Enzyme Induction by L-Buthionine(S,R)-Sulfoximine in Cultured Mouse Hepatoma Cells

Abstract

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-1 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The hepatotoxic metabolite of acetaminophen directly activates the Keap1-Nrf2 cell defense system

“…20 BSO activated Nrf2 only when cellular GSH was depleted to 10%-15% of basal levels, a finding that is consistent with previous reports that only substantial depletion of GSH activates the Nrf2-ARE pathway 33,34 and our previous work showing that a more modest depletion of GSH by BSO in vivo (to 40% of basal levels) is not sufficient to activate Nrf2. 18 There was no evidence for direct chemical modification of Keap1-V5 by BSO, consistent with its known biochemical mechanism of action 20 and chemical structure.…”

Site-directed mutagenesis of cysteine to serine in the DNA binding regionof Nrf2 decreases its capacity to upregulate antioxidant responseelement-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase1 gene

“…These results demonstrate the importance of cysteine 506 in redox regulation of Nrf2. This is also supported by previous observations that AREmediated gene expression is sensitive to sulfhydryl modifying agents, such as glutathione, and related molecules (Li and Jaiswal, 1994;Bergelson et al, 1994;Shertzer et al, 1995).…”

Analysis of target cell susceptibility as a basis for the development of a chemoprotective strategy against benzene-induced hematotoxicities.

“…Treatment with BSO resulted in a significant induction in QR activity in the rat stromal cells and the TPA-differentiated ML-1 cells. Shertzer et al (32) recently reported a similar effect of BSO in mouse Hepa-lclc7 cells. Overall, the data presented in Table 3 are consistent with the concept that QR and (36).…”