Investigation of pulmonary inflammatory responses following intratracheal instillation of and inhalation exposure to polypropylene microplastics

Abstract

Background: Microplastics have been detected in the atmosphere as well as in the ocean, and there is concern about their biological effects in the lungs. We conducted a short-term inhalation exposure and intratracheal instillation using rats to evaluate lung disorders related to microplastics. We conducted an inhalation exposure of polypropylene fine powder at a low concentration of 2 mg/m³ and a high concentration of 10 mg/m³ on 8-week-old male Fischer 344 rats for 6 h a day, 5 days a week for 4 weeks. We also conducted an intratracheal instillation of polypropylene at a low dose of 0.2 mg/rat and a high dose of 1.0 mg/rat on 12-week-old male Fischer 344 rats. Rats were dissected from 3 days to 6 months after both exposures, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected to analyze lung inflammation and lung injury.

Results: Both exposures to polypropylene induced a persistent influx of inflammatory cells and expression of CINC-1, CINC-2, and MPO in BALF from 1 month after exposure. Genetic analysis showed a significant increase in inflammation-related factors for up to 6 months. The low concentration in the inhalation exposure of polypropylene also induced mild lung inflammation.

Conclusion: These findings suggest that inhaled polypropylene, which is a microplastic, induces persistent lung inflammation and has the potential for lung disorder. Exposure to 2 mg/m³ induced inflammatory changes and was thought to be the Lowest Observed Adverse Effect Level (LOAEL) for acute effects of polypropylene. However, considering the concentration of microplastics in a real general environment, the risk of environmental hazards to humans may be low.

Keywords: Inhalation exposure; Intratracheal instillation; Microplastics; Polypropylene; Pulmonary toxicity; Rat.

Fig. 1

Characterization of polypropylene aerosol and suspension. SEM observation of polypropylene aerosol (A). Particle size distribution of polypropylene in aerosol (B). SEM observation of polypropylene suspension (C). Particle size distribution of polypropylene in suspension by dynamic light scattering (D)

Fig. 2

Cell analysis and cytokine concentration in BALF following inhalation exposure of polypropylene. Total cell counts in BALF (A). Neutrophil count in BALF (B). Percentage of neutrophils in BALF (C). LDH activity in BALF (D). Concentration of CINC-1 in BALF (E). Concentration of CINC-2 in BALF (F). Concentration of HO-1 in BALF (G). Inhalation exposure of polypropylene induced persistent influx of inflammatory cells and expression of CINC-1, CINC-2, and MPO in BALF from 1 month after exposure. Data are presented as mean ± SD for n = 5/group (*p < 0.05, **p < 0.01)

Fig. 3

Cell analysis and cytokine concentration in BALF following intratracheal instillation of polypropylene. Total cell counts in BALF (A). Neutrophil count in BALF (B). Percentage of neutrophils in BALF (C). LDH activity in BALF (D). Concentration of CINC-1 in BALF (E). Concentration of CINC-2 in BALF (F). Concentration of HO-1 in BALF (G). Inhalation exposure of polypropylene induced persistent influx of inflammatory cells and expression of CINC-1, CINC-2, and MPO in BALF from 1 month after instillation. Data are presented as mean ± SD for n = 4–5/group (*p < 0.05, **p < 0.01)

Fig. 4

Hematoxylin and eosin staining of lung sections following inhalation exposure of polypropylene at each time course (A), and comparison of polarized light observation in the lung at 3 months after exposure in the high concentration group (B). Persistent mild inflammation, mainly neutrophils and alveolar macrophages, was observed until 1 month after exposure. Aggregation of inflammatory cells was observed at 3 months after exposure in the high-concentration group (Black arrow). (Scale bar: 100 µm)

Fig. 5

Hematoxylin and eosin staining of lung sections following intratracheal instillation of polypropylene at each time course. Persistent mild inflammation, mainly neutrophils and alveolar macrophages, was observed until 3 months after exposure. (Scale bar: 100 µm)

Fig. 6

Results of microarray and Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) on genes that were highly expressed in the microarray analysis in lung tissue after inhalation exposure of polypropylene. Clustered heatmaps showing the expression patterns based on mRNA whose expression ratio is more than twice that of the control among genes whose expression level is above the median value in inhalation exposure (A) and intratracheal instillation (B), respectively. qRT-PCR in lung tissue in inhalation exposure (C). Red and blue represent high and low expression in each exposure group, and color density indicating levels of fold change was displayed in the heatmaps. Persistent increases of genes at 1–6 months after exposure in the high-concentration group in qRT-PCR analysis. Data are presented as mean ± SD for n = 5/group (*p < 0.05, **p < 0.01)