Expression of Zebra Fish Aromatase cyp19a and cyp19b Genes in Response to the Ligands of Estrogen Receptor and Aryl Hydrocarbon Receptor

Many endocrine-disrupting chemicals act via estrogen receptor (ER) or aryl hydrocarbon receptor (AhR). To investigate the interference between ER and AhR, we studied the effects of 17β-estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of zebra fish cyp19a (zfcyp19a) and cyp19b (zfcyp19b) genes, encoding aromatase P450, an important steroidogenic enzyme. In vivo (mRNA quantification in exposed zebra fish larvae) and in vitro (activity of zfcyp19-luciferase reporter genes in cell cultures in response to chemicals and zebra fish transcription factors) assays were used. None of the treatments affected zfcyp19a, excluding the slight upregulation by E2 observed in vitro. Strong upregulation of zfcyp19b by E2 in both assays was downregulated by TCDD. This effect could be rescued by the addition of an AhR antagonist. Antiestrogenic effect of TCDD on the zfcyp19b expression in the brain was also observed on the protein level, assessed by immunohistochemistry. TCDD alone did not affect zfcyp19b expression in vivo or promoter activity in the presence of zebra fish AhR2 and AhR nuclear translocator 2b (ARNT2b) in vitro. However, in the presence of zebra fish ERα, AhR2, and ARNT2b, TCDD led to a slight upregulation of promoter activity, which was eliminated by either an ER or AhR antagonist. Studies with mutated reporter gene constructs indicated that both mechanisms of TCDD action in vitro were independent of dioxin-responsive elements (DREs) predicted in the promoter. This study shows the usefulness of in vivo zebra fish larvae and in vitro zfcyp19b reporter gene assays for evaluation of estrogenic chemical actions, provides data on the functionality of DREs predicted in zfcyp19 promoters and shows the effects of cross talk between ER and AhR on zfcyp19b expression. The antiestrogenic effect of TCDD demonstrated raises further concerns about the neuroendocrine effects of AhR ligand

An individual-based model of Zebrafish population dynamics accounting for energy dynamics

International audienceDeveloping population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebra-fish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level

Future water quality monitoring--adapting tools to deal with mixtures of pollutants in water resource management

Environmental quality monitoring of water resources is challenged with providing the basis for safeguarding the environment against adverse biological effects of anthropogenic chemical contamination from diffuse and point sources. While current regulatory efforts focus on monitoring and assessing a few legacy chemicals, many more anthropogenic chemicals can be detected simultaneously in our aquatic resources. However, exposure to chemical mixtures does not necessarily translate into adverse biological effects nor clearly shows whether mitigation measures are needed. Thus, the question which mixtures are present and which have associated combined effects becomes central for defining adequate monitoring and assessment strategies. Here we describe the vision of the international, EU-funded project SOLUTIONS, where three routes are explored to link the occurrence of chemical mixtures at specific sites to the assessment of adverse biological combination effects. First of all, multi-residue target and non-target screening techniques covering a broader range of anticipated chemicals co-occurring in the environment are being developed. By improving sensitivity and detection limits for known bioactive compounds of concern, new analytical chemistry data for multiple components can be obtained and used to characterise priority mixtures. This information on chemical occurrence will be used to predict mixture toxicity and to derive combined effect estimates suitable for advancing environmental quality standards. Secondly, bioanalytical tools will be explored to provide aggregate bioactivity measures integrating all components that produce common (adverse) outcomes even for mixtures of varying compositions. The ambition is to provide comprehensive arrays of effect-based tools and trait-based field observations that link multiple chemical exposures to various environmental protection goals more directly and to provide improved in situ observations for impact assessment of mixtures. Thirdly, effect-directed analysis (EDA) will be applied to identify major drivers of mixture toxicity. Refinements of EDA include the use of statistical approaches with monitoring information for guidance of experimental EDA studies. These three approaches will be explored using case studies at the Danube and Rhine river basins as well as rivers of the Iberian Peninsula. The synthesis of findings will be organised to provide guidance for future solution-oriented environmental monitoring and explore more systematic ways to assess mixture exposures and combination effects in future water quality monitoring.</p

Co-Carriage of Metal and Antibiotic Resistance Genes in Sewage Associated Staphylococci

Controlling spread of resistance genes from wastewater to aquatic systems requires more knowledge on how resistance genes are acquired and transmitted. Whole genomic sequences from sewage-associated staphylococcus isolates (20 S. aureus, 2 Staphylococcus warneri, and 2 Staphylococcus delphini) were analyzed for the presence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). Plasmid sequences were identified in each isolate to investigate co-carriage of ARGs and MRGs within. BLASTN analysis showed that 67% of the isolates carried more than one ARG. The carriage of multiple plasmids was observed more in CC5 than CC8 S. aureus strains. Plasmid exchange was observed in all staphylococcus species except the two S. delphini isolates that carried multiple MRGs, no ARGs, and no plasmids. 85% of S. aureus isolates carried the blaZ gene, 76% co-carried blaZ with cadD and cadX, with 62% of these isolates carrying blaZ, cadD, and cadX on the same plasmid. The co-carriage of ARGs and MRGs in S. warneri isolates, and carriage of MRGs in S. delphini, without plasmids suggests non-conjugative transmission routes for gene acquisition. More studies are required that focus on the transduction and transformation routes of transmission to prevent interspecies exchange of ARGs and MRGs in sewage-associated systems

Identifying Metal Resistance Genes in Staphylococcus Species Isolated from Wastewater and Streams Receiving Treated Effluent

Staphylococcus aureus and other staphylococci share mobile genetic elements that code for resistance to antibiotics and metals, allowing them to survive outside of their hosts in the environment, and become less sensitive to environmental factors and more resistant to antimicrobial agents. This study investigated the presence of select metal resistance genes (MRGs) originating from S. aureus and other staphylococci utilizing whole genomic sequencing and BlastN analysis of isolate genetic sequences obtained by selective enrichment of sewage-impacted samples over a 2-year period. Results from 26 isolate sequences recovered from 2 wastewater treatment plants, and creek sediments upstream and downstream, showed that MRGs carried in treatment plant influent S. aureus isolates were present in effluent Staphylococcus warneri isolates. The same MRGs were found in Enterococcus faecalis isolates recovered from the receiving creek sediments downstream of the plant effluent. The presence of MRGs specific to Staphylococcus delphini was identified in S. warneri, S. aureus, and E. faecalis isolates, indicative of horizontal gene transfer. This study has identified the potential role of coagulase-negative S. warneri in supporting the environmental spread of MRGs from coagulase-positive staphylococcus species present in sewage influent to bacteria in the receiving stream sediments, and highlights the need to control the spread of resistance genes of S. aureus by increasing treatment effectiveness for removing gram-positive bacteria and transference vectors

Effect of ethinylestradiol (EE2) on GnRH system in zebrafish (Danio rerio) exposed at adult and larval stages.

Poste

Azole fungicides in zebrafish: new effects for old molecules

Azole is a class of diverse compounds discovered several decades ago and essentially used as antifungals in agriculture and medicine. Their primary mode of action is to inhibit the fungal enzyme 14α-demethylase, which produces ergosterol, an important component of the cell membranes of fungi. Despite this specific mode of action, azoles are also characterized by their capacity to disrupt the endocrine system of vertebrate through multiple mechanisms notably by altering steroidogenesis, a key physiological process responsible for the biosynthesis of steroidal hormones. For instance, azole compounds affect both expression and enzymatic activities of several steroidogenic enzymes in vertebrate models, including fish, leading to reproductive disorders. Because of their uses, their presence in the aquatic environment (surface waters of rivers, lakes and estuaries; sewage sludge) has been recently reported in different industrialized countries raising the need to assess hazard and risk posed to aquatic organisms. In this context, several experiments have been performed to explore the effects of the pharmaceutical azole, clotrimazole, on the endocrine system in the zebrafish. In males, we found that clotrimazole was able to affect the testicular physiology by affecting steroidogenesis, androgen release and spermatogenesis (Hinfray et al., 2011, Baudiffier et al. 2012, 2013). However, the most striking effect was observed in females. Indeed, we found that exposure of adult female zebrafish to clotrimazole led to a dramatic masculinisation as revealed by the complete sex-reversal of the phenotypic sex. Remarkably, this sex-reversal occurred rapidly leading to well-differentiated testicular tissue after 42 days of exposure. By using cyp19a1a-GFP transgenic zebrafish, we further demonstrated that clotrimazole led to a time-dependent inhibition of GFP expression in ovary which preceded the histological differentiation of testis demonstrating the crucial role played by aromatase in the process of masculinisation. Altogether, our study demonstrates that clotrimazole significantly affect the gonad endocrinology and physiology of fish revealing new and striking effects on its ability to reverse the phenotypic sex of adult female. Based on our data, it is clear that further studies are needed to address the issue raised by the presence of azoles in the aquatic environment as regards to their potential impact on wild population of fish

Azole fungicides in zebrafish: new effects for old molecules

International audienceAzole is a class of diverse compounds discovered several decades ago and essentially used as antifungals in agriculture and medicine. Their primary mode of action is to inhibit the fungal enzyme 14α-demethylase, which produces ergosterol, an important component of the cell membranes of fungi. Despite this specific mode of action, azoles are also characterized by their capacity to disrupt the endocrine system of vertebrate through multiple mechanisms notably by altering steroidogenesis, a key physiological process responsible for the biosynthesis of steroidal hormones. For instance, azole compounds affect both expression and enzymatic activities of several steroidogenic enzymes in vertebrate models, including fish, leading to reproductive disorders. Because of their uses, their presence in the aquatic environment (surface waters of rivers, lakes and estuaries; sewage sludge) has been recently reported in different industrialized countries raising the need to assess hazard and risk posed to aquatic organisms. In this context, several experiments have been performed to explore the effects of the pharmaceutical azole, clotrimazole, on the endocrine system in the zebrafish. In males, we found that clotrimazole was able to affect the testicular physiology by affecting steroidogenesis, androgen release and spermatogenesis (Hinfray et al., 2011, Baudiffier et al. 2012, 2013). However, the most striking effect was observed in females. Indeed, we found that exposure of adult female zebrafish to clotrimazole led to a dramatic masculinisation as revealed by the complete sex-reversal of the phenotypic sex. Remarkably, this sex-reversal occurred rapidly leading to well-differentiated testicular tissue after 42 days of exposure. By using cyp19a1a-GFP transgenic zebrafish, we further demonstrated that clotrimazole led to a time-dependent inhibition of GFP expression in ovary which preceded the histological differentiation of testis demonstrating the crucial role played by aromatase in the process of masculinisation. Altogether, our study demonstrates that clotrimazole significantly affect the gonad endocrinology and physiology of fish revealing new and striking effects on its ability to reverse the phenotypic sex of adult female. Based on our data, it is clear that further studies are needed to address the issue raised by the presence of azoles in the aquatic environment as regards to their potential impact on wild population of fish