Zaki, Mohamed M.; Ibrahem, Sali O.; Shenouda, Helpies D.; El Salam Morsy, Sherifa A.; Faruk, Eman M.
Recently, antiandrogenic compounds have been recognized as endocrine disruptors because of their hormone-like activities . The endocrine disruptors are thought to act through many mechanisms, such as a decrease in the synthesis of androgen by exerting effects on the pituitary–gonadal axis and or blocking the androgen receptor (AR) .
Some researchers have classified antiandrogens into steroidal and nonsteroidal compounds . Eleuxin is a nonsteroidal antiandrogenic anilide compound that does not have estrogenic, progestational, or androgenic activities . It is believed to act by inhibiting the uptake of androgens by blocking ARs in the target tissue. However, some data have indicated that it might reduce the synthesis of androgens and or increase their metabolism to inactive molecules . Eleuxin prevents adrenal androgens, which normally account for about 9% of circulating androgens, from binding to ARs in the prostatic gland and in prostate cancer cells [5,6].
In castrated rats, eleuxin antagonized the effects of testosterone, testosterone propionate, androstenedione, and importantly, dihydrotestosterone on ventral prostate and seminal vesicle weights. Eleuxin had antiandrogenic potency in rats and dogs on the male secondary sex structures as in previously used cyproterone acetate (CPA) (a potent steroidal antiandrogenic compound) [7,8].
However, eleuxin differed from CAP in two important aspects: (a) unlike CPA, which had progestational as well as other hormonal activities (e.g. antigonadotropic and adrenocorticotropic hormone-suppressing activity), eleuxin did not exert a hormonal effect [3,7] and (b) whereas CPA has a molecular structure that is steroidal in nature that of flutamide is nonsteroidal. Eleuxin is used in association with castration in the treatment of metastatic prostatic carcinoma . Eleuxin is indicated for patients who have responded to or are beginning treatment with an analog of luteinizing hormone-releasing hormone. Some authors have reported that a combination of eleuxin (flutamide) with a luteinizing hormone-releasing hormone agonist was the only therapy shown to prolong life in patients with advanced prostate cancer .
Materials and methods
The drug used in this study was eleuxin (flutamide), which is available as tablets (Schering Corporation, Sigma menofia quoisna Egypt). Each tablet contains 250mg; eleuxin was dissolved in 50ml of a vehicle of sesame oil: ethanol (2:1) so that 1ml of a prepared solution contained 5mg eleuxin . The dose administered to each rat was calculated according to its body weight (67.5mg/kg body weight) . The control groups were given the dissolving mixture through an orogastric tube, which was introduced deep inside the rat's mouth .
Thirty adult male albino rats with body weights ranging between 150 and 205g were used throughout the experiment and received a balanced diet with free access to water. All animal procedures were performed according to approved protocols and in accordance with the recommendations for the proper care and use of laboratory animals. The animals were divided into the following groups:
Control group I: This group included six adult male albino rats that were administered a mixture of sesame oil and ethyl alcohol daily according to their body weight for 2 weeks (group Ia), 4 weeks (group Ib), and 6weeks (group Ic).
Experimental group II: This group included 24 adult male albino rats that were divided into three subgroups. Subgroup IIa (eight rats) was administered the daily therapeutic dose of the drug (67.5mg/kg body weight) for 2 weeks. Subgroup IIb (eight rats) was administered the daily therapeutic dose of the drug for 4 weeks. Subgroup IIc (eight rats) was administered the daily therapeutic dose of the drug for 6 weeks.
At the end of each experiment, the rats were weighted and then sacrificed by a high dose of ether. Sections from prostates were prepared for histological examination using an H&E  and PAS (histochemical) reaction  and immunohistochemistry for ARs and estrogen receptors (ERs) .
The polyclonal (rabbit) anti-AR and the anti-ER primary antibody (PA1-111A) (Affinity Bioreagents Inc., Colorado, USA, Schering-plough ESSEX Chemie Ag Offices 11 Sizostris ST., ELKourba, Heliopolis, Cairo) were produced by immunizing New Zealand white rabbits with a synthetic peptide corresponding to the N-terminal 21 amino acids of mice, rats, and human AR. The sections were incubated with the avidin–biotin–peroxidase complex (ABC reagent) (Oncogene Research Products, Agilent Technologies country USA) for 30min. The peroxides enzyme would bind to the biotin on the secondary antibody had specifically bound to its antigen: the AR. The peroxidase in the ABC complex transformed the DAB substrate into an insoluble dark brown precipitate, which was deposited around the binding site of the primary antibody. Thus, DAB localized the site of the antigen (AR) within the tissue section. The presence of peroxidase was assessed by the addition of a substrate–chromogen solution (reagent D). The presence of peroxidase was assessed by the addition of a substrate–chromogen solution (reagent D).
In the case of the anti-ER, peroxidase catalyzed the substrate (hydrogen peroxide) and transformed the chromogen (fast red) into a red deposit, which indicated the location of the antigen.
Quantitative morphometric measurements
Area % of positive AR and ER immunoreactive cells in the prostate was estimated using the ‘OlympusBX40, DOT med's Shipping Quote Service’ image analyzer computer system at the Histology Department, Faculty of Medicine, Cairo University. Measurements were performed within 10 nonoverlapping fields for each animal at ×400 magnification.
The data obtained from the image analyzer were analyzed using the statistical software SPSS for Windows; version 16.0 software, Chicago, IL, the parameters were tested using the Student t-test and the results were considered significant when P value was less than 0.05.
Macroscopic and statistical results
In the present work, there were no deaths throughout the experimental period. The administration of eleuxin led to a significant decrease in the body weight in all experimental subgroups as shown in Table 1 and Histogram 1.
H&E sections of the prostate of control animals included different types of acini and fibromuscular stroma. The acini had variable sizes and shapes. the epithelial lining was mainly formed of pseudostratified columnar ciliated epithelium. The height of the epithelium varied according to the functional state of the gland from cuboidal to columnar to pseudostratification with acidophilic secretion (Figs 1 and 2). The PAS reaction was strongly positive in the basal laminae below the epithelium and in the apical or the luminal border of the cells (Fig. 3). In subgroup IIa, the most characteristic finding was an apparent decrease in epithelial height in some areas of acini; some acini showed papillary projections whereas other acini appeared as a solid cord of cells (nonfunctional acini) (Fig. 4). A decrease in the PAS reaction in the secretion was observed with an irregular apparently thick basement membrane especially in the folded area (Fig. 5). In subgroup IIb, some acini showed cystic dilatation with a decrease in epithelial height whereas others were atrophied (Fig. 6). Most of the acini showed no secretion whereas others showed a positive PAS reaction inside the lumen (Fig. 7). In subgroup IIc, most of the acini were more dilated and irregular in shape with no folding compared with the control group; in addition, there was a decrease in epithelial height and for acini an apparent decrease in the cell number with dilated and congested blood vessels (Fig. 8). The acini showed no PAS-positive secretion, with an irregular apparent thick basement membrane especially in the folded area (Fig. 9).
Immunohistochemical detection of androgen and estrogen receptors in the prostate
In the control groups, a section of the prostate showed strong-positive AR immunoreactivity in the nuclei of acinar cells (Fig. 10). None of the cells of the prostate showed any positive ER immunoreactivity (Fig. 11).
In subgroup IIa, there was a decrease in immunoreactivity in the acinar epithelial cells and in the cells of the fibromuscular stroma compared with the control group (Fig. 12). In subgroup IIb, a marked decrease in immunoreactivity was observed in the acinar epithelial cells (Fig. 13). In subgroup IIc, the positive nuclear AR immunoreactivity was weaker in the acinar epithelial cells and in the cells of the fibromuscular stroma (Fig. 14). In subgroup IIa, the intensity of ER immunoreactivity was slightly increased compared with the control group and the prostate showed a weak red color of weak immunoreactivity in the lining epithelium of acini. The cells of the fibromuscular stroma showed a weak ER immunoreactivity (Fig. 15). In subgroup IIb, a moderate positive nuclear ER immunoreactivity was observed in the acinar epithelial cells and a weak reaction was observed in the cells of the fibromuscular stroma (Fig. 16). In subgroup IIc, compared with the control group, the intensity of ER immunostaining was markedly increased. The prostate showed a red color of immunoreactivity in the lining epithelium of acini. The cells of the fibromuscular stroma showed a weak ER immunoreactivity (Fig. 17). Thus, there was a significant decrease in AR immunoreactivity in all the experimental groups and a significant increase in ER immunoreactivity in all the experimental groups Tables 2 and 3 Histograms 2, 3.
In mammals, antiandrogens affect sexual differentiation and development, causing feminization and demasculinization of male offspring dosed in utero during sensitive developmental stages [17,18].
It is generally known that estrogens are synthesized and act on the testis of mammals including humans. The site of estradiol synthesis in the testis switches to Leydig cells during neonatal development, where the aromatase enzyme is present .
The effect on body weight found in this study was in agreement with the result obtained by Bonkhoff and Remberger , who found that flutamide led to reduced weight gain in their experimental animals compared with the control group. The development and activity of the prostate were dependent on stimulation by the testicular hormones. In the absence of the testis, the prostate failed to develop . In our study, early changes in acini were represented by folding of some areas of acini whereas other acini were rudimentary when treated for 2 weeks. Cystic dilatation of acini with a decrease in the epithelial height was observed in acini treated for 4 weeks. Highly dilated and irregular acini with no stroma in between were observed after 6 weeks of treatment and there was an apparent decrease in the cell number with dilated and congested blood vessels because of the separation of the extracellular matrix element.
Some investigators have studied the process of tissue atrophy in the prostates of mice. They found that the height of the epithelium was reduced to one-half after castrations and that the diameter of the glandular acini also increased rapidly . They concluded that the prostatic epithelium was very sensitive to androgen deprivation. Similar results have been obtained by other researchers [22,23].
Another histological finding obtained in this work in subgroup IIb was the increased amount of interacinar fibromuscular tissue; actually, this increase might have been because of the atrophy of the prostatic acini. This reaction of the interacinar tissue was explained by other researcher in which the stromal remodeling following androgen ablation is also variable [3,24]. They observed that the thickness of the interacinar was almost doubled during castration. The same finding was also obtained by Horn and colleagues [25,26].
A weak PAS reaction was observed in the basal laminae of the acinar epithelium and the secretory material with an irregular apparent thick basement membrane, especially in the folded area. This weak reaction in the apical parts of the cells probably resulted from the decreased supranuclear secretory granules, secondary to the antiandrogenic action of flutamide. A similar result was obtained by Ekman , who found that castration resulted in loss of cell polarity accompanied by alterations in all cell organoids. It has been suggested previously that changes in the composition of the basement membrane and other components of connective tissue markedly influence cell proliferation and differentiation in the prostate, and that these alterations may be of fundamental importance in the etiology and progression of pathological processes .
In this study, the prostates of the rats of subgroup IIb and IIc showed the same histological changes as those in subgroup IIa, but the changes in subgroup IIb and IIc were much more exaggerated and much more diffuse in their distribution. The prostatic acini showed cystic changes with a decrease in epithelial height and some acini were in the form of solid cords and rudimentary.
In the sections of prostate in the present study, by light microscopic examination, although the degree AR immunoreactivity seemed to be strongly positive in the acinar epithelial cells of experimental subgroup IIa, the nuclear AR immunoreactivity decreased in the three experimental subgroups (P < 0.001), which was statistically very highly significant compared with the control group. This decrease was most marked in subgroup IIc. It appeared that flutamide as an antiandrogen removed the effect of testosterone away from the ARs, an effect that simulated castration. Many authors have shown that androgen deprivation by castration was associated with a reduction in the immunodetected AR content to the extent that the nuclear immunostaining was completely lost [29,30].
In the sections of prostate in the present study, the ER immunoreactivity was positive in the acinar epithelial cells of experimental subgroups IIa and IIc; the nuclear ER immunoreactivity increased in the three experimental subgroups (P < 0.005), which was statistically significant compared with the control group. This increase was most marked in subgroup IIc. Some researchers have reported that ER in the male genital system have moderate to mild ER values, which is in agreement with our results [31,32]. The blockage of testosterone receptors by flutamide could result in an increase in the amount of free testosterone . Another explanation for the increase in ER could be [34,35] that flutamide, being a competitor for testosterone, inhibits testosterone occupancy of ET, rendering them free for immunoreactivity. Some researchers have reported that ER expression is localized to epithelium and stroma, which is in agreement with our present study [36,37].
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Conflicts of interest
There are no conflicts of interest.
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