Indonesian Betel Quid and Oral Cancer

Purpose Oral cancer (OC) is considered as a public health problem that carries significant morbidity and mortality. The disease represents a group of conditions with a range of sites and varied etiology. Indonesia has insufficient data of epidemiology studies in relation with oral pre-malignant disorders (OPMD) or OC prevalence and their risk factors. It is also important to understand the knowledge of GDPs in recognizing OC risk factors, early symptoms of OC, as well as in performing conventional oral examination. Without a comprehensive understanding of these factors, efforts to prevent, detect and manage this disease are likely to be ineffective in terms of outcomes and use of resources. Hypothesis and Aim The overall hypothesis was that Indonesia has a high prevalence of OPMD or OC that are strongly associated with the actual found risk factors. The overall aims of the epidemiology studies were to understand the prevalence and risk factors of OC and OPMD among Indonesians as well as to assess Indonesian GDP’s knowledge in risk factors for OC and in performing COE. The aims of the laboratory studies were to identify antioxidants activities and phytochemical components of Indonesian BQ using HPLC, LCMS, and GC-MS, as well as to know the pathobiological response of Indonesian BQ components on oral keratinocyte (OKF-6) and oral fibroblast (MMF-1). Materials and Methods A cross-sectional study was undertaken using a pre and post-training questionnaire and training intervention with both theory and practical based. The study conducted in five different provinces of Indonesia; Aceh, Banda Aceh (BA); Bandung, West Java (WJ); special district Jakarta, Jakarta (JKT); Pontianak, West Kalimantan (WK); and Sorong, West Papua (WP). The local Dental Association or Faculty of Dentistry invited the GDPs to attend a one-day standardized education program of oral malignant and OPMDs detection and 3 days hospital-based or community service-based practice. A cross-sectional study was undertaken in multiple geographical areas within five Indonesian provinces, namely West Java (WJ), Jakarta (JKT), West Papua (WP), West Kalimantan (WK) and Banda Aceh (BA) regions. Individuals attending community health services, dental hospitals and selected villages were recruited. Volunteer respondents answered a previously validated extensive questionnaire that included socio-demographic factors, oral hygiene practices, diet, adverse oral habits and frequency of dental visits. An oral examination was undertaken using standardized WHO methodology. Data were analyzed using ANOVA, Chi-Square, and logistic regression to assess association between risk factors and mucosal disease. The total phenolic content (TPC), antioxidant activity (by mean of ferric reducing antioxidant power, FRAP), radical scavenging activity (DPPH test), phenolics profile and arecoline content in different components of BQ which were AN, Leaf or SI, Husk, and blended BQ (BQ mixture). BQ mixture were a blending form contained AN, Leaf or SI and slaked lime. Samples were imported from 4 major regions of Indonesia, namely: Banda Aceh (BA), North Sumatra (NS), West Kalimantan (WK) and West Papua (WP). The highest TPC, FRAP, and DPPH values were detected in AN samples compared to other BQ components, while samples from WP region were of higher values compared to the other regions. High performance liquid chromatography - Mass Spectrometry (LC-MS) analysis was used to identified and quantified polyphenols and arecoline. The gas chromatography mass spectrometry (GC-MS) platform was used to analyse the alkaloids present in betel quid originated from four different regions of Indonesia, Banda Aceh (BA), North Sumatra (NS), West Kalimantan (WK), and West Papua (WP). Further, our study profiled the plant metabolites present in different components of betel quid, including areca nuts, betel leaf, stem inflorescence and betel quid as whole originated from each region. Fluorescein diacetate assay (FDA) was used as a cell cytotoxicity test of BQ components on oral keratinocytes (OKF-6) and oral fibroblast (MMF-1) cells. The cytotoxicity effects of areca nut (AN), leaf of piper betle (Leaf) or stem inflorescence of piper betle (SI), areca husk (Husk) and whole BQ Mixture was tested on the growth of oral keratinocytes (OKF-6) and oral primary fibroblasts (MMF-1). On the basis of their chemical characteristics, BQ from Banda Aceh (BA) and West Papua (WP) regions were selected for in vitro testing. Results 177 GDPs were registered in the one-day standardised training and 3 days practice. The highest number of registered GDPs was from WJ region (n=63), followed by GDPs from BA (n=44), JKT (n=27), WK (N=23), and WP (20) region respectively in rank. A total of 144 (81.37%) GDPs sent back their response to the post-training questionnaire 2-6 weeks after attending the training. Female respondents predominated with 157 (88.7%) returning the pre-training questionnaire and 126 (87.5%) returning the post-training questionnaire. Only 20 (11.3%) and 18 (12.5%) male GDPs answered the pre- and post-training questionnaire, respectively. Most of Indonesian GDPs demonstrated inadequate knowledge, awareness, and confidence in performing COE. The OC training could positively increase their knowledge and confidence of early screening. A total of 973 respondents were enrolled (WJ 35.5%; JKT 13.3%, WP 18.3%, WK 9%; BA 23.9%). Smoking (14.8%), BQ chewing (12.6%) and alcohol drinking (4%) varied geographically. An OPMD was detected in 14.3% respondents and 0.2% had history of OC. Leukoplakia was the most common OPMD (8.6%), while the prevalence of erythroplakia/erythroleukoplakia was 0.6%, and oral submucous fibrosis (OSMF) 1.9%. Leukoplakia was the most common type OPMD found in the survey most likely due to high number of smoking habit related. Overall, there was a strong correlation between the prevalence of OPMD and risk factors for OC. In particular, high use of BQ chewing in WK and WP, related to high prevalence of OPMD. The knowledge of OC risk factors, oral hygiene behaviour, ethnicities and low-income SES group were significantly associated with OPMD. High performance liquid chromatography - Mass Spectrometry (LC-MS) analysis showed that Husk contains the widest range of polyphenols, including hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavonols and stilbenes. Catechin and epicatechin were the main polyphenols detected in BQ, and they were present at the highest concentrations in WP-AN sample. Arecoline was detected in all AN and BQ mix samples and was significantly correlated with catechin and epicatechin, and significantly negatively correlated with p-hydroxybenzoic acid. The current study is the first to extensively characterise the chemical composition of BQ and provides insight for the interactions of BQ alkaloids and phenolics in the development of oral submucous fibrosis and oral cancer. Three types of alkaloids (arecoline, arecaidine, and guvacoline) were successfully identified using GC-MS. However, guvacine could not be analysed due to low volatility. This currently also profiled the metabolites in different BQ components and identified high concentrations of benzenoid and terpense, in stem inflorescence samples, especially the carcinogenic constitute, safrole. Interestingly, high concentrations of sesquiterpenes, such as gamma-cadinene, spatulenol, alpha-copaene, linalool, alpha-gurjunene, gamma-amorphene were also identified in stem inflorescence. The study found that WP-AN exhibited cytotoxic effect on OKF-6 at a concentration of 100 micro g/ml, as measured by a 50% reduction of cell viability (IC50), while BA-AN exhibited cytotoxic effect at a higher concentration (500 micro g/ml) after 2 days of incubation. WP-SI also showed cytotoxic effect at 500 micro g/ml after 2 days incubation. In contrast, the extract of Leaf, BQ Mixture and Husk did not exhibit cytotoxic effect for up to 3 days of incubation. The exposure of BQ components to MMF-1 showed no cytotoxicity at any concentration assessed. Incubation of MMF-1 with BA-AN and WP-AN led to an increase of cell proliferation (1 day) followed by a decline of cell growth (2 days) in a dose and time dependent manner. Thus, the results of our study showed distinctive cytotoxic profiles in stromal and epithelial cells of the oral cavity induced by BQ components. Conclusion A previously underestimated high prevalence of OPMD in Indonesia strongly correlates with OC risk behaviour. There is an urgent need for primary prevention of OC programs in Indonesia. GDP knowledge of OC risk factors and COE is important to be addressed, as the lack of their knowledge and awareness can impact to delaying OC early screening and diagnosis. Therefore, improving the national curriculum in Indonesia on OC is more likely the best way for GDPs to have sufficient knowledge and confidence regarding OC before graduation. The ripeness of AN is directly related to the amount of both polyphenols and arecoline. The unripe AN contained a higher concentration of polyphenols and arecoline compared to ripe AN, suggesting that consuming the ripe AN could lower the potential of developing OSMF. Further, our finding that safrole was detected in betel stem inflorescence (SI) as well as husk. This finding suggests that adding betel SI and husk into BQ compound could raise the risk of having OSMF and oral cancer even more as safrole is a possible potential human carcinogen

Keywords

Oral cancer, betel quid, oral pre-malignant disorders (OPMD), oral cancer risk factors, HPLC, LC-MS, GC-MS, Total phenolic content, Antioxidant activities, FRAP, DPPH, alkaloids, arecoline, arecaidine, guvacoline, guvacine, polyphenols, cell citotoxicity of betel quid.

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