Modulatory effects of Azadirachta indica on benzo(a)pyrene-induced forestomach tumorigenesis in mice

Abstract

AIM: To evaluate the chemopreventive effects of aqueous Azadirachta indica (A indica) leaf extract (AAILE) against benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis in Balb/c mice.

METHODS: Female Balb/c mice were divided into four groups of 10-12 animals each. For induction of forestomach tumors, starting from d 14 of the experi-ment, mice of B(a)P and B(a)P + A indica groups were given intra-gastric instillations of B(a)P (40 mg/kg), twice a week for four weeks. Mice of A indica and B(a)P + A indica groups were orally administered with AAILE (100 mg/kg), two weeks prior to B(a)P instillations till the end of the experiment. After 22 wk of the first B(a)P instillation, mice were sacrificed and the forestomachs were analyzed for development of tumors, scanning electron microscopy (SEM) and histopathology.

RESULTS: Tumor incidence was observed to be 100% in mice that received only B(a)P. However, treatment with AAILE reduced the tumor incidence by 58.4% as observed in mice of B(a)P + A indica group when compared to that of B(a)P group. Similarly, the tumor burden and multiplicity were seen to decrease by 87.3% and 69.6% respectively in mice of B(a)P + A indica group when compared to those of B(a)P group. Scanning electron microscopy analysis showed that AAILE treatment itself did not cause any abnormalities on the surface architecture of forestomach epithelium. In tumorous forestomach, surface disruption was observed. Over the forestomach tumors of B(a)P group of mice certain rounded structures were seen in addition to closely placed tongue-shaped squamous cells. Interestingly, these rounded structures were not observed in B(a)P + A indica group of mice. Histopathalogically, the tumors were identical and diagnosed to be papillomas. Mice from control and A indica groups of mice did not develop any forestomach tumors and showed normal histo-architecture.

CONCLUSION: The present data suggest that A indica exerts chemopreventive effects against B(a)P-induced forestomach tumors in murine model. Because of lack of toxicity and ubiquitous bioavailability, A indica may play a promising role in future drug discovery and development as far as chemoprevention of cancer is concerned.

Key Words: Azadirachta indica; Benzo(a)pyrene; Chemoprevention; Histopathology; Scanning electron microscopy

INTRODUCTION

Recent estimates show that gastric cancer is the fourth most frequent type of cancer and the second most prevalent cause of cancer deaths worldwide[1]. Human beings are exposed to a multitude of carcinogens in their environment, and majority of the cancers are considered to be chemically-induced[2]. Polycyclic aromatic hydrocarbons (PAHs) are environmental carcinogens present in the atmosphere from combustion sources such as diesel exhaust, cigarette smoke, residential heating processes, refuse burning, industrial coke production, volcanic eruption, and oil contamination by effluents and oil spills[3]. They can also be generated by the pyrolysis of amino acids, fatty acids and carbohydrates during cooking process[4]. After inhalation, ingestion is the second most important exposure route for PAHs to enter the human body[5]. Stomach is the organ where engulfed/ingested materials come into prolonged contact with gastro-intestinal (GI) mucosa[6]. So it is quite reasonable to assume that stomach has high risk of developing cancer from carcinogens entering the GI tract. Benzo(a)pyrene [B(a)P], a potent pro-carcinogen employed in initiating stomach cancer, is the prototypical and best characterized member of PAHs family of chemical carcinogens[7]. It has been shown to induce tumors of skin, lung, mammary glands and forestomach tissues of various experimental animals[8]. Since B(a)P is an omnipresent environmental pollutant and is believed to be a risk factor for human chemical carcinogenesis, it is important to identify the naturally occurring/synthetic agents that could modulate B(a)P-induced tumorigenesis[9].

In experimental studies as well as pre-clinical systems, several drugs have been discovered to combat cancer, but are not much in use because of their undesirable toxic side effects[10]. Moreover, most of the synthetic chemotherapeutic drugs available today are cytotoxic, immuno-supressants and may cause a variety of side effects in the normal organs of the body[11]. Prevention is an effective strategy to control the incidence of cancer. Recent progress in molecular biology and pharmacology enhances the likelihood that cancer prevention will increasingly rely on chemoprevention[12]. The later is a means of cancer control, in which the occurrence of disease as a consequence of exposure to carcinogen can be slowed, blocked or reversed by the administration of one or more naturally occurring or synthetic compounds[13]. Chemoprevention also deals with the chemotherapy of pre-cancerous lesions, which are called pre-invasive neoplasia, dysplasia or intra-epithelial neoplasia depending on the organ system[14]. It has been observed that the synthetic chemopreventive agents produce toxic side effects, which have limited their extensive use[15]. Plants, vegetables as well as medicinal herbs used in the folk and traditional system of medicine have been accepted currently as one of the main source of cancer chemopreventive drug discovery and development[16]. Ayurveda, the Indian traditional system of medicine, which dates back many centuries, uses many herbal extracts to cure a variety of diseases including cancer[17]. There is a growing interest in evaluation of chemopreventive efficacy of plants used in Ayurveda and one of such plants is A indica commonly called Neem.

A indica leaf extract has been reported to be non-toxic, non-mutagenic and possesses immuno-stimulant, hepato-protective, anti-oxidant, anti-genotoxic and anti-cancer activities[18]. The anticancer activities of A indica leaf extract have attracted much of the attention of research community in last few years. Subapriya et al[19] have observed chemopreventive activity of ethanolic A indica leaf extract against N-methyl-N-nitro-N-nitroso-guanidine-induced gastric carcinogenesis in rats. Dasgupta et al[9] have reported the chemopreventive effects of ethanolic A indica leaf extract against DMBA-induced TPA promoted skin and B(a)P-induced forestomach papillomagenesis. AAILE has been shown to ameliorate 4-nitroquinoline 1-oxide-induced oral tumorigenesis in male Wister rats[20] and 7-12 dimethylbenz(a)anthracine (DMBA)-induced buccal pouch carcinogenesis in Syrian hamsters[21]. Inhibitory effect of AAILE against DMBA-induced skin carcinogenesis in mice has recently been reported from our research laboratory[22].

Keeping in view the above mentioned reports, the present study was designed to evaluate the modulatory effects of AAILE, if any, on B(a)P-induced forestomach tumorigenesis in Balb/c mice. Tumor incidence, burden and multiplicity were taken as an index for chemopreventive tumor response. In addition to histopathology, scanning electron microscopy (SEM) of forestomach tumors was also done in an attempt to examine the surface morphology of tumorous forestomach. To the best of our knowledge the SEM of tumorous forestomach of Balb/c mice has not been reported yet.

MATERIALS AND METHODS

Materials

B(a)P was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Olive oil used as a vehicle for B(a)P, and all other chemicals utilized were obtained from reputed local firms (India) and were of the highest purity grade. Capsules containing 100 mg of spray dried AAILE were obtained from a recognized herbal company (Dabur India Ltd). The contents of the capsules were dissolved in double-distilled water immediately before its oral administration to the mice in order to attain the required doses as described by Balasenthil et al[21]. The solution obtained after dissolving AAILE was covered with dark paper in order to protect the same from direct exposure to light.

Animal experiment

Random-bred virgin female Balb/c mice (6-8 wk old) procured from Central Animal House, Panjab University, Chandigarh were housed in polypropylene cages bedded with sterilized rice husk. All the mice were allowed to have free access to clean drinking water and standard animal pellet diet formulated on scientific backgrounds (Ashirwad Industries, Tirpari, Kharar, Distt. Ropar, Punjab) throughout the experiment. All the experimental protocols were approved by the Institutional Ethics Committee (Panjab University, Chandigarh, India) and conducted according to the Indian National Science Academy Guidelines for the use and care of experimental animals. Before the commencement of various treatments, mice were kept for acclimatization to experimental conditions for one week and the final day of their acclimatization period was considered as day 0 of the experiment. Mice were randomly divided into four groups (10-12 mice per group) namely control group, B(a)P group, A indica group and B(a)P + A indica group. Mice of B(a)P and B(a)P + A indica groups were given B(a)P for induction of forestomach tumor as described by Wattenberg et al[23], with minor modification. Briefly, each mouse received intra-gastric instillations of 40 mg B(a)P/kg in 0.2 mL olive oil, twice a week for four weeks from d 14 to d 42 of the experiment (a total of eight B(a)P instillations per mouse). Also, the animals of control and A indica groups received 0.2 mL of only olive oil in the same schedule. Mice of A indica and B(a)P + A indica groups were orally administered (using blunt tipped canula and a syringe) with AAILE at a dose of 100 mg/ kg thrice a week on alternate days throughout the experiment as described by Balasenthil et al[21]. Mice from all the groups were observed for alterations in body weight and diet and water consumption. After 22 wk of the first B(a)P instillation, mice from all the groups were sacrificed by cervical dislocation under light ether anesthesia. The stomachs were carefully removed and processed for different parameters.

Chemopreventive tumor response

The stomachs were kept in expanded state by injecting them with 40 g/L buffered formaldehyde solution. Twenty-four hours later, the stomachs were split longitudinally and the forestomachs were carefully observed under the dissecting microscope for the presence of tumors. Forestomach tumors (1 mm in diameter) were counted/recorded for each mouse. The chemopreventive tumor response was assessed on the basis of tumor incidence, burden and multiplicity that were calculated as follows: tumor incidence: percentage number of animals having tumors; tumor burden: total number of tumors counted/total number of mice; tumor multiplicity: total number of tumors counted /number of tumor bearing mice.

Scanning electron microscopy

For SEM studies, the forestomachs from all the groups of mice were fixed in 4% gluteraldehyde prepared in 200 mmol/L phosphate buffer (pH 7.2) for two hours followed by two washings in phosphate buffer. After this, the tissues were dehydrated in ascending grades of acetone, i.e. 30%, 50%, 70%, 90% and absolute acetone (for 30 min per grade). Tissues were then immersed in a mixture of acetone and amyl acetate (1:1, V/V) followed by amyl acetate alone (30 min each). Following this, the tissues were subjected to critical point drying (CPD) to get rid of remaining water. After CPD, the tissues were pasted on metallic stubs and further trimmed as per requirement. The stubs were placed inside the sputter for gold coating (15 min). These were then viewed under scanning electron microscope of Jeol 2601, installed at Regional Sophisticated Instrumentation Center, Panjab University, Chandigarh.

Histopathology

Formalin fixed tissues were processed for HE staining using the conventional laboratory procedure. Briefly, the tissues were dehydrated through ascending grades of alcohol, cleared in benzene and embedded in low melting point paraffin. Sections of 5 micrometer thick were cut, placed serially on clean glass slides and then deparaffinised through descending grades of alcohol. Three contiguous sections were made from each forestomach tissue and stained with HE for evaluation under light microscope. The slides were coded, so that the particular sample identity was not known while performing assessment.

Statistical analysis

Statistical significance of differences for tumor burden and multiplicity was analyzed by Student’s t-test. Comparison was carried out between B(a)P and B(a)P + A indica groups.

RESULTS

In the present study, it was noticed that the diet and water consumption by the mice in all the groups studied was approximately same during the entire experimental span (data not shown). Mice from all the groups showed normal body growth and gained weights when compared to their respective initial body weights (Table 1). It was observed that the mice from B(a)P and B(a)P + A indica groups developed forestomach tumors, whereas none of the mice from control & A indica groups developed forestomach tumors (Figures 1A-C). Tumor incidence was observed to be 100% in B(a)P group of mice. However, the tumor incidence was found to be 41.6% in the mice of B(a)P + A indica group, i.e. the mice that received AAILE treatments along with B(a)P instillations. This experiment clearly demonstrated that AAILE potentially reduced the tumor incidence by 58.4 %. The tumor burden was observed to decrease significantly (approximately 7.88 fold) upon AAILE treatment when the mice of B(a)P + A indica group were compared to that of B(a)P group. Similarly, the tumor multiplicity was observed to decrease (approximately 2.59 folds) upon AAILE treatment as seen in mice of B(a)P + A indica group when compared to that of B(a)P group (Table 1). In another similar investigation, Dasgupta et al[9] reported that the administration of ethanolic neem leaf could reduce the tumor incidence by 39% during B(a)P-induced forestomach tumorigenesis in Swiss albino mice.

Table 1 Modulatory effects of AAILE on B(a)P-induced forestomach tumorigenesis in Balb/c mice.

Groups	Body mass (g)		Tumor incidence	Tumor burden	Tumor multiplicity	No. of Tumors (Min-Max)
	Initial	Final
Control	22.1 ± 1.51	27.6 ± 1.64	0 % (0/10)	-	-	-
B(a)P	22.6 ± 1.68	26.5 ± 2.24	100 % (12/12)	7.25 ± 2.80	7.25 ± 2.80	5-14
A indica	22.4 ± 1.78	27.8 ± 1.94	0 % (0/10)	-	-	-
B(a)P + A indica	22.3 ± 1.64	27.1 ± 1.64	41.6% (5/12)	0.92 ± 1.24 b	2.20 ± 0.84 b	0-3

^bP < 0.001 vs B(a)P.

Figure 1 Gross anatomy of forestomach showing normal surface (A), tumorous surface in mice that received only B(a)P (B), and lesser number of tumors in mice that received AAILE along with B(a)P (C).

Forestomach of control group as well as A indica group of mice showed normal surface morphology with distinct grooves and ridges (Figures 3A and 3C). Mucous sheath at certain places and tongue-shaped keratinized squamous epithelial cells arranged in layers like tiles were observed[24] (Figures 3B and 3D). These observations suggested that AAILE treatment itself could not cause any abnormalities over the epithelium. In forestomach of tumor bearing mice, surface disruption was observed at the sites of tumor development (Figures 3E and 3G). In forestomach tumors of the mice that received only B(a)P, within the closely placed squamous epithelial cells, in addition to tongue-shaped cells certain rounded to irregularly shaped structures were seen (Figure 3F). The observed rounded structures could be transformations of tumorous cells. However in tumors of the mice that received AAILE treatments along with B(a)P instillations, such structures were not seen (Figure 3H). Their absence in the forestomach tumors of B(a)P + A indica group of mice could be attributed to the different cytotoxic effects of the phytochemical components present in AAILE.

Figure 3 SEM of forestomach of mice. A: Control mice showing grooves and ridges X 200; B: Control mice showing tongue-shaped squamous epithelium X 600; C: Mice treated with only AAILE showing grooves and ridges X 200; D: Mice treated with only AAILE showing squamous epithelial cells X 600; E: Mice received only B(a)P showing ruptured surface X 200; F: Mice received only B(a)P showing certain rounded structures in addition to tongue-shaped squamous epithelial cells X 600; G: Mice received AAILE along with B(a)P instillations showing surface rupturing X 200; H: Mice received AAILE along with B(a)P instillations showing tongue shaped squamous epithelial cells X 600.

Histopathological analysis showed that forestomach of mice from control and A indica groups depicted normal histoarchitecture with distinct muscular layers, sumucosa and squamous mucosa (Figure 2A). The tumors developed in B(a)P and B(a)P + A indica groups were observed to be similar and diagnosed as papillomas with endophytic growth in the form of papillary projections having fibro-vascular core lined by stratified squamous epithelium (Figure 2B). Though the tumors developed in B(a)P as well as in B(a)P + A indica were similar histopathologically, our observations clearly suggested the chemopreventive effects of AAILE against B(a)P induced forestomach tumorigenesis.

Figure 2 Histo-micrograph of forestomach. A: Control mice showing squamous mucosa, sub-mucosa and muscular layers X 20; B: Papillary projections, disrupted submucosa and muscular layers X 20.

DISCUSSION

Stomach cancer is the second most prevalent malignancy in the world and chemoprevention has evolved as an effective strategy to combat this dreadful disease[25]. Fruits, vegetables, vitamins, common beverages and several medicinal plants/herbs with diversified pharmacological properties have been shown to be a rich source of cancer chemopreventive agents[26]. A indica, one of the most versatile traditional medicinal plants, has been known to possess a wide spectrum of medicinal properties and is used in Ayurveda to cure several ailments including cancer. In the present piece of research work, an attempt was made to evaluate the modulatory effects of AAILE against B(a)P-induced forestomach tumorigenesis.

B(a)P, an extremely potent pro-carcinogen, is metabolized by biotransformation enzymes to a variety of metabolites that are responsible for initiating tumorigenesis[27]. Biotransformation enzymes have broadly been divided into two categories namely phase-I and phase-II biotransformation enzymes. The former constitutes cytochrome P-450 based mono-oxygenase system which is responsible for initiating conversion of pro-carcinogens to several of their metabolites including ultimate carcinogens. Glutathione-S-transferase (GST) is a major phase II detoxifying enzyme that primarily functions in catalyzing the active carcinogenic metabolites to endogenous ligand-reduced glutathione (GSH) favoring their elimination from the body of the organisms[28]. The balance between the phase I carcinogen-activating enzymes and the phase II detoxifying enzymes is critical to determining an individual's risk for cancer[29]. There is substantial evidence that chemopreventive agents including medicinal plants exert their anti-carcinogenic effects by modulation of phase I and phase II xenobiotic biotransformation enzymes[30]. A significant reduction in the activity of cytochrome P450 and cytochrome b₅ (phase I enzymes) in hepatic tissue of mice upon AAILE treatment has recently been reported from our laboratory[31]. Ethanolic A indica leaf extract can exert down-regulatory effect on the activity of cytochrome P450 and cytochrome b₅ in some organs like liver, kidney and forestomach of mice[9]. Maintaining rats on diets containing neem flowers has been reported to decrease the activity of cytochrome P-450 in hepatic tissue that have been correlated with inhibition of aflatoxin B-1- and DMBA-induced liver and mammary gland carcinogenesis respectively[32]. A indica leaf extract can also enhance the activity of GST as well as GSH contents (phase II components) in different tissues of a number of experimental animal models[9,21,31]. These observations suggest that the AAILE treatment decreases the metabolic activation with simultaneous increase in the detoxification of B(a)P. Therefore the chemopreventive effects of AAILE observed in the present study could be ascribed, at least in part, to its ability to modulate certain components of carcinogen biotransformation system.

Reactive oxygen species (ROS) are widely generated in biological systems either by normal metabolic pathways or as a consequence of exposure to chemical carcinogens leading to oxidative stress that may further result in membrane dysfunction, protein inactivation, DNA damage ultimately contributing to the process of carcinogenesis[33]. For protection against the deleterious effects of these ROS, organisms have developed a sophisticated antioxidant defense system which has enzymatic as well as non-enzymatic components. The antioxidant defense system includes enzymes like glutathione peroxidase (GPx), catalase, glutathione reductase (GR), GST and superoxide dismutase (SOD), whereas non-enzymatic components include non-protein thiol GSH, uric acid, some trace metals and certain vitamins such as ascorbic acid and alpha-tocopherol, etc[34]. A indica leaf extract has been shown to enhance the activities of all the above mentioned antioxidant defense enzymes and GSH contents in various tissues of mice and rats[9,19]. Increased antioxidant defense upon AAILE treatment could lower the ROS-mediated damage at the initiation as well as progression/promotion phase of the process of tumorigenesis.

Nimbin is the first phyto-chemical compound that has been isolated from A indica seed oil[35] and now appro-ximately 140 different phytochemicals have been identified and isolated from various parts of this plant[36]. The anti-carcinogenic effects of this tree are also attributed to its antioxidant phytochemicals such as flavonoids[37]. Quercetin and kaemferol are the flavonoids present in neem leaf that have been documented to retard carcinogenesis at initiation, as well as promotion phases of carcinogenesis by virtue of their radical scavenging properties[38,39]. Although we did not carry out quantitation and characterization of individual components from AAILE in this study, the contribution of antioxidant phytochemicals of AAILE in reducing the extent of tumorigenesis can not be ruled out.

Yadav et al[40] showed that mitotic activity can be inhibited by A indica leaf extract. Baral et al[41] reported that the growth of Ehrlich carcinoma and B16 melanoma cells is reduced significantly after administration of A indica leaf extract. Limonin 17β-D-glucopyranoside, a limonoid found in neem, can inhibit DMBA-induced oral carcinogenesis[42]. Azadirone 1, a limonoid constituent of A indica possesses cytotoxic activity against breast, melanoma and prostate cancer cell lines[43]. Cytotoxicity of azadirachtin A has been reported in human glioblastoma cell lines by Akudugu et al[44]. Nimbolide and 28-deoxonimbolide have been identified as cytotoxic constituents of A indica leaves[45]. Intra-peritoneal administration of some isolated polysaccharides from A indica is active against sarcoma -180 ascites tumor cells in mice[46]. In another investigation Petit et al[47] reported that some active components of A indica show cytotoxic effects against murine P_-388 lymphocytic leukemia. In the present study, since AAILE was given throughout the experiment, the cytotoxic effects of its active constituents against pre-cancerous / tumorous cells could not be denied.

In in-vivo experiments, it has been observed that A indica leaf extract treatment does not alter the activity of cellular lactate dehydrogenase (an indicator of cell damage) in the hepatic tissues, suggesting that the leaf extract itself does not exert any adverse effects[9]. Also, it is generally believed that the crude therapeutic preparations are more effective and less toxic than the isolated active components[48] or their respective synthetic counterparts because they contain the total family of medicinal compounds (known and unknown) just as they are found in their natural source and hence offer less risk of side effects. In crude preparations, perhaps, the other components present in addition to the active components may affect the effects of the active components. These known and unknown components might act as synergists for the therapeutic effects and antagonists for the side effects of the active components as well as the other toxic components in the crude preparation[49].

The present data together with reports available in literature, suggest that A indica has immense che-mopreventive potentials as far as B(a)P-induced fores-tomach tumorigenesis is concerned. A indica-based therapeutic preparations have already been used in the Indian traditional system of medicine for several ailments. Previous experimental investigations along with the present study support the rational use of these preparations for chemoprevention of certain types of cancers. Because of lack of toxicity and ubiquitous distribution of this plant in nature, A indica seems to be a valuable plant source for use in modern chemopreventive drug discovery and development.