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

据报告，含有高浓度二恶英和/或多氯联苯的野生动物的生殖和免疫系统受到影响。近年来，与甲状腺激素具有结构相似性的有机卤化合物作为阻燃剂得到了广泛的应用，其中一些化合物在环境样品中被检测到。这些事实表明，这些有机卤素化合物可能作为环境污染物对野生动物和人类产生影响。在这项工作中，我们构建了生物测定系统，利用甲状腺激素受体的转录激活作为生物标志物。首先，我们建立了甲状腺激素受体α基因稳定转染的人细胞系，命名为HeLaTR细胞。然后在T3存在或不存在的情况下，将其中在荧光素酶基因上游插入甲状腺激素反应元件（DR 4）的DR 4-SV-Luc质粒瞬时转染到HeLaTR细胞中。荧光素酶活性测定采用 ...更多信息 使用细胞裂解物，发现T3使荧光素酶活性增加4至5倍。在向该生物测定系统中加入各种有机卤素化合物如环境污染物后，测定每种细胞裂解物中的荧光素酶活性，以评价有机卤素化合物对甲状腺激素系统的影响。PCB的代谢产物羟基化PCB可降低T3诱导的HeLaTR细胞荧光素酶活性。与此相反，二恶英，六环十二烷，这是用于阻燃剂，除草醚，已被用于农药增强荧光素酶活性的T3诱导。二碘联苯（DIB）作为手机光电导体，可诱导荧光素酶活性，增强T3作用。DIB还可通过雌激素反应元件增强雌激素受体的转录激活作用。我们还通过DNA微阵列测定了T3加入HeLaTR细胞后的基因表达谱，结果表明加入T3后，通过4-1BB的凋亡信号被激活。少

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