Mechanism for human-specific hepatotoxicity and development of prediction system

为了确定人类特异性肝毒性的机制并开发预测系统，使用了氟替卡松和胆汁酸模型。使用人β-内酰胺酶表达的微粒体分析氟替卡松的代谢。FLU-1-N-OH（N-[4-硝基-3-（三氟甲基）苯基]羟胺）是氟他胺诱导肝毒性的候选代谢中间体，由人CYP 3A 4从FLU-1产生。在小鼠中，FLU-1 N-羟基化主要由不同于Cyp 3a亚型的Cyp亚型催化。此外，通过用FLU-1和TCPOBOP联合处理建立小鼠肝毒性模型。本研究采用小鼠肝毒性模型，通过胆汁酸与药物的相互作用，探讨氟鲁米胺肝毒性的机制。胆酸（CA）或氨苄青霉素治疗不会引起肝毒性，但CA和氨苄青霉素联合治疗会引起小鼠严重的肝毒性。这些结果表明CA和氨苄青霉素之间的相互作用参与肝毒性的存在。另一方面，胆酸处理在类佛尼酯X受体（FXR）缺失小鼠中引起肝毒性。在与1.25%胆固醇（Chol）共同治疗后，与CA饮食对照组相比，喂食0.5%胆酸饮食的FXR缺失小鼠的血清ALT和ALP活性显著降低。在喂食CA加Chol饲料的FXR基因敲除小鼠中，通过原位袢法估计的回肠胆汁酸吸收能力和回肠Asbt蛋白含量明显降低。这些结果表明胆固醇对胆汁酸诱导的毒性具有保护作用，并且还表明回肠胆汁酸吸收存在FXR非依赖性抑制机制。

To identify the mechanism for human-specific hepatotoxicity and develop the prediction system, flutamide and bile acid models were used. The metabolism of flutamide was analyzed using human CYP expressed microsomes. FLU-1-N-OH (N-[4-nitro-3-(trifluoromethyl)phenyl]hydroxylamine) which is a candidate of metabolic intermediate for flutamide-induced hepatotoxicity was produced from FLU-1 by human CYP3A4. In mice, FLU-1 N-hydroxylation was mainly catalyzed by Cyp isoforms different from Cyp3a isoforms. Furthermore, mouse hepatotoxicity model was established by co-treatment with FLU-1 and TCPOBOP. We are analyzing the mechanism for fulutamide-induced hepatotoxicity using the mouse model.To analyze the mechamism for hepatotoxicity through the interaction between endogenous compound (bile acid) and drug, C57/BL6 male mice were treated with cholic acid and ampicillin. Cholic acid (CA) or ampicillin treatment did not cause hepatotoxicity, but CA and ampicillin co-treatment cause severe hepatotoxicity in the mice. These results suggest the presence of the interaction between CA and ampicilin involved in hepatotoxicity. On the other hand, cholic acid treatment causes hepatotoxicity in fornesoid X receptor (FXR)-null mice. After co-treatment with 1.25% cholesterol (Chol), serum ALT and ALP activities were significantly decreased in FXR-null mice fed a 0.5% cholic acid diet, as compared to those of the CA diet controls. Clear decreases were observed on ileal bile acid absorption capacity estimated by a in situ loop method and contents of ileal Asbt protein in FXR-null mice fed the CA plus Chol diet. These results indicate a protective role of cholesterol against bile acid-induced toxicity and also suggest the presence of an FXR-independent suppressive mechanism of ileal bile acid absorption.