镉对大鼠子宫雌激素受体影响的体外研究

目的研究镉对子宫雌激素受体结合容量影响,并评价其环境内分泌干扰作用。方法制备大鼠子宫细胞质雌激素受体;运用正交实验设计L16(45)]进行单剂量配基结合试验研究两因素——镉的浓度和预先孵育时间对雌二醇(E2)结合雌激素受体(ER)的最大结合容量(Bmax)影响;用竞争结合抑制试验,计算镉的IC50值,并以己烯雌酚为参照品计算相对亲和力(RBA),研究镉与ER的结合能力。结果体外不同浓度的镉(0、10-3、10-5、10-7mol/L)与孵育不同时间(0、30、60、90min)对E2与ER的最大结合位点数(Bmax,单位为pmol/mg蛋白)的改变均无统计学意义(P>0.05),同时也未见体外不同浓度的镉与孵育不同时间有交互作用,差异无统计学意义(P>0.05);其中0、10-3、10-5、10-7mol/L浓度的镉孵育0min的Bmax分别为203.15±75.16、203.41±22.78、220.82±45.35、209.10±49.66;孵育30min的Bmax分别为215.67±92.97、139.79±53.78、205.27±23.60、172.63±55.09;孵育60min的Bmax分别为197.11±50.68、203.24±66.33、183.92±31.89、183.33±32.70;孵育90min的Bmax分别为229.69±76.88、175.70±70.28、164.26±24.46、150.78±65.97。氯化镉的半数抑制浓度(IC50)为10-4～10-3mol/L之间,Cd与ER结合的RBA约为DES的10-6～10-7。结论镉对E2与子宫雌

Effect of cadmium on estrogen receptor from rat uterus in vitro

OBJECTIVE: To investigate the effect of cadmium (Cd) on estrogen receptor and to assess its endocrine disrupting action. METHODS: The estrogen receptor rich supernatant was prepared from the ovariectomized Sprague-Dawley rats. The effects of cadmium on estrogen binding were performed using a sing-dose ligand-binding assay. Extract from uterus were treated with various concentrations of cadmium (0, 10(-3), 10(-5) or 10(-7) mol/L) for various pre-incubation time (0, 30, 60, 90 min) by means of orthogonal experimental design with orthogonal layout of L16(4(5)) (the experiment was repeated for 5 times). In addition to the radioinert competitor, each assay included a zero tube and a DES standard curve for quality control purpose. Data for cadmium and the DES standard curve were plotted as percent 3H]-E2 bound versus log (molar concentration), and the IC50 for cadmium was determined. The RBA for cadmium was calculated by dividing the IC50 of DES in terms of the IC50 of cadmium. RESULTS: Cadmium could not block the binding of estradiol to the receptor because hormone binding did not change with increasing cadmium concentration or increasing preincubation time. The results showed that the binding of 3H]-estradiol to uterine cytosols was not significant (P > 0.05). The Bmax (its unit is pmol/mg protein) of various concentrations of cadmium (0, 10(-3), 10(-5) or 10(-7) mol/L) for pre-incubating 0 min is 203.15 +/- 75.16, 203.41 +/- 22.78, 220.82 +/- 45.35, 209.10 +/- 49.66 respectively; The Bmax of them for pre-incubating 30 min is 215.67 +/- 92.97, 139.79 +/- 53.78, 205.27 +/- 23.60, 172.63 +/- 55.09 respectively. The Bmax of them for pre-incubating 60 min is 197.11 +/- 50.68, 203.24 +/- 66.33, 183.92 +/- 31.89, 183.33 +/- 32.70, respectively. The Bmax of them for pre-incubating 90 min is 229.69 +/- 76.88, 175.70 +/- 70.28, 164.26 +/- 24.46, 150.78 +/- 65.97 respectively. Mean IC50 for cadmium is 10(-4) - 10(-3) M. If the affinities of DES binding to estrogen receptors was taken to be 100%, the relative binding affinities of cadmium was 10(-6) - 10(-7). The results indicated that cadmium had only a very poor affinity with estrogen receptor. CONCLUSION: In vitro assay cadmium did not have distinct disrupting effect on binding of estradiol to estrogen receptors from rat uterine.