Sample preparation
A sample of Polypropylene was purchased from SEISHIN ENTERPRISE Co.,Ltd. (PPW-5J, Tokyo, Japan). Polypropylene is a white, easily scattered powder, with a specific gravity of approximately 0.9. No additives are used in the process of synthesizing polypropylene. Images of scanning electron microscopy (SEM) by HITACHI S-4500 (Hi-tachi, Ltd., Tokyo, Japan) of polypropylene were obtained by sampling aerosol in an inhalation chamber and by drying the dispersion, respectively. Polypropylene suspended in 0.4 ml distilled water including 0.1% Tween 80 was used for the intratracheal instillation. The size distribution of the polypropylene particles in the dispersion was measured by Dynamic Light Scattering (DLS) (ZEN1600, Malvern Panalytical, Ltd., Malvern, UK) and a dried sample of the dispersion was observed by SEM.
Animals
Male Fischer 344 rats (Inhalation exposure: 6 weeks old, Intratracheal instillation: 10 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were kept in the Laboratory Animal Research Center of the University of Occupational and Environmental Health for 2 weeks with access to free-feeding of commercial diet and water. All procedures and animal handling were done according to the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan (animal studies ethics clearance proposal number; AE20-015 and AE22-023).
Inhalation exposure
The inhalation system consisted of a dust generator, exposure chambers (volume; 0.57 m3), and gas-liquid-solid separators, as described by Tanaka et al. [48]. To obtain a constant concentration, the polypropylene was mixed with fluidizing particles (small glass beads with a diameter of 250 𝜇m) and polypropylenes close to each other were prevented from agglutinating. The mixture was fed into a hopper and transported smoothly via a continuous screw feed into a fluidized bed. Dry air flow was blown through the fluidized bed to transport the polypropylene to the exposure chamber while leaving behind the fluidizing particles. Only the glass beads remained in the fluidized bed because they were heavier than the polypropylene. The polypropylene aerosol weight concentration in the exposure chamber was measured daily by the isokinetic suction of air though a glass fiber filter beside the chamber. The glass fiber filter was weighed before and after the measurements in accordance with the standard method in JIS-Z8808, and the measured daily exposure weight concentration was determined as mg/m3. The particle size distribution for number concentration of the aerosols inside the exposure chamber was also measured in the diluted condition repeatedly using a particle size spectrometer (model 1000XP WPS, MSP Corp., Shoreview, MN). The weight concentration distribution of polypropylene in the inhalation chamber was measured by an Andersen Sampler (AN-200, Dylec Inc., Ibaraki, Japan). The sampling using a membrane filter was also performed to off-line analysis using SEM. After exposure for 4 weeks, the rats were dissected at 3 days, 1 month and 3 months, under anesthesia by isoflurane (VIATRIS Japan, Tokyo, Japan) inhalation.
Intratracheal instillation
Doses of 0.2 mg (0.8 mg/kg BW) and 1.0 mg (4.0 mg/kg BW) of polypropylene suspended in 0.4 ml distilled water including 0.1% Tween 80 were administered to the lungs of rats (12 weeks old) in single intratracheal instillations. The rats were intratracheally instilled under anesthesia by sevoflurane (VIATRIS Japan, Tokyo, Japan) inhalation. Briefly, a laryngeal extension was performed using a laryngoscope blade (MAC1, Rudolf Riester GmbH, Jungingen, Germany), and an animal feeding needle (KN-348, Natsume Seisa-kusho Co., Ltd., Tokyo, Japan) was inserted directly into the trachea, and the suspension was manually injected. Then, 3 ml of air twice with a syringe from the animal feeding needle was inserted into the trachea. The rats were then allowed to awaken spontaneously and were observed periodically. The control group received distilled water including 0.1% Tween 80 water suspension. The rats were dissected at 3 days, 1 week, 1 month, 3 months and 6 months after the instillation under anesthesia by isoflurane (VIATRIS Japan, Tokyo, Japan) inhalation.
Animals following inhalation and intratracheal instillation
There were 5 rats in each exposure and control group at each time point. Body and lung weights were measured under anesthesia by isoflurane (Pfizer Japan, Tokyo, Japan) inhalation, and then, at autopsy, blood was removed from the abdominal aorta and the lungs were perfused with normal saline. The right lungs were repeatedly inflated with normal saline under a pressure of 20 cm H2O, following fluid recovery two times, while the left main bronchus was clamped. Between 7 and 14 mL of the recovered fluid (BALF) was collected in collection tubes by free fall, and then the right and left lungs were divided. The homogenized third lobes of the right lungs were used for cDNA microarray after recovery of BALF. The left lungs were inflated and fixed by 10% formaldehyde under a pressure of 25 cm H2O for use in histopathological evaluation.
Cytospin analysis of inflammatory cells and measurement of inflammation related markers in BALF
BALF was centrifuged at 400 g at 4°C for 15 minutes, and the supernatant was transferred to a new tube for measurement of total protein, lactate dehydrogenase (LDH) and cytokines. The pellets were washed by suspension with polymorphonuclear leukocyte (PMN) Buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM Na2HPO4, 1.5 mM KH2PO4 and 5.6 mM C6H12O6) and centrifuged at 400 g at 4°C for 15 minutes. After removal of the supernatant, the pellets were resuspended with 1mL of PMN (poly-morphonuclear leukocyte) Buffer. The number of cells in the BALF was counted by ADAM-MC (AR BROWN CO., LTD, Tokyo, Japan), and the cells were splashed on a slide glass using cytospin, and fixed and stained with Diff-Quik (Sysmex CO., Kobe, Hyogo, Japan), after which the number of neutrophils and alveolar macrophages were counted by microscopic observation. The released LDH activity in the BALF supernatant was measured by a Cytotoxicity Detection KitPLUS (LDH) (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany) according to the manufacturer’s instructions. LDH activity was estimated using a standard curve obtained from known concentrations of recombinant LDH from rabbit muscle (Oriental Yeast Co., ltd., Tokyo, Japan). Concentrations of CINC-1 and CINC-2 in the BALF were measured by ELISA kits, #RCN100, #RCN200 (R&D Systems, Minneapolis, MN, USA), ELISA kits, respectively. The concentrations of rat MPO proteins in the BALF samples in all of the examinations were measured by ELISA kits, HK105 (Hycult Biotech, The Netherlands). All measurements were performed according to the manufacturer’s instructions.
Total RNA Extraction
The third lobes of the right lungs (n = 5 per group per time point) were homogenized while using a QIAzol lysis reagent with a TissueRupotor (Qiagen, Hilden, Ger-many). Total RNA from the homogenates was extracted using a miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).
Affymetrix Whole Transcript Expression Arrays Methods
The Affymetrix Whole Transcript Expression array process was executed according to the manufacturer's protocol (GeneChip Whole TranscriptPLUS reagent Kit). cDNA was synthesized using the GeneChip WT (Whole Transcript) Amplificationkit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit. Approximately 5.5 µg of labeled DNA target was hybridized to the Affymetrix GeneChip Rat Clariom-Schip at 45 ℃ for 16 hours. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix® GeneChip™ Command Console software. We created a heatmap using Transcriptome Analysis Console (TAC) version 4.0 (Thermo Fisher Scientific Inc., Waltham, MA, USA). We analyzed the related genes that were found to be elevated in the polypropylene using the above microarray data for Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis via the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 2021 (https://david.ncifcrf.gov/home.jsp).
Validation of gene expression data using quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed as described previously 15. Briefly, the total RNA extracted from the lungs at each observation point in each group was transcribed into cDNA (High-Capacity cDNA Reverse Transcription Kit, Thermo Fisher Scientific Inc., MA, USA). qRT-PCR assays were performed while using TaqMan (TaqMan Gene Ex-pression Assays, Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocol. Gene expression data were analyzed by the comparative cycle time (ΔΔCT) method. The Assays-on-Demand TaqMan probes and primer pair were CD177 (Assay ID Rn01454812_m1), Interleukin (IL)-17a (Assay ID Rn01757168_m1), Lipokine 2 (LCN2) (Assay ID Rn00590612_m1), IL-4 (Assay ID Rn01456866_m1), IL-13 (Assay ID Rn00587615_m1), CXCL1 (Assay ID Rn00578225_m1), IL-5 (Assay ID Rn01459975_m1), CXCL6 (Assay ID Rn00573587_g1) and IL-1β (Assay ID 00580432_m1). All experiments were performed in aStepOnePlusTM Real-Time PCR Systems (Thermo Fisher Scientific Inc., MA, USA). All expression data were normalized to endogenous control β-actin expression (Assay ID Rn00667869_m1) and calculated relative to their gene expression in each negative control.
Histopathology
Formaldehyde-fixed left lung tissue was embedded in paraffin, sectioned at a thick-ness of 4 µm, and then stained with hematoxylin and eosin (HE) staining.
Statistical analysis
Statistical analysis was carried out using IBM® SPSS® software (IBM Corporation, Chicago, IL, USA). P values < 0.05 were considered statistically significant. Dunnett’s test was used appropriately to detect individual differences between those rats exposed to the polypropylene samples and the controls.