Inhalation exposure to a tire-derived pollutant (6-PPD) triggers lung injury and mitochondrial dysfunction in mice

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD)-a commonly used rubber antioxidant-is primarily released into the environment through tire wear particles, raising concerns about its potential respiratory health effects. However, little is known of its direct effects. This study investigated the pulmonary toxicity of 6-PPD in mice administered 5 or 10 mg/kg via intratracheal instillation for 7 or 14 days. No treatment-related mortality or significant changes in body weight were observed. However, lung weight significantly increased in all 6-PPD-treated groups, indicating lung tissue alterations. Bronchoalveolar lavage fluid analysis revealed dose-dependent increases in inflammatory cells, including macrophages, lymphocytes, neutrophils, and eosinophils, suggesting both acute and sustained inflammation. Furthermore, pro-inflammatory cytokines, including IL-6 and TNF-alpha, were significantly elevated, confirming a robust inflammatory response. Histopathological evaluation showed inflammatory infiltration, foamy alveolar macrophages, bronchial epithelial degeneration, and granulomatous inflammation, confirming 6-PPD-induced pulmonary injury. Mitochondrial protein expression analysis demonstrated that 6-PPD drives increased mitochondrial stress, leading to both mitophagy and autophagy. This stress was functionally supported by a notable reduction in Complex I activity and a corresponding decrease in cellular ATP levels, highlighting severe mitochondrial dysfunction. These findings demonstrate that 6-PPD induces considerable pulmonary inflammation (IL-6/TNF-alpha), histopathological alterations, and mitochondrial dysfunction (Complex I activity and ATP). The disruption of mitochondrial homeostasis may also contribute to sustained oxidative stress and chronic lung inflammation. These results highlight the potential respiratory risks associated with environmental exposure to 6-PPD. Although the doses used in this study exceed typical environmental levels, our results could provide a relevant model for high-level human occupational exposures to 6-PPD in humans.