Zebra Fish Embryo Assay for Toxicological riSk assessment: case of endocrine disruptors acting on brain aromatase – FEATS

Much research efforts have focused on endocrine disruptors (EDs) actions on the reproductive axis, while little research examined their effects on steroid-sensitive brain structures and functions. Notwithstanding, experimental animal data and epidemiological studies support links between developmental exposures to EDs and disruption of neuroendocrine circuits and brain functions that could result in increased neurodevelopmental and neurocognitive disorders. However, there is a lack of knowledge about the mechanisms and effects of EDs on brain development and behavior, which precludes to correctly assess the hazard and risks of EDs targeting brain.

Previous studies conducted by members of FEATS have provided a better understanding of the expression, regulation and disruption of the estrogen-regulated cyp19a1b gene encoding aroB, in the brain of fish. They successfully developed the mechanism-based in vivo EASZY assay. This bioassay, validated at OECD level, is a highly sensitive, specific, non-invasive screening assay allowing for a reliable quantification of the estrogenic activities of substances. Based on EASZY, they demonstrated that one of the most striking effects of EDs in fish is their impact on the radial glial cell (RGCs)- specific expression of aroB. RGCs are stem cells in the central nervous system and play a crucial role in neurogenesis and brain repair, giving birth to neurons in developing as well as adult fish.

The toxicological consequences of aroB disruption remain poorly explored. Defining the consequences of this alteration is a prerequisite to assess the risk posed by contaminants acting on aroB in the developping brain. Modulation of aroB expression and activity is associated with altered neurogenic activity in several models including fish, rodent and human. However, this link is limited to very few compounds and it is advisable to determine whether such consequences could occur following exposure to environmental compounds known to affect aroB expression and/or activity. In addition, there is very limited knowledge as to whether alteration of aroB expression leads to significant changes in the concentration of neuroestrogens and other neurosteroids in the developing brain, and whether such changes would lead to altered brain development and behavior. The identification of such functional links between molecular initiating events and individual responses is essential to increase our knowledge on the role of aroB 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, FEATS is an interdisciplinary project that aims to study the short- and long-term toxicological consequences induced by EDs acting on aroB during early developmental stage. Our strategy will be based on experiments with new and original engineered zebrafish models (cyp19a1b-GFP, cyp19a1b-GFP-Casper, knock-out cyp19a1b) exposed to a panel of substances selected a priori for their known effect on the expression aroB to assess their impact on neurosteroid synthesis, neurogenesis, neuroplasticity and behavior. It will rely on complementary approaches combining sensitive chemical analyzes for xenobiotics and neurosteroids quantification, 2D/3D fluorescence imaging, single cell transcriptomics in parallel to forefront behavioral analysis in embryos, larvae and adults. The objective is to provide a consistent set of data to provide the establishment of functional and quantitative relationships between aroB disruption and adverse effects through the development of mathematical models (i.e., PBPK, AOP) for a better risk assessment of EDs. Overall, FEATS aims to address both fundamental and regulatory challenges to improve risk assessment of chemical acting at the brain level.