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.     

Carcinogenicity of betel quid. III. Enhancement of 4-nitroquinoline-1-oxide- and N-2-fluorenylacetamide-induced carcinogenesis in rats by subsequent administration of betel nut

The effect of betel nut on chemical carcinogenesis in the upper digestive tract and liver was examined in two different experimental models with ACI rats. The incidences of neoplasms and preneoplastic lesions of the tongue in animals given 5 ppm 4-nitroquinoline-1-oxide (4-NQO; CAS: 56-57-5) in the drinking water for 16 weeks and followed by 20% betel nut in the diet for 40 weeks were significantly higher than those in animals given 4-NQO alone. No enhancing effect from betel nut on the incidences of neoplastic and preneoplastic lesions in the upper digestive tract was found in animals administered 4-NQO for 12 weeks. The number of altered liver cell foci in rats given 200 ppm N-2-fluorenylacetamide (FAA; CAS: 53-96-3) in the diet for 8 weeks and followed by the betel nut diet for 16 weeks was significantly greater than that in animals fed the FAA diet alone. These results indicate enhancing effects of dietary administration of betel nut on oral carcinogenesis by 4-NQO and hepatocarcinogenesis initiated by FAA.     

A study of betel quid carcinogenesis. 3. 3-(Methylnitrosamino)-propionitrile, a powerful carcinogen in F344 rats

3-(Methylnitrosamino)propionitrile (MNPN) is formed in vitrounder mild nitrosation conditions from the major areca alkaloidarecoline. It appears likely that MNPN is generated during betelquid chewing especially when tobacco is added to the quid. Uponsubcutaneous injection of 1.1 mmol of MNPN in 60 subdoses, allof the 15 male and of the 15 female F344 rats developed tumorswithin 24 weeks. Twenty-six of the rats had tumors in two differentorgans at least. Twenty-seven rats had esophageal tumors, 21nasal tumors, 11 had tongue tumors and two animals had eitherpharyngeal carcinomas or papillomas of the forestomach. No tumorswere observed in the solvent control group. These data indicatethat MNPN is a potent carcinogen.     

Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis

Head and neck cancer (HNC) is one of the 10 most frequent cancers worldwide, with an estimated over 500 000 new cases being diagnosed annually. The overall 5-year survival rate in patients with HNC is one of the lowest among common malignant neoplasms and has not significantly changed during the last two decades. Oral cavity squamous cell carcinoma (OSCC) shares part of HNC and has been reported to be increasing in the betel quid chewing area in recent years. During 2006, OSCC has become the sixth most common type of cancer in Taiwan, and it is also the fourth most common type of cancer among men. It follows that this type of cancer wreaks a high social and personal cost. Environmental carcinogens such as betel quid chewing, tobacco smoking and alcohol drinking have been identified as major risk factors for head and neck cancer. There is growing interest in understanding the relationship between genetic susceptibility and the prevalent environmental carcinogens for HNC prevention. Within this review, we discuss the molecular and cellular aspects of HNC carcinogenesis in Taiwan, an endemic betel quid chewing area. Knowledge of molecular carcinogenesis of HNC may provide critical clues for diagnosis, prognosis, individualization of therapy and molecular therapeutics. ( Cancer Sci 2008; 99: 1507–1514)     

Up-regulation of matrix metalloproteinase-8 by betel quid extract and arecoline and its role in 2D motility

Betel quid (BQ) and matrix metalloproteinase-8 (MMP-8) play roles in oral diseases. Here, we analyzed the regulation of MMP-8 by BQ and its effect on cell migration. We found that BQ extract (BQE) increased the secretion of an 85 kDa caseinolytic proteinase, specifically precipitated by an anti-MMP-8 antibody, in the culture medium of OECM-1, an oral squamous cell carcinoma (OSCC) cell line. BQE also stimulated MMP-8 secretion in an esophageal carcinoma cell line, CE81T/VGH, in a dose-dependent manner, and MMP-8 protein was maximally expressed at 24 h after BQE treatment in OECM-1. The BQE-induced MMP-8 expression was dose-dependently inhibited by PD98059. Arecoline, the major alkaloid of areca nut, was tested to dose-dependently up-regulate MMP-8 protein level. Moreover, both arecoline- (4.7-fold) and BQE-selected (5.5-fold) CE81T/VGH cells expressed higher MMP-8 protein level and exhibited enhanced two-dimensional (2D) motility (p = 0.009 in both cells) than parental cells. The enhanced motility of arecoline- (p = 0.006) and BQE-selected (p = 0.002) cells was both specifically blocked by an anti-MMP-8 antibody. We conclude that BQ may accelerate tumor migration by stimulating MMP-8 expression through MEK pathway in at least some carcinomas of the upper aerodigestive tract. Furthermore, arecoline may be one of the positive MMP-8 regulators among BQ ingredients.     

A study of betel quid carcinogenesis--VIII. Carcinogenicity of 3-(methylnitrosamino)propionaldehyde in F344 rats.

In assays of Areca-specific N-nitrosamines, 3-(methylnitrosamino)propionaldehyde (MNPA) exhibits higher cytotoxicity than nitrosoguvacine (NGC), nitrosoguvacoline (NG) and 3-(methylnitrosamino)propionitrile (MNPN). NGC is not mutagenic. However, NG is a weak carcinogen in F344 rats while MNPN is a potent carcinogen; MNPA had thus far not been tested. In this study MNPA was injected s.c. at a dose of 6.57 mg three times weekly for 15 weeks (total dose 2.6 mmol/rat). During the 100 weeks of the bioassay, the treated F344 rats, and especially the females, showed significantly less weight gain than the control animals, indicating high toxicity for MNPA at the tested dose. Upon termination of the bioassay, the MNPA-treated animals were found to have tumors of the lung, liver, nasal cavity, forestomach and kidneys. The control animals showed no tumors in these organs. The incidence of lung tumors in the MNPA group was statistically significant (P less than 0.025). The results of this study show that MNPA is a carcinogen in F344 rats.     

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.     

A review of betel quid chewing, oral cancer and precancer in Mainland China

On the Chinese mainland, betel quid (BQ) chewing is common in the Hunan and Hainan provinces. The BQ chewing habit in Hunan consists of dried husks and betel nuts, which are sold as industrially packaged, areca nut-based products. In Hainan, the fresh nut is chewed. Tobacco is not added. Reported prevalence of BQ chewing in Hunan province is high (64.5-82.7%). Oral diseases associated with BQ chewing are oral submucous fibrosis (OSF), oral leukoplakia (OL) and oral cancer. Reported prevalence of OSF among BQ chewers ranges from 0.9% to 4.7%. People most commonly affected are between the ages of 30 and 39 years, and 40 and 49 years. The reported prevalence of OL in Hainan ranges from 2.1% to 2.5%. In BQ chewers who also smoke, the reported prevalence is 20.3%. The prevalence of OL in Hunan province ranges from 0.1% to 0.5%. The prevalence of oral cancer among BQ chewers is low, ranging from 0.02% to 0.05%. In cases of OSF, reported prevalence is 2.6% and 1.2%. Presently, data on prevalence of BQ chewing in southern provinces of Mainland China is limited. BQ chewing habits, however, seem to differ between geographic areas. Future case-control studies are necessary to evaluate the risk for oral cancer and other associated oral mucosal diseases resulting from variations in BQ chewing habits.     

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.     

The mRNA profile of genes in betel quid chewing oral cancer patients

Oral cancer is one of the most common types of human cancer in the world. Although the risk factors for oral cancer are well-recognized in different countries, the molecular mechanism responsible for this malignancy remains elusive particularly in the countries where betel quid chewing is prevalent. The cDNA microarray analysis was used to analyse the mRNA expression patterns of 1177 genes in ten oral cancer patients with betel quid chewing history. Eighty-four genes involving cell adhesion, cell shape, growth, apoptosis, angiogenesis, metastasis, and metabolism were deregulated. Although the expression profile of these genes was shared by certain clinical patients, there was no significant association of the expression profile with clinical staging. Functional implication of four validated genes including caspase-1, STAT-1, COX-2 and pleiotrophin was discussed. This study provides pilot data for understanding the pathogenesis of oral cancer in countries like Taiwan where betel quid chewing is prevalent.     

Dietary phenolics and betel nut extracts as modifiers of N-nitrosation in rat and man

Polyphenolic compounds (PPC) isolated from betel nuts and some dietary PPC were examined for their modifying effects on N-nitrosation in vitro and in vivo. The formation of N-nitrosodiethylamine (NDEA) and N-nitrosoproline (NPRO) was either enhanced or inhibited by PPC from betel nuts, depending on (1) the structure of the PPC, (2) the pH of the reaction medium, (3) the relative concentrations of nitrite and PPC, and (4) the nature of the nitrosatable amino compounds. Both catalysis and inhibition of endogenous nitrosation of proline were observed in rats, although to a lesser extent than in vitro. Caffeic and ferulic acids, as well as the PPC-containing beverages tea and coffee, exerted inhibitory effects on endogenous formation of NPRO in two human subjects. These results demonstrate that PPC can modify the yield of endogenously formed N-nitroso compounds, and may thus effect the carcinogen burden in man.     

CYP2A6 gene deletion reduces oral cancer risk in betel quid chewers in Sri Lanka

We investigated the relationship between inter-individual difference in CYP2A6 genotype and susceptibility to oral cancer among habitual betel quid chewers in a Sri Lanka population. A total of 286 subjects showing oral malignant or premalignant lesions and 135 control subjects with no lesions were analyzed. The frequency of homozygotes for CYP2A6*4C mutation, a gene deletion type of polymorphism, was significantly lower in the case subjects than the controls. The odds ratio (OR) of the group homozygous for the deletion was significantly lower and calculated to be 0.14 (95% CI; 0.03-0.72). In the allelic base analysis, there was also a significant decrease in the OR of the deletion allele. Our data suggest that deficient CYP2A6 activity due to genetic polymorphism reduces oral cancer risk in betel quid chewers.     

Glutathione S-transferase M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers.

Oral cancer is the most common cancer in males and third most common in females in India, the main causative agent being the use of chewing tobacco with or without betel quid (BQ). However, nothing is known about the role of the host metabolic genes in oral cancer in ethnic Indian population. In this study, the prevalence of GSTM1 and GSTT1 null genotypes (GSTM1*2 and GSTT1*2) in oral premalignant leukoplakia cases and controls was ascertained in genomic DNA by a multiplex PCR technique. Biopsies taken from 98 oral leukoplakia patients and exfoliated cells from 82 healthy controls both of Indian ethnicity were analysed. GSTM1*1 (active) was present in 83% and GSTT1*1 (active) was present in 78% of all control subjects, while prevalence of GSTM1*2 and GSTT1*2 null genotypes was significantly higher among oral leukoplakia cases. The prevalence of GSTM1*2 in leukoplakia cases was 81.6% compared with 17% in controls [odds ratio (OR), 22; 95% confidence interval (CI), 1047] and GSTT1*2 was 75.5% in the cases versus 22% in controls (OR, 11; 95% CI, 5-22). Combined null genotypes of GSTM1 and GSTT1 prevailed in 60.2% of the cases with none detected in controls. Glutathione S-transferase M1 and T1 enzymes are both known to catalyse detoxification of reactive oxygen species, lipid peroxidation products and tobacco-derived carcinogens that have been found in the saliva of BQ/tobacco chewers. Our results, still requiring confirmation by a larger study, demonstrate that the null genotypes of both GSTM1 and GSTT1 increase with high penetrance, separately or in combination, the risk for developing leukoplakia in an Indian ethnic population.     

Endogenous nitrosation in the oral cavity of chewers while chewing betel quid with or without tobacco

In order to evaluate endogenous nitrosation in the oral cavity of chewers of betel quid with tobacco (BQT) or without tobacco (BQ), saliva samples were collected from healthy male volunteers after chewing sequentially (i) unmodified BQT or BQ, (ii) BQT or BQ to which proline has been added, and (iii) BQT or BQ to which proline and ascorbic acid had been added. Samples were collected over 20 min and analysed for N-nitrosoproline (NPRO), tobacco-specific nitrosamines (TSNA) and areca nut-specific nitrosamines using gas chromatography-thermal energy analysis, arecoline and nicotine using gas chromatography-nitrogen phosphorus-specific detector, and for nitrite and thiocyanate. When results were expressed as a ratio of NPRO (ng/ml) to nicotine (micrograms/ml), all BQT chewers had increased NPRO contents after chewing BQT with proline. For BQ chewers, when the results were expressed as a ratio of NPRO (ng/ml) to arecoline (micrograms/ml), a similar increase in NPRO content was observed. However, the presence of ascorbic acid inhibited the increased nitrosation in only four out of ten BQT chewers and in five out of ten BQ chewers; in the rest of the samples, its presence enhanced the levels of NPRO. N'-Nitrosoanatabine (NAT) and N-nitrosoguvacoline (NGCO) levels decreased significantly in saliva of chewers of BQT in the presence of ascorbic acid, suggesting inhibition of their formation. In-vitro nitrosation of BQT/BQ with proline and proline plus ascorbic acid showed a similar pattern of nitrosation at salivary pH. The study confirmed previous results that certain nitrosamines are formed during the chewing of BQT/BQ.     

Chromosomal alterations affecting the 1cen-1q12 region in buccal mucosal cells of betel quid chewers detected using multicolor fluorescence in situ hybridization

Epidemiological studies have shown that a high incidence of oral cancers is associated with chewing betel quid. Since chromosomal aberrations are involved in many types of cancers, we investigated whether increased frequencies of chromosomal alterations could be detected in the oral mucosa cells of betel quid chewers as compared to non-chewers. Due to the difficulty in culturing these epithelial cells, we used multicolor FISH with adjacent DNA probes to detect hyperdiploidy and breakage/exchanges affecting the 1cen-q12 region in interphase cells. Buccal mucosa cells from 19 male betel quid chewers and 23 non-chewers were hybridized and 1000 cells per donor were evaluated. A highly significant increase in the frequency of breakage affecting 1cen-1q12 region was observed in the mucosa cells of the chewers as compared to the non-chewers. A good correlation was also seen between breakage and duration of chewing. A modest increase in hyperdiploidy for chromosome 1 was also observed among chewers who had used betel quid for many years. These results indicate that this FISH approach can be useful for human biomonitoring, particularly for detecting alterations in non-dividing cells.      

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.      

Low incidence of p53 mutations in betel quid and tobacco chewing-associated oral squamous carcinoma from India

Mutations of the p53 tumor suppressor gene have been found to be the single most frequent event in human cancers. In India and other southeast Asian countries tobacco chewing with betel quid was attributed to be the major factor in oral carcinogenesis. We have analyzed 72 untreated primary oral squamous cell carcinomas (SCCs) for mutations in the tumor suppressor gene p53 exons 4-9 by PCR-SSCP and DNA sequencing. Sequencing analysis revealed 16 missense mutations, one silent mutation in codon 307 and four A to G substitution polymorphism in codon 213. The incidence of p53 mutation was 21% (15 of 72) excluding the polymorphism and the silent mutation. Eight mutations were clustered in codons 266-282 of exon 8. Of the total mutation events 37.5% were G to A transitions and 31.3% were G to T transversions. These results indicate the possible involvement of tobacco derived nitrosamines and their adducts in the genesis of oral cancer among Indians.