Burcham P C, Harman A W
Department of Pharmacology, Queen Elizabeth II Medical Centre, University of Western Australia, Nedlands.
J Biol Chem. 1991 Mar 15;266(8):5049-54.
Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.
将分离的小鼠肝细胞暴露于对乙酰氨基酚的毒性浓度（5 mM）会导致线粒体呼吸装置受损。通过测量对乙酰氨基酚暴露后洋地黄皂苷透化肝细胞中位点特异性底物刺激的呼吸作用，来研究这种损伤的性质。能量偶联位点2处由琥珀酸刺激的呼吸作用对抑制最为敏感，1小时后降低了47%。由NADH连接的底物（位点1）支持的呼吸作用也有所降低，但程度较小，而抗坏血酸+N，N，N'，N'-四甲基对苯二胺（TMPD）支持的呼吸作用速率没有降低（位点3）。线粒体呼吸功能的丧失伴随着胞质区室中ATP水平和ATP/ADP比值的降低，并且在胞质溶胶和线粒体中还原型谷胱甘肽丧失之前就已出现。所有这些效应都在细胞膜完整性丧失之前就已发生。对乙酰氨基酚的假定有毒代谢产物N-乙酰对苯醌亚胺（NAPQI）在分离的肝线粒体中产生了类似的呼吸功能障碍模式。由琥珀酸和NADH连接的底物刺激的呼吸作用对50 microM NAPQI非常敏感，而抗坏血酸+TMPD支持的呼吸作用不受影响。使用亚线粒体颗粒进一步研究了NAPQI与呼吸链之间的相互作用。发现琥珀酸脱氢酶（与呼吸复合体II相关）对NAPQI非常敏感，而NADH脱氢酶（呼吸复合体I）受到的抑制程度较小。我们的结果表明，由于NAPQI对呼吸链的攻击，导致利用琥珀酸和NADH连接底物的能力丧失，从而在对乙酰氨基酚肝毒性中引起能量稳态的破坏。
