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|Year : 2009 | Volume
| Issue : 2 | Page : 146--150
Implications of free radicals and antioxidant levels in carcinoma of the breast: A never-ending battle for survival
RJ Sinha1, R Singh2, S Mehrotra3, RK Singh2,
1 Department of Urology, CSMMU (formerly KGMU), Lucknow, (U. P.), India
2 Department of Biochemistry, CSMMU (formerly KGMU), Lucknow, (U. P.), India
3 Department of Obstetrics and Gynaecology, CSMMU (formerly KGMU), Lucknow, (U. P.), India
R J Sinha
Department of Urology, CSMMU (formerly KGMU), Lucknow, (U. P.)
Background: Under normal circumstances, there is a steady balance between the production of oxygen derived free radicals and their destruction by the cellular antioxidant system inside the human body. However, any imbalance between the levels of these oxidants and antioxidants might cause DNA damage and may lead to cancer development. The aim of this study was to evaluate the level of antioxidants and free radicals in blood and tissue of cancer patients and compare these levels at different TNM stages to derive the possible role of free radicals and antioxidant enzymes in the etiology of breast cancer. Materials and Methods: This study includes 30 patients suffering from cancer breast and 20 patients as controls who had benign breast diseases. Circulating lipid peroxide (Malonyldialdehyde [MDA]) levels and activities of the defensive enzymes (Superoxide Dismutase [SOD] and Catalase [CAT]) were estimated in the blood and breast tissue of these patients. Results: Increased levels of free radicals and low levels of antioxidants were observed in malignant tissue. An elevated lipid peroxide concentration was found in the tissue of all the cancer breast patients as evidenced by an increase in the mean MDA level seen with increasing TNM stage of carcinoma breast. Levels of antioxidants SOD and CAT were decreased in cancer patients. Conclusion: The results of our study suggest that free radical activity is enhanced in cancer breast patients while the antioxidant defense mechanism is weakened. This activity is enhanced with the increasing severity of cancer as depicted in different TNM stages of breast cancer.
|How to cite this article:|
Sinha R J, Singh R, Mehrotra S, Singh R K. Implications of free radicals and antioxidant levels in carcinoma of the breast: A never-ending battle for survival.Indian J Cancer 2009;46:146-150
|How to cite this URL:|
Sinha R J, Singh R, Mehrotra S, Singh R K. Implications of free radicals and antioxidant levels in carcinoma of the breast: A never-ending battle for survival. Indian J Cancer [serial online] 2009 [cited 2013 May 19 ];46:146-150
Available from: http://www.indianjcancer.com/text.asp?2009/46/2/146/49153
Free radicals can be traced back to 3-5 billion years ago when the basic components of life were being produced by the free radicals with the help of solar reaction. Now the same free radicals responsible for the initiation of life have become a threat to our very existence of life. Experimental investigations as well as clinical and epidemiological findings have provided evidence supporting the role of reactive oxygen metabolites or free radicals such as singlet oxygen O 2 - , superoxide anions (O 2 ), hydrogen peroxide (H2 O2 ) and hydroxyl radical in the etiology of cancer.  Certain aldehydes such as Malonyldialdehyde (MDA), the end product of lipid peroxidation arising from free radical degeneration of polyunsaturated fatty acids can cause cross linking in lipids, proteins and nucleic acids leading to cellular damage.
Human body is equipped with certain antioxidants (scavenging enzymes) such as Superoxide Dismutase (SOD) and Catalase (CAT) which can counteract the deleterious actions of these reactive oxygen species and protect against cellular and molecular damage.  Disruption of this delicate balance between the free radicals and the antioxidants may cause cellular damage and trigger carcinogenesis.
The aim of this study was to evaluate the level of antioxidants and free radicals in blood and tissue of cancer patients and compare these levels at different TNM stages to derive the possible role of free radicals and antioxidant enzymes in the etiology of breast cancer by providing evidence that the balance between scavenging enzymes and free radicals is disrupted in cancer breast patients.
A prospective study was undertaken to assess the level of free radicals and its scavenging enzymes in patients suffering from cancer of the breast. Since it was not possible to study all the antioxidant enzymes and free radicals due to economic and logistic constraints; levels of MDA were assessed to determine lipid peroxidation and free radical activity and levels of SOD and CAT were used as representative of antioxidant activity in the body.
Materials and Methods
The study was divided into 2 groups: (1) control group: included 20 patients with benign breast diseases and (2) study group: comprised of 30 patients having breast malignancy. Informed consent was taken from the participants at the time of inclusion in this study. Ethical clearance for this study was obtained from the institutional ethics committee and was in accordance with the Declaration of Helsinki.
Patients were subjected to detailed history and examination. Investigations included routine laboratory tests with renal function tests and liver function tests. Specific investigations like mammography, Fine Needle Aspiration Cytology (FNAC), excisional biopsy and axillary lymph node biopsy were done as and when indicated. Specific radiological investigations like X-ray chest, Intravenous Urography (IVU), CT scan and MRI were performed for metastatic workup if required.
MDA, CAT and SOD levels were estimated in the plasma (blood samples taken just before the surgery) and breast tissue (after the surgery) for the estimation of free radical and antioxidant levels.
The patients included in this study did not consume tobacco in any form and nor did they suffer from any concomitant disease like diabetes mellitus, chronic liver disease, rheumatoid arthritis or any other prolonged illness which could have caused conflict with the results of this study. They were not on prolonged medication of any kind which could have resulted in discrepancy during estimation of MDA, CAT and SOD levels. Patients on captopril (containing -SH [thiol] group) were excluded from this study.
Procedure of free radical and antioxidant measurement
Histopathological diagnosis of the tissue was confirmed in the pathology department. From the control group, 5 ml of venous blood was collected in disposable plastic syringe previously rinsed with Heparin. Samples were transported in ice packed flask to the Department of Biochemistry and kept at - 2º to 0 º C. 1 gm of tissue was collected from patients operated for benign breast conditions e.g. fibroadenoma, lipoma and transported to the Department of Biochemistry in ice pack. Similar method was used for patients in the study group.
Estimation of MDA (lipid peroxide) was done according to the method of Ohkawa et al. with modifications as described by Sanocka et al. using thiobarbituric acid.  Standard absorbance of MDA (2.5 nmol) was used to calculate the amount of lipid peroxide in the samples and results were expressed as nmol of MDA/ml plasma and nmol/gm tissue.
SOD activity was evaluated as per the method of McCord and Fridovich.  The unit of enzyme activity was defined as the amount of enzyme required to inhibit the optical density at 560 nm of Nitro Blue Tetrazolium (NBT) reduction by 50% in one minute under the assay conditions and results were expressed as units/ml RBC in plasma and U/mg protein in tissue.
CAT activity was determined as per the method of Aebi and Suter.  Results were expressed as U/ml RBC in plasma and U/mg protein in tissue; 1 unit of CAT decomposes 1.0 mM of hydrogen peroxide per minute under specified conditions.
The data was entered in the MS-Excel computer program and all the analysis were carried out using SPSS (Ver.15.0) statistical program. Results were expressed as Mean ± SD. The statistical significance of difference between the various groups was determined by using the students't' test; P > 0.05 = not significant (NS); P P value between 0.05 to 0.001 = moderately significant, P P P P P P 
Screening programme is not very effective in this country and the population on the whole is ignorant about breast self-examination. This leads to late detection and carries poor prognosis due to advanced stage of the breast carcinoma. Maximum number of cases belonged to stage IV (40%) which might have been due to the above mentioned fact and also because this study was conducted in a tertiary medical center and a referral hospital which attracts terminal cases.
Lipid peroxidation is a free radical mediated phenomenon in biological tissues where poly unsaturated fatty acids are generally abundant and is one of the most frequently used parameters for assessing the involvement of free radicals in cell damage. The probable reason for the elevated level of serum lipid peroxide in breast carcinoma may be due to defective antioxidant system which leads to the accumulation of lipid peroxides in cancer tissue which are released into the blood stream.  Accurate measurement of lipid peroxide products is quite difficult due to their rapid degradation in vitro . The thiobarbituric acid assay is one of the most popular and easiest methods to use as an index of lipid peroxidation and free radical mediated cellular injury in biological samples.
In this study, patients with cancer exhibited higher levels of MDA, both in tissues and serum (p ,,
Ray et al . in their study on breast cancer demonstrated high levels of MDA in females with breast cancer (12.87 ± 4.13 µM/ml) as compared to control (10.21± 2.91 µM/ml) and the difference was found to be statistically significant (p  We also discovered that the levels of MDA are higher in females suffering from breast cancer as compared to the control group consisting of benign breast disease patients.
In another recent study, Rajneesh et al. also observed increased level of lipid peroxidation in the plasma of patients of breast cancer.  The levels of antioxidants (SOD and CAT) increased in the patients of breast cancer in this study compared to control group which is in contrast to our study where the levels were low in study group as compared to the control group. This might have happened due to the fact that their patients were in TNM stage 2 and 3 and perhaps detected earlier in the course of their disease; so, the anti-oxidant level might have risen initially to meet the challenge of carcinogenesis before final consumption which might occur in the late stages of this disease; which perhaps occurred in our subset of patients.
The measurement of different oxidation products such as conjugated dienes, lipid hydroperoxides formed during the initiation and propagation stages of lipid peroxidation in addition to MDA have also been recommended as useful endpoints. We only assessed MDA levels as an indicator of lipid peroxidation; this might be a possible limitation of this study.
SOD and CAT are considered primary antioxidant enzymes, since they are involved in direct elimination of reactive oxygen metabolites. [13-16] They also act as anti-carcinogens and inhibitors at initiation and promotion/transformation stage in carcinogenesis. Mutation caused by potassium superoxide in mammalian cells is blocked by SOD.  Plasma DNA strand scission caused by xanthine/xanthine oxidase is prevented by SOD and catalase enzymes.  Catalase also prevents chromosomal aberration caused by hypoxanthine/xanthine oxidase in Chinese hamster cells.  It also prevents the onset of spontaneous neoplastic transformation in mouse fibroblast and epidermal keratinocytes.  Bellisola et al . measured liver catalase activity in 22 subjects and compared it with that of 13 patients suffering from hepatocellular carcinoma and found that catalase was reduced significantly in human tissue as well. 
In our study, SOD and CAT levels were found to be low in all cancer patients as compared to controls [Table 1]. Fridovich and Tayarani have demonstrated in their respective studies that the reduction in SOD activity increases the toxic effects of O2 - and this might lead to severe cellular damage. ,,
Mehrotra et al. in their study also observed high levels of MDA and low levels of SOD and CAT in patients of cancer cervix which is in sync with our observations. 
The results of our study provide strong evidence regarding the definitive role of free radicals in breast malignancy. More studies are required to confirm these results because our study was conducted in a small number of patients, over a short period of time and was limited to a small geographical area.
There is an elevation in plasma lipid peroxide concentrations and a depletion of the anti-oxidant defense potential in patients of breast carcinoma. This difference is more evident with increasing severity of the disease as depicted by the levels in different TNM stages of cancer breast. It is not certain however, whether lower concentrations of the antioxidant enzymes lead to carcinogenesis or they are merely the result of the disease process. Further epidemiological, molecular and clinical studies may answer this question and help in establishing the possible therapeutic role of antioxidants in cancer chemo-prevention.
The authors would like to acknowledge the help and support of Dr N B Singh (Former Professor and Head, Department of Surgery, GSVM Medical College, Kanpur, [U.P.], India); Dr Rajeev Bhargava (Professor and Head, Department of Surgery, GSVM Medical College, Kanpur, [U.P.], India) and Dr Asha Agarwal (Professor, Department of Pathology, GSVM Medical College, Kanpur, [U.P.], India).
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