Embryonic exposures to chemicals acting on brain aromatase lead to different locomotor effects in zebrafish larvae.

Pathways underlying neurodevelopmental effects of endocrine disruptors (EDs) remain poorly known. Expression of brain aromatase (aroB), responsible for estrogen production in the brain of teleosts, is regulated by estrogenic EDs and could play a role in their behavioral effects. We exposed zebrafish eleutheroembryos (0-120 h post-fertilization) to various concentrations of 16 estrogenic chemicals (incl. bisphenols and contraceptives), and of 2 aroB inhibitors. Behavior was monitored using a photomotor response test procedure. Both aroB inhibitors (clotrimazole and prochloraz) and a total of 6 estrogenic EDs induced significant behavioral alterations, including DM-BPA, BPC and BPS-MPE, three bisphenol substitutes which behavioral effects were, to our knowledge, previously unknown. However, no consensus was reported on the effects among tested substances. It appears that behavioral changes could not be linked to groups of substances defined by their specificity or potency to modulate aroB expression, or by their structure. Altogether, behavioral effects of estrogenic EDs in 120 h post-fertilization larvae appear unrelated to aroB but are nonetheless not to be neglected in the context of environmental safety.

ReadEDTest: A tool to assess the readiness of in vitro test methods under development for identifying endocrine disruptors.

Growing evidence shows that endocrine disruptors (EDs), known to affect the reproductive system, may also disturb other hormone-regulated functions leading to cancers, neurodevelopmental defects, metabolic and immune diseases. To reduce exposure to EDs and limit their health effects, development of screening and mechanism-based assays to identify EDs is encouraged. Nevertheless, the crucial validation step of test methods by regulatory bodies is a time- and resource-consuming process. One of the main raisons of this long duration process is that method developers, mainly researchers, are not fully aware of the regulatory needs to validate a test. We propose an online self-assessment questionnaire (SAQ) called ReadEDTest easy to be used by all researchers. The aim of ReadEDTest is to speed up the validation process by assessing readiness criteria of in vitro and fish embryo ED test methods under development. The SAQ is divided into 7 sections and 13 sub-sections containing essential information requested by the validating bodies. The readiness of the tests can be assessed by specific score limits for each sub-section. Results are displayed via a graphical representation to help identification of the sub-sections having sufficient or insufficient information. The relevance of the proposed innovative tool was supported using two test methods already validated by the OECD and four under development test methods.

PBTK modeled perfluoroalkyl acid kinetics in zebrafish eleutheroembryos suggests impacts on bioconcentrations by chorion porosity dynamics.

The zebrafish eleutheroembryo (zfe) is widely used as a model to characterize the toxicity of chemicals. However, analytical methods are still missing to measure organ concentrations. Therefore, physiologically-based toxicokinetic (PBTK) modeling may overcome current limitations to help understand the relationship between toxic effects and internal exposure in various organs. A previous PBTK model has been updated to include the chorionic transport barrier and its permeabilization, hatching dynamics within a zfe population over development, and active mediated transport mechanisms. The zfe PBTK model has been calibrated using measured time-dependent internal concentrations of PFBA, PFHxS, PFOA, and PFOS in a zfe population and evaluated using external datasets from the literature. Calibration was successful with 96% of the predictions falling within a 2-fold range of the observed concentrations. The external dataset was correctly estimated with about 50% of the predictions falling within a factor of 3 of the observed data and 10% of the predictions are out of the 10-fold error. The calibrated model suggested that active mediated transport differs between PFAS with a sulfonic and carboxylic acid functional end groups. This PBTK model predicts well the fate of PFAS with various physicochemical properties in zfe. Therefore, this model may improve the use of zfe as an alternative model in toxicokinetic-toxicodynamic studies and help to refine and reduce zfe-based experiments, while giving insights into the internal kinetics of chemicals.

Interlaboratory prevalidation of a new in vitro transcriptional activation assay for the screening of (anti-)androgenic activity of chemicals using the UALH-hAR cell line.

We report an interlaboratory evaluation of a recently developed androgen receptor (AR) transactivation assay using the UALH-hAR reporter cell line that stably expresses the luciferase gene under the transcriptional control of androgen receptor elements (AREs) with no glucocorticoid receptor (GR) crosstalk. Herein, a two-step prevalidation study involving three laboratories was conducted to assess performance criteria of the method such as transferability as well as robustness, sensitivity, and specificity. The first step consisted in the validation of the transfer of the cell line to participant laboratories through the testing of three reference chemicals: the AR agonist dihydrotestosterone, the AR antagonist hydroxyflutamide and the glucocorticoid dexamethasone. Secondly, a blinded study was conducted by screening a selection of ten chemicals, including four AR agonists, five AR antagonists, and one non-active chemical. All test compounds yielded the same activity profiles in all laboratories. The logEC50 (agonist assay) or logIC50 (antagonist assay) were in the same range, with intra-laboratory coefficients of variation (CVs) of 0.1-3.4% and interlaboratory CVs of 1-4%, indicating very good within- and between-laboratory reproducibility. Our results were consistent with literature and regulatory data (OECD TG458). Overall, this interlaboratory study demonstrated that the UALH-hAR assay is transferable, produces reliable, accurate and specific (anti)androgenic activity of chemicals, and can be considered for further regulatory validation.

A PBPK model to evaluate zebrafish eleutheroembryos' actual exposure: bisphenol A and analogs' (AF, F, and S) case studies.

The zebrafish eleutheroembryo model is increasingly used to assess the toxicity and developmental adverse effects of xenobiotics. However, the actual exposure is seldom measured (poorly accessible), while a predictive model could estimate these concentrations. The predictions with a new eleutheroembryo physiologically based pharmacokinetic (PBPK) model have been evaluated using datasets obtained from literature data for several bisphenols. The model simulated the toxicokinetics of bisphenols A (BPA), AF, F, and S through the eleutheroembryo tissues while considering the body and organ growth. We further improved the predictions by adding dynamic flows through the embryo and/or its chorion, impact of experimental temperature, metabolic clearance, and saturation of the absorption by Bayesian calibration. The model structure was determined using the BPA dataset and generalized to the other bisphenols. This model revealed the central role of the chorion in the compound uptake in the first 48 h post-fertilization. The predictions for the BPA substitutes estimated by our PBPK model were compared to available toxicokinetics data for zebrafish embryos, and 63% and 88% of them were within a twofold and fivefold error intervals of the corresponding experimental values, respectively. This model provides a tool to design new eleutheroembryo assays and evaluate the actual exposure.

Combinatorial pathway disruption is a powerful approach to delineate metabolic impacts of endocrine disruptors.

The prevalence of metabolic diseases, such as obesity, diabetes, metabolic syndrome and chronic liver diseases among others, has been rising for several years. Epidemiology and mechanistic (in vivo, in vitro and in silico) toxicology have recently provided compelling evidence implicating the chemical environment in the pathogenesis of these diseases. In this review, we will describe the biological processes that contribute to the development of metabolic diseases targeted by metabolic disruptors, and will propose an integrated pathophysiological vision of their effects on several organs. With regard to these pathomechanisms, we will discuss the needs, and the stakes of evolving the testing and assessment of endocrine disruptors to improve the prevention and management of metabolic diseases that have become a global epidemic since the end of last century.

Hypericum lanceolatum Lam. Medicinal Plant: Potential Toxicity and Therapeutic Effects Based on a Zebrafish Model.

Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders.

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 strains 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.

Human and Zebrafish Nuclear Progesterone Receptors Are Differently Activated by Manifold Progestins.

The environmental risk of natural and synthetic ligands of the nuclear progesterone receptor (nPR) has been pointed out, however there is still a lack of mechanistic information regarding their ability to interact with nuclear PR in aquatic species. To identify possible interspecies differences, we assessed in vitro the ability of manifold progestins to transactivate zebrafish (zf) and human (h) PRs, using two established reporter cell lines, U2OS-zfPR and HELN-hPR, respectively. Reference ligands highlighted some differences between the two receptors. The reference human agonist ligands promegestone and progesterone induced luciferase activity in both cell lines in a concentration-dependent manner, whereas the natural zebrafish progestin 17α,20ß-dihydroxy-4-pregnen-3-one activated zfPR but not hPR. The potent human PR antagonist mifepristone (RU486) blocked PR-induced luciferase in both cell models but with different potencies. In addition, a set of 22 synthetic progestins were screened on the two cell lines. Interestingly, all of the tested compounds activated hPR in the HELN-hPR cell line, whereas the majority of them acted as zfPR antagonists in U2OS-zfPR. Such zfPR-specific response was further confirmed in zebrafish liver cells. This study provides novel information regarding the activity of a large set of progestins on human and zebrafish PR and highlights major interspecies differences in their activity, which may result in differential effects of progestins between fish and humans.

Estrogenic activity of surface waters using zebrafish- and human-based in vitro assays: The Danube as a case-study.

Most in vitro reporter gene assays used to assess estrogenic contamination are based on human estrogen receptor α (hERα) activation. However, fish bioassays can have distinct response to estrogenic chemicals and mixtures, questioning the relevance of human-based bioassays for assessing risk to this species. In this study, zebrafish liver cells stably expressing zebrafish ERß2 (ZELHß2) and human breast cancer cells expressing hERα (MELN) were used to quantify the estrogenic activity of 25 surface water samples of the Danube River, for which chemicals have been previously quantified. Most samples had a low estrogenic activity below 0.1 ng/L 17ß-estradiol-equivalents that was more often detected by MELN cells, while ZELHß2 response tend to be lower than predicted based on the chemicals identified. Nevertheless, both bioassays quantified well a higher estrogenic activity at two sites, which was confirmed in vivo using a transgenic zebrafish assay. The results are discussed considering the effect-based trigger values proposed for water quality monitoring.

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.

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 (TG 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 172.6 µg/L PCZ 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. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

Neurodevelopmental effects of natural and synthetic ligands of estrogen and progesterone receptors in zebrafish eleutheroembryos.

Natural and synthetic estrogens and progestins are widely used in human and veterinary medicine and are detected in waste and surface waters. Our previous studies have clearly shown that a number of these substances targets the brain to induce the estrogen-regulated brain aromatase expression but the consequences on brain development remain virtually unexplored. The aim of the present study was therefore to investigate the effect of estradiol (E2), progesterone (P4) and norethindrone (NOR), a 19-nortestosterone progestin, on zebrafish larval neurogenesis. We first demonstrated using real-time quantitative PCR that nuclear estrogen and progesterone receptor brain expression is impacted by E2, P4 and NOR. We brought evidence that brain proliferative and apoptotic activities were differentially affected depending on the steroidal hormone studied, the concentration of steroids and the region investigated. Our findings demonstrate for the first time that steroid compounds released in aquatic environment have the capacity to disrupt key cellular events involved in brain development in zebrafish embryos further questioning the short- and long-term consequences of this disruption on the physiology and behavior of organisms.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Combined effects of environmental xeno-estrogens within multi-component mixtures: Comparison of in vitro human- and zebrafish-based estrogenicity bioassays.

Some recent studies showed that in vitro bioassays based on fish or human estrogen receptor (ER) activation may have distinct responses to environmental samples, highlighting the need to better understand bioassay-specific ER response to environmental mixtures. For this purpose, we investigated a 12-compound mixture in two mixture ratios (M1 and M2) on zebrafish (zf) liver cells stably expressing zfERα (ZELHα cells) or zfERß2 (ZELHß2 cells) and on human ER-reporter gene (MELN) cells. The mixture included the well-known ER ligands bisphenol A (BPA) and genistein (GEN), and other compounds representatives of a freshwater background contamination. In this context, the study aimed at assessing the robustness of concentration addition (CA) model and the potential confounding influence of other chemicals by testing subgroups of ER activators, ER inhibitors or ER activators and inhibitors combined. Individual chemical testing showed a higher prevalence of ER inhibitors in zebrafish than human cells (e.g. propiconazole), and some chemicals inhibited zfER but activated hER response (e.g. benzo(a)pyrene, triphenylphosphate). The estrogenic activity of M1 and M2 was well predicted by CA in MELN cells, whereas it was significantly lower than predicted in ZELHß2 cells, contrasting with the additive effects observed for BPA and GEN binary mixtures. When testing the subgroups of ER activators and inhibitors combined, the deviation from additivity in ZELHß2 cells was caused by zebrafish-specific inhibiting chemicals. This study provides novel information on the ability of environmental pollutants to interfere with zfER signalling and shows that non-estrogenic chemicals can influence the response to a mixture of xeno-estrogens in a bioassay-specific manner.

Metformin in Reproductive Biology.

Initially produced in Europe in 1958, metformin is still one of the most widely prescribed drugs to treat type II diabetes and other comorbidities associated with insulin resistance. Metformin has been shown to improve fertility outcomes in females with insulin resistance associated with polycystic ovary syndrome (PCOS) and in obese males with reduced fertility. Metformin treatment reinstates menstrual cyclicity, decreases the incidence of cesareans, and limits the number of premature births. Notably, metformin reduces steroid levels in conditions associated with hyperandrogenism (e.g., PCOS and precocious puberty) in females and improves fertility of adult men with metabolic syndrome through increased testosterone production. While the therapeutical use of metformin is considered to be safe, in the last 10 years some epidemiological studies have described phenotypic differences after prenatal exposure to metformin. The goals of this review are to briefly summarize the current knowledge on metformin focusing on its effects on the female and male reproductive organs, safety concerns, including the potential for modulating fetal imprinting via epigenetics.

An integrative approach combining passive sampling, bioassays, and effect-directed analysis to assess the impact of wastewater effluent.

Wastewater treatment plant (WWTP) effluents are major sources of endocrine-disrupting chemicals (EDCs) and other chemicals of toxicological concern for the aquatic environment. In the present study, we used an integrated strategy combining passive sampling (Chemcatcher®), developmental toxicity, and mechanism-based in vitro and in vivo bioassays to monitor the impacts of a WWTP on a river. In vitro screening revealed the WWTP effluent as a source of estrogen, glucocorticoid, and aryl hydrocarbon (AhR) receptor-mediated activities impacting the downstream river site where significant activities were also measured, albeit to a lesser extent than in the effluent. Effect-directed analysis of the effluent successfully identified the presence of potent estrogens (estrone, 17α-ethinylestradiol, and 17ß-estradiol) and glucocorticoids (clobetasol propionate and fluticasone propionate) as the major contributors to the observed in vitro activities, even though other unidentified active chemicals were likely present. The impact of the WWTP was also assessed using zebrafish embryo assays, highlighting its ability to induce estrogenic response through up-regulation of the aromatase promoter-dependent reporter gene in the transgenic (cyp19a1b-green fluorescent protein [GFP]) zebrafish assay and to generate teratogenic effects at nonlethal concentrations in the zebrafish embryo toxicity test. The present study argues for the use of such an integrated approach, combining passive sampling, bioassays, and effect-directed analysis, to comprehensively identify endocrine active compounds and associated hazards of WTTP effluents. Environ Toxicol Chem 2018;37:2079-2088. © 2018 SETAC.