6PPD exposure reduced the melanin deposition by inhibiting tyrosinase activity in larval zebrafish

Abstract

N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), a critical antioxidant in rubber manufacturing, has raised global concerns due to its environmental persistence and toxic transformation products. While its developmental toxicity is increasingly recognized, the mechanisms underlying melanogenesis disruption in fish remain poorly understood. In this study, we systematically investigated 6PPD's effects on melanin synthesis in zebrafish embryos and murine melanoma cells (B16F10). Larval zebrafish were exposed to 10 and 100 μg/L 6PPD exhibited dose-dependent reductions in melanin deposition, suppressed tyrosinase activity and downregulation of melanogenesis-related genes (tyr, mitfa, trp1, trp2, and dct). Notably, the inhibition of tyrosinase activity was closely linked to behavioral impairments, as larvae exposed to 100 μg/L 6PPD showed significantly decreased swimming distance and velocity under light-dark cycles. Molecular docking revealed strong binding affinities between 6PPD and the key melanogenic proteins (Dct, Tyr, Tyrp1a, and Tyrp1b), suggesting direct interference with enzymatic function. Parallel experiments in B16F10 cells confirmed cross-species melanin suppression, with reduced gene expression and melanin synthesis-related protein (Mitfa and Tyr) at subtoxic concentrations (0.5 and 2.5 mg/L), which was also verified by Masson-Fontana staining. These findings demonstrated that 6PPD disrupted conserved melanogenic pathways by targeting enzymatic activity, posing ecological risks to aquatic and terrestrial organisms.