The metabolism of benzene and phenol by a reconstituted purified phenobarbital-induced rat liver mixed function oxidase system.

Abstract

During its long history of extensive industrial use, chronic exposure of humans to benzene has been associated with blood disorders, such as aplastic anemia and leukemia. It has been our aim to study the link between the metabolism of benzene and the mechanism by which it produces bone marrow toxicity (Snyder et al., 1967, 1977, 1982). The mixed function oxidases, a family of hemoprotein cytochrome P-450 enzymes located in the smooth endoplasmic reticulum of liver as well as most other tissues, was shown by Gonasun et al. (1973) to play a key role in the metabolism of benzene. Studies by Jerina and Daly (1974) and Tunek et al. (1978) strongly supported the concept that the formation of benzene oxide is the principal first step in benzene metabolism. However, Ingelman-Sundberg and Hagbjork (1982) have suggested that the hydroxylation may occur via the insertion of a hydroxyl free radical, postulated to be generated from an “iron-catalyzed cytochrome P-450-dependent Haber Weiss reaction.” Gorsky and Coon (1984) have demonstrated that the pathway by which benzene is metabolized is directly related to the concentration utilized in the in vitro reaction, i.e. with very low concentrations the reaction is mediated by a free hydroxyl-radical mechanism and at higher concentrations of benzene, in the range of its KM, by direct cytochrome P-450-mediated oxidation. These two concepts could be reconciled if (1) there were two (or more) cytochromes P-450 involved in the hydroxylation, one with the ability to form an epoxide intermediate and another generating free radicals through a Haber Weiss mechanism, or (2) the cytochrome P-450 responsible for epoxidation also functioned as a NADPH oxidase, thereby, producing H2O2 which subsequently generated the hydroxyl radicals. Post and Snyder (1983) recently reported evidence which suggested that there are at least two different rat liver mixed function oxidases active in the hydroxylation of benzene. Using a reconstituted purified mixed function oxidase system from phenobarbital-induced rat liver, we have studied the initial hydroxylation step in benzene metabolism leading to phenol formation and the subsquent conversion of phenol to polyhydroxylated metabolites.