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Cytogenetic surveillance of tobacco-areca nut (mava) chewers, including patients with oral cancers and premalignant conditions
41
Mutation Research, 261 (1991) 41-49 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100129R
MUTGEN 01689
Cytogenetic surveillance of tobacco-areca nut (mava) chewers, including patients with oral cancers and premalignant conditions Siddharth G. Adhvaryu, Bhavana J. Dave and Amit H. Trivedi Cell Biology Division, Department of Cancer Biology, Gujarat Cancer and Research Institute, Ahmedabad - 380 016 (India) (Received 3 September 1990) (Revision received 12 March 1991) (Accepted 14 March 1991)
Keywords: Tobacco plus areca nut (mava) chewing; Sister-chromatid exchange; Chromosome aberration; Micronucleus; Human lymphocytes; Buccal mucosa cells
Summary Three cytogenetic endpoints were studied in non-chewing healthy controls and 3 groups of tobaccoareca nut chewers, viz. normal chewers, chewers with oral submucous fibrosis and chewers with oral cancer. Frequencies of sister-chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and of micronucleated cells in exfoliated buccal mucosa were evaluated. All the parameters showed statistically significant elevations in all 3 groups of chewers compared to the controls. The data indicate possible application of the parameters as sensitive endpoints for monitoring tobacco-areca nut chewers, the group of individuals at higher risk of developing oral cancer, the commonest cancer among Indian males.
Consuming tobacco is the major avoidable cause of human morbidity and mortality. A number of epidemiological studies have established a close correlation between the consumption of tobacco in various forms and a higher incidence of site-specific cancers (Jussawala and Deshpande, 1971; Sanghvi, 1981). The ill effects of smoking have been studied in great depth, however, tobacco is carcinogenic irrespective of the mode of its consumption. As a result of these findings, coupled with reported harmful effects of passive smoking, a strong public opinion against tobacco
Correspondence: Dr. Siddharth G. Adhvaryu, Gujarat Cancer and Research Institute, Ahmedabad - 380 016 (India).
smoking has been generated. This has partly culminated in the increasing acceptance of smokeless tobacco, under the pretext of being a safer alternative (Chassin et al., 1985; Schaefer et al., 1985). Chewing of tobacco as an ingredient of betel quid (areca nut, tobacco, catechu, lime and some flavouring ingredients wrapped in betel leaf (Piper betel Linn.)), is a widespread habit in India and South-East Asia. This habit has been held responsible for oral cavity cancers, which form almost one third of total cancer cases in India (WHO, 1984). In Gujarat, a part of Western India, a mixture of sun-cured unflavoured tobacco, dried pieces of areca nut and lime (locally known as maya), which does not contain betel
17 M 19M 23 M 23F 24M 24F 25 M 25M 26M 28 F 35 M 49 F 55F 58F 60 F
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+0.325 +0.319 +0.411 +_0.400 +0.550 +_0.380 +_0.315 +_0.465 +_0.298 + 0.420 +0.510 +- 0.387 +-0.346 +_0.228 +_0.207
6.185 +- 0.088
6.04 6.35 5.66 6.46 6.31 5.86 6.63 6.28 5.70 6.19 6.36 6.95 6.06 5.79 6.14
Controls 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Subject 21 M 24M 25 M 26M 27M 33M 34 M 35M 37M 37 M 40 M 46 F 50M 56M 56 M
Age/Sex +0.453 +_0.516 +0.432 +_0.448 +_0.228 _+0.375 +_0.286 +_0.585 +_0.503 +_0.584 +-0.405 ± 0.408 +-0.288 +_0.430 +_0.549
7.219 +- 0.22• a
9.63 7.51 6.24 5.86 6.80 6.65 7.28 7.13 7.57 7.33 7.28 6.54 6.69 7.95 7.83
NC 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Subject
NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients. a p <~0.001 compared to controls. b p < 0.001 compared to NC. c p < 0.01 compared to OSMF.
Mean
Age/Sex
Subject 17 M 20M 21 M 23M 24M 24M 28 M 28M 29M 32 M 33 M 36 M 37M 43M 60 F
Age/Sex
_+0.568 +_0.399 +_0.345 +0.335 +_0.319 +-0.264 +_0.315 +_0.400 +_0.375 _+0.377 +0.249 +-0.287 +-0.478 +_0.504 +0.392 7.617 ± O. 183 a
8.38 6.21 6.55 7.53 8.35 7.33 7.62 7.44 8.11 8.07 6.66 7.36 7.75 8.87 8.02
OSMF
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Subject
22 M 23M 34 M 34M 35M 40F 40 F 45M 50M 50 F 50 F 50 F 55M 64M 70 M
Age/Sex
_+0.384 + 0.369 _+0.477 + 0.487 _+0.233 +0.553 +0.501 + 0.276 + 0.247 +0.431 +- 0.405 + 0.388 _+0.412 + 0.542 +- 0.426 8.500 + 0.248 a,b,c
7.45 8.05 9.61 8.62 6.77 9.99 9.18 8.34 7.44 8,82 7.96 7.73 8.62 10.36 8.56
OC
I N D I V I D U A L F R E Q U E N C I E S O F S I S T E R - C H R O M A T I D E X C H A N G E S (SCE) P E R C E L L ( M E A N +_SE) IN B L O O D L Y M P H O C Y T E S O F T O B A C C O - A R E C A N U T (MAVA) C H E W E R S A N D C O N T R O L P E R S O N S
TABLE 1
#0
43 leaf or any other ingredient, is chewed very commonly. It is a mixture of ingredients well known for their carcinogenic/cocarcinogenic properties. The problem of oral cancers associated with tobacco-areca nut chewing is likely to assume very serious dimensions, as it is gaining acceptance among adolescents and women. Equally alarming is a recent report showing that genotoxic effects of betel quid chewing were more pronounced in pregnant women (Ghosh and Ghosh, 1988). These facts present an urgent need of evolving parameters for monitoring the genotoxic hazards of the tobacco-areca nut chewing habit. Cytogenetic endpoints, such as sister-chromatid exchange (SCE), chromosome aberration (CA) and micronucleated cell (MNC) frequencies, have been advocated as sensitive parameters for assessing genotoxic effects of chemical or physical mutagens. When employed in combination, the data collected would provide more comprehensive information (Carrano and Natarajan, 1988; Gebhart, 1981; Hsu, 1982; Stich and Rosin, 1984). We have therefore selected scoring of CA and SCE frequencies in peripheral blood lymphocytes (PBLs) and scoring of MNC frequencies in exfoliated buccal mucosa cells of 4 different groups of individuals. Such an experimental design was expected to provide useful information about possible genotoxic effects of the tobaccoareca nut (mava) chewing habit on target as well as non-target tissues. Materials and methods
Selection of subjects Altogether we studied 60 non-smoking teetotalers, including 45 chewing a mixture of tobacco, areca nut and lime only (hereafter referred as chewers) and 15 healthy non-chewing individuals as controls. The 45 chewers were further classifiable as (1) normal chewers (NC) without any clinically detectable change in oral mucosa, (2) chewers suffering from oral submucous fibrosis (OSMF), an oral premalignant condition and (3) chewers with oral cancers (OC), squamous cell carcinoma of the oral cavity. All 3 groups of chewers include individuals who chewed a mixture of tobacco, areca nut and lime (locally known as mava, which is not a betel quid) with compara-
ble frequency and duration (average 6 chews/ day). An equal number of individuals, who had never consumed tobacco or areca nut in any form, had had no viral disease or antibiotic therapy during the last 6 months and who had normal oral mucosa, were included as controls. The OSMF patients and OC patients were histologically confirmed cases, however, blood samples were collected before any anticancer therapy was started. Informed consent was obtained from all the individuals studied before sample collection. Blood collection and culture were done in 9 batches. The controls and the normal chewers were pre-identified from the staff members of our hospital and adjoining institutions. Whenever appropriate OC a n d / o r OSMF patients were available, the sampling was done on the same day and cultures were set with at least one control and one normal chewer sample.
Culture procedure PBL culture procedures were essentially the same as detailed earlier (Adhvaryu et al., 1985). Briefly, aseptically collected heparinised whole blood (1.0 ml) was added to 7.0 ml growth medium comprised of 80% MEM (Earle's base and nonessential amino acids) and 20% newborn calf serum. PHA-M at 3% and 5-bromodeoxyuridine (BrdU) at 2.0 /~g/ml final concentration were added. Following colchicine treatment (0.3 # g / m l ) for the final 3 h, cultures were harvested at 72 h after the usual hypotonic (0.56% KCI) and fixation (3 : 1 methanol : acetic acid) protocol. Air-dried slides were stained by the fluorescence plus Giemsa (FPG) method for sister-chromatid differential staining which helped to identify ceils in the M1, M2 and M3 phases of cell division. A minimum of 100 well-spread metaphase cells in M1 phase were scored per sample for CA analysis. The identification of CA was carried out as recommended by WHO (1985). 25 metaphase ceils in M2 phase were counted per sample for SCE scoring.
Cytological preparations and micronucleus staining procedure After cleaning the mouth thoroughly by repeated gargling with drinking water, the cheek mucosa was scraped with a blunt spatula and
44
smears of exfoliated buccal mucosa cells were prepared on clean glass slides. Cells were fixed with 1:3 acetic acid:methanol fixative for 15 min. The cells were stained using the Feulgen plus fast green method with minor modifications. Briefly, after hydrolysis with 5 N HC1 for 20 min at room temperature, cells were stained with Schiff's reagent for 2 h. Following 3 changes of SO 2 water, the cells were counterstained with 0.5% alcoholic fast green, dried and mounted with DPX. A minimum of 1000 cells was screened from each individual for examining the presence of micronuclei. Micronucleus identification was done according to the criteria detailed by Sarto et al. (1987). The assay was not carried out for oral cancer patients as it was not feasible to obtain scorable smears due to poor hygienic condition of the oral cavity and because of abundant necrotic cells in the preparations. For all 3 parameters studied, slides were coded and read blindly by 2 microscopists. The person responsible for coding distributed the slides in such a way that both microscopists had almost comparable numbers of controls and patients in each group. Results for the 4 groups examined, were compared with each other. Student's t-test was applied to check the statistical significance of the results. Results
Table 1 provides mean lymphocytic S C E / c e l l values for all 4 groups studied. For controls the mean S C E / c e l l value was 6.185 + 0.088 (mean + SE), whereas the values were 7.219 + 0.221, 7.617 + 0.183 and 8.500 + 0.248 for NC, OSMF patients and OC patients, respectively. All 3 groups of tobacco-areca nut (maya) chewers had a statistically significant elevation in mean S C E / c e l l values compared to the controls ( p < 0.001), who did not consume tobacco or areca nut in any form. Among the chewers, OSMF patients had a marginally elevated mean frequency compared to NC, whereas OC patients had a statistically significant elevation in mean SCE value compared to both NC ( p < 0.001) and OSMF patients ( p < 0.01). The metaphases scored for SCEs were further separated according to the number of SCEs
- -
CONTROLS
m
15
J
k NC
15
Vk OSMF
O"
0C 15
5
. . . .
"~0 .
.
.
.
.
15"
Fig. 1. Percentage distribution of metaphases according to the number of SCEs. NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients.
(Fig. 1). As visualised in the figure, with the chewing habit and progression from normal to oral cancer, a shift is observed towards a greater number of cells with higher SCEs, thus resulting in higher mean SCE frequencies. Details of lymphocytic CA analysis are presented in Tables 2 and 3. The C A / c e l l values varied from 0.03 to 0.08 in controls, providing a mean of 0.050 + 0.004 (mean + SE). Among the chewers, the C A / c e l l frequencies varied from 0.06 to 0.16 in NC giving a mean value of 0.097 + 0.007. For OSMF patients, the values ranged from 0.08 to 0.20 yielding a mean value of 0.127
45
+ 0.009 and in OC patients CA values from 0.09 to 0.20 resulted in a mean of 0.144 + 0.009. The increase in CA frequency (including gaps) in all 3 groups of chewers was statistically significant ( p < 0.001) when compared with controls. The difference between CA frequencies of NC and OSMF patients ( p < 0.02) as well as NC and OC patients was statistically significant ( p < 0.001). Chromatid aberrations, the majority of which were gaps, were more frequent than chromosome-type aberrations. Interchanges were not observed in controls, but were found in all 3 groups of chewers. Even after excluding gaps from aberrations, elevations in C A / c e l l value in all 3 groups
of chewers were significant when compared to the controls (Table 3). Table 4 details the frequency of percent MNC observed in the smeared exfoliated buccal mucosa ceils of different individuals. NC and OSMF patients had 0.74% and 0.75% MNC, respectively. These elevations in the frequency of MNC compared to the control value of 0.19% MNC were statistically significant ( p < 0.001).
Discussion In India tobacco-areca nut chewing is very popular and has been strongly implicated in the
TABLE 2 I N D I V I D U A L F R E Q U E N C I E S OF C H R O M O S O M E A B E R R A T I O N S P E R C E L L ( M E A N + S E ) IN B L O O D L Y M P H O CYTES O F T O B A C C O - A R E C A N U T (MAVA) C H E W E R S A N D C O N T R O L P E R S O N S Subject
Controls
Subject
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.04 0.04 0.04 0.05 0.03 0.03 0.05 0.05 0.06 0.06 0.05 0.04 0.07 0.08 0.06
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Mean
0.050_+0.004
+0.0196 _+0.0196 -+0.0196 _+0.0218 _+0.0171 -+0.0171 _+0.0218 _+0.0218 -+0.0276 _+0.0238 _+0.0218 _+0.0196 -+0.0292 +0.0306 -+0.0238
NC 0.08 0.09 0.10 0.06 0.16 0.09 0.08 0.08 0.05 0.09 0.12 0.13 0.14 0.11 0.08
Subject +0.0351 _+0.0349 _+0.0300 _+0.0237 +0.0441 -+0.0286 _+0.0306 _+0.0271 -+0.0218 -+0.0349 _+0.0354 +0.0429 _+0.0401 -+0.0343 -+0.0271
OSMF
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
0.15 0.13 0.08 0.10 0.16 0.11 0.13 0.14 0.09 0.14 0.19 0.11 0.20 0.08 0.10
* 0.097_+0.007 a
Subject _+0.0384 _+0.0448 _+0.0271 _+0.0300 -+0.0504 -+0.0467 _+0.0391 +0.0400 _+0.0349 -+0.0400 -+0.0463 -+0.0313 _+0.0469 -+0.0271 -+0.0300
OC
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
** 0.127-+0.009 a.b
0.12 0.20 0.15 0.10 0.10 0.16 0.12 0.14 0.09 0.14 0.19 0.20 0.13 0.18 0.14
_+0.0382 +0.0490 _+0.0528 _+0.0360 +0.0332 -+0.0484 _+0.0354 -+0.0566 +0.0286 _+0.0375 -+0.0468 -+0.0658 _+0.0447 +0.0477 -+0.0422
* 0.144-+0.009 a,c
NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients. P values including gaps: a p < 0.001 compared to controls; b p < 0.02 compared to NC; c p < 0.001 compared to NC. P values excluding gaps: * p < 0.01 compared to controls; ** p < 0.001 compared to controls. TABLE 3 N U M B E R O F D I F F E R E N T TYPES O F C H R O M O S O M E A B E R R A T I O N S P E R 100 CELLS IN B L O O D L Y M P H O C Y T E S O F T O B A C C O - A R E C A N U T (MAVA) C H E W E R S A N D C O N T R O L P E R S O N S Group
Controls NC OSMF OC
Total aberrations
Chromatid-type G
B
I
Chromosome-type G
B
Dm
5.0 9.7 12.7 14.4
3.7 6.4 8.0 10.6
0.9 2.2 3.0 2.5
0.1 0.1 0.1
0.3 1.1 1.5 0.8
0.1 0.1 0.1
0.1 0.2
D
R
NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients. G, gap; B, break; I, interchange; Dm, double minute; D, dicentric; R, ring.
46 TABLE 4 INDIVIDUAL FREQUENCIES (%) OF MICRONUCLEATED BUCCAL MUCOSA CELLS AMONG TOBACCOARECA NUT (MAVA) CHEWERS AND CONTROL PERSONS Subject Controls Subject NC 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.2 0.3 0.3 0.1 0.1 0.2 0.3 0.1 0.3 0.3 0.1 0.1 0.2 0.2 0.1
Mean _+SE
0.193 +0.022
1
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
0.8 1.2 0.4 1.1 0.5 0.9 0.7 0.9 0.5 0.7 0.6 0.7 1.0 0.8 0.3 0.740 +0.064 *
Subject OSMF 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
0.8 0.7 0.3 1.8 0.4 0.2 0.8 1.1 0.6 1.0 0.7 0.5 0.8 0.6 1.0 0.753 +0.097 *
NC, normal chewers; OSMF, oral submucous fibrosis patients. * p < 0.001 compared to controls. causation of oral premalignant and malignant diseases. Considerable interindividual variations have been observed regarding dose, duration of chewing mava and occurrence of the disease. Hence, a great deal of human morbidity and mortality can be prevented by watchful monitoring. In the present study, it was attempted to explore the possible utility of a combination of 3 cytogenetic endpoints in assessing the extent of genomic damage caused by the habit of mava chewing on target as well as non-target tissues. Previously we reported elevated lymphocytic SCEs in normal chewers and chewers suffering from OSMF (Adhvaryu et al., 1986). Ghosh and Ghosh (1984, 1988) reported similar results among betel-quid chewers. Most striking in their studies was the SCE elevation in pregnant women. Stich et al. (1982a, b) also studied chewers from India and reported a significant elevation of MNC frequencies in exfoliated buccal mucosa cells. They attributed this to the genotoxic effects of tobacco a n d / o r areca nut alkaloids. Arecoline, the major areca nut alkaloid, and areca nut extracts have
been reported to increase SCE and CA frequencies in animal studies (Panigrahi and Rao, 1982, 1986) as well as in the in vitro system (Stich et al., 1981, 1983). Though nicotine, which is a major tobacco alkaloid, is by itself a weak clastogen, it induces SCEs in C H O cells (Riebe and Westphal, 1983). Many of the tobacco-areca nut-specific nitrosamines are detected in the saliva of chewers (Nair et al., 1985; Wenke et al., 1984; Sipahimalani et al., 1984; Prokopczyk et al., 1987) and some of these are potent mutagens and carcinogens (IARC, 1985). SCE elevations have also been documented in PBLs of smokers (Sarto et al., 1985; Husum et al., 1982; Ardito et al., 1980). The SCE elevations observed in the present study are thus in accordance with the reports of others. However, in an ongoing work, individuals consuming only areca nut also exhibited higher lymphocytic SCEs than controls. The subjects included in the present study consumed a mixture of tobacco and areca nut. Since alkaloids of both these substances also exert genotoxic effects individually, both of them should be considered responsible for the elevated SCE frequencies in mava chewers. To the best of our knowledge, CAs have not been reported previously for tobacco a n d / o r areca nut chewers. The lymphocytic CA rates were significantly higher in all 3 groups of chewers than in the controls. This increase can also be explained on the basis of the clastogenic potential of tobacco and areca nut alkaloids. However, since increases in SCE and CA frequencies have been recorded in different premalignant and malignant conditions (Adhvaryu et al., 1986, 1988a, b; Murty et al., 1985, 1986), the elevations in SCEs and CAs among OSMF patients and OC patients may be due to the habit, the occurrence of the disease, or a combination of both. In the present study, the OC patients were older (mean age 44.1 years) than the other groups (mean ages 32.7, 36.5 and 30.3 years for controls, NC and OSMF patients, respectively). Reports regarding the effect of age and sex on the frequencies of SCEs and CAs are contradictory (Carrano and Moore, 1982; Livingstone et al., 1983; Husum et al., 1986; Margolin and Shelby, 1985; Steenland et al., 1986). Here, due to the small sample size in each group, the evaluation of
47 the effect of these confounding factors will not be meaningful. However, it was observed during previous studies in our laboratory that age and sex have no influence on the frequency of the cytogenetic endpoints in question. Scoring of MNC in exfoliated oral mucosa ceils is technically less demanding than scoring of SCE and CA frequencies, but this simple assay is flawed with several shortcomings. Most important is the difficulty in obtaining scorable smears from most OC patients. This is because of ulcerated areas in the oral cavity resulting in poorer oral hygiene. Secondly, different laboratories utilising this parameter have employed different criteria for MN scoring, making interlaboratory comparisons difficult. Stich et al. (1982b) have reported elevated frequencies of MNC in the buccal mucosa cells of tobacco-areca nut chewers, but they did not observe any such elevation in smears obtained from smokers and those consuming alcohol or both (Stich and Rosin, 1983). Similarly, Mandard et al. (1987) also reported a negative MN test in patients with upper digestive tract cancer and controls, both consuming alcohol and tobacco. Sarto et al. (1987) proposed more stringent criteria for MN scoring and reported a highly positive MN test in smokers. We have followed the criteria suggested by them. The frequencies of MNC were almost 4 times higher in NC and OSMF patients. As already stated, the MN assay was not conducted in OC patients; however, considering the reports of Stich et al. on tobaccoareca nut chewers and that of Sarto et al. o n smokers, the validity of the MN test, being noninvasive and less demanding, is very high. All 3 parameters studied, viz. frequency of lymphocytic SCE and CA and MNC in exfoliated oral mucosa cells, showed statistically significant elevations in all 3 groups of tobacco-areca nut (mava) chewers compared to controls. Considering the commonness of the chewing habit, the number of 45 chewers (15 each, in 3 groups) may sound insufficient; however, it should be emphasised here that the individuals in all 3 groups (1) chewed the mixture with comparable f r e q u e n c y / duration, (2) had no concomitant habit of smoking or alcohol consumption and (3) were not engaged in any occupation which might affect the parameters studied. Chewing tobacco has been
causally associated with oral cancers; however, looking to the elevated frequencies of CA and SCE in PBLs, the possibility of damage caused to other systems/tissues should also be considered, as is the case with smoking which causes lung cancer but also elevates the risk of cancer at other sites like the bladder, pancreas etc. SCE frequencies were higher in OC patients compared to NC and OSMF patients, whereas CA frequencies were higher in OSMF patients compared to NC. It is recommended that, when studying individuals exposed to potential genotoxins, both parameters be employed in combination. Further, the MN assay, being less demanding and providing vital information about the genotoxic effect on the target tissue, would prove to be most useful for monitoring tobacco consumers, not only in estimating the extent of genomic damage caused by the habit, but also in appraisal of the advantages of either quitting the habit or, as already documented by Stich et al. (1984, 1985), for assessing the benefits of chemopreventive measures.
Acknowledgements The authors are thankful to Dr. N.L. Patel, The Director, Gujarat Cancer and Research Institute and to the Head, Department of Cancer Biology, for the facilities provided. Dr. P.K. Dayal, Government Dental College and Hospital, and staff members of Department of Surgical Oncology, Gujarat Cancer and Research Institute, are gratefully acknowledged for their interest and cooperation.
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48 Balar (1988b) Increased frequency of sister chromatid exchanges in lymphocytes of breast cancer patients, Int. J. Cancer, 41,394-398. Ardito, G., L. Lamberti, E. Ansaldi and P. Ponzetto (1980) Sister chromatid exchanges in cigarette-smoking human females and their newborns, Mutation Res., 78, 209-212. Carrano, A.V., and D.H. Moore II (1982) The rationale and methodology for quantifying sister chromatid exchange in humans, in: J.A. Heddle (Ed.), Mutagenicity: New Horizons in Genetic Toxicology, Academic Press, New York, pp. 267-304. Carrano, A.V., and A.T. Natarajan (1988) Considerations for population monitoring using cytogenetic techniques, Mutation Res., 204, 379-406. Chassin, L., C. Presson, S.J. Sherman, L. McLaughlin and D. Gioia (1985) Psychological correlates of adolescent smokeless tobacco use, Addict. Behav., 10, 431-435. Gebhart, E. (1981) Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing, Hum. Genet., 58, 235-254. Ghosh, P.K., and R. Ghosh (1988) Effect of betel chewing on the frequency of sister chromatid exchanges in pregnant women and women using oral contraceptives, Cancer Genet. Cytogenet., 32, 211-215. Ghosh, R., and P.K. Ghosh (1984) Sister chromatid exchanges in betel and tobacco chewers, Mutation Res., 139, 79-81. Hedner, K., B. Hogstedt, A.-M. Kolnig, E. Mark-Vendel, B. Strombeck and F. Mitelman (1982) Sister chromatid exchange and structural chromosome aberrations in relation to age and sex, Hum. Genet., 62, 305-309. Hsu, T.C. (1982) Introduction, in: T.C. Hsu (Ed.), Cytogenetic Assays of Environmental Mutagens, Allanheld Osmun, Totowa, N J, pp. 1-9. Husum, B., H.C. Wulf and E. Niebuhr (1982) Increased sister chromatid exchange frequency in lymphocytes in healthy cigarette smokers, Hereditas, 96, 85-88. Husum, B., H.C. Wulf and E. Niebuhr (1986) Sister chromatid exchange frequency correlates with age, sex and cigarette smoking in a 5-year material of 533 healthy adults, Hereditas, 105, 17-21. International Agency for Research on Cancer (1985) Tobacco habits other than smoking; betel-quid and areca-nut chewing; and some related nitrosamines, IARC Monogr., 37, 205-267. Jussawala, D.J., and V.A. Deshpande (1971) Evaluation of cancer risk in tobacco chewers and smokers: an epidemiological assessment, Cancer, 28, 244-252. Livingston, G.K., L.A. Cannon, D.T. Bishop, P. Johnson and R.M. Fineman (1983) Sister chromatid exchange: variation of age, sex, smoking and breast cancer status, Cancer Genet. Cytogenet., 8, 289-299. Mandard, A.M., F. Duigou, J. Marnay, P.H. Masson, QuiSong-Lang, Yi-Jing-Shu, P. Barrellier and G. Lebigot (1987) Analysis of the results of the micronucleus test in patients presenting upper digestive tract cancers and in non-cancerous subjects, Int. J. Cancer, 39, 442-444. Margolin, B.H., and M.D. Shelby (1985) Sister chromatid exchanges: a reexamination of the evidence for sex and
race differences in humans, Environ. Mutagen., 7, Suppl. 4, 63-72. Murty, V.V.V.S., A.B. Mitra and U.K. Luthra (1985) Spontaneous chromosomal aberrations in patients with precancerous and cancerous lesions of the cervix uteri, Cancer Genet. Cytogenet., 17, 347-354. Murty, V.V.V.S., A.B. Mitra, U.K. Luthra and I.P. Singh (1986) Sister chromatid exchanges in patients with precancerous and cancerous lesions of cervix uteri, Hum. Genet., 72, 37-42. Nair, J., H. Ohshima, M. Freisen, A. Croisy, S.V. Bhide and H. Bartsch (1985) Tobacco-specific and betel nut-specific N-nitroso compounds: occurrence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid, Carcinogenesis, 6, 295-303. Panigrahi, G.B., and A.R. Rao (1982) Chromosome breaking ability of arecoline, a major betel nut alkaloid, in mouse bone-marrow cells in vivo, Mutation Res., 103, 197-204. Panigrahi, G.B., and A.R. Rao (1986) Study of the genotoxicity of the total aqueous extract of betel nut and its tannin, Carcinogenesis, 7, 37-39. Prokopczyk, K.B., A. Rivension, P. Bertinato, K.D. Brunnemann and D. Hoffmann (1987) 3-(Methylnitrosamino)-propionitrile: occurrence in saliva of betel quid chewers; carcinogenicity and DNA methylation in F344 rats, Cancer Res., 47, 67-71. Riebe, M., and K. Westphal (1983) Studies on the induction of sister chromatid exchanges in Chinese hamster ovary cells by various tobacco alkaloids, Mutation Res., 124, 281-286. Sanghvi, L.D. (1981) Cancer epidemiology; the Indian scene, J. Cancer Res. Clin. Oncol., 91, 1-14. Sarto, F., M. Faccioli, L. Cominato and A.G. Levis (1985) Aging and smoking increase the frequency of sister chromatid exchanges (SCE) in man, Mutation Res., 144, 183187. Sarto, F., S. Finotto, L. Giacomelli, D. Mazzotti, R. Tomanin and A.G. Levis (1987) The micronucleus assay in exfoliated cells of the human buccal mucosa, Mutagenesis, 2, 11-17. Schaefer, S.D., A.H. Anderson, E.D. Glover and A.G. Christen (1985) Pattern of use and incidence of smokeless tobacco consumption in school-age children, Arch. Otolaryngol. Head Neck Surg., 111,639-642. Sipahimalani, A.T., M.S. Chadha, S.V. Bhide, A.I. Pratap and J. Nair (1984) Detection of N-nitrosamines in the saliva of habitual chewers of tobacco, Food Chem. Toxicol., 22, 261-264. Stich, H.F., and M.P. Rosin (1983) Quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells, Int. J. Cancer, 31,305-308. Stich, H.F., and M.P. Rosin (1984) Micronuclei in exfoliated human cells as a tool for studies in cancer risk and cancer intervention, Cancer Lett., 22, 241-253. Stich, H.F., W. Stich and P.P.S. Lam (1981) Potentiation of genotoxicity by concurrent application of compounds found in betel quid: arecoline, eugenol, quercetin, chlorogenic
49 acid and Mn 2+, Mutation Res., 90, 355-363. Stich, H.F., J.R. Curtis and B.B. Parida (1982a) Application of the micronucleus test to exfoliated cells of high cancer risk groups: tobacco chewers, Int. J. Cancer, 30, 553-559. Stich, H.F., W. Stich and B.B. Parida (1982b) Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk of oral cancer: betel quid chewers, Cancer Lett., 17, 125-134. Stich, H.F., B.A. Bohm, K. Chatterjee and J. Sailo (1983) The role of saliva-borne mutagens and carcinogens in the etiology of oral and esophageal carcinomas of betel nut and tobacco chewers, in: H.F. Stich (Ed.), Carcinogens and Mutagens in the Environment, Naturally Occurring Compounds: Epidemiology and Distribution, Vol. 3, CRC Press, Boca Raton, FL, pp. 48-58. Stich, H.F., W. Stich, M.P. Rosin and H.O. Vallejera (1984)
Use of micronucleus test to monitor the effect of vitamin A, beta-carotene and canthaxanthin on the buccal mucosa of betel nut/tobacco chewers, Int. J. Cancer, 34, 745-750. Stich, H.F., A.P. Hornby and B.P. Dunn (1985) A pilot beta-carotene intervention trial with Inuits using smokeless tobacco, Int. J. Cancer, 36, 321-327. Wenke, G., K.D. Brunnemann, D. Hoffmann and S.V. Bhide (1984) A study on betel quid carcinogenesis. 4. Analysis of the saliva of betel chewers: a preliminary report, J. Cancer Res. Clin. Oncol., 108, 110-113. WHO (1984) Meeting Report Control of oral cancer in developing countries, Bull. WHO, 62, 817-830. WHO (1985) In vitro cytogenetics and sister chromatid exchange, in: Guide to Short-Term Tests for Detecting Mutagenic and Carcinogenic Chemicals, Environ. Health Criteria 51, World Health Oraganization, Geneva, pp. 57-67.
Mutation Research, 261 (1991) 41-49 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100129R
MUTGEN 01689
Cytogenetic surveillance of tobacco-areca nut (mava) chewers, including patients with oral cancers and premalignant conditions Siddharth G. Adhvaryu, Bhavana J. Dave and Amit H. Trivedi Cell Biology Division, Department of Cancer Biology, Gujarat Cancer and Research Institute, Ahmedabad - 380 016 (India) (Received 3 September 1990) (Revision received 12 March 1991) (Accepted 14 March 1991)
Keywords: Tobacco plus areca nut (mava) chewing; Sister-chromatid exchange; Chromosome aberration; Micronucleus; Human lymphocytes; Buccal mucosa cells
Summary Three cytogenetic endpoints were studied in non-chewing healthy controls and 3 groups of tobaccoareca nut chewers, viz. normal chewers, chewers with oral submucous fibrosis and chewers with oral cancer. Frequencies of sister-chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and of micronucleated cells in exfoliated buccal mucosa were evaluated. All the parameters showed statistically significant elevations in all 3 groups of chewers compared to the controls. The data indicate possible application of the parameters as sensitive endpoints for monitoring tobacco-areca nut chewers, the group of individuals at higher risk of developing oral cancer, the commonest cancer among Indian males.
Consuming tobacco is the major avoidable cause of human morbidity and mortality. A number of epidemiological studies have established a close correlation between the consumption of tobacco in various forms and a higher incidence of site-specific cancers (Jussawala and Deshpande, 1971; Sanghvi, 1981). The ill effects of smoking have been studied in great depth, however, tobacco is carcinogenic irrespective of the mode of its consumption. As a result of these findings, coupled with reported harmful effects of passive smoking, a strong public opinion against tobacco
Correspondence: Dr. Siddharth G. Adhvaryu, Gujarat Cancer and Research Institute, Ahmedabad - 380 016 (India).
smoking has been generated. This has partly culminated in the increasing acceptance of smokeless tobacco, under the pretext of being a safer alternative (Chassin et al., 1985; Schaefer et al., 1985). Chewing of tobacco as an ingredient of betel quid (areca nut, tobacco, catechu, lime and some flavouring ingredients wrapped in betel leaf (Piper betel Linn.)), is a widespread habit in India and South-East Asia. This habit has been held responsible for oral cavity cancers, which form almost one third of total cancer cases in India (WHO, 1984). In Gujarat, a part of Western India, a mixture of sun-cured unflavoured tobacco, dried pieces of areca nut and lime (locally known as maya), which does not contain betel
17 M 19M 23 M 23F 24M 24F 25 M 25M 26M 28 F 35 M 49 F 55F 58F 60 F
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
+0.325 +0.319 +0.411 +_0.400 +0.550 +_0.380 +_0.315 +_0.465 +_0.298 + 0.420 +0.510 +- 0.387 +-0.346 +_0.228 +_0.207
6.185 +- 0.088
6.04 6.35 5.66 6.46 6.31 5.86 6.63 6.28 5.70 6.19 6.36 6.95 6.06 5.79 6.14
Controls 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Subject 21 M 24M 25 M 26M 27M 33M 34 M 35M 37M 37 M 40 M 46 F 50M 56M 56 M
Age/Sex +0.453 +_0.516 +0.432 +_0.448 +_0.228 _+0.375 +_0.286 +_0.585 +_0.503 +_0.584 +-0.405 ± 0.408 +-0.288 +_0.430 +_0.549
7.219 +- 0.22• a
9.63 7.51 6.24 5.86 6.80 6.65 7.28 7.13 7.57 7.33 7.28 6.54 6.69 7.95 7.83
NC 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Subject
NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients. a p <~0.001 compared to controls. b p < 0.001 compared to NC. c p < 0.01 compared to OSMF.
Mean
Age/Sex
Subject 17 M 20M 21 M 23M 24M 24M 28 M 28M 29M 32 M 33 M 36 M 37M 43M 60 F
Age/Sex
_+0.568 +_0.399 +_0.345 +0.335 +_0.319 +-0.264 +_0.315 +_0.400 +_0.375 _+0.377 +0.249 +-0.287 +-0.478 +_0.504 +0.392 7.617 ± O. 183 a
8.38 6.21 6.55 7.53 8.35 7.33 7.62 7.44 8.11 8.07 6.66 7.36 7.75 8.87 8.02
OSMF
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Subject
22 M 23M 34 M 34M 35M 40F 40 F 45M 50M 50 F 50 F 50 F 55M 64M 70 M
Age/Sex
_+0.384 + 0.369 _+0.477 + 0.487 _+0.233 +0.553 +0.501 + 0.276 + 0.247 +0.431 +- 0.405 + 0.388 _+0.412 + 0.542 +- 0.426 8.500 + 0.248 a,b,c
7.45 8.05 9.61 8.62 6.77 9.99 9.18 8.34 7.44 8,82 7.96 7.73 8.62 10.36 8.56
OC
I N D I V I D U A L F R E Q U E N C I E S O F S I S T E R - C H R O M A T I D E X C H A N G E S (SCE) P E R C E L L ( M E A N +_SE) IN B L O O D L Y M P H O C Y T E S O F T O B A C C O - A R E C A N U T (MAVA) C H E W E R S A N D C O N T R O L P E R S O N S
TABLE 1
#0
43 leaf or any other ingredient, is chewed very commonly. It is a mixture of ingredients well known for their carcinogenic/cocarcinogenic properties. The problem of oral cancers associated with tobacco-areca nut chewing is likely to assume very serious dimensions, as it is gaining acceptance among adolescents and women. Equally alarming is a recent report showing that genotoxic effects of betel quid chewing were more pronounced in pregnant women (Ghosh and Ghosh, 1988). These facts present an urgent need of evolving parameters for monitoring the genotoxic hazards of the tobacco-areca nut chewing habit. Cytogenetic endpoints, such as sister-chromatid exchange (SCE), chromosome aberration (CA) and micronucleated cell (MNC) frequencies, have been advocated as sensitive parameters for assessing genotoxic effects of chemical or physical mutagens. When employed in combination, the data collected would provide more comprehensive information (Carrano and Natarajan, 1988; Gebhart, 1981; Hsu, 1982; Stich and Rosin, 1984). We have therefore selected scoring of CA and SCE frequencies in peripheral blood lymphocytes (PBLs) and scoring of MNC frequencies in exfoliated buccal mucosa cells of 4 different groups of individuals. Such an experimental design was expected to provide useful information about possible genotoxic effects of the tobaccoareca nut (mava) chewing habit on target as well as non-target tissues. Materials and methods
Selection of subjects Altogether we studied 60 non-smoking teetotalers, including 45 chewing a mixture of tobacco, areca nut and lime only (hereafter referred as chewers) and 15 healthy non-chewing individuals as controls. The 45 chewers were further classifiable as (1) normal chewers (NC) without any clinically detectable change in oral mucosa, (2) chewers suffering from oral submucous fibrosis (OSMF), an oral premalignant condition and (3) chewers with oral cancers (OC), squamous cell carcinoma of the oral cavity. All 3 groups of chewers include individuals who chewed a mixture of tobacco, areca nut and lime (locally known as mava, which is not a betel quid) with compara-
ble frequency and duration (average 6 chews/ day). An equal number of individuals, who had never consumed tobacco or areca nut in any form, had had no viral disease or antibiotic therapy during the last 6 months and who had normal oral mucosa, were included as controls. The OSMF patients and OC patients were histologically confirmed cases, however, blood samples were collected before any anticancer therapy was started. Informed consent was obtained from all the individuals studied before sample collection. Blood collection and culture were done in 9 batches. The controls and the normal chewers were pre-identified from the staff members of our hospital and adjoining institutions. Whenever appropriate OC a n d / o r OSMF patients were available, the sampling was done on the same day and cultures were set with at least one control and one normal chewer sample.
Culture procedure PBL culture procedures were essentially the same as detailed earlier (Adhvaryu et al., 1985). Briefly, aseptically collected heparinised whole blood (1.0 ml) was added to 7.0 ml growth medium comprised of 80% MEM (Earle's base and nonessential amino acids) and 20% newborn calf serum. PHA-M at 3% and 5-bromodeoxyuridine (BrdU) at 2.0 /~g/ml final concentration were added. Following colchicine treatment (0.3 # g / m l ) for the final 3 h, cultures were harvested at 72 h after the usual hypotonic (0.56% KCI) and fixation (3 : 1 methanol : acetic acid) protocol. Air-dried slides were stained by the fluorescence plus Giemsa (FPG) method for sister-chromatid differential staining which helped to identify ceils in the M1, M2 and M3 phases of cell division. A minimum of 100 well-spread metaphase cells in M1 phase were scored per sample for CA analysis. The identification of CA was carried out as recommended by WHO (1985). 25 metaphase ceils in M2 phase were counted per sample for SCE scoring.
Cytological preparations and micronucleus staining procedure After cleaning the mouth thoroughly by repeated gargling with drinking water, the cheek mucosa was scraped with a blunt spatula and
44
smears of exfoliated buccal mucosa cells were prepared on clean glass slides. Cells were fixed with 1:3 acetic acid:methanol fixative for 15 min. The cells were stained using the Feulgen plus fast green method with minor modifications. Briefly, after hydrolysis with 5 N HC1 for 20 min at room temperature, cells were stained with Schiff's reagent for 2 h. Following 3 changes of SO 2 water, the cells were counterstained with 0.5% alcoholic fast green, dried and mounted with DPX. A minimum of 1000 cells was screened from each individual for examining the presence of micronuclei. Micronucleus identification was done according to the criteria detailed by Sarto et al. (1987). The assay was not carried out for oral cancer patients as it was not feasible to obtain scorable smears due to poor hygienic condition of the oral cavity and because of abundant necrotic cells in the preparations. For all 3 parameters studied, slides were coded and read blindly by 2 microscopists. The person responsible for coding distributed the slides in such a way that both microscopists had almost comparable numbers of controls and patients in each group. Results for the 4 groups examined, were compared with each other. Student's t-test was applied to check the statistical significance of the results. Results
Table 1 provides mean lymphocytic S C E / c e l l values for all 4 groups studied. For controls the mean S C E / c e l l value was 6.185 + 0.088 (mean + SE), whereas the values were 7.219 + 0.221, 7.617 + 0.183 and 8.500 + 0.248 for NC, OSMF patients and OC patients, respectively. All 3 groups of tobacco-areca nut (maya) chewers had a statistically significant elevation in mean S C E / c e l l values compared to the controls ( p < 0.001), who did not consume tobacco or areca nut in any form. Among the chewers, OSMF patients had a marginally elevated mean frequency compared to NC, whereas OC patients had a statistically significant elevation in mean SCE value compared to both NC ( p < 0.001) and OSMF patients ( p < 0.01). The metaphases scored for SCEs were further separated according to the number of SCEs
- -
CONTROLS
m
15
J
k NC
15
Vk OSMF
O"
0C 15
5
. . . .
"~0 .
.
.
.
.
15"
Fig. 1. Percentage distribution of metaphases according to the number of SCEs. NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients.
(Fig. 1). As visualised in the figure, with the chewing habit and progression from normal to oral cancer, a shift is observed towards a greater number of cells with higher SCEs, thus resulting in higher mean SCE frequencies. Details of lymphocytic CA analysis are presented in Tables 2 and 3. The C A / c e l l values varied from 0.03 to 0.08 in controls, providing a mean of 0.050 + 0.004 (mean + SE). Among the chewers, the C A / c e l l frequencies varied from 0.06 to 0.16 in NC giving a mean value of 0.097 + 0.007. For OSMF patients, the values ranged from 0.08 to 0.20 yielding a mean value of 0.127
45
+ 0.009 and in OC patients CA values from 0.09 to 0.20 resulted in a mean of 0.144 + 0.009. The increase in CA frequency (including gaps) in all 3 groups of chewers was statistically significant ( p < 0.001) when compared with controls. The difference between CA frequencies of NC and OSMF patients ( p < 0.02) as well as NC and OC patients was statistically significant ( p < 0.001). Chromatid aberrations, the majority of which were gaps, were more frequent than chromosome-type aberrations. Interchanges were not observed in controls, but were found in all 3 groups of chewers. Even after excluding gaps from aberrations, elevations in C A / c e l l value in all 3 groups
of chewers were significant when compared to the controls (Table 3). Table 4 details the frequency of percent MNC observed in the smeared exfoliated buccal mucosa ceils of different individuals. NC and OSMF patients had 0.74% and 0.75% MNC, respectively. These elevations in the frequency of MNC compared to the control value of 0.19% MNC were statistically significant ( p < 0.001).
Discussion In India tobacco-areca nut chewing is very popular and has been strongly implicated in the
TABLE 2 I N D I V I D U A L F R E Q U E N C I E S OF C H R O M O S O M E A B E R R A T I O N S P E R C E L L ( M E A N + S E ) IN B L O O D L Y M P H O CYTES O F T O B A C C O - A R E C A N U T (MAVA) C H E W E R S A N D C O N T R O L P E R S O N S Subject
Controls
Subject
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.04 0.04 0.04 0.05 0.03 0.03 0.05 0.05 0.06 0.06 0.05 0.04 0.07 0.08 0.06
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Mean
0.050_+0.004
+0.0196 _+0.0196 -+0.0196 _+0.0218 _+0.0171 -+0.0171 _+0.0218 _+0.0218 -+0.0276 _+0.0238 _+0.0218 _+0.0196 -+0.0292 +0.0306 -+0.0238
NC 0.08 0.09 0.10 0.06 0.16 0.09 0.08 0.08 0.05 0.09 0.12 0.13 0.14 0.11 0.08
Subject +0.0351 _+0.0349 _+0.0300 _+0.0237 +0.0441 -+0.0286 _+0.0306 _+0.0271 -+0.0218 -+0.0349 _+0.0354 +0.0429 _+0.0401 -+0.0343 -+0.0271
OSMF
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
0.15 0.13 0.08 0.10 0.16 0.11 0.13 0.14 0.09 0.14 0.19 0.11 0.20 0.08 0.10
* 0.097_+0.007 a
Subject _+0.0384 _+0.0448 _+0.0271 _+0.0300 -+0.0504 -+0.0467 _+0.0391 +0.0400 _+0.0349 -+0.0400 -+0.0463 -+0.0313 _+0.0469 -+0.0271 -+0.0300
OC
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
** 0.127-+0.009 a.b
0.12 0.20 0.15 0.10 0.10 0.16 0.12 0.14 0.09 0.14 0.19 0.20 0.13 0.18 0.14
_+0.0382 +0.0490 _+0.0528 _+0.0360 +0.0332 -+0.0484 _+0.0354 -+0.0566 +0.0286 _+0.0375 -+0.0468 -+0.0658 _+0.0447 +0.0477 -+0.0422
* 0.144-+0.009 a,c
NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients. P values including gaps: a p < 0.001 compared to controls; b p < 0.02 compared to NC; c p < 0.001 compared to NC. P values excluding gaps: * p < 0.01 compared to controls; ** p < 0.001 compared to controls. TABLE 3 N U M B E R O F D I F F E R E N T TYPES O F C H R O M O S O M E A B E R R A T I O N S P E R 100 CELLS IN B L O O D L Y M P H O C Y T E S O F T O B A C C O - A R E C A N U T (MAVA) C H E W E R S A N D C O N T R O L P E R S O N S Group
Controls NC OSMF OC
Total aberrations
Chromatid-type G
B
I
Chromosome-type G
B
Dm
5.0 9.7 12.7 14.4
3.7 6.4 8.0 10.6
0.9 2.2 3.0 2.5
0.1 0.1 0.1
0.3 1.1 1.5 0.8
0.1 0.1 0.1
0.1 0.2
D
R
NC, normal chewers; OSMF, oral submucous fibrosis patients; OC, oral cancer patients. G, gap; B, break; I, interchange; Dm, double minute; D, dicentric; R, ring.
46 TABLE 4 INDIVIDUAL FREQUENCIES (%) OF MICRONUCLEATED BUCCAL MUCOSA CELLS AMONG TOBACCOARECA NUT (MAVA) CHEWERS AND CONTROL PERSONS Subject Controls Subject NC 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0.2 0.3 0.3 0.1 0.1 0.2 0.3 0.1 0.3 0.3 0.1 0.1 0.2 0.2 0.1
Mean _+SE
0.193 +0.022
1
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
0.8 1.2 0.4 1.1 0.5 0.9 0.7 0.9 0.5 0.7 0.6 0.7 1.0 0.8 0.3 0.740 +0.064 *
Subject OSMF 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
0.8 0.7 0.3 1.8 0.4 0.2 0.8 1.1 0.6 1.0 0.7 0.5 0.8 0.6 1.0 0.753 +0.097 *
NC, normal chewers; OSMF, oral submucous fibrosis patients. * p < 0.001 compared to controls. causation of oral premalignant and malignant diseases. Considerable interindividual variations have been observed regarding dose, duration of chewing mava and occurrence of the disease. Hence, a great deal of human morbidity and mortality can be prevented by watchful monitoring. In the present study, it was attempted to explore the possible utility of a combination of 3 cytogenetic endpoints in assessing the extent of genomic damage caused by the habit of mava chewing on target as well as non-target tissues. Previously we reported elevated lymphocytic SCEs in normal chewers and chewers suffering from OSMF (Adhvaryu et al., 1986). Ghosh and Ghosh (1984, 1988) reported similar results among betel-quid chewers. Most striking in their studies was the SCE elevation in pregnant women. Stich et al. (1982a, b) also studied chewers from India and reported a significant elevation of MNC frequencies in exfoliated buccal mucosa cells. They attributed this to the genotoxic effects of tobacco a n d / o r areca nut alkaloids. Arecoline, the major areca nut alkaloid, and areca nut extracts have
been reported to increase SCE and CA frequencies in animal studies (Panigrahi and Rao, 1982, 1986) as well as in the in vitro system (Stich et al., 1981, 1983). Though nicotine, which is a major tobacco alkaloid, is by itself a weak clastogen, it induces SCEs in C H O cells (Riebe and Westphal, 1983). Many of the tobacco-areca nut-specific nitrosamines are detected in the saliva of chewers (Nair et al., 1985; Wenke et al., 1984; Sipahimalani et al., 1984; Prokopczyk et al., 1987) and some of these are potent mutagens and carcinogens (IARC, 1985). SCE elevations have also been documented in PBLs of smokers (Sarto et al., 1985; Husum et al., 1982; Ardito et al., 1980). The SCE elevations observed in the present study are thus in accordance with the reports of others. However, in an ongoing work, individuals consuming only areca nut also exhibited higher lymphocytic SCEs than controls. The subjects included in the present study consumed a mixture of tobacco and areca nut. Since alkaloids of both these substances also exert genotoxic effects individually, both of them should be considered responsible for the elevated SCE frequencies in mava chewers. To the best of our knowledge, CAs have not been reported previously for tobacco a n d / o r areca nut chewers. The lymphocytic CA rates were significantly higher in all 3 groups of chewers than in the controls. This increase can also be explained on the basis of the clastogenic potential of tobacco and areca nut alkaloids. However, since increases in SCE and CA frequencies have been recorded in different premalignant and malignant conditions (Adhvaryu et al., 1986, 1988a, b; Murty et al., 1985, 1986), the elevations in SCEs and CAs among OSMF patients and OC patients may be due to the habit, the occurrence of the disease, or a combination of both. In the present study, the OC patients were older (mean age 44.1 years) than the other groups (mean ages 32.7, 36.5 and 30.3 years for controls, NC and OSMF patients, respectively). Reports regarding the effect of age and sex on the frequencies of SCEs and CAs are contradictory (Carrano and Moore, 1982; Livingstone et al., 1983; Husum et al., 1986; Margolin and Shelby, 1985; Steenland et al., 1986). Here, due to the small sample size in each group, the evaluation of
47 the effect of these confounding factors will not be meaningful. However, it was observed during previous studies in our laboratory that age and sex have no influence on the frequency of the cytogenetic endpoints in question. Scoring of MNC in exfoliated oral mucosa ceils is technically less demanding than scoring of SCE and CA frequencies, but this simple assay is flawed with several shortcomings. Most important is the difficulty in obtaining scorable smears from most OC patients. This is because of ulcerated areas in the oral cavity resulting in poorer oral hygiene. Secondly, different laboratories utilising this parameter have employed different criteria for MN scoring, making interlaboratory comparisons difficult. Stich et al. (1982b) have reported elevated frequencies of MNC in the buccal mucosa cells of tobacco-areca nut chewers, but they did not observe any such elevation in smears obtained from smokers and those consuming alcohol or both (Stich and Rosin, 1983). Similarly, Mandard et al. (1987) also reported a negative MN test in patients with upper digestive tract cancer and controls, both consuming alcohol and tobacco. Sarto et al. (1987) proposed more stringent criteria for MN scoring and reported a highly positive MN test in smokers. We have followed the criteria suggested by them. The frequencies of MNC were almost 4 times higher in NC and OSMF patients. As already stated, the MN assay was not conducted in OC patients; however, considering the reports of Stich et al. on tobaccoareca nut chewers and that of Sarto et al. o n smokers, the validity of the MN test, being noninvasive and less demanding, is very high. All 3 parameters studied, viz. frequency of lymphocytic SCE and CA and MNC in exfoliated oral mucosa cells, showed statistically significant elevations in all 3 groups of tobacco-areca nut (mava) chewers compared to controls. Considering the commonness of the chewing habit, the number of 45 chewers (15 each, in 3 groups) may sound insufficient; however, it should be emphasised here that the individuals in all 3 groups (1) chewed the mixture with comparable f r e q u e n c y / duration, (2) had no concomitant habit of smoking or alcohol consumption and (3) were not engaged in any occupation which might affect the parameters studied. Chewing tobacco has been
causally associated with oral cancers; however, looking to the elevated frequencies of CA and SCE in PBLs, the possibility of damage caused to other systems/tissues should also be considered, as is the case with smoking which causes lung cancer but also elevates the risk of cancer at other sites like the bladder, pancreas etc. SCE frequencies were higher in OC patients compared to NC and OSMF patients, whereas CA frequencies were higher in OSMF patients compared to NC. It is recommended that, when studying individuals exposed to potential genotoxins, both parameters be employed in combination. Further, the MN assay, being less demanding and providing vital information about the genotoxic effect on the target tissue, would prove to be most useful for monitoring tobacco consumers, not only in estimating the extent of genomic damage caused by the habit, but also in appraisal of the advantages of either quitting the habit or, as already documented by Stich et al. (1984, 1985), for assessing the benefits of chemopreventive measures.
Acknowledgements The authors are thankful to Dr. N.L. Patel, The Director, Gujarat Cancer and Research Institute and to the Head, Department of Cancer Biology, for the facilities provided. Dr. P.K. Dayal, Government Dental College and Hospital, and staff members of Department of Surgical Oncology, Gujarat Cancer and Research Institute, are gratefully acknowledged for their interest and cooperation.
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