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 hepatoma Hepa-1c1c7 cells. BSO, a nonelectrophilic inhibitor of gamma-glutamylcysteine synthetase (GCS), is routinely used to examine the toxicological implications of GSH depletion. Exposure to BSO for 24 h produced a 75-85% depletion of GSH levels, proportional to the inhibition of GCS activity, as well as small increases in the UDP-glucuronosyltransferase (UGT, 60%) and glutathione transferase (GST, 30%) enzyme activities in Hepa-1 wild-type (wt) cells. However, for the NAD(P)H:menadione oxidoreductase (NMO1) and cytosolic aldehyde dehydrogenase class 3 (AHD4) enzyme activities, BSO produced larger increases (110% and 170%, respectively). The mechanisms of NMO1 and AHD4 induction were examined further. In Hepa-l wt cells, NMO1 and AHD4 activities were increased by the aromatic hydrocarbon inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and by the electrophile tert-butylhydroquinone (tBHQ), known inducing agents for these enzymes. However, NMO1 and AHD4 were induced in Ah receptor nuclear translocation-defective mutant (c4) cells by BSO and tBHQ, but not by TCDD, suggesting that the induction by BSO and tBHQ is not Ah receptor-mediated. In wt cells, N-acetylcysteine produced 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 examined, suggesting that GSH levels per se, rather than change in overall thiol status, might be mediating increased NMO1 activity. The increase in NMO1 was accompanied by increased mRNA; actinomycin D prevented the increases in both NMO1 enzyme activity and mRNA levels. Conversely, N-acetylcysteine significantly increased AHD4 activity in the absence, but not the presence, of BSO. Although no increase in AHD4 mRNA was observed following BSO treatment, actinomycin D partially prevented the increase in enzyme activity. These results suggest that GSH depletion 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 the increased NMO1, and possibly in part for the increased AHD4 activity. A model is proposed whereby transcription of genes under the control 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 protein(s). We propose that the collection of genes that are under partial control of an EpRE be termed the ''electrophile response element'' [EpRE] gene battery.