6PPD and its metabolites induce locomotor dysfunction in zebrafish through dopaminergic disruption with brain accumulation

Tire wear particles release the antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidative products, notably 4-hydroxydiphenylamine (4-HDPA) and 6PPD-quinone (6PPD-Q), which pose unrecognized neurotoxic hazards to aquatic life. This study demonstrates that seven-day exposure of zebrafish larvae to environmentally relevant concentrations (30 and 300 µg/L) of these compounds leads to dose-dependent locomotor deficits, with up to a 42 % reduction in swimming speed at 300 µg/L. LC-MS/MS analysis confirmed accumulation of 6PPD, 4-HDPA, and 6PPD-Q in brain tissue, where they provoked oxidative stress, elevated reactive oxygen species, and dysregulated key antioxidant enzymes. Critically, dopaminergic dysfunction emerged as a central mechanism: downregulation of tyrosine hydroxylase (th2) and dopamine transporter (dat) expression coincided with depletion of brain dopamine and behavioral impairments. Suppression of corticotropin-releasing hormone further indicated broad neuroendocrine disruption. Despite preserved motor neuron morphology, apoptotic neuronal death increased, triggering compensatory neurotrophic and anti-apoptotic gene responses. By delineating this adverse outcome pathway, from chemical exposure and brain accumulation to oxidative injury, dopaminergic collapse, and locomotor impairment, our findings provide the mechanistic evidence of tire-derived antioxidant neurotoxicity in fish. These results underscore the urgent need to reassess 6PPD use in tire formulations, inform regulatory policies on tire wear particle emissions, and guide the design of safer alternative antioxidants to protect aquatic ecosystems.