Fd Chem. Toxic. Vol. 30, No. 12, pp. 1045-1049,1992

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G E N O T O X I C I T Y OF ' G U D A K H U ' , A T O B A C C O P R E P A R A T I O N . II. IN H A B I T U A L USERS R. K. DAS* and B. C. DASH Environmental Mutagenesis Unit, School of Life Sciences, Sambalpur University, Jyotivihar-768019, Orissa, India (Accepted 10 August 1992)

Abstract--The genotoxic potential of 'gudakhu', a paste-like tobacco preparation that is used widely in Orissa, India, was evaluated using the micronucleus test in exfoliated cells of the buccal mucosa. Cells from 120 habitual users and from 102 non-users were examined. The incidence of micronuclei (MN) was increased in the mucosa cells of users, and the increase was significant in those who had used gudakhu for more than 5 yr. The increased incidence of MN was significantlycorrelated with the period of use of gndakhu, as well as with the frequency of daily use. There were no significant differences between the results for men and women.

INTRODUCTION Gudakhu is a paste-like tobacco preparation that is widely used in the state of Orissa, India; here every third person is habituated to it. There is now an alarming spread of its use to other parts of the country. The ingredients of gudakhu are tobacco (finely cut chewing variety 10%), lime (7%), molasses (35%), red soil (a particular type of locally available reddish soil containing oxides of iron, aluminium and several other metals, 28%) and water (20%). During use it is rubbed over the teeth and gum with a finger-tip for 10--15min; some users swallow the saliva-mixed extracts, whereas others spit it away. Highly addicted persons use gudakhu 20 times or more a day and every time about 2 g is used. Rural people are more addicted to this habit than are those in urban areas; among the urbanites this habit is substituted, particularly during office hours, by smoking, snuff dipping and tobacco chewing. Sometimes children as young as l0 yr old have been found to use gudakhu. India has the dubious distinction of having the highest incidence of cancer of the oropharyngeai cavity in the world. Oropharyngeal cancers account for one-third of all cancer cases in India (WHO, 1984). The use of tobacco and tobacco products has been found to be responsible for a variety of cancers in humans (Doll and Peto, 1981; IARC Working Group, 1985 and 1986). In a WHO report (WHO, 1984) about 90% of oral cancer cases in South-east Asia are attributed to tobacco use. Epidemiological as well as experimental studies have conclusively proved the involvement of smokeless tobacco in the aetiology of oral cancer (Claxton et al., 1989; Hecht

*To whom correspondence should be addressed. MN = micronuclei; MN-cells= micronucleated cells; SCE = sister chromatid exchange.

Abbreviations:

and Hoffmann, 1988; IARC Working Group, 1985; Lofroth, 1989; Mehta et al., 1981; Sanghvi, 1981; Tsuda and Kurashima, 1991). Although gudakhu is a potential carcinogen, unfortunately no data are available on the incidence of cancer due to use of this variety of tobacco preparation alone. That somatic mutations play an important role in the induction of cancer is well established. The mutagenic and clastogenic potential of smokeless tobacco and a variety of tobacco preparations, such as snuff (finely cut moist tobacco), khaini (tobacco plus slaked lime) and nass (tobacco plus slaked lime and oil) have also been demonstrated in bacteria, experimental mammals and in habitual users (Dash and Das, 1992; Ghosh and Ghosh, 1984; Kulkarni et al., 1987; Shah et al., 1985; Shirname et al., 1984; Shirname-More, 1991; Stich and Anders, 1989; Stich et al., 1982a,b; Stich and Rosin, 1983). However, our knowledge on such an effect of gudakhu is extremely limited. So far as we are aware, the report of Stich and Anders (1989) included some data on the incidence of micronuclei (MN) in the buccal mucosa cells and the level of tobacco-specific nitrosamines in the saliva of gudakhu users. However, the number of subjects (7) is given only for the nitrosamine study, and no details are given of the history of gudakhu use by the study participants. Very recently we have reported the induction of chromosome aberrations, MN and sister chromatid exchanges (SCEs) in the mouse in vivo after single or repeated treatment with an acetone extract of gudakhu (Dash and Das, 1992). All this prompted us to make a comprehensive study of the possible genotoxic effect of this widely used tobacco preparation in habitual users--a study that is more relevant from the point of view of risk assessment. We have examined the buccal mucosa cells of 120 habitual users of gudakhu, who had a history of use ranging from 1 to 42 yr, in order to assess the incidence of induction of MN.

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1046

R.K. DAS and B. C. DASH MATERIALS AND METHODS

Subjects. A total of 222 healthy subjects (120 users and 102 non-users) from Sambalpur (Orissa) were examined. About 75% of them lived in rural areas; the rest lived in semi-towns and towns. Before collecting the samples, we asked each subject about their lifestyle, habits of food consumption, occupation, infection, X-ray exposure, medication, etc. Individuals who had recently had a viral infection or been exposed to X-ray or were under medication for some disease(s) were excluded. Persons having other confounding habits such as drinking alcohol, smoking or chewing betel quid and tobacco were also excluded. The user group included 59 men and 61 women; their ages ranged from 17 to 70yr (mean age, 39.98 yr). The period of use of gudakhu ranged from 1 to 42 yr. Among the non-users (62 men and 40 women) ages ranged from 16 to 65 yr (mean 31.25 yr). The nonuser group came from the same localities as the habitual users. However, the non-users were very puritanical, having no habit of drinking alcohol, or chewing or smoking tobacco. Sampling and scoring. Before sampling, each individual was asked to rinse his/her mouth thoroughly with water. The exfoliated cells were obtained by scraping the buccal mucosa cells of the cheek with a moistened wooden spatula. Scraping was done separately for left and right cheeks of each individual. The scraped cells were taken onto pre-cleaned slides and smears were prepared separately for left and right cheeks. The smears were air-dried for 10 min, fixed in ethanol-acetic acid (3 : 1, v/v) for 15 min and allowed to air-dry again. The smears were then stained by the

Feulgen reaction and counter-stained with Fast Green (0.5% alcoholic), mainly following the technique of Stich et al. (1982a). The slides were randomized and scored by a single observer. From each individual, 1000 intact cells were examined (500 from each cheek) under x 600 magnification, and when micronucleated cells were located they were examined under x 1000 magnification. Criteria suggested by Sarto et al. (1987) to recognize MN were followed. Dead or degenerating cells (karyolysis, karyorrhexis, nuclear fragmentation) were not considered. Nuclear blebbings (micronucleus-like structure connected with the main nucleus with a bridge) were also excluded. MN equal to or smaller than one-fifth of the main nucleus only were assumed to have resulted from chromosome breakage and considered. The number of micronucleated cells (MN-cells), irrespective of the number of MN present in a cell, and the total number of MN observed were recorded. Statistical analysis. The data were sorted for convenience in statistical analysis into various groups on the basis of(a) sex (males and females), (b) period (yr) of use (~< 5 yr, 6-10 yr, 11-20 yr and > 20 yr), and (c) frequency of daily use (~ 10 times). The mean value (with SEM) for each category was calculated from individual values under that category. Mean values obtained from users were compared with those of non-users by one-way analysis of variance (ANOVA). The nested ANOVA test (Sokal and Rohlf, 1969) was conducted taking data for sex versus period of use and sex versus frequency of daily use in order to determine whether the data were homogeneous. The contribution of two

Table 1. Frequency o f distribution o f micronucleated buccal m u c o s a cells in non-users and different categories of users o f g u d a k h u Use status

Period of use (yr)

Non-users

--

Users

1-5

Sex M F M

F

6-10

M

F

11-20

M

F

>20

M

F

Frequency of daily use

No. o f subjects

M e a n age (yr)

Age range (yr)

M N in buccal mucosa cells

--1-5 6-10 > 10 1-5 6-10 > 10 1-5 6-10 > 10 1-5 6-10 > 10 1-5 6-10 > 10 1-5 6-10 > 10 1-5 6-10 >10 1-5 6 - 10 > 10

62 40 2 3 1 2 1 2 4 3 2 2 2 2 13 5 2 11 6 1 14 8 2 12 16 4

30.50 31.78 36.5 22.0 19 30.5 24 27 37.5 31.33 33.5 28 26.5 25 36.07 37.2 35.5 36.64 39.67 38 48.36 45.25 51.5 47.73 46.69 45.25

17~65 16-50 3043 17 29 29 32 -26-28 29-42 25-30 2740 18 38 25-28 20-30 27-58 2947 2447 2847 32-54 -25 70 29 55 40-63 39~60 40-58 4348

MN-cells

Total no.

0.37 _+ 0.04 0.32 + 0.04 0.45 ,+ 0.11 0.40 + 0.05 0.40 + 0.00 0.70 + 0.21 0.70 + 0.00 0.65 i 0.11 0.90 ,+ 0.22*** 1.00_+0.16"** 1.00 ,+ 0.07*** 0,80_+0.14"* 0.85 -+ 0.04*** 1.05 ,+ 0.32*** 1.38 + 0.15*** 1.16+_0,12"** 1.50 -+ 0,35*** 1.38_+0.19"** 1.87 -+ 0.21 *** 1.80 + 0.00"** 1.77 ,+ 0,16"** 1.63_+0,15"** 2.30_+0,14"** 2.03+0.19"** 1.89 ,+ 0,18"** 2.33 ± 0.48***

0.39 +_ 0.04 0.33 ,+ 0.04 (I.45 ,+ 0. I 1 {I.40 + 0.05 0.50 + 0.00 0.70 + 0.21 0.90 + 0.00 0.70 _+ 0.07 1.05 + 0.25*** 1.17+0.09"** I. 10 ± 0.00"** 0.95_+0.20* 0.85 ,+ 0.04* 1.20 + 0.35*** 1.50 ,+ 0.16"** 1,34_+0.11"** 1.70 ,+ 0.35*** 1.56-+0.21"** 1.98 _+ 0.23*** 1.80 + 0.00"** 1.94,+0.16"** 1.91 ,+0.16"** 2,40,+0.14"** 2.55_+0.11"** 2.16 _+ 0.22*** 2.55 ,+ 0.45***

MN-cells = micronucleated cells M N = micronuclei Values are m e a n s per 100 cells (_.+SEM). Values m a r k e d with asterisks are significantly ( A N O V A test) higher than the corresponding value for the controls (non-users): * P < 0.05; **P < 0.01; ***P < 0.001.

Genotoxicity of gudakhu in habitual users Table 2. Incidence of mieronuclei in bueeal mucosa cells of control individuals (non-users) of different ages Age No. of Age Mean group (yr)

subjects (males + females)

range (yr)

age (yr)

MN-cclIs (mean %)*

~ 0.1).

o~ "~

./o

2.5

o

o° 2.0 i~ -- ,-~ 1.5

O _

~_ ~ ¢n o

= O ~

6O

[]

Non - users

I

Users

• • o~ ~

j/.

o o o o

1.0

/.. 0.5 I

80 --

/o

~J

, ~ o ~ /

RESULTS M N were round or elliptical in shape. The affected cells usually contained one M N ; cells with more than one M N were also observed, but less frequently. The data on the incidence of M N in the buccal mucosa cells of gudakhu users and non-users are summarized in Table 1. Since statistical analysis did not reveal any significant difference between the data for left and right cheeks they have been pooled. A m o n g the non-users, there was no statistically significant difference between males and females in the frequencies of M N cells ( A N O V A test; F = 1.00, P > 0.1). In order to investigate the influence of age o f the subjects on the frequency o f M N , the nonusers, irrespective of sex, were divided arbitrarily into four age groups, ~