Regulation of [Ah] gene battery enzymes and glutathione levels by 5,10-dihydroindeno[l,2-b]indole in mouse hepatoma cell lines

The murine aromatic hydrocarbon ([Ah]) gene battery consists of at least six genes that code for two functionalizing (Phase I) enzymes and four non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptorprotein. Studies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT1*O6) and NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the synthetic antioxidant 5,10-dihydroindeno[l,2-b]indole (DHII). The induction of [Ah] gene battery enzymes and the levels of reduced glutathione (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nuclear translocation-defective mutant (c4). DHII and TCDD increased the activities of ethoxyresorufin Odeethylase, an indicator of CYP1A1 activity, as well as NMO1, UGT1*O6, cytosolic aldehyde dehydrogenase class 3 and glutathione S-transferase form Al in wt cells, but had little or no induction effect in c37 or c4 cells. DHII and TCDD differed in their effects on GSH levels; while DHII increased GSH levels 3-fold in wt, but not at all in c37 or c4 cells, TCDD had no effect on GSH levels in any cell type. However, GSH levels were enhanced in both wt and c4 cells by tert-butyl hydroquinone (TBHQ). LButhionine S,R-sulfoximine, an inhibitor of γ-glutamylcysteine synthetase, prevented DHII-induced increases in wt cell GSH. The increase in GSH levels occurred after 8 h, while the induction of enzymes occurred within 4 h. The induction of the higher GSH levels in wt cells by DHII and TBHQ correlated with increases in intracellular levels of the GSH precursor thiol cysteine, as well as with increased activities of γ-glutamylcysteine synthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-mediated GSH increases in c4 cells were accompanied by increased γ-glutamylcysteine synthetase activity with no change in intracellular cysteine concentration. The results suggest that DHII induction of [Ah] gene battery enzymes requires a functional Ah receptor, but not the functional gene product CYP1A1. Furthermore, metabolism, possibly via CYP1A1, appears to be required for DHII to enhance intracellular levels of cysteine and GCS activity that result in higher GSH levels.