1. Introduction

Oral cancer (OC) is the most common cancer in South Asia and accounts for 40% of all malignancies in the Indian Subcontinent ^[1]. Several oral lesions such as leukoplakia (OL), erythroplakia and lichen planus carry an increased risk for malignant transformation to OC in the oral cavity ^[2]. Smoking and alcohol intake are the best defined and extensively researched risk factors for OC ^[3]. DNA damage, occurring as the result of detrimental effects of certain agents, in large part by the products of oxidative metabolism and in particular by reactive oxygen species (ROS), are probably the most potential spontaneous mutagenic events ^[4]. The scavenging of the ROS in physiological system such as blood and tissues is carried out by enzymatic and non-enzymatic antioxidants ^[5]. The enzymatic antioxidant includes superoxide dismutase, catalase, glutathione peroxidase. The non-enzymatic anti-oxidants include lipid soluble vitamins, vitamin E, vitamin A, water soluble vitamin C and glutathione ^[5].

Glutathione (GSH) acts as both, a nucleophile and a reductant, and can therefore react with electrophilic or oxidizing species before the latter interact with more critical cellular constituents such as nucleic acids and proteins ^[6]. Glutathione directly scavenges free radicals, and also serves as a substrate for glutathione peroxidases and glutathione S-transferases during the detoxification of ROS ^{[7, 8]}. Recently studies have been carried out on glutathione levels in patients with diabetes, dental caries and head and neck squamous cell carcinoma ^{[3, 9, 10]}. The aim of our study was to evaluate the levels of serum glutathione in oral leukoplakia and oral squamous cell carcinoma patients.

2. Methods

The study involved 75 subjects with age range of 20 to 65 years reporting to the Department of Oral Medicine and Radiology. Twenty-five clinically diagnosed and histopathologically confirmed cases of oral leukoplakia formed the first group (OL). Twenty-five histopathologically confirmed of oral cancer (OC) cases were enrolled into the second group. Twenty-five age and sex matched healthy controls formed the third group (HC). Each of the study groups had five female subjects. Patients with diabetes mellitus and patients with history of previous malignancy were also excluded from the study. 5ml of venous blood was collected from the antecubital vein by venipuncture. Serum GSH levels, were determined by the method of Beutler ^[11]. 100µL of filtered serum was diluted with 1mL of distilled water. 500µL of the diluted serum was taken and to this 2mL of phosphate solution and 250µL of DTNB [5,5-Dithiobis (2 nitrobenzoic acid)] reagent were added. Simultaneously (use capital letter) a blank was maintained containing 200µL of distilled water, 300µL of precipitating solution, 2mL of phosphate solution and 250µL of DTNB. The intensity of yellow colour formed was sphectrophotometricallyread at 412nm against blank using “Cystronics 106”(wavelength 340-960nm). The optical densities obtained were plotted against the standard graph. The concentration of glutathione was calculated graphically and multiplied with respective dilution factors and the total glutathione in the sample was expressed as mmol/L. The data obtained were subjected to statististical analysis using SPSS version 17 software. One-way ANOVA was used to compare the data among the groups and the differences were considered statiscally significant if P values were 0.05 or less.

3. Results

The mean serum glutathione in group OL was 01.04 ± 0.22mmol/L . Whereas in group OC the mean serum glutathione level was 0.94 ± 0.27mmol/L. The mean serum glutathione level of the group HC was 1.88 ± 0.36mmol/L. The mean serum glutathione levels of the male subjects in study groups OL, OC and HC were 0.94 ± 0.28mmol/L, 0.72 ± 0.28mmol/L and 1.97 ± 0.17mmol/L respectively (Figure 1). Whereas the mean serum glutathione levels of the female subjects in study groups OL, OC and HC were 0.91 ± 0.07mmol/L, 0.86 ± 0.19 mmol/L and 1.74 ± 0.27mmol/L respectively (Figure 2). The serum glutathione levels of female subjects were lower than male subjects but not statistically significant. When the mean serum glutathione level of group OL was compared to group HC a statistically significant difference (p=0.001) was observed. The mean serum glutathione level of group OC was higher than group OL but the difference was not statistically significant (p=0.1). The mean serum glutathione level of OC group was significantly lower (p=0.001) than the HC group (Figure 3).

Figure 1. Mean serum glutathione levels in male study subjects

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Figure 2. Mean serum glutathione levels in female study subjects

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Figure 3. Intercomparison of mean serum glutathione levels in the study groups

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4. Discussion

Glutathione (GSH) is a major intracellular antioxidant and hence plays a major role in cancer prevention ^[12]. GSH acts as first line of defense against oxidative stress ^[13]. Few studies have shown a reduced occurrence of polycyclic aromatic hydrocarbon induced oral cancer in experimental animals fed with diet rich in glutathione ^{[14, 15]}. GSH detoxifies carcinogens and also regulates immune system by mitogenic response and lymphocytic proliferation ^{[16, 17]}. Several serum studies have revealed decrease in the levels of plasma GSH levels in oral cancer ^[18]. In one Indian study the levels of plasma glutathione consistently reduced in the advanced stages of oral cancer when compared to initial stages ^[19]. Utilization of the glutathione by tumor tissues or by excessive oxidation in circulation were the probable reason for the depletion of gluthione in plasma ^[19]. Recently few studies have been carried out on salivary glutathione levels in smokers and in patients with dental caries and oral cancer ^{[3, 5, 20]}. These studies showed alterations in glutathione levels in study groups when compared to controls. In our study we evaluated serum glutathione in oral cancer and precancer patients. We found reduced glutathione levels in both oral cancer and precancer study groups. This depletion could be due to an imbalance in the free-radical and antioxidant homeostasis in the body fluids that occurs during carcinogenesis. Although statistically insignificant, the serum glutathione levels in oral cancer patients were higher than patients with leukoplakia. The results obtained from this study suggests that the altered levels of serum glutathione could be used as an ancillary screening tool for individuals with risk of oral cancer.

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