Areca Quid Research Articles

Evaluation of a self-rating screening test for areca quid abusers in Taiwan

Areca quid chewing is a popular habit and areca is a well-known ethnopsychopharmalogic agent in southeast Asia. While the chewing habit is legal and also socially acceptable in many places of Taiwan, the public health problem of high oral cancer incidence has remained a priority on the health care list in our local health department. Helping areca quid chewers to reduce or even stop the habit will be paramount in the oral cancer prevention programme. Hence, in order to identify the appropriate strategy for stopping the chewing habit, it is important to distinguish whether an areca quid chewer has reached the level of substance abuse. In accordance with the Diagnostic and Statistical manual of Mental disorder (fourth edition, DSM-IV), we developed a specific self-report questionnaire modified from the famous SCAN system, DSM-IV and ICD-10. The initial screening test for areca quid abusers had 52 questions. Its components included the onset age and frequency, subjective craving and feeling, social problems, physical problems, oral symptoms, psychological and abstinence-related problems, the motivation and capacity to abstain, and demographic data. The answers were divided into ‘Yes’ or ‘No’. One hundred and twenty-five areca quid users (53 men, 72 women) were recruited. The abusers tended to have older age, less education, and higher daily consumption of areca quid. There were no differences on motivation to quit chewing (abstinence) between abusers and non-abusers. There were no statistical differences on tobacco-smoking and alcohol-drinking behavior. Based on the statistical analysis of receiver operation characteristic (ROC) curves, 11 questions were chosen for the Self-report Screening Test for Areca quid Abuser (SSTAA). An areca quid chewer's answers with a score of 4 or more in these 11 questions would be considered an areca quid abuser. The modified process of SSTAA is performed for the evaluation of the native culture-related substance user. At this current stage, the SSTAA developed from the study is the first instrument for screening areca quid abuser.

Prevalence and related risk factors of areca quid chewing among junior high students in eastern Taiwan

Areca quid chewing is a serious problem in eastern Taiwan. The prevalence and related risk factors of areca quid chewing among junior high students were studied. Group 1 consisted of 896 non-aboriginal students and group 2 of 565 aboriginal students. The students were asked to fill out a questionnaire anonymously. Among the non-aboriginal students, 2.2% were found as current areca quid chewers, 16.4% had tried but not become regular users, and 2.2% had used it regularly and quit, whereas 9.7% of aboriginal students were current chewers, and 32.7% had tried it but had not become regular users, and 7.4% had used it regularly and quit. The prevalence of areca chewing is significantly different between boys and girls in the non-aboriginal group. A logistic regression analysis indicated that a non-aboriginal student who is a male, grows areca at home, is a tobacco smoker, an alcohol drinker, and has family members and close friends as chewers is more likely to chew areca quid than others. Aboriginal students who are chewers are likely to grow areca at home, are tobacco smokers and alcohol drinkers, and have family members and close friends as chewers. Based on the findings of the study, the need for a health promotion program was identified. Areca quid is not only detrimental to people's health, but also to the environment in which people live.

Elevated vimentin expression in buccal mucosal fibroblasts by arecoline in vitro as a possible pathogenesis for oral submucous fibrosis

Areca quid chewing is strongly correlated with oral submucous fibrosis (OSF) in Taiwan. The cytotoxicity of arecoline, a major areca nut alkaloid, on human oral fibroblasts has been extensively studied. To date, however, there has been little research exploring the possible effects of arecoline on cytoskeleton components. In this study, in addition to conducting a cytotoxicity assay, we examine the effect of arecoline on vimentin, an intermediate filament, and its expression in human buccal mucosal fibroblasts on exposure to various levels of arecoline (0–200 μg/ml) for 48 h. At a concentration above 50 μg/ml, arecoline demonstrated dose-dependent cytotoxicity ( P<0.05) for cultured fibroblasts. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, we demonstrated dose-dependent elevation of 57 kDa cytoskeletal-protein levels for arecoline. Evidence from immunoblotting assay indicated this 57 kDa cytoskeletal protein was vimentin. The increase in vimentin with arecoline exposure corresponded to that noted for fibroblasts cultured from OSF patients. Immunohistochemical assay also revealed that vimentin expression was much higher for OSF specimens than for normal buccal mucosa. We suggest these results may advance understanding of the possible pathogenesis for submucous fibrosis through the transformation of normal buccal mucosa as a result of areca quid chewing.

Prevalence and related risk factors of areca quid chewing among junior high students in eastern Taiwan

Areca quid chewing is a serious problem in eastern Taiwan. The prevalence and related risk factors of areca quid chewing among junior high students were studied. Group 1 consisted of 896 non-aboriginal students and group 2 of 565 aboriginal students. The students were asked to fill out a questionnaire anonymously. Among the non-aboriginal students, 2.2% were found as current areca quid chewers, 16.4% had tried but not become regular users, and 2.2% had used it regularly and quit, whereas 9.7% of aboriginal students were current chewers, and 32.7% had tried it but had not become regular users, and 7.4% had used it regularly and quit. The prevalence of areca chewing is significantly different between boys and girls in the non-aboriginal group. A logistic regression analysis indicated that a non-aboriginal student who is a male, grows areca at home, is a tobacco smoker, an alcohol drinker, and has family members and close friends as chewers is more likely to chew areca quid than others. Aboriginal students who are chewers are likely to grow areca at home, are tobacco smokers and alcohol drinkers, and have family members and close friends as chewers. Based on the findings of the study, the need for a health promotion program was identified. Areca quid is not only detrimental to people's health, but also to the environment in which people live.

HYDROXYL RADICAL FORMATION AND OXIDATIVE DNA DAMAGE INDUCED BY ARECA QUID IN VIVO

Chewing areca quid (AQ) has been implicated as a major risk factor for the development of oral squamous-cell carcinoma (OSCC). Recent studies have suggested that AQ-generated reactive oxygen species (ROS) is one of the contributing factors for oral carcinogenesis. However, the AQ used in Taiwan is different from that used in other countries. This study is designed to test whether ROS are generated and the consequent effects in locally prepared AQ in vivo. We measured the hydroxyl radical formation, as represented by the presence of o - and m -tyrosine in saliva from volunteers who chewed AQ containing 20 mg phenylalanine. Their saliva contained significantly higher amounts ( p < .05) of o - and m -tyrosine as compared to the controls. In addition, chewing AQ containing Piper betle inflorescence generated higher amounts of m -tyrosine, but not o -tyrosine, in saliva than did chewing AQ containing betel leaf. We further tested the oxidative DNA damaging effect of the reconstituted AQ, as evidenced by the elevation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels, in hamster buccal pouch. Following daily painting for 14 d, the 8-OH-dG level in hamster buccal pouch is significantly elevated ( p < .05) in the AQ-treated group versus the controls. These findings demonstrate that ROS, such as hydroxyl radical, are formed in the human oral cavity during AQ chewing, and chewing such prepared AQ might cause oxidative DNA damage to the surrounding tissues.

Increased tissue inhibitor of metalloproteinase-1 expression and inhibition of gelatinase A activity in buccal mucosal fibroblasts by arecoline as possible mechanisms for oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of tissue inhibitors of metalloproteinase (TIMPs) and matrix metalloproteinases (MMPs) in the pathogenesis of OSF. In the present study, we examined the activity of TIMPs from cells cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP or MMP production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 expression at the concentration level under 20 microg/ml in a dose-dependent manner. The amount of TIMP-1 was about 2.7 fold at a concentration level of 10 microg/ml compared with control. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production at the concentration level of 40 microg/ml. The gelatinolytic activity of MMP-2 was about 54% at a concentration level of 80 microg/ml compared with control. Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF.

Prognostic role of p27(Kip1) expression in oral squamous cell carcinoma in Taiwan.

The cyclin-dependent kinase inhibitor p27(Kip1) can inhibit the G1 to S transition of the cell cycle and is a putative tumor suppressor. Decreased expression of p27(Kip1) protein has been correlated with poor prognosis in a variety of human tumors. We examined the expression of p27(Kip1) in oral squamous cell carcinoma (SCC), epithelial dysplasia (ED) and normal oral mucosa (NOM) using antibodies to p27(Kip1). Positive p27(Kip1) nuclear staining was detected in all the specimems from ED and NOM, whereas positive p27(Kip1) staining was observed in 16 of the 63 (25%) cases of oral SCC. The labeling index for p27(Kip1) protein was significantly reduced from NOM through ED to oral SCCs, indicating that changes of p27(Kip1) protein expression may be an early event in oral carcinogenesis in Taiwan. The Kaplan-Meier analysis showed patients with p27(Kip1)-positive tumors had significantly higher overall survival than those with p27(Kip1)-negative tumors in a total of 63 patients (P=0.015) and 47 patients with areca quid chewing habit (P=0.026). Multivariate analysis showed decreased p27(Kip1) protein expression was an independent significant predictor of poor overall survival in the patients with oral SCCs. These results indicate that p27(Kip1) protein expression may serve as a putative new adjuvant prognostic marker for routine assessment of oral SCC patients.

Prognostic role of p21WAF1 expression in areca quid chewing and smoking-associated oral squamous cell carcinoma in Taiwan.

Alterations in p21WAF1 protein expression have been observed in a wide variety of human cancers by immunohistochemistry, and both decreased and increased levels of p21WAF1 protein expression have been shown to correlate with poor prognosis. To examine the relation between p21WAF1 protein expression and prognosis in oral squamous cell carcinomas (SCCs), we performed an immunohistochemical study with antip21WAF1 antibody on 43 oral SCCs. Immunostaining results were then correlated with p53 protein levels, clinicopathological parameters of the tumors and overall patient survival. Of the 43 patients, 31 (72%) had tumors with positive p21WAF1 nuclear staining and 27 (63%) had tumors with p53 nuclear staining. There was no significant correlation between p21WAF1 and p53 protein expressions and both mutant p53-containing oral SCCs overexpressed p21WAF1 protein. In addition, no significant correlation was found between the p21WAF1 expression and the patients' age, sex, oral habit, cancer location, or primary tumor TNM status at the time of initial presentation. The Kaplan-Meier analysis showed a significant correlation between p21WAF1 protein overexpression and poor patient overall survival (P = 0.049). When p53 and p21WAF1 were evaluated together, the 5-year overall survival was lowest in p53(+)-p21WAF1(+) patients and highest in p53(-)-p21WAF1(-) patients (P = 0.057). Combined evaluation of p21WAF1 and p53 expressions may be useful in estimating the prognosis of patients with oral SCCs in Taiwan.

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

MDM2 expression in areca quid chewing‐associated oral squamous cell carcinomas in Taiwan

MDM2 (murine double minute gene 2) overexpression has been implicated in the pathogenesis of human tumors via inhibition of the p53 tumor suppressor protein. To investigate the potential involvement of MDM2 overexpression in the pathogenesis of oral squamous cell carcinomas (SCCs) in Taiwan, we examined the expression of MDM2 protein and its relationship to p53 protein levels in 52 oral SCCs using antibodies to MDM2 and p53. Of the 52 patients, 36 (69 %) had tumors with positive MDM2 nuclear staining and 32 (61%) had tumors with p53 nuclear staining. Co-expression of MDM2 protein and p53 was detected in 25 (48%) cases; and 9 (17%) tumors showed neither MDM2 protein nor p53 staining. A significant correlation was observed between MDM2 protein and p53 expression in 38 cases with an areca quid (AQ) chewing habit (P=0.032). No significant correlation was found between the degree of MDM2 protein staining and the patients' ages, sex, cancer location, clinical staging, primary tumor TNM status or histological differentiation of SCC at the time of initial presentation. Kaplan-Meier analysis showed that either MDM2 protein expression or co-expression of p53 and MDM2 protein did not relate significantly to patient overall survival. Nevertheless, the high prevalence of MDM2 protein overexpression found in this study suggest that MDM2 may also participate in the carcinogenesis of AQ chewing-associated oral SCCs in Taiwan.

Induction of the c-jun protooncogene expression by areca nut extract and arecoline on oral mucosal fibroblasts.

To investigate the mechanisms of areca quid (AQ)-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in human oral mucosal fibroblasts after exposure to areca nut extracts (ANE) or arecoline. We found that treatment of cells with 200 microg/ml ANE or 10 microg/ml arecoline for 1 h induced about three-fold increase in c-jun mRNA levels. This increase was transient and the level of c-jun mRNAs returned rapidly to control levels thereafter. However, ANE and arecoline did not induce c-fos mRNA expression at detectable levels. During AQ chewing, oral mucosal cells are continuously stimulated by ANE and arecoline. Persistent induction of the c-jun protooncogene by ANE and arecoline may be one of the mechanisms in the carcinogenesis of oral squamous cell carcinoma in Taiwan. Furthermore, we observed that pre-incubation of cells with either N-acetyl-cysteine [a glutathione (GSH) precursor] or L-buthionine-S,R-sulfoximine (a specific inhibitor of GSH biosynthesis) had a minimal effect on arecoline-induced c-jun expression. Therefore, arecoline-induced c-jun expression is independent of GSH depletion.

Expression of p53 protein correlates with decreased survival in patients with areca quid chewing and smoking-associated oral squamous cell carcinomas in Taiwan.

Expression of p53 protein was examined in oral squamous cell carcinoma (SCC) from patients who were areca quid (AQ) chewers and/or tobacco smokers, using anti-p53 antibodies with an immunoperoxidase technique. Positive p53 stain was observed in 47 of 81 (58%) cases of oral SCC. p53 overexpression was found to be higher in patients without AQ chewing and smoking habits than in patients with these two habits (80% vs 52%, P=0.076). No significant correlation was found between p53 expression and the patients' age, sex, cancer location, clinical staging, primary tumor TNM status, or histological differentiation of SCC. The Kaplan-Meier analysis showed that the prognosis for patients with p53-negative tumors was significantly better than that for patients with p53-positive tumors (P<0.05). A significant correlation was also observed between positive lymph node status and poor prognosis (P<0.05). These results suggest that p53 may serve as an adjuvant marker of poor survival in patients with oral SCCs in Taiwan.