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Lipid Peroxidation & Antioxidants Status in Patients With Oral Squamous Cell Carcinoma

Posted on: Friday, 31 March 2006, 06:00 CST

By Manoharan, S; Kolanjiappan, K; Suresh, K; Panjamurthy, K

Background & objectives: Overproduction of lipid peroxidation byproducts and disturbances in antioxidant defense system have been implicated in the pathogenesis of several diseases including oral cancer. Though several studies have been done on the level of lipid peroxidation and antioxidants in oral cancer patients, there are no reports in patients with various clinical stages of oral squamous cell carcinoma. We carried out this study to assess the level of oxidative stress in oral cancer patients with various clinical stages.

Methods: Blood samples of 48 adult male oral cancer patients with various clinical stages of oral cancer (stage II to stage IV, 16 of each) and 16 age and sex matched healthy subjects were collected. Plasma and erythrocytes levels of thiobarbituric acid reactive substances (TBARS), vitamin E, reduced glutathione (GSH), and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were assayed using specific colorimetric methods. The statistical comparisons were performed by one way analysis of variance (ANOVA) followed by Student's t-test.

Results: Elevated lipid peroxidation and decline in non- enzymatic and enzymatic antioxidants status were noticed in oral cancer patients as compared to healthy subjects. The TBARS levels were gradually increased whereas antioxidants were gradually reduced from stage II to stage IV of oral cancer patients.

Interpretation & conclusion: The altered lipid peroxidation in plasma and erythrocytes of oral cancer patients may be related to their compensatory changes in the antioxidants defense system.

Key words Antioxidants - lipid peroxidation - oral cancer

Oral cancer is a major form of cancer worldwide and is one of the most common malignancy in India accounting for 30-40 per cent of all cancers1,2. Squamous cell carcinoma of the oral cavity is responsible for considerable morbidity and mortality in India where 60,000 new cases of oral cancer are reported to occur every year3. Tobacco chewing with betel quid, tobacco smoking and alcohol consumption are the most important aetiological factors associated with the incidence of oral cancer in India4.

Reactive oxygen species plays an effective role in the pathogenesis of different pathological diseases including cancer5,6. Free radical induced lipid peroxidation causes a loss of cell homeostasis by modifying the structure and functions of cell membrane. The most important characteristic of lipid peroxidation is to cause a considerable DNA-MDA adducts by interacting with cellular DNA7,8. However, mammalian cells possess elaborate antioxidant defense mechanisms to neutralize the deleterious effects of free radical induced lipid peroxidation9'10.

Enzymatic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)] and non-enzymatic antioxidants [vitamin E, reduced glutathione (GSH)] act synergistically with one another to detoxify the effects of lipid peroxidation. We have earlier demonstrated the status of lipid peroxidation and antioxidant defense mechanism in plasma and erythrocytes of oral cancer patients11,12. Subapriya et al13 have reported enhanced lipid peroxidation with decline in antioxidants in venous blood of patients with oral squamous cell carcinoma at different intraoral sites. However, there are no reports on lipid peroxidation and antioxidant status in patients with various clinical stages of oral squamous cell carcinoma. The present study was therefore, undertaken to evaluate the status of lipid peroxidation and antioxidants in plasma and erythrocytes of oral cancer patients with various clinical stages.

Material & Methods

Blood samples were obtained consecutively from 48 male oral cancer patients admitted during 2001-2003, in Raja Muthiah Dental College and Hospital (RMDC&H), Annamalai University, Annamalai Nagar, Tamil Nadu, India. The patients were categorized into three different groups of 16 each (stages II, III and IV) according to TNM (tumour, node, metastasis) system of cancer classification14. Sixteen age matched healthy males were also investigated as control. Appropriate control subjects were selected in and around Annamalai Nagar. The subjects who were diagnosed as cancerfree were categorised as control subjects. The controls were healthy subjects not habituated to tobacco chewing and smoking and were of the same age, sex and socio-economic strata as the oral cancer patients. The subjects were ranging in age from 40-60 yr (control: 51.3 4.6; oral cancer patients: 53.2 5.1). As only 16 patients with stage IV carcinoma could be admitted and included during the study period the number in other subgroups with stages II and III and control group was also restricted to 16 in each group. So the first 16 patients with stage II or III were included. Study protocol was approved by the ethics committee of RMDC & H.

Oral tumour tissues obtained at the time of surgery from buccal mucosa of patients were immediately fixed in 10 per cent formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The tumours were histopathologically confirmed as well, moderately and poorly differentiated squamous cell carcinoma15.

Blood samples (6 ml) were obtained from patients and controls by venous arm puncture and the plasma was separated by centrifugation at 1000 g for 15 min. After plasma separation, the buffy coat was removed and the packed cells were washed three times with physiological saline. A known volume of erythrocytes was lysed with hypotonie phosphate buffer, pH 7.4. The haemolysate was separated by centrifugation at 3500 g for 15 min at 2C. The erythrocyte membrane was prepared by the method of Dodge et al16 with a change in buffer according to Quist17. TBARS and antioxidants estimations were carried out in plasma, erythrocytes and erythrocyte membranes of healthy subjects and oral cancer patients.

Thiobarbituric acid reactive substances (TEARS) released from the endogenous lipoperoxides, reflecting the lipid peroxidation processes, were assayed in plasma18, and in erythrocytes and erythrocytes membranes19. Vitamin E level was determined by the method of Desai20.

The reduced glutathione was determined by the method of Beutler and Kelly21. The technique involves protein precipitation by meta- phosphoric acid and spectrophotometric assay at 412 nm of the yellow derivative obtained by the reaction of the supernatant with 5- 5'dithiobis-2-nitrobenzoic acid. The erythrocyte membrane protein was measured by the method of Lowry et al22.

Cytoplasmic Cu/Zn superoxide dismutase activity was assayed by the method of Kakkar et aP. One unit of enzyme is taken as the amount of enzyme required to give 50 per cent inhibition of nitro blue tetrazolium (NBT) reduction. The activity of catalase was assayed by the method of Sinha24, based on the utilization of H2O1 by the enzyme. The colour developed was read at 620 nm. One unit of the enzyme is expressed as moles of H^sub 2^O^sub 2^ utilized per minute. Glutathione peroxidase activity was assayed by the method of Rotruck et al25 based on the utilization of reduced glutathione by the enzyme. One unit of enzyme is expressed as moles of GSH utilized per minute.

Statistical analysis: The statistical comparisons were performed by one way analysis of variance (ANOVA) followed by Student's t- test.

Results & Discussion

A total of 48 patients and 16 control subjects were selected for the study. The patients were categorized into three groups of 16 each, based on their clinical stages of tumour (Stages II, III. and IV). according to TNM system of cancer classification. The habit of tobacco chewing, smoking and alcohol consumption were common in oral cancer patients. The clinical status of patients as confirmed by histopathological examination was found to be welldifferentiated, moderately differentiated and poorly differentiated squamous cell carcinoma. Healthy subjects were not habituated to tobacco chewing, smoking and alcoholic consumption, and were free from any systemic diseases. Both normal subjects and oral cancer patients were males ranging in age from 40-60 yr (control: 51.3 4.6; oral cancer patients: 53.2 5.1).

Table I. TBARS levels in plasma, erythrocytes. and erythrocytc membranes of healthy subjects and oral cancer patients with various clinical stages

The level of TBARS was significantly increased in plasma, erythrocytes and erythrocyte membranes of oral cancer patients as compared to healthy subjects, and were gradually increased from stage II to stage IV of oral cancer patients (Table I). The levels of vitamin E and reduced glutathione were significantly decreased in oral cancer patients as compared to healthy subjects, and were gradually decreased from stage II to stage IV of oral cancer patients (Table II). The activities of superoxide dismutase. catalase and glutathione peroxidase were also significantly decreased in oral cancer patients as compared to healthy subjects, and were gradually decreased from stage II to stage IV of oral cancer patients (Table III).

Byproducts of lipid peroxidation cause marked alteration in the structural integrity and function of cell membranes2'1. Lipid peroxidation byproducts formed under physiological and pathological conditions are scavenged by non enzymatic and enzymatic antioxidants27. An imbalance betw\een antioxidant defense mechanism and lipid peroxidation processes results in cell and tissue damage28,29. Lower levels of plasma and erythrocyte membrane vitamin E have been reported in various pathological conditions including oral cancer11,30. A positive correlation between vitamin E and lipid peroxidation has been reported in oral cancer patients11,12. Enhanced lipid peroxidation with decline in antioxidants has been reported in venous blood of oral cancer patients and patients with oral squamous cell carcinoma at different intraoral sites11"13. Kolanjiappan et al31 have reported an increase in vitamin E and glutathione levels in tumour tissues of patients with various clinical stages of oral squamous cell carcinoma. Buzby et al32 suggested that host tumour cells sequester essential nutrients from the circulation to meet the demand of growing tumour. Thus, the observed decrease in vitamin E and reduced glutathione in plasma and erythrocytes of oral cancer patients can be due to utilization of these antioxidants by tumour tissues or to compromise the excessive oxidative stress in circulation.

Table II. Plasma and erythrocytes vitamin E and reduced glutathione levels in healthy subjects and oral cancer patients with various clinical stages

Table III. Plasma and erythrocyte lysate glutathione peroxidase, superoxide dismutase and catalase activities in healthy subjects and oral cancer patients with various clinical stages

The antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase serve as the backbone of cellular antioxidant defense mechanism3'. Lowered activities of these enzymes have been reported in various pathological conditions including oral carcinogenesis"14. Our results support these observations.

In the present study the levels of circulating TEARS and antioxidants were found to be associated with the tumour stages of patients. Measurement of lipid peroxidation and antioxidants in circulation of oral cancer patients may thus be helpful in assessing the tumour stages of oral squamous cell carcinoma.

Acknowledgment

Authors thank Dr C.R. Ramachandran, Dean, Faculty of Dentistry. Raja Muthiah Dental College and Hospital (RMDC&H). Annamalai University for his help in categorizing oral cancer patients and carrying out histopathological studies.

References

1. Notani PN. Global variation in cancer incidence and mortality. Curr Sd 2001: 81 : 465-74.

2. Moore SR. Johnson NW. Pierce AM. Wilson DF. The epidemiology of mouth cancer: a review of global incidence. Oral Dis 2000: 6 : 65- 74.

3. Gupta PC, Nandakumar A. Oral cancer scene in India. Oral Dis 1999; 5 : 1-2.

4. Rao DN. Ganesh B. Rao RS. Desai PB. Risk assessment of tobacco, alcohol and diet in oral cancer - A case control study. Int J Cancer 1994; 58 : 469-73.

5. Halliwell B. Mechanisms involved in the generation of free radicals. Patlwl Biol (Paris) 1996: 44 : 6-13.

6. Kehrer JP. Free radicals as mediators of tissue injury and disease. CnV Rev Toxicol 1993; 23 : 21-48.

7. Abidi S. AIi A. Role of oxygen free radicals in the pathogenesis and etiology of cancer. Cancer LeIt 1999; 142 : 1-9.

8. van Ginkel G. Sevanian A. Lipid peroxidation-induced membrane structural alterations. Methods Enzyiol 1994; 233 : 273-88.

9. Klaunig JE. Xu Y. Isenberg JS. Bachowski S, Kolaja KL, Jiang J, et al. The role of oxidative stress in chemical carcinogenesis. Environ Health Perspect 1998; 106 (Suppl) : 289-95.

10. Barber DA. Harris SR. Oxygen free radicals and antioxidants: a review. Am Plianu 1994: 34 : 26-35.

11. Manoharan S. Shreeram S, Nagini S. Life style can induce lipid peroxidation and compromise of antioxidant defence mechanisms in the erythrocytes of oral cancer patients. Med Sd Rex 1996; 24 : 397-400.

12. Manoharan S. Nagini S. Lipid peroxidation and antioxidant status in oral cancer patients. Med Sd Res 1994: 22 : 291-2.

13. Subapriya R. Kumaraguruparan R. Ramachundran CR, Nagini S. Oxidant-antioxidant status in patients with oral squamous cell carcinomas at different intraoral sites. CUn Biochem 2002; 35 : 489- 93.

14. Sobin LH. Wittenkind CH. editors. International Union Against Cancer (UICC): TNM classification of malignant tumours. 5th ed. New York: Wiley Liss Publications; 1997 p. 17-42.

15. Kannan S. Kartha CC. Chandran GJ. Pilloi MR. Sudha L. Nalinakumari KR. et a/. Ultrastructure of oral squamous cell carcinoma: A comparitive analysis of different histological types. Eur J Cancer B Oral Oncol 1994; 30 : 32-42.

16. Dodge JT. Mitchell C. Hanahan DJ. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biopliyx 1963: 100 : 119-30.

17. Quist EH. Regulation of erythrocyte membrane shape by calcium ion. Biochem Biophys Rex Commun 1980; 92 : 631-7.

18. Yagi K. Lipid peroxides and human diseases. Chem Phys Lipids 1987; 45 : 337-51.

19. Donnan SK. The thiobarbituric acid test applied to tissues from rats treated in various ways. J Bio/ Chem 1950: 182 : 415-9.

20. Desai FD. Vitamin E analysis, methods for animal tissues. Met/ was Enzymo/ 1984: 105 : 138-45.

21. Beutler E. Kelly BM. The effect of sodium nitrite on red cell GSH. Experientia 1963; 19 : 96-7.

22. Lowry OH. Rosebrough NJ. Farr AL. Randall RJ. Protein measurement with Folin's phenol reagent. J Biol Chem 1951 ; 193 : 265-75.

23. Kakkar P, Das B. Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984: 21 : 130-2.

24. Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972; 47 : 389-94.

25.Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: biochemical roles as a component of glutathione peroxidase. Science 1973; 779 : 588-90.

26. Kolanjiappan K, Manoharan S, Kayalvizhi M. Measurement of erythrocyte lipids, lipid peroxidation, antioxidants and osmotic fragility in cervical cancer patients. Clin CMm Acta 2002; 326 : 143- 9.

27. Gutteridge JMC. Lipid peroxidation and antioxidants as biomarkers of tissue damage. CHn Chem 1995; 41 : 1819-28.

28. Nagini S, Saroja M. Circulating lipid peroxides and antioxidants as biomarkers of tumour burden in patients with oral squamous cell carcinoma. J Biochem MoI Biol Biophys 2001; 5 : 55-9.

29. Guyton KZ, Kensler TW. Oxidative mechanisms in carcinogenesis. Br Med Bull 1993; 49 : 523-44.

30. Halliwell B. Free radicals, antioxidants and human disease: curiosity, cause or consequence? Lancet 1994; 344 : 721-4.

31. Kolanjiappan K, Ramachandran CR, Manoharan S. Biochemical changes in tumour tissues of oral cancer patients. Clin Biochem 2003; 36 : 61-5.

32. Buzby GP, Mullen JL, Stein TP, Miller EE, Hobbs CL, Rosato EF. Host-tumor interaction and nutrient supply. Cancer 1980; 45 : 2940-8.

33. Sun Y. Free radicals, antioxidant enzymes, and carcinogenesis. Free Radie Biol Med 1990; 8 : 583-99.

34. Mates JM. Perez-Gomez C, Nunez de Castro I. Antioxidant enzymes and human diseases. Clin Biochem 1999; 32 : 595-603.

S. Manoharan, K. Kolanjiappan, K. Suresh & K. Panjamurthy

Department of Biochemistry, Annamalai Universitv, Annamalai Nagar, India

Received July 19, 2005

Reprint requests: Dr S. Manoharan, Senior Lecturer, Department of Biochemistry, Faculty of Science Annamalai University, Annamalai Nagar 608002, India

e-mail: manshisak@yahoo.com

Copyright Indian Council of Medical Research Dec 2005

Source: Indian Journal of Medical Research

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