Effects of the Antifungal Agent Itraconazole on Proliferative Changes of the Forestomach Mucosa in Alloxan-induced Diabetic Rats

Abstract

Alloxan-induced diabetic rats frequently exhibit proliferative lesions of squamous hyperplasia accompanied by chronic inflammation and Candida albicans infection in the forestomach, and some lesions progress to squamous cell carcinoma (SCC). Candida infection causes not only hyperplastic changes with inflammation but might also lead to SCC in human oral mucosa. Thus, the present study was conducted to examine the effects of the antifungal agent itraconazole (ITCZ) on proliferative and inflammatory changes of the forestomach in alloxan-induced diabetic WBN/Kob rats. Diabetes was induced by alloxan at fifteen weeks of age. Rats were allocated to three groups at forty-five weeks of age and were given ITCZ by gavage 0 (vehicle control), 5, and 10 mg/kg/day for four weeks, and they were sacrificed at the sixty-fifth week of age. Mucosal hyperplastic changes were consistently accompanied by inflammation and Candida infections in the 0 mg/kg group. These lesions were reduced by ITCZ (0 mg/kg; 100%, 5 mg/kg; 53.5%, 10 mg/kg; 61.5%). Squamous cell carcinoma was detected in three rats from the 0 mg/kg, but only one rat from the 10 mg/kg dose groups in this study. Itraconazole reduced the degree of mucosal hyperplasia, inflammatory changes, and Candida infection. Therefore, C. albicans infection was an important factor in pathogenesis of mucosal proliferation and inflammation.

Introduction

Recently, we reported that long-lasting diabetic conditions accelerated by alloxan (AL), a non-genotoxic diabetogenic chemical, frequently induced proliferative changes of the forestomach that progressed to squamous cell carcinoma (SCC) in rats, and recommended this experimental system as a suitable model for the study of the inflammation-related carcinogenesis (Kodama et al. 2006; Sano et al. 2008). In this model, hyperplastic changes were consistently accompanied by chronic suppurative inflammation of the mucosal epithelium with fungal infection. Fungi showing dimorphism, such as yeast and mycelial forms, were identified as Candida albicans (C. albicans) (Kodama et al. 2006).

Candida infection is well known to occur in immunocompromised hosts affected by various diseases such as diabetes mellitus and acquired immunodeficiency syndrome (AIDS) and those undergoing immunosuppressive therapy. Candida infection of the human oral mucosa causes not only chronic hyperplastic candidosis, characterized by thickening of the epithelium associated with acute and chronic inflammation, but might also lead to malignant change (Barrett, Kingsmill, and Speight. 1998; Renstrup 1970; Sitheeque and Samaranayake 2003). C. albicans may also lead the progression to carcinoma in vitro (Williams et al. 2001) and has been shown to have a promoter function of oral mucosal tumor in in vivo carcinogenesis studies (O’Grady and Reade 1992). Therefore, C. albicans infection in a prolonged diabetic state may be deeply involved in AL-induced proliferative lesions, including inflammatory changes.

C. albicans selectively up-regulates cyclooxygenase-2 (COX-2), but not COX-1 (Deva et al. 2003). Cyclooxygenase-2 was shown to be involved in human upper aerodigestive tract carcinogenesis (Mohan and Epstein 2003), and COX-2 overexpression was found in mucosal hyperplastic lesions of the rat forestomach induced by butylated hydroxyanisole or caffeic acid treatment (Kaneko et al. 2002). Our previous report also demonstrated that AL-induced proliferative lesions have elevated expression of COX-2 with C. albicans infection (Kodama et al. 2006). C. albicans might enhance COX-2 expression of mucosal hyperplastic and neoplastic lesions in AL-induced diabetic rats.

Oral administration of azole antifungals including itraconazole (ITCZ) is recognized as a clinical treatment for upper alimentary tract Candida infection (Blatchford 1990; de Repentigny and Ratelle 1996). In this study, we evaluated the effect of the antifungal agent ITCZ on C. albicans infection, proliferative changes, inflammatory lesions, and COX-2 expression of the forestomach mucosa in AL-induced diabetic rats.

Materials and Methods

Animals and Diets

Female WBN/Kob rats were obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan). They were reared in a barrier-sustained animal room maintained at a temperature of 24°C ± 2°C and a relative humidity of 60% ± 20%, with twelve-hour light/dark cycles and ventilated at least twelve times per hour with sterilized fresh air. All rats were housed and reared in aluminum mesh cages. To protect against infection, the cages were changed once or more each week. Rats were given a pellet diet (CRF-1; Oriental Yeast, Tokyo, Japan) and chlorinated water ad libitum.

All procedures for animal handling and experimental treatments were in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Committee for Animal Experiments of Setsunan University and the Japanese Association for Laboratory Animal Science. The level of care provided to the animals met the basic requirements outlined in the National Institutes of Health guidelines (ILAR 2000; NIH 1996).

Glucosuria and Glycemia Monitoring

Fresh urine samples were collected in metabolism cages. Urinary glucose levels were measured semiquantitatively, using a urine test paper (Wako Pure Chemical Industries, Osaka, Japan) every day from day 1 to day 3 after AL dosing, once every week for one month after the first week, and once every month thereafter from the fresh urine obtained from AL-induced diabetic rats. Blood glucose levels were also measured semiquantitatively by the glucose oxidase method (Glutest E; Sanwa Kagaku, Aichi, Japan) once every month from the fourth week after dosing, using blood samples from the tail vein. Samples of blood from the tail vein and fresh urine were collected from 1:00 to 4:00 p.m.

Experimental Design

The experimental design is shown in Figure 1. A total of sixty female WBN/Kob rats were divided into three groups at fifteen weeks of age. At this time, all rats were given a single dose of AL (Sigma-Aldrich Japan, Tokyo, Japan) via the tail vein at a dosage level of 40 mg/kg body weight. The concentrations were set up as a given dose at which a rat survives for a long period after developing diabetes symptoms, and which induces glucosuria continuously. Each group of twenty rats were divided into three groups at forty-five weeks of age, and the rats were given ITCZ (kindly provided by Janssen Pharmaceutical K. K., Tokyo, Japan) by gavage 0 (vehicle control), 5, and 10 mg/kg/day in polyethylene glycol for four weeks. A dose of ITCZ was used to suppress Candida infection in a clinical and experimental study (Blatchford 1990; de Repentigny and Ratelle 1996; Ishibashi et al. 2007). Rats were sacrificed at the sixty-fifth week of age for histopathologic examination.

Histopathological Analysis

Moribund animals (a total of twenty rats) from each group were sacrificed and necropsied along with dead animals during the examination period, and some of their organs were unavailable for histopathological examination because of cannibalism or autolysis. The remaining forty rats were killed by exsanguination from the abdominal aorta under deep anesthesia at the end of each scheduled period, or when they began to exhibit a moribund condition. The entire alimentary tract was immediately removed following necropsy. The organs of the forty rats were immersed in 10% phosphate-buffered formalin solution immediately after necropsy.

Fixed organs were trimmed, dehydrated by automated processor, and embedded in paraffin. Sections (4 μm thick) of tissue specimens were stained with hematoxylin and eosin and periodic acid–Schiff reaction (PAS) for histopathological examination. Some sections were stained with Congo red for confirmation of eosinophils (David et al. 2009). The severity of the proliferative lesions in the forestomach squamous epithelium was judged from the thickness of the mucosal epithelium, described in the previous report as follows: −, equivalent with control; +, slight change; ++, moderate change; +++, severe change (Kodama et al. 2006).

Immunohistochemical Analysis

Sections containing carcinomatous tissue were examined for the presence of COX-2. In addition, immunohistochemical confirmation of C. albicans was conducted in representative forestomach sections. The sections were deparaffinized in xylene and rehydrated through graded ethanol at room temperature. Rehydrated sections were digested by pepsin for twenty min at 37°C to retrieve antigen. Solutions and washes were prepared between various steps using 0.05 M Tris-buffered saline (TBS, pH 7.6) with 0.01% Tween 20. Nonspecific endogenous peroxidase activity was blocked by exposure to 0.03% hydrogen peroxide in 100% methanol for five minutes, and masking was conducted with 1% bovine serum albumin or 5% normal goat serum in phosphate-buffered saline for five minutes at room temperature. Incubation was carried out overnight at 4°C with primary antibodies, such as anti-myeloperoxidase (diluted 1:600, A0398; DAKO, Japan), anti–COX-2 (diluted 1:200, C22420; BD Pharmingen, Franklin Lakes, NJ, USA), and anti C. albicans (diluted 1:400, MAB806; Chemicon, Temecula, CA, USA) mouse monoclonal antibodies. The slides were subsequently rinsed with TBS plus Tween 20, treated for thirty minutes at room temperature with Histofine simple stain rat MAX PO (R) and (M) (Nichirei, Tokyo, Japan), rinsed with TBS plus Tween 20, incubated in diaminobenzidine solution containing 0.01% hydrogen peroxide for the peroxidase coloring reaction, and counter-stained with Mayer’s hematoxylin. Staining was negatively controlled by substituting rabbit serum or mouse isotype immunoglobulin, diluted to the same concentration, for the primary antibody.

The severity of the C. albicans infection in the forestomach was judged from immunohistochemical staining and the PAS reaction as follows: −, no infection; +, slight infection; + +, moderate infection; and +++, severe infection. Expression of COX-2 in the forestomach was judged from the immunohistochemistry as follows: −, COX-2 was positive in lamina muscularis mucosa; +, COX-2 was moderately positive in the cytoplasm of the spindle mesenchymal cells and mononuclear inflammatory cells in the lamina propria; ++, COX-2 was moderately positive in lamina propria and superficial mucosal cells.

Quantitation of Cell Proliferation

Immunohistochemical staining for proliferating cell nuclear antigen (PCNA, diluted 1:200, PC10; DAKO Co., Kyoto, Japan) was performed. This immunohistochemistry was done using a stain system kit DAKO LSAB 2 kit/HRP (DAKO Japan). Slides were subsequently reviewed in a blinded fashion by the pathologist. The proliferation index was estimated as the percentage of PCNA-labeled nuclei/1000 squamous cells near the limiting ridge. Staining was negatively controlled by substituting mouse isotype immunoglobulin, diluted to the same concentration, for the primary antibody.

Statistical Analysis

The Mann-Whitney U test was used to compare the frequency data.

Results

Glucosuria, Glycemia, and General Condition Monitoring

Severe hyperglycemia (>300 mg/dL) and glucosuria (>500 mg/dL) in all rats continued for approximately fifty weeks from the day after injection of AL to the last monitoring day before the scheduled necropsy or death of the rat. Blood glucose levels in individual surviving animals and the mean blood glucose data of each group are shown in Tables 1 and 2.

The mean body weights of each group decreased within several days after the AL injection but gradually increased thereafter, and the final mean body weight of each group is shown in Table 1.

Three moribund rats and one found dead in the 0 mg/kg group, three moribund rats and three found dead in the 5 mg/kg group, and three moribund rats and two found dead in the 10 mg/kg group were necropsied or sacrificed, respectively, between one to forty-five weeks of age. Each moribund rat and the rat found dead in the 0 mg/kg group, one moribund rat of the 5 mg/kg group and two found dead in the 10 mg/kg group were necropsied or sacrificed, respectively, between forty-five to sixty-two weeks of age. The cause of the moribund condition or death was attributed to malignant lymphoma, urinary tract infection, and ketoacidosis resulting from a severe diabetic condition.

Histopathology

The individual animal data about histopathological examination are shown in Table 2. The degree of hyperplasia of the mucosal squamous epithelium varied from slight to severe in the forestomach and was observed in fourteen rats of the 0 mg/kg group (100%), seven from the 5 mg/kg group (53.8%), and eight from the 10 mg/kg group (61.5%). Squamous cell carcinoma was detected in three rats in the 0 mg/kg group and one rat from the 10 mg/kg group (Table 3). The incidence and severity of inflammation and proliferative change were apparently reduced in both ITCZ-treated groups, although the dose response effects were not obvious (Table 3, Figure 2A). The stratified epithelium under hyperplasia increased its height and cell layer with the thickening of the cornified layer. The connective tissue stalk was elongated according to the increased thickness of the epithelial layer, but papillary protrusion of the mucosal surface was inconspicuous (Figure 2B). All SCC were diagnosed as well-differentiated type (Table 3, Figure 2C), and tumor cells focally invaded the submucosa. Accumulation of granulated leukocytes in the mucosa and infiltration of mononuclear cells in the lamina propria were invariably accompanied by proliferative changes and erosion or ulceration of the mucosal epithelium in the forestomach (Table 3). Granulated leukocytes were confirmed as neutrophils by negative staining for Congo Red and immunopositive reaction for myeloperoxidase. Inflammatory cells mainly consisted of plasma cells and lymphocytes infiltrated into the submucosa just under the proliferative epithelial lesions (Table 3). Furthermore, filamentous fungi showing dimorphism such as yeast and the mycelial form (Figure 3A) and/or rod-shaped bacterial colonies were usually observed in the suppurative inflammatory lesions of the hyperplastic mucosa. The fungus infection rate was closely related to the severity of inflammatory lesions and mucosal proliferation. All fungi such as yeast and the mycelial form were positive for C. albicans antibody (Figure 3B). The incidence and severity of C. albicans infection were significantly reduced in both ITCZ-treated groups (Table 3).

In organs other than the forestomach, the proliferative changes of the mucosal squamous epithelium accompanied by inflammatory changes were often observed in the central part of the tongue and posterior parts of the esophagus.

Immunohistochemical Analysis for COX-2 Expression and PCNA

The COX-2 positivity score was significantly higher in the 0 mg/kg group than in the 5 and 10 mg/kg ITCZ-treated groups (Table 3). Cyclooxygenase-2 was slightly to strongly positive in the cytoplasm of the spindle-shaped mesenchymal cells and mononuclear inflammatory cells in the lamina propria near suppurative inflammatory lesions of the hyperplastic mucosa, with Candida infection in all members of the 0 mg/kg group (Figures 4 and 5A). In addition, epithelial cells adjacent to a purulent inflammatory area undergoing erosive or ulcerative changes were also weakly to moderately positive for COX-2 expression (Figures 4, 5B, 5D and 5F). Meanwhile, in 46.2% of the 5 mg/kg and 38.6% of the 10 mg/kg ITCZ-treated groups, COX-2 was negative according to the method for quantitative scoring (Figures 5C and 5E).

The PCNA-positive indices of the forestomach are shown in Figure 6. The PCNA-positive cells of the forestomach were increased with the severity of hyperplasia in each group (Figure 7). The mean PCNA-positive index of the 5 and 10 mg/kg ITCZ-treated groups (19.5% and 18.5%, respectively) was significantly lower than that of the AL group (31.5%). The PCNA-labeling index of the 5 mg/kg ITCZ-treated group and 10 mg/kg ITCZ-treated group was comparable.

Discussion

The present study revealed that ITCZ, an antifungal agent, reduced the severity of mucosal hyperplasia, inflammatory changes, and fungal infection of the forestomach. These data strongly suggest that C. albicans infection is an important factor in this inflammation-related carcinogenic mechanism. However, inflammatory lesions, proliferative changes, and Candida infection were not completely abrogated by ITCZ at these dosage levels. Also, in our experimental design it was not possible to prove that all animals were infected with Candida before ITCZ-treatment, and there was no evidence to suggest whether re-infection could have occurred between after ITCZ-treatment and the sacrifice of the animals.

A recent epidemiological study suggested a significant correlation between oral SCC and the degree of oral yeast carriage in 223 human patients (McCullough et al. 2002). Candida infection of human patients may also lead to malignant change from primary mucosal hyperplasia (Barrett, Kingsmill, and Speight 1998; Renstrup 1970; Sitheeque and Samaranayake 2003). C. albicans also has the potential to lead the progression to carcinoma in vitro (Williams et al. 2001). However, the role of potential virulence factors of the infecting C. albicans as causative agents of malignant change remains unclear. Certain C. albicans biotypes are capable of catalyzing carcinogenic nitrosamine N-nitrosobenzylmethylamine from its precursors (Krogh, Hald, and Holmstrup 1987). N-nitrosobenzylmethylamine has been demonstrated to be capable of inducing tumors in the tongue, esophagus, and forestomach of rats (Lijinsky et al. 1982). Thus, nitrosamines produced by C. albicans may play an important role in this carcinogenesis. Another researcher has suggested that infection by C. albicans is more important for carcinogenesis, because it has a promoter function in relation to the chemically induced oral mucosal tumor (O’Grady and Reade 1992). The consistent colonization of the lingual mucosa of the rat by artificial inoculation of candidal organisms could lead to chronic hyperplastic candidosis, but further progression to SCC has never been observed (Russell and Jones 1975). In our experimental system, squamous cell hyperplasia served to develop SCC in the forestomach of three rats of the 0 mg/kg group and was suppressed by the treatment with ITCZ. In addition, the PCNA-positive index for cell proliferation, ITCZ treatment significantly suppressed cell proliferation in the forestomach. Although there was no apparent dose response in ITCZ-treated groups in this study, our present data suggest that C. albicans infection is related to squamous cell proliferation and carcinogenesis.

The clear mechanism of the exaggerated infectious sensitivity to Candida in the forestomach is unknown in AL-treated diabetic rats. Uncontrolled diabetes mellitus is well known to predispose individuals to oral candidiasis. In experimental animals, a single application of C. albicans on the tongue of streptozotocin-treated diabetic rats enables the study of long-term mycotic lesions of the lingual mucosa (Dourov and Coremans-Pelseneer 1987). The polymorphonuclear leukocytes of diabetic patients have a lower chemotactic index and decreased phagocytosis, and it is well known that diabetic patients are susceptible to inflammation of the skin and intestinal organs through readily infectious conditions (Sentochnik and Eliopoulos 2005). Thus, diabetic status may be most likely to induce visceral mycosis in diabetic rats.

Epidemiological studies have indicated that type 1 and type 2 diabetes are risk factors for oral premalignant lesions such as leukoplakia and oral SCC (Ujpal et al. 2004). In experimental animals, type 1 diabetes possibly increases the risk of oral SCC according to chemical carcinogenesis studies (Vairaktaris et al. 2007). Maintenance of a severe diabetic condition also likely enhances the proliferative lesions and may have caused the malignant transformation in our study. C. albicans may lead to the progression to carcinoma in vitro (Williams et al. 2001) and has been shown to have a promoter function in in vivo carcinogenesis studies (O’Grady and Reade 1992). Thus, the promoter function of C. albicans might be enhanced by the diabetic condition in the present study.

Furthermore, the surface expression of phospholipomannan, which is a phylogenic trait of C. albicans, directly initiates tumor necrosis factor (TNF) production through an interaction with toll-like receptor 2 (TLR2) (Jouault et al. 2003; Netea et al. 2002). C. albicans also has the potential to selectively lead activation of COX-2 via activation of nuclear factor-κB (NF-κB) and mitogen-associated protein kinase (MAPKs). Toll-like receptor 2 plays a critical role in these signal transduction pathway induced by C. albicans (Deva et al. 2003). In addition, TNF may also induce the infiltration of COX-2–expressing macrophages and neutrophils in rat colon carcinogenesis (Popivanova et al. 2008). In the present study, COX-2 was strongly expressed in the mesenchymal cells and inflammatory cells and was weakly positive in the hyperplastic epithelium near the acute inflammatory lesions. Itraconazole-treatment of AL-induced diabetic rats significantly decreased both COX-2 expression and C. albicans infection. Therefore, C. albicans might enhance COX-2 expression through the TLR pathway in our model.

In conclusion, ITCZ reduces the forestomach inflammatory lesions, proliferative changes, and C. albicans infection in AL-induced diabetic rats. It became clear that the inflammation-related proliferative lesions were deeply involved in the C. albicans infection in this model.