Peripheral lymphocyte DNA damage and oxidative stress in patients with ulcerative colitis.

Abstract

Ulcerative colitis (UC) is a fairly common chronic inflammatory disorder. Chronic inflammation may contribute to the risk of colorectal cancer through the accumulation of specific products resulting from DNA damage. Previous studies reported that DNA damage and oxidative stress play a significant role in the pathophysiology of UC, but the results are inconsistent.
In the present study, we investigated peripheral DNA damage and oxidative stress in patients with UC.
The study included 20 patients with UC and 20 controls. Peripheral lymphocyte DNA damage was measured using the alkaline comet assay. Plasma total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI) were determined.
DNA damage levels, TOS, and OSI were significantly higher in patients with UC than in controls (P <0.001 for all parameters), while TAC was significantly lower (P <0.001). DNA damage was significantly correlated with TOS, TAC, and OSI (r = 0.604, P <0.001; r = -0.593, P <0.001; and r = 0.716, P <0.001, respectively). Moreover, TAC levels were significantly correlated with TOS and OSI (r = 0.604, P <0.001; r = -0.399, P <0.05; and r = -0.513, P <0.05, respectively).
Our results show that increased peripheral DNA damage and oxidative stress seem to be associated with decreased antioxidant levels and thus may in part contribute to the development of colorectal cancer associated with UC.

Full text

Page 1

ORIGINAL ARTICLE  Peripheral lymphocyte DNA damage and oxidative stress...1
IntroductIon Ulcerative colitis (UC) and
Crohn’s disease (CD) are the two major types of
inflammatory bowel disease (IBD). UC is an idio‑
pathic disease characterized by mucosal inflam‑
mation of the colon.1 The incidence of UC varies
depending on geography and is most common in
Western countries, including the United States.2
It is predominately a disease of late adolescence
and early adulthood, with the highest incidence in
the third decade of life.2 The pathogenesis of UC
remains uncertain. Numerous factors including
immuno logic abnormalities, genetic influences,
environmental agents, alterations in the colon‑
ic barrier function, bacterial and viral infections,
altered colonic microflora, as well as nutrition
and psychosocial factors account for the initia‑
tion and enhancement of chronic inflammatory
response in patients with UC.3,4
The mucosal lesion in IBD is characterized by
dense inflammatory cell infiltrate that contains
mainly neutrophils, macrophages, and lympho‑
cytes. Increased number of inflammatory cells
orIGInAL ArtIcLE
Peripheral lymphocyte DNA damage
and oxidative stress in patients
with ulcerative colitis
Mehmet Aslan1, Yaşar Nazligul2, Cengiz Bolukbas3, Fusun F. Bolukbas3, Mehmet Horoz4,
Ahmet C. Dulger5, Fatih M. Erdur1, Hakim Celik6, Abdurrahim Kocyigit6
1 Yuzuncu Yil University, Medical Faculty, Department of Internal Medicine, Van, Turkey
2 Kecioren Education and Research Hospital, Department of Gastroenterology, Ankara, Turkey
3 Harran University, Medical Faculty, Department of Gastroenterology, Sanliurfa, Turkey
4 Harran University, Medical Faculty, Department of Internal Medicine, Sanliurfa, Turkey
5 Yuzuncu Yil University, Medical Faculty, Department of Gastroenterology, Van, Turkey
6 Harran University, Medical Faculty, Department of Clinical Biochemistry, Sanliurfa, Turkey
Correspondence to:
Mehmet Aslan, MD, PhD, Yuzuncu
Yil University, Medical Faculty,
Department of Internal Medicine,
65 400, Van, Turkey,
phone: +90-432 -216 -7362,
fax: +90-432 -216 -7519,
e -mail: m.aslan301@mynet.com
Received: April 5, 2011.
Revision accepted: June 16, 2011.
Conflict of inter est: none declared.
Pol Arch Med Wewn. 2011;
121 (7-8): xx -xx
Copyright by Medycyna Praktyczna,
Kraków 2011
AbstrAct
IntroductIon Ulcerative colitis (UC) is a fairly common chronic inflammatory disorder. Chronic inflam‑
mation may contribute to the risk of colorectal cancer through the accumulation of specific products
resulting from DNA damage. Previous studies reported that DNA damage and oxidative stress play
a significant role in the pathophysiology of UC, but the results are inconsistent.
objEctIvEs In the present study, we investigated peripheral DNA damage and oxidative stress in
patients with UC.
PAtIEnts And mEthods The study included 20 patients with UC and 20 controls. Peripheral lymphocyte
DNA damage was measured using the alkaline comet assay. Plasma total antioxidant capacity (TAC),
total oxidant status (TOS), and oxidative stress index (OSI) were determined.
rEsuLts DNA damage levels, TOS, and OSI were significantly higher in patients with UC than in
controls (P <0.001 for all para meters), while TAC was significantly lower (P <0.001). DNA damage
was significantly correlated with TOS, TAC, and OSI (r = 0.604, P <0.001; r = –0.593, P <0.001;
and r = 0.716, P <0.001, respectively). Moreover, TAC levels were significantly correlated with TOS and OSI
(r = 0.604, P <0.001; r = –0.399, P <0.05; and r = –0.513, P <0.05, respectively).
concLusIons Our results show that increased peripheral DNA damage and oxidative stress seem to
be associated with decreased antioxidant levels and thus may in part contribute to the development of
colorectal cancer associated with UC.
KEy words
comet assay,
peripheral lymphocyte
DNA damage, total
antioxidant status,
total oxidant status,
ulcerative colitis

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POLSKIE ARCHIWUM MEDYCYNY WEWNĘTRZNEJ  011; 121 (7-8)
2
diet at the time of the study. Average follow ‑up
for patients with UC was 10.2 ±2.3 months.
Diagnosis of UC was established on the ba‑
sis of clinical, radio logic, endoscopic, and
histopatho logical findings. The disease activi‑
ty for UC patients was assessed as described by
Rachmilewitz.22 In addition, an increase in C ‑re‑
active protein (CRP) and erythrocyte sedimenta‑
tion rate (ESR), platelet and leukocyte counts, and
a decrease in hemo globin and serum albumin lev‑
els were considered as indicative of disease activi‑
ty.23 Patients with UC were allowed to use mesala‑
mine or sulfasalazine, but these medications were
stopped 24 hours before sigmoidoscopy.
Patients with UC were excluded if they had
a history of alcohol abuse, smoking habit, intra‑
venous drug abuse, pregnancy, micronutrient
supplements, fish ‑oil supplement in the previous
month, cryoglobulinemia, human immune defi‑
ciency virus infection, active infection, hyperten‑
sion, diabetes, hyperlipidemia, chronic respirato‑
ry insufficiency, rheumatoid arthritis, cirrhosis,
renal disease, coronary heart disease, cerebrovas‑
cular disease, or malignant tumor(s).
Control subjects were free of gastrointestinal
symptoms and had normal colonoscopy. Control
subjects were excluded if they had a history of
alcohol abuse, smoking habit, intravenous drug
abuse, pregnancy, micronutrient supplements,
fish ‑oil supplement in the previous month, cryo‑
globulinemia, human immune deficiency virus in‑
fection, active infection, hypertension, diabetes,
hyperlipidemia, chronic respiratory insufficien‑
cy, rheumatoid arthritis, cirrhosis, renal disease,
coronary heart disease, cerebrovascular disease,
or malignant tumor.
The study was conducted according to the Hel‑
sinki Declaration as revised in 1989 and was ap‑
proved by the local ethics committee. All subjects
provided their written informed consent to par‑
ticipate in the study.
measurement of the total antioxidant capacity Plas‑
ma total antioxidant capacity (TAC) levels were
determined using an auto mated method devel‑
oped by Erel.24 The results are expressed as mmol
Trolox equiv./l.
measurement of the total oxidant status Plasma to‑
tal oxidant status (TOS) levels were determined
using a novel auto mated method developed by
Erel.25 The results are expressed as µmol H2O2
equiv./l.
calculation of the oxidative stress index The oxi‑
dative stress index (OSI) was defined as the ratio
of the TOS to TAC levels.26 OSI (arbitrary unit)
= TOS (µmol H2O2 equiv./l)/TAC (mmol Trolox
equiv./l).
dnA damage determination by the alkaline comet
assay After an overnight fasting, 6 ml of pe‑
ripheral blood sample from each subject was with‑
drawn into a heparinized tube, kept on ice and
and tissue oxidative injury may result from the re‑
lease of proinflammatory cytokines in the mucosa.
Previous studies have shown that oxidative stress
causes an increase in the levels of proinflamma‑
tory cytokines.4,5 During chronic inflammation,
when sustained production of reactive oxygen
species (ROS) occurs, antioxidant defenses can
weaken, resulting in oxidative stress.5 Increased
production of reactive oxygen and nitrogen spe‑
cies has been observed after in vitro stimulation
of the whole colonic mucosa, mucosal macrophag‑
es, and peripheral blood monocytes of IBD pa‑
tients.6,7 Antioxidant enzymes are known to pre‑
vent the promotion of carcinogenesis. It has been
suggested that a defect in mucosal antioxidant de‑
fenses is an etio logical factor in IBD.8,9
Chronic inflammation has long been recognized
to be associated with increased risk of human can‑
cer at various sites, for example UC with the risk
of colorectal cancer.10,11 With long ‑standing
UC,12 dysplastic changes occur frequently and
a dysplasia ‑carcinoma sequence is generally ac‑
cepted.13 Oxidative stress can arise when the pro‑
duction of ROS exceeds the cellular antioxidant
capacity, resulting in damage to cellular macro‑
molecules such as DNA, proteins, and lipids.14
Oxidative stress has been shown to play a signif‑
icant role in inflammation ‑associated cancero‑
genesis via DNA damage.15 Also, several studies
have suggested that genomic damage plays a ma‑
jor role in the number of chronic diseases and pro‑
cesses, including various cancers, cardiovascular
and neurodegenerative diseases, inflammation/
infection, and aging.16,17 Oxidative stress result‑
ing in DNA damage is considered to be the most
common insult affecting the genome.18
It is generally thought that DNA damage in
lymphocytes can reflect the level of oxidative
stress in the body.19 The comet assay was original‑
ly developed to measure DNA strand breaks with
high sensitivity.19 Previous studies have shown
that DNA damage is significant in the pathophys‑
iology of UC,8,20 but the results are controversial.
Furthermore, there are no data concerning pe‑
ripheral DNA damage and oxidative stress in UC
patients. Therefore, we aimed to investigate ox‑
idative stress and DNA damage level in periph‑
eral blood lymphocytes using the comet assay in
this patient group.
PAtIEnts And mEthods The study involved
20 patients with UC (11 with active disease and
9 in remission) and 20 controls. Patients with
active form had a documented diagnosis of UC.
At the time of the study, these patients experi‑
enced symptoms of bloody diarrhea, urgency, and
cramps and demonstrated typical sigmoidoscopic
and histo logic features of active UC as described
previously.21 Patients with inactive UC had a doc‑
umented history of active UC but were currently
symptom ‑free. They had normal mucosa on sig‑
moidoscopy and histo logical features typical of
inactive UC. All subjects consumed their usual

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ORIGINAL ARTICLE  Peripheral lymphocyte DNA damage and oxidative stress...3
statistical analysis The results were expressed as
mean ± standard deviation. Qualitative variables
were assessed by the χ2 test. Parametric variables
were compared using the Student t test. Correla‑
tion analyses were performed using the Pearson’s
correlation test. P <0.05 was considered statisti‑
cally significant. The analysis was performed with
the SPSS 11.0 statistical software.
rEsuLts The demographic, clinical, and labo‑
ratory data of patients with UC and controls are
presented in tAbLE 1. There were no statistical‑
ly significant differences between patients with
UC and the control group with respect to age,
sex, and body mass index (P >0.05). In addition,
the average follow ‑up for patients with UC was
10.2 ±2.3 months.
ESR, CRP levels, albumin levels, as well as
platelet and leukocyte counts were higher in
patients with UC than in controls (P <0.05,
P <0.05, P <0.05, P <0.05, and P <0.01, respec‑
tively) (tAbLE 1).
Peripheral lymphocyte DNA damage, TOS lev‑
els, and OSI values were significantly higher in pa‑
tients with UC than in controls (P <0.001), while
TAC levels were significantly lower (P <0.001)
(tAbLE 2). When patients with UC were divided ac‑
cording to disease activity, no differences were ob‑
served between active UC and inactive UC with re‑
spect to peripheral lymphocyte DNA damage, TOS
levels, TAC levels, and OSI values (P >0.05).
Peripheral lymphocyte DNA damage was sig‑
nificantly correlated with TOS levels, TAC levels,
and OSI values (r = 0.604, P <0.001; r = –0.593,
P <0.001, r = 0.716, P <0.001; respectively). In
addition, TAC levels were significantly correlat‑
ed with TOS levels and OSI values (r = –0.604,
P <0.001; r = –0.399, P <0.05; respectively).
We observed that peripheral lymphocyte
DNA damage in UC patients was significanlty
correlated with CRP levels, ESR, albumin lev‑
els, and platelet and leukocyte counts (r = 0.544,
P <0.001; r = 0.725, P <0.05; r = –0.357, P <0.05;
r = 0.525, P <0.001; r = 0.655, P <0.001; respec‑
tively). TAC was significantly correlated with CRP,
ESR, albumin levels, platelet and leukocyte counts
(r = –0.719, P <0.001; r = –0.686, P <0.05; r =
lymphocyte isolation for the comet assay per‑
formed within 2 hours as described elsewhere.27
The endogenous DNA damage in lymphocytes
was analyzed by the alkaline comet assay accord‑
ing to Singh et al.,28 with a minor modification.
Thus, lymphocytes were embedded in 0.7% low
melting point agarose in phosphate buffer saline
at 37oC at the final concentration of 104 cells/
ml. Then, 80 µl of this cellular suspension was
spread on roughened slides that had previously
been coated with 1.0% hot (60oC) normal melting
point agarose, covered with a cover slip at 4oC for
at least 5 minutes to allow the agarose to solidify,
and transferred to a humidified box after remov‑
al of the cover slips. The slides were allowed again
to solidify for 5 minutes at 4oC in a moist box. Af‑
ter removing the cover slips, the slides were sub‑
mersed in freshly prepared cold (4oC) lysing so‑
lution (2.5M NaCl, 100mM EDTA ‑2Na; 10 mM
Tris–HCl, pH 10–10.5; 1% Triton X ‑100 and 10%
dimethyl sulfoxide (DMSO) added immediate‑
ly before use) for at least 1 hour. The slides were
then immersed in freshly prepared alkaline elec‑
trophoresis buffer (0.3 mol/l NaOH and 1 mmol/l
Na2ETDA, pH >13) at 4oC for unwinding (40 min)
and then they were subjected to electrophoresis
(25 V/300 mA, 25 min). All of the above steps
were conducted under red light and without direct
light in order to prevent additional DNA damage.
After electrophoresis, the slides were stained with
ethidium bromide (2 µ/ml in distilled H2O; 70 µl/
slide), covered with a cover slip and analyzed us‑
ing a fluorescence microscope (Nikon). Images
of 50 randomly selected cells (25 cells from each
of the two replicate slides) were analyzed visual‑
ly from each subject as described elsewhere.27,29
Each image was classified according to the inten‑
sity of the fluorescence in the comet tail and was
given a value of either 0–3 or 4 (from class 0 [no
damage] to class 4 [maximum damage]), so that
the total scores of slide could be between 0 and
200 arbitrary units. All procedures were complet‑
ed by the same staff and DNA damage was de‑
tected by a single observer who was not aware
of the diagnosis.
tAbLE 1 Characteristics of the study group
Parameters
age, y
sex (women/men)
body mass index, kg/m2
CRP, mg/l
ESR, mm/h
platelet count, 103/mm3
leukocyte count 103/mm3
albumin, g/dl
Patient group (n = 20)
34 ±7
11/9
20.3 ±2.1
4.7 ±2.8
65.8 ±23.5
395.3 ±137.8
11.9 ±4.3
3.5 ±0.7
Control group (n = 20)
36 ±10
10/10
21.4 ±1.1
1.9 ±0.8
17.3 ±7.5
302.3 ±91.5
6.6 ±1.9
4.3 ±0.6
P
NS
NS
NS
0.05
0.05
0.05
0.01
0.05
Values are presented as mean ± standard deviation
Abbreviations: CRP – C‑reactive protein, ESR – erythrocyte sedimentation rate, NS – nonsignificant

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POLSKIE ARCHIWUM MEDYCYNY WEWNĘTRZNEJ  011; 121 (7-8)
4
used to monitor environmentally induced genet‑
ic damage by a variety of methods, such as mi‑
cronucleus, chromosome aberration, and sister‑
‑chromatid exchange assays.34 One of the useful
methods of quantifying DNA damage is the sin‑
gle cell gel electrophoresis (comet) assay.35 Be‑
cause of its simplicity and sensitivity, the comet
assay has quickly gained acceptance as a geno‑
toxicity assay.35 It has been reported that strand
breaks arise from DNA damage generated by ox‑
idative stress.36 We therefore used this method
to measure DNA damage in circulating mononu‑
clear leukocytes.
The comet assay is a well ‑established genotox‑
icity test that is easy to perform. It is quick, sim‑
ple, and the observations are made at the lev‑
el of single cells.27 It is an extremely useful tool
that is used to assess the extent of endogenous
DNA damage. The comet assay can potentially
estimate DNA damage at the level of single cells
and provide information on DNA damage in in‑
dividual cells.37,38
To our knowledge, there have been no data con‑
cerning the oxidative stress and DNA damage lev‑
el in peripheral blood lymphocytes measured us‑
ing the comet assay in patients with UC, partic‑
ularly in relation to oxidative stress. In the pres‑
ent study, we for the first time observed that UC
patients have increased peripheral DNA damage
levels, TOS levels, and OSI values along with de‑
creased TAC levels. Furthermore, when patients
with UC were divided according to the disease ac‑
tivity, no differences were observed between ac‑
tive UC and inactive UC with respect to periph‑
eral lymphocyte DNA damage, TOS, OSI, and
TAC. Moreover, we found that peripheral lym‑
phocyte DNA damage was significanlty corre‑
lated with CRP, ESR, albumin levels, as well as
platelet and leukocyte counts in patients with
UC. Finally, these findings suggest that increased
peripheral DNA damage may be related to acti‑
vated phagocytic leukocytes generating excess
amounts of ROS with subsequent increase in ox‑
idative stress.
Previous studies have shown that DNA dam‑
age is involved in the pathophysiology of UC but
the results are controversial.8,20 Lih ‑Brody et al.8
measured 8 ‑OHdG concentration in the intesti‑
nal mucosa of the samples with UC. They reported
no significant differences in the levels of 8 ‑OHdG
between UC samples and controls. On the other
hand, D’Incà et al.20 investigated the role of free
0.307, P <0.05; r = 0.504, P <0.001; r = 0.577,
P <0.001; respectively).
In patients with UC, TOS and OSI were signif‑
icantly correlated with CRP (r = 0.416, P <0.001,
r = 0.410, P <0.05, respectively), ESR (r = 0.421,
P <0.001, r = 0.544, P <0.05, respectively), al‑
bumin levels (r = –0.529, P <0.001, r = –0.466,
P <0.05, respectively), platelet count (r = 0.519,
P <0.001, r = 0.526, P <0.05, respectively), and
leukocyte count (r = 0.623, P <0.001, r = 0.349,
P <0.05, respectively).
There were no correlations between the dura‑
tion of the disease and DNA damage, TOS, OSI,
and TAC in UC patients (P >0.05).
dIscussIon UC is a chronic inflammatory dis‑
ease. Increasing attention has been recently giv‑
en to the role of reactive oxygen species (ROS) in
the pathogenesis of UC, because an inflamed in‑
testine is exposed to oxidative stress generated by
infiltrating macrophages and neutrophils within
the lamina propia.30 On the other hand, it is well
known that an increased cancer risk occurs in
tissues undergoing chronic inflammation. Many
cancers develop at the sites of infection, chronic
irritation, and inflammation.11
The colon is more sensitive to oxidative dam‑
age because of the relatively low amount of anti‑
oxidants available in the mucosa. The high lev‑
els of DNA damage in IBD could represent a sig‑
nificant source of genomic instability. The accu‑
mulation of ROS could cause damage to specif‑
ic genes involved in cell growth or differentia‑
tion or could cause changes in antioxidant en‑
zyme activities.20
Oxidative stress has been well documented in
UC with increased ROS levels and decreased anti‑
oxidant levels in the inflamed mucosa, which ulti‑
mately contribute to chronic tissue damage.31 In‑
creased ROS in the cell may lead to severe dam‑
age to macromolecules, for example DNA, caus‑
ing base changes, strand breaks, and the altered
expression of oncogenes.32 Increased oxidative
stress and lipid peroxidation causing DNA dam‑
age and disturbance of cell signalling pathways,
are implicated in human cancers, neurodegener‑
ative diseases, and aging processes.16,17,33
Oxidative damage to DNA is particularly im‑
portant because there is growing recognition that
such damage can initiate and promote carcino‑
genesis.33 During the last decade, human periph‑
eral mononuclear leukocytes have been widely
tAbLE 2 Peripheral DNA damage and oxidative stress levels in patients with ulcerative colitis and in controls
Parameters
total antioxidant capacity, mmol Trolox equiv./l
total oxidant status, µmol H2O2 equiv./l
oxidative stress index, arbitrary unit
DNA damage, arbitrary units
Patient group (n = 20)
0.92 ±0.41
7.84 ±1.79
0.63 ±0.16
69.90 ±13.89
Control group (n = 20)
1.35 ±0.12
5.11 ±1.39
0.34 ±0.11
30.20 ±8.70
P
0.001
0.001
0.001
0.001
Values are presented as mean ± standard deviation

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ORIGINAL ARTICLE  Peripheral lymphocyte DNA damage and oxidative stress...5
7 Simmonds NJ, Allen RE, Stevens TRJ, et al. Chemiluminescence as‑
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radical ‑mediated oxidative DNA damage during
inflammation determined in patients with UC by
measuring 8 ‑OhdG.20 They observed that mucosal
8 ‑OHdG concentrations were significantly in‑
creased in patients with both UC and dysplasia.
Antioxidant defense mechanisms are decreased
in UC, presumably due to excessive inflamma‑
tion.8,9 Recent studies have shown that the mu‑
cosa of patients with active UC has lower con‑
tent of superoxide dismutase.8,39 Beno et al.40 ob‑
served a significant increase in GSH ‑Px activity
in inflamed mucosa in a study on patients with
active and inactive UC. However, Durak et al.41
reported no significant difference in the levels
of tissue GSH ‑Px between patients with UC and
controls. They observed that tissue malonyldial‑
dehyde (MDA) levels in the patient group were
significantly lower indicating that there was no
oxidative stress in the mucosa and that defense
mechanism was not impaired.41 In line with
the study by Bhaskar et al.,42 plasma MDA levels
were not different between the patient and con‑
trol groups. On the other hand, Bhaskar et al.42
observed that mucosal activities of antioxidant
enzymes were not different between patients with
UC and the control group. MDA levels, the end
product of lipid peroxidation, were not increased
in patients or in controls.42
Conventional drugs for treating UC in humans,
such as 5 ‑aminosalicylate (5 ‑ASA), are potent
ROS scavengers.43 Recent studies have shown that
treatment of UC patients with 5 ‑ASA compounds
can prevent the development of carcinoma.44
Our results indicate that increased peripher‑
al DNA damage and oxidative stress are associ‑
ated with decreased antioxidant levels and thus
may be one of the pathophysio logic mechanisms
in the development of UC ‑associated colorectal
cancer. Further studies on a larger group of pa‑
tients are needed to confirm the mechanisms un‑
derlying the association of increased peripher‑
al DNA damage and decreased antioxidant lev‑
els with the development of colorectal cancer in
UC patients.
Acknowledgments The authors thank the staff
of the Harran University Clinical Biochemistry
for their generous and friendly assistance at each
stage of the study.
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