Areca-nut toxicity in cultured human buccal epithelial cells.

In cultured human buccal epithelial cells, at doses of 3-540 micrograms/ml, areca-nut extract significantly decreased viability, as determined by colony-forming efficiency, clonal growth rate, ability to take up neutral red and ability to exclude trypan blue, and also caused significant formation of DNA single-strand breaks and DNA protein cross-links. Comparisons of the areca nut-related compounds, 3-(N-nitrosomethylamino)propion-aldehyde (NMPA), 3-(N-nitrosomethylamino)propionitrile (NMPN), N-nitrosoguvacoline, N-nitrosoguvacine, arecoline, arecaidine, guvacoline and guvacine, in terms of the above endpoints, indicate that NMPA is ten times more cytopathic to buccal cells than the other agents on a molar basis. Because metabolism of NMPA can potentially yield several reactive breakdown products, including aldehydes, this study indicates that both the parent compound and its metabolites may contribute to the observed pathobiological effects. Taken together, the observed pathobiological effects of areca-nut extract and certain related compounds in cultured human buccal epithelial cells indicate that these agents may contribute to the oral carcinogenicity associated with chewing betel quid.     

Oral lesions, genotoxicity and nitrosamines in betel quid chewers with no obvious increase in oral cancer risk

A link between the generation of areca nut-related N-nitrosamines in the saliva, the induction of genotoxic damage in the oral mucosa, as judged by an increase in micronucleated exfoliated cells (MEC), and a low incidence of oral cancer was studied in 2 population groups characterized by their habit of chewing quids without tobacco: Guamanians, who chew areca nuts (Areca catechu) with or without the addition of betel leaf (Piper betle); Taiwanese, who use areca nut, betel leaf or inference and slaked lime. The levels of N-nitrosoguvacoline (NG) in the saliva of chewers of fresh green areca nuts were very high (70.8 ng/ml) as compared to those reported for individuals using the more complex Indian betel quids (0.91 ng/ml or 5.6 ng/ml). None of the other areca nut-related nitrosamines (N-nitrosoguvacine (NGC), 3-(methylnitrosamino)propionitrile (MNPN) and 3-(methylnitrosamino)propionaldehyde (MNPA)) were detected in the saliva of Taiwanese betel quid chewers. The addition of slaked lime to the areca nut enhances the formation of NG during a chewing session. The frequency of MEC did not increase in the oral mucosa of areca nut chewers who do not use slaked lime, but showed a small but significant elevation in individuals using lime-containing quids. The elevation of MEC in Taiwanese, who are at low risk for oral cancer, is relatively small as compared to that found in chewers of Indian betel quids (pan), who show a highly elevated oral cancer risk. The results seem to suggest that NG may play only a minor role, if any, in the etiology of oral cancer among betel quid chewers.     

A study of betel quid carcinogenesis. II. Formation of N-nitrosamines during betel quid chewing

In model studies, nitrosation of the major areca alkaloid, arecoline, leads to the formation of N-nitrosoguvacoline, 3-(methylnitrosamino)propionitrile (MNPN), 3-(methylnitrosamino)propionaldehyde and two unknown N-nitrosamines. MNPN is a strong carcinogen in Fischer 344 rats. After subcutaneous injection of 1.1 mmol MNPN in 60 doses, all 15 male and 15 female rats developed tumours within 24 weeks; multiple tumours occurred in 26 of the rats. Eighty-seven percent of the animals had tumours of the oesophagus, 70% had nasal cavity tumours, 37% had tumours of the tongue, 7% tumours of the pharynx and 7% tumors of the forestomach. At the dose used, male and female rats showed no significant difference in tumour incidence or site of tumours. The formation of MNPN during betel quid chewing, although likely, has not yet been proven, while the areca-derived N-nitrosamine, N-nitrosoguvacoline (NG), has been found in the saliva of betel quid chewers at levels of 2.2-348 micrograms/L. N-Nitrosoguvacoline levels were higher in the saliva of chewers who used betel quid together with tobacco. The saliva of these chewers also contained tobacco-specific N-nitrosamines.     

Tobacco-specific 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

In order to evaluate exposure of betel quid chewers to N-nitrosocompounds, saliva and urine samples were collected from chewersof betel quid with or without tobacco, from tobacco chewers,from cigarette smokers and from people with no such habit, andwere analysed for the presence of N-nitrosamines by gas chromatographycoupled with Thermal Energy Analyzer and alkaloids derived frombetel nut and tobacco by capillary gas chromatography fittedwith nitrogen-phosphorous selective detector. The levels ofthe betel nut-specific nitrosamines, N-nitrosoguvacoline andN-nitrososoguvacine (the latter being detected for the firsttime in saliva), ranged from 0 to 7.1 and 0 to 30.4 ng/ml, respectively.High levels of tobacco-specific nitrosamines were detected inthe saliva of chewers of betel quid with tobacco and in thatof chewers of tobacco, ranging from 1.6 to 59.7 (N'-nitrosonornicotine),1.0 to 51.7 (N'-nitrosoanatabine) and 0 to 2.3 [4-(methyl-nitrosamino)-1-(3-pyridyl)-l-butanone]ng/ml. Urinary concentrations of certain N-nitrosamino acids,including N-nitrosoproline, were determined as a possible indexof exposure to nitroso compounds and their precursors in thestudy groups: no clear difference was observed. The betel nut-specificalkaloid, arecoline, was present at high levels in the salivaof betel quid chewers with or without tobacco. Nicotine andcotinine were also detected in saliva and urine of chewers oftobacco and of betel quid with tobacco. In order to assess whetherN-nitroso compounds are formed in vivo in the oral cavity duringchewing or in the stomach after swallowing the quids, the levelsof N-nitroso compounds in betel quid extracts were determinedbefore and after nitrosation at pH 7.4 and 2.1. The resultsindicate that N-nitroso compounds could easily be formed invivo. The possible role of N-nitroso compounds in the causationof cancer of the upper alimentary tract in betel quid chewersis discussed.     

A study of betel quid carcinogenesis. 1. On the in vitro N-nitrosation of arecoline

Betel quid chewing is strongly associated with cancer of theoral cavity. Extracts of betel quid are tumorigenic in the experimentalanimal, but thus far, not a single carcinogen has been detectedin the tobacco free quid. This study is based on the hypothesisthat during chewing, arecoline, the major alkaloid of the betelnut, gives rise to carcinogenic N-nitros-amines. In vitro experimentsreported here have shown that N-nitrosation of arecoline leadsto N-nitrosoguvacoline (NG), 3-(methylnitrosamino)propionitrile(MNPN) and 3-(methyI-nitrosoamino)propionaldehyde. Although,according to an earlier study, NG is most likely not carcinogenic,MNPN is suspected to be a relatively strong animal carcinogenbased on bioassays with its lower homologue. The conditionsprevailing in the oral cavity of betel quid chewers are likelyto favor the formation of these three nitrosamines.     

Effect of lime composition on the formation of reactive oxygen species from areca nut extract in vitro

Lime, representative of that used by betel quid chewers, was collected in a region of Papua New Guinea where the incidence of oral cancer is high. The free calcium hydroxide content and pH of 25 lime samples were highly correlated with the generation of reactive oxygen species from areca nut extract in vitro, and DNA damage in vitro, measured as 8-hydroxy-2'-deoxyguanosine. Fe2+ and Mg2+ levels in the lime samples were too low to modify formation of reactive oxygen species, but hydrogen peroxide formation was almost entirely inhibited by addition of Mg2+ to the reaction mixture. These results suggest that the calcium hydroxide content of lime in the presence of areca nut is primarily responsible for the formation of reactive oxygen species which might cause oxidative damage in the DNA of buccal mucosa cells of betel quid chewers.     

Cytotoxic Effects of Betel Quid and Areca Nut Aqueous Extracts on Mouse Fibroblast,Human Mouth-Ordinary-Epithelium 1 and Human Oral Squamous Cell Carcinoma Cell Lines

Background: Betel quid chewing is more common among the older generation in rural areas of Malaysia. Oral cancer in Asia has been associated with the habit of chewing betel quid and areca nut. Objective:  This study aims to investigate the cytotoxic effects of betel quid and areca nut extracts on the fibroblast (L929), mouth-ordinary-epithelium 1 (MOE1) and oral squamous cell carcinoma (HSC-2) cell lines. Methods: L929, MOE1 and HSC-2 cells were treated with 0.1, 0.2 and 0.4 g/ml of betel quid and areca nut extracts for 24, 48 and 72 h. MTT assay was performed to assess the cell viability. Results: Both extracts, regardless of concentration, significantly reduced the cell viability of L929 compared with the control (P<0.05). Cell viability of MOE1 was significantly enhanced by all betel quid concentrations compared with the control (P<0.05). By contrast, 0.4 g/ml of areca nut extract significantly reduced the cell viability of MOE1 at 48 and 72 h of incubation. Cell viability of HSC-2 was significantly lowered by all areca nut extracts, but 0.4 g/ml of betel quid significantly increased the cell viability of HSC-2 (P<0.05). Conclusion: Areca nut extract is cytotoxic to L929 and HSC-2, whereas the lower concentrations of areca nut extract significantly increased the cell viability of MOE1 compared to the higher concentration and control group. Although betel quid extract is cytotoxic to L929, the same effect is not observed in MOE1 and HSC-2 cell lines. Further investigations are needed to clarify the mechanism of action.     

Mutagenicity of betel quid and its ingredients using mammalian test systems

The mutagenic potential of betel quid and its ingredients (knowncolloquially as PAN) were tested in two short term mutagenicityassays, the micronucleus test and a mammalian gene mutationtest. Betel quid with tobacco, tobacco, betel nut and one ofits alkaloids arecoline were positive in both tests. Extractsof betel quid and arecaidine (a metabolite of arecoline presentin Betel nut) were negative. The data presented correlate wellwith our previous tumorigenicity data on these compounds.     

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water- based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well- established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.      

Promoting activity of betel quid ingredients and their inhibition by retinol

Ingredients of betel quids, which have been linked to the high incidence of precancerous oral lesions and oral cancers, were examined for their promoting activity. Aqueous extracts were tested using the bovine papillomavirus (BPV) DNA transformation assay, which consists of cultured C3H/10T1/2 cells transfected with the plasmid pdPBV-1 as targets, and the frequency of transformed foci as endpoints. Areca nut extracts enhanced the formation of BPV DNA-induced transformed foci approximately tenfold. No promoting activity was detected in two samples of chewing tobacco examined. The addition of retinol to the areca nut extract inhibited its tumour promoting effect in a dose-dependent manner, completely abolishing the promoting activity at a dose of 10(-6) M. The experimental results are compared with epidemiological data on oral cancer incidences among chewers of different areca nut/tobacco mixtures and with the chemopreventive effect of vitamin A administered to betel quid chewers.     

Role of areca nut consumption in the cause of oral cancers. A cytogenetic assessment.

BACKGROUND. Cytogenetic studies, framed to assess the possible genomic damage caused by areca nut consumption (without tobacco and not as a component of betel quid), were performed among areca nut chewers, which included normal people who chew areca nuts, patients with oral submucous fibrosis, and patients with oral cancer, and healthy nonchewing controls. RESULTS. The analysis showed statistically significant increases in the frequencies of sister chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and the percentage of micronucleated cells in exfoliated cells of buccal mucosa among all three groups of chewers when compared with those of the controls. CONCLUSIONS. The current data, the first of this type among only areca nut chewers, highlight that this popular masticatory is erroneously considered "safe" and that it increases the genomic damage even when chewed without tobacco. The data also signify that, henceforth, in cytogenetic biomonitoring, areca nut consumption also should be considered as one of the confounding factors.     

Chromosome-damaging activity of saliva of betel nut and tobacco chewers

Saliva of volunteers chewing betel quid, cured betel nut (Areca catechu), betel leaves (Piper betle), a mixture of quid ingredients (dried betel nut flakes, catechu, cardamon, lime, copra and menthol) and Indian tobacco was collected and examined for its genotoxic activity. Chromosome aberrations (chromatid breaks and chromatid exchanges) in Chinese hamster ovary (CHO) cells were used to estimate the genotoxic effect. No detectable levels of clastogenic activity were observed in the saliva of non-chewing individuals. After 5 min of chewing betel quid, betel nut, betel leaves, quid ingredients and Indian tobacco, the saliva samples showed relatively potent clastogenic activities. The addition of transition metals Mn²⁺ and Cu²⁺ to the saliva samples of betel nut and Indian tobacco chewers enhanced their clastogenic activities, whereas Fe³⁺ increased the clastogenicity of the betel nut saliva but decreased the genotoxic effect of the saliva of Indian tobacco chewers. After removal of the betel quid or its components from the mouth, the clastogenic activity disappeared within 5 min. The western-type chewing tobacco did not produce a genotoxic activity in the saliva of chewers. A possible association between the genotoxicity in the saliva of betel quid chewers and the development of oral, pharyngeal and esophageal carcinomas is discussed.     

Use of the micronucleus test to monitor the effect of vitamin A, beta-carotene and canthaxanthin on the buccal mucosa of betel nut/tobacco chewers

The frequency of exfoliated cells with micronuclei in buccal swabs was used to estimate the protective effect of vitamin A, beta-carotene and canthaxanthin (4,4'-diketo-beta-carotene) on the buccal mucosa of betel (areca) nut/tobacco chewers. Micronuclei were scored on exfoliated cells taken by swabbing and stained with the Feulgen reaction and fast green. The betel (areca) nut/tobacco chewers served as their own controls. Prior to the administration of vitamin A and beta-carotene, the examined betel quid chewers had elevated frequencies of micronucleated buccal mucosa cells, averaging 4.03% +/- 1.24 SD (n = 26) and 3.43% +/- 1.22 SD (n = 25), respectively. The frequency of micronucleated buccal mucosa cells in non-chewers and non-smokers was 0.51% (n = 52). Following a 9-week ingestion of vitamin A (150,000 IU/week) and beta-carotene (180 mg/week in 6 capsules), the frequency of micronucleated cells decreased significantly (p less than 0.001) to 1.70% and 1.16%, respectively. No significant shift in the frequencies of micronucleated cells was observed following the intake of canthaxanthin (180 mg/week in 6 capsules) for 9 weeks or that of a placebo. The lack of protective activity of canthaxanthin, which is a good trapper of oxygen singlets but cannot be converted into vitamin A, suggests that vitamin A and beta-carotene exert their inhibitory effect on the formation of micronuclei by a mechanism not involving the scavenging of free radicals. The efficacy of beta-carotene as an inhibitor of micronucleated cell formation, the lack of toxicity, and its availability from a multitude of dietary sources should focus attention on this carotenoid as a promising chemopreventive agent.     

Tobacco (Kretek) Smoking,Betel Quid Chewing and Risk of Oral Cancer in a Selected Jakarta Population

Purpose: This study aimed to determine the association between tobacco consumption (kretek) and betel quidchewing with oral cancer risk. Materials and Methods: A total of 81 cases of oral cancers were matched with162 controls in this hospital-based study. Information on sociodemographic characteristics and details of riskhabits (duration, frequency and type of tobacco consumption and betel quid chewing) were collected. Associationbetween smoking and betel quid chewing with oral cancer were analysed using conditional logistic regression.Results: Slightly more than half of the cases (55.6%) were smokers where 88.9% of them smoked kretek. Afteradjusting for confounders, smokers have two fold increased risk, while the risk for kretek consumers and thosesmoking for more than 10 years was increased to almost three-fold. Prevalence of betel quid chewing among casesand controls was low (7.4% and 1.9% respectively). Chewing of at least one quid per day, and quid combinationof betel leaf, areca nut, lime and tobacco conferred a 5-6 fold increased risk. Conclusions: Smoking is positivelyassociated with oral cancer risk. A similar direct association was also seen among betel quid chewers.     

Chewing areca nut, betel quid, oral snuff, cigarette smoking and the risk of oesophageal squamous-cell carcinoma in South Asians: a multicentre case-control study

Oesophageal cancer remains an important public health problem worldwide. This multicentre matched case-control study examined the chewing areca nut alone, betel quid with tobacco, oral snuff (snuff dipping) and cigarette smoking as the risk factors for oesophageal squamous-cell carcinoma. We enrolled 91 cases of oesophageal squamous-cell carcinoma and 364 matched controls from three tertiary-care hospitals in Karachi, Pakistan. A structured questionnaire was used to collect the data through face-to-face interview of the participants. Multivariable conditional logistic regression model showed that after adjusting for the effect of ethnicity, ever chewed areca nut alone (adjusted matched odds ratio (mOR(adj))=3.7; 95% confidence interval (CI): 1.6-8.5), ever chewed betel quid with tobacco (mOR(adj)=12.8; 95% CI: 6.3-26.2), ever practiced snuff dipping (mOR(adj)=4.3; 95% CI: 1.6-11.7) and ever smoked cigarettes (mOR(adj)=2.9; 95% CI: 1.4-5.9) were significantly and independently associated with oesophageal squamous-cell carcinoma status. The adjusted summary population attributable risk (PAR) percent for all four substances together was 67.0. Furthermore, despite incomplete synergy, there was manifold increase in the risk of oesophageal squamous-cell carcinoma, if the respondents ever smoked cigarettes and ever chewed betel quid with tobacco (mOR(adj)=21.4; 95% CI: 6.3-72.4) or if they ever smoked cigarettes and ever practiced snuff dipping (mOR(adj)=14.4; 95% CI: 2.3-91.1). The adjusted PAR (%) was higher for the dual practice of smoking cigarettes and chewing betel quid with tobacco (64.3) than the dual practice of smoking cigarettes and snuff dipping (32.2). Public awareness to curtail the addiction to these substances may result in a substantial reduction in the incidence of oesophageal squamous-cell carcinoma and related mortality in this and similar settings.     

Oral submucous fibrosis: review on aetiology and pathogenesis

Data from recent epidemiological studies provide overwhelming evidence that areca nut is the main aetiological factor for OSF. A clear dose-dependent relationship was observed for both frequency and duration of chewing areca nut (without tobacco) in the development of OSF. Commercially freeze dried products such as pan masala, Guthka and mawa (areca and lime) have high concentrates of areca nut per chew and appear to cause OSF more rapidly than by self prepared conventional betel quid that contain smaller amounts of areca nut. It is logical to hypothesise that the increased collagen synthesis or reduced collagen degradation as possible mechanisms in the development of the disease. There are numerous biological pathways involved in the above processes and, it is likely that the normal regulatory mechanisms are either down regulated or up regulated at different stages of the disease. Among the chemical constituents, alkaloids from areca nut are the most important biologically whilst tannin may have a synergistic role. These chemicals appear to interfere with the molecular processes of deposition and/or degradation of extracellular matrix molecules such as collagen. In vitro studies on human fibroblasts using areca extracts or chemically purified arecoline support the theory of fibroblastic proliferation and increased collagen formation that is also demonstrable histologically in human OSF tissues. The copper content of areca nut is high and the possible role of copper as a mediator of fibrosis is supported by the demonstration of up regulation of lysyl oxidase in OSF biopsies. It has been postulated that areca nut may also induce the development of the disease by increased levels of cytokines in the lamina propria. Increased and continuous deposition of extracellular matrix may take place as a result of disruption of the equilibrium between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMP). Current evidence implicates collagen-related genes in the susceptibility and pathogenesis of OSF. The individual mechanisms operating at various stages of the disease-initial, intermediate and advanced-need further study in order to propose appropriate therapeutic interventions.     

Modifying influences of betel quid ingredients on B(a)P-induced carcinogenesis in the buccal pouch of hamster

The present study evaluates the incidence of tumors in hamster buccal pouches following short-term (10 days) and long-term (6 months) topical exposures to graded doses of benzo(a)pyrene, B(a)P (25 micrograms, 50 micrograms and 100 micrograms per pouch either daily for 10 days or thrice weekly for 6 months) alone or in combination with extract of tobacco (1 mg/pouch, twice daily), betel nut (1 mg/pouch, twice daily) or betel leaf (5 mg/pouch, twice daily). Given alone, the three doses of B(a)P respectively yielded, 6 months after the last treatment, 4%, 8.7% and 16.7% tumors in the short-term study, and 20%, 35% and 61% tumors in the long-term study. Short-term treatments with individual ingredients of betel quid did not produce any tumors while long-term treatments produced tumors only with tobacco (17.6%) and betel nut (10.5%). When B(a)P, and betel quid ingredients were painted concomitantly for 10 days, there was, depending upon the dose of B(a)P, complete or partial suppression of tumor production. But when B(a)P-plus-tobacco or B(a)P-plus-betel nut treatments were given for 6 months, there was a considerable increase in tumor incidence. Betel leaf extract, in both short-term and long-term studies, expressed its inhibitory influence on B(a)P-induced tumorigenesis.     

Roles of CYP1A1 and CYP2E1 Gene Polymorphisms in Oral Submucous Fibrosis

Background: Oral submucous fibrosis (OSF) is a precancerous condition with a 4 to13% malignant transformation rate. Related to the habit of areca nut chewing it is mainly prevalent in Southeast Asian countries where the habit of betel quid chewing is frequently practised. On chewing, alkaloids and polyphenols are released which undergo nitrosation and give rise to Nnitrosamines which are cytotoxic agents. CYP450 is a microsomal enzyme group which metabolizes various endogenous and exogenous chemicals including those released by areca nut chewing. CYP1A1 plays a central role in metabolic activation of these xenobiotics, whereas CYP2E1 metabolizes nitrosamines and tannins. Polymorphisms in genes that code for these enzymes may alter their expression or function and may therefore affect an individuals susceptibility regarding OSF and oral cancer. The present study was therefore undertaken to investigate the association of polymorphisms in CYP1A1 m2 and CYP2E1 (RsaI/PstI) sites with risk of OSF among areca nut chewers in the Northern India population. A total of 95 histopathologically confirmed cases of OSF with history of areca nut chewing not less than 1 year and 80, age and sex matched controls without any clinical signs and symptoms of OSF with areca nut chewing habit not less than 1 year were enrolled. DNA was extracted from peripheral blood samples and polymorphisms were analyzed by PCRRFLP method. Gene polymorphism of CYP1A1 at NcoI site was observed to be significantly higher (p 0.016) in cases of OSF when compared to controls. Association of CYP1A1 gene polymorphism at NcoI site and the risk of OSF (Odds Ratio 2.275) was also observed to be significant. However, no such association was observed for the CYP2E1 gene polymorphism (Odds Ratio 0.815). Our results suggest that the CYP1A1 gene polymorphism at the NcoI site confers an increased risk for OSF.     

Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk for oral cancer: betel quid chewers

The micronucleus test was applied to buccal mucosa cells of 2 population groups at high risk for oral cancer: Khasis of the northeastern hill region of India, who eat raw betel nuts together with betel leaves and lime, and residents of the state of Orissa (India), who chew betel quids consisting mainly of perfumed tobacco, dried betel nut, betel leaf, lime and several spices. Micronuclei were scored on Feulgen/fast green-stained smear preparations of exfoliated cells obtained by scraping the surface of the buccal mucosa. All 17 raw betel nut eaters and all 20 chewers of betel quids had significantly elevated frequencies of micronucleated mucosa cells over nonchewing controls of comparable ethnic background and dietary habits. The frequencies of micronucleated exfoliated cells were higher at the site within the oral cavity where the quid was kept compared to those at the opposite buccal wall. The micronuclei frequency was lower among individuals chewing a raw betel nut, betel leaf and lime mixture compared to those using tobacco,-betel nut-, lime- and betel leaf-containing quids. Micronuclei frequencies in exfoliated human cells seem to represent a useful 'internal dosimeter' for estimating exposure to genotoxic, and by implication, carcinogenic agents in the tissue from which cancers will develop.     

Role of areca nut in betel quid-associated chemical carcinogenesis: current awareness and future perspectives.

Betel quid (BQ)-chewing is a popular oral habit with potential links to the occurrence of oral cancer. Many of the literature-based studies reveal that areca nut (AN) extract may demonstrate mutagenic and genotoxic effects, in addition to inducing preneoplastic as well as neoplastic lesions in experimental animals. Areca nut should, thus, be highly suspected as a human carcinogen. Toxicity studies relating to AN-contained polyphenols and tannins are not conclusive, with both carcinogenic and anti-carcinogenic effects being reported. The mutagenicity and genotoxicity of areca alkaloids has been detected by many short-term assays. However, their genotoxicity to oral fibroblasts and keratinocytes, the target cells of BQ, has not been identified. It would thus appear that AN toxicity is not completely due to its polyphenol, tannin and alkaloid content. The single agent which is responsible for AN carcinogenicity awaits further clarification. Reactive oxygen species produced during auto-oxidation of AN polyphenols in the BQ-chewer's saliva, are crucial in the initiation and promotion of oral cancer. Nitrosation of areca alkaloids also produces AN-specific nitrosamines, that have been demonstrated to be mutagenic, genotoxic and are capable of inducing tumors in experimental animals. Arecaidine and AN extract are further suggested to be tumor promoters. Antioxidants such as glutathione and N-acetyl-L-cysteine can potentially prevent such AN-elicited cytotoxicity. Further studies are needed to delineate the metabolism of AN ingredient and their roles in the multi-step chemical carcinogenesis, in order to enhance the success of the future chemoprevention of oral cancer and oral submucous fibrosis.