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Estrogenic chemicals are widely reported to be present in the environment. Their chlorinated derivatives are considered to be produced through the chlorination process in water purification and sewage treatment plants. In this study, several chlorinated derivatives of estrogens and flavonoids, including phytoestrogens, were synthesized by the reaction with hypochlorous acid, and their estrogenic activities were investigated using a devised GFP expression system in human breast carcinoma MCF7 cells. The chlorinated derivatives were less estrogenic than the parent compounds. The EC(50) ranking of estrogen-related compounds was 17β-estradiol (E2)>4-ClE2>estrone (E1)>4-ClE1>10-Cl-1,4-estradiene-3,17-dione (10-Cl-3,17-dione)>2-ClE2>2-ClE1. 2,4-diClE2, 2,4-diClE1, and 2,4,16,16-tetraClE1 showed lower or no estrogenic activity. Genistein and daidzein are well known as phytoestrogens. 6,8-diCl-genistein, 3',8-diCl-daidzein, (+)-6,8-diCl-naringenin, and 6,8-diCl-apigenin showed lower estrogenic activity than their parent compounds. 3',5',8-triCl-daidzein exhibited no estrogenic activity. No activity was detected in chrysin, (+)-catechin, and their chlorinated derivatives. Similar results were obtained in a cell proliferation assay using MCF7 cells.

Alkylphenol ethoxylates, widely used non-ionic surfactants, are biodegraded into alkylphenols such as nonylphenol (NP) and t-octylphenol (OP), short-chain ethoxylates such as NP-monoethoxylate (NP1EO) and NP-diethoxylate (NP2EO), and alkylphenoxy carboxylic acids such as 4-t-octylphenoxyacetic acid (OP1EC). Bisphenol S (BPS) is more heat-stable and photo-resistant than bisphenol A (BPA), and therefore replaces BPA. These chemicals could be chlorinated during wastewater treatment. We synthesized these compounds and their chlorinated derivatives to estimate their estrogenic activities using a GFP expression system. The EC50 ranking of NP-related compounds was NP > ClNP > diClNP > NP1EO > ClNP1EO > NP2EO. The estrogenic activity of OP1EC was 10 times less potent than parent OP. Furthermore, BPS showed comparable estrogenic activity with BPA. The EC50 ranking of BPS-related compounds was BPA ≥ BPS > triClBPS > diClBPS > ClBPS. Other tested BPS derivatives had no estrogenic activity. Chlorination of the tested chemicals did not enhance their estrogenic activity, in contrast to certain chlorinated BPAs. Thus, our results demonstrated that chlorinated derivatives of NP, OP, and BPS, even if artificially produced during wastewater processing, were less estrogenic than their parent chemicals, known as endocrine disruptors.

Green fluorescent protein (GFP) has been used extensively as a novel non-invasive reporter in the investigation of issues such as promoter function, transformation marker recognition, and sub-cellular localization analysis. A plant-optimized synthetic GFP modified via the replacement of the serine to threonine at position 65 [sGFP(S65T)] has achieved high levels of expression with no toxic side-effects in many plants. It would be useful to develop a non-invasive selection system based only upon GFP expression levels. A trapping vector was constructed using an sGFP(S65T) and a quantitative fluorescence imaging system was used for non-invasive screening of trapped tobacco cultured cells. Putative trapped lines could be identified using this system. Moreover, the GFP products from the candidate trapped lines could be quantitatively visualized directly via SDS-PAGE using the fluorescence imaging system. We conclude that the non-invasive selection system described here could be a powerful new tool for plant biotechnology.

A green fluorescent protein (GFP)-reporter vector regulated by an estrogen response element (ERE) was constructed and transfected into human breast carcinoma MCF7 cells. Stable transfectants were selected and their GFP fluorescence intensity was measured using a quantitative fluorescent imaging system. 17β-estradiol (E(2)) and bisphenol A (BPA) induced a dose-dependent increase in GFP intensity in the cells, reaching maximum response at 5×10(-10) and 10(-5) M, respectively. Using this GFP expression system, we examined the estrogenicity of mono-, di-, tri-, and tetra-chlorinated BPAs, which were detected in wastewater from waste-paper recycling plants using sodium hypochlorite as a bleaching agent. 3-ClBPA and 3,3'-diClBPA showed similar estrogenicities, effective at lower concentrations than parent BPA. On the other hand, the maximum activities of BPA and 3,3',5-triClBPA, whose EC(50) were similar, were higher than other chlorinated BPAs. This is the first demonstration of the estrogenicity of chlorinated BPAs. Since polychlorinated BPAs were not easily biodegraded, chlorinated BPAs might be more severe endocrine disruptors than BPA.

Chlorinated polycyclic aromatic hydrocarbons (ClPAHs), which are a series of halogenated aromatic hydrocarbons, have been found in the environment. The primary step in their metabolic activation seems to be associated with aryl hydrocarbon receptor (AhR)-mediated induction of the cytochrome P450 (CYP) 1 family, although the evidence remains unclear. In this study, we first investigated the effects of five ClPAHs with three to five rings and the corresponding parent PAHs on the expression of CYP1A1 and 1B1 in human breast cancer MCF-7 cells. For the targeted ClPAHs, Western blot analysis of ClPAH-induced CYP1A1 and 1B1 showed an enhancement in activities in comparison with induction by the corresponding parent PAHs, and the effects of chlorination were especially prominent in phenanthrene. In a further study, using 6-chlorobenzo[a]pyrene (6-ClBaP), cotreatment with 17β-estradiol showed an increase in the expression of CYP1B1 mRNA but not CYP1A1 mRNA. Since the AhR ligand has been reported to induce formation of an AhR-estrogen receptor (ER) complex, which stimulates transcription of ER target genes, the effects of ClPAHs in MCF-7 cells transfected with estrogen response elements-regulated green fluorescent protein (GFP) reporter genes were also investigated in this study. 6-ClBaP induced a dose-dependent increase in GFP expression related to ER signaling through AhR activation in the cells, but 3,9,10-trichlorophenanthrene (3,9,10-Cl3ClPhe) did not, despite its ability to activate AhR. Furthermore, we investigated the effect of ClPAHs on the expression of the endogenous ER-responsive genes, cathepsin D, in MCF-7 cells. 6-ClBaP stimulated expression of the ER-responsive genes but 3,9,10-Cl3ClPhe did not, as in the GFP expression system. These results suggest that estrogenic action mediated ER signaling through AhR activation does not necessarily occur for every ligand that can activate AhR. © 2009 Wiley Periodicals, Inc. Environ Toxicol, 2010.

Chlorinated derivatives of estrone (E1) in the effluent of a municipal sewage treatment plant located in Shizuoka prefecture, Japan were detected by gas chromatography/mass spectrometry using electron impact in selected ion monitoring (GC/MS-EI-SIM) analysis. The concentrations of E1, 2-chloroestrone, 4-chloroestrone and 2,4-dichloroestrone in the effluent sample collected in December 2005 were 60.0 ng l −1 , 4.0 ng l −1 , 14.5 ng l −1 , and 9.8 ng l −1 , respectively. In the effluent sample taken in June 2005, 2,4-dichloroestrone was detected at 5.6 ng l −1 along with 17.6 ng l −1 of E1. However, only E1 was detected at 5.9 ng l −1 in the sample in May 2005. To elucidate the behavior of E1 during the disinfection process with sodium hypochlorite in the sewage treatment plant, we carried out a reaction of E1 with sodium hypochlorite in buffer solutions at pH 7 and 9. As E1 was consumed rapidly, chlorinated estrones were produced and relatively fast formation of 2-chloroestrone, 4-chloroestrone, and 2,4-dichloroestrone was observed. Furthermore, 1,4-estradiene-3,17-dione derivatives were formed from the reaction between 2,4-dichloroestrone and sodium hypochlorite.

Bisphenol A (BPA), an estrogenic endocrine disrupting chemical, has been reported to affect embryos and alter their postnatal development. In the present study, we measured the concentrations of BPA in human colostrum by a competitive enzyme-linked immunosorbent assay (ELISA) with the aim of understanding the present status of BPA burden in human breast milk in Shizuoka, Japan. Human colostral samples were collected from 101 healthy mothers within three days after delivery. The BPA concentrations of colostral samples were estimated by ELISA after the acetonitrile extraction and solid phase extraction column purification. BPA in 101 samples was detected in the concentration range of 1-7 ng ml(-1). The mean concentration of BPA was 3.41+/-0.13 (mean+/-SD) ng ml(-1). This is the first demonstration as to what BPA concentrations are in human colostrum. The BPA concentrations in colostrum were higher than those in blood sera samples obtained from healthy women in a previous study. In our study, there was no significant correlation between the concentrations of BPA in colostrum and the age and parity of mothers.

The physiologically active metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (DVD), is a potent inducer of cell differentiation in human myeloid leukemia cells. In the present study, we examined changes in gene expression during DVD-induced cell differentiation of promyelocytic HL-60 cells employing a DNA microarray technique. The results identified 7 up-regulated and 9 down-regulated genes with a change greater than 1.5-fold after the DVD-treatment for both 2 and 6 days. Seven of these genes were further examined by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that findings obtained from the DNA microarray analysis and RT-PCR are generally comparable with each other. Gene expression of the subunits of eukaryotic translation initiation factor 2 was then examined by methods including RT-PCR and real-time PCR. The results indicated the suppression of these genes, suggesting a linkage to differentiation-associated growth inhibition of these cells.

Dou-chi, a traditional soybean food fermented with Aspergillus sp., is usually used as a seasoning in Chinese food, and has also been used as a folk medicine in China and Taiwan. As 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavengers, four phenol compounds, one isoflavanone, eight isoflavones and one 4-pyrone have been isolated from dou-chi. Among these fourteen compounds, 3'-hydroxydaidzein, dihydrodaidzein and a 4-pyrone compound have not yet been isolated from soybean miso. The structure of the novel 4-pyrone compound, 3-((E)-2-carboxyethenyl)-5-(4-hydroxyphenyl)-4-pyrone-2-carboxylic acid was elucidated by using the same compound as that obtained from the biotransformation of daidzein. 3'-Hydroxydaidzein showed as high DPPH radical-scavenging activity as that of alpha-tocopherol, and 6-hydroxydaidzein had mushroom tyrosinase inhibitory activity with an IC(50) value of 10 muM. The order of estrogenic activity is as follows: genisteindaidzein3'-hydroxydaidzein8-hydroxygenistein, using a green fluorescent protein expression system. Furthermore, the contents of isoflavones in the fermentation process of dou-chi were measured.

Tea catechins exert many biological effects, including anticancer and antibacterial activities. Also, it is reported that some plant flavonoids exhibit estrogenic activity. In this study, we investigated estrogenic or antiestrogenic activities of catechins in HeLa cells transiently transfected with an estrogen response element (ERE)-regulated luciferase reporter and an estrogen receptor (ER) alpha or ERbeta expression vector. Catechins alone did not induce luciferase (luc) activity in either of the ERs. Addition of 17beta-estradiol (E2) plus epicatechin gallate (ECG) or epigallocatechin gallate (EGCG) at 5 x 10(-6) M resulted in significant decreases in the ERalpha-mediated luc activity compared with that of E2 alone. On the contrary, lower concentrations significantly increased the E2-induced luc activity. Similar effects were observed with tamoxifen. The ERbeta-mediated estrogenic activities were stimulated by catechins. In conclusion, some catechins, particularly EGCG, were antiestrogenic for ERalpha at higher doses, and co-estrogenic for ERalpha at lower doses and for ERbeta. The lower doses were found in human plasma after tea-drinking. In addition, some catechins may be antiendocrine disruptors because they suppressed bisphenol A-induced luc activities.

Transcriptional regulation of the gene for the myeloid calcium binding protein, MRP14, was investigated in human monocytic leukemia cell lines. The MRP14 gene was not expressed in monoblastic ML-1 cells, promonocytic U-937 cells, or promyelocytic HL-60 cells. On the other hand, the gene was expressed in monocytic THP-1 cells and in the HL-60 cells treated with 1,25-dihydroxyvitamin D3 (VD3). The level of MRP14 in VD3-treated HL-60 cells was two-fold higher than that in THP-1 cells. Among several known transcription factor binding motifs, nuclear protein(s) of VD3-treated HL-60 cells and THP-1 cells bound to the CCAAT/enhancer binding protein (C/EBP)-binding motif that was located in the upstream region of the MRP14 gene (-81), as evidenced by the competitive gel mobility-shift assay. An antibody for C/EBP alpha super-shifted the nucleoprotein complex in THP-1 cells but not in the VD3-treated HL-60 cells, whereas an antibody for C/EBP beta blocked the formation of the complex with the nuclear factor of the HL-60 cells but not with that of THP-1 cells. An anti-C/EBP delta antibody had no effect on the complex in either cell. Thus, it was concluded that C/EBP alpha and -beta were able to bind to the C/EBP motif, and that C/EBP alpha bound to the motif in THP-1 cells and C/EBP beta bound to that in the VD3-treated HL-60 cells. Furthermore, to examine the transcriptional activity of the C/EBP motif, we transfected several constructed luciferase reporter DNAs into HL-60 cells and THP-1 cells. The luciferase activity of the C/EBP motif in HL-60 cells was increased by VD3 treatment. The C/EBP motif in the MRP14 gene was confirmed to function as a regulatory region in VD3-treated HL-60 cells and THP-1 cells by the assay. Since C/EBP beta was also detected in VD3-untreated HL-60 cells by immunoblotting, VD3 activated C/EBP beta to bind to the motif, probably through post-translational modification.