Lund B O, Miller D M, Woods J S
Department of Environmental Health, University of Washington, Seattle 98195.
Biochem Pharmacol. 1993 May 25;45(10):2017-24. doi: 10.1016/0006-2952(93)90012-l.
Studies were undertaken to investigate the principal actions underlying mercury-induced oxidative stress in the kidney. Mitochondria from kidneys of rats treated with HgCl2 (1.5 mg/kg i.p.) demonstrated a 2-fold increase in hydrogen peroxide (H2O2) formation for up to 6 hr following Hg(II) treatment using succinate as the electron transport chain substrate. No increase in H2O2 formation was observed when NAD-linked substrates (malate/glutamate) were used, suggesting that Hg(II) affects H2O2 formation principally at the ubiquinone-cytochrome b region of the mitochondrial respiratory chain in vivo. Together with increased H2O2 formation, mitochondrial glutathione (GSH) content was depleted by more than 50% following Hg(II) treatment, whereas formation of thiobarbiturate reactive substances (TBARS), indicative of mitochondrial lipid peroxidation, was increased by 68%. Studies in vivo revealed a significant concentration-related depolarization of the inner mitochondrial membrane following the addition of Hg(II) to mitochondria isolated from kidneys of untreated rats. This effect was accompanied by significantly increased H2O2 formation, GSH depletion and TBARS formation linked to both NADH dehydrogenase (rotenone-inhibited) and ubiquinone-cytochrome b (antimycin-inhibited) regions of the electron transport chain. Oxidation of pyridine nucleotides (NAD[P]H) was also observed in mitochondria incubated with Hg(II) in vitro. In further studies in vitro, the potential role of Ca2+ in Hg(II)-induced mitochondrial oxidative stress was investigated. Ca2+ alone (30-400 nmol/mg protein) produced no increase in H2O2 and only a slight increase in TBARS formation when incubated with kidney mitochondria isolated from untreated rats. However, Ca2+ significantly increased H2O2 and TBARS formation elicited by Hg(II) at the ubiquinone-cytochrome b region of the mitochondrial electron transport chain, whereas TBARS formation was decreased significantly when the Ca2+ uptake inhibitors, ruthenium red or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), were included with Hg(II) in the reaction mixtures. These findings support the view that Hg(II) causes depolarization of the mitochondrial inner membrane with consequent increased H2O2 formation. These events, coupled with Hg(II)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant stress condition characterized by increased susceptibility of mitochondrial membranes to iron-dependent lipid peroxidation (TBARS formation). Since increased H2O2 formation, GSH depletion and lipid peroxidation were also observed in vivo following Hg(II) treatment, these events may underlie oxidative tissue damage caused by mercury compounds. Moreover, Hg(II)-induced alterations in mitochondrial Ca2+ homeostasis may exacerbate Hg(II)-induced oxidative stress in kidney cells.
开展了多项研究以探究汞诱导肾脏氧化应激的主要作用机制。用氯化汞（1.5毫克/千克腹腔注射）处理的大鼠肾脏中的线粒体，在以琥珀酸作为电子传递链底物进行汞（II）处理后的6小时内，过氧化氢（H2O2）生成量增加了2倍。当使用与NAD相关的底物（苹果酸/谷氨酸）时，未观察到H2O2生成量增加，这表明汞（II）在体内主要在线粒体呼吸链的泛醌 - 细胞色素b区域影响H2O2的生成。随着H2O2生成量增加，汞（II）处理后线粒体谷胱甘肽（GSH）含量减少了50%以上，而作为线粒体脂质过氧化指标的硫代巴比妥酸反应性物质（TBARS）的生成量增加了68%。体内研究显示，向从未经处理的大鼠肾脏分离的线粒体中添加汞（II）后，线粒体内膜出现了与浓度相关的显著去极化。这种效应伴随着与电子传递链的NADH脱氢酶（鱼藤酮抑制）和泛醌 - 细胞色素b（抗霉素抑制）区域相关的H2O2生成量显著增加、GSH耗竭以及TBARS生成。在体外与汞（II）一起孵育的线粒体中也观察到了吡啶核苷酸（NAD[P]H）的氧化。在进一步的体外研究中，研究了Ca2+在汞（II）诱导的线粒体氧化应激中的潜在作用。单独的Ca2+（30 - 400纳摩尔/毫克蛋白质）与从未经处理的大鼠分离的肾脏线粒体一起孵育时，H2O2生成量没有增加，TBARS生成量仅略有增加。然而，Ca2+显著增加了汞（II）在线粒体电子传递链泛醌 - 细胞色素b区域引发的H2O2和TBARS生成，而当反应混合物中加入Ca2+摄取抑制剂钌红或[乙二胺双（氧乙基腈）]四乙酸（EGTA）与汞（II）一起时，TBARS生成量显著降低。这些发现支持了汞（II）导致线粒体内膜去极化并随之增加H2O2生成的观点。这些事件，再加上汞（II）介导的GSH耗竭和吡啶核苷酸氧化，产生了一种氧化应激状态，其特征是线粒体膜对铁依赖性脂质过氧化（TBARS生成）的敏感性增加。由于在汞（II）处理后的体内也观察到了H2O2生成量增加、GSH耗竭和脂质过氧化，这些事件可能是汞化合物引起氧化组织损伤的基础。此外，汞（II）诱导的线粒体Ca2+稳态改变可能会加剧汞（II）诱导的肾脏细胞氧化应激。
