Free radicals hasten head and neck cancer risk: A study of total oxidant, total antioxidant, DNA damage, and histological gradeAK Singh1, P Pandey2, M Tewari1, HP Pandey3, IS Gambhir2, HS Shukla1
1 Department of Surgical Oncology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0022-3859.180555
Source of Support: None, Conflict of Interest: None
Background: Free radicals such as reactive oxygen species (ROS), which induce oxidative stress, are the main contributors to head and neck carcinogenesis (HNC). The present study was conducted with the aim to assess the oxidant/antioxidant status and DNA damage analysis in head and neck cancer/control patients. Materials and Methods: This prospective study was conducted on 60 patients with biopsy-proven HNC and 17 patients of head and neck disease (HND). The total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were determined by novel automatic colorimetric methods from tissue homogenate. DNA damage analysis was determined by single cell gel electrophoresis (SCGE). Results: The mean age of the study cohort was 46.65 ± 14.84 years for HNC patients, while it was 49.41 ± 13.00 years for HND patients. There were no significant differences found between the two groups with respect to demographic presentation except tobacco addiction. The association between oxidative stress parameters and DNA damage analysis with study group revealed the following. (A) DNA damage - tissue homogenate TOS and OSI were significantly higher in HNC subjects than in HND (16.06 ± 1.78 AU vs 7.86 ± 5.97 AU, P < 0.001; 53.00 ± 40.61 vs 19.67 ± 21.90, P < 0.01; 7.221 ± 5.80 vs 2.40 ± 2.54, P < 0.01, respectively), while TAS was significantly decreased. (B) Aggressive histological features were identified, more commonly with higher TOS and lower TAS [probability (P) = 0.002, relative risk (RR) = 11.838, 95% confidence interval CI = 2.514-55.730 and P = 0.043, RR = 0.271, 95% CI = 0.077-0.960, respectively]. Conclusion: The increase in free radicals may be the event that led to the reduction of antioxidant status in HNC, thus explaining the oxidative damage of DNA and the severity of disease. Increased OSI represents a general mechanism in its pathogenesis.
Keywords: Antioxidants, DNA damage, head and neck cancer (HNC), oxidative stress
Head and neck cancer (HNC) is the sixth most common malignancy in the world, with an annual worldwide incidence of over 600,000 cases per year and 350,000 deaths per year.  According to National Cancer Registry Programme of the Indian Council of Medical Research (ICMR), more than 1300 Indians die every day due to cancer.  The absolute number of cancer deaths in India is projected to increase because of population growth and increasing life expectancy. Cancers of the head and neck include cancers of the buccal cavity, head and neck subset, larynx, pharynx, thyroid, salivary glands, and nose/nasal passages.
Cellular oxidative damage is a well-established general mechanism of cell and tissue injury that is primarily caused by free radicals and reactive oxygen species (ROS). Low levels of ROS are indispensable in many biochemical processes;  however, overproduction and/or inadequate removal of ROS can result in oxidative stress, which is characterized as an imbalance between the formation of active oxygen metabolites and the rate at which they are scavenged by enzymatic and nonenzymatic antioxidants.  Oxidative stress can participate in the pathogenesis and complications of many diseases including cancer.  ROS-induced DNA damage not only initiate tumorigenesis but also aid cancer progression. ,
Total antioxidant status (TAS) measures the peroxyl-scavenging capacity of the extracellular antioxidant system, comprised of sulfydryl groups (mostly albumin), urate, ascorbate, carotenoids, retinol, α-tocopherol, bilirubin, and proteins. TAS reflects the residual antioxidant capacity after the neutralization of ROS. ,
Free radicals are implicated in the pathogenesis of a multistage process of carcinogenesis. They are proposed to cause DNA base alterations, strand breaks, damage to the tumor suppressor genes, and an enhanced expression of the protooncogenes. ,,, It has been proposed that DNA damage induced by ROS may contribute to increased mutation rates, genome instability, apoptosis and associated tissue regeneration, and cell proliferation.  However, to best of our knowledge, the oxidative/antioxidative systems in the carcinogenesis of HNC and their relationships to DNA damage is limited. To address this, we evaluated the levels of TAS, total oxidant status (TOS), and oxidative stress index (OSI) in HNC and head and neck disease (HND) and analyzed the relationship among these oxidative stress parameters, DNA damage, and HNC risk.
Patients and sample specimens
The study was carried out at the Institute of Medical Sciences, Banaras Hindu University (BHU), Varanasi, India. The study protocol was approved by the Institutional Ethics Committee. Informed consent was obtained from each individual who attended the Department of Surgical Oncology, SS Hospital, BHU, during the period from February 2012 to January 2014 for treatment. Initially a series of 40 HNC and 37 HND patients were recruited, but after histopathological confirmation this number finally turned out to be 60 and 17 patients, respectively. Patients with cancer in regions other than the head and neck, those with any systemic diseases, and those with infections were excluded from the study. Tissue samples were obtained immediately after resection, snap-frozen, and stored in liquid nitrogen for assay.
Measurement of TAS, TOS, OSI from tissue homogenate
Tissue lysate obtained after homogenization and centrifugation was used for measurement at an equimolar concentration of 3 mg/mL. TOS and TAS were measured using the novel automated colorimetric method of Erel. , TOS measurement is based on the oxidization of the ferrous ion-o-dianisidine complex to ferric ion by oxidants of the sample in acidic medium, which forms a colored complex with xylenol orange. Results were expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol H 2 O 2 Eq/L). TAS measurement is based on the bleaching of the characteristic color of a more stable 2, 2´-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) radical cation by antioxidants. Results were expressed in millimole Trolox (Cayman Chemical Company) equivalent per liter.  The OSI value was determined as:  OSI (arbitrary unit) = [TOS (μmol H 2 O 2 Eq/L)/TAS (μmol Trolox Eq/L)] ×100.
DNA damage determination by single cell gel electrophoresis (SCGE)/COMET Assay
Tumor tissue minced in 1 mL hydroxyethyl piperazineethanesulfonic acid (HEPES)-buffered medium without serum containing 20 mol ethylene di amine tetra acetic acid (EDTA)/10% di methyl sulphoxide (DMSO), allowed to settle, and removed. The SCGE was carried out under alkaline conditions, as per standard protocol described by Singh et al.,  with modifications. 
To prevent additional DNA damage, all steps were conducted under yellow light or in the dark. Furthermore, to avoid possible position effects during electrophoresis, two parallel replicate slides per sample were prepared and processed in different electrophoretic runs. Slides were examined at 250× magnification using a fluorescence microscope (Olympus BX43 Fluorescence, Olympus America Inc.), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm. To avoid the potential variability, one well-trained scorer performed all the scorings of comets. As a measure of DNA damage, the percentage amount of tail and head was used [Figure 1]. It was calculated from the midpoint of the head and presented in micrometers.
All statistical analyses were performed using SPSS for Windows, version 16.0 (SPSS, Chicago, IL, USA). Chi-square test was used to compare categorical variables between the groups. The independent sample t-test and Mann-Whitney U test were used to compare continuous variables between the two groups. Multivariate logistic regression and receiver operating characteristic (ROC) curve analysis was performed to evaluate the association and sensitivity of TOS, TAS, and addiction to tobacco (either chewing or smoking) with histological grade of HNC. A two-sided P value <0.05 was considered statistically significant.
Demographic presentation of HNC and HND patients
Demographic and clinical data of patients with HNC and HND are shown in [Table 1]. The mean age of the study cohort of HNC patients was 46.65 ± 14.84 years, while it was 49.41 ± 13.00 years for HND patients. There were no significant differences between the two groups with respect to age, body mass index (BMI), body surface area (BSA), gender, religion, residence, and educational status. The preponderance of the study showed a significant positive association between the use of tobacco and HNC. The overall percentages of tobacco-addicted HNC and HND patients were 70% and 35.3%, respectively, and are depicted in [Figure 2].
Association of oxidative stress and DNA damage with study group
The association of oxidative stress parameters and DNA damage analysis with the study group is laid out in [Table 2]. The value of DNA damage (i.e., percent of comet tail) was found to be significantly higher in HNC subjects compared to HND (16.06 ± 1.78 AU vs 7.86 ± 5.97 AU; P < 0.001) subjects. Tissue homogenate TOS and OSI were higher in HNC than in HND (53.00 ± 40.61 vs 19.67 ± 21.90, P < 0.01; 7.221 ± 5.80 vs 2.40 ± 2.54, P < 0.01, respectively) subjects. The tissue homogenate TAS level in HNC was lower than in HND (748.33 ± 112.38 vs 809.41 ± 70.28, P < 0.05).
Relative risk prediction of histological grade with TOS, TAS, and addiction to tobacco through logistic regression and ROC curve analyses
Aggressive histological features (dependent variable), specifically, poorly and moderately differentiated grades of HNC, have been identified more commonly with (independent variables) higher level of TOS, lower TAS, and addiction to tobacco. Poorly and moderately differentiated histological grade has persisted on multinomial logistic regression analysis [Table 3], suggesting that higher TOS level (>30 μmol H 2 O 2 /L) [probability (P) = 0.002, relative risk (RR) = 11.838, 95% CI = 2.514-55.730] and lower TAS level (≤800 μmol Trolox equivalent/L) (P = 0.043, RR = 0.271, 95% CI = 0.077-0.960) have been significantly found to elevate the relative risk of HNC in comparison to lower TOS (≤30 μmol H 2 O 2 /L) and higher TAS (>800 μmol Trolox equivalent/L). Addiction to tobacco has shown 25% higher relative risk of HNC, but not up to significance.
ROC curve analysis [Figure 3] estimates sensitivity and specificity for predicted probability to test variables (TOS, TAS, addiction to tobacco) with HNC. The area under the ROC curve was 0.756 (standard error (SE) 0.062) with 95% CI 0.664-0.879 and P < 0.001.
The present study revealed the incidence of HNC increases in patients who are/have: a) addicted to tobacco b) elevated level of OSI, and c) decreased level of antioxidant. Although most studies have focused on the role of lifestyle-related factors in the incidence of cancer, recent reports suggest that these risk factors can also influence prognosis and survival rate after diagnosis. Our study analyzed a comparison of cases and controls with demographic factors as well as biochemical and molecular approaches with selected HNC risk.
The baseline characteristics of this study are listed in [Table 1]. Tobacco use was ascertained in 70.0% of the cases, compared to only 35.3% in the control group. Risk of HNC was significantly higher with use of tobacco/smoking as compared to the HND group. It is considered that the smoke from cigarettes has 4000 chemicals, 40 of which have carcinogenic potential. It has been shown that cigarette smoke contains prooxidants that are capable of initiating the process of lipid peroxidation and depleting levels of antioxidants from the diet. , In contrast, there is epidemiological evidence that demonstrates the protective effect of diet in some populations. ,,
The present study is in concordance with previous studies that revealed that free radicals were associated with increased risk of HNC. The data in [Table 2] indicate that DNA damage in tissues was significantly higher in patients with HNC than in HND. In addition, DNA damage was related to severity of disease in patients. Oxidative damage to DNA has been associated with a number of pathologies including neoplastic, neurodegenerative, cardiovascular, and autoimmune diseases.  We found decreased TAS levels, increased TOS levels, and increased DNA damage in HNC patients. DNA damage is associated with oxidative stress. Therefore, this suggests that DNA damage may be related with insufficient antioxidant capacity and excessive ROS generation, which contributes to the pathogenesis of the disease in HNC patients. Thus, the finding supports the hypothesis of oxidative stress involvement in the malignant process in the head and neck. Several works explore the levels of oxidative stress in patients with oral cancer ,, and most of them quantified the products of lipid peroxidation (mainly malondialdehyde) and contrasted them with the activity of antioxidant enzymes or exogenous antioxidants levels in blood or even saliva. The results agree that there is an imbalance between the high amount of free radicals and insufficient antioxidant activity. In addition, some researchers have observed that high levels of lipid peroxidation combined with low levels of thiols and antioxidant status correlate with a poor survival rate in patients with oral cancer. 
Thus, an in-depth understanding of the intricate molecular mechanisms underlying tumor initiation and progression in cells disturbing the oxidant/antioxidant equilibrium is a critical step toward the development of more effective strategies for prevention, early detection, and treatment of HNC, which disproportionately affects the Indian population.
Incorporating multivariate analysis, this study proves that disturbed oxidant/antioxidant equilibrium is significantly associated with the presence of advanced histological grade. We also report elevated risk with higher TOS and poorer TAS for advanced histological grade of HNC (P = 0.002, RR) = 11.838, 95% CI=2.514-55.730 and P = 0.043, RR = 0.271, 95% CI = 0.077-0.960, respectively). Dormandy  has proposed a close relationship between free radical activity and malignancy. Catalase, peroxidase, and superoxide dismutase (SOD) can all act as scavenging enzymes, destroying the free radicals and H 2 O 2 . The greater decline in SOD might spare O 2 , which has supporting evidence from other studies.  Additionally, the activities of catalase and peroxidase showed a greater decline, suggesting a greater accumulation of H 2 O 2 . This might also be responsible for degradative reactions in the tissues, including membrane damage via lipid peroxidation.  Gupta et al.  demonstrated that a reduction in several antioxidant defense mechanisms correlates with the emergence of the malignant phenotype. The low activities of these antioxidant enzymes observed in our study might be due to the depletion of the antioxidant defense system. This could occur as a consequence of overwhelming free radicals, as evidenced by the elevated levels of lipid peroxides in the circulation of oral cavity cancer patients.
Thus, this study provides ample evidence to support the fact that an elevated level of free radicals and decreased antioxidant capacity are linked to increased risk of HNC.
In conclusion, this study explored some differences related to free radicals and antioxidant status in the presentation of HNC. Oxidative stress is increased and antioxidant defenses are compromised in patients with HNC. A weak antioxidant defense system makes the mucosal cells more vulnerable to the genotoxic effect of ROS. This creates an intracellular environment more favorable for DNA damage and disease progression. These results advocate that elevated free radical generation could be an important prognostic factor in HNC.
Funding support for this work was from sources including the Dr. D. S. Kothari Post Doctoral Fellowship [No.F.4/2006 (BSR)/13-581/2012(BSR)] and SRF [Dev/scholarship (UGC-JRF-324)/S-01] from the University Grants Commission. We thank the Institute of Medical Sciences, Banaras Hindu University, Varanasi, India for providing the infrastructure and other research facilities.
Financial support and sponsorship
This study is supported by a research grant from the University Grants Commission, India, availed through Banaras Hindu University and by a DSK-Post Doctoral Fellowship. Medical writing was not used for the preparation of the article. The authors declare that all of them have made substantial contributions toward the writing of this article.
Conflicts of interest
Accordingly, there is no conflict of interest arising whatsoever with this article.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]