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Original Article

Assessment of the redox profile and

oxidative DNA damage (8-OHdG) in
squamous cell carcinoma of head and neck
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. Pearsons 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.
KEY WORDS: 8-OHdG, reactive nitrogen species, reactive oxygen species, squamous cell carcinoma of head and neck, total antioxidant

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.[1,2] 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 (O2), hydrogen peroxide (H2O2),
hydroxyl radicals (OH), malondialdehyde, and
nitric oxide are involved in the multistep process of
carcinogenesis. The free radicals increase the level

Anil Kumar,
Mohan C. Pant1,
Hirdya S. Singh2,
Division, CSIR, Indian
Institute of Toxicology
Research, 1Ch. S.M.
Medical University,
Lucknow, 2Ch. Charan
Singh University
Meerut, India
For correspondence:
Dr. Shashi
Scientist F and
Head, CSIR Indian
Institute of Toxicology
Research, 80 Mahatma
Gandhi Marg,
Lucknow 226001,
E-mail: skhandelwal_

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Website: www.cancerjournal.net
DOI: 10.4103/0973-1482.98980
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Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2

Kumar, et al.: Oxidative stress and cancer

of proteins degradation products such as kinins and activate

the arachidonic acid.[3] The free radical-induced oxidative stress
is associated with a variety of chronic degenerative diseases,
including cancer, diabetes, cardiovascular diseases, and
Alzheimers disease as well as in aging. The free radicals can
induce several kinds of DNA damage including strand breakage,
base modification and DNAprotein 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.[4,5] The 8-OHdG was first reported by Kasai
et al.[6] 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.[7,8] 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 highrisk HPV E6 proteins target p53 for proteosomal degradation,
whereas E7 expression results in p53 stabilization, but inhibits
its transcriptional activity.[9]
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 -tocopherol (vitamin
E), vitamin C, phytochemicals, carotenoids and glutathione.
Glutathione (GSH) is a ubiquitous thiol-containing tripeptide
(L--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.[10]
GSH degrades hydrogen peroxide and singlet oxygen before
they are converted to a hydroxyl radical. Pastore et al.[11]
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.[12-14]
The aim of this study was to evaluate oxidantantioxidant
Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2

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.
This casecontrol study was approved by the ethical
committees 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, 2575 years) and mean age of the controls was 51
(range, 2260 years). Subjects having regular smoking habits
and smoking index (cigarettes/day 365) of more than
730[15] and regular smokeless tobacco chewers with chewing
index more than 365[16] (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.
Table 1: Clinical and the socio-demographic details of the
N = 90
Healthy individuals
Tobacco habituates
Smokers + chewers
HNSCC patients
Tobacco habituates
Oral region (Tongue, buccal mucosa, cheek,
Neck region (Larynx, Pharynx, etc.)

2560 years
N = 100
2575 years

Kumar, et al.: Oxidative stress and cancer

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).[17] OPT reacts with both glutathione amino and sulphydryl
groups, yielding a cyclic highly fluorescent product. Briefly, 25
l each of plasma was added in a microplate and the volume
was made up to 200 l 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 37C, 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.[18] 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.[19,20] Briefly, 25 l of plasma was added in
a microplate, and the volume was made up to 200 l with
PBS. Twenty-five microliters of freshly prepared DCF-H
(500 M 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.[21] Briefly,
10 l of plasma was added in a microplate and the volume
made up to 100 l 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 37C, 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 avidinbiotin 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 l of whole blood, an equal volume of buffer A (0.32
M sucrose, 10 mM TrisHCl, 5 mM MgCl2, 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 4C, the pellets were dissolved in 800 l buffer A
and 1200 l cold sterile distilled water. The sample was again
centrifuged at 3500 rpm for 15 min at 4C and the pellets
(white or cream in color) were re-suspended in 1 ml buffer B
(20 mM TrisHCl, 4 mM Na2EDTA, 100 mM NaCl, adjusted to
pH 7.4) and 100 l of 10% SDS. Ten microliters of Proteinase
K (20 mg/ml, freshly prepared) was then added and further
incubated overnight at 37C.
After overnight incubation with Proteinase K, 250 l 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 l of TrisHCl,
pH 8.5 and was kept at 37C 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 g/l), isolated from blood, were taken in a
microcentrifuge tube, mixed with 5 l of ARP solution and
incubated for 1 h at 37C, 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.
Statistical analysis
The data were statistically analyzed using SPSS statistical
software (Version 12). Students t-test was performed to
compare levels between controls and patients. Pearsons
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.
Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2

Kumar, et al.: Oxidative stress and cancer

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 M, whereas in the SCCHN
patients, levels dropped to 1.40 M. A ~1.2-fold reduction
(P < 0.001) in TAC was evident in the plasma.
Similarly, GSH levels in both saliva and blood of SCCHN patients
indicated a lowering pattern. A ~1.7-fold reduction in GSH
suggests an oxidantantioxidant imbalance in the cancer patients.
GSH values in blood fell to 4.40 M from 2.54 M in controls.
In contrast, ROS and RNS were found to be elevated in SCCHN
patients. The NO2 levels in blood, increased by 1.7-fold. The
NO2 values were 62.68 M when compared to 37.21 M in
controls. The ROS values in blood also showed a substantial
~3.0-fold increase. Control ROS levels of 3.20 M increased to
9.50 M in the patients.

0 2

62.68 6.94
3.20 0.35

2.54 0.38

9.20 0.57


15. 00 1.64


9.50 0.92

37.21 4.65

4.40 0.52

1.40 0.23

1.63 0.28

4 6 8 10 12 14 16 18 40 50 60 70 80

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 Students t-test
[Figure 1].

Pearsons 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 3 demonstrates the salivary TAC values in nonhabituates, 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.
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 NO2, lowered GSH, and suppressed
TAC levels [Table 3].
The blood 8-OHdG levels were although elevated in all


All values are expressed as mean S.E.,*p<0.05 , **p<0.01, ***p<0.001 vs.

control, by Students t test

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 M, 8-OHdG as number of apurinic sites/105 bp.

Table 2: Comparison between plasma antioxidant and

oxidant levels in squamous cell carcinoma of head and
neck patients
Correlation between biomarkers
Plasma ROS vs. GSH plasma
Plasma RNS vs. 8-OHdG leucocytes



Table 3: Levels of the plasma oxidative stress determinants in controls and SCCHN cases

TAC mM (mean SE)

GSH M (mean SE)
ROS M (mean SE)
RNS M (mean SE)
8-OHdG AP sites/105
(mean SE)

1.83 0.10 1.50 0.17
5.12 0.48 3.16 0.95
2.49 0.65 8.47 1.02
31.74 0.30 40.09 1.28
5.83 0.39 9.11 0.21

1.67 0.03* 1.24 0.02*
4.40 0.52* 2.48 0.61*
3.24 0.72* 9.22 1.44*
35.98 7.54* 65.17 3.80*
7.33 0.71 13.28 2.36

Smokers + chewers
1.55 0.03
1.26 0.02 1.48 0.04* 1.33 0.05*
4.06 0.77* 2.37 0.78* 3.87 0.42* 2.33 0.47*
3.35 1.39* 9.97 1.61* 3.87 0.88* 10.71 1.42*
38.43 2.37* 66.77 14.34* 43.33 3.01* 76.67 8.18**
9.55 0.77 14.12 1.59 10.84 0.88 16.08 2.82

**P < 0.001, *P < 0.05, vs. control by one-way ANOVA

Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2


Kumar, et al.: Oxidative stress and cancer

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.
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.[12-14,22,23] 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.[24] The ROS and RNS are
involved in initiation and promotion of carcinogenesis through
DNA damage.[25,26] for example, NO-mediated inhibition of base
excision DNA repair may potentiate oxidative DNA damage in
cells and could be relevant to carcinogenesis.[27] Likewise Van
Wijk et al.[28] 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.[29] 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.[30]
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.[31]
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.[32,33] 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.[34,35]
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.[14,23,36-38] Glutathione, another antioxidant, is also
found to be lower in cancer patients in comparison to
healthy controls.[23,39] Farias et al.[36] 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), -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.[40,41] However, some of the
studies reported an increase in the levels of GSH in cancer
patients.[42,43] 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 NO2 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 oxidantantioxidant 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.
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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Cite this article as: 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.
Source of Support: Nil, Conflict of Interest: None declared.