Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice

Experimental and Toxicologic Pathology 62 (2010) 643–651

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Didecyldimethylammonium chloride induces pulmonary inflammation and fibrosis in mice Aya Ohnuma a, Toshinori Yoshida a,, Haruka Tajima a, Tomoki Fukuyama b, Koichi Hayashi b, Satoru Yamaguchi c, Ryoichi Ohtsuka c, Junya Sasaki a, Junko Fukumori a, Mariko Tomita a, Sayuri Kojima a, Naofumi Takahashi a, Yukiko Takeuchi a, Maki Kuwahara a, Makio Takeda c, Tadashi Kosaka b, Nobuaki Nakashima a, Takanori Harada d a

Laboratory of Pathology, Toxicology Division, Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan Laboratory of Acute Toxicology and Immunotoxicology, Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan Laboratory of Molecular Toxicology, Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan d Toxicology Division, Institute of Environmental Toxicology, Uchimoriya-machi 4321, Joso, Ibaraki 303-0043, Japan b c

a r t i c l e in fo

abstract

Article history: Received 21 April 2009 Accepted 24 August 2009

Didecyldimethylammonium chloride (DDAC) is used worldwide as a germicide, in antiseptics, and as a wood preservative, and can cause adverse pulmonary disease in humans. However, the pulmonary toxicity of DDAC has not yet been thoroughly investigated. Mice were intratracheally instilled with DDAC to the lung and the bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess dose- and time-related pulmonary injury. Exposure to 1500 mg/kg of DDAC caused severe morbidity with pulmonary congestive oedema. When the BAL fluid from survivors was examined on day 3 after treatment, exposure to 150 mg/kg of DDAC caused weakly induced inflammation, and exposure to 15 mg/kg did not cause any visible effects. Next, we observed pulmonary changes that occurred up to day 20 after 150 mg/kg of DDAC exposure. Pulmonary inflammation peaked on day 7 and was confirmed by expression of interleukin-6, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP)1a, MIP-1b, and regulated upon activation, normal T-cell expressed and secreted in the BAL fluid; these changes were accompanied by altered gene expression of their chemokine (C–C motif) receptor (Ccr) 1, Ccr2, Ccr3, and Ccr5. Cytotoxicity evoked by DDAC was related to the inflammatory changes and was confirmed by an in vitro study using isolated mouse lung fibroblasts. The inflammatory phase was accompanied or followed by pulmonary remodeling, i.e., fibrosis, which was evident in the mRNA expression of type I procollagen. These results suggest that administering DDAC by intratracheal instillation causes pulmonary injury in mice, and occupational exposure to DDAC might be a potential hazard to human health. & 2009 Elsevier GmbH. All rights reserved.

Keywords: Alkyl ammonium Lung Chemokine Receptor Fibrosis Type I procollagen

Introduction DDAC [C10H21N(CH3)2C10H21  Cl], a representative dialkyl-quaternary ammonium compound (QAC), is used as a detergent in wood preservatives, as a disinfectant against pathogens, and in other applications (Dickey, 2003; Kanazawa et al., 1994; Skaliy et al., 1980; Walsh et al., 2003). This QAC is most commonly used as an antisapstain fungicide and is employed in the forestry industry to prevent the growth of fungi that can result in dark stains on softwood lumbera (Gray et al., 2005; Juergensen et al., 2000). DDAC-treated wood has increased resistance against termite attack (Hwang et al., 2007). At present, DDAC-containing

 Corresponding author. Tel.: + 81 297 274521; fax: + 81 297 274518.

E-mail address: [email protected] (T. Yoshida). 0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.08.007

wood preservatives have been useful alternatives for hazardous chromated copper arsenate (CCA), which is one of the most commonly used waterborne wood preservatives worldwide (Dickey, 2003). DDAC formulations are also added directly to water in swimming pools, spas, and humidifiers and used in institutional, commercial, industrial, and residential settings by fogging, flood, immersion, wiping, mopping, aerosol spray, and low and high-pressure spray (USEPA, 2006). The final concentrations 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). The action of DDAC in relation to the cell membrane causes the leakage of the intracellular molecules (Yoshimatsu and Hiyama,

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2007), together with autolysis and the subsequent death of Escherichia coli and Staphylococcus aureus (Ioannou et al., 2007). Occupational exposure to QACs including DDAC, therefore, has been known to cause contact dermatitis and conjunctivitis (Dejobert et al., 1997) and asthma (Bernstein et al., 1994; Burge and Richardson, 1994; Purohit et al., 2000) among professionals working in healthcare and cleaning. Although DDAC dose not seem to contaminate the indoor hospital atmosphere during the disinfection process, it can contaminate working atmospheres if it is put in suspension by aerosolisation (Vincent et al., 2007). It is to be expected that the same health concerns exist for veterinary professionals, as DDAC is a useful disinfectant in preventing the entry and spread of infectious disease agents including enveloped

and non-enveloped viruses in domestic animals (Shirai et al., 1997, 2000). The toxicology information currently available for DDAC is limited (BIBRA, 1990; USEPA, 2006). The mode of action of DDAC hypothetically provides human heath concerns, particularly if people have been unknowingly exposed to this QAC or have experienced prolonged workplace exposure. Our preliminary exposure to DDAC to the lung suggested that this QAC is potentially proinflammatory in C57BL/6J mice; thus we hypothesized that DDAC causes inflammation, followed by fibrosis in this strain, which is sensitive to bleomycin-induced fibrosis (Schrier et al., 1983). Our initial aim was to understand the threats to the respiratory system; hence, we investigated whether the

Table 1 Primer set used. Gene

Gene bank acc.

Primers

Product (bp)

Ccr1

NM_009912.4

F: 50 -AGCATGACATTCTGCTCAGCTCTC-30 R: 50 -GTGCTCAGATCATGAAGCCTATTCC-30

78

Ccr2

NM_009915

F: 50 -GAGGCTGTCAGGACTGAGTGAGA-30 R: 50 -ATTTGAGAGCCCTGCTCACTTTC-30

88

Ccr3

NM_009914.4

F: 50 -GGCACTATGCAAATAACCCATGAA-30 R: 50 -GGGTCTGTGTGCCAGAATAATGAA-30

179

Ccr5

NM_009917.5

F: 50 -CTACCACACCGGGACTGTGAAAC-30 R: 50 -TCAAACTATGGAAACAGCCCTCATC-30

107

Col1a1

NM_007742

F: 50 -TGGACCAGCAGACTGGCAAC-30 R: 50 -CCACAAGGGTGCTGTAGGTGAA-30

108

Col1a2

NM_007743

F: 50 -GCACCACTTGTGGCTTCTGACTA-30 R: 50 -ACCTCAGTTCGTGTCAGCCTTG-30

142

Rps18

NM_011296

F: 50 -AGGATGTGAAGGATGGGAAG-30 R: 50 -ACGAAGGCCCCAAAAGTG-30

128

Fig. 1. DDAC recruits inflammatory cells in the BAL fluid. Mice were intratracheally instilled with 150 mg/kg of DDAC, and BAL fluid was collected on days 7, 13, or 20 after the treatment. Control mice (Control) received the vehicle were sacrificed on day 20 after the treatment. (A) Total cell, (B) macrophage, (C) neutrophil, and (D) lymphocyte counts. Values are represented as mean and SEM (n= 4 or n= 5 per group). *Po 0.05, when compared with the control group, as demonstrated by Steel’s multiple comparison test.

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instillation of DDAC in the lungs resulted in adverse pulmonary injury in mice.

Materials and method Animal treatment Male C57BL/6J mice were obtained from Charles River, Japan. The mice were housed in cages maintained under suitable conditions with regard to temperature (24 1C 72 1C), humidity (55%715%), ventilation (continuous circulation of fresh air), and illumination (a 12-h light/dark cycle). Each mouse was maintained in a separate cage, and each 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 82.2% using the Kjeldahl method to determine the

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amount of nitrogen, and was 87.2% from analysing the chloride ion using silver nitrogen titrimetry; water concentration was 1.3%, and the contents of the other were 11.5% or 16.5%. In the present study, the actual concentrations for the preparation of dosing solutions were normalized by the purity of DDAC, 87.2%. For the dose-related study, the mice (aged 8–12 weeks; n=3 per group) were anesthetised with 50 mg/kg of pentobarbital. Next, DDAC dissolved in PBS was intratracheally instilled into the lung at concentrations of 1000 ppm, 100 ppm, or 10 ppm which consisted of 1500, 150, or 15 mg/kg, respectively, because we set that the volume administered was 1.5 mL/kg. The intratracheal instillation was performed as previously described (Bogaerts and Durville-van der, 1972). Survivors treated with DDAC and controls treated with PBS were sacrificed 3 days after administration. For the time-course study, mice who intratracheally received 150 mg/kg of DDAC (n=4–5) in PBS were sacrificed on days 7, 13, or 20 after administration. Control mice who received PBS were sacrificed on day 20 after administration. These doses of DDAC were suitable for evaluating the toxicity, because 1000 and 100 ppm of DDAC (the purity: 50%) were used for a skin patch test in a DDAC-sensitive patient (Dejobert et al., 1997), and 0.5–26320 ppm of DDAC have been achieved from variable formulations (USEPA, 2006).

Fig. 2. DDAC induces cytokine and chemokine expression in the BAL fluid. Mice were treated as described in Fig. 1, and subjected to cytokine and chemokine analyses for IL-6 (A), MCP-1 (B), MIP-1a (C), MIP-1b (D), and RANTES (E) in the BAL fluid. Values are represented as mean and SEM (n= 4 or n= 5 per group). *P o0.05, when compared with the control group, as demonstrated by Steel’s multiple comparison test.

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Tissue preparation and bronchoalveolar lavage Lavage of the lung was performed thrice via the trachea, using 1 mL of cold PBS. The BAL supernatants obtained during the first stage (approximately 0.7–0.8 mL) were kept aside to assess the protein concentration, lactate dehydrogenase (LDH) activity, and cytokine/chemokine expression therein using the methods described below. The BAL samples were pooled and the total cell count was determined using the ADVIA120 Hematology System (Bayer Corporation). The samples were cytocentrifuged and ¨ stained with May–Grunwald–Giemsa, after which the leukocyte count was determined under a light microscope (400  ); the count was found to be 300 cells. The aliquots that were initially kept aside were frozen and stored at  80 1C. Furthermore, the right and middle lobes of the lungs were resected and frozen, and stored at 80 1C. The left lobe was infused with low-meltingpoint agarose and then fixed with 10% neutral-buffered formalin. The formalin-fixed lungs were routinely processed and embedded in paraffin. Cytokine and chemokine expression In the BAL fluid, interleukin-6 (IL-6), monocyte chemotactic protein-1(MCP-1), macrophage inflammatory protein (MIP)-1a, MIP-1b, and regulated upon activation, normal T-cell expressed and secreted (RANTES) were measured using a flow cytometric bead array assay kit (BD Biosciences), in accordance with the manufacturer’s protocol. Histopathology The paraffin-embedded lung tissues were sectioned and stained with haematoxylin and eosin (H&E) and with Masson’s

trichrome stain. Immunohistochemical analysis for a-smooth muscle actin (a-SMA) was performed using a previously reported method (Takeuchi et al., 2002). RT-PCR Total RNA was isolated from the right and middle lobes of the lung using the RNeasy Mini Kit (Qiagen), and 100 ng of this sample was reverse-transcribed in a 50-mL reaction mixture using the TaqMans Reverse transcription Reagents (Applied Biosystems), according to the manufacturer’s protocol. PCR primers (listed in Table 1) for the genes encoding the following proteins were purchased from Takara Bio Inc., Japan: chemokine (C–C motif) receptor 1 (Ccr1), Ccr2, Ccr3, Ccr5, collagen 1a1 (Col1a1) and 1a2 (Col1a2), and ribosomal protein S18 (Rps18). The corresponding cDNAs were amplified by ABI PRISMs 7700 (Applied Biosystems) with a primer set and Power CYBRs 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 and the data acquired for each sample was normalised to the expression levels recorded for the housekeeping gene Rps18. RT-PCR was repeated at least twice to obtain consistent results from same samples. Cell culture For isolation of mouse lung fibroblast (MLF), the mice were anesthetised with ether and the lungs were washed with cold PBS through the heart and pulmonary artery. The lungs were minced to about 1-mm3 pieces with 1 mg/ml of collagenase type IV (Worthington Biochemical Co.) and incubated at 37 1C for 40 min. The suspension was passed through a 75-mm mesh to remove fragments and was resuspended in IMDM (Invitrogen) containing

Fig. 3. DDAC alters expression of Ccr mRNA in the lung. Mice were treated as described in Fig. 1, and subjected to RT-PCR for Ccr1 (A), Ccr2 (B), Ccr3 (C), and Ccr5 (D) in the lung. Values are represented as mean and SEM (n= 4 or n= 5 per group). *Po 0.05, when compared with the control group, as demonstrated by Dunnett’s (B–D) or Steel’s (A) multiple comparison test.

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100 U/mL of penicillin and 100 mg/mL of streptomycin, and 10% foetal bovine serum. The isolated cells were seeded into 100-mm dish and incubated at 37 1C in a humidified 5% CO2 incubator; the culture medium was changed twice a week. The isolated cells were used for in vitro experiments at passes 5 and 6. MTT assay Cytotoxicity was determined by an MTT metabolism assay according to a previously reported method (Okeson et al., 2003). Briefly, the isolated cells were seeded into a 98-well plate (100 mL/ well) at 2  104/well, allowed to reach confluent, and were treated with DDAC dissolved in PBS at indicated doses. After 24 h, the cells were exposed to 20 mL of fresh MTT solution (5 mg/mL, SigmaAldrich) for 3 h, and were washed with dimethyl sulfoxide. Absorbance was measured at 540 nm using a microplate reader. Cell death was expressed as absorbance relative to the absorbance of the control. Individual experiments were repeated thrice in triplicate. Statistical analyses Data are represented as the mean and standard deviation (SEM). The experimental data were analysed by one-way ANOVA followed by Dunnett’s multiple comparison test or Kruskal–Wallis one-way ANOVA followed by Steel’s multiple comparison test. A P-value less than 0.05 was considered significant.

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expressed collagen, as demonstrated by a Masson trichrome stain (Figs. 5B and D), and were associated with proliferation of myofibroblast, as revealed by a-SMA immunostaining (Figs. 5B and D). On days 13, inflammatory responses and myofibroblast proliferation seemed to be reduced (Figs. 5E and F), but collagen production remained evident as shown with a Masson trichrome stain (Fig. 5F). To further assess collagen production in the lung, we performed real-time RT-PCR for Col1a1 and Col1a2. Col1a1 and Col1a2 mRNA reached a peak on day 13, and returned to the control level on day 20 (Figs. 6A and B). In consistent, lung injury became improved on day 20 (Figs. 5G and H). In vitro study of mouse lung fibroblasts Cytotoxicity against pathogens by DDAC exposure is crucial to prevent their growth. Our in vivo study showed that DDAC enhanced LDH activity and enhanced protein in the BAL fluid as demonstrated above (Figs. 4A and B). To elucidate in vitro cytotoxicity, we isolated MLF which are useful and valuable tools for investigating cell function and the response contributing to pulmonary fibrosis (Kapoun et al., 2006; Kuang et al., 2002). We assessed cytotoxicity in the isolated MLF with an MTT assay, showing that DDAC treatment clearly reduced cell viability in a dose-related manner (Fig. 7).

Results Dose-related study in vivo Initially, we intratracheally instilled DDAC in the lung to determine a dose-related response. A high dose (1500 mg/kg) of DDAC treatment caused severe morbidity with pulmonary congestive oedema. The mid dose (150 mg/kg) of DDAC moderately increased inflammation, while the low dose (15 mg/kg) did not, these results were based on counting the inflamed cells in the BAL fluid obtained from survivors on day 3 after instillation (data not shown). Therefore, we selected 150 mg/kg of DDAC for the following time-course study. Time-course study in vivo We assessed pulmonary inflammation using recovered BAL fluid from animals sacrificed on days 7, 13, or 20 after administration of 150 mg/kg of DDAC. The numbers of total cells (Fig. 1A), macrophages (Fig. 1B), neutrophils (Fig. 1C), and lymphocytes (Fig. 1D) rose on day 7, and decreased thereafter. Neutrophil and lymphocyte counts, however, remained higher on day 20 than the control group (Figs. 1C and D). The increase in inflammatory cell counts on day 7 was associated with a significant increase in the expression of IL-6, MCP-1, MIP-1a, MIP-1b, and RANTES in the BAL fluid (Figs. 2A–E). The expression level of MCP-1 was considerably higher than that of the other cytokines and chemokines. Interestingly, the mRNA levels of their receptors Ccr1, Ccr2, and Ccr5 in the lung were increased on Day 7; in contrast, the mRNA level of Ccr3 was decreased (Figs. 3A–D). The inflammatory changes were consistent with cytotoxicity, as demonstrated by protein concentration (Fig. 4A) and LDH activity (Fig. 4B) in the BAL fluid. On histological examination, we found focal inflammation in the alveolar spaces and ducts, along with evidence of fibrosis on day 7 in animals exposed to 150 mg/kg of DDAC but not in control animals (Figs. 5A and C). Fibrotic tissues

Fig. 4. DDAC induces cytotoxicity in the BAL fluid. Mice were treated as described in Fig. 1, and subjected to cytotoxicity analysis by demonstrating protein concentration (A) and LDH activity (B) in the BAL fluid. LDH activity was represented as a ratio relative to the activity in the control samples. Values are represented as mean and SEM (n= 4 or n=5 per group). *Po 0.05, when compared with the control group, as demonstrated by Steel’s multiple comparison test.

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Fig. 5. DDAC induces inflammation and fibrosis in the lungs. Mice were treated as described in Fig. 1. Lung tissue samples were subjected to histopathological examination. Representative images of the lung stained with H&E (A, C, E, and G) and Masson trichrome staining (B, D, F, and H) from the vehicle control (A and B) and DDAC-treated mice on day 7 (C and D), 13 (E and F), or 20 (G and H) (n =4 or n= 5 per group). Scale bar = 100 mm (A, C, E, and G), 50 mm (B, D, F, and H). Inset (B, D, F, and H): Representative images of the lung immunohistochemically stained with anti-a-SMA antibody from the vehicle control (B) and DDAC-treated mice on day 7 (D), 13 (F), or 20 (H) (n= 4 or n= 5 per group). Scale bar =20 mm.

Discussion The results presented here are important to our understanding of DDAC – one of the most popular QACs – as a cytotoxic, proinflammatory, and profibrogenic substance following a single instillation of this QAC at a sublethal dose level (150 mg/kg) to the lungs of mice. The inflammatory changes caused by DDAC administration were probably associated with cytotoxicity because both were evident at the same time point (day 7) after DDAC exposure. DDAC is a membrane-active agent, which is rapidly taken up via the cytoplasmic membrane – independently

of the treatment temperature – and kills bacterial cells 5 min after treatment (Ioannou et al., 2007). Together with critical membrane damage, autolysis is initiated in cells and is thought to be due to the breakdown of RNA materials by activated RNases, and this coincides with potassium depletion from cells (Ioannou et al., 2007). Intriguingly, Pseudomonas fluorescents degrade DDAC to produce decyldimethylamine and demethylamine (Nishihara et al., 2000), which is particularly toxic to the respiratory systems of rats and mice (Buckley et al., 1985). We speculate that DDAC or its metabolites directly activate or kill alveolar macrophages and parenchymal cells, adopting a similar mode of action against

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Fig. 7. DDAC induces dose-dependent cytotoxicity in mouse lung fibroblasts. Mouse lung fibroblasts were plated in a 96-well plate at 2  104/well and were incubated overnight in an incubator. On the following day the cells were treated with different doses of DDAC. After 24 h, viable cells were determined by MTT assay, measured spectrophotometrically at 540 nm. Cytotoxicity was quantified relative to the control cells. Results were obtained from three experiments and the bars on the curved line denote SEM.

Fig. 6. DDAC induces type I procollagen mRNA in the lung. Mice were treated as described in Fig. 1, and subjected to real-time RT-PCR for type I procollagen, Col1a1 (A) and Col1a2 (B), normalised to the levels of Rps18 mRNA. Values are represented as mean and SEM (n= 4 or n= 5 per group). *P o0.05, when compared with the control group, as demonstrated by Dunnett’s multiple comparison test.

bacteria. The idea of direct action is consistent with in vitro data on cytotoxicity, as demonstrated by an MTT assay. Furthermore, cytotoxicity is considered to be associated with an increased risk of death from pulmonary congestive oedema which was demonstrated at a lethal dose level (1500 mg/kg). A similar finding has been reported in dead rats aspirated with cationic surfactant benzalkonium chloride, which is a mixture of alkylbenzyldimethyl-ammonium chlorides [C6H5CH2N(CH3)2CnH2n+1  Cl] (Xue et al., 2004). A BAL cytokine analysis showed that the concentration of a typical proinflammatory cytokine IL-6; Th1 chemokines MIP-1a, MIP-1b and RANTES; and Th2 acquiescent chemokine MCP-1 were increased by DDAC administration. We found that MCP-1 was strongly induced in DDAC-instilled mice and this is consistent with responses to pulmonary irritants including cigarette smoke (Bracke et al., 2006). MCP-1 enhances Th2 cell acquisition, monocyte, NK cell, and basophil chemotaxis, and mast cell mediator release (Yoshida and Tuder, 2007). A receptor of MCP1, CCR2, which was upregulated by DDAC administration, has been known to be highly expressed in circulating dendritic cells and monocytes, mobilising into the tissue by the expression of MCP-1

in cells of blood vessel linings (Vanbervliet et al., 2002). Dendritic cells posses various other receptors, CCR1 for RANTES, CCR5 for MIP-1a and MIP-1b, and RANTES (Toews, 1991); both receptors were also upregulated by DDAC administration in this study, as noted in bleomycin-treated mice (Ishida et al., 2007; Tokuda et al., 2000). Indeed, dendritic cells migrate to BAL fluid and alveolar parenchyma in response to cigarette smoke and/or ovalbumin, which also recruit CD4 + and CD8 + T-cells (D’hulst et al., 2005; Moerloose et al., 2005). Similar regulation could occur in DDACinstilled mice, because lymphocyte recruitment was noted on not only day 7 but also day 20 after administration. A decrease in the Ccr3 mRNA level in our study is inconsistent with the finding that bleomycin stimulates the expression of the Ccr3 mRNA in CBA/J mice with a maximal effect on day 3 after of treatment and its level continued to rise on days 7 and 14 when compared with the control groups (Huaux et al., 2005). However, a reduction of receptor expression might reflect its ligand-induced internalization. For instance, RANTES treatment induces internalization of CCR3 and decreases the protein and mRNA levels; those are involved in regulating activation of eosinophils (Dulkys et al., 2001; Zimmermann et al., 1999). Accordingly, DDAC is suggestive of mediating acquired immunity in the lung, and this hypothesis is consistent with the epidemiological data that QACs is one of risk factor of asthma in human (Bernstein et al., 1994; Burge and Richardson, 1994; Purohit et al., 2000). MCP-1 is also involved in fibrosis via the regulation of profibrotic cytokine generation and matrix formation (Agostini and Gurrieri, 2006). This belief is supported by the fact that a MCP-1 receptor, CCR2 plays a critical role in modulating the fibrotic process, as CCR2-knockout mice are protected from fibrosis induced by fluorescein isothiocyanate (FITC) or bleomycin (Gharaee-Kermani et al., 2003; Moore et al., 2001). This protective effect is associated with suppressed macrophage infiltration and macrophage-derived matrix proteinase production (Okuma et al., 2004). Interestingly, MCP-1 stimulates isolated lung fibroblasts to produce procollagen that is dependent on transforming growth factor-b1 (TGF-b1), a factor responsible for fibrogenesis (GharaeeKermani et al., 1996; Hogaboam et al., 1999). Additionally, other chemokines such as MIP-1a, MIP-1b, and/or RANTES are involved in pulmonary inflammation and fibrosis induced by IL-13 (Lee

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¨ a¨ et al., 2006), mustard gas (Emad et al., 2006), wood dust (Ma¨ att and Emad, 2007), and bleomycin (Ishida et al., 2007; Tokuda et al., 2000). Bleomycin or FITC-induced lung injury is a well-known model of pulmonary fibrosis in human. In response to either chemical, evidence is considered to begin with an initiating event or injury, followed by cytokine expression and inflammatory cell recruitment in the 1st week; fibrotic responses occur from the 2nd week to the 4th week (Huaux et al., 2005; Ishida et al., 2007; Moore et al., 2001; Tokuda et al., 2000; Yara et al, 2001). In our current protocol, a similar sequence of pulmonary changes was demonstrated by DDAC administration; it seemed that the fibrotic response is more rapid but weaker than belomycin or FITC treatment. Since it remains unclear how DDAC initiates and promotes pulmonary fibrosis, followed by repairing responses, further studies are needed to clarify the contribution of profibrotic factors as well as antifibrotic factors. This unique property of pulmonary responses would be helpful in better understanding pulmonary fibrotic diseases in human. In addition, we need to explore the possibility that DDAC may influence acquired immunity in pulmonary diseases, especially asthma, because epidemiological data suggested that QACs is one of risk factor of asthma in human. Considering residential or occupational use and workplace exposure to DDAC, its toxic effect may cause a health problem that is of significant importance to humans.

Acknowledgement We are very grateful to Yuko Chiba, Mutsumi Kumagai, Chizuko Tomiyama, Takako Kazami, Yukie Jibiki, Satoshi Akema, Haruka Miura, and Kayoko Iijima for their assistance in tissue preparation and special staining. References Agostini C, Gurrieri C. Chemokine/cytokine cocktail in idiopathic pulmonary fibrosis. Proc Am Thorac Soc 2006;3:357–63. Bernstein JA, Stauder T, Bernstein DI, Bernstein IL. A combined respiratory and cutaneous hypersensitivity syndrome induced by work exposure to quaternary amines. J Allergy Clin Immunol 1994;94:257–9. BIBRA. Toxicity profile didecyldimethylammonium chloride. Carshalton, UK: BIBRA International Ltd.; 1990. Bogaerts WJ, Durville-van der Oord. Immunization of mice against encephalomyocarditis virus. II. Intraperitoneal and respiratory immunization with ultraviolet-inactivated vaccine: effect of Bordetella pertussis extract on the immune response. Infect Immun 1972;6:513–17. Bracke KR, D’hulst AI, Maes T, Moerloose KB, Demedts IK, Lebecque S, et al. Cigarette smoke-induced pulmonary inflammation and emphysema are attenuated in CCR6-deficient mice. J Immunol 2006;177:4350–9. Buckley LA, Morgan KT, Swenberg JA, James RA, Hamm TE, Barrow CS. The toxicity of dimethylamine in F-344 rats and B6C3F1 mice following a 1-year inhalation exposure. Fundam Appl Toxicol 1985;5:341–52. 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. 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. D’hulst AI, Vermaelen KY, Brusselle GG, Joos GF, Pauwels RA. Time course of cigarette smoke-induced pulmonary inflammation in mice. Eur Respir J 2005;26:204–13. Dickey P. Guidelines for selecting wood preservatives. For The San Francisco Department of the Environment, September 9, 2003 [The guidelines are available at /http://www.sfenvironment.org/downloads/library/preservatives. pdfS]. ¨ ¨ S, Kapp A, et al. IL-3 induces Dulkys Y, Kluthe C, Buschermohle T, Barg I, Knoss down-regulation of CCR3 protein and mRNA in human eosinophils. J Immunol 2001;167:3443–53. Emad A, Emad V. Elevated levels of MCP-1, MIP-alpha and MIP-1 beta in the bronchoalveolar lavage (BAL) fluid of patients with mustard gas-induced pulmonary fibrosis. Toxicology 2007;240:60–9. Gharaee-Kermani M, Denholm EM, Phan SH. Co-stimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte

chemoattractant protein-1 via specific receptors. J Biol Chem 1996 17779–84. Gharaee-Kermani M, McCullumsmith RE, Charo IF, Kunkel SL, Phan SH. CCchemokine receptor 2 required for bleomycin-induced pulmonary fibrosis. Cytokine 2003;24:266–76. Gray MA, Peake SJ, Farrell AP, Bruch R. Acute didecyl dimethyl ammonium chloride toxicity to larval lake sturgeon, Acipenser fulvescens Rafinesque, walleye Sander vitreus Mitchill, and northern pike, Esox lucius Linnaeus. Bull Environ Contam Toxicol 2005;75:890–6. Hogaboam CM, Bone-Larson CL, Lipinski S, Lukacs NW, Chensue SW, Strieter RM, et al. Differential monocyte chemoattractant protein-1 and chemokine receptor 2 expression by murine lung fibroblasts derived from Th1- and Th2-type pulmonary granuloma models. J Immunol 1999;163:2193–201. Huaux F, Gharaee-Kermani M, Liu T, Morel V, McGarry B, Ullenbruch M, et al. Role of eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycininduced lung injury and fibrosis. Am J Pathol 2005;167:1485–96. Hwang WJ, Kartal SN, Yoshimura T, Imamura Y. Synergistic effect of heartwood extractives and quaternary ammonium compounds on termite resistance of treated wood. Pest Manag Sci 2007;63:90–5. Ioannou CJ, Hanlon GW, Denyer SP. Action of disinfectant quaternary ammonium compounds against Staphylococcus aureus. Antimicrob Agents Chemother 2007;51:296–306. Ishida Y, Kimura A, Kondo T, Hayashi T, Ueno M, Takakura N, et al. Essential roles of the CC chemokine ligand 3-CC chemokine receptor 5 axis in bleomycininduced pulmonary fibrosis through regulation of macrophage and fibrocyte infiltration. Am J Pathol 2007;170:843–54. Japanese Association for Laboratory Animal Science. Guideline for animal experimentation. Exp Anim 1987;36:285–8 (In Japanese). Juergensen L, Busnarda J, Caux P-Y, Kent RA. Fate, behavior, and aquatic toxicity of the fungicide DDAC in the Canadian environment. Environ Toxicol 2000;15:174–200. Kanazawa A, Ikeda T, Endo T. Synthesis and antimicrobial activity of dimethyl- and trimethyl-substituted phosphonium salts with alkyl chains of various lengths. Antimicrob Agents Chemother 1994;38:945–52. Kapoun AM, Gaspar NJ, Wang Y, Damm D, Liu YW, O’young G, et al. Transforming growth factor-beta receptor type 1 (TGFbRI) kinase activity but not p38 activation is required for TGFbRI-induced myofibroblast differentiation and profibrotic gene expression. Mol Pharmacol 2006;70:518–31. Kuang PP, Lucey E, Rishikof DC, Humphries DE, Bronsnick D, Goldstein RH. NF-kB induced by IL-1b inhibits elastin transcription and myofibroblast phenotype. Am J Physiol Cell Physiol 2002;283:C58–65. Lee PJ, Zhang X, Shan P, Ma B, Lee CG, Homer RJ, et al. ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo. J Clin Invest 2006;116:163–73. ¨ a¨ J, Lehto M, Leino M, Tillander S, Haapakoski R, Majuri ML, et al. Ma¨ att Mechanisms of particle-induced pulmonary inflammation in a mouse model: exposure to wood dust. Toxicol Sci 2006;93:96–104. Moerloose KB, Pauwels RA, Joos GF. Short-term cigarette smoke exposure enhances allergic airway inflammation in mice. Am J Respir Crit Care Med 2005;172: 168–172. Moore BB, Paine R, Christensen PJ, Moore TA, Sitterding S, Ngan R, et al. Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol 2001;167:4368–77. Nishihara T, Okamoto T, Nishiyama N. Biodegradation of didecyldimethylammonium chloride by Pseudomonas fluorescens TN4 isolated from activated sludge. J Appl Microbiol 2000;88:641–7. Okeson CD, Riley MR, Fernandez A, Wendt JO. Impact of the composition of combustion generated fine particles on epithelial cell toxicity: influences of metals on metabolism. Chemosphere 2003;51:1121–8. Okuma T, Terasaki Y, Kaikita K, Kobayashi H, Kuziel WA, Kawasuji M, et al. C-C chemokine receptor 2 (CCR2) deficiency improves bleomycin-induced pulmonary fibrosis by attenuation of both macrophage infiltration and production of macrophage-derived matrix metalloproteinases. J Pathol 2004 594–604. Purohit A, Kopferschmitt-Kubler MC, Moreau C, Popin E, Blaumeiser M, Pauli G. Quaternary ammonium compounds and occupational asthma. Int Arch Occup Environ Health 2000;73:423–7. Shirai J, Kanno T, Inoue T, Mitsubayashi S, Seki R. Effects of quaternary ammonium compounds with 0.1% sodium hydroxide on swine vesicular disease virus. J Vet Med Sci 1997;59:323–8. Shirai J, Kanno T, Tsuchiya Y, Mitsubayashi S, Seki R. Effects of chlorine, iodine, and quaternary ammonium compound disinfectants on several exotic disease viruses. J Vet Med Sci 2000;62:85–92. Schrier DJ, Kunkel RG, Phan SH. The role of strain variation in murine bleomycininduced pulmonary fibrosis. Am Rev Respir Dis 1983;127:63–6. Skaliy P, Thompson TA, Gorman GW, Morris GK, McEachern HV, Mackel DC. Laboratory studies of disinfectants against Legionella pneumophila. Appl Environ Microbiol 1980;40:697–700. Takeuchi Y, Yoshida T, Chiba Y, Kuwahara M, Maita K, Harada T. Pulmonary sequestration with ectopic pancreatic tissue in a Wistar Hannover GALAS rat. Toxicol Pathol 2002;30:288–91. Toews GB. Pulmonary dendritic cells: sentinels of lung-associated lymphoid tissues. Am J Respir Cell Mol Biol 1991;4:204–5. Tokuda A, Itakura M, Onai N, Kimura H, Kuriyama T, Matsushima K. Pivotal role of CCR1-positive leukocytes in bleomycin-induced lung fibrosis in mice. J Immunol 2000;64:2745–51.

A. Ohnuma et al. / Experimental and Toxicologic Pathology 62 (2010) 643–651

United States Environmental Protection Agency. Reregistration eligibility decision for aliphatic alkyl quaternaries (DDAC), August 2006. [The guidelines are available at /http://www.epa.gov/oppsrrd1/REDs/ddac_red.pdfS]. Vanbervliet B, Homey B, Durand I, Massacrier C, A¨ıt-Yahia S, de Bouteiller O, et al. Sequential involvement of CCR2 and CCR6 ligands for immature dendritic cell recruitment: possible role at inflamed epithelial surfaces. Eur J Immunol 2002;32:231–42. Vincent G, Kopferschmitt-Kubler MC, Mirabel P, Pauli G, Millet M. Sampling and analysis of quaternary ammonium compounds (QACs) traces in indoor atmosphere. Environ Monit Assess 2007;133:25–30. Walsh SE, Maillard JY, Russell AD, Catrenich CE, Charbonneau DL, Bartolo RG. Activity and mechanisms of action of selected biocidal agents on Grampositive and -negative bacteria. J Appl Microbiol 2003;94:240–7.

651

Xue Y, Hieda Y, Saito Y, Nomura T, Fujihara J, Takayama K, et al. Distribution and disposition of benzalkonium chloride following various routes of administration in rats. Toxicol Lett 2004;148:113–23. Yara S, Kawakami K, Kudeken N, Tohyama M, Teruya K, Chinen T, et al. FTS reduces bleomycin-induced cytokine and chemokine production and inhibits pulmonary fibrosis in mice. Clin Exp Immunol 2001;124:77–85. Yoshida T, Tuder RM. Pathobiology of cigarette smoke-induced chronic obstructive pulmonary disease. Physiol Rev 2007;87:1047–82. Yoshimatsu T, Hiyama K. Mechanism of the action of didecyldimethylammonium chloride (DDAC) against Escherichia coil and morphological changes of the cells. Biocontrol Sci 2007;12:93–9. Zimmermann N, Conkright JJ, Rothenberg ME. CC chemokine receptor-3 undergoes prolonged ligand-induced internalization. J Biol Chem 1999;274:12611–18.