Enantioselective Metabolism Influences PCB Developmental Neurotoxicity

描述（由申请方提供）：多氯联苯（PCB）同系物具有多个邻位氯取代基，是兰尼碱受体（RyR）的强效致敏剂，该活性被认为有助于与围产期PCB暴露相关的发育神经毒性。这些同系物中有许多显示出轴向手性，并作为外消旋体（两种阻转异构体的比例为1：1）存在于工业多氯联苯混合物中。然而，这两种对映体的比例在体内的变化，可能是由于涉及细胞色素（CYP）P450酶的对映体选择性过程。新出现的数据表明，显着的差异，在对映体富集的手性多氯联苯在人群中。这一点，当考虑到我们的初步数据表明，PCB阻转异构体差异敏化兰尼碱受体（RyR），提出了一个问题，是否对映体富集影响PCB暴露后的不良神经发育结果的风险。我们提出了三个具体的目标来测试的假设，手性PCB同系物进行对映选择性生物转化催化P450酶和这些差异的生物转化影响神经发育终点。在目标1中，RyR介导的发育神经毒性机制的对映体特异性将在体外进行表征。纯PCB阻转异构体对树突形态的影响将在原代大鼠海马神经元-神经胶质细胞共培养物中定量，并与细胞PCB水平相关。负责RyR 1和RyR 2通道复合物的对映体特异性激活的分子机制将使用生物化学，生物物理和细胞分析进行研究。在目标2中，物种和异构体依赖的对映选择性结合和代谢的纯PCB阻转异构体的P450酶将使用小鼠和人的微粒体和组织切片，以及重组人P450酶进行研究。将使用P450抑制剂鉴定微粒体中负责多氯联苯对映体选择性代谢的P450亚型。目的3将证实，在体内，P450酶的代谢是负责对映体富集的PCB和PCB 136阻转异构体引起对映体选择性RyR介导的发育神经毒性。首先，将在小鼠中开发药代动力学模型，以检查多氯联苯的对映体选择性处置中代谢的作用。随后的研究将确定围产期暴露于手性多氯联苯是否会导致对映体富集依赖的影响海马表达和功能的RyR和树突状树枝。这些研究将为了解与人类接触对发育中的神经系统具有高度毒性的手性多氯联苯同系物相关的风险做出根本性贡献，并将深入了解手性在许多手性污染物（如农药和增塑剂）的处置和毒性中的作用。考虑到人类基因多态性的广泛性，拟议的研究也可能表明未来的调查基因-环境相互作用，调节易感性多氯联苯发育神经毒性。 公共卫生相关性：发育暴露于手性多氯联苯（PCBs）可能会导致神经发育毒性，通过干扰树突的生长和可塑性，通过涉及兰尼碱受体的对映体特异性敏化机制。拟议的研究的目标是调查如何在手性多氯联苯的对映体选择性处置在怀孕的小鼠影响暴露后代的神经发育终点的差异。由于手性多氯联苯的对映体比例在人群中变化很大，因此拟议的研究将为了解人类接触手性多氯联苯同系物的风险做出根本性贡献，这些同系物对发育中的神经系统具有高度毒性，并将深入了解手性在广泛的其他有机污染物（如许多农药和增塑剂）的处置和毒性中的作用。

DESCRIPTION (provided by applicant): Polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substituents are potent sensitizers of the ryanodine receptor (RyR) and this activity is thought to contribute to the developmental neurotoxicity associated with perinatal PCB exposure. Many of these congeners display axial chirality and are present in industrial PCB mixtures as a racemate (a 1:1 ratio of both atropisomers). However, the ratio of the two enantiomers changes in vivo, probably due to enantioselective processes involving cytochrome (CYP) P450 enzymes. Emerging data suggest significant variability in the enantiomeric enrichment of chiral PCBs in the human population. This, when considered in light of our preliminary data demonstrating that PCB atropisomers differentially sensitize the ryanodine receptor (RyR), raises the question of whether enantiomeric enrichment influences the risk for adverse neurodevelopmental outcomes following PCB exposure. We propose three specific aims to test the hypothesis that chiral PCB congeners undergo enantioselective biotransformation catalyzed by P450 enzymes and that these differences in biotransformation influence neurodevelopmental endpoints. In Aim 1, the enantiospecificity of RyR-mediated mechanisms of developmental neurotoxicity will be characterized in vitro. The effects of pure PCB atropisomers on dendritic morphology will be quantified in primary rat hippocampal neuron-glia co-cultures and correlated with cellular PCB levels. The molecular mechanisms responsible for enantiospecific activation of RyR1 and RyR2 channel complexes will be investigated using biochemical, biophysical and cellular analyses. In Aim 2, the species and isoform-dependent enantioselective binding and metabolism of pure PCB atropisomers by P450 enzymes will be investigated using murine and human microsomes and tissue slices, as well as recombinant human P450 enzymes. The P450 isoforms responsible for the enantioselective metabolism of PCBs in microsomes will be identified using P450 inhibitors. Aim 3 will confirm, in vivo, that metabolism by P450 enzymes is responsible for enantiomeric enrichment of PCBs and that PCB 136 atropisomers cause enantioselective RyR-mediated developmental neurotoxicity. First, a pharmacokinetic model will be developed, in mice, to examine the role of metabolism in the enantioselective disposition of PCBs. Subsequent studies will determine whether perinatal exposure to chiral PCBs causes enantiomeric enrichment-dependent effects on hippocampal expression and function of RyR and dendritic arborization. These studies will make a fundamental contribution to understanding the risk associated with human exposure to chiral PCB congeners that are highly toxic to the developing nervous system and will provide an insight into the role of chirality in the disposition and toxicity of many chiral pollutants, such as pesticides and plasticizers. Given the extensive polymorphism in human CYP genes, the proposed studies may also suggest future investigations into gene-environment interactions that modulate susceptibility to PCB developmental neurotoxicity. PUBLIC HEALTH RELEVANCE: Developmental exposures to chiral polychlorinated biphenyls (PCBs) may cause neurodevelopmental toxicity by interfering with dendritic growth and plasticity via mechanisms involving the enantiospecific sensitization of ryanodine receptors. The goal of the proposed research is to investigate how differences in the enantioselective disposition of chiral PCBs in pregnant mice influence neurodevelopmental endpoints in exposed offspring. Because the enantiomer ratio of chiral PCBs is highly variable in human populations, the proposed studies will make a fundamental contribution to understanding the risk associated with human exposure to chiral PCB congeners that are highly toxic to the developing nervous system and will provide an insight into the role of chirality in the disposition and toxicity of a broad range of other organic pollutants, such as many pesticides and plasticizers.