Sea bass (Dicentrarchus labrax) : a model organism for assessing multi-level responses to estrogenic chemicals in marine surface waters

Resumo apresentado sob poster ao 5th International Symposium of Fish Endocrinology, CAstellon, Spain, Setember 5-9, 2004.There is growing concern that aquatic wildlife in surface waters of the European Union is exposed to natural and man-made chemicals that have the ability to mimic estrogens and lead to reproductive dysfunction. Estrogenic responses in fish are the net result of complex chains of events involving the uptake, distribution and metabolism of test agents until they interact with their target sites. Typically these aspects cannot be modelled in short-term cell-based assays, only studies with vertebrates offer the opportunity to assess potential interactions of test compounds at higher organisational levels. The most widely studied biological response in fish to environmental estrogens is the production of vitellogenin (Vtg). However, few studies have attempted to link this endpoint with effects on xenobiotic biotransformation enzymes and genotoxic responses. This work is part of a study focusing on the combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms. As test organism the sea bass (Dicentrarchus labrax) was selected, a common species in European marine systems. Juveniles were exposed under a flow-through system for 14 days to the natural estrogen 17ß-estradiol and ethynylestradiol. Actual chemical concentrations in the water-column were determined by gas chromatography with ion trap detection. Effects at subcellular level were analysed using Vtg as a reference endpoint [1]. Its relevance is evaluated by further investigations on liver 7-ethoxyresorufin-O-deethylase (EROD) activity and erythrocytic nuclear abnormalities [2]. These measurements were integrated with organism level endpoints (i.e. condition factor, hepatossomatic index) to provide evidence for cause-effect of estrogenic contamination. The general suitability of the sea bass as a model organism for the screening of estrogenic chemicals in the marine environment is discussed.Comissão Europeia (CE) - ACE, EVK1-CT-2001-100

Sea bass (Dicentrarchus labrax) : a model organism for the screening of estrogenic chemicals in marine surface waters?

Society of Environmental Toxicology and Chemistry - SETAC Europe 14th Annual Meeting, Prague, Czech Republic, April 2004.There is growing concern that aquatic wildlife in surface waters of the European Union is exposed to natural and man-made chemicals that have the ability to mimic estrogens and lead to reproductive dysfunction. Estrogenic responses in fish are the net result of complex chains of events involving the uptake, distribution and metabolism of test agents until they interact with their target sites. Typically these aspects cannot be modelled in short-term cell-based assays, only studies with vertebrates offer the opportunity to assess potential interactions of test compounds at higher organisational levels. However, studies with endocrine disrupting chemicals have been performed mainly with freshwater organisms. The sensitivity of a marine fish species to different estrogenic chemicals was investigated under chronic exposure conditions. This work is part of a study focusing on the combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms (ACE, EVK1-CT-2001-100). As test organism the sea bass (Dicentrarchus labrax) was selected, a common species in European marine systems. Juveniles were exposed under a flow-through system for 14 days for a set of reference chemicals (17Ã -estradiol, ethynylestradiol, nonylphenol, octylphenol, bisphenol A). Effects at subcellular level were analysed using vitellogenesis as endpoint. Its relevance is evaluated by further investigations about the individual fitness (condition factor, hepatossomatic index), as well as the liver cytochrome P450 activity. The general suitability of the sea bass as a model organism for the screening of estrogenic chemicals in the marine environment is discussed.Comissãoo Europeia (CE) - ACE, EVK1-CT-2001-100

High resolution effect-directed analysis of steroid hormone (ant)agonists in surface and wastewater quality monitoring

Monitoring of chemical water quality is extremely challenging due to the large variety of compounds and the presence of biologically active compounds with unknown chemical identity. Previously, we developed a high resolution Effect-Directed Analysis (EDA) platform that combines liquid chromatography with high resolution mass spectrometry and parallel bioassay detection. In this study, the platform is combined with CALUX bioassays for (anti)androgenic, estrogenic and glucocorticoid activities, and the performance of the platform is evaluated. It appeared to render very repeatable results, with high recoveries of spiked compounds and high consistency between the mass spectrometric and bioassay results. Application of the platform to wastewater treatment plant effluent and surface water samples led to the identification of several compounds contributing to the measured activities. Eventually, a workflow is proposed for the application of the platform in a routine monitoring context. The workflow divides the platform into four phases, of which one to all can be performed depending on the research question and the results obtained. This allows one to make a balance between the effort put into the platform and the certainty and depth by which active compounds will be identified. The EDA platform is a valuable tool to identify unknown bioactive compounds, both in an academic setting as in the context of legislative, governmental or routine monitoring

Sea bass (Dicentrarchus labrax) as a potential bioindicator of estrogenic contamination in marine surface waters

Resumo apresentado sob poster apresentado ao 5th International Symposium on Fish Endocrinology, September, 2004, Castellon, Spain.Exposure of aquatic wildlife in surface waters to (xeno-)estrogens is known to cause reproductive dysfunction. Estrogenic responses in fish are the net result of complex chains of events that will depend on a number of factors, such as bioavailability, bioconcentration/bioaccumulation, and biotransformation. Most of known estrogenic chemicals are lipophilic and hydrophobic and therefore have a strong potential to accumulate in aquatic biota. Therefore, determining environmental exposures may be very difficult and not be particularly meaningful. As test organism the sea bass (Dicentrarchus labrax) was selected, a common species in European marine systems. This work is part of a study focusing on the combination effects of mixtures of estrogenic chemicals in marine and freshwater organisms. Juvenile sea bass were used in order to analyse the bioconcentration and distribution among different tissues of the chemical residues of a set of reference estrogenic chemicals such as 17ß-estradiol (E2), ethynylestradiol (EE2), nonylphenol (NP), octylphenol (OP), bisphenol-A (BPA). Fish were exposed for a period of two weeks to environmentally relevant levels of these compounds, after which liver, bile, muscle, gill and kidney were collected and analyzed. Actual concentrations of E2, EE2 and BPA seawater in the tanks were determined by either gas chromatography with ion trap detection or HPLC coupled to diode array detection. In bile, levels of BPA were determined according to a method presented earlier by Houtman et al. (13th Annual Meeting SETAC Europe, 2003). Actual NP and OP concentrations in both water and tissues were determined by HPLC-ESI-MS according to recently developed methods by Pojana et al. (J. Anal. Chem., in press). Bioconcentration and distribution of residual compounds in tissues were correlated to the levels of plasma vitellogenin (results are presented also at this conference) and to actual exposure concentrations. The general suitability of the sea bass as a bioindicator of estrogenic contamination in the marine environment is discussed.Comissão Europeia (CE) - ACE, EVK1-CT-2001-100

The ACE Project: a synopsis of in vivo studies to predict estrogenic mixture effects in freshwater and marine fish

Society of Environmental Toxicology and Chemistry - SETAC Europe 15th Annual Meeting, Lille, France, May 2005.This work is part of the ACE project (ACE, EVK1-CT-2001-100) which aim is to investigate multi-component mixtures of estrogenic compounds in aquatic ecosystems. Here we present a synopsis of in vivo data related with the joint estrogenic action of five estrogenic compounds (17ß-estradiol, ethynylestradiol, nonylphenol, octylphenol and bisphenol-A) on vitellogenesis in fathead minnow (Pimephales promelas) and sea bass (Dicentrarchus labrax). The studies were conducted with freshwater adult males and marine juveniles under flow through exposure conditions for two weeks. In the first step, fish were exposed to the five compounds individually in order to generate concentration- response curves. Therefore mixture effects were predicted on the basis of the potency of each compound by using the model of concentration addition (CA). Finally, the compounds were tested as a mixture at equipotent concentrations, and the observed mixture effects were compared to the predictions. The mixture studies showed an good agreement between observed and predicted effects and provided evidence that CA can be used as a predictive tool for the effect assessment of mixtures of (xeno)estrogens in freshwater or marine ecosystems. The differences/limitations of running in vivo mixture studies with freshwater and marine species will be discussed.Comissão Europeia (CE) - ACE project - ACE, EVK1-CT-2001-100

Mixture Risk Assessment of Complex Real-Life Mixtures—The PANORAMIX Project

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union’s Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising ‘real-life’ chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children’s development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.Horizon 2020 research and innovation programme, the Green Deal project PANORAMIX Grant Agreement No. 10103663

Analysis of Lipid Metabolism, Immune Function, and Neurobehavior in Adult C57BL/6JxFVB Mice After Developmental Exposure to di (2-ethylhexyl) Phthalate

Background: Developmental exposure to di (2-ethylhexyl) phthalate (DEHP) has been implicated in the onset of metabolic syndrome later in life. Alterations in neurobehavior and immune functions are also affected by phthalate exposure and may be linked to the metabolic changes caused by developmental exposure to DEHP.Objectives: Our goal was to study the effects of developmental exposure to DEHP in the context of metabolic syndrome by integrating different parameters to assess metabolic, neurobehavioral, and immune functions in one model.Methods: Female C57BL/6J mice were exposed to DEHP through the diet during gestation and lactation at doses ranging from 3.3 to 100,000 μg/kg body weight/day (μkd). During a 1-year follow-up period, a wide set of metabolic parameters was assessed in the F1 offspring, including weekly body weight measurements, food consumption, physical activity, glucose homeostasis, serum lipids, and endocrine profile. In addition, neurobehavioral and immune functions were assessed by sweet preference test, object recognition test, acute phase protein, and cytokines production. Animals were challenged with a high fat diet (HFD) in the last 9 weeks of the study.Results: Increased free fatty acids (FFA) and, high density lipoprotein (HDL-C) were observed in serum, together with a decrease in glycated hemoglobin levels in blood of 1-year old male DEHP-exposed offspring after HFD challenge. For the most sensitive endpoint measured (FFA), a lower bound of the 90%-confidence interval for benchmark dose (BMD) at a critical effect size of 5% (BMDL) of 2,160 μkd was calculated. No persistent changes in body weight or fat mass were observed. At 33,000 μkd altered performance was found in the object recognition test in males and changes in interferon (IFN)γ production were observed in females.Conclusions: Developmental exposure to DEHP combined with HFD in adulthood led to changes in lipid metabolism and neurobehavior in male offspring and cytokine production in female offspring. Our findings contribute to the evidence that DEHP is a developmental dyslipidemic chemical, however, more research is needed to further characterize adverse health outcomes and the mechanisms of action associated with the observed sex-specific effects

Effect-Directed Analysis of Municipal Landfill Soil Reveals Novel Developmental Toxicants in the Zebrafish Danio rerio

Effect-directed analysis (EDA) is an approach used to identify (unknown) contaminants in complex samples which cause toxicity, using a combination of biology and chemistry. The goal of this work was to apply EDA to identify developmental toxicants in soil samples collected from a former municipal landfill site. Soil samples were extracted, fractionated, and tested for developmental effects with an embryotoxicity assay in the zebrafish Danio rerio. Gas chromatograph mass selective detection (GC-MSD) chemical screening was used to reveal candidate developmental toxicants in fractions showing effects. In a parallel study, liquid chromatography-hybrid linear ion trap Orbitrap mass spectrometry was also applied to one polar subfraction (Hoogenboom et al. J. Chromatogr. A2009, 1216, 510-519). EDA resulted in the identification of a number of previously unknown developmental toxicants, which were confirmed to be present in soil by GC-MS. These included 11H-benzo[b]fluorene, 9-methylacridine, 4-azapyrene, and 2-phenylquinoline, as well as one known developmental toxicant (retene). This work revealed the presence of novel contaminants in the environment that may affect vertebrate development, which are not subject to monitoring or regulation under current soil quality assessment guidelines. © 2011 American Chemical Society

Identification strategy for unknown pollutants using high-resolution mass spectrometry: Androgen-disrupting compounds identified through effect-directed analysis

Effect-directed analysis has been applied to a river sediment sample of concern to identify the compounds responsible for the observed effects in an in vitro (anti-)androgenicity assay. For identification after non-target analysis performed on a high-resolution LTQ-Orbitrap, we developed a de novo identification strategy including physico-chemical parameters derived from the effect-directed analysis approach. With this identification strategy, we were able to handle the immense amount of data produced by non-target accurate mass analysis. The effect-directed analysis approach, together with the identification strategy, led to the successful identification of eight androgen-disrupting compounds belonging to very diverse compound classes: an oxygenated polyaromatic hydrocarbon, organophosphates, musks, and steroids. This is one of the first studies in the field of environmental analysis dealing with the difficult task of handling the large amount of data produced from non-target analysis. The combination of bioassay activity assessment, accurate mass measurement, and the identification and confirmation strategy is a promising approach for future identification of environmental key toxicants that are not included as priority pollutants in monitoring programs