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Kinetic Analysis of the Metabolism of Benzo(a)pyrene to Phenols, Dihydrodiols, and Quinones by High-Pressure Chromatography Compared to Analysis by Aryl Hydrocarbon Hydroxylase Assay, and the Effect of Enzyme Induction¹

Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology,³ Pasadena, California 91125 [S. K. Y.], and Chemistry Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014 [J. K. S., E. V. P., H. V. G.]

High-pressure liquid chromatography was used to analyze the rate of benzo(a)pyrene metabolite formation by liver microsomes from control and 3-methylcholanthrenetreated rats. The relative amounts of each metabolite formed were determined with several concentrations of microsomal mixed-function oxidases. The specific activity, i.e., amount formed per mg protein per min, was found to be constant for the formation of 3-hydroxybenzo(a)pyrene and 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene. The specific activity for the formation of 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene was higher at high microsomal enzyme concentration. The formation of 9-hydroxybenzo(a)pyrene, however, did not increase with greater amounts of microsomes. The data indicate that 9-hydroxybenzo(a)pyrene is a nonenzymatic product derived from a reactive intermediate, probably benzo(a)pyrene-9,10-oxide. The relatively constant specific activity for the formation of 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene with several enzyme concentrations suggests that the K-region epoxide, benzo(a)pyrene-4,5-oxide, is the most stable of the benzo(a)pyrene epoxide intermediates.

The relative percentages of each metabolite fraction found are as follows: 3-hydroxybenzo(a)pyrene, 36; 9-hydroxybenzo(a)pyrene, 3 to 13; 9,10-dihydro-9,10-dihydroxybenzo(a)pyrene, 15 to 25; 4,5-dihydro-4,5-dihydroxybenzo(a)pyrene, 8; 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, 12 to 14; benzo(a)pyrene quinones, 14 to 17. Induction of the enzyme system by 3-methylcholanthrene increases the amount of each metabolite formed to a different extent. The amount of 9,10-dihydro-9,10-dihydroxy- and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene formed are markedly increased relative to the increase in the other metabolites. Thus the induction of the enzyme may specifically alter pathways of metabolism relevant to carcinogenesis. This study also makes a detailed comparison between the results obtained by high-pressure liquid chromatography analysis and the standard aryl hydrocarbon hydroxylase assay and further develops the chromatographic analysis of benzo(a)pyrene metabolites.

¹ This research was supported in part by grants to Professor John D. Baldeschwieler from the National Science Foundation (Grant 38855X and NIH Grant GM 21111-02).

² Visiting Associate in the Chemistry Branch, National Cancer Institute, NIH from November 1973 to March 1974.

³ Contribution 5031.

Received 5/ 5/75. Accepted 9/19/75.

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