## 4 Discussion  
The present study found that DEHP and its metabolite MEHP, at non- and low-cytotoxic concentrations, inhibited myogenesis, which may further cause immature muscle fiber development, in the immortalized mouse and primary human muscle cell models. The mechanistic investigation indicated that both DEHP and MEHP markedly downregulated the Akt-regulated myogenic signaling molecules in C2C12 myoblasts and HSMPCs during differentiation. Our data show that there was no cytotoxicity in DEHP (1-1000 \(\upmu\)M)-treated C2C12 myoblasts under differentiation medium for 96 h. This result is consistent with the report by [PERSON] et al., in which there was no cytotoxicity of DEHP (10-1000 \(\upmu\)m) on C2C12 cells [18]. Nevertheless, we further found that its metabolite MEHP displayed greater cytotoxicity at concentrations of \(>\)100 \(\upmu\)M. Our data also showed that, at non-cytotoxic concentrations, DEHP (10-100 \(\upmu\)M) and MEHP (10-50 \(\upmu\)M) significantly suppressed myoblast differentiation and myotube formation in C2C12 myoblasts and HSMPCs. These concentrations for DEHP and MEHP seem to be relevant to human exposure in bloods, which will be discussed later. The levels of DEHP in indoor air of the Tokyo Metropolitan area have been shown to be up to 1000-fold higher than ambient levels in Japan [28]. It has been found that the concentrations of DEHP are 72 \(\pm\) 13 \(\upmu\)g/mL and 668 \(\pm\) 96 \(\upmu\)g/mL after 8 h and 24 h, respectively, in a cyclosporine solution stored in a PVC bag at room temperature [29]. In blood transfusions, the replacement blood DEHP levels have been detected to range from 10 to 650 \(\upmu\)g/mL (about 25-1600 \(\upmu\)M) [8]. A prospective and comparative clinical study showed that the mean maximum plasma level of DEHP was 8.3 \(\pm\) 5.7 \(\upmu\)g/mL at any time in a group of 28 consecutive term infants during an extracorporeal membrane  
Figure 6: The changes in myogenesis-related protein expression in primary HSMPCs treated with both DEHP and MEHP. Under differentiation medium (DM), HSMPCs treated with DEHP (**A**) or MEHP (**B**) (10–100 \(\upmu\)M) were observed. GM indicated as growth medium. The analysis of protein expression for myogenin, myosin heavy chain (MHC), phospho-Akt, and Akt was performed using Western blot. An internal control using \(\beta\)-actin was performed. The densitometric analysis of proteins via Western blot was performed. Data are presented as means \(\pm\) SEM (at least three independent experiments, triplicate repeats in each); \(p<\) 0.05 indicates significance compared to the control (*). oxygenation therapy [30]. A study of newborn infant plasticizer exposure showed that plasma DEHP levels were 3.4-1.1 \(\upmu\)g/mL in the individual infants, and MEHP levels were 2.4-15.1 \(\upmu\)g/mL in the corresponding samples [31]. Therefore, the test concentrations of DEHP/MEHP in the current study may be the blood concentrations to which human is likely to be exposed. The major processes involved in skeletal myogenic differentiation are withdrawing myoblasts from the cell cycle, expressing myotube-specific genes, and the formation of multinucleated myotube [32; 33]. Several factors can regulate myogenic differentiation, including transcription factors of myogenic basic helix-loop-helix family, such as MyoD, myogenin, myf5, and MRF4, and myocyte enhancer factor 2. These factors can further regulate muscle-specific gene/protein expression, including MHC and creatine kinase expression [34; 35]. Moreover, the signaling of phosphatidylinositol 3-kinase (PI3K)/Akt contributes to controlling the expression of muscle genes/proteins during myogenesis [27; 36]. The inhibition of the Akt-dependent signaling pathway has been demonstrated to contribute to the inhibitory effect of low-dose As\({}_{2}\)O\({}_{3}\) on skeletal myogenic differentiation [22]. The defects in insulin activation of the PI3K/Akt signaling pathway has been shown in the skeletal muscle biopsies from young men with low birth weights [37].