MODULATION OF DETOXIFICATION GENE-EXPRESSION IN HUMAN COLON HT29 CELLS BY GLUTATHIONE-S-TRANSFERASE INHIBITORS

We investigated the effects of glutathione-S-transferase (GST) inhibit or treatment on human colon HT29 cell mRNA levels of dihydrodiol dehyd rogenase (DDH), glyoxalase I, and gamma-glutamylcysteine synthetase. T ime- and concentration-dependent increases in both DDH and gamma-gluta mylcysteine synthetase mRNAs resulted from treatment with ethacrynic a cid, ethacrynic acid/glutathione conjugate, and T.199 (gamma-glutamyl- S-(benzyl)-cysteinyl-R(-)-phenyl glycine diethyl ester), a selective G ST pi inhibitor. In contrast, glutathione analogue GST alpha- and GST mu-selective inhibitors did not induce expression of these genes. Trea tment with ethacrynic acid or T.199 had no effect on the mRNA levels o f the glutathione-dependent glyoxalase I gene. Pretreatment of cells w ith buthionine-DL-sulfoximine, a gamma-glutamylcysteine synthetase inh ibitor and glutathione depleter, coupled with ethacrynic acid, ethacry nic acid/glutathione conjugate, or T.199 resulted in greater levels of gamma-glutamylcysteine synthetase and DDH induction compared with sin gle treatments. Treatment with buthionine-DL-sulfoximine alone resulte d in modest increases in both gamma-glutamylcysteine synthetase and DD H expression. Analyses of DDH induction by both differential Northern hybridization with specific oligonucleotides as probes and reverse tra nscriptase-polymerase chain reaction amplification of products, follow ed by diagnostic restriction digestion with endonucleases, showed that ethacrynic acid induced multiple DDH transcripts in HT29 cells and hu man HepG2 and SKHep1 hepatoma cells. Possible induction mechanisms inc lude the alteration of sulfhydryl status by the electrophilic properti es of EA or by elevations of endogenously generated oxidative stress v ia transient removal of GST pi from the cytosolic GST pool.