|Year : 2018 | Volume
| Issue : 6 | Page : 1285-1290
Chemopreventive potential of vanillic acid against 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis
Anbalagan Vinoth1, Raju Kowsalya2
1 Department of Biochemistry and Biotechnology, Annamalai University, Chidambaram, Tamil Nadu, India
2 Department of Biochemistry, Government Arts College for Women, Krishnagiri, Tamil Nadu, India
|Date of Web Publication||28-Nov-2018|
Department of Biochemistry, Government Arts College for Women, Krishnagiri - 635 001, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Objectives: The aim of this study was to evaluate the chemopreventive potential of vanillic acid against 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch oral carcinogenesis.
Materials and Methods: Determine the tumor incidence, tumor volume and burden, assessment of the status of Phase I and Phase II detoxification enzymes were measured in the liver and buccal mucosa of hamsters using specific colorimetric methods.
Results: One hundred percent tumor formation was observed in DMBA alone treated hamsters. Phase I and Phase II detoxification enzymes status were significantly altered DMBA-induced oral carcinogenesis. Vanillic acid (200 mg/kg bw p.o) significantly restored the biochemical variables of liver and buccal mucosa in DMBA + vanillic acid treated hamsters to near normal range compared with DMBA alone treated hamsters.
Conclusion: The present study thus shows chemopreventive potential of vanillic acid in DMBA-induced hamster buccal pouch carcinogenesis. Vanillic acid improves the phase I and phase II detoxification enzymes in DMBA treated hamsters.
Keywords: Vanillic acid, chemoprevention, 7,12-dimethylbenz(a)anthracene, detoxification
|How to cite this article:|
Vinoth A, Kowsalya R. Chemopreventive potential of vanillic acid against 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. J Can Res Ther 2018;14:1285-90
|How to cite this URL:|
Vinoth A, Kowsalya R. Chemopreventive potential of vanillic acid against 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. J Can Res Ther [serial online] 2018 [cited 2020 Jul 2];14:1285-90. Available from: http://www.cancerjournal.net/text.asp?2018/14/6/1285/191057
| > Introduction|| |
Oral cancer is one of the common neoplasms worldwide and it is a common malignancy in developing countries particularly India. Oral cancer accounts for 30–40% out of all cancer. Oral cancer incidence in India is high and four times folded than other countryside. Tobacco and alcohol consumption is main promoters/initiators of oral cancer in India. Ninety percent of oral cancer is oral squamous cell carcinoma, histologically proved.
Oral cancer is the eight rank of all cancer types worldwide and in the region of the USA in its rate approximately 2.5% all cancer. In the last three decades, 10-year survival rate is 51% and represents the survival rate could not be changed in over 30 years. Late diagnosis and knowledge deficiency about oral cancer risks and symptoms of oral cancer, it attributed the low oral cancer survival rate. Secondary primary tumors may repetition 5–8 years of 22–42% oral cancer patients.
Squamous cell carcinoma of the buccal mucosa is a tremendous candidate cancer for measurement of chemoprevention, oral administration of chemopreventive agents, results can be seen during experimental period, and alteration or suppression of genes or gene products participated in oral malignancies represents molecular targets oppose which chemopreventive method can be examined and confirmed. Syrian golden hamsters used as an oral cancer experimental model since 1954 and instead of more studies on prevention of oral squamous cell carcinoma by several chemopreventive entities. The previous studies reported that the Golden Syrian hamster model has been developing oral cancer, these tumors determined and characterize few of the genetic modifications appearing in premature and mature development of oral squamous cell carcinoma. Several morphological and physiological characteristics those are observed in human oral squamous tumors are too observed in chemically induced hamster buccal pouch tumors. Hamster buccal pouch tumors and human oral squamous cell carcinoma tumors are both histologically and molecularly have the similarity and that may help as feasible biomarkers. Chemically induced hamster buccal pouch tumors are shown genetic events that are very similar to human oral malignancy.
Chemoprevention is a novel, possible, and remarkable path to elucidate the anticancer effects of natural and synthetic compounds. Profound articles evidently proved chemopreventive agents that inhibit or down regulate the tumor formation and progression through biochemically and molecular level.
The liver is the first-string for the change of integrity of xenobiotics, plays an important role in the alteration of the carcinogenic process. Determination of xenobiotic biotransformation enzymes such as cytochrome P450, cytochrome b5, glutathione-S-transferase (GST), glutathione reductase (GR), and reduced glutathione (GSH) in liver and buccal mucosa give the help to understand between the chemoprevention and chemopreventive entities potentials.,
Vanillic acid is an oxidized form of vanillin and used as a flavoring agent, vanillic acid shows high free radical scavenging property compares to the vanillin. Vanillic acid highly present in the roots of Angelica sinensis, it could be used in Traditional Chinese Medicine. Stanely Mainzen Prince et al., reported that protective effect of vanillic acid and its free radical scavenging, antioxidant, and anti-inflammatory activities in isoproterenol-induced cardiotoxic rats. The present study deals to evaluate the chemopreventive potentials of vanillic acid against 7,12-dimethylbenz(a)anthracene (DMBA) stimulated oral cancer in the buccal pouch of the Golden Syrian hamster.
| > Materials and Methods|| |
Chemicals and reagents
DMBA and vanillic acid was procured from Sigma-Aldrich Chemicals Pvt. Ltd., Bengaluru, Karnataka, India. All other chemicals used were of analytical grade obtained from HiMedia Laboratories, Bengaluru, Karnataka, India.
Twenty-four male Golden Syrian hamsters (8-week-old and 80–120g weighing) were procured from National Institute of Nutrition, Telangana, India. Experimental animals are maintained in the Central Animal House, RMMC and H, Annamalai University, Annamalainagar, Tamil Nadu, India. Experimental animals are housed in polypropylene cages and supplemented with standard pellet diet and water ad libitum. Animals were kept in a controlled environment with a 12 h light and 12 h dark cycle.
A total number of 24 experimental hamsters divided into four groups of six hamsters in each group. Group I hamsters as control and liquid paraffin only applied in their left buccal pouch of hamsters in the whole experimental period. Group II and III hamsters were painted with 0.5% DMBA in liquid paraffin thrice times a week for 14 weeks on their left buccal pouches of hamsters. No other treatment is given to the Group II hamsters. Vanillic acid at a dose of 200 mg/kg bw was orally administrated to Group III hamsters. Group IV hamsters treated with vanillic acid (200 mg/kg bw) alone all over the experimental session. The experiment was completed at the end of the 16th week all the animals were sacrificed by cervical dislocation. Biochemical estimations were performed in the liver and buccal mucosa of control and experimental hamsters. For histopathological examinations, buccal mucosa tissues were kept in 10% formalin and continuously developed and embedded with paraffin, serial 2–3 μm sections were cut in rotary microtome and stained with hematoxylin and eosin.
Induction of oral squamous cell carcinogenesis
0.5% DMBA was painted in the buccal pouches of Group II and Group III hamsters, three times a week for 14 weeks. Total tumors were estimated by macroscopically and tumor incidence was analyzed using Vernier caliper.
Determine the Phase I and Phase II detoxification enzymes. End of the experimental session, hamsters was sacrificed, liver, and buccal mucosa tissue samples are rinsed with ice-cold saline and samples were homogenized then used for biochemical assessment. Cytochrome P450 and cytochrome b5, were assayed in liver and buccal mucosa according to the methods of Omura and Sato, respectively. Status of GST in the buccal mucosa was determined by Habig et al. GR activity was assayed using the method employed by Carlberg and Mannervik. Reduced GSH in the liver and buccal mucosa activity was assayed by the method of Beutler and Kelly. The activity of DT-diaphorase in the liver was estimated by Ernster. The level of oxidized glutathione (GSSG) level in the buccal mucosa was described by the method of Tietze.
Statistical analysis and software details
All data are expressed as mean ± standard deviation (n = 6). One-way analysis of variance followed by Duncan's multiple range test (DMRT) was employed to determine the statistical difference among experimental groups. The P < 0.05 among experimental groups results regarded as statistically significant.
| > Results|| |
Incidence of oral neoplasm
Tumor incidence of hamsters was shown in [Table 1] and [Figure 1]. We found 100% tumor incidence in DMBA alone treated hamsters and it is confirmed by histopathologically as well-differentiated squamous cell carcinoma. Using of Vernier caliper to measure the tumor incidence in the diameter of each tumor. We observed no tumor formation in vanillic acid administrated and DMBA painted hamsters (Group III). Vanillic acid (200 mg/kg bw) prevents the tumor formation and decrease the risk rate of oral squamous cell carcinoma development.
|Table 1: Incidence of oral neoplasm and histopathological changes in the buccal pouch of hamsters in the control and experimental groups (n=6)|
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|Figure 1: Gross appearance of buccal mucosa in control and experimental hamsters in each group. (a and d) Control and vanillic acid alone: Normal buccal pouch. (b) 7,12-dimethylbenz(a)anthracene alone: Well differentiated squamous cell carcinoma. (c) 7,12-dimethylbenz(a)anthracene + vanillic acid: Precancerous oral epithelial layers|
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The histopathological features observed in the buccal mucosa of the control and treated animals in each group are shown in [Figure 2]. Control (Group I) and vanillic acid alone (Group IV) treated hamsters buccal mucosa hematoxylin and eosin (H and E) stained sections shows normal epithelium. No dysplastic change in the deeper stroma is unremarkable. DMBA alone (Group II) treated hamsters buccal mucosa H and E sections shows squamous epithelium with acanthosis, hyperkeratosis, and dysplastic changes with dyskeratosis (keratin pearl formation) and infiltration into deeper tissue and well differentiated squamous cell carcinoma. H and E stained sections of DMBA and vanillic acid-treated (Group III) hamsters shows mild dysplastic changes.
|Figure 2: Hematoxylin and Eosin-stained sections of hamster buccal pouch 14 weeks after commencement of 7,12-dimethylbenz(a)anthracene treatment. (a and d) Control and vanillic acid alone: Normal epithelium, (b) 7,12-dimethylbenz(a)anthracene alone: Well differentiated squamous cell carcinoma, (c) 7,12-dimethylbenz(a)anthracene + vanillic acid: Mild dysplasia|
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Levels of liver Phase I and Phase II detoxification agents
The levels of liver Phase I parameters (cytochrome P450 and cytochrome b5) and Phase II parameters (GSH, GR, and DT-diaphorase) was given in [Figure 3]. Vanillic acid alone (Group IV) treated hamsters did not show any significant variation as compared to control groups. DMBA alone (Group II) treated hamsters showed significantly increased Phase I detoxification enzymes while decreased Phase II detoxification enzymes in the liver. DMBA painted and vanillic acid administrated (Group III) hamsters reverts the levels of Phase I and Phase II detoxification enzymes into nigh to normal level in the liver.
|Figure 3: Status of Phase I and Phase II detoxification agents in the liver of control and experimental animals in each group (n = 6). Values are expressed as mean ± Standard deviation values that do not share a common superscript between two groups differ significantly at P < 0.05 (Duncan's multiple range test). X – micromoles of cytochrome P450; Y – micromoles of cytochrome b5; C – micromoles of 2, 6-dichlorophenol reduced/min. GR = Glutathione reductase, GSH = Glutathione|
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Levels of buccal mucosa Phase I and Phase II detoxification agents
The levels of liver Phase I parameters (cytochrome P450 and cytochrome b5) and Phase II parameters (GST, GSSG, and GSH) were given in [Figure 4]. Cytochrome P450, cytochrome b5, GST, and GSH were significantly increased meanwhile GSSG levels were lower levels in the DMBA alone treated hamsters (Group II) as compared to control hamsters. DMBA painted and vanillic acid administrated (Group III) hamsters reverts the levels of GSH, GST, and GSSG into near normal level. Vanillic acid alone treated animals (Group IV) no variance in the levels of GSH, GST, and GSSG as compared to control group hamsters (Group I).
|Figure 4: Status of Phase I and Phase II detoxification agents in the buccal mucosa of control and experimental animals in each group (n = 6). Values are expressed as mean ± standard deviation values that do not share a common superscript between two groups different significantly at P < 0.05 (Duncan's multiple range test). X – micromoles of cytochrome P450; Y – micromoles of cytochrome b5; A – micromoles of 1-chloro 2, 4 dinitrobenzene/reduced glutathione conjugate formed/min. GSSG = Oxidized glutathione, GSH = Glutathione, GST = Glutathione-S-transferase|
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| > Discussion|| |
Worldwide oral cancer is the common and eight most cancer. Oral carcinogenesis is a multifactorial disease, antecedent by different premalignant lesions. Hamster buccal mucosa precancerous and cancerous lesions are closely mimics to the human oral cancer. DMBA a site specific carcinogen frequently used as a carcinogen to develop tumors in the buccal pouches of Golden Syrian hamsters. Profound studies clearly revealed that DMBA-induced oral carcinogenesis be similar to human oral cancer. We found 100% tumor level in DMBA alone treated hamsters in the end of the experimental session. We observed tumor incidence, tumor volume, and tumor burden in experimental hamsters. Severe hyperplasia, hyperkeratosis, and dysplasia were determined in the DMBA alone treated hamsters. DMBA and vanillic acid treated hamsters revealed severe hyperkeratosis, hyperplasia, and mild dysplasia. Results of this study proposed that vanillic acid suppresses tumor formation through DMBA-induced experimental oral carcinogenesis.
Overall survival rate of oral cancer is not considerably changed over the past three decades, because in spite of finding and treatment. In consequence of, primary prevention by surcease of tobacco and alcohol consumption, in the company of dietary supplement/chemoprevention is of essential importance. In addition, this disease given a chance for secondary prevention, described as hindrance of recurrent, or new secondary primary oral malignant tumors. Certainly, 20% oral squamous cell carcinoma patients will show reappearance in 18 months postsurgery and 22–44% of patients will exhibit with secondary primary tumors within 5–8 years.
Previous studies reported that number of chemopreventive entities prevents progression of tumors in the hamster buccal pouch carcinogenesis as induced earlier, during, or later by DMBA. Chemopreventive agents have different mechanisms including detoxification metabolites, induction of Phase II enzymes, free radical scavenging, control of proliferation, inhibition of angiogenesis, upregulation of apoptotic markers, and suppression of DNA adduct formation.,
Natural and synthetic compounds peculiarly efficiently chemoprevention of hamster buccal pouch carcinogenesis. DMBA induced tumors in hamsters, thus tumors was suppressed or inhibited or controlled by several agents such as green tea polyphenols, retinyl acetate, Ocimum sanctum, 18-β-glycyrrhetinic acid, lupeol, cromolyn, piperine, apigenin and different synthetic compounds. Our results are lend credence to these results.
The histopathological study of buccal pouches shows the establishment of well-differentiated squamous cell carcinoma, in hamsters treated with DMBA only. OSCC was detected by precancerous lesions such as hyperplasia and dysplasia and keratosis (56–70 days). Vanillic acid and DMBA treated hamsters unveiled that the vanillic acid is very effectually decrease the number of early lesions, as induced earlier, during, or later by DMBA treatment. Multiplicity of dysplasia was decreased to near normal levels and tumor formation was not seen in DMBA + vanillic acid administrated hamsters.
During cancer including oral cancer, carcinogens metabolically activated in the liver. Activated metabolites are presented in the liver due to the upregulated performance of Phase I detoxification enzymes and lesser performance of Phase II detoxification enzymes. We observed increased activity of Phase I detoxification enzymes and lesser activity of Phase II detoxification enzymes in the DMBA alone treated hamsters. Imbalance between the Phase I and Phase II enzymes may possibly result in malignant development. Detoxification enzymes are regulated during the cells are exposed to carcinogens including DMBA., Our findings are lending credence to these results.
| > Conclusion|| |
Vanillic acid at a dose of 200 mg/kg/bw improved the levels of Phase I and Phase II enzymes in the liver and buccal mucosa of hamsters treated with DMBA.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]