Development Of A Suite Of Bioassays To Detect Endocrine Disrupting Chemicals (EDCs) And Evaluate EDC Synergy

Abstract

The freshwater system is modified by human influences, and with the human population increasing over the next few years, it is hard to ignore the increasing anthropogenic pressure on our freshwater. Endocrine disrupting compounds (EDCs) are emerging chemical pollutants found in the freshwater system that have gained both public and scientific interest because of potential harmful effects on humans and wildlife. These natural and synthetic compounds are capable of disrupting endocrine system in organisms by mimicking or inhibiting the effect of endogenous hormones. Currently, EDCs are commonly screened by chemical-based analyses (e.g. LC-MS, GC-MS) as they provide the lowest detection limit. Analytical chemistry techniques do not, however, describe synergistic influences that may arise in the presence of multiple compounds. Bioassays provide quantitative and qualitative responses to EDCs that may be an alternative to chemical analyses. Bioassays do not identify the specific chemical structures, but rather, identify the presence of molecules with estrogenic potential. Bioassays detect combined effects of multiple xenbiotic substances similar to the real-world situation. Yeast oestrogen screen (YES) is a rapid and cost effective cellular-based bioassay capable of detecting oestrogenic compounds with high level of sensitivity comparable to chemical analysis. The YES assay was established using recombinant yeast cells encoding the human oestrogen receptor α (hERα) and an oestrogen-inducible β-galactosidase reporter. Addition of oestrogenic compounds gives a concentration dependent expression of reporter protein that is detectable using a chemiluminescent substrate and microplate luminometry. A whole organism assay using transgenic zebrafish may not have the equivalent oestrogenic sensitivity compared to an in vitro bioassay, but it is cost effective and expected to detect the physiological effects of oestrogenic compounds with a measureable endpoint that could potentially reflect to the real world conditions. With the newly develop transgenic zebrafish line, it is anticipated that levels of oestrogenic compounds can be detected by the expression of green fluorescent protein (GFP). The primary focus of this project is to: 1) Investigate potential synergistic and inhibitory effects by mixing two oestrogenic compounds with specific oestrogenicity in the YES assay, and 2) construct a transgenic zebrafish line expressing GFP-tagged to the oestrogen inducible Vtg promoter. A mixture of known-estrogenic compounds, 17a-Ethinylestradiol (EE2), Nonylphenol (NP) and Carbamazepine (CBZ) in YES assay showed no synergistic correlation, but displayed an inhibitory response. However, potential synergistic effect can be observed at low concentrations. Following a strategy from previous study, a transgenic zebrfish expressing GFP-tagged vitellogenin protein was successfully constructed.