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

全世界的注意力主要集中在观察野生动物和人类种群的生殖中断，这可能是由于接触干扰人体激素信号系统的化学品（所谓的内分泌干扰化学品; EDCs）。最近，暴露于EDCs的更广泛的健康影响已经在人群中提出，包括与心血管疾病和肥胖的关联。大多数关于内分泌干扰物的研究都集中在模拟雌性激素雌激素的化学物质上，有大量证据表明，暴露于雌激素化学物质会导致男性精子数量和质量减少，并导致各种生殖癌症。最近，拮抗雄性激素（雄激素）作用的化学物质（所谓的抗雄激素）与生殖系统畸形有关。这类化学品在环境中非常普遍，已成为主要的环境问题。我们花了3年时间开发了一种基因工程斑马鱼，它具有检测雌激素的方便反应系统（它们在受影响的组织中发出绿色荧光）。这为我们提供了一个先进的模型，用于研究雌激素化学品在体内的作用，并对其作用机制和整体健康影响进行评估。本研究旨在开发一种新型的转基因生物传感器鱼，用于检测雄激素和抗雄激素活性。我们将在斑马鱼（casper）的透明品系中进行这一研究，从而使我们能够研究所有鱼类生命阶段中的（抗）雄激素化学物质，即使是成年鱼，其中通常天然色素沉着会掩盖绿色荧光。我们将在这些转基因鱼中使用的荧光蛋白称为Kaede，其将发出绿色荧光，但在暴露于UV光时变为红色，这将特别有助于分离Kaede蛋白的背景表达（由于体内存在天然雄激素）和研究化学混合物效应。在成功地设计了我们的（抗）雄激素反应斑马鱼后，我们将使它们（胚胎/幼虫和成虫）暴露于一系列环境抗雄激素（和雄激素），以建立全身反应并识别受影响的靶组织。这些研究还将评估哪些生命阶段对暴露于（抗）雄激素最敏感/最脆弱。我们将进一步研究选定的靶组织和雄激素和抗雄激素化学物质的全基因组反应（数千个基因同时反应），以详细确定受影响的基因，途径和过程。然后，我们将应用我们的荧光鱼来研究混合物效应，以研究身体靶组织的叠加效应和其他相互作用效应。最后，我们将研究应用我们的斑马鱼来测试真实的世界和复杂的环境样品（例如废水处理厂的废水）中是否存在（抗）雄激素活性。总的来说，这项工作将帮助我们了解不同的（抗）雄激素如何在自然环境中产生不同的影响和毒性。该项目的研究结果将对广泛的社会阶层产生相当大的兴趣和潜在的利益，包括以下方面：-医学界，更好地了解天然雄激素和抗雄激素污染物对健康的作用;- 化学和制药工业，用于化学筛选和测试，以避免所生产的化学品中意外的雄激素和抗雄激素活性，以及开发用于治疗疗法的更有效和/或选择性的抗雄激素药物，-工业和政府监管机构、环境保护团体和更广泛的公众，以更好地保护环境和人类免受EDCs的影响。

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.