REGULATION OF [AH] GENE BATTERY ENZYMES AND GLUTATHIONE LEVELS BY 5,10-DIHYDROINDENO[1,2-B]INDOLE IN MOUSE HEPATOMA-CELL LINES

The murine aromatic hydrocarbon ([Ah]) gene battery consists of at lea st six genes that code for two functionalizing (Phase I) enzymes and f our non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibe nzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptor protein. St udies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT106) an d NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the s ynthetic antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII). The induc tion of [Ah] gene battery enzymes and the levels of reduced glutathion e (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt ), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nucle ar translocation-defective mutant (c4). DHII and TCDD increased the ac tivities of ethoxyresorufin O-deethylase, an indicator of CYP1A1 activ ity, as well as NMO1, UGT106 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 thei r 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 i n any cell type. However, GSH levels were enhanced in both wt and c4 c ells by tert-butyl hydroquinone (TBHQ). L-Buthionine S,R-sulfoximine, an inhibitor of gamma-glutamyl-cysteine synthetase, prevented DHII-ind uced increases in wt cell GSH. The increase in GSH levels occurred aft er 8 h, while the induction of enzymes occurred within 4 h. The induct ion of the higher GSH levels in wt cells by DHII and TBHQ correlated w ith increases in intracellular levels of the GSH precursor thiol cyste ine, as well as with increased activities of gamma-glutamylcysteine sy nthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-med iated GSH increases in c4 cells were accompanied by increased gamma-gl utamylcysteine synthetase activity with no change in intracellular cys teine concentration. The results suggest that DHII induction of [Ah] g ene battery enzymes requires a functional Ah receptor, but not the fun ctional gene product CYP1A1. Furthermore, metabolism, possibly via CYP 1A1, appears to be required for DHII to enhance intracellular levels o f cysteine and GCS activity that result in higher GSH levels.