Exploring the targets and mechanisms of 6PPD-Q-induced cardiotoxicity: A network toxicology and molecular docking study.

Abstract

6PPD-quinone (6PPD-Q), a transformation product of the tire antioxidant 6PPD, is an emerging traffic-related contaminant with well-documented ecotoxicity in aquatic species. Its recent detection in airborne particulate matter, road dust, and human urine has raised concerns about possible human health effects. However, its cardiotoxicity in mammalian cells remains poorly understood. Here, we integrated network toxicology, molecular modeling, and in vitro experiments to investigate the effects of 6PPD-Q on the human AC16 cardiomyocyte cell line and the underlying mechanisms. Network toxicology identified 144 putative targets associated with 6PPD-Q-induced cardiotoxicity, with TP53 ranked as the top hub gene, followed by TRAF6, MAPK1, SQSTM1, TNF, and IL6. Enrichment analysis highlighted oxidative stress, inflammation, apoptosis, and autophagy regulation as key biological processes. Molecular docking suggested potential interactions between 6PPD-Q and these hub proteins, while molecular dynamics simulation supported the dynamic stability of the predicted 6PPD-Q-p53 complex. Experimentally, 6PPD-Q induced marked ROS accumulation in AC16 cells, with an IC50 of 13.92 μM after 24 h exposure. It also promoted p53 phosphorylation at Ser15 without increasing total p53 protein or TP53 mRNA expression, indicating stress-associated post-translational activation of p53. This response was accompanied by TRAF6 upregulation, enhanced MAPK1 phosphorylation, and increased IL6 and TNF expression and secretion, supporting the activation of inflammatory stress signaling. Functionally, 6PPD-Q triggered mitochondrial apoptosis, as indicated by mitochondrial membrane potential loss, an increased Bax/Bcl-2 ratio, and activation of cleaved caspase-9 and cleaved caspase-3. In addition, 6PPD-Q disrupted autophagy homeostasis, as evidenced by LC3-II accumulation, p62 depletion, reduced SQSTM1 mRNA expression, increased yellow LC3 puncta, and decreased red-only puncta, suggesting autophagosome accumulation with impaired late-stage autophagic flux. Pharmacological inhibition of p53 by Pifithrin-α (PFTα) attenuated 6PPD-Q-induced inflammatory responses, MAPK1 phosphorylation, mitochondrial apoptosis, and SQSTM1 transcriptional suppression, supporting the functional involvement of p53-related stress signaling. Collectively, these findings show that 6PPD-Q induces cardiomyocyte injury through a p53-associated stress network linking oxidative stress, inflammatory signaling, mitochondrial apoptosis, and autophagy dysregulation, providing mechanistic insight into its potential cardiovascular relevance.