USE THE VIAL EQUILIBRATION TECHNIQUE FOR DETERMINATION OF METABOLIC-RATE CONSTANTS FOR DICHLOROMETHANE

Metabolism of methylene chloride, or dichloromethane (DCM), plays a ke y role in determining the kinetics and carcinogenicity of the halocarb on. The objectives of this study were: to evaluate and optimize the vi al equilibration technique, originally described by Sate and Nakajima (1979a), in order to characterize the hepatic metabolism of DCM by Spr ague-Dawley rats; to employ different hepatic microsomal preparations to examine buffer effects on DCM metabolism; and to assess the relativ e importance and metabolic constants of the mixed-function oxidase (MF O) and glutathione (GSH) S-transferase (CST) metabolic pathways. A cru de liver homogenate (20% w/v) was prepared from perfused livers of mal e Sprague-Dawley (S-D) rats (275-325 g). A 30% glycerol buffer was fou nd to significantly inhibit DCM metabolism, while 0.25 M sucrose buffe r containing 10 mM EDTA and 1.15% KCl did not. DCM was incubated with the liver 10,000g supernatant or microsomes and cofactors in sealed he adspace vials. Disappearance of DCM, as a measure of the chemical's me tabolism, was monitored by headspace gas chromatography. Different tri als were conducted to elucidate time-, enzyme-, and substrate-activity relationships, The scaled-up K-m and V-max values for the microsomal fraction were quite similar to optimized in vivo values reported by ot her investigators. In the current study, DCM appeared to be metabolize d preferentially by cytochrome P450 IIE1, since substrates (e.g., pyra zole, ethanol, and glycerol) for this isozyme completely inhibited DCM metabolism, Thus, glycerol should not be used as a P450 stabilizer fo r preparation or storage of microsomes. Phorone pretreatment caused ma rked hepatic GSH depletion, but had little effect on the overall rate of DCM metabolism. Quantitatively, the GST pathway in the cytosol play ed a very minor role in DCM metabolism. It was not possible to accurat ely calculate metabolic constants for this pathway in S-D rats. The vi al equilibration technique, as described here, is a relatively simple and reliable method, which should be broadly applicable for measuring the microsomal metabolism of DCM and other VOCs. (C) 1996 Academic Pre ss, Inc.