* Department of Endocrine, Reproductive and Developmental Toxicology, CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 27709; and
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843
Received March 30, 2001; accepted June 11, 2001
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ABSTRACT |
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Key Words: methoxychlor; HPTE; estrogen; estrogen receptor (ER
); estrogen receptor ß (ERß); androgen receptor (AR); microarray; gene expression.
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INTRODUCTION |
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MXC is metabolized in vivo into mono- and bisphenol demethylated derivatives; however, the bisphenol metabolite, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), is believed to be responsible for the in vivo estrogenic action of MXC (Bulger et al., 1978; Kupfer and Bulger, 1987
). HPTE is uterotropic in a 3-day bioassay (Shelby et al., 1996
) and stimulates proliferation of uterine leiomyoma cells in vitro (Hodges et al., 2000
). We have shown that HPTE binds directly to the ER and AR in vitro (Gaido et al.1999
); however, the transcriptional effect of this binding is receptor-specific. HPTE is an ER
agonist and ERß and androgen receptor (AR) antagonist (Gaido et al., 2000
). The physiological effects of MXC treatment in vivo may be due to actions mediated by HPTE binding to ER
, ERß, or AR. To date, only genes that are regulated similarly by E2 and HPTE, such as gonadotropin-releasing hormone (Roy et al., 1999
) and uterine insulin-like growth factor 1 (Klotz et al., 2000
), have been identified.
In this study, we utilized the unique properties of MXC as a model system to identify differential gene expression profiles that may relate to its receptor-specific activity. HPTE was used instead of MXC to eliminate mixtures of compounds and metabolites. The dose of E2 was selected to induce a slightly stronger uterotropic response to that seen with a uterotropic dose of HPTE, based upon a previous study (Shelby et al., 1996). Sexually immature female mice were treated with either E2 (50 µg/kg) or HPTE (500 mg/kg) for 3 days, uterine and ovarian tissues were obtained, and extracted mRNA from these tissues was analyzed by microarray and real-time PCR for changes in gene expression. A combined dose of E2 and HPTE (E2 + HPTE) was administered to examine HPTE antagonism of ERß-mediated gene regulation by E2, and a flutamide (FLU) treatment group (45 mg/kg) was included to identify genes regulated through AR antagonism. The unique and overlapping patterns of gene expression produced by E2 or HPTE treatment were linked to the reproductive and developmental effects of E2 and MXC observed in treated rodents.
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MATERIALS AND METHODS |
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Animals.
This study was approved by the Institutional Animal Care and Use Committee at CIIT, and the CIIT animal-care unit is accredited by the American Association for Accreditation of Laboratory Animal Care, with humidity- and temperature-controlled rooms, maintained on a 12-h light-dark cycle. All procedures followed the criteria of the National Research Council for the care and use of laboratory animals in research. C57BL6/J mice were obtained from Taconic (Germantown, NY) at postnatal day 21 (weaning). Upon arrival, animals were assigned to treatment groups by randomization of body weights. Rodent diet NIH-07 and reverse-osmosis water was provided ad libitum, and animals were acclimatized for at least 48 h prior to dosing.
Study design.
Sexually immature female mice received daily subcutaneous injections of either vehicle (corn oil), E2 (50 µg/kg), HPTE (500 mg/kg), E2 + HPTE, or FLU (45 mg/kg) for 3 days. Three animals were included in each treatment group. On the morning of day 4, animals were euthanized by cervical dislocation and target tissues were harvested. Body weights were measured, as well as wet weights for uterus and ovaries. Tissues were submerged in the RNA preservative reagent RNA later (Ambion, Austin, TX), kept at 4°C overnight, and then stored at 80°C until processed for RNA isolation.
cDNA array analysis.
Total RNA from mouse tissues was isolated using the Totally-RNA kit and subsequently DNase-treated with RNase-free DNase, both reagents from Ambion (Austin, TX). Reverse-transcription (RT) reactions were performed with 5 µg of total RNA, (P-32)-dATP, and Superscript-II MMLV reverse transcriptase (Life Technologies, Gaithersburg, MD) for 30 min at 50°C. Following purification, probes were added to each Clontech (Palo Alto, CA) Atlas Mouse cDNA expression array (588 genes) or stress/tox array membrane (140 genes), with hybridization and washing performed according to manufacturer's instructions. Membranes were exposed to film for 13 days to acquire optimum images for analysis with Clontech Atlas Image software (Palo Alto, CA) and pairwise comparisons of log2 transformed raw data. Array analysis was performed on one animal per treatment group for both the expression membrane and the stress/tox membrane.
Real-time RT-PCR.
Changes in gene expression were confirmed and quantified using real time RT-PCR on the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Total RNA (2.5 µg) from each animal tissue was aliquoted in quadruplicate for RT using the TaqMan Reverse Transcription Reagents (Applied Biosystems) and SuperScript-II reverse transcriptase (Life Technologies), with one aliquot designated to receive no enzyme, according to manufacturer's instructions. PCR was performed using SYBR Green PCR Core Reagents (Applied Biosystems) according to manufacturer's instructions, with one modification: reaction volume was adjusted to 25 µl. Primer sets were selected by the Primer Express software package, production of a single PCR product was confirmed using gel electrophoresis, and primer efficiency was determined according to manufacturer's recommended protocol (Applied Biosystems). Sequences of gene primer sets are given in Table 1. RT-PCR analysis was performed on all 3 animals in each treatment group, in triplicate. Quality of RT reactions was confirmed by comparison of triplicate RT versus no enzyme control for each RNA sample, using the GAPDH primer set according to manufacturer's protocols (Applied Biosystems).
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RESULTS |
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Insulin-like growth factor 1A and insulin-like growth factor-binding protein 5 genes are downregulated in the uterus but not in the ovary.
In the uterus, insulin-like growth factor 1A (IGF1A) expression was reduced 65 and 64% by E2 or HPTE, respectively (Fig. 4A). Likewise, insulin-like growth factor-binding protein 5 expression was downregulated 81 and 51% by E2 or HPTE, respectively. E2 + HPTE treatment showed the same pattern for regulation of IGF1A and IGFBP5 as the compounds alone. Again, these are most likely ER
-mediated responses. In the ovary, E2 decreased IGFBP5 expression by 38% (p = 0.08), and HPTE had a slight effect in reversing the repression of IGFBP5 by E2 when given at the same time (Fig. 4B
). For these genes, FLU had no effect on expression in either the uterus or the ovary.
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GPX3 mRNA was upregulated by E2 in both the uterus and the ovary, but not affected by HPTE (Fig. 5). As in the uterus, cotreatment with E2 + HPTE decreased induction compared with E2 treatment alone, although it was not statistically significant in the ovary.
Cathepsin B gene is differentially regulated and steroid metabolism is significantly downregulated through the Cyp17 gene in the ovary.
Cathepsin B (CTSB) was upregulated 75 and 49% by E2 or HPTE, respectively, and upregulated 54% with combined E2 + HPTE treatment in the uterus (Fig. 6A). This could also be an ER
-mediated response. In the ovary, CTSB is also upregulated by E2 (45% above control); however, HPTE does not upregulate CTSB (Fig. 6B
). In fact, E2 + HPTE combined treatment results in significantly lower expression of CTSB than observed for E2 alone. Interestingly, FLU treatment downregulates CTSB by 27% in the uterus and 35% in the ovary. This could be an ERß-mediated response, since HPTE is an ERß antagonist (Gaido et al.1999
) and ERß is highly expressed in the ovary, specifically in the granulosa cells (Sar and Welsch, 1999
). However, it could also be an antiandrogenic response, since FLU treatment downregulated CTSB in the ovary, and AR co-localizes with ERß in granulosa cells (Pelletier et al.2000
).
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Differential regulation of cFMS and STAT1 mRNAs in the uterus and JunD mRNA in the ovary.
Two genes showing up-regulation after treatment with both E2 and HPTE on the microarray were c-FMS proto-oncogene and signal transducer and activator of transcription 1 (STAT1) in the uterus. RT-PCR analysis of these gene changes showed no significant change with E2 (Fig. 7A), although HPTE induced cFMS more than 100% (p = 0.08). Two animals in this treatment group responded well (146 and 258% above control), while one did not respond (data not shown). FLU treatment downregulated STAT1 expression by 76%, a significant change that was not predicted by the array analysis (data not shown).
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DISCUSSION |
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The decrease in IGF1A and IGFBP5 mRNA expression with E2 or HPTE treatment in the uterus was unexpected (Fig. 4A). Estrogen increases IGF1 in the adult ovx rodent uterus (Carlsson and Billig, 1991
; Ghahary and Murphy 1989
; Kapur et al., 1992
; Murphy and Friesen 1988
; Murphy et al., 1987
) through what has been determined to be an ER
-dependent mechanism (Kahlert et al., 2000
; Klotz et al., 2000
). Adolescent rodents have high levels of circulating growth hormone in preparation for puberty, resulting in high IGF1 in the uterus (Murphy et al., 1987
) compared to the low levels of IGF1 in adult ovariectomized rodents (Carlsson and Billig, 1991
). This difference in IGF expression between immature and adult mice may account for the difference seen with this study compared with previously published reports using mature mice. The decreased IGF1 mRNA levels after treatment with E2 may be due to decreased ER
in the uterus. This effect was also seen in the ovary, where a slight decrease in ER
mRNA levels with E2 treatment corresponded with a slight decrease in IGF1A and IGFBP5 gene expression (though not significant; Fig. 4B
). In the ovary, IGF1 amplifies FSH action in granulosa cells, thereby determining the ovulatory fate of follicles (Adashi et al., 1997
). In developmental studies with E2 and MXC, reduced fertility in adult animals may result from repressed IGF1 mRNA and FSH levels in immature animals, thereby preventing ovulation and causing the buildup of large atretic follicles (Chapin et al., 1997
).
Clus mRNA is associated with tissue remodeling (loss) in the uterus (Brown et al., 1995) and is repressed in rat endometrial tissue by treatment with E2 (Wunsche et al., 1998
). Our data confirm this result, which is not unexpected given the uterotropic response observed in our animals after 3 days of treatment with E2 and HPTE (Fig. 1
). This finding is also observed with MXC treatment (Eroschenko, 1991
; Swartz et al., 1994
; Walters et al., 1993
). Induction of GPX activity (but not mRNA) in the uterus and ovary by E2 has previously been reported (Ohwada et al., 1996
). The time course of estrogen-induced uterotropic response positively correlates with increased GPX and glutathione reductase activities in rats (Suojanen et al., 1980
), and GPX activity in the human ovary is significantly decreased in premenopausal women in comparison to menopausal women, when circulating estradiol levels are markedly reduced (Okatani et al., 1993
). It is thought that lipid peroxides may be produced during cell growth and DNA synthesis that is induced by E2 treatment (Ohwada et al., 1996
). Since HPTE treatment does not cause this increase in GPX3, it is either a unique effect of E2, or it may be an HPTE-inhibited response, given the slight attenuation of the E2 response in both the uterus and the ovary with HPTE cotreatment. Conversely, GST activity is inhibited by quinoid metabolites of estrogen (Chang et al., 1998
), and GST levels are low in uterine tumors induced by E2 and testosterone (Hudson et al., 1998
). In the ovary, GST activity is induced with E2 treatment (Singh and Pandey, 1996
). Our data confirm these findings, and the lack of regulation by HPTE treatment indicates that this effect is unique to E2.
CTSB secretion is induced by E2 in rat uterine tissue (Pietras and Szego, 1975), but regulation in ovarian cells has not previously been reported. Although the effects observed in this study are small, CTSB is localized to developing follicles in the ovary, only a small fraction of our tissue sample, and its expression increases in granulosa cells during follicle maturation (Oksjoki et al., 2001
). Given the localization of ERß to ovarian granulosa cells, the reversal of the E2 regulation of CTSB with HPTE cotreatment may be an ERß-mediated effect. Since E2 regulates its own synthesis through a negative feedback mechanism acting on the hypothalamic-pituitary axis, inhibition of Cyp17 by E2 in the ovary is likely an LH-mediated response. The regulation of Cyp17 by HPTE is more complex because of the competing ER
agonist activity and the antiandrogenic activity, in which Cyp17 is upregulated by FLU (Fig. 6B
). The antiandrogenic effect may occur in the granulosa cells, which express AR and FSH receptors as well as secreting inhibin. Inhibin participates in a paracrine mechanism that locally amplifies androgen synthesis (Hillier et al., 1994
), thereby up-regulating Cyp17.
In this study, microarray and RT-PCR analysis have shown that several gene families appear to be regulated by E2 or HPTE in some unique, but also overlapping, patterns. These gene changes are mediated either through the steroid receptors present in the given tissue, through the steroid receptors in another tissue leading to a down-stream effect in the tissue examined, or through an alternate undetermined mechanism. Without coordinate immunohistochemistry analyses that co-localizes the proteins, it is impossible to be absolutely certain of the particular mechanism of action. Microarray analyses are useful for this type of study, but they are not without limitations. Qualitative information derived from microarrays should not be confused with the quantitative data generated from standard molecular techniques, such as real-time RT-PCR, that measure relative gene expression levels. The modest changes in mRNA expression levels observed for several genes were far below what was predicted by microarray analysis. Two examples are cFMS and STAT1, which exhibited 34-fold changes in uterine expression in mice treated with E2 or HPTE and analyzed by microarray (data not shown); however, these changes were not confirmed by RT-PCR analysis (Fig. 7A). Biological variability in the expression of these two genes in the uterus is probably the cause of this discrepancy. Our array analysis was performed on only one animal from each treatment group, whereas the quantitative RT-PCR was performed on all 3 animals per treatment group. Had the array analysis been performed on all 3 animals and then averaged treatment groups compared to each other, the resulting gene ratios would likely have been more reliable for quantitation.
This is the first study to explore ligand-specific patterns of gene regulation through ER, ERß, and AR as determined by microarray analysis and confirmed with quantitative RT-PCR. Many xenoestrogens, such as MXC and HPTE, exhibit some agonist, partial agonist, or antagonist properties through ER binding that result in tissue-specific regulation of gene expression. Results from this study highlight how the expression of specific receptors in a given tissue or cell type, and as yet undetermined interaction of the receptors and their corresponding cofactor proteins, can affect the biological consequence of exposure during developmental, prepubertal, or adult life stage. Gene profiling is a useful tool to explore differences between xenobiotic chemicals and the hormones that they mimic, and it can also provide valuable information and insight into the biological mechanisms that result in toxicological pathologies.
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ACKNOWLEDGMENTS |
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NOTES |
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