4-壬基酚、双酚A对雌性SD幼鼠生殖器官拟雌激素样作用的联合效应

[目的]检测4-壬基酚（4-NP）、双酚A（BPA）对幼雌sD大鼠拟雌激素样作用的联合效应。[方法]选取4-NP、BPA为染毒物质;以21d雌性SD大鼠为实验对象,共分9组,每组8只。分组为：4-NP高、中、低剂量组（200、100、50mg／kg）,BPA高、中、低剂量组（600、400、200mg／kg）,混合组（50mg／kg4-NP＋200mg／kgBPA）,阳性对照组,溶剂对照组。连续喂饲3d,末次给药后24h处死。以子宫湿重、脏器系数、子宫内膜及平滑肌增生程度和细胞增殖核抗原（PCNA）的蛋白表达作为检测指标,采用单因素方差分析方法进行统计。[结果]高、中剂量的4-NP和BPA能够使染毒动物的子宫湿重及脏器系数明显增加（P〈0.01）;4-NP和BPA高、中、低剂量都能够使子宫内膜上皮、平滑肌增厚,PCNA蛋白表达增强（P〈0.05）,并且具有一定的剂量-效应关系。最大无效应剂量的4-NP（50mg／kg）和BPA（200mg／kg）混合喂饲后能够使染毒动物的子宫湿重及脏器系数明显增加;能够使子宫内膜上皮、平滑肌增厚,PCNA蛋白表达增强（P〈0.05）。[结论]4-NP、BPA具有拟雌激素样作用,干扰内分泌系统;两种物质混合后可以发挥联合效应。     

染镉未成年雌性大鼠的雌激素样作用研究

目的评价镉的雌激素样活性。方法SD未成年雌性大鼠随机分为6组(阴性对照组、0.5、1.0、2.0、4.0mg/kgCd2 组及β-雌二醇阳性对照组,皮下注射染毒,每天1次,共3d,第4天时剖杀观察子宫湿重,子宫内膜腔上皮厚度,子宫内膜增殖细胞核抗原(PCNA)表达。结果各剂量染镉组大鼠子宫增重、子宫内膜腔上皮厚度及PCNA阳性表达各项指标与阴性对照组比较差异未见统计学意义(P>0.05)。结论在该实验条件下,并未显示镉具有雌激素样活性。     

莠去津对雌性幼年大鼠生殖系统发育的毒性研究

为研究莠去津(ATR)对雌性幼年大鼠生殖系统发育的毒性作用,将48只健康初断乳(出生17 d)SPF级SD雌性大鼠随机分为4组,分别为对照组和低(50 mg/kg)、中(100 mg/kg)、高(200 mg/kg)剂量莠去津染毒组,每组12只.采用经口灌胃方式进行染毒,染毒容量为20 ml/kg,每天一次,连续染毒22 d.观察并记录大鼠阴道开口数和时间,计算阴道开口率;并对子宫进行病理学观察和PCNA免疫组织化学染色.结果显示,与对照组比较,高剂量莠去津染毒组大鼠的阴道开口率降低,阴道开口时间延迟;子宫横切面及子宫腔较小,子宫壁较薄,且定位于子宫内膜上皮细胞核的PCNA阳性数明显减少.提示莠去津对幼年雌性SD大鼠的生殖系统发育具有抑制作用.     

子宫营养试验检测对-壬基酚的雌激素样活性

目的 :检测对 -壬基酚 (p- NP)的雌激素样活性。方法 :采用子宫营养试验 ,以使此法作为我国筛查环境内分泌干扰物 (EEDs)的常规方法。未成年 SD雌性大鼠 (2 1d～ 2 2 d) 10 0只 ,分为 p- NP 6 0 m g/ kg、 12 0 m g/ kg和 2 4 0 m g/ kg3个剂量组 ,溶剂对照组 (花生油 )和阳性对照组 (苯甲酸雌二醇 ,E2 B) 0 .4 m g/ kg,每天一次灌胃给药 ,连续给 3d和 7d。于末次给药的第 2 4 h处死动物 ,取子宫。结果 :3d和 7d两个时段 ,E2 B、 p- NP 12 0 mg/ kg和 2 4 0 mg/ kg 2个剂量组子宫湿重、子宫 /体重比、平滑肌厚度和宫腔上皮高度与溶剂对照组相比均增高 (P<0 .0 1) ,且有明显的剂量效应关系。给 p- NP7d的未成年 SD雌性大鼠阴道开口时间提前。结论 :p- NP具有弱雌激素样活性 ,3d幼雌 SD大鼠子宫营养试验在我国作为EEDs常规筛检方法是可行的     

双酚A和对-壬基酚雌激素受体的竞争性结合

目的　用雌激素受体竞争性结合试验检测双酚A(BPA)、对 -壬基酚 (p -NP)的雌激素样活性。 方法　将SD大鼠进行人工卵巢去除术 ,饥饿受体 14d后提取子宫胞浆雌激素受体 (ER) ,受体蛋白定量 ,作雌二醇 (E2 )非标记配体饱和分析 ,以确定E2 的IC50 。随后进行BPA和 p -NP的竞争性取代实验 ,观察BPA ,p -NP是否能与3 H -E2竞争性结合大鼠子宫胞浆ER。结果　E2 的IC50 是 1× 10 -9mol/L ,BPA、p -NP的IC50 分别为 2 0 8× 10 -5mol/L和2 6 6R10 -5mol/L ,BPA、p -NP的相对结合亲和力系数 (RBA)分别为 0 0 0 4 8和 0 0 0 38,BPA与ER的结合能力稍高于 p -NP。 结论　BPA和 p -NP结合ER是发挥其雌激素样效应机制中的重要因素     

染镉对雌性大鼠卵巢、子宫雌激素受体及热休克蛋白90α表达的影响

目的研究镉对卵巢子宫内膜雌激素受体(ER)及HSP 90α表达的影响,探讨其内分泌干扰作用机制。方法40只成年雌性Wistar大鼠按体重分层随机分为4组,每组10只,镉(Cd2+)染毒剂量分别为0、1.25、2.5、5.0 mg Cd2+/(kg.d),皮下注射,每周5 d连续4周。于动情间期将大鼠处死;用免疫组化染色法观察卵巢间质和子宫内膜ER表达以及子宫内膜HSP 90α表达。结果高剂量组卵巢间质ER表达显著降低于其他各剂量组(P<0.05);各染毒组子宫内膜ER表达与对照组差异无统计学意义(P>0.05),其中高、中剂量组显著低于低剂量组(P<0.05);各染毒组子宫内膜HSP 90α表达与对照组差异无统计学意义(P>0.05)。结论本实验条件下,高剂量组染镉可使卵巢间质ER表达显著下降,而各染毒组子宫内膜ER表达与对照组比较差异无统计学意义,其在镉的内分泌干扰作用机制中镉对ER表达影响的意义值得进一步研究;镉不会通过增加子宫内膜HSP 90α的表达来干扰内分泌系统。     

子宫增重法检测壬基酚、双酚A雌激素样活性及其联合作用

目的应用子宫增重法检测壬基酚(NP)、双酚A(BPA)的雌激素样活性并评价其联合作用。方法选择性未成熟雌性昆明种小鼠110只,体重10~13g,按体重随机分为11组:NP240mg/kg、120mg/kg、60mg/kg剂量组,BPA480mg/kg、240mg/kg、120mg/kg剂量组,联合240mg/kgBPA 120mg/kgNP、120mg/kgBPA 60mg/kgNP、60mg/kgBPA 30mg/kgNP剂量组,阴性对照组、阳性对照组(苯甲酸雌二醇1·47μg/10g),皮下注射染毒3d,称子宫湿重,计算子宫重量系数。用金正均法评价联合作用的性质。结果NP和BPA可致子宫重量系数增大,呈剂量-效应关系(分别为r=0·8192,P<0·01;r=0·6898,P<0·01);两者联合染毒致子宫重量系数增大,亦呈剂量-效应关系(r=0·5465,P<0·01)。各染毒组与阴性对照组比较,差异有统计学意义(P<0·05或P<0·01),其联合作用性质在混合低剂量组呈协同作用(Q=1·41),混合中、高剂量组呈相加作用(Q=0·88,Q=0·87)。结论NP、BPA具有雌激素样活性,其联合作用性质在较低剂量呈协同作用,较高剂量呈相加作用。     

镉对去卵巢大鼠子宫增重及血清性激素水平的影响

目的 研究镉对去卵巢大鼠子宫增重效应及血清性激素水平的影响.方法 28天龄健康雌性SD大鼠40只,切除双侧卵巢后按体重分层,分为4组,即阴性对照组(超纯水)、低镉组(0.12 mg/kg)、高镉组(1.2 mg/kg)、阳性对照组(0.03 mg/kg的1713-雌二醇).各组大鼠均按1 ml/kg腹腔注射染毒,每天1次,连续3 d.染毒结束后称子宫湿重,计算子宫脏器系数;采用放免法测定血清雌二醇(E2)、孕酮(P4)、卵泡刺激素(FSU)、黄体生成素(LH)水平.结果 高镉组、阳性对照组子宫湿重与子宫脏器系数均高于阴性对照组,差异有统计学意义(P＜0.05,P＜0.01).与阴性对照组比较,高镉组大鼠血清E2与P4水平降低,差异有统计学意义(P＜0.05);阳性对照组血清E2与P4水平升高,差异有统计学意义(P＜0.01);低镉组大鼠血清FSH与LH水平降低,差异有统计学意义(P＜0.05,P＜0.01);阳性对照组血清LH水平升高,差异有统计学意义(P＜0.01).结论 在本实验条件下,镉具有子宫增重效应,并能通过非性腺组织发挥内分泌干扰作用.     

农药联苯菊酯对大鼠内分泌干扰作用的研究

目的通过大鼠子宫增重试验和Hershberger试验研究农药联苯菊酯(BIF)的内分泌干扰作用。方法子宫增重试验中,60只雌性SD大鼠随机分为6组,每组10只:BIF低、中、高剂量组分别经口灌胃给予1.47、4.41和13.23mg/kgBW的BIF;溶剂对照组经口灌胃给予玉米油;17α-乙炔雌二醇(EE)经口阳性组经口灌胃给予1.0μg/kg BW的EE,EE经皮阳性组经口灌胃给予玉米油并皮下注射0.6μg/kg BW的EE。连续给药3天后,测定大鼠子宫湿重、干重,计算其子宫系数,并观察病理变化。在Hershberger试验中,60只去势雄性SD大鼠随机分为6组,每组10只:BIF低、中、高剂量组分别经口灌胃给予1.4、4.2和12.6mg/kg BW的BIF,阳性对照组经口灌胃给予3.0mg/kg BW的氟他胺,溶剂对照组和丙酸睾酮对照组(TP组)经口灌胃给予玉米油,除溶剂对照组外,其余各组均皮下注射给予0.2mg/kgBW丙酸睾酮。所有剂量组连续给药10天后,称量腹侧前列腺(VP)、精囊腺和凝固腺(SVCG)、肛提肌加球海绵体肌(LABC)、阴茎头(GP)、尿道球腺(COW)、肝、肾、肾上腺的重量,计算其脏器系数,测定血清中三碘甲腺原氨酸(T3)和甲状腺素(T4)水平。结果子宫增重试验中,与溶剂对照组相比,BIF高剂量组子宫湿重系数和干重系数显著增加(P<0.05)。组织病理学检查发现,BIF中、高剂量组子宫内膜增生、增厚,子宫内膜上皮细胞高度增高(P<0.05)。Hershberger试验,与TP组相比,BIF高剂量组VP、LABC重量显著减轻,T3、T4水平显著降低(P<0.05)。结论子宫增重试验和Hershberger试验提示BIF具有潜在的内分泌干扰作用。     

3,4-双(4’-氨基呋咱基-3’)氧化呋咱对大鼠亚急性经口毒性实验研究

为评价3,4-双(4’-氨基呋咱基-3’)氧化呋咱(DATF)的亚急性经口毒性,将40只健康SPF级SD大鼠按体重随机分为4组,分别为对照(4%淀粉溶液)组和77.5、155、310 mg/kg DATF染毒组,每组10只,雌雄各半。采用灌胃方式染毒28 d。结果显示,与对照组相比,155、310 mg/kg DATF染毒组雄性大鼠与310 mg/kg DATF染毒组雌性大鼠总食物利用率降低;各剂量DATF染毒组大鼠血清胆固醇含量均较高,乳酸脱氢酶活力均较低;155、310 mg/kg DATF染毒组雄性大鼠肝、肾上腺的脏器系数以及各剂量DATF染毒组雌性大鼠肝的脏器系数和155、310 mg/kg DATF染毒组雌性大鼠脾的脏器系数及310 mg/kg DATF染毒组雌性大鼠肾的脏器系数均较高。提示DATF对大鼠肝脏、脾脏和肾脏具有一定的损伤作用。     

Immature rat uterotrophic assay of bisphenol A

We used the immature rat uterotrophic assay to determine the estrogenicity of bisphenol A (BPA). We administered BPA (in sesame oil) to rats subcutaneously (sc; 0, 8, 40, and 160 mg/kg/day) or orally (0, 40, 160, and 800 mg/kg/day) for 3 days beginning on postnatal day (PND) 18; rats were sacrificed 24 hr after the last administration. Uterine wet, blotted, and relative weights increased in all groups given BPA sc. After oral administration, uterine relative weight increased in 160 and 800 mg/kg BPA groups, and wet and blotted weights increased in the 800 mg/kg BPA group. Plasma concentrations of BPA at 1 hr after the last administration were detected in all groups given BPA sc and in groups given 160 and 800 mg/kg BPA orally, with a dose-response effect. The study was then reproduced under the same conditions. After sc injections, uterine wet and blotted weights increased in the 40 and 160 mg/kg BPA groups, and relative weight increased in all groups given BPA sc. By contrast, uterine wet, blotted, and relative weights increased only in the 160 and 800 mg/kg oral BPA groups. Also, to examine time-course changes in uterine weight, we administered BPA (in sesame oil) sc from PND 18 to PND 20 for 3 days at doses of 0, 8, 40, and 160 mg/kg/day; uterine weights were then measured at 6, 12, 18, and 24 hr after the last administration. Uterine wet, blotted, and relative weights increased in all BPA groups at 6 and 24 hr and in 40 and 160 mg/kg BPA groups at 12 hr. By contrast, at 18 hr, uterine wet, blotted, and relative blotted weights increased in all BPA groups and relative wet weight increased in 40 and 160 mg/kg BPA groups. The percentage increases in uterine wet and relative weights of 40 and 160 mg/kg BPA groups at 6 hr were higher than those at 24 hr relative to the controls, but the coefficient of variation in these weights in the group given 8 mg/kg BPA at 24 hr was smaller than that at 6 hr. These findings demonstrate BPA-induced uterotrophy in the immature uterotrophic assay in rats administered 8 mg/kg/day sc and in rats given 160 mg/kg/day orally, and suggest that the autopsy at 24 hr after the last administration is suitable.     

Antifertility effect of aqueous extract of seeds of Cassia fistula in female rats

Oral administration of aqueous extract of seeds of Cassia fistula to mated female rats from day 1-5 of pregnancy at the doses of 100 and 200 mg/kg body weight resulted in 57.14% and 71.43% prevention of pregnancy, respectively, whereas 100% pregnancy inhibition was noted at 500 mg/kg bw. In the uterine bioassay test carried out in immature bilaterally ovariectomized female rats, aqueous extract of seeds of Cassia fistula (100 mg/kg bw) increased the uterine wet weight (p<0.05) and luminal epithelial cell height (p<0.001) but did not induce premature opening of the vagina. This suggests a mild estrogenic activity of the extract. However, when the extract was administered conjointly with estradiol valerate (EDV, 0.1 mg/kg bw), it significantly (p<0.001) prevented the estrogen-induced uterotrophic effect, thus showing an antiestrogenic nature of the extract in the presence of a strong estrogen.     

Uterotrophic activity of bisphenol A in the immature rat.

Bisphenol A (BPA) is active in immature AP rat uterotrophic assays when evaluated using either the oral or the subcutaneous (sc) injection routes of exposure (three daily administrations of 400-800 mg/kg BPA). Premature vaginal opening was seen for 8 of 14 animals exposed to 600 and 800 mg/kg BPA by sc injection. Vaginal opening was not produced by BPA in the gavage studies. These results are consistent with those of Dodds and Lawson [Nature 137:96 (1936)] who found that BPA induces persistent vaginal cornification in ovariectomized rats exposed to three twice-daily injections of 85 mg/kg BPA (total daily dose 170 mg/kg), but they conflict with the reported inactivity of BPA in the immature mouse uterotrophic assay. The uterotrophic activity of diethylstilbestrol in the rat is also established (0.04 mg/kg/day for three days).     

The mouse uterotrophic assay: a reevaluation of its validity in assessing the estrogenicity of bisphenol A

The prevalence of synthetic chemicals in our environment that are capable of mimicking the female hormone estrogen is a growing concern. One such chemical, bisphenol A (BPA), has been shown to leach from a variety of resin-based and plastic products, including dental sealants and food and beverage containers, in concentrations that are sufficient to induce cell proliferation in vitro. The response to BPA in vivo has been varied; thus the aims of this study were to investigate a) whether BPA has an estrogenic effect in CD-1 mice, a strain that is useful for developmental studies; and b) whether the uterotrophic assay is a valid means of determining the estrogenicity of BPA by comparing it with other end points measured in the uterus. Immature female CD-1 mice were exposed to BPA in concentrations ranging from 0.1 to 100 mg/kg body weight for 3 days. Results showed that BPA induced a significant increase in the height of luminal epithelial cells within the uterus at concentrations of 5, 75, and 100 mg/kg and that BPA induced lactoferrin at concentrations of 75 and 100 mg/kg. A uterotrophic response (increase in uterine wet weight) was induced by 100 mg/kg BPA only. Further, the proportion of mice showing vaginal opening was greater after exposure to 0.1 and 100 mg/kg BPA, relative to the control animals and those receiving intermediate doses of BPA. These results demonstrate that BPA induces changes in the mouse uterus that differ depending on the exposure dose and the end point measured, and reveal that certain tissue effects show a nonmonotonic relationship with dose. These data also demonstrate that BPA induces estrogenic changes in the uterus of the CD-1 mouse, and highlight the need to reevaluate the validity of the mouse uterotrophic assay as an end point for determining the estrogenicity of suspected environmental estrogens.     

Genistein selectively inhibits estrogen-induced cell proliferation and other responses to hormone stimulation in the prepubertal rat uterus

Sex hormone replacement therapy helps improve quality of life in climacteric women. However, estrogen-induced cell proliferation in the uterus and mammary gland increases the risk for cancer in these organs. The lower incidence of mammary cancer in Asian women than in western women has been attributed to high intake of soy isoflavones, including genistein. Our previous work in the prepubertal rat uterus model showed that genistein (0.5 mg/kg body weight subcutaneously) caused an estradiol-like hypertrophy in myometrial and uterine luminal epithelial cells and an increase in RNA content in luminal epithelium; however, it did not induce cell proliferation, uterine eosinophilia, or endometrial edema. The present study investigated, in the same animal model, the effect of genistein administration (0.5 mg/kg body weight subcutaneously) before treatment with estradiol-17β (0.33 mg/kg body weight subcutaneously) on uterine responses that were not induced by genistein. Pretreatment with this phytoestrogen completely inhibited estradiol-induced mitoses in uterine luminal epithelium, endometrial stroma, and myometrium and partially inhibited estradiol-induced uterine eosinophilia and endometrial edema. These findings indicate that genistein protects against estrogen-induced cell proliferation in the uterus and suggest that future studies should investigate the possibility of using this agent to decrease the risk for uterine cancer after hormone replacement therapy in climacteric women.     

Flaxseed does not enhance the estrogenic effect of low-dose estrogen therapy on markers of uterine health in ovariectomized rats

Abstract Flaxseed (FS) is an oilseed rich in phytoestrogens and n-3 polyunsaturated fatty acids, compounds that may attenuate bone loss during aging. We previously demonstrated using the ovariectomized (OVX) rat model of postmenopausal osteoporosis that 10% dietary FS combined with low-dose estrogen therapy (LD) preserves vertebral bone mass and strength more so than either treatment alone. However, it was prudent to also consider the effect of this intervention on uterine tissue as LD, and possibly FS, may have estrogenic, and thus negative, effects on uterine tissue. The present study investigated if FS enhances the estrogenic effect of LD on markers of uterine health in OVX rats. Three-month-old rats were randomized to groups: (1) SHAM, (2) OVX, (3) OVX+FS, (4) OVX+LD, or (5) OVX+FS+LD. Ground FS was added to the AIN-93M diet (100?g/kg of diet), and LD was delivered by subcutaneous implant (0.42?μg of 17β-estradiol/kg of body weight/day) to mimic LD in postmenopausal women. After 12 weeks, histological analyses of uterine tissue demonstrated flattened or cuboidal luminal epithelia organized in a single layer in the OVX group, while FS, LD, and FS+LD induced a single layer of elongated luminal epithelia, columnar in shape. The SHAM group had the greatest epithelial mass. Cell proliferation was similar among all OVX groups. Therefore FS and FS+LD similarly induce estrogen-like effects on the morphology of luminal epithelia that are weaker than in the SHAM group without inducing cell proliferation in OVX rats. Thus, FS does not enhance the estrogenic effect of LD on markers of uterine health in OVX rats.     

Protective effect of Cordyceps militaris extract against bisphenol A induced reproductive damage

This study aimed to investigate the protective effects of Cordyceps militaris (C. militaris) against reproductive damage induced by bisphenol A (BPA). Rats were administrated 200 mg/kg BPA for 4 weeks and treated with C. militaris (200, 400, and 800 mg/kg body weight/day). By the end of the fourth week, the level of oxidative damage, sperm parameters, hormone levels, and histopathological changes were examined. In the group that only received BPA, there was a signi?cant decrease in body weight compared with the normal control (NC) group. C. militaris signi?cantly alleviated the BPA-induced reproductive damage by increasing testicular superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and glutathione (GSH); as well as by reducing serum malondialdehyde (MDA). C. militaris not only obviously enhanced the levels of serum LH and T, but it also improved the sperm count and motility compared to the BPA-treated group. These results suggest that C. militaris could be used as a potential natural substance for preventing BPA induced reproductive damage.

Abbreviations BPA: bisphenol A; SOD: superoxide dismutase; GSH: glutathione; GSH-PX: glutathione peroxidase; MDA: malondialdehyde; ROS: reactive oxygen species; T: testosterone; LH: luteinizing hormone; FSH: follicle-stimulating hormone; UPLC: ultra performance liquid chromatography; RIA: radioimmunoassay; qRT-PCR: quantitative real time PCR; NC: normal control group; BPA: 200 mg/kg BPA administered group; H: 800 mg/kg C. militaris extract administered group; LB, MB, and HB: 200 mg/kg BPA + 200 mg/kg, 400 mg/kg, and 800 mg/kg C. militaris administered group, respectively; VeB: 200 mg/kg BPA + 300 mg/kg Vitamin E administered group; Star: steroidogenic acute regulatory protein; 3β-HSD: 3beta-hydroxyl-delta-5-steroid dehydrogenase; CYP11A1: cytochrome P 450 family 11 subfamily A member 1; CYP17A1: cytochrome P 450 family 17 subfamily A member 1     

The immature mouse is a suitable model for detection of estrogenicity in the uterotropic bioassay

The traditional rodent uterotropic response assay has been incorporated into the U.S. Environmental Protection Agency's screening and testing program for environmental endocrine-disrupting chemicals (EDCs). While much effort continues to focus on determining protocol variables, few studies compare uterotropic responses in rats, a species commonly used in toxicologic testing, with other rodent species. In this study, we compared uterine responses in immature outbred CD-1 mice and Sprague-Dawley rats. After three daily subcutaneous injections with 17beta-estradiol (0.1-500 microg/kg/day), immature mice and rats demonstrated a similar dose-response increase in absolute uterine wet weight and uterine weight:body weight ratio. Further, morphologic and biochemical parameters of estrogenicity, including uterine epithelial cell height and number, gland number, and induction of estrogen-responsive proteins lactoferrin and complement C3, mirror wet weight increases. We conclude that mice are as well suited as rats for the uterotropic bioassay. Because of the advantages of using mice, including lower costs, less space required, and smaller amounts of compound needed for tests, mice should be given appropriate consideration in testing paradigms for EDCs.