Monochloramine induced DNA fragmentation in gastric cell line
MKN45
Hidekazu
Suzuki1,
Koichi
Seto2,
Mikiji
Mori1,
Masayuki
Suzuki3,
Soichiro
Miura4, and
Hiromasa
Ishii1
1 Department of Internal
Medicine, School of Medicine, Keio University, Shinjuku-ku, Tokyo
160-8582; 2 Department of
Pharmacology, Central Research Laboratory, Zeria Pharmaceutical
Co., Ltd., Saitama 369-0100;
3 Department of
Gastroenterology, National Tokyo Medical Center, Meguro-ku, Tokyo
152-0021; and 4 Second
Department of Internal Medicine, National Defense Medical College,
Saitama 359-8513, Japan
 |
ABSTRACT |
Monochloramine
(NH2Cl) is known to be one of the
virulence factors in Helicobacter
pylori-associated gastric mucosal
injury. The present study was designed to examine
NH2Cl-evoked DNA fragmentation in
the gastric epithelial cell line MKN45.
NH2Cl was produced by mixing
NH3 with sodium hypochlorite
(NaClO). MKN45 cells were exposed to
NH2Cl,
NH3, or NaClO in Hanks' balanced
salt solution. DNA cleavage was evaluated quantitatively by
photometeric enzyme immunoassay for the in vitro determination of
cytoplasmic mono- and oligonucleosomes. Damage to the plasma membrane
was assessed by measuring the activity of lactate dehydrogenase in the
supernatants. Separately, DNA ladder formation was performed to confirm
the incidence of DNA fragmentation.
NH2Cl (0.001-0.01 mM)
significantly increased the cytoplasmic mono- and oligonucleosomes,
suggesting the incidence of DNA cleavage. The DNA ladder was clearly
evoked by NH2Cl.
NH2Cl induced a DNA fragmentation,
one of the important aspects in apoptosis, in the gastric cell line
MKN45.
Helicobacter
pylori; leukocyte; mononucleosome; oligonucleosome; lactate dehydrogenase
 |
INTRODUCTION |
HELICOBACTER pylori
(H.
pylori) is a gram-negative
microaerophilic bacterium that infects >50% of the human population.
It is now generally acknowledged that
H. pylori infection is the major cause of
acute and chronic gastritis and peptic ulcer disease (12). Although
colonization of the gastric mucosa by
H. pylori is also reported to play an
important role in the pathogenesis of gastric cancer (13), the
mechanism of malignant transformation remains obscure. Oxygen free
radicals derived from H. pylori-activated neutrophils are also
known to be one of the virulence factors in the course of gastric
mucosal injury.
Myeloperoxidase in neutrophils catalyzes the oxidation of chloride by
H2O2
to yield hypochlorous acid (HClO). The interaction between
H. pylori-derived
NH3 and HClO produces
monochloramine (NH2Cl), which is
reported to be exceptionally reactive and toxic because of its high
lipophilic property and low molecular weight. We previously found (18)
that H. pylori directly elicited human neutrophils and then led to the gastric mucosal cell injury mediated by
NH2Cl. The value of
luminol-dependent chemiluminescence, which depicts the contents of
HClO, an NH2Cl precursor, is
increased in the H. pylori-colonized gastric mucosa
(16). We have previously reported (17) that
NH2Cl induced gastric cellular DNA
double-strand break and chromatin condensation.
Apoptosis is a physiological suicide mechanism that preserves
homeostasis, in which cell death naturally occurs during normal tissue
turnover (21). In general, cells undergoing apoptosis display profound
structural changes, including a rapid blebbing of the plasma membrane
and nuclear disintegration. The nuclear collapse is associated with
extensive damage to chromatin and DNA cleavage into oligonucleosomal
length DNA fragments after activation of calcium-dependent endogenous
endonuclease (3). In patients chronically infected with
H. pylori, increased numbers of apoptotic
epithelial cells were found along with increased numbers of
proliferating epithelial cells (6, 10). Accelerated rates of
proliferation may be the stimulus to increased apoptosis, or vice
versa, and an imbalance between apoptosis and proliferation may explain
the diverse clinical outcomes of infection, including neoplasia (19).
The present study demonstrates quantitatively the effect of
NH2Cl on the level of DNA cleavage
in the gastric cell line MKN45.
 |
MATERIALS AND METHODS |
Reagents.
RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin sulfate,
and Hanks' balanced salt solution (HBSS) were obtained from GIBCO BRL
(Rockville, MD). Sodium hypochlorite (NaClO) and NH3 solution were obtained from
Kanto Chemical (Tokyo, Japan). Taurine was obtained from Sigma Chemical
(St. Louis, MO).
Cells.
In the present set of experiments, we used the gastric epithelial cell
line MKN45 (Japanese Cancer Research Bank no. 0254), because this cell
line is adherent enough to assess the cytoplasmic release of mono- and
oligonucleosomes in the present protocol and does not constitutively
express the proinflammatory chemokine interleukin-8, which may itself
cause apoptosis (1), as observed in KATO III cells (2).
The MKN45 cell line used for this study was obtained from Japan Health
Sciences Foundation (Osaka, Japan). Cells were maintained in RPMI 1640 containing 10% FBS, 2 mM
L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin sulfate. The cells were rinsed
with PBS and then transferred in HBSS at a concentration of
106 cells/well. Separate sets of
cell cultures were pretreated with the different concentrations of
taurine.
Preparation of oxidant solutions.
NaClO and NH3 solution were
prepared, and NH2Cl solution was
produced by mixing 100 mM NaClO with 1.3-fold volume of 100 mM NH3 solution. The concentration of
NH2Cl in the mixture was measured photometrically by the optical density of 242 nm. The
NH2Cl concentration of these
original mixtures ranged between 40 and 50 mM. With the appropriate
dilution, stock solutions at 0.1, 1, and 10 mM
NH2Cl were prepared as were
suspensions with the comparable concentrations of
NH3 or NaClO.
Cell incubation.
Cells grown to confluent condition for several days were incubated with
HBSS containing NH2Cl,
NH3, or NaClO for 5 h. Then the supernatant of the cell cultures were sampled
for the measurement of lactate dehydrogenase (LDH)
activity. The cells were lysed and used for the analysis
of cytoplasmic mono- and oligonucleosomes.
In separate sets of experiments, human neutrophils were isolated from
the peripheral blood of one healthy volunteer with the use of standard
dextran sedimentation and gradient separation on Histoplaque-1077
(Sigma Chemical). This procedure yields a 95% viable (trypan blue
exclusion) and 98% pure (acetic acid-crystal violet staining)
neutrophil population (18). Some fractions of isolated neutrophils were
preincubated with sodium azide
(NaN3; 0.3 mM), a myeloperoxidase
inhibitor, to inactivate the hypochlorous anion generation by
neutrophils. MKN45 cells were incubated with neutrophils (1.0 × 107 cells/ml) in the presence or
absence of NH3 (0.0001 mM) for 5 h. To test the NH2Cl scavenger, we
applied taurine (0.0001 mM) to some wells of cell mixtures. Then the
cells were lysed and used for the analysis of cytoplasmic mono- and
oligonucleosomes.
Assay for cytoplasmic mono- and oligonucleosomes.
Apoptosis was evaluated by photometeric enzyme immunoassay of
cytoplasmic mono- and oligonucleosomes (histone-associated DNA fragments) determination (Boehringer Mannheim). Briefly, cells treated
with several inducers were harvested, and an aliquot of the cell
suspension was transferred into a streptavidin-precoated 96-well
microplate. Then biotinylated anti-histone antibody, which binds to
histone I, histone IIA, histone IIB, histone III, or histone IV, and
peroxidase-labeled anti-DNA antibody were added. Because mitochondrial
DNA is not covered by histones (14), the present value of
cytoplasmic mono- and oligonucleosomes specifically addresses the
damage of nuclear DNA, which is covered by histones. Two hours later,
the supernatant was discarded and then incubated with substrate
solutions. The absorbance (405 nm) was analyzed by a microplate
reader (Bio-Rad Laboratories, Hercules, CA).
Assay for LDH activity.
Plasma membrane damage, which is linked to necrosis, was evaluated by
measuring the LDH activity in the cell supernatant by formazan
colorimetric methods (LDH-CII test; Wako Pure Chemical, Osaka,
Japan) (4).
DNA electrophoresis.
DNA fragmentation was assessed by DNA agarose gel electrophoresis,
which was performed as described previously (15). Briefly, cells were
incubated with 0.001 mM NH2Cl or
HBSS alone for 5 h and harvested by using lysis buffer [50 mM
Tris · HCl, pH 7.8, 10 mM EDTA-Na, and 0.5% (wt/vol)
sodium N-lauroyl sarcosinate], and then cell numbers were counted. The cell suspension
(2 × 106 cells) was pelleted
by centrifugation at 500 g for 5 min
and resuspended with lysis buffer. RNase A (1 mg/ml) was added in cell
suspension, incubated at 50°C for 30 min, and then incubated with
proteinase K (1 mg/ml) at 50°C for 30 min. Each sample was resuspended in the gel loading buffer. Then 18 µl of each sample solution were loaded to a 1.5% agarose gel and electrophoresed at 100 V for 1 h.
Statistical analysis.
Statistical analysis was performed using one-way ANOVA and compared by
Fisher's multiple comparison test. P < 0.05 was considered significant.
 |
RESULTS |
Figure 1 shows the levels of cytoplasmic
mono- and oligonucleosomes under incubation with various concentrations
of NH2Cl for 5 h. The levels of
cytoplasmic mono- and oligonucleosomes incubated with
NH2Cl were significantly increased
compared with the control (0 mM). Cytoplasmic mono- and oligonucleosome
levels after incubation with 0.001 and 0.01 mM
NH2Cl were significantly higher
than those after incubation with 0.1 mM
NH2Cl.

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Fig. 1.
Levels of cytoplasmic mono- and oligonucleosomes under incubation with
various concentrations of monochloramine
(NH2Cl) for 5 h. Values are means ± SE. * P < 0.05 compared
with control (0 mM
NH2Cl).
P < 0.05 compared with
0.01 mM NH2Cl.
A405 nm, Absorption at 405 nm.
|
|
Such an increase in the levels of cytoplasmic mono- and
oligonucleosomes at 0.001 mM NH2Cl
was significantly attenuated by taurine at a
concentration of
0.0003 mM (Fig. 2). This
suggests that the effect of NH2Cl
on the cytoplasmic mono- and oligonucleosome levels was inhibited by at
least a 0.3-fold greater concentration of taurine.
Incubation for 5 h with NH3 as
well as NaClO at a concentration of 0.001 mM did not evoke levels of
cytoplasmic mono- and oligonucleosomes similar to those evoked by
NH2Cl (Fig. 3), suggesting this phenomenon was mainly
induced by NH2Cl and not by its
substrates, NH3 and NaClO.

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Fig. 2.
Effect of taurine on levels of cytoplasmic mono- and oligonucleosomes
evoked by 0.001 mM NH2Cl. Values
are means ± SE. * P < 0.05 compared with control. P < 0.05 compared with 0.001 mM
NH2Cl alone.
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Fig. 3.
Levels of cytoplasmic mono- and oligonucleosomes evoked by
NH2Cl,
NH3, or sodium hypochlorite
(NaClO) at a concentration of 0.001 mM. Values are means ± SE.
* P < 0.05 compared with
NH2Cl.
|
|
Figure 4A
shows the DNA ladder formation after a 5-h treatment with (Fig.
4A,
lane
2) or without (Fig.
4A,
lane
1) 0.001 mM NH2Cl.
NH2Cl enhanced the levels of DNA
ladder formation (Fig. 4A,
lane
2). Figure
4B depicts the DNA ladder formation of
KATO III cells under the same conditions as MKN45 cells. KATO III cells did not evoke clear DNA ladder formation with 0.001 mM
NH2Cl.

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Fig. 4.
A: DNA ladder formation of
MKN45 cells after 5-h treatment without
(lane
1) or with 0.001 mM
NH2Cl
(lane
2).
NH2Cl enhanced levels of DNA
ladder formation (lane
2).
B: DNA ladder
formation of KATO III cells after 5-h treatment without
(lane
1) or with 0.001 mM
NH2Cl
(lane
2).
NH2Cl enhanced levels of DNA
ladder formation (lane
2). Lane at
far
left shows
Hae III markers made by digesting
X174 DNA to completion with Hae
III. From top to
bottom, DNA fragments indicate 1,353, 1,078, 872, 603, 310, 281, 234, 194, 118, and 72 bp.
|
|
The level of LDH activity in the supernatant of MKN45
culture is shown in Fig. Although LDH
activity level did not significantly increase at an
NH2Cl concentration of 0.001 or
0.01 mM after 5 h of incubation, it was significantly increased at 0.1 mM NH2Cl. This phenomenon,
together with the above-mentioned fact that mono- and oligonucleosome
release was less at 0.1 mM NH2Cl
than at 0.001 or 0.01 mM (Fig. 1), suggests that
NH2Cl at a concentration of 0.1 mM
had already evoked direct damage to the cell membrane and that mono-
and oligonucleosomes were further released from the cytosol to the
extracellular space. The incubation for 5 h with NH3 as well as NaClO at a
concentration of 0.1 mM did not evoke levels of LDH activity similar to
those evoked by NH2Cl (Fig. 6), suggesting this phenomenon was
also induced by NH2Cl, not by its
substrates, NH3 or NaClO.

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Fig. 5.
Levels of lactate dehydrogenase (LDH) activity in supernatant of MKN45
culture after incubation with
NH2Cl for 5 h. Values are means ± SE. * P < 0.05 compared
with control (0 mM NH2Cl).
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Fig. 6.
Levels of LDH activity in supernatant of MKN45 culture after treatment
with NH2Cl,
NH3, or NaClO at a concentration
of 0.1 mM. Values are means ± SE.
* P < 0.05 compared with
NH2Cl.
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|
Figure 7 shows the levels of
cytoplasmic mono- and oligonucleosomes under incubation with
neutrophils for 5 h. Although the levels of cytoplasmic mono- and
oligonucleosomes were slightly but significantly increased with the
addition of neutrophils even in the absence of
NH3, the values were further
increased in the presence of NH3
(0.0001 mM). Such an increase in the levels of cytoplasmic mono- and
oligonucleosomes under incubation with neutrophils in the presence of
NH3 was significantly attenuated
by taurine (0.0001 mM). The pretreatment of neutrophils with
NaN3 attenuated significantly such
an increase in the levels of cytoplasmic mono- and oligonucleosomes
under incubation with neutrophils in the presence of
NH3.

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Fig. 7.
Levels of cytoplasmic mono- and oligonucleosomes under incubation with
neutrophils (PMN; 1.0 × 107
cells/ml) for 5 h. Values are means ± SE.
NH3 (0.0001 mM) or taurine (0.0001 mM) was applied to some wells of cell mixtures. Some fractions of PMN
were pretreated with sodium azide
(NaN3; 0.3 mM).
* P < 0.001 compared with
control (no PMN, no NH3).
** P < 0.001 compared with PMN
(no NH3).
P < 0.001 compared with
PMN + NH3.
|
|
 |
DISCUSSION |
The present investigation demonstrated that
NH2Cl leads to DNA fragmentation
of MKN45 cells (Figs. 1 and 4). Treatment with taurine significantly
attenuated the DNA fragmentation evoked by
NH2Cl (Fig. 2). Neither
NH3 nor NaClO evoked levels of DNA fragmentation that were higher than those evoked by
NH2Cl (Fig. 3). The injurious
effect was mainly due to the specific effect of
NH2Cl. The DNA fragmentation of
MKN45 cells was also demonstrated by the mixture of isolated human
neutrophils with NH3 and
attenuated by neutrophil inactivation by
NaN3 or treatment with taurine
(Fig. 7).
We previously reported that the high level of chromatin condensation
was evoked by the treatment of rabbit gastric mucosal cells or KATO III
cells with 0.1 mM NH2Cl (17). In
that study (17), the level of chromatin condensation was significantly higher in NH2Cl-treated cells than
in NH3- or HClO-treated cells. Although it has been estimated that activated neutrophils can generate
OCl
and
NH2Cl in the range of 0.1-0.6
mM (11, 18), the mucosal epithelial concentration at the site distant
from the surface of activated neutrophils should be <0.1
mM. In the present study, we examined the effect of lower
concentrations of NH2Cl (0.001 and
0.01 mM) from the viewpoint of DNA fragmentation (cytoplasmic mono- and
oligonucleosomes) as well as the direct cell membrane damage (LDH
activity) and obtained the results that although a lower concentration
of NH2Cl (0.001 and 0.01 mM)
evoked DNA fragmentation without cell membrane damage, a higher
concentration (0.1 mM) evoked the direct cell membrane damage
compatible with necrotic cell death rather than DNA fragmentation. As
shown in Fig. 4B, although DNA
fragmentation was not clearly induced in KATO III cells with
NH2Cl, it was evoked significantly
in MKN45 cells (Fig. 4A). These
observations support the present selection of MKN45 for evaluating the
DNA fragmentation.
Although Chen et al. (7) recently reported that
H. pylori directly induced gastric
epithelial apoptosis by a Bak (a Bcl-2 family protein)-dependent
pathway, other independent regulators of apoptosis may also be
important in gastric mucosa. Among them, recruited leukocytes as well
as their products, excessive amounts of oxygen free radicals exhibited
in the damaged gastric mucosa (20), can become candidates for the
inducer of apoptosis. In a previous study (9), a positive correlation
was found between oxygen free radical production and the
H. pylori status of patients. There was
even a positive association between mucosal oxygen free radical
production and quantitative histological and microbiological H. pylori assessments (8). We have
previously reported (17, 18) that the level of gastric cell membrane
damage assessed by the cytotoxicity assay using the extracellular
release of
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and its
level of DNA double-strand break and chromatin condensation of KATO III
cells as well as of isolated rabbit gastric mucosal cells were higher
with NH2Cl treatment than with
HClO or NH3. The present study
further examined the incidence of DNA cleavage by the levels of
cytoplasmic mono- and oligonucleosomes and DNA ladder formation, which
could be strongly related to DNA fragmentation, one of the aspects of
apoptosis.
With regard to oxidative DNA damage in
H. pylori-associated gastric mucosal
injury, Baik et al. (5) recently reported that the
8-hydroxy-2'-deoxyguanosine content, a marker of oxidative DNA
damage, of the gastric mucosal DNA of the
H. pylori-positive group was 2.3-fold
higher than that of the H. pylori-negative group. This finding
(5) of higher levels of oxidative DNA damage in the gastric mucosa
during the early phase of H. pylori infection supports the
hypothesis that the oxygen radicals persistently produced in the
gastric mucosa due to H. pylori infection are the driving force
that transforms the chronic gastritis ultimately into gastric cancer.
If cell death is the major effect of oxygen radicals on the rapidly
proliferating gastric epithelial stem cells, the extensive gastric
atrophy would be encountered. Mutation on the DNA in the stem cells
induced by the challenge of oxygen free radicals might lead to
intestinal metaplasia, dysplasia, and neoplasia in the long term.
In conclusion, NH2Cl originating
from H. pylori-infected gastric mucosa
remarkably induces DNA fragmentation, which is comparable to apoptosis,
at its pathophysiological concentration (0.001-0.01 mM) in gastric
epithelial cells, suggesting the possible involvement of
NH2Cl in gastric epithelial
apoptosis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Tomoyuki Yoneta, Department of Pharmacology, Zeria
Central Research Laboratory, for providing technical information.
 |
FOOTNOTES |
A portion of this study was presented at the Third Annual Meeting of
the Japanese Research Society for Helicobacter
pylori-Related Gastroduodenal Diseases, in Beppu,
Japan, on July 19, 1997.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: H. Ishii, Dept. of Internal Medicine,
School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo
160-8582, Japan.
Received 16 March 1998; accepted in final form 19 June 1998.
 |
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