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Didecyldimethylammonium chloride induces pulmonary fibrosis in association with TGF-β signaling in mice
Experimental and Toxicologic Pathology 65 (2013) 1003–1009
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Didecyldimethylammonium chloride induces pulmonary fibrosis in association with TGF- signaling in mice Aya Ohnuma-Koyama a , Toshinori Yoshida a,∗ , Haruka Tajima-Horiuchi a , Naofumi Takahashi a , Satoru Yamaguchi b , Ryoichi Ohtsuka b , Yukiko Takeuchi-Kashimoto a , Maki Kuwahara a , Makio Takeda b , Nobuaki Nakashima a , Takanori Harada c a
Laboratory of Pathology, Toxicology Division, The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan Laboratory of Molecular Toxicology, Toxicology Division, The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan c The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan b
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Article history: Received 16 October 2012 Accepted 18 February 2013 Keywords: Dialkyl-quaternary ammonium Transforming growth factor- Bone morphogenetic protein SMAD Pulmonary fibrosis
a b s t r a c t Didecyldimethylammonium chloride (DDAC) is a representative dialkyl-quaternary ammonium compound that is used as a disinfectant against several pathogens and is also used in commercial, industrial, and residential settings. We previously investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, and found that DDAC causes pulmonary injury, which is associated with altered antioxidant antimicrobial responses; the inflammatory phase is accompanied or followed by fibrotic response. The present study was conducted to monitor transforming growth factor- (TGF-) signaling in pulmonary fibrosis induced by DDAC. Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment to measure TGF- signaling. In order to further evaluate TGF signaling, we treated isolated mouse lung fibroblasts with DDAC. Fibrotic foci were observed in the lungs on day 3, and were widely extended on day 7, with evidence of increased ␣-smooth muscle actinpositive mesenchymal cells and upregulation of Type I procollagen mRNA. Developing fibrotic foci were likely associated with increased expression of Tgf-ˇ1 mRNA, in addition to decreased expression of Bone morphogenetic protein-7 mRNA. In fibrotic lung samples, the expression of phosphorylated SMAD2/3 was considerably higher than that of phosphorylated SMAD1/5. In isolated lung fibroblasts, the mRNA levels of Tgf-ˇ1 were specifically increased by DDAC treatment, which prolonged phosphorylation of SMAD2/3. These effects were abolished by treatment with SD208 – a TGF-RI kinase inhibitor. The results suggest that DDAC induces pulmonary fibrosis in association with TGF- signaling. © 2013 Elsevier GmbH. All rights reserved.
1. Introduction Didecyldimethylammonium chloride [DDAC; C10 H21 N(CH3 )2 C10 H21 ·Cl] is a representative dialkyl-quaternary ammonium compound (QAC) that is used as a disinfectant against several pathogens such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa (Walsh et al., 2003; Ioannou et al., 2007), Legionella pneumophilia (Skaliy et al., 1980), Stachybotrys chartarum (Micales-Glaeser et al., 2004), and even against several enveloped and non-enveloped viruses (Argy et al., 1999; Shirai et al., 2000). DDAC is added directly to water in swimming pools, spas, and humidifiers, and is also used in institutional, commercial, industrial, and residential settings by fogging, flooding, immersion, wiping, mopping, aerosol sprays, and low- and high-pressure spraying (Dickey, 2003; US EPA. 2006). The final concentrations
∗ Corresponding author. Tel.: +81 297 27 4521; fax: +81 297 27 4518. E-mail address: [email protected] (T. Yoshida). 0940-2993/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2013.02.003
of DDAC achieved are as follows: 32–1800 ppm for treatment of industrial recalculating water system; 0.5–2 ppm in swimming pool water; 5–938 ppm in decorative fountains and water displays; 26320 ppm for application by fog in hatcheries; 234–2400 ppm for application in homes; 240–2400 ppm for application in hospitals and day care centers (USEPA, 2006). Nearly 2700 incidents, most of which are dermal, ocular, and inhalation irritation, were reported to the Office of Pesticide Programs Incident Data System and the California Department of Pesticide Regulation in association with exposure to end-use products containing QAC (1982–2004) (US EPA. 2006). Occupational exposure to QACs, including DDAC, has been known to cause contact dermatitis, conjunctivitis (Dejobert et al., 1997), and asthma (Bernstein et al., 1994; Burge and Richardson, 1994) among professionals working in healthcare and sanitation. Limited data on respiratory toxicity is available from studies that one of QAC, benzalkonium chloride (BAC), induces pulmonary injury after inhalation (Swiercz et al., 2008) or oral administration (mediated by aspiration of a small amount into the bronchi)
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(Xue et al., 2004) in rats. These data, however, did not demonstrate the precise toxic effects on respiratory systems by QAC. Therefore, we accurately investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, because intratracheal instillation is a suitable tool with which to evaluate the pulmonary toxicity of compounds relative to its inhalation effects (Warheit et al., 2007). We found that DDAC causes pulmonary injury and inflammation, which are associated with altered antioxidant antimicrobial responses and expression of several chemokines and their receptors; the inflammatory phase is accompanied or followed by fibrotic response (Ohnuma et al., 2010, 2011). Despite these observations, the underlying mechanisms of pulmonary fibrosis observed in DDAC-treated mice remain unclear. Transforming growth factor- (TGF-) superfamily is a key regulator of extracellular matrix (ECM) assembly and remodelling (Piek et al., 1999; Verrecchia and Mauviel, 2007). TGF- isoforms, TGF-1, -2, and -3, have the ability to induce the expression of ECM proteins in mesenchymal cells. Cellular TGF-1 mRNA levels were higher than cellular TGF-2 and TGF-3 mRNA levels in idiopathic pulmonary fibrosis (IPF) patients (Koli et al., 2006) and bleomycin-treated mice (Coker et al., 1997). Signaling by TGF- family members occurs via type I and II receptors (TGF-RI and II) (Piek et al., 1999; Verrecchia and Mauviel, 2007). Upon phosphorylation by activated TGF-RI (ALK5), TGF- activates SMAD2/3 to form heteromeric complexes with SMAD4, which are translocated into the nucleus, where they function as transcription factors. On the other hand, bone morphogenetic protein (BMP) member, one of the TGF- superfamily, activates SMAD1/5 via BMP receptors (Bobik, 2006); the roles of, particularly, BMP-7 and BMP-4, and that of gremlin, an antagonist of BMP signalling, in pulmonary fibrosis has been investigated. In normal human lung fibroblast, BMP-4 reduces TGF-1-mediated ECM production, and BMP-7 inhibits TGF-1-induced myofibroblast transformation (Pegorier et al., 2010). TGF-1 induces GREMLIN mRNA expression in normal human bronchial epithelial cells (Myllärniemi et al., 2008), and expression of GREMLIN enhances TGF-1-induced epithelialto-mesenchymal transition (EMT) in A549 lung epithelial cells (Koli et al., 2006). Further in order to assess the mechanism of DDCA-induced pulmonary fibrosis, we investigated whether DDAC instillation to the lungs results in pulmonary injuries associated with TGF- and BMP signaling. We also treated isolated fibroblast-like cells from mouse lungs with DDAC, in order to elucidate the involvement of TGF- and BMP in the pathogenesis of pulmonary fibrosis.
2. Materials and methods
isopropyl alcohol for purification of DDAC, was not identified in the formulation. The mice (aged 8–12 weeks) were anaesthetised with 50 mg/kg of pentobarbital and were subjected to intratracheal instillation of DDAC dissolved in PBS (150 g/kg) into the lungs, as described previously (Ohnuma et al., 2010). One hundred fifty g/kg of DDAC was consisted with 100 ppm, because we set that the volume administered was 1.5 mL/kg. The dose of DDAC were suitable for evaluating the toxicity, because 1000 ppm of DDAC (the purity: 50%) was used for a skin patch test in a DDAC-sensitive patient (Dejobert et al., 1997), and 0.5–26,320 ppm of DDAC have been achieved from variable formulations (USEPA, 2006). Mice (n = 3–5 per group) were sacrificed on days 1, 3, or 7 after administration. Control mice, which received PBS, were sacrificed on day 7 after administration. 2.2. Tissue preparation The right and middle lobes of the lungs were resected, frozen, and stored at −80 ◦ C. The left lobe was infused with low-meltingpoint agarose, and was then fixed with 10% neutral-buffered formalin (Ohnuma et al., 2010). The formalin-fixed lungs were routinely processed and embedded in paraffin. 2.3. Histopathological and immunohistochemical analyses The paraffin-embedded lung tissues were sectioned and stained with haematoxylin and eosin (H&E), as well as Masson’s trichrome stain. Immunohistochemical analysis for ␣-smooth muscle actin (␣-SMA) was performed using a previously reported method (Ohnuma et al., 2010). Horseradish peroxidase-labeled anti-human ␣-SMA monoclonal antibodies (1A4) was obtained from Dako. 2.4. Reverse transcriptase-polymerase chain reaction Total RNA was isolated from the right and middle lobes of the lungs and cell samples described below, and reverse transcriptasepolymerase chain reaction (RT-PCR) was performed as previously described (Ohnuma et al., 2010). PCR primers (listed in Supplemental Table 1) for the genes encoding the following proteins were purchased from Takara Bio Inc., Japan: Tgf-ˇ1, -ˇ2, and -ˇ3; Gremlin 1 (Grem1); Bmp-2, -4, and -7; Collagen 1˛1 (Col1˛1) and 1˛2 (Col1˛2); and Ribosomal protein S18 (Rps18). The corresponding cDNAs were amplified by ABI PRISM® 7700 (Applied Biosystems) with a primer set and Power CYBR® Green PCR Master Mix (Applied Biosystems). All measurements were performed in duplicate. The levels of active gene expression were determined with the help of a standard curve. The data acquired for each sample were normalised to the expression levels recorded for the housekeeping gene Rps18.
2.1. Animal treatment 2.5. Western blotting Male C57BL/6J mice were obtained from Charles River, Japan. The mice were housed in cages, which were maintained under suitable conditions in terms of temperature (22 ± 2 ◦ C), humidity (50% ± 20%), ventilation (continuous circulation of fresh air), and illumination (a 12-h light/dark cycle). Each mouse was maintained in a separate cage and had ad libitum access to a pellet diet (Oriental MF; Oriental Yeast Co.) and tap water. All animals were handled in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science (JALAS, 1987). The current study was conducted in accordance with the Code of Ethics for Animal Experimentation of this institute. DDAC was purchased from Wako Pure Chemical. The purity of DDAC was 87.2% from analysis of chloride ions using silver nitrogen titrimetry; water concentration was 1.3%, and concentration of the other components (possibly unreactive ingredients, intermediates, or degraded DDAC) was 16.5% (Ohnuma et al., 2011). A solvent,
The right and middle lobes and cell samples described below were homogenised in a phosphorylation lysis buffer, and the extract was processed for western blotting as previously described (Tajima et al., 2010). Primary antibodies against phospho-Smad2 (ser465/467), Smad2, phospho-Smad1/5 (ser463/465), and Smad5 were obtained from Cell Signaling Technology. Antibodies against -actin and tubulin were obtained from Sigma–Aldrich and Abcam, respectively. Anti-pig vimentin monoclonal antibodies (V9) (Dako) was also applied. 2.6. Cell culture Mouse lung fibroblasts (MLFs) were isolated and cultured in IMDM (Invitrogen) as previously described (Ohnuma et al., 2010). Briefly, fibroblasts were isolated from the lung of normal mice
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with collagenase digestion, and cultured in IMDM (Invitrogen) containing 10% foetal bovine serum (FBS). Ten% FBS was suitable for the in vitro experiments, as lower concentration of FBS impaired growth of MLFs. MLFs were used for in vitro experiments at passes 5 and 6. MLFs were seeded into 100-mm dishes (8 × 104 cells/dish), and they reached 80–90% confluence. For RNA and protein analyses, MLFs were incubated with 0, 2, or 5 nM DDAC for 3 h, and 1 and 2 days, as previously described (Ohnuma et al., 2010). To assess the effect of SD208 – a TGF-RI (ALK5) kinase inhibitor (Sigma-Aldrich) – MLFs were treated with 5 nM DDAC for 24 h and then washed with PBS 3 times. MLFs were then incubated with 400 nM SD208 for 24 h and subjected to protein analyses as previously described (Kapoun et al., 2006). Individual experiment was repeated at least thrice. 2.7. Statistical analyses Data are represented as mean and standard error (SE). The data were analysed by Dunnett’s multiple comparison tests between the control group and treatment group at each time point. A p-value below 0.05 or 0.01 was considered significant. 3. Results 3.1. DDAC induces pulmonary fibrosis Histopathological analysis did not show treatment-related changes in the lungs of control mice when the intratracheal vehicle (PBS) was instilled into the lungs (Fig. 1(A)). On day 1 after exposure to DDAC, no bronchiolar or alveolar lesions were evident (Fig. 1(B)), despite the cytotoxicity demonstrated in the bronchoalveolar lavage (BAL) fluid as previously reported (Ohnuma et al., 2011). On days 3 and 7, mice exhibited fibrotic lesions in their lungs; their distribution and severity increased in a timedependent manner (Fig. 1(C) and (D)). The fibrotic lesions consisted
Fig. 1. DDAC induces fibrosis in the lung. Mice were intratracheally instilled with DDAC or vehicle control (Control), and lung samples were collected 1, 3, or 7 days after treatment for histopathology. Representative images of the lung stained with H&E (A–D) and Masson trichrome (E), and immuhistochemically stained for ␣-SMA (F) from the vehicle control (A) and DDAC-treated mice at 1 (B), 3 (C), and 7 (D–F) days. No abnormalities are detected in the control group (A) and treated group 1 day after treatment (B). Fibrotic lesions are observed 3 days after treatment (C), followed by profound development of pulmonary fibrosis 7 days after treatment (D). At higher magnification of fibrotic lesions, deposition of collagen fibres (E) and accumulation of myofibroblasts (F) are evident 7 days after treatment. n = 3–4 per group. Scale bar = 100 (A–D) and 20 m (E,F).
Fig. 2. DDAC induces mRNA of Tgf-s, Bmps, Grem1, and type I procollagen in lung tissues. Mice were treated as described in Fig. 1 and lung tissues were subjected to real-time RT-PCR for Tgf-ˇ1, -ˇ2, and -ˇ3 (A); Col1˛1 (B) and Col1˛2 (C); Bmp-2 (D), -4 (E), and -7 (F); and Grem1 (G). The data were normalised to Rps18 mRNA. Values are presented as the mean and SE (n = 3–5 per group). *p < 0.05 and **p < 0.01, as determined by Dunnett’s multiple comparison test.
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of high amounts of collagen fibres (Fig. 1(E)) and proliferation of myofibroblasts, which tested positive for ␣-SMA (Fig. 1(F)). Infiltrated inflammatory cells consisted of macrophages, neutrophils, and lymphocytes: neutrohpils were evidently infiltrated on day 3 and macrophages and lymphocytes were profoundly increased on day 7. A small lymphoid follicle was observed in the fibrotic lesions (Fig. 1(D)), but no lymphocyte cuffing was present in the adjacent airways and vesicles.
3.2. TGF-ˇ/SMAD signaling in DDAC-induced pulmonary fibrosis in vivo We examined whether pulmonary fibrotic lesions were associated with expression of Tgf-ˇ1, -ˇ2, or -ˇ3; Bmp-2, -4, or -7; or Grem1 mRNA. We found a 2-fold increasein Tgf-ˇ1 mRNA in the lung samples on day 7 after exposure to DDAC (p < 0.05); Tgf-ˇ2 and -ˇ3 mRNA were not increased at any time point (Fig. 2(A)). Consistent with the expression of Tgf-ˇ1, both Col1˛1 and Col1˛2 mRNA were highly expressed on day 7 (Col1˛1, p < 0.05; Col1˛2, p < 0.01) (Fig. 2(B) and (C)). Bmp-2 mRNA was temporally expressed on day 1 (p < 0.01) (Fig. 2(D)), and expression of Bmp-7, but not Bmp-4, was reduced throughout the observation period (Bmp-7: p < 0.05 on days 1 and 7; p < 0.01 on day 3) (Fig. 2(E) and (F)). Interestingly, the mRNA level of Grem1 was high, but not significantly high, on day 7 (Fig. 2(G)). In order to elucidate the activation of TGF- signaling in treated lungs, phosphorylated SMAD2/3 was analysed by western blotting. We found that phosphorylated SMAD2/3 was profound in the lung samples on days 3 and 7 in a time-dependent manner compared to phosphorylated SMAD1/5 (Fig. 3). VIMENTIN expression on day 7, which might correspond to mesenchymal cell proliferation, seemed to be associated with phosphorylation of SMAD2/3. No clear
Fig. 3. DDAC enhances Smad phosphorylation in the lung. Mice were treated as described in Fig. 1, and subjected to western blotting. Representative images of western blotting for phosphorylated Smad2/3, total Smad2/3, phosphorylated Smad1/5, total Smad5, ␣-SMA, vimentin, and tubulin (an internal control). Stripped membranes for phosphorylated proteins were probed with total protein and tubulin; for ␣-SMA, membranes were probed with vimentin and tubulin (n = 3 per group). Higher expression of ␣-SMA in control group than those in treated group was considered to be non-specific.
time-related changes were detected in ␣-SMA and TUBULIN (an internal control) expression. The results suggested that TGF-/SMAD2/3 signaling is associated with DDAC-induced fibrogenesis. 3.3. TGF-ˇ/SMAD signaling in DDAC-treated mouse lung fibroblasts in vitro In order to evaluate the regulation of members of the TGF family in DDAC-treated mesenchymal cells, we isolated MLFs and treated them with DDAC. Tgf-ˇ1, -ˇ2, and -ˇ3, Bmp-4, and Grem1 mRNA expression was quantified using real-time RT-PCR.
Fig. 4. DDAC alters Tgf-ˇs and Bmp-4 mRNA levels in MLFs. Isolated MLFs were plated in 100-mm dishes at 8 × 104 cells/well and incubated overnight in an incubator. On the following day, the cells were treated with different doses of DDAC. After 3 h, and 1 and 2 days, cells were collected for real-time RT-PCR of Tgf-ˇ1 (A), -ˇ2 (B), and -ˇ3 (C), and Bmp-4 (D). The data were normalised to Rps18 mRNA. As compared to the control level, Tgf-ˇ1 mRNA level was increased in both a time- and dose-dependent manner (A), whereas Tgf-ˇ2 (B) and -ˇ3 (C), and Bmp-4 (D) mRNA levels were reduced. Values are presented as the mean and SE, which were obtained from four individual experiments. *p < 0.05 and **p < 0.01, as determined by Dunnett’s multiple comparison test at each time point.
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Fig. 5. Time-dependent reduction of expression of Grem1 mRNA level in MLFs. Isolated MLFs were treated as described in Fig. 4. After 3 h, and 1 and 2 days, cells were collected for real-time RT-PCR of Grem1. The data were normalised to Rps18 mRNA. Grem1 mRNA level was reduced in a time-related manner without dependence on the DDAC concentration. Values are presented as the mean and SE, which were obtained from four individual experiments. *p < 0.05 and **p < 0.01, as determined by Dunnett’s multiple comparison test at each time point.
Tgf-ˇ1 mRNA expression was increased in both a time- and dosedependent manner compared to the control level (Fig. 4(A): 2 M DDAC, p < 0.01 on day 2; 5 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2), whereas Tgf-ˇ2 (Fig. 4(B); 2 M DDAC, p < 0.01 on day 2; 5 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2) and -ˇ3 (Fig. 4(C): 2 M and 5 M DDAC, p < 0.01 on days 1 and 2), and Bmp-4 (Fig. 4(D); 2 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2; 5 M DDAC, p < 0.01 on day 2) mRNA levels were considerably reduced. Grem1 mRNA level was higher in DDAC treatment than the control level but reduced in a time-related manner without dependence on the DDAC concentration (Fig. 5; 2 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2). Bmp-2 and -7 mRNA were not detected in MLFs. DDAC weakly cancelled the time-dependent reduction of phosphorylation of SMAD2/3 (Fig. 6). The basal level of Bmp-4 mRNA appeared to increase in a time-dependent manner (Fig. 4(D)), but no significant increase of phosphorylated SMAD1/5 was observed during the treatment period (Fig. 6). Pretreatment with SD208 – a TGF-RI (ALK5) kinase inhibitor – attenuated DDACinduced ␣-SMA and VIMENTIN expression via suppression of the phosphorylation of SMAD2/3 (Fig. 7). 4. Discussion A number of environmental and occupational agents play a role in the pathogenesis of chronic lung disease such as pulmonary fibrosis, and COPD (Yoshida and Tuder, 2007; Yoshida et al., 2011). DDAC formulations are diluted in water to treat premises and equipment by fogging, aerosol sprays, and/or low- and highpressure spraying (US EPA. 2006). Vincent et al. reported that DDAC
Fig. 6. DDAC weakly cancels a time-related reduction of phospo-Smad2/3 in MLFs. Isolated MLFs were treated as described in Fig. 4, using a dose of 5 M DDAC. Cells were collected for western blotting of phosphorylated Smad2/3, total Smad2/3, phosphorylated Smad1/5, total Smad5, ␣-SMA, vimentin, and tubulin. Results were obtained from three individual experiments. Highly nonspecific phosphorylation of Smad 2/3 was noted 3 h after, independent on DDAC treatment.
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Fig. 7. DDAC enhances SMAD phosphorylation via TGF-RI (ALK5) in MLFs. Isolated MLFs were treated with 5 M DDAC for 24 h as described in Fig. 4 and then incubated for another 24 h in the presence or absence of SD208 – a TGF-RI (ALK5) kinase inhibitor. Cells were collected for western blotting of phosphorylated Smad2/3, total Smad2/3, phosphorylated Smad1/5, total Smad5, ␣-SMA, vimentin, and tubulin. Pretreatment with SD208 attenuated DDAC-induced phosphorylation of Smad2/3 and ␣-SMA and vimentin expression. Results were obtained from three individual experiments.
would not contaminate the indoor hospital atmosphere during the disinfection process because of its insignificant volatility; however, it could contaminate working atmospheres when it is put in suspension by aerosolisation (Vincent et al., 2007). Therefore, the study was designed to determine the direct effect of DDAC on the respiratory system. Inhaled QACs cause pulmonary dysfunction including occupational asthma (Bernstein et al., 1994; Burge and Richardson, 1994; Swiercz et al., 2008); however, a key question is whether irritated QACs cause pulmonary fibrotic responses following inducible cytotoxicity and inflammation. Our findings indicated that DDAC causes the development of pulmonary fibrotic lesions following single instillation of DDAC to the lungs in mice; the change appears to be associated with TGF- and BMP signaling. Impaired BMP and its antagonist gremlin signaling have not been found in the mouse bleomycin model, one of the most common model of pulmonary fibrosis (Myllärniemi et al., 2008). However, the signaling is an important regulator for the development of human interstitial lung diseases (ILDs) (Koli et al., 2006; Pegorier et al., 2010). Therefore, our data suggested that intratracheally applying DDAC to the lung might have an impact on the investigation of lung fibrosis. We previously reported that the inflammatory phase was accompanied by remodeling (i.e. fibrosis), which was evident in Masson trichrome stains and mRNA expression of type I collagen (Ohnuma et al., 2010). In the present study, we attempted to clarify the molecular targets of DDAC-induced pulmonary fibrosis in mice. Our data suggest that TGF-1 and SMAD2/3 signaling might play a role in fibrogenic lungs, which is consistent with the observation in several lines of animal models for pulmonary fibrosis (Bonniaud et al., 2004; Zhao et al., 2002). This finding was confirmed by in vitro experiments that showed that DDAC increased TGF-1/SMAD2/3 signaling via TGF-RI (ALK5), and appeared to act as a maintainer of myofibroblast differentiation in isolated MLFs. This was demonstrated by using a TGF-RI (ALK5) kinase inhibitor, SD208, which inhibits TGF--induced phosphorylation of SMAD2/3 and myofibroblast transformation, as reported in human lung fibroblasts (Kapoun et al., 2006). The activation of myofibroblasts is a critical event in the pathogenesis of pulmonary fibrosis, because myofibroblasts represent the predominant source of heightened ECM (Hinz et al., 2007). TGF-1 has been mainly examined in pulmonary fibrosis, but other members of the TGF- family have not been fully investigated. Real-time RT-PCR revealed that induction of TGF-ˇ1 is associated with increases in type I collagens, Col1␣1 and Col1˛2, in fibrogenic lungs. However, Tgf-ˇ2 and -ˇ3 were not highly expressed in fibrogenic lungs. This finding is consistent with a previous report on bleomycin-treated mice (Coker et al., 1997). In IPF patients, cellular TGF-ˇ1 mRNA levels are higher than TGF-ˇ2 and
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-ˇ3 levels (Koli et al., 2006). Active TGF-1 is a major cytokine that stimulates the transcription of Col1␣2 genes, which are determined by the binding of SMAD3/4 to the TbRE cis-element in the proximal promoter (Zhang et al., 2000). TGF-2 and -3 have a potential for in vitro fibrogenic activity, which is the same as or higher than the in vitro activity of TGF-1 (Eickelberg et al., 1999). Specific expression of Tgf-ˇ1 by DDAC treatment in vivo and in vitro suggested that this growth factor plays an important role on mesenchymal cell response to a cytotoxic stimulus. We provided evidence that BMPs could be involved in DDACinduced pulmonary damage. Bmp-7 mRNA was persistently reduced after DDAC instillation, suggesting that BMP-7 is a negative regulator of fibrosis. Indeed, BMP-7 has been known to be an antagonist against TGF-1-dependent fibrogenic activity in mouse pulmonary myofibroblasts (Izumi et al., 2006). However, the role of BMP-7 in fibrosis is controversial, as BMP-7 treatment significantly reduces collagen deposition in asbestos-treated mice (Myllärniemi et al., 2008) but not in bleomycin-treated mice (Murray et al., 2008). Because the levels of Bmp-2, but not Bmp-4, were temporally increased after DDAC instillation, they might be mediated by cellular stresses, such as inflammatory stimuli, oxidative stress (Csiszar et al., 2006), and/or hypoxia (Takahashi et al., 2006). No alteration of BMP-4 levels has been reported in lung samples of IPF patients, but IPF fibroblasts are less responsive to exogenous BMP-4 (Koli et al., 2006). According to our in vitro findings, Bmp-4 mRNA levels under control media were increased in a time-dependent manner, but treatment with DDAC cancelled this background upregulation. The downregulation of Bmp-4 by DDAC seems to be consistent with the idea that BMP-4 inhibits growth and induces apoptosis in myofibroblasts (Koli et al., 2006). Lately, it is reported that BMP-4 reduces TGF-1-mediated ECM production, while BMP-7 inhibits TGF-1-induced myofibroblast transformation in normal human lung fibroblast (Pegorier et al., 2010). Because of lack of expression of Bmp-7 in isolated MLFs, further experiments will be required using epithelial cell and/or other cells to clarify the role of BMPs in the progression of pulmonary fibrosis after the intratracheal instillation of DDAC. The upregulation of gremlin (a BMP inhibitor) in our lung samples, is supported by previous findings that TGF-1 induces GREMLIN expression in A549 lung epithelial cells, and that IPF fibroblasts and their lung samples have a high level of GREMLIN mRNA and its protein (Koli et al., 2006; Myllärniemi et al., 2008). Pulmonary Gremlin mRNA levels are also upregulated in asbestostreated mice (Myllärniemi et al., 2008). Farkas et al. (2011) show that transient Gremlin overexpression results in alveolar epithelial injury, followed by modest lung tissue fibrosis that is partly reversible. In our experiment, however, there were individual variation in Grem1 expression in vivo, and DDAC treatment did not affect time-related reduction of its expression in vitro. At this point, the role of Grem1, as well as BMP-4, and -7 remains controversial and their function on DDAC-mediated response in vitro and in vivo should be further investigated. A series of our experiments indicate that exposure to DDAC provides evidence of a three phase model of wound repair: injury, inflammation, and repair (Wilson and Wynn, 2009). DDAC increases LDH activity as well as protein concentration in the BAL fluid (as cytotoxicity markers), recruits inflammatory cells, together with expression of several chemokine and cytokines in the BAL fluid (as inflammation markers), and enhances expressions of surfactant protein D and hem oxygenase-1 mRNAs in the lung tissues (as repair responses) (Ohnuma et al., 2010, 2011). A dysregulated healing gradually evolves into a fibrotic response through pathogenic processes such as EMT and proteolytic imbalance. EMT is critical for the production of myofibroblast in an established model of pulmonary fibrosis, overexpression of active TGF-1 (Kim et al., 2006), but not bleomycin model (Rock et al., 2011), based on the experiments with labelling lung epithelial cells to follow their
fate. Gremlin expression in DDAC-treated mice leads to speculations of its role in EMT, as overexpression of GREMLIN enhances TGF-1mediated EMT in A549 lung epithelial cells (Koli et al., 2006). Matrix proteinases (MMPs) degrade components of ECM and the activities of MMPs are controlled by tissue inhibitors of MMP (TIMPs), a process that is essential for a regulated healing (Lagente et al., 2005). MMPs such as MMP-2 and -9 and/or TIMPs such as TIMP-1 and -2 are increased after belomycin and asbestos exposure in mice (Swiderski et al., 1998; Tan et al., 2006), suggesting that imbalance between MMPs and TIMPs could play a role in excessive ECM production and abnormal matrix remodeling. These raise a question of how DDAC dysregulates healing process via EMT and proteolytic imbalance. Our work demonstrate a clearer understanding of the pathobiology of DDAC in pulmonary fibrosis, which might be associated with TGF- and SMAD signaling and to some extent by BMP, as reported in asbestos-, but not bleomycin-, treated lungs (Murray et al., 2008; Myllärniemi et al., 2008). Since the bleomycin-treated mice might be not a suitable model for investigating the role of BMP and gremlin, investigations of the signaling in DDAC-treated lungs would make important advances of ILDs. We doses not conclude at present that DDAC causes a severe form of ILD, because DDAC-induced fibrosis appears to be reversible following a single instillation to the lung (Ohnuma et al., 2010). The reversible effect seems to be similar to the study of transient overexpression of Gremlin (Farkas et al., 2011). A repeated dose study of DDAC is required to confirm the potential reversibility of the pulmonary fibrosis. The present study provides a better understanding of the health problems associated with environmental contaminants in lung disease. Acknowledgements We would like to thank Yuko Chiba, Mutsumi Kumagai, Takako Kazami, Yukie Sakano, Satoshi Akema, Chizuko Tomiyama, and Kayoko Iijima for their assistance in tissue preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etp.2013.02.003. References Argy G, Bricout F, d’Hermies F, Cheymol A. Study of prophylaxis by didecyl dimethyl ammonium chloride against herpes simplex virus infection in nude mice. Comptes Rendus de l Academie des Sciences Serie III, Sciences de la Vie 1999;322:863–70. Bernstein JA, Stauder T, Bernstein DI, Bernstein IL. A combined respiratory and cutaneous hypersensitivity syndrome induced by work exposure to quaternary amines. Journal of Allergy and Clinical Immunology 1994;94:257–9. Bobik A. Transforming growth factor-s and vascular disorders. Arteriosclerosis, Thrombosis, and Vascular Biology 2006;26:1712–20. Bonniaud P, Kolb M, Galt T, Robertson J, Robbins C, Stampfli M, Lavery C, Margetts PJ, Roberts AB, Gauldie J. Smad3 null mice develop airspace enlargement and are resistant to TGF--mediated pulmonary fibrosis. Journal of Immunology 2004;173:2099–108. Burge PS, Richardson MN. Occupational asthma due to indirect exposure to lauryl dimethyl benzyl ammonium chloride used in a floor cleaner. Thorax 1994;49:842–3. Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois RM, Jeffery PK, McAnulty RJ. Transforming growth factors-1, -2, and -3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis. American Journal of Pathology 1997;150:981–91. Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G, Edwards JG, Wolin MS, Ungvari Z. Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype. American Journal of Pathology 2006;168:629–38. Dejobert Y, Martin P, Piette F, Thomas P, Bergoend H. Contact dermatitis from didecyldimethylammonium chloride and bis-(aminopropyl)-lauryl amine in a detergent-disinfectant used in hospital. Contact Dermatitis 1997;37:95–6. Dickey P. Guidelines for selecting wood preservatives. For the San Francisco department of the environment; 2003, The guidelines are available at http://www.sfenvironment.org/downloads/library/preservatives.pdf
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Didecyldimethylammonium chloride induces pulmonary fibrosis in association with TGF- signaling in mice Aya Ohnuma-Koyama a , Toshinori Yoshida a,∗ , Haruka Tajima-Horiuchi a , Naofumi Takahashi a , Satoru Yamaguchi b , Ryoichi Ohtsuka b , Yukiko Takeuchi-Kashimoto a , Maki Kuwahara a , Makio Takeda b , Nobuaki Nakashima a , Takanori Harada c a
Laboratory of Pathology, Toxicology Division, The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan Laboratory of Molecular Toxicology, Toxicology Division, The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan c The Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan b
a r t i c l e
i n f o
Article history: Received 16 October 2012 Accepted 18 February 2013 Keywords: Dialkyl-quaternary ammonium Transforming growth factor- Bone morphogenetic protein SMAD Pulmonary fibrosis
a b s t r a c t Didecyldimethylammonium chloride (DDAC) is a representative dialkyl-quaternary ammonium compound that is used as a disinfectant against several pathogens and is also used in commercial, industrial, and residential settings. We previously investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, and found that DDAC causes pulmonary injury, which is associated with altered antioxidant antimicrobial responses; the inflammatory phase is accompanied or followed by fibrotic response. The present study was conducted to monitor transforming growth factor- (TGF-) signaling in pulmonary fibrosis induced by DDAC. Mice were intratracheally instilled with DDAC and sacrificed 1, 3, or 7 days after treatment to measure TGF- signaling. In order to further evaluate TGF signaling, we treated isolated mouse lung fibroblasts with DDAC. Fibrotic foci were observed in the lungs on day 3, and were widely extended on day 7, with evidence of increased ␣-smooth muscle actinpositive mesenchymal cells and upregulation of Type I procollagen mRNA. Developing fibrotic foci were likely associated with increased expression of Tgf-ˇ1 mRNA, in addition to decreased expression of Bone morphogenetic protein-7 mRNA. In fibrotic lung samples, the expression of phosphorylated SMAD2/3 was considerably higher than that of phosphorylated SMAD1/5. In isolated lung fibroblasts, the mRNA levels of Tgf-ˇ1 were specifically increased by DDAC treatment, which prolonged phosphorylation of SMAD2/3. These effects were abolished by treatment with SD208 – a TGF-RI kinase inhibitor. The results suggest that DDAC induces pulmonary fibrosis in association with TGF- signaling. © 2013 Elsevier GmbH. All rights reserved.
1. Introduction Didecyldimethylammonium chloride [DDAC; C10 H21 N(CH3 )2 C10 H21 ·Cl] is a representative dialkyl-quaternary ammonium compound (QAC) that is used as a disinfectant against several pathogens such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa (Walsh et al., 2003; Ioannou et al., 2007), Legionella pneumophilia (Skaliy et al., 1980), Stachybotrys chartarum (Micales-Glaeser et al., 2004), and even against several enveloped and non-enveloped viruses (Argy et al., 1999; Shirai et al., 2000). DDAC is added directly to water in swimming pools, spas, and humidifiers, and is also used in institutional, commercial, industrial, and residential settings by fogging, flooding, immersion, wiping, mopping, aerosol sprays, and low- and high-pressure spraying (Dickey, 2003; US EPA. 2006). The final concentrations
∗ Corresponding author. Tel.: +81 297 27 4521; fax: +81 297 27 4518. E-mail address: [email protected] (T. Yoshida). 0940-2993/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.etp.2013.02.003
of DDAC achieved are as follows: 32–1800 ppm for treatment of industrial recalculating water system; 0.5–2 ppm in swimming pool water; 5–938 ppm in decorative fountains and water displays; 26320 ppm for application by fog in hatcheries; 234–2400 ppm for application in homes; 240–2400 ppm for application in hospitals and day care centers (USEPA, 2006). Nearly 2700 incidents, most of which are dermal, ocular, and inhalation irritation, were reported to the Office of Pesticide Programs Incident Data System and the California Department of Pesticide Regulation in association with exposure to end-use products containing QAC (1982–2004) (US EPA. 2006). Occupational exposure to QACs, including DDAC, has been known to cause contact dermatitis, conjunctivitis (Dejobert et al., 1997), and asthma (Bernstein et al., 1994; Burge and Richardson, 1994) among professionals working in healthcare and sanitation. Limited data on respiratory toxicity is available from studies that one of QAC, benzalkonium chloride (BAC), induces pulmonary injury after inhalation (Swiercz et al., 2008) or oral administration (mediated by aspiration of a small amount into the bronchi)
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(Xue et al., 2004) in rats. These data, however, did not demonstrate the precise toxic effects on respiratory systems by QAC. Therefore, we accurately investigated toxicity on air way system following single instillation of DDAC to the lungs in mice, because intratracheal instillation is a suitable tool with which to evaluate the pulmonary toxicity of compounds relative to its inhalation effects (Warheit et al., 2007). We found that DDAC causes pulmonary injury and inflammation, which are associated with altered antioxidant antimicrobial responses and expression of several chemokines and their receptors; the inflammatory phase is accompanied or followed by fibrotic response (Ohnuma et al., 2010, 2011). Despite these observations, the underlying mechanisms of pulmonary fibrosis observed in DDAC-treated mice remain unclear. Transforming growth factor- (TGF-) superfamily is a key regulator of extracellular matrix (ECM) assembly and remodelling (Piek et al., 1999; Verrecchia and Mauviel, 2007). TGF- isoforms, TGF-1, -2, and -3, have the ability to induce the expression of ECM proteins in mesenchymal cells. Cellular TGF-1 mRNA levels were higher than cellular TGF-2 and TGF-3 mRNA levels in idiopathic pulmonary fibrosis (IPF) patients (Koli et al., 2006) and bleomycin-treated mice (Coker et al., 1997). Signaling by TGF- family members occurs via type I and II receptors (TGF-RI and II) (Piek et al., 1999; Verrecchia and Mauviel, 2007). Upon phosphorylation by activated TGF-RI (ALK5), TGF- activates SMAD2/3 to form heteromeric complexes with SMAD4, which are translocated into the nucleus, where they function as transcription factors. On the other hand, bone morphogenetic protein (BMP) member, one of the TGF- superfamily, activates SMAD1/5 via BMP receptors (Bobik, 2006); the roles of, particularly, BMP-7 and BMP-4, and that of gremlin, an antagonist of BMP signalling, in pulmonary fibrosis has been investigated. In normal human lung fibroblast, BMP-4 reduces TGF-1-mediated ECM production, and BMP-7 inhibits TGF-1-induced myofibroblast transformation (Pegorier et al., 2010). TGF-1 induces GREMLIN mRNA expression in normal human bronchial epithelial cells (Myllärniemi et al., 2008), and expression of GREMLIN enhances TGF-1-induced epithelialto-mesenchymal transition (EMT) in A549 lung epithelial cells (Koli et al., 2006). Further in order to assess the mechanism of DDCA-induced pulmonary fibrosis, we investigated whether DDAC instillation to the lungs results in pulmonary injuries associated with TGF- and BMP signaling. We also treated isolated fibroblast-like cells from mouse lungs with DDAC, in order to elucidate the involvement of TGF- and BMP in the pathogenesis of pulmonary fibrosis.
2. Materials and methods
isopropyl alcohol for purification of DDAC, was not identified in the formulation. The mice (aged 8–12 weeks) were anaesthetised with 50 mg/kg of pentobarbital and were subjected to intratracheal instillation of DDAC dissolved in PBS (150 g/kg) into the lungs, as described previously (Ohnuma et al., 2010). One hundred fifty g/kg of DDAC was consisted with 100 ppm, because we set that the volume administered was 1.5 mL/kg. The dose of DDAC were suitable for evaluating the toxicity, because 1000 ppm of DDAC (the purity: 50%) was used for a skin patch test in a DDAC-sensitive patient (Dejobert et al., 1997), and 0.5–26,320 ppm of DDAC have been achieved from variable formulations (USEPA, 2006). Mice (n = 3–5 per group) were sacrificed on days 1, 3, or 7 after administration. Control mice, which received PBS, were sacrificed on day 7 after administration. 2.2. Tissue preparation The right and middle lobes of the lungs were resected, frozen, and stored at −80 ◦ C. The left lobe was infused with low-meltingpoint agarose, and was then fixed with 10% neutral-buffered formalin (Ohnuma et al., 2010). The formalin-fixed lungs were routinely processed and embedded in paraffin. 2.3. Histopathological and immunohistochemical analyses The paraffin-embedded lung tissues were sectioned and stained with haematoxylin and eosin (H&E), as well as Masson’s trichrome stain. Immunohistochemical analysis for ␣-smooth muscle actin (␣-SMA) was performed using a previously reported method (Ohnuma et al., 2010). Horseradish peroxidase-labeled anti-human ␣-SMA monoclonal antibodies (1A4) was obtained from Dako. 2.4. Reverse transcriptase-polymerase chain reaction Total RNA was isolated from the right and middle lobes of the lungs and cell samples described below, and reverse transcriptasepolymerase chain reaction (RT-PCR) was performed as previously described (Ohnuma et al., 2010). PCR primers (listed in Supplemental Table 1) for the genes encoding the following proteins were purchased from Takara Bio Inc., Japan: Tgf-ˇ1, -ˇ2, and -ˇ3; Gremlin 1 (Grem1); Bmp-2, -4, and -7; Collagen 1˛1 (Col1˛1) and 1˛2 (Col1˛2); and Ribosomal protein S18 (Rps18). The corresponding cDNAs were amplified by ABI PRISM® 7700 (Applied Biosystems) with a primer set and Power CYBR® Green PCR Master Mix (Applied Biosystems). All measurements were performed in duplicate. The levels of active gene expression were determined with the help of a standard curve. The data acquired for each sample were normalised to the expression levels recorded for the housekeeping gene Rps18.
2.1. Animal treatment 2.5. Western blotting Male C57BL/6J mice were obtained from Charles River, Japan. The mice were housed in cages, which were maintained under suitable conditions in terms of temperature (22 ± 2 ◦ C), humidity (50% ± 20%), ventilation (continuous circulation of fresh air), and illumination (a 12-h light/dark cycle). Each mouse was maintained in a separate cage and had ad libitum access to a pellet diet (Oriental MF; Oriental Yeast Co.) and tap water. All animals were handled in accordance with the Guidelines for Animal Experimentation issued by the Japanese Association for Laboratory Animal Science (JALAS, 1987). The current study was conducted in accordance with the Code of Ethics for Animal Experimentation of this institute. DDAC was purchased from Wako Pure Chemical. The purity of DDAC was 87.2% from analysis of chloride ions using silver nitrogen titrimetry; water concentration was 1.3%, and concentration of the other components (possibly unreactive ingredients, intermediates, or degraded DDAC) was 16.5% (Ohnuma et al., 2011). A solvent,
The right and middle lobes and cell samples described below were homogenised in a phosphorylation lysis buffer, and the extract was processed for western blotting as previously described (Tajima et al., 2010). Primary antibodies against phospho-Smad2 (ser465/467), Smad2, phospho-Smad1/5 (ser463/465), and Smad5 were obtained from Cell Signaling Technology. Antibodies against -actin and tubulin were obtained from Sigma–Aldrich and Abcam, respectively. Anti-pig vimentin monoclonal antibodies (V9) (Dako) was also applied. 2.6. Cell culture Mouse lung fibroblasts (MLFs) were isolated and cultured in IMDM (Invitrogen) as previously described (Ohnuma et al., 2010). Briefly, fibroblasts were isolated from the lung of normal mice
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with collagenase digestion, and cultured in IMDM (Invitrogen) containing 10% foetal bovine serum (FBS). Ten% FBS was suitable for the in vitro experiments, as lower concentration of FBS impaired growth of MLFs. MLFs were used for in vitro experiments at passes 5 and 6. MLFs were seeded into 100-mm dishes (8 × 104 cells/dish), and they reached 80–90% confluence. For RNA and protein analyses, MLFs were incubated with 0, 2, or 5 nM DDAC for 3 h, and 1 and 2 days, as previously described (Ohnuma et al., 2010). To assess the effect of SD208 – a TGF-RI (ALK5) kinase inhibitor (Sigma-Aldrich) – MLFs were treated with 5 nM DDAC for 24 h and then washed with PBS 3 times. MLFs were then incubated with 400 nM SD208 for 24 h and subjected to protein analyses as previously described (Kapoun et al., 2006). Individual experiment was repeated at least thrice. 2.7. Statistical analyses Data are represented as mean and standard error (SE). The data were analysed by Dunnett’s multiple comparison tests between the control group and treatment group at each time point. A p-value below 0.05 or 0.01 was considered significant. 3. Results 3.1. DDAC induces pulmonary fibrosis Histopathological analysis did not show treatment-related changes in the lungs of control mice when the intratracheal vehicle (PBS) was instilled into the lungs (Fig. 1(A)). On day 1 after exposure to DDAC, no bronchiolar or alveolar lesions were evident (Fig. 1(B)), despite the cytotoxicity demonstrated in the bronchoalveolar lavage (BAL) fluid as previously reported (Ohnuma et al., 2011). On days 3 and 7, mice exhibited fibrotic lesions in their lungs; their distribution and severity increased in a timedependent manner (Fig. 1(C) and (D)). The fibrotic lesions consisted
Fig. 1. DDAC induces fibrosis in the lung. Mice were intratracheally instilled with DDAC or vehicle control (Control), and lung samples were collected 1, 3, or 7 days after treatment for histopathology. Representative images of the lung stained with H&E (A–D) and Masson trichrome (E), and immuhistochemically stained for ␣-SMA (F) from the vehicle control (A) and DDAC-treated mice at 1 (B), 3 (C), and 7 (D–F) days. No abnormalities are detected in the control group (A) and treated group 1 day after treatment (B). Fibrotic lesions are observed 3 days after treatment (C), followed by profound development of pulmonary fibrosis 7 days after treatment (D). At higher magnification of fibrotic lesions, deposition of collagen fibres (E) and accumulation of myofibroblasts (F) are evident 7 days after treatment. n = 3–4 per group. Scale bar = 100 (A–D) and 20 m (E,F).
Fig. 2. DDAC induces mRNA of Tgf-s, Bmps, Grem1, and type I procollagen in lung tissues. Mice were treated as described in Fig. 1 and lung tissues were subjected to real-time RT-PCR for Tgf-ˇ1, -ˇ2, and -ˇ3 (A); Col1˛1 (B) and Col1˛2 (C); Bmp-2 (D), -4 (E), and -7 (F); and Grem1 (G). The data were normalised to Rps18 mRNA. Values are presented as the mean and SE (n = 3–5 per group). *p < 0.05 and **p < 0.01, as determined by Dunnett’s multiple comparison test.
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of high amounts of collagen fibres (Fig. 1(E)) and proliferation of myofibroblasts, which tested positive for ␣-SMA (Fig. 1(F)). Infiltrated inflammatory cells consisted of macrophages, neutrophils, and lymphocytes: neutrohpils were evidently infiltrated on day 3 and macrophages and lymphocytes were profoundly increased on day 7. A small lymphoid follicle was observed in the fibrotic lesions (Fig. 1(D)), but no lymphocyte cuffing was present in the adjacent airways and vesicles.
3.2. TGF-ˇ/SMAD signaling in DDAC-induced pulmonary fibrosis in vivo We examined whether pulmonary fibrotic lesions were associated with expression of Tgf-ˇ1, -ˇ2, or -ˇ3; Bmp-2, -4, or -7; or Grem1 mRNA. We found a 2-fold increasein Tgf-ˇ1 mRNA in the lung samples on day 7 after exposure to DDAC (p < 0.05); Tgf-ˇ2 and -ˇ3 mRNA were not increased at any time point (Fig. 2(A)). Consistent with the expression of Tgf-ˇ1, both Col1˛1 and Col1˛2 mRNA were highly expressed on day 7 (Col1˛1, p < 0.05; Col1˛2, p < 0.01) (Fig. 2(B) and (C)). Bmp-2 mRNA was temporally expressed on day 1 (p < 0.01) (Fig. 2(D)), and expression of Bmp-7, but not Bmp-4, was reduced throughout the observation period (Bmp-7: p < 0.05 on days 1 and 7; p < 0.01 on day 3) (Fig. 2(E) and (F)). Interestingly, the mRNA level of Grem1 was high, but not significantly high, on day 7 (Fig. 2(G)). In order to elucidate the activation of TGF- signaling in treated lungs, phosphorylated SMAD2/3 was analysed by western blotting. We found that phosphorylated SMAD2/3 was profound in the lung samples on days 3 and 7 in a time-dependent manner compared to phosphorylated SMAD1/5 (Fig. 3). VIMENTIN expression on day 7, which might correspond to mesenchymal cell proliferation, seemed to be associated with phosphorylation of SMAD2/3. No clear
Fig. 3. DDAC enhances Smad phosphorylation in the lung. Mice were treated as described in Fig. 1, and subjected to western blotting. Representative images of western blotting for phosphorylated Smad2/3, total Smad2/3, phosphorylated Smad1/5, total Smad5, ␣-SMA, vimentin, and tubulin (an internal control). Stripped membranes for phosphorylated proteins were probed with total protein and tubulin; for ␣-SMA, membranes were probed with vimentin and tubulin (n = 3 per group). Higher expression of ␣-SMA in control group than those in treated group was considered to be non-specific.
time-related changes were detected in ␣-SMA and TUBULIN (an internal control) expression. The results suggested that TGF-/SMAD2/3 signaling is associated with DDAC-induced fibrogenesis. 3.3. TGF-ˇ/SMAD signaling in DDAC-treated mouse lung fibroblasts in vitro In order to evaluate the regulation of members of the TGF family in DDAC-treated mesenchymal cells, we isolated MLFs and treated them with DDAC. Tgf-ˇ1, -ˇ2, and -ˇ3, Bmp-4, and Grem1 mRNA expression was quantified using real-time RT-PCR.
Fig. 4. DDAC alters Tgf-ˇs and Bmp-4 mRNA levels in MLFs. Isolated MLFs were plated in 100-mm dishes at 8 × 104 cells/well and incubated overnight in an incubator. On the following day, the cells were treated with different doses of DDAC. After 3 h, and 1 and 2 days, cells were collected for real-time RT-PCR of Tgf-ˇ1 (A), -ˇ2 (B), and -ˇ3 (C), and Bmp-4 (D). The data were normalised to Rps18 mRNA. As compared to the control level, Tgf-ˇ1 mRNA level was increased in both a time- and dose-dependent manner (A), whereas Tgf-ˇ2 (B) and -ˇ3 (C), and Bmp-4 (D) mRNA levels were reduced. Values are presented as the mean and SE, which were obtained from four individual experiments. *p < 0.05 and **p < 0.01, as determined by Dunnett’s multiple comparison test at each time point.
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Fig. 5. Time-dependent reduction of expression of Grem1 mRNA level in MLFs. Isolated MLFs were treated as described in Fig. 4. After 3 h, and 1 and 2 days, cells were collected for real-time RT-PCR of Grem1. The data were normalised to Rps18 mRNA. Grem1 mRNA level was reduced in a time-related manner without dependence on the DDAC concentration. Values are presented as the mean and SE, which were obtained from four individual experiments. *p < 0.05 and **p < 0.01, as determined by Dunnett’s multiple comparison test at each time point.
Tgf-ˇ1 mRNA expression was increased in both a time- and dosedependent manner compared to the control level (Fig. 4(A): 2 M DDAC, p < 0.01 on day 2; 5 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2), whereas Tgf-ˇ2 (Fig. 4(B); 2 M DDAC, p < 0.01 on day 2; 5 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2) and -ˇ3 (Fig. 4(C): 2 M and 5 M DDAC, p < 0.01 on days 1 and 2), and Bmp-4 (Fig. 4(D); 2 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2; 5 M DDAC, p < 0.01 on day 2) mRNA levels were considerably reduced. Grem1 mRNA level was higher in DDAC treatment than the control level but reduced in a time-related manner without dependence on the DDAC concentration (Fig. 5; 2 M DDAC, p < 0.05 on day 1 and p < 0.01 on day 2). Bmp-2 and -7 mRNA were not detected in MLFs. DDAC weakly cancelled the time-dependent reduction of phosphorylation of SMAD2/3 (Fig. 6). The basal level of Bmp-4 mRNA appeared to increase in a time-dependent manner (Fig. 4(D)), but no significant increase of phosphorylated SMAD1/5 was observed during the treatment period (Fig. 6). Pretreatment with SD208 – a TGF-RI (ALK5) kinase inhibitor – attenuated DDACinduced ␣-SMA and VIMENTIN expression via suppression of the phosphorylation of SMAD2/3 (Fig. 7). 4. Discussion A number of environmental and occupational agents play a role in the pathogenesis of chronic lung disease such as pulmonary fibrosis, and COPD (Yoshida and Tuder, 2007; Yoshida et al., 2011). DDAC formulations are diluted in water to treat premises and equipment by fogging, aerosol sprays, and/or low- and highpressure spraying (US EPA. 2006). Vincent et al. reported that DDAC
Fig. 6. DDAC weakly cancels a time-related reduction of phospo-Smad2/3 in MLFs. Isolated MLFs were treated as described in Fig. 4, using a dose of 5 M DDAC. Cells were collected for western blotting of phosphorylated Smad2/3, total Smad2/3, phosphorylated Smad1/5, total Smad5, ␣-SMA, vimentin, and tubulin. Results were obtained from three individual experiments. Highly nonspecific phosphorylation of Smad 2/3 was noted 3 h after, independent on DDAC treatment.
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Fig. 7. DDAC enhances SMAD phosphorylation via TGF-RI (ALK5) in MLFs. Isolated MLFs were treated with 5 M DDAC for 24 h as described in Fig. 4 and then incubated for another 24 h in the presence or absence of SD208 – a TGF-RI (ALK5) kinase inhibitor. Cells were collected for western blotting of phosphorylated Smad2/3, total Smad2/3, phosphorylated Smad1/5, total Smad5, ␣-SMA, vimentin, and tubulin. Pretreatment with SD208 attenuated DDAC-induced phosphorylation of Smad2/3 and ␣-SMA and vimentin expression. Results were obtained from three individual experiments.
would not contaminate the indoor hospital atmosphere during the disinfection process because of its insignificant volatility; however, it could contaminate working atmospheres when it is put in suspension by aerosolisation (Vincent et al., 2007). Therefore, the study was designed to determine the direct effect of DDAC on the respiratory system. Inhaled QACs cause pulmonary dysfunction including occupational asthma (Bernstein et al., 1994; Burge and Richardson, 1994; Swiercz et al., 2008); however, a key question is whether irritated QACs cause pulmonary fibrotic responses following inducible cytotoxicity and inflammation. Our findings indicated that DDAC causes the development of pulmonary fibrotic lesions following single instillation of DDAC to the lungs in mice; the change appears to be associated with TGF- and BMP signaling. Impaired BMP and its antagonist gremlin signaling have not been found in the mouse bleomycin model, one of the most common model of pulmonary fibrosis (Myllärniemi et al., 2008). However, the signaling is an important regulator for the development of human interstitial lung diseases (ILDs) (Koli et al., 2006; Pegorier et al., 2010). Therefore, our data suggested that intratracheally applying DDAC to the lung might have an impact on the investigation of lung fibrosis. We previously reported that the inflammatory phase was accompanied by remodeling (i.e. fibrosis), which was evident in Masson trichrome stains and mRNA expression of type I collagen (Ohnuma et al., 2010). In the present study, we attempted to clarify the molecular targets of DDAC-induced pulmonary fibrosis in mice. Our data suggest that TGF-1 and SMAD2/3 signaling might play a role in fibrogenic lungs, which is consistent with the observation in several lines of animal models for pulmonary fibrosis (Bonniaud et al., 2004; Zhao et al., 2002). This finding was confirmed by in vitro experiments that showed that DDAC increased TGF-1/SMAD2/3 signaling via TGF-RI (ALK5), and appeared to act as a maintainer of myofibroblast differentiation in isolated MLFs. This was demonstrated by using a TGF-RI (ALK5) kinase inhibitor, SD208, which inhibits TGF--induced phosphorylation of SMAD2/3 and myofibroblast transformation, as reported in human lung fibroblasts (Kapoun et al., 2006). The activation of myofibroblasts is a critical event in the pathogenesis of pulmonary fibrosis, because myofibroblasts represent the predominant source of heightened ECM (Hinz et al., 2007). TGF-1 has been mainly examined in pulmonary fibrosis, but other members of the TGF- family have not been fully investigated. Real-time RT-PCR revealed that induction of TGF-ˇ1 is associated with increases in type I collagens, Col1␣1 and Col1˛2, in fibrogenic lungs. However, Tgf-ˇ2 and -ˇ3 were not highly expressed in fibrogenic lungs. This finding is consistent with a previous report on bleomycin-treated mice (Coker et al., 1997). In IPF patients, cellular TGF-ˇ1 mRNA levels are higher than TGF-ˇ2 and
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-ˇ3 levels (Koli et al., 2006). Active TGF-1 is a major cytokine that stimulates the transcription of Col1␣2 genes, which are determined by the binding of SMAD3/4 to the TbRE cis-element in the proximal promoter (Zhang et al., 2000). TGF-2 and -3 have a potential for in vitro fibrogenic activity, which is the same as or higher than the in vitro activity of TGF-1 (Eickelberg et al., 1999). Specific expression of Tgf-ˇ1 by DDAC treatment in vivo and in vitro suggested that this growth factor plays an important role on mesenchymal cell response to a cytotoxic stimulus. We provided evidence that BMPs could be involved in DDACinduced pulmonary damage. Bmp-7 mRNA was persistently reduced after DDAC instillation, suggesting that BMP-7 is a negative regulator of fibrosis. Indeed, BMP-7 has been known to be an antagonist against TGF-1-dependent fibrogenic activity in mouse pulmonary myofibroblasts (Izumi et al., 2006). However, the role of BMP-7 in fibrosis is controversial, as BMP-7 treatment significantly reduces collagen deposition in asbestos-treated mice (Myllärniemi et al., 2008) but not in bleomycin-treated mice (Murray et al., 2008). Because the levels of Bmp-2, but not Bmp-4, were temporally increased after DDAC instillation, they might be mediated by cellular stresses, such as inflammatory stimuli, oxidative stress (Csiszar et al., 2006), and/or hypoxia (Takahashi et al., 2006). No alteration of BMP-4 levels has been reported in lung samples of IPF patients, but IPF fibroblasts are less responsive to exogenous BMP-4 (Koli et al., 2006). According to our in vitro findings, Bmp-4 mRNA levels under control media were increased in a time-dependent manner, but treatment with DDAC cancelled this background upregulation. The downregulation of Bmp-4 by DDAC seems to be consistent with the idea that BMP-4 inhibits growth and induces apoptosis in myofibroblasts (Koli et al., 2006). Lately, it is reported that BMP-4 reduces TGF-1-mediated ECM production, while BMP-7 inhibits TGF-1-induced myofibroblast transformation in normal human lung fibroblast (Pegorier et al., 2010). Because of lack of expression of Bmp-7 in isolated MLFs, further experiments will be required using epithelial cell and/or other cells to clarify the role of BMPs in the progression of pulmonary fibrosis after the intratracheal instillation of DDAC. The upregulation of gremlin (a BMP inhibitor) in our lung samples, is supported by previous findings that TGF-1 induces GREMLIN expression in A549 lung epithelial cells, and that IPF fibroblasts and their lung samples have a high level of GREMLIN mRNA and its protein (Koli et al., 2006; Myllärniemi et al., 2008). Pulmonary Gremlin mRNA levels are also upregulated in asbestostreated mice (Myllärniemi et al., 2008). Farkas et al. (2011) show that transient Gremlin overexpression results in alveolar epithelial injury, followed by modest lung tissue fibrosis that is partly reversible. In our experiment, however, there were individual variation in Grem1 expression in vivo, and DDAC treatment did not affect time-related reduction of its expression in vitro. At this point, the role of Grem1, as well as BMP-4, and -7 remains controversial and their function on DDAC-mediated response in vitro and in vivo should be further investigated. A series of our experiments indicate that exposure to DDAC provides evidence of a three phase model of wound repair: injury, inflammation, and repair (Wilson and Wynn, 2009). DDAC increases LDH activity as well as protein concentration in the BAL fluid (as cytotoxicity markers), recruits inflammatory cells, together with expression of several chemokine and cytokines in the BAL fluid (as inflammation markers), and enhances expressions of surfactant protein D and hem oxygenase-1 mRNAs in the lung tissues (as repair responses) (Ohnuma et al., 2010, 2011). A dysregulated healing gradually evolves into a fibrotic response through pathogenic processes such as EMT and proteolytic imbalance. EMT is critical for the production of myofibroblast in an established model of pulmonary fibrosis, overexpression of active TGF-1 (Kim et al., 2006), but not bleomycin model (Rock et al., 2011), based on the experiments with labelling lung epithelial cells to follow their
fate. Gremlin expression in DDAC-treated mice leads to speculations of its role in EMT, as overexpression of GREMLIN enhances TGF-1mediated EMT in A549 lung epithelial cells (Koli et al., 2006). Matrix proteinases (MMPs) degrade components of ECM and the activities of MMPs are controlled by tissue inhibitors of MMP (TIMPs), a process that is essential for a regulated healing (Lagente et al., 2005). MMPs such as MMP-2 and -9 and/or TIMPs such as TIMP-1 and -2 are increased after belomycin and asbestos exposure in mice (Swiderski et al., 1998; Tan et al., 2006), suggesting that imbalance between MMPs and TIMPs could play a role in excessive ECM production and abnormal matrix remodeling. These raise a question of how DDAC dysregulates healing process via EMT and proteolytic imbalance. Our work demonstrate a clearer understanding of the pathobiology of DDAC in pulmonary fibrosis, which might be associated with TGF- and SMAD signaling and to some extent by BMP, as reported in asbestos-, but not bleomycin-, treated lungs (Murray et al., 2008; Myllärniemi et al., 2008). Since the bleomycin-treated mice might be not a suitable model for investigating the role of BMP and gremlin, investigations of the signaling in DDAC-treated lungs would make important advances of ILDs. We doses not conclude at present that DDAC causes a severe form of ILD, because DDAC-induced fibrosis appears to be reversible following a single instillation to the lung (Ohnuma et al., 2010). The reversible effect seems to be similar to the study of transient overexpression of Gremlin (Farkas et al., 2011). A repeated dose study of DDAC is required to confirm the potential reversibility of the pulmonary fibrosis. The present study provides a better understanding of the health problems associated with environmental contaminants in lung disease. Acknowledgements We would like to thank Yuko Chiba, Mutsumi Kumagai, Takako Kazami, Yukie Sakano, Satoshi Akema, Chizuko Tomiyama, and Kayoko Iijima for their assistance in tissue preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etp.2013.02.003. References Argy G, Bricout F, d’Hermies F, Cheymol A. Study of prophylaxis by didecyl dimethyl ammonium chloride against herpes simplex virus infection in nude mice. Comptes Rendus de l Academie des Sciences Serie III, Sciences de la Vie 1999;322:863–70. Bernstein JA, Stauder T, Bernstein DI, Bernstein IL. A combined respiratory and cutaneous hypersensitivity syndrome induced by work exposure to quaternary amines. Journal of Allergy and Clinical Immunology 1994;94:257–9. Bobik A. Transforming growth factor-s and vascular disorders. Arteriosclerosis, Thrombosis, and Vascular Biology 2006;26:1712–20. Bonniaud P, Kolb M, Galt T, Robertson J, Robbins C, Stampfli M, Lavery C, Margetts PJ, Roberts AB, Gauldie J. Smad3 null mice develop airspace enlargement and are resistant to TGF--mediated pulmonary fibrosis. Journal of Immunology 2004;173:2099–108. Burge PS, Richardson MN. Occupational asthma due to indirect exposure to lauryl dimethyl benzyl ammonium chloride used in a floor cleaner. Thorax 1994;49:842–3. Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois RM, Jeffery PK, McAnulty RJ. Transforming growth factors-1, -2, and -3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin-induced lung fibrosis. American Journal of Pathology 1997;150:981–91. Csiszar A, Ahmad M, Smith KE, Labinskyy N, Gao Q, Kaley G, Edwards JG, Wolin MS, Ungvari Z. Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype. American Journal of Pathology 2006;168:629–38. Dejobert Y, Martin P, Piette F, Thomas P, Bergoend H. Contact dermatitis from didecyldimethylammonium chloride and bis-(aminopropyl)-lauryl amine in a detergent-disinfectant used in hospital. Contact Dermatitis 1997;37:95–6. Dickey P. Guidelines for selecting wood preservatives. For the San Francisco department of the environment; 2003, The guidelines are available at http://www.sfenvironment.org/downloads/library/preservatives.pdf
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