Toxicology in Vitro 28 (2014) 327–333
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Penta- and octa-bromodiphenyl ethers promote proinflammatory protein expression in human bronchial epithelial cells in vitro Eiko Koike a,⇑, Rie Yanagisawa a, Hidetaka Takigami b, Hirohisa Takano c a
Center for Environmental Health Sciences, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan c Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto 615-8530, Japan b
a r t i c l e
i n f o
Article history: Received 14 June 2013 Accepted 23 October 2013 Available online 30 October 2013 Keywords: Brominated flame retardants Polybrominated diphenyl ethers Bronchial epithelial cells Proinflammatory cytokines Epidermal growth factor receptor
a b s t r a c t Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in consumer products. Humans can be exposed to PBDEs mainly through the inhalation of air or dust. Thus, PBDEs can affect respiratory and immune systems. In the present study, we investigated whether PBDEs stimulate bronchial epithelial cells. We examined commercial penta-BDE (DE-71), octa-BDE (DE-79), and decaBDE (DE-83R). Human bronchial epithelial cells (BEAS-2B) were exposed to each PBDE for 24 h. Subsequently, the expression of intercellular adhesion molecule-1 (ICAM-1) and proinflammatory cytokines were investigated. DE-71 and DE-79, but not DE-83R, significantly increased the expression of ICAM-1, interleukin-6 (IL-6), and IL-8 in BEAS-2B. Because these remarkable effects were observed with DE-71, we further investigated the underlying intracellular mechanisms. DE-71 promoted epidermal growth factor receptor (EGFR) phosphorylation. Inhibitors of EGFR-selective tyrosine kinase and p38 mitogenactivated protein kinase effectively blocked the increase of IL-6 and IL-8. Furthermore, antagonists of thyroid hormone receptor and aryl hydrocarbon receptor significantly suppressed the increase in IL-6 and/or IL-8 production. In conclusion, penta- and octa-BDE, but not deca-BDE, might promote the expression of proinflammatory proteins in bronchial epithelial cells possibly by activating protein kinases and/ or stimulating nuclear receptors related to subsequent activation of transcriptional factors. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Brominated flame retardants (BFRs) are used in industrial and consumer products, including electronic products, textiles, and building materials, to prevent fire-related injury and property damage (Birnbaum and Staskal, 2004). Major BFRs are polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane, and tetrabromobisphenol A (Birnbaum and Staskal, 2004). BFRs are used as additive or reactive components. Additive BFRs, including PBDEs, are simply blended with polymers and are not chemically bound to products; thus, additive BFRs can easily leach into the environment (Alaee et al., 2003). In particular, PBDEs have been observed in indoor dust in many countries (Abdallah et al., 2007;
Abbreviations: AhR, aryl hydrocarbon receptor; BFRs, brominated flame retardants; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, EGF receptor; ELISA, enzyme linked immunosorbent assay; ER, estrogen receptor; HBCD, hexabromocyclododecane; ICAM, intercellular adhesion molecule; IL, interleukin; MAPK, mitogen-activated protein kinase; PBDEs, polybrominated diphenyl ethers; STAT, signal transducer and activator of transcription; TR, thyroid hormone receptor. ⇑ Corresponding author. Tel./fax: +81 29 850 2334. E-mail address: [email protected] (E. Koike). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.10.014
Batterman et al., 2010; Sjodin et al., 2008; Stapleton et al., 2005). A positive relationship was also found between PBDE concentrations in household dust and in human plasma (Karlsson et al., 2007) and breast milk (Wu et al., 2007). Besides BFRs, other chemicals such as phthalate plasticizers are present in indoor air and dust and coexist with allergens such as house dust mites. Epidemiological studies have suggested associations between phthalate exposure at home and the risk of asthma and allergies (Jaakkola and Knight, 2008). Experimental studies have also shown that phthalates can aggravate allergic diseases and/or responses in animal models (Koike et al., 2010; Larsen and Nielsen, 2007; Takano et al., 2006) and in vitro (Koike et al., 2009, 2010; Rakkestad et al., 2010). Thus, individuals with respiratory and allergy-related diseases may have a high sensitivity for indoor chemical pollutants. PBDEs include the commercial products of penta-, octa-, and deca-BDE. Each product is a mixture composed of several PBDE congeners. PBDEs are structurally similar to polychlorinated biphenyls and resist degradation in the environment. Because of their accumulation in human tissues (Hites, 2004; Inoue et al., 2006; Sjodin et al., 2003) and potential toxicity (Birnbaum and Staskal, 2004; Viberg et al., 2004), penta- and octa-BDE are banned in some
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countries or have been voluntarily withdrawn. However, their component congeners are still detected in humans and the environment. Recent concerns regarding the possible adverse health effects of BFRs have focused on their potential endocrine-disrupting effects and developmental neurotoxicity (Costa et al., 2008; Talsness et al., 2009). Because humans can be exposed to BFRs by inhaling indoor air or dust, it is important to determine the effects of BFRs on respiratory and immune systems. Recently, we examined the effects of BFRs, including PBDEs, on immune cells in atopic prone NC/Nga mice and demonstrated that these BFRs can aggravate immune/allergic responses by enhancing antigen presentationrelated molecule expression and IL-4 production in splenocytes (Koike et al., 2013). However, the effects of BFRs on respiratory and immune systems have not been completely elucidated. Activated airway epithelial cells release hematopoietic and proinflammatory cytokines that initiate immune responses and, therefore, play an essential role in respiratory diseases such as asthma. For instance, air pollutants (particulate matters and organic chemicals) can aggravate respiratory diseases by proinflammatory cytokine secretion from airway epithelial cells (Dergham et al., 2012; Fischader et al., 2008; Ghio et al., 2009). In this study, we investigated whether PBDEs affect the expression of proinflammatory proteins such as intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), and IL-8 in bronchial epithelial cells in vitro. In addition, we examined the effects of PBDEs on the signaling pathways such as activation of inflammatory-related protein kinase to evaluate the intracellular mechanisms. 2. Materials and methods
2.2. Cell culture and treatments The normal human bronchial epithelial cell line, BEAS-2B, was obtained from the European Collection of Cell Cultures (Health Protection Agency Culture Collection, Salisbury, UK). Cells were maintained in LHC-9 medium (Invitrogen™, Life Technologies Co., Carlsbad, CA, USA) in a collagen I coated culture dish (BD Biosciences, Bedford, MA, USA) at 37 °C and 5% CO2/95% air atmosphere. The cells (2 104/cm2) were seeded in a collagen I coated plate or dish and were allowed to grow to semiconfluence for three days. The culture medium was removed, and the cells were exposed to each PBDE (0.01–10 lg/ml) or 0.1% DMSO (control) in medium. The range of DE-83R concentration was limited from 0.01 to 6 lg/ml because of its solubility. We examined protein kinase activity using epidermal growth factor receptor (EGFR)-selective tyrosine kinase inhibitor (AG1478; Sigma–Aldrich), p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580; Sigma–Aldrich), and MEK inhibitor (PD98059; Sigma–Aldrich). Cells were pretreated with these protein kinase inhibitors (20 lM) for 1 h and were then exposed to DE-71 (3 lg/ml) for 24 h in the presence of these protein kinase inhibitors (10 lM). In the same manner, we examined the role of the nuclear receptor using thyroid hormone receptor (TR) antagonist (1–850; Merck KGaA, Darmstadt, Germany), aryl hydrocarbon receptor (AhR) antagonist (CH-223191; Merck KGaA), and estrogen receptor (ER) antagonist (ICI 182,780; Sigma–Aldrich). The cells were pretreated with 1–850 (10 lM), CH-223191 (10 lM), and ICI 182,780 (10 nM) for 1 h and then exposed to DE-71 (5 lg/ml) for 24 h in the presence of these nuclear receptor antagonists at half of the above-mentioned concentration. All procedures were approved by the Institutional Review Board of the National Institute for Environmental Studies.
2.1. Chemicals 2.3. Cell viability/proliferation assay Commercial penta-BDE mixture (DE-71), octa-BDE mixture (DE-79), and deca-BDE mixture (DE-83R) (Wellington Laboratories Inc., Guelph, Ontario, Canada) were used. Fig. 1a shows the structures of PBDEs. Each PBDE was dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich Co., St. Louis, MO, USA) and diluted with culture medium. Final concentration of DMSO was 0.1% in all experiments.
O
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Cells grown in a collagen I coated 96-well plate were exposed to PBDEs (0.01–10 lg/ml) for 24 h. After exposure, cells were incubated with tetrazolium salt WST-1 reagent (Takara Bio Inc., Otsu, Shiga, Japan) for 2 h. WST-1 is cleaved to soluble formazan in viable cells through a complex cellular mechanism, and the amount of formazan dye formed directly correlates to the number of viable cells. After the incubation period, we measured absorbance at 450 nm. Results are represented as a percentage of the control value.
Cells grown in a collagen I coated 12-well plate were exposed to PBDEs (0.01–10 lg/ml) for 24 h. After exposure, cells were collected by treatment with 0.25% trypsin/EDTA (GibcoÒ, Life Technologies Co.) and were washed with phosphate-buffered saline containing 0.3% bovine serum albumin and 0.05% sodium azide. The cells were incubated in the buffer for 30 min on ice with an optimal amount of anti-human ICAM-1 antibody (CD54; PE-conjugated, BioLegend, San Diego, CA, USA). Fluorescence was measured on FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). For each sample, fluorescence data was collected from 10,000 cells and the mean fluorescence intensity were analyzed by FLOWJO (Tree Star Inc., Ashland, OR, USA).
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BFRs concentration (µg/ml) Fig. 1. Chemical structure of PBDEs (a) and cytotoxicity of PBDEs on BEAS-2B (b). Cells were exposed to each PBDE (0.01–10 lg/ml) for 24 h. Cell viability/proliferation is indicated as a percentage of the control. Data are mean ± SEM of triplicate and are representative of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, DE71 versus control; p < 0.01, DE-79 versus control.
2.5. Quantitation of cytokines in culture supernatants Cells grown in a collagen I coated 12-well plate were exposed to PBDEs (0.01–10 lg/ml) for 24 h. After exposure, the culture supernatant was collected and was stored at 80 °C until measurement. The levels of IL-6, IL-8 (Thermo Scientific, Rockford, IL, USA), and
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soluble ICAM-1 (Bender MedSystems, Vienna, Austria) in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Detection limits for IL-6, IL-8, and soluble ICAM-1 were 1, 2, and 60 pg/ml, respectively. For EGF measurement, the culture medium was changed into LHC basal medium after exposure to remove EGF in LHC-9 medium. The cells were then incubated with LHC basal medium for 3 h, and the culture supernatant was collected and was stored at 80 °C until measurement. EGF content was also measured by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions, and the detection limit was 0.7 pg/ml. 2.6. Immunoassay for EGFR phosphorylation Cells grown in a collagen I coated 96-well plate were exposed to DE-71 (10 lg/ml) for 15 and 30 min. After exposure, protein kinase phosphorylation of EGFR was detected by cell-based ELISA FACE (Active Motif Japan, Shinjuku-Ku, Tokyo, Japan) according to the manufacturer’s instructions. In brief, cells were fixed with 4% formaldehyde in a culture plate and inactivated with endogenous peroxidase activity with 1% H2O2 and 0.1% NaN3. After blocking, total- and phospho-EGFR protein levels were examined. Binding of primary antibodies was performed overnight at 4 °C. After colorimetric reactions, absorbance was measured at 450 nm with a reference wavelength of 655 nm. The relative number of cells in each well was determined by Crystal Violet, which gives an OD595 reading. The total- and phospho-EGFR signals at OD450 were normalized for cell number by dividing the OD450 reading by the OD595 reading for each well. 2.7. Statistical analysis Each experiment was performed using triplicate cultures, and three to five independent experiments were repeated. The data were represented as mean ± SEM. Significance of intergroup variations was determined by one-way ANOVA or Kruskal–Wallis analysis. Differences among groups were analyzed using Dunnett’s multiple comparison test (Excel Statistics 2010, Social Survey Research Information Co., Ltd., Shinjuku-Ku, Tokyo, Japan). p < 0.05 was considered statistically significant.
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with the control (Fig. 2b: DE-71 and DE-79: 10 lg/ml, p < 0.01; Fig. 2c: DE-71 and DE-79: 1, 10 lg/ml, p < 0.01). We further examined whether DE-71 and DE-79 stimulated EGF production. As Fig. 2d indicates, DE-71 significantly induced EGF production compared with the control (p < 0.01), and DE-79 tended to increase the production. 3.3. Intracellular signaling pathways in BEAS-2B exposed to DE-71 BEAS-2B showed remarkable effects on exposure to DE-71. Therefore, we further investigated their intracellular mechanisms. EGFR-mediated signaling pathways play a crucial role in the epithelial inflammatory response. Compared with the control, DE-71 significantly promoted EGFR phosphorylation in 15 min (Fig. 3: p < 0.05), but the total EGFR expression was unaffected by DE-71 exposure. Significant changes in expression were not observed with exposures greater than 30 min (data not shown). AG1478 effectively blocked DE-71-induced increases of IL-6 and IL-8 production (Fig. 4a and b: DE-71 with AG1478 versus DE-71 without an inhibitor, p < 0.01), whereas AG1478 blocked IL-6 and IL-8 production at the control levels (Fig. 4a and b: control with inhibitor versus control without an inhibitor, p < 0.01). We also investigated the subsequent signal transducer, MAPK. SB203580 effectively blocked the increase of IL-6 and IL-8 production in the same manner as AG1478 (Fig. 4a and b: DE-71 with SB203580 versus DE-71 without an inhibitor, p < 0.01). SB203580 also blocked IL-6 and IL-8 production at the control levels (Fig. 4a and b: control with an inhibitor versus control without an inhibitor, p < 0.01). PD98059 did not affect IL-6 production (Fig. 4a) but suppressed the DE-71-induced increase of IL-8 production (Fig. 4b: DE-71 with PD98059 versus DE-71 without an inhibitor, p < 0.01). PD98059 also suppressed IL-8 production at the control levels (Fig. 4a and b: control with an inhibitor versus control without an inhibitor, p < 0.01). CH-223191significantly suppressed DE-71-induced increase in IL-6 and IL-8 production (Fig. 5a: DE-71 with CH-223191 versus DE-71 without an antagonist, p < 0.05; Fig. 5b: DE-71 with CH223191 versus DE-71 without an antagonist, p < 0.01). 1–850 tended to suppress the increase in IL-6 production (Fig. 5a) and significantly suppressed the increase in IL-8 production (Fig. 5b: DE71 with 1–850 versus DE-71 without an antagonist, p < 0.01). ICI 182,780 did not affect those increases (Fig. 5a and b).
3. Results 4. Discussion 3.1. Cytotoxicity of PBDEs on BEAS-2B We examined the cell viability and proliferation of BEAS-2B following a 24-h exposure to each PBDE. DE-71 and DE-79 significantly increased cell proliferation compared with the control (Fig. 1b: DE-71: 0.1 lg/ml, p < 0.05; DE-71: 1, 3, 10 lg/ml and DE-79: 3, 10 lg/ml, p < 0.01). DE-83R slightly increased cell proliferation, but this was not significant. None of the PBDEs showed cytotoxic effects on BEAS-2B. 3.2. Expression of proinflammatory proteins in BEAS-2B exposed to PBDEs We examined the expressions of ICAM-1 and cytokines (IL-6 and IL-8) in BEAS-2B. DE-71 and DE-79, but not DE-83R, significantly increased ICAM-1 expression intensity compared with the control (Fig. 2a: DE-71: 1 lg/ml, p < 0.05; DE-71: 10 lg/ml and DE-79: 0.1, 1, 10 lg/ml, p < 0.01). Although soluble ICAM-1 production tended to increase with each PBDE exposure, the trend was not significant (data not shown). DE-71 and DE-79, but not DE-83R, significantly increased IL-6 and IL-8 production compared
In the present study, we investigated whether PBDEs stimulate bronchial epithelial cells in vitro. We show that penta- and octaBDE, but not deca-BDE, can promote the expression of proinflammatory proteins possibly by activating signaling pathways that include EGFR and by subsequent activation of transcriptional factors. As environmental pollutants, the potential adverse health effects of BFRs and PBDEs are concerning. Recent studies have demonstrated their potential endocrine-disrupting effects and developmental neurotoxicity (Costa et al., 2008; Talsness et al., 2009). However, the effects of BFRs on respiratory and immune systems and their underlying molecular mechanisms have not been elucidated. In this study, we focus on the effects of PBDEs on the inflammatory response in BEAS-2B and their underlying mechanisms. In BEAS-2B, penta- and octa-BDE, but not deca-BDE, significantly increased cell proliferation and the expression of proinflammatory proteins such as ICAM-1, IL-6, and IL-8 (Figs. 1b and 2a–c). These mediators play an important role in the immune system and in promoting inflammatory responses. IL-6 shows various
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Fig. 2. Effects of PBDEs on the expression of proinflammatory proteins in BEAS-2B. Expression intensity (mean fluorescence intensity) of ICAM-1 (a) and production level of IL-6 (b) and IL-8 (c) in BEAS-2B were measured after 24-h exposure to each PBDE (0.1–10 lg/ml). For EGF production (d), the culture medium was changed into LHC basal medium after exposure to 10 lg/ml of DE-71 or DE-79, and the cells were incubated for 3 h. Subsequently, the production level of EGF was measured. Data are mean ± SEM of triplicate cultures and are representative of three to five independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, PBDE versus control.
physiological functions such as promoting IL-8 production, the expression of cell adhesion molecules (including ICAM-1), and plasma cell differentiation. IL-8 is a chemokine that recruits inflammatory cells, and ICAM-1 also recruits inflammatory cells by signal-transducing functions. We observed penta- and octaBDE stimulated EGF production by BEAS-2B (Fig. 2d), which may have caused the increase in cell proliferation. In addition, we observed remarkable effects with penta-BDE. In general, less brominated PBDEs like penta-BDE are considered more toxic than octa-BDE and deca-BDE because of their high affinity for lipids that results in accumulation in human and animals (Siddiqi et al., 2003). Penta-BDE also enhances the production of reactive oxygen
species in human neutrophils in vitro, whereas octa-BDE, decaBDE, and the non-brominated diphenyl ether cannot do the same (Reistad and Mariussen, 2005). Although verification by further examinations including comparison of more molecules is needed, we suspect the toxic characteristics of PBDEs might be associated with brominated levels. We investigated the intracellular mechanisms that mediate the effects of penta-BDE. We observed that penta-BDE promoted EGFR phosphorylation (Fig. 3). AG1478 and SB203580 effectively blocked the increase in IL-6 and IL-8 production, whereas PD98059 partly affected only the increase in IL-8 production (Fig. 4). AG1478 also effectively blocked the increase in ICAM-1 expression by
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Fig. 5. Effects of nuclear receptor antagonists on DE-71-induced cytokine production in BEAS-2B. TR antagonist 1–850, AhR antagonist CH-223191, and ER antagonist ICI 182,780 were used. Cells were pretreated with these antagonists for 1 h and exposed to DE-71 for 24 h in the presence of those antagonists. Subsequently, the expression of production level of IL-6 (a) and IL-8 (b) were measured. Data are the mean ± SEM of triplicate cultures and are representative of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, DE-71 versus control; # p < 0.05, ##p < 0.01, antagonist (+) versus antagonist ().
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Fig. 3. Effects of DE-71 on EGFR phosphorylation in BEAS-2B. BEAS-2B was exposed to 10 lg/ml of DE-71 for 15 min. Subsequently, the total- and phospho-EGFR protein signals at OD450 were normalized for cell number by dividing the OD450 reading for a given well by the OD595 reading for the same well. Data are mean ± SEM of triplicate cultures and are representative of three independent experiments. ⁄p < 0.05, DE-71 versus control.
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+ inhibitor Fig. 4. Effects of protein kinase inhibitors on DE-71-induced cytokine production in BEAS-2B. EGFR-selective tyrosine kinase inhibitor AG1478, p38 MAPK inhibitor SB203580, and MEK inhibitor PD98059 were used. Cells were pretreated with those protein kinase inhibitors for 1 h and were exposed to DE-71 for 24 h in the presence of those protein kinase inhibitors. Subsequently, the production level of IL-6 (a) and IL-8 (b) were measured. Data are mean ± SEM of triplicate cultures and are representative of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, DE-71 versus control; ##p < 0.01, inhibitor (+) versus inhibitor ().
penta-BDE (data not shown). In addition, we observed similar results with octa-BDE (data not shown). AG1478, SB203580, and PD98059 also blocked IL-6 and/or IL-8 production at the control levels (Fig. 4a and b). It may be caused by culture medium LHC-9 containing EGF. EGF in the medium can stimulate EGFR on BEAS2B and subsequent signaling pathway such as MAPK. Therefore, these signal related proinflammatory proteins such as IL-6 and IL-8 were also observed in control levels and these signal inhibitors affected control levels. However, penta-BDE promoted EGFR phos-
phorylation (Fig. 3) and we observed suppressive effects of AG1478 and SB203580 on penta-BDE-increased proinflammatory cytokine production. These results suggest that activation of EGFR-specific tyrosine kinase and MAPK might be important pathways for the increased production of proinflammatory cytokines with exposure to less brominated PBDEs. Activation of these protein kinases can subsequently induce several transcriptional factors related to inflammation. Nuclear factor-kappa B is related to the induction of various proinflammatory proteins, including ICAM-1, IL-6, and IL-8 (Cybulsky and Gimbrone, 1991; Monaco and Paleolog, 2004). Activator protein 1 is related to the regulation of cellular processes such as differentiation, proliferation, and apoptosis. Signal transducer and activator of transcription (STAT) are latent cytoplasmic proteins that regulate cellular functions and are activated in response to cytokines and growth factors. IL-6 and EGF signal induce activation of STAT3 through the activation of JAK (JAK-STAT pathway) (Heinrich et al., 1998). Our preliminary studies suggestes that penta-PDE might promote the activation of these transcriptional factors (data not shown). It is suggested that endocrine-disrupting chemicals affect cytokine production (Yano et al., 2003) that may be partly caused by a nuclear receptor-mediated response and nuclear factor activation. BFRs can disrupt thyroid, androgen, and estrogenic signaling both in vivo and in vitro (Legler and Brouwer, 2003). In fact, PBDEs can bind to TR and alter the thyroid hormone balance by disrupting
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brain development (Siddiqi et al., 2003). PBDEs can also bind to ER (Meerts et al., 2001) and cytosolic AhR (Siddiqi et al., 2003). Moreover, our preliminary study suggests that penta-BDE has the ligand activity for TR, AhR, and ER. Fig. 5a and b shows nuclear receptor antagonists, 1–850 and CH-223191, significantly suppressed the increase in IL-6 and/or IL-8 production by penta-BDE in BEAS-2B, although ICI 182,780 did not affect those increases. Therefore, it is possible that PBDEs affect nuclear receptors such as TR and AhR, and might promote inflammatory responses in bronchial epithelial cells. The present study demonstrates that deca-BDE does not affect bronchial epithelial cells. In contrast, our previous study indicated that deca-BDE can stimulate immune cells in the same manner as penta- and octa-BDE (Koike et al., 2013). The different effects on these cells may be associated with membrane permeability, receptor activity, sensitivity, or cellular metabolism. It is also important to elucidate such effects on different types of cells and cell to cell interaction as a result of the effects of BFRs. Another problem is the possible breakdown of deca-BDE into more toxic, lighter PBDE congeners. For these reasons, further investigation on the effects of deca-BDE is also necessary. In our hypothesized scheme, we considered that some PBDEs directly activate protein kinases such as MAPK or activate the protein kinases by stimulating EGFR. The second proposed mechanism would promote proinflammatory proteins such as IL-6 and IL-8 by activating transcriptional factors. Another pathway is the nuclear receptor-mediated response and transcriptional factor activation. Then, the promotion of the expression of proinflammatory proteins in bronchial epithelial cells would stimulate inflammatory responses that might contribute to respiratory and immune diseases. Our previous reports (Koike et al., 2009, 2010, 2013), in addition to the present study, show that plasticizers and BFRs might affect immune cells and bronchial epithelial cells. Hence, it is necessary to pay attention to the adverse effects on respiratory diseases caused by indoor chemical pollutants and associated epidemiological studies are needed. Future experimental studies should include bronchial epithelial cell interactions with immune cells. It is also important to evaluate toxicological effects of indoor chemical pollutants including BFRs in the low concentration and long term exposure that reflect their environmental level. In conclusion, penta- and octa-BDE, but not deca-BDE, promoted the expression of proinflammatory proteins such as IL-6 and IL-8 in bronchial epithelial cells. Remarkable effects were observed with penta-BDE exposure, which might occur partly by the EGFR-related signaling pathways and/or nuclear receptormediated pathways that would lead to stimulation of transcriptional factors. Thus, PBDEs might promote inflammatory responses by stimulating respiratory and immune systems. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by an Environment Research and Technology Development Fund (K2314) from the Ministry of the Environment, and in part by grants from the National Institute for Environmental Studies. The authors would like to thank Ms. Satomi Abe for technical assistance and Enago (http://www.enago.jp) for the English language review. References Abdallah, M.A.-E., Harrad, S., Ibarra, C., Diamond, M., Melymuk, L., Robson, M., Covaci, A., 2007. Hexabromocyclododecanes in indoor dust from Canada, the
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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Penta- and octa-bromodiphenyl ethers promote proinflammatory protein expression in human bronchial epithelial cells in vitro Eiko Koike a,⇑, Rie Yanagisawa a, Hidetaka Takigami b, Hirohisa Takano c a
Center for Environmental Health Sciences, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan c Graduate School of Engineering, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto 615-8530, Japan b
a r t i c l e
i n f o
Article history: Received 14 June 2013 Accepted 23 October 2013 Available online 30 October 2013 Keywords: Brominated flame retardants Polybrominated diphenyl ethers Bronchial epithelial cells Proinflammatory cytokines Epidermal growth factor receptor
a b s t r a c t Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in consumer products. Humans can be exposed to PBDEs mainly through the inhalation of air or dust. Thus, PBDEs can affect respiratory and immune systems. In the present study, we investigated whether PBDEs stimulate bronchial epithelial cells. We examined commercial penta-BDE (DE-71), octa-BDE (DE-79), and decaBDE (DE-83R). Human bronchial epithelial cells (BEAS-2B) were exposed to each PBDE for 24 h. Subsequently, the expression of intercellular adhesion molecule-1 (ICAM-1) and proinflammatory cytokines were investigated. DE-71 and DE-79, but not DE-83R, significantly increased the expression of ICAM-1, interleukin-6 (IL-6), and IL-8 in BEAS-2B. Because these remarkable effects were observed with DE-71, we further investigated the underlying intracellular mechanisms. DE-71 promoted epidermal growth factor receptor (EGFR) phosphorylation. Inhibitors of EGFR-selective tyrosine kinase and p38 mitogenactivated protein kinase effectively blocked the increase of IL-6 and IL-8. Furthermore, antagonists of thyroid hormone receptor and aryl hydrocarbon receptor significantly suppressed the increase in IL-6 and/or IL-8 production. In conclusion, penta- and octa-BDE, but not deca-BDE, might promote the expression of proinflammatory proteins in bronchial epithelial cells possibly by activating protein kinases and/ or stimulating nuclear receptors related to subsequent activation of transcriptional factors. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Brominated flame retardants (BFRs) are used in industrial and consumer products, including electronic products, textiles, and building materials, to prevent fire-related injury and property damage (Birnbaum and Staskal, 2004). Major BFRs are polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane, and tetrabromobisphenol A (Birnbaum and Staskal, 2004). BFRs are used as additive or reactive components. Additive BFRs, including PBDEs, are simply blended with polymers and are not chemically bound to products; thus, additive BFRs can easily leach into the environment (Alaee et al., 2003). In particular, PBDEs have been observed in indoor dust in many countries (Abdallah et al., 2007;
Abbreviations: AhR, aryl hydrocarbon receptor; BFRs, brominated flame retardants; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, EGF receptor; ELISA, enzyme linked immunosorbent assay; ER, estrogen receptor; HBCD, hexabromocyclododecane; ICAM, intercellular adhesion molecule; IL, interleukin; MAPK, mitogen-activated protein kinase; PBDEs, polybrominated diphenyl ethers; STAT, signal transducer and activator of transcription; TR, thyroid hormone receptor. ⇑ Corresponding author. Tel./fax: +81 29 850 2334. E-mail address: [email protected] (E. Koike). 0887-2333/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2013.10.014
Batterman et al., 2010; Sjodin et al., 2008; Stapleton et al., 2005). A positive relationship was also found between PBDE concentrations in household dust and in human plasma (Karlsson et al., 2007) and breast milk (Wu et al., 2007). Besides BFRs, other chemicals such as phthalate plasticizers are present in indoor air and dust and coexist with allergens such as house dust mites. Epidemiological studies have suggested associations between phthalate exposure at home and the risk of asthma and allergies (Jaakkola and Knight, 2008). Experimental studies have also shown that phthalates can aggravate allergic diseases and/or responses in animal models (Koike et al., 2010; Larsen and Nielsen, 2007; Takano et al., 2006) and in vitro (Koike et al., 2009, 2010; Rakkestad et al., 2010). Thus, individuals with respiratory and allergy-related diseases may have a high sensitivity for indoor chemical pollutants. PBDEs include the commercial products of penta-, octa-, and deca-BDE. Each product is a mixture composed of several PBDE congeners. PBDEs are structurally similar to polychlorinated biphenyls and resist degradation in the environment. Because of their accumulation in human tissues (Hites, 2004; Inoue et al., 2006; Sjodin et al., 2003) and potential toxicity (Birnbaum and Staskal, 2004; Viberg et al., 2004), penta- and octa-BDE are banned in some
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countries or have been voluntarily withdrawn. However, their component congeners are still detected in humans and the environment. Recent concerns regarding the possible adverse health effects of BFRs have focused on their potential endocrine-disrupting effects and developmental neurotoxicity (Costa et al., 2008; Talsness et al., 2009). Because humans can be exposed to BFRs by inhaling indoor air or dust, it is important to determine the effects of BFRs on respiratory and immune systems. Recently, we examined the effects of BFRs, including PBDEs, on immune cells in atopic prone NC/Nga mice and demonstrated that these BFRs can aggravate immune/allergic responses by enhancing antigen presentationrelated molecule expression and IL-4 production in splenocytes (Koike et al., 2013). However, the effects of BFRs on respiratory and immune systems have not been completely elucidated. Activated airway epithelial cells release hematopoietic and proinflammatory cytokines that initiate immune responses and, therefore, play an essential role in respiratory diseases such as asthma. For instance, air pollutants (particulate matters and organic chemicals) can aggravate respiratory diseases by proinflammatory cytokine secretion from airway epithelial cells (Dergham et al., 2012; Fischader et al., 2008; Ghio et al., 2009). In this study, we investigated whether PBDEs affect the expression of proinflammatory proteins such as intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), and IL-8 in bronchial epithelial cells in vitro. In addition, we examined the effects of PBDEs on the signaling pathways such as activation of inflammatory-related protein kinase to evaluate the intracellular mechanisms. 2. Materials and methods
2.2. Cell culture and treatments The normal human bronchial epithelial cell line, BEAS-2B, was obtained from the European Collection of Cell Cultures (Health Protection Agency Culture Collection, Salisbury, UK). Cells were maintained in LHC-9 medium (Invitrogen™, Life Technologies Co., Carlsbad, CA, USA) in a collagen I coated culture dish (BD Biosciences, Bedford, MA, USA) at 37 °C and 5% CO2/95% air atmosphere. The cells (2 104/cm2) were seeded in a collagen I coated plate or dish and were allowed to grow to semiconfluence for three days. The culture medium was removed, and the cells were exposed to each PBDE (0.01–10 lg/ml) or 0.1% DMSO (control) in medium. The range of DE-83R concentration was limited from 0.01 to 6 lg/ml because of its solubility. We examined protein kinase activity using epidermal growth factor receptor (EGFR)-selective tyrosine kinase inhibitor (AG1478; Sigma–Aldrich), p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580; Sigma–Aldrich), and MEK inhibitor (PD98059; Sigma–Aldrich). Cells were pretreated with these protein kinase inhibitors (20 lM) for 1 h and were then exposed to DE-71 (3 lg/ml) for 24 h in the presence of these protein kinase inhibitors (10 lM). In the same manner, we examined the role of the nuclear receptor using thyroid hormone receptor (TR) antagonist (1–850; Merck KGaA, Darmstadt, Germany), aryl hydrocarbon receptor (AhR) antagonist (CH-223191; Merck KGaA), and estrogen receptor (ER) antagonist (ICI 182,780; Sigma–Aldrich). The cells were pretreated with 1–850 (10 lM), CH-223191 (10 lM), and ICI 182,780 (10 nM) for 1 h and then exposed to DE-71 (5 lg/ml) for 24 h in the presence of these nuclear receptor antagonists at half of the above-mentioned concentration. All procedures were approved by the Institutional Review Board of the National Institute for Environmental Studies.
2.1. Chemicals 2.3. Cell viability/proliferation assay Commercial penta-BDE mixture (DE-71), octa-BDE mixture (DE-79), and deca-BDE mixture (DE-83R) (Wellington Laboratories Inc., Guelph, Ontario, Canada) were used. Fig. 1a shows the structures of PBDEs. Each PBDE was dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich Co., St. Louis, MO, USA) and diluted with culture medium. Final concentration of DMSO was 0.1% in all experiments.
O
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Cells grown in a collagen I coated 96-well plate were exposed to PBDEs (0.01–10 lg/ml) for 24 h. After exposure, cells were incubated with tetrazolium salt WST-1 reagent (Takara Bio Inc., Otsu, Shiga, Japan) for 2 h. WST-1 is cleaved to soluble formazan in viable cells through a complex cellular mechanism, and the amount of formazan dye formed directly correlates to the number of viable cells. After the incubation period, we measured absorbance at 450 nm. Results are represented as a percentage of the control value.
Cells grown in a collagen I coated 12-well plate were exposed to PBDEs (0.01–10 lg/ml) for 24 h. After exposure, cells were collected by treatment with 0.25% trypsin/EDTA (GibcoÒ, Life Technologies Co.) and were washed with phosphate-buffered saline containing 0.3% bovine serum albumin and 0.05% sodium azide. The cells were incubated in the buffer for 30 min on ice with an optimal amount of anti-human ICAM-1 antibody (CD54; PE-conjugated, BioLegend, San Diego, CA, USA). Fluorescence was measured on FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). For each sample, fluorescence data was collected from 10,000 cells and the mean fluorescence intensity were analyzed by FLOWJO (Tree Star Inc., Ashland, OR, USA).
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BFRs concentration (µg/ml) Fig. 1. Chemical structure of PBDEs (a) and cytotoxicity of PBDEs on BEAS-2B (b). Cells were exposed to each PBDE (0.01–10 lg/ml) for 24 h. Cell viability/proliferation is indicated as a percentage of the control. Data are mean ± SEM of triplicate and are representative of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, DE71 versus control; p < 0.01, DE-79 versus control.
2.5. Quantitation of cytokines in culture supernatants Cells grown in a collagen I coated 12-well plate were exposed to PBDEs (0.01–10 lg/ml) for 24 h. After exposure, the culture supernatant was collected and was stored at 80 °C until measurement. The levels of IL-6, IL-8 (Thermo Scientific, Rockford, IL, USA), and
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soluble ICAM-1 (Bender MedSystems, Vienna, Austria) in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Detection limits for IL-6, IL-8, and soluble ICAM-1 were 1, 2, and 60 pg/ml, respectively. For EGF measurement, the culture medium was changed into LHC basal medium after exposure to remove EGF in LHC-9 medium. The cells were then incubated with LHC basal medium for 3 h, and the culture supernatant was collected and was stored at 80 °C until measurement. EGF content was also measured by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions, and the detection limit was 0.7 pg/ml. 2.6. Immunoassay for EGFR phosphorylation Cells grown in a collagen I coated 96-well plate were exposed to DE-71 (10 lg/ml) for 15 and 30 min. After exposure, protein kinase phosphorylation of EGFR was detected by cell-based ELISA FACE (Active Motif Japan, Shinjuku-Ku, Tokyo, Japan) according to the manufacturer’s instructions. In brief, cells were fixed with 4% formaldehyde in a culture plate and inactivated with endogenous peroxidase activity with 1% H2O2 and 0.1% NaN3. After blocking, total- and phospho-EGFR protein levels were examined. Binding of primary antibodies was performed overnight at 4 °C. After colorimetric reactions, absorbance was measured at 450 nm with a reference wavelength of 655 nm. The relative number of cells in each well was determined by Crystal Violet, which gives an OD595 reading. The total- and phospho-EGFR signals at OD450 were normalized for cell number by dividing the OD450 reading by the OD595 reading for each well. 2.7. Statistical analysis Each experiment was performed using triplicate cultures, and three to five independent experiments were repeated. The data were represented as mean ± SEM. Significance of intergroup variations was determined by one-way ANOVA or Kruskal–Wallis analysis. Differences among groups were analyzed using Dunnett’s multiple comparison test (Excel Statistics 2010, Social Survey Research Information Co., Ltd., Shinjuku-Ku, Tokyo, Japan). p < 0.05 was considered statistically significant.
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with the control (Fig. 2b: DE-71 and DE-79: 10 lg/ml, p < 0.01; Fig. 2c: DE-71 and DE-79: 1, 10 lg/ml, p < 0.01). We further examined whether DE-71 and DE-79 stimulated EGF production. As Fig. 2d indicates, DE-71 significantly induced EGF production compared with the control (p < 0.01), and DE-79 tended to increase the production. 3.3. Intracellular signaling pathways in BEAS-2B exposed to DE-71 BEAS-2B showed remarkable effects on exposure to DE-71. Therefore, we further investigated their intracellular mechanisms. EGFR-mediated signaling pathways play a crucial role in the epithelial inflammatory response. Compared with the control, DE-71 significantly promoted EGFR phosphorylation in 15 min (Fig. 3: p < 0.05), but the total EGFR expression was unaffected by DE-71 exposure. Significant changes in expression were not observed with exposures greater than 30 min (data not shown). AG1478 effectively blocked DE-71-induced increases of IL-6 and IL-8 production (Fig. 4a and b: DE-71 with AG1478 versus DE-71 without an inhibitor, p < 0.01), whereas AG1478 blocked IL-6 and IL-8 production at the control levels (Fig. 4a and b: control with inhibitor versus control without an inhibitor, p < 0.01). We also investigated the subsequent signal transducer, MAPK. SB203580 effectively blocked the increase of IL-6 and IL-8 production in the same manner as AG1478 (Fig. 4a and b: DE-71 with SB203580 versus DE-71 without an inhibitor, p < 0.01). SB203580 also blocked IL-6 and IL-8 production at the control levels (Fig. 4a and b: control with an inhibitor versus control without an inhibitor, p < 0.01). PD98059 did not affect IL-6 production (Fig. 4a) but suppressed the DE-71-induced increase of IL-8 production (Fig. 4b: DE-71 with PD98059 versus DE-71 without an inhibitor, p < 0.01). PD98059 also suppressed IL-8 production at the control levels (Fig. 4a and b: control with an inhibitor versus control without an inhibitor, p < 0.01). CH-223191significantly suppressed DE-71-induced increase in IL-6 and IL-8 production (Fig. 5a: DE-71 with CH-223191 versus DE-71 without an antagonist, p < 0.05; Fig. 5b: DE-71 with CH223191 versus DE-71 without an antagonist, p < 0.01). 1–850 tended to suppress the increase in IL-6 production (Fig. 5a) and significantly suppressed the increase in IL-8 production (Fig. 5b: DE71 with 1–850 versus DE-71 without an antagonist, p < 0.01). ICI 182,780 did not affect those increases (Fig. 5a and b).
3. Results 4. Discussion 3.1. Cytotoxicity of PBDEs on BEAS-2B We examined the cell viability and proliferation of BEAS-2B following a 24-h exposure to each PBDE. DE-71 and DE-79 significantly increased cell proliferation compared with the control (Fig. 1b: DE-71: 0.1 lg/ml, p < 0.05; DE-71: 1, 3, 10 lg/ml and DE-79: 3, 10 lg/ml, p < 0.01). DE-83R slightly increased cell proliferation, but this was not significant. None of the PBDEs showed cytotoxic effects on BEAS-2B. 3.2. Expression of proinflammatory proteins in BEAS-2B exposed to PBDEs We examined the expressions of ICAM-1 and cytokines (IL-6 and IL-8) in BEAS-2B. DE-71 and DE-79, but not DE-83R, significantly increased ICAM-1 expression intensity compared with the control (Fig. 2a: DE-71: 1 lg/ml, p < 0.05; DE-71: 10 lg/ml and DE-79: 0.1, 1, 10 lg/ml, p < 0.01). Although soluble ICAM-1 production tended to increase with each PBDE exposure, the trend was not significant (data not shown). DE-71 and DE-79, but not DE-83R, significantly increased IL-6 and IL-8 production compared
In the present study, we investigated whether PBDEs stimulate bronchial epithelial cells in vitro. We show that penta- and octaBDE, but not deca-BDE, can promote the expression of proinflammatory proteins possibly by activating signaling pathways that include EGFR and by subsequent activation of transcriptional factors. As environmental pollutants, the potential adverse health effects of BFRs and PBDEs are concerning. Recent studies have demonstrated their potential endocrine-disrupting effects and developmental neurotoxicity (Costa et al., 2008; Talsness et al., 2009). However, the effects of BFRs on respiratory and immune systems and their underlying molecular mechanisms have not been elucidated. In this study, we focus on the effects of PBDEs on the inflammatory response in BEAS-2B and their underlying mechanisms. In BEAS-2B, penta- and octa-BDE, but not deca-BDE, significantly increased cell proliferation and the expression of proinflammatory proteins such as ICAM-1, IL-6, and IL-8 (Figs. 1b and 2a–c). These mediators play an important role in the immune system and in promoting inflammatory responses. IL-6 shows various
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Fig. 2. Effects of PBDEs on the expression of proinflammatory proteins in BEAS-2B. Expression intensity (mean fluorescence intensity) of ICAM-1 (a) and production level of IL-6 (b) and IL-8 (c) in BEAS-2B were measured after 24-h exposure to each PBDE (0.1–10 lg/ml). For EGF production (d), the culture medium was changed into LHC basal medium after exposure to 10 lg/ml of DE-71 or DE-79, and the cells were incubated for 3 h. Subsequently, the production level of EGF was measured. Data are mean ± SEM of triplicate cultures and are representative of three to five independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, PBDE versus control.
physiological functions such as promoting IL-8 production, the expression of cell adhesion molecules (including ICAM-1), and plasma cell differentiation. IL-8 is a chemokine that recruits inflammatory cells, and ICAM-1 also recruits inflammatory cells by signal-transducing functions. We observed penta- and octaBDE stimulated EGF production by BEAS-2B (Fig. 2d), which may have caused the increase in cell proliferation. In addition, we observed remarkable effects with penta-BDE. In general, less brominated PBDEs like penta-BDE are considered more toxic than octa-BDE and deca-BDE because of their high affinity for lipids that results in accumulation in human and animals (Siddiqi et al., 2003). Penta-BDE also enhances the production of reactive oxygen
species in human neutrophils in vitro, whereas octa-BDE, decaBDE, and the non-brominated diphenyl ether cannot do the same (Reistad and Mariussen, 2005). Although verification by further examinations including comparison of more molecules is needed, we suspect the toxic characteristics of PBDEs might be associated with brominated levels. We investigated the intracellular mechanisms that mediate the effects of penta-BDE. We observed that penta-BDE promoted EGFR phosphorylation (Fig. 3). AG1478 and SB203580 effectively blocked the increase in IL-6 and IL-8 production, whereas PD98059 partly affected only the increase in IL-8 production (Fig. 4). AG1478 also effectively blocked the increase in ICAM-1 expression by
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Fig. 5. Effects of nuclear receptor antagonists on DE-71-induced cytokine production in BEAS-2B. TR antagonist 1–850, AhR antagonist CH-223191, and ER antagonist ICI 182,780 were used. Cells were pretreated with these antagonists for 1 h and exposed to DE-71 for 24 h in the presence of those antagonists. Subsequently, the expression of production level of IL-6 (a) and IL-8 (b) were measured. Data are the mean ± SEM of triplicate cultures and are representative of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, DE-71 versus control; # p < 0.05, ##p < 0.01, antagonist (+) versus antagonist ().
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Protein expression (OD450/OD595)
7
##
*
## ##
## ##
0
+ inhibitor Fig. 4. Effects of protein kinase inhibitors on DE-71-induced cytokine production in BEAS-2B. EGFR-selective tyrosine kinase inhibitor AG1478, p38 MAPK inhibitor SB203580, and MEK inhibitor PD98059 were used. Cells were pretreated with those protein kinase inhibitors for 1 h and were exposed to DE-71 for 24 h in the presence of those protein kinase inhibitors. Subsequently, the production level of IL-6 (a) and IL-8 (b) were measured. Data are mean ± SEM of triplicate cultures and are representative of three independent experiments. ⁄p < 0.05, ⁄⁄p < 0.01, DE-71 versus control; ##p < 0.01, inhibitor (+) versus inhibitor ().
penta-BDE (data not shown). In addition, we observed similar results with octa-BDE (data not shown). AG1478, SB203580, and PD98059 also blocked IL-6 and/or IL-8 production at the control levels (Fig. 4a and b). It may be caused by culture medium LHC-9 containing EGF. EGF in the medium can stimulate EGFR on BEAS2B and subsequent signaling pathway such as MAPK. Therefore, these signal related proinflammatory proteins such as IL-6 and IL-8 were also observed in control levels and these signal inhibitors affected control levels. However, penta-BDE promoted EGFR phos-
phorylation (Fig. 3) and we observed suppressive effects of AG1478 and SB203580 on penta-BDE-increased proinflammatory cytokine production. These results suggest that activation of EGFR-specific tyrosine kinase and MAPK might be important pathways for the increased production of proinflammatory cytokines with exposure to less brominated PBDEs. Activation of these protein kinases can subsequently induce several transcriptional factors related to inflammation. Nuclear factor-kappa B is related to the induction of various proinflammatory proteins, including ICAM-1, IL-6, and IL-8 (Cybulsky and Gimbrone, 1991; Monaco and Paleolog, 2004). Activator protein 1 is related to the regulation of cellular processes such as differentiation, proliferation, and apoptosis. Signal transducer and activator of transcription (STAT) are latent cytoplasmic proteins that regulate cellular functions and are activated in response to cytokines and growth factors. IL-6 and EGF signal induce activation of STAT3 through the activation of JAK (JAK-STAT pathway) (Heinrich et al., 1998). Our preliminary studies suggestes that penta-PDE might promote the activation of these transcriptional factors (data not shown). It is suggested that endocrine-disrupting chemicals affect cytokine production (Yano et al., 2003) that may be partly caused by a nuclear receptor-mediated response and nuclear factor activation. BFRs can disrupt thyroid, androgen, and estrogenic signaling both in vivo and in vitro (Legler and Brouwer, 2003). In fact, PBDEs can bind to TR and alter the thyroid hormone balance by disrupting
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brain development (Siddiqi et al., 2003). PBDEs can also bind to ER (Meerts et al., 2001) and cytosolic AhR (Siddiqi et al., 2003). Moreover, our preliminary study suggests that penta-BDE has the ligand activity for TR, AhR, and ER. Fig. 5a and b shows nuclear receptor antagonists, 1–850 and CH-223191, significantly suppressed the increase in IL-6 and/or IL-8 production by penta-BDE in BEAS-2B, although ICI 182,780 did not affect those increases. Therefore, it is possible that PBDEs affect nuclear receptors such as TR and AhR, and might promote inflammatory responses in bronchial epithelial cells. The present study demonstrates that deca-BDE does not affect bronchial epithelial cells. In contrast, our previous study indicated that deca-BDE can stimulate immune cells in the same manner as penta- and octa-BDE (Koike et al., 2013). The different effects on these cells may be associated with membrane permeability, receptor activity, sensitivity, or cellular metabolism. It is also important to elucidate such effects on different types of cells and cell to cell interaction as a result of the effects of BFRs. Another problem is the possible breakdown of deca-BDE into more toxic, lighter PBDE congeners. For these reasons, further investigation on the effects of deca-BDE is also necessary. In our hypothesized scheme, we considered that some PBDEs directly activate protein kinases such as MAPK or activate the protein kinases by stimulating EGFR. The second proposed mechanism would promote proinflammatory proteins such as IL-6 and IL-8 by activating transcriptional factors. Another pathway is the nuclear receptor-mediated response and transcriptional factor activation. Then, the promotion of the expression of proinflammatory proteins in bronchial epithelial cells would stimulate inflammatory responses that might contribute to respiratory and immune diseases. Our previous reports (Koike et al., 2009, 2010, 2013), in addition to the present study, show that plasticizers and BFRs might affect immune cells and bronchial epithelial cells. Hence, it is necessary to pay attention to the adverse effects on respiratory diseases caused by indoor chemical pollutants and associated epidemiological studies are needed. Future experimental studies should include bronchial epithelial cell interactions with immune cells. It is also important to evaluate toxicological effects of indoor chemical pollutants including BFRs in the low concentration and long term exposure that reflect their environmental level. In conclusion, penta- and octa-BDE, but not deca-BDE, promoted the expression of proinflammatory proteins such as IL-6 and IL-8 in bronchial epithelial cells. Remarkable effects were observed with penta-BDE exposure, which might occur partly by the EGFR-related signaling pathways and/or nuclear receptormediated pathways that would lead to stimulation of transcriptional factors. Thus, PBDEs might promote inflammatory responses by stimulating respiratory and immune systems. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by an Environment Research and Technology Development Fund (K2314) from the Ministry of the Environment, and in part by grants from the National Institute for Environmental Studies. The authors would like to thank Ms. Satomi Abe for technical assistance and Enago (http://www.enago.jp) for the English language review. References Abdallah, M.A.-E., Harrad, S., Ibarra, C., Diamond, M., Melymuk, L., Robson, M., Covaci, A., 2007. Hexabromocyclododecanes in indoor dust from Canada, the
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