MECHANISMS OF PROTECTION FROM MENADIONE TOXICITY BY 5,10-DIHYDROINDENO[1,2,-B]INDOLE IN A SENSITIVE AND RESISTANT MOUSE HEPATOCYTE LINE

Established cell lines derived from newborn livers of c14CoS/c14CoS an d c(ch)/c(ch) 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 gl utathione (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 hepatocyte s. This report describes how the 14CoS/14CoS and ch/ch cell lines prov ide a valuable experimental system to distinguish the mechanism of che moprotection by DHII from menadione toxicity. The addition of 25 muM D HII produced a time-dependent decrease in menadione-mediated cell deat h in 14CoS/14CoS cells, with little effect on ch/ch cell viability. Th e maximum protective effect occurred at 24 hr, although the concentrat ion of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24 hr), as well as induction of four enzymes involved in the detoxification a nd excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, qui none reductase), glutathione reductase, glutathione transferase (GST1A 1), and UDP glucuronosyltransferase (UGT106), with 24-hr maximum indu ction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione pe roxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent sup eroxide dismutase, and NADPH cytochrome c oxidoreductase) were not ind uced 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 typ es against the menadione-mediated depletion of GSH. and the increase i n percent (oxidized glutathione GSSG), an indicator of oxidative stres s. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properti es. Rather, it acts by inducing a protective enzyme profile that mitig ates redox cycling and facilitates excretion of menadione.