Chlorpyrifos abolished C2C12 myoblast cell proliferation and differentiation via mitochondrial stress

Chlorpyrifos (CPF) is a widely used organophosphate insecticide reported to contaminate agricultural products, and is absorbed through the gastrointestinal mucosa, respiratory epithelium, and skin. To date, knowledge about the effect of CPF on skeletal muscle stem cells responsible for muscle formation and regeneration is still limited. Hence, this study aimed to investigate the effects of CPF on skeletal muscle stem cell proliferation, differentiation, and mitochondrial stress.

This study used the C2C12 myoblast cell line as a model for skeletal muscle stem cells. The myoblasts were treated with CPF at 0-100 µM for 24-72 h. Cell viability and proliferation were determined by MTT assay, cell counting, Ki-67 immunostaining, and flow cytometry. Reactive oxygen species (ROS) production was determined by H₂DCFDA assay, and mitochondrial stress-related gene expression determined by real-time polymerase chain reaction. Differentiated myotube formation was measured by immunostaining and western blotting.

Treatment with 50-100 µM CPF significantly decreased myoblast cell viability and cell proliferation 24 h after treatment. Flow cytometry revealed that CPF significantly decreased cells in G0/G1, but increased cell accumulation at Sub G0/G1, S, and G2/M cell cycle phases. In addition, CPF significantly increased ROS production and downregulated mitochondrial-related genes OPA-1, Mfn-1, Mfn-2, and Pink-1, but upregulated Cyt-c expression leading to caspase-3 activation. Moreover, 10-25 µM CPF significantly diminished myoblast differentiation by decreasing both the number and size of multinucleated myotubes. The myoblast differentiation markers myosin heavy chain and myogenin (but not MyoD) also decreased with CPF treatment. A possible mechanism of myoblast proliferation and differentiation inhibition by CPF may occur through inhibition of Akt phosphorylation.

CPF potentially abrogated myoblast proliferation and differentiation by inhibiting Akt phosphorylation. These findings raise concerns about the potential adverse effects of CPF contamination in agricultural products on consumers.