Metabolic Activation of Vinyl Chloride by Rat Liver Microsomes: Low-Dose Kinetics and Involvement of Cytochrome P450 2E1

Abstract

The metabolism and pharmacokinetics of vinyl chloride (VC) have been extensively studied in rodents and humans, but the maximum velocity ( V max) and Michaelis constant ( K m) for the activation of VC by microsomal monooxygenases in vitro have not yet been determined. Using a new sensitive assay, the epoxidation of VC by rat liver microsomes (adult Sprague–Dawley) at concentrations from 1 ppm to 10 6 ppm in the gas phase was measured. In the assay, the reactive VC metabolites chloroethylene oxide and 2-chloroacetaldehyde were trapped with excess cAMP, yielding 1, N 6-etheno-cAMP (ϵcAMP) which was quantitated by HPLC fluorimetry. The trapping efficiency of electrophilic VC metabolites by cAMP was close to 10%. The specificity of the method was confirmed by purification of ϵcAMP 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 between air and the microsomal suspension. Activation of VC by rat liver microsomes followed Michaelis–Menten kinetics with K m = 7.42 ± 0.37 (±SD) μM and V max = 4674 ± 46 pmol · mg protein −1 · min −1. Inhibitor studies and immunoinhibition assays showed that VC was activated by cytochrome P450 (CYP) 2E1 down to 1 ppm in the air phase. Based on the metabolic parameters determined, the uptake of VC by rats in vivo can be accurately predicted.