Mechanisms of Drug-Induced Liver Disease

今年，我们提供了更完整的氟烷的三氟乙酰化蛋白加合物（TFAPA）可能导致肝损伤的超微结构证据。采用的方法是用肝毒性剂量的氟烷治疗雌性Balb/cJ小鼠。 12小时后，肝毒性早期的时间点，从氟烷处理和未处理的小鼠中取出肝片。 将碎片化学固定用于常规透射电子显微镜（TEM），以研究氟烷剂量引起的亚细胞超微结构的任何变化。 透射电镜图像显示许多肝细胞的超微结构变化。氟烷处理小鼠的肝细胞比对照小鼠的肝细胞含有更多的脂滴，更多的受损线粒体和更多的扩张的滑面内质网。各种形态学变化表明线粒体损伤。这些变化包括基质中的小透明区域、明显的肿胀、致密的絮状体、内外膜分离和细胞膜的凝聚。将一些肝片固定，冷冻保护并冷冻用于冷冻免疫金标记，以使用TFAPA特异性抗血清在肝细胞中定位TFAPA。氟烷处理后形态正常的肝细胞在整个细胞上具有稀疏的免疫金标记。 相比之下，许多肝细胞在内质网、细胞质和较小程度的细胞核上被金标记。这些肝细胞显示脂滴增加和一些受损的线粒体。然而，在这些肝细胞中的大多数线粒体上标记稀疏，包括一些具有形态学损伤的线粒体。 一些浓缩的线粒体以及过氧化物酶体和自溶酶体被标记为类似于周围的细胞质。我们的研究结果表明，氟烷处理后12小时，TFAPA集中在受损肝细胞的几个隔室，但在未受损和一些受损的线粒体中的浓度要低得多。 这表明，尽管线粒体损伤是肝毒性的早期迹象，但氟烷诱导的线粒体损伤可能通过对其他细胞质细胞器的作用间接发生。 基于先前的研究结果，即氟烷毒性中的ER应激是肝细胞死亡的前兆，我们进一步研究了氟烷治疗后一段时间内ER应激和线粒体功能障碍的生物标志物。 我们评估了ER应激细胞死亡途径CHOP在肝细胞死亡中的作用，并确定ER应激信号在已知对氟烷诱导的肝损伤敏感的鼠品系中特别增加。 结论：氟烷诱导的肝损伤的小鼠模型继续提供关于亚细胞位点的重要信息，其中药物的蛋白加合物可能不仅由氟烷引起，而且由其他药物引起的肝损伤。

This year we provide more complete ultrastructural evidence of sites where trifluoroacetylated proteins adducts (TFAPA) of halothane may cause liver injury. The approach taken was to treat female Balb/cJ mice with a hepatotoxic dose of halothane. After 12 hours, a time point early in hepatotoxicity, liver pieces were removed from halothane treated and untreated mice. Pieces were chemically fixed for conventional transmission electron microscopy (TEM) to investigate any changes in subcellular ultrastructure resulting from the halothane dose. TEM images indicated ultrastructural changes in many hepatocytes. Hepatocytes from halothane treated mice contained more lipid droplets, more damaged mitochondria and more dilated smooth endoplasmic reticulum than hepatocytes from control mice. Various morphological changes indicated mitochondrial damage. These included small lucent regions in the matrix, overt swelling, dense flocculent bodies, separation of the inner and outer membrane and condensation of the mitochondrion. Some pieces of liver were fixed, cryoprotected and frozen for cryo-immunogold labeling to localize TFAPA in the hepatocytes, using TFAPA specific antiserum. Hepatocytes that appeared morphologically normal after the halothane treatment had sparse immunogold label over the entire cell. In contrast, many hepatocytes were heavily labeled with gold over the endoplasmic reticulum, cytoplasm and to a lesser extent, the nuclei. These hepatocytes showed increased lipid droplets and some damaged mitochondria. However, the labeling was sparse over most of the mitochondria in these hepatocytes, including some mitochondria with morphological damage. Some condensed mitochondria as well as peroxisomes and autolysosomes were labeled similar to surrounding cytoplasm. Our results indicate that 12 hours after treatment with halothane, TFAPA are concentrated in several compartments in the damaged hepatocytes, but are at much lower concentrations in undamaged and some damaged mitochondria. This suggests the possibility that, although mitochondrial damage is an early sign of hepatotoxicity, halothane-induced damage of mitochondria may occur indirectly through action on other cytoplasmic organelles. Based on previous findings that ER stress in halothane toxicity is a precursor to hepatocellular death, we further investigated biomarkers of ER stress and mitochondrial dysfunction over a time course following halothane treatment. We assessed the role of the ER-stress cell death pathway CHOP in hepatocellular death and determined that ER stress signaling is particularly increased in murine strains known to be susceptible to halothane-induced liver injury. Conclusion: The murine model of halothane-induced liver injury continues to provide important information concerning subcellular sites where protein adducts of drugs may initiate liver injury caused not only by halothane, but also by other drugs.