Usages des Infrastructures de Données Géographiques par les acteurs côtiers publics français

oeuvre collective de processus et d'outils qui visent à favoriser leur connaissance et leur gestion est actuellement primordiale. Les Infrastructures de Données Géographiques (IDG) ont pour but de faciliter la mise à disposition et l'accès aux données géographiques. Elles peuvent contribuer à la Gestion Intégrée des Zones Côtières (GIZC) à travers les systèmes d'informations, les politiques et les dispositions institutionnelles qu'elles rassemblent. Mais leur contribution effective dépend des réponses qu'elles apportent aux acteurs côtiers. Notre démarche se base sur un questionnaire en ligne diffusé en octobre 2012 et qui a pour but d'évaluer les usages et les pratiques effectives des IDG françaises. Les points de vue des utilisateurs ou producteurs d'information géographique de la sphère publique, travaillant sur les territoires côtiers sont analysés selon une double approche : statistique et structurale. Les premiers résultats permettent de saisir la diversité des acteurs côtiers et la variabilité de leurs opinions concernant la contribution des IDG à la gestion du littoral

A comparison of behavior, reproductive parameters and response to pollutant between wild-type, transgenic and mutant zebrafish: could they all be considered the same "zebrafish" ?

International audienceRegulatory assays available from the OECD for the testing of chemicals, such as endocrine disrupting chemicals (EDC) testing (TG229, TG230), can be performed with zebrafish without any recommendation regarding the strain that should be used. In addition to endpoints defined in the TG, transgenic fish could inform on mechanisms underlying EDC effects. By avoiding interference with pigmentation during imaging, use of casper background may improve tests sensitivity. In the present study, we compared 4 zebrafish lines (wild type AB, casper, cyp19a1a-eGFP and cyp19a1a-eGFP-casper) to determine whether transgene insertion and mutations could modify their basal physiology and their response to pollutant, thereby impeding their use for EDC testing. We thus compared the behavior (anxiety, sociability) and reproductive parameters (circulating vitellogenin (VTG) concentrations, female fecundity, egg viability) of control fish of the 4 lines and evaluated their responses to a model pollutant, e.g. clotrimazole. Sociability was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the novel tank diving test. No difference was observed between lines for either sociability 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 4 lines studied. Clotrimazole exposure (28 days; 250 µg/L) had similar effects in the 4 zebrafish lines despite a slightly higher sensitivity in wild type AB. Clotrimazole led to a masculinization of one part of the females, an inhibition of VTG, an inhibition of aromatase A expression (GFP measurement and qPCR) and an induction of 11-ketotestosterone. In summary, no critical difference in the behavior, reproductive parameters and response to pollutant was found between the 4 zebrafish lines, indicating that the transgene insertion and the mutations did not modify basal physiology of the fish. Therefore, their use in OECD tests for the study of EDC appears to be relevant. Further work will be conducted on strains sensitivity by evaluating dose-response effects of pollutants

A comparison of behavior, reproductive parameters and response to pollutant between wild-type, transgenic and mutant zebrafish: could they all be considered the same "zebrafish" ?

International audienceRegulatory assays available from the OECD for the testing of chemicals, such as endocrine disrupting chemicals (EDC) testing (TG229, TG230), can be performed with zebrafish without any recommendation regarding the strain that should be used. In addition to endpoints defined in the TG, transgenic fish could inform on mechanisms underlying EDC effects. By avoiding interference with pigmentation during imaging, use of casper background may improve tests sensitivity. In the present study, we compared 4 zebrafish lines (wild type AB, casper, cyp19a1a-eGFP and cyp19a1a-eGFP-casper) to determine whether transgene insertion and mutations could modify their basal physiology and their response to pollutant, thereby impeding their use for EDC testing. We thus compared the behavior (anxiety, sociability) and reproductive parameters (circulating vitellogenin (VTG) concentrations, female fecundity, egg viability) of control fish of the 4 lines and evaluated their responses to a model pollutant, e.g. clotrimazole. Sociability was evaluated by monitoring the time spent near congeners in a shuttle box while anxiety was evaluated using the novel tank diving test. No difference was observed between lines for either sociability 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 4 lines studied. Clotrimazole exposure (28 days; 250 µg/L) had similar effects in the 4 zebrafish lines despite a slightly higher sensitivity in wild type AB. Clotrimazole led to a masculinization of one part of the females, an inhibition of VTG, an inhibition of aromatase A expression (GFP measurement and qPCR) and an induction of 11-ketotestosterone. In summary, no critical difference in the behavior, reproductive parameters and response to pollutant was found between the 4 zebrafish lines, indicating that the transgene insertion and the mutations did not modify basal physiology of the fish. Therefore, their use in OECD tests for the study of EDC appears to be relevant. Further work will be conducted on strains sensitivity by evaluating dose-response effects of pollutants

A novel transgenic zebrafish line (cyp19a1a-GFP) : refinement of an OECD test guideline for evaluating the effects of prochloraz on aromatase gene expression and reproduction

Concern about the effects of endocrine disrupting chemicals (EDCs) on reproductive health has stimulated the development of mechanism-based models and assays. In this regard, transgenic fish are powerful biological models that can provide mechanistic information regarding the endocrine activity of test chemicals. The objective of this study is to assess the feasibility of using a new biological model in an OECD fish screening assay to provide additional mechanistic information, as compared to wild-type models. In the present study, we used a transgenic zebrafish line cyp19a1a-GFP expressing GFP under the control of the “ovarian” aromatase promoter. Based on the OECD Fish Short Term Reproduction Assay (TG229), the effect of a prochloraz (PCZ; 3, 30 and 300µg/L), was assessed on “classical” endpoints. Additionally, fluorescence of ovarian GFP was monitored in vivo at 4 times from T0, to determine the baseline level of fluorescence, to T21 days of exposure to determine the kinetic of perturbation. After 21 days of exposure, a significant decrease in the number of eggs laid per female per day was observed at 300 µg/L. PCZ led to a concentration-dependent inhibition of circulating vitellogenin concentrations in females most probably reflecting the decreasing E2 synthesis due to inhibition of ovarian aromatase activity. GFP intensities were similar over treatment groups at 7 and 14 days of exposure but significantly increased after 21 days of exposure at 30 and 300 µg/L. A similar profile was observed on the endogenous cyp19a1a gene expression analyzed by qPCR. The overexpression of the aromatase gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities consistent with the literature on PCZ. Thus, the cyp19a1a-GFP transgenic zebrafish line allows in vivo non-invasive monitoring over time of the GFP fluorescence of the ovary, providing information on aromatase expression and its perturbation. The protocol developed for GFP measures has no observable effects on reproduction and survival of fish. Moreover, it allows evaluation of EDCs effects over time and concentrations. Taken together, our findings highlight that the transgenic cyp19a1a-GFP model is a sensitive and relevant tool, which can bring complementary data without increasing the number of individuals or costs of the experiments

Refinement of an OECD test guideline for evaluating the effects of endocrine disrupting chemicals on aromatase gene expression and reproduction using novel transgenic cyp19a1a-GFP zebrafish

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study our objective was to refine the OECD Fish Short Term Reproduction Assay (TG229) using a new transgenic zebrafish line expressing GFP under the control of the cyp19a1a promoter, to provide additional mechanistic information. For this purpose, we exposed this zebrafish line using prochloraz (PCZ), one of the TG229 reference molecule, at 3, 30 and 300 µg/L. In addition to “classical” endpoints, the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. At the end of the experiment, we found that PCZ significantly decreased the number of eggs laid per female per day at 300 µg/L and the concentrations of vitellogenin in females, most probably reflecting the decreasing E2 synthesis due to inhibition of the ovarian aromatase activities. Additional information on the mode of action of PCZ were provided through the measurement of GFP intensities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 30 and 300 µg/L. A similar profile was observed for cyp19a1a expression (qPCR) thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-GFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information of the time- and concentration-dependent effect on a key target gene through in vivo GFP imaging without increasing the number of individuals or costs of the experiments

Estrogenic activities in water and sediment from French rivers using zebrafish-based bioassays

Over the last decade, assessment of exposure of fish to aquatic endocrine disrupting chemicals (EDCs) and their effects on endocrine functions has become a major issue in ecotoxicology. In the present study, we explore the potential use of newly developed zebrafish (zf)-based in vitro and in vivo bioassays to detect estrogenic compounds in aquatic environment. For this purpose we use stable zf-estrogen receptor (zfER) subtypes (zfERa, zfERb1, zfERb2) in the zebrafish liver (ZFL) cell line and transgenic cyp19a1b-GFP zf embryos to screen organic extracts of sediment and water (using polar organic compounds integrative samplers - POCIS) sampled from 19 French river sites. Results showed no detection of estrogenic activities in sediment extracts by zf-based in vitro bioassays. Unlike zfERs in vitro bioassays, human receptor (hERa) in vitro bioassay was able to detect estrogenic activities in sediment extracts. However, POCIS-based bio-monitoring provide much more significant information on zf-based in vitro bioassays as estrogenic activities were mainly detected for most of the river sites. High numbers of POCIS extracts were found to be more active on zfERb2 than on zfERa. The responsiveness of zfERb2 to environmental samples is important as this estrogen receptor subtype is present in fish species but not in humans. In addition, SPE-based fractionation of POCIS extracts allowed distinguishing fractions that were active on zfERb2 from those active on hERa. These results suggest a significant inter-assay difference (human MCF-7 versus zebrafish ZFL cells). Moreover, we found similar estrogenic activities on in vivo bioassay by sediment and POCIS extracts as observed on in vitro bioassays. Complementarily, this in vivo assay will allow taking into account the bioavailability and pharmacodynamics of estrogen mimicking compounds to enhance the efficiency and the accuracy of EDCs testing strategies. In summary, this study reports for the first time the use of fish-based bioassays, supporting the significance of using both in vitro and in vivo fish models to detect species-specific active contaminants in aquatic environment

A novel transgenic zebrafish line (cyp19a1a-GFP) : refinement of an OECD test guideline for evaluating the effects of prochloraz on aromatase gene expression and reproduction

Concern about the effects of endocrine disrupting chemicals (EDCs) on reproductive health has stimulated the development of mechanism-based models and assays. In this regard, transgenic fish are powerful biological models that can provide mechanistic information regarding the endocrine activity of test chemicals. The objective of this study is to assess the feasibility of using a new biological model in an OECD fish screening assay to provide additional mechanistic information, as compared to wild-type models. In the present study, we used a transgenic zebrafish line cyp19a1a-GFP expressing GFP under the control of the “ovarian” aromatase promoter. Based on the OECD Fish Short Term Reproduction Assay (TG229), the effect of a prochloraz (PCZ; 3, 30 and 300µg/L), was assessed on “classical” endpoints. Additionally, fluorescence of ovarian GFP was monitored in vivo at 4 times from T0, to determine the baseline level of fluorescence, to T21 days of exposure to determine the kinetic of perturbation. After 21 days of exposure, a significant decrease in the number of eggs laid per female per day was observed at 300 µg/L. PCZ led to a concentration-dependent inhibition of circulating vitellogenin concentrations in females most probably reflecting the decreasing E2 synthesis due to inhibition of ovarian aromatase activity. GFP intensities were similar over treatment groups at 7 and 14 days of exposure but significantly increased after 21 days of exposure at 30 and 300 µg/L. A similar profile was observed on the endogenous cyp19a1a gene expression analyzed by qPCR. The overexpression of the aromatase gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities consistent with the literature on PCZ. Thus, the cyp19a1a-GFP transgenic zebrafish line allows in vivo non-invasive monitoring over time of the GFP fluorescence of the ovary, providing information on aromatase expression and its perturbation. The protocol developed for GFP measures has no observable effects on reproduction and survival of fish. Moreover, it allows evaluation of EDCs effects over time and concentrations. Taken together, our findings highlight that the transgenic cyp19a1a-GFP model is a sensitive and relevant tool, which can bring complementary data without increasing the number of individuals or costs of the experiments

Refinement of an OECD test guideline for evaluating the effects of EDCs on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (Test Guideline 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that the highest PCZ concentration significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. These experiments showed that the physiological responses observed in control and PCZ-exposed cyp19a1aeGFP transgenic zebrafish (E2, VTG, reproduction) are similar to those of wild-type zebrafish, both qualitatively and quantitatively. Besides, the cyp19a1a-eGFP model reliably informs on the time- and concentrationdependent effects of PCZ on ovarian aromatase through in vivo GFP fluorescence of the ovaries, thereby providing novel mechanistic information without increasing the number of animals needed. Overall, the cyp19a1a-eGFP transgenic zebrafish line proved to be a relevant model to study the effects of EDCs in fish, which might also be wisely used to refine mechanism-based assays such as OECD TG 229

Corrigendum to ‘Refinement of an OECD test guideline for evaluating the effects of endocrine disrupting chemicals on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish’ [Aquat. Toxicol. 220 (2020) 105403](S0166445X19308392)(10.1016/j.aquatox.2020.105403)

The authors deeply regret that errors occurred in the original above article. Corrections follow below. - In the 2.7. Vitellogenin ELISA section of the Materials and methods section, the sentence “Standard / samples were pre-incubated … overnight at 4°C.” should read as “Standard / samples were pre-incubated with the primary antibody (1:1 with DR-264 zebrafish anti-VTG antibody diluted to 1:500, Biosense Laboratories, Norway) overnight at 4°C.” - In Figure 2, the authors have inadvertently forgot to apply dilution factors to the circulating E2 concentrations. The correct E2 concentrations can be found on the corrected figure bellow. [Formula presented] - In the 3.4. Circulating estradiol concentrations section of the Results section, the sentence “Nevertheless, a 48% inhibition was seen…concentration (172.6 µg/L)” should now read as “Nevertheless, a 40% inhibition was seen between the fish in the different control groups and the fish exposed to the highest concentration (PCZ 172.6 μg/l).” - In the 3.5. Circulating vitellogenin concentrations section of the Results section, the sentence “A significant decrease was measured in fish … (PCZ 172.6 μg/l).” should read as “A significant decrease was measured in fish exposed to the highest concentration of PCZ, with a fall from 23.40 ± 5.11 mg/ml (DMSO) to 6.10 ± 0.74 mg/ml (PCZ 172.6 μg/l).” - In Figure 3, the unit reported on the Y-axis for the vitellogenin concentrations in mg/ml should read as ng/ml. - In the 4.1. Effect of PCZ on classical endpoints of the OECD TG 229 in cyp19a1a-eGFP zebrafish of the Discussion section, the sentence “From a quantitative point of view, the data….(Doering et al., 2019)” should now read as “From a quantitative point of view, the data we obtained in the cyp19a1a-eGFP zebrafish (40 % decrease of estradiol concentration in the high concentration of PCZ compared to control females, leading to a 65 % decrease of circulating VTG and finally to 60 % less eggs produced) are in perfect adequation with the quantitative relationships existing between all key events of the AOP recently described for wildtype zebrafish (Doering et al., 2019).” These corrections do not affect the conclusions of the article in any way. The authors would like to apologise for any inconvenience caused. DOI of original article: https://doi.org/10.1016/j.aquatox.2020.105403 Nathalie Hinfray © 202