Gut microbiota-associated metabolic alterations contribute to 6PPD-Q-induced intestinal barrier dysfunction in mice

ABSTRACT N- (1,3-Dimethylbutyl)- N ′-phenyl- p -phenylenediamine quinone (6PPD-Q), a ubiquitous tire-derived contaminant detected in environmental matrices and human samples, poses potential intestinal health risks to mammals. However, the underlying mechanisms remain poorly understood. C57BL/6 mice were orally exposed to 6PPD-Q (4 mg/kg, once every 3 days for 28 days) with or without broad-spectrum antibiotic pretreatment to deplete gut microbiota. 16S rRNA sequencing, untargeted metabolomics, immunofluorescence, and periodic acid-Schiff staining were performed to characterize microbial, metabolic, and intestinal barrier alterations. 6PPD-Q exposure induced gut dysbiosis characterized by enrichment of Lachnospiraceae and depletion of Akkermansiaceae and Bacteroidaceae accompanied by alterations in tryptophan and nicotinate-nicotinamide metabolism, including reduced fecal serotonin and indole-3-propionic acid levels. Furthermore, 6PPD-Q exposure impaired intestinal barrier integrity, as evidenced by altered expression of tight junction proteins (ZO-1 and claudin-1), reduced goblet cell density, and decreased MUC2 expression. Notably, antibiotic-mediated microbiota depletion attenuated 6PPD-Q-associated microbial, metabolic, and intestinal barrier alterations, suggesting that gut microbiota contribute to 6PPD-Q-associated intestinal responses. Collectively, this study provides mechanistic insights into microbiota-associated mechanisms underlying 6PPD-Q-induced intestinal barrier alterations and advances understanding of potential health implications of tire-derived emerging contaminants. IMPORTANCE 6PPD-Q, a primary oxidation product of the rubber antioxidant 6PPD, is an emerging environmental pollutant widely detected in human blood and urine. While its systemic toxicity has been recognized, the role of the gut microbiota in mediating 6PPD-Q-induced intestinal barrier disruption remains largely unexplored. Here, we demonstrate that gut microbiota plays an important modulatory role in 6PPD-Q-induced intestinal barrier impairment using antibiotic depletion and mouse models. Multi-omics analyses revealed that 6PPD-Q altered the composition of gut commensals and disrupted metabolic homeostasis, while functional validation confirmed its impact on epithelial integrity. Our findings identify the gut microbiota as a key modulator of 6PPD-Q toxicity and provide novel mechanistic insights into the environmental health risks of tire-derived pollutants.