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a Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
b Department of Anatomy, Physiological Sciences and Radiology, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina 27606
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we reported that a toxicant exposure to the dam during early lactation increased the incidence of lateral prostate inflammation in the male offspring [7]. This effect may have resulted from a transient period of hyperprolactinemia just prior to puberty. In the rat, the period prior to puberty may be a second sensitive period for prostate growth, with the first growth period for branching and morphogenesis being the first few days following birth [8]. Prolactin (PRL) values in the male rat are low between postnatal Day (PND) 15 and 20, increase rapidly and peak around PND 25, then decrease slightly to a relatively constant level from PND 30 to 50 [9]. Similarly, studies in humans have also shown a sharp rise in circulating PRL levels at the time of sexual maturation [10]. Negro-Vilar and coworkers [9] correlated this PRL surge in the rat with the beginning of a rapid growth period for the prostate. In addition, estradiol levels in the male rat are high at birth, fall to low levels immediately after birth, rise from PND 12 to a peak on PND 19, and then decrease to low levels through adulthood [11].
We hypothesized that a transient increase in PRL from PND 22 to 32 would augment the levels of PRL that rise naturally to a peak on PND 25, and that this increase in PRL might result in alterations in the adult prostate. Specifically, we predicted that the lateral prostate would be affected, because PRL has been correlated with lateral prostate growth [12–14] and inflammation [15] in the adult rat. Tangbanluekal and Robinette [15] observed a dose-response relationship between the administration of exogenous PRL and the severity of lateral prostate inflammation.
The critical periods for developmental exposures and the hormonal involvement in the induction of prostatitis remain to be identified. In humans, nonbacterial prostatitis with undefined etiology has become a significant clinical problem [16] and has been associated with infertility (e.g., [17, 18]). Inflammation has been noted by histological examination in other prostate diseases, such as benign hyperplasia and adenocarcinoma where the contribution of the inflammatory response to these processes remains to be clearly elucidated [19]. The extent to which altered hormonal regulation of the prostate is involved in these diseases is unclear; however, a recent report by Leav et al. [20] indicated that PRL action plays a role in the development and maintenance of the human prostate and may also participate in early neoplastic transformation of the gland.
To test our hypothesis, we pharmacologically induced an increase in circulating PRL during the prepubertal period (PND 22 to 32) by administering several different compounds that can increase PRL release, including pimozide (PIM), estradiol-17ß (E2), and bisphenol A (BPA). PIM is a dopamine antagonist that increases PRL secretion from the pituitary [21]. E2 stimulates pituitary PRL synthesis and secretion in the adult rat, in part by reducing the release of dopamine from hypothalamic neurons [21]. Therefore, it seems plausible that compounds with estrogenic activity given prepubertally could act indirectly on the adult prostate by increasing the synthesis and release of pituitary PRL during the prepubertal period. Several environmental compounds have also been reported to show estrogenic activity and may be able to increase PRL secretion. For example, BPA is a monomer of polycarbonate plastics and a constituent of epoxy and polystyrene resins that are used in the food packaging industry and in dentistry, which has been reported to be weakly estrogenic and to stimulate PRL release in vitro and in vivo [22].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Timed-pregnant female Wistar rats were purchased from Charles River Laboratories in Raleigh, NC. Animals were received on gestation Day 13 and were housed one per cage in an American Association for the Accreditation of Laboratory Animal Care accredited facility at 22°C. All animals were maintained on a 12L:12D photoperiod (lights-on 0500 h) and were provided with food and water ad libitum. Temperature and humidity were regulated at 20–24°C and 40–50%, respectively. The day of parturition was designated as PND 0. The offspring were weaned at 22 days of age and placed in individual cages until they were killed. Each treatment group consisted of males from separate litters. Wistar rats were selected because they have been reported to have a low incidence of prostatitis in younger animals and a moderate sensitivity for its development [23].
Dosing
On PND 22, Silastic tubing (Scientific Products brand medical grade silicone tubing 0.062 in. i.d.; 0.125 in. o.d.; 0.032 wall thickness) implants containing either sesame oil (control) or E2 (Sigma Chemical Co., St. Louis, MO; 8 mm long filled with 4 mg/ml E2 in sesame oil) were placed subdermally in the flank while the rats were under a general anesthetic (ketamine/xylazine). Some of the estradiol-implanted males also received two injections of either 0.417 mg/kg of bromocriptine (Sigma) in saline s.c. or saline alone each day at 0900 and 1600 h on PND 22 to 32. Others received s.c. injections of sesame oil as a control, 20 mg/kg of PIM in sesame oil, or 50 mg/kg of BPA in sesame oil at 0900 h on PND 22 to 32. The sesame oil and estradiol implants were removed on the afternoon of PND 32 while the males were again anesthetized using ketamine/xylazine.
Necropsy and Examination of Prostates
On PND 29, the eighth day of dosing, six animals from each dose group were killed by decapitation 1 h after dosing (1000 h). This morning hour was selected to determine the effects of the treatments on basal PRL secretion (5 h after lights-on), prior to the afternoon rise [24]. Rats were decapitated within 15 sec of removal from their home cage for accurate assessment of the PRL concentration. Blood was collected for determination of serum PRL and estradiol. At 120 days of age, the male offspring were killed by decapitation within 15 sec of removal from their cage at approximately 0900 h for collection of blood, anterior pituitary, testes, and the ventral and lateral prostate. The lateral lobe was carefully separated from the dorsal prostate. Body weights were taken the day before animals were killed. The clotted blood was centrifuged at 1260 x g for 30 min, and the serum was then harvested and stored frozen at -80°C for PRL assay. The anterior pituitaries were removed, frozen on dry ice, and stored at -80°C for subsequent hormonal analyses. The extent of inflammation in the ventral and lateral lobes of the prostate was visually inspected without knowledge of treatment and given a subjective score (0 to 3: 0, no inflammation; 1, mild inflammation; 2, moderate inflammation; and 3, severe inflammation). This score was used to determine subsequent dilutions for the DNA assay. The left lateral and ventral prostates were removed, weighed, and immediately frozen on dry ice and stored at -80°C until analyzed for myeloperoxidase (MPO) and DNA. The right lateral prostate was fixed in 10% neutral buffered formalin for 24 h and then transferred to 70% ethanol for later histological processing.
RIAs
After animals were killed at 29 and 120 days of age, serum and anterior pituitary PRL were quantified by RIA. The assay was performed using the following materials supplied by the National Hormone and Pituitary Program (Rockville, MD): iodination preparation I-6, reference preparation RP-3, and antiserum S-9. Iodination material was radiolabeled with 125I (Dupont/New England Nuclear, Boston, MA) by a modification of the chloramine-T method of Greenwood et al. [25]. Labeled PRL was separated from unreacted iodide by gel filtration chromatography as described previously [26].
Sample serum and pituitary homogenate were pipetted with appropriate dilutions to a final assay volume of 500 µl with 100 mM phosphate buffer containing 1% BSA. Standard reference PRL was serially diluted for standards of 10 ng/ml to 0.313 ng/ml. Primary antiserum (200 µl) at a dilution of 1:437 500 in 100 mM potassium phosphate, 76.8 mM EDTA, 1% BSA, and 3% normal rabbit serum was pipetted into each assay tube, vortexed, and incubated at 5°C for 24 h; 100 µl of 125I-PRL was added to each tube, vortexed, and incubated for 24 h. Second antibody (goat anti-rabbit gamma globulin [Calbiochem, San Diego, CA] at a dilution of 1 U/100 µl) was then added, vortexed, and incubated for 24 h. The samples were then centrifuged at 1260 x g for 30 min; the supernate was aspirated, and the sample tube with pellet was counted on a gamma counter. Intra- and interassay coefficients of variation for the PRL assays were 2.86% and 9.1%, respectively.
An estradiol Coat-a-Count kit from Diagnostic Products Corporation (DPC: Los Angeles, CA) was used to determine the estradiol content of the serum collected from the control-injected, the estradiol, and the estradiol+bromocriptine (E+B) groups on PND 29. Control samples obtained from DPC were included in the estradiol assay, and the values obtained were within the ranges provided by the supplier.
DNA Assay
Total DNA was performed by a modification of the method of Labarca and Paigen [27]. The left lateral prostates were homogenized with an Ultra-turrax T25 (IKA-Labortechnik, Staufen, Germany) in a final volume of 10 ml with a phosphate buffer containing 0.05 M sodium phosphate and 2 M sodium chloride at a pH of 7.4. Samples were vortexed for 5 min and centrifuged at 1260 x g for 15 min. A 600-µl sample of the supernate was removed, and appropriate aliquots (50–200 µl depending on visual inflammation score) were brought to a final volume of 4 ml with DNA assay buffer containing 0.8 µg bisbenzimide (Sigma). Samples were then read on a DyNA Quant 200 fluorometer (Hoefer Pharmacia, San Francisco, CA) at an excitation wavelength of 350 nm and an emission wavelength of 456 nm. Calf thymus DNA standards (Sigma), ranging from 0 to 9.6 µg/tube, were used to generate a standard curve for determination of unknown sample amounts.
MPO Assay
An MPO assay was performed on all left lateral prostates. MPO is an enzyme secreted by neutrophils, and it is also found at lower concentrations in monocytes and macrophages. This enzyme catalyzes the oxidation of electron donors by hydrogen peroxide. The MPO activity from this assay is directly proportional to the number of neutrophils seen in histologic sections [28]. It has advantages over histological examination in that it provides a readily quantifiable assessment of neutrophil infiltration at the whole organ level.
Once the sample of supernate was taken for the DNA assay, as described above, 600 µl of 8.33% hexadecyltrimethylammonium bromide (HTAB) buffer (50 mM sodium phosphate, pH 5.4) was added to the tube containing the remaining DNA supernatant and the pelleted cell fragments, followed by vortexing for 2 min. Hexadecyltrimethylammonium bromide is a detergent that releases MPO from the primary granules of the neutrophil [29]. The suspension was then centrifuged at 1260 x g for 15 min, and 1 ml of the supernate was diluted with 1.5 ml of 0.5% HTAB; 200 hundred µl of the diluted supernatant was brought to 1580 µl with 100 mM sodium phosphate buffer. Then 200 µl of 16 mM 3,3',5,5'-tetramethyl-benzidine (Sigma) and 20 µl of 30 mM hydrogen peroxide solution (Sigma) were added to the sample in a cuvette and read at a wavelength of 655 nm on a Beckman (Palo Alto, CA) DU 650 Spectrophotometer for 2 min at 30-sec intervals. The rate was then used to calculate the final concentration of MPO (MPO units per milligram) in each sample. The small amount of MPO activity present in the 600-µl aliquot removed for DNA content was similarly determined and added to the amount found in the residual supernatant/cell debris fraction to give a total MPO/mg tissue.
Histology
The right lateral prostate was placed in 10% formalin at necropsy and 24 h later transferred to a 70% ethanol solution. The tissue was then submitted to Experimental Pathology Laboratory (Research Triangle Park, NC) for processing and histopathological evaluation. Following paraffin embedding, the first complete 10-µm section from the flat (urethral) side of the prostate tissue was stained with hematoxylin and eosin for evaluation. The sections were examined and rated for the relative degree of severity of inflammatory, degenerative, and proliferative changes on a scale from 1 to 5 (1, minimal; 2, slight/mild; 3, moderate; 4, moderately severe; 5, severe/high). Cellular infiltration in the prostate was defined by cell type (mononuclear or polymorphonuclear cell) and by location (interstitial or lumen).
Statistics
Tissue weights, PRL, and DNA data were examined for statistical significance by ANOVA, and comparisons among individual treatment conditions within each time period were further examined by Dunnett's t-test for multiple comparisons using INSTAT (San Diego, CA). The incidence of inflammation in the right lateral prostates examined by histology and MPO assay were analyzed using the Fisher Exact Test of Probability.
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RESULTS |
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Ventral prostate weights at 120 days are shown in Figure 2. The ventral prostate weights in all treatment groups did not differ from those of comparable controls. However, an increase in the weight of the lateral prostates was observed in the males exposed to 20 mg/kg of PIM and 50 mg/kg of BPA from PND 22 to 32 as compared to the vehicle-injected controls (P < 0.01 and P < 0.05, respectively; Fig. 3). There were no differences in the weights of the lateral prostates in the E or E+B treatments as compared to the controls with sesame oil implants. Body weights and testes weights in all treatment groups were not different from those of comparable controls at 120 days (data not shown).
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According to the results of the MPO assay, there was a significant increase in the percentage of males with lateral prostate inflammation in the PIM and the E group as compared to their appropriate controls (P < 0.01 and P < 0.05, respectively; Fig. 4; inflammation determined as three times the mean MPO concentration in the historical controls or 0.042 MPO/mg). There was a trend for an increase in the percentage of males with inflammation in the BPA group, but it was not significant. Administration of 0.417 mg/kg of bromocriptine to the estradiol-implanted males from PND 22 to 32 resulted in a dramatic reduction in the proportion of males with lateral prostate inflammation. There was also a significant increase in the severity of the inflammation (as determined by the mean MPO content per milligram tissue) in the PIM and E groups as compared to their respective controls (P < 0.05; Fig. 5). The mean MPO of the BPA group and the E+B group was not statistically different from that of their appropriate controls. A regression analysis of the mean serum PRL level 1 h after dosing on PND 29 and the mean MPO concentrations at 120 days of age (one point for each treatment) revealed a correlation between the two values (r = 0.931).
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Histological evaluations of the lateral prostates revealed that the E and PIM groups had an increased incidence of stromal mononuclear (primarily lymphocytes and macrophage) and luminal polymorphonuclear cellular infiltrate (neutrophils), whereas the BPA group had only an increased amount of luminal polymorphonuclear cellular infiltrate that was milder than that seen with E or PIM treatment (Table 2 and Fig. 6). There is some discrepancy between the MPO and histological data with respect to the neutrophil content in the BPA group. However, because the MPO assay detects and quantifies the neutrophil content of the tissue as a whole, it is not surprising to find some variability between the MPO data and the pathological data, which are determined in only one section of the tissue.
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As shown in Figure 7, there was a significant increase in the total DNA of the lateral prostates in the PIM and estradiol groups (P < 0.01 and P < 0.05, respectively). There was also an increase in DNA concentration (µg/mg) in the lateral prostates of the PIM males as compared to the injected controls (P < 0.01; Fig. 8). These effects on DNA in the PIM and E groups are likely due to the increase in the number of inflammatory cells, in addition to the increased weight response of the lateral prostate to PIM treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As mentioned previously, studies using adult male rats have shown that periods of hyperprolactinemia, induced with either estradiol or ovine PRL, can result in lateral prostate inflammation [15, 30]. The inflammation appears to be a direct effect of elevated PRL, which was demonstrated by a reversal of the estrogen-induced inflammation in rats that were concurrently treated with bromocriptine and by the establishment of a dose response in severity of inflammation with increasing doses of ovine PRL. However, the exact biochemical mechanism of this effect of PRL on lateral prostate inflammation remains to be determined. Moreover, the inflammation induced by ovine PRL appeared to be similar to the spontaneous prostatitis seen in the lateral prostates of aging Lewis, Copenhagen, and Wistar rats [31, 32] that show the presence of a stromal mononuclear and luminal polymorphonuclear cellular infiltrate. In addition, this type of inflammation has been shown to precede fibromuscular growth [30], similar to that seen in human benign prostatic hyperplasia (BPH), with several studies indicating that neutrophils and lymphocytes are involved in fibroblast proliferation [33] and the synthesis of collagen [34]. Inflammation is noted frequently in human BPH specimens, with one study reporting that 98% of the specimens analyzed had an inflammatory infiltrate [35]; but no correlation has been made between the inflammatory cells and the stromal proliferation.
In the present study, the mean serum PRL levels 1 h after dosing on PND 29 appear to be correlated with the amount of neutrophil infiltrate in the lateral prostate at 120 days of age, with the highest serum PRL in the PIM group, followed by the estradiol and then the BPA group. However, further investigation is needed to characterize this relationship better within the same individual male. The inflammation observed in the PIM and estradiol groups was identified as stromal mononuclear and luminal polymorphonuclear, which is very similar to the type mentioned previously in the aging rat. The BPA group, however, showed only an increased incidence of luminal polymorphonuclear cells. Therefore, it is possible that the estradiol and PIM groups may later have developed fibromuscular proliferation, like the condition in human BPH as mentioned above, if the animals had been examined at an age older than 4 mo. The effect of estradiol on lateral prostate inflammation was reversed by cotreatment with bromocriptine on PND 22–32, indicating that the effect of estradiol was mediated indirectly through its effects on PRL synthesis and secretion.
It has previously been shown that ectopic pituitary implants or exposure to ovine PRL in immature rats will increase ventral and dorsolateral prostate weight [36, 37]. This increase was believed to be due to an increase in the number of androgen receptors in the prostate [37]. The immature male rat's prostate is believed to be most sensitive to changes in PRL release, because the 20-day-old rat prostate has the highest specific binding of PRL [38], and PRL normally rises sharply at 25 days of age in the rat [9]. In the present study, we observed a significant increase in the lateral prostate weight in the PIM and BPA males in comparison to their vehicle-injected controls. The mean lateral prostate weight of the E males was not significantly different from the control, suggesting that in spite of an E2-elevated PRL, the steroid hormone may also have an opposing effect on the prostate, either directly or indirectly by the suppression of LH and androgen release.
BPA has been estimated to be 10 000-fold less potent as an estrogen receptor agonist than E2 [39]. As mentioned previously, this compound has been found to stimulate PRL release from rat anterior pituitary cells in vitro in a manner similar to estradiol, but at a 1000- to 500-fold lower potency [40]. These investigators also demonstrated that BPA produced an increase in PRL release in vivo in Fisher 344 but not Sprague-Dawley female rats. The dose of BPA used in the present study increased PRL 210% above the injected control value. It is possible that the low estrogenicity of BPA is having a direct effect on the weight of the lateral prostate that is the opposite of the effect of the high levels released from the E2 implants. This type of effect of exposure to weak estrogens has been shown previously. Low doses of estrogen prenatally can induce an increase in the tissue weight of the mouse prostate, while high doses can cause decreased size [41]. Another example of a paradoxical low-dose stimulation phenomenon was found with a low neonatal dose of dihydrotestosterone or progesterone. Low doses of both compounds were more effective in increasing the unstimulated weight of the lateral prostate than higher doses [5].
In summary, the results of the present study demonstrate that transient pubertal increases in PRL secretion induced pharmacologically or with an estrogenic environmental compound can result in an increased incidence of lateral prostate inflammation at 120 days of age. Because the rat's dorsal and lateral prostate lobes are the most homologous to the human prostate [42], the increase in lateral prostate weight in the PIM and BPA treatment groups and the increase in incidence of lateral prostate inflammation in the PIM, BPA, and E groups may be relevant to exposures in humans. This may be especially true for BPA, since it has been identified in the liquid of foods preserved in lacquer-coated cans [43] and has been found in the saliva of children following treatment of teeth with BPA-containing sealant [44]. The results of this study gain added significance as it has been recently shown that PRL may play a role in the development and maintenance of the human prostate and also in the etiology of neoplastic transformation of the gland [20].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Tammy E. Stoker, MD-72, U.S. EPA, NHEERL, 72 Alexander Dr., Research Triangle Park, NC 27711. FAX: 919 541 5138; stoker.tammy{at}epa.gov
Accepted: August 10, 1999.
Received: June 17, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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