|Year : 2012 | Volume
| Issue : 2 | Page : 254-259
Assessment of the redox profile and oxidative DNA damage (8-OHdG) in squamous cell carcinoma of head and neck
Anil Kumar1, Mohan C Pant2, Hirdya S Singh3, Shashi Khandelwal1
1 Immunotoxicology Division, CSIR, Indian Institute of Toxicology Research, Lucknow, India
2 Ch. S.M. Medical University, Lucknow, India
3 Ch. Charan Singh University Meerut, India
|Date of Web Publication||26-Jul-2012|
Scientist F and Head, CSIR - Indian Institute of Toxicology Research, 80 Mahatma Gandhi Marg, Lucknow 226001
Source of Support: None, Conflict of Interest: None
Background: In developing countries especially in south Asia, there are growing habits of consumption of tobacco and its products in various forms. These are known to generate a strong free radical environment and when the free radicals overwhelm the antioxidant system, they may lead to degeneration of cellular components and mutations.
Aim: The aim of this study is to assess the levels of oxidative stress determinants, which may be one of the critical factors in head and neck cancer development.
Materials and Methods: This study included 100 consenting SCCHN patients and 90 matched healthy controls and we assessed the total antioxidant capacity (TAC), glutathione (GSH), free radicals (RNS, ROS) and oxidative DNA adduct (8-OHdG).
Results: We observed a substantial rise in reactive oxygen species (ROS, ~3.0-fold) and reactive nitrogen species (RNS, ~1.7-fold), together with significant lowering in TAC (~1.2-fold) and GSH (~1.7-fold) was observed. The 8-OHdG levels were also found to be significantly (P < 0.05) higher in patients in comparison to controls. Pearson's correlation between blood ROS and GSH were found to be negatively correlated -0.38 (P < 0.01) and RNS and DNA damage positively correlated 0.44 (P < 0.01).
Conclusion: Our present results demonstrate significant Redox imbalance in cancer patients suggesting their paramount importance in the development of SCCHN. The 8-OHdG could be the potential biomarker for evaluating risk of SCCHN. To develop new approaches of SCCHN prevention, there is a need of detailed study and better understanding of the molecular mechanisms underlying oxidative stress and DNA damage.
Keywords: 8-OHdG, reactive nitrogen species, reactive oxygen species, squamous cell carcinoma of head and neck, total antioxidant capacity
|How to cite this article:|
Kumar A, Pant MC, Singh HS, Khandelwal S. Assessment of the redox profile and oxidative DNA damage (8-OHdG) in squamous cell carcinoma of head and neck. J Can Res Ther 2012;8:254-9
|How to cite this URL:|
Kumar A, Pant MC, Singh HS, Khandelwal S. Assessment of the redox profile and oxidative DNA damage (8-OHdG) in squamous cell carcinoma of head and neck. J Can Res Ther [serial online] 2012 [cited 2019 Nov 17];8:254-9. Available from: http://www.cancerjournal.net/text.asp?2012/8/2/254/98980
| > Introduction|| |
Squamous cell carcinoma of head and neck (SCCHN) is globally the sixth most common cancer,which includes cancer of oral cavity, larynx, and pharynx. In developing countries especially in south Asia, there are growing habits of consumption of tobacco and its products in the form of surti, bidi, cigarette, khaine, and pan masala. The tobacco and tobacco products are known to generate a strong free radical environment, when the free radicals overwhelm the antioxidant system, they may lead to degeneration of cellular components and mutations.
Free radicals are the molecules which contain an unpaired electron in their outer shell and are generated endogenously during various cellular metabolic activities and exogenously by number of harmful compounds, tobacco and tobacco products. In human cells, mitochondria are the major intracellular source of reactive oxygen species (ROS) generation. , These molecules are highly reactive and can react with any molecule that comes in contact. In a biological system, they easily react with nucleic acids, proteins and lipids. Out of these, DNA is a major target of these free radicals and it is well established that the development of cancer is associated with change in genetic material. In some of the biological reactions, the free radicals play a vital role especially in the case of defense against microbial pathogens. This role occurs by low concentration of these molecules. However, when the level of free radicals go up as in the case of parasitic infection, inflammatory disease and cancer, these free radicals work like a fanatic, damage cellular components, eventually resulting in degeneration or mutation. It is evident from the literature that free radical species such as reactive nitrogen species (RNS), reactive oxygen species (ROS), and reactive oxygen metabolites such as superoxide anions (O 2− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (OH• ), malondialdehyde, and nitric oxide are involved in the multistep process of carcinogenesis. The free radicals increase the level of proteins degradation products such as kinins and activate the arachidonic acid.  The free radical-induced oxidative stress is associated with a variety of chronic degenerative diseases, including cancer, diabetes, cardiovascular diseases, and Alzheimer's disease as well as in aging. The free radicals can induce several kinds of DNA damage including strand breakage, base modification and DNA-protein cross-linkage. They can react with cell membrane fatty acids and form lipid peroxides.
8-Hydroxy-2?-deoxyguanine (8-OHdG) is one of the major oxidative modified DNA base products, which may lead to G:C to T:A transversions. , The 8-OHdG was first reported by Kasai et al.  to be formed on interaction of hydroxyl radical (OH• ) and singlet oxygen photodynamic action with DNA. Several studies have reported the higher content of 8-OHdG in cancer tissue in comparison to normal tissue. Another risk factor which can also contribute to head and neck cancer is the human papilloma virus (HPV). The HPV infects the epithelial cells of skin and mucosa, such as the mouth, throat, tongue, tonsils, vagina, penis and anus. Infection with the virus occurs when these areas come into contact with the virus, allowing it to transfer between epithelial cells. It is recognized as the major risk factor in about 60% of head and neck cancer, particularly among young subjects with no tobacco or alcohol history. , All HPV-positive cases express viral E6 and E7 oncoproteins which lack specific DNA binding activity, but can still associate with transcription factor complexes, such as p53 and E2F and alter their transcriptional activity. The high-risk HPV E6 proteins target p53 for proteosomal degradation, whereas E7 expression results in p53 stabilization, but inhibits its transcriptional activity. 
On the other hand, all organisms possess a range of enzymatic and nonenzymatic antioxidant systems, which neutralize a free radical molecule to a non-free-radical molecule. The enzymes included in the antioxidant system are glutathione peroxidase, glutathione reductase, catalase, thioredoxin reductase, superoxide dismutase, heme oxygenase and biliverdin reductase. The nonenzymatic part includes antioxidants and free radical scavengers, such as α-tocopherol (vitamin E), vitamin C, phytochemicals, carotenoids and glutathione. Glutathione (GSH) is a ubiquitous thiol-containing tripeptide (l-γ-glutamyl-l-cysteinylglycine), which plays a central role in cell. It is a critical factor in protecting organisms against toxicity and disease since it provides reducing capacity for several reactions and plays an important role in the detoxification of hydrogen peroxide and other free radicals.  GSH degrades hydrogen peroxide and singlet oxygen before they are converted to a hydroxyl radical. Pastore et al.  suggested that GSH in the nucleus is involved in mechanisms that are necessary for DNA repair and expression.
In recent past, the role of oxidant and antioxidant system in development of cancer has gained importance. Higher oxidants and lower antioxidant activities in blood of cancer cases suggest their significance in progression of disease. ,,
The aim of this study was to evaluate oxidant-antioxidant related parameters (ROS, RNS, GSH), antioxidant capacity (TAC) and oxidative DNA adduct (8-OHdG) in plasma of patients with SCCHN and their respective controls to understand the relationship between the levels of oxidants and antioxidants in pathogenesis of the disease.
| > Materials and Methods|| |
This case-control study was approved by the ethical committee's concerned institute and medical university for clinical research. The protocol confirmed to the provisions of declaration of Helsinki in 1995. Prior to collection of samples, an informed consent was obtained from the study subjects, for inclusion in the study and subject anonymity was ensured. A total of 100 newly diagnosed patients with biopsy proven SCCHN prior to any chemoradiotherapy and 90 healthy control subjects were included. The mean age of SCCHN patients was 53 (range, 25-75 years) and mean age of the controls was 51 (range, 22-60 years). Subjects having regular smoking habits and smoking index (cigarettes/day × 365) of more than 730  and regular smokeless tobacco chewers with chewing index more than 365  (CY = frequency of tobacco chewed/kept/day × 365) were considered in the category of smokers and tobacco chewers, respectively. All the subjects belonged to the same socioeconomic group [Table 1]. Two milliliters of the blood sample was collected in 3.4% sodium citrate (pH 7.6) vial. The blood samples were immediately kept in ice till further use. One milliliter blood was centrifuged at 2500 rpm for 15 min at 4 °C, to separate plasma and remaining one ml was used for DNA isolation.
Total antioxidant capacity (TAC), glutathione (GSH), reactive oxygen species (ROS) and reactive nitrogen species (RNS) were analyzed in plasma of all the subjects.
Total antioxidant capacity
Total antioxidant capacity was measured using an Antioxidant Assay Kit (Cayman Chemical Company, USA). Theoretically, the assay relies on the ability of antioxidants in the sample to inhibit oxidation of ABTS (2,2?-azino-di-(3-ethybenzthiazoline sulphonate)) by metmyoglobin, which can be monitored by reading the absorbance at 750 nm. The capacity of antioxidants in the sample to prevent ABTS oxidation is compared with that of Trolox and quantified as millimolar Trolox equivalents.
Ten microliters of plasma (20 times diluted) was taken in a microplate and the procedure followed according to the manual. The absorbance of ABTS oxidation was measured at 750 nm on a plate reader (BMG FLUOstar Omega).
Glutathione in plasma was evaluated by using o-phthaldialdehyde (OPT).  OPT reacts with both glutathione amino and sulphydryl groups, yielding a cyclic highly fluorescent product. Briefly, 25 μl each of plasma was added in a microplate and the volume was made up to 200 μl with HEPES buffer (0.1 M, pH 7.4). Ten microliters of o-phthaldialdehyde (OPT, 100 mM)) was then added and after 10 min incubation at 37°C, the fluorescence was measured at 360 nm excitation and 460 nm emission on a plate reader (BMG FLUOstar Omega). Glutathione reduced was used as standard, for quantification of GSH.
Reactive oxygen species
The oxygen free radicals were measured with the help of 2?,7?,-dichlorofluorescein diacetate (DCF-DA), a fluorescent probe, which on interaction with ROS yields highly fluorescent DCF.  In cells DCF-DA diffuses through the cell membrane and is subsequently deacetylated by intracellular esterases to nonfluorescent DCF-H, while in the cell free system, DCF-DA on treatment with 0.1M NaOH for 30 min at room temperature gets converted into nonfluorescent DCF-H, which reacts with free radicals and produce fluorescent DFC. , Briefly, 25 μl of plasma was added in a microplate, and the volume was made up to 200 μl with PBS. Twenty-five microliters of freshly prepared DCF-H (500 μM final concentration) was then added to each well and after 1 h incubation at 37 °C, the florescence was measured at 485 nm excitation and 528 nm emission on a plate reader (BMG FLUOstar Omega). 2?,7?-Dichlorofluorescein was used as standard, for quantification of total ROS.
Reactive nitrogen species
The nitrite present in plasma reacts with sulfanilamide and N-(naphthyl)ethylenediamine to produce a red color.  Briefly, 10 μl of plasma was added in a microplate and the volume made up to 100 μl with PBS. Fifty microliters of Griess Reagent I and of Griess Reagent II were then added to each well and after 10 min incubation at 37°C, the absorbance was measured at 550 nm on a plate reader (BMG FLUOstar Omega). For the quantification of total nitrite in plasma, sodium nitrite solution was used as standard.
Oxidative DNA damage
The oxidative DNA damage (8-OHdG) was quantified using a DNA Damage Quantification Kit (BioVision, USA). After treating DNA containing abasic sites (AP) with aldehyde reactive probe reagent (ARP), AP sites were tagged with biotin residues which can be quantified using the avidin-biotin assay followed by colorimetric detection.
Genomic DNA from whole blood was isolated using the lab protocol of Laura-Lee Boodram, Department of Life Sciences, The University of West Indies, with minor modifications. To 400 μl of whole blood, an equal volume of buffer A (0.32 M sucrose, 10 mM Tris-HCl, 5 mM MgCl 2 , 1% Triton X-100, adjusted to pH 7.6) and two volumes of cold sterile distilled water were added and after gently vortexing for 30 s, it was kept on ice for 3 min. Following centrifugation at 3500 rpm for 15 min at 4°C, the pellets were dissolved in 800 μl buffer A and 1200 μl cold sterile distilled water. The sample was again centrifuged at 3500 rpm for 15 min at 4°C and the pellets (white or cream in color) were re-suspended in 1 ml buffer B (20 mM Tris-HCl, 4 mM Na 2 EDTA, 100 mM NaCl, adjusted to pH 7.4) and 100 μl of 10% SDS. Ten microliters of Proteinase K (20 mg/ml, freshly prepared) was then added and further incubated overnight at 37°C.
After overnight incubation with Proteinase K, 250 μl of 6 M NaCl was added and after vigorous shaking for 15 s, the samples were centrifuged at 2500 rpm for 15 min. Pellets weres discarded and the supernatant was taken in a separate tube and double volume of cold ethanol (100%) was added to it, inverting the tube seven to eight times to precipitate DNA. The precipitated DNA was resuspended in 200 μl of Tris-HCl, pH 8.5 and was kept at 37°C to dissolve. The NanoDrop Spectrophotometer (ND 1000 V3.3.1) was used to measure the amount and purity of DNA. Five microliters of a highly purified DNA sample (0.1 μg/μl), isolated from blood, were taken in a microcentrifuge tube, mixed with 5 μl of ARP solution and incubated for 1 h at 37°C, to tag AP sites of DNA. Assay was carried out according to the manual provided. 40 ARP-DNA Standard (40 ARP sites per 105 bp) was used for quantification of AP sites in samples to determine the level of DNA damage.
The data were statistically analyzed using SPSS statistical software (Version 12). Student's 't'-test was performed to compare levels between controls and patients. Pearson's correlation was carried out to study the association between the various oxidants and antioxidants. Differences between groups and variables were analyzed for significance using an one-way ANOVA test using GraphPad PRISM 5 software (CA, USA). The difference was considered statistically significant when 'P' value were 0.05 or less.
| > Results|| |
The overall profile of TAC, GSH, RNS and ROS of controls and SCCHN patients is given in [Figure 1]. Total antioxidant capacity of the patients as evaluated in blood was found to be substantially suppressed in comparison to controls. The TAC value in blood of controls was 1.63 μM, whereas in the SCCHN patients, levels dropped to 1.40 μM. A ~1.2-fold reduction (P < 0.001) in TAC was evident in the plasma.
|Figure 1: Plasma TAC, GSH RNS and ROS in controls (n=90) and HNSCC patients (n=100), 8-OHdG in blood cells of controls (n=50) and HNSCC patients (n=50),The level of TAC is expressed as mM, GSH, RNS and ROS as μM, 8-OHdG as number of apurinic sites/105 bp.|
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Similarly, GSH levels in both saliva and blood of SCCHN patients indicated a lowering pattern. A ~1.7-fold reduction in GSH suggests an oxidant-antioxidant imbalance in the cancer patients. GSH values in blood fell to 4.40 μM from 2.54 μM in controls.
In contrast, ROS and RNS were found to be elevated in SCCHN patients. The NO 2 levels in blood, increased by 1.7-fold. The NO 2 values were 62.68 μM when compared to 37.21 μM in controls. The ROS values in blood also showed a substantial ~3.0-fold increase. Control ROS levels of 3.20 μM increased to 9.50 μM in the patients.
The level of oxidized DNA expressed as 8-OHdG was also observed to rise significantly in blood. A 1.6-fold rise in blood failed to show any statistical significance by Student's 't'-test [Figure 1].
Pearson's correlation analysis was performed to study correlation between blood ROS and GSH and found to be negatively correlated −0.38 (P < 0.01) and RNS and DNA damage were positively correlated, 0.44 (P < 0.01), as shown in [Table 2]. The blood determinations when compared in groups habituated to tobacco, smoking and chewing provide a clear representation of the lifestyle effects.
|Table 2: Comparison between plasma antioxidant and oxidant levels in squamous cell carcinoma of head and|
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[Table 3] demonstrates the salivary TAC values in non-habituates, smokers, tobacco chewers and in both with smoking and tobacco chewing habits. The patients group comprising of smokers and smokers and tobacco chewers showed a significant decrease in the TAC level (P < 0.05). Tobacco chewers, although, exhibited suppressed TAC levels did not show any statistical significance.
|Table 3: Levels of the plasma oxidative stress determinants in controls and SCCHN cases|
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Glutathione levels as depicted in [Table 3] showed that the GSH levels both in control habituates and case habituates failed to show any statistical significance. However, significance of P < 0.05 observed in controls smokers versus patients with smoking and tobacco chewing habits was evident.
The ROS levels in blood of control habituates versus SCCHN habituates as shown in [Table 3] indicated high significance (P < 0.01) in control smokers versus patients with smoking and tobacco chewing habits, also exhibited significance of P < 0.01.
Regarding the RNS values in blood, when controls and patients with smoking and chewing habits were compared, highly significant increase by ~2-fold (P < 0.01) was observed. In another set of comparisons, between control smokers versus patients smoking and chewing habits, significance of the level of P < 0.05 suggested that patients with smoking and chewing habits revealed a higher NO 2 , lowered GSH, and suppressed TAC levels [Table 3].
The blood 8-OHdG levels were although elevated in all habituated patients, no statistical significance was achieved in any of the case groups [Table 3]. The overall redox profile of blood clearly demonstrates oxidative stress determinants including 8-OHdG levels, to be highly altered in SCCHN.
| > Discussion|| |
In this study, we assess the levels of oxidants as well as antioxidants in human blood plasma, which forms the frontline defense to encounter various oxidants present in tobacco smoke, tobacco chewing, alcohol and food. It is evident that the higher level of oxidants inside the body or cell may lead to serious consequences, several neurodegerative, parasitic diseases and cancer. ,,,, The imbalance between the levels of oxidants and antioxidants may implicate in the pathogenesis of SCCHN. The basic concept is that the free radicals damage cellular materials which could result in activation or altering normal cells into malignant ones.  The ROS and RNS are involved in initiation and promotion of carcinogenesis through DNA damage. , for example, NO-mediated inhibition of base excision DNA repair may potentiate oxidative DNA damage in cells and could be relevant to carcinogenesis.  Likewise Van Wijk et al.  suggested that the metabolism of ROS in cancer cells is drastically altered with evidence favoring at least two mechanisms; cancer cells produce large amounts of ROS compared to non-neoplastic cells and secondly, suppression of the antioxidant system in cancer cells. An oxygen free radical interaction with DNA can break its strands or delete a base.
The free radical-induced genetic alterations such as mutations and chromosomal rearrangement can lead to initiation and progression of carcinogenesis. Mutations can occur through misrepair or due to incorrect replication, while chromosomal rearrangements can result from strand breakage misrepair.  The increase in replication errors can initiate additional oncogene activation and tumor suppressor gene inactivation, ultimately contributing to malignancy. Free radical-induced cytotoxicity may also contribute to the initiation of carcinogenesis by depleting the normal cell population, Promoting clonal expansion of more resistant initiated cells and thus increasing the probability of mutation. 
Oxidative DNA damage can affect carcinogenesis by modulation of gene expression and altered gene expression can lead to the stimulation of growth signals and proliferation.  It is also evident that ROS may stimulate signal transduction pathways, for example, protein kinase and poly(ADP ribosylation), c-Raf-1 and ras pathways. , In addition to 8-OHdG production, an accumulation of intracellular ROS and/or RNS can induce point mutation in the DNA, thus disrupting the expression and function of several tumor-suppressing genes such as RAS and p53, which might contribute to the pathogenesis of hepatocellular, lung and gastric cancer. , The 8-oxoGua induced aberrant modifications in adjacent DNA, a hypothesized mechanism, can significantly contribute to the genetic instability and metastatic potential of tumor cells.
On the other hand, the antioxidant system includes a variety of antioxidants including vitamins, Carotenoids, flavonoids and glutathione. They scavenge free radicals and protect the cells from harmful oxidants. Depletion in levels of antioxidants in the body may lead to harmful consequences including cancer. The association between cancer and inadequate levels of antioxidant capacity has been reported. ,,,, Glutathione, another antioxidant, is also found to be lower in cancer patients in comparison to healthy controls. , Farias et al.  claimed that there is no significant difference in levels of GSH in breast cancer patients. The glutathione participates as nonenzymatic antioxidant. The other nonenzymatic antioxidants include ascorbic acid (vitamin C), α-tocopherol (vitamin E), carotenoids and flavonoids. Another antioxidant studied in this study, is glutathione, present in all mammalian tissues and is a critical factor in protecting organisms against toxicity and disease. Several studies reported that the decrease in GSH is associated with the development of cancer. , However, some of the studies reported an increase in the levels of GSH in cancer patients. , GSH also prevents oxyradical damage, and thus, blood GSH level may serve as an indicator of GSH status and disease risk in human subjects.
These findings indicate an imbalance in the oxidant-antioxidant status that results in reduced TAC and GSH and enhanced production of ROS and NO 2 in head and neck cancer patients. This discrepancy in the redox status appears to have a marked effect on DNA oxidation (DNA adduct), one of the causative factors for oral cancer development. Since the alteration in the oxidant-antioxidant profile is more prominent in those patients with both smoking and chewing habits, it is imperative to believe that lifestyle habits do play a central role in the onset of SCCHN.
Our results also support the idea that oxidative stress plays a role in the development of head and neck cancer. The 8-OHdG could be a potential biomarker in evaluating the risk of SCCHN. To develop new approaches of SCCHN prevention, we need further studies and better understanding of the molecular mechanisms underlying oxidative stress and DNA damage.
| > Acknowledgments|| |
The authors are grateful to the Director, CSIR - Indian Institute of Toxicology Research, Lucknow, for his keen interest and support in carrying out the study. AK is thankful to UGC, New Delhi, for providing a Senior Research Fellowship. Financial support from CSIR - Network Project SIP-08 is gratefully acknowledged. CSIR-IITR communication number is 3014.
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[Table 1], [Table 2], [Table 3]
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