Assaying uptake of endocrine disruptor compounds in zebrafish embryos and larvae

Abstract

To study the effects of environmental endocrine disruptors (EEDs) on aquatic animals, embryos and larvae are typically incubated in water containing defined concentrations of EEDs. However, the amount of EED uptake into the animal is often difficult to determine. Using radiolabeled estradiol ([3H]E2), we previously developed a rapid, straightforward assay to measure estradiol uptake from water into zebrafish embryos and larvae. Here, we extend this approach to measure the uptake of two additional EEDs, bisphenol A (BPA) and ethinyl estradiol (EE2). As with E2, the uptake of each compound by individual larvae was low (< 6%), and increased with increasing concentration, duration, and developmental stage. We found that E2 and EE2 had similar uptake under equivalent exposure conditions, while BPA had comparatively lower uptake. One application of this assay is to test factors that influence EED uptake or efflux. It has been suggested that persistent organic pollutants (POPs) inhibit ABC transporters that may normally efflux EEDs and their metabolites, inducing toxicity in aquatic organisms. We measured [3H]E2 levels in zebrafish in the presence or absence of the POP PDBE-100, and cyclosporine A, a known inhibitor of ABC transporters. Neither chemical significantly affected [3H]E2 levels in zebrafish, suggesting that zebrafish maintain estradiol efflux in the presence of PDBE-100, independently of cyclosporine A-responsive transporters. These uptake results will be a valuable reference for EED exposure studies in developing zebrafish, and provide a rapid assay to screen for chemicals that influence estrogen-like EED levels in vivo.