METABOLIC-ACTIVATION OF VINYL-CHLORIDE BY RAT-LIVER MICROSOMES - LOW-DOSE KINETICS AND INVOLVEMENT OF CYTOCHROME-P450 2E1

The metabolism and pharmacokinetics of vinyl chloride (VC) have been e xtensively studied in rodents and humans, but the maximum velocity (V- max) and Michaelis constant (K-m) for the activation of VC by microsom al monooxygenases in vitro have not yet been determined. Using a new s ensitive assay, the epoxidation of VC by rat liver microsomes (adult S prague-Dawley) at concentrations from 1 ppm to 10(6) ppm in the gas ph ase was measured. In the assay, the reactive VC metabolites chloroethy lene oxide and 2-chloroacetaldehyde were trapped with excess cAMP, yie lding 1,N-6-etheno-cAMP (epsilon cAMP) which was quantitated by HPLC f luorimetry. The trapping efficiency of electrophilic VC metabolites by CAMP was close to 10%. The specificity of the method was confirmed by purification of epsilon cAMP on an immunogel. The VC concentration in the gas phase was measured by GC/flame ionization detection, while in the aqueous phase it was calculated from the partition coefficient be tween air and the microsomal suspension. Activation of VC by rat liver microsomes followed Michaelis-Menten kinetics with K-m = 7.42 +/- 0.3 7 (+/-SD) mu M and V-max = 4674 +/- 46 pmol.mg protein(-1).min(-1). In hibitor studies and immunoinhibition assays showed that VC was by cyto chrome P450 (CYP) 2E1 down to 1 ppm in the air phase. Based on the met abolic parameters determined, the uptake of VC by rats in vivo can be accurately predicted. (C) 1998 Elsevier Science Inc.