Estrogen-related receptor γ disruption of source water and drinking water treatment processes extrac

Available online at ww.sciencedirect.comJOURNAL OFENVIRONMENTALScienceDirectSCIENCES巴ISSN 101-0742JES 门Jourmal of Erironmneatal Sciences 2011, 23(2) 301-306www.jesc.sc.cnLLEstrogen-related receptor γ disruption of source water and drinking watertreatment processes extractsNa Lil, Weiwei Jiang' , Kaifeng Rao' , Mei Ma', Zijian Wang'.",Satyanarayanan Senthik Kumaran2I. Research Centerfor Eco-Envirormental Sciences, Chinese Academy of Sciences, Bejing 100085, China. E-mail: linazone@ 163.com2. Unit of Toxiclogs. Bharathiar Universis, Coimbalore 641046, IndiaReceived 31 March 2010; revised 19 November 2010; acepted 24 November 2010AbstractEnvironmental chemicals in drinking water can impact human health through nuclear receptors. Addionally, estrogen-relatedreceptors (ERRs) are vulnerable to endocrine-disrupting effects. To date, however, ERR disruption of drinking water potency hasnot been reported. We used ERRY two-hybrid yeast assay to screen ERRY disrupting activities in a drinking water treatment plant(DWTP) located in north China and in source water from a reservoir, focusing on agonistic, antagonistic, and inverse agonistic activityto 4-hydroxytamoxifen (4-OHT). Water treatment processes in the DWTP consisted of pre chlorination, coagulation, coal and sandfilration, activated carbon fltration, and secondary chlorination processes. Samples were extracted by solid phase extraction. Resultsshowed that ERRγ antagonistic activities were found in all sample extracts, but agonistic and inverse agonistic activity to 4-0HTwas not found. When calibrated with the toxic equivalent of 4-OHT, antagonistic efluent effects ranged from 3.4 to 33.1 ug/L. In thetreatment processes, secondary chlorination was efective in removing ERRY antagonists, but the coagulation process led to significantlyincreased ERRγ antagonistic activity. The drinking water treatment processes removed 73.5% of ERRγ antagonists. To our knowledge,the occurrence of ERRγ disruption activities on source and drinking water in vitro had not been reported previously. It is vital, therefore,to increase our understanding of ERRy disruping activities in drinking water.Key words: drinking water; estrogen receptor, estrogen-related receptor; two-hybrid yeast; solid phase extractionDOI: 10.1016/S1001-0742(10)60406-8Citation: Li N, Jiang W w, Rao K F, Ma M, Wang ZJ, Kumaran S s, 2011. Estrogen. related reeptor r disuption of source water anddrinking water treatment processes extracts. Joural of Environmental Sciences, 23(2): 301-306IntroductionWhile research has focused on the disruption activityof estrogen receptors (ERs) in water in recent years, farOver the last several decades, an increasing number ofless attention has been paid to identifying compoundsenvironmental contaminants have been found to disruptwith estrogen-related receptors (ERRs) disrupting activity.endocrine systerms in wildlife and humans (Sonnenschein Increasing evidence from in vivo and in vitro stud-and Soto, 1998). These endocrine disrupting chemicals ies demonstrates, however, that ERRs are vulnerable to(EDCs) can interact with human nuclear receptors (NRs)，endocrine-disrupting effects (Horard and Vanacker, 2003)interfering with the endocrine system, causing develop- and are possibly disrupted by environmental chemicalsmental degeneration, reducing fecundity, and leading to (Takayanagi et al, 2006). Recent research has also doc-an increase in human breast cancer (Colbom et al., 1993;umented a correlation between ERs and ERRs in breastCrews et al, 2000). Recently, EDCs have emerged as a cancer patients (Ariazi et al,. 2002), indicating that ERRmajor water quality concerm as they can interfere with disrupting chemicals may play an important role in breastendocrine systems when found at certain concentrations tumors. Therefore, the rate of elimination of ERRs disrupt-in drinking water (Scruggs et al., 2005). Many EDCs areing substances during the treatment process of drinkingbiologically active at very low concentrations and have water and assessing the disrupting potency of surface waterbeen detected in surface water (Heberer et al, 2002) and resour中国煤化-lental importance.drinking water (Stackelberg et al, 2004). This is of concemSi, ERRs have beenas many conventional treatment processes are ineffectiveclassitYHCN M H Gptor subfamily asin completely removing EDCs from water (Johnson et al, no enaogenousuganas nave Deen laentified. Both ERRs2007).and ERs have a high degree of amino acid sequencesimilarity and identity in their DNA-binding (DBD) and●Corresponding auhor. E-mail: wangiz@rces.ac.cn02Joumal of Enironmental Sciences 2011, 23(2) 301- 306/NaLi etal.Vol. 23ligand-binding (LBD) domains (Horard and Vanacker,ASource water2003). ERRs can bind to functional estrogen responseelements (EREs) in ERs target genes, suggesting possiblesimilarities between ERRs and ERs action (Takayanagiet al., 2006). ERRY and ERs functional crosstalk systemsBPre- chlorinationmight explain low-dose effects of environmental estrogenbisphenol A (Takayanagi et al, 2006). Of the three ERRtypes, ERRa, ERRB and ERRγ (Giguere et al, 1988;Hong et al, 1999), ERRγ is essential for the develop-CCoagulationment of the hypothalamic-hypophyseal-adrenocortical axis(Wilson et al, 1993; Luo et al, 1994), and is expressedin a number of human adult and fetal tissues includingthe brain, skeletal muscle, heart, kidney, and retina. SomeDCoal and sand flrationsynthetic chemicals including bisphenol A, diethylstilbe-strol, 4 nonylphenol and some phytoestrogens can bind tohuman ERRy (Tremblay et al, 2001; Coward et al, 2001;Greschik et al, 2004; Takayanagi et al, 2006), which haveEActivated carbonbeen detected in many environment samples and drinkingwater. Assessing the environmental pollutants interferingwith ERRY is, therefore, of great importance.We previously developed novel screening methods forFSecondary chlorinationchemicals with ERRγ disrupting properties using a yeasttwo-hybrid system, and found that some pesticides hadFig. 1 Flow scheme of the treatment processes and sampling locationsERRγ disrupting activity (Li et al, 2008a). Although(A-F).many endocrine disrupting chemicals can survive drinkingamber glass bottle. The bottle was washed three times withwater treatment (Westerhoff et al, 2005), litle is knowndeionized water before sample ollection. Approximatelyabout the fate of ERRY disrupting chemicals in drinking2 mL/L (V/V) of methanol was added to each samplewater and their possible effects on human health. In theimmediately after sampling to suppress possible bioticpresent study we assessed, therefore, the ERRγ mediatedactivity. All samples were stored at 4°C and treated withineffects in source and drinking waters using the yeast two-8 hr after sampling.hybrid system. As ERRY shows very high constitutiveWater samples and procedural blank (Mini-Q water,activity without ligand addition, the disrupting activities18.22) were filtered with glass fiber filters (0.45 μm,of drinking water extracts were tested with and with-Whatman, England) to remove insoluble materials. Solidout the standard antagonist4-hydroxytamoxifen (4-OHT)phase extraction (SPE) was then performed using 500 mg(Takayanagi et al, 2006) using ERRy-GRIP1 yeast byOasis HLB carridges (Waters, USA) conditioned accord-measuring the change of -galactosidase activity.ing to the manufacturer's directions. The cartridges wereforced under vacuum at a flow rate of approximately 61 Materials and methodsmL/min.' The cartridges were kept under vacuum aspirationfor 5 min to dry off any residual water, and then washedtwice with 5 mL of hexane/dichloromethane (7/3, V/V),1.1 Chemicalstwice with 5 mL of tert-buty methyl ether, twice with 5 mLHigh performance liquid chromatography (HPLC)of dichloromethane/methanol (9/1, V/V), and once with 5grade dichloromethane, hexane, methanol, and tert -butylmL of methanol at a flow rate of 1 mL/min. The extractsmethyl ether were obtained from Fisher Scientific (Fairwere then combined and filtered by anhydrous sodiumLawn, USA). The 4-hydroxytamoxifen (4-OHT, 98%) andsulphate to remove water and evaporated to dryness indimethyl sulfoxide (DMSO, 99.5%) were obtained froma rotary evaporator (R- 200, Buchi, France) at 40°C to 2Sigma Chemical (USA). For all chemicals, stock solutionsmL. The dehydrated extract was then dried under gentlewere prepared in DMSO.nitrogen flow and reconstituted in 0.1 mL of DMSO andused for bioassay immediately.1.2 Sample collection and processingSampling was conducted in May 2007 at a drinking1.3 Bioassaywater treatment plant (DWTP) in north China. The DWTPThe bioassay was conducted using yeast strain hERRγ-had four treatment lines with a total capacity of 1,500,000 GRIP1 (Li et al., 2008a). AI1 assays were conducted in atm3 per day. Samples were collected from line two, whichleast中国煤化工ruded the sample, theprocessed the source water from a reservoir. The DWTPpositgonistic activity), theconsisted of pre-chlorination, coagulation, coal and sandnegatCNMHGocedural blank. Testfilration, activated carbon filtration, and secondary chlori-samples (5 μL) of serial dilutions were combined with 995nation (Fig. I).μL of medium containing 5x103 yeast cells/mL, resultingEach water sample (20L) was collected in a pre-cleanedin a test culture in which the volume of DMSO did notNo.2Estrogen-related receptor y disruption of source water and drinking water treatment processes extracts03exceed 1.0% of the total volume. Control assays were80performed for all field blank and laboratory blank samplesand found to be lower than the detection limit.Test cultures (200 μL) were transferred into each well of更6(a 96- well plate and incubated at 30°C with vigorous orbitalshaking (800 r/min) on a titer plate shaker (Heidolph8 40TITRAMAX 1000, Hamburg, Germany) for 2 hr. The30cell density of the culture was then measured at 600-nmwavelength (TECAN GENios A-5002, Salzburg, Austria).20After that, 50 μ of test culture was transferred to a new1096-well plate and after addition of 120 μL of Z- buffer (ing/L) Na2HPO4-7H2O 16.1; NaH2PO4H2O 5.5; KCl 0.75;1E-9IE-8 1E-7IE-6 IE-5IE-4MgSO4.7H2O 0.246) and 20 μ山of chloroform, the assays4-OHT concentation (molL)were carefully mixed (vortex 25 sec) and preincubatedFig.2 Inhibition of high constituive galactosidase activity byfor 5 min at 30°C. The enzyme reaction was started4-hydroxytamoxifen (4-OHT) in yeast strain ERRyGRIPI. Chemicalby adding 40 μL of o-nitrophenyl-B-D-galactopyranosideantagonist activity is represented as inhibition activity percentage relative(13.3 mmol/L, dissolved in Z-buffer), then incubated atto high constitutive activity in yeast strain ERRy-GRIPI. Values are30°C on a titer plate shaker for 60 min. The reactionsaverage士standard error (n = 3).were terminated by the addition of 100 山of Na2CO3(1 mo/L). After centrifugaion at 12,000 xg for 15 min 2 Results and discussion(Sigma Laborzentifugen 2K15, Osterode, Germany), 200μ of the supermatant was transferred into a new 96-well 2.1 Response to known ERRy antagonist 4-OHTplate and the 0D420 nm was determined.The antagonistic activity of varying 4-OHT concen-The B-galactosidase activity (u) was calculated accord-trations was measured (Fig. 2). The 4-OHT antagonisticing to the following Eqs. (1) and (2):activity in a concentration-dependent manner was similarto that previously reported (Li et al, 2008a). The halfu= Cs/txVxDx ODs(1) maximal effective concentration (EC50) value of 4-OHTwas 8.0 x 10-6 mol/L.2.2 Response to drinking water extractsCs= 10-6(As-AB)1εxd(2)Drinking water extracts did not increase B-galactosidaseactivity in the yeast assay compared with the negative con-trol. However, all extracts had ERRγ antagonistic activitieswhere, Cs: the concentration of o-nitrophenol in the en- that inhibited β-galactosidase expression. Removal rates ofzyme assay reaction mix, t: incubation duration of the ERRY antagonistic activities of B, C, D, E, and F treatmentenzyme reaction, V: volume of the test culture, D: dilutionprocesses were -28.9%, -155.0%，60.3%, -44.7% andfactor, ODs: OD600 of test culture, As: OD420 of the en- 73.5% at ftyfold raw water concentration compared withzyme supermatant of the sample, Ag: OD420 of the enzyme the reservoir water (step A) (Fig. 3). When calibratedreaction supematant of the blank, ε: ε for o-nitrophenolregarding the toxic equivalent (TEQ) of 4-0HT (Fig. 2),in the enzyme assay reaction mix, and d: diameter of the values ranged from 3.4 to 33.1 ug/L 4-0HT (Fig. 4).cuvette (Routledge and Sumpter, 1996; Gaido et al., 1997;After tested samples were incubated with 4-0HT(1.0xLi et al, 2008b).To exclude false results caused by cytotoxicity, cell via-25 rbility was determined spectrophotometrically as a changein cell density (OD600) in the assay medium. The procedu-:o-ral blank was tested at the same concentration to monitorfor a false-positive result. Detailed steps are described爸5-elsewhere (Li et al., 2008c). .1.4 Data analysis0一Results were performed on the toxic equivalent (TEQ)approach (Qiao et al., 2006). The dose-response curve onthe inhibition of β-galactosidase expression by 4-0HT isdescribed in our previous work (Li et al., 2008a). The中国煤化工Negative Positive Emptyextract responses were calibrated according to the dose-response curve of 4-OHT to obtain the bioassay derivedFig.3"THC NMH G。; in yeast srain ERRy-equivalent concentrations (TEQ). If nccessary, the extract GRIPI (ffy times concentrated of raw water). Values are average 士was diluted to fit the linear part of tbe dose-response curve standard eror (n = 3). Negative: dimethyl sulfoxide (DMSO); Positive:4-OHT; Empyy: procedural blank.for 4-OHT.304Jourmal of Environmental Sciences 2011, 23(2) 301-306/NaLi et al.Vol. 23that source water centained many compounds that bind toERRγ. Among the processes, .secondary chlorination was.effective in removing ERRY disrupting substances, but co-agulation led to a significant increase in ERRY disruptingactivity. Many previous studies have found that coagu-lation, flocculation, and precipitation were ineffective atremoving dissolved organic contaminants, especially forlow molecular weight compounds (Temes et al, 2002).Additionally, pre- chlorination was responsible for higherconcentrations of disinfection byproducts including manyorganic chemicals (Simpson and Hayes, 1998), and coag-ulation caused small organic molecules to increase (Luo etal., 1998). In the present study, activated carbon showedSampling sitesBioassay derived 4-0HT equivalence in yeast strain ERRy-no obvious removal efect. Although activated carbon hasGRIP1 for sample ERRY antagonistic activity. Values are average 土been effective at removing organic contaminants, removalstandard erTor (1n=.3).capacity was limited by contact time, competition fromnatural organic matter, contaminant solubility, and carbon25 rtype (Kolpin et al, 2002; Boyd et al, 2003). Other studieshave also stated that several compounds were detectablein carbon effluent, and removal efficiency of activatedcarbon was largely dependent on water quality (Snyderet al, 2007). Compared with source water, DWTP canremove 73.4% of ERRY disrupting substances, indicating10that ERRy antagonist elimination in the DWTP was in-complete. Considering some ERRγ disrupting substancesremained in the final efluent, it is possible that the outletmight be harmful to human health.An increasing number of organic compounds have beenE F Negative Positive Emptydetected in surface waters, raising concerms about theSampling sitscontamination of water resources as it is sometimes nec-Fig5 Effect of test samples on inverse agonist activity of 4-OHT inessary to produce drinking water from polluted surfaceyeast strain ERRy-GRIPI for ERRY antagonistic activity at ftfy-fold waters (Heberer et al., 2002). However, complete removalconcentration. Sample inverse agonist activity is represented as inhibitionof EDCs is not possible through conventional wastewateractivity percentage relative to the high constitutive activity in yeast straintreatment and a significant fraction of EDCs may beERRy-GRIP1. Values are average土standard error (n = 3). Negative:released into the aquatic environment (Halling-Sorensen,dimecthyl sulfoxide (DMSO) Positive: 4-OHT.1998; Daughton and Temes, 1999; Joss et al, 2004). The10-5 mol/L), which inhibited about 20% ERRγ constitutivedischarge of ERRY disrupting chemicals in water must,activity in the recombined yeast, 4-OHT's inverse activity therefore, be understood.was not suppressed (Fig. 5).Considering the important role ERRs play in humanhealth (Ariazi et al, 2002), asessing the rate of elimi-2.3 Discussionnation of ERR disrupting substances during DWTPs is ofMultiple contaminants such as pharmaceuicals, steroid considerable importance. To date, however, no report onhormones, unregulated pesticides, flame retardants, rocketERR disrupting activity in DWTPs has been presented.fuel chemicals, plasticizers, detergents, and stain repel-Results from this study suggested that ERRγ antagonisticlants have been found in source and drinking watersactivity in drinking water may biologically impact humans,(Kolpin et al., 2002; Barmes et al, 2008; Focazio et al, and ERRY antagonists in drinking water acting via more2008).Although they are at low levels in water, considering than one mechanism might contribute to biological efectsonly trace amounts of natural hormones can afect theon organisms and contribute to a wide range of hormon-body, there is growing interest in understanding the fate al and/or anti-hormonal efects in vivo through dferentof EDCs during drinking water treatment (Benoti et al, pathways (Molina-Molina et al, 2006). Although ERRs2009). As contaminant removal by applied water treatmentplay an important role in breast cancer (Ariazi et al,is often incomplete, natural waters contain many dissolved2002), for example, current information is insuficient forchemicals that afect ecosystems and impact drinkingdetermining whether ERRY antagonistic activity levels inwater supplies (Boyd et al, 2003),drinki中国煤化工endocrine system.In the present work, ERRY antagonistic activities were Furthein vivo studies, tofound in the reservoir and DWTP. When calibrated to the assessTHC N M H Gndocrine disruptionTEQ of 4-OHT, values ranged from 3.4 to 33.1 ug/L. in humans. basea on current knowledge, it is clear thatThe ERRY antagonistic activities found in reservoir sourceensuring a safe and sustainable water supply will be anwater with 4-0HT equivalent was 13.0 ug/L, suggestingincreasingly great challenge. 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