Study Of Endocrine Disruptor Screening Methods System And Assessment About Soy Isoflavone Safety

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(E₂) 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 E₂ was 22 nM. In E-SCREEN assay, compared with control, the OD value of E₂ 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 E₂ 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 SafetyObjectives: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.