“A CYTOMORPHOMETRIC STUDY ON ORAL MUCOSAL CHANGES AMONG USERS OF TOBACCO WITH BETEL LEAF AND TOBACCO WITHOUT BETEL LEAF” By DR AHAMMED NOUFAL M Dissertation submitted to the Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore In partial fulfillment of the requirements for the degree of MASTER OF DENTAL SURGERY In BRANCH VI: ORAL PATHOLOGY AND MICROBIOLOGY Under the guidance of Dr Maji Jose Professor Department of Oral Pathology and Microbiology Yenepoya Dental College and Hospital Mangalore 2006 – 2009 I RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES DECLARATION BY THE CANDIDATE I hereby declare that this dissertation / thesis entitled “A CYTOMORPHOMETRIC STUDY ON ORAL MUCOSAL CHANGES AMONG USERS OF TOBACCO WITH BETEL LEAF AND TOBACCO WITHOUT BETEL LEAF” is a bonafide and genuine research work carried out by me under the guidance of Dr Maji Jose, Professor, Department of Oral Pathology and Microbiology, Yenepoya Dental College & Hospital, Mangalore. Date: Signature of the candidate Place: Mangalore Dr Ahammed Noufal M II CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled “A CYTOMORPHOMETRIC STUDY ON ORAL MUCOSAL CHANGES AMONG USERS OF TOBACCO WITH BETEL LEAF AND TOBACCO WITHOUT BETEL LEAF” is a bonafide research work done by DR AHAMMED NOUFAL M in partial fulfillment of the requirement for the degree of MASTER OF DENTAL SURGERY in Oral Pathology & Microbiology. Date: Signature of the Guide Place: Mangalore Dr Maji Jose Professor Department of Oral Pathology & Microbiology Yenepoya Dental College & Hospital Mangalore – 575018 III ENDORSEMENT BY THE HOD, PRINCIPAL / HEAD OF THE INSTITUTION This is to certify that the dissertation entitled “A CYTOMORPHOMETRIC STUDY ON ORAL MUCOSAL CHANGES AMONG USERS OF TOBACCO WITH BETEL LEAF AND TOBACCO WITHOUT BETEL LEAF” is a bonafide research work done by DR AHAMMED NOUFAL M under the guidance of Dr Maji Jose, Professor, Department of Oral Pathology and Microbiology, Yenepoya Dental College & Hospital, Mangalore. Seal & Signature of HOD Seal & Signature of Principal Dr Vidya M Dr B H Sripathi Rao Professor & Head Principal Dept. of Oral Pathology & Microbiology Yenepoya Dental College Mangalore Date: Date: Place: Mangalore Place: Mangalore IV COPYRIGHT Declaration by the Candidate I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall have the right to preserve, use and disseminate this dissertation / thesis in print or electronic format for academic / research purpose. Date: Signature of the candidate Place: Mangalore Dr Ahammed Noufal M © Rajiv Gandhi University of Health Sciences, Karnataka V ACKNOWLEDGMENT First and foremost let me thank Almighty GOD. I can't think of words that would begin to describe my gratitude for his bounteous blessings and his protective gaze which watches over me all the time and every time. I would like to express my deep sense of gratitude reverence to my teacher Dr Vidya M, Professor and Head, Department of Oral and Maxillofacial Pathology, Yenepoya Dental College, Mangalore for her unquestionable guidance, tolerant nature and mammoth patience towards me and who has constantly encouraged me to put in my best effort in completing this dissertation. Her relentless encouragement & pursuit for perfection and class has instilled in me a sense of purpose and always to aim high. It is with supreme sincerity and deep sense of appreciation that I express my indebtedness and sincere thanks to my esteemed teacher and guide Dr Maji Jose, Professor, Department of Oral and Maxillofacial Pathology for her constant encouragement, support, understanding, valuable guidance and thought provoking ideas in the execution of this dissertation. I shall forever remain indebted to her for the overwhelming help and meticulous care in correcting my mistakes with her valuable advices. I would like to express my thanks to Prof Dr B.H Sripathi Rao, Principal Yenepoya Dental College, and Prof Dr Sham Bhat, Vice Principal, Yenepoya Dental College for supporting me in conducting this study. A word of thanks to Dr Laxmikanth Chatra, Head of the Department of Oral Medicine & Radiology, Yenepoya Dental College for allowing me in taking up the cases related to this thesis. VI A thankfully acknowledge the support and encouragement and valuable suggestion and support provided by Dr Rajeesh Mohammed, Dr Nandaprasad, Dr Bhavana and Dr Hemanth, Assistant Professors, Department of Oral and Maxillofacial Pathology, Yenepoya Dental College & Hospital under whose guidance this work was done and who were always there with encouraging guidance, detailed and constructive comments, and for their important support accommodating attitude at every step of this work. I am also thankful to my other teachers Dr Joshi V R & Dr Meera Mary Nevis, Readers, Department of Oral Pathology & Microbiology, also a word of thanks to Dr Hezal, Lecturer, Department of Oral Pathology & Microbiology, Yenepoya Dental College & Hospital. I would like to extend my appreciation to Ms. Neevan D’souza, Lecturer / Statistician, Department of community medicine for her valuable assistance in statistically analyzing this thesis. Words are in adequate to express my gratitude to Dr. Girish, who the one encouraged me to do PG in oral pathology. I would like to express my thanks to Mrs. Jayalakshmi Shetty, Lab Technician, Department of Oral Pathology & Microbiology, for her help and support in lab techniques during my post graduation course. A word of appreciation to Mrs. Mabel and Ms. Meera for their assistance and cooperation during this study. VII My Co-PG’s Dr Faraz, Dr Pavitra and Dr Anil deserve heartfelt accolades for their unending cooperation and support throughout my postgraduate course. My senior colleagues, Dr Vani, Dr Vidya, Dr Spoorthi, Dr Bhavana, Dr Hemanth & Dr Kavitha G and to my juniors Dr Jubin, Dr Shannawaz, Dr Kavitha K, Dr Ashik, Dr Shakil, Dr Kartikay, Dr Charan and Dr Afnan supported me in my thesis work. I want to thank them for all their support and cooperation. My love and thanks to my brother Haris. M for his friendly support, encouragement and love throughout my life. I have a special envy and thanks to my roommates: Dr Mohasin A and Dr Mahmood Moothedath, who have always been so encouraging, supporting and understanding during this work. “Nothing in my life could be credited solely to me. I owe it all to my Family.” Finally I can’t think how to thank my mother and father, for thanks seem too inadequate a word when I regard them. My very existence today, is because of their love, trust, prayers and much more for which I can’t find the right words. Also I owe so much to my wife Mrs. Shamleena for her care and love during this project when I need it most. Noufal Date: Place: Mangalore VIII LIST OF ABBREVIATIONS BC - Before Christ BCM - Betel Chewer’s Mucosa BLE - Betel Leaf Extract BQ - Betel Quid BQC - Betel Quid Chewing BQC - Betel Quid Chewing CA - Cytoplasmic Area CD - Cell Diameter IARC- International Agency for Research on Cancer NA - Nuclear Area NAB - N-nitrosoanabasine ND - Nuclear Diameter NNK - 4-(methylenitrosamino)-1-(3-pyridyl)-1-butanone NNN - N- nitrsamine nicotine OPD - Out Patient Department OSF - Oral Submucous Fibrosis PAHs - Polycyclic aromatic carcinogens SCC- Squamous Cell Carcinoma ST - Smokeless Tobacco TSNA - Tobacco Specific N-nitrosamine UK - United Kingdom IX ABSTRACT Use of tobacco is very popular among Asian countries specially India, Sri Lanka, Pakistan etc. Tobacco is generally consumed in two forms such as smoking and smokeless form. In smokeless type itself there are two forms, tobacco with betel leaf and commercial tobacco products without betel leaf. Tobacco in any form is harmful and may induce various oral mucosal changes; But the degree and nature of these changes would be different in individuals who use tobacco with betel leaf, which can be correlated with some protective effect of betel leaf. In this study we assessed the oral mucosal changes in (no=30 each) individuals who are using tobacco with betel leaf and those who are using commercial tobacco products without betel leaf and compared the observations with 30 healthy controls. Mucosal changes were assessed clinically and also in cytological smears taken with the help of cytobrush from the buccal mucosa or mucosal lesions whenever present. After staining the smears with RAPID- PAP (papanicolaou stain kit), the cell diameter (CD) and nuclear diameter (ND) of exfoliated cells were measured using computerized image analysis soft ware. From the above measurements, nuclear cytoplasmic ratio also was calculated. The results obtained were analysed using statistical test ANOVA. Results showed that the incidence of mucosal lesions were more in individuals who used commercial tobacco products than the traditional tobacco users with betel leaf and they had developed the lesions within relatively shorter period after beginning of the habits. Alteration in nuclear and cell diameter was also observed in users of both forms of X tobacco compared to healthy controls. But the alterations were relatively significant in commercial tobacco product users compared to the other group. Key words: Betel quid; Commercial tobacco products; Exfoliative cytology; Image analysis _____________________________________________________________ XI CONTENTS PAGE NO. _____________________________________________________________ INTRODUCTION 1 -3 AIMS & OBJECTIVES 4 REVIEW OF LITERATURE 5 - 46 METHODOLOGY 47 - 57 RESULTS 58 - 73 DISCUSSION 74 - 81 CONCLUSION 82 SUMMARY 83 BIBLIOGRAPHY 84 -96 ANNEXURES 97 - 101 ____________________________________________________________ XII FIGURES PAGE NO. ________________________________________________ 01. TRADITIONAL TOBACCO 51 02. COMMERCIALLY AVAILABLE TOBACCO PRODUCTS 51 03. ORAL EXAMINATION ARMEMENTARIUM 52 04. SMEAR TAKING WITH THE HELP OF CYTO BRUSH 52 05. ORAL SUB MUCOUS FIBROSIS 53 06. LEUKOPLAKIA 53 07. TOBACCO POUCH KERATOSIS 53 08. CHEMICAL BURN 54 09. CHEWERS MUCOSA 54 10. LICHENOID REACTION 54 11. PAP KIT 55 12. TRINOCULAR RESEARCH MICROSCOPE WITH COMPUTER 55 13. EXFOLIATED CELLS OF BUCCAL MUCOSA 56 14. PROCEDURE ADOPTED FOR MEASURING 57 ND AND CD (50%ZOOM) 15. SHOWING LINE DRAWN FOR MEASURING 57 CD AND ND (100% ZOOM) _____________________________________________________________ XIII TABLES PAGE NO. 1. AGE AND SEX DISTRIBUTION OF THE TRADITIONAL AND COMMERCIAL TOBACCO USERS 63 2. COMPARISON BETWEEN TYPE OF TOBACCO USERS AND DEVELOPMENT OF LESION 64 3. CORRELATION OF FREQUENCY AND DURATION OF TRADITIONAL TOBACCO USE 64 4. CORRELATION OF FREQUENCY AND DURATION OF COMMERCIAL TOBACCO USE 65 5. CORRELATIONS OF TRADITIONAL TOBACCO PER DAY AND DURATION OF USE 65 6. MEAN COMPARISON OF NUMBER OF OMMERCIAL& TRADITIONAL TOBACCO PER DAY AND DURATION (YEARS) 66 7. NUCLEAR DIAMETER BETWEEN THREE GROUPS 66 8. POST HOC TESTS. MULTIPLE COMPARISONS (TURKEY HSD) 67 9. CELL DIAMETER BETWEEN THREE GROUPS 68 10. POST HOC TESTS. MULTIPLE COMPARISONS (TUKEY HSD) 68 ____________________________________________________________ XIV GRAPHS PAGE NO. ________________________________________________ 01. AGE DISTRIBUTION OF THE STUDY POPULATION (TRADITIONAL) 69 02. AGE DISTRIBUTION OF THE STUDY POPULATION (COMMERCIAL) 69 03. DISTRIBUTION OF DIFFERENT LESIONS BY TRADITIONAL TOBACCO USERS 70 04. DISTRIBUTION OF DIFFERENT LESIONS BY COMMERCIAL TOBACCO USERS 70 05. SCATTER DIAGRAM SHOWING CORRELATION OF FREQUENCY AND DURATION OF TRADITIONAL TOBACCO USE 71 06. SCATTER DIAGRAM SHOWING WEAK CORRELATION BETWEEN FREQUENCY AND DURATION OF COMMERCIAL TOBACCO USE 71 07. FREQUENCY OF COMMERCIAL TOBACCO USE AND DEVELOPMENT OF LESIONS 72 08. MEAN COMPARISON OF USE OF TRADITIONAL TOBACCO AND COMMERCIAL PACKETS PER DAY & DURATION OF USE 72 09. SHOWING MEAN NUCLEAR DIAMETER BETWEEN 3 GROUPS 73 10. SHOWING MEANCYTOPLASMIC DIAMETER BETWEEN 3 GROUPS 73 XV Introduction INTRODUCTION Tobacco is a menace that has grabbed millions of people all over the world, cutting across the nation and social barriers. Tobacco was introduced into India by Portuguese traders in the late 16th or early 17th century. Since then tobacco use has spread with remarkable rapidity seeping into all sections of the society. In India, people consume tobacco in different forms. Betel quid chewing is one of the most popular Tobacco habit among Indians1, where tobacco is used along with betel leaf, arecanut, slaked lime etc. Betel quid chewing is socially accepted in India and is often given on social occasions as a traditional custom. Betel leaf which is used in the quid has many valuable effects. Earlier studies indicated that betel leaf offers some protection against tobacco induced carcinogenesis2. One of the commercial replacements for betel quid is Pan Masala and Guthka where tobacco along with other ingredients is dispensed in ready to use packets. The packaging revolution has made tobacco products portable, cheap and convenient, with the added advantage of a long shelf-life. Because of these reasons use of commercial tobacco product have become highly prevalent among youngsters. The smoking and chewing of tobacco alone or with betel leaf, has a significant detrimental impact on oral tissues. Tobacco contains chemical constituents which are 1 Introduction known carcinogens. The habit of tobacco consumption has been clearly associated with potentially malignant lesions and conditions and the use is considered as the major etiological factor for oral cancer development, accounting for 30-40 % of all cancer cases in India. Studies have reported various mucosal changes related to tobacco habits, ranging from relatively harmless chewers mucosa to most dangerous Oral cancer. As a rule any mucosal change in tobacco users should be dealt with due seriousness and examined histopathologically. Presently biopsy is the diagnostic test of choice for oral pre malignant and malignant lesions. Studies have shown that oral cytology also may provide an important adjuvant in the assessment of the patients with potentially cancerous lesions3. Exfoliative cytology offers a simple and non invasive technique that can be repeated frequently with little discomfort to the patients. Quantitative parameters such as nuclear size, cell size, nuclear to cytoplasmic ratio, nuclear shape, nuclear discontinuity, optical density and nuclear texture can be evaluated collectively in order to establish the diagnosis of malignancy. Of these parameters, nuclear and cytoplasmic area and nuclear to cytoplasmic ratio have been shown to be significant in the diagnosis of oral lesions4. The reduction in cell diameter and increase in nuclear diameter could be early indications of malignant change. Application of quantitative techniques to smear obtained from oral premalignant and malignant lesions could possibly improve the diagnostic value of oral exfoliative cytology. 2 Introduction The present study was undertaken to assess the mucosal changes in individuals who are using tobacco with betel leaf and those who are using tobacco without betel leaf with respect to the duration and also to assess the changes of cell diameter (CD) and nuclear diameter (ND) of exfoliated cells from the buccal mucosa/ mucosal lesions in those with the above mentioned habits. 3 Aims and Objectives AIMS AND OBJECTIVES 1. Identification and recording of various oral changes among the people who chew tobacco with betel leaf and tobacco without betel leaf. 2. Study of cellular changes of exfoliated cells collected from the buccal mucosa or lesion (if present) of subjects chewing tobacco with betel leaf and tobacco without betel leaf. 3. Morphometric study of exfoliated cells taken from the buccal mucosa or lesion (if present) of subjects chewing tobacco with betel leaf and tobacco without betel leaf. 4. Comparison of cellular and morphometric changes of exfoliated cells of subject chewing tobacco with and without betel leaf. 4 Review of Literature REVIEW OF LITERATURE TOBACCO: Tobacco is an agricultural product processed from the fresh leaves of plants in the genus Nicotiana. Tobacco has been growing on both American continents since about 6000 BC and began being used by native cultures at about 3000 BC. It has been smoked in one form or another since about 2000 BC. There are drawings of ancient Mayans smoking cigars from about 1400 BC5. A ceremonial gift of dried tobacco leaves given to Columbus by Native Americans in 1492 led to the introduction of tobacco into the rest of the world. It arrived in India in the 16th century; a sample was presented to the Emperor Akbar, who patronized smoking, rapidly spreading the habit in the sub-continent. An attempt to ban it in 1619 had little effect, as the revenues from tobacco were already considerable. Now tobacco is grown in many part of our country and is consumed by millions of people in all socioeconomic status. Tobacco is consumed in many forms. All methods of tobacco consumption result in varying quantities of nicotine being absorbed into the user's bloodstream. Over time, tolerance and dependence develop. Absorption quantity, frequency, and speed seem to have a direct relationship with how strong a dependence (or addiction) and tolerance, if any, might be created5. Many countries set a minimum smoking age, regulating the 5 Review of Literature purchase and use of tobacco products. According to the World Health Organization, tobacco smoke is the second biggest cause of death worldwide, just between hunger and malaria, having killed 100 million people in the 20th century, and predicted to kill one billion in the 21st century. "For each 1,000 tons of tobacco produced, about 1,000 people eventually will die. Lifelong smokers on average have a 50 percent chance of dying from tobacco-related illnesses, with half of these dying before the age of 70" TOBACCO RELATED HABITS: Tobacco is commercially available in dried, cured, and natural forms. The most common usage is smoking in the form of cigarette, Beedi or cigar. In addition, it can be smoked in a stem pipe, water pipe, or hookah. Tobacco can also be chewed, "dipped" (placed between the cheek and gum), or sniffed into the nose as finely powdered snuff. Tobacco is one of the important ingredients of Gutka which is available in ready-to-eat pouches. TOBACCO SMOKING: Cigarettes, Beedi and cigar A cigarette (French "small cigar", from cigar + -ette) is a product consumed through smoking and manufactured out of cured and finely cut tobacco leaves and reconstituted tobacco, often combined with other additives, then rolled or stuffed into a paper-wrapped 6 Review of Literature cylinder (generally less than 120 mm in length and 10 mm in diameter). A beedi (also spelled bidi or biri) is a thin, often flavored, South Asian cigarette made of tobacco wrapped in a tendu (or temburini; Diospyros melonoxylon) leaf, and secured with colored thread at one end. Tobacco content in beedies is 10-20% and, unlike regular cigarettes, beedies do not contain added chemicals like all tobacco products, use can cause various cancers. A cigarette is distinguished from a cigar by its smaller size, use of processed leaf, and white paper wrapping. Cigars are typically composed entirely of whole-leaf tobacco. Smokeless form There are two main types of smokeless tobacco: chewing tobacco and snuff. Chewing tobacco in the form of loose leaf, cut or shredded, is universally available. Snuff for applying, dipping or sucking could be moist or is commercially available as portion bag packed products. Finely powdered tobacco marketed as dry snuff is used nasally by few population groups. Worldwide several names are used to denote different ST products: plug, gutkha, khiwam, khaini, iq’milk, zarda, naswar, nass, chimo, toombak, shamma, gudhaku, gul, mishri, maras and moist snus.6 Tobacco is one of the important ingredients of Gutka which is available in ready-to-eat pouches. Smokeless tobacco is sometimes called "spit" or "spitting" tobacco because people spit out the tobacco juices and saliva that build up in the mouth5. 7 Review of Literature Snuff: Snuff is a generic term used for finely ground tobacco which is packaged as dry, moist, or in sachets (tea bag-like pouches). Typically, the user places a pinch or dip between the cheek and gum. Originally the term referred only to dry snuff, a fine tan dust popular mainly in the eighteenth century. This is often called "Scotch Snuff", a folk-etymology derivation of the scorching process used to dry the cured tobacco by the factory. Snuff powder originated in the UK town of Great Harwood and was famously ground in the town's monument prior to local distribution and transport further up north to Scotland. Snuff has been found to be beneficial in some cases of hay fever due to the fact that the snuff may prevent allergens from getting to the mucus membrane within the nose. Creamy snuff is a tobacco paste, consisting of tobacco, clove oil, glycerin, spearmint, menthol, and camphor, and sold in a toothpaste tube. It is marketed mainly to women in India, and is known by the brand names Ipco (made by Asha Industries), Denobac, Tona, Ganesh. It is locally known as "mishri" in some parts of Maharashtra. According to the U.S. NIH-sponsored 2002 Smokeless Tobacco Fact Sheet, it is marketed as a dentifrice. The same factsheet also mentions that it is "often used to clean teeth". The manufacturer recommends letting the paste linger in the mouth before rinsing5. 8 Review of Literature Chewing tobacco: Chewing is one of the oldest ways of consuming tobacco leaves. Native Americans in both North and South America chewed the leaves of the plant, frequently mixed with lime. Chewing tobacco is available in loose leaf, plug, or twist forms, with the user putting a wad of tobacco inside the cheek. Chewing betel quid is one of the commonest traditional form of tobacco use among Indian villagers. The BQ is a mixture of areca nut (Areca catechu), catechu (Acacia catechu) and slaked lime (calcium oxide and calcium hydroxide) wrapped in a betel leaf (Piper betle) (Figure1). Condiments, sweetening agents and spices may be added according to individual preferences. In India, most habitual chewers of betel quid add tobacco. In some countries, such as Papua New Guinea and China, tobacco is not added5. The lime acts to keep the active ingredient in its freebase or alkaline form, thus enabling it to enter the bloodstream via sublingual absorption. The areca nut contains the alkaloid arecoline, which promotes salivation (the saliva is stained red), and is itself a stimulant. For several thousand years Indian villagers have been using betel leaf as one of the major ingredient in quid. In India, the betel and areca play an important role in Indian culture, especially among Hindus. All the traditional ceremonies governing the lives of Hindus use betel and areca. The bitter poultice is a acquired taste, and although it is not clear why 9 Review of Literature the people of the pacific originally began to chew betel nut, the habit has been passed down through the generations and now provides a cultural link to their past7. The betel plant is attractive, aromatic slender creeper, with glossy heart shaped, smooth, shining, and long stalked leaves with pointed apex. Betel is bit sour in taste, but is a popular mouth freshener. It is stimulant, digestive and carminative, anti flatulent, anti inflammatory, invigorating, anti phlegmatic, pain reliever antiseptic and a breathfreshener Eating betel leaves with clove relieves cold & cough. It eliminates foul smell and is an antiseptic7. According to Hakeem Hashmi, outstanding Unani physician, betel leaves have been used from ancient times as an aromatic stimulant and anti flatulent. It is useful in preventing secretion or bleeding and is an aphrodisiac; betel leaf is used in several common house hold remedies. Recent Studies have shown that betel leaf extract (BLE) is antimutagenic against standard mutagen and tobacco specific N-nitrosamine (TSNA), N- nitrsamine nicotine (NNN) and 4-(methyle nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). It is also indicated that BLE has a promising anticarcinogenic role to play in tobacco induced cancer2. The Betel (piper betle) is a spice whose leaves have medicinal properties. The active ingredient of betel oil which is obtained from the leaves, are primarily a class of allylbenzene compounds. Though particular emphasis has been placed on chavibetol, it 10 Review of Literature also contains chavicol, estragole, euginol, methyl eoginol and hydroxycatecol. Several terpnes and terpenoids are present in the betel oil as well. There are two monoterpenes, pcymene and terpinene, and two mono terpenoids, eucalyptol and carvacrol. Additionally, here are two sesquiterpenes, cadinene and caryophylene7. Betel chewing is a part of many Asian and Pacific cultures and is often chewed at ceremonies and gatherings, and preparation techniques vary from region to region. The nut is slivered or grated, often flavoured with spices according to local tradition, and usually wrapped in a betel leaf, along with some lime (calcium oxide or calcium hydroxide) to better extract the alkaloids7. Some people also chew tobacco with betel nut. After about 20 minutes of chewing, the fibrous residue which remains of the nut is spat on the street, where it remains visible due to its characteristic bright red pigment. The commercial available betel quid chewing substitutes are pan masala and gutkha. Pan masala is basically a preparation of areca nut, catechu, cardamon, lime and a number of natural and artificial perfuming and flavouring materials. Gutkha is a variant of pan masala, in which in addition to these ingredients flavoured chewing tobacco is added. Both products are often sweetened to enhance the taste. 11 Review of Literature CHEMICAL CONSTITUENTS (CARCINOGENS) IN TOBACCO Smokeless tobacco (ST) products contain a large array of carcinogens. The main carcinogens in betel quid, pan masala and gutkha are derived from their ingredients areca nut, lime, catechu and tobacco. Although carcinogens present in betel quid, pan masala or gutkha have not been systemically analysed. Studies of the ingredients and their mixtures provide indications of the carcinogenic potential of these products. Benzo[a]pyrene and other polycyclic aromatic carcinogens (PAHs) are the most important carcinogenic agents in cigarette smoke but unburnt tobacco contain 28 carcinogens (cancer causing agents)of which the most harmful carcinogens are the tobacco specific nitrosamines (TSNA's). The metabolites of nitrosamines, such as carcinogenic TSNAs N'-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamino)-1-(3pyridyl)-1-butanone (NNK) and N-nitrosoanabasine (NAB), as well as the volatile nitrosamines N-nitrosodimethylamine and N-nitrosodiethylamine, have been detected in the saliva of chewers of tobacco (Wenke et al, 19848; Nair et al, 19859, 198710a; Bhide et al, 198611. Other cancer-causing substances include formaldehyde, acetaldehyde, crotonaldeyde, hydrazine, arsenic, nickel, cadmium, benzopyrene and polonium (which give off radiation).These agents are known to cause toxic effects particularly cancer, and 12 Review of Literature other cellular and DNA changes, either at the local placement sites or by indirect systemic effects. Another element found in smokeless tobacco is nicotine. Nicotine is absorbed by smokeless tobacco users at a rate 2 to 3 times higher than that of cigarette smokers, facilitating rapid addiction. Also, the nicotine stays in the bloodstream for a longer time. It has been reported that some chewing tobacco products actually contain microscopic abrasives which speed the absorption of nicotine, and carcinogens into the cell membranes. While not considered the primary cause of tobacco related malignancy, nicotine is responsible for the addiction of people to tobacco products, and the following long term use. Chewing of tobacco with BQ results in high exposure to carcinogenic tobacco-specific nitrosamines (TSNAs), to 1000 µg/day (Nair et al, 199912), compared with 20 µg/day in smokers (Hoffmann and Hecht, 198513), as well as leading to exposure to nitrosamines derived from areca nut alkaloids. In betel quid chewers, several carcinogens (Volatile nitrosamines and tobacco-specific nitrosamines) are derived from tobacco but also from areca nut (Hoffmann et al, 199414) can also be formed endogenously from abundant precursors during chewing. Secondary and tertiary amines present in areca nut 13 Review of Literature and tobacco can be nitrosated during BQ chewing when they react with available nitrite in the presence of catalysts such as thiocyanate (Nair et al, 1985, 1987a) 9, 10. SMOKELESS TOBACCO HABITS - GLOBAL INCIDENCE AND TRENDS: Chewing of BQ and areca nut is an ancient custom in several parts of south-east Asia, the south Pacific islands and Taiwan. This practice dates back several thousand years and is deeply entrenched in the culture of the population. BQ chewing was already a socially well accepted practice and the introduction of tobacco reinforced this practice. Betel leaf is perishable and preparation of BQ is somewhat complex or requires visits to shops selling pan/BQ. With the emergence of commercial pan masala and gutkha about three decades ago, not only did the Indian market witness massive growth in the sales of smokeless tobacco and areca nut products, but also a huge worldwide export market developed. The packaging revolution has made these products portable, cheap and convenient, with the added advantage of a long shelf-life. Tobacco products which were usually consumed by a small section of the population are today part of the modern urban and rural lifestyle. 14 Review of Literature The first major manufacturer of pan masala and gutkha presents their strategy as “to prepare convenient anytime”, anywhere substitute for pan give some respectability to a habit that was considered low in image by the genteel’. The product was put on the market in 1985 as 4 g sachets. Today sachets and bulk packages are produced and sold in India and exported to markets in the USA, Europe, the Middle East, Australia and many other countries. Pan masala is basically a preparation of areca nut, catechu, cardamon, lime and a number of natural and artificial perfuming and flavouring materials. Gutkha is a variant of pan masala, in which in addition to these ingredients flavoured chewing tobacco is added. Both products are often sweetened to enhance the taste15. Promoted by a slick, high profile advertising campaign and aggressive marketing, pan masala and gutkha have become very popular with all sections of Indian society, including school children. For most children, teenagers and women, cigarette smoking still remains taboo in India. It is consumed much like chewing tobacco, and like chewing tobacco it is considered responsible for oral cancer and other severe negative health effects. Some packaging does not mention tobacco as an ingredient, and some brands are pitched as candies - featuring packaging with children's faces and are brightly colored. Some are chocolate-flavored, and some are marketed as breath fresheners. These alternative tobacco products are often advertised as being safer than conventional cigarettes, leading to a much higher frequency 15 Review of Literature of use, so that these younger chewers constitute an alarming avant garde for a new epidemic of oral cancer. Further, these habits and preparations have spread to Europe and the USA, wherever there are Asian migrant communities15. Although the actual prevalence of this habit is unknown, its popularity can be gauged by commercial estimates valuing the Indian market for pan masala and gutkha at several hundred million US dollars (Gupta, 1999)16. These products are typically consumed throughout the day. A number of small surveys conducted in schools and colleges in several states of India have shown that 13–50% of students chew pan masala and gutkha on a regular basis (Gupta and Ray, 2003)17. A large proportion of migrant ethnic groups resident in the UK practice various chewing habits (Warnakulasuriya et al, 2002)18; population studies conducted among Asian ethnic groups in the UK suggest that chewing habits are prevalent in 14–15% of 11– 15 yr old children, with pan masala having the highest average frequency of use (Farrand et al, 2001)19. Areca nut chewing is an addictive habit (Chu, 2001)20 and evidence from the UK shows that the use of pan masala and gutkha is also addictive (Winstock, 2002) 21. An extensive survey was conducted by Gupta& Ray 22 : Epidemiology of Betel Quid Usage, covering studies In 1991, 13.3% of the interviewees in a survey aged 15 years and 16 Review of Literature above reported chewing betel quid (only 2.8% chewed daily) and the age distribution of chewing behaviour suggested that more young people chewed it at the time of the survey than in the past. Increasing betel quid usage among adolescents in Taiwan has been investigated in other studies More than half (53.6%) of the habitual chewers first tried it with a family member, most often the father or grandfather. In other school surveys in Taiwan, betel quid use was more common among boys than girls. It was also more common among students who smoked, consumed alcohol and had friends who chewed betel quid22. In Taiwan, a green unripe areca nut of the size of an olive is often used with betel inflorescence or betel leaves. The habit is more common among men than women (9.8% versus 1.6%) and starts in childhood (around 12 years). In the pacific island of Palau, a prevalence study conducted in 1995 on 2 states, 5 to 74 years, with an age distribution similar to that of the whole population, found that 72% of males and 80% of females chewed areca nut(betel quid), 80% of whom incorporated tobacco in their quid23. Betel quid chewing (BQC) is prevalent among elderly Cambodian women and is associated with a wide variety of oral mucosal lesions24. In a community-based study, 17 Review of Literature 32.6% of women and 0.8% of men over the age of 15 years chewed betel quid. Most of the women chewers were over the age of 39 and the men over the age of 5025. In Indonesia, betel quid is chewed first and then a large wad of finely cut tobacco is used to clean the teeth. It is then kept in the mouth for a while26. In the 1960s, a set of house-to-house surveys were conducted in India where in over 50,000 individuals of 15 years of age and above with roughly equal numbers of males and females in 5 disparate districts in 4 states (Andhra Pradesh, Bihar, Gujarat, Kerala) were included in the study. This study showed a range of betel quid usage prevalence of 3.3% to 37%27. In Ernakulam, Kerala, where the highest prevalence had been found, when it was studied again in the late 1970s and early 1980s, over 30% of both men and women aged 15 years and older chewed betel quid, almost always with tobacco28,29. A survey was conducted during 1992 to 1994 in Mumbai, India, among 99,598 residents 35 years and older, belonging to the middle and lower socioeconomic strata. Areca nut use was 29.7% among women and 37.8% among men, almost all with tobacco, while smokeless tobacco habits were reported by 57.1% women and 45.7% men30. In other population-based, rural and urban habit surveys reported from India, Nepal and Pakistan over the last 2½ decades, between 20% and 40% of the population 15 years of age and 18 Review of Literature above were betel quid or areca nut chewers. The surveyed areas were located in the Indian states of Karnataka and Maharashtra, as well as in Karachi, Pakistan and 3 region of Nepal31-35. In addition to men and women, betel quid chewing is also a major habit among youth. A community based survey among youth aged 5 to 20 years in a small fishing community in Kerala, betel quid-tobacco chewing was regularly practiced by 27.4% boys (14.3% occasionally) and 1.6% girls (11.3% occasionally)36. In a fishing community on Baba Island of Karachi, 74.2% of students in a probability sample of 160 primary school students were using areca nut products, mostly sweetened areca nut and betel quid37. A major change in betel quid/areca nut use occurred in India when an industrially manufactured mixture of areca nut, lime, a catechin-containing substance, sandalwood fragrance and tobacco was introduced to the market in small Aluminium foil sachets. This product was termed gutka. The same product without tobacco was termed pan masala. Most companies manufacture gutka as well as pan masala and market both products with the same brand name and packaging. A number of unpublished surveys conducted in schools and colleges in India have shown that pan masala and gutka are commonly chewed by children and youth, especially in Gujarat, 19 Review of Literature Maharashtra and Bihar. Street children have been commonly found to chew gutka all day long. In a survey of 1200 students from junior and degree colleges of Maharashtra, 9.9% took pan masala and 9.6% chewed gutka. A survey conducted in 1998 among 400 male medical students in Patna, Bihar, India (out of a total of 509) revealed that about 12.5% were regular users of gutka and 27.5% used pan masala occasionally, while 30.5% used other smokeless tobacco products not containing areca nut38. A telephone survey was used to study betel quid chewing behavior of Kaohsiung residents aged 15 and above in early 1991 by Chen JW & Shaw JH(1996)3913.3% reported that they chewed betel quids and most of them (145/154) were men. Among the chewers, 21.4% indicated that they chewed it daily; all were men. Age distribution of the chewing behavior suggested that more young people chewed it at the time of the survey than had been the case in the past. The demographic distributions of betel quid chewing behavior were similar to the characteristics of tobacco smoking in Taiwan. The degree of concurrence of these two behaviors was very high. Nearly 9O% of the subjects believed that betel quid chewing would cause more harm than good, or it would cause only harm, to their health. 20 Review of Literature Studies have been conducted by and Ying-Chin Ko et al (1992)40 and Yang MS et al (1996) 41to evaluate the Prevalence of betel quid chewing in Taiwan: In the junior high school 1.9 % of students including all grades (13-15 years old) and both sexes was found to be a current betel quid chewer and 14%, was an ex-chewer. Whereas 10.2% of vocational school students (16-18 years old) was a current chewer and 31% was an exchewer. The prevalence of betel chewing was significantly higher among boys than girls; a high proportion of chewers were also a smoker and alcohol drinker. Kow-T C et al (2001) 42 conducted a study on Tobacco, Betel Quid, Alcohol, and Illicit Drug Use Among 13- to 35-Year-Olds in I-Lan, Rural Taiwan: Prevalence and Risk Factors; The prevalence of betel quid chewing was 15.5% among males, compared with 1.3% among females, and was 1.3% among those aged 13 to 18 years compared with 8.1% among adults. The prevalence of betel quid chewing increased with age was higher among those with lower levels of education than among those with higher levels .Students were less likely and manual workers more likely to be betel quid chewers than were professionals. Among current betel quid chewers, the mean age for starting chewing was 20 years (range=8–35 years). Gupta PC & Ray CS (2003) 17 presented a paper on smokeless tobacco and health in India and south Asia: South Asia is a major producer and net exporter of tobacco. Over 21 Review of Literature one-third of tobacco consumed regionally is smokeless. Traditional forms like betel quid, tobacco with lime and tobacco tooth powder are commonly used and the use of new products is increasing, not only among men but also among children, teenagers, women of reproductive age, medical and dental students and in the South Asian diaspora. Smokeless tobacco users studied prospectively in India had age-adjusted relative risks for premature mortality of 1.2-1.96 (men) and 1.3 (women). Current male chewers of betel quid with tobacco in case-control studies in India had relative risks of oral cancer varying between 1.8-5.8 and relative risks for oesophageal cancer of 2.1-3.2. Oral submucous fibrosis is increasing due to the use of processed areca nut products, many containing tobacco. Pregnant women in India who used smokeless tobacco have a threefold increased risk of stillbirth and a two- to threefold increased risk of having a low birth weight infant17. A study was conducted by Kumar et al (2006) 43 on tobacco habit in northern India: in the survey chewing was prevalent in (74.5%), smoking in 59.3%, rubbing in (7.45%) and snuffing in (0.9%). women significantly preferred smokeless tobacco and perceived social barrier for smoking. Gutka consumption was significantly higher in youngsters(<25 yrs)most subjects used tobacco 7-24/day. Majority users started consuming tobacco before 21 years and about (22%) before 15 years. Majority users (53.6%) did not procure tobacco from a fixed shop. The commonest context of tobacco use was with any refreshment (78%) of the (74.5%) tobacco chewers, half rotated the 22 Review of Literature quid in their mouth, (97.2%) later pat it out, (2.1%) swallowed it and (4.7%) admitted to sleep with the quid in the mouth. ORAL LESIONS RELATED TO TOBACCO HABITS A recent workshop in Kuala Lumpur it was proposed that quid-related lesions should be categorized conceptually into two categories: first, those that are diffusely outlined and second, those localized at the site where a quid is regularly, placed. Additional or expanded criteria and guidelines were proposed to define, describe or identify lesions such as chewer’s mucosa, areca nut chewer’s lesion, of oral submucous fibrosis and other quid-related lesions. A new clinical entity, betel-quid lichenoid lesion, was also proposed to describe an oral lichen planus like lesion associated with the betel quid habit44. Jens J et al (1984) 45done a pilot survey of oral mucosa in betel nut chewers on Hianan islands of the People’s Republic of china: 100 people on Hianan islands were studied for their smoking and chewing habits and their condition of their oral mucosa. 95% of the study population chewed betel nut. In two men a small commissural leukoplakia was found in three women clinical and histologic changes pointed toward oral submucous fibrosis. 23 Review of Literature A study by Reichart et al (1987) 46 conducted in (1979-1984) on Precancerous and other oral mucosal lesions related to chewing, smoking and drinking habits in Thailand. The lesions of the oral mucosa (Leukoedema, preleukoplakia, leukoplakia and chewer's mucosa) were recorded. Chewing of betel and miang was more prevalent among older people; these habits seem to have lost their attraction for the younger people47. A positive correlation could be demonstrated between some mucosal lesions (Leukoedema, chewer's mucosa) and some smoking and chewing habits, Chewer's mucosa, the most frequent lesion among the examined individuals. A study conducted by Sow-Yeh Chen (1989) 48 on Effects of smokeless tobacco on the buccal mucosa of HMT rats: Fifteen male and 15 female HMT rats, 6 months of age, received weekly applications of smokeless tobacco to the buccal mucosa for one year. Hyper orthokeratosis, acanthosis, numerous binucleate spinous cells, and subepithelial connective tissue hyalinization were observed, whereas verrucous carcinoma and squamous cell carcinoma were not seen. Results indicate that the effects of smokeless tobacco on the rat buccal mucosa are similar to those observed in humans and that the mitotic process can be disturbed by tobacco treatment. Axell T et al (1990) 49 conducted a study on Prevalence of oral soft tissue lesions in outpatients at two Malaysian and Thai dental schools. In their study one case of a squamous cell carcinoma was found in a 45-yr-old Indian woman in Malaysia who had been 24 Review of Literature chewing betel with tobacco daily for many years. High prevalence figures were found for lichen planus, 3.8% in Thailand and 2.1% in Malaysia. In Thailand and Malaysia three cases each (1.3%) of tobacco associated leukoplakias were found. In Malaysia an additional idiopathic leukoplakia was registered. Another study by Saraswathi TR et al (1995) 50 on prevalence of oral lesions in relation to habits: cross-sectional study in south India stated oral submucous fibrosis was the most prevalent lesion among those who chewed panmasala or Gutka or betel quid with or without tobacco. In an article Reichart PA et al (1996) 51on Betel chewer's mucosa in elderly Cambodian Women (range 39 to 80 years) who chewed betel quid were examined for oral mucosal lesions, in particular betel chewer's mucosa. The average duration of betel quid chewing was 15.5±12.8 years. The average number of daily betel quids was 5.4±2.9; forty women (39.2%) used betel quids overnight. Thirty-eight (37.3%) did not show any oral mucosal lesion. Sixty-two (60.8%) showed betel chewer's mucosa. Homogeneous leukoplakia was found in three women (2.9%), Out of 130 sites affected by chewer's mucosa, the buccal mucosa was the most frequently involved (n = 68). In thirty-two subjects more than one location was affected. The presence of a lesion was significantly associated with the duration of the habit and the number of betel quids per day. 25 Review of Literature A study conducted by Zain RB et al (1997) 52 on a population based survey of oral mucosal lesions of a representative sample of the entire population of Malaysia. The age in the sample ranged from 25 to 115 years. The sample comprised 40.2% males and 59.85% females; 55.8% were Malays, 29.4% Chinese, lO.O% Indians and 1.2% other ethnic groups. Oral mucosal lesions were detected in (9.7%) subjects, (0.04%) had oral cancer, (1.4%) had lesions or conditions that may be precancerous (leukoplakia, erythroplakia, submucous fibrosis and lichen planus) and (1.6%) had betel chewer's mucosa. The prevalence of oral precancer was highest amongst Indians (4.0%) and other Bumiputras (the indigenous people of Sabah and Sarawak) (2.5%) while the lowest prevalence was amongst the Chinese (0.5%). In a study conducted by Tomar et al (1997) 53 to identify oral mucosal smokeless tobacco lesions among adolescents in the United States: Smokeless tobacco lesions (leukoplakia, erythroplakia, submucous fibrosis, lichen planus and betel chewer's mucosa)were detected in 1.5% of the students, including 2.9% of males and 0.1% of females. The lesions were more prevalent among whites (2.0%) than among AfricanAmericans (0.2%). Among white males, current snuff use was the strongest correlate of lesions, followed by current chewing tobacco use. Lesions were strongly associated with duration, monthly frequency, and daily minutes of use of snuff and chewing tobacco. This study showed very little evidence that the use of alcohol or cigarettes may increase the risk of smokeless tobacco lesions. 26 Review of Literature Mhhta et al (1997) 54 reported a prevalence of 0.12% of oral cancer and of 6.7% of leukoplakia among 101 761 villagers in Maharashtra, India. In Sweden, the prevalence of oral cancer in 20333 individuals was found to be low (<0.01%) while leukoplakia was found to be high (3.6"%) when compared with the current study. Reports from countries in the Pacific Asia region were found to be on smaller samples. A high leukoplakia prevalence of 2.5%i (compared with the current study) was found in a mass screening of 3131 Japanese. In Cambodia, the prevalence of oral cancer was found to be 0.1% and of leukoplakia 1.1% in 1319 individuals. An investigation by Shah N & Sharma PP (1998) 55 on role of chewing and smoking habits in the etiology of oral submucous fibrosis (OSF): a case-control study. It was found that chewing of areca nut/quid or pan masala (a commercial preparation of areca nuts, lime, catechu and undisclosed colouring, quid; flavouring and sweetening agents) was directly related to OSF. Also, pan masala was chewed by a comparatively younger age group and was associated with OSF changes earlier than areca nut/quid chewing. However, chewing or smoking tobacco with various other chewing habits did not increase the risk of developing OSF. It was also found that frequency of chewing rather than the total duration of the habit was directly correlated to OSF. 27 Review of Literature Johnson et al (1998) 56 conducted a study on development of smokeless tobacco- induced oral mucosal lesions: This study examined clinical and inflammatory mediator parameters during the development of snuff induced mucosal lesions. Nineteen smokeless tobacco users placed moist snuff at designated new placement sites over either 2-or 7-day period. By day 2, the predominant clinical alteration was an erythematous reaction, and one third of the subjects demonstrated white striation in combination with erythema or ulceration. By 7 days, 56% of the subjects displayed white striated lesions. Reichart PA & Philipsen HP (1998) 57 presented a paper on Betel chewer’s mucosa – a review: Betel chewer’s mucosa (BCM) was first described and defined in 1971. Its clinical appearance is characterised by a brownish-red discolouration of the oral mucosa with an irregular epithelial surface that has a tendency to desquamate or peel off. The buccal mucosa is most frequently affected. The prevalence of BCM varies between 0.2% and 60% in different studies from South and Southeast Asia. Women are more frequently affected than men. Betel chewer’s mucosa may be found together with other oral mucosal lesions such as Leukoedema, leukoplakia and ulceration. The histological features are characteristic. The epithelium is often hyperplastic, and brownish amorphous material derived from the betel quid may be demonstrated not only on the epithelial surface but also intra- and inter-cellularly. Ballooning of epithelial cells may occur. The etiology is traumatic and possibly chemical. Betel chewer’s mucosa is most likely not precancerous. 28 Review of Literature Betel chewer’s mucosa was originally defined as: ‘‘a condition of the oral mucosa where, because of either direct action of the quid or due to traumatic effect of chewing, or both, there is a tendency of desquamation or peeling off of the oral epithelium. Loose and detached tags of the tissue can also be seen and felt. The underlying area assumes a pseudo membranous or wrinkled appearance. The area may also show evidence of incorporation of the quid in the form of yellowish encrustations. It has been noticed that this condition can be scraped off.’’ The most conspicuous clinical finding is a brownishred discolouration of the affected oral mucosa, predominantly the buccal mucosa. The coloured material stems from the betel quid and is composed of calcium hydroxide and polyphenols, which on the teeth becomes black due to polymerisation. The discoloration of the oral mucosa may vary considerably depending on the number of betel quids chewed per day and the composition of the chew .The brownish material is not easily removed or scraped off. The mucosal surface is irregular, rough, macerated and some epithelial tags may be seen. Areas of desquamation often show a reddish background due to partial or total loss of epithelium and to hyperemia. Mucosa affected by BCM is usually diffusely outlined. Depending on the position of the quid being chewed, the BCM may be predominantly uni- or bi-laterally located. In some cases the oral mucosa may have a dried-out appearance. How long the discolouration of the oral mucosa lasts after cessation of the chewing habit is presently unknown. 29 Review of Literature In a study of Northern Thai hill tribes and indigenous Thai, an overall BCM prevalence rate of 13.1% was found. In another study from Thailand of miang (fermented tea leaves) and betel quid chewing, BCM was also recorded (7%). Betel chewer’s mucosa was also found in a study on oral mucosal lesions among adults in Malaysia at a prevalence rate of 1.6% among 11,697 subjects aged 25 years and above. Ikeda et al (1995)50 reported the prevalence of BCM in a selected Cambodian population to be 0.2%. In another selected study on BCM in elderly Cambodian women, BCM was found in 60.8% of the subjects. Philipsen et al. (unpublished) studied BCM in Sri Lankan tea estate workers and found a prevalence rate of 56.1% among chewers; the lesion was more frequent among women (60.6%) compared to men (45.5%). The enormous differences (0.2–60%) in prevalence rates of BCM in different studies may be due to selection procedures (highly selected versus unselected populations), sampling methods, age profiles, different interpretation of the definition of BCM, and insufficient calibration of examiners. It has been shown in several studies that BCM occurs more frequently in women than in men despite the fact that in one unpublished study men were the heavier chewers. Betel quid chewing has long been shown to be a habit of the elderly and this has been confirmed for the occurrence of BCM. Among 102 rural Cambodian women between the 30 Review of Literature ages of 39 and 80 years, BCM was observed more frequently in those over 60 years of age. Duration of the betel quid habit also seems to be a major factor. Since the betel quid is most often placed in the lower buccal sulci, the most frequent location for BCM is the buccal mucosa. Nearly 60% of BCM in an unpublished study from Sri Lanka were located to the cheek mucosa, with the edentulous alveolar process, tongue (mainly margins) and labial mucosa in descending order of frequency. A similar distribution of locations was found by Reichart et al(1998)57 with the buccal mucosa, the lower buccal sulcus, the lateral border of the tongue, the hard palate and the upper lip being the most common sites of BCM in elderly Cambodian women. Betel chewer’s mucosa is often found together with other mucosal lesions such as leukoedema, oral leukoplakia or pre-leukoplakia and oral ulcerations. Reichart PA et al(1999)58 conducted a study on Betel quid-associated oral lesions and oral Candida species in a female Cambodian Cohort: They have investigated lesions of the oral mucosa likely to be associated with BQC habit in individuals with average habit duration of 10 years (range 10 months+/-30 years). They have observed oral lesions such as betel chewer’s mucosa (85.4%), oral leukoplakia (8.3%), leukoedema (37.5%) and oral lichen planus (4.2%). Oral candidiasis was not seen in BQ-chewers58. 31 Review of Literature A study conducted by Ling et al (2001)59 to know the Association between betel quid chewing, periodontal status and periodontal pathogens. The periodontal status of 34 betel quid chewers and 32 non-betel quid chewers were compared. A significantly higher prevalence of bleeding on probing was found in betel quid chewers than non-chewers among the subjects with higher plaque level, greater gingival inflammation, deeper probing depth or greater attachment loss. Also, the results suggested that betel quid chewers may harbor higher levels of infection with A. actinomycetemcomitans and P. gingivalis than non-betel quid chewers. Sally JL et al (2002) 60 conducted a study on Smokeless Tobacco Habits and Oral Mucosal Lesions in Dental Patients (1992). During the study they collected data on tobacco use habits and recorded oral health status in 245 male Smokeless Tobacco users aged 15 to 77. Results showed that 78.6 percent of Smokeless Tobacco users had observable oral lesions, 23.6 percent of which were in the most clinically advanced category. Of the lesions noted, 85 percent were in the same location the patient identified as his primary area of smokeless tobacco placement. In a comparison sample of 223 nonusers of Smokeless Tobacco with the same age distribution, only 6.3 percent had observable lesions. An article by Avon (2004)61 on Oral Mucosal Lesions Associated with Use of Quid stated that quid induced lichenoid oral lesion has been reported exclusively among users 32 Review of Literature of betel quid. It resembles oral lichen planus, but there are certain specific differences. The quid-induced lesion is characterized by the presence of fine, white, wavy, parallel lines that do not overlap or criss cross, are not elevated and in some instances radiate from a central erythematous area. The lesion generally occurs at the site of placement of the quid. This lesion was originally described as a lichen-planus-like lesion, but it is now termed a betel-quid lichenoid lesion. This lesion may regress with decrease in the frequency or duration of quid use or a change in the site of placement of the quid. There may be complete regression if the quid habit is given up. A study conducted by Ching-HC et al (2005)62 on Oral precancerous disorders associated with areca quid chewing, smoking, and alcohol drinking in southern Taiwan. 136 precancerous lesions and conditions were detected among 1075 subjects (12.7%). The analysis of the spectrum of oral precancerous disorders detected, leukoplakia (n = 80), OSF (n = 17) and verrucous lesions (n =9), demonstrated. The synergistic effect of smoking and areca quid chewing habit on leukoplakia and OSF was demonstrated. A study conducted by Yang et al (2005)63 to identify the incidence rates of oral cancer and oral pre-cancerous lesions in a Taiwanese aboriginal community, discovered cancer and pre-cancerous lesions in areca/betel quid chewers. The age-standardized incidence rates for quid lesion, oral submucous fibrosis (OSF) and squamous cell carcinoma (SCC) 33 Review of Literature were 267.0, 374.1 and 146.2 per 100 000 person-years, respectively, for areca/betel quid chewers. A pilot study is conducted by Anwar et al (2005) 64 aimed at investigating attitudes and practices concerning gutka use in a town in India(Chitrakoot),124 consecutively attending subjects (103 males and 21 females) at a charity-run dental clinic in Chitrakoot, India, were interviewed using a semi-structured questionnaire. Main findings: 57 (46%) of the respondents, of whom there were more males than females (103 v. 21), reported current gutka use. Of the 57 gutka chewers, 36 also chewed paan with tobacco and 20 smoked cigarettes. They concluded that Gutka chewing involved almost half of the study population and proportionately more males than females. The habit was endemic within families. Many gutka chewers also smoked chewed paan or drank alcohol, representing a high-risk behaviour for oral cancer. Proportionately more non-chewers were aware of the link with cancer. There are clear implications for health promotion in India. Another study by Ahmad (2006) 65 on an etiological and epidemiological study of oral submucous fibrosis (OSMF) has been done in Patna, Bihar. Total 157 cases of OSMF and 135 control subjects were selected for study in the period of 2002-2004. It was observed that male: female ratio was 2.7: 1. The youngest case of OSMF was 11 year old and the oldest one was 54 years of age. Maximum numbers of cases were belonging to 21-40 years of age and they were belonging to low or middle socioeconomic class. Most of the 34 Review of Literature OSMF cases used heavy spices and chillies, whereas control mild spices and chillies. Gutkha was the most commonly used by the OSMF cases only 3 per cent did not use any gutkha or other areca nut product where as 80 per cent control did not have any chewing habit. The OSMF cases used gutkha and other products 2-10 pouches per day and kept in the mouth for 2-10 minutes and they were using since 2-4 years. Most of the OSMF cases kept gutkha in the buccal vestibule or they chewed and swallowed it, only a small number of patients chewed and spitted it out. It was also observed that OSMF developed on one side of the buccal vestibule where they kept the chew and other side was normal. A study was done by Ariyawardana et al (2006) 66 on Effect of betel chewing, tobacco smoking and alcohol consumption on oral submucous fibrosis: a case–control study in Sri Lanka Betel chewing was the only significantly associated factor in the aetiology of OSMF (OR = 171.83, 95% CI: 36.35–812.25). There were no interaction effects of chewing, smoking and alcohol consumption in the causation of OSMF. Warnakulasuriya & Ralhan (2007)67 published a paper on Clinical, pathological, cellular and molecular lesions caused by oral smokeless tobacco – a review: Epidemiological evidence for a significantly increased risk of oral cancer in ST users was reviewed. The main categories of snuff or chewing tobacco induced oral mucosal soft tissue lesions reported are Oral squamous cell carcinoma, Verrucuos carcinoma, 35 Review of Literature Leukoplakia, Erythroplakia, Snuff Dippers’ lesion/snuff-induced lesion, Tobacco and lime users’ lesion. Sundstrom et al (2007) 68 described the clinical features of 23 oral cancers in snuff dipping Swedish males (age range 39–52 years). Hirsch et al69 reported eight oral cancer cases in Swedish snuff-dippers. Seven of this series were elderly male and had used snuff for longer than 20 years. Zatterstrom et al70 described a further case of well differentiated oral carcinoma in a 90-year-old Swedish man who had consumed snuff. Schildt et Al71reported 410 oral cancer cases in a case–control study in Sweden. There were 106 active users of oral snuff and 28 were ex-users. Among their cases 32.6% people had some lifetime exposure to oral snuff. Most common tumor site was the lip. An increased risk was found for lip cancer among ex-snuff users. From the United States McGuirt and Wray72 described the clinical profile of 116 patients with oral cavity cancer who were exclusive users of Smokeless Tobacco with no exposure to smoked tobacco or alcohol. The average age of the case-series was 78.4 years and average period of consumption was 55.5 years. Females were predominant (1:23 male to female ratio). A second primary tumor developed in the oral cavity of 18% (21 of 116) suggesting field cancerization. Forty-five of 91 who were followed up died of or with cancer. 36 Review of Literature McGuirt73 earlier described a series of 76 patients with oral cancers who were all Smokeless Tobacco users. In this series 57 patients reported exclusive snuff use. Females were again predominant (1:3). Common lesion sites were alveolar ridge (32%) and buccal cavity (47%). Winn et al74 in 30 snuff users, who were nonsmokers, with cancer of the gum or buccal mucosa demonstrated a significant trend for the reported period of snuff use and their cancers. The risk increased with increasing length of exposure, with risk greatest for anatomic sites where the product was held in contact with oral mucosa. Toombak dipping – a form of snuff used in Sudan – is implicated as a toxic product causing oral cancer. According to International Agency for Research on Cancer (IARC) Betel-quid and arecanut chewing (Volume 37 and Supplement 7), the evidence for the carcinogenicity to humans of betel quid with tobacco was evaluated as sufficient; the evidence for betel quid without tobacco was evaluated as inadequate. Many more studies now provide evidence for the carcinogenicity of betel quid without tobacco for oral cancer and for betel quid with tobacco for cancers of the oral cavity, pharynx and oesophagus75. Chewers of betel quid in India and the Philippines had elevated frequencies of micronucleated cells in their buccal mucosa. The proportion of micronucleated exfoliated cells is related to the site within the oral cavity where the betel quid is kept habitually and 37 Review of Literature to the number of betel quids chewed per day. The proportion can be reduced by administration for two to three months of vitamin A or β-carotene or a mixture of the two. The risk for chewers of betel quid without tobacco was statistically significant in one study each from India, Pakistan and Taiwan, China, after stratifying for betel-quid chewing with tobacco, tobacco smoking and alcohol use75. The risk for cancer of the esophagus was significantly increased among chewers of betel quid in five case–control studies, four from India and one from Taiwan, China. This evidence comes from studies investigating populations that chew betel quid with and without tobacco. Significantly increased risks persisted in two studies that provided results stratified for smoking and alcohol intake75. A few case–control studies were reported for cancers of the larynx, stomach, lung and cervix. The results for cancers of the stomach and cervix were suggestive of an association with chewing betel quid. According to International Agency for Research on Cancer (IARC) in case–control, cross-sectional and cohort studies, chewing betel quid was strongly associated with leukoplakia. In several studies, oral submucous fibrosis was reported to occur among chewers of areca nut only, chewers of betel quid without tobacco and chewers of betel quid with tobacco, and the observed relative risk was usually extremely high. Oral 38 Review of Literature leukoplakia shows a strong association with habits of betel-quid chewing in India. Follow-up studies showed high risks for malignant transformation of leukoplakia and oral submucous fibrosis75. Several large-scale population-based epidemiological studies have been conducted in other countries. Among these are studies from India where there was a comparatively high prevalence of oral precancer. Smith et al76 reported a prevalence of oral cancer of 0.05'%) and of leukoplakia of 11.68% among 57 518 industrial workers in Gujarat, India. A study conducted by Lars Salonen et al (2004) 77 The relationship between tobacco habits and mucosal lesions was analyzed and the time needed for treatment of the lesions was estimated. A positive correlation could be demonstrated between tobacco use and leukoplakia, frictional white lesion, coated tongue, hairy tongue and excessive melanin pigmentation. Robertson et al (2007)78 in the US had noted that 46% of current users of ST (all professional baseball players) had oral mucosal lesions located primarily at sites where ST quid was placed. Using Greer and Poulson criteria for the detection of ST lesions among 245 male patients aged 15–77 years attending a dental practice in Oregan. Little et al. recorded a high prevalence of mucosal lesions (78.6%), a quarter of which were in the most clinically advanced category (grade 3). 39 Review of Literature Kaugars et al79 investigated oral lesions that persisted for at least 7 days after discontinuation of ST use. Among white males in this group (mean age 29.3 years) 45 of 347 (13%) had mucosal alterations consistent with ST use. An oral lesion in tobacco and lime users in Maharastra, India was described by Bhonsle et al (2007)80 this mucosal lesion coincided with the placement of the quid and could be scrapped off leaving a raw surface. Tobacco and lime mixture also called Khaini is usually retained in the anterior part of the mouth rather than chewed. Among Nepalese the habit is associated with white and red patches with rippled/fissured surface characteristics. Nass made with local tobacco (partly cured), ash and lime used in Central Asian Republics of the former Soviet Republic and parts of Pakistan is significantly associated with the risk of oral leukoplakia. In 118 current nass users in Uzbekistan, the associated risk for oral leukoplakia (corrected for smoking and alcohol) was 3.9 (CI 2.6– 5.7). 40 Review of Literature CYTOLOGY AND IMAGE ANALYSIS : Ogden et al (1997) 81published a paper on Oral exfoliative cylology review of methods of assessment: they have opined that the use of oral exfoliative cylology in clinical practice is declined due to the subjective nature of its interpretation and because there may be only a small number of abnormal cells identifiable in a smear. The more recent application of quantitative techniques, together with advances in immunocytochemistry have refined the potential role of cytology, stimulating a reappraisal of its value in the diagnosis of oral cancer. In 1957 REAGAN et al82. Undertook a more extensive study on exfoliated cells. They examined 20.000 malignant cells and 11.000 normal cells and observed a significant difference between normal and malignant cells. In 1963, Goldsby & Staats83 suggested that NA. CA and N:C ratios were important factors to consider in assessing normal exfoliated cells from the oral cavity. Since then, few researchers have quantitatively assessed oral mucosal smears. In 1985, Cowpe et al84. Published a quantitative assessment of exfoliated cells collected from the oral Cavity using a planimetry method, based on the calculation of NA and CA values, of cells. They found that 50 cells were sufficient to provide a consistent assessment of a variety of oral mucosal sites. It was proposed that such values might 41 Review of Literature define a baseline for comparison with pathological smears. Literatures also reveal a similar study which was done by JOHNSTON85 in 1952. Image analysis was concluded to be a more appropriate method for evaluating oral smears, even though the technique can be time consuming. Moves towards the use of more fully automated image analysis systems in assessing oral smears would be expected to improve speed of measurement and. hopefully, its accuracy by increasing the number of cells measured in each smear. Efforts so far have been hampered by difficulties in avoiding the assessment of extraneous and fragmented cellular debris and thus the need for careful smear preparation. Modern image analysis encompasses morphometry, densitometry, neural networks and expert systems. Neural networks have been applied with some success to the assessment of smears taken from normal and dysplastic oral mucosa. Three hundred and forty-eight smears were collected from clinically normal and abnormal buccal mucosa and their NA and CA areas measured. A neural network differentiated between normal non-dysplastic mucosa and dysplastic/malignant mucosa. Ramaesh et al (1998)86 conducted a study on Cytomorphometric analysis of squames obtained from normal oral mucosa and lesions of oral leukoplakia and squamous cell carcinoma. Cell and nuclear diameters (CD and ND) were measured in squames obtained from normal buccal mucosa and lesions of oral leukoplakia and squamous carcinoma (SCC). They have reported a positive correlation between the ND and CD for normal 42 Review of Literature buccal mucosa and lesions with no epithelial dysplasia and insignificant correlations in lesions with epithelial dysplasia and SCC lesions. From the results obtained they have suggested that that ND and CD could possibly be sensitive parameters in the diagnosis of oral premalignant and malignant lesions. In a study by Ramaesh et al (1999)87 on the effect of tobacco smoking and of betel chewing with tobacco on the buccal mucosa, the effect of tobacco use on the buccal mucosa has been assessed by cytomorphometry. Cell and nuclear diameters (CD, ND) of exfoliated oral squames were measured in tobacco smokers (S), betel chewers with tobacco (C) and those with a combined habit (S+C). Non-users (NU) served as controls. The results showed a significant reduction for CD in tobacco chewers and those with combined habits and an increase for ND in all three habit groups, compared to the controls. This study shows that the use of tobacco influences the cytomorphology of the normal buccal mucosa. According to these authors, Betel chewing with tobacco influences the ND and CD, while smoking influences only the ND. Shabana AHM et al (1987)88 conducted a study in 100 specimen for evaluating the size and shape of the cells in the basal cell layer using interactive image analysis system(IBAS1). They took four groups of white lesions they are traumatic keratosis, lichen planus, leukoplakia, risk group and two control group are normal mucosa and squamous cell carcinoma. They observed area, perimeter and maximum diameter of the nuclei from 43 Review of Literature lesional group and control group. They interpreted that nucleus in squamous cell carcinoma was twice as large as in normal mucosa. A substantial increase in the dimensions of cell and the nucleus was found in the risk group lesions. They concluded that the size of the basal cells and its nucleus could be of diagnostic value for lesions with a high risk of malignant transformation. Maha AS et al (1988) 89 conducted study on the value of image analysis in predicting the malignant potential of oral epithelial lesions showing either hyperplasia or dysplasia. . For all these cases they measured the nucleus features (Shape-Area, Perimeter and Form factor) stain (Average, total and adjusted stain) were assessed. They analyzed mean, standard deviation and interquartile range and used for linear stepwise discriminate systems with the above values. They concluded that the malignant potential of the lesions that later transformed could be predicted with 87.5% accuracy. Study was conducted by Cooper R. John, Hellquist B. Henrik and Michaels Leslie (1991)90to investigate the size of the intermediate cells in verrucous carcinoma and squamous papilloma and squamous cell carcinoma by means of image analysis. The results showed that the area of cells in the malphighian layer in verrucous carcinoma was larger than that in squamous papilloma. The lower nuclear/cytoplasmic ratio in verrucous carcinoma was contrary to the expectation of an increased ratio in malignant cells. Squamous cell carcinoma 44 Review of Literature had a greater variation in cell size than did verrucous carcinomas indicating that morphometry of the cell size is not a useful diagnostic tool for squamous cell carcinoma. Masahide Ikeguchi et al (1999)91 had suggested that nuclear profiles such as size of the nuclear area or nuclear shape as useful indicators of prognosis in various cancers. Using computerized morphometry, the authors evaluated the usefulness of nuclear morphometric analysis as a prognostic indicator in gastric cancer.Tthey concluded in their study that nuclear morphometry should be introduced as a useful morphologic prognostic marker for patients with advanced gastric cancer as nuclear area was closely correlated to malignant potential of gastric cancer. A study by Mollaoglu et al (2000)92 on Cytomorphologic Analysis of Papanicolaou Stained Smears Collected from Floor of the Mouth Mucosa in Patients with or without Oral Malignancy. Cytology has been found to be a reliable and accurate technique when used to diagnose early malignant transformation in areas of the body where visual examination is difficult or impossible. Thus, automated instruments, capable of objective and quantitative cell analysis, have been used in descriptive morphology, for the assessment of tumour cell heterogeneity. Smears were collected from a group of 33 patients presenting with squamous cell carcinoma. Smears collected from the tumours displayed a statistically significant elevation in mean NA (p<0.01) and reduction in mean CA (p<0.001) when compared with normal smears from patients with no oral lesions. In 45 Review of Literature conclusion, cytomorphologic assessment of the Papanicolaou stained oral smears collected from suspicious lesions was found to be a significant diagnostic factor in relation to malignancy. Alberti Sandra et al (2003)93 had conduced cytomorphometric study on exfoliative cytology smears of the oral mucosa in type II diabetic patients. The nuclear (NA) and cytoplasmic (CA) areas were evaluated from 50 integral cells predominant in each oral site by the use of the KS 300™ image analysis system (Carl Zeiss, Germany), by which the cytoplasmic/nuclear ratio (C/N) was calculated. Morphometric study on cytological smears of normal oral mucosa using high-resolution image analyser was done by Andreas Neher et al (2004)94 as possible approach for the early detection of laryngopharyngeal cancers. 3000 cell nuclei were automatically measured using a high-resolution image analyser (CytoSavant Oncometrics, Vancouver, BC,Canada). The classifier reached an overall performance of 72.7%sensitivity, 82.4% specificity. Anuradha and Shivapatahasundaram (2007)95 had done an image analysis of normal exfoliated gingival cells, to evaluate the nuclear diameter (ND), cell diameter (CD) and nuclear-cytoplasmic ratio (N:C) and their variation with age and sex in normal gingival smears. The cell and nuclear diameters were measured using image analysis software (KS lite 2.0). 46 Results RESULTS The present study was carried out in the Department of Oral and Maxillofacial Pathology, Yenepoya Dental College Hospital, Mangalore. Individuals participated in this study were divided into 3 groups of 30 patients each. The group1 comprised of traditional tobacco users, the group 2 those individuals who were using commercial tobacco products and the group 3 normal healthy controls who had no Tobacco related habits. Among the traditional tobacco users 14 were males and 16 were females. They belong to the age group ranging from 32 to 67 years with an average age of 44.47 years. It showed almost equal predilection among both the genders. (Table 1) In this study most of the Commercial tobacco products users were males (29/30). We have also observed that the habit of use of commercial tobacco products was more prevalent among younger age group ranging from 18 to 42 years with average age of 22.4 years when compared to traditional tobacco users (44.47 years). (Table 1, Graph 1 & 2) Out of 30 individual examined with habits of traditional tobacco use 11 individuals presented with mucosal changes while 19 were without any lesions. Lesions identified 58 Results were lichenoid reaction (4), chemical burn (3), chewer’s mucosa (2), and oral submucous fibrosis (2). (Table 2, Graph 3) Of the 30 commercial tobacco products users, 23 showed some form of mucosal changes related to habits. Only 7 people were free of any lesions. The lesions were different from those of traditional betel quid users and lesions were chewer’s mucosa (8), lichenoid lesion (3), leukoplakia (3), oral submucous fibrosis (9), and tobacco pouch keratosis (1). The distribution of different lesions by tobacco without betel leaf is shown in (Table 2, graph 4) In our study we have analyzed the type of tobacco used, the frequency and the duration of the habit and the related mucosal lesions. A positive correlation between duration and frequency in traditional tobacco users were observed. When these observations were analyzed using Karl pearson correlation (r = .789, P=<.0005) the results were found to be statistically significant. (i.e.) As the duration increases the frequency of traditional tobacco use also increases. But there was a weak correlation between duration and frequency of use of commercial tobacco (r=.161, P= .396) which was statistically not significant. (Table 3, 4 and Graph 5, 6) Although the frequency of use of traditional tobacco was increasing over a period of years, we have observed relatively less mucosal changes among these groups, when compared to that observed in commercial tobacco users. The frequency, duration and 59 Results associated lesions in 30 individuals who consumed tobacco with betel leafs are tabulated in Graph 7 Mean comparison between the use of betel quid & Commercial packets per day & duration of use was made. It showed that the mean use of betel quid/day was 5.53 Nos. for the duration of 13.50 years. 5.87 No. of Commercial packets per day were used in a mean duration of 4.5 years. (Table 6, Graph 8) Smears taken from the study group and control group were stained using PAP technique and the cellular changes of exfoliated cells were studied. Most of the exfoliated cells from the buccal mucosa of individuals with use of traditional and commercial tobacco were superficial cells; however some intermediate cells were also evident. On gross examination these cells did not show significant morphological variation from those of controls. To measure the CD and ND of the exfoliated cells, image analysis soft ware was used. Statistical analysis was done using SPSS Version-15 statistical software and excel. The ND and CD were analyzed for three groups by using statistical test 1-way ANOVA & Turkey multiple comparison tests. 60 Results The nuclear diameter of exfoliated cells in traditional tobacco users ranged from 4.26 to 5.91 µm with a mean of 4.99 µm. The same cells showed cell diameter which ranged between 22.87 to 37.89 µm with an average mean of 30.27 µm. (Table 6, Graph 9) In commercial tobacco product users, the exfoliated cells had nuclear diameter ranging from 4.84 to 8.46 µm with a mean of 5.60 µm and the cell diameter which ranged between 21.25 to 34.94 µm with a mean of 27.88 µm (Table 7,8,Graph 9) Mean CD and ND in both group 1 & 2 were more compared to individuals without tobacco habits (31.9 and 4.81 µm respectively) (Table 9, 10, Graph 10) When the nuclear diameter was compared between the three groups a statistically significant difference was observed between group 1&2 and 2&3 with the P value of 0.054 and 0.008 respectively. But when the same was compared between 1&3, it showed a P value of 0.765 which was not significant. Similarly When the cell diameter was compared between the three groups a statistically difference was observed between group 1&2, 2&3 with the P value of 0.037 and < 0.0005 respectively. But when the same was compared between 1&3, it showed a P value of 0.208which was not significant. 61 Results The ratio between CD & ND in exfoliated cells from group 1 individuals was -1: 6, while that of group 2 was 1:4.5. This was relatively high compared to those of control group which had the ratio of 1:7. 62 Results Table 1: Age and sex distribution of the traditional and commercial tobacco users Group Traditional commercial Age groups Number 31-35 3 36-40 3 41-45 4 46-50 5 51- 55 4 56-60 6 61-above 5 15-20 3 21-25 12 26-30 8 31-35 3 36- above 4 63 Male Female Total 29 1 30 14 16 30 Results Table 2: Comparison between type of tobacco users & development of lesion Tobacco users * LESION Crosstabulation Tobacco users commercial Traditional Total Count % within Tobacco users % within LESION % of Total Count % within Tobacco users % within LESION % of Total Count % within Tobacco users % within LESION % of Total Chi-square=9.774 LESION present absent 23 7 76.7% 23.3% 67.6% 26.9% 38.3% 11.7% 11 19 36.7% 63.3% 32.4% 73.1% 18.3% 31.7% 34 26 56.7% 43.3% 100.0% 100.0% 56.7% 43.3% Total 30 100.0% 50.0% 50.0% 30 100.0% 50.0% 50.0% 60 100.0% 100.0% 100.0% P-value=.004 Table 3: Scatter diagram showing correlation of frequency and duration of traditional tobacco use Correlations No. of traditional tobacco/day Duration/years Pearson Correlation .630** P-value <.0005 N 30 ** Correlation is significant at the 0.01 level Inference: There is significant positive correlation between frequency & duration of tobacco use. 64 Results Table 4: Scatter diagram showing correlation of frequency and duration of commercial tobacco use No. of commercial packets per day duration of use Pearson Correlation .161 P-value .396 N 30 There is a weak correlation between duration of use and frequency of use of commercial tobacco and it is not statistically significant (r=.161, p=.396) Table 5: Correlations of traditional tobacco per day and duration of use No. of Traditional tobacco per day duration of use Pearson Correlation .789(**) P-value <.0005 N 30 Correlation is significant at the 0.01 level (2-tailed) There is high correlation between duration and frequency. As the duration of use of traditional tobacco is increasing the frequency of use of traditional tobacco is also increasing (r = .789, P=<.0005) and is statistically significant. 65 Results Table 6: Mean comparison of number of commercial&traditional tobacco per day and duration (years) Mean Std. Deviation No. of Traditional tobacco/ day 5.53 3.758 Traditional tobacco duration(years)of use 13.50 8.254 No. of commercial packets /day 5.87 3.401 commercial packets duration of use 4.50 2.209 Total commercial packets used 10049.67 9385.214 Table 7: Nuclear diameter between three groups Nuclear Diameter N Mean Std. Deviation Minimum Maximum Commercial 30 5.6049613 1.28274823 3.82274 8.46274 Traditional 30 4.9971127 .85804950 2.95686 6.46112 Control 30 4.8174927 .78353676 3.59326 6.45218 Total 90 5.1398556 1.04446931 2.95686 8.46274 F=5.117 P=0.008 One way ANOVA shows ND is significantly more in traditional and commercial (P- Value =.008) 66 Results Table 8: Post Hoc Tests Multiple Comparisons (Turkey HSD) (I) Group (J) Group Mean Difference (I-J) P-value Commercial Traditional .60784867 .054 Control .78746867(*) .008 Control .17962000 .766 Traditional * The mean difference is significant at the .05 level. Turkey multiple test shows commercial and betel quid are not significant (P- value=.054). Commercial group is significantly different from control group (P = .008) Betel quid is not significantly different from control 2Commercial vs 1traditional P= .054 (not significant) 2Commercial vs 3control P= .008 (higly significant) 1Traditional vs 3control P= .765 (not significant 67 Results Table 9: Cell diameter between three groups Cell Diameter N Mean Std. Deviation Minimum Maximum Commercial 30 27.889785 3.7604796 21.2578 34.9431 Traditional 30 30.275687 3.8105107 22.8717 37.8986 Control 30 31.902003 3.4836589 24.0410 36.3787 Total 90 30.022492 4.0048685 21.2578 37.8986 F=8.984 P=0.001 There is significant difference in CD between3 groups (P- value = .001) Table 10: Post Hoc Tests Multiple Comparisons (Tukey HSD) (I) Group (J) Group Mean Difference (I-J) P-value Commercial Traditional -2.3859027(*) .037 Control -4.0122180(*) <.0005 Commercial 2.3859027(*) .037 Control -1.6263153 .208 Commercial 4.0122180(*) <.0005 Traditional 1.6263153 .208 Traditional Control * The mean difference is significant at the .05 level. Commercial vs traditional P = .037 (significant) Commercial vs control P = <.0005 ( very higly significant) Traditional vs control P = .208 (not significant) There is a significant difference between commercial and betel quid (p= .037) There is also significant difference between commercial and control (p<.0005). 68 Results Graph 1: Age distribution of the study population (Traditional) 7 No.of people 6 5 4 3 2 1 0 31-35 36-40 41-45 46-50 51- 55 56-60 61above Age distribution Graph 2: Age distribution of the study population (commercial) 14 No.of people 12 10 8 6 4 2 0 15-20 21-25 26-30 Age (yrs) 69 31-35 36above Results Graph 3: Distribution of different lesions by traditional tobacco users 2 Oral submucous fibrosis 3 chemical burn lichenoid reaction 4 chewers mucosa 2 0 1 2 3 4 5 No.of lesions Graph 4: Distribution of different lesions by commercial tobacco users Tobacco pouch keratosis 1 Oral submucous fibrosis 9 Leukoplakia 3 Lichenoid reaction 3 Chewers mucosa 8 0 2 4 6 No. of lesions 70 8 10 Results No.of traditional tobacco/day Graph 5: Scatter diagram showing correlation of frequency and duration of traditional tobacco use 20 18 16 14 12 10 8 6 4 2 0 0 40 10 20 30 duration of use (yrs) No.of commercial packets per day Graph 6: Scatter diagram showing weak correlation between frequency and duration of commercial tobacco use 16 14 12 10 8 6 4 2 0 0 2 4 6 duration of use (yrs) 71 8 10 12 Results Graph 7: Frequency of commercial tobacco use and development of lesions 4 3.5 No.of lesions 3 Chewers mucosa tobacco pouch keratosis lichenoid reaction Osmf 2.5 2 1.5 1 Leukoplakia 0.5 0 3000-6000 6000-9000 9000-12000 12000-15000 15000above No.of quids used Graph 8: Mean comparison of use of Traditional tobacco & Commercial packets per day & duration of use 14 12 Mean 10 8 use per day 6 duration of use 4 2 0 betel duid Commercial packets 72 Results Graph 9: Graph showing nuclear diameter between 3 groups Nuclear diameter(microns) 8 7 6 5 4 3 2 1 0 Commercial packet Betel duid Control Graph 10: Graph showing cytoplasmic diameter between 3 groups 33 31.90 Cell diameter (microns) 32 31 30.28 30 29 28 27.89 27 26 25 Commercial Betel duid 73 Control Discussion DISCUSSION Oral cavity is a miniature screen of whole body and a lesion which is present in oral cavity may indicate the total health or habits of the individual. Oral cavity and oral mucosa is the first site to get exposed to any substances including noxious substance like tobacco products. Tobacco related habits are highly prevalent among Indian people. It is highly alarming that tobacco products which were usually consumed by a small section of the population are today part of the modern urban and rural lifestyle and even children of school going age are getting into these habits. It is well known that chemical constituents of tobacco are carcinogenic and cause various mucosal lesions ranging from chewers mucosa to potentially malignant lesions which may progress to oral cancer. Early detection and cessation in the habits can save people from life threatening tobacco related oral malignancies. The study was performed in individuals with two popular smokeless tobacco habits prevalent in Indian population such as traditional tobacco use i.e tobacco used along with betel leaf and use of commercially available tobacco products. Along with, normal healthy individuals without any tobacco related habits were also included as controls. In each group 30 individuals were selected to participate in the study. Among the traditional tobacco users participated in this study 14 were males and 16 were females, while most of the commercial tobacco product users were males (29/30). 74 Discussion The prevalence rate of males and females who consumed commercial tobacco was 96.6% and 3.3% respectively. Our observations in the present study are consistent with previous studies which reported that areca nut and betel quid with tobacco was a more common habit in females than in males; however, more males tend to use commercial tobacco product as compared to females.96 Significant male predominance among commercial tobacco product users could be due to easy access for males to the shops and social barrier that prevent females from buying such product from small shops where tobacco products are sold. It seems that females prefer the traditional form of habit where quid can be prepared at home. While analyzing the age distribution in this study, we have seen that people using commercial tobacco products belong to much younger age group (average age of 22.4 years) compared to traditional tobacco users (average age of 44.47 years). This result was coinciding with the study of Kumar et al43 who reported that Gutka consumption was significantly higher in youngsters (<25 yrs) where as in traditional tobacco users it starts over 30 years. The high prevalence of commercial tobacco product used among youngsters could be because they are influenced by the marketing strategies of manufacturers. Betel leaf is perishable and preparation of BQ is somewhat complex. The packaging revolution has made tobacco products portable, cheap and convenient, with the added advantage of a long shelf-life, which could have made these individuals get attracted to an easy replacement which is available in ready to use packets. Our 75 Discussion observations are slightly different from the results obtained after a set of house-to-house surveys conducted in 4 states (Andhra Pradesh, Bihar, Gujarat, Kerala) in India 1960s, where in tobacco quid habits were observed in individuals of 15 years and above27. The difference in observation could be due to the fact that he trend of using commercial products would not have become highly prevalent in 1960s. Out of 30 traditional tobacco users, oral mucosal lesions were seen only in 11 individuals, (36.6%) while 23 individuals out of 30 commercial product users presented with some form of mucosal changes, with a prevalence rate of 76.7%. This observation is consistent with the study of Robertson et al.78 who reported the marked prevalence rate of 78.6%. The frequency and duration was correlated in relation to the lesion and it was seen that the lesions were present in those patients who consumed more than 5 betel quids per day for minimum of 15 years. This result was consistent with previous reports (Ying-Chin Ko et al49, Reichart PA et al 56 ). When a similar comparison was made among the commercial tobacco product users, we have observed mucosal lesions had developed within a relatively shorter period of time with and less frequency, among this group. The above observations in this study is consistent with those of Sinor PN et al (1990)97 who reported that chewing of tobacco with arecanut , betel leaves and lime results in later onset of the disease. 76 Discussion Lesions identified in traditional tobacco users were lichenoid reaction (4), chemical burn (3), chewer’s mucosa (2), and oral submucous fibrosis (2). The lesions in commercial tobacco product users were different from those of traditional betel quid users and lesions were chewer’s mucosa (8), lichenoid lesion (3), leukoplakia (3), oral submucous fibrosis (9), and tobacco pouch keratosis (1). The above results point towards the more harmful effect of commercial tobacco products. This could be because of the processing techniques used, additives and artificial perfuming and flavoring materials added to enhance the taste. Less prevalence of lesions in individuals using tobacco as a traditional preparation along with betel leaf can be attributed to protective effect of the betel leaf. It is reported that betel leaf is rich in antioxidants and also have anticarcinogenic properties 2. The prevalence of lesions is always related to the frequency and duration of the habits. In our study the frequency and duration of habits were different in each individual. So we tried to correlate the types of lesions with the approximate No. of tobacco product they used by multiplying the number of packets used per day and the duration of usage. The usage of packets was divided in the range groups of 3000 each for our convenience. Statistically it was analyzed that as the usage of No. of packets increases there was gradual progression of lesions from relatively harmless chewers mucosa to more dangerous potentially malignant lesions. 77 Discussion In our study we have found that the incidence of progression of oral lesions is faster in commercial tobacco users than traditional tobacco users. Lesions in commercial tobacco users started at a very young age of approximately 15 years when compared to traditional tobacco users were the lesions was seen starting at an approximate age of 30 years. This result was coinciding with the study of Kumar et al43 in which it was recorded that Gutka consumption was significantly higher in youngsters (<25 yrs) where as in traditional tobacco users it starts over 30 years. This might be because of the anti oxidant, protective and medicinal benefits of betel leaf used in betel quid. Smears were taken from the buccal mucosa, the preferred site for taking smear as stated by Ramaesh et al (1998)86. These smears were stained using PAP technique and the cellular changes of exfoliated cells were studied. We counted 50 cells from each patient similar to a study done by Ogden GR et al (1997)81.where he stated that 50 cells were sufficient to provide a consistent assessment for a variety of oral mucosal changes. Most of the exfoliated cells from the buccal mucosa of individuals with use of traditional and commercial tobacco were superficial cells; however some intermediate cells were also evident. These cells did not show significant morphological variation from those of controls. 78 Discussion We have calculated the CD and ND of 50 randomly selected cells from each smear in order to assess the alteration in the cells more precisely. We agree with Ramaesh T et al who suggested that of various cellular and nuclear parameters, nuclear and cytoplasmic area and nuclear to cytoplasmic ratio are significant in the diagnosis of oral lesions4. To minimize the human errors in recording computerized image analysis soft ware was used for this purpose. The results obtained were statistically analyzed using SPSS Vir 15 statistical software and excel. The cytomorphometric analysis of the cells, reveled the following findings. The nuclear diameter of exfoliated cells in traditional tobacco users ranged from 4.26 to 5.91 µm with a mean of 4.99 µm. The same exfoliated cells showed cell diameter which ranged between 22.87 to 37.89 µm with an average mean of 30.27 µm.when it was statistically compared using 1-way ANOVA & Turkey multiple comparison tests. In commercial tobacco product users, the exfoliated cells had nuclear diameter ranging from 4.84 to 8.46 µm with a mean of 5.60 µm and the cell diameter which ranged between 21.25 to 34.94 µm with a mean of 27.88 µm Mean CD and ND in both group 1 & 2 were more compared to individuals without tobacco habits (31.9 and 4.81 µm respectively) 79 Discussion This study shows that the use of smokeless tobacco in any form influences the ND and CD of the buccal mucosal cells. This observation is consistent with the report of Ramaesh et al (1998, 99)86, 87 When the nuclear diameter was compared between the three groups a statistically significant difference was observed between group 1&2 and 2&3 with the P value of 0.054 and 0.008 respectively. But when the same was compared between 1&3, it showed a P value of 0.765 which was not significant. This point out that tobacco use in any form brings about alteration in the nuclear size, which indicates detrimental effect of tobacco on oral mucosal cells. This nuclear alteration was more significant in commercial tobacco product users compared to traditional form of tobacco users with betel leaf. Similarly result was obtained when the cell diameter was compared between the three groups. Statistically difference was observed between group 1&2, 2&3 with the P value of 0.037 and < 0.0005 respectively. But when the same was compared between 1&3, it showed a P value of 0.208which was not significant. The ratio between CD & ND in exfoliated cells from group 1 individuals was 1: 6, while that of group 2 was 1:4.5. This was relatively high compared to those of control group which had the ratio of 1:7. As with the ND and CD the alteration in nuclear cytoplasmic ratio was also relatively more in commercial tobacco users. We could not make a 80 Discussion comparison of our results with respect to CD and ND and nuclear cytoplasmic ratio in traditional and commercial tobacco users due to lack of availability of similar study I the literature. In our study we have observed commercial tobacco product use is highly prevalent among young men while traditional tobacco use was more common among relatively older people and was equally distributed among men and women. Oral mucosal changes were noted in both groups, however was more prevailing among commercial tobacco product users. Similarly cellular changes such as alteration in ND and CD and nuclear cytoplasmic ratio were appreciated in both the groups while the alterations were more significant in commercial tobacco product users. The above observation related to variation in the cellular changes could be due to more dangerous effects of commercial tobacco products or due to protective effect of betel leaf in traditional tobacco habits. The role of arecanut which is the main ingredient of most of the commercial products such as gutka also should be considered. To rule out the above possibilities further studies are required including more sample size, standardized parameters and advanced techniques. 81 Conclusion Conclusion In our study we have observed commercial tobacco product use is highly prevalent among young men while traditional tobacco use was more common among relatively older people and was equally distributed among men and women. This difference in age and sex prediction between the two groups point to the changing trend in tobacco habits. This could be due to the packaging revolution that has made tobacco products portable, cheap and convenient, with the added advantage of a long shelf-life. Oral mucosal changes were noted in both groups, however found to be more prevailing among commercial tobacco product users. Similarly cellular changes such as alteration in ND and CD and nuclear cytoplasmic ratio were appreciated in both the groups while the alterations were more significant in commercial tobacco product users. The above observation related to variation in the cellular changes could be due to more dangerous effects of commercial tobacco products or due to protective effect of betel leaf in traditional tobacco habits. 82 Summary SUMMARY The present study was done in the Department of Oral and Maxillofacial Pathology, Yenepoya Dental College Hospital, Mangalore. The study was done among individuals with two different forms of smokeless tobacco habits, such as traditional tobacco use and use of commercial tobacco products in an attempt to compare the mucosal changes and cellular changes among them. From the study we have obtained the following informations. 1. Traditional tobacco use was more among older age group with almost equal predilection among both the genders. 2. Use of commercial tobacco products was more prevalent among young males 3. Mucosal changes were observed in both groups. But the changes were more prevalent and developed within a shorter duration after the beginning of the habits in commercial tobacco products users. 4. Alteration in ND and CD and nuclear cytoplasmic ratio was observed in both the groups compared to healthy controls. The alteration was more significant among commercial tobacco product users than traditional tobacco users. 83 Bibliography BIBLIOGRAPHY 1. Yang MS, Wen JK, Ko YC, Su IH. Prevalence and related risk factors of betel quid chewing by adolescent students in Southern Taiwan. J Oral Pathol Med 1996; 25: 69-71. 2. Padma PR, Lalitha VS, Amonkar AJ, Bhide SV. Anticarcinogenic effect of betel leaf extract against tobacco carcinogens. Cancer Lett. 1989 Jun; 45(3):195-202. 3. Ogden GR, Cowpe JD, Wight AJ. 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No: OP NO: NAME: AGE/SEX INCOME: OCCUPATION: ADRESS: CHIEF COMPLAINT AND DURATION: HISTORY OF PRESENT ILLNESS: RELEVENT MEDICAL HISTORY: PAST DENTAL HISTORY: FAMILY HISTORY: PERSONAL HISTORY: 1. ORAL HYGIENE HABITS 97 DATE: ANNEXURE 2. DIETARY HABITS 3. ADVERSE ORAL HABITS 4. DO YOU USE TOBACCO IN ANY FORMS? YES NO IF YES 5. TYPE OF TOBACCO USED: SMOKING CHEWING 6. IF IT’S IN A CHEWABLE FORM: TRADITIONAL COMMERCIALY AVAILABLE IF COMMERCIALLY AVAILABLE BRAND FREQUENCY/DAY INGREDIENTS: MAIN INGREDIENT FREQUENCY/DAY AGE STARTED ARECANUT ALONE ARECANUT WITH BETEL LEAF ARECANUT WITH TOBACCO BETEL LEAF WITH TOBACCO BETEL LEAF WITH ARECANUT,TOBACCO 98 DURATION IN YEARS ANNEXURE &LIME TOBACCO ALONE OTHERS:SPECIFY 7. DO YOU HAVE THE HABIT OF KEEPING THE CHEWED PRODUCT IN THE ORAL CAVITY FOR A LONG TIME? SITE HOW LONG 8. DO YOU SPIT OR SWALLOW THE JUICE OBTAINED? SPIT SWALLOW 9. SMOKING HABIT TYPE DURATION FREQUENCY/DAY EX SMOKER 10. GENERAL EXAMINATION: BUILT: WELL/MODERATE/POOR NOURISHMENT WELL/MODERATE/POOR 11. EXTRA ORAL EAMINATION: 12. INTRA ORAL EXAMINATION: LABIAL MUCOSA: NAD BUCCAL MUCOSA NAD HARD PALATE: WL WL UL UL PIG PIG 99 ANY OTHER ANY OTHER ANNEXURE NAD SOFT PALATE: WL UL PIG ANY OTHER NAD VESTIBULAR AREA: WL UL PIG ANY OTHER WL UL PIG ANY OTHER NAD ALVEOLAR MUCOSA: WL UL PIG ANY OTHER NAD FLOOR OF THE MOUTH: WL UL PIG ANY OTHER NAD WL UL PIG ANY OTHER NAD WL UL PIG ANY OTHER NAD TONGUE: GINGIVA: 13. HARD TISSUE EXAMINATION: DMFT 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 _____________________________________________________ 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 ATTRITON/ ABRATION/ EROSION STAINS: 14. PERIODONTAL STATUS: IF ABNORMAL MUCOSAL CHANGES ARE PRESENT: 1. DURATION 100 ANNEXURE 2. ASSOCIATED SYMPTOMS: BURNING PAIN DIFFICULTY IN OPENING THE MOUTH INTOLERENCE TO SPICY FOOD 3. RATE OF PROGRESSION: SLOW RAPID 4. SITE: 5. EXTENT / SIZE: 6. NATURE OF THE LESION: ULCER REDDISH PATCH WHITE PATCH BLANCHING OTHERS 7. PALPATORY FINDINGS: TEXTURE PAIN BLEEDING PROVISIONAL DIAGNOSIS: DIFFERENCIAL DIAGNOSIS: INVESTIGATION: FINAL DIAGNOSIS: 101 OTHERS
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