Gossypol acetic acid affects locomotor activity by inducing mitophagy-related ferroptosis and apoptosis in zebrafish

Gossypol acetic acid (GAA), a medicinal form of gossypol, is a natural phenolic compound found in cottonseed, which possesses various biological activities, such as anti-fertility, antiviral, anti-inflammatory, antibacterial, and anticancer properties. However, its potential risks to aquatic organisms are poorly investigated. Here, the toxic effects on locomotor activity and the underlying mechanism were assessed in vitro and in vivo, using the zebrafish model and PC12 cells. The results showed that after treatment from 6 to 120 h post fertilization, 0.8 μM GAA significantly reduced the locomotor activity of zebrafish larvae. In addition, 0.6 and 0.8 μM GAA disrupted the dopamine neurons and the central nervous system, further inhibiting swimming activity. Furthermore, the muscle sarcomeres and hair cells of zebrafish larvae were affected after GAA exposure. Moreover, after GAA exposure, Fe2+ accumulated in the 0.6 and 0.8 μM GAA groups, and reactive oxygen species (ROS) increased, especially in the head region. In addition, apoptosis could be observed in both the head and tail muscle regions. RT-qPCR results showed that GAA dysregulated the mRNA expression of the genes correlated to mitophagy, oxidative stress, and ferroptosis pathways. The in vitro study using PC12 cells also showed that GAA could induce mitochondrial membrane potential rise, ROS generation, apoptosis, and ferroptosis. These results indicate that GAA affects locomotor activity by disrupting the nervous system and muscle in zebrafish, and its toxicity is closely related to mitochondrial dysfunction, oxidative stress, apoptosis, and ferroptosis.