###### Abstract  
The relationship between plasticizer di(2-ethylhexyl) phthalate (DEHP) and low birth weight in neonates has been reported. Immature muscle differentiation may be involved in low birth weight. The myotoxic characteristics of chemicals have been observed in differentiating immortalized and primary muscle cells. Here, we explored the myotoxic effects of DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in vitro using the immortalized mouse skeletal myoblasts C2C12 and primary human skeletal muscle progenitor cell (HSMPC) models. We found that both DEHP and MEHP at the concentrations of 10-100 uM, which were non- and low-cytotoxicity concentrations, significantly and dose-dependently inhibited the creatine kinase activity, myotube formation with multiple nuclei, and myogenin and myosin heavy chain (muscle differentiation markers) protein expression in C2C12 and HSMPCs under differentiation medium. Both DEHP and MEHP significantly decreased Akt phosphorylation in C2C12 and HSMPCs during differentiation. Taken together, DEHP and its metabolite MEHP are capable of inhibiting Akt-regulated myogenesis in myoblasts/myogenic progenitors during differentiation. These findings suggest the possibility of DEHP as an environmental risk factor affecting skeletal myogenic differentiation. Moreover, these in vitro muscle cell models may be a possible alternative method to animal myotoxicity testing.  
plasticizer phthalates; DEHP; MEHP; Akt signaling; myogenesis +
Footnote †: journal: Medical Research, China Medical University Hospital, China Medical University, Taipei 406, Taiwan  
metabolite MEHP and four major secondary metabolites could be significantly detected in urine [7]. DEHP has been reported to possess the ability to interfere with the reproductive and developmental systems [8; 9]. Based on the analysis of urine phthalate levels in Danish children exposed to a phthalate-enriched indoor environment, DEHP was found to have the highest total daily intake [10].  
A low birth weight is a marker of poor fetal development and growth [11]. An epidemiological study has shown that prenatal di-n-butyl phthalate and DEHP exposure contribute to low birth weight in Chinese newborns [12]. A relationship between prenatal exposure to phthalates and preterm delivery in Chinese women has also been reported [13]. In seriously low-birth-weight infants, a significant negative correlation between the sum of DEHP metabolites and birth weight have been demonstrated [14]. These studies indicate that DEHP exposure may contribute to low birth weight. A reduction in myogenesis has been demonstrated to be reflected in the low birth weight of intrauterine growth-restricted (IUGR) fetal piglets [15] and sheep [16]. [PERSON] et al. also found that myogenesis in human muscle stem cells cultured from the low-birth-weight individuals is retarded [17]. DEHP has been shown to inhibit myogenesis in C2C12 myoblasts [18]. However, it is still necessary to further explore the detailed effects and possible mechanisms of DEHP, and its metabolite MEHP, on skeletal myogenic differentiation.  
The myotoxic characteristics of chemicals on differentiating immortalized and primary muscle cells have been observed in vitro [19; 20; 21; 22; 23]. Here, we aimed to study the myotoxic effects of both DEHP and MEHP on the myogenesis using immortalized mouse C2C12 skeletal myoblast and primary human skeletal muscle progenitor cell (HSMPC) models.