Synergistic Effect of Betel Quid,Tobacco, and Alcohol in Cigarette Smoking Induced Micronuclei in a Population of Arunachal Pradesh,India

Genotoxicity is one of the important endpoints for risk assessment of various lifestyle factors. The present study examined the synergistic effect of tobacco, betel quid, and alcohol in cigarette smoking induced micronuclei (MN) in the buccal epithelia of exposed individuals. Analysis of MN frequency and nuclear abnormalities (binucleated, karyorrhectic, karyolitic, and pyknotic cells) was performed in the exfoliated buccal cells of 110 habituates and compared to a control group matched for gender, age, and habit. A significant increase in the frequency of MN was found in smokers and alcohol, betel quid, and tobacco users compared to the control group. Tobacco, alcohol, and betel quid seem to potentiate the effect of cigarette smoking induced MN formation in the buccal epithelium. Smoking alone significantly increased the number of karyorrhexis cells in the buccal epithelium and combined exposure of all four test substances significantly increased the number of karyorrhexis and pycnotic cells. The findings indicate a synergistic effect between smoking, betel quid, tobacco, and alcohol in MN induction and cell death in buccal cells of exposed individuals.     

Safrole-DNA adducts in tissues from esophageal cancer patients: clues to areca-related esophageal carcinogenesis

Epidemiological studies have demonstrated that areca quid chewing can be an independent risk factor for developing esophageal cancer. However, no studies are available to elucidate the mechanisms of how areca induces carcinogenesis in the esophagus. Since the areca nut in Taiwan contains a high concentration of safrole, a well-known carcinogenic agent, we analyzed safrole-DNA adducts by the 32P-postlabelling method in tissue specimens from esophageal cancer patients. In total, we evaluated 47 patients with esophageal cancer (16 areca chewers and 31 non-chewers) who underwent esophagectomy at the National Taiwan University Hospital between 1996 and 2002. Of the individuals with a history of habitual areca chewing (14 cigarette smokers and two non-smokers), one of the tumor tissue samples and five of the normal esophageal mucosa samples were positive for safrole-DNA adducts. All patients positive for safrole-DNA adducts were also cigarette smokers. Such adducts could not be found in patients who did not chew areca, irrespective of their habits of alcohol consumption or cigarette smoking (p<0.001, comparing the areca chewers with non-chewers). The genotoxicity of safrole was also tested in vitro in three esophageal cell lines and four cultures of primary esophageal keratinocytes. In two of the esophageal keratinocyte cultures, adduct formation was increased by treatment with safrole after induction of cytochrome P450 by 3-methyl-cholanthrene. This paper provides the first observation of how areca induces esophageal carcinogenesis, i.e., through the genotoxicity of safrole, a component of the areca juice.     

Safrole–DNA adducts in tissues from esophageal cancer patients: clues to areca-related esophageal carcinogenesis

Epidemiological studies have demonstrated that areca quid chewing can be an independent risk factor for developing esophageal cancer. However, no studies are available to elucidate the mechanisms of how areca induces carcinogenesis in the esophagus. Since the areca nut in Taiwan contains a high concentration of safrole, a well-known carcinogenic agent, we analyzed safrole–DNA adducts by the ³²P-postlabelling method in tissue specimens from esophageal cancer patients. In total, we evaluated 47 patients with esophageal cancer (16 areca chewers and 31 non-chewers) who underwent esophagectomy at the National Taiwan University Hospital between 1996 and 2002. Of the individuals with a history of habitual areca chewing (14 cigarette smokers and two non-smokers), one of the tumor tissue samples and five of the normal esophageal mucosa samples were positive for safrole–DNA adducts. All patients positive for safrole–DNA adducts were also cigarette smokers. Such adducts could not be found in patients who did not chew areca, irrespective of their habits of alcohol consumption or cigarette smoking (p < 0.001, comparing the areca chewers with non-chewers). The genotoxicity of safrole was also tested in vitro in three esophageal cell lines and four cultures of primary esophageal keratinocytes. In two of the esophageal keratinocyte cultures, adduct formation was increased by treatment with safrole after induction of cytochrome P450 by 3-methyl-cholanthrene. This paper provides the first observation of how areca induces esophageal carcinogenesis, i.e., through the genotoxicity of safrole, a component of the areca juice.     

Epidemiology of areca (betel) nut use in the mariana islands: Findings from the University of Guam/University of Hawai`i cancer center partnership program

BackgroundAreca (betel) nut is considered a Group 1 human carcinogen shown to be associated with other chronic diseases in addition to cancer. This paper describes the areca (betel) nut chewing trend in Guam, and health behaviors of chewers in Guam and Saipan.MethodsThe areca (betel) nut module in the Guam Behavioral Risk Factor Surveillance Survey was used to calculate the 5-year (2011–2015) chewing trend. To assess the association between areca (betel) nut chewing and health risks in the Mariana Islands, a cross-section of 300 chewers, ≥18 years old, were recruited from households in Guam and Saipan. Self-reported socio-demographics, oral health behaviors, chronic disease status, diet, and physical activity were collected. Anthropometry was measured. Only areca (betel) nut-specific and demographic information were collected from youth chewers in the household.ResultsThe 5-year areca (betel) nut chewing prevalence in Guam was 11% and increased among Non-Chamorros, primarily other Micronesians, from 2011 (7%) to 2015 (13%). In the household survey, most adult chewers (46%) preferred areca nut with betel leaf, slaked lime, and tobacco. Most youth chewers (48%) preferred areca nut only. Common adult chronic conditions included diabetes (14%), hypertension (26%), and obesity (58%).ConclusionThe 5-year areca (betel) nut chewing prevalence in Guam is comparable to the world estimate (10–20%), though rising among Non-Chamorros. Adult and youth chewers may be at an increased risk for oral cancer. Adult chewers have an increased risk of other chronic health conditions. Cancer prevention and intervention strategies should incorporate all aspects of health.     

Areca (betel) nut chewing practices of adults and health behaviors of their children in the Freely Associated States,Micronesia: Findings from the Children’s Healthy Living (CHL) Program

BackgroundChewing areca (betel) nut has been deemed carcinogenic. The practice has become a public health concern in Micronesia. The Children's Healthy Living (CHL) Program included an areca (betel) nut questionnaire in a survey of household characteristics in the Freely Associated States (FAS). This paper describes areca (betel) nut chewing practices of adults and the health behaviors of their children.MethodsA cross-section of 1200 children (2–8 year-olds) and their caregivers in Chuuk, Kosrae, Pohnpei, Republic of Palau, Republic of the Marshall Islands (RMI), and Yap were recruited. Socio-demographics, adult areca (betel) nut chewing practices, and other health behaviors of children and adults were assessed. Child anthropometric measurements were collected to estimate weight status.ResultsThe FAS areca (betel) nut chewing prevalence was 42%, ranging from 3% (RMI) to 94% (Yap). Among chewers, 84% added tobacco, 97% added slaked lime, 85% added betel leaf, and 24% mixed the components with alcohol. Among FAS children, 95% practiced daily teeth-brushing and 53% visited the dentist annually. Compared to non-chewing households, areca (betel) nut chewing households were more likely to have very young children enrolled, more highly educated adults, and members that used tobacco and alcohol.ConclusionThe FAS areca (betel) nut chewing prevalence (42%) is above the world prevalence of 10–20%, with wide variability across the islands. The oral health findings in this study may inform future oral cancer prevention programs or policies. Regular monitoring of areca (betel) nut use is needed to measure the impact of such programs or policies.     

Betel Nut Chewing Is Strongly Associated With General and Central Obesity in Chinese Male Middle‐aged Adults

Betel nut chewing has been reported to increase the risk of cardiovascular disease and all‐cause mortality. The reason is unclear. In this study, we investigated the association between betel nut chewing and general obesity (BMI ≥25 kg/m²) and central obesity (waist circumference (WC) ≥90 cm). A total of 1,049 male subjects, aged ≥40 years, were recruited from Taichung city in Taiwan in 2004. The relationships between betel nut chewing and general and central obesity were studied by multiple linear and logistic regression analyses. The prevalence of current and former betel nut chewing was 7.0 and 10.5% in our male Taiwanese cohort. Current/former betel nut chewers had a higher prevalence of general and central obesity when compared with individuals who had never chewed betel nut. Adjusted for age, diabetes, hypertension, lipids, smoking, alcohol drinking, physical activity, income, and education level, the odds ratios (ORs; 95% confidence intervals) of general and central obesity among the lower consumption of betel nut chewers were 1.78 (1.07, 2.96) and 1.19 (0.70, 2.02), respectively, compared to 2.01 (1.18, 3.41) and 1.89 (1.10, 3.23), respectively, among higher consumption chewers compared to individuals who had never chewed betel nut. The increasing ORs of general and central obesity with higher betel nut consumption revealed dose–response effects. Using multiple linear regression analyses, after adjusting for potential confounders, betel nut consumption was statistically significantly associated with BMI and WC. In conclusion, betel nut chewing was independently associated with general and central obesity in Taiwanese men. Dose–response effects of the association between betel nut consumption and general obesity as well as central obesity were found.     

Characterization of the psychological, physiological and EEG profile of acute betel quid intoxication in naïve subjects

Betel quid use and abuse is wide spread in Asia but the physiological basis of intoxication and addiction are unknown. In subjects na?ve to the habit of betel quid intoxication, the psychological and physiological profile of intoxication has never been reported. We compared the effect of chewing gum or chewing betel quid, and subsequent betel quid intoxication, on psychological assessment, prospective time interval estimation, numerical and character digit span, computerized 2 choice tests and mental tasks such as reading and mathematics with concurrent monitoring of ECG, EEG and face temperature in healthy, non-sleep deprived, male subjects na?ve to the habit of chewing betel quid. Betel quid intoxication, dose dependently induced tachycardia (max 30 bpm) and elevated face temperature (0.7°C) (P<0.001) above the effects observed in response to chewing gum (max 12 bpm and 0.3°C) in 12 subjects. Gross behavioral indices of working memory such as numerical or character digit span in 8 subjects, or simple visual-motor performance such as reaction speed or accuracy in a two choice scenario in 8 subjects were not affected by betel quid intoxication. Betel quid intoxication strongly influenced the psychological aspects of perception such as slowing of the prospective perception of passage of a 1 minute time interval in 8 subjects (P<0.05) and perceived increased arousal (P<0.01) and perceived decreased ability to think (P<0.05) in 31 subjects. The EEG spectral profile recorded from mental states associated with open and closed eyes, and mental tasks such as reading and eyes closed mental arithmetic were significantly modified (P<0.05) relative to chewing gum by betel quid intoxication in 10 subjects. The prevalence of betel quid consumption across a range of social and work settings warrants greater investigation of this widespread but largely under researched drug.     

Atherogenic effect of Arecoline: A computational study

There are over 600 million people worldwide covering Asian and Oceanic countries including India have the habit of chewing areca nut as masticator in different forms. Arecoline (C(8)H(13)NO(2)) has been reported as one of the abundant constituents of areca nut. A good number of scientific publications have made Arecoline responsible for oral cancer. Based on observation from clinical situation in North East India, one of the most betel quid chewing region of the country, we suspected a link between consumption of areca nut and Cerebro Vascular Disease like stroke. Therefore, we considered Low Density Lipoprotein (LDL) receptor as target and Arecoline as ligand and studied ligand -target interaction using computational tools. Also we considered High Density Lipoprotein (HDL) receptor as another target to see if Arecoline has any binding potential with it over and above LDL receptor. Docking result indicated that Arecoline and Cholesterol both, have affinity towards extracellular domain of Human LDL receptor but affinity of Arecoline is much higher (-12.3560.) than that of Cholesterol(-0.1810). Docking of Arecoline and 1, 2-Hexyl-1- cyclopentanone thiosemicarbazone (thiosemicarbazone) with Bovine HDL receptor showed that Arecoline also has the potential (Score, -6.2690Kcal/Mol) to block HDL receptor though its potential is less than that (score, -10.0509 Kcal/Mol) of control (thiosemicarbazone). We, therefore, suggest that by inhibiting endocytosis of LDL cholesterol because of blocking LDL receptor function and also by preventing LDL cholesterol uptake by liver from blood because of interference with HDL receptor, Arecoline may contribute to atherosclerosis. The study therefore, indicates a positive correlation between chewing of betel quid and Cerebro Vascular Disease.     

The Magnitude of Tobacco Smoking-Betel Quid Chewing-Alcohol Drinking Interaction Effect on Oral Cancer in South-East Asia. A Meta-Analysis of Observational Studies

Tobacco smoking, betel quid chewing and alcohol drinking are oral cancer risk factors. Observational studies unanimously report that oral cancer risk in smoking-drinking-chewing exposed subjects is exceptionally high. However, none of them assessed the fractions of this risk attributable to the three individual risk factors and to the smoking-drinking-chewing interaction. The present study sought to assess the magnitude of the smoking-drinking-chewing interaction effect on oral cancer. A meta-analysis of observational South-East Asian studies which reported oral cancer odds ratios (ORs) stratified for smoking-drinking-chewing exposures was performed. The pooled ORs were estimated and controlled for quality, heterogeneity, publication bias and inclusion criteria. The smoking-drinking-chewing interaction effect was estimated through the pooled Relative Excess Risk due to Interaction (RERI, excess risk in smoking-drinking-chewing exposed individuals with respect to the risk expected from the addition of the three individual risks of smoking, drinking and chewing). Fourteen studies were included with low between-study heterogeneity. The pooled ORs for smoking, drinking, chewing, smoking-drinking-chewing, respectively were 3.6 (95% confidence interval −95% CI, 1.9–7.0), 2.2 (95% CI, 1.6–3.0), 7.9 (95% CI, 6.7–9.3), 40.1 (95% CI, 35.1–45.8). The pooled RERI was 28.4 (95% CI, 22.9–33.7). Among smoking-drinking-chewing subjects, the individual effects accounted for 6.7% (smoking), 3.1% (drinking), 17.7% (chewing) of the risk, while the interaction effect accounted for the remaining 72.6%. These data suggest that 44,200 oral cancer cases in South-East Asia annually occur among smoking-drinking-chewing exposed subjects and 40,400 of these are exclusively associated with the interaction effect. Effective oral cancer control policies must consider concurrent tobacco smoking, alcohol drinking, betel quid chewing usages as a unique unhealthy lifestyle.     

Evaluation of frequency of micronucleated oral mucosa cells as a marker for genotoxic damage in chewers of betel quid with or without tobacco.

The frequency of micronucleated cells (MNC) derived from exfoliated human oral mucosal cells has been measured to assess genotoxic damage in chewers of betel quid with tobacco (BQT) and tobacco with lime (T). Significantly elevated frequencies of MNC were observed in the exposed groups (BQT = 4.83 +/- 0.70; T = 5.20 +/- 0.66 per 1000 cells) compared to the control group (C = 2.59 +/- 0.37) although the levels observed were lower than those reported in the literature. No correlation was seen between age, duration and frequency of habits and the frequency of MNC in the 2 habit groups. Clastogenic agents in betel quid possibly involved in micronucleus formation are discussed.     

Characterization of the Psychological,Physiological and EEG Profile of Acute Betel Quid Intoxication in Na?ve Subjects

Betel quid use and abuse is wide spread in Asia but the physiological basis of intoxication and addiction are unknown. In subjects naïve to the habit of betel quid intoxication, the psychological and physiological profile of intoxication has never been reported. We compared the effect of chewing gum or chewing betel quid, and subsequent betel quid intoxication, on psychological assessment, prospective time interval estimation, numerical and character digit span, computerized 2 choice tests and mental tasks such as reading and mathematics with concurrent monitoring of ECG, EEG and face temperature in healthy, non-sleep deprived, male subjects naïve to the habit of chewing betel quid. Betel quid intoxication, dose dependently induced tachycardia (max 30 bpm) and elevated face temperature (0.7°C) (P<0.001) above the effects observed in response to chewing gum (max 12 bpm and 0.3°C) in 12 subjects. Gross behavioral indices of working memory such as numerical or character digit span in 8 subjects, or simple visual-motor performance such as reaction speed or accuracy in a two choice scenario in 8 subjects were not affected by betel quid intoxication. Betel quid intoxication strongly influenced the psychological aspects of perception such as slowing of the prospective perception of passage of a 1 minute time interval in 8 subjects (P<0.05) and perceived increased arousal (P<0.01) and perceived decreased ability to think (P<0.05) in 31 subjects. The EEG spectral profile recorded from mental states associated with open and closed eyes, and mental tasks such as reading and eyes closed mental arithmetic were significantly modified (P<0.05) relative to chewing gum by betel quid intoxication in 10 subjects. The prevalence of betel quid consumption across a range of social and work settings warrants greater investigation of this widespread but largely under researched drug.     

Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.     

Systematic Review and Meta-Analysis of Association of Smokeless Tobacco and of Betel Quid without Tobacco with Incidence of Oral Cancer in South Asia and the Pacific

Aim

This systematic review and meta-analysis aimed to critically appraised data from comparable studies leading to quantitative assessment of any independent association between use of oral smokeless tobacco in any form, of betel quid without tobacco and of areca nut with incidence of oral cancer in South Asia and the Pacific.

Methods

Studies (case control and/or cohort) were identified by searching Pub Med, CINAHL and Cochrane databases through June 2013 using the keywords oral cancer: chewing tobacco; smokeless tobacco; betel quid; betel quid without tobacco; areca nut; Asia, the Pacific and the reference lists of retrieved articles. A random effects model was used to compute adjusted summary OR_RE for the main effect of these habits along with their corresponding 95% confidence intervals. To quantify the impact of between-study heterogeneity on adjusted main-effect summary OR_RE, Higgins'' H and I₂ statistics along with their 95% uncertainty intervals were used. Funnel plots and Egger''s test were used to evaluate publication bias.

Results

Meta-analysis of fifteen case–control studies (4,553 cases; 8,632 controls) and four cohort studies (15,342) which met our inclusion criteria showed that chewing tobacco is significantly and independently associated with an increased risk of squamous-cell carcinoma of the oral cavity (adjusted main-effect summary for case- control studies OR_RE = 7.46; 95% CI = 5.86–9.50, P<0.001), (adjusted main-effect summary for cohort studies RR = 5.48; 95% CI = 2.56–11.71, P<0.001). Furthermore, meta-analysis of fifteen case control studies (4,648 cases; 7,847 controls) has shown betel quid without tobacco to have an independent positive association with oral cancer, with OR = 2.82 (95% CI = 2.35–3.40, P<0.001). This is presumably due to the carcinogenicity of areca nut. There was no significant publication bias.

Conclusion

There is convincing evidence that smokeless (aka chewing) tobacco, often used as a component of betel quid, and betel quid without tobacco, are both strong and independent risk factors for oral cancer in these populations. However, studies with better separation of the types of tobacco and the ways in which it is used, and studies with sufficient power to quantify dose-response relationships are still needed.     

A comparative biomonitoring study of populations residing in regions with low and high risk of lung cancer using the chromosome aberration and the micronucleus tests

Chromosome aberration (CA) and micronucleus (MN) tests were performed in peripheral blood lymphocytes from people residing in two districts of Chiang Mai, Thailand, a high-risk area, Saraphi (n=107), where the lung cancer incidence is three-fold higher than in a low-risk area, Chom Thong (n=118). The percentage of cells with CAs was significantly lower in the Saraphi population than in the Chom Thong population (0.47+/-0.91 versus 1.04+/-1.18, P=0.0001) as was the percentage of CAs (0.49+/-0.91 versus 1.08+/-1.21, P<0.0001) and the mitotic indices (1.25+/-0.44 versus 1.33+/-0.33, P=0.025). The frequency of MN in binucleated (BN) cells, however, was significantly higher in the Saraphi population (12.01+/-3.57 versus 9.99+/-3.11, P<0.0001) as was the percentage of BN cells with MN (1.14+/-0.31 versus 0.93+/-0.23, P<0.0001). There was no difference in the nuclear division indices (1.49+/-0.07 versus 1.47+/-0.11, P=0.1759) between the two populations. With regard to the effect of confounding factors, it was found that cigarette smoking influenced both CA and MN frequencies, and that the chewing of fermented tea leaves or betel nuts affected CA and sex affected MN frequencies. An increasing of CA and MN frequencies were seen in smokers and chewers over non-smokers and non-chewers, with CA frequencies being higher in Chom Thong smokers and chewers and MN frequency being higher in Saraphi smokers. However, pesticide exposure and alcohol consumption had no impact on CA and MN frequencies. Due to the conflicting results obtained in the two tests, we cannot make a clear statement regarding the potential effects of the environmental exposures in the two study populations.     

Formation of reactive oxygen species and of 8-hydroxydeoxyguanosine in DNA in vitro with betel quid ingredients

The formation of reactive oxygen species (ROS) from betel quid ingredients, namely areca nut, catechu and tobacco, was studied using a chemiluminescence (CL) technique. Aqueous extracts of areca nut and catechu were capable of generating superoxide anion and hydrogen peroxide at pH greater than 9.5. The formation of O2 was enhanced by Fe2+, Fe3+ and Cu2+ but inhibited by Mn2+. Tobacco extract failed to generate ROS under similar conditions. Saliva was found to inhibit both O2 and H2O2 formation from betel quid ingredients. Upon incubation of DNA at alkaline pH with areca nut extract and Fe3+ or catechu, 8-hydroxydeoxyguanosine was formed as quantified by high performance liquid chromatography (HPLC)/electrochemical detection. The data suggest a possible role of reactive oxygen species in the etiology of oral cancer in betel quid chewers.     

Epigenetic,Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

Background

Esophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes.

Methodology/Principal Findings

The study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models.

Conclusion

Our study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.     

IL‐1β Promotes Malignant Transformation and Tumor Aggressiveness in Oral Cancer

Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β can be induced by tobacco and betel quid‐related carcinogens, and participates in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. 230: 875–884, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Measurement and characterization of micronuclei in exfoliated human cells by fluorescence in situ hybridization with a centromeric probe

The micronucleus (MN) assay in human exfoliated cells has been widely used to detect the genotoxic effects of environmental mutagens, infectious agents and heriditary diseases. Substantial variability characterizes the MN frequencies reported by different research groups. One reason for this may be the restricted resolution power of the Feulgen-Fast-Green staining that is routinely used. Here we describe a new version of the MN assay that employs fluorescent propidium iodide staining along with fluorescence in situ hybridization (FISH) with a centromeric probe. Buccal and urothelial cells were collected from 5 healthy unexposed female volunteers and 55 000 cells analyzed for MN frequency and abnormal nuclear events. The Feulgen-Fast-Green and the new fluorescent staining produced very similar results. The frequency of MN in buccal cells was 0.145±0.118% and in urothelial cells 0.083±0.074%. No correlation was found between the frequencies of MN in the two types of exfoliated cells. FISH with a centrometric probe allowed MN containing whole chromosomes with a centromere to be differentiated from those containing only acentric fragments. The former appear as a result of chromosome lagging in mitosis, while those without a centromere are due to chromosome breakage. In urothelial cells 43% of MN were centromere-negative and in buccal cells — 44%. Fluorescent staining provided more accurate scoring of degenerative cells than standard Feulgen-Fast-Green staining. The combined frequency of pycnotic cells, “broken eggs” and cells with fragmented nuclei did not exceed 2%, while that of karyorrhexis and karyolysis together was as high as 21%. Significant interindividual variability was found in the frequency of cells with karyolysis and karyorrhexis. Thus, the new version of micronucleus assay allows for MN to be scored more precisely, the mechanism of MN formation to be determined and abnormal nuclear events to be readily identified in exfoliated human cells. It is therefore ideal for studying genotoxicity in human populations using exfoliated cells from the mouth, bladder and nose.     

Safrole-DNA adducts in human peripheral blood--an association with areca quid chewing and CYP2E1 polymorphisms

It has been recently demonstrated that safrole (4-allyl-1,2-methylenedioxybenzene)-DNA adducts are present in oral cancer tissue from patients who have chewed areca quid (AQ) containing high concentration of safrole. In this study, the presence of safrole-DNA adducts in peripheral white blood cells from 88 subjects with a known AQ chewing history and 161 matched controls were studied with the aim of identifying the adducts as a biomarker for safrole exposure. This study also analyzed the correlation between the level of safrole-DNA adducts and polymorphism of the CYP2E1 gene, alone and in combination with the GST M1 and GST T1-deletion polymorphisms. The results demonstrated the presence of safrole-DNA adducts in 83 (94.32%) of the DNA samples from subjects with current AQ chewing history and 21 (13.04%) of the control samples without known AQ chewing habit ( [Formula: see text] ). Individuals with at least one CYP2E1 c2 allele had a significant higher frequency of safrole-DNA adducts (odds ratio (OR), 4.00; 95% confidence interval (CI), 1.03-15.53) than those with the CYP2E1 c1c1 genotype while chewing less than 20 areca quids per day. In conclusion, this study demonstrates the presence of safrole-DNA adducts in peripheral blood lymphocytes (PBL), and the presence of these safrole-DNA adducts is correlated with AQ chewing. In addition, the CYP2E1 would seem to play an important role in the modulation of safrole-DNA adduct formation.     

Micronucleus induction and FISH analysis in buccal cells and lymphocytes of nurses administering antineoplastic drugs

A genotoxic effect for antineoplastic drugs, in particular micronucleus induction, has been shown in several studies. The aim of our study was to assess genotoxic effects in nurses administering different mixtures of antineoplastic drugs in an oncology hospital by evaluating the frequency of micronuclei in exfoliated buccal cells and blood lymphocytes by use of the standard micronucleus (MN) test and by identifying, by means of FISH analysis with centromeric probes, the mechanism of micronucleus induction (clastogenic or aneugenic). The study group comprised 23 nurses, 10 of whom worked in the day-care hospital and 13 in the ward. Twenty healthy subjects were selected as controls. Pan-centromeric FISH analysis was performed on lymphocytes from a selected group of nurses (12/23 subjects) characterized by higher MN frequencies as observed by standard Giemsa staining. A significant increase of micronucleus frequency compared with controls was found in exfoliated buccal cells of both groups of nurses: day-care hospital nurses 0.92 versus 0.45 (p=0.034) and ward nurses 0.94 versus 0.45 (p=0.051). An increase, although not statistically significant, of mean MN frequency was also found by the MN standard test on lymphocytes of the day-care hospital nurses (10.9 versus 7.5; p=0.056), while no differences were found in ward nurses (8.15 versus 7.5; p=0.56). We found that the administration of antineoplastic drugs by nurses in ward units induced a higher frequency of FISH MN+ (43% of subjects) than in the day-care hospital (20%). This was associated with the micronucleus size percentage. This finding could be correlated with the different compositions of administered mixtures of antineoplastic drugs: in ward units the mixtures contained drugs, such as vinorelbine, that were absent in the mixtures administered in the day-care hospital. Our results show genetic damage induced by administration of antineoplastic drugs, particularly in exfoliated buccal cells. This result suggests the useful application of this non-invasive sampling to evaluate genotoxic effects of occupational exposure to mixtures of inhalable chemicals at low doses.