Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,- b]indole in a sensitive and resistant mouse hepatocyte line

Abstract

Established cell lines derived from newborn livers of c 14CoS c 14CoS and 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 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 25μM 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 24 hr), 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 mitigates redox cycling and facilitates excretion of menadione.