Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: No apparent synergism
Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: No apparent synergism
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DOI：
10.1210/en.138.4.1520
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发表时间：
1997-04-01
期刊：
影响因子：
4.8
通讯作者：
Safe, S
中科院分区：
文献类型：
--
作者：
Ramamoorthy, K;Wang, F;Safe, S
在21日龄B6C3F1小鼠子宫、MCF-7人乳腺癌细胞和基于酵母的报告基因检测中，研究了狄氏剂、毒虫芬以及这两种化合物的等摩尔混合物（狄氏剂/毒虫芬）的雌激素活性。17 β -雌二醇（E(2)） （0.0053 kg/day x3）处理小鼠子宫湿重、过氧化物酶活性和孕酮受体结合分别增加3.1倍、4.8倍和7.8倍。相比之下，2.5、15和60 μ mol/Kg （x3）剂量的毒杀芬、狄氏剂或狄氏/毒杀芬（等摩尔）处理没有显著诱导E(2)诱导反应的剂量依赖性增加。在以[H-3]E(2)为放射配体的竞争性结合实验中，有机氯农药单独和二元混合物均未与小鼠子宫雌激素受体（En）结合。在平行研究中，通过细胞增殖试验和测定氯霉素乙酰转移酶（CAT）在MCF-7细胞中的诱导活性，MCF-7细胞瞬时转染了含有雌激素应答的5'-促进区（来自大鼠肌酸激酶B和人组织蛋白酶D基因）的质粒，从而确定了雌激素a的活性。E，使瞬时转染肌酸激酶B的MCF-7细胞中CAT活性增加24倍，瞬时转染人组织蛋白酶D构建体的细胞中CAT活性增加3.8倍。用狄氏剂、毒杀芬或狄氏剂加毒杀芬10(-8)-10(-5)M的等摩尔混合物处理MCF-7细胞，在两种质粒的瞬时转染实验中都没有显著诱导细胞增殖或CAT活性。通过将MCF-7细胞与10(-9)M [3H]E孵育，在存在或不存在2 × 10（-7) M未标记的E(2)以确定非特异性结合），毒杀芬（10(-5)M），狄氏剂（10(-5)M）和等摩尔浓度的狄氏剂加毒杀芬混合物（10(-5)M），来确定有机氯农药的相对竞争性结合。在全细胞提取物中观察到的[H-3]E(2)的结合被未标记的E所取代，而有机氯农药酸二元混合物表现出最小或无法检测到的竞争结合活性。E(2)在用人内质网和p-gal报告基因上游的双雌激素反应元件转化的酵母中诱导了5000倍的p-半乳糖苷酶（p-gal）活性。用10(-6)-10(-4)M氯丹、狄氏剂、毒杀芬或等摩尔的狄氏剂/毒杀芬的混合物处理没有诱导活性，而10(-4)M硫丹则使β -gal活性增加2000倍。己烯雌酚在小鼠内质网和β -gal报告基因上游的单一雌激素反应元件转化的酵母中引起20倍的活性增加。狄氏剂、氯丹、毒杀芬和硫丹在2.5 × 10(-5) M浓度下可诱导活性增加1.5至4倍。在2.5 × 10(-5) M或2.5 × 10(-4) M浓度下，这些农药的任何等摩尔二元混合物均未观察到协同反应。本研究结果表明，在小鼠子宫、MCF-7人乳腺癌细胞和基于酵母的报告基因试验中，狄氏剂和毒杀芬的活性都很低，并且与这两种化合物的二元混合物没有观察到协同作用。
The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17 beta-estradiol (E(2)) (0.0053 kg/day x3) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 mu mol/Kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose dependent increase in ally of the E(2)-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (En) in a competitive binding assay using [H-3]E(2) as the radioligand. In parallel studies, estrogenic a activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 5'-promotel regions from the rat creatine kinase B and human cathepsin D genes. E, caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene 10(-8)-10(-5) M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 10(-9) M [3H]E, in the presence or absence of 2 x 10(-7) M unlabeled E(2) ito determine nonspecific binding), toxaphene (10(-5) M), dieldrin (10(-5) M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10(-5) M). The binding observed for [H-3]E(2) in the whole cell extracts was displaced by unlabeled E,, whereas the organochlorine pesticides acid binary mixture exhibited minimal to nondetectable competitive binding activity. E(2) caused a 5000-fold induction of P-galactosidase (p-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the P-gal reporter gene. Treatment with 10(-6)-10(-4) M chlordane, dicldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10(-4) M endosulfan caused a 2000-fold increase in beta-gal activity. Diethylstilbcstrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the beta-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in actitity at a concentration of 2.5x10(-5) M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10(-5) M or 2.5 x 10(-4) M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds.