Breast cancer affects nearly one million women per year worldwide [1
]. However, with better investigation tools, a better understanding of tumor biology, and increasing public awareness, more cases are being diagnosed at earlier stages. Still, it accounts for the highest morbidity and mortality in the female population. Benign breast diseases are non-cancerous breast conditions which are at least ten times more common than breast malignancies and account for 50% of all breast biopsies performed; hence, their diagnosis, prognosis, and proper treatment is probably as important as other chronic malignancies [2
]. Benign breast diseases are a heterogeneous group of lesions that are very common abnormalities of breast. Common benign lesions such as fibroadenoma, fibrocystic disease, breast abscess, ductectasia, and mastitis, as well as their relationship to the development of subsequent breast carcinogenesis, need to be more investigated.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS), distinctive characteristics of cancer [3
], can damage cells. If persistent, this damage may lead to base substitution, deletion, and strand fragmentation, which may inactivate tumor suppressor genes or increase the expression of proto-oncogenes within cells. Besides that, these free radicals can modify many intracellular signalling pathways via growth factor receptors and transcription factors which may modulate inflammation, angiogenesis, and cell proliferation pathways and provide a favourable environment for tumor growth [4
].Previous studies have revealed the role of potential risk factors such as advancing age, early menarche, late menopause, late age at first birth, and a family history of breast cancer, as well as the imbalance in oncogenes and tumor suppressor genes in breast cancer [9
] but few reports on the oxidant-antioxidant profile in the serum of patients suffering from breast carcinoma and benign breast diseases exist [12
The present study investigates and provides validation of the alterations in the biochemical parameters of pro-oxidants and antioxidants using biochemical and cell-based assays in the serum of breast disease patients with respect to controls. We studied in vivo DNA damage (8-hydroxy-2-deoxyguanosine) and non-enzymatic antioxidants (i.e., vitamin A, vitamin C, vitamin E, total antioxidant capacity, and cell proliferation index) in order to determine their role in breast diseases etiology, and their application to the diagnosis of breast cancer risk groups.
Reactive oxygen or reactive nitrogen species can damage DNA in many ways: (1) they can form a single or double-strand break, and (2) they can modify nitrogenous bases and induce cross links.
When cells are not able to rectify these damages, they undergo necrosis, show replication errors, or develop increased cell proliferation, angiogenesis, and genomic instability, which ultimately results in the onset of a variety of diseases, including breast cancer [21
]. The most abundant by-product produced by these consequences is 8-hydroxy-2-deoxyguanosine; therefore, measurement of its levels may be applied to evaluate the load of oxidative DNA damage in the cell. This value could be important in understanding the role of oxidative stress in breast cancer development and disease intervention. In the present study, we observed significantly higher 8-OHdG serum levels in the patients suffering from breast carcinoma in comparison with benign breast disease patients and the control group. A few studies conducted by our research group have also reported an elevated level of this oxidatively modified biomolecule in breast carcinogenesis and benign breast diseases, which might be involved in the initiation of breast carcinogenesis [24
]. Our study also indicates that the increased level of 8-OHdG in breast cancer patients was associated with disease progression and advancing to higher stages of breast carcinogenesis [25
]. Its significantly higher level was also estimated in the benign breast diseases group in comparison to the control group.
Free radical generation and the damage caused by it in the cell is usually controlled by a large number of antioxidant systems that initiate a protection mechanism against free radicals. In the present study, a significant reduction in total antioxidant status and in non-enzymatic antioxidant profile in the malignant group in suggests and increased utilization of serum antioxidants in response to an enhanced level of oxidative damage production in those patients. The group of patients with benign breast diseases also followed a very similar significant (p
< 0.05) pattern of alteration of antioxidant levels in comparison to healthy control subjects. Total antioxidant activity is a measure of the scavenging capacity of the cell defense system. Depleted levels of enzymatic and non-enzymatic antioxidant protective mechanisms have also been documented in a wide variety of malignancies including breast malignancy [27
]. However, very few evidences regarding the depleted level of enzymatic and non-enzymatic antioxidant levels in fibroadenoma and other benign lesions have been available until now. Owing to the importance of antioxidants in our body, a large variety of testing methods have been proposed and applied. The colorimetric methods used by us have been validated and tested by others to quantify antioxidants levels. The relevance, advantages, and limitations of these methods have been critically discussed with respect to their chemistry and the mechanisms of antioxidant activity [33
]. Apart from all these methods including the gold standard methods, the results of the present study are reproducible, and a paper has been published with regards to the estimation of antioxidants [33
Vitamin A, a biomolecule well known for its natural antioxidant properties, plays an important role in the cellular function, development, and maintenance of normal visual acuity [34
]. It has been demonstrated that it acts together with vitamin C and vitamin E to protect cells against oxidative damage [30
]. Vitamin C, or ascorbic acid, is a water-soluble chain-breaking antioxidant [32
] that strongly inhibits lipid peroxidation, the oxidation of glutathione, and other enzymes [35
] by directly reacting with free radicals such as superoxide, hydroxyl radicals, and singlet oxygen. This interaction promotes recycling of α-tocopherol radicals and regeneration of α-tocopherol [38
]. Vitamin E is the major lipid-soluble antioxidant present in lipid membranes and human plasma lipoproteins [39
]. It exists in eight different isoforms, of which α-tocopherol is the most biologically active form. The main function of vitamin E is to inhibit apoptosis and to stabilize biological membranes [40
]. Alpha-tocopherol also functions in vivo as a strong protector, mainly against lipid peroxidation, and prevents nitrosamine formation [38
]. In our study, a strong negative correlation was found between an increased level of oxidative stress and a depleted level of non-enzymatic antioxidants, which results in redox imbalance and may be associated with the advancement of breast diseases.
Proliferation rates can also increase our understanding about diagnosis, prognosis, and recurrence of almost all cancers and can be used to guide treatment strategies in clinical practice. Carcinogenesis is associated with various epigenetic mechanisms which can ultimately alter cellular communication and gene expression controlling cell proliferation, differentiation, and apoptosis. An increased proliferation correlates strongly with poor prognosis. In our study, the development and continued growth of breast cancer involves significantly altered rates of cell proliferation index. 8-OHdG has been suggested as a modulator of signalling pathways related to cell proliferation and apoptosis that lead to breast cancer initiation and progression [42
]. Yano et al. also found that vitamin E can suppress lung tumorigenesis by inhibiting cell proliferation at the initial stages of the disease [43
]. Phenobarbital (PB) activates the transcription factor nuclear factor kappa beta (NF-κB), and dietary vitamin E effectively inhibits PB-induced NF-κB DNA binding and leads to a decreased level of cell proliferation [44
]. Vitamin C can also inhibit the proliferation of A549 cells (adenocarcinomic human alveolar basal epithelial cells) in G0/G1 and S phases [45
]. However, the observations with vitamin A intake are not unanimous. Small doses of vitamin A or β-carotene are suggested to prevent cancer, but in high doses this biomolecule has toxic effects [46
]. The anti-cancer effects of β-carotene are such that it can elicit an anti-proliferative effect through the retinoid acid receptor (RAR) and retinoid X receptor (RXR), which can, in turn, regulate retinoid-mediated gene expression and transcription, thereby hampering cell proliferation [46
]. Our study also describes a significant positive correlation between the levels of 8-OHdG and cell proliferation activity. Diminished levels of antioxidants showed a significant negative correlation with cell proliferation activity, which is in agreement with previous studies where antioxidants were found to inhibit the modulation of gene expression and cell proliferation [30
]. Maalouf et al. found that, when vitamin E and its acetate analog were applied to cells at times before and after ultraviolet B (UVB) radiation-induced DNA fragmentation, a significant increase in the percentage of viable cells and a concomitant decrease in the number of apoptotic cells was noted, which indicates that vitamin E and its acetate analog have the potential to modulate the cellular response to UVB partly through their action on NF-κB activation [48
]. Similarly, in another experiment carried out by Duarte et al., a vitamin C derivative, ascorbic acid 2-phosphate, was used to treat contact-inhibited populations of primary human dermal fibroblasts which showed faster repair of oxidatively damaged DNA bases [49
]. Bagchi et al. also assessed the protective role of vitamins C, vitamin E and grape seed proanthocyanidin extract (GSPE) against smokeless tobacco (STE)-induced oxidative stress (DNA damage) in normal human oral keratinocytes (NHOK) cells. Protection values of ~11%, 26%, 28%, and 50% were recorded following the incubation with vitamin C, vitamin E, a combination of vitamins C and E, and GSPE, respectively [50
Multinomial logistic regression was used to study association studies. The results of this study identify the contribution of the selected biomarkers; using odds ratios and the associated confidence interval, it was found that subjects with increased levels of oxidative DNA damage and cell proliferation activity or a reduced level of antioxidant defense might be at higher risk of developing breast cancer.
Taken together, these findings reveal an alteration in the tumor microenvironment due to changes in DNA damage markers and antioxidant levels. Despite the exciting advances in the field of free radical research, such applications need to be further investigated on a larger sample and in follow-up cases. Moreover, the involvement of oxidative stress in the pathogenesis of other human disorders, including various chronic malignancies, limits its usefulness as a screening tool. One of the various challenges in this field is how oxidative stress-induced cancer-related signaling pathways can be targeted for drug development. These findings provide a scientific basis for designing a treatment modality along with antioxidants supplementation; however, it cannot be generalized due to the heterogeneity and the different body requirements of every individual.