The Degree of Cross-linking of Polyacrylic Acid Affects the Fibrogenicity in Rat Lungs

The Degree of Cross-linking of Polyacrylic Acid Affects the Fibrogenicity in Rat Lungs | 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 Article The Degree of Cross-linking of Polyacrylic Acid Affects the Fibrogenicity in Rat Lungs Taisuke Tomonaga, Hiroto Izumi, Chinatsu Nishida, Kazuma Sato, and 12 more This is a preprint; it has not been peer reviewed by a journal.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-4704450/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Background : Polyacrylic acid (PAA) with different concentrations of cross-linker was instilled into the trachea of ​​rats to examine the effect of PAA crosslink density on lung disorders. Methods: F344 rats were intratracheally exposed to low and high doses of PAA with cross-linker concentrations of 0.1, 1.0, and 5.0% (CL0.1%, CL1.0%, and CL5.0%, respectively). Rats were sacrificed at 3 days, 1 week, 1 month, 3 months, and 6 months after exposure. Results: PAA with different cross-linker concentrations caused an increase in neutrophil influx, cytokine-induced neutrophils, and chemotactic factor (CINC) in bronchoalveolar lavage fluid (BALF) from 3 days to 1 week after instillation. Lactate dehydrogenase (LDH) activity in BALF and heme oxygenase-1 (HO-1) release in lung tissue were higher in the CL0.1% exposure group during the acute phase. Lung histopathological findings also showed that severe fibrotic changes induced by CL0.1% were greater than those observed in CL1.0% and CL5.0% exposure during the observation period. Conclusions: CL0.1% was associated with more severe lung fibrosis, and a decrease in lung fibrosis was observed with increasing cross-linker concentrations, suggesting that the cross-link density of PAA is a physicochemical feature that affects lung disorders. Health sciences/Diseases/Respiratory tract diseases Physical sciences/Chemistry/Organic chemistry Polyacrylic acid Cross-link Intratracheal instillation pulmonary toxicity Fibrosis rat Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background The types of respirable dust are broadly categorized into inorganic and organic substances. Pneumoconiosis, a well-known respiratory disease caused by respirable dust, is caused mainly by inorganic substances such as silica and asbestos, and is known to cause chronic inflammation, emphysematous changes fibrosis, and even lung cancer [ 1 , 2 ]. Organic substances, on the other hand, cause allergic diseases such as bronchial asthma and hypersensitivity pneumonitis, but they have not been thought to cause pulmonary fibrosis such as pneumoconiosis. In recent years, however, it has been reported that organic substances cause inflammation and fibrosis. In South Korea, there were many cases of progressive interstitial pneumonia among people who used disinfectants in humidifiers, which became a social problem [ 3 , 4 ]. In the United States, there were reports of e-cigarette users who developed acute respiratory failure [ 5 , 6 ]. In Japan, inflammation and fibrosis of interstitium around the airways have been reported in workers who handle water-absorbing cross-linked acrylic acid-based polymer compounds, which are used as intermediates for various products in daily life, such as diapers, cosmetics, shampoos, cosmetics, and food additives [ 7 – 10 ]. In the case of those workers, fibrosis progressed quickly, about two years after exposure to acrylic acid-based polymers, so the progression was faster than in lung damage caused by asbestos and crystalline silica, which are typical inorganic substances [ 7 – 9 ]. In animal exposure examinations, intratracheal instillation and inhalation exposure have been conducted with this acrylic acid-based polymer, and it has been reported that it causes lung disorders, including rapidly progressing fibrosis, similar to human cases [ 9 , 11 , 12 ]. In spite of these lines of evidence, however, the pathogenesis of lung disorders caused by acrylic acid-based polymers has not been elucidated. PolyAcrylic acid (PAA) is composed of repeating chemical structural formulas of acrylic acid monomers, and the linear repeating structures of the monomers are intricately entangled. A cross-linker is used to cause the cross-linking of multiple linear molecules through covalent bonds. The cross-linking becomes more polymerized and forms a three-dimensional structure with complex meshes, and as the molecular weight and cross-linked structure increase, thickening and swelling by absorbing water are caused [ 13 – 15 ]. It is thought that the thickening and swelling properties associated with water absorption cause lung disorders. We have previously conducted intratracheal installations on rats using PAAs with different molecular weights and cross-linked structures [ 16 , 17 ]. When evaluating the effects of the presence or absence of a cross-linked structure, it was observed that PAAs with a cross-linked structure caused higher lung injury and fibrogenicity than linear PAAs without a cross-linked structure [ 16 ]. This cross-linked structure is thought to be involved in biological effects since it is considered that water absorption and thickening properties change in a complicated manner by changing the density of the cross-linked structure. In order to investigate the effects of PAAs with different cross-linking densities on lung disorders, we synthesized three types of PAAs with different cross-linking densities by varying the concentration of cross-linker and intratracheally instilling them into rats. Results Characterizations of polyacrylic acid with different degrees of cross-linking The fundamental characteristics of different cross-linking concentrations of PAA are summarized in Table 1 . Figure 1 shows the scanning electron microscopy (SEM) by HITACHI S-4500 (Hitachi, Ltd., Tokyo, Japan) of the powder of the PAAs (Fig. 1 ). We examined the fundamental characteristics of the PAAs by preparing the molecular dispersion as follows: the polymers were dissolved in 0.1 M carbonate-bicarbonate buffer, then the solutions were alkalized with 2N NaOH and then neutralized with 1N HCl. The PAA without a cross-linking reagent in the same synthetic process had an average molecular weight (MW) of 50.4×10 5 as measured by gel permeation chromatography (GPC) (a Prominence 501 system coupled with Dawn-Heleos-Ⅱ(Wyatt Technology Europe GmbH, Dernbach, Rheinland-Pfalz, Germany)) using GF-7MHQ (Showa Denko K.K., Tokyo, Japan) with 0.1M carbonate-bicarbonate buffer as the eluent [ 18 , 19 ]. The PAAs with cross-linking could not be measured, however, because the molecular weight was too large. On the other hand, the radius of gyration (Rg) in molecular dispersion measured by GPC was increased in a cross-linker concentration-dependent manner. Rg indicates the extent of a polymer, which is influenced by its molecular weight and polymer structure. Body and lung weights Body and lung weights increased dose-dependently at each observation point in all of the PAA-exposure groups (Figure. 2AB). There was a significant increase in body weight in CL0.1% and CL1.0% at 3 days or 1 week after exposure (Figure. 2A). A significant increase in lung weights was sustained throughout the observation period in all of the PAA-exposure groups compared to the control group (Figure. 2B). During the acute phase, there was less lung swelling in the high-dose group in a cross-linking-concentration dependent manner (Figure. 2C). Cell analysis and cell injury markers in bronchoalveolar lavage fluid (BALF) The results of inflammatory cell counts in BALF are shown in Fig. 3 . There were significant increases in the number of total cells in all of the 1 mg-exposure groups from 3 days to 1 month after exposure compared to each control group, and a significant increase in the number of total cells was observed until 3 months after exposure in the CL0.1% and CL5.0% -high exposure groups (Fig. 3 A). The number and percentage of neutrophils were significantly increased from 3 days to 1 week after exposure in CL0.1% and CL1.0%, and the exposure to CL5.0% induced a persistent increase until 1 month (Fig. 3 B, 3 C). In the acute phase, the increase in inflammatory cells in BALF was similar, regardless of the degree of cross-linking of PAA, but there was a difference in the persistence of increased inflammatory cells in BALF in CL0.1%, CL1.0%, and CL5.0%. Measurements of inflammatory markers in BALF and lung tissue The concentrations of cytokine-induced neutrophil chemoattractant (CINC) -1 and CINC-2 in BALF, and the concentrations of heme oxygenase (HO)-1 in lung tissue following the intratracheal instillation of PAAs are shown in Fig. 4 . CINC-1 and CINC-2 were increased in the exposure groups from 3 days to 1 week after exposure. HO-1 in lung tissue showed a tendency of significant increase in the 1.0 mg exposure group until 3 months after exposure in CL0.1% and CL5.0%, but at 1 month and 3 months after exposure HO-1 was more increased in CL5.0% than in CL0.1%. Cell injury markers in BALF The results of lactate dehydrogenase (LDH) activity and total protein in BALF are shown in Fig. 5 . The results of LDH activity and the concentration of total protein in all of the exposure groups also showed tendencies of increase from 3 days to 1 month after exposure compared to each control group. The increase of the LDH activity was highest in CL0.1% and lowest in CL5.0%, showing cross-linking concentration-dependent changes (Fig. 5 A). The increase in total protein concentration also tended to be lowest in CL5.0% (Fig. 5 B). Histopathological features in the lung Representative histopathological findings of the lungs at 3 days, 1 month, and 6 months after exposure to PAA with different cross-linking concentrations are shown in Fig. 6 . There was inflammatory cell infiltration, mainly by neutrophils, into the alveoli, which was evident in a dose-dependent manner at 3 days after exposure. Inflammatory changes with fibrosis were observed from 3 days to 1 week after exposure, suggesting severe lung disorders. In particular, inflammatory cell aggregation was more pronounced in the 1.0 mg CL0.1% and CL1.0% exposure groups than in the CL5.0% group (Fig. 6 A). In the evaluation by inflammation score, a dose-dependent increase in the score was observed in CL0.1%, CL1.0%, and CL5.0%. The CL0.1% 1.0 mg exposure group tended to have a higher score than the CL5.0% group (Fig. 6 B). On the other hand, the inflammation score in the CL5.0% 1.0 mg exposure group was persistently increased compared to the CL0.1% and CL1.0% groups. Fibrosis in the CL0.1% 1.0 mg group, as compared to CL1.0% and CL5.0%, was the most severe and progressed the fastest, and the extent of fibrosis decreased in a cross-linker concentration-dependent manner (Fig. 7 A). Ashcroft score assessment showed a dose-dependent increase in scores with PAA exposure. The CL0.1% 1.0 mg exposure group had a higher score than the CL1.0% and CL5.0% groups (Fig. 7 B). The relationship between lung fibrosis and lung injury The relationship between pulmonary fibrosis and lung injury is shown in Fig. 8 . The data used is from 3 days to 1 month after exposure. LDH activity and total protein concentration as lung injury markers showed good correlations with the Ashcroft score, a fibrosis marker, suggesting that lung injury is related to the progression of fibrosis. Discussion In this study, we used PAAs synthesized with different concentrations of cross-linkers, and intratracheal instillation was conducted on rats. Intratracheal instillation was used to evaluate the pulmonary toxicity of these PAAs. We estimated the human exposure equivalent to the maximum dose of 1 mg in this study by using the following formula that had been used previously [ 20 ]: (amount of PAA) = (exposure concentration of particle) × (tidal volume) × (breathing frequency)× (exposure hours in a day) × (particle deposition fraction) Assuming that the lung deposition rate (0.1) of PAA is the same for humans and rats if humans were exposed to the TLV-TWA (Threshold Limit Value - Time-Weighted Average from American Conference of Governmental Industrial Hygienist; ACGIH) level of general dust (3 mg/m 3 ), this would correspond to an exposure period of approximately 926 days (calculation in rat and human under assumption of tidal volume 2.1 and 625 mL/times; breathing frequency volume 102 and 12 times/min; and exposure hours in day 6 hours (lung weights of rats and humans are set at 1 g and 1000 g, respectively)). Assuming that a worker continued to work under conditions of 3 mg/m 3 (8 hours a day, 5 days a week), a high dose of 1 mg instilled into the trachea would be equivalent to an exposure period of approximately 3.6 years. In actual worker cases, lung disorder has been reported after exposure to acrylic acid-based polymers for more than two years [ 8 ]; we considered that the level of exposure in the present study corresponded to the real environment. In this study, pulmonary inflammation progressed to severe fibrosis sooner with CL0.1% than with CL1.0% or CL5.0%. Considering that inflammation leads to subsequent fibrosis in lung disorders caused by inorganic substances [ 21 , 22 ], it is thought that PAAs have a strong fibrogenicity after inflammation. The development of fibrosis after acute inflammation has been observed in animal exposure studies and clinical manifestations, and is common in LPS exposure models that induce acute inflammation and in ARDS with acute respiratory failure [ 23 – 25 ]. Acute respiratory failure in animal models due to LPS or bleomycin causes severe inflammation of the lungs that ends in 1–2 weeks and causes fibrosis without long-term inflammation [ 24 , 25 ]. In the worker cases, fibrosis was observed about two years after the start of exposure to an acrylic acid-based polymer [ 10 ] and it progressed faster than with asbestos or silica. Considering that acrylic acid-based polymers used in products often demand water-soluble applications and the cross-linkers are generally used at a concentration of about 0.1%, the early observation of fibrosis in the CL0.1% in this study is not inconsistent with the pathology in humans. On the other hand, fibrosis progressed only mildly with the CL5.0%, but inflammation tended to persist. Considering that persistent inflammation leads to subsequent fibrosis in lung disorders caused by inorganic substances [ 20 , 26 ], there is a possibility that, in the long term, fibrosis may progress or lead to irreversible lesions such as tumors. The inflammatory and fibrotic potential of the CL5.0% may be lower than that of CL0.1% and CL1.0%, but it is considered to be high among general inhalable chemicals. The relationship between the crosslink density of the PAAs and fibrosis is considered to be as follows. It is known that the water absorption of PAA increases when the polymer has a cross-linked structure, but the water absorption decreases when the crosslink density increases beyond a certain level [ 14 , 27 , 28 ]. The water absorbency of PAA may cause biological hyperosmotic stress on an organism. Hyperosmotic stress on an organism is reportedly associated with various pathological conditions such as induction of inflammatory cytokines and apoptosis [ 29 – 32 ]. Schwartz et al. reported that when three epithelial cell lines (colorectal HT29, bladder T24, and lung A549) were exposed to glycol-derived compounds, the presence of hyperosmolar concentrations induced significant production of the proinflammatory cytokines IL-6, IL-8, TNF-α, and IL-1β in all cell lines [ 31 ]. Singh and Ramarao also reported that the culture medium of RAW cells with mannitol induced cell death in an osmotic pressure-dependent manner [ 32 ]. In our study, Fibrosis was most severe at 0.1% CL, which is considered to be the highest water absorbency and stress, and fibrosis potential was reduced as the cross-linker concentration increased. Considering that a previous comparison of the pulmonary fibrotic potential of non-cross-linked and cross-linked (0.1% or less cross-linker) PAAs showed stronger fibrosis in the cross-linked PAA [ 16 ], it is thought that the increased water absorption caused by the change in crosslink density causes fibrosis. Furthermore, in the acute phase of this study, the LDH activity and total protein concentration in BALF, which are indicators of lung injury, were most elevated in the CL0.1% and tended to be the lowest in the CL5.0%. There are reports that severe lung injury leads to the progression of fibrosis. It has been reported in inhalation exposure and intratracheal instillation of MWCNT in mice that the increase in LDH in BALF and the amount of MWCNT deposition and fibrosis level showed a similar tendency of increase [ 33 , 34 ]. In an intratracheal instillation of bleomycin in mice, the increase in LDH activity in BALF and the progression of fibrosis were observed in a dose-dependent manner [ 35 ]. In the present study, a correlation was observed between lung injury and fibrosis from 3 days to 1 month after instillation (Fig. 8 ), so it is possible that the CL0.1%, which caused the highest lung injury, may have progressed to fibrosis early in the tissue repair process. As mentioned above, considering that the increased water absorption caused by changes in crosslink density leads to fibrosis, it is speculated that lung injury due to increased water absorption contributed to the progression of fibrosis (Fig. 9 ). In this study, we analyzed the effects of PAAs with different cross-linking densities on the lungs by varying the cross-linker concentration. CL0.1% tended to cause the strongest fibrosis in the acute phase, and a decrease in pulmonary fibrogenicity was observed as the cross-linker concentration was increased. It is thought that the water absorbency of PAA influences lung disorders since fibrogenicity decreases with increasing cross-linking concentration. Taken together, it is suggested that the crosslink density of PAA is a physicochemical feature that influences lung disorders. Methods Sample polymer Using a previous method [ 36 ], we synthesized a sample of PAA by polymerization of the acrylic acid monomer adding different cross-linker concentrations (0.1 mol%, 1.0 mol%, and 5.0 mol%) and labeled them CL0.1%, CL1.0%, and CL5.0%, respectively. The details of the preparations are described in the Supplementary Information. PAA, a white, easily scattered powder, was mixed with distilled water and slowly stirred for 40 minutes (Mag-Mixer MF820 or MD300, Yamato Scientific Co., Ltd., Tokyo, Japan). Animals Male Fischer 344 rats (8 weeks old) (The Jackson Laboratory Japan, Inc., Kanagawa, Japan) were acclimated for 4 weeks at the Laboratory Animal Research Center of the University of Occupational and Environmental Health, Japan with commercial food and water available ad libitum. All procedures and animal handling were performed under the guidelines described in the "Japanese Guide for the Care and Use of Laboratory Animals" and approved by the Animal Experiment Committee of the University of Occupational and Environmental Health, Japan (Animal Research Ethics Approval Proposal No.; AE17-009). All methods were performed in accordance with the relevant guidelines ( https://arriveguidelines.org ). Intratracheal instillation Doses of 0.2 mg (0.8 mg/kg BW) and 1.0 mg (4.0 mg/kg BW) of PAA suspended in 0.4 mL of distilled water at different cross-linker concentrations were administered by single intratracheal instillation into the lungs of rats (12 weeks old). Rats were instilled intratracheally under sevoflurane (Viatris Inc., Canonsburg, PA, USA) inhalation anesthesia. Briefly, the laryngeal extension was performed using a laryngoscope blade (MAC1, Rudolf Riester GmbH, Jungingen, Germany), and an animal feeding needle (KN-348, Natsume Seisakusho Co., Ltd., Tokyo, Japan) was inserted directly into the trachea and the suspension was instilled manually. Next, 3 mL of air was inserted into the trachea twice with a syringe from the animal feeding needle. The rats were then spontaneously awakened and observed periodically. Single intratracheal instillations of 0.2 mg and 1.0 mg of PAA at different cross-linker concentrations were administered at different times, for a total of three single intratracheal instillations. A control group was established for each intratracheal instillation, with distilled water administered to the control group; doses of 0.2 mg and 1.0 mg per rat were used for the PAA intratracheal instillation. These maximum doses were set to avoid overloading the lungs in anticipation of human exposure [ 11 ]. Animals following intratracheal instillation There were five rats in each exposure and control group at each time point. Animals were dissected at 3 days, 1 week, 1 month, 3 months, and 6 months after intratracheal instillation under isoflurane (Viatris Inc., Canonsburg, PA, USA) inhalation anesthesia. Body weight was measured, then blood was removed from the heart at autopsy. Lungs were extracted from the body, lung weights were measured, and the lungs were perfused with saline solution. With the left main bronchus clamped, the right lung was repeatedly inflated with saline at a pressure of 20 cm H 2 O after two fluid collections. Seven to 14 mL of the fluid (BALF) was collected in collection tubes by free fall, and then the right and left lungs were separated. The homogenized third lobe of the right lung after BALF collection was used for measurement of HO-1. The left lung was inflated and fixed with 10% formaldehyde under pressure of 25 cm H 2 O for histopathological evaluation. Cytospin analysis of inflammatory cells and measurement of inflammation-related markers in BALF The BALF was centrifuged at 400 g for 15 min at 4°C, and the supernatant was transferred to a new tube for determination of total protein, lactate dehydrogenase (LDH) and cytokines. Pellets were washed in polymorphonuclear leukocyte (PMN) Buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 and 5.6 mM C 6 H 12 O 6 ) in suspension and centrifuged at 400 g at 4°C for 15 minutes. After the removal of the supernatant, the pellet was resuspended in 1 mL of PMN buffer. Cell counts in BALF were measured with ADAM-MC (AR BROWN CO., LTD., Tokyo, Japan), cells were spread on glass slides with cytospin, fixed, stained with Diff-Quik (Sysmex CO., Kobe, Hyogo, Japan), and the number of neutrophils and alveolar macrophages was measured by microscopic observation. LDH activities released into the BALF supernatant were measured with the Cytotoxicity Detection KitPLUS (LDH) (Roche Diagnostics GmbH, Mannheim, North Rhine-Westphalia, Germany) according to the manufacturer's instructions. LDH activity was estimated using a standard curve obtained from known concentrations of recombinant LDH derived from rabbit muscle (Oriental Yeast Co., ltd., Tokyo, Japan). Protein concentrations in BALF supernatants were determined using the Pierce™ 660 nm Protein Assay (Thermo Scientific Inc., Rockford, IL, USA). Ford, Illinois, USA). Measurement of chemokine in BALF and HO-1 in lung tissue Concentrations of CINC-1 and CINC-2 in BALF were measured with the ELISA kits #RCN100 and #RCN200 (R&D Systems, Minneapolis, MN, USA), respectively. All measurements were performed according to the manufacturer's instructions. The third lobe of the right lung was cultured in T-PER tissue containing protein inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA) and cOmplete Mini (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany), and homogenized in Protein Extraction Reagent (Thermo Scientific Inc., Rockford, IL, USA) and centrifuged (20,400 g at 4°C for 10 min). Protein concentrations in the supernatant were determined with Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific Inc., Rockford, IL, USA), using bovine serum albumin as a standard. HO-1 measurements with the Elisa kit ADI-EKS-810A (Enzo Life Sciences, Farmingdale, NY, USA) were corrected by the protein concentration in the supernatant to calculate the final HO-1 concentration in the lung tissue. Histopathology Formaldehyde-fixed lung tissue was embedded in paraffin, sectioned at a thickness of 4 µm, and then stained with hematoxylin and eosin (HE) and Masson trichrome (MT) staining. The lung inflammation and fibrosis were examined using the inflammatory cell infiltration score [ 37 ] and the Ashcroft score [ 38 ], respectively, according to previous reports [ 11 , 37 ]. Briefly, the inflammatory cell infiltration score was calculated by scoring the degree of inflammatory cell infiltration in the lung tissue as none (0), minimal (0.5), mild (1), moderate (2), or severe (3). The mean and standard deviation of the scores were calculated for each group. Pulmonary fibrosis was assessed by scoring histopathological findings in the lungs on a scale of 0 to 8 using the modified Ashcroft score, with the mean and standard deviation calculated for each group. Statistical analysis Statistical analysis was performed using IBM® SPSS® software (IBM Corporation, Chicago, IL, USA). p-values < 0.05 were determined to be statistically significant. The Dunnett and Tukey's honestly significant difference (HSD) tests were appropriately used to detect individual differences between individuals exposed to different cross-linker concentrations of PAA samples and the control group. Construct validity was measured using Spearman’s rank correlation coefficients (ρ) between the Ashcroft scores as lung fibrosis indicators and lung injury markers. Declarations Acknowledgments This work was supported by JSPS KAKENHI Grant Number JP21H04855. The authors would like to thank Sumiyo Kuramoto, Tomoko Watanabe, Rika Takai, Mayumi Tashiro, Yuno Ariyoshi, and Tomoko Morimoto for technical support with the experiments. Authors’ contributions TT: Writing – original draft, Validation, Methodology, Investigation, Conceptualization. HI: Methodology, Investigation, Conceptualization. CN: Validation, Methodology, Data curation. KS (Kazuma Sato): Visualization, Methodology. YN: Methodology, Formal analysis. TM: Methodology, Formal analysis. YH: Methodology, Formal analysis. KW: Methodology, Formal analysis. HH: Methodology, Formal analysis. TK: Methodology, Formal analysis. KS (Kazuo Sakurai): Writing – review & editing, Supervision, Validation, Conceptualization. JT: Methodology, Data curation. AM: Methodology, Data curation. KY (Kei Yamasaki): Methodology, Formal analysis. KY (Kazuhiro Yatera): Writing – review & editing, Supervision, Validation. YM: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Funding This work was supported by JSPS KAKENHI Grant Number JP21H04855. Availability of data and material The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All procedures and animal handling were done according to the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan (animal studies ethics clearance proposal number; AE17-009). All methods were also performed in accordance with the relevant guidelines (https://arriveguidelines.org). Consent for publication Not required as no human data is presented. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author details 1 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. 2 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. 3 Department of Respiratory Medicine, 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 Department of Chemistry and Biochemistry, The University of Kitakyushu. 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan. 6 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 References Leung CC, Yu ITS, Chen W. 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Differential expression of EC-SOD, Mn-SOD and CuZn-SOD in rat lung exposed to crystalline silica. J Occup Health. 2007;49:242–8. MARSHALL R, BELLINGAN G, LAURENT G. The acute respiratory distress syndrome: fibrosis in the fast lane. Thorax. 1998;53:815–7. Kim S-N, Lee J-S, Yang H-S, Cho J-W, Kwon S-J, Kim Y-B, et al. Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicol Res. 2010;26:217–22. de Souza Xavier Costa N, Ribeiro Júnior G, dos Santos Alemany AA, Belotti L, Zati DH, Frota Cavalcante M, et al. Early and late pulmonary effects of nebulized LPS in mice: An acute lung injury model. Palaniyar N, editor. PLoS One. 2017;12:e0185474. Morimoto Y, Izumi H, Yoshiura Y, Tomonaga T, Lee B-W, Okada T, et al. Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles. Nanotoxicology. 2016;10:607–18. Yang J, Liang W, He X, Su Y, Wang F, Wang T, et al. Experimental Synthesis of Polyacrylic-Type Superabsorbent Polymer and Analysis of Its Internal Curing Performances. Fluid Dyn Mater Process. 2022;18:15–27. M.J.A.D. ZM, Kabiri K. Superabsorbent Polymer Materials: A Review. 2008. Available from: https://api.semanticscholar.org/CorpusID:55246346 Brocker C, Thompson DC, Vasiliou V. The role of hyperosmotic stress in inflammation and disease. Biomol Concepts. 2012;3:345–64. Kültz D. Hyperosmolality triggers oxidative damage in kidney cells. Proc Natl Acad Sci. 2004;101:9177–8. Schwartz L, Guais A, Pooya M, Abolhassani M. Is inflammation a consequence of extracellular hyperosmolarity? J Inflamm. 2009;6:21. Singh RP, Ramarao P. Accumulated Polymer Degradation Products as Effector Molecules in Cytotoxicity of Polymeric Nanoparticles. Toxicol Sci. 2013;136:131–43. Porter DW, Hubbs AF, Chen BT, McKinney W, Mercer RR, Wolfarth MG, et al. Acute pulmonary dose–responses to inhaled multi-walled carbon nanotubes. Nanotoxicology. 2012;7:1179–94. Snyder-Talkington BN, Dong C, Porter DW, Ducatman B, Wolfarth MG, Andrew M, et al. Multiwalled carbon nanotube-induced pulmonary inflammatory and fibrotic responses and genomic changes following aspiration exposure in mice: A 1-year postexposure study. J Toxicol Environ Heal Part A. 2016;79:352–66. Kim S-N, Lee J-S, Yang H-S, Cho J-W, Kwon S-J, Kim Y-B, et al. Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicol Res. 2010;26:217–22. Takano S, Ono R, Sakurai K. A surprisingly narrow particle size distribution for polyacrylic acid nanospheres produced by precipitation polymerization and revealed by small-angle X-ray scattering. Polym J. 2023;55:1387–91. Nishida C, Izumi H, Tomonaga T, Takeshita J, Wang K-Y, Yamasaki K, et al. Predictive Biomarkers for the Ranking of Pulmonary Toxicity of Nanomaterials. Nanomaterials. 2020;10:2032. Hübner R-H, Gitter W, Eddine El Mokhtari N, Mathiak M, Both M, Bolte H, et al. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 2008;44:507–17. Additional Declarations No competing interests reported. Supplementary Files Additionalfile.docx Additional file Figure S1. Overview of the synthesis of PAA with different cross-linker concentrations. Acrylic acid was used as a monomer. Ethylene glycol diacrylate, a bifunctional monomer, was used as a cross-linker (concentration: 0.1 mol%, 1.0 mol%, 5.0 mol%). Acrylic acid monomer and α,α'-Azobisisobutyronitrile (AIBN, KANTO CHEMICAL CO., INC.) were dissolved in a mixture of Ethyl acetate and Ethylene carbonate (mixture ratio 7:3) and bubbled with nitrogen. The mixture was bubbled with nitrogen for 30 minutes. The reaction was then carried out at 60°C for 3 hours to obtain spherical PAA particles. The resulting particles were purified by centrifugation in acetonitrile three times. After that, the solvent was removed by vacuum drying to obtain cross-linked PAA particles. Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Nov, 2024 Reviews received at journal 29 Oct, 2024 Reviews received at journal 19 Oct, 2024 Reviewers agreed at journal 09 Oct, 2024 Reviewers agreed at journal 09 Oct, 2024 Reviewers invited by journal 09 Oct, 2024 Editor assigned by journal 03 Oct, 2024 Editor invited by journal 27 Sep, 2024 Submission checks completed at journal 09 Jul, 2024 First submitted to journal 08 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4704450","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":325919230,"identity":"e7e8db64-77ad-48e5-a1ba-6c6e8958a52f","order_by":0,"name":"Taisuke Tomonaga","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYHAD5gMMDGwgBg9M5ABWdTwMzDAmWwLJWngM0LVgB/YM/Acf3ai4Iy/vfubrhg9lDIn97L0HmHkY7OQZGM9itQZoC7NxzplnhhvP5G67OeMcQ+LMnnMJQC3Jhg0M5xKwapF/zCad23aYcWND7rbbvG0MiRtu5Jj/BpoEVH7GAIctYC32G/vfPLv9F6Tl/hsDoC31BLUkzpfIYbvNCLaFB6TlMG4tB5iNgX45nLxB4pnZzZ5zEsYze3IMGOcYHDdsw+EX9gbGh49zKg7bzu9PfnbjR5mNbD870PA3FdXy/BLYQwwODCDSEjAuMI4kzuDVwSDfgCHE34NfyygYBaNgFIwUAACZR1zIIDQVEAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Occupational and Environmental Health","correspondingAuthor":true,"prefix":"","firstName":"Taisuke","middleName":"","lastName":"Tomonaga","suffix":""},{"id":325919231,"identity":"4750acf7-f4aa-4d23-9782-01c9ad0ee728","order_by":1,"name":"Hiroto Izumi","email":"","orcid":"","institution":"University of Occupational and Environmental 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Kitakyushu","correspondingAuthor":false,"prefix":"","firstName":"Kazuo","middleName":"","lastName":"Sakurai","suffix":""},{"id":325919243,"identity":"bd266a37-dd1d-4a72-85a6-2a04c9311e25","order_by":11,"name":"Jun-ichi Takeshita","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jun-ichi","middleName":"","lastName":"Takeshita","suffix":""},{"id":325919245,"identity":"92069217-28b2-4076-853b-728426d50550","order_by":12,"name":"Akihiro Moriyama","email":"","orcid":"","institution":"National Institute of Advanced Industrial Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Moriyama","suffix":""},{"id":325919247,"identity":"57bff286-c879-4bec-b806-31efae12b29f","order_by":13,"name":"Kei Yamasaki","email":"","orcid":"","institution":"University of Occupational and Environmental Health","correspondingAuthor":false,"prefix":"","firstName":"Kei","middleName":"","lastName":"Yamasaki","suffix":""},{"id":325919251,"identity":"6bb9c2a2-2f46-4891-bc46-7f10afca9289","order_by":14,"name":"Kazuhiro Yatera","email":"","orcid":"","institution":"University of Occupational and Environmental Health","correspondingAuthor":false,"prefix":"","firstName":"Kazuhiro","middleName":"","lastName":"Yatera","suffix":""},{"id":325919253,"identity":"1df8bb26-ee9f-4b64-b237-19eb8adcac3e","order_by":15,"name":"Yasuo Morimoto","email":"","orcid":"","institution":"University of Occupational and Environmental Health","correspondingAuthor":false,"prefix":"","firstName":"Yasuo","middleName":"","lastName":"Morimoto","suffix":""}],"badges":[],"createdAt":"2024-07-08 09:44:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4704450/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4704450/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-87174-6","type":"published","date":"2025-01-28T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61583459,"identity":"5dba847e-6f3f-4550-838e-2899fa6c75ee","added_by":"auto","created_at":"2024-08-01 13:52:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":378531,"visible":true,"origin":"","legend":"\u003cp\u003eThe images of scanning electron microscopy of the powder of polyacrylic acid with different concentrations of cross-linker.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/8593fc846a6121c6f0855660.png"},{"id":61584064,"identity":"62e50a46-a91e-48c1-9ce8-ae2332234da3","added_by":"auto","created_at":"2024-08-01 14:00:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168724,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of Body weight and lung findings after the instillation. (A) Body weight at each time after the instillation of PAAs with different cross-linker concentrations. (B) Lung weight at each time after the instillation of PAAs with different cross-linker concentrations. (C) Macro findings at 1 week after the instillation. There was a significant increase in body weight in CL0.1% and CL1.0% at 3 days or 1 week after exposure. In all PAA-exposure groups, a significant increase in lung weights was sustained throughout the observation period compared to the control group. The lungs in the CL0.1% and CL1.0% exposed groups showed swelling at 1 week after intratracheal instillation. Data are presented as mean ±SD for n= 5/group (vs each negative control * p \u0026lt; 0.05, ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/144eb82ec31d503edd562d9c.png"},{"id":61583451,"identity":"c33af593-0010-462a-9f40-7168ff63c055","added_by":"auto","created_at":"2024-08-01 13:52:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44924,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of cell number in BALF following intratracheal instillation of PAAs with different cross-linker concentrations. (A) total cell number in BALF. (B) neutrophil count in BALF. (C) percentage of neutrophils in BALF. Inflammatory cell count in BALF in all of the exposed groups were higher than those in the control groups in a dose-dependent manner at 3 days to 1 week after exposure. Data are presented as mean ±SD for n= 5/group (vs each negative control * p \u0026lt; 0.05, ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/dd4a589696420bc91cd4c869.png"},{"id":61584067,"identity":"b1ca54cc-f0a7-4b0a-868e-b89c4ed8aa01","added_by":"auto","created_at":"2024-08-01 14:00:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41356,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of cytokines in BALF and oxidative stress in lung tissue after intratracheal instillation. (A) CINC-1 concentration in BALF. (B) CINC-2 concentration in BALF. (C) HO-1 concentration in lung tissue. There was an increase of CINC-1 and CINC-2 in BALF in all exposure groups from 3 days to 1 week. HO-1 in lung tissue as an oxidative stress marker increased in a dose-dependent manner from 3 days to 1 month in all exposure groups, and had a tendency of increase in the 1.0 mg exposure group of both CL0.1% and CL5.0% until 3 months after exposure. Data are presented as mean ±SD for n= 5/group (vs each negative control * p \u0026lt; 0.05, ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/fc94c3343d286f5bd9ec016b.png"},{"id":61584063,"identity":"eaf566d9-837a-425a-b082-91dfeba41474","added_by":"auto","created_at":"2024-08-01 14:00:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30236,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of released LDH activity and total protein in BALF following intratracheal instillation of PAAs with different cross-linker concentrations. (A) Released LDH activity in BALF. (B) Concentration of total protein in BALF. Released LDH activity in BALF in CL0.1% groups was higher than CL1.0% and CL5.0% groups at 3 days to 1 month after exposure, but there was a persistent increase of LDH activity and total protein concentration from 1 month to 3 months. Data are presented as mean ±SD for n= 5/group (vs each negative control * p \u0026lt; 0.05, ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/1a984a9681c0f5d35d5e3dca.png"},{"id":61584066,"identity":"77bc6918-cff5-42cb-a5e2-2aacce30c407","added_by":"auto","created_at":"2024-08-01 14:00:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":528348,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological findings of HE staining in lung-exposed PAAs with different cross-linker concentrations. (A) Histopathological findings in the lung at 3 days, 1 month, and 6 months after exposure of CL0.1%, CL1.0%, and CL5.0%. (B) Inflammation score in histopathological findings in the lung. There were severe inflammatory cell infiltrations into the alveoli, mainly neutrophils, which were remarkable in the lung in a dose-dependent manner 3 days after exposure to all of the PAAs. Inflammatory changes with fibrosis were observed from 3 days to 1 week after exposure (vs each negative control * p \u0026lt; 0.05, ** p \u0026lt; 0.01, and vs each 1.0 mg exposure group # p \u0026lt; 0.05, ## p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/208b07d621fabc124aa1f90c.png"},{"id":61583454,"identity":"490598a1-b3d6-4fef-bf77-8884b649dbfd","added_by":"auto","created_at":"2024-08-01 13:52:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":501964,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological findings of MT staining in lung-exposed PAAs with different cross-linker concentrations. (A) Histopathological findings in the lung at 3 days, 1 month, and 6 months after exposure of CL0.1%, CL1.0%, and CL5.0%. (B) Ashcroft score, which is the indicated fibrosis score, in histopathological findings in the lung. There was fibrosis in the 1.0 mg exposure group of all of the PAAs, whereas fibrosis was more spread in the 1.0 mg exposure group of CL0.1％. Evaluation by the Ashcroft score showed a dose-dependent increase in the score in the exposure of CL0.1%, CL1.0%, and CL5.0%. There were higher scores in the 1.0 mg exposure group of CL0.1％ than in those of CL1.0％ and CL5.0%. Data are presented as mean ±SD for n= 5/group (vs each negative control * p \u0026lt; 0.05, ** p \u0026lt; 0.01, and vs each 1.0 mg exposure group # p \u0026lt; 0.05, ## p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/927fa78accde59e46a10b275.png"},{"id":61583458,"identity":"669b1f33-f21a-448f-a00f-a880144474d6","added_by":"auto","created_at":"2024-08-01 13:52:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":42354,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between lung fibrosis and lung injury from 3 days to 1 month after exposure to PAAs. (A) The relationship between lung fibrosis and release LDH activity. (B) The relationship between lung fibrosis and total protein concentration. There were good correlations between lung fibrosis and LDH activity or total protein as lung injury indicators from 3 days to 1 month after exposure. Values of ρ are Spearman’s rank\u003c/p\u003e\n\u003cp\u003ecorrelation coefficient for the data at each time after exposure PAAs (significance level * p \u0026lt; 0.05, ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/3f99d88196027492a1571a56.png"},{"id":61583460,"identity":"15b2ca90-37ad-475a-88c5-030b5ac68449","added_by":"auto","created_at":"2024-08-01 13:52:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":189136,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of the results of this study. PAA causes changes in osmotic pressure due to water absorption, and differences in water absorption by PAA with different degrees of cross-linking may have affected the differences in fibrosis via cell injury.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/10e578580393ec0dc7cf6436.png"},{"id":75351470,"identity":"6cd83470-8141-4405-b742-65f4fce9b5c6","added_by":"auto","created_at":"2025-02-03 16:11:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2690892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/99e6a18f-45f2-4bee-926f-6e95317cd54f.pdf"},{"id":61584065,"identity":"51d89433-a489-40ec-9fcf-7e4ad894b7bc","added_by":"auto","created_at":"2024-08-01 14:00:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure S1. Overview of the synthesis of PAA with different cross-linker concentrations. Acrylic acid was used as a monomer. Ethylene glycol diacrylate, a bifunctional monomer, was used as a cross-linker (concentration: 0.1 mol%, 1.0 mol%, 5.0 mol%). Acrylic acid monomer and α,α'-Azobisisobutyronitrile (AIBN, KANTO CHEMICAL CO., INC.) were dissolved in a mixture of Ethyl acetate and Ethylene carbonate (mixture ratio 7:3) and bubbled with nitrogen. The mixture was bubbled with nitrogen for 30 minutes. The reaction was then carried out at 60°C for 3 hours to obtain spherical PAA particles. The resulting particles were purified by centrifugation in acetonitrile three times. After that, the solvent was removed by vacuum drying to obtain cross-linked PAA particles.\u003c/p\u003e","description":"","filename":"Additionalfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4704450/v1/47afd9b5552927a9c2990fd2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Degree of Cross-linking of Polyacrylic Acid Affects the Fibrogenicity in Rat Lungs","fulltext":[{"header":"Background","content":"\u003cp\u003eThe types of respirable dust are broadly categorized into inorganic and organic substances. Pneumoconiosis, a well-known respiratory disease caused by respirable dust, is caused mainly by inorganic substances such as silica and asbestos, and is known to cause chronic inflammation, emphysematous changes fibrosis, and even lung cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Organic substances, on the other hand, cause allergic diseases such as bronchial asthma and hypersensitivity pneumonitis, but they have not been thought to cause pulmonary fibrosis such as pneumoconiosis. In recent years, however, it has been reported that organic substances cause inflammation and fibrosis. In South Korea, there were many cases of progressive interstitial pneumonia among people who used disinfectants in humidifiers, which became a social problem [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the United States, there were reports of e-cigarette users who developed acute respiratory failure [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In Japan, inflammation and fibrosis of interstitium around the airways have been reported in workers who handle water-absorbing cross-linked acrylic acid-based polymer compounds, which are used as intermediates for various products in daily life, such as diapers, cosmetics, shampoos, cosmetics, and food additives [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the case of those workers, fibrosis progressed quickly, about two years after exposure to acrylic acid-based polymers, so the progression was faster than in lung damage caused by asbestos and crystalline silica, which are typical inorganic substances [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In animal exposure examinations, intratracheal instillation and inhalation exposure have been conducted with this acrylic acid-based polymer, and it has been reported that it causes lung disorders, including rapidly progressing fibrosis, similar to human cases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In spite of these lines of evidence, however, the pathogenesis of lung disorders caused by acrylic acid-based polymers has not been elucidated.\u003c/p\u003e \u003cp\u003ePolyAcrylic acid (PAA) is composed of repeating chemical structural formulas of acrylic acid monomers, and the linear repeating structures of the monomers are intricately entangled. A cross-linker is used to cause the cross-linking of multiple linear molecules through covalent bonds. The cross-linking becomes more polymerized and forms a three-dimensional structure with complex meshes, and as the molecular weight and cross-linked structure increase, thickening and swelling by absorbing water are caused [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is thought that the thickening and swelling properties associated with water absorption cause lung disorders.\u003c/p\u003e \u003cp\u003eWe have previously conducted intratracheal installations on rats using PAAs with different molecular weights and cross-linked structures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When evaluating the effects of the presence or absence of a cross-linked structure, it was observed that PAAs with a cross-linked structure caused higher lung injury and fibrogenicity than linear PAAs without a cross-linked structure [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This cross-linked structure is thought to be involved in biological effects since it is considered that water absorption and thickening properties change in a complicated manner by changing the density of the cross-linked structure. In order to investigate the effects of PAAs with different cross-linking densities on lung disorders, we synthesized three types of PAAs with different cross-linking densities by varying the concentration of cross-linker and intratracheally instilling them into rats.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCharacterizations of polyacrylic acid with different degrees of cross-linking\u003c/h2\u003e\n \u003cp\u003eThe fundamental characteristics of different cross-linking concentrations of PAA are summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the scanning electron microscopy (SEM) by HITACHI S-4500 (Hitachi, Ltd., Tokyo, Japan) of the powder of the PAAs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). We examined the fundamental characteristics of the PAAs by preparing the molecular dispersion as follows: the polymers were dissolved in 0.1 M carbonate-bicarbonate buffer, then the solutions were alkalized with 2N NaOH and then neutralized with 1N HCl. The PAA without a cross-linking reagent in the same synthetic process had an average molecular weight (MW) of 50.4×10\u003csup\u003e5\u003c/sup\u003e as measured by gel permeation chromatography (GPC) (a Prominence 501 system coupled with Dawn-Heleos-Ⅱ(Wyatt Technology Europe GmbH, Dernbach, Rheinland-Pfalz, Germany)) using GF-7MHQ (Showa Denko K.K., Tokyo, Japan) with 0.1M carbonate-bicarbonate buffer as the eluent [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. The PAAs with cross-linking could not be measured, however, because the molecular weight was too large. On the other hand, the radius of gyration (Rg) in molecular dispersion measured by GPC was increased in a cross-linker concentration-dependent manner. Rg indicates the extent of a polymer, which is influenced by its molecular weight and polymer structure.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eBody and lung weights\u003c/h2\u003e\n \u003cp\u003eBody and lung weights increased dose-dependently at each observation point in all of the PAA-exposure groups (Figure. 2AB). There was a significant increase in body weight in CL0.1% and CL1.0% at 3 days or 1 week after exposure (Figure. 2A). A significant increase in lung weights was sustained throughout the observation period in all of the PAA-exposure groups compared to the control group (Figure. 2B). During the acute phase, there was less lung swelling in the high-dose group in a cross-linking-concentration dependent manner (Figure. 2C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eCell analysis and cell injury markers in bronchoalveolar lavage fluid (BALF)\u003c/h2\u003e\n \u003cp\u003eThe results of inflammatory cell counts in BALF are shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. There were significant increases in the number of total cells in all of the 1 mg-exposure groups from 3 days to 1 month after exposure compared to each control group, and a significant increase in the number of total cells was observed until 3 months after exposure in the CL0.1% and CL5.0% -high exposure groups (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The number and percentage of neutrophils were significantly increased from 3 days to 1 week after exposure in CL0.1% and CL1.0%, and the exposure to CL5.0% induced a persistent increase until 1 month (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). In the acute phase, the increase in inflammatory cells in BALF was similar, regardless of the degree of cross-linking of PAA, but there was a difference in the persistence of increased inflammatory cells in BALF in CL0.1%, CL1.0%, and CL5.0%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eMeasurements of inflammatory markers in BALF and lung tissue\u003c/h2\u003e\n \u003cp\u003eThe concentrations of cytokine-induced neutrophil chemoattractant (CINC) -1 and CINC-2 in BALF, and the concentrations of heme oxygenase (HO)-1 in lung tissue following the intratracheal instillation of PAAs are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. CINC-1 and CINC-2 were increased in the exposure groups from 3 days to 1 week after exposure. HO-1 in lung tissue showed a tendency of significant increase in the 1.0 mg exposure group until 3 months after exposure in CL0.1% and CL5.0%, but at 1 month and 3 months after exposure HO-1 was more increased in CL5.0% than in CL0.1%.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eCell injury markers in BALF\u003c/h2\u003e\n \u003cp\u003eThe results of lactate dehydrogenase (LDH) activity and total protein in BALF are shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e. The results of LDH activity and the concentration of total protein in all of the exposure groups also showed tendencies of increase from 3 days to 1 month after exposure compared to each control group. The increase of the LDH activity was highest in CL0.1% and lowest in CL5.0%, showing cross-linking concentration-dependent changes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). The increase in total protein concentration also tended to be lowest in CL5.0% (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eHistopathological features in the lung\u003c/h2\u003e\n \u003cp\u003eRepresentative histopathological findings of the lungs at 3 days, 1 month, and 6 months after exposure to PAA with different cross-linking concentrations are shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. There was inflammatory cell infiltration, mainly by neutrophils, into the alveoli, which was evident in a dose-dependent manner at 3 days after exposure. Inflammatory changes with fibrosis were observed from 3 days to 1 week after exposure, suggesting severe lung disorders. In particular, inflammatory cell aggregation was more pronounced in the 1.0 mg CL0.1% and CL1.0% exposure groups than in the CL5.0% group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). In the evaluation by inflammation score, a dose-dependent increase in the score was observed in CL0.1%, CL1.0%, and CL5.0%. The CL0.1% 1.0 mg exposure group tended to have a higher score than the CL5.0% group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). On the other hand, the inflammation score in the CL5.0% 1.0 mg exposure group was persistently increased compared to the CL0.1% and CL1.0% groups. Fibrosis in the CL0.1% 1.0 mg group, as compared to CL1.0% and CL5.0%, was the most severe and progressed the fastest, and the extent of fibrosis decreased in a cross-linker concentration-dependent manner (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Ashcroft score assessment showed a dose-dependent increase in scores with PAA exposure. The CL0.1% 1.0 mg exposure group had a higher score than the CL1.0% and CL5.0% groups (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003eThe relationship between lung fibrosis and lung injury\u003c/h2\u003e\n \u003cp\u003eThe relationship between pulmonary fibrosis and lung injury is shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. The data used is from 3 days to 1 month after exposure. LDH activity and total protein concentration as lung injury markers showed good correlations with the Ashcroft score, a fibrosis marker, suggesting that lung injury is related to the progression of fibrosis.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used PAAs synthesized with different concentrations of cross-linkers, and intratracheal instillation was conducted on rats. Intratracheal instillation was used to evaluate the pulmonary toxicity of these PAAs. We estimated the human exposure equivalent to the maximum dose of 1 mg in this study by using the following formula that had been used previously [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e(amount of PAA) = (exposure concentration of particle) × (tidal volume) ×\u003c/p\u003e \u003cp\u003e(breathing frequency)× (exposure hours in a day) ×\u003c/p\u003e \u003cp\u003e(particle deposition fraction)\u003c/p\u003e \u003cp\u003eAssuming that the lung deposition rate (0.1) of PAA is the same for humans and rats if humans were exposed to the TLV-TWA (Threshold Limit Value - Time-Weighted Average from American Conference of Governmental Industrial Hygienist; ACGIH) level of general dust (3 mg/m\u003csup\u003e3\u003c/sup\u003e), this would correspond to an exposure period of approximately 926 days (calculation in rat and human under assumption of tidal volume 2.1 and 625 mL/times; breathing frequency volume 102 and 12 times/min; and exposure hours in day 6 hours (lung weights of rats and humans are set at 1 g and 1000 g, respectively)). Assuming that a worker continued to work under conditions of 3 mg/m\u003csup\u003e3\u003c/sup\u003e (8 hours a day, 5 days a week), a high dose of 1 mg instilled into the trachea would be equivalent to an exposure period of approximately 3.6 years. In actual worker cases, lung disorder has been reported after exposure to acrylic acid-based polymers for more than two years [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; we considered that the level of exposure in the present study corresponded to the real environment.\u003c/p\u003e \u003cp\u003eIn this study, pulmonary inflammation progressed to severe fibrosis sooner with CL0.1% than with CL1.0% or CL5.0%. Considering that inflammation leads to subsequent fibrosis in lung disorders caused by inorganic substances [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], it is thought that PAAs have a strong fibrogenicity after inflammation. The development of fibrosis after acute inflammation has been observed in animal exposure studies and clinical manifestations, and is common in LPS exposure models that induce acute inflammation and in ARDS with acute respiratory failure [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e–\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Acute respiratory failure in animal models due to LPS or bleomycin causes severe inflammation of the lungs that ends in 1–2 weeks and causes fibrosis without long-term inflammation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the worker cases, fibrosis was observed about two years after the start of exposure to an acrylic acid-based polymer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and it progressed faster than with asbestos or silica. Considering that acrylic acid-based polymers used in products often demand water-soluble applications and the cross-linkers are generally used at a concentration of about 0.1%, the early observation of fibrosis in the CL0.1% in this study is not inconsistent with the pathology in humans. On the other hand, fibrosis progressed only mildly with the CL5.0%, but inflammation tended to persist. Considering that persistent inflammation leads to subsequent fibrosis in lung disorders caused by inorganic substances [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], there is a possibility that, in the long term, fibrosis may progress or lead to irreversible lesions such as tumors. The inflammatory and fibrotic potential of the CL5.0% may be lower than that of CL0.1% and CL1.0%, but it is considered to be high among general inhalable chemicals.\u003c/p\u003e \u003cp\u003eThe relationship between the crosslink density of the PAAs and fibrosis is considered to be as follows. It is known that the water absorption of PAA increases when the polymer has a cross-linked structure, but the water absorption decreases when the crosslink density increases beyond a certain level [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The water absorbency of PAA may cause biological hyperosmotic stress on an organism. Hyperosmotic stress on an organism is reportedly associated with various pathological conditions such as induction of inflammatory cytokines and apoptosis [\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e–\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Schwartz et al. reported that when three epithelial cell lines (colorectal HT29, bladder T24, and lung A549) were exposed to glycol-derived compounds, the presence of hyperosmolar concentrations induced significant production of the proinflammatory cytokines IL-6, IL-8, TNF-α, and IL-1β in all cell lines [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Singh and Ramarao also reported that the culture medium of RAW cells with mannitol induced cell death in an osmotic pressure-dependent manner [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, Fibrosis was most severe at 0.1% CL, which is considered to be the highest water absorbency and stress, and fibrosis potential was reduced as the cross-linker concentration increased. Considering that a previous comparison of the pulmonary fibrotic potential of non-cross-linked and cross-linked (0.1% or less cross-linker) PAAs showed stronger fibrosis in the cross-linked PAA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], it is thought that the increased water absorption caused by the change in crosslink density causes fibrosis.\u003c/p\u003e \u003cp\u003eFurthermore, in the acute phase of this study, the LDH activity and total protein concentration in BALF, which are indicators of lung injury, were most elevated in the CL0.1% and tended to be the lowest in the CL5.0%. There are reports that severe lung injury leads to the progression of fibrosis. It has been reported in inhalation exposure and intratracheal instillation of MWCNT in mice that the increase in LDH in BALF and the amount of MWCNT deposition and fibrosis level showed a similar tendency of increase [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In an intratracheal instillation of bleomycin in mice, the increase in LDH activity in BALF and the progression of fibrosis were observed in a dose-dependent manner [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the present study, a correlation was observed between lung injury and fibrosis from 3 days to 1 month after instillation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e), so it is possible that the CL0.1%, which caused the highest lung injury, may have progressed to fibrosis early in the tissue repair process. As mentioned above, considering that the increased water absorption caused by changes in crosslink density leads to fibrosis, it is speculated that lung injury due to increased water absorption contributed to the progression of fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we analyzed the effects of PAAs with different cross-linking densities on the lungs by varying the cross-linker concentration. CL0.1% tended to cause the strongest fibrosis in the acute phase, and a decrease in pulmonary fibrogenicity was observed as the cross-linker concentration was increased. It is thought that the water absorbency of PAA influences lung disorders since fibrogenicity decreases with increasing cross-linking concentration. Taken together, it is suggested that the crosslink density of PAA is a physicochemical feature that influences lung disorders.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003ch2\u003eSample polymer\u003c/h2\u003e\u003cp\u003eUsing a previous method [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], we synthesized a sample of PAA by polymerization of the acrylic acid monomer adding different cross-linker concentrations (0.1 mol%, 1.0 mol%, and 5.0 mol%) and labeled them CL0.1%, CL1.0%, and CL5.0%, respectively. The details of the preparations are described in the Supplementary Information. PAA, a white, easily scattered powder, was mixed with distilled water and slowly stirred for 40 minutes (Mag-Mixer MF820 or MD300, Yamato Scientific Co., Ltd., Tokyo, Japan).\u003c/p\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eMale Fischer 344 rats (8 weeks old) (The Jackson Laboratory Japan, Inc., Kanagawa, Japan) were acclimated for 4 weeks at the Laboratory Animal Research Center of the University of Occupational and Environmental Health, Japan with commercial food and water available ad libitum. All procedures and animal handling were performed under the guidelines described in the \"Japanese Guide for the Care and Use of Laboratory Animals\" and approved by the Animal Experiment Committee of the University of Occupational and Environmental Health, Japan (Animal Research Ethics Approval Proposal No.; AE17-009). All methods were performed in accordance with the relevant guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003eIntratracheal instillation\u003c/h2\u003e\u003cp\u003eDoses of 0.2 mg (0.8 mg/kg BW) and 1.0 mg (4.0 mg/kg BW) of PAA suspended in 0.4 mL of distilled water at different cross-linker concentrations were administered by single intratracheal instillation into the lungs of rats (12 weeks old). Rats were instilled intratracheally under sevoflurane (Viatris Inc., Canonsburg, PA, USA) inhalation anesthesia. Briefly, the laryngeal extension was performed using a laryngoscope blade (MAC1, Rudolf Riester GmbH, Jungingen, Germany), and an animal feeding needle (KN-348, Natsume Seisakusho Co., Ltd., Tokyo, Japan) was inserted directly into the trachea and the suspension was instilled manually. Next, 3 mL of air was inserted into the trachea twice with a syringe from the animal feeding needle. The rats were then spontaneously awakened and observed periodically. Single intratracheal instillations of 0.2 mg and 1.0 mg of PAA at different cross-linker concentrations were administered at different times, for a total of three single intratracheal instillations. A control group was established for each intratracheal instillation, with distilled water administered to the control group; doses of 0.2 mg and 1.0 mg per rat were used for the PAA intratracheal instillation. These maximum doses were set to avoid overloading the lungs in anticipation of human exposure [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eAnimals following intratracheal instillation\u003c/h2\u003e\u003cp\u003eThere were five rats in each exposure and control group at each time point. Animals were dissected at 3 days, 1 week, 1 month, 3 months, and 6 months after intratracheal instillation under isoflurane (Viatris Inc., Canonsburg, PA, USA) inhalation anesthesia. Body weight was measured, then blood was removed from the heart at autopsy. Lungs were extracted from the body, lung weights were measured, and the lungs were perfused with saline solution. With the left main bronchus clamped, the right lung was repeatedly inflated with saline at a pressure of 20 cm H\u003csub\u003e2\u003c/sub\u003eO after two fluid collections. Seven to 14 mL of the fluid (BALF) was collected in collection tubes by free fall, and then the right and left lungs were separated. The homogenized third lobe of the right lung after BALF collection was used for measurement of HO-1. The left lung was inflated and fixed with 10% formaldehyde under pressure of 25 cm H\u003csub\u003e2\u003c/sub\u003eO for histopathological evaluation.\u003c/p\u003e\u003ch2\u003eCytospin analysis of inflammatory cells and measurement of inflammation-related markers in BALF\u003c/h2\u003e\u003cp\u003eThe BALF was centrifuged at 400 g for 15 min at 4°C, and the supernatant was transferred to a new tube for determination of total protein, lactate dehydrogenase (LDH) and cytokines. Pellets were washed in polymorphonuclear leukocyte (PMN) Buffer (137.9 mM NaCl, 2.7 mM KCl, 8.2 mM 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) in suspension and centrifuged at 400 g at 4°C for 15 minutes. After the removal of the supernatant, the pellet was resuspended in 1 mL of PMN buffer. Cell counts in BALF were measured with ADAM-MC (AR BROWN CO., LTD., Tokyo, Japan), cells were spread on glass slides with cytospin, fixed, stained with Diff-Quik (Sysmex CO., Kobe, Hyogo, Japan), and the number of neutrophils and alveolar macrophages was measured by microscopic observation. LDH activities released into the BALF supernatant were measured with the Cytotoxicity Detection KitPLUS (LDH) (Roche Diagnostics GmbH, Mannheim, North Rhine-Westphalia, Germany) according to the manufacturer's instructions. LDH activity was estimated using a standard curve obtained from known concentrations of recombinant LDH derived from rabbit muscle (Oriental Yeast Co., ltd., Tokyo, Japan). Protein concentrations in BALF supernatants were determined using the Pierce™ 660 nm Protein Assay (Thermo Scientific Inc., Rockford, IL, USA). Ford, Illinois, USA).\u003c/p\u003e\u003ch2\u003eMeasurement of chemokine in BALF and HO-1 in lung tissue\u003c/h2\u003e\u003cp\u003eConcentrations of CINC-1 and CINC-2 in BALF were measured with the ELISA kits #RCN100 and #RCN200 (R\u0026amp;D Systems, Minneapolis, MN, USA), respectively. All measurements were performed according to the manufacturer's instructions. The third lobe of the right lung was cultured in T-PER tissue containing protein inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA) and cOmplete Mini (Roche Diagnostics GmbH, Mannheim, Nordrhein-Westfalen, Germany), and homogenized in Protein Extraction Reagent (Thermo Scientific Inc., Rockford, IL, USA) and centrifuged (20,400 g at 4°C for 10 min). Protein concentrations in the supernatant were determined with Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific Inc., Rockford, IL, USA), using bovine serum albumin as a standard. HO-1 measurements with the Elisa kit ADI-EKS-810A (Enzo Life Sciences, Farmingdale, NY, USA) were corrected by the protein concentration in the supernatant to calculate the final HO-1 concentration in the lung tissue.\u003c/p\u003e\u003ch2\u003eHistopathology\u003c/h2\u003e\u003cp\u003eFormaldehyde-fixed lung tissue was embedded in paraffin, sectioned at a thickness of 4 µm, and then stained with hematoxylin and eosin (HE) and Masson trichrome (MT) staining. The lung inflammation and fibrosis were examined using the inflammatory cell infiltration score [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and the Ashcroft score [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], respectively, according to previous reports [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Briefly, the inflammatory cell infiltration score was calculated by scoring the degree of inflammatory cell infiltration in the lung tissue as none (0), minimal (0.5), mild (1), moderate (2), or severe (3). The mean and standard deviation of the scores were calculated for each group. Pulmonary fibrosis was assessed by scoring histopathological findings in the lungs on a scale of 0 to 8 using the modified Ashcroft score, with the mean and standard deviation calculated for each group.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using IBM® SPSS® software (IBM Corporation, Chicago, IL, USA). p-values \u0026lt; 0.05 were determined to be statistically significant. The Dunnett and Tukey's honestly significant difference (HSD) tests were appropriately used to detect individual differences between individuals exposed to different cross-linker concentrations of PAA samples and the control group. Construct validity was measured using Spearman’s rank correlation coefficients (ρ) between the Ashcroft scores as lung fibrosis indicators and lung injury markers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number JP21H04855. The authors would like to thank Sumiyo Kuramoto, Tomoko Watanabe, Rika Takai, Mayumi Tashiro, Yuno Ariyoshi, and Tomoko Morimoto for technical support with the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTT: Writing \u0026ndash; original draft, Validation, Methodology, Investigation, Conceptualization. HI: Methodology, Investigation, Conceptualization. CN: Validation, Methodology, Data curation. KS (Kazuma Sato): Visualization, Methodology. YN: Methodology, Formal analysis. TM: Methodology, Formal analysis. YH: Methodology, Formal analysis. KW: Methodology, Formal analysis. HH: Methodology, Formal analysis. TK: Methodology, Formal analysis. KS (Kazuo Sakurai): Writing \u0026ndash; review \u0026amp; editing, Supervision, Validation, Conceptualization. JT: Methodology, Data curation. AM: Methodology, Data curation. KY (Kei Yamasaki): Methodology, Formal analysis. KY (Kazuhiro Yatera): Writing \u0026ndash; review \u0026amp; editing, Supervision, Validation. YM: Writing \u0026ndash; review \u0026amp; editing, Supervision, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Number JP21H04855.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures and animal handling were done according to the guidelines described in the Japanese Guide for the Care and Use of Laboratory Animals as approved by the Animal Care and Use Committee, University of Occupational and Environmental Health, Japan (animal studies ethics clearance proposal number; AE17-009).\u0026nbsp;All methods were also performed in accordance with the relevant guidelines (https://arriveguidelines.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required as no human data is presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eDepartment 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. \u003csup\u003e2\u003c/sup\u003eDepartment 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. \u003csup\u003e3\u003c/sup\u003eDepartment of Respiratory Medicine, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan. \u003csup\u003e4\u0026nbsp;\u003c/sup\u003eShared-Use Research Center, School of Medicine, University of Occupational and Environmental Health, Japan. 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan. \u003csup\u003e5\u0026nbsp;\u003c/sup\u003eDepartment of Chemistry and Biochemistry, The University of Kitakyushu. 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan. \u003csup\u003e6\u0026nbsp;\u003c/sup\u003eResearch 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\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeung CC, Yu ITS, Chen W. 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(In Japanese)\u003c/li\u003e\n\u003cli\u003eNODA M, TAKAKURA K, OKADA S, FUJII S, NAKAMURA Y, URAHAMA Y. Influences of Crosslinking Degree and Test Rate on The Mechanical Properties of Polyacrylic Pressure-Sensitive Adhesive. J Adhes Soc Japan. 2017;53:268\u0026ndash;75. (In Japanese)\u003c/li\u003e\n\u003cli\u003eMaitra J, Shukla VK. Cross-linking in Hydrogels - A Review. 2014. Available from: https://api.semanticscholar.org/CorpusID:26756895\u003c/li\u003e\n\u003cli\u003eTomonaga T, Nishida C, Izumi H, Kawai N, Wang K-Y, Higashi H, et al. Crosslinked Structure of Polyacrylic Acid Affects Pulmonary Fibrogenicity in Rats. Int J Mol Sci. 2022;23:13870. \u003c/li\u003e\n\u003cli\u003eNishida C, Izumi H, Tomonaga T, Wang K-Y, Higashi H, Takeshita J-I, et al. Effect of Different Molecular Weights of Polyacrylic Acid on Rat Lung Following Intratracheal Instillation. Int J Mol Sci. 2022;23:10345. \u003c/li\u003e\n\u003cli\u003eSumiya K, Matsunaga T, Tanaka M, Mochizuki S, Sakurai K. 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Comparison of pulmonary inflammatory responses following intratracheal instillation and inhalation of nanoparticles. Nanotoxicology. 2016;10:607\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eYang J, Liang W, He X, Su Y, Wang F, Wang T, et al. Experimental Synthesis of Polyacrylic-Type Superabsorbent Polymer and Analysis of Its Internal Curing Performances. Fluid Dyn Mater Process. 2022;18:15\u0026ndash;27. \u003c/li\u003e\n\u003cli\u003eM.J.A.D. ZM, Kabiri K. Superabsorbent Polymer Materials: A Review. 2008. Available from: https://api.semanticscholar.org/CorpusID:55246346\u003c/li\u003e\n\u003cli\u003eBrocker C, Thompson DC, Vasiliou V. The role of hyperosmotic stress in inflammation and disease. Biomol Concepts. 2012;3:345\u0026ndash;64. \u003c/li\u003e\n\u003cli\u003eK\u0026uuml;ltz D. Hyperosmolality triggers oxidative damage in kidney cells. Proc Natl Acad Sci. 2004;101:9177\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eSchwartz L, Guais A, Pooya M, Abolhassani M. Is inflammation a consequence of extracellular hyperosmolarity? J Inflamm. 2009;6:21. \u003c/li\u003e\n\u003cli\u003eSingh RP, Ramarao P. Accumulated Polymer Degradation Products as Effector Molecules in Cytotoxicity of Polymeric Nanoparticles. Toxicol Sci. 2013;136:131\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003ePorter DW, Hubbs AF, Chen BT, McKinney W, Mercer RR, Wolfarth MG, et al. Acute pulmonary dose\u0026ndash;responses to inhaled multi-walled carbon nanotubes. Nanotoxicology. 2012;7:1179\u0026ndash;94. \u003c/li\u003e\n\u003cli\u003eSnyder-Talkington BN, Dong C, Porter DW, Ducatman B, Wolfarth MG, Andrew M, et al. Multiwalled carbon nanotube-induced pulmonary inflammatory and fibrotic responses and genomic changes following aspiration exposure in mice: A 1-year postexposure study. J Toxicol Environ Heal Part A. 2016;79:352\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eKim S-N, Lee J-S, Yang H-S, Cho J-W, Kwon S-J, Kim Y-B, et al. Dose-response Effects of Bleomycin on Inflammation and Pulmonary Fibrosis in Mice. Toxicol Res. 2010;26:217\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003eTakano S, Ono R, Sakurai K. A surprisingly narrow particle size distribution for polyacrylic acid nanospheres produced by precipitation polymerization and revealed by small-angle X-ray scattering. Polym J. 2023;55:1387\u0026ndash;91. \u003c/li\u003e\n\u003cli\u003eNishida C, Izumi H, Tomonaga T, Takeshita J, Wang K-Y, Yamasaki K, et al. Predictive Biomarkers for the Ranking of Pulmonary Toxicity of Nanomaterials. Nanomaterials. 2020;10:2032. \u003c/li\u003e\n\u003cli\u003eH\u0026uuml;bner R-H, Gitter W, Eddine El Mokhtari N, Mathiak M, Both M, Bolte H, et al. Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques. 2008;44:507\u0026ndash;17. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Polyacrylic acid, Cross-link, Intratracheal instillation, pulmonary toxicity, Fibrosis, rat","lastPublishedDoi":"10.21203/rs.3.rs-4704450/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4704450/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Polyacrylic acid (PAA) with different concentrations of cross-linker was instilled into the trachea of ​​rats to examine the effect of PAA crosslink density on lung disorders. Methods: F344 rats were intratracheally exposed to low and high doses of PAA with cross-linker concentrations of 0.1, 1.0, and 5.0% (CL0.1%, CL1.0%, and CL5.0%, respectively). Rats were sacrificed at 3 days, 1 week, 1 month, 3 months, and 6 months after exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e PAA with different cross-linker concentrations caused an increase in neutrophil influx, cytokine-induced neutrophils, and chemotactic factor (CINC) in bronchoalveolar lavage fluid (BALF) from 3 days to 1 week after instillation. Lactate dehydrogenase (LDH) activity in BALF and heme oxygenase-1 (HO-1) release in lung tissue were higher in the CL0.1% exposure group during the acute phase. Lung histopathological findings also showed that severe fibrotic changes induced by CL0.1% were greater than those observed in CL1.0% and CL5.0% exposure during the observation period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e CL0.1% was associated with more severe lung fibrosis, and a decrease in lung fibrosis was observed with increasing cross-linker concentrations, suggesting that the cross-link density of PAA is a physicochemical feature that affects lung disorders.\u003c/p\u003e","manuscriptTitle":"The Degree of Cross-linking of Polyacrylic Acid Affects the Fibrogenicity in Rat Lungs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-01 13:52:03","doi":"10.21203/rs.3.rs-4704450/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-05T04:31:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-29T17:53:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-20T02:44:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148399550467283698691575327814420381331","date":"2024-10-09T15:24:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118768069080288273241288022591490936651","date":"2024-10-09T13:39:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-09T11:03:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-03T10:28:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-27T14:46:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-09T12:56:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-08T09:42:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3832bdf-b123-4905-9ca2-d0cc5def7384","owner":[],"postedDate":"August 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34483558,"name":"Health sciences/Diseases/Respiratory tract diseases"},{"id":34483559,"name":"Physical sciences/Chemistry/Organic chemistry"}],"tags":[],"updatedAt":"2025-02-03T16:07:34+00:00","versionOfRecord":{"articleIdentity":"rs-4704450","link":"https://doi.org/10.1038/s41598-025-87174-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-01-28 15:57:18","publishedOnDateReadable":"January 28th, 2025"},"versionCreatedAt":"2024-08-01 13:52:03","video":"","vorDoi":"10.1038/s41598-025-87174-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-87174-6","workflowStages":[]},"version":"v1","identity":"rs-4704450","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4704450","identity":"rs-4704450","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}