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. 2010 Jun;1(3):146-55.
doi: 10.1007/s12672-010-0015-9.

In Utero Exposure to Diethylstilbestrol (DES) or bisphenol-A (BPA) Increases EZH2 Expression in the Mammary Gland: An Epigenetic Mechanism Linking Endocrine Disruptors to Breast Cancer

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Free PMC article

In Utero Exposure to Diethylstilbestrol (DES) or bisphenol-A (BPA) Increases EZH2 Expression in the Mammary Gland: An Epigenetic Mechanism Linking Endocrine Disruptors to Breast Cancer

Leo F Doherty et al. Horm Cancer. .
Free PMC article

Abstract

Diethylstilbestrol (DES) and bisphenol-A (BPA) are estrogen-like endocrine-disrupting chemicals that induce persistent epigenetic changes in the developing uterus. However, DES exposure in utero is also associated with an increased risk of breast cancer in adult women. Similarly, fetal exposure to BPA induces neoplastic changes in mammary tissue of mice. We hypothesized that epigenetic alterations would precede the increased risk of breast neoplasia after in utero exposure to endocrine disruptors. Enhancer of Zeste Homolog 2 (EZH2) is a histone methyltransferase that has been linked to breast cancer risk and epigenetic regulation of tumorigenesis. We examined the effect of BPA and DES on EZH2 expression and function in MCF-7 cells and in mammary glands of mice exposed in utero. DES and BPA treatment approximated human exposure. EZH2 functional activity was assessed by measuring histone H3 trimethylation. Treatment of MCF-7 cells with DES or BPA led to a 3- and 2-fold increase in EZH2 mRNA expression, respectively (p < 0.05) as well as increased EZH2 protein expression. Mice exposed to DES in utero showed a >2-fold increase in EZH2 expression in adult mammary tissue compared with controls (p < 0.05). EZH2 protein was elevated in mammary tissue of mice exposed to DES or BPA. Histone H3 trimethylation was increased in MCF-7 cells treated with BPA or DES. Similarly, mice exposed to BPA or DES in utero showed increased mammary histone H3 trimethylation. Developmental programming of EZH2 is a novel mechanism by which in utero exposure to endocrine disruptors leads to epigenetic regulation of the mammary gland.

Figures

Fig. 1
Fig. 1
Structure of diethylstilbestrol (DES) and bisphenol-A (BPA) structure of the endocrine-disrupting compounds diethylstilbestrol and bisphenol-A.
Fig. 2
Fig. 2
DES and BPA exposure in vitro induce EZH2 mRNA expression in human mammary cells. a Quantitative RT-PCR performed on MCF-7 cells treated with DES at five concentrations. Treatment with 5 × 10−8 and 5 × 10−7M DES resulted in a greater than 2-fold increase in EZH2 mRNA compared with controls. Treatment with 5 × 10−6M DES caused a 4-fold increase in EZH2 mRNA expression. Results are representative of three independent experiments performed in triplicate. *p<0.05. b Quantitative RT-PCR performed on MCF-7 cells treated with four concentrations of BPA. Treatment with 2.5 × 10−6 and 2.5 × 10−5M BPA resulted in greater than 2-fold increases in EZH2 mRNA when compared with controls. Results are representative of three independent experiments performed in triplicate. *p<0.05.
Fig. 3
Fig. 3
DES and BPA exposure in vitro increase EZH2 protein expression in human mammary cells. a Western blotting demonstrates that BPA and DES treatment of MCF-7 cells for 48 h results in increases in EZH2. EZH2 was present in low, but detectable, amounts in vehicle-treated control cells. DES-treated cells showed an increase in EZH2. BPA-treated cells also showed an increase in EZH2. β-Actin was used to as a loading control. b Densitometric analysis of Western blots confirm increased EZH2 expression in BPA and DES-treated MCF-7 cells. Values were normalized to β-actin to obtain relative densitometric intensity. *p<0.05.
Fig. 4
Fig. 4
DES and BPA exposure in vitro increase net EZH2 functional activity in mammary cells. a Western blot demonstrates that in vitro BPA and DES exposure results in increases in EZH2 protein function, assessed by blotting with antibody specific to histone H3 (tri methyl), the target of EZH2 methyltransferase activity. Histone H3 (tri methyl K27) was low, but detectable, in vehicle-treated control cells. Treatment with DES increased histone H3 (tri methyl K27). BPA-treated cells also showed an increase in histone H3 (tri methyl K27). Total histone H3 protein expression was not changed by treatment with DES or BPA. b Densitometric analysis of Western blots confirm increased histone H3 (tri methyl K27) in BPA and DES-treated MCF-7 cells. Values were normalized to β-actin to obtain relative densitometric intensity. *p<0.05.
Fig. 5
Fig. 5
Plasma BPA concentration in treated animals and controls as determined by mass spectroscopy. a Representative LC/MS chromatography from untreated and BPA-treated mice sampled 1 h after administration of BPA. Top panel demonstrates quantification of BPA in control mice. Bottom panel shows quantification of BPA after treatment. b Plasma BPA concentration was determined after administration of BPA or vehicle control to pregnant mice. Plasma was obtained at 1, 6, 12, and 18 h after BPA administration.
Fig. 6
Fig. 6
DES, but not BPA, exposure in utero increases EZH2 mRNA expression in adult murine mammary tissue. Quantitative RT-PCR was performed in mammary tissue from 6-week-old mice after in utero exposure to DES or BPA during days 9–26 of gestation. DES treatment resulted in a 2-fold increase in EZH2 mRNA expression when compared with control mice. BPA treatment did not alter expression of EZH2 mRNA. Results are representative of three independent experiments using five animals in each treatment group in each experiment. *p<0.05.
Fig. 7
Fig. 7
DES and BPA exposure in utero increases EZH2 protein levels in adult murine mammary tissue. a Western blot demonstrating that in utero BPA or DES exposure during days 9–26 of gestation increases EZH2 protein within mammary tissue of 6-week-old female mice. EZH2 was present in low, but detectable, amounts in control animals. DES-treated animals showed an increase in EZH2. BPA-treated animals also showed an increase in EZH2. b Densitometric analysis of Western blots confirm increased EZH2 expression in adult murine mammary tissue subsequent to in utero BPA or DES exposure. Values were normalized to β-actin to obtain relative densitometric intensity. *p<0.05.
Fig. 8
Fig. 8
DES and BPA exposure in utero increase net EZH2 functional activity in adult murine mammary tissue. a Western blot demonstrates that in utero BPA and DES exposure during days 9–26 of gestation increases EZH2 protein function, assessed by blotting with antibody specific to histone H3 (tri methyl), the target of EZH2 methyltransferase activity. Histone H3 (tri methyl K27) was low, but detectable, in control mammary glands. Treatment with DES increased histone H3 (tri methyl K27). BPA-treated mice also showed an increase in histone H3 (tri methyl K27). Total histone H3 protein expression was not changed by treatment with DES or BPA. b Densitometric analysis of Western blots confirm increased histone H3 (tri methyl K27) in adult murine mammary tissue subsequent to in utero BPA or DES exposure. Values were normalized to β-actin to obtain relative densitometric intensity. *p<0.05.

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