GOD” who illuminated me the desire that “to let go you don’t need the strength, what you need is faith and understanding of God’s will.”
I acknowledge my deep sense of gratitude to the Dean Prof.Dr. (Capt).S.Gokulanathan, B.Sc.,MDS., Vivekanandha Dental College for Women, for immense support for my Post Graduate Curriculum.
My genuine and deep hearted thanks the Principal Dr.N.Balan MDS., Vivekanandha Dental College for Women, for the help in pursuing the various research activities.
I take pride in acknowledging the insightful guidance of my respected Head of the Department, Prof.Dr.N.Ganapathy,MDS.,Professor, Department of Oral Pathology, Vivekanandha Dental College for Women, for providing me the opportunity and more thankful to him for the patience on me during my tough times in completing this study.
I am blessed to have the Associate Professor Dr.T.Maheswaran, MDS, MBA; as my Guide for his sustenance throughout my postgraduate curriculum. He has been there providing his heartfelt support and guidance at all times and has given me invaluable guidance, inspiration and suggestions in my quest for knowledge. He has given me all the freedom to pursue my research, while silently and non-obtrusively ensuring that I stay on course and do not deviate from the core of my research.
Without his able guidance, this dissertation would not have been possible. I shall eternally be grateful to him for his assistance.
I acknowledge my Associate Professors Dr.V.Ilayaraja MDS, Dr.J.DineshShankar MDS, Dr.T.R.Yoithap Prabhunath MDS and Senior Lecturers, Dr.A.Yamunadevi MDS and
I extend my sincere thanks to Biochemistry department Prof.Dr.SachuPhilip, Prof.Dr.K.M.Tullanithi, Dr.S.Ayappan for their valuable suggestions and support in completing this study.
I should thank fervently Dr. Rachel Sarah Vinodhini, Dr.R.Iswariya for the unremitted help and holding me in every minute during the progress of my work. I am always indebted for their extreme moral support all through my work.
I acknowledge my batch mates Dr.T.KeerthiPriyadharshini, Dr.U.Maheshwari and my juniors Dr. Gayathri, Dr. Jisha George, Dr. Porkodi Sudha, Dr. Renuka Devi and Dr. Swathi Raman in pursuing my work.
I am grateful to Oral medicine Postgraduate students Dr.Edwina, Dr.Divya kanimozhi, for their kindly help in sample collection.
My acknowledgement would be incomplete without thanking the biggest source of my strength. I have no words to thank the sacrifices of my beloved Parent Mr.Y.A.Shanmugam, Mrs.P.Anbarasi and my even-tempered Sister Miss.S.Sharmila B.E., in chasing my dissertation work. I thank them for putting up with me in difficult moments where I felt stumped and for goading me on to follow my dream of getting this degree. This would not have been possible without their unwavering and unselfish love and support given to me at all times.
my childhood days.
It would be inappropriate if I omit to mention my gratefulness to my friends Dr.E.Dhivya, Dr.V.Karthika, Dr.N.Preethi, Dr.T.SriVandhana, Dr.J.Karthick and M.Jagadeeshwaran for their constant support and whole hearted solidarity.
I acclaim my seniors Dr.Meenalochani, Dr.Nagendran for their extreme support in completing this study.
Finally yet importantly I am pleased to thank all the Research scholars, my juniors and each and everyone to incite me every day in completing the work with utmost satisfication.
S.No Contents
01 Age distribution
02 The average tail length of Group-I 03 The average tail length of Group-II 04 The average tail length of Group III-a 05 The average tail length of Group III-b
06 Comparison of average tail length and standard deviation 07 The difference in various groups by Post-hoc test
08 Comparison of average tail length within the groups 09 Gender and average tail length
10 Duration of tobacco usage
11 Average tail length and the duration of tobacco habit in Group-II 12 Average tail length and the duration of tobacco habit in Group III-a
13 Average tail length and the duration of tobacco habit in Group III-b
14 Forms of tobacco usage and average tail length in Group II 15 Forms of tobacco usage with average tail length in Group III-a
16 Forms of tobacco usage with average tail length in Group III-b 17 Average tail length and type of lesion in Group III-a
18 Average tail length and type of lesion in Group III-b
S.No Contents 01 Sample collection
02 Centrifuge
03 Agarose
04 Micropipette
05 Mechanical vibrator
06 Microwave oven
07 Clear agarose solution 08 Slide preparation 09 Stock solutions 10 Lysis buffers 11 Working solution 12 Electrophoresis
13 Power pack
14 Neutralization buffer
15 Staining
16 Fluorescent Microscope 17 Image analysis
18 Normal cytological smear with Ethidium bromide staining 19 Microscopy of Group -I
20 Microscopy of Group II 21 Microscopy of Group III-a 22 Microscopy of Group III-b
S. No. TITLE PAGE No.
1. INTRODUCTION 1
2. AIM AND OBJECTIVES 6
3. REVIEW OF LITERATURE 7
4. MATERIALS AND METHODS 37
5. RESULTS 47
6. DISCUSSION 63
7. SUMMARY 71
8. CONCLUSION
72
9. REFERENCES 73
1
INTRODUCTION
The use of tobacco continues to be the leading preventable cause of death in the world.1 The burden of disease and death that tobacco imposes on the public's health is very extensive.World Health Organization reported that tobacco kills more than 5 million people which is greater than the combined mortality of Tuberculosis, Malaria and AIDS combined. It is estimated that by the year of 2030, death rate due to tobacco will increase to eight million per year in the world.2
Tobacco is consumed in various forms, though smoking of manufactured cigarettes is the most prevalent form of its use. The emergence of widespread cigar use particularly among adolescents in both males and females has been reported in the past decade in the United States. Cigars have higher total nicotine content than cigarettes do and can deliver nicotine both through smoke and through direct oral contact with the tobacco wrapper. Cheroots are small cigars made of heavy bodied tobacco. Bidi smoking is a popular form of tobacco use in south Asia, accounting for one-third of the tobacco produced in India for smoking. Pipe smoking also entertained in various parts of Asian countries.3
The usage of tobacco affects the general health of the patient like ischemic heart disease, respiratory tract infections, cerebro-vascular disease and lung cancers.4 Our oral cavity is prone for myriad of changes with aging due to environmental and life style associated factors. Tobacco is one such factor that leads to oral diseases ranging from staining of teeth, periodontal problems and the life threatening oral cancer.5 The epidemiological surveys established that the smokers are five to nine times more prone for oral cancer development and risk may increase to as much as 17 times greater for the heavy smokers.6
2
It has been observed by the researchers that most oral cancers are followed by visible clinical changes in the oral mucosa usually in the form of white or red patch (two-step process of cancer development). Diagnosis of such potentially malignant disorders has the potential of not only decreasing the occurrence of oral cancer but also in improving the survival of those who develop it.7 Tobacco is the major component that changes the chemical composition of the cells and the structure of deoxyribonucleic acid (DNA). Deoxyribonucleic acid is one of the key targets of many carcinogens.8
Indirect-acting carcinogens like tobacco are unreactive, water-insoluble compounds. They can act as carcinogen only after conversion to ultimate carcinogens by introduction of electrophilic centers. Such metabolic activation of carcinogens is activated by enzymes that are normal body constituents. The enzymes Cytochrome P450 catalyze addition of an oxygen atom to the carcinogen, increasing its water solubility and converting it to a form that can be easily excreted. This
‘metabolic detoxification’ process is further assisted by phase 2 enzymes, which convert the oxygenated carcinogen to a form that is highly soluble in water. This is one of the protective mechanisms in the body. Some of P450 intermediates are electron deficient and they may form DNA adducts. These multiple adducts lost the capability of DNA repair and undergoes mutation. These mutation leads to changes in tumor suppressor genes and leads to cancer.9
There are several forms of DNA damage like single strand break, double strand breaks, cyclobutane pyrimidine dimers, 6-4 photoproducts by various photochemical processes including ultraviolet radiation. These changes can be identified by various techniques like Polymerase Chain Reaction, Comet assay, Halo assay, Terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate Nick End
3
Labelling assay, HPLC-Electrospray tandem mass spectrometry, Fluorescence in situ hybridization, Flow cytometry, annexin V labelling, immunological assays including immunofluorescent and Chemiluminescence thymine dimer detection, immunohistochemical assay, Enzyme-linked immunosorbent assay, Radio immunoassay, Gas chromatography-mass spectrometry and electrochemical methodisation.10,11
Among the various techniques, in the year 1984, Ostling and Johansson were the first to quantify the DNA double strand breaks using microgel electrophoresis technique known as single cell gel electrophoresis technique or the Comet assay. In the year 1988, Singh et al, using alkaline conditions identified single strand and the double strand breaks as well as alkali-labile sites expressed as frank strand breaks in the DNA.8
The assay is now, a well established, simple, versatile, rapid, visual, and a sensitive, extensively used tool to assess DNA damage and repair, quantitatively as well qualitatively in individual cell populations. 8 Some other lesions of DNA damage such as DNA cross linking (e.g. thymidine dimers) and oxidative DNA damage may also be assessed using lesion-specific antibodies or specific DNA repair enzymes in the Comet assay. It has gained wide acceptance as a valuable tool in fundamental DNA damage and repair studies, genotoxicity testing and human biomonitoring.11
Comet assay is the non-invasive technique of measuring the DNA damage. It helps to assess the DNA damage caused by radiation, during carcinogenesis, to determine the genotoxicity and to analyse the irradiated foods. The principle behind the comet assay is the breaks in the super coiling of DNA so that loops extend outside.13 The damaged DNA tends to migrate during electrophoresis in which shortest
4
fragment travel the farthest. The undamaged DNA that appears as round intact shape is referred to as ‘Head’ and the DNA fragments that migrate toward the anode is the
‘Tail’. When the procedure is standardized and validated, it helps in identifying the diseases and the changes occur due to oxidative stress, occupational exposure, to find the effectiveness of medical treatment.12The head containing the high-molecular- weight DNA and the comet tail containing the leading ends of migrating fragments were measured from digitized images using software.13
Comet assay can be done in epithelial cells, peripheral blood leukocytes, nasal cells, Lens epithelial cells, corneal cells. Lymphocytes are most commonly used to identify the changes that occur due to the long term exposure of carcinogens.
Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as biomarkers; furthermore, 80% of solid cancers are of epithelial origin. Epithelial cells are characterized by common structural features (specifically, their arrangement into cohesive sheets), but have diverse functions that are made possible by many specialized adaptations.14
Many of the physical properties of epithelial cells are dependent upon their attachment to one another, which is mediated by several types of cell junctions. The specialized functions of epithelial cells are mediated through both structural modifications of their surfaces and internal modifications, which adapt cells to fulfill their specific roles, ranging from absorption to secretion to serving as a barrier.14
Buccal epithelial cells have direct contact with the carcinogens during the tobacco usage and the identification of DNA damage would be useful and appropriate.11 The buccal mucosal cells are also used in micronuclei assay and help to
5
identify the pyknotic nuclei, loss of nuclear material. Few cells have other forms such as binucleated stage with two nuclei in the same cytoplasm, form of nuclear bud or
“broken egg” or form small micronuclei (MN) near nuclei in the same cytoplasm.
These biomarkers of genome damage and cell death can be observed in both the lymphocyte and buccal cell systems, and thus helps in assessment of genome damage.15
Few published reports are available in the scientific literature regarding the comet assay in the buccal epithelial cells and also no investigation was found regarding the comparison of DNA damage using comet assay between the lesional and the non-lesional site of the oral mucosa.
6
AIM & OBJECTIVES
Aim
To assess the DNA damage in the tobacco associated human buccal cells using comet assay.
Objectives
I) To assess the DNA damage in the buccal cells of i) Individuals with no history of tobacco usage.
ii) Individuals with tobacco usage without oral lesions.
iii) The lesional site of the individuals with tobacco associated oral lesions.
iv) Non-lesional site of the individuals with tobacco associated oral lesions.
II) To compare the DNA damage among the buccal cells between lesional and non-lesional site of individuals with tobacco usage.
7
REVIEW OF LITERATURE
Martin et al., 1993 reviewed the single cell gel electrophoresis. The comet assay was developed by Ostling and Johansson in 1984 and modified by Singh et al in the year of 1988. It is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. The development of comet assay particularly alkaline versions development, protocols and analysis of the DNA were described. The potential application and the principles determining the behaviour of the single cell gel electrophoresis technique was detailed in the review.16
Valverde et al., 1997 analysed the DNA damage in leukocytes, buccal epithelial cells, nasal epithelial cells of the individuals exposed to air pollution in Mexico city using comet assay. The study group includes 42 students in the medical school. The average age of the sample group was 19 years with 24 females and 18males. The significant difference was not found in the smokers who smoke less than 10 cigarettes per day. The significant difference was found in the nasal epithelial cells and the lymphocytes. There doesnot exist the mean difference in the buccal epithelial cells. Since nasal cells are the first target for the environmental pollutants, they can be used for the biomarker studies but several studies should be done to identify the genotoxic effects due to pollutants.17
Rojas et al., 1999 reviewed the method of single cell gel electrophoresis and its principles, methodology and applications in different fields. The principles, basic methodology with variations were explained. The various technical variables like lysis buffer, pH, voltage, amperage and unwinding conventional DNA electrophoretic techniques, electrophoresis buffer composition, pH and the various parameters for the analysis of DNA damage were reviewed.
8
The applications of comet assay include genotoxicology, clinical area, DNA repair studies, environmental biomonitoring and human monitoring. The comet assay gained importance when used along with the fluorescence in situ hybridization to identify the genotoxicity.18
Lodish et al., 2000 reviewed the various changes in the DNA sequences and explained about the chemical characteristic of electrophilic and P450 mechanisms. The changes in the DNA sequence result from copying errors and the effects of various physical and chemical agents, or carcinogens. All carcinogens are mutagens by altering one or more nucleotides in DNA. The mismatch repair, bacterial and eukaryotic DNA repair systems was extensively reviewed. DNA-repair mechanisms that are ineffective or error-prone may perpetuate mutations. This is a major way by which DNA damage, caused by radiation or chemical carcinogens leading to tumor formation. Cellular DNA-repair processes have been implicated both in protecting against and contributing to the progression of cancer. 19
Moller et al., 2000 evaluated the effect of comet assay in analyzing the risk of individuals in the biomontoring studies. The confounding factors and the occupational risk factors that lead to DNA damage were reviewed. The age, gender and smoking status are used as criteria in the selection of populations. The information on the level of exercise, infection, and diet on the day of the sampling was recorded. In the biomonitoring studies, comet assay found to be the suitable technique in evaluating the buccal cells and nasal epithelial cells exposed to DNA damage. Samples from exposed and unexposed populations should be taken at the same time to avoid seasonal variation. The comet assay is described a suitable and fast test for DNA-damaging potential in biomonitoring studies. 20
9
Marnett et al., 2000 described that the major reason for DNA damage during carcinogenesis. The oxyradicals that play an important role in the progression of cancer were reviewed. The various methods like P-post labelling were developed to detect adducts that arising from the exogenous carcinogens. Oxyradicals affect the signal transduction pathways and the transcription factors that alter the cell kinetics. The various adducts affect the DNA damage and the lipid peroxidation, however many research needed for the identification of intercellular and the intracellular signal transduction role in the carcinogenesis process.21
R.J. Albertini et al., 2000 proposed the guidelines for monitoring the group of individuals exposed to genotoxic agents and its effect on humans as carcinogens. The concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents was proposed. It serves as the guidelines and serves as a check-list for evaluating the methodology and results of completed studies. The main emphasis is on continuous exposures, such as those that occur in occupational environmental settings, but an effort is also made to give guidance in situations where unexpected short term exposures have occurred. These guidelines are not intended to be extensive reviews of each assay or how they are performed. The most important genotoxic endpoints and the numerical chromosomal aberrations were assessed using classical chromosomal aberration analysis like micronuclei, DNA damage adducts, strand breaks, protein adducts and biochemical electrophoretic assays.22
Tice et al., 2000 state the guidelines for in vivo and in vitro genetic toxicology testing related to single cell gel electrophoresis. The methodology, analysis, interpretation of results for in vivo and invitro genetic toxicology testing were reviewed. The comet assay has many advantages over the other geno-toxicity assays.
10
The advantages were demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The other related methodologies included were the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS, the use of repair enzymes or antibodies to detect specific classes of DNA damage, the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The expert panel mentioned that the minimal experimental and methodological standards were needed to get the results of Comet studies, to be accepted as valid by knowledgeable scientists and by regulatory agencies.23
Williams et al., 2000 reviewed the molecular pathogenesis of oral squamous cell carcinoma. It is a multistep process in which multiple genetic events occur that alter the normal functions of oncogenes and tumour suppressor genes. This can result in increased production of growth factors or numbers of cell surface receptors, enhanced intracellular messenger signalling, and/or increased production of transcription factors.
The progression of the disease leads to a cell phenotype capable of increased cell proliferation, with loss of cell cohesion, and the ability to infiltrate local tissue and spread to distant sites. The recent advances in the understanding of the molecular control of these various pathways will allow more prompt diagnosis and assessment of prognosis. This lead the way for more novel approaches to treatment.24
Nadin et al., 2001 identified an alternative stain, silver nitrate for comet assay.
The comet assay was found to be the best technique for the identification of DNA repair and base-level DNA damage. The lymphocytes were used to identify the alternative stain for fluorescent staining using comet assay. The fluorescent stains have
11
the disadvantage of high cost and needs immediate scoring. The silver nitrate stain was found to be the best alternative for fluorescent stain and helps in the long term storage of slides with the ease of visualization in the conventional microscope. The modifications in the silver nitrate stain increases the sensitivity and the reliability of the assay.25
Kuper et al., 2002 discussed the tobacco use and its role in carcinogenesis.
The use of various forms of tobacco and its impact on the public were reviewed. A wide variety of tobacco products exist, of which cigarettes are most frequently consumed by the individuals. Tobacco products contain more than 50 established or identified carcinogens. It increases the risk of cancer by causing mutations that disrupt cell cycle regulation, or through their effect on the immune or endocrine systems.
Certain factors such as genes, diet and environmental exposures may alter susceptibility to cancer in tobacco users. The 15% of all cancers are estimated to be attributable to smoking. The most common pathway of metabolic activation that results in adduct formation in the pathogenesis of cancer was outlined. There are also many genes that confer an increased risk of cancer. The intake of tobacco varies in different countries and the practice of smoking cessation reduces the incidence of cancer to larger extent.26 Wilkins et al., 2002 studied the radiation induced apoptosis in human lymphocytes using flow cytometry with Annexin V and propidium iodide versus the comet assay. The neutral comet assay was used to measure double- stranded DNA breaks, but it has also been used to identify the apoptosis based on its characteristic DNA fragmentation patterns. Comet assay was used to evaluate the various apoptotic pathway with the help of lymphocytes radiated by ionizing radiation.
It was found that annexin-V and propidium iodide was used for detection of earlier stages of apoptosis, late apoptosis, and an apoptotic or necrotic phase, where as comet
12
assay was used for assessing the apoptosis at the final stages of apoptosis. The flow cytometry found to be better method to analyse the apoptosis than other methods.27 Martinet et al., 2002 found that there was an increased DNA damage in patient with atherosclerosis. The study was done to find if the oxidative DNA damage and repair mechanisms lead to atherosclerotic plaques. The result of their study concluded that there was increased DNA damage with severity of formation of atherosclerotic plaques. They described that comet assay has an advantage of identifying DNA damage in carcinogenesis as well as in atherogenesis. The various DNA base pair repair, poly (ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated in immunohistochemistry and western blotting.28
Eren et al., 2002 evaluated the changes in the buccal epithelial cells after 18 days of chlorhexidine mouth wash usage using the single cell gel electrophoresis technique. The study was done in the thirteen dental students aged 21-29years without any systemic complaint. One hundred cells per subject were analysed for the DNA damage. A statistical increase was observed in the damaged buccal and blood cells after the Chlorhexidine application. The mean grade of damage in buccal cells was statistically different from that in blood cells. The mean DNA damage of lymphocytes was more than that of the buccal epithelial cells provided that chlorhexidine was not absorbed much in the oral tissues. The measure of DNA damage is transient as the chlorhexidine doesnot accumulate in the body. The changes detected earlier after the use of chlorhexidine could be reversible. 29
GP Pfeifer et al., 2002 estimated the tobacco use and role of p53 mutation in the pathogenesis of cancer. It was estimated that the use of tobacco suppresses the p53
13
by specific binding of benzopyrene that causes permanent DNA damage. The mutation of p53 is often higher in the smokers when compared to non-smokers. The p53 mutational patterns are different between smokers and nonsmokers with an excess of G to T transversions in smoking-associated cancers in lung cancers. The prevalence of G to T transversions is 30% in smokers’ lung cancer but only 12% in lung cancers of nonsmokers. The similar finding was found in laryngeal type of cancer. This type of mutation is infrequent in most other tumors aside from hepatocellular carcinoma.
However, they concluded that the effect of exogenous events in carcinogenesis differs at molecular level and several approaches needed for the future research in the role of environmental mutagens in the role of carcinogenesis.11
Patrick et al., 2003 reviewed the molecular biology of mucosal field cancerization of the head and neck. The concept of clonality and the molecular techniques used by researchers to elucidate clonal relationships between cell populations was discussed. The occurrence of multiple foci of premalignant or malignant disease in the upper aerodigestive tract was focused. The different studies that have attempted to look at the question of clonality in these lesions were explored.
The genetic progression involved in the development of head and neck squamous cell carcinoma (HNSCC) were described. The possible therapeutic interventions in this disease and the future areas of research that have impact on these observations were highlighted.30
Braakhuis et al., 2003, reviewed about the concept of field cancerization. The concept of “field cancerization” was first introduced by Slaughter et al. in 1953 when studying the presence of histologically abnormal tissue surrounding oral squamous cell carcinoma. The development of multiple primary tumors and locally recurrent cancer was proposed. The carcinogenesis model in which the development of a field with
14
genetically altered cells plays a central role supports the recent concept. The characteristics of the genetically altered field were described. In the tumor-adjacent
“normal tissue” and surgical margins to assess the presence of a field lesion molecular analysis was done. LOH, microsatellite alterations, chromosomal instability, and mutations in the TP53 gene were studied and the changes are detected by DNA amplification techniques, immunohistochemistry, and in situ hybridization. By measuring LOH, a quantitative analysis has been performed on HNSCC, and it was shown that at least one-third (10 of 28) of unselected tumors have tumor-associated genetic alterations in a biopsy taken from the macroscopically adjacent normal mucosa to the tumor.31
Tulkia Bose in 2003 found that the tumours show more chromosomal instability in the peripheral blood lymphocytes studied invitro. The study was conducted in 250 patients with various types of malignancies. Chromosome plates were classified using standard ISCN nomenclature. Karyotypes were considered abnormal when they showed acrocentric associations, premature centromeric division or Aneuploidies. It was concluded that many of the solid tumors show cytogenetic changes in the leukocytes with chromosomal instability. The cytogenetic changes may also be due to the effect of environmental mutagen in multiple tissues. The inheritance of chromosomal instability through some of the genes involved in DNA repair and synthesis provided the proper introduction on which environmental mutagens causes the final damage or sometimes the changes also might indicate the effect of environmental mutagen in multiple tissues. 32
N.H. Kleinsasser et al., 2004 assessed the cytotoxicity and genotoxicity of dental materials in human lymphocytes by microgel single cell gel electrophoresis. The xenobiotics investigated in the study were 2-hydroxyethylmethacrylate (HEMA),
15
triethyl- eneglycoldimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A-glycidyl methacrylate (Bis-GMA) with N-methyl-N0-nitro-N-nitrosogu- anidine and dimethyl sulfoxide. At higher concentration levels, the methacrylates HEMA, TEGDMA, Bis-GMA have significant and less enhancement of DNA migration in the comet assay. The inclusion of the test systems in the epithelial structure of buccal mucosal cells and DNA repair studies were recommended in the nearby future.33
Chandna et al., 2004 studied the apoptotic cells by a modification of the original neutral comet assay developed by Ostling and Johansson to detect various stages of DNA fragmentation. This assay involves cell lysis with anionic detergents at nearly neutral pH without high salt concentrations. BMG-1 human glioma cells were induced apoptosis by treating with a large dose of etoposide, and comets were prepared after different durations of treatment. The different shapes of DNA fragmentation induced during apoptosis were evaluated. The shapes of comet of six distinct shapes were distinguished into three types. Comet assay helps in differentiating the various stages of disease. It requires only small number of cells for DNA fragmentation analysis and found to be useful tool for pathophysiological conditions and DNA damage.34
Vinies et al., 2004 mentioned in the review that the tobacco smoke could be the cause of cancer along with the epidemiological evidence of International Agency for research on Cancer in 1986. There was a high coherence exists between the epidemiologic evidence and biologic evidence involving measurements of carcinogenic metabolites. The mortality of life depends on smoking habits. The extent of young individuals become cigarette smokers over the next few decades will strongly affect
16
mortality in the middle and second half of their life period. By preventing the young individuals from smoking will prevent cancer to greater extent in the future.35
Benzie et al., 2005 reviewed the application of different types of comet assay with its role in the nutraceutical research. Nutraceuticals that protect DNA from oxidant challenge promote DNA repair and they may have potential health benefits with lower risk of age-related disease. However, a sensitive and reliable laboratory tool to investigate DNA damage, protection and repair was needed to investigate them. The DNA repair can be reduced by the use of antioxidants. Comet assay will be the powerful tool in studying the effect of nutraceutical on DNA damage.36
Szeto et al., 2005 mentioned the comet assay as an important biomonitoring tool. However, they need alternative tool for assessing the DNA damage. They described that epithelial cells are the most common sites of malignant transformation; it would be useful if a comet assay model employing an easily accessible epithelial cell type. Buccal cells found to be the better alternative for lymphocytes in assessing the earlier invasion. The various intra- and inter-individual baseline DNA damage in buccal cells was investigated. The basic protocol for the comet assay was summarized. The framework for this evaluation will facilitate the use of the buccal cell comet assay model as an alternative test to the lymphocyte model for assessing genotoxicity and genoprotection in human trials.37
Hoffmann et al., 2005 analysed the effect of damaging on peripheral blood cells as measured by the comet assay. They discussed that the effect was more pronounced when smoking was considered as a genotoxic exposure and only expressed to a lesser extent in the group of studies considering smoking as a potential confounding factor in an occupational study. The positive outcome of the meta-analysis
17
depends on the minority of studies using arbitrary scores. The various statistical analyses that were used in these studies to identify the genetic damage were described.38
Olive et al., 2006 presented the protocol for the comet assay. The procedure for the comet assay, a gel electrophoresis-based method can be used to measure DNA damage in individual eukaryotic cells. The materials, methodology and the troubleshooting error that occur during the application of comet assay were explained.
The DNA damage and its repair in single-cell suspensions made from yeast, protozoa, plants, invertebrates and mammals can also be studied using the comet assay. The measured variation in DNA damage and repair capacity within a population of mammalian cells, comet assay can be used from human and sentinel animal biomonitoring. The protocol can be completed in time fewer than 24 h.39
Zhao X et al., 2006 conducted a randomized, double-blind, placebo-controlled intervention study. Thirty-seven healthy, nonsmoking postmenopausal women aged 50- 70 yrs were randomly allotted to 1 of 5 groups and were instructed to take a daily dose of mixed carotenoids for 56 days. Plasma carotenoid concentrations were analyzed by using HPLC, and lymphocyte DNA damage was measured by using the comet assay.
At the end of 57th day carotenoid intake group have significantly less DNA damage.
The carotenoid supplements were much useful. It helps to evaluate the patient after treatment in postmenopausal elderly women.40
Mehrotra et al., 2006 reviewed the various stages of carcinoma, epidemiology and the etiology. Various forms of tobacco, betel quid, viruses, Candida, alcohol found to be the most important etiological factor. Genomic instability, tumor suppressor genes, cytokine activity, apoptosis, neovascularization were detailed as the most
18
important role in the pathogenesis of cancer. Various proteomic array technologies, Lab-on-chip, Laser capture microdissection are the new recent advances in the diagnosis of premalignant lesions and cancer. The study of the carcinogenic process of the head and neck, including continued analysis of new genetic alterations, along with their temporal sequencing during initiation, promotion and progression, will allow us to identify new diagnostic and prognostic factors, which will provide a promising basis for the application of more effective treatments.41
Moller et al., 2006 discussed the importance of comet assay in genotoxicity in humans. The comet assay can be used with increasing popularity to investigate the level of DNA damage in terms of strand breaks and alkaline labile sites in biomonitoring studies. The comet assay is the simple and rapid method to detect the genotoxicity in humans. The incubation of bacterial glycosylase/endonuclease enzymes, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are identified as additional DNA migration. Comet assay can be used for multi –laboratory validation trial. The data regarding the level of exercise, infection, and diet should be recorded on the day of the sampling.42
Proia et al., 2006 reviewed from the literature about the association of smoking and smokeless tobacco buccal cell mutation and their association with the cancer. Many authors made an attempt to identify the tobacco smoke exposure and its relation to oral cancer. Tobacco-associated genetic mutations and non-genetic changes includes micronuclei, bacterial adherence, genetic mutations, DNA polymorphisms, carcinogen-DNA adducts and chromosomal abnormalities. The buccal cell assays was quite useful in identifying the genetic and non genetic changes in tobacco users thus helps in promoting smoking cessation and cancer prevention. The buccal cell micronuclei found to be the unique biomarker in clinicopathological investigations to
19
detect cancer. The buccal cells are useful not only for characterizing the molecular mechanisms underlying tobacco-associated oral cancers but also as exfoliative cells that express various changes that offers a unique biomarkers for the early detection of oral cancer. 43
Debolina Pal et al., 2007 conducted an epidemiological study in the influence of regular black tea consumption on tobacco associated DNA damage and HPV prevalence in human oral mucosa. The comet assay in the patients consuming black tea with tobacco users, oral cancer patients and their association with HPV was correlated.
The regular consumption of black tea reduces the tobacco associated DNA damage.
There was no association found to be seen between the HPV and tenure of tobacco habit, age and the tea drinking.44
D.J. McKenna et al., 2008 elaborated that the comet assay has been widely used to detect the cellular responses to DNA damage and has found its uses in genotoxicity studies, bio-monitoring, ecological testing and in the study of human disease. The application of comet assay to study the DNA damage and repair associated with cancer was discussed. The potential of the assay as a tool for indicating an individual’s tumour sensitivity to radiation and to various chemotherapeutic drugs in various genotoxic studies was examined. The advantages and the drawbacks of these studies were addressed. In addition comet assay found to be the best tool in assessing the tumour hypoxia to surgical treatment and survival.45
Saran et al., 2008 validated the biomarker needed for identifying the risk of precancer. The risk assessment of oral cancer in precancerous patients by using micronuclei, comet assay and challenge assay were analysed. The study includes 129 patients with cancer, 138 untreated people with untreated precancerous stage and 176
20
control patients. Micronuclei and comet assay was done in the buccal mucosal cells and challenge assay in blood lymphocytes. Challenge comet assay was done in the lymphocytes using MNNG sensitivity of DNA after DNA repair. The stepwise increase in DNA damage from control group to precancer and from precancer to cancerous patients by interobserver and intra-observer variability were noted.46
Christofoletti et al., 2009 explained the application of comet assay in erythrocytes of Oreochromis niloticus. The erythrocytes of Oreochromis niloticus are good genotoxicity indicators through the comet assay, an efficient, rapid and sensitive method to detect the DNA damage. The comet assay was as an essential tool in evaluating the DNA damage and it helps in comparing the technique at two different pH. It was revealed that pH 12.1 is the best, although both pHs, 12.1 and pH > 13 can be used with adaptations under electrophoresis conditions. The correlation between the silver nitrate and the ethidium bromide staining was found. The comets were visually analysed and scored. The use of silver staining over ethidium bromide without the special equipment was suggested.47
Sellapa et al., 2009 mentioned that many polymorphisms affect the different DNA repair genes and modulate the cancer. The study was done to correlate the relation between polymorphisms in the DNA repair gene XRCC1399 and hOGG1326 genotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) and risk of cancer development. It was conducted in 156 smokeless tobacco users and 70 controls without significant exposure to mutagens. The occupational, smoking, and medical histories were encountered with the questionnaires.
The relationship between the DNA polymorphism and the cancer developments was analysed by using micronuclei, comet assay and chromosomal aberrations. The significant difference between the smokeless tobacco users and the controls using the
21
micronuclei assay was found. There exists the relationship between the XRCC1399 Gln/Gln and hOGG1326 Ser/Ser genotypes with DNA damage level in subjects with smokeless tobacco exposure. The study to evaluate the genotoxic effect of smokeless tobacco use was recommended to identify the factor that promote carcinogenesis.48 Kumaravel et al., 2009 described the methods, advantages and disadvantages in using comet assay. The selection of different parameters, staining methods along with inter-laboratory validation and methodologies was suggested to make the study more scientific and acceptable. The good analysis and measurements depends on the slide, staining and the parameter for scoring. The Comet Assay as a reliable method for fundamental biological research, in addition to hazard and risk assessment in the field of genetic toxicology. 49
Lodovici et al., 2009 evaluated the carcinogenic-DNA adducts as biomarkers of tobacco smoke exposure. The significant difference between the use of tobacco and the incidence of cancer was found but not all the smokers are susceptible to cancer. The genetic pleomorphism also plays an important role in the occurrence of cancer. The future studies and research will be developed in evaluating the cancer through the potential modulators of biomarkers.50
Manikantan et al., 2009 found DNA damage in the peripheral blood lymphocytes of 74 workers in photocopying industry. The toxic components emitted from the photocopiers and low frequency electromagnetic fields were analysed. The significant result of DNA damage between years of exposure, smoking, age, gender, alcohol consumption and high level of DNA damage was obtained. In comparing the gender wise distribution, male have much more genetic damage than the female population. The findings of their study postulated that occupational exposure to toxic
22
components from photocopy machines could cause genomic damage in the workers along with other etiological factors. The workers should be educated for the damage that occur due to the exposure of photocopying machines and also the protective measures to avoid it.51
Kawaguchi et al., 2010 identified in their study that comet assay as a sensitive procedure in detecting genotoxicity. The comet assay has low power to detect the low level of genotoxic potential than micronuclei but it has the advantage that it can be applied to any cell type. It was postulated that comet assay can be used in any type of cells where as the micronuclei can be done only in the mitoticaly active cell population.
They also provided the suggestion that low power potential of comet assay can be enhanced by using DNA resynthesis inhibitors.52
Baricevic et al., 2010 done an invitro study in which they examined the genotoxic effect of casting alloys in patients using removable and fixed appliances for more than five years. The study was done in 55years aged subjects and 30 wearer of prosthodontic appliances and 25 controls. The sample was scrapped of from the buccal mucosa of the subjects and processed for analysis. The cell viability was assessed using trypan blue exclusion test and found there was high comet assay parameter in the group wearing metal appliances. In vivo evaluation of dental materials with respect to genotoxicity helpful in enhancing the biocompatibility.53
Hovhannisyan et al., 2010 discussed the importance of comet assay, micronuclei in combination with FISH technique. The potential of two methods enhanced by FISH technique was described. The comet assay in addition with FISH technique helps in recognition of DNA damage and repair and when used along with micronuclei test chromosomal target substances can be identified. The advantages,
23
limitations of Comet –FISH and MN- FISH in genotoxic studies were briefly stated.
The combination of all these technique helps to clarify the behaviour and organization of DNA within the comet. The technique offers the identification of DNA damage and repair of cancer relevant genes for measuring transcription-coupled repair, identification of genome targets of action of various genotoxic agents, including anti- tumour preparations.54
Salmani et al., 2011 in-vivo study of peripheral blood mononuclear cells as a biomarker of oxidative stress to estimate the DNA damage. The base level DNA damage was examined in the blood collected by venipuncture and lancet method. It has an advantage of using comet assay to estimate the damage in the blood collected via the lancet. It is the non invasive technique and requires minimum amount of blood. The small amount of blood collected by the lancet can be used and stored without the addition of the cryopreservative without any substantial changes in DNA. The comet assay can be done for various bio-monitoring studies by simple sample collection, storage and analyses were described in detail.55
Zamani et al., 2011 compared the micronuclei frequencies of smokers and non-smokers in Turkish young adults from three different tissues (peripheral blood lymphocytes, buccal mucosa and exfoliative urothelial cells) at the same time.
Peripheral blood lymphocytes were stained using the Giemsa stain for 5 to 7 minutes.
The buccal mucosa and the urothelial cells were stained using the Fuelgen reaction counterstained with fast green. The variation in the rate higher in urothelial cells than the blood lymphocytes and the buccal mucosa was found. The correlation exists between the smoking and the bladder cancer even after the cessation of smoking. The early education of smoking on health will prevent the habit.56
24
Kaur et al., 2011 found DNA damage in occupationally exposed Punjab farmers to pesticides. They assessed the level of DNA damage in 210 occupationally exposed Punjab farmers along with 50 matched control subjects. Results have shown a significant increase in the level of DNA damage in the exposed workers as compared to controls and no variation found to exist regarding age, smoking, drinking and dietary habits. There exists DNA damage in 35.7% of the workers and 65.29% sprayers did not show any damage. This may be due to the protective mechanism working against the toxic exposure or sometimes they could not be detected by this assay as it detect single strand breaks and alkali-labile sites.57
Ram et al., 2011 reviewed the risk factors and the molecular pathogenesis of oral cancer. The literature search was carried out in NCBI Pubmed database using keywords oral cancer, Risk factor, epidemiology and patho. The basic information was also obtained from textbook and medical university websites. Tobacco, alcohol, Viruses, syphilis, Candida, nutritional factors found to be the major risk factors. The genetic susceptibility and polymorphisms found to be the key role in the etio- pathogenesis of cancer. The development of oral cancer is a multistep process involving the accumulation of genetic and epigenetic alterations in key regulatory genes. Experimental pathological studies of oral cancer in animal models and direct molecular genetic analysis of oral cancer subjects in recent times have revealed a substantial amount of knowledge on specific gene alterations or other genetic mechanisms involved in initiation and subsequent progression. The easier diagnosis, better prognostication and efficient therapeutic management should be followed for the prevention.58
25
Mukherjee et al., 2011 evaluated the DNA damage in oral precancerous lesions and squamous cell carcinoma patients by using comet assay. They collected the peripheral blood by venepuncture method and SCGE performed. The study was done in the 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls). The mean tail length between diagnostic groups and between the people having different oral habits was compared. DNA damage found to be greater in leukoplakia and squamous cell carcinoma and lesser in OSMF. There found to be greater DNA damage in patients with any form of oral habits when compared to the control with no habits.59
De Oliveira et al., 2011 evaluated the DNA damage in paint industry workers.
The sample was collected from the blood lymphocytes and found a significant high level of DNA damage in the paint industry workers when compared to the control group. The hippuric acid level in the urine samples were analysed using high performance liquid chromatography (HPLC) with UV-VIS detector. The chronic occupational exposure to paints causes more genetic damage. The study concluded that the mutagenicity of cells were low but the cytogenetic damage indicates the occupational health hazard of paint industry workers.60
Soma et al., 2012 found nicotine free extracts as a barrier for tumoriogenesis.
The study was conducted in four lines of human cells treated with nicotine free tobacco extract and the MRC-5 and CCD841 non-cancerous cells treated with the tobacco extract showed hypersensitivity, apoptosis and cell cycle arrest in S and G2/M phases.
They mentioned that tobacco contains many chemicals of medicinal use and promote apoptosis in oral squamous cell carcinoma. It was said that nicotine free extract contains the bioactive compounds which act as a chemoprevention agent and helps to target the cancer cells at precancerous stages. The bioactive compounds in the nicotine-
26
free tobacco extract should be identified to encounter the signalling pathway against carcinogenesis.61
Dodani et al., 2012 studied the periodontal findings in patients with Oral Submucous fibrosis and Comet assay of affected gingival epithelial cells. The study was conducted in 100 oral submucous fibrosis patients and 89 without OSMF. The gingival parameters were analysed, comet assay was done by scraping the gingival epithelial cells. The gingival recession, probing depth in the periodontitis patients were recorded. There was an increased tail length in the epithelial cells of gingiva with oral submucous fibrosis when compared with the control group. The control group was noted to have less DNA damage than the oral submucous fibrosis patients.62
Orta et al., 2012 done a study and found the effect of aging on micronuclei frequency was found till 50years of age. The study was conducted in the lymphocytes of 30 healthy volunteers of male and female of same age group. The micronuclei, nuclear buds, nuclear bridging was evaluated to identify the effect of aging other than the micronuclei. The nuclear buds after H2O2 treatment were higher than the nuclear buds before treatment. The micronuclei frequencies induced by H202 and proliferative capacity didn’t differ with an increasing age. They postulated the reason that the decrease in H2O2-induced micronuclei frequencies compared to spontaneous Micronuclei frequencies may be stimulating an apoptosis by H2O2 treatment leading to underscore cells. It require further studies for the investigation.63
Sharma et al., 2012 evaluated the precancerous genetic damage biomarkers in minimally invasive tissue of professional sports. The study was ensured in the Players professionally active in their sport from 3 to 11 years on a daily training session of 4h/day. The players have significantly increased level of genetic damage in both
27
hockey and base-ball. The mean tail length of the hockey player found to be higher than the other team. There was no difference exist among gender. The DNA migration was found to be greater in the hockey players.64
Bansal et al., 2012 estimated the micronuclei in the tobacco users of Punjabi population. The study was conducted in the 75 healthy male subjects. The significant level of increased micronuclei frequency in smokeless tobacco than in smokers was found. The habituated smokeless tobacco and the smokers have higher risk of cancerous change than the non smokers. The micronuclei can be used as a biomarker of genotoxicity and helps in analysing the cytogenetic changes. The correlation between the micronuclei and the genetic damage acts as an effective alternative for metaphase analysis.65
Mukunda et al., 2013 reviewed the various methods available to detect the apoptotic cells. Microscopic analysis can provide information of cell surface, cell-cell interactions. The various techniques to correlate apoptotic cells to their phenotype were discussed. The accurate detection of apoptosis in various stages was described.
Dysregulation of apoptosis can play a primary or secondary role leading to cancer whereas excessive apoptosis contributes to neuron degeneration, autoimmunity, AIDS, and ischemia. The techniques for detecting apoptotic cells will allow the development of more effective, higher specific and therefore better-tolerable therapeutic approaches.
It helps to assess the apoptotic index, a known indicator of prognosis and metastasis of various treatment modalities.66
Shetty et al., 2013 conducted a study in 50 subjects of age group of 20-50 years to assess genotoxicity in the peripheral lymphocytes of smokers. Out of which they recruited 30 subjects of smokers and found no significant difference in the analysis
28
of DNA damage between smokers and non-smokers. The better standardized protocol was advised for better validation of the comet assay. It requires large number of samples for the better conclusive results.67
Li et al., 2013 carried out an invitro study to identify the method to detect the radiosensitivity of tumour cells. Many biological markers for the simultaneous determination results in a more accurate identification of the radiosensitivity of tumor cells. They identified that PCR helps in identifying the deletion and the comet assay used along with PCR helps in detecting the three tumour cell lines HepG2, EC-9706 and MCF-7. The most notable differences among these three tumor cell lines were observed by comet assay following 8–Gy γ-ray irradiation. A combined method of nested PCR and comet assay, is the most effective and accurate method in evaluating the radiosensitivity of tumor cells.68
Jyoti et al., 2013 assessed the DNA damage in the pan masala, gutkha chewers and the smokers. The study group includes 300 individuals which comprises of various groups. The study comprises 300 individuals of which 50 individuals were gutkha chewers along with smoking, 50 individuals were pan masala chewers along with smoking, 50 individuals were gutkha chewers, 50 individuals were pan masala chewers, 50 individuals were smokers and 50 individuals were non-users (control) or not having any addiction The mean tail length was greater in the gutkha chewers along with the smoking was noted. The greater DNA damage was reported in pan masala and gutkha than the smokers. Pan masala, Gutkha were found to be more genotoxic than the other forms.69
Bhat et al., 2013 had done a study to evaluate the attitude, practices and perceived barriers of smoking cessation among Dentists in Udaipur city, Rajasthan.
29
Tobacco is one of the important causes of morbidity and mortality. Our health professionals should have a major role in promoting the smoking cessation. Most of the dentist had good attitude towards smoking cessation and everyone have a thought that it was the responsibility but only few have adequate knowledge of practising smoking cessation and referral for it. The lack of cooperation among the patient and lack of time was found to be the most important barriers for smoking cessation.1
Bhagawath et al., 2013 carried out a study and found the DNA damage in buccal mucosal cells of patients with oral squamous cell carcinoma by single cell gel electrophoresis. The study includes the patients with oral squamous cell carcinoma and 30 patients with normal buccal mucosa as a control group. The buccal smear was taken from the individuals and the collected cells were centrifuged and single-cell gel electrophoresis (SCGE) assay was performed. DNA damage was visualized under a fluorescent microscope. The statistically significant step wise increase in DNA damage levels related to TNM staging was noted. The assessment of DNA damage will help in assessing the prognosis.70
Silva et al., 2013, found the DNA damage in COPD patients and correlation with polymorphism of repair genes. Fifty-one COPD patients, treated at Santa Cruz Hospital by the Research Group for Health Rehabilitation, Santa Cruz do Sul, RS, Brazil were included in this study. COPD was diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease guidelines (GOLD) using clinical history, physical examination, and presence of airflow obstruction, defined as a ratio of forced expiratory volume in one second (FEV1) to forced vital capacity (FVC) less than 70% of predicted value. The peripheral blood was used for estimating the DNA damage by comet assay and polymorphism by PCR. The residual damage was high in COPD
30
patients with risk allele in the four genes. The polymorphisms such as XCCR1, XCCR2, XCCR3, XCCR4 was suggested to have direct association with the COPD. 71 Sridhar et al., 2014 reviewed the epidemiology, control and prevention of tobacco induced oral mucosal lesions in India. Oral cancer is one of the leading causes of morbidity and mortality especially in developing countries. Tobacco consumption in smokeless and smoking form along with alcohol is found to be the primary risk factors.
Tobacco is a major health challenge with various tobacco products available for use which are known to have harmful effects on the oral mucosa. The oral lesions caused by tobacco are inclusive of those that are less likely to progression to cancer. Few lesions in the oral cavity have increased tendency to develop into cancer and cancerous lesions. Prevention and control of tobacco induced oral mucosal lesions is the prime requisite currently and mainly involves measures undertaken at primary, secondary and tertiary levels. Primary prevention plays an important role in tobacco induced lesions and steps can be taken at policy level, community as well as individual level. The epidemiological data of tobacco induced oral mucosal lesions in India available in the literature with an overview on various strategies for their prevention and control were reviewed.5
Janbaz et al., 2014 reviewed the risk for oral cancer from smokeless tobacco.
The various forms of smokeless tobacco were discussed. Various carcinogenic agents are tobacco-specific nitrosamines (TSNAs), polonium, formaldehyde, cadmium, lead, and benzopyrene. The combustion of the smokeless tobacco results in the synthesis of a chemical mixture containing a highly reactive oxygen and smokers show low plasma levels of antioxidant vitamins. In the management for oral cancer, the resistance and tolerance to the existing drugs has led to decreased efficacy. The attempts have been made to overcome this problem by increasing the drug delivery to the target site by the
31
use of polymers through nanotechnology and synthesis of new drugs, either by the use of proteomics or synthesis from lactic acid bacteria or marine microorganisms.72
Khan et al., 2014 compared the genetic damage that occurs in precancerous and cancerous patients by micronuclei and comet assay. The study was done in 260 patients including the leukoplakia, lichen planus, OSMF, Oral squamous cell carcinoma and the normal. They mentioned that leukoplakia patients have more DNA damage than the oral squamous cell carcinoma. This suggests that oral leukoplakia have more tendency of malignant transformation. The gender wise difference in the DNA damage was not found. The greater number of micronuclei had seen increasing from the normal group to precancerous and oral cancerous patients. Only few studies have been done in the buccal cells and the procedure found to be much erroneous and needs more validation. 73
Goncalves et al., 2014 analysed the genotoxic and cytotoxic effects due to orthodontic brackets when subjected to both the HepG2 and HOK cell lines. The stainless steel brackets are also genotoxic. The MTT reduction assay was used for analysing the cytotoxicity, comet assay was used to identify double strand breaks and micronuclei assay to verify the DNA damage.74
Jurel et al., 2014 reviewed the importance of early diagnosis of oral cancer and explained that before the clinical changes to occur, some genetic changes occur subclinically. Molecular biology is highly promising in detecting the alterations at a molecular level much before they are seen under a microscope and much before clinical changes occur. Molecular studies serve as the basis by which we will eventually be able not only to evaluate the clinical assessment and classification of oral lesions but also predict malignant potential of oral lesions, thus reducing the incidence and increasing
32
the scope for early diagnosis and treatment. The various changes that occur during the pathogenesis were discussed and they put forth that new developments should be made in the investigating field of oral cancer. It is now very important to create a localized, collaborative network of scientist from different specialties, each with new or established interest in oral cancer, for scientific vigor and rapid improvements in investigating the oral cancer research. 75
Manjunath et al., 2014 reviewed the update on field cancerization. The hypothesizes is that the entire epithelial surface of the upper aerodigestive tract has an increased risk for the development of premalignant lesions, because of multiple genetic abnormalities in the whole tissue region. Cancer begins with multiple cumulative epigenetic and genetic alterations that sequentially transform a cell or a group of cells in a particular organ. These early genetic events may lead to the clonal expansion of preneoplastic daughter cells in a particular tumor field. Subsequent genomic changes in some of these cells drive them toward the malignant phenotype. These transformed cells are diagnosed histopathologically as cancers, leading changes in the cell morphology.76
Guttikonda et al., 2014 evaluated the DNA damage in the peripheral blood leukocytes of the tobacco associated individuals. The DNA damage in smokers with clinically normal mucosa and in oral carcinoma was analysed. The significant mean DNA damage was found from normal mucosa, tobacco users with normal mucosa and tobacco users with oral cancer patients. They inferred that by evaluating the DNA damage the predisposition of cancer development may be identified.77
Rojas et al., 2014 explained the importance of comet assay in epithelial cells of nasal, corneal, buccal and tear ductal cells. They explained various methodologies,
