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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2016 Feb 15;97(1):27–36. doi: 10.1111/iep.12164

Effect of bisphenol A on morphology, apoptosis and proliferation in the resting mammary gland of the adult albino rat

Marwa A A Ibrahim 1, Reda H Elbakry 1, Naglaa A Bayomy 1,2,✉
PMCID: PMC4840247  PMID: 26877094

Summary

Bisphenol A (BPA) is a synthetic oestrogen that is extensively used in a wide range of daily used plastic products. This makes it one of the environmental chemicals that may have impact on human health. Due to its oestrogenic effect, BPA might affect the mammary gland. This study aimed to investigate the influence of BPA on the histological structure of the mammary gland of the adult female albino rat and its effect on epithelial cell proliferation and apoptosis status, in addition to its possible modulating effect on estrogen receptor expression. Thirty female adult albino rats were divided into control and experimental groups. The rats in the experimental group were gavaged with 5 mg/kg BPA daily for 8 weeks. The mammary glands were dissected and processed for histological and immunohistochemical stains for Ki‐67, activated caspase‐3 and estrogen receptor alpha (ER‐α). BPA induced an increase in the number and size of the acini and ducts in the mammary gland of treated rats with hyperplasia of their lining epithelial cells. The collagen fibre content was significantly increased in the connective tissue stroma separating the ducts. Immunohistochemical results showed a significant increase in Ki‐67 and caspase‐3, but a non‐significant increase in ER‐α expression. Bisphenol A induced structural changes and affected the proliferation rate of mammary glands, so it might be one of the predisposing factors for breast cancer.

Keywords: bisphenol A, caspase‐3, ER‐α, histology, Ki 67, mammary gland

Bisphenol A (BPA), a synthetic oestrogen, is the building block of polycarbonate plastic. It is broadly used in a wide range of products including toys, baby bottles, drinking bottles, food containers, water pipes and medical devices (Welshons et al. 2006). Plastics are used extensively in modern life, and hence, BPA is liberated into the surrounding environment either directly or due to incomplete polymerization under normal conditions. Acidic, basic or heated environments speed up the hydrolysis of the ester bonds linking BPA monomers, thus promoting the release of BPA and enhancing its rate of migration. Owing to this leaching out, the BPA is found in streams, rivers and drinking water. The principal source of exposure occurs through food and drink (Lakind & Naiman 2011; Loganathan & Kannan 2011).

Despite the fact that endogenous oestrogen is more potent than xenoestrogen, the latter is more hazardous due to its prolonged existence in the environment and resistance to chemical or enzymatic degradation (Teilmann et al. 2002). In humans the BPA is absorbed rapidly following ingestion and then converted to a number of metabolites in the liver, mainly BPA glucuronide (Kuch & Ballschmiter 2001; Kamrin 2004; Fenichel et al. 2013).

BPA influences a wide diversity of physiological functions involved in development, reproduction and metabolism. These effects are mainly attributable to its oestrogen‐like action as it binds the same cellular receptors, thus changing the hormonal balance of the body (Valentino et al. 2013). It has been strongly suggested that BPA exposure is linked to many diseases which are influenced by the variation in oestrogen level, such as ovarian disease and breast cancer. In addition, a relationship between body fat distribution in women and BPA blood levels has been proposed (Carwile & Michels 2011).

Ki‐67 is a nuclear protein essential for cellular proliferation, and is associated with ribosomal RNA transcription. It can be detected within the cell nucleus during interphase. Ki‐67 is present throughout the active phases of the cell cycle, but is absent in resting cells. (Scholzen & Gerdes 2000; Bullwinkel et al. 2006). Ki‐67 has proven to be a useful marker of proliferation that correlates strongly with in vivo labelling of the S‐phase of with bromodeoxyuridine (BrdU) in tissues that are undergoing renewal and regeneration, and thus it is considered to be an excellent index if one wishes to determine the growth fraction of a given cell population (Muskhelishvili et al. 2003).

Cysteine‐aspartic proteases (caspases) are a family of cysteine proteases that have essential roles in apoptosis. Two classes of caspases are associated with the apoptotic process: initiator caspases and effector caspases. Initiator caspases cleave inactive pro‐forms of effector caspases. Activated effector caspases cleave other protein substrates inside the cell to elicit the apoptotic process (Gregersen 2007). Inadequacy of apoptosis is believed to be one of the underlying mechanisms in tumour development (Pettigrew & Cotter 2009).

Estrogen receptors are nuclear receptors present inside the cells of different tissue types. These receptors are the target of oestrogen hormone. The activated receptors regulate the activity of different genes (Levin 2005). There are two forms of estrogen receptors, alpha and beta receptors (ER‐α and ER‐β), which are differentially distributed in the tissues. ER‐α is found in breast cancer cells, the endometrium, the ovarian stromal cells and the hypothalamus. ER‐β is detected in benign breast tissue, ovarian granulosa cells, the kidney, the brain, the bone and the heart (Babiker et al. 2002; Shaaban et al. 2003; Yaghmaie et al. 2005). Both receptors have varying affinity for different ligands. Attachment of oestrogen to the ER provokes the proliferation of mammary cells, with a consequent augmentation in cell division and DNA replication. Thus it may be linked to tumour formation under certain conditions (Deroo & Korach 2006).

There is an increasing rate of breast cancer worldwide and there are several environmental factors that have been implicated in this increase. This study aimed to investigate the influence one such factor, BPA, on the histological structure of the resting mammary gland of the adult female albino rat and to explore its effect on epithelial cell proliferation and apoptosis status, in addition to its possible modulating effect on ER expression.

Materials and methods

Thirty female albino rats aged eight weeks and weighing 150–200 grams each were used in this study. The animals were housed in appropriate cages and fed with a commercial rodent chow (ad libitum) and tap water. They were kept on a 12‐h light/12‐h dark cycle before the experiment and throughout the study period. They were acclimatized to the laboratory conditions for two weeks prior to the initiation of the experiment.

The rats were divided into two groups: Group I (control group): 20 rats were subdivided into two subgroups: negative control group, where 10 rats were kept without any treatment and vehicle control group, where another 10 rats were gavaged with 1 ml of commercial corn oil daily for 8 weeks. Group II (experimental group): 10 rats were gavaged with 5 mg BPA/kg body weight dissolved in 1 ml of commercial corn oil daily for 8 weeks. Previous studies revealed that chronic exposure to this dose altered some biological endpoints (Welshons et al. 2006; Braniste et al. 2010). Bisphenol A (BPA), cat# 239658, was purchased from Sigma‐Aldrich Chemical Co (St. Louis, MO, USA).

All rats were anesthetized 24 h after the last dose by an intraperitoneal injection of pentobarbital (50 mg/kg body weight) (Zhang et al. 2015). The animals were then perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4. The mammary glands were dissected out and processed for:

Light microscopic examination

Mammary gland specimens were immersed in 4% paraformaldehyde for 24 h and processed for paraffin sections of 5 μm thickness. Sections were stained with haematoxylin and eosin (H&E) and Mallory's trichrome stain (Bancroft & Layton 2012).

Immunohistochemistry

Immunohistochemical staining was performed for Ki‐67, activated caspase‐3 and ER‐α. Sections were treated with xylene to remove paraffin and rehydrated through a series of alcohols, washed with phosphate‐buffered saline (PBS) and then microwaved in citrate buffer (pH 6) for antigen retrieval. Non‐specific binding was blocked by incubation with normal goat serum for 1 h. The sections were incubated overnight at 4°C in a humid chamber with a monoclonal antibody against Ki‐67 and activated caspase‐3 in a dilution of 1:100 (NeoMarkers; Lab Vision, Fremont CA, USA) and polyclonal anti‐ER‐α (Thermo Fisher Scientific, Waltham, MA, USA) in a dilution of 1:400 using the streptavidin–peroxidase method according to the manufacturer's protocol. Corresponding biotinylated secondary antibody was applied to the sections for 1 h in a humidified chamber at room temperature. Slides were rinsed with PBS, and the sections were then counterstained with Mayer's haematoxylin. Positive controls for Ki‐67 were from the normal tonsil, for caspase‐3 from the placenta and for ER‐α from the breast carcinomas. Negative control was made by the exclusion of the primary antibody (Kobayashi et al. 2013).

Morphometric and quantitative analysis

Leica Qwin 500 C Image analyzer computer system (Leica Imaging System LTD., Cambridge, UK) at Central Research Lab, Tanta Faculty of Medicine, Egypt, was used to obtain the morphometric data in this study. Ten non‐overlapping fields in the slides of each animal of each group were examined.

  1. The area of collagen fibre content in the Mallory's trichrome‐stained sections (estimate area %/20 μm2 frame) per slide (five slides in each group) was examined a magnification of 400.

  2. The number of Ki‐67‐immunostained cells (proliferation index), were counted (×400) and the result expressed as a percentage of the total cells counted (the number of labelled cells × 100/the total cell number).

  3. The number of caspase‐3‐positive epithelial cells was counted (×400) the result was expressed as a percentage of the total cells counted (the number of labelled cells × 100/the total cell number).

  4. In the mammary gland was estimated (×400). It was measured by using the colour detect menu and in relation to a standard measuring frame.

Statistical analysis

Student's t‐test was used for statistical evaluation of the data using the statistical package for social sciences (version 11.5; SPSS Inc., Chicago, IL, USA) statistical analysis software. All values were expressed as mean ± standard deviation. The level of significance was set at P ≤ 0.05.

Ethical approval

All the procedures were performed according to the Guide for Care and Use of Laboratory Animals and were approved (No. 3585/06/14) by the Local Ethics Committee of Faculty of Medicine, Tanta University, Egypt.

Results

There were no deaths recorded throughout the experimental period.

Histological results

Haematoxylin and eosin stains

Light microscopic examination of H&E‐stained sections of the rat mammary gland from both the control subgroups showed normal histological architecture of resting mammary gland with scattered small‐ and medium‐sized mammary ducts and a few acini surrounded by an abundant content of adipose connective tissues in which small blood vessels were observed (Figure 1). Ducts and acini were lined by a single layer and sometimes bilayer of cubical epithelium. Individual cells had a moderate amount of slightly basophilic cytoplasm (Figure 2).

Figure 1.

Figure 1

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A photomicrograph of a section in the mammary gland of the control group showing few scattered small‐ and medium‐sized mammary ducts (thin arrow) and few acini (thick arrow) surrounded by an abundant content of adipose connective tissues containing small blood vessels (asterisk) (H&E, ×200, scale bar: 40 μm).

Figure 2.

Figure 2

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A higher‐magnification photomicrograph of the previous one showing ducts (thin arrows) and acini (thick arrows) lined by a single layer or bilayer of cubical epithelium. Individual cells have a moderate amount of slightly basophilic cytoplasm. An abundant content of adipose connective tissues is observed with the small blood vessels (asterisk) (H&E, ×400, scale bar: 20 μm).

BPA‐treated group showed an increase in the number and size of ducts and acini. The nuclei of the epithelium were variable in size and shape and appeared very dark (Figures 3 and 4). The ducts were enlarged with a massive increase in the number of lining epithelial cells (Figure 5). The stroma of BPA‐treated group also exhibited morphologic changes in the extracellular matrix. A dense stroma layer was formed around mammary epithelial structures, and a fibroblastic/cellular stroma replaced the normal adipose tissue of the mammary gland. In addition, huge fat cells were observed in the adipose connective tissue (Figures 3, 4, 5, 6). Mononuclear inflammatory cell infiltration and dilated congested blood vessels were also observed (Figures 3, 4 and 6).

Figure 3.

Figure 3

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing numerous wide ducts (thin arrows) and acini (thick arrows) with dense cellular stroma (arrow heads) in between. Note the presence of mononuclear cellular infiltration (notched arrow) (H&E, ×200, scale bar: 40 μm).

Figure 4.

Figure 4

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A higher‐magnification photomicrograph of the previous one showing numerous ducts (thin arrows) and acini (thick arrows). The nuclei of the epithelium are variable in size and shape and appear very dark‐stained. A dense stroma (arrow head) is observed around the mammary epithelial structures. Mononuclear cellular infiltration (notched arrow) and dilated congested blood vessels (curved arrow) are observed (H&E, ×400, scale bar: 20 μm).

Figure 5.

Figure 5

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing the mammary ducts with a massive increase in the number of lining epithelial cells (thin arrow) and acini (thick arrow) with variable‐shaped dark‐stained nuclei. Note the huge fat cells in the adipose connective tissues (asterisks) (H&E, ×400, scale bar: 20 μm).

Figure 6.

Figure 6

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing mononuclear cellular infiltration (notched arrow) and dilated congested blood vessels (curved arrow) within the dense cellular adipose connective tissues (arrow head) (H&E, ×400, scale bar: 20 μm).

Mallory's trichrome stain

Mallory's trichrome‐stained sections of the mammary gland of the control group showed few blue‐stained collagen fibres in the connective tissue stroma between and around the ducts (Figure 7). The sections examined from the BPA‐treated group showed an abundant content of collagen fibres in the connective tissue stroma separating the ducts (Figure 8). Morphometric analysis was consistent with this data and showed a significant increase in the collagen fibre content in the BPA‐treated group compared with the control group (Table 1, Figure 9a).

Figure 7.

Figure 7

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A photomicrograph of a section in the mammary gland of the control group showing few collagen fibres surrounding the ducts (arrows) (Mallory's trichrome, ×400, scale bar: 20 μm).

Figure 8.

Figure 8

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing an abundant content of collagen fibres (arrows) in between the ducts and acini with apparently reduced fatty tissue formed of the huge fat cells (asterisks) (Mallory's trichrome, ×400, scale bar: 20 μm).

Table 1.

Morphometric and quantitative analysis of the effects of BPA treatment on mammary gland specimens

Parameters Control BPA‐treated group Significance P < 0.05
Collagen fibre volume fraction (%) 2.41 ± 0.55 7.18 ± 0.63 <0.0001
Epithelial cell proliferation index (%) 6.33 ± 0.42 23.65 ± 1.57 <0.0001
Epithelial cell apoptosis index (%) 4.59 ± 0.36 21.78 ± 1.09 <0.0001
ER‐α immunoexpression (optical density) 19.31 ± 3.93 23.01 ± 4.48 >0.05
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Data are expressed as mean ± standard deviation, P value = probability of chance, P < 0.05 is significant, tested by using Student's t‐test.

Figure 9.

Figure 9

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(a) Collagen fibres volume fraction %. (b, c) Proliferation and apoptotic index. (d) Immunoexpression (optical density) of ER‐α. Tested using Student's t‐test; *Significant at P < 0.05.

Immunohistochemical results

Ki 67 antigen immunostaining

Immunostained mammary gland sections with Ki‐67 of the control group showed a few epithelial cells exhibiting a positive nuclear and/or cytoplasmic reaction for Ki‐67 in the form of a brown colour (Figure 10). In the BPA‐treated group, many epithelial cells exhibited a positive nuclear and/or cytoplasmic Ki‐67 immunostaining (Figure 11). Quantitative analysis confirmed a significantly higher Ki‐67 index in the BPA‐treated group when compared with the control group (Table 1, Figure 9b).

Figure 10.

Figure 10

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A photomicrograph of a section in the mammary gland of the control group showing few cells with Ki‐67‐positive nuclear and/or cytoplasmic immunoreaction in the form of a brown colour (arrows) in both epithelial cells lining the ducts and the adipose connective tissues (Ki‐67 immunostaining, ×400, scale bar: 20 μm; inset ×1000, scale bar: 10 μm).

Figure 11.

Figure 11

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing many cells with Ki‐67‐positive nuclear and/or cytoplasmic immunoreaction in the form of a brown colour (arrows) in both the epithelial cells lining the ducts and the adipose connective tissues (Ki‐67 immunostaining, ×400, scale bar: 20 μm; inset ×1000, scale bar: 10 μm).

Activated caspase‐3 antigen immunostaining

Immunostained mammary gland sections with activated caspase‐3 of the control group showed a few epithelial cells exhibiting a positive cytoplasmic reaction for caspase‐3 in the form of a brown colour (Figure 12). In the BPA‐treated group, many epithelial cells exhibited a positive cytoplasmic caspase‐3 immunostaining (Figure 13). Quantitative analysis showed that the percentage of caspase‐3‐positive cells in the BPA‐treated group was significantly increased as compared with the control group (Table 1, Figure 9c).

Figure 12.

Figure 12

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A photomicrograph of a section in the mammary gland of the control group showing few cells with caspase‐3‐positive cytoplasmic immunoreaction in the form of a brown colour both in the epithelial cells lining the ducts or the acini and in the adipose connective tissues (caspase‐3 immunostaining, ×400, scale bar: 20 μm; inset ×1000, scale bar: 10 μm).

Figure 13.

Figure 13

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing abundant cells with caspase‐3‐positive cytoplasmic immunoreaction in the form of a brown colour (arrows) both in epithelial cells lining the ducts or the acini and in the adipose connective tissues (caspase‐3 immunostaining, ×400, scale bar: 20 μm; inset ×1000, scale bar: 10 μm).

Estrogen receptor alpha (ER‐α)

In the control group, the sections of the mammary gland showed a strong positive ER immunoreactivity in the nuclei and/or cytoplasm of duct cells and in the cells of the fibromuscular stroma in the form of a brown colour (Figure 14). ER immunostaining in the BPA‐treated group was apparently more intense in the nuclei and/or cytoplasm of the duct epithelial cells and in the cells of the fibromuscular stroma compared with the control group (Figure 15). Morphometric analysis of the optical density of ER‐α‐positive cells in the BPA‐treated group showed a non‐significant increase when compared with the control group (Table 1, Figure 9d).

Figure 14.

Figure 14

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A photomicrograph of a section in the mammary gland of the control group showing a strong ER‐alpha‐positive nuclear and/or cytoplasmic immunoreaction in the form of a brown colour (arrows) both in the epithelial cells lining the ducts or the acini and in the adipose connective tissues (ER‐alpha immunostaining, ×400, scale bar: 20 μm; inset ×1000, scale bar: 10 μm).

Figure 15.

Figure 15

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A photomicrograph of a section in the mammary gland of the BPA‐treated group showing relatively intense ER‐alpha‐positive nuclear and/or cytoplasmic immunoreaction in the form of a brown colour (arrows) in both the epithelial cells lining the ducts or the acini and in the adipose connective tissues (ER‐alpha immunostaining, ×400, scale bar: 20 μm; inset ×1000, scale bar: 10 μm).

Discussion

During the past few decades there has been increasing awareness of the environmental impact of chemical pollutants. These chemicals impede the endocrine equilibrium of the body causing deleterious effects to many organs (Sikka & Wang 2008). Bisphenol A (BPA), an oestrogenic monomer, is used in enormous quantities each year (Liao & Kannan 2011). It has been proposed to cause changes in the structure and development of the mammary gland that could lead to malignant changes. Thus, it might have a role in the development of breast cancer (Moral et al. 2008; Rochefort 2013).

In the present study, the effect of BPA on the histological structure of the mammary gland of the adult female rats was examined. BPA caused an increase in the number of the ducts and acini of the mammary gland with hyperplasia of their lining epithelium. Collagen fibres showed an increase in both amount and distribution at the expense of the fat cells. These results were consistent with some previous studies that examined the effects of BPA on the mammary gland of different rodents, where their results showed a significant increase in the percentage of terminal ducts and terminal end buds (Markey et al. 2001; Muñoz‐de‐Toro et al. 2005). In addition, there were hyperplastic changes in the duct system in the form of excessive proliferation of the lining epithelium with enlarged variable‐shaped dark‐stained nuclei. Some authors have reported that female rats that were exposed prenatally to BPA showed an increase in the percentage of hyperplastic ducts, which could be related to the development of neoplastic lesions later on (Murray et al. 2007). Studies of Popa et al. (2014) showed that pubertal exposure to BPA increases the differentiation of the mammary gland structures and increases the proliferation of the epithelial cells. Exposing other species, such as monkeys, to BPA showed mammary duct hyperplasia and intraductal epithelial proliferation leading to an increase in the density of mammary buds and advanced development of the gland in comparison with the control group (Tharp et al. 2012).

Immunohistochemical staining for Ki‐67 was used to confirm the observed epithelial proliferation. Its expression showed a statistically significant increase in the BPA‐treated group. This was consistent with a previous work documenting a significant increase in the average number of cells per unit area of the epithelium in the BPA‐exposed animals as compared with the control ones (Vandenberg et al. 2007). Increased proliferation upon BPA exposure is supposed to be caused by the upregulation of proteins such as p16 and cyclin E, which are known to give rise to cellular senescence and induce proliferation (Qin et al. 2012). Although Ki‐67 is classically described as a nuclear protein strictly associated with cell proliferation, a peculiar cytoplasmic expression was observed in the control specimens. A similar finding was described in the normal tissue by Ciulla et al. (2009) who proposed that cytoplasmic expression of Ki67 might be a functional phenomenon that is shared by normal tissues undergoing postnatal remodelling. Moreover, the cytoplasmic expression of Ki‐67 detected on the treatment with BPA in this study has so far been only described in relation to high grades of mammary tumours (Faratian et al. 2009). Although the underlying mechanism of such expression is not yet elucidated, cross‐reactivity with other proteins, technical artefacts and Ki67 relocalization within the cell during the cell cycle were all proposed by Leonardo et al. (2007).

An altered amount and distribution of collagen fibres and subsequent reduced fat cells were observed in the current study. A dense stroma was formed around the mammary epithelial structures with mononuclear cellular infiltrate replacing the normal adipose tissue. These results were in agreement with previous studies documenting that BPA exposure promoted the maturation of the fat pad and altered the localization of collagen mainly in the peritubular area (Durando et al. 2007; Vandenberg et al. 2007). These fatty tissue changes are probably related to the effect of BPA on estrogen receptor (ER), mainly ER‐α, which has an inhibitory effect on adipocyte number and lipogenesis (Heine et al. 2000; Cooke et al. 2001). Some authors have hypothesized that these changes were due to either a decrease in the total number of cells or an increase in the amount of fat‐containing vacuoles stored in the cytoplasm of these cells. They postulated that apoptosis may play a pivotal role in the decreased cellular density of the developing fat pad of the BPA‐exposed females (Muñoz‐de‐Toro et al. 2005). Another contributing mechanism could be the central effect of oestrogen in suppressing the fat deposition in adults. It might also be a result of reducing the body weight resulting in decrease in the fat deposition (Nunez et al. 2001; Al‐Hiyasat et al. 2002).

Immunohistochemical staining for activated caspase‐3 showed expression in the control mammary tissues. This could be due to the predominance of ductal structures and the presence of mammary involution processes in the resting unstimulated mammary tissue (Belli et al. 2010). However, the BPA‐treated group showed a statistically significant increase in the number of caspase‐3‐positive cells in comparison with that of the control. Similarly, increased apoptosis with elevated expression of caspase‐3 was detected in the testes of mice pups exposed to BPA during pregnancy and lactation (Liu et al. 2013). Moreover, similar findings were observed in the liver of rats treated with BPA (Xia et al. 2014). Nevertheless, other researchers revealed that the mode of cell death due to BPA changed from apoptosis to necrosis and this was also associated with caspase activation (Li et al. 2012).

Immunohistochemical staining for ER‐α revealed no significant upregulation in the BPA‐treated group compared to the control group. This result was in accordance with the work of others suggesting that the effect of BPA on the expression of ER‐α in the stroma or the epithelial ducts of the mammary glands was not significant (Vandenberg et al. 2007). On the contrary, some authors reported a significant decrease in ER‐α expression in the hypothalamus of the juvenile and adult female rat upon BPA exposure (Rebuli et al. 2013). In the current study, additional cytoplasmic expression of ER‐α was observed. Such finding could be attributed to the ER‐α non‐nuclear signalling functions, which might be related to carcinogenic predisposition (Welsh et al. 2012). Although it has been well known that BPA is an endocrine‐disrupting agent, the principal mechanisms of its impact has not been fully established as yet. It has been proposed that the BPA might influence enzymatic activity and metabolism. It could also induce alteration in hormone receptor numbers and gene activity in target tissues (Richter et al. 2007).

Previous studies suggested that BPA acts through the activation of cell signalling pathways especially at very low concentrations. It has been documented that BPA causes an upregulation of vascular endothelial growth factor (VEGF) expression in cells by an ER‐dependent mechanism. VEGF augments the capillary permeability and breast tumour angiogenesis (Buteau‐Lozano et al. 2008). However, the most commonly accepted mechanism to explain the effect of BPA is its action as a selective ER modulator (Rochefort 2013). Many experimental studies in rodents have suggested that the BPA directly acts in the mammary cells through ER‐α resulting in a change in the endocrine environment of the mammary gland, leading to an increase in angiogenesis. This might explain the high frequency of preneoplastic lesions found later in life (Welshons et al. 2006). Additionally, ER‐α expression has been proposed to serve a role in modulating mammary tumorigenesis through defining its ratio to ER‐β expression. Benign lesions in which ER‐α is increased relative to ER‐β are at a significant risk of progression into an invasive breast cancer (Shaaban et al. 2003). Dairkee et al. (2013) attributed the deleterious effect of BPA on non‐malignant human breast epithelial cells in vitro to the increase in that particular ratio.

According to the results of this study, we propose that BPA mainly targets the proliferation process rather than modulating the estrogen receptor activity. It causes the replacement of the normal adipose tissue of the mammary gland with a cellular stroma with classical inflammatory signs. Taken together, these BPA‐induced changes set up the stage for a prominent and permanent mammary gland lesion which, with uncontrollable exposure to that chemical, could be related eventually to an increased incidence of breast cancer.

Conflict of interest

The authors of this manuscript have no conflict of interests to declare.

Funding source

This work was not supported by any funds from any organization.

Acknowledgements

The authors are grateful to the Histology Department, Faculty of Medicine, Tanta University, Egypt, for providing facilities and technical support during the study.

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