Aliment Pharmacol Ther 2003; 18 (Suppl. 1): 139–145.
Effects of antioxidative agents on apoptosis induced by
ischaemia-reperfusion in rat intestinal mucosa
M. KOJIMA, R. IWAKIR I, B. WU, T. FU JISE, K. WATA NABE, T. LIN, S. A MEMOR I, H. SAK ATA,
R. SH IMODA , T. OGUZU, A . OO TANI, S. TSUNA DA & K . FUJIMOTO
Department of Internal Medicine and Gastrointestinal Endoscopy, Saga Medical School, Saga, Japan
SUMMARY
Background & aim: We have previously demonstrated
that ischaemia-reperfusion induces apoptosis in the
intestinal mucosa. To evaluate that reactive oxygen
species enhanced intestinal apoptosis after ischaemiareperfusion, we examined whether antioxidants reduced
apoptosis.
Methods: Rats were infused through a duodenal tube
with antioxidative agents, glutathione, rebamipide and
dymethylsulfoxide during 2 h before an ischaemic
insult. The superior mesenteric artery was occluded for
INTRODUCTION
We previously demonstrated that occlusion of the
superior mesenteric artery (SMA) followed by reperfusion
caused injury to the intestinal mucosa, and that the
injury to the intestinal mucosa was characterized by
apoptosis, cell death.1–5 Reactive oxygen species, such as
superoxide anion, hydrogen peroxide, and organic
peroxides, are generated by cells after ischaemiareperfusion (I ⁄ R), and these compounds have been
implicated as signalling molecules in apoptosis in the
gastrointestinal tract.6–8 Gastric mucosal injury after I ⁄ R
was related to apoptosis enhanced by lipid peroxidation9, 10 which was different from ethanol induced
mucosal injury.9
Correspondence to: Professor K. Fujimoto, Department of Internal Medicine
and Gastrointestinal Endoscopy, Saga Medical School, 5-1-1 Nabeshima,
Saga, Saga 849-8501, Japan.
E-mail: [email protected]
2003 Copyright Blackwell Publishing Ltd
60 min, followed by 60 min reperfusion. Apoptosis was
evaluated by percentage fragmented DNA (fragmented
DNA ⁄ total DNA) and immunochemical staining.
Results: Increase in apoptosis in the intestinal mucosa
after ischaemia-reperfusion was attenuated by intraduodenal infusion of antioxidative agents, but was not
completely abolished.
Conclusion: Scavenging effects of the antioxidative
agents attenuated increases in intestinal apoptosis,
indicating that oxidative stress after ischaemiareperfusion plays an important role in induction of
apoptosis in the intestinal mucosa.
Intestinal mucosa is an organ sensitive to I ⁄ R,
and reactive oxygen species might play important
roles on I ⁄ R induced injury.11–13 The aim of this
experiment is to elucidate whether reactive oxygen
species are correlated to apoptosis in the rat intestinal
mucosa after I ⁄ R. For this purpose, we evaluated
effects of antioxidative agents, a reduced form of
glutathione (GSH), rebamipide and dymethylsulfoxide
(DMSO) on intestinal mucosal apoptosis induced by
I ⁄ R.
MATERIALS AND METHODS
Surgery
Male Sprague–Dawley rats (250–300 g) were used in
this study. The animals were housed in a room
illuminated from 8:00 to 20:00 (12 h light-dark
cycle). The rats were allowed access to water and
food ad libitum. Laparotomies were performed under
139
140
M. KOJIMA et al.
halothane anaesthesia. We previously demonstrated
that the occlusion of the SMA markedly reduced the
blood flow to the jejunum and ileum1 and that
apoptosis in the intestinal mucosa peaked during
60 min of reperfusion after a 60-min occlusion of
the SMA.2 In this study, the SMA was occluded
for 60 min with a microbulldog clamp, and was
followed by 60 min of reperfusion as demonstrated in
the previous study.1, 2 In sham-operated rats, the
SMA was isolated but was not occluded. A silicone
infusion tube was introduced 2 cm down the
duodenum through the fundus of the stomach before
I ⁄ R.14
Collection of intestinal mucosa
After I ⁄ R, the animals were anaesthetized and then
euthanized. The entire small intestine was carefully
removed and placed on ice. The oral 10 cm of the
intestine (duodenum) was removed, and the rest of
divided into two equal segments, representing the
proximal (jejunum) and distal (ileum) segments. Each
segment was rinsed thoroughly with physiological
saline and was opened on its antimesenteric border
longitudinally to expose the intestinal epithelium. The
mucosal layer was harvested by gently scraping the
epithelium with a glass slide.
DNA fragmentation assay
The mucosal scrapings were processed immediately
after collection to minimize nonspecific DNA fragmentation. The amount of fragmented DNA was determined
as previously described with modification.2, 15 Mucosal
scrapings were homogenized in a lysis buffer (pH 8.0)
10 times their volume. This buffer consisted of 5 mm
tris (hydroxymetlye) aminomethane (Tris)-HCl, 20 mm
ethylenediaminetetraacetic acid (EDTA), and 0.5%
(wt ⁄ vol) t-octylphenoxypolyethoxyethanol. One mL
aliquots of each sample were centrifuged for 20 min
at 27 000 g to separate the intact chromatin (pellet)
from the fragmented DNA (supernatant).15, 16 The
supernatant was decanted and saved, and the pellet
was re-suspended in 1 mL of Tris buffer (pH 8.0) with
10 mm Tris-HCl and 1 mm EDTA. The pellet and
supernatant fractions were assayed for DNA content,
using a diphenylamine reaction and the results
expressed as a percentage of the fragmented DNA
divided by total DNA.
Purification of mucosal DNA and agarose gel electrophoresis
DNA was extracted from the 27 000 g fraction.16
Fragmented DNA from the various fractions was
extracted sequentially by a phenol-chloroform-isoamyl
alcohol mixture (25:24:1, vol:vol:vol) to remove
protein. The protein-free DNA extracts were treated
with 100% ethanol in 0.1 m sodium acetate at )20 C
overnight to purify the DNA. The precipitated DNA was
washed with 70% ethanol and re-suspended in Tris
buffer (pH 8.0) with 10 mm Tris-HCl and 10 mm EDTA.
DNA samples were incubated with 100 lg ⁄ mL ribonuclease for 15 min at 37 C to remove RNA. Resolving
agarose gel electrophoresis was performed with 1.5% gel
strength containing 1.0 lg ⁄ mL ethidium bromide.
Depending on the experiment, 20 lg DNA was put
into each well. DNA standards (0.5 lg per well) were
included to identify the size of the DNA fragments.
Electrophoresis was performed for 2 h at 70 V, and DNA
was observed under ultraviolet fluorescent lighting.
Immunohistochemical staining
Tissue samples were removed from the jejunum and
ileum, and were immediately fixed in 10% neutral
buffered formalin. The samples were then embedded in
paraffin and sectioned. Fragmented DNA was stained by
the terminal deoxynucleotidyl transferase (TdT)mediated dUDP-biotin nick end labelling (TUNEL)
method17 with modification using an Apop Tag Kit
(Oncor, Gaithersburg, MD, USA). The specimens were
dewaxed and immersed in phosphate-buffered saline
containing 0.3% hydrogen peroxide for 10 min at room
temperature and then incubated with 20 lg ⁄ mL proteinase K for 15 min at room temperature. Seventy-five
micro-litres of equilibration buffer was applied directly
on to the specimens for 10 min at room temperature,
followed by 55 lL of TdT enzyme and incubation at
37 C for 1 h. The reaction was terminated by transferring the slides to a prewarmed stop ⁄ wash buffer for
30 min at 37 C. The specimens were covered with a
few drops of rabbit serum, incubated for 20 min at room
temperature, and then covered with 55 lL of antidigoxigenin peroxidase and incubated for 30 min at
room temperature. The specimens were then soaked in
Tris buffer containing 0.02% diaminobenzidine and
0.02% hydrogen peroxide for 1 min to develop colour.
Finally, the specimens were counterstained by immersing them in haematoxylin.
2003 Copyright Blackwell Publishing Ltd, Aliment Pharmacol Ther 18 (Suppl. 1), 139–145
ANTIOXIDANT AND INTESTINAL APOPTOSIS
Glutathione (L-c-glutamyl-L-cycteinylglycine)
concentration measurement
Reduced form of glutathione (GSH) and oxidative form
of glutathione (glutathione disulphide: GSSG) were
measured. Mucosal scrapings of jejunum were added
with cold 0.1 m phosphate buffer (pH 7.0) or cold
20 mm N-ethylmaleimide-phosphate buffer (pH 7.0)
and homogenized. One millilitre of each homogenized
mucosal scraping sample was added with 0.6 N
HCl4–1 mm EDTA and kept cold for 10 min, then
centrifuged for 10 min at 3000 g. The supernatant
was collected for measurement of GSH and GSSG with
5,5-dithiobis-(2-nitrobenzoic acid).18
Effect of GSH on intestinal apoptosis after I ⁄ R
Rats were intraperitoneally pretreated with buthionine[s,r]-sulfoximine (BSO) at a dose of 8 mmol ⁄ kg 2 h
before I ⁄ R in order to deplete the endogenous GSH pool.
Rats were infused with 20 mL of 1.0% GSH through a
duodenal tube during the 2 h just before I ⁄ R. Six groups
were tested; sham pre-treated with vehicle; sham pretreated with BSO; sham + GSH pre-treated with BSO;
I ⁄ R pre-treated with vehicle; I ⁄ R pre-treated with BSO;
and I ⁄ R + GSH pre-treated with BSO. Six rats were
tested in each group.
Effect of rebamipide and DMSO on intestinal apoptosis
after I ⁄ R
Rats were infused with 20 mL of test solutions through a
duodenal tube for the 2 h just before I ⁄ R. Rebamipide
(Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan) was
dissolved in 0.5% carboxymethyl cellulose. Seven groups
were tested; sham + vehicle; sham + 100 mg ⁄ kg
rebamipide; I ⁄ R + vehicle; I ⁄ R + 3 mg ⁄ kg rebamipide;
I ⁄ R + 30 mg ⁄ kg rebamipide; I ⁄ R + 100 mg ⁄ kg rebamipide; I ⁄ R + 20 mm ⁄ kg DMSO. Six rats were tested in
each group.
Statistics
Results are expressed as mean ± S.E. Data were evaluated by a one-way analysis of variance in which
multiple comparisons were carried out by the method of
least significant difference. Differences were considered
significant if the probability of the difference occurring
by chance was less than 5 in 100 (P < 0.05).
141
RESULTS
Effect of GSH on intestinal apoptosis after I ⁄ R
The percentage of fragmented DNA to total DNA in the
rat duodenal and jejunal mucosa after 60 min of
ischaemia followed by 60 min of reperfusion is shown
in Table 1. Neither pre-treatment with BSO nor GSH
infusion had any effect on intestinal apoptosis in shamoperated rats. The percentage of fragmented DNA in the
jejunal mucosa increased significantly in the I ⁄ R rats
pre-treated with a vehicle compared with the shamoperated rats (P < 0.01). BSO pre-treatment itself had
no effect on increased apoptosis induced by I ⁄ R. Intraluminal infusion of GSH with BSO pre-treatment
attenuated increased apoptosis in the jejunal mucosa
after I ⁄ R (P < 0.01), but GSH could not completely
suppress the increased apoptosis. An increased dose of
GSH had no further attenuation effect on apoptosis
induced by I ⁄ R compared with the dose tested in this
study (data not shown). As shown in Table 2,
GSH ⁄ GSSG ratio decreased in the I ⁄ R rat pre-treated
with BSO (P < 0.01), and this decrease was recovered
by infusion of GSH through the duodenal tube. In
contrast to the jejunum, I ⁄ R with occlusion of SMA did
not increase apoptosis in the duodenal mucosa, where
the blood flow was not reduced by the occlusion of
the SMA.1
Table 1. Effect of intraluminal infusion of glutathione (GSH) on
intestinal apoptosis induced by ischaemia-reperfusion (I ⁄ R) in rat
intestinal mucosa
Duodenal mucosa Jejunal mucosa
(% fragmented
(% fragmented
DNA)
DNA)
Sham-operated rats
Pretreated with vehicle
Pretreated with BSO
Pretreated with BSO + GSH
I ⁄ R rats
Pretreated with vehicle
Pretreated with BSO
Pretreated with BSO + GSH
4.2 ± 0.8
3.7 ± 1.9
4.4 ± 1.6
6.3 ± 2.5
5.0 ± 1.6
4.4 ± 1.4
6.0 ± 2.2
7.0 ± 2.5
5.6 ± 1.9
26.7 ± 2.5a
29.0 ± 6.5a
14.4 ± 3.0a,
b, c
Values are mean ± S.E. Six rats were tested in each group.
a
P < 0.01, compared with sham operated controls; bP < 0.01, compared with I ⁄ R rats pre-treated with vehicle; cP < 0.01, compared
with I ⁄ R rats pre-treated with BSO.
Rats were intraperitoneally pre-treated with buthionine-[s, r]-sulfoximine (BSO) at a dose of 8 mmol ⁄ kg 2 h before I ⁄ R and were infused
with 20 mL of 1.0% GSH through a duodenal tube during the 2 h just
before I ⁄ R.
2003 Copyright Blackwell Publishing Ltd, Aliment Pharmacol Ther 18 (Suppl. 1), 139–145
M. KOJIMA et al.
142
Table 2. GSH ⁄ GSSG ratio in jejunal mucosa after ischaemiareperfusion (I ⁄ R)
Sham pre-treated
with vehicle
I ⁄ R pre-treated
with BSO
I ⁄ R + GSH
pre-treated with BSO
124.8 ± 21.6
18.3 ± 8.3a
104.2 ± 18.2
Values are mean ± S.E. Six rats were tested in each group.
a
P < 0.01, compared with sham pre-treated with vehicle.
Rats were intraperitoneally pre-treated with buthionine-[s, r]-sulfoximine (BSO) at a dose of 8 mmol ⁄ kg 2 h before I ⁄ R and were infused
with 20 mL of 1.0% GSH through a duodenal tube during the 2 h just
before I ⁄ R.
of apoptosis19, 20 although they were not useful for
quantitative analysis because the same doses of fragmented DNA were placed on to each lane.
Immunohistochemical staining (TUNEL) of the small
intestine showed that in the I ⁄ R group pretreated with
the vehicle alone, there was an increase in the number
of apoptotic cells and a marked destruction of the
structure with mucosal erosion and oedema (Figure 2A). In contrast, in the GSH infused group, the
Resolving agarose gel electrophoresis was performed
to evaluate the nature of the fragmented DNA in the
jejunal mucosa after I ⁄ R. As shown in Figure 1 (lanes 1
and 2), agarose gel electrophoresis of the fragmented
DNA obtained from the jejunal after I ⁄ R revealed
distinct DNA ladders. These ladders were characteristic
1
2
3
4
5
6
7
3530 bp
1910 bp
1353 bp
872 bp
603 bp
Figure 1. Agarose gel electrophoresis of fragmented DNA from
jejunal mucosa after ischaemia-reperfusion (I ⁄ R). Twenty micrograms of fragmented DNA were loaded. Lanes 1–5, I ⁄ R + GSH
pre-treated with BSO, I ⁄ R + vehicle pre-treated with BSO, I ⁄ R +
rebamipide (100 mg ⁄ kg), I ⁄ R + rebamipide (30 mg ⁄ kg), and
I ⁄ R + vehicle, respectively. The ladder, which is characteristic of
apoptosis, was clearly shown on each lane; however, this method
evaluated the quality of fragmented DNA, but not the quantity of
fragmented DNA. Lanes 6 and 7 contained marker DNA from
·174 Hae III and lambda EcoRIII digest (Wako Pure Chemical,
Tokyo, Japan), respectively.
Figure 2. Light micrographs of jejunum stained by terminal
deoxynucleotidyl transferase-mediated dUDP-biotin nick end
labelling method following ischaemia-reperfusion (I ⁄ R). (A) I ⁄ R
pretreated with vehicle. (B) I ⁄ R + GSH pre-treated with BSO.
Magnifications: ·100.
2003 Copyright Blackwell Publishing Ltd, Aliment Pharmacol Ther 18 (Suppl. 1), 139–145
ANTIOXIDANT AND INTESTINAL APOPTOSIS
number of apoptotic cells and the destruction of the
structure of the intestinal mucosa were lower compared
with the I ⁄ R group (Figure 2B).
Effect of rebamipide and DMSO on intestinal apoptosis
after I ⁄ R
Table 3 shows that the increase in the percentage of
fragmented DNA of the jejunal mucosa was reduced by
rebamipide at doses of 30 and 100 mg ⁄ kg (P < 0.01 in
each), although rebamipide itself (100 mg ⁄ kg) had no
effect on jejunal apoptosis. Rebamipide (100 mg ⁄ kg)
attenuated I ⁄ R induced apoptosis in BSO pre-treated
rats as same as vehicle pre-treated rats (data not
shown). An increased dose of rebamipide had no further
attenuation effect on apoptosis induced by I ⁄ R (data not
shown). Infusion of DMSO attenuated the increased
apoptosis in the jejunal mucosa after I ⁄ R.
As shown in Figure 1, agarose gel electrophoresis of
the fragmented DNA obtained from the jejunal after I ⁄ R
revealed distinct DNA ladders; lane 3: rebamipide
100 mg ⁄ kg, lane 4: rebamipide 30 mg ⁄ kg (DMSO, data
not shown). TUNEL showed that the numbers of
apoptotic cells and the level of destruction of the
structure of the intestinal mucosa were lower in the
rebamipide and DMSO groups compared with the I ⁄ R
group treated with vehicle (data not shown).
Table 3. Effect of rebamipide and dymethylsulfoxide (DMSO) on
intestinal apoptosis induced by ischaemia-reperfusion (I ⁄ R) in rat
intestinal mucosa
Duodenal mucosa
(% fragmented DNA)
I ⁄ R rats
Vehicle
6.9
Rebamipide:
3 mg ⁄ kg
4.9
30 mg ⁄ kg
6.9
100 mg ⁄ kg
5.1
DMSO 20 mm ⁄ kg 4.1
Sham-operated rats
Vehicle
3.9
Rebamipide:
100 mg ⁄ kg
4.3
Jejunal mucosa
(% fragmented DNA)
± 3.3
28.4 ± 7.1a
±
±
±
±
24.6
17.4
18.9
13.1
2.2
1.8
2.1
1.9
±
±
±
±
3.3a
3.2a,
4.3a,
3.6a,
± 1.3
5.7 ± 2.0
± 1.8
4.8 ± 1.7
b
b
b
Values are mean ± S.E. Six rats were tested in each group.
a
P < 0.01 compared with sham operated controls; bP < 0.01, compared with I ⁄ R rats infused with vehicle.
Rats were infused with 20 mL of test solutions through a duodenal
tube during the 2 h just before I ⁄ R.
143
DISCUSSION
Apoptosis is an active energy-dependent mode of cell
death that is caused by physiological signals and ⁄ or
pathological cytotoxic stimuli such as free-radicals, FAS,
tumour necrosis factor a, tumour necrosis factor related
apoptosis inducing ligands, NO, a change in ionic channels of the cellular membrane, and growth factor
withdrawal.5, 21–27 Previous studies have demonstrated
that ischaemia with occlusion of SMA following reperfusion causes injury and apoptosis in small intestinal
mucosa.1–5, 28 This study showed that antioxidative
agents reduced apoptosis after I ⁄ R; indicating that
oxidative stress with production of reactive oxygen
species was related to an increase in apoptosis following
I ⁄ R.
GSH is the major cellular reductant for the glutathioneperoxidase-catalysed elimination of organic and lipid
hydroperoxides.29 Previous studies in rats and mice
demonstrated that exogenous GSH is absorbed intact
from the intestinal surface in vivo to supplement cellular
GSH pools30, 31 indicating that intestinal uptake of
glutathione plays a part in maintaining mucosal GSH
status and promotes intestinal hydroperoxide detoxidation.32, 33 In this study, we performed I ⁄ R experiments
under conditions in which mucosal GSH was depleted
pre-treated with BSO. As a result, the supplementation
of exogenous GSH attenuated I ⁄ R induced intestinal
apoptosis by restoration of mucosal endogenous GSH.
Rebamipide has been used for treatment of gastric
ulcers. Pre-treatment with rebamipide inhibited injury
in the rat gastric mucosa induced by increase in freeradicals caused by platelet activating factor.34 Rebamipide reduced gastric mucosal injury caused by diethyl
dithiocarbamate that chelated Cu from superoxide
dismutase.35 Rebamipide decreased gastric mucosal
injury induced by I ⁄ R and ⁄ or hydrogen peroxide
(H2O2) in rats.36 The scavenging effect of rebamipide
on free-radicals was demonstrated by electron spin
resonance.37 Judging from these facts, the attenuation
effect of rebamipide on intestinal apoptosis after I ⁄ R in
this experiment was caused by, at least in part, an
antioxidative effect of this reagent. This result suggested
that rebamipide might have therapeutic effects on
intestinal diseases related to increase in free-radicals
and ⁄ or apoptosis such as inflammatory bowel diseases.
In this study, DMSO attenuated I ⁄ R induced intestinal
apoptosis. DMSO is also an antioxidant that solubilizes
and delivers hydrophobic phospholipase inhibitors.38, 39
2003 Copyright Blackwell Publishing Ltd, Aliment Pharmacol Ther 18 (Suppl. 1), 139–145
144
M. KOJIMA et al.
In this study, intestinal apoptosis induced by I ⁄ R was
attenuated by the antioxidative agents, GSH, rebamipide and DMSO. Pre-treatment with these agents,
however, could not eradicate apoptosis completely after
I ⁄ R, and this suggested that factors other than free
radicals might be related to the development of
apoptosis.
15
16
17
REFERENCES
18
1 Fujimoto K, Price VH, Granger DN, Specian R, Bergstedt S,
Tso P. Effect of ischemia-reperfusion on lipid digestion and
absorption in rat intestine. Am J Physiol 1991; 261: G595–
602.
2 Noda T, Iwakiri R, Fujimoto K, Matsuo S, Aw TY.
Programmed cell death induced by ischemia-reperfusion
in the rat intestinal mucosa. Am J Physiol 1998; 274:
G270–6.
3 Yoshida T, Iwakiri R, Noda T, et al. Histaminergic effect on
apoptosis of rat small intestinal mucosa after ischemiareperfusion. Dig Dis Sci 2000; 45: 1138–44.
4 Fukuyama K, Iwakiri R, Noda T, et al. Apoptosis induced by
ischemia-reperfusion and fasting in gastric mucosa compared
with small intestine in rats. Dig Dis Sci 2001; 46: 545–9.
5 Wu B, Iwakiri R, Tsunada S, et al. iNOS enhanced rat intestinal apoptosis after ischemia-reperfusion. Free Rad Bio Med
2002; 33: 649–58.
6 Ramachandran A, Madesh M, Balasubramanian KA. Apoptosis in the intestinal epithelium: its relevance in normal and
pathophysiological conditions. J Gastroenterol Hepatol 2000;
15: 109–20.
7 Tarnawski AS, Szabo I. Apoptosis-programmed cell death and
its relevance to gastrointestinal epithelium: survival signal
from matrix. Gastroenterology 2001; 120: 294–9.
8 Wang TG, Gotoh Y, Jennings MH, Rhoads CA, Aw TY. Lipid
hyroxide-induced apoptosis in human colonic CaCo-2 cells is
associated with early loss of cellular redox balance. FASEB J
2000; 14: 1567–76.
9 Gregory SS, David WM, James MC, Jose CB, Thomas AM.
Gastric injury induced by ethanol and ischemia-reperfusion in
the rat; differing roles for lipid peroxidation and oxygen radicals. Dig Dis Sci 1996; 41: 1157–64.
10 Ohta Y, Kobayashi T, Nishida K, Ishiguro I. Relationship
between changes of active oxygen metabolism and blood flow
and formation, progression, and recovery of lesions in gastric
mucosa of rats with a single treatment of compound 48 ⁄ 80, a
mast cell degranulator. Dig Dis Sci 1997; 42: 1221–32.
11 Granger DN, Korthuis RJ. Physiologic mechanisms of postischemic tissue injury. Ann Rev Physiol 1995; 57: 311–32.
12 Grisham MB. Oxidants and free radicals in inflammatory
bowel disease. Lancet 1994; 344: 859–61.
13 Kurtel H, Fujimoto K, Zimmerman BJ, Granger DN, Tso P.
Ischemia-reperfusion-induced mucosal dysfunction: role of
neutrophils. Am J Physiol 1991; 261: G490–6.
14 Fujimoto K, Imamura I, Granger DN, Wada H, Sakata T, Tso
P. Histamine and histidine decarboxylase are correlated with
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
mucosal repair in rat small intestine after ischemiareperfusion. J Clin Invest 1992; 89: 126–33.
Aw TY, Nicotera P, Manzo L, Orrenius S. Tributylin stimulates
apoptosis in rat thymocytes. Arch Biochem Biophy 1990;
283: 46–50.
Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is
associated with endogenous endonuclease activation. Nature
1980; 284: 555–6.
Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA
fragmentation. J Cell Biol 1992; 119: 493–50.
Owens CWI, Belcher RV. A colorimetric micro-method for
determination of glutathione. Biochem J 1966; 94: 705–11.
Guh JH, Hwang TL, Ko FN, Chueh SC, Lai MK, Teng CM.
Antiproliferative effect in human prostatic smooth muscle
cells by nitric oxide donor. Mol Pharmacol 1998; 53: 467–74.
Hashimoto S, Ochs RL, Rosen F, et al. Chondrocyte-derived
apoptotic bodies and calcification of articular cartilage. Proc
Natl Acad Sci USA 1998; 95: 3094–9.
Steller H. Mechanisms and genes of cellular suicide. Science
1995; 267: 1445–9.
Thompson CB. Apoptosis in the pathogenesis and treatment of
disease. Science 1995; 267: 1456–62.
Thornberry NA, Lazebnik Y. Caspase: enemies within. Science
1998; 281: 1312–6.
Barros LF, Stutzin A, Calixto A, et al. Nonselective cation
channels as effectors free radical-induced rat liver cell necrosis. Hepatology 2001; 33: 114–22.
Chai JC, Wu JW, Kyin S, Wang X, Shi Y. Structural and
biochemical basis of apoptotic activation by Smac ⁄ DIABLO.
Nature 2000; 406: 855–62.
Pinkoski MJ, Brunner T, Green DR, Lin T. Fas and Fas ligand
in gut and liver. Am J Phyiol 2000; 278: G354–6.
Iwakiri R, Gotoh Y, Noda H, et al. Programmed cell death in
rat intestine: effect of feeding and fasting. Scand J Gastroenterol 2001; 36: 39–47.
Ikeda H, Suzuki Y, Suzuki K, et al. Apoptosis is a major mode
of cell death caused by ischemia and ischemia ⁄ reperfusion
injury to the rat intestinal epithelium. Gut 1998; 42: 530–7.
Chance B, Sies H, Boveris A. Hydrogenperoxide metabolism in
mammalian organs. Physiol Rev 1979; 59: 527–605.
Hagen TM, Wierzbicka GT, Bowman BB, Aw TY, Jones DP.
Fate of dietary glutathione: disposition in the gastrointestinal
tract. Am J Physiol 1990; 259: G530–5.
Aw TY, Wierzbicka GT, Jones DP. Oral glutathione enhances
tissue glutathione in vivo. Chem Biol Interact 1991; 80: 89–
97.
Aw TY, Marianne WW. Intestinal absorption and lymphatic
transport of peroxidized lipid in rats: effect of exogenous GSH.
Am J Physiol 1992; 263: G665–72.
Noda T, Iwakiri R, Fujimoto K, Aw TY. Induction of mild
intrsavellular redox imbalance inhibits proliferation of CaCo-2
cell. FASEB J 2001; 15: 1231–9.
Kokura S, Yoshikawa T, Naito Y, et al. Effects of rebamipide, a
novel anti-ulcer agent, on gastric mucosal injury induced by
platelet-activating factor in rats. Dig Dis Sci 1997; 42: 2566–
71.
2003 Copyright Blackwell Publishing Ltd, Aliment Pharmacol Ther 18 (Suppl. 1), 139–145
ANTIOXIDANT AND INTESTINAL APOPTOSIS
35 Ogino K, Hobara T, Ishiyama H, et al. Antiulcer mechanism of
action of rebamipide, a novel on diethyldithiocarbamateinduced antral gastric ulcers in rats. Eur J Pharmacol 1992;
212: 9–13.
36 Sakurai K, Yamasaki K. Protective effect of rebamipide against
hydrogen peroxide-induced hemorrhagic mucosal lesion in rat
stomach. Jpn J Pharmacol 1994; 64: 229–34.
37 Yoshikawa T, Naito Y, Kondo M. Free radical scavenging
activity of the novel anti-ulcer agent rebamipide studied by
145
electron spin resonance. Arzneimittelforschung 1993; 43:
363–6.
38 Phillis JW, Estevez AY, O’Regan MH. Protective effects of
the free radical scavengers, dimethylsulfoxide and ethanol,
in cerebral ischemia in gerbils. Neurosci Lett 1998; 244:
109–11.
39 Iwakiri R, Sakemi T, Fujimoto K. Dimethylsulfoxide for renal
dysfunction caused by systemic amyloidosis complicating
Crohn’s disease. Gastroenterology 1999; 117: 1031–2.
2003 Copyright Blackwell Publishing Ltd, Aliment Pharmacol Ther 18 (Suppl. 1), 139–145