3.3.4(a)(i) Preparation of high and low concentration crude aqueous arecanut and betel quid filtered. 3.3.5(a) Morphological changes of MOE1 and HSC-2 cells and nuclei treated with raw aqueous areca nut. 242 4.3.3(c)(i) Expression of selected genes in MOE1 and HSC-2 cells treated with areca nut filtered extract…….
Background
Oral cancer is reported to be the third and fifth most common malignancies in Indian women and men, respectively (Azizah et al., 2019). The main risk factors for oral cancer are modifiable and lifestyle related, namely excessive alcohol consumption, tobacco smoking and betel chewing (Warnakulasuriya, 2010; Winn et al., 2015). The practice of betel quid chewing is strongly influenced by cultural and demographic differences in South, Southeast, and East Asia (Lee et al., 2011).
In Kelantan, a state with 0.3% Indian population, 22.9% of Malay oral cancer patients reportedly chewed betel quid (Razak et al., 2009). Areca nut chemical compounds include polyphenols (flavonols, tannins), alkaloids, carbohydrates, fats and some crude fibers (Sharan et al., 2012; Gupta and Johnson, 2014; Chen et al., 2017). However, there have also been attempts to use alkaloid from areca nut as an anticancer drug (Sari et al., 2018; Sari, 2021).
Cellular glutathione depletion has also been reported in arecoline-induced cytotoxicity (Sundqvist et al., 1989; Chang et al., 2001).
Justification of the study
Objectives
General Objective
Specific Objectives
The shared genes expressed by both cell lines may serve as potential gene biomarkers for oral cancer related to betel quid etiology. To analyze and compare the Gene Ontology (GO) terms and enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways of DEGs treated by MOE1 and HSC-2 cell lines with crude aqueous areca nut and betel-quid-filtered extracts using g:Profiler toolkit .
Research questions
Research hypothesis
Global prevalence of betel quid chewing
Studies conducted in Singapore and Vietnam reported a prevalence of betel quid chewing of 6.4% and 6.7%, respectively (Kuek et al., 1990; Reichart and Nguyen, 2008). A cross-sectional survey in Myanmar found that 52% of respondents chewed betel liquid on a regular basis. Two hundred and forty of the 284 current betel quid chewers used tobacco to chew their betel quid (Zaw et al., 2016).
The study by Lee et al. 2012b) in Indonesia revealed that the prevalence of betel quid was 12% and 46.8% among men and women, respectively. Another study in Jakarta, Indonesia found that the prevalence of betel quid chewing was 9.3% (Amtha et al., 2014). Prevalence of betel quid chewing among South Asian immigrants in Leicester, United Kingdom reported that betel quid chewing was common among first-generation Asian immigrants, with the highest prevalence among Jains, followed by Muslims and Hindus. In second-generation Asian immigrants, the prevalence of betel quid chewing was highest among Muslims, followed by Hindus and Jains (Vora et al., 2000).
Qorannoon biroo hedduunis godaantota Eeshiyaa biratti babal’ina daakuun betel quid irratti xiyyeeffataniiru.
Malaysian prevalence of betel quid chewing
Betel quid chewing and general health
The International Agency for Research on Cancer classifies betel quid with and without added tobacco as carcinogenic (IARC and may increase the risk of hepatocellular carcinoma, breast cancer and colorectal polyps (Wang et al., 2003; Kaushal et al., 2010; Chen et al. al., 2018).Numerous studies have shown that chewing betel quid with or without tobacco is associated with the risk of several systemic diseases, including metabolic and cardiovascular diseases (Zhang et al., 2010; Yamada et al., 2013; Khan et al. al., 2014; Yen et al., 2016). Nicotine and arecoline in tobacco and betel quid can cause dyslipidemia and hypertension, leading to cardiovascular disease (Zhang et al., 2010). Some studies have found that betel quid consumption is associated with central obesity, diabetes mellitus, cirrhosis, goiter and hepatic steatosis (Mannan et al., 2000; Tseng, 2010; Paulino et al., 2015; Bleibel and Saleem, 2018; Chu et al., 2018).
Betel quid with tobacco use has been associated with an increased risk of adverse birth outcomes, including preterm birth, low birth weight, and shorter birth length (Yang et al., 2008; Berger et al., 2016).
Betel quid chewing and oral health
Betel quid chewing effects on teeth, periodontium, and bone
Chewing betel quid on a regular basis is known to be detrimental to the teeth of betel quid chewers due to the hard, fibrous nature of betel quid (Anand et al., 2014; The clinical study by Ilyas et al. 2015) revealed that participants who chewed betel quid were more likely to have tooth wear and missing teeth than those who did not chew betel quid. Dental sensitivity has also been associated with betel quid chewers, which may be due to enamel loss and exposure of the underlying dentine (Yeh, 1997).
Effect of betel quid chewing on the periodontal viz. probing bleeding, pocket probing depth, dental plaque index and gingival recession was significantly greater among betel quid chewers than non-chewers (Javed et al., 2013; Giri et al. also reported). that betel quid chewing significantly increased radiographic alveolar bone loss. It has been speculated that masticatory forces generated during habitual betel quid chewing may increase temporomandibular joint degeneration (Trivedy et al., 2002; Nawaz, 2015).
Betel quid chewing and oral potentially malignant disorders (OPMDs) (OPMDs)
Oral erythroplakia has been identified as having the highest rates of malignant transformation (Villa et al., 2011). The pathogenesis of OSF related to nutmeg, a component of betel quid, is more frequently discussed in the literature than other OPMDs. Evidence for the role of betel nut use, with or without tobacco, in increasing the risk of developing OSF is based on many previous case reports, prospective cohort studies, as well as several case-control and cross-sectional studies that are performed. in many countries including India, Pakistan, Sri Lanka, Taiwan and Malaysia.
In Malaysia, Indians and indigenous people of East Malaysia had the highest prevalence of precancerous lesions, including leukoplakia, erythroplakia, submucosal fibrosis and lichen planus and may be attributed to betel consumption (Zain et al., 1997b). OSF is an insidious, chronic disease that affects any part of the oral cavity and sometimes the pharynx (Arora et al., 2014). Its pathogenesis is mainly related to alkaloids from areca nuts, resulting in abnormal collagen synthesis and degradation (Ahmad et al., 2006).
Betel quid chewing and oral cancer
Betel quid composition
- Areca nut
- Betel leaf
- Slaked lime
- Betel inflorescence
The fruit of Areca catechu is oval in shape with a pointed top (Figure 2.1: C). When the fruit is unripe, it is green; when ripe, it is orange-yellow (IARC, 2004). The seed has a distinct astringent and slightly bitter taste and can be eaten at various stages of maturity (IARC, 2004). Nuts can be consumed fresh, dried or sun-dried, baked or in the oven (IARC, 2004). Slaked lime is sold in Asian markets as a paste that is mixed with water (IARC, 2004).
Apart from the leaf, other parts of the betel vine, such as the stem, inflorescence or catkins, are consumed while chewing betel quid (IARC, 2004). Consumption of inflorescences is widespread in some countries, including Taiwan and Melanesia, where they are added to quid to give an aromatic flavor (IARC, 2004). It is made without further processing from sun-dried and partially fermented Nicotiana rustica and Nicotiana tabacum (IARC, 2004).
Slaked lime and areca nut slices placed on betel leaf, and (F): Dried, shredded tobacco leaves.
Betel quid preparation and consumption
Generally, the areca nut is used as one of the additives in betel quid, but it can be chewed alone without any other ingredients. Nevertheless, there is limited data available on the use of areca nuts without other ingredients. However, the Chamorros people of Micronesia would swallow the chewed areca nut (with or without betel leaf) and they do not add slaked lime or tobacco (Paulino et al., 2011).
The traditional quid in Malaysia is made with slaked lime, areca nut and flavoring ingredients wrapped in a betel leaf (IARC, 2004). The betel leaf can be young or mature, while the areca nut is sometimes consumed fresh. The additional ingredient could be tobacco and gambir, the former being more commonly used (Tan et al., 2000).
Gambir is made by boiling the leaves and bark of Uncaria gambir, a member of the Rubiaceae family (Sazwi et al., 2013).
Phytochemical compounds of betel quid and areca nut
Phytochemical analysis using GC-MS
GC-MS is a hyphenated technique that is extremely compliant and is the most widely used technique for quantification and identification (Padma et al., 2019). By interpreting and comparing the spectra with the known organic compounds in a complex mixture, the unknown organic compounds can be identified (Yonzone et al., 2012). Gas chromatography (GC) is used to separate different compounds in a complicated mixture, while mass spectrometry (MS) is used to identify and quantify the active compound's specific structural information (Iordache et al., 2009).
The mobile phase of GC consists of a gas carrier, while the stationary phase is a solid layer that supports the inside of a tube known as a column (Hussain and Maqbool, 2014). During sample travel in the column, the separation of different chemical features between different molecules in a mixture of their relative affinity usually takes place in the stationary phase (Hussain and Maqbool, 2014). The selectivity of capillary columns is influenced by column size and factors such as length, diameter and film thickness (Sahil et al., 2011). The molecules are kept in the column and elute at different times, which is called the retention. time (RT). The downstream mass spectrometer will then be able to independently capture, ionize, accelerate and identify the ionized molecules (Hussain and Maqbool, 2014).
By splitting each molecule into ionized fragments and identifying these fragments based on their mass-to-change (m/z) ratio, the identified and quantified chemicals are analyzed by MS (Kanthal et al., 2014).
![Figure 2.2 Schematic diagram of GC-MS system. [Adapted from Sutherland (2018)]](https://thumb-ap.123doks.com/thumbv2/azpdforg/1500256.168684/54.892.170.787.257.546/figure-schematic-diagram-gc-ms-adapted-sutherland.webp)
Cellular and molecular effects of betel quid and areca nut
ROS and oxidative stress induction
In response to areca nut-induced oxidative stress, many studies highlighted the regulation of many genes through various biological processes and pathways, including hypoxia-inducible factor-1 (HIF-1), autophagy, cell cycle arrest, vascular endothelial growth factor (VEGF). ) and glutathione (Smith and Fornace, 1996; Lu et al., 2010; Ji et al., 2014; Kwon et al., 2019). ROS is directly involved in the tumor initiation process through genotoxicity (Nair et al., 1990; Sundqvist and Grafström, 1992). As a result, cancer cells have higher amounts of molecules that scavenge ROS as an adaptive response (Samanta et al., 2016; Ciccarese and Ciminale, 2017).
In addition, Khan et al. 2015) highlighted that areca nut extracts induced ROS in human keratinocytes. Oral KB carcinoma cells exposed to areca nut extract can also produce ROS (Chang et al., 2001b). A similar response is also shown by OSCC when treated with areca nut (Lu et al., 2010).
GSH is an antioxidant that acts as a free radical scavenger and a detoxifying agent in cells (Traverso et al., 2013; Haenen and Bast, 2014; Nimse and Pal, 2015).
