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

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

Long-term performance and microbial dynamics of an up-flow fixed bed reactor established for the biodegradation of fluorobenzene

An up-flow fixed bed reactor (UFBR) was established to investigate the biodegradation of fluorobenzene (FB) under a number of operating conditions, which included variation in the concentration of FB in the feed stream (up to 180 mg l−1) and temporary suspension of feeding. Degradation of FB was followed for a period of 8 months under a continuous flow regime. During the operation of the UFBR, FB was never detected in the reactor effluent, being biodegraded by the microbial biofilm or adsorbed to the granular activated carbon (GAC). Biodegradation of FB was observed from the beginning of the reactor operation, and overall, it accounted for 50% of the total amount fed to the bioreactor. High organic loads of FB (210–260 mg d−1 dm−3) were found to affect the biological removal efficiency, possibly due to an inhibitory effect caused by the higher FB concentrations fed to the bioreactor (149–179 mg l−1). When FB feeding was suspended for 1 month, biodegradation continued, indicating that the adsorbed FB became bioavailable. Biofilm bacterial dynamics were followed throughout the UFBR operation by denaturing gradient gel electrophoresis and plate-counting techniques, showing that a quite stable community was found in the bioreactor, and this was mainly attributed to the high selective pressure exerted by the presence of FB

Assessment of hydrocarbon pollution in NW Iberian Peninsula using bioaccumulation and molecular biomarkers in Mytilus galloprovincialis

Society of Environmental Toxicology and Chemistry - SETAC Europe 15th Annual Meeting, Lille, France, May 2005.Polycyclic aromatic hydrocarbons (PAH´s) are ubiquitous contaminants in marine environment as a result of uncontrolled spills, river transport, surface runoff and atmospheric deposition. A significant amount of industrial activity including shipping and oil refining is located along the NW Iberian Peninsula coast. The use of exposure biomarkers holds out promise due to the incipient state of the cost-effective methodologies for diagnosis and monitoring of oil pollution. This work presents the preliminary results concerning the identification of a set of biomarkers for an early warning detection of PAH toxicity. The bivalve Mytillus galloprovincialis was selected due to its ubiquitous distribution along coastline, being used as sentinels in pollution monitoring. This species has also an important value. Four locations in the vicinity of industrial wastewater discharges along the NW Iberian coast were selected and compared with a nearby (reference) site for (i) measurements of PAH body burdens and (ii) levels of enzyme activity: catalase (CAT), superoxide-dismutase (SOD), glutathione peroxidase (GPx), gluthathione S-transferase (GST) and Na+/K+ATPase (ATPase). The results will be discussed on the basis of their potential in providing additional evidence for discriminate between well known polluted and unpolluted sites

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

Time course and mechanisms of left ventricular systolic and diastolic dysfunction in monocrotaline-induced pulmonary hypertension

Although pulmonary hypertension (PH) selectively overloads the right ventricle (RV), neuroendocrine activation and intrinsic myocardial dysfunction have been described in the left ventricle (LV). In order to establish the timing of LV dysfunction development in PH and to clarify underlying molecular changes, Wistar rats were studied 4 and 6 weeks after subcutaneous injection of monocrotaline (MCT) 60 mg/kg (MCT-4, n = 11; MCT-6, n = 11) or vehicle (Ctrl-4, n = 11; Ctrl-6, n = 11). Acute single beat stepwise increases of systolic pressure were performed from baseline to isovolumetric (LVPiso). This hemodynamic stress was used to detect early changes in LV performance. Neurohumoral activation was evaluated by measuring angiotensin-converting enzyme (ACE) and endothelin-1 (ET-1) LV mRNA levels. Cardiomyocyte apoptosis was evaluated by TUNEL assay. Extracellular matrix composition was evaluated by tenascin-C mRNA levels and interstitial collagen content. Myosin heavy chain (MHC) composition of the LV was studied by protein quantification. MCT treatment increased RV pressures and RV/LV weight ratio, without changing LV end-diastolic pressures or dimensions. Baseline LV dysfunction were present only in MCT-6 rats. Afterload elevations prolonged tau and upward-shifted end-diastolic pressure dimension relations in MCT-4 and even more in MCT-6. MHC-isoform switch, ACE upregulation and cardiomyocyte apoptosis were present in both MCT groups. Rats with severe PH develop LV dysfunction associated with ET-1 and tenascin-C overexpression. Diastolic dysfunction, however, could be elicited at earlier stages in response to hemodynamic stress, when only LV molecular changes, such as MHC isoform switch, ACE upregulation, and myocardial apoptosis were present.Supported by Portuguese grants from FCT (POCI/SAU-FCF/60803/2004 and POCI/SAU-MMO/61547/2004) through Cardiovascular R&D Unit (FCT No. 51/94)