Enhanced content bio-imaging tools: realising the potential for high-throughput zebrafish bioassays

Abstract

Abstract Estrogenic endocrine disrupting chemicals (EEDCs) are environmental contaminants that can alter hormone signalling in both humans and wildlife, exerting their action through estrogen receptors (ERs). A wealth of evidence has indicated that EEDCs are capable of producing a broad range of adverse outcomes by interacting with, and disrupting, the normal functioning of the estrogen system. Fish are particularly vulnerable to endocrine disruption due to EEDCs being frequently discharged into waterways. With more than 900 chemicals identified as being endocrine disruptors, of which ~200 may exert estrogenic effects, there is an urgent need for screening processes that can assess the estrogenic potential of chemicals in order to avoid human and environmental health risks. In vivo models capable of demonstrating the physiological effects of EEDCs hold great utility for understanding the potential health impacts of estrogens, and transgenic (TG) zebrafish (Danio rerio) models are particularly well-suited for the screening of EEDCs via bioimaging approaches. The pigment-free estrogen-responsive ERE:GFP:Casper model represents a promising transgenic line for qualifying and quantifying EEDC-induced fluorescence responses in larval fish and is amenable to high-throughput screening (HTS). We optimised a medium-throughput semi-automated in vivo bioimaging assay using the model, while simultaneously generating important data concerning estrogen-driven responses to an EEDC (EE2). Through refinement of assay parameters, including the use of various image-masking and pixel-thresholding approaches, controlled-breeding to reduce genetic variability and standardised larval orientation for image acquisition, we established the most sensitive and robust approaches for screening of the EE2-exposed model using a semi-automated imaging modality. Our optimised assay was capable of detecting a significant GFP response in 4 day old zebrafish larvae at an environmentally relevant (5ng/L) concentration of EE2. These specifications were then adopted for investigating the influence of varying incubation temperatures (24, 28 and 32°C) on EE2-exposed ERE:GFP:Casper larval growth and GFP responses. This analysis provided information concerning the potential for an EEDC to interact with temperature in a fish model, with important implications for subsequent interpretation of results. We screened the same animal over a series of timepoints generating valuable data concerning estrogen-induced fluorescence responses and specific larval growth. Incubation temperature was found to have a significant effect on GFP induction, both alone and in interaction with EE2. The findings of this thesis help to outline an improved approach for further development of higher-throughput in vivo estrogenic screening of a transgenic zebrafish model.