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Similarities in the Endocrine-Disrupting Potencies of Indoor Dust and Flame Retardants by Using Human Osteosarcoma (U2OS) Cell-Based Reporter Gene Assays Go Suzuki,*,† Nguyen Minh Tue,‡ Govindan Malarvannan,‡ Agus Sudaryanto,‡ Shin Takahashi,‡ Shinsuke Tanabe,‡ Shin-ichi Sakai,§ Abraham Brouwer,∥ Naoto Uramaru,⊥ Shigeyuki Kitamura,⊥ and Hidetaka Takigami† †

Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, Tsukuba, Japan, Tsukuba 305-8506, Japan ‡ Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan § Environment Preservation Center, Kyoto University, Kyoto 606-8501, Japan ∥ BioDetection Systems, 1098 XH Amsterdam, The Netherlands ⊥ Nihon Pharmaceutical University, Kita-adachi 362-0806, Japan S Supporting Information *

ABSTRACT: Indoor dust is a sink for many kinds of pollutants, including flame retardants (FRs), plasticizers, and their contaminants and degradation products. These pollutants can be migrated to indoor dust from household items such as televisions and computers. To reveal high-priority end points of and contaminant candidates in indoor dust, using CALUX reporter gene assays based on human osteosarcoma (U2OS) cell lines, we evaluated and characterized the endocrine-disrupting potencies of crude extracts of indoor dust collected from Japan (n = 8), the United States (n = 21), Vietnam (n = 10), the Philippines (n = 17), and Indonesia (n = 10) and for 23 selected FRs. The CALUX reporter gene assays used were specific for compounds interacting with the human androgen receptor (AR), estrogen receptor α (ERα), progesterone receptor (PR), glucocorticoid receptor (GR), and peroxisome proliferator-activated receptor γ2 (PPARγ2). Indoor dust extracts were agonistic to ERα, GR, and PPARγ2 and antagonistic against AR, PR, GR, and PPARγ2. In comparison, a majority of FRs was agonistic to ERα and PPARγ2 only, and some FRs demonstrated receptor-specific antagonism against all tested nuclear receptors. Hierarchical clustering clearly indicated that agonism of ERα and antagonism of AR and PR were common, frequently detected end points for indoor dust and tested FRs. Given our previous results regarding the concentrations of FRs in indoor dust and in light of our current results, candidate contributors to these effects include not only internationally controlled brominated FRs but also alternatives such as some phosphorus-containing FRs. In the context of indoor pollution, high-frequency effects of FRs such as agonism of ERα and antagonism of AR and PR are candidate high-priority end points for further investigation.

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INTRODUCTION

indoor dust, as an excellent indicator of important contaminants, on chemical risk management throughout the lifecycle of product.

Because all resources are limited, society needs to promote a mindset of material cycling, which is based on appropriate management of the useful resources and toxic substances that emerge throughout a product’s lifecycle. This approach means that we have to not only recycle useful resources but also remove toxic substances from products. Recently our attention has focused on the evaluation of © 2013 American Chemical Society

Received: Revised: Accepted: Published: 2898

November 18, 2012 February 4, 2013 February 11, 2013 February 12, 2013 dx.doi.org/10.1021/es304691a | Environ. Sci. Technol. 2013, 47, 2898−2908

Environmental Science & Technology

Article

Indoor dust is a known sink for many kinds of pollutants1 and likely is one of the major routes of exposure in children, especially infants and toddlers, although actual exposure levels vary greatly according to individual lifestyle and activity patterns. House dust recently has been reported to be a key source of polybrominated diphenylethers (PBDEs), which are important flame retardants (FRs) in plastics, polyurethane foams, and textiles. These chemicals are contained in household items such as televisions, computers, and flameproof curtains and have led to the exposure of children2−4 due to their release during the weathering of these products.5,6 In addition, serum concentrations of 1,2,5,6,9,10hexabromocyclododecane (HBCD), another major FR, in adults were correlated with exposure to indoor dust but not dietary ingestion,7 indicating that indoor dust is also important for HBCD exposure in humans. Furthermore, phosphorus-containing flame retardants (PFRs) have recently been detected in indoor dust and air, likely because restrictions on the use of persistent organic pollutants (POPs) and the manufacture of tetra- to heptabrominated BDEs have led to increased use of alternative flame retardants, including PFRs.8−14 Concentrations of PFRs including triphenyl phosphate (TPHP), tris(1,3dichloroisopropyl) phosphate (TDCIPP), and tris(2-butoxyethyl) phosphate (TBOEP) in indoor dust tended to be higher than those of brominated FRs (BFRs) such as PBDEs. Moreover, it is highly possible that various additional compounds (e.g., plasticizers, antifoaming agents) and related compounds (e.g., degradation products, impurities) can also be migrated from household items to indoor dust. Therefore, effective chemical assessment is needed to reveal toxicologically important contaminants in indoor dust. To determine priority pollutants for assessment from the list of chemicals manufactured and distributed worldwide, we have focused on using in vitro bioassays and instrumental analyses to indicate the presence and activity profiles of potential pollutants in various environmental matrices. Applying such an effect-based approachcalled ‘bioassay-directed chemical analysis’ (reviewed in Schuetzle and Lewtas15) or ‘effect-directed analysis’ (reviewed in Brack et al.16)to indoor dust is a useful step toward the integration of effect and exposure analyses for improved chemical risk management throughout a product’s lifecycle. For example, the use of the dioxin-responsive chemicalactivated luciferase gene expression (DR-CALUX) assay combined with high-performance liquid chromatography (HPLC) and gas chromatography-high resolution mass spectrometry (GC-HRMS) clearly showed the prevalence of polybrominated dibenzofurans (PBDFs), which are known degradation products of17 and impurities in18 PBDEs, in indoor dust.19 In the current study, we used a panel of human-cell-based CALUX reporter gene assays to evaluate steroid-hormonedisrupting potency [that is, human androgen receptor (AR), estrogen receptor α (ERα), progesterone receptor (PR), and glucocorticoid receptor (GR)-mediated activities] and lipid metabolism and immune function-disrupting potency [human peroxisome proliferator-activated receptor γ2 (PPARγ2) -mediated activity] as markers of in vitro toxicity. Previous studies clearly indicated that BFRs including PBDEs classified as POPs (POPs-PBDEs) have nuclear hormone-receptor-mediated activities.20,21 As a first step in bioassay-directed chemical analysis, an effect-based approach toward evaluating indoor dust, we need to identify the toxicologically important, high-priority candidate end points and contaminants in indoor dust. To this end, we characterized the endocrine-disrupting potencies of indoor dust

from Japan, the United States, Vietnam, the Philippines, and Indonesia and of selected BFRs and PFRs. We then evaluated the data for possible similarities in the endocrine-disrupting potencies between indoor dust and FRs.

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MATERIALS AND METHODS Collection and Preparation of Indoor-Dust Samples. The dust samples used in the current study were collected during 2005 through 2009 from homes in the United States (n = 21), Vietnam (n = 10), the Philippines (n = 17), and Indonesia (n = 10), and from homes (n = 4) and offices (n = 4) in Japan. Dust samples from the United States and Japan were collected by using vacuum cleaners; those from other countries were collected by using brooms. Indoor dust was transferred from the vacuum cleaner bags to a stainless steel sieve (pore size, 95%), 17β-estradiol (E2; ≥98%), progesterone (PRO; >98%), dexamethasone (DEX; ≥97%), and rosiglitazone (ROS; >98%) were used as reference agonists for AR-, ERα-, PR-, GR-, and PPARγ2CALUX in the current study. Flutamide (FLU; >99%), tamoxifen (TAM; ≥99%), and GW9662 (≥98%) were used as reference antagonists for AR-, ERα-, and PPARγ2-CALUX. Mifepristone (RU486; ≥98%) was used as a reference antagonist in PR- and GR-CALUX assays. DHT, PRO, T3, and ROS were obtained from WAKO (Osaka, Japan). E2, DEX, FLU, TAM, RU486, and GW9662 were purchased from Sigma-Aldrich (Tokyo, Japan). All compounds were dissolved in DMSO. We tested 7 BFRs, including POPs-PBDEs, and 16 PFRs. The structures of tested compounds are shown in Figure S1; the abbreviations of the tested FRs follow the convention of Bergman et al.25 BDE-47 (purity, >98%) and -99 (>98%) were synthesized according to the methods of Teclechiel et al.26 and Orn et al.27 BDE-100 (≥98%), -183 (≥98%), -209 (≥98%), tetrabromobisphenol A (TBBPA, ≥98%), triethyl phosphate 2899

dx.doi.org/10.1021/es304691a | Environ. Sci. Technol. 2013, 47, 2898−2908


Article

reference agonist at the concentration of 100 × EC50 level in the same exposure medium. Antagonistic effects were considered to be receptor-specific when mitigated by coexposure to a high dose of the reference agonist. In the current study, we statistically evaluated the mitigation effect by using a two-sample t-test, providing that the data showed normal distribution (version 11.0, SigmaPlot, Systat Software, San Jose, CA). Calculation for EC50 and IC50 of FRs. When the data were appropriate for this application, dose−response curves of agonists and antagonists were generated for calculation of the median effective concentration (EC50) or inhibitory concentration (IC50) of FRs. Dose−response curves for these compounds were depicted as ligand-binding, sigmoidal dose− response (that is, variable slope) plots by using SigmaPlot (version 11.0, Systat Software): y = min + (max − min)/(1 + 10(log a1 −x) × a2), where y is the measured luciferase activity; x is the compound concentration; min (approximately 0%) is the luciferase activity of the DMSO vehicle-only control or the maximal concentration of reference antagonist for agonistic or antagonistic testing, respectively; max (approximately 100%) is the luciferase activity due to the maximal concentration of reference agonist or DMSO vehicle control for agonistic or antagonistic testing, respectively; a1 is the EC50 or IC50 of the compound; and a2 is the Hillslope (slope at the midpoint of the curve). Hierarchical Cluster Analysis. As preliminary data analysis for clustering analysis, composite indoor-dust extracts were ranked in order of their endocrine-disrupting potency to include samples that had no such activity. For each agonistic or antagonistic activity, composite dust samples were ranked in descending order of the dust dose in the wells that showed a response corresponding to about 5% induction (in assays of agonistic effects) or 20% suppression (in assays of antagonistic effects); these data were used to determine the 5% relative effective concentration (REC5) or 20% relative inhibitory concentration (RIC20), as shown in the Supporting Information (Table S1). In addition, the concentrations of FRs that were equivalent to the REC5 and RIC20 were ranked, as shown in the Supporting Information (see Table S2). These values were used for the hierarchical cluster analysis. After preliminary data analysis, indoor-dust samples, FRs, and their detected end points were grouped hierarchically according to their endocrine-disrupting profiles by using an agglomerative method, which clustered compounds in light of the distance between pairs of observations and the linkage criterion.34 We used the Euclidean distance as the method for measuring distance and Ward’s linkage as our linkage criterion for the hierarchical clustering generated by using SYSTAT 13 for Windows (Systat Software, Chicago, IL, USA).

(TEP, ≥99%), triphenyl phosphate (TPHP, ≥99%), and 1,2,5,6,9,10-hexabromocyclododecane (γ-HBCD, ≥98%) were obtained from Accu Standard (New Haven, CT, USA). Tris(2chloroethyl)phosphate (TCEP, ≥97%) and tris(2chloroisopropyl)phosphate (TCIPP, ≥99%) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Trimethyl phosphate (TMP, ≥99%), tris(propyl) phosphate (TPP, ≥97%), tris(butyl) phosphate (TNBP, ≥97%), tris(1,3-dichloroisopropyl) phosphate (TDCIPP), tris(2-butoxyethyl) phosphate (TBOEP), tris(2-ethylhexyl) phosphate (TEHP), and tricresyl phosphate (TMPP, ≥98%) were purchased from WAKO. Tris(2,6-dimethylphenyl) phosphate (2,6-TXP, ≥99%), trioctyl phosphate (TOP, ≥96%), tris(2-isopropylpenyl)phosphate (2TIPPP, ≥99%), tris(3-isopropylpenyl)phosphate (3-TIPPP, ≥99%), and tris(4-isopropylpenyl)phosphate (4-TIPPP, ≥94%) were obtained from Hayashi Pure Chemical Industries (Osaka, Japan). All compounds were dissolved in DMSO to a final concentration of 1.0 × 10−2 M. CALUX Assays of Endocrine-Disrupting Activity. AR-, ERα-, PR-, GR-, and PPARg2-CALUX cell lineshuman osteosarcoma cell lines that were stably cotransfected with individual target human-receptor-regulated luciferase gene constructs (U2OS-luc cells)were developed by BioDetection Systems (Amsterdam, The Netherlands). The assay procedures have been described previously.20,28−33 Briefly, U2OS-luc cells were cultured at 37 °C under 5% CO2 and high humidity in DF (1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 [DMEM−F12]) medium supplemented with 7.5% fetal calf serum (FCS). U2OS-luc cells were seeded into microplates with DF medium (without phenol red) supplemented with stripped (dextran-coated and charcoal-treated) FCS. After 24 h of incubation, the medium was replaced with medium containing stripped FCS and reference chemicals with or without indoor dust extracts or FRs for agonistic and antagonistic response testing, as described following. After 24 h of exposure, the medium was removed, and the cells were lysed in Triton lysis buffer. Luciferin solution was added, and luciferase activity was measured by using a luminometer (Atto, Tokyo, Japan). All measurements were conducted in 3 wells; experiments were performed at least in duplicate for dust extract and in triplicate for FRs. Evaluation of Agonistic Activity. CALUX cells were exposed to reference hormone-receptor agonists, indoor-dust extracts, or FRs. Levels of luciferase induction were expressed in terms of percentage of the maximal level of induction by the respective reference agonist. Indoor-dust extracts and FRs showing more than 5% induction and dose-dependent responses were defined as positive agonistics. Evaluation of Antagonistic Activity. We performed 3 experiments to test the antagonistic activity of various compounds. In the first, CALUX cells were exposed to reference hormone-receptor antagonists, dust extracts, or FRs in conjunction with the respective reference agonists at the EC50 level. For this experiment, luciferase induction was expressed in terms of percentage of the luciferase activity in cells exposed to the DMSO vehicle-only control at the EC50 concentration of the reference agonist. When the induction level decreased by 20% and showed dose-dependency, we then evaluated the cytotoxicity of the compound in the same exposure medium by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as described elsewhere.33 Finally, in the absence of a cytotoxic effect in the MTT assay, the assay was rerun and included reference antagonists, dust extracts, or FRs as well as

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RESULTS AND DISCUSSION Agonistic and antagonistic testing using CALUX assays performed had been well validated with respective reference compounds (see the section of Validation of CALUX assays for testing agonistic and antagonistic ef fects in Supporting Information, Figures S2 and S3). Therefore, we concluded that the panel of CALUX assays used in the current study was appropriate and efficient for evaluating the endocrine-disrupting potencies of indoor-dust samples and of selected FRs. Indoor-Dust Extracts. Agonistic Testing. Several of the indoor-dust extracts that we tested showed agonistic dose− responses on ERα-, GR-, and PPARγ2-CALUX cells (Figure S4 A, B, and C). However, none of the extracts were agonistic in AR2900



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particularly potent GR agonistic effects (Figure S4 B). JPN HD showed only weak PPARγ2-agonistic activity in the current study (Figure S4 C). Our data indicated that ERα agonistic activity was detected frequently in indoor dust. Kennedy et al.35 recently reported that the ER-agonistic activity of indoor (office) air was more about 1000 times more potent than that of outdoor air, suggesting that indoor air is a potential source of estrogenic activity. This previous finding is consistent with our current results because the ERα-agonistic activity of JPN OD was higher than that of other indoor-dust extracts. Importantly, despite the collection of indoor dust samples from homes rather than offices, US HD samples tended to have higher ERα agonistic activity than did the other indoor dusts. For more than a decade, ER agonistic activities have been investigated in various environmental matrices, including surface water;36 suspended particulate matter in water;36,37 influent, effluent, and sludge from wastewater treatment plants;36 effluents from industries, hospitals, and sewage treatment plants;33 freshwater and marine sediment;37−39 runoff and leachate from land-applied biosolids;40 and outdoor air particulate.41,42 In contrast, few studies have evaluated the GR- and PPARγ2-agonistic activity in environmental samples because of fewer detection systems, although van der Linden et al.33 reported GR-agonist activation in effluents from industries, hospitals, and sewage treatment plants. Antagonistic Testing. Receptor-specific antagonistic activities against AR, PR, GR, and PPARγ2 but not ERα were evaluated for several of the indoor-dust samples (Figure S5 A, B, C, and D). The dose of indoor dust that achieved RIC20 and dose-dependent responses are indicated in Table 1. An example of potent antagonistic indoor dusts in this study was shown in Figure 2. Most of the indoor-dust extracts showed AR-, PR-, and PPARγ2antagonistic activity. In particular, all indoor-dust extracts except IND HD2 had AR- and PR-antagonistic activity with dosedependent responses. In addition, JPN HD, JPN OD, US HD1 through 3, and PHL HD1 through 3 tended to be more potent antagonists than did any of the VN or IND HD samples; in fact, IND HD2 had no AR- or PR-antagonistic at all. PPARγ2-specific inhibitory effects were detected in the JPN HD, JPN OD, US HD, and PHL HD extracts, which also demonstrated AR- and PR-antagonistic activity. In comparison, only JPN dust extracts showed dose-dependent GR-antagonistic responses, indicating location-specific end points. Our results indicated that AR- and PR- antagonistic activity were detected frequently in indoor dust. Various AR- and PRantagonistic responses in extracts of various environmental

or PR-CALUX assays. The concentrations of indoor-dust that yielded REC5 values as well as dose-dependent responses are indicated in Table 1. An example of potent agonistic indoor dusts Table 1. Agonistic and Antagonistic Effects of Extracts of Pooled Indoor-Dust Samples Collected from Japan (JPN), the United States (US), Vietnam (VN), the Philippines (PHL), and Indonesia (IND) on AR, ERα, PR, GR, and PPARγ2c dose eliciting agonistic effectsa

dose eliciting antagonistic effectsb

dust extract

ERα

GR

PPARγ2

AR

PR

GR

PPARγ2

JPN HD JPN OD US HD1 US HD2 US HD3 US HD4 VN HD1 VN HD2 PHL HD1 PHL HD2 PHL HD3 IND HD1 IND HD2

12 12 38 38 12 39 110 110 70 69 72 140 NE

NE NE 110 NE 40 100 NE NE NE NE NE NE NE

120 NE NE NE NE NE NE NE NE NE NE NE NE

120 120 38 38 38 120 110 110 70 69 72 140 NE

39 38 38 38 38 120 110 39 23 69 72 140 NE

120 120 NE NE NE NE NE NE NE NE NE NE NE

120 120 11 110 120 120 NE NE 70 69 72 NE NE

a The concentration (μg/well) of indoor dust necessary to achieve 5% of the maximal response of the respective reference agonist. bThe concentration (μg/well) of indoor dust necessary to achieve 20% inhibition of the activity induced by respective antagonist. cHD, house dust; NE, no effect at about 70 μg/well for PHL samples and about 100 μg/well for samples from JPN, US, VN, and IND; OD, office dust.

in this study was shown in Figure 1. In particular, all indoor-dust extracts except for IND HD2 had ERα-agonistic activity (Figure S4 A), and the samples JPN OD and US HD1 through HD3 all had marked E2-like activity (Figure 1). Furthermore, the results suggest that some of the compounds in the US HD3 extract seem to be complete ERα agonists. In contrast, JPN HD likely contained partial ERα agonists in light of its dose−response curves (Figure S4 A). Although dose−responses from other indoor-dust extracts were weaker than those from JPN and US dust samples, all extracts except IND HD2 were judged as inducing agonistic responses because they all induced ERαagonistic activity greater than 5% of the maximal activity of E2. For GR agonistic activity, all US dust extracts except HD2 exhibited DEX-like activity, and compounds in US HD3 had

Figure 1. Detected potent agonistic indoor dusts collected from Japan (JPN) and the United States (US) on ERα-CALUX cells. Values represent the mean ± SD from repeated assays conducted in this study at least. REC5 (the 5% relative effective concentration), agonist concentration indicating 5% induction; HD, house dust; OD, office dust. 2901



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Figure 2. Detected potent antagonistic indoor dusts collected from Japan (JPN) and the United States (US) on AR- and PR-CALUX cells. The black bar shows the result for coexposure of indoor dust extract and respective reference agonist at EC50 level to the cells, which indicate no cytotoxicity. The gray bar shows the result for coexposure of indoor dust extract and respective reference agonist at EC50 level × 100 to the cells. Values represent the mean ± SD from three to five independent assays conducted in this study. RIC20 (20% relative inhibitory concentration), antagonist concentration indicating 80% induction; HD, house dust; OD, office dust. *Compared with results for coexposure experiment of dust extracts and respective reference agonists at the EC50 level.

have showed no ER-agonistic activity in the MVLN assay. Even though 2,6-TXPnot POPs-PBDEswas the most potent ERα agonist in the current study, its REP was about 10,000-fold lower than that of the natural ligand, E2. In addition, 2 of the 23 FRs tested (BDE-47 and TBBPA) induced PPARγ2-agonistic activity that was greater than 5% of the maximal activity of the antidiabetic drug ROS, a well-known agonist of PPARγ, in the PPARγ2-CALUX assay. Specifically the REC5 value of both was 1.0 × 10−5 M, which is 1000-fold lower than that of ROS. Similar weak PPARγ-agonistic activity has previously been reported for TBBPA but not BDE-47.52 Antagonistic Testing. Some of the FRs showed receptorspecific antagonistic activities against all of the nuclear receptors that we tested (Figure S7); the RIC20 values of these antagonistic compounds are indicated in Table 2. An example of detected antagonistic FRs in this study was shown in Figure 4. Specifically, 15 of the 23 FRs exerted AR-antagonistic activity that exceeded the 20% of the maximal activity of FLU in the ARCALUX assay (Figure S7 A). Ranked in order of the REPs based on the RIC20 values, the AR-antagonistic activities of the FRs are as follows: BDE-100 > BDE-47, BDE-99 > γ-HBCD, TDCIPP, 2,6-TXP, 2-TIPPP > BDE-183, TPHP, TMPP > TBBPA, TNBP, TCIPP, 3-TIPPP, 4-TIPPP. AR-antagonistic potencies similar to those in the current study have been reported previously for the BFRs BDE-47, -100, and -99;20,21,53 γ-HBCD;20 and TBBPA.54 The AR-antagonistic activity of the PFR TCIPP has been reported,55 although the previously determined REP to FLU for TCIPP was 100-fold lower than that in the current study. Although TEHP was reported to induced AR antagonistic activity in AR-CALUX cells,55 this compound lacked dose− response effects in our current study. BDE-100 showed the highest AR-antagonistic effect in the current study, with an RIC20 of 3.0 × 10−8 M and an REP that was about 3 times that of the reference AR-antagonist drug FLU. Furthermore, the ARantagonistic potencies of 15 of the 23 FRs were quite strong. In particular, REPs to FLU for BDE-47, BDE-99, γ-HBCD, TDCIPP, 2,6-TXP, and 2-TIPPP were greater than or equal to 0.1 (Table 2). We noted that 16 of the 23 FRs exerted PR-antagonistic activity greater than 20% of the maximal activity of RU486 in the PR-CALUX assay (Figure S7 B). The PR-antagonistic activity of these 16 FRs, ranked in order of the REPs based on their RIC20 values, is as follows: BDE-47, BDE-99, BDE-100, γ-HBCD,

samples have been reported previously, including AR-antagonistic43 and PR-antagonistic44 effects of air extracts, antiandrogenic effects of traffic exhaust particles,45 and AR-antagonistic responses to water46 and sediment38,46,47 samples. To our knowledge, the present study revealed, for the first time, the ERα-, GR- and PPARγ2-agonistic activities and the AR-, PR-, GR-, and PPARγ2-antagonistic effects of indoor dust collected from homes and offices. FRs. Agonistic Testing. Some of the BFRs and PFRs evaluated demonstrated agonistic dose−response effects on ERα- and PPARγ2-CALUX cells but not other cell lines (Figure S6 A and B); the REC5 and EC50 values of these agonistic compounds are indicated in Table 2. An example of detected agonistic FRs in this study was shown in Figure 3. Specifically, 9 of 23 FRs tested exerted ERα-agonistic activity at greater than the 5% of the maximal activity of E2 in the ERαCALUX assay. The ERα-agonistic activity [ranked in order of the relative potency (REPs) determined from the REC5 value] of these FRs is as follows: 2,6-TXP > BDE-47, BDE-100, TPHP, TCP > BDE-99, TDCIPP > TBBPA, TBP. In regard to BFRs, similar ERα-agonistic potency has been reported for BDE-47 and -100 in 3 independent studies,20,21,48 for BDE-99 in 1 study,48 and for TBBPA in 2 studies.49,50 However few previous studies have addressed the ERαagonistic activity of the PFRs we tested here. TDCIPP, TPHP, and TMPP had no ERα-agonistic activity at the maximal doses (2.3 × 10−5 M, 3.1 × 10−5 M, and 2.7 × 10−5 M, respectively) tested on MVLN cells,50 which are MCF7 human breast carcinoma cells that are stably transfected with an estrogenreceptor-controlled luciferase reporter gene. The difference in outcome between our current study and the one cited likely is due primarily to the differences in sensitivity between the ERαCALUX and MVLN assays. In fact, the EC50 of E2 in the ERαCALUX assay (4.2 × 10−12 M, Figure S2) in the current study is 20 times lower than that from the MVLN assay (8.3 × 10−11 M51). Considering the differences in sensitivity between the assays, the REC5 values of TDCIPP, TPHP, and TMPP in the MVLN assayas estimated according to the REC5 values from the ERαCALUX assaywould be 6.0 × 10−5 M, 2.0 × 10−5 M, and 2.0 × 10−5 M, respectively. Because the REC5 values estimated for the MVLN assay are equivalent to or higher than the previously mentioned maximal doses, TDCIPP, TPHP, and TMPP may 2902


2903

−12

4.2 × 10 ― ― ― ― ― NC NC 5.1 × 10−6 NC NC NC NC NC NC NC NC NC NC NC NC 3.3 × 10−6 NC NC 8.3 × 10−8 NC NC NC NC

EC50

EC50 ― 5.2 × 10−8 ― ― ― ― NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

PPARγ2 ― 1.0 × 10−8 ― ― ― ― 1.0 × 10−5 NE NE NE NE NE 1.0 × 10−5 NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE

REC5 ― ― 1.0 × 10−7 ― ― ― 3.0 × 10−7 3.0 × 10−7 3.0 × 10−8 3.0 × 10−6 NE 1.0 × 10−6 1.0 × 10−5 NE NE NE 1.0 × 10−5 NE 1.0 × 10−5 1.0 × 10−6 NE 3.0 × 10−6 NE 3.0 × 10−6 1.0 × 10−6 NE 1.0 × 10−6 1.0 × 10−5 1.0 × 10−5

RIC20

AR ― ― 2.8 × 10−7 ― ― ― 5.2 × 10−7 9.7 × 10−7 9.8 × 10−8 3.3 × 10−6 NC NC NC NC NC NC NC NC NC 1.9 × 10−6 NC 5.8 × 10−6 NC 4.1 × 10−6 2.2 × 10−6 NC 3.5 × 10−6 NC NC

IC50

IC50 ― ― ― 3.3 × 10−8 ― ― NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

ERα ― ― ― 1.0 × 10−7 ― ― NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE 1.0 × 10−5 NE NE NE 3.0 × 10−6 NE 1.0 × 10−5

RIC20 ― ― ― ― 7.3 × 10−11 ― 3.0 × 10−7 3.0 × 10−7 3.0 × 10−7 1.0 × 10−6 NE 3.0 × 10−7 1.0 × 10−5 NE NE NE 3.0 × 10−6 NE 3.0 × 10−6 3.0 × 10−7 1.0 × 10−5 1.0 × 10−6 NE 3.0 × 10−7 1.0 × 10−6 NE 1.0 × 10−6 1.0 × 10−6 3.0 × 10−6

RIC20

PR ― ― ― ― 7.9 × 10−11 ― 1.2 × 10−6 1.1 × 10−6 3.7 × 10−7 1.5 × 10−6 NC 3.8 × 10−7 NC NC NC NC 4.2 × 10−6 NC 1.1 × 10−5 8.5 × 10−7 NC 1.9 × 10−6 NC 1.4 × 10−6 1.5 × 10−6 NC 3.0 × 10−6 3.5 × 10−6 NC

IC50

antagonistic effects (M)

― ― ― ― 1.0 × 10−9 ― NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE 1.0 × 10−5 NE NE NE 1.0 × 10−5 NE 1.0 × 10−5

RIC20

GR ― ― ― ― 2.2 × 10−9 ― NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

IC50

IC50 ― ― ― ― ― 8.4 × 10−10 NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC 2.9 × 10−6

PPARγ2 ― ― ― ― ― 3.0 × 10−10 NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE 1.0 × 10−6

RIC20

NE, no effect at 1.0 × 10−5 M; NC, not calculated due to lack of a complete dose−response curve; REC5 (the 5% relative effective concentration), agonist concentration indicating 5% induction; RIC20 (20% relative inhibitory concentration), antagonist concentration indicating 80% induction.

a

1.0 × 10 ― ― ― ― ― 1.0 × 10−6 3.0 × 10−6 1.0 × 10−6 NE NE NE 1.0 × 10−5 NE NE NE 1.0 × 10−5 NE NE 3.0 × 10−6 NE 1.0 × 10−6 NE 1.0 × 10−6 1.0 × 10−8 NE NE NE NE

E2 ROS FLU TAM RU486 GW9662 BDE-47 BDE-99 BDE-100 BDE-183 BDE-209 γ-HBCD TBBPA TMP TEP TPrP TNBP TCEP TCIPP TDCIPP TBOEP TPHP TEHP TMPP 2,6-TXP TOP 2-TIPPP 3-TIPPP 4-TIPPP

ERα

−12

REC5

compounds

agonistic effects (M)

Table 2. Agonistic and Antagonistic Effects of Selected Flame Retardants (FRs) on AR, ERα, PR, GR, and PPARγ2a

Environmental Science & Technology Article



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Figure 3. Detected agonistic dose−responses for BDE-47, TDCIPP, TPHP, and 2,6-TXP on ERα-CALUX cells. Values represent the mean ± SD from three independent assays conducted in this study. REC5 (the 5% relative effective concentration), agonist concentration indicating 5% induction.

Figure 4. Detected antagonistic dose−responses for TDCIPP and TPHP on (A) AR- and (B) PR-CALUX cells. Values represent the mean ± SD from three independent assays conducted in this study. Receptor specificity of all antagonistic effects were confirmed by coexposure to a high dose of the reference agonist in this study (not shown). RIC20 (20% relative inhibitory concentration), antagonist concentration indicating 80% induction.

ERα antagonist, in the current study (1.0 × 10−7 M) is about 17 times higher than that reported previously (6.0 × 10−9 M21). The ER-antagonistic activities for BDE-183 and γ-HBCD that were reported previously20 were obtained from reporter gene assays using the T47D human breast cancer cell line which has not only ERα but also ERβ, whereas our data resulted from ERα-CALUX assays, which incorporate the U2OS human osteosarcoma cell line. The current study is the first to indicate that TDCIPP, TPHP, 2,6-TXP, TNBP and TMPP are ERα agonists. For antagonistic FRs results, various AR- and PR-antagonists among the tested FRs (other than those mentioned earlier) have been so defined for the first time in the current study. Furthermore, we also initially indicated that TEHP and 2-TIPPP have ERα- and GRantagonistic activity, whereas 4-TIPPP was antagonistic toward ERα, GR, and PPARγ2. Hierarchical Clustering. Indoor Dusts. We used the results of the characterization of the endocrine-disrupting potencies of the indoor-dust samples from various countries to allocate them into 3 groups (Figure 5). Subcluster I included samples from Japan, the United States, and the Philippines that had potent agonism of ERα and antagonism of AR, PR, and PPARγ2. Subcluster II contained all of the samples from Vietnam and one from Indonesia; all of these extracts showed weak ERα-agonistic and AR- and PR-antagonistic effects. Subcluster III was the Indonesian composite extract that lacked any agonistic and antagonistic activities. The endocrine-disrupting activity profiles of the composite indoor-dust extracts tended to be similar between countries, suggesting a common indoor-pollution issue perhaps derived from the use of products containing chemicals such as flame-retardants and plastic additives.

TDCIPP, TMPP > BDE-183, TPHP, 2,6-TXP, 2-TIPPP, 3TIPPP > TBBPA, TBOEP, TNBP, TCIPP, 4-TIPPP. Hamers et al.20 have reported similar PR antagonistic potencies for BDE100, -183, and γ-HBCD but not BDE-47 and -99. The PRantagonistic effects of BDE-47 and -99 that we obtained here were more potent than those of Hamers et al.20 Although Li et al.49 reported that TBBPA had potent PR antagonistic activity (RIC20: 7.8 × 10−8 M) in a yeast two-hybrid assay, the RIC20 that we obtained is about 100-fold higher. This difference may reflect differences in the in vitro assays used. The REP of the most potent PR-antagonist among the FRs we tested, BDE-100, was about to 1/4000th that of the abortion drug RU486. In contrast to the results regarding AR- and PR-antagonism, only a few FRs induced antagonistic activity greater than the 20% criterion in the ERα-, GR-, and PPARγ2-CALUX assays (Figure S7 C, D, and E). The REPs of TEHP, 2-TIPPP, and 4-TIPPP during testing for ERα-antagonism were 100-, 30-, and 100-fold lower than that of the breast cancer prevention drug TAM. In comparison, the GR-antagonistic activity of these same compounds, as represented by their REPs, was 10,000-fold lower than that of RU486 which blocks anti-inflammatory effects. 4-TIPPP is about 3000 times less potent an inhibitor of PPARγ2 than is GW9662, the reference PPARγ antagonist. Several of the FRs we tested were POPs-PBDEs. BDE-99, BDE-183, and γ-HBCD demonstrated no ERα antagonistic effect in our hands, although these compounds all reportedly had antiestrogenic potency in previous studies.20,21 In particular, Kojima et al.21 indicated that the RIC20 of BDE-99 was 5.7 × 10−6 M on ERα antagonistic testing. Differences in the sensitivities of the assays used may have led to these apparent differences in ERα-antagonism. In fact, the RIC20 value of TAM, the reference 2904



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Figure 5. Hierarchical clustering for indoor dust samples collected from Japan (JPN), the United States (US), the Philippines (PHL), Vietnam (VN), and Indonesia (IND) according to their endocrine-disrupting potencies. HD, house dust; OD, office dust.

Figure 6. Hierarchical clustering for tested FRs according to their endocrine-disrupting potencies.

FRs. Similar to what we did for the indoor-dust extracts, we divided the FRs into three groups based on their endocrinedisrupting effects (Figure 6). Subcluster I comprised compounds including BDE-209 and TBOEP that had no or only weak activity. Subcluster II contained compounds such as γ-HBCD, BDE-183, and TBBPA that showed weak antagonism of AR and PR. Subcluster III included compounds with potent agonism of ERα and antagonism of AR and PR. Note that TDCIPP and TPHP, which were detected at ppm-levels in indoor dust from Japan,10,13 Sweden,11 the United States,12 and Belgium14 were grouped with POP-PBDEs (e.g., BDE-47, BDE-99, and BDE100), suggesting similarities in their potential reproductive toxicities. Importantly, estimated alternative FRs such as a part of PFRs (e.g., TDCIPP and TPHP) had analogous effects with POP-PBDEs, which indicate that alternative FRs may not have intended to reduce their toxicity. In fact, a recent review article clearly indicated that potentially replacement flame-retardants have large data gaps concerning their published PBT (Persistence, Bioaccumulation, and Toxicity) properties even if these compounds are currently being marketed.56 However, we anticipate that, based on a comparison of persistence and bioaccumulation potentials which undergo abiotic degradation and biotic metabolism, FRs tested in this study would have lower in vivo toxicity than POPPBDEs. Detected End Points. Cluster analysis according to detected end points of endocrine disruption divided the effects of the tested composite indoor-dust samples and FRs into 2 large subclusters (Figure 7). For composite dust samples, those in subcluster I had ERα agonism and antagonism for AR, PR, and PPARγ2, and those in subcluster II were characterized by other end points. For FRs, subcluster I comprised those compounds with ERα agonism and antagonism for AR and PR, whereas subcluster II FRs had other end points. Overall, subcluster I comprised samples with high-frequency end points, compared with subcluster II, which contained low-frequency or undetected end points. This clustering analysis indicates that the highfrequency end points of endocrine disruption are comparable

among the indoor-dust samples and FRs tested in the current study. Implications of the Results from the Current Study. Our results clearly indicated similarities in the endocrine-disrupting potencies between indoor dust and FRs. In the context of indoor pollution due to FRs, high-frequency end points such as agonism of ERα and antagonism of AR and PR likely are high-priority end points that should be addressed in the next step of the bioassaydirected chemical analysis approach as applied to indoor dust. In addition to POP-PBDEs, γ-HBCD, TDCIPP, and TPHP might be candidate contributors to this pollution, in light of our current results and previous findings regarding the high concentrations of these compounds in indoor dust.12,13 These future efforts should identify and quantify the compounds that are responsible for the observed AR and PR antagonism and ERα agonism to determine priorities for subsequent source identification and in vivo toxicity testing for risk assessment. We also hope bioassaydirected chemical analysis based on application of chemical fractionation reveals mixture effects such as masking effect47 and implicates the need of further research about risk assessment for an exposure to a chemical mixture. In addition, the toxicologic importance of PPARγ2 antagonists needs to be elucidated, because these activities tended to be detected frequently in indoor dust, although our current results suggest that the tested FRs may not be substantial contributors to the effects measured (Table 1 and 2). Furthermore, the GR-agonists and -antagonists in indoor dust collected from the United States and Japan should be addressed in future bioassay-directed chemical analysis, even though the toxicologic importance of these compounds is unclear. In conclusion, by using in vitro toxicologic testing, we revealed several candidate high-priority end points and contaminants in indoor dust. Our application of a panel of human-cell-based CALUX reporter gene assays effectively identified various contaminants among FRs and in indoordust samples that should be studied further and managed throughout the lifecycles of products that include these contaminants. 2905



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Figure 7. Hierarchical clustering for the detetected end points for endocrine disruption of indoor dust and tested FRs.

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indoor dust. Environ. Sci. Technol. 2009, 43, 1437−1442, DOI: 10.1021/ es802599d. (6) Webster, T. F.; Harrad, S.; Millette, J. R.; Holbrook, R. D.; Davis, J. M.; Stapleton, H. M.; Allen, J. G.; McClean, M. D.; Ibarra, C.; Abdallah, M. A.; Covaci, A. Identifying transfer mechanisms and sources of decabromodiphenyl ether (BDE 209) in indoor environments using environmental forensic microscopy. Environ. Sci. Technol. 2009, 43, 3067−3072, DOI: 10.1021/es803139w. (7) Roosens, L.; Abdallah, M. A.; Harrad, S.; Neels, H.; Covaci, A. Exposure to hexabromocyclododecanes (HBCDs) via dust ingestion, but not diet, correlates with concentrations in human serum: preliminary results. Environ. Health Perspect. 2009, 117, 1707−1712, DOI: 10.1289/ehp.0900869. (8) Brommer, S.; Harrad, S.; Van den Eede, N.; Covaci, A. Concentrations of organophosphate esters and brominated flame retardants in German indoor dust samples. J. Environ. Monit. 2012, 14, 2482−2487, DOI: 10.1039/c2em30303e. (9) García, M.; Rodríguez, I.; Cela, R. Microwave-assisted extraction of organophosphate flame retardants and plasticizers from indoor dust samples. J. Chromatogr., A 2007, 1152, 280−286, DOI: 10.1016/ j.chroma.2006.11.046. (10) Kanazawa, A.; Saito, I.; Araki, A.; Takeda, M.; Ma, M.; Saijo, Y.; Kishi, R. Association between indoor exposure to semi-volatile organic compounds and building-related symptoms among the occupants of residential dwellings. Indoor Air 2010, 20, 72−84, DOI: 10.1111/j.16000668.2009.00629.x. (11) Marklund, A.; Andersson, B.; Haglund, P. Screening of organophosphorus compounds and their distribution in various indoor environments. Chemosphere 2003, 53, 1137−1146, DOI: 10.1016/ S0045-6535(03)00666-0. (12) Stapleton, H. M.; Klosterhaus, S.; Eagle, S.; Fuh, J.; Meeker, J. D.; Blum, A.; Webster, T. F. Detection of organophosphate flame retardants in furniture foam and U.S. house dust. Environ. Sci. Technol. 2009, 43, 7490−7495, DOI: 10.1021/es9014019. (13) Takigami, H.; Suzuki, G.; Hirai, Y.; Ishikawa, Y.; Sunami, M.; Sakai, S. Flame retardants in indoor dust and air of a hotel in Japan. Environ. Int. 2009, 35, 688−693, DOI: 10.1016/j.envint.2008.12.007. (14) Van den Eede, N.; Dirtu, A. C.; Ali, N.; Neels, H.; Covaci, A. Multi-residue method for the determination of brominated and organophosphate flame retardants in indoor dust. Talanta 2012, 89, 292−300, DOI: 10.1016/j.talanta.2011.12.031. (15) Schuetzle, D.; Lewtas, J. Bioassay-directed chemical analysis in environmental research. Anal. Chem. 1987, 58, 1060A−1075A. (16) Brack, W.; Klamer, H. J.; López de Alda, M.; Barceló, D. Effectdirected analysis of key toxicants in European river basins a review.

ASSOCIATED CONTENT

S Supporting Information *

Additional experimental information containing seven figures and two tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-29-850-2205. Fax: +81-29-850-2269. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the technical support of Ms. Chieko Michinaka of the National Institute for Environmental Studies (Japan). We thank Prof. Kurunthachalam Kannan of the State University of New York at Albany for providing indoor dust collected from the United States. This research was supported by Grants-in-Aid for Young Scientists (A) (no. 23681011) and Scientific Research (S) (no. 20221003) from the Japan Society for the Promotion of Science.

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