Screening method to identify environmental endocrine disruptors mediated by PPARs

描述（由申请人提供）：美国使用的化学品超过80，000种，每年引入的新化学品超过2，000种（NRC，1984年; OTA，1995年）。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，但其中大多数化学品的影响尚不清楚。特别令人关注的是，特别是在儿童中，那些可能影响内分泌系统的药物。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存在显著水平，并有可能影响代谢紊乱，包括肥胖症、糖尿病、慢性炎症性疾病和心血管疾病。目前可用的识别PPAR激动剂的体外筛选试验测量受体结合或全基因组表达。这些检测方法显然提供了大量信息，但前者没有提供生物活性方面的信息，后者缺乏特异性，可能难以转化为对人类健康的影响，所需技术在临床研究中的可移植性有限，需要进行深入的统计分析。此外，PPAR反应是复杂的，难以解释因果关系。PPAR依赖性反应不仅极其多样，而且以种属特异性和组织特异性方式发生。反应可受激动剂浓度及其组合以及特异性结合亲和力的影响。鉴于这些现有的限制，我们建议建立一种高通量的体外细胞培养筛选试验，以确定基因表达的变化，发生在关键的生物学途径，从假定的环境PPAR激动剂使用低密度聚焦阵列卡和选定的人类细胞系，包括HepaRG和THP 1细胞，（肝细胞和单核细胞细胞系，分别）。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，反映了人体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。这种假设驱动的测试方案的重点是确定可能产生的激动剂， 与代谢紊乱相关的健康影响，如高胆固醇血症、肥胖症、2型糖尿病和慢性炎性疾病。这些研究的结果将有助于确定内分泌干扰物，这些干扰物通过其作为过氧化物酶体增殖物激活剂的能力有可能影响人类健康。它还可以用于鉴定用于治疗代谢紊乱的特异性和有效的候选治疗剂。

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.