Introduction {#s1}
============

The incidence of cardiovascular disease (CVD) has increased in many countries, and CVD has been the leading underlying cause of death worldwide since 2000 ([@c45]). CVD contributed to one in every three deaths in the United States in 2008 ([@c26]) and to two in every five deaths in China in 2014 ([@c8]). More than 80% of CVD can be attributed to modifiable and nongenetic factors, and lifestyle choices such as smoking, diet, and exercise were considered as major environmental influences on CVD ([@c4]). Recent disparate lines of evidences together indicate that exposure to chemical pollutants plays a larger role in the etiology of CVD than previously thought ([@c3]). The adverse effects of fine particles present in ambient air and metal on CVD have been demonstrated in epidemiological and animal studies ([@c3], [@c4]; [@c6]). However, significant gaps remain in our understanding of the environmental factors that affect cardiovascular health ([@c2]).

Atherosclerosis is a major cause of CVD. Foam cell (i.e., cholesterol-laden macrophages) formation within the artery wall can facilitate adventitia angiogenesis and the buildup of necrotic pools, thereby playing a key role in atherosclerosis pathogenesis ([@c35]). Liver X receptors (LXRs, including the $\text{LXR}\alpha$ and $\text{LXR}\beta$ subtypes) regulate foam cell formation by functioning as cholesterol sensors that regulate cholesterol efflux to achieve a balance between cholesterol influx and efflux in foam cells ([@c13]; [@c24]; [@c46]). Recent studies showed that treatment of atherosclerotic mice with a synthetic LXR agonist (GW3965) inhibited foam cell formation and promoted regression of atherosclerotic plaques ([@c16]) and that macrophage-specific deletion of LXRs in mice enhanced atherosclerosis ([@c33]). Due to the central role played by LXRs in atherosclerosis pathogenesis, chemicals that can activate LXRs have been designed as drugs to treat atherosclerosis ([@c16]; [@c34]). Thus, it is possible that pollutants with LXR-antagonist activities may induce atherosclerosis by promoting foam cell formation. These findings led us to postulate that human exposure to pollutants with LXR-antagonist activities would induce atherosclerosis by promoting foam cell formation.

To test this hypothesis, this study was designed to determine *a*) whether LXR-antagonist chemicals are present in indoor dust (a well-known carrier of various pollutants and human exposure matrix) ([@c32]; [@c18]; [@c11]), and *b*) whether these LXR-antagonist environmental chemicals can promote foam cell formation and consequently induce atherosclerosis in an experimental animal model.

Materials and Methods {#s2}
=====================

Chemicals and Reagents {#s2.1}
----------------------

The names and sources of chemicals and reagents used in the study are listed in Table S1. Dose solutions of SR9238, triphenyl phosphate (TPHP), *p*-nitrophenol (PNP), and 2-ethylhexyl diphenyl phosphate (EHDPP) were prepared in dimethyl sulfoxide (DMSO) on the basis of their molarity.

Indoor Dust Sample Collection and Extraction {#s2.2}
--------------------------------------------

The indoor dust used in this study was collected using brooms from the main living area (floor and the top of furniture) of 29 Chinese homes. To prevent contamination between different households, a new broom was used for sample collection from each household. The sampling locations were chosen based on convenience. As shown in Table S2, samples were obtained from cities in various regions of China including Beijing, Yiyang, Zhengzhou, Fuzhou Shanghai, Guangzhou, and Chongqing. The homes varied in age and size and were inhabited by persons of varying incomes (see Table S2). For example, the monthly income of the family ranged from $< 5,000$ to 20,000 RMB, the size of the house ranged from $\sim 20$ to $185{\, m}^{2}$, the age of the house ranged from $< 5$ to 10--30 y, the age of the furniture ranged from $< 5$ to 10--30 y, and the distance from the house to the nearest road ranged from $\sim 20$ to $800\; m$. A dust standard reference material (SRM2585) was purchased from the National Institute of Standards and Technology. The indoor dust was collected, wrapped in aluminum foil, and stored at $- 20{^\circ}C$ until extraction. As much as possible, impurities (clips, small stones, human and animal hair, and wood chips) were removed from the dust sample by using tweezers. The dust samples were then extracted as described previously ([@c11]). In brief, $0.1\; g$ of a dust sample was extracted with $5\;{mL}$ acetone/hexane (1:1, vol/vol), followed by shaking for 20 min on an orbital shaker and then sonicating for 15 min. After centrifugation at $4,000\text{ \ rpm}$ for 10 min, the acetone/hexane extract was transferred to a clean glass bottle. The residue was subjected to extraction twice, and the organic solvent extracts were combined. The $7.5\text{-}{mL}$ extracts were evaporated to near dryness under a gentle stream of high purity nitrogen and reconstituted in $7.5\;{mL}$ methanol (MeOH) for determining the concentrations of TPHP and EHDPP. The residual extracts of $7.5\;{mL}$ were evaporated to near dryness and reconstituted in $200\;\mu L$ DMSO to form a $500\text{-}{mg}$ dust/mL DMSO solution for the yeast two-hybrid assay. A $10\text{-}\mu L$ aliquot of the DMSO solution from 11 samples (Samples 1--11; listed in Table S2) was then composited to create a mixture sample for screening the indoor dust for LXR ligands. Glass tubes used during the extraction process were pretreated at 500°C for 6 h to prevent background contaminations. The sample collection was approved by the ethics committee of Peking University, and informed consent was obtained from each homeowner.

Screening LXR Ligands Using a Pull-Down Assay and a Nontarget Chemical Analysis {#s2.3}
-------------------------------------------------------------------------------

In the pull-down assay, the full-length human $\text{LXR}\alpha$ gene was amplified by polymerase chain reaction (PCR) using KOD FX Neo DNA polymerase A plasmid vector $\text{pCMX-hLXR}\alpha$, kindly provided by Dr. D. Mangelsdorf from the University of Texas Southwestern Medical Center, was used as a template. After preincubation at 94°C for 2 min, PCR was performed with 40 cycles of 98°C (10 s), 58°C (30 s), and 72°C (60 s). Primers used were as follows:

1.  forward 5′-GCTCGGTACCCTCGAAATGTCCTTGTGGCTGGG-3′

2.  reverse 5′-ATTCGGATCCCTCGATCATTCGTGCACATCCCAG-3′.

The amplified circular DNA (cDNA) was subcloned into the pCold-trigger factor (TF) vector predigested with EcoRI using the In-Fusion HD Cloning Kit according to the manufacturer's instructions. In brief, the $50\text{ \ ng}$ of predigested plasmid and the $100\text{ \ ng}$ of amplified cDNA were mixed with $2\;\mu L$ premix solution (In-Fusion HD Cloning Kit), and then the mixture was adjusted to $10\;\mu L$ using double-distilled water. After incubation at 50°C for 15 min, the reaction mixture was transformed into *Escherichia coil* (*E. coli*) $\text{DH}5\alpha$-competent cells by heat shock transformation (the mixture was incubated on ice for 30 min, at 42°C for 90 s, and then transferred to ice for another 3 min). The expression plasmids of $\text{pCold-His-TF-LXR}\alpha$ or pCold-His-TF were transfected into *E. coli* BL21 cells by heat shock transformation. The expression of His-TF or $\text{His-TF-LXR}\alpha$ was induced with $0.4\text{ \ mM}$ isopropyl $\beta$-[d]{.smallcaps} thiogalactoside at 20°C for 18 h. The recombinant proteins were purified with a nickel-affinity column (${Ni}^{2 +}\text{-charged}$ HiTrap chelating high-performance column from GE Healthcare), followed by a Hitrap desalting (GE Healthcare) to remove imidazole. The expression of $\text{His-TF-LXR}\alpha$ was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see Figure S1A) with 4% stacking and 15% resolving, and the gel was stained with Coomassie® blue (Imperial Chemical). For the pull-down assay, $20\;\mu M$ $\text{His-TF-LXR}\alpha$ ($n = 6$) and $20\;\mu M$ His-TF ($n = 6$) was spiked with $5\;\mu L$ of house dust extract and incubated in $2\text{-}{mL}$ glass tubes) at 4°C for 1 h. After adding $100\;\mu L$ of His-select nickel magnetic agarose beads (Beaver Life Science) to each tube, the mixture was incubated for 2 h in a shaker at approximately $170\text{ \ rpm}$ at 4°C. After removing the supernatant using a magnetic separator, the residues were washed twice with $1\;{mL}$ buffer ($20\text{ \ mM}$ Tris and $20\text{ \ mM}$ NaCl; pH 8.0), and $\text{LXR}\alpha$ protein was then eluted from the His-select nickel magnetic agarose beads with $1\;{mL}$ buffer ($20\text{ \ mM}$ Tris, $500\text{ \ mM}$ NaCl, and $250\text{ \ mM}$ imidazole; pH 8.0). This protein elution step was performed in triplicate. The chemicals bound to the $\text{LXR}\alpha$ protein were extracted by adding ethyl acetate ($7\;{mL}$) and formic acid (FA; $60\;\mu L$), followed by shaking on an orbital shaker for 15 min and centrifuging at $4,000\text{ \ rpm}$ for 5 min. The extracts were concentrated to near dryness under a gentle stream of high-purity nitrogen and reconstituted in MeOH for nontarget chemical analysis. A negative control experiment was performed using the same protocol but with His-TF protein instead of $\text{His-TF-LXR}\alpha$ protein. We used freshly purified $\text{LXR}\alpha$ protein to pull down LXR ligands from the indoor dust extracts. The efficiency of the recombinant $\text{LXR}\alpha$ to pull down chemicals was confirmed using the positive $\text{LXR}\alpha$ ligand TO901317 at 0.25-, 2.5-, 25-, and $250\text{-}\mu g/L$ concentrations, and the recoveries of TO901317 were $100\% \pm 3\%$, $85\% \pm 5\%$, $80\% \pm 0.7\%$, and $92\% \pm 4\%$, respectively (see Figure S1B). The average percentage of pulled down TO901317 of the spiked concentration in the $\text{His-TF-LXR}\alpha$ group was 89%, and the average percentage of pulled down TO901317 of the spiked concentration in the control group was 10% (see Figure S1C).

A Dionex UltiMate 3000 Ultra high-performance liquid chromatography--quaternary (UHPLC-Q)-Exactive™ mass spectrometer (MS) system (Thermo Fisher Scientific) was used for the nontargeted chemical analysis. Separation of chemicals was achieved by use of a C18 column ($1.7\;\mu m$, $2.1\;{mm} \times 100\;{mm}$; Waters). Milli-Q® water (A phase) and MeOH (B phase) were employed as the mobile phases. In 0--3 min, the B phase increased from 5% to 60%. Then in 3--13 min, the B phase increased from 60% to 100%, and was held static for 3 min. Finally the B phase fell back to the initial condition, which was held for 4 min for equilibration. The sample injection volume, flow rate, column temperature, and sample compartment temperature were set to $5\;\mu L$, $0.30\;{mL}/\text{min}$, 40°C, and 5°C, respectively. In the mode of full scan MS/data-dependent (dd) MS2, the full scan resolution was set at 70,000, and the ddMS2 resolution was set at 17,500. In the full scan and ddMS2, the automatic gain control values were $3 \times 10^{6}$ and $5 \times 10^{4}$, and maximum injection time values were set at 100 ms and 80 ms, respectively. The isolation window of precursor ions was set to $1.0\; m/z$. The TopN was set to 5, where N represents the top most abundant product ions. The step-normalized collision energy was set to 10, 30, and 50. The parameters for electrospray ionization mode were set as follows: the spray voltages of $3\text{ \ kV}$ and $3.5\text{ \ kV}$ were for negative and positive modes, respectively; the capillary temperature was maintained at 320°C; and the sheath gas flow and auxiliary gas flow were set to 35 Arb and 10 Arb, respectively. The S-lens radio frequency (rf) level was 50.

The data sets were deconvoluted and aligned using Progenesis® QI (Waters). The initial and final retention times were set at 2 and 16.2 min, respectively, and peaks with 5,000 minimum intensity in the negative mode and 10,000 minimum intensity in the positive mode were used for further analysis. Adducts considered in this experiment included $\left\lbrack {{M\text{-}H}_{2}O\text{-}H} \right\rbrack^{-}$, $\left\lbrack {M\text{-}H} \right\rbrack^{-}$, $\left\lbrack {M + {Cl}} \right\rbrack^{-}$, and $\left\lbrack {M + \text{FA\ -}H} \right\rbrack^{-}$ in the negative mode and $\left\lbrack {{M + H\text{-}H}_{2}O} \right\rbrack^{+}$, $\left\lbrack {M + H} \right\rbrack^{+}$, $\left\lbrack {M + \text{NH}}_{4} \right\rbrack^{+}$, $\left\lbrack {M + {Na}} \right\rbrack^{+}$, and $\left\lbrack {M + K} \right\rbrack^{+}$ in the positive mode. To differentiate the chemicals between the $\text{His-TF-LXR}\alpha$ and control groups, principal component analysis (PCA) was performed based on the aligned peak list using $\text{SIMCA-}P +$ (version 12.0; Umetrics). The fold change for each feature matched across samples was calculated as the ratio of the area of each peak in the $\text{His-TF-LXR}\alpha$ group to the area of each peak in the control group. Peaks with a fold change of $> 3$ ($p < 0.05$ with Student's *t*-test) in the $\text{His-TF-LXR}\alpha$ group compared with the fold change in the control group were considered as potential ligands of $\text{LXR}\alpha$. These chemicals were manually checked using formula exact mass and retention time. Formulas were further calculated by use of Progenesis® QI based on the extract mass at the mass error of $5\text{ \ ppm}$, element numbers of $C_{50}H_{200}N_{10}O_{10}P_{5}S_{5}{Cl}_{10}{Br}_{10}F_{10}$, and the isotopic difference of $< 10\%$. Then the database of mzCloud (<https://www.mzcloud.org/>), which provides accurate mass spectra based on high resolution mass spectrometry (HRMS), was used to identify the structures of the LXR ligands in the indoor dust samples, the mass tolerance was set to $5\text{ \ ppm}$, and peaks with MS/MS spectra containing at least two product ions matched with the database were selected for further evaluation. The standards of TPHP, EHDPP, and PNP (see Table S1) were purchased for obtaining their mass spectra and the chromatograms that were compared with those of the dust samples to ultimately confirm their structures.

To determine the concentrations of TPHP and EHDPP in the indoor dust extracts, the UHPLC-Q Exactive™ MS was operated in positive ionization mode, and the instrumental method was the same as the nontarget chemical analysis. As an internal standard, $\text{TPHP-}d_{15}$ was added to the indoor dust extracts at $5\text{ \ ng}/{mL}$. Linearity was valid between 0.1 and $100\text{ \ ng}/{mL}$, and regression coefficients of 7-point standard curves were always $> 0.99$. Because low levels of TPHP and EHDPP were detectable in blank samples, the limit of detection of TPHP and EHDPP were set as the average blank signal plus three times the standard deviation (SD) ($0.05\text{ \ ng}/{mL}$ for TPHP and $0.01\text{ \ ng}/{mL}$ for EHDPP). The contribution was calculated as the ratio of the concentration determined by the instrumental method and the concentration equivalent to the inhibition based on *in vitro* bioassay. As described previously ([@c39]), the equivalent concentration was calculated by a single-point estimation at the maximum dust dose ($500\;\mu g$ indoor dust/mL) when the inhibition of a sample was less than 20%, otherwise the multiple-point estimation method was used to derivate the TPHP equivalent concentration by fitting a regression model to the dose--response relationship.

Assessment of LXR Activity {#s2.4}
--------------------------

The LXR-agonistic and -antagonistic activities of the indoor dust samples and identified chemicals were assessed by a yeast two-hybrid assay using human LXRs and coactivator transcription intermediary factor 2 in *Saccharomyces cerevisiae* Y187. The yeast expression plasmids, pGBT9 and pGAD424, were purchased from Clontech/Takara-Bio. The ligand-binding domains (LBDs) of $\text{LXR}\alpha$ (residues 205--447) and $\text{LXR}\beta$ (residues 219--460) and the receptor interaction domains (RIDs) of transcriptional intermediary factor 2 (TIF2) (residues 483--1,090) were amplified from human total RNA (Clontech/Takara-Bio) by reverse transcription (RT)-PCR. The following primers used were designed and purchased from Sigma-Aldrich: $\text{LXR}\alpha$ LBD: forward 5ʹ-CGGAGGAGTGTGTCCTGTCAGA-3′, reverse 5′-TCATTCGTGACGCCCAGATCTC-3′; $\text{LXR}\beta$ LBD: forward 5′-AGGGAGCAGGCTCCTTTCTGAA-3′, reverse 5′-TCACTCGTGGACGCCCAGATCTC-3′; TIF2 RID: forward 5′-TCCGAATCCCACCCAGTCAG-3′, reverse 5′-TAAGGCTCTATCAATCTCCTCCAG-3′. The RT-PCR was performed by AmpliTaq Gold® DNA polymerase (Thermo Fisher Scientific) as follows: $\text{LXR}\alpha$ LBD and $\text{LXR}\beta$ LBD: 95°C for 5 min, 35 cycles of denaturation at 95°C for 15 s, 60°C for 15 s, and 72°C for 60 s; TIF2 RID: 95°C for 5 min, 35 cycles of denaturation at 95°C for 15 s, 58°C for 15 s, and 72°C for 120 s. The amplified $\text{LXR}\alpha$ LBD or $\text{LXR}\beta$ LBD and the TIF2 RID fragments were subcloned into pGBT9 digested with SmaI-BamHI (Takala Bio) and pGAD424 digested with EcoRI-BamHI (Takara Bio), respectively. The ligation reaction was performed using T4 DNA ligase with a DNA ligation kit (Takara Bio) by incubating at 16°C for 1 h. The yeast expression plasmids for fusion protein of GAL4 DNA-binding domain (DBD)---LXR-LBD ($\text{pGBT}9\text{-LXR}\alpha$ and $\text{pGBT}9\text{-LXR}\beta$) consisted of LXR-LBD residues coupled to the GAL4 DBD in pGBT9. The yeast expression plasmid for fusion protein of GAL4 activation domain (AD)--TIF2 RID (pGAD-TIF2) consisted of the TIF2-RID residues coupled to the GAL4 AD in pGAD424. Yeast cells were transformed by the lithium acetate (LiAc) method. In brief, yeast cells were precultured in yeast extract peptone dextrose (YPDA) medium (Clontech/Takara-Bio). At 18 h after preculture, yeast cells were grown to optical density ($\left. \text{OD} \right)_{595} = 0.4–0.6$. The cells were resuspended in a polyethylene glycol (PEG) LiAc solution ($0.1\; M$ LiAc, 40% wt/vol PEG 4000, $10\text{ \ mM}$ Tris-HCl, $1\text{ \ mM}$ EDTA; pH 7.5) and incubated at 30°C for 30 min. Then the cells were transfected with $\text{pGBT}9\text{-LXR}\alpha$ (or $\text{pGBT}9\text{-LXR}\beta$) and pGAD-TIF2 by heat shock (42°C for 15 min). After heat shock, the cells were centrifuged at $3,000\text{ \ rpm}$ for 3 min at room temperature. The centrifuged cells were resuspended in distilled water, and then were centrifuged at $3,000\text{ \ rpm}$ for 3 min at room temperature. The centrifuged cells were then resuspended in a LiAc solution ($0.1\; M$ LiAc, $10\text{ \ mM}$ Tris-HCl, $1\text{ \ mM}$ EDTA; pH 7.5) and plated onto the agar plate. The transfected cells were selected on synthetic defined (SD) minimal medium (for details, see Table S3) solidified with agar at 30°C for 2--3 d. The yeast two-hybrid experiment was carried out as described previously ([@c47]). Briefly, transformed yeast cells were cultured ($\sim 15\; h$, 30°C, with shaking) in SD medium (for details, see Table S3). After the cell suspension ($50\;\mu L$) and chemicals or house dust extracts ($2.5\;\mu L$, in DMSO) were added to the SD medium ($200\;\mu L$), the culture mixtures were incubated at 30°C for 4 h with shaking. Then the culture mixtures ($150\;\mu L$) were removed for determining the absorbance at $595\text{ \ nm}$ by a microplate reader (Thermo Fisher Scientific). The residual cells were lysed with $200\;\mu L$ Z buffer (for details, see Table S3). After incubation at 30°C for 20 min, $40\;\mu L$ of 2-nitrophenyl-b-[d]{.smallcaps}-galactoside ($4\;{mg}/{mL}$) was added and reacted at 30°C for 30 min. The reaction was terminated by adding $100\;\mu L$ sodium carbonate ($1\; M$). Finally, the mixtures ($150\;\mu L$) were added into a 96-well microplate for detecting the absorbance at 415 and $570\text{ \ nm}$. Then $\beta\text{-galactosidase}$ activity was calculated by [Equation 1](#d1){ref-type="disp-formula"}: $$\beta\text{-galactosidase\ activity} = \left( OD_{415} - 1.75 \times OD_{570} \right)/\left( {OD_{595}} \right)$$where $\text{OD}_{595}$, $\text{OD}_{415}$, and $\text{OD}_{570}$ represent the absorbance at 595, 415, and $570\text{ \ nm}$, respectively. DMSO (1%) was used as the control. The LXR agonist TO901317 and the LXR antagonist SR9238 were used as positive controls. The stock solution of TO901317 was diluted by a 3-fold serial dilution with DMSO, and 11 concentrations ($0.05–3,000\text{ \ nM}$) were prepared ($n = 3$ per group). Half-maximal effective concentration ($\text{EC}_{50}$) values were determined to be $100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$. The stock solution of SR9238 was diluted by a 2-fold serial dilution with DMSO, and 11 concentrations ($24.4–25,000\text{ \ nM}$) were prepared ($n = 3$ per group). The half-maximal inhibitory concentration ($\text{IC}_{50}$) values were determined to be $824.7\text{ \ nM}$ for $\text{LXR}\alpha$ and $446.4\text{ \ nM}$ for $\text{LXR}\beta$. To determine whether indoor dust extracts possessed LXR agonistic or antagonistic activity, indoor dust extracts were diluted by a 3-fold serial dilution with DMSO, and five various concentrations ($6–500\;\mu g/{mL}$) were further prepared ($n = 3$ per group). The inhibition (%) was calculated by [Equation 2](#d2){ref-type="disp-formula"}: $$\text{Inhibition\  \  }\% = \left( \frac{\left\lbrack {A - B} \right\rbrack}{A} \right) \times 100$$where A represents the $\beta\text{-galactosidase}$ activity induced by TO901317 at $100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$, and B represents the $\beta\text{-galactosidase}$ activity after co-exposing to house dust extract at different concentrations and TO901317 at $100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$. To determine whether the identified chemicals possessed LXR-agonistic or -antagonistic activity, standards of TPHP, EHDPP, and PNP were tested. Stock solutions were diluted by a 2-fold serial dilution with DMSO, and 11 various concentrations ($24.4–25,000\text{ \ nM}$ for PNP and $48.8–50,000\text{ \ nM}$ for TPHP and EHDPP) were further prepared ($n = 3$ per group). To determine whether the identified chemicals possessed LXR-antagonistic activity, TO901317 at $\text{EC}_{50}$ ($100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$) along with the diluted chemicals was added to the medium. The final DMSO concentration was 1% in the medium. The $\text{IC}_{50}$ was calculated based on the sigmoidal dose--effect curves using Prism software (version 5; Graphpad Inc.).

Cell Cultures and Treatments {#s2.5}
----------------------------

RAW264.7 cells were obtained from the National Infrastructure of Cell Line Resource of Beijing, China, and the cells used for the experiment were cultured for at least three passages. Dulbecco's Modified Eagle's Medium (DMEM)-F12 medium containing 10% fetal bovine serum (FBS) was employed to culture the RAW264.7 cells. RAW264.7 cells were incubated at 37°C with 5% carbon dioxide. The RAW264.7 cells were plated at $2.5 \times 10^{5}$ cells/well (6-well plates), $6.0 \times 10^{4}$ cells/well (24-well plates), and $10^{4}$ cells/well (96-well plates). The RAW264.7 cells were plated and grown overnight; then the medium was removed and fresh DMEM-F12 contained with 0.1% DMSO (control; $n = 3$) or TPHP at the concentrations of $1–20,000\text{ \ nM}$ ($n = 3$ per group) was added. For the LXRs reactivated experiment, RAW264.7 cells were seeded for 24 h and then the cells were treated simultaneously with TPHP ($10,000\text{ \ nM}$, $n = 3$) and TO901317 at 5, 10, or $100\text{ \ nM}$ in DMSO ($n = 3$ per group) or with 0.1% DMSO (control; $n = 3$). Then the cells were used for the quantitative reverse transcription (qRT)-PCR, cellular cholesterol efflux assay, and foam cell formation experiment.

Cell Viability {#s2.6}
--------------

Cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay based on the manufacturer's instructions ($n = 3$ per group). Luminescence was assessed using an LB 941 TriStar multimode microplate reader (Berthold Technologies).

Quantitative Reverse Transcription--Polymerase Chain Reaction {#s2.7}
-------------------------------------------------------------

Total RNA was isolated from RAW264.7 cells and mouse peritoneal macrophages using TRIzol reagent according to the manufacturer's instructions. Then a Nanovue Plus™ spectrophotometer (GE Healthcare Life Science) was used to analyze the concentration, quality, and purity of the RNA. Total RNA ($1\;\mu g$) was mixed with oligo deoxythymine (dT) ($1\;\mu L$) at 70°C for 5 min and then combined with a mixture containing Moloney murine leukemia virus (MMLV) reverse transcriptase ($1\;\mu L$), deoxy-ribonucleoside triphosphate (dNTP) ($1.25\;\mu L$), Recombinant RNasin® Ribonuclease Inhibitor (Promega) ($0.65\;\mu L$), and MMLV buffer ($5\;\mu L$) at 42°C for 60 min. The SYBR® Green PCR Kit (TOYOBO) was used for RT-PCR analysis. Real-time fluorescence detection was performed using the StepOnePlus™ sequence detection system (Applied Biosystems). Relative gene expression was evaluated by the $2^{- \Delta\Delta\text{Ct}}$ method, as suggested by Applied Biosystems. The following primers were designed and purchased from Sangon Biotech:

1.  actin: 5′-CCTGGCACCCAGCACAAT-3′ and 5′-GCCGATCCACACACGGAGTACT-3′ ATP-binding cassette transporter A1 (*ABCA1*): 5′-CCCAGAGCAAAAAGGGACTC-3′ and 5′-GGTCATCATCACTTTGGTCCTTG-3′

2.  ATP-binding cassette transporter G1 (*ABCG1*): 5′-CAAGACCCTTTTGAAAGGGATCTC-3′ and 5′-GCCAGAATATTCATGAGTGTGGAC-3′.

Cellular Cholesterol Efflux Assay {#s2.8}
---------------------------------

RAW264.7 cells or mouse peritoneal macrophages were plated in 96-well plates at $10^{4}$ cells/well and cultured with DMEM-F12 containing 10% FBS and $1\;\mu g/{mL}$ $22\text{-}{\lbrack{N\text{-}\left( {7\text{-nitrobenz-}2\text{-oxa-}1,3\text{-diazol-}4\text{-yl}} \right)\text{amino}}\rbrack}\text{-}23,24\text{-bisnor-}5\text{-cholen-}3\beta\text{-Ol}$ (NBD-cholesterol) for 24 h. Subsequently, the medium was completely removed and fresh DMEM-F12 containing 10% FBS and 0.1% DMSO (control; $n = 3$) or TPHP at the concentration of $1–20,000\text{ \ nM}$ ($n = 3$ per group) was added. After a further 24-h incubation, the cells were washed with phosphate-buffered saline (PBS) and then incubated with serum-free medium containing ApoA-I ($20\;\mu g/{mL}$) or high-density lipoprotein (HDL; $50\;\mu g/{mL}$) for 4 h. Then the medium was collected, and the cells were lysed with a cell lysis buffer. NBD-cholesterol in the medium and cell lysis buffer were measured, respectively, using a Tecan Infinite® M1000Pro Microplate Reader (Tecan Group Ltd.) at $485\text{-nm}$ excitation wavelength and $535\text{-nm}$ emission wavelength. Cholesterol efflux was expressed as a percentage of fluorescence in the medium relative to the total amount of fluorescence.

Foam Cell Formation {#s2.9}
-------------------

RAW264.7 cells or mouse peritoneal macrophages were plated in 24-well plates at $6.0 \times 10^{4}$ cells/well and grown overnight. Then the medium was removed and fresh DMEM-F12 containing 10% FBS, $20\;\mu g/{mL}$ oxidized low-density lipoprotein (ox-LDL), and 0.1% DMSO (control; $n = 3$) or TPHP at the concentration of $1–20,000\text{ \ nM}$ ($n = 3$ per group) was added. The cells were washed with PBS, followed by fixing in 4% paraformaldehyde for 30 min and staining with boron-dipyrromethene (BODIPY) 493/503 ($20\;\mu g/{mL}$) and 4′,6-diamidino-2-phenylindole (DAPI; $100\text{ \ ng}/{mL}$) for 10 min. Foam cell formation was analyzed by high-content microscopy using ImageXpress® Micro XL (Molecular Devices). From each well, 25 image fields were scanned, and MetaXpress® (Molecular Devices) was used to quantify compartmentalized changes in the fluorescence of each cell.

Animals and Exposure {#s2.10}
--------------------

All experiments related to animals were approved by the institutional animal care and use committee of Peking University and followed the guidelines for animal experiments of the university as well as for experimental animals in China. The biological effect size of the animal experiments was based on the previous paper ([@c22]). C57BL/6 (12 weeks old) and $\text{ApoE}^{- / -}$ mice (12 weeks old) were obtained from the Beijing Vital River Laboratory Animal Technology Company. Mice used in the experiments were acclimated to the housing unit 2 weeks before they were used for experiments, and they were randomly assigned to control or TPHP treatment groups. Five animals were housed to a cage, and mice were acclimatized to the controlled environment (temperature: $22 \pm 2{^\circ}C$; relative humidity: 40--60%; artificial lighting: 12 h:12 h light/dark). Mice received diet and drinking water *ad libitum*. Body weight (BW) was measured every week during the dosing period. Forty-five C57BL/6 mice and 27 $\text{ApoE}^{- / -}$ mice were used in this study. Animal protocol 1: Male C57BL/6 mice (five mice per group) were fed a basic diet (Laboratory Animal Center of the Academy of Military Medical Sciences, China). On Day 0, mice were stimulated with paraffin oil by intraperitoneal injection. From Day 1 to Day 7, mice were exposed to corn oil (control) or TPHP (dissolved in corn oil) at 40 or $200\;{mg}/{kg}$ BW by daily oral gavage. The volume of the corn oil or TPHP in corn oil administered was $5\;{mL}/{kg}$ BW. On Day 8, after the mice were anesthetized by sodium pentobarbital via intramuscular injection, peritoneal macrophages were harvested from peritoneal lavage with $4\;{mL}$ pre-warmed DMEM-F12 (containing 10% FBS) using a 25-gauge needle; 5--10 min later, $3–3.5\;{mL}$ peritoneal fluid was collected, and the cells were isolated by centrifuge at $1,000\text{ \ rpm}$ for 5 min. The isolated cells were cultured in DMEM-F12 containing 10% FBS. After 4 h, the medium was changed, and the cells were cultured overnight. The cells were then used for the qRT-PCR, cellular cholesterol efflux assay, and foam cell formation experiment. The exposure period used in the present study was based on previous studies, in which peritoneal macrophages were harvested from mice treated with chemicals for 4--14 d ([@c19]; [@c27]). Considering that this study assessed the effects of short-term exposure to TPHP, whereas humans are exposed to TPHP over a lifelong period, the present study used exposure doses that were higher than that of human exposure from house dust. Animal protocol 2: Because wild-type mice are highly resistant to atherosclerosis, a well-established atherosclerosis model, the $\text{ApoE}^{- / -}$ mouse, was used in the study. The male $\text{ApoE}^{- / -}$ mice (nine mice per group) were fed a high-fat diet (D12108C; Research Diets Inc.) and exposed to corn oil (control) or TPHP (dissolved in corn oil) at 10 or $40\;{mg}/{kg}$ BW for 13 weeks by oral gavage. Mice were sacrificed 24 h after the last day of exposure with an overdose of sodium pentobarbital via intraperitoneal injection. Because the formation of atherosclerosis lesions required a long time, the exposure period used in the present study was based on previous studies in which the effects of chemicals on formation of atherosclerosis lesions were observed after an exposure period of 12--15 weeks ([@c19]; [@c22]; [@c29]). The doses of TPHP used in the $\text{ApoE}^{- / -}$ model mouse referred to previous studies in which mice were treated with TPHP at $50–300\;{mg}/{kg}$ BW to observe significant adverse effects, including decreased testosterone levels and hypertriglyceridemia ([@c7]; [@c21]). Although the adverse effects were observed in mice exposed to a higher dose than the human exposure dose in those studies, epidemiological studies have associated TPHP exposure with decreased sperm concentration and hypertriglyceridemia ([@c20]; [@c48]).

Plasma Lipids and Atherosclerotic Lesion Analysis {#s2.11}
-------------------------------------------------

Treated $\text{ApoE}^{- / -}$ mice ($n = 9$ per group) were fasted overnight, and heart puncture was used to collect the blood soon after each animal was sacrificed. The blood samples were then centrifuged ($1,000 \times g$, 10 min, 4°C) to separate the plasma, and the plasma was stored at $- 80{^\circ}C$. The total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels in the plasma were measured using Roche Diagnostics COBAS MIRA, following the manufacturer's instructions. For atherosclerotic lesion analysis, nine mice per group were evaluated. The aortas from the base of the ascending aorta to the iliac bifurcation were separated, and the aortic roots with the heart were harvested. For *en face* aorta analyses, the aortic tree was perfused with PBS, and the aorta was then dissected *en bloc* from the root to the iliac bifurcation by removing minor branching arteries and fat tissue. The aortic lumen was opened with a longitudinal incision and then immediately fixed in 4% paraformaldehyde. After 24 h of fixation, aortic lipids were stained with Oil Red O, and the stained aortas were photographed. The percentage of aortic area stained with Oil Red O was determined using the image analysis software Image J software ([@c31]). For histological analysis of the aortic roots, PBS-perfused aortas and hearts were fixed in 4% paraformaldehyde/PBS for 5 h, followed by incubation in 30% sucrose for at least 20 h at 4°C. Hearts were cut in half, and the upper part was embedded in O.C.T. (Coolaber Science & Technology) for the frozen sections. Three serial $6\text{-}\mu m$ sections were cut from the point of appearance of the aortic valve leaflets from the frozen hearts, and the sections were stained with Oil Red O. An equal number of aortic valve sites (three slides per mouse) were analyzed in both the corn oil (control) and TPHP-exposed mice. The stained surface area in the sections was quantified using ImageJ software.

Immunofluorescence Staining {#s2.12}
---------------------------

Slides (three slides per mouse) loaded with frozen $6\text{-}\mu m$ sections were rinsed in PBS, followed by quenching of endogenous peroxidase in 3% hydrogen peroxide for 30 min and permeation by 0.25% Triton™ X-100 in PBS for 20 min. The slides were incubated with blocking reagent (ZSGB-BIO, ZLI-9,022) for 30 min at room temperature. Antigens were then successively detected using the Opal 7-Color IHC Kit (NEL797001KT) according to the manufacturer's instructions. Briefly, slides were incubated with macrophage antibody (MOMA)-2 (Abcam; ab33451, 1:300, Opal 650) and $\alpha\text{-Smooth}$ Muscle Actin ($\text{SMC}\alpha\text{-Actin}$; CST, 19245, 1:500, Opal 520) for 2 h in a humidified chamber at 37°C, followed by detection using the horseradish peroxidase (HRP)-conjugated secondary antibody (GBI Labs; Polink-1 HRP polymer detection kit) and fluorophore-conjugated TSA® TSA-fluors (PerkinElmer; Opal 7-color IHC Kit, NEL797001KT, 1:100, 20--60 s), after which the primary and secondary antibodies were thoroughly eluted in glycine SDS pH 2 buffer for 40 min at 50°C. Nuclei were subsequently visualized with DAPI (1:2,000), and the slides were coverslipped using ProLong® Gold antifade mountant (ThermoFisher; P36934).

Statistical Analysis {#s2.13}
--------------------

One-way analysis of variance (ANOVA) followed by a post hoc Dunnett's test was performed using SPSS (version 16.0; SPSS Inc.) to determine the statistical significance of the results. Student's *t*-test was used to analyze the data of the pull-down assay combined with the chemical analysis. For the animal experiments, the Mann-Whitney *U*-test was applied. Results with $p < 0.05$ were considered to be statistically significant. The number of replicates is indicated in the figure legends. The nontarget chemical analysis, yeast two-hybrid assay, cell viability, qRT-PCR, cellular cholesterol efflux assay, and foam cell formation experiment were performed at least three times. For the experiments with the $\text{ApoE}^{- / -}$ mouse model, experiments were performed twice with $n = 4$ and 5.

Results {#s3}
=======

Identification of LXR Ligands in Indoor Dust {#s3.1}
--------------------------------------------

LXR-agonistic and -antagonistic activities of extracts of 11 indoor dust samples (Samples 1--11) were assessed using a yeast two-hybrid assay. TO901317, the LXR-agonist positive control, induced $\beta\text{-galactosidase}$ activity in a dose-dependent manner in both $\text{LXR}\alpha$ and $\text{LXR}\beta$ assays, and its $\text{EC}_{50}$ was $100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$ (see Figure S2). In a screening of indoor dust samples, we unexpectedly discovered that 9 of the 11 indoor dust samples showed dose-dependent LXR-antagonist activity induced by TO901317 in $\text{LXR}\alpha$ and $\text{LXR}\beta$ assays ([Figure 1A,B](#f1){ref-type="fig"}). However, no indoor dust samples showed LXR-agonist activity ([Figure 1C,D](#f1){ref-type="fig"}). Seven indoor dust samples (i.e., Samples 5--11) completely inhibited $\beta\text{-galactosidase}$ activity in an $\text{LXR}\alpha$ assay at the concentration of $500\;\mu g$ indoor dust/mL. Four samples (i.e., Samples 2, 3, 10, and 11) inhibited approximately 50% of the $\beta\text{-galactosidase}$ activity in an $\text{LXR}\beta$ assay.

![Yeast two-hybrid assay to evaluate LXR-agonistic and -antagonistic activity ($\text{means} \pm \text{SDs}$) of indoor dust samples. The (A) $\text{LXR}\alpha$- and (B) $\text{LXR}\beta$-antagonistic activity of indoor dust samples (Samples 1--11). LXR-positive agonist, TO901317 ($100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$) along with indoor dust sample extracts were added to the medium. Inhibition percentage was estimated as the $\beta\text{-galactosidase}$ inhibition compared with TO901317-treated group ($100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$) by [Equation 2](#d2){ref-type="disp-formula"} in the main text. The (C) $\text{LXR}\alpha$- and (D) $\text{LXR}\beta$-agonistic activity of indoor dust samples (Samples 1--11). One percent DMSO and TO901317 ($100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$) were used as the negative control and the positive control, respectively. $n = 3$. Note: LXR, liver X receptor; SD, standard deviation.](ehp-127-117003-g001){#f1}

To identify chemicals with LXR-binding activity, we combined a $\text{His-LXR}\alpha$ pull-down assay with a nontarget HRMS method. Using freshly purified $\text{LXR}\alpha$ protein (see Figure S1), LXRs ligands were pulled down from the indoor dust extracts. The elution extracts from the control and the $\text{His-TF-LXR}\alpha$ group were analyzed to identify $\text{LXR}\alpha$ ligands in indoor dust samples using nontarget HRMS chemical analysis. PCA of the spectral data revealed that samples exhibiting similar variances were clustered together, with the control and $\text{His-TF-LXR}\alpha$ groups showing notable differences (see Figure S3). These results suggest that $\text{LXR}\alpha$ specifically pulled down LXR ligands from the extracts of indoor dust. The 195 peaks in the $\text{His-TF-LXR}\alpha$ group showed a 3-fold higher abundance than those in the control group ($p < 0.05$) ([Figure 2A](#f2){ref-type="fig"}). Based on the mass error and isotopic difference, the molecular formulas of 147 peaks could be calculated using Progenesis® QI software, and the formulas with the highest scores are shown in Excel Table S1. Of the obtained peaks, the mass spectra of three chromatographic peaks at retention times of 3.75, 7.65, and 10.49 min ([Figure 2B](#f2){ref-type="fig"}), were well matched with the respective mass spectra of PNP and two organophosphate flame retardants (OPFRs)---TPHP, and EHDPP--- in the mzCloud mass spectrum database ([Figure 2C--E](#f2){ref-type="fig"}). TPHP, EHDPP, and PNP were further confirmed by comparing the retention times and MS/MS spectra to those of standards (see Figures S4 and S5).

![Identification of LXR ligands from indoor dust samples. Identification of differentiated peaks between the His-TF (control) and $\text{His-TF-LXR}\alpha$ group by nontarget chemical analysis using high-resolution mass spectrometry. (A) Only peaks exhibiting 3-fold higher abundance in $\text{His-TF-LXR}\alpha$ group than the control ($p < 0.05$; analyzed by Student's *t*-test) in volcano plot were considered to be potential ligands (closed circles) ($n = 6$). (B) Extracted chromatograms of triphenyl phosphate (TPHP), 2-ethylhexyl diphenyl phosphate (EHDPP), and *p*-nitrophenol (PNP) in the extract of indoor dust mixture sample of Samples 1--11. Mass spectra of (C) TPHP, (D) EHDPP, and (E) PNP in the extract of indoor dust mixture sample of Samples 1--11. Note: LXR, liver X receptor; TF, trigger factor.](ehp-127-117003-g002){#f2}

LXR-Agonist and -Antagonist Activities of Identified Chemicals {#s3.2}
--------------------------------------------------------------

The LXR-agonist and -antagonist activities of the three identified chemicals were evaluated using yeast two-hybrid assays. PNP showed weak LXR-agonist activity, with $\text{EC}_{50}$ values of $14,020\text{ \ nM}$ for $\text{LXR}\alpha$ and $15,090\text{ \ nM}$ for $\text{LXR}\beta$. TPHP and EHDPP significantly inhibited $\beta\text{-galactosidase}$ activity induced by $100\text{ \ nM}$ TO901317 in an $\text{LXR}\alpha$ yeast assay, with $\text{IC}_{50}$ values of $1,477\text{ \ nM}$ \[95% confidence interval (CI): 1,037, $2,103\text{ \ nM}$\] and $5,691\text{ \ nM}$ (95% CI: 3,658, $8,853\text{ \ nM}$), respectively ([Figure 3A](#f3){ref-type="fig"}). TPHP and EHDPP also significantly inhibited $\beta\text{-galactosidase}$ activity induced by $220\text{ \ nM}$ TO901317 in an $\text{LXR}\beta$ yeast assay, with $\text{IC}_{50}$ values of $762\text{ \ nM}$ (95% CI: 229.9, $1,622\text{ \ nM}$) and $2,165\text{ \ nM}$ (95% CI: 1,622, $4,233\text{ \ nM}$), respectively ([Figure 3B](#f3){ref-type="fig"}). TPHP was found to possess especially strong $\text{LXR}\alpha$- and $\text{LXR}\beta$-antagonist activity, similar to SR9238, a positive antagonist of LXRs (SR9238 $\text{IC}_{50}$: $824.7\text{ \ nM}$ for $\text{LXR}\alpha$ and $446.4\text{ \ nM}$ for $\text{LXR}\beta$) ([Figure 3A,B](#f3){ref-type="fig"}).

![Yeast two-hybrid assay to evaluate LXR-antagonistic activity ($\text{means} \pm \text{SDs}$) of TPHP and EHDPP. The (A) $\text{LXR}\alpha$- and (B) $\text{LXR}\beta$-antagonistic activity of TPHP and EHDPP in comparison with that of the positive LXR antagonist, SR9238. The LXR-positive agonist TO901317 ($100\text{ \ nM}$ for $\text{LXR}\alpha$ and $220\text{ \ nM}$ for $\text{LXR}\beta$) along with TPHP, EHDPP, or SR9238 were added to the medium. $n = 3$. Note: EHDPP, 2-ethylhexyl diphenyl phosphate; LXR, liver X receptor; SD, standard deviation; TPHP, triphenyl phosphate.](ehp-127-117003-g003){#f3}

LXR-antagonist activities were also observed in the indoor dust samples from Shanghai, Guangzhou, and Chongqing. Of the 18 indoor dust samples, 12 samples also showed dose-dependent LXR-antagonist activity induced by TO901317 in $\text{LXR}\alpha$ and $\text{LXR}\beta$ assays (see Figure S6). The TPHP and EHDPP concentrations in the indoor dust extracts tested in the present study were determined to estimate their contribution to total LXR-antagonist activity. TPHP and EHDPP concentrations were $0.13–12.28\;\mu g/g$ and $0.01–0.61\;\mu g/g$, respectively (see Table S4). TPHP explained 0.19--51.07% of the $\text{LXR}\alpha$-antagonist activity and 0.37--32.60% of the $\text{LXR}\beta$-antagonist activity of indoor dust; 0.01--25.02% of the $\text{LXR}\alpha$-antagonist activity and 0.01--22.92% of the $\text{LXR}\beta$-antagonist activity of indoor dust were contributed by EHDPP (see Table S4). The dust standard reference material SRM2585 also exhibited LXR-antagonist activity, and the inhibition could be observed at $167\;\mu g$ indoor dust/mL, and 28.4% and 46.8% of the $\beta\text{-galactosidase}$ activity of $\text{LXR}\alpha$ and $\text{LXR}\beta$ were inhibited at $500\;\mu g$ indoor dust/mL. TPHP and EHDPP contributed 33.83% and 3.01% of the $\text{LXR}\alpha$-antagonist activity, and 12.68% and 1.55% of the $\text{LXR}\beta$-antagonist activity of SRM2585.

Treatment of Macrophages with TPHP and Evaluation of Foam Cell Formation {#s3.3}
------------------------------------------------------------------------

Because TPHP exhibited the strongest LXR-antagonist activity, we tested the variation in the mRNA levels of two representative direct LXR target genes, *ABCA1* and *ABCG1* ([@c30]; [@c43]), in RAW264.7 macrophages exposed to TPHP. Confluent RAW264.7 macrophages showed no significant loss of cell viability after 24-h exposure to TPHP at $1–20,000\text{ \ nM}$ concentrations (see Figure S7). The *ABCG1* gene expression was significantly down-regulated by $0.87 \pm 0.05$-, $0.80 \pm 0.06$-, $0.52 \pm 0.02$-, $0.42 \pm 0.05$-, and $0.28 \pm 0.02\text{-fold}$ in the 100 ($p < 0.05$), 1,000 ($p < 0.05$), 5,000 ($p < 0.01$), 10,000 ($p < 0.01$), and $20,000\text{ \ nM}$ ($p < 0.01$) TPHP-exposure groups, respectively, relative to the control group, indicating a dose--response relationship. Significant down-regulation of *ABCA1* was also observed in 5,000, 10,000, and $20,000\text{ \ nM}$ TPHP-exposure groups by $0.49 \pm 0.06$-, $0.38 \pm 0.01$-, and $0.30 \pm 0.03\text{-fold}$, respectively, relative to the control group ($p < 0.01$) ([Figure 4A,B](#f4){ref-type="fig"}).

![Gene expression ($\text{means} \pm \text{SDs}$) of LXR target genes, cholesterol efflux, and foam cell formation in RAW264.7 cells treated with TPHP. Quantitative analysis of (A) *ABCA1* and (B) *ABCG1* gene expression in RAW264.7 macrophages treated with TPHP. Data are expressed relative to the levels of vehicle-treated cells, which were set to 1. Cholesterol efflux in the presence of (C) HDL or (D) ApoA-I from RAW264.7 macrophages loaded with NBD-cholesterol and treated with DMSO or TPHP at concentration ranges from $1\text{ \ nM}$ to $20,000\text{ \ nM}$ and no significant loss of cell viability was observed (see Figure S7). Relative NBD-cholesterol efflux to HDL or ApoA-I induced by TPHP was calculated. (E) BODIPY 493/503 staining of RAW264.7 macrophages treated with ox-LDL, and exposed with (E) DMSO or (F) TPHP at $1–20,000\text{ \ nM}$. (G) Quantitative analysis of foam cell formation in RAW264.7 macrophages treated with TPHP. Quantitative analysis of (H) *ABCA1* and (I) *ABCG1* gene expression in RAW264.7 macrophages treated with TPHP ($10,000\text{ \ nM}$) and co-exposed to TO901317 at $5–100\text{ \ nM}$. Data are expressed relative to the levels of vehicle-treated cells, which were set to 1. $n = 3$. Analyzed by one-way ANOVA. Indicated values are significantly different from vehicle/control value. Note: ANOVA, analysis of variance; BODIPY, boron-dipyrromethene; DMSO, dimethyl sulfoxide; EHDPP, 2-ethylhexyl diphenyl phosphate; HDL, high-density-lipoprotein; LXR, liver X receptor; NBD, $22\text{-}{\lbrack{N\text{-}\left( {7\text{-nitrobenz-}2\text{-oxa-}1,3\text{-diazol-}4\text{-yl}} \right)\text{amino}}\rbrack}\text{-}23,24\text{-bisnor-}5\text{-cholen-}3\beta\text{-Ol}$; ox-LDL, oxidized low-density-lipoprotein; SD, standard deviation; TPHP, triphenyl phosphate. \*$p < 0.05$. \*\*$p < 0.01$.](ehp-127-117003-g004){#f4}

We further assessed the effects of TPHP on NBD-cholesterol efflux from RAW264.7 macrophages and found that the cholesterol efflux mediated by ApoA-I and HDL was significantly inhibited by $100–20,000\text{ \ nM}$ and $1,000–20,000\text{ \ nM}$ TPHP, respectively ([Figure 4C,D](#f4){ref-type="fig"}). The effects of TPHP on foam cell formation were further investigated in RAW264.7 macrophages. The intensity of foam cell staining in RAW264.7 macrophages was significantly greater by $1.64 \pm 0.02$-, $1.67 \pm 0.14$-, $1.78 \pm 0.31$-, $2.08 \pm 0.45$-, and $2.09 \pm 0.52\text{-fold}$ in the 100 ($p < 0.01$), 1,000 ($p < 0.01$), 5,000 ($p < 0.05$), 10,000 ($p < 0.05$), and $20,000\text{ \ nM}$ ($p < 0.05$) TPHP-exposure groups, respectively, relative to that in the control group ([Figure 4E--G](#f4){ref-type="fig"}). *ABCA1* and *ABCG1* gene expression in RAW264.7 macrophages exposed to TPHP at $10,000\text{ \ nM}$ was dose-dependently higher in TO901317-treated cells than in cells treated with TPHP alone ([Figure 4H,I](#f4){ref-type="fig"}).

*ABCA1* and *ABCG1* gene expression in peritoneal macrophages isolated from mice exposed to TPHP at $200\;{mg}/{kg}$ BW for 7 d was also significantly down-regulated by $0.51 \pm 0.07$- and $0.30 \pm 0.08\text{-fold}$, respectively, compared with the control ($p < 0.01$) ([Figure 5A,B)](#f5){ref-type="fig"}. Significant inhibition of ApoA-I- and HDL-mediated cholesterol efflux was observed in $200\;{mg}/{kg}$ BW TPHP exposure ([Figure 5C,D](#f5){ref-type="fig"}), and foam cell staining was higher by $2.88 \pm 0.95\text{-fold}$ compared with that in the control ($p < 0.05$) ([Figure 5E](#f5){ref-type="fig"}).

![Gene expression ($\text{means} \pm \text{SDs}$) of LXR target genes, cholesterol efflux, and foam cell formation in peritoneal macrophages from animals treated with vehicle or TPHP for 7 d. C57BL/6 mice were exposed to TPHP at 40 and $200\;{mg}/{kg}$ BW for 7 d, and the peritoneal macrophages were isolated. Quantitative analysis of (A) *ABCA1* and (B) *ABCG1* gene expressions in peritoneal macrophages. Data are expressed relative to the levels of vehicle/control group, which was set to 1. Cholesterol efflux in the presence of (C) HDL or (D) ApoA-I from peritoneal macrophages loaded with NBD-cholesterol. Relative NBD-cholesterol efflux to HDL or ApoA-I induced by TPHP was calculated. (E) Foam cell formation of peritoneal macrophages treated with ox-LDL. $n = 5$. Analyzed by one-way ANOVA. Indicated values are significantly different from vehicle/control value. Note: ANOVA, analysis of variance; BW, body weight; EHDPP, 2-ethylhexyl diphenyl phosphate; HDL, high-density-lipoprotein; LXR, liver X receptor; NBD, $22\text{-}{\lbrack{N\text{-}\left( {7\text{-nitrobenz-}2\text{-oxa-}1,3\text{-diazol-}4\text{-yl}} \right)\text{amino}}\rbrack}\text{-}23,24\text{-bisnor-}5\text{-cholen-}3\beta\text{-Ol}$; ox-LDL, oxidized low-density-lipoprotein; TPHP, triphenyl phosphate. \*$p < 0.05$. \*\*$p < 0.01$.](ehp-127-117003-g005){#f5}

Atherosclerotic Lesion Formation in the $\text{ApoE}^{- / -}$ Mouse Model {#s3.4}
-------------------------------------------------------------------------

Foam cells play a central role in atherosclerosis development ([@c35]). To demonstrate whether TPHP could exacerbate atherosclerosis, we exposed $\text{ApoE}^{- / -}$ mice to TPHP at 10 and $40\;{mg}/{kg}$ BW for 13 weeks. No significant differences in BW were observed (see Figure S8). However, *en face* analysis of the whole atherosclerotic lesion and the entire aorta showed a stark contrast between the control and TPHP-exposure groups ([Figure 6A](#f6){ref-type="fig"}). *En face* lipid staining showed the area of the atherosclerotic lesion in the 10 and $40\;{mg}/{kg}$ BW TPHP-exposure groups were 41% and 83% higher than that of the control group ($p < 0.01$), respectively ([Figure 6B](#f6){ref-type="fig"}). Because atherosclerotic lesions generally develop first in the aortic root, we also analyzed lesion areas in the aortic root. Quantification of the area covered by the atherosclerotic lesion around the aortic root revealed a significantly higher lesion area in the TPHP-exposure groups than that of the control ($p < 0.01$) ([Figure 6C,D](#f6){ref-type="fig"}). In order to further characterize the atherosclerotic lesions, we used immunofluorescence staining of the aortic root sections to determine the contents of MOMA-2--positive macrophages and $\text{SMC}\alpha\text{-Actin}$--positive smooth muscle cells (SMCs) in the atherosclerotic lesions ([Figure 7A](#f7){ref-type="fig"}). A significantly higher macrophage abundance was observed in the aortic roots of TPHP-exposed mice. Specifically, the macrophage content was 1.9- and 3.1-fold higher in the 10 ($p < 0.05$) and $40\;{mg}/{kg}$ ($p < 0.01$) BW TPHP-exposure groups than in the control group ([Figure 7B](#f7){ref-type="fig"}). However, for the abundance of SMCs in the aortic roots, a significant difference was not found between the TPHP-exposed groups and the control ([Figure 7C](#f7){ref-type="fig"}). Further analyses found that the serum TC and TG levels were significantly higher in the $40\;{mg}/{kg}$ BW TPHP-exposure group than in the control group ([Table 1](#t1){ref-type="table"}). However, we also found that LDL-C and HDL-C levels of the two exposure groups were not significantly different from those in the control group.

![Atherosclerotic plaque formation in $\text{ApoE}^{–/–}$ mice treated with vehicle or TPHP for 13 weeks. (A) Representative *en face* Oil red O staining of aortas of mice exposed to TPHP compared with the control group. (B) The plaque area is presented as lesion percentage (of whole aorta) in the graph ($n = 9$). (C) Representative Oil red O staining of the aortic root sections. (D) The graph represents the quantification of the area positive for lipid staining $n = 9$). Analyzed by Mann-Whitney *U*-test. Indicated values are significantly different from vehicle/control value. Note: TPHP, triphenyl phosphate. \*$p < 0.05$. \*\*$p < 0.01$.](ehp-127-117003-g006){#f6}

![Characterization of macrophage and smooth muscle cell content ($\text{means} \pm \text{SDs}$) of atherosclerosis lesion in $\text{ApoE}^{–/–}$ mice treated with vehicle or TPHP for 13 weeks. (A) Representative histology of the aortic root sections stained with DAPI, smooth muscle cells marker ($\text{SMC}\alpha\text{-Actin}$), and macrophage marker (MOMA-2). The graphs represent the quantification of the relative positive area of cellular elements (B) MOMA-2 and (C) $\text{SMC}\alpha\text{-Actin}$. Bar: $100\;\mu m$. $n = 9$). Analyzed by Mann-Whitney *U*-test. Indicated values are significantly different from vehicle/control value. Note: DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation; TPHP, triphenyl phosphate. \*$p < 0.05$. \*\*$p < 0.01$.](ehp-127-117003-g007){#f7}

###### 

Plasma lipid levels \[$\text{mean} \pm \text{SE}$ (mmol/L)\] in $\text{ApoE}^{–/–}$ mice after 13-week treatment of TPHP.

Table 1 lists levels for controls; the corresponding values for cholesterol, triglycerides, HDL-C, and LDL-C are listed in the other columns.

                    Cholesterol                                       Triglycerides                                    HDL-C             LDL-C
  ----------------- ------------------------------------------------- ------------------------------------------------ ----------------- ------------------
  Control           $23.19 \pm 1.21$                                  $0.77 \pm 0.06$                                  $1.22 \pm 0.04$   $10.16 \pm 0.36$
  $10\;{mg}/{kg}$   $26.15 \pm 1.34$                                  $0.92 \pm 0.06$                                  $1.13 \pm 0.05$   $10.53 \pm 0.32$
  $40\;{mg}/{kg}$   $28.83 \pm 1.90$[\*](#TF2){ref-type="table-fn"}   $1.06 \pm 0.12$[\*](#TF2){ref-type="table-fn"}   $1.29 \pm 0.05$   $10.27 \pm 0.22$

Note: $n = 9$ per group. HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SE, standard error; TPHP, triphenyl phosphate.

$p < 0.05$ (analyzed by Mann-Whitney *U*-test) indicate values significantly different from vehicle/control value.

Discussion {#s4}
==========

To date, few toxicological studies have investigated the LXR-antagonist activity of environmental samples. This study revealed that among 29 indoor house dust samples from nine cities in China, 21 exhibited dose-dependent LXR-antagonist activity, suggesting that LXR-antagonist activity in indoor dust may be relatively common. Because environmental samples such as indoor dust are chemically complex and contain a multitude of organic contaminants, the identification of specific chemicals in indoor dust that possess LXR-antagonist activity is challenging. We used an $\text{LXR}\alpha$ pull-down assay combined with a nontarget HRMS method to isolate and identify those chemicals that could interact with $\text{LXR}\alpha$ from indoor dust. In this study, 147 chemicals with $\text{LXR}\alpha$-binding activity were found in indoor dust. Although many putative LXR ligands could not be identified because the recorded spectra of standards in available mass spectra database are still limited, two OPFRs, namely TPHP and EHDPP, were identified as LXR antagonists in house dust samples from Chinese cities. TPHP exhibited strong $\text{LXR}\alpha$- and $\text{LXR}\beta$-antagonistic activity, similar to that of SR9238, a positive antagonist of LXRs ([Figure 3A,B](#f3){ref-type="fig"}). TPHP and EHDPP are widely used as flame retardants and plasticizers in various consumer products (including soft foams, paints, and wallpaper) and building materials ([@c44]). TPHP and EHDPP have been detected in indoor dust, and TPHP is one of the most predominant pollutants in the indoor dust samples collected between January and March 2002 in Tokyo, where it was found at concentrations as high as $20,700\;\mu g/g$ ([@c28]). In our study, TPHP was found to explain 0.19--51.07% of the $\text{LXR}\alpha$-antagonist activity and 0.37--32.60% of the $\text{LXR}\beta$-antagonist activity of indoor dust (see Table S4). EHDPP explained 0.01--25.02% of the $\text{LXR}\alpha$-antagonist activity and 0.01--22.92% of the $\text{LXR}\beta$-antagonist activity of indoor dust (see Table S4). Thus, other contaminants contributed a large part of the LXR-antagonist activity of indoor dust, and further identification is needed.

Although the synthetic LXR agonist GW3965 has been reported to inhibit foam cell formation in both macrophages and in the atherosclerosis mouse model ([@c16]) and chemicals that can activate LXRs are designed to treat atherosclerosis, the LXR activity of organic pollutants and associated atherosclerosis risk have not yet been assessed. This study revealed that TPHP promoted foam cell formation in RAW264.7 macrophages ([Figure 4E](#f4){ref-type="fig"}) and in mice peritoneal macrophages. Foam cell formation may be attributable to TPHP-inhibited cholesterol efflux ([Figure 4C,D](#f4){ref-type="fig"}) given that attenuating excess cellular cholesterol efflux has previously been reported to be the causal factor for foam cell formation ([@c10]). *ABCA1* and *ABCG1* gene expressions in macrophages play a critical role in the efflux of excess cellular cholesterol to Apo acceptors such as ApoA-I and HDL ([@c30]; [@c43]), and the selective inactivation of *ABCA1* and *ABCG1* in macrophages causes an imbalance between uptake and ABCA1 and ABCG1-mediated cholesterol efflux ([@c14]; [@c38]; [@c42], [@c41]). In the present study, TPHP down-regulated *ABCA1* and *ABCG1* expression in macrophages in a dose--response manner ([Figure 4A,B](#f4){ref-type="fig"}), demonstrating that the TPHP-induced foam cell formation might be mediated by LXRs given that *ABCA1* and *ABCG1* are downstream target genes of LXRs in macrophages ([@c30]; [@c43]).

In this study, a well-established atherosclerosis model, the $\text{ApoE}^{- / -}$ mouse, was used to evaluate the atherosclerotic activity of TPHP *in vivo.* The development from initial fatty streaks to complex lesions observed in $\text{ApoE}^{- / -}$ mice resembles that in human, whereas wild-type mice are highly resistant to atherosclerosis ([@c5]). In $\text{ApoE}^{- / -}$ mice exposed to 10 and $40\;{mg}/{kg}$ BW of TPHP, *en face* lipid staining revealed significant promotion of the atherosclerotic lesion and plaque area in the aortic roots ([Figure 6](#f6){ref-type="fig"}). TPHP exposure at $40\;{mg}/{kg}$ BW significantly promoted plasma TC and TG levels ([Table 1](#t1){ref-type="table"}). Although the alterations in plasma lipid profiles might have contributed to atherosclerosis development in mouse, it may be insufficient to explain the promotion in atherosclerotic lesions because substantial changes in TC and TG levels occurred only in the $40\;{mg}/{kg}$ BW TPHP-exposure group. It has been reported that TPHP is the agonist of peroxisome proliferator-activated receptor-$\gamma$ ($\text{PPAR}\gamma$) ([@c15]; [@c25]). $\text{PPAR}\gamma$ plays an essential role in regulating adipogenesis ([@c36]), and TPHP has been shown to promote adipogenesis in human primary preadipocytes ([@c37]). In addition to adipogenesis, obesity has been also observed after *in utero* and lactational exposure to TPHP and was reported to result from modulation of the gut microbiome composition of mice ([@c40]). In this study, no significant BW change was observed in $\text{ApoE}^{- / -}$ mice (fed a high-fat diet) after 13-week exposure to TPHP, compared with the control. This may be due to the fact that the absence of ApoE would reduce the BW and induce some of the obesity-associated metabolic complications, including impaired glucose tolerance and insulin resistance in obese models, given that ApoE has been shown to play a key role in energy metabolism in adipose tissue ([@c9]; [@c12]; [@c17]; [@c23]).

Our results demonstrate that TPHP exposure promoted foam cell formation in RAW264.7 macrophages and in peritoneal macrophages isolated from TPHP treatment mice ([Figures 4E](#f4){ref-type="fig"} and [5E](#f5){ref-type="fig"}). The foam cells facilitate adventitia angiogenesis and the buildup of necrotic pools, thereby leading to atherosclerosis exacerbation and plaque instability ([@c1]). The importance of foam cells in atherosclerosis has been demonstrated by the lack of atherosclerotic lesions in the macrophage-deficient mouse ([@c30]). Foam cells play a central role in atherosclerosis development and are prominent cellular elements in atherosclerotic plaque pathogenesis ([@c35]). Analysis of the atherosclerosis lesion composition also revealed a significantly higher macrophage content in the aortic valves of mice exposed to 10 and $40\;{mg}/{kg}$ BW TPHP in the $\text{ApoE}^{- / -}$ mouse model ([Figure 7](#f7){ref-type="fig"}). These results collectively suggest that the TPHP-promoted atherosclerosis in the $\text{ApoE}^{- / -}$ mouse model is largely attributable to foam cell formation within the artery wall mediated by LXR pathways.

This work assessed the LXR-antagonist activity of TPHP *in vitro* and *in vivo* in an atherosclerosis mouse model and demonstrated that TPHP may pose an atherosclerosis risk in the mouse. Moreover, TPHP contributed only a part of the LXR-antagonist activity of indoor dust (see Table S4), leading us to suspect that indoor dust contains other chemicals with LXR-antagonist activity and that these chemicals also may heighten atherosclerosis risk. Ultimately, observations of atherosclerosis induced by chemical pollutants via LXR-signaling pathways in the present paper promote the understanding of the environmental chemicals that might also affect cardiovascular health in humans and illustrate that a comprehensive assessment of the LXR-inactivating potential should be an important part of chemical management to ensure the protection of human health.

Supplementary Material
======================

###### 

Click here for additional data file.

###### 

Click here for additional data file.

###### 

Click here for additional data file.

We thank X. Li and G. Li from Core Facilities, School of Life Sciences, Peking University for assistance with the ImageXpress® Micro XL and AKTA pure protein purification system. This study received financial support from the International S&T Cooperation Program of China (2016YFE0117800) and the National Natural Science Foundation of China (21737001, 21577001, and 41821005).

Supplemental Material is available online (<https://doi.org/10.1289/EHP5039>).

The authors declare they have no actual or potential competing financial interests.

**Note to readers with disabilities:** *EHP* strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in *EHP* articles may not conform to [508 standards](http://ehp.niehs.nih.gov/accessibility/) due to the complexity of the information being presented. If you need assistance accessing journal content, please contact <ehponline@niehs.nih.gov>. Our staff will work with you to assess and meet your accessibility needs within 3 working days.
