Support Care Cancer (2007) 15:1399–1405
DOI 10.1007/s00520-007-0266-3
ORIGINAL ARTICLE
Oxidative stress in lymphocytes, neutrophils, and serum
of oral cavity cancer patients: modulatory array
of L-glutamine
Subhasis Das & Santanu Kar Mahapatra & N. Gautam &
Amrita Das & Somenath Roy
Received: 22 January 2007 / Accepted: 2 May 2007 / Published online: 26 June 2007
# Springer-Verlag 2007
Abstract
Objectives Our aim was to assess the oxidative stress and
ameliorative effect of L-glutamine in serum, neutrophils,
and lymphocytes of oral cancer patients by measuring the
levels of malondialdehyde (MDA) and antioxidants.
Materials and methods This study has been conducted on
serum and specific blood cells in adult, male oral cancer
patients (stage III-6, stage IV-42) and normal subjects of an
equal number of age and sex-matched disease-free healthy
subjects. The levels of lipid peroxidation and antioxidant
enzymes were assayed using spectrophotometric methods.
Results MDA levels were elevated, and antioxidant enzyme
status was decreased significantly in all groups of cancer
patients simultaneously, but after supplementation of
“glutammune” (66.66% L-glutamine), oxidative stress has
been alleviated to some extent; especially, it has repaired
the glutathione cascade system.
Conclusion We conclude that oxidative stress is due to the
enhanced lipid peroxidation and decrease in antioxidant
enzymes, and it can be restored with dietary supplementa-
tion of L-glutamine related drug.
Keywords Oral cavity cancer . Lipid peroxidaton .
Antioxidant enzymes . Oxidative equilibrium . L-Glutamine
S. Das : S. Kar Mahapatra : N. Gautam : A. Das : S. Roy (*)
Immunology and Microbiology Laboratory,
Department of Human Physiology with Community Health,
Vidyasagar University,
Midnapore 721 102 West Bengal, India
e-mail: somenathroy@hotmail.com
Introduction
Oral cancer is the sixth most frequent cancer in the world;
some of the highest rates are in developing countries where up
to 25% of all malignancies are found in the oral cavity [37].
Tobacco is the predominant cause of this disease [18, 33, 48].
Alcohol use is a risk factor that acts synergistically with
tobacco. In India, the use of chewing tobacco and betel quid,
usually mixed with other toxins such as slaked lime, is a
common custom that causes oral cancer, which is responsible
for 50–90% of cases worldwide [12, 39, 41]. Smoking is the
main etiology of oral cancer and generates oxygen free
radicals in the oral cavity. Free radicals have been implicated
in apoptosis and in DNA damage, inducing alteration of the
cell cycle [35]. Alkaline saliva generated by chewing betel
quid plays an important role in cigarette-related nicotine-
induced DNA damage, and reactive oxygen species (ROS)
may be involved in generating this DNA damage [49].
ROS can cause DNA base alterations, strand breaks,
damage to tumor suppressor genes, and enhanced expres-
sion of proto-oncogenes [8]. ROS-induced mutation could
also arise from protein damage and attack on lipids, which
then initiate lipid peroxidation. It should be noted, however,
that the development of human 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 [6].
The burst of ROS has been implicated in the development
of oral cavity cancer in tobacco chewers and smokers [45].
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 [43].
1400 Support Care Cancer (2007) 15:1399–1405

The successful control of oral cavity cancer will depend on Materials and methods
its prevention. Considerable evidence exists, suggesting that
the role of enzymatic and non-enzymatic antioxidant defense Selection of human subjects
systems protect cell against ROS produced during normal
metabolism and after an oxidative insult. Antioxidant defense Control group (group I) of volunteers were only those who
systems work cooperatively to alleviate the oxidative stress were almost in the same age group (40–70 years), unlike the
caused by enhanced free radical production. Any changes in experimental groups, and proved to be in normal state of health
one of these systems may break this equilibrium and cause and free from any signs of chronic disease by the careful
cellular damages and, ultimately, malignant transformation clinical examinations with similar socioeconomic back-
[13, 47]. It has been reported that ROS production is grounds. They were non-smokers and not consuming alcohol.
pathologically high in advance-stage cancer patients [27, 28]. Control subjects were not receiving any drug on a long-term
Glutathione peroxidase (GPx), one of the major antioxidant basis.
enzymes in glutathione antioxidant defense system, showed The subjects (age group 40–70 years) were suffering from
significantly lower value in cancer patients as compared to oral cavity cancer, divided into three groups, ie., group II (just
control [27, 28, 38]. Moreover, these anti-oxidants, both 1 day after operation), group III (14 days after operation), and
natural and synthetic, neutralize metabolic products (includ- group IV (supplemented group, supplement was given for
ing reactive oxygen species), interfere with activation of 14 days just after the operation). After experimental period,
procarcinogens, prevent binding of carcinogens to DNA, heparinized blood and serum were collected from Royd
inhibit chromosome aberrations, restrain replication of the Nursing Home, 5B Royd Street, Kolkata, India and analyzed
transformed cell, suppress actions of cancer promoters, and in Immunology and Microbiology Laboratory, Vidyasagar
may even induce regression of precancerous oral lesions [12]. University, Midnapur, West Bengal, India. All the carcinomas
A number of nutrients zinc, epigallcatechin galatee, were graded as well-differentiated squamous cell carcinoma.
omega-3 polyunsaturated fatty acids, probiotics all act The ethical committee of the Vidyasagar University approved
differently to modulate the immune response, but all seem the study, and informed consent of all the subjects was
to have the potential to protect the host from the development obtained. None of the patients and control subjects had
of cancer and its progression [5]. Glutamine is a precursor for concomitant diseases such as diabetes mellitus, liver disease,
nucleotide synthesis, a substrate for liver gluconeogenesis and and rheumatoid arthritis. The clinical characteristics of oral
an important fuel source for cells (such as gastrointestinal cavity cancer patients were shown in Table 1.
epithelia, lymphocytes, fibroblasts, and reticulocytes) that
have rapid turnover [42]. Visceral organs, enterocytes, T Experimental design
lymphocytes, and macrophages utilize glutamine mobilized
from muscle and plasma as a source of fuel [8]. In critically Group I—control (normal healthy subjects)
ill patients, glutamine supplementation may reduce morbidity Group II—cancer patients (just 1 day after operation)
and mortality [32]. Studies in animal models and emerging
clinical trials suggest the benefits of enteral or parenteral
glutamine supplementation. Although some studies show no Table 1 Clinical characteristics of oral cavity cancer patients
apparent benefit, glutamine supplementation of parenteral
nutrition, enteral diets, or drinking water improves animal Characteristics Number of patients
survival, decreases infectious morbidity, and enhances gut Group II Group III Group IV
mucosal repair in models of chemotherapy and irradiation,
sepsis, and inflammation [51]. Glutamine-enriched nutrition Patients 16 16 16
attenuates depletion of the key antioxidant reduced glutathi- Male/female 14/2 15/1 15/1
one (GSH) in plasma, liver, and gut after chemotherapy, Age (years): 55.3±7.2 55.7±9.1 58.2±10.4
mean±SEM
sepsis, and gut ischemia/reperfusion and upregulates systemic
Stage
and tissue immune function in animals [9, 44, 51, 52]. III 2 3 1
Considering the fact that glutamine is vital for the rapidly IV 14 13 15
dividing cells like lymphocytes, it is not surprising that its Tumor location
depletion contributes to the development of immunosuppres- Buccal mucosa 9 10 12
sion ill patients [25]. Anterior two 4 5 3
Our aim of this study was to assess the degree of thirds of the tongue
oxidative stress and alleviative role of “glutammune” Retromolar area 3 1 1
Body weight: mean±SEM 65.89± 63.28± 68.27±
(66.66% L-glutamine) in serum and immunocompetent
11.49 8.7 11.92
cells of oral cavity cancer patients.
Support Care Cancer (2007) 15:1399–1405
Group III—cancer patients (14 days after operation)
Group IV—cancer patients (supplemented after opera-
tion for 14 days): The dose of “glutammune” per day per
patient was 30 g orally, which contains 20 g L-glutamine.
The dose and duration was selected as per the previous
report by many researchers [1, 2, 10, 14]
Chemicals and reagents
All fine chemicals were obtained from Sigma Chemical,
USA. Other chemicals used were analytical grade and
obtained locally. L-Glutamine was obtained as a brand name
of “glutammune” from Claris Lifesciences Limited, Corpo-
rate Towers, Ellisbridge, Ahmedabad #380 006, India.
Collection of blood samples and separation of serum,
neutrophil, and lymphocytes
Fasting blood samples were collected from all groups of
individuals. Serum was obtained by centrifugation at 1500×g
for 15 min of blood samples taken without anticoagulant.
Serum was kept at −86°C for further analysis.
Heparinized blood samples were used for the separation
of neutrophils and lymphocytes. Neutrophils and lympho-
cytes were isolated from heparinized blood using standard
isolation techniques [21]. The pellets of neutrophils and
lymphocytes were lysed in a hypotonic solution (kept for
45 min at 37°C) and stored at −86°C until biochemical
estimations [40].
Biochemical assays
Malondialdehyde level was measured according to the
method of Ohkawa et al. [36]. Reduced glutathione content
was measured as described by modified procedure of
Grifith [15]. Glutathione disulfide content was estimated
according to the method described by Grifith [15]. The
catalase activity was measured according to the methodol-
ogy of Beers and Sizer [4]. The activity of superoxide
dismutase was measured according to the method of
Marklund and Marklund [29]. The activity of glutathione-s-
Table 2 Lipid peroxidation and antioxidant enzyme status of lymphocytes
Groups MDA GSH
(nmol/mg protein) (μg/mg protein)
Group I 0.83±0.035 28.17±1.68
Group II 1.48±0.081* 15.52±0.94*
Group III 1.99±0.035* 6.31±0.61*
Group IV 1.31±0.069** 17.95±0.88**
Values are expressed as mean±SEM for n=16.
*P≤0.05 compared to control (group I)
**P≤0.05 compared to patients 14 days after surgery (group III)
1401
transferase was assayed as described by Habig et al. [17].
Total protein content was estimated by the method of Lowry
et al. [24].
Statistical analysis
The data were expressed as mean±standard error. Compar-
isons of the means of group I, group II, group III, and group
IV were made by model I analysis of variance test with
(using a statistical package, Origin 6.1, Northamption, MA
01060, USA) multiple comparison t test, P<0.05 as a limit
of significance.
Results
The findings of the study are presented in Table 2 of control
(normal healthy subjects), different groups of cancer
patients (groups II and III), and cancer patients with
supplementation of “glutammune” (L-glutamine).
Result shows that the extent of lipid peroxidation, as
evidenced by lymphocytes, neutrophils, and serum malon-
dialdehyde (MDA), was significantly increased in the both
group II and group III oral cancer patients (P<0.05)
compared to control subjects, whereas supplemented group
revealed the significantly decreased MDA level (P<0.05)
as compared to group III patients, but it did not show the
reversible effect.
The enzymatic antioxidants profile in the circulation of
oral cancer patients and control subjects was measured. The
levels of GSH and oxidized glutathione (GSSG) in
lymphocytes, neutrophils, and serum were significantly
lower and simultaneously decreased in both group II and
group III oral cancer patients (P<0.05) when compared to
the control subjects. On the other hand, supplemented
group shows significant (P<0.05) increase in both GSH
and GSSG levels. More interestingly, glutamine increases
the GSH levels rather than GSSG. A significant decrease
(P<0.05) in the activities of superoxide dismutase (SOD),
glutathione-S-transferase (GST), and catalase (CAT) in the
lymphocytes, neutrophils, and serum were seen in the two
GSSG CAT (nmol min−1 SOD (U min−1 GST (nmol min−1
(μg/mg protein) mg−1 protein) mg−1 protein) mg−1 protein)
36.96±1.64 28.16±0.21 46.51±0.31 365.26±13.35
28.27±0.82* 17.22±0.15* 32.89±0.33* 268.74±6.37*
22.79±1.58* 14.59±0.18* 29.51±0.29* 177.87±9.29*
25.98±1.45** 15.76±0.22 31.55±0.24 292.19±10.31**
1402
groups of oral cancer patients as compared to control
subjects. Maximum significant decrement has been seen in
the case of catalase activity. “Glutammune”-supplemented
group shows maximum effect on GST activity, which is
near to control level, but SOD and CAT did not response
significantly on the “glutammune”-supplemented lympho-
cytes, neutrophils, and serum.
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, irradiations, and
the genetic makeup of the individual play a role in
carcinogenesis. ROS are found to be involved in both
initiation and promotion of multi-step carcinogenesis. They
can cause DNA damage, activate pro-carcinogenesis, initiate
lipid peroxidation, inactivate enzyme systems, and alter the
cellular antioxidants defense system [46]. High levels of
oxidative stress result in peroxidation of membrane lipids,
with the generation of peroxides that can decompose to
multiple mutagenic carbonyl products. They are considered to
be mutagenic and carcinogenic [50]. They can also modulate
the expression of genes related to tumor promotion [7].
In our study (Tables 2 and 3), we observed significantly
simultaneous increase in MDA levels in serum, neutrophil,
and lymphocytes of both the two groups (II and III) as
compared to control. On the other hand, supplemented
group showed significantly decreased level of MDA in
comparison to group III, except neutrophil, but it did not
show the reversible effect. Elevated levels of lipid perox-
idation product support the hypothesis that the cancer cells
produce large amount of free radicals [8] and that there
exist a relationship between free radical activity and
malignancy [11]. So, specifically oxidative stress provides
evidence of the relationship between lipid peroxidation and
oral cavity cancer. On the contrary, supplemented group
showed reduced levels of MDA, which may be due to few
increased antioxidant activity in the system out of the four
antioxidants (GSH, CAT, SOD, and GST).
Table 3 Lipid peroxidation and antioxidant enzyme status of neutrophils
Groups MDA GSH GSSG
(nmol/mg protein) μg/mg protein) (μg/mg protein)
Group I 1.4±0.021 25.83±1.09 31.55±1.31
Group II 2.14±0.063* 14.12±0.64* 24.36±0.54*
Group III 2.43±0.061* 5.16±0.33* 16.59±0.83*
Group IV 2.25±0.057** 16.91±0.71** 20.66±0.98**
Values are expressed as mean±SEM for n=16.
*P≤0.05 compared to control (group I)
**P≤0.05 compared to patients 14 days after surgery (group III)
Support Care Cancer (2007) 15:1399–1405
Antioxidants have been shown to inhibit both initiation
and promotion in carcinogenesis and counteract cell
immunization and transformation [34]. Cellular antioxidant
enzymes and free radical scavengers protect a cell against
toxic oxygen radicals. GSH, an important non-protein thiol,
in conjugation with GST and glutathione peroxidase (GPx),
plays a significant role in protecting cells by scavenging
ROS [31]. A wide variety of oxidizing molecules such as
ROS and/or depleting agents can alter the glutathione redox
state, a key compound in the regulation of body redox
homeostasis. The glutathione redox state is normally
maintained by the activity of GSH-depleting (GPx) and
-replenishing enzymes (GR) [3, 22]. A significant depletion
of lymphocytes, neutrophils, and plasma GSH observed in
our study reflects enhanced pro-oxidant milieu of the
system and correlates with the increased lipid peroxides in
the circulation of oral cavity cancer patients, whereas
supplemented group showed significantly increased level
of GSH, GSSG, and activity of GST when compared to
groups II and III of cancer patients. This increased level of
enzymes in glutathione system, especially GSH, may help
to minimize the MDA levels in the supplemented group.
Antioxidant enzymes such as SOD and CAT provide
the first line of cellular defense against toxic-free radicals.
These enzymes react directly with oxygen-free radicals to
yield non-radical products. These enzymes prevent H2O2-
mediated intracellular DNA damage, which is thought to
be a prerequisite for carcinogenesis [30]. 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 [19]. Gupta et al. [16]
demonstrated that reduction in several antioxidant defense
mechanisms correlates with the emergence of the malig-
nant phenotype. The low activities of these antioxidant
enzymes observed in our study during the group II and
group III of cancer patients might be due to the depletion
of the antioxidant defense system. This could occur as a
consequence of overwhelming free radicals, as evidenced
CAT (nmol min−1 SOD (U min−1 mg−1 GST (nmol min−1
mg−1 protein) protein) mg−1 protein)
22.16±0.29 38.92±0.36 311.99±11.38
13.62±0.18* 27.29±0.35* 225.57±10.09*
10.09±0.12* 23.51±0.39* 150.9±9.27*
12.76±0.12 24.97±0.29 248.18±11.36**
Support Care Cancer (2007) 15:1399–1405 1403

Table 4 Lipid peroxidation and antioxidant enzyme status of serum

Groups MDA GSH GSSG CAT (mmol min−1 SOD (U min−1 GST (μmol min−1
(nmol/mg protein) (μg/mg protein) (μg/mg protein) mg−1 protein) mg−1 protein) mg−1 protein)

Group I 9.49±0.939 0.63±0.047 0.23±0.077 0.55±0.054 1.22±0.065 571.95±24.28

Group II 21.31±1.987* 0.51±0.054* 0.88±0.076* 0.24±0.036* 0.82±0.034* 420.38±21.029*
Group III 45.46±4.167* 0.26±0.047* 0.57±0.047* 0.19±0.018* 0.57±0.049* 318.70±13.459*
Group IV 23.60±1.886** 0.64±0.054** 0.96±0.119** 0.29±0.032 0.76±0.044 477.99±19.95**

Values are expressed as mean±SEM for n=16.

*P≤0.05 compared to control (group I)
**P≤0.05 compared to patients 14 days after surgery (group III)

by the elevated levels of lipid peroxides in the circulation of
oral cavity cancer patients. Reports on CAT activity in cancer
are contradictory. Both increase [20] and decrease [23, 26,
38] in CAT activity have been reported previously. Reduction
in CAT and SOD activity as observed in this study might be due
to increased endogenous production of the superoxide anion, as
evidenced by increased MDA. But in the supplemented group,
it has been revealed that significantly increased CAT and SOD
activity minimizes the MDA level near to the group II subjects,
but not to the normal level (Table 4).
From this study, it could be concluded that oxidative
stress is increased (as evidenced by elevated levels of
lipid peroxidation products), and antioxidant defenses are
depleted (as evidenced by depletion of enzymatic anti-
oxidants) in patients with oral cavity cancer. A weak
antioxidant defense system makes the macromolecules of
immunocompetent cells and serum more vulnerable to the
genotoxic effect of ROS. This creates an intracellular
environment more favorable for DNA damage and
disease progression. “Glutammune”, a rich source of L-
glutamine (66.66%), ameliorate the oxidative stress in
some extent (Fig. 1). However, the degree of effectiveness
with which the redox balance can be restored with dietary
supplements including “glutammune” and “glutammune”-
related drugs so that cancer patients can be benefited.
Finally, more research can be continued in this field to
know the actual mechanism and development of a suitable
drug. Studies on these lines are presently in progress.

Fig. 1 Oxidative stress during

the oral cavity cancer and the Cigarette smoke,
Tobacco products DECREASE THE CANCER
role of L-glutamine to reduce the
potential cancer progression and other factors PROGRASSION

Improved immune
Initiation of lipid peroxidation
functions
and weak antioxidant enzyme

Decreased genotoxic effect of ROS

OXIDATIVE STRESS leads to carcinogenic deactivation

Boost up the antioxidant

Genotoxic effect of ROS leads to defense system especially
carcinogenic activation glutathione system

L-glutamine
supplementation

ORAL CAVITY CANCER

1404
Acknowledgment The authors are very much grateful to Dr. Sk.
Saidul Islam, maxillofacial onco and microvascular surgeon, Royd
Nursing Home, Kolkata for kind cooperation and Claris Lifesciences
Limited, Corporate Towers, Ellisbridge, Ahmedabad, 380 006, India
for providing the “glutammune” free of cost.
References
1. Akobeng AK, Miller V, Stanton J et al (2000) Double-blind
randomized controlled trial of glutamine-enriched polymeric diet
in the treatment of active Crohn’s disease. J Pediatr Gastroenterol
Nutr 30:78–84
2. Akobeng AK, Miller V, Thomas AG, Richmond K (2000)
Glutamine supplementation and intestinal permeability in Crohn’s
disease. J Parenter Enteral Nutr 24:196
3. Ames BN, Shigenega MK, Hagen TM (1993) Oxidant, antioxi-
dant and degenerative diseases of ageing. Proc Natl Acad Sci
USA 90:7915–7922
4. Beers RF, Sizer IW (1952) A spectrophotometric method for
measuring the breakdown of hydrogen peroxide by catalase. J Biol
Chem 195:133–140
5. Bingisser RM, Tilbrook PA, Holt PG, Kees UR (1998) Macro-
phage-derived nitric oxide regulates T-cell activation via revers-
ible disruption of the Jak3/STAT5 signaling pathway. J Immunol
160:5729–5734
6. Burdon RH (1995) Superoxide and hydrogen peroxide in relation
to mammalian cell proliferation. Free Radic Biol Med 18:775–794
7. Cerutti PA (1985) Pro-oxidant status and tumor promotion.
Science 227:375–381
8. Cerutti PA (1994) Oxy-radicals and cancer. Lancet 344:862–863
9. Darmaun D (2000) Role of glutamine depletion in severe illness.
Diabetes Nutr Metab 13:25–30
10. Den Hond E, Hiele M, Peeters M, Ghoos Y, Rutgeerts P (1999)
Effect of long-term oral glutamine supplements on small intestinal
permeability in patients with Crohn’s disease. J Parenter Enteral
Nutr 23:7–11
11. Dormandy TI (1983) An approach to free radicals. Lancet 1:1010–
1014
12. Enwonwu CO, Meeks VI (1995) Bionutrition and oral cancer in
humans. Crit Rev Oral Biol Med 6(1):5–17
13. Garewal H (1995) Antioxidants in oral cancer. Am J Clin Nutr 62
(Suppl):1410S–1416S
14. Gayle K, Savy RD (1997) Enteral glutamine supplementation:
clinical review and practical guidelines. Nutr Clin Prac 12
(6):259–262
15. Grifith OW (1980) Determination of glutathione and glutathione
sulfide using glutathione reductase and 2-vinyl pyridine. Anal
Biochem 106:207
16. Gupta A, Batts B, Kwei KA, Dvorakova K, Stratton SP, Briehl
MM et al (2001) Attenuation of catalase activity in the malignant
phenotype plays a functional role in an in vitro model for tumor
progression. Cancer Lett 73:115–125
17. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-
transferases: the first enzymatic step in mercapyuric acid
formation. J Biol Chem 249:7130–7139
18. Hasnis E, Reznick AZ, Pollack S, Klein Y, Nagler RM (2004)
Synergistic effect of cigarette smoke and saliva on lymphocytes—
the mediatory role of volatile aldehydes and redox active iron and
the possible implications for oral cancer. Int J Biochem Cell Biol
36(5):826–839
19. Hassan HM, Fridovich I (1978) Superoxide radical and the
oxygen enhancement of the toxicity of paraquat in E. coli. J Biol
Chem 253:8143–8148
Support Care Cancer (2007) 15:1399–1405
20. Hristozov D, Gadjeva V, Vlaykova T, Dimitrov G (2001)
Evaluation of oxidative stress in patients with cancer. Arch
Physiol Biochem 109:331–336
21. Hudson L, Hay FC (1991) Practical immunology. Blackwell
Scientific Publications, Oxford University Press, pp 21–22
22. Kong Q, Lillehei KO (1998) Antioxidants inhibitors for cancer
therapy. Med Hypotheses 51:405–409
23. Kumaraguruparan R, Subapriya R, Kabalimoorthy J, Nagini S
(2002) Antioxidant profile in the circulation of patients with
fibroadenoma and adenocarcinoma of the breast. Clin Biochem
35:275–279
24. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein
measurement with the Folin phenol reagent. J Biol Chem
193:255–275
25. Malmberg KJ, Lenkei R, Petersson M et al (2002) A short-term
dietary supplementation of high doses of vitamin E increases T
helper 1 cytokine production in patients with advanced colorectal
cancer. Clin Cancer Res 8:772–778
26. Manju V, Sailaja JK, Nalini N (2002) Circulating lipid perox-
idation and antioxidant status in cervical cancer patients: a case-
control study. Clin Biochem 35:621–625
27. Mantovani G, Maccio A, Madeddu C, Mura L, Gramignano G,
Lusso MR, Mulas C, Mudu MC, Murgia V, Camboni P, Massa E,
Ferreli L, Contu P, Rinaldi A, Sanjust E, Atzei D, Elsener B
(2002) Quantitative evaluation of oxidative stress, chronic
inflammatory indexes and leptin in cancer patients: correlation
with stage and performance status. Int J Cancer 98:84–91
28. Mantovani G, Maccio A, Madeddu C, Mura L, Massa E,
Gramignano G, Lusso MR, Murgia V, Camboni P, Ferreli L
(2002) Reactive oxygen species, antioxidant mechanisms and
serum cytokine levels in cancer patients: impact of an antioxidant
treatment. J Cell Mol Med 6(4):570–582
29. Marklund S, Marklund G (1974) Involvement of superoxide anion
radical in autoxidation of pyrogallol and a convenient assay of
superoxide dismutase. Eur J Biochem 47:469–474
30. Mates JM, Perez-Gomez C, Nunez de Castro I (1999) Antioxidant
enzymes and human disease. Clin Biochem 32:595–603
31. Meister A (1988) Glutathione metabolism and its selective
modification. J Biochem 263:17205–17208
32. Melis GC, Wengel N, Boelens PG, Leeuwen P (2004) Glutamine:
recent developments in research on the clinical significance of
glutamine. Curr Opin Clin Nutr Metab Care 7(1):59–70
33. Muir CS, Kirk R (1990) Betel, tobacco and cancer of the mouth.
Br J Cancer 14:597–608
34. Oberley LW, Oberley TD (1988) Role of antioxidant enzymes in
cell immortalization and transformation. Mol Cell Biochem
84:147–153
35. Oda D, Nguyen MP, Royack GA, Tong DC (2001) H2O2 oxidative
damage in cultured oral epithelial cells: the effect of short-term
vitamin C exposure. Anticancer Res 21(4A):2719–2724
36. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in
animal tissues by thiobarbituric acid reaction. Anal Biochem
95:351–358
37. Parkin SM, Laara E, Muir CS (1988) Estimates of the worldwide
frequency of sixteen major cancers. Int J Cancer 41:184–197
38. Ray G, Batra S, Shukla NK, Deo S, Raina V, Ashok S, Husain
SKI (2000) Lipid peroxidation, free radical production and
antioxidant status in breast cancer. Breast Cancer Res Treat
59:163–170
39. Reznick AZ, Klein I, Eiserich JP, Cross CE, Nagler RM (2003)
Inhibition of oral peroxidase activity by cigarette smoke: in vivo
and in vitro studies. Free Radic Biol Med 34(3):377–384
40. Sandhu SKI, Kaur G (2002) Alterations in oxidative stress
scavenger system in aging rat brain and lymphocytes. Bioger-
ontology 3:161–173
Support Care Cancer (2007) 15:1399–1405
41. Sankaranarayana R (1990) Oral cancer in India: an epidemio-
logic and clinical review. Oral Surg Oral Med Oral Pathol
69:325–330
42. Sieja K (1998) Selenium (Se) deficiency in women with ovarian
cancer undergoing chemotherapy and the influence of supplemen-
tation with this micro-element on biochemical parameters.
Pharmazie 53(7):473–476
43. Singh A, Singh SP (1997) Modulatory potential of smokeless
tobacco on the garlic, mace or black mustard-altered hepatic
detoxication system enzymes sulphydryl contents and lipid
peroxidation in murine system. Cancer Lett 118:109–114
44. Souba WW (1993) Intestinal glutamine metabolism and nutrition.
J Nutr Biochem 4:2–9
45. Stitch HF, Anders F (1989) The involvement of reactive oxygen
species in oral cancer of betel quid-tobacco chewers. Mutat Res
214:47–61
46. Sun Y (1990) Free radicals, antioxidant enzymes and carcinogen-
esis. Free Radic Biol Med 8:583–599
47. Syed Sultan Beevi S, Muzib Hassanal Rasheed A, Geetha A
(2004) Evaluation of oxidative stress and nitric oxide levels in
1405
patients with oral cavity cancer. Jpn J Clin Oncol 34(7):379–
385
48. US Department of Health and Human Services (1982) The health
consequences of smoking and cancer. A report of the Surgeon
General. US Government Printing Office, Washington, DC
[DDHS publication (P511) no. 5107]
49. Wu HJ, Chi CW, Liu TY (2005) Effect of nicotine-induced DNA
damage and oxidative stress. J Toxicol Environ Health 68(17–
18):1511–1523
50. Zhang Y, Chen SY, Hsu T, Santella RM (2002) Immunohisto-
chemical detection of malondialdehyde-DNA adducts in human
oral mucosa cells. Carcinogenesis 23:207–211
51. Ziegler TR, Bazargan, N, Leader LM, Martindale RG (2000)
Glutamine and the intestinal tract. Curr Opin Clin Nutr Metab
Care 3:355–362
52. Ziegler TR, Puckett AB, Griffiths DP, Galloway JR (1997)
Interactions between nutrients and growth factors in cellular
growth and tissue repair. In: Ziegler TR, Pierce GF, Herdon DN
(eds) Growth factors and wound healing: basic science and
potential clinical applications. Springer, New York, pp 104–150