Inhalation Exposure to Cross-linked Polyacrylic Acid Induces Pulmonary Disorders

Inhalation Exposure to Cross-linked Polyacrylic Acid Induces Pulmonary Disorders | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhalation Exposure to Cross-linked Polyacrylic Acid Induces Pulmonary Disorders Yasuyuki Higashi, Chinatsu Nishida, Hiroto Izumi, Kazuma Sato, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4787568/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background : Organic polymers are used widely in daily necessities, foods, and medicines, but recent reports show that cross-linked polyacrylic acid (CL-PAA), an organic polymer, induces severe lung disease. Whereas most previous toxicity studies of CL-PAA were intratracheal instillation studies, we conducted inhalation exposure studies to corroborate those findings. Methods : We conducted 5-day (subacute) and 13-week (subchronic) inhalation exposure studies using CL-PAA. In the subacute inhalation study, male F344 rats inhaled 0.2 mg/m 3 , 2.0 mg/m 3 , or 20 mg/m 3 of CL-PAA for 6 hours/day for 5 days. Rats were sacrificed 3 days and 1 month after the exposure. In the subchronic inhalation study, male F344 rats inhaled 0.2 mg/m 3 or 2.0 mg/ m 3 of CL-PAA for 6 hours/day for 5 days/week for 13 weeks. Rats were sacrificed from 3 days to 6 months after exposure. We also investigated the mechanism of pulmonary disorders by conducting an additional subacute inhalation exposure (CL-PAA 20 mg/m 3 ) study, in which the antioxidant N-acetylcysteine (NAC) (200 mg/kg) was injected intraperitoneally. The rats in that study were sacrificed on the day after the exposure. Results : Subacute inhalation exposure resulted in concentration-dependent increases in neutrophil influx, total protein, lactate dehydrogenase (LDH), cytokine-induced neutrophil chemoattractant (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in lung tissue. Histopathological findings in the lung tissue also showed neutrophil infiltration in a concentration-dependent manner. Subchronic inhalation exposure resulted in persistent increases in total protein in BALF and HO-1 in lung tissue. Histopathological findings in the lung tissue showed persistent neutrophil infiltration and fibrosis. NAC administration decreased the number of neutrophils, total protein, LDH, and CINC in the BALF, and HO-1 in the lung tissue. Histopathological findings of the lung tissue also showed that NAC administration improved neutrophil infiltration. Conclusion : Inhalation of CL-PAA for 5 days induced concentration-dependent inflammation in the rats' lungs. Inhalation of CL-PAA for 13 weeks induced persistent inflammation and fibrosis in the rats' lungs. We considered that the no observed adverse effect level (NOAEL) for chronic pulmonary disorders was 0.2 mg/m 3 . Furthermore, oxidative stress was associated with CL-PAA-induced pulmonary inflammation, and NAC administration attenuated the pulmonary inflammation. cross-linked polyacrylic acid inhalation exposure oxidative stress N-acetylcysteine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Background Polyacrylic acid (PAA) is a general term for organic polymer compounds whose monomer is the organic material acrylic acid (C 3 H 4 O 2 ). Their viscosity and water absorbency increase when they are cross-linked (cross-linked polyacrylic acid; CL-PAA). They are used in a variety of applications due to differences in their properties, including as additives in daily necessities, food, and medicines; therefore, PAA is currently one of the most widely used thickening agents [ 1 ]. Organic materials are conventionally thought to be unlikely to cause pneumoconiosis by inhalation and to rarely cause the severe pulmonary fibrosis that is caused by inorganic materials such as crystalline silica and chrysotile, but in recent years there have been reports of organic materials inducing progressive pulmonary fibrosis [ 2 ]. There were reports in Japan in 2017 of a high incidence of pulmonary disorders among workers at a company that manufactures PAAs, which are organic materials [ 3 ]. In response to those reports, animal exposure studies on PAA have been conducted in Japan, and it has been reported that PAA has strong inflammatory and fibrotic potential depending on its physicochemical properties [ 4 – 9 ]. The mechanism of pulmonary injury caused by chemicals is considered to be that macrophages that phagocytose foreign materials release cytokines, which recruit inflammatory cells such as neutrophils, induce neutrophilic inflammation [ 10 , 11 ]. The neutrophils then induce oxidative stress by generating reactive oxygen species (ROS), such as free radicals, leading to pulmonary inflammation and fibrosis. Oxidative stress induces further inflammation and alveolar epithelial damage [ 12 ] and induces pulmonary fibrosis due to excessive epithelial-mesenchymal transition (EMT)-induced fibroblast activation and abnormal repair during the process of alveolar epithelial repair [ 13 ]. Oxidative stress is therefore ​​​​​​​thought to play an important role in chemical-induced pulmonary disorders. Most animal experimental reports of pulmonary disorders caused by PAAs so far were based on intratracheal instillation. Inhalation exposure studies provide important information for evaluating the biological effects on humans because the dust​​​​​​​ deposition patterns and amounts are physiological, and the findings obtained from inhalation exposure studies are similar to the pathological conditions caused by exposure in humans [ 14 ]. For that reason, we conducted subacute and subchronic inhalation exposure studies of CL-PAA in rats to investigate the effects on the lungs of CL-PAA, a cross-linked type commonly used as a thickening agent, and analyzed the factors related to inflammation, fibrosis, and oxidative stress. 2. Materials and Methods 2.1 Sample polymer We used CL-PAA with an average Mv ~ 450,000 (Sigma-Aldrich Co. LLC., St. Louis, MO, USA). The fundamental characteristics of the CL-PAA are summarized in Table 1 . The weight average molecular weight (Mw), number average molecular weight (Mn), polydispersity index (PDI), and radius of gyration (Rg) of CL-PAA were measured by gel permeation chromatography (GPC) (Prominence 501 system, SHOKO SCIENCE, Kanagawa, Japan), and their values were 768,000, 414,000, and 1.85, respectively. The mass median aerodynamic diameter (MMAD) in aerosols determined by an Andersen sampler (AN-200; TOKYO DYLEC CORP., Shinjyuku, Japan) was 1.9 µm (σg; geometric standard deviation = 2.0), 2.2 µm (σg = 1.9), and 2.5 µm (σg = 2.0) for 0.2 mg/m 3 , 2.0 mg/m 3 , and 20 mg/m 3 , respectively. Figure 1 shows scanning electron microscopy (SEM) images of the polymer used in these studies, obtained by a HITACHI S-4500 (Hitachi, Ltd., Tokyo, Japan) instrument. The bulk PAA powder and the suspended PAA in distilled water are shown in Figs. 1 A and 1 B, respectively. Table 1. Physiochemical characterization of the polymer used in these studies. 2.2 Animals Male Fischer 344 rats (8 weeks old) that were exposed to CL-PAA were purchased from Charles River Laboratories International, Inc., Kanagawa, Japan. The animals were housed at the Laboratory Animal Research Center of the University of Occupational and Environmental Health for four weeks with free access to commercial feed and water. All of the procedures and animal handling procedures were performed in accordance with the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan (animal studies ethics clearance proposal number; AE18-003). 2.3 Inhalation exposure and intraperitoneal injection The inhalation exposure studies involved the use of a dust feeder composed of a table feeder, an ejector (DF-3; SIBATA Scientific Technology Ltd., Saitama, Japan), and a whole-body aerosol exposure chamber (AD-8506046; SIBATA Scientific Technology Ltd., Saitama, Japan), as shown in Fig. 2 . The CL-PAA particles were delivered by the ejector to the rats in a whole-body exposure chamber with air. The mass concentration of aerosols in the exposure chamber was sometimes monitored by sampling the weight of the particles and the volume of the air. We performed three inhalation exposure studies with this device: subacute inhalation, subchronic inhalation, and subacute inhalation and intraperitoneal injection (Fig. 3 ). In the subacute inhalation study, the rats were exposed to 0.2 mg/m 3 , 2.0 mg/ m 3 , or 20 mg/m 3 CL-PAA for 6 hours/day for 5 days/week for 1 week. This study was conducted with reference to other short-term inhalation studies (STIS) [ 15 ]. The 0.2 mg/m 3 and 2.0 mg/m 3 exposures were administered on the same schedule, while the 20 mg/m 3 exposure was administered on a different schedule, and a control group was established for each schedule. The control group received clean air. The subchronic inhalation treatment group was exposed to 0.2 mg/m 3 and 2.0 mg/m 3 CL-PAA for 6 hours/day for 5 days/week for 13 weeks. This was conducted with reference to the Organisation for Economic Co-operation and Development (OECD) TG413, the OECD guidelines for testing chemicals [ 16 ]. The control group received clean air. Finally, four groups were subjected to subacute inhalation and intraperitoneal injection: group one, "Control", received normal saline intraperitoneally 1 hour before inhaling clean air; group two, "NAC", received N-acetylcysteine (NAC) intraperitoneally 1 hour before inhaling clean air; group three, "CL-PAA", received normal saline intraperitoneally 1 hour before inhaling 20 mg/m 3 CL-PAA; and group four, "CL-PAA + NAC", received NAC intraperitoneally 1 hour before inhaling 20 mg/m 3 CL-PAA. The volume of saline administered intraperitoneally was 1 ml, and NAC was administered at 200 mg/kg (48.78 mg NAC, calculated based on the average body weight of the rats in groups two and four, which was 243.9 g) dissolved in 1 ml of normal saline. Inhalation exposure was conducted for 6 hours/day, and intraperitoneal injection and inhalation exposure were conducted for 5 consecutive days. The actual analyzed mass concentrations were 0.26 ± 0.03 mg/m 3 , 2.1 ± 0.1 mg/m 3 , and 20.5 ± 0.6 mg/m 3 in the subacute inhalation study (Fig. 4 A), 0.26 ± 0.04 mg/m 3 and 2.4 ± 0.4 mg/m 3 in the subchronic inhalation study (Fig. 4 B), and 20.5 ± 0.6 mg/m 3 in the subacute inhalation and intraperitoneal injection study (Fig. 4 C), respectively. 2.4 Animals following inhalation exposure There were 5 rats in the exposure groups and the control group at each time point. Rats were sacrificed at each time point under anesthesia by inhalation of isoflurane (Pfizer Japan, Tokyo, Japan). In the subacute inhalation study, rats were sacrificed at 3 days and 1 month after the end of inhalation. In the subchronic inhalation study, rats were sacrificed at 3 days, 1 month, 3 months, and 6 months after the end of inhalation. In the subacute inhalation and intraperitoneal injection studies, rats were sacrificed on the day after the end of inhalation and intraperitoneal injection. All rats were sacrificed under anesthesia by inhalation of isoflurane (Pfizer Japan, Tokyo, Japan). Body and lung weights were measured at autopsy. Blood was removed from the abdominal aorta, and the lungs were perfused with normal saline. The right lung was repeatedly infused with normal saline under a pressure of 20 cmH 2 O, followed by fluid recovery two times, while the left main bronchus was clamped. Between 5 and 15 mL of the recovered fluid (bronchoalveolar lavage fluid; BALF) was collected by free fall into tubes, and then the right and left lungs were divided. The third lobe of the right lung after recovery from the BALF was homogenized and used for the evaluation of heme oxygenase-1 (HO-1). The left lungs were inflated and fixed with 10% formaldehyde under a pressure of 25 cmH 2 O for histopathological evaluation. 2.5 Cytospin analysis of inflammatory cells and measurement of total protein and lactate dehydrogenase (LDH) levels in BALF The BALF was centrifuged at 400 g at 4°C for 15 minutes, and the supernatant was transferred to a new tube to measure total protein and LDH levels. The pellets were washed with a suspension of polymorphonuclear leukocyte (PMN) buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 and 5.6 mM C 6 H 12 O 6 ) and centrifuged at 400 g at 4°C for 15 minutes. After removal of the supernatant by decanting, the pellets were resuspended in 1 mL of PMN buffer. The number of cells in the BALF was counted by ADAM-MC™ (AR BROWN Co., Ltd., Tokyo, Japan), an automated cell counting device, and the cells were splashed and fixed onto a glass slide using cytospin and stained with Diff-Quik® (Sysmex Co., Kobe, Hyogo, Japan), after which the number of neutrophils and alveolar macrophages were counted by microscopic observation. Total protein in the BALF was measured by a Pierce™ 660 nm Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL, USA) and calculated using a standard curve obtained from the Albumin Standard (Thermo Fisher Scientific Inc., Rockford, IL, USA). LDH levels in the BALF supernatant were measured with a Cytotoxicity Detection Kit PLUS (LDH) (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany) according to the manufacturer’s instructions. LDH levels were calculated using a standard curve obtained from known concentrations of recombinant LDH from rabbit muscle (Oriental Yeast Co., Ltd., Tokyo, Japan). 2.6 Measurement of chemokines in BALF and HO-1 in lung tissue The concentrations in the BALF of cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2, which are neutrophil chemotactic factors in rats, were measured by the enzyme-linked immunosorbent assay (ELISA) kits, #RCN100 and #RCN200 (R&D Systems, Minneapolis, MN, USA), respectively. The third lobe of the right lung was homogenized with a tissue protein extraction reagent (T-PER) (Thermo Fisher Scientific Inc., Rockford, IL, USA) containing P8340 (Sigma-Aldrich, St. Louis, MO, USA), protein inhibitor cocktails, cOmplete™ Mini (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany), and protease inhibitor cocktails, and then centrifuged (20,400 g at 4°C for 10 minutes). The protein concentration in the homogenized lung tissue extracts was measured by a Pierce™ 660 nm Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL, USA) and calculated using a standard curve obtained from the Albumin Standard (Thermo Fisher Scientific Inc., Rockford, IL, USA). The total protein concentration in the lung tissue was adjusted to a final concentration of 500 mg/mL to measure HO-1, an index of oxidative stress, which was measured by the ELISA kit ADI-EKS-810A (Enzo Life Sciences, Farmingdale, NY, USA). 2.7 Total RNA Extraction The third lobe of the right lung (n = 5 per group per time point) was homogenized with QIAzol lysis reagent. Total RNA from the homogenates was extracted using a miRNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The purity and quantity of the RNA were evaluated with a NanoDrop instrument. The quality of the RNA was evaluated with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). 2.8 Affymetrix Whole Transcript Expression Array Methods cDNA was synthesized using the GeneChip Whole Transcript (WT) Amplification Kit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase (TdT) using a GeneChip WT terminal labeling kit. Approximately 5.5 µg of labeled DNA target was hybridized to the Affymetrix GeneChip Array at 45˚ for 16 hours. The hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). The probe cell intensity data computation and CEL file generation were performed using Affymetrix® GeneChip™ Command Console® software. We performed two analyses. The first was an analysis of genes whose expression was increased by subchronic inhalation exposure to CL-PAA (fc 2-fold or more in the 2.0 mg/m 3 group vs 0 mg/m 3 group), and the second was an analysis of genes whose expression was increased by subacute inhalation exposure to CL-PAA (fc 2-fold or more in the CL-PAA group vs control group) and decreased by NAC administration (fc -1.5 or less in the CL-PAA + NAC group vs CL-PAA group). We analyzed the genes related to CL-PAA exposure and NAC administration 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 ). 2.9 Histopathology Formaldehyde-fixed lung tissue was embedded in paraffin and sectioned at a thickness of 4 µm for hematoxylin and eosin (HE) and Masson trichrome (MT) staining. The slides were assessed for histological changes by a board-certified pathologist. The Ashcroft scale was used to assess the degree of pulmonary fibrosis [ 17 ]. We scored the MT-stained specimens at ×10 magnification with the Ashcroft scale, as described in another report [ 18 ]. The Ashcroft scale, briefly explained, is a lung section graded from 0 (normal lung) to 8 (most severe fibrosis), with the grades summed and divided by the number of fields. 2.10 Statistical analysis Statistical analysis was performed using IBM SPSS Statistics ver. 27 (IBM Corp., Armonk, NY, USA) and carried out using Student’s t-test and the Holm-Bonferroni correction in the subacute inhalation study, with Dunnett’s test in the subchronic inhalation study, and with Tukey’s test in the subacute inhalation and intraperitoneal injection study, with differences of p < 0.05 considered to be statistically significant. The data are presented as the means ± SDs (* p < 0.05, ** p < 0.01). 3. Results 3.1 Body and lung weight Figure 5 shows the body weight and relative lung weight of the rats. 1) Subacute inhalation study There was a statistically significant difference in body weight only in the 0.2 mg/m 3 group at 1 month postexposure (Fig. 5 A). There was a statistically significant increase in the relative lung weight (lung weight/body weight) in the 2.0 mg/m 3 group at 3 days postexposure and in the 20 mg/m 3 group at 3 days and 1 month postexposure (Fig. 5 B). 2) Subchronic inhalation study There were no significant differences in body weight among the groups (Fig. 5 C). There was a statistically significant increase in the relative lung weight only in the 2.0 mg/m 3 group from 3 days to 3 months postexposure (Fig. 5 D). 3) Subacute inhalation and intraperitoneal injection study There were no significant differences in body weight among the groups (Fig. 5 E). There was a statistically significant increase in the relative lung weight in the CL-PAA and CL-PAA + NAC group compared to the control and the NAC group (Fig. 5 F). 3.2 Cell analysis and total protein and lactate dehydrogenase (LDH) levels in BALF 1) Subacute inhalation study Figure 6 shows the inflammatory cell counts and total protein and LDH levels in the BALF in the subacute inhalation study. There was a statistically significant increase in the total cell count in the 2.0 mg/m 3 group at 3 days postexposure and in the 20 mg/m 3 group at 3 days and 1 month postexposure (Fig. 6 A). There was a statistically significant concentration-dependent increase in the neutrophil count and percentage of neutrophils at 3 days postexposure. There were no significant differences in the neutrophil count or percentage of neutrophils at 1 month postexposure (Fig. 6 B and 6 C). There was a statistically significant increase in the macrophage count in the 2.0 mg/m 3 group at 3 days postexposure and in the 20 mg/m 3 group at 3 days and 1 month postexposure (Fig. 6 D). There was a statistically significant increase in the total protein content in the 2.0 mg/m 3 group and in the 20 mg/m 3 group at 3 days postexposure (Fig. 6 E). There was a statistically significant increase in LDH levels in the 2.0 mg/m 3 group at 3 days postexposure and in the 20 mg/m 3 group at 3 days and 1 month postexposure (Fig. 6 F). 2) Subchronic inhalation study Figure 7 shows the inflammatory cell counts and total protein and LDH levels in the BALF in the subchronic inhalation study. There was a statistically significant increase in the total cell count, neutrophil count, and percentage of neutrophils in the 2.0 mg/m 3 group at 3 days postexposure (Fig. 7 A-C). There was a statistically significant increase in the macrophage count and total protein content in the 2.0 mg/m 3 group at 3 days and 1 month postexposure (Fig. 7 D, 7 E). There was a statistically significant increase in LDH levels in the 2.0 mg/m 3 group at 3 days postexposure (Fig. 7 F). There were no significant differences in the inflammatory cell count or total protein or LDH levels in the BALF of the 0.2 mg/m 3 group during the observation period (Fig. 7 A-F). 3) Subacute inhalation and intraperitoneal injection study Figure 8 shows the inflammatory cell count and total protein and LDH levels in the BALF in the subacute inhalation and intraperitoneal injection study. There was a statistically significant increase in the total cell count, neutrophil count, percentage of neutrophils, macrophage count, total protein, and LDH levels in the exposed groups. There was a statistically significant decrease in the total cell count, neutrophil count, macrophage count, total protein content, and LDH level, which were increased by CL-PAA exposure following the intraperitoneal injection of NAC (Fig. 8 A-F). 3.3 Concentration of CINCs in BALF and concentration of HO-1 in lung tissue 1) Subacute inhalation study Figure 9 shows the concentrations of CINC-1 and CINC-2 in the BALF and of HO-1 in the lung tissue in the subacute inhalation study. There was a statistically significant increase in the concentration of CINC-1 in the exposure groups at 3 days postexposure (Fig. 9 A). There was a statistically significant increase in the concentration of CINC-2 in the exposure groups at 3 days postexposure and in the 20 mg/m 3 group at 1 month postexposure (Fig. 9 B). There was a statistically significant concentration-dependent increase in the concentration of HO-1 in the exposure groups at 3 days postexposure, and there was a statistically significant increase in the 20 mg/m 3 group at 1 month postexposure (Fig. 9 C). 2) Subchronic inhalation study Figure 10 shows the concentrations of CINC-1 and CINC-2 in the BALF and of HO-1 in the lung tissue in the subchronic inhalation study. There was a statistically significant increase in the concentration of CINCs in the 2.0 mg/m 3 group at 3 days postexposure (Fig. 10 A, 10 B). There was a statistically significant increase in the concentration of HO-1 in the 2.0 mg/m 3 group at 3 days and 1 month postexposure (Fig. 10 C). There were no significant differences in the concentrations of CINCs in the BALF or HO-1 in the lung tissue in the 0.2 mg/m 3 group during the observation period (Fig. 10 A-C). 3) Subacute inhalation and intraperitoneal injection study Figure 11 shows the concentrations of CINC-1 and CINC-2 in the BALF and of HO-1 in the lung tissue in the subacute inhalation and intraperitoneal injection study. There was a statistically significant increase in the concentration of CINCs and HO-1 in the exposed groups. The concentrations of CINCs and HO-1, which were elevated by CL-PAA exposure, were statistically significantly decreased by intraperitoneal injection of NAC (Fig. 11 A-C). 3.4 Histopathological features of the lung 1) Subacute inhalation study Figure 12 shows the representative histopathological findings in the lungs in the subacute inhalation study. There was neutrophil and macrophage infiltration in the alveoli and interstitium in the exposure group at 3 days postexposure. There was inflammatory cell infiltration only in the 20 mg/m 3 group at 1 month postexposure. No fibroblasts or collagen fibers were observed during the observation period (Fig. 12 A, 12 B). 2) Subchronic inhalation study Figure 13 shows the representative histopathological findings in the lungs in the subchronic inhalation study. There was neutrophilic inflammatory cell infiltration in the alveoli and interstitium from 3 days to 3 months, and fibroblasts and collagen fibers were observed from 3 days to 1 month in the 2.0 mg/m 3 group. There were no obvious inflammatory cell infiltration, fibroblasts, or collagen fibers in the 0.2 mg/m 3 group during the observation period (Fig. 13 A-D). The Ashcroft scale, an indicator of fibrosis, showed a significant increase only in the 2.0 mg/m 3 group from 3 days to 1 month (Fig. 13 E). 3) Subacute inhalation and intraperitoneal injection study Figure 14 shows representative histopathological findings in the lungs in the subacute inhalation and intraperitoneal injection study. There was neutrophilic inflammatory cell infiltration in the alveoli and interstitium in the inhalation of CL-PAA 20 mg/m 3 group. These changes were reduced by the administration of NAC. No fibroblasts or collagen fibers were observed during the observation period (Fig. 14 ). 3.5 Microarray analysis and KEGG pathway analysis 1) Subchronic inhalation study We conducted a microarray analysis of mRNA in the lung tissue at 1 month after the subchronic inhalation exposure. Among the 23,188 genes that were found to be expressed, 488 genes were expressed more than twice as much as in the negative control group. Among those 488, there were 288 genes with a global normalization value of 25 or higher, and genes related to inflammation and fibrosis were observed at the top (Table 2 ). We evaluated the effects of inhaling CL-PAA on the lungs by conducting a pathway analysis using 288 genes whose expression was more than twice that of the negative control group after inhalation exposure. We identified pathways related to innate immunity, such as phagosome and neutrophil extracellular trap (NET) formation, and pathways related to inflammation and fibrosis, such as cytokine-cytokine receptor interaction and extracellular matrix (ECM)-receptor interaction (Table 3 ). Table 2 Comparison of the expression of the top 10 genes after subchronic inhalation exposure Gene symbol Gene description Fold charge Zbtb16 Zinc finger and BTB domain containing 16 22.69 Retnla Resistin like alpha 13.77 Rspo3 R-spondin 3 10.98 Mmp12 Matrix metallopeptidase 12 10.39 LOC100909700* CD177 antigen-like 8.28 Slc26a4 Slc26a4 8.11 Lgals3 Galectin-3 protein 7.86 Cd177 CD177 molecule 6.99 Ighg Immunoglobulin constant heavy G chain 6.81 Gfra3 GDNF family receptor alpha 3 6.31 Note: *LOC100909700 (tentatively renamed CD177 in the rat genome database) Table 3 Results of KEGG pathway analysis after subchronic inhalation exposure Terms of KEGG pathway Gene Counts % P Value Benjamini Tuberculosis 15 6.4 0.00000025 0.000058 Rheumatoid arthritis 9 3.8 0.000032 0.0038 Osteoclast differentiation 10 4.3 0.000062 0.0049 Phagosome 11 4.7 0.00034 0.020 Leishmaniasis 7 3.0 0.00055 0.026 Lysosome 9 3.8 0.00071 0.028 Complement and coagulation cascades 7 3.0 0.0016 0.054 Amoebiasis 7 3.0 0.0026 0.078 Apoptosis 8 3.4 0.0036 0.087 Protein digestion and absorption 7 3.0 0.0038 0.087 Pertussis 6 2.6 0.0041 0.087 Cytokine-cytokine receptor interaction 11 4.7 0.0059 0.12 Systemic lupus erythematosus 7 3.0 0.0076 0.14 Staphylococcus aureus infection 6 2.6 0.015 0.25 Thyroid cancer 4 1.7 0.017 0.26 Neutrophil extracellular trap formation 8 3.4 0.018 0.26 Viral protein interaction with cytokine and cytokine receptor 5 2.1 0.035 0.48 ECM-receptor interaction 5 2.1 0.039 0.51 Small cell lung cancer 5 2.1 0.044 0.54 Alcoholic liver disease 6 2.6 0.046 0.54 2) Subacute inhalation and intraperitoneal injection study We conducted a microarray analysis of mRNA in the lung tissue 1 day after the subacute inhalation exposure and intraperitoneal injection. Among the 23,188 genes that were found to be expressed, there were 827 genes whose expression increased in response to subacute inhalation exposure to CL-PAA (fc 2-fold or greater in the CL-PAA group than in the control group, with a global normalization value of 25 or greater). Among those 827 genes, 186 exhibited decreased expression following NAC administration (fc -1.5-fold or less in the CL-PAA + NAC group than in the CL-PAA group, with a global normalization value of 25 or greater). Genes related to inflammation and oxidative stress were observed at the top (Table 4 ). We evaluated the effects of CL-PAA inhalation on the lungs by conducting a pathway analysis using 186 genes extracted under the conditions described above. We identified pathways related to innate immunity, such as the phagosome and Toll-like receptor signaling pathways, and pathways related to inflammation, such as cytokine-cytokine receptor interaction (Table 5 ). Table 4 Comparison of the expression of the top 10 genes after subacute inhalation exposure and intraperitoneal injection of NAC Gene symbol Gene description Fold charge 1 Fold charge 2 Zbtb16 Zinc finger and BTB domain containing 16 6.89 -11.59 Cxcl13 chemokine (C-X-C motif) ligand 13 3.43 -4.05 Bpifb1 BPI fold containing family B member 1 7.71 -3.69 Hpx Hemopexin 10.69 -3.60 Ighg Immunoglobulin constant heavy G chain 2.58 -3.59 Olr1 Oxidized low density lipoprotein receptor 1 2.06 -3.54 Ly6al Lymphocyte antigen 6 complex, locus A-like 7.78 -3.08 Hk3 Hexokinase 3 3.17 -2.92 Irf7 Interferon regulatory factor 7 2.74 -2.88 Enpp3 Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 2.02 -2.71 Table 5 Results of KEGG pathway analysis after subacute inhalation exposure and intraperitoneal injection of NAC Terms of KEGG pathway Gene Counts % P Value Benjamini Complement and coagulation cascades 9 5.7 0.0000012 0.0002 Amoebiasis 7 4.5 0.00025 0.02 Pertussis 6 3.8 0.00055 0.03 Phagosome 8 5.1 0.0016 0.066 Staphylococcus aureus infection 6 3.8 0.0023 0.074 Tuberculosis 7 4.5 0.0058 0.16 Viral protein interaction with cytokine and cytokine receptor 5 3.2 0.0078 0.18 Toll-like receptor signaling pathway 5 3.2 0.012 0.23 Cytokine-cytokine receptor interaction 8 5.1 0.012 0.23 HIF-1 signaling pathway 5 3.2 0.021 0.34 Starch and sucrose metabolism 3 1.9 0.035 0.52 Measles 5 3.2 0.043 0.53 Neomycin, kanamycin and gentamicin biosynthesis 2 1.3 0.045 0.53 Biosynthesis of nucleotide sugars 3 1.9 0.046 0.53 4. Discussion We considered (1) pulmonary disorders in a subacute inhalation exposure study, (2) pulmonary disorders in a subchronic inhalation exposure study, and (3) the mechanism of pulmonary disorders caused by CL-PAA. 1) Pulmonary disorders in the subacute inhalation exposure study We conducted a 5-day subacute inhalation exposure study to examine the acute effects of CL-PAA. The exposure concentrations for the subacute inhalation exposure study were set at 0, 0.2, 2.0, and 20 mg/m 3 , because the 8-h time-weighted average (TWA) of personal exposure concentrations to acrylic acid-based polymers at the site where pulmonary disorder incidents occur is 0.4 to 7.6 mg/m 3 [ 3 ], and the threshold limit value (TLV)-TWA of respirable dust set by the American Conference of Governmental Industrial Hygienists (ACGIH) is 3 mg/m 3 , and we considered that it was necessary to cover these ranges. We observed concentration-dependent neutrophil-centered pulmonary inflammation in the rats’ lungs from 0.2 mg/m 3 when rats were exposed to CL-PAA via subacute inhalation. In a report on a subacute inhalation exposure study of materials with a known extent of lung damage ability, when rats were exposed to multiwalled carbon nanotubes (MWCNTs), a material with high pulmonary toxicity, via inhalation (0.1, 0.5, 2.5 mg/m 3 ) for five days, neutrophilic inflammation and pulmonary injury were observed at exposure concentrations of 0.5 mg/m 3 or higher [ 19 ]. Similarly, cerium dioxide (CeO 2 ), a material with high pulmonary toxicity, also caused neutrophilic inflammation in the lungs at exposure concentrations of 0.5 mg/m 3 or higher [ 20 ]. In contrast, nano-titanium dioxide (TiO 2 ), a material with low pulmonary toxicity, caused transient pulmonary injury at a concentration of 2 mg/m 3 for five days, but no neutrophilic inflammation was observed [ 21 ], and the inhalation of amorphous silica (10 or 100 mg/m 3 ) for three days caused only transient pulmonary inflammation [ 22 ]. Comparing to pulmonary inflammation with known toxic materials, the CL-PAA used in the present study caused pulmonary inflammation even at a low concentration of 0.2 mg/m 3 , suggesting that it has a high potential for inducing pulmonary inflammation in the acute phase. We investigated the expression of the neutrophilic chemokines [ 23 ] CINC-1 and CINC-2, which are associated with the mechanism of neutrophilic inflammation in the lungs following subacute inhalation exposure to CL-PAA, and we observed a concentration-dependent increase in CINC-1 and CINC-2 levels in the BALF at 0.2 mg/m 3 . In our previous study of four-week inhalation exposure to inorganic materials, CINC-1 and CINC-2 levels in the BALF increased, accompanied by pulmonary neutrophilic inflammation, following exposure to nickel oxide (NiO) and CeO 2 nanoparticles [ 24 ]. Although CL-PAA is an organic material and the mechanism of pulmonary inflammation caused by inorganic materials may not be comparable in general, the phenomenon that the CINC levels increased with neutrophil inflammation suggested that CINC is associated with neutrophil infiltration in CL-PAA, similar to neutrophil infiltration in inorganic materials. We also investigated oxidative stress following subacute inhalation exposure to CL-PAA by measuring the expression of HO-1, an oxidative stress response protein, and observed a concentration-dependent increase in HO-1 levels in lung tissue from the concentration of 0.2 mg/m 3 . There have also been reports of increased HO-1 expression in other dust-exposed pulmonary inflammation models. Nakashima et al. reported that in a pulmonary inflammation model induced by crystalline silica, ROS activated the extracellular signal-regulated kinase (ERK) signaling pathway and induced HO-1 expression [ 25 ]. In our previous study of four-week inhalation exposure to inorganic materials, we also observed that inhalation exposure to NiO nanoparticles induced pulmonary inflammation, which was accompanied by an increase in HO-1 levels in lung tissue [ 26 ]. These findings suggest that oxidative stress is associated with CL-PAA-induced pulmonary inflammation, similar to inorganic material-induced pulmonary inflammation. 2) Pulmonary disorders in the subchronic inhalation exposure study We conducted a 13-week subchronic inhalation exposure study to examine the chronic effects of CL-PAA at concentrations of 0, 0.2, and 2.0 mg/m 3 . Persistent pulmonary inflammation and fibrosis were observed in the rats' lungs at the 2.0 mg/m 3 concentration, whereas no obvious responses were observed at the 0.2 mg/m 3 concentration. The concentrations of CINC-1 and CINC-2 in BALF and of HO-1 in lung tissue were elevated at a concentration of 2.0 mg/m 3 , suggesting that CINC-1, CINC-2, and HO-1 are associated with the pathology of subchronic exposure as well as subacute exposure. In another subchronic inhalation exposure study, when rats were exposed to MWCNTs (0.1, 0.4, 1.5, and 6 mg/m 3 ) for 13 weeks, pulmonary inflammation and fibrosis were observed at exposure concentrations of 0.4 mg/m 3 or higher [ 27 ], and CeO 2 (0.1, 0.3, 1.0, and 3.0 mg/m 3 ) caused neutrophilic pulmonary inflammation and pulmonary injury at exposure concentrations of 1.0 mg/m 3 or higher [ 28 ]. In contrast, in a 13-week inhalation exposure study using toner, a material with low pulmonary toxicity, there was no significant increase in neutrophilic inflammation, cytokine, or HO-1 levels during the observation period even at an exposure concentration of 4 mg/m 3 [ 29 ], and in ultrafine-TiO 2 (0.5, 2, 10 mg/m 3 ), there was no pulmonary injury, although transient neutrophilic inflammation was observed at a concentration of 2 mg/m 3 [ 30 ]. Considering that inflammation and fibrosis persisted in the present study, we consider that the CL-PAA we used has chronic pulmonary inflammation and fibrosis potential. In the subchronic inhalation exposure, the mild pulmonary inflammation observed in the subacute inhalation exposure at a concentration of 0.2 mg/m 3 was not observed despite continuous exposure. This may have been because the body of the rats had become habituated to CL-PAA through continued inhalation. Similar phenomena have been observed with other chemicals. In an experiment in which rats were exposed to ozone, an increase in the number of neutrophils and total protein concentration in BALF was observed after two days of exposure, but these changes disappeared after four days of exposure [ 31 ]. In an experiment in which humans were exposed to ozone for five consecutive days, the forced vital capacity decreased after three days of exposure but recovered after subsequent exposure [ 32 ], and, in zinc oxide (ZnO) inhalation exposure studies in mice and humans, the initial neutrophil infiltration was reduced during the exposure period [ 33 , 34 ]. Although the characteristics of chemical materials that cause inflammation to disappear upon repeated exposure are unclear, habituation was thought to have occurred in the present study with CL-PAA. Regarding the exposure concentration in the present study, we calculated the amount of inhaled material accumulated in the lungs using the following formula: deposition rate × exposure concentration × exposure time (duration) × respiratory volume (which is the amount of CL-PAA) = deposition fraction × exposure concentration of particle × exposure minutes in one day × days of exposure × tidal volume × breathing frequency [ 35 ]. Assuming a deposition fraction of 0.1, a rat tidal volume of 2.1 mL/time, and a respiratory rate of 102 times/min, the lung accumulation amounts at exposure concentrations of 0.2 mg/m 3 and 2.0 mg/m 3 for 13 weeks of subchronic inhalation exposure were calculated as 0.1 and 1.0 mg, respectively. Furthermore, if those values are converted to exposure periods in humans, and assuming that CL-PAA is deposited at the same rate in both rats and humans (particle deposition efficiency of 0.1, amount of deposited material/1 g of rat lung weight), the human tidal volume is 625 mL/time, the respiratory rate is 12 times/min, the working time is 8 hours/day and 5 days/week, and the exposure concentration is 3 mg/m 3 , then in the 13-week subchronic inhalation exposure in rats in this study, the exposure periods in humans would be 4.6 months and 3.5 years, respectively, at concentrations of 0.2 mg/m 3 and 2.0 mg/m 3 . Some workers handling CL-PAA have developed fibrosis and interstitial pneumonia within as little as 3 years [ 3 ]; therefore, the observation of pulmonary fibrosis at an exposure concentration of 2.0 mg/m 3 is considered to be an exposure concentration that can reflect the level of pulmonary damage in humans. The physicochemical properties of the CL-PAA used in these studies had a molecular weight of 765,000 and a cross-linked structure. CL-PAA, which has a molecular weight in this range, is generally used as a thickener, particularly as a stabilizer for emulsions and suspensions in cosmetics, pharmaceuticals, and chemicals [ 36 , 37 ]. Based on their physical properties, the pulmonary toxicity of the commonly used CL-PAA and the CL-PAA used in these studies were considered to be similar. It has been reported that molecular weight is one of the characteristics that determines the pulmonary toxicity of PAA, and pulmonary inflammation and fibrosis potential increase with increasing molecular weight. When PAA of different molecular weights was intratracheally instilled in rats to examine pulmonary damage, PAA with a larger molecular weight of 598,000 caused persistent pulmonary inflammation and fibrosis, while PAA with a smaller molecular weight of 30,900 showed only a transient reaction [ 6 ]. Another CL-PAA with a molecular weight greater than 1 million also induced persistent inflammation and fibrosis in rats' lungs when intratracheally instilled [ 4 , 5 ]. Considering that a molecular weight of at least six hundred thousand is sufficient to cause persistent pulmonary damage, CL-PAA, which is commonly used as a thickening agent, has a similar molecular weight and is therefore likely to cause pulmonary damage. 3) The mechanism of pulmonary disorders caused by CL-PAA In the subacute inhalation exposure study of CL-PAA at 20 mg/m 3 , intraperitoneal injection of NAC, which has antioxidant properties [ 38 ], suppressed pulmonary inflammation, cell damage, cytokine production, and HO-1 expression induced by CL-PAA exposure. NAC is a thiol with reducing properties that neutralizes and eliminates ROS, such as hydroxyl radicals and hydrogen peroxide, thereby mitigating oxidative stress and suppressing inflammation [ 39 – 41 ]. The anti-inflammatory effects of NAC have also been reported in other dust-exposed animal models. In a rat silicosis model induced by intratracheal instillation of SiO 2 powder, the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) [ 42 , 43 ] and HO-1, which are activated by ROS, were suppressed by intraperitoneal injection of NAC, and pulmonary inflammation was suppressed accordingly [ 44 ]. It has also been reported that the anti-inflammatory mechanism of NAC acts through the inhibition of nuclear factor-kappa B (NF-κB) [ 45 ], a cytokine transcription factor [ 40 ]. In a rat model of bleomycin (BLM)-induced pulmonary inflammation and fibrosis, NAC administration reduced serum NF-κB levels and pulmonary inflammation [ 41 ]. Considering that the transcription factor of CINC-1 is NF-κB [ 46 ], it was suggested that the anti-inflammatory effect of NAC is associated with the suppression of NF-κB activation. We performed a comprehensive analysis of mRNA extracted from lung tissue to investigate the mechanism of inflammation suppression. The results showed that genes whose expression was increased by subacute inhalation exposure to 20 mg/m 3 CL-PAA and whose expression was decreased by the administration of NAC included genes related to subacute inflammation and oxidative stress (Table 4 ). KEGG pathway analysis of the extracted genes revealed pathways related to innate immunity, such as phagosome and Toll-like receptor signaling, and pathways related to cytokines. Pathways related to innate immunity and cytokines have also been extracted in other animal models of particle exposure. The pathway for phagosome was extracted from the lung tissue of mice exposed to PM2.5 inhalation [ 47 ], and pathways for cytokine-cytokine receptor interaction and viral protein interaction with cytokine and cytokine receptor were extracted from the lung tissue of rats intratracheally instilled with lipopolysaccharide (LPS) [ 48 ]. Related to the above, in our previous intratracheal instillation study of another CL-PAA we extracted cytokine-related pathways, such as cytokine-cytokine receptor interaction and chemokine signaling pathway, by KEGG pathway analysis of genes obtained from lung tissue [ 4 ]; our findings suggested that the inflammation and fibrosis induced by oxidative stress due to CL-PAA exposure are associated with particle phagocytosis and cytokines. Conclusions We performed 5-day and 13-week inhalation exposure studies in rats to examine the acute and chronic pulmonary injury potential of CL-PAA. Inhalation exposure for 5 days induced concentration-dependent inflammation in the rats' lungs. Inhalation exposure for 13 weeks induced persistent inflammation and fibrosis in the rats' lungs. Based on the results of the subchronic inhalation exposure study, we determined that the no observed adverse effect level (NOAEL) for CL-PAA for chronic pulmonary damage, such as inflammation and fibrosis, was 0.2 mg/m 3 . Furthermore, oxidative stress was associated with pulmonary inflammation caused by inhalation exposure to CL-PAA, and the administration of NAC attenuated pulmonary inflammation. Abbreviations ACGIH American Conference of Governmental Industrial Hygienists ANOVA analysis of variance BALF bronchoalveolar lavage fluid CeO 2 cerium dioxide CINC cytokine-induced neutrophil chemoattractant CL-PAA cross-linked polyacrylic acid DAVID Database for Annotation,Visualization and Integrated Discovery ECM extracellular matrix ELISA enzyme-linked immune-sorbent assay EMT epithelial-mesenchymal transition ERK extracellular signal-regulated kinase GPC gel permeation chromatography HE hematoxylin and eosin HO-1 heme oxygenase-1 KEGG Kyoto Encyclopedia of Genes and Genomes LDH lactate dehydrogenase LPS lipopolysaccharide MMAD mass median aerodynamic diameter Mn number average molecular weight MT masson trichrome Mw weight average molecular weight MWCNT multiwalled carbon nanotubes NAC N-acetylcysteine NET neutrophil extracellular trap NF-κB nuclear factor-kappa B NiO nickel oxide NOAEL no observed adverse effect level Nrf2 nuclear factor-erythroid 2-related factor 2 OECD Organisation for Economic Co-operation and Development PAA polyacrylic acid PDI polydispersity index PMN polymorphonuclear leukocyte Rg radius of gyration ROS reactive oxygen species SEM scanning electron microscopy STIS short-term inhalation studies TdT terminal deoxynucleotidyl transferase TiO 2 titanium dioxide TLV threshold limit value T-PER tissue protein extraction reagent TWA time-weighted-average WT Whole Transcript ZnO zinc oxide Declarations Ethics approval and consent to participate All procedures and animal handling procedures were performed 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: AE18-003). Consent for publication Not required as no human data presented. Availability of data and materials The datasets used during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Industrial Disease Clinical Research Grants from the Ministry of Health, Labour, and Welfare (Grant number 200101-01) and JSPS KAKENHI Grant Number 22K10499. Authors’ contributions Authors YH, CN, TT, HI, KY (Kazuhiro Yatera), and YM were responsible for the study design and writing of the manuscript. Authors YH, CN, TT, HI, KS (Kazuma Sato), NK, TM, KY (Kei Yamasaki), KW, HH, AM, JT, TK, KS (Kazuo Sakurai), KY (Kazuhiro Yatera), and YM were responsible for the data and analysis. Authors YH, CN, TT, HI, KS (Kazuma Sato), NK, TM, HH, TK, KS (Kazuo Sakurai), and YM performed the experiments. All the authors read and approved the final manuscript. Acknowledgements The authors would like to thank Sumiyo Kuramoto, Tomoko Watanabe, Rika Takai, Mayumi Tashiro, Yuno Ariyoshi, Kumiko Matsuyama, and Tomoko Morimoto for their technical support with the experiments. Authors’ information 1) Department of Respiratory Medicine, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan 2) Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan 3) Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan 4) Shared-Use Research Center, School of Medicine, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan 5) Research Institute of Science for Safety and Sustainability, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan. 16-1 Onogawa, Tsukuba, Ibaraki, 305-8569, Japan 6) Department of Chemistry and Biochemistry, The University of Kitakyushu. 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan References Chen X, Huang W, He B, Zhang Y. Synthesis and Application of Tackifying Dispersant Poly (Vinyl Alcohol-Acrylic Acid-Triallyl Cyanate). Polym (Basel). 2022;14. Park S, Lee K, Lee EJ, Lee SY, In KH, Kim H-K, et al. Humidifier disinfectant-associated interstitial lung disease in an animal model induced by polyhexamethylene guanidine aerosol. Am J Respir Crit Care Med. 2014;190:706–8. Kishimoto T, Okamoto K, Koda S, Ono M, Umeda Y, Yamano S et al. Respiratory disease in workers handling cross-linked water-soluble acrylic acid polymer. PLoS ONE. 2023;18. Nishida C, Tomonaga T, Izumi H, Wang KY, Higashi H, Ishidao T et al. Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation. Part Fibre Toxicol. 2022;19. Higashi Y, Morimoto Y, Nishida C, Tomonaga T, Izumi H, Wang KY et al. Pulmonary disorder induced by cross-linked polyacrylic acid. J Occup Health. 2022;64. Nishida C, Izumi H, Tomonaga T, Wang KY, Higashi H, Takeshita JI et al. Effect of Different Molecular Weights of Polyacrylic Acid on Rat Lung Following Intratracheal Instillation. Int J Mol Sci. 2022;23. Tomonaga T, Nishida C, Izumi H, Kawai N, Wang KY, Higashi H et al. Crosslinked Structure of Polyacrylic Acid Affects Pulmonary Fibrogenicity in Rats. Int J Mol Sci. 2022;23. Morimoto T, Izumi H, Tomonaga T, Nishida C, Kawai N, Higashi Y et al. The Effects of Endoplasmic Reticulum Stress via Intratracheal Instillation of Water-Soluble Acrylic Acid Polymer on the Lungs of Rats. Int J Mol Sci. 2024;25. Higashi Y, Nishida C, Tomonaga T, Izumi H, Kawai N, Morimoto T et al. Intratracheal instillation of polyacrylic acid induced pulmonary fibrosis with elevated transforming growth factor-β1 and connective tissue growth factor. Toxicology. 2024;506. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8:958–69. Arora S, Dev K, Agarwal B, Das P, Syed MA. Macrophages: Their role, activation and polarization in pulmonary diseases. Immunobiology. 2018;223:383–96. Chen PJ, Chen SH, Chen YL, Wang YH, Lin CY, Chen CH et al. Ribociclib leverages phosphodiesterase 4 inhibition in the treatment of neutrophilic inflammation and acute respiratory distress syndrome. J Adv Res. 2024. Yamada A, Aki T, Unuma K, Funakoshi T, Uemura K. Paraquat induces epithelial-mesenchymal transition-like cellular response resulting in fibrogenesis and the prevention of apoptosis in human pulmonary epithelial cells. PLoS ONE. 2015;10. Johnson N. The limitations of inhalation, intratracheal, and intracoelomic routes of administration for identifying hazardous fibrous materials. Fiber Toxicol. 1993;D:43–72. Landsiedel R, Ma-Hock L, Hofmann T, Wiemann M, Strauss V, Treumann S et al. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part Fibre Toxicol. 2014;11. OECD/OCDE 413 OECD GUIDELINES FOR THE TESTING OF CHEMICALS 90-DAY. (SUBCHRONIC) INHALATION TOXICITY STUDY. 2018. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988;41:467–70. Hübner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, et al. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 2008;44:507–17. Ma-Hock L, Strauss V, Treumann S, Küttler K, Wohlleben W, Hofmann T et al. Comparative inhalation toxicity of multi-wall carbon nanotubes, graphene, graphite nanoplatelets and low surface carbon black. Part Fibre Toxicol. 2013;10. Landsiedel R, Ma-Hock L, Hofmann T, Wiemann M, Strauss V, Treumann S et al. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part Fibre Toxicol. 2014;11. Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, Van Ravenzwaay B, et al. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance. Inhal Toxicol. 2009;21:102–18. Warheit DB, Mchugh TA. Hartsky1 MA. Differential pulmonary responses in rats inhaling crystalline, colloidal or amorphous silica dusts. Scand J Work Environ Health. 1995;21:19–21. Handa O, Naito Y, Yoshikawa T. Rat Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) in Inflammation. J Clin Biochem Nutr. 2006;38:51–8. Tomonaga T, Izumi H, Oyabu T, Lee BW, Kubo M, Shimada M, et al. Assessment of cytokine-induced neutrophil chemoattractants as biomarkers for prediction of pulmonary toxicity of nanomaterials. Nanomaterials. 2020;10:1–18. Nakashima K, Sato T, Shigemori S, Shimosato T, Shinkai M, Kaneko T. Regulatory role of heme oxygenase-1 in silica-induced lung injury. Respir Res. 2018;19. Horie M, Yoshiura Y, Izumi H, Oyabu T, Tomonaga T, Okada T et al. Comparison of the pulmonary oxidative stress caused by intratracheal instillation and inhalation of NiO nanoparticles when equivalent amounts of NiO are retained in the lung. Antioxidants. 2016;5. Pauluhn J. Subchronic 13-week inhalation exposure of rats to multiwalled carbon nanotubes: Toxic effects are determined by density of agglomerate structures, not fibrillar structures. Toxicol Sci. 2009;113:226–42. Schwotzer D, Ernst H, Schaudien D, Kock H, Pohlmann G, Dasenbrock C et al. Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats. Part Fibre Toxicol. 2017;14. Morimoto Y, Oyabu T, Horie M, Kambara T, Izumi H, Kuroda E, et al. Pulmonary toxicity of printer toner following inhalation and intratracheal instillation. Inhal Toxicol. 2013;25:679–90. Bermudez E, Mangum JB, Wong BA, Asgharian B, Hext PM, Warheit DB, et al. Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol Sci. 2004;77:347–57. Henriquez AR, Snow SJ, Dye JA, Schladweiler MC, Alewel DI, Miller CN et al. The contribution of the neuroendocrine system to adaption after repeated daily ozone exposure in rats. Toxicol Appl Pharmacol. 2022;447. Farrell BP, Kerr HD, Kulle TJ, Sauder LR, Young JL. Adaptation in human subjects to the effects of inhaled ozone after repeated exposure. Am Rev Respir Dis. 1979;119:725–30. Wesselkamper SC, Chen C, Gordon T. Development of Pulmonary Tolerance in Mice Exposed to Zinc Oxide Fumes. Toxicol Sci. 2001;60:144–51. Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, Sparer J, et al. Characterization of clinical tolerance to inhaled zinc oxide in naive subjects and sheet metal workers. J Occup Environ Med. 2000;42:1085–91. Martins V, Cruz Minguillón M, Moreno T, Querol X, de Miguel E, Capdevila M, et al. Deposition of aerosol particles from a subway microenvironment in the human respiratory tract. J Aerosol Sci. 2015;90:103–13. Jassal M, Bajaj P. Developments in acrylic - based thickeners as substitute of emulsion thickeners for pigment printing. Indian J Fibre Text Reseach. 2001;26:143–55. Horiuchi T. Physico-Chemical Properties of Water Soluble Polymers in Aqueous Solution. J Surf Finish Soc Jpn. 2009;60:746–53. Kerksick C, Willoughby D. The Antioxidant Role of Glutathione and N-Acetyl-Cysteine Supplements and Exercise-Induced Oxidative Stress. J Int Soc Sports Nutr. 2005;2. Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. CMLS, Cell Mol Life Sci. 2003;60:6–20. Šalamon Š, Kramar B, Marolt TP, Poljšak B, Milisav I. Medical and dietary uses of n-acetylcysteine. Antioxidants. 2019;8. Mansour HH, Omran MM, Hasan HF, El kiki SM. Modulation of bleomycin-induced oxidative stress and pulmonary fibrosis by N-acetylcysteine in rats via AMPK/SIRT1/NF-κβ. Clin Exp Pharmacol Physiol. 2020;47:1943–52. Ma F, Luo S, Lu C, Jiang X, Chen K, Deng J et al. The role of Nrf2 in periodontal disease by regulating lipid peroxidation, inflammation and apoptosis. Front Endocrinol (Lausanne). 2022;13. Hayes JD, McMahon M. NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci. 2009;34:176–88. Tang M, Yang Z, Liu J, Zhang X, Guan L, Liu X et al. Combined intervention with N-acetylcysteine and desipramine alleviated silicosis development by regulating the Nrf2/HO-1 and ASMase/ceramide signaling pathways. Ecotoxicol Environ Saf. 2022;242. Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2. Handa O, Naito Y, Takagi T, Shimozawa M, Kokura S, Yoshida N, et al. Tumor Necrosis Factor-α-Induced Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) Production by Rat Gastric Epithelial Cells: Role of Reactive Oxygen Species and Nuclear Factor-κB. J Pharmacol Exp Ther. 2004;309:670–6. Liu S, Zhang W, Zhang F, Roepstorff P, Yang F, Lu Z et al. TMT-based quantitative proteomics analysis reveals airborne PM2.5-induced pulmonary fibrosis. Int J Environ Res Public Health. 2019;16. Xie Y, Luo J, Hu W, Ye C, Ren P, Wang Y et al. Whole Transcriptomic Analysis of Key Genes and Signaling Pathways in Endogenous ARDS. Dis Markers. 2022;2022. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 01 Aug, 2024 Submission checks completed at journal 23 Jul, 2024 First submitted to journal 23 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Morimoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYPACGyBOADEOQAV4CGpJI13LYXQteIDB8ePPJD7mnI/mZ09gk/hQcYfB4ADzww8MMndwazmTYyY5c9vt3Jk9D9gkZ5x5BtTCZizBwPMMt5YDOWzSvEAtG24kABlth+s3HGAwA/rlMG4t558/A2o5l7sfrOXfYaAh7N/wa7mRYAbUciB3gwRISwNICw9+WyRvvDG2nLktOXfGmYfNljOOHWaQPMxTLJGAxy9859Mf3vi4zS63vz354I0PNYcZ+I63b/zwsQd3iCkcgDMZGyA0MxAn9hzAohgC5Buwi//ArWUUjIJRMApGHAAA0LJa5lXxlAsAAAAASUVORK5CYII=","orcid":"","institution":"University of Occupational and Environmental Health Japan","correspondingAuthor":true,"prefix":"","firstName":"Yasuo","middleName":"","lastName":"Morimoto","suffix":""}],"badges":[],"createdAt":"2024-07-23 09:48:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4787568/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4787568/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62878938,"identity":"0f2f362b-8f11-4ff3-a393-abdc70687155","added_by":"auto","created_at":"2024-08-20 14:26:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":718952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM) images of the polymer used in these study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/25e2eb97f5acd4f0de311728.png"},{"id":62879628,"identity":"00aaa18e-425f-4808-afef-3a91656af1d6","added_by":"auto","created_at":"2024-08-20 14:34:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55490,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhalation exposure system for CL-PAA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/1bc39d1bec497e0545d4528a.png"},{"id":62877309,"identity":"ed49ba15-4eae-49c9-be78-9c56cdc26e67","added_by":"auto","created_at":"2024-08-20 14:10:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhalation exposure schedule for CL-PAA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/126444dcedca7f17b27845e3.png"},{"id":62878936,"identity":"3547ce54-e8ce-4d91-90bf-dd657551b8be","added_by":"auto","created_at":"2024-08-20 14:26:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhalation exposure concentration of CL-PAA\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/5b4faf76a5fea0f4059a8013.png"},{"id":62877313,"identity":"3def0373-93fc-44a8-8cae-6ccddfd4782c","added_by":"auto","created_at":"2024-08-20 14:10:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":293781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBody weight and relative lung weight (lung weight/body weight)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/266e96c5a708157d3e748155.png"},{"id":62877310,"identity":"acb729f7-ce11-4a7d-a94d-fc3faefd79cb","added_by":"auto","created_at":"2024-08-20 14:10:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of cell counts and total protein and LDH levels in BALF following subacute inhalation.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/60e5fbf58a08222b1744ec0b.png"},{"id":62877317,"identity":"d148327b-ec08-49e4-9f29-69cdce97de45","added_by":"auto","created_at":"2024-08-20 14:10:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":131733,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of cell counts and total protein and LDH levels in BALF following subchronic inhalation.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/2549fbff6391b2aa82d67460.png"},{"id":62877319,"identity":"f4a5711f-0b26-4e5a-b2f2-a720754207b4","added_by":"auto","created_at":"2024-08-20 14:10:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":161502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of cell counts and total protein and LDH levels in BALF following subacute inhalation and intraperitoneal injection.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/f75c676663e1cfbddf144d50.png"},{"id":62877322,"identity":"7d80361b-94d8-4eda-be62-a92ba1b6d796","added_by":"auto","created_at":"2024-08-20 14:10:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":136727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of chemokines in BALF and HO-1 in lung tissue following subacute inhalation.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/19e62d64c09845bc9fa4f693.png"},{"id":62878092,"identity":"7f9259a8-6800-4b7a-b9b1-6ddbd01c5a02","added_by":"auto","created_at":"2024-08-20 14:18:57","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":118765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of chemokines in BALF and HO-1 in lung tissue following subchronic inhalation.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/c8634e23bc0e9d609883ed43.png"},{"id":62878090,"identity":"1c31c711-0c78-4200-bbb7-8fef538b5ef6","added_by":"auto","created_at":"2024-08-20 14:18:57","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":131306,"visible":true,"origin":"","legend":"","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/c8dcfabea84039d5c9e89b39.png"},{"id":62877321,"identity":"ce466e9a-2926-4f4d-abe2-25ce61ba12c5","added_by":"auto","created_at":"2024-08-20 14:10:58","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":723386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological findings following subacute inhalation (HE and MT staining).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/91d94efe87c2f6438adffc77.png"},{"id":62878095,"identity":"9e4858a4-d8ba-47a5-9f4a-167cb6a6c02c","added_by":"auto","created_at":"2024-08-20 14:18:58","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1365418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological findings following subchronic inhalation (HE and MT staining).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/8f01272555d17a0e620a036a.png"},{"id":62877318,"identity":"157d463e-8e56-498d-a12e-220fce033e85","added_by":"auto","created_at":"2024-08-20 14:10:58","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1087862,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological findings following subacute inhalation and intraperitoneal injection (HE and MT staining).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/e2a76904c1b979be53da1396.png"},{"id":62880719,"identity":"b94542b6-57a7-4b9c-ae1c-dd05fa61815f","added_by":"auto","created_at":"2024-08-20 14:43:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6260741,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4787568/v1/08f41068-a9db-41f4-b061-b40309bd1e31.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhalation Exposure to Cross-linked Polyacrylic Acid Induces Pulmonary Disorders","fulltext":[{"header":"1. Background","content":"\u003cp\u003ePolyacrylic acid (PAA) is a general term for organic polymer compounds whose monomer is the organic material acrylic acid (C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). Their viscosity and water absorbency increase when they are cross-linked (cross-linked polyacrylic acid; CL-PAA). They are used in a variety of applications due to differences in their properties, including as additives in daily necessities, food, and medicines; therefore, PAA is currently one of the most widely used thickening agents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Organic materials are conventionally thought to be unlikely to cause pneumoconiosis by inhalation and to rarely cause the severe pulmonary fibrosis that is caused by inorganic materials such as crystalline silica and chrysotile, but in recent years there have been reports of organic materials inducing progressive pulmonary fibrosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. There were reports in Japan in 2017 of a high incidence of pulmonary disorders among workers at a company that manufactures PAAs, which are organic materials [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In response to those reports, animal exposure studies on PAA have been conducted in Japan, and it has been reported that PAA has strong inflammatory and fibrotic potential depending on its physicochemical properties [\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe mechanism of pulmonary injury caused by chemicals is considered to be that macrophages that phagocytose foreign materials release cytokines, which recruit inflammatory cells such as neutrophils, induce neutrophilic inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The neutrophils then induce oxidative stress by generating reactive oxygen species (ROS), such as free radicals, leading to pulmonary inflammation and fibrosis. Oxidative stress induces further inflammation and alveolar epithelial damage [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and induces pulmonary fibrosis due to excessive epithelial-mesenchymal transition (EMT)-induced fibroblast activation and abnormal repair during the process of alveolar epithelial repair [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Oxidative stress is therefore ​​​​​​​thought to play an important role in chemical-induced pulmonary disorders.\u003c/p\u003e \u003cp\u003eMost animal experimental reports of pulmonary disorders caused by PAAs so far were based on intratracheal instillation. Inhalation exposure studies provide important information for evaluating the biological effects on humans because the dust​​​​​​​ deposition patterns and amounts are physiological, and the findings obtained from inhalation exposure studies are similar to the pathological conditions caused by exposure in humans [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For that reason, we conducted subacute and subchronic inhalation exposure studies of CL-PAA in rats to investigate the effects on the lungs of CL-PAA, a cross-linked type commonly used as a thickening agent, and analyzed the factors related to inflammation, fibrosis, and oxidative stress.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Sample polymer\u003c/h2\u003e\n \u003cp\u003eWe used CL-PAA with an average Mv\u0026thinsp;~\u0026thinsp;450,000 (Sigma-Aldrich Co. LLC., St. Louis, MO, USA). The fundamental characteristics of the CL-PAA are summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The weight average molecular weight (Mw), number average molecular weight (Mn), polydispersity index (PDI), and radius of gyration (Rg) of CL-PAA were measured by gel permeation chromatography (GPC) (Prominence 501 system, SHOKO SCIENCE, Kanagawa, Japan), and their values were 768,000, 414,000, and 1.85, respectively. The mass median aerodynamic diameter (MMAD) in aerosols determined by an Andersen sampler (AN-200; TOKYO DYLEC CORP., Shinjyuku, Japan) was 1.9 \u0026micro;m (\u0026sigma;g; geometric standard deviation\u0026thinsp;=\u0026thinsp;2.0), 2.2 \u0026micro;m (\u0026sigma;g\u0026thinsp;=\u0026thinsp;1.9), and 2.5 \u0026micro;m (\u0026sigma;g\u0026thinsp;=\u0026thinsp;2.0) for 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e, 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e, and 20 mg/m\u003csup\u003e3\u003c/sup\u003e, respectively. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows scanning electron microscopy (SEM) images of the polymer used in these studies, obtained by a HITACHI S-4500 (Hitachi, Ltd., Tokyo, Japan) instrument. The bulk PAA powder and the suspended PAA in distilled water are shown in Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, respectively.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1. Physiochemical characterization of the polymer used in these studies.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cimg 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\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Animals\u003c/h2\u003e\n \u003cp\u003eMale Fischer 344 rats (8 weeks old) that were exposed to CL-PAA were purchased from Charles River Laboratories International, Inc., Kanagawa, Japan. The animals were housed at the Laboratory Animal Research Center of the University of Occupational and Environmental Health for four weeks with free access to commercial feed and water. All of the procedures and animal handling procedures were performed in accordance with the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan (animal studies ethics clearance proposal number; AE18-003).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Inhalation exposure and intraperitoneal injection\u003c/h2\u003e\n \u003cp\u003eThe inhalation exposure studies involved the use of a dust feeder composed of a table feeder, an ejector (DF-3; SIBATA Scientific Technology Ltd., Saitama, Japan), and a whole-body aerosol exposure chamber (AD-8506046; SIBATA Scientific Technology Ltd., Saitama, Japan), as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The CL-PAA particles were delivered by the ejector to the rats in a whole-body exposure chamber with air. The mass concentration of aerosols in the exposure chamber was sometimes monitored by sampling the weight of the particles and the volume of the air. We performed three inhalation exposure studies with this device: subacute inhalation, subchronic inhalation, and subacute inhalation and intraperitoneal injection (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIn the subacute inhalation study, the rats were exposed to 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e, 2.0 mg/ m\u003csup\u003e3\u003c/sup\u003e, or 20 mg/m\u003csup\u003e3\u003c/sup\u003e CL-PAA for 6 hours/day for 5 days/week for 1 week. This study was conducted with reference to other short-term inhalation studies (STIS) [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e]. The 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e and 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e exposures were administered on the same schedule, while the 20 mg/m\u003csup\u003e3\u003c/sup\u003e exposure was administered on a different schedule, and a control group was established for each schedule. The control group received clean air.\u003c/p\u003e\n \u003cp\u003eThe subchronic inhalation treatment group was exposed to 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e and 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e CL-PAA for 6 hours/day for 5 days/week for 13 weeks. This was conducted with reference to the Organisation for Economic Co-operation and Development (OECD) TG413, the OECD guidelines for testing chemicals [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. The control group received clean air.\u003c/p\u003e\n \u003cp\u003eFinally, four groups were subjected to subacute inhalation and intraperitoneal injection: group one, \u0026quot;Control\u0026quot;, received normal saline intraperitoneally 1 hour before inhaling clean air; group two, \u0026quot;NAC\u0026quot;, received N-acetylcysteine (NAC) intraperitoneally 1 hour before inhaling clean air; group three, \u0026quot;CL-PAA\u0026quot;, received normal saline intraperitoneally 1 hour before inhaling 20 mg/m\u003csup\u003e3\u003c/sup\u003e CL-PAA; and group four, \u0026quot;CL-PAA\u0026thinsp;+\u0026thinsp;NAC\u0026quot;, received NAC intraperitoneally 1 hour before inhaling 20 mg/m\u003csup\u003e3\u003c/sup\u003e CL-PAA. The volume of saline administered intraperitoneally was 1 ml, and NAC was administered at 200 mg/kg (48.78 mg NAC, calculated based on the average body weight of the rats in groups two and four, which was 243.9 g) dissolved in 1 ml of normal saline. Inhalation exposure was conducted for 6 hours/day, and intraperitoneal injection and inhalation exposure were conducted for 5 consecutive days. The actual analyzed mass concentrations were 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/m\u003csup\u003e3\u003c/sup\u003e, 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mg/m\u003csup\u003e3\u003c/sup\u003e, and 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mg/m\u003csup\u003e3\u003c/sup\u003e in the subacute inhalation study (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), 0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mg/m\u003csup\u003e3\u003c/sup\u003e and 2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg/m\u003csup\u003e3\u003c/sup\u003e in the subchronic inhalation study (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB), and 20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mg/m\u003csup\u003e3\u003c/sup\u003e in the subacute inhalation and intraperitoneal injection study (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Animals following inhalation exposure\u003c/h2\u003e\n \u003cp\u003eThere were 5 rats in the exposure groups and the control group at each time point. Rats were sacrificed at each time point under anesthesia by inhalation of isoflurane (Pfizer Japan, Tokyo, Japan). In the subacute inhalation study, rats were sacrificed at 3 days and 1 month after the end of inhalation. In the subchronic inhalation study, rats were sacrificed at 3 days, 1 month, 3 months, and 6 months after the end of inhalation. In the subacute inhalation and intraperitoneal injection studies, rats were sacrificed on the day after the end of inhalation and intraperitoneal injection. All rats were sacrificed under anesthesia by inhalation of isoflurane (Pfizer Japan, Tokyo, Japan). Body and lung weights were measured at autopsy. Blood was removed from the abdominal aorta, and the lungs were perfused with normal saline. The right lung was repeatedly infused with normal saline under a pressure of 20 cmH\u003csub\u003e2\u003c/sub\u003eO, followed by fluid recovery two times, while the left main bronchus was clamped. Between 5 and 15 mL of the recovered fluid (bronchoalveolar lavage fluid; BALF) was collected by free fall into tubes, and then the right and left lungs were divided. The third lobe of the right lung after recovery from the BALF was homogenized and used for the evaluation of heme oxygenase-1 (HO-1). The left lungs were inflated and fixed with 10% formaldehyde under a pressure of 25 cmH\u003csub\u003e2\u003c/sub\u003eO for histopathological evaluation.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.5 Cytospin analysis of inflammatory cells and measurement of total protein and lactate dehydrogenase (LDH) levels in BALF\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe BALF was centrifuged at 400 g at 4\u0026deg;C for 15 minutes, and the supernatant was transferred to a new tube to measure total protein and LDH levels. The pellets were washed with a suspension of polymorphonuclear leukocyte (PMN) buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.5 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and 5.6 mM C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e) and centrifuged at 400 g at 4\u0026deg;C for 15 minutes. After removal of the supernatant by decanting, the pellets were resuspended in 1 mL of PMN buffer. The number of cells in the BALF was counted by ADAM-MC\u0026trade; (AR BROWN Co., Ltd., Tokyo, Japan), an automated cell counting device, and the cells were splashed and fixed onto a glass slide using cytospin and stained with Diff-Quik\u0026reg; (Sysmex Co., Kobe, Hyogo, Japan), after which the number of neutrophils and alveolar macrophages were counted by microscopic observation. Total protein in the BALF was measured by a Pierce\u0026trade; 660 nm Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL, USA) and calculated using a standard curve obtained from the Albumin Standard (Thermo Fisher Scientific Inc., Rockford, IL, USA). LDH levels in the BALF supernatant were measured with a Cytotoxicity Detection Kit\u003csup\u003ePLUS\u003c/sup\u003e (LDH) (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany) according to the manufacturer\u0026rsquo;s instructions. LDH levels were calculated using a standard curve obtained from known concentrations of recombinant LDH from rabbit muscle (Oriental Yeast Co., Ltd., Tokyo, Japan).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Measurement of chemokines in BALF and HO-1 in lung tissue\u003c/h2\u003e\n \u003cp\u003eThe concentrations in the BALF of cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2, which are neutrophil chemotactic factors in rats, were measured by the enzyme-linked immunosorbent assay (ELISA) kits, #RCN100 and #RCN200 (R\u0026amp;D Systems, Minneapolis, MN, USA), respectively. The third lobe of the right lung was homogenized with a tissue protein extraction reagent (T-PER) (Thermo Fisher Scientific Inc., Rockford, IL, USA) containing P8340 (Sigma-Aldrich, St. Louis, MO, USA), protein inhibitor cocktails, cOmplete\u0026trade; Mini (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany), and protease inhibitor cocktails, and then centrifuged (20,400 g at 4\u0026deg;C for 10 minutes). The protein concentration in the homogenized lung tissue extracts was measured by a Pierce\u0026trade; 660 nm Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL, USA) and calculated using a standard curve obtained from the Albumin Standard (Thermo Fisher Scientific Inc., Rockford, IL, USA). The total protein concentration in the lung tissue was adjusted to a final concentration of 500 mg/mL to measure HO-1, an index of oxidative stress, which was measured by the ELISA kit ADI-EKS-810A (Enzo Life Sciences, Farmingdale, NY, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Total RNA Extraction\u003c/h2\u003e\n \u003cp\u003eThe third lobe of the right lung (n\u0026thinsp;=\u0026thinsp;5 per group per time point) was homogenized with QIAzol lysis reagent. Total RNA from the homogenates was extracted using a miRNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer\u0026rsquo;s instructions. The purity and quantity of the RNA were evaluated with a NanoDrop instrument. The quality of the RNA was evaluated with a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Affymetrix Whole Transcript Expression Array Methods\u003c/h2\u003e\n \u003cp\u003ecDNA was synthesized using the GeneChip Whole Transcript (WT) Amplification Kit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase (TdT) using a GeneChip WT terminal labeling kit. Approximately 5.5 \u0026micro;g of labeled DNA target was hybridized to the Affymetrix GeneChip Array at 45˚ for 16 hours. The hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). The probe cell intensity data computation and CEL file generation were performed using Affymetrix\u0026reg; GeneChip\u0026trade; Command Console\u0026reg; software.\u003c/p\u003e\n \u003cp\u003eWe performed two analyses. The first was an analysis of genes whose expression was increased by subchronic inhalation exposure to CL-PAA (fc 2-fold or more in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group vs 0 mg/m\u003csup\u003e3\u003c/sup\u003e group), and the second was an analysis of genes whose expression was increased by subacute inhalation exposure to CL-PAA (fc 2-fold or more in the CL-PAA group vs control group) and decreased by NAC administration (fc -1.5 or less in the CL-PAA\u0026thinsp;+\u0026thinsp;NAC group vs CL-PAA group). We analyzed the genes related to CL-PAA exposure and NAC administration 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 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/home.jsp\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Histopathology\u003c/h2\u003e\n \u003cp\u003eFormaldehyde-fixed lung tissue was embedded in paraffin and sectioned at a thickness of 4 \u0026micro;m for hematoxylin and eosin (HE) and Masson trichrome (MT) staining. The slides were assessed for histological changes by a board-certified pathologist. The Ashcroft scale was used to assess the degree of pulmonary fibrosis [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. We scored the MT-stained specimens at \u0026times;10 magnification with the Ashcroft scale, as described in another report [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. The Ashcroft scale, briefly explained, is a lung section graded from 0 (normal lung) to 8 (most severe fibrosis), with the grades summed and divided by the number of fields.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using IBM SPSS Statistics ver. 27 (IBM Corp., Armonk, NY, USA) and carried out using Student\u0026rsquo;s t-test and the Holm-Bonferroni correction in the subacute inhalation study, with Dunnett\u0026rsquo;s test in the subchronic inhalation study, and with Tukey\u0026rsquo;s test in the subacute inhalation and intraperitoneal injection study, with differences of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered to be statistically significant. The data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs (* \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Body and lung weight\u003c/h2\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the body weight and relative lung weight of the rats.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e1) Subacute inhalation study\u003c/h3\u003e\n\u003cp\u003eThere was a statistically significant difference in body weight only in the 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e group at 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). There was a statistically significant increase in the relative lung weight (lung weight/body weight) in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure and in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days and 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003e2) Subchronic inhalation study\u003c/h3\u003e\n\u003cp\u003eThere were no significant differences in body weight among the groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). There was a statistically significant increase in the relative lung weight only in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group from 3 days to 3 months postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003e3) Subacute inhalation and intraperitoneal injection study\u003c/h3\u003e\n\u003cp\u003eThere were no significant differences in body weight among the groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). There was a statistically significant increase in the relative lung weight in the CL-PAA and CL-PAA\u0026thinsp;+\u0026thinsp;NAC group compared to the control and the NAC group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Cell analysis and total protein and lactate dehydrogenase (LDH) levels in BALF\u003c/h2\u003e\n\u003c/div\u003e\n\u003ch3\u003e1) Subacute inhalation study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows the inflammatory cell counts and total protein and LDH levels in the BALF in the subacute inhalation study. There was a statistically significant increase in the total cell count in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure and in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days and 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). There was a statistically significant concentration-dependent increase in the neutrophil count and percentage of neutrophils at 3 days postexposure. There were no significant differences in the neutrophil count or percentage of neutrophils at 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). There was a statistically significant increase in the macrophage count in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure and in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days and 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). There was a statistically significant increase in the total protein content in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group and in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). There was a statistically significant increase in LDH levels in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure and in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days and 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\n\u003ch3\u003e2) Subchronic inhalation study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e shows the inflammatory cell counts and total protein and LDH levels in the BALF in the subchronic inhalation study. There was a statistically significant increase in the total cell count, neutrophil count, and percentage of neutrophils in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). There was a statistically significant increase in the macrophage count and total protein content in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days and 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE). There was a statistically significant increase in LDH levels in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF). There were no significant differences in the inflammatory cell count or total protein or LDH levels in the BALF of the 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e group during the observation period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA-F).\u003c/p\u003e\n\u003ch3\u003e3) Subacute inhalation and intraperitoneal injection study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e shows the inflammatory cell count and total protein and LDH levels in the BALF in the subacute inhalation and intraperitoneal injection study. There was a statistically significant increase in the total cell count, neutrophil count, percentage of neutrophils, macrophage count, total protein, and LDH levels in the exposed groups. There was a statistically significant decrease in the total cell count, neutrophil count, macrophage count, total protein content, and LDH level, which were increased by CL-PAA exposure following the intraperitoneal injection of NAC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA-F).\u003c/p\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Concentration of CINCs in BALF and concentration of HO-1 in lung tissue\u003c/h2\u003e\n\u003c/div\u003e\n\u003ch3\u003e1) Subacute inhalation study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e shows the concentrations of CINC-1 and CINC-2 in the BALF and of HO-1 in the lung tissue in the subacute inhalation study. There was a statistically significant increase in the concentration of CINC-1 in the exposure groups at 3 days postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). There was a statistically significant increase in the concentration of CINC-2 in the exposure groups at 3 days postexposure and in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB). There was a statistically significant concentration-dependent increase in the concentration of HO-1 in the exposure groups at 3 days postexposure, and there was a statistically significant increase in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003e2) Subchronic inhalation study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e shows the concentrations of CINC-1 and CINC-2 in the BALF and of HO-1 in the lung tissue in the subchronic inhalation study. There was a statistically significant increase in the concentration of CINCs in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eB). There was a statistically significant increase in the concentration of HO-1 in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group at 3 days and 1 month postexposure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eC). There were no significant differences in the concentrations of CINCs in the BALF or HO-1 in the lung tissue in the 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e group during the observation period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eA-C).\u003c/p\u003e\n\u003ch3\u003e3) Subacute inhalation and intraperitoneal injection study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e shows the concentrations of CINC-1 and CINC-2 in the BALF and of HO-1 in the lung tissue in the subacute inhalation and intraperitoneal injection study. There was a statistically significant increase in the concentration of CINCs and HO-1 in the exposed groups. The concentrations of CINCs and HO-1, which were elevated by CL-PAA exposure, were statistically significantly decreased by intraperitoneal injection of NAC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA-C).\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Histopathological features of the lung\u003c/h2\u003e\n\u003c/div\u003e\n\u003ch3\u003e1) Subacute inhalation study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e shows the representative histopathological findings in the lungs in the subacute inhalation study. There was neutrophil and macrophage infiltration in the alveoli and interstitium in the exposure group at 3 days postexposure. There was inflammatory cell infiltration only in the 20 mg/m\u003csup\u003e3\u003c/sup\u003e group at 1 month postexposure. No fibroblasts or collagen fibers were observed during the observation period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003e2) Subchronic inhalation study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e shows the representative histopathological findings in the lungs in the subchronic inhalation study. There was neutrophilic inflammatory cell infiltration in the alveoli and interstitium from 3 days to 3 months, and fibroblasts and collagen fibers were observed from 3 days to 1 month in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group. There were no obvious inflammatory cell infiltration, fibroblasts, or collagen fibers in the 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e group during the observation period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003eA-D). The Ashcroft scale, an indicator of fibrosis, showed a significant increase only in the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e group from 3 days to 1 month (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003eE).\u003c/p\u003e\n\u003ch3\u003e3) Subacute inhalation and intraperitoneal injection study\u003c/h3\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e shows representative histopathological findings in the lungs in the subacute inhalation and intraperitoneal injection study. There was neutrophilic inflammatory cell infiltration in the alveoli and interstitium in the inhalation of CL-PAA 20 mg/m\u003csup\u003e3\u003c/sup\u003e group. These changes were reduced by the administration of NAC. No fibroblasts or collagen fibers were observed during the observation period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Microarray analysis and KEGG pathway analysis\u003c/h2\u003e\n\u003c/div\u003e\n\u003ch3\u003e1) Subchronic inhalation study\u003c/h3\u003e\n\u003cp\u003eWe conducted a microarray analysis of mRNA in the lung tissue at 1 month after the subchronic inhalation exposure. Among the 23,188 genes that were found to be expressed, 488 genes were expressed more than twice as much as in the negative control group. Among those 488, there were 288 genes with a global normalization value of 25 or higher, and genes related to inflammation and fibrosis were observed at the top (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). We evaluated the effects of inhaling CL-PAA on the lungs by conducting a pathway analysis using 288 genes whose expression was more than twice that of the negative control group after inhalation exposure. We identified pathways related to innate immunity, such as phagosome and neutrophil extracellular trap (NET) formation, and pathways related to inflammation and fibrosis, such as cytokine-cytokine receptor interaction and extracellular matrix (ECM)-receptor interaction (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of the expression of the top 10 genes after subchronic inhalation exposure\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene symbol\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene description\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold charge\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZbtb16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZinc finger and BTB domain containing 16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22.69\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRetnla\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eResistin like alpha\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.77\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRspo3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eR-spondin 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.98\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMmp12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMatrix metallopeptidase 12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.39\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLOC100909700*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCD177 antigen-like\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.28\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSlc26a4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSlc26a4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.11\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLgals3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGalectin-3 protein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.86\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCd177\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCD177 molecule\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.99\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIghg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImmunoglobulin constant heavy G chain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.81\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGfra3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGDNF family receptor alpha 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.31\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003eNote: *LOC100909700 (tentatively renamed CD177 in the rat genome database)\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eResults of KEGG pathway analysis after subchronic inhalation exposure\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerms of KEGG pathway\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene Counts\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP Value\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBenjamini\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTuberculosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00000025\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.000058\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eRheumatoid arthritis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.000032\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0038\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOsteoclast differentiation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.000062\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0049\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePhagosome\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00034\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.020\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLeishmaniasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00055\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.026\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLysosome\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00071\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.028\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eComplement and coagulation cascades\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.054\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAmoebiasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0026\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.078\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eApoptosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0036\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.087\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eProtein digestion and absorption\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0038\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.087\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePertussis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0041\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.087\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCytokine-cytokine receptor interaction\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0059\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.12\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSystemic lupus erythematosus\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0076\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.14\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStaphylococcus aureus infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.25\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eThyroid cancer\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.017\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNeutrophil extracellular trap formation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.018\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.26\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eViral protein interaction with cytokine and cytokine receptor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.48\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eECM-receptor interaction\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.039\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.51\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSmall cell lung cancer\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.044\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.54\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAlcoholic liver disease\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.046\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.54\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003e2) Subacute inhalation and intraperitoneal injection study\u003c/h3\u003e\n\u003cp\u003eWe conducted a microarray analysis of mRNA in the lung tissue 1 day after the subacute inhalation exposure and intraperitoneal injection. Among the 23,188 genes that were found to be expressed, there were 827 genes whose expression increased in response to subacute inhalation exposure to CL-PAA (fc 2-fold or greater in the CL-PAA group than in the control group, with a global normalization value of 25 or greater). Among those 827 genes, 186 exhibited decreased expression following NAC administration (fc -1.5-fold or less in the CL-PAA\u0026thinsp;+\u0026thinsp;NAC group than in the CL-PAA group, with a global normalization value of 25 or greater). Genes related to inflammation and oxidative stress were observed at the top (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). We evaluated the effects of CL-PAA inhalation on the lungs by conducting a pathway analysis using 186 genes extracted under the conditions described above. We identified pathways related to innate immunity, such as the phagosome and Toll-like receptor signaling pathways, and pathways related to inflammation, such as cytokine-cytokine receptor interaction (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eComparison of the expression of the top 10 genes after subacute inhalation exposure and intraperitoneal injection of NAC\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene symbol\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene description\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold charge 1\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eFold charge 2\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZbtb16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eZinc finger and BTB domain containing 16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.89\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-11.59\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCxcl13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003echemokine (C-X-C motif) ligand 13\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-4.05\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBpifb1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBPI fold containing family B member 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.71\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-3.69\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHpx\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHemopexin\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-3.60\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIghg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eImmunoglobulin constant heavy G chain\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.58\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-3.59\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOlr1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOxidized low density lipoprotein receptor 1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-3.54\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLy6al\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLymphocyte antigen 6 complex, locus A-like\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.78\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-3.08\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHk3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHexokinase 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-2.92\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIrf7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eInterferon regulatory factor 7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-2.88\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEnpp3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eEctonucleotide Pyrophosphatase/Phosphodiesterase 3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-2.71\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eResults of KEGG pathway analysis after subacute inhalation exposure and intraperitoneal injection of NAC\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTerms of KEGG pathway\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGene Counts\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eP Value\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eBenjamini\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eComplement and coagulation cascades\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0000012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0002\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAmoebiasis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00025\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.02\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePertussis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.00055\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePhagosome\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0016\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.066\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStaphylococcus aureus infection\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0023\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.074\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTuberculosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0058\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eViral protein interaction with cytokine and cytokine receptor\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0078\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.18\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eToll-like receptor signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCytokine-cytokine receptor interaction\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.23\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHIF-1 signaling pathway\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.021\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.34\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStarch and sucrose metabolism\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.52\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMeasles\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.043\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.53\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNeomycin, kanamycin and gentamicin biosynthesis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.53\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBiosynthesis of nucleotide sugars\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.046\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.53\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eWe considered (1) pulmonary disorders in a subacute inhalation exposure study, (2) pulmonary disorders in a subchronic inhalation exposure study, and (3) the mechanism of pulmonary disorders caused by CL-PAA.\u003c/p\u003e\n\u003cp\u003e1) Pulmonary disorders in the subacute inhalation exposure study\u003c/p\u003e\n\u003cp\u003eWe conducted a 5-day subacute inhalation exposure study to examine the acute effects of CL-PAA. The exposure concentrations for the subacute inhalation exposure study were set at 0, 0.2, 2.0, and 20 mg/m\u003csup\u003e3\u003c/sup\u003e, because the 8-h time-weighted average (TWA) of personal exposure concentrations to acrylic acid-based polymers at the site where pulmonary disorder incidents occur is 0.4 to 7.6 mg/m\u003csup\u003e3\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e], and the threshold limit value (TLV)-TWA of respirable dust set by the American Conference of Governmental Industrial Hygienists (ACGIH) is 3 mg/m\u003csup\u003e3\u003c/sup\u003e, and we considered that it was necessary to cover these ranges. We observed concentration-dependent neutrophil-centered pulmonary inflammation in the rats’ lungs from 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e when rats were exposed to CL-PAA via subacute inhalation. In a report on a subacute inhalation exposure study of materials with a known extent of lung damage ability, when rats were exposed to multiwalled carbon nanotubes (MWCNTs), a material with high pulmonary toxicity, via inhalation (0.1, 0.5, 2.5 mg/m\u003csup\u003e3\u003c/sup\u003e) for five days, neutrophilic inflammation and pulmonary injury were observed at exposure concentrations of 0.5 mg/m\u003csup\u003e3\u003c/sup\u003e or higher [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Similarly, cerium dioxide (CeO\u003csub\u003e2\u003c/sub\u003e), a material with high pulmonary toxicity, also caused neutrophilic inflammation in the lungs at exposure concentrations of 0.5 mg/m\u003csup\u003e3\u003c/sup\u003e or higher [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. In contrast, nano-titanium dioxide (TiO\u003csub\u003e2\u003c/sub\u003e), a material with low pulmonary toxicity, caused transient pulmonary injury at a concentration of 2 mg/m\u003csup\u003e3\u003c/sup\u003e for five days, but no neutrophilic inflammation was observed [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], and the inhalation of amorphous silica (10 or 100 mg/m\u003csup\u003e3\u003c/sup\u003e) for three days caused only transient pulmonary inflammation [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. Comparing to pulmonary inflammation with known toxic materials, the CL-PAA used in the present study caused pulmonary inflammation even at a low concentration of 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e, suggesting that it has a high potential for inducing pulmonary inflammation in the acute phase.\u003c/p\u003e\n\u003cp\u003eWe investigated the expression of the neutrophilic chemokines [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e] CINC-1 and CINC-2, which are associated with the mechanism of neutrophilic inflammation in the lungs following subacute inhalation exposure to CL-PAA, and we observed a concentration-dependent increase in CINC-1 and CINC-2 levels in the BALF at 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e. In our previous study of four-week inhalation exposure to inorganic materials, CINC-1 and CINC-2 levels in the BALF increased, accompanied by pulmonary neutrophilic inflammation, following exposure to nickel oxide (NiO) and CeO\u003csub\u003e2\u003c/sub\u003e nanoparticles [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although CL-PAA is an organic material and the mechanism of pulmonary inflammation caused by inorganic materials may not be comparable in general, the phenomenon that the CINC levels increased with neutrophil inflammation suggested that CINC is associated with neutrophil infiltration in CL-PAA, similar to neutrophil infiltration in inorganic materials.\u003c/p\u003e\n\u003cp\u003eWe also investigated oxidative stress following subacute inhalation exposure to CL-PAA by measuring the expression of HO-1, an oxidative stress response protein, and observed a concentration-dependent increase in HO-1 levels in lung tissue from the concentration of 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e. There have also been reports of increased HO-1 expression in other dust-exposed pulmonary inflammation models. Nakashima et al. reported that in a pulmonary inflammation model induced by crystalline silica, ROS activated the extracellular signal-regulated kinase (ERK) signaling pathway and induced HO-1 expression [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our previous study of four-week inhalation exposure to inorganic materials, we also observed that inhalation exposure to NiO nanoparticles induced pulmonary inflammation, which was accompanied by an increase in HO-1 levels in lung tissue [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. These findings suggest that oxidative stress is associated with CL-PAA-induced pulmonary inflammation, similar to inorganic material-induced pulmonary inflammation.\u003c/p\u003e\n\u003cp\u003e2) Pulmonary disorders in the subchronic inhalation exposure study\u003c/p\u003e\n\u003cp\u003eWe conducted a 13-week subchronic inhalation exposure study to examine the chronic effects of CL-PAA at concentrations of 0, 0.2, and 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e. Persistent pulmonary inflammation and fibrosis were observed in the rats' lungs at the 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e concentration, whereas no obvious responses were observed at the 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e concentration. The concentrations of CINC-1 and CINC-2 in BALF and of HO-1 in lung tissue were elevated at a concentration of 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e, suggesting that CINC-1, CINC-2, and HO-1 are associated with the pathology of subchronic exposure as well as subacute exposure. In another subchronic inhalation exposure study, when rats were exposed to MWCNTs (0.1, 0.4, 1.5, and 6 mg/m\u003csup\u003e3\u003c/sup\u003e) for 13 weeks, pulmonary inflammation and fibrosis were observed at exposure concentrations of 0.4 mg/m\u003csup\u003e3\u003c/sup\u003e or higher [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e], and CeO\u003csub\u003e2\u003c/sub\u003e (0.1, 0.3, 1.0, and 3.0 mg/m\u003csup\u003e3\u003c/sup\u003e) caused neutrophilic pulmonary inflammation and pulmonary injury at exposure concentrations of 1.0 mg/m\u003csup\u003e3\u003c/sup\u003e or higher [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. In contrast, in a 13-week inhalation exposure study using toner, a material with low pulmonary toxicity, there was no significant increase in neutrophilic inflammation, cytokine, or HO-1 levels during the observation period even at an exposure concentration of 4 mg/m\u003csup\u003e3\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e], and in ultrafine-TiO\u003csub\u003e2\u003c/sub\u003e (0.5, 2, 10 mg/m\u003csup\u003e3\u003c/sup\u003e), there was no pulmonary injury, although transient neutrophilic inflammation was observed at a concentration of 2 mg/m\u003csup\u003e3\u003c/sup\u003e [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. Considering that inflammation and fibrosis persisted in the present study, we consider that the CL-PAA we used has chronic pulmonary inflammation and fibrosis potential.\u003c/p\u003e\n\u003cp\u003eIn the subchronic inhalation exposure, the mild pulmonary inflammation observed in the subacute inhalation exposure at a concentration of 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e was not observed despite continuous exposure. This may have been because the body of the rats had become habituated to CL-PAA through continued inhalation. Similar phenomena have been observed with other chemicals. In an experiment in which rats were exposed to ozone, an increase in the number of neutrophils and total protein concentration in BALF was observed after two days of exposure, but these changes disappeared after four days of exposure [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. In an experiment in which humans were exposed to ozone for five consecutive days, the forced vital capacity decreased after three days of exposure but recovered after subsequent exposure [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], and, in zinc oxide (ZnO) inhalation exposure studies in mice and humans, the initial neutrophil infiltration was reduced during the exposure period [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although the characteristics of chemical materials that cause inflammation to disappear upon repeated exposure are unclear, habituation was thought to have occurred in the present study with CL-PAA.\u003c/p\u003e\n\u003cp\u003eRegarding the exposure concentration in the present study, we calculated the amount of inhaled material accumulated in the lungs using the following formula: deposition rate × exposure concentration × exposure time (duration) × respiratory volume (which is the amount of CL-PAA) = deposition fraction × exposure concentration of particle × exposure minutes in one day × days of exposure × tidal volume × breathing frequency [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Assuming a deposition fraction of 0.1, a rat tidal volume of 2.1 mL/time, and a respiratory rate of 102 times/min, the lung accumulation amounts at exposure concentrations of 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e and 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e for 13 weeks of subchronic inhalation exposure were calculated as 0.1 and 1.0 mg, respectively. Furthermore, if those values are converted to exposure periods in humans, and assuming that CL-PAA is deposited at the same rate in both rats and humans (particle deposition efficiency of 0.1, amount of deposited material/1 g of rat lung weight), the human tidal volume is 625 mL/time, the respiratory rate is 12 times/min, the working time is 8 hours/day and 5 days/week, and the exposure concentration is 3 mg/m\u003csup\u003e3\u003c/sup\u003e, then in the 13-week subchronic inhalation exposure in rats in this study, the exposure periods in humans would be 4.6 months and 3.5 years, respectively, at concentrations of 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e and 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e. Some workers handling CL-PAA have developed fibrosis and interstitial pneumonia within as little as 3 years [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]; therefore, the observation of pulmonary fibrosis at an exposure concentration of 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e is considered to be an exposure concentration that can reflect the level of pulmonary damage in humans.\u003c/p\u003e\n\u003cp\u003eThe physicochemical properties of the CL-PAA used in these studies had a molecular weight of 765,000 and a cross-linked structure. CL-PAA, which has a molecular weight in this range, is generally used as a thickener, particularly as a stabilizer for emulsions and suspensions in cosmetics, pharmaceuticals, and chemicals [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. Based on their physical properties, the pulmonary toxicity of the commonly used CL-PAA and the CL-PAA used in these studies were considered to be similar. It has been reported that molecular weight is one of the characteristics that determines the pulmonary toxicity of PAA, and pulmonary inflammation and fibrosis potential increase with increasing molecular weight. When PAA of different molecular weights was intratracheally instilled in rats to examine pulmonary damage, PAA with a larger molecular weight of 598,000 caused persistent pulmonary inflammation and fibrosis, while PAA with a smaller molecular weight of 30,900 showed only a transient reaction [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]. Another CL-PAA with a molecular weight greater than 1\u0026nbsp;million also induced persistent inflammation and fibrosis in rats' lungs when intratracheally instilled [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Considering that a molecular weight of at least six hundred thousand is sufficient to cause persistent pulmonary damage, CL-PAA, which is commonly used as a thickening agent, has a similar molecular weight and is therefore likely to cause pulmonary damage.\u003c/p\u003e\n\u003cp\u003e3) The mechanism of pulmonary disorders caused by CL-PAA\u003c/p\u003e\n\u003cp\u003eIn the subacute inhalation exposure study of CL-PAA at 20 mg/m\u003csup\u003e3\u003c/sup\u003e, intraperitoneal injection of NAC, which has antioxidant properties [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e], suppressed pulmonary inflammation, cell damage, cytokine production, and HO-1 expression induced by CL-PAA exposure. NAC is a thiol with reducing properties that neutralizes and eliminates ROS, such as hydroxyl radicals and hydrogen peroxide, thereby mitigating oxidative stress and suppressing inflammation [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. The anti-inflammatory effects of NAC have also been reported in other dust-exposed animal models. In a rat silicosis model induced by intratracheal instillation of SiO\u003csub\u003e2\u003c/sub\u003e powder, the transcription factors nuclear factor-erythroid 2-related factor 2 (Nrf2) [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e] and HO-1, which are activated by ROS, were suppressed by intraperitoneal injection of NAC, and pulmonary inflammation was suppressed accordingly [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. It has also been reported that the anti-inflammatory mechanism of NAC acts through the inhibition of nuclear factor-kappa B (NF-κB) [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e], a cytokine transcription factor [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. In a rat model of bleomycin (BLM)-induced pulmonary inflammation and fibrosis, NAC administration reduced serum NF-κB levels and pulmonary inflammation [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Considering that the transcription factor of CINC-1 is NF-κB [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e], it was suggested that the anti-inflammatory effect of NAC is associated with the suppression of NF-κB activation.\u003c/p\u003e\n\u003cp\u003eWe performed a comprehensive analysis of mRNA extracted from lung tissue to investigate the mechanism of inflammation suppression. The results showed that genes whose expression was increased by subacute inhalation exposure to 20 mg/m\u003csup\u003e3\u003c/sup\u003e CL-PAA and whose expression was decreased by the administration of NAC included genes related to subacute inflammation and oxidative stress (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). KEGG pathway analysis of the extracted genes revealed pathways related to innate immunity, such as phagosome and Toll-like receptor signaling, and pathways related to cytokines. Pathways related to innate immunity and cytokines have also been extracted in other animal models of particle exposure. The pathway for phagosome was extracted from the lung tissue of mice exposed to PM2.5 inhalation [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e], and pathways for cytokine-cytokine receptor interaction and viral protein interaction with cytokine and cytokine receptor were extracted from the lung tissue of rats intratracheally instilled with lipopolysaccharide (LPS) [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. Related to the above, in our previous intratracheal instillation study of another CL-PAA we extracted cytokine-related pathways, such as cytokine-cytokine receptor interaction and chemokine signaling pathway, by KEGG pathway analysis of genes obtained from lung tissue [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]; our findings suggested that the inflammation and fibrosis induced by oxidative stress due to CL-PAA exposure are associated with particle phagocytosis and cytokines.\u003c/p\u003e\n\n"},{"header":"Conclusions","content":"\u003cp\u003eWe performed 5-day and 13-week inhalation exposure studies in rats to examine the acute and chronic pulmonary injury potential of CL-PAA. Inhalation exposure for 5 days induced concentration-dependent inflammation in the rats' lungs. Inhalation exposure for 13 weeks induced persistent inflammation and fibrosis in the rats' lungs. Based on the results of the subchronic inhalation exposure study, we determined that the no observed adverse effect level (NOAEL) for CL-PAA for chronic pulmonary damage, such as inflammation and fibrosis, was 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e. Furthermore, oxidative stress was associated with pulmonary inflammation caused by inhalation exposure to CL-PAA, and the administration of NAC attenuated pulmonary inflammation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACGIH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmerican Conference of Governmental Industrial Hygienists\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanalysis of variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBALF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebronchoalveolar lavage fluid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCeO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecerium dioxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCINC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytokine-induced neutrophil chemoattractant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCL-PAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecross-linked polyacrylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAVID\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDatabase for Annotation,Visualization and Integrated Discovery\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme-linked immune-sorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eepithelial-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eextracellular signal-regulated kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egel permeation chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHO-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eheme oxygenase-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLPS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elipopolysaccharide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emass median aerodynamic diameter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMn\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enumber average molecular weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emasson trichrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMw\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eweight average molecular weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMWCNT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emultiwalled carbon nanotubes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN-acetylcysteine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutrophil extracellular trap\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor-kappa B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNiO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enickel oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNOAEL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eno observed adverse effect level\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNrf2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor-erythroid 2-related factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOECD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOrganisation for Economic Co-operation and Development\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyacrylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolydispersity index\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymorphonuclear leukocyte\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradius of gyration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003escanning electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eshort-term inhalation studies\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTdT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eterminal deoxynucleotidyl transferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTiO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etitanium dioxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethreshold limit value\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eT-PER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etissue protein extraction reagent\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTWA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etime-weighted-average\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole Transcript\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eZnO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ezinc oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures and animal handling procedures were performed 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: AE18-003).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required as no human data presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Industrial Disease Clinical Research Grants from the Ministry of Health, Labour, and Welfare (Grant number 200101-01) and\u0026nbsp;JSPS KAKENHI Grant Number 22K10499.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors YH, CN, TT, HI, KY (Kazuhiro Yatera), and YM were responsible for the study design and writing of the manuscript. Authors YH, CN, TT, HI, KS (Kazuma Sato), NK, TM, KY (Kei Yamasaki), KW, HH, AM, JT, TK, KS (Kazuo Sakurai), KY (Kazuhiro Yatera), and YM were responsible for the data and analysis. Authors YH, CN, TT, HI, KS (Kazuma Sato), NK, TM, HH, TK, KS (Kazuo Sakurai), and YM performed the experiments. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Sumiyo Kuramoto, Tomoko Watanabe, Rika Takai, Mayumi Tashiro, Yuno Ariyoshi, Kumiko Matsuyama, and Tomoko Morimoto for their technical support with the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1) Department of Respiratory Medicine, University of Occupational and Environmental Health, Japan.\u0026nbsp;1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan\u003c/p\u003e\n\u003cp\u003e2) Department of Occupational Pneumology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan.\u0026nbsp;1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan\u003c/p\u003e\n\u003cp\u003e3) Department of Environmental Health Engineering, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan\u003c/p\u003e\n\u003cp\u003e4)\u0026nbsp;Shared-Use Research Center, School of Medicine, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan\u003c/p\u003e\n\u003cp\u003e5)\u0026nbsp;Research Institute of Science for Safety and Sustainability, National Institute of Advanced\u0026nbsp;Industrial Science and Technology (AIST), Tsukuba, Japan. 16-1 Onogawa, Tsukuba, Ibaraki,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e305-8569, Japan\u003c/p\u003e\n\u003cp\u003e6) Department of Chemistry and Biochemistry, The University of Kitakyushu. 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen X, Huang W, He B, Zhang Y. Synthesis and Application of Tackifying Dispersant Poly (Vinyl Alcohol-Acrylic Acid-Triallyl Cyanate). Polym (Basel). 2022;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark S, Lee K, Lee EJ, Lee SY, In KH, Kim H-K, et al. Humidifier disinfectant-associated interstitial lung disease in an animal model induced by polyhexamethylene guanidine aerosol. Am J Respir Crit Care Med. 2014;190:706\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKishimoto T, Okamoto K, Koda S, Ono M, Umeda Y, Yamano S et al. Respiratory disease in workers handling cross-linked water-soluble acrylic acid polymer. PLoS ONE. 2023;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishida C, Tomonaga T, Izumi H, Wang KY, Higashi H, Ishidao T et al. Inflammogenic effect of polyacrylic acid in rat lung following intratracheal instillation. Part Fibre Toxicol. 2022;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHigashi Y, Morimoto Y, Nishida C, Tomonaga T, Izumi H, Wang KY et al. Pulmonary disorder induced by cross-linked polyacrylic acid. J Occup Health. 2022;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishida C, Izumi H, Tomonaga T, Wang KY, Higashi H, Takeshita JI et al. Effect of Different Molecular Weights of Polyacrylic Acid on Rat Lung Following Intratracheal Instillation. Int J Mol Sci. 2022;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomonaga T, Nishida C, Izumi H, Kawai N, Wang KY, Higashi H et al. Crosslinked Structure of Polyacrylic Acid Affects Pulmonary Fibrogenicity in Rats. Int J Mol Sci. 2022;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorimoto T, Izumi H, Tomonaga T, Nishida C, Kawai N, Higashi Y et al. The Effects of Endoplasmic Reticulum Stress via Intratracheal Instillation of Water-Soluble Acrylic Acid Polymer on the Lungs of Rats. Int J Mol Sci. 2024;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHigashi Y, Nishida C, Tomonaga T, Izumi H, Kawai N, Morimoto T et al. Intratracheal instillation of polyacrylic acid induced pulmonary fibrosis with elevated transforming growth factor-β1 and connective tissue growth factor. Toxicology. 2024;506.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8:958\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArora S, Dev K, Agarwal B, Das P, Syed MA. Macrophages: Their role, activation and polarization in pulmonary diseases. Immunobiology. 2018;223:383\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen PJ, Chen SH, Chen YL, Wang YH, Lin CY, Chen CH et al. Ribociclib leverages phosphodiesterase 4 inhibition in the treatment of neutrophilic inflammation and acute respiratory distress syndrome. J Adv Res. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamada A, Aki T, Unuma K, Funakoshi T, Uemura K. Paraquat induces epithelial-mesenchymal transition-like cellular response resulting in fibrogenesis and the prevention of apoptosis in human pulmonary epithelial cells. PLoS ONE. 2015;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson N. The limitations of inhalation, intratracheal, and intracoelomic routes of administration for identifying hazardous fibrous materials. Fiber Toxicol. 1993;D:43\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandsiedel R, Ma-Hock L, Hofmann T, Wiemann M, Strauss V, Treumann S et al. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part Fibre Toxicol. 2014;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOECD/OCDE 413 OECD GUIDELINES FOR THE TESTING OF CHEMICALS 90-DAY. (SUBCHRONIC) INHALATION TOXICITY STUDY. 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988;41:467\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026uuml;bner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, et al. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 2008;44:507\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa-Hock L, Strauss V, Treumann S, K\u0026uuml;ttler K, Wohlleben W, Hofmann T et al. Comparative inhalation toxicity of multi-wall carbon nanotubes, graphene, graphite nanoplatelets and low surface carbon black. Part Fibre Toxicol. 2013;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandsiedel R, Ma-Hock L, Hofmann T, Wiemann M, Strauss V, Treumann S et al. Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials. Part Fibre Toxicol. 2014;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, Van Ravenzwaay B, et al. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance. Inhal Toxicol. 2009;21:102\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarheit DB, Mchugh TA. Hartsky1 MA. Differential pulmonary responses in rats inhaling crystalline, colloidal or amorphous silica dusts. Scand J Work Environ Health. 1995;21:19\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanda O, Naito Y, Yoshikawa T. Rat Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) in Inflammation. J Clin Biochem Nutr. 2006;38:51\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomonaga T, Izumi H, Oyabu T, Lee BW, Kubo M, Shimada M, et al. Assessment of cytokine-induced neutrophil chemoattractants as biomarkers for prediction of pulmonary toxicity of nanomaterials. Nanomaterials. 2020;10:1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakashima K, Sato T, Shigemori S, Shimosato T, Shinkai M, Kaneko T. Regulatory role of heme oxygenase-1 in silica-induced lung injury. Respir Res. 2018;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorie M, Yoshiura Y, Izumi H, Oyabu T, Tomonaga T, Okada T et al. Comparison of the pulmonary oxidative stress caused by intratracheal instillation and inhalation of NiO nanoparticles when equivalent amounts of NiO are retained in the lung. Antioxidants. 2016;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePauluhn J. Subchronic 13-week inhalation exposure of rats to multiwalled carbon nanotubes: Toxic effects are determined by density of agglomerate structures, not fibrillar structures. Toxicol Sci. 2009;113:226\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwotzer D, Ernst H, Schaudien D, Kock H, Pohlmann G, Dasenbrock C et al. Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats. Part Fibre Toxicol. 2017;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorimoto Y, Oyabu T, Horie M, Kambara T, Izumi H, Kuroda E, et al. Pulmonary toxicity of printer toner following inhalation and intratracheal instillation. Inhal Toxicol. 2013;25:679\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBermudez E, Mangum JB, Wong BA, Asgharian B, Hext PM, Warheit DB, et al. Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol Sci. 2004;77:347\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenriquez AR, Snow SJ, Dye JA, Schladweiler MC, Alewel DI, Miller CN et al. The contribution of the neuroendocrine system to adaption after repeated daily ozone exposure in rats. Toxicol Appl Pharmacol. 2022;447.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarrell BP, Kerr HD, Kulle TJ, Sauder LR, Young JL. Adaptation in human subjects to the effects of inhaled ozone after repeated exposure. Am Rev Respir Dis. 1979;119:725\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWesselkamper SC, Chen C, Gordon T. Development of Pulmonary Tolerance in Mice Exposed to Zinc Oxide Fumes. Toxicol Sci. 2001;60:144\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFine JM, Gordon T, Chen LC, Kinney P, Falcone G, Sparer J, et al. Characterization of clinical tolerance to inhaled zinc oxide in naive subjects and sheet metal workers. J Occup Environ Med. 2000;42:1085\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartins V, Cruz Minguill\u0026oacute;n M, Moreno T, Querol X, de Miguel E, Capdevila M, et al. Deposition of aerosol particles from a subway microenvironment in the human respiratory tract. J Aerosol Sci. 2015;90:103\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJassal M, Bajaj P. Developments in acrylic - based thickeners as substitute of emulsion thickeners for pigment printing. Indian J Fibre Text Reseach. 2001;26:143\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoriuchi T. Physico-Chemical Properties of Water Soluble Polymers in Aqueous Solution. J Surf Finish Soc Jpn. 2009;60:746\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKerksick C, Willoughby D. The Antioxidant Role of Glutathione and N-Acetyl-Cysteine Supplements and Exercise-Induced Oxidative Stress. J Int Soc Sports Nutr. 2005;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. CMLS, Cell Mol Life Sci. 2003;60:6\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŠalamon Š, Kramar B, Marolt TP, Poljšak B, Milisav I. Medical and dietary uses of n-acetylcysteine. Antioxidants. 2019;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMansour HH, Omran MM, Hasan HF, El kiki SM. Modulation of bleomycin-induced oxidative stress and pulmonary fibrosis by N-acetylcysteine in rats via AMPK/SIRT1/NF-κβ. Clin Exp Pharmacol Physiol. 2020;47:1943\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa F, Luo S, Lu C, Jiang X, Chen K, Deng J et al. The role of Nrf2 in periodontal disease by regulating lipid peroxidation, inflammation and apoptosis. Front Endocrinol (Lausanne). 2022;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayes JD, McMahon M. NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci. 2009;34:176\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang M, Yang Z, Liu J, Zhang X, Guan L, Liu X et al. Combined intervention with N-acetylcysteine and desipramine alleviated silicosis development by regulating the Nrf2/HO-1 and ASMase/ceramide signaling pathways. Ecotoxicol Environ Saf. 2022;242.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanda O, Naito Y, Takagi T, Shimozawa M, Kokura S, Yoshida N, et al. Tumor Necrosis Factor-α-Induced Cytokine-Induced Neutrophil Chemoattractant-1 (CINC-1) Production by Rat Gastric Epithelial Cells: Role of Reactive Oxygen Species and Nuclear Factor-κB. J Pharmacol Exp Ther. 2004;309:670\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Zhang W, Zhang F, Roepstorff P, Yang F, Lu Z et al. TMT-based quantitative proteomics analysis reveals airborne PM2.5-induced pulmonary fibrosis. Int J Environ Res Public Health. 2019;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, Luo J, Hu W, Ye C, Ren P, Wang Y et al. Whole Transcriptomic Analysis of Key Genes and Signaling Pathways in Endogenous ARDS. Dis Markers. 2022;2022.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"particle-and-fibre-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pftx","sideBox":"Learn more about [Particle and Fibre Toxicology](http://particleandfibretoxicology.biomedcentral.com)","snPcode":"12989","submissionUrl":"https://submission.nature.com/new-submission/12989/3","title":"Particle and Fibre Toxicology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cross-linked polyacrylic acid, inhalation exposure, oxidative stress, N-acetylcysteine","lastPublishedDoi":"10.21203/rs.3.rs-4787568/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4787568/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Organic polymers are used widely in daily necessities, foods, and medicines, but recent reports show that cross-linked polyacrylic acid (CL-PAA), an organic polymer, induces severe lung disease. Whereas most previous toxicity studies of CL-PAA were intratracheal instillation studies, we conducted inhalation exposure studies to corroborate those findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We conducted 5-day (subacute) and 13-week (subchronic) inhalation exposure studies using CL-PAA. In the subacute inhalation study, male F344 rats inhaled 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e, 2.0 mg/m\u003csup\u003e3\u003c/sup\u003e, or 20 mg/m\u003csup\u003e3\u003c/sup\u003e of CL-PAA for 6 hours/day for 5 days. Rats were sacrificed 3 days and 1 month after the exposure. In the subchronic inhalation study, male F344 rats inhaled 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e or 2.0 mg/ m\u003csup\u003e3\u003c/sup\u003e of CL-PAA for 6 hours/day for 5 days/week for 13 weeks. Rats were sacrificed from 3 days to 6 months after exposure. We also investigated the mechanism of pulmonary disorders by conducting an additional subacute inhalation exposure (CL-PAA 20 mg/m\u003csup\u003e3\u003c/sup\u003e) study, in which the antioxidant N-acetylcysteine (NAC) (200 mg/kg) was injected intraperitoneally. The rats in that study were sacrificed on the day after the exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Subacute inhalation exposure resulted in concentration-dependent increases in neutrophil influx, total protein, lactate dehydrogenase (LDH), cytokine-induced neutrophil chemoattractant (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in lung tissue. Histopathological findings in the lung tissue also showed neutrophil infiltration in a concentration-dependent manner.\u003c/p\u003e\n\u003cp\u003eSubchronic inhalation exposure resulted in persistent increases in total protein in BALF and HO-1 in lung tissue. Histopathological findings in the lung tissue showed persistent neutrophil infiltration and fibrosis. NAC administration decreased the number of neutrophils, total protein, LDH, and CINC in the BALF, and HO-1 in the lung tissue. Histopathological findings of the lung tissue also showed that NAC administration improved neutrophil infiltration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Inhalation of CL-PAA for 5 days induced concentration-dependent inflammation in the rats' lungs. Inhalation of CL-PAA for 13 weeks induced persistent inflammation and fibrosis in the rats' lungs. We considered that the no observed adverse effect level (NOAEL) for chronic pulmonary disorders was 0.2 mg/m\u003csup\u003e3\u003c/sup\u003e. Furthermore, oxidative stress was associated with CL-PAA-induced pulmonary inflammation, and NAC administration attenuated the pulmonary inflammation.\u003c/p\u003e","manuscriptTitle":"Inhalation Exposure to Cross-linked Polyacrylic Acid Induces Pulmonary Disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-20 14:10:52","doi":"10.21203/rs.3.rs-4787568/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-08-02T02:45:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-23T21:01:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Particle and Fibre Toxicology","date":"2024-07-23T09:47:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"particle-and-fibre-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pftx","sideBox":"Learn more about [Particle and Fibre Toxicology](http://particleandfibretoxicology.biomedcentral.com)","snPcode":"12989","submissionUrl":"https://submission.nature.com/new-submission/12989/3","title":"Particle and Fibre Toxicology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9815935e-15c8-4eb3-a719-ea44292aa825","owner":[],"postedDate":"August 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T11:57:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-20 14:10:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4787568","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4787568","identity":"rs-4787568","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}