Skip Navigation LinksHome > June 2012 - Volume 35 - Issue 2 > Does eleuxin exert toxic effects on the prostate of adult ma...
The Egyptian Journal of Histology:
doi: 10.1097/01.EHX.0000414675.37365.63
Original articles

Does eleuxin exert toxic effects on the prostate of adult male albino rats? Histological and immunohistochemical study

Zaki, Mohamed M.; Ibrahem, Sali O.; Shenouda, Helpies D.; El Salam Morsy, Sherifa A.; Faruk, Eman M.

Free Access
Article Outline
Collapse Box

Author Information

Department of Histology and Cell Biology, Faculty of Medicine, Benha University, Benha, Egypt

Correspondence to Eman M. Faruk Tel: +20 112 015 8166; fax: 0133243190; E-mail: em5an76@yahoo.com

Received October 3, 2011

Accepted January 20, 2012

Collapse Box

Abstract

Background: Eulexin (flutamide) is a pure nonsteroidal antiandrogen drug widely used in the treatment of prostatic cancer, hirsutism, and acne vulgaris.

Aim of the work: The aim of this work is to determine the histological and immunohistochemical effects of eleuxin on the prostate of adult albino rats.

Materials and methods: Thirty adult male albino rats were used and divided into a control group of six rats and an experimental group of 24 rats. This group was further subdivided into three subgroups (eight rats each). Each rat of the experimental subgroups received a daily dose of 67.5 mg/kg body weight of flutamide by the oral route. Animals were weighed and sacrificed after 2, 4, and 6 weeks. The prostate specimens were subjected to H&E, PAS reaction, and immunohistochemistry for androgen receptor (AR) and estrogen receptor (ER) localization. Statistical analysis of body weight and area % of the immunoreactivity of ARs and ERs was carried out.

Results: Eleuxin induced a significant reduction in the body weight of the rats of the experimental subgroups. Moreover, the experimental groups showed a significant decrease in the immunoreactivity of ARs and an increase in the immunoreactivity of the ERs. Early changes in acini were represented by folding of some areas of acini, whereas other acini were rudimentary after 2 weeks of treatment. Cystic dilatation of acini with a decrease in 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 acini decrease in the cell number with dilated and congested blood vessels. There was a decreased PAS reaction in the apical parts of epithelial cells with an irregular apparent thick basement membrane especially in the folded area.

Conclusion: Eleuxin exerted many histological effects on the prostate of adult albino rats. Further study is required to determine the effect of flutamide on the function of the male reproductive system, especially fertility.

Back to Top | Article Outline

Introduction

Recently, antiandrogenic compounds have been recognized as endocrine disruptors because of their hormone-like activities [1]. 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) [2].

Some researchers have classified antiandrogens into steroidal and nonsteroidal compounds [3]. Eleuxin is a nonsteroidal antiandrogenic anilide compound that does not have estrogenic, progestational, or androgenic activities [1]. 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 [4]. 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 [9]. 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 [10].

Back to Top | Article Outline

Materials and methods

Drugs

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 [11]. The dose administered to each rat was calculated according to its body weight (67.5mg/kg body weight) [12]. The control groups were given the dissolving mixture through an orogastric tube, which was introduced deep inside the rat's mouth [13].

Back to Top | Article Outline
Animal grouping

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.

Back to Top | Article Outline
Experimental parameters

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 [14] and PAS (histochemical) reaction [15] and immunohistochemistry for ARs and estrogen receptors (ERs) [16].

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.

Back to Top | Article Outline
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.

Back to Top | Article Outline
Statistical analysis

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.

Back to Top | Article Outline

Results

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.

Histogram 1
Histogram 1
Image Tools
Table 1
Table 1
Image Tools
Back to Top | Article Outline
Histological results

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).

Figure 1
Figure 1
Image Tools
Figure 2
Figure 2
Image Tools
Figure 3
Figure 3
Image Tools
Figure 4
Figure 4
Image Tools
Figure 5
Figure 5
Image Tools
Figure 6
Figure 6
Image Tools
Figure 7
Figure 7
Image Tools
Figure 8
Figure 8
Image Tools
Figure 9
Figure 9
Image Tools
Back to Top | Article Outline
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).

Figure 10
Figure 10
Image Tools
Figure 11
Figure 11
Image Tools

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.

Figure 12
Figure 12
Image Tools
Figure 13
Figure 13
Image Tools
Figure 14
Figure 14
Image Tools
Figure 15
Figure 15
Image Tools
Figure 16
Figure 16
Image Tools
Figure 17
Figure 17
Image Tools
Histogram 2
Histogram 2
Image Tools
Histogram 3
Histogram 3
Image Tools
Table 2
Table 2
Image Tools
Table 3
Table 3
Image Tools
Back to Top | Article Outline

Discussion

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 [19].

The effect on body weight found in this study was in agreement with the result obtained by Bonkhoff and Remberger [20], 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 [19]. 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 [21]. 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 [27], 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 [28].

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 [33]. 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].

Back to Top | Article Outline
Acknowledgements
Table. No title avai...
Table. No title avai...
Image Tools
Back to Top | Article Outline
Conflicts of interest

There are no conflicts of interest.

Back to Top | Article Outline

References

Sharpe RM. Pathways of endocrine disruption during male sexual differentiation and masculinization. Best Pract Res Clin Endocrinol Metab. 2006;20:91–110

Metzdorff SB, Dalgaard M, Christiansen S, Axelstad M, Hass U, Kiersgaard MK, et al. Dysgenesis and histological changes of genitals and perturbations of gene expression in male rats after in utero exposure to antiandrogen mixtures. Toxicol Sci. 2007;98:87–98

Beers MH, Berkow R The Merck Manual of Diagnosis and Therapy. 200618th ed. Merck & Co., Inc., Whitehouse Station, NJ, USA. [online] Prostate Cancer.

Miyata K, Yabushita S, Sano M, Miyashita K, Okuno Y, Matsuo M. Effects of perinatal exposure to flutamide on sex hormone responsiveness in F1 male rats. J Toxicol Sci. 2003;28:149–163

Song GS, Seo JT. Relationship between ambient temperature and heat flux in the scrotal skin. Int J Androl. 2009;32:288–294

Song GS, Seo JT. Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses. Int J Androl. 2009;32:288–294

Brandes D, Gyorkey F, Groth DP. Fine structural and histochemical study of the effect of castration on the rat prostatic complex. I. The coagulating gland. Lab Invest. 1962;11:339–350

Williams K, McKinnell C, Saunders PT, Walker M, Fisher JS, Turner KJ, et al. Neonatal exposure to potent and environmental oestrogens and abnormalities of the male reproductive system in the rat: evidence for importance of the androgen-oestrogen balance and assessment of the relevance to man. Hum Reprod Update. 2001;7:236–247

Anderson J. The role of antiandrogen monotherapy in the treatment of prostate cancer. BJU Int. 2003;91:455–461

Zhang M, Latham DE, Delaney MA, Chakravarti A. Survivin mediates resistance to antiandrogen therapy in prostate cancer. Oncogene. 2005;24:2474–2482

Abdelnabi MA, Ottinger MA. Hypothalamic indolamines during embryonic development and effects of steroid exposure. Gen Comp Endocrinol. 2003;130:13–19

Marchlewicz M, Wiszniewska B, Kurzawa R, Wenda Rózewicka L. Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis. Folia Histochem Cytobiol. 2004;42:19–27

Greco TL, Furlow JD, Duello TM, Gorski J. Immunodetection of estrogen receptors in fetal and neonatal male mouse reproductive tracts. Endocrinology. 1992;130:421–429

Pandini G, Mineo R, Frasca F, Roberts CT Jr, Marcelli M, Vigneri R, Belfiore A. Androgens up-regulate the insulin-like growth factor-I receptor in prostate cancer cells. Cancer Res. 2005;65:1849–1857

Iczkowski KA, Sun EL, Gondos B. Morphometric study of the prepubertal rabbit testis: germ cell numbers and seminiferous tubule dimensions. Am J Anat. 1991;190:266–272

Bancroft JD, Gamble MBancroft JD, Gamble M. The haematoxylin and eosin Theory and practice of histological techniques. 20076th ed. London Churchill Livingstone:99–112 In: , pp.

Drury Wallington SF Histological techniques and their diagnostic applications. 1980 London Churchill Livingstone

Janssen PJ, Brinkmann AO, Boersma WJ, van der Kwast TH. Immunohistochemical detection of the androgen receptor with monoclonal antibody F39.4 in routinely processed, paraffin-embedded human tissues after microwave pre-treatment. J Histochem Cytochem. 1994;42:1169–1175

Bercovich Z, Kahana C. Degradation of antizyme inhibitor, an ornithine decarboxylase homologous protein, is ubiquitin-dependent and is inhibited by antizyme. J Biol Chem. 2004;279:54097–54102

Bonkhoff H, Remberger K. Morphogenetic aspects of normal and abnormal prostatic growth. Pathol Res Pract. 1995;191:833–835

De Carvalho HF, Vilamaior PS, Taboga SR. Elastic system of the rat ventral prostate and its modifications following orchiectomy. Prostate. 1997;32:27–34

Moore RW, Rudy TA, Lin TM, Ko K, Peterson RE. Abnormalities of sexual development in male rats with in utero and lactational exposure to the antiandrogenic plasticizer di(2-ethylhexyl) phthalate. Environ Health Perspect. 2001;109:229–237

Chang CS, Kokontis J, Liao ST. Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science. 1988;240:324–326

Góes RM, Zanetoni C, Tomiosso TC, Ribeiro DL, Taboga SR. Surgical and chemical castration induce differential histological response in prostate lobes of Mongolian gerbil. Micron. 2007;38:231–236

Horn R, Pastor LM, Moreno E, Calvo A, Ganteras M, Pallares J. Morphological and morphometric study of early changes in the ageing golden hamster testis. J Anat. 1996;188:109–117

Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, et al. Perinatal exposure to the fungicide prochloraz feminizes the male rat offspring. Toxicol Sci. 2005;85:886–897

Ekman P. The prostate as an endocrine organ: androgens and estrogens. Prostate Suppl. 2000;10:14–18

Tuxhorn JA, Ayala GE, Rowley DR. Reactive stroma in prostate cancer progression. J Urol. 2001;166:2472–2483

Mylchreest E, Sar M, Cattley RC, Foster PM. Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol Appl Pharmacol. 1999;156:81–95

Hotchkiss AK, Ankley GT, Wilson VS, Hartig PC, Durhan EJ, Jensen KM, et al. Of mice and men (and mosquitofish): antiandrogens and androgens in the environment. Bioscience. 2008;58:1037–1050

Carreau S, Delalande C, Silandre D, Bourguiba S, Lambard S. Aromatase and estrogen receptors in male reproduction. Mol Cell Endocrinol. 2006;246:65–68

Fawcett DWFawcett DW. Male reproductive system A textbook of histology. 199412th ed. London Hodder Arnold Publishers:768–815 In: , pp.

Rosenfield RL. Hirsutism. N Engl J Med. 2005;353:2578–2588

Snell RSSnell RS. The abdomen Clinical anatomy for medical students. 20006th ed. London Lippincott Williams & Wilkins:133–183 In: , pp.

Anahara R, Toyama Y, Mori C. Flutamide induces ultrastructural changes in spermatids and the ectoplasmic specialization between the sertoli cell and spermatids in mouse testes. Reprod Toxicol. 2004;18:589–596

Fix C, Jordan C, Cano P, Walker WH. Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in sertoli cells. Proc Natl Acad Sci USA. 2004;101:10919–10924

Gray LEJ, Wilson VS, Stoker T, Lambright C, Furr J, Noriega N, et al. Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals. Int J Androl. 2006;29:96–104

Keywords:

eleuxin; histological methods; immunohistochemistry for AR and ER; prostate

© 2012 The Egyptian Journal of Histology

Login

Search for Similar Articles
You may search for similar articles that contain these same keywords or you may modify the keyword list to augment your search.