Investigation of ammonia-induced lethal toxicity toward ion regulation in zebrafish embryos

Ammonia is an environmental pollutant that is toxic to all aquatic animals. However, the mechanism of ammonia toxicity toward the ion regulatory function of early-stage fish has not been fully documented. We addressed this issue using zebrafish embryos as a model. We hypothesized that ammonia might impair ion regulation by inducing oxidative stress, mitochondrial dysfunction, and cell death of epidermal ionocytes and keratinocytes in zebrafish embryos. After exposure to various concentrations (10- 30 mM) of NH₄Cl for 96 h, mortality increased up to 50 % and 100 % at 25 and 30 mM, respectively. Whole-embryo sodium, potassium, and calcium contents decreased at ≥10 mM, suggesting dysfunction of ion regulation. Numbers of H⁺-ATPase-rich (HR) cells and Na⁺/K⁺-ATPase-rich (NaR) cells (two ionocyte subtypes) were not significantly altered at 15 or 20 mM, while the mitochondrial abundance significantly decreased and reactive oxygen species (ROS) levels significantly increased in ionocytes. Moreover, caspase-3-dependent apoptosis was found in epidermal keratinocytes. Whole-embryo transcript levels of several genes involved in ion regulation, antioxidation, and apoptosis were upregulated after ammonia exposure. In conclusion, ammonia exposure was shown to induce oxidative stress and mitochondrial dysfunction in ionocytes and apoptosis in keratinocytes, thereby impairing ion regulation and ultimately leading to the death of zebrafish embryos.