35 Comparative evaluation of ROS–NLRP3 inflammasome-mediated pulmonary toxicity induced by primary and secondary microplastics

Abstract Micro- and nanoplastics (MNPs) have emerged as global environmental pollutants, raising growing concerns about their potential respiratory toxicity. MNPs are classified into primary forms, which are manufactured directly, and secondary forms, which originate from the fragmentation and environmental aging of larger plastic debris. Although most previous studies have focused only on primary MNPs, information regarding the toxicity of environmentally relevant secondary MNPs remains limited. Hence, this study aimed to comparatively evaluate in vitro and in vivo toxicities of primary and secondary polystyrene (PS) MNPs. In vitro, secondary PS induced greater cytotoxicity than primary PS at 24 h post-treatment, which was associated with cellular reactive oxygen species (ROS) accumulation, NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, and subsequent interleukin (IL)-1β release. These processes are known to be triggered by ROS production, lysosomal damage, and frustrated phagocytosis. Moreover, conditioned medium from PS-exposed macrophages did not directly cause cytotoxicity in epithelial cells but markedly enhanced cytokine expression, indicating indirect inflammatory signalling. In a single pharyngeal aspiration exposure study using BALB/c mice, secondary PS showed higher levels of pulmonary inflammatory parameters in bronchoalveolar lavage fluid, including lactate dehydrogenase, total protein, cytokines, and the numbers of total cells and neutrophils, compared with primary PS at 24 h post-exposure. Furthermore, UV irradiation of the particle surface enhanced intrinsic ROS generation, thereby amplifying the subsequent toxic and inflammatory responses. Collectively, these findings provide mechanistic insights into how primary and secondary microplastics differentially induce respiratory inflammation and contribute to the safety assessment of airborne microplastics.