Effects of zinc acexamate (NAS-501) on superoxide radicals and lipid peroxidation of rat gastric mucosa

Article abstract of DOI:10.1159/000028283

Zinc acexamate (NAS-501), an anti-ulcer agent, has been reported to prevent various acute experimental gastric mucosal lesions and duodenal ulcers in rats. In order to clarify the mechanisms by which NAS-501 exhibits the anti-ulcer effects, we investigated the anti-oxidative effects of NAS-501 in vitro and in vivo. NAS-501 significantly reduced the superoxide radical-dependent chemiluminescence, generated by hypoxanthine-xanthine oxidase, rat neutrophils and guinea-pig macrophages in vitro. These in vitro effects were also confirmed by electron spin resonance using a 5,5-dimethyl-1-pyrroline-N-oxide spin-trapping method. In addition, NAS-501 significantly inhibited lipid peroxidation induced by increasing concentrations of Fe²⁺/ascorbate in rat gastric mucosal homogenate in vitro. Oral administration of NAS-501 (30 mg/kg) significantly inhibited production of thiobarbituric acid-reactive substance in rat gastric mucosa following per os instillation of 60% ethanol in 150 mmol/l HCI in vivo. These results suggest that NAS-501 exhibits the preventive effect from acute gastric mucosal lesions by the anti-oxidative activity.

Full text of DOI:10.1159/000028283

Original Paper
Pharmacology 1999;58:209–219
Received: May 14, 1998
Accepted: October 17, 1998
EffectsofZincAcexamate(NAS-501)
on Superoxide Radicals and Lipid
PeroxidationofRatGastricMucosa
Yasuhiro Tsutsui Yukio Nakamura Shiho Yamaguchi
Nobuyo Kawanaka Makoto Sato
Pharmaceutical Research Center, Nissho Corporation, Osaka, Japan
KeyWords
in vitro. Oral administration of NAS-501
(30 mg/kg) significantly inhibited production
of thiobarbituric acid-reactive substance in
rat gastric mucosa following per os instilla-
tion of 60% ethanol in 150 mmol/l HCl in vivo.
These results suggest that NAS-501 exhibits
the preventive effect from acute gastric mu-
Zinc acexamate W Superoxide radical W Lipid
peroxidation W Acute gastric mucosal injury W
Inflammatory cell
Abstract
Zinc acexamate (NAS-501), an anti-ulcer cosal lesions by the anti-oxidative activity.
agent, has been reported to prevent various
acute experimental gastric mucosal lesions
and duodenal ulcers in rats. In order to clarify
the mechanisms by which NAS-501 exhibits
the anti-ulcer effects, we investigated the
anti-oxidative effects of NAS-501 in vitro and
Introduction
Recent studies have demonstrated that He-
in vivo. NAS-501 significantly reduced the licobacter pylori- and nonsteroidal anti-in-
superoxide radical-dependent chemilumi- flammatory drug-induced gastropathies and
nescence, generated by hypoxanthine-xan- active chronic gastritis are localized in areas
thine oxidase, rat neutrophils and guinea-pig of inflammation dominated by infiltration
macrophages in vitro. These in vitro effects with inflammatory cells such as neutrophils,
were also confirmed by electron spin reso- monocytes and macrophages [1–3]. These in-
nance using a 5,5-dimethyl-1-pyrroline-N-ox- flammatory cells activated in local areas gen-
ide spin-trapping method. In addition, NAS- erate reactive oxygen species and cytokines [4,
5
01 significantly inhibited lipid peroxidation 5]. In particular, superoxide radicals have
2+
induced by increasing concentrations of Fe / been proposed to play important roles in tis-
ascorbate in rat gastric mucosal homogenate sue injury, because superoxide radical gener-
©
1999 S. Karger AG, Basel
Yasuhiro Tsutsui
ABC
Fax + 41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
0031–7012/99/0584–0209$17.50/0
Pharmaceutical Research Center, Nissho Corporation
2-32-4, Shinzaike, Settsu, Osaka 566-0055 (Japan)
Tel. +81 66 827 0920, Fax +81 66 827 0892
E-Mail tsutsui_alab@star.nissho.co.jp
Accessible online at:
http://BioMedNet.com/karger

ated in vivo is converted into the other more poxanthine (HX), allopurinol and bovine serum albu-
min (Sigma, St. Louis, Mo., USA); superoxide dismu-
tase (SOD) and xanthine oxidase (XO; Boehringer
Mannheim, Mannheim, Germany); 5,5-dimethyl-1-
pyroline-N-oxide (DMPO; Labotec, Tokyo, Japan);
reactive oxygen species, and gives more tissue
injury [6]. Thus a number of studies have sug-
gested a possible contribution of free radicals
in the development of digestive injury.
diethylenetriamine pentaacetic acid (DETAPAC), ·-
Zinc, a cofactor of more than 200 metal- tocopherol, NADPH and 3-t-butyl-4-hydroxyanisole
(BHA; Nacalai Tesque, Kyoto, Japan); malonalde-
loenzymes, has been known to play an impor-
tant role in the DNA and RNA synthesis [7],
the stabilization of biological membranes [8,
hyde bis(dimethylacetal) (MDA; Aldrich, Milwaukee,
Wisc., USA); Diff-Quik® (International Reagents,
Kobe, Japan); sucralfate (Chugai Pharmaceutical, To-
9
], wound healing [10, 11] and protection kyo, Japan). Polaprezinc was synthesized in our labo-
of lipid peroxidation [12]. Zinc acexamate ratory. Zinc acexamate (NAS-501) was supplied from
Vinas (Barcelona, Spain). Rabbit anti-rat neutrophil
(NAS-501), an anti-ulcer agent, has been re-
serum was prepared as described by Murakami et al.
6].
ported to prevent various experimental acute
gastric mucosal lesions and duodenal ulcers in
rats [13]. Polaprezinc, a zinc complex of car-
nosine, has also been reported to have anti-
oxidative activity [14], membrane-stabilizing
activity [15], and wound-healing activity [16].
This drug is more effective than zinc sulfate,
[
Preparation of Neutrophils and Macrophages
Rat peritoneal neutrophils were harvested 15–16 h
after intraperitoneal injection of 100 ml/kg saline con-
taining 0.3% oyster glycogen. Peritoneal macrophages
of guinea pigs were obtained 4 days after intraperitone-
al injection of 25 ml/animal paraffin liquid. After
carnosine or a combination of zinc sulfate and removing erythrocytes by hypotonic lysis, the peritone-
carnosine on anti-oxidative effects in vivo al neutrophils or macrophages were washed twice and
resuspended in Hanks’ balanced salt solution, then
[
17].
kept on ice until assay. The purity and viability of these
cells were both more than 95%, as determined by Diff
Quik staining and the trypan blue dye exclusion test,
In the present study, we investigated the
anti-oxidative effects of NAS-501 in compari-
son with polaprezinc in vitro and in vivo to respectively. NAS-501 (0.001–10 mmol/l) did not sig-
determine the mechanism by which NAS-501 nificantly change the viability of these cells, as deter-
®
mined by the trypan blue dye exclusion test.
protects the acute gastric mucosal lesions.
Measurement of Superoxide Radicals Generated
by the HX-XO System, Neutrophils or
Materials and Methods
Macrophages Using the CLA-Dependent
Chemiluminescence Method
Animals
Superoxide radicals derived from the HX-XO sys-
tem, neutrophils or macrophages were assayed using
the CLA-chemiluminescence method, as described by
Sugioka et al. [18]. In the HX-XO system, the reaction
mixture containing 150 Ìl of 125 mmol/l Tris-HCl
buffer (pH 6.7) with 50 Ìmol/l HX, 1 Ìmol/l CLA and
various concentrations of test agents was preincubated
for 2 min at 25°C, then the reaction was initiated by
addition of 50 Ìl of 2 ! 10 untis/ml XO. After incu-
bating for 1 min at 25°C, total chemiluminescence
intensity was measured for 0.5 min with a luminome-
ter (model 20e, Turner, USA).
Male Jbc:Sprague-Dawley rats, weighing 200–
00 g, and male Hartley guinea pigs, weighing 320–
70 g, from Keari (Osaka, Japan) were used. The ani-
mals were kept at 23 B2°C under a 12-hour light-dark
cycle. They were kept in individual cages with raised
mesh bottoms to prevent coprophagy. They were de-
prived of food, but allowed free access to tap water for
3
3
–
3
2
4 h before the in vivo experiments.
Drugs
The following chemicals were obtained from com-
mercial sources: 2-methyl-6-phenyl-3,7-dihydro-imi-
dazo[1,2-a]pyrazin-3-one (CLA; Tokyo Kasei Co., To-
kyo, Japan); N-formyl-methionyl-leucyl-phenylalanine
For measurement of superoxide radicals generated
by neutrophils or macrophages, 150 Ìl of 50 mmol/l
6
Tris-HCl buffer (pH 6.7) containing 10 cells/ml cell
(FMLP), phorbol 12-myristate 13-acetate (PMA), hy-
suspension, 1 Ìmol/l CLA and various concentrations
210
Pharmacology 1999;58:209–219
Tsutsui/Nakamura/Yamaguchi/
Kawanaka/Sato

of test agents was preincubated at 37°C for 5 min, then
0 Ìl of stimulant (10–6 mol/l FMLP or 250 ng/ml
Effect of NAS-501 on NADPH Oxidation in Rat
Neutrophils
5
PMA) was added to generate superoxide radicals.
Maximum intensity of chemiluminescence was mea-
sured for 3 min at 37°C with a luminometer.
In order to confirm suppressive effect of NAS-501
on the generation of superoxide radicals, the rate of
NADPH oxidation was measured [21, 22]. Rat neutro-
phils were isolated as described above. The neutrophils
7
Measurement of Superoxide Radicals Generated
by the HX-XO System, Neutrophils or
Macrophages Using the Electron Spin Resonance
(a final cell concentration of 2 ! 10 cells/ml in RPMI
1640) were prewarmed for 5 min at 37°C and acti-
vated by the addition of PMA at a final concentration
of 1 Ìg/ml. After 5 min, the activation was terminated
by addition of 5 vol of ice-cold 0.34 mol/l sucrose. The
cells were centrifuged at 200 g for 10 min at 4°C,
resuspended in 1 ml of distilled water and immediately
disrupted by sonication four times for 4 s on melting
ice. Then, 1 ml of ice-cold 0.68 mol/l sucrose was add-
ed to sonicate, and unbroken cells and cell debris were
removed by centrifugation at 500 g for 10 min at 4°C.
The supernatant was centrifuged at 27,000 g for
30 min at 4°C. The final pellet was resuspended at
(ESR) Spin-Trapping Method
Superoxide radicals were assayed by the ESR spin-
trapping method using DMPO as a spin-trapping agent
[19]. In the HX-XO system, 150 Ìl of 50 mmol/l Tris-
HCl buffer (pH 6.7) containing 0.5 mmol/l HX,
0
.1 mmol/l DETAPAC, 0.44 mol/l DMPO and various
–
3
concentrations of test agents was mixed with 5 ! 10
units/ml XO. The mixture was transferred into a stan-
dard quartz flat cell (Labotec, Tokyo, Japan) and the
ESR spectra of DMPO-OOH, the spin-trapped adduct
of superoxide radical, was measured by the ESR spec-
trometer (JEOL-JES-FE1X ESR spectrometer, JEOL,
Tokyo, Japan). The conditions of the spectrometer
were as follows: microwave power 10 mW; magnetic
field 3,320 B 100 G; modulation frequency 100 kHz,
6
a concentration equivalent to 4 ! 10 cells/ml in
0.34 mol/l sucrose. Reaction mixtures were prepared
in a total volume of 0.5 ml, using 50 mmol/l Tris HCl
buffer (pH 7.0) with 1 mmol/l sodium azide. The tubes
containing enzyme fraction were prewarmed for 3 min
at 25°C and after addition of 50 Ìl of NAS-501 (0.01–
1 mmol/l) or above buffer (as a control) incubated for a
further 5 min. Enzyme reaction was initiated by add-
ing 50 Ìl of NADPH (0.2 mmol/l) and absorbance at
340 nm was measured for 5 min at 25°C.
1
2
G; amplitude 6.3 ! 100; response 0.1 s; sweep time
min; temperature 25°C.
For the measurement of superoxide radicals gener-
ated by neutrophils and macrophages, 150 Ìl of reac-
tion medium containing 106 cells/ml cell suspension
and various concentrations of test agents was pre-
incubated for 2 min, then 0.1 mmol/l DETAPAC,
Assay of Lipid Peroxidation in Homogenate of Rat
Gastric Mucosa in vitro
36.8 mmol/l DMPO and stimulant (250 ng/ml PMA)
were added to the mixture. The reaction mixture was
transferred into a quartz cell and the signal of DMPO-
OOH was measured using the ESR spectrometer 4 min
after addition of PMA. The conditions of the spec-
trometer were as follows: microwave power 20 mW;
magnetic field 3,326 B 100 G; modulation frequency
Homogenate of rat gastric mucosa was prepared as
follows: the gastric mucosa of 24-hour fasted normal
rat was removed by a glass slide and homogenized in a
Potter homogenizer at a concentration of 100 mg wet
tissue in 1.8 ml of 10 mmol/l Tris-HCl buffer (pH 6.7).
Five hundred microliters of the homogenate was
added to a reaction mixture containing 0.5 ml of
10 mmol/l Tris-HCl buffer (pH 6.7), 0.1 mmol/l L-
1
00 kHz, 1 G; amplitude 2.0 ! 100; response 0.1 s;
sweep time 2 min; temperature 25°C.
3 2 2
ascorbic acid, 0.1 mmol/l FeCl , 1 mmol/l H O and
Effects of NAS-501 and Allopurinol on the Activity
of XO
various concentrations of test agents, then the mixture
was incubated for 3 h at 37°C. The reaction was termi-
nated by adding 50 Ìl of 100 mmol/l BHA. Thiobarbi-
turic acid-reactive substances (TBARS) in the superna-
tant (1,100 g, 10 min) of the reaction mixture were
measured by the method of Ohkawa et al. [23] using
MDA as an external standard.
In order to rule out a possible effect of NAS-501 on
XO activity, uric acid generated during superoxide
radical generation in the xanthine-XO system was
measured [20]. Enzyme reaction was initiated by add-
ing XO (16.4 ! 10–3 units/ml) to the reaction mixture
containing 0.1 mmol/l xanthine, 0.01% H
units/ml catalase and various concentrations of NAS-
01 or allopurinol, then absorbance at 293 nm was
2 2
O , 43.3
Assay of TBARS in Rat Gastric Mucosa following
HCl/Ethanol Instillation in vivo
5
measured for 1 min at 25°C.
Acute gastric injury was produced by HCl/ethanol
instillation according to the method of Mizui and
Anti-Oxidative Activity of Zinc
Acexamate
Pharmacology 1999;58:209–219
211

Fig. 1. Effects of NAS-501, SOD and allopurinol on
HX-XO-derived superoxide radicals in the CLA-
chemiluminescence method. Data are expressed as the
mean B SEM for 3 assays.
Fig. 2. Effects of NAS-501 and allopurinol on XO
activity. The generation of uric acid in the presence of
various concentrations of NAS-501 ([) or allopurinol
(P) was measured to assess XO activity. Each point
represents the mean B SEM for 3 assays. Significant
difference from the control: ** p ! 0.01.
Doteuchi [24]. The 24-hour fasted rats were given
1
1
50 mmol/l HCl/60% ethanol orally at a volume of
ml/200 g body weight 30 min after the administra-
tion of NAS-501, polaprezinc and sucralfate. SOD
45,000 units/kg, i.v.) and allopurinol (30 mg/kg, i.p.)
Results
(
were given 5 or 60 min before HCl/ethanol instillation,
respectively. Anti-neutrophil serum was given intrave-
nously at a dose of 0.5 ml/kg 2 h before HCl/ethanol
instillation. Each control animal received the vehicle
Effects of NAS-501 on HX-XO-Derived
Superoxide Radicals in the
CLA-Chemiluminescence Method
(
0.5% carboxymethylcellulose in saline for NAS-501,
Figure 1 shows the effects of NAS-501,
SOD and allopurinol on HX-XO-derived su-
peroxide radicals in the CLA-chemilumines-
cence method. CLA-dependent chemilumi-
nescence in the HX-XO system was sup-
polaprezinc and sucralfate; saline for SOD and allopu-
rinol; preimmune rabbit serum for anti-neutrophil se-
rum) alone. Sixty minutes after the instillation of HCl/
ethanol, the rats were killed by exsanguination via the
abdominal aorta, and the stomach was removed. The
gastric mucosa was scrapped with a glass slide and pressed by NAS-501 at concentrations of 1–
homogenized at a concentration of 100 mg wet tissue
1
0 Ìmol/l. SOD and allopurinol also inhibited
in 0.9 ml of 1.15% KCl. TBARS in gastric mucosa was
measured, as described above. The level of TBARS is
expressed as picomoles of MDA per milligram of pro-
tein. Protein was determined according to the method
of Lowry et al. [25].
CLA-dependent chemiluminescence. To rule
out a direct effect of NAS-501 on XO activity,
we determined the effect of NAS-501 on uric
acid generation by a xanthine-XO system in
comparison with allopurinol (fig. 2). Allopuri-
nol significantly inhibited XO activity at con-
centrations of 3 ! 10–6 to 10 mol/l, while
NAS-501 had no effect within the concentra-
tion range 10–7 to 10 mol/l. These results
Statistical Analyses
The data are presented as the means B SEM. Sta-
tistical significance was estimated by Dunnett’s multi-
ple comparison test, or Student’s t test, and values of
p ! 0.05 were regarded as significant.
–4
–3
212
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Fig. 3. Effects of NAS-501 and SOD on the generation of superoxide radicals in FMLP (P)-
and PMA ([)-stimulated neutrophils of rats. Superoxide radicals were measured by the CLA-
chemiluminescence method. Data are expressed as the mean B SEM for 3 assays.
indicate that NAS-501 scavenges the superox-
ide radical without inhibiting the activity of
XO.
Effect of NAS-501 on NADPH Oxidation
in Rat Neutrophils
The particulate fractions from activated
neutrophils are known to generated superox-
ide radicals in the presence of NADPH [26,
27]. In this study, we measured a decrease in
absorbance at 340 nm by NADPH oxidation.
As shown in figure 5, NAS-501 inhibited oxi-
Effects of NAS-501 on Neutrophils or
Macrophage-Generated Superoxide
Radicals in the CLA-Chemiluminescence
Method
The effects of NAS-501 and SOD on dation of NADPH in a concentration-depen-
FMLP- or PMA-induced production of super- dent manner.
oxide radicals by rat neutrophils were studied
by the CLA-chemiluminescence method. As
shown in figure 3, NAS-501, as well as SOD,
inhibited chemiluminescence induced by
either FMLP or PMA in a concentration-
dependent manner.
Effects of NAS-501 on HX-XO- or
Cell-Derived Superoxide Radicals in the
ESR Spin-Trapping Method
The in vitro effect of NAS-501 on superox-
ide radicals was also studied by the ESR spin-
The effects of NAS-501 on FMLP- or trapping method. As shown in figure 6, the
PMA-induced production of superoxide rad- DMPO-OOH signal (the hyperfine splittings;
ß
Á
icals by guinea-pig macrophages were also AN = 14.28 G, AH = 11.28 G, and AH
=
examined by the CLA-chemiluminescence 1.27 G) appeared, when XO was added to the
method. As shown in figure 4, NAS-501 in- reaction mixture of the HX-XO system or
hibited FMLP- or PMA-induced chemilumi- PMA to that of neutrophils or macrophages.
nescence in a concentration-dependent man- The intensity of the DMPO-OOH signal in
ner.
the HX-XO system was suppressed in the
Anti-Oxidative Activity of Zinc
Acexamate
Pharmacology 1999;58:209–219
213

Fig. 4. Effects of NAS-501 on the generation of super-
oxide radical in FMLP (P)- or PMA ([)-stimulated
macrophages of guinea pigs. Superoxide radicals were
measured by the CLA-chemiluminescence method.
Data are expressed as the mean B SEM for 3 assays.
Fig. 5. Effect of NAS-501 on NADPH oxidation in rat
neutrophils. The rate of NADPH oxidation in the pres-
ence of various concentrations of NAS-501 was record-
ed at 340 nm at 25°C. Data are expressed as the mean
B SEM for 3 assays.
presence of NAS-501 (1 mmol/l) and SOD. only at a high concentration (10 mmol/l),
NAS-501 (1 mmol/l) or SOD also reduced the while ·-tocopherol exhibited a potent effect at
DMPO-OOH signal intensity that was in- concentrations of 0.03–10 mmol/l.
duced by neutrophils or macrophages. These
results indicate that NAS-501 has a suppres-
sive activity on the production of superoxide
radicals by neutrophils, macrophages or an
ability to scavenge these radicals.
Effects of NAS-501 on the Lipid
Peroxidation of Rat Gastric Mucosa by
Acute Gastric Injury in vivo
Effects of NAS-501 on the lipid peroxida-
tion of gastric mucosa were studied in rats
with acute gastric injury. Acute gastric injury
was induced by an instillation of HCl/ethanol
after administration of NAS-501 or other
Effects of NAS-501 on the Lipid
Peroxidation of Rat Gastric Mucosal
Homogenate in vitro
Effects of NAS-501 on lipid peroxidation drugs. TBARS levels in gastric mucosa of the
were examined in vitro using rat gastric mu- non-treated group significantly elevated 5–
cosal homogenate by measuring the levels of 60 min after instillation of HCl/ethanol in a
TBARS in comparison to those of polaprezinc time-dependent manner. Elevation of muco-
and ·-tocopherol. As shown in figure 7, NAS- sal TBARS 60 min after instillation was com-
5
01 significantly inhibited lipid peroxidation pletely inhibited by NAS-501 at a dose of
of mucosal homogenate at concentrations of 30 mg/kg (fig. 8, 9). A similar effect was ob-
.3–10 mmol/l. Polaprezinc revealed an effect tained by sucralfate (300 mg/kg, p.o.) or SOD
0
214
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Tsutsui/Nakamura/Yamaguchi/
Kawanaka/Sato

Fig. 6. Scavenging effects of NAS-501 and SOD on the formation of DMPO spin-trapped
adduct by ESR spectrometer. Superoxide radicals were generated by the HX-XO system,
PMA-stimulated neutrophils or PMA-stimulated macrophages, then the ESR spectra of
DMPO-OOH, the spin-trapped adduct of superoxide radical, was measured by the ESR spec-
trometer. A = Control; B = NAS-501 (1 mmol/l), and C = SOD (100 mmol/l in the HX-XO
system and macrophages, and 10 nmol/l in neutrophils).
(
(
45,000 units/kg, i.v.), but not by polaprezinc
10–100 mg/kg, p.o.) or allopurinol (30 mg/
Discussion
kg, i.p.). Interestingly, an intravenous injec-
NAS-501 has been reported to prevent var-
tion of rabbit anti-rat neutrophil serum exhib- ious acute experimental gastric mucosal le-
ited an inhibitory effect on mucosal lipid sions and duodenal ulcers in the rat [13].
peroxidation by a HCl/ethanol instillation, However, the mechanisms of anti-ulcer action
suggesting an involvement of neutrophils in of NAS-501 has not yet been clarified. Since
this model.
reactive oxygen species generated by ethanol,
Anti-Oxidative Activity of Zinc
Acexamate
Pharmacology 1999;58:209–219
215

Fig. 7. Effects of NAS-501, polaprezinc and ·-tocopherol on lipid peroxidation of rat gastric
mucosal homogenate in vitro. Gastric mucosal homogenate was incubated for 3 h at 37°C
with Fe2+/ascorbate and various concentrations of NAS-501 (P), polaprezinc ([) or ·-toco-
pherol (o). TBARS were assayed as described in the text. Each point represents the mean B
SEM for 3 assays. Significant difference from the control: * p ! 0.05; ** p ! 0.01.
nonsteroidal anti-inflammatory drugs and
Helicobacter pylori have been recognized as
the partial cause of gastric ulcer or gastritis [5,
28], we have studied the anti-oxidative activi-
ty of NAS-501 in vitro and in vivo.
The ability of NAS-501 to scavenge super-
oxide radicals was tested in the HX-XO sys-
tem in vitro using the CLA-chemilumines-
cence method. NAS-501 revealed an inhibito-
ry effect on the HX-XO-induced chemilumi-
nescence without inhibiting XO activity, indi-
cating that NAS-501 has a scavenging effect
on superoxide radicals. This scavenging effect
of NAS-501 was also confirmed by the ESR
spin-trapping method. Since there is much
evidence suggesting that the inflammatory
cells, such as neutrophils, monocytes and
Fig. 8. Changes in the lipid peroxidation of rat gastric
mucosa after HCl/ethanol instillation by treatment
with NAS-501 (P, 30 mg/kg) or vehicle ([, 0.5% car- macrophages, are sources of superoxide radi-
boxymethylcellulose in saline). Each point represents cals in gastritis [1–3], we examined the effects
the mean B SEM for 6 rats. Significant difference
from the values of before HCl/ethanol instillation:
of NAS-501 on superoxide radicals generated
by activated neutrophils or macrophages us-
ing two different methods: the CLA-chemilu-
minescence method, and the ESR spin-trap-
*
p ! 0.05; ** p ! 0.01.
216
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Fig. 9. Effects of NAS-501, polaprezinc, sucralfate,
SOD, ALP and anti-neutrophil serum on the lipid
peroxidation of rat gastric mucosa following HCl/etha-
nol instillation. One hour after instillation of 60% eth-
anol in 150 mmol/l HCl, animals were sacrificed, and
the TBARS levels in gastric mucosa were measured as
described in the text. NAS-501, polaprezinc, sucralfate
and control (0.5% carboxymethylcellulose in saline)
were given orally 30 min before HCl/ethanol instilla-
tion. SOD (45,000 units/kg, i.v.) and allopurinol
(30 mg/kg, i.p.) were given 5 or 60 min before HCl/eth-
anol instillation, respectively. Anti-neutrophil serum
or preimmune rabbit serum was given intravenously at
a dose of 0.5 ml/kg 2 h before HCl/ethanol instillation.
Each column represents the mean B SEM for 5 rats.
Significant difference from the control: * p ! 0.05;
** p ! 0.01.
ping method. Either method demonstrated suggested that NAS-501 prevented the muco-
that NAS-501 was effective to reduce produc- sal membrane from lipid peroxidation by in-
tion of superoxide radicals from these inflam- hibiting a conversion of peroxide into more
matory cells. Furthermore, NAS-501 also ex- reactive hydroxyl radicals or scavenging inju-
hibited an inhibitory effect on the NADPH rious radicals.
oxidation by the superoxide radical generat-
In order to clarify whether NAS-501 exhib-
ing enzyme, NADPH oxidase in rat neutro- its an anti-ulcer action by preventing lipid
phils. These results suggest that an anti-oxida- peroxidation of gastric mucosa from injurious
tive activity of NAS-501 depends upon its radicals, we studied the anti-oxidative effect
ability not only to scavenge superoxide radi- of NAS-501 in experimentally induced acute
cals but also to inhibit the generation of super- gastric mucosal injury. We used HCl/ethanol
oxide radicals.
as an acute necrotizing agent in this model,
It is well documented that the lipid peroxi- since NAS-501 has been demonstrated to be
dation by superoxide radicals or hydrogen effective in this model [13], and the gastric
peroxide in vivo depends upon a conversion mucosal injury induced by ethanol is associat-
of these peroxides into more reactive species, ed with active oxygen species [32, 33]. We
such as hydroxyl radicals [29, 30]. In fact, observed that an instillation of HCl/ethanol
Dudeja and Brastitus [31] demonstrated that resulted in elevation of TBARS levels in gas-
hydroxyl radicals play an important role in tric mucosa, and this elevation was inhibited
2+
lipid peroxidation induced by the Fe /ascor- by an intravenous injection of anti-neutrophil
bate system in tissue homogenate. Thus it was serum at a dose of 0.5 ml/kg; the numbers of
Anti-Oxidative Activity of Zinc
Acexamate
Pharmacology 1999;58:209–219
217

neutrophils in the peripheral blood were sup- effects of these drugs seems to be due to a dif-
pressed to about 10% of those in preimmune ference in their potencies of anti-oxidative
serum-treated animals for up to 12 h (data not activity, as suggested by the following evi-
shown). Pretreatment with NAS-501 at a dose dence: first, NAS-501 reduced the amount of
of 30 mg/kg resulted in complete inhibition of superoxide radicals generated by PMA-stimu-
HCl/ethanol-induced lipid peroxidation in lated neutrophils, while polaprezinc does not
gastric mucosa. NAS-501 at a dose of 30 mg/ [14], and second, NAS-501 inhibited lipid pe-
kg, but not 10 mg/kg, exhibited a potent anti- roxidation of gastric mucosal homogenate in
oxidative action as well as a protective action vitro at lower concentrations than those of
on the HCl/ethanol-induced lesions, suggest- polaprezinc. In contrast to polaprezinc, su-
ing that the in vivo effect of NAS-501 on gas- cralfate inhibited elevation of TBARS levels
tric lesion may depend on its anti-oxidative in gastric mucosa at 300 mg/kg. This observa-
effect. A concentration of NAS-501 in gas- tion might be supported by a previous re-
tric contents after an oral administration of ported that AlCl protected cultured rat gas-
3
3
3
0 mg/kg was estimated as approximately tric mucosal cells from 15% ethanol-induced
0 mmol/l that was sufficient for exhibiting damage in which oxygen free radicals partici-
an anti-oxidative activity in vitro, supporting pate [34].
that the protective action of NAS-501 on gas-
In conclusion, we demonstrated that NAS-
tric lesions is responsible for its anti-oxidative 501 has an anti-oxidative activity by reducing
effect. However, a possible effect of NAS-501 superoxide radical production in vitro. NAS-
on neutrophil infiltration remains to be clari- 501 effectively inhibited lipid peroxidation
fied. Further studies will be necessary to re- injury by HCl/ethanol instillation, probably
solve the effect of NAS-501 on leukocyte che- by inhibiting production of oxygen radicals
motaxis.
Polaprezinc, an organic zinc compound
from activated neutrophils in injured tissues.
similar to NAS-501, has been reported to be a
more effective anti-oxidative agent than inor-
ganic zinc substance [17]. However, this drug
failed to prevent lipid peroxidation of gastric
mucosa by HCl/ethanol instillation even at a
dose of 100 mg/kg, while NAS-501 was effec-
Acknowledgements
We wish to thank Prof. Dr. H. Sayo, Kobe Gakuin
University, Kobe, Japan, for analyses by ESR spec-
trometer and his advice, and Prof. Dr. H. Okamoto,
Kobe Gakuin University, for critically reviewing the
tive at 30 mg/kg. This difference in the in vivo manuscript.
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