A new alternative method using cyp3a65 expression in transgenic zebrafish embryos to assess metabolic...

Metabolic endocrine-disrupting chemicals (MDCs) contribute to the development and increasing incidence of metabolic disorders, highlighting the need for relevant assays to identify them. Several alternative models to mammals, notably the zebrafish, have been developed, but none of these assays account for the modes of action and effects of MDCs on the intestine. This study aimed to 1) establish and characterize a transgenic embryo model expressing GFP under the control of the zebrafish cyp3a65 gene, ortholog of the human cyp3a4; 2) set up an original zebrafish embryo-based bioassay to study the Effects of Metabolic Endocrine disRuptors in Gut of zebrafish Embryos (EMERGE). Spatiotemporal expression of cyp3a65-GFP and temporal expression of endogenous cyp3a65 was characterized using fluorescence imaging of GFP, immunohistochemistry and RT-qPCR, under both normal and exposed conditions to clotrimazole and TCDD (respectively zfPXR and zfAhR2 agonists). Then, twenty-two chemicals were screened for their potential activity using the EMERGE assay. We report an early and dynamic expression of cyp3a65 in the developing intestine which is disrupted in a time and concentration dependent manner by zfPXR and zfAhR2 agonists. Moreover, we report that various environmental chemicals can significantly up- or down regulate cyp3a65 expression, most of them being newly identified as targeting the intestine and disrupt this critical metabolic enzyme. In conclusion, EMERGE represents a new easy to use assay to screen metabolic disrupting activity of chemicals in a non-mammalian model. The data collected suggest it could provide valuable insights into the effects of substances on cyp3a4 in humans and may emerge as a valuable assay for assessing human-relevant effects.