Mechanisms of Drug-Induced Liver Disease

药物性肝病的机制

• 批准号: 8939855
• 负责人: Lance R Pohl
• 金额: $ 25.1万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939855
• 关键词: Acetaminophen; Aftercare; Animal Model; Biological Markers; Cell Death; Cell Nucleus; Cells; Cessation of life; Clinical Trials; Cytoplasm; Cytoplasmic Organelle; Development; Dose; Electron Microscopy; Endoplasmic Reticulum; FarGo; Female; Freezing; Gold; Halothane; Hepatitis; Hepatocyte; Hepatotoxicity; Hour; Human; Image; Immune Sera; Immunoelectron Microscopy; Inhalation Anesthetics; Label; Lipids; Liver; Marketing; Membrane; Mitochondria; Modeling; Mus; Pathway interactions; Patients; Pharmaceutical Preparations; Physical condensation; Protein Synthesis Inhibition; Proteins; Relative (related person); Reporting; Risk Factors; Role; Signal Transduction; Site; Smooth Endoplasmic Reticulum; Stress; Swelling; Techniques; Time; Toxic effect; Translating; Transmission Electron Microscopy; adduct; base; drug induced liver disease; endoplasmic reticulum stress; liver injury; mitochondrial dysfunction; peroxisome; pre-clinical; response

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.

今年，我们提供了更完整的超微结构证据，证明氟烷的三氟乙酰化蛋白加合物 (TFAPA) 可能导致肝损伤。采取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，即肝毒性早期的时间点，从氟烷治疗和未治疗的小鼠中取出肝脏碎片。 对碎片进行化学固定，用于传统透射电子显微镜 (TEM)，以研究氟烷剂量引起的亚细胞超微结构的任何变化。 TEM 图像显示许多肝细胞的超微结构变化。与对照小鼠的肝细胞相比，氟烷处理小鼠的肝细胞含有更多的脂滴、更多受损的线粒体和更多扩张的光滑内质网。各种形态变化表明线粒体损伤。这些包括基质中的小透明区域、明显的膨胀、致密的絮状体、内膜和外膜的分离以及线粒体的浓缩。将一些肝脏碎片固定、冷冻保护并冷冻进行冷冻免疫金标记，使用 TFAPA 特异性抗血清将 TFAPA 定位在肝细胞中。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，许多肝细胞的内质网、细胞质以及较小程度的细胞核都被大量金标记。这些肝细胞显示出增加的脂滴和一些受损的线粒体。然而，这些肝细胞中的大多数线粒体上的标记都很稀疏，包括一些形态受损的线粒体。 一些浓缩的线粒体以及过氧化物酶体和自溶酶体的标记与周围的细胞质相似。我们的结果表明，用氟烷处理 12 小时后，TFAPA 集中在受损肝细胞的几个区室中，但在未受损和一些受损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。 基于先前的发现，即氟烷毒性中的内质网应激是肝细胞死亡的先兆，我们进一步研究了氟烷治疗后一段时间内内质网应激和线粒体功能障碍的生物标志物。 我们评估了内质网应激细胞死亡途径 CHOP 在肝细胞死亡中的作用，并确定内质网应激信号在已知易受氟烷诱导的肝损伤的小鼠品系中尤其增加。 结论：氟烷引起的肝损伤小鼠模型继续提供有关药物蛋白加合物可能引发肝损伤的亚细胞位点的重要信息，这些肝损伤不仅由氟烷引起，而且还由其他药物引起。

项目成果

Pathologic role of stressed-induced glucocorticoids in drug-induced liver injury in mice.

• DOI: 10.1016/j.bbrc.2010.05.126
• 发表时间: 2010-07-02
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Masson, Mary Jane;Collins, Lindsay A.;Carpenter, Leah D.;Graf, Mary L.;Ryan, Pauline M.;Bourdi, Mohammed;Pohl, Lance R.
• 通讯作者: Pohl, Lance R.