Pesticide exposure of pregnant women in Guadeloupe: Ability of a food frequency questionnaire to estimate blood concentration of chlordecone

Context Chlordecone, an environmentally persistent organochlorine insecticide used intensively in banana culture in the French West Indies until 1993, has permanently polluted soils and contaminated foodstuffs. Consumption of contaminated food is the main source of exposure nowadays. We sought to identify main contributors to blood chlordecone concentration (BCC) and to validate an exposure indicator based on food intakes.Material and methods We used a food frequency questionnaire (FFQ) completed by a sample of 194 pregnant women to estimate their dietary exposure to chlordecone and compared it to blood levels. In a first approach, chlordecone daily intake was estimated as the product of daily eaten quantity of 214 foodstuffs, multiplied by their chlordecone content, and summed over all items. We then predicted individual blood chlordecone concentration with empirical weight regression models based on frequency of food consumption, and without contamination data.Results Among the 191 subjects who had BCC determination, 146 (76%) had detectable values and mean BCC was 0.86†ng/mL (range < LOD-13.2). Mean per capita dietary intake of chlordecone was estimated at 3.3†[mu]g/day (range: 0.1-22.2). Blood chlordecone levels were significantly correlated with food exposure predicted from the empirical weight models (r=0.47, p<0.0001) and, to a lesser extent, with chlordecone intake estimated from food consumption and food contamination data (r=0.20, p=0.007). Main contributors to chlordecone exposure included seafood, root vegetables, and Cucurbitaceous.Conclusion These results show that the Timoun FFQ provides valid estimates of chlordecone exposure. Estimates from empirical weight models correlated better with blood levels of chlordecone than did estimates from the dietary intake assessment

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project

Impacts et mécanismes d'action de dérégulateurs endocriniens sur le gastéropode dulçaquicole hermaphrodite Lymnaea stagnalis (Linnaeus, 1758)

Adverse impacts exerted by molecules called endocrine disruptors on reproduction have been extensively described in vertebrates. However, endocrine-disrupting chemicals might alter reproduction of invertebrate species, at lower concentrations or through different modes of action, with consequences on population growth and stability. Among invertebrates, molluscs have been shown to be very sensitive to endocrine disruptors and were therefore proposed as model species for the development of OECD guideline for the testing and the assessment of reprotoxic effect of chemicals, including EDCs. The hermaphrodite gastropod species Lymnaea stagnalis is a candidate species for the development of such guidelines. In this context, the aim of this thesis is to provide a better understanding of the impacts and the mechanisms of action of endocrine disrupting chemicals on the reproduction of L. stagnalis. The chemicals tested in the present work were chosen based on their interactions with the endocrine system of vertebrates. Testosterone is the natural ligand of the androgen receptor in vertebrates. Three different molecules were selected for their antagonism of the androgen receptor in vertebrates: the synthetic steroid cyproterone acetate, the dicarbamide fungicide vinclozolin and the organophosphate insecticide fenitrothion. Chlordecone is an organochlorinated pesticide, which binds oestrogen receptors in vertebrates. Finally, two organotin compounds, triphenyltin and tributyltin, are able to induce the development of imposex, imposition of male sex organs in females, in gonochoric gastropod molluscs. Results of the present work are divided into two main sections. Firstly, we investigated the impacts of the selected chemicals on the reproduction of L. stagnalis. Our results displayed that TBT, TPT, and chlordecone are able to induce adverse impacts on the reproduction of L. stagnalis through a reduction of the cumulative number of eggs and clutches produced per individual. The assessment of the quality of eggs produced showed that exposure to the chemicals, except TPT, increases the frequency of abnormal eggs laid. The impacts on egg quality were observed at lower concentrations than those affecting the classical reproductive endpoints assessed in reprotoxicity tests in this species. Our results highlight that the implementation of egg quality as an endpoint should increase the sensitivity of the reprotoxicity test with L. stagnalis. The second section of results aims at providing new insights on the mechanisms of action of putative endocrine disruptors in L. stagnalis. Results from chapter 3 and 4 allowed us to select chemical treatments (testosterone, tributyltin, cyproterone acetate, and chlordecone) that induced adverse effects, by interacting with the endocrine system, on the reproduction of L. stagnalis. In Chapter 5, we analysed endogenous concentrations of testosterone, in its free and esterified forms, in the reproductive organs of L. stagnalis. Exposure to waterborne testosterone has confirmed that molluscs can take up this steroid from environmental media and that a homeostatic regulation of endogenous concentrations occurs through the esterification of steroids to fatty acid esters. Moreover, our results support that testosterone is endogenously synthesised in L. stagnalis, as it has been suggested in other mollusc species. However, the impacts on the reproduction and the modifications of endogenous concentrations of testosterone reported in this thesis could not be associated. In chapter 6, proteomic experiments were used to identify proteins differently expressed in reproductive organs of L. stagnalis exposed to testosterone, tributyltin, cyproterone acetate, and chlordecone. Modifications in the expression of three proteins involved in L. stagnalis reproduction (i.e., ovipostatin, yolk ferritin and PIWI) could be associated with the alterations of reproductive endpoints reported in chapters 3 and 4, therefore suggesting possible endocrine disruption mechanisms. Altogether, the results obtained in the present work provide evidences of interactions between chemicals and the endocrine system of this hermaphrodite species. Moreover, the additional sensitive reproductive endpoints described in this thesis (i.e., egg quality) as well as the identification of particular proteins (i.e., ovipostatin, yolk ferritin and PIWI) and their differential expression patterns following chemical exposure, provide reliable tools to screen potency of toxicants to be endocrine disruptors in a partial life cycle test on the freshwater gastropod Lymnaea stagnalis.Des molécules connues pour leur capacité d’interférer avec le système endocrinien des vertébrés peuvent engendrer, à de faibles concentrations, des effets néfastes sur la reproduction des invertébrés. Ces produits, appelés perturbateurs endocriniens, peuvent à terme avoir des conséquences préjudiciables sur la croissance et la survie des populations exposées. Parmi les espèces d’invertébrés touchées par les perturbateurs endocriniens, les mollusques voient leur reproduction affectée par de nombreuses molécules à des concentrations inférieures que celles atteignant d’autres phyla. Les mollusques ont donc été proposés comme espèce-modèle pour l’évaluation de la reprotoxicité des produits chimiques, dont font partie les perturbateurs endocriniens, dans les guidelines développées par l’OCDE. Lymnaea stagnalis, une espèce de gastéropode dulçaquicole hermaphrodite, a été proposée comme espèce de référence pour le développement de ce type de test. Le premier objectif de cette thèse consiste à définir les impacts de différentes molécules, choisies sur base de leurs interactions avec le système endocrinien des vertébrés, sur la reproduction de L. stagnalis. Son deuxième objectif vise à apporter une meilleure compréhension des mécanismes d’action de ces perturbateurs endocriniens. Dans ce travail, nous avons testé plusieurs produits choisis sur base de leurs interactions avec le système endocrinien des vertébrés. La testostérone est le ligand naturel du récepteur aux androgènes. Trois autres molécules sont des antagonistes avérés de ce récepteur : l’acétate de cyprotérone qui est un stéroïde synthétique, la vinclozoline qui est un fongicide dicarbamide, et le fénitrothion qui est un insecticide organophosphoré. La chlordécone, un pesticide organochloré, peut se lier au récepteur aux oestrogènes. Enfin, les deux organoétains, triphenyltin (TPT) and tributyltin (TBT), peuvent induire une masculinisation des femelles, consistant en la formation additionnelle de caractères masculins (l’imposexe) chez les gastéropodes prosobranches. Les résultats de ce travail sont divisés en deux volets. Le premier regroupe les résultats obtenus sur les impacts de ces substances sur la reproduction de L. stagnalis. Le nombre total d’œufs (fécondité) et de pontes (oviposition) produits par individu sur toute la période d’exposition à ces molécules nous a permis d’évaluer l’impact des différentes substances. De plus, l’analyse de la qualité des œufs produits a montré que toutes les molécules testées, excepté le TPT, peuvent augmenter la fréquence des œufs malformés produits par les individus exposés. Les œufs polyembryonés (contenant plusieurs embryons par œuf) sont les plus fréquemment produits parmi tous les œufs malformés. Etant donné que ces effets sur la qualité des œufs sont observés à des concentrations plus faibles que celles affectant l’oviposition et la fécondité, l’inclusion de ce paramètre lors de l’analyse des impacts de reprotoxicité sur L. stagnalis pourrait améliorer la sensibilité des tests, particulièrement pour l’étude des perturbateurs endocriniens. Le second volet des résultats, présentés dans les chapitres 3 et 4, nous a permis de mieux comprendre les mécanismes d’action des molécules induisant des effets sur la reproduction de L. stagnalis suite à des interactions probables avec le système endocrinien de cette espèce. Dans le chapitre 5, nous avons analysé les impacts de la testostérone, de l’acétate de cyprotérone, de la chlordécone et du tributylétain sur les concentrations en testostérone sous forme libre et estérifiée présentes dans les tissus d’individus exposés pendant 21 jours. La testostérone est une hormone stéroïdienne de « type vertébré » qui a été isolée dans les tissus de nombreuses espèces de mollusques. Nos résultats ont confirmé que les lymnées peuvent incorporer ce stéroïde depuis le milieu environnant. De plus, chez cette espèce comme chez d’autres espèces de mollusques, une régulation homéostatique des concentrations en testostérone permet de stocker, dans les tissus adipeux, les excès de testostérone en forme libre sous une forme estérifiée. Cependant, dans le premier volet des résultats, les impacts de ces molécules sur la reproduction n’ont pu être mis en relation avec les modifications des concentrations en testostérone. Dès lors, le rôle joué par cette hormone stéroïde dans la reproduction de la lymnée n’a pu être élucidé. Dans le chapitre 6, des analyses protéomiques (2D-DIGE, western blot) ont été réalisées afin d’identifier les impacts de la testostérone, de l’acétate de cyprotérone, de la chlordécone et du tributylétain sur l’expression de protéines extraites des tissus reproducteurs des individus exposés pendant 21 jours. Ces techniques d’analyse nous ont permis d’identifier des protéines qui jouent un rôle dans la reproduction de cette espèce (yolk ferritin, ovipostatin et PIWI). Les altérations d’expression de ces protéines pourraient expliquer les effets de ces molécules sur la reproduction de L. stagnalis observés dans les chapitres précédents. En intégrant les résultats obtenus au cours de cette thèse, il apparaît que les produits testés peuvent induire des impacts néfastes sur la reproduction en interagissant avec le système endocrinien de Lymnaea stagnalis. De plus, l’étude de la qualité des œufs, ainsi que l’analyse de protéines impliquées dans la reproduction, constituent indéniablement des outils utiles pour évaluer la reprotoxicité de ces molécules chez Lymnaea stagnalis

Esterification of vertebrate like steroids in molluscs: A target of endocrine disruptors?

Alterations of the reproductive organs of gastropod molluscs exposed to pollutants have been reported in natural populations for more than 40 years. In some cases these impacts have been linked to exposure to endocrine disrupting chemicals (EDCs), which are known to induce adverse impacts on vertebrates, mainly by direct binding to steroid receptors or by altering hormone synthesis. Investigations on the mechanisms of action of endocrine disruptors in molluscs show that EDCs induce modifications of endogenous titres of androgens (e.g., testosterone, androstenedione) and oestrogens (e.g., 17ß-oestradiol). Alterations of the activity of enzymes related to steroid metabolism (i.e., cytochrome P450 aromatase, acyltransferases) are also often observed. In bivalves and gastropods, fatty acid esterification of steroids might constitute the major regulation of androgen and oestrogen homeostasis. The present review indicates that metabolism of steroid hormones to fatty acid esters might be a target of synthetic EDCs. Alterations of this process would impact the concentrations of free, potentially bioactive, form of steroids

Selection and ranking method for currently used pesticides (CUPs) monitoring in ambient air

Chronic exposure to pesticides can induce adverse human health effects. Even though ingestion is considered as the main exposure pathway, it is now suggested that inhalation might also be important not only in rural but also in urban locations. Therefore, assessment of currently used pesticides (CUPs) concentrations in ambient air is important for better understanding of human exposure through inhalation and potential health effects. Analytical methods do not allow assessing ambient air concentration of all the CUPs registered. Designing a cost-effective and a fitted-for-purpose monitoring strategy at the local/regional scale must therefore rely on a methodology allowing targeting CUPs by a ranking approach accounting for the most relevant selection criteria. In this study, after a first selection, a ranking method is used to identify most relevant CUPs for ambient air assessment in Wallonia, Belgium. This method took into account not only toxicological endpoints but also national and regional data on sales and uses along with other uses criteria. Moreover, probability to detect CUPs in ambient air was investigated using international, national, and regional studies and physicochemphysicochemical properties. The ranking method used three main criteria (i.e., chronic toxicity, sales and uses, and presence in ambient air), which are divided in 17 sub-criteria, to provide the most accurate identification of CUPs that might be measured in ambient air and that might impact human health. After final selection based on analytical methods, 43 CUPs were further submitted to analytical method development

Testosterone levels and fecundity in the hermaphroditic aquatic snail Lymnaea stagnalis exposed to testosterone and endocrine disruptors

Endocrine disruptors are known to alter endogenous free and/or esterified levels of androgenic and estrogenic steroid hormones in aquatic molluscs. However, there is still a controversy on the origin of steroids in these animals. In this study, free and esterified testosterone concentrations were measured in the hermaphroditic aquatic gastropod Lymnaea stagnalis exposed to molecules known for their androgenic (testosterone and tributyltin), anti-androgenic (cyproterone-acetate) and estrogenic (chlordecone) properties by reference to their mode of action in vertebrates. In parallel, snail oviposition and fecundity were followed over the 21-day exposure period. Testosterone exposure resulted in increased esterified testosterone levels whereas free testosterone concentrations remained stable. In contrast, cyproterone-acetate increased significantly the free form of testosterone with no changes in the esterified form whereas chlordecone showed a tendency to reduce, though not significantly, esterified testosterone concentrations without changes in free testosterone levels. Finally, tributyltin did not alter testosterone homeostasis. The production of egg-clutches and eggs was significantly reduced only in the snails exposed to the highest concentrations of chlordecone (19.6 µg/L) and tributyltin (94.2 ng Sn/L). Overall, this study demonstrates that uptake of testosterone from the exposure medium occurs in L. stagnalis. Moreover, it shows that cyproterone-acetate and, to a lesser extent, chlordecone can alter endogenous testosterone levels in this freshwater snail. However, relationship between hormonal changes and snail reproduction has not been established

Impacts of androgenic and antiandrogenic substances on the freshwater pulmonate gastropod Lymnaea stagnalis

Knowledge on the impacts of endocrine disruptors on gastropods is scarce and their mechanism of action poorly understood especially the impacts of androgenic and antiandrogenic compounds. In this study effects of 5 androgenics and antiandrogenics endocrine disruptors were investigated on the reproduction and life traits of the freshwater pulmonate gastropod Lymnaea stagnalis. The chemicals tested were Tributyltin, Cyproterone acetate, Methyltestosterone, Vinclozolin and Fenitrothion. Tributyltin was used as androgenic positive control. The other compounds tested are androgenic and antiandrogenic compounds either steroids or non steroids. Adult snails were exposed to 3 concentrations of each chemical for 30 days. The size and reproductive organs development were monitored throughout exposure. The number of clutches, the number of eggs per clutch were assessed during the first 10 days. The clutches were kept individually in clear water except for the clutches of the eighth day which were divided in two. Half was kept individually in clear water and the other half was individually reared in contaminated water following the same exposure as their parents. The percentage of hatching per clutch of these clutches was measured. The development size, mortality, and development of reproductive organs of the juveniles were assessed until their first clutch was laid