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Formation of Glutathione Conjugates by Reactive Metabolites of Vinylidene Chloride in Microsomes and Isolated Hepatocytes¹

Departments of Pharmacology [D. C. L.] and Biochemistry [M. J. M., F. P. G.] and Center in Molecular Toxicology [D. C. L., M. J. M., F. P. G.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Oxidation of the vinyl halide carcinogen and hepatotoxin vinylidene chloride (VDC) by microsomal cytochrome P-450 yields 2,2-dichloroacetaldehyde, 2-chloroacetyl chloride, 2-chloroacetic acid, and 1,1-dichloroethylene oxide. The roles of these metabolites in covalent modification of proteins and reduced glutathione (GSH) were examined. 2-Chloroacetyl chloride reacted with model thiols at least 10³-fold faster than did 1,1-dichloroethylene oxide and at least 10⁵-fold faster than did 2,2-dichloroacetaldehyde or 2-chloroacetic acid. Microsomal covalent binding of [¹⁴C]VDC was inhibited by GSH but not by lysine, suggesting that protein thiols, rather than amino groups, are major targets. Liver microsomes catalyzed the formation of three GSH:VDC metabolite conjugates, identified as S-(2,2-dichloro-1-hydroxy)ethylglutathione, 2-(S-glutathionyl)acetate, and S-(2-glutathionyl)acetylglutathione, a novel conjugate containing both stable (thioether) and labile (thioester) linkages. The latter two conjugates also were formed in isolated rat hepatocytes and measurable amounts of 2-(S-glutathionyl)acetate were released into the incubation medium. Both 2-(S-glutathionyl)acetate and S-(2-glutathionyl)acetylglutathione were formed with [³⁵S]GSH added to the hepatic medium, indicating that reactive VDC metabolites are capable of crossing the plasma membrane to react with extracellular targets. Unlabeled S-(2-glutathionyl)-acetylglutathione underwent carbonyl substitution with added [³⁵S]GSH, suggesting that this conjugate may participate in modification of protein thiols. This conjugate also underwent hydrolysis with a half-life of approximately 3 hr. GSH:VDC metabolite conjugates may serve as accessible models for labile covalent adducts formed between VDC metabolites and protein thiols.

¹ This work was supported by USPHS Grants ES 02205 and ES 00267 and by Vanderbilt University Research Council Grant 361024.

² Recipient of a Pharmaceutical Manufacturer's Association Foundation Advanced Predoctoral Fellowship. Present address: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331.

³ Burroughs Wellcome Scholar in Toxicology (1983 to 1988). To whom requests for reprints should be addressed, at Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.

Received 5/21/84. Accepted 9/27/84.

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