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Cell Biology International (2007) 31, 459–472 (Printed in Great Britain)
Experimental alcoholism and pathogenesis of prostatic diseases in UChB rats
Eduardo Marcelo Cåndidoa, César Alexandre Fabrega Carvalhoa, Francisco Eduardo Martinezb and Valéria Helena Alves Cagnona*
aDepartment of Anatomy, Institute of Biology, State University of Campinas—UNICAMP, P.O. Box 6109, Campinas, 13083970 São Paulo, Brazil
bDepartment of Anatomy, Institute of Biosciences, State University of São Paulo—UNESP, Botucatu, São Paulo, Brazil


Abstract

Previous studies have shown that long-term alcohol treatment has negative effects on prostatic stromal–epithelial interaction. Thus, the aim of the present study was to analyze the histochemical, immunohistochemical and ultrastructural alterations that occur in the prostatic stroma and epithelium of rats submitted to chronic alcohol ingestion and alcohol abstinence, as well as to establish the relationship between these changes and prostatic diseases. Thirty male rats (10 Wistar and 20 UChB rats) were divided into three experimental groups: the control group received tap water, the alcoholic group received ethanol diluted to 10° G.L. for 150days, and the abstinent group received the same liquid diet as the alcoholic group up to 120days of treatment and only tap water for 30days thereafter. At the end of treatment, all animals were sacrificed and the ventral lobe of the prostate was removed and processed for histochemical, immunohistochemical and ultrastructural analyses. In addition, plasma testosterone levels were measured. The results showed prostatic intraepithelial neoplasia, infolding of the epithelium towards the stroma, stromal hypertrophy and the presence of inflammatory cells in alcoholic animals. In the abstinent group, alterations were noted mainly in the stromal area. In conclusion, ethanol triggers alterations in prostatic epithelial and stromal compartments, affecting the stromal microenvironment and predisposing the organ to pathological processes.


Keywords: Alcoholism, UChB rat, Prostate, Stromal–epithelial interaction, Morphology.

*Corresponding author. Tel.: +55 19 3788 6102; fax: +55 19 3289 3124.


1 Introduction

Alcoholism is a disease found worldwide and is one of the most frequent clinical diagnoses, causing morbidity and premature death (Caces et al., 1995; Campbell et al., 1996). In the United States, excessive alcohol consumption is the third main preventable cause of death and is associated with adverse health consequences such as liver cirrhosis and different types of cancer (Centers for Disease Control and Prevention (CDC), 2004).

Alcohol and its metabolites cause generalized disturbances in various organ systems such as the nervous, digestive, urinary and male reproductive systems, including the accessory sex glands (Marks and Wright, 1978; Martinez et al., 2001a,b; Gomes et al., 2002).

Animal models have contributed to the elucidation of many biological, biochemical, physiological and morphological aspects involved in the habit of consumption of alcoholic beverages by humans. Various experimental studies conducted on mice (Erwin et al., 1980) and rats (Tabakoff and Ritzmann, 1979; Waller et al., 1983) have emphasized the alcohol preference of some rodent lines which showed rapid metabolic adaptation to the initial effects of ethanol ingestion. This is the case of UChB rats which present acute and rapid tolerance to ethanol (Tampier and Mardones, 1999). These rats, originating from Wistar rats, have gone through decades of selection by inbred crossings between animals characterized by low and high voluntary 10% ethanol consumption, called UChA and UChB rats, respectively (Martinez et al., 2001a).

In the male reproductive system, one of the consequences of alcoholism is hypogonadism which is observed both in man and in laboratory animals (Bannister and Losowsky, 1987). Ethanol is believed to act directly on the gonads by altering the synthesis of testicular testosterone (Gordon et al., 1976; Ellingboe and Varanelli, 1979; Rivier and Vale, 1983; Anderson et al., 1989; Saxena et al., 1990; Tadic et al., 2000), and also indirectly through changes in the hypothalamus-pituitary-gonadal axis (Gavaler et al., 1983). In the ventral prostate of rodents, both a direct and indirect effect of ethanol on the glandular epithelium have been suggested (Martinez et al., 1993).

The harmful effect of alcohol on the male reproductive system has been demonstrated using various methods for the induction of alcoholism. The main morphological alterations were damage of testicular cells, reduction in the diameter of the seminiferous tubules, depression of serum testosterone levels, reduction in the weight of accessory sex glands, and significant decrease in the height of secretory epithelial cells of the seminal vesicles and ventral, lateral and dorsal prostate (Van Thiel et al., 1979; Semczuk and Rzeszowska, 1981; Willis et al., 1983; Anderson et al., 1985; Salonen and Huhtaniemi, 1990; Cagnon et al., 1996, 1998, 2001; Martinez et al., 1997; Garcia et al., 1999). In the accessory sex glands, deleterious effects on organelles involved in secretory processes can be emphasized, such as dilatation of the cisternae of the granular endoplasmic reticulum (GER) and Golgi complex, rupture of the microvilli, and accumulation of lipid droplets in the cytoplasm of epithelial cells (Gavaler et al., 1983; Cagnon et al., 1996, 1998, 2001; Martinez et al., 1997; Garcia et al., 1999).

The prostate is a male accessory sex gland that raises interest among researchers because it is frequently affected by different pathologies such as benign prostatic hyperplasia and cancer (Guess, 2001). Epidemiological studies have demonstrated increase in the frequency of prostate cancer since 1998, which in many cases leads to the death of the patient (Landis et al., 1998). Prostatic diseases, especially carcinoma, are known to be of an endocrine nature (Morton et al., 1996) and their frequency increases with age (Davies and Eaton, 1991).

The development and maintenance of morphofunctional activity of the prostate are mainly regulated by testosterone, in addition to other hormones such as estrogen, whose mechanism of glandular action has not been completely established (Cunha et al., 1987, 2003; Droller, 1997; Risbridger et al., 2003).

In rodents, the prostate is a complex gland formed by three pairs of lobes: ventral, lateral and dorsal lobes (Langworthy, 1965; Purinton et al., 1973; Vaalasti and Hervonen, 1979; Jesik et al., 1982; Prins, 1992). The ventral prostatic acini are lined with simple epithelium with tall columnar cells intermingled by basal cells resting on a distinct basement membrane (Cavazos, 1975). The prostatic stroma, on the other hand, is a complex arrangement of stromal cells and extracellular matrix associated with growth factors, regulatory molecules and remodeling enzymes. Stromal cells and extracellular matrix characterize a microenvironment that regulates the growth and functional differentiation of adjacent cells, with each of these components playing an important role in the maintenance of tissue form and function (Narbaitz, 1975; Labat-Robert et al., 1990; Tuxhorn et al., 2001). In addition, epithelial–stromal interaction plays an important role in maintaining the structure and function of the prostate gland (Ekman, 2000). Recent studies have demonstrated that epithelial–stromal interaction is the principal factor in the progression of prostate carcinoma (Tuxhorn et al., 2001; Cunha and Matrisian, 2002).

The consequences of alcohol toxicity on the prostate are complex and doubts remain regarding the morphophysiology of the stroma and its interaction with epithelial cells in response to chronic alcoholism. Thus, the aim of the present study was to analyze the morphophysiological alterations that occur in the secretory and stromal portion of the ventral lobe of the prostate of UChB rats in response to abusive alcohol use and further alcohol abstinence, as well as to associate these results with pathogenesis of the prostatic diseases.

2 Material and methods

2.1 Animals and procedures

Ten Wistar rats (control group) and 20 UChB rats (alcoholic and abstinent groups) aged 3months were used. The control group received tap water as the liquid diet, the alcoholic group received ethanol diluted to 10° Gay Lussac (G.L., 10ml ethanol/100ml solution—Martinez et al., 2001a) for 150days, and the abstinent group received 10° G.L. ethanol for 120days and then tap water like the control group for more 30days. All animals received Nuvilab® CR chow ad libitum as the solid diet. It should be noted that 1g solid diet supplies 2.7kcal of energy and 1g ethanol contains 7.1kcal. Liquid intake was measured daily. Solid intake and body weight were measured weekly. The variation of the body weight was calculated by means of final body weight minus initial body weight from each animal of the group. At the end of each treatment period, the animals of each group were weighed, anesthetized with Francotar®/Virbaxyl® (Virbac, Brazil) (1:1) and then submitted to cardiac puncture to obtain blood samples for hormone measurement. The testes, seminal vesicles, coagulation glands were collected and weighed on a Sartorius 2434 analytical scale. The ventral lobes of the prostate (intermediate/distal regions) were collected, by means of stereo-microscopy (DFV Vasconcelos S/A), and analyzed by histochemistry, immunohistochemistry and transmission and scanning electron microscopy.

2.2 Light microscopy

The ventral prostate was collected, fixed by immersion in Bouin's solution and formaldehyde 10%, embedded in paraplast (Paraplast Plus, Brazil) and methacrylate resin (Historesin Embedding Kit, Leica, USA), cut into 3-μm thick sections (five sections/animal), and submitted to staining procedures: (a) hematoxylin and eosin (H&E), (b) Picrossirius red (Junqueira et al., 1979), and (c) Gomori's silver impregnation for reticulin (Vilamaior et al., 2000). The thin sections were photographed using a Nikon Eclipse E-400 photomicroscope.

2.3 Immunohistochemistry

Prostate samples were embedded in tissue freezing medium (TBS, Durham, NC), frozen in liquid nitrogen, and cut into 7-μm thick sections with a Microm cryostat. The sections were collected, fixed with ice-cold methanol (3min) and acetone (3min), and air-dried for 30min, followed by fixation in 4% paraformaldehyde for 10min and rinsing with PBS. Next, the specimens were incubated with blocking solution (4% BSA and 1% Triton X-100, in PBS; Sigma, St. Louis, MO) for 1h at room temperature to block nonspecific binding. Primary rabbit anti-type I and anti-type III collagen antibodies (Chemicon International Inc., Temecula, CA) were diluted in blocking solution according to manufacturer instructions and applied to the sections overnight at 4°C. The slides were washed three times for 5min with PBS and the material was incubated with a secondary fluorescein-conjugated antibody (anti-rabbit IgG, FICT, Sigma) diluted 1:100 in blocking solution. The sections were mounted in DABCO (Sigma) solution and observed under a confocal microscope (BioRad MRC1024UV).

2.4 Stereological procedures

Five rats from each one of the experimental groups were used. Epithelial, luminal and stromal areas of the ventral lobe of the prostate were measured (25 fields per group; 5 fields per animal). The fields were chosen randomly from sections stained for light microscopy. The stromal area was defined as the non-acinar tissue in the fields. The microscopic image was acquired with 200× magnification using a calibrated eyepiece (Filar Micrometer, Nikon). The areas were measured using the Image-Pro Express 4.0 computerized image analysis system. The tool used to outline the acini was the Irregular or Freeform AOI Tool. Cellular, cytoplasmic and nuclear volumes were measured from sections stained for light microscopy. Nuclear volume was determined by means of the average of 100 measurements per group, in other words, 20 measurements per animal. Long (D) and short (d) axes were measured and the mean volume was calculated considering nuclei as ellipsoids. The average of the 20 measurements of the long and short axes was used in the formulae: V=4/3π(d/2)2·D/2, to determinate the nuclear volume. The sections were analyzed with an ocular micrometer coupled to a Zeiss microscope equipped with a 100× objective. For the determination of cytoplasmic volume, an eyepiece with a 400 grid coupled to a 100× objective was used. Points on the cytoplasm and nuclei were counted from 10 areas per group and the cytoplasmic and nuclear fractions were obtained. These data and the nuclear volume were used to estimate the cytoplasmic volume for each animal (Weibel, 1979). Cell volume was calculated by summing the nuclear and cytoplasmic volumes.

2.5 Transmission electron microscopy

Five animals per group were perfused with 2.5% glutaraldehyde in 0.1M phosphate buffer through the left ventricle (Sprando, 1990). The ventral lobe of the prostate was collected and fixed by immersion in the same fixative. The material was then dehydrated in a graded acetone series, the tissue was embedded in Araldite resin (Polysciences, USA), and 0.5-μm thick sections were stained with Toluidine blue and prepared for light microscopy in order to choose specific areas for transmission electron microscopy analysis. Ultrathin sections were obtained with an LKB ultramicrotome and contrasted with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). Electron micrographs were obtained with a LEO 906 electron microscope.

2.6 Scanning electron microscopy

Specimens were prepared as described for transmission electron microscopy up to the last dehydration step in absolute acetone. The material was then submitted to critical point drying in liquid CO2 using a CPD 030 Balzers apparatus. The specimens were coated with gold using an SCD 050 Balzers sputtering device and observed under a JEOL JSM-5880LV scanning microscope.

2.7 Hormone measurement

Blood samples from five animals of each group were used for the determination of testosterone by RIA using the Coat-a-Count Total Testosterone Kit (Diagnostic Products Corp., Los Angeles, CA). Blood was collected at the end of the 150-day experiment.

2.8 Statistical analysis

Data were analyzed statistically by analysis of variance and the Tukey multiple range test, with the level of significance set at 5% (Norman and Streiner, 1994).

3 Results

3.1 Nutritional analysis and body weight

No difference in body weight gain was observed between the three experimental groups. On the other hand, liquid and solid intakes were lower in the alcoholic group and higher in the abstinent group compared to the control group (Table 1). The consumption of 10% ethanol differed between alcoholic and abstinent animals, with a mean intake of 2.76g ethanol/kg body weight/day in the alcoholic group and of 2.05g ethanol/kg body weight/day in the abstinent group.


Table 1.

Mean ± standard deviation of body and organ weights (testis, seminal vesicle and coagulation gland), water and chow intake, and hormone measurement

Variable
Groups
ControlAlcoholicAbstinent
Variation of body weight (g)149.0 ± 28.4a126.3 ± 24.0a142.3 ± 30.6a
Chow intake (g/day)27.4 ± 1.0a,b25.6 ± 1.9a29.2 ± 2.3b
Water intake (ml/day)44.8 ± 4.9a,b40.6 ± 3.7a48.0 ± 3.6b
Testis (g)2.05 ± 0.17b1.59 ± 0.15a1.67 ± 0.20a
Seminal vesicle (g)1.08 ± 0.17a1.07 ± 0.15a0.90 ± 0.22a
Coagulation gland (g)0.12 ± 0.02a0.11 ± 0.02a0.11 ± 0.02a
Hormone measurement (ng/ml)2.86 ± 0.78b1.41 ± 0.50a1.88 ± 0.77a,b


The testes were significantly reduced in the alcoholic and abstinent groups compared to the control group. The weight of the seminal vesicles and coagulating glands was lower than in the control group, although this difference was not statistically significant (Table 1).

3.2 Light microscopy, immunohistochemistry and stereology

3.2.1 Control group

The prostatic ventral lobe was characterized by acini of different sizes and infolded mucosa (Fig. 1A, B, D). The secretory epithelium was simple with tall columnar cells and a basal nucleus (Fig. 1A, C). The prostatic stroma showed thin and short type I collagen fibers (Figs. 1B, C and 2A) often underlying the epithelium and intermingled with smooth muscle cells. In addition, reticular fibers (type III collagen) were observed underlying the epithelium around prostatic acini (Figs. 1D and 2D). The epithelial glandular area was approximately two times larger than the stromal area (Table 2).


Fig. 1

Photomicrographs of the ventral prostate of control (A–D), alcoholic (E–H, M, N) and abstinent rats (I–L). Control group: (A) Folded prostatic acinus surrounded by a thin layer of smooth muscle cells (arrowhead). H&E, ×215. (B) Prostatic stroma containing thin and scarce collagen fibers (arrowheads). Picrosirius–hematoxylin, ×100. (C) Simple secretory epithelium (Ep) with tall columnar cells. Thin collagen fibers adjacent to the epithelium (arrow). Picrosirius–hematoxylin, ×430. (D) Reticular fibers (wide arrow) accompanying the smooth muscle bundles that surround the prostatic acini. Gomori reticulin, ×430. Alcoholic group: (E) Acini showing reduced lumen (L) and areas of epithelial cell proliferation (Ep). Glandular stroma with inflammatory cells (star). H&E, ×215. (F) Acini showing poorly folded mucosa. Accumulation of collagen fibers in the prostatic stroma (arrow). Note the increase in the smooth muscle cell layer around the acini that showed cell involution (asterisks). Picrosirius–hematoxylin, ×100. (G) Epithelial atrophy (Ep). Apparent increase in the amount of collagen fibers (arrow). Presence of inflammatory cells (star). Picrosirius–hematoxylin, ×430. (H) Epithelial evagination (star) and stromal accumulation of reticular fibers with an undulated aspect. L, glandular lumen. Gomori reticulin, ×430. Abstinent group: (I) Simple columnar secretory epithelium surrounded by a thick layer of smooth muscle cells (empty arrowhead). H&E, ×215. (J) Prostatic stroma (St) containing large amounts of collagen fibers (arrow). Bv, blood vessel. Picrosirius–hematoxylin, ×100. (K) Columnar epithelial cells showing recovery of cell volume (Ep). Stroma with accumulation of collagen fibers (short arrow). Inflammatory cells among collagen fibers (long arrow). Picrosirius–hematoxylin, ×430. (L) Folded secretory epithelium. Note the presence of thick reticular fibers (wide arrow). Gomori reticulin, ×430. Alcoholic group: (M and N) Prostatic acini showing intraepithelial neoplasia (arrows). St, prostatic stroma; L, glandular lumen. Picrosirius–hematoxylin, ×215 and×430, respectively.


Fig. 2

Prostatic ventral lobe. Immunohistochemistry for type I collagen fibers (A–C). (A) Control group. Thin sheet of collagen I fibers (arrow) underlying the secretory epithelium and around blood vessels. (B) Alcoholic group. Accumulation of collagen I (arrow) underlying the secretory epithelium and around micro-acini. (C) Abstinent group. Collagen I fibers (wide arrow) underlying the secretory epithelium. Stromal accumulation of collagen I fibers (arrow). Prostatic ventral lobe. Immunohistochemistry for collagen type III fibers (D–F). (D) Control group. Stroma containing small agglomerates of type III fibers (arrows). (E) Alcoholic group. Collagen III distributed both at the base (arrow) of the epithelium and at other points (wide arrows) of the prostatic stroma. (F) Abstinent group. Collagen type III fibers (arrows) throughout the prostatic stroma. Note the epithelial evagination. Bar=200X.


Table 2.

Mean ± standard deviation of epithelial, stromal and luminal areas and nuclear, cytoplasmic and cell volumes

Variable
Groups
ControlAlcoholicAbstinent
Epithelial area (μm2)132.103 ± 19.103b72.103 ± 14.103a87.103 ± 9.103a
Stromal area (μm2)74.103 ± 13.103a118.103 ± 18.103b97.103 ± 13.103ab
Luminal area (μm2)157.103 ± 25.103a173.103 ± 31.103a179.103 ± 22.103a
Nuclear volume (μm3)100.8 ± 7.7a90.0 ± 7.7a96.8 ± 3.2a
Cytoplasmic volume (μm3)552.4 ± 87.1b358.1 ± 76.1a452.0 ± 55.7ab
Cell volume (μm3)653.3 ± 92.4b448.1 ± 78.0a548.9 ± 54.8ab




3.2.2 Alcoholic group

The prostatic acini were smaller and contained less infolded mucosa than observed for the control group (Fig. 1F, G and Table 2). A drastic reduction in the total volume of secretory epithelial cells was noted, with the nucleus occupying a large part of the cytoplasm (Fig. 1G and Table 2). Focal areas of secretory epithelial cell stratification were observed within acini showing variation in nuclear size and shape without nucleolomegaly, besides intact basal cell layer and basement membrane characterizing the low-grade of prostatic intraepithelial neoplasia (PIN) (Fig. 1M,N). The secretory epithelium showed evaginations consisting of cell masses projecting to the stroma, but the basement membrane was intact (Fig. 1H). Enlargement of collagen fibers (type I collagen) was noted in the prostatic stroma, indicating hypertrophy of stromal fibrillar elements not only in the underlying epithelium but also throughout the stromal area (Figs. 1F, G and 2B). In addition, an increase in smooth muscle cell layers was observed around the acini, especially in the case of structural disorganization (Fig. 1F). Cellular and fibrillar elements of the glandular stroma were increased, as demonstrated by 0.6 times larger stromal area than epithelial one (Table 2). Reticular fibers (type III collagen) were increased in thickness, showing an undulated aspect (Figs. 1H and 2E). These fibers were found throughout the stromal area. In addition, focuses of inflammatory cells were frequently observed in the glandular stroma (Fig. 1E), mainly in areas without high epithelial reduction.

3.2.3 Abstinent group

The cell volume was higher than that obtained for the alcoholic group, showing cellular volume recovery (Fig. 1I and Table 2). Nevertheless, enlargement of collagen fibers (type I collagen) was noted close to the epithelium and around all prostatic stromal areas as observed in the alcoholic group (Figs. 1J, K and 2C). An increase in the reticular fibers (type III collagen) was noted but was not as evident as in the alcoholic group (Figs. 1L and 2F). Therefore, the distribution of reticular fibers close to epithelial cells was similar to that observed in the control group (Fig. 1L). Inflammatory cells were intermingled with fibrillar elements, especially collagen fibers (Fig. 1K). Areas with epithelial cell stratification and epithelial evagination were observed as in the alcoholic group, but these morphological characteristics were less frequent. The glandular epithelial area was 0.8 times smaller than the stromal one (Table 2). The luminal area did not differed significantly among the three experimental groups (Table 2).

3.3 Scanning and transmission electron microscopy

3.3.1 Control group

The prostatic epithelium consisted of tall columnar cells with a basal nucleus and a clearly visible nucleolus (Fig. 3A, B). The cellular cytoplasm showed GER with parallel and flattened cisternae (Fig. 3A, B) and a well-developed Golgi complex (Fig. 3E) in the perinuclear and supranuclear regions. Secretory vacuoles containing secretion granules were identified in the apical cytoplasm (Fig. 3C). Basal cells were noted between epithelial cells resting on a clearly visible and intact basal lamina (Fig. 3D). Thin smooth muscle cells underlying the secretory epithelium were observed in the glandular stroma (Fig. 3D).


Fig. 3

Electronmicrographs of the ventral prostate of control rats. (A and B) General view of the secretory epithelium. Simple epithelium with tall columnar cells intermingled with basal cells (Bc). Oval nucleus (N) with regular contours. Note the granular endoplasmic reticulum (GER) cisternae in the perinuclear cytoplasm containing material of low electron density. L, lumen. Bar=1.0μm. (C) Detail of the apical region. Short and scattered microvilli (MV) cover the cell surface. Secretory vacuoles containing granules of different electron densities are present. (D) Detail of the basal region showing a basal cell (Bc) resting on a clearly visible basal lamina (black arrow). Stroma (St) containing smooth muscle cells (SM). Bar=1.0μm. (E) In the supranuclear region, note the Golgi complexes with flattened and thin vesicles arranged in a parallel fashion (thick black arrow). Bar=1.0μm. (F) Detail of the luminal surface of the secretory epithelium. Note the integrity of the secretory epithelium with reduced intercellular space between cells (white arrow) and the regular distribution of microvilli on the convex apical surface of these cells. Secretion granules (asterisk). Bar=10μm.


A close juxtaposition of cells was observed on the epithelial luminal surface (Fig. 3F). All epithelial cells presented uniformly distributed microvilli covering the cell surface (Fig. 3F). In addition, accumulation of glandular secretion could be noted (Fig. 3F).

3.3.2 Alcoholic group

The secretory epithelium consisting of cuboidal cells and irregular basal nuclei was atrophic and showed a reduction of cell cytoplasmic volume (Fig. 4A). Proliferation of epithelial cells and intercellular spacing with PIN-like morphology were observed in some glandular regions (Fig. 4B). Dilatation of GER and Golgi complex cisternae were noted in the supranuclear region (Fig. 4C and E, respectively). Occasional points of discontinuity of the basal lamina were noted, in addition to an irregular distribution of subepithelial collagen fibers (Fig. 4D). In the apical region, some secretory vacuoles were observed (Fig. 4C).


Fig. 4

Electronmicrographs of the ventral prostate of alcoholic rats. (A) Atrophied epithelium with involuted cells. Basal nuclei (N) with irregular contours. Intercellular spacing indicated by asterisk. L, lumen. Bar=1.0μm. (B) Occurrence of prostatic intraepithelial neoplasia (PIN). N, irregularly shaped nuclei. Intercellular spacing indicated by asterisk. St, prostatic stroma. Bar=1.0μm. (C) Details of the supranuclear and apical regions. Dilatation of granular endoplasmic reticulum (GER) cisternae. Eventual secretory vacuoles of different electron densities (V) in the apical region of the cell. Small and scattered microvilli (MV) cover the cell surface. L, glandular lumen. Bar=1.0μm. (D) Detail of the basal region. Apparent discontinuity of the basal lamina. Stroma (St) containing scattered collagen bundles (Col). Irregular smooth muscle cell (wide arrow) showing a spiny aspect. N, epithelial cell nucleus. Bar=1.0μm. (E) Detail of supranuclear region. Dilatation of Golgi complex (thick arrow). Bar=1.0μm. (F) Clearly visible intercellular spacing (arrow). Simple epithelium with a spherical nucleus (N). Clearly visible layer of smooth muscle cells (SM). Bar=10μm.


The intercellular spacing was increased in these animals (Fig. 4F). However, the convex form of the apical cell and the distribution of microvilli on the luminal surface were similar to those of the control group (Fig. 4F).

3.3.3 Abstinent group

Abstinent animals showed recovery of prostatic epithelial cell volume compared to alcoholic animals. The basal nucleus recovered its regular shape comparable to that of the control group (Fig. 5A). In the supranuclear region, the GER cisternae were flattened but some of them were dilated, characterizing intermediate morphological aspects between the control and alcoholic groups (Fig. 5B). Golgi complex were dilated (Fig. 5B). In addition, secretory vacuoles were identified in the cell apical cytoplasm (Fig. 5C). Nevertheless, the prostatic stroma continued to be hypertrophic (Fig. 5D).


Fig. 5

Electronmicrographs of the ventral prostate of abstinent rats. (A) General view of the secretory epithelium with columnar cells. Basal nucleus (N) with regular contours. Secretory vacuoles (V) in the apical region of the cell. Small microvilli (Mv) cover the cell surface. SM, smooth muscle cells; L, lumen. Bar=1.0μm. (B) Detail of the supranuclear region. Note the granular endoplasmic reticulum (GER) with flattened cisternae. Some of them were dilated (arrowhead). Dilatation of the Golgi complex (thick arrow). N, cell nucleus. Bar=1.0μm. (C) Apical region. Dilatation of GER. V, secretory vacuoles. (D) Detail of the stromal region. Visible basal lamina (arrow). Voluminous stroma (St) with a large number of cells. Smooth muscle cells (SM) showing projections with small vacuoles (v). Bar=1.0μm. (E) Regular epithelium with juxtaposed cells (arrow). Convex apical surface consisting of cells covered in a continuous fashion with microvilli. Note the simple epithelium, nuclei (N) with irregular contours and a thick layer of smooth muscle cells (SM) in the adjacent stroma. Collagen bundles in the prostatic stroma (asterisk). Bar=10μm.


As in the control group, the prostatic cells of abstinent animals were closely juxtaposed, reducing the intercellular space (Fig. 5E). The microvilli were uniformly distributed on the luminal cell surface as observed for the other groups (Fig. 5E).

3.4 Hormone analysis

A significant reduction of serum total testosterone levels was observed not only in the alcoholic group (1.41ng/ml), but also in the abstinent group (1.88ng/ml) compared to control animals (2.86ng/ml). This reduction was more prominent in the alcoholic group.

4 Discussion

In the present study, atrophy of male reproductive organs such as testes, seminal vesicles and coagulation glands was observed in the alcoholic and abstinent groups compared to control animals, with hypogonadism being significant in the alcohol-treated groups compared to control. In contrast, the weights of the seminal vesicles and coagulation glands of alcohol-treated animals, although lower, did not differ significantly from those obtained from control animals. Atrophy of the testes and accessory sex glands has been described in chronically alcohol-treated animals. According to Tadic et al. (2000), Wistar rats showed marked testicular reduction after ethanol ingestion for 3 months. Furthermore, Martinez et al. (2001a), studying UChB rats voluntarily consuming 10% ethanol for 150days, confirmed the occurrence of testicular atrophy. In that study, the authors found no difference in seminal vesicle weight between UChB rats and control animals. Cagnon et al. (2001) observed no marked differences either in the weight of the seminal vesicles and coagulation glands between control mice (C57B1/6J) and mice treated with 6% ethanol for 120days despite significant atrophy of the glandular secretory compartment in the latter. Thus, it can be concluded that in alcoholic rats ethanol exerts negative effects not only on the macroscopy of the gonads but also on that of the accessory sex glands.

The present results showed that all experimental animals gained body weight throughout the experimental period. Although those values were lower in the alcoholic and abstinent groups compared to the control group, this difference was not statistically significant. In addition, total calorie intake from the ration was numerically higher in the control and abstinent groups than in alcoholic animals, with mean daily intakes of 149.6kcal/kg0.75, 157.6kcal/kg0.75 and 138.4kcal/kg0.75, respectively. According to the National Research Council (1995), the daily energy requirement for the maintenance of body functions of adult rats is approximately 110kcal of digestible energy per kg0.75 body weight. In the literature, several morphological studies conducted on alcoholic animals, especially involuntarily alcohol consuming ones, have raised concern regarding the nutritional status of these animals. These studies demonstrated that chronic alcoholic animals presented deficient weight gain, with weight gain being numerically lower than in controls (Cicero and Badger, 1977; Klassen and Persaud, 1978; de Oliveira and Ferreira, 1987). Campana et al. (1975) reported that in the state of protein malnutrition the animals presented disturbances such as hair loss or altered hair distribution, diarrhea and edema, in addition to marked weight loss. Furthermore, Mardones and Quintanilla (1996) demonstrated a natural tendency of UChB and UChA rats to ingest alcohol based on the administration of bromocriptine, which acts on ethanol satiety in rats. In that experiment, a specific reduction in alcohol consumption was observed during treatment with the drug, without alterations in ration intake. Ethanol consumption was resumed after the discontinuation of bromocriptine treatment. Thus, it was concluded that the amounts of energy ingested by the animals studied were adequate for the development and maintenance of body requirements. Also, they showed voluntary ethanol consumption, a fact that qualified them as an excellent model for the study of chronic alcoholism.

Chronic alcoholic animals showed marked structural and ultrastructural alterations in both secretory epithelium and glandular stroma. These changes included epithelial atrophy, low-grade PIN, epithelial evaginations projecting to the stroma, alterations in the biomembrane system of organelles involved in the glandular secretory process, an increase and irregular distribution of subepithelial collagen fibers (types I and III), altered smooth muscle morphology and presence of inflammatory cells in the prostatic stroma. Experimental studies employing different alcohol doses and treatment times have demonstrated the damaging effects of abusive ethanol use on the accessory sex glands, including epithelial atrophy and alterations in the cell organelles involved in the secretory process (Cagnon et al., 1998, 2001; Martinez et al., 2001a,b). On the other hand, no prostatic focal intraepithelial proliferation was observed in these studies. The term PIN (Prostatic Intraepithelial Neoplasia) is usually used to describe high-grade PIN. However, we have two forms of PIN, low-grade PIN and high-grade PIN. The first type (low-grade PIN) is characterized by epithelial cell crowding and stratification with irregular spacing, marked variation in nuclear size and shape without nucleolomegaly, besides normal chromatin and intact basal cell layer and basement membrane (Häggman et al., 1997; Bostwick et al., 2003). The high-grade PIN shows, similar to low-grade PIN, more crowding and stratification into four patterns: tufting, micropapillary, cribriform and flat. Moreover, high-grade PIN presents nucleus enlargement, with some size and shape variation, increased density and clumping of chromatin, large and prominent nucleoli and it may show some points of disruption of the basal cell layer and basement membrane (Häggman et al., 1997; Bostwick et al., 2003). Actually, PIN has been clinically indicated as a precursor lesion of invasive adenocarcinoma (Davidson et al., 1995; Häggman et al., 1997; Xie et al., 2000; Alberts and Blute, 2001). The high clinical incidence of prostatic cancer identified in positive biopsies obtained from patients with PIN, especially high grade PIN, confirms the tendency of PIN to progress to malignancy (Davidson et al., 1995; Häggman et al., 1997; Xie et al., 2000). Thus, it can be concluded that ethanol not only was a harmful drug to the glandular secretory process but also caused pathogenesis of prostatic lesions which could be associated with later processes of glandular malignancy.

In the specialized literature, the stromal microenvironment is known to be dynamic and to directly influence the differentiation of prostatic epithelial cells and glandular growth and function, in addition to actively participating in tissue repair in response to injuries (Tuxhorn et al., 2001; Cunha et al., 2002). Furthermore, epithelial–stromal interaction plays an important role in the maintenance of the structure and functioning of the prostate gland (Ekman, 2000). It is known that an imbalance in the interaction of the glandular compartments favors the onset and development of prostate carcinoma (Cunha et al., 2003). According to several investigators, stromal cells together with tumor cells respond to androgens and growth factors, leading to the interruption of epithelial–stromal homeostasis, an event that definitely triggers processes of cell growth, angiogenesis, apoptosis, and tumor metastases (Wong et al., 2000; Cunha et al., 2001, 2002).

Vilamaior et al. (2000), investigating the prostate gland of rats after castration, reported that smooth muscle cells showed an altered phenotype in response to reduced serum testosterone levels, with this alteration not only involving the synthesis and secretion of extracellular matrix components but also the active remodeling of fibrillar components of the stroma. An altered smooth muscle cell phenotype was also observed by other investigators both in remodeling tissues in response to different injuries and in prostate carcinomas. Such alterations characterizes these cells as myofibroblasts, which are secretory cells of an intermediate phenotype between fibroblasts and smooth muscle cells (Tuxhorn et al., 2001, 2002; Cunha et al., 2003). So it can be concluded that in the present study, abusive ethanol consumption caused changes in smooth muscle cells which assumed a secretory phenotype and it could be responsible for the hypertrophy of stromal fibrillar elements.

The presence of inflammatory cells in the prostate stroma of alcoholic animals has not been emphasized in the literature. However, the prostate chronic inflammatory processes are associated with both postatrophic hyperplasia and simple focal atrophy (Billis and Magna, 2003). According to Billis (2000), chronic prostatitis is generally caused by bacteria. However, among the different types of nonspecific chronic prostatitis the noninfectious forms are the most frequent and are a constant finding in benign prostatic hyperplasia. This author also reported that this process of inflammation is the result of extravasation of prostatic secretion into the stroma after obstruction of the ducts. Current studies have demonstrated a relationship between inflammatory infiltrates and tumor maintenance and progression (Wilson and Balkwill, 2002). According to Lin and Pollard, 2004, inflammatory cells, especially leukocytes, when present in the tumor microenvironment, promote the production of diverse growth factors, proteases and angiogenic mediators, thus permitting tumor maintenance and progression. Thus, the presence of inflammatory cells concomitantly with the occurrence of PIN and stromal hypertrophy observed in the present study suggests an active participation of these cells in the destructuring of the epithelial–stromal interaction. On the other hand, the occurrence of inflammatory cells might have been secondary to the abusive use of alcohol, originating from the process of epithelial atrophy and stromal hypertrophy which probably caused extravasation of prostatic secretion into the stroma, indicating the possible presence of an inflammatory process.

Abstinent animals showed quantitative volume recovery of the secretory epithelium. However, similar to the alcoholic group, the prostatic stroma was found to be hypertrophic and foci of inflammatory cells were present. On the other hand, low-grade PIN and epithelial evaginations were observed only occasionally. Cagnon et al. (1998), studying Wistar rats submitted to experimental chronic alcoholism for periods ranging from 60 to 300days, observed recovery of epithelial height in the alcoholic group after interruption of ethanol ingestion for 60days, similar to the present findings. However, the same authors noted that the alterations in the organelles involved in the glandular secretory process persisted. With respect to stromal alterations, studies correlating the effects of alcoholism with changes in the prostatic stroma are scarce. However, the fact that no complete recovery of prostatic tissue was observed in abstinent animals indicates continuous glandular disorganization.

Hormone measurement revealed a significant decrease in serum testosterone levels in both alcoholic and abstinent rats compared to control animals. Different investigators have shown a reduction of serum testosterone levels in animals submitted to the ingestion of various alcohol doses, with these alterations being attributed to a direct action of ethanol on tissues as well as to an indirect action through imbalance of the hypothalamus–pituitary–gonadal axis (Van Thiel and Lester, 1979; Tadic et al., 2000). According to Himwich (1957), the direct action of ethanol occurs firstly after alcohol absorption by gastrointestinal tract. Secondly, it is transported through hepatic-portal system and lately it is distributed to different organs by means of circulatory system. Martinez et al. (2001a) reported a significant reduction of serum testosterone in UChB rats voluntarily consuming 10% ethanol compared to control UChA and Wistar rats. Tadic et al. (2000) also observed a significant decline in serum testosterone in Wistar rats submitted to chronic alcoholism for periods ranging from 30 to 90days. These authors characterized a direct relationship between the testicular atrophy of these animals and plasma testosterone levels. Thus, it can be concluded that the decreased testosterone levels caused by alcohol intakes compromised the balance of the hypothalamic–pituitary–gonadal axis, corresponding to indirect effect of the ethanol on the prostate. Moreover, taking into consideration the literature, it could be suggested that probably there is also a direct effect of the alcohol on the prostatic gland by means of circulatory system. Therefore, both of them could contribute to the disorganization of prostatic glandular tissue.

Recent epidemiological studies have been conducted to correlate the effects of alcoholism with the possibility or an increased risk of developing prostate cancer (Albertsen and Gronbaek, 2002), but no direct relationship was observed. However, the present results indicate that ethanol causes intensive stromal disorganization characterizing the interruption of epithelial–stromal homeostasis, a fact that points out this drug as a possible element of the focal epithelial proliferation which could predispose the organ to early processes of glandular malignancy.

Acknowledgment

This study was supported by CAPES.

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Received 28 July 2006/24 September 2006; accepted 5 November 2006

doi:10.1016/j.cellbi.2006.11.009


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)