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Abstract

Background

Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a well-recognized, potentially malignant condition of the oral cavity associated with betel quid chewing. Betel quid chewing is a popular oral habit in India and shows strong association in the incidence of OSMF. The objective of the study was to determine the levels of trace elements, pro-oxidants and reactive oxygen species (ROS) in saliva of betel quid chewers and OSMF patients, which may help in the diagnosis of cancer progression in the oral cavity.

Methods

A total of 35 cases of OSMF and 35 cases of healthy individuals were included in the present study. The salivary status of ROS, pro-oxidants and some trace elements was studied in OSMF patients and normal healthy individuals.

Results

The levels of lipid peroxides (P < 0.001), conjugated dienes (P < 0.01), hydroxyl radicals (P < 0.01), superoxide dismutase (P < 0.05), copper (P < 0.05), calcium (P < 0.01), magnesium (NS), potassium (P < 0.05) and iron (P < 0.05) in OSMF patients were elevated when compared with normal healthy individuals. The levels of hydrogen peroxide (P < 0.05) and sodium (P < 0.01) in OSMF patients were found to be decreased when compared with control subjects. A significant alteration was noticed after supplementing with α-tocopherol in oral precancerous patients.

Conclusion

These parameters may help in the detection of the severity of oral diseases.

Introduction

Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a well-recognized, potentially malignant condition of the oral cavity mainly associated with betel chewing. Betel quid chewing is a popular habit in India, South Africa and other southeastern Asian countries.¹ The areca nut component of betel nut quid plays a major role in the pathogenesis of OSMF.^2–4 The rate of occurrence of oral carcinoma and other precancerous-stage-like OSMF is still increasing because of the non-existence of sophisticated diagnostic and therapeutic approaches. OSMF is characterized by a generalized submucosal fibrosis of the oral soft tissue resulting in morbid rigidity and progressive inability to open the mouth.^5,6

Lipid peroxidation has been shown to cause a profound alteration in the structural integrity and functions of the cell membrane. Chemical compounds are capable of generating potential toxic oxygen species (ROS) or free radicals which are referred to as pro-oxidants. In a normal cell, there is an appropriate pro-oxidant–antioxidant balance. This balance can be shifted to pro-oxidant when the production of free radicals is increased, and can result in serious cell damage.^7,8 There are many potential advantages of using a saliva sample instead of serum for the assessment of systemic diseases. Genetic biomarkers isolated in saliva may help in diagnosing oral cancer.⁹ The above findings may help scientists to focus on saliva, which is a non-invasive procedure to determine biochemical markers for the detection of oral cancer at an early precancerous stage. In addition, blood concentrations of many components are reflected in saliva. A number of studies have suggested that salivary proteins may be potentially valuable for the diagnosis or prognosis of human diseases like oral cancer.^10,11 Diagnosis of precancerous stages of the oral cavity is a difficult task for several reasons; the most important one is the lack of symptoms. Therefore, the development of simple, accurate, rapid and cost-effective methods to detect neoplastic lesions is required. Saliva can be used as a diagnostic tool for different oral and systemic diseases. Various studies have been carried out to screen the clinical usefulness of certain biochemical and physiological changes for early diagnosis and management of oral cancer.

Materials and methods

Study design and case selection

Thirty-five cases of OSMF (M:F ratio, 16:19; age 30–55 y) who were attending the dental clinic of Sri Ramachandra University were selected for the present study. Control subjects consisted of 35 age- and gender-matched healthy individuals (M:F ratio, 18:17; age, 30–58 y). They were not on any medication and were not known to have diabetes, hypertension or any other illness. α-Tocopherol (Bio-E, Chennai, Tamil Nadu, India; 400 IU/d¹²) was given for 15 d to OSMF patients as a vitamin supplement. All patients gave informed consent prior to their inclusion in the study. Patients were selected for this research following examination by the dentist and histopathological confirmation of OSMF by the pathologist. This study was conducted with the approval of the Ethical Committee of the hospital.

Sample collection and storage

The participants had to refrain from eating and drinking water for a minimum of 90 min before saliva collection. The whole resting saliva was collected in a wide-mouthed graduated tube over a 30-min period according to the method of Navazesh et al. ¹³ from healthy individuals and from OSMF patients before and after supplementation with α-tocopherol. Saliva samples were collected and centrifuged at 1200 g for 10 min at 4°C. The supernatant of centrifuged saliva was transferred into a sterile disposable container and stored at −20°C until analysis. All the samples were analysed within seven days of collection. Biochemical parameters such as lipid peroxides (LPO),¹⁴ conjugated dienes,¹⁵ hydroxyl radicals¹⁶ and hydrogen peroxides¹⁷ were analysed using standard procedures. The superoxide dismutase (SOD) assessment was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase, which reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl tetrazolium chloride to form a red formazan dye. The SOD activity was measured by the degree of inhibition of this reaction. The method is a modification of the nitro blue tetrazolium assay. 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl-2-tetrazolium-5-chloride [XTT]) is reduced by the superoxide anion oxygen generated by xanthine oxidase. Formazan is read at 470 nm.¹⁸ SOD inhibits this reaction by scavenging the anion oxygen. One unit of the enzyme is defined as the amount of enzyme needed for 50% inhibition of absorption in the absence of the enzyme.¹⁹ The SOD activity was expressed as U/L. Trace elements were analysed using an atomic absorption spectrophotometer (PerkinElmer 2380, PerkinElmer, Waltham, MA, USA).

Statistical analysis

Statistical analysis was performed using a paired Student's t-test. Values were expressed as means ± SEM and P < 0.05 was considered as significant.

Results

The demographic variables are shown in Table 1. In the present study, the frequency of occurrence of OSMF was highest in patients who had the habit of chewing tobacco with areca nut (32%), followed by patients who had the habit of gutkha (26%) in the study. The frequency of occurrence of OSMF was observed to be minimum in the case of other habits. All patients complained of burning sensation (100%); difficulty in opening the mouth was reported by around 80% of the patients; difficulty in swallowing and speech was reported by 20% and 11%, respectively.

Table 1

Demographic variables of oral submucous fibrosis (OSMF) patients in the study group

Sl no.	Description	Number of cases	Percentage
1	Sex-wise distribution
	OSMF patients
	Male	16	46
	Female	19	54
2	Habits
	Areca nut with tobacco	11	32
	Betel quid	4	11
	Gutkha	9	26
	Gutkha + beedi	5	14
	Gutkha + alcohol	4	11
	Gutkha + beedi + alcohol	2	6
3	Chief complaints
	Burning sensation	35	100
	Difficulty in opening the mouth	28	80
	Difficulty in swallowing	7	20
	Difficulty in speech	3	11

The levels of LPO (P < 0.001), conjugated dienes (P < 0.01), hydroxyl radicals (P < 0.01) and SOD (P < 0.05) in OSMF patients were significantly increased when compared with normal healthy individuals, whereas hydrogen peroxide (P < 0.05) levels in OSMF patients were significantly decreased when compared with normal healthy individuals. A significant change in LPO (P < 0.01), conjugated dienes (P < 0.05), hydroxyl radicals (P < 0.001), SOD (P < 0.05) and hydrogen peroxide (P < 0.05) was noticed in OSMF patients after α-tocopherol supplementation (Table 2).

Table 2

Levels of pro-oxidants and free radicals in saliva of normal healthy individuals and oral submucous fibrosis (OSMF) patients (n = 35)

Parameters	Normal healthy individuals	OSMF patients (before treatment)	OSMF patients (after treatment)
Lipid peroxides (nmol/mL)	620 ± 52	980 ± 96***	740 ± 82**
Conjugated dienes (units/mL)	10 ± 2.2	28 ± 4.8**	16 ± 1.9*
Hydroxyl radicals (nmol/mL)	16 ± 1.8	206 ± 19.2**	65 ± 7.02***
Hydrogen peroxide (nmol/mL)	538 ± 60.1	408 ± 39*	502 ± 16*
Superoxide dismutase (U/L)	2.08 ± 0.50	7.89 ± 0.76*	3.69 ± 0.82*

Statistically significant values of OSMF patients were compared with normal healthy individuals. *P < 0.05; **P < 0.01; ***P < 0.001

The levels of trace elements such as calcium (P < 0.01), magnesium, potassium (P < 0.05), copper (P < 0.05) and iron (P < 0.05) were elevated, whereas the level of sodium (P < 0.05) in OSMF patients was found to be decreased when compared with normal healthy individuals. No significant change in the level of zinc was found in OSMF patients. There was a significant change in the levels of sodium (P < 0.05), potassium (P < 0.05) and copper (P < 0.05) in OSMF patients after supplementing with α-tocopherol (Table 3).

Table 3

Levels of trace elements in saliva of normal healthy individuals and OSMF patients (n = 35)

Parameters (ppm/L)	Normal healthy individuals	OSMF patients (before treatment)	OSMF patients (after treatment)
Sodium	65.03 ± 7.2	40.58 ± 4.8**	59.82 ± 6.1**
Potassium	60.32 ± 5.9	77.58 ± 7.9**	69.63 ± 7.2**
Calcium	52.6 ± 5.8	67.2 ± 6.8**	56.8 ± 5.8*
Magnesium	8.6 ± 9.1	12.8 ± 1.4*	9.2 ± 0.93^NS
Zinc	0.15 ± 0.017	0.13 ± 0.014^NS	0.14 ± 0.015^NS
Iron	0.1 ± 0.02	0.4 ± 0.05*	0.26 ± 0.033*
Copper	0.15 ± 0.016	0.13 ± 0.015*	0.14 ± 0.015*

Statistically significant values of OSMF patients were compared with normal healthy individuals. *P < 0.05; **P < 0.01. NS, non-significant

Discussion

Free radicals are ROS, which are found at a significantly high level relative to disease progression. Some micronutrients act as antioxidants, which are found in different sites including body fluids, tissues and even in cell membranes. Researchers working on cancer have significant experience with free radical biology, which is more important for the phenomenon of tumour metastasis and angiogenesis. LPO are important free radicals, which lead to cell membrane destruction. LPO and lipid hydroperoxides are easily scavenged or neutralized immediately by certain antioxidant enzymes such as glutathione peroxidase and non-enzymatic antioxidants such as vitamin C and E and reduced glutathione. Due to the over-production of LPO in the precancerous condition,²⁰ the available antioxidants in the system are unable to neutralize and, hence, the levels of LPO in OSMF patients are elevated when compared with normal healthy individuals. The high rates of membrane damage results in the elevated levels of low-molecular-weight conjugated dienes in OSMF patients which was observed in this study.

Hydroxyl radicals are generated in the presence of high iron/copper content. An increased level of hydroxyl radicals targets the lipid bilayer of the cell membrane, predominantly causing peroxidation and hydroperoxidation of lipids, which in turn leads to cell damage.²¹ High levels of hydroxyl radical were observed in saliva of OSMF patients, which might be due to high copper content in OSMF patients. Hydrogen peroxide is extremely sensitive and is easily degradable. The degraded products can react with metal ions, which have a strong effect on the oxidation and reduction process. The increased level of hydroxyl radicals is due to the degradation of excess amounts of hydrogen peroxide in OSMF patients.²² SOD plays a key role in the protection of various tissues against oxidative damage, and a raised SOD level is a sensitive marker of increased ROS production.^23,24 Erythrocyte SOD is of special importance to red cells in the protection against superoxide production due to haemoglobin autoxidation.²⁵ On the other hand, erythrocyte SOD has been recognized as a potent antioxidant for other tissues, since the anion channel allows the transport of superoxide and other radicals into red cells.²⁶ As a consequence, fluctuation in the erythrocyte SOD activity may reflect changes in ROS generation not only in red cells but also in other tissues. The SOD activity increase in OSMF patients may be the body's first defense mechanism for the neutralization of free radical generation due to their lifestyle habits. Our present data are in good agreement with the findings of others.²⁷ α-Tocopherol supplementation showed a significant change in SOD levels, which might be due to the action of the membrane antioxidant α-tocopherol.

A significant increase in potassium level and a significant decrease in sodium level found in OSMF patients might be due to the activation of ion channels of Na⁺/K⁺ of the cell through the membrane and also the imbalance of Na⁺/K⁺-dependent ATPases.²⁸ Chewing areca nuts for 5–30 min significantly increases the soluble copper levels in oral fluids. These increased levels of soluble copper support the hypothesis that copper acts as an initiating factor in OSMF by stimulating fibrogenesis through up-regulation of oxidation activity.²⁹ The increased levels of copper in OSMF patients were due to the continuous exposure of areca nut, which led to a higher level of copper in saliva of OSMF patients. The increased copper level in OSMF patients may enhance the formation of OH^• radicals from H₂O₂.³⁰ These hydroxyl radicals enhance the lipid peroxidation that ultimately results in DNA adduct formation in the cell, which in turn leads to carcinogenesis of the oral cavity.³¹ The copper content enhances the lysyl oxidases, which in turn enhance the cross-linking of collagen fibres in oral mucosal tissues.³²

The high calcium content and the insignificant increase in magnesium content found in saliva of OSMF patients may be due to membrane damage that leaks out calcium and magnesium ions from cellular content. The iron level also enhances the level of hydroxyl radicals which was elevated in OSMF, which in turn develops cancer by DNA adduct formation.³³ No significant changes in the levels of zinc were found in OSMF patients.

The alteration in the levels of free radicals, lipid peroxidation products and some trace elements in OSMF patients treated with α-tocopherol might be due to its anti-oxidant property which helps in quenching the free radical generated in the oral precancerous condition. Antioxidant vitamins have been known to cause extensive morphological changes in tumorigenic acinar cells and also to cause significant growth inhibition in vitro.³⁴ The effect of α-tocopherol on the improvement of saliva flow rate and on oral mucosal tissue changes in radiation-treated oral cancer patients has been studied before.²⁸

Preliminary data indicate the protective role of antioxidant supplementation in preventing precancerous lesions. Saliva, being non-invasive and easy to collect, can be used to assess malondialdehyde and antioxidant status of patients with an oral lesion. Further studies should be performed on a large scale to clarify the importance and role of antioxidant vitamins in oral diseases.

DECLARATIONS

Competing interests: None.

Funding: This research was a post-graduate project study. Hence no funding is involved.

Ethical approval: The ethics committee of the Hospital approved this study.

Guarantor: SC.

Contributorship: SC and MB were involved in protocol development, gaining ethical approval, patient recruitment and data analysis. JH researched literature and conceived the study. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

Acknowledgements: The authors gratefully acknowledge the Management and Dean of Faculties of Sri Ramachandra University, Chennai, India for providing the facility and samples for this research work.

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Effect of α- tocopherol on salivary reactive oxygen species and trace elements in oral submucous fibrosis

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Study design and case selection

Sample collection and storage

Statistical analysis

Results

Discussion

DECLARATIONS

References