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.

Oral submucous fibrosis (OSF) is a debilitating irreversible fibrotic condition of the oral cavity. It is characterized by inflammation and ultimately results in trismus. Patients face difficulty in speaking, swallowing and chewing due to restricted mouth opening (trismus). This disease is also categorized as an oral premalignant disorder (OPMD). Recent reports cite a conversion rate of 10% from OSF to oral squamous cell carcinoma (OSCC). Epidemiological studies and case reports over the years have correlated the habit of chewing areca nut (Areca catechu) to the manifestation of OSF. It is a major cause of concern in the South and South East Asian parts of the world where areca nut is cultivated and routinely consumed. There are an estimated 700 million areca nut chewers around the globe with 0.5% of the population in the Indian subcontinent being affected by OSF due to this habit. Previous studies have reported differential gene expression profile and up regulation of the pro-fibrotic transforming growth factor-β (TGF-β) pathway in OSF. However, detailed molecular mechanisms for the pathogenesis of this disease are still unclear despite our knowledge about the etiological agent (areca nut) responsible for its progression. Therefore, to gain insights into the etiopathogeneses of OSF, following objectives were undertaken:  To study the gene expression changes induced by areca nut and pro-fibrotic cytokine TGF-β in primary fibroblast cells, and their implications in OSF.  To elucidate the mechanism of TGF-β signal activation in epithelial cells by areca nut. Fibroblast cells are the effectors in all fibrotic disorders. Therefore, it is essential to study the response of this cell type in fibrosis. With prior knowledge of the activation of TGF-β pathway in OSF and the etiological agent of this disease being areca nut; we wanted to study the differential gene response of fibroblasts to these two agents. For this purpose, human primary gingival fibroblasts (hGF) were used as a model system to study the global gene expression profile regulated by areca nut and/or TGF-β. hGF cells were treated with sub-cytotoxic dose of areca nut (5 µg/ml) with and without TGF-β (5 ng/ml) for 72 hours and microarray was performed. The results revealed 4666 genes being differentially regulated by areca nut in hGF cells while TGF-β regulated 1214 genes. Both of them together differentially regulated 5752 genes. 413 genes which were commonly regulated by areca nut and TGF-β were observed to have enhanced regulation with a combined treatment of areca nut, together with TGF-β. This result pointed towards the potential role of both areca nut and TGF-β in modulating fibroblast response. To further assess the role of areca nut in OSF manifestation, we first compared the transcriptome profile induced by it in epithelial cells with fibroblast cells. Areca nut was found to induce differential response in these two cell types which corroborates with the disease pathology wherein; epithelial atrophy is observed and conversely fibroblasts are proliferative. To extend these observations we compared the areca nut induced profile in epithelial cells with OSF differential profile and found that a majority of the genes regulated by areca nut which were common with OSF are regulated by TGF-β. Similarly, areca nut and TGF-β regulated profile in fibroblast cells overlapped significantly with OSF profile. Additionally, areca nut and TGF-β treatment positively enriched matrix associated and metabolic pathways among others which are reported to be differentially regulated in OSF. These observations also highlighted the importance of combined actions of areca nut and TGF-β in OSF manifestation. To test the physiological importance of combined actions of areca nut and TGF-β in the context of OSF; activation of fibroblasts by these treatments was assessed. Treatment of fibroblasts with areca nut and TGF-β enhanced the expression of myofibroblast markers αSMA and γSMA with a concomitant increase in the contractile property when compared to areca nut or TGF-β treatment alone. Further, we observed that areca nut did not regulate any of the TGF-β ligands or receptors in fibroblasts, whereas it induced TGF-β2 in epithelial cells. Therefore, this invoked a possible epithelial-mesenchymal interaction that may exist in OSF pathogenesis. To test this possibility in-vitro, epithelial cells were treated with areca nut and the secretome of these cells was put on hGF cells to study the regulation of fibrosis associated genes. This treatment enhanced the regulation of fibroblast activation markers (αSMA and γSMA) as compared to direct areca nut treatment. This increase in regulation was abrogated when induction of TGF-β2 was compromised in epithelial cells. Similar results were obtained for the regulation of other genes (TGM-2, THBS-1, EDN1, LOXL3, PLOD2, TMEPAI, TGFBI, CTGF, BMP1, LMIK1). Therefore, we concluded that TGF-β which is secreted in response to areca nut by epithelial cells influences fibroblasts in combination with areca nut to enhance fibrosis response. Furthermore, the secretome of untreated epithelial cells was found to down regulate the basal expression of fibrosis related genes in fibroblasts, invoking a role for epithelial secretome in regulating the fibrosis progression. Our data highlighted the importance of TGF-β’s influence on fibroblast response in OSF, but the mechanism for the regulation of this cytokine was not known. Areca nut did not induce TGF-β ligands in fibroblast as discussed above, but previous data from our group had reported areca nut mediated up regulation of TGF-β2 in epithelial cells. Therefore, we further elucidated the mechanistic details for this induction using immortalized keratinocytes (HaCaT and HPL1D) and correlated these in OSF tissues. The kinetics of the induction of TGF-β signaling by areca nut (5 µg/ml) in epithelial cells was established. Areca nut induced TGF-β2 transcript, protein and activated the canonical signaling (pSMAD2/3) at 2 hours post treatment, which persisted till 24 hours. The regulation of TGF-β2 mRNA at 2 hours was dependent on active transcription but was independent of protein translation whereas the activation of signaling (pSMAD2) required both transcription and translation at this time point. This warranted probing for the role of TβR-I in the activation of TGF-β signal by areca nut. A small molecule inhibitor was used to abrogate the kinase activity of TβR-I. Areca nut induced TGF-β2 mRNA at 2 hours even in the presence of TβR-I inhibitor whereas the induction was compromised at 24 hours although the activation of SMAD2 at both 2 and 24 hours was compromised in the presence of TβR-I. This result signified that induction of TGF-β signaling was dependent on the TβR-I activity at early and late time points, but the transcription of the ligand was independent of the receptor activity at early time point. These results indicated the activation of some other pathway by areca nut which could regulate the transcription of TGF-β2 and thereby activate TGF-β signaling in epithelial cells. To explore this possibility, a panel of pathway inhibitors was used and only JNK inhibitor compromised areca nut induced TGF-β2 mRNA and pSMAD2. The results were corroborated by transient knockdown of JNK1 and JNK2. Further, JNK was phosphorylated at 30 minutes to 2 hours by areca nut treatment on epithelial cells. This activation was found to be independent of TβR-I activity. In good correlation, activated JNK1/2 was also detected in OSF tissues and was not detectable in normal tissues. Since JNK activation was found to be a pre-requisite for areca nut induced TGF-β signal activation; we further explored the mechanism of JNK activation by areca nut itself. Areca nut mediated activation of JNK was found to be dependent on muscarinic acid receptor, Ca2+/CAMKII and ROS. Inhibition of these significantly compromised areca nut induced pJNK. In line with this, inhibition of muscarinic acid receptor activity, CAMKII and ROS also abrogated areca nut mediated induction of TGF-β2 mRNA and pSMAD2. The regulation of TGF-β signaling by areca nut in epithelial cells was dependent on transcription, and JNK activity was essential for this. We further sought to explore transcription factors which were regulated by JNK and therefore could possibly induce TGF-β2 promoter activity. ATF2 and c-Jun transcription factors were found to be induced at 30 minutes by areca nut and this up regulation also persisted till 24 hours. Further, activation of both ATF2 and c-Jun was dependent on JNK but independent of TβR-I activity. Moreover, areca nut treatment induced translocation of these phoshorylated transcription factors in the nucleus of epithelial cells. Additionally, pATF2 and p-c-Jun were enriched on TGF-β2 promoter after 2 hours of treatment by areca nut. To investigate the importance of this enrichment and regulation of TGF-β signal activation by areca nut, we transiently knocked down these proteins and studied the regulation of TGF-β2. Areca nut induced TGF-β2 mRNA and pSMAD2 was abrogated upon ATF2 and c-Jun knockdown, implicating JNK mediated activation of ATF2 and c-Jun in areca nut induced TGF- β signaling. To explore the significance of this mechanism in OSF, immunohistochemical staining for pATF2 and p-c-Jun was performed on OSF and normal tissues. Both the transcription factors were found in the nuclei of OSF tissues whereas their expression was not detected in normal tissues. This expression also correlated with the previously reported activation of SMAD2 in OSF tissues by our group. Hence, ATF2 and c-Jun were observed to be important in areca nut mediated TGF-β signaling in OSF. In conclusion, the work described in this thesis provides mechanistic details into OSF etiopathogenesis. Combined actions of areca nut and TGF-β induced a response in fibroblasts akin to OSF. Our results advocate a role for epithelial secreted factors in influencing fibroblast response in both normal and disease (OSF) conditions. Further, importance of TGF-β in OSF has been elucidated in terms of enhancing the fibroblast response to areca nut. We have also elucidated the mechanism for areca nut mediated activation of TGF-β signaling and have identified the contribution of JNK/ATF2/Jun axis in this process. This work can impact the management of oral submucous fibrosis by providing newer targets for treatment.

Doctor Scientiae (Odontology) - DSc(Odont) / Investigations of pathoses of the oral cavity encompass a relatively wide spectrum of diseases, abnormalities, tumours and tumour-like conditions affecting and occurring in the dental hard tissues and supportive structures, the bony skeleton of the face and the soft tissues of the. mouth. It involves a study of the normal - oral biology - and the abnormal - oral pathology. Oral pathology is a relatively new specialized field of dental science and practice. In South Africa, prior to the nineteen-fifties, research in oral pathology was primarily directed towards dental disease. Two people - Julius Staz of the University of the Witwatersrand and Tony Ockerse of the University of Pretoria - were the doyens in this field and made major contributions to dental science. Staz reported on the status of dental caries and tumorous malformations of teeth and Ockerse on the prevalence and severity of fluorosis in South Africa. During the fifties a second generation of dental surgeons, who were interested in soft tissue, bone and tumour pathology, emerged. They ,were Bertie Cohen, George Baikie, Mervyn Shear and John Lemmer who, at that time, were all from the University of the Witwatersrand. Bertie Cohen later joined the Royal College of Surgeons of England. Mervyn Shear led the field with his research on cysts of the oral cavity. The practice of oral pathology, moulded on anatomical pathology, was established in the early sixties and Mervyn Shear and the author, from the University of Pretoria, became known as oral pathologists. Research at that early stage comprised clinical and histological observations of oral lesions, diseases, tumours and tumour-like conditions. Observation techniques became more sophisticated during the sixties and seventies with the advent of histochemistry and electronmicroscopy. The next major development which blossomed in the seventies and early eighties was the application of epidemiological methods in the study of disease. Epidemiological principles enabled the correct recording of profiles of oral pathoses in the community. Much was learnt about the prevalence and distribution of oral conditions. The application and use of experimental models, especially laboratory animals, became popular in the eighties. Amongst others, a germfree animal unit was established in the Faculty of Dentistry of the University of Stellenbosch enabling workers to study the microbiological aetiology of dental and oral disease. Morphological observations of tumours and mucosal lesions were further enhanced during this period with the development of immunocytochemistry Experimental cell studies by means of cell culture techniques, commenced late in the eighties and was established in the early nineties. These models fostered molecular biology techniques which have become useful tools for the investigation of the aetiology of disease at a cellular and molecular level. At present molecular techniques are also popular in other spheres of oral pathology such as microbiological, immunological and oncological research. The author's first contact with oral pathology as a subject, forming an important and interesting part of dentistry, was the prescribed textbook "Oral and Dental Diseases", 2nd ed., 1951., by HH Stone of the University of Liverpool in the United Kingdom. Subsequently an enduring interest in the subject and research was cultivated by three teachers and colleagues, Ivor Kramer, Robert Bradlow and Mervyn Shear. Ivor Kramer, Professor of Oral Pathology in the Eastman Dental Institute of the University of London was a superb postgraduate teacher of oral pathology, and revelled in research. The Dean of the Institute, Professor Sir Robert Bradlow was a clinician and splendid diagnostician. He correlated the clinical and histopathological features of oral diseases. These two teachers set the course in oral pathology for the author during his postgraduate studies. In the sixties, after a spell at the University of Pretoria, the author joined Professor Mervyn Shear at the University of Witwatersrand. It was here that the author could further his skills of presenting lectures and research papers in an orderely manner and strengthen his love of research. The research carried out by the author reflects to a large extent the development of research in oral pathology in South Africa since 1960.. It includes studies of diseases and lesions of the oral mucosa, the dental hard tissues, tumours of the oral cavity and jaws and forensic odonto-stomatology. To date 139 articles have been published and accepted in scientific journals of which I was the first or co-author. The research presented here, however, comprises only those studies related to pathoses of the oral mucosa as it occurs in South Africa. Fifty-four papers and two abstracts are submitted. The papers are grouped into two divisions which include studies on (I) normal human oral and ectocervical mucosa and (II), those related to pathoses of the oral mucosa. The latter is subdivided into sections on: the profile of lesions of the oral mucosa in the community; cytological, clinical and morphological features of lesions of the oral mucosa; and studies on the aetiology of lesions of the oral mucosa. Each division and section is preceded by a declaration as to the contribution of the author or co-authors and a précis of the aims, objects and research findings. In the introduction of the précis statements are made explaining the aims of the study. These statements are not referenced because they appear in the respective articles.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.