Application of in vivo and in vitro assays for studying the chemosensitizing potential of environmental chemicals in zebrafish embryos

BIECZYNSKI F.; BURKHARDT-MEDICKE, K.; SCHOLZ, S.; LUCKENBACH, T.

Innsbruck

Congreso; 7th FEBS Special Meeting on ABC Proteins; 2018

Federation of European Biochemical Societies

In zebrafish embryos the uptake of chemicals from the water is controlled by an Abcb4 ortholog. Man-made chemicals can be abundant in aquatic environments and we were interested, to which extent such chemicals interfere with efflux transporter activity acting as chemosensitizers. We applied ATPase assays with recombinant zebrafish Abcb4 protein generated with the baculovirus system for obtaining indication for interaction of test compounds with the efflux transporter. Assays were run with concentration series of test compounds. From more than 30 environmental pollutants, comprising agro-chemicals, industrial chemicals, ingredients of personal care products and drugs, 20 caused either stimulation or inhibition of the basal Abcb4-ATPase activity and/or inhibition of the verapamil-stimulated ATPase activity. This indicates that interaction of environmental pollutants with zebrafish Abcb4 is prevalent. The finding that a majority of the compounds (almost 20) inhibited the verapamil-stimulated ATPase activity indicates that chemosensitizer action could be a common effect. For determining how potent these chemicals are as chemosensitizers in vivo we are currently establishing a dye accumulation assay with live zebrafish embryos. Embryos at 72 hours post fertilization are exposed to chemicals along with rhodamine B, a fluorescent substrate of zebrafish Abcb4, and the dye accumulated in the embryo tissue is quantified using the VAST (Vertebrate Automated Screening Technology) platform, which allows taking bright field and fluorescent images of live embryos in an automated way. If a test compound interferes with the efflux transporter the fluorescence signal increases, as more dye accumulates in the embryo tissue. So far, we applied cyclosporine A, a known inhibitor of zebrafish Abcb4, which in a concentration-dependent way caused an up to 2- to 3-fold increase of the fluorescence signal in the embryo tissue.