Sodium arsenite delays the differentiation of C2C12 mouse myoblast cells and alters methylation patterns on the transcription factor myogenin.
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.
Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634, USA.
Amanda A
AA
Steffens
Gia-Ming
GM
Hong
Lisa J
LJ
Bain
D000818:Animals; D018053:Arsenites / Q000633:toxicity*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D019175:DNA Methylation / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D064326:MEF2 Transcription Factors; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D032446:Myoblasts / Q000187:drug effects* / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000187:drug effects / Q000235:genetics; D018008:Myogenin / Q000187:drug effects* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D017670:Sodium Compounds / Q000633:toxicity*
D018053:Arsenites; D064326:MEF2 Transcription Factors; C578041:Mef2c protein, mouse; D018006:Myogenic Regulatory Factors; D018008:Myogenin; D012333:RNA, Messenger; D017670:Sodium Compounds; C017947:sodium arsenite