* Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361763 Republic of Korea; Department of Obstetrics and Gynecology, British Columbia Children's and Women's Hospital, British Columbia Research Institute for Children's and Women's Health, University of British Columbia, Vancouver, BC, V6H 3V5 Canada
2 To whom correspondence should be addressed at Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361763, Republic of Korea. Fax: +82-43-267-3150. E-mail: ebjeung{at}chungbuk.ac.kr
Received September 27, 2004; accepted October 20, 2004
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ABSTRACT |
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Key Words: calbindin-D9k; endocrine disruptor; estradiol; progesterone.
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INTRODUCTION |
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Recent research has demonstrated that environmental chemicals caused endocrine disruption through agonistic or antagonistic effects on steroid hormone receptors (Sumida et al., 2001). Endocrine disruptors (EDs) have been proposed to bind to one of the nuclear estrogen receptors (ERs), and xenoestrogens and phytoestrogens, except for genistein, showed an equal binding affinity to both ER subtypes; nevertheless it is unclear that this is their major or only mode of actions (Gutendorf and Westendorf, 2001
). Examples of suspected environmental estrogenic chemicals include polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT), diethylstilbestrol (DES), 4-tert-octylphenol (OP), 4-nonylphenol (NP), and bisphenol A (BPA) (An et al., 2003a
; Sumida et al., 2001
). In addition, these chemicals have been demonstrated to exhibit similar affinities to steroid receptors, with the inhibition of receptor binding affinity concentrations of 0.7 µM for ER and 1.23.8 µM for PR, respectively (Laws et al., 2000
). Alkylphenols, such as OP and NP, are derived from alkylphenol ethoxylates. Bisphenol is a monomer on polycarbonate plastics and a constituent of the epoxy and polystyrene resins used extensively in food packaging and dental sealants (Jorgensen et al., 2000
; Witorsch, 2002
). The mechanism of action of these compounds has not been established in all cases, but the resulting effect is consistent with ER-mediated action (Arnold et al., 1996
; Nagel et al., 1997
; White et al., 1994
). The effects of NP and OP are due to their direct interaction with ER (Odum et al., 2001
; Routledge and Sumpter, 1997
). The differences in the biological potency and binding affinity of OP, NP, and BPA to ER have been demonstrated in a recent study (Jorgensen et al., 2000
). The ER can bind to a variety of nonsteroidal compounds that are structurally similar to the alkyl-substituted phenol of E2 (Hu and Aizawa, 2003
). Alkylphenolic compounds exhibit a weak ER binding affinity, 1/1,000 to 1/1,000,000fold lower than endogenous E2, and they appear to be weak agonists for PR (Witorsch, 2002
). Previous studies demonstrated that a significant increase in CaBP-9k mRNA and protein was observed in the uterus when immature rats were treated with OP, NP, or BPA (An et al., 2002
; 2003a
), and in the fetal uterus following maternal injection of rats (Hong et al., 2003
).
A pure antagonist for ER and ERß, ICI 182,780, blocked the effect of E2 and BPA (Papaconstantinou et al., 2001
), and controlled ER and ER-regulated responses in the uterus (Cowley et al., 1997
; Das et al., 1998
). ICI 182,780 downregulated ER and antagonized E2-induced increases in uterine PR. In addition, ICI 182,780 antagonized E2-induced and BPA-induced effects on the localization of heat shock protein 90
in vitro (Papaconstantinou et al., 2001
). RU486 has a high affinity to PR and glucocorticoid receptor (Mahajan and London, 1997
). After RU486 binds to target receptors, receptor interaction with heat-shock protein 90 and p53 is strengthened, but there is no interaction with DNA at the hormone response element (Mahajan and London, 1997
). The phenyl-aminodimethyl group at the 11-ß and 17-
position of the steroidal skeleton and the carboxyl side chain of RU486 correlated with an antagonist activity of progesterone (Leonhardt and Edwards, 2002
). In other words, RU486, an 11-ß substituted nor-steroid containing a 17-
propylnyl group, is used as an antiprogestin agent (He et al., 1999
). RU486, an antiprogesterone and antiglucocorticoid, antagonizes not only PR-mediated transactivation but also ER transactivation via PR inhibition (Papaconstantinou et al., 2001
).
Because EDs were expected to have the properties of both estrogenic and weak progestogenic activities, the effect of EDs in the induction of CaBP-9k expression has not been elucidated yet in a mouse in vivo model. Thus, we investigated the effect of EDs on the expression of CaBP-9k mRNA and protein compared to that of endogenous E2 and P4 in the uterus of immature mice. To specify an estrogenicity or progestogenicity of EDs, both RU486, a PR antagonist, and ICI 182,780, an ER antagonist, were employed. This study describes a novel in vivo model of immature mice used for the first time to detect estrogenicity and progestogenicity.
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MATERIALS AND METHODS |
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To investigate the effective dose of EDs in the uterus, the immature mice were treated with increasing doses of OP, NP, and BPA in a dose-dependent manner (Fig. 2). In the first experiment, nine groups of five animals (14 days old) were injected subcutaneously (s.c., 0.1 ml per mouse) with OP, NP, or BPA at three different doses: 100, 250, and 500 mg/kg body weight (BW) per day. These EDs were administered in a corn oil vehicle, once per day for 3 days. The positive groups of mice (5 of each treatment) were given s.c. injections with E2 (10 µg/kg), P4 (200 mg/kg), E2 plus P4, or corn oil alone as a vehicle. All mice were euthanized at 24 h after the final injection. To examine the expression level of CaBP-9k, the immature mice were treated with EDs for 3 days and euthanized in a time-dependent fashion (Fig. 2). In the second experiment, four groups of 30 animals (14 days old) were injected s.c. with corn oil, OP, NP, or BPA (500 mg/kg BW) daily for 3 days. Five animals from each group were euthanized at 3, 6, 12, 24, 48, and 72 h after the final injection. In addition, to specify steroid hormonelike activity of EDs, the third experiment was performed as shown in Figure 2. After treatment with RU486 (300 mg/kg BW) or ICI 182,780 (10 mg/kg BW) 1 h before injection, 10 groups of five animals (14 days old) were injected s.c. with E2 (10 µg/kg), P4 (200 mg/kg), OP, NP, or BPA (500 mg/kg BW) daily for 3 days. The mice were euthanized 24 h after final injection.
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Real-time polymerase chain reaction (PCR). The standard curve was generated for a standard RNA preparation by serial dilution (1, 1/10, 1/100, 1/1,000, 0). The Real-Time PCR reaction was carried out in a 25 µl final volume containing 5 µl of 5x Taq DNA polymerase (TaKaRa Bio Inc.), 2.5 µl of diluted (1:30,000) SYBR Green (TaKaRa Bio Inc.), 0.5 µl of each of forward and reverse primers, 2 µl of cDNA, and distilled water up to 25 µl. The oligonucleotide sequences of primers employed to detect mouse CaBP-9k mRNA, were 5'-GCA AAA TGT GTG CTG AGA A-3' (sense) and 5'-GGA ACT CCT TCT TCC TGA CT-3' (antisense). Primers for the 1A gene were: 5'-GAT ATA GCA TTC CCA CGA ATA-3' (sense) and 5'-GGG CTT TTG CTC ATG TGT CAT-3' (antisense). Polymerase chain reaction amplification using the Smart Cycle System (TaKaRa Bio Inc.) began with an initial denaturation at 95°C for 30 s. Each of the 35 amplification cycles consisted of denaturation at 95°C for 5 s, annealing at 55°C for 7 s, and extension at 72°C for 12 s. Relative expression levels of each sample were calculated based on the Cycle threshold (Ct) and monitored for amplification curve. The PCR amplification curves were evaluated by fluorescence of the double-stranded DNA-specific dye, SYBR Green, versus the amount of standardized PCR product. Expression of CaBP-9k was normalized to IA mRNA.
Western blot analysis. To measure the expression level of CaBP-9k protein by EDs, we performed Western blot analysis as described previously (Hong et al., 2003). Briefly, immature female mice were euthanized, the uteri were isolated, and protein was extracted with Pro-prep (Intron Co, Seoul, South Korea) according to the manufacturer's instructions. The amount of protein was determined using a Bradford assay (Bio-Rad Laboratories) (Bradford, 1976
). Cytosolic protein (40 µg) was separated by 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Rockville, MD) with a Semi-dry Transfer Cell (Bio-Rad Laboratories) according to the manufacturer's protocol. Membranes were incubated in 4% (w/v) skim milk dissolved in PBS-T buffer (137 mM NaCl, 2 mM KCl, 10 mM phosphate buffer and 5% (v/v) Tween-20) for 4 h at room temperature to block nonspecific binding. Membranes were then incubated with a rabbit polyclonal antibody (Swant, Switzerland, 1:3,000) specific for mouse CaBP-9k, and washed in PBS-T buffer. Incubation with the secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase diluted 1:3,000 in PBS-T containing 3% (w/v) bovine serum albumin) was for 1 h at room temperature. Protein bands were visualized with the ECL chemiluminescent system (Amersham Pharmacia Biotech) and autoradiography. The expression of CaBP-9k was quantitated by densitometry (NIH image beta 3).
Data analysis. Data are presented as the mean ± S.D. Data were analyzed by the nonparametric Kruskal-Wallis test, followed by Dunnett's test for five-pair comparisons. Each value of Dunnett's test was converted to rank for statistical analysis. All statistical analyses were performed with the Statistical Analysis System (SAS Institute, Cary, NC); p < 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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In the rat uterus, CaBP-9k expression is completely repressed during diestrus when E2 concentration is low, and increased at proestrus in response to the rise in plasma E2 (Krisinger et al., 1992; L'Horset et al., 1994
). Treatment of rats with E2 significantly increased CaBP-9k mRNA and protein in the uterus (An et al., 2002
; 2003a
; Elger et al., 2000
), indicating that E2 is an important factor in the regulation of CaBP-9k in the uterus of rats. It is interesting that the regulation of CaBP-9k by P4 has been demonstrated in the mouse uterus, but its regulation by E2 has not (Nie et al., 2000
). It has been shown that E2 treatment alone did not affect CaBP-9k mRNA expression in the uteri of ovariectomized mice (Nie et al., 2000
; Tatsumi et al., 1999
). This difference between E2 and P4 suggests that distinct mechanisms exist in the regulation of the CaBP-9k gene during the estrous cycles of mice and rats (Nie et al., 2000
). Taken together, these previous findings indicate that P4 is a major factor in the regulation of CaBP-9k gene during the estrous cycle in mice, and CaBP-9k is mainly regulated by P4, not E2, in the uterus of mice (Tatsumi et al., 1999
). In the present study, it is not surprising that treatment with P4 only, but not E2, resulted in the induction of CaBP-9k mRNA and protein and a combination of E2 and P4 enhanced P4-induced CaBP-9k expression in the uterus of immature mice, suggesting that P4 is a dominant factor in the regulation of CaBP-9k and that E2 may enhance P4 effect on its gene expression through activation of PR.
There is increasing evidence that man-made chemicals in the environment may interfere with the human body's complex and carefully regulated hormonal system (Crisp et al., 1998). These synthetic chemicals can disrupt the endocrine system in diverse ways; for example, they not only mimic or block endogenous hormones but also alter hormonal levels, affecting the functions of various tissues (Crisp et al., 1998
; Gaido et al., 1997
). Examples of suspected environmental estrogenic chemicals include polychlorinated hydroxybiphenyls, DDT, kepine, methoxychlor, and BPA. Alkylphenolic compounds are environmentally persistent and have an estrogenic activity (Laws et al., 2000
; Sumpter and Jobling, 1993
; White et al., 1994
). In our previous studies, we elucidated the effect of estrogenic compounds, OP, NP, and BPA on the expression of CaBP-9k mRNA and protein as a biomarker in the uterus of immature rats (An et al., 2002
; 2003a
). EDs can replace endogenous hormones in the uterus, so a high dose of EDs resulted in the induction of CaBP-9k mRNA and protein. Using an immature rat model, we demonstrated for the first time that maternally injected estrogenic compounds (OP, NP, and BPA) caused an increased CaBP-9k mRNA and/or protein in the maternal tissues (uterus and placenta) and the fetal uterus during late pregnancy, suggesting that for fetal health, the placenta may not be a reliable barrier against some estrogenic compounds in a rat model (Hong et al., 2003
, 2004a
). In addition, maternally injected estrogenic compounds may be transferred to neonates through breast milk, thus affecting uterine function, as shown by the induction of CaBP-9k gene expression in the neonatal uterus (Hong et al., 2004b
). Estrogenic compounds exhibit affinities to ER and PR and bind to both steroid receptors in vitro binding assay (Laws et al., 2000
). In our previous studies, estrogenic induction of uterine CaBP-9k mRNA and protein by environmental phenol products, OP, NP, and BPA were analyzed using an immature rat model; however, no model system was available to detect a progestogenic effect of EDs on the induction of CaBP-9k mRNA and protein (An et al., 2003a
; Hong et al., 2003
). An immature mouse model is an excellent system because CaBP-9k expression in the uterus is regulated in a P4-dependent manner (Nie et al., 2000
). The present study demonstrated that estrogenic chemicals resulted in the induction of CaBP-9k mRNA and protein in a dose-dependent and time-dependent manner as only P4 did. These results indicate that these estrogenic compounds OP, NP, and BPA have a progestogenic activity in the uterus of immature mice, implicating the risk assessment of these chemicals.
It is of interest that RU486 significantly reversed EDs-induced CaBP-9k mRNA expression, in part, indicating that EDs may have a specific progestogenic effect through PR in this tissue. On the other hand, treatment of immature mice with ICI 182,780 completely blocked OP-, NP-, and BPA-induced CaBP-9k expression in the uterus compared to the vehicle level. In addition, the effect of P4 on the expression of CaBP-9k was completely blocked by ICI 182,780, although E2 itself did not have any effect on the expression of CaBP-9k. These results suggest that ICI 182,780 may block both P4-induced CaBP-9k mRNA and induction of CaBP-9k mRNA itself in the uterus of immature mice. RU486 (Mifepristone) is an 11-beta-dimethyl-amino-phenyl derivative of norethindrone with a high affinity to PR and glucocorticoid receptors. Because RU486 has a higher binding affinity to PR than does P4, this antagonist effectively blocks the action of P4 (Koide, 1998). ICI 182,780 binds to ER with high affinity and completely inhibits the effects of E2 on the growth of rat uterus, on the growth of MCF-7 cells in vitro, and on E2-stimulated breast tumor and endometrial tumor growth in the nude mouse (Koide, 1998
; Wakeling and Bowler, 1988a
, 1988b
; Wakeling et al., 1991
). In addition, ICI 182,780 alone has no estrogenic activity in the induction of CaBP-9k gene expression (Blin et al., 1995
). In the present study, ED-induced CaBP-9k mRNA and protein was reversed in part or completely by treatments with RU486, a P4 antagonist, or ICI 182,780, an E2 antagonist, suggesting that these synthetic chemicals may have both progestogenic and estrogenic properties. The previous work showed that treatment of pregnant mice with RU486 decreased uterine CaBP-9k mRNA expression, suggesting that P4 upregulated CaBP-9k gene in this tissue (An et al., 2003b
). Taken together, these results demonstrated a novel finding that OP, NP, and BPA may have progestogenic activity in the uterus of immature mice and that the expression of CaBP-9k mRNA may be completely blocked by ICI 182,780 in this tissue. However, it is not clear how ICI 182,780 blocks EDs-induced CaBP-9k mRNA expression in this tissue because ICI 182,780 is a complete antagonist of ER, which warrants further study.
In conclusion, treatment of immature mice with OP-, NP-, and BPA-induced CaBP-9k mRNA and protein in a dose-dependent and time-dependent manner in the uterus of immature mice. Treatment of the mice with ICI 182,780 and EDs or RU486 and EDs reversed EDs-induced CaBP-9k mRNA and protein levels in this tissue, suggesting that EDs may have both progestogenic and estrogenic properties through PR and ER in this in vivo model. These results validate a novel in vivo model of immature mice for the first time to detect activities of estrogenic and progestogenic EDs through the induction of uterine CaBP-9k mRNA and protein.
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NOTES |
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ACKNOWLEDGMENTS |
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