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Carcinogenic and genotoxic effects of the tobacco substitute pan masala: present status and likely future impact on the Indian population
Cancer
Treatment
Reviews
(1996)
22,345-354
Carcinogenic and genotoxic effects of the tobacco substitute pan masala: present status and likely future impact on the Indian population A. H. Trivedi*, S. R. Bakshi*,
D. B. Balart, P. M. Shah*, V. B. R. Dinavahill
D. D. PateI%, R. K. Patel*,
* Cell Biology Division, Department of Cancer Biology, t Departments of Pathology and Cancer Biology, * Department of Medical Oncology, 5 Department of Surgical Oncology and 11Depattment of Community Oncology, The Gujarat Cancer & Research Institute, NCH Campus, Asarwa, Ahmedabad 380016, India
Introduction The human population is inevitably exposed to a number of chemical mutagens/ carcinogens either accidentally, occupationally or by life style. Almost 80-90% of cancers are attributable to such factors. Nevertheless, cancers which are linked with the exposure to carcinogen by life style can be largely avoided by restricting the exposure. Among the risk factors associated with personal habits, tobacco consumption seems to be the most important. A wide spectrum of tobacco products are available for human consumption. They can be classified into two fundamental patterns, i.e. tobacco smoking and oral use of unburnt tobacco (smokeless tobacco). Irrespective of its mode of consumption, tobacco is carcinogenic. A close correlation between the manner of its consumption and site of subsequent cancers has also been observed (I-3). Morbidity due to tobacco-related cancers in India is 48% in men and 20% in women, with an overall estimate of 33% for the two sexes (4). Thus, in view of the popularity and diversity of tobacco habits in India, and the severity of associated ill-effects on health, studies on tobacco-related products have become a priority research area for India. The present paper outlines the health hazards from the oral use of smokeless tobacco in India, and focuses specifically on the possible harmful effects of ‘pan masala’, a new substitute for betel quid and tobacco chewing.
Types of smokeless
tobacco
in use
Tobacco was introduced in India about 400 years ago, primarily as a substance for smoking. Later on, it became an ingredient of betel quid which has traditionally consisted of betel leaf, pieces of areca nut, lime, sweetening, flavouring agents etc. (5). Currently, smokeless tobacco is consumed in a variety of ways ranging from chewing tobacco alone, tobacco with lime, tobacco with 0305-7372/96/050345
0 1996
+ 10 $12.00/O 345
W.B.
Saunders
Company
Ltd
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A. Ii. TRIVEDI ETAL.
areca nut and lime, tobacco as an ingredient of betel quid (colloquially termed as ‘pan’), snuff (powdered tobacco), masheri (pyrolysed tobacco product) etc. In recent years, the trend for chewing betel quid, tobacco and areca nut has shifted to a new product ‘pan masala’ which was introduced in around 1975 and is commercially marketed under various brand names.,At almost every ‘pan shop’, it is available in small pouches containing about 4-5g of the material. Partly under the pretext of being a safer alternative but also because of the opinion against tobacco smoking, pan masala has gained acceptance even among women and children who generally refrain from smoking and other tobacco habits. Behavioural differences, which include convenience, detectability, duration and frequency of use, seem to play an important role in the adoption of this product. The advertisements in print and the media have linked the use of pan masala to social and general public functions. Peer influence, emulating the elders and a desire to match various successful personalities are some of the reasons for its popularity among the literate upper and middle classes of urban population.
Composition
of pan masala
The number of constituents and their concentrations in pan masala varies from brand to brand, and exact details are not available. However, depending on taste and content, there are three main types of pan masala, namely: (1) pan masala plain; (2) sweet pan masala; and (3) pan masala with tobacco-also known as gutkha. As mentioned on its packaging, pan masala is a dry mixture of various ingredients, mainly areca nut, catechu, lime, menthol, sandal oil, spices and flavours (unspecified). Thus, pan masala has all the major ingredients of betel quid except betel leaf. Areca nut accounts for about 70-60% of total weight, catechu forms about IO%, lime about 1% and the remainder includes various spices and flavouring agents. Dry dates are often added to make sweet pan masala, while the varieties of pan masala with tobacco (gutkha) also contain powdered tobacco of uncertain quantity and quality. The tobacco used in pan masala may be either raw tobacco or a processed form such as ‘Zarda’, prepared by cutting tobacco leaves into small pieces, boiling them in water with lime and spices until evaporation, followed by drying and colouring with vegetable dyes (2). Tricker and Preussmann (6) estimated the levels of various tobaccospecific nitrosamines (TSNA), including N-nitrosodiethanolamine, and volatile and non-volatile N-nitroso compounds, in zarda samples obtained from both India and a zarda-chewing Indian community in London (U.K.). They reported very high concentrations of TSNAs in zarda. Moreover, they identified for the first time the presence of pre-formed N-nitrosoethylmethylamine as well as the non-volatile compounds N-nitrososarcosine, N-nitrosoazotidine-4-carboxylic acid and N-nitrosothiazolidine-4-carboxylic acid in the tobacco products. Chemical analysis of five different types of pan masala (plain as well as with tobacco) has been carried out at the National Institute of Occupational Health, Ahmedabad, India (7). The results showed the presence of polycyclic aromatic hydrocarbons, nitrosamines, toxic metals (such as Pb, Cd and Ni) and residual pesticides in pan masala.
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Biological Epidemiological
MASALA
data on harmful
effects
347
of pan masala
studies
Reports on epidemiological studies associating consumption of pan masala (with or without tobacco) with oral cancer are not yet available. Recently, a baseline survey about cancer awareness and the prevalence of various habits was conducted in the Panchmahals district of Gujarat State, India during 1994-95 by the Community Oncology Department of the Gujarat Cancer & Research Institute. It was observed that among the different habits prevailing in the community, 4.5% individuals admitted to the habit of chewing tobaccofree pan masala. The habit was predominant in the youngsters of ~25 years of age, and decreased drastically with increasing age. Similar studies were also conducted in the Banaskantha district and Bharuch district during the same time. A comparable proportion of pan masala chewers; i.e. about 4%, is expected (unpubl. data). While evaluating serum markers in patients with oral submucous fibrosis, Anuradha and Shyamala Devi (8) have reported that patients who chewed pan masala had a shorter duration history of chewing as compared to those who chewed betel nut and betel nut + betel leaf, and hence suggested that pan masala has an acute effect. However, unequivocal epidemiological evidence causally associating pan masala chewing with oral cavity cancer is not yet available.
Experimental
studies
Animal studies. Very few studies have been reported on carcinogenic effects of pan masala in animals. Sinha (9) observed dysplasia in 95% of albino rats after application of a paste of pan masala with tobacco to the entire buccal mucosa, as compared to 14% of control rats. Khrime et al. (IO) have observed mild leukoplakia and submucous fibrosis in the oral cavity of albino rats following painting with instant preparations of betel nut (pan masala). After a period of 6 months exposure to the paste of pan masala, mild to moderate loss of nuclear polarity and increases in karatoses, parakeratoses, inflammatory cell infiltration and vascularity were noted as compared to the control group. Sarma et a/. (11) have assessed the acute and chronic toxicity of pan masala (betel quid without betel leaf) in rats, and reported that chronic feeding of pan masala impaired the liver function and decreased relative weights of the gonads and brain. Mukherjee et a/. (12) have conducted cytogenetic analysis of meiotic metaphase I germ cells in male mice following oral feeding of pan masala, and identified abnormalities of head morphology in spermatazoa. They also reported a significant increase in the frequency of X-Y univalents and breaks at high doses of pan masala. The frequency of sperm head abnormalities was significantly increased at all doses tested. In another study, Mukherjee and Giri (13) have reported a significant dose-related increase in SCE frequency in male mice after intraperitoneal injection of an aqueous suspension of pan masala. Kashyap et a/. (7) have carried out toxicological evaluation of pan masala using albino Swiss mice. A significant increase in chromosomal abnormalities in bone marrow cells was observed following single intraperitoneal injection of pan
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masala at relatively low doses in the range of 6-200 mg/kg body weight. Oral administration also caused somatic chromosome aberrations as well as sperm head abnormalities. A dose-related increase in chromosome aberration frequency was also observed in bone marrow cells of mice fed with either plain pan masala or pan masala with tobacco (14).
Short-term tests. In view of the presence of various mutagenic and nonmutagenic constituents in pan masala, an investigation of its genotoxic potential is necessary for the safety evaluation of its consumption. The identification of carcinogens by long-term animal experiments has always been the system of choice. However, the shortcomings of this assay are the cost and the time involved. These have led to searches for alternative methods. Numerous shortterm tests have been developed and are being used successfully for detecting the ability of chemicals to cause alterations in genetic material and thereby to play a crucial role in causing cancer. Compounds which exhibit carcinogenic activity generally also exhibit genotoxic activity (15). Thus, genetic t.oxicology has major application in determining the outcome of exposure to genotoxic carcinogens. It is relatively simple to use the induction of mutation in bacteria as a very sensitive indirect indicator of DNA damage. A substance found to be mutagenic in properly conducted bacterial mutation assays may be regarded as potentially genotoxic to mammals until proved otherwise. It has also been suggested that in genetic toxicology studies, the in vitro cytogenetic assays should take a central role in a test battery (16). Short-term assays like the micronucleus (MN) test, chromosome aberration (CA) analysis and sister chromatid exchange (SCE) analysis are sensitive and well-established cytogenetic markers of DNA damage. Both the in vitro and in vivo induction of a micronucleus by carcinogens and mutagens are indicators of genomic damage (17-19). Stich et a/. (20-22) have widely applied the MN assay at a population level for studying the effects of tobacco/areca nut chewing habits on the buccal mucosa. The test permits a non-invasive assessment of human tissues as well as an opportunity for repeated samplings which appear to be ethically and emotionally acceptable. Compared to MN test, metaphase analysis for CAfrequency has been considered as a more sensitive method for identifying clastogenic compounds (23). Genomic damage, caused by exposure to harmful environmental agents, which may ultimately increase the risk of cancer, can be assessed in terms of CA frequency in the peripheral blood lymphocytes (24). The misrepaired chromosomal lesions, which escape being detected as CA, can be detected effectively by studying SCE frequencies. Sister chromatid exchange analyses are reciprocal exchanges between sister chromatids. Cells with SCEs are capable of subsequent growth, and hence SCE elevation may be a more valid indicator of heritable genomic damage. However, the SCE assay should be considered as a complement to, rather than a substitute for, CA analysis (25). Induction of SCE showed good correlation with mutation frequency and cell transformation in several cell lines (26,271. Increased SCE rates have been reported in cells of individuals exposed to various environmental mutagens/carcinogens, e.g. in cigarette smokers (26,291, and in betel and tobacco chewers (30). Taking into consideration all the above facts,
PAN
MASALA
results of these assays have been included of pan masala.
349
to evaluate
the genotoxic
potential
(I) Bacterial test system. Bagwe et a/. (31) have evaluated the mutagenicity of pan masala after aqueous extraction, hot aqueous:ethanolic extraction, hot chloroform extraction, ethanolic extraction and pre-treatment of extracts with nitrite at acidic pH using the Salmonella/mammalian microsome test (Ames assay) and two tester strains of Salmonella typhimurium, TA98 and TAIOO. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation. Using tester strains TA98 and TAIOO, Polasa et al. (32) found a significant dose-dependent elevation of revertants with extracts of pan masala. (2) Mammalian test system: The effects of aqueous and organic (dimethyl sulphoxide) extracts of plain pan masala (PM) as well as pan masala with tobacco (PM-T) were tested on Chinese hamster ovary (CHO) cells employing three cytogenetic endpoints viz. CA, SCE and percent micronucleated cells (%MNC). The frequencies of all three endpoints were found to be significantly increased by an aqueous extract of pan masala in a dose-dependent manner in cultures treated without metabolic activation. However, addition of S9 activation system resulted in suppression of chromosomal damage (33). Similarly, the genotoxic potential of dimethyl sulphoxide (DMSO) extracts were also tested in the presence or absence of a metabolic activation system (34). In cultures without metabolic activation, the extracts were found to significantly increase the frequency of all three parameters tested; however, those with activation elicited a weak response. The data suggested, therefore, that pan masala (without and with tobacco) contains water-soluble as well as DMSO-soluble direct-acting mutagens. A dose-dependent increase in CA frequency following treatment with pan masala with and without tobacco in combination with ethanol indicated that alcohol consumption may potentially elevate the risk of oral cancer among pan masala chewers (35). Human
studies
The pharmacological handling and metabolic conversion of a compound(s) in human beings too often complicates the extrapolation of carcinogenesis data from in vitro assays to the heterogeneous human population. Furthermore, for predicting genotoxicity of a carcinogen, it has been suggested that in vivo genotoxicity data would serve well to substantiate the in vitro data (36). A significantly higher frequency of micronucleated cells in exfoliated buccal mucosa (a target tissue), and high frequencies of CA and SCE in peripheral blood lymphocytes (a non-target tissue) have been observed in users of both plain pan masala and pan masala with tobacco when compared with a control population (37,381. Relevant
biological
data on major constituents
of pan masala
The genotoxic effects of pan masala can be attributed mainly (and, of course, to tobacco in the case of tobacco-containing
to the areca nut varieties of pan
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A. H. TRIVEDI ETAL.
masala). Areca nut is a major constituent of pan masala (70-80% by weight). Several epidemiological studies have revealed an association between areca nut consumption and the occurrence of pre-malignant and malignant oral diseases (2,39). A significantly higher frequency of % MNC in exfoliated buccal mucosa, and increased frequencies of CA and SCE in the lymphocytes of chewers of areca nut per se (40,411 as well as in chewers of tobacco with areca nut (421, have been reported. The clastogenicity of the saliva of areca nut chewers to CHO cells has also been repot-ted (43). Several animal experiments have provided evidence of the carcinogenicity of areca nut extracts and its tannins (44,451. Genotoxic effects of areca nut on CHO cells (461, mouse bone marrow cells, V79 Chinese hamster cells (47) and mouse lymphocytes (48) have been documented. Some of the areca nut specific alkaloids have been found to possess transforming (49) and clastogenic properties (46,501. Wenke and Hoffmann (51) have reported the conversion of these areca nut specific alkaloids to nitrosamines, and some of them have even been detected in the saliva and urine of betel quid chewers (52,531. The tobacco present in ‘gutkha’ varieties of pan masala is one of the major reasons for the addiction observed among chewers of pan masala with tobacco. The International Agency for Research on Cancer (2) has reviewed the exposure data, experimental data and human data available on tobacco habits other than smoking. The literature available clearly indicated that oral use of smokeless tobacco is playing a major role in causation of cancer of the oral cavity. The present authors have also carried out in vitro short-term assays with: (I) aqueous extract of unprocessed tobacco (Nicotiana tabacum); (2) nicotine, a major alkaloid of tobacco; and (3) nicotine plus arecoline, a combination of the major alkaloids of tobacco and areca nut. It was observed that aqueous extract of tobacco is genotoxic to CHO cells (54). Secondly, nicotine, in addition to being addictive, is genotoxic (55), and combined application of nicotine and arecoline results in more severe effects than nicotine alone (56). The observations of cytogenetic monitoring of smokeless tobacco users also clearly suggested that the habit causes genomic damage to the oral mucosa (a target tissue) as well as to the peripheral blood lymphocytes (a non-target tissue) (57). The results of evaluation of chromosomedamaging effects of urine concentrates from tobacco plus areca nut chewers indicated that besides the oral cavity, mutagens/carcinogens present in tobacco and areca nut might also be playing a causative role in cancer of the urinary bladder (58). Details of spices and flavouring agents used in the preparation of pan masala are not always revealed by the manufacturers. However, the occasional use of synthetic flavours like musk ambrette and musk xylene, to improve the taste and flavour of zarda, is well known. Nair et al. (59) have detected both of these agents in the saliva of chewers of betal quid with tobacco. Mutagenicity of these agents has also been reported in the Salmonella/mammalian microsome test (59). The role of lime in the development of oral cancer remains a mystery. However, it causes local irritation to the mucosa, and hyperplasia has been observed following the application of lime to the cheek pouch of hamsters (60). Catechu, another ingredient of pan masala, contains 2-10% catechin (61) and has hepatoprotective effects (62). Monteith (63) demonstrated the ability of
PAN
MASALA
351
catechin to alter the metabolic pathway of 2-acetyl-aminofluorene (AAF) in rat hepatocytes. Nagabhushan et a/. (64) observed that catechin inhibited mutagenicity of B(a)P or DMBA, and also inhibited the in vitro binding of 3HB(a)P metabolites to DNA in a dose-dependent manner. It has also been reported to inhibit some of the mutagenicity of tobacco (65). On the other hand, Giri et al. have reported genotoxicity (66). In addition to all these facts, the quality of the basic ingredients mixed for preparing pan masala is also an important factor. Fungal infection, if any, to tobacco and/or areca nut may affect the ultimate toxicity of the mixture. When the areca nuts are infected with Aspergillus flavus or Aspergillus niger (67-69), they are likely to contain aflatoxins, which have known genotoxic/carcinogenic effects (70). Overall, these data indicate that pan masala has carcinogenic potential and may be causally associated with oral cavity cancers.
Implications
of present
knowledge
and future
direction
Unlike betel quid, which is almost always freshly prepared, pan masala is a dry powdered mixture of various ingredients, mainly areca nut and tobacco. Thus, it represents a complex mixture of harmful constituents. As mentioned earlier, there has been irrefutable evidence that the use of tobacco is a major health hazard. It is also very clear from the abovementioned experimental data that pan masala has the ability to cause DNA damage in bacterial and in vitro mammalian test systems in animal models as well as among pan masala chewers. However, there is currently a paucity of epidemiological data causally associating oral pre-cancerous and cancerous lesions with pan masala chewing habits. Moreover, the degree of transformation of pre-cancerous lesions to cancerous lesions among pan masala chewers, and knowledge about the factors associated with it are also lacking. However, genotoxicity and carcinogeneicity data on areca nut and tobacco, ingredients of pan masala, provide enough reasons to consider pan masala chewing as a harmful habit and a likely association with cancer of the oral cavity. Since a long latent period may exist between exposure and manifestation of the disease, the harmful effect of a habit will be observed only after a prolonged period of time. Hence, close surveillance and follow-up epidemiological and molecular biology studies will definitely prove to be one of the significant contributions in the field. Such studies are currently under consideration at the authors’ institute which will help the relevant authorities to take more realistic steps towards controlling the sale and indiscriminate use of pan masala.
Acknowledgement The authors thank the Indian financial support.
Council
of Medical
Research,
India for providing
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ETAL.
References 1. IARC (1987) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Soppl. 7, Overall Evaluations of Carcinogenicity: An Updating of /ARC Monographs Volumes I-42. Lyon: International Agency for Research on Cancer, pp.357381. 2. IARC (1985) Monooraohs on the Evaluation of the Carcinoaenic Risk of Chemicals to Humans, Vol. 37. Be& Quid&b Areca Nut Cheving and Some Nitro:amines. Lyon: International Agent; for Research on Cancer. 3. Sanghvi, L. D. (1981) Cancer epidemiology: The Indian scene. J. Cancer Res. C/in. Oncol. 91: 1-14. 4. Sanghvi, L. D. (1989) Tobacco related cancers. In: Sanghvi, L. D. & Notani, P. N., eds, Tobacco and Health: The Indian Scene. Bombay, pp. 9-15. 5. Gupta, P. C. (1992) Smokeless tobacco use in India. Smokeless Tobacco or Health-An International Perspective. N/H Publication No. 93-3461, pp. 19-25. 6. Tricker, A. R. & Preussmann, R. (1988) The occurrence of N-nitro compounds in zarda tobacco. Cancer Left. 42: 113-118. 7. Kashyap, S. K., Nigam, S. K., Karnik, A. B. et a/. (1989) Toxicological evaluation of pan masala. Annual Report (7989-90). National Institute of Occupational Health (Indian Council of Medical Research),.Ahmedabad, India, pp. 60-66. 8. Anuradha. C. D. & Shvamala Devi. C. S. (1993) Serum orotein. ascorbic acid & iron &tissue collagen in oral submhcous fibrosis-a preliminary study. Ind. 2. Med. Res. (6) 98: 147-151. 9. Sinha, B. K. (1991) Fibronectin levels and their correlation with histopathological changes following application of instant ‘pan masala with tobacco’ on buccal mucosa of albino rats. Thesis submitted to Postgraduate Institute of Medical Education and Research, Chandigarh, India. 10. Khrime, R. D., Mehra, Y. N., Mann, S. B. S., Mehta, S. K. & Chakraborti, R. N. (1991) Effect of instant preparation of betel nut (pan masala) on the oral mucosa of albino rats. Ind. J. Med. Res. (6) 94: 119-124. 11. Sarma, A. B., Chakrabatti, J., Chakrabarti A. et a/. (1992) Evaluation of pan masala for toxic effects on liver and other organs. food Chem. Toxicol. 30: 161-163. 12. Mukherjee, A., Chakrabarti, J., Chakrabarti, A., Banerjee, T. & Sarma, A. (1991) Effect of ‘pan masala’ on the germ cells of male mice. Cancer Lett. 58: 161-165. 13. Mukherjee, A. & Giri, A. K. (1991) Sister chromatid exchange induced by pan masala (a betel quid ingredient) in male mice in vivo. Food Chem. Toxicol29: 401-403. 14. Patel, R. K., Jaju, R. J., Bakshi, S. R. et a/. (1996) Pan masala induces chromosome aberrations in mouse bone marrow cells. GCR/ Bulletin 3: 63-67. 15. Tennant, R. W., Margolin, B. H., Shelby, M. D. et a/. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science 236: 933-941. 16. Natarajan, A. T. 81 Obe, G. (1982) Mutagenicity testing with cultured mammalian cells: Cytogenetic assays. In: Heddle, J. A., ed., Mutagenicity: New Horizons in Genetic Toxicology. New York: Academic Press, pp. 171-213. 17. Blakey, D. H., Duncan, A. M. V., Wargovich, M. J., Goldberg, M. T., Bruce, W. R. & Heddle, J. A. (1985) Detection of nuclear anomalies in the colonic epithelium of the mouse. Cancer Res. 45: 242-249. 18. Heddle, J. A., Raj, A. S. & Krepinsky A. B. (1981) The micronucleus assay. II In vitro. In: Stich H. F. & San, R. H. C., eds., Short-term Tests for Chemical Carcinogens. Berlin, Heidelberg: Springer Verlag, pp. 250-254. 19. Ronen, A. & Heddle, J. A. (1984) Site-specific induction of nuclear anomalies (apoptotic bodies and micronuclei) by carcinogens in mice. Cancer Res. 44: 1536-1540. 20. Stich, H. F. (1986) The use of micronuclei in tracing the genotoxic damage in the oral mucosa of tobacco users. In: Hoffman, D. & Harris, C. C., eds., Mechanisms in Tobacco Carcinogenesis. New York: Banbury Report No. 23, Cold Spring Harbor Laboratory, pp. 99-111. 21. Stich, H. F., Acton, A. B. & Palcic, 6. (1990) Towards an automated micronucleus assay as an internal dosimeter for carcinogen-exposed human population groups. In: Band, P., ed., Recent Results in Cancer Research, Vol. 120, Occupational Cancer Epidemiology. Berlin, Heidelberg, New York: Springer Verlag, pp. 94-105. 22. Stich, H. F., Bohm, B. A., Chatterjee, K. & Sailo, J. (1983) The role of saliva-borne mutagens and carcinogens in the etiology of oral and esophageal carcinomas of betel nut and tobacco chewers. In: Stich, H. F., ed., Carcinogens and Mutagens in the Environment, Vol. 3, Naturally Occurring Compounds: Epidemiology and Distribution. Boca Raton, FL: CRC Press, pp. 43-58. 23. Kliesch, U. & Adler, I. D. (1983) Chromosome analysis and micronucleus test in mouse bone marrow are not equally sensitive as shown by results with 5 chemical mutagens. Mutat Res. 113: 340-346.
PANMASALA 24. 25.
26. 27.
28. 29.
30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
40. 41.
42.
43. 44. 45.
46. 47. 48. 49. 50. 51. 52.
353
Carrano, A. V. & Natarajan, A. T. (1988) Considerations for population monitoring using cvtoaenetic techniaues. Mufat. Res. 204: 379-406. Per,, P., Henderson, L. & Kirkland, D. (1984) Sister chromatid exchange in cultured cell. in: Dean, 6. J., ed., Report of the LJKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part II, UKEMS, U.K., pp. 89-121. Popescu, N. C., Amsbaugh, S. C. 61 DiPaolo, J. A. (1981) Relationship of carcinogen induced sister chromatid exchange and neoplastic cell transformation. Int. J. Cancer26 71-77. Popescu, N. C., Amsbaugh, S. C. & DiPaolo, J. A. (1984) Correlation of morphological transformation to sister chromatid exchanges induced by split dosesof chemical or physical carcinogens on cultured Syrian hamster cells. Cancer Res. 44: 1933-1938. Ghosh, R. & Ghosh, P. K. (1987) The effect of tobacco smoking on the frequency of sister chromatid exchanges in human lymphocyte chromosomes. Cancer Genet. Cytogenet27: 15-19. Husum, B., Wulf, H. C. & Niebuhr, E. (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. Ghosh, R. & Ghosh, P. K. (1984) Sister chromatid exchanges in betel and tobacco chewers. Motat. Res. 139: 79-81. Bagwe, A. N., Ganu, U. K., Gokhale, S. V. & Bhisey, R. A. (1990) Evaluation of the mutagenicity of ‘pan masala’, a chewing substitute widely used in India. Motat. Res. 241: 349-354. Polasa, K., Babu, S. & Shenolikar, I. S. (1993) Dose-dependent genotoxic effect of pan masala and areca nut in the Salmonella typhimurium assay. Food Chem. Toxicol. 31: 439-442. Jaju, R. J., Patel, R. K., Bakshi, S. R., Trivedi, A. H., Dave, B. J. 61 Adhvaryu, S. G. (1992) Chromosome damaging effects of pan masala. Cancer Lett. 65: 221-226. Patel, R. K., Jaju, R. J., Bakshi, S. R., Trivedi, A. H., Dave, B. J. & Adhvaryu, S. G. (1994) Pan masala-a genotoxic menace. Mutat. Res. 320: 245-249. Patel, R. K., Trivedi, A. H., Jaju, R. J., Adhvaryu, S. G. 81 Baiar, D. B. (1994) Ethanol potentiates the clastoaenicitv of oan masala-an in vitro experience. Carcinooenesis 15: 2017-2021. Carrano, &. V. & Natarajan, A. T. (1988) Considerations for population monitoring using cytogenetic techniques. Mutat. Res. 204: 379-406. Dave, B. J., Trivedi, A. H. & Adhvaryu, S. G. (1991) Cytogenetic studies reveal increased genomic damage among pan masala consumers. Mutagenesis 6: 159-164. Trivedi, A. H. (1992) A Study on Carcinogenic Potentials of Tobacco. Ph.D. Thesis, M.S. University of Baroda, Baroda, India, pp. 148-179. Gupta, P. C., Mehta, F. S., Daftary, D. K. et a/. (1980) Incidence rates of oral cancer and natural history of oral precancerous lesions in a 10 year follow-up study of Indian villagers. Common. Dent. Oral Epidemiol. 8: 287-333. Dave, B. J., Trivedi, A. H. & Adhvaryu S. G. (1992) Role of areca nut consumption in the cause of oral cancers. Cancer 70: 1017-1023. Stich, H. F., Stich, W. & Parida, B. B. (1982) Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk from oral cancer: Betel quid chewers. Cancer Lett. 17:125-134. Adhvaryu, S. G., Dave, B. J. & Trivedi, A. H. (1991) Cytogenetic surveillance of tobacco-areca nut (mava) chewers, including patients with oral cancers and premalignant conditions. Mutat. Res. 261:41-49. Stich, H. F. 81 Stich, W. (1982) Chromosome damaging activity of saliva of betel nut and tobacco chewers. Cancer Lett. 15: 193-202. Shirname, L. P., Menon, N. M., Nair, J. & Bhide, S. V. (1983) Correlation of mutagenicity and tumorigenicity of betel quid and its ingredients. Nuts: Cancer 5: 87-91. Shivapurkar, N. M., Ranadive, S. N., Gothoskar, S. V., Bhide, S. V. & Ranadive, K. J. (1980) Tumorigenic effect of aqueous and polyphenolic fractions of betel nut in Swiss strain mice. Ind. J. Exp. Biol. 18: 1159-1161. Dave, B. J., Trivedi, A. H. & Adhvaryu S. G. (1992) In vitro genotoxic effects of areca nut extract and arecoline. J. Cancer Res. Clin. Oncol. 118: 283-288. Shirname, L. P., Menon, M. M. & Bhide, S. V. (1984) Mutagenicity of betel-quid and its ingredients using mammalian test systems. Carcinogenesis 5: 501-503. Panigrahi, G. 8. & Rao, A. R. (1986) Study of the genotoxicity of the total aqueous extract of betel nut and its tannin. Carcinogenesis 7: 37-39. Ashby J., Styles, J. A. & Boyland, E. (1979) Betel nuts, arecaidine and oral cancer. Lancet i: 112. Panigrahi, G. B. & Rao, A. R. (1982) Chromosome breaking ability of arecoline, a major betelnut alkaloid, in mouse bone-marrow cells in vivo. Mutat. Res. 103: 197-204. Wenke, G. & Hoffman, D. (1983) A study of betel quid carcinogenesis. 1. On the in vitro Nnitrosation of arecoline. Carcinogenesis 4: 169-172. Nair J., Ohshima H., Friesen, M. Croisy, A., Bhide, S. V. & Bartsch, H. (1985) Tobacco specific
354
53.
54. 55. 56. 57. 58.
59.
60. 61.
62. 63. 64. 65. 66. 67. 68. 69. 70.
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ETAL.
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. Caminogenesis 6: 295-303. Wenke, G., Brunnemann, K. D., Hoffmann, D. & Bhide, S. V. (1984) A study of betel quid carcinogenesis IV. Analysis of the saliva of betel chewers: A preliminary report. .I. Cancer Res. C/in. Oncol. 108: 110-113. Trivedi, A. H., Dave, B. J. 81 Adhvaryu, S. G. (1993) Genotoxic effects of tobacco extract on Chinese hamster ovary cells. Cancer Lett. 70: 107-112. Trivedi, A. H., Dave, B. J. & Adhvaryu, S. G. (1990) Assessment of genotoxicity of nicotine employing in vitro mammalian test system. Cancer Letf. 54: 89-94. Trivedi, A. H., Dave, B. J. & Adhvaryu, S. G. (1993) Genotoxic effects of nicotine in combination with arecoline on CHO cells. Cancer Letf. 74: 105-110. Trivedi, A. H., Dave, B. J. &Adhvaryu, S. G. (1993) Monitoring of smokeless tobacco consumers using cytogenetic endpoints. Anticancer Res. 13: 2245-2249. Trivedi, A. H., Roy, S. K., Jaju, R. J., Patel, R. K., Adhvaryu, S. G. & Balar, D. B. (1995) Urine of tobacco/areca nut chewers causes genomic damage in Chinese hamster ovary cells. Carcinogenesis 16: 205-208. Nair, J., Oshima, H., Malaveille, C. et al. (1966) Identification, occurrence and mutagenicity in Salmonella typhimurium of two synthetic nitroarenes, musk ambrette and musk xylene, in Indian chewing tobacco and betel quid. Food Chem. Toxicol. 24: 27-31. Dunham, L. J., Muir, C. S. & Hamner, J. J. E. Ill (1966) Epithelial atypia in hamster cheek pouches treated repeatedly with calcium hydroxide. Br. J. Cancer 20: 588-593. IARC (1983) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 31, Some Food Additives, and Naturally Occurring Substances. Lyon: International Agency for Research on Cancer, pp. 171-178.213-219. Lapis, K., Jeney, A., Divald, A., Vajta, G., Zalatnai, A. & Schaff, Z. (1986) Experimental studies on the effect of hepatoprotective compounds. Tokai J. Exp. C/in. Med. 11: 135-145. Monteith, D. K. (1990) Catechin inhibition of mutagenesis and alteration of DNA binding of 2acetyl-aminofluorene in rat hepatocytes. Motat. Res. 246: 151-158. Nagbhushan, M., Amonkar, A. J., Nair, U. J. et a/. Catechin as an antimutagen: its mode of action. J. Cancer Res. C/in. Oncol. 114: 177-182. Nagabhushan, M. & Bhide, S. V. (1988) Anti-mutagenicity of catechin against environmental mutagens. Mutagenesis 3: 293-296. Giri A. K., Banerjee, T. S. & Talukdar, G. (1987) Induction of sister chromatid exchange and dominant lethal mutation by ‘katha’ (catechu) in male mice. Cancer Lett. 36: 189-198. Borle, R. M. & Gupta, D. S. (1987) Fungal contamination of Areca nut. Ind. J. Pathol. Microbial. 30: 357-360. Mahdihassan, S. (1981) Aspergillus flavus as the probable carcinogen in betel nuts. Hamdard Medicos 24: 139-I 34. Mahdihassan, S. & Rabia, Z. (1988) Coloured illustrations of betel nuts infected with a cancer producing fungus. Hamdard Medicus 31: 35-36. tARC (1982) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 4, Chemicals, Industrial Processes and Industries Associated with Cancer in Humans (/ARC Monographs Vol. 1-291. Lyon: International Agency for Research on Cancer.
Treatment
Reviews
(1996)
22,345-354
Carcinogenic and genotoxic effects of the tobacco substitute pan masala: present status and likely future impact on the Indian population A. H. Trivedi*, S. R. Bakshi*,
D. B. Balart, P. M. Shah*, V. B. R. Dinavahill
D. D. PateI%, R. K. Patel*,
* Cell Biology Division, Department of Cancer Biology, t Departments of Pathology and Cancer Biology, * Department of Medical Oncology, 5 Department of Surgical Oncology and 11Depattment of Community Oncology, The Gujarat Cancer & Research Institute, NCH Campus, Asarwa, Ahmedabad 380016, India
Introduction The human population is inevitably exposed to a number of chemical mutagens/ carcinogens either accidentally, occupationally or by life style. Almost 80-90% of cancers are attributable to such factors. Nevertheless, cancers which are linked with the exposure to carcinogen by life style can be largely avoided by restricting the exposure. Among the risk factors associated with personal habits, tobacco consumption seems to be the most important. A wide spectrum of tobacco products are available for human consumption. They can be classified into two fundamental patterns, i.e. tobacco smoking and oral use of unburnt tobacco (smokeless tobacco). Irrespective of its mode of consumption, tobacco is carcinogenic. A close correlation between the manner of its consumption and site of subsequent cancers has also been observed (I-3). Morbidity due to tobacco-related cancers in India is 48% in men and 20% in women, with an overall estimate of 33% for the two sexes (4). Thus, in view of the popularity and diversity of tobacco habits in India, and the severity of associated ill-effects on health, studies on tobacco-related products have become a priority research area for India. The present paper outlines the health hazards from the oral use of smokeless tobacco in India, and focuses specifically on the possible harmful effects of ‘pan masala’, a new substitute for betel quid and tobacco chewing.
Types of smokeless
tobacco
in use
Tobacco was introduced in India about 400 years ago, primarily as a substance for smoking. Later on, it became an ingredient of betel quid which has traditionally consisted of betel leaf, pieces of areca nut, lime, sweetening, flavouring agents etc. (5). Currently, smokeless tobacco is consumed in a variety of ways ranging from chewing tobacco alone, tobacco with lime, tobacco with 0305-7372/96/050345
0 1996
+ 10 $12.00/O 345
W.B.
Saunders
Company
Ltd
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A. Ii. TRIVEDI ETAL.
areca nut and lime, tobacco as an ingredient of betel quid (colloquially termed as ‘pan’), snuff (powdered tobacco), masheri (pyrolysed tobacco product) etc. In recent years, the trend for chewing betel quid, tobacco and areca nut has shifted to a new product ‘pan masala’ which was introduced in around 1975 and is commercially marketed under various brand names.,At almost every ‘pan shop’, it is available in small pouches containing about 4-5g of the material. Partly under the pretext of being a safer alternative but also because of the opinion against tobacco smoking, pan masala has gained acceptance even among women and children who generally refrain from smoking and other tobacco habits. Behavioural differences, which include convenience, detectability, duration and frequency of use, seem to play an important role in the adoption of this product. The advertisements in print and the media have linked the use of pan masala to social and general public functions. Peer influence, emulating the elders and a desire to match various successful personalities are some of the reasons for its popularity among the literate upper and middle classes of urban population.
Composition
of pan masala
The number of constituents and their concentrations in pan masala varies from brand to brand, and exact details are not available. However, depending on taste and content, there are three main types of pan masala, namely: (1) pan masala plain; (2) sweet pan masala; and (3) pan masala with tobacco-also known as gutkha. As mentioned on its packaging, pan masala is a dry mixture of various ingredients, mainly areca nut, catechu, lime, menthol, sandal oil, spices and flavours (unspecified). Thus, pan masala has all the major ingredients of betel quid except betel leaf. Areca nut accounts for about 70-60% of total weight, catechu forms about IO%, lime about 1% and the remainder includes various spices and flavouring agents. Dry dates are often added to make sweet pan masala, while the varieties of pan masala with tobacco (gutkha) also contain powdered tobacco of uncertain quantity and quality. The tobacco used in pan masala may be either raw tobacco or a processed form such as ‘Zarda’, prepared by cutting tobacco leaves into small pieces, boiling them in water with lime and spices until evaporation, followed by drying and colouring with vegetable dyes (2). Tricker and Preussmann (6) estimated the levels of various tobaccospecific nitrosamines (TSNA), including N-nitrosodiethanolamine, and volatile and non-volatile N-nitroso compounds, in zarda samples obtained from both India and a zarda-chewing Indian community in London (U.K.). They reported very high concentrations of TSNAs in zarda. Moreover, they identified for the first time the presence of pre-formed N-nitrosoethylmethylamine as well as the non-volatile compounds N-nitrososarcosine, N-nitrosoazotidine-4-carboxylic acid and N-nitrosothiazolidine-4-carboxylic acid in the tobacco products. Chemical analysis of five different types of pan masala (plain as well as with tobacco) has been carried out at the National Institute of Occupational Health, Ahmedabad, India (7). The results showed the presence of polycyclic aromatic hydrocarbons, nitrosamines, toxic metals (such as Pb, Cd and Ni) and residual pesticides in pan masala.
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Biological Epidemiological
MASALA
data on harmful
effects
347
of pan masala
studies
Reports on epidemiological studies associating consumption of pan masala (with or without tobacco) with oral cancer are not yet available. Recently, a baseline survey about cancer awareness and the prevalence of various habits was conducted in the Panchmahals district of Gujarat State, India during 1994-95 by the Community Oncology Department of the Gujarat Cancer & Research Institute. It was observed that among the different habits prevailing in the community, 4.5% individuals admitted to the habit of chewing tobaccofree pan masala. The habit was predominant in the youngsters of ~25 years of age, and decreased drastically with increasing age. Similar studies were also conducted in the Banaskantha district and Bharuch district during the same time. A comparable proportion of pan masala chewers; i.e. about 4%, is expected (unpubl. data). While evaluating serum markers in patients with oral submucous fibrosis, Anuradha and Shyamala Devi (8) have reported that patients who chewed pan masala had a shorter duration history of chewing as compared to those who chewed betel nut and betel nut + betel leaf, and hence suggested that pan masala has an acute effect. However, unequivocal epidemiological evidence causally associating pan masala chewing with oral cavity cancer is not yet available.
Experimental
studies
Animal studies. Very few studies have been reported on carcinogenic effects of pan masala in animals. Sinha (9) observed dysplasia in 95% of albino rats after application of a paste of pan masala with tobacco to the entire buccal mucosa, as compared to 14% of control rats. Khrime et al. (IO) have observed mild leukoplakia and submucous fibrosis in the oral cavity of albino rats following painting with instant preparations of betel nut (pan masala). After a period of 6 months exposure to the paste of pan masala, mild to moderate loss of nuclear polarity and increases in karatoses, parakeratoses, inflammatory cell infiltration and vascularity were noted as compared to the control group. Sarma et a/. (11) have assessed the acute and chronic toxicity of pan masala (betel quid without betel leaf) in rats, and reported that chronic feeding of pan masala impaired the liver function and decreased relative weights of the gonads and brain. Mukherjee et a/. (12) have conducted cytogenetic analysis of meiotic metaphase I germ cells in male mice following oral feeding of pan masala, and identified abnormalities of head morphology in spermatazoa. They also reported a significant increase in the frequency of X-Y univalents and breaks at high doses of pan masala. The frequency of sperm head abnormalities was significantly increased at all doses tested. In another study, Mukherjee and Giri (13) have reported a significant dose-related increase in SCE frequency in male mice after intraperitoneal injection of an aqueous suspension of pan masala. Kashyap et a/. (7) have carried out toxicological evaluation of pan masala using albino Swiss mice. A significant increase in chromosomal abnormalities in bone marrow cells was observed following single intraperitoneal injection of pan
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masala at relatively low doses in the range of 6-200 mg/kg body weight. Oral administration also caused somatic chromosome aberrations as well as sperm head abnormalities. A dose-related increase in chromosome aberration frequency was also observed in bone marrow cells of mice fed with either plain pan masala or pan masala with tobacco (14).
Short-term tests. In view of the presence of various mutagenic and nonmutagenic constituents in pan masala, an investigation of its genotoxic potential is necessary for the safety evaluation of its consumption. The identification of carcinogens by long-term animal experiments has always been the system of choice. However, the shortcomings of this assay are the cost and the time involved. These have led to searches for alternative methods. Numerous shortterm tests have been developed and are being used successfully for detecting the ability of chemicals to cause alterations in genetic material and thereby to play a crucial role in causing cancer. Compounds which exhibit carcinogenic activity generally also exhibit genotoxic activity (15). Thus, genetic t.oxicology has major application in determining the outcome of exposure to genotoxic carcinogens. It is relatively simple to use the induction of mutation in bacteria as a very sensitive indirect indicator of DNA damage. A substance found to be mutagenic in properly conducted bacterial mutation assays may be regarded as potentially genotoxic to mammals until proved otherwise. It has also been suggested that in genetic toxicology studies, the in vitro cytogenetic assays should take a central role in a test battery (16). Short-term assays like the micronucleus (MN) test, chromosome aberration (CA) analysis and sister chromatid exchange (SCE) analysis are sensitive and well-established cytogenetic markers of DNA damage. Both the in vitro and in vivo induction of a micronucleus by carcinogens and mutagens are indicators of genomic damage (17-19). Stich et a/. (20-22) have widely applied the MN assay at a population level for studying the effects of tobacco/areca nut chewing habits on the buccal mucosa. The test permits a non-invasive assessment of human tissues as well as an opportunity for repeated samplings which appear to be ethically and emotionally acceptable. Compared to MN test, metaphase analysis for CAfrequency has been considered as a more sensitive method for identifying clastogenic compounds (23). Genomic damage, caused by exposure to harmful environmental agents, which may ultimately increase the risk of cancer, can be assessed in terms of CA frequency in the peripheral blood lymphocytes (24). The misrepaired chromosomal lesions, which escape being detected as CA, can be detected effectively by studying SCE frequencies. Sister chromatid exchange analyses are reciprocal exchanges between sister chromatids. Cells with SCEs are capable of subsequent growth, and hence SCE elevation may be a more valid indicator of heritable genomic damage. However, the SCE assay should be considered as a complement to, rather than a substitute for, CA analysis (25). Induction of SCE showed good correlation with mutation frequency and cell transformation in several cell lines (26,271. Increased SCE rates have been reported in cells of individuals exposed to various environmental mutagens/carcinogens, e.g. in cigarette smokers (26,291, and in betel and tobacco chewers (30). Taking into consideration all the above facts,
PAN
MASALA
results of these assays have been included of pan masala.
349
to evaluate
the genotoxic
potential
(I) Bacterial test system. Bagwe et a/. (31) have evaluated the mutagenicity of pan masala after aqueous extraction, hot aqueous:ethanolic extraction, hot chloroform extraction, ethanolic extraction and pre-treatment of extracts with nitrite at acidic pH using the Salmonella/mammalian microsome test (Ames assay) and two tester strains of Salmonella typhimurium, TA98 and TAIOO. Only the ethanolic extract elicited a weak mutagenic response in strain TA98 without metabolic activation. Using tester strains TA98 and TAIOO, Polasa et al. (32) found a significant dose-dependent elevation of revertants with extracts of pan masala. (2) Mammalian test system: The effects of aqueous and organic (dimethyl sulphoxide) extracts of plain pan masala (PM) as well as pan masala with tobacco (PM-T) were tested on Chinese hamster ovary (CHO) cells employing three cytogenetic endpoints viz. CA, SCE and percent micronucleated cells (%MNC). The frequencies of all three endpoints were found to be significantly increased by an aqueous extract of pan masala in a dose-dependent manner in cultures treated without metabolic activation. However, addition of S9 activation system resulted in suppression of chromosomal damage (33). Similarly, the genotoxic potential of dimethyl sulphoxide (DMSO) extracts were also tested in the presence or absence of a metabolic activation system (34). In cultures without metabolic activation, the extracts were found to significantly increase the frequency of all three parameters tested; however, those with activation elicited a weak response. The data suggested, therefore, that pan masala (without and with tobacco) contains water-soluble as well as DMSO-soluble direct-acting mutagens. A dose-dependent increase in CA frequency following treatment with pan masala with and without tobacco in combination with ethanol indicated that alcohol consumption may potentially elevate the risk of oral cancer among pan masala chewers (35). Human
studies
The pharmacological handling and metabolic conversion of a compound(s) in human beings too often complicates the extrapolation of carcinogenesis data from in vitro assays to the heterogeneous human population. Furthermore, for predicting genotoxicity of a carcinogen, it has been suggested that in vivo genotoxicity data would serve well to substantiate the in vitro data (36). A significantly higher frequency of micronucleated cells in exfoliated buccal mucosa (a target tissue), and high frequencies of CA and SCE in peripheral blood lymphocytes (a non-target tissue) have been observed in users of both plain pan masala and pan masala with tobacco when compared with a control population (37,381. Relevant
biological
data on major constituents
of pan masala
The genotoxic effects of pan masala can be attributed mainly (and, of course, to tobacco in the case of tobacco-containing
to the areca nut varieties of pan
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A. H. TRIVEDI ETAL.
masala). Areca nut is a major constituent of pan masala (70-80% by weight). Several epidemiological studies have revealed an association between areca nut consumption and the occurrence of pre-malignant and malignant oral diseases (2,39). A significantly higher frequency of % MNC in exfoliated buccal mucosa, and increased frequencies of CA and SCE in the lymphocytes of chewers of areca nut per se (40,411 as well as in chewers of tobacco with areca nut (421, have been reported. The clastogenicity of the saliva of areca nut chewers to CHO cells has also been repot-ted (43). Several animal experiments have provided evidence of the carcinogenicity of areca nut extracts and its tannins (44,451. Genotoxic effects of areca nut on CHO cells (461, mouse bone marrow cells, V79 Chinese hamster cells (47) and mouse lymphocytes (48) have been documented. Some of the areca nut specific alkaloids have been found to possess transforming (49) and clastogenic properties (46,501. Wenke and Hoffmann (51) have reported the conversion of these areca nut specific alkaloids to nitrosamines, and some of them have even been detected in the saliva and urine of betel quid chewers (52,531. The tobacco present in ‘gutkha’ varieties of pan masala is one of the major reasons for the addiction observed among chewers of pan masala with tobacco. The International Agency for Research on Cancer (2) has reviewed the exposure data, experimental data and human data available on tobacco habits other than smoking. The literature available clearly indicated that oral use of smokeless tobacco is playing a major role in causation of cancer of the oral cavity. The present authors have also carried out in vitro short-term assays with: (I) aqueous extract of unprocessed tobacco (Nicotiana tabacum); (2) nicotine, a major alkaloid of tobacco; and (3) nicotine plus arecoline, a combination of the major alkaloids of tobacco and areca nut. It was observed that aqueous extract of tobacco is genotoxic to CHO cells (54). Secondly, nicotine, in addition to being addictive, is genotoxic (55), and combined application of nicotine and arecoline results in more severe effects than nicotine alone (56). The observations of cytogenetic monitoring of smokeless tobacco users also clearly suggested that the habit causes genomic damage to the oral mucosa (a target tissue) as well as to the peripheral blood lymphocytes (a non-target tissue) (57). The results of evaluation of chromosomedamaging effects of urine concentrates from tobacco plus areca nut chewers indicated that besides the oral cavity, mutagens/carcinogens present in tobacco and areca nut might also be playing a causative role in cancer of the urinary bladder (58). Details of spices and flavouring agents used in the preparation of pan masala are not always revealed by the manufacturers. However, the occasional use of synthetic flavours like musk ambrette and musk xylene, to improve the taste and flavour of zarda, is well known. Nair et al. (59) have detected both of these agents in the saliva of chewers of betal quid with tobacco. Mutagenicity of these agents has also been reported in the Salmonella/mammalian microsome test (59). The role of lime in the development of oral cancer remains a mystery. However, it causes local irritation to the mucosa, and hyperplasia has been observed following the application of lime to the cheek pouch of hamsters (60). Catechu, another ingredient of pan masala, contains 2-10% catechin (61) and has hepatoprotective effects (62). Monteith (63) demonstrated the ability of
PAN
MASALA
351
catechin to alter the metabolic pathway of 2-acetyl-aminofluorene (AAF) in rat hepatocytes. Nagabhushan et a/. (64) observed that catechin inhibited mutagenicity of B(a)P or DMBA, and also inhibited the in vitro binding of 3HB(a)P metabolites to DNA in a dose-dependent manner. It has also been reported to inhibit some of the mutagenicity of tobacco (65). On the other hand, Giri et al. have reported genotoxicity (66). In addition to all these facts, the quality of the basic ingredients mixed for preparing pan masala is also an important factor. Fungal infection, if any, to tobacco and/or areca nut may affect the ultimate toxicity of the mixture. When the areca nuts are infected with Aspergillus flavus or Aspergillus niger (67-69), they are likely to contain aflatoxins, which have known genotoxic/carcinogenic effects (70). Overall, these data indicate that pan masala has carcinogenic potential and may be causally associated with oral cavity cancers.
Implications
of present
knowledge
and future
direction
Unlike betel quid, which is almost always freshly prepared, pan masala is a dry powdered mixture of various ingredients, mainly areca nut and tobacco. Thus, it represents a complex mixture of harmful constituents. As mentioned earlier, there has been irrefutable evidence that the use of tobacco is a major health hazard. It is also very clear from the abovementioned experimental data that pan masala has the ability to cause DNA damage in bacterial and in vitro mammalian test systems in animal models as well as among pan masala chewers. However, there is currently a paucity of epidemiological data causally associating oral pre-cancerous and cancerous lesions with pan masala chewing habits. Moreover, the degree of transformation of pre-cancerous lesions to cancerous lesions among pan masala chewers, and knowledge about the factors associated with it are also lacking. However, genotoxicity and carcinogeneicity data on areca nut and tobacco, ingredients of pan masala, provide enough reasons to consider pan masala chewing as a harmful habit and a likely association with cancer of the oral cavity. Since a long latent period may exist between exposure and manifestation of the disease, the harmful effect of a habit will be observed only after a prolonged period of time. Hence, close surveillance and follow-up epidemiological and molecular biology studies will definitely prove to be one of the significant contributions in the field. Such studies are currently under consideration at the authors’ institute which will help the relevant authorities to take more realistic steps towards controlling the sale and indiscriminate use of pan masala.
Acknowledgement The authors thank the Indian financial support.
Council
of Medical
Research,
India for providing
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References 1. IARC (1987) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Soppl. 7, Overall Evaluations of Carcinogenicity: An Updating of /ARC Monographs Volumes I-42. Lyon: International Agency for Research on Cancer, pp.357381. 2. IARC (1985) Monooraohs on the Evaluation of the Carcinoaenic Risk of Chemicals to Humans, Vol. 37. Be& Quid&b Areca Nut Cheving and Some Nitro:amines. Lyon: International Agent; for Research on Cancer. 3. Sanghvi, L. D. (1981) Cancer epidemiology: The Indian scene. J. Cancer Res. C/in. Oncol. 91: 1-14. 4. Sanghvi, L. D. (1989) Tobacco related cancers. In: Sanghvi, L. D. & Notani, P. N., eds, Tobacco and Health: The Indian Scene. Bombay, pp. 9-15. 5. Gupta, P. C. (1992) Smokeless tobacco use in India. Smokeless Tobacco or Health-An International Perspective. N/H Publication No. 93-3461, pp. 19-25. 6. Tricker, A. R. & Preussmann, R. (1988) The occurrence of N-nitro compounds in zarda tobacco. Cancer Left. 42: 113-118. 7. Kashyap, S. K., Nigam, S. K., Karnik, A. B. et a/. (1989) Toxicological evaluation of pan masala. Annual Report (7989-90). National Institute of Occupational Health (Indian Council of Medical Research),.Ahmedabad, India, pp. 60-66. 8. Anuradha. C. D. & Shvamala Devi. C. S. (1993) Serum orotein. ascorbic acid & iron &tissue collagen in oral submhcous fibrosis-a preliminary study. Ind. 2. Med. Res. (6) 98: 147-151. 9. Sinha, B. K. (1991) Fibronectin levels and their correlation with histopathological changes following application of instant ‘pan masala with tobacco’ on buccal mucosa of albino rats. Thesis submitted to Postgraduate Institute of Medical Education and Research, Chandigarh, India. 10. Khrime, R. D., Mehra, Y. N., Mann, S. B. S., Mehta, S. K. & Chakraborti, R. N. (1991) Effect of instant preparation of betel nut (pan masala) on the oral mucosa of albino rats. Ind. J. Med. Res. (6) 94: 119-124. 11. Sarma, A. B., Chakrabatti, J., Chakrabarti A. et a/. (1992) Evaluation of pan masala for toxic effects on liver and other organs. food Chem. Toxicol. 30: 161-163. 12. Mukherjee, A., Chakrabarti, J., Chakrabarti, A., Banerjee, T. & Sarma, A. (1991) Effect of ‘pan masala’ on the germ cells of male mice. Cancer Lett. 58: 161-165. 13. Mukherjee, A. & Giri, A. K. (1991) Sister chromatid exchange induced by pan masala (a betel quid ingredient) in male mice in vivo. Food Chem. Toxicol29: 401-403. 14. Patel, R. K., Jaju, R. J., Bakshi, S. R. et a/. (1996) Pan masala induces chromosome aberrations in mouse bone marrow cells. GCR/ Bulletin 3: 63-67. 15. Tennant, R. W., Margolin, B. H., Shelby, M. D. et a/. (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays. Science 236: 933-941. 16. Natarajan, A. T. 81 Obe, G. (1982) Mutagenicity testing with cultured mammalian cells: Cytogenetic assays. In: Heddle, J. A., ed., Mutagenicity: New Horizons in Genetic Toxicology. New York: Academic Press, pp. 171-213. 17. Blakey, D. H., Duncan, A. M. V., Wargovich, M. J., Goldberg, M. T., Bruce, W. R. & Heddle, J. A. (1985) Detection of nuclear anomalies in the colonic epithelium of the mouse. Cancer Res. 45: 242-249. 18. Heddle, J. A., Raj, A. S. & Krepinsky A. B. (1981) The micronucleus assay. II In vitro. In: Stich H. F. & San, R. H. C., eds., Short-term Tests for Chemical Carcinogens. Berlin, Heidelberg: Springer Verlag, pp. 250-254. 19. Ronen, A. & Heddle, J. A. (1984) Site-specific induction of nuclear anomalies (apoptotic bodies and micronuclei) by carcinogens in mice. Cancer Res. 44: 1536-1540. 20. Stich, H. F. (1986) The use of micronuclei in tracing the genotoxic damage in the oral mucosa of tobacco users. In: Hoffman, D. & Harris, C. C., eds., Mechanisms in Tobacco Carcinogenesis. New York: Banbury Report No. 23, Cold Spring Harbor Laboratory, pp. 99-111. 21. Stich, H. F., Acton, A. B. & Palcic, 6. (1990) Towards an automated micronucleus assay as an internal dosimeter for carcinogen-exposed human population groups. In: Band, P., ed., Recent Results in Cancer Research, Vol. 120, Occupational Cancer Epidemiology. Berlin, Heidelberg, New York: Springer Verlag, pp. 94-105. 22. Stich, H. F., Bohm, B. A., Chatterjee, K. & Sailo, J. (1983) The role of saliva-borne mutagens and carcinogens in the etiology of oral and esophageal carcinomas of betel nut and tobacco chewers. In: Stich, H. F., ed., Carcinogens and Mutagens in the Environment, Vol. 3, Naturally Occurring Compounds: Epidemiology and Distribution. Boca Raton, FL: CRC Press, pp. 43-58. 23. Kliesch, U. & Adler, I. D. (1983) Chromosome analysis and micronucleus test in mouse bone marrow are not equally sensitive as shown by results with 5 chemical mutagens. Mutat Res. 113: 340-346.
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26. 27.
28. 29.
30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
40. 41.
42.
43. 44. 45.
46. 47. 48. 49. 50. 51. 52.
353
Carrano, A. V. & Natarajan, A. T. (1988) Considerations for population monitoring using cvtoaenetic techniaues. Mufat. Res. 204: 379-406. Per,, P., Henderson, L. & Kirkland, D. (1984) Sister chromatid exchange in cultured cell. in: Dean, 6. J., ed., Report of the LJKEMS Sub-committee on Guidelines for Mutagenicity Testing. Part II, UKEMS, U.K., pp. 89-121. Popescu, N. C., Amsbaugh, S. C. 61 DiPaolo, J. A. (1981) Relationship of carcinogen induced sister chromatid exchange and neoplastic cell transformation. Int. J. Cancer26 71-77. Popescu, N. C., Amsbaugh, S. C. & DiPaolo, J. A. (1984) Correlation of morphological transformation to sister chromatid exchanges induced by split dosesof chemical or physical carcinogens on cultured Syrian hamster cells. Cancer Res. 44: 1933-1938. Ghosh, R. & Ghosh, P. K. (1987) The effect of tobacco smoking on the frequency of sister chromatid exchanges in human lymphocyte chromosomes. Cancer Genet. Cytogenet27: 15-19. Husum, B., Wulf, H. C. & Niebuhr, E. (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. Ghosh, R. & Ghosh, P. K. (1984) Sister chromatid exchanges in betel and tobacco chewers. Motat. Res. 139: 79-81. Bagwe, A. N., Ganu, U. K., Gokhale, S. V. & Bhisey, R. A. (1990) Evaluation of the mutagenicity of ‘pan masala’, a chewing substitute widely used in India. Motat. Res. 241: 349-354. Polasa, K., Babu, S. & Shenolikar, I. S. (1993) Dose-dependent genotoxic effect of pan masala and areca nut in the Salmonella typhimurium assay. Food Chem. Toxicol. 31: 439-442. Jaju, R. J., Patel, R. K., Bakshi, S. R., Trivedi, A. H., Dave, B. J. 61 Adhvaryu, S. G. (1992) Chromosome damaging effects of pan masala. Cancer Lett. 65: 221-226. Patel, R. K., Jaju, R. J., Bakshi, S. R., Trivedi, A. H., Dave, B. J. & Adhvaryu, S. G. (1994) Pan masala-a genotoxic menace. Mutat. Res. 320: 245-249. Patel, R. K., Trivedi, A. H., Jaju, R. J., Adhvaryu, S. G. 81 Baiar, D. B. (1994) Ethanol potentiates the clastoaenicitv of oan masala-an in vitro experience. Carcinooenesis 15: 2017-2021. Carrano, &. V. & Natarajan, A. T. (1988) Considerations for population monitoring using cytogenetic techniques. Mutat. Res. 204: 379-406. Dave, B. J., Trivedi, A. H. & Adhvaryu, S. G. (1991) Cytogenetic studies reveal increased genomic damage among pan masala consumers. Mutagenesis 6: 159-164. Trivedi, A. H. (1992) A Study on Carcinogenic Potentials of Tobacco. Ph.D. Thesis, M.S. University of Baroda, Baroda, India, pp. 148-179. Gupta, P. C., Mehta, F. S., Daftary, D. K. et a/. (1980) Incidence rates of oral cancer and natural history of oral precancerous lesions in a 10 year follow-up study of Indian villagers. Common. Dent. Oral Epidemiol. 8: 287-333. Dave, B. J., Trivedi, A. H. & Adhvaryu S. G. (1992) Role of areca nut consumption in the cause of oral cancers. Cancer 70: 1017-1023. Stich, H. F., Stich, W. & Parida, B. B. (1982) Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk from oral cancer: Betel quid chewers. Cancer Lett. 17:125-134. Adhvaryu, S. G., Dave, B. J. & Trivedi, A. H. (1991) Cytogenetic surveillance of tobacco-areca nut (mava) chewers, including patients with oral cancers and premalignant conditions. Mutat. Res. 261:41-49. Stich, H. F. 81 Stich, W. (1982) Chromosome damaging activity of saliva of betel nut and tobacco chewers. Cancer Lett. 15: 193-202. Shirname, L. P., Menon, N. M., Nair, J. & Bhide, S. V. (1983) Correlation of mutagenicity and tumorigenicity of betel quid and its ingredients. Nuts: Cancer 5: 87-91. Shivapurkar, N. M., Ranadive, S. N., Gothoskar, S. V., Bhide, S. V. & Ranadive, K. J. (1980) Tumorigenic effect of aqueous and polyphenolic fractions of betel nut in Swiss strain mice. Ind. J. Exp. Biol. 18: 1159-1161. Dave, B. J., Trivedi, A. H. & Adhvaryu S. G. (1992) In vitro genotoxic effects of areca nut extract and arecoline. J. Cancer Res. Clin. Oncol. 118: 283-288. Shirname, L. P., Menon, M. M. & Bhide, S. V. (1984) Mutagenicity of betel-quid and its ingredients using mammalian test systems. Carcinogenesis 5: 501-503. Panigrahi, G. 8. & Rao, A. R. (1986) Study of the genotoxicity of the total aqueous extract of betel nut and its tannin. Carcinogenesis 7: 37-39. Ashby J., Styles, J. A. & Boyland, E. (1979) Betel nuts, arecaidine and oral cancer. Lancet i: 112. Panigrahi, G. B. & Rao, A. R. (1982) Chromosome breaking ability of arecoline, a major betelnut alkaloid, in mouse bone-marrow cells in vivo. Mutat. Res. 103: 197-204. Wenke, G. & Hoffman, D. (1983) A study of betel quid carcinogenesis. 1. On the in vitro Nnitrosation of arecoline. Carcinogenesis 4: 169-172. Nair J., Ohshima H., Friesen, M. Croisy, A., Bhide, S. V. & Bartsch, H. (1985) Tobacco specific
354
53.
54. 55. 56. 57. 58.
59.
60. 61.
62. 63. 64. 65. 66. 67. 68. 69. 70.
A. H. TRIVEDI
ETAL.
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. Caminogenesis 6: 295-303. Wenke, G., Brunnemann, K. D., Hoffmann, D. & Bhide, S. V. (1984) A study of betel quid carcinogenesis IV. Analysis of the saliva of betel chewers: A preliminary report. .I. Cancer Res. C/in. Oncol. 108: 110-113. Trivedi, A. H., Dave, B. J. 81 Adhvaryu, S. G. (1993) Genotoxic effects of tobacco extract on Chinese hamster ovary cells. Cancer Lett. 70: 107-112. Trivedi, A. H., Dave, B. J. & Adhvaryu, S. G. (1990) Assessment of genotoxicity of nicotine employing in vitro mammalian test system. Cancer Letf. 54: 89-94. Trivedi, A. H., Dave, B. J. & Adhvaryu, S. G. (1993) Genotoxic effects of nicotine in combination with arecoline on CHO cells. Cancer Letf. 74: 105-110. Trivedi, A. H., Dave, B. J. &Adhvaryu, S. G. (1993) Monitoring of smokeless tobacco consumers using cytogenetic endpoints. Anticancer Res. 13: 2245-2249. Trivedi, A. H., Roy, S. K., Jaju, R. J., Patel, R. K., Adhvaryu, S. G. & Balar, D. B. (1995) Urine of tobacco/areca nut chewers causes genomic damage in Chinese hamster ovary cells. Carcinogenesis 16: 205-208. Nair, J., Oshima, H., Malaveille, C. et al. (1966) Identification, occurrence and mutagenicity in Salmonella typhimurium of two synthetic nitroarenes, musk ambrette and musk xylene, in Indian chewing tobacco and betel quid. Food Chem. Toxicol. 24: 27-31. Dunham, L. J., Muir, C. S. & Hamner, J. J. E. Ill (1966) Epithelial atypia in hamster cheek pouches treated repeatedly with calcium hydroxide. Br. J. Cancer 20: 588-593. IARC (1983) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 31, Some Food Additives, and Naturally Occurring Substances. Lyon: International Agency for Research on Cancer, pp. 171-178.213-219. Lapis, K., Jeney, A., Divald, A., Vajta, G., Zalatnai, A. & Schaff, Z. (1986) Experimental studies on the effect of hepatoprotective compounds. Tokai J. Exp. C/in. Med. 11: 135-145. Monteith, D. K. (1990) Catechin inhibition of mutagenesis and alteration of DNA binding of 2acetyl-aminofluorene in rat hepatocytes. Motat. Res. 246: 151-158. Nagbhushan, M., Amonkar, A. J., Nair, U. J. et a/. Catechin as an antimutagen: its mode of action. J. Cancer Res. C/in. Oncol. 114: 177-182. Nagabhushan, M. & Bhide, S. V. (1988) Anti-mutagenicity of catechin against environmental mutagens. Mutagenesis 3: 293-296. Giri A. K., Banerjee, T. S. & Talukdar, G. (1987) Induction of sister chromatid exchange and dominant lethal mutation by ‘katha’ (catechu) in male mice. Cancer Lett. 36: 189-198. Borle, R. M. & Gupta, D. S. (1987) Fungal contamination of Areca nut. Ind. J. Pathol. Microbial. 30: 357-360. Mahdihassan, S. (1981) Aspergillus flavus as the probable carcinogen in betel nuts. Hamdard Medicos 24: 139-I 34. Mahdihassan, S. & Rabia, Z. (1988) Coloured illustrations of betel nuts infected with a cancer producing fungus. Hamdard Medicus 31: 35-36. tARC (1982) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 4, Chemicals, Industrial Processes and Industries Associated with Cancer in Humans (/ARC Monographs Vol. 1-291. Lyon: International Agency for Research on Cancer.