Determination of bisphenol A and related substitutes/analogues in human breast milk using gas chromatography-tandem mass spectrometry.

Bisphenol A (BPA) is an industrial chemical widely used in the production of polycarbonate and epoxy resins. Identified as an endocrine-disrupting chemical (EDC), BPA is a matter of existing or ongoing restrictive regulations and then is increasingly being replaced by other analogues used as BPA's substitutes. Human biomonitoring studies focusing on both BPA and emerging related analogues consequently appear as a requirement either for documenting the efficiency of regulatory actions toward BPA and for fuelling incoming risk assessment studies toward BPA's substitutes. In particular, the increasing concern about the late effects consecutive to early exposures naturally identify human breast milk as a target biological matrix of interest for priority exposure assessment focused on critical sub-populations such as pregnant women, fetuses, and/or newborns. In this context, an accurate and sensitive analytical method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed for the quantification of 18 "BPA-like" compounds in breast milk samples at trace levels (<0.05 µg kg(-1)). The method includes a preliminary protein precipitation step followed by two successive solid-phase extraction (SPE) stages. Quantification of the targeted compounds was achieved according to the isotopic dilution method using (13)C12-BPA as internal standard. The method was validated according to current EU guidelines and criteria. Linearity (R (2)) was better than 0.99 for each molecule within the concentration range 0-5 µg kg(-1). The detection and quantification limits ranged from 0.001 to 0.030 µg kg(-1) and from 0.002 to 0.050 µg kg(-1), respectively. The analytical method was successfully applied to the first set of human breast milk samples (n = 30) originating from French women in the Region Pays-de-la-Loire. The measured levels of BPA were found in the

Characterization of pregnant women exposure to halogenated parabens and bisphenols through water consumption.

Exposure of pregnant women to endocrine disruptor compounds, such as parabens and bisphenol A is of concern for fetal transition. Their halogenated degradation products, mainly coming from water treatment plans, could be problematic as well, depending on their occurrence in drinking water in the first place. Thus, 25 halogenated compounds were synthesised in order to investigate 60 substances (Bisphenols, parabens and their degradation products) in 325 drinking water samples coming from a French cohort study of pregnant women. Analysis was performed by tandem mass spectrometry coupled to gas chromatography (GC-MS/MS) after SPE extraction and derivation of the contaminants. Results indicate that parabens (methylparaben, n-propylparaben, ethylparaben and n-butylparaben), bisphenols S, A and F, and their degradation product, 4-hydroxybenzoic acid, were detected up to several hundred ng/L in drinking water, with detection frequencies between 16% and 88%. Regarding halogenated degradation products, the highest detection frequencies were found for monochlorinated products (about 50% for 2-chlorobisphenol A), which were quantified up to several tens of ng/L. Such analytical approaches with broader spectrum of monitoring (i.e. chemical hazards and their degradation products) constitute in the beginning of a solution to exhaustively answer the questions related to the characterization of the human chemical exposome.

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis.

BACKGROUND: Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 µM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models. METHODS: Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 µM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 µM BPA (~ 500 µg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 µM and 0.038 µM respectively. Mice grafted with second trimester testes received 0.5 and 50 µg/kg/day BPA by oral gavage for 5 weeks. RESULTS: With first trimester human testes, using the hFeTA model, 10 µM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice. CONCLUSIONS: Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.

Urinary signature of pig carcasses with boar taint by liquid chromatography-high-resolution mass spectrometry.

Boar taint is an offensive odour that can occur while cooking pork or pork products and is identified in some uncastrated male pigs that have reached puberty. It is widely held that boar taint is the result of the accumulation in back fat of two malodorous compounds: androstenone and skatole. The purpose of this study is to assess a mass spectrometry-based metabolomics strategy to investigate the metabolic profile of urine samples from pig carcasses presenting low (untainted) and high (tainted) levels of androstenone and skatole in back fat. Urine samples were analysed by LC-ESI(+)-HRMS. Discrimination between tainted and untainted animals was observed by the application of multivariate statistical analysis, which allowed candidate urinary biomarkers to be highlighted. These urinary metabolites were positively correlated to androstenone and skatole levels in back fat. Therefore, the study suggests that the measurement of these urinary metabolites might provide information with regard to androstenone and skatole levels in live pigs.

An investigation of the endocrine-disruptive effects of bisphenol a in human and rat fetal testes.

Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5-17.5 days post-coitum) and humans (8-12 gestational weeks) and under different culture conditions. BPA concentrations of 10(-8)M and 10(-5)M for 72 h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10-5M suppressed it in the Wistar strain. The suppressive effects at 10-5M were seen as early as 24h and 48 h in both strains. BPA at 10(-7)-10(-5)M for 72 h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10(-8)M, 10(-7)M, and 10(-5)M for 72 h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.

Assessment of dietary exposure to bisphenol A in the French population with a special focus on risk characterisation for pregnant French women.

Bisphenol A (BPA) is used in a wide variety of products and objects for consumers use (digital media such as CD's and DVD's, sport equipment, food and beverage containers, medical equipment). Here, we demonstrate the ubiquitous presence of this contaminant in foods with a background level of contamination of less than 5 µg/kg in 85% of the 1498 analysed samples. High levels of contamination (up to 400 µg/kg) were found in some foods of animal origin. We used a probabilistic approach to calculate dietary exposure from French individual consumption data for infants under 36 months, children and adolescents from 3 to 17 years, adults over 18 years and pregnant women. The estimated average dietary exposure ranged from 0.12 to 0.14 µg/kg body weight per day (bw/d) for infants, from 0.05 to 0.06 µg/kg bw/d for children and adolescents, from 0.038 to 0.040 µg/kg bw/d for adults and from 0.05 to 0.06 µg/kg bw/d for pregnant women. The main sources of exposure were canned foods (50% of the total exposure), products of animal origin (20%) and 30% as a background level. Based on the toxicological values set by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) for pregnant women, the risk was non negligible. Thus, we simulated scenarios to study the influence of cans and/or food of animal origin on the BPA-related risk for this specific population.

Determination of MRL regulated corticosteroids in liver from various species using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC).

Dexamethasone, betamethasone, prednisolone and methylprednisolone are corticosteroids widely used in animal husbandry. These compounds are licensed for therapy in veterinary practices while their use for growth promoting practices, mainly in combination with other growth promoters, is prohibited within the European Union. In order to protect the consumer, maximum residue limits (MRLs) have been set by the European Community in liver to 2.0 µg kg(-1) (dexamethasone and betamethasone) and 10.0 µg kg(-1) (prednisolone and methylprednisolone) for different species. The major challenges in the analysis of dexamethasone and betamethasone consist in performing an efficient separation of both isomers and in detecting and identifying all the molecules according to the regulatory requirements fixed in Commission Decision 2002/657/EC. In this context, an UHPLC-MS/MS method with a short runtime (7 min) and using the SRM acquisition mode was developed and validated. An efficient selectivity of the sample preparation combined with a high sensitivity of the measurement system allowed identifying and quantifying the four corticosteroids of interest in this complex biological matrix. Signals obtained were found very repeatable, even at very low concentration levels with an unambiguous identification of the compounds. The performance limits of the method have been validated according to the regulatory requirements and the method has been successfully applied to the confirmation of incurred liver samples.

Feed or food responsible for the presence of low-level thiouracil in urine of livestock and humans?

In recent years, questions have been raised on the possible semi-endogenous status of the alleged xenobiotic thyreostatic drug thiouracil; thiouracil has been detected in the urine of various animals (livestock and domesticated) at concentrations between 1 and 10 µg L(-1) and also in human urine. Although several studies suggest Brassicaceae-derived feed as potential origin, no traces of thiouracil have been detected in feed so far. Therefore, the aim of this study was to elucidate the origin of thiouracil in the urine of livestock and humans. To this purpose various Brassicaceae feed and food sources (e.g., rapeseed, rapeseed coarse meal, cabbage, cauliflower, broccoli) were investigated for the presence of thiouracil. In addition, the impact of the Brassicaceae-related ß-thioglucosidase enzyme was evaluated. This myrosinase enzyme appeared to be crucial, because without its catalyzed hydrolysis no thiouracil could be detected in the various Brassicaceae-derived samples. Therefore, a sample pretreatment with incorporated enzymatic hydrolysis was developed after ensuring the quality performance of the extracted myrosinase mixture with a single-point glucose assay. Upon enzymatic hydrolysis and LC-MS(2) analysis, thiouracil was successfully detected in samples of traditional rapeseed, rapeseed-'00' variety coarse meal (values of erucic acid <2% and glucosinolates <25 µmol g(-1)), and rapeseed cake at 1.5, 1.6, and 0.4 µg kg(-1), respectively. As for the food samples, broccoli and cauliflower displayed thiouracil concentrations of 6.0 and <1.0 µg kg(-1), respectively. To the best of the authors' knowledge this study is the first to report the presence of naturally occurring thiouracil in feed and food samples. Future research should investigate the pathway of thiouracil formation and identify its possible precursors.

Toward a criterion for suspect thiouracil administration in animal husbandry.

Thyreostats are growth-promoters banned in Europe since 1981. The identification of thiouracil (TU) in animal biological matrices can, however, no longer be systematically interpreted as a consequence of illegal administration. Indeed, some experimental results have indicated a causal link between cruciferous-based diet and the presence of TU in urine of bovines. The present study aims at investigating, on a large scale (n > 1300), the natural occurrence of thiouracil in urine samples collected from different animal species. TU was identified in main breeding animal species: bovine, porcine and ovine. The natural distribution of TU allowed proposing threshold values to differentiate compliant from suspect urine samples. Suggested values are 5.7 and 9.1 µg l(-1) in male adult bovines (6-24 months), 3.1 and 8.1 µg l(-1) in female adult bovines (6-24 months), 7.3 and 17.7 µg l(-1) in calves (<6 months), 3.9 and 8.8 µg l(-1) in female bovines (>24 months), and 2.9 and 4.1 µg l(-1) in porcines at a 95 and 99% confidence level, respectively.

Development and validation of a multi-residue method for the detection of a wide range of hormonal anabolic compounds in hair using gas chromatography-tandem mass spectrometry.

The monitoring of anabolic steroid residues in hair is undoubtedly one of the most efficient strategies to demonstrate the long-term administration of these molecules in meat production animals. A multi-residue sample preparation procedure was developed and validated for 28 steroids. A 100 mg hair sample was grinded into powder and extracted at 50 degrees C with methanol. After acidic hydrolysis and extraction with ethyl acetate, phenolsteroids, such as estrogens, resorcyclic acid lactones and stilbens in one hand, are separated from androgens and progestagens in the other hand. Solid phase extractions were performed before applying a specific derivatisation for each compound sub-group. Detection and identification were achieved using gas chromatography-tandem mass spectrometry with acquisition in the selected reaction monitoring mode after electron ionisation. The method was validated according to the 2002/657/EC guideline. Decision limits (CCalpha) for main steroids were in the 0.1-10 microg kg(-1) range.

Unambiguous identification of thiouracil residue in urine collected in non-treated bovine by tandem and high-resolution mass spectrometry.

Thyrostats are banned compounds in Europe since 1981 (directive 81/602/EC) because of their carcinogenic and teratogenic properties. However, the occurrence of thiouracil (TU) in bovine urines from national monitoring plans with quantifications in the range 1-10 microg . L(-1) occasionally raises the question of its origin which might either be the consequence of an illegal administration or the result of 'endogenous' production. In order to definitively and unambiguously identify the so-called thiouracil signal in non-treated bovine urines, independent mass spectrometry (MS) approaches have been used. Different reagents (3-IBBr, 3-BrBBr and PFBBr) were used to derivatise and to extract TU from urine samples and characterisation of the residues was performed by means of different MS approaches [LC/(ESI-)MS/MS, GC/(EI+)MS/MS and HRMS (EI and NCI)]. These combined strategies allowed for an independent and confident identification of TU in bovine urine samples collected from animals never treated with any thyrostatic drugs. This result is of prime importance for laboratories and risk managers involved in the field of forbidden growth promoters control: detection of TU residue in bovine urine will have to be carefully considered as a non-systematic proof of illegal administration.