Oral squamous cell carcinoma (OSCC) is now the forth leading cause of male cancer mortality in Taiwan. The betel quid (BQ) chewing is the main cause OSCC in Taiwan. This study was aimed to clone the CDK2-associated protein 1 (CDK2AP1) complete CDs and characterization of its expression profiles as well as protein sublocalization in oral cancers. The human CDK2AP1 gene is 1.6 Kbp in length, mapped to 12q24.31 and encodes for a 12.4 kDa polypeptide. Human CDK2AP1 protein interacts with DNA polymerase alpha/primase resulting in negative regulation of the rate of initiation of DNA replication. It has been reported that differential CDK2AP1 expression, with decreased or absent expression in microsatellite-unstable (MSI+) colorectal cancer (CRC) cell lines, suggesting that loss of CDK2AP1 protein expression is a characteristic of malignant transformation in MSI+CRC. The role of CDK2AP1 in the onset or progression of oral cancer is still unknown. In this study, we firstly extracted RNA from 45 patients¡¦ specimens. Then, we cloned CDK2AP1 CDs from stomach carcinoma cell line (Scm1) and subcloned into various protein expression vectors for further examining CDK2AP1 subcellular localization in HeLa cell. Polyclonal CDK2AP1 antibody was prepared. We have demonstrated that the CDK2AP1 protein locates in both cytoplasm and nucleus by immunofluorescence analysis. In addition, we examined the CDK2AP1 mRNA expression profiles in oral cancer specimens by quantitative RT-PCR. The results showed that the expression of CDK2AP1 mRNA in oral cancer tissues were higher than those in normal oral tissues. Furthermore, we have determined and compared the CDK2AP1 protein in both oral cancer and normal tissues by immunoblotting analysis and immunohistochemical (IHC) analysis. The results from both immunoblotting and IHC were consistent with the results from quantitative RT-PCR. CDK2AP1 protein expression was higher in oral cancer tissues than in normal oral tissues.

Oral cancer is now the fourth leading cause of male cancer mortality in Taiwan. Betel quid chewing is one of the main causes of oral cancer in Taiwan. CDK2AP1 is a growth suppressor gene that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12CDK2AP1 protein is reduced and/or lost in oral cancers. Mutations in microsatellite-like sequence of CDK2AP1 gene in microsatellite instability colorectal cancer are associated with down-regulated CDK2AP1 transcription. This mutation was due to down-regulation of one DNA repair protein, MLH1. In order to understand whether CDK2AP1 mRNA and protein expression levels associate with betel-chewing oral cancers, we firstly analyzed 44 oral cancer specimens (normal and tumor, in pairs) by quantitative reverse transcription polymerase chain reaction and Western blotting. Immunohistochemistry was used to examine p12CDK2AP1 protein expression in another 167 buccal mucosa squamous cell carcinoma tissues. We have demonstrated that the expression levels of CDK2AP1 mRNAs were slightly higher in normal oral tissues than those in tumor tissues (P>0.05). Similarly, p12CDK2AP1 and CDK2 protein expression levels were up-regulated in oral cancer tissues than in normal tissues by Western blot analysis (P<0.05). Among Ca9-22, CAL27, SAS and in betel-chewing oral cancer cells TW2.6 and human normal skin keratinolial cells (HaCaT) that we examined, p12CDK2AP1 and CDK2 proteins were detected to be highest expressed in Ca9-22 and TW2.6 cells, respectively, when compared to HaCaT cells. Immunocytochemistry indicated p12CDK2AP1 expressed in nucleus and cytoplasm in Ca9-22, CAL27, SAS and HaCaT cells, however predominant present in nucleus in TW2.6 cells. On the other hand, immunohistochemistry demonstrated that nuclear (P=0.157) and cytoplasmic p12CDK2AP1 (P=0.350) in 167 patients with buccal mucosa squamous cell carcinoma were slightly down-regulated. Reduction of nuclear p12CDK2AP1 was not significantly correlated with any clinicopathologic characteristics or prognosis. Direct sequencing indicated that lack of microsatellite-like instability of CDK2AP1 3¡¦-UTR in four oral cancer cell lines, HaCaT and six patients with down-regulated MLH1 protein. In conclusion, we demonstrated that: (1) p12CDK2AP1 was located in both the nucleus and cytoplasm in most oral cancer cell lines and HaCaT cells but predominate present in the nucleus in betel-chewing oral cancer cells, TW2.6; (2) Reduction of nuclear p12CDK2AP1 in buccal mucosa squamous cell carcinoma tissues were identified, however, not significantly correlated with any clinicopathologic characteristics, prognosis or betel chewing; (3) In six patients with down-regulated MLH1, lack of micorsatellite-like instability in the CDK2AP1 3¡¦-UTR region has been found.

Clinical and epidemiological studies support a strong association between chronic inflammation and cancer. Inflammatory related cytokines, such as IL-1£\, IL-1RN, IL-1£], IL-4, IL-6, IL-8, IL-10, TNF-£\ and TGF£]-1, might play important role in carcinogenesis of oral squamous cell carcinoma (OSCC). Two case-control studies were carried out to evaluate the association of 16 various polymorphisms of 9 inflammatory-related genes with the risk for OSCC and the risk for betel quid (BQ)-related oral precancer lesions (OPL) and BQ-related OSCC. Then the association between various IL-1B C-511T/T-31C haplotypes with plasma levels of IL-1£] was evaluated. One case-contol study included 363 OSCC case patients and 487 healthy controls as well as the other case-control study included 227 BQ-related OSCC cases, 116 BQ-related OPL patients and 209 BQ-related controls. All subjects were recruited and genotyped by use of the PCR-RFLP techniques or TaqMan real-time PCR method from November 2003 and May 2007 at Kaohsiung Veterans General Hospital. Then, 9 OSCC case patients, 9 OPL patients, and 9 controls were selected and matched on sex, age as well as the quantity of BQ-chewing, alcohol drinking, and cigarette smoking for evaluation of plasma levels of IL-1£] by use of ELISA. In the single locus analysis, the variant genotype of RP1RP2 or RP2RP2 (VS. RP1RP1) of IL-4 intron 3 VNTR (AOR = 0.67, 95% CI = 0.45-0.99; AOR = 0.65, 95% CI = 0.45-0.95), TA or AA (VS. TT) genotype of IL-8 T-251A (AOR = 1.55, 95% CI = 1.05-2.30; AOR = 2.50, 95% CI = 1.46-4.27), TT (VS. CC) genotype of IL-8 C+781T (AOR = 2.01; 95% CI = 1.11-3.63), and GA combined with AA (VS. GG) genotype of TNFA G-308A (AOR = 0.40; 95% CI = 0.25-0.66) were associated with risk of OSCC, as compare with those genotypes of healthy controls. However, CC (VS. TT) genotype of IL-10 T-819C (AOR = 0.24, 95% CI = 0.08-0.74) and CC (VS. AA) genotype of IL-10 A-592C (AOR = 0.25, 95% CI = 0.08-0.79) were significantly associated with reduced risk of BQ-related OPL, as compared with those genotypes of BQ controls. In addition, the variant genotype of 2/2 or 1/2 (VS. 1/1) of IL-1RN intron2 VNTR (AOR = 0.11, 95% CI = 0.01-0.97; AOR = 0.48, 95% CI = 0.27-0.87), TC (VS TT) genotype of IL-1B T-31C (AOR = 1.82, 95% CI = 1.14-2.92), AA (VS. TT) genotype of IL-8 T-251A (AOR = 1.92, 95% CI = 1.01-3.66), GG (VS. TT) genotype of IL-8 T+396G (AOR = 2.18; 95% CI = 1.12-4.21), and GA combined with AA (VS. GG) genotype of TNFA G-308A (AOR = 0.46, 95% CI = 0.27-0.79) were significantly related with risk of BQ-related OSCC, as compared with BQ controls. Moreover, CC (VS. TT) genotype of IL-10 T-819C (AOR = 3.33, 95% CI = 1.07-10.42) was associated with increased risk of BQ-related OSCC, as compared with those genotypes of BQ-related OPL. In the haplotype analysis, -590C/RP2 (VS. -590T/RP1) haplotype of IL-4 (AOR = 0.69; 95% CI = 0.49-0.98) and -251A/+781T (VS. -251T/+781C) haplotype of IL-8 (AOR = 1.57, 95% CI = 1.19-2.06) were related with risk of OSCC, as compared with those haplotypes of healthy controls. However, -511C/-31C (VS. -511C/-31T) haplotype of IL-1B (AOR = 0.00, 95% CI = 0.00-0.01) and -1082A/-819C/-592C (VS. -1082A/-819T/-592A) haplotype of IL-10 (AOR = 0.66, 95% CI = 0.44-0.98) were strongly associated with reduced risk of BQ-related OPL, as compared with those genotypes BQ controls. In addition, -889C/2/-511C/-31T or -889T/1/-511C/-31T or -889T/1/-511T/-31C (VS. -889C/1/-511C/-31T) haplotypes of IL-1 family genes (AOR = 0.47, 95% CI = 0.26-0.87; AOR = 0.31, 95% CI = 0.13-0.73; AOR = 3.26; 95% CI = 1.34-7.93) were associated with risk of BQ-related OSCC, as compared with those genotypes of BQ controls. On the contrary, -511T/-31T (VS. -511C/-31T) haplotype of IL-1B (AOR = 0.34, 95% CI = 0.12-0.97) and -889T/1/-511C/-31T (VS. -889C/1/-511C/-31T) haplotype of IL-1 family genes (AOR = 0.27, 95% CI = 0.10-0.71) were associated with reduced risk of BQ-related OSCC, as compared with those haplotypes of BQ-related OPL. Finally, in the stratification analysis, the combined effects of three genes (IL-8 T-251A, IL-4 intron 3 VNTR and TNFA G-308A) had a significantly increased risk of OSCC among male, older group (¡Ö50 years old), Fukienece combined with Aborigine population, never-BQ chewers, never as well as heavy smokers, or light and heavy drinkers, compared with healthy controls. The results suggested that gene-environment combined effect were associated with the risk of OSCC. In ELISA assay, plasma IL-1£] levels in BQ-related OSCC and OPL were found significantly higher than those in haplotypes for IL-1B -511T/-31C and IL-1B -511T/-31T compared with the combined effects of IL-1B -511C/-31C and IL-1B -511C/-31T. In conclusion, polymorphisms of IL-1RN, IL-4 and TNFA were associated with the decreased risk of OSCC or BQ-related OPL, but IL-8 was with increased risk of OSCC or BQ-related OPL. Furthermore, polymorphisms of IL-1B and IL-10 had contrary effect between BQ-related OSCC and OPL. Additionally, plasma levels of IL-1£] was correlated with various IL-1B C-511T/T-31C haplotypes but not with correlated with the status of disease.

Betel quid¡]BQ¡^chewing is recognized as a major risk factor for oral precancerous lesions¡]OPLs¡^in Taiwan. The compositions of Betel quid could cause DNA damage. X-ray repair cross complementing Group 1¡]XRCC1¡^plays a crucial role in the process of DNA repair. Polymorphisms in XRCC1 gene may affect DNA repairing ability and modulate the susceptibility of OPLs. The aim of this study was to investigate the association of XRCC1 genetic variants with the risk of BQ-related oral precancerous lesions, including oral leukoplakia¡]OL¡^ and oral submucous fibrosis ( OSF ). A total of 449 males¡]169 OL cases, 82 OSF cases, and 208 healthy controls¡^who habitually chewed BQ were recruited. The genotypes were determined by PCR-RFLP and TaqMan real-time assays. The C allele and T/C+C/C genotypes at XRCC1 -77 were associated with the reduced risk of OL ( AOR=0.54, 95%CI:0.34-0.85 and AOR=0.47, 95%CI:0.28-0.78, respectively ). The 399Gln allele and 399 Arg/Gln+Gln/Gln genotypes were associated with the increased risk of OL¡]AOR=1.94; 95%CI: 1.41-2.67 and AOR=2.64; 95%CI: 1.73-4.03, respectively¡^and OSF ( AOR=1.67; 95%CI: 1.11-2.49 and AOR=2.30; 95%CI: 1.35-3.91, respectively ). The haplotypes or diplotypes contain fewer risk alleles¡]-77T or 399Gln¡^ were with lower risk of OL¡]both Ptrend<0.001¡^and OSF (Ptrend=0.056 and Ptrend=0.040, respectively). In conclusion, our results suggest that polymorphisms of XRCC1 at -77 and 399 may be associated with the risk of OPLs.

It is estimated that over 30 million people are exposed to arsenic from drinking contaminated groundwater in Bangladesh. Furthermore, due to the use of contaminated water for irrigation purposes, arsenic and other toxic elements are entering the food chain of Bangladeshis. In this thesis, the total levels of toxic elements (As, Cd, Pb) and essential elements (Mn, Se, Zn) in 1,120 samples of Bangladeshi foods (including rice, vegetables, fish) and non-foods (betel quid and baked clay) imported into the United Kingdom were determined. From this analysis, it is concluded that Bangladeshis are exposed to high levels of toxic elements. Inorganic arsenic levels in Bangladeshi rice can be very high, especially from regions with high arsenic in groundwater. However, there is a lack of studies in the literature regarding arsenic levels in rice from regions in Bangladesh with relatively low levels of arsenic in the groundwater. Therefore, rice from one such region (Sylhet district) was analysed. The results indicated that boro (mean 71.7 µg/kg) and aman (mean 85.7 µg/kg) rice from Sylhet contained between 2 to 4-fold lower levels of arsenic, compared to other regions of Bangladesh thus far reported in the literature. Arsenic speciation was carried out on a selection of rice (aromatic and non-aromatic) from Sylhet region and this revealed 70% (mean value) as inorganic arsenic (AsIII and AsV), which is similar to rice grown in other regions of Bangladesh. Importantly, it was found that the arsenic levels of aromatic rice (mean 48.5 µg/kg) from Sylhet region was over 40% lower than that of non-aromatic rice (mean 81 µg/kg). The aromatic rice also contained higher levels of essential elements (such as Se and Zn). It was calculated that for an individual consuming 0.5 kg of rice per day, switching from consumption of non-aromatic rice to aromatic rice would increase Se and Zn intake by 46% and 23% respectively. Arsenic speciation was also carried out on other Bangladeshi food and non-food items, including fish, betel quid and baked clay, to obtain a better insight into exposure to toxic arsenic species. High levels of arsenic (range 3.8-13.1 mg/kg) and lead (range 21-26.7 mg/kg) were detected in the baked clay samples, which are consumed by some Bangladeshi women in an ancient practice known as geophagy. The efficiency of arsenic extraction from baked clay was 33% of the total arsenic present and the main arsenic species present was inorganic AsV (100%); AsIII was not detected in these samples. Millions of Bangladeshis chew betel quid and this contained predominantly AsIII species (extraction efficiency was 100%). Arsenic and lead intake from eating baked clay could exceed the provisional maximum tolerable daily intake (PMTDI) by 2- and 5-fold respectively. For the first time, arsenic speciation in Bangladeshi fish is reported. Hilsha, which is a very popular fish in Bangladesh, contained 2.55 mg/day (mean value) of total arsenic. Extraction efficiencies (%) were 59 – 89 for fish flesh, over 69% of arsenic present in the extract was dimethyl arsenic acid (DMA) species with about 11% arsenobetaine (AsBet) and 19% arsenosugar. These studies reveal that rice, betel quids and baked clay can be a significant source of exposure to inorganic arsenic and DMA in Bangladeshis. Exposure to cadmium is linked with kidney disease and over 20 million people in Bangladesh suffer from chronic kidney disease. Results obtained showed that the daily intake of cadmium by the Bangladeshi population from baked clay (mean 17 µg/day), rice (mean 18.6 µg/day) and certain leafy vegetables (mean 12 µg/day) was higher total daily intake compared to other countries. Surprisingly, puffed rice, which is commonly consumed by Bangladeshis, contained much higher levels of cadmium (mean 67.9 µg/kg) and lead (mean 98 µg/kg), compared to uncooked rice (cadmium, 37.2 µg/kg; lead, 18.9 µg/kg). This may be related to the illegal practice of using urea for whitening puffed rice in Bangladesh. Exposure to manganese in the Bangladeshi population through drinking water has been previously highlighted as a possible health problem, although the intake from foods and non-food has not been reported. The daily manganese intake by Bangladeshis was calculated to be 20.3 mg/day, which is higher than any other country in the world thus far reported. Betel quid components have high levels of manganese and this was reflected by higher urinary manganese the mean urinary Mn levels in chewers (1.93 µg/L, SD 1.8) was significantly higher (3.1 fold; P = 0.009) compared to non-chewers (0.62 µg/L, SD 0.4). Bangladeshi women who eat baked clay and chew betel quids are likely to be exposed to high levels of arsenic, lead and other toxic elements. This is particularly of concern for pregnant women as these metals can be transferred to the unborn baby through the placenta. For assessing the risk of exposure to toxic elements versus intake of essential elements from the same foods, a Food Toxicity Scale (FTS) was devised in order to identify foods that are beneficial or harmful. FTS values were obtained by calculating the toxic elements : essential elements ratio (As:Se, As:Zn etc) and the value obtained subsequently multiplied by toxic element concentration and the quantity of the particular food consumed per day. The higher the FTS value, the greater the risk of exposure to harmful elements. Rice and leafy vegetables had the highest FTS values, partly because large quantities of these foods are consumed. However, lentils and animal products (such as small fish) had relatively lower FTS values compared to other foods. Total daily intake of arsenic (306 µg/day), selenium (90.4 µg/day), cadmium (34.6 µg/day), lead (74.4 µg/day), manganese (20.3 mg/day) and zinc (11.2 mg/day) in the Bangladeshi population was calculated. The intake of arsenic and manganese exceeds the PMTDI for these elements. Water was the highest source of arsenic exposure in Bangladeshis followed by rice. For cadmium and lead, rice and leafy vegetables were the key contributors to the daily intake. The results presented in this thesis show that Bangladeshis are exposed to high levels of toxic elements and how modifications can be made to their diet to not only reduce their exposure to toxic elements but also increase the intake of essential elements. This could be achieved by a combination of the following: (i) reducing the intake of rice (by about 50%); (ii) switching to eating aromatic rice; (iii) increasing the intake of animal products (meat, fish etc.); (iv) decreasing the intake of certain leafy vegetables; and (v) stopping or reducing the practice of eating baked clay and chewing betel quid.

Magister Scientiae Dentium - MSc(Dent) / Betel nut/quid chewing is a habit that is commonly practiced in the Indian subcontinent. This age-old social habit is still practiced by Indians in Durban, Kwazulu Natal (South Africa). The betel nut/quid is prepared in a variety of ways. The quid may be prepared with or without tobacco. This habit is said to be associated with the development of premalignant lesions,namely, Oral Submucous Fibrosis (OSF) which increases the susceptibility for malignancy of the oral mucosa and the foregut. The aim of this study was to investigate the prevalence of betel nut/quid chewing (with or without tobacco), the associated habits (smoking and alcohol consumption) and awareness of the harmful effects of the chewing habit among Indians in Durban, KwaZulu-Natal.A cross-sectional study design was chosen utilising a self-administered questionnaire and semi-structured interviews to collect data. Consenting participants were requested to complete a self-administered, structured questionnaire. The study population included any person in the Durban area who chewed betel nut/quid/tobacco. Only persons willingly and who consented to be part of the study, were included. The sample size was based on convenience. People were approached at the pan shops, leisure markets, traditional functions and at the dental practice the researcher operated at. A total of 101 respondents were interviewed.A significantly higher proportion of females chewed betel nut/quid from the total of the respondents. The results showed that the habit is increasingly practiced in the younger age group (20-39 years). There was evidence to show that the chewing habit is used more by the employed than the unemployed (p=0.055). Of the sample population, 78% were born in South Africa and the rest were immigrants from Pakistan, India and Dubai. All respondents from the migrant community were males. The most important reasons for chewing betel nut were for enjoyment and at special functions. More than two third indicated family members (aunts,uncles and cousins) influence as a reason for chewing, in comparison to influences by parents or grandparents. The study also indicated that parents were far more likely to influence betel nut chewing if grandparents did so (p-value= 0.000). In addition, the study revealed that family members (aunts, uncles and cousins) were far more likely to influence betel nut chewing if parents did so (p=0.000).The most popular ingredients chewed were betel nut, betel leaf, lime and pan masala and the most popular combinations were betel nut/lime/betel leaf quid preparation, betel nut alone,betel nut/betel leaf/lime/tobacco/pan masala and betel nut/betel leaf/lime/pan masala. Two thirds of the respondents do not know that betel nut chewing is harmful to their health, thus indicating a lack of awareness on the risks associated with the chewing habit, and the majority have not attempted to give up the habit. Most of the respondents retained their chewing habits after being informed about the risks. A little more than half the study population reported neither smoking nor drinking.The present study found that betel nut/quid chewing habits continue to be enjoyed by many people and most are unaware of the hazardous effects of the habit. More younger people are using the habit as compared to previous studies. This is probably because it is an affordable and easily accessible habit. It is recommended that aggressive awareness programmes on the harmful effects of betel nut/quid chewing be developed, similar to that for smoking cessation.Government health warnings need to be instituted, for example, by having written warnings on packagings. Taxes need to be imposed on the betel nut and condiments thereby reducing access to most people. Age restrictions need to be imposed on purchasing of the betel nut/quid thus making access difficult for the children.

Most oral squamous cell carcinoma (OSCC) is believed to develop via a multistep process of cumulative gene damage in epithelial cells. Increasing incidence of OSCC and evidence that traditional risk factors may not be responsible directed us to investigate the prevalence of virus in pre- and malignant samples.The integration of the DNA from human papillomavirus (HPV), Epstein-Barr virus (EBV), and Herpes simplex (HSV) into the human genome is associated with the expression of oncogenes and the down-regulation of tumor-suppressor genes in OSCC carcinogenesis. This thesis compared samples from India and Sudan, two countries on two continents having a documented high incidence of oral cancer, with specimens from Sweden, with its known low incidence of oral cancer. Each region has, in addition to smoking, a unique non-smoked tobacco habits with documented carcinogenic effects. These countries also typify areas of low and high socioeconomic living conditions with their expected impact on disease development. The study populations were selected from tobacco users and nonusers with OSCC, oral sub-mucous fibrosis (India), oral lichen planus (Sweden), oral leukoplakia with and without dysplasia and snuff-induced lesions (Sweden and Sudan). An expedient method was developed for extracting DNA from old formalin-fixed and paraffin-embedded biopsies. The prevalence of HPV, EBV, and HSV was investigated using PCR/DNA sequencing and southern blot hybridization analysis. We found HPV and EBV to be most prevalent in samples of tissue characterized as normal, with decreasing prevalence in dysplastic and malignant lesions. This intriguing finding that prevalence decreases as neoplastic development proceeds warrants further investigation. Our data do not at first sight support the conclusion that viruses and tobacco use jointly interact with cell mechanisms in the development of oral cancer.