EPOXIDATION OF ACRYLONITRILE BY RAT AND HUMAN CYTOCHROMES P450

The cytochromes P450 (P450) involved in the epoxidation of the rat carcinogen acrylonitrile (ACN) to the mutagen 2-cyanoethylene oxide (CEO) have been investigated in hepatic microsomes from F-344 rats and humans. Induction of P450 2E1 by acetone treatment increased the V-max for rat microsomal CEO formation 5-fold, while ACN treatment had little effect. Treatment with beta-naphthoflavone, dexamethasone, and phenobarbital had little effect upon V-max but increased the K-M 3- to 5-fold. The P450 ligand 1-phenylimidazole and substrate ethanol were potent inhibitors of ACN epoxidation after all treatments. 2-(Diethylamino)ethyl 2,2-diphenylvalerate (SKF 525A; 0.1 mM) was not an effective inhibitor with microsomes from untreated or acetone-treated rats, but inhibited similar to 50 % following dexamethasone or phenobarbital treatment. Antibodies to P450 2E1 inhibited >85% of the ACN epoxidation activity in microsomes from untreated or beta-naphthoflavone- or acetone-treated rats, but only produced 40% and 60% inhibition following dexamethasone or phenobarbital treatments, respectively. These results indicate that P450 2E1 is the major catalyst of ACN epoxidation in untreated rats and that other forms of P450 can also epoxidize ACN. Diethyldithiocarbamate (0.1 mM) was a potent irreversible inhibitor of ACN epoxidation after all of the induction treatments, indicating that it is not specific for P450 2E1. Chlorzoxazone (2 mM) produced 75-90% inhibition after all of the induction treatments, indicating that it interacts with several rodent P450 isoforms in addition to 2E1. Human hepatic microsomes (n = 6) epoxidized ACN with V-max's ranging from 129 to 315 pmol of CEO formed/(min.mg of protein) and K-M's from 12 to 18 mu M. The fidelity between the inhibition of ACN epoxidation by anti-P450 2E1 and chlorzoxazone (similar to 60% inhibition) is consistent with a specificity of chlorzoxazone for human P450 2E1. In contrast, diethyldithiocarbamate produced >95% irreversible inhibition of the epoxidation reaction. SKF 525A produced 20-35% inhibition of the human microsomal epoxidation reaction. These results show that human P450 2E1 is a major catalyst of ACN epoxidation, but that other forms of human cytochromes P450 are involved in ACN epoxidation to a greater extent than in untreated rats.