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. 2013 May 10;14(7):564–573. doi: 10.4161/cbt.24599

Exposure to estrogen and ionizing radiation causes epigenetic dysregulation, activation of mitogen-activated protein kinase pathways, and genome instability in the mammary gland of ACI rats

Kristy Kutanzi 1,†, Olga Kovalchuk 1,*
PMCID: PMC3742486  PMID: 23792640

Abstract

The impact of environmental mutagens and carcinogens on the mammary gland has recently received a lot of attention. Among the most generally accepted carcinogenic agents identified as factors that may increase breast cancer incidence are ionizing radiation and elevated estrogen levels. However, the molecular mechanisms of mammary gland aberrations associated with radiation and estrogen exposure still need to be further elucidated, especially the interplay between elevated hormone levels and radiation. Therefore, in the present study, we investigated molecular changes induced in rat mammary gland tissue by estrogen, ionizing radiation, and the combined action of these two carcinogens using a well-established ACI rat model. We found that continuous exposure of intact female ACI rats to elevated levels of estrogen or to both estrogen and radiation resulted in significant hyperproliferative changes in rat mammary glands. In contrast, radiation exposure alone did not induce hyperplasia. Interestingly, despite the obvious disparity in mammary gland morphology, we did not detect significant differences in the levels of genomic methylation among animals exposed to estrogen, radiation, or both agents together. Specifically, we observed a significant global genomic hypomethylation at 6 weeks of exposure. However, by 12 and 18 weeks, the levels of global DNA methylation returned to those of age-matched controls. We also found that combined exposure to radiation and estrogen significantly altered the levels of histone H3 and H4 methylation and acetylation. Most importantly, we for the first time demonstrated that estrogen and radiation exposure caused a significant induction of p42/44 MAPK and p38 pathways that was paralleled by elevated levels of H3S10 phosphorylation, a well-established biomarker of genome and chromosome instability. The precise role of MAPK pathways and their inter-relationship with H3S10 phosphorylation and genome instability in mammary gland tissues needs to be explored further.

Keywords: epigenetics, estrogen, ionizing radiation, DNA methylation, histones, breast cancer, mitogen-activated protein kinase pathways

Introduction

Breast cancer is the most commonly diagnosed malignancy in women and the leading cause of death among women between the ages of 35 to 55.1,2 It is estimated that only 5% of new sporadic breast cancer cases are attributed to abnormal function of susceptibility genes, while the etiology of the remaining 95% of cases remains unclear. The emerging evidence suggests the crucial role of environmental mutagens and carcinogens in breast cancer etiology.3,4

Among the most generally accepted environmental carcinogenic agents identified as factors that may increase breast cancer risk are ionizing radiation (IR) and elevated estrogen levels.5,6 Indeed, the results of numerous epidemiological studies have strongly established an increased breast cancer incidence in atomic bomb survivors and women exposed to various diagnostic and therapeutic irradiation.5,7-9 For instance, an elevated incidence of breast cancer has been reported in patients with scoliosis and tuberculosis,7 women treated for benign breast disease,10 and in cancer survivors who received radiation therapy.5 The average dose of IR-exposure linked to breast carcinogenesis ranges between 0.2 and 20 Gy.5 Additionally, experimental in vitro and in vivo studies have also established that IR can alter the functioning of normal mammary gland epithelial cells and trigger their neoplastic transformation.11,12 However, the exact nature of these radiation effects on the mammary gland needs to be further explored.

Additionally, recent epidemiological studies have also convincingly proved a causative role of estrogen in human breast cancer development, especially in young premenopausal women.13 More importantly, women with elevated estrogen levels are considered to be a high-risk group for breast cancer development14,15 and are likely to be exposed to diagnostic radiation procedures more frequently. Similarly, many patients with estrogen-induced breast cancer undergo radiation treatment and are exposed to relatively high X-ray doses to the healthy breast. In vitro application of both IR and estrogen induces genome instability and also leads to malignant transformation of normal breast epithelial cells.12 Genomic instability is an important feature of almost all human cancers, including breast cancer.16 However, the underlying mechanisms behind radiation- and estrogen-induced mammary gland genome instability and carcinogenesis, especially the effects of combined exposure to estrogen and IR on mammary glands, are not fully understood and remain to be elucidated.

Epigenetic changes, including DNA methylation and histone modifications and variations in gene expression have been associated with changes in the molecular function of breast cells and development of breast cancer.17-19 Furthermore, it is believed that epigenetic alterations in breast cancer are more prominent than genetic changes,20 and these epigenetic alterations may predispose cells to genomic instability and acquisition of genetic changes during carcinogenesis.21,22 The altered genomic methylation pattern is a well-known epigenetic feature of cancer cells23 with regional hyper- and hypo-methylation of specific genes being very important in breast carcinogenesis.17,18,24 Despite recent advances in uncovering breast cancer-related epigenetic abnormalities, the extent and timing of epigenetic dysregulation induced in the mammary gland in response to different carcinogenic agents remains poorly understood.

In light of these considerations, the goal of the present study was to investigate the association between morphological and molecular changes induced in the rat mammary gland by exposure to estrogen, radiation and the combination of these two carcinogens.

Results

Estrogen- and radiation-induced morphological changes in rat mammary glands

Exposure of ACI rats to constitutively elevated levels of E2 significantly altered mammary gland morphology, specifically elevated estrogen caused extensive cell proliferation (Fig. 1). The increased levels of cell proliferation were noted as early as 6 weeks after E2-exposure and persisted further for 12 and 18 weeks of treatment (Fig. 1). Interestingly, after 18 weeks, the severity of these hyperproliferative changes decreased slightly compared with the 6- and 12-week values and persisted through 12 and 18 weeks (Fig. 1B). Similar changes were detected in the mammary glands of ACI rats exposed to both E2 and IR (Fig. 1B). In contrast to E2- and E2 + IR-treated animals, no hyperproliferative changes were found in mammary glands of IR-exposed animals (Fig. 1B). However, it was noted that the mammary glands of IR-exposed ACI rats exhibited noticeable fibrotic changes characterized by general loss of functional units at 12 and 18 weeks.

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Figure 1. Estrogen- and radiation-induced morphological changes in rat mammary glands. (A) Representative images as viewed at 10× magnification. (1) control; (2) mild hyperplasia; (3) moderate hyperplasia; (4) severe hyperplasia. (B) Histopathology score data are presented as means ± SD relative to age-matched controls; n = 6. *Significantly different from the control at that same time point. E2, estrogen exposed-group; IR, radiation-exposed group; E2 + IR, estrogen and carcinogenesis-exposed groups. Scoring was done using a following scheme: 1, normal; 2, mild hyperplasia; 3, moderate hyperplasia; 4, moderate/severe hyperplasia; 5, severe hyperplasia.

Levels of global DNA methylation in the rat mammary glands exposed to estrogen and radiation

To investigate the role of epigenetic changes in rat mammary gland tissues in response to E2 exposure, IR, and the combined action of E2 and IR, we first analyzed changes in the levels of global DNA methylation in rat mammary gland tissues of control and treated rats using a sensitive HpaII-based cytosine extension assay that measures the proportion of unmethylated CCGG sites in the genome. DNA methylation patterns exhibit a degree of plasticity, which can be reflective of the cellular response to environmental stimuli, including chemical and physical agents.25 We observed a significant increase in the [3H]dCTP incorporation (P < 0.05), indicative of global genome hypomethylation at 6 weeks, a stage marked by extensive hyperproliferative changes in mammary glands of E2- and E2 and IR-treated groups (Fig. 2). Interestingly, we also detected a significant 2.5-fold loss of global methylation as compared with control age-matched rats (P < 0.05) in response to IR treatment alone (Fig. 2). However, at later times (12 and 18 weeks), the extent of global DNA methylation in all groups was not significantly different from that in the age-matched control rats.

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Figure 2. Altered levels of global DNA methylation in the mammary gland tissue of estrogen- and radiation-exposed rats. Levels of global DNA hypomethylation were measured by the DNA cytosine extension assay. The bars represent relative levels of global DNA methylation where an increase in H3-dCTP indicates a decrease in global methylation in the mammary gland tissues of rats exposed to E2, IR, and E2 + IR, six weeks after exposure. White bars, controls; gray, E2-exposed animals; striped, IR-exposed animals; black, E2 + IR-exposed animals. Data are presented as means ± SEM relative to age-matched controls; n = 6. *Significantly different from the control at that same time point.

Expression of DNA methyltransferases in the rat mammary glands exposed to estrogen and radiation

DNA methyltransferases, DNMT1 and DNMT3A, are key cellular enzymes responsible for maintaining proper DNA methylation in mammalian cells. Any changes in cellular levels of these proteins and/or their activity may alter DNA methylation. Hence, we assessed whether or not the observed changes in DNA methylation were accompanied by altered expression of Dnmt1 and Dnmt3a genes. Figure 3A shows that E2 exposure resulted in a significant increase in the number of DNMT1-positive cells at 6, 12, and 18 weeks after exposure. This increase was most pronounced after 18 weeks of E2 treatment. At that time, the number of DNMT1-positive cells in the mammary glands of E2-treated rats was over 4 times greater (P < 0.05) than that observed in control rats (Fig. 3A). Similarly to E2 exposure, combined treatment with E2 and IR led to a significant increase in the number of DNMT1-positive cells after 6, 12, and 18 weeks of exposure. Contrarily, IR alone did not affect the number of DNMT1-positive cells after 18 weeks of exposure (Fig. 3A).

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Figure 3. Expression of DNA methyltransferases in the mammary glands of estrogen- and radiation-exposed rats. Levels of DNA methyltransferases DNMT1 (A) and DNMT3A (B) were determined by immunohistochemical analysis and presented as an average number of cells with intense positive immunostaining. Abbreviations as defined in Figure 1. Data presented as means ± SEM (n = 6). *Significantly different from the age-matched control. White bars, controls; gray, E2-treatment; striped, IR-treatment; black, E2 + IR treatment.

E2 exposure and combined exposure to E2 and IR also resulted in a pronounced and statistically significant increase in the number of DNMT3a-positive cells after 6 and 12 weeks of treatment, while IR alone did not cause significant changes in the number of DNMT3A positive cells over the 18-week period (Fig. 3B).

Expression of DNA repair proteins in the rat mammary glands exposed to estrogen and radiation

Another factor that may affect the status of DNA methylation is the integrity of DNA.26,27 It is well established that E2 and IR are strong DNA damaging agents that cause a variety of DNA lesions, including DNA strand breakage and oxidative DNA damage.28-30 In light of these considerations, we investigated whether or not altered DNA methylation in the mammary glands of ACI rats exposed to E2-, IR-, and E2 + IR may be associated with compromised DNA integrity.

Currently, it is widely accepted that altered levels of proteins in the base excision repair (BER) pathway, a key pathway involved in the repair of oxidative DNA damage, are sensitive in vivo markers of oxidative DNA damage.31Figure 4A shows that exposure to E2 for 6 weeks induced a significant (P < 0.05) 1.3- and 1.2-fold increase in the levels of APE1 and Polβ proteins, respectively. Likewise, combined exposure to E2 and IR also resulted in the significantly elevated levels of APE1 and Polβ proteins after 6 weeks of treatment. At later times (after 12 and 18 weeks of treatment), the levels of APE and Polβ proteins in the mammary glands of E2- and E2 + IR-exposed rats diminished as compared with the control values. In contrast, IR exposure alone did not change the levels of these BER proteins in the mammary glands.

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Figure 4. Dysregulation of DNA repair enzymes in the mammary gland tissues of rats exposed to estrogen, radiation and combined action of both carcinogens. (A) Western blot analysis of base excision repair enzymes, APE1 and Polβ. (B) Western blot analysis of non-homologous end joining enzymes NBS1 and Ku70. Abbreviations as defined in Figure 1 Representative western immunoblots of (A) Ape1 and DNA Pol. β proteins; and (B) NBS1 and Ku70 proteins from two independent technical repeats. Protein levels were normalized to β-actin. Data presented as mean values ± SEM (n = 6). *Significantly different from the age-matched control. CT, control, E2, estrogen, IR, irradiated, E2 + IR. White bars, controls; gray, E2 treatment; striped, IR treatment; black, E2 + IR treatment.

The NBS1 protein is part of a nuclear multi-protein complex composed also of MRE11 and RAD50 (the MRN complex) which plays a crucial role in response to DNA double-strand breaks (DSBs) as well as in DNA strand break repair by homologous recombination and non-homologous end joining (NHEJ). Importantly, in mammals, most double-strand breaks are repaired by NHEJ.32,33 Ku70 is essential for NHEJ and is induced in mammalian cells exposed to ionizing radiation.34 Therefore, we measured the levels of NBS1 and Ku70 in the mammary glands of control rats and rats exposed to E2, IR, and both E2 and IR. Figure 4B shows that E2 exposure and combined exposure to E2 and IR substantially increased cellular levels of NBS1 and slightly, but statistically significantly increased the levels of Ku70 after 6 weeks. At later times (after 12 and 18 weeks of treatment for Ku70 and after 18 weeks of treatment for NBS1), the levels of these proteins decreased as compared with control rats, somewhat similarly to the levels of APE and Polβ.

Phosphorylation of histone H3 serine 10 in mammary glands of estrogen- and radiation-exposed rats

DNA methylation is intimately associated with alterations in the other components of chromatin structure, primarily with histone modifications.35 Histone modifications, including acetylation, methylation, phosphorylation, and ubiquitination, are important epigenetic modifications for the regulation of gene transcription and overall genome stability.36-40 Therefore, we investigated the effects of exposure to E2, IR, and E2 + IR on the levels of histone H3 and H4 methylation and acetylation. E2 exposure caused a significant increase in the levels of H3K4me3 (a modification associated with chromatin relaxation and activation of gene expression) after 12 and 18 weeks of treatment as compared with control rats. IR exposure alone affected the level of H3K4me3 in the 12-week group only. However, combined exposure to E2 and IR resulted in a strong and significant increase in the level of H3K4me3 after 12 and 18 weeks, respectively. Additionally, we have evaluated the status of histone H4 acetylation at lysine residues 5, 8, and 12, another epigenetic marker associated with gene activation. Exposure to E2 resulted in a significant increase in H4K5, H4K8 and H4K12 acetylation after 6, 12, and 18 weeks of estrogen treatment. Changes similar in magnitude were induced by combined exposure to E2 and IR, whereas IR exposure alone had no effect on H4K12 acetylation levels (Fig. 5). Notably, IR exposure affected levels of H4K5 acetylation 6 and 12 weeks after exposure. It also influenced H4K8 acetylation 12 weeks after exposure.

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Figure 5. Levels of histone methylation and acetylation in the mammary glands of estrogen- and radiation-exposed rats. Immunohistochemical analysis of (A) H3K4me3, (B) H3K9me3, (C) H4K20me3 histone marks, (D) AcH4K5, (E) AcH4K8, and (F) AcH4K12. Data presented as means ± SEM (n = 6). *Significantly different from age-matched controls. White bars, controls; gray, E2 treatment; striped, IR treatment; black, E2 + IR treatment. Abbreviations as defined in Figure 1. Data are presented as means ± SEM relative to age-matched controls; n = 6. *Significantly different from the control at that same time point.

In addition, we studied the effects of E2, IR, and E2 + IR exposure on the levels of H3S10 phosphorylation in rat mammary glands. Figure 5 shows that exposure to E2 caused a small but statistically significant increase in H3S10 phosphorylation after 6 weeks. At later times (12 and 18 weeks), the levels of H3S10 phosphorylation in rat mammary glands further increased and were 2.9 and 3.0 times greater, respectively, than in control rats. Contrarily, IR treatment resulted in a significant 4.3- and 1.4-fold decrease in the levels of H3S10 phosphorylation after 6 and 18 weeks of exposure, respectively. Interestingly, at 12 weeks after exposure, IR caused a 1.9-fold increase in the levels of H3S10 phosphorylation. The combined application of E2 and IR led to a statistically significant 1.9, 4.1, and 3.2 times increase in the levels of H3S10 phosphorylation in rat mammary gland tissues (Fig. 6).

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Figure 6. Phosphorylation of histone H3 serine 10 in the mammary glands of estrogen- and radiation-exposed rats. White bars, controls; gray, E2-exposed animals; striped, IR-exposed animals; black, E2 + IR-exposed animals. Abbreviations as defined in Figure 1. Data are presented as means ± SEM relative to age-matched controls; n = 6. *Significantly different from the control at that same time point. The right panel shows a representative immunostain of the control mammary gland tissue and in the mammary gland tissue 18 weeks after exposure of animals to estrogen and irradiation (40×).

Alterations in the mitogen-activated protein kinase (MAPK) pathways in the mammary glands of estrogen- and radiation-exposed rats

The results of several studies have linked the increased level of histone H3S10 phosphorylation with the induction of MAPK pathways.41-43 Indeed, Figure 7A shows that E2 exposure resulted in the upregulation of p-p42/44 MAPK after 12 and 18 weeks of treatment. IR alone and exposure to both E2 and IR caused a significant induction of p-p42/44 MAPK at 6, 12, and 18 weeks of treatment. Similarly, exposure to IR resulted in increased levels of phosphorylated p38 protein at 6, 12, and 18 weeks after irradiation (Fig. 7B). Likewise, E2 exposure led to increased p-p38 levels after 12 and 18 weeks of treatment. Contrarily, combined exposure to both E2 and IR did not affect p-p38 levels in the mammary glands of exposed rats. Interestingly, we did not observe any statistically significant changes in the levels of JNK phosphorylation after treatment of ACI rats with E2, IR, and E2 + IR (data not shown).

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Figure 7. Alterations in the mitogen-activated protein kinase (MAPK) pathways in the mammary glands of estrogen- and radiation-exposed rats. (A) Western blot analysis of p-p42/44 MAPK. (B) Western blot analysis of p-p38. Sample loading was normalized to β-actin. Abbreviations as defined in Figure 1. White bars, controls; gray, E2-exposed animals; striped, IR-exposed animals and black, E2 + IR, exposed animals. Data are presented as means ± SEM relative to age-matched controls; n = 6. *Significantly different from the control at that same time point.

Discussion

Currently, breast cancer is the most common malignancy among North American women.1,2 Most breast cancer patients undergo radiation diagnostics and are also treated with radiotherapy. In addition to being an important treatment modality, IR is a potent tumor-causing agent that has been linked to breast cancer development. However, the exact molecular changes induced by IR exposure in mammary gland tissues remain largely unknown. In addition, the interplay between elevated estrogen levels and the magnitude of IR responses in the mammary gland has to be defined.

The ACI rat provides a unique opportunity for studying breast carcinogenesis, as estrogen-induced mammary carcinogenesis in the female ACI rat exhibits remarkably similar histopathological features and hormone-responsiveness as observed in humans.44-48Considering that few other rat strains exhibit this propensity to develop mammary cancer in response to continuous E2 treatment, it appears that the ACI rat possesses a unique genetic and epigenetic background that underlies its susceptibility to elevated estrogen levels.49-51 Furthermore, the ACI rat has low levels of spontaneous and radiation-induced mammary tumors, thus it is an ideal model to analyze the combined effects of radiation and estrogen.

In the present study, we demonstrated that two very different carcinogens, estrogen and radiation, applied either separately or in conjunction, exert numerous cellular and molecular effects on the mammary gland of female ACI rats. This was evidenced by estrogen-driven morphological alterations, deregulation of critical cellular epigenetic processes, and altered cell signaling and DNA repair pathways. Importantly, we have identified several key differences in the extent and timing of these changes in response to two known carcinogens.

The results of the present study demonstrated that despite the obvious disparity in morphology induced by E2 and IR, there was no significant differences in the levels of genomic methylation among animals exposed to E2, IR and E2 + IR. Specifically, E2, IR, and both carcinogens together caused a significant increase in global DNA hypomethylation only at six weeks after treatment. Interestingly, the extent of DNA methylation returned to normal levels by 12 and 18 weeks after exposure. Similar findings were reported by Starland-Davenport and colleagues (2010)52 in a recent independent study using the ACI model rat of breast carcinogenesis.

Several possible explanations exist for a comparable loss of global DNA methylation at 6 weeks, including DNA damage and/or a DNA repair-based mechanism. It has been demonstrated previously that DNA damage can interfere with the methylating ability of DNA methyltransferases by stalling DNA methyltransferase at sites of lesions.53,54 Furthermore, during DNA repair synthesis, polymerases incorporate cytidine but not methylcytidine, thus the presence of DNA lesions and activation of DNA repair mechanisms may also contribute to DNA hypomethylation. The results of our study that demonstrate a close association between the induction of DNA repair enzymes (Fig. 4) and loss of global DNA methylation (Fig. 2) after 6 weeks of exposure support these suggestions.

Alternatively, DNA hypomethylation may arise as a byproduct of estrogen-driven cell hyperproliferation. In this case, DNA methyltransferases may be overwhelmed by the rate of replication synthesis, such that they are unable to maintain methylation patterns on the nascent strands. As one might expect, we observed a significant induction of DNMTs, especially DNMT3A, in the estrogen-treated groups, which may serve as a cellular compensatory mechanism. The observation that IR, in conjunction with exposure to elevated E2, did not contribute to a more pronounced loss of DNA methylation than exposure to either carcinogen alone is very interesting and deserves attention in the future. Also an increase in DNA methylation levels in the mammary glands of exposed rats from a relatively hypomethylated state at 6 weeks to unchanged levels at 12 and 18 weeks further affirms the concept of an overburdened methylation machinery (at 6 weeks). However, an increased expression of DNMTs may initiate aberrant gene-specific de novo methylation events and result in gene silencing. Indeed, a persistent upregulation of DNMTs has been reported to play a significant role in transcriptional silencing of gene expression by hypermethylating the promoter CpG islands during breast carcinogenesis.52,55,56 These findings strongly correlate with our current observations of the induction of DNMT1 and DNMT3A expression in the E2- and E2 + IR-treated groups.

In addition to DNA methylation changes, exposure of ACI rats to E2 and IR, applied either separately or in conjunction, resulted in noticeable histone modification changes, especially alterations of histone H3S10 phosphorylation levels. Phosphorylation of histone H3S10 has been implicated in gene activation and, most importantly, in controlling genome stability.57 Generally, phosphorylation of H3S10 is crucial for proper chromosome condensation and segregation, with nearly all H3 molecules phosphorylated at this residue during the entry into mitosis. Overall, phosphorylation of H3S10 is therefore regarded as a marker of mitosis.58,59 The presence of a greater percentage of cells expressing this marker, especially in the highly proliferative estrogen group, is consistent with this interpretation. Increased levels of pH3S10, therefore, are consistent with the observed hyperplasia.

Furthermore, H3 histone phosphorylation may play an important part in genome instability. Recent data suggest that H3 histone phosphorylation is a major contributing factor for genome and chromosome instability and thus may play a role in cellular transformation and carcinogenesis.60,61 Indeed, our results that demonstrated a substantial increase in histone H3S10 phosphorylation correspond to previous findings of overexpression of Aurora-A and chromosomal instability during breast carcinogenesis in ACI rats.62

Furthermore, previous studies have demonstrated that genotoxic DNA-damaging agents can induce phosphorylation of H3S10 through the activation of the MAPK pathway.43 There are four distinct MAPK cascades that include the extracellular signal-regulated kinase (p42/44 ERK)/MAPK 1 and 2 pathway (p42/44 MAPK), the p38 pathway, the c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway, and the mitogen-activated protein kinase/ERK5 pathway. MAPK pathways transmit signals that partake in control of cell proliferation and cell death. Recent studies have reported that breast cancer frequently exhibits an activation of MAPK pathways.63-65 Most importantly, MAPK pathways have been implicated in genotoxic stress responses. Indeed, exposure to breast carcinogens, such as radiation and estrogen, has been shown to activate MAPK pathways.66-68 The activated MAPK pathways may in turn influence H3S10 phosphorylation. Here, we demonstrate a good correlation between H3S10 phosphorylation and activation of the p42/44 MAPK and p38 MAPK pathways. This correlation may suggest that estrogen- and radiation-induced activation of p42/44 MAPK and p38 may act in H3S10 phosphorylation.

Notwithstanding, the precise role of MAPK pathways and their inter-relationship with H3S10 phosphorylation, genome instability and genotoxic stress-induced changes in the mammary gland tissue has to be further delineated.

Another interesting outcome of this study is an observation of the age-related changed in the levels of DNMT1, DNMT3A, and acetyl H4K5 and H4K8 that were seen in the untreated control groups. Even though analysis of the age-related changes in the rat mammary glands was not the focus of this study, these findings could not be overlooked. We noted that the levels of DNMT1, DNMT3A, and acetyl H4K5 and H4K8 were much higher in the 18 weeks groups as compared with the 6 weeks groups. This may be due to the normal aging process, and possibly, due to the physiological changes in the levels of hormones. Future studies are clearly needed to analyze the effects of genotoxic stressors on mammary gland as a function of animal age. Furthermore, it would be of special interest to analyze the activity of DNMTs and HDACs, as well as any peculiarities of transcriptional and post-transcriptional regulation of their expression.

Notably, changes induced by radiation exposure alone were less prominent than those induced by estrogen and the combination of both carcinogens. Primarily, radiation is a DNA damaging agent which causes breaks in DNA and a wide array of base damages. Here, animals received 3 Gy of X-rays to the whole body, yet, even at this moderate dose, not every cell in the mammary gland has been traversed by radiation. Interestingly, cells that have been traversed by radiation can spread the distress signal to the neighboring unexposed naïve cells. This phenomenon is known as a bystander effect. Bystander effects are associated with genome instability and carcinogenesis. They occur within the exposed organs or tissues in vivo. Yet, the contribution of bystander effects to mammary gland radiation responses has never been addressed. In the future, it would be interesting to determine if the observed molecular changes in the irradiated mammary gland tissue are produced by exposed cells, by bystander cells or by both types of cells. With the new technology available for tissue microdissections such experiments will become possible. Furthermore, it would be interesting and important to determine the occurrence and contribution of radiation-induced bystander effects in combination with elevated estrogen exposure.

Importantly, the radiation and estrogen-induced molecular changes were detected in non-cancerous mammary glands that exhibited mild to severe benign hyperplasia suggesting that these early molecular changes may be important events that may drive the cancer predisposition and early stages of carcinogenesis.69 Therefore, future studies are needed to dissect the role of the aforementioned epigenetic and signaling changes in mammary gland carcinogenesis.

In summary, the results of the current study confirm our hypothesis that estrogen- and radiation-induced changes in mammary gland tissues are mediated, at least in part, by perturbations in the epigenome. Exposure to these two carcinogens, either separately or in conjunction, induces morphological changes that are paralleled by altered DNA methylation, histone modification changes and imbalances in proliferation and apoptosis. The observed epigenetic alterations may contribute to genomic instability. Further research is required to understand the impact of dose and administration on the carcinogenic potential of these agents, as well as to examine their effects when combined with other carcinogens. This is especially important for radiation, since IR effects may be direct as well as indirect. Thus, our study provides an important roadmap for the future analysis of IR and estrogen-induced breast carcinogenesis.

Materials and Methods

Animals, treatment, and tissue preparations

Female ACI rats were purchased from Harlan Spraque-Dawley, Inc. (Indianapolis, IN). The animals were housed two per cage in a temperature-controlled (24 °C) room with a 12-h light–dark cycle and given ad libitum access to water and NIH-31 diet. At 8 weeks of age, the rats were randomly allocated into four groups of 18 rats each: (1) sham-treated (control group); (2) estrogen-treated (E2); (3) IR-treated (IR); and (4) estrogen plus IR-treated (E2 + IR). Estrogen-treated rats were implanted with estrogen constant release mini-pellets (7.5 mg/90 d release; Innovative Research of America Inc.) subcutaneously in the shoulder region44. Animals from the irradiated groups were exposed to a single dose of 3 Gy of X-rays one week later (90 kVp, 5 mA). Six rats per group were humanely euthanized using an overdose of CO2 after 6, 12, and 18 weeks of treatment. All experimental procedures were performed in accordance with animal study protocols approved by the Institutional Animal Care and Use Committee.

The paired caudal inguinal mammary glands (and fat pad) were excised from the overlying skin. One gland was frozen immediately in liquid nitrogen and stored at -80 °C for subsequent analyses. The contralateral gland and fat pad were used for histopathology.

Histopathology

Mammary gland and fat pad were carefully spread onto a 5 × 8 cm glass slide, and excess fat and other tissues were trimmed. The gland was then placed flat in a cassette in toto. This provided a histological specimen with frontal (coronal) plane orientation, in which the gland profile is comparable to that of a mammary whole mount. This orientation allows more clear and complete visualization of an arbrorizing pattern of the duct system and associated alveoli than is achievable using a transverse section of the gland. The specimens were then fixed in 10% neutral buffered formalin for 48 h, processed, embedded in paraffin, sectioned at 4 microns and mounted on glass slides. Tissue sections were stained with hematoxylin and eosin using a standard protocol. The morphological changes in the mammary gland of animals from control and experimental groups were independently assessed by two pathologists in a blinded fashion. A semi-quantitative system was used to record the severity of epithelial hyperplasia of the mammary gland.

Global DNA methylation analysis

Genomic DNA was isolated from rat mammary tissue using the Qiagen DNeasyTM Kit (Qiagen) according to the manufacturer’s instructions. The extent of global DNA methylation was evaluated with a well-established radiolabeled [3H]-dCTP extension assay as previously described.70

Western blot analysis of protein expression

Total protein was extracted from mammary tissue using 1% sodium dodecyl sulfate (SDS) and sonication to homogenize the tissue. Equal amounts of proteins (20 μg) were separated by SDS-polyacrylamide electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Baie d’Urfé, Quebéc). The membranes were probed with primary antibodies against p-p42/44 MAPK, p-p38, p-JNK (1:500, Cell Signaling Technology), KU70 (1:500, Santa Cruz Biotechnology), apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (Polβ), nijmegen breakage syndrome 1 (NBS1), and β-actin (1:1000, Abcam). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and the ECL Plus immunoblotting detection system (Amersham) were used to reveal antibody binding. Chemiluminescence was detected with Biomax MR films (Eastman Kodak). All membranes were stained with Coomassie Blue (BioRad) to confirm equal protein loading. Signals were quantified using NIH ImageJ 1.63 Software and related to loading controls. Experiments were repeated twice to ensure reproducibility.

Immunohistochemistry

Following pathological examination, the tissues were assembled into tissue microarrays (TMAs) with 4.5 mm cores. TMAs (Pantomics, Inc.) offer great benefits to perform fast and efficient analysis using less tissue and reagents.

The tissue sections were fixed to the slides by baking at 60 °C for 1 h, deparaffinized, rehydrated, and steamed in antigen retrieval citrate buffer (pH 6.0) (DAKO). Endogenous peroxidase activity was quenched in 3% H2O2 and the slides were blocked in 3% goat serum (Santa Cruz Biotechnology). The slides were probed with primary antibodies against histone H3 lysine 4 (H3K4) and H3K9 trimethylation (1:500; Abcam), histone H4K5, H4K8, H4K12 acetylation, histone H3 serine 10 (H3S10) phosphorylation (1:300; Cell Signaling Biotechnology), and DNA methyltransferases DNMT1 and DNMT3A (1:200; Santa Cruz Biotechnology). Binding was detected using avidin-biotinylated horseradish peroxidase and visualized with 3,3′-diaminobenzidine (DAB) (ABC Staining System; Santa Cruz Biotechnology). The tissues were counterstained with hematoxylin (Santa Cruz Biotechnology). Staining for histone modifications was scored in a blinded fashion in at least 5 high power fields in each of the six animals per group.

Statistical analysis

Statistical analysis was conducted using the Student t test. P values < 0.05 were considered significant. Statistical significance was only calculated within each group of exposures compared with the 6, 12, and 18 week untreated controls (not compared between groups).

Acknowledgments

Work was supported by the Alberta Cancer Research Institute and Canadian Breast Cancer Foundation Operating grants to OK. KK was a recipient of the Vanier Canada Graduate Scholarship, National Science and Engineering Research Council Graduate Scholarship, Queen Elizabeth II Graduate Scholarship, and the Alberta Heritage for Medical Research Scholarship. We are thankful to Rocio Rodriguez-Juarez and Igor Koturbash for technical assistance, to Dr Roderick T Bronson and Dr Langxing Pan for their help and advice with histopathology, to Dr Igor Pogribny for useful discussions, and to Dr Valentina Titova for careful proofreading of this manuscript.

Glossary

Abbreviations:

E2

17-β estradiol

(E2)

estrogen

IR

ionizing radiation

DNMT

DNA methyltransferase

HMT

histone methyltransferase

IHC

immunohistochemistry (IHC)

H4

Histone

H3

Histone 3

K

lysine

S

serine

me3

trimethylation

Ac

acetylated

p

phosphorylated

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Footnotes

References

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