Development and validation of an in vitro model to explore mechanisms of skeletal muscle toxicity

The validation of in vitro skeletal muscle models may play a pivotal role in capturing safety endpoints early within the research and development process. Therefore, the primary aim of this project was to investigate the extent of translation from an in vivo rat model to an in vitro skeletal muscle model, using a toxicogenomics approach. To this end, the mechanisms of toxicity of three novel sulfonyl isoxazoline (SI) herbicides (two triazoles and one phenyl) developed by Syngenta were investigated in vivo and in vitro. In vivo histopathology studies identified striated muscle as the target organ of SI triazole toxicity, and the stomach and liver as the target organs of SI phenyl toxicity. Mechanistic toxicogenomics was carried out on liver, heart and skeletal muscle tissues from rats treated with sub-toxic doses of the SI triazoles (177 and 197) and phenyl (907) compounds for 28 days. The biological processes perturbed by SI triazoles included mitochondrial dysfunction, oxidative stress, energy metabolism, cell death, protein regulation and cell cycle. In contrast, perturbation of cholesterol biosynthesis was identified as the SI phenyl mechanism of toxicity. Using an in vitro rat skeletal muscle cell line (L6), it was demonstrated that the SI triazoles induced mitochondrial dysfunction, mitochondrial superoxide production, cell cycle arrest, hypertrophy and apoptosis. These in vitro results were consistent with the in vivo toxicogenomics data, providing validation that these models may predict skeletal muscle toxicity. To increase detection of xenobiotic-induced mitochondrial effects in skeletal muscle, L6 cells were forced to rely on mitochondrial oxidative phosphorylation by substituting galactose for high glucose in the growth media. In galactose-grown cells, oxygen consumption was increased, glycolysis was repressed and susceptibility to mitochondrial toxicants correspondingly increased. Future work should aim to further develop the L6 model to better mimic the in vivo model using 3D and microfluidic technologies.