Toxicology Letters 245 (2016) 31–39

Effects of primary metabolites of organophosphate flame retardants ontranscriptional activity via human nuclear receptors

Hiroyuki Kojimaa,*, Shinji Takeuchia, Nele Van den Eedeb, Adrian Covacib

aHokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japanb Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium

H I G H L I G H T S

� Nuclear receptor activities of 12 OPFR-metabolites were studied by cell-based assays.� HO-m-TPHP and HO-p-TPHP showed more potent ERa/b agonistic activity than did TPHP.� These HO-TPHPs also acted as PXR agonists as well as ERb, AR and GR antagonists.� Diester OPFR-metabolites and BCIPHIPP did not show any receptor activity.

A R T I C L E I N F O

Article history:Received 13 September 2015Received in revised form 1 December 2015Accepted 11 January 2016Available online 14 January 2016

Keywords:Flame retardantsHumanNuclear receptorOrganophosphateReporter gene assayUrinary metabolite

A B S T R A C T

Organophosphate flame retardants (OPFRs) have been used in a wide variety of applications and detectedin several environmental matrices, including indoor air and dust. Continuous human exposure to thesechemicals is of growing concern. In this study, the agonistic and/or antagonistic activities of 12 primaryOPFR-metabolites against ten human nuclear receptors were examined using cell-based transcriptionalassays, and compared to those of their parent compounds. As a result, 3-hydroxylphenyl diphenylphosphate and 4-hydroxylphenyl diphenyl phosphate showed more potent estrogen receptor a (ERa)and ERb agonistic activity than did their parent, triphenyl phosphate (TPHP). In addition, thesehydroxylated TPHP-metabolites also showed ERb antagonistic activity at higher concentrations andexhibited pregnane X receptor (PXR) agonistic activity as well as androgen receptor (AR) andglucocorticoid receptor (GR) antagonistic activities at similar levels to those of TPHP. Bis(2-butoxyethyl)30-hydroxy-2-butoxyethyl phosphate and 2-hydroxyethyl bis(2-butoxyethyl) phosphate act as PXRagonists at similar levels to their parent, tris(2-butoxyethyl) phosphate. On the other hand, seven diesterOPFR-metabolites and 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate did not show any receptoractivity. Taken together, these results suggest that hydroxylated TPHP-metabolites show increasedestrogenicity compared to the parent compound, whereas the diester OPFR-metabolites may havelimited nuclear receptor activity compared to their parent triester OPFRs.

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1. Introduction

The phasing-out of polybrominated diphenyl ethers (PBDEs)saw an increase in the use of organophosphate flame retardants(OPFRs) as additives to flame retardants and plasticizers in avariety of applications, such as building materials, textiles andelectric appliances, resulting in their widespread environmentaldispersion (Reemtsma et al., 2008; van der Veen and de Boer,

* Corresponding author. Fax: +81 11 736 9476.E-mail address: kojima@iph.pref.hokkaido.jp (H. Kojima).

http://dx.doi.org/10.1016/j.toxlet.2016.01.0040378-4274/ã 2016 Elsevier Ireland Ltd. All rights reserved.

2012). Many studies have reported that various OPFRs are widelydistributed in both indoor and outdoor environments (Hartmannet al., 2004; Marklund et al., 2003; Takigami et al., 2009; Stapletonet al., 2009). In particular, the presence of OPFRs in indoor dust hasbeen worldwide reported, with several OPFRs, such as tris(2-butoxyethyl) phosphate (TBOEP), tris(1-chloro-2-propyl) phos-phate (TCIPP), triphenyl phosphate (TPHP), tris(2-chloroethyl)phosphate (TCEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP),and tri-n-butyl phosphate (TNBP), predominately found in indoordust from residential houses and offices (Tajima et al., 2014; Vanden Eede et al., 2011). The concentrations of OPFRs detected inindoor dust in recent years have been higher than those of PBDEs

32 H. Kojima et al. / Toxicology Letters 245 (2016) 31–39

(Ali et al., 2012; Stapleton et al., 2012). As TCEP, TDCIPP, TCIPP andTBOEP have been shown to be carcinogenic or possibly carcino-genic in animal studies (WHO, 1998, 2000), continuous humanexposure to these OPFRs is of growing concern.

Two previous studies using rodents showed that TDCIPP isquickly metabolized to bis(1,3-dichloro-2-propyl) phosphate(BDCIPP), which is excreted primarily in the urine (Lynn et al.,1981; Nomeir et al., 1981). Recent human biomonitoring studieshave also reported that OPFRs are readily metabolized, viahydrolysis or hydroxylation, and excreted in the urine as theirdialkyl and diaryl compounds, which function as biomarkers forOPFR exposure (Butt et al., 2014; Cooper et al., 2011; Dodson et al.,2014; Van den Eede et al., 2013b). Van den Eede et al. (2015b)reported that 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phos-phate (BCIPHIPP), diphenyl phosphate (DPHP), and BDCIPP weredetected in >95% of human urine samples and bis(2-butoxyethyl)2-hydroxyethyl phosphate (BBOEHEP) was found in >80% of those.In particular, DPHP, a primary metabolite of TPHP, was frequentlydetected at ppb levels in human urine (Cooper et al., 2011; Meekeret al., 2013; Reemtsma et al., 2011; Schindler et al., 2009).Thus, although various OPFR-metabolites are frequently detectedin human urine and are probably quickly eliminated from thehuman body, it remains unclear whether these metabolites haveadverse effects on human health during their residence time in thebody.

Fig. 1. Chemical structures of the 12 OPFR-metabolites used in the present study.*HO-m-TPHP was confirmed as a metabolite of TPHP in chicken embryonic hepatocytes (S

The nuclear receptor superfamily is comprised of ligand-inducible transcription factors that specifically regulate theexpression of target genes involved in metabolism, development,and reproduction (McKenna et al., 1999). Their primary function isto mediate the transcriptional response in target cells to hormones,such as sex steroids, adrenal steroids, vitamin D3, and thyroid andretinoid hormones, in addition to a variety of other metabolicligands. Recent in vitro studies have suggested that several OPFRsact as endocrine disruptors via nuclear receptors, includinghormone receptors (Kojima et al., 2013; Suzuki et al., 2013). Inparticular, our previous study using cell-based transactivationassays revealed that several OPFRs have agonistic and/or antago-nistic activity against estrogen receptors (ERs), androgen receptor(AR), glucocorticoid receptor (GR) and pregnane X receptor (PXR)(Kojima et al., 2013). In addition, other studies have shown thatTPHP could act as an activator of the peroxisome proliferators-activated receptor (PPAR)g to induce adipogenesis (Belcher et al.,2014; Pillai et al., 2014). In the present study, we characterized theagonistic and antagonistic activity of twelve metabolites of TBOEP,TCIPP, TPHP, TCEP, TDCIPP, and TNBP, which are predominatelyfound in indoor dust (Tajima et al., 2014; Van den Eede et al., 2011),against the following human nuclear receptors; ERa/b, AR, GR,TRa1, retinoic acid receptor (RAR)a, retinoic X receptor (RXR)a,PPARa/g and PXR. In this article, we provide the first evidence thathydroxylated TPHP-metabolites might possess multiple effects on

u et al., 2015), but not in in vitro metabolizing study using human liver preparations.

H. Kojima et al. / Toxicology Letters 245 (2016) 31–39 33

transcriptional activity via human nuclear receptors, whereasdealkylated OPFR-metabolites do not show these activities.

2. Materials and methods

2.1. Chemicals, biochemicals and cells

Di-n-butyl phosphate (DNBP; >97% pure), diphenyl phosphate(DPHP; >99% pure) and TPHP (>99% pure) were purchased fromSigma–Aldrich (Bornem, Belgium). TDCIPP, TCIPP, TBOEP, TNBP,and TCEP (each >99% pure) were purchased from Wako PureChemical Industries, Ltd. (Wako; Osaka, Japan). Bis(2-chloro-1-methylethyl) phosphate (BCIPP; >91% pure), bis(2-chloroethyl)phosphate (BCEP; >97.5% pure), bis(2-butoxyethyl) phosphate(BBOEP; >97.5% pure), and BDCIPP (>97.5% pure) were synthesizedin the Max Planck Institute (Göttingen, Germany). 4-Hydroxyl-phenyl diphenyl phosphate (HO-p-TPHP; >90% pure), 4-hydrox-ylphenyl phenyl phosphate (HO-DPHP; >90% pure), bis(2-butoxyethyl) 30-hydroxy-2-butoxyethyl phosphate (HO-TBOEP;>90% pure), BBOEHEP (>95% pure), and BCIPHIPP (>90% pure)were also synthesized in the Max Planck Institute. 3-Hydrox-ylphenyl diphenyl phosphate (HO-m-TPHP; >90% pure) was kindlyprovided from Dr. Heather Stapleton (Duke University, USA). Thechemical structures of the 12 OPFR-metabolites and their parentcompounds are shown in Fig. 1.

17b-Estradiol (E2; >97% pure), 5a-dihydrotestosterone (DHT;95% pure), hydrocortisone (HC; >98% pure), 9-cis retinoic acid (9-cis RA; 98% pure), and hydroxyflutamide (HF; >99% pure) werepurchased from Wako. 3,30,5-Triiodo-L-thyronine (T3; 99% pure), alltrans-retinoic acid (at-RA; >98% pure), rifampicin (RIF; >97% pure),ciprofibrate (>99% pure), rosiglitazone (>99% pure), and mifepris-tone (RU-486; 98% pure) were purchased from Sigma–Aldrich (St.Louis, MO, USA). Dimethyl sulfoxide (DMSO) was used as a vehicleand purchased from Wako, and all compounds used were dissolvedin DMSO at a concentration of 1 �10�2M.

Dulbecco’s modified Eagle’s medium (D-MEM), and D-MEM plusHam’s F-12 nutrient mixture (D-MEM/F-12) were obtained fromInvitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) andcharcoal/dextran-treated FBS (CD-FBS) were obtained from Hyclone(Logan, UT, USA). The penicillin-streptomycin solution (antibiotics)wasobtainedfromDainippon Pharmaceutical Co.Ltd. (Osaka, Japan).Bovine serum albumin (BSA) and 4-methylumbelliferyl-b-D-galac-toside (4-MUG) were obtained from Sigma-Aldrich.

Fig. 2. Estrogenic effects of TPHP and its metabolites in the hERa and hERb transactivahuman ERa (a) or ERb (b) as well as a reporter-responsive firefly luciferase plasmid and1 �10�7 to 3 �10�5M of TPHP or its metabolites. The firefly luciferase activity was normalindependent experiments and are presented as the percentage of the response, taking

CHO-K1 cells and simian kidney COS-7 cells were obtained fromthe American Type Culture Collection. CHO-K1 cells weremaintained in D-MEM/F-12 supplemented with 5% FBS andantibiotics, and COS-7 cells were maintained in D-MEM supple-mented with 10% FBS and antibiotics.

2.2. Plasmids

The expression plasmids, pcDNAERa, pcDNAERb, pZeoSV2AR,pSG5-GR, pZeo-TRa1, pcDNA-RXRa, pcDNA-RARa, pSG5-hPXR,pcDNA-PPARa, and pTransExpress-PPARg1, encoding full-lengthreceptor proteins, as well as the reporter plasmids, pGL3-tkERE,pIND-ARE, pGRE-tk-Luc, pIND-TREpal, pXREM-3A4-Luc, and pGL4-10xAOXluc2, were prepared as previously described (Kojima et al.,2009, 2011, 2013). The internal control plasmid, pCMVb-Gal, waspurchased from Clontech (Palo Alto, CA, USA).

2.3. Reporter gene assays

For detection of human ERa, ERb, AR, GR, TRa1, RARa, andRXRa activity, we plated the CHO-K1 cells in 96-well microtiterplates (Nalge, Nunc, Denmark) at a density of 8,400 cells per well inphenol red-free D-MEM/F-12 containing 5% CD-FBS (completemedium) one day before transfection. COS-7 cells (8400 cells/well)in D-MEM containing 10% CD-FBS were used as host cells for thedetection of human PXR, PPARa, and PPARg1 activity. These cellswere transiently transfected with the receptor expression plasmid,the reporter plasmid, and the internal control plasmid (pCMVb-Gal) using the FuGENE 6 Transfection Reagent (Promega, Madison,WI, USA) (Kojima et al., 2009, 2011, 2013). After a 3-h transfectionperiod, cells were dosed with various concentrations of the testcompounds or with 0.3% DMSO (vehicle control) in completemedium. To avoid any cytotoxic effects associated with the testcompounds, assays were performed for test compounds atconcentrations of less than 3 � 10�5M. After an incubation periodof 24 h, cells were rinsed with phosphate-buffered saline (pH 7.4)and lysed with passive lysis buffer (50 ml/well; Promega).

We measured firefly luciferase activity using a MiniLumat LB9506 luminometer (Berthold, Wildbad, Germany) in one reactiontube with a 5-ml aliquot of the cell lysate using the Luciferase AssaySystem (Promega), according to the manufacturer’s instructions.Luciferase activity was normalized against the b-galactosidaseactivity for each treatment. Results are expressed as means � SD

tion assays. CHO cells were transiently transfected with an expression plasmid for a constitutively active b-galactosidase expression plasmid. Cells were treated withized based on the b-galactosidase activity. Values represent the means � SD of three

the activity obtained using 1 �10�9M E2 as 100%.

34 H. Kojima et al. / Toxicology Letters 245 (2016) 31–39

from at least three independent experiments performed intriplicate.

2.4. b-Galactosidase activity assay

The measurement of b-galactosidase activity was performedusing a fluorescence method as described in our previous paper(Takeuchi et al., 2005).

2.5. Evaluation of agonistic and antagonistic activities

In order to estimate the potency of the receptor agonisticactivities of the tested compounds, the luminescence intensity ofthe assay was represented in a dose-response curve. Theconcentration of the compound equal to 20% of the maximalresponse of the positive control was obtained from the dose-response curve of the luminescence intensity and expressed as anREC20 value (20% relative effective concentration). The results forthe receptor antagonistic activities of each compound wereexpressed as RIC20 values (20% relative inhibitory concentration),which is the concentration of the test compounds showing 20%inhibition of the activities induced by the endogenous hormones.These values can be compared to those of the positive controls andother compounds described in previous our reports (Kojima et al.,2009, 2011, 2013).

2.6. Statistical analysis

An analysis of variance (ANOVA) followed by Bonferronicorrection was used to evaluate the differences in transcriptionallevels between the control group and each of the chemical groupsin the ERb, AR and GR antagonist assays. The level of significance

Table 1Comparison of agonistic and/or antagonistic activities against ERa, ERb, AR, GR, and P

Compounds Agonistic activity; REC20a (�SD, mM)

ERa ERb PXR

E2 2.5 � 0.3 (pM) 5.3 � 0.5 (pM) –

RIF – – 0.31 � 0TAM – – –

HF – – –

RU-486 – – –

TBOEP N.E.c N.E. 3.1 � 0.3HO-TBOEP N.E. N.E. 14 � 2

BBOEP N.E. N.E. N.E.

BBOEHEP N.E. N.E. 28 � 3

TPHP 4.6 � 0.2 7.3 � 0.4 2.8 � 0.5HO-m-TPHP 1.7 � 0.2 3.8 � 0.8 2.2 � 0.1HO-p-TPHP 0.29 � 0.02 0.30 � 0.03 4.7 � 0.1DPHP N.E. N.E. N.E.

HO-DPHP N.E. N.E. N.E.

TCIPP N.E. N.E. 4.9 � 2.0BCIPHIPP N.E. N.E. N.E.

BCIPP N.E. N.E. N.E.

TNBP N.E. N.E. 9.4 � 0.5DNBP N.E. N.E. N.E.

TCEP N.E. N.E. N.E.

BCEP N.E. N.E. N.E.

TDCIPP N.E. N.E. 1.4 � 0.5BDCIPP N.E. N.E. N.E.

a 20% relative effective concentration; the concentration of the test compound show1 �10�5M RIF via PXR, respectively.

b 20% relative inhibitory concentration; the concentration of the test compound showiAR, or 3 �10�8M HC via GR, respectively.

c No effect (REC20 or RIC20 > 1 �105M).

was set at p < 0.05. Data were presented as the mean � SD of threetriplicate experiments.

3. Results

3.1. Agonistic activities of the OPFR-metabolites against ERa/b, AR, GR,and TRa1

We found that HO-m-TPHP and HO-p-TPHP among the 12 OPFR-metabolites tested induced ERa- and ERb-mediated estrogenicactivity of greater than 20% that of the E2–induced maximalactivity. However, none of the 12 OPFR-metabolites showed AR, GRor TRa1 agonistic activity in these assays (data not shown).

Fig. 2a shows the dose-response curves of ERa agonisticactivities for TPHP and four TPHP-metabolites. HO-m-TPHP andHO-p-TPHP showed more potent ERa agonistic activity than didtheir precursor TPHP, whereas DPHP and HO-DPHP did not showany ERa agonistic activity. The order of the relative ERa agonisticactivity of these compounds was HO-p-TPHP > HO-m-TPHP >TPHP > DPHP = HO-DPHP. From a comparison of REC20 values,the estrogenic activity via ERa of HO-p-TPHP, the most potent ERaagonist in this study, was about 100,000-fold lower than that of E2,but over 10-fold higher than that of TPHP (Table 1).

Fig. 2b shows the dose-response curves of ERb agonisticactivities for TPHP and four TPHP-metabolites. These resultsindicated that HO-m-TPHP, HO-p-TPHP, and TPHP could act as ERbagonists while DPHP and HO-DPHP didn't show any ERb agonisticactivity, as also seen in above results for the ERa agonistic assay. Acomparison of REC20 values revealed that the estrogenic activityvia ERb of HO-p-TPHP was about 57,000-fold lower than that of E2,but HO-m-TPHP and HO-p-TPHP induced about 2- and 20-foldhigher responses, respectively, than did TPHP (Table 1).

XR between OPFRs and their metabolites.

Antagonistic activity; RIC20b (�SD, mM)

ERb AR GR

– – –

.05 – – –

0.006 � 0.001 – –

– 0.018 � 0.005 –

– – 0.057 � 0.007

N.E. N.E. N.E.N.E. N.E. N.E.N.E. N.E. N.E.N.E. N.E. N.E.

N.E 11 � 3 12 � 5 15 � 3 12 � 2 12 � 2 5.4 � 1.4 12 � 1 4.3 � 2.2

N.E. N.E. N.E.N.E. N.E. N.E.

N.E. N.E. N.E.N.E. N.E. N.E.N.E. N.E. N.E.

N.E. 24 � 3 22 � 3N.E. N.E. N.E.

N.E. N.E. N.E.N.E. N.E. N.E.

N.E. 1.9 � 1.5 16 � 4N.E. N.E. N.E.

ing 20% of the agonistic activity of 1 �109M E2 via ERa, 1 �10�9M E2 via ERb, or

ng 20% of the antagonistic activity against 1 �1010M E2 via ERb, 1 �10�10M DHT via

Fig. 3. Antagonistic activities of TPHP-hydroxylated metabolites against hERb, hAR and hGR. CHO cells were transiently transfected with an expression plasmid for humanERb (a), AR (b) or GR (c) as well as a reporter-responsive firefly luciferase plasmid and a constitutively active b-galactosidase expression plasmid. In ERb antagonist assay (a),cells were treated with 1 �10�7M to 3 � 10�5M of TPHP or its hydroxylated metabolites, or with 1 �10�9M to 1 �10�7M of tamoxifen (TAM) in the presence of 1 �10�10M E2to detect ERb antagonistic activity. In AR antagonist assay (b), cells were treated with 1 �10�7M to 3 � 10�5M of TPHP or its hydroxylated metabolites, or with 1 �10�9M to1 �10�7M of hydroxyflutamide (HF) in the presence of 1 �10�10M DHT to detect AR antagonistic activity. In GR antagonist assay (c), cells were treated with 1 �10�7M to3 � 10�5M of TPHP or its hydroxylated metabolites, or with 1 �10�8M to 1 �10�6M of RU-486 in the presence of 3 � 10�8M HC to detect GR antagonistic activity. The fireflyluciferase activity was normalized based on the b-galactosidase activity. Values represent the means � SD of three independent experiments and are presented as thepercentage of the response, taking the activity obtained using 1 �10�10M E2 (a), 1 �10�10M DHT (b) or 3 � 10�8M HC (c) as 100%.*Significant difference (p < 0.05; ANOVA) from 1 �10�10M E2 (a), 1 �10�10M DHT (b) or 3 � 10�8M HC (c) alone.

H. Kojima et al. / Toxicology Letters 245 (2016) 31–39 35

0

20

40

60

80

100

120

1401x10 -6 M 3x10-6 M 1x10-5 M 3x10 -5 M

Fig. 4. Comparison of hPXR agonistic activities between 6 OPFRs and their metabolites. COS-7 cells were transiently transfected with an expression plasmid for human PXR aswell as a reporter-responsive firefly luciferase plasmid and a constitutively active b-galactosidase expression plasmid. Cells were treated with 1 �10�6M to 3 � 10�5M ofOPFRs, their metabolites, or rifampicin (RF) to detect PXR agonistic activity. The firefly luciferase activity was normalized based on the b-galactosidase activity. Valuesrepresent the means � SD of three independent experiments and are presented as the percentage of the response, taking the activity obtained using 1 �10�5M RF as 100%.

0

20

40

60

80

100

120

TPHP HO-m- TPHP HO-p-TPHPDPHP HO-DPHP Rosiglitazone

Conce ntration , Log (M)-9 -8 -7 -6 -5 -4

Fig. 5. Effects of TPHP and its 4 metabolites on transcriptional activity via hPPARg.COS-7 cells were transiently transfected with an expression plasmid for humanPPARg as well as a reporter-responsive firefly luciferase plasmid and a constitutivelyactive b-galactosidase expression plasmid. Cells were treated with 1 �10�8M to3 � 10�5M of TPHP or its 4 metabolites, or with 1 �10�9M to 1 �10�5M ofrosiglitazone to detect PPARg agonistic activity. The firefly luciferase activity wasnormalized based on the b-galactosidase activity. Values represent the means � SDof three independent experiments and are presented as the percentage of theresponse, taking the activity obtained using 1 �10�6M rosiglitazone as 100%.

36 H. Kojima et al. / Toxicology Letters 245 (2016) 31–39

3.2. Antagonistic activities of the OPFR-metabolites against ERa/b, AR,GR, and TRa1

We found that HO-m-TPHP and HO-p-TPHP among the 12 OPFR-metabolites tested had antagonistic activity against ERb, AR andGR. However, none of the 12 OPFR-metabolites showed antago-nistic activity against ERa or TRa1 (data not shown).

Fig. 3a shows the dose-response curves of ERb antagonisticactivities for HO-m-TPHP, HO-p-TPHP, TPHP and TAM, a well-known ER antagonist. HO-m-TPHP and HO-p-TPHP inhibited ERb-mediated estrogenic activity of greater than 20% that of the activityinduced by 1 �10�10M E2, while TPHP showed slight ERbantagonistic activity of less than 20% that of the activity inducedby 1 �10�10M E2. Although HO-m-TPHP and HO-p-TPHP were ERbagonists, they could also act as ERb antagonists at higherconcentrations. As shown in Table 1, the RIC20 value for HO-p-TPHP, the most potent ERb antagonist in this study, was5.4 �10�6M and its antiestrogenic activity via ERb was 900-foldlower than that of TAM (RIC20: 6.0 � 10�9M).

Fig. 3b shows the AR antagonistic effects of HO-m-TPHP and HO-p-TPHP at high concentrations as well as that of TPHP. A comparisonof RIC20 values showed that the anti-androgenic activities of thehydroxylated TPHP-metabolites and TPHP were about 650-foldlower than that of HF, a known AR antagonist (Table 1).

Fig. 3c shows that HO-m-TPHP and HO-p-TPHP as well as TPHPhad weak inhibitory effects on the GR-mediated transcriptionalactivity induced by 3 �10�8M of HC. A comparison of RIC20 valuesrevealed that the GR antagonistic activity of HO-p-TPHP, the mostpotent GR antagonist in this study, was an estimated 75-fold lowerthan that of RU-486, a known GR antagonist (Table 1).

Based on their b-galactocidase activity, except for TPHP, HO-m-TPHP and HO-p-TPHP, which were slightly cytotoxic at aconcentration of 3 � 10�5M, none of the six OPFRs or 12 OPFR-metabolites tested in this study showed any cytotoxic effectsagainst CHO-K1 cells at the doses tested (�3 �10�5M).

3.3. Agonistic activities of the OPFR-metabolites against RARa, RXRa,PXR, and PPARa/g1

We found that HO-TBOEP, BBOEHEP, HO-m-TPHP and HO-p-TPHP among the 12 OPFR-metabolites possess PXR agonistic

activity. However, none of the 12 OPFR-metabolites showed RARa,RXRa, PPARa or PPARg1 agonistic activity in these assays (data notshown).

Fig. 4 shows the dose-response curves of HO-TBOEP, BBOEHEP,HO-m-TPHP and HO-p-TPHP showing PXR agonistic activitygreater than 20% that of the maximum activity of RIF. The REC20

values of the 12 OPFR-metabolites and their precursors for PXRagonistic activity are given in Table 1. The relative potencies of theirPXR agonistic activities were as follows: TDCIPP > TBOEP > HO-m-TPHP > TPHP > HO-p-TPHP > TNBP > HO-TBOEP > BBOEHEP. Com-parisons of their REC20 values suggested that their agonistic

H. Kojima et al. / Toxicology Letters 245 (2016) 31–39 37

activities against PXR were between 4.5- and 90.3-fold lower thanthat of RIF (Table 1).

None of the 12 OPFR-metabolites induced PPARg1 agonisticactivity greater than 20% that of the maximum activity ofrosiglitazone. Fig. 5 shows the dose-response curves of TPHP, fourTPHP-metabolites and rosiglitazone in the PPARg1 assay. AlthoughTPHP showed week PPARg1 agonistic activity of about 15% that ofthe maximum response of rosiglitazone, the activities of the twohydroxylated TPHP metabolites (HO-m-TPHP and HO-p-TPHP)were less potent than that of TPHP. On the other hand, the TPHP-derived diaryl compounds, DPHP and HO-DPHP, did not show anyPPARg1 agonistic activity.

Based on their b-galactosidase activity, except for HO-m-TPHPand HO-p-TPHP, which were slightly cytotoxic at a concentration of3 � 10�5M, none of the twelve OPFR-metabolites in this studyshowed any cytotoxic effects against COS-7 cells at the doses tested(� 3 � 10�5M).

4. Discussion

Recently, environmental pollution by OPFRs has been ofincreasing concern in terms of public health and the need forrisk assessment (van der Veen and de Boer, 2012). Our previousstudy provided evidence that several OPFRs show potentialendocrine disruption activity via human nuclear receptors (Kojimaet al., 2013). In this study, to further elucidate the endocrine-disrupting properties of OPFRs in humans, we characterizedactivities of 12 primary metabolites (Fig. 1) derived from TBOEP,TCIPP, TPHP, TCEP, TDCIPP, and TNBP against 10 nuclear receptorsusing cell-based reporter gene assays with CHO-K1 and COS-7 cells. The results summarized in Table 1 reveal that twohydroxylated TPHP-metabolites among the 12 compounds testedexhibited ERa/b and PXR agonistic activity as well as ERb, AR, andGR antagonistic activity, while two TBOEP-metabolites showedonly PXR agonistic activity. However, it was also found that none ofthe 12 OPFR-metabolites, together with the 11 OPFRs reported inour previous paper (Kojima et al., 2013), possess TRa1, RARa, RXRa,or PPARa/g activity.

The metabolization of TPHP by human liver preparations hasbeen reported to produce several primary metabolites, HO-p-TPHP,DPHP and HO-DPHP, through the hydroxylation and O-deal-kylation of TPHP (Fig. 1) (Sasaki et al., 1984; Van den Eede et al.,2013a). Recent studies have shown that in vitro metabolism ofTPHP using chicken embryonic hepatocytes induced the formationof HO-m-TPHP, together with DPHP and HO-p-TPHP (Su et al., 2014,2015), although HO-m-TPHP has not been confirmed as ametabolite formed by human liver enzymes. However, as HO-m-TPHP is present as an impurity/hydrolysis product of the flameretardant resorcinol bis(diphenylphosphate) (RDP), humans couldbe directly exposed to this compound (Ballesteros-Gómez et al.,2015). Several analytical studies have shown that DPHP wasfrequently detected as a major metabolite of TPHP at levels in orderof a few ng/ml in human urine samples (Cooper et al., 2011; Meekeret al., 2013; Reemtsma et al., 2011; Schindler et al., 2009). In theassays of the 12 OPFR-metabolites against nuclear receptors, HO-m-TPHP and HO-p-TPHP showed multiple ERa, ERb, and PXRagonistic activities as well as ERb, AR and GR antagonistic activity.In particular, HO-p-TPHP activated ERa and ERb at lowerconcentrations than did the parent, TPHP, and HO-m-TPHP (Fig.2a and b). This suggests that a para-substituted OH groupconnected to a benzene ring may be important for the inductionof high levels of estrogen activity, a notion also supported byseveral studies reporting that hydroxylated polychlorinatedbiphenyls (HO-PCBs) with para-substituted HO-groups possessthe most potent estrogenic effects via ERa, followed by meta- andortho-substituted HO-PCBs (Arulmozhiraja et al., 2005; Kitamura

et al., 2005). Additionally, this result implies that the metabolismof TPHP to HO-p-TPHP and HO-m-TPHP by oxidation enzymesenhances their endocrine-disrupting effects, such as estrogenicactivity, suggesting metabolic activation against ERs. Interestingly,we found that HO-p-TPHP and HO-m-TPHP induce ERa- and ERb-mediated transcriptional activities, but simultaneously inhibitERb-, AR- and GR-mediated transcriptional activities (Fig. 3a–c,Table 1). Such an activity pattern in same the assay systems usingCHO-K1 cells is similar to those of 20,30,40,50-tetrachlorobiphenyl-2-ol (20-HO-CB61) and 20,3,30,40,50-pentachlorobiphenyl-4-ol (40-HO-CB106) (Takeuchi et al., 2011) and those of butylbenzyl phthalate(plasticizer) and the methoxychlor (insecticide) metabolite, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) (Takeuchi et al.,2005). These findings, together with those of the present study,indicate that chemicals showing such pleiotropic effects mayunexpectedly exist in the environment. Furthermore, hydroxylatedTPHP-metabolites also showed PXR agonistic activity similar tothat of TPHP (Fig. 4). Thus, HO-p-TPHP and HO-m-TPHP are thoughtto act as modulators against various nuclear receptors. On theother hand, esterase-dealkylated DPHP and HO-DPHP did notexhibit any receptor activity. These results suggest that TPHP andhydroxylated TPHP-metabolites (triaryl phosphates) have multipleand pleiotropic effects against nuclear receptors whereas DPHPand HO-DPHP (diaryl phosphates) were inactive against nuclearreceptors. This difference might reflect the fact that diarylphosphates show decreased hydrophobicity, an important factorin their interactions with nuclear receptor, compared to triarylphosphates.

TBOEP is an OPFR produced in large volumes and used in abroad range of applications, and is detectable at high concen-trations in indoor dust (Tajima et al., 2014; Van den Eede et al.,2011). TBOEP-metabolizing studies using human liver preparationsrevealed the presence of several primary metabolites, such asBBOEP, HO-TBOEP and BBOEHEP (Fig.1) (Van den Eede et al., 2013a,2015a). BBOEHEP, in particular, is frequently detected in humanurine (Van den Eede et al., 2015b). Although our previous studyshowed that the parent TBOEP has only PXR agonistic activityamong various nuclear receptor assays (Kojima et al., 2013), wefound that HO-TBOEP and BBOEHEP exhibited weaker PXRagonistic activity than did TBOEP, while an O-dealkylatedmetabolite, BBOEP, did not show any activity (Fig. 4). This impliesthat TBOEP-OH and BBOEHEP are structurally similar to TBOEP,allowing them to act as PXR activators, whereas the structuralproperties of BBOEP might lead to reduction in its binding to PXR(Fig. 1).

Recent studies have reported that TDCIPP was detected inindoor dust in homes and work environments (Meeker andStapleton, 2010; Van den Eede et al., 2011). In addition to an in vivostudy using rats (Lynn et al., 1981), in vitro metabolic studies usinghuman liver microsomes or hepatocytes reported the formation ofBDCIPP as a major metabolite of TDCIPP through its O-dealkylation(Abdallah et al., 2015; Van den Eede et al., 2013a). Several humanmonitoring studies have reported that BDCIPP is frequentlydetected in human urine samples (Carignan et al., 2013; Cooperet al., 2011; Hoffman et al., 2014). Although TDCIPP itself has ARand GR antagonistic activities as well as PXR agonistic activity(Kojima et al., 2013), the present study indicated that BDCIPP didnot possess any receptor activity, including AR antagonistic activity(Table 1). This suggests that BDCIPP, formed through O-deal-kylation of TDCIPP, does not affect the nuclear receptor activity, asin the case of DPHP and BBOEP.

The in vitro biotransformation of TCIPP, TCEP and TNBP byhuman liver preparations showed that these compounds arepredominately metabolized to BCIPP, BCEP and DNBP, respectively(Van den Eede et al., 2013a). In this study, we also examined thenuclear receptor activity of the dechlorinated and hydroxylated

38 H. Kojima et al. / Toxicology Letters 245 (2016) 31–39

TCIPP-metabolite, BCIPHIPP. The results revealed that these fourmetabolites did not exhibit any nuclear receptor activity, althoughTCIPP is a PXR agonist, and TNBP is a PXR agonist as well as an ARand GR antagonist (Kojima et al., 2013). These results suggest thatthe diester phosphates BCIPP, BCEP and DNBP lose their activityagainst nuclear receptors as also seen in DPHP, BBOEP and BDCIPP.These decreases in receptor activity commonly observed in thediester phosphates appear to resemble those with regard to PXRagonistic activity observed in several oxon-forms of phosphorusinsecticides (Kojima et al., 2011). Thus, the metabolization of atriester phosphate (R3-P = O) to a diester phosphate (R2-P(¼O)��OH) by liver esterase increases the hydrophilicity of the OPFRs,might consequently lead to the loss of nuclear receptor activity.

We also found that none of the OPFRs or their metabolitespossesses TRa1, RARa, RXRa, or PPARa/g activity. On the otherhand, more recent studies have shown that TPHP and otherenvironmental chemicals, including brominated flame retardants,can act as PPARg agonists to induce adipogenesis (Pillai et al., 2014;Fang et al., 2015a,b). Based on COS-7 reporter gene and BMS2 cellprotein expression assays together with lipid accumulation, Pillaiet al. (2014) reported that FM550 and TPHP have PPARg agonisticactivity. Fang et al. (2015b) showed that several of compounds inindoor dust can potentially activate PPARg1 on the basis of a highlysensitive reporter assay with a human embryo kidney 293H cellline containing PPARg1 ligand-binding domain/GAL4 DNA-bindingdomain. In this study, we showed that TPHP-metabolites had littlePPARg1 agonistic activity, although the parent TPHP showedPPARg1 activity at a level 15% that of the maximum activity ofrosiglitazone (Fig. 5). Our reporter assay in which a full-lengthPPARg1 protein was expressed in COS-7 cells may be less sensitiveby one order of magnitude than the assay reported by Fang et al.(2015b), but is as sensitive as the assays reported in other studies(Belcher et al., 2014; Pillai et al., 2014; Suzuki et al., 2013).Nevertheless, our findings suggest that TPHP is a weakPPARg1 activator, and its metabolization to hydroxylated anddealkylated TPHP lead to a further decrease in PPARg1 agonisticactivity.

5. Conclusions

We demonstrated for first time that monohydroxylated TPHPs(HO-m-TPHP and HO-p-TPHP) possessed ERa/b and PXR agonisticactivities as well as ERb, AR and GR antagonistic activities, whereasnone of the diester OPFR-metabolites tested showed any receptoractivity. The diverse effects of these OPFR-metabolites againstnuclear receptors might be due to their structural properties.

Conflict of interest

The authors declare that there are no conflict of interest.

Acknowledgements

The authors thank Dr. Heather Stapleton (Duke University, USA)for the generous donation of the HO-m-TPHP standard. This studywas supported by JSPS KAKENHI Grant Numbers21590675,15K08791.

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