Malondialdehyde levels in oral sub mucous fibrosis: a clinicopathological and biochemical study.

BACKGROUND: Oral submucous fibrosis is one of the most commonly occurring potentially malignant disorders in the South-East Asia. Levels of lipid peroxidation product malondialdehyde have been recently correlated with clinical grades of oral Submucous fibrosis. AIMS: The aims of this study were to estimate the levels of malondialdehyde in serum, saliva and tissue in patients with oral submucous fibrosis, to correlate change in levels of malodialdehyde with the histopathological grading.
MATERIALS AND METHODS: The study group comprised of 65 clinically diagnosed and histopathologically confirmed cases of oral submucous fibrosis, 21 age and sex matched controls were also enrolled into the study. The serum saliva and tissue samples in the study groups were evaluated by the thiobarbutric acid reactive substances.
RESULTS: There was a significant difference between the serum and salivary malondialdehyde among the histopathological grades of oral submucous fibrosis. Tissue malonaldehyde levels were significantly higher as the grading progressed but tissue levels in grade 3 oral submucous fibrosis were lower than the controls.
CONCLUSION: This decrease in tissue malonaldehyde could possibly be associated to collagen cross linking occurring during the advanced stages of oral submucous fibrosis.

Introduction

Oral submucous fibrosis (OSMF) is a fibrotic condition of the oral cavity and is always associated with chronic epithelial inflammation and progressive deposition of collagenous extracellular matrix (ECM) proteins in the subepithelial layer of the buccal mucosa.[1] The disease is seen in those from Indian subcontinent and from many parts of South-East Asia such as Taiwan.[2] Numerous etiologies have been suggested for the pathogenesis of this condition that include consumption of chillies, nutritional deficiency, chewing of arecanut, genetic susceptibility, altered salivary constituents, autoimmunity and collagen disorders.[2] Current evidence implicates collagen-related genes in the susceptibility and pathogenesis of OSMF.[3]

The cytotoxic effects of the chewing tobacco including pan masalas are mediated through the production of the reactive oxygen species (ROS).[4] ROS induced lipid peroxidation causes a loss of cell homeostasis by modifying the structure and functions of cell membrane.[5] The most important characteristic of lipid peroxidation is to cause a considerable deoxyribose-nucleic acid malondialdehyde (DNA-MDA) adducts by interacting with cellular DNA.[6] Increased levels of MDA reported in individuals consuming large quantities of meat and fish.[7] We conducted a study to determine the levels of serum, salivary and tissue MDA in patients with OSMF. The influence of dietary pattern and habit frequency on the level of MDA has also been investigated.

Materials and Methods

Eighty-six patients between the age range of 20 and 40 years, reporting to the department of oral medicine and radiology in a dental college in south India were enrolled into the study. The study subjects included 65 histopathologically confirmed cases of OSMF and 21 age and sex matched healthy controls who required to undergo frenectomy or operculectomy. A detailed case history which included diet pattern and habit index was taken from each subject in the study. Subjects with any other long-term systemic illness and long term medication were excluded from the study. Five milliliter of unstimulated saliva was obtained by spit method after following standard precollection protocol. Five milliliter of venous blood was obtained from the antecubital vein, centifudged and stored. Tissue obtained during frenectomy, operculectomy from healthy controls and sectioned biopsy specimens from the OSMF patients were dissolved by the nitric acid method. The malonaldehyde content of the dissolved tissue, serum and saliva of the study subjects were evaluated by the TBARS procedure and sphectrophotometrically determined. The data obtained was subjected to statistical analysis using the SPSS version 17 software.

Results

Of the 65 subjects in the control group, 22, 20, and 23 were categorized under grade 1, 2, and 3, respectively [Figures 1a–c] after histopathological examination. The mean serum, salivary, and tissue malondialdehyde level of control group was significantly lower (P<0.001) than the cases [Table 1]. Increased levels of serum and salivary malondiadehyde was observed in all three study groups as the grading progressed but tissue malondialdehyde levels were lower in grade 3 OSMF (0.0244±0.01043 nmol/mg) compared to controls (0.0255±0.00593 nmol/mg). There was positive correlation between serum and salivary MDA levels, in case and control groups but negative correlation between serum MDA when compared to tissue levels. Salivary MDA had no significant correlation with tissue levels in case and control groups [Table 2]. There was no significant difference between the frequency of habits (gutka chewing) and mean serum, salivary, and tissue levels of MDA. No significant difference was observed in diet pattern (predominantly vegetarian/nonvegetarian) between cases and controls. When intercomparison of the diet status was done among the groups, no significant difference was observed [Table 3].

Figure 1

(a) Photomicrograph (×40) showing loose thin and thick fibers; (b) photomicrograph (×40) showing loose thin or thick fibers with partial hyalinization; (c) photomicrograph (×40) showing complete hyalinization

Table 1

Comparion of malonaldehyde levels between cases and controls (Student's unpaired t-test)

Table 2

Correlation of serum, salivary, and tissue levels in cases and control

Table 3

Intergroup comparison of diet pattern

Discussion

So far studies have been conducted on serum and salivary levels of lipid peroxidation end product malondialdehyde in cancer and precancerous conditions.[589] Most of the studies report of increase in the levels of MDA in oral cancer and OSMF.[8-10] Similar observations were reported in our study. A study were subjects were graded according to the classification by Bhat and Dholakia revealed significant elevation in serum MDA levels as grading progressed.[1011] We have also analyzed the tissue levels of MDA in different histopathological grades of OSMF (based on grading proposed by Kiran et al.).[2] Tissue levels of MDA were consistently higher in Grade 1 and Grade 2 OSMF when compared to controls. Elevated tissue MDA levels have been observed in experimental animals when exposed to cigarette smoke and ischemic conditions.[1213] Significantly higher levels of tissue MDA were detected in tumor tissues of breast cancer patients.[14] Tissue levels of MDA were reduced in Grade 3 OSMF when compared to controls. Possible reason could be due to utilization of malondialdehyde in crosslinking of collagen.[1516] It has been shown by an amino acid analysis that malonaldehyde reacts in a significant way on lysine and tyrosine residues.[16] Previous studies have revealed fibroblast stimulating properties of malondialdehyde in culture.[17] Addition of MDA to cultured fibroblasts increased collagen production by 2–3 times.[18] We used nitric acid dissolution method for tissue dissolution and thiobarbutric acid method (TBARS) for evaluation of malondialdehyde. Similar nitric acid dissolution method was used for determination of tissue malondialdehyde in zinc deficient rats.[19] We also investigated the dietary pattern and habit frequency of the subjects; however, no relevant information was obtained.

Conclusion

The decreased levels of tissue MDA in the advanced histopathological grades of OSMF could be probably due its utilization in pro-fibroblastic and collagen cross linking activities. However this finding requires further research which will enhance current knowledge about pathogenesis and progression of OSMF.