Xenobiotic metabolism, cancer chemoprevention and cancer biomarkers

PPARpha在胆汁酸(BA)动态平衡中的作用开始显现。用有效的PPARpha激动剂Wy-14,643(Wy)和全球代谢组学来确定肝细胞PPARpha在BA稳态调节中的作用，揭示了超过FXR的BA稳态控制的新水平。WY处理的野生型小鼠的血清BA水平显著升高，而Pparnull和PpardHep小鼠的血清BA水平没有明显升高。基因表达分析表明，PPARpha激活(1)下调负责BAS进入肝脏的牛磺胆酸钠转运多肽和有机离子转运多肽1和4的表达；(2)降低将BA从肝细胞输送到胆小管的胆盐输出泵的表达；(3)上调从肝细胞向门静脉输送BA的多药耐药相关蛋白3和4的表达。此外，WY治疗后血清、肝脏和胆汁中胆酸和牛磺胆酸的组成显著增加，这与编码甾醇12α-羟基酶的Cyp8b1基因表达上调有关。WY在Ppara-dHep小鼠和Ppara-null小鼠之间的作用是相同的。肝细胞PPARpha不仅通过直接转录调节调控BA的合成和转运，而且还通过与肝脏FXR信号的串扰来调控BA的合成和运输。这些发现强调了肝细胞PPARpha在BA动态平衡控制中的关键作用。BA也与某些药物引起的肝毒性有关，吴茱萸次碱(RUT)、吴茱萸碱(EOD)和脱氢吴茱萸碱(DHed)是从吴茱萸中分离出来的三种主要生物活性吲哚喹唑啉生物碱，吴茱萸是一种广泛使用的中药。在这里，这些类似物激活芳香烃受体(AHR)的构效关系是通过利用AhR缺失(AhR-Null)小鼠、原代肝细胞培养、荧光素酶报告基因分析、硅胶配基对接研究和代谢组学来探索的。体外小鼠原代肝细胞AHR靶基因的信使核糖核酸分析和肝癌细胞株荧光素酶报告基因的检测均表明，RUT、EOD和DHed均能显著激活AHR，其作用顺序为RUT、EOD和DHed。配体对接分析表明，N-14位的甲基取代是影响AHR活性的关键因素。在体内，EOD口服吸收差，不能激活AHR，而RUT和DHT显著上调野生型小鼠肝脏AHR基因的表达，但在AhR缺失小鼠中不表达。此外，在使用的剂量下，RUT、EOD和DHED没有肝毒性；然而，RUT和DHED以AHR依赖的方式破坏BA的动态平衡。这些结果表明，这些类似物的N-14位甲基及其药代动力学行为是AHR激活的主要决定因素，提示在临床应用时应注意监测BA的代谢。代谢组学用于研究化学诱导的肝脏毒性。雷公藤甲素是雷公藤的主要活性成分。F，是中国治疗自身免疫性疾病的处方。临床应用中观察到的最严重的不良反应之一是肝毒性，但其作用机制尚不清楚。采用LC/MS代谢组学分析方法研究雷公藤甲素对小鼠血清和肝脏代谢的影响。采用雷公藤甲素灌胃的方法建立小鼠急性肝损伤模型，应用血清生化和肝组织学分析评价其毒性程度。多变量数据分析被用来调查代谢变化。使用投影值中的变量重要性和学生t检验来识别潜在的代谢物。共观察到30种代谢物在雷公藤甲素处理后发生了显着变化，其中29种代谢物的丰度与毒性的严重程度相关。通路分析表明，雷公藤甲素肝毒性的机制与谷胱甘肽代谢、三羧酸循环、嘌呤代谢、甘油磷脂代谢、牛磺酸和次牛磺酸代谢、泛酸和辅酶A生物合成、嘧啶代谢和氨基酸代谢等多条代谢途径的改变有关。目前的研究为雷公藤甲素诱导的肝毒性的代谢改变提供了新的机械性见解。

The role of PPARalpha in bile acid (BA) homeostasis is beginning to emerge. Ppara-null and hepatocyte-specific Ppara-null (Ppara-dHep) as well as the respective wild-type mice were treated with the potent PPARalpha agonist Wy-14,643 (Wy) and global metabolomics performed to determine the role of hepatocyte PPARalpha in the regulation of BA homeostasis revealing a new level of control of BA homeostasis beyond FXR. Levels of all serum BA were markedly elevated in Wy-treated wild-type mice but not in Ppara-null and Ppara-dHep mice. Gene expression analysis showed that PPARalpha activation (1) down-regulated the expression of sodium-taurocholate acid transporting polypeptide and organic ion transporting polypeptide 1 and 4, responsible for the uptake of BAs into the liver; (2) decreased the expression of bile salt export pump transporting BA from hepatocytes into the bile canaliculus; (3) upregulated the expression of multidrug resistance-associated protein 3 and 4 transporting BA from hepatocytes into the portal vein. Moreover, there was a notable increase in the compositions of serum, hepatic and biliary cholic acid and taurocholic acid following Wy treatment, which correlated with the upregulated expression of the Cyp8b1 gene encoding sterol 12alpha-hydroxylase. The effects of Wy were identical between the Ppara-dHep and Ppara-null mice. Hepatocyte PPARalpha controlled BA synthesis and transport not only via direct transcriptional regulation but also via crosstalk with hepatic FXR signaling. These findings underscore a key role for hepatocyte PPARalpha in the control of BA homeostasis. BA are also involved in hepatotoxicity induced by certain drugs, Rutaecarpine (RUT), evodiamine (EOD), and dehydroevodiamine (DHED) are the three main bioactive indoloquinazoline alkaloids isolated from Euodia rutaecarpa, a widely prescribed traditional Chinese medicine. Here, the structure-activity relationships of these analogs for aryl hydrocarbon receptor (AHR) activation were explored by use of Ahr-deficient (Ahr-null) mice, primary hepatocyte cultures, luciferase reporter gene assays, in silico ligand-docking studies, and metabolomics. In vitro, both mRNA analysis of AHR target genes in mouse primary hepatocytes and luciferase reporter assays in hepatocarcinoma cell lines demonstrated that RUT, EOD, and DHED significantly activated AHR, with an efficacy order of RUT DHED EOD. Ligand-docking analysis predicted that the methyl substitute at the N-14 atom was a key factor affecting AHR activation. In vivo, EOD was poorly orally absorbed and failed to activate AHR, whereas RUT and DHED markedly upregulated expression of the hepatic AHR gene battery in wild-type mice, but not in Ahr-null mice. Furthermore, RUT, EOD, and DHED were not hepatotoxic at the doses used; however, RUT and DHED disrupted BA homeostasis in an AHR-dependent manner. These findings revealed that the methyl group at the N-14 atom of these analogs and their pharmacokinetic behaviors were the main determinants for AHR activation, and suggest that attention should be given to monitoring BA metabolism in the clinical use of E. rutaecarpa. Metabolomics was used to study chemically-induced liver toxicity. Triptolide, a major active constitute of Tripterygium wilfordii Hook. F, is prescribed for the treatment of autoimmune diseases in China. One of its most severe adverse effects observed in the clinical use is hepatotoxicity, but the mechanism remained unknown. LC/MS-based metabolomic analysis was employed to characterize the metabolic changes in serum and liver induced by triptolide in mice. Mice were administered triptolide by gavage to establish the acute liver injury model, and serum biochemical and liver histological analyses applied to assess the degree of toxicity. Multivariate data analyses were performed to investigate the metabolic alterations. Potential metabolites were identified using variable importance in the projection values and Student's t-test. A total of 30 metabolites were observed that were significantly changed by triptolide treatment and the abundance of 29 metabolites was correlated with the severity of toxicity. Pathway analysis indicated that the mechanism of triptolide-induced hepatotoxicity was related to alterations in multiple metabolic pathways, including glutathione metabolism, tricarboxylic acid cycle, purine metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism and amino acid metabolism. The current study provides new mechanistic insights into the metabolic alterations that lead to triptolide-induced hepatotoxicity.