Quantification of steroids and endocrine disrupting chemicals in rat ovaries by LC-MS/MS for reproductive toxicology assessment

International audienceReproductive function is controlled by a finely tuned balance of androgens and estrogens. Environmental toxicants, notably endocrine disrupting chemicals (EDCs), appear to be involved in the disruption of hormonal balance in several studies. To further describe the effects of selected EDCs on steroid secretion in female rats, we aim to simultaneously investigate the EDC concentration and the sex hormone balance in the ovaries. Therefore, an effective method has been developed for the quantification of the sex steroid hormones (testosterone, androstenedione, estradiol, and estrone) and four endocrine disrupting chemicals (bisphenol A, atrazine, and the active metabolites of methoxychlor and vinclozolin) in rat ovaries. The sample preparation procedure is based on the so-called "quick, easy, cheap, effective, rugged, and safe" approach, and an analytical method was developed to quantify these compounds with low detection limits by liquid chromatography coupled with a tandem mass spectrometer. This analytical method, applied to rat ovary samples following subacute EDC exposure, revealed some new findings for toxicological evaluation. In particular, we showed that EDCs with the same described in vitro mechanisms of action have different effects on the gonadal steroid balance. These results highlight the need to develop an integrative evaluation with the simultaneous measurement of EDCs and numerous steroids for good risk assessment

Quantification of pharmaceuticals and endocrine disruptors in river sediments: development and validation of a QuEChERS-based extraction.

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Quantification of pharmaceuticals and endocrine disruptors in river sediments: development and validation of a QuEChERS based extraction.

communication par afficheInternational audienceAccessing exposure concentrations can be challenging for the analyst as environmental concentrations and toxicological or eco-toxicological effect induced quantities are often not consistent with analysis capacities. To encounter those limitations analysts use pre-concentration and purification steps like Accelerated Solvent Extraction (ASE) and Solid Phase Extraction (SPE) to extract and analyse pharmaceuticals or hormones at the lower or sub ng/g level from solid matrices. A recent extraction method known as Quick Easy Cheap Effective Rugged and Safe has been developed for the analysis of pesticides in food matrices. This method has already been extended to various matrices like rats' serum and gonads in our laboratory. We developed and validated a multiresidue analytical method with QuEChERS extraction for the screening of multi-families of pharmaceuticals and endocrine disruptors in river sediments. The molecules were chosen among the priority pollutants listed in a prioritisation for French surface waters, in the Water Framework Directive and within the potential or confirmed EDCs. This selection of 13 pharmaceuticals includes β-blockers, anti-inflammatories, antibiotics, anxiolitics, anti-depressants and anti-analgesics 24 EDCs includes pesticides, alkylphenols, hormones and phenolic derivates and. Quantification is performed on a 3200QTrap tandem mass spectrometer (MS/MS) coupled to Liquid Chromatography (LC) Agilent 1200 system with a Kinetex XB-C18 column (100x2.1mm, 1.7μm). Extraction was optimised to get acceptable recoveries (>60%) and the linearity of the method has been verified by statistical means. Acceptable Relative Standard Deviations of both injection and preparation steps (<20 to 30%) were calculated for intraday injections of 3 independent solutions. Repetition of a calibration over three days allowed us to calculate good interday RSD. Limits of quantification were determined by injection of 5 independent standard solutions and were consistent with environmental concentrations

Approche en chimie environnementale : de la quantification ciblée de contaminnats à l'évaluation de la contamination du milieu aquatique par une approche non ciblée.

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Determination of endocrine disruptors and endogenic androgens and estrogens in rat serum by high-performance liquid chromatography–tandem mass spectrometry

International audienceTo simultaneously measure some targeted endocrine disruptors and several forms of sex hormones in rat serum, an accurate analytical procedure was developed. First, a comparison between a polymeric-based solid-phase extraction (SPE) and a micro-extraction by packed sorbent was performed to choose the optimal method to extract and concentrate the analytes: bisphenol A, atrazine, vinclozolin metabolite, testosterone, androstenedione, estrone, estradiol, estrone-sulfate and glucuronide and estradiol-sulfate and glucuronide. The analyses were then performed by high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) with electrospray ionisation in positive and negative modes. The protocol based on SPE was validated using the ICH/2005 guidelines. The validation demonstrated good performance in terms of linearity (R2 > 0.99), recovery (71–90%) and repeatability (relative standard deviation: 1–18%). The method was sensitive with LOQ comprised between 0.1 and 0.4 ng/ml for androgens and between 0.098 and 10.2 ng/ml for estrogens. The results obtained on the serum of rats exposed to the targeted endocrine disruptors showed the suitability of this analytical strategy

Survey regarding the occurrence of selected organic micropollutants in the groundwaters of overseas departments

The authors wish to acknowledge INERIS (Institut National de l'Environnement Industriel et des Risques) for their technical support and the field technicians for the providing the samples.International audienceTo collect a complete dataset regarding the occurrence of organic substances in groundwater, this study presents the examination of 66 organic contaminants in the groundwater of overseas departments, including pesticides, pharmaceutical compounds, hormones and some industrial substances. The selective and sensitive analytical methods are described. These techniques begin with solid-phase extraction (SPE) followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatographymass spectrometry (GC-ToF-MS). The paper summarises the analytical results from 40 sampling points collected during two campaigns in Guadeloupe, Martinique, Reunion, Mayotte and Guiana, representing 80 samples. Of the 66 target substances, 36 were determined at least once. Among the most frequently detected are bisphenol A (frequency, 96 %; max., 7,400 ng/L), caffeine (frequency, 91 %; max., 1,240 ng/L), pentachlorophenol (frequency, 55 %; max., 418 ng/L), and carbamazepine (frequency, 56 %; max., 22 ng/L). The results do not put in evidence that the origin of the sample or climatic characteristics of these regions influence the dilution and release of micropollutants

Characterization of endocrine-disrupting chemicals based on hormonal balance disruption in male and female adult rats

International audienceReproductive functions are controlled by a finely tuned balance between estrogens and androgens. To further characterize the gonadal pathways leading to hormonal balance disruption by atrazine, vinclozolin, methoxychlor, and bisphenol A in rat, we investigated their effects in male and female young adult animals. Specifically, we assessed reproductive tract alterations, sex hormone balance in serum and gonads, tissue dosimetry, and mRNA expression. Remarkably, we observed different aromatase regulation profiles between animals with similar estrogen-to-androgen ratios but with different chemical treatments. For example, increased estrogen-to-androgen ratios in atrazine-treated females could be partly linked to aromatase upregulation, while in methoxychlor- and bisphenol A-treated females, peripheral mechanisms such as conjugation/deconjugation processes might be more likely to elevate estrogen levels. In vinclozolin-treated animals, the decreased estrogen-to-androgen ratios reported might be due to an increase of peripheral (adrenal) steroidogenesis. Thus, measurement of many endpoints is necessary for good risk assessment. Highlights Hormonal balance as a relevant endpoint for reproductive toxicology assessment. Characterization of gonadal pathways involved in endocrine disruption. Discrepancy between in vitro chemicals classification and in vivo reality. Several well chosen complementary endpoints are required for good risk assessment

How to evaluate pollution, degradation and metabolisation of multi-families of pharmaceuticals and endocrine disruptors in multiple compartments of the surface water media?

communication par afficheInternational audienceEvaluations like risks or effects assessments of the presence of various xenobiotics in the environment show a continuously growing interest since the past decades. To establish models for degradation of a family of pharmaceuticals and thus evaluate its impact on the environment, scientists need good analytical methods to reinforce databases. While literature focuses on single matrices or single family, this work aims to develop an integrative approach of the surface water media: water, sediments and biota. Those methods are combined to relatively simple and short time-consuming extraction techniques investigated for various matrices for the 20 various pharmaceuticals and 28 Endocrine Disruptor. NanoLC-MS/MS method has already been applied to a benthic model exposed for eco-toxicological study. All presented LC-MS/MS, automated online-SPE-LC-MS/MS and nanoLC-MS/MS methods have been validated following ICH recommendations. They allow rapid quantification of multiple pharmaceuticals and endocrine disruptors at environmental levels and have been for the study of real ecosystems in French rivers. In parallel thanks to passive samplers calibrated in the laboratory and validated in situ with PRCs, real time weighted average concentration can be determined in surface water. Exposing the benthic model to those concentrations should allow not only the quantification of accumulated and metabolised compounds in biota but also degradation products and coefficient partitioning between water and sediments. Those studies even could correlate fate to effects observed on benthic models. Further investigations with TOF spectrometers should allow non-targeted analysis for identification of new by-products, degradation products or metabolites. Although this analytical challenge is needed for risk and effects assessments on environment and populations, it is really time-consuming for the analyst. Prioritisation of substances is crucial to select representative pollutants to help scientists building models. This project opens the discussion for assessments of multi-families of various ubiquitous pollutants with a relatively large panel of analysis for different matrices of the same water surface media

How to evaluate pollution, degradation and metabolisation of multi-families of pharmaceuticals and endocrine disruptors in multiple compartments of water surface media.

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Comparaison des extractions MEPS et SPE pour la quantification par LC-MS/MS d'androgènes et d'oestrogènes sous forme libres et conjugués dans du sérum de rat.

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