Dioxin-like Potency of HO- and MeO- Analogues of PBDEs ... · AhR receptor was assayed by use of a commercial kit (Promega Corporation, Madison, WI) in a microplate reader (BioTek
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Dioxin-like Potency of HO- and MeO- Analogues of PBDEs’ thePotential Risk through Consumption of Fish from Eastern ChinaGuanyong Su,† Jie Xia,† Hongling Liu,† Michael H. W. Lam,§ Hongxia Yu,*,† John P. Giesy,†,‡,§
and Xiaowei Zhang*,†
†State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China‡State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat CheeAvenue, Kowloon, Hong Kong SAR, China§Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
*S Supporting Information
ABSTRACT: Polybrominated diphenyl ethers (PBDEs) and theiranalogues, such as hydroxylated PBDE (HO-PBDEs) andmethoxylated PBDE (MeO-PBDEs) are of interest due to theirwide distribution, bioaccumulation and potential toxicity to humansand wildlife. While information on the toxicity/biological potenciesof PBDEs was available, information on analogues of PBDEs waslimited. Dioxin-like toxicity of 34 PBDEs analogues was evaluated byuse of the H4IIE-luc, rat hepatoma transactivation bioassay in 384-well plate format at concentrations ranging from 0 to 10 000 ng/mL.Among the 34 target analogues of PBDEs studied here, 19 activatedthe aryl hydrocarbon receptor (AhR) and induced significant dioxin-like responses in H4IIE-luc cells. Efficacies of the analogues ofPBDEs ranged from 5.0% to 101.8% of the maximum responsecaused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD-max) and their respective 2,3,7,8-TCDD potency factors (RePH4IIE‑luc)ranged from 7.35 × 10−12 to 4.00 × 10−4, some of which were equal to or more potent than some mono-ortho-substituted PCBs(TEF-WHO = 3 × 10−5). HO-PBDEs exhibited greater dioxin-like activity than did the corresponding MeO-PBDEs. Analogues ofPBDEs were detected mostly in marine organisms. Of these 11 detected analogues of PBDEs, 6 were found to have measurabledioxin-like potency. Though some analogues of PBDEs exhibited significant dioxin-like potency as measured by responses of theH4IIE-luc transactivation assay, concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents(PBDEs analoguesTEQH4IIE‑luc), calculated as the sum of the product of concentrations of individual PBDE and their RePH4IIE‑luc,were less than the tolerance limit proposed by European Union and the oral reference dose (RfD) derived by U.S. EnvironmentalProtection Agency, respectively. (Hazard Quotients (HQ) < 0.005) Additional investigations should be conducted to evaluatethe toxic potencies of these chemicals, especially for 2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeO-BDE-47,which had been detected in other environmental media, including human blood.
1. INTRODUCTION
Due to their performance and cost-effectiveness, polybromi-nated diphenyl ethers (PBDEs) have been used for many yearsas flame retardants in various commercial products, such asfurniture, textiles, plastics, paints, and electronic appliances.1,2
Due to their persistence and potential to bioaccumulate,3
PBDEs have been detected in various environmental matrixesand concentrations have been increasing continuously.4
Hydroxylated polybrominated diphenyl ethers (HO-PBDEs)and methoxylated polybrominated diphenyl ethers (MeO-PBDEs) have been observed in tissues of wildlife and humansand have been suggested to be biotransformation products ofPBDEs,.5,6 This is especially true for 6-HO-BDE-47, 5-HO-BDE-47, and 5′-HO-BDE-99. Concerns have been raised aboutthe potential toxicity of these PBDEs analogues and theirmodes of molecular toxicity.
Previous studies have shown that PBDEs and their analoguescan interact with some endocrine nuclear receptors such asestrogen receptors (ER), androgen receptor (AR) and thyroidhormone receptor (ThR). Furthermore, HO-PBDEs weremore potent than their postulated precursor PBDEs andcorresponding MeO-PBDEs.7−11 Because of its structuralsimilarity to other polyhalogenated aromatic hydrocarbonssuch as polychorinated biphenyls (PCBs), PBDEs have beensuggested to be potential agonists of the Aryl hydrocarbonreceptor (AhR). To test this hypothesis, several in vivo or invitro experiments have been conducted, and a weak response of
Received: June 12, 2012Revised: August 20, 2012Accepted: September 6, 2012Published: September 6, 2012
AhR has been observed.12,13 However, the presence ofbrominated furans, which were impurities in PBDEs was thelikely reason for these apparent potencies.14,15 PBDEs did notactivate the AhR, but AhR-mediated effects of tetrachlorodi-benzo-p-dioxin (TCDD) could be reduced during coexposureto PBDEs and TCDD. This chemical activity effect is likely dueto the fact that PBDEs can interact with the AhR but not bindwith sufficient avidity to produce AhR-mediated signaling.However, an investigation of the potency of the HO- and MeO-analogues of PBDEs had not been conducted.These two classes of analogues of PBDEs have been detected
in various environments media, including human blood.5,6,16,17
MeO-PBDEs have been known to be produced naturally bymarine organisms.18,19 There are contradictory reports onsources of HO-PBDEs analogues. Both in vitro and in vivoexposures have shown that HO-PBDEs might be formed due tobiotransformation of various PBDEs.20 However, recentresearch has demonstrated that HO-PBDEs, especially 6-HO-BDE-47, can also be generated from naturally occurring MeO-PBDEs.21−23 Specifically, demethylation of 6-MeO-BDE-47 wasthe primary transformation pathway that resulted in formationof 6-HO-BDE-47 in the small fish, Japanese medaka, while thepreviously hypothesized formation of HO-PBDEs fromsynthetic BDE-47 did not occur.21
Here we report the first evidence that analogues of PBDEshave measurable potency as AhR-agonists and might elicitdioxin-like toxicity. Concentrations of PBDE and theiranalogues were determined in freshwater and marine fishesfrom East China. Finally, the potential risk of these analogues ofPBDEs through dioxin-like mechanism was assessed.
-99, -85, -154, -153, -138, -183, -190), C13-BDE-139, C13-2-HO-BDE-99 and 13C-PCB-178 used for quantification werepurchased from Cambridge Isotope Laboratories (Andover,MA). Analogues of PBDEs, including 19 HO-PBDEs and 15MeO-PBDEs (Figure 1), were synthesized in the Departmentof Biology and Chemistry of City University of Hong Kongfollowing the previously published methods.24 Purities of thesynthesized compounds were determined to be higher than98%. The results of proton NMR and electrospray LC-MS/MSof the intermediates and end products, the synthesis procedurewas confirmed to not generate brominated dioxin and/orfurans.8
2.2. H4IIE-luc Cell Culture and Bioassay. For the firsttime a high throughput, 384-well plate method was used todetermine the relative potencies of various PBDE and theiranalogues. Rat hepatoma cells that had been stably transfected
Figure 1. Structures of 34 PBDEs analogs. (19 HO-PBDEs are marked with a red frame, and 15 MeO-PBDEs are marked with a dark-blue frame.).
with an AhR-responsive luciferase reporter gene construct(H4IIE-luc) was used to study AhR activity of PBDEsanalogues.25,26 Potencies of individual analogues of PBDEswere determined by use of previously published methods.27,28
Cells were cultured in Dulbecco’s Modified Eagle Medium(DMEM) medium at 37 °C with 5% CO2 and 99% humidity.On the first day, 79 μL of cell solution at a concentration of 7.5× 104 cells/mL was added to each well of a 384-well plates. Toavoid cross-contamination, each chemical treatment wasbordered by one blank column. A volume of 79 μL of mediumwas also added into each well of blank columns. On the secondday, cells were dosed with serial dilution of chemicals stocksolutions (2 × 106 ng/mL) with dimethylmethane (DMSO) assolvent. Stock solutions were diluted with cell culture mediumby 20-fold, and then 0.8 μL of the diluted solution was addedinto each well of 384-well plate to make to a final dose at 0.5%v/v. Three replicates were conducted per treatment, includingTCDD standards. Each control and each standard concen-tration were averaged for all plates within a given experiment.For chemicals and TCDD standards, 7 (0−10 000 ng/mL) and10 (0−1.61 ng/mL) concentrations were used, respectively. Onthe fifth day, cells were lysed and luciferase activity mediated byAhR receptor was assayed by use of a commercial kit (PromegaCorporation, Madison, WI) in a microplate reader (BioTekInstruments Inc., Winooski, VT).2.3. Sampling. Six fishes (the sharpbelly (Hemicculter
leuciclus), the yellow catfish (Pelteobagrus fulvidraco), thecrucian carp (Carassius auratus), the bigmouth grenadieranchovy (Coilia macrognathos bleeker), the oriental sheatfish(Silurus spp), and the common carp (Cyprinus carpio)) werecollected from the lower Yangtze River. Five marine fishes (therazor clam (Sinonovacula constrzcta), the spotted sicklefish(Drepane punctata), the elongate ilisha (Ilisha elongate), the big-eyed flathead (Suggrundus meerdervoortii), the small yellowcroaker (Pseudosciaena polyactis)) were collected from YellowSea. All samples were transported to the lab on ice and weremaintained intact at −20 °C until dissection for subsequentidentification and quantification of PBDEs and their analogues.Details of the samples are given in Supporting Information (SI)(Table S1).
2.4. Chemical Analysis. Details of the instrumentalanalyses, including “Identification and Quantification ofPBDEs and their analogues”, “Instrument Conditions”, “QualityAssurance/Quality Control”, and “TEQBIO testing for eachbiological sample” are provided in the SI.
2.5. Data Analysis. Relative potency factors (RePs),expressed as g TCDD/g chemical, were calculated for eachanalogue of PBDEs as the quotient of the 20% effectconcentration (EC20) for TCDD divided by the EC20 ofindividual PBDE and analogues of PBDEs.29 TCCD equiv-alents (TEQ) for each sample were calculated as the sum of theproduct of concentrations of individual analogues of PBDEs bytheir respective RePs as follows:
∑= ×=
TEQ concentration RePn
i
i i
1
Following EPA superfund guidance terminology, hazardquotients (HQ) were calculated as the ratio of the exposureestimate to effects concentration considered to represent a“safe” environmental concentration or dose. For quantification,statistical analyses were performed by use of SPSS 13.0 forWindows (SPSS Inc., Chicago, IL). Spearman rank correlationwas used to examine the strength of associations betweenparameters (including mass and length of individual fish, theconcentrations of individual target compounds). Mann−Whitney U nonparametric tests were used to compare thedifference between/among groups. Concentrations of analytesin fishes are presented as the mean and range. Figures weregenerated with ChemBioDraw Ultra 11.0 (Figure 1), MicrosoftOffice Excel 2007 (Figure 2 and SI Figure S3), OriginPro 8(Figure 2) or with R software (version 2.14.1) (SI Figures S1and S2). The R code for these analyses is available uponrequest.
3. RESULTS3.1. Method Robustness. A 384-well plate format for the
H4IIE-luc assay was used here for the first time, and therobustness of this modified method was evaluated. Exposure ofH4IIE-luc cells to AhR agonists results in induction of luciferaseactivity that is a function of duration of exposure, dose, and
Figure 2. Dioxin-like effects of 34 PBDEs analogs in the AhR transactivation assay using stable H4IIE-luc reporter cells. Cells were treated with0.0024−10 mg/L of 34 target PBDEs analogs. Values represent mean ± SD of three independent experiments and are presented as the percentage ofthe response, compared with 100% activity defined as the maximum activity achieved with TCDD.
strength of binding of ligands to the AhR (Figure 2). Eachpoint of the curve represents the mean of three replicates andits standard error, and also represents the ratio of meanluciferase response relative to the maximum response to 2,3,7,8-TCDD (TCDDmax). The mean EC50 for luciferase inductionby 2,3,7,8-TCDD was 5.4 ± 0.7 pg/mL (16.9 ± 2.2 pM). Whenthe maximal response induced by chemicals exceeded thestandard deviation (expressed as % TCDDmax) of the meanDMSO blank response (0% TCDDmax) by at least 3-fold, thechemical was deemed to have significant AhR-mediatedpotency.3.2. Relative Potencies of Analogues of PBDEs
Relative to 2,3,7,8-TCDD. Among the HO-PBDEs tested,68% (13 of 19 HO-PBDEs) exhibited significant AhR-mediatedpotency relative to 2,3,7,8-TCDD in H4IIE-luc cells. The OH-BDE's that exhibited significant potency included 6′-Cl-2′-HO-BDE-7, 2′-HO-BDE-28, 2′-HO-BDE-68, 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HO-BDE-85, 6-HO-BDE-90, 2-HO-BDE-123, 4-HO-BDE-90, 6-HO-BDE-137, 3-HO-BDE-100, 2′-HO-BDE-66, and 2′-HO-BDE-25. (Figure 2) At the maximal testedconcentration of 10 000 ng/mL, 6′-Cl-2′-HO-BDE-7, 2′-HO-BDE-28, 6-HO-BDE-47, and 6-HO-BDE-85 caused significantcytotoxicity to H4IIE-luc cells, and the TCCD-max for thesechemicals was 2500 ng/mL. (SI Table S3) Similarly, 6 of the 15MeO-PBDEs that were tested exhibited significant AhR-mediated potency. These MeO-PBDEs included 2′-MeO-BDE-28, 6-MeO-BDE-47, 5-Cl-6-MeO-BDE-47, 6-MeO-BDE-85, 2-MeO-BDE-123, and 6-MeO-BDE-137, which accountedfor 40% of the tested MeO-PBDEs. (Figure 2) Dose−responsecurves of four PBDEs analogues that exceeded 50% TCDD-max, including 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HO-BDE-137, and 5-Cl-6-MeO-BDE-47, were also fitted, whichindicated that these chemicals exhibited significant, concen-tration-dependent, AhR-mediated potency as determined inH4IIE-luc cells (SI Figure S1).3.3. Concentrations of PBDEs and their Analogues.
3.3.1. Freshwater Fishes. Concentrations of 13 PBDEs and 34PBDEs analogues were quantified in six fishes from the YangtzeRiver, China. Eleven PBDEs (BDE-17, BDE-71, BDE-47, BDE-66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153, BDE-183,and BDE-190), and two analogues of PBDEs (2′-MeO-BDE-68and 6-MeO-BDE-47), were detected. (SI Figure S3 and Table1) The analogues of PBDEs, 2′-MeO-BDE-68, and 6-MeO-BDE-47, were detected only in bigmouth grenadier anchovy,which is a migratory fishes that resides in the Yangtze Riverestuary and migrate back to the Yangtze River to spawn.Concentrations of ∑PBDEs ranged from 1.8 to 1.4 × 102 ng/glipid with mean and median values of 6.8 × 101 ng/g lipid and6.9 × 101 ng/g lipid, respectively. Concentrations of fourPBDEs congers, including BDE-47, BDE-154, BDE-153, andBDE-183, were detected most frequently and exhibited thegreatest concentrations and percentages of the four individualcongeners that contributed to total PBDE (∑PBDEs) werecalculated: BDE-47: 9.0%; BDE-154: 20.5%; BDE-153: 34.0%;BDE-183: 25.7%.3.3.2. Marine Fishes. Concentrations of 13 PBDEs and 34
PBDEs analogues were quantified in five fishes from the YellowSea, China. Eleven PBDEs (BDE-17, BDE-28, BDE-71, BDE-47, BDE-66, BDE-100, BDE-99, BDE-85, BDE-154, BDE-153,and BDE-183), 8 MeO-PBDEs (2′-MeO-BDE-28, 2′-MeO-BDE-68, 6-MeO-BDE-47, 6-MeO-BDE-90, 3-MeO-BDE-100,2-MeO-BDE-123, 6′-MeO-BDE-17, and 4-MeO-BDE-90) and3 HO-PBDEs (6-HO-BDE-47, 2′-HO-BDE-68, and 4-HO-
BDE-90), were detected. (SI Figure S3 and Table 1)Concentration of ∑PBDEs in marine fish (1.8−2.3 × 101
ng/g lipid with mean of 1.2 ng/g lipid) was 56.7 fold lower thanthose in freshwater fish. These results suggest that fishes in theYangtze River were more affected by synthetic chemicals thanthose from the marine environment. Unlike freshwaterorganisms, analogues of PBDEs were detected in each of themarine fishes, especially 2′-MeO-BDE-68, 6-MeO-BDE-47, and4-HO-BDE-90, which were detected in 80% of samples.
3.4. TEQCHEM and TEQBIO in Samples. In order to assessthe potential for adverse effects of PBDEs and their analogueson humans and wildlife in the future, PBDEs analoguesTEQH4IIE(TEQCHEM) for each organism were calculated as the sum ofthe product of concentrations of individual analogues of PBDEsby their respective RePs, which ranged from NA (NA meansnot achieved) to 1.88 × 10−4 pg.g−1 wet weight (Table 1).Among these testing organisms, the analoguesTEQH4IIE in spottedsickle fish was highest. Raw extractTEQH4IIE (TEQBIO) were testedto be from 0.86 to 6.60 pg pg.g−1 wet weight (Table 1). Theratio of TEQCHEM and TEQBIO (TEQCHEM/ TEQBIO) werecalculated to be from NA to 7.77 × 10−5.
4. DISCUSSIONSlight alterations in structures of chemicals can alter thepotency to bind to biomolecules. Based on 6 homologous pairsof HO- and MeO-substitued BDE, including 2′-HO-BDE-28and 2′-MeO-BDE-28, 6-HO-BDE-47, and 6-MeO-BDE-47, 5-Cl-6-HO-BDE-47 and 5-Cl-6-MeO-BDE-47, 6-HO-BDE-85and 6-MeO-BDE-85, 2-HO-BDE-123 and 2-MeO-BDE-123,6-HO-BDE-137 and 6-MeO-BDE-137, the maximum responserelative to TCDDmax caused by HO-PBDEs was greater thanthat caused by MeO-PBDEs, which indicated that HO-PBDEsexhibited greater potencies to induce AhR activity than didMeO-PBDEs. The maximum potency of four chemicals,including 6-HO-BDE-47, 5-Cl-6-HO-BDE-47, 6-HO-BDE-137, and 5-Cl-6-MeO-BDE-47, exceeded 50% of TCDDmax,even though the respective analogous PBDEs did not result insignificant activation of the AhR-mediated responses. (SI FigureS1) These results are consistent with the observation thataddition of a MeO- or HO group can result in greater potencyas AhR agonists.30 This conclusion is supported by comparingrelative potencies of BDE-47, 6-MeO-BDE-47 and 6-HO-BDE-47.31
ReP values were calculated for each of the HO- and MeO-substituted BDE relative to 2,3,7,8-TCDD (SI Table S3).ReP-H4IIE of dioxin-like analogues of PBDEs ranged from 7.35× 10−12 to 4.00 × 10−4. ReP-H4IIE for 6-MeO-BDE-85, 6′-Cl-2′-HO-BDE-7, 5-Cl-6-MeO-BDE-47, 6-HO-BDE-47, 6-HO-BDE-137, 6-HO-BDE-85, and 5-Cl-6-HO-BDE-47 ranged from 2.56× 10−5 to 4.00 × 10−4, which were equal to or greater than2,3,7,8-TCDD Equivalents suggested by the World HealthOrganization (TEFWHO) reported for mono-ortho-substitutedPCBs, which were assigned a value of 3 × 10−5. Of thesubstituted analogues studied here, 5-Cl-6-HO-BDE-47 ex-hibited the greatest relative potency, which was almost equal tothat for OCDD and OCDF.Concentrations of analogues of PBDEs, regardless of
whether they are natural products or come from syntheticBDE, are greater in marine organisms.18,22 For this reason,marine organisms might pose greater risks to humans via thediet than would freshwater organisms. Among those 11detected analogues of PBDEs, 6 exhibited AhR-mediatedpotency. These included: 2′-HO-BDE-68, 2-MeO-BDE-123,
2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeO-BDE-47. Among those analogues of PBDEs that were detectedin fishes, 2′-MeO-BDE-68, 6-MeO-BDE-47, 2′-MeO-BDE-28,6-HO-BDE-47, and 2′-HO-BDE-68 had been previouslyidentified and confirmed to be natural products of eithermarine sponges or their associated filamentous cyanobacteria,red or green algae.23 6-MeO-BDE-90 and 6′-MeO-BDE-17have been observed in marine wildlife, but have not beenclassified as natural products. 4-HO-BDE-90 has been detectedin blood serum of humans.6 For these four substituted BDEthat have been observed to occur in algae or sponges, 2′-HO-BDE-28 and 6-HO-BDE-85 have been determined to be ofnatural origin.23 Analogues of PBDEs might be concentrated inthe marine environment by fishes. These results wereconsistent with previously published results.19 Concentrationsof ∑PBDEs in organisms studied here, were generally less thanthose in biota from other locations around the world, but equalto those reported by Gao et al (mean: 44.04 ng/g lipid).32
Analogues of PBDEs were identified to be naturally occurringAhR ligands. Generally, AhR ligands were classified into twocategories: synthetic and naturally occurring. PCBs PCDD/Fsand PAHs had been known to be synthetic, dioxin-likecompounds. However, recent work has focused on naturallyoccurring compounds with the hope of identifying endogenousligands. After exposure to PBDEs analogues, AhR of H4IIEcells was activated, which indicated that PBDEs analoguesshould also be listed as naturally occurring dioxin-likecompounds. Most importantly, PBDEs analogues, especiallyMeO-PBDEs, can be accumulated by organisms because oftheir large solubility in lipids,23 unlike the other naturallyoccurring dioxin-like compounds, derivatives of tryptophan33 ortetrapyrroles.34
Based on the calculated PBDEs analoguesTEQH4IIE, risks posed bymarine organisms were greater than freshwater fishes. Takinginto account risk related to consumption of fishes, theEuropean Union (EU) had proposed tolerance limits of 8 pgTEQWHO g−1 wet weight for fish and fishery products,35 whichis relatively greater than that in the marine fishes studied here.Assuming that a 60 kg adult would eat 1 kg sea fish, the totaldaily dietary intake (TDI) from PBDEs analogues wasestimated to be approximately 3.13 × 10−3 pg PBDEs analogues-
TEQH4IIE‑luc /kg bw-day), which is also less than the oralreference dose (RfD) of 7 × 10−1 pg/kg-day for TCDD derivedby U.S. Environmental Protection Agency.36 This RfD wasbased on the results of two epidemiologic studies: spermconcentration and motility in men, and thyroid stimulatinghormone levels in newborn infants. The HQ from PBDEsanalogues in marine fishes was calculated to be 0.005. Thoughconcentrations of PBDEs analoguesTEQH4IIE in individual fisheswere less than the reported tolerance limits, humans could beexposed to some analogues of PBDEs that are of natural originvia seafood and thus should be further evaluated in vivo fortheir toxicity.
Raw extractTEQH4IIE (TEQBIO) for each biological sample wasdetermined to be from 0.86 to 6.60 pg pg.g−1 wet weight, whichwas lower than samples from other locations37,38 and less thanthe tolerance limit of 8 pg TEQ g−1 wet weight for dioxin anddioxin-like compounds in fish proposed by the EuropeanUnion. The ratio of TEQCHEM and TEQBIO (TEQCHEM/TEQBIO) were calculated to be from NA to 7.77 × 10−5. Theseresults suggested that the contribution of PBDEs analogues-
TEQH4IIE‑luc to the total TEQ in fishes was very little, nomore than 0.1%, though some OH- and MeO- analogues do
exhibit significant concentration-dependent AhR-mediatedpotency.Thirty eight OH-PBDE and 25 MeO-PBDEs have been
detected in the environment or tissues of humans (SI Figure S2and Table S4). Of the 63 analogues of PBDE, 15 exhibitedmeasurable dioxin-like potency. Four analogues, including 2′-MeO-BDE-28, 4-HO-BDE-90, 6-HO-BDE-47, and 6-MeO-BDE-47, have been detected in various environmental samples,human samples and this study, and exhibit significant AhRagonist potency as measured in H4IIE-luc cells. This isespecially true for 6-HO-BDE-47 and 6-MeO-BDE-47, whichhave been shown to be of natural origin in marine organismsand have been quantified in various environment media. Sinceanalogues of PBDEs exhibited dioxin-like potency andconcentrations in the environment that are sufficient to causeadverse effects, these chemicals should be considered whenassessing the total potency of mixtures in the environment.
5. PERSPECTIVEFor the first time, it has been reported that naturally occurringanalogues of PBDEs could exhibit measurable AhR-mediatedpotency. While the ReP values of the OH- and MeO-analoguesof PBDE are in general less than those of PCDD/DF andPCBs, they can contribute significant proportions of the totalconcentration of AhR-mediated potency in marine organisms.In China, wild fish are considered beneficial to human healthand marine algae and plants are thought to be nutritionally rich,and thus relatively large quantities are consumed by people. It isstill too early to reject this “ancient wisdom”, however,additional work should be conducted to assess the balancebetween the toxicity and benefit of these compounds naturallyoccurred in dietary source in East China.
■ ASSOCIATED CONTENT*S Supporting InformationSupporting Information includes additional information asnoted in the text including Tables S1−S4 and Figures S1−S3.This material is available free of charge via the Internet athttp://pubs.acs.org.
■ ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (Nos. 20737001 and 20977047) andNational Science and Technology Major Project (No.2008ZX08526-003). The research was also supported by agrant from National Natural Science Foundation of China(No.21007025) and from Major State Basic Research Develop-ment Program (No. 2008CB418102). G.S. was supported theShanghai Tongji Gao Tingyao Environmental Science &Technology Development Foundation (STGEF). J.P.G. wassupported by the program of 2012 “High Level ForeignExperts” (#GDW20123200120) funded by the State Admin-istration of Foreign Experts Affairs, the P.R. China. J.P.G. wasalso supported by the Canada Research Chair program, an atlarge Chair Professorship at the Department of Biology and
Chemistry and State Key Laboratory in Marine Pollution, CityUniversity of Hong Kong, The Einstein Professor Program ofthe Chinese Academy of Sciences.
■ REFERENCES(1) Zhou, J. J.; Zeng, Z. R. Novel fiber coated with beta-cyclodextrinderivatives used for headspace solid-phase microextraction ofephedrine and methamphetamine in human urine. Anal. Chim. Acta2006, 556 (2), 400−406.(2) Lau, F. K.; Charles, M. J.; Cahill, T. M. Evaluation of gas-strippingmethods for the determination of Henry’s law constants forpolybrominated diphenyl ethers and polychlorinated biphenyls. J.Chem. Eng. Data 2006, 51 (3), 871−878.(3) Meerts, I.; van Zanden, J. J.; Luijks, E. A. C.; van Leeuwen-Bol, I.;Marsh, G.; Jakobsson, E.; Bergman, A.; Brouwer, A. Potentcompetitive interactions of some brominated flame retardants andrelated compounds with human transthyretin in vitro. Toxicol. Sci.2000, 56 (1), 95−104.(4) Noren, K.; Meironyte, D. Certain organochlorine and organo-bromine contaminants in Swedish human milk in perspective of past20−30 years. Chemosphere 2000, 40 (9−11), 1111−1123.(5) Qiu, X. H.; Bigsby, R. M.; Hites, R. A. Hydroxylated metabolitesof polybrominated diphenyl ethers in human blood samples from theUnited States. Environ. Health Persp. 2009, 117 (1), 93−98.(6) Athanasiadou, M.; Cuadra, S. N.; Marsh, G.; Bergman, A.;Jakobsson, K. Polybrominated diphenyl ethers (PBDEs) andbioaccumulative hydroxylated PBDE metabolites in young humansfrom Managua, Nicaragua. Environ. Health Perspect. 2008, 116 (3),400−408.(7) Meerts, I.; Letcher, R. J.; Hoving, S.; Marsh, G.; Bergman, A.;Lemmen, J. G.; van der Burg, B.; Brouwer, A. In vitro estrogenicity ofpolybrominated diphenyl ethers, hydroxylated PBDEs, and poly-brominated bisphenol A compounds. Environ. Health Perspect. 2001,109 (4), 399−407.(8) He, Y.; Murphy, M. B.; Yu, R. M. K.; Lam, M. H. W.; Hecker, M.;Giesy, J. P.; Wu, R. S. S.; Lam, P. K. S. Effects of 20 PBDE metaboliteson steroidogenesis in the H295R cell line. Toxicol. Lett. 2008, 176 (3),230−238.(9) Kojima, H.; Takeuchi, S.; Uramaru, N.; Sugihara, K.; Yoshida, T.;Kitamura, S. Nuclear hormone receptor activity of polybrominateddiphenyl ethers and their hydroxylated and methoxylated metabolitesin transactivation assays using chinese hamster ovary cells. Environ.Health Persp. 2009, 117 (8), 1210−1218.(10) Wan, Y.; Jones, P. D.; Wiseman, S.; Chang, H.; Chorney, D.;Kannan, K.; Zhang, K.; Hu, J. Y.; Khim, J. S.; Tanabe, S.; Lam, M. H.;Giesy, J. P. Contribution of synthetic and naturally occurringorganobromine compounds to bromine mass in marine organisms.Environ. Sci. Technol. 2010, 44 (16), 6068−73.(11) Su, G.; Zhang, X.; Liu, H.; Giesy, J. P.; Lam, M. H.; Lam, P. K.;Siddiqui, M. A.; Musarrat, J.; Al-Khedhairy, A.; Yu, H. ToxicogenomicMechanisms of 6-HO-BDE-47, 6-MeO-BDE-47, and BDE-47 in E. coli.Environ. Sci. Technol. 2012, 46 (2), 1185−91.(12) Luthe, G.; Jacobus, J. A.; Robertson, L. W. Receptor interactionsby polybrominated diphenyl ethers versus polychlorinated biphenyls:A theoretical structure-activity assessment. Environ. Toxicol. Pharmacol.2008, 25 (2), 202−10.(13) Kuiper, R.; Murk, A.; Leonards, P.; Grinwis, G.; Van den Berg,M.; Vos, J. In vivo and in vitro Ah-receptor activation by commercialand fractionated pentabromodiphenylether using zebrafish (Daniorerio) and the DR-CALUX assay. Chemosphere 2006, 79 (4), 366−375.(14) Peters, A. K.; van Londen, K.; Bergman, A.; Bohonowych, J.;Denison, M. S.; van den Berg, M.; Sanderson, J. T. Effects ofpolybrominated diphenyl ethers on basal and TCDD-inducedethoxyresorufin activity and cytochrome P450−1A1 expression inMCF-7, HepG2, and H4IIE cells. Toxicol. Sci. 2004, 82 (2), 488−96.(15) Peters, A. K.; Sanderson, J. T.; Bergman, A.; van den Berg, M.Antagonism of TCDD-induced ethoxyresorufin-O-deethylation activ-ity by polybrominated diphenyl ethers (PBDEs) in primary
cynomolgus monkey (Macaca fascicularis) hepatocytes. Toxicol. Lett.2006, 164 (2), 123−32.(16) Zota, A. R.; Park, J. S.; Wang, Y.; Petreas, M.; Zoeller, R. T.;Woodruff, T. J. Polybrominated diphenyl ethers, hydroxylatedpolybrominated diphenyl ethers, and measures of thyroid function insecond trimester pregnant women in California. Environ. Sci. Technol.2011, 45 (18), 7896−905.(17) Zhang, K.; Wan, Y.; Jones, P. D.; Wiseman, S.; Giesy, J. P.; Hu,J. Occurrences and fates of hydroxylated polybrominated diphenylethers in marine sediments in relation to trophodynamics. Environ. Sci.Technol. 2012, 46, 2148−2155.(18) Teuten, E. L.; Xu, L.; Reddy, C. M. Two abundantbioaccumulated halogenated compounds are natural products. Science2005, 307 (5711), 917−20.(19) Su, G. Y.; Gao, Z. S.; Yu, Y.; Ge, J. C.; Wei, S.; Feng, J. F.; Liu, F.Y.; Giesy, J. P.; Lam, M. H.; Yu, H. X. Polybrominated diphenyl ethersand their methoxylated metabolites in anchovy (Coilia sp.) from theYangtze River Delta, China. Environ. Sci. Pollut. Res. Int. 2010, 17 (3),634−42.(20) Zeng, Y. H.; Luo, X. J.; Chen, H. S.; Yu, L. H.; Chen, S. J.; Mai,B. X. Gastrointestinal absorption, metabolic debromination, andhydroxylation of three commercial polybrominated diphenyl ethermixtures by common carp. Environ. Toxicol. Chem. 2012, 31 (4), 731−8.(21) Wan, Y.; Liu, F. Y.; Wiseman, S.; Zhang, X. W.; Chang, H.;Hecker, M.; Jones, P. D.; Lam, M. H. W.; Giesy, J. P. Interconversionof hydroxylated and methoxylated polybrominated diphenyl ethers inJapanese medaka. Environ. Sci. Technol. 2010, 44 (22), 8729−8735.(22) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X. W.; Jones, P. D.;Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J. Y.; Lam, M. H. W.; Giesy, J.P. Origin of hydroxylated brominated diphenyl ethers: Naturalcompounds or man-made flame retardants? Environ. Sci. Technol.2009, 43 (19), 7536−7542.(23) Wiseman, S. B.; Wan, Y.; Chang, H.; Zhang, X.; Hecker, M.;Jones, P. D.; Giesy, J. P. Polybrominated diphenyl ethers and theirhydroxylated/methoxylated analogs: Environmental sources, metabolicrelationships, and relative toxicities. Mar. Pollut. Bull. 2011, 63 (5−12),179−88.(24) Marsh, G.; Stenutz, R.; Bergman, A. Synthesis of hydroxylatedand methoxylated polybrominated diphenyl ethers - Natural productsand potential polybrominated diphenyl ether metabolites. Eur. J. Org.Chem. 2003, 14, 2566−2576.(25) El-Fouly, M. H.; Richter, C.; Giesy, J. P.; Denison, M. S.Production of a novel recombinant cell line for use as a bioassaysystem for detection of 2,3,7,8-tetrachlorodibenzi-p-dioxin-like chem-icals. Environ. Toxicol. Chem. 1994, 13 (10), 1581−1588.(26) Garrison, P. M.; Tullis, K.; Aarts, J. M. M. J. G.; Brouwer, A.;Giesy, J. P.; Denison., M. S. Species-specific recombinant cell lines asbioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam. Appl. Toxicol. 1996, 30, 194−203.(27) Sanderson, J. T.; Aarts, J. M.; Brouwer, A.; Froese, K. L.;Denison, M. S.; Giesy, J. P. Comparison of Ah receptor-mediatedluciferase and ethoxyresorufin-O-deethylase induction in H4IIE cells:Implications for their use as bioanalytical tools for the detection ofpolyhalogenated aromatic hydrocarbons. Toxicol. Appl. Pharmacol.1996, 137 (2), 316−25.(28) Tillitt, D. E.; Giesy, J. P.; Ankley, G. T. Characterization of theH4IIE rat hepatoma cell bioassay as a tool for assessing toxic potencyof planer halogenated hydrocarbons in environmental samples.Environ. Sci. Technol. 1991, 25, 87−92.(29) Villeneuve, D. L.; Blankenship, A. L.; Giesy, J. P. Derivation andapplication of relative potency estimates based on in vitro bioassayresults. Environ. Toxicol. Chem. 2000, 19, 2835−2843.(30) Canton, R. F.; Sanderson, J. T.; Letcher, R. J.; Bergman, A.; vanden Berg, M. Inhibition and induction of aromatase (CYP19) activityby brominated flame retardants in H295R human adrenocorticalcarcinoma cells. Toxicol. Sci. 2005, 88 (2), 447−55.(31) Wahl, M.; Lahni, B.; Guenther, R.; Kuch, B.; Yang, L.; Straehle,U.; Strack, S.; Weiss, C. A technical mixture of 2,2′,4,4′-tetrabromo
diphenyl ether (BDE47) and brominated furans triggers arylhydrocarbon receptor (AhR) mediated gene expression and toxicity.Chemosphere 2008, 73 (2), 209−15.(32) Gao, Z.; Xu, J.; Xian, Q.; Feng, J.; Chen, X.; Yu, H.,Polybrominated diphenyl ethers (PBDEs) in aquatic biota from thelower reach of the Yangtze River, East China. Chemosphere 2009,chemosphere.2009.01.065.(33) McKinney, J. A.; Turel, B.; Winge, I.; Knappskog, P. M.; Haavik,J. Functional properties of missense variants of human tryptophanhydroxylase 2. Hum. Mutat. 2009, 30 (5), 787−94.(34) Airado-Rodriguez, D.; Intawiwat, N.; Skaret, J.; Wold, J. P. Effectof naturally occurring tetrapyrroles on photooxidation in cow’s milk. J.Agric. Food Chem. 2011, 59 (8), 3905−14.(35) Regulation, C. amending Regulation (EC) No 466/2001 settingmaximum levels for certain contaminants in foodstuffs as regardsdioxins and dioxin-like PCBs. Off. J. Eur. Union 2006, L32/34−38.(36) Agency, E. P., EPA’s reanalysis of key issues related to dioxintoxicity and response to nas comments, Volume 1In support ofsummary information on the Integrated Risk Information System(IRIS). 2012, L32/34-38.(37) Giesy, J. P.; Jude, D. J.; Tillitt, D. E.; Gale, R. W.; Meadows, J.C.; Zajieck, J. L.; Peterman, P. H.; Verbrugge, D. A.; Sanderson, J. T.;Schwartz, T. R.; Tuchman, M. L. Polychlorinated dibenzo-p-dioxins,dibenzofurans, biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxinequivalents in fishes from Saginaw Bay, Michigan. Environ. Toxicol.Chem. 1997, 16 (4), 713−724.(38) So, M. K.; Zhang, X.; Giesy, J. P.; Fung, C. N.; Fong, H. W.;Zheng, J.; Kramer, M. J.; Yoo, H.; Lam, P. K. Organochlorines anddioxin-like compounds in green-lipped mussels Perna viridis fromHong Kong mariculture zones. Mar. Pollut. Bull. 2005, 51 (8−12),677−87.
21 Marine Sponges and Fish Samples 2'-MeO-BDE68, 6-MeO-BDE47, 2,2'-diMeO-BB80, 2',6-diMeO-BDE68, 2'-OH-BDE68, 6-OH-BDE47,
2,2'-diOH-BB80, 2',6-diOH-BDE68
26
1. Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X. W.; Jones, P. D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J. Y.; Lam, M. H. W.; Giesy, J. P., Origin of Hydroxylated Brominated Diphenyl Ethers:
Natural Compounds or Man-Made Flame Retardants? Environ Sci Technol 2009, 43, (19), 7536-7542.
2. Su, G. Y.; Gao, Z. S.; Yu, Y.; Ge, J. C.; Wei, S.; Feng, J. F.; Liu, F. Y.; Giesy, J. P.; Lam, M. H.; Yu, H. X., Polybrominated diphenyl ethers and their methoxylated metabolites in anchovy (Coilia
sp.) from the Yangtze River Delta, China. Environ Sci Pollut Res Int 2010, 17, (3), 634-42.
3. So, M. K.; Zhang, X.; Giesy, J. P.; Fung, C. N.; Fong, H. W.; Zheng, J.; Kramer, M. J.; Yoo, H.; Lam, P. K., Organochlorines and dioxin-like compounds in green-lipped mussels Perna viridis
from Hong Kong mariculture zones. Mar Pollut Bull 2005, 51, (8-12), 677-87.
4. Koh, C. H.; Khim, J. S.; Kannan, K.; Villeneuve, D. L.; Senthilkumar, K.; Giesy, J. P., Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), biphenyls (PCBs), and polycyclic
aromatic hydrocarbons (PAHs) and 2,3,7,8-TCDD equivalents (TEQs) in sediment from the Hyeongsan River, Korea. Environ Pollut 2004, 132, (3), 489-501.
5. Villeneuve, D. L.; Khim, J. S.; Kannan, K.; Giesy, J. P., Relative potencies of individual polycyclic aromatic hydrocarbons to induce dioxinlike and estrogenic responses in three cell lines.
Environ Toxicol 2002, 17, (2), 128-37.
6. Athanasiadou, M.; Cuadra, S. N.; Marsh, G.; Bergman, A.; Jakobsson, K., Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDE metabolites in young humans
from Managua, Nicaragua. Environmental Health Perspectives 2008, 116, (3), 400-408.
7. Lacorte, S.; Ikonomou, M. G., Occurrence and congener specific profiles of polybrominated diphenyl ethers and their hydroxylated and methoxylated derivatives in breast milk from
Catalonia. Chemosphere 2009, 74, (3), 412-20.
8. Qiu, X. H.; Bigsby, R. M.; Hites, R. A., Hydroxylated Metabolites of Polybrominated Diphenyl Ethers in Human Blood Samples from the United States. Environmental Health Perspectives
2009, 117, (1), 93-98.
9. Zota, A. R.; Park, J. S.; Wang, Y.; Petreas, M.; Zoeller, R. T.; Woodruff, T. J., Polybrominated diphenyl ethers, hydroxylated polybrominated diphenyl ethers, and measures of thyroid
function in second trimester pregnant women in California. Environ Sci Technol 2011, 45, (18), 7896-905.
10. Wan, Y.; Choi, K.; Kim, S.; Ji, K.; Chang, H.; Wiseman, S.; Jones, P. D.; Khim, J. S.; Park, S.; Park, J.; Lam, M. H.; Giesy, J. P., Hydroxylated polybrominated diphenyl ethers and bisphenol A in
pregnant women and their matching fetuses: placental transfer and potential risks. Environ Sci Technol 2010, 44, (13), 5233-9.
11. Marsh, G.; Athanasiadou, M.; Bergman, A.; Asplund, L., Identification of hydroxylated and methoxylated polybrominated diphenyl ethers in Baltic Sea salmon (Salmo salar) blood.
Environ Sci Technol 2004, 38, (1), 10-8.
12. Valters, K.; Li, H.; Alaee, M.; D'Sa, I.; Marsh, G.; Bergman, A.; Letcher, R. J., Polybrominated diphenyl ethers and hydroxylated and methoxylated brominated and chlorinated analogues in
the plasma of fish from the Detroit River. Environ Sci Technol 2005, 39, (15), 5612-9.
13. Verreault, J.; Gabrielsen, G. W.; Chu, S.; Muir, D. C.; Andersen, M.; Hamaed, A.; Letcher, R. J., Flame retardants and methoxylated and hydroxylated polybrominated diphenyl ethers in
two Norwegian Arctic top predators: glaucous gulls and polar bears. Environ Sci Technol 2005, 39, (16), 6021-8.
14. McKinney, M. A.; Cesh, L. S.; Elliott, J. E.; Williams, T. D.; Garcelon, D. K.; Letcher, R. J., Brominated flame retardants and halogenated phenolic compounds in North American west coast
bald eaglet (Haliaeetus leucocephalus) plasma. Environ Sci Technol 2006, 40, (20), 6275-81.
15. Kelly, B. C.; Ikonomou, M. G.; Blair, J. D.; Gobas, F. A., Hydroxylated and methoxylated polybrominated diphenyl ethers in a Canadian Arctic marine food web. Environ Sci Technol 2008, 42,
(19), 7069-77.
16. Weijs, L.; Das, K.; Siebert, U.; van Elk, N.; Jauniaux, T.; Neels, H.; Blust, R.; Covaci, A., Concentrations of chlorinated and brominated contaminants and their metabolites in serum of
harbour seals and harbour porpoises. Environ Int 2009, 35, (6), 842-50.
17. Liu, J.; Luo, X. J.; Yu, L. H.; He, M. J.; Chen, S. J.; Mai, B. X., Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyles (PCBs), hydroxylated and methoxylated-PBDEs, and
methylsulfonyl-PCBs in bird serum from South China. Arch Environ Contam Toxicol 2010, 59, (3), 492-501.
18. Nomiyama, K.; Uchiyama, Y.; Horiuchi, S.; Eguchi, A.; Mizukawa, H.; Hirata, S. H.; Shinohara, R.; Tanabe, S., Organohalogen compounds and their metabolites in the blood of Japanese
amberjack (Seriola quinqueradiata) and scalloped hammerhead shark (Sphyrna lewini) from Japanese coastal waters. Chemosphere 2011, 85, (3), 315-21.
19. Houde, M.; Pacepavicius, G.; Darling, C.; Fair, P. A.; Alaee, M.; Bossart, G. D.; Solomon, K. R.; Letcher, R. J.; Bergman, A.; Marsh, G.; Muir, D. C., Polybrominated diphenyl ethers and their
hydroxylated analogs in plasma of bottlenose dolphins (Tursiops truncatus) from the United States east coast. Environ Toxicol Chem 2009, 28, (10), 2061-8.
20. Routti, H.; Letcher, R. J.; Chu, S.; Van Bavel, B.; Gabrielsen, G. W., Polybrominated diphenyl ethers and their hydroxylated analogues in ringed seals (Phoca hispida) from Svalbard and the
Baltic Sea. Environ Sci Technol 2009, 43, (10), 3494-9.
21. Ueno, D.; Darling, C.; Alaee, M.; Pacepavicius, G.; Teixeira, C.; Campbell, L.; Letcher, R. J.; Bergman, A.; Marsh, G.; Muir, D., Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in
the abiotic environment: surface water and precipitation from Ontario, Canada. Environ Sci Technol 2008, 42, (5), 1657-64.
22. Zhang, K.; Wan, Y.; Jones, P. D.; Wiseman, S.; Giesy, J. P.; Hu, J., Occurrences and Fates of Hydroxylated Polybrominated Diphenyl Ethers in Marine Sediments in Relation to
Trophodynamics. Environ Sci Technol 2012.
23. Malmvarn, A.; Zebuhr, Y.; Kautsky, L.; Bergman, K.; Asplund, L., Hydroxylated and methoxylated polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins in red alga and
cyanobacteria living in the Baltic Sea. Chemosphere 2008, 72, (6), 910-6.
24. Hazuki, M.; Kei, N.; Susumu, N.; Shu-ji, Y.; Terutake, H.; Yutaka, T.; Miyuki, Y.; Shinsuke, T., Accumulation Features of Organohalogen Metabolites in the Blood of Japanese Terrestrial
Mammals. Interdisciplinary Studies on Environmental Chemistry—Environmental Pollution and Ecotoxicology 2012, 203-210.
25. Ryden, A.; Nestor, G.; Jakobsson, K.; Marsh, G., Synthesis and tentative identification of novel polybrominated diphenyl ether metabolites in human blood. Chemosphere 2012, 88, (10),
1227-34.
26. Haraguchi, K.; Kato, Y.; Ohta, C.; Koga, N.; Endo, T., Marine sponge: a potential source for methoxylated polybrominated diphenyl ethers in the Asia-Pacific food web. J Agric Food Chem
2011, 59, (24), 13102-9.
Supporting Figure 1 Responses and a fitted curve for TCDD and 4 analogues of PBDEs that resulted in luciferase expression that exceeded 50 % of
TCDD-max. Individual values and mean are plotted along with the fitted curve.
Supporting Figure 2 Number of PBDEs analogues detected in our study (marked with red “This Study”), with a dioxin-like activity (marked with
green “Dioxin-like Activity”), detected in the previous publications (marked with blue “Environment Samples”), and detected in human tissues
(marked with pink “Human Tissues”).
Supporting Figure 3 Profiles of concentrations of PBDEs (A) and HO- and MeO-analogues of PBDEs (B) in fishes from the Yangtze River and marine