食品中内分泌干扰物筛选方法体系与大豆异黄酮安全性评价研究
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摘要
第一部分:食品中内分泌干扰物筛选方法体系研究
目的:建立食品中内分泌干扰物筛选方法体系,探索筛选模型的敏感终点。作为筛选食品新资源、食品添加剂、食品污染物等可能具有内分泌干扰作用物质的方法,为进行食品内分泌干扰物的风险评估提供科学资料和依据。
研究内容:本研究的筛选方法体系包括体外和体内筛选方法部分。体外筛选方法包括:雌激素受体竞争结合试验,从分子水平筛选雌激样活性物质;E-SCREEN试验从细胞水平筛选雌激素样活性物质;卵黄蛋白原(VTG)间接的检测雌素样活性物质;类固醇合成试验从器官水平检测雄激素和或抗雄激素活性物质。体内筛选方法包括:啮齿动物子宫试验用于雌激素和或抗雌激素活性物质的筛选;HERSHBERGER试验用于雄激素和或抗雄激素活性物质的筛选。
方法:体外受体竞争结合试验用荧光偏振法,阳性物采用雌二醇(E_2),E_2浓度分别为2650、530、106、21.2、4.25、0.85、0.17、0.032和0.0064 nmol/l。E-SCREEN试验采用雌激素受体阳性的MCF-7细胞,2.5 mM E_2作为阳性物,用MTT法测定细胞增殖情况。卵黄蛋白原(VTG)测定用酶联免疫反应法,检测转入生长激素的转基因鲤鱼和亲本鲤鱼的VTG水平。类固醇合成试验用睾丸切片法,人类促绒毛膜性腺激素(hCG)作为促进睾酮合成的诱导物,每组均有hCG(+hCG)激发和无hCG(-hCG)两个亚组,阳性物用250mM氨鲁米特(Aminoglutethimide,AMI),阴性对照用10μM非那雄胺(Finasteride,FIN,有英文均统一)),分别与0、1、2、3小时收集培养液,最后测定睾酮(T)的水平。啮齿动物子宫试验用未成年的青春期前大鼠,阳性物用3.0μg/kg E2,和0.1、0.3、0.6和1.0g/kg·bw的白藜芦醇,连续三天灌胃给予受试物;实验结束称量子宫的湿重和干重,对卵巢、子宫和阴道进行组织病理学检查。HERSHBERGER(大小写统一)试验用4-5周未成年SD大鼠,阳性物用3 mg/kgFIN,连续灌胃10天;试验结束称量腹侧前列腺、精囊和凝结腺、肛提肌(LA)加球海绵体肌、阴茎头和尿道球腺重量,并测定促黄体激素(LH)和T。
结果:受体竞争结合试验荧光偏振的最大值和最小值的差值为111mP,E_2的IC50为22nM。在E-SCREEN实验中,2.5μM的雌二醇组OD值比对照组升高并达到统计学显著性水平。卵黄蛋白原测定结果显示,和亲本鲤鱼相比,转入生长激素基因的鲤鱼生长激素水平无明显变化。类固醇合成(睾丸切片法)试验:各组的睾酮水平随培养时间增加而增加,其中每组的hCG激发均比无hCG亚组的睾酮水平高,AMI+hCG组平均T浓度低于hCG激发对照组,FIN组和对照组相比无显著性差异。啮齿动物子宫试验:E2组子宫干重、湿重及其脏器系数显著升高;E2组的卵巢、子宫和阴道病理学均为显著增生改变;1.0g/kg bw白藜芦醇剂量组的子宫轻度增生,0.3、0.6和1.0g/kg bw白藜芦醇剂量组的阴道轻度增生无剂量-反应关系。Hershberger试验:TP+FLU组和TP组相比,前列腺、精囊腺、阴茎、肛提肌/球体海绵体肌、尿道球腺等重量明显降低;TP+FLU组和TP组相比,LH水平明显升高,LH/T比值明显升高。
结论:本研究成功建立了体外和体内筛选方法,有效利用这两类方法,分别从不同的水平筛选内分泌干扰物,即有助于提高筛选效率、结余筛选成本,更有助于提高筛选的灵敏性和特异性。体外筛选模型包括:受体结合试验、E-SCREEN、类固醇合成筛选试验;体内筛选模型包括:啮齿类动物子宫试验;HERSHBERGER试验。
第二部分：大豆异黄酮安全性评价研究
目的：对大豆异黄酮(SIF)的毒理学安全性进行研究和评价，并重点针对内分泌干扰毒性进行系统研究；同时探索大豆异黄酮的毒理学机制，为大豆异黄酮作为食品资源的合理应用提供科学依据。
方法:急性毒性试验选用Sprague Dawley(SD)大鼠，以SIF为受试物，采用最大耐受量(MTD)法，实验剂量为10 g/kg BW。
遗传毒性试验：Ames试验以染料木素(GEN)为受试物，设置0.008、0.04、0.2、1.0、5.0 mg/皿的5个剂量水平；小鼠(KM)精子畸形(包括精子线粒体获能试验)试验和小鼠骨髓细胞微核试验以SIF为受试物，试验设置0.2、0.5、1.5和4.5 g/kg BW四个剂量水平。
内分泌干扰毒性试验：E-SCREEN试验以GEN为受试物(剂量分别为1、0.5、0.25、0.125、0.0625、0.03125、0.016、0.008和0.004 mM)；啮齿动物子宫试验以SIF和GEN为受试物，SIF剂量分别为0.1、0.2、0.5和1.5 g/kg BW，GEN剂量分别为0.05、0.1、0.25和0.75 g/kg BW；HERSHBERGER试验选用SD大鼠，以SIF为受试物，剂量分别为0.2、0.5、1.5和4.5 g/kg BW。
亚慢性毒性试验：选用SD大鼠，连续13周灌胃给予SIF，剂量分别为0.2、0.5、1.5和4.5 g/kg BW剂量，每周观察动物的活动情况，并记录体重和进食量，试验结束后测定尿常规、血液学、血生化、凝血功能、内分泌指标(包括总睾酮、雌二醇、甲状腺素和促甲状腺素)、DNA损伤(彗星试验)，脏器组织病理观察和核增值因子以及雌激素受体的免疫组织化学分析。
结果和结论:大鼠急性毒性试验：SIF对雌、雄性大鼠的MTD大于10.0g/kgBW，因此大豆异黄酮属于实际无毒物。
三项遗传毒性试验：Ames试验：未见SIF有致基因突变作用；小鼠骨髓嗜多染红细胞微核试验：SIF对小鼠体细胞染色体无致断裂作用；小鼠精子畸形和精子活力试验：SIF对小鼠精子畸形发生率无影响，4.5 g/kg剂量组红色荧光精子所占比例明显降低。因此本研究未见SIF遗传毒性，但是4.5 g/kg BW SIF抑制精子线粒体获能。
内分泌干扰毒性研究：E-SCREEN试验：0.0625 mM剂量组OD值明显增高：子宫增重试验：与对照组比较，SIF和GEN剂量组子宫重量和脏器系数显著增加，GEN和SIF 4个剂量组的阴道均为增生改变：HERSHBERGER试验：与对照组比较，1.5和4.5 g/kg SIF剂量组的阴茎头/肾脏、尿道球脂/肾脏以及4．5g/kg剂量组肛提肌/肾脏均明显降低；和TP组相比，1.5 g/kg组的LH/T明显升高。因此SIF具有雌激素样活性，1.5 g/kg BW和4.5 g/kg BW SIT产生间接抗雄激素作用并降低大鼠的体重。
亚慢性毒性试验：1.5和4.5 g/kg剂量组体重增长明显滞后于对照组体重；进食量从第1周开始雄性1.5和4.5 g/kg剂量组的进食量明显低于对照组；血常规和尿常规均无明显变化：雌性1.5和4.5 g/kg的CHO和LDL-C和4.5 g/kg的HDL-C明显降低，0.2、0.5和1.5g/kg剂量组的HDL-C/LDL-C明显升高。雄性4个受试物剂量组的TO明显低于对照组，4个剂量组的HDL-C明显高于对照组。尿常规未见明显变化。雌性大鼠4.5 g/kg BW剂量组T3和T3/T4明显高于对照组，TSH/T4和TSH/T3各受试物具有下降的趋势。雄性大鼠0.5、1.5和4.5 g/kg BW剂量组总睾酮明显低于对照组；4.5 g/kg剂量组的宰杀体重、肝脏、肾脏、心脏和附睾重量以及1.5 g/kg剂量组的心脏重量均明显低于对照组，4.5 g/kg饲喂大鼠90天后雌性动物阴道壁增厚、上皮增生，雄性动物乳腺发育并泌乳：雌性大鼠各剂量组外周血淋巴细胞彗星试验的彗星惯量、彗星分布矩和尾惯量均比对照组降低：雌性4.5g/kg剂量组阴道上皮细胞PCNA的表达水平和ER-α表达水平高于对照组。因此SIF的毒性具有性别差异，体现为对雌性大鼠：影响血生化的脂类指标(降低胆固醇水平)，降低DNA的氧化损伤，高剂量SIF(4.5 g/kg BW)干扰甲状腺素水平，导致阴道增生，NOAEL为1.5 g/kg BW；对雄性大鼠：SIF影响血生化的脂类代谢指标降(甘油三脂水平)，降低体重，降低睾酮水平，促进乳腺增生并泌乳，NOAEL为0.2 g/kg BW。
Objectives: To establish endocrine disrupter screening system, to explore sensitive endocrine disrupter screening endpoint. The methods can be used to screen endocrine disruptors in novel sources of food, food additives, contaminations in food, further more to provide information and basal for risk assessment.
Content: to establish endocrine disrupter screening system, including in vitro and in vivo methods. In vitro method including: In vitro estrogen receptor(ER beta) competitor assays, to screen estrogenic activity amterials from molecular level; E-SCREEN to screen estrogenic activity amterials from cellular level; Vitellogenin(VTG) was used to screen estrogenic activity amterials indirectly; steroidogenesis screening assay was used to screen agonist and/or antagonist materials. In vivo methods including: rodent uterotrophic assay was used to screen estrogenic and/or anti-estrogenic activity amterials; HERSHBERGER assay was used to screen agonist and/or antagonist materials.
Methods: In vitro estrogen receptor(ER beta) competitor assays, fluorescence polarization value was measured, estradiol(E_2) used as positive control(the concentration were 2650, 530, 106, 21.2, 4.25, 0.85, 0.17, 0.032, 0.0064 nmol/l). In E-SCREEN assay, proliferation of MCF-7 cells was measured with MTT method, 2.5mM estradiol used as positive control. Vitellogenin(VTG) was measured with ELISA kit, in transgenic carp and wild carp. In steroidogenesis screening assay, sectioned testis assay was used, hCG as stimulation factor, there were hCG stimulating and no hCG stimulating subgroup in every group, 250 mM aminoglutethimide as positive substance and 10μM finasteride as negative substance, the endpoint of testosterone was measured in 0, 1, 2, 3 hour. In rodent uterotrophic assay using immature SD rats, 3.0μg/kg estradiol as positive control, 0.1, 0.3, 0.6 and 1.0g/kg bw Trans-resveratrol, administered orally to the animal for 3 consecutive day; in the end of the test, uterine wet and blotted weight, histopathology of ovary, uterus and vaginal were measured. HERSHBERGER assay was carried out in immature SD rats aged 4-5 weeks, 3.0 mg/kg flutamide(Flu) as positive control orally adminastrated, 0.4 mg/kg Testosterone Propionate(TP) subcutaneous adminastrated, which was given to every groups, all groups were administered for 10 consecutive day; in the end of test,animals were anesthetized , weight of ventral Prostate (VP), seminal vesicles together with coagulating gland (SV and CG),levator ani(LA), bulbocavernous muscles (BC), glans penis (GP) , bulbourethral glands (BG) were measured, testosterone(T) and luteinizing hormone(LH) levels were measured.
Results: In estrogen receptor(ER beta) competitor assays, the difference between max and min polarization was 111 mP, IC50 of E_2 was 22 nM. In E-SCREEN assay, compared with control, the OD value of E_2 group increased sigmficantly. There was no difference between transgenic(with growth hormone gene) carp and wild carp, in VTG level. In steroidogenesis screening assay, the concentration of testosterone increased with time in all group; concentration of testosterone was higher in hCG stimulation group than no stimulation group; concentration of testosterone was lower in AMI+ hCG group than hCG group. In rodent uterotrophic assay E_2 induce uterine weight increase significantly; histopathology of ovary, uterus and vagina showed proliferation in E2 group significantly; uterine histopathologies showed that 1.0 g/kg bw trans-resveratrol induce slight proliferation; 0.3, 0.6 and 1.0 g/kg bw trans-resveratrol induce slight vagina proliferation without dose-response relationship. In HERSHBERGER assay, the weights of ventral Prostate (VP), seminal vesicles together with coagulating gland (SV and CG),levator ani(LA), bulbocavernous muscles (BC), glans penis (GP) , bulbourethral glands (BG) decreased significantly in TP+FLU group than TP group; LH and ratio of LH/T increase in TP+FLU group than TP group.
Conclusion: In our study, in vitro and in vitro screening methods were established, it is benefit to improve efficiency of screening and also to decrease cost of screening, further more it can improve sensitivity and specialty, to use two methods together.
In vitro estrogen receptor(ER beta) competitor assays, E-SCREEN assay, Vitellogenin(VTG) measurement, steroidogenesis screening assay, rodent uterotrophic assay, HERSHBERGER were established successfully.
Study about Assessment of Soy Isoflavone Safety
Objectives:
To evaluate toxicological safly of soy isoflavones(SIF), and to study endoscrine disrupting toxicity; to study the toxicological mechanism of SIF, finally to provide scientific proof for reasonable usage of SIF as novel source in food.
Method:
In acute toxicity test with Sprague Dawley (SD) ,the maximal tolerated dose (MTD) of 10 g/kg BW SIF was used.
Genetic toxicology studies were conducted in Ames test with 0.008、0.04、0.2、1.0、5.0 mg/plate, KM mouse sperm abnormality test (including sperm capacitation state) and micronucleus test in bone marrow cells, with 0.2, 0.5, 1.5 and 4.5 g/kg BW SIF.
Endocrine disrupting toxicity studies were conducted in E-SCREEN(with 1、0.5、0.25、0.125、0.0625、0.03125、0.016、0.008 and 0.004 mM genistein(GEN)), rodent uterotrophic assay(with 0.1、0.2、0.5 and 1.5 g/kg BW SIF, 0.05、0.1、0.25 and 0.75 g/kg BW GEN), HERSHBERGER assay was carried out, with 0.2, 0.5, 1.5 and 4.5 g/kg soy isoflavones, in immature male SD rats aged 4-5 weeks.
In 13-week feed study, SD rats (10 male and 10 female each group) were gavaged 0.2, 0.5, 1.5, 4.5 g/kg BW SIF, for consecutive 13 weeks, clinical observations, body weight and food consumption were performed weekly. At the end of the study, urinalysis, hematology, clinical chemistry, endocrine parameter (including total testosterone, estrogen, follicle-stimulating hormone, thyroxine), DNA damage (comet assay), pathological and histopathological examinations of PCNA and ER-αwere performed.
Results and Conclusions:
In acute toxicity test, MTD of SIF was more than 10 g/kg BW, thus SIF belong to no toxicity materials.
Genetic toxicities: No genetic toxicities were found in Ames test, sperm abnormality test and micronucleus test, ratio of red fluorescent sperm decreased in 4.5 g/kg BW(P<0.05); thus SIF belong to no genetic toxicity materials, but 4.5 g/kg BW SIF inhibit capacitation state of mitochondrion in sperm tail.
Endocrine disrupting toxicity: In E-SCREEN test, value of OD in0.0625 mM genistein groups increased significantly(P<0.05). In rodent uterotrophic assay, compared with control group, both SIF and GEN induced uterine weight increasing significantly; histopathology of uterus and vagina showed that both SIF and GEN induce proliferation; there were dose-response relationship. In HERSHBERGER assay, compared with control group, body weight decreased in 0.50,1.50 and 4.5 g/kg dose of SIF (P<0.05); the ratio of of glans penis/kidney and bulbourethral glands/kidney were found to be significantly decreased in 1.50 and 4.5 g/kg BW groups (P<0.05); the ratio of levator ani muscles/kidney decreased in in 4.5 g/kg BW group (P<0.05); luteinizing hormone (LH) and ratio of LH/testosterone (T) increase in 1.5 g/kg BW group (P<0.05). Our studies indicated that SIF has estrogenic activity, antagonist activity.
In 13-week feed study, no mortality, ophthalmic abnormalities or treatment-related clinical observations were found during the study; in male rats, as compared with the control group, significantly lower body weights and food consumption were observed in 1.5 and 4.5 g/kg BW groups(P<0.05); in clinical chemistry tests, triglyceride was significantly decreased and high-density lipoprotein cholesterol was significantly increased in all SIF-treated groups(P<0.05); total testosterone levels were significantly lower in 0.50,1.50 and 4.5 g/kg BW dose groups than the control group, microscopic examination showed that the mammary glands exhibited hyperplastic and excreted latex in 4.5 g/kg BW group. In female rats, compared with the control group,cholesterol and LDL-C decreased in the 1.5 and 4.5 g/kg BW/day SIF groups, while HDL-C decreased only in the 4.5 g/kg BW/day SIF group, the vaginas were hyperplastic in the 4.5 g/kg BW/day SIF group. Comet assay showed that inertia, distributing square and tail inertia decreased significantly(P<0.05). The PCNA and ER-αof vaginas increased significantly, There were no other treatment-related macroscopic or microscopic lesions. Our results indicated that SIF affected blood biochemistry, induce estrogenic effects, both in male and female; but there were sexual differences. In male rats intake a large amounts of SIF caused antiandrogenic effects. In female rats intake a large amounts of SIF can disrupt hypothyroid hormone metabolism. In our study, the NOAEL of male was 0.2 g/kg BW, the NOAEL of female was 1.5 g/kg BW. Our results indicated that SIF at high dosages caused significant endocrine disruption.
目的:建立食品中内分泌干扰物筛选方法体系,探索筛选模型的敏感终点。作为筛选食品新资源、食品添加剂、食品污染物等可能具有内分泌干扰作用物质的方法,为进行食品内分泌干扰物的风险评估提供科学资料和依据。
研究内容:本研究的筛选方法体系包括体外和体内筛选方法部分。体外筛选方法包括:雌激素受体竞争结合试验,从分子水平筛选雌激样活性物质;E-SCREEN试验从细胞水平筛选雌激素样活性物质;卵黄蛋白原(VTG)间接的检测雌素样活性物质;类固醇合成试验从器官水平检测雄激素和或抗雄激素活性物质。体内筛选方法包括:啮齿动物子宫试验用于雌激素和或抗雌激素活性物质的筛选;HERSHBERGER试验用于雄激素和或抗雄激素活性物质的筛选。
方法:体外受体竞争结合试验用荧光偏振法,阳性物采用雌二醇(E_2),E_2浓度分别为2650、530、106、21.2、4.25、0.85、0.17、0.032和0.0064 nmol/l。E-SCREEN试验采用雌激素受体阳性的MCF-7细胞,2.5 mM E_2作为阳性物,用MTT法测定细胞增殖情况。卵黄蛋白原(VTG)测定用酶联免疫反应法,检测转入生长激素的转基因鲤鱼和亲本鲤鱼的VTG水平。类固醇合成试验用睾丸切片法,人类促绒毛膜性腺激素(hCG)作为促进睾酮合成的诱导物,每组均有hCG(+hCG)激发和无hCG(-hCG)两个亚组,阳性物用250mM氨鲁米特(Aminoglutethimide,AMI),阴性对照用10μM非那雄胺(Finasteride,FIN,有英文均统一)),分别与0、1、2、3小时收集培养液,最后测定睾酮(T)的水平。啮齿动物子宫试验用未成年的青春期前大鼠,阳性物用3.0μg/kg E2,和0.1、0.3、0.6和1.0g/kg·bw的白藜芦醇,连续三天灌胃给予受试物;实验结束称量子宫的湿重和干重,对卵巢、子宫和阴道进行组织病理学检查。HERSHBERGER(大小写统一)试验用4-5周未成年SD大鼠,阳性物用3 mg/kgFIN,连续灌胃10天;试验结束称量腹侧前列腺、精囊和凝结腺、肛提肌(LA)加球海绵体肌、阴茎头和尿道球腺重量,并测定促黄体激素(LH)和T。
结果:受体竞争结合试验荧光偏振的最大值和最小值的差值为111mP,E_2的IC50为22nM。在E-SCREEN实验中,2.5μM的雌二醇组OD值比对照组升高并达到统计学显著性水平。卵黄蛋白原测定结果显示,和亲本鲤鱼相比,转入生长激素基因的鲤鱼生长激素水平无明显变化。类固醇合成(睾丸切片法)试验:各组的睾酮水平随培养时间增加而增加,其中每组的hCG激发均比无hCG亚组的睾酮水平高,AMI+hCG组平均T浓度低于hCG激发对照组,FIN组和对照组相比无显著性差异。啮齿动物子宫试验:E2组子宫干重、湿重及其脏器系数显著升高;E2组的卵巢、子宫和阴道病理学均为显著增生改变;1.0g/kg bw白藜芦醇剂量组的子宫轻度增生,0.3、0.6和1.0g/kg bw白藜芦醇剂量组的阴道轻度增生无剂量-反应关系。Hershberger试验:TP+FLU组和TP组相比,前列腺、精囊腺、阴茎、肛提肌/球体海绵体肌、尿道球腺等重量明显降低;TP+FLU组和TP组相比,LH水平明显升高,LH/T比值明显升高。
结论:本研究成功建立了体外和体内筛选方法,有效利用这两类方法,分别从不同的水平筛选内分泌干扰物,即有助于提高筛选效率、结余筛选成本,更有助于提高筛选的灵敏性和特异性。体外筛选模型包括:受体结合试验、E-SCREEN、类固醇合成筛选试验;体内筛选模型包括:啮齿类动物子宫试验;HERSHBERGER试验。
第二部分：大豆异黄酮安全性评价研究
目的：对大豆异黄酮(SIF)的毒理学安全性进行研究和评价，并重点针对内分泌干扰毒性进行系统研究；同时探索大豆异黄酮的毒理学机制，为大豆异黄酮作为食品资源的合理应用提供科学依据。
方法:急性毒性试验选用Sprague Dawley(SD)大鼠，以SIF为受试物，采用最大耐受量(MTD)法，实验剂量为10 g/kg BW。
遗传毒性试验：Ames试验以染料木素(GEN)为受试物，设置0.008、0.04、0.2、1.0、5.0 mg/皿的5个剂量水平；小鼠(KM)精子畸形(包括精子线粒体获能试验)试验和小鼠骨髓细胞微核试验以SIF为受试物，试验设置0.2、0.5、1.5和4.5 g/kg BW四个剂量水平。
内分泌干扰毒性试验：E-SCREEN试验以GEN为受试物(剂量分别为1、0.5、0.25、0.125、0.0625、0.03125、0.016、0.008和0.004 mM)；啮齿动物子宫试验以SIF和GEN为受试物，SIF剂量分别为0.1、0.2、0.5和1.5 g/kg BW，GEN剂量分别为0.05、0.1、0.25和0.75 g/kg BW；HERSHBERGER试验选用SD大鼠，以SIF为受试物，剂量分别为0.2、0.5、1.5和4.5 g/kg BW。
亚慢性毒性试验：选用SD大鼠，连续13周灌胃给予SIF，剂量分别为0.2、0.5、1.5和4.5 g/kg BW剂量，每周观察动物的活动情况，并记录体重和进食量，试验结束后测定尿常规、血液学、血生化、凝血功能、内分泌指标(包括总睾酮、雌二醇、甲状腺素和促甲状腺素)、DNA损伤(彗星试验)，脏器组织病理观察和核增值因子以及雌激素受体的免疫组织化学分析。
结果和结论:大鼠急性毒性试验：SIF对雌、雄性大鼠的MTD大于10.0g/kgBW，因此大豆异黄酮属于实际无毒物。
三项遗传毒性试验：Ames试验：未见SIF有致基因突变作用；小鼠骨髓嗜多染红细胞微核试验：SIF对小鼠体细胞染色体无致断裂作用；小鼠精子畸形和精子活力试验：SIF对小鼠精子畸形发生率无影响，4.5 g/kg剂量组红色荧光精子所占比例明显降低。因此本研究未见SIF遗传毒性，但是4.5 g/kg BW SIF抑制精子线粒体获能。
内分泌干扰毒性研究：E-SCREEN试验：0.0625 mM剂量组OD值明显增高：子宫增重试验：与对照组比较，SIF和GEN剂量组子宫重量和脏器系数显著增加，GEN和SIF 4个剂量组的阴道均为增生改变：HERSHBERGER试验：与对照组比较，1.5和4.5 g/kg SIF剂量组的阴茎头/肾脏、尿道球脂/肾脏以及4．5g/kg剂量组肛提肌/肾脏均明显降低；和TP组相比，1.5 g/kg组的LH/T明显升高。因此SIF具有雌激素样活性，1.5 g/kg BW和4.5 g/kg BW SIT产生间接抗雄激素作用并降低大鼠的体重。
亚慢性毒性试验：1.5和4.5 g/kg剂量组体重增长明显滞后于对照组体重；进食量从第1周开始雄性1.5和4.5 g/kg剂量组的进食量明显低于对照组；血常规和尿常规均无明显变化：雌性1.5和4.5 g/kg的CHO和LDL-C和4.5 g/kg的HDL-C明显降低，0.2、0.5和1.5g/kg剂量组的HDL-C/LDL-C明显升高。雄性4个受试物剂量组的TO明显低于对照组，4个剂量组的HDL-C明显高于对照组。尿常规未见明显变化。雌性大鼠4.5 g/kg BW剂量组T3和T3/T4明显高于对照组，TSH/T4和TSH/T3各受试物具有下降的趋势。雄性大鼠0.5、1.5和4.5 g/kg BW剂量组总睾酮明显低于对照组；4.5 g/kg剂量组的宰杀体重、肝脏、肾脏、心脏和附睾重量以及1.5 g/kg剂量组的心脏重量均明显低于对照组，4.5 g/kg饲喂大鼠90天后雌性动物阴道壁增厚、上皮增生，雄性动物乳腺发育并泌乳：雌性大鼠各剂量组外周血淋巴细胞彗星试验的彗星惯量、彗星分布矩和尾惯量均比对照组降低：雌性4.5g/kg剂量组阴道上皮细胞PCNA的表达水平和ER-α表达水平高于对照组。因此SIF的毒性具有性别差异，体现为对雌性大鼠：影响血生化的脂类指标(降低胆固醇水平)，降低DNA的氧化损伤，高剂量SIF(4.5 g/kg BW)干扰甲状腺素水平，导致阴道增生，NOAEL为1.5 g/kg BW；对雄性大鼠：SIF影响血生化的脂类代谢指标降(甘油三脂水平)，降低体重，降低睾酮水平，促进乳腺增生并泌乳，NOAEL为0.2 g/kg BW。
Objectives: To establish endocrine disrupter screening system, to explore sensitive endocrine disrupter screening endpoint. The methods can be used to screen endocrine disruptors in novel sources of food, food additives, contaminations in food, further more to provide information and basal for risk assessment.
Content: to establish endocrine disrupter screening system, including in vitro and in vivo methods. In vitro method including: In vitro estrogen receptor(ER beta) competitor assays, to screen estrogenic activity amterials from molecular level; E-SCREEN to screen estrogenic activity amterials from cellular level; Vitellogenin(VTG) was used to screen estrogenic activity amterials indirectly; steroidogenesis screening assay was used to screen agonist and/or antagonist materials. In vivo methods including: rodent uterotrophic assay was used to screen estrogenic and/or anti-estrogenic activity amterials; HERSHBERGER assay was used to screen agonist and/or antagonist materials.
Methods: In vitro estrogen receptor(ER beta) competitor assays, fluorescence polarization value was measured, estradiol(E_2) used as positive control(the concentration were 2650, 530, 106, 21.2, 4.25, 0.85, 0.17, 0.032, 0.0064 nmol/l). In E-SCREEN assay, proliferation of MCF-7 cells was measured with MTT method, 2.5mM estradiol used as positive control. Vitellogenin(VTG) was measured with ELISA kit, in transgenic carp and wild carp. In steroidogenesis screening assay, sectioned testis assay was used, hCG as stimulation factor, there were hCG stimulating and no hCG stimulating subgroup in every group, 250 mM aminoglutethimide as positive substance and 10μM finasteride as negative substance, the endpoint of testosterone was measured in 0, 1, 2, 3 hour. In rodent uterotrophic assay using immature SD rats, 3.0μg/kg estradiol as positive control, 0.1, 0.3, 0.6 and 1.0g/kg bw Trans-resveratrol, administered orally to the animal for 3 consecutive day; in the end of the test, uterine wet and blotted weight, histopathology of ovary, uterus and vaginal were measured. HERSHBERGER assay was carried out in immature SD rats aged 4-5 weeks, 3.0 mg/kg flutamide(Flu) as positive control orally adminastrated, 0.4 mg/kg Testosterone Propionate(TP) subcutaneous adminastrated, which was given to every groups, all groups were administered for 10 consecutive day; in the end of test,animals were anesthetized , weight of ventral Prostate (VP), seminal vesicles together with coagulating gland (SV and CG),levator ani(LA), bulbocavernous muscles (BC), glans penis (GP) , bulbourethral glands (BG) were measured, testosterone(T) and luteinizing hormone(LH) levels were measured.
Results: In estrogen receptor(ER beta) competitor assays, the difference between max and min polarization was 111 mP, IC50 of E_2 was 22 nM. In E-SCREEN assay, compared with control, the OD value of E_2 group increased sigmficantly. There was no difference between transgenic(with growth hormone gene) carp and wild carp, in VTG level. In steroidogenesis screening assay, the concentration of testosterone increased with time in all group; concentration of testosterone was higher in hCG stimulation group than no stimulation group; concentration of testosterone was lower in AMI+ hCG group than hCG group. In rodent uterotrophic assay E_2 induce uterine weight increase significantly; histopathology of ovary, uterus and vagina showed proliferation in E2 group significantly; uterine histopathologies showed that 1.0 g/kg bw trans-resveratrol induce slight proliferation; 0.3, 0.6 and 1.0 g/kg bw trans-resveratrol induce slight vagina proliferation without dose-response relationship. In HERSHBERGER assay, the weights of ventral Prostate (VP), seminal vesicles together with coagulating gland (SV and CG),levator ani(LA), bulbocavernous muscles (BC), glans penis (GP) , bulbourethral glands (BG) decreased significantly in TP+FLU group than TP group; LH and ratio of LH/T increase in TP+FLU group than TP group.
Conclusion: In our study, in vitro and in vitro screening methods were established, it is benefit to improve efficiency of screening and also to decrease cost of screening, further more it can improve sensitivity and specialty, to use two methods together.
In vitro estrogen receptor(ER beta) competitor assays, E-SCREEN assay, Vitellogenin(VTG) measurement, steroidogenesis screening assay, rodent uterotrophic assay, HERSHBERGER were established successfully.
Study about Assessment of Soy Isoflavone Safety
Objectives:
To evaluate toxicological safly of soy isoflavones(SIF), and to study endoscrine disrupting toxicity; to study the toxicological mechanism of SIF, finally to provide scientific proof for reasonable usage of SIF as novel source in food.
Method:
In acute toxicity test with Sprague Dawley (SD) ,the maximal tolerated dose (MTD) of 10 g/kg BW SIF was used.
Genetic toxicology studies were conducted in Ames test with 0.008、0.04、0.2、1.0、5.0 mg/plate, KM mouse sperm abnormality test (including sperm capacitation state) and micronucleus test in bone marrow cells, with 0.2, 0.5, 1.5 and 4.5 g/kg BW SIF.
Endocrine disrupting toxicity studies were conducted in E-SCREEN(with 1、0.5、0.25、0.125、0.0625、0.03125、0.016、0.008 and 0.004 mM genistein(GEN)), rodent uterotrophic assay(with 0.1、0.2、0.5 and 1.5 g/kg BW SIF, 0.05、0.1、0.25 and 0.75 g/kg BW GEN), HERSHBERGER assay was carried out, with 0.2, 0.5, 1.5 and 4.5 g/kg soy isoflavones, in immature male SD rats aged 4-5 weeks.
In 13-week feed study, SD rats (10 male and 10 female each group) were gavaged 0.2, 0.5, 1.5, 4.5 g/kg BW SIF, for consecutive 13 weeks, clinical observations, body weight and food consumption were performed weekly. At the end of the study, urinalysis, hematology, clinical chemistry, endocrine parameter (including total testosterone, estrogen, follicle-stimulating hormone, thyroxine), DNA damage (comet assay), pathological and histopathological examinations of PCNA and ER-αwere performed.
Results and Conclusions:
In acute toxicity test, MTD of SIF was more than 10 g/kg BW, thus SIF belong to no toxicity materials.
Genetic toxicities: No genetic toxicities were found in Ames test, sperm abnormality test and micronucleus test, ratio of red fluorescent sperm decreased in 4.5 g/kg BW(P<0.05); thus SIF belong to no genetic toxicity materials, but 4.5 g/kg BW SIF inhibit capacitation state of mitochondrion in sperm tail.
Endocrine disrupting toxicity: In E-SCREEN test, value of OD in0.0625 mM genistein groups increased significantly(P<0.05). In rodent uterotrophic assay, compared with control group, both SIF and GEN induced uterine weight increasing significantly; histopathology of uterus and vagina showed that both SIF and GEN induce proliferation; there were dose-response relationship. In HERSHBERGER assay, compared with control group, body weight decreased in 0.50,1.50 and 4.5 g/kg dose of SIF (P<0.05); the ratio of of glans penis/kidney and bulbourethral glands/kidney were found to be significantly decreased in 1.50 and 4.5 g/kg BW groups (P<0.05); the ratio of levator ani muscles/kidney decreased in in 4.5 g/kg BW group (P<0.05); luteinizing hormone (LH) and ratio of LH/testosterone (T) increase in 1.5 g/kg BW group (P<0.05). Our studies indicated that SIF has estrogenic activity, antagonist activity.
In 13-week feed study, no mortality, ophthalmic abnormalities or treatment-related clinical observations were found during the study; in male rats, as compared with the control group, significantly lower body weights and food consumption were observed in 1.5 and 4.5 g/kg BW groups(P<0.05); in clinical chemistry tests, triglyceride was significantly decreased and high-density lipoprotein cholesterol was significantly increased in all SIF-treated groups(P<0.05); total testosterone levels were significantly lower in 0.50,1.50 and 4.5 g/kg BW dose groups than the control group, microscopic examination showed that the mammary glands exhibited hyperplastic and excreted latex in 4.5 g/kg BW group. In female rats, compared with the control group,cholesterol and LDL-C decreased in the 1.5 and 4.5 g/kg BW/day SIF groups, while HDL-C decreased only in the 4.5 g/kg BW/day SIF group, the vaginas were hyperplastic in the 4.5 g/kg BW/day SIF group. Comet assay showed that inertia, distributing square and tail inertia decreased significantly(P<0.05). The PCNA and ER-αof vaginas increased significantly, There were no other treatment-related macroscopic or microscopic lesions. Our results indicated that SIF affected blood biochemistry, induce estrogenic effects, both in male and female; but there were sexual differences. In male rats intake a large amounts of SIF caused antiandrogenic effects. In female rats intake a large amounts of SIF can disrupt hypothyroid hormone metabolism. In our study, the NOAEL of male was 0.2 g/kg BW, the NOAEL of female was 1.5 g/kg BW. Our results indicated that SIF at high dosages caused significant endocrine disruption.
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14 Montani C,Penza M,Jeremic M,Biasiotto G,La Sala G,De Felici M,Ciana P,Maggi A,Di Lorenzo D.Genistein is an efficient estrogen in the whole-body throughout mouse development.Toxicol Sci.2008,103(1):57-67.
15 Wang W W,Smith E D,Stuff J E,et al.Chelesterol-lowering effects of soy protein in normcholeterolemic and hypercholesterolemic men.Am J Clin,1998,68(suppl):1385-1389.
16 Hsieh CY,Santell RC,Haslam SZ,Helferich WG.Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer(MCF-7) cells in vitro and in vivo.Cancer Res.1998,58(17):3833-8.
17 Von S,Montano M,Montano M.Prostate enlargement in mice due to feta exposure to low doses of estodiol or diethylstilbestroland opposite effects at high doses[J].Proc Natl Acad Sci,1997,94(2):2056-2061.
18 Home R,Brouwers E,Al-Maharik.Time-resolved fluroimmunoassay of plasma and urine O-desmethyl angolensm[J].Journal of steroid biochemistry & molevular biology,2002,81:353-361.
19 Bowey E,Adlereut Z,Rowland I.Metabolism of isoflavones and lignans by the gut microflora:a study in germ-free and human flora associated rats[J].Food and Chemical Toxicology,2003,41(5):631-636.
20 Maubach J,Bracke M,Heyerick A.Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography[J].Journal of Chromotography B,2003,784(1):137-144.
21 荣顺兴,金泰,周袁芬,等.子宫增重试验的病理学研究[J].中华劳动卫生职业病杂志,2006,24(3):172-174.
22 Delclos K,Bucci T,Lomax L,Latendresse J,Warbritton A,Weis CC,Newbold RR.Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.Reprod Toxicol.2001 Nov-Dec;15(6):647-63.
23 Ajdzanovic V,Sosic-Jurjevic B,Filipovic B,Trifunovic S,Manojlovic-Stojanoski M,Sekulic M,Milosevic VL.Genistein-Induced Histomorphometric and Hormone Secreting Changes in the Adrenal Cortex in Middle-Aged Rats.Exp Biol Med (Maywood).2008 Dec 8.
24 Basak S,Pookot D,Noonan E,Dahiya R.Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function.Mol Cancer Ther.2008Oct;7(10):3195-202.
25 Hancock K,Coleman E,Tao Y,Morrison E,Braden T,Kemppainen B,Akingbemi B.Genistein decreases androgen biosynthesis in rat Leydig cells by.interference with luteinizing hormone-dependent signaling.Toxicol Lett.2008 Nov 18.
26 Tao J,Zhang Y,Li S,Sun W,Soong T.Tyrosine kinase-independent inhibition by genistein on spermatogenic T-type calcium channels attenuates mouse sperm motility and acrosome reaction.Cell Calcium.2008 Sep 11.
27 Huang Y,Pan L,Xia X,Feng Y,Jiang C,Cui Y.Long-term effects of phytoestrogen daidzein on penile cavemosal structures in adult rats.Urology.2008Jul;72(1):220-4.
28 李丽,刘兆平,严卫星.大豆异黄酮毒性作用研究进展.国外医学卫生学分册,2005,32(6):338-342.
29 Huang Y,Zeng J,Huang Y,Qiu F,Ye H,Wang S.Antagonistic effect of 3'-daidzein sulfonate sodium on prostatic hyperplasia in mice.Zhonghua Nan Ke Xue.2007 May;13(5):387-90.
30 Michael M,Erich W,Alberto D,Friedlieb P,James A,Jochen B.Acute,subchronic and chronic safety studies with genistein in rots.Food and Chemical Toxicology.2006,44:56-80.
31 Okazaki K,Okazaki S,Nakamura H,Kitamura Y,Hatayama K,Wakabayashi S,Tsuda T,Katsumata T,Nishikawa A.and Hirose M.A repeated 28-day oral dose toxicity study of genistein in rats,based on the 'Enhanced OECD Test Guideline 407'for screening endocrine-disrupting chemicals.Arch Toxicol 2002;76:553-9.
32 Weber K,Setchell K,Stocco D.Dietary soy-phytoestrogens decrease testosterone levels and prostate weight without altering LH,prostate 5alpha-reductase or testicular steroidogenic acute regulatory peptide levels inadult male Sprague-Dawley rats.J Endocrinol,2001,170(3):591-9.
33 Gray J,Nunez A,Siegel L.Effects of testosterone on body weight and adipose tissue:role of aromatization.Physiol Behavior,1979,23(3):465-9.
34 迟晓星,张涛,崔洪斌.大豆异黄酮对青年雌性大鼠的副作用研究[J]食品科技,2008,(02):246-248.
35 NTP Toxicology and Carcinogenesis Studies of Genistein(CAS No.446-72-0) in Sprague-Dawley Rats(Feed Study).Natl Toxicol Program Tech Rep Ser.2008,545):1-240.
36 Olive PL,Ranath J,Durand R.hererogenicity in radiation induced DNA damage and repair in tumour and normal cell same a suredusing the comet assay.Radiation Res,1990,122:86-94.
37 Whitaker SJ,Powell SN,McMillan TJ.Molecular assays of radiation-induced DNA damage.Eur J Cancer.1991;27(7):922-8.
38 Klaude M,Eriksson S,Nygren J,Ahnstr(o|¨)m G.The comet assay:mechanisms and technical considerations.Mutat Res.1996 Jun 12;363(2):89-96.
39 Fuhler S,Wolf HU.Detection of DNA-crosslinking agents with the alkaline comet assay.Environ Mol Mutagen,1996,27:196-201.
40 衡正昌,王涛,张遵真.彗星试验检测环境致癌物的敏感性评价,癌变·畸变·突变,1999,6(11):316.
41 Wijeratne SS,Cuppett SL.J Agdc Food Chem.Soy isoflavones protect the intestine from lipid hydroperoxide mediated oxidative damage.2007 Nov 28;55(24):9811-6.
42 Oh HY,Lim S,Lee JM,Kim DY,Ann ES,Yoon S.A combination of soy isoflavone supplementation and exercise improves lipid profiles and protects antioxidant defense-systems against exercise-induced oxidative stress in ovariectomized rats.Biofactors.2007;29(4):175-85
43 逢晓云,沈金芳,贡沁燕.大豆异黄酮对大鼠血脂的影响及其体内外抗氧化作用研究.中国临床药理学与治疗学2008,13(1):62-67.
44 Anderson JW,Jhonstone BM,Cook N.Meta-analysis of the effects of soy protein intake on serum lipids.N Engl J Med,1995,333:276-282.
45 Ascenscio C,Torres N,Isoard F,Gomez F,Hemandez R,Tovar A.Soy protein affects serum insulin and hepatic SREBP-1 mRNA and reduces fatty liver in rats.J Nutr,2004,134:522-529.
46 Xiao C,Mei J,Wood C.Effect of soy proteins and isoflavones on lipid metabolism and involved gene expression.Front Biosci,2008,13:2660-2673.
47 NCTR.1.Genistein:Evaluation of reproductive effects over multiple generations [Volume1]and the chronic effects[Volume Ⅱ]of exposure during various life states.Jefferson AR:National Center for Toxicological Research;2005.
48 R.Michael McClain,Erich Wolz b,Alberto Davidovich,Friedlieb Pfannkuch,James A.Edwards,Jochen Bausch.Acute,subchronic and chronic safety studies with genistein in rats.Food and Chemical Toxicology.2006,44:56-80.
49 Adlercreutz H,Markkanen H,Watanabe S.Plasma concen- trations of phyto-oestrogens in Japanese men.Lancet,1993,342(8 881):1 209-10.
50 Ntp-Cerhr Expert Panel Report On The Reproductive And Developmental Toxicity Of Soy Formula.April 2006 Ntp-Cerhr-Soy Formula-06.
51 翟凤英,杨晓光主编.中国居民营养与健康状况调查报告之二—膳食与营养素摄入状况(2002).人民卫生出版社.27.
52 史桂玲,马欣,杨军,孙家莉.豆制品中大豆异黄酮的含量比较.华西药学杂志2006,21(3):305-306.
1 Guerrero-Bosagna CM,Sabat P,Valdovinos FS,Valladares LE,Clark SJ.Epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice.BMC Physiol.2008 Sep 15;8:17.
2 Hughes CL,Liu G.Beall S,Foster WG,Davis V.Effects of genistein or soy milk during late gestation and lactation on adult uterine organization in the rat.Exp Biol Med(Maywood).2004 Jan;229(1):108-17.
3 Casanova M,You L,Gaido KW,Archibeque-Engle S,Janszen DB,Heck HA.Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro.Toxicol Sci.1999 Oct;51(2):236-44.
4 Newbold RR,Banks EP,Bullock B,Jefferson WN.Uterine adenocarcinoma in mice treated neonatally with genistein.Cancer Res.2001 Jun 1;61(11):4325-8.
5 Delclos KB,Bucci TJ,Lomax LG,Latendresse JR,Warbritton A,Weis CC,Newbold RR.Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley)rats.Reprod Toxicol.2001 Nov-Dec;15(6):647-63.
6 Jefferson WN,Couse JF,Padilla-Banks E,Korach KS,Newbold RR.Neonatal exposure to genistein induces estrogen receptor(ER)alpha expression and multioocyte follicles in the maturing mouse ovary:evidence for ERbeta-mediated and nonestrogenic actions.Biol Reprod.2002 Oct;67(4):1285-96.
7 Nikaido Y,et al.[J].Reprod Toxicol,2004,18:803-811
8 National Toxicology Program.NTP Multigenerational Reproductive Study of Genistein(CAS No.446-72-0) in Sprague-Dawley Rats(Feed Study).Natl Toxicol Program Tech Rep Ser.2008 Mar;(539):1-266.
9 Takagi H,Shibutani M,Lee KY,Lee HC,Nishihara M,Uneyama C,Takigami S,Mitsumori K,Hirose M.Lack of modifying effects of genistein on disruption of the reproductive system by perinatal dietary exposure to ethinylestradiol in rats.Reprod Toxicol.2004 Jul;18(5):687-700.
10 East,J.The effect of genistein on the fertility of mice.J Endocdnol 1955;13:94-100.
11 Thuillier R,Wang Y,Culty M.Prenatal exposure to estrogenic compounds alters the expression pattern of platelet-dedved growth factor receptors alpha and beta in neonatal rat testis:identification of gonocytes as targets of estrogen exposure.Biol Reprod.2003 Mar;68(3):867-80.
12 NCTR.1.Genistein:Evaluation of reproductive effects over multiple generations [Volume 1]and the chronic effects[Volume Ⅱ]of exposure during various life states.Jefferson AR:National Center for Toxicological Research;2005.
13 迟晓星,张涛,崔洪斌.大豆异黄酮对青年雌性大鼠的副作用研究[J]食品科技,2008,(02):246-248.
14 NTP Toxicology and Carcinogenesis Studies of Genistein(CAS No.446-72-0) in Sprague-Dawley Rats(Feed Study).Natl Toxicol Program Tech Rep Ser.2008 Jan;(545):1-240.
15 Basak S,Pookot D,Noonan EJ,Dahiya R.Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function.Mol Cancer Ther.2008Oct;7(10):3195-202.
16 Hancock KD,Coleman ES,Tao YX,Morrison EE,Braden TD,Kemppainen BW,Akingbemi BT.Genistein decreases androgen biosynthesis in rat Leydig cells by interference with luteinizing hormone-dependent signaling.Toxicol Lett.2008 Nov 18.
17 Ajdzanovic V,Sosic-Jurjevic B,Filipovic B,Trifunovic S,Manojlovic-Stojanoski M,Sekulic M,Milosevic VL.Genistein-Induced Histomorphometric and Hormone Secreting Changes in the Adrenal Cortex in Middle-Aged Rats.Exp Biol Med (Maywood).2008 Dec 8.
18 Tao J,Zhang Y,Li S,Sun W,Soong TW.Tyrosine kinase-independent inhibition by genistein on spermatogenic T-type calcium channels attenuates mouse sperm motility and acrosome reaction.Cell Calcium.2008 Sep 11.
19 Huang Y,Pan L,Xia X,Feng Y,Jiang C,Cui Y.Long-term effects of phytoestrogen daidzein on penile cavernosal structures in adult rats.Urology.2008Jul;72(1):220-4.
20 李丽,刘兆平,严卫星.大豆异黄酮毒性作用研究进展.国外医学卫生学分册,2005,32(6):338-342.
21 Huang YS,Zeng J,Huang YP,Qiu F,Ye HY,Wang SR.Antagonistic effect of 3'-daidzein sulfonate sodium on prostatic hyperplasia in mice.Zhonghua Nan Ke Xue.2007 May;13(5):387-90.
22 Okazaki,K.,Okazaki,S.,Nakamura,H.,Kitamura,Y.,Hatayama,K.,Wakabayashi,S.,Tsuda,T.,Katsumata,T.,Nishikawa,A.and Hirose,M.A repeated 28-day oral dose toxicity study of genistein in rats,based on the 'Enhanced OECD Test Guideline 407' for screening endocrine-disrupting chemicals.Arch Toxicol 2002;76:553-9.
23 R.Michael McClain,Erich Wolz b,Alberto Davidovich,Friedlieb Pfannkuch,James A.Edwards,Jochen Bausch.Acute,subchronic and chronic safety studies with genistein in rats.Food and Chemical Toxicology.2006,44:56-80.
24 NTP-CERHR EXPERT PANEL REPORT on the REPRODUCTIVE and DEVELOPMENTAL TOXICITY of SOY FORMULA.April 2006NTP-CERHR-SOY FORMULA-06
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1 Murata M,Midorikawa K,Koh M,Umezawa K,Kawanishi S.Genistein and daidzein induce cell proliferation and their metabolites cause oxidative DNA damage in relation to isoflavone-induced cancer of estrogen-sensitive organs.Biochemistry.2004,43(9):2569-77.
2 Daniel R,Doerge,Daniel M,Sheehan.Goitrogenic and Estrogenic Activity of Soy Isoflavones.Environmental Health Perspectives,2002,110(supplement 3):349-353.
3 Takagi H,Shibutani M,Lee K,Lee H,Nishihara M,Uneyama C,Takigami S,Mitsumori K,Hirose M.Lack of modifying effects of genistein on disruption of the reproductive system by perinatal dietary exposure to ethinylestradiol in rats.Reprod Toxicol.2004 Jul;18(5):687-700.
4 Newbold RR,Banks EP,Bullock B,Jefferson WN.Uterine adenocarcinoma in mice treated neonatally with genistein.Cancer Res.2001,61(11):4325-8.
5 Michael McClain R,Wolz E,Davidovich A,at el,Food Chem Toxicol.Acute,subchronic and chronic safety studies with genistein in rats 2006,44(1):56-80.
6 许建宁,王全凯,崔涛,罗玉姝,王四旺.染料木素对大鼠生殖毒性的实验研究(Ⅳ)围产期毒性,中草药,2004,35(11):1279-1282.
7 许建宁,王全凯,李忠生,崔涛,罗玉姝,王四旺,郑玉新.染料木素对大鼠生殖毒性的实验研究(Ⅲ)致畸敏感期毒性,中草药,2004,35(8):914-915.
8 许建宁,王全凯,崔涛,罗玉姝,王四旺,郑玉新.染料木素对大鼠生殖毒性的实验研究(Ⅱ)一般生殖毒性.中草药,2003,34(9):830-832.
9 Series on testing and assessment number 59.report of the validation of the updated test guideline 407:repeat dose 28-day oral toxicity study in laboratory rats.ENV/JM/MONO,2006,26.
10 石岩,毛焕新,李毅.大豆异黄酮粉致突变作用的研究.吉林医学,2008,29(11):924-926.
11 Lynch A,Harvey J,Aylott M,Nicholas E,Burman M,Siddiqui A,Walker S,Rees R.Mutagenesis.Investigations into the concept of a threshold for topoisomerase inhibitor-induced clastogenicity.2003 Jul;18(4):345-53.
12 Michael McClain R,Wolz E,Davidovich A,Bausch J.Genetic toxicity studies with genistein.Food Chem Toxicol.2006,44(1):42-55.
13 Snyder RD,Gillies PJ.Reduction of genistein clastogenicity in Chinese hamster V79 cells by daidzein and other flavonoids.Food Chem Toxicol.2003,41(10):1291-8.
14 Montani C,Penza M,Jeremic M,Biasiotto G,La Sala G,De Felici M,Ciana P,Maggi A,Di Lorenzo D.Genistein is an efficient estrogen in the whole-body throughout mouse development.Toxicol Sci.2008,103(1):57-67.
15 Wang W W,Smith E D,Stuff J E,et al.Chelesterol-lowering effects of soy protein in normcholeterolemic and hypercholesterolemic men.Am J Clin,1998,68(suppl):1385-1389.
16 Hsieh CY,Santell RC,Haslam SZ,Helferich WG.Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer(MCF-7) cells in vitro and in vivo.Cancer Res.1998,58(17):3833-8.
17 Von S,Montano M,Montano M.Prostate enlargement in mice due to feta exposure to low doses of estodiol or diethylstilbestroland opposite effects at high doses[J].Proc Natl Acad Sci,1997,94(2):2056-2061.
18 Home R,Brouwers E,Al-Maharik.Time-resolved fluroimmunoassay of plasma and urine O-desmethyl angolensm[J].Journal of steroid biochemistry & molevular biology,2002,81:353-361.
19 Bowey E,Adlereut Z,Rowland I.Metabolism of isoflavones and lignans by the gut microflora:a study in germ-free and human flora associated rats[J].Food and Chemical Toxicology,2003,41(5):631-636.
20 Maubach J,Bracke M,Heyerick A.Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography[J].Journal of Chromotography B,2003,784(1):137-144.
21 荣顺兴,金泰,周袁芬,等.子宫增重试验的病理学研究[J].中华劳动卫生职业病杂志,2006,24(3):172-174.
22 Delclos K,Bucci T,Lomax L,Latendresse J,Warbritton A,Weis CC,Newbold RR.Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.Reprod Toxicol.2001 Nov-Dec;15(6):647-63.
23 Ajdzanovic V,Sosic-Jurjevic B,Filipovic B,Trifunovic S,Manojlovic-Stojanoski M,Sekulic M,Milosevic VL.Genistein-Induced Histomorphometric and Hormone Secreting Changes in the Adrenal Cortex in Middle-Aged Rats.Exp Biol Med (Maywood).2008 Dec 8.
24 Basak S,Pookot D,Noonan E,Dahiya R.Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function.Mol Cancer Ther.2008Oct;7(10):3195-202.
25 Hancock K,Coleman E,Tao Y,Morrison E,Braden T,Kemppainen B,Akingbemi B.Genistein decreases androgen biosynthesis in rat Leydig cells by.interference with luteinizing hormone-dependent signaling.Toxicol Lett.2008 Nov 18.
26 Tao J,Zhang Y,Li S,Sun W,Soong T.Tyrosine kinase-independent inhibition by genistein on spermatogenic T-type calcium channels attenuates mouse sperm motility and acrosome reaction.Cell Calcium.2008 Sep 11.
27 Huang Y,Pan L,Xia X,Feng Y,Jiang C,Cui Y.Long-term effects of phytoestrogen daidzein on penile cavemosal structures in adult rats.Urology.2008Jul;72(1):220-4.
28 李丽,刘兆平,严卫星.大豆异黄酮毒性作用研究进展.国外医学卫生学分册,2005,32(6):338-342.
29 Huang Y,Zeng J,Huang Y,Qiu F,Ye H,Wang S.Antagonistic effect of 3'-daidzein sulfonate sodium on prostatic hyperplasia in mice.Zhonghua Nan Ke Xue.2007 May;13(5):387-90.
30 Michael M,Erich W,Alberto D,Friedlieb P,James A,Jochen B.Acute,subchronic and chronic safety studies with genistein in rots.Food and Chemical Toxicology.2006,44:56-80.
31 Okazaki K,Okazaki S,Nakamura H,Kitamura Y,Hatayama K,Wakabayashi S,Tsuda T,Katsumata T,Nishikawa A.and Hirose M.A repeated 28-day oral dose toxicity study of genistein in rats,based on the 'Enhanced OECD Test Guideline 407'for screening endocrine-disrupting chemicals.Arch Toxicol 2002;76:553-9.
32 Weber K,Setchell K,Stocco D.Dietary soy-phytoestrogens decrease testosterone levels and prostate weight without altering LH,prostate 5alpha-reductase or testicular steroidogenic acute regulatory peptide levels inadult male Sprague-Dawley rats.J Endocrinol,2001,170(3):591-9.
33 Gray J,Nunez A,Siegel L.Effects of testosterone on body weight and adipose tissue:role of aromatization.Physiol Behavior,1979,23(3):465-9.
34 迟晓星,张涛,崔洪斌.大豆异黄酮对青年雌性大鼠的副作用研究[J]食品科技,2008,(02):246-248.
35 NTP Toxicology and Carcinogenesis Studies of Genistein(CAS No.446-72-0) in Sprague-Dawley Rats(Feed Study).Natl Toxicol Program Tech Rep Ser.2008,545):1-240.
36 Olive PL,Ranath J,Durand R.hererogenicity in radiation induced DNA damage and repair in tumour and normal cell same a suredusing the comet assay.Radiation Res,1990,122:86-94.
37 Whitaker SJ,Powell SN,McMillan TJ.Molecular assays of radiation-induced DNA damage.Eur J Cancer.1991;27(7):922-8.
38 Klaude M,Eriksson S,Nygren J,Ahnstr(o|¨)m G.The comet assay:mechanisms and technical considerations.Mutat Res.1996 Jun 12;363(2):89-96.
39 Fuhler S,Wolf HU.Detection of DNA-crosslinking agents with the alkaline comet assay.Environ Mol Mutagen,1996,27:196-201.
40 衡正昌,王涛,张遵真.彗星试验检测环境致癌物的敏感性评价,癌变·畸变·突变,1999,6(11):316.
41 Wijeratne SS,Cuppett SL.J Agdc Food Chem.Soy isoflavones protect the intestine from lipid hydroperoxide mediated oxidative damage.2007 Nov 28;55(24):9811-6.
42 Oh HY,Lim S,Lee JM,Kim DY,Ann ES,Yoon S.A combination of soy isoflavone supplementation and exercise improves lipid profiles and protects antioxidant defense-systems against exercise-induced oxidative stress in ovariectomized rats.Biofactors.2007;29(4):175-85
43 逢晓云,沈金芳,贡沁燕.大豆异黄酮对大鼠血脂的影响及其体内外抗氧化作用研究.中国临床药理学与治疗学2008,13(1):62-67.
44 Anderson JW,Jhonstone BM,Cook N.Meta-analysis of the effects of soy protein intake on serum lipids.N Engl J Med,1995,333:276-282.
45 Ascenscio C,Torres N,Isoard F,Gomez F,Hemandez R,Tovar A.Soy protein affects serum insulin and hepatic SREBP-1 mRNA and reduces fatty liver in rats.J Nutr,2004,134:522-529.
46 Xiao C,Mei J,Wood C.Effect of soy proteins and isoflavones on lipid metabolism and involved gene expression.Front Biosci,2008,13:2660-2673.
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