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671 Procymidone: Characterisation of estrogen-like activity in the MCF-7 human breast cancer cell line
s180
Poster Session P34. Endocrine disrupters
dose. Relative weights of ventral prostate (VP), seminal vesicles (SV), gland penis (GP), levator ani and bulbocavernosus muscles (LABC), and Cowper’s glands (CG) were statistically significantly increased 1.9-, 2.7-, 1.5-, 2.2-, and 2.1-fold compared to control values. Administration of the positive control testosterone propionate (TP, 0.4 mg/kg b.w.) by subcutaneous injection strongly and highly significantly increased the relative weight of VP, SV, GP, LABC, and CP 5.6-, 11.1-, 1.6-, 2.7-, and 4.9-fold. VIN (0 - 3 - 10 - 30 - 100 mg/kg b.w.), orally administered to TP-supplemented rats (0.4 mg/kg b.w. by subcutaneous injection) for ten days dose-dependently decreased the relative weights of all androgen-sensitive tissues. Statistically significant changes were obtained at 30 mg/kg b.w. and above. At the high dose, weight reduction to 48% (VP), 44% (SV), 85% (GP), 58% (LABC), and 68% (CG) of control values was observed. Flutamide (3 mg/kg b.w.), the positive control, orally administered to TP-supplemented rats, reduced the corresponding relative tissue weights even more strongly to 33% (VP), 28% (SV), 79% (GP), 51% (LABC), and 50% (CG) of control tissue weights. Our data suggest that the Hershberger assay is suitable to screen for compounds with affinity for the androgen receptor. Further testing also including methyltestosterone, p, p’-DDE, linuron, procymidone and finasteride in a world wide effort will provide comprehensive information on the properties of the Hershberger assay. This study was performed in collaboration with OECD and was sponsored by CEFIC-EMSG. 670
A COMMON MODE OF TOXICOLOGICAL ACTIONS IN ENDOCRINE DISRUPTERS
B.M. Lee, S.M. Choi. Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Chunchun-Dong 300, Suwon, Kyonggi-Do, South Korea A common mode of toxicological actions was investigated for the 48 endocrine disrupting chemicals (EDCs) classified by Centers for Disease Control and Prevention (CDC). Based on a literature survey, 24 EDCs out of 48 were shown to be positive and 3 EDCs were negative, in terms of generating free radicals, and for twenty one, information was not available (NA) as to free radical generation. Therefore, generation of free radicals (measured by malondialdehyde, MDA) was examined for the remaining 21 EDCs, not reported in the literature and 3 negatives as to free radical generation. Mice were i.p. treated with each of the total 23 EDCs(except one, which is commercially unavailable) at various doses and MDA was measured in the liver. Twenty out of 23 EDCs significantly increased MDA production (p<0.05) and only three EDCs were shown to be non-peroxidative. Therefore, a total of 44 out of the 47 EDCs (94%) reported or tested was determined to be positive with respect to free radical generation. Our results indicate that 94% of the 48 EDCs generate free radicals and this feature may be useful for the investigation of a common mechanism of EDCs, method development for the screening of EDCs, and a chemoprevention strategy. (This work was supported by the brain Korea 21 project 2003 and a grant from NITR/Korea FDA.) 671
PROCYMIDONE: CHARACTERISATION OF ESTROGEN-LIKE ACTIVITY IN THE MCF-7 HUMAN BREAST CANCER CELL LINE
E. Chiesara, R. Fumagalli, M. Ferraris, S. Frigerio, L. Marabini, S. Radice. Department of Pharmacology, Chemotherapy and Medical Toxicology “E. Trabucchi”, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy Procymidone, a dicarboximide fungicide, is known to modify sexual differentiation in vivo and in vitro, and has been shown to induce vitellogenin synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the estrogen-like activity of procymidone in the MCF-7 (ER+) human breast cancer cell line, using 17β-estradiol 1nM as the positive control. The estrogenic effects were assessed using a number of specific endpoints, including the mitogenic time-dependent effect measured by means of a pro-
liferation assay (MTT), which can also discriminate cytotoxic and non-cytotoxic concentrations; a quantitative assay (ELISA) to measure the estrogen-regulated secretorial protein pS2; and microscopy to evaluate the development of foci which are preneoplastic cell aggregates superimposed on a monolayer background that appear in post-confluent cultures and are also characteristic of malignant transformation. Our data show that procymidone 100 µM stimulates constant cell proliferation up to the ninth day, which peaks between day 10 and day 13, and subsequently returns to baseline levels. The results clearly confirm the potent foci promoting action of procymidone 100 µM, which also induces the pS2 content of MCF-7. We conclude that procymidone has estrogen-like activity in this human cell model by means of a still unknown mechanism of action through ligand-dependent or ligand-independent activation of the ER pathway. 672
ESTABLISHMENT OF CALUX BIOASSAY SYSTEM FOR ESTROGENS AND DIOXINS
Y.Y. Sheen 1 , K.E. Joung 1 , K.N. Min 1 , J.Y. Kim 1 , K. Gu 2 , S. Paik 2 , S. Hong 3 , S. Kang 3 , H. Kim 3 , S. Cho 3 . 1 College of Pharmacy, Ewha Womans University, Seoul 120–750, Korea, 2 College of Medicine Hanyang University, Seoul, 133–792, Korea 3 College of Medicine Seoul National University, Seoul, 110–744, Korea In order to establish the rapid and easy-to-perform methods using ERE-MCF-7 cells by luciferase assay. MCF-7 stable cells which are stably transfected with phERE-Luc were treated with many chemicals and then luciferase activity were determined. Estradiol (E2) and synthetic estrogen, diethylstylbesterol (DES) were induced luciferase activity in dose dependent manner ranging 20–30 folds over that of control, and their activities were blocked by Tam treatment. 29 Flavonoids and 5 curcumin derivatives were tested in this system. Their E2 equivalent concentrations (EEQs) were calculated as a concentration of E2 that resulted in the same luciferase reading of test compound from the dose response curve. And also of Kumho river of Korea showed 0.77 pM EEQ in upstrean and 7.7pM EEQ in downstream. Kum River of Korea showed 3.5pM and 1.7pM EEQ in upstream and downstream respectively. Mankyung River of Korea showed 61fM and 0.41 pM EEQ in upstream and downstream respectively. Miho Stream of Korea showed 0.2pM EEQ only in the upstream. In this study, we attempted to identify the possible association between dioxin like compounds (such as TCDD, PCDDs, PCDFs, and PCBs) and the occurrence and severity of endometriosis using CALUX (Chemically Activated LUciferase eXpression) bioassay method. We analyzed the serum levels of dioxin like compounds in the endometriosis patients (n=46) and control patients with similar symptoms (n=14). Among them, adipose tissues of 10 cases were analyzed by high resolution GC/MS for validation of CALUX bioassay. The CALUX TEQs significantly correlated with the total TEQs determine by GC/MS (r2 = 0.96). So we demonstrated that CALUX bioassay is a rapid, sensitive and quantitative assay for biomonitoring of dioxin like compounds from small volume of blood. This study showed statistically significant association between exposure to dioxin like compounds and the occurrence of endometriosis (p < 0.003). The mean TEQ of control patient was 0.144 µg TEQ/L and the mean TEQ of endometriosis patient was 0.321 µg TEQ/L. After adjusting confounding factor, we found that the higher stage of the endometriosis, the higher level of CALUX TEQ. The TEQs of endometriosis I, II, III, and IV was 0.213 µg TEQ/L, 0.284 µg TEQ/L, 0.352 µg TEQ/L, and 0.450 µg TEQ/L, respectively. (Supported by grant from KFDA of Korea) 673
EXPRESSION PATTERN AND PROMOTER ANALYSIS OF MEDAKA CHORIOGENIN AS A BIOMARKER OF ENVIRONMENTAL ESTROGEN
Chulwoo Lee, Eung-Roh Park, Jin-Gyun Na, Deok-Gil Rhee, Moon-Soon Lee. Environmental Risk Research Dept., National Institute of Environmental Research (NIER), Incheon, KOREA In teleost fish, medaka (Oryzias latipes), the inner layer of egg envelope comprises two groups of subunits, designated ZI-1,2
Poster Session P34. Endocrine disrupters
dose. Relative weights of ventral prostate (VP), seminal vesicles (SV), gland penis (GP), levator ani and bulbocavernosus muscles (LABC), and Cowper’s glands (CG) were statistically significantly increased 1.9-, 2.7-, 1.5-, 2.2-, and 2.1-fold compared to control values. Administration of the positive control testosterone propionate (TP, 0.4 mg/kg b.w.) by subcutaneous injection strongly and highly significantly increased the relative weight of VP, SV, GP, LABC, and CP 5.6-, 11.1-, 1.6-, 2.7-, and 4.9-fold. VIN (0 - 3 - 10 - 30 - 100 mg/kg b.w.), orally administered to TP-supplemented rats (0.4 mg/kg b.w. by subcutaneous injection) for ten days dose-dependently decreased the relative weights of all androgen-sensitive tissues. Statistically significant changes were obtained at 30 mg/kg b.w. and above. At the high dose, weight reduction to 48% (VP), 44% (SV), 85% (GP), 58% (LABC), and 68% (CG) of control values was observed. Flutamide (3 mg/kg b.w.), the positive control, orally administered to TP-supplemented rats, reduced the corresponding relative tissue weights even more strongly to 33% (VP), 28% (SV), 79% (GP), 51% (LABC), and 50% (CG) of control tissue weights. Our data suggest that the Hershberger assay is suitable to screen for compounds with affinity for the androgen receptor. Further testing also including methyltestosterone, p, p’-DDE, linuron, procymidone and finasteride in a world wide effort will provide comprehensive information on the properties of the Hershberger assay. This study was performed in collaboration with OECD and was sponsored by CEFIC-EMSG. 670
A COMMON MODE OF TOXICOLOGICAL ACTIONS IN ENDOCRINE DISRUPTERS
B.M. Lee, S.M. Choi. Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Chunchun-Dong 300, Suwon, Kyonggi-Do, South Korea A common mode of toxicological actions was investigated for the 48 endocrine disrupting chemicals (EDCs) classified by Centers for Disease Control and Prevention (CDC). Based on a literature survey, 24 EDCs out of 48 were shown to be positive and 3 EDCs were negative, in terms of generating free radicals, and for twenty one, information was not available (NA) as to free radical generation. Therefore, generation of free radicals (measured by malondialdehyde, MDA) was examined for the remaining 21 EDCs, not reported in the literature and 3 negatives as to free radical generation. Mice were i.p. treated with each of the total 23 EDCs(except one, which is commercially unavailable) at various doses and MDA was measured in the liver. Twenty out of 23 EDCs significantly increased MDA production (p<0.05) and only three EDCs were shown to be non-peroxidative. Therefore, a total of 44 out of the 47 EDCs (94%) reported or tested was determined to be positive with respect to free radical generation. Our results indicate that 94% of the 48 EDCs generate free radicals and this feature may be useful for the investigation of a common mechanism of EDCs, method development for the screening of EDCs, and a chemoprevention strategy. (This work was supported by the brain Korea 21 project 2003 and a grant from NITR/Korea FDA.) 671
PROCYMIDONE: CHARACTERISATION OF ESTROGEN-LIKE ACTIVITY IN THE MCF-7 HUMAN BREAST CANCER CELL LINE
E. Chiesara, R. Fumagalli, M. Ferraris, S. Frigerio, L. Marabini, S. Radice. Department of Pharmacology, Chemotherapy and Medical Toxicology “E. Trabucchi”, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy Procymidone, a dicarboximide fungicide, is known to modify sexual differentiation in vivo and in vitro, and has been shown to induce vitellogenin synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the estrogen-like activity of procymidone in the MCF-7 (ER+) human breast cancer cell line, using 17β-estradiol 1nM as the positive control. The estrogenic effects were assessed using a number of specific endpoints, including the mitogenic time-dependent effect measured by means of a pro-
liferation assay (MTT), which can also discriminate cytotoxic and non-cytotoxic concentrations; a quantitative assay (ELISA) to measure the estrogen-regulated secretorial protein pS2; and microscopy to evaluate the development of foci which are preneoplastic cell aggregates superimposed on a monolayer background that appear in post-confluent cultures and are also characteristic of malignant transformation. Our data show that procymidone 100 µM stimulates constant cell proliferation up to the ninth day, which peaks between day 10 and day 13, and subsequently returns to baseline levels. The results clearly confirm the potent foci promoting action of procymidone 100 µM, which also induces the pS2 content of MCF-7. We conclude that procymidone has estrogen-like activity in this human cell model by means of a still unknown mechanism of action through ligand-dependent or ligand-independent activation of the ER pathway. 672
ESTABLISHMENT OF CALUX BIOASSAY SYSTEM FOR ESTROGENS AND DIOXINS
Y.Y. Sheen 1 , K.E. Joung 1 , K.N. Min 1 , J.Y. Kim 1 , K. Gu 2 , S. Paik 2 , S. Hong 3 , S. Kang 3 , H. Kim 3 , S. Cho 3 . 1 College of Pharmacy, Ewha Womans University, Seoul 120–750, Korea, 2 College of Medicine Hanyang University, Seoul, 133–792, Korea 3 College of Medicine Seoul National University, Seoul, 110–744, Korea In order to establish the rapid and easy-to-perform methods using ERE-MCF-7 cells by luciferase assay. MCF-7 stable cells which are stably transfected with phERE-Luc were treated with many chemicals and then luciferase activity were determined. Estradiol (E2) and synthetic estrogen, diethylstylbesterol (DES) were induced luciferase activity in dose dependent manner ranging 20–30 folds over that of control, and their activities were blocked by Tam treatment. 29 Flavonoids and 5 curcumin derivatives were tested in this system. Their E2 equivalent concentrations (EEQs) were calculated as a concentration of E2 that resulted in the same luciferase reading of test compound from the dose response curve. And also of Kumho river of Korea showed 0.77 pM EEQ in upstrean and 7.7pM EEQ in downstream. Kum River of Korea showed 3.5pM and 1.7pM EEQ in upstream and downstream respectively. Mankyung River of Korea showed 61fM and 0.41 pM EEQ in upstream and downstream respectively. Miho Stream of Korea showed 0.2pM EEQ only in the upstream. In this study, we attempted to identify the possible association between dioxin like compounds (such as TCDD, PCDDs, PCDFs, and PCBs) and the occurrence and severity of endometriosis using CALUX (Chemically Activated LUciferase eXpression) bioassay method. We analyzed the serum levels of dioxin like compounds in the endometriosis patients (n=46) and control patients with similar symptoms (n=14). Among them, adipose tissues of 10 cases were analyzed by high resolution GC/MS for validation of CALUX bioassay. The CALUX TEQs significantly correlated with the total TEQs determine by GC/MS (r2 = 0.96). So we demonstrated that CALUX bioassay is a rapid, sensitive and quantitative assay for biomonitoring of dioxin like compounds from small volume of blood. This study showed statistically significant association between exposure to dioxin like compounds and the occurrence of endometriosis (p < 0.003). The mean TEQ of control patient was 0.144 µg TEQ/L and the mean TEQ of endometriosis patient was 0.321 µg TEQ/L. After adjusting confounding factor, we found that the higher stage of the endometriosis, the higher level of CALUX TEQ. The TEQs of endometriosis I, II, III, and IV was 0.213 µg TEQ/L, 0.284 µg TEQ/L, 0.352 µg TEQ/L, and 0.450 µg TEQ/L, respectively. (Supported by grant from KFDA of Korea) 673
EXPRESSION PATTERN AND PROMOTER ANALYSIS OF MEDAKA CHORIOGENIN AS A BIOMARKER OF ENVIRONMENTAL ESTROGEN
Chulwoo Lee, Eung-Roh Park, Jin-Gyun Na, Deok-Gil Rhee, Moon-Soon Lee. Environmental Risk Research Dept., National Institute of Environmental Research (NIER), Incheon, KOREA In teleost fish, medaka (Oryzias latipes), the inner layer of egg envelope comprises two groups of subunits, designated ZI-1,2