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Regular Article |
a Laboratorio de Endocrinología y Tumores Hormonodependientes, Faculty of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina
b Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111-1800
ABSTRACT
Environmental estrogens (xenoestrogens) are chemicals that bind to estrogen receptor, mimic estrogenic actions, and may have adverse effects on both human and wildlife health. Bisphenol A (BPA), a monomer used in the manufacture of epoxy resins and polycarbonate has estrogenic activity. In male rodents prenatal exposure to BPA resulted in modifications at the genital tract level. Our objective was to examine the effects of in utero exposure to low, environmentally relevant levels, of the xenoestrogen BPA on proliferation and differentiation of epithelial and stromal cells on the prepubertal rat ventral prostate. To characterize the periductal stromal cells phenotype the expression of vimentin and smooth muscle
-actin was evaluated. Androgen receptor (AR) and prostatic acid phosphatase (PAP) expression were also evaluated in epithelial and stromal compartments. Prenatal exposure to BPA increases the fibroblastic:smooth muscle cells ratio and decreases the number of AR-positive cells of periductal stroma of the ventral prostate. In contrast, no differences in AR expression were observed in epithelial cells between control and BPA-treated groups. No changes in proliferation patterns were observed in epithelial and stromal compartments; however, the expression of PAP was diminished in prostate ductal secretory cells of rats in utero exposed to BPA. Our results suggest that prenatal exposure to BPA altered the differentiation pattern of periductal stromal cells of the ventral prostate. These findings are significant in light of the data on human prostate cancers where alterations in the stroma compartment may enhance the invasive and/or malignant potential of the nascent tumor.
androgen receptor, early development, environment, male reproductive tract, prostate
INTRODUCTION
Environmental estrogens (xenoestrogens) are a diverse group of chemicals that bind to estrogen receptor, mimic estrogenic actions, and may have adverse effects on both human and wildlife health and reproductive performance [1, 2]. Bisphenol A (BPA), a monomer used in the manufacture of epoxy resins and polycarbonate, is a xenoestrogen. BPA binds to estrogen receptors and ß, induces progesterone receptors, and promotes MCF-7 breast cancer cell proliferation [3]. In vivo administration of BPA to ovariectomized Fischer 344 rats induced cell proliferation in the epithelia of the uterus and vagina and hyperprolactinemia [4, 5]. Human exposure to BPA is not insignificant, as microgram amounts of BPA were detected in liquid from canned vegetables [6] and in the saliva of patients treated with dental sealant [7].
Estrogen administration to rodents during early development leads to persistent alterations in the growth and function of the prostate gland [8, 9]. Neonatal estrogenization of rodents is being used as a model to evaluate the role of exogenous and endogenous estrogens as a predisposing factor for prostatic diseases later in life [10]. Prenatal exposure to estrogens (i.e., 17ß-estradiol, diethylstilbestrol) affects the size and weight of the mouse prostate gland; the dose-response curve for this end point follows an inverted U-shaped dose-response curve whereby lower doses result in larger effects [8]. In addition, prenatal exposure to BPA and arochlor modified anogenital distance (AGD), increased prostate size, and decreased epididymal weight [11, 12]. These results suggest that elevated estrogen levels during fetal life may affect the expression of genes involved in the morphogenesis of the gland and, in turn, result in persistent changes in the histological architecture of the gland.
The stromal microenvironment is an important determinant in the progression from a normal prostatic epithelium to an invasive carcinoma [13, 14]. Alterations in the stromal compartment of prostatic tumors may enhance the invasive and/or malignant potential of the nascent epithelial tumor [13]. A growing body of evidence suggests that epigenetic influences, such as elevated estrogen levels during critical periods in the development of the prostate gland, may predispose for abnormal function and disease in later life [8].
The objective of this investigation was to examine the effects of in utero exposure to low, environmentally relevant levels of BPA on the pattern of proliferation and differentiation of epithelial and stromal cells on the prepubertal rat ventral prostate.
MATERIALS AND METHODS
Animals and Experimental Design
Sexually mature female rats of an inbred Wistar-derived strain bred at the Department of Human Physiology (Santa Fe, Argentina) were used. Animals were maintained under controlled environment (22 ± 2°C; lights-on from 0600 to 2000 h) and had free access to pellet laboratory chow (Nutric, Córdoba, Argentina) and tap water supplied from glass bottles.
All rats were handled in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals issued by the U.S. National Academy of Sciences.
Animals were placed into three experimental groups: dimethylsulfoxide (DMSO) vehicle-treated (control), 25 µg/kg body weight/day of BPA (25-BPA), and 250 µg/kg body weight/day of BPA (250-BPA).
Females in proestrous were caged overnight with males of proven fertility. The day that sperm were found in the vagina was designated Day 1 (D1) of pregnancy. Timed-pregnant rats were assigned to each group (n = 4 mothers per treatment group) and then individually housed in stainless steel cages. In our colony, delivery occurs on D23 between 1230 and 1400 h [15]. On gestation D8, a miniature osmotic pump (model 1002; Alza Corp., Palo Alto, CA) was inserted s.c. over the spine caudal to the scapula. Osmotic pumps were filled with one of two doses of BPA (Sigma Chemical Co., St. Louis, MO) dissolved in DMSO (Sigma) or only with DMSO as a vehicle in control rats. BPA or its vehicle was administered continuously from D8 of gestation to day of parturition (D23). Dose levels for BPA were 25 µg/kg/day or 250 µg/kg/day. These solutions were released at a rate of 0.25 µl/h. No signs of acute or chronic toxicity were observed and no significant differences in weight gain were recorded during gestation between BPA and controls mothers. There were no differences in litter sizes and pups body weight either at birth or at weaning. Moreover, sex ratios of the litters were comparable in the three groups and AGD distance measured at birth and at Postnatal Day 4 did not differ among groups.
After parturition, pups were weighed, sexed according to AGD, and litters of eight pups (preferably four males and four females) were left with lactating mothers until weaning on Postnatal Day 22. Males from a single mother were assigned to one of three treatment groups; siblings were excluded in the same experimental group. At weaning, pups were individually identified by ear tag and housed in groups of four according to one of three treatments. On Postnatal Day 30, all pups were injected with bromodeoxyuridine (BrdU; Sigma) (6 mg/100 g body weight in 1.5 ml PBS, i.p.) and killed by decapitation 2 h later.
Ventral prostates were microdissected under a Leica GZ6 Series (Leica Inc., Buffalo, NY) dissecting microscope, collected, and fixed by immersion in 10% buffered formalin for 6 h at room temperature (RT). Fixed tissues were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. Serial sections (5 µm in thickness) of ventral prostate were mounted on 3-aminopropyl triethoxysilane (Sigma)-coated slides and dried for 24 h at 37°C. For each prostate specimen, three sections separated at 20-µm intervals were evaluated. In order to secure uniformity between sections of each animal a nonparametric analysis of variance between sections of the same specimen was performed.
Immunohistochemistry
Bromodeoxyuridine incorporation into proliferating cells was determined immunohistochemically using the protocol described by Kass et al. [16]. Briefly, slides were cleared with xylene and rehydrated in a descending series of ethanol solutions. Then, microwave pretreatment (antigen retrieval method) was performed; immediately after, the slides were subjected to acid hydrolysis by incubation in 2 N HCl for 30 min at 37°C followed by 0.1 M borate buffer (pH 8.5) for 10 min. To block the endogenous peroxidase activity, slides were treated with H2O2 in methanol for 15 min. Sections were incubated 30 min with 10% normal goat serum to block nonspecific binding. Overnight incubation at 4°C in a humidified chamber with diluted monoclonal antibody to BrdU was performed (see Table 1), followed by incubation with a biotinylated secondary antibody (Sigma) and streptavidin-peroxidase complex (Sigma). Diaminobenzidine (DAB) (Sigma) was used as chromogen substrate for 10 min at RT, and rinsed in running water. Harris hematoxylin (Biopur, Rosario, Argentina) was used as counterstaining solution. Samples were mounted with permanent mounting medium (PMyR; Buenos Aires, Argentina).
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Expression of several markers was evaluated by immunohistochemistry [17] to characterize the cellular phenotype. The procedure was similar to the above mentioned for BrdU incorporation skipping the acid hydrolysis step. Primary antibody incubation was done at 4°C during 14–16 h (Table 1). Visualization of antigens was achieved using DAB and sections were stained with Mayer hematoxylin (Biopur) as a blue nuclear counterstain.
Each immunohistochemical run included positive and negative controls. In the negative control slides the primary antibody was replaced with nonimmune mouse or rabbit serum (Sigma).
Bromodeoxyuridine Incorporation Index
BrdU incorporation was determined quantitatively in the epithelial (basal + glandular) and stromal cell nuclei (periductal and interductal). The periductal stroma zone was defined like a ring area of 18 µm wide around the ducts (from basement membrane towards the outer layers). Figure 1 shows a representative field where all the evaluated areas are present.
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Twenty representative fields in each section were scored using a Dplan 40x objective lens (Olympus Optical Co., Ltd., Tokyo, Japan) and a graticule placed in the eyepiece. All immunostained epithelial and stromal nuclei in the defined regions, regardless of intensity, were scored as positive. Positive cells were expressed as the percent ratio of total number of epithelial or stromal cells evaluated in the ventral prostate.
Image Analysis and Morphometry
Image analysis was performed using Image Pro-Plus 4.1.0.1 system (Media Cybernetics, Silver Spring, MD). Images were recorded by a Sony ExwaveHAM color video camera (Sony Electronics Inc., Sony Drive, Park Ridge, NJ), attached to an Olympus BH2 microscope (illumination: 12-V tungsten-halogen lamp, 100 W, equipped with a stabilized light source; Olympus Optical Co.), using a Dplan 100x objective (numerical aperture = 1.25). The microscope was set up properly for Koehler illumination; a reference image of an empty field for the correction of unequal illumination (shading correction) was recorded as well as calibration of the measurement system with a reference slide was done before any measurement started. The resolution of the images was set to 640 x 480 pixels, and the final screen resolution was a 0.103 µm/pixel.
The expression of the cytoskeletal proteins was quantified in the periductal stroma (same area as it was defined for quantitation of BrdU expression). Microscopic fields covering the whole periductal stromal areas (between 40 and 50 fields) from each section were recorded. Means for each rat were calculated and used for statistical analysis. Using Auto-Pro macro language, an automated standard sequence operation was performed to measure the percentage of the reference periductal area (relative area) occupied by vimentin or smooth muscle -actin (-SMA) cells. In this automated analysis process, the relative area evaluation was measured through color segmentation analysis that extracts objects by locating all objects of the specified color(s) and setting everything else to black.
For estrogen (ER)- and androgen receptor (AR)-positive (+) cell characterization, two sections were evaluated for each prostate specimen and 30 representative fields in each section were scored using a Dplan 40x objective. All immunostained epithelial and stromal nuclei (periductal and interductal), regardless of intensity, were scored as positive. Positive cells were expressed as the percent ratio of total number of epithelial or stromal cells measured in the studied area of the ventral prostate.
In order to obtain quantitative data about prostatic acid phosphatase (PAP) expression in ductal epithelial cells, two sections were evaluated for each prostate specimen and 30 representative fields in each section were digitalized and recorded using a Dplan 40x objective. Using Auto-Pro macro language, an automated sequence operation was created to measure the optical density (OD). In this automated analysis process, the images of immunostained slides were converted to an 8-bit gray scale, and the operator calibrated the gray level so that the background staining of the histological slide was regarded as zero. The OD was measured as an average gray, being equal to the sum of the gray intensity of each pixel divided by the number of pixels measured. All epithelial cells with positive OD values were considered as PAP-positive cells. Results were expressed as the percent ratio of total number of epithelial cells measured in the studied area.
Statistics
Differences between groups were evaluated by the Kruskal-Wallis one-way analysis of variance. Probabilities were assigned using the Mann-Whitney U-test [18].
RESULTS
Stromal Cell Characterization and Proliferative Activity
The ductal-stroma architecture of the 30-days prepubertal control glands resembled that of an adult ventral prostate. No differences were found in the BrdU labeling index in the epithelial and stromal compartments between BPA-treated and control groups (see Table 2).
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In control animals, the periductal ring was occupied proximally by a layer of several cells thick and in the distal region of ducts it thinned to two to three cells. These cells were vimentin or -SMA(+), indicating that the periductal stroma was composed of fibroblastic and smooth muscle cells (Fig. 2, A and B). In control animals the periductal-staining pattern for vimentin was discontinuous in proximal and distal regions while the -SMA(+) cells formed a continuous ring around the ducts (Fig. 2, A and B). In the BPA-treated groups the vimentin(+) cells (fibroblasts) showed a uniform, continuous halo around the proximal and distal ducts; the halo was thicker around the proximal ducts than around the distal ones (Fig. 2E). In contrast the -SMA(+) cells formed a discontinuous layer in both distal and proximal ducts; these zones were thinner than in control groups (Fig. 2F). As shown in Figure 2E, in treated groups the stromal cellular population that maintains close contact with the ductal basal membrane was mainly made up of fibroblastic cells rather than smooth muscle cells.
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Figure 3 shows relative areas occupied by vimentin(+) cells and -SMA(+) cells in the periductal compartment of the proximal regions of rat ventral prostates in different experimental conditions. In BPA-treated groups the total thickness of the periductal stromal cells ring in proximal and distal regions was similar to controls. However, in proximal and distal regions the relative area occupied by vimentin(+) cells in the periductal ring was significantly larger than in the control group (Fig. 2, E versus A). The relative area occupied by the -SMA(+) cells was smaller in BPA-treated groups than in controls (Fig. 2, F versus B). No differences were observed in the relatives areas occupied by vimentin(+) or -SMA(+) cells between 25-BPA- and 250-BPA-treated groups.
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Androgen Receptor and ER Distribution
Epithelial cells (basal and glandular) were strongly stained for AR in proximal and distal prostate regions in all groups studied (Fig. 2, C and G), and no statistical differences were found among them (Table 2). In the periductal stroma of control groups most cells were AR(+), and the AR staining intensity was higher in smooth muscle cells than in fibroblasts. In BPA-treated groups significantly smaller quantities of AR(+) stromal cells were found in proximal and distal regions (Fig. 2G). Table 2 and Figure 4A shows the percentage of periductal stromal AR(+) cells in different experimental conditions. Thirty percent of the interductal fibroblastic cells were AR(+), and no differences were found between BPA-treated and control groups (see Table 2).
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Epithelial and stromal compartments were negative for ER in all groups studied (Table 2).
Prostate Acidic Phosphatase Expression
PAP immunostaining was restricted to glandular epithelial cells of proximal and distal ducts. No expression was found in the stromal compartment and the signal:background ratio was high in the control group. In control animals most epithelial cells (Fig. 4B) were PAP(+), and PAP expression covered all the cytoplasm; however, the apical regions were more heavily stained than basal regions. This polarized arrangement fits the secretory nature of PAP protein. In contrast, in BPA-exposed animals the presence of PAP was significantly lower and it was restricted to a pale, apical, discontinuous staining in only a few epithelial cells (Fig. 2, D versus H). The number of PAP(+) cells was significantly reduced in BPA-treated animals and no differences were observed between 25-BPA and 250-BPA groups (Fig. 4B).
DISCUSSION
Most studies on the effect of exogenous estrogens on the development of the rat prostate have been carried out using high doses that were administered postnatally [9, 19, 20]. However, data from recent reports suggested that the sensitivity to estrogens is also significantly increased during prenatal development [12, 21]. Positional effects were also reported, whereby male fetuses that are placed between two females in the uterus showed an increased area of developing prostatic epithelial buds [22] when compared to males that develop between males. These effects are believed to be due to the higher estrogen levels in the males placed between two females. Recently, an increasing number of reports are showing deleterious effects of low, environmentally relevant doses of xenoestrogens on the development and function of rodent reproductive organs [11, 12, 23–25]. These studies have utilized different species, strains, doses, and routes of administration. However, the low-dose studies provided limited information concerning histoarchitectural changes underlying anatomical malformations and increased prostate weight [8, 11, 12, 26, 27]. The present study shows that prenatal exposure to low environmentally relevant doses of BPA affects histoarchitecture and epithelial secretory pattern of PAP, suggesting alterations in glandular cell function at puberty.
In the normal developing prostate, periductal mesenchymal cells differentiate to form a multicellular layer of smooth muscle cells and a thin layer of fibroblasts that maintain intimate contact with the basal membrane of ductal epithelium [28, 29]. Our data show that prenatal exposure to BPA altered the differentiation pattern of the periductal stromal cells of the rat ventral prostate. In BPA-treated groups the presence of a thick layer of vimentin(+)--SMA(-) cells in the periductal zone contrasts with the multicellular smooth muscle vimentin(-)--SMA(+) layer observed in control animals at Postnatal Day 30. The presence of the thicker layer of fibroblasts in BPA-treated animals could be the result of either a modified proliferation:apoptosis ratio in early stages of development or altered differentiation of periductal mesenchymal cells. No differences in the BrdU index were recorded; however, a change in proliferative-apoptotic rates before this age cannot be ruled out. A reduced number of smooth muscle cells was observed in the periductal stroma of proximal and distal regions of BPA-treated rats. In addition, a significantly lower percentage of AR(+) stromal cells were found in proximal and distal regions of the BPA-exposed animals. These observations are in agreement with those reported in neonatally estrogenized rats [9, 19]. Whereas it has been shown that neonatal exposure to high doses of estradiol-induced ER expression in periductal smooth muscle cells of developing prostate lobes [30]; in this study, no expression of ER was observed either in epithelial or stromal compartments of the BPA-exposed group. Further studies will be performed to elucidate the ontogeny of ER in prenatally BPA-exposed animals.
A fully developed prostatic epithelium is composed of two major cell types: the secretory epithelial cells and the basal-stem cells [31]. Both types of cells express AR during early development between birth and the onset of puberty [32]; however, only the columnar, luminal, well-differentiated cells possess apical, secretory granules containing PAP [31]. Lower expression of PAP observed in BPA-treated groups could be mediated by a direct effect of BPA in the columnar epithelial cells, or an indirect consequence of primary events in the stroma [13, 20]. Signals from the stroma are believed to be critical in determining the decision of epithelial cells to undergo proliferation, apoptosis, or differentiation. The decreased expression of AR in periductal stromal cells may affect the androgen-signaling pathway and this results in lower expression of PAP, although a direct effect of BPA on the epithelial cells could not be ruled out.
Morphological and functional prostate changes observed in our study could be the result of a direct and/or indirect effect of in utero exposure to BPA. Recent reports have shown that estrogen and xenoestrogen have a direct effect on prostate growth when explants were exposed to estrogens in vitro [12, 33]. We cannot rule out, however, that BPA may also be disrupting the hypothalamic-pituitary-testicular axis of these fetuses. Observations that perinatal exposure of rat to low BPA doses alters estrous cyclicity and reduces serum LH support an indirect effect by these compounds [21]. A series of studies done by Hayward et al. [34, 35] has shown that after castration, as a result of changing androgen levels, prostatic smooth muscle cells transformed into fibroblasts expressing AR and vimentin. It has been reported that BPA is both an estrogen and an antiandrogen [36]; however, in our study AGD, a marker of androgenic status [37, 38], was not modified. In order to gain a better insight into the mechanisms by which BPA affects morphology and secretory function of the prostate, we are now evaluating the effect of castration and androgen administration in epithelial and stromal prostatic cells of prenatal BPA-exposed males.
In summary, in utero exposure to environmentally relevant levels of BPA diminished the expression of AR and altered the phenotype of periductal stromal cells in 30-day-old rats. In addition, decreased expression of PAP was observed in epithelial cells, suggesting alterations in prostatic functional activity. Because tissue organization was altered, decreased PAP expression could be due to the disruption of communications among periductal stroma and epithelial cells. From the tissue organization field theory perspective, carcinogenesis results from the disruption of cell-to-cell communication involving the parenchyma and its stroma [39]. Our findings are significant in light of the data on human prostate cancers where alterations in the stroma compartment may enhance the invasive and/or malignant potential of the nascent tumor [13]. Further studies will be conducted to evaluate whether these BPA-induced alterations may directly lead or predispose to aberrant and tumor growth of the prostate later in life.
ACKNOWLEDGMENTS
We are very grateful to Mr. Juan C. Villarreal and Mr. Juan Grant for technical assistance and animal care, and to Dr. Maricel V. Maffini (Tufts University School of Medicine, Boston, MA) for her advice on evaluating the specificity of primary antibodies.
FOOTNOTES
1 This study was supported by grants from the Argentine National Council for Science and Technology (CONICET) (PIP 528/98), the Argentine National Agency for the Promotion of Science and Technology (ANPCyT) (PICT-99 13-7002), the Argentine Ministery of Health (R. Carrillo-A. Oñativia award), and a Yamagiwa-Yoshida Memorial UICC International Cancer Study Grant. J.G.R. is a recipient of an R. Carrillo-A. Oñativia fellowship; J.V. is a Fellow and E.H.L. is Career Investigator of the CONICET. 
2 Correspondence: Enrique H. Luque, Laboratorio de Endocrinología y Tumores Hormonodependientes, Faculty of Biochemistry and Biological Sciences, Casilla de Correo 242, (3000) Santa Fe, Argentina. FAX: 54 342 4550944; eluque{at}fbcb.unl.edu.ar
Accepted: June 4, 2001.
Received: April 11, 2001.
REFERENCES
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