Effect of betel chewing on the frequency of sister chromatid exchanges in pregnant women and women using oral contraceptives

Effect of betel chewing on the frequency of sister chromatid exchanges in pregnant women and women using oral contraceptives

Effect of Betel Chewing on the Frequency of Sister Chromatid Exchanges in Pregnant Women and Women Using Oral Contraceptives P. K. Ghosh and Reita Gho...

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Effect of Betel Chewing on the Frequency of Sister Chromatid Exchanges in Pregnant Women and Women Using Oral Contraceptives P. K. Ghosh and Reita Ghosh

ABSTRACT The incidence of sister chromatid exchange (SCE) was investigated in the lymphocyte chromosomes of betel chewing and non-chewing normal women, pregnant women, and women using oral contraceptives. The frequency of SCE was found to be 7.82 +- 0.24 and 8.27 +_ 0.27 in non-chewing pregnant women and women using oral contraceptives respectively, which were significantly higher than the mean value of 5.21 +_ 0.18 observed in non-chewing normal women. Betel chewing induced higher SCE in pregnant women and women using oral contraceptives, the frequencies being 11.79 +_ 0.38 and 12.51 -'- 0.44, respectively, which were significantly higher than the SCE frequency of 6.28 +_ 0.21 found in normal betel chewing females.

INTRODUCTION Betel leaf (Piper betel Linn) is a plant product which, together with other ingredients, is widely chewed in India. The essential components chewed with betel leaf are areca nut, slaked lime, catechu, and often tobacco. The carcinogenic potential of betel is well established [1]. A dimethyl sulphoxide extract of betel leaf showed a carcinogenic effect on the mucosa of hamster buccal pouch [2]. The c]astogenic effect of betel leaf has been demonstrated recently. An increase in the frequency of chromatid aberrations was observed when the betel leaf extract was added to lymphocyte cultures [3]. The frequency of sister chromatid exchanges (SCE) is now believed to be a more sensitive indicator of genetic damage than chromosome aberration [4]. Elevated frequency of SCE have also been observed in betel chewers

lab The incidence of oral cancer is around 8% of all cancers reported in Indian females [6]. The high incidence of oral cancer has long been associated with the habit of chewing betel leaf with other ingredients [7]. Pregnant women are known to be more prone to genetic damage, compared with normal women, due to the altered levels of certain sex hormones during pregnancy [8]. These sex hormones present in pregnant females play an important role in increasing the sensitivity of their

From

the Cytogenetics Laboratory, Department of Anthropology,Universityof Delhi, Delhi-110007,In-

dia. Address requests for reprints to Dr. P. K. Ghosh, Reader, Deportment of Anthropology, University of Delhi, Delhi-110007, India. Received April 10, 1987; accepted December 10, 1987.

211 © 1988 Elsevier Science PublishingCo., Inc. 52 Vanderhih Ave., New York, NY 10017

Cancer Genet Cytogenet32:211-215 (1988] 0165-4608/88/$03.50

212

P.K. Ghosh and R. Ghosh

lymphocytes to genetic damage [9]. The effect of betel chewing on the frequency of SCE was therefore investigated in pregnant women and women using oral contraceptives, in whom the hormonal profile is k n o w n to be different from normal women. MATERIALS AND METHODS Peripheral blood samples were obtained from 129 healthy adult females who were not occupationally exposed to mutagens. They were non-smokers without recent viral infections or radiation exposure. The first group included non-chewers consisting of 20 normal women, 24 pregnant women, and 27 women using oral contraceptives. The second group included betel chewers consisting of 16 control women, 19 pregnant women, and 23 oral contraceptive users. All pregnant women were in their last trimester of pregnancy. A combination of ethenyl estradiol and d-norgestrel has been taken by women using oral contraceptives for periods ranging from 5 to 26 months (mean, 17 months). Chewing habits were 15.2 --- 6.4 (range, 6-29) betel leaves per day for normal women, 14.5 _+ 5.8 {range, 4-25) betel leaves per day for pregnant women, and 14.8 +_ 6.1 (range, 8-31) betel leaves per day for women using oral contraceptives. Whole blood lymphocytes from all these individuals were cultured in TC 199 m e d i u m supplemented with 20% fetal calf serum, phytohemagglutinin, and antibiotics. Bromodeoxyuridine was added to a concentration of 3 v.g/ml cultured medium. Cells were grown in the dark at 37°C for 72 hours. At 70 hours, colcemid was added to a final concentration of 0.1 ~g/ml. After hypotonic treatment with KCI and fixation in methanol/acetic acid (3:1). The slides were air-dryed. Differential staining of sister chromatids were obtained according to the technique of Wolff and Perry [10] and Sugiyama et al. I l l ] with slight modification. One-day-old slides were stained with 5.0 v.g/ml Hoechst 33258 in the dark for 30 minutes. They were washed with distilled water, mounted in 2 × SSC with a cover slip and exposed to sunlight for 2 hours. They were washed again with distilled water and stained in 5% Giemsa. SCE was scored blindly in 50 M2 cells per individual. RESULTS Data on mean frequency of SCE per cell in betel chewing and non-chewing normal women, pregnant women, and oral contraceptive users are shown in Table 1. Nonchewing pregnant women had a mean SCE per cell of 7.82 +_ 0.24, whereas, nonchewing oral contraceptive users had a mean SCE per cell of 8.27 -+ 0.27. This value was significantly higher as found in normal non-chewing women (5.21 +_ 0.18). Betel chewing pregnant women and oral contraceptive users, on the other hand, had a mean frequency of SCE of 11.79 - 0.38 and 12.51 +_ 0.44, respectively, which was significantly higher than the mean SCE frequency of 6.28 x 0.21 found in betel chewing normal women. There was significant correlation between the n u m b e r of betel leaves chewed and the mean SCE frequency (r = 0.523, p < 0.01]. DISCUSSION Changes in the level of certain sex hormones, such as progesterone and estrogen are known to occur during pregnancy, especially in the last trimester [12-14]. Sex hormones, when administered in vivo, have been shown to induce chromosome aberrations [15-18]. Women using an estrogen-progesterone combination contraceptive exhibited either an increased frequency in SCE [9] or no change [191. SCE frequency has been found to be enhanced in pregnant women in the last trimester of pregnancy and also in the lymphocyte cultures of normal w o m e n to which the sex hor-

213

Effect of Betel on SCE

Table 1

Series number 1

2

3

Mean frequencies of SCE in betel chewing and non-chewing normal women, pregnant women, and women using oral contraceptives

Group Normal women Non-chewers Chewers Pregnant women Non-chewers Chewers Oral contraceptive Non-chewers Chewers

SCE per (:ell

Number of subjects

Mean age (yr)

Mean ~. SE

Range

20 16

36.9 35.8

5.21 + 0 . 1 8 6 . 2 8 +_ 0.21

0-11 0--16

24 19

32.5 33.2

7 . 8 2 +_ 0 . 2 4 1 1 . 7 9 __ 0 . 3 8

0-17 2-21

27 23

34.1 34.7

8 . 2 7 _+ 0 . 2 7 12.51 _+ 0 . 4 4

1-19 3--22

users

I)ifferences between i'~Oll-chewing normal w o m e n a n d pregnant w o m e n , between non-cbewiilg llorma[ wolnen a n d oral contraceptive users are significant (p < 0.001l by S t u d e n t ' s t-test. Differences between n o n - c h e w i n g normal ;vomen anti betel c h e w i n g normal women, between n n n - c h e w i n g pregnant w o m e n and betel c h e w i n g pregnant women, between non-che,,ving oral contraceptive users a n d betel c h e w i n g oral contraceptive users are significant (p < t).001) by Student's t-test. Differences between betel c h e w i n g normal ,,','omen a n d betel c h e w i n g pregnant women, between betel c h e w i n g normal w o m e n and betel c h e w i n g oral contraceptive users are significant [p < 0.0011 by Student's t-test.

mones progestrone, estrogen, and h u m a n chorionic gonadotrophin have been exogenously a d d e d [20]. The increased frequency of spontaneous SCE in pregnant women and women using oral contraceptives observed in the present study, thus, may be due to the altered hormonal profile in these women, compared with normal controls. The observation of increased frequency of SCE in betel chewing normal women, pregnant women, and w o m e n using oral contraceptives indicates that betel induces SCE. This is in agreement with our previous study where an increase in the frequency of SCE were observed in betel chewers [5]. However, the extent of SCE increase induced by betel chewing in the present study was found to be much more pronounced in pregnant women and women using oral contraceptives, compared with normal women. Betel chewing pregnant women and oral contraceptive users showed an elevation of the mean SCE frequency of 3.97 and 4.24 SCE compared with non-chewing females in contrast to an increase of mean values of only 1.07 SCE in betel chewing control women. This finding may imply that chromosomes from pregnant women and women using oral contraceptives are more susceptible to DNA damage induced by betel chewing compared with normal women. This observation may be related to the different hormonal environment. There are indications that a higher level of sex hormones in pregnant women and those using oral contraceptives may increase the sensitivity of lymphocytes to genetic damage [21, 22]. The frequency of X-ray induced c h r o m o s o m e aberrations was found to be significantly higher in pregnant muntjac compared with normal controls [23[. I,ymphocytes of pregnant women and oral contraceptive users have been found to be more susceptible to SCE induction by diethylstilbestrol and mitomycin-C [24, 25[. The incidence of oral cancer has been found to be as high as 34.9% of all cancers reported from India [26]. ]'his high incidence of oral cancer has been ascribed to the habit of betel chewing [27-33]. According to Jussawalla and Deshpande [34], the betel chewers showed a three-fold greater risk of developing oral cancer than non-chewers. Presence of high levels of sex hormones during pregnancy may also initiate processes leading to an enhanced growth of certain cancers due to increased

214

P . K . G h o s h a n d R. G h o s h

s e n s i t i v i t y of l y m p h o c y t e s to m u t a g e n s / c a r c i n o g e n s [ 3 5 - 3 8 ] . SCE are good i n d i c a tors of g e n e t i c d a m a g e a n d t h e r e e x i s t s a r e l a t i o n s h i p b e t w e e n i n c r e a s e d SCE rates a n d a l t e r e d m u t a t i o n f r e q u e n c y [39]. I n c r e a s e d SCE f r e q u e n c y o b s e r v e d in b e t e l c h e w i n g p r e g n a n t w o m e n a n d oral c o n t r a c e p t i v e u s e r s m a y be d u e to t h e m u t a g e n i c a c t i v i t y of betel o n t h e o n e h a n d a n d e s t r o g e n or its m e t a b o l i t e s o n t h e o t h e r . It s e e m s t h a t t h e c a r c i n o g e n i c p o t e n t i a l of b e t e l is e n h a n c e d by t h e e l e v a t e d h o r m o n a l profile in p r e g n a n t w o m e n a n d oral c o n t r a c e p t i v e users. E p i d e m i o l o g i c s t u d i e s m a y tell us w h e t h e r t h e r e is a n i n c r e a s e d l i k e l i h o o d of d e v e l o p i n g c a n c e r in b e t e l c h e w ing p r e g n a n t w o m e n a n d oral c o n t r a c e p t i v e u s e r s d u e to t h e i n c r e a s e d s e n s i t i v i t y of t h e i r l y m p h o c y t e s to g e n e t i c d a m a g e c o m p a r e d w i t h n o n - c h e w i n g p r e g n a n t w o m e n a n d oral c o n t r a c e p t i v e users. Supported ill part by a grant from the Indian Council of Medical Research (No. 5/12(7)85BMS-II).

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Effect of Betel o n SCE

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