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Activation of Human Peroxisome Proliferator-Activated Nuclear Receptors
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(PPARγ1) by Semi-Volatile Compounds (SVOCs) and Chemical Mixtures in
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Indoor Dust Mingliang Fang1, Thomas F. Webster2, and Heather M. Stapleton1,*
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Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, United
States; 2
Department of Environmental Health, Boston University School of Public Health, Boston,
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Massachusetts 02118, United States
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*Corresponding author:
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Heather Stapleton, PhD
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Nicholas School of the Environment
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Duke University
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LSRC, Box 90328
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Durham, NC 27708
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Phone: 919-613-8717
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Fax: 919-684-8741
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Email:
[email protected] 18 19 20 21 22 23 1 ACS Paragon Plus Environment
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ABSTRACT
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Recently, we reported that several semi-volatile compounds (SVOCs) were competitive
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ligands for human peroxisome proliferator-activated nuclear receptor gamma (PPARγ1). We also
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observed significant binding from chemicals extracted from house dust at a concentration of 3
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mg dust /mL in the dosing medium. To follow up on this study, a commercially available
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reporter gene assay (GeneBLAzer PPARγ1 non-DA Assay, Invitrogen) was used to investigate
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the PPARγ1 activation by 30 common SVOCs (e.g., brominated flame retardants,
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organophosphates, and phthalates) and in house dust extracts. 28 SVOCs or their metabolites
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were either confirmed or for the first time were found to be weak or moderate PPARγ1 agonists.
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We also observed activation in 15 of 25 dust extracts examined. In some cases, activation was as
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high as 50% of the activation of the positive control (rosiglitazone). Furthermore, there was a
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significant and positive correlation (r = 0.7, p < 0.003) between data collected from this reporter
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assay and our previous ligand binding assay tested on the same dust extracts. Our results suggest
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that many SVOCs ubiquitous in house dust, or their metabolites, are possible PPARγ1 agonists.
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Also, chemical mixtures present in house dust at environmentally relevant levels can activate
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human PPARγ1 in a transfected cell culture system, and further research is needed to identify the
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primary chemical(s) driving this activity.
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INTRODUCTION
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The peroxisome proliferator-activated nuclear receptor gamma (PPARγ) is a master nuclear
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receptor that regulates lipid metabolism, cell proliferation, apoptosis, and differentiation. PPARγ
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have two isoforms, i.e., PPARγ1 and γ2; the former is expressed in virtually all tissues, including
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heart, muscle, colon, kidney, and pancreas, while the latter is primarily expressed in adipose
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tissue (30 amino acids longer than γ1)1. Activation of PPARγ, especially γ2, regulates adipocyte
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gene expression and may be a key factor for obesity2. Recently, many environmental
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contaminants have been shown to activate PPARγ, leading to increased adipogenesis in cell
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cultures and sometimes in vivo3, 4. Those chemicals include several organotins (tributyltin (TBT)
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and triphenyltin (TPT)), and mono(2-ethylhexyl) phthalate bis(2-ethylhexyl) (MEHP) (a
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metabolite of the phthalate DEHP), both of which were shown to upregulate and stimulate
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PPARγ3, 4. Several recent studies have suggested that flame retardants (FRs) and phthalates
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might represent important classes of compounds that could bind to and activate PPARγ. For
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example, 2,2′,6,6′-tetrabromo bisphenol (TBBPA),3,3’,5,5’-tetrachlorobisphenol A (TCBPA)
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and triphenyl phosphate (TPP), were identified as partial agonists of PPARγ5, 6. In another study,
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benzyl butyl phthalate (BzBP) and butyl paraben showed significant activation of PPARγ and
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adipogenesis using cell culture assays7. Thus there has been a lot of interest in identifying new
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“chemical obesogens” and investigating their potential health effects.
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In our recent study, more than 20 semi-volatile organic compounds (SVOCs), primarily
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including flame retardants (FRs) and some of their metabolites, were tested for PPARγ binding
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potential using a fluorescence polarization ligand binding assay (PolarScreenTM PPARγ1-
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competitor assay kit, Invitrogen)8. We found that several organophosophate compounds such as
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tributylphosphate (TBuP) and tris(2-butoxyethyl) phosphate (TBOEP) showed significant
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binding potential at the highest dose level tested. Several of the polybrominated diphenyl ethers
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(PBDE) metabolites (i.e., hydroxylated PBDEs and halogenated phenols) also effectively bound
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to PPARγ. As for the house dust extracts, 21 of 24 dust samples tested showed significant
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PPARγ binding potency at a concentration of 3 mg dust equivalent quantity (DEQ)/mL.
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However, ligand binding does not necessarily indicate agonism of the receptor, leading to
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transcriptional events. Therefore, it is of great interest to investigate whether those identified
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possible PPARγ ligands or the chemical mixtures in house dust, can activate human PPARγ. 3 ACS Paragon Plus Environment
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To follow up on our previous research, PPARγ activation using a cell-based reporter assay
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was used in this study. Chemicals that were previously identified as possible PPARγ ligands
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were tested here for PPARγ1 activation. Furthermore, several other groups of chemicals such as
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phthalates and triaryl phosphates that are ubiquitous in house dust were also included. The aryl
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phosphates that contain isopropyl or tert-butyl substitutions are similar to TPP in chemical
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structure, and are used as important components in FRs and plasticizers. Therefore, we included
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these chemicals in this study on PPARγ1 activation to investigate the structure-dependent
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activity. In addition, PPARγ1 activation by house dust extracts was also examined. As many
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SVOCs such as phthalates and FRs are not chemically bound to their commercial products, they
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migrate out over time and have a high accumulation in dust due to their physicochemical
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properties (high octanol-air partitioning coefficient Log KOA). According to the US EPA
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Exposure Factors Handbook (2009), children ingest approximately 50 mg of dust per day, and
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dust ingestion has been increasingly identified as an important exposure pathway for the uptake
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of SVOCs (e.g., PBDEs) in the home, especially for the toddlers and infants who spend most of
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their time (>95%) in the indoor environment10, 11. Therefore, investigating PPARγ activation
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using environmentally relevant house dust samples could be of more value in estimating real-
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world exposure and possible health effects.
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MATERIALS AND METHODS
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Tested Compounds. The abbreviation, structures and supplier of all the tested compounds are
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shown in Table 1, Figure S1, and Text S1 in Supporting Information (SI). In general, these
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chemicals included tri-aryl organophosphates, FM550 (and their metabolites), 2,2',4,4'-
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tetrabromodiphenyl ether (BDE47 and its metabolites), phthalates, halogenated phenols and
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bisphenols. The tested compounds were mostly SVOCs, which were either identified as PPARγ
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ligands in our previous study8 or have high abundances in indoor dust (e.g., phthalates). The type
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II diabetes drug rosiglitazone was used as a positive control. A possible endogenous PPARγ
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ligand 15-Deoxy-D12,14-prostaglandin J2 (15d-PJG2) was also run for comparison.
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House Dust Extracts. House dust extracts were from our previous PPARγ binding assay8. In
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brief, indoor dust samples were investigator-collected from the main living areas of homes for
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Group A11 and D12. Dust samples in Group B were collected from gymnastics studios13. Dust 4 ACS Paragon Plus Environment
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samples in Group C were investigator-collected from office environments14, and Group E were
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participant-collected dust samples from the main living area using a similar method as reported
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in Hoffman et al. (2014)15. All dust samples were extracted with acetone:hexane (1:1, v/v) using
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sonication, and then concentrated, filtered, cleaned by gel permeation chromatography [GPC,
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Environgel GPC system (Waters, Milford, CA, USA)] and reconstituted in DMSO. A final stock
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with a concentration approximately 2,000 mg DEQ dust/mL DMSO was prepared for PPARγ1
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reporter assay.
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PPARγ1 Reporter Assay. A commercially available reporter gene assay (GeneBLAzer
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PPARγ1 non-DA Assay, Invitrogen) was used to investigate the PPARγ1 activation of groups of
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possible PPARγ ligands and house dust extracts. At least six different dosing levels were
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prepared for each chemical or dust extract and triplicate analyses were conducted for each dose
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level. The details of the assay are fully described in the Supporting Information. An amalar blue
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assay, which was prepared from resazurin, was used for the cell viability test.
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Data Analysis. After subtraction of fluorescence background from cell-free wells, the
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response ratio (RR) of fluorescence intensity at 460 versus 530 nm (designated as 460:530 nm)
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was calculated. All of the observed PPARγ1 activation by the chemicals or house dust extracts
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was normalized to the maximal response of rosiglitazone, and this activation percentage
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(Activation%) was used to describe the potency/efficacy of the samples. Activation% was
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calculated using the following equation:
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Activation% = (RRCompound – RRDMSO)/(RRrosiglitazone–RRDMSO)*100
[1];
RRCompound, RRDMSO and RRrosiglitazone were the florescence response ratio of 460:530 nm in
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the tested compounds, DMSO control and maximal response of rosiglitazone; respectively.
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For most tested chemicals and dust extracts, the activation% was less than 30%. As suggested for
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weak agonists7, an activation threshold (LAT) is proposed based on the limit of quantification
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(LOQ) used in analytical chemistry techniques to assure the biological meaning of statistically
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significant effects. In this study, LAT is based on the variation of the DMSO control and
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calculated as the average DMSO value + 10*SD of the solvent control over all experiments. The
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result showed that activation% less than 7% was not thought to be biologically different from the 5 ACS Paragon Plus Environment
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DMSO control. To compare the potency/efficacy between compounds, maximal activation%
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(ATVmax), the concentration inducing the maximal activation [ATV]max, non-observable adverse
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effect level (NOAEL), and the concentration inducing 15% (EC15) and 20% (EC20) activation
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was reported in this study. Specifically, EC15 and EC20 were used for the potency and maximal
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activation was used to describe the efficacy. For the mixture such as ITP, FM550 and tert-butyl
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phenyl diphenyl phosphate (BPDP), the weighted average of the molecular weight was
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calculated based on the composition. The statistical analyses and quality control are detailed in
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the Text S3.
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RESULTS
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PPARγ1 reporter assay performance. Rosiglitazone was used as a positive control and
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showed a significant dose-response curve in the transactivation assay (Figure 1a). The calculated
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EC50 was approximately 5.5 nM (RSD10 µM and those
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points were discarded in the dose-response curve fit. In some cases, there was no evident
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difference between the control and treated cells when evaluated for viability using the Alamar
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Blue assay. However, the proliferation of cell appeared to be hindered under microscopic
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examination (see Figure S2); cells were characterized by smaller proliferation colonies compared
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to control samples. Morphologically, HEK293 cells exposed to high dosed chemicals or high
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concentrations of dust extracts displayed an enlarged round shape, losing cell-cell contact, in 6 ACS Paragon Plus Environment
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contrast with control cultures dominated by elongated star-shaped cell morphology. A similar
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observation has been reported in a previous study18.
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Organophosphates. PPARγ1 activation varied greatly with slight changes in the chemical
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structure. As shown in Figure 1b, the maximal activation of those chemicals followed the order:
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TBPP~TPPi~Mono-ITP~Di-ITP
