Mechanisms Of Drug-induced Toxicities

我们继续研究药物性肝病(DILD)的分子基础，这是一种罕见但往往危及生命的毒性。它是急性肝功能衰竭的主要原因，也是药物退出临床使用的主要原因。我们假设这种疾病的特殊性质部分是由于缺乏肝脏保护因子。今年，人们从以下几个方面对这一想法进行了探索： 1.用IL-13中和抗体对肝毒性剂量的对乙酰氨基酚(APAP)小鼠进行预处理，观察IL-13对DILD的保护作用。APAP治疗后24小时，中和抗体显著加重肝损伤。这一发现得到了证实，表明IL-13基因敲除小鼠比野生型小鼠更容易受到APAP诱导的肝损伤。 2.探讨了N-乙酰-L-半胱氨酸对APAP所致大鼠肝损伤的保护作用机制。这是通过研究NAC从APAP治疗引起的肝损伤中拯救小鼠后全基因组范围内肝脏mRNA表达的变化来完成的。基因芯片分析显示，NAC处理诱导了几个潜在的保肝因子的表达增加，包括热休克蛋白、急性期蛋白和细胞周期调节基因。 3.利用同位素编码亲和标签(ICAT)质谱仪，比较耐药株(SJL)和敏感株(C57BL/6)小鼠肝脏蛋白质组对APAP诱导的肝损伤的影响，探讨DILD的多源性。结果显示，SJL小鼠体内有几种蛋白质水平较高，这些蛋白质可能会保护肝脏免受损伤。这些物质包括过氧化还蛋白、应激反应蛋白和肝脏生长因子。C57BL/6小鼠肝脏线粒体蛋白也丢失，支持线粒体是APAP反应性代谢产物的靶点，在APAP诱导的肝损伤中起重要作用的假说。 4.目前的证据表明，DILD通常是由肝脏药物-蛋白质加合物引起的过敏反应(药物性变态反应性肝炎，DIAH)引起的。Diah的发生率很低，并且无法在动物身上复制，这表明耐受机制可能会阻止Diah在大多数人和动物中发生。我们已经获得了支持这一想法的证据。当小鼠接受APAP治疗时，对淋巴器官(胸腺、脾和淋巴结)的组织学和流式细胞术分析显示，T和B细胞区都有显著的淋巴细胞溶解，这部分归因于细胞凋亡。在没有明显肝毒性的情况下，没有明显的淋巴细胞溶解迹象。淋巴器官中细胞的减少与侵入肝脏的单个核细胞的增加有关，这些细胞因子的水平降低。这些发现表明，肝毒性剂量的APAP后广泛的淋巴细胞溶解可能抑制了对APAP蛋白加合物的适应性免疫反应，从而解释了大多数患者缺乏APAP诱导的过敏反应。类似的过程也可能发生在其他导致肝脏损伤的药物上。

We have continued our studies of the molecular basis of drug-induced liver disease (DILD), which is a rare but often life-threatening toxicity. It is the major cause of acute liver failure and a principal reason drugs are withdrawn from clinical use. We have hypothesized that the idiosyncratic nature of this disease is due in part to a deficiency in hepatoprotective factors. This idea has been explored in the following ways this year: 1. We tested whether IL-13 can protect against DILD by pretreating mice with IL-13 neutralizing antibody prior to the administration of a hepatotoxic dose of acetaminophen (APAP). The neutralizing antibody significantly exacerbated liver injury up to 24 hours after APAP treatment. This finding was confirmed by showing that IL-13 knockout mice were more susceptible than wild type mice to APAP-induced liver injury. 2. We explored the mechanism of N-acetyl-L-cysteine (NAC) protection against APAP-induced liver disease, an antidote used in clinical medicine. This was done by investigating the genome-wide changes in hepatic mRNA expression following NAC rescue of mice from liver injury caused by APAP treatment. Microarray analyses revealed that NAC treatment induced the increased expression of several potential hepatoprotectic factors including heat shock proteins, acute phase phase proteins, and cell cycle regulation genes. 3. We investigated the polygenicity of DILD by comparing the liver proteomes of resistant (SJL) and susceptible (C57Bl/6) strains of mice to APAP-induced liver injury with the use of isotope-coded affinity tag (ICAT) mass spectrometry. The results revealed that SJL mice had higher levels of several proteins that could potentially protect the liver from injury. These included peroxiredoxins, stress response proteins, and hepatic growth factors. There also was a loss of mitochondrial proteins from the livers of the C57Bl/6 mice, supporting the hypothesis that mitochondria is a target of the reactive metabolite of APAP and has a significant role in APAP-induced liver injury. 4. Current evidence indicates that DILD is often caused by an allergic response (drug-induced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The low incidence of DIAH and inability to reproduce it in animals suggests that tolerogenic mechanisms may prevent DIAH from occurring in most people and animals. We have obtained evidence to support this idea. When mice were treated with APAP, histological and flow cytometric analyses of the lymphoid organs (thymus, spleen, and lymph nodes) revealed a marked lymphocytolysis in both the T- and B-cell zone areas that was attributed in part to apoptosis. No signs of lymphocytolysis were apparent in the absence of significant hepatotoxicity. The decrease in cells in the lymphoid organs was associated with an increase of mononuclear cells infiltrating the liver that had depressed levels of several cytokines. These findings suggest that the widespread lymphocytolysis following a hepatotoxic dose of APAP may inhibit an adaptive immune response to APAP-protein adducts, thereby accounting for the lack of APAP-induced allergic reactions in most patients. Similar processes may occur with other drugs that cause liver injury.