Xenobiotic-Metabolizing Enzymes

CYP2E1表达和活性的生物标志物：CYP2E1是许多低分子量毒物代谢激活的关键酶，也是氧化应激的重要贡献者。无创监测体内CYP2E1活性的方法对于研究CYP2E1在化学诱导的毒性和应激相关疾病中的作用具有重要价值。在本研究中，通过比较野生型、CYP2E1缺失型和CYP2E1人源化小鼠的尿液代谢组，采用UPLC-ESI-QTOFMS鉴定CYP2E1的代谢物生物标志物。尿液代谢物多变量模型代谢组学分析显示，在尿液代谢物多变量模型中，cyp2e1缺失小鼠与野生型和cyp2e1人源化小鼠明显分离。随后，2-哌啶酮被鉴定为尿液代谢物，与三种小鼠系的CYP2E1活性呈负相关。野生型和cyp2e1缺失小鼠回交，以及小鼠血清中2-哌啶酮的靶向分析，证实了2-哌啶酮的基因型依赖性。cyp2e1缺失小鼠体内2-哌啶酮的积累主要是由于2-哌啶酮的生物合成和降解发生了变化，因为与野生型小鼠相比，尸胺向2-哌啶酮的转化更高，而cyp2e1缺失小鼠体内2-哌啶酮向6-羟基-2-哌啶酮的代谢较低。总体而言，非靶向代谢组学分析确定了尿中2-哌啶酮浓度与CYP2E1表达和活性之间的相关性，从而提供了一种无创代谢物生物标志物，可用于监测CYP2E1活性。异烟肼通过CYP2E1代谢导致胆汁淤积：异烟肼是预防和治疗结核病的一线药物。已知异烟肼对CYP2E1的抑制-诱导具有双相作用，也被认为参与了异烟肼诱导的肝毒性。然而，CYP2E1参与异烟肼引起的肝毒性的全部程度和机制仍有待深入研究。本研究将异烟肼给予野生型和cyp2e1缺失小鼠，研究异烟肼的体内潜在毒性。结果表明，异烟唑对野生型小鼠和cyp2e1缺失小鼠均无肝毒性作用，但使野生型小鼠血清胆固醇、甘油三酯和肝胆酸升高，并使野生型小鼠游离脂肪酸丰度降低，而cyp2e1缺失小鼠无肝毒性作用。代谢组学分析表明，野生型小鼠异烟肼代谢物的产生增加，胆汁酸、胆汁酸代谢物、肉毒碱和肉毒碱衍生物的丰度增加；这些在cyp2e1缺失的小鼠中没有观察到。此外，野生型小鼠胆汁酸合成酶减少，胆汁酸转运蛋白显著增加。最后，针对异烟肼代谢物对野生型小鼠进行治疗，导致胆固醇、甘油三酯和游离脂肪酸的类似变化。这些发现表明，虽然CYP2E1与异烟肼诱导的肝毒性无关，但异烟肼代谢物可能通过增强胆酸积累和线粒体β氧化，在异烟肼诱导的胆汁淤积中发挥作用。肿瘤细胞的生物标志物代谢组学方法：优化了基于LC/ ms的代谢组学方案，用于从Panc-1癌细胞系中提取代谢物。比较了纯乙腈、甲醇、甲醇/氯仿/H2O、甲醇/氯仿/乙腈等不同提取方法对细胞内代谢物的提取效率。并用UPLC-QTOF-MS评价了亲水相互作用色谱法（HILIC）和反相液相色谱法（RPLC）的分离效率。胰蛋白酶/EDTA治疗导致大量代谢物泄漏，证明其不足以用于代谢组学研究。采用液氮闪灭后直接刮取的方法来收获Panc-1细胞，这样可以在提取前保存样品。选择甲醇/氯仿/水为最佳提取溶剂，提取的细胞内特征物数量最多，重现性最好。HILIC对Panc-1细胞的胞内代谢物具有较好的分辨率。该优化方法对多种细胞代谢物具有较高的灵敏度和重复性，可应用于进一步的肿瘤细胞系全球代谢组学研究。鳞状细胞癌在异种照相中的生长：uplc - esi - qtofms为基础的代谢组学用于探索小鼠肿瘤生长的代谢特征。收集对照小鼠和注射鳞状细胞癌（SCCVII）肿瘤细胞小鼠的尿液样本作为时间的函数。当肿瘤达到2厘米时，所有小鼠均被杀死，并采集血液和肝脏样本。荷瘤小鼠的尿液代谢产物己烯酰甘氨酸、烟酰胺1-氧化物和11beta， 20α -二羟基-3-氧opregn-4-烯-21-酸升高，不对称二甲基精氨酸（氧化应激的生物标志物）也升高。有趣的是，SCCVII肿瘤生长导致肝脏肿大，白蛋白/球蛋白比值降低，血清甘油三酯升高，提示肝功能障碍。SCCVII荷瘤小鼠和对照小鼠之间肝脏代谢物的变化证实了肝损伤的存在。肝脏mRNA分析显示，SCCVII荷瘤小鼠的炎症因子、肿瘤坏死因子α和转化生长因子β表达增强，荷瘤小鼠的细胞色素P450表达降低。此外，参与脂肪酸氧化的基因减少，表明SCCVII荷瘤小鼠的脂肪酸氧化受损。此外，在SCCVII荷瘤小鼠中观察到磷脂代谢激活和三羧酸循环中断。这些数据表明，肿瘤生长施加了导致肝功能障碍的全局炎症反应，并强调使用代谢组学来暂时检查这些变化，并可能使用代谢物变化来监测肿瘤治疗反应。硫氧还蛋白和抗氧化剂在药物性肝毒性中的作用：谷胱甘肽在减轻亲电生成药物（如对乙酰氨基酚）的有害作用方面的重要性已得到证实。然而，其他抗氧化系统的作用，如硫氧还蛋白，尚未研究。硒蛋白硫氧还蛋白还原酶1 （Txnrd1）在降低高剂量对乙酰氨基酚引起的凋亡信号调节激酶1和c-Jun n -末端激酶途径的激活中起重要作用。因此，我们研究了Txnrd1在对乙酰氨基酚引起的肝毒性中的作用。产生肝脏特异性Txnrd1基因敲除小鼠，并用毒性剂量的对乙酰氨基酚处理。在对乙酰氨基酚治疗后1小时，对照组和肝脏特异性Txnrd1敲除小鼠的总谷胱甘肽和线粒体谷胱甘肽水平降低。然而，在对乙酰氨基酚处理6小时后，肝脏特异性Txnrd1敲除小鼠对对乙酰氨基酚毒性产生抗性。研究发现，在肝脏特异性Txnrd1基因敲除小鼠中，对乙酰氨基酚治疗前后，许多核因子红细胞2相关因子2靶基因和蛋白的代偿性上调。然而，c-Jun n端激酶在对乙酰氨基酚处理的对照小鼠中被磷酸化的程度相似。这些结果表明，Txnrd1基因敲除小鼠主要通过核因子2相关因子2的激活来启动外源解毒。

BIOMARKERS FOR CYP2E1 EXPRESSION AND ACTIVITY: CYP2E1 is a key enzyme in the metabolic activation of many low molecular weight toxicants and also an important contributor to oxidative stress. A noninvasive method to monitor CYP2E1 activity in vivo would be of great value for studying the role of CYP2E1 in chemical-induced toxicities and stress-related diseases. In this study, UPLC-ESI-QTOFMS was used to identify a metabolite biomarker of CYP2E1 through comparing the urine metabolomes of wild-type, Cyp2e1-null, and CYP2E1-humanized mice. Metabolomic analysis with multivariate models of urine metabolites revealed a clear separation of Cyp2e1-null mice from wild-type and CYP2E1-humanized mice in the multivariate models of urine metabolomes. Subsequently, 2-piperidone was identified as a urinary metabolite that inversely correlated to the CYP2E1 activity in the three mouse lines. Backcrossing of wild-type and Cyp2e1-null mice, together with targeted analysis of 2-piperidone in mouse serum, confirmed the genotype dependency of 2-piperidone. The accumulation of 2-piperidone in the Cyp2e1-null mice was mainly caused by the changes in the biosynthesis and degradation of 2-piperidone because compared with the wild-type mice, the conversion of cadaverine to 2-piperidone was higher, whereas the metabolism of 2-piperidone to 6-hydroxy-2-piperidone was lower in the Cyp2e1-null mice. Overall, untargeted metabolomic analysis identified a correlation between 2-piperidone concentrations in urine and the expression and activity of CYP2E1, thus providing a noninvasive metabolite biomarker that can be potentially used in to monitor CYP2E1 activity. METABOLISM OF ISONIAZID BY CYP2E1 LEADS TO CHOLESTASIS: Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition-induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria beta-oxidation. BIOMARKERS METABOLOMICS METHODS FOR TUMOR CELLS: An LC/MS-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the Panc-1 cancer cell line. Different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further global metabolomics studies on cancer cell lines. FOR SQUAMOUS CELL CARCINOMA GROWTH IN XENOGRAPHS: UPLC-ESI-QTOFMS-based metabolomics was used to explore metabolic signatures of tumor growth in mice. Urine samples were collected from control mice and mice injected with squamous cell carcinoma (SCCVII) tumor cells as a function of time. When tumors reached 2 cm, all mice were killed and blood and liver samples collected. The urine metabolites hexanoylglycine, nicotinamide 1-oxide, and 11beta, 20alpha-dihydroxy-3-oxopregn-4-en-21-oic acid, were elevated in tumor-bearing mice as was asymmetric dimethylarginine, a biomarker for oxidative stress. Interestingly, SCCVII tumor growth resulted in hepatomegaly, reduced albumin/globulin ratios, and elevated serum triglycerides suggesting liver dysfunction. Alterations in liver metabolites between SCCVII tumor-bearing and control mice confirmed the presence of liver injury. Hepatic mRNA analysis indicated that inflammatory cytokines, tumor necrosis factor alpha and transforming growth factor beta were enhanced in SCCVII tumor-bearing mice and cytochromes P450 expression were decreased in tumor-bearing mice. Further, genes involved in fatty acid oxidation were decreased, suggesting impaired fatty acid oxidation in SCCVII tumor-bearing mice. Additionally, activated phospholipid metabolism and disrupted tricarboxylic acid cycle were observed in SCCVII tumor-bearing mice. These data suggest that tumor growth imposes a global inflammatory response that results in liver dysfunction and underscores the use of metabolomics to temporally examine these changes and potentially use metabolite changes to monitor tumor treatment response. ROLE OF THIOREDOXIN AND ANTIOXIDANTS IN DRUG-INDUCED HEPATOTOXICITY: The importance of glutathione in mitigating the deleterious effects of electrophile generating drugs such as acetaminophen is well established. However, the role of other antioxidant systems, such as that provided by thioredoxin, has not been studied. Selenoprotein thioredoxin reductase 1 (Txnrd1) is important for attenuating activation of the apoptosis signaling-regulating kinase 1 and the c-Jun N-terminal kinase pathway caused by high doses of acetaminophen. Therefore, the role of Txnrd1 in acetaminophen -induced hepatotoxicity was investigated. Liver-specific Txnrd1 knockout mice were generated and treated with a toxic dose of acetaminophen. At 1 h post- acetaminophen treatment, total and mitochondrial glutathione levels in control and liver-specific Txnrd1 knockout mice were depleted. However, at 6 h post- acetaminophen treatment, liver-specific Txnrd1 knockout mice were resistant to acetaminophen toxicity. A compensatory up-regulation of many of the nuclear factor erythroid 2-related factor 2 target genes and proteins in liver-specific Txnrd1 knockout mice with and without acetaminophen treatment was found. Yet, c-Jun N-terminal kinase was phosphorylated to a similar extent in acetaminophen-treated control mice. These results suggest that Txnrd1 knockout mice mice are primed for xenobiotic detoxication primarily through nuclear factor erythroid 2-related factor 2 activation.