The molecular mechanisms of toxicity responses such as inflammation and ROS production to PP nanoplastic stimulation were investigated both in vivo and in vitro. Our results showed increased inflammatory cells, cytokines, and chemokines in the BALF of PP-instilled mice, alongside higher ROS production in the lung tissues, as compared to the VC group. Histopathological analysis of the lung tissue of PP-instilled mice revealed lung damage, including the infiltration of inflammatory cells in the perivascular/parenchymal space, alveolar epithelial hyperplasia, and foamy macrophage aggregates. In vitro investigation revealed depolarisation of the mitochondrial membrane potential, decreased ATP levels, and ROS production as a result of PP stimulation. PP was also found to induce the production of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and cell death, with an increase in the levels of p-p38 and p-NF-κB protein observed both in vivo and in vitro. Interestingly, p38 and ROS inhibition regulated toxic responses such as inflammatory cytokines and cell death. These results suggest that PP nanoplastic stimulation may contribute to the pathogenesis of inflammation within the respiratory system via NF-κB signaling, which is associated with mitochondrial damage (Fig. 11).
With advancements in technology, the exposure to airborne microparticles such as PM in daily life has been increasing. Microparticles including nanoplastic can form as a result of industrial processes, with other sources including traffic or road construction [23,24,25,26]. Fragments and fibres are the most commonly found forms of plastic in different environments worldwide [27]. According to a recent report, the human body may be exposed to an average of 0.1–5 g of microplastics per week through different modes such as ingestion and inhalation [28]. Previous studies hypothesised that the major route of human exposure to microplastics was through ingestion. However, recent studies have reported that airborne microplastics through inhalation may be another major route [29, 30]. Airborne microplastic particles have been measured in various regions including indoor and outdoor environments, with particles of approximately 9.80/m3 on average, the inhalation rates were predicted to be 15 m3/day, with an average rate of annual inhalation exposure of 53,700 particles per person [29, 31]. In a few exposure models, humans were predicted to inhale 6.5–8.97 μg/kg/day of microplastics, and the rate of exposure to microplastics through inhalation, with infants inhaling approximately 3–50-fold higher levels than adults [32]. We observed a pulmonary toxic response including inflammation response and ROS production following 2.5 mg/kg/day of PP nanoplastic exposure, which is shown that correlate to approximately 300-fold the daily human exposure rate for adults and sixfold the dose for infants [32]. These results suggest that further studies on the effects of long-term exposure to microplastics on the lungs are needed, considering that microplastics accumulate in the lungs through continuous and long-term inhalation pathways in the human body.
Inhalation exposure to microplastic in human can be influenced by factors such as the rate of uptake, translocation, and accumulation for the sizes, shapes, doses, and surface functionalization [33]. Microplastics in natural matrices are most often found to have irregular shapes including fragments and fibres and can be deposited in the interior parts of the lung because of their small aerodynamic diameter (particle size expressed in terms of settling rate) [27]. Recent studies reported that microplastics have been identified within all lung regions (upper, middle, and lower) of humans, with the majority being fibrous and fragmented (microplastics with dimensions as small as 4 μm) in shape [10]. Asbestos fibres with lengths ranging from 50 to 200 μm have been found in the alveolar cavity despite their large size, which indicates infiltration into the alveolar region of the lung [34]. Previous studies reported that exposure to airborne microplastics occurring naturally poses might be a risk to the human body through accumulation of lung tissue upon inhalation [7]. However, in vivo studies are lacking on the inhalation toxicity of naturally occurring microplastics with different shapes, including fragments, fibres, and beads. We manufactured PP nanoparticles (irregular in shape and average size 0.66 ± 0.27 μm) by pyrolysis using PP beads. We showed that PP nanoplastics had a spherical shape but formed irregular fragments (Fig. 1a). We observed that inflammatory cellular changes in BALF of PP nanoplastic-instilled mice. Histopathological analysis in 2.5 and 5 mg/kg PP nanoplastic-instilled mice revealed foamy macrophage aggregates in the alveolar space, indicating direct alveolar infiltration of PP nanoplastic. These results show that PP nanoplastic stimulation can cause inflammatory responses through alveolar infiltration; however, further studies are needed to determine the mechanism of lung inflow and injury according to the shape of nanoplastic.
Previous studies have demonstrated that the inhalation of various nanoparticles can lead to bronchial epithelial injury by epithelial barrier infiltration both in vivo and in vitro [22, 35, 36]. Through inhalation, airborne nanoplastics may potentially pose a threat to the health of the human respiratory system. However, only a limited number of in vitro studies have been conducted on the inhalation toxicity of micro and nanoplastic exposure [12]. Previous studies investigated the effects of microplastic exposure for various polymers on lung epithelial cells. It has been reported that Polyvinyl chloride (PVC) microplastic (2.5 mg/mL) stimulation increases the level of inflammatory cytokines in a time-dependent manner. However, exposure to 0.625 mg/mL PVC showed no effect until 48 h [37]. PET and PS nanoplastic exposure for 24 h resulted in ROS production and cytotoxicity owing to the cellular uptake of particles in A549 cells [38, 39]. In other studies, the exposure of A549 cells to PS nanoplastics over 24 h led to the accumulation of the particles, which led to the production of inflammatory cytokines and oxidative stress, with ROS production and lipid peroxidation [40]. However, it has been reported that 4.5 mg/mL PP microplastics (size < 20 μm and 25–200 μm) stimulation did not show any toxicity effect for 48 h [41]. PP has been investigated in several toxicological and physiological studies owing to its use in various disposable products. However, toxicity studies focusing on microplastic inhalation are lacking. Recent reports have suggested that microplastic (1–5 μm in size) exposure can result in infiltration of the lung tissue through the respiratory route, and that nanoplastics can infiltrate the alveolus region [12]. Microplastic (< 10 μm) reduced epithelial uptake by the mucociliary clearance mechanism in the lung, whereas particles (< 1 μm) uptake through the epithelium is possible [6]. Therefore, the inhalation toxicity assessment of PP microplastics should consider nanosized plastics both in vitro and in vivo. Our results showed that ROS production, inflammatory cytokine levels, and cytotoxicity were significantly increased in A549 cells exposed to 4 mg/mL of PP nanoplastic compared to those in control cells (Fig. 7), and alveolar epithelial hyperplasia and inflammatory cell infiltration were observed in PP nanoplastic-instilled mice (2.5 and 5 mg/kg) (Fig. 3). In contrast, the cytotoxicity in 1 and 2 mg/mL PP nanoplastic stimulation were slightly increased compared to the VC, but did not show significant increase. These results suggest that PP nanoplastics from long-term exposure and dose might contribute to the pulmonary toxic response through lung epithelium injury.
Our results showed that the number of inflammatory cells, including macrophages, neutrophils, and lymphocytes in the BALF of PP nanoplastic-instilled mice (2.5 and 5 mg/kg) increased significantly as compared with that in the VC group. Neutrophils, which are the most common leukocytes and are essential first responders during the initial phases of inflammation, were predominant in the BALF of PP-instilled mice (Fig. 2a). The granulation and activation of neutrophils causes pulmonary inflammation via the release of various inflammatory cytokines and chemokines [42,43,44]. Especially, helper T (Th) cytokines play important roles in inflammatory responses in pulmonary diseases such as asthma and pulmonary fibrosis [45, 46]. Previous studies reported that IL-17 overexpression and neutrophil accumulation in BALF of mice with diesel exhaust particulates (DEP)-induced lung inflammation were observed and that the Th17 pathway might be involved in DEP-induced inflammation [23]. We investigated the gene expression patterns after PP exposure to reveal the molecular mechanism associated with PP-induced lung inflammation. We observed that the expressions of Th17 signaling pathway-associated genes including those encoding C–C motif chemokine ligand (CCL) 2, CCL12, CCL17, CXCL1, and CXCL5 were increased in the lung tissue of PP-instilled mice. We presume that, PP stimulation might have induced lung inflammation through the Th17 signaling pathway (Additional file 2: Table S1). Recently, nanoparticles such as TiO2, CeO2, and ZnO have been reported to activate neutrophil degranulation, inducing inflammatory tissue injury [47,48,49]. PM-instilled mice have previously been shown to suffer from an increase in the number of neutrophils followed by the release of inflammatory cytokines and chemokines [50]. Interestingly, our results showed that inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, MCP-1, and CXCL1/KC in the BALF of PP-instilled mice (2.5 or 5 mg/kg) increased significantly as compared with those in the VC group, and the histopathological results of PP-instilled mice showed inflammatory cell infiltration (Figs. 2b–f, 3). These results indicate that PP nanoplastic stimulation may contribute to neutrophilic lung inflammation.
Oxidative stress and endoplasmic reticulum (ER) stress resulting from cellular organelle injury caused by exposure to environmental factors such as chemicals and pathogens can lead to various diseases including pulmonary diseases via abnormal inflammation and immune responses [19, 20, 51, 52]. Our results show that PP nanoplastic-exposed A549 cells had significantly increased ROS production and levels of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD)1, SOD2, and glutathione peroxidase-1 (GPX1) (Additional file 1: Figure S1). However, the protein levels of ER stress markers such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) were unchanged (Additional file 1: Figure S2). Interestingly, nuclear factor erythroid-2-related factor 2 (Nrf2) protein levels (total and nuclear) in PP-exposed A549 cells were significantly increased compared to the control, which might have regulated the expression of antioxidant proteins that protect against oxidative damage (Additional file 1: Figure S3). Although more studies focusing on the mechanism of mitochondrial damage by uptake, translocation, and accumulation for PP nanoplastic exposure, are required, we speculate that oxidative stress may be an indirect result of mitochondrial damage. Previous studies have reported that nanoparticles can damage mitochondria, and induce toxicity [53,54,55,56]. Recent studies have reported that ultrafine dust exposure induced oxidative stress and mitochondrial damage in bronchial epithelial cells (BEAS-2B) and monocyte/macrophage cell lines (RAW 264.7 cells) [53]. NH2-PS stimulation induces mitochondrial dysfunction, leading to decreased ATP levels, DNA degradation, and a decline in the mitochondrial membrane potential of BEAS-2B and RAW 264.7 cells [54]. Our results showed that PP nanoplastic exposure induces mitochondrial dysfunctions such as mitochondrial depolarization and decreases ATP levels (Fig. 5). Interestingly, DRP1 proteins merged into the damaged mitochondrial regions (Fig. 6), which might be a quality control mechanism for preserving a healthy mitochondrial network via fission [57]. These results suggest that PP nanoplastic stimulation causes mitochondrial damage and that long-term PP nanoplastic exposure may potentially lead to mitochondrial diseases.
Recent studies have reported that airborne microplastics including nanoplastic cause inflammation through various pathogenesis, such as dust overload, oxidative stress, and cytotoxicity [58,59,60,61]. In particular, ROS overproduction by particle exposure-induced inflammation and cytotoxicity is mediated by the release of cytokines and inflammatory mediators due to the translocation of nuclear factor NF-κB in cell signaling pathways [62,63,64]. Various studies have reported that oxidative stress causes NF-κB activation via the phosphorylation of MAPKs such as p38, ERK, and JNK, which regulate important cellular processes such as proliferation, stress responses, apoptosis, and immune defence [65,66,67,68,69]. Recent studies have reported that the persistent activation of p38 significantly contributes to the pathogenesis of Th2 low neutrophilic inflammation, which is associated with severe asthmatic phenotypes [70]. In asthmatic patients, activated p38 MAPK contributed to TNF-α secretion from natural killer (NK) cells stimulated by IL-12, and the secretion of IL-6, IL-8, and MCP-1 were also partially dependent upon p38 activation [71, 72]. Interestingly, recent studies have reported that apoptosis was induced through p38 signaling by hydrogen peroxide, which is used as an oxidative stress inducer [73]. We demonstrated that PP nanoplastic stimulation caused inflammatory response, oxidative stress, and cell death. Interestingly, we also observed that the inflammatory cytokines and cell death induced by PP nanoplastic stimulation were reduced by p38 and ROS inhibitors (Fig. 11). These results suggest that PP nanoplastic stimulation may contribute to pulmonary toxic response via the p38 MAPK and NF-κB signalling pathways.
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