Enan E, Lasley B, Stewart D, Overstreet J, Vandevoort C A
Department of Environmental Toxicology, University of California, Davis 95616-8615, USA.
Reprod Toxicol. 1996 May-Jun;10(3):191-8. doi: 10.1016/0890-6238(96)00021-4.
This study examined the changes in cellular glucose uptake, cAMP-dependent protein kinase (PKA), and progesterone production induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human luteinizing granulosa cells (LGCs) in culture. The role of Ah receptor on TCDD-mediated toxicity in human LGCs was investigated. Treatment of human LGCs with TCDD produced a time- and dose-dependent decrease in the cellular uptake of glucose. The Vmax and the K(m) of glucose transport were decreased by TCDD treatment. Furthermore, cytochalasin B, a specific inhibitor of facilitative glucose transporter proteins, totally abolished the portion of glucose transport activity that is sensitive to TCDD. Pretreatment of the cells with the Ah receptor blockers 4,7-phenanthroline and alpha-naphthoflavone antagonised the effect of TCDD on 3H-Me-glucose uptake. Structure-activity relationship studies with TCDD and three dioxin congeners revealed a rank order for their potency in the inhibition of glucose transport as follows: TCDD > 1,2,3,7,8-PCDD > 1,2,4,7,8-PCDD > 2,7-DCDD. Such a rank order is consistent with the previously determined biological activity of TCDD and the other dioxin congeners. Treatment of cells for 48 h with 10 nM TCDD substantially reduced PKA and progesterone production. The inhibitory effect of TCDD on progesterone production was more pronounced in the presence of insulin (10 micrograms/mL) and D-glucose (13.3 mM). However, cytochalasin B abolished the effect of TCDD on progesterone production. Forskolin (adenylate cyclase activator) abolished the effect of TCDD on glucose uptake and progesterone production but it did not affect the action of TCDD on PKA activity. A relationship between glucose transporting activity and progesterone production in human LGCs treated with TCDD is indicated by several lines of evidence: a) cytochalasin B downregulated glucose transporting activity and progesterone production, b) insulin plus D-glucose downregulated glucose uptake and amplified the negative effect of TCDD on progesterone production, and c) forskolin abolished the negative effect of TCDD on glucose transporting activity and on progesterone production. From the present data we conclude that glucose transporting activity can be used as a sensitive biomarker to detect the very early response to TCDD in human steroid-producing cells and that effect of TCDD on steroid production is mediated through the cAMP-dependent protein kinase.
本研究检测了2,3,7,8-四氯二苯并对二噁英（TCDD）对培养的人黄体化颗粒细胞（LGCs）中细胞葡萄糖摄取、环磷酸腺苷依赖性蛋白激酶（PKA）和孕酮产生的影响。研究了芳烃受体（Ah受体）在TCDD介导的人LGCs毒性中的作用。用TCDD处理人LGCs导致细胞葡萄糖摄取呈时间和剂量依赖性下降。TCDD处理降低了葡萄糖转运的最大速率（Vmax）和米氏常数（Km）。此外，促葡萄糖转运蛋白的特异性抑制剂细胞松弛素B完全消除了对TCDD敏感的葡萄糖转运活性部分。用Ah受体阻滞剂4,7-菲咯啉和α-萘黄酮预处理细胞可拮抗TCDD对3H-Me-葡萄糖摄取的影响。对TCDD和三种二噁英同系物的构效关系研究揭示了它们抑制葡萄糖转运的效力顺序如下：TCDD > 1,2,3,7,8-五氯二苯并对二噁英（PCDD）> 1,2,4,7,8-PCDD > 2,7-二氯二苯并对二噁英（DCDD）。这样的效力顺序与先前确定的TCDD和其他二噁英同系物的生物活性一致。用10 nM TCDD处理细胞48小时可显著降低PKA和孕酮的产生。在存在胰岛素（10微克/毫升）和D-葡萄糖（13.3毫摩尔）的情况下，TCDD对孕酮产生的抑制作用更明显。然而，细胞松弛素B消除了TCDD对孕酮产生的影响。福斯可林（腺苷酸环化酶激活剂）消除了TCDD对葡萄糖摄取和孕酮产生的影响，但不影响TCDD对PKA活性的作用。几条证据表明了TCDD处理的人LGCs中葡萄糖转运活性与孕酮产生之间的关系：a）细胞松弛素B下调葡萄糖转运活性和孕酮产生；b）胰岛素加D-葡萄糖下调葡萄糖摄取并放大TCDD对孕酮产生的负面影响；c）福斯可林消除了TCDD对葡萄糖转运活性和孕酮产生的负面影响。根据目前的数据，我们得出结论，葡萄糖转运活性可作为一种敏感的生物标志物，用于检测人类固醇生成细胞对TCDD的早期反应，并且TCDD对类固醇产生的影响是通过环磷酸腺苷依赖性蛋白激酶介导的。
