ASSESSMENT OF SERUM PARAOXONASE-1 ENZYME ACTIVITY , MALONDIALDEHYDE AND VITAMIN-C IN ORAL PREMALIGNANCIES

Oral premalignant lesions also known as potentially malignant disorders are a group of disease or syndromes which if left untreated can lead to oral cancer [1]. This way it carries a great significance in Indian perspective, as oral cancer shares about 1316% of the cancer load of India [2]. It is seen that maximum oral cancers are squamous cell cancers, where some of them can arise from an apparently normal mucosa while others arise from some clinically obvious premalignant lesions [3]. In Indian context, in contrary to that of western countries, these diseases are seen to be maximally coexistent with life style related problems like smoking, chewing tobacco, paan, betel nut, ghutkha, alcohol drinking and so on [4]. These known addiction substances can induce


INTRODUCTION
Oral premalignant lesions also known as potentially malignant disorders are a group of disease or syndromes which if left untreated can lead to oral cancer [1]. This way it carries a great significance in Indian perspective, as oral cancer shares about 13-16% of the cancer load of India [2]. It is seen that maximum oral cancers are squamous cell cancers, where some of them can arise from an apparently normal mucosa while others arise from some clinically obvious premalignant lesions [3]. In Indian context, in contrary to that of western countries, these diseases are seen to be maximally coexistent with life style related problems like smoking, chewing tobacco, paan, betel nut, ghutkha, alcohol drinking and so on [4]. These known addiction substances can induce Study location: The study was carried out in Dept. of Biochemistry in collaboration with Dept. of ENT at MGIMS, Sewagram.
Sample selection: Patients with oral premalignant lesions and diseased controls were recruited after obtaining their written consent in regional language.
Sample size: A total number of 63 samples.
Inclusion criteria: Cases of premalignancies were chosen totally on clinical diagnosis. They were recruited within the age group of 20 to 60years without any sex differentiation. Controls were taken considering the exclusion criteria Exclusion criteria: with no history of smoking and any past or present history of oral premalignancy were excluded from the study.
Grouping: Subjects were mainly divided into two group: Group 2: Test group, clinically according to the severity each of them were again divided into different subgroups. OSMF cases were divided into clinical Grades of I, II & III and OL group was divided in Homogenous and Non-Homogenous clinically.
Methodology: Clinical and demographic data was collected regarding their smoking habits and other risk behaviors by proforma method. About 10 ml venous blood was collected from participants in plain (for serum) and EDTA (for plasma) vials. Serum was used for PON-1, MDA estimation and plasma for Vitamin C.
Estimation of PON-1 Activity: Serum Paraoxonase 1 activity was measured spectrophotometrically (ELICO) according to Gan et al. (1991) [9] with some modifications. Using Paraoxon (Paraoxon Ethyl of Sigma-Eldrich) as a substrate, concentration of the product p-nitrophenol was measured at 420 nm using molar extinction coefficient of 18,290 M-1 cm-1 at pH 10.
Estimation of Malondialdehyde (MDA): Serum MDA level was measured according to Buege & Aust SD (1978) [10]. MDA in serumreacts with thiobarbituric acid (TBA) (Sigma-Eldrich) to form a colored product, absorbance of which was measured spectrophotometrically (ELICO) at 535 nm. The malondialdehyde concentration of the sample was calculated using the extinction coefficient of 1.56 x 105 M-1 cm-1.
Estimation of Vitamin C: Plasma Vitamin C was measured according to Aye Kyaw, (1978) [11]. The acid phototungstate used in this method serves as plasma protein precipitant as well as ascorbic acid extractant and color developing agent, as it gets reduced to tungstate blue by ascorbic acid. The blue color was measured in spectrophotometer (ELICO) at 700 nm. Absorbancewas read is read against blank, constituted with distilled water (instead of plasma), and subjected to all treatments simultaneously as test samples. For every set, a standard (Sigma-Eldrich) and blank are run through the procedure.

Statistical analysis:
For comparison in the groups we used unpaired t test, ANOVA, TUKEY HSD and for correlation analysis we used Pearson's Correlation Test. A 'p-value' less than 0.05 was considered significant.

RESULTS
OSMF cases were divided into clinical Grades of I, II & III (after Anil K Ghom) [12] and OL group was divided in Homogenous and Non-Homogenous clinically (after S Warnakulasuriya et al) [13] .
There were 5, 7 & 8 subjects in the subgroups of Grades of I, Grade II & Grade III OSMF accordingly. Likewise, in Oral Leukoplakia there were 12 subjects in Non-homogenous subgroup and 10 subjects in Homogenous leukoplakia subgroups. Tobacco related addiction like Paan, Kharra, tobacco chew, smoking were the common addiction habits in both the premalignant groups with a mean age addicting of 6 years.

DISCUSSION
The most commonly found PMD are OSMF, OL and erythroplakia [14] . In our study we got two groups of premalignancies i.e. OSMF and Oral Leukoplakia. Clinically, on inspection of oral mucosa, one can find a chronic fibrotic change in OSMF and white patches that cannot be characterized otherwise in Oral Leukoplakia. The malignant transformation rate for OSMF is 7.6% in17 years of follow up and for OL it is about 13.6% globally [15,16]. In India, according to various studies, there is a strikingly varied prevalence of both OSMF (0.03 to 3.2%) and OL (0.2% to 4.9%) [17,18].
Oral Premalignancies are found to share common etiological insults and common geographical distribution like Oral Cancers in India [19]. Thus, in these backgrounds mentioned and moreover, the absence of an established etiopathogenic mode and inability to predict a malignant transformation, makes oral premalignancy an important candidate to be investigated in line of oral cancer, at least in Indian perspective.
Talking about molecular mechanisms behind, oxidative stress is a known culprit behind cancerogenesis and amongst the various mechanisms that our body has developed to fight back, Paraoxonase -1 enzyme is one of them. PON -1, a hydrolytic enzyme with a wide range of substrate, gathered a significant interest as a protein that is mainly responsible for most of the antioxidant properties of High Density Lipoprotein (HDL). Its beneficial role against atherosclerosis is already well documented. PON-1 gene is mainly expressed in liver and it is transported in the circulation in association with lipoproteins and get delivered to the multiple other sites who doesn't syn-thesise PON-1 by themselves. PON-1 by interacting with Apo-A binds to HDL and gets circulated along the blood circulation. An increased accumulation of PON-1 in the media and intima of the aorta in atherosclerotic arties is attributed to macrophages which also connects the link of PON-1 to lipid peroxidation. Thus, it is seen that PON-1 has a capacity to protect our cells from lipid peroxidation [20]. By virtue of antioxidant property PON-1 enzyme achieved a heavily suspected role in pathogenesis of OSCC, which makes it an important candidate to be investigated in Oral Premalignancies too. In our study, wefound a significant decrease in serum PON-1 activity in both the premalignancies than the control group (Table1). Even in oral leucoplakia group mean serum PON-1 activity was significantly lower (P<0.05) than OSMF (Table1). A gradual decrease in the mean serum PON-    Table 3). Probably this is due to small sample size of these subgroups. However, interestingly, there was a statistically significant decrease (P<0.05) in PON-1 activity from Homogenous to Non-Homogenous Leukoplakia subgroups (P<0.05) (Table2, Table 3).Smokers are known to have a significant decrease in PON-1 activity along with higher propensity to have oxidized Low-Density Lipoprotein (LDL) and HDL. But the molecular mechanism is not clear till now [21]. Table 4. Correlation between serum PON-1 activity with MDA and Vitamin C in the study groups

Fig 1: Scatter Plot showing Correlation between serum PON-1 activity with MDA and Vitamin C
The addiction habits of our study population substantiate our findings of decreased serum PON-1 activity, andpositively indicates that the decrease in the protective antioxidant role of PON-1 is playing some role in their progression.
Reactive Oxygen species degrades polyunsaturated lipids forming malondialdehyde. This reactive aldehyde by its electrophilic properties produces advanced lipoxidation end products (ALE) by causing toxic stress in cells. This ALE is widely used as a biomarker to measure lipid peroxidation related oxidative stress in organisms.In this study, we found a grad- Frie et al showed that plasma ascorbic acid is the only endogenous antioxidant which is able to completely protect the lipids from detectable peroxidative damage induced by aqueous peroxyl radicals [25]. It acts as a co-antioxidant that regenerates α-tocopherol from α-tocopheroxy radical generated during the scavenging of free radicals. In this study we have estimated the antioxidant vitamins in plasma expecting that circulating vitamins are better reflected than their tissue/cellular levels. We found a statistically significant decrease in the mean plasma Ascorbic Acid in Oral PMD from controls ( Table 1). The decrease was also significant in OL than OSMF (P<0.05) ( Table 1). Except for Grade I and Grade II OSMF Vitamin C level was significantly lower (P<0.05) from less severe to more severe clinical subgroup of premalignancies ( Table 2, Table 3). Our overall findings are in agreement with those of Dr. Sarita Basu [26]. Vitamin C by its oxidizing properties reacts with superoxide produced from normal cellular metabolism; this in turn inhibits formation of nitrosamines during protein digestion and prevents damage to DNA and other cellular proteins. But very little is documented regarding the ability of Ascorbic Acid to maintain the oral mucosal integrity. In a study conducted by V. Touvinen et al, a significant higher percentage of Oral Leukoplakia than any other Oral Premalignancies were found in Vitamin C deficient cases [27].
Again, in OSMF, it was found that there was an increase in the production of highly cross linked insoluble collagen Type I, increase loss of soluble procollagen type III and collagen type VI. This cross-linking is attributed to the upregulation of lysyl oxidase, playing a crucial role in the development of Grade II OSMF from Grade I [28]. A decrease of such in plasma Vitamin C level probably indicates to its more utilization towards collagen synthesis. This hypothesis is also strengthened by our findings of significant reduction of plasma vitamin C level in oral premalignancies than controls. PON-1 is also found to be negatively correlating with MDA and positively correlating with Vita-min C in both OSMF and Oral Leukoplakia groups. In OSMF, PON-1 had fair negative correlation with MDA and in Leukoplakia it was rather fair positively correlated with Vitamin C. Despite these minor differences, it can be aptly said that overall finding suggests that decreased PON-1 activity was associated with increased oxidative stress parameters (increased MDA, decreased Vitamin C) in Oral Premalignant Disease patients.

CONCLUSION
From this finding it can be envisaged and concluded that the interplay between decreased serum PON-1 activity and increased in oxidative stress do play some role in the etiopathogenesis and progression of Oral Premalignant Diseases. Further large-scale studies for genetic susceptibility, PON-1 gene polymorphism, PON-1 activity in non -tobacco related oral Premalignancies are needed to substantiate our findings.