Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio)

International audienceMany chemicals in the aquatic environment are able to adversely affect in vitro brain and ovarian aromatase expression/activity. However, it remains to be determined if these substances elicit in vivo effect in fish. With the view to further understanding possible effects of endocrine disrupting chemicals (EDCs) on aromatase function, we first developed methods to measure brain and ovarian aromatase expression/activity in a model species, the zebrafish, and assessed the effect of estradiol (E2) and androstatrienedione (ATD), a steroidal aromatase inhibitor. We showed that CYP19b gene was predominantly expressed in the brain whereas in the ovary CYP19a mRNA level was predominant. Moreover, aromatase activities (AA) were higher in brain than in ovary. In adult zebrafish, E2 treatment had no effect on aromatase expression/activity in brain, whereas at larval stage, E2 strongly triggered CYP19b expression. In the ovaries, E2 led to a complete inhibition of both CYP19a expression and AA. Exposure to ATD led to a total inhibition of both brain and ovarian AA but had no effect on CYP19 transcripts abundance. Together, these results provide relevant knowledge concerning the characterization of aromatase in the zebrafish, and reinforce the idea that brain and ovarian aromatase are promising markers of EDCs in fish and deserve further in vivo studies

Intérêt des réponses immunitaires dans le cadre des études de biosurveillance

Immune parameters are interesting in biomonitoring studies as they reflect environmental contamination and fish health status. Nevertheless, fish immunomarkers could be influenced by many environmental and interindividual parameters. All these confounding factors limit the interpretation of variation between sites and may disturb the routine environmental risk assessment. Characterizing the remaining natural sources of biomarker variability could allow discriminating responses induced by pollutant exposure and background noise. In this way, establishment of reference values, that consider the major sources of variation, may be of great interest for environmental monitoring studies. Moreover, connection has been established between the destabilization of the fish immune system caused by sublethal doses of contaminants and a defect in fish pathogen resistance. In this way, we proposed to develop a stress on stress responses by using some bacterial endotoxin, which mimic pathogen effect, and chemical exposure. This method can be a promising approach for the assessment of pollutant effects in aquatic ecosystems by determining biological threshold values. This biological threshold values could indicate the real capacity of fish to respond during pathogenic conditionLes écosystèmes sont dépendants des conditions de vie qui leur sont fournies : toute pollution y engendre des effets néfastes. De ce fait, il est primordial de pouvoir statuer sur l’état chimique et écologique des masses d’eau afin de caractériser le risque environnemental induit et de prendre des décisions en conséquence sur sa gestion. Néanmoins, la réglementation actuelle, ne s’appuyant que sur le caractère a posteriori d’une dégradation, s’avère insuffisamment prédictive de l’effet à long terme des modifications d’un écosystème. En complément des méthodes conventionnelles de détermination de la qualité des masses d’eau (état chimique et écologique), d’autres approches basées sur les mécanismes d’action des polluants permettent une évaluation plus précoce et plus explicative de l’impact de la contamination sur les organismes. En effet, les contaminants sont susceptibles de causer, directement ou indirectement, des effets nuisibles sur les principaux systèmes physiologiques comprenant les systèmes endocriniens, reproducteurs, nerveux et immunitaires. De tels effets peuvent remettre en cause la croissance, la reproduction, voire la survie des organismes sur lesquels ils s’appliquent. Ainsi, une approche biologique basée sur l’étude des biomarqueurs, permet de rendre compte des effets potentiels sur les individus. Même si les biomarqueurs ne permettent pas une identification formelle des substances responsables, de par leur prédictivité, ils présentent une place importante en complément des approches « substances » et « écologiques

Influence of a multistress (cadmium and temperature) on the reproduction of the three-spined stickleback (Gasterosteus aculeatus)

Human activities introduce significant amounts of organic and inorganic pollutants (trace metals) into aquatic environments. However, this contamination occurs within a large context that concerns the scientific community since the last decades: global warming. The complex nature of the interactions between toxic substances and temperature presents new challenges to improve the risk assessment for aquatic environments. Thus, the study of these interactions proves an essential priority for toxicologists. This study aims to evaluate the effect of chronic exposure to both chemical (cadmium) and physical stress (temperature) on the reproduction of a river fish widely used in ecotoxicology: three-spined stickleback (Gasterosteus aculeatus). For this, juvenile sticklebacks were exposed to 1µg.L-1 of cadmium for 4 months in semi-static conditions at two different temperatures, i.e. 16°C and 21°C. Water quality and cadmium concentration in the water were monitored throughout the experiment. Parameters such as somatic indices (height, weight, GSI) were measured at 15, 60, 90 and 120 days interval after cadmium exposure. Reproductive capacity was evaluated by performing several rounds of in vitro fertilization and monitoring of the offspring. The fertilization rate, mortality and birth defects were evaluated until the end of the larval stage (21 days post fertilization “dpf”). In total, 20 in vitro fertilizations were performed during this experiment with different results for each group. A decrease in fertilization rate was observed under the effect of temperature 21°C (84.79 ± 12%), which seems to be accentuated by the presence of cadmium (77.26 ± 31%). Similarly, the rate of embryonic and larval mortality seems to follow the same trend, with a rather large effect of temperature, especially in the presence of cadmium. Temperature 21°C in presence of cadmium delays the moment of hatching about two days compared to controls (10.5 ± 0.71 dpf Vs 8.67 ± 1.15 dpf). Several malformations were observed in all groups including the control group with predominance in groups from parents exposed to cadmium at 16°C (11.79 ± 10.8%). Our results showed fairly significant and worrying effects of a very low dose of cadmium (1µg.L-1) on the parents and their offsprings. Most studies reported in the literature on the effects of cadmium were demonstrated with much higher doses than used in our study. Further experiments are needed to complete and validate these results

Use of transgenic zebrafish models to study the endocrine effects of natural and synthetic progestins

International audienc

A comparison of behavioral and reproductive parameters between wild-type, transgenic and mutant zebrafish: Could they all be considered the same “zebrafish” for reglementary assays on endocrine disruption?

Transgenic zebrafish models are efficiently used to study the effects of endocrine disrupting chemicals (EDC); thereby informing on their mechanisms of action. However, given the reported differences between zebrafish strains at the genetical, physiological and behavioral levels; care should be taken before using these transgenic models for EDC testing. In the present study, we undertook a set of experiments in different transgenic and/or mutant zebrafish lines of interest for EDC testing: casper, cyp19a1a-eGFP, cyp19a1a-eGFP-casper, cyp11c1-eGFP, cyp11c1-eGFP-casper. Some behavioral traits, and some biochemical and reproductive physiological endpoints commonly used in EDC testing were assessed and compared to those obtained in WT AB zebrafish to ensure that transgene insertion and/or mutations do not negatively modify basal reproductive physiology or behavior of the fish. Behavioral traits considered as anxiety and sociality have been monitored. Sociality was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the Novel tank diving test. No critical difference was observed between strains for either sociality or anxiety level. Concerning reproduction, no significant difference in the number of eggs laid per female, in the viability of eggs or in the female circulating VTG concentrations was noted between the 5 transgenic/mutants and the WT AB zebrafish studied. In summary, the transgene insertion and the mutations had no influence on the endpoints measured in basal conditions. These results were a prerequisite to the use of these transgenic/mutant models for EDC testing. Next step will be to determine the sensitivity of these biological models to chemical exposure to accurately validate their use in existing fish assays for EDC testing

The three spined-stickleback's digestive enzymes as new biomarkers in environmental monitoring: effect of cadmium and temperature

The three-spined stickleback, is a freshwater, brackish, and marine fish species with large distribution throughout the northern Hemisphere. This pollution-tolerant fish is present in most European streams and small rivers but also coastal and estuarian areas, which make it a suitable candidate for environmental monitoring and risk assessment of aquatic ecosystems. Several biomarkers have been developed for this animal model. This study aims at characterizing and developing a new category of biomarkers in relation with energy acquisition, in sticklebacks. Among these parameters, digestive capacities of many invertebrates and vertebrates aquatic organisms have demonstrated a good sensitivity to contaminants. Thus, in this context, the most important digestive enzymes were firstly characterized in this sentinel species, according to it diet, in optimal living conditions. Secondly male and female juvenile sticklebacks were exposed for 3 months in semi-static conditions to cadmium at 1µg.L-1 (chemical stress), and two temperature values 16°C and 21°C (physical stress). Water quality and cadmium concentration in the water were also monitored throughout the experiment. Parameters such as somatic indexes (height, weight, fulton’s condition index) and digestive enzymes (trypsin, intestinal alkaline phosphatase and amylase) were measured at 15, 60 and 90 days interval after cadmium exposure. The results have demonstrated a significant decrease in biometric parameters, from the 3rd month, especially among groups subjected to a temperature of 21 ° C. Furthermore, we observed an important alteration of the activity of the different enzymes over time and especially for trypsin and alkaline phosphatase in individuals exposed to cadmium at 21°C suggesting interactions between chemical and physical stresses on biological responses. For the first time, the digestive enzymes of the three-spined stickleback are reported as new environmental biomarkers

The three spined-stickleback's digestive enzymes as new biomarkers in environmental monitoring: effect of cadmium and temperature

The three-spined stickleback, is a freshwater, brackish, and marine fish species with large distribution throughout the northern Hemisphere. This pollution-tolerant fish is present in most European streams and small rivers but also coastal and estuarian areas, which make it a suitable candidate for environmental monitoring and risk assessment of aquatic ecosystems. Several biomarkers have been developed for this animal model. This study aims at characterizing and developing a new category of biomarkers in relation with energy acquisition, in sticklebacks. Among these parameters, digestive capacities of many invertebrates and vertebrates aquatic organisms have demonstrated a good sensitivity to contaminants. Thus, in this context, the most important digestive enzymes were firstly characterized in this sentinel species, according to it diet, in optimal living conditions. Secondly male and female juvenile sticklebacks were exposed for 3 months in semi-static conditions to cadmium at 1µg.L-1 (chemical stress), and two temperature values 16°C and 21°C (physical stress). Water quality and cadmium concentration in the water were also monitored throughout the experiment. Parameters such as somatic indexes (height, weight, fulton’s condition index) and digestive enzymes (trypsin, intestinal alkaline phosphatase and amylase) were measured at 15, 60 and 90 days interval after cadmium exposure. The results have demonstrated a significant decrease in biometric parameters, from the 3rd month, especially among groups subjected to a temperature of 21 ° C. Furthermore, we observed an important alteration of the activity of the different enzymes over time and especially for trypsin and alkaline phosphatase in individuals exposed to cadmium at 21°C suggesting interactions between chemical and physical stresses on biological responses. For the first time, the digestive enzymes of the three-spined stickleback are reported as new environmental biomarkers

Toward identification of fish specific estrogens through effect-directed analysis based on zebrafish in vitro and in vivo reporter gene assays

Estrogens are present in all vertebrates, from telesots to mammals, which can bind to and activate estrogen receptors (ERs) through a conserved mechanism. However, existence of cross-species differences as regards ER subtypes and the binding affinity of (xeno)estrogens on ERs, as well as the cellular context may lead to differential ER activation by environmental contaminants when assessing hazard on aquatic organisms. For this purpose, we previously developed, characterized and demonstrated the functionality of stable zebrafish (zf)-ER subtypes (zfERa, zfERb1, zfERb2) in the zebrafish liver (ZFL) cell line and transgenic cyp19a1b-GFP zf embryosto quantify estrogenic activity in complex mixtures. In the present study, we used zebrafish (zf)-based in vitro and in vivo bioassays to assess estrogenic activity in surface waters by investigating 20 French river sites using polar organic chemical integrative sampling (POCIS). Simultaneously, the human MELN bioassay (MCF-7 cells-based, expressing hERa) was used to study cross-species differences. POCIS-based bio-monitoring provide significant information on zf-based in vitro bioassays as zfERs and hERa assays significantly differed at some sites, identifying zf-specific estrogenic activities in surface waters. Higher numbers of POCIS extracts were found to be more active on zfERbs than on zfERa, which is in line with the better sensitivity of zfERbs to steroid estrogens. The responsiveness of zfERb2 to environmental samples is important as this ER subtype is present in fish species but not in humans. In complementary to in vitro assays, the estrogenic activities at the organism level were observed using zf embryo assay at sites that were highly active on in vitro bioassays. These in vivo results are relevant for hazard assessment as they reveal that detected active compounds target radial glial cells expressing brain aromatase which are known to be involved in neurogenesis. In addition, combining these zf-based tools within an EDA approach, we were able to isolate fractions that were active only on zfERb2 subtype, hence reinforcing the hypothesis of species-specificity for, yet unknown, ER ligands. Further investigation using mass spectrometry techniques is foreseen to identify fish-specific active compounds which will allow improving bioassay-based environmental risk assessment towards aquatic species