Difenoconazole induces cardiovascular toxicity through oxidative stress-mediated apoptosis in early life stages of zebrafish (Danio rerio)

Difenoconazole (DIF), a common broad-spectrum triazole fungicide, is associated with an increased risk of cardiovascular diseases. Unfortunately, little attention has been paid to the mechanisms underlying this association. In this study, zebrafish embryos were exposed to DIF (0, 0.3, 0.6 and 1.2 mg/L) from 4 to 96 h post fertilization (hpf) and cardiovascular toxicity was evaluated. Our results showed that DIF decreased hatching rate, survival rate and heart rate, with increased malformation rate. Cardiovascular deformities are the most prominent, including pericardial edema, abnormal cardiac structure and disrupted vascular pattern in two transgenic zebrafish models (myl7:egfp and fli1:egfp). DIF exacerbated oxidative stress by via accumulation of reactive oxygen species (ROS) and inhibition of antioxidant enzyme. Cardiovascular apoptosis was triggered through increased expression of p53, bcl-2, bax and caspase 9, while DIF suppressed the transcription of key genes involved in calcium signaling and cardiac muscle contraction. These adverse outcomes were restored by the antioxidant N-acetyl-L-cysteine (NAC), indicating that oxidative stress played a crucial role in DIF-induced cardiovascular toxicity caused by apoptosis and inhibition of cardiac muscle contraction. Taken together, this study revealed the key role of oxidative stress in DIF-induced cardiovascular toxicity and provided novel insights into strategies to mitigate its toxicity.