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Communications Aryl Hydrocarbon Receptor-Mediated Effects of Chlorinated Polycyclic Aromatic Hydrocarbons Takeshi Ohura,* Maki Morita, Masakazu Makino, Takashi Amagai, and Kayoko Shimoi Institute for EnVironmental Sciences, UniVersity of Shizuoka,52-1 Yada, Shizuoka 422-8526, Japan ReceiVed May 1, 2007
Chlorinated polycyclic aromatic hydrocarbons (ClPAHs) with 3–5 rings are ubiquitous environmental contaminants. However, toxicities of ClPAHs remain unclear. In this study, aryl hydrocarbon receptor (AhR)-mediated activities of ClPAHs were investigated by using a yeast assay system. All environmentally relevant 18 ClPAHs showed the AhR activities in the test; the activities were elevated with the number of chlorine atoms on the lower molecular weight PAH (∼three-ring and fluoranthene derivatives) but not for higher molecular weight ClPAHs (>four-ring). The similar trends were also observed in certain ClPAHs-induced cytochrome P450 1A1 expression in MCF-7 cells. The structure–activity relationship between the AhR activity and the corresponding solvent accessible surface area of ClPAHs revealed a parabolic relationship, with approximately 350 Å2/molecule as the optimal dimensions as the ligand for binding to AhR. These findings indicate that the spatial dimensions of ClPAHs apparently influence their ability to activate the AhR. Finally, we discussed the toxicity of exposure to ClPAHs based on the AhR activities, estimated that it would be approximately 30–50 times higher than that of dioxins. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that are formed by the incomplete combustion of organic compounds (1–3). Exposure to PAHcontaining substances increases the risk of cancer in humans to be mediated through aryl hydrocarbon receptor (AhR) activation (4, 5). The ligand-bound AhR, a cytosolic ligand-activated transcription factor complex, activates the transcription of genes related to the drug-metabolizing enzymes such as cytochrome P450 (CYP) and glutathione S-transferase. The most characterized pathway involves translocation of the activated AhR to the nucleus, where it binds with the AhR nuclear translocator protein (Arnt) to form a heterodimer. Binding of the heterodimer leads to transcriptional modulation of genes that contain a xenobiotic responsive element (XRE) (6). To promote PAH toxicity, interaction with AhR seems to be an essential mechanism. A series of structurally related aromatics, halogenated aromatic hydrocarbons (HAHs) such as polychlorinated dibenzop-dioxins/dibenzofurans (PCDDs/Fs) and polychlorinated biphenyls (PCBs), are widespread in the environment as anthropogenic by-products and mixtures for commercial applications (7). The presence of these compounds has prompted great concern about their adverse human effects in terms of mortality, embryotoxicity, immunotoxicity, and carcinogenicity, which are also mediated through AhR (6, 8–10). In particular, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent AhR ligand and has been used as a reference standard for hazard and risk assessment of these environmental and dietary contaminants (11). * To whom correspondence should be addressed. Tel: +81 54 264 5789. Fax: +81 54 264 5798. E-mail:
[email protected].
Figure 1. Structures of ClPAH derivatives.
Chlorinated PAHs with 3–5 rings (ClPAHs, Figure 1) recently have been found in air, automobile exhaust, snow, tap water, and kraft pulp mill products and wastes (reviewed in ref 12). ClPAHs are treated as structural hybrids of PAHs and HAHs; such hybrids include dioxins and PCBs and can themselves have adverse biologic effects. However, toxicologic information on ClPAHs currently is very limited, whereas mutagenic effects using the Salmonella assay have been investigated for some ClPAHs (13, 14). Hence, the association between ClPAHs and AhR as it pertains to the biologic toxicities of ClPAHs remains unclear. Here, we performed a receptor-specific yeast assay to examine the human AhR ligand-binding activity of environmentally relevant 18 ClPAHs with 3–5 rings. In addition, we also used these data to analyze the structure–activity relationship. Finally, the risk of human exposure to ClPAHs was also discussed. This report is the first to describe the AhR-mediated effects of ClPAHs.
10.1021/tx700148b CCC: $37.00 2007 American Chemical Society Published on Web 08/21/2007
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Table 1. AhR-Mediated Activities of ClPAHs and Corresponding Parent PAHs and Their Related Physicochemical Parameters AhR-mediated activity compound
abbreviation
anthracene phenanthrene fluoranthene pyrene chrysene benz[a]anthracene benzo[a]pyrene 1-chloroanthracene 2-chloroanthracene 9-chloroanthracene 9,10-dichloroanthracene 9-chlorophenanthrene 1,9-dichlorophenanthrene 3,9-dichlorophenanthrene 9,10-dichlorophenanthrene 3,9,10-trichlorophenanthrene 3-chlorofluoranthene 8-chlorofluoranthene 3,8-dichlorofluoranthene 6-chlorochrysene 6,12-dichlorochrysene 1-chloropyrene 7-chlorobenz[a]anthracene 7,12-dichlorobenz[a]anthracene 6-chlorobenzo[a]pyrene
Ant Phe Fluor Py Chry BaA BaP 1-ClAnt 2-ClAnt 9-ClAnt 9,10-Cl2Ant 9-ClPhe 1,9-Cl2Phe 3,9-Cl2Phe 9,10-Cl2Phe 3,9,10-Cl3Phe 3-ClFluor 8-ClFluor 3,8-Cl2Fluor 6-ClChry 6,12-Cl2Chry 1-ClPy 7-ClBaA 7,12-Cl2BaA 6-ClBaP
rings MW EC50 (µM) 3 3 4 4 4 4 5 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 5
178 178 202 202 228 228 252 212 212 212 246 212 246 246 246 280 236 236 270 262 296 236 262 296 286
9.38 15.1 6.41 1.27 0.03 0.05 0.06 1.78 0.65 2.51 0.32 2.07 0.54 0.20 0.39 0.08 0.38 0.35 0.01 0.03 2.35 0.66 0.08 0.66 0.69
Experimental Section Chemicals. In light of their environmental and toxicologic relevance, we selected seven PAH species as reference substances and chlorinated each by a procedure described previously (15). Indeed, N-chlorosuccinimide (∼100 mg) was added to each PAH (∼150 mg) in carbonated propylene (10 mL); the mixture was maintained at 100 °C for 2 h. The reactant for each PAH was fractionated by HPLC (column, COSMOSIL 5C18-AR; eluent, methanol), and the fractions corresponding to the dominant peaks were isolated and analyzed by GC/MS and 1H NMR spectroscopy (500 MHz, CDCl3). Because a small amount of impurities including parent PAH can contribute to the following AhR assay, the purities of the 18 synthesized ClPAHs were confirmed by GC/MS by area, resulting in >95%. The ClPAHs evaluated were as follows: 1-chloroanthracene (1ClAnt), 2-chloroanthracene (2-ClAnt), 9-chloroanthracene (9ClAnt), 9,10-dichloroanthracene (9,10-Cl2Ant), 9-chlorophenanthrene (9-ClPhe), 1,9-dichlorophenanthrene (1,9-Cl2Phe), 3,9dichlorophenanthrene (3,9-Cl2Phe), 9,10-dichlorophenanthrene (9,10-Cl2Phe), 3,9,10-trichlorophenanthrene (3,9,10-Cl3Phe), 3-chlorofluoranthene (3-ClFluor), 8-chlorofluoranthene (8ClFluor), 3,8-dichlorofluoranthene (3,8-Cl2Fluor), 6-chlorochrysene (6-ClChry), 6,12-dichlorochrysene (6,12-Cl2Chry), 1-chloropyrene (1-ClPy), 7-chlorobenz[a]anthracene (7-ClBaA), 7,12-dichlorobenz[a]anthracene (7,12-Cl2BaA), and 6-chlorobenzo[a]pyrene (6-ClBaP). The ring numbers, molecular weights, and structural derivatives of these ClPAHs are presented in Table 1 and Figure 1, respectively. These ClPAHs and the seven parent PAHs [anthracene (Ant), phenanthrene (Phe), fluoranthene (Fluor), chrysene (Chry), pyrene (Py), benz[a]anthracene (BaA), and BaP] were dissolved in dimethyl sulfoxide (DMSO; 10-2 M) before their use in the following experiments. AhR Activity in Yeast Assay. Saccharomyces cereVisiae YCM3, which expresses human AhR and Arnt and carries a lacZ reporter plasmid containing the aryl hydrocarbon response element (XRE) system (16–18), was provided by Dr. C. A. Miller, III (Tulane University, New Orleans, LA). The assay procedure was essentially the same as described by Miller (16). YCM3 cells were grown overnight at 30 °C in
physicochemical parameter 2
REPBaP SAS (Å /molecule) EHOMO (eV) ELUMO (eV) dipole moment (µ) 0.01 0.004 0.01 0.05 2.5 1.4 1 0.04 0.10 0.03 0.20 0.03 0.12 0.32 0.16 0.77 0.17 0.18 5.7 2.1 0.03 0.10 0.83 0.10 0.09
280 280.4 289.2 276.4 333.2 335.9 329.7 306.3 308.0 303.5 326.6 305.5 330.9 334.2 325.8 354.5 315.9 323.2 349.8 358.9 384.5 306.0 357.2 376.8 352.9
-0.201 -0.220 -0.223 -0.206 -0.213 -0.205 -0.198 -0.207 -0.206 -0.206 -0.212 -0.226 -0.231 -0.229 -0.231 -0.233 -0.227 -0.227 -0.229 -0.217 -0.221 -0.210 -0.211 -0.215 -0.203
-0.065 -0.042 -0.071 -0.061 -0.053 -0.063 -0.071 -0.073 -0.071 -0.075 -0.084 -0.052 -0.060 -0.058 -0.060 -0.066 -0.079 -0.077 -0.084 -0.061 -0.068 -0.069 -0.072 -0.081 -0.080
0.002 0.060 0.414 0.003 0.005 0.124 0.054 2.483 2.833 2.462 0.002 2.531 4.085 0.652 3.844 2.391 2.188 3.231 0.802 2.537 0.020 2.829 2.584 0.518 2.861
a synthetic complete medium containing 2% glucose and lacking tryptophan. The following day, test chemicals dissolved in DMSO, 5 µL of the overnight culture, and 195 µL of medium containing 2% galactose were mixed in a 96 well microplate, with subsequent incubation for 18 h at 30 °C. The optical densities of the wells were determined by reading the absorbance at a wavelength of 595 nm. A 30 µL aliquot of the suspension in each well was added to 120 µL of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM MgCl2, 10 mM KCl, 2 mM dithiothreitol, and 0.2% sarcosyl, adjusted to pH 7), and the reaction was started by adding 50 µL of o-nitrophenol-βgalactopyranoside (4 mg/ml solution in Z-buffer), followed by incubation for 60 min at 37 °C. The absorbance of o-nitrophenol was read at 405 nm. The β-galactosidase activity (referred to as lacZ units) was calculated by use of the following formula: absorbance at 405 nm × 1000/(absorbance at 595 nm × mL of well suspension added × min of reaction time). Cell Culture. MCF-7 cells were kindly provided by Dr. H. Hagenmaier (University of Tuebingen, Germany). MCF-7 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 0.1 mg/mL kanamycin, and 0.1 mg/mL ampicillin at 37 °C under a humidified atmosphere of 5% CO2. CYP1A1 mRNA Expression. Subconfluent MCF-7 cells were exposed to 2 µM ClPAH and the parent PAH in the assay medium (phenol red-free DMEM supplemented with 10% charcoal-dextran-treated FBS) and were collected after incubation at 37 °C for 12 h. Total RNA was isolated using RNeasy plus Mini kit (Qiagen GmbH, Hilden, Germany) and quantified by measuring the absorbance at 260 and 280 nm. The RNA sample was added to 20 µL of reaction mixture containing random hexamers, MuLv Reverse Transcriptase, RNase inhibitors, 25 mM MgCl2, 10 × PCR Buffer II (Applied Biosystems, Foster City, CA), and 10 mM dNTPmix (Promega Co., Madison, WI). Synthesis of cDNA was performed at 42 °C for 60 min, and the reverse transcription reaction was stopped by heating to 99 °C for 5 min followed by chilling on ice. Expression of CYP1A1 mRNA was measured using the Gene Amp PCR System 2400 (Perkin Elmer Inc., Winter Street
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Waltham, MA) according to the method previously reported with some modifications (19). Briefly, the cDNA of CYP1A1 was denatured at 95 °C for 5 min and cycled immediately for 30 cycles, denatured at 94 °C for 30 s, annealed at 49 °C for 30 s, and extended at 72 °C for 1 min. Primer sequences and conditions for CYP1A1 were as described by Iwanari et al. (19). The level of CYP1A1 mRNA was normalized to the level of GAPDH mRNA. Molecular Structure Calculation. We used GaussView W version 2.1 (20) for three-dimensional molecular modeling of the ClPAHs, and their molecular structures were optimized by using Gaussian R 98W version 5.4 (21). The calculation was performed on the basis of density functional theory in terms of B3LYP as the exchange and the correlation functions and the 3-21* basis set. We used the solvent accessible surface area (SAS), showing spatial dimensions, as the geometrical descriptor to reveal the structure–activity relationship of the ClPAHs, and the SAS was calculated under DMSO phase defined by the polarized continuum model (22). Other physicochemical characteristics, including dipole moment (µ), the energy level of the highest occupied molecular orbital (EHOMO), and the lowest unoccupied molecular orbital (ELUMO), were calculated for the optimized molecular structure (23). Data Analysis. Data are presented as means ( standard error. To determine the 50% effective concentration (EC50), dose–response curves were fitted (sigmoid fit) by using GraphPad Prism 4.0 (GraphPad Software, CA). The EC50 was calculated by determining the concentration at which 50% of the maximum intensity was reached from the sigmoidal fit equation. On the basis of the EC50 value, the relative potency of the AhR activity for each ClPAH was calculated as EC50 (BaP)/EC50 (test compound).
Results AhR Activity of ClPAHs. We individually assessed the activities of AhR as an agonist of 3–5 ring ClPAHs and their corresponding parent compounds in lacZ reporter gene assays using yeast YCM3 cells, which carried human AhR and Arnt genes. All target ClPAHs, as well as PAHs known to be AhR ligands, showed appreciable dose-dependent increases in lacZ units in this assay system; the dose–response curves ranging from 0.1 nM to 100 µM ClPAHs are presented in Figure S2 of the Supporting Information, and the potencies of these compounds are presented as EC50 values in Table 1. In addition, to compare the potencies of ClPAHs with that of BaP, an exogenous potent AhR ligand, we calculated the relative activity normalized from the EC50 value of BaP, showed as relative potency (REPBaP) (Table 1). The activities for Chry and BaA, large four-ring PAHs and potent AhR ligands, were comparable to that of BaP but were lower for the four-ring PAHs Py and Fluor and even less for compounds that had fewer than four rings, such as Ant and Phe. Among ClPAHs, 3,8-Cl2Fluor and 6-ClChry were the most potent AhR ligands, having activities that were about 2.0 and 5.7 times higher than that of BaP, respectively (Table 1). The activities of 3,9,10-Cl3Phe and 7-ClBaA (0.77 and 0.83 times that of BaP, respectively) were also relatively high among those of the ClPAHs (Table 1). The relative activities of the remaining ClPAHs ranged from 0.03 to 0.32 times that of BaP (Table 1). CYP1A1 Expression. In light of the evidence of AhR ligand activities of ClPAHs, we also investigated CYP1A1 mRNA expression in MCF-7 cells treated with lower or higher molecular weight ClPAHs and the parent PAHs. For chlorophenanthrene derivatives at a concentration of 2 µM, the
Figure 2. Effects of chlorophenanthrene derivatives and 6-ClBaP and the corresponding parent PAH on CYP1A1 mRNA levels. MCF-7 cells were treated with 2 µM ClPAH or PAH for 12 h. Levels of CYP1A1 and GAPDH mRNA were measured by RT-PCR, normalized to that of GAPDH, and compared to 0.1% DMSO as control; n ) 3; bars, SE.
CYP1A1 mRNA expression trended to elevate with an increase in the number of chlorine atoms on Phe (Figure 2). However, chlorination of BaP (6-ClBaP) decreased the expression as compared to BaP (Figure 2). These trends were quite similar to those observed in the yeast AhR assay. Relationships between AhR Activity and the Structure of ClPAHs. Comparison of the number of chlorine atoms on each PAH skeleton and the corresponding AhR activity of the compound revealed some interesting trends. For relatively low molecular weight ClPAHs (e.g., three-ring ClPAHs and ClFluor), the AhR activity tended to increase with the number of chlorine atoms on the corresponding parent PAH skeleton (Table 1 and Figure S2 of the Supporting Information). In contrast, the AhR activity of relatively high molecular weight ClPAHs (i.e., >four-ring ClPAHs) tended to decrease as the number of chlorine atoms on the corresponding parent PAH skeleton increased (Table 1 and Figure S2 of the Supporting Information). These findings suggest that the AhR activities of ClPAHs are strongly dependent on the spatial dimensions of the molecule. We evaluated the variability of activity in light of structural differences by using quantitative structure–activity relationship (QSAR) models. Previous studies using QSAR have demonstrated that the interaction between haloganated aromatic xenobiotics and AhR is a charge-transfer type and that the toxins appear to act as electron acceptors in the charge-transfer complex (24, 25). For polychlorinated naphthalenes, it has also been reported that the atomic charges of the fused carbons and the number of chlorine atoms are important determinants for the affinities to the AhR (10). In the present study for QSAR evaluation, we used relative SAS as one of the descriptors defining the spatial area size of the compounds. In addition to this analysis, we also compared the relationships between AhR activity and three physicochemical descriptors: EHOMO, ELUMO, and dipole moment (see Table 1). The log(1/EC50) as a measure of AhR activity and the SAS of ClPAH and PAHs for which SAS was e350 Å2/molecule showed a significant positive correlation [log(1/EC50) ) 0.0354 × SAS - 13.925, R2 ) 0.790, P < 0.01] (Figure 3A). Furthermore, for ClPAHs for which SAS was >350 Å2/molecule, such as 3,9,10-Cl3Phe, 7-Cl, and 7,12-Cl2BaA, 6-Cl and 6,12-Cl2Chry, and 6-ClBaP, the AhR activity and SAS showed a negative correlation [log(1/EC50) ) -0.0472 × SAS + 14.815, R2 ) 0.564, P ) 0.051] (Figure 3A). On the other hand, there was no significant correlation
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Figure 3. Relationship between AhR activity (calculated as log(1/EC50) of each ClPAH and PAH and the physicochemical parameter of SAS analysis (A), EHOMO (B), ELUMO (C), and dipole moment (D). The values for the physicochemical parameters of each compound used are listed in Table 1.
between log(1/EC50) and EHOMO, ELUMO, or dipole moment for any compound tested (Figure 2B–D).
Discussion ClPAHs are recently identified ubiquitous environmental contaminants (12). AhR-mediated effects and other related biological effects of HAHs are not unclear. As expected, all 3–5-ring ClPAHs tested showed AhR ligand activities in the yeast assay (Table 1). The potency tended to depend on the structural dimension of the compound. The similar trends were also observed in certain ClPAHs-induced cytochrome P450 1A1 expression in MCF-7 cells (Figure 2), suggesting that AhR-mediated transcriptional activities of ClPAHs could occur in MCF-7 cells. The relationships between structures and activities were confirmed by QSAR analysis using SAS as the molecular geometrical descriptor; the optimal structural dimension of the ligand for AhR was approximately 350 Å2/molecule (Figure 3A). The binding pocket of human AhR can accept planar ligand with maximal dimensions of 14 Å × 12 Å × 5 Å (26); the optimal value that we calculated by using AhR activities and SAS analysis is appropriate and consistent with this previous finding. To evaluate the risk of human exposure to ClPAHs, it is important to demonstrate the relationship between residue levels in the environment and toxicity. Most of the adverse effects of HAHs are thought to be mediated through AhR activation. Therefore, the toxic potencies of these compounds can be expressed in terms of relationship between congener-specific concentrations and a relative potency (REP), which is specific to an experiment, or a TCDD equivalency factor (TEF), which is consensus-derived from many relative potency values (27). We established the REP values of each ClPAH relative to BaP (REPBaP) in a yeast assay system (Table 1). In a similar system, the intensity of the AhR-associated toxicity of TCDD was 60fold higher than that of BaP (28). Therefore, the overall toxicity or toxic equivalents (TEQs) of a mixture of ClPAHs relative to TCDD can be defined as follows:
TEQ )
∑ [Ci] × REPBaPi ⁄ 60
(1)
where Ci represents the concentration of an individual ClPAH. Although the available data concerning environmental occur-
rences are currently limited, our group has investigated the atmospheric concentrations intensively. AhR activities associated with 17 of the 18 ClPAHs (except 3,8-Cl2Fluor) that we evaluated were detected in gaseous and particulate matter in urban air in Japan in 2005 and ranged from 0.75 pg/m3 for 3,9,10-Cl3Phe to 233 pg/m3 for 9-ClPhe (29); according to equation 1, the TEQ for the detected ClPAHs is 1.18 pg TEQ/ m3. In comparison, the annual concentrations of dioxins at three sites near our university (sampling site of ambient ClPAHs) ranged from 0.024 to 0.043 pg TEQ/m3 in 2005 (30). Comparison of both ambient concentrations shows that the toxicity of exposure to ClPAHs would be approximately 30–50 times higher than that of dioxins. The estimation raises the problem that the adverse effects of exposure to ClPAHs deserve further attention. In addition, Ohtake et al. reported that the molecular mechanism underlying the estrogen-related action of TCDD could be due to estrogen receptor (ER) R and ER β signaling by association with a ligand-activated AhR/Arnt heterodimer (31). This finding implies that such AhR-activated environmental contaminants also have the potential to induce adverse estrogenrelated actions, so that further study will be needed to clarify such toxicities of ClPAHs. Acknowledgment. We thank Dr. Charles A. Miller III of the Department of Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine, for kindly supplying us with the YCM3 strain and Dr. R. KurutoNiwa for help with MCF-7 cell care. This work was supported in part by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (14780416) and by the Steel Industry Foundation for the Advancement of Environmental Protection Technology (C-31-55), awarded to T.O. Supporting Information Available: Dose–response curves of AhR-mediated activity for ClPAHs and relative intensity of AhR activity of each ClPAH derivative normalized by that of the corresponding parent PAH. These materials are available free of charge via the Internet at http://pubs.acs.org.
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