Development of bioassay system for evaluating the (anti-) thyroid hormone-like activity by various environmental contaminants

开发生物测定系统，用于评估各种环境污染物的（抗）甲状腺激素样活性

基本信息

批准号：
14570303
负责人：
OKABE Toshiko
金额：
$ 1.86万
依托单位：
Yokohama City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

It had been reported that reproductive and immune systems were affected in wildlife containing high concentrations of dioxins and/or polychlorinated biphenyls (PCBs). Recently the organohalogen compounds which have structural similarities with thyroid hormone have been used widely as flame retardants and some of them are detected in environmental samples. From these facts, it has been suspected that these organohalogen compounds may affect on wildlife as well as human beings as environmental contaminants. In this work, we constructed the bioassay system using transcriptional activation by thyroid hormone receptor as biomarkers. First, we established human cell line in which thyroid hormone receptor α gene was stable transfected and designated HeLaTR cells. Then DR4-SV-Luc plasmid in which thyroid hormone response element (DR4) was inserted upstream of luciferase gene was transfected into HeLaTR cells transiently in the presence or absence of T3. Luciferase activities were determined us … More ing cell lysate and it was found that T3 increased luciferase activity four to five fold. After addition of various organohalogen compounds such as environmental contaminants to this bioassay system, luciferase activities in each cell lysates were determined to evaluate the effects on thyroid hormone system by organohalogen compounds. One hydroxylated PCB, which was the metabolite of PCB turned to reduce the luciferase activity which was induced by T3 in HeLaTR cells. In contrast, dioxin, hexacyclododecane which is used for flame retardant, and nitrofen which had been used for pesticide augmented the luciferase activities induced by T3. Diiodobiphenyl (DIB) which is used for photoconductor of cellular phone could induce luciferase activity and augment the T3 action. Furthermore, DIB could augment the transcriptional activation by estrogen receptor through estrogen response element. We also determined the gene expression profile after addition of T3 into HeLaTR cells by DNA microarray, and showed that apoptosis signal through 4-1BB was activated after addition of T3. Less

据报道，在含有高浓度的二恶英和/或多氯联苯（PCB）的野生动植物中，生殖和免疫系统受到影响。最近，与甲状腺荷烯具有结构相似性的有机脂蛋白化合物已被广泛用作阻燃剂，其中一些在环境样品中被检测到。从这些事实来看，人们怀疑这些有机催化剂化合物可能会影响野生动植物以及人类作为环境污染物的影响。在这项工作中，我们使用甲状腺激素受体作为生物标志物的转录激活构建了生物测定系统。首先，我们建立了人类细胞系，其中甲状腺激素受体α基因被稳定转染并指定为HELATR细胞。然后将甲状腺激素反应元件（DR4）插入荧光素酶基因上游的DR4-SV-LUC质粒在存在或不存在T3的情况下插入荧光素酶基因上游的HELATR细胞。确定了荧光素酶活性……更多的细胞裂解物，发现T3增加了荧光素酶活性四到五倍。在添加了各种有机脂蛋白化合物（例如环境污染物）中，确定了每个细胞裂解液中的荧光素酶活性，以评估通过有机糖化化合物对甲状腺激素系统的影响。一种羟基化的PCB，即PCB的代谢产物，以减少Helatr细胞中T3诱导的荧光素酶活性。相比之下，用于阻燃剂的二恶英，己糖环烷和已用于农药的亚硝酸增强了T3诱导的荧光素酶活性。用于细胞手机光电的二二苯基（DIB）可以诱导荧光素酶活性并增强T3动作。此外，DIB可以通过雌激素反应元件来增加雌激素受体的转录激活。我们还通过DNA微阵列确定了将T3添加到HELATR细胞中后的基因表达谱，并表明在添加T3后激活了通过4-1BB的凋亡信号。较少的