Addiction Biology (2002) 7, 115±125 ARECA NUT SYMPOSIUM The oral health consequences of chewing areca nut C. R. TRIVEDY,1 G. CRAIG2 & S. WARNAKULASURIYA1 1 Department of Oral Pathology and Medicine, WHO Collaborating Center for Oral Cancer and Precancer, The Guy’s, King’s and St Thomas’ Medical and Dental Schools of King’s College, London & 2Department of Oral Pathology, School of Clinical Dentistry, Sheffield, UK Abstract Deleterious effects of areca nut on oral soft tissues are published extensively in the dental literature. Its effects on dental caries and periodontal tissues, two major oral diseases, are less well researched. Areca-induced lichenoid lesions mainly on buccal mucosa or tongue are reported at quid retained sites. In chronic chewers a condition known as betel chewer’s mucosa, a discoloured areca nut-encrusted change, is often found where the quid particles are retained. Areca nut chewing is implicated in oral leukoplakia and submucous fibrosis, both of which are potentially malignant in the oral cavity. Oral cancer often arises from such precancerous changes in Asian populations. In 1985 the International Agency for Research on Cancer concluded that there is limited evidence to conclude that areca chewing may directly lead to oral cancer. There is, however, new information linking oral cancer to pan chewing without tobacco, suggesting a strong cancer risk associated with this habit. Public health measures to quit areca use are recommended to control disabling conditions such as submucous fibrosis and oral cancer among Asian populations. Introduction Areca nut may be consumed either on its own or more commonly in association with other ingredients such as tobacco, lime, catechu and other spices wrapped in a betel leaf and referred to as a betel quid or pan.1 Recently the trend has been to chew processed areca products known as pan masalas. These contain a mixture of areca, catechu and slaked lime and may also on occasion contain tobacco. Chewing areca nut on a habitual basis is known to be deleterious to human health.2 A growing body of evidence over the last 40 years, mainly in the form of large-scale epidemiological and experimental studies, has shown that even when consumed in the absence of tobacco or lime areca may have potentially harmful effects on the oral cavity. These effects can be divided into two broad categories: those affecting the dental hard tissues, which include teeth, their supporting periodontium and the temporomandibular joint and the soft tissues, which make up the mucosa that lines the oral cavity. Effects on hard tissues Dental attrition The main effects of areca on the hard tissues are on the teeth. The habitual chewing of areca may result in severe wear of incisal and occlusal tooth Correspondence to: Prof. S. Warnakulasuriya, Department of Oral Pathology and Medicine, King’s Dental Institute, Denmark Hill Campus, London SE5 9RW, UK. ISSN 1355-6215 print/ISSN 1369-1600 online/02/010115-11 € Society for the Study of Addiction to Alcohol and Other Drugs DOI: 10.1080/13556210120091482 Carfax Publishing, Taylor & Francis Ltd 116 C. R. Trivedy et al. surfaces, particularly the enamel covering. The loss of enamel may also expose the underlying dentine and as this is softer than enamel wears at an increased rate. The exposure of dentine may also result in dentinal sensitivity. The degree of attrition is dependent upon several factors, which include the consistency (hardness) of the areca, the frequency of chewing and the duration of the habit. Root fractures have also been demonstrated in chronic areca chewers and this is likely to be a consequence of the increased masticatory load that is placed upon the teeth and is not direct effect of areca.3 Areca staining Among areca chewers, extrinsic staining of teeth due to areca deposits is often observed particularly when good oral hygiene prophylaxis is lacking and where regular dental care is minimal. Dental caries It has been suggested that areca chewing may confer protection against dental caries. Epidemiological studies carried out in South East Asia suggest that the prevalence of dental caries in areca chewers is lower than in non-chewers.4±6 Some investigators, however, have shown that there is no difference in the prevalence of dental caries between areca chewers and non-chewers in other Asian populations.7,8 Although little is known about the cariostatic properties of areca it has been suggested that the betel stain, which often coats the surface of the teeth, may act as a protective varnish. 9 There is also in vitro evidence that suggests that the tannin content of areca may have antimicrobial properties and this may contribute to the cariostatic role of areca.10 In addition, chronic chewers also have marked attrition of cusps of teeth leading to loss of occlusal pits and fissures, which may reduce the risk of pit and fissure caries by eliminating potential stagnation areas. The increased production of sclerosed dentine in response to attrition may confer protection against microbial invasion. Furthermore, the process of chewing itself brings copious amounts of saliva to the mouth and in the presence of added slaked lime may increase the pH in the oral environment; this may act as a buffer against acid formed in plaque on teeth. Temporomandibular joint (TMJ) pathology The masticatory forces generated during chewing areca may be transmitted to the TMJ and subsequently may give rise to joint arthrosis. However, there is no reliable data to substantiate an increased prevalence in areca users and further studies are required. Furthermore, symptoms such as trismus are common to both TMJ pathology and to other oral disorders associated with areca chewing such as oral submucous fibrosis. It is difficult to distinguish a direct effect on the TMJ from the fibrotic involvement of the oral musculature that may contribute to limitation of mouth opening in chronic chewers. Effects on soft tissues Periodontal disease In vitro studies have demonstrated that areca extracts containing arecoline inhibit growth and attachment of, and protein synthesis in, human cultured periodontal fibroblasts.11,12 On the basis of these findings the investigators proposed that areca may be cytotoxic to periodontal fibroblasts and may exacerbate pre-existing periodontal disease as well as impair periodontal reattachment. Some investigators have shown that loss of periodontal attachment and calculus formation is greater in areca chewers.13 However, it is difficult to interpret such studies, as there are several confounding variables such as the level of oral hygiene, dietary factors, general health and dental status, not to mention tobacco smoking, which may have a significant influence on periodontal status. Furthermore, as the majority of chewers are in the Indian subcontinent, where oral health education is limited, periodontal health may be compromised even in non-areca chewers. It is therefore difficult to ascertain the biological effects of areca on periodontal health but in view of the recent in vitro evidence further clinical and experimental studies are necessary. Lichenoid lesions Areca-induced lichenoid lesions, mainly on buccal mucosa or tongue, have been reported at sites of quid application. 14 This is considered to be a type IV contact hypersensitivity-type lesion but resembles oral lichen planus clinically. The main detectable features are that it is found at the site of quid placement in areca chewers and may be unilateral in nature. Fine wavy keratotic lines are Oral health consequences of chewing areca nut 117 Table 1. Prevalence of betel chewer’s mucosa in different populations Reference Country Year Number Prevalence % 16 17 18 19 Cambodia Malaysia Cambodia Thailand 1996 1995 1995 1987 102 850 1319 1866 60.8 21.9 <1 13.1 seen to radiate from a central red/atrophic area and, interestingly, the keratotic striae do not criss-cross and are parallel to each other. The histology is suggestive of a lichenoid reaction and the lesion is noted to resolve following cessation of areca use. Betel chewer’s mucosa (BCM) This condition was first described by Mehta et al. 15 and is characterized by a brownish-red discolouration of the oral mucosa. This discolouration is often accompanied by encrustation of the affected mucosa with quid particles, which are not easily removed, and with a tendency for desquamation and peeling. The underlying area assumes a wrinkled appearance. The lesion is usually localized and associated with the site of quid placement in the buccal cavity. The lesion is associated strongly with the habit of betel quid chewing, particularly in elderly women who have been chronic chewers for long durations.16 Several epidemiological studies have shown that the prevalence of betel chewer’s mucosa may vary between 0.2% and 60.8% in different South East Asian populations (Table 1). Histologically, betel chewer’s mucosa shows epithelial hyperplasia, which is encrusted with an amorphous deposit. This reacts positively to von Kossa staining suggesting that these granules, which are located both intra- and intercellularly, may contain calcium from the calcium hydroxide in slaked lime.20 A ballooning effect of oral keratinocytes is noted. The presence of the human papilloma virus (HPV) subtypes 11, 16 and 18 has also been demonstrated in BCM but the significance of this is not fully understood. 21 Although the exact mechanisms underlying the development of this condition are not fully understood it is thought that the chemical and traumatic effects of the betel quid on the oral mucosa may be significant factors. Furthermore, there is also evidence that the presence of tobacco in the quid is not essential for the development of BCM.22 At present BCM is not considered to be potentially malignant, although the condition often co-exists with other mucosal lesions such as leukoplakia and oral submucous fibrosis, which are well known for their potential for malignant change. Oral leukoplakia Leukoplakia can be defined as a predominantly white patch or plaque on the oral mucosa that cannot be characterized clinically or pathologically as any other disease and is not associated with any other physical or chemical agent except tobacco.23 Based on clinical appearance, leukoplakia can be divided into several subtypes: homogeneous (white), speckled (red/white), nodular or verrucous leukoplakia. This condition is well known for its potential for malignant change and transformation rates between 0.1 and 17.5% have been quoted in the literature. 24 The figures for malignant transformation based on population studies reported from India25 are much lower than those reported from Europe and the United States based on hospital series, but this may be due to a selection bias. There is also evidence that the clinical presentation (site and type) may influence the risk potential for malignant change. The speckled lesions carry a greater risk for malignant change in comparison to the homogeneous white plaques.26 In biopsies in addition to the presence of an amorphous brown-staining von Kossa positive layer on the surface, parakeratosis and atrophy of the covering oral epithelium are reported in areca chewers.27 In another study, 14% of leukoplakia biopsies obtained from areca chewers demonstrated cellular atypia amounting to epithelial dysplasia. 28 Histopathology of an areca nutassociated oral mucosal lesion presenting clinically as a white/red patch and demonstrating severe epithelial dysplasia is shown in Fig 1. 118 C. R. Trivedy et al. 1.94±156.27) for areca nut chewers.33 Furthermore, the authors demonstrated that the cessation of areca chewing resulted in resolution of 62% of leukoplakias, suggesting that areca on its own is a significant aetiological factor in the development of leukoplakia. Further evidence of its relationship with areca chewing has come from the increased prevalence of this condition in subjects who suffer from oral submucous fibrosis, which is associated strongly with the habit of areca chewing.34 Figure 1. Severe epithelial dysplasia in a `speckled leukoplakia’ arising in the lateral border of tongue mucosa in a long-term tobacco and areca nut user; male aged 47 years; H&E ´100. Although there is considerable controversy and debate about how to define oral leukoplakia there is little doubt that both tobacco, in any form, and areca nut use are major risk factors for developing this condition.29,30 In Ernakulum, India a 10-year follow-up study recorded that the incidence of leukoplakia in a group of chewers, which consisted mainly of pan chewing, was 2.5 per 1000 people.25 Warnakulasuriya31 reviewed four case±control studies that examined relative risk of oral leukoplakia in betel quid chewers. In one of the studies, chewing areca (in betel quid without tobacco) raised the odds ratio (OR) to 5 compared with non-chewers (OR = 1); adding tobacco to the quid further raised the relative risk by at least threefold compared with non-tobacco users.32 More recently, the results of a case± control study conducted in Taiwan, where areca is chewed without tobacco, found the odds ratio for developing leukoplakia was 17.43 (95% CI Oral submucous fibrosis (OSF) This is a chronic disorder characterized by fibrosis of the lining mucosa of the upper digestive tract involving the oral cavity, oro- and hypopharynx and the upper third of the oesophagus.35 The fibrosis involves the lamina propria and the submucosa and may often extend into the underlying musculature resulting in the deposition of dense fibrous bands, which give rise to the limited mouth opening which is a hallmark of this disorder. Warnakulasuriya36 defined a series of symptoms and subjective signs which are characteristic of OSF to be employed as the clinical criteria required to make a diagnosis of OSF (Table 2). This symptomatology of OSF was adopted at a consensus workshop held to clarify the nomenclature of areca quid-associated lesions and conditions.1 Histopathology of OSF is illustrated in Figs 2 and 3 in long-standing areca chewers, with underlying fibrosis of lamina propria being the pathognomic feature. Epithelial atrophy often associated with OSF is seen in Fig. 2 and accompanied epithelial dysplasia in Fig. 3. The potentially malignant nature of this condition has been well documented. A malignant transformation rate of 7.6% over a period of 10 Table 2. Clinical features of OSF Early Late Blanching of mucosa Intolerance to spicy food Petechiae Depapillation of tongue Oral ulceration Leathery mucosa Taste disturbance Fibrous bands Trismus Flattening of palate Hockey-stick uvula Reduced tongue mobility Xerostomia Keratosis Source: Refs 1 & 36. Oral health consequences of chewing areca nut 119 Figure 3. Severe epithelial dysplasia in the retromolar mucosa of a 59 year-old female with oral submucous fibrosis and a long history of both tobacco and areca nut usage; same patient as shown in Fig. 2B but 16 years later; H&E ´20. Figure 2. (A) Epithelial atrophy and subepithelial fibrosis extending into the underlying voluntary muscle fibres in the buccal mucosa of 49 year-old male with a long history of oral submucous fibrosis; H&E ´10 (B) Epithelial atrophy and subepithelial fibrosis in the floor of mouth mucosa from a 43 year-old female with extensive oral submucous fibrosis and a long history of both tobacco and areca nut usage; H&E ´50. United States but there have been several case reports, describing OSF in some habitues who continue to chew areca following migration.50±52 It is now accepted that chewing areca is the single most important aetiological factor for developing OSF.53,54 Other causes which have been proposed in the past but have not been substantiated include excessive consumption of chilies, autoimmune reaction and nutritional factors, particularly iron deficiency. Much of the evidence implicating areca has been derived from epidemiological data arising largely from India, Pakistan and South Africa. Many observational studies on OSF patients have recorded a high frequency of the areca chewing habit in the OSF subjects (close to 100%) compared to that of the general popula- Table 3. The global epidemiology of OSF years was described in an Indian cohort37 and the relative risk for malignant transformation may be as high as 397.3.38 Although OSF was first described by Schwartz39 in a series of Indian women living in East Africa there are descriptions of a similar condition occurring in betel chewers in early texts dating back to 1908.40 Since Schwartz’s publication, OSF has been detected in several epidemiological studies conducted mainly in India, among Indians living in south Africa, among Chinese or in other south Asian populations (Table 3). At present there are few data on the prevalence of OSF among Asians living in Europe and the Country/Ref. India 41 42 43 44 45 46 South Africa 47 48 China 49 Sample no. 10 000 5000 10 071 35 000 101 761 5018 1000 2058 11 046 Prevalence % 0.51 1.22 0.16 0.59 0.07±0.4 3.2 0.5 3.4 3.03 120 C. R. Trivedy et al. Table 4. Relative risk (RR) of developing of OSF in areca chewers Cases Controls Relative risk (RR) India 164 5018 56 India 200 200 57 Pakistan 157 157 58 India 60 60 59 Taiwan 35 100 60.6 (areca) 75.6 (Mawa) 489.1 (pan masala) 136.5 (areca) 154.0 (areca only) 64.0 (betel quid + tobacco) 32.0 (betel quid) 29.9 (areca) 106.4 (Mawa) 43.8 (betel quid) Reference Country 46 tion.48,49,55 Several case±control studies have also shown that there is an increased risk of developing OSF in subjects consuming areca products (Table 4). The relative risk was noted to rise with an increasing frequency of the areca chewing habit, suggesting a dose±response relationship. 57,58 An interventional study conducted over a 10-year follow-up period in India showed a decrease in the incidence of OSF in the trial cohort compared with a control population as a result of cessation of areca use.60 However, the authors point out that OSF is emerging as an new epidemic in India due to rising trends in per capita areca consumption. 46 Although there is good evidence to support the role of areca as the major risk factor in the development of OSF, the mechanisms by which this occurs are not fully understood. In vitro studies have shown that areca nut alkaloids such as arecoline and its hydrolysed product arecaidine may stimulate cultured fibroblasts to proliferate and synthesize collagen.61,62 In addition flavonoids within the nut have also been shown to increase the stabilization of collagen by enhancing the cross-linking of collagen, thereby increasing the resistance to degradation by collagenases.63 However, subsequent in vitro studies have failed to show similar effects of arecoline on cultured OSF fibroblasts.64,65 Furthermore, recent studies have shown that arecoline inhibits collagen synthesis and fibroblast proliferation in vitro, suggesting that arecoline may have cytotoxic properties. 12,66,67 The disparity of results from in vitro studies suggests that the areca may contain other agents in addition to arecoline which are important in the pathogenesis of OSF; the role of arecoline, therefore, needs further evaluation. There has been recent interest in the role of copper in the pathogenesis of this disorder and raised copper concentrations have been shown in products containing areca nut in comparison to other nut-based snacks.68 Chewing areca for up to 20 minutes releases significant amounts of soluble copper to the whole mouth fluid.69 Furthermore, there is evidence to show that mucosal biopsies taken from OSF subjects contain a higher copper concentration than those taken from controls.70 This has led to the hypothesis that the increased tissue copper may increase the activity of the enzyme lysyl oxidase, which is a copper-dependent enzyme that has been implicated in the pathogenesis of several fibrotic disorders, including OSF.71,72 Further support for this theory comes from studies which demonstrate that inorganic copper salts in vitro significantly increase the production of collagen by oral fibroblasts.73 Very few animal models of OSF have been described. Khrime et al. 74 demonstrated histopathological changes consistent with OSF in albino rats whose skin was subjected to repeated exposure to commercially available areca products. Earlier studies, however, found that the application of arecoline to the palates of B12-deficient rats did not give rise to any features which were suggestive of OSF.75 Application of arecaidine to hamster cheek pouch also failed to show any microscopic changes suggestive of fibrosis. 76 The fact that only a small proportion of subjects who chew areca actually develop OSF raises the possibility that there may be a genetic predisposition for this disorder. There is limited evidence in the literature to support that people Oral health consequences of chewing areca nut 121 of this disorder. Figure 4 illustrates a case of squamous cell carcinoma arising in a case of OSF. Figure 4. Micro-invasive, well-differentiated squamous cell carcinoma arising in the ventral tongue mucosa of the patient shown in Fig’s 2b and 3; now aged 63 years and still using both tobacco and areca nut; H&E ´25. of Bangladeshi origin develop OSF although they indulge in areca habits. HLA studies conducted on OSF have shown divergent results and a study of a cohort of UK-based OSF patients found an increased frequency of DR3, A10 and B7,77 whereas a study conducted on a Pakistani population found an increased frequency of CW2 and DR1.78 In contrast, studies conducted in Asians living in South Africa found no HLA-associated susceptibility in OSF.79 The evidence from the literature strongly implicates areca in the aetiopathogenesis of OSF although further studies are warranted to uncover the mechanisms underlying the pathological processes in relation to the development of the fibrosis and transformation to squamous cell carcinoma, which can be considered as the most serious oral health sequence Oral squamous cell carcinoma (OSCC) There is historical evidence dating back nearly a century that suggests that the areca nut may be involved in the development of OSCC.40,80,81 Although it is widely accepted that the presence of tobacco in betel quid plays an important role in the pathogenesis of oral squamous cell carcinoma the carcinogenic potential of areca in the absence of other ingredients is less clear.82There are epidemiological data from several case±control studies that confirm that the habit of betel quid chewing increases the relative risk of developing OSCC.83 The description of the chewing habit has not been explicit in some of these studies. Some of the relevant studies with estimated relative risk are listed in Table 5. The bulk of the data in the literature suggests that the addition of tobacco to the quid increases its carcinogenic potential.88±90 Areca (pan) chewing without tobacco causing oral cancer has been highlighted in a few recent studies. In one South African study, 68% of cheek cancers and 84% of tongue cancers were found in subjects consuming areca without tobacco.87 Furthermore, there is new evidence which suggest that areca in the absence of tobacco may be an independent risk factor for the development of oral SCC.84 In addition to human data there are also a large number of experimental studies, which have attempted to evaluate the carcinogenic potential Table 5. Relative risk of developing oral squamous cell carcinoma (SCC) in relation to chewing habits Reference Country Cases Controls Habit RR for SCC 33 84 Taiwan Pakistan 60 79 100 149 85 Taiwan 40 160 86 87 Taiwan South Africa 107 143 200 735 Areca nut Pan with tobacco Pan without tobacco Betel quid* Duration (years): 1±20 21±40 40 + Frequency (/day) 1±9 10±20 > 20 Betel quid* Areca nut Areca nut + tobacco 3.79 (95% CI 1.20±12.24) 8.42 9.9 58.4 (95% CI 7.6±447.6) 17.1 (95% CI 1.8±161.9) 108.4 (95% CI 11.9±987.9) 403.5 (95%CI 20.8±7843.0) 28.3 (95% CI 3.3± 240.7) 61.9 (95% CI 7.9±487.2) 294.1(95%CI 16.5±5233.6 123 43.9 (95% CI 18.6±103.6) 47.4 (95% CI 20.3±110.5) *The betel quid used in Taiwan is predominantly areca nut based. 122 C. R. Trivedy et al. of areca and its derivatives both in vivo and in vitro. Jeng et al. 91 in a recent review examined the mutagenecity and genotoxicity of areca alkaloids to target cells in the oral mucosa. In vivo studies Several animal studies have confirmed that areca products and derivatives such as arecoline- and areca- derived nitrosamines have the ability to induce neoplastic changes in experimental models. Early studies found that the application of arecaidine to the oral mucosa of experimental animals failed to have any carcinogenic effects, but when supplemented with a known promoter such as croton oil resulted in cellular damage,76 whereas other studies have shown that the application of areca products to the oral mucosa of animals results in histological changes which may be indicative of DNA damage.92,93 In addition to the local effects of areca on the oral mucosa there is also evidence that suggests that areca, when applied to the skin of animal models or included in the feed, may cause neoplastic changes in sites distant from the oral cavity such as the foregut, liver, kidneys and lung.94±97 In vitro studies There are also data in the form of in vitro studies which have been conducted on Chinese hamster ovary (CHO) cells and mammalian cell lines that suggest that areca extract may possess cytotoxic and genotoxic effects.67,98±100 There are also limited data from mutagenicity assays conducted on bacteria (Staphyllococcus typhi) which suggest that commercially available products containing (pan areca masalas) have mutagenic properties. 101,102 Human studies Among areca chewers possible genomic damage caused by areca nut (without tobacco) was confirmed in cytogentic studies.103 Conclusion It is clear from the literature that areca may have significant effects upon the hard and soft tissues of the oral cavity. Its alleged beneficial effects on dental caries and the possible damage to the periodontium need further evaluation. Based on many population studies conducted in Asia the role of areca in the pathogenesis of betel chewer’s mucosa, oral leukoplakia and oral submucous fibrosus is well established. Both leukoplakia and submucous fibrosis are potentially malignant conditions in the oral cavity. At the time of the last review by the International Agency for Research on Cancer in 1985 information available on the carcinogenic role of areca was limited. New information from several population studies, however, suggest that areca on its own may play an aetiological role in the causation of oral cancer. Public health education against areca nut use is essential to control oral cancer in emerging market economies and among Asian migrant communities in other parts of the globe. References 1. Zain RB, Ikeda N, Gupta PC et al. Oral mucosal lesions associated with betel quid, areca nut and tobacco chewing habits: consensus from a workshop held in Kuala Lumpur, Malaysia, November 25±27, 1996. J Oral Pathol Med 1999;28: 1±4. 2. Trivedy C, Warnakulasuriya S, Peters TJ. Areca nuts have deleterious effects. Br Med J 1999;318:1287. 3. Yeh CJ. Fatigue root fracture: a spontaneous root fracture in non-endodontically treated teeth. Br Dent J 1997;182: 261±266. 4. Moller IJ, Pindborg JJ, Effendi I. The relation between betel chewing and dental caries. Scand J Dent Res 1977;85:64±70. 5. Schamschula RG, Adkins BR, Barmes DR, Charlton G. Betel chewing and caries experience in New Guinea. Community Dent Oral Epidemiol 1977;5:284±6. 6. Nigam P, Srivastava AB. Betel chewing and dental decay. Fed Oper Dent 1990;1:36±8. 7. Reichart P, Gehring FJ. Streptococcus mutans and caries prevalence in Lisu and Karen of Northern Thailand. Dent Res 1984;63:56±8. 8. Williams SA, Summers RM, Ahmed IH, Prendergast MJ. Caries experience, tooth loss and oral health related behaviours among Bangladeshi women resident in West Yorkshire, UK. Community Dent Health 1996;13:150±6. 9. Howden GF. The cariostatic effects of betel chewing. PNG Med J 1984;27:123±31. 10. de Miranda CM, van Wyk CW, van der Biji P, Basson NJ. The effect of areca nut on salivary and selected organisms. Int Dent J 1996;46:350±6. 11. Jeng JH, Hahn LJ, Lin BR, Hsieh CC, Chan CP, Chang MC. Effects of areca nut, inflorescence piper betle extracts and arecoline on cytotoxicity, total and unscheduled DNA synthesis in cultured gingival keratinocytes. J Oral Pathol Med 1999;28:64±71. Oral health consequences of chewing areca nut 12. Chang MC, Kuo MY, Hahn LJ, Hsieh CC, Lin SK, Jeng JH. Areca nut extract inhibits the growth, attachment, and matrix protein synthesis of cultured human gingival fibroblasts. J Periodontol 1998;69:1092±7. 13. Anerud A, Loe H, Boysen H. The natural history and clinical course of calculus formation in man. J Clin Periodontol 1991;18:160±70. 14. Daftary DK, Bhonsle RB, Murti PR, Pindborg JJ, Mehta FS. An oral lichen planus-like lesion in Indian betel-tobacco chewers. Scand J Dent Res 1980;88:244±9. 15. Mehta FS, Pindborg JJ Hamner III JE. Oral cancer and precancerous conditions in India. Copenhagen: Munksgaard; 1971, p. 17. 16. Reichart PA, Schmidtberg W, Scheifele CH. Betel chewer’s mucosa in elderly Cambodian women. J Oral Pathol Med 1996;25:367 ±70. 17. Zain R, Ikeda N, Yaacob MBH. Oral mucosal lesions survey of adults in Malaysia. Kuala Lampur: Ministry of Health, Malaysia; 1995. 18. Ikeda N, Handa Y, Khim SP et al. Prevalence study of oral mucosal lesions in a selected Cambodian population. J Oral Pathol Med 1995; 23;49±54. 19. Reichart PA, Mohr U, Srisuwan S, Gerlings H, Theetranont C, Kangwanpong T. Precancerous and other oral mucosal lesions related to chewing, smoking and drinking habits in Thailand. Community Dent Oral Epidemiol 1987;15: 152±60. 20. Reichart PA, Philipsen HP. Betel chewer’s mucosa Ða review. J Oral Pathol Med 1998;27: 239±42. 21. Maeda H, Ikeda N, Zain R et al. Detection of human papillomaviruses in betel chewer’s mucose and leukoplakias of Cambodian patients. Proceedings of the 2nd Asian Pacific Workshop for Oral Mucosal Lesions, 5±8 November 1995, Chiang Mai, Thailand. P. 3, abstract 45. 22. Philipsen HP, Ramanathan J, Mendis BRRN. Betel chewer’s mucosa in Sri Lankan tea estate workers (unpublished data). 23. Axell T, Holmstrup P, Kramer IRH, Pindborg JJ, Shear M. International seminar on oral leukoplakia and associated lesions related to tobacco habits. Community Dent Oral Epidemiol 1984; 12:145±54. 24. Van der Wall I, Schepman KP, van der Meij EH, Smeele LE. Oral leukoplakia: a clinicopathological review. Oral Oncol 1997;33: 291±301. 25. Gupta PC, Mehta FS, Daftary DK et al. Incidence rates of oral cancer and natural history of oral precancerous lesions in a 10-year follow-up study of Indian villagers. Community Dent Oral Epidemiol 1980;8:287±333. 26. Mehta FS, Daftary DK, Shroff BC, Sanghvi LD. Clinical and histologic study of oral leukoplakia in relation to habits. A five-year follow-up. Oral Surg Oral Med Oral Pathol 1969;28:372±88. 27. Lee KW, Chin CT. The effects of betel-nut chewing on the buccal mucosa: a histological study. Br J Cancer 1970;24:433 ±41. 123 28. Tennakoon GE, Bartlett GC. Effect of betel chewing on the oral mucosa. Br J Cancer 1969;23:39±43. 29. Mehta FS, Hamner J III. Leukoplakia in tobacco related oral mucosal lesions and conditions in India. Bombay: Basic Dental Research Unit; 1993, pp. 28±46. 30. Johnson NW, Ranasinghe AW, Warnakulasuriya KAAS. Potentially malignant lesions and conditions of the mouth and oropharynx: natural historyÐcellular and molecular markers of risk. Eur J Cancer Prev 1993;2:31±51. 31. Warnakulasuriya S. The role of betel-quid in oral carcinogenesis. In: Bedi R, Jones P, editors. Betelquid and tobacco chewing among the Bangladeshi community in the United Kingdom. London: Centre for Transcultural Health; 1995, pp. 61±9. 32. Warnakulasuriya S. Smoking and chewing habits in Sri Lanka: implications for oral cancer and precancer. In: Gupta PC, Hamner JE, Murti PR editors. Control of tobacco-related cancers and other diseases. International Symposium. Bombay: Oxford University Press;1992, pp. 113±18. 33. Shiu MN, Chen TH, Chang SH, Hahn LJ. Risk factors for leukoplakia and malignant transformation to oral carcinoma: a leukoplakia cohort in Taiwan. Br J Cancer 2000;82:1871±4. 34. Pindborg JJ. Is submucous fibrosis a precancerous condition in the oral cavity? Int Dent J 1972;22:474 ±80. 35. Johnson NW, Maher R, Trivedy C, Warnakulasuriya S. The clinical condition and pathology of oral submucous fibrosis. Oral Dis 1997;3:278±9. 36. Warnakulasuriya S. Semi-quantitative clinical description of oral submucous fibrosis. Ann Dent 1987;46:18±21. 37. Murti PR, Bhonsle RB, Pindborg JJ et al. Malignant transformation rate in oral submucous fibrosis over a 17-year period. Community Dent Oral Epdemiol 1985;13:340 ±1. 38. Gupta PC, Nandakumar A. Oral cancer scene in India. Oral Dis 1999;5:1 ±2. 39. Schwartz J. Atrophica idiopathica tropica mucosa oris. Proceedings of the 11th International Dental Congress, London, 1952. 40. Fells A. Cancer of the mouth in Southern India. A summary of 1700 cases. Br Med J 1908; 2: 1428±31. 41. Pindborg JJ, Chawla TN, Misra RK, Nagpaul RK, Gupta VK. Frequency of oral carcinoma, leukloplakia, leukokeratosis, leukoedema, submucous fibrosis, and lichen planus in 10,000 Indians in Lucknow, Uttar Pradesh, India. Preliminary report. J Dent Res 1965;44:615. 42. Zachariah J, Mathew B, Varma NAR, Iqbal AM, Pindborg JJ. Frequency of oral mucosal lesions among 5,000 individuals in Trivandrum, South India. Preliminary report. J All Indian Dent Assoc 1966;38:290±4. 43. Pindborg JJ, Mehta FS, Gupta PC, Daftary DK. Prevalence of oral submucous fibrosis among 50,915 Indian villagers. Br J Cancer 1968;22: 646±54. 124 C. R. Trivedy et al. 44. Wahi PN, Kapur VL, Luthra RK, Seth RK, Arora GD. Epidemiological study of precancerous lesions of the oral cavity: a preliminary report. Indian J Med Res 1970;38:1361±91. 45. Mehta FS, Gupta PC, Daftary DK, Pindborg JJ, Choksi SK. An epidemiological study of oral cancer and precancerous conditions among 101,761 villagers in Maharashtra, India. Int J Cancer 1972;10:134 ±41. 46. Gupta PC, Sinor PN, Bhonsle RB, Pawar VS, Mehta HC. Oral Submucous fibrosis in India: a new epidemic? Natl Med J India 1998;11: 113±16. 47. Dockrat I, Shear M. Oral submucous fibrosis in Natal. Proc Int Acad Oral Pathol 1969;57±63. 48. Seedat HA, van Wyk CW. Betel chewing and dietary habits of chewers without and with submucous fibrosis and with concomitant oral cancer. S Afr Med J 1988;74:572 ±5. 49. Tang JG, Jian XF, Gao ML, Ling TY, Zhang KH. Epidemiological survey of oral submucous fibrosis in Xiangtan City, Hunan Province, China. Community Dent Oral Epidemiol 1997;25:177±80. 50. Moos KF, Madan DK. Submucous fibrosis. Br Dent J 1968;124:313 ±17. 51. McGurk M, Craig GT. Oral submucous fibrosis: two cases of malignant transformation in Asian immigrants to the United Kingdom. Br J Oral Maxillofac Surg 1984;22:56±64. 52. Canniff JP, Harvey W, Harris M. Oral submucous fibrosis: its pathogenesis and management. Br Dent J 1986;160:429 ±34. 53. Murti PR, Bhonsle RB, Gupta PC et al. Etiology of oral submucous fibrosis with special reference to the role of areca nut chewing. J Oral Pathol Med 1995;24:145±52. 54. Warnakulasuriya KAAS, Trivedy C, Maher R, Johnson NW. Aetiology of oral submucous fibrosis. Oral Dis 1997;3:286±7. 55. Shear M, Lemmer J, Dockrat I. Oral submucous fibrosis in South African Indians: an epidemiological study. S Afr J Med Sci 1967;32:41±6. 56. Hazare VK, Goel RR, Gupta PC. Oral submucous fibrosis, areca nut and pan masala use: a case±control study. Natl Med J India. 1998;11:299. 57. Maher R, Lee AJ, Warnakulasuriya KAAS et al. Role of areca nut in the causation of oral submucous fibrosis: a case-control studying in Pakistan. J Oral Pathol Med 1994;23:65±9. 58. Sinor PN, Gupta PC, Murti PR et al. A case± control study of oral submucous fibrosis with special reference to the etiologic role of areca nut. J Oral Pathol Med 1990;19:94±8. 59. Shiau YY, Kwan HW. Submucous fibrosis in Taiwan. Oral Surg Oral Med Oral Pathol 1979;47:453 ±7. 60. Murti PR, Gupta PC, Bhonsle RB, Daftary DK, Mehta FS, Pindborg JJ. Effect on the incidence of oral submucous fibrosis of intervention in the areca nut chewing habit. J Oral Pathol Med 1990;19:99±100. 61. Canniff JP, Harvey W. The aetiology of oral submucous fibrosis: the stimulation of collagen 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. synthesis by extracts of areca nut. Int J Oral Surg 1981;10(Suppl. 1):163±7. Harvey W, Scutt A, Meghji S, Canniff JP. Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids. Arch Oral Biol 1986;31:45±9. Scutt A, Meghji S, Canniff JP, Harvey W. Stabilisation of collagen by betel nut polyphenols as a mechanism in oral submucous fibrosis. Experientia 1987;43:391 ±3. Meghji S, Scutt A, Harvey W, Canniff JP. An invitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue. Arch Oral Biol 1987;32: 213±15. van Wyk CW, Olivier A, Hoal-van Helden EG, Grobler-Rabie AF. Growth of oral and skin fibroblasts from patients with oral submucous fibrosis. J Oral Pathol Med 1995;24:349±53. van Wyk CW, Olivier A, de Miranda CM, van der Bijl P, Grobler-Rabie A. Observations on the effect of areca nut extracts on oral fibroblast proliferation. J Oral Pathol Med 1994;23:145±8. Jeng JH, Lan WH, Hahn LJ, Hsieh CC, Kuo MY. Inhibition of the migration, attachment, spreading, growth and collagen synthesis of human gingival fibroblasts by arecoline, a major areca alkaloid, in vitro. J Oral Pathol Med 1996;25:371±5. Trivedy C, Baldwin D, Warnakulasuriya S, Johnson NW, Peters T. Copper content in Areca catechu (betel nut) products and oral submucous fibrosis. Lancet 1997;347:1447. Trivedy C, Warnakulasuriya S, Hazarey VK, Johnson NW. The role of copper in the aetiopathogenesis of oral submucous fibrosis. In: Varma AK, editor. Oral oncology, vol. VI. New Delhi: Macmillan Press; 1999, pp. 7±10. Trivedy CR, Warnakulasuriya KA, Peters TJ, Senkus R, Hazarey VK, Johnson NWJ. Raised tissue copper levels in oral submucous fibrosis. J Oral Pathol Med. 2000;29: 241±8. Ma RH, Tsai CC, Shieh TY. Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan. J Oral Pathol Med 1995;24:407±12. Trivedy C, Warnakulasuriya S, Hazarey VK, Tasvassoli M, Sommer P, Johnson NW. The upregulation of lysyl oxidase in oral submucous fibrosis and squamous cell carcinoma. J Oral Pathol Med 1999;28: 246±51. Trivedy CR, Meghji S, Warnakulasuriya KAAS, Johnson NW, Harris NW. Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis. J Oral Pathol Med 2001;30:465 ±70. Khrime RD, Mehra YN, Mann SB, Mehta SK, Chakraborti RN. Effect of instant preparation of betel nut (pan masala) on the oral mucosa of albino rats. Indian J Med Res 1991;94:119 ±24. Sirsat SM, Khanolkar VR. The effect of arecoline on the palatal and buccal mucosa of the Wistar rat: an optical and electron microscope study. Indian J Med Sci 1962;16:198 ±202. Oral health consequences of chewing areca nut 76. MacDonald DG. Effects of arecaidine application to hamster cheek pouch. J Oral Med 1986;41:269 ±70. 77. Canniff JP, Batchelor JR, Dodi IA, Harvey W. HLA-typing in oral submucous fibrosis. Tissue Antigens 1985;26:138±42. 78. Maher R. Clinical and etiological aspects of oral submucous fibrosis. MPhil thesis. University of London, 1999. 79. van Wyk CW, Grobler-Rabie AF, Martell RW, Hammond MG. HLA-antigens in oral submucous fibrosis. J Oral Pathol Med 1994;23:23±7. 80. Balaram AP. The use of betel nut: a cause of cancer in Malabar. Indian Med Gaz 1902;37:414. 81. Orr IM. Oral cancer in betel chewer’s in Travancore, its aetiology, pathology and treatment. Lancet 1933;2:575±80. 82. International Agency for Research on Cancer. IARC monograph on the evaluation of carcinogenic risk of chemicals to humans: tobacco habits other than smoking: betel quid and areca-nut chewing; and some related nitrosamines, vol. 37. Lyon: IARC; 1985. 83. Thomas S, Wilson A. A quantitative evaluation of the etiological role of betel-quid in oral carcinogenesis. Eur J Cancer Oral Oncol 1993;29B;265±71. 84. Merchant A, Husain SS, Hosain M et al. Paan without tobacco: an independent risk factor for oral cancer. Int J Cancer 2000;86:128±31. 85. Lu CT, Yen YY, Ho CS et al. A case±control study of oral cancer in Changhua County, Taiwan. J Oral Pathol Med 1996;25: 245±8. 86. Ko YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J Oral Pathol Med 1995;24:450 ±3. 87. van Wyk CW, Stander I, Padayachee A, GroblerRabie AF. The areca nut chewing habit and oral squamous cell carcinoma in South African Indians. A retrospective study. S Afr Med J 1993;83:425±9. 88. Jafarey NA, Mahmood Z, Zaidi SHM. Habits and dietary pattern of cases of carcinoma of the oral cavity and oropharynx. J Pak Med Assoc 1977;27:340 ±3. 89. Hiriyama T. An epidemiological study of oral and pharyngeal cancer in Central and South-East Asia. Bull WHO 1966;34:41±69. 90. Gupta PC, Pindborg JJ, Mehta FS. Comparison of carcinogenicity of betel quid with and without tobacco: an epidemiological review. Ecol Dis 125 1982;1:213±19. 91. Jeng JH, Chang MC, Hahn LJ. Role of areca nut in betel quid associated chemical carcinogenesis: current awareness and future perspectives. Eur J Cancer Oral Oncol 1999;37B:477±92. 92. Jin YT, Tsai ST, Wong TY, Chen FF, Chen RM. Studies on promoting activity of Taiwan betel quid ingredients in hamster buccal pouch carcinogenesis. Eur J Cancer Oral Oncol 1996;32B:343±6. 93. Saikia JR, Schneeweiss FH, Sharan RN. Arecoline-induced changes of poly-ADP-ribosylation of cellular proteins and its influence on chromatin organization. Cancer Lett 1999;139:59 ±65. 94. Tanaka T, Mori H, Fujii M, Takashi M, Hirono I. Carcinogenicty examination of betel betel quid. II. Effect of vitamin A deficiency on rats fed semipurified diet containing betel nut and calcium hydroxide. Nutr Cancer 1983;4:260±6. 95. Nishikawa A, Prokopczyk B, Rivenson A, Zang E, Hoffmann D. A study of betel quid carcinogenesis-VIII. Carcinogenicity of 3-(methylnitrosamino) propionaldehyde in F344 rats. Carcinogenesis. 1992;13:369 ±72. 96. Bhide SV, Shivapurkar NM, Gothoskar SV, Ranadive KJ. Carcinogenicity of betel quid ingredients: feeding mice with aqueous extract and the polyphenol fraction of betel nut. Br J Cancer 1979;40:922±6. 97. Bhisey RA, Ramchandani AG, D’Souza AV, Borges AM, Notani PN. Long-term carcinogenicity of pan masalas in Swiss mice. Int J Cancer 1999;83:679±84. 98. Adhvaryu SG, Dave BJ, Trivedi AH. An in vitro assessment of the genotoxic potential of pan masalas. Indian J Med Res 1989;90:131 ±4. 99. Fang C, Liu S, Sheng Z, Li Z, Tang F. Study on the cytotoxic and DNA damaging effects in oral mucosal fibroblasts by areca nut extract. Hunan I Ko Ta Hsueh Hsueh Pao 1997;22:105 ±8. 100. Liu TY, Chen CL, Chi CW. Oxidative damage to DNA induced by areca nut extract. Mutat Res 1996;367: 25±31. 101. Shirname LP, Menon MM, Nair J, Bhide SV. Correlation of mutagenicity and tumorigenicity of betel quid and its ingredients. Nutr Cancer 1983;5:87±91. 102. Polasa K, Babu S; Shenolikar IS. Dose-dependent genotoxic effect of pan masala and areca nut in the Salmonella typhimurium assay. Food Chem Toxicol 1993;31:439 ±42. 103. Dave BJ, Trivedi AH, Adhvaryu SG. Role of areca nut consumption in the cause of oral cancers. Cancer 1992;70:1017 ±23.