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Formation in Isolated Rat Liver Microsomes and Nuclei of Benzo(a)pyrene Metabolites That Bind to DNA¹

Department of Forensic Medicine, Karolinska Institutet, S-104 01 Stockholm, Sweden

The hepatic nuclear fraction isolated from 3-methylcholanthrene (MC)-treated rats contained enhanced levels of cytochrome P-450 and aryl hydrocarbon hydroxylase [benzo(a)pyrene (BP) monooxygenase], whereas the activities of epoxide hydrase and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and the concentration of cytochrome b₅ were not altered. The metabolite pattern of BP was investigated by using high-pressure liquid chromatography and was found to be similar in nuclei and microsomes from MC-treated rats.

After incubation of the nuclear fraction with [³H]BP and reduced nicotinamide adenine dinucleotide phosphate, radioactivity was found to be associated with nuclear DNA and the extent of binding was markedly enhanced by pretreatment of the animals with MC. Binding was strongly inhibited by -naphthoflavone but was not influenced by 1,1,1-trichloropropene-2,3-oxide, an inhibitor of epoxide hydrase. In the presence of microsomes from MC-treated rats, increased binding of BP to DNA was observed in nuclei from both control and MC-treated rats; moreover, when the nuclear DNA was replaced by a corresponding amount of calf thymus DNA, the extent of binding was severalfold enhanced.

In contrast to nuclei from control rats, the nuclear fraction from MC-treated rats showed an increase in bound radioactivity when incubated with a microsome-free supernatant, obtained by incubating microsomes from MC-treated rats with [³H]BP. The increase in extent of binding was eliminated in the presence of menadione or -naphthoflavone.

It is suggested that under the conditions used here the following different processes may have contributed to the total incorporation of BP products into nuclear DNA: (a) formation of DNA-binding products derived from BP by nuclear aryl hydrocarbon hydroxylase; (b) formation of DNA-binding products from microsomal BP metabolites by nuclear aryl hydrocarbon hydroxylase; and (c) direct transfer of reactive microsomal metabolites to nuclear DNA.

¹ This study was supported by NIH Contract 1 CP 33363, "Studies on Polycyclic Hydrocarbon Metabolism in the Respiratory Tract." Part of this study has been reported in preliminary form at the "Symposium on Polynuclear Aromatic Hydrocarbons," Columbus, Ohio, October 15 to 17, 1975. Vadi, H., Jernström, B., and Orrenius, S. Recent Studies on Benzo(a)pyrene Metabolism in Rat Liver and Lung. In: R. Freudenthal and P. W. Jones (eds.), Polynuclear Aromatic Hydrocarbons, pp. 45–61. New York: Raven Press, 1976.

Received 4/12/76. Accepted 8/11/76.