Assessing Indoor Dust Interference with Human Nuclear Hormone Receptors in Cell-Based Luciferase Reporter Assays
2021; National Institute of Environmental Health Sciences; Volume: 129; Issue: 4 Linguagem: Inglês
10.1289/ehp8054
ISSN1552-9924
AutoresAnna S. Young, Thomas Zoeller, Russ Hauser, Tamarra James‐Todd, Brent A. Coull, Peter Behnisch, Abraham Brouwer, Hongkai Zhu, Kurunthachalam Kannan, Joseph G. Allen,
Tópico(s)Indoor Air Quality and Microbial Exposure
ResumoVol. 129, No. 4 ResearchOpen AccessAssessing Indoor Dust Interference with Human Nuclear Hormone Receptors in Cell-Based Luciferase Reporter Assays Anna S. Young, Thomas Zoeller, Russ Hauser, Tamarra James-Todd, Brent A. Coull, Peter A. Behnisch, Abraham Brouwer, Hongkai Zhu, Kurunthachalam Kannan, and Joseph G. Allen Anna S. Young Address correspondence to Anna S. Young, 401 Park Dr., 4W Suite 405, Boston, MA 02215 USA. Email: E-mail Address: [email protected] Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Department of Population Health Sciences, Harvard Graduate School of Arts and Sciences, Cambridge, Massachusetts, USA , Thomas Zoeller Department of Biology, University of Massachusetts Amherst, Amherst, Massachusetts, USA , Russ Hauser Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA , Tamarra James-Todd Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA , Brent A. Coull Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA , Peter A. Behnisch BioDetection Systems, Amsterdam, Netherlands , Abraham Brouwer BioDetection Systems, Amsterdam, Netherlands , Hongkai Zhu Department of Pediatrics and Department of Environmental Medicine, New York University School of Medicine, New York, New York, USA , Kurunthachalam Kannan Department of Pediatrics and Department of Environmental Medicine, New York University School of Medicine, New York, New York, USA , and Joseph G. Allen Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Published:14 April 2021CID: 047010https://doi.org/10.1289/EHP8054AboutSectionsPDF Supplemental Materials ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractBackground:Per- and polyfluoroalkyl substances (PFAS), organophosphate esters (OPEs), and polybrominated diphenyl ethers (PBDEs) are hormone-disrupting chemicals that migrate from building materials into air and dust.Objectives:We aimed to quantify the hormonal activities of 46 dust samples and identify chemicals driving the observed activities.Methods:We evaluated associations between hormonal activities of extracted dust in five cell-based luciferase reporter assays and dust concentrations of 42 measured PFAS, OPEs, and PBDEs, transformed as either raw or potency-weighted concentrations based on Tox21 high-throughput screening data.Results:All dust samples were hormonally active, showing antagonistic activity toward peroxisome proliferator-activated receptor (PPARγ2) (100%; 46 of 46 samples), thyroid hormone receptor (TRβ) (89%; 41 samples), and androgen receptor (AR) (87%; 40 samples); agonist activity on estrogen receptor (ERα) (96%; 44 samples); and binding competition with thyroxine (T4) on serum transporter transthyretin (TTR) (98%; 45 samples). Effects were observed with as little as 4μg of extracted dust. In regression models for each chemical class, interquartile range increases in potency-weighted or unknown-potency chemical concentrations were associated with higher hormonal activities of dust extracts (potency-weighted: ΣPFAS–TRβ, ↑28%, p<0.05; ΣOPEs–TRβ, ↑27%, p=0.08; ΣPBDEs–TRβ, ↑20%, p<0.05; ΣPBDEs–ERα, ↑7.7%, p=0.08; unknown-potency: ΣOPEs–TTR, ↑34%, p<0.05; ΣOPEs–AR, ↑13%, p=0.06), adjusted for chemicals with active, inactive, and unknown Tox21 designations.Discussion:All indoor dust samples exhibited hormonal activities, which were associated with PFAS, PBDE, and OPE levels. Reporter gene cell-based assays are relatively inexpensive, health-relevant evaluations of toxic loads of chemical mixtures that building occupants are exposed to. https://doi.org/10.1289/EHP8054IntroductionMaterials inside buildings contain many hormone-disrupting chemicals, including flame retardants (FRs) and per- and polyfluoroalkyl substances (PFAS) (Lucattini et al. 2018). As unbound additives, these chemicals can leach out of products and accumulate in the dust (Allen et al. 2008b; Mitro et al. 2016; Rauert et al. 2014; Tokranov et al. 2019) that we unintentionally ingest and breathe (Johnson-Restrepo and Kannan 2009; Poothong et al. 2020; Xu et al. 2016). In fact, FRs and PFAS have been detected in the urine or blood of over 90% of Americans (Calafat et al. 2007; Ospina et al. 2018; Sjödin et al. 2008).PFAS are a class of over 4,700 extremely persistent chemicals (OECD 2018) applied as stain-, grease-, or water-resistant coatings to carpet, furniture, clothing, cookware, and disposable food packaging (Sunderland et al. 2019). PFAS are associated with adverse human health effects on thyroid function (Rappazzo et al. 2017; Xiao et al. 2020), metabolism (including overweight/obesity, diabetes, insulin resistance, and high cholesterol) (Lin et al. 2019; Sunderland et al. 2019), fetal development (Liew et al. 2018; Xiao et al. 2020), and the immune system (Rappazzo et al. 2017), and possibly kidney and testicular cancer (Barry et al. 2013; Stanifer et al. 2018). Even though two of the most widely known toxic PFAS were voluntarily phased out of production by manufacturers in the United States starting in the early 2000s, the numerous replacement PFAS are also of concern to human health (Wang et al. 2013, 2015, 2017).Chemical FRs have been added to foam furniture, carpet, electronics, and building insulation (Cooper et al. 2016; Jinhui et al. 2017; Kemmlein et al. 2003). One type, polybrominated diphenyl ethers (PBDEs), were largely phased out by 2013 in the United States (Dodson et al. 2012). However, old PBDE-containing products are still in use after decades and can be recycled into new products (Abbasi et al. 2015). In addition, organophosphate esters (OPEs) are often used as PBDE replacements and as plasticizers. Research has found the adverse health effects to include thyroid dysfunction, poor pregnancy outcomes, infertility, and impairment of cognitive or reproductive development for both PBDEs (Allen et al. 2016; Boas et al. 2012; Choi et al. 2019; Czerska et al. 2013; Johnson et al. 2013; Linares et al. 2015; Mumford et al. 2015; Vuong et al. 2018) and, more recently, OPEs (Carignan et al. 2017, 2018; Doherty et al. 2019a, 2019b; Meeker and Stapleton 2010; Messerlian et al. 2018; Preston et al. 2017; Wang et al. 2020).There is considerable evidence that PFAS, PBDEs, and OPEs are hormone-disrupting chemicals. Because nuclear hormone receptors regulate critical genes, their signaling disruption can lead to reproductive (e.g., infertility), developmental (e.g., abnormal fetal growth), and metabolic (e.g., obesity or diabetes) diseases. Certain PFAS and PBDEs or OPEs have been shown to activate estrogen receptor α (ERα) (Du et al. 2013; Hamers et al. 2006; Ren and Guo 2013; Suzuki et al. 2013); suppress peroxisome proliferator-activated receptor γ (PPARγ) (U.S. EPA 2019; Wen et al. 2016), androgen receptor (AR) (Hamers et al. 2006; Klopčič et al. 2016; Orton et al. 2014; Suzuki et al. 2013), and thyroid hormone receptor β (TRβ) (Du et al. 2013; Klopčič et al. 2016; Kollitz et al. 2018; Ren and Guo 2013) and to interfere with thyroid hormone serum transport (Hamers et al. 2006; Rosenmai et al. 2021; Weiss et al. 2009). ERα regulates the development and maintenance of breast and uterine tissue, as well as the cardiovascular system, female reproductive cycle, and bone density (Delfosse et al. 2015; Grimaldi et al. 2015). AR plays an important role in male sexual development differentiation and spermatogenesis (Delfosse et al. 2015; Grimaldi et al. 2015). PPARγ regulates fat storage, lipid metabolism, and insulin sensitivity and can produce anti-inflammatory effects (Delfosse et al. 2015; Grimaldi et al. 2015). TRβ is crucial for normal development, growth, metabolism, and brain function (Grimaldi et al. 2015). Serum transporter transthyretin (TTR) is important for delivering the thyroid hormone thyroxine (T4) across the blood–brain barrier and placenta (Grimm et al. 2013). When a chemical competitively binds to TTR instead, free T4 is more readily eliminated from the body, and that chemical could move into important target tissues (Grimm et al. 2013; Ishihara et al. 2003).Cell-based assays are an emerging method to quantify the total activation or suppression of hormone receptors by complex environmental mixtures of hormone-disrupting chemicals. Compared with traditional targeted laboratory approaches that measure each chemical in a mixture individually, cell-based assays of dust are inexpensive, rapid, and statistically simple to model. Hormonal activities in assays of dust also reflect the combined effects from co-exposures of all hormone-disrupting chemicals in the sample, including unmeasurable chemicals and unknown regrettable substitutes. The assays account for any mixture effects, such as when a chemical's effect is triggered, enhanced, or reduced in the presence of another chemical (Kollitz et al. 2018; Vandermarken et al. 2016).Few studies have measured the activities of dust toward nuclear hormone receptors using cell-based assays (Chou et al. 2015; Fang et al. 2015; Hamers et al. 2020; Kassotis et al. 2019; Suzuki et al. 2007; Vandermarken et al. 2016). For example, Suzuki et al. (2013) reported that certain measured PBDEs or OPEs in household dust were probable contributors to ERα activation and AR suppression. Kollitz et al. (2018) found significant correlations between PBDE or OPE levels and TRβ antagonism in household dust even though the 12 measured FRs were not active when tested in isolation, demonstrating possible mixture effects and influence from unmeasured chemicals. To our knowledge, there are currently no published studies relating PFAS concentrations to bioactivities in dust.Hormone receptor activity of a mixture is a function of not only each chemical's concentration, but also its potency. The increase in available high-throughput screening assay data, such as the Tox21 database for individual potencies of almost 10,000 chemicals (U.S. EPA 2019; Huang et al. 2016), has recently enabled studies to integrate information on chemical concentrations and their respective potencies in order to identify key contaminants driving the total bioactivities of water samples (Blackwell et al. 2017). To our knowledge, this type of potency-weighted exposure evaluation using high-throughput screening data has not been done with chemicals in dust.The objectives of this study were to a) quantify hormonal activities of 46 indoor dust samples; b) identify associations between measured PFAS, PBDE, and OPE chemicals and hormonal activities of dust; and c) evaluate potency-weighted chemical concentration calculations as a method to determine which measured chemicals are driving the observed effects of dust mixtures.MethodsStudy DesignWe collected dust samples from 46 rooms across 21 buildings at a university in the United States during January to March 2019. The rooms included 22 common spaces, 6 office suites, and 18 classrooms distributed across different renovation statuses. Approximately half of the samples (n=22) were collected from rooms renovated between 2017 and 2019, with furniture and carpet specified to be free of all chemical FRs and PFAS. The remaining samples (n=24) were collected from carpeted rooms that constituted a similar distribution of room types as the other 22 samples and that had been renovated with conventional furniture as recently as possible. Building construction years ranged from 1863 to 2018 (median=1966) and years of last renovation ranged from 2001 to 2019 (median=2017). Further details about the rooms and sample collection are provided in our previous manuscript (Young et al. 2021).Dust Sample CollectionWe collected a dust sample within each third of each room in order to have sufficient dust mass for three different laboratories (one for chemicals, one for all the cell-based assays, and one for other research). For each sample, we vacuumed all floor surfaces within that one-third sampling area, including underneath furniture, for 10 min. We vacuumed dust into a cellulose extraction thimble (Whatman International) secured with a nitrile rubber O-ring (McMaster-Carr) in a crevice tool attached to a vacuum cleaner (Dyson CY18), following a previously published protocol (Allen et al. 2008a). Thus, the dust came into contact only with the crevice tools, which were cleaned with hot water and isopropyl alcohol between samples. The thimbles were stored in polypropylene centrifuge tubes in polyethylene bags at −13°C until analysis. For field blanks, we carried four unopened centrifuge tubes with thimbles to field sites on various sampling days.Cell-Based Luciferase Reporter Gene AssaysThe dust samples and field blanks were analyzed in chemically activated luciferase gene expression (CALUX) assays by BioDetection Systems. Based on known or suspected mechanisms of human toxicity for PFAS and FRs, we chose the following assays: antagonism of TRβ, AR, and PPARγ2; agonism of ERα; and interference of T4 binding to transthyretin. We initially tested 10 samples for PPARγ agonism too, but no agonism was detected, so we did not proceed further (Figure S2bi,bj).These luciferase reporter gene assays employed human female osteosarcoma cell lines (U2OS) stably transfected with the firefly luciferase reporter gene, whose expression was controlled by activation of specific nuclear hormone receptors under study (Sonneveld et al. 2005). When any agonistic chemicals in the dust extract activated a specific receptor, it would trigger expression of luciferase, and that enzyme produced light (luminescence) in the presence of added luciferin substrate. By contrast, antagonistic chemicals would have inhibited the added agonist from activating the receptor, thereby reducing light induction. The intensity of light was measured with a luminometer and was directly proportional to the degree of receptor activation. In the TTR-T4 interference assay, the chemicals competed with a fixed concentration of T4 to bind the transport protein TTR, and some T4 would be replaced. The amount of T4 still bound to TTR was then separated out, and its activation of TRβ was quantified in the TRβ agonism assay.As demonstrated in Figure S1, the measured luminescence in a particular assay was benchmarked against a reference compound to calculate a final result for each sample in units of microgram equivalents per gram. This unit can be interpreted as for a given mass (in grams) of dust, the mass (in micrograms) of the reference compound that produces the same level of activity. The reference compounds were potent and selective agonists or antagonists and measured alongside the samples in the agonism or antagonism assays, respectively (Table 1). A full dose–response curve for each reference compound was fitted from the activities of eight separate serial dilutions using the Hill equation. Then, the benchmarked activities (in microgram equivalents per gram) of the dust extracts were calculated by interpolating a certain concentration of dust extract onto the calculated calibration curve of the reference compound. For agonistic activity for each dust extract, we used the data point for the lowest sample concentration that produced a response above the limit of quantification (LOQ). The result was then the ratio of the reference compound concentration in medium to the sample concentration at that same measured response level. Whereas an actual measured point was used for the sample concentration, the reference concentration was interpolated from the calculated dose–response curve. For antagonism, we used the lowest sample concentration that produced the highest response 20% inhibition) of the reference compound. The result was then the ratio of the reference to sample concentration at that measured response level for that sample. Although different response levels were used in the calculations for different samples, the results were comparable because of the interpolation onto the reference curve and because the chosen response levels were targeted to be within the linear range of the reference curve. Dose–response curves shifted further to the left indicated higher potency.Table 1 Summary statistics for the hormonal activities of 46 indoor dust samples in luciferase reporter gene assays.Table 1 has eight columns, namely, Assay end point, Abbreviation, Reference compound, Percentage detected, Geometric mean (geometric standard deviation), Median, Range among detected, and Units.Assay end pointAbbreviationReference compoundPercentage detectedGM (GSD)MedianRange among detectedUnitsPeroxisome proliferator-activated receptor γ2 antagonismPPARγGW9662 (chemical)100% (46 of 46)0.554 (1.92)0.5800.150–2.90μg-eq/gEstrogen receptor α agonismER17β-estradiol (natural hormone)96% (44 of 46)2.21 (2.38)1.760.287–22.0ng-eq/gThyroid hormone receptor β antagonismTRDeoxynivalenol (mycotoxin)89% (41 of 46)68.7 (2.30)80.812.8–370μg-eq/gAndrogen receptor antagonismARFlutamide (medication)87% (40 of 46)105 (2.26)10427.5–434μg-eq/gThyroid hormone transport interferenceTTR-T4Perfluorooctanoate (PFAS chemical)98% (45 of 46)104 (2.90)14115.2–626μg-eq/gNote: Activities by sample can be found in Table S2 and Figure S2. AR, androgen receptor; ER, estrogen receptor; GM, geometric mean; GSD, geometric standard deviation; PFAS, per- and polyfluoroalkyl substances; PPARγ2, peroxisome proliferator-activated receptor; TR, transport protein; TTR-T4, transthyretin–thyroxine.Exposure of Cell-Based Assays to Dust ExtractsTo prepare the dust samples for the cell assays, the samples were first sieved with a 1-mm mesh and extracted by accelerated solvent extraction (ASE) using hexane and acetone (1:1, vol/vol). An average 0.43g of dust was extracted for each sample. After gentle evaporation under nitrogen, the hexane/acetone extracts were dissolved in 100μL of dimethyl sulfoxide (DMSO). Five-point serial dilutions (1×, 3×, 10×, 30×, and 100×) of each final extract were then prepared in DMSO. The 3× dilution was made with 8μL of 1× dilution plus 16μL DMSO, and the subsequent dilutions were made with 36μL DMSO plus 4μL of a prior level of dilution (using the 1× dilution to make the 10× dilution, using the 3× dilution to make the 30×, using the 10× to make the 100×, using the 30× to make the 300×, etc.). The final DMSO concentration during exposure of the cells to the prepared serial dilutions was 0.1% in the hormone receptor assays, based on 1μL of the dilution in 1mL of medium.For the nuclear hormone receptor assays, CALUX cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) without phenol-red (#31331-028; Gibco) supplemented with 7.5% charcoal-stripped fetal calf serum (FCS), nonessential amino acids (#11140-03; Gibco) and 10U/mL penicillin and 10μg/mL streptomycin (culture medium). Cells were maintained at standardized conditions (37°C, 5% CO2, high humidity) and subcultured every 3–4 d.For agonistic analysis, cultured CALUX cells were trypsinized and resuspended in assay medium (DMEM/F12 medium supplemented with 5% charcoal-stripped FCS, nonessential amino acids, and 10U/mL penicillin and 10μg/mL streptomycin) at a final concentration of 1×105 cells/mL. Assay medium for TRβ antagonism analyses did not contain dextran-coated charcoal-stripped FCS. Resuspended cells were seeded in 96-well plates and incubated for 16–24 h under standardized conditions. Following incubation, the medium was removed, and the cells were incubated with 200μL of exposure medium containing assay medium supplemented with the dilution series of dust extract or reference compound in 0.1% DMSO. All dilutions were tested in triplicate. Following 24±2h of incubation of exposed cells under standardized conditions, the incubation plates were removed from the incubator, the exposure medium was removed, and the cells were lysed using 30μL/well of a Triton-lysis buffer. Luciferase activity in cellular lysates was measured for 4 s using a luminometer (TriStar LB941; Berthold).For antagonistic analysis, the procedure described above was followed with the exception that the exposure medium was supplemented with the nonsaturating, half maximal effective concentration (EC50) of an agonist [triiodothyronine (T3) (#T2877; Sigma-Aldrich), rosiglitazone (#71740; Cayman Chemical Company), and dihydrotestosterone (#S4757; Selleckchem) for TRβ, PPARγ, and AR assays, respectively]. Antagonistic compounds were expected to compete with binding of that agonist to its respective receptor, resulting in a reduction in light emission.For the TTR-T4 binding interference analysis, we used a combination of the TTR-T4 competition assay and the TRβ assay to determine the amount of T4 still bound to TTR after exposure. Serial sample dilutions in DMSO were incubated in Tris buffer (pH 8.0; 200μL transferred from a mixture of 140μL of Tris buffer with 500μL of serum-free medium) overnight at 4°C in the presence of TTR (0.058μM) and a fixed concentration of T4 (0.052μM). The final concentration of DMSO in the incubations was 3.2%. Following incubation, TTR-bound T4 was separated from free T4 by placing the incubation mixture on cooled Bio-Gel P-6DG columns (#150-0739; Bio-Rad) after which the columns were centrifuged for 1 min at 120×g. The eluate (TTR-bound T4) was added to the assay medium, after which TRβ CALUX cells were exposed for 24 h, as described above. The procedure for the TTR-T4 assay is presented in Figure 2 of the manuscript by Collet et al. (2019).Before evaluating the samples in the CALUX nuclear hormone receptor assays, prepared serial dilutions (with 1% DMSO) were first evaluated for cytotoxicity (cell line #83; BioDetection Systems). Unlike other assays, this cell line continuously expressed luciferase, and any cell death reduced the amount of light emitted, which we measured with the luminometer. Sample extract dilutions that caused a 20% reduction in light were considered cytotoxic and were excluded from assessment because the lack of cell viability in the test system could be misinterpreted as antagonism (van der Linden et al. 2014). The cytotoxicity assay was conducted with a cell density of 10,000 cells/mL and treatment length of 24 h.To quantify CALUX analysis results, full dose–response dilution series of the assay-specific reference compounds were included on each plate. The reference compounds were 17β-estradiol (#E2758; Sigma-Aldrich) for ERα agonism, flutamide for AR antagonism (#F9397; Sigma-Aldrich), GW9662 for PPARγ antagonism (#70785-50; Cayman), deoxynivalenol for TRβ antagonism (#D0156; Sigma-Aldrich), perfluorooctanoate (#171468; Sigma-Aldrich) for TTR-T4 binding interference, and tributyltin acetate (#8216500050; Merck) for cytotoxicity.The method LOQs for antagonism were defined as the concentration of reference compound resulting in 80% of its maximal response. For agonism, the LOQs were calculated as the average of the DMSO solvent blank plus 10 times the standard deviation of the triplicate measurements of the solvent blank. Each plate had separate solvent blanks, so different samples could have a slightly different LOQ depending on which plate they were analyzed on. For samples with no dilutions producing a response above the LOQ, the LOQ was reported, corrected to represent the first dilution not showing cytotoxicity if needed. The average LOQs for the assays were 0.099ng-eq/g-dust for ERα agonism, 8.3μg-eq/g-dust for AR antagonism, 13μg-eq/g-dust for TRβ antagonism, 0.022μg-eq/g-dust for PPARγ antagonism, and 0.94μg-eq/g-dust for TTR-T4 binding interference, without taking into account the cytotoxicity of certain sample dilutions. The catalog numbers for the assay cell lines are BioDetection Systems #44 for AR antagonism, #60 for ERα agonism, #82 for PPARγ antagonism, and #88 for TRβ antagonism.For quality assurance and quality control, all dust sample extracts, reference compound series, and solvent blanks were analyzed in triplicate with an acceptable maximum coefficient of variation defined as <15%. Each plate contained its own reference compound series and solvent blanks. The four field blanks were almost all below the LOQ for all five assays (plus cytotoxicity) or otherwise well below the minimum detected response of the samples, except that one field blank had a detected response against TRβ that was about half the median of the samples (all three other blanks had responses below the LOQ) (Table S1). We subtracted average field blank responses from the sample responses, as described in the "Statistical Analyses" section. More details are described in previous studies for the CALUX assay procedures for AR antagonism and ERα agonism (Sonneveld et al. 2005), PPARγ antagonism (Gijsbers et al. 2011), TRβ antagonism, and TTR-T4 binding interference (Collet et al. 2019).Chemical Analyses of DustThe dust samples and field blanks were analyzed for 15 PFAS (Kim and Kannan 2007), 19 OPEs (Kim et al. 2019), and 8 PBDEs (Johnson-Restrepo and Kannan 2009) by following published protocols. Specifically, the measured PFAS were perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorohexanoate (PFHxA), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonamide (FOSA), perfluoroheptanoate (PFHpA), perfluoropentanoate (PFPeA), perfluorononanoate (PFNA), perfluorobutane sulfonate (PFBS), perfluorodecane sulfonate (PFDS), perfluorobutanoate (PFBA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnDA), perfluorododecanoate (PFDoDA), and n-methyl perfluorooctane sulfonamidoacetic acid (N-MeFOSAA). PBDE analytes included congeners 28, 47, 99, 100, 153, 154, 183, and 209. The OPE analytes were tris(2-butoxyethyl) phosphate (TBOEP), tris(1-chloro-2-propyl) phosphate (TCIPP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), triphenyl phosphate (TPHP), tris(2-chloroethyl) phosphate (TCEP), 2-ethylhexyl diphenyl phosphate (EHDPP), isodecyl diphenyl phosphate (IDDP), tri-iso-butyl phosphate (TIBP), tripropyl phosphate (TPP), cresyl diphenyl phosphate (CDPP), tert-butylphenyl diphenyl phosphate (BPDP), tri-n-butyl phosphate (TNBP), tetrakis(2-chloroethyl) dichloroisopentyl diphosphate (V6), bisphenol A bis(diphenyl phosphate) (BDP), resorcinol bis(diphenyl phosphate) (RDP), tris(2-ethylhexyl) phosphate (TEHP), tris(methylphenyl) phosphate (TMPP), triethyl phosphate (TEP), and tris(p-tert-butylphenyl) phosphate (TBPHP).First, the dust samples were sieved through a 150-μm stainless steel mesh. Then, the samples (0.2–0.5g) were spiked with 30 ng each of labeled surrogate standard mixture and extracted using methanol (3mL) with mechanical oscillation (1 h) followed by ultrasonication (30 min). The resultant extracts were centrifuged (3,500×g, 10 min) and transferred into new polypropylene tubes. The extraction procedure was repeated twice with acetonitrile (3mL) and ethyl acetate (3mL), and then the extracts were combined and evaporated to 3mL under a gentle stream of nitrogen and divided into three aliquots for analysis of PFAS, OPEs, and PBDEs. The aliquots were evaporated to near dryness and were reconstituted with 200μL of different solvents: methanol for PFAS, water:methanol (4:6; vol/vol) for OPEs, and hexane for PBDEs. The extracts were filtered through 0.2-mm nylon filters into glass vials prior to instrumental analysis.OPEs were analyzed with high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) triple quadrupole mass spectrometry (ESI-MS/MS), using electrospray positive ionization multiple reaction monitoring. PBDEs were analyzed using a gas chromatographer coupled with a mass spectrometer (GC-MS) under electron impact ionization mode. PFAS were analyzed using HPLC coupled with ESI-MS/MS. Target PFAS were monitored by multiple reaction monitoring mode under negative ionization. Limits of detection ranged from 0.1–0.8 ng/g for OPEs, 0.09–4.5 ng/g for PBDEs, and 0.06–1.5 ng/g for PFAS.Chemical concentrations in the field blanks were all either below the LOD or far below measured concentrations in dust samples. Duplicate analysis of seven dust samples showed that median relative percentage differences were 0% (range: −62 to 190%) for PFAS, 0% (range: −96 to 52%) for OPEs, and −3.2% (range: −50 to 80%) for PBDEs. This variability likely reflects the natural heterogeneity of dust. Another publication describes the laboratory analyses and results of the chemical analyses in depth (Young et al. 2021).Potency-Weighted Concentrations of ChemicalsTo account for different degrees of hormonal activity of individual chemicals present in dust mixtures, we used previously published information to calculate relative potency factors (RPFs), which are weights for each chemical based on its bioactivity in a given assay compared with other chemicals. For the four antagonism or agonism end points, we downloaded Tox21 data on the in vitro toxicity screening of thousands of chemicals from the U.S. Environmental Protection Agency's ToxCast Chemistry Dashboard (Huang et al. 2016). We chose one reporter gene assay per end point based on relevance, availability, and cell line sensitivity: "TOX21_PPARg_BLA_antagonist_ratio" (beta-lactamase reporter; human embryonic kidney cells), "TOX21_TR_LUC_GH3_Antagonist" (luciferase reporter; rat pituitary tumor cells), "TOX21_AR_LUC_MDAKB2_Antagonist_0.5nM_R1881" (luciferase; human breast cells), and "TOX21_ERa_LUC_VM7_Agonist" (luciferase; human ovarian cancer cells), respectively.As measures of potency in the Tox21 tests, we used activity concentrations at cutoff (ACCs) because they are point-of-departure estimates based on the potency of a chemical at a threshold that is predefined for all chemicals for the given assay. By contrast, more traditional chem
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