Risk assessment of developmental and neurotoxicity by the flavoring agent perillaldehyde: NAC (N-acetylcysteine) mitigation of oxidative stress-mediated inhibition of the Nrf2 pathway.

Perillaldehyde (PAE), a prevalent flavoring agent, has raised safety concerns due to conflicting evidence regarding its toxicity. This study provides a comprehensive assessment of the developmental and neurotoxic effects of PAE in zebrafish, elucidating the underlying mechanisms of its toxicity. Results showed that PAE affected the viability and hatching rate of zebrafish at 96 h postfertilization with the 50 % lethal concentration (LC50) of 7.975 mg/L. Furthermore, exposed‌ to a non-lethal concentration of 4 mg/L PAE induced a spectrum of morphological abnormalities, such as pericardial edema, delayed yolk sac absorption, reduced body length, and microphthalmia. Behavioral observations revealed that PAE reduced motor ability, and was accompanied by an increase in spontaneous turning angle and angular velocity. Using the TG(elav13:EGFP) transgenic model, we observed the number of newborn neurons was reduced, indicating that PAE induced obvious neurotoxic effects. Additionally, this concentration facilitated the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), concomitantly decreasing the activity of antioxidant enzymes. QRT-PCR analysis revealed that PAE down-regulated Nestin and Neurogenin1 gene expression, up-regulated Glipr1a and Nox1 gene expression, and inhibited the Nrf2/HO-1 pathway. Notably, co-administration of N-acetylcysteine (NAC), an inhibitor of oxidative stress, mitigated oxidative stress levels and partially ameliorated the neurotoxicity. These findings suggest that oxidative stress is the primary mediator of PAE-induced neurotoxicity. This study provides crucial insights for the safe application of PAE.