Toxicological effects of polycyclic aromatic hydrocarbons and their derivatives on respiratory cells

Atmospheric Environment xxx (2014) 1e8

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Atmospheric Environment journal homepage: www.elsevier.com/locate/atmosenv

Toxicological effects of polycyclic aromatic hydrocarbons and their derivatives on respiratory cells Eiko Koike a, *, Rie Yanagisawa a, Hirohisa Takano b a b

Center for Environmental Health Sciences, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan Graduate School of Engineering, Kyoto University, Kyoto Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8530, Japan

h i g h l i g h t s
Different toxicological effects related to chemical structure were observed.
Pyrene, 1-NP, and 1-AP increased proinflammatory protein expression in BEAS-2B.
The increase might be mediated by protein kinases and nuclear receptors.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 August 2013 Received in revised form 31 March 2014 Accepted 4 April 2014 Available online xxx

Polycyclic aromatic hydrocarbons (PAHs) are found in ambient aerosols and particulate matter. Experimental studies have shown that PAHs and related chemicals can induce toxicological effects. The present study aimed to investigate the effects of PAHs and their derivatives on the respiratory and immune systems and the underlying mechanisms. The human bronchial epithelial cell line BEAS-2B was exposed to PAHs and their derivatives, and the cytotoxicity and proinflammatory protein expression were then investigated. A cytotoxic effect was observed in BEAS-2B exposed to PAH derivatives such as naphthoquinone (NQ), phenanthrenequinone (PQ), 1-nitropyrene (1-NP), and 1-aminopyrene (1-AP). In addition, 1,2-NQ and 9,10-PQ showed more effective cytotoxicity than 1,4-NQ and 1,4-PQ, respectively. Pyrene showed a weak cytotoxic effect. On the other hand, naphthalene and phenanthrene showed no significant effects. Pyrene, 1-NP, and 1-AP also increased intercellular adhesion molecule-1 expression and interleukin-6 production in BEAS-2B. The increase was partly suppressed by protein kinase inhibitors such as the epidermal growth factor receptor-selective tyrosine kinase inhibitor and nuclear receptor antagonists such as the thyroid hormone receptor antagonist. The present study suggests that the toxicological effects of chemicals may be related to the different activities resulting from their structures, such as numbers of benzene rings and functional groups. Furthermore, the chemical-induced increase in proinflammatory protein expression in bronchial epithelial cells was possibly a result of the activation of protein kinase pathways and nuclear receptors. The increase may partly contribute to the adverse health effects of atmospheric PAHs. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Polycyclic aromatic hydrocarbons Bronchial epithelial cells Proinflammatory cytokines Intracellular signaling pathway

1. Introduction

Abbreviations: AhR, aryl hydrocarbon receptor; AP, aminopyrene; DEP, diesel exhaust particles; DMSO, dimethyl sulfoxide; E2, b-estradiol; EGF, epidermal growth factor; EGFR, EGF receptor; ELISA, enzyme-linked immunosorbent assay; ER, estrogen receptor; ICAM, intercellular adhesion molecule; IL, interleukin; MAPK, mitogen-activated protein kinase; b-NF, b-naphthoflavone; NFkB, nuclear factorkappa B; NP, nitropyrene; NQ, naphthoquinone; PAHs, polycyclic aromatic hydrocarbons; PM, particulate matters; PQ, phenanthrenequinone; ROS, reactive oxygen species; SEM, standard error of the mean; T3, 3,30 ,5-Triiodo-L-thyronine; TR, thyroid hormone receptor. * Corresponding author. E-mail address: [email protected] (E. Koike).

Aerosols in urban air consist of gases and particulate matters (PM), including the carbonaceous core, metals, organic chemicals, nitrate, sulfate, and biological organisms, and they may affect human health. Previous epidemiological studies have indicated that PM may be a risk factor for the development/exacerbation of allergic, respiratory, and cardiovascular diseases (Hart et al., 2011; Jenerowicz et al., 2012; Sacks et al., 2011; Sint et al., 2008) and that fine PM levels are associated with increased morbidities and mortalities from respiratory and cardiovascular diseases (Brook et al., 2010; Dockery et al., 1993; Pope et al., 1995). In the

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atmosphere, aerosol PM can react with oxidants such as ozone and nitrogen dioxide. The reaction induces transformation in the chemical composition and properties that may contribute to a change in toxicity (George and Abbatt, 2010). However, little is known about the relative effects of chemical composition and properties in aerosols and PM. One of the major sources of urban air pollutants is assumed to be diesel exhaust particles (DEP). Many experimental studies have suggested that PM or DEP can enhance inflammatory responses, particularly those mediated by organic chemical components such as polycyclic aromatic hydrocarbons (PAHs) in vivo (Diaz Sanchez, 1997; Hiyoshi et al., 2005; Podechard et al., 2008; Yanagisawa et al., 2006) and in vitro (Baulig et al., 2009; Kawasaki et al., 2001; Schober et al., 2007; Tsien et al., 1997; Umannova et al., 2011). A number of papers have characterized ambient air samples and have measured concentrations of PAHs and derivatives. For example, it has been reported that the maximum concentration of phenanthrene and pyrene in roadside air is 129.4 ng/m3 and 21.5 ng/m3, respectively (Ho et al., 2002) and the mean concentration of phenanthrene and pyrene in wintertime urban air is 43.5 ng/m3 and 17.63 ng/m3, respectively (Andreou and Rapsomanikis, 2009). PAHs can generate reactive oxygen species (ROS), which induce lipid peroxidation and DNA damage (Fu et al., 2012). Oxidative stress may be one of the factors responsible for the inflammation. It has been suggested that PM exposure induces oxidative damage and increases the gene expression and/or protein secretion of proinflammatory cytokines in airway epithelial cells (Dergham et al., 2012). Our previous studies have also shown that organic chemical components in DEP can induce cytotoxicity and/or the activation of alveolar epithelial cells and immune cells through oxidative stress (Koike et al., 2004; Koike and Kobayashi, 2005; Shima et al., 2006). Organic compounds adsorbed on DEP may have adverse health effects; however, these compounds comprise various chemicals, including very complex oxygenated chemicals. Thus, we previously investigated the chemical and biological characteristics of fractionated organic compounds and demonstrated that the n-hexane-insoluble fraction in DEP contains many functional groups related to oxygenation, such as hydroxyl, carbonyl, and nitro groups. This fraction has shown a high oxidative capacity, and it strongly contributes to the induction of oxidative stress, cytotoxicity, and inflammatory response in vivo and in vitro (Shima et al., 2006). Reactive PAH derivatives are generated from PAHs by oxidative reactions in the atmosphere and biological metabolism. PAH derivatives may have a functional group that is a key factor in PAH toxicity. However, the effects of PAH derivatives on respiratory and immune systems have not been completely elucidated. We therefore focused on the differences in the effects of PAHs and their derivatives on respiratory cells and immune cells. Our recent study examined the effects of these chemicals on mouse immune cells such as splenocytes and bone marrow-derived dendritic cells. It has been demonstrated that some PAH derivatives can increase the expression of the antigen presentation-related molecule CD86, proliferation of antigen-stimulated cells, and/or production of Th2 cytokine interleukin (IL)-4 in splenocytes (Takano et al., 2014). However, these effects were relatively weak and there were no significant effects on dendritic cells. Activated airway epithelial cells can release hematopoietic and proinflammatory cytokines, which initiate immune responses. Airway epithelial cellmediated reactions may play an essential role in the toxicity of PAHs and their derivatives on respiratory and immune systems. The present study aimed to determine whether PAHs and their derivatives affect the expression of proinflammatory proteins in bronchial epithelial cells and the underlying intracellular mechanisms in vitro.

2. Materials and methods 2.1. PAHs and their derivatives Naphthalene (SigmaeAldrich, St. Louis, MO, USA), 1,2naphthoquinone (1,2-NQ; Tokyo Chemical Industry, Chuo-ku, Tokyo, Japan), 1,4-NQ (Tokyo Chemical Industry), phenanthrene (Tokyo Chemical Industry), 9,10-phenanthrenequinone (9,10-PQ; SigmaeAldrich), 1,4-PQ (Chiron, Trondheim, Norway), pyrene (SigmaeAldrich), 1-nitropyrene (1-NP; SigmaeAldrich Co.), and 1aminopyrene (1-AP; SigmaeAldrich Co.) were used in the present study. Fig. 1 shows the structures of the chemicals. Each chemical was dissolved in dimethyl sulfoxide (DMSO; SigmaeAldrich) and diluted with the culture medium. The final concentration of DMSO in all experiments was 0.1%. 2.2. Cell culture and treatments The normal human bronchial epithelial cell line BEAS-2B was obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were maintained in LHC-9 medium (Invitrogen, 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 allowed to grow to semi-confluence for 3 days. The culture medium was removed, and the cells were exposed to each chemical (0.01e 10 mM) or 0.1% DMSO (control) in the medium for 24 h. 2.3. Cell viability assay Cells grown in a collagen I-coated 96-well plate were exposed to PAHs and their derivatives as described above. The cells were then incubated with the tetrazolium salt WST-1 reagent (Takara Bio, Shiga, Japan) for 2 h. WST-1 is cleaved to soluble formazan in viable cells through a complex mechanism, and the amount of formazan

Fig. 1. Chemical structure of PAHs and their derivatives Naphthalene, 1,2-NQ, 1,4-NQ, phenanthrene, 9,10-PQ, 1,4-PQ, pyrene, 1-NP, and 1-AP were examined.

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dye formed directly correlates with the number of viable cells. After incubation, absorbance at 450 nm was measured. Results are represented as a percentage of the control value. 2.4. Flow cytometry Cells grown in a collagen I-coated 12-well plate were exposed to PAHs and their derivatives as described above. After exposure, the cells were collected by treatment with 0.25% trypsin/EDTA (Gibco, Carlsbad, CA, USA) and 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 antihuman intercellular adhesion molecule-1 (ICAM-1) antibody (CD54; PE-conjugated, BioLegend, San Diego, CA, USA). Fluorescence was measured on FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). For each sample, fluorescence data were collected from 10,000 cells, and the mean fluorescence intensity was analyzed. 2.5. Quantitation of cytokines in culture supernatants Cells grown in a collagen I-coated 12-well plate were exposed to PAHs and their derivatives as described above. Following this, the culture supernatant was collected and stored at 80  C until measurement. Levels of IL-6 and IL-8 (Thermo Scientific, Rockford, IL, USA) in the culture supernatant were measured by enzymelinked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Limits of detection for IL-6 and IL-8 were 1 and 2 pg/mL, respectively. For epidermal growth factor (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 stored at 80  C until measurement. The EGF content was also measured by ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, and the limit of detection was 0.7 pg/mL. 2.6. Activation analyses using specific signaling pathway inhibitors To investigate the role of EGF receptor (EGFR)-related protein kinase pathways, an EGFR-selective tyrosine kinase inhibitor (AG1478; SigmaeAldrich), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB 203580; SigmaeAldrich), and an MEK inhibitor (PD98059; SigmaeAldrich) were used. The cells were pretreated with these protein kinase inhibitors (20 mM) for 1 h and then exposed to 1-AP (10 mM) for 24 h in the presence of these protein kinase inhibitors (10 mM). To investigate the role of the nuclear receptor, a thyroid hormone receptor (TR) antagonist (1e850; Merck KGaA, Darmstadt, Germany), aryl hydrocarbon receptor (AhR) antagonist (CH223191; Merck KGaA), and an estrogen receptor (ER) antagonist (ICI 182,780; SigmaeAldrich) were used. The cells were pretreated with 1e850 (10 mM), CH-223191 (10 mM), and ICI 182,780 (10 nM) for 1 h and then exposed to 1-AP (10 mM) for 24 h in the presence of these nuclear receptor antagonists at half of the abovementioned concentration. 2.7. Nuclear receptoreligand binding assay To investigate the ligand activity of PAHs and their derivatives for nuclear receptors, interactions between nuclear receptors (TRab and ERab) and the test chemicals were measured with duplicate samples using the EnBio nuclear receptor cofactor assay system according to the manufacturer’s protocol (Fujikura Kasei, Tokyo, Japan). In brief, test chemicals and the positive control were

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dissolved in DMSO. 3,30 ,5-Triiodo-L-thyronine (T3) and b-estradiol (E2) were used as positive controls for the agonists of TR and ER, respectively. Test chemicals and positive controls were incubated with each nuclear receptor in a cofactor-coated plate. After washing the plate, a detection antibody was applied to the wells, and the plate was incubated. After washing the plate again, a substrate solution was added and which the absorbance was measured at 450 nm. Ligand activity for TR and ER was shown as the absorbance of test chemical minus the absorbance of solvent control. Ligand activity of the test chemicals for AhR was measured by a reporter gene assay using CROMIS AhR, a yeast (Saccharomyces cerevisiae) bioassay kit, according to the manufacturer’s protocol (NAGASE, Tokyo, Japan). This recombinant yeast co-expresses human AhR and Arnt cDNA under the control of the GAL1eGAL10 bidirectional promoter in the pUCura3 plasmid. Test chemicals and b-naphthoflavone (b-NF; positive control) were dissolved in DMSO. Test chemicals and b-NF were incubated with the recombinant yeast in a plate. After incubation, the cell densities of the samples were determined by reading their absorbance at 595 nm. Following this, the samples were incubated with the substrate for b-galactosidase, and the b-galactosidase activity was determined by reading their absorbance at 540 nm. AhR ligand activity was calculated as follows: (absorbance of test chemical at 540 nm/absorbance of test chemical at 595 nm)  (absorbance of solvent control at 540 nm/ absorbance of solvent control at 595 nm). 2.8. Statistical analyses Each experiment of cytotoxicity and inflammatory protein expression was performed using triplicate cultures, and 2e4 independent experiments were repeated. Data are the mean  standard error of the mean (SEM). The significance of intergroup variations was determined by 1-way analysis of variance or KruskaleWallis analyses. Differences among groups were analyzed using Dunnett’s multiple comparison test or Steel’s multiple comparison test (Excel Statistics 2010, Social Survey Research Information, Tokyo, Japan). A p value < 0.05 was considered significant. 3. Results 3.1. Cytotoxicity of PAHs and their derivatives on BEAS-2B viability Naphthalene and phenanthrene did not affect cell viability. Although low concentrations of NQ and PQ induced the viability, higher concentrations decreased it (Fig. 2a and b). 1,2-NQ and 9,10PQ showed stronger cytotoxicity than 1,4-NQ and 1,4-PQ, respectively. Compared with the control, pyrene at 10 mM tended to decrease the viability; however, there was no statistical difference. 1-NP and 1-AP at 10 mM significantly decreased the viability (Fig. 2c). 3.2. Expression of proinflammatory proteins in BEAS-2B exposed to PAHs and their derivatives As representative data, the expression of ICAM-1 and IL-6 in BEAS-2B after exposure to pyrene and derivatives is shown in Fig. 3. Compared with the control, pyrene, 1-NP, and 1-AP significantly increased ICAM-1 expression intensity (Fig. 3a). 1-AP also significantly increased IL-6 production. Pyrene and 1-NP tended to increase IL-6 production; however, it was not statistically significant (Fig. 3b). In addition, compared with the control, PQ tended to increase ICAM-1 expression intensity and IL-6 production. Naphthalene, NQ, and phenanthrene did not stimulate the expression of these proteins. IL-8 production was not altered by PAHs (naphthalene, phenanthrene, and pyrene) and was decreased by PAH

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Fig. 2. Cytotoxicity of chemicals on BEAS-2B Cells were exposed to each chemical (0.01e10 mM) for 24 h. Cell viability is denoted as a percentage of the control. Data are the mean  SEM of triplicate cultures and representative of 2e4 independent experiments. (a) ##p < 0.01, 1,2-NQ vs. control; {p < 0.05, {{p < 0.01, 1,4-NQ vs. control; yp < 0.05, yyp < 0.01, 1,2-NQ vs. naphthalene; zp < 0.05, zzp < 0.01, 1,4-NQ vs. naphthalene; xxp < 0.01, 1,2-NQ vs. 1,4-NQ, (b) **p < 0.01, phenanthrene vs. control; #p < 0.05, ##p < 0.01, 9,10PQ vs. control; {{p < 0.01, 1,4-PQ vs. control; yyp < 0.01, 9,10-PQ vs. phenanthrene; zp < 0.05, zzp < 0.01, 1,4-PQ vs. phenanthrene; xp < 0.05, xxp < 0.01, 9,10-PQ vs. 1,4-PQ, (c) ##p < 0.01, 1-NP vs. control; {p < 0.05, 1-AP vs. control; yp < 0.05, 1-NP vs. pyrene; zp < 0.05, 1-AP vs. pyrene; xp < 0.05, xxp < 0.01, 1-NP vs. 1-AP.

derivatives (NQ, PQ, 1-NP, and 1-AP). These chemicals did not affect EGF production. A summary of the results is shown in Table 1. 3.3. Intracellular signaling pathways in BEAS-2B exposed to 1-AP Intracellular mechanisms underlying the increase in protein expression of ICAM-1 and IL-6 as a result of 1-AP were investigated because this PAH derivative significantly affected the BEAS-2B cell line. In the examination using protein kinase inhibitors, AG1478 partially but significantly suppressed 1-AP-induced increases in ICAM-1 expression intensity and completely suppressed IL-6 production (Fig. 4a and b). SB 203580 also significantly suppressed the increase in IL-6 production (Fig. 4b). PD98059 did not block the expression of ICAM-1 and IL-6 (Fig. 4a and b). At the control levels, these inhibitors affected ICAM-1 expression intensity and/or IL-6 production (Fig. 4a and b). In the examination using nuclear receptor antagonists, 1e850 and ICI 182,780 slightly but significantly suppressed 1-AP-induced increase in ICAM-1 expression intensity (Fig. 5a). 1-AP-induced increase in IL-6 production was partially but significantly suppressed by treatment with 1e850, CH-223191, and ICI 182,780 (Fig. 5b). At the control levels, ICAM-1 expression intensity was not altered; however, IL-6 production was suppressed by these antagonists (Fig. 5a and b). 3.4. Ligand activity of pyrene, 1-NP, and 1-AP for nuclear receptors The ligand activity for TRab, ERab, and AhR was investigated, and the activity of 1-AP was compared with that of pyrene and 1NP. The ligand activity for TR and ER was observed in pyrene and 1-AP (Fig. 6a and b). In particular, the ligand activity for TRa, TRb, and ERb was increased in a 1-AP concentration-dependent manner (Fig. 6c and d). 1-NP did not exhibit the activity. In contrast, AhR ligand activity was observed in not only pyrene and 1-AP but also 1NP, and 1-NP showed the most effective ligand activity (Fig. 6e). 4. Discussion Fig. 3. Effect of pyrene, 1-NP, and 1-AP on proinflammatory protein expression in BEAS-2B Expression intensity (mean fluorescence intensity) of ICAM-1 (a) and production level of IL-6 (b) were measured after 24-h exposure to pyrene, 1-NP, and 1-AP (10 mM). Data are the mean  SEM of triplicate cultures and are representative of 3 independent experiments. *p < 0.05, **p < 0.01 vs. control.

In the present study, we investigated the effects of PAHs and their derivatives on bronchial epithelial cells in vitro. We found that PAH derivatives (NQ, PQ, 1-NP, and 1-AP) and pyrene could induce

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E. Koike et al. / Atmospheric Environment xxx (2014) 1e8 Table 1 Summary of toxicological effects of PAHs and their derivatives on BEAS-2B. Chemicals

Naphthalene 1,2-NQ (ortho) 1,4-NQ (para) Phenanthrene 9,10-PQ (ortho) 1,4-PQ (para) Pyrene 1-NP 1-AP

Cytotoxicity (decrease of cell viability)

Protein expression ICAM-1

IL-6

IL-8

EGF

e þ(ortho > para) þ e þ(ortho > para) þ Weak þ þ

e e e e b b [ [ [

e e e e b b b b [

e Y Y e Y Y e Y Y

e e e e e e e e e

þ Effective cytotoxicity;  no effect; [ significant increase; Y significant decrease; b increasing trend.

cytotoxicity; however, no cytotoxic effects of naphthalene and phenanthrene were observed. Pyrene, 1-NP, and 1-AP also increased inflammatory protein expression. The number of benzene rings and active functional groups may be key factors in the cytotoxicity and inflammatory effects of PAHs on bronchial

Fig. 4. Effect of protein kinase inhibitors on 1-AP-increased expression of proinflammatory proteins in BEAS-2B The EGFR-selective tyrosine kinase inhibitor AG1478, p38 MAPK inhibitor SB 203580, and MEK inhibitor PD98059 were used. Cells were pretreated with the abovementioned protein kinase inhibitors for 1 h and exposed to 1-AP (10 mM) for 24 h in the presence of these protein kinase inhibitors. Subsequently, the expression of ICAM-1 (a) and production of IL-6 (b) were measured. Data are the mean  SEM of triplicate cultures and are representative of 2 independent experiments. *p < 0.05, **p < 0.01, 1-AP vs. control; #p < 0.05, ##p < 0.01, cells treated with inhibitor vs. cells not treated with inhibitor.

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epithelial cells. The increase in inflammatory protein was partly suppressed by some protein kinase inhibitors and nuclear receptor antagonists such as an EGFR-selective tyrosine kinase inhibitor and TR antagonist. Recent studies have investigated the effects of chemical compounds such as organic extracts of PM or DEP, particularly those of PAH-related chemicals (Baulig et al., 2009; Diaz Sanchez, 1997; Kawasaki et al., 2001; Yanagisawa et al., 2006). However, the different effects of PAHs and their derivatives on respiratory and immune systems are not completely understood. In the present study, we focused on the toxicological effects related to cytotoxicity and inflammation in different PAHs and their derivatives on immune systems and the underlying molecular mechanisms. Exposure to NQ and PQ but not naphthalene and phenanthrene induced cytotoxic effects on BEAS-2B (Fig. 2a and b). Furthermore, ortho-quinoid PAHs (1,2-NQ and 9,10-PQ) had stronger cytotoxic effects than para-quinoid PAHs (1,4-NQ and 1,4-PQ). Pyrene showed a weak cytotoxic effect, and 1-NP and 1-AP significantly induced the cytotoxic effect (Fig. 2c). Previously, we also examined ROS generation using dichlorodihydrofluorescein diacetate (Koike, 2013). The oxidative capacity of NQ and PQ was strongly observed. Moreover, ortho-isomers showed a stronger capacity than para-isomers in a cell-free system. Naphthalene, phenanthrene, pyrene, 1-NP, and 1-AP did not show oxidative capacity. A report has also suggested that ortho-quinoid PAHs induce stronger

Fig. 5. Effect of nuclear receptor antagonists on 1-AP-increased expression of proinflammatory proteins in BEAS-2B TR antagonist 1e850, AhR antagonist CH-223191, and ER antagonist ICI 182,780 were used. Cells were pretreated with these antagonists for 1 h and exposed to 1-AP (10 mM) for 24 h in the presence of those antagonists. Subsequently, the expression of ICAM-1 (a) and production level of IL-6 (b) were measured. Data are the mean  SEM of triplicate cultures and are representative of 2 independent experiments. **p < 0.01, 1-AP vs. control; #p < 0.05, ##p < 0.01, cells treated with antagonist vs. cells not treated with antagonist.

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Fig. 6. Nuclear receptor ligand activity of 1-AP compared with that of pyrene and 1-NP Ligand activity for TRab (a, c) and ERab (b, d) was assessed by the nuclear receptor cofactor assay. This value was calculated as follows: (absorbance of test chemical at 450 nm)  (absorbance of solvent control at 450 nm). AhR ligand activity (e) was assessed by the reporter gene assay, and the value was calculated as follows: (absorbance of test chemical at 540 nm/absorbance of test chemical at 595 nm)  (absorbance of solvent control at 540 nm/ absorbance of solvent control at 595 nm). Data are the mean (a, b) and mean  SEM (cee) of duplicate sample and are representative of 2 independent experiments.

cytotoxic effects than para-quinoid PAHs by ROS generation through a redox cycle in lung epithelial cells (Motoyama et al., 2009). Therefore, chemical-mediated cytotoxicity may be consistent with oxidative capacity. However, other factors also exist because pyrene, 1-NP, and 1-AP showed a cytotoxic tendency without oxidative capacity. Some PAH derivatives (PQ, 1-NP, and 1-AP) and pyrene increased the intensity of ICAM-1 expression and IL-6 production in BEAS-2B. Naphthalene, phenanthrene, and NQ did not affect proinflammatory protein expression. These differences may have

been caused by oxidative capacity and by intracellular metabolism, permeability of the cell membrane, and receptor binding. Pyrene, 1-NP, and 1-AP increased proinflammatory protein expression (Fig. 3a and b). In particular, 1-AP significantly increased such expression. These proteins play an important part in the immune system. IL-6 shows various physiological functions such as proliferation and expression of cell adhesion molecules (including ICAM-1) on immune cells and differentiation of plasma cells. ICAM-1 recruits inflammatory cells by signal transduction.

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Next, we investigated the intracellular mechanisms underlying the effects of 1-AP on the expression of proinflammatory substances in BEAS-2B. EGFR tyrosine kinase and the subsequent signal transducer MAPK play a crucial role in the inflammatory response. In the evaluation of protein kinase activity, an EGFR-specific tyrosine kinase inhibitor (AG1478) and p38 MAPK inhibitor (SB 203580) effectively suppressed the increase in IL-6 production (Fig. 4). AG1478 also suppressed the increase in ICAM-1 expression. The MEK inhibitor PD98059 did not show the suppressive effects. In addition, we observed similar results with pyrene and 1-NP (data not shown). AG1478, SB 203580, and PD98059 also suppressed IL-6 production at the control levels (Fig. 4b), which may be because of the presence of EGF in the culture medium LHC-9. EGF can stimulate EGFR and the subsequent signaling pathway such as MAPK and could have induced the expression of proinflammatory proteins such as IL-6. Therefore, it is likely that these signal inhibitors affected control levels. However, we did observe suppressive effects of AG1478 and SB 203580 on 1-AP-increased IL-6 production, suggesting that the activation of EGFR-specific tyrosine kinase and p38 MAPK may be important pathways for the increased expression of proinflammatory proteins exposed to PAHs and related chemicals. The activation of these protein kinases can subsequently induce several transcriptional factors such as nuclear factor-kappa B (NFkB). NFkB is related to the induction of expression of various proinflammatory proteins, including ICAM-1 and IL-6 (Cybulsky and Gimbrone, 1991; Monaco and Paleolog, 2004). An organic extract of DEP induced the expression of proinflammatory cytokines through the activation of the p38 MAPK pathway and NFkB in bronchial epithelial cells (Kawasaki et al., 2001). It has been demonstrated that proinflammatory cytokines and environmental PAH may interact to promote DNA damage and the inflammatory response through the activation of p38 MAPK in alveolar epithelial type-II cells (Umannova et al., 2011). Furthermore, nuclear receptors play a role as transcriptional factors and regulate the expression of specific genes to control the development, homeostasis, and metabolism of cells. Nuclear receptors may be a trigger for adaptive responses to various environmental stimuli. It has been suggested that exposure to endocrine-disrupting chemicals such as PAHs may be a risk factor for adverse health effects through the disruption of nuclear receptors such as AhR and ER (Swedenborg and Pongratz, 2010). AhR ligands such as PAHs have been also found to induce antiestrogenic activity (Navas and Segner, 2000) and estrogenic activity (Ohtake et al., 2003). Reports have also shown that endocrine-disrupting chemicals affect cytokine production (Yano et al., 2003). Benzo[a] pyrene has shown AhR-dependent proinflammatory cytokine induction in macrophages that may lead to lung inflammation (Podechard et al., 2008). The effects of TR ligands on immune responses have not been completely elucidated; however, the TR ligand T3 has been shown to stimulate the maturation and function of dendritic cells (Mascanfroni et al., 2008). Therefore, certain types of PAHs and related chemicals affect protein kinase pathways and/ or nuclear receptors that may lead to the activation of bronchial epithelial cells and immune cells. In the evaluation of nuclear receptor activity, we found that antagonists for TR, AhR, and ER (1e850, CH-223191, and ICI 182,780) significantly suppressed the increase in IL-6 production by 1-AP in BEAS-2B (Fig. 5b). 1e850 and ICI 182,780 also slightly but significantly suppressed the 1-AP-mediated increase in ICAM-1 expression (Fig. 5a). We also examined the ligand activity of 1-AP, pyrene, and 1-NP for nuclear receptors and observed that 1-AP binds to TRab and ERb (Fig. 6aed). The activity of pyrene and 1NP was weak or not observed. AhR ligand activity was observed in pyrene, 1-NP, and 1-AP, and the most effective chemical was 1-NP (Fig. 6e). Therefore, some types of PAHs and related chemicals may

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affect nuclear receptors that partly contribute to proinflammatory protein expression in bronchial epithelial cells. We also observed the activation of immune cells by some PAH derivatives and pyrene (Takano et al., 2014). Naphthalene and phenanthrene did not affect immune cells. Thus, the number of benzene rings and active functional groups may be key factors in the inflammatory effects of PAHs on immune cells and bronchial epithelial cells. PAH derivatives (NQ, PQ, 1-NP, and 1-AP) and pyrene may promote inflammatory responses by stimulating respiratory and immune systems. Activated airway epithelial cells release proinflammatory cytokines such as IL-6 and may initiate or promote immune responses. A more detailed investigation involving the interaction between bronchial epithelial cells and immune cells is necessary. Furthermore, our previous study has shown that organic chemical components of DEP, including PAHs and derivatives, aggravate allergic airway inflammation (Yanagisawa et al., 2006). Future experimental studies should include the effects of PAHs and derivatives in vivo and ex vivo examination to elucidate the major toxic chemicals in the atmospheric environment. 5. Conclusions Some PAH derivatives (PQ, 1-NP, and 1-AP) and pyrene can induce cytotoxicity and increase proinflammatory protein expression in human bronchial epithelial cells. The toxicological effects of PAHs and their derivatives may be related to the different activities resulting from their chemical structures, including the numbers of benzene rings and functional groups. Furthermore, the chemicalinduced increase in proinflammatory protein expression, possibly through the activation of protein kinase pathways and nuclear receptors, may partly contribute to the adverse health effects of atmospheric PAHs. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (MEXT KAKENHI 20120014) and in part by grants from the National Institute for Environmental Studies (1115AA082). The authors would like to thank Ms. Satomi Abe for technical assistance and Enago (www.enago.jp) for the English language review. References Andreou, G., Rapsomanikis, S., 2009. Polycyclic aromatic hydrocarbons and their oxygenated derivatives in the urban atmosphere of Athens. Journal of Hazardous Materials 172, 363e373. Baulig, A., Singh, S., Marchand, A., Schins, R., Barouki, R., Garlatti, M., Marano, F., Baeza-Squiban, A., 2009. Role of Paris PM2.5 components in the proinflammatory response induced in airway epithelial cells. Toxicology 261, 126e135. Brook, R., Rajagopalan, S., Pope, C.A., Brook, J., Bhatnagar, A., Diez Roux, A., Holguin, F., Hong, Y., Luepker, R., Mittleman, M., Peters, A., Siscovick, D., Smith, S., Whitsel, L., Kaufman, J., 2010. Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 121, 2331e2378. Cybulsky, M.I., Gimbrone, M.A., 1991. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251, 788e791. Dergham, M., Lepers, C., Verdin, A., Billet, S., Cazier, F., Courcot, D., Shirali, P., Garçon, G., 2012. Prooxidant and proinflammatory potency of air pollution particulate matter (PM₂.₅₋₀.₃) produced in rural, urban, or industrial surroundings in human bronchial epithelial cells (BEAS-2B). Chemical Research in Toxicology 25, 904e919. Diaz Sanchez, D., 1997. The role of diesel exhaust particles and their associated polyaromatic hydrocarbons in the induction of allergic airway disease. Allergy 52, 52e56.

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