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Table of Contents   
ORIGINAL RESEARCH  
Year : 2022  |  Volume : 33  |  Issue : 3  |  Page : 287-291
A comparative investigation of the activity of superoxide dismutase in patients with leukoplakia and healthy controls


1 Department of Oral Pathology, PSM College of Dental Sciences and Research, Thrissur, Kerala, India
2 Department of Oral Pathology, Royal Dental College, Palakkad, Kerala, India
3 Division of Biochemistry, Malabar Cancer Research Centre, Thalassery, Kerala, India
4 Department of Biochemistry, Royal Dental College, Palakkad, Kerala, India

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Date of Submission15-Oct-2021
Date of Decision25-Aug-2022
Date of Acceptance05-Sep-2022
Date of Web Publication17-Jan-2023
 

   Abstract 


Context and Aim: Oral leukoplakia has recently been re-defined as “a predominantly white lesion of the oral mucosa that cannot be characterised as any other definable lesion”. Superoxide dismutase (SOD) is one of the enzymatic anti-oxidants which form the first line of defence in the cell. There are very limited data available on the analysis of SOD in leukoplakia patients. Therefore, the present study was planned to analyse anti-oxidant SOD levels in leukoplakia patients. Methods and Material: The study group consisted of 29 subjects of oral leukoplakia, and the control group consisted of 25 healthy individuals. All the subjects were evaluated for SOD enzyme in plasma. Statistical Analysis Used: The results for each determinant were calculated using one-way ANOVA test, Student 't' test, Pearson Chi square test, and 't' test for equality of means wherever applicable. Results: The study revealed that among the different clinical entities of oral leukoplakia, the enzymatic anti-oxidant SOD is shown to have decreased in all forms of oral leukoplakia, but without any statistical significance. A statistically highly significant decrease (at P < 0.0001) of SOD in oral leukoplakia patients compared to normal healthy patients was observed in the present study. Conclusions: The study revealed a difference in the free-radical activity and oxidative stress in blood of leukoplakia patients compared to healthy patients, which is reflected by the variation in the levels of blood SOD, thus showing vulnerability to cellular damage and tendency towards further malignant changes.

Keywords: Oral leukoplakia, reactive oxygen species (ROS), superoxide dismutase

How to cite this article:
Kuthoor JM, Sunil EA, Raghavan SE, Purushothaman BP. A comparative investigation of the activity of superoxide dismutase in patients with leukoplakia and healthy controls. Indian J Dent Res 2022;33:287-91

How to cite this URL:
Kuthoor JM, Sunil EA, Raghavan SE, Purushothaman BP. A comparative investigation of the activity of superoxide dismutase in patients with leukoplakia and healthy controls. Indian J Dent Res [serial online] 2022 [cited 2023 Feb 5];33:287-91. Available from: https://www.ijdr.in/text.asp?2022/33/3/287/367870



   Introduction Top


Cancer is the greatest social fear because it is one of the leading causes of death in the globe. Free radicals are charged atoms as they have a surplus or deficient number of electrons in their atomic orbit. A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. The presence of unpaired electrons results in certain common properties that are shared by most radicals. Many radicals are unstable and highly reactive. They can either donate an electron to or accept an electron from other molecules, therefore behaving as oxidants or reductants.[1] When there is an excess or overload of these free radicals, they cause oxidative stress and cellular damage.[2]

This oxidative stress causes harm to lipids and proteins, thereby damaging the cell membranes, and alters the genetic code, leading to DNA damage.[3] Oxygen itself is a totally harmless molecule as in its ground state, it has two unpaired electrons with parallel spin, which makes it paramagnetic and, hence, unlikely to participate in reactions with organic molecules unless it is activated.[4] An antioxidant is defined as any molecule or substance present in low concentration that is capable of stabilising or de-activating free radicals before they attack the cells.[5] The antioxidant can be enzymatic or non-enzymatic and is found both exogenously and endogenously. They are seen intra-cellular, extracellular, and in most body fluids. Antioxidants are produced endogenously in the body during normal metabolism or may be obtained exogenously from diet or dietary supplements.

Enzymatic antioxidants are superoxide dismutase (SOD), catalase, glutathione, glutathione reductase, glutathione peroxidases, and glutathione S-transferases. Non-enzymatic antioxidants are further divided into natural and synthetic antioxidants.[6]

SOD is the first enzyme that catalyses the portioning or dismutation of harmful superoxide (O2-) into either ordinary oxygen (O2) or H2O2. H2O2 may also have a damaging effect on the cells, but catalases split H2O2 into water and oxygen. Oral leukoplakia is by far the most common potentially malignant disorder (PMD) of the oral mucosa. It is commonly defined as “a white patch or plaque that cannot be characterised clinically or pathologically as any other disease and is not associated with any physical or chemical causative agent except the use of tobacco”. The malignant transformation rates of oral leukoplakia range from 0.13 to 17.5% depending on the clinical sub-types of leukoplakia. Among the clinical sub-types, proliferative verrucous leukoplakia (PVL), erythroleukoplakia, and speckled leukoplakia carry a greater risk of malignant transformation where PVL carries 40–50% malignant transformation rate. Thus, our study is an attempt to assess the level of anti-oxidant enzyme SOD in plasma samples of patients with oral leukoplakia as well as normal controls and find out that salivary SOD can be used as a biomarker for early detection, medication, and management of leukoplakia, allowing it to be prevented and the risk of malignant transformation to be reduced.


   Subjects and Methods Top


The study was conducted among 54 randomly selected subjects presenting to the out-patient department of Royal Dental College and included two groups which consisted of 29 subjects of leukoplakia and 25 healthy controls. The Institutional Ethics Committee approval was obtained prior to the start of the study, and informed consent was taken from all the participants included in the study (RDC/ADMN/2016/560). All the subjects were explained about the objectives of the study.

Selection of Subjects

A total of 54 chosen subjects were divided into two study groups, namely, Group A (cases) and Group B (controls).

Inclusion criteria: A total of 29 patients clinically diagnosed with leukoplakia and confirmed by histopathology were included in Group A. Moreover, 25 randomly selected normal subjects were included in Group B.

Exclusion criteria: Patients with any systemic disease and a previous history of treatment for the same; patients with any other pre-malignant lesions such as lichen planus, lupus erythematosus, and oral submucous fibrosis; patients diagnosed or treated for oral cancer; and patients on anti-oxidant supplements were excluded from the study.

Method of collecting data

The subjects selected for the study were instructed verbally regarding the study and were given written instructions elaborating on the procedures of the current study with a special instruction not to consume tobacco at least 8–10 hours prior to the collection of the blood samples. The leukoplakic lesions from any part of the oral mucosa were selected. The leukoplakic lesions were classified as homogeneous and non-homogeneous sub-types. Under aseptic conditions, 5 ml of venous blood samples was collected to sterile vials pre-coated with ethylenediaminetetraacetic acid from the subjects between 8 and 10 a.m. in the morning. Plasma was separated by centrifugation at 3,000 rpm in a cooling centrifuge at 4°C for 10 minutes and stored in a deep freezer at -80°C for not more than 1–2 days before assay. Estimation of SOD was determined using a commercially available Caymans SOD kit (Cayman Chemicals, USA), and samples were processed on Nanodrop at 440–460 nm.

Principle of reaction in Cayman's superoxide dismutase assay kit

The Cayman SOD assay kit used in this study works on the principle that superoxide radicals formed by the xanthine oxidase and hypoxanthine are detected by tetrazolium salt.[7],[8] One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. SOD activity is standardised using the cytochrome C and xanthine oxidase coupled assay and was read by Nanodrop (Thermo Nano Drop Lite, USA).

Statistical analysis

The preliminary data obtained were sub-divided into determinants of relevant criteria and then subjected to statistical analysis using SPSS version 18 software. The results for each determinant were calculated using one-way ANOVA test, Student 't' test, Pearson Chi square test, and 't' test for equality of means wherever applicable. The readings were expressed as mean ± SD (standard deviation). The P value of <0.05 was considered to be statistically significant.


   Results Top


Frequency of leukoplakia site distribution and SOD Level

Among the 29 subjects in our study, it was noted that the most common site of leukoplakia was buccal mucosa and the least common site was labial mucosa. A total of 21 subjects had leukoplakia in the buccal mucosa (72.4%), followed by five subjects with leukoplakia on commisures (17.2%), two with leukoplakia on the tongue (6.9%), and one subject of leukoplakia on the labial mucosa (3.4%).

We tabulated the level of SOD in leukoplakia from different intra-oral sites and expressed it as mean standard deviation. The mean SOD of leukoplakia on the buccal mucosa was 0.050 ± 0.012; for commissures, it was 0.051 ± 0.009; the mean SOD of the tongue was found to be 0.070 ± 0.014; and because there was only one subject with leukoplakia on labial mucosa, the mean was 0.05900 [Table 1] and [Table 2].
Table 1: Frequency of subjects' leukoplakia site distribution and SOD level

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Table 2: Chi square value for site-wise distribution of leukoplakia

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Frequency distribution and SOD levels of homogeneous and non-homogeneous leukoplakia

In our study, we noted that 25 subjects had homogeneous leukoplakia as the most common presentation (86.2%), whereas the remainder of four subjects had a non-homogeneous leukoplakia speckled variant (13.8%) [Table 3]. Student t test for the same group did not show statistical significance between homogeneous and non-homogeneous leukoplakia.
Table 3: Frequency distribution and SOD levels of homogeneous and non-homogeneous leukoplakia in subjects

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Comparison of SOD levels in cases and controls

The SOD levels in subjects (group A) and controls (group B) are tabulated and compared [Table 4]. Student t test values for comparing SOD activity levels in subjects and controls showed a highly statistical significant P value of 0.0001.
Table 4: Comparison of SOD levels in subjects and controls

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Comparison between SOD levels in males and females of cases and controls

In this study, we noted that subjects in the group A (cases) were in the age group of 40–49 years (37.9%), followed by 60–70 years (24.1%), 50–59 years (24.1%), and 30–39 years (13.8%). However, most of the subjects of group B (controls) were in the age group of 40–49 years (36%), followed by 60–70 years (20%), 50–59 years (16%), and 30–39 years (28%). The findings showed that 20.4% of all the subjects belong to the age group of 30–39. Most patients who participated in the study belong to age group of 40–49, which accounts for 37.0% of total subjects, followed by 22.2% in the age group of 60–70 years and 20.4% in the age group of 50–59 years [Figure 1].
Figure 1: Comparison between SOD levels in males and females of subjects and controls

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From [Figure 1], 20.4% of all the subjects belong to the age group of 30–39. Most patients who participated in the study belong to age group of 40–49, which accounts for 37.0% of total subjects, followed by 22.2% in the age group of 60–70 years and 20.4% in the age group of 50–59 years. The Chi square value for the same showed that the patients belonging to the age group of 40–49 years had 1.884 times more chances of getting leukoplakia than the other age groups; however, no significant relation across the age groups was noticed with a P value of 0.597.


   Discussion Top


Leukoplakia is the most common pre-malignant disorder of the oral cavity. Leukoplakia may progress eventually into oral squamous cell carcinoma (OSCC). OSCC is the most common malignancy accounting for 92–95% of all intra-oral malignancies. Although the aetiology of OSCC and its potential precursor leukoplakia is thought to be multifactorial, it is mostly attributed to the use of tobacco, especially in people younger than 40 years of age.[9],[10]

SOD, an important endogenous antioxidant, forms the first line of defence against the superoxide radicals and protects the cells by converting it to O2 and H2O2 almost instantly, preventing damage.[11] SOD available in all body fluids including blood samples is increasingly used for its evaluation. SOD in plasma is much more stable than in serum, and hence, plasma is an ideal source for its assay.[12]

Based on the above facts, we conducted our current study by assessing the levels of plasma SOD in 29 leukoplakia patients and compared it with 25 normal subjects. The findings of our study revealed that the majority of patients with leukoplakia were in the age group of 40–49 years (37.9%). The findings of our studies correlate with previous studies of Srivastava et al.[13] This is in accordance with the fact that although leukoplakia may occur in a wide age group ranging from 20–70 years, its prevalence is more common in the middle age group, that is, 40–50 years, and the risk increases with age.[14] We noticed that subjects with leukoplakia were mostly males (72.4%) as compared to females (27.6%) with a ratio of 3:2. The result of our study is consistent with previous studies of Gurudath S.[15]

Tobacco in the smoke form is considered to be more dangerous as well as carcinogenic, and around 80% of cases of leukoplakia are known to be smokers rather than chewers. Although there is a slight rise in smoking habit among females, it is still more common in males. This is what reflected in our study as well, which showed that all 29 patients with leukoplakia were using tobacco. Twenty patients consumed tobacco in the smoke form, whereas the remaining nine used the smokeless form. However, we noted that all the females with tobacco habit used only the smokeless form of tobacco.

The findings of our study showed that leukoplakia was more common in the buccal mucosa and the least common in the labial mucosa. Even with contradicting findings in various studies, buccal mucosa is the predominant site of occurrence of leukoplakia. Our study mostly included smokers rather than chewers, and among nine chewers, one male patient had the habit of placing quid in the labial vestibule. This is the single case of leukoplakia in labial mucosa noted in this study.

In one study conducted by Akhlaq et al.,[16] the levels of salivary SOD were significantly lower in the sample of oral submucous fibrosis (OSMF) and lower levels of SOD (mU/L) were the indication of a decrease in mouth opening among the cases of OSMF.

We observed that 86.2% was of homogeneous type of leukoplakia and was the most common form and the remainder of 13.8% was of non-homogeneous type. It is a widely accepted fact that the exact reason for the high chance of occurrence of homogeneous leukoplakia is not known and thought to be because of interplay of many factors. This might be because of use of different forms of tobacco products, duration and frequency of tobacco consumption, tobacco use associated with alcohol consumption, and chewing of tobacco mixed with or without slaked lime.

Shetty reported that the levels of salivary SOD decreased in oral leukoplakia and oral cancer patients when compared to healthy controls. The results suggested that salivary SOD levels could be a useful bio-marker in oral carcinogenesis.[17] The toxic radicals formed by tobacco are first encountered by the enzymatic anti-oxidant SOD, which forms the first line of defence. SOD counteracts the superoxide anion radical, thereby diminishing the toxic effects of hydroxyl and hydrogen peroxide radicals. In our study, the levels of SOD were estimated in subjects and controls and then compared. We found a decrease in the SOD levels of subjects as compared to the controls. Thus, an extremely significant level of plasma SOD in individual samples of subjects and controls was obtained.

The decrease in the levels of SOD in subjects suggested that patients were more prone to tobacco-induced oxidative stress because of the corresponding increase in loads of free radicals. The SOD enzymes are utilized in order to scavenge the free radicals, thus resulting in its depletion. Such depletion of antioxidants causes an imbalance in oxidant and antioxidant statuses, leading to oxidative damage in cellular structures, a key event in carcinogenesis. A low level of oxidative stress is thought to encourage cell proliferation and/or hinder apoptosis. All these events are capable of promoting tumour growth.[18]

As an extension to our study, we also compared the levels of SOD in homogeneous leukoplakia and non-homogeneous leukoplakia occurring inside different oral sites in males and females and in smokers and non-smokers. The comparison of mean plasma SOD levels in patients with homogeneous and non-homogeneous leukoplakia in our study did not show any statistical significance. We noted that the mean SOD level was decreased in homogeneous leukoplakia compared to that in non-homogeneous leukoplakia. To the best of our knowledge, we did not come across this parameter assessment in any previous studies. The results may be more accurate if a larger sample size is used. Many studies conducted to assess the difference in levels of SOD among smokers and nonsmokers have yielded varying results ranging from a significant difference in the levels of SOD, whereas many have also shown no difference. This contradictory finding may be attributed to the form of tobacco, frequency of tobacco, and the site of quid placement. There were some limitations to the study. It is suggested that in the future, a comparative crosssectional study with a larger number of subjects and controls from multiple clinical sites be performed. As a result, salivary SOD can be employed as a biomarker for early detection, therapy, and management of leukoplakia, allowing it to be stopped at an early stage and minimising the risk of malignant transformation.


   Conclusion Top


The deleterious effects of ROS are counteracted by the human body by well-equipped enzymatic and non-enzymatic anti-oxidants. Endogenous antioxidants are depleted during the course of neutralising ROS; hence, dietary supplementations are required. Dietary supplementation with antioxidants is a healthy adjunct to traditional health principles in the modern age to maintain a healthy life style.

Acknowledgements

We acknowledge the kind help and support from Oral Pathology Department, Royal Dental College; Biochemistry and Physiology Departments of Amala Cancer Research Centre and College of Veterinary And Animal Sciences respectively in completion of the study.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Binitha Ponnamparambil Purushothaman
Department of Biochemistry, Royal Dental College, Chalissery, Palakkad - 679536, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_1009_21

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