNA by OPP or phenylhydroquinone in the presence of microsomal activation system matched with those major adducts observed in deoxyguanosine 3'-phosphate or DNA reacted with pure phenylbenzoquinone. These data demonstrate that OPP or phenylhydroquinone, a hydroxylated metabolite of OPP, is able to bind covalently to DNA in the presence of a microsomal cytochrome P450 activation system. Phenylbenzoquinone is one of the DNA-binding metabolite(s) of OPP. It is concluded that OPP is genotoxic in an in vitro system and genotoxicity produced by OPP-reactive metabolites may play a role in OPP-induced cellular toxicity or cancer.