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Selectivity of Rat and Human Glutathione S-Transferases in Activation of Ethylene Dibromide by Glutathione Conjugation and DNA Binding and Induction of Unscheduled DNA Synthesis in Human Hepatocytes¹

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 [J. L. C., P. B. I., M. J. M., F. P. G.], and CRC Molecular Toxicology Research Group, Middlesex School of Medicine, Cleveland St., London, W1P 6DB, United Kingdom [D. J. M., B. K.]

The major DNA adduct formed by the carcinogen ethylene dibromide (EDB) is S-[2-(N⁷-guanyl)ethyl]glutathione. This adduct results from the glutathione S-transferase (GST)-catalyzed conjugation of EDB with glutathione (GSH), which generates an episulfonium ion capable of reacting with cellular nucleophiles. Purified rat and human GST enzymes were compared for their ability to conjugate EDB with GSH and displayed high selectivity. Of the six forms of rat GST tested, conjugation was catalyzed by the class enzyme 2-2 and, to a lesser extent, by the µ class enzyme 3-3. Of the three classes of cytosolic human GST, EDB conjugation was catalyzed by the class enzymes. Three dimers of the human class (_x-_x, _x-_y, and _y-_y) were separated by chromatofocusing. The _x-_x preparation demonstrated the highest specific activity. Rat microsomal GST had negligible activity for the conjugation of EDB with GSH. The levels of EDB-DNA adducts formed in rat and human hepatocytes were compared. DNA was isolated from both rat and human hepatocytes incubated with 0.5 mM EDB, and the level of DNA adduct formation in the human samples was about 40% of that in the rat hepatocytes. EDB concentration-dependent unscheduled DNA synthesis was demonstrated in isolated human hepatocytes. Concurrent treatment of the hepatocytes with diethylmaleate to deplete intracellular GSH inhibited EDB-induced unscheduled DNA synthesis. These results indicate that EDB alkylates DNA in human hepatocytes and that enzymatic repair of adducts may occur. The results of experiments done in rat and human systems using both purified GST enzymes and intact hepatocytes imply that the genotoxic pathway of EDB metabolism in rats and humans is similar.

¹ Supported in part by USPHS Grants CA 44353, ES 00267, ES 01590, and ES 03272.

² Present address: Pfizer Central Research, Drug Metabolism Department, Eastern Point Rd., Groton, CT 06340.

³ Present address: Department of Biochemistry, School of Dentistry, Oregon Health Sciences University, Portland, OR 97201.

⁴ To whom requests for reprints should be addressed.

Received 9/ 7/89. Revised 2/ 1/90.

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