Tris(1-chloro-2-propyl) phosphate (TCIPP) is an emerging contaminant which is ubiquitous in the indoor and outdoor environment. Moreover, its presence in human body fluids and biota has been ...evidenced. Since no quantitative data exist on the biotransformation or stability of TCIPP in the human body, we performed an in vitro incubation of TCIPP with human liver microsomes (HLM) and human serum (HS). Two metabolites, namely bis(2-chloro-isopropyl) phosphate (BCIPP) and bis(1-chloro-2-propyl) 1-hydroxy-2-propyl phosphate (BCIPHIPP), were quantified in a kinetic study using HLM or HS (only BCIPP, the hydrolysis product) and LC-MS. The Michaelis–Menten model fitted best the NADPH-dependent formation of BCIPHIPP and BCIPP in HLM, with respective VMAX of 154 ± 4 and 1470 ± 110 pmol/min/mg protein and respective apparent Km of 80.2 ± 4.4 and 96.1 ± 14.5 μM. Hydrolases, which are naturally present in HLM, were also involved in the production of BCIPP. A HS paraoxonase assay could not detect any BCIPP formation above 38.6 ± 10.8 pmol/min/μL serum. Our data indicate that BCIPP is the major metabolite of TCIPP formed in the liver. To our knowledge, this is the first quantitative assessment of the stability of TCIPP in tissues of humans or any other species. Further research is needed to confirm whether these biotransformation reactions are associated with a decrease or increase in toxicity.
•We have incubated TCIPP with human liver microsomes (HLM) and human serum.•A hydrolysis product (BCIPP) and dechlorination metabolite (BCIPHIPP) were quantified.•BCIPP was formed by oxidative enzymes and hydrolases in HLM.•Production of BCIPP was eight times faster compared to BCIPHIPP in HLM.•Serum enzymes did not show any detectable activity in production of BCIPP.
Phosphate flame retardants (PFRs) are ubiquitously detected in indoor environments. Despite increasing health concerns pertaining to PFR exposure, few epidemiological studies have examined PFR ...exposure and its effect on children's allergies.
To investigate the association between PFRs in house dust, their metabolites in urine, and symptoms of wheeze and allergies among school-aged children.
A total of 128 elementary school-aged children were enrolled. House dust samples were collected from upper-surface objects. Urine samples were collected from the first morning void. Levels of 11 PFRs in dust and 14 PFR metabolites in urine were measured. Parent-reported symptoms of wheeze, rhinoconjunctivitis, and eczema were evaluated using the International Study of Asthma and Allergies in Childhood questionnaire. The odds ratios (ORs) of the Ln transformed PFR concentrations and categorical values were calculated using a logistic regression model adjusted for sex, grade, dampness index, annual house income, and creatinine level (for PFR metabolites only).
The prevalence rates of wheeze, rhinoconjunctivitis, and eczema were 22.7%, 36.7%, and 28.1%, respectively. A significant association between tris(1,3-dichloroisopropyl) phosphate (TDCIPP) in dust and eczema was observed: OR (95% confidence interval), 1.44 (1.13–1.82) (>limit of detection (LOD) vs <LOD). The ORs for rhinoconjunctivitis (OR = 5.01 1.53–16.5) and for at least one symptom of allergy (OR = 3.87 1.22–12.3) in the 4th quartile of Σtris(2-chloro-isopropyl) phosphate (TCIPP) metabolites was significantly higher than those in the 1st quartile, with significant p-values for trend (Ptrend) (0.013 and 0.024, respectively). A high OR of 2.86 (1.04–7.85) (>LOD vs <LOD) was found for hydroxy tris(2-butoxyethyl) phosphate (TBOEP)-OH and eczema. OR of the 3rd tertile of bis (1,3-dichloro-2-propyl) phosphate (BDCIPP) was higher than the 1st tertile as a reference for at least one symptom (OR = 3.91 1.25–12.3), with a significant Ptrend = 0.020.
We found that TDCIPP in house dust, and metabolites of TDCIPP, TBOEP and TCIPP were associated with children's allergic symptoms. Despite some limitations of this study, these results indicate that children's exposure to PFR may impact their allergic symptoms.
•The house dust of 128 children's homes showed 11 phosphate flame retardants (PFRs).•Fourteen PFR metabolites were determined in children's urine.•Correlation was observed between increased levels of TDCIPP in dust and eczema.•Dose response relationships are observed for ΣuTCIPP and rhinoconjunctivitis.•Dose response relationships are seen with BDCIPP and at least one allergic symptom.
This is the first study investigating the in vitro metabolism of α-, β-, and γ-hexabromocyclododecane (HBCD) stereoisomers in humans and providing semiquantitative metabolism data. Human liver ...microsomes were incubated with individual racemic mixtures and with individual stereoisomers of α-, β-, and γ-HBCDs, the hydroxylated metabolites formed were analyzed by liquid chromatography–tandem mass spectrometry, and the value of the intrinsic in vitro clearance (Clint,vitro) was calculated. Several mono- and dihydroxylated metabolites of α-, β-, and γ-HBCDs were formed, with mono-OH-HBCDs being the major metabolites. No stereoisomerization of any of the six α-, β-, and γ-HBCD isomers catalyzed by cytochrome P450 (CYP) enzymes occurred. The value of Clint,vitro of α-HBCDs was significantly lower than that of β-HBCDs, which, in turn, was significantly lower than that of γ-HBCDs (p < 0.05). Such differences were explained by the significantly lower values of Clint,vitro of each α-HBCD stereoisomer than those of the β- and γ-HBCD stereoisomers. In addition, significantly lower values of Clint,vitro of the (−) over the (+)α- and β-HBCD stereoisomers, but not γ-HBCDs, were obtained. Our data offer a possible explanation of the enrichment of α-HBCDs over β- and γ-HBCDs on the one hand and, on the other hand, of (−)α-HBCDs over (+)α-HBCDs previously reported in human samples. It also offers information about the mechanism resulting in such enrichments, the stereoisomer-selective metabolism of α-, β-, and γ-HBCDs catalyzed by CYPs with the lack of stereoisomerization.
Aryl phosphate flame retardants (aryl-PFRs), such as triphenyl phosphate (TPHP) and 2-ethylhexyl diphenyl phosphate (EHDPHP), are emerging contaminants that can exhibit toxic properties, including ...severe aquatic toxicity and endocrine disruptive effects. Monitoring exposure to aryl-PFRs through specific biomarkers is necessary to assess the health risk associated with chronic exposure. Hydrolytic serum enzymes could play an important role in the formation of the hydrolysis product diphenyl phosphate (DPHP), the seemingly most abundant in vivo biomarker of TPHP in urine. Here, we assess whether serum enzymes have an impact on the toxicokinetics of TPHP and EHDPHP and on the contribution of both aryl-PFRs to in vivo DPHP levels. TPHP and EHDPHP were incubated separately with pooled human serum to measure the formation of hydrolysis products DPHP and 2-ethylhexyl phenyl phosphate (EHPHP) by liquid chromatography-tandem mass spectrometry. Clearance of TPHP and EHDPHP was 70 and 8.6 mL/min/L serum (as measured by formation of DPHP and EHPHP, respectively). No discernible amount of DPHP was produced from EHDPHP by serum hydrolases. Our results suggest that serum hydrolases can significantly contribute to the in vivo levels of DPHP formed from TPHP and can play an important role in the toxicokinetics, toxicity, and selection of biomarkers for aryl-PFRs.
Indoor environments contain a wide range of new chemicals such as phosphate flame retardants and plasticizers (PFRs). Despite recent epidemiological evidence suggesting that children might be ...affected by widespread exposure to PFRs, questions remain about the various exposure pathways to these chemicals. Therefore, the aim of this study was to investigate exposure to PFRs by measuring the concentrations a set of urinary metabolites for schoolchildren from Japan (n = 128) and associating them with house dust concentrations and housing characteristics. Detectable concentrations of both diaryl and dialkyl phosphates (DAPs) and hydroxylated metabolites (HO-PFRs) were found in urine samples of almost all children. 2-Hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP) was the most frequently detected metabolite (98%) followed by 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP, 95%) and tris(chloroethyl) phosphate (TCEP). Next to BBOEHEP, two other metabolites of tris(2-butoxyethyl) phosphate (TBOEP) were also frequently detected. Significant correlations of moderate strength were found between parent compounds detected in high concentrations in house dust (TBOEP, tris(2-chloroisopropyl) phosphate (TCIPP)) and their corresponding metabolites, suggesting that dust is a primary exposure source for these PFRs. Several personal and housing characteristics, such as gender, income, and the use of PVC and ventilation were associated with metabolite concentrations in multivariate linear regression. Overall, this study showed that Japanese schoolchildren are exposed to a wide range of PFRs.
•High concentrations of TBOEP metabolites compared to other studies.•At least one metabolite of each investigated parent PFR was frequently detected.•Significant correlations were found between dust and urine for TBOEP and TCIPP.•Several characteristics such as gender, income, and the use of PVC and ventilation were associated with PFR metabolites.
A young woman with a history of several suicide attempts was admitted to the hospital after suspicion of a new intoxication without definite identification of the causing agent. The patient had a ...high anion gap metabolic acidosis (HAGMA) with respiratory compensation, a lactate gap and an osmolar gap at admission. Initial toxicological screening showed no abnormalities except for a weak positive gamma-hydroxy butyric acid (GHB) enzymatic screen in urine. This finding could not be confirmed using chromatographic analysis nor be explained by the presence of known cross-reacting substances like ethanol. In this case, falsely elevated urinary GHB screening was caused by the ingestion of ethylene glycol. To confirm that the interference was due to ethylene glycol or its metabolites, we performed a spiking experiment. Cross reactivity was linked to ethylene glycol and was low in our experiments (0.1-0.2%). Substantial amounts of ethylene glycol are required to slightly elevated GHB results, depending on the endogenous cutoff used. We can conclude that ethylene glycol can give rise to falsely elevated urinary GHB levels at ethylene glycol concentrations that are typically found in intoxications.
Phthalate esters and phosphate flame retardants and plasticizers (PFRs) are both used as plasticizers and are commonly detected in indoor environments. Although both phthalates and PFRs are known to ...be associated with children's wheeze and allergic symptoms, there have been no previous studies examining the effects of mixtures of these exposures.
To investigate the association between exposure to mixtures of phthalate esters and PFRs, and wheeze and allergic symptoms among school-aged children.
A total of 128 elementary school-aged children were enrolled. Metabolites of 3 phthalate esters and 7 PFRs were measured in urine samples. Parent-reported symptoms of wheeze, rhinoconjunctivitis, and eczema were evaluated using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. In the primary model, we created a phthalate ester and PFR mixture exposure index, and estimated odds ratios (ORs) using weighted quantile sum (WQS) regression and quantile g (qg)-computation. The two highest chemicals according to qg-computation weight %s were combined to create a combination high × high exposure estimate, with ORs calculated using the “low × low” exposure group as the reference category. Concentrations of each metabolite were corrected by multiplying this value by the sex- and body size-Standardised creatinine concentration and dividing by the observed creatinine value. All models were adjusted for sex, grade, dampness index and annual house income.
The odds ratio of rhinoconjunctivitis for the association between exposure to chemical mixtures according to the WQS index positive models was; OR = 2.60 (95% confidence interval CI: 1.38-5.14). However, wheeze and eczema of the WQS index positive model, none of the WQS index negative models or qg-computation result yielded statistically significant results. Combined exposure to the two highest WQS weight %s of “high-high” ΣTCIPP and ΣTPHP was associated with an increased prevalence of rhino-conjunctivitis, OR = 5.78 (1.81–18.43) to the “low × low” group.
Significant associations of mixed exposures to phthalates and PFRs and increased prevalence of rhinoconjunctivitis was found among elementary school-aged children in the WQS positive model. Mixed exposures were not associated with any of allergic symptoms in the WQS negative model or qg-computation approach. However, the combined effects of exposure to two PFRs suggested an additive and/or multiplicative interaction, potentially increasing the prevalence of rhinoconjunctivitis. A further study with a larger sample size is needed to confirm these results.
•Metabolites of three phthalates and seven PFRs were measured in children's urine.•Associations between mixture and combined of chemicals with the prevalence of allergic symptoms were examined.•Mixture of chemicals was not associated with the prevalence of any allergy symptoms.•Combined ΣTCIPP and ΣTPHP was associated with a higher prevalence of rhinoconjunctivitis.
In 2015, nine laboratories from Belgium, USA, Canada, China, and Australia participated in an interlaboratory exercise to quantify metabolites of organophosphate ester (OPE) contaminants in pooled ...human urine. Pooled human urine available as SRM 3673 (Organic contaminants in non-smokers’ urine) was obtained from the U.S. National Institute of Standards and Technology and was analyzed for its content of OPE metabolites. Each participating laboratory received 10 mL sample and used its own validated method and standards to report the concentrations of the OPE metabolites of its choice. Four OPE metabolites were consistently measured by most laboratories and they were the following diesters: bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), diphenyl phosphate (DPHP), bis(2-chloroethyl) phosphate (BCEP), and bis(1-chloro-2-propyl) phosphate (BCIPP). Concentrations of other OPE metabolites in SRM 3673 were also reported but are only considered as informative values since they were measured by three laboratories at most. All laboratories used liquid chromatography with tandem mass spectrometry (LC-MS/MS) with or without solid-phase extraction (SPE). This is the first study to report indicative values for OPE metabolites in a human urine Standard Reference Material. It is expected that these indicative values obtained for these four metabolites will be used as quality control to ensure compatibility of results in biomonitoring studies and by other researchers who validate their own methods for the quantification of OPE metabolites in human urine.
•Nine labs have participated to the collaborative study on organophosphate ester metabolites.•Indicative values in pooled human urine SRM 3673 have been established.•Four OPE metabolites (BDCIPP, DPHP, BCEP, and BCIPP) were consistently measured.•All laboratories used their own methods based on LC-MS/MS.
•Fractionation and optimised analysis improves the metabolomic coverage.•The lipidome was analysed with a tailored RP-LC gradient for each ionisation mode.•HILIC-LC has better separation properties ...for neutral and basic polar compounds.•Anionic polar compounds were better retained and separated with ion paring RP-LC.•A four-run analysis provides coverage of 2200 high precision metabolites (mRSD<10%).
Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC–MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible.
In this study, we optimised the LC–MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites.
The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column.
Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure.
By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC–MS metabolomics studies.