ENZYME-INDUCTION BY L-BUTHIONINE (S,R)-SULFOXIMINE IN CULTURED MOUSE HEPATOMA-CELLS

Induction of Phase II enzymes of the [Ah] gene battery by L-buthionine (S,R)-sulfoximine (BSO) and other agents was examined in mouse hepato ma Hepa-1c1c7 cells. BSO, a nonelectrophilic inhibitor of gamma-glutam ylcysteine synthetase (GCS), is routinely used to examine the toxicolo gical implications of GSH depletion. Exposure to BSO for 24 h produced a 75-85% depletion of GSH levels, proportional to the inhibition of G CS activity, as well as small increases in the UDP-glucuronosyltransfe rase (UGT, 60%) and glutathione transferase (GST, 30%) enzyme activiti es in Hepa-1 wild-type (wt) cells. However, for the NAD(P)H:menadione oxidoreductase (NMO1) and cytosolic aldehyde dehydrogenase class 3 (AH D4) enzyme activities, BSO produced larger increases (110% and 170%, r espectively). The mechanisms of NMO1 and AHD4 induction were examined further. In Hepa-l wt cells, NMO1 and AHD4 activities were increased b y the aromatic hydrocarbon inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and by the electrophile tert-butylhydroquinone (tBHQ), known i nducing agents for these enzymes. However, NMO1 and AHD4 were induced in Ah receptor nuclear translocation-defective mutant (c4) cells by BS O and tBHQ, but not by TCDD, suggesting that the induction by BSO and tBHQ is not Ah receptor-mediated. In wt cells, N-acetylcysteine produc ed a concentration-dependent increase in intracellular cysteine levels , but not GSH levels, in the absence or presence of BSO. Furthermore, N-acetylcysteine had no effect on NMO1 activity under any conditions e xamined, suggesting that GSH levels per se, rather than change in over all thiol status, might be mediating increased NMO1 activity. The incr ease in NMO1 was accompanied by increased mRNA; actinomycin D prevente d the increases in both NMO1 enzyme activity and mRNA levels. Converse ly, N-acetylcysteine significantly increased AHD4 activity in the abse nce, but not the presence, of BSO. Although no increase in AHD4 mRNA w as observed following BSO treatment, actinomycin D partially prevented the increase in enzyme activity. These results suggest that GSH deple tion by BSO might be correlated with inducing NMO1 and AHD4 activities . Furthermore, the data suggest that the electrophile response element (EpRE)-binding complex of the Nmol gene might differ from that of the Ahd4 gene. Transcriptional activation appears to be responsible for t he increased NMO1, and possibly in part for the increased AHD4 activit y. A model is proposed whereby transcription of genes under the contro l of an EpRE, such as that found in the regulatory region of all four [Ah] battery Phase II genes, might be controlled by the state of redox -active cysteinyl sulfur(s) contained in one or more EpRE-binding prot ein(s). We propose that the collection of genes that are under partial control of an EpRE be termed the ''electrophile response element'' [E pRE] gene battery.