vitamin-k-semiquinone-radical and indenoindole

We have studied the response of genes in the dioxin-inducible [Ah] battery to three compounds that protect mouse hepatoma cells (Hepa-1c7c7 wild-type, wt) against menadione toxicity. Pretreatment of wt cells with 25 microM 5,10-dihydroindenol[1,2-b]indole (DHII), 25 microM tert-butylhydroquinone (tBHO) or 10 microM menadione itself, generated substantial protection against toxicity produced by subsequent menadione exposure. The gene response was examined in wt cells, and three mutant lines: CYP1A1 metabolism-deficient (c37 or P1-); nuclear translocation-impaired (c4 or nt-); and AHR-deficient (c2 or r-, containing < 10% of normal functional receptor levels). DHII treatment of wt cells for 12 hr markedly elevated the enzyme activities and mRNA levels of genes in the [Ah] battery: aryl hydrocarbon hydroxylase (Cyp1a1), NAD(P)H:menadione oxidoreductase (Nmol), cytosolic aldehyde dehydrogenase class 3 (Ahd4), and UDP-glucuronosyltransferase form 1*06 (Ugt1*06). Treatment of the c4 and c2 cells with DHII failed to induce mRNA levels of the genes, indicating that induction of the [Ah] gene battery by DHII is aromatic hydrocarbon receptor (AHR)-mediated. On the other hand, neither tBHO nor menadione caused increases in CYPlAl mRNA, but tBHQ significantly enhanced the NMO1, AHD4, and UGT1*06 mRNA levels in all three mutant cell lines. In conclusion, we expect one or more putative electrophile response elements (EpRE), previously found in the regulatory regions of the murine Nmol, Ahd4, and ugt1*06 genes, to be functional in responding to phenolic antioxidants.

Topics: Aldehyde Dehydrogenase; Animals; Antioxidants; Cytochrome P-450 CYP1A2; Cytochrome P-450 Enzyme System; Enzyme Induction; Glucuronosyltransferase; Hydroquinones; Indoles; Mice; NAD(P)H Dehydrogenase (Quinone); Oxidoreductases; Receptors, Aryl Hydrocarbon; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Vitamin K

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.

Topics: Animals; Animals, Newborn; Cell Line; Glutathione; Indoles; Liver; Mice; Mice, Mutant Strains; NAD(P)H Dehydrogenase (Quinone); Superoxides; Vitamin K