Asia-Pacific Journal of Public Health

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1 Asia-Pacific Journal of Public Health Assessment of Genetic Damage Among Chewers of Mixture Containing Mainly Areca Nut and Tobacco Mayur S Joshi, Yogendra Verma, Anil K. Gautam, Vijay K. Shivgotra, Girish Parmar and Sunil Kumar Asia Pac J Public Health : 852 originally published online 13 September 2011 DOI: / The online version of this article can be found at: Published by: On behalf of: Asia-Pacific Academic Consortium for Public Health Additional services and information for Asia-Pacific Journal of Public Health can be found at: Alerts: Subscriptions: Reprints: Permissions: Citations: >> Version of Record - Dec 4, 2011 Proof - Sep 13, 2011 What is This?

2 419838APHXXX / Joshi et alasia-pacific Journal of Public Health Assessment of Genetic Damage Among Chewers of Mixture Containing Mainly Areca Nut and Tobacco Asia-Pacific Journal of Public Health 23(6) APJPH Reprints and permission: sagepub.com/journalspermissions.nav DOI: / Mayur S Joshi, MSc 1, Yogendra Verma, MSc 1, Anil K. Gautam, MSc 1, Vijay K. Shivgotra, PhD 1, Girish Parmar, MD, PhD 2, and Sunil Kumar, MSc, PhD 1 Abstract Chewing mixture containing areca nut and tobacco is believed to be associated with oral cancer. Habit of chewing such mixture is prevalent among South Asian countries. This study aimed to evaluate the genotoxic effect of areca nut and tobacco on human lymphocytes. Peripheral blood from 107 subjects (nonchewers, 48; chewers, 59, including 20 subjects with oral submucous fibrosis [OSMF]) analyzed by cytokinesis-block micronucleus (CBMN) and alkaline comet assay. Nuclear anomalies, namely, binucleated cells with micronuclei (BN MN), total MN, nucleoplasmic bridge, and nuclear buds were higher in chewers whereas elevation in BN MN and total MN were significant among subjects with OSMF than nonchewers. DNA damage assessed by comet assay showed increased percentage of Tail DNA, Tail moment, and Olive tail moment among chewers as well as OSMF subjects. Significant positive correlation was observed between induction of CBMN and consumption of quids per day (r =.280, P =.033). Results suggested cytotoxic and genotoxic potential of mixture containing areca nut and tobacco. Keywords areca nut, tobacco, comet assay, DNA damage, cytokinesis-block micronucleus (CBMN), oral submucous fibrosis (OSMF) Introduction The chewing habit associated with the use of areca nut and tobacco is more prevalent in South Asian countries and is spreading to Western countries among settled Asian migrant communities. 1 Recently, current trends of tobacco use and implications of increasing rise in adolescent smoking in the Southeast Asian region have been revealed by Al-Sadat et al. 2 Oral cancer is one of the most 1 National Institute of Occupational Health, Ahmedabad, India 2 Government Dental College and Hospital, Ahmedabad, India Corresponding Author: Sunil Kumar, Division of Reproductive and Cytotoxicology, National Institute of Occupational Health, Ahmedabad , India sunilnioh@gmail.com

3 Joshi et al. 853 common cancers in South and Southeast Asian countries. 3 In fact, there are several types of chewing habits featuring the use of betel quid (fresh betel leaf, fresh areca nut, slaked lime, catechu with or without tobacco), Kheni (tobacco with lime), Mawa (mixture of areca nut, tobacco, and slaked lime), pan masala plain (areca nut, catechu, lime, cardamom, and unspecified flavoring agents, etc), and pan masala with tobacco (gutkha). The habit of chewing areca nut and tobacco might be responsible for higher incidence of oral cancers in this part of the world, which form almost one third of the total cancer cases in India. 4 Review of studies on these chewing mixtures also revealed that it is likely to be carcinogenic, as tobacco and areca nut have carcinogenic potential, and both have encompassing addictive potential leading to dependence on chewing mixture. 5 Areca nut components, especially an alkaloid called arecoline, plays a major role in the pathogenesis of oral submucous fibrosis (OSMF) by causing an abnormal increase in collagen production. 6 Tobacco when chewed leaches out mutagen/carcinogens in the oral cavity, which act on oral mucosa. Furthermore, evidence is also increasing showing the association between areca nut use causing OSMF, restricted mouth opening due to stiffening of the oral mucosa, and development of fibrous bands. OSMF is not reversible, is precancerous, and has been shown to have an association with areca nut and tobacco chewing. 7 Several cytogenetic methods are used for the biological monitoring of populations exposed to mutagenic and carcinogenic agents. The cytokinesis-blocked micronucleus (CBMN) assay in human lymphocytes is one of the most commonly used techniques for assessing cytogenetic damage. 8 Inhibition of cytokinesis by cytochalasin B allows distinction between the dividing as well as the nondividing cells after treatment and thereby prevents the confounding effects caused by the differences in cell division kinetics. 9,10 The CBMN assay is also designated as the CBMN cytome or CBMN Cyt assay. 9 Micronuclei are acentric fragments or complete chromosomes that, during a mitotic cycle, fail to attach to the mitotic spindle and are excluded from the nucleus. Different mechanisms may be involved in formation of MN, including chromosome breakage and spindle disruption. 11,12 CBMN assay is a comprehensive method for measuring chromosome breakage, DNA misrepair, and chromosome loss. It is now also used to measure nucleoplasmic bridge (NPB), a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair and to measure N buds (nuclear buds), a biomarker of gene amplification. 9 The comet assay (single cell gel electrophoresis or SCGE) received attention in recent years because it is a quick, simple, sensitive, reliable, and fairly inexpensive way to measure DNA damage at the level of individual cells. The comet assay allows the detection of DNA alterations of diverse kinds, such as double-stranded breaks, single-stranded breaks, alkali-labile sites, incomplete repair sites, and cross-links. 13 In the SCGE assay, cells are embedded in agarose, lysed, and electrophoresis carried out under low voltage, so that fragmented and relaxed DNA migrate farther than intact or cross-linked DNA, resembling the image of a comet. The extent of migration of the tail of the comet is related to increased DNA damage. Biological markers such as CBMN and DNA damage could be useful to assess cytogenetic damage induced by areca nut and tobacco chewing. Because cancer can take from years to decades to develop, it is ethically incorrect to perform prospective epidemiological studies. Therefore, elevation in MN and DNA damage might be useful as an early biomarker of disease and exposure. Hence this study was carried out to assess the genotoxic potential of chewing mixture (containing areca nut and tobacco) by employing CBMN (Cytome) and alkaline comet assay. Materials and Methods Study Subjects The subjects were enrolled randomly among apparently healthy patients attending the outpatient department of Government Dental College and Hospital, Ahmedabad, India and a total of 107

4 854 Asia-Pacific Journal of Public Health 23(6) Table 1. Characteristics of Chewers, Subjects With Oral Submucous Fibrosis (OSMF), and Nonchewers Group Nonchewers Chewers Chewers With OSMF Subjects Male Female Total Age (years) a All subjects ± ± ± 2.18 a Values are mean ± SE. subjects were classified as chewers (n = 59), among them 20 subjects with OSMF diagnosed clinically, and nonchewers (n = 48). The mean frequency and duration of chewing material among the chewers was 6.00 ± 0.52 quids/day and 9.00 ± 0.81 years, respectively. General characteristics of the study subjects are listed in Table 1. The number of male subjects in all groups was higher than females indicating male preponderance over females. The mean age of the nonchewers, chewers, and subjects with OSMF was 36.0 ± 1.93, 34.0 ± 1.36, and 32.0 ± 2.18 years, respectively. Study Ethics This is part of a major study for which ethical approval has been obtained from the institutional ethics committee. Informed consent of each subject was taken after explaining the aims and objectives of the study. Sampling Peripheral blood (2 ml) was collected from each subject by venipuncture into a heparinized disposable vacutainer, placed in ice to prevent exogenous damage and processed for CBMN and comet assay. Lymphocyte cultures and MN analysis. Lymphocyte cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum, 1% antibiotics (penicillin and streptomycin) and 1% l-glutamine. Lymphocytes were stimulated by addition of 1% phytohemagglutinin and incubated for 72 hours at 37 C. A cytochalasin-b solution was prepared in dimethylsulfoxide at a final concentration of 6 µg/ ml 14,15 and added to the cultures after 44 hours of incubation to arrest cytokinesis. At 72 hours incubation, the cultures were harvested by centrifugation at 1000 rpm for 10 minutes. The cell pellet was treated with a hypotonic solution (1 to 2 minutes in 0.56% KCl at 37 C). Cells were then centrifuged and Carnoy s fixative was gently added. Cells were washed by fixative repeated twice and the resulting cells were resuspended in a small volume of fixative solution and dropped onto the clean micro slides. Finally, cells were stained with 10% Giemsa in Sorenson buffer, ph 6.8, for 10 minutes. To determine the frequency of binucleated cells with micronuclei (BN MN) and the total number of MN in lymphocytes, a total of 1000 binucleated cells with well-preserved cytoplasm were scored per subject. In addition, NPBs and N buds in binucleated cells were also counted as reported by Fenech et al. 16 Observation was carried out using a large field light microscope (DMLA, Leica, Germany).

5 Joshi et al. 855 DNA damage by alkaline comet assay. Comet assay was performed on whole blood under alkaline conditions following the procedure of Speit and Hartmann. 17 Ten-microliter blood samples were mixed with 80 µl of 0.5% low melting point agarose (37 C) prepared in Ca 2+ /Mg 2+ -free PBS and pipetted onto a 1% normal melting point agarose precoated slide. After keeping the slide on a chilled plate for 10 minutes, the cover slip was gently removed, and the slides were kept into the freshly prepared cold lysis solution (2.5 M NaCl, 100 mm ethylenediamine tetraacetic acid [EDTA], 10 mm Tris, 10% dimethyl sulfoxide, 1% Triton X-100, ph 10) for 2 hours at 4 C in dark. After draining the lysis solution, the slides were placed in a horizontal electrophoresis tank containing freshly prepared electrophoresis buffer (300 mm NaOH and 1 mm EDTA, ph > 13). The slides were then left in the alkaline buffer solution for 20 minutes to allow the unwinding of DNA before electrophoresis. Electrophoresis was carried out for 30 minutes by applying an electric field of 25 V and adjusting the current to 300 ma. After electrophoresis, the slides were drained, placed on a tray, and flooded slowly with 3 changes of neutralization buffer (0.4 M Tris HCl buffer, ph 7.5) for 5 minutes each and stained with ethidium bromide. Slides were scored using an image analysis system attached to a fluorescence microscope (DMLA, Leica, Germany) equipped with appropriate filters. The microscope was connected to a computer through a charge coupled device camera to transport images. The measurement (tail length; % tail DNA; tail moment [TM]; and olive tail moment [OTM]) was performed using public domain PC image analysis program CASP software. 18 Statistical Analysis All statistical analyses were performed using SPSS software (version 16.0). The mean values of the data of chewers, subjects with OSMF, and nonchewers were compared using Analysis of covariance by considering age as a covariate followed by Bonferroni adjustment test. To find out the relationship between different continuous parameter, Pearson correlation coefficient analysis was used. For all the comparisons, P <.05 was considered statistically significant. Results Percentage frequency of BN MN, total MN, NPB, and N buds in nonchewers, chewers, and subjects with OSMF are summarized in Table 2. One-way analysis of covariance revealed that the chewing has significant positive role on levels of BN MN (P =.001), total MN (P =.043), and NPB (P =.006). Games Howell post hoc test revealed that average percentile of BN MN for the OSMF group was significantly higher as compared with nonchewers and chewers. Elevation in BN MN was also higher but statistically nonsignificant among chewers with respect to nonchewers. Also, total MN in the OSMF group was significantly higher (P =.037) than in the nonchewers group, but there was no difference between nonchewers and chewers. Moreover, the NPB was higher in both subjects with OSMF and in chewers but it was statistically significant only in chewers (P =.004) with respect to nonchewers. The single-stranded DNA damage in individual cells is shown in Figures 1A to 1D. One-way analysis of covariance revealed that chewing has the significant positive role in the tail DNA, TM, and OTM among the study groups. Furthermore, the multiple comparisons test Games Howell post hoc test was applied to check the significance between the groups. It was observed that tail DNA (7.596 ± 0.774), TM ( ± 1.846), and OTM (6.458 ± 1.160) were significantly higher among chewers than among nonchewers (tail DNA [4.451 ± 0.699, P =.010], TM [6.056 ± 1.666, P =.007], and OTM [2.944 ± 0.746, P =.006]). Furthermore, tail DNA, TM, and OTM were also higher among subjects with OSMF with respect to nonchewers, but the difference did not reach statistical significance. This might be because of the lower number of subjects

6 856 Asia-Pacific Journal of Public Health 23(6) Table 2. Age-Adjusted Frequency of BN MN, MN, NPB, and N buds in nonchewers, chewers, and OSMF Subjects a Nonchewers Chewers OSMF Subjects F P BN MN b ± ± ± c,d Total MN e ± ± ± c NPB ± ± c ± N buds ± ± ± Abbreviations: BN MN, binucleated cells with micronuclei; MN, total micronucleui; NPB, nucleoplasmic bridge; N buds, nuclear buds; OSMF, oral submucous fibrosis. a Values are presented as mean ± standard error. b Total counts of MN in 1000 binucleated cells. c Values compared with nonchewers. d Values compared with chewers. e Total count of MN in binucleated and multinucleated cells. Figure 1. A. % Tail DNA; amount of DNA in the comet tail (pixels); B. Tail length (pixels); C. Tail moment (Tail DNA% x tail length) (arbitrary units); D. Olive Tail Moment ( Tail DNA% x distance between the center of the tail) (arbitrary units); (a) p = (b) p = (c) p = Chewers compared with non-chewers

7 Joshi et al. 857 Table 3. Correlation Between the Percentage BN MN and OTM as Well as Their Relationship With the Chewing Frequency per Day and Duration in Years OTM Chewing Frequency (Quids/Day) Duration (Years) BN MN % r a.121 P OTM r P Abbreviations: BN MN, binucleated cells with micronuclei; OTM, olive tail moment. a Correlation is significant at the.05 level (2-tailed analysis). as compared with chewers. The tail length increased nonsignificantly in both chewers and in subjects with OSMF. A positive correlation (r =.145, P =.146) was observed between frequency of MN and NPBs. OTM and BN MN were positively correlated (r =.118, P =.229). A significant positive correlation was found between chewing frequency (quids per day) with BN MN (r =.280, P =.033), and positive (nonsignificant) relationship between chewing duration and BN MN (r =.120, P =.367; Table 3). However, age did not show relationship with micronucleus frequency and comet value. Discussion The present study describes the comparison of micronuclei frequencies and DNA damage in chewers of mixture of areca nut and tobacco, subjects with OSMF, and nonchewers. It had been indicated that DNA damage is a prerequisite for development of malignancies as well as increase in damage points to reduced repair capacity is a risk for progression. 19 The results of this comprehensive cross-sectional study provide the evidence that areca nut and tobacco are responsible for the increased micronuclei and DNA damage among the chewers of mixture containing these ingredients. The study also indicated that all the subjects with OSMF were chewers of mixture containing areca nut with tobacco indicating the role of chewing these in OSMF and also risk for further progression of the disease. Earlier, on the basis of house-to-house survey, Ali et al 20 had suggested that occurrence of oral precancerous lesions is related to betel quid chewing. Ariyawardana et al 21 had also shown a strong association of betel quid chewing (including tobacco as an ingredient) with the causation of OSMF. OSMF is a high-risk precancerous condition 22 with a malignant transformation rate of about 7.6%. 23 Earlier, it was also suggested that areca nut chewing could be one of the most important etiologic factors in OSMF. 24 A prospective study had shown that MN in peripheral blood lymphocytes is a valid biomarker for predicting an increased cancer risk in humans. 25 Milić et al 26 investigated chromosomal damage in workers occupationally exposed to tobacco dust and indicated that the micronucleus frequency by CBMN and sister chromatid exchange were more reliable indicators of genome damage in monitoring chronically exposed subjects. The CBMN assay is a genotoxicity assay, which can be used to detect a variety of chromosomal damage endpoints that reflect chromosomal breakage, chromosome rearrangements, and gene amplification. The frequency of NPBs was significantly higher in chewers as compared with nonchewers, whereas MN were significantly higher in chewers and in subjects with OSMF as compared with nonchewers, which may

8 858 Asia-Pacific Journal of Public Health 23(6) suggest a higher level of genetic instability among the chewers. Earlier, it has been reported that frequencies of sister chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and micronucleated cells in exfoliated buccal mucosa were significantly elevated in tobacco areca nut chewers and subjects with OSMF. 27 Earlier experimental study from this laboratory also observed increased frequency of MN in bone marrow cells of mice exposed to pan masala plain and pan masala gutkha. 28 Bonassi et al 29 also observed that heavy smokers group showed a significant increase in genotoxic damage as measured by the micronucleus assay (CBMN) in lymphocytes. It has been shown that the comet assay can be used as a cost-effective biomonitoring tool to assess damage and identify the individuals at risk for a progressive pathology and even to malignancy. 19 The comet assay further revealed a statistically significant increase in the level of DNA damage among chewers as compared with the nonchewers. Sardas et al 30 had studied the genotoxicity in the peripheral lymphocytes of maras powder (smokeless tobacco) users, cigarette smokers, and nonsmokers using comet assay. It was found that the oral use of smokeless tobacco has the potential to cause genotoxic hazard, which is even higher than the DNA damage observed in cigarette smokers. 30 A positive correlation was found between total MN with TM and total MN with OTM, and also significant correlation was found between MN and consumption in terms of frequency per day. Positive correlation was also found between the BN MN and the OTM. This shows that chewing areca nut and tobacco induces DNA damage in lymphocyte of chewers. It has also been suggested in a recent study that cyto-genotoxicity of cigarette smoke condensates from different brands of cigarettes varied greatly and comet assay might be a sensitive assay in assessing the genotoxicity induced by cigarette smoke condensates. 31 In conclusion, an increased MN frequency and DNA damage was observed among the chewers, which might be associated with greater risk of precancerous lesions, that is, OSMF. Also the CBMN assay and comet assay can serve as early biomarkers to study the DNA damage induced by mixture containing areca nut and tobacco. Further studies are needed to look for the genetic susceptibility of the individuals leading to the oral cancers as fraction of chewers develops cancer, because areca nut and tobacco chewing is the most prevalent habit in Southeast Asian countries and also oral cancer in this region. Thus, it is necessary to adopt the information education communication strategy to spread awareness about the ill effects of tobacco and areca nut in the community. Acknowledgments Authors are thankful to the Director, National Institute of Occupational Health, for the encouragement. Authors are also thankful to Dr Shyam Agarwal and Dr Priyanka Patel for their assistance. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The financial support from the Indian Council of Medical Research, New Delhi, India in the form of a research grant to SK is gratefully acknowledged. References 1. Panchamukhi PR, Woolery T, Nayantara SN. Economics of bidis in India. In: Gupta PC, Asma S, eds. Bidi Smoking and Public Health. New Delhi, India: Ministry of Health and Family Welfare, Government of India; 2008:

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