High-pressure liquid chromatographic analysis of benzo(alpha)pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane.

Abstract

Summary High-pressure liquid chromatography is a rapid and efficient method for the separation of benzo(a)pyrene metabolites. This new technique readily separates eight benzo(a)pyrene metabolites from the parent hydrocarbon. These are three dihydrodiols (9,10-, 7,8-, and 4,5-dihydrodihydroxybenzo(a)pyrene), three quinones (benzo(a)pyrene-1,6-dione, -3,6-dione, and -6,12-dione) and two phenols (9- and 3-hydroxybenzo(a)pyrene). This method was applied to the study of the mechanism of inhibitor action on the microsomal benzo(a)pyrene metabolism. 7,8-Benzoflavone, an inhibitor of aryl hydrocarbon hydroxylase, inhibits the covalent binding of benzo(a)pyrene to DNA and inhibits the formation of each of the metabolites. The lack of selective inhibition suggests that the 7,8-benzoflavone acts on the oxidase or a prior component of the microsomal electron chain. 1,2-Epoxy-3,3,3-trichloropropane inhibits benzo(a)pyrene disappearance, which also suggests an inhibitory effect on oxidase activity. This compound, however, also stimulates formation of benzo(a)pyrene binding to DNA, reduces the ratio of 3-hydroxybenzo(a)pyrene to 9-hydroxybenzo(a)pyrene formation, and completely eliminates the formation of the three dihydrodiols. This selective effect suggests a specific inhibition of hydrase activity with a lesser effect on oxidase function.