Enantioselective Metabolism Influences PCB Developmental Neurotoxicity

描述(由申请人提供)：具有多个邻位氯取代基的多氯联苯(PCB)同系物是兰尼定受体(RyR)的有效增敏剂，这种活性被认为与围产期接触多氯联苯有关的发育神经毒性有关。其中许多同系物表现出轴向手性，并以外消旋形式存在于工业多氯联苯混合物中(两种阿托异构体的比例为1：1)。然而，这两种对映体的比例在体内发生变化，可能是由于涉及细胞色素(CYP)P450酶的对映选择性过程。新出现的数据表明，在人类群体中手性多氯联苯的对映体浓缩具有显著的变异性。考虑到我们的初步数据显示，多氯联苯阿托品对兰尼定受体(RyR)有不同的敏化作用，这就提出了一个问题，即对映体浓缩是否会影响多氯联苯暴露后不良神经发育结果的风险。我们提出了三个特定的目标来检验这一假说，即手性多氯联苯同系物经历P450酶催化的对映选择性生物转化，以及这些生物转化过程中的差异影响神经发育终点。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表征。纯多氯联苯阿托品对树突形态的影响将在原代培养的大鼠海马神经元-神经胶质细胞中进行量化，并与细胞多氯联苯水平相关联。RyR1和RyR2通道复合体对映体特异性激活的分子机制将通过生化、生物物理和细胞分析进行研究。在目标2中，将使用小鼠和人的微粒体和组织切片以及重组人的P450酶来研究纯的多氯联苯对映体与P450酶的种类和异构体依赖的对映选择性结合和代谢。负责微生物体中多氯联苯对映体选择性代谢的P450异构体将使用P450抑制剂进行鉴定。AIM 3将在体内证实，P450酶的代谢是多氯联苯对映体浓缩的原因，而多氯联苯136阿托品引起对映体选择性RyR介导的发育神经毒性。首先，将在小鼠身上建立一个药代动力学模型，以检验代谢在多氯联苯对映体选择性处置中的作用。后续的研究将确定围产期接触手性多氯联苯是否会导致对映体浓缩依赖的影响，影响海马区RyR的表达和功能以及树突的分枝。这些研究将有助于了解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的风险，并将深入了解手性在许多手性污染物(如杀虫剂和增塑剂)的处置和毒性中所起的作用。鉴于人类CYP基因的广泛多态性，拟议的研究还可能建议进一步研究基因-环境相互作用调节多氯联苯发育性神经毒性的易感性。 公共卫生相关性：发育过程中暴露于手性多氯联苯(PCbs)可能会通过兰尼定受体的对映特异性敏化机制干扰树突状细胞的生长和可塑性，从而导致神经发育毒性。这项拟议研究的目的是调查怀孕小鼠手性多氯联苯对映体选择性处置的差异如何影响暴露于该物质的后代的神经发育终点。由于手性多氯联苯的对映体比例在人类群体中是高度可变的，拟议的研究将有助于了解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的风险，并将深入了解手性在许多其他有机污染物(如许多杀虫剂和增塑剂)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.