Liu R M, Sainsbury M, Tabor M W, Shertzer H G
Department of Environmental Health, University of Cincinnati Medical Center, OH 45267-0056.
Biochem Pharmacol. 1993 Oct 19;46(8):1491-9. doi: 10.1016/0006-2952(93)90117-f.
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.
已证明，从c14CoS/c14CoS和cch/cch小鼠新生肝脏中获得的细胞系对甲萘醌毒性具有遗传抗性（14CoS/14CoS细胞）或敏感性（ch/ch细胞）。这些差异部分归因于14CoS/14CoS细胞中相对较高水平的还原型谷胱甘肽（GSH）和NAD(P)H：甲萘醌氧化还原酶（NMO1）活性。吲哚类膜稳定抗氧化剂5,10-二氢茚并[1,2-b]吲哚（DHII）先前已被证明在体内和原代大鼠肝细胞中可抵御各种肝毒性物质。本报告描述了14CoS/14CoS和ch/ch细胞系如何提供一个有价值的实验系统，以区分DHII对甲萘醌毒性的化学保护机制。添加25μM DHII可使14CoS/14CoS细胞中甲萘醌介导的细胞死亡呈时间依赖性减少，而对ch/ch细胞活力影响很小。尽管DHII浓度在48小时内保持恒定，但最大保护作用在24小时出现。DHII的保护作用与谷胱甘肽水平升高（24小时时增加234%）以及诱导参与甲萘醌解毒和排泄的四种酶相关：NAD(P)H：甲萘醌氧化还原酶（NMO1，醌还原酶）、谷胱甘肽还原酶、谷胱甘肽转移酶（GST1A1）和UDP葡萄糖醛酸基转移酶（UGT1*06），24小时时最大诱导率分别为707%、201%、171%和198%。其他不直接参与甲萘醌代谢的生物转化酶（谷胱甘肽过氧化物酶、细胞色素P4501A1和P4501A2、铜锌依赖性超氧化物歧化酶和NADPH细胞色素c氧化还原酶）未被DHII诱导。仅在14CoS/14CoS细胞中，DHII可将甲萘醌刺激的超氧化物产生抑制50%，且该抑制需要24小时预孵育。用DHII预处理还可保护两种细胞类型免受甲萘醌介导的GSH消耗以及氧化应激指标（氧化型谷胱甘肽GSSG）百分比增加的影响。这些结果表明，DHII并非凭借其抗氧化或膜稳定特性来抵御甲萘醌毒性。相反，它通过诱导一种保护性酶谱来发挥作用，该酶谱可减轻氧化还原循环并促进甲萘醌的排泄。
