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ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 1  |  Page : 46-53

Chemopreventive potential of chrysin in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis


Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India

Date of Submission25-Nov-2011
Date of Acceptance24-Dec-2011
Date of Web Publication6-Feb-2013

Correspondence Address:
Shanmugam Manoharan
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.106993

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   Abstract 

Introduction: Chemoprevention, an emerging, appealing, and innovative approach in experimental oncology, deals with the inhibition, prevention or suppression of carcinogenesis, using natural products or synthetic derivatives. The aim of the present study is to investigate the chemopreventive potential of chrysin during 7,12-dimethylbenza[a]anthracene (DMBA) - induced hamster buccal pouch carcinogenesis. Materials and Methods: Oral squamous cell carcinoma was developed in the buccal pouch of Syrian golden hamsters by painting them with 0.5 percent DMBA in liquid paraffin thrice a week, for 14 weeks. The status of lipid peroxidation, antioxidants, and phase I and II detoxification agents were utilized as biochemical end points, to assess the chemopreventive efficacy of chrysin in DMBA-induced hamster buccal pouch carcinogenesis. Results: In the present study, 100% tumor formation with marked abnormalities in the status of lipid peroxidation, antioxidants, and detoxification agents was noticed in hamsters treated with DMBA alone. Oral administration of chrysin at a dose of 250 mg/kg bw to DMBA-treated hamsters significantly reduced the tumor incidence and tumor size as well as reverted the status of the above-mentioned biochemical markers during DMBA-induced hamster buccal pouch carcinogenesis. Conclusion: Chrysin has the potential to delay rather than inhibit tumor formation as evidenced by the tumor formation in two of the DMBA + chrysin-treated hamsters, during DMBA-induced hamster buccal pouch carcinogenesis.

Keywords: Antioxidants, chrysin, detoxification enzymes, lipid peroxidation, oral cancer


How to cite this article:
Karthikeyan S, Srinivasan R, Wani SA, Manoharan S. Chemopreventive potential of chrysin in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis 2013;3:46-53

How to cite this URL:
Karthikeyan S, Srinivasan R, Wani SA, Manoharan S. Chemopreventive potential of chrysin in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2020 Jan 25];3:46-53. Available from: http://www.ijnpnd.com/text.asp?2013/3/1/46/106993


   Introduction Top


Oral carcinogenesis, a multistep process, occurs due to accumulation of multiple genetic alterations that modify the normal functions of proto-oncogenes and tumor suppressor genes. [1] Although oral cancer occurs anywhere in the oral cavity, the most common sites are the cheek, tongue, and floor of the mouth. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of all oral cancers. [2] Despite significant improvements in early diagnosis and treatment, the five-year survival rate for advanced cancer has not drastically improved for the past three decades. Survival rates for stage 1 cancers are, however, 90%, and early detection of oral cancer may increase the survival outcome of the patients. [3] Oral cancer, the fifth most common cancer worldwide, affects 500,000 new cases and causes 120,000 deaths every year. Around 39,400 new oral cancer cases and 7,900 deaths due to this cancer have been reported in USA by the year 2010. Oral cancer accounts for 3-4% of all cancers in Western Europe and every year more than 4,000 and 3,000 new oral cancer cases are reported in UK and Canada, respectively. [4] The annual incidence as well as death rate due to oral cancer was very high in India, Srilanka, and Pakistan, where this form of cancer accounts for 40-50% of all cancers. [5] The etiology of oral cancer is multifactorial, and the genetic, environmental, social, and behavioral effects are implicated in the pathogenesis of oral cancer. Tobacco smoke and alcohol are the major risk factors of oral cancer. Immunodeficiency, infection with human papilloma virus types 16 and 18, and chronic exposure to sunlight are also identified as possible risk factors of oral cancer development. [6]

7,12-dimethylbenz(a)anthracene (DMBA), a site-specific procarcinogen, is commonly used to develop oral carcinogenesis in golden Syrian hamsters. Oral tumors that are developed in hamster's buccal mucosa have a close resemblance to that of human oral carcinoma, both histologically and morphologically. DMBA-induced oral carcinogenesis is therefore commonly utilized to study the chemopreventive potential of natural and synthetic agents. [7]

The liver plays a pivotal role in the metabolism of carcinogenic substances. The metabolic activation of carcinogens are catalyzed by phase I detoxification agents (cytochrome P 450 and cytochrome b 5 ) and the resultant carcinogenic metabolites are excreted by phase II detoxification agents [Glutathione-S-transferase (GST) and Glutathione reductase (GR)], by conjugating them with reduced glutathione (GSH) or glucuronic acid. Cytochrome P 450 and cytochrome b 5 are the key enzymes responsible for the metabolic activation of DMBA. [8] Cytochrome P 450 enzymes metabolize lipophilic compounds to more polar products, which are then acted upon by the phase II enzymes such as GST and GR, to enhance their polarity and assist in their excretion. [9] Microsomal cytochrome b 5 is a ubiquitous 15.2 kDa hemeprotein, implicated in a number of cellular processes such as fatty acid desaturation during metabolism, steroid hormone biosynthesis, and methemoglobin reduction. [10] GSTs are a family of enzymes that catalyze the conjugation of reactive oxygen species with GSH, and thereby, facilitate the excretion of reactive metabolites. [11],[12] GR maintains the levels of GSH in the cells by catalyzing the NADPH-dependent reduction of glutathione disulfide to glutathione. [13] DT-diaphorase [(DTD) (NAD[P]H:Quinone oxidoreductase)], is an electron transfer flavoprotein that oxidizes NADH or NADPH to NAD + or NADP + with quinine as a hydrogen acceptor. DT-diaphorase protects the cell from oxidative damage by preventing oxy-radical formation. [14] Estimation of the status of phase I and phase II detoxification agents in the liver could help to assess the chemopreventive potential of the test compound. [15],[16]

The reactive oxygen species (ROS) can cause destruction of lipids, particularly polyunsaturated fatty acids (PUFA), and they mediate a chain reaction known as lipid peroxidation. ROS-mediated lipid peroxidation has been implicated in the pathogenesis of several cancers including oral cancer, by damaging DNA, lipids, and proteins. The human body, however, has a wonderful protective enzymatic [SOD (superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase)] and non-enzymatic [GSH (reduced glutathione) and Vitamin E] antioxidant defense mechanism to combat the deleterious effects of ROS. [17] Oxidative stress thus occurs in the body only when there is an imbalance in the oxidant and antioxidant status. Lipid peroxidation and an antioxidant status are used as non-specific biomarkers of several cancers including oral carcinogenesis. [18] Overproduction of ROS and impaired antioxidant defense mechanisms were reported in human and experimental oral carcinogenesis. [19]

Medicinal plants serve as an important resource for the investigation of therapeutically useful compounds. [20],[21] Chrysin [(5,7-dihydroxyflavone) [Figure 1]] is the major active constituent of Oroxylum indicum. It is also found in propolis, blue passion flower, vegetables, and fruits. Chrysin and its derivatives possess a wide spectrum of biological activities, including antioxidant, anti-viral, and anti-inflammatory properties. [22] Chrysin exhibited anti-cancer potential in a diverse range of human cancer cells (in vitro) and in animal bearing tumors (in vivo). [23] The anti-tumoral effect of chrysin has been reported in murine cell lines (LM3, mammary adenocarcinoma; B16-F0, melanoma) and human cell lines (HeLa, cervical adenocarcinoma; KB, oropharyngeal carcinoma). [24] Chrysin exhibited the protective effect against human immunodeficiency virus activation in models of latent infection. [25] Chrysin induced apoptosis in HeLa cervical cancer cells, U937, HL-60, and L1210 leukemia cells. [26] There are, however, no studies on the chemopreventive potential of chrysin in DMBA-induced hamster buccal pouch carcinogenesis. The present study is therefore designed to study the chemopreventive effect of chrysin in DMBA-induced hamster buccal pouch carcinogenesis.
Figure 1: Chemical structure of chrysin (5,7-dihydroxyflavone)

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   Materials and Methods Top


Chemicals

7,12-dimethylbenz(a)anthracene (DMBA), chrysin, and other biochemicals, such as, reduced glutathione, reduced nicotinamide adenine dinucleotide, and 1,1', 3, 3'-tetramethoxypropane, were obtained from Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, India. Thiobarbituric acid (TBA), trichloroacetic acid, 2,4-dinitrophenylhydrazine (DNPH), 5,5'-dithiobis (2-nitro benzoic acid) (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), cysteine hydrochloride, and sodium metaarsenate were purchased from Hi-media Laboratories Mumbai, India. All other reagents used were of analytical grade.

Animals

Male golden Syrian hamsters, 8-10 weeks old, weighing 80-120 g were purchased from the National Institute of Nutrition, Hyderabad, India, and were maintained in the Central Animal House, at the Rajah Muthiah Medical College and Hospital, Annamalai University. The animals were housed in polypropylene cages and provided standard pellet diet and water ad libitum. The animals were maintained under controlled conditions of temperature and humidity with a 12-hour light/ dark cycle. Institutional Animal Ethics Committee (Register number 160/1999/CPCSEA), Annamalai University, Annamalai Nagar, India, approved the experimental design (Proposal No. 813, dated: 20.04.2011). The animals were maintained as per the principles and guidelines of the Ethical Committee for Animal Care of the Annamalai University, in accordance with Indian National Law on animal care and use.

Experimental design

A total of 24 hamsters were randomized into four groups of six hamsters each. Group I hamsters served as the control and were painted with liquid paraffin thrice a week for 14 weeks on their left buccal pouches. Group II and III hamsters were painted with 0.5% DMBA in liquid paraffin thrice a week for 14 weeks on their left buccal pouches. Group II animals received no other treatment. Group III hamsters were given chrysin orally, at a dose of 250 mg/kg bw/day, starting one week before the exposure to the carcinogen and continued on days alternate to DMBA painting, until the sacrifice of the hamsters. Group IV hamsters received oral administration of chrysin (250 mg/kg bw) alone throughout the experimental period. The experiment was terminated at the end of the sixteenth week and all animals were sacrificed by cervical dislocation. For a histopathological examination, the buccal mucosa tissues were fixed in 10% formalin and routinely processed and embedded with paraffin, 2-3 μm sections were cut in a rotary microtome and stained with hematoxylin and eosin.

Induction of oral squamous cell carcinoma

Oral squamous cell carcinoma was developed in the buccal pouch of Syrian golden hamsters by painting with 0.5% DMBA in liquid paraffin thrice a week, for 14 weeks.

Biochemical estimations

Biochemical estimations were carried out in the plasma, liver, and buccal mucosa of control and experimental hamsters in each group. The protein content was determined by the method of Lowry et al. [27] Cytochrome P 450 and cytochrome b 5 in the liver and buccal mucosa was measured by the method of Omura and Sato. [28] The activity of glutathione-s-transferase, glutathione reductase, and DT-Diaphorase was determined by the methods of Habig et al, [29] Carlberg and Mannervik, [30] and Ernster, [31] respectively. The reduced glutathione level was determined by the method of Beutler and Kelley. [32] Thiobarbituric acid reactive substances (TBARS) in plasma and buccal mucosa were assayed by the method of Yagi [33] and Ohkawa et al.,[34] respectively. Superoxide dismutase, catalase, and glutathione peroxidase activities were determined by the method of Kakkar et al., [35] Sinha, [36] and Rotruck et al.,[37] respectively. Vitamin E was estimated in the plasma by the method of Palan [38] based on the classical Emmerie Engle reaction. The concentration of total vitamin E in the tissues was estimated by the method of Desai et al. [39]

Statistical analysis

The values were expressed as mean ± SD. The statistical comparisons were performed by one way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT), using SPSS version 12.0 for windows (SPSS Inc. Chicago; http:// www.spss. com). The values were considered to be statistically significant if the P value was less than 0.05.


   Results Top


[Table 1] shows the incidence of oral neoplasm in control and experimental hamsters in each group. One hundred percent tumor formation with mean tumor volume (203.58 mm 3 ) and tumor burden (508.95 mm 3 ) was observed in hamsters treated with DMBA alone [Figure 2]. Oral administration of chrysin at a dose of 250 mg/kg bw significantly prevented tumor incidence, tumor volume, and tumor burden in DMBA-treated hamster. No tumors were observed in the control hamsters and chrysin alone administered hamsters.
Figure 2: Gross appearance of exophytic tumors in the buccal pouch of hamsters treated with DMBA alone

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Table 1: Incidence of oral neoplasm in control and experimental hamsters in each group (n=6)

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Tumor volume was measured using the formula, V = (4/3)π[D 1 /2][D 2 /2][D 3 /2] where D 1 , D 2, and D 3 are the three diameters (mm 3 ) of the tumor. [40] Tumor burden was calculated by multiplying tumor volume and the number of tumors per animal

Values that do not share a common superscript in the same row differ significantly at P < 0.05 (DMRT).

The histopathological features observed in the buccal mucosa of the control and experimental hamsters in each group are shown in [Table 2] and [Figure 3]. Severe hyperkeratosis, hyperplasia, dysplasia, and well-differentiated squamous cell carcinoma were noticed in the buccal mucosa of hamsters treated with DMBA alone. Although well-differentiated squamous cell carcinoma was seen in two of the six DMBA + chrysin treated hamsters, the incidence of tumor formation was less, and the size of the tumor, tumor volume, and burden was also significantly decreased in the buccal pouches. The severity of histopathological changes was also significantly reduced in the DMBA + chrysin-treated hamsters. Hamsters administered with chrysin alone showed well-defined and intact epithelial layers similar to that of the control hamsters.
Figure 3: Histopathological changes in the buccal mucosa of control and experimental hamsters in each group. a and d-Photomicrographs showing well-defined buccal pouch epithelium from the control and chrysin-alone hamsters (X40), b-Photomicrographs showing well-differentiated squamous cell carcinoma with keratin pearls in hamsters treated with DMBA alone (X40), c-Photomicrographs showing moderate dysplastic epithelium in hamsters treated with DMBA+chrysin (X40)

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Table 2: Histological changes in oral buccal mucosa of control and experimental hamsters in each group (n=6)

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The status of phase I (cytochrome P 450 and cytochrome b 5 ) and phase II (GSH, GST, GR, and DTD) detoxification agents in the livers of the control and experimental hamsters in each group are shown in [Figure 4]. The status of phase I detoxification agents were significantly increased, whereas, that of phase II detoxification agents were decreased in the liver of hamsters treated with DMBA alone, as compared to control hamsters. Oral administration of chrysin to DMBA-treated animals reverted the status of phase I and phase II detoxification agents to the near normal range in the liver. No significant difference was noticed in the status of the liver detoxification cascade between control hamsters and hamsters administered with chrysin alone.
Figure 4: Status of phase I and phase II detoxification agents in the liver of control and experimental hamsters in each group. Values are expressed as mean ± SD (n = 6). Values that are not sharing a common superscript letter between groups differ significantly at p < 0.05 (DMRT). X-Micromoles of cytochrome P450, Y-Micromoles of cytochrome b5, A-Micromoles of NADPH oxidized per minute, B-Micromoles of CDNB/GSH conjugate formed per minute, C-Micromoles of 2,6-dichlorophenol reduced per minute

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[Figure 5] shows the status of phase I (cytochrome P 450 and cytochrome b 5 ) and phase II (GSH and GST) detoxification agents in the buccal mucosa of control and experimental hamsters in each group. The status of phase I (cytochrome P 450 and cytochrome b 5 ) and phase II (GSH and GST) detoxification agents were significantly increased in the tumor-bearing hamsters, as compared to the control hamsters. Oral administration of chrysin to DMBA-treated animals reverted the status of phase I and phase II detoxification agents to the near normal range. Hamsters treated with chrysin alone showed no significant difference in the status of GSH and GST as compared to control hamsters.
Figure 5: Status of phase I and phase II detoxification agents in the buccal mucosa of control and experimental hamsters in each group. Values are expressed as mean ± SD (n = 6). Values that do not share a common superscript between groups differ significantly at P < 0.05 (DMRT). X-Micromoles of cytochrome P450, Y-Micromoles of cytochrome b5, A-Micromoles of 1-chloro-2,4-dinitrobenzene (CDNB)-GSH conjugate formed per minute

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The status of thiobarbituric acid reactive substances (TBARS) and antioxidants (SOD, CAT, GPx, GSH, and vitamin E) in the plasma of control and experimental hamsters in each group are shown in [Figure 6]. The concentration of TBARS was increased, whereas, the status of antioxidants was significantly decreased in tumor-bearing hamsters, as compared to control hamsters. Oral administration of chrysin to DMBA-treated hamsters reverted the concentration of TBARS and antioxidants in the plasma. Hamsters treated with chrysin alone showed no significant difference in TBARS and antioxidant status as compared to control hamsters.
Figure 6: Status of TBARS and antioxidants in the plasma of control and experimental hamsters in each group. Values are expressed as mean ± SD (n=6). Values that do not share a common superscript between groups differ significantly at P < 0.05 (DMRT). A-The amount of enzyme required to inhibit 50% NBT reduction, B-Micromoles of hydrogen peroxide utilized/second, C-Micromoles of glutathione utilized/minute

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The status of TBARS and antioxidants (SOD, CAT, GPx, GSH, and vitamin E) in the buccal mucosa of control and experimental hamsters in each group are shown in [Figure 7]. Lowered TBARS levels and disturbances in antioxidant status (GPx, GSH, and vitamin E were increased; SOD and CAT were decreased) were observed in tumor-bearing hamsters as compared to control hamsters. Oral administration of chrysin to DMBA-treated hamsters reverted the concentration of TBARS and antioxidants in the buccal mucosa. Hamsters treated with chrysin alone showed no significant difference in TBARS and antioxidant status as compared to control hamsters.
Figure 7: Status of TBARS and antioxidants in the buccal mucosa of control and experimental hamsters in each group. Values are expressed as mean ± SD (n=6). Values that do not share a common superscript between groups differ significantly at P < 0.05 (DMRT). A-The amount of enzyme required to inhibit 50% NBT reduction, B-Micromoles of hydrogen peroxide utilized/second, C-Micromoles of glutathione utilized/minute

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   Discussion Top


The present study has investigated the chemopreventive potential of chrysin in DMBA-induced hamster buccal pouch carcinogenesis. The percentage of tumor-bearing animals and tumor size was monitored during the experimental period in DMBA-treated and DMBA + chrysin treated hamsters. The status of phase I and phase II detoxification agents, lipid peroxidation, and antioxidants were utilized as biochemical end points, to assess the chemopreventive potential of chrysin during DMBA-induced oral carcinogenesis. In the present study, 100% oral tumor formation (well-differentiated squamous cell carcinoma) with severe dysplasia was noticed in hamsters treated with DMBA alone. The tumor tissues from the buccal pouches of golden Syrian hamsters showed pleomorphic hyperchromatic nuclei with epithelial pearl formation. Oral administration of chrysin to DMBA-treated hamsters significantly reduced oral tumor formation and decreased the severity of histopathological changes. The present results thus suggest that chrysin might have inhibited abnormal cell proliferation during DMBA-induced oral carcinogenesis.

It has been well-documented that chemopreventive agents facilitate the excretion of carcinogenic metabolites either by inactivating the metabolic activation of carcinogens or by stimulating the activities of phase II detoxification enzymes, mainly glutathione-S-transferase. [7],[41] Even as phase I enzymes are involved in the metabolic activation of carcinogenic agents, phase II enzymes are involved in the detoxification of carcinogenic agents. [42] In the present study, the activities of phase I enzymes were increased, whereas, phase II detoxification agents were decreased in the liver of hamsters treated with DMBA alone. The present results thus suggest that the carcinogenic metabolite of DMBA, dihydrodiol epoxide, was excessively generated during the metabolic activation of DMBA, which impaired the activities of phase II detoxification agents. Buccal mucosa phase I and II detoxification agents were significantly increased in hamsters treated with DMBA alone. Extensive studies reported that increase in the activities of phase I and II enzymes was probably due to repeated carcinogenic exposure in the buccal mucosa. [43] Our results supported these findings. Oral administration of chrysin at a dose of 250 mg/kg bw reverted the status of phase I and phase II detoxification enzymes in the liver and buccal mucosa of DMBA-treated hamsters. The present results suggest that chrysin modulated the activities of phase I and II enzymes in favor of the excretion of carcinogenic metabolites during DMBA-induced hamster buccal pouch carcinogenesis.

In the present study, an increase in plasma TBARS and decrease in antioxidants were noticed in hamsters treated with DMBA alone. Increase in plasma TBARS and decrease in antioxidants confirmed oxidative stress in hamsters treated with DMBA alone. Increased plasma TBARS could be due to overproduction of lipid peroxidation by-products during DMBA metabolic activation as well as from the damaged host tissues, with subsequent leakage into the plasma. [44] Lowered levels of non-enzymatic and enzymatic antioxidants are probably due to their utilization by the tumor tissues or to combat the deleterious effects of lipid peroxidation by-products respectively. [45] Diminished lipid peroxidation and disturbed antioxidants were noticed in the tumor tissues of hamsters treated with DMBA alone as compared to normal buccal mucosa tissues. Low content of PUFA and abnormal cell proliferation in tumor tissues were suggested as factors responsible for decreased levels of lipid peroxidation by-products. [46] Our results were in line with these findings. Increase in GSH and GPx activity in tumor tissues was probably due to their regulatory effects in cell proliferation. [47] Lowered activities of SOD and CAT were well-documented in several cancerous conditions including oral cancer. [48] Our results corroborated these observations. Oral administration of chrysin to DMBA-treated hamsters improved the status of lipid peroxidation and antioxidants in the plasma and buccal mucosa, which suggested that chrysin might have been involved in maintaining the oxidant and antioxidant balance during DMBA-induced hamster buccal pouch carcinogenesis. The antioxidant property of chrysin was probably due to its phenolic hydroxyl groups at positions 5 and 7 in the chemical structure. [24] The present study thus demonstrated the chemopreventive potential of chrysin in DMBA-induced hamster buccal pouch carcinogenesis. The overall results of the present study concluded that chrysin had the potential to delay the tumor formation rather than inhibiting tumor formation in the buccal mucosa during DMBA-induced hamster buccal pouch carcinogenesis.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]


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