Mechanisms of Arsenic-Induced Developmental Toxicity

描述（由申请人提供）：砷是一个巨大的公共卫生问题，因为它是世界许多地方饮用水的污染物。最近的一些流行病学研究表明，砷暴露与不良发育结果有关，如死胎、自然流产、新生儿死亡、出生体重低和肌肉组织使用延迟。对啮齿动物的研究也表明，怀孕母鼠暴露于砷的后代有早产死亡，低出生体重和运动活动的变化增加。使用一个模式鱼类称为mummichogs，我们的实验室已经表明，在配子发生过程中暴露于230ppb砷的后代发育异常，这与涉及肌肉组织的基因，如肌球蛋白轻链2，原肌球蛋白和小清蛋白的上调。我们还证明，暴露于20 nM砷的C2C12肌细胞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相关。因此，我们建议使用C2C12细胞系研究砷暴露后发育异常和肌生成变化增加的机制。我们的假设是，参与骨骼肌发育的转录因子的不适当表达，再加上改变基因甲基化，是一种机制，部分负责改变肌肉发生和砷暴露后代的发育异常。我们将通过将C2C12成肌细胞暴露于浓度增加的砷中来测试这一假设，以通过实时PCR和免疫印迹来研究分化、多核化、肌肉特异性基因和生肌转录因子表达的时间和剂量依赖性变化。为了研究缺乏分化的机制，将检查DNA甲基化、甲基转移酶活性和表达以及甲基化前体。最后，甲基化诱导剂和抑制剂，以及甲基转移酶基因的转染将用于确定是否可以改变成肌表型和成肌蛋白表达。我们的实验室是很好的定位进行拟议的研究，因为我们有经验，在检查砷对发展的影响，并对差异基因和蛋白质表达。我们以前一直在研究表型和基因表达的变化后，砷暴露在一个模型鱼类，现在希望进一步我们在体内的研究结果，通过使用细胞模型来研究砷的作用机制，因为它涉及到改变发展和改变肌发生。公共卫生相关性：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.