Development and Application of Transgenic Biosensor Zebrafish for Assessing the Environmental Health Impacts of Anti-Androgens

Major worldwide attention has focused on the observations of disruptions of reproduction in both wildlife and human populations that can result from exposure to chemicals that interfere with the body's hormone signalling systems (so-called endocrine disrupting chemicals; EDCs). More recently, wider health effects for exposure to EDCs have been suggested in the human population, including associations with cardiovascular disease and obesity. Most studies on EDCs have focused on chemicals that mimic the female hormone oestrogen and there is substantial evidence that exposure to oestrogenic chemicals can result in reduced sperm count and quality in males, and cause various reproductive cancers. More recently chemicals that antagonise male sex hormones (androgens) action (so called anti-androgens) have been associated with malformations of the reproductive system. This class of chemicals is very widespread in the environment and has become of major environmental concern. We have spent 3 years developing a genetically engineered zebrafish with a convenient response system for detecting oestrogens (they fluoresce green in affected tissues) . This has provided us with an advanced model for studying where oestrogenic chemicals act in the body and for making assessments on their mechanisms of action and overall health effects.. In this project, we aim to develop novel transgenic biosensor fish for detecting androgen and anti-androgen activity. .We will do this in a transparent strain of zebrafish (casper ) allowing us to study (anti-)androgenic chemicals in all fish life stages, even adults, where normally natural pigmentation would mask the green fluorescence. The fluorescing protein we will use in these transgenic fish is called Kaede which will fluoresces green, but on exposure to UV light changes to a red colour and this will be especially useful to separate background expression of Kaede protein (due to the presence of natural androgens in the body) and for studies into chemical mixture effects. Having successfully engineered our (anti-)androgenic responsive zebrafish, we will expose them (embryos/larvae and adults) to a series of environmental anti-androgens (and androgens) to establish whole body responses and identify target tissues affected . These studies will also assess which life stages are the most sensitive/vulnerable to exposure to (anti)androgens. We will further study whole genome responses (responses of thousands of genes simultaneously) for selected target tissues and androgenic and anti-androgenic chemicals to identify in detail the genes, pathways and processes that are affected. We will then apply our fluorescing fish to investigate mixture effects to investigate for additive and other interactive effects across body target tissues. Finally, we will investigate the application of our zebrafish to test fort he presence of (anti-)androgenic activity in real world and complex environmental samples (for example effluents from wastewater treatment works). Collectively this work will help us understand how different (anti-) androgens confer different effects and toxicities in the natural environment. Findings from this project will be of considerable interest and potential benefit to a very wide section of the community including the following: - the medical profession, for better understanding the roles of natural androgens and anti-androgenic contaminants on health; - chemical and pharmaceutical industries, for chemical screening and testing to avoid unintended androgenic and anti-androgenic activity in produced chemicals, and the development of more effective and/or selective anti-androgenic pharmaceuticals for treatment therapies, - industry and government regulatory bodies, environment protection groups and the wider public to better protect the environment and humans from the effects of EDCs.