Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems |
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Authors: | A L Shen W E Fahl C R Jefcoate |
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Institution: | Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706 USA |
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Abstract: | Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrene-treated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxides) and of epoxide derivatives of BP-9-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:1 to 3:1 between 30 and 100 μm BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated. |
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