The Role of Cyp2b in Toxicant Metabolism and Sensitivity

说明(申请人提供)：细胞色素P450(Cyps)是许多化学物质解毒的关键酶，包括类固醇、胆汁酸、药物和环境化学物质。Cyp2b被认为是被遗忘的CYP，因为人们对其底物特异性知之甚少，它在肝脏中的表达水平低于其他一些解毒的CYP，而且FDA没有强制要求在药物代谢测试中使用重组Cyp2b。然而，Cyp2b是高度可诱导的、高度多态的、性别主导的，最近的研究表明，它的表达水平高于人们曾经认为的水平，特别是在西班牙裔美国人中。我们将研究这种CYP在环境毒物代谢中的作用，确定低Cyp2b的个体是否对作为Cyp2b底物的特定毒物敏感，并评估毒物抑制CYP是否可能导致代谢紊乱，如肥胖。我们最近开发了一种Cyp2b基因敲除小鼠，它使用RNAi抑制所有五个小鼠Cyp2b成员的表达。这些小鼠表现出较低的Cyp2b蛋白和活性，而强大的Cyp2b诱导剂TCPOBOP无法胜过shRNA介导的抑制。在目标1中，我们将使用这个小鼠模型，使用未经处理和TCPOBOP处理的野生型和Cyp2b-KD小鼠的微生物体来估计Cyp2b代谢。在目标2中，我们将测试Cyp2b-KD小鼠是否对由Cyp2b代谢的特定毒物，如壬基酚或对硫磷敏感。最后，在以TCPOBOP或玉米油为载体的IP注射后，最近的初步数据显示，Cyp2b-KD小鼠而不是野生型小鼠无法适当地从肝脏中净化不饱和脂肪酸。这表明Cyp2b在脂质动态平衡中起着重要作用。在目标3中，我们将测试Cyp2b活性低的小鼠是否容易受到高脂饮食(西方饮食)导致的肝脏脂肪变性(脂肪肝)和肥胖的影响。如果Cyp2b-KD小鼠对肥胖易感，这将表明Cyp2b抑制剂可能是环境肥胖的原因。总之，这个项目的目的是利用我们实验室最近开发的一个新的小鼠模型来确定Cyp2b在体内环境毒物的新陈代谢、命运和毒性效应中的作用。 与公共健康相关：细胞色素P450(Cyps)是许多化学物质解毒的关键酶，包括类固醇、胆汁酸、药物和环境化学物质。Cyp2b被认为是被遗忘的CYP，因为它在肝脏中的表达水平低于其他一些解毒的CYP。然而，它具有很高的诱导性，最近的研究表明，它的表达水平比人们曾经认为的更高。我们将研究这种CYP在环境毒物代谢中的作用，确定低Cyp2b个体是否对特定的毒物敏感，并评估毒物对CYP的抑制是否可能导致代谢紊乱。

DESCRIPTION (provided by applicant): The cytochrome P450s (CYPs) are key enzymes involved in the detoxification of numerous chemicals including steroids, bile acids, pharmaceuticals, and environmental chemicals. Cyp2b is considered the forgotten CYP because not as much is known about its substrate specificity, it is expressed at lower levels in the liver than some other detoxification CYPs, and the FDA does not mandate the use of recombinant Cyp2b in drug metabolism testing. However, Cyp2b is highly inducible, highly polymorphic, gender predominant, and recent work shows it is expressed at higher levels than once thought, especially in hispanics. We will investigate the role of this CYP in the metabolism of environmental toxicants, determine whether individuals with low Cyp2b are sensitive to specific toxicants that are Cyp2b substrates, and evaluate whether CYP inhibition by toxicants could potentially lead to metabolic disorders such as obesity. We have recently developed a Cyp2b-knockdown mouse using RNAi that represses the expression of all five murine Cyp2b members. These mice demonstrate lower Cyp2b protein and activity and TCPOBOP the potent Cyp2b inducer is unable to out-compete the shRNA-mediated repression. In aim 1, we will use this mouse model to estimate Cyp2b metabolism using microsomes from untreated and TCPOBOP-treated wild-type and Cyp2b-KD mice. In aim 2, we will test whether Cyp2b-KD mice are susceptible to specific toxicants such as nonylphenol or parathion that are metabolized by Cyp2b. Last, recent preliminary data following ip injections with TCPOBOP or corn oil as a carrier revealed that Cyp2b-KD mice but not wild-type mice are unable to properly depurate the unsaturated fatty acids from the liver. This suggests an important role for Cyp2b in lipid homeostasis. In aim 3, we will test whether mice with low Cyp2b activity are susceptible to high-fat diet (Western diet) induced hepatic steatosis (fatty liver) and obesity. If Cyp2b-KD mice are susceptible to obesity, this would suggest that Cyp2b inhibitors may act as environmental obesogens. Overall, the purpose of this project is determine the role of Cyp2b in the metabolism, fate, and toxic effects of environmental toxicants in vivo using a novel murine model recently developed in our laboratory. PUBLIC HEALTH RELEVANCE: The cytochrome P450s (CYPs) are key enzymes involved in the detoxification of numerous chemicals including steroids, bile acids, pharmaceuticals, and environmental chemicals. Cyp2b is considered the forgotten CYP because it is expressed at lower levels in the liver than some other detoxification CYPs. However, it is highly inducible and recent work shows it is expressed at higher levels than once thought. We will investigate the role of this CYP in the metabolism of environmental toxicants, determine whether individuals with low Cyp2b are sensitive to specific toxicants, and evaluate whether CYP inhibition by toxicants could potentially lead to metabolic disorders.