2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) MODULATES FUNCTION OF HUMAN LUTEINIZING GRANULOSA-CELLS VIA CAMP SIGNALING AND EARLY REDUCTION OF GLUCOSE TRANSPORTING ACTIVITY

This study examined the changes in cellular glucose uptake, cAMP-depen dent 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 toxi city in human LGCs was investigated. Treatment of human LGCs with TCDD produced a time- and dose-dependent decrease in the cellular uptake o f glucose. The V-max and the K-m of glucose transport were decreased b y TCDD treatment. Furthermore, cytochalasin B, a specific inhibitor of facilitative glucose transporter proteins, totally abolished the port ion of glucose transport activity that is sensitive to TCDD. Pretreatm ent of the cells with the Ah receptor blockers 4,7-phenanthroline and alpha-naphthoflavone antagonised the effect of TCDD on H-3-Me-glucose uptake. Structure-activity relationship studies with TCDD and three di oxin congeners revealed a rank order for their potency in the inhibiti on 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 d etermined 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 progeste rone production was more pronounced in the presence of insulin (10 mu g/mL) and D-glucose (13.3 mM). However, cytochalasin B abolished the e ffect of TCDD on progesterone production. Forskolin (adenylate cyclase activator) abolished the effect of TCDD on glucose uptake and progest erone production but it did not affect the action of TCDD on PKA activ ity. A relationship between glucose transporting activity and progeste rone production in human LGCs treated with TCDD is indicated by severa l lines of evidence: a) cytochalasin B downregulated glucose transport ing activity and progesterone production, b) insulin plus D-glucose do wnregulated glucose uptake and amplified the negative effect of TCDD o n progesterone production, and c) forskolin abolished the negative eff ect of TCDD on glucose transporting activity and on progesterone produ ction. From the present data we conclude that glucose transporting act ivity can be used as a sensitive biomarker to detect the very early re sponse to TCDD in human steroid-producing cells and that effect of TCD D on steroid production is mediated through the cAMP-dependent protein kinase.