• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.9
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• pp.1486-1491
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• 2004

The estrogenicities of six heavy metal compounds, which contaminate frequently in foods, were assayed using a combination of in vitro and in vivo assays. The assays were 1) estrogen receptor dependent transcriptional expression assay, 2) E-screen assay and, 3) the uterotropic assay in mice. The chemicals studied were 17$\beta$ -estradiol, diethylstilbestrol (DES), arsenic oxide, bis(tri-n-butyltin), cadmium chloride, chromium chloride, lead acetate, and mercuric chloride. Using the estrogen receptor dependent transcriptional expression assay, the following estrogenicity ranking was measured: bis(tri-n-butyltin) > cadmium chloride > chromium chloride >> mercuric chloride >lead acetate = arsenic oxide. Using E-screen test, the following estrogenicity ranking was measured: bis(tri-n-butyltin) > cadmium chloride > chromium chloride >> mercuric chloride > lead acetate = arsenic oxide. Results from the uterotropic assay showed that bis(tri-n-butyltin), cadmium chloride, chromium chloride caused an increase in uterine wet weight, while lead acetate, mercuric chloride, and arsenic oxide failed to do so. Bis(tri-n-butyltin), cadmium chloride and chromium chloride showed the highest estrogenicity in three assay systems. Recent studies suggesting that bis(tri-n-butyltin), cadmium chloride have estrogenicities are compatible with the present finding. Furthermore, our study is suggesting that chromium chloride may be estrogenic. The results demonstrate that this three level-assay combination (transcriptional activation, cell proliferation, and an in vivo effect in an estrogen-responsive tissue) could serve as a useful method to assess the estrogenicity of heavy metals.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.168-168
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• 2002

To establish a test protocol for the rodent 20-day thyroid/pubertal assay, flutamide and diethylstilbestrol (DES) were administered to intact male Sprague-Dawley rats from postnatal day 33 for 20 days. Flutamide (1, 5, and 25 mg/kg/day) or DES (10, 20, and 40 ug/kg/day) was given once daily by oral gavage to immature male rats. Prepuce separation was significantly delayed in flutamide group and in DES group. One day after the last dose, the rats were killed and pituitary, thyroid, and reproductive organs were removed and weighed. Flutamide treatment resulted in a significant reduction in the weights of epididymides, ventral prostate, seminal vesicles plus coagulating glands and fluid (SVCGF), levator ani. bulbocarvenus muscles (LABC), Cowper's glands, and glans penis. The weight of adrenal glands decreased at % mg/kg/day, while testes and any other organ weights were unaffected. No microscopic changes were observed in the thyroid glands. Serum levels of testosterone wert significantly increased in the flutamide-treated groups and serum levels of estradiol were also increased. A significant reduction in the weights of testes, epididymides, ventral prostate, SVCGF, LABC, Cowpers glands, and glans penis of DES treated group. Serum testosterone and LH decreased significantly in DES group. Decrease of estradiol was observed, but not significant. These results indicate that flutamide and DES delay puberty in the male rat and its mode of action appears to be via altered secretion of steroids, which subsequently affect the development of the reproductive tract. (Supported by the grant from NITR/Korea FDA for Endocrine Disrupter Research.)

• Safety and Health at Work
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• v.2 no.1
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• pp.1-8
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• 2011

The knowledge on the etiology of breast cancer has advanced substantially in recent years, and several etiological factors are now firmly established. However, very few new discoveries have been made in relation to occupational risk factors. The International Agency for Research on Cancer has evaluated over 900 different exposures or agents to-date to determine whether they are carcinogenic to humans. These evaluations are published as a series of Monographs (www.iarc.fr). For breast cancer the following substances have been classified as "carcinogenic to humans" (Group 1): alcoholic beverages, exposure to diethylstilbestrol, estrogen-progestogen contraceptives, estrogen-progestogen hormone replacement therapy and exposure to X-radiation and gamma-radiation (in special populations such as atomic bomb survivors, medical patients, and in-utero exposure). Ethylene oxide is also classified as a Group 1 carcinogen, although the evidence for carcinogenicity in epidemiologic studies, and specifically for the human breast, is limited. The classification "probably carcinogenic to humans" (Group 2A) includes estrogen hormone replacement therapy, tobacco smoking, and shift work involving circadian disruption, including work as a flight attendant. If the association between shift work and breast cancer, the most common female cancer, is confirmed, shift work could become the leading cause of occupational cancer in women.

• Toxicological Research
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• v.16 no.2
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• pp.109-116
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• 2000

The purposes of our study were to optimize the conditions of the screening and testing methods for endocrine disruptors, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the sensitivity between these assays currently undergoing validation. Two in vitro test systems, MCF-7 cells proliferation (E-screen assay) and competitive binding to estrogen receptors (ER) were selected to evaluate the estrogenic effects. 17$\beta$-Estradiol (E2) and diethylstilbestrol (DES) were used as a positive control in vitro test. Also, E2 and ethinyl estradiol (EE) were used as a positive control in vivo uterotrophic assay. In in vitro test, E2 and DES showed a strong estrogenic response at concentration of 1.0 nM. In uterotrophic assay, E2 (0.3 $\mu\textrm{g}$/kg) and EE (0.3 $\mu\textrm{g}$/kg) produced a significant increase in uterus and vagina weight in both immature and ovariectomized rats. Although we did not com-pared the specificity between in vivo and in vitro assays, these assay systems may serve as a good tool for endocrine disruptors screening methods. Our data indicate that these assay systems exhibit some difference in their sensitivity to the same estrogenic compounds. Therefore, as a first rapid screening assay for estrogenic activity qf unknown chemicals, at least two assay systems should probably be carried out with a view of high sensitivity and standardization conditions. Also, a careful validation tests are necessary to obtain a reasonable degree of reproducibility.

• Toxicological Research
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• v.16 no.4
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• pp.275-284
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• 2000

The organ culture model of the whole mammary gland has many advantages for the study of branching morphogenesis and biological characteristics, including tumorigenesis. Prior to whole gland organ culture, rats were treated with 7,12-dimethylbenz[a]anthracene (DMBA) or N-methyl-N-nitrosourea (MNU) for one week. The tramdorming effect and the morphological changes were assessed by the whole mount preparations and histopathological examination in terminal end buds (TEB), terminal ducts (TD), alveolar buds (AB), alveolar lobules (AL) and hyperplastic alveolar nodules (HAN) of the mammary gland. Grossfindings of the mammary glands at dissection were higher branching morphogenesis and larger volume in carcinogen-treated groups than in carcinogen-non-treated groups. Results of the whole mount method were coincided with those of the histopathological observations. Circular TEB, normally maintained AB, AL, and high cellular density were more frequently observed in carcinogen-treated groups than in carcinogen-nan-treated groups. Histopathologically, as a preneoplastic marker, HAN was maintained only in mammary organ culture of the carcinogen-treated groups. These findings suggest that in vivo trans-formation effects by carcinogens persisted during the mammary organ culture. These results were more characteristic in DMBA than in MNU-treated group. Ducts and terminal ducts appeared to have lost morphology during their growths in case of without diethylstilbestrol (DES). The fact that in vitro organ culture without DES was resulted in abnormal ductular morphogenesis confirms that DES is a physiological regulator of ductular epithelial cell growth.

• Clinical and Experimental Reproductive Medicine
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• v.16 no.2
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• pp.131-138
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• 1989

The present study was designed to develop a new method for the determination of estrogen receptor in porcine uterus using frozen sections. Cryostat sections were incubated with $^3H$-estradiol($^3H$-$E_2$) in the presence or absence of diethylstilbestrol(DES) and the radioactivity of 3H-E2 bound to estrogen receptor(ER) was detected. The level of specific estrogen receptor was determined by Scatchard analysis. The highest ratio of specific binding against total binding was achieved in 3 sec. tions(5mm x 5mm) which was corresponded to lOO${\mu}$/ml protein concentration. Optimal binding was obtained during incubation with $^3H$-$E_2$ for 30 minutes at 23$^{\circ}C$ after treatment of sections with acetone for 20 seconds. Three time-washing of sections was proved to be appropriate for the removal of unbound 3H-E2. 200-fold molar excess of DES was substituted for the binding of $^3H$-$E_2$ to ER sufficiently(binding efficiency of 54.8%). ER was saturated with 4nM of $^3H$-$E_2$ and its dissociation constant was 0.1nM. ER assay using frozen sections(Histological radioreceptor assay, HRRA) was significantly correlated with radioreceptor assay for estradiol(RRA, 0.976 , p

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.112-112
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• 2001

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that activates the aryl hydrocarbon receptor (AhR) and disrupts multiple endocrine signaling pathways by enhancing ligand metabolism, altering hormone synthesis, down regulating receptor levels, and interfering with gene transcription. And TCDD-mediated gene transactivation via the AhR has been shown to be dependent upon estrogen receptor (ER) expression in human breast cancer cells. In the present study, we have examined the effect of natural estrogen, phytoestrognes and environmental estrogens on the regulation of CYP1A1 gene expression in MCF-7 human breast cancer cell line. that ER and AhR are co-expressed. pCYP1A1 -luc reporter gene was transiently transfected into MCF-7 cells. These cells were treated with various chemicals and then luciferase assay was carried out. 17be1a-estradiol significantly inhibited TCDD stimulated luciferase activity dose dependently and this inhibition was partially recovered by concomitant treatment of tamoxifen. 17beta-estradiol metabolites, 2-hydroxyestradiol and 16alpha-estriol resulted in less potent inhibitory effect than estradiol and synthetic estrogen, diethylstilbestrol (DES) showed no effect on CYP1A1 gene expression. This study demonstrated that estrogen down-regulated TCDD stimulated CYP1A1 expression via ER mediation. And we have found out that several flavonoids such as genistein, kaempferol, daidzein, naringenin, and alkylphenols such as nonylphenol, 4-octylphenol and resveratrol also inhibited TCDD induced CYP1A1 expression like estrogen.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.1
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• pp.18-23
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• 2011

Objective: Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. This report examined the expression of Prx isotype I in the rat ovary after hormone treatment. Methods: Immature rats were injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) to induce the growth of multiple preovulatory follicles and 10 IU of human chorionic gonadotropin (hCG) to induce ovulation. Immature rats were also treated with diethylstilbestrol (DES), an estrogen analogue, to induce the growth of multiple immature follicles. Northern blot analysis was performed to detect gene expression. Cell-type specific localization of Prx I mRNA were detected by in situ hybridization analysis. Results: During follicle development, ovarian Prx I gene expression was detected in 3-day-old rats and had increased in 21-day-old rats. The levels of Prx I mRNA slightly declined one to two days following treatment with DES. A gradual increase in Prx I gene expression was observed in ovaries obtained from PMSG-treated immature rats. Furthermore, hCG treatment of PMSG-primed rats resulted in a gradual stimulation of Prx I mRNA levels by 24 hours (2.1-fold increase) following treatment, which remained high until 72 hours following treatment. In situ hybridization analysis revealed the expression of the Prx I gene in the granulosa cells of PMSG-primed ovaries and in the corpora lutea of ovaries stimulated with hCG for 72 hours. Conclusion: These results demonstrate the gonadotropin and granulosa cell-specific stimulation of Prx I gene expression, suggesting its role as a local regulator of follicle development.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.1
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• pp.47-55
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• 2003

Objectives: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. Methods: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at $21{\sim}23$ days of age. Ovaries were collected $1{\sim}3$ days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for $1{\sim}2$ days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 IU PMSG. Some mice received a single intraperitoneal injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. Results: Treatment of immature mice with diethylstilbestrol (DES) for $24{\sim}48$ h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for $24{\sim}48$ h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at $6{\sim}9$ h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. Conclusion: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

• Environmental Analysis Health and Toxicology
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• v.20 no.3 s.50
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• pp.195-203
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• 2005

Cytochrome P45O 17$\alpha$-hydroxylase (CYPI 7) and cytorhrome P45O aromata.ie (CYPI 9) are key steroidogenic enzymes in androgen and estrogen synthesis. ThiL study evaluated the effects of nonylphenol (NP) on CYP17 and CYP19 expression in the ovary of Sprague-Dawley rats. All female rats were administered orally with the vehicle (control, corn oil), diethylstilbestrol (DES, 5.0 $\mu$g/kg) and NP (50, 100, or 200 mg/kg/day), which was startinB when they were weaned at 21 days of age for 20 days. Twenty four hours after final dose, the animals were anelthetized with ether. Significant decreases in the uterus (wet weight) were observed with 5.0 $\mu$g/kg/day DES (78$\%$, of control) and 200 mg/kg/day NP (62$\%$ of control), respectively Additionally, ovarian weight was significantly decreased with 5.0 $\mu$g/kg/day DES (63$\%$ of control) and 200 mg/kg/day NP (72$\%$ of control). The serum estradiol levels were sligHtly lower in DES and high dose NP treatment groups, but the 74 levels were not affected by DES and NP. The expression of the ovarian CYP19 gene increased with low doses (50 and 100 mg/kg/day) of NP. while DES and high dose oi NP (200 mg/kg/day) did not affect on the CYP19 mRNA levels. In contrast to the CYP19 gene, the CYP17 gene expreLsion level was significantly down-regulated by the DES and 200 mg/ks/day NP. This result suggestE that NP inhibits ovarian estrogen synthelis by supprelsing CYP17 mRNA efprelsion, And different mechanisml might exist for the expression of Lteroidogenic CYP17 and CYP19 genes in the ovary of Sprague-Dawley rats in response to NP.