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An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
ORIGINAL RESEARCH Table of Contents   
Year : 2004  |  Volume : 8  |  Issue : 2  |  Page : 82-85
 

Estimation of antioxidants in oral exfoliated cells


1 Department of Oral and Moxillofacial Pathology, Meenakshi Ammal Dental College & Hospital, Chennai, India
2 Department of Biochomistry, Meenakshi Ammal Dental College & Hospital, Chennai, India

Correspondence Address:
B Sivapathasundharam
Department of Oral and Maxillofacial Pathology, Meenakshi Ammal Dental College & Hospital, Alapakkam Main Road, Modduravoyal, Chennai - 600 095
India
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Source of Support: None, Conflict of Interest: None


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How to cite this article:
Gururaj N, Sivapathasundharam B, Sumathy S. Estimation of antioxidants in oral exfoliated cells. J Oral Maxillofac Pathol 2004;8:82-5

How to cite this URL:
Gururaj N, Sivapathasundharam B, Sumathy S. Estimation of antioxidants in oral exfoliated cells. J Oral Maxillofac Pathol [serial online] 2004 [cited 2019 Sep 22];8:82-5. Available from: http://www.jomfp.in/text.asp?2004/8/2/82/40971



   Introduction Top


Anti oxidants arc substances that when present in low concentrations compared to that of oxidisahlc substrates signiticantlc delay or inhibit the oxidation of that substrate [1] . A free radical can be defined as any molecular species capable of independent existence that containing an unpaired electron in an atomic orbital [1] .

Recent years have witnesses the role of free radical oxidative damage in human diseases and aging. Free radical oxidative stress has a probable role in the pathogenesis of variety of diseases including pre cancerous oral lesions, Various natural antioxidant enzymes. vitamins and even synthetic agents with antioxidant properties have a potential role in treatment of various diseases including cancer.

Many of the free radicals are highly reactive which cannot be measured by any means and have a very short life (10 6 seconds or less) in biological systems, When compared to this, antioxidants produced against the free radicals can be measured Air estimated from body fluids blood etc. Numerous assays have been described to measure various free radical products or antioxidant status and none of method is ideal for its estimation [2],[3],[4],[5],[6] . Most of the investigators used blood, body fluids and tissues to estimate antioxidant status both in healthy and diseased individuals.

Oral epithelium renews itself rapidly and it sheds off its superficial cells into the oral cavity through the process known as desquamation. These cells reflect the physiological or pathological changes of underlying tissue, which can be studied both quantitatively and quantitatively [7] . Here we attempted a different approach to estimate antioxidants in oral exfoliated epithelial cells obtained by exfoliative cytology in both clinically healthy non-smokers and smokers.


   Materials and Methods Top


15 clinically healthy non-smokers of same age group were selected who were not on any medication or any, antioxidant supplements and considered as normal control group.

15 clinically healthy smokers without any oro mucosal changes and not on any medication or antioxidants supplements were selected as the study group from the out patient department of Meenakshi Ammal Dental College Chennai. All of them are smoking; 10 cigarettes per day for 2 years.

The subjects were requested to gargle the mouth with chlorhexidine mouthwash. The buccal mucosa was scrapped with the help of wet wooden spatula and the scrapings were collected immediately with help of saline in a closed airtight test tube and send for laboratory.


   Preparation of Cell Lysate Top


The cells were washed 3 times with 0.9% saline and centrifuged for 10 minutes at 3000 rpm after each wash. A known volume of ice-cold distilled water was added to washed cells for cell lysis. This was allowed to stand for an hour at 4 C. The cell lysate was used for the estimation of glutathione reductase (GSH ) & superoxide dismutase (SOD). The estimations were carried out within 6 hours of sampling.

GSH: The total reduced glutathione was determined by the method of Moron et al (3).

Procedure

To the 0 5 ml cell lysate. O.5ml of 5% tri carboxylic acid (TCA) was added to precipitate the protein. The solution was mixed and centrifuged, to 0.5ml of Supernatant, 2ml of 0.2m phosphate buffer (pH-8.0),0.5ml of 0.6mm dithiobisnitro benzoic acid (DTNB) in 0.2m phosphate buffer was added. The deep yellow colour developed was read at 412nm in a spectrophotometer against a blank containing 5% TCA instead of sample Reduced glutathione was used as standard (The amount of glutathione in cell lysate was expressed as mg,Of GSH).

SOD: The enzyme was assayed according to the method Marklund & marklund [4] . The degree of inhibition of auto-oxidation of Pyrogallol, at an alkaline pH by SOD was used as a measure of the enzyme activity.

Procedure

To 0.5ml of cell lysate, 0.25ml of absolute ethanol 0.15ml of CHCI3 were added. After 15 minutes of shaking in a mechanical shaker, the suspension was centrifuged & the supernatant obtained constituted the enzyme extract.

For control, the reaction mixture contains 2ml of 0.1 ml TrisHel buffer (pH 8.2). 0.5ml of 2mm pyrogallol & 1.5ml distilled water. Initially the rate anti oxidation of pyrogallol was noted at an interval of one minute for 3minutes.

For test, the reaction mixture contains 2ml of 0.0.5m TrisHel Buffer (pH 7.4), 0.5ml of enzyme preparation. 0.5 ml pyrogallol & 1 ml water. The rate of inhibition of pyrogallol auto oxidation after the addition of the enzyme was added.

The enzyme activity was expressed in terms of units ml of cell lysate. One unit corresponded to the amount of enzyme that inhibited the auto oxidation reaction by 50% 1 Unit = 50%reduction.


   Results Top


[Table 1]


   Discussion Top


Cancer is the culmination of a multistep process that occurs over a period of several years or decades. The underlying cause is thought to be DNA damage, much of which is oxidative in nature. These oxidative processes, the mechanisms of which are not clearly understood, occur during the promotional stage of carcinogencsis [8] . Hence.antioxidants may be able to cause regression of premaligant lesions or inhibit their progression into cancer.

Oxidative stress, arising as a result of an imbalance between free radical production and antioxidant defences, is associated with damage to a wide range of molecular species including lipids, proteins, and nucleic acids [5] . Reactive oxygen species may interact with and modify cellular protein, lipid, and DNA, which result in altered target cells function. The accumulation of oxidative damage has been implicated in both acute and chronic cell injuries including possible participation in the formation of cancer. Acute oxidative injury may produce selective cell death and a conipensatory increase in cell proliferation. This stimulus may result in the formation of newly initiated prencoplastic cells and/ or enhance the selective clonal expansion of latent initialed preneoplastic cells.

Similarly , sub-lethal acute oxidative injury May produce un-repaired DNA damage and results in the formation of new mutations and, potentially, newly initiated cells, in contrast, sustained chronic oxidative injury may lead to a nonlethal modification of normal cellular growth control mechanisms. It can therefore be said that the measurement of endogenous antioxidant levels is of great importance because the resultant values may be used as indicators of future health.

We have decided to study the anti oxidant status of oral exfoliated cells collected by exfoliative cytology in smokers and non-smokers which is a non-invasive procedure to predict the oxidativc status of the tissue intracellularly. In our study. Glutathione reductase and Superoxide dismulase were present in both groups but the level of the enzymes was significantly reduced in smokers when compared to non-smokers.

The exact mechanism by which the free radicals are liberated and how the antioxidants are protecting the damage are not known. The most important free radicals in many disease states are oxygen derivatives, particularly superoxide and the hydroxyl radical. Radical formation in the body occurs by several mechanisms, involving both endogenous and environmental factors. Superoxide is produced by the addition of a Single electron to oxygen, and several mechanisms exist by which superoxide can be produced in vivo [1] .

Several molecules, including adrenaline flavine nucleotides, thiol compounds. and glucose, can oxidise m the presence of oxygen to produce superoxide, and these reactions arc greatly acccerated by the presence of transition metals such as iron or copper. There might also be continuous production of superoxide by vascular endothelium to neutralise nitric oxide, production of superoxide by other cells to regulate cell growth and differentiation, and the production of superoxide by phagocytic cells during the respiratory burst [1],[9] .

Any biological system generating superoxide will also produce hydrogen peroxide as a result of a spontaneous dismutation reaction. In addition, several enzymatic reactions, including that catalysed by glycolate oxidase and D-amino acid oxidise, might produce hydrogen peroxide directly. Hydrogen peroxide is not a free radical itself, but is usually included under the general heading of reactive oxygen species (ROS). It is a weak oxidising agent that might directly damage proteins and enzymes containing reactive thiol groups. However, its most vital property is the ability to cross cell membranes freely, which superoxide generally cannot do. Therefore, hydrogen peroxide formed in one location might diffuse a considerable distance before decomposing to yield the highly reactive hydroxyl radical. which is likely to mediate must of the toxic effects ascribed to hydrogen peroxide. Therefore hydrogen peroxide acts as a conduit to transmit free radical induced damage across cell compartments and between cells. In the presence of hydrogen peroxide myeloperoxidase will generate hypochlorous acid and singlet oxygen a reaction that plays an important role in the killing of bacteria by phagocytes.

The hydroxyl radical (OH) or a closely related species, is probably the final mediator of Most free radical induce tissue damage. Although hydroxyl radical formation can occur in several ways, by far the most important mechanism in vivo is likely to be the transition metal catalysed decomposition of superoxide and hydrogen peroxide.

The most important transition metal in human disease is iron and copper. These elements play a key role in the production of hydroxyl radicals in vivo. Hydrogen peroxide can react with iron II (or copper 1) to generate the hydroxyl radial a reaction first described by Fenton in 1984 [1],[9] :

Fe 2- + H 2 O 2 → Fe 3+ + OH + OH

Although free radical production occurs as at consequence of the endogenous reactions described above and plays an important role in normal cellular function, it is important to remember that exogenous environmental factors can also promote radical formation. Ultraviolet light will lead to the formation of singles oxygen and other reactive oxygen species in the skin. Atmospheric pollutants such as ozone and nitrogen dioxide lead to radical formation and antioxidant depletion in the bronchoalveolar lining fluid and this may exacerbate respiratory disease, cigarette smoke contains millintolar amounts of free radicals, along with other toxins.

The human body has several mechanisms to counteract the damage caused by free radicals and other reactive oxygen species [8] . These act on different oxidants as well as , in different cellular compartments.One important line of defence is a system of enzynmes. including Glutathione peroxidases. superoxide dismutases and catalase. which decrease concentrations of the most harmful oxidants in the tissues. Several essential minerals including Selenium, copper, manganese and zinc arc necessary for the formation or activity of these enzymes. Hence, if the nutritional supply of these minerals is inadequate, enzymatic defence against free radical may be impaired.

The second line of defence against free radical damage is the presence of antioxidants. An antioxidant is molecule stables enough to donate an election to a rampaging free radical and neutralize it, thus reducing its capacity to damage.

Antioxidant defence systems can be divided into three main groups [1] :

1. Antioxidaut enzymes

E.g. Superoxide dismutases, Catalase. Glutathione peroxidase and Caoruloplasmin.

2. Chains breaking antioxidants

Eg.Tocopherols and Carotenoids.

3. Transition metal binding proteins

E.G. Transferrin and Ferritin

Catalase was the first antioxidant enzyme to be characterized and catalyses the two stage conversion of hydrogen peroxide to water and oxygen.

Glutathione peroxidases catalyze the oxidation of Glutalhione at the expense of a hydroperoxide, which might be hydrogen peroxide or another species such as lipid hydroperoxide. The predominant subcellular distribution is in the cytosol and mitochondria, suggesting that Glutathione peroxidase is the main scavenger of hydrogen peroxide in these subcellular compartments. The activity of the enzyme is dependent on the constant availability of reduced Glutathione [1] .

Glutalhione reductase is a flavine nucleotide dependent enzyme and has a similar tissue distribution to glutathione peroxidase. The superoxide dismutases catalyse the dismutation of superoxide to hydrogen peuxide:

O 2 +0 2 + 2H → H 2 O 2 + O 2

Superoxide dismutase catalyzes the dismutation of super oxide to hydrogen peroxide

When ever a free radical interacts with other molecule secondary radicals may be generated and produce more radical species. E.g. Lipid peroxidation. This can be corrected by retinoids and tocopherol and vitamin C.

Though the antioxidant levels in the exfoliated cells are present in both non-smokers and smokers, the reduction in the level of antioxidants in smokers may be due to tobacco induced free radical liberation. It indicates that the cells of the smokers are under oxidative stress and prone for oxidative damage. We have not compared our values with blood levels because our aim is to know whether the antioxidant can be estimated front the exfoliated cells biochemically and to compare the values with the non-smokers and smokers. In our study we have found out that the levels of antioxidants are reduced in smokers and it is consistent with the earlier studies conducted in patients with oro mucosal diseases like sub mucous fibrosis [10] . This can be also used as a marker in field cancerization.

The estimation of antioxidants in the blood may not suggest the exact reason for the changes in their levels, as free radicals liberated due to various reasons and will be reflected by the blood values. But measurements of intra cellular antioxidant levels, front oral exfoliated cells can give an idea about the tissue status that is the reason for its clinical and hisological change. Our study shows the evidence of oxidative damage to the cells in smokers confirmed by its antioxidant status, even before the onset of any clinical change.

Collection of exfoliated cells is a simple, noninvasive, and easy procedure and demonstrate abnormalities of the epithelial tissue even before they are clinically demonstrable can be used to predict the future status of the tissue at an early stage. which is very much useful to prevent in reversible tissue damage and also beneficial for the patients, As thus is non-invasive procedure. it can be performed at every stage of the disease, for any number of times, also useful after the initiation of treatment to follow up and asses the prognosis. using this technique further study in a larger sample alone with their blood levels can confirm its accuracy and its applicability.

Thus evidence of oxidative stress should be detectable beefore the onset of tissue damage and augmentation of antioxidant status at an early stage should either prevent or greatly reduce the tissue damage.

 
   References Top

1.Young. IS & Woodside. Jv: Antioxidant is health & disease. j, clin. Pathol, 2001; 54: 176-186  Back to cited text no. 1    
2.Young IS,: Measurement of total antioxidant capacity J. clin .path. 2001:54:3 9.  Back to cited text no. 2    
3.Moron et al. Levels of' Glutathione. Glutathione reductate, Glutathione S-transterase activities in rat long &- liver. Biochem. Biophys. Acta: 1979. vol.582, 6 7-78.  Back to cited text no. 3    
4.Marklund & Marklund. Involvement of the super oxide anion radical in the auto oxidation of pyrogallol a convenient assay for SOD. European J Biochem 1974, Vol47,469-174  Back to cited text no. 4    
5.Cowan. C.G CaIwell E.I.L, young I.S. Mckillop D.J and Lamey P.J. Antioxidant status of oral august tissue and plasma levels in smokers and non-smokers. J. Oral Path & med. 1999,28.36-36. 3 ).  Back to cited text no. 5    
6.Komatsu et al. Immunohisto chemical detection of human gastro intestinal glutathione pcroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies, J. Histochemistry & Cytochemistry.200I. Vol 49(6): 759- 766.  Back to cited text no. 6    
7.Saraswatht. TR. Sivapathasundram. B. Umadevi. Einstein. T. Benin A. (2002): Oral exfoliative cytology. Monograph published at national PG training conventions. Indian assoctation of oral & maxillofaciaI pathologists, Chennai.  Back to cited text no. 7    
8.Baghi, K and Puri. S. Free radical & antioxidants in health & disease Nutrition Vol. 1(_2)350-360,  Back to cited text no. 8    
9.Das U N free radicals: Biology & relevance to disease. JAPI 199(1. Vol. 38, no.7495-497.  Back to cited text no. 9    
10.Hazarey V.K Vatdhya S.M. Estimation of Serum antioxidant enzyme superoxide dismutase & glutathione peroxidase in oral submucous fibrosis. a biochemical study. JOMFP Vol.7 12)2003(44-45)  Back to cited text no. 10    



 
 
    Tables

  [Table 1]



 

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    Introduction
    Materials and Me...
    Preparation of C...
    Results
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