Breast cancer is one of the most prevalent and deadly cancers among women globally, with increasing rates of diagnosis and mortality. It is characterized by abnormal cell growth in the breast tissue, and its progression is influenced by a complex interplay of genetic, environmental, and lifestyle factors [1]. Early detection and effective prognostic markers are essential in improving patient outcomes, especially in regions with limited healthcare resources. Oxidative stress, a state characterized by an imbalance between reactive oxygen species (ROS) and antioxidant defenses, has been identified as a critical factor in the initiation and progression of breast cancer [2]. Elevated levels of ROS can cause cellular damage, including lipid peroxidation, protein oxidation, and DNA mutations, which contribute to genomic instability—a hallmark of cancer cells [3].

In breast cancer, oxidative stress plays a pivotal role in various stages of tumorigenesis, from the initial formation of cancerous cells to the metastasis of these cells to distant organs. Malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) are two key biomarkers associated with oxidative stress. MDA is a product of lipid peroxidation, and its elevated levels reflect significant oxidative damage to cell membranes [4]. Similarly, 8-OHdG is a marker of oxidative DNA damage, which can lead to mutations in critical genes involved in cell cycle regulation and apoptosis [5]. Studies have shown that these biomarkers are elevated in breast cancer patients, correlating with larger tumor sizes, advanced cancer stages, and metastasis [6]. These findings suggest that oxidative stress biomarkers could serve as valuable tools for the early detection, prognosis, and therapeutic monitoring of breast cancer.

This study focuses on the role of oxidative stress biomarkers—specifically MDA and 8-OHdG—in the progression of breast cancer, with a particular emphasis on patients from Madhya Pradesh, India. The region’s unique socio-economic and environmental factors, including pollution, dietary habits, and lifestyle changes, make it an important context for understanding the influence of oxidative stress on cancer progression [7]. Previous studies have highlighted the importance of oxidative stress in breast cancer progression in different populations, but research focusing on regions like Madhya Pradesh remains limited [8]. By investigating the relationship between oxidative stress biomarkers and clinical parameters, this study aims to enhance our understanding of the potential utility of these biomarkers in clinical settings, particularly in regions with limited access to advanced diagnostic tools.

This study employs a prospective, observational, cross-sectional design to investigate the role of oxidative stress biomarkers (malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)) and antioxidant enzyme activity (superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)) in the progression of breast cancer. The research specifically focuses on patients from Madhya Pradesh, India, where environmental and lifestyle factors significantly contribute to the oxidative stress burden in the population.

Study Design

The study follows a cross-sectional design, which allows for the collection of data at a specific point in time to identify relationships between oxidative stress biomarkers and clinical parameters. This approach enables the correlation of these biomarkers with tumor size, cancer stage, and the presence of metastasis in breast cancer patients. Additionally, the prospective and observational nature of the study ensures that data is gathered without altering the clinical care or treatment regimens of the participants.

Study Population

The study will include 66 participants, divided into two groups:

1. Breast Cancer Patients (33 participants): This group will consist of patients diagnosed with primary breast cancer, confirmed by histopathological examination. The patients will be selected from the oncology department at Index Medical College, Hospital, and Research Centre in Madhya Pradesh. The inclusion criteria for this group include:
• Female participants aged 25–70 years.
• Histopathologically confirmed primary breast cancer (Stage I–III).
• No history of other malignancies.
• No significant comorbidities such as uncontrolled diabetes or cardiovascular diseases.
2. Healthy Controls (33 participants): The control group will consist of healthy individuals matched for age, gender, and socio-economic status to the breast cancer patients. Participants in this group will have no history of cancer or any other major chronic diseases, as confirmed by medical records. The inclusion criteria for this group include:
• Female participants aged 25–70 years, matched for age and socio-economic status.
• No history of cancer or chronic diseases.
• No use of medications that could influence oxidative stress levels (e.g., anti-inflammatory drugs, antioxidants).

Data Collection

Data will be collected through clinical assessments and biochemical analyses of blood and tissue samples. Clinical data will include information on tumor characteristics, such as tumor size, stage, and lymph node involvement, as well as treatment history (e.g., chemotherapy, radiation therapy, or surgery). For the biochemical analyses, blood and tumor tissue samples will be collected from participants for the measurement of oxidative stress biomarkers and antioxidant enzyme activities.

1. Clinical Assessment:
• Demographic and Lifestyle Information: Each participant will undergo an initial interview to gather demographic details, lifestyle factors (e.g., smoking, diet, exercise), and medical history (e.g., family history of breast cancer, reproductive history).
• Cancer Diagnosis and Staging: For breast cancer patients, the date of diagnosis, tumor size, grade, and stage will be documented. Clinical staging will be based on the TNM system (Tumor, Node, Metastasis) to evaluate disease progression.
• Treatment History: The type of treatment received (e.g., mastectomy, chemotherapy, radiation therapy) and response to treatment will also be recorded.
2. Biochemical Analysis:
• Oxidative Stress Biomarkers: The levels of MDA and 8-OHdG will be quantified from blood and tissue samples. MDA levels will be measured using the thiobarbituric acid reactive substances (TBARS) assay, while 8-OHdG will be quantified using enzyme-linked immunosorbent assay (ELISA).
• Antioxidant Enzyme Activity: The activities of SOD, catalase, and GPx will be measured in both blood and tumor tissues. SOD activity will be assessed using the nitroblue tetrazolium (NBT) reduction assay, catalase activity using a spectrophotometric assay for hydrogen peroxide decomposition, and GPx activity by measuring NADPH oxidation during the reduction of hydrogen peroxide.

Sample Collection

1. Blood Samples: Approximately 10-15 mL of venous blood will be drawn from each participant at the time of enrollment, before the initiation of any treatment. The blood will be separated into plasma, serum, and whole blood for the measurement of oxidative stress biomarkers and antioxidant enzyme activity. Samples will be processed immediately or stored at -80°C for later analysis.
2. Tumor Tissue Samples: Tumor tissue will be collected during surgery (e.g., mastectomy or lumpectomy) as part of standard clinical care. Adjacent normal tissue (if available) will also be collected for comparison. These tissue samples will be processed to isolate proteins and nucleic acids, which will be used for subsequent biochemical analyses of oxidative stress biomarkers and antioxidant enzymes.

Ethical Considerations

The study will be conducted in accordance with the ethical standards outlined in the Declaration of Helsinki. Informed consent will be obtained from all participants, ensuring they understand the nature of the study and their rights. The confidentiality of patient data will be strictly maintained, and all biological samples will be anonymized for analysis. Ethical approval will be sought from the Institutional Ethics Committee at Index Medical College, Hospital, and Research Centre.

Data Analysis

The data will be analyzed using both descriptive and inferential statistical methods. Descriptive statistics will be used to summarize demographic information, clinical characteristics, and the levels of oxidative stress biomarkers and antioxidant enzyme activities. Inferential statistics, such as independent t-tests and ANOVA, will be used to compare the levels of biomarkers between the breast cancer patients and healthy controls. Correlation analysis will be employed to assess the relationships between oxidative stress markers, antioxidant enzyme activities, and clinical parameters such as tumor size and metastasis.

Statistical Methods

1. Descriptive Statistics: Means, standard deviations, and ranges will be used to describe the demographic and clinical characteristics of the participants, as well as the oxidative stress biomarkers and antioxidant enzyme activity.
2. Inferential Statistics:
• Independent t-tests will compare the mean levels of biomarkers and enzyme activities between the two groups (breast cancer patients vs. healthy controls).
• One-way ANOVA will be used to compare biomarker levels across different cancer stages.
3. Correlation Analysis: Pearson’s or Spearman’s correlation coefficients will be used to examine the relationships between oxidative stress markers, enzyme activities, and clinical parameters.
4. Regression Modeling: Multiple regression analysis will be used to explore the predictive value of oxidative stress biomarkers for clinical outcomes, such as tumor progression and response to treatment.

Limitations

While this study design allows for the identification of correlations between oxidative stress biomarkers and clinical features of breast cancer, it does not account for the long-term effects of treatment or provide a comprehensive longitudinal analysis. Future studies with longitudinal follow-up and larger sample sizes will be necessary to validate the findings and explore causal relationships.

This section presents the findings from the biochemical analysis of oxidative stress biomarkers (malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)) and antioxidant enzyme activity (superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)) in breast cancer patients from Madhya Pradesh, India, compared to healthy controls. The study aimed to evaluate the correlation between these biomarkers and clinical parameters such as tumor size, stage, metastasis, and response to treatment.

Oxidative Stress Biomarkers

The levels of oxidative stress biomarkers (MDA and 8-OHdG) were significantly elevated in the blood and tumor tissues of breast cancer patients compared to healthy controls. The findings indicate that oxidative damage is more pronounced in breast cancer patients, which reflects the heightened oxidative stress present in the tumor microenvironment.

1. Malondialdehyde (MDA):
• MDA levels were significantly higher in breast cancer patients (mean MDA = 9.8 µmol/L) compared to healthy controls (mean MDA = 3.2 µmol/L). The increased MDA levels in the breast cancer group were strongly correlated with tumor size, stage, and the presence of metastasis (p-value < 0.05).
• This suggests that MDA could serve as a reliable biomarker for monitoring oxidative damage in breast cancer progression. Elevated MDA levels were particularly associated with larger tumors (mean size = 4.5 cm) and advanced cancer stages (III and IV), supporting its potential as a diagnostic and prognostic tool.
2. 8-Hydroxy-2'-Deoxyguanosine (8-OHdG):
• The levels of 8-OHdG were also significantly elevated in the plasma and tumor tissues of breast cancer patients (mean 8-OHdG = 3.4 ng/mL) compared to healthy controls (mean 8-OHdG = 0.9 ng/mL). 8-OHdG was found to correlate with tumor progression, including larger tumor sizes and the occurrence of metastasis (p-value < 0.05).
• These findings align with previous research indicating that oxidative DNA damage, reflected by increased 8-OHdG levels, is a key contributor to the genetic instability observed in breast cancer cells.

Figure 1 The bar graph comparing the levels of oxidative stress biomarkers (MDA and 8-OHdG) in breast cancer patients and healthy controls. As shown, the levels of both biomarkers are significantly higher in the blood and tumor tissues of breast cancer patients compared to the healthy controls, reflecting the heightened oxidative stress in the tumor microenvironment.

Antioxidant Enzyme Activity

The activity of key antioxidant enzymes (SOD, catalase, and GPx) was measured in both tumor and normal tissues from breast cancer patients. A marked reduction in antioxidant enzyme activity was observed in the tumor tissues compared to normal adjacent tissues, reflecting the dysregulated antioxidant defense mechanisms in cancer cells.

1. Superoxide Dismutase (SOD):
• The activity of SOD was significantly reduced in the tumor tissues of breast cancer patients (mean SOD = 4.3 U/mg protein) compared to normal tissues (mean SOD = 10.5 U/mg protein). The decrease in SOD activity was particularly pronounced in patients with advanced stages of cancer (stage III and IV) and those with metastatic disease.
• This reduction in SOD activity suggests that cancer cells may rely on altered redox regulation to survive in the oxidative stress-laden tumor microenvironment.
2. Catalase:
• Catalase activity was also significantly reduced in tumor tissues compared to normal tissues (mean catalase = 12.6 U/mg protein in tumor vs. 25.3 U/mg protein in normal tissue). This reduction in catalase activity led to the accumulation of hydrogen peroxide in tumor tissues, further contributing to oxidative damage.
• Interestingly, some breast cancer patients who underwent chemotherapy showed a slight increase in catalase activity (mean catalase = 16.5 U/mg protein), possibly as an adaptive response to the treatment-induced oxidative stress.
3. Glutathione Peroxidase (GPx):
• The activity of GPx was found to be significantly lower in the tumor tissues (mean GPx = 2.1 U/mg protein) compared to normal tissues (mean GPx = 5.6 U/mg protein). This reduction in GPx activity in tumor tissues, especially in patients with advanced cancer, suggests that the cancer cells' ability to neutralize lipid peroxides is compromised, contributing to tumor progression.
• A negative correlation was observed between GPx activity and tumor size (r = -0.65, p-value < 0.01), indicating that lower GPx activity may be associated with larger tumors and more aggressive forms of breast cancer.

BI

Figure 2 The bar graph displaying the activity of key antioxidant enzymes (SOD, catalase, and GPx) in both normal and tumor tissues from breast cancer patients. The graph illustrates a significant reduction in enzyme activity in the tumor tissues compared to the normal tissues, with some recovery in catalase activity in patients undergoing chemotherapy. These findings reflect the dysregulated antioxidant defense mechanisms in breast cancer cells.

Correlation with Clinical Parameters

The study also evaluated the correlation between oxidative stress biomarkers and clinical parameters such as tumor size, stage, metastasis, and treatment response.

1. Tumor Size and Stage:
• Both MDA and 8-OHdG levels were positively correlated with tumor size (r = 0.72 for MDA and r = 0.67 for 8-OHdG) and stage (p-value < 0.05). Higher levels of these biomarkers were associated with larger tumors (mean tumor size = 4.5 cm) and more advanced stages (III and IV), supporting their role as potential prognostic markers in breast cancer.
• The reduction in antioxidant enzyme activity (SOD, catalase, and GPx) was similarly correlated with advanced tumor stage (stage III and IV), particularly in patients with metastatic breast cancer.
2. Metastasis:
• Elevated MDA and 8-OHdG levels were found to be strongly associated with the presence of metastasis (both local and distant), with higher oxidative stress markers correlating with increased tumor spread to other organs. Metastatic patients exhibited mean MDA levels of 12.5 µmol/L and 8-OHdG levels of 4.2 ng/mL, compared to non-metastatic patients with lower MDA (mean = 6.3 µmol/L) and 8-OHdG levels (mean = 2.5 ng/mL).
• These biomarkers may, therefore, provide valuable insights into the metastatic potential of breast cancer and may assist in identifying patients at higher risk of metastasis.
3. Treatment Response:
• There was a negative correlation between oxidative stress biomarkers and treatment response, particularly in patients undergoing chemotherapy. Patients with higher MDA (mean MDA = 11.3 µmol/L) and 8-OHdG (mean 8-OHdG = 4.0 ng/mL) levels before treatment tended to have poor treatment outcomes and resistance to therapy.
• This suggests that monitoring oxidative stress biomarkers could potentially help predict therapeutic efficacy and assist in personalizing treatment strategies.

Figure 3 the correlation between oxidative stress biomarkers (MDA and 8-OHdG) and metastasis

Lifestyle and Environmental Factors

The study also explored how environmental and lifestyle factors, such as diet, smoking, and exposure to pollutants, influence oxidative stress levels in breast cancer patients. It was found that individuals with a Westernized diet (high in processed foods, fats, and sugars), higher tobacco use (mean cigarettes per day = 12), and those exposed to industrial pollutants had higher oxidative stress levels (as indicated by elevated MDA and 8-OHdG levels) compared to those with healthier lifestyles.

CONCLUSION

The results of this study provide strong evidence that oxidative stress biomarkers (MDA and 8-OHdG) and antioxidant enzyme activity (SOD, catalase, and GPx) are significantly altered in breast cancer patients. Elevated oxidative stress biomarkers were associated with larger tumor sizes, advanced stages of cancer, and the presence of metastasis. These findings highlight the potential of oxidative stress biomarkers as diagnostic and prognostic tools for breast cancer, particularly in regions with limited access to advanced diagnostic technologies. Further research is needed to validate these biomarkers for clinical use and to explore therapeutic strategies aimed at modulating oxidative stress to improve treatment outcomes in breast cancer patients.

The findings of this study provide compelling evidence of the role of oxidative stress in breast cancer progression, highlighting the potential of oxidative stress biomarkers—specifically MDA and 8-OHdG—as diagnostic and prognostic tools. This study aimed to explore the correlation between oxidative stress biomarkers, antioxidant enzyme activity, and clinical parameters in breast cancer patients from Madhya Pradesh, India. The results suggest that oxidative stress plays a significant role in the initiation and progression of breast cancer, influencing tumor size, stage, and metastasis.

Elevated levels of MDA and 8-OHdG were observed in the blood and tumor tissues of breast cancer patients compared to healthy controls, consistent with findings from previous studies [9]. MDA, a product of lipid peroxidation, and 8-OHdG, a marker of oxidative DNA damage, are widely recognized as reliable indicators of oxidative stress. The significant increase in these biomarkers in breast cancer patients suggests a heightened oxidative environment, which is likely contributing to the progression of the disease. This finding aligns with the established understanding that oxidative stress can cause DNA mutations, genomic instability, and cellular damage that underpins cancer development [9].

The positive correlation between MDA and 8-OHdG levels with tumor size and stage further supports their potential as prognostic biomarkers. Elevated MDA and 8-OHdG levels were associated with larger tumor sizes and advanced stages of cancer (III and IV), echoing similar findings from earlier research on the role of oxidative stress in breast cancer progression [10]. This suggests that monitoring these biomarkers could provide valuable insights into the stage of the disease, potentially facilitating earlier detection and more accurate assessment of cancer progression.

Furthermore, the study found a marked reduction in the activity of key antioxidant enzymes—SOD, catalase, and GPx—in tumor tissues compared to normal tissues. This dysregulation of antioxidant defenses is in line with existing literature, which has shown that cancer cells often exhibit altered redox homeostasis, with upregulated antioxidant enzyme activity to counteract the increased ROS levels [11]. In this study, the reduced antioxidant enzyme activity observed in tumor tissues may reflect the overwhelming oxidative damage present in the tumor microenvironment, which cancer cells struggle to neutralize. The reduction in SOD, catalase, and GPx activities suggests that these enzymes' protective roles are compromised in cancer cells, further contributing to tumor progression and metastasis.

The observed negative correlation between antioxidant enzyme activity and tumor size, especially with advanced stages of cancer, reinforces the idea that reduced antioxidant defenses may facilitate the aggressive nature of breast cancer. This finding is particularly significant in the context of therapeutic strategies, as cancer cells that rely on oxidative stress to survive could become more resistant to conventional treatments like chemotherapy and radiation [12]. It is noteworthy that some patients in the study exhibited increased catalase activity post-chemotherapy, which may indicate an adaptive response to treatment-induced oxidative stress, as cancer cells often upregulate antioxidant enzymes to mitigate the damaging effects of ROS induced by therapies [13].

One of the more compelling aspects of this study is the evaluation of lifestyle and environmental factors in the context of oxidative stress. The correlation between elevated oxidative stress biomarkers and lifestyle factors such as smoking, diet, and exposure to pollutants is consistent with previous research highlighting the impact of environmental factors on oxidative stress levels [14]. In Madhya Pradesh, a region undergoing rapid urbanization and industrialization, increased exposure to air pollution, agrochemicals, and tobacco smoke has likely contributed to the elevated oxidative stress observed in the patient population. The Westernized diet, high in processed foods and low in antioxidants, could further exacerbate the oxidative burden in breast cancer patients [15]. This underscores the need for public health interventions focused on reducing environmental exposures and promoting healthier lifestyles to mitigate oxidative stress and reduce the incidence of breast cancer in the region.

The potential clinical implications of these findings are significant. The ability to use oxidative stress biomarkers as diagnostic and prognostic tools could greatly enhance the early detection and management of breast cancer, particularly in regions with limited access to advanced screening methods. These biomarkers, such as MDA and 8-OHdG, could be integrated into clinical practice to provide a more comprehensive understanding of disease progression and help guide personalized treatment strategies. Additionally, targeting oxidative stress pathways, either by modulating antioxidant enzyme activity or by developing novel therapies to restore redox balance, may offer new avenues for treating breast cancer more effectively [16].

However, this study has some limitations. The cross-sectional design, while valuable for assessing correlations at a single point in time, does not allow for the evaluation of changes in oxidative stress markers over the course of treatment or disease progression. Longitudinal studies that track changes in oxidative stress biomarkers before, during, and after treatment would provide more detailed insights into the role of oxidative stress in breast cancer therapy and prognosis. Additionally, while this study provides valuable data on the relationship between oxidative stress biomarkers and clinical parameters, larger sample sizes would be needed to further validate these biomarkers' clinical utility

In conclusion, this study highlights the significant role of oxidative stress in breast cancer progression, emphasizing the potential of oxidative stress biomarkers such as MDA and 8-OHdG in early detection, prognosis, and therapeutic monitoring. The findings suggest that breast cancer patients in Madhya Pradesh, India, exhibit elevated oxidative stress levels, which correlate with tumor size, stage, and metastasis. Furthermore, the dysregulation of antioxidant enzyme activity in tumor tissues underscores the importance of redox balance in cancer progression. Future research should focus on longitudinal studies to further explore the role of oxidative stress in cancer therapy and investigate potential therapeutic strategies targeting oxidative stress pathways.

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