Diester Metabolites Research Articles

Unravelling bioaccumulation, depletion and metabolism of organophosphate triesters in laying hens: Insight of in vivo biotransformation assisted by diester metabolites

Organophosphate triesters (tri-OPEs) threaten human health through dietary exposure, but little is known about their feed-to-food transfer and in vivo behavior in farm animals. Herein 135 laying hens were fed with contaminated feed (control group, low-level group and high-level group) to elucidate the bioaccumulation, distribution, and metabolism of the six most commonly reported tri-OPEs. The storage (breast muscle), metabolism and mobilization (liver and blood) and non-invasive (feather) tissues were collected. The exposure-increase (D1∼14) and depuration-decrease (D15∼42) trends indicated that feed exposure caused tri-OPE accumulation in animal tissues. Tissue-specific and moiety-specific behavior was observed for tri-OPEs. The highest transfer factor (TF) and transfer rate (TR) were observed in liver (TF: 14.8%∼82.3%; TR: 4.40%∼24.5%), followed by feather, breast muscle, and blood. Tris(2-chloroisopropyl) phosphate (TCIPP) had the longest half-life in feather (72.2 days), while triphenyl phosphate (TPhP) showed the shortest half-life in liver (0.41 days). Tri-OPEs’ major metabolites (organophosphate diesters, di-OPEs) were simultaneously studied, which exhibited dose-dependent and time-dependent variations following administration. In breast muscle, the inclusion of di-OPEs resulted in TF increases of 735%, 1108%, 798%, and 286% than considering TCIPP, tributyl phosphate, tris(2-butoxyethyl) phosphate and tris(2-ethylhexyl) phosphate alone. Feather was more of a proxy of birds’ long-term exposure to tri-OPEs, while short-term exposure was better reflected by di-OPEs. Both experimental and in silico modeling methods validated aryl-functional group facilitated the initial accumulation and metabolism of TPhP in the avian liver compared to other moiety-substituted tri-OPEs.

In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

Characterization of the phosphotriesterase capable of hydrolyzing aryl-organophosphate flame retardants.

A related group of phosphotriesters known as organophosphate flame retardants (OPFRs) has become emerging contaminants due to its worldwide use. The lack of an easily hydrolysable bond renders OPFRs inert to the well-known phosphotriesterases capable of hydrolyzing the neurotoxic organophosphates. An OPFRs phosphotriesterase gene stpte was cloned from plasmid pStJH of strain Sphingopyxis terrae subsp. terrae YC-JH3 and was heterologously expressed in Escherichia coli. The recombinant protein St-PTE was purified and analyzed.St-PTE showed the highest catalytic activity at pH 8.5 and 35°C.The optimal substrate for St-PTE is triphenyl phosphate, with kcat/Km of 5.03 × 106M-1s-1, two orders of magnitude higher than those of tricresyl phosphate (4.17 × 104M-1s-1), 2-ethylhexyl diphenyl phosphate (2.03 × 104M-1s-1) and resorcinol bis(diphenyl phosphate) (6.30 × 104M-1s-1).St-PTE could break the P-O bond of tri-esters and convert aryl-OPFRs into their corresponding di-ester metabolites, including polymers of resorcinol bis(diphenyl phosphate). Mediated by transposase, the gene of OPFRs phosphotriesterase could be transferred horizontally among closely related strains of Sphingomonas, Sphingobium and Sphingopyxis. KEY POINTS: • St-PTE from Sphingopyxis terrae subsp. terrae YC-JH3 could hydrolyze aryl-OPFRs. • Metabolites of RBDPP hydrolyzed by phosphotriesterase were identified. • St-PTE could hydrolyze the P-O cleavage of dimer and trimer of RBDPP. • Phosphotriesterase genes transfer among Sphingomonadaceae mediated by transposase.

Deposition, bioaccumulation and depletion of organophosphate triesters (tri-OPEs) and their organophosphate diester metabolites (di-OPEs) from feed to laying hens’ eggs

Although organophosphate triesters’ (tri-OPEs) large application and potential toxicity has aroused public concern of human dietary exposure, no study has evaluated their deposition, bioaccumulation and depletion in farm animals yet, limiting our understanding about their behavior and further regulation. In this study, we fed 135 laying hens (control group, low-level group (LLG) and high-level group (HLG)) with chlorinated alkyl-, alkyl- and aryl-tri-OPEs for 14 days and followed by a 28-day depuration period to investigate in ovo transfer. Feeding at environmentally-relevant levels, concentrations of tri-OPEs clearly increased during exposure period and decreased during the depuration period in eggs. Tri-OPEs were 5.95–10.8 times higher in HLG feed than LLG feed, but only 1.41–2.52 times higher in HLG eggs than LLG eggs. Half-lives of tri-OPEs in eggs were between 11.3 and 106 d and transfer rates were between 0.22% and 4.22%. As the major metabolites, organophosphate diesters (di-OPEs) were simultaneously studied. Taking di-OPEs into consideration would increase the transfer rate by 64.1%. Both tri-OPEs and di-OPEs mainly deposited in egg albumen. The molecular docking showed that strong combination between OPEs and egg albumen might be responsible for the maternal transfer. The average ratios of di-OPEs to tri-OPEs (Rdi/tri) were 16.7% and 67.2% in LLG and HLG albumen, and were 438% and 517% in LLG and HLG yolk, respectively. Chlorinated alkyl-di-OPEs showed higher in ovo transfer than other di-OPEs. Different distribution patterns of tri-OPEs and di-OPEs were controlled by combination to egg protein, egg formation, metabolic organs and congener-specific properties.

Sex-specific associations between maternal exposure to parabens, phenols and phthalates during pregnancy and birth size outcomes in offspring.

Current evidence on the effects of prenatal exposure to endocrine disrupting chemicals on birth size remains largely inconclusive. We aimed to investigate sex-specific associations between maternal exposure to parabens, phenols and phthalates during pregnancy and birth weight, length and head/abdominal circumferences. We performed a prospective study of 88 pregnant women who underwent amniocentesis in the period 2012 to 2014. Maternal urine samples were collected during pregnancy in weeks 12 to 36 (median: 18 weeks). The concentrations of parabens, phenols and individual phthalate diester metabolites were analyzed by isotope-diluted liquid chromatography-tandem mass spectrometry and osmolality adjusted. Linear regression models estimated the associations between urinary levels of selected compounds (tertile(T2-T3)medium/high versus T1low exposure) and birth size, stratified by offspring sex. A total of three parabens, two phenols, four individual phthalate metabolites and four sums of diester metabolites were detectable above limits of detection in at least 60% of urine samples. Overall, we observed few statistically significant associations, but medium/high exposure to bisphenol A (BPA) in male offspring was associated with statistically significant lower birth size across most outcomes [birth weight: -428 g (95% CI -756 to -99.4); birth length: -1.76 cm (95% CI -3.28 to -0.25); abdominal circumference: -1.97 cm (95% CI -3.55 to -0.39)]. Similarly, medium/high exposure to methyl paraben (MeP) in male offspring was associated with lower birth weight (-661 g, 95% CI -1251 to -70.7) and length (-3.11 cm, 95% CI -5.76 to -0.46) compared to low exposure. None of these associations were statistically significant in female offspring. Across all compounds, individual exposures were associated with more negative estimates of birth weight for male than for female offspring. Our study indicates that prenatal exposure to BPA and MeP may negatively affect birth size outcomes, with a possible sex effect. Given the small sample size, these findings need to be replicated in future larger studies.

Computational Insight into Biotransformation Profiles of Organophosphorus Flame Retardants to Their Diester Metabolites by Cytochrome P450.

Biotransformation of organophosphorus flame retardants (OPFRs) mediated by cytochrome P450 enzymes (CYPs) has a potential correlation with their toxicological effects on humans. In this work, we employed five typical OPFRs including tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), tris(1-chloro-2-propyl) phosphate (TCIPP), tri(2-chloroethyl) phosphate (TCEP), triethyl phosphate (TEP), and 2-ethylhexyl diphenyl phosphate (EHDPHP), and performed density functional theory (DFT) calculations to clarify the CYP-catalyzed biotransformation of five OPFRs to their diester metabolites. The DFT results show that the reaction mechanism consists of Cα-hydroxylation and O-dealkylation steps, and the biotransformation activities of five OPFRs may follow the order of TCEP ≈ TEP ≈ EHDPHP > TCIPP > TDCIPP. We further performed molecular dynamics (MD) simulations to unravel the binding interactions of five OPFRs in the CYP3A4 isoform. Binding mode analyses demonstrate that CYP3A4-mediated metabolism of TDCIPP, TCIPP, TCEP, and TEP can produce the diester metabolites, while EHDPHP metabolism may generate para-hydroxyEHDPHP as the primary metabolite. Moreover, the EHDPHP and TDCIPP have higher binding potential to CYP3A4 than TCIPP, TCEP, and TEP. This work reports the biotransformation profiles and binding features of five OPFRs in CYP, which can provide meaningful clues for the further studies of the metabolic fates of OPFRs and toxicological effects associated with the relevant metabolites.

A simple method for simultaneous determination of organophosphate esters and their diester metabolites in dairy products and human milk by using solid-phase extraction coupled to liquid chromatography-tandem mass spectrometry.

Organophosphate esters (OPEs) and their diester metabolites have been frequently found in various environmental matrices and regarded as emerging environmental pollutants, whereas data on their occurrence in foods and human matrices are still limited. In this study, a novel and simple procedure was developed to simultaneously determine 14 OPEs and 6 diester metabolites in dairy products and human milk. After enzymatic hydrolysis by β-glucuronidase/arylsulfatase, a freeze-dried milk sample was extracted with acetonitrile and purified by solid-phase extraction. Subsequently, all target compounds were determined by HPLC-ESI-MS/MS. Linearity, limits of detection (LODs), recovery, precision, and matrix effects of the proposed methodology were validated, and the parameters of HPLC-ESI-MS/MS were optimized. LODs for OPEs and their diester metabolites were from 0.001 to 0.02 ng/mL, and limits of quantification (LOQs) were 0.01-0.3 ng/mL. Average recoveries at two spiked levels ranged between 67.3 and 121%, with relative standard deviation lower than 20.7%. A test for matrix effects showed that most analytes presented signal suppression, and isotopically labeled ISs were essential for compensating for the matrix effects. Finally, OPEs and their metabolites both showed high detecting frequencies in real samples, which indicated that these emerging pollutants were ubiquitous in foods and the human body, and the impact of the diester metabolites on population exposure must be included in exposure assessment.

Organophosphate esters and their diester metabolites in infant formulas and baby supplementary foods collected in Beijing, China: Occurrence and the implications for infant exposure

Organophosphate esters (OPEs) have been extensively used as flame retardants and/or plasticizers and they found to be ubiquitous in various environmental matrices along with the gradual phase-out of brominated flame retardants (BFRs). Moreover, their main metabolites, organophosphate di-esters (di-OPEs), were also frequently detected. However, few studies focused on the occurrence of OPEs and di-OPEs in foods. In this study, fourteen OPEs and five di-OPEs were measured in infant formula and baby supplementary food (BSF) collected in Beijing, China. Most OPEs and di-OPEs presented high detection frequencies, which indicated their ubiquity in baby foods. The concentrations of ∑14OPEs in the 75 infant formula samples ranged from 0.79 to 159 ng/g, with a median of 23.2 ng/g, and in which triphenyl phosphate (TPhP) was the most abundant compound. The concentrations of ∑14OPEs in the 32 BSF samples were 4.42–115 ng/g (median: 19.5 ng/g), and tri(3-chloropropyl) phosphate (TCIPP) was predominant. Moreover, no significant difference was observed between OPE levels in infant formula and BSF. The median concentrations of Σ5di-OPEs in infant formula and BSF were 3.39 and 5.43 ng/g, respectively. However, no significantly correlation was observed between concentrations of di-OPEs and their parent compounds, which indicated they have different sources. The median estimated dietary intakes (EDIs) of the ∑14OPEs were from 165 to 383 ng/kg bodyweight (bw)/day for infants via infant formula feeding, and were from 429 to 470 ng/kg bw/day via BSF feeding. A comparison to corresponding reference dose (RfD) suggested that dietary intakes of OPEs to Beijing infants via formula/BSF consumption were still unable to cause significant health concerns. However, EDIs of OPEs for infants were found to be significantly higher than that for Chinese adults, and dietary intake might be the predominant OPE intake pathway for infants. To our knowledge, this is the first study to investigate OPEs and their metabolites in baby foods.

Organophosphate ester metabolites in human breast milk determined by online solid phase extraction coupled to high pressure liquid chromatography tandem mass spectrometry.

The analysis of metabolites of organophosphate esters (OPEs) in human breast milk is essential to evaluate OPE and OPE metabolite exposure of newborns. In the current study, an analytical method which only needs a small amount of breast milk (100μl) was developed and validated for six diester metabolites and three hydroxylated metabolites applying salt-induced liquid-liquid extraction (SI-LLE) and dispersive solid phase extraction (d-SPE) for sample preparation and online solid phase extraction coupled to high pressure chromatography tandem mass spectrometry (online-SPE-HPLC-MS/MS) for quantitative measurement. The final method consisted of an extraction with formic acid (FA)/acetonitrile (1:200, v/v) and a cleanup with C18 d-SPE. The final extracts were trapped on a C18 cartridge with application of a wash step of 2ml 0.1% FA milli-Q/methanol (98:2, v/v). Method detection limits (MDLs) ranging from 21.7ng/l for BBOEHEP to 500ng/l for BCIPP and average recoveries ranging from 58% for 5-OH-EHDPHP to 120% for BCIPP were achieved. Thirty-three breast milk samples from the LINC (Linking EDCs in maternal Nutrition to Child health) cohort collected in three distinct areas in The Netherlands were analyzed using the validated method. BCEP, BCIPP, BCIPHPP, BDCIPP, and 5-OH-EHDPHP were not detected in any of the samples, while BBOEP was the most frequently detected metabolite with a concentration range of <MDL to l.47ng/ml, followed by DPhP and BBOEHEP, detected in ranges of <MDL to 0.09 and <MDL to 0.027ng/ml. The results indicated that OPEs entering the human body are only to a limited extent excreted via breast milk.

Legacy and emerging semi-volatile organic compounds in sentinel fish from an arctic formerly used defense site in Alaska

The Arctic is subject to long-range atmospheric deposition of globally-distilled semi-volatile organic compounds (SVOCs) that bioaccumulate and biomagnify in lipid-rich food webs. In addition, locally contaminated sites may also contribute SVOCs to the arctic environment. Specifically, Alaska has hundreds of formerly used defense (FUD) sites, many of which are co-located with Alaska Native villages in remote parts of the state. The purpose of this study was to investigate the extent of SVOC contamination on Alaska’s St. Lawrence Island through the analysis of sentinel fish, the ninespine stickleback (Pungitius pungitius), collected from Troutman Lake located within the watershed of an FUD site and adjacent to the Yupik community of Gambell. We measured the concentrations of legacy and emerging SVOCs in 303 fish samples (81 composites), including polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organophosphate esters (OPEs) and their diester metabolites, and per- and poly-fluoroalkyl substances (PFAS). PBDEs and PCBs were the most abundant SVOC groups found in stickleback with ΣPBDE and ΣPCB median concentrations of 25.8 and 10.9 ng/g ww, respectively, followed by PFAS (median ΣPFAS 7.22 ng/g ww). ΣOPE and ΣOPE metabolite concentrations were lower with median concentrations of 4.97 and 1.18 ng/g ww, respectively. Chemical patterns and distributions based on correlations and comparison with SVOC concentrations in stickleback from other parts of the island suggest strong local sources of PCBs, PBDEs, and PFAS on St. Lawrence Island.

Occurrence and ecological implications of organophosphate triesters and diester degradation products in wastewater, river water, and tap water

The occurrence and composition profiles of 13 triester organophosphate flame retardants and their three diester metabolites in river water, wastewater, and tap water in China were studied. Most target organophosphate esters (OPEs) were found in water samples, with average concentrations of 787 ng/L for triethyl phosphate (TEP) and 0.1 ng/L for tripropyl phosphate (TPP) in wastewater, 1.48 × 103 ng/L for TEP and 0.12 ng/L for tripentyl phosphate (TPeP) in river water, and 15.5 ng/L for tris(2-chloroethyl) phosphate (TCEP) and 0.08 ng/L for tritolyl phosphate (TMPP) in tap water. TEP was the most abundant compound among the detected OPEs in all water types. The exposure of zebrafish embryos showed negligible effects of TEP, triphenyl phosphate (TPHP), and diphenyl phosphate (DPHP), while mixed solutions that mimic river water and wastewater composition disturbed the development of embryos and led to the altered transcription of genes relating to the hypothalamic-pituitary-thyroid (HPT) axis. In addition, the binding affinity between OPEs and a thyroid hormone receptor (TRβ) protein was further investigated by molecular docking modeling, which helped to estimate the effects of OPEs on TRβ. This research provides experimental and theoretical evidence for the ecotoxicological effects of OPEs in aquatic environments.

The environment behavior of organophosphate esters (OPEs) and di-esters in wheat (Triticum aestivum L.): Uptake mechanism, in vivo hydrolysis and subcellular distribution

To reveal the metabolic characteristic of organophosphate esters (OPEs) in plants, uptake efficiency, subcellular distribution and hydrolysis of OPEs and their hydrolysis metabolites in wheat (Triticum aestivum L.) were investigated by hydroponic experiments. Tris(2-chloroethyl) phosphate (TCEP) and bis(2-chloroethyl) phosphate (BCEP) were prone to transporting to shoots by transpiration stream, with the translocation factors of 6.2 and 21 for TCEP and BCEP, respectively, as greater than 40% of TCEP and BECP were distributed in the cell sap due to their hydrophilicity. Hydrophobic OPEs (i.e. tri-n-butyl phosphate [TnBP] and triphenyl phosphate [TPhP]), and their hydrolysis metabolites (di-n-butyl phosphate [DnBP] and diphenyl phosphate [DPhP]) were stored in roots, resulting in low translocation capacity in wheat. As organophosphate diesters with relatively high proportions in cell walls (70–84% of DnBP and 41–43% of DPhP) are difficult to being transmembrane transported due to electrical repulsion of the cytomembrane, it is supposed that cell walls could be a main location for in vivo hydrolysis of OPEs. For DnBP, absorption by roots after in vitro hydrolysis of TnBP in hydroponic media was also an important source in wheat. Inhibition experiments showed that it is usually a non-energy-consuming process for root uptake of OPEs and their hydrolysis metabolites. The uptake of OPEs (i.e. TCEP, TnBP, and TPhP) and DPhP were facilitated diffusion mediated by aquaporins or anion channels, while uptake of BCEP and DnBP were simple diffusion processes. This study illustrated the distribution characteristics and translocation capacity of OPEs and their diester metabolites at the subcellular level.

Estimating renal and hepatic clearance rates of organophosphate esters in humans: Impacts of intrinsic metabolism and binding affinity with plasma proteins

The renal and hepatic clearance rates of organophosphate esters (OPEs) in humans were estimated. Six OPEs and their corresponding diester metabolites (mOPEs) were quantified respectively in 30 paired human plasma and urine samples collected in Hengshui, Hebei province, China. The renal clearance rate (CLrenal) of triphenyl phosphate (TPHP), tris(chloroethyl) phosphate (TCEP) and tris(1,3-dichloro-isopropyl) phosphate (TDCIPP) was estimated to be 68.9, 50.9 and 33.3 mL/kg/day, respectively, while it was not calculated for other three OPEs due to the low detection frequencies in human samples. To estimate the clearance rates of the target OPEs, hepatic clearance rates (CLh) of OPEs were extrapolated from their in vitro intrinsic clearance data in human liver microsomes (CLHLM). The calculated CLh values of TCEP and TDCIPP were comparable to their CLrenal, indicating that the in vitro extrapolation method was suitable for estimating the clearance rates of OPEs. The higher binding affinity of TDCIPP with plasma proteins could reduce its renal clearance. The estimated half-lives of Cl-OPEs in human were longer than those of the aryl- and alkyl-OPEs. This study provided a feasible in vitro method to predict the clearance and half-lives of OPEs in human, which is significant for their accurate health risk assessment.

In vitro metabolic activation of triphenyl phosphate leading to the formation of glutathione conjugates by rat liver microsomes

The present study investigated the metabolism of the flame retardant and plasticizer chemical, triphenyl phosphate (TPHP), in a rat liver microsome-based in vitro assay with glutathione (GSH) in order to elucidate metabolic pathways leading to formation of conjugates. A highly sensitive and efficient method was developed for the detection and characterization of GSH reactive metabolites using LC-Q-TOF-MS/MS both in the negative and positive electrospray ionization modes. Seven GSH conjugates formed as a result of microsomal incubation, which were identified as S-conjugates based on MS/MS spectra, and confirmed by subsequent time-dependent incubation assays. With the exception of hydrolysis reactions leading to formation of a diester metabolite, diphenyl phosphate (DPHP), the results demonstrated that Phase I epoxidation on phenyl ring of TPHP leading to mono- and di-hydroxylated TPHP metabolites, which can further conjugate with GSH. Depending on hydroxylated TPHP formation, an o-hydroquinone intermediate formed in vitro via Phase I metabolism, and the o-benzoquinone form reacted with GSH and also formed GSH conjugates. The present study showed that via hydroxylated TPHP Phase I formation that GSH conjugates are important Phase II metabolites for TPHP metabolism in vitro. Some GSH conjugates may be valuable candidate biomarkers for monitoring TPHP exposure in biota.

Urinary biomarkers for assessment of human exposure to monomeric aryl phosphate flame retardants

While monomeric aryl organophosphate flame retardants (m-aryl-OPFRs) are used worldwide in a variety of consumer products, specific biomarkers for epidemiologic studies are lacking. To explore the potential of urinary hydroxylated metabolites of m-aryl-OPFRs as the biomarkers, we detected triphenyl phosphate (TPHP), 2-ethylhexyl diphenyl phosphate (EHDPP), and tricresyl phosphate (TCrP) in 259 whole blood samples and their 5 hydroxylated and 2 diester metabolites in the paired urine samples from the general population. 2-Ethyl-5-hydroxyhexyl diphenyl phosphate (5-OH-EHDPP), 4-hydroxyphenyl diphenyl phosphate (4-OH-TPHP), and 3-hydroxy-4-methylphenyl di-p-tolyl phosphate (3-OH-MDTP) were detected in >80% of urine samples after enzymatic hydrolysis of conjugates, and their concentrations showed significant positive correlations with the blood concentrations of their corresponding parent compounds, respectively. To characterize the temporal reliability, the m-aryl-OPFRs metabolites were also determined in urine samples repeated nine times from six volunteers over 3 months. Urinary 5-OH-EHDPP showed strong temporal reliability (creatinine-corrected intraclass correlation coefficients (ICCs), 0.77; 95% confidence interval [CI], 0.58 to 0.90), and urinary 3-OH-MDTP (creatinine-corrected ICC, 0.52; 95% CI, 0.37 to 0.87) and 4-OH-TPHP (0.56; 95% CI, 0.32 to 0.80) showed moderate-to-strong temporal reliability, while relatively weak temporal reliability was found for urinary DPHP (creatinine-corrected ICC, 0.37; 95% CI, 0.12 to 0.62). This study confirmed specific, reliable, and frequently detected biomarkers for TPHP and EHDPP and developed new biomarker of TCrP for future epidemiological research on health effects of m-aryl-OPFRs.

Metabolites of organophosphate esters in urine from the United States: Concentrations, temporal variability, and exposure assessment

Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers in consumer products, which contributes to widespread exposure of humans. OPE diester metabolites in urine have been used as biomarkers of human exposure to these chemicals. Little is known, however, about occurrence and temporal variability in urinary concentrations of OPE metabolites in humans. In this study, 11 OPE metabolites were measured in 213 urine samples collected from 19 volunteers from Albany, New York, United States, at 3-day intervals for five weeks to investigate temporal variability in urinary concentrations. Diphenyl phosphate (DPHP) and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) were the major OPE metabolites, detected in all urine samples at specific gravity (SG)-adjusted concentrations (geometric mean, GM) of 1060 and 414 pg/mL and creatinine (Cr)-adjusted concentration (GM) of 404 and 156 ng/g, respectively. Inter-day variability in urinary OPE metabolite concentrations in 19 individuals was evaluated by intraclass correlation coefficients (ICCs). The inter-day variability in Cr-adjusted OPE metabolite concentrations (ICC: 0.31–0.67) was lower than those of SG-adjusted (ICC: 0.19–0.71) and unadjusted urinary concentrations (ICC: 0.24–0.74). BDCIPP (ICC: 0.68) and bis(2-chloroethyl) phosphate (BCEP) (ICC: 0.67) concentrations showed a moderate-to-high reliability over the sampling period, whereas the other nine OPE metabolites exhibited a moderate reliability (ICC: 0.31–0.55). Urine samples were further stratified by gender, age, ethnicity, and body mass index (BMI). The concentrations of BDCIPP and DPHP were significantly lower in males with normal BMI (BMI: 18.5–25 kg/m2) than in females and other BMI categories (p < 0.01). Relatively high ICCs, indicating low inter-day variability, were observed for males (ICC: 0.35–0.71) of 30–40 years of age (ICC: 0.34–0.87) with normal BMI (ICC: 0.28–0.64). The daily exposure doses to OPEs were estimated from urinary concentrations of corresponding OPE metabolites. The estimated doses of triphenyl phosphate (TPHP) and triethyl phosphate (TEP), based on median urinary concentrations of their metabolites, were 19.4 and 24.0 ng/kg bw/day, and the exposure dose to ∑OPEs was estimated at 65.3 ng/kg bw/day. Overall, our results indicate a high ICC for Cr-adjusted urinary concentrations of 11 OPE metabolites in urine.

Metabolic Mechanism of Aryl Phosphorus Flame Retardants by Cytochromes P450: A Combined Experimental and Computational Study on Triphenyl Phosphate

Understanding metabolic mechanisms is critical and remains a difficult task in the risk assessment of emerging pollutants. Triphenyl phosphate (TPHP), a widely used aryl phosphorus flame retardant (aryl-PFR), has been frequently detected in the environment, and its major metabolite was considered as diphenyl phosphate (DPHP). However, knowledge of the mechanism for TPHP leading to DPHP and other metabolites is lacking. Our in vitro study shows that TPHP is metabolized into its diester metabolite DPHP and mono- and dihydroxylated metabolites by cytochromes P450 (CYP) in human liver microsomes, while CYP1A2 and CYP2E1 isoforms are mainly involved in such processes. Molecular docking gives the conformation for TPHP binding with the active species Compound I (an iron IV-oxo heme cation radical) in specific CYP isoforms, showing that the aromatic ring of TPHP is likely to undergo metabolism. Quantum chemical calculations have shown that the dominant reaction channel is the O-addition of Compound I onto the aromatic ring of TPHP, followed by a hydrogen-shuttle mechanism leading to ortho-hydroxy-TPHP as the main monohydroxylated metabolite; the subsequent H-abstraction-OH-rebound reaction acting on ortho-hydroxy-TPHP yields the meta- and ipso-position quinol intermediates, while the former of which can be metabolized into dihydroxy-TPHP by fast protonation, and the latter species needs to go through type-I ipso-substitution and fast protonation to be evolved into DPHP. We envision that the identified mechanisms may give inspiration for studying the metabolism of several other aryl-PFRs by CYP.

Organophosphate esters in East Greenland polar bears and ringed seals: Adipose tissue concentrations and in vitro depletion and metabolite formation

East Greenland is a contamination “hot spot” for long-range transported anthropogenic chemicals, including organophosphate esters (OPEs). High concentrations of OPEs have been reported in arctic air while very little is known for wildlife where OPE tissue residues levels appear to be strongly influenced by biotransformation. In the present study, the hepatic in vitro metabolism of six environmentally relevant organophosphate (OP) triesters and corresponding OP diester formation were investigated in East Greenland polar bears (PBs) and ringed seals (RSs). The in vitro metabolism assay results were compared to adipose levels in field samples from the same individuals. In vitro OP triester metabolism was generally rapid and structure-dependent, where PBs metabolized OPEs more rapidly than RSs. Exceptions were the lack of triethyl phosphate (TEP) metabolism and slow metabolism of tris(2-ethylhexyl) phosphate (TEHP) in both species. OP diester metabolites were also formed with the exception of TEP which was not metabolized at all. Tris(1,3-dichloro-2-propyl) phosphate was completely converted to its corresponding diester. However, the mass balances showed that OP diester formation corresponding to TEHP, tri(n-butyl) phosphate, and tris(2-butyoxyethyl) phosphate did not account for 100% of the OP triester depletion, which indicated alternate pathways of OP triester metabolism had occurred. Triphenyl phosphate was completely converted to its OP diester metabolite in PBs but not in RSs suggesting species-specific differences. The results demonstrated that OP triester bioaccumulation and fate in PBs versus their RS prey is substantially influenced by biotransformation.

Occurrence, Removal, and Environmental Emission of Organophosphate Flame Retardants/Plasticizers in a Wastewater Treatment Plant in New York State.

The occurrence and fate of 14 triester organophosphate flame retardants (OPFRs) and plasticizers and their two diester metabolites were investigated in a wastewater treatment plant (WWTP) in the Albany area of New York State. All target OPFRs were found in wastewater, with average concentrations that ranged from 20.1 ng/L for tris(methylphenyl) phosphate (TMPP) to 30 100 ng/L for tris(2-butoxyethyl)phosphate (TBOEP) in influents and from 7.68 ng/L for TMPP to 12 600 ng/L for TBOEP in final effluents. TBOEP was the dominant compound in influents (max: 69 500 ng/L) followed in decreasing order by tris(1-chloro-2-propyl)phosphate (TCIPP; max: 14 500 ng/L), bis(1,3-dichloro-2-propyl)phosphate (BDCIPP; max: 4550 ng/L), tris(1,3-dichloro-2-propyl)phosphate (TDCIPP; max: 3150 ng/L) and tris(2-chloroethyl)phosphate (TCEP; max: 8450 ng/L). The fraction of TMPP sorbed to suspended particulate matter (SPM) was 56.4% of the total mass in wastewater, which was the highest among the target chemicals analyzed. The average concentrations of OPFRs in sludge were between 4.14 ng/g dw for tripropyl phosphate (TPP) and 7290 ng/g dw for TBOEP; for ash, they were between 2.17 ng/g dw for TMPP and 427 ng/g dw for triphenyl phosphate (TPhP). The mass loadings of OPFRs into the WWTP ranged from 0.02 mg/day/person for TPP to 28.7 mg/day/person for TBOEP, whereas the emission from the WWTP ranged from 0.01 mg/day/person for 2-ethylhexyl diphenyl phosphate (EHDPP) to 5.12 mg/day/person for TCIPP. The removal efficiencies for OPFRs were slightly above 60% for TMPP, TBOEP, and tris(2-ethylhexyl)phosphate (TEHP) whereas those for other OPFRs were <40% (TPhP and BDCIPP) to negative values, suggesting incomplete removal in WWTPs.

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay

The in vitro biotransformation and kinetics of six organophosphate triester (OPE) flame retardants were investigated in herring gulls (Larus argentatus) from the Great Lakes using a hepatic microsomal metabolism assay. Administration of each individual OPE (tri-n-butyl phosphate (TNBP), tris(2-butoxyethyl) phosphate (TBOEP), triphenyl phosphate (TPHP), triethyl phosphate (TEP), tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-chloroisopropyl) phosphate (TCIPP)) to the in vitro assay (concentration range 0.01 to 10μM) resulted in rapid depletion with the exception of TEP. Following the Michaelis-Menten enzyme kinetics model, a preliminary 2-minute incubation period was used to estimate the Vmax (±SE) values (i.e., the maximal rate of reaction for a saturated enzyme system), which ranged from 5.0±0.4 (TPHP) to 29±18pmol/min/mg protein (TBOEP), as well as the KM (±SE) values (i.e., the OPE concentration corresponding to one half of the Vmax), which ranged from 9.8±1 (TPHP) to 189±135nM (TBOEP). Biotransformation assays over a 100-minute incubation period revealed that TNBP was metabolized most rapidly (with a depletion rate of 73±4pmol/min/mg protein), followed by TBOEP (53±8pmol/min/mg), TCIPP (27±1pmol/min/mg), TPHP (22±2pmol/min/mg) and TDCIPP (8±1pmol/min/mg). In vitro biotransformation of OP triesters was clearly structure-dependent where non-halogenated alkyl OP triesters were metabolized more rapidly than halogenated alkyl triesters. Halogenated OP triesters were transformed to their respective diesters more efficiently relative to non-halogenated OP triesters. To our knowledge, this is the first study to investigate OP triester metabolism and OP diester formation in an avian or wildlife model system, which is important to understand the fate and biological activity of OPEs in an exposed organism.