Unité mixte de recherche
Stress Environnementaux et BIOsurveillance des milieux aquatiques
Chez les poissons, le test Easzy a permis de montrer que de nombreux PE sont capables de perturber, au stade embryonnaire, l’expression d’une enzyme, cytochrome P450 aromatase B, responsable de la synthèse des neuroestrogènes dans les cellules gliales radiaires. Ces cellules jouent un rôle important dans le développement du cerveau puisqu’elles donnent naissance à de nouvelles cellules dont certaines se différencient en neurones. La stratégie du projet FEATS repose sur la conduite d’expérimentations sur des modèles de poissons zèbres exposés à un panel de substances a priori connues pour avoir des effets sur l’expression du cytochrome P450 aromatase B afin d’explorer les effets sur le développement du cerveau et du comportement.
Concerns about the effects of endocrine disruptors (EDs) on wildlife and humans developmental and reproductive health have stimulated important research programs leading to a better understanding of their modes of action and effects at individual and population levels. In parallel to this basic research, the development and implementation of screening and testing procedures for EDs have become important aims notably within REACH regulatory framework. Despite important progress, there is still a need to improve risk assessment of EDs through the implementation of efficient screening and testing procedures providing relevant information for subsequent management measures. In that respect, studies conducted by members of the present consortium through national and international research projects (including the ANR projects NEED and PROOFS), have provided a better understanding of the expression, regulation and disturbance in the central nervous system of the estrogen-regulated cyp19a1b gene encoding brain aromatase (aroB) in fish. As a result, a wealth of molecular tools and in vitro or in vivo assays have been developed including the cutting-edge mechanism-based in vivo EASZY assay (Detection of Endocrine Active Substances acting on aroB through estrogen receptors using transgenic cyp19a1b-GFP Zebrafish embrYos) . This bioassay, now under validation at OECD level, is a highly sensitive, specific, non-invasive screening assay allowing for a reliable quantification of the estrogenic activities of substances alone and in mixture. Importantly, it is also the only assay that provides information on the neuroendocrine effects of chemicals targeting radial glial cells in a vertebrate model. We were the first to show that numerous substances (e.g. natural steroids, pharmaceuticals, industrial pollutants) target some neuron progenitors (radial glial cells) to disrupt aroB expression in the developing brain [1,2,3].
However, the toxicological consequences of such disruptions remain poorly explored. Modulations of aromatase expression and activity have been associated with altered neurogenic activity in several models including fish, rodent and human. However, this information is limited to very few compounds (e.g., estradiol) and it would be advisable to determine whether such effects could occur in case of exposure to other compounds similarly affecting aroB expression and/or activity. Similarly, we have very limited knowledge as to whether an altered aromatase expression leads to significant changes in the concentration of neuro-estrogens or other neurosteroids in the developing brain, and whether such changes could lead to altered brain development and individual behavior. The establishment of such functional links between molecular initiating events and individual responses are essential to increase our knowledge on the role of aromatase on brain development and on toxicological consequences of early disruption of aroB. It is also critical in the regulatory context of EDs, since information about both adverse effects and their underlying mechanisms (described as an adverse outcome pathway, AOP) are required criteria.
In this context, the ambition of FEATS is to fill knowledge gaps on the role of aroB and to produce a comprehensive dataset on the mode of action of EDs on brain development. FEATS is a unique opportunity to go a step-forward to elucidate the toxicological consequences of aroB disruption and develop mathematical models for predictive (eco)toxicology. It will help in elaborating efficient and functional strategies for assessing the risk of substances acting as neuro-EDs through the development of a quantitative AOP. To this end, the project FEATS is structured into complementary and closely interacting WPs (Fig. 1).
A detailed understanding of the molecular mechanism leading to short- and long-term effects upon exposure to EDs is essential for the risk assessment of substances acting on aroB expression/activity. Already established zebrafish models for aroB, including the well-characterized transgenic cyp19a1b-GFP zebrafish line and newly developed knock-out cyp19a1b (KO-cyp19a1b) zebrafish will be used in all WPs. Early exposures (during embryo-larval stages) will be performed in several partner’s facilities for downstream analyses and/or sample dispatch. Exposed individuals or samples will be used to investigate steroids environment in the developing larvae (WP3) and to improve knowledge of aroB in response to estrogen signal on neurogenesis, neuroplasticity (WP4) and its role in mediating behavior changes (WP5). These WPs are pivotal in the context of FEATS as their outcomes along with quantitative data acquired using powerful imagery methods (WP6) will bring a comprehensive dataset to establish a physiologically based pharmacokinetic model (PBPK) in the frame of the integrative WP7.
Figure 1. FEATS is structured into complementary and closely related WPs aiming at investigating the toxicological consequences mediated by aroB disturbance and to propose mathematical tools for a better risk assessment of EDs.
SEBIO is involved in WP1, WP2 and WP7
WP1. Coordination (Ineris): The project coordinator will be responsible for organizing regular meeting between all members of the consortium, compiling periodic and final reports for delivery to ANR.
WP2. Biological tools: engineering zebrafish models for aroB (Ineris): The objectives are i) to improve the methodological procedures to increase screening capacity of the EASZY assay through the use of microplate methods (Irset) and automatic processing of the larvae using VAST system (L’Oreal), ii) to use these improved methods to quantify aroB disruption by neuro-EDs for modelling and iii) to assess the long-term effects of an early exposure through in vivo measurement of GFP in juvenile and adult fish using recently established Casper-cyp19a1b-GFP line (devoid of pigments). Data produced will be combined with data already acquired with cyp19a1b-GFP [e.g., 1,2,3] and a number of chemicals as input for modelling (WP7).
WP7. Toxicokinetic and dynamic modeling of neuro-endocrine disruptors in zebrafish embryo (Ineris): The first model developed will be a physiologically based pharmacokinetic (PBPK) model of the zebrafish embryo to refine the analysis of the EASZY assay (WP2) and improve the understanding of the neuro-endocrine disruption. This model will be able to predict the intra-cellular concentrations of xenobiotics as a function of time and dose, following exposure of the whole embryo. A PBPK model for adult zebrafish has already been developed and tested for a panel of EDs at Ineris . In this WP, it will be extended to the embryo-larval stage using quantitative data acquired by imagery methods (WP6) to set up the physiological parameters, and sensitive chemical analysis of xenobiotics (WP3) to evaluate toxicokinetic predictions. The second model will be a quantitative AOP model of the aroB disruption based on the intra-cellular concentrations predicted by the PBPK model. This model will be calibrated using the concentrations of neurosteroids in developing zebrafish determined (WP3) using dynamic differential equation system or Bayesian network. Finally, the models developed could be integrated later in an existing modelling framework to extrapolate how neuro-EDs could impact the individual fitness and behaviour (data from WP5) and population dynamics.