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Profiling of antioxidant superoxide dismutase in saliva of oral submucous fibrosis patients to categorize its diagnosis in varying stages.

Byline: Yuthicka Sirohi - Email: yuthickasirohi@gmail.com, Devi Charan Shetty, Aadithya B Urs and Harish Chandra Rai

Abstract

Background: Oral submucous fibrosis is a premalignant condition in Indian and South-East Asia. Role of oxidant- antioxidant in causation and progression of cancer and pre cancers is known. Reactive oxygen species are generated in the oral cavity during chewing areca nut, the major etiological agent in oral submucous fibrosis.

Objectives: To see the alterations in the salivary superoxide dismutase levels in various clinical and histopathological grades of oral submucous fibrosis.

Materials and Methods: Unstimulated saliva was collected from 25 oral submucous fibrosis patients and age and gender matched controls. The saliva was assessed for superoxide dismutase value by spectrophotometric method using assay kit (Bio Vision Catalog # K335-100). The oral submucous fibrosis cases were grouped into clinical stages and histopathological grades and superoxide dismutase values were compared in different clinical stages and histopathological grades.

Results: The superoxide dismutase levels were reduced in oral submucous fibrosis as compared to controls. A steady decline in the levels was seen as the clinical stage and histopathological grade of oral submucous fibrosis advanced. Conclusions: Salivary superoxide dismutase levels can be alternatively used as a surrogate marker for the diagnosis of oral submucous fibrosis.

Policy message: Oral physicians should advise the pan chewers to regularly check their salivary superoxide dismutase levels so as to ease the early diagnosis of oral submucous fibrosis.

Key words: Oral submucous fibrosis, reactive oxygen species, superoxide dismutase, saliva.

Introduction

Oral submucous fibrosis (OSMF) is a chronic fibrosing disease of oral cavity and oropharynx leading to limitation in mouth opening. Its pathogenesis is not well established, but is believed to be multifactorial. The chewing of betel quid (containing areca nut, tobacco and slaked lime) has been recognized as one of the most important risk factors for oral submucous fibrosis1.

Reactive oxygen species are generated in the oral cavity during chewing betel quid (areca nut) which is rich in Fe2+ and Cu2+ and raised tissue copper levels have demonstrated in oral submucous fibrosis tissues. Reactive oxygen species act by initiating lipid peroxidation2 while enzymatic antioxidant superoxide dismutase plays a key role in detoxification of superoxide anion radical and hence diminishes the toxic effects of this radical and other free radicals like hydrogen peroxide and hydroxyl radicals from secondary reactions2,3.

The diagnosis and prognosis of oral submucous fibrosis is done through biopsy which is invasive, time consuming and causes psychological trauma. As oral submucous fibrosis is a precancerous condition and not a precancerous lesion, therefore, it is important to select an appropriate site for biopsy where histopathological features are present4. Moreover, routine histopathological examination depends on the expertise of the examiner. Therefore, it is important that the diagnostic tests are simple, fast, less invasive and easily interpreted. They should give a reliable diagnosis, prognosis and be useful in monitoring the response to treatment.

The mean serum superoxide dismutase levels in oral submucous fibrosis patients were found to be decreased (mean superoxide dismutase 86.63+-20.36 U/ml) as compared to the healthy control group (mean superoxide dismutase 1.50+-0.30 U/ml). The authors correlated the results with the premalignant potential of oral submucous fibrosis4.

This study was designed to measure the levels of superoxide dismutase in saliva of oral submucous fibrosis patients and hypothesize its role in pathogenesis and progression of oral submucous fibrosis.

Materials and Methods

Subjects clinically suspicious of having oral submucous fibrosis who were routinely visiting the OPD of ITS-Centre for Dental Studies and Research, Ghaziabad, were selected for the study. Age and gender matched healthy individuals without any habit histories were taken as controls to assess the variation in superoxide dismutase levels between oral submucous fibrosis patients and healthy individuals. Informed consent was taken from them followed by history recording. All patients were males between the ages of 18-35 years and were free of any systemic disease and had not received any therapy for oral submucous fibrosis. The patients were grouped clinically according to Ranganathan et al (2004) classification5. After establishing clinical diagnosis and one hour after the food intake, patients were asked to generate saliva in their mouth and spit it in a wide test tube for 10 minutes.

The saliva was immediately centrifuged at 4000 rpm for 10 minutes and the resulting supernatant was used for further biochemical analysis. A punch biopsy of the cheek was simultaneously done from an appropriate site. Histopathologically the tissue was fixed, processed and graded according to Sirsat and Pindborg (1967)6 classification by a panel of three oral pathologists.

Superoxide dismutase was estimated using assay kit (Bio Vision Catalog # K335-100). This kit utilizes WST-1 that produced a water soluble formazan dye upon reduction with superoxide anion. The rate of reduction with a superoxide anion was linearly related to xanthine oxidase activity, and is inhibited by superoxide dismutase.

The inhibition activity of superoxide dismutase was determined by a colourimetric method using a spectrophotometer at wavelength of 450 nm.

The data was analyzed with one-way analysis of variance (ANOVA) test.

Results

Twenty male patients with oral mucosal fibrosis and twenty controls were included in the study. Superoxide dismutase levels in saliva were compared between patients and controls. Superoxide dismutase levels were lower in patients (0.70 U/ml) than in controls (0.98U/ml) (Figure-1).

The levels of superoxide dismutase were compared within different clinical and histopathological grades of oral submucous fibrosis and a steady decline in superoxide dismutase levels was seen with the advancement of disease (Figure-2). On application of ANOVA a statistically significant decrease in superoxide dismutase levels were observed in various clinical stages and histopathological grades of oral submucous fibrosis (Table-1 and 2).

Table 1: Assessment of SOD levels in various clinical stages of oral submucous fibrosis.

Clinical Stage###Mean###SD###F###p value of

###SOD levels###value###Sig (2tailed)###

Stage I (n=7)###0.8343###0.03409

Stage II (n=12)###0.6642###0.03965###92.43###0.00 1

Stage III (n=6)###0.46###0.07694

Table 2: Assessment of SOD levels in various histopathological grades of oral submucous fibrosis.

Histopatho logic###Mean###SD###F###p value of

Grade###SOD levels###value###Sig (2 tailed)

Early (n=8)###0.77###0.10

Moderately###9.767###0.001

advanced(n=14)###0.64###0.11

Early (n=3)###0.46###0.11

Discussion

Superoxide dismutase levels were low in patients with oral mucosal fibrosis and a steady decrease in the levels was found with the advancement of disease in concordance with previous studies6,7. The decline in superoxide dismutase levels showed a statistically significant correlation between the clinical stages of oral submucous fibrosis. However, no significant correlation was found between the superoxide dismutase levels and histopathological stages in the present study. The enzymatic serum superoxide dismutase levels in oral submucous fibrosis patients did not show any significant change in any stage of oral submucous fibrosis8.

Oral submucous fibrosis is one of the prevalent premalignant conditions in Indian and South-East Asian population. It is easy to diagnose but difficult to manage because of its progressive nature even after cessation of the adverse habits1.

The etiopathogenesis of oral submucous fibrosis is multifactorial with a very strong correlation between betel quid chewing and development of disease with a dose dependant relationship for both frequency and duration of chewing areca nut (without tobacoo)6. Substantial amounts of reactive oxygen species like superoxide anion and hydrogen peroxide are produced in the oral cavity during chewing of areca nut and catechu at a pH greater than 9.5. This areca nut-induced production of reactive oxygen species is enhanced by Fe2+, Fe3+ and Cu2+, but is inhibited by Mn2+ (2). These radicals can transfer their unpaired electron to oxygen to give free superoxide which is essential in this system to prevent oxidative stress. The superoxide dismutase catalyzes the dismutation of superoxide to hydrogen peroxide9.

O2 + O2 ------ H2O2 + O2

The hydrogen peroxide is then removed by glutathione peroxidase or catalase and this oxidative stress, arising as a result of imbalance between free radical production and antioxidant defences causes damage to a wide range of molecular species including lipids, proteins and nucleic acids. Antioxidants like, superoxide dismutase when present in low concentrations, as compared, to that of an oxidisable substrate, significantly delays or inhibits the oxidation of that substrate9. These antioxidants prevent lipid peroxidation, protein damage and modification of DNA bases or as chain breaking antioxidants that receive or donate electrons to the free radical with formation of stable products or as transition metal binding proteins which act by sequestering Fe or Cu, so that they are not available to drive the formation of hydroxyl radical8. The serum levels of antioxidants like superoxide dismutase and catalase are decreased in oral squamous cell carinoma and oral submucous fibrosis7,11.

The current study sheds further light on the role of saliva and oxidative stress in the pathogenesis of oral submucous fibrosis. As saliva can be easily obtained, tested and monitored for its antioxidants, nitrosation products, oxidized DNA, proteins, etc) therefore, using this marker one can help plan an early intervention, with local therapeutic agents acting as antioxidants that can be easily applied to the mucosa.

References

1. Pillai R, Balaram P, Reddiar KS. Pathogenesis of oral submucous fibrosis. Relationship to risk factors associated with oral cancer. Cancer 1992; 69: 2011-7.

2. Jeng JH, Chang MC, Hahn LJ. Role of areca nut in betel- quid associated chemical carcinogenesis: current awareness and future perspectives. Oral Oncol 2001; 37: 477-92.

3. Nair UJ, Floyd RA, Nair J, Bussachini V, Friesen M, Bartsch H. Formation of reactive oxygen species and 8- OH-Dg IN DNA in vitro with betel quid ingredients. Chem-Biol Interact 1987; 63: 157-69.

4. Uikey AK, Hazarey VK, Vaidhya SM. Estimation of serum antioxidant enzymes superoxide dismutase and glutathione peroxidase in oral submucous fibrosis: a biochemical study. J Oral Maxillofac Pathol 2003; 4: 44-5.

5. Ranganathan K, Uma Devi M, Joshua E, Kirankumar K, Saraswathi S. Oral submucous fibrosis: a case control study in Chennai, South India. J Oral Pathol Med 2004; 33: 274-7.

6. Sirsat SM, Pindborg JJ. The vascular response in early and advanced oral submucous fibrosis. Acta Pathol Microbial Scand 1967; 70: 179-84.

7. J Carlsoon. Salivary peroxidase: an important part of our defense against oxygen toxicity. J Oral Pathol 1987; 16: 412-6.

8. Gupta S, Reddy MVR, Harinath BC. Role of oxidative stress and antioxidants in aetiopathogenesis and management of oral submucous fibrosis. Indian J Clin Biochem 2004; 19: 138-41.

9. Suryakant BM, Tupkari JV, Barpander SR. An estimation of serum malondialdehyde, superoxide dismutase and Vitamin A in oral submucous fibrosis and its clinicopathologic correlation. J Oral Maxillofac Pathol 2007; 11: 23-7.

10. Rajendran R. Oral submucous fibrosis: etiology, pathogenesis, and future research. WHO Bulletin OMS 1994; 72: 985-95.

11. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol 2001; 54: 176-86.

Corresponding Author: Yuthicka Sirohi, Department of Oral Pathology, ITS-Centre for Dental Studies and Research (CDSR) Ghaziabad, Uttar Pradesh, India .
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Publication:Pakistan Journal of Medical Research
Article Type:Report
Date:Sep 30, 2011
Words:1828
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