Methimazole disrupted skeletal ossification and muscle fiber transition in Bufo gargarizans larvae

Methimazole (MMI) is an emerging endocrine disrupting chemical (EDC) due to its increasing use in the treatment of thyrotoxicosis (hyperthyroidism), but its potential impact on amphibian development remains largely unexplored. In the present study, the effects of 8 mg/L MMI and 1 μg/L thyroxine (T4) exposure on skeletal ossification and muscle development in Bufo gargarizans tadpoles were comprehensively investigated by double skeletal staining, histological analysis and RNA sequencing. Our results indicated that MMI treatment down-regulated the expression levels of ossification-related genes (e.g., BMPs, MMPs, and Wnt9a) in cartilage, thereby delaying chondrocyte apoptosis and inhibiting hindlimb ossification. Muscle sarcomere was elongated in both the MMI and T4 treatment groups, which may lead to muscle weakness and consequently affect land motion. Additionally, we evaluated the expression levels of fast muscle-related genes (TNNI2 and TNNT3) and slow muscle-related genes (TNNI1 and TNNT1), revealing an opposite trend in the transition from fast to slow muscle after T4 and MMI exposures. In conclusion, these findings fill the data gap regarding MMI contamination in aquatic environments by revealing the negative effects of MMI on amphibian bone and muscle development. Future studies should address the toxicity of EDCs to wildlife and inform aquatic ecosystem conservation strategies.