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 hepatocytes.
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 alpha class enzyme 2-2 and, to a lesser extent, by the mu class enzyme 3-3. Of the three classes of cytosolic human GST, EDB conjugation was catalyzed by the alpha class enzymes. Three dimers of the human alpha class (alpha x-alpha x, alpha x-alpha y, and alpha y-alpha y) were separated by chromatofocusing. The alpha x-alpha 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.
Cmarik
J L
JL
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Inskeep
P B
PB
Meredith
M J
MJ
Meyer
D J
DJ
Ketterer
B
B
Guengerich
F P
FP
D000818:Animals; D001711:Biotransformation; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D015946:Ethylene Dibromide / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity
D015946:Ethylene Dibromide; D004247:DNA; D005982:Glutathione Transferase; D005978:Glutathione