Abstract
BackgroundThimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells.Methodology/Principal FindingsThe study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125–500 nM) for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Aktser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml) increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells.Conclusions/SignificanceOur results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway.
Highlights
Thimerosal is a water-soluble derivative of thiosalicylic acid
Conclusions/Significance: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and causes apoptosis via inhibition of Phosphoinositide 3-kinase (PI3K)/Akt/survivin signaling followed by activation of the mitochondrial apoptotic pathway
After exposure to 51.3%63.2% (250 nM) and 27.4%62.2% (500 nM) thimerosal, 50.6% of the cells were in S phase, whereas only 10.9% of the cells were in G2/M phase and 38.5% were in G0/G1 phase
Summary
Thimerosal is a water-soluble derivative of thiosalicylic acid. Due to its antimicrobial properties, it is widely used as a preservative in vaccines, ophthalmic products and cosmetics [1]. The safety of thimerosal has recently been questioned based on a number of studies that indicate to its possible risk of toxicity [2,3,4,5]. Thimerosal has been classified as the second most common allergen after nickel [14,15,16,17,18], and been shown to induce epithelial cytotoxicity via oxidative stress in HeLa S epithelial cells and apoptosis in human SCM1 gastric cancer cells via activation of the p38 MAP kinase and caspase-3 [19]. We investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells
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