Environmental-related concentrations of 6PPD-quinone drives intestinal toxicity via neutrophil extracellular traps: A novel mechanism of rubber-additive contamination.

The ubiquitous distribution of 6PPD-quinone (6PPD-Q), a transformation product of the widely used tire antioxidant 6PPD, across various environmental matrices has raised growing concern over its potential health risks. Nevertheless, the involvement of neutrophils in 6PPD-Q-induced biotoxicity remains poorly understood, and its key effector pathways, along with the associated molecular regulatory networks, have yet to be fully characterized. In this study, male mice exposed to 6PPD-Q (0.2 or 2 μg/kg/day) were found to develop colonic inflammation, mucosal layer attenuation and loss of tight junction proteins. These pathological changes were associated with the formation of neutrophil extracellular traps (NETs) and gut microbiota dysbiosis, including alterations in Ruminococcus, Coprostanoligenes, and Brachybacterium. Correlation analyses showed that Ruminococcus was positively linked to NETs-related markers, while Brachybacterium correlated inversely with inflammation and positively with intestinal barrier function. Further analysis revealed that 6PPD-Q triggers NETs formation through a p38-dependent pathway. Flow cytometry demonstrated that supernatant from 6PPD-Q-treated neutrophils increased apoptosis in colonic epithelial cells by ~9.9%, which corresponded to elevated NLRP3 expression and inflammasome activation. These effects were reversed upon DNase I treatment. Moreover, in neutrophil-epithelial coculture model, the presence of neutrophils further augmented 6PPD-Q-induced colonocyte apoptosis by an additional 5.5%. Collectively, these findings indicate that 6PPD-Q elicits both direct intestinal damage and indirect injury mediated by p38-driven NETs formation. This study underscores the role of NETs-mediated mechanisms in 6PPD-Q toxicity and provides mechanistic basis for preventive strategies aimed at mitigating its health risks.