Screening method to identify environmental endocrine disruptors mediated by PPARs

描述（由申请人提供）：在美国有超过80,000种化学品在使用，每年有超过2,000种新化学品被引入（NRC, 1984； OTA, 1995）。虽然这些化学品中可能对人类健康或环境构成重大风险的相对较少，但其中大多数的影响是未知的。特别值得关注的是那些可能影响内分泌系统的药物，尤其是对儿童而言。通过过氧化物酶体增殖物激活受体（PPARs）发挥作用的内分泌干扰物的潜在健康意义正受到越来越多的关注。其中许多在婴儿和儿童中含量很高，有可能影响代谢紊乱，包括肥胖、糖尿病、慢性炎症性疾病和心血管疾病。目前用于鉴定PPAR激动剂的体外筛选试验可测量受体结合或全基因组表达。这些分析显然提供了信息，但前者没有提供关于生物活动的信息，后者缺乏特异性，难以转化为对人类健康的影响，所需技术用于临床研究的可移植性有限，需要进行深入的统计分析。此外，PPAR反应很复杂，很难用因果关系来解释。ppar依赖性反应不仅极其多样化，而且以物种和组织特异性的方式发生。该反应可受激动剂浓度及其组合以及特定结合亲和力的影响。考虑到这些目前的局限性，我们建议建立一个高通量的体外细胞培养筛选实验，利用低密度聚焦阵列卡和选定的人类细胞系，包括HepaRG和THP1细胞（分别是肝细胞和单核细胞系），鉴定由假定的环境PPAR激动剂引起的关键生物学途径的基因表达变化。先前发表的研究和本文提供的初步数据表明，这些细胞系对PPAR激动剂的激活有反应，反映了人类暴露后发生的生物学反应，并提供了足够的灵敏度，表明成功的可能性很高。这个假设驱动的测试方案的重点是确定可能产生的激动剂

DESCRIPTION (provided by applicant): Over 80,000 chemicals are in use in the United States and over 2,000 new ones are introduced annually (NRC, 1984; OTA, 1995). While relatively few of these chemicals are likely to pose a significant risk to human health or the environment, the effects of most of them are unknown. Of particular concern, particularly in children, are those that may affect the endocrine system. The potential health significance of endocrine disruptors that act through peroxisome proliferator- activated receptors (PPARs) is receiving more attention. Many of these are found at significant levels in infants and children and have the potential of influencing metabolic disorders including obesity, diabetes, chronic inflammatory diseases and cardiovascular disease. In vitro screening tests to identify PPAR agonists currently available measure receptor binding or whole genome expression. These assays are clearly informative but the former provides no information on biological activities and the latter lacks specificity and can be difficult to translate to human health effects, having limied portability of the required technology to clinical studies and requiring intensive statistical analyses. Furthermore, PPAR responses are complex and difficult to interpret in terms of causality. PPAR-dependent responses are not only extremely diverse, but occur in both a species- and tissue-specific manner. The response can be influenced by the agonist concentration and combinations thereof as well as specific binding affinities. Given these current limitations, we propose to establish a high throughput in vitro cell culture screening assay to identify gene expression changes in key biological pathways that occur from putative environmental PPAR agonists using a low-density focused array card and selected human cell lines including HepaRG and THP1 cells, (hepatocyte and monocytes cell lines, respectively). Previous published studies and preliminary data provided here suggests that these cell lines respond to activation by PPAR agonists, reflect the biological responses that occur following human exposures and provide sufficient sensitivity indicating a high likelihood for success. The focus of this hypothesis-driven testing scheme is to identify agonists that can potentially produce health effects related to metabolic disorders such as hypercholesterolemia, obesity, type 2 diabetes and chronic inflammatory diseases. Results from these studies will help identify endocrine disruptors that have the potential to affect human health through their ability to serve as PPAR agonists. It can also be used to identify specific and potent candidate therapeutics for the treatment of metabolic disorders.