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Sister chromatid exchanges in patients with oral submucous fibrosis
Sister Chromatid Exchanges in Patients with Oral Submucous Fibrosis P. K. Ghosh, Radha Madhavi, Mohan Guntur, and Reita Ghosh
ABSTRACT: The incidence of sister chromatid exchange (SCE) was investigated in the lymphocyte chromosomes of 45 patients with oral submucous fibrosis and 56 age- and sex-matched nansmoking controls. The frequency of SCE was 9.26 ± 2.15 in patients with oral submucous fibrosis, which was significantly higher than the mean SCE value of 5.49 + 1.24 observed in normal controls. The frequency of SCE in patients with oral submucous fibrosis addicted to the habit of betel with tobacco chewing, "bidi'/cigarette smoking and combined habits of chewing and smoking of tobacco were 8.12 ± 1.69, 9.43 ± 1,87, and 10.06 ± 2.28, respectively. These values were also significantly higher as compared with the SCE values observed in normal calltro]s.
INTRODUCTION S u b m u c o u s fibrosis is a chronic disease affecting the mucosa of any part of the oral cavity and occasionally extending into the pharynx and esophagus. Onset of the condition is insidious and often is of 2 to 5 years duration. The most c o m m o n initial symptom is a b u r n i n g sensation in the mouth, often experienced when the patient is eating spicy food. The condition is ahvays accompanied by an inflammatory reaction. Patients often experience difficulty in eating, chewing, swallowing, and speaking [1]. Clinically, s u b m u c o u s fibrosis causes whitish, blanched depigmented areas of the oral mucosa [2]. The disease occurs mainly among Indians, but sporadic cases have been observed in other countries of East Asia [3]. S u b m u c o u s fibrosis of the oral cavity has recently been associated with an increased t e n d e n c y for local cancer development [4]. The precancerous nature of submucous fibrosis was first m e n t i o n e d by Paymaster [5]. In a survey in South India, s u b m u c o u s fibrosis was observed in 40% of 100 consecutive oral cancer patients [6l. Follow-up studies in India have clearly demonstrated that about one-third of the patients with oral s u b m u c o u s fibrosis develop squamous cell carcinoma [7]. All this evidence clearly indicates the precancerous nature of subnmcous fibrosis. The etiological factors responsible for initiation of oral malignancy are not precisely known. However, it has been argued that the habit of tobacco chewing compared with tobacco smoking plays an important role in the transformation of submucous fibrosis through distinct preneoplastic changes to carcinoma of the oral cavity. Because chromosomal instability has long been associated with neoplastic disorders [8] and the quantitative assay of sister chromatid exchange (SCE) provides an easy, From the CytogeneticsLaboratory, Departmentof Anthropology,Universityof Delhi. Delhi, India. Address reprint requests to: Dr. P. K. Ghosh, Reader, Department of Anthrapoh)gy, University of Delhi, Delhi-110007, India. Received March 2, 1989; accepted/une 30, 1989.
197 (~21990 Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas,New York, NY 10010
Cancer Genet Cytogenet44:197 201 (1990) 0165-4608/90/$03.50
198
P.K. Ghosh et al.
rapid, and sensitive method for studying c h r o m o s o m e / D N A instability and its subsequent repair processes [9], an attempt was made in the present study to investigate SCE levels in the peripheral blood of patients with submucous fibrosis and to correlate them with the habits of different forms of tobacco usage.
MATERIALS AND METHODS Peripheral blood samples were collected from 45 cases of oral submucous fibrosis. S u b m u c o u s fibrosis was diagnosed solely on clinical grounds and only w h e n the patients exhibited the presence of palpable fibrous bands. The control group consisted of 56 age-matched normal individuals with no history of tobacco usage or radiation or viral exposure. The mean age was 41.53 years (range 24 to 58 years) for i n d i v i d u a l s with oral s u b m u c o u s fibrosis and 39.25 years (range 22 to 55 years) for the controls. Detailed history regarding the different habits of tobacco usage ( " b i d i ' / cigarette smoking and betel/tobacco chewing) were taken from each patient with oral s u b m u co u s fibrosis. The past history with regard to oral symptoms was also collected from each patient. All the individuals studied in the present investigation were males. Whole blood l y m p h o c y t e s from all individuals were cultured in TC-199 m e d i u m s u p p l e m e n t e d with 20% fetal calf serum, phytohemagglutinin, and antibiotics. Brom o d e o x y u r i d i n e (BrdU) was added to a concentration of 3/zg/mL and the 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 /zg/ml. After hypotonic treatment with KC1 and fixation in methanol : acetic acid (3 : 1), the slides were prepared by air drying. Differential staining of sister chromatids were obtained according to the technique of Wolff and Perry [10] and Sugiyama et al. [11] with slight modifications. One-day-old slides were stained with 5.0/~g/ml Hoechst 33258 in the dark for 30 minutes. They were washed with distilled water, m o u n t e d in 2 x SSC with a coverslip, and exposed to sunlight for 2 hours. They were again washed with distilled water and stained in 5% Giemsa. SCEs were scored blindly in 50 second division metaphase cells per individual. The differences b e tw e e n the controls and the patients with submucous fibrosis were statistically evaluated with the paired t test.
RESULTS Data on the mean frequency of SCE per cell in patients with oral submucous fibrosis and normal controls are s h o w n in Table 1. Patients with oral submucous fibrosis had a mean SCE frequency of 9.26 -+ 2.15, w h i c h was significantly higher than the mean
Table 1
Mean frequencies of SCE in patients with oral submucous fibrosis in relation to different habits of tobacco usage
Series
Group/tobacco habits
1
Normal controls/no tobacco habits Oral submucous fibrosis a) Betel with tobacco chewing b) Bidi/cigarette smoking c) Combined habits ~'
2
No. of subjects
Mean age (yr}
SCE per cell Mean ± SD (range)
56
39.25
5.49 ± 1.24 (0-11)
45 11 13 21
41.53 39.95 41.26 42.18
9.26 8.12 9.43 10.06
± ± ± ±
2.15 (1-20) 1.69 (1-16) 1.87 (2-18} 2.28 (3-20)
dPersons addicted to both tobacco chewing and smoking. Differences between series 1 and 2. l and 2a, l and 2b, 1 and 2c were signii~cant {p < 0.001} by Student's t test.
SCE in Oral S u b m u c o u s Fibrosis
199
SCE value of 5.49 -+ 1.24 observed in normal controls (p < 0.001). The mean SCE frequency in patients with oral submucous fibrosis who were addicted to the single habit of betel with tobacco chewing or bidi/cigarette smoking or to c o m b i n e d habits of c h e w i n g and smoking tobacco was 8.12 -+ 1.69, 9.43 -+ 1.87, and 10.06 -+ 2.28, respectively. These values were also significantly higher as c o m p a r e d with the SCE values observed in normal controls (p < 0.001). There was significant correlation between n u m b e r of betel leaves with tobacco chewed and mean SCE frequency (r = 0.65; p < 0.001) and also the number of cigarettes or bidis smoked and the mean SCE frequency (r = 0.68;p p < 0.001). DISCUSSION
Oral cancer presents a major health problem in India. The buccal cavity and p h a r y n x are the areas most frequently involved by cancer and constitute 34.9% of the total cancers in India [12]. Tobacco usage is an important etiological factor in development of cancer of oral e p i t h e l i u m [13]. In India, tobacco is smoked in the form of cigarettes in urban areas, whereas in rural areas, particularly in the lower socioeconomic classes, the prevalent habit is bidi smoking. The other important form of tobacco usage in India is betel chewing, w h i c h is practiced in a variety of ways. Every patient with oral s u b m u c o u s fibrosis studied in the present investigation was addicted to either one form of tobacco usage or the other: 24.4% of the patients with oral s u b m u c o u s fibrosis were a d d i c t e d to betel with tobacco chewing, 28.9% smoked tobacco in the form of bidi or cigarettes, and 46.7% were habituated to the combination of tobacco chewing and tobacco smoking. Thus, the increase in frequency of SCEs observed in patients with oral submucous fibrosis may be attributed to their a d d i c t i o n to tobacco habits, as has been w i d e l y reported [14-19]. The SCE frequencies in patients with oral s u b m u c o u s fibrosis varied according to the mode of tobacco usage. The single habits of betel/tobacco chewing and bidi/cigarette smoking and the c o m b i n e d habits of chewing and smoking tobacco i n d u c e d a mean elevation of 2.63, 3.94, and 4.57 SCEs, respectively, as c o m p a r e d with the SCE values observed in normal controls. Thus, the c o m b i n e d habits of tobacco smoking and chewing may i n d u c e more c h r o m o s o m a l instability as c o m p a r e d with the single habits of betel with tobacco chewing or tobacco smoking alone. About 250,000 persons in India are believed to have submncous fibrosis. Epidemiological studies on the prevalence of submucons fibrosis have been c o n d u c t e d by Pindborg et al. [20] and Zachariah et al. [21], who examined 35,000 urban Indians seeking a d m i s s i o n to clinics of dental colleges of various hospitals of India and reported the frequency of s u b m u c o u s fibrosis to range from 0.2% to 1.2%. The etiology of s u b m u c o u s fibrosis is still unknown. The most likely explanation is that chili, a spicy ingredient of Indian food, may play a role in its onset. Sirsat and Khanolkar [2] d e m o n s t r a t e d e x p e r i m e n t a l l y that painting rat palates with capsicum, the active ingredient of chili, causes a connective tissue response similar to the changes observed in h u m a n s with s u b m u c o u s fibrosis. Patients with oral s u b m u c o n s fibrosis are often characterized by epithelial atypia. Lemmer and Shear [22] reported the incidence of atypia to be 16.6% in patients with s u b m u c o u s fibrosis. In a series of surveys, Pindborg et al. [6, 23, 24] found the frequency of epithelial atypia to range from 7% to 23.8% in patients with oral submucous fibrosis. Similar figures were reported by Seedat [25], who found atypia in 23% of 49 s p e c i m e n s of s u b m u c o u s fibrosis. The extent to w h i c h this high rate of atypia indicates the premalignant potential of oral submucous fibrosis is not precisely known, especially because atypia may not be a permanent feature. In a follow-up of patients with gastric dysplasia, A n d e r s s o n et al. [26] reported that atypia disappeared after 10 to 63 months. Furthermore, submucous fibrosis has been reported in
200
P . K . G h o s h et al.
p a t i e n t s w h o n e v e r c h e w e d t o b a c c o / b e t e l n u t [27, 28]. S o m e p a t i e n t s s h o w e d s e v e r e early changes in the mucosa with minimal betel nut contact, whereas in others this c h a n g e w a s n o t i n d u c e d d e s p i t e p r o l o n g e d b e t e l n u t c h e w i n g [29]. T h i s i n d i c a t e s a v a r y i n g s u s c e p t i b i l i t y a m o n g c h e w e r s w h i c h m a y be g e n e t i c a l l y d e t e r m i n e d . T h a t t h i s m a y b e t r u e w a s r e c e n t l y r e p o r t e d b y C a n n i f e t a ] . [301, w h o p e r f o r m e d HLAt y p i n g i n 50 p a t i e n t s w i t h oral s u b m u c o u s fibrosis. T h e y r e p o r t e d t h a t h a p l o t y p e s A10, B7, a n d DR3 s h o w e d a s i g n i f i c a n t e l e v a t i o n a n d h a p l o t y p e DRT w a s s n b s t a n tially d e c r e a s e d i n t h e s e p a t i e n t s . A s y s t e m a t i c l o n g - t e r m f o l l o w - u p of p a t i e n t s w i t h oral s u b n m c o u s fibrosis w i t h a n d w i t h o u t t h e h a b i t of t o b a c c o / b e t e l n u t c h e w i n g w i t h r e s p e c t to e p i t h e l i a l a t y p i a in r e l a t i o n to c h r o m o s o m a l i n s t a b i l i t y m a y h e l p us u n d e r s t a n d t h e p r o c e s s e s i n v o l v e d in i n i t i a t i o n of oral c a r c i n o g e n e s i s .
REFERENCES 1. Pindborg lJ, Chawla TN, Srivastava AN, Gupta D, Mehrotra ML (1964): Clinical aspects of oral submucous fibrosis. Acta Odont Scand 22:679-691. 2. Sirsat SM, Khanolkar VR (1962): Submucous fibrosis of the palate and pillars of the Fauces. lnd J Med Sci 16:189-197. 3. Pindborg JJ, Sirsat SM (1966): Oral submucous fibrosis. Oral Surg 22:764-779. 4. Pindborg JJ (1980): Oral cancer and precancer. John Wright and Sons, Bristol. 5. Paymaster JC (1956): Cancer of the buccal mucosa: A clinical study of 650 cases in Indian patients. Cancer 9:431 435. 6. Pindborg JJ (1972): Is submucous fibrosis a precancerous condition in the oral cavity? lnt Dent J 22:474-480. 7. Michaels L (1984): Precancerous states in the head and neck. In: Precancerous States, RL Carter, ed. Oxford University Press, London, pp. 129-160. 8. German J (1972): Genes which increase chromosomal instability in somatic cells and predispose to cancer. Prog Med Genet 8:61-101. 9. Wo{ff S (19771: Sister chromatid exchange. Annu Rev Genet 11:183-201. 10. Wolff S, Perry P (1974): Differential Giemsa staining of sister chromatids and the sister chromatid exchanges without autoradiography. Chromosmna 48:34"i-353. 11. Sugiyama T, Togo K, Kano Y (1976): Mechanism of differential Giemsa method for sister chromatids. Nature 259:59-6t). 12. Paymaster JC (1964): Cancer and its distribution in India. Cancer 17:1026-1034. 13. Sanghvi LD (1981): Cancer epidemiology: The Indian scene. J Cancer Res Clin Oncol 99:114. 14. Ghosh R, Ghosh PK (1984): Sister chromatid exchanges in betel and tobacco chewers. Mutat Res 139:79-81. 15. Ghosh R, Ghosh PK (1987): The effect of tobacco smoking on the frequency of sister chromatid exchanges in h u m a n lymphocyte chromosomes. Cancer Genet Cytogenet 27:15-19. 16. Ghosh PK, Ghosh R (1988): Effect of betel chewing on the frequency of sister chromatid exchanges in pregnant women and women using oral contraceptives. Cancer Genet Cytogenet 32:211 216. 17. Ghosh R, Sharma JK, Ghosh PK {1988): Sister chromatid exchanges in the lymphocytes of patients with oral leukoplakia. Cancer Genet Cytogenet 36:177-182. 18. Livingstone GK, Fineman RM (1983): Correlation of h u m a n lymphocyte SCE frequency with smoking history. Mutat Res 119:59-64. 19. Meiying C, Jivjin X, Xianting Z {1982}: Comparative studies on spontaneous and mitomycin-C induced sister chromatid exchanges in smokers and non-smokers. Mutat Res 105:195-200. 20. Pindborg JJ, Chawla TN, Misra RK, Nagpaul RK, Gupta VK (1965): Frequency of oral carcinoma, leukoplakia, leukokeratosis, leukoedema, submucous fibrosis and lichen planus in 10,000 Indians in Lucknow, Uttar Pradesh, India: Preliminary report. J Dent Res 44:615.
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203_
21. Zachariah J, Mathew B, Verma NAR, Iqbal AM, Pindborg JJ (1966): Frequency of oral mucosal lesions among 5000 individuals in Trivandrum, South India. J Ind Dent Assoc 38:290-294. 22. Lemner J, Shear M (1968): Precancerous and cancerous lesions of the mouth. J Dent Assoc S Afr 23:274-285. 23. Pindborg JJ (1966): Oral submucous fibrosis as a precancerous condition. J Dent Res 45:546 553. 24. Pindborg JJ, Mehta FS, Daftary DK (1970): Occurrence of epithelial atypia in 51 Indian villagers with oral submucous fibrosis. Br I Cancer 24:253 257. 25. Seedat HA (1986): Oral submucous fibrosis in Durban, Natal: A study of its epidemiology, aetiology and morphological features. Thesis, University of Stellenbosch, pp. 118 122. 26. Andersson AP, Lavritsen KB, West F, Johansen A (1987): Dysplasia in gastric: mucosa: Prognostic significance. Acta Chir Scand 153:29-31. 27. Paissat DK (1981): Oral submucous fibrosis. Int I Oral Surg 10:307-312. 28. Seedat HA, Van Wyk CW (1988): Submucous fibrosis in non-betel nut chewing subjects. I Biol Buccale 16:3-6. 29. Seedat HA, Van Wyk CW (1988): The oral features of betel nut chewers without submucous fibrosis. J Biol Buccale 16:123-138. 30. Cannif JP, Batchelor JR, Dodi IA, Harvey W (1985): HLA-typing in oral submucous fibrosis. Tissue Antigens 26:138-142.
ABSTRACT: The incidence of sister chromatid exchange (SCE) was investigated in the lymphocyte chromosomes of 45 patients with oral submucous fibrosis and 56 age- and sex-matched nansmoking controls. The frequency of SCE was 9.26 ± 2.15 in patients with oral submucous fibrosis, which was significantly higher than the mean SCE value of 5.49 + 1.24 observed in normal controls. The frequency of SCE in patients with oral submucous fibrosis addicted to the habit of betel with tobacco chewing, "bidi'/cigarette smoking and combined habits of chewing and smoking of tobacco were 8.12 ± 1.69, 9.43 ± 1,87, and 10.06 ± 2.28, respectively. These values were also significantly higher as compared with the SCE values observed in normal calltro]s.
INTRODUCTION S u b m u c o u s fibrosis is a chronic disease affecting the mucosa of any part of the oral cavity and occasionally extending into the pharynx and esophagus. Onset of the condition is insidious and often is of 2 to 5 years duration. The most c o m m o n initial symptom is a b u r n i n g sensation in the mouth, often experienced when the patient is eating spicy food. The condition is ahvays accompanied by an inflammatory reaction. Patients often experience difficulty in eating, chewing, swallowing, and speaking [1]. Clinically, s u b m u c o u s fibrosis causes whitish, blanched depigmented areas of the oral mucosa [2]. The disease occurs mainly among Indians, but sporadic cases have been observed in other countries of East Asia [3]. S u b m u c o u s fibrosis of the oral cavity has recently been associated with an increased t e n d e n c y for local cancer development [4]. The precancerous nature of submucous fibrosis was first m e n t i o n e d by Paymaster [5]. In a survey in South India, s u b m u c o u s fibrosis was observed in 40% of 100 consecutive oral cancer patients [6l. Follow-up studies in India have clearly demonstrated that about one-third of the patients with oral s u b m u c o u s fibrosis develop squamous cell carcinoma [7]. All this evidence clearly indicates the precancerous nature of subnmcous fibrosis. The etiological factors responsible for initiation of oral malignancy are not precisely known. However, it has been argued that the habit of tobacco chewing compared with tobacco smoking plays an important role in the transformation of submucous fibrosis through distinct preneoplastic changes to carcinoma of the oral cavity. Because chromosomal instability has long been associated with neoplastic disorders [8] and the quantitative assay of sister chromatid exchange (SCE) provides an easy, From the CytogeneticsLaboratory, Departmentof Anthropology,Universityof Delhi. Delhi, India. Address reprint requests to: Dr. P. K. Ghosh, Reader, Department of Anthrapoh)gy, University of Delhi, Delhi-110007, India. Received March 2, 1989; accepted/une 30, 1989.
197 (~21990 Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas,New York, NY 10010
Cancer Genet Cytogenet44:197 201 (1990) 0165-4608/90/$03.50
198
P.K. Ghosh et al.
rapid, and sensitive method for studying c h r o m o s o m e / D N A instability and its subsequent repair processes [9], an attempt was made in the present study to investigate SCE levels in the peripheral blood of patients with submucous fibrosis and to correlate them with the habits of different forms of tobacco usage.
MATERIALS AND METHODS Peripheral blood samples were collected from 45 cases of oral submucous fibrosis. S u b m u c o u s fibrosis was diagnosed solely on clinical grounds and only w h e n the patients exhibited the presence of palpable fibrous bands. The control group consisted of 56 age-matched normal individuals with no history of tobacco usage or radiation or viral exposure. The mean age was 41.53 years (range 24 to 58 years) for i n d i v i d u a l s with oral s u b m u c o u s fibrosis and 39.25 years (range 22 to 55 years) for the controls. Detailed history regarding the different habits of tobacco usage ( " b i d i ' / cigarette smoking and betel/tobacco chewing) were taken from each patient with oral s u b m u co u s fibrosis. The past history with regard to oral symptoms was also collected from each patient. All the individuals studied in the present investigation were males. Whole blood l y m p h o c y t e s from all individuals were cultured in TC-199 m e d i u m s u p p l e m e n t e d with 20% fetal calf serum, phytohemagglutinin, and antibiotics. Brom o d e o x y u r i d i n e (BrdU) was added to a concentration of 3/zg/mL and the 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 /zg/ml. After hypotonic treatment with KC1 and fixation in methanol : acetic acid (3 : 1), the slides were prepared by air drying. Differential staining of sister chromatids were obtained according to the technique of Wolff and Perry [10] and Sugiyama et al. [11] with slight modifications. One-day-old slides were stained with 5.0/~g/ml Hoechst 33258 in the dark for 30 minutes. They were washed with distilled water, m o u n t e d in 2 x SSC with a coverslip, and exposed to sunlight for 2 hours. They were again washed with distilled water and stained in 5% Giemsa. SCEs were scored blindly in 50 second division metaphase cells per individual. The differences b e tw e e n the controls and the patients with submucous fibrosis were statistically evaluated with the paired t test.
RESULTS Data on the mean frequency of SCE per cell in patients with oral submucous fibrosis and normal controls are s h o w n in Table 1. Patients with oral submucous fibrosis had a mean SCE frequency of 9.26 -+ 2.15, w h i c h was significantly higher than the mean
Table 1
Mean frequencies of SCE in patients with oral submucous fibrosis in relation to different habits of tobacco usage
Series
Group/tobacco habits
1
Normal controls/no tobacco habits Oral submucous fibrosis a) Betel with tobacco chewing b) Bidi/cigarette smoking c) Combined habits ~'
2
No. of subjects
Mean age (yr}
SCE per cell Mean ± SD (range)
56
39.25
5.49 ± 1.24 (0-11)
45 11 13 21
41.53 39.95 41.26 42.18
9.26 8.12 9.43 10.06
± ± ± ±
2.15 (1-20) 1.69 (1-16) 1.87 (2-18} 2.28 (3-20)
dPersons addicted to both tobacco chewing and smoking. Differences between series 1 and 2. l and 2a, l and 2b, 1 and 2c were signii~cant {p < 0.001} by Student's t test.
SCE in Oral S u b m u c o u s Fibrosis
199
SCE value of 5.49 -+ 1.24 observed in normal controls (p < 0.001). The mean SCE frequency in patients with oral submucous fibrosis who were addicted to the single habit of betel with tobacco chewing or bidi/cigarette smoking or to c o m b i n e d habits of c h e w i n g and smoking tobacco was 8.12 -+ 1.69, 9.43 -+ 1.87, and 10.06 -+ 2.28, respectively. These values were also significantly higher as c o m p a r e d with the SCE values observed in normal controls (p < 0.001). There was significant correlation between n u m b e r of betel leaves with tobacco chewed and mean SCE frequency (r = 0.65; p < 0.001) and also the number of cigarettes or bidis smoked and the mean SCE frequency (r = 0.68;p p < 0.001). DISCUSSION
Oral cancer presents a major health problem in India. The buccal cavity and p h a r y n x are the areas most frequently involved by cancer and constitute 34.9% of the total cancers in India [12]. Tobacco usage is an important etiological factor in development of cancer of oral e p i t h e l i u m [13]. In India, tobacco is smoked in the form of cigarettes in urban areas, whereas in rural areas, particularly in the lower socioeconomic classes, the prevalent habit is bidi smoking. The other important form of tobacco usage in India is betel chewing, w h i c h is practiced in a variety of ways. Every patient with oral s u b m u c o u s fibrosis studied in the present investigation was addicted to either one form of tobacco usage or the other: 24.4% of the patients with oral s u b m u c o u s fibrosis were a d d i c t e d to betel with tobacco chewing, 28.9% smoked tobacco in the form of bidi or cigarettes, and 46.7% were habituated to the combination of tobacco chewing and tobacco smoking. Thus, the increase in frequency of SCEs observed in patients with oral submucous fibrosis may be attributed to their a d d i c t i o n to tobacco habits, as has been w i d e l y reported [14-19]. The SCE frequencies in patients with oral s u b m u c o u s fibrosis varied according to the mode of tobacco usage. The single habits of betel/tobacco chewing and bidi/cigarette smoking and the c o m b i n e d habits of chewing and smoking tobacco i n d u c e d a mean elevation of 2.63, 3.94, and 4.57 SCEs, respectively, as c o m p a r e d with the SCE values observed in normal controls. Thus, the c o m b i n e d habits of tobacco smoking and chewing may i n d u c e more c h r o m o s o m a l instability as c o m p a r e d with the single habits of betel with tobacco chewing or tobacco smoking alone. About 250,000 persons in India are believed to have submncous fibrosis. Epidemiological studies on the prevalence of submucons fibrosis have been c o n d u c t e d by Pindborg et al. [20] and Zachariah et al. [21], who examined 35,000 urban Indians seeking a d m i s s i o n to clinics of dental colleges of various hospitals of India and reported the frequency of s u b m u c o u s fibrosis to range from 0.2% to 1.2%. The etiology of s u b m u c o u s fibrosis is still unknown. The most likely explanation is that chili, a spicy ingredient of Indian food, may play a role in its onset. Sirsat and Khanolkar [2] d e m o n s t r a t e d e x p e r i m e n t a l l y that painting rat palates with capsicum, the active ingredient of chili, causes a connective tissue response similar to the changes observed in h u m a n s with s u b m u c o u s fibrosis. Patients with oral s u b m u c o n s fibrosis are often characterized by epithelial atypia. Lemmer and Shear [22] reported the incidence of atypia to be 16.6% in patients with s u b m u c o u s fibrosis. In a series of surveys, Pindborg et al. [6, 23, 24] found the frequency of epithelial atypia to range from 7% to 23.8% in patients with oral submucous fibrosis. Similar figures were reported by Seedat [25], who found atypia in 23% of 49 s p e c i m e n s of s u b m u c o u s fibrosis. The extent to w h i c h this high rate of atypia indicates the premalignant potential of oral submucous fibrosis is not precisely known, especially because atypia may not be a permanent feature. In a follow-up of patients with gastric dysplasia, A n d e r s s o n et al. [26] reported that atypia disappeared after 10 to 63 months. Furthermore, submucous fibrosis has been reported in
200
P . K . G h o s h et al.
p a t i e n t s w h o n e v e r c h e w e d t o b a c c o / b e t e l n u t [27, 28]. S o m e p a t i e n t s s h o w e d s e v e r e early changes in the mucosa with minimal betel nut contact, whereas in others this c h a n g e w a s n o t i n d u c e d d e s p i t e p r o l o n g e d b e t e l n u t c h e w i n g [29]. T h i s i n d i c a t e s a v a r y i n g s u s c e p t i b i l i t y a m o n g c h e w e r s w h i c h m a y be g e n e t i c a l l y d e t e r m i n e d . T h a t t h i s m a y b e t r u e w a s r e c e n t l y r e p o r t e d b y C a n n i f e t a ] . [301, w h o p e r f o r m e d HLAt y p i n g i n 50 p a t i e n t s w i t h oral s u b m u c o u s fibrosis. T h e y r e p o r t e d t h a t h a p l o t y p e s A10, B7, a n d DR3 s h o w e d a s i g n i f i c a n t e l e v a t i o n a n d h a p l o t y p e DRT w a s s n b s t a n tially d e c r e a s e d i n t h e s e p a t i e n t s . A s y s t e m a t i c l o n g - t e r m f o l l o w - u p of p a t i e n t s w i t h oral s u b n m c o u s fibrosis w i t h a n d w i t h o u t t h e h a b i t of t o b a c c o / b e t e l n u t c h e w i n g w i t h r e s p e c t to e p i t h e l i a l a t y p i a in r e l a t i o n to c h r o m o s o m a l i n s t a b i l i t y m a y h e l p us u n d e r s t a n d t h e p r o c e s s e s i n v o l v e d in i n i t i a t i o n of oral c a r c i n o g e n e s i s .
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