Combined exposure to 6PPD and 6PPD-Q induced neurotoxic responses in zebrafish and SH-SY5Y cells: Evidence from neurotransmitter disruption, oxidative damage, and apoptosis

The rubber antioxidant N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) and its quinone derivative (6PPD-Q) are coexisting environmental contaminants with documented neurobehavioral effects. However, the neurotoxic consequences arising from their combined exposure remain unclear. In this study, adult zebrafish and SH-SY5Y cells were used to investigate the neurotoxic effects associated with co-exposure to 6PPD and 6PPD-Q. In zebrafish, 100 μg/L 6PPD+100 μg/L 6PPD-Q increased time spent and distance traveled in the non-reward area of the T-maze after 28 d exposure, accompanied by pathological damage in brain tissue, including reduced neuronal density, decreased Nissl bodies, and apoptosis. 6PPD-Q exacerbated oxidative damage and the decreased levels of neurotransmitters induced by 6PPD. Metabolomics implicated disruptions in neuroactive ligand-receptor interaction and citrate cycle. Transcriptomic analysis further identified dysregulation in oxidative stress, cell death, and nervous system processes related pathways, such as Peroxisome, Axon guidance, and PI3K-Akt pathway. In SH-SY5Y cells, co-exposure reduced cell viability and produced predominantly synergistic effects across concentration combinations tested. 6PPD-Q aggravated mitochondrial damage and enhanced the protein expression levels related to apoptosis induced by 6PPD, including caspase-3 and bax/bcl-2. Moreover, co-exposure inhibited the PI3K-AKT pathway, which might exacerbate neurotransmitter disturbance and apoptosis. The findings enrich the understanding of neurological health risks linked to 6PPD and 6PPD-Q, highlighting the importance of preventive strategies to mitigate the exposure risks.