Journal of Cancer Research and Therapeutics Medknow Publications on behalf of the Association of Radiation Oncologists of India (AROI) ISSN: 0973-1482 EISSN: 1998-4138 Vol. 6, Num. 4, 2010, pp. 487-491

Journal of Cancer Research and Therapeutics, Vol. 6, No. 4, October-December, 2010, pp. 487-491

Original Article

Estimation of serum malondialdehyde in oral cancer and precancer and its association with healthy individuals, gender, alcohol, and tobacco abuse

Revant H Chole¹, Ranjitkumar N Patil², Anjan Basak³, Kamlesh Palandurkar³, Rahul Bhowate⁴

¹ Department of Oral Medicine and Radiology, Peoples Dental Academy, Bhopal, Madhya Pradesh, India
² Department of Oral Medicine and Radiology, KGMC, Lucknow, India
³ Department of Biochemistry, JN Medical College, Wardha, Maharashtra, India
⁴ Department of Oral Medicine and Radiology, SP Dental College, Wardha, Maharashtra, India

Correspondence Address: Revant H Chole, Department of Oral Medicine and Radiology, Peoples Dental Academy, Bhopal, Madhya Pradesh, India, drchole@rediffmail.com

Code Number: dv10122

PMID: 21358086

DOI: 10.4103/0973-1482.77106

Abstract

Keywords: Malondialdehyde, oral cancer, oral precancer

Introduction

The walls of biological cells are composed of cell membranes. They are composed of a double layer of lipid (fats and fatty compounds) with proteins dispersed throughout. Free radicals generated by a partial reduction of oxygen pose a serious hazard to tissues and vital organs, especially membrane lipids, connective tissues, and the nucleic acids of cells. Free radicals and reactive oxygen species (ROS) (like hydrogen peroxide ions, hydroxyl radical and superoxide anion which are highly reactive due to the presence of unpaired valence shell electrons) interact with lipids, DNA, and proteins which degrade proteins and promote DNA-strand breakage and damage to other genomic structures including the integrity of polyunsaturated fatty acids. As cell membranes are high in polyunsaturated fatty acids and this makes them prone to free radical attack, which in turn can affect the homeostatic environment of the cell. ^[1],[2] Lipid peroxidation is a chain reaction providing a continuous supply of free radicals that initiate further peroxidation ^[3] in which many damaging aldehydes are formed particularly malondialdehyde (MDA), propanedial, 4-hydroxynonenal (4-HNE), etc. MDA is a major metabolite of arachidonic acid (20:4) [fatty acid with 20 carbons and four double-bonds]. It is well known that MDA serves as a reliable marker of free radical-mediated lipid peroxidation. It is one of the important indicators of free radical-mediated tissue injury. It participates in a variety of chemical and biological reactions including covalent binding to protein, RNA, and DNA. The endogenous formation of MDA during intracellular oxidative stress and its reaction with biologically important macromolecules makes MDA-DNA adducts a suitable biomarker of endogenous DNA damage. ^[4] ROS are constantly produced during normal aerobic metabolism and are safely removed by a variety of biological antioxidants. Antioxidant protection is never 100% efficient; thus, mechanisms of repair are of key importance for survival. When pro-oxidants increase or antioxidants fail, a situation of oxidative stress (caused by an imbalance between the production of ROS and a biological system′s ability to readily detoxify the reactive intermediates or easily repair the resulting damage) ensues that leads to excessive molecular damage and tissue injury. ^[5] Thus there is a need to measure the oxidative stress in normal individuals, in oral precancerous lesions and conditions, and in oral cancer. Limited research has been done in this area of dentistry. Hence the purpose of this paper is to determine the serum level of MDA in oral cancer and precancer.

Material and Methods

The patients of oral cancer, precancer, and normal healthy controls were selected from the Department of Oral Diagnosis, Medicine and Radiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences, Sawangi, Wardha. After obtaining an informed consent, the patients were examined thoroughly and detailed case history of individuals was recorded on the case history proforma. Inclusion criteria: clinically and histopathologically diagnosed cases of oral cancer and precancer. Exclusion criteria: persons having any underlying systemic diseases and previously treated cases for oral cancer and precancer were not included in this study.

The patients were divided into following groups:

• First group: 30 individuals with clinically and histopathologically diagnosed cases of oral cancer without any underlying systemic disease
• Second group: 30 individuals with clinically and histopathologically diagnosed cases of oral precancer without any underlying systemic disease
• Third group: 30 healthy individuals as control not having any oral lesions or any underlying systemic disease.

From each individual 5 ml of blood was collected. Serum was separated by centrifuging blood sample at 3000 rpm for 5 min within 15 min of collection. This study was approved by the institutional ethical committee of Datta Meghe Institute of Medical Sciences, Sawangi, Wardha. Serum level of MDA was measured according to the method of Ohkawa et al (1979). ^[6] MDA level was determined by thiobarbituric acid reactive substances (TBARS) in serum, based on reaction between MDA and thiobarbituric acid. 1.5 ml of 0.8% thiobarbituric acid was added to 1 ml of serum sample. Then 0.4 ml of 8.1% sodium dodecyl sulfate and 1.5 ml of acetic acid were added. The mixture was finally made upto 5 ml with distilled water and placed in a hot water bath at 95 ⁰ C for 1 h. After cooling, 1.0 ml of distilled water and 5 ml of the mixture of n-butanol and pyridine (15:1, v/v) were added. The mixture was vortexed and after centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer (upper layer) was measured by the spectrophotometer at 532 nm against blank using distilled water. Thiobarbituric acid when allowed to react with MDA aerobically formed a colored complex [MDA - (TBA)2 complex] which was measured by the spectrophotometer. MDA concentration (measured as TBARS) was calculated as μmol/ml. Serum MDA level was estimated before undergoing treatment.

Results

The mean MDA level in the control group was found to be 5.107±2.32 ηmol/ml, whereas the mean MDA level in oral precancer and oral cancer was found to be 9.33 ± 4.89 ηmol/ml and 14.34 ± 1.43 ηmol/ml, respectively. There was statistically significant increase in serum MDA levels in serum of the oral precancer and oral cancer patients compared with the control group [Table - 5]. The t-calculated value between serum MDA level in WDSCC and MDSCC is 0.021, between WDSCC and PDSCC is 0.1927; and between MDSCC and PDSCC is 0.1331; and it is statistically non-significant (P>0.05).

In the control group without habits, the mean MDA level in males was found to be 3.77 ηmol/ml and in females was found to be 4.79 ηmol/ml; and this difference was statistically not significant (P > 0.05). In the oral precancer group, the mean MDA level in males was found to be 9.40 ηmol/ml and in females was found to be 8.43 ηmol/ml; and this difference was statistically not significant (P > 0.05). In the oral cancer group, the mean MDA level in males was found to be 14.805 ηmol/ml and in females was found to be 13.93 ηmol/ml; and this difference was statistically not significant (P > 0.05).

Discussion

Clinical research in the area of lipid peroxidation has been hampered by the lack of a valid biomarker. One of the most frequently used biomarkers providing an indication of the overall lipid peroxidation level is the concentration of MDA, one of the several byproducts of lipid peroxidation processes. ^[7] MDA is a naturally occurring endogenous product of lipid peroxidation and prostaglandin biosynthesis, but is mutagenic and tumorigenic. The MDA value in blood is a measure for the ability of the body to handle the oxidative stress it is exposed to. The literature shows an increase in MDA level in patients with cancerous changes in oral mucosa as compared to normal individuals [Table - 6]. This study included 30 subjects with oral cancer, 30 subjects with oral precancer, and 30 normal individuals as controls. The literature shows that the process of carcinogenesis occurs by generation of ROS, which act by initiating lipid peroxidation. The present study has revealed an intriguing aspect of tumor biochemistry. This study showed significant differences between serum MDA levels in oral precancer (9.33 ± 4.89 ηmol/ml), oral cancer (14.34 ± 1.43 ηmol/ml), and healthy controls (5.107 ± 2.32 ηmol/ml) (P < 0.05). Serum MDA increased in oral precancer and cancer patients as compared to normal healthy individuals. Few studies showed that MAD levels were significantly elevated in oral squamous cell carcinoma patients as compared with the healthy controls. ^[8],[9] In our study in the oral cancer group, MDA levels in males (14.805 ηmol/ml) were more as compared to females (13.93 ηmol/ml) but the results were not statistically significant (P > 0.05).

MDA levels showed a marked increase in cancers of other parts of the body as compared to oral cancer. According to Sahin et al^[10] MDA levels increased significantly in lung cancer (20.5 ± 7.9 ηmol/ml) as compared to healthy controls (12.6 ± 7.1 ηmol/ml) (P < 0.001). Bakan E et al^[11] stated that the levels of plasma MDA were significantly higher in patients with gastric cancer (7.6 ± 1.4 ηmol/ml) as compared with the healthy controls (5.4 ± 1 ηmol/ml). In a study on cervical carcinoma by Gitanjali et al, ^[12] there was no statistically significant difference in the mean serum MDA levels of healthy controls (3.40 ± 0.75 ηmol/ml) and patients with cervical carcinoma (3.54 ± 1.89 ηmol/ml. Other than cancer, literature has reported increased MDA levels in chronic obstructive pulmonary disease, H. pylori-infected gastric mucosa, ^[13] during cardiopulmonary bypass ^[14] and during hemodialysis. ^[15],[16]

This study found that the degree of differentiation of oral squamous cell carcinoma was inversely proportional to lipid peroxidation. MDA levels in well-differentiated squamous cell carcinoma [14.81 ± 1.54 ηmol/ml] was greater as compared to moderately differentiated [14.68 ± 1.80 ηmol/ml] and poorly differentiated squamous cell carcinoma [13.2 ± 0.54 ηmol/ml], but this difference was statistically not significant (P > 0.05). Thus there was no correlation in lipid peroxidation between degrees of differentiation of malignant oral lesions. These results correlated with study of Salzman et al^[17] who showed a negative correlation of MDA and tumor grade.

Though radiotherapy is one of the clinical means by which oral cancer can be treated, many biochemical complications, such as damage to cellular DNA and membrane structures can occur. Sabitha et al^[18] showed a significant increase in MDA levels in untreated and radiation-treated oral cancer patients when compared with normal subjects. Radiation-treated patients had higher MDA levels (0.792 ± 0.1157 ηmol/l) than untreated patients (0.598 ± 0.169 ηmol/l) (P < 0.001). According to Gupta et al^[19] MDA levels in oral sub mucous fibrosis (3.3 + 0.4 ηmol/ml) were more as compared to healthy controls (2.4 + 0.5) (P < 0.001). In our study serum MDA levels was found to be increased in oral precancer [9.33 (± 0.89) ηmol/ml] as compared to normal individuals [5.107 (±0.4247) ηmol/ml] and the increase was statistically significant (P < 0.05). This shows possibility of role of ROS in aetiopathogenesis of oral precancer.

An attempt was made to study the gender related changes in lipid peroxidation in normal healthy individuals. Males (3.77 ηmol/ml) had less MDA level than females (4.79 ηmol/ml), but these results were not statistically significant (P > 0.05). These results were in contrast with Knight et al^[20] whose study showed that males have higher MDA concentrations in plasma [0.60 (0.21) μ mol/l] than do females [0.54 (0.20) μ mol/l] (P < 0.05); and Richard et al^[21] who showed that there existed no sex-related difference for TBARS: 2.57 (0.28) in males versus 2.44 (0.20) μ mol/l in females.

Further attempt was made to study the association of lipid peroxidation and the habit of either betel nut or betel leaf chewing, or tobacco chewing or smoking, in normal subjects. Ten of the thirty healthy subjects had the habit of either betel nut or betel leaf chewing, or tobacco chewing or smoking. The MDA level was increased in these subjects (7.162 ± 2.233 ηmol/ml) as compared to the 20 subjects having none of these habits (4.08 ± 1.609 ηmol/ml) and the increase was statistically significant (P < 0.05).

Cigarette smoke, in both the gaseous phase and condensed particles, contains alkenes, nitrosamines, aromatic, and heterocyclic hydrocarbons and amines. In addition, it is an excellent source of ROS, such as hydroxyradicals, superoxides, and peroxides that are capable of initiating or promoting oxidative damage. Sahin et al^[10] has reported increased MDA levels in smoker (22.5 ± 8.0 ηmol/ml) than nonsmoker (16.1 ± 6.7 ηmol/ml) (P < 0.001) in patients with lung cancer. Reznick et al^[22] elucidated the outcome of interaction of between cigarette smoke and oral salivary peroxidase in smokers and nonsmokers. After smoking a single cigarette, a sharp drop of oral peroxidase activity was observed in both groups: 42.5% in smokers and 58.5% in nonsmokers (P < 0.05). After 30 min, the level of activity returned to 90-100% of the presmoking level, presumably due to the secretion of new saliva into the oral cavity. The oral peroxidase activity loss was accompanied by increased carbonylation of the salivary proteins, an indicator of the oxidative damage to proteins. According to Nielsen et al^[7] daily smokers had a slightly higher average concentration of plasma MDA than nonsmokers, and plasma MDA correlated with daily exposure to cigarette smoke. Morrow ^[23] proved that plasma levels of lipid peroxidation product (F ₂ -isoprostanes) were significantly higher in the smokers than in the nonsmokers. One study by Craig et al^[24] was in contrast with the above studies, which stated that there was no strong relation between lipid peroxide level and cigarette smoke exposure, suggesting that certain interactions related to oxidation status are not measurable in the serum compartment. They further stated that serum copper was the major determinant of serum lipid peroxidation status, indicating that it contributes to lipid peroxidation in vivo. Nielsen et al^[7] demonstrated a positive correlation between plasma MDA and weekly alcohol consumption. No major interaction occurred between gender and age. Men had slightly but significantly higher plasma MDA concentrations than women.

Reaction of the ROS with cellular DNA results in oxidative damage which is considered to be crucial in cancer development, thus providing an additional possible mechanism for the apparent association between smoking and mouth, lung, pharynx, esophagus, bladder, and cervical cancers. Niedernhofer et al^[25] proved that MDA-induced DNA damage is mutagenic in human cells. Zhang et al^[26] showed that MDA-DNA adducts may serve a biomarker of DNA damage by lipid peroxidation induced endogenously or exogenously in oral mucosal cells. Leuratti et al^[27] showed that MDA-deoxyguanosine (M1-dG), a DNA adduct derived from lipid peroxidation plays a role in human colorectal carcinogenesis, in combination with other genetic and environmental factors. The body has evolved its own natural free radical scavengers, which include the antioxidant vitamins (Vitamin A and beta-carotene, several of the B-complex vitamins, Vitamin C and Vitamin E), the mineral selenium and the enzyme systems superoxide dismutase, glutathione peroxidase, and catalase. Damage from free radicals can be prevented and even reversed if there are sufficient concentrations of antioxidants, which work individually and together in the body. Antioxidants inhibit the formation as well as propagation of free radicals. Gitanjali et al^[12] and Gupta et al^[19] has shown that administration of antioxidant vitamins in cancer patients, decreased the MDA level. From this study we concluded that serum MDA level increases in oral cancer and oral precancer as compared to normal healthy individuals, which may lead to their early detection. Increased serum MDA level indicates a change from normal to a high risk yet non-cancerous state, would serve as a valuable marker for both preventive and clinical intervention, and may deserve further investigation for the early diagnosis, treatment, and prognosis. Oxidative stress (increased MDA level) is more intense in oral cancer and precancer compared to normal healthy individuals, which indicates the role of antioxidant therapy as an adjunct, in the treatment of oral precancer and cancer.

References

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