In-Depth Investigation into the Neurotoxic and Neuroglioma Risks of 6PPD Exposure in Zebrafish Larvae: Benchmark Dose Analysis, Molecular Mechanisms, and Adult Pathological Effects

As an antioxidant widely used in tire rubber, (1,3-dimethylbutyl)-<i>N</i>'-phenyl-<i>p</i>-phenylenediamine (6PPD) has been recognized as an emerging contaminant with unclear neurotoxic potential. Using zebrafish (<i>Danio rerio</i>) as a model, we systematically evaluated the neurodevelopmental, behavioral, and molecular effects of 6PPD at environmentally relevant (5 μg/L) and sublethal (300 μg/L) concentrations. 6PPD exposure caused developmental abnormalities, including microphthalmia, exophthalmos, cardiac malformations, and hypopigmentation. Notably, benchmark dose (BMD) modeling identified the eye as a sensitive target, with microphthalmia likely impairing retinal development, photoreception, and circadian regulation. 6PPD increased neuronal apoptosis; impaired central nervous system and lateral line development; and, at high doses, reduced vitality, rhythmicity, and responsiveness. Corticotropin-releasing hormone receptor 1 (CRHR1), a synaptic receptor enhancing glutamatergic and suppressing GABAergic signaling, was identified as a potential target mediating neuronal hyperexcitability and hyperactivity at low doses. Consistently, behavioral tests confirmed a biphasic hormetic response: hyperactivity at low doses and hypoactivity at high doses. Furthermore, transcriptomic analysis, immunohistochemical staining, and xenograft tumor assays revealed a close association between 6PPD exposure and gliomagenesis, suggesting its potential risk of inducing glioma. Overall, these findings provide the first integrated phenotypic, behavioral, and molecular evidence of 6PPD-induced neurotoxicity and highlight CRHR1-mediated hormetic responses as critical considerations for risk assessment.