Micronucleus investigation in human buccal epithelial cells of gutkha users
Smita Jyoti1, Saif Khan2, Mohammad Afzal1, Yasir Hasan Siddique1
1 Department of Zoology, Human Genetics and Toxicology Laboratory, Section of Genetics, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of Periodontics and Community Dentistry, Dr Z.A. Dental College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
|Date of Submission||06-Feb-2012|
|Date of Acceptance||07-Mar-2012|
|Date of Web Publication||28-Aug-2012|
Yasir Hasan Siddique
Department of Zoology, Human Genetics and Toxicology Laboratory, Section of Genetics, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Gutkha is a cheap and convenient betel quid substitute, which is popular among all age groups. Various studies reveal its carcinogenic nature that leads to oral submucosus fibrosis and increases the chances of oral cancer. The micronucleus (MN) assay in exfoliated mucosal cells is a useful method for observing genetic damage in humans.
Aim: To observe the genotoxic effect of gutkha on human buccal epithelial cells.
Materials and Methods: The MN assay was performed to assess the frequency of MN in human buccal epithelial cells. The study comprises 60 individuals of which 30 individuals were gutkha chewers and another 30 were nonusers (control). The MN frequency was scored to estimate the genotoxic damage.
Results: In gutkha users, the frequency of MN was highly significant (17.4 ± 0.944) as compared with nonusers (control) groups (4.53 ± 0.331) (P < 0.001).
Conclusions: The MN assay in human buccal epithelial cells is a useful and minimally invasive method for monitoring genetic damage in humans. Asignificantly higher frequency of micronucleated cells are found among gutkha users.
Keywords: Buccal, epithelial cells, gutkha, micronuclei
|How to cite this article:|
Jyoti S, Khan S, Afzal M, Siddique YH. Micronucleus investigation in human buccal epithelial cells of gutkha users. Adv Biomed Res 2012;1:35
|How to cite this URL:|
Jyoti S, Khan S, Afzal M, Siddique YH. Micronucleus investigation in human buccal epithelial cells of gutkha users. Adv Biomed Res [serial online] 2012 [cited 2019 Sep 17];1:35. Available from: http://www.advbiores.net/text.asp?2012/1/1/35/100128
| Introduction|| |
Gutkha and pan masala are in more demand among all age groups. It is revealed that betel quid chewing with or without tobacco are carcinogenic in humans. , Gutkha is the mixture of areca nut, catechu, lime, cardamom, spices, unspecified flavouring agents, and tobacco. Gutkha is supposed to be responsible for a number of oral diseases and has addictive effects that leads to the addiction due to the presence of areca nut and tobacco.  Areca nut is a main component of gutkha, which is responsible for oral submucous fibrosis (OSMF).  OSMF is uncurable disease, and finally leads to oral cancer.  After long time of smoking, adverse effects are seen but in case of gutkha users, OSMF develops within a very short span of time.  The intake of gutkha and OSMF is very common in young persons.  Areca nut increases the chances of formation of precancerous lesion and OSMF. Micronuclei are small chromatin bodies that appear in the cytoplasm by the condensation of acrocentric chromosomal fragments or by whole chromosomes, lagging behind during cell division. Thus, it is the only biomarker that allows the simultaneous evaluation of both clastogenic and aneugenic effects in a wide range of cells, that are easily detected in interphase cells.  MN assay has been used as a biomarker of genetic damage in buccal mucosa cells. , An elevated micronucleated cell frequency is found in the buccal mucosal epithelium of areca nut chewers.  The aqueous extract of N-nitroso compounds related to areca nut, that is, 3-(methylnitrosamino) proprionitrile is highly cytotoxic and genotoxic in cultured human buccal epithelial cells, and enhance the induction of tumors in betel quid chewers.  The MN assay in buccal cells can be used to detect cancerous or precancerous lesions and also to monitor the effects of a number of chemopreventive agents. , In the present study, the effect of gutkha was studied on the micronucleus (MN) frequency in buccal epithelial cells.
Aims and Objectives
The present study showed the frequency variation of MN in the chewers and nonchewers of gutkha by performing MN assay.
| Materials and Methods|| |
The study comprised 60 male individuals out of whom 30 individuals were having the habit of chewing gutkha (cases), these were compared with the remaining 30 individuals who were nonusers (control: Those who did not involve in any addiction). A written consent was taken from each individual, and the samples were taken from the Department of Ziauddin Ahmed Dental College and Hospital, A.M.U. Aligarh, U.P. The period of the study was almost 6 months.
Trizma hydrochloride (Tris-HCl), ethylene diamine tetraacetic acid (EDTA) from SRL, India. Giemsa stain, sodium chloride, methanol, and sodium hydroxide pellets from Merck (India). The buffer solution was prepared by dissolving 0.1 M EDTA, 0.001 M Tris-HCl and 0.02 M NaCl in a sterile 1 L distilled water. The pH of the buffer was adjusted to 7.0 with NaOH.
Oral Mucosa Cell Collection and Processing
Oral mucosa cells were collected from each subject using a soft toothbrush gently from the oral mucosa of cheeks.  The brush was then swirled into a centrifuge tube containing a buffer solution of pH 7.0, thereby creating a cell suspension. The cells were washed three times by centrifugation at 1500 rpm for 10 min in the buffer solution.  About 15 mL of buffer in a 30 mL conical tube was used in every washing step. About 50-100 μL of the cell suspension was laid and spread on clean, preheated (37°C) glass slide and allowed to air dry for 5-10 min. The slides were fixed in methanol, stained with 5% Giemsa and observed under microscope.  A total of 2000 oral mucosal cells were scored per individual.
Statistical analysis was carried out by Student's t test using commercial software Statistica Soft Inc.
| Results|| |
MN frequency among individuals having chewing habit was found to be 4 times higher (21.3 ± 1.788) as compared with the control (4.56 ± 0.331) [Table 1]. The number of micronucleated cells in the controls and cases were 4.53 ± 0.331 and 17.4 ± 0.944, respectively [Table 1]. The distribution of micronucleated cells is given in [Table 2] and [Figure 1]. The age distribution of cases and controls is given in [Table 3]. Among users, the youngest was of 12 years and the oldest one was of 65 years of age. [Figure 2], [Figure 3], [Figure 4] and [Figure 5] shows the MN in buccal epithelial cells of the users.
|Table 1: Total micronucleus frequency per 2000 cells per individual in the buccal region of 30 cases and 30 controls|
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| Discussion|| |
MN has been used since 1937 as an indicator of genotoxicity.  Studies on MN frequencies support that MN as a product of early events in human carcinogenic processes, particularly in oral regions. ,, MN test is especially used for the identification of preclinical steps of the cancer.  Various studies from 1985 till date have shown significant increase in micronucleated frequency in betel quid chewers as compared with healthy individuals.  The present study shows the higher frequency of MN among the users of gutkha, this was proved by previous studies. , The main carcinogens in gutkha is derived from their ingredients areca nut, catechu, and tobacco. Tobacco-specific nitrosamines are formed due to chewing of gutkha.  That leads to exposure of buccal cells to volatile nitrosamines derived from areca nut alkaloids.  A high level of nitrite and nitrate reductase activity have been reported in the saliva of gutkha chewers.  Swallowing of the quid leads to the nitrosation of secondary and tertiary amines due to the acidic pH of stomach. Urinary levels of N-nitrosoproline were 4- to 6.5-fold higher in gutkha chewers. , Aqueous extracts of areca nut and catechu responsible for the generation of reactive oxygen species that cause the genotoxic damage in buccal epithelial cells.  Variations in the number of micronucleated cells may be affected by the ingredients in the quid, the number of quids per day and different lifestyles, gender, age, and food habits.  We observed the difference in the frequencies of micronucleated cells in the control group, which may be due to the different food habits of the population groups. Individuals ingest various types of chemicals in their daily diet, which was the reason for the variable levels of micronucleated cells.  The duration of addiction of the chewing habit in the present study of 30 individuals was in average of 1-20 years and their frequency was 2-18 pouches/day. A majority of degenerative and developmental diseases are caused by genomic damage, which is produced by environmental exposure of radiation, chemicals, micronutrient deficiency, and lifestyle factors, for example, alcohol, smoking, drugs, gutkha, pan masala and stress. So it is important to biomonitoring, identifying, and treatment of diseases caused by, or associated with genetic damage. The MN assay in buccal cells serves as an excellent biomarker.  A supplement of vitamins and beta-carotene found to be an effective measure used for reduction in the number of micronucleated cell frequency in healthy chewers as well as precancerous lesions. 
| Conclusions|| |
This study reveals that gutkha is highly genotoxic and responsible for oral cancer in near future, so it is important to increase the awareness programs to inform and educate the public regarding the adverse health consequences and possible cancer risk associated with gutkha.
| Acknowledgments|| |
The authors are thankful to the Council of Science and Technology (CST/D-3908), Lucknow, UP, for awarding the project titled "Genotoxicity assessment in exfoliated Mucosal cells of Pan masala and Gutkha Chewers." We are also thankful to the Chairman, Department of Zoology, for providing laboratory facilities and to the Chairman, Department of Periodontics and Community Dentistry, for the support in providing the samples.
| References|| |
|1.||IARC. Monographs on the Evaluation of Carcinogenic Risks of Chemicals to Humans, tobacco Habits Other than Smoking; Betel quid and Areca-nut Chewing; and Some Related Nitrosamines. Vol. 37. Lyon: IARC; 1985. |
|2.||IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans, tobacco Habits Other than Smoking: Betel quid and Areca-nut Chewing. Vol. 85. Lyon: IARC; 2004. |
|3.||Kumar S. Panmasala chewing induces deterioration in oral health and its implications in carcinogenesis. Toxicol Mech Methods 2008;18:665-77. |
|4.||Tilakaratne WM, Klinikowski MF. Review on aetiology and pathogenesis. Oral Oncol 2005;42:561-8. |
|5.||Murti PR, Bhonsle RB, Pindborg JJ, Daftary DK, Gupta PC, Mehta FS. Malignant transformation rate in oral submucous fibrosis over a 17-year period. Community Dent Oral Epidemiol 1985;13:340-1. |
|6.||Babu S, Sesikeran B, Bhat RV. Oral fibrosis among teenagers chewing tobacco, areca nut, and pan masala. Lancet 1996;348:692. |
|7.||Gupta PC, Sinor PN, Bhonsle RB. Oral submucous fibrosis in India: A new epidemic? Natl Med J India 1998;11:113-6. |
|8.||Znaor A, Fuciæ A, Strnad M, Barkoviæ D, Škara M, Hozo I. Micronuclei in Peripheral Blood Lymphocytes as a Possible Cancer Risk Biomarker: A Cohort Study of Occupationally Exposed Workers in Croatia. Croat Med J 2003;44:441-6. |
|9.||Stich HF, Rosin MP. Quantitating the synergistic effect of smoking alcohol consumption with the micronucleus test on human buccal mucosa cells. Int J Cancer 1983;31:305-8. |
|10.||Speit G, Schmid O. Local genotoxic effects of Formaldehyde in humans measured by the micronucleus test with exfoliated epithelial cells. Mutat Res 2006;613:1-9. |
|11.||Stich HF, Stich W. Chromosome damaging activity of saliva of betel nut and tobacco chewers. Cancer Lett 1982;15:193-202. |
|12.||Sundqvist K, Liu Y, Nair J, Bartsch H, Arvidson K, Grafstrom RC. Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells. Cancer Res 1989;49:294-8. |
|13.||Stich HF, Rosin MP. Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention. Cancer Lett 1984;22:241-53. |
|14.||Stich HF, Dunn BP. DNA adducts, micronuclei and leukoplakias as intermediate endpoints in intervention trials. IARC Sci Publ 1988;89:137-45. |
|15.||Surrallés J, Autio K, Nylund L, Jarventaus H, Norppa H, Veidebaum T, et al. Molecular cytogenetic analysis of buccal cells and lymphocytes from benzene-exposed workers. Carcinogenesis 1997;18:817-23. |
|16.||Rajeswari N, Ahuja YR, Malini U, Chandrashekar S, Balakrishna N, Rao KV, et al. Risk assessment in first-degree female relatives of breast cancer patients using the alkaline Comet assay. Carcinogenesis 2000;21:557-61. |
|17.||Heddle JA, Hite M, Kirkhart B, Mavournin K, MacGregor JT, Newell GW, et al. The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res 1983;123:61-118. |
|18.||Stich HF, Curtis JR, Parida BB. Application of the micronucleus test to exfoliated cells of high cancer risk groups: Tobacco chewers. Int J Cancer 1982;30:553-9. |
|19.||Roberts DM. Comparative cytology of the oral cavities of snuff users. Acta Cytol 1997;41:1008 -14. |
|20.||Ramirez A, Gattas GJF, Carvalho MB, Rapopport A, Saldanha PH. Clinical implications of micronuclei frequency as a biomonitor for alcoholic patients with oral carcinomas. Oral Oncol 1999;6:199-204. |
|21.||Nair U, Obe G, Nair J, Maru GB, Bhide SV, Pieper R, et al. Evaluation of frequency of micronucleated oral mucosa cells as a marker for genotoxic damage in chewers of betel quid with or without tobacco. Mutat Res 1991;261:163-8. |
|22.||Gandhi, Kaur R. Cytogenetic studies in exfoliated cells of high cancer risk groups, pan masala chewers. Hum Ecol 2000;9:221-8. |
|23.||Siddique YH, Ara G, Beg T, Afzal M. Micronucleus investigation in oral mucosal cells of gutkha/pan masala chewers. Indian Biologist 2008;40:1-5. |
|24.||Nair UJ, Nair J, Mathew B, Bartsch H. Glutathione S-transferasen M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers. Carcinogenesis 1999;20:743-8. |
|25.||Wenke G, Rivenson A, Brunnemann KD, Hoffmann D, Bhide SV. A study of betel quid carcinogenesis. II. Formation of N nitrosamines during betel quid chewing. In: O'Neill IK, von Borstel RC, Miller CT, Long J, Bartsch H, editors. N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer. Lyon: IARC: IARC Scientific Publications no. 57; 1984. p. 859-66. |
|26.||Murdia US, Mehta FJ, Bhide SV. Nitrate reductase activity and nitrite levels in the saliva of habitual users of various tobacco products. Food Chem Toxicol 1982;20:269-71. |
|27.||Nair J, Ohshima H, Pignatelli B, Friesen M, Calmels S, Bartsch H. Modifiers of endogenous carcinogen formation: Studies on in vivo nitrosation in tobacco users. In: Hoffmann D, Harris CC, editors. Mechanisms in Tobacco Carcinogenesis, Banbury Report 23. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory Press; 1986. p. 45-61. |
|28.||Chakradeo PP, Nair J, Bhide SV. Endogenous formation of N-nitrosoproline and other N-nitrosamino acids in tobacco users. Cancer Lett 1994;86:187-94. |
|29.||Nair UJ, Floyd RA, Nair J, Bussachini V, Friesen M, Bartsch H. Formation of reactive oxygen species and of 8- hydroxydeoxyguanosine in DNA in vitro with betel quid ingredients. Chem Biol Interact 1987;63:157-69. |
|30.||Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, et al. The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: The HUMN project perspective on current status and knowledge gaps. Mutat Res 2008;659:93-108. |
|31.||Kamboj M, Mahajan S. Micronucleus- an upcoming marker of genotoxic damage. Clin Oral Investig 2007;11:121-6. |
|32.||Tolbert PE, Shy CM, Allen JW. Micronucleus and other nuclear anomalies in buccal smears: Methods development. Mutat Res 1992;271::69-77. |
|33.||Van Schooten FJ, Besaratinia A, De Flora S, Agostini FD, Izzotti A, Camoirano A, et al. Effects of oral administration of N-acetyl-L-cysteine: A multi-biomarker study in smokers. Cancer Epidemiol Biomarkers Prev 2002;11:167-75. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]
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