• Toxicological Research
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• v.18 no.1
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• pp.59-64
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• 2002

Substantial evidences have been accumulated about the hormone-like effects of exogenous substances such as pesticides and industrial chemicals during past years. The effects of these substances on the endocrine system are believed to be either enhancing or reducing of various endocrine action. It is necessary to identify putative causal agents by the batter system and to assess their ability to disrupt the endocrine system. A variety of in vitro and In vivo approaches have been used to determine the androgenic effects of environmental chemicals. To establish the method for assessment of the putative endocrine disruptors with androgenic activity, we carried out the cell proliferation assay by MTS method after treatment with the various concentration of testosterone in LNCaP cells (human prostatic cancer cell line) and also observed the expression of androgen-related genes by quantitative RT-PCR. In the cell proliferation assay, the results showed that the grouth of LNCaP cells increased within level of at least 10pM testosterone. We measured by quantitative RT-PCR method on the effects of testosterone on mRNA expression of androgen receptor (AR), prostate-specific antigen (PSA), bone morphogenetic protein (BMP) and BMP receptor (BMPR) In LNCaP cells. The results demonstrated that mRNA expression of PSA and BMPR-IB was observed differently within level of at least 0.01 pM testosterone compared with non-treated control. These observations suggest that the detection of PSA and BMPR-IB mRNA by the quantitative RT-PCR in LNCaP cells is very sensitive method to identify the endocrine disruptors to have the androgenic effects.

• Journal of Korean Society of Water and Wastewater
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• v.20 no.6
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• pp.893-904
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• 2006

To improve sensitivity of biosensor as yeast two-hybrid detection system for estrogenic activity of suspected chemicals, we tested effects of several combinations of the bait and fish components in the two-hybrid system on Saccharomyces cerevisiae inducted a chromosome-integrated lacZ reporter gene that was under the control of CYC1 promoter and the upstream Gal4p-binding element $UAS_{GAL}$. The bait components that were fused with the Gal4p DNA binding domain are full-length human estrogen receptor ${\alpha}$ and its ligand-binding domain. The fish components that were fused with the Gal4p transcriptional activation domain were nuclear receptor-binding domains of co-activators SRC1 and TIF2. We found that the combination of the full-length human estrogen receptor ${\alpha}$ with the nuclear receptor-binding domain of co-activator SRC1 was most effective for the estrogen-dependent induction of reporter activity among the two-hybrid systems so far reported. The relative strength of transcriptional activation by representative natural and xenobiotic chemicals was well correlated with their estrogenic potency that had been reported with other assay systems.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1563-1570
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• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• Environmental Mutagens and Carcinogens
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• v.22 no.3
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• pp.175-182
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• 2002

Endocrine disruptors (EDs) are the chemicals that affect endocrine systems through activation or inhibition of steroid hormone response. It is necessary to have a good system to evaluate rapidly and accurately endocrine-disrupting activities of suspected chemicals and their degradation products. The key targets of EDs are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We constructed a co-expression system of Gal4p DNA binding domain (DBD)- ligand binding domain of human estrogen receptor $\alpha$ or $\beta$, and Gal4p transactivation domain (TAD)-co-activator AIB-1, SRC-1 or TIF-2 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of CYC1 promoter and Gal4p binding site (GAL4 upstream activating sequence, GAL4$_{UAS}$). Expression of this reporter gene was dependent on the presence of estrogen or EDs in the culture medium. We found that the two-hybrid system with combination of the hER$\beta$ LBD and co-activator SRC-1 was most effective in the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by those chemicals correlated with their estrogenic activities measured by other assay systems, indicating that this assay system is efficient and reliable for measuring estrogenic activity. The data in this research demonstrated that the yeast detection system using steroid hormone receptor and co-activator is a useful tool for identifying chemicals that interact with steroid receptors.s.

• Development and Reproduction
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• v.13 no.4
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• pp.217-226
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• 2009

Effects of sera from several fish species on monolayer formation, viability and functions of catfish hepatocytes were investigated to establish a primary hepatocyte culture system for screening endocrine disruptors. Hepatocytes of Korean catfish (Silurus asotus) were attached and formed monolayer using the media supplemented with their own serum or sera from eel and tilapia, but not with fetal bovine serum (FBS). The amount of fish sera (0.5~3%) for monolayer culture of the catfish hepatocytes was less than 1/10 of FBS (5~20%) that is commonly used for primary culture of hepatocytes of other species. The results indicate that FBS can be replaced with sera from some fish species and the fish sera are more effective than FBS in maintaining the shape and functions of the hepatocytes. The primary culture of catfish hepatocytes was maintained monolayer with fish sera for at least 10 days, which makes possible to be used for screening the activities of endocrine disruptors. In conclusion, the primary culture system of hepatocytes with fish sera in the present study could be a useful tool for screening and studying endocrine disruptors.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2002.10a
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• pp.119-121
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• 2002

인류는 1,500 만 종류에 달하는 화학물질을 합성 혹은 분리ㆍ동정 해왔다. 그리고 현재 약 10 만 종류 정도의 화학물질이 상업적으로 이용되고 있다. 이들 화학물질 중에 유기염소화합물, 공업용화합물, 농약류, 유기취소(bromine)화합물, 중금속 및 유기금속, 식물 및 합성에스트로겐 등을 포함한 수십 종의 화합물이 내분비계장애물질 (Endocrine Disruptors, EDs)로서 생각, 혹은 의심되고 있다. 그러나, 대부분의 다른 화합물에 대해서는 그것들이 내분비계장애 작용을 가지고 있는지에 대한 충분한 지견이 얻어지지 않고 있어 이들 화학물질의 내분비계장애 작용을 평가하기 위해서는 신속 또는 간편한 검출계의 확립은 매우 중요하다.

• Toxicological Research
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• v.21 no.4
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• pp.333-338
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• 2005

Many of thyroid hormones disrupting chemicals induce effects via interaction with thyroid hormone and retinoic acid receptors and responsive elements intrinsic in target cells. We studied thyroid hormones disrupting effects of food additives and contaminants including BHA, BHT, ethoxyquin, propionic acid, sorbic acid, benzoic acid, CPM, aflatoxin B1, cadmium chloride, genistein, TCDD, PCBs and TDBE in recombinant HeLa cells containing plasmid construct for thyroxin responsive elements. The limit of response of the recombinant cells to T3 and T4 was $1\times10^{-12}\;M$. BHA. genistein, cadmium and TBDE were interacted with thyroid receptors with dose-responsive pattern. In addition, BHA, BHT, ethoxyquin, propionic acid, benzoic acid, sorbic acid, and TBDE showed synergism while cadmium chloride antagonism for T3-induced activity. This study elucidates that recombinant HeLa cell is sensitive and high-throughput system for the detection of chemicals that induce thyroid hormonal disruption via thyroid hormone receptors and responsive elements. Also this study raised suspect of BHA. BHT, ethoxyquin, propionic acid, benzoic acid, sorbic acid, TBDE, genisteine and cadmium chloride as thyroid hormonal system disruptors.

• Applied Biological Chemistry
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• v.49 no.3
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• pp.254-258
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• 2006

Vitellogenin has been known as a potent biomarker protein for the estrogenic activity in fish exposed to endocrine disruptors. In this study, a piezoelectric immunosensor making use of an anticarp vitellogenin antibody and an AT-cut quartz crystal microbalance as the biological component and transducer was prepared, followed by its application to the analysis of carp vitellogenin as follows. Antibody immobilization was conducted by chemisorption of a thiolated antibody with a heterobifunctional thiolation cross-linker, sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate. The reaction buffer for the immunosensor system was optimized as 0.1 M sodium phosphate (pH 7.4). Concentration-dependent sensor responses were obtained in the vitellogenin concentrations ranging from 0.4864 to 486.4000 nM, with a linear correlation between vitellogenin concentration and frequency shift in double-logarithmic scale. The limit of detection of the immunosensor for carp vitellogenin was presumed as 0.4864 nM.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.378-384
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• 2012

In aquatic invertebrates, particularly marine gastropods, organotin compounds induce irreversible sexual abnormality in females, which is termed imposex, at very low concentrations. Organotin compounds are agonists for nuclear receptors such as RXRs and $PPAR{\gamma}$. However, the imposex phenomenon has not been reported to act as an antagonist on estrogen receptors in other species, including vertebrates and invertebrates. In order to gain insights into the antagonistic activity of organotin compounds on estrogen receptors (ERs), we examined the inhibitive effect of these compounds on estradiol-dependent ${\beta}$-galactosidase activity using the yeast two-hybrid detection system consisting of a combination of the human estrogen receptor ($hER{\beta}$) ligand-binding domain and the co-activator steroid receptor co-activator-1 (SRC1). Tributyltin-hydroxide (TBT-OH) and triphenyltin-chlorine (TPT-Cl) exhibited an inhibitive effect on $E_2$-dependent transcriptional activity, similar to antagonistic chemicals such as 4-hydroxytamoxifen (OHT) or ICI 182,780, at a very low concentration of $10^{-14}$ M TBT or $10^{-10}$ M TPT, respectively. The yeast growth and transcriptional activity with transcriptional factor GAL4 did not exhibit any effect at the tested concentration of TBT or TPT. Moreover, the yeast two-hybrid system using the interaction between p53 and the T antigen of SV40 large did not describe any effect at the tested concentration of OHT or ICI 182,780. However, the interaction between p53 and T antigen was inhibited at a TBT or TPT concentration of $10^{-9}$ M, respectively. These results indicate that TBT and TPT act as inhibitors of ER-dependent reporter gene transcriptional activation and of the interaction between $hER{\beta}$ LBD and the co-activator SRC1 in the yeast two-hybrid system. Consequently, our data could partly explain the occurrence of organotin compound-induced imposex on the endocrine system of mammals, including humans.

• Journal of Environmental Health Sciences
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• v.36 no.6
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• pp.510-517
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• 2010

Phthalates, such as di (2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP) have been proved to be teratogenics and endocrine disruptors, metabolized rapidly and excreted in the urine. In this study, a simultaneous analytical method for 10 phthalate metabolites, MnBP, MiBP, MBzP, MCHP, MEHP, MEHHP, MEOHP, MnOP, MiNP and MiDP, in human urines, based on switching system with on-line pretreatment column using HPLC-MS/MS has been developed. This method was validated according to the guideline of bioanalytical method validation of National Institute of Toxicological Research. Limits of detection range between 0.2 and 0.9 ng/ml for 10 phthalate metabolites. The calibration curves showed linearity in the range 0.997~0.999, and the results of the intra- and inter-day validations were in the range from 0.4 to 14.7% RSD and from 0.3 to 9.4% RSD, respectively. Recoveries of phthalate metabolites varied from 87.0 to 116.1%. This analytical method showed high accuracy and stable precision for all metabolites, and seems to be suitable for biomonitoring of phthalates in human urine.