Abstract
Tobacco exposure is an established risk factor for head and neck squamous cell carcinoma. Individuals vary in their susceptibility to the insults of tobacco carcinogens based in part on their abilities to metabolically activate or detoxify these compounds. Biomarkers are becoming available that can be used to assess the susceptibility of an individual to tobacco related cancers. In this study we investigated the effects of tobacco on peripheral lymphocytes in the identification of susceptible individuals and established oral squamous cell carcinoma cases which might provide a powerful deterrant to the use of tobacco products

Introduction
Tobacco in both smoking and smokeless forms have been associated with oral cancer for many decades.1Tobacco products are complex mixtures of chemical constituents, many of which have carcinogenic or toxic properties.2,3 Cigarette smoke contains at least 3500 identified chemical constituents, of which 40 are accepted carcinogens and some of these carcinogens are also found in unburned processed tobacco, which is used in the manufacturing of smokeless tobacco products. The major carcinogen in tobacco belongs to the family of nicotine derived nitrosoamines collectively called “tobacco specific nitrosamines”. Polynuclear aromatic hydrocarbons such as Benzo[a]pyrene and aromatic amines are the other carcinogens found in tobacco. These agents are foreign chemicals. The response of mammalian systems to such chemicals is to metabolize them, mainly through enzyme-catalyzed reactions. These metabolic reactions detoxifies most of these chemicals and facilitates excretion through urine or feces. However, some of these reactions can result in the generation of electrophilic (electron-seeking or positively charged) intermediates that can react readily with nucleophilic sites in critical cellular molecules such as DNA, RNA and protein. These electrophilic intermediates are called ultimate carcinogens, and their covalent reaction products with DNA, RNA, or protein are called adducts.
The formation of specific DNA adducts is thought to be a critical step in carcinogenesis because these adducts, if unrepaired, can lead to miscoding during DNA replication, resulting in somatic mutations and derangement of normal growth controls. The formation of specific DNA adducts and the resulting mutations is considered to be responsible for activation of oncogenes or inactivation of tumor suppressor genes – the two major events thought to be involved in the multistep carcinogenic process.
The metabolic activation and detoxification processes ultimately controls the levels of particular carcinogen DNA adducts that form in tissues of people exposed to tobacco products. High levels of miscoding adducts that persist unrepaired in relevant genes will lead to a higher probability of critical permanent mutations. Thus the balance between metabolic activation and detoxification should be one factor influencing the probability that an individual will develop cancer on exposure to tobacco. Quantitation of metabolically activated carcinogens that reach target macromolecules is one way of assessing the ability of an individual to activate a carcinogen. One of the most extensively employed method to assess genetic effects of such exposures to tobacco carcinogens has been the analysis of chromosomal alterations in stimulated blood lymphocytes of exposed persons.
Cytogenetic alterations in cultured peripheral blood lymphocytes, such as chromosomal aberrations, Sister Chromatid Exchanges, and micronuclei, have for many years been applied as biomarkers of genotoxic exposure and early effects of genotoxic carcinogens.4,5 These cytogenetic alterations in lymphocytes are considered surrogate endpoints in surrogate cells, presumed to represent the more specific chromosomal alterations important in carcinogenesis in target tissues.5 The fact that tobacco contains established carcinogens which are genotoxic and capable of inducing chromosomal damage, it was therefore considered to study the frequencies of numerical and unstable structural chromosomal aberrations in the peripheral lymphocytes of individuals who were exposed to various forms of tobacco (tobacco users) and also in oral squamous cell carcinoma patients.

Materials and methods
Subjects
The study groups consisted of 15 histopathologically diagnosed oral squamous cell carcinoma individuals, 15 tobacco users both in smoking and smokeless forms and 5 healthy non-tobacco users. The characteristics of the individuals in the study group were as follows:

(i) Oral carcinoma individuals included histopathologically diagnosed 9 cases of well differentiated squamous cell carcinoma, 4 cases of moderately differentiated squamous cell carcinoma and 2 cases of poorly differentiated squamous cell carcinoma. Individuals with recurrence or those undergoing/undergone radiotherapy were excluded from the study.
(ii) Smokers had a smoking period of atleast 10 years without intervening pauses before the sampling time and smoking atleast 12 cigaretes a day
(iii) Smokeless tobacco users had used tobacco along with betel quid for almost 9-10 hours a day
(iv) Healthy non-tobacco users had refrained from any form of tobacco usage and should have had no family history of any genetic disorders

Cell culture
Peripheral blood was drawn into heparinized syringes and lymphocyte cultures were obtained using media containing RPMI with L- Glutamine without sodium bicarbonate, 20% fetal calf serum (sigma), Phytohaemagglutinin M (Pan Biotech) and antibiotics. The dividing lymphocytes were arrested at metaphase by addition of colcemid (PanBiotech; 0.5μg/ml) at the end of 64 hours. Following centrifugation at 1000RPM, cells were subjected to hypotonic shock using 0.075M KCl followed by fixation with a fixative containing methanol:acetic acid (3:1). Fixed cells were then dropped on cooled glass slides from a distance to ensure better spreading of chromosomes. Following air-drying slides were stained with Giemsa and analysed for the presence of numerical and structural aberrations.

Microscopy
Analysis of numerical and structural aberrations were made under light microscope on these Giemsa-stained preparations. A minimum of 30metaphases were scored for each individual. Numerical aberrations included the diploid status of each spread (hypodiploidy/hyperdiploidy) while unstable alterations like dicentrics, acentrics, minutes and ring chromosomes were assessed for structural aberrations.

Statistical analysis
The obtained data was analyzed by applying the SPSS Package 16.0 software and a p-value <0.05 was considered significant.
Results

The age and sex distribution for the study population are presented in table I. 150 cells were scored for the controls, 462 cells for Heavy tobacco users and 468 cells for squamous cell carcinoma patients.

Table 1: Distribution by age and sex – study population
HTU – Heavy Tobacco Users;	SCC-Squamous cell carcinoma

Numerical aberrations in the study population

Table II shows the numerical status of the chromosomes in the controls, tobacco users and squamous cell carcinoma subjects. All of the cells in the control group exhibited the normal diploid set of chromosomes while 3 out of 462 cells scored in tobacco users and 10 out of 468 cells scored in oral squamous cell carcinoma patients(SCC) showed hypodiploidy (45 chromosomes).

Table II: Frequency of numerical aberrations in the study population

On comparison of hypodiploidy status of cells in the  study population (table III) a p-value of 0.039 showed statistically significant results.

Table III: Frequency of hypodiploid cells in the study population

Frequencies of structural aberrations in the study population

Table IV and Graph I shows 0.65% dicentric chromosomes in a total of 462 cells in tobacco users and 2.14% of dicentrics in a total of 468 cells of the squamous cell carcinoma individuals. Ring chromosomes were seen only in oral squamous cell carcinoma patients group where 1.28% of a total of 468 cells exhibited ring structure. Acentrics or minutes type of aberrations were not present in any of the groups in the study population.

Table IV: Frequency of structural aberrations in the peripheral lymphocytes of study population

Graph I: Distribution of structural aberrations in the study population

Comparison of dicentric frequency in the study population (Table V) showed statistically significant results in the study population with a p-value of 0.039 while comparison of ring chromosome (Table VI) gave a p-value of 0.020 which was considered significant.

Table V: Comparison of dicentric frequency in the peripheral lymphocytes of study population

Table VI: Comparison  of ring frequency in the peripheral lymphocytes of study population

Discussion
The number of chromosomes in a normal cell is diploid i.e., 46 chromosomes. Based on the modal number of chromosomes, the numerical aberrations can be classified as hypodiploid (<46 chromosomes), diploid (46 chromosomes) or hyperdiploid (>46 chromosomes). Several cytogenetic studies have quantified unstable type of chromosomal aberrations such as dicentrics, acentrics, ring chromosomes and minutes in peripheral blood lymphocytes of normal subjects, smokers and carcinoma patients.6,7 Several studies 8,9 have shown that numerical and structural aberrations are usually associated with birth defects and human neoplasms.

In the present study, the frequency of numerical and structural chromosomal aberrations in human peripheral blood lymphocytes of healthy individuals, tobacco users and histopathologically diagnosed oral squamous cell carcinoma patients using conventional Giemsa stained karyotyping procedure was determined.

Numerical aberrations in the study population
The frequency of numerical aberrations seen in the study population was that all of the healthy controls showed diploid set of chromosomes while 3 of the 462 scored in heavy tobacco users and 10 out of 468 cells scored in oral squamous cell carcinoma patients showed hypodiploidy. These hypodiploid cells showed 45 number of chromosomes which generally arises from the loss of a whole chromosome, unbalanced translocations or the formation of dicentric chromosomes. In our study the reason for the hypodilpoid status was the occurrence of dicentrics in all of the cases. Our studies confer with that of Ravindran et al.,10 who found 12 numerical aberrations in a total of 75 cases. Hyperdiploidy was not seen in any of the three groups and our results on hyperdiploidy did not confer with that of Van Dieman et al.,11 who had demonstrated a higher frequency of hyperdiploidy in smokers. The reason for this may be that the number of metaphases scored were less in our study when compared to their study and also their study had demonstrated hyperdiploidy utilizing the more precise FISH techniques as compared to Giemsa stained preparations that was used in our study.

Structural aberrations in the study population
Cytogenetic studies quantifying unstable chromosomal aberrations such as dicentrics, acentrics, ring chromosomes and minutes have been reported in the peripheral lymphocytes of normal subjects, smokers and carcinoma patients.11 In our study the controls showed no structural chromosomal aberrations while dicentrics (Figure 1) were observed in heavy tobacco usersand at a higher frequency in oral squamous cell carcinoma patients. Ring chromosomes(Figure 2) were seen to occur only in oral squamous cell carcinoma patients at a statistically significant level. Van Dieman et al., in their study11 have shown a 2-fold higher frequency of dicentrics and ring chromosomes in 42% of smokers when compared to non smokers and this higher frequency of chromosomal aberrations could be attributed to inclusion of only smokers in their study and studies have shown that tobacco smoke carcinogens causes DNA double strand breaks at a higher level or that the DNA damage repairing ability could be less in smokers compared to that of tobacco chewers who constituted a significant number in our study. Also the occurrence of statistically significant number of ring chromosomes in squamous cell carcinoma patients supports the findings of Schantz and Hsu12 who have shown a higher degree of chromosomal mutagen sensitivity in head and neck cancer patients.

Studies13,14 show that the karyotypes seen in cells of oral squamous cell carcinomas are complex, often triploid and are composed of multiple numerical and structural abnormalities, including deletions, balanced and unbalanced translocations, isochromosomes, dicentric chromosomes and homogenously staining regions. The most striking manifestations of genetic instability in cancer cells is the variation observed in the karyotypes of different cells, even within the same tumour. William S Saunders15 in their observation on the source of chromosomal instability cultured cells derived from human oral squamous cell carcinoma have supported the fact that theses cell-to-cell variations in chromosome structure and numbers in cancer cells result from (i) dicentric chromosomes forming anaphase bridges and (ii) specific structural defects in the spindle and chromosomal seggregational machinery. Though it may be argued that the mutagen sensitivity of peripheral bood lymphocytes does not reflect mutagen sensitivity in the target tissue. Denissenko et al.,16 had found a similar adduct pattern in different cell types: a)Hela cells b) bronchial cells c) normal human fibroblasts and d) peripheral blood lymphocytes, as seen in target tissues suggesting that the same adduct pattern is probably present in different target cells for oral tissue transformation.

Conclusion
The results of the present study supports the fact17 that the chromosomal aberration frequency in peripheral blood lymphocytes may be an indicator of cancer risk in heavy tobacco users and that the increased frequency of ring chromosomes seen in the undifferentiated squamous cell carcinoma cases observed suggests a definite pattern as the grade of the carcinoma increases and a strong genetic instability.

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Dhanuja M ,* Ramesh V,**Premalatha B .*** Department of Oral Pathology and Microbiology, Mahatma Gandhi Post Graduate Institute Of  Dental Sciences, Pondicherry