Keller B J, Yamanaka H, Liang D C, Thurman R G
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Toxicol Appl Pharmacol. 1990 Jun 15;104(2):259-66. doi: 10.1016/0041-008x(90)90300-j.
Toxicity of clofibrate, a hypolipidemic drug, was assessed in livers from fasted rats perfused in both the anterograde and the retrograde directions. Oxygen uptake decreased steadily following infusion of clofibrate (15 mM) and was diminished by about 40% in 15 min. Cell damage, assessed by the appearance of lactate dehydrogenase (LDH) in the effluent perfusate, began within 20 min. Maximal values for LDH release into perfusate were around 250 U/g/hr after perfusion with clofibrate for 40 min. Inhibition of oxygen uptake and release of LDH into the perfusate was dose-dependent (half-maximal effect = ca. 12 mM clofibrate). Nearly 90% of hepatocytes in oxygen-rich, periportal regions but only about 30% in oxygen-poor, pericentral areas took up trypan blue, an indicator of irreversible cell death, following perfusion with clofibrate in the anterograde direction. In contrast, when livers were perfused in the retrograde direction, 85% of cells in upstream, oxygen-rich pericentral regions were damaged whereas only about 30% in downstream areas were stained. When local oxygen tension was lowered by reducing the flow rate to one-quarter of normal, trypan blue uptake in periportal areas was diminished nearly completely (ca. 5% of cells were stained). Incubation in vitro of isolated cylinders of periportal and pericentral tissue with clofibrate at 800 or 200 microM oxygen led to about three times greater LDH release in incubations carried out at high than at low oxygen tension. This experiment led us to rule out the involvement of clofibrate delivery in the mechanism of zone-specific toxicity. Subsequently, local rates of oxygen uptake were measured using miniature oxygen electrodes placed on the liver surface. Clofibrate decreased oxygen uptake about 30% in oxygen-rich, periportal regions of the liver lobule, yet had no effect on respiration in downstream, pericentral areas. These phenomena can best be explained by a direct effect of clofibrate on active mitochondria in periportal regions of the liver lobule where oxygen uptake predominates, since state 3 but not state 4 rates of respiration were inhibited by clofibrate in isolated mitochondria (half-maximal effect = ca. 1.8 mM clofibrate). Thus, toxicity of clofibrate in upstream, periportal areas of the liver lobule is dependent on local oxygen tension and affects actively respiring mitochondria. This may lead to local cell death and be responsible for initiating a sequence of events leading to the well-known carcinogenic effects of this compound.
对降血脂药物氯贝丁酯的毒性进行了评估，实验对象是在顺行和逆行灌注条件下的禁食大鼠肝脏。注入氯贝丁酯（15 mM）后，氧摄取量持续下降，15分钟内减少了约40%。通过流出灌注液中乳酸脱氢酶（LDH）的出现来评估细胞损伤，损伤在20分钟内开始。用氯贝丁酯灌注40分钟后，灌注液中LDH释放的最大值约为250 U/g/小时。氧摄取的抑制和LDH向灌注液中的释放呈剂量依赖性（半数最大效应 = 约12 mM氯贝丁酯）。顺行灌注氯贝丁酯后，富含氧气的门静脉周围区域近90%的肝细胞摄取了台盼蓝，台盼蓝是不可逆细胞死亡的指标，但缺氧的中央静脉周围区域只有约30%的肝细胞摄取了台盼蓝。相反，当肝脏逆行灌注时，上游富含氧气的中央静脉周围区域85%的细胞受损，而下游区域只有约30%的细胞被染色。当流速降至正常的四分之一从而降低局部氧张力时，门静脉周围区域的台盼蓝摄取几乎完全减少（约5%的细胞被染色）。在800或200 microM氧气条件下，将分离的门静脉周围和中央静脉周围组织圆柱体与氯贝丁酯进行体外孵育，结果显示，在高氧张力下孵育时LDH的释放量比低氧张力下高出约三倍。该实验使我们排除了氯贝丁酯递送参与区域特异性毒性机制的可能性。随后，使用置于肝脏表面的微型氧电极测量局部氧摄取率。氯贝丁酯使肝小叶富含氧气的门静脉周围区域的氧摄取量降低了约30%，但对下游中央静脉周围区域的呼吸没有影响。这些现象最好通过氯贝丁酯对肝小叶门静脉周围区域活跃线粒体的直接作用来解释，在该区域氧摄取占主导地位，因为在分离的线粒体中，氯贝丁酯抑制了呼吸的状态3速率而非状态4速率（半数最大效应 = 约1.8 mM氯贝丁酯）。因此，氯贝丁酯在肝小叶上游门静脉周围区域的毒性取决于局部氧张力，并影响活跃呼吸的线粒体。这可能导致局部细胞死亡，并引发一系列事件，从而导致该化合物众所周知的致癌作用。
