Cytochrome P-450-mediated activation of procarcinogens and promutagens to DNA-damaging products by measuring expression of umu gene in Salmonella typhimurium TA1535/pSK1002

Abstract

A simple and sensitive procedure for the determination of cytochrome P-450 (P-450)-mediated activation of chemical procarcinogens and promutagens to DNA-damaging products has been developed using a method measuring the expression of the umu gene in Salmonella typhimurium TA1535/pSK1002, which is based upon the initial procedures as described by Oda et al. [Mutation Res. 147, 219 (1985)]. The chemicals examined were a variety of potent carcinogenic and mutagenic compounds including heterocyclic aromatic amines, aromatic amines, polycyclic aromatic hydrocarbons and aflatoxin B1. These chemicals were incubated with rat liver microsomes or a reconstituted monooxygenase system containing three forms of purified P-450 in the presence of a bacterial tester strain, and the induced-umu gene expression was determined by measuring the beta-galactosidase activity produced by fusion gene in the cells. The activity was increased linearly for at least 2 hr with an initial lag time of 30 min and was dependent on the concentrations of P-450 in the reaction mixture. Thus, the metabolic activation of these compounds by P-450 could be compared on a basis of the specific beta-galactosidase activity/min/nmol P-450. Among three forms of P-450, two isozymes induced by 3-methylcholanthrene were found to be more active in catalyzing the metabolic activation of most of the chemicals examined than a form of P-450 which is induced by phenobarbital. Data also showed that a high spin form of P-450 isolated from 3-methylcholanthrene-treated rats had a profound role in the activation of procarcinogens and promutagens. This conclusion was based on the results of catalytic activities by three forms of P-450 in a reconstituted monooxygenase system, and on the effects of specific antibodies against these P-450s on the reactions catalyzed by liver microsomes.