In the present study, total number of 50 patients were involved Group I - 20 patients belonging to control, Group II- study group consisting of 10 patients with oral pre-cancer (oral leukoplakia) (sub group A) and 20 patients with oral carcinoma oral squamous cell carcinoma-OSCC- subgroup B.

The subject patients were selected and physical examination with routine investigation including complete blood examination and reports were done and were declared free from any other systemic diseases. The mean age of the patients was between 45.1y to 52.8 y. Signed informed consent on an institutionally approved document was obtained from all the participants. The study was approved by the human ethics committee of India. The patients selected for oral leukoplakia or OSCC had a habit of smoking, chewing or both habits together. The patients who had come for dental examination of carious tooth were selected for control group and for selecting the patients with oral leukoplakia, patients were clinically examined, and criteria for selection was evidence of white lesion in the oral mucosa more than 5mm in diameter, non scrapable and the lesion is raised, firm, mildly indurated and on histopathological examination the leukoplakia showed dysplastic changes in the epithelium. The patients suffering from oral carcinomas were clinically and histologically confirmed as squamous cell carcinoma.

Estimation of lipid peroxides was done in control and study groups including oral leukoplakia and OSCC. A needle puncture was made with the help of 2ml sterile disposable syringe and 2ml of venous blood from ante cubital vein were collected. The blood was transferred to sterile test tube and left for clotting. The clotted blood sample was centrifuged at 3000rpm for 5min and obtained serum sample was used.

Unstimulated saliva was collected and the sample was then centrifuged at 3000rpm at 4^oC for 5min and saliva free of large particle debris were used for estimation of lipid peroxide in saliva.

Normal tissues were collected from gingival biopsy from normal persons free from disease who accompanied the patients who came for treatment. For the study group patients, toluidene blue (1%) staining of the lesion was done and areas of maximum staining were chosen for biopsy in order to specifically limit the incision to areas of dysplasia [4]. The tissues which were collected were made to two sections and transferred in bottles containing normal saline and the other one containing 10% formalin and sent for histopathological examination using routine staining procedure. The fresh tissue samples were homogenized with phosphate buffer and processed immediately.

Lipid peroxidation was estimated as evidenced by formation of thiobarbituric acid reactive substances TBARs. TBARs were assessed and described by Okhawa et al., [10]. The pink coloured chromogen formed by the reaction of thiobarbituric acid with MDA malondialdehyde breakdown products of lipid peroxidation was read at 532 nm [8]. All the MDA values were expressed in nmol/ml.

Descriptive statistics included mean and standard deviation which were estimated from the sample for each study group. Mean values were compared among different study groups by students independent t-test of one-way-ANOVA approximately. Multiple range tests by Tukey-HSD procedure was employed to identify the significant groups, if p-values in one-way-ANOVA was significant. Students paired t-test was used to test the significance of difference in mean values between mediums, within each study group.

In the present study, p<0.05 was considered as the level of significance.

[Table/Fig-1]

The levels of lipid peroxidation were recorded with reference to the released MDA and the results in saliva, serum and tissue were compared with control group and study groups with oral leukoplakia and OSCC.

The control values of mean lipid peroxides in saliva were 0.349± 0.09 but the values in saliva were increased in patients with leukoplakia which was 0.651±0.08. This increase was statistically significant with p-value <0.0001.The control values of mean lipid peroxides in serum was 0.712±0.13, which were increased in patients with leukoplakia as 1.346±0.26 which was statistically significant (p<0.0001). The control values of mean lipid peroxides in tissue were 0.59±0.13 and the values were increased in patients with leukoplakia as 0.79±0.09 which was also statistically significant (p<0.0001).

In saliva, the mean lipid peroxides (MDA) values in patients with OSCC was 1.007±0.16, which was increased when compared to control group which was statistically significant (p<0.0001). In serum, the mean lipid peroxide value in patients with OSCC was 1.824±0.55, which was also increased when compared to control group and was statistically significant (p<0.0001). Similarly, in tissue also the values were increased in patients with OSCC which was 1.115±0.12 which was also statistically significant. Thus, lipid peroxidation in all the mediums (saliva, serum and tissue) was more in patients suffering with oral leukoplakia and OSCC when compared with control group.

[Table/Fig-2]

The present study also included the evaluation of mean lipid peroxides in males and female patients. The value of mean lipid peroxides were 0.347±0.10 in saliva, 0.717±0.12 in serum and 0.576±0.15 in tissue in males of the control group. In females of the control group, values were recorded and were 0.353±0.08 in saliva, 0.703±0.15in serum and 0.611±0.11 in tissue.

(i)The mean saliva lipid peroxides in males in patients suffering with oral leukoplakia was 0.661±0.08 and mean value in females in the same study group was 0.629±0.08. On comparison, it was not statistically significant as there was only a slight variation between males and females.

(ii) In serum, the mean lipid peroxides in males in patients with oral leukoplakia was 1.273±0.17 and in females it was 1.515±0.41. In this also there was a mild variation which was not statistically significant.

(iii) In tissue, the mean lipid peroxides in oral leukoplakia patients were 0.794±0.09 in males and 0.780±0.11 in females which was also statistically not significant.

(i)In saliva, in male patients with OSCC, the mean MDA values were 0.964±0.16 and in female patients it was 1.087±0.14.

(ii) In serum of male patients with OSCC, the mean MDA values were 1.814±0.46 and in female patients, the values were 1.844±0.74.

(iii) In tissue of male patients with OSCC, the values were 1.104±0.12 and in female patients, it was 1.134±0.12

The mean values of lipid peroxides in saliva, serum and tissue in male and female patients with OSCC were found to be only a slight variation which was not statistically significant. Also in the mean value of MDA in males with oral leukoplakia and OSCC compared to control and female patients with oral leukoplakia and OSCC compared to control, there was slight variation which was not statistically significant.

[Table/Fig-3&4]

In leukoplakia, the mean value of lipid peroxides in smokers in saliva, serum and tissue were compared with non smokers in all the medium and was found to be statistically non significant. Also, in OSCC, the mean values of lipid peroxides among smokers were compared with non smokers in all the mediums and was found to be non significant.

In leukoplakia the patients with chewing habit showed mild variation between patients with non chewing habit in the entire medium. In OSCC also, the patients with chewing habit showed slight variation between patients with non-chewing habit. In both study groups, the values were statistically non significant. On comparison of mean values of lipid peroxides among smokers and chewers the values were increased in patients with OSCC than in patients with leukoplakia

The results show that the lipid peroxide mean values in saliva, serum and tissue is more in patients with leukoplakia and OSCC compared to the control group. The mean lipid peroxide values among study group is more in OSCC than oral leukoplakia in saliva, serum and tissue. The lipid peroxide value in saliva, serum and tissue in males and female patients with oral leukoplakia and OSCC were not much significant when compared with control, but the values were higher in OSCC compared to oral leukoplakia in both males and females. In smoking and chewing habit also, the mean lipid peroxide values were increased in patients with OSCC than oral leukoplakia.

The aerobic life cycle, oxygen free radicals are formed in normal cell metabolism from molecular O₂, even though body has anti oxidant defenses, oxygen free radicals can cause constant damage to oxidizable molecules which are repaired or replaced in a dynamic equilibrium. When there is oxidative stress either in the form of over production of oxygen free radicals or from the deficiency of anti oxidant defense or repair mechanism, there can be irreversible tissue injury [11], (rheumatoid arthritis, ischemic heart disease, several auto immune disorders and cancer) [12].

The important exogenous cause of oxidative stress associated with oral pre-cancerous and cancer is the use of tobacco. Continuous exposure to carcinogenic benzopyrene and nitrosamine present in tobacco and areca nut predisposes the mucosal surface to malignant transformation. Tobacco smoking has been reported to stimulate H₂O₂ and hydroxyl radicals and chewing of betel quid with generate DNA maging oxygen free radicals causing chromosomal damage [13]. In the present study, patients with oral pre-cancer(oral leukoplakia) has been taken as oral leukoplakia is the best known precursor lesion and most common potentially transforming malignant lesion of the oral cavity. The risk of malignant transformation of leukoplakia with dysplasia has been reported as high as 43% [14].

Free radical chain reaction leads to lipid peroxidation which causes degeneration of cell membrane. Lipid peroxides are disintegrated quickly and forms reactive carbon compounds. Among these MDA is an important reactor carbon compound which is used as an indicator of lipid peroxidation [15].

The levels of MDA were evaluated in saliva, serum and tissue in this study. Saliva , a heterogenous fluid, apart from cleaning , lubricating, and buffering properties also acts as a first line of defense against free radical mediated oxidative stress involving a variety of reactions including lipid peroxidation [16]. So, saliva was used as a biological sample in monitoring lipid peroxidation in the present study. The MDA values were higher in patients with oral leukoplakia and oral cancer compared to normal patients. Interestingly, this clearly shows the relationship between free radical metabolism and malignancy [7].

The increase in lipid peroxidation in abnormally proliferating cells leads to an increase in serum lipid peroxides in precancer and cancer patients. Increase in MDA in oral cancer patients might be due to decomposition products of polysaturated fatty acids of biomembrane which are released in the blood. Such results have been reported in breast cancer patients also [17]. Similar statistically significant MDA levels were observed in tissue samples which is in contrast to many studies [13,18,19] wherein the inverse relationship of TBARS to tumour progression was attributed to cellular proliferation [13], decreased concentration of free fatty acids In tumour tissue and decreased levels of phospholipids [18]. However, in the present study, the positive correlation between TBARS and tumour tissue as compared to normal mucosa could be attributed to greater cyclooxygenase activity resulting from inflammatory mediators involved in tumour progression [20].

Thus, the patients with oral cancer showed increased levels of MDA when compared to patients with oral pre-cancer. This could be attributed to the initiation and promotion of multistage carcinogenesis induced by free radicals which were increased in oral cancer [11]. Free radicals and radical mediated lipid peroxidation reaction can alter according to sex, as per the study by Bast et al., [21], but in our study, these were not much variations in sex, which was similar to the study by Bulent et al., [22].

In patients with tobacco chewing and smoking, the mean MDA values are increased as tobacco consumption is positively co related with accumulation of free radicals leading to lipid peroxidation [23]. In the present study, increase in salivary concentration of MDA is co related with increase in serum. These findings reinforce the concept that saliva is a viable, easy to obtain and relatively risk free diagnostic biofluid for assessing biomarkers.

As there is an imbalance in the body defense mechanism, the anti oxidant activity is reduced in patients with oral cancer and pre cancer. Various studies have proved the role of vitamins and other anti oxidants in primary prevention of oral cancer. So, further emphasis should be given in various plant based anti oxidants which can detoxify the deleterious free radicals and may be also beneficial in chemoprevention of oral cancer. The role of saliva as a viable and relatively risk free biofluid warrants more attention and research into finding out more biomarkers for assessing biochemical changes of various chronic ailments like cancer, which affects the human body.

The results of this study highlight the already established role of anti oxidants in the chemotherapeutics of oral pre cancer and oral cancer. However, the role of saliva as an easily available and non invasively extractable biofluid, which is highlighted in this study assumes significance in the light of the fact that the salivary values of lipid peroxides obtained corresponded with the values obtained in serum and tissue. Therefore, future studies aimed at standardizing values of salivary MDA would provide clinicians an easily available biofluid to prognostically assess results of anti oxidant cancer therapy.