Jpn J Clin Oncol 2004;34(7)379–385

Original Articles

Evaluation of Oxidative Stress and Nitric Oxide Levels in Patients

with Oral Cavity Cancer
S. Syed Sultan Beevi1, A. Muzib Hassanal Rasheed2 and A. Geetha3
1Department of Physiology and Biochemistry, Ragas Dental College and Hospital, Chennai, Tamil Nadu, India,
2Department of Surgical Oncology, Government Royapettah Hospital, Chennai, Tamil Nadu, India and 3Department
of Biochemistry, Bharathi Womens’ College, Chennai, Tamil Nadu, India
Received October 13, 2003; accepted March 31, 2004

Objective: The aim of this study was to evaluate the magnitude of oxidative stress and levels

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of nitric oxide in patients with oral cavity cancer by analyzing the levels of lipid peroxidation
products, antioxidants and nitric oxide products.
Methods: This prospective study was conducted on 15 patients with biopsy proven squamous
cell cancer of the oral cavity with clinical stage III/IV and an equal number of age and sex
matched healthy subjects. The levels of lipid peroxidation products, antioxidants and nitric
oxide products were determined by colorimetric methods.
Results: Lipid peroxidation products like lipid hydroperoxide (LHP) and malondialdehyde
(MDA) and nitric oxide products like nitrite (NO2–), nitrate (NO3–) and total nitrite (TNO2–) were
significantly elevated, whereas enzymatic and non-enzymatic antioxidants were significantly
lowered in oral cavity cancer patients when compared to normal healthy subjects.
Conclusions: Enhanced lipid peroxidation with concomitant decrease in antioxidants is indic-
ative of oxidative stress that provides evidence of the relationship between lipid peroxidation
and oral cavity cancer. Increased nitric oxide production represents a general mechanism in its
pathogenesis.

Key words: oral cavity cancer – nitric oxide – lipid peroxidation – antioxidants

INTRODUCTION It should be noted, however, that the development of human

Oral cavity cancer is an important cancer globally and is one of
the ten most frequent cancers worldwide (1). Tobacco is the
primary etiological factor in its development, other factors
being alcohol, genetic predisposition and a diet lacking in
micronutrients.
The proposal that reactive oxygen species (ROS) such as
superoxide radicals (O2•), hydroxyl radicals (OH•) and hydro-
gen peroxide (H2O2) play a key role in human cancer develop-
ment has gained much support recently. They have been shown
to possess several characteristics of carcinogens (2). ROS can
cause DNA base alterations, strand breaks, damage to tumor
suppressor genes and enhanced expression of protooncogenes
(2). ROS-induced mutation could also arise from protein dam-
age and attack on lipids, which then initiate lipid peroxidation.
For reprints and all correspondence: S. Syed Sultan Beevi, Department of
Physiology and Biochemistry, Ragas Dental College and Hospital, 2/102, East
Coast Road, Chennai, Tamil Nadu, India. E-mail: fathuus@rediffmail.com
cancer is multifactorial, depending on several factors including
the extent of DNA damage, effectiveness of antioxidant
defense, DNA repair system and growth promoting effect of
ROS (3).
The burst of ROS has been implicated in the development
of oral cavity cancer in tobacco chewers and smokers (4).
Tobacco consumption in any form has been demonstrated to
have carcinogenic, teratogenic and genotoxic effects and is
positively correlated with accumulation of DNA damage.
Tobacco is therefore believed to directly induce cellular DNA
damages in the human oral cavity (5). Oxidative modification
of nucleic acids by ROS could result in the transformation of
normal cells into malignant cells (6). ROS induced lipid perox-
idation has been implicated in malignant transformation (7).
The prime targets of peroxidation by ROS are the polyunsatu-
rated fatty acids (PUFA) in the membrane lipids. Furthermore,
the decomposition of these peroxidized lipids yields a variety
of end products, including lipid hydroperoxides (LHP) and
malondialdehyde (MDA). The levels of these lipid peroxides

© 2004 Foundation for Promotion of Cancer Research

380 Oxidative stress in oral cancer
indicate the extent of lipid peroxidation in general and serve as
markers of cellular damages due to free radicals.
Enzymatic and non-enzymatic antioxidant defense systems
include superoxide dismutase (SOD), catalase (CAT), gluta-
thione peroxidase (GPx), reduced glutathione (GSH), vitamins
E, C and A and β carotene. They protect cells against ROS pro-
duced during normal metabolism and after an oxidative insult.
Antioxidant defense systems work cooperatively to alleviate
the oxidative stress caused by enhanced free radical produc-
tion. Any changes in one of these systems may break this
equilibrium and cause cellular damages and ultimately malig-
nant transformation.
Nitric oxide (NO•) is a free radical, an uncharged molecule
with an unpaired electron. NO• plays multiple roles in both
intracellular and extracellular signaling mechanisms (8). This
highly reactive, yet simple molecule is produced in the body by
the isoenzyme nitric oxide synthase (NOS) using L-arginine as
a substrate. Three isoforms of NOS have been characterized.
Two of them are constitutive NOS (cNOS) and the third is
inducible (iNOS) by endotoxins and cytokines (9).
Reaction of NO• with oxygen or other free radicals generates
reactive nitrogen species (RNS), which cause multiple bio-
logical effects (10). NO• is either cytostatic or cytotoxic, inter-
acting with a number of molecular targets within cells. Cells
within different tissues display varying responses to NO•,
which may relate to the presence of cellular antioxidants such
as GSH, GPx, CAT and SOD (11).
Overexpression of NOS in chronic inflammation can lead to
genotoxicity. NO• may mediate DNA damage through the for-
mation of carcinogenic nitrosamines, generation of RNS and
inhibition of DNA damage repair mechanism. It can thus be
considered as a tumor initiating agent (12). However, NO• may
also have an impact on other stages of cancer development.
These effects of NO• are broad, with its involvement ranging
from cellular transformation and formation of neoplastic
lesions to the regulation of various other aspects of tumor
biology (11).
NO• plays an important role in host defense and homeostasis
when generated at a low level for a brief period of time, but
becomes genotoxic and mutagenic when generated at higher
concentrations for prolonged periods of time. Thus, the bio-
logical outcome of the NO• mediated effects is complex and
depends on the internal and external environment of the target
and generation sites of the cells as well as the concentration of
NO• generated.
The aim of this study was to evaluate the extent of oxidative
stress and the levels of nitric oxide in oral cavity cancer
patients by analyzing the levels of lipid peroxidation products,
antioxidants and nitric oxide products.
SUBJECTS AND METHODS
PATIENTS
This prospective study was conducted on 15 patients (12
males, 3 females) with biopsy proven squamous cell carcinoma
Table 1. Clinical characteristics of patients
Characteristics No. of patients
Patients 15
Male/Female 12/3
Age (years) (range) 56.2 (33–72)
Stage
III 2
IV 13
Grade
I (well differentiated SCC) 15
Tumor location
Anterior 2/3rd of the tongue 4
Buccal mucosa 7
Soft palate 1
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Alveolus 2
Retromolar area 1
of the oral cavity with clinical stage III/IV, registered at the
Department of Oncology, Government Royapettah Hospital,
Chennai. All the patients included in the investigation were
either smokers or tobacco chewers. The control groups
consisted of 15 age and sex matched non-smoking healthy
volunteers (12 males, 3 females) from similar socioeconomic
backgrounds. The age range was 33 to 72 years (mean ± SD
56.2 ± 11.8 years) for both patients and controls. The clinical
characteristics of patients (sex, age, disease localization and
stage) are shown in Table 1. Tumors were classified according
to the UICC criteria (13). Two patients were in stage III and 13
patients in stage IV. All the carcinomas were graded as well-
differentiated squamous cell carcinoma. The ethical committee
of the institution approved the study, and informed consent of
all the subjects was obtained. None of the patients and control
subjects had concomitant diseases such as diabetes mellitus,
liver disease and rheumatoid arthritis. All the patients and the
control subjects included in this study were not on any medical
treatment including supplementation of antioxidants. All the
subjects underwent a thorough dental checkup. The subjects
who had poor oral hygiene had dental scaling, removal of
caries-affected teeth and were treated with antibiotics and
mouth cleaning agents for a period of one week. In such cases,
blood samples were collected 15 days after the completion of
the dental treatment.
COLLECTION OF SAMPLES
Taking aseptic precautions, blood samples (approximately 10
ml) were collected in appropriate sterile vials by venous arm
puncture after overnight fasting. Five milliliters of blood was
collected with EDTA as an anticoagulant for erythrocyte
preparation and plasma. Another 5 ml of blood was collected
without anticoagulant for the separation of serum. Plasma
and sera were separated by centrifugation at 1000 g for 15 min.

Table 2. Levels (µmol/ml) of LHP and (nmol/ml) of MDA in plasma of oral
cancer patients and control subjects (mean ± SD)
LHP MDA
Control group (n = 15) 290.57 ± 48.70 2.02 ± 0.23
Patient group (n = 15) 496.29 ± 19.51* 5.57 ± 0.97*
P value 0.0000 0.0000
*P < 0.001, when compared with control group
After the separation of plasma, the buffy coat was removed
and the packed cells were washed thrice with 0.89% saline. A
known volume of erythrocytes was lysed with deionized water.
The hemolysate was separated by centrifugation at 2500 g for
15 min at 2 °C. Biochemical estimations were carried out im-
mediately.
A portion of the oral mucosa was excised from oral cancer
cavity patients by punch biopsy and washed immediately in
physiological saline. Tissues were preserved in 0.9% formalin
saline. Thin tissue sections were fixed and stained with eosin–
hemotoxylin mixture. The stained tissue smear was observed
through medical microscope under 100× magnification.
BIOCHEMICAL MEASUREMENTS
The estimation of MDA in plasma was done by the method of
Draper and Hadley (14). The color produced by the reaction of
thiobarbituric acid with MDA was measured colorimetrically
at 533 nm. The results were expressed as nmoles/ml plasma.
LHP in plasma was estimated by the method of Jiang et al.
(15). This method is based on the ability of H2O2 to oxidize
ferrous ion under acidic condition in the presence of xylenol
orange. The resultant chromophore was measured colorimetri-
cally at 560 nm. The LHP level was expressed as µmoles/ml
plasma.
SOD was assayed in RBC hemolysate by the method of
Misra and Fridovich (16). This method is based on the ability
of SOD to inhibit autoxidation of epinephrine under specific
conditions. One unit of SOD is defined as the amount of
enzyme required to cause 50% inhibition of epinephrine
autoxidation. Enzyme activity was reported as units/100 mg
protein in erythrocyte lysate.
CAT was assayed in RBC hemolysate by the method of
Clairborne (17). The color produced by the reaction of H2O2
with dichromate in acetic acid was measured colorimetrically
at 620 nm. Enzyme activity was expressed as µmoles of H2O2
decomposed/min/mg protein.
Glutathione peroxidase (GPx) was assayed in RBC hemo-
lysate by the method of Flohe and Gunzler (18) and based on
the reaction of the residual GSH with 5,5′,dithiobisnitroben-
zoic acid to form a complex that absorbs at 412 nm. Enzyme
activity was expressed as nmoles of GSH oxidized/min/mg
protein.
GSH was estimated in plasma by the method of Thomas and
Skrinska (19), which is based on the reaction of GSH with
Jpn J Clin Oncol 2004;34(7) 381
Table 3. Activities (units/100 mg protein) of SOD, (units/mg protein) of GPx
and CAT in erythrocytes of oral cancer patients and control subjects (mean ±
SD)
SOD GPx CAT
Control group (n = 15) 21.35 ± 2.80 13.80 ± 1.22 33.63 ± 2.59
Patient group (n = 15) 10.07 ± 2.93* 3.33 ± 0.80* 14.44 ± 1.63*
P value 0.0000 0.0000 0.0000
*P < 0.001, when compared with control group
5,5′,dithiobisnitrobenzoic acid to form a complex that absorbs
at 412 nm. The results were expressed as mg/dl plasma.
Vitamin E was estimated in serum by the method of Baker
and Frank (20), which is based on the reduction of ferric to
ferrous ions by tocopherol, which then forms a red colored
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complex with 2,2′,bipyridyl that is read at 520 nm. The level
of vitamin E was expressed as mg/l serum.
Vitamin C in plasma was analyzed by the method of Jacob
(21). This method is based on the oxidation of ascorbic acid to
dehydroascorbic acid, which when heated with dinitrophenyl-
hydrazine forms a colored complex with absorption maxima at
520 nm. The results were expressed as mg/dl plasma.
Serum nitric oxide was measured in terms of its products,
nitrite and nitrate, by the method of Griess modified by Fiddler
(22). This method is based on a two-step process. The first step
is the conversion of nitrate to nitrite using tin metal powder and
the second is the addition of sulphanilamide and N (-naphthyl)
ethylenediamine (Griess reagent). This converts nitrite into a
deep purple azo compound, which was measured colorimetri-
cally at 540 nm. Nitric oxide products were expressed as
µmoles/dl serum.
Protein in RBC hemolysate was estimated by the method of
Biuret (23). This method is based on the reaction of Biuret rea-
gent with peptide bonds of proteins. The violet color produced
was measured at 540 nm.
STATISTICAL ANALYSIS
The findings were expressed as the mean ± standard deviation.
The data were analyzed with Student’s independent t test. All
statistical analyses were performed with the program Statisti-
cal Package for the Social Science (SPSS for Windows, Ver-
sion 10.0). A P value of <0.05 was accepted as statistically
significant.
RESULTS
Our findings on the assessed parameters in oral cavity cancer
patients and healthy control subjects are shown in Tables 2, 3,
4 and 5.
Table 2 shows the levels of plasma LHP and MDA in control
subjects and oral cavity cancer patients. The extent of lipid
peroxidation as evidenced by plasma LHP and MDA was
382 Oxidative stress in oral cancer

Table 4. Levels (mg/dl) of GSH and vitamin C in plasma and (mg/l) of

vitamin E in serum of oral cancer patients and control subjects (mean ± SD)

GSH Vitamin C Vitamin E

Control group (n = 15) 10.02 ± 0.55 1.80 ± 0.32 7.71 ± 0.81
Patient group (n = 15) 3.09 ± 0.53* 0.51 ± 0.09* 1.40 ± 0.32*
P value 0.0000 0.0000 0.0000

*P < 0.001, when compared with control group

Table 5. Levels (µmol/dl) of NO2–, NO3– and TNO• in serum of oral cancer
patients and control subjects (mean ± SD)

NO2– NO3– TNO•

Control group (n = 15) 0.25 ± 0.04 0.27 ± 0.09 0.52 ± 0.13
Patient group (n = 15) 0.50 ± 0.12* 0.47 ± 0.11* 0.97 ± 0.23* Figure 2. Photomicrograph of the oral mucosa from oral cavity cancer patient
shows well-marked inflammatory regions, characterized by accumulation of

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P value 0.0000001 0.000007 0.000007 lymphocytes.

*P < 0.001, when compared with control group

significantly increased in the oral cavity cancer patients
(P < 0.001), compared to control subjects.
The enzymatic antioxidants profile in the circulation of oral
cavity cancer patients and control subjects is presented in Table
3. A decrease in the activities of SOD, GPx and CAT in the
erythrocyte lysate was seen in the oral cavity cancer patients as
compared to control subjects. The difference approached a
statistical significance of P < 0.001.
Table 4 presents the non-enzymatic antioxidants in the con-
trol subjects and oral cavity cancer patients. The levels of GSH
and vitamin C in plasma and vitamin E in serum were signifi-
cantly lower in the oral cavity cancer patients (P < 0.001), when
compared to the control subjects.
Table 5 shows the levels of NO• in terms of NO2–, NO3– and
TNO• in the control subjects and oral cavity cancer patients.
The levels were found to be significantly elevated in the oral
cancer patients group (P < 0.001), as compared to the control
subjects.
Histopathology reports are shown in Figs 1, 2 and 3. Figure
1 shows oral mucosa from a control subject, which reveals
stratified squamous epithelium with normal cellular architec-
ture. Histopathological studies of the oral mucosa excised from
the oral cavity cancer patients show the presence of well-
marked inflammatory regions, characterized by the accumula-
tion of lymphocytes (Fig. 2) and several areas of necrosis (Fig.
3) in the squamous epithelium.
DISCUSSION
Cancer is essentially an event occurring at the gene level and
the ultimate step resulting in carcinogenesis is DNA damage.
Multiple factors such as viruses, chemicals, irradiation and the

Figure 1. Photomicrograph of the oral mucosa from control shows stratified Figure 3. Photomicrograph of the oral mucosa from oral cavity cancer patient
squamous epithelium with normal cellular architecture. shows several areas of necrosis in the squamous epithelium.

genetic makeup of the individual play a role in carcinogenesis.
ROS and RNS are two important agents of DNA damage.
Damage to the DNA by ROS/RNS is believed to occur natu-
rally as well, since low steady-state levels of base damage
products have been detected in human nuclear DNA. The
magnitude of this damage (called oxidative stress or oxidative
damage) depends not only on ROS/RNS levels but also on the
body’s defense mechanisms against them mediated by various
cellular antioxidants. Disruption of this delicate oxidant/anti-
oxidant balance in the body seems to play a causative role in
carcinogenesis.
There are increasing evidences to support the role of oxida-
tive stress in several human pathological conditions such as
rheumatoid arthritis, ischemic heart disease, several auto-
immune disorders and cancer.
ROS/RNS are found to be involved in both initiation and pro-
motion of multistep carcinogenesis. They can cause DNA
damage, activate procarcinogens, initiate lipid peroxidation,
inactivate enzyme systems and alter the cellular antioxidant
defense system (24).
High levels of oxidative stress result in peroxidation of mem-
brane lipids with the generation of peroxides that can decom-
pose to multiple mutagenic carbonyl products. LHP and MDA
are well-characterized lipid peroxidation end products. They
are considered to be mutagenic and carcinogenic (25). They
can also modulate the expression of genes related to tumor pro-
motion (26). The level of LHP and MDA reflect the extent of
lipid peroxidation.
In our study, we observed increased systemic levels of LHP
and MDA in patients with oral cavity cancer, which could be
attributed to increased formation or inadequate clearance of
free radicals by the cellular antioxidants. Elevated levels of
lipid peroxidation products support the hypothesis that the
cancer cells produce large amount of free radicals (2) and that
there exists a relationship between free radical activity and
malignancy (27). Observations similar to our findings have
been reported in studies on various human cancers (28–30).
Antioxidants have been shown to inhibit both initiation and
promotion in carcinogenesis and counteract cell immortaliza-
tion and transformation (31). Activities of different antioxi-
dants show different patterns during neoplastic transformation
and tumor cells exhibit abnormal activities of antioxidant
enzymes, when compared to their appropriate normal cell
counterparts (24).
Cellular antioxidant enzymes and free radical scavengers
protect a cell against toxic oxygen radicals. GSH, an important
non-protein thiol, in conjugation with glutathione transferase
(GST) and GPx, plays a significant role in protecting cells by
scavenging ROS (32). A significant depletion of plasma GSH
observed in our study reflects enhanced pro-oxidant milieu of
the cells and correlates with the increased lipid peroxides in the
circulation of oral cavity cancer patients.
Antioxidant enzymes such as SOD, CAT and GPx provide
the first line of cellular defense against toxic free radicals.
These enzymes react directly with oxygen free radicals to yield
non-radical products. Selenium dependent GPx removes both
Jpn J Clin Oncol 2004;34(7) 383
H2O2 and LHP using GSH. This prevents H2O2 mediated intra-
cellular DNA damage, which is thought to be a prerequisite for
carcinogenesis (33). Oxidative damage to the cell membrane
has been reported to inactivate GPx (34). SOD metabolizes
free radicals and dismutates superoxide anions (O2•) to H2O2
and protects the cells against O2• mediated lipid peroxidation.
CAT acts on H2O2 by decomposing it, thereby neutralizing its
toxicity. It has been reported that superoxide radicals inhibit
catalase activity and H2O2 suppresses SOD activity in the cell
(35). Gupta et al. (36) demonstrated that reduction in several
antioxidant defense mechanisms correlates with the emergence
of the malignant phenotype. The low activities of these antioxi-
dant enzymes observed in our study might be due to the deple-
tion 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. Reports on CAT activity in cancer are
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contradictory. Both increase (37) and decrease (28,29,38) in
CAT activity have been reported previously. Reduction in CAT
activity as observed in this study might be due to increased
endogenous production of the superoxide anion, as evidenced
by increased LHP and MDA, or increased nitric oxide end
products, or decreased activity of GPx and SOD or all of these
factors. Furthermore, it might also be due to a higher magni-
tude of oxidative stress, since all our patients were in advanced
clinical stages (stage III/IV) with a large tumor burden.
A strong synergism exists between GSH, vitamin E and
vitamin C. Vitamin E is an important antioxidant in the
lipid domain. It is present in both erythrocyte membrane
and plasma. It is readily exchanged between erythrocytes and
plasma, with a balance in favor of plasma. Vitamin C is an
important extracellular antioxidant that disappears faster than
other antioxidants when plasma is exposed to oxygen free
radicals. Vitamin C spares GSH and together with vitamin
E prevents the oxidation of GSH. Since regeneration of both
vitamin E and vitamin C requires GSH, diminished level of
plasma GSH in oral cavity cancer patients might be responsible
for the low levels of these antioxidants.
The role of nitric oxide (NO•) is multidimensional. It func-
tions as an intracellular messenger and is also implicated as a
deleterious agent in various pathophysiological conditions
including cancer, inflammatory conditions and autoimmune
diseases. Chronic inflammation can lead to the production of
NO•, which in turn has the potential to mediate DNA damage
directly, or indirectly through the generation of more persistent
RNS (12).
Serum levels of nitrite (NO2–) and nitrate (NO3–) are used to
estimate the level of NO• formation, since NO• is highly unsta-
ble and has a very short half-life. Evidence of the role of NO•
in carcinogenesis is provided by the fact that both cNOS and
iNOS are detected in various human cancers (39,40). Biopsy
samples of human breast cancer show the presence of greater
expression of iNOS in a high-grade tumor, which tends to be
more invasive (9). Our study is the first to undertake the evalu-
ation of serum nitric oxide levels in human oral cavity cancer.
We observed significantly higher NO• end products in the
384 Oxidative stress in oral cancer
serum of oral cavity cancer patients. This could result from a
generalized increased NO• synthesis throughout the body of
the cancer patient or reflect increased NO• degradation pro-
moted by oxidative stress. Observations similar to ours have
been reported previously in gastric, colorectal, hepatocellular
and breast cancer (41–44).
Histopathological examinations of the oral mucosa revealed
necrosis and inflammatory changes. The oxidative stress might
have resulted in the abnormal changes evidenced in the histo-
logical examinations. Lipid peroxidation and oxidative stress
have been reported to be among the causes of tissue damage
and inflammation (30).
From this study, it could be concluded that oxidative stress is
increased (as evidenced by elevated levels of lipid peroxidation
products and nitric oxide products) and antioxidant defenses
are compromised (as evidenced by depletion of enzymatic and
non-enzymatic antioxidants) in patients with oral cavity can-
cer. 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. Studies have shown that a
diet rich in antioxidants may alleviate oxidative stress to some
extent (45). However, the degree of effectiveness with which
the antioxidant system can be restored with dietary modifica-
tions and nutritional supplements, so that cancer patients can
actually be benefited remains to be elucidated. Since the role of
NO• in tumorigenesis is multidimensional, an elaborate study
with a larger sample size is needed to ascertain the actual role
of NO• and RNS in the initiation and promotion of carcino-
genesis.
Acknowledgment
The authors wish to thank the Pathology Department of Ragas
Dental College and Hospital for evaluation of the histopathol-
ogy slides.
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