Mechanisms of Arsenic-Induced Developmental Toxicity

描述(申请人提供)：砷是一个巨大的公共卫生问题，因为它是世界许多地区饮用水的污染物。最近的一些流行病学研究表明，砷暴露与不利的发育结果相关，如死产、自然流产、新生儿死亡、低出生体重和肌肉系统使用延迟。对啮齿动物的研究还表明，暴露在砷中的怀孕母鼠的后代会增加早产死亡、低出生体重和运动活动的变化。使用一种名为木乃伊米猪的模式鱼类，我们的实验室已经显示出在配子发生期间暴露于230 ppb砷的后代出现发育异常，这与涉及肌肉组织的基因上调有关，如肌球蛋白轻链2、原肌球蛋白和小白蛋白。我们还证明了C2C12心肌细胞暴露在20 nm砷中导致成肌细胞分化延迟，这与肌生成素表达减少和甲基转移酶表达减少有关。因此，我们建议使用C2C12细胞株来研究砷暴露后发育异常增加和肌肉生成变化的机制。我们的假设是，参与骨骼肌发育的转录因子的不适当表达，加上基因甲基化的改变，是砷暴露的后代肌肉发生改变和发育异常的部分原因。我们将通过将C2C12成肌细胞暴露在不断增加的砷浓度中来检验这一假说，以实时荧光聚合酶链式反应和免疫印迹法研究分化、多核、肌肉特异性基因和成肌转录因子表达的时间和剂量依赖性的变化。为了探讨缺乏分化的机制，将对DNA甲基化、甲基转移酶活性和表达以及甲基化前体进行检测。最后，将使用甲基化诱导剂和抑制物，以及甲基转移酶基因的转染来确定是否可以改变成肌表型和成肌蛋白的表达。我们的实验室能够很好地开展拟议的研究，因为我们在研究砷对发育的影响以及对差异基因和蛋白质表达的影响方面有经验。我们之前曾在一个模式鱼类物种中检测过砷暴露后的表型和基因表达变化，现在希望通过使用细胞模型来研究砷的作用机制，因为它与发育改变和肌肉发生改变有关，从而进一步推动我们在体内的发现。与公众健康相关：这项工作的最终好处是评估环境上现实的砷暴露如何影响发展的机制，并帮助检查饮用水砷标准是否保护人类健康。

DESCRIPTION (provided by applicant): Arsenic is an enormous public health problem as it is a contaminant of drinking water in many parts of the world. A number of recent epidemiological studies have correlated arsenic exposure with adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. Studies in rodents also demonstrate that offspring of pregnant dams exposed to arsenic have increases in preterm death, low birth weight, and changes in locomotor activity. Using a model fish species termed mummichogs, our laboratory has shown developmental abnormalities in offspring exposed to 230ppb arsenic during gametogenesis, which correlated with an upregulation of genes involved the musculature, such as myosin light chain 2, tropomyosin, and parvalbumin. We have also demonstrated that exposure of C2C12 myocyte cells to 20nM arsenic resulted in a delay in myoblast differentiation that correlated to a reduction in myogenin expression and a reduction in methyltransferase expression. Thus, we propose to investigate the mechanisms underlying the increase in developmental abnormalities and changes in myogenesis after exposure to arsenic using the C2C12 cell line. Our hypothesis is that inappropriate expression of transcription factors involved in skeletal muscle development, coupled with altered gene methylation, is a mechanism partly responsible for altered myogenesis and the developmental abnormalities in arsenic-exposed offspring. We will test this hypothesis by exposing C2C12 myoblast cells to increasing concentrations of arsenic to investigate the time and dose-dependent changes in differentiation, multinucleation, muscle-specific genes, and myogenic transcription factor expression by real-time PCR and immunoblotting. To investigate the mechanisms responsible for the lack of differentiation, DNA methylation, methyltransferase activity and expression, and methylation precursors will be examined. Finally, methylation inducers and inhibitors, and transfection of methyltransferase genes will be used to determine whether the myogenic phenotype and myogenic protein expression can be altered. Our laboratory is well positioned to carry out the proposed studies because we have experience in examining the effects of arsenic on development, and on differential gene and protein expression. We have previously been examining phenotypic and gene expression changes after arsenic exposure in a model fish species, and now wish to further our in vivo findings by using a cell model to examine arsenic's mechanisms of action as it relates to altered development and altered myogenesis. PUBLIC HEALTH RELEVANCE: The ultimate benefit of this work is to assess the mechanisms of how environmentally realistic arsenic exposure impacts development, and help to examine whether the drinking water standard for arsenic is protective of human health.