From the Departments of Physiology and
¶ Department of Immunology, Indian Institute of Chemical
Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India
Received for publication, October 9, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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The mechanism of the antiulcer effect of
omeprazole was studied placing emphasis on its role to block oxidative
damage and apoptosis during ulceration. Dose-response studies on
gastroprotection in stress and indomethacin-induced ulcer and
inhibition of pylorus ligation-induced acid secretion indicate that
omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent
mechanism for its antiulcer effect. Time course studies on
gastroprotection and acid reduction also indicate that omeprazole
almost completely blocks lesions at 1 h when acid inhibition is
partial. The severity of lesions correlates well with the increased
level of endogenous hydroxyl radical (·OH), which when scavenged
by dimethyl sulfoxide causes around 90% reduction of the lesions,
indicating that ·OH plays a major role in gastric damage.
Omeprazole blocks stress-induced increased generation of ·OH and
associated lipid peroxidation and protein oxidation, indicating that
its antioxidant role plays a major part in preventing oxidative damage.
Omeprazole also prevents stress-induced DNA fragmentation, suggesting
its antiapoptotic role to block cell death during ulceration. The
oxidative damage of DNA by ·OH generated in vitro is
also protected by omeprazole or its analogue, lansoprazole.
Lansoprazole when incubated in a ·OH-generating system scavenges
·OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak at
m/z 385, which is 16 mass units higher than
that of lansoprazole (m/z 369). The product
shows no additional aromatic proton signal for aromatic hydroxylation
in 1H NMR. The product absorbing at 278 nm shows no
alkaline shift for phenols, thereby excluding the formation of
hydroxylansoprazole. The product is assigned to lansoprazole sulfone
formed by the addition of one oxygen atom at the sulfur center
following attack by the ·OH. Thus, omeprazole plays a
significant role in gastroprotection by acting as a potent antioxidant
and antiapoptotic molecule.
Proton pump inhibitors (1) such as omeprazole, lansoprazole,
pantoprazole, and rabeprazole are extensively used for therapeutic control of acid-related disorders including
gastroesophageal reflux disease and
Zollinger-Ellison syndrome and for peptic-ulcer disease caused by
stress (stress-related erosive syndrome), nonsteroidal antiinflammatory
drugs, and Helicobacter pylori infection (2-5). These
compounds share a common structural motif contributed by a substituted
pyridylmethylsulfinyl benzimidazole (Fig.
1). Inhibition of gastric acid secretion
by these compounds is considered to be an important step to control the
disorders (6). Proton pump inhibitors inhibit acid secretion by
irreversibly interacting with the H+-K+-ATPase,
the terminal proton pump of the parietal cell (7, 8). In the acid space
of the secreting parietal cell or in the vicinity of the enzyme, these
compounds are converted to thiophilic sulfenamide or sulfenic acid,
which reacts mainly with the Cys-813 residue in the catalytic subunit
of the H+-K+-ATPase, which is critical for
enzyme inactivation (5). Although omeprazole, the primary member of the
proton pump inhibitors, has been extensively used to control these
disorders (2), lansoprazole, the second member of the substituted
benzimidazole containing a trifluoroethoxy group, has also been used
more recently (4).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Chemical structure of proton pump
inhibitors.
The role of acid in gastroduodenal pathogenesis has been extensively studied. Although gastric ulcer patients show normal or reduced level of acid secretion, duodenal ulcer patients usually secrete more acid (9, 10). In fact, "no acid, no ulcer" is the dictum for duodenal ulcer. Because 30% of patients having duodenal ulcer and very few patients with gastric ulcer are hyperchlorohydric (9), clearly factors other than acid are involved in the pathogenesis of gastroduodenal ulcer. Although the secreted acid itself is not sufficient for ulcer formation, its corrosive property and increased peptic activity is sufficient to aggravate the ulcer. Even the normal rate of acid secretion may cause ulceration in the breached mucosa when some gastroprotective factors are lost. Hence, acid suppression by omeprazole is a common practice to control gastroduodenal lesions (2, 5). Suppression of intragastric acid also helps in the healing of ulcer (11). In animals, the role of acid in gastric lesions has been studied using some animal models such as stress or nonsteroidal antiinflammatory drug-induced gastric ulcer. Stress itself inhibits gastric acid secretion through a central nervous reflex mechanism (12). Restraint cold stress or restraint water immersion stress induces gastric lesions, which are associated with a decreased or normal level of acid secretion (13, 14). Because restraint or water immersion stress significantly decreases acid secretion induced by pylorus ligation (14), acid plays a minor role in stress ulcer. Administration of antacids to neutralize secreted acid does not protect stress ulcer (15), suggesting that factors other than acid are involved in ulcer formation. However, in indomethacin-induced gastric damage, acidity may be increased because of decreased biosynthesis of prostaglandin (16, 17). Because acidity as high as 0.6 M HCl can experimentally produce gastric lesions (18), mild irritants like 0.35 M HCl prevents gastric damage caused by stronger necrotizing agent through "adaptive cytoprotection" mediated by increased formation of prostaglandin (19).
It is now generally agreed that gastric lesions develop when the
delicate balance between some gastroprotective and aggressive factors
is lost. Although the cellular and molecular basis of gastric mucosal
defense against gastrodamaging factors are known (20), the mechanism of
mucosal damage by the aggressive factors is not fully clear today.
Stress (13, 21, 22), nonsteroidal antiinflammatory drugs (23), and
H. pylori (24) cause mucosal damage through a number of
mechanisms, of which some reactive oxygen species
(ROS)1 such as O and release of cytochrome c to
activate caspase-3-like protease are involved in apoptotic cell death
in indomethacin ulcer (41, 42). Apoptosis also occurs because of nitric
oxide production through induction of nitric-oxide synthase by H. pylori (45). Involvement of ROS and oxidative damage of DNA and
DNA fragmentation have also been evident in apoptotic cell death in
gastric mucosal injury (39, 41, 45-47).
Although omeprazole is believed to offer its antiulcer activity through
acid suppression (1, 2, 5) by inactivating the
H+-K+-ATPase (5, 7, 8, 52), very little is
known regarding its role in controlling oxidative damage and apoptotic
cell death of the gastric mucosa during ulceration. The role of acid
suppression effect of omeprazole on gastroprotection against some
necrotizing agents (ethanol, acidified aspirin, hypertonic saline, 0.6 M HCl) has been studied earlier (18, 53), where evidence
has been provided to show that acid inhibition plays no significant
role on the gastroprotective effect of omeprazole. Moreover, omeprazole neither stimulates prostaglandin biosynthesis nor increases bicarbonate secretion to offer gastroprotection (18, 53). Thus, omeprazole exerts
its antiulcer activity through some other mechanism that has not been explored yet. Using animal models of stress and
indomethacin-induced gastric lesions and pylorus ligation-induced
acid secretion, evidence has been presented in this paper to show that
the gastroprotective effect of omeprazole is not mediated through its
acid inhibitory effect. Further evidence has been presented to show
that endogenous ·OH plays one of the major roles in gastric
lesions and that omeprazole acts as a potent antioxidant to scavenge
the endogenous ·OH, thereby preventing the oxidative damage by
increased lipid peroxidation and protein oxidation. Moreover, it offers
an antiapoptotic effect by blocking DNA fragmentation during
ulceration. Evidence has also been presented to show that omeprazole or
lansoprazole blocks ·OH-induced oxidative damage of DNA by
scavenging ·OH in vitro. Analysis of the major
oxidation product of lansoprazole indicates that this antioxidant
activity is due to scavenging of ·OH to form an oxygenated
product that is assigned to lansoprazole sulfone. The studies thus
provide new insights on the mechanism of the antiulcer effect of proton
pump inhibitors.
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MATERIALS AND METHODS |
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Drugs and Chemicals--
Omeprazole was a kind gift from Dr. W. Beil (Medizinische Hochschule, Hannover, Germany). Lansoprazole,
melatonin, -phenyl N-tert-butylnitrone (PBN),
thiobarbituric acid, ethidium bromide, ascorbic acid,
2,4-dinitrophenylhydrazine, collagenase type 1A, Pronase E, proteinase
K, RNase, catalase, guanidine HCl, Fast Blue BB salt,
tetraethoxypropane, benzenesulfinic acid, and
5,5- dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Sigma. Desferrioxamine was obtained from Ciba Geigy Ltd. Vitamin E
(
-tocopheryl acetate), Me2SO, and TLC plates coated with
silica gel 60 F254 were procured from Merck.
Animals Used-- Sprague-Dawley rats (200-250g) of both control and experimental groups kept separately in controlled condition were fasted for 24 h with water ad libitum. The control group received the vehicle only while the experimental group received omeprazole intraperitoneally 30 min prior to restraint cold stress or indomethacin administration for gastric ulceration or pylorus ligation for acid secretion. Animal experiments (n = 8-30) were carried out following the guidelines of the animal ethics committee of the institute. Human gastric mucosal biopsy specimens were obtained from the Cancer Centre Welfare Home and Research Institute (Kolkata, India) following approval by the human ethics committee of the institute.
Restraint Cold Stress-induced Gastric Ulceration-- The rats were immobilized under light ether anesthesia and subjected to cold (4 ± 1 °C) stress for 3.5 h (13). The severity of mucosal lesions was scored as ulcer index as follows: 0 = no pathology; 1 = a small ulcer (1-2 mm); 2 = a medium ulcer (3-4 mm); 4 = a large ulcer (5-6 mm); and 8 = larger ulcer (>6 mm). The sum of the total scores divided by the number of animals was expressed as the mean ulcer index (13). Luminal acid content was determined by titration with 1 mM NaOH using an autoburette pH stat system from Radiometer (Copenhagen, Denmark) (13).
Indomethacin-induced Gastric Ulceration-- The rats were orally fed with indomethacin at 48 mg/kg of body weight. After 4 h, the animals were killed, and gastric lesions in the mucosa (54) were scored and expressed as ulcer index as follows: 0 = no pathology; 1 = one pinhead ulcer. The sum of the total scores divided by the number of animals gives the ulcer index.
Pylorus Ligation-induced Gastric Acid Secretion-- Hypersecretion was induced in rats by pylorus ligation (55) under light anesthesia with ketamin (12 mg/kg of body weight). The animals were killed 2.5 h after ligation, and the clarified gastric fluid volume was determined and titrated for acid content with 1 mM NaOH (13).
Measurement of Lipid Peroxidation as an Index of Oxidative Damage-- Lipid peroxidation products of the mitochondrial membrane fraction of fundic stomach homogenate were determined (22, 32, 56) as thiobarbituric acid-reactive substances. The fundic stomach from control, stress-ulcerated, and omeprazole (4 mg/kg)-pretreated stressed rats was homogenized in ice-cold 0.9% saline in a Potter-Elvehjem glass homogenizer for 45 s to get 5% homogenate. One ml of the mitochondrial membrane fraction obtained after differential centrifugation (22) was allowed to react with 2 ml of trichloroacetic acid-thiobarbituric acid-HCl reagent containing 0.01% butylated hydroxytoluene, heated in a boiling water bath for 15 min, cooled, and centrifuged, and the supernatant was used for thiobarbituric acid-reactive substance determination at 535 nm using tetraethoxypropane as standard.
Measurement of Protein Carbonyl Content as an Index of Oxidative
Damage--
Protein oxidation was measured as carbonyl content (57) in
the low speed supernatant of the fundic stomach homogenate (32). The
fundic stomach from control, stress-ulcerated, and omeprazole (8 mg/kg)
pretreated stressed rats was homogenized in 50 mM sodium phosphate buffer, pH 7.4, in a Potter-Elvehjem glass homogenizer for
45 s to get 10% homogenate. After centrifugation at 600 × g for 10 min, the proteins from 0.8 ml of the supernatant
were precipitated with 5% trichloroacetic acid and allowed to react with 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine for 1 h. After precipitation with 10% trichloroacetic acid, the protein was
washed thrice with a mixture of ethanol-ethyl acetate (1:1), dissolved in 0.6 ml of a solution containing 6 M guanidine HCl in 20 mM potassium phosphate adjusted to pH 2.3 with
trifluoroacetic acid, and centrifuged, and the supernatant was used for
measurement of carbonyl content at 362 nm ( = 22000 M
1 cm
1).
Measurement of Endogenous ·OH-- Hydroxyl radical generated in the gastric mucosa was measured using Me2SO as ·OH scavenger (58-60). Briefly, the control group was kept at room temperature without any stress after administration (intraperitoneally) of 1 ml of Me2SO. The second group received the same amount of Me2SO 30 min before the onset of restraint cold stress. The third group received omeprazole (8 mg/kg intraperitoneally) 30 min prior to Me2SO administration and were then subjected to stress. After 3.5 h of stress, the animals were killed, and fundic stomach was processed for the extraction of methanesulfinic acid formed by reaction of ·OH with Me2SO. Methanesulfinic acid was allowed to react with the Fast Blue BB salt to yield an yellow chromophore that was measured at 425 nm using benzenesulfinic acid as standard.
Measurement of DNA Damage in Vivo as an Index of
Apoptosis--
To study DNA fragmentation as an index of apoptosis,
DNA was isolated from fundic mucosal surface epithelial cells of normal rats and rats subjected to restraint cold stress without or after pretreatment with omeprazole (8 mg/kg). Fundic mucosa (~1.5 g) from
three animals was scraped, minced separately in PBS-E (50 mM sodium phosphate buffer containing 0.9% saline and 20 mM EDTA, pH 8), washed twice with PBS-E, and finally
suspended in 2 ml of PBS-E containing 0.5 mg/ml collagenase. The
suspension was incubated at 37 °C for 1 h with stirring,
followed by the addition of Pronase E (1 mg/ml), and further incubated
for 15 min at 37 °C. It was centrifuged at 1000 rpm for 5 min. The
pellet was dispersed and incubated with 2 ml of a lysis buffer
containing 50 mM Tris-Cl, pH 8, 20 mM EDTA, 10 mM NaCl, and 1% w/v SDS for
15 min. It was centrifuged at 14,000 × g for 15 min,
and DNA was isolated from the lysate by a phenol-chloroform extraction
procedure (61). DNA was dissolved in 10 mM
Tris-Cl
, pH 8, containing 1 mM EDTA by gentle
shaking at 65 °C. Residual contaminating RNA was removed by
incubating the DNA solution with 1 µg/ml DNase-free RNase at 37 °C
for 1 h followed by 0.1 mg/ml proteinase K for 3 h.
Phenol-chloroform extraction was repeated to obtain purified DNA that
was dissolved in 10 mM Tris-Cl
buffer, pH 8, containing 1 mM EDTA. To study DNA fragmentation, DNA was
loaded on to a 1.5% agarose gel. Electrophoresis was carried out at
100 V for 1.5 h in TBE (90 mM Tris borate, 2 mM EDTA, pH 8) buffer, and DNA was visualized by UV
exposure after staining with ethidium bromide.
Measurement of Reactive Oxygen Species Mediated Oxidative Damage
of DNA in Vitro and Its Protection by Omeprazole or
Lansoprazole--
To study the antioxidant effect of omeprazole or
lansoprazole, ·OH-mediated oxidative damage of DNA isolated from
rat mucosal surface epithelial cells or from human gastric mucosal
biopsy specimen was studied in absence or presence of omeprazole or
lansoprazole. For isolation of human gastric mucosal DNA, the minced
mucosa (1 g) was digested with 12 ml of the digestion buffer (100 mM NaCl, 10 mM Tris-Cl, pH 8.0, 25 mM EDTA, 0.5% SDS, 0.1 µg/ml proteinase K, and 1 µg/ml RNase) by incubating in a shaker bath at 52 °C for 15 h. DNA was extracted from the lysate after phenol-chloroform extraction as described (61). Rat DNA (~200 ng) or human DNA (~300 ng) was
incubated in a ·OH-generating system containing 100 mM sodium phosphate buffer, pH 7.4, 0.2 mM
CuSO4, and 1 mM ascorbate (59) in a total
volume of 30 µl for a period of 30 min at 37 °C in presence or
absence of omeprazole or lansoprazole. The reaction was stopped by the addition of 1 µg of catalase, and electrophoresis was carried out in
a 2% agarose gel.
Scavenging of ·OH by Lansoprazole-- Hydroxyl radical was generated in vitro in the Cu2+-ascorbate system (59, 60) and quantitated as described by Babbs and Steiner (58). The assay system contained in a final volume of 1 ml: 50 mM sodium phosphate buffer, pH 7.4, 0.2 mM CuCl2, 2 mM ascorbate, and 2 mM Me2SO in the absence and presence of lansoprazole. After incubation at 37 °C for 1 h, the reaction was stopped with 0.5 mM EDTA, and the methanesulfinic acid formed was extracted and allowed to react with Fast Blue BB salt for quantitation as described (58).
Isolation of ·OH Mediated Oxidation Product of Lansoprazole-- Because crystalline lansoprazole is readily available commercially, this experiment was carried out with lansoprazole instead of omeprazole with the aim of isolating the ·OH-mediated oxidation product of lansoprazole, if the latter scavenges ·OH. Lansoprazole (0.2 mM) was incubated at 37 °C for 3.5 h with 0.2 mM CuCl2 and 2 mM ascorbate in the presence of 10 mM phosphate buffer, pH 7.4, in a final volume of 400 ml. The content was evaporated in a Eyela N-N series rotary vacuum evaporator, and the residue was extracted repeatedly with chloroform followed by methanol. A control system containing 0.2 mM lansoprazole in 10 mM phosphate buffer was incubated under similar conditions without a ·OH-generating system and subjected to the same extraction procedure to find out whether any aerial oxidation occurs or not. The major oxidation product was isolated from the methanol extract after separation by preparative TLC on plate (8 × 18 cm) coated with silica gel 60 F254 using chloroform:methanol (90:10) as the mobile phase. The compounds were detected by spraying with iodine vapor. The major oxidation product was recovered from the TLC plate by elution with methanol and was further purified by Waters HPLC system using Waters 15 µ 100 Å Deltapak-C18 semipreparative column (7.8 × 300 mm) eluted with methanol: water (80:20) at a flow rate of 1 ml/min. The absorbance was monitored at 285 nm.
Analysis of Oxidation Product of Lansoprazole-- The HPLC-purified major oxidation product of lansoprazole was dissolved in CDCl3, and the 1H NMR spectrum was recorded in a Bruker 300 MHz NMR spectrometer. Molecular weight was determined by electron impact (EI+) mass spectrometry using Jeol JMS 600 mass spectrometer. The UV-visible spectrum was recorded in a Shimadzu UV-1601 spectrophotometer.
Statistical Analysis--
All of the data were expressed as the
means ± S.E. The significance was calculated using a Student's
t test.
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RESULTS |
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Differential Effect of Omeprazole in Blocking Gastric Ulcer and
Gastric Acid Secretion--
To investigate whether omeprazole blocks
gastric lesions through an independent mechanism other than the
inhibition of acid secretion, the dose-dependent effect of
omeprazole was studied both on stress- and indomethacin-induced gastric
ulceration and pylorus ligation-induced gastric acid secretion. Fig.
2A indicates that omeprazole
dose-dependently blocks both stress and
indomethacin-induced gastric lesions showing nearly 90% inhibition at
8 and 16 mg/kg, respectively. More than 90% of the animals showed no
gastric lesion at all. In contrast, omeprazole blocks pylorus
ligation-induced acid secretion at a higher dose, causing nearly 90%
inhibition at 20 mg/kg (Fig. 2B). However, one significant
finding is evident from the dose-response patterns for blocking gastric
damage and acid secretion. At the dose of 2.5 mg/kg, omeprazole cannot
block acid secretion at all, whereas at a slightly lower dose of 2 mg/kg, omeprazole blocks stress ulcer by 70%, and indomethacin
ulcer by 50%. The efficacy of omeprazole in blocking gastric damage and acid secretion can be more accurately determined by the potency (ED50) calculation from the dose-response profiles. Whereas
the ED50 values for inhibiting stress and
indomethacin-induced gastric lesions are 0.8 and 2 mg/kg, respectively,
that for the induced acid secretion is 3.25 mg/kg. The data indicate
that omeprazole blocks gastric lesions through a mechanism independent
of its role on acid secretion. The relationship between
gastroprotection and acid inhibition by omeprazole has been further
clarified from the time course studies of inhibition as shown in Table
I. The results indicate that at the
initial period of 1 h when acid secretion is inhibited by 50%
only, gastroprotection by omeprazole is almost complete, showing around
90% inhibition of the gastric lesions caused by stress or
indomethacin. At later time periods of 2.5 and 3.5 h,
gastroprotection remains more or less at the same level when acid
inhibition is increased to 80%. It is thus clear that omeprazole can
offer gastroprotection almost completely even when it cannot completely
block acid secretion. In other words, omeprazole-induced gastroprotection is not decreased with relatively higher rate of acid
secretion. Omeprazole thus protects gastric lesions through mechanisms
other than acid inhibition.
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Role of Hydroxyl Radical on Gastric Ulceration--
To assess
whether endogenous ·OH plays any significant role on the
development of gastric lesions, the effect of Me2SO, a
specific ·OH scavenger (58) was studied on both stress- and
indomethacin-induced gastric lesions. The data indicate that
Me2SO causes 87% protection of stress ulcer (Fig.
3A) and 94% protection of
indomethacin ulcer (Fig. 3B) without affecting the luminal
acid content. The results indicate that ·OH plays one of the
major roles in stress- or indomethacin-induced gastric lesions. Fig.
4 further shows that
time-dependent severity of gastric lesions (ulcer index)
correlates well with the increased generation of ·OH and not
with the luminal acid content, suggesting that ·OH plays a
significant role in the gastric damage.
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Effect of Omeprazole on ·OH-mediated Oxidative Damage of the Gastric Mucosa-- Because ·OH is one of the major causative factors for gastric ulceration and creates oxidative damage by increased membrane lipid peroxidation and protein oxidation (22), the effect of omeprazole was therefore studied on these two parameters. Table II shows that omeprazole blocks stress-induced generation of ·OH and at the same time completely prevents radical-induced increased lipid peroxidation and protein oxidation. Omeprazole thus blocks gastric oxidative damage by acting as an antioxidant through scavenging of endogenous ·OH. The potency of omeprazole as an antioxidant to block gastric lesions was compared with some natural and synthetic antioxidants having antiulcer activity. The dose-response profiles clearly indicate that omeprazole (Fig. 5A) is more potent than the naturally occurring antioxidants such as vitamin E or melatonin (Fig. 5B) or some synthetic antioxidants such as desferrioxamine (Fig. 5C), a transition metal ion chelator to prevent ·OH generation, or PBN (Fig. 5D), a radical scavenger.
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Effect of Omeprazole on DNA Damage of the Mucosal Cell as an
Indication of Apoptosis--
Apoptotic cell death is associated with
DNA fragmentation, and oxidative attack is thought to be one of the
underlying mechanisms. Because restraint cold stress causes extensive
damage of the surface epithelium with numerous cells sloughed off into
the gastric lumen because of cell death (13), it is interesting to
investigate whether this process is associated with apoptotic cell
death or not. Fig. 6 shows that
stress-induced gastric epithelial cell damage is associated with DNA
fragmentation showing typical DNA ladder (lane 2), an index
of cell apoptosis. However, omeprazole pretreatment can completely
block stress-induced DNA fragmentation (lane 3), suggesting
its antiapoptotic role to prevent cell death during ulceration.
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Antioxidant Role of Omeprazole and Lansoprazole in Blocking
Oxidative DNA Damage in Vitro--
Oxidative damage of DNA can be
studied in vitro when incubated in a ·OH-generating
system. Fig. 7A shows that rat
gastric mucosal cell DNA (lane 1), when incubated in the
Cu2+-ascorbate-mediated ·OH-generating system, is
completely fragmented into small pieces so that the main DNA band
(lane 1) is not observed at all in lane 2. DNA
can be completely protected from the oxidative damage by catalase
(lane 7), suggesting the involvement of
H2O2 in the process. Protection is also evident
with the spin trap DMPO (lane 8), suggesting the generation
of the radical species. The data indicate that DNA is oxidatively
damaged by ·OH generated from H2O2 in
presence of Cu2+ and ascorbate (reducing equivalent of
O
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Effect of Lansoprazole on the Level of ·OH in
Vitro--
The protection of the oxidative DNA damage by omeprazole or
lansoprazole may be explained as being due to its direct scavenging action on ·OH so that DNA is spared from the radical attack. To
study the ·OH scavenging action, ·OH generation was
measured in vitro in the Cu2+-ascorbate system
in the absence and presence of lansoprazole. Fig.
8 indicates that lansoprazole can
directly decrease the level of ·OH in a
concentration-dependent manner showing 90% inhibition at 2 mM. Because lansoprazole does not decompose
H2O2 or chelate Cu2+ (data not
shown), the effect is not due to the decreased generation of ·OH
from endogenous H2O2 through metal-catalyzed
Haber-Weiss reaction.
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Identification and Isolation of Oxidation Product of
Lansoprazole--
To investigate the possibility for scavenging of
·OH by lansoprazole to form an oxidation product, lansoprazole
was incubated in the Cu2+-ascorbate system, and the mixture
after extraction with chloroform followed by methanol was subjected to
TLC (Fig. 9A). Lane
1 shows the single spot of commercial lansoprazole used in this
study. Lane 2, on the other hand, shows the formation of at
least four oxidation products of lansoprazole, of which spot 4 is the
major oxidation product. None of these products were detected when
lansoprazole was incubated only in phosphate buffer (not shown). This
major oxidation product was isolated from the preparative TLC plate and
subjected to HPLC (Fig. 9B). The chromatogram (Fig.
9B, tracing 1) shows a major peak of the product
preceded by a number of small peaks probably contributed by some
impurities from the silica gel. These impurities were removed by
isolating the compound in the major peak by HPLC. The HPLC-purified
product shows more than 95% purity as evidenced by HPLC profile shown
in tracing 2 of Fig. 9B. The product when run in
TLC (Fig. 9A, lane 3) shows one single spot
exactly matching with the Rf value of spot
4.
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Characterization of the Oxidation Product of Lansoprazole--
The
HPLC-purified oxidation product of lansoprazole when analyzed by
EI+ mass spectroscopy (Fig.
10) shows a clear molecular ion peak at m/z 385, which was 16 mass units higher than that
of lansoprazole (m/z 369 not shown). This
indicates that the compound is an oxidation product of lansoprazole
involving the addition of one oxygen atom. The oxidation product
absorbs at 278 nm (Fig. 11,
tracing 1) because of the presence of the benzene ring. The
addition of alkali does not cause any alkaline shift to the higher
wavelength (Fig. 11, tracing 2) characteristic to phenol,
indicating that no hydroxylation occurs at the benzene ring to form
hydroxylansoprazole. When compared with lansoprazole, no additional
aromatic proton signal was detectable in the oxidation product by
1H NMR (not shown). Thus, the oxidation product having a
molecular ion mass of 385 is an oxygenated species of lansoprazole,
formed by scavenging of ·OH.
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DISCUSSION |
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The salient points of the present studies are that omeprazole blocks stress and indomethacin-induced gastric lesions through mechanism independent of its role on acid secretion. Omeprazole can protect ulcer at a dose that does not inhibit acid secretion. Time course studies on gastroprotection and acid inhibition further indicate that omeprazole can almost completely block gastric lesions at the initial period when acid secretion is not completely inhibited. On the other hand, Larsson and co-workers (18, 53) showed that intravenous doses of omeprazole that block acid secretion cannot protect ethanol-induced gastric lesions, suggesting that acid inhibition plays no significant role on gastroprotective effect of omeprazole, which is observed after oral administration of the drug, presumably through its local action. Although this observation is opposite to ours because of the different experimental design and different models of ulcer and acid secretion used, nevertheless, it is clear from both these studies that omeprazole offers gastroprotection through a mechanism other than acid inhibition. Second, evidence has been provided to show that majority of the gastric lesions is caused by endogenous ·OH, as revealed by almost complete (~90%) protection by Me2SO, a specific ·OH scavenger (58). This is further supported by the finding that time-dependent severity of gastric lesions correlates well with the increase in endogenous ·OH and has no correlation with the luminal acid content. Almost complete protection by other antioxidants like melatonin, desferrioxamine, and PBN further strengthens the view that ·OH plays one of the major roles in the development of gastric lesions. Third, omeprazole scavenges the endogenous ·OH and thus blocks radical-induced oxidative damage of the membrane lipid and proteins. Fourth, DNA damage and fragmentation, an indication of apoptotic cell death during ulceration, is also protected by omeprazole. Omeprazole or lansoprazole also protects ·OH-mediated oxidative damage of DNA in vitro. These studies indicate that omeprazole blocks gastric lesions by acting as an antioxidant and antiapoptotic compound. Finally, using lansoprazole as an analogue of omeprazole, evidence has been provided to show that lansoprazole scavenges ·OH to form lansoprazole sulfone as a major oxidation product.
Gastric mucosal integrity is maintained by a dynamic process of cell
death and cell proliferation. Among various factors involved in gastric
mucosal lesions, oxidative damage (13, 22, 25-37) and apoptotic cell
death (39-51) play significant roles in the loss of gastric mucosal
integrity caused by various aggressive factors. In other words, lesions
develop when oxidative damage and apoptosis predominate over the
healing process (62) by cell proliferation where the role of various
growth factors, nitric oxide, endothelin, angiogenesis,
mitogen-activated protein kinases, and oncogene (c-myc,
c-Ha-ras, and c-fos) expression has been demonstrated (63-73). The modern approach of understanding the mechanism of the antiulcer effect of omeprazole should therefore be
directed toward exploring its plausible role in preventing oxidative
damage and apoptosis as well as on the promotion of healing process by
cell proliferation. As far our knowledge goes, this is the first
evidence to show that omeprazole blocks gastric lesions by preventing
oxidative damage and apoptosis of the gastric mucosal cells. Although
omeprazole blocks ulceration at a lower dose (<2.5 mg/kg) without
inhibiting acid secretion, suggesting its independent antiulcer
activity, at higher doses its additional antisecretory action
definitely exerts beneficial effect by preventing aggravation of the
wound, thereby helping the healing process by cell proliferation. The
question arises as to how omeprazole offers antiulcer activity
independent of acid secretion. Our studies indicate that omeprazole is
highly effective in blocking membrane lipid peroxidation and protein
oxidation, which occur because of oxidative damage by ROS especially by
·OH (13, 22, 25-37). Omeprazole can scavenge the endogenous ·OH and thus prevents oxidative damage and gastric lesions. By blocking oxidative damage through lipid peroxidation and protein oxidation, omeprazole prevents loss of membrane permeability and dysfunction of the cellular proteins, leading to survival of the functionally active cells. Many natural and synthetic compounds are
known to offer antiulcer effect by acting as antioxidants. Melatonin (a
pineal hormone), vitamin E, PBN, or desferrioxamine directly or
indirectly decreases the endogenous level of ·OH to block
gastric ulcer (22, 59). Comparative bioefficacy studies indicate that
omeprazole is superior to these antioxidants in blocking gastric
lesions. However, the most important effect of omeprazole lies in its
novel antiapoptotic role during ulceration, as evidenced by prevention
of DNA fragmentation in vivo. Apoptosis of mucosal cells
occurs almost in all types of gastric ulcer (39-51) where DNA damage
and fragmentation occur by various aggressive factors (39, 41, 45-47).
Using histological section and terminal deoxynucleotidyltransferase biotin-dUTP nick end labeling (TUNEL) staining technique (39), gastric mucosal cell apoptosis was detected up
to 4 h after stress, following which cell proliferation was found
to be significantly increased to promote mucosal healing (39).
Moreover, apoptosis is triggered by the up-regulation of
apoptosis-promoting Bax mRNA and down-regulation of the
antiapoptotic Bcl-2 mRNA expression (39). We have, however, directly
demonstrated stress-induced DNA fragmentation in the surface epithelial
cell and the beneficial role of omeprazole to block it, thereby
preventing apoptotic cell death and gastric lesions. It is not clear
yet how differential expression of Bax and Bcl-2 proteins controls apoptosis. However, decreased gastric mucosal blood flow (39) leads to
the ischemic condition to generate ROS through alteration of
antioxidant systems of gastric mucosa (13, 22), which may cause
apoptosis through oxidative damage of DNA (39, 45, 46). However,
ischemia may also cause apoptosis through other mechanisms such as
involvement of Bcl-2, Bax, and c-Fos proteins (74, 75). Excessive
generation of nitric oxide by gastric mucosal-inducible nitric-oxide
synthase by stress also promotes apoptosis through increased formation
of ROS (76, 77). That ROS can cause oxidative damage of DNA isolated
from both rat and human gastric mucosal epithelial cells has been
evident from our in vitro studies where incubation of DNA
with an ·OH-generating system (22, 59, 78) causes extensive DNA
degradation, which is sensitive to catalase and DMPO. Both omeprazole
and lansoprazole have a unique capacity to block this oxidative damage,
indicating its potent antioxidant role to protect DNA from the attack
of ·OH. This could be achieved if omeprazole or lansoprazole can
directly scavenge the ·OH to form oxidation product.
Lansoprazole when incubated in the ·OH-generating system, can in
fact diminish the level of ·OH by its direct scavenging action.
This is evident by the observation that incubation of lansoprazole in
the ·OH-generating system produces four oxidation products, of
which the major one shows the addition of 16 mass units
(m/z 385) over the mass of lansoprazole
(m/z 369), indicating incorporation of an oxygen
atom into lansoprazole. Omeprazole and lansoprazole undergo oxidation
in cytochrome P-450 systems to produce hydroxyomeprazole or
hydroxylansoprazole and omeprazole sulfone or lansoprazole sulfone
(79-81). Hydroxylation in the benzene ring of lansoprazole in our
system does not occur because the oxidation product absorbing at 278 nm
does not show the characteristic alkali shift for the formation of
phenol. Absence of any additional aromatic proton signal in the
1H NMR spectrum also negates the possibility of the
formation of phenolic group in the oxidation product. Thus, addition of
an oxygen atom has occurred at the sulfur atom of the lansoprazole to
form lansoprazole sulfone. The possible mechanism of formation of
lansoprazole sulfone from lansoprazole by scavenging ·OH is
shown in Fig. 12. Lansoprazole is
converted to a highly reactive sulfur centered radical intermediate by
scavenging the ·OH, and the intermediate is stabilized to form
lansoprazole sulfone by further incorporation of ·OH at the
sulfur radical with the elimination of one molecule of
water.
|
The present study thus provides the first direct evidence for the
antioxidant and antiapoptotic role of omeprazole in preventing gastric
ulceration by scavenging endogenous ·OH. This is in contrast to
the earlier observation that omeprazole neither protects
indomethacin-induced gastric damage nor decreases apoptotic DNA
fragmentation (41). Although a large number of reports (including the
present study) indicate that omeprazole prevents indomethacin-induced
gastric damage, the inability of omeprazole to block gastric lesions
and associated apoptosis (41) is not clear, and hence the conclusion
that omeprazole does not possess antiapoptotic property to block
indomethacin ulcer (41) remains to be verified. However, a question may
arise as to what percentage of total gastroprotection by omeprazole is
mediated through block of apoptosis and its antioxidant action by
scavenging ·OH. Because quantitation of total gastroprotection
by omeprazole is difficult to assess because it has morphological,
cellular, biochemical and pharmacological parameters, extensive studies are required to answer this question. Because gastric ulceration is a
multifactorial process (24, 39, 41), it is possible that
gastroprotective effect of omeprazole may partially be mediated through
other mechanisms also. Recently omeprazole has been shown to prevent
compound 48/80 (mast cell degranulator)-induced gastric lesions (with
no acid secretion) by acting as an antiinflammatory agent and also by
preventing neutrophil infiltration, activation, and associated mucosal
damage (82). Thus, omeprazole may have multiple modes of action.
Although no unifying concept has developed yet on the mechanism of
gastric mucosal damage caused by various ulcerogens, it will be
interesting to investigate whether omeprazole has a common molecular
target for gastroprotection. Because apoptosis (30-51) and reactive
oxygen species (13, 22, 25-37, 83, 84) play significant roles in
mucosal damage, it is conceivable that antiapoptotic and antioxidant
role of omeprazole play a major part in the total gastroprotection.
These novel actions of omeprazole are of particular clinical
significance for the control of gastroduodenal ulcer by this class of
proton pump inhibitors.
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FOOTNOTES |
---|
* 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. Section 1734 solely to indicate this fact.
§ Supported by a Senior Research Fellowship from Council of Scientific and Industrial Research (New Delhi).
To whom correspondence should be addressed. Tel.:
91-33-24733491 or 91-33-24733493; Fax: 91-33-24730284 or
91-33-24735197; E-mail: ranajitb@yahoo.com.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M210328200
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ABBREVIATIONS |
---|
The abbreviations used are:
ROS, reactive oxygen
species;
PBN, -phenyl N-tert-butylnitrone;
DMPO, 5,5-dimethyl-1-pyrroline N-oxide;
HPLC, high
performance liquid chromatography.
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