The mechanism of bromobenzene-induced cytotoxicity studied with isolated hepatocytes
The mechanism of bromobenzene-induced cytotoxicity studied with isolated hepatocytes
复制标题
分离肝细胞研究溴苯诱导的细胞毒性机制
作者：
H. Thor;S. Orrenius
新鲜分离的大鼠肝细胞将溴苯代谢为高反应性环氧化物，该环氧化物通过与谷胱甘肽 (GSH) 结合而失活。在谷胱甘肽耗尽的细胞中观察到细胞损伤增加，而这些细胞反过来又通过促进谷胱甘肽再合成而免受溴苯毒性。这是在培养基中存在半胱氨酸或蛋氨酸的情况下实现的。扫描电子显微镜显示，溴苯诱导的细胞毒性与肝细胞超微结构外观的显着变化有关。正常绒毛表面结构的丧失是损伤的早期迹象，随后形成气泡、破裂，最终导致细胞碎裂。肝细胞代谢溴苯不仅会导致 GSH 消耗，还会导致其他低分子亲核试剂（包括辅酶 NAD(H)、NADP(H) 和辅酶 A）水平降低。它还与蛋白质结合代谢物的形成和抑制某些酶活性，例如葡萄糖-6-磷酸脱氢酶和谷胱甘肽还原酶活性。当肝细胞与溴苯一起孵育时，O2 吸收率降低。长时间培养（3 小时）后，向培养基中添加葡萄糖、乳酸或琥珀酸盐后，O2 吸收受到刺激，但不会恢复正常。在底物诱导、ADP 刺激的 O2 摄取和琥珀酸细胞色素 c 还原酶活性方面，从暴露于溴苯的肝细胞中分离的线粒体与从对照细胞中分离的线粒体相当。此外，ATP 的损失是溴苯诱导的细胞毒性的晚期事件。溴苯诱导的细胞毒性的许多早期迹象可能与质膜渗透性的变化有关。这些包括绒毛表面结构的丧失和起泡、NADH、台盼蓝和琥珀酸盐渗透以及钾从细胞中渗漏。此外，某些氨基酸（例如蛋氨酸）的摄取受到抑制，并且分离的肝质膜组分与肝微粒体、NADPH和溴苯一起孵育会导致Mg2+依赖性、Na+、K+刺激的ATP酶活性失活。因此，质膜似乎可能是溴苯诱导的细胞毒性的主要靶标。然而，损伤是否是由反应性代谢物对关键质膜成分的直接攻击介导的，仍有待确定。
Freshly isolated rat hepatocytes metabolize bromobenzene to a highly reactive epoxide which is inactivated by conjugation with glutathione (GSH). Increased cell damage is observed with GSH-depleted cells which, in turn, are protected from bromobenzene toxicity by facilitated GSH resynthesis. This is achieved in the presence of cysteine or methionine in the medium.Scanning electron microscopy reveals that bromobenzene-induced cytotoxicity is associated with marked changes in the ultrastructural appearance of the hepatocytes. A loss of the normal villous surface structure is an early sign of damage which is followed by formation of blebs, rupture and, eventually, fragmentation of the cells.Metabolism of bromobenzene by hepatocytes does not only lead to GSH depletion but also to decreased levels of other low-molecular nucleophiles including the coenzymes NAD(H), NADP(H) and coenzyme A. It is also associated with the formation of protein-bound metabolites and inhibition of certain enzyme activities, e g glucose-6-phosphate dehydrogenase and glutathione reductase activities.The rate of O2 uptake is decreased when hepatocytes are incubated with bromobenzene. After prolonged incubation (3 h) O2 uptake is stimulated, but not restored to normal, upon addition of glucose, lactate or succinate to the medium. Mitochondria isolated from bromobenzene-exposed hepatocytes are comparable to those isolated from control cells in regards to substrate-induced, ADP-stimulated O2 uptake and succinate-cytochrome c reductase activity. Further, loss of ATP is a late event in bromobenzene-induced cytotoxicity.Many of the early signs of bromobenzene-induced cytotoxicity may be related to a change in permeability properties of the plasma membrane. These include loss of villous surface structure and blebbing, NADH, trypan blue and succinate penetration, and potassium leakage from the cells. Moreover, the uptake of certain amino acids, e g methionine, is inhibited and the incubation of an isolated hepatic plasma membrane fraction with liver microsomes, NADPH and bromobenzene results in inactivation of Mg2+-dependent, Na+, K+-stimulated ATPase activity.Thus, it appears that the plasma membrane may be a primary target in bromobenzene-induced cytotoxicity. Whether the damage is mediated by a direct attack of the reactive metabolite on critical plasma membrane component(s) remains, however, to be established.