6-PPD induces mitochondrial dysfunction and reduces healthspan and lifespan through SKN-1 in Caenorhabditis elegans

The synthetic antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) is widely used in rubber products, but its toxicity in non-aquatic animals remains poorly understood. We investigated 6-PPD toxicity in mouse embryonic fibroblasts (MEFs) and Caenorhabditis elegans (C. elegans). In MEFs, 6-PPD caused dose-dependent increases in cell death, apoptosis, and mitochondrial dysfunction, along with elevated reactive oxygen species (ROS). In C. elegans, 6-PPD exposure led to higher mortality, delayed development, reduced reproduction, and shortened lifespan. Mitochondrial impairment, increased ROS, decreased mobility, and weakened stress tolerance were also observed. High-performance liquid chromatography (HPLC) analysis detected trace amounts of 6-PPD in C. elegans, whereas 6-PPD Quinone (6-PPDQ) was not detected within the analytical range. Furthermore, we found that 6-PPD upregulated the conserved transcription factor skinhead-1 (SKN-1/Nrf2) transcription factor. While SKN-1 was necessary for the reduced lifespan and early mortality caused by 6-PPD exposure, its loss failed to prevent the mitochondrial defects induced by 6-PPD exposure. Notably, SKN-1 gain-of-function mutations under basal conditions impaired mitochondrial function, and 6-PPD exposure further exacerbated these defects. These findings indicate that 6-PPD disrupts mitochondria through both SKN-1-dependent and SKN-1-independent mechanisms. Overall, our study demonstrates that 6-PPD impairs development, reproduction, healthspan, and lifespan through the SKN-1/Nrf2 pathway, with mitochondrial dysfunction mediated by both SKN-1/Nrf2-dependent and independent mechanisms.