Article

Reactive Nitrogen Species Mediate DNA Damage in Helicobacter pylori‐Infected Gastric Mucosa

Department of Gastroenterology and Hepatology, Division of Clinical Medicine and Biochemical Sciences, Mie University Graduate School of Medicine, Mie, Japan.
Helicobacter (Impact Factor: 4.11). 12/2009; 14(6):552-8. DOI: 10.1111/j.1523-5378.2009.00719.x
Source: PubMed
ABSTRACT
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can play an important role in cellular injury and carcinogenesis of gastric epithelial cells infected with Helicobacter pylori. 8-OH-deoxy guanosine (8-OHdG) and 8-nitroguanine (8-NG) are markers for ROS- and RNS-mediated DNA oxidation, respectively. In this study, RNS-mediated DNA damage in gastric mucosa was observed directly using a newly developed antibody to 8-NG to clarify how H. pylori infection causes nitrative DNA damage to gastric epithelial cells.
Immunohistochemistry with anti-8-OHdG and anti-8-NG antibodies was performed on gastric tissue samples from 45 patients (25 men and 20 women) with H. pylori-positive gastritis and 19 patients (11 men and 8 women) exhibiting successful H. pylori eradication. Histologic factors for gastric mucosal inflammation were graded according to the guidelines of the Updated Sydney system.
In corpus mucosa, 8-OHdG and 8-NG production were significantly associated with the degree of glandular atrophy, infiltration of chronic inflammatory cells and intestinal metaplasia in the glandular epithelial cells. Successful H. pylori eradication resulted in a significant reduction of chronic inflammatory cell infiltration and neutrophilic activity. Mean 8-OHdG production was lower after H. pylori eradication in both corpus and antral mucosa (p = .022 and .049, respectively). However, the reduction in 8-NG exhibited was more pronounced than the reduction of 8-OhdG (p = .004 and .007, respectively).
Helicobacter pylori infection can induce inflammatory cells infiltration, which evokes DNA damage of gastric epithelial cells through ROS and RNS production. 8-NG might be a more sensitive biomarker than 8-OHdG for H. pylori-induced DNA damage in gastric mucosa.

Full-text

Available from: Yoshinao Kobayashi
Reactive Nitrogen Species Mediate DNA Damage in Helicobacter
pylori-Infected Gastric Mucosa
Masaki Katsurahara,* Yoshinao Kobayashi,* Motoh Iwasa,* Ning Ma,
Hiroyuki Inoue,* Naoki Fujita,*
Kyosuke Tanaka,* Noriyuki Horiki,* Esteban C. Gabazza* and Yoshiyuki Takei*
*Department of Gastroenterology and Hepatology, Division of Clinical Medicine and Biochemical Sciences, Mie University Graduate School of
Medicine, Mie, Japan,
Faculty of Health Sciences, Suzuka University of Medical Science, Mie, Japan
Introduction
Helicobacter pylori infection of the stomach occurs in
roughly half of the human population worldwide.
Understanding of the etiology of gastritis has been pro-
foundly altered by the discovery of H. pylori, which is
now recognized as the major cause of chronic gastritis.
Chronic nonatrophic gastritis, chronic atrophic gastritis,
intestinal metaplasia (IM), and dysplasia represent an
inflammatory sequence towards the intestinal type of
gastric carcinoma [1].
Helicobacter pylori induces the recruitment and activa-
tion of monocytes and neutrophils. These cells produce
reactive oxygen species (ROS), such as superoxide anion
radical, hydrogen peroxide, and subsequently hydroxyl
radical [2,3]. Increased levels of ROS are evident in
H. pylori-infected gastric mucosa [4,5]. In parallel with
ROS production, inducible nitric oxide synthase (iNOS)
is expressed in gastric mucosa with H. pylori infection
and the increase in iNOS expression is closely correlated
with inflammatory cell infiltration in the gastric mucosa
[6]. iNOS catalyzes the production of NO
, which
Keywords
Helicobacter pylori, oxidative stress, nitrative
stress, reactive nitrogen species,
8-nitroguanine, chronic gastritis, eradication.
Reprint requests to: Yoshinao Kobayashi,
Department of Gastroenterology and
Hepatology, Division of Clinical Medicine and
Biochemical Sciences, Mie University Graduate
school of Medicine, 2-174 Edobashi, Tsu-city,
Mie 514-8507, Japan.
E-mail: yoshinao@clin.medic.mie-u.ac.jp
Abstract
Background: Reactive oxygen species (ROS) and reactive nitrogen species
(RNS) can play an important role in cellular injury and carcinogenesis of
gastric epithelial cells infected with Helicobacter pylori. 8-OH-deoxy guanosine
(8-OHdG) and 8-nitroguanine (8-NG) are markers for ROS- and RNS-
mediated DNA oxidation, respectively. In this study, RNS-mediated DNA
damage in gastric mucosa was observed directly using a newly developed
antibody to 8-NG to clarify how H. pylori infection causes nitrative DNA
damage to gastric epithelial cells.
Methods: Immunohistochemistry with anti-8-OHdG and anti-8-NG antibod-
ies was performed on gastric tissue samples from 45 patients (25 men and
20 women) with H. pylori-positive gastritis and 19 patients (11 men and 8
women) exhibiting successful H. pylori eradication. Histologic factors for
gastric mucosal inflammation were graded according to the guidelines of the
Updated Sydney system.
Results: In corpus mucosa, 8-OHdG and 8-NG production were significantly
associated with the degree of glandular atrophy, infiltration of chronic
inflammatory cells and intestinal metaplasia in the glandular epithelial cells.
Successful H. pylori eradication resulted in a significant reduction of chronic
inflammatory cell infiltration and neutrophilic activity. Mean 8-OHdG
production was lower after H. pylori eradication in both corpus and antral
mucosa (p = .022 and .049, respectively). However, the reduction in 8-NG
exhibited was more pronounced than the reduction of 8-OhdG (p = .004
and .007, respectively).
Conclusions: Helicobacter pylori infection can induce inflammatory cells
infiltration, which evokes DNA damage of gastric epithelial cells through
ROS and RNS production. 8-NG might be a more sensitive biomarker than
8-OHdG for H. pylori-induced DNA damage in gastric mucosa.
Helicobacter ISSN 1523-5378
552 ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 552–558
Page 1
generates various reactive nitrogen species (RNS) [7],
such as nitrogen oxides and peroxynitrite [8,9]. The
enhancement of ROS- or RNS-mediated DNA damage
leads to a G:C-to-T:A transversion, which is one of the
important mechanisms for carcinogenesis [10,11].
8-OH-deoxy guanosine (8-OHdG) is a commonly
used and highly sensitive marker for ROS-mediated
DNA oxidation in gastric mucosa [12]. Many studies
have demonstrated a positive association between
8-OHdG formation and H. pylori infection. However, to
date, only a few studies have reported the direct obser-
vation of RNS-mediated damage in gastric mucosa
[13,14]. We have developed an antibody to 8-nitrogua-
nine (8-NG) [15], which enabled direct observation of
nitrative DNA damage. We hypothesized that RNS-
mediated DNA damage is important in the pathogenesis
of gastritis and development of gastric cancer. In this
study, we analyzed the production of 8-NG in parallel
with 8-OH-dG in gastric mucosa infected with H. pylori
to clarify how H. pylori infection causes nitrative DNA
damage to gastric epithelial cells.
Patients and Methods
Subjects
Forty-five patients with H. pylori-positive gastritis (25
men and 20 women; age range 22–79 years; mean age
55 years) and 19 patients exhibiting successful H. pylori
eradication (11 men and 8 women; age range 22–72;
mean age 56 years) were enrolled. They visited Mie
University Hospital (Tsu, Japan) between April 2005
and March 2007. The enrollment criteria were as
follows: evidence of H. pylori infection, provision of
informed consent including endoscopic examination
with collection of three corpus and three antral speci-
mens, and absence of malignant legions in the gastric
and duodenal mucosa determined by endoscopy. In
patients who received eradication therapy, endoscopic
examination was performed before and after therapy.
The interval between the end of therapy and
endoscopic examination was 3–13 months (mean
6.8 months). The study was approved by The Ethics
Committee of Mie University.
Anti-H. pylori Eradication Therapy
Treatment for H. pylori eradication was a triple therapy
consisting of omeprazole (40 mg b.i.d.), amoxicillin (1 g
b.i.d.) and clarithromycin (500 mg b.i.d.). In three
patients, metronidazole (500 mg t.i.d.) was adminis-
trated in place of amoxicillin. Each drug was taken for
7 days.
Histopathlogic Assessment
Formalin-fixed, paraffin-embedded archival biopsies
from the gastric corpus and antrum were examined.
Sections (4 lm thick) were stained using hematoxylin-
eosin for histologic evaluation and a modified Giemsa
stain was used for the identification of H. pylori.
Histologic factors for gastric mucosal inflammation and
bacterial density were graded according to the
guidelines of the Updated Sydney system [16]. In brief,
the system systematically graded glandular atrophy,
infiltration of chronic inflammatory cells, neutrophilic
activity, IM, and density of H. pylori. Each feature was
evaluated as absent, mild, moderate or severe,
corresponding to a score of 0, 1, 2, and 3, respectively.
Immunohistochemistry
The immunohistochemical procedures were performed
on 4 lm-thick serial sections mounted on poly-
l-lysine-coated plates. Immunostaining for 8-OHdG
and 8-NG detection was performed using an Envision
Plus kit (Dako Japan Co. Ltd, Kyoto, Japan). Briefly,
after deparaffinization and inactivation of endogenous
peroxidases, nonspecific immunoreactivity was blocked,
and the sections were incubated with mouse monoclo-
nal anti-8-OHdG antibody (dilution 1 : 500; Japan
Institute for the Control of Aging, Fukuroi, Japan) or
rabbit polyclonal anti-NG antibody [15] (dilution
1 : 500) for 1 h at 4!C. As the secondary antibody for
anti-8-OHdG, a horseradish peroxidase (HRP)-labeled
goat anti-mouse IgG antibody was used. For anti-
8-NG, an HRP-labeled sheep antibody to rabbit IgG
was used. The degree of immunoreactivity was then
evaluated and scored according to the percentage of
positive cells as follows: 0–10% (score 0), 10–25%
(score 1), 25–50% (score 2), 50–75 (score 3), and
>75% (score 4).
Histopathology and immunohistochemistry results
were reviewed blindly by three gastrointestinal patholo-
gists. They did not receive any information about clini-
cal setting or the diagnoses of the other pathologists.
High concordance with <10% variation was obtained in
the evaluation of histopathology and immunostaining.
Statistical analysis
Spearman’s rank correlation coefficient was used to
analyze the relationship between histologic factors
and intensity of 8-OHdG or 8-NG immunostaining.
Wilcoxon test was used for statistical analysis of
histologic factors and 8-OHdG and 8-NG production
before and after treatment.
Katsurahara et al. Nitrative DNA Damage in H. pylori Infection
ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 552–558 553
Page 2
Results
Relationship Between 8-OHdG Production and
Histology of Gastritis
Immunohistochemical staining revealed a brown-
colored positive immunoreactivity for 8-OHdG pre-
dominantly localized in the nuclei of foveolar cells,
neutrophils, and lymphocytes (Fig. 1A). In corpus
mucosa, 8-OHdG production was significantly associ-
ated with the degree of glandular atrophy (rs = 0.490,
p = .001 by Spearman’s rank correlation coefficient),
infiltration of chronic inflammatory cells (rs = 0.458,
p = .003) and IM (rs = 0.447, p = .003) in the glandular
epithelial cells, while 8-OHdG in antral epithelia did
not show any correlation with either of these histologic
factors (Table 1). In corpus mucosa, 8-OHdG production
in chronic inflammatory cells was significantly associ-
ated with the degree of glandular atrophy (rs = 0.337,
p = .027), infiltration of chronic inflammatory cells
(rs = 0.364, p = .017), and IM (rs = 0.385, 0.011). In
antral mucosa, 8-OHdG was significantly associated
with the degree of glandular atrophy (rs = 0.349,
p = .024) and IM (rs = 0.435, 0.005), but not with the
degree of infiltration of chronic inflammatory cells.
Relationship Between Accumulation of 8-NG and
Histology of Gastritis
Positive immunoreactivity for 8-NG was predominantly
localized in the nuclei and cytosol of foveolar cells,
neutrophils, and lymphocytes (Fig. 1B). In corpus
mucosa, 8-NG production in the glandular epithelial
cells was significantly associated with the degree of
glandular atrophy (rs = 0.383, p = .014 by Spearman’s
rank correlation coefficient), infiltration of chronic
inflammatory cells (rs = 0.406, p = .009) and IM
(rs = 0.484, p = .002), while 8-NG in antral epithelia
was significantly correlated only with glandular atrophy
(rs = 0.320, p = .043) (Table 2). In corpus mucosa,
8-NG production in chronic inflammatory cells was
significantly associated with the degree of glandular
atrophy (rs = 0.343, p = .028), infiltration of chronic
inflammatory cells (rs = 0.342, p = .046) and IM
(rs = 0.416, p = .008), whereas in antrum mucosa,
8-NG production in chronic inflammatory cells did not
show any significant correlation with histologic factors
of gastritis.
Effect of H. pylori Eradication Treatment on
Gastritis
Successful H. pylori eradication was associated with a
significant reduction of chronic inflammatory cell infil-
tration (corpus; p = .034 vs pre-treatment, antrum;
p = .007 vs pre-treatment by Wilcoxon test) and
neutrophilic activity (corpus; p < .001 vs pre-treatment,
antrum; p < .001) (Table 3).
Effect of H. pylori Eradication Treatment
on 8-OHdG Production
Reduction of 8-OHdG productions in corpus and
antrum epithelia was observed in 57.9% (11 19) and
52.6% (10 19) patients, respectively (Fig. 2A,B). After
eradication, the mean 8-OHdG score was significantly
reduced from 1.6 to 0.8 (50%, p = .022 vs pre-
treatment by Wilcoxon test) in corpus mucosa, and
from 1.6 to 0.9 (56.3%, p = .049) in antral mucosa.
Despite the complete eradication of H. pylori, reduced
8-OHdG production by infiltrating inflammatory cells in
corpus and antrum was found only in 26.3% (5 19)
and 36.8% (7 19) patients, respectively (Fig. 2C,D).
Mean 8-OHdG immunoreactivity in inflammatory cells
was not significantly decreased after the treatment.
Effect of H. pylori Eradication Treatment on 8-NG
Production
In the patients who received eradication therapy,
68.4% (13 19) and 78.9% (15 19) were positive for
8-NG in corpus and antral mucosa before therapy,
respectively. After eradication, 8-NG production was
reduced in 12 (92.3%) of the 13 patients who were
positive in corpus mucosa before treatment and in all
(100%) of the 15 patients who were positive in antral
mucosa before treatment (Fig. 3A,B). After eradication,
the mean 8-NG score was significantly reduced from
1.4 to 0.5 (35.7%, p = .004 vs pre-treatment by Wilco-
xon test) in corpus mucosa and from 1.5 to 0.7 (46.7%,
p = .007) in antral mucosa. The effect of eradication on
mucosal immunoreactivity of 8-NG was greater than
AB
Figure 1 Immunohistochemical detection of 8-OH-deoxy guanosine
(8-OHdG) and 8-nitroguanine (8-NG). Positive immunoreactivity for
8-OHdG (A) and 8-NG (B) predominantly localized in the nuclei of
foveolar cells, neutrophils, and lymphocytes. Original magnification
was ·400.
Nitrative DNA Damage in H. pylori Infection Katsurahara et al.
554 ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 552–558
Page 3
that of 8-OHdG. Conversely, after H. pylori eradication,
reduced 8-NG production by infiltrating inflammatory
cells in corpus and antrum was found only in 7
(36.8%) and 4 (21.1%) patients, respectively
(Fig. 3C,D). The mean 8-NG immunoreactivity in
inflammatory cells did not significantly decrease after
the treatment.
Relationship Between 8-OHdG and 8-NG
8-Nitroguanine production was strongly correlated with
8-OHdG production in corpus epithelial cells
(rs = 0.871, p < .001 by Spearman’s rank correlation
coefficient), corpus inflammatory cells (rs = 0.799
p < .001), antral epithelial cells (rs = 0.833, p < .001),
and antral inflammatory cells (rs = 0.806, p < .001).
Discussion
The Agency for Research on Cancer (IARC) concluded in
1994 that H. pylori had a causal linkage to gastric
carcinogenesis. Gastric cancer is the fourth most
common cancer and is the second leading cause of
cancer-related deaths, resulting in approximately
700,000 deaths worldwide in 2002 [17]. Gastric carcino-
genesis is a complex, multi-step and multi-factorial
event, characterized by progressive cytohistologic dedif-
ferentiation. Helicobacter pylori can play an important role
in cellular injury and carcinogenesis of gastric epithelial
cells. The results from this study demonstrated an
increase in oxidative and nitrative DNA damage in the
setting of H. pylori infection and underlined the
Table 2 Relationship between histologic factors and intensity of 8-NG
8-NG
Gastric gland
epithelial cells
Inflammatory
cells
rs p rs p
Corpus
Glandular atrophy 0.383 .014 0.343 .028
Infiltration by chronic
inflammatory cells
0.406 .009 0.312 .046
Neutrophilic activity 0.219 .161 0.152 .330
IM 0.484 .002 0.416 .008
Density of H. pylori )0.002 .890 0.035 .823
Antrum
Glandular atrophy 0.320 .043 0.251 .112
Infiltration by chronic
inflammatory cells
0.140 .376 0.230 .146
Neutrophilic activity )0.101 .523 )0.055 .728
IM 0.305 .053 0.269 .089
Density of H. pylori 0.046 .770 )0.079 .618
Gastritis and H. pylori density were graded according to the Updated
Sydney System [19]. Spearman’s rank correlation coefficient was used
for analysis.
8-NG, 8-nitroguanine; IM, intestinal metaplasia.
Table 1 Relationship between histologic factors and intensity of
8-OHdG
8-OHdG
Gastric gland
epithelial cells
Inflammatory
cells
rs p rs p
Corpus
Glandular atrophy 0.490 .001 0.337 .027
Infiltration by chronic
inflammatory cells
0.458 .003 0.364 .017
Neutrophilic activity 0.205 .179 0.167 .274
IM 0.447 .003 0.385 .011
Density of H. pylori )0.024 .877 )0.024 .874
Antrum
Glandular atrophy 0.257 .095 0.349 .024
Infiltration by chronic
inflammatory cells
0.247 .109 0.217 .160
Neutrophilic activity 0.064 .681 0.038 .807
IM 0.295 .056 0.435 .005
Density of H. pylori 0.040 .796 )0.130 .398
Gastritis and H. pylori density were graded according to the Updated
Sydney System [19]. Spearman’s rank correlation coefficient was used
for analysis.
8-OHdG, 8-OH-deoxy guanosine; IM, intestinal metaplasia.
Table 3 Effect of treatment for H. pylori eradication on gastritis
Eradication treatment
pBefore After
Corpus
Glandular atrophy 1.6 ± 0.5 1.5 ± 0.5 .414
Infiltration by chronic
inflammatory cells
1.6 ± 0.5 1.2 ± 0.4 .034
Neutrophilic activity 1.7 ± 0.9 0.3 ± 0.5 <.001
IM 0.1 ± 0.3 0.1 ± 0.3 1
Density of H. pylori 1.4 ± 0.9 0 ± 0 <.001
Antrum
Glandular atrophy 2.2 ± 0.5 2.1 ± 0.5 .317
Infiltration by chronic
inflammatory cells
2.0 ± 0.8 1.3 ± 0.5 .007
Neutrophilic activity 1.6 ± 1.0 0.3 ± 0.5 <.001
IM 0.5 ± 0.8 0.2 ± 0.7 .160
Density of H. pylori 1.6 ± 1.0 0 ± 0 <.001
IM, intestinal metaplasia.
Katsurahara et al. Nitrative DNA Damage in H. pylori Infection
ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 552–558 555
Page 4
importance of adequate treatment. Of interest, gastric
mucosal atrophy and IM, which represent early epithelial
lesions in the sequence leading from H. pylori gastritis to
gastric carcinoma, correlated significantly with immuno-
reactivity for both DNA adducts in both corpus and antral
gastric mucosa. This suggests a possible role of such ad-
ducts in early gastric carcinogenesis. A follow-up study of
cases with these mitogenic events for developing gastric
adenocarcinoma may help further elucidate this poten-
tial association.
Recent studies have revealed an essential role of
cagA-positive H. pylori in the development of gastric
carcinoma. It has been reported that Cag-A-positive
patients showed higher scores for gastritis and greater
oxidative DNA damage [18]. Approximately, 30–40% of
H. pylori strains isolated in Western countries are
cagA-negative, whereas almost all of the East Asian
strains are cagA-positive. When interpreting the data of
this study, it should be noted that the enrolled subjects
were all Japanese.
CagA protein [19–21], DNA methylation [22,23], and
induction of cytidine deaminase [24] are key determi-
nants for the development of gastric cancer [25];
however, direct DNA damage by ROS and RNS adducts
can also play an important role in gastric carcinogene-
sis. Enhanced production of ROS was found in biopsied
samples from the duodenum and stomach with H. pylori
infection [2,26]. Davis et al. [2] reported a positive
association between ROS production and the infective
load of H. pylori. In addition, they reported that H. pylori
infection induced ROS production and DNA fragmenta-
tion in a primary culture system of human gastric
mucosal cells. This evidence strongly supports a positive
association between H. pylori infection and ROS produc-
tion. RNS-mediated pathways have also been identified
as an important mechanism for DNA damage. iNOS is
induced in inflammatory cells through activation of sig-
nal transducer and activator of transcription (STAT) and
nuclear factor-kappa B (NF-jB) [28], leading to NO
production. NO
reacts with O
2
)
to produce ONOO
)
,
p = .022
AB
CD
p = .049
1.6 ± 1.3 0.8 ± 1.1 1.6 ± 1.2 0.9 ± 1.1
1.1 ± 1.51.2 ± 1.21.2 ± 1.51.0 ± 1.2
Eradication treatment
Eradication treatment
p = .749
p = .681
4
3
1
2
0
1
Intensity of 8OHdG staining
4
3
1
2
0
1
Intensity of 8OHdG staining
4
3
1
2
0
1
Intensity of 8OHdG staining
4
3
1
2
0
1
Intensity of 8OHdG staining
Before After
Before After Before After
Eradication treatment
Before After
Eradication treatment
Figure 2 Effect of Helicobacter pylori eradication treatment on immu-
noreactivity of 8-OH-deoxy guanosine (8-OHdG). (A) Corpus epithelial
cells, (B) antral epithelial cells, (C) corpus inflammatory cells, (D) antral
epithelial cells. Wilcoxon test was used for statistical comparison of
8-OHdG production before and after treatment.
p = .004
AB
CD
p = .007
1.4 ± 1.2 0.5 ± 0.9
1.2 ± 1.5 1.0 ± 1.5 1.4 ± 1.5 1.4 ± 1.4
1.5 ± 1.1 0.7 ± 1.0
4
3
1
2
Intensity of 8-nitroguanine staining
0
4
3
1
2
Intensity of 8-nitroguanine staining
0
4
3
1
2
Intensity of 8-nitroguanine staining
0
4
3
1
2
Intensity of 8-nitroguanine staining
0
p = .431 p = 1.000
Before After
Eradication treatment
Before After
Eradication treatment
Before After
Eradication treatment
Before After
Eradication treatment
Figure 3 Effect of Helicobacter pylori eradication treatment on immu-
noreactivity of 8-nitroguanine (8-NG). (A) Corpus epithelial cells, (B)
antral epithelial cells, (C) corpus inflammatory cells, (D) antral epithelial
cells. Wilcoxon test was used for statistical comparison of 8-NG
production before and after treatment.
Nitrative DNA Damage in H. pylori Infection Katsurahara et al.
556 ª 2009 Blackwell Publishing Ltd, Helicobacter 14: 552–558
Page 5
which results in nitrative and oxidative damage to
nucleic acids [29]. Furthermore, NO
itself has various
tumor-enhancing effects; it increases angiogenesis and
blood flow [30], prevents apoptotic cell death [31], acti-
vates cyclooxygenase-2 [32], and suppresses the
immune system [33]. Recently, Naito et al. reported
that the expression of iNOS and interleukin-8 mRNA
was enhanced in H. pylori-induced atrophic gastritis
[34]. Further investigations are necessary to elucidate
the precise mechanism involved in human gastric NO
production and the role that nitrative DNA damage
plays in H. pylori-induced gastric carcinogenesis.
This study clearly showed that H. pylori eradication
resulted in a significant improvement of cellular infil-
tration of neutrophils and lymphocytes. The degree of
8-OHdG and 8-NG accumulation in gastric epithelial
cells was reduced after eradication therapy. This
evidence suggests that H. pylori infection can facilitate
the infiltration of such inflammatory cells, which evoke
DNA damage to the gastric epithelial cells through ROS
and RNS production. However, in this study, the reduc-
tion in 8-OHdG and 8-NG after H. pylori eradication
was observed only in epithelial cells and not in inflam-
matory cells. Although there is no obvious explanation
for this finding, we suspect that H. pylori in direct
contact with gastric epithelia resulted in the strong
correlation between production of DNA adducts and
histologic changes of the gastric mucosa; i.e. Cag-
A-positive H. pylori directly injects Cag-A protein into
gastric epithelial cells via the type IV secretion system,
which enhances ROS production [2,35]. Conversely,
DNA adduct levels in circulating inflammatory cells can
be influenced by oxidative and nitrative stress from
many other organs besides the stomach, which would
diminish the correlation between DNA adducts produc-
tion in those cells and gastric mucosal inflammation.
We found that with successful eradication, the reduc-
tion of 8-NG immunoreactivity was greater than that of
8-OHdG, possibly indicating higher sensitivity for 8-NG
as a marker for H. pylori-induced cell damage in gastric
mucosa. Interestingly, ROS and RNS production was
more strongly correlated with the degrees of corpus
inflammation than with antral inflammation. Farinati
et al. reported that DNA adduct levels in the antral
mucosa were unaffected by H. pylori eradication [36],
which supports our results. It is therefore possible that
the antral mucosa might be exposed to a wider variety
of exogenous factors that can lead to cell damage, for
example, through stimulation by ingested foods. As a
consequence, grading inflammation in the body
can be more useful for predicting H. pylori-related
gastric carcinogenesis than grading of antral inflamma-
tion. It should be noted that not all patients with
H. pylori-induced gastritis develop gastric cancer. Accu-
mulated DNA damage is repaired by base excision
repair pathways including 8-oxoguanine DNA glycosi-
dase. Genetic polymorphisms of DNA repair genes have
been reported [37]. It is therefore possible that gastric
carcinogenesis through oxidative and nitrative DNA
damage may depend, at least in part, on individual sus-
ceptibility to ROS and RNS.
This study demonstrated a positive relationship
between nitrative DNA damage and gastric mucosal
inflammation with H. pylori infection. Helicobacter pylori-
induced gastritis was more strongly associated with
nitrative DNA damage than 8-OHdG. 8-NG might be
more sensitive biomarker for H. pylori-induced DNA
damage in gastric mucosa. 8-NG can be useful for direct
observation of nitrative DNA damage in H. pylori-
infected gastric mucosa and it can be useful for under-
standing the mechanism of H. pylori-induced gastric
carcinogenesis.
Acknowledgements and Disclosures
We thank Mrs D’Alessandro-Gabazza CN (Department of
Gastroenterology and Hepatology, Mie University Graduate
School of Medicine) for technical assistance. We also thank
Prof. Kawanishi S (Faculty of Pharmacoceutical Sciences,
Suzuka University of Medical Science) and Dr Ichiro Imoto
(Department of Gastroenterology and Hepatology, Mie Univer-
sity Graduate School of Medicine) for helpful discussions.
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  • Source
    • "Moreover, a characteristic event in H. pylori-associated gastritis is that the production of large amounts of ROS and reactive nitrogen species (RNS) likely leads to an imbalance between removing the production of ROS/RNS and antioxidant defenses, which is the main cause of chronic gastritis [4,32,33]. ROS and RNS can cause DNA damage and induce gastric carcinogenesis in gastric mucosa infected by H. pylori [34]. Excessive production of ROS/RNS causes irreversible damage and destruction to a variety of cellular components, including proteins, nucleic acids, and lipids [35,36]. "
    [Show abstract] [Hide abstract] ABSTRACT: Helicobacter pylori (H. pylori)-induced oxidative stress has been shown to play a very important role in the inflammation of the gastric mucosa and increases the risk of developing gastric cancer. Resveratrol has many biological functions and activities, including antioxidant and anti-inflammatory effect. The purpose of this study was to probe whether resveratrol inhibits H. pylori-induced gastric inflammation and to elucidate the underlying mechanisms of any effect in mice. A mouse model of H. pylori infection was established via oral inoculation with H. pylori. After one week, mice were administered resveratrol (100 mg/kg body weight/day) orally for six weeks. The mRNA and protein levels of iNOS and IL-8 were assessed using RT-PCR, Western blot and ELISA. The expression levels of IκBα and phosphorylated IκBα (which embodies the level and activation of NF-κB), Heme Oxygenase-1 (HO-1; a potent antioxidant enzyme) and nuclear factor-erythroid 2 related factor 2 (Nrf2) were determined using Western blot, and lipid peroxide (LPO) level and myeloperoxidase (MPO) activity were examined using an MPO colorimetric activity assay, thiobarbituric acid reaction, and histological-grade using HE staining of the gastric mucosa. The results showed that resveratrol improved the histological infiltration score and decreased LPO level and MPO activity in the gastric mucosa. Resveratrol down-regulated the H. pylori-induced mRNA transcription and protein expression levels of IL-8 and iNOS, suppressed H. pylori-induced phosphorylation of IκBα, and increased the levels of HO-1 and Nrf2. In conclusion, resveratrol treatment exerted significant effects against oxidative stress and inflammation in H. pylori-infected mucosa through the suppression of IL-8, iNOS, and NF-κB, and moreover through the activation of the Nrf2/HO-1 pathway.
    Preview · Article · Nov 2015 · International Journal of Molecular Sciences
  • Source
    • "Infection with H. pylori can induce gastric pre-malignancies by recruiting bone marrow-derived cells (BMDCs) [9, 10]. H. pylori can cause DNA double-strand breaks directly [11], and cause DNA damage indirectly by stimulating the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [12, 13] or by increasing the activity of cytidine deaminase [14]. Hypermethylation as well as subsequent downregulation of tumor suppressor genes is an important epigenetic mechanism in H. pylori-related gastric carcinogenesis [15]. "
    [Show abstract] [Hide abstract] ABSTRACT: A section of gastric cancers presents nuclear β-catenin accumulation correlated with H. pylori infection. H. pylori stimulate Wnt/β-catenin pathway by activating oncogenic c-Met and epidermal growth factor receptor (EGFR), or by inhibiting tumor suppressor Runx3 and Trefoil factor 1 (TFF1). H. pylori also trigger Wnt/β-catenin pathway by recruiting macrophages. Moreover, Wnt/β-catenin pathway is found involved in H. pylori-induced gastric cancer stem cell generation. Recently, by using gastroids, researchers have further revealed that H. pylori induce gastric epithelial cell proliferation through β-catenin. These findings indicate that Wnt/β-catenin is an oncogenic pathway activated by H. pylori. Therefore, this pathway is a potential therapy target for H. pylori-related gastric cancer.
    Preview · Article · Sep 2015 · Oncotarget
  • Source
    • "It is known that Gram-negative bacteria , through their external lipopolysaccharides, induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) production that can contribute to genotoxicity. Indeed, studies indicated that ROS and RNS participate in DNA damage during infection by Gram-negative Helicobacter pylori bacteria [37, 38]. It is possible that ROS and RNS might have a role in DNA DSB induced by P. aeruginosa . "
    Full-text · Dataset · Aug 2015
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