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区饮用水的污染物。最近的一些流行病学研究已将砷暴露与不良发育结果（如死产、自然流产、新生儿死亡率、低出生体重和肌肉组织使用延迟）联系起来。对啮齿动物的研究也表明，暴露于砷的怀孕鼠的后代早产死亡、低出生体重和运动活动的变化增加。利用一种名为mummichogs的模型鱼类，我们的实验室发现，在配子发生过程中，暴露于230ppb砷的后代发育异常，这与肌肉组织相关的基因上调有关，如肌凝蛋白轻链2、原肌凝蛋白和小蛋白。我们还证明，将C2C12肌细胞暴露于20nM的砷中会导致成肌细胞分化的延迟，这与肌原素表达的减少和甲基转移酶表达的减少有关。因此，我们建议利用C2C12细胞系研究暴露于砷后发育异常增加和肌肉发生变化的机制。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，是砷暴露后代肌肉发生改变和发育异常的部分原因。为了验证这一假设，我们将C2C12成肌细胞暴露于浓度增加的砷环境中，通过实时荧光定量PCR和免疫印迹技术研究其分化、多核、肌肉特异性基因和肌源性转录因子表达的时间和剂量依赖性变化。为了研究分化缺乏的机制，将研究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肌源性表型和肌源性蛋白表达。我们的实验室有能力开展拟议的研究，因为我们在检查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。我们之前一直在研究砷暴露后模型鱼类的表型和基因表达变化，现在希望通过使用细胞模型来进一步研究我们在体内的发现，因为它与改变的发育和改变的肌肉发生有关。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，并有助于检查砷的饮用水标准是否对人类健康有保护作用。

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.