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. 2008 Aug;89(4):276–283. doi: 10.1111/j.1365-2613.2008.00591.x

Malignant lesions in the ventral prostate of alloxan-induced diabetic rats

Daniele Lisboa Ribeiro *, Silvio Fernando Guideti Marques †, Sandra Alberti †, César Tadeu Spadella †, Antônio José Manzato ‡, Sebastião Roberto Taboga §, Nishtman Dizeyi , Per-Anders Abrahamsson , Rejane Maira Góes §
PMCID: PMC2525783  PMID: 18715471

Abstract

The aim of this study was to evaluate the changes caused by chronic diabetes in the rat ventral prostate and to establish a correlation between diabetes and the development of prostatic lesions. Male rats received alloxan (42 mg/kg b.w.) to induce diabetes. Ninety days after diabetes diagnosis, animals were sacrificed and the ventral prostate was removed and prepared for general and immunohistochemical analyses. The total area showing different types of lesions was estimated. Diabetes led to a decrease in the body and prostatic weights, as well as in testosterone levels. The prostate morphology and stereology showed high variation in the diabetic group. Some animals had light changes; the great majority had an intense epithelial atrophy; and other rats showed premalignant and malignant lesions in the prostate. Such epithelial atrophy was, in some samples, combined with chronic inflammation, similar to proliferative inflammatory atrophy (PIA). The diabetic group also presented high incidence of prostatitis, adenocarcinoma and prostatic intra-epithelial neoplasia (PIN). Samples with adenocarcinoma had poorly differentiated acini with high levels of cellular proliferation and nuclear atypia. These lesions exhibited an invasive feature showing Bcl-2-positive cells and interruptions in the basement membrane. An association of PIA, PIN and adenocarcinoma was detected in one sample. Reduced androgen levels have a synergic effect to insulin dysfunction promoting negative effects in the rat prostate. Diabetic individuals had a high incidence of prostatitis, and this inflammation could stimulate the incidence of other forms of prostatic pathology.

Keywords: alloxan, diabetes, inflammation, intra-epithelial neoplasia, prostate atrophy

Diabetes Mellitus is a chronic disease which affects the metabolism of proteins, carbohydrates and lipids. The major characteristic is hyperglycaemia as a consequence of abnormal secretion of insulin in the pancreas (type I) or inefficient action of insulin in the target tissues (type II) (Robbins 1989). It is estimated that there are 150 million diabetic people in the world and the projection of the World Health Organization is 366 million by the year 2030 (Barros et al. 2006). This disorder promotes adverse effects in all organic systems. Diabetes exerts a negative action on the neuroendocrine axis and those effects can enhance the action of diabetes on other organs that are dependent on the axis, for example, male gonads and accessory glands. It is well established that low testosterone levels are related to diabetes and they can influence the morphology of reproductive accessory glands (Soudamani et al. 2005).

The impact of diabetes on the prostate is still a controversial issue. Morphological and quantitative studies in rodents (Cagnon et al. 2000) indicated that changes caused in the ventral prostate by experimental and spontaneous diabetes are similar to those described for castration (Goes et al. 2007; Oliveira et al. 2007). Furthermore, it has been shown that besides the aforementioned drastic structural alterations, diabetes is also related to the incidence of prostatic intra-epithelial neoplasia (Ribeiro et al. 2006). However, the relationship between diabetes and prostate cancer is not clear (Ilic et al. 1996; Weiderpass et al. 2002).

Prostate cancer is still a considerable source of mortality among men around the world (Palapattu et al. 2004). The factors which determine the risk for prostate cancer are still poorly understood but it has been recently described that a chronic process such as inflammation could be responsible for the development of cancer in this gland. Furthermore, chronic inflammation is frequently associated with focal atrophy and post-atrophic hyperplasia of the prostate (Tomas et al. 2007). The term proliferative inflammatory atrophy (PIA) was proposed by De Marzo et al. (2003) to unify the atrophic processes that are highly proliferative and occur in the prostatic epithelium. Histological studies suggest that PIA could be a precursor or a risk factor for prostate cancer (De Marzo et al. 2007). Besides morphological and immunophenotypical observations, there is also genetic evidence for the potential role of PIA as a precursor of malignant tumours (Faith et al. 2005).

It is interesting to note that diabetes causes hormonal changes and plays an important role in prostate physiology and it can also cause damage to the immunological system, thus facilitating the development of chronic inflammation. Thus, it is necessary to study the effects of diabetes on prostate morphophysiology focusing on the lesions that can occur in this gland. Regarding the controversial correlation between diabetes and prostate cancer and the lack of studies focusing on prostatic biology in diabetic individuals, the purpose of the present work was to evaluate the changes caused by chronic diabetes induced in the rat ventral prostate through histological, stereological and immunohistochemical methods.

Material and Methods

Experimental diabetes induction

Seventy Wistar rats 3-month-old male, weighting 200–300 g were used in this experiment. Diabetes was induced by a single injection of 42 mg/kg alloxan diluted in physiological solution after 12 h of fasting, according to Lerco et al. (2003). Animals received 100 μl of the drug solution in the tail vein (experimental group, n = 55) or just the vehicle solution (control group animals, n = 15). Blood glucose was measured between 1 and 15 days after injection using the glucose oxidase method (Accu-Chek Active; Roche Diagnostics, Mannheim, Germany). Animals with glucose levels up to 200 mg/dl were considered diabetic. Three months after diabetes onset, all the animals were anaesthetized by CO2 inhalation and immediately killed by decapitatation and their prostate removed. Body weight was monitored throughout the experiment. Animal handling and experiments were performed according to the ethical guidelines of the São Paulo State University (UNESP, publication No. 17/07-CEEA), following the Guide for Care and Use of Laboratory Animals (NIH).

Serum hormonal levels

Blood was collected by cardiac puncture immediately before death. Plasma was separated by centrifugation and stored at −20°C for subsequent assays. Serum quantification of testosterone was done using the Modular Analyzer for Immunoassay of Chemiluminescence ECI (Johnson & Johnson, Langhorne, PA, USA) (Weeks & Woodhead 1984). Five animals were used for each group and the test was performed in triplicate. The intra- and inter-assay variations were 4.6% and 4.3% respectively.

Light microscopy

The prostates were weighed and fixed by immersion in Karnovsky fixative (5% parformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2) for 24 h. After fixation, the tissues were dehydrated in ethanol series, embedded in glycol methacrylate resin (Leica historesin embedding kit, Leica, Nussloch, Germany) or paraplast and sectioned at 3 μm on a Leica automatic rotatory microtome (Leica RM2155). Sections were stained with haematoxylin–eosin and Gömöri Reticulin for general studies. The analyses were made in a Zeiss-Jenamed light microscope (Carl-Zeiss, Jena, Germany), coupled with a semi-automatic image analysis system (image-pro plus; ©Media Cybernetics version 4.5 for Windows software, MD, USA).

Immunohistochemical detection of tenascin and Bcl-2

Some fragments were fixed in formaldeyde 4% for immunohistochemistry analysis. The antigen retrievel was performed at 92 °C with citrate buffer pH 5.0 for 20 min. Briefly, slides were treated with H2O2 3% in methanol, for 20 min, to quench the endogenous peroxidase activity and incubated in horse serum 1% in PBS for 1 h, to block non-specific binding. Primary antibodies: rabbit anti-human laminin (ab7463, Abcam, Cambridge, UK), mouse anti-human tenascin (IQ012, FKBiotek, Brazil) and rabbit anti-human Bcl-2 (SC-783, Santa Cruz, CA, USA) were incubated (1:200) in a humidified chamber overnight, at 4 °C. Then, sections were incubated for 45 min with a biotinylated secondary antibody, followed by peroxidase-labelled avidin/biotin solution reaction (NovoStain Super ABC Kit, Novocastra, Newcastle-upon-Tyne, UK) for 45 min. Finally, they were exposed to the peroxidase substrate DAB for 3 min, stained in methyl green or haematoxylin and mounted.

Stereological and statistical analysis

Paraffin sections were used to estimate the volume of components of the prostate: epithelium, lumen, smooth muscle cells and stroma. For each animal at least two different fragments were used and from those fragments, 20 different and consecutive fields were observed at the objective of 20X. The percentage of each component was calculated using the Weibel method of counting points (Weibel 1963), performed according to Ribeiro et al. (2006). The quantitative data were analysed by non-parametric Student's t-test, linear correlation of Pearson and the significance level between groups was evaluated by non-parametric tests: Mann–Whitney and Kruskal–Wallis using Minitab programme.

Multiplicity of prostatic lesions

Histopathological lesions detected in the rat ventral prostate of diabetic animals were classified according to the classification system proposed by Shappel et al. (2004): simple atrophy, PIA, prostatic intra-epithelial neoplasia (PIN), adenocarcinoma and inflammation. The digitized images of histological sections from three different fragments per animal were acquired as described above and employed to quantify the multiplicity of lesions (% of the tissue). Image analysis (image j 1.34; Wayne Rasband, Research Services Branch, National Institute of Health, Bethesda, MD, USA) was used to line up the area corresponding to lesions and the total area of the histological section. The remaining areas without these parameters were considered normal and the sum of all parameters was always 100%.

Results

Because of the very high glucose levels, about 25% of alloxan-treated animals survived after 3 months of diabetes. Alloxan-induced diabetes caused a significant decrease in body and prostatic weight, as well as a significant increase in the glucose levels (Table 1). The relative weight of the ventral prostate and testosterone serum levels also decreased after 3 months of diabetes (Table 1). Statistical analyses showed a strong inverse correlation between body weight and glucose levels (−0.73) and a weak inverse correlation between prostate weight and glucose levels (−0.55) in diabetic animals.

Table 1.

Mean values of glucose (mg/dl) and serum testosterone levels (ng/dl), variation of body weight (BW) (g) and relative ventral prostate weight (RPW) from control and diabetic group

Control Diabetic P-value
Glucose levels 89.29 412* 0.0000
Testosterone levels 82.2 26.4* 0.0327
BW 200 −36* 0.0000
RPW 0.0014 0.0008* 0.0002
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*

Significant difference between groups. P < 0.05.

Light microscopy and stereology

In control animals, the intermediary portion of the ventral prostate showed a typical tubulo-alveolar organization, with well-developed acini and different degrees of epithelial folds (Figure 1a). The presence of a wide acinar lumen and the evidence of conspicuous Golgi complex in the apical portion of the epithelium point to a high level of secretory activity in the prostate of these animals (Figure 1b). The sub-epithelial stroma exhibited a diffuse and discontinuous layer of smooth muscle cells associated with fibroblasts and other components of connective tissue (Figure 1b).

Figure 1.

Figure 1

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Photomicrographs of the ventral prostate from control (a and b) and diabetic rats (c–f). H&E. (a) Large acini with folded epithelium. (b) Typical prismatic epithelium (e) constituted by high columnar cells with prominent Golgi complex (*). The smooth muscle layer is thin with long and flat nuclei (s). (c) Ventral prostate of diabetic rat showing characteristics similar to the control with epithelial folds in addition to some gland atrophy (arrows). (d) Detail of the prismatic epithelium (e) showing cells smaller than controls. Golgi complex area (*). The stroma(s) showed clear decrease in the muscle layer thickness. (e) Prostatic atrophy. Acini had bigger luminal space (a), secretion accumulation and decreased epithelium. (f) Detail of the epithelial atrophy consisting predominantly of flat cells with small nuclei and distinct loss of cytoplasm (arrows). Stromal space between the acini was loose with reduced muscle layer(s) Scale bars: A, C, E: 200 μm; B, D, F: 10 μm.

The light microscopy analysis evidenced three patterns of histological response to diabetes. Mild histological alterations were detected in a small percentage (22%) of animals which had glandular features very similar to the control ones. Aside from some points of atrophy, most acini had epithelial folding (Figure 1c), high columnar cells, normal secretion activity and stroma with a high degree of similarity to the control animals (Figure 1d). Most diabetic animals (64%) showed intense atrophy in the prostate. Acini had dilated lumen interspersed with loose stroma (Figures 1e, 4a). The epithelial cells exhibited a high degree of atrophy that showed greater width than height and decreased volume of cytoplasm, but still indicated secretory activity (Figure 1f). Among diabetic animals, 35% showed drastic alterations in prostate histology caused by premalignant and malignant lesions, exhibiting PIA, PIN, adenocarcinoma and granulomatous inflammation (Figure 4).

Figure 4.

Figure 4

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Photomicrographs of prostatic lesions in diabetic rats. Staining: H&E (a–g), Gomori's reticulin (h). (a) Simple atrophy. (b) Prostatitis: inflammation in the stroma consisting of lymphocyte cells (ii) and larger number of macrophages (arrow). Acini (a). (c) PIA lesion is represented by atrophic acini associated with intense inflammation (ii). (d) Prostatic intra-epithelial neoplasia (PIN) in a pseudocribriforme pattern. (e) Adenocarcinoma showing poor differentiated acini (arrows) and intense cell proliferation related to inflammation areas. (f) Malignant cells (arrow head) invading adipose tissue surrounding prostate (at). (g) Poorly differentiated acini in detail showing disorganization of the epithelium and high morphological variation, cells with large nuclei, numerous and prominent nucleoli (arrows). (h) Reticular fibers are not continuous and indicate interruption in the basement membrane (white arrow). Scale bars: a, d, e: 50 μm; b: 25 μm; c: 100 μm; f, g, h: 10 μm.

When the stereological data of diabetic animals were plotted on an integrated graphic representation of epithelium, lumen and muscle stroma, the control group forms a cluster represented by high epithelial and mild luminal volume (Figure 2). On the other hand, the diabetic group was clustered in two subgroups located at opposite extremes of the graph, reflecting very distinct patterns of histological changes (Figure 2). One subgroup had a considerable decrease in the epithelial volume and large luminal amplitude which reveals an atrophic response caused by diabetes induction. The other subgroup was located on the opposite side because of decreased frequency of epithelium and an almost complete lack of luminal space, representing a drastic difference between control and diabetic animals with prostatic atrophy (Figure 2).

Figure 2.

Figure 2

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Variations in the relative frequency (%) of tissue compartments of the ventral prostate in control (n = 5; •) and diabetic (n = 9; Inline graphic) animals. Ep, acinar epithelium; L, lumen and SMC, smooth muscle cells. The points represent the mean of each individual. Note that diabetic group was separated into two sub-groups according to its high morphological variation in the prostate. The parameters epithelium and lumen were statistically different between control and diabetic group (P =0.018 and 0.003 respectively. Kruskal–Wallis test).

Multiplicity of lesions

About 64% of all diabetic animals had simple atrophy and PIN, 7% showed PIA and 35% had adenocarcinoma. In most cases, areas with normal glands were located together with simple atrophy which occupied about 20% of the total gland (Figures 3 and 4a). PIA occurred in concomitant area with 13% of PIN and 81% of adenocarcinoma. PIN lesions occupied an average of 4% and adenocarcinoma represented 23% of the gland (Figure 3). There was a frequent association of PIN with atrophic epithelium and some adenocarcinoma areas.

Figure 3.

Figure 3

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Graph for multiplicity of the lesions (atrophy, PIA, PIN, adenocarcinoma and inflammation) compared with normal areas in the ventral prostate of control and diabetic rats.

Morphological features of predominant lesions

The atrophy was predominant in all diabetic samples. There were large acini, drastic reduction in the epithelial height with flat cells and evident loss of cytoplasm (Figure 4a). Inflammation occurred in 29% of diabetic animals and all of them were represented by infiltrate of lymphocytes and eosinophils, except the animal with PIA where the atrophy was related to drastic inflammation characterized by high numbers of macrophages (Figure 4b). PIA lesions were classified as proliferative epithelium which exhibited morphological features of simple atrophy associated with dense inflammatory areas (Figure 4c). PIN lesions were frequent and had the papillary and/or pseudocribiform pattern (Figure 4d). Adenocarcinoma exhibited poorly differentiated gland in some cases (Figure 4e) and invasion in the surrounding adipose tissue (Figure 4f). These tumours showed atypical cells, nuclear and nucleolar morphological variation (Figure 4g) and interruptions in the basement membrane (Figure 4h), which demonstrate the invasiveness of these lesions.

Immunohistochemistry

The control group prostate did not express Bcl-2. In contrast, all diabetic groups with simple gland atrophy did express Bcl-2 (Figure 5a and b). All suspect lesions including PIA and adenocarcinoma were positive for Bcl-2 (Figure 5c and d). The immunohistochemistry for tenascin in the prostate of control group demonstrated light reaction below the epithelium and in some blood vessels (Figure 5e). There was intense staining for tenascin throughout the area surrounding tumours especially in the sub-epithelial region, in the diabetic animals with prostatic lesions (Figure 5f). Immunostaining for laminin showed evident interruptions in the basement membrane of malignant acini when compared to control ones (Figure 5g and h).

Figure 5.

Figure 5

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Immunohistochemistry of the ventral prostate of control (a, e, g) and diabetic rats (b, c, d, f, h). (a) Control animals showed no positive reaction for Bcl-2 in the ventral prostate. (b) Diabetic group with prostatic simple atrophy also did not exhibit Bcl-2 expression, except in some lymphocytes. (c and d) Positive staining for Bcl-2 in the lesions suspected for malignancy: PIA (c) and adenocarcinoma (d). (e) Light positive staining for tenascin in the control prostate. (f) Intense immunoreaction for tenascin surrounding lesions indicates the presence of reactive stroma. (g and h) Laminin immunostaining showed intact basement membrane in control (g) and its disarrangement in the malignant lesions of diabetic animals (h). The arrows indicate the point of the basement membrane disruption. Scale bars: a, b, e, f: 100 μm; c, d, g, h: 25 μm.

Discussion

Prostatic weight, as well as testosterone serum levels decreased significantly in the diabetic group. Androgens are crucial for cell proliferation, differentiation and normal functioning of the prostate. Beside androgens, other hormones such as insulin, glucocorticoid and oestrogens also have an impact on this gland since it has receptors for these hormones (Cleutjens 1997). A recent study has shown that diabetes influences the differentiation and maturation of the pubertal rat prostate negatively and decreases its weight and epithelial volume. Moreover, it has been demonstrated that diabetes can reduce the number of androgen receptors in the prostate (Barros et al. 2006). Thus, the loss of prostatic weight shown in the present work is in line with the literature (Cagnon et al. 2000; Ribeiro et al. 2006) and confirms the negative role of diabetes in this reproductive gland.

This study showed a high variation in the prostatic morphology of diabetic rats. Considering the histological and stereological data, some diabetic animals had light changes; a great majority had an intense epithelial atrophy; and other rats showed premalignant and malignant lesions in the prostate. This data confirmed the high degree of atrophy in the ventral prostate caused by experimentally induced diabetes. It is interesting to note that prostatic atrophy observed in chronic diabetes herein, especially the epithelial type, is an event that resembles other androgen deficiency situations, such as castration (Goes et al. 2007; Oliveira et al. 2007). However, while the epithelial atrophy resultant from androgenic suppression is similar to the involution process that shows morphology similar to undifferentiated prostate and decreased secretion, the atrophic pattern observed in most of the chronic diabetic animals, is very distinct with extremely flat epithelial cells that maintained secretory activity. Previous reports about testosterone supplementation in diabetic animals revealed that the androgen does not completely establish changes in the reproductive system, while insulin replacement can reverse prostatic involution and serum levels of testosterone (Yono et al., 2005). Our morphological data reinforce this idea because they show clear evidence that the effects of diabetes do not result only from androgen decrease but are also related to insulin. It is possible that the negative effects of androgen deficiency could be intensified by absence of insulin since it presents a stimulatory effect on the normal prostatic morphophysiology.

Prostatic lesions occurred in 35% of diabetic animals. These lesions were classified as PIA, PIN and adenocarcinoma. The immunohistochemistry for Bcl-2, the presence of intense proliferation and points of basement membrane interruption together with the poor differentiation of some acini indicate that these lesions have malignant features. The bcl-2 is a proto-oncogene family that plays a central role in the regulation of apoptosis. The protein product encoded by bcl-2 promotes cell survival by effectively suppressing apoptosis in diverse cellular settings (Wang et al. 2004; Yoshino et al. 2006). The potential predictive value of Bcl-2 for determining the malignancy has been demonstrated in other human tumours, including breast cancer and head and neck cancer (Mackey et al. 1998). Overexpression of Bcl-2 was observed in numerous neoplastic prostate tissues and was found to protect prostate cancer cells against apoptosis in vitro and to conferring resistance to androgen depletion in vivo (Huang et al. 2003). These findings prove that an abnormal balance between anti-apoptotic and pro-apoptotic molecules may affect tumour development and/or progression showing the importance of Bcl-2 as a malignant marker. In spite of this malignant features described herein for diabetic animals, data have shown no positive relation between diabetes and prostate cancer development (Weiderpass et al. 2002). Furthermore, insulin acts as a growth factor for cell proliferation and stimulates IGF release which also has a stimulant effect on tumours. Thus, the absence of insulin in diabetic animals should be a protective factor against tumour (Calton et al. 2007). In contrast, other reports suggest a possible positive correlation between diabetes and benign hyperplasia, urinary symptoms and prostate cancer (Will et al. 1999; Michel et al. 2000). Therefore, the correlation of diabetes and prostate cancer is still controversial and warrants further elucidation.

The area presenting prostatitis was larger in diabetic animals compared to control ones and occupied most of the gland. A recent work showed that alloxan-induced diabetes promoted a high incidence of stomach infections and this infection would act as a precursor for carcinogenesis in this organ (Kodama et al. 2006). It is well known that high levels of glucose caused by diabetes can block the correct functioning of immunological cells and for this reason diabetic patients have recurrent infections (Calvet & Yoshikawa 2001). Furthermore, the immune system is damaged in diabetic individuals, and it could explain the high incidence of prostatitis in diabetic rats. The inflammation process may induce carcinogenesis through morphological and genetic damage in the cells, and it also can create a microenvironment rich in cytokines and growth factors increasing cell proliferation (Palapattu et al. 2004). According to recent models of prostatic carcinogenesis, epithelial proliferative cells from PIA express high levels of glutathione S-transferase (GSTP1), an enzyme which inactivates carcinogens in the cells (Nelson et al., 2003). The high expression of GSTP1 acts as a defence against oxidative damage in the genome. Areas with inflammation surrounding PIA can induce mutations in GSTP1 genes in atrophic epithelial cells rendering them vulnerable to oxidants, thus damaging DNA and promoting neoplasic transformation to develop PIN lesions. In a next step, cell mutation in PIN performs a malignant progression (Karainov et al. 2007). All these studies show that PIA is more frequent in cases of adenocarcinoma suggesting an active role in tumour promotion. But this progression from PIA to PIN and subsequent tumour development is still debated (Tomas et al. 2007).

Our results demonstrated the incidence of malignant lesions in the prostate gland of diabetic animals. However, a possible toxic role of alloxan in tissues other than the pancreas cannot be excluded. Previous studies showed that alloxan promotes toxic effects in the cell membrane of hepatocytes (Bilic 1975) while others have suggested that alloxan is not toxic in hepatocytes as the high levels of glucose can protect these cells from membrane damage (Harmam & Fischer 1982). Thus, the toxic role of alloxan in other tissues remains unproven.

The high morphological variation observed in the diabetic group indicates that the immunological function and the deficiency of insulin and testosterone presents action that varies among individuals. Thus, it is possible to assume that those with high incidence of inflammation caused by diabetes could develop subsequent premalignant and malignant lesions in the prostate, while the remained diabetic individuals without prostatitis may develop only simple atrophy resultant from androgenic decrease.

Acknowledgments

This work was supported by Coordinating Body for Training-Capes, scholarship (PDEE-BEX0146/07-2) and Foundation for Scientific Research of Sao Paulo State-FAPESP. The authors would like to thank to Mr Luiz Roberto Faleiros Jr for the technical support.

Conflict of interest

The authors declare that there is no conflict of interest associated with this manuscript.

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