Potential mechanism of delayed endochondral ossification and muscle atrophy induced by nitrate exposure in Bufo gargarizans tadpoles.

Nitrate pollution in aquatic ecosystems is a pervasive and growing problem globally, which can pose a substantial threat to amphibian survival. The shift in amphibian locomotive behaviors, specifically from tadpole tail swimming to adult limbs movement, are essential for facilitating physiological adaptations required for terrestrial survival. However, there is a question as to whether nitrate exposure affects amphibian motor systems. For this purpose, morphological, histological, transmission electron microscopic and transcriptomic analyses were performed on the hindlimb bones and muscles of Bufo gargarizans tadpoles exposed to different concentrations of nitrate-nitrogen (0, 10, and 100 mg/L NO3-N). Our studies showed that by inhibiting chondrocyte apoptosis, NO3-N treatment potentially delays hypertrophy progression, and may thereby delay the progression of endochondral ossification. Meanwhile, muscle atrophy occurred after exposure to NO3-N. In addition, transcriptome analysis indicated that NO3-N inhibited the endochondral ossification process by regulating the gene expression levels associated with thyroid hormone metabolism (THM) and endochondral ossification (EO) by affecting the expression of genes linked to mitochondrial complex (MC), oxidative stress (OS), ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP). Collectively, this article shed light on the underlying molecular mechanisms responsible for the perturbations caused by NO3-N on the bones and muscles, highlighted the adverse effects of NO3-N pollution on the locomotor function of amphibians, and provided fresh perspectives for evaluating the potential ecotoxicity of nitrate pollution to aquatic organisms.