Levels of Blood Organophosphorus Flame Retardants andAssociation with Changes in Human Sphingolipid HomeostasisFanrong Zhao,† Yi Wan,† Haoqi Zhao,† Wenxin Hu,† Di Mu,† Thomas F. Webster,‡ and Jianying Hu*,†

†Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China‡Department of Environmental Health, Boston University School of Public Health, Boston, Massachusetts 02118, United States

*S Supporting Information

ABSTRACT: While a recent toxicological study has shown that organophosphorus flameretardants (OPFRs) may disrupt sphingolipid homeostasis, epidemiologic evidence is currentlylacking. In this study, a total of 257 participants were recruited from Shenzhen, China. ElevenOPFRs were for the first time simultaneously determined in the human blood samples byultraperformance liquid chromatography and tandem mass spectrometry. Six OPFRs, tributylphosphate (TNBP), 2-ethylhexyl diphenyl phosphate (EHDPP), tris(2-chloroisopropyl)phosphate (TCIPP), tris(2-butoxyethyl) phosphate (TBOEP), triethyl phosphate (TEP),and TPHP, were detectable in at least 90% of participants, with median concentrations of 37.8,1.22, 0.71, 0.54, 0.49, and 0.43 ng/mL, respectively. Sphingomyelin (SM) levels in the highestquartile of EHDPP, TPHP, TNBP, TBOEP, TEP, and TCIPP were 45.3% [95% confidenceinterval; 38.1%, 53.0%], 51.9% (45.5%, 58.6%), 153.6% (145.1%, 162.3%), 20.6% (14.5%,27.0%), 59.0% (52.1%, 66.2%), and 62.8% (55.2%, 70.6%) higher than those in the lowestquartile, respectively, after adjusting for covariates. Sphingosine 1-phosphate (S1P) levels in thehighest quartile of EHDPP, TPHP, and TNBP were 36% (−39%, −33%), 16% (−19%, −14%), and 36% (−38%, −33%) lowerthan those in the lowest quartile, respectively. A similar pattern emerged when exposures were modeled continuously. We for thefirst time found the associations between OPFRs and changes in human sphingolipid homeostasis.

■ INTRODUCTION

Organophosphorus flame retardants (OPFRs) are used asplasticizers, antifoaming agents, and additives in floor polishes,glue, lubricants, food packaging, and hydraulic fluids.1,2 Theusage of OPFRs as alternative and replacement flame retardantshas increased significantly with the production phase-out andregulation of some brominated flame retardants, with annualglobal production currently reaching approximately 200000 t.3

OPFRs have been detected in various environmental media,including sediment, sewage water, drinking water, dust, indoorair, and biological samples.1,4−9 Humans may be exposed toOPFRs through various exposure pathways, including drinkingwater, food, indoor air, and indoor dust.1,2,10 Adverse healthissues, including skin irritation, carcinogenicity, dermatitis,neurotoxicity, and hemolytic, reproductive, and cardiac effects,have been observed in animals exposed to OPFRs,2,11−15 andexposure to triphenyl phosphate (TPHP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) in house dust has beenassociated with hormone levels and semen quality in men.15

The cardiotoxicity of OPFRs is of concern. Increased leftventricular wall thickening, which is suggestive of poorcardiovascular performance, has been observed in male ratsexposed to Firemaster 550 (FM 550) containing TPHP as amajor chemical.14 The impacts of some OPFRs on cardiaclooping and function have also been observed in zebrafishduring embryogenesis.16 In cardiovascular functions, sphingo-lipid homeostasis is of vital importance, because sphingolipidsand biosynthetic intermediates, including sphingomyelin (SM),

ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (S1P), play essential roles as both structuralcomponents of cell membranes and signaling molecules thatregulate cardiac development and barrier function of thevasculature.17 Sphingolipid levels are tightly controlled by themetabolic interplay of the de novo and recycling pathways(Figure S1). In particular, the recycling metabolic pathway ofsphingolipids, including SM, Cer, Sph, and S1P, is thedominant pathway for regulating the homeostasis ofsphingolipid in most tissues,18,19 and any imbalance can causestress to the cell and lead to cardiovascular disease.19 A growingbody of animal and epidemiological evidence has shown that anelevated SM plasma level is associated with atherosclerosis, andsphingolipid homeostasis disorders are implicated in thepathogenesis of atherosclerosis.19−21 A recent study assessedsphingolipid homeostasis in mice exposed to TPHP, in whichsphingolipid homeostasis was disrupted as characterized bysignificant increases in the levels of SM and decreases in thelevels of its metabolite, Cer, and precursor, sphinganine.22

However, to the best of our knowledge, there have been nostudies of the association of OPFRs with the disruption ofsphingolipid homeostasis in humans.

Received: May 18, 2016Revised: July 15, 2016Accepted: July 19, 2016Published: July 19, 2016

Article

pubs.acs.org/est

© 2016 American Chemical Society 8896 DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

While various OPFRs have been detected in milk, adiposetissue, and seminal fluid and their metabolites have beendetected in human urine,1,9,23−26 only one paper made anattempt to assess nine OPFRs in human plasma, and onlyTPHP was detected when the authors developed a gaschromatography−nitrogen phosphorus detector (GC−NPD)method.9 In this paper, 14 OPFRs were analyzed in 257 humanblood samples using a sensitive ultraperformance liquidchromatography and tandem mass spectrometry (UPLC−ESI-MS/MS) method. The associations between OPFRexposure levels and changes in sphingolipid levels (SM, Cer,Sph, and S1P) were assessed to explore the potential impacts ofOPFRs on human sphingolipid homeostasis.

■ MATERIALS AND METHODSStudy Population and Blood Collection. A total of 327

residents between 20 and 50 years of age were recruited fromthe general population in Shenzhen, China, in November 2012.The study was approved by the Human Ethics Committee ofPeking University (IRB00001052-12058). Participants came toa mobile center for a physical examination and to provide bloodand urine samples. All participants signed informed consentwhen they were enrolled. Volunteer participants were asked tofill out interview questionnaires by trained interviewers. Allsubjects were asked to fast for 10 h overnight before collectionof the fasting blood sample. Blood samples were collected intoheparinized brown glass bottles (CNW Technologies GmbH)and stored at −80 °C prior to extraction.We collected data on potential confounding variables

through questionnaires. Our models included a number ofcovariates that are important predictors of cardiovasculardisease (CVD): personal characteristics [age, gender, andbody mass index (BMI, calculated as weight in kilogramsdivided by height in square meters)], socioeconomic status(SES, household income mainly considered), and lifestylehabits (dietary structure, alcohol intake, and tobacco use). Ofthe 327 participants, we obtained 296 blood samples withmatching questionnaires (Figure S2).Materials. Purities of all analytical standards used in this

study were ≥95%. Standards tris(2-chloroethyl) phosphate(TCEP), tripropyl phosphate (TPrP), triisopropyl phosphate(TiPP), tris(2-chloroisopropyl) phosphate (TCIPP), tributylphosphate (TNBP), and tricresyl phosphate (TMPP) werepurchased from Sigma-Aldrich (St. Louis, MO). Triethylphosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate(TDCIPP), tris(2-butoxyethyl) phosphate (TBOEP), 2-ethyl-hexyl diphenyl phosphate (EHDPP), and tris(2-ethylhexyl)phosphate (TEHP) were purchased from TCI Corp. (Tokyo,Japan). Tris(2,3-dibromopropyl) phosphate (TDBPP) wassupplied by AccuStandard Inc. (New Haven, CT). BisphenolA bis(diphenyl phosphate) (BPA-BDPP) was purchased fromToronto Research Chemicals Inc. (Toronto, ON). SM, Cer,Sph, and S1P were purchased from Avanti Polar Lipids(Alabaster, AL). The stable isotope-labeled standards, includingtriethyl-d15 phosphate (TEP-d15), tri-n-propyl-d21 phosphate(TPrP-d21), and triphenyl-d15 phosphate (TPHP-d15), weresupplied by C/D/N Isotopes Inc. (Pointe-Claire, QC). Tri-n-butyl-d27 phosphate (TNBP-d27) was purchased from Cam-bridge Isotope Laboratories Inc. (Tewksbury, MA). Tris(2-chloroethyl)-d12 phosphate (TCEP-d12) and tris(1,3-dichloro-2-propyl)-d15 phosphate (TDCIPP-d15) were obtained fromToronto Research Chemicals Inc. Tri-p-cresyl-d21 phosphate(TMPP-d21) and tris(2-ethylhexyl)-d51 phosphate (TEHP-d51)

were supplied by Hayashi Pure Chemical Industries, Ltd.(Osaka, Japan). Internal standards SM-d31, Cer-d31, Sph-d7, andS1P-d7 were purchased from Avanti Polar Lipids. Solvents,including n-hexane, ethyl acetate, and methanol (MeOH), wereof pesticide residue grade and obtained from Fisher Chemicals.Acetone and dichloromethane (pesticide residue grade) werepurchased from Mallinckrodt Baker Inc. (Phillipsburg, NJ).Formic acid (HPLC grade) was from Dikma Technologies Inc.Sep-Pak Silica (3 cm3, 200 mg) and Sep-Pak C18 (3 cm3, 200mg) solid phase extraction (SPE) cartridges were purchasedfrom Waters (Milford, MA). Lithium-heparin blood collectiontubes were from Corning Inc. (Tewksbury, MA). Ultrapurewater was prepared using a Milli-Q Synthesis water purificationsystem (Millipore, Bedford, MA). A full list of the 14 targetcompounds, along with their full chemical names andstructures, can be found in Figure S3.

Analysis of OPFRs. Blood samples were thawed on ice andprepared immediately for analysis. Each whole blood sample(0.4 mL) was transferred into an 8 mL glass centrifuge tube,and 20 μL of an internal standard solution (10 ng/mL for eachsurrogate) was added. Ethyl acetate (2 mL) was added to thesample, which was shaken for 20 min on an orbital shaker andcentrifuged at 4000 rpm for 10 min, and the ethyl acetate layerwas then transferred to a clean glass bottle. Extraction from theresidue was repeated twice, and the organic layers werecombined, concentrated to near dryness under a gentle streamof nitrogen, and redissolved in 500 μL of n-hexane. Theconcentrated extract was loaded on a silica cartridgepreconditioned with 6 mL of a hexane/acetone solvent [1:1(v/v)], 3 mL of a hexane/dichloromethane solvent [7:3 (v/v)],and 3 mL of hexane. After the cartridge was rinsed with 3 mL ofhexane and 3 mL of a hexane/dichloromethane solvent [7:3 (v/v)], the target analytes were eluted with 3 mL of an n-hexane/acetone solvent [1:1 (v/v)]. The extracts were then dried andredissolved in 2 mL of methanol to pass through a C18-SPEcartridge preconditioned with 6 mL of methanol. The filtratewas collected and evaporated to dryness under a gentle streamof nitrogen and reconstituted with 100 μL of methanol forUPLC−MS/MS analysis.Analysis of OPFRs was performed using a Waters Acquity

UPLC system. All OPFRs were separated using a WatersAcquity UPLC BEH C8 column (2.1 mm × 100 mm × 1.8μm) preceded by a Waters Acquity UPLC BEH C18 guardcolumn (2.1 mm × 50 mm × 1.7 μm). The column wasmaintained at 40 °C, with a flow rate of 0.2 mL/min and aninjection volume of 5 μL. Methanol (A) and ultrapure watercontaining 0.1% (v/v) formic acid (B) were used as the mobilephases. Detailed information about the UPLC gradientconditions is shown in the Supporting Information.Mass spectrometry was performed using a Waters Micromass

Quattro Premier XE triple-quadrupole instrument detectorequipped with an electrospray ionization source (Micromass,Manchester, U.K.) in positive ion mode. The optimizedparameters were as follows: source temperature, 110 °C;desolvation temperature, 350 °C; capillary voltage, 3.50 kV;desolvation gas flow, 800 L/h; cone gas flow, 50 L/h; andmultiplier, 650 V. Finally, MS/MS data were acquired inmultiple-reaction monitoring (MRM) mode, and time-segmented scanning in four functions was used on the basisof the chromatographic separation of target compounds tomaximize detection sensitivity. The MS/MS parameters for theanalytes, including their precursors and product ions, conevoltage, and collision energy, are summarized in Table S1.

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8897

All analytical procedures were checked for accuracy,precision, reproducibility, linearity, blank contamination, matrixspikes, method limits of detection (LODs), and limits ofquantification (LOQs). Matrix-spiked recoveries of individualOPFRs through the analytical procedure were estimated byspiking the target analytes at 5, 50, and 100 ng/mL for TNBP,1, 10, and 20 ng/mL for EHDPP, TCEP, and TCIPP, 0.25, 1,and 10 ng/mL for TEP, and 0.1, 1, and 10 ng/mL for theothers into sample matrices (n = 6). The low concentrations ofspiked analytes were similar to those in samples. The methodrecoveries of all target analytes were calculated by subtractingbackground concentrations in nonspiked samples from spikedsamples, and the recoveries ranged from 74% to 98% (with aRSD of 3−15%), 73% to 101% (RSD of 2−7%), and 73% to99% (RSD of 2−11%) for the low, medium, and highconcentrations, respectively. The relative standard deviation(RSD) was used to evaluate precision. The interday precisionswere calculated on the basis of the means of six spiked samplesat three different levels over 5 days, and the interday precisionsof all substances were within 15%. To prevent possiblespecimen contamination, only pretreated glassware (500 °C,6 h) was used throughout the study, and aluminized paper wasused in all plastic seals to minimize possible contamination ofthe samples during sampling, storage, transport, and extraction.The SPE cartridges, silica, and C18 were prerinsed with an n-hexane/acetone solvent [1:1 (v/v)] and MeOH prior to use tominimize the contamination of the SPE procedure. For eachbatch of 20 samples analyzed, two procedural blanks wereprocessed. Procedural blanks were prepared by substitution of0.4 mL of Milli-Q water for blood, followed by passage throughthe entire analytical procedure. TEP, TCEP, TCIPP, TPHP,TDCIPP, TNBP, TMPP, TBOEP, EHDPP, BPA-BDPP, andTEHP were detected in the procedural blanks at concentrationsof 0.076 ± 0.010, 0.106 ± 0.031, 0.53 ± 0.062, 0.084 ± 0.007,0.052 ± 0.007, 0.64 ± 0.17, 0.082 ± 0.006, 0.012 ± 0.005,0.024 ± 0.004, 0.018 ± 0.002, and 0.016 ± 0.002 ng/mL,respectively. Field blanks (n = 6) were performed bytransferring 8 mL of Milli-Q water (with the same vacuumtubes used for collecting blood samples) into samplingcontainers, storing them, and processing them as samples.The concentrations of target compounds in field blanks werealmost equal to those in procedural blanks. Calibration curvesof standards of target analytes were calculated with aconcentration series of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and100 ng/mL, except for TNBP and TCEP (0.1, 0.5, 1, 5, 10, 50,100, 500, and 1000 ng/mL). All calibration curves providedadequate linearity (r2 > 0.995), and the signal-to-noise ratios forthe lowest concentration on calibration curves of OPFRs were13−98. Concentrations of TCEP and TNBP in two sampleswere reanalyzed after diluting the extracts 4−10-fold due to theextremely high concentrations.Identification of the target analytes was accomplished by

comparing the retention time (within 2%) and ratio (within20%) of the two selected precursor ion-produced iontransitions with those of the standards. Quantification wasaccomplished using the MRM transitions. To automaticallycorrect for the losses of analytes during extraction or samplepreparation and to compensate for variations in instrumentalresponse from injection to injection, quantification of theanalytes was achieved using an internal standard method withcalibration against standard solutions. TEP-d15 was used as thesurrogate standard for TEP, TCEP-d12 for TCEP and TCIPP,TDCIPP-d15 for TDCIPP and TDBPP, TPrP-d21 for TiPP and

TPrP, TPHP-d15 for TPHP and EHDPP, TNBP-d27 for TNBP,TMPP-d21 for TMPP, and TEHP-d51 for TBOEP, BPA-BDPP,and TEHP. For chemicals with detectable blank contamination,the LODs and LOQs were calculated as 3 and 10 times thestandard deviations of procedural blanks, respectively, and finalconcentrations were calculated by the initial concentrationssubtracting the blank values. For TiPP, TPrP, and TDBPP,which were not detected in the blanks, LODs and LOQs werecalculated on the basis of signal-to-noise ratios of 3 and 10 inmatrix-spiked samples, respectively. The LODs and LOQs were0.004−0.52 and 0.02−1.73 ng/mL, respectively (Table S2),which are significantly lower than the LODs of OPFRs inhuman plasma (0.2−1.8 ng/mL) published in a previouspaper,27 showing high sensitivity using the developed method.

Analysis of Sphingolipids. Sphingolipids were analyzedby UPLC−MS/MS using the method previously reported withminor modifications.16,17 Briefly, after blood samples (100 μLfor each test) were loaded into a 1.5 mL centrifuge tube,MeOH (880 μL) and 20 μL of an internal standard solution(2000 ng/mL for each) were added to the same tube, whichwas then shaken vigorously for 1 min. The concentrated extract(1 mL) was stored at −20 °C for 24 h, and most of theprecipitated or suspended lipids were easily removed byfiltration. After centrifugation at 12000 rpm for 15 min, thesupernatant was collected and analysis of shingolipid wasperformed with a Waters Acquity UPLC system. A WatersAcquity UPLC BEH phenyl column (2.1 mm × 100 mm × 1.8μm) was used for chromatographic separation. Methanol (A)and ultrapure water containing 0.5% formic acid (B) were usedas the mobile phases. The gradient started at 10% A and thenincreased linearly to 60% in 6 min and to 100% at 6 min andheld for 2 min, followed by a decrease to the initial conditionsof 10% A and held for 2 min to allow for equilibration. Thecolumn was maintained at 40 °C, with a flow rate of 0.3 mL/min and an injection volume of 5 μL. Quantification ofsphingolipids was accomplished using the MRM transitions.The optimized MS/MS parameters for the analyses, includingprecursor and product ions, cone voltage, and collision energy,are listed in Table S3.Mean recoveries of SM-d31, Cer-d3, Sph-d7, and S1P-d7 were

88 ± 9%, 91 ± 8%, 106 ± 4%, and 103 ± 6%, respectively, byspiking internal standards into blood samples at low, median,and high levels (100, 500, and 1000 ng/mL for SM-d31; 1000,5000, and 10000 ng/mL for Cer-d31; 200, 1000, and 2000 ng/mL for Sph-d7; and 2000, 10000, and 20000 ng/mL for S1P-d7)(n = 6 for each level). The LOQs for internal standards of SM,Cer, Sph, and S1P in matrix-spiked samples were estimated tobe 7.3, 40, 12, and 25 ng/mL, respectively, on the basis of thepeak-to-peak noise of the baseline and on the basis of a minimalsignal-to-noise value of 10. Calibration curves of standards oftarget analytes were calculated with a concentration series of 50,100, 500, 1000, 2000, 5000, 10000, and 50000 ng/mL. Allcalibration curves showed strong linearity (correlation co-efficients of >0.99) with good precision (RSD of ≤5%).

Statistical Analysis. Basic descriptive statistics werederived for population characteristics, blood sphingolipid levels,and blood OPFR levels. Whole blood samples with non-detectable OPFR concentrations were assigned a value as theLOQ divided by the square root of 2. Spearman correlationcoefficients were calculated to assess bivariate relationshipsbetween OPFR concentrations and sphingolipid levels.Sphingolipid concentrations were transformed to the naturallog for statistical analyses as they were log-normally distributed.

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8898

For OPFRs detected at >90%, we used multivariate linearregression to assess associations between OPFR concentrationsand sphingolipid levels, while adjusting for all the relevantcovariates (age, gender, BMI, SES, dietary structure, alcoholintake, and tobacco use). Quartile variables were constructedfor individual OPFRs in multivariate linear regression models.We presented effect estimates for each quartile compared withthe first quartile and their corresponding 95% confidenceintervals (CIs). Tests for trends in quartile analyses wereperformed by treating the OPFR category as a linear predictorin the models. To further confirm the associations betweensphingolipid and OPFR concentrations, OPFRs were alsomodeled as continuous predictors.All analyses excluded those with a history of cardiovascular

events (coronary event or stroke) or heart failure. In addition,subjects who reported current use of medications or who weremissing this variable were also excluded. The details of peoplein the exclusion group are shown in Figure S2.Data analysis was performed using SPSS, version 22.0 (IBM,

Corp.). Models were adjusted for relevant covariates. For alltests, an α level of 0.05 was chosen; p values of <0.05 wereconsidered statistically significant in all statistical models.

■ RESULTS AND DISCUSSIONPopulation Characteristics. Of the 296 participants, we

excluded 9% (n = 27) who reported recent medication use, 2%(n = 6) with a history of cardiovascular events, 2% (n = 6) whowere missing this variable, and 1% (n = 2) with highconcentrations of TCEP and TNBP that may due to exposurecaused by their occupation. Thus, the final study populationconsisted of 255 people, including 154 men and 101 women(Figure S2). Participants were all of Chinese origin (participantand both parents born in China), and the mean age was 29.7 ±5.0 years (range of 20−50 years). Most participants had a BMIwithin the range of 15.6−31.6 kg/m2 (median of 21.5 kg/m2).

Of the participants, 73% were nonsmokers and 87% had a lightweekly intake of alcohol. Information about the distribution ofcovariates is listed in Table 1, and sphingolipid levels are listedin Table 2.

Concentrations of Organophosphorus Flame Retard-ants (OPFRs). In this study, we analyzed 14 OPFRs in humanblood using the LLE−SPE−UPLC−MS/MS method, and all14 OPFRs except for TiPP, TPrP, and TDBPP were detected.The typical MRM chromatograms of detected targetcompounds are shown in Figure 1. TNBP, EHDPP, TCIPP,TBOEP, TEP, and TPHP were detectable in >90% ofparticipants; TEHP, TMPP, TCEP, TDCIPP, and BPA-BDPP were detected in 71.6%, 66.1%, 63.0%, 47.1%, and41.2% of participants, respectively (Table 3). Among the 11detected OPFRs, TNBP was the most abundant with a medianvalue of 37.8 ng/mL (<LOD − 758 ng/mL), which was ∼30-fold higher than that of EHDPP, the OPFR with the secondhighest level with a median concentration of 1.22 ng/mL. Arelatively high concentration of TNBP (758 ng/mL) wasdetected in the blood sample from one person who wasworking in a shoemaking factory. Because OPFRs are used intextiles, rubber, and glues,2 occupation exposure may lead tosuch high concentrations. The third highest level was that ofTCIPP with a median concentration of 0.71 ng/mL, followedby those of TBOEP (0.54 ng/mL), TEP (0.49 ng/mL), andTPHP (0.43 ng/mL). Although TCEP had a relatively low

Table 1. Characteristics of the Final Study Population (n = 255)

characteristic mean ± SD median (25th, 75th) range

age (years) 29.7 ± 5.0 29 (26, 32) 20−50height (cm) 165.5 ± 7.5 166 (160, 171) 150−186weight (kg) 60.4 ± 12.1 60.0 (50.0, 69.0) 38.0−98.0BMI (kg/m2) 21.9 ± 3.1 21.5 (19.5, 23.9) 15.6−31.6

gender 154 men (60%) 101 women (40%)SES (<3000a Chinese yuan per month) 77 (30%)alcohol consumption (drinks per week, past month) 221 (87%) lighter drinkers (<2) 28 (11%) moderate drinker (2−4) 6 (2%) heavy drinkers (>5)smoking status 56 (22%) current 13 (5%) former 186 (73%) never

Dietary Structure

rice intake (g/meal) 50 100 150 200 ≥25027 (11%) 117 (46%) 80 (31%) 19 (7%) 12 (5%)

meat (meals per week) 0 1 2 3 4 5 6 ≥77% 55% 18% 8% 4% 2% 4% 2%

seafood (meals per week) 0 1 2 3 4 5 6 73% 13% 9% 10% 9% 13% 6% 9%8 9 10 11 12 13 14 154% 0% 11% 0% 4% 0% 9% 1%

egg intake (per week) 0 1 2 3 4 5 6 710% 25% 26% 13% 7% 8% 3% 2%8 ≥92% 4%

milk intake (L/week) 0 0−1 >146% 41% 13%

aA household income of <3000 RMB per month.

Table 2. Sphingolipid Levels of the Study Population

sphingolipid mean ± SD median (25th, 75th) range

SM (ng/mL) 414 ± 185 413 (260, 527) 186.7−1114Cer (ng/mL) 1507 ± 1073 1392 (921, 1637) 707−8552Sph (ng/mL) 186 ± 65 176 (145, 202) 59.7−515S1P (ng/mL) 2520 ± 875 2427 (1862, 3053) 583−5825

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8899

detection frequency and a low median concentration below theLOQ, its highest concentration reached 2438 ng/mL. Such ahigh exposure level of TCEP for a person who was a worker inthe storage of a hotel may be related to his occupation. Onlyone paper has identified nine OPFRs (TEP, TPrP, TNBP,TCEP, TCIPP, TPHP, TMPP, TBOEP, and TEHP) in humanplasma, but only TPHP was detected at relatively highconcentration of 0.13−0.15 μg/g, much higher than thatdetected in the work presented here.9 This is possibly due tothe relatively low sensitivity of the analytical method, of whichLODs were 0.07−4 ng/g, much higher than those (0.007−0.52ng/mL) in the work presented here.

Because of the different structures (alkyl chains, halogenatedalkyl chains, and aromatic functions), applications, and sources,the Spearman correlation coefficients among individual OPFRshad different ranges (Table S4). Three chlorinated OPFRs(TCEP, TCIPP, and TDCIPP) were moderately correlated toeach other (Spearman correlation coefficients ranged from0.289 to 0.475). Eight non-halogen OPFRs except for TEP andBPA-BDPP were moderately correlated with one another.TEHP and TMPP were the most strongly correlated, with aSpearman correlation coefficient of 0.645, and EHDPP andTBOEP were the least correlated (Spearman correlationcoefficient of 0.170). TPHP was positively correlated with allother detected OPFRs (Spearman correlation coefficientsranged from 0.175 to 0.469). Such correlations among theOPFRs in blood samples were likely related to externalexposure via various routes,1,7,28,29 while the pharmacokineticsof OPFRs would also affect the concentration of OPFRs inblood considering their obviously different physicochemicalproperties, as exemplified by wide range of logKow values from0.80 for TEP to 9.49 for TEHP.

Associations of OPFRs with Sphingolipids. Multivariatelinear regression adjusted for relevant confounders (age,gender, BMI, SES, dietary structure, alcohol intake, andtobacco use) by treating OPFRs as categorical predictors wasused to assess the associations between the concentrations ofOPFRs and sphingolipid levels. The results, for the six OPFRs(TNBP, EHDPP, TCIPP, TBOEP, TEP, and TPHP) detectedat >90%, are shown in Figure 2. While no strong trendsemerged in the Cer analyses for all six OPFRs (Figure 2A),their concentrations of six OPFRs were all significantlyassociated with the increase in SM levels. The SM levels inthe highest quartile of EHDPP, TPHP, TNBP, TBOEP, TEP,and TCIPP were 45.3% (95% CI, 38.1%, 53.0%; p < 0.001),51.9% (95% CI, 45.5%, 58.6%; p < 0.001), 153.6% (95% CI,145.1%, 162.3%; p < 0.001), 20.6% (95% CI, 14.5%, 27.0%; p <0.001), 59.0% (95% CI, 52.1%, 66.2%; p < 0.001), and 62.8%(95% CI, 55.2%, 70.6%; p < 0.001) higher than those of thelowest quartile, respectively (Figure S4B). SM appeared toincrease linearly across the quartiles of OPFR exposure forEHDPP (p trend of <0.001), TPHP (p trend of 0.001), TNBP(p trend of <0.001), TBOEP (p trend of 0.028), TEP (p trendof <0.001), and TCIPP (p trend of <0.001) (Figure 2B). Suchpositive association between OPFRs and the SM level has beenalso found in rats exposed to TPHP.22 The S1P level in thehighest quartile of EHDPP was 36% lower (95% CI, −39%,

Figure 1. UPLC−MS/MS MRM chromatograms of the ions selectedfor identification of analytes detected in blood samples.

Table 3. Concentrationsa (nanograms per milliliter) of Analytes in Whole Blood Samples (n = 257)

analyte detection frequency (%) 5% 25% median 75% 95% range

TEP 96.1 0.10 0.34 0.49 0.80 2.60 ND−4.59TCEP 63.0 ND ND <LOQ 0.32 2.76 ND−2438TCIPP 90.3 ND <LOQ 0.71 1.15 1.85 ND−21.61TPHP 98.4 0.18 0.34 0.43 0.53 0.72 ND−1.21TDCIPP 47.1 ND ND ND 0.10 0.31 ND−3.41TNBP 99.6 1.87 5.33 37.8 46.3 62.0 ND−758TMPP 66.1 ND ND 0.09 0.13 0.20 ND−0.32TBOEP 98.1 0.24 0.38 0.54 0.72 1.61 ND−16.0EHDPP 100.0 0.78 1.00 1.22 1.44 1.95 0.60−3.13BPA-BDPP 41.2 ND ND ND 0.02 0.07 ND−0.23TEHP 71.6 ND ND 0.04 0.05 0.10 ND−0.36

aReported values were calculated by subtracting the mean blanks from the initial concentrations. TiPP, TPrP, and TDBPP were not detected in anysamples.

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8900

−33%; p < 0.001) than that in the lowest quartile, 16% lower(95% CI, −19%, −14%; p < 0.001) than that in the highestTPHP quartile, and 36% lower (95% CI, −38%, −33%; p <0.001) than that in the highest TNBP quartile (Figure S4C).After adjustment for confounders, concentrations of EHDPP (ptrend of <0.001), TPHP (p trend of <0.005), and TNBP (ptrend of <0.001) were significantly associated with S1P levels inthe categorical models (Figure 2C). We found a negativeassociation between Sph levels and EHDPP (p trend of 0.031)and a positive association between Sph levels and TEP (p trendof 0.006) and TCIPP (p trend of 0.005) in the categoricalmodel analyses (Figure 2D). The Sph level in the highestquartile of EHDPP was 11.6% lower (95% CI, −12.7%,−10.5%; p < 0.001) than that in the lowest quartile, and therewas a 16.5% (95% CI, 14.9%, 18.2%; p < 0.001) increase forTEP and a 25.7% (95% CI, 23.6%, 27.8%; p < 0.001) increasefor TCIPP (Figure S4D).To further confirm the associations between sphingolipid

and OPFR concentrations, OPFRs were also modeled ascontinuous predictors as exemplified by EHDPP in Figure 3.After all confounders had been adjusted, the change insphingolipid level per 100 pg/mL increase of a OPFR wasestimated. Large changes of both SM and S1P levels wereobserved in EHDPP and TPHP. The increases in SM per 100pg/mL increase of EHDPP and TPHP were estimated to be3.5% (95% CI, 1.9%, 5.2%) and 4.9% (95% CI, 1.0%, 8.8%),and the decreases in S1P level per 100 pg/mL increase ofEHDPP and TPHP were 3.0% (95% CI, −4.1%, −1.9%) and3.8% (95% CI, −6.5%, −1.2%), respectively (Table 4).Studies using rat and human liver microsomes suggest that

OPFRs are metabolized rapidly to diesters and othermetabolites with short half-lives of the parent compounds inblood, and of their metabolites in urine.10,25 A single bloodsample was measured in this study, which could lead to

considerable day-to-day variation in blood. However, a singlehuman urine sample could moderately reflect former exposureto OPFRs over a relatively long period of time as observed inthe reliability of urinary concentrations of BDCPP and DPhPover 3 months.10 In addition, the relatively large number ofparticipants used for investigating the association betweenOPFR exposure and changes in sphingolipid homeostasis mayhelp detect statistically significant associations.OPFRs or metabolites might interact with enzymes

responsible for synthesizing and degrading sphingolipids,therefore leading to disruption of sphingolipid homeostasisconsidering the relatively similar structures between OPFRs (or

Figure 2. Levels of blood sphingolipids by quartiles of OPFRs. Quartile 1 (Q1) is the lowest and Q4 the highest. All models adjusted for BMI,gender, age, socioeconomic status (SES, household income mainly considered), and lifestyle habits, including dietary structure, alcohol intake, andtobacco use. p trends of sphingolipid across the quartiles of OPFR are presented.

Figure 3. Scatter plot of ln-transformed sphingolipid levels andEHDPP concentrations in human blood. The regression results forother OPFRs are listed in Table S6.

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8901

metabolites) and the sphingolipids with phosphate (SM andS1P). There is evidence that TPHP could inhibit carboxylester-ase and disrupt hepatic lipid metabolism, which resulted in anincrease in the level of SM.21 This study for the first timeprovided epidemiologic evidence of the association betweenOPFR concentrations in blood and changes in sphingolipidhomeostasis. The levels of sphingolipids are tightly regulated toensure sphingolipid homeostasis, and perturbations tosphingolipid metabolism cause cellular stress and organismalpathology.17 In animal and epidemiological studies, elevatedlevels of SM are thought to play a role in increasing the extentof atherosclerosis in veins.19,30,31 S1P also plays a major role incardiovascular physiology, because it mediates cardioprotectionfrom ischemia/reperfusion injury and is involved in remodeling,proliferation, and differentiation of cardiac fibroblasts.32,33

Thus, sustained exposure to OPFRs may pose a human riskof atherosclerosis via the potential disruption of sphingolipidhomeostasis. While toxicology data of OPFRs relevant to theend points of atherosclerosis are limited, increased leftventricular wall thickening, which is suggestive of poorcardiovascular performance, has been observed in male ratsexposed to Firemaster 550 (FM 550) containing TPHP as amajor chemical.14 The impacts of TPHP on cardiac loopingand function have also been observed in zebrafish duringembryogenesis.16 The finding of this study is a significant steptoward improving the existing data on human health risksrelated to OPFRs, while the associations identified in this studycannot be thought to be causal as a result of the limitation ofthe inherent nature of cross-sectional design. Further study ofthe cause and effect between human OPFR exposure and healthoutcomes is needed.Overall, we for the first time simultaneously detected 11

OPFRs in blood in a large nonoccupational population andfound associations between concentrations of OPFRs and thelevels of sphingolipids in human blood. This study providedclues for future epidemiological and toxicological research. Inparticular, additional studies are needed to shed light on themechanism by which OPFRs affect sphingolipid homeostasisand the association of OPFR exposure with atherosclerosis inhumans.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.est.6b02474.

Information about UPLC gradient conditions; calcu-lations of the percentage change (%) in sphingolipidlevels in Q4 relative to Q1; calculations of regressioncoefficients of the percentage change (%) in sphingolipidlevels per 100 pg/mL increase in OPFRs; optimized

instrumental and MRM conditions of target analytes andsurrogate standards; optimized instrumental and MRMconditions of sphingolipids; limits of detection (LODs),limits of quantification (LOQs), and recoveries of targetanalytes in human blood; Spearman correlations betweenblood OPFR concentrations (n = 255); regressioncoefficients of OPFRs in the unadjusted continuouslinear models; regression coefficients (95% CIs) ofOPFRs in the adjusted continuous multivariate linearmodels; the sphingolipid synthetic pathway; determi-nation of final sample sizes for analyses and the numberof participants who met the different exclusion criteriaand did not exhibit covariates; structures of the 14 targetcompounds along with their full chemical names andabbreviations; and percent changes in sphingolipid levelswith increasing quartile of OPFR exposure (PDF)

■ AUTHOR INFORMATIONCorresponding Author*College of Urban and Environmental Sciences, PekingUniversity, Beijing 100871, China. Telephone and fax: 86-10-62765520. E-mail: hujy@urban.pku.edu.cn.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support from the National Natural ScienceFoundation of China (21577001), and the National SpecialFunding Project for Water Pollution Control and Managementof China (2014ZX07405001) is gratefully acknowledged.

■ REFERENCES(1) Sundkvist, A. M.; Olofsson, U.; Haglund, P. Organophosphorusflame retardants and plasticizers in marine and fresh water biota and inhuman milk. J. Environ. Monit. 2010, 12, 943−951.(2) van der Veen, I.; de Boer, J. Phosphorus flame retardants:properties, production, environmental occurrence, toxicity andanalysis. Chemosphere 2012, 88, 1119−1153.(3) Greaves, A. K.; Letcher, R. J. Comparative body compartmentcomposition and in ovo transfer of organophosphate flame retardantsin north American Great Lakes herring gulls. Environ. Sci. Technol.2014, 48, 7942−7950.(4) Andresen, J. A.; Grundmann, A.; Bester, K. Organophosphorusflame retardants and plasticisers in surface waters. Sci. Total Environ.2004, 332, 155−166.(5) Marklund, A.; Andersson, B.; Haglund, P. Organophosphorusflame retardants and plasticizers in air from various indoorenvironments. J. Environ. Monit. 2005, 7, 814−819.(6) Marklund, A.; Andersson, B.; Haglund, P. Screening oforganophosphorus compounds and their distribution in various indoorenvironments. Chemosphere 2003, 53, 1137−1146.

Table 4. Percentage Change (95% CI)a in Sphingolipid Levels per 100 pg/mL Increase in OPFR Concentration

SMb Cerb Sphb S1Pb

EHDPP 3.5% (1.9%, 5.2%) 2.2% (−0.1%, 4.4%) −1.2% (−2.3%, −0.1%) −3.0% (−4.1%, −1.9%)TPHP 4.9% (1.0%, 8.8%) 2.5% (−2.7%, 7.7%) −2.8% (−5.4%, −0.3%) −3.8% (−6.5%, −1.2%)TNBP 0.09% (0.07%, 0.11%) 0.0% (−0.03%, 0.03%) 0.02% (0.00%, 0.03%) −0.02% (−0.04%, −0.01%)TBOEP 0.5% (−0.1%, 1.1%) 0.3% (−0.5%, 1.0%) −0.3% (−0.7%, 0.5%) −0.3% (−0.7%, 0.1%)TEP 1.9% (1.1%, 2.6%) −2.5% (−3.5%, −1.5%) 1.0% (0.5%, 1.5%) - 0.04% (−0.6%, 0.5%)TCIPP 0.5% (0.1%, 1.0%) −0.1% (−0.7%, 0.5%) 0.3% (0.01%, 0.6%) 0.02% (−0.3%, 0.3%)

aAll models are adjusted for age, gender, BMI, SES, and lifestyle habits, including dietary structure, alcohol intake, and tobacco use. The calculationsof back-transformed regression coefficients are given in the Supporting Information. bVariable ln-transformed in statistical analysis. We excludedvalues identified as influential points and outliers from the population.

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8902

(7) Van den Eede, N.; Dirtu, A. C.; Neels, H.; Covaci, A. Analyticaldevelopments and preliminary assessment of human exposure toorganophosphate flame retardants from indoor dust. Environ. Int.2011, 37, 454−461.(8) Rodriguez, I.; Calvo, F.; Quintana, J. B.; Rubi, E.; Rodil, R.; Cela,R. Suitability of solid-phase microextraction for the determination oforganophosphate flame retardants and plasticizers in water samples. J.Chromatogr. A 2006, 1108, 158−165.(9) Jonsson, O. B.; Dyremark, E.; Nilsson, U. L. Development of amicroporous membrane liquid-liquid extractor for organophosphateesters in human blood plasma: identification of triphenyl phosphateand octyl diphenyl phosphate in donor plasma. J. Chromatogr. BBiomed. Appl. 2001, 755, 157−164.(10) Meeker, J. D.; Stapleton, H. M. House dust concentrations oforganophosphate flame retardants in relation to hormone levels andsemen quality parameters. Environ. Health Perspect. 2009, 118, 318−323.(11) Matthews, H. B.; Eustis, S. L.; Haseman, J. Toxicity andcarcinogenicity of chronic exposure to tris(2-chloroethyl)phosphate.Fundam. Appl. Toxicol. 1993, 20, 477−485.(12) Camarasa, J. G.; Serra-Baldrich, E. Allergic contact dermatitisfrom triphenyl phosphate. Contact Dermatitis 1992, 26, 264−5.(13) Moller, A.; Xie, Z.; Caba, A.; Sturm, R.; Ebinghaus, R.Organophosphorus flame retardants and plasticizers in the atmosphereof the North Sea. Environ. Pollut. 2011, 159, 3660−3665.(14) Patisaul, H. B.; Roberts, S. C.; Mabrey, N.; Mc Caffrey, K. A.;Gear, R. B.; Braun, J.; Belcher, S. M.; Stapleton, H. M. Accumulationand endocrine disrupting effects of the flame retardant mixturefiremaster 550 in rats: an exploratory assessment. J. Biochem. Mol.Toxicol. 2013, 27, 124−136.(15) Meeker, J. D.; Cooper, E. M.; Stapleton, H. M.; Hauser, R.Urinary metabolites of organophosphate flame retardants: temporalvariability and correlations with house dust concentrations. Environ.Health Persp. 2013, 121, 580−585.(16) McGee, S. P.; Konstantinov, A.; Stapleton, H. M.; Volz, D. C.Aryl phosphate esters within a major pentaBDE replacement productinduce cardiotoxicity in developing zebrafish embryos: potential role ofthe aryl hydrocarbon receptor. Toxicol. Sci. 2013, 133, 144−156.(17) Breslow, D. K.; Weissman, J. S. Membranes in balance:mechanisms of sphingolipid homeostasis. Mol. Cell 2010, 40, 267−279.(18) Hait, N. C.; Allegood, J.; Maceyka, M.; Strub, G. M.; Harikumar,K. B.; Singh, S. K.; Luo, C.; Marmorstein, R.; Kordula, T.; Milstien, S.;Spiegel, S. Regulation of histone acetylation in the nucleus bysphingosine-1-phosphate. Science 2009, 325, 1254−7.(19) Hornemann, T.; Worgall, T. S. Sphingolipids and athero-sclerosis. Atherosclerosis 2013, 226, 16−28.(20) Samad, F.; Hester, K. D.; Yang, G.; Hannun, Y. A.; Bielawski, J.Altered adipose and plasma sphingolipid metabolism in obesity - Apotential mechanism for cardiovascular and metabolic risk. Diabetes2006, 55, 2579−2587.(21) Chatterjee, S.; Kolmakova, A.; Miller, M. The role of thephospholipid sphingomyelin in heart disease. Curr. Opin. Invest. Drugs2006, 7, 219−228.(22) Morris, P. J.; Medina-Cleghorn, D.; Heslin, A.; King, S. M.; Orr,J.; Mulvihill, M. M.; Krauss, R. M.; Nomura, D. K. Organophosphorusflame retardants inhibit specific liver carboxylesterases and causeserum hypertriglyceridemia. ACS Chem. Biol. 2014, 9, 1097−1103.(23) Lebel, G. L.; Williams, D. T. Determination of halogenatedcontaminants in human adipose-tissue. J. Assoc. Offic. Anal. Chem.1986, 69, 451−458.(24) Hudec, T.; Thean, J.; Kuehl, D.; Dougherty, R. C. Tris-(dichloropropyl)phosphate, a mutagenic flame-retardant - frequentoccurrence in human seminal plasma. Science 1981, 211, 951−952.(25) Cooper, E. M.; Covaci, A.; van Nuijs, A. L. N.; Webster, T. F.;Stapleton, H. M. Analysis of the flame retardant metabolites bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl phosphate(DPP) in urine using liquid chromatography−tandem massspectrometry. Anal. Bioanal. Chem. 2011, 401, 2123−2132.

(26) Van den Eede, N.; Neels, H.; Jorens, P. G.; Covaci, A. Analysisof organophosphate flame retardant diester metabolites in humanurine by liquid chromatography electrospray ionisation tandem massspectrometry. J. Chromatogr. A 2013, 1303, 48−53.(27) Amini, N.; Crescenzi, C. Feasibility of an on-line restrictedaccess material/liquid chromatography/tandem mass spectrometrymethod in the rapid and sensitive determination of organophosphorustriesters in human blood plasma. J. Chromatogr. B: Anal. Technol.Biomed. Life Sci. 2003, 795, 245−256.(28) Kim, J.-W.; Isobe, T.; Sudaryanto, A.; Malarvannan, G.; Chang,K.-H.; Muto, M.; Prudente, M.; Tanabe, S. Organophosphorus flameretardants in house dust from the Philippines: occurrence andassessment of human exposure. Environ. Sci. Pollut. Res. 2013, 20,812−822.(29) Li, J.; Yu, N.; Zhang, B.; Jin, L.; Li, M.; Hu, M.; Zhang, X.; Wei,S.; Yu, H. Occurrence of organophosphate flame retardants in drinkingwater from China. Water Res. 2014, 54, 53−61.(30) Taniguchi, M.; Okazaki, T. The role of sphingomyelin andsphingomyelin synthases in cell death, proliferation and migration-from cell and animal models to human disorders. Biochim. Biophys.Acta, Mol. Cell Biol. Lipids 2014, 1841, 692−703.(31) Nelson, J.; Jiang, X. C.; Tabas, I.; Tall, A.; Shea, S. Plasmasphingomyelin and subclinical atherosclerosis: Findings from themulti-ethnic study of atherosclerosis. Am. J. Epidemiol. 2006, 163,903−912.(32) Means, C. K.; Brown, J. H. Sphingosine-1-phosphate receptorsignalling in the heart. Cardiovasc. Res. 2008, 82, 193−200.(33) Mann, D. L. Sphingosine 1-phosphate as a therapeutic target inheart failure: more questions than answers. Circulation 2012, 125,2692−2694.

Environmental Science & Technology Article

DOI: 10.1021/acs.est.6b02474Environ. Sci. Technol. 2016, 50, 8896−8903

8903