Elucidation of Mechanism of Drug-Induced Liver Injury Based on Danger Hypothesis

对炎性肠病的动物模型进行炎性条件下肝功能的表征。用100 mg/kg三硝基苯磺酸（TNBS）对大鼠进行结肠内给药，诱导出血性结肠炎。结肠炎伴随门静脉内毒素、白细胞介素-6和一氧化氮代谢产物水平升高，肝脏CYP 3A 2含量和活性降低。尼美舒利，一个优先的考克斯-2抑制剂保护大鼠与TNBS结肠炎对下调肝脏CYP 3A 2。多粘菌素B（可中和内毒素）、姜黄素（具有抗炎特性）和氯化钆（可灭活巨噬细胞）可减弱CYP 3A 2的下调。这些数据表明，TNBS-结肠炎大鼠受损结肠泄漏的内源性物质激活枯否细胞，导致肝脏P450下调。因此，提出肠源性炎症刺激在药物诱导的肝脏效应中表现为“危险信号”。在其他研究中，通过测量与不同动物种属肝微粒体的代谢依赖性共价结合，评估了共价加合物在双氯芬酸肝毒性中的作用。与NADPH孵育后，大鼠和小鼠中[14 C]双氯芬酸衍生的放射性与微粒体蛋白的共价结合高于人体。与双氯芬酸的5-羟基化类似，雄性大鼠中的代谢依赖性共价结合高于雌性大鼠，而人体中的该活性非常低。降低共价结合的GSH在雄性小鼠中比在雄性大鼠中更有效。因此，在小鼠中，反应性代谢物可以有效地到达P450以外的蛋白质，并可能导致肝毒性，这已通过在小鼠中进行的双氯芬酸诱导的肝损伤的体内研究证实。这些发现表明，与特定靶点的共价结合和随后的炎症刺激是药物诱导的肝毒性的原因。

Animal models of inflammatory bowel disease were subjected to characterization of liver function under inflammatory conditions. Rats were treated intracolonically with 100 mg/kg trinitrobenzene sulfonic acid (TNBS), which induced hemorrhagic colitis. The colitis accompanies appearance of higher levels of portal endotoxin, interleukin-6 and nitric oxide metabolites, and decreases in contents and activities for hepatic CYP3A2. Nimesulide, a preferential COX-2 inhibitor protected rats with TNBS-colitis against the down-regulation of hepatic CYP3A2. Polymyxin B, which neutralizes endotoxin, curcumin, which has anti-inflammatory properties, and gadolinium chloride, which inactivates macrophages, attenuated the down-regulation of CYP3A2. These data suggest that endogenous substances leaked from damaged colon in the rats with TNBS-colitis activate Kupffer cells, leading to down-regulation of hepatic P450s. It is thus proposed that gut-derived inflammatory stimuli behave as "danger signal" in drug-induced liver effects. In other studies, role of covalent adduct in diclofenac hepatotoxicity was assessed by measuring metabolism-dependent covalent binding to hepatic microsomes from various animal species. Covalent binding [14C]diclofenac- derived radioactivity to microsomal protein after incubation with NADPH was higher in rats and mice than in humans. Similar to diclofenac '5-hydroxylation, the metabolism-dependent covalent binding was higher in male rats than in females, whereas this activity in human is very low. GSH, which lowers covalent binding, was more effective in male mice than in male rats. Thus, reactive metabolites can reach proteins other than P450 effectively in mice and may lead to hepatotoxicity, which was confirmed by in vivo study developing diclofenac-induced liver injury in mice. These findings suggest that both covalent binding to the specific targets and subsequent inflammatory stimuli are responsible for drug-induced hepatotoxicity.