Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634, USA.
Toxicol Appl Pharmacol. 2011 Jan 15;250(2):154-61. doi: 10.1016/j.taap.2010.10.006. Epub 2010 Oct 18.
Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.
流行病学研究表明，砷暴露与癌症、皮肤病以及自发性流产、新生儿死亡率、低出生体重和肌肉使用延迟等不良发育结果有关。本研究使用 C2C12 小鼠成肌细胞来研究低浓度的砷是否会改变其分化为肌管的过程，这表明砷可能是一种发育毒物。将成肌细胞暴露于 20 nM 亚砷酸钠中，使其分化为肌管，并使用 QPCR 和免疫荧光法研究肌肉特异性转录因子肌生成素(myogenin)以及原肌球蛋白(tropomyosin)、细胞因子信号转导抑制因子 3(Socs3)、前列腺素 I2 合成(Ptgis)和肌细胞增强因子 2(Mef2)的表达。将 C2C12 细胞暴露于 20 nM 亚砷酸钠会延迟分化过程，这表现为多核肌管的数量显著减少，myogenin mRNA 表达减少，以及表达肌生成素蛋白的细胞核总数减少。参与肌管形成的 mRNA，如 Ptgis 和 Mef2 mRNA 的表达也在分化过程中显著降低了 1.6 倍和 4 倍。通过 Mef2 的免疫荧光证实了这一点，Mef2 的核转位减少了 2.6 倍。通过甲基化特异性 PCR 和亚硫酸氢盐基因组测序检查了 myogenin 启动子区域(-473 至+90)的甲基化模式变化。在砷暴露细胞中，发现-236 和-126 bp 处的 CpG 发生超甲基化，而-207 bp 处的 CpG 发生低甲基化。这项研究表明，20 nM 亚砷酸钠可以通过降低转录因子 myogenin 和 Mef2c 的表达来改变成肌细胞的分化，这可能是由于启动子甲基化模式的改变。肌肉分化的延迟可能导致发育异常。
