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.     

Betel quid chewing and oral cancer: experimental studies on hamsters

Betel quid ingredients--betel nut, betel leaf, lime, catechu and tobacco--were tested separately and in various combinations for carcinogenicity, using hamster cheek pouch as the experimental site. The four modes of administration used were (1) tri-weekly painting of the cheek pouch with aqueous extracts of test materials, (2) deposition of replaceable wax pellets containing the test material, (3) gelatin capsules containing the powdered material and (4) insertion of natural material into the pouch for trauma and direct exposure. Untreated controls and standard carcinogen DMBA-treated controls were also maintained. A total of 317 young adult golden Syrian hamsters (Mesocricetus auratus) used for the experiments were killed in two age groups: 6-12 months and 13-24 months, only when signs of general debility were observed. In the untreated controls, animals were free of any malignancy. In the experimental series, various betel quid ingredient combinations under test induced both oral and gastric lesions ranging from massive atypia and precancerous lesions to frank carcinomas. Maximum lesions were observed in the groups receiving betel nut, lime and tobacco combinations and in the polyphenol fraction of betel nut containing major tannins. The mode of administration of test material resulted in distinct differences; tri-weekly paintings giving oral lesions in the range of 22-23% and gastric lesions 39-48%; the same material given either through the replaceable gelatin capsule or in natural form induced 69% oral lesions and 63 to 82% gastric lesions. Overall evaluation of the data of all the four series confirms the potent carcinogenicity of betel nut, particularly its tannin-containing polyphenolic fraction and its combination with lime and tobacco. Maximum oral lesions induced in the hamsters by continuous exposure to capsules and natural material, highlight the direct relationship of frequency of chewing in habitual chewers with oral carcinogenesis. The high incidence of gastric (forestomach) lesions invites special attention.     

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.     

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.     

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.     

Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A

Fishermen from Kerala (India) who chewed tobacco-containing betel quids daily (17.2 +/- 9.6 quids per day) and had well-developed oral leukoplakias with elevated frequencies of micronucleated cells participated in a short-term intervention trial. Beta-carotene (180 mg/week) (Group I), beta-carotene (180 mg/week) plus vitamin A (100,000 IU/week) (Group II), and placebo (Group III) capsules were given twice weekly for 6 months under strict supervision. The remission of oral leukoplakias, the inhibition of new leukoplakias, and the reduction of micronucleated oral mucosal cells were recorded at the 3rd and 6th months of the trial period. After 3 months, the frequency of micronucleated cells was significantly reduced in Group I (from 4.09% to 1.1% in areas of leukoplakia, and from 4.1% to 1.0% in the normal mucosa). At this time, remission of oral leukoplakias did not differ significantly from that observed in the placebo group. After 6 months of treatment, remission of leukoplakias in Group I (14.8%) and Group II (27.5%) differed significantly from that seen in Group III (3.0%). The development of new leukoplakias during the 6-month period was strongly inhibited in Group II (7.8%), and to a lesser degree in Group I (14.8%), as compared to Group III (21.2%). During the trial period, all participants continued to chew tobacco-containing betel quids in their accustomed manner. Thus, remission and inhibition of new oral leukoplakias and reduction of micronucleated mucosal cells occurred in the groups receiving beta-carotene and beta-carotene plus vitamin A during the continuous presence of carcinogens derived from tobacco and areca nut.     

Betel quid chewing as a risk factor for hepatocellular carcinoma: a case-control study

The role of betel quid chewing in the aetiology of hepatocellular carcinoma (HCC) was evaluated in a case-control study including 263 pairs of age- and sex-matched HCC patients and healthy controls. Serum hepatitis B surface antigen (HBsAg), and antibodies to hepatitis C virus (anti-HCV) were determined, and standardized personal interview conducted using a structured questionnaire. Multivariate analysis indicated that betel quid chewing (odds ratio (OR), 3.49; 95% confidence interval (CI), 1.74-6.96), HBsAg (OR, 16.69; 95% CI, 9.92-28.07), anti-HCV (OR, 38.57; 95% CI, 18.15-81.96), and educational duration of less than 10 years (OR, 1.71; 95% CI, 1.05-2.78) are independent risk factors of HCC. In addition, there was an additive interaction between betel quid chewing and chronic infection with either hepatitis B virus (synergy index, 5.37) or hepatitis C virus (synergy index, 1.66). Moreover, risk on HCC increased as duration of betel quid chewing increased, or amount of betel quid consumed (each P for trend < 0.0001).     

Response of oral leukoplakias to the administration of vitamin A

Tobacco/betel nut chewers (Kerala, India) with well-developed oral leukoplakias were chosen for a short-term intervention trial of vitamin A therapy. Participants were randomly distributed into two groups, one receiving 200,000 IU vitamin A per week (0.14 mg/kg body wt/per day) for 6 months, and the other receiving placebo capsules. Their cancer-causing habit, which can be quantitated (an average of 13.1 betel quids/day, 26.1 min/quid), did not change during the trial period. The 6-month oral administration of vitamin A caused complete remission in 57.1% of participants, and a total suppression of the development of new leukoplakias in all chewers receiving vitamin A (n = 21), as compared to 3% and 21%, respectively, in the placebo group (n = 33). The results were substantiated by examining the histological and cytological changes on small biopsies which were taken at the onset and at the completion of the trial period. Over the 6-month period of vitamin A administration, the number of layers of spinous cells decreased in 85% of the participants, the loss of polarity of basal cells was reduced from 72.2% to 22.2% of chewers, subepidermal lymphocytic infiltration was greatly diminished from 66.7% to 5.5% of chewers, and nuclei with condensed chromatin disappeared from the epidermal layer (72.2% before to 0% at the end of the trial).     

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.     

Piper betel Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion

Since antiquity, Piper betel Linn (betel vine; family Piperaceae) has been an important medicinal agent in the various traditional and folk systems of medicine in Southeast Asia countries. The leaves are the most valued plant part and in the past were routinely used as a chewing agent to prevent halitosis. The leaves are also supposed to harden the gum, conserve the teeth and to prevent indigestion, bronchitis, constipation, congestion, coughs and asthma. Innumerable scientific studies have validated the ethnomedicinal claims. Betel leaves are an integral component of the betel quid that consists of areca nut (Areca catechu Linn.), tobacco (Nicotiana tabacum L) and slaked lime; a highly abused agent with carcinogenic properties. Regular chewing of betel quid is associated mainly with oral cancer and detail studies with individual constituents of the quid have shown that both tobacco and areca nut are carcinogenic, while slaked lime is shown to promote the process of carcinogenesis. However unlike other constituents of the betel quid, the betel leaves devoid carcinogenic effects and on the contrary possesses cancer preventive effects including against the carcinogens present in tobacco. This review for the first time provides information on cancer preventive effects and also addresses the various mechanisms which might be involved.     

Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing

The habit of betel quid chewing, common in South-East Asia andthe South Pacific islands, is causally associated with an increasedrisk of oral cancer. Reactive oxygen species formed from polyphenolicbetel quid ingredients and lime at alkaline pH have been implicatedas the agents responsible for DNA and tissue damage. To determinewhether hydroxyl radical (HO) is generated in the human oralcavity during chewing of betel quid, the formation of o- andm-tyrosine from L-phenylalanine was measured, Both o- and m-tyrosinewere formed in vitro in the presence of extracts of areca nutand/or catechu, transition metal ions such as Cu²⁺ and Fe²⁺and lime or sodium carbonate (alkaline pH). Omission of anyof these ingredients from the reaction mixture significantlyreduced the yield of tyrosines. Hydroxyl radical scavengerssuch as ethanol, D-mannitol and dimethylsulfoxide inhibitedthe phenylalanine oxidation in a dose-dependent fashion. Fivevolunteers chewed betel quid consisting of betel leaf, arecanut, catechu and slaked lime (without tobacco). Their saliva,collected after chewing betel quid, contained high concentrationsof p-tyrosine, but no appreciable amounts of o- or m-tyrosine.Saliva samples from the same subjects after chewing betel quidto which 20 mg phenylalanine had been added contained o- andm-tyrosine at concentrations ranging from 1010 to 3000 nM andfrom 1110 to 3140 nM respectively. These levels were significantlyhigher (P< 0.005) than those of subjects who kept phenylalaninein the oral cavity without betel quid, which ranged from 14to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine.These studies clearly demonstrate that the HO radical is formedin the human oral cavity during betel quid chewing and is probablyimplicated in the genetic damage that has been observed in oralepithelial cells of chewers.     

The Risk of Oral Cancer among Different Categories of Exposure to Tobacco Smoking in Sri Lanka

Background: The global incidence of oral squamous cell carcinoma (OSCC) is on the rise with no improvement seen in survival rates. Tobacco consumption varies depending on geographic location, ethnicity and culture. The present case-controlled study aimed to determine the relative risk of OSCC for different tobacco consumption patterns in a selected Sri Lankan population. Methods: One hundred and five patients with histopathologically confirmed OSCC attending the National Cancer Institute (Apeksha Hospital) of Sri Lanka and 210 age and gender-matched controls from the community responded to an interviewer-administered questionnaire regarding their smoking and betel-quid chewing (with/ without smokeless tobacco) habits were included in the study. The odds ratios (OR) and 95% confidence intervals (CI) were calculated. p<0.05 was considered as statistically significant. Results: The overall risk of OSCC increased 2.93-fold for smokers. Those smoking two packets of cigarettes or more per day (OR=5.56; 95% CI-2.822-10.984; p=0.000) had more than double the risk of OSCC than those smoking 1-2 packets per day. Smoking for more than 20 years had a 3.4-fold risk of OSCC. Consumption of betel quid containing tobacco (smokeless tobacco) had a 4.26-fold higher risk for OSCC (OR=4.26; 95% CI-2.21-8.21; p=0.000), and the risk increased when all four ingredients (betel leaf, slaked lime, areca nut, and tobacco) were consumed together (OR=4.26; 95% CI-2.34-7.74; p=0.000). The combined effect from concurrent smoking and betel chewing emerged as the highest risk for OSCC (OR=15.34) which significantly exceeded the risks evident for the two habits practised in isolation from each other. Conclusions: Use of smokeless tobacco, consumption of all four ingredients together, duration of smoking, the number of cigarettes smoked per day and combined consumption of betel quid and smoking are significant risk factors in the development of OSCC among Sri Lankans.     

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.     

Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells

Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.     

Experimental studies on betel nut and tobacco carcinogenicity.

Sun-dried Mangalore betel nut extracts in water and in DMSO, and sun-cured Vadakkan tobacco extract in DMSO, were tested for their carcinogenic potency. Inbred Swiss and C17 mice and golden hamsters were used for the experiments. Control animals treated with either DMSO or water did not show any changes at the sites of administration. On subcutaneous administration of betel nut extract, 60% of Swiss mice developed transplantable fibrosarcomas at the site of injection. Skin application of DMSO extracts of tobacco and of betel nut separately did not result in skin lesions in C17 mice; but when a mixed DMSO extract of tobacco and betel nut was used, skin papilloma and epidermoid carcinoma developed in some animals. Similarly, hamster cheek pouches painted with a DMSO extract of tobacco alone did not develop malignant atypia whereas those painted with a DMSO extract of betel nut showed early malignant changes. DMSO extract of a mixture of tobacco and betel nut positively increased the incidence of early malignant changes in the hamster cheek pouch, indicating the enhancing effect of betel nut in carcinogenesis.     

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.     

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.     

Carcinogenicity of betel quid ingredients: feeding mice with aqueous extract and the polyphenol fraction of betel nut

Male mice of inbred strains Swiss and C17 were fed daily 5 times a week by intragastric tube 0.1 ml of betel-nut aqueous extract, betel-leaf aqueous extract and the polyphenol fraction of betel nut. Male mice of corresponding strains fed 0.1 ml of distilled water served as controls. Treated and control mice were kept under observation and killed when moribund. Betel-nut aqueous extract induced tumours of the gastrointestinal tract in 58% Swiss mice and 25% C17 mice. The polyphenol fraction by the same route induced tumours at other sites in 17% of the mice. Betel-leaf aqueous extract failed to induce any tumour in the treated mice, which supports an earlier report of the lack of any carcinogenic principle in betel leaf, an essential constituent of betel quid. Results are discussed in relation to the relevant literature.     

3-(Methylnitrosamino)propionitrile: occurrence in saliva of betel quid chewers, carcinogenicity, and DNA methylation in F344 rats

3-(Methylnitrosamino)propionitrile (MNPN), a potent carcinogen in F344 rats, was detected for the first time in the saliva of betel quid chewers at levels ranging from 0.5 to 11.4 micrograms/liter. The tumorigenic properties of MNPN and its potential to methylate DNA in F344 rats were evaluated. Groups of 21 male and 21 female rats were given 60 s.c. injections over a 20-week period (total doses 0.055 and 0.23 mmol per rat). The experiment was terminated after 106 weeks. MNPN at the higher dose induced 18 (86%) malignant tumors of the nasal cavity in male and 15 (71%) in female rats. The lower dose induced nine (43%) liver tumors. Groups of four or five male F344 rats were treated with a single s.c. or i.v. injection of MNPN (0.4 mmol/kg). MNPN was also administered to rats by swabbing the oral cavity (2.21 mmol/kg). The levels of 7-methylguanine and O6-methylguanine, formed 0.5-36 h after treatment, were measured in the liver, nasal mucosa, esophagus, and oral issues. The highest levels of methylated guanines were detected in the nasal cavity independent of the route of administration. The results of this study demonstrate that MNPN is present in the saliva of betel quid chewers and is a potent carcinogen in F344 rats.     

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.