Sample polymer
Using a previous method [36], we synthesized a sample of PAA by polymerization of the acrylic acid monomer adding different cross-linker concentrations (0.1 mol%, 1.0 mol%, and 5.0 mol%) and labeled them CL0.1%, CL1.0%, and CL5.0%, respectively. The details of the preparations are described in the Supplementary Information. PAA, a white, easily scattered powder, was mixed with distilled water and slowly stirred for 40 minutes (Mag-Mixer MF820 or MD300, Yamato Scientific Co., Ltd., Tokyo, Japan).
Animals
Male Fischer 344 rats (8 weeks old) (The Jackson Laboratory Japan, Inc., Kanagawa, Japan) were acclimated for 4 weeks at the Laboratory Animal Research Center of the University of Occupational and Environmental Health, Japan with commercial food and water available ad libitum. All procedures and animal handling were performed under the guidelines described in the "Japanese Guide for the Care and Use of Laboratory Animals" and approved by the Animal Experiment Committee of the University of Occupational and Environmental Health, Japan (Animal Research Ethics Approval Proposal No.; AE17-009). All methods were performed in accordance with the relevant guidelines (https://arriveguidelines.org).
Intratracheal instillation
Doses of 0.2 mg (0.8 mg/kg BW) and 1.0 mg (4.0 mg/kg BW) of PAA suspended in 0.4 mL of distilled water at different cross-linker concentrations were administered by single intratracheal instillation into the lungs of rats (12 weeks old). Rats were instilled intratracheally under sevoflurane (Viatris Inc., Canonsburg, PA, USA) inhalation anesthesia. Briefly, the laryngeal extension was performed using a laryngoscope blade (MAC1, Rudolf Riester GmbH, Jungingen, Germany), and an animal feeding needle (KN-348, Natsume Seisakusho Co., Ltd., Tokyo, Japan) was inserted directly into the trachea and the suspension was instilled manually. Next, 3 mL of air was inserted into the trachea twice with a syringe from the animal feeding needle. The rats were then spontaneously awakened and observed periodically. Single intratracheal instillations of 0.2 mg and 1.0 mg of PAA at different cross-linker concentrations were administered at different times, for a total of three single intratracheal instillations. A control group was established for each intratracheal instillation, with distilled water administered to the control group; doses of 0.2 mg and 1.0 mg per rat were used for the PAA intratracheal instillation. These maximum doses were set to avoid overloading the lungs in anticipation of human exposure [11].
Animals following intratracheal instillation
There were five rats in each exposure and control group at each time point. Animals were dissected at 3 days, 1 week, 1 month, 3 months, and 6 months after intratracheal instillation under isoflurane (Viatris Inc., Canonsburg, PA, USA) inhalation anesthesia. Body weight was measured, then blood was removed from the heart at autopsy. Lungs were extracted from the body, lung weights were measured, and the lungs were perfused with saline solution. With the left main bronchus clamped, the right lung was repeatedly inflated with saline at a pressure of 20 cm H2O after two fluid collections. Seven to 14 mL of the fluid (BALF) was collected in collection tubes by free fall, and then the right and left lungs were separated. The homogenized third lobe of the right lung after BALF collection was used for measurement of HO-1. The left lung was inflated and fixed with 10% formaldehyde under pressure of 25 cm H2O for histopathological evaluation.
Cytospin analysis of inflammatory cells and measurement of inflammation-related markers in BALF
The BALF was centrifuged at 400 g for 15 min at 4°C, and the supernatant was transferred to a new tube for determination of total protein, lactate dehydrogenase (LDH) and cytokines. Pellets were washed in 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) in suspension and centrifuged at 400 g at 4°C for 15 minutes. After the removal of the supernatant, the pellet was resuspended in 1 mL of PMN buffer. Cell counts in BALF were measured with ADAM-MC (AR BROWN CO., LTD., Tokyo, Japan), cells were spread on glass slides with cytospin, fixed, stained with Diff-Quik (Sysmex CO., Kobe, Hyogo, Japan), and the number of neutrophils and alveolar macrophages was measured by microscopic observation. LDH activities released into the BALF supernatant were measured with the Cytotoxicity Detection KitPLUS (LDH) (Roche Diagnostics GmbH, Mannheim, North Rhine-Westphalia, Germany) according to the manufacturer's instructions. LDH activity was estimated using a standard curve obtained from known concentrations of recombinant LDH derived from rabbit muscle (Oriental Yeast Co., ltd., Tokyo, Japan). Protein concentrations in BALF supernatants were determined using the Pierce™ 660 nm Protein Assay (Thermo Scientific Inc., Rockford, IL, USA). Ford, Illinois, USA).
Measurement of chemokine in BALF and HO-1 in lung tissue
Concentrations of CINC-1 and CINC-2 in BALF were measured with the ELISA kits #RCN100 and #RCN200 (R&D Systems, Minneapolis, MN, USA), respectively. All measurements were performed according to the manufacturer's instructions. The third lobe of the right lung was cultured in T-PER tissue containing protein inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA) and cOmplete Mini (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany), and homogenized in Protein Extraction Reagent (Thermo Scientific Inc., Rockford, IL, USA) and centrifuged (20,400 g at 4°C for 10 min). Protein concentrations in the supernatant were determined with Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific Inc., Rockford, IL, USA), using bovine serum albumin as a standard. HO-1 measurements with the Elisa kit ADI-EKS-810A (Enzo Life Sciences, Farmingdale, NY, USA) were corrected by the protein concentration in the supernatant to calculate the final HO-1 concentration in the lung tissue.
Histopathology
Formaldehyde-fixed lung tissue was embedded in paraffin, sectioned at a thickness of 4 µm, and then stained with hematoxylin and eosin (HE) and Masson trichrome (MT) staining. The lung inflammation and fibrosis were examined using the inflammatory cell infiltration score [37] and the Ashcroft score [38], respectively, according to previous reports [11, 37]. Briefly, the inflammatory cell infiltration score was calculated by scoring the degree of inflammatory cell infiltration in the lung tissue as none (0), minimal (0.5), mild (1), moderate (2), or severe (3). The mean and standard deviation of the scores were calculated for each group. Pulmonary fibrosis was assessed by scoring histopathological findings in the lungs on a scale of 0 to 8 using the modified Ashcroft score, with the mean and standard deviation calculated for each group.
Statistical analysis
Statistical analysis was performed using IBM® SPSS® software (IBM Corporation, Chicago, IL, USA). p-values < 0.05 were determined to be statistically significant. The Dunnett and Tukey's honestly significant difference (HSD) tests were appropriately used to detect individual differences between individuals exposed to different cross-linker concentrations of PAA samples and the control group. Construct validity was measured using Spearman’s rank correlation coefficients (ρ) between the Ashcroft scores as lung fibrosis indicators and lung injury markers.