Assessment of oxidative stress and antioxidant profiles in patients with breast carcinoma.
Breast carcinoma is one of the most common neoplasms in women and is the leading cause of cancer related deaths worldwide (Rajneesh et al, 2008). It refers to cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules. Oxidative stress is a term used to denote the imbalance between the concentrations of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) and the antioxidant defence mechanisms in the body (Badid et al, 2009). Reactive oxygen species (ROS) or free radicals are generated as by-products of normal cell metabolism. They can be produced and act inside the cell or they can be generated within the cell and released to extra cellular space (Frei, 1994). Free radicals may attack polyunsaturated fatty acids within membranes, forming peroxyl radicals. These newly formed free radicals can then attack adjacent fatty acids within membranes causing a chain reaction of lipid peroxidation. The lipid hydroperoxide end products are also harmful, and may be responsible for some of the overall effect, which can lead to tissue and organ damage.
In healthy individuals, antioxidants protect components of the body against free-radical damage (Halliwell, 1997). Antioxidants react with the free radicals to neutralize their effect by donating electrons, thereby forming much less reactive radicals. This system consists of numerous enzymatic and non-enzymatic low molecular weight components that scavenge the free radicals formed within the cell. Thus scavenger antioxidants are important not only because they react with free radicals directly but also because they act synergistically with one another (Freisleben and Packer, 1993). The present study was planned to evaluate oxidative stress and antioxidant status in the pathobiology of breast cancer patients.
Materials and Methods
A group of fifty clinically diagnosed breast cancer patients from various hospitals of Tamil Nadu who had not undergone any previous treatment for their tumours was taken. The 50 patients chosen were catogorised into 2 groups namely, non-metastatic and metastatic breast cancer. Post-treatment group consisted of 25 samples comprising of patients who had undergone either chemotherapy or radiotherapy for their disease (Table 1). Informed consent was obtained from every patient. Control consisted of 25 members of the public with no prior history of breast cancer or other cancer related diseases.
Sample Collection and Methods
Blood samples were collected by venous arm puncture into heparinised tubes and plasma was separated by centrifugation at 3000 rpm for 15 minutes. Lipid peroxidation was estimated as evidenced by the formation of TBARS (Thiobarbituric acid Reactive Substance) by the method of Yagi (1987). Plasma was deproteinised with phosphotungstic acid and the precipitate was treated with thiobarbituric acid at 90[degrees]C for I hour. Absorbance of pink coloured complex formed was measured at 535nm. Superoxide dismutase (SOD) activity was assayed at 520nm by the method of Kakkar et al, 1984 which was based on the inhibition of NADH- phenazine methosulphate nitroblue tetrazolium formation. The activity of catalase (CAT) was assayed by the method of Sinha (1972) based on the utilisation of [H.sub.2][O.sub.2] by the enzyme and the colour developed was read at 620nm. The activity of glutathione peroxidase (GPX) was estimated according to the method of Rotruck et al, (1973). Glutathione S transferace (GST) was estimated by the method of Habig et al. (1994) which was measured by following the increase in absorbance at 340 nm using 1-chloro 2,4 dinitrobenzene (CDNB) as the substrate.
Vitamin E was estimated by the method of Palan et al. (1973) which involves the conversion of ferric ions to ferrous ions by a-tocopherol and the formation of red coloured complex with 2, 2 dipyridyl measured at 520nm. Vitamin C level was estimated by the method of Omaye et al, (1979) and the dehydroascorbic acid formed by the oxidation of ascorbic acid by copper on treatment with 2,4 dinitro phenyl hydrazene, was measured at 525nm. Reduced glutathione (GSH) was measured at an absorbance of 412 nm according to the method of Beutler and Kelley (1963).
The data obtained in the present study was subjected to statistical analysis. Standard deviation was done to obtain accuracy. Students t test was used to compare the significance of means, between control and experimental groups at 5% level.
The extent of lipid peroxidation and the antioxidant profile in breast cancer patients before and after treatment is represented in table 2. The concentration of TBARS in breast cancer patients was significantly elevated than that of the healthy subjects. Patients with metastatic disease have shown marked increase in TBARS than those with non metastatic disease.
The concentrations of enzymatic antioxidants namely, SOD, CAT, GPX and GST were all found to be significantly elevated in breast cancer patients than that of the healthy subjects. When compared between non-metastatic disease and metastatic disease, the values were found to be significantly elevated in metastatic breast carcinoma.
In case of non-enzymatic antioxidants, GSH was found to be significantly increased in breast cancer patients, with marked elevation found in metastatic group. Vitamin C was found to be significantly reduced, whereas in vitamin E level no change was observed in breast carcinoma patients.
In post treatment group TBARS, SOD, CAT and GST were found to be significantly elevated than the normal subjects; Vitamin C was significantly elevated than the normal subjects whereas in GPX, GSH and vitamin E no significant differences were observed. However significant differences have been found in the pre and post-treatment values of TBARS, SOD, GPX and GSH.
Oxidative stress is defined as a pervasive condition of increased and/or inadequate removal of Reactive oxygen species (ROS) (Irshad and Chaudhuri , 2002). In the present study TBARS content was found to be significantly increased in breast cancer patients when compared with the healthy subjects (table 2). This coincides with the findings of other researchers (Yeh et al., 2005; Rajneesh et al., 2008). Similar result was also obtained by Gonenc et al. (2006) in blood and tissue of malignant breast tumour and benign breast disease. In this study significant elevation has been found in metastatic disease than in those of non metastatic disease. ROS can specifically damage certain protease inhibitors including [[alpha].sub.1]-proteinase inhibitor, [[alpha].sub.2]-macroglobulin, PAI and [[alpha].sub.2]-plasmin inhibitor by oxidising methionine residues at the active site. Inactivation of these protein inhibitors could induce enhanced action of proteases such as elastase, plasminogen activator and plasmin. They may facilitate tumour invasion and metastasis (Szatrowski and Nathan, 1991).
Antioxidants react with the free radicals to neutralize their effect by donating electrons, thereby forming much less reactive radicals. In the present study, the concentrations of enzymatic antioxidants namely, SOD, CAT, GPX and GST were all found to be significantly elevated in breast cancer patients than that of the healthy subjects. Similar findings have been documented by several workers (Rajneesh et al., 2008; Skrzydlewska et al., 2001 and Iscan et al, 2002). Increased SOD mRNA expression was observed in cancer samples from patients with carcinoma of breast (Li et al., 1998). Higher activity of CAT has been documented in tumour cell lines compared to controls( Ripple and Henry, 1997). GST which is involved in the detoxification of electrophilic toxins and carcinogens is increased in most of the human tumours studied. Similarly a significant increase in GPX in tumours has also been reported by Iscan et al.(2002).
In the present study, three non-enzymatic antioxidants were considered namely GSH, vitamin C and vitamin A. GSH was found to be significantly elevated in breast cancer patients. Over expression of GSH have been reported in both animal and human tumours by other workers (Rajneesh et al., 2008; Skrzydlewska et. al, 2001; Yang et al., 1997). In this study Vitamin C were significantly reduced in breast cancer patients than that of the healthy subjects which is in accordance to the study made by Badid et al. (2009) in over-weight women with breast cancer. They have reported that the plasma levels of vitamin C and E could reflect their high utilisation rate, suggesting that these vitamins may be used to reduce oxidative stress in breast cancer patients. Alternately it is also possible that reduced vitamin C and E concentrations reflects low intake, which resulted in decreased antioxidant defence systems in breast cancer patients. However in this study, no significant difference was observed in plasma vitamin E concentration.
In the present study , the lipid peroxidation and the antioxidant response after treatment differed significantly in the examined groups. Increased activity of SOD and CAT, vitamin C as well as reduced GSH, were sufficient for the elimination of elevated free radical and [H.sub.2][O.sub.2] production and suppression of the oxidative stress. But the activity of GPx and GSH concentration were found to be significantly lowered when compared with the pre treatment level. Based on this it would appear that the decreased production capacity of [H.sub.2][O.sub.2] elimination might be a cause of the detected oxidative damage. Hydrogen peroxide in the increased concentration, apart from causing direct oxidative damage, can generate extremely reactive hydroxyl radicals through interaction with transition metals (Stohss and Bagchi, 1995). Kasapovic et al. (2009) in a similar study reported that response to radiotherapy involves age related decrease in antioxidants capacity for the elimination of [H.sub.2][O.sub.2] thus causing the occurrence of oxidative damage to blood cells in progressive stages of breast carcinoma.
The summary of the findings of the present study revealed the following:
1. Significant increase in oxidative stress was found in non-metastatic and metastatic breast cancer patients as well as in post-treatment group as evidenced by increase in TBARS
2. Enhanced production of enzymatic antioxidants was observed in all the three groups
3. Among non-enzymatic antioxidants, significant increase in GSH and reduction in vitamin C levels were observed in all 3 groups when compared with the healthy subjects
4. Generally to counterbalance the oxidative stress, enhanced production of major antioxidants except vitamin C and vitamin E was observed
5. The above conditions may give the tumour cells a selective growth advantage over the normal counterparts
 Badid, N., Ahmed, F.Z.B., Merzouk, H., Belbraouet, S., Mokhtari, N., Merzouk, S.A., Benhabib, R., Hamzaoui, D. and Narce, 2009, "Oxidant, antioxidant status, lipids and hormonal profile in overweight women with breast cancer," Pathol. Oncol. Res., 16(2), pp 159-167.
 Beutler, E. and Kelley, B.M., 1963, "The effect of sodium nitrate on RBC glutathione," Experientia, 19, pp 96-97.
 Dalle-Donne I., Rossi R., Colombo R., Giustarini D. and Milazani A., 2006, "Biomarkers of oxidative stress in human diseases," Clin. Chem, 52, pp 60123.
 Frei B., 1994, "Reactive oxygen species and antioxidant vitamins: mechanism of action," Am. J. Med., 97, pp 5-13.
 Gonenc, A., Erten, D. and Aslan, S., 2006, "Lipid peroxidation and antioxidant status in blood and tissue of malignant breast tumour and benign breast disease," Cell Biol. Int., 30, pp 376-380.
 Halliwell B., 1997, "Antioxidants and human disease: a general introduction," Nutrition Reviews, 55, pp 44-52.
 Iscan, M., Coban, T., Cok, I., 2002, "The organochlorine pesticide residues and antioxidant enzyme activities in human breast tumour: is there any association?," Breast cancer Res. Treat., 72, pp 173.
 Ishad M. and Chaudhuri P.S., 2000, "Oxidant-antioxidant system: Role and significance in human body," Ind. J. Exp. Biol., 40, pp 1233-1239.
 Kakkar, P., Das, B. and Visvanathan, P.N., 1984, "A modified spectrophotometric assay for superoxide dismutase," Indian. J. Biophys., 21, pp 130-132.
 Kaspovic, J., Pejic, S., Todorovic, A., Stojilkovic, V., Jelic, L.R. and Pajovic, S.B., 2009, "Antioxidants in breast cancer patients of different ages after radiotherapy," Arch. Biol. Sci., 61(1), pp 23-28.
 Omaye, S.T., Turnbull, T.D. and Sauberlich, H.E., 1979, "Selected method for the determination of vitamin E in animal cells tissues and fluids," In: Mc Cormic, D.B., Wright, D.L. Eds., Methods Enzymol., 62, pp 3-11.
 Palan, P.R., Milkhail, B.S., Basin, J. and Romney, S.L., 1973, "Plasma levels of antioxidants beta carotene and tocopherol in uterine cervix dysplasia and cancer," Nutr. Cancer, 15, pp 13-20.
 Rajneesh, C.P., Manimaran, A., Sasikala, K.R. and Adaikappan, P., 2008, "Lipid peroxidation and antioxidant status in patients with breast cancer," Singapore Med. J., 49(8), pp 640-643.
 Ripple, M.O. and Henry, W.F., 1997, "Proxidant antioxidant shift induced by androgen treatment of human prostrate carcinoma cells," J. Natl. Cancer Inst., 89, pp 40-48.
 Rotruck, J.T., Pope, A.L., Ganther, H.T., Swanson, A.B., Hafemann, D.G. and Hockstra, W.G., 1973, Selenium: biochemical role as a component of glutathione peroxidase," Science, 179, pp 588-590.
 Shina, K.A., 1972, "Colorimetric assay of catalase," Ann. Biochem., 17, pp 389-394.
 Skrzydlewska, E., Stankiewicz, A., Sulkowska, M., Sulkowski, S. and Kasacka, I., 2001, "Antioxidant status and lipid peroxidation in colorectal cancer," J. Toxicol. Environ. Health, 64, pp 213-222.
 Stohs, S.J. and Bagchi, D., 1995, "Oxidative mechanism in the toxicity of metal ions," Free Radic. Biol. Med., 18, pp 321-336.
 Szatrowski, T.P. and Nathan, C.F., 1991, "Production of large amounts of hydrogen peroxide in human tumour cells," Cancer Res., 51, pp 794- 798.
 Yagi, K., 1987, "Lipid peroxides and human diseases," Chem. Phys. Lipids, 45, pp 337-351.
 Yang, C.R., Ou, Y.C. and Kou, J.H., 1997, "Intracellular glutathione content of urothelial cancer in correlation to chemotherapy response," Cancer Lett., 119, pp 157- 162.
 Yeh, C.C., Hou, M.F. and Tsai, S.M., 2005, "Superoxide anion radical, lipid peroxides and antioxidant status in blood of patients with breast cancer," Clin. Chem. Acta, 361, pp 104-111.
A. Sheeba Christina (1) and G. Swamidoss Daniel (2)
(1) Assistant Professor, Department of Biotechnology, PRIST University--East Campus, Yagappa Chavady, Thanjavur-614 904, Tamil Nadu, India E-mail: firstname.lastname@example.org
(2) Dean of Life Sciences and HOD Microbiology, PRIST University-East Campus, Yagappa Chavady, Thanjavur-614 904 Tamil Nadu, India
Table 1: General characteristics of breast cancer patients. S.No. Parameter 1 Age group 30-70 2 Age at menarche 12-16 3 Menopausal status Pre menopausal 45 Post menopausal 30 4 Clinical status Non- metastatic breast carcinoma 25 Metastatic breast carcinoma 25 Post-treatment group 25 Table 2: Oxidative stress and antioxidant profile in healthy subjects and breast cancer patients. S. Parameters Healthy Non- No. subjects Metastatic Breast cancer 1 Oxidative TBARS 120.69 129.92 stress (mmol/ml) (10.77) (12.55) * 2 SOD 17.38 20.07 (U/ml) (2.38) (2.84) * 3 Enzymatic CAT 7.84 9.13 antioxidants (U/ml) (0.98) (1.76) * 4 GPx 17.36 24.84 (U/l) (1.51) (2.64) * 5 GST 2.23 4.07 (U/ml) (0.77) (1.00) * 6 GSH 7.39 16.18 Non- enzymatic (mg/dl) (0.66) (1.75) * 7 antioxidants Vitamin C 7.43 5.22 (mg/dl) (0.82) (0.88) * 8 Vitamin E 1.27 1.16 (mg/dl) (0.35) (0.29) (NS) S. Metastatic Post- No. breast cancer treatment 1 138.84 133.76 (48.37) * (9.85) * 2 23.10 22.61 (2.64) * (4.27) * 3 10.13 9.75 (1.08) * (1.45) * 4 26.58 17.45 (2.02) * (1.54) (NS) 5 5.21 4.29 (0.91) * (1.11) * 6 18.04 7.68 (1.61) * (1.54) (NS) 7 4.66 6.58 (0.89) * (1.30) * 8 1.02 1.28 (0.36) (NS) (0.42) (NS) The values are expressed as mean (S.D) from 25 subjects in each group. * Values are statistically significant at P < 0.05 NS- not significant
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|Author:||Christina, A. Sheeba; Daniel, G. Swamidoss|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Feb 1, 2011|
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