CYP1A1 GENE AND ENVIRONMENTAL TOXICITY

该项目的长期目标是了解细胞色素P450 1A1(CYP1A1)在环境污染物引起的毒性中的作用。Cyp1a1具有广泛的组织分布，在妊娠早期就有表达，并受ah受体(AHR)的转录调控。环境污染物苯并[a]芘(BaP)和二恶英是AHR的配体。已知这些污染物引起的毒性是由AHR介导的，BaP需要代谢激活，而二恶英的代谢可以忽略不计。越来越多的证据表明，CYP1A1也参与了二恶英的毒性作用。越来越清楚的是，在最基本的条件下，化学品的环境毒性通过两条途径发生：[a)活性中间体与细胞大分子的共价结合和/或[b]最终影响特定类型细胞命运的信号转导通路的激活。随着我们最近成功地建立了Cyp1a1(-/-)基因敲除小鼠系，我们现在处于独特的地位，可以描述组织和细胞类型特异性的CyP1A1与AHR依赖和非独立的由代谢(BaP)和非代谢(Dioxin)AHR配体引起的毒性模式。其他人的工作明确表明，大约15%到25%以上(取决于组织)的CYP1A1位于线粒体膜内膜(Mt1A1)，其余的位于内质网，即微粒体(Mc1A1)，并且mt1A1和mc1A1在诱导性和底物特异性方面存在明显差异。细胞凋亡级联中的早期事件(细胞色素c释放，bcl2功能)也发生在线粒体膜内。因此，CYP1A1依赖的毒性很大程度上可以归因于酶的一个亚细胞位置而不是另一个亚细胞位置。我们的假设是，BAP和二恶英对多种组织的毒性主要是细胞色素P1A1功能的结果。因此，在下一个资助期，我们将[1]评估Cyp1a1(-/-)基因敲除小鼠与野生型Cyp1a1(+/+)基因敲除小鼠在[a]骨髓、[b]肝脏和[c]发育胚胎中由BaP和二恶英诱导的差异毒性；以及[2]仅产生含线粒体CYP1A1的(Mt1A1)和含微粒体的CYP1A1(Mc1A1)敲入小鼠系，以进一步剖析肝脏毒性的机制。这些研究将极大地增强我们对环境污染物引起的mtCYP1A1和mcCYP1A1介导的毒性的理解，并可能导致设计药物来抵御饮食中，特别是吸烟者和职业暴露者的这种CYP1A1底物和AHR配体的毒性。

The long-term goal of this project is to understand the role of cytochrome P450 1A1 (CYP1A1) in toxicity caused by environmental pollutants. Cyp1a1 has a broad tissue distribution, is expressed very early in gestation, and is transcriptionally regulated by the Ah receptor (AHR). The environmental contaminants benzo[a]pyrene (BaP) and dioxin are ligands for the AHR. Toxicity caused by these pollutants is known to be AHR-mediated, BaP requiring metabolic activation and dioxin negligibly metabolized. Evidence is accumulating that CYP1A1 also mediates the toxicity of dioxin. It is increasingly clear that, in the most basic of terms, environmental toxicity of chemicals occurs via two routes: [a] covalent binding of reactive intermediates to cellular macromolecules and/or [b] activation of signal transduction pathways that ultimately influence the fate of specific cell types. With our recent success in making the Cyp1a1 (-/-) knockout mouse line, we are now in the unique position to delineate tissue- and cell type-specific CYP1A1- versus AHR-dependent and - independent modes of toxicity elicited by metabolized (BaP) and nonmetabolized (dioxin) AHR ligands. The work of others has unequivocally shown that about 15% to more than 25% (depending on the tissue) of CYP1A1 is located in the inner mitochondrial membrane (mt1A1), the remaining in the endoplasmic reticulum, i.e. microsomes (mc1A1), and that clear differences in the inducibility profile and substrate specificity exist between mt1A1 and mc1A1. Events very early in the apoptosis cascade (cytochrome c release, BCL2 function) also occur in the inner mitochondrial membrane. Thus, CYP1A1-dependent toxicity may be largely ascribed to one subcellular location of the enzyme versus another. Our hypothesis is that Bap and dioxin-induced toxicity of various tissues is primarily he result of CYP1A1 function. For the next funding period we therefore will [1] assess in the Cyp1a1 (-/-) knockout mouse, compared with the Cyp1a1 (+/+) wild-type, differential toxicity induced by BaP versus dioxin in the [a] bone marrow, [b] liver, and [c]developing embryo; and [2] generate exclusively mitochondrial CYP1A1-containing (mt1A1) and exclusively microsomal CYP1A1 - containing (mc1A1) knock-in mouse lines to dissect the mechanism of liver toxicity further. These studies will greatly enhance our understanding of mtCYP1A1 versus mcCYP1A1- mediated toxicity caused by environmental pollutants and perhaps lead to the design of drugs to shield against such toxicity of CYP1A1 substrates and AHR ligands, in the diet and especially for cigarette smokers and occupationally-exposed workers.