Anxiety-like behavior and social stress in adult zebrafish induced by 6PPD: Insights from single-cell RNA sequencing

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), a common rubber antioxidant, has raised global concern due to its widespread presence in the environment and its toxicity to multiple organs in aquatic organisms. However, whether 6PPD causes neurotoxicity and its underlying mechanisms remain unclear. In this study, adult zebrafish were exposed to 20 μg/L of 6PPD for 28 days to investigate the behavioral and molecular mechanisms underlying 6PPD-induced neurotoxicity. The results showed that zebrafish exposed to 6PPD exhibited anxiety-like behaviors and reduced social interactions. Single-cell RNA sequencing (scRNA-seq) of the whole-brain tissue revealed that although cellular diversity was maintained, 6PPD disrupted cerebral homeostasis, resulting in decreased abundance of immune cells, neural progenitor cells (NPCs), and neurons, alongside an increase in astrocytes. Mechanistically, 6PPD disturbed the balance between excitatory and inhibitory neurons and downregulated key neuromodulators (e.g., oxt, kiss1, and th), contributing to behavioral abnormalities. Additionally, 6PPD altered NPCs fate by inhibiting neuronal differentiation through cell cycle arrest and sustained Notch activation, thereby suppressing neurogenesis. In summary, our study reveals that chronic exposure to 6PPD impairs neurobehavioral function by disrupting cerebral cellular homeostasis, compromising neuronal communication, and suppressing neurogenesis, highlighting its potential hazard to neurological health in aquatic organisms and underscoring the need for greater environmental risk assessments.