Polaprezinc has been shown to exert an anti-oxidant property in a tube experiment, protect gastric mucosa from experimental ulcerations in vivo, and accelerate the healing of gastric ulcer in humans.
Polaprezinc has been shown to exert an anti-oxidant property in a tube experiment, protect gastric mucosa from experimental ulcerations in vivo, and accelerate the healing of gastric ulcer in humans.
To examine a possible protective effect of polaprezinc on oxidant-mediated injury in primary monolayer cultures of rat gastric fundic mucosa.
Cytotoxicity was quantified by measuring 51Cr release. Whether or not polaprezinc exerts an antioxidant property was investigated by determining the effect of this agent on hydrogen peroxide (H2O2)-induced injury. The effects of polaprezinc on superoxide (O2–·) generation as well as on ethanol (EtOH)-induced injury were also examined. Generation of O2–· was assessed by the reduction in cytochrome c.
H2O2 caused a time- and dose-dependent increase in 51Cr release. The dose-response curve of 51Cr release by H2O2 shifted to the right in the presence of polaprezinc. Polaprezinc, at submillimolar concentrations, prevented H2O2-induced 51Cr release. EtOH also caused a dose-dependent increase in 51Cr release, which was prevented by the addition of polaprezinc. The incubation of cells with EtOH caused an increase in cytochrome c reduction, as the concentrations of EtOH increased. Polaprezinc inhibited EtOH-induced cytochrome c reduction. Protection by polaprezinc was microscopically associated with the prevention of monolayer disruption.
Polaprezinc is antioxidative and directly protects gastric mucosal cells from noxious agents through its antioxidant properties in vitro. This finding may provide the theoretical basis for the usage of an antiulcer drug with antioxidant properties for the treatment of gastric inflammation, such as that induced by ethanol.
Polaprezinc [N-(3-aminopropionyl)- L-histidinato zinc], a novel antiulcer agent developed in Japan,1 is a chelate compound consisting of zinc ion and L-carnosine (Figure 1 ).2 It has been shown that this agent protects the gastric mucosa against experimental ulcerations in vivo1–6 and accelerates the healing of gastric ulcers in humans7 as well as in rats in vivo.8 This property of polaprezinc may be accounted for by its stimulation of mucus production, antioxidant property and by the maintenance of the gastric mucosal barrier,3, 6, 9 although the precise mechanisms remain obscure.
A number of studies have shown that reactive oxygen species (ROS) are involved in the pathogenesis of a variety of gastroduodenal diseases, such as those induced by ischaemia/reperfusion,10 ethanol (EtOH)11, 12 or nonsteroidal anti-inflammatory drugs (NSAIDs).13, 14Helicobacter pylori causes type B chronic gastritis,15 which becomes a long-standing and life-long gastritis if it is not eradicated.16 In gastric mucosa with H. pylori infection, active inflammation with infiltration of neutrophils in the acute stage of the infection and of macrophages/monocytes, lymphocytes and plasma cells in the chronic stages are seen in the lamina propria of the stomach.17, 18 Although it remains unclear how H. pylori infection induces mucosal inflammation and injury, it is highly likely that inflammatory cell infiltration is responsible for the injury. These neutrophils and macrophages/monocytes produce large quantities of ROS that could cause DNA damage in the adjacent cells19, 20 and ultimately lead to mucosal injury. Therefore, it is of great interest to examine whether an antiulcer agent with an antioxidant property, such as polaprezinc, protects the gastric mucosal cells from oxidants in vitro independently of vascular, neural or hormonal factors.
We have previously demonstrated that extracellular hydrogen peroxide (H2O2) induces cellular injury,21 and that EtOH-induced cytolysis is attributable in part to the generation of ROS in cultured rat gastric mucosal cells.22 In the present study, we investigated: (i) whether polaprezinc exerts a protective effect on H2O2-induced cellular injury; (ii) whether this agent protects against EtOH; and (iii) whether polaprezinc has the ability to scavenge ROS in these cells.
Seven- to 10-day old rats of either sex (Sprague–Dawley) were obtained from Dohken (Ibaraki, Japan). Collagenase (Collagenase A from Clostridium histolyticum), Coon’s modified Ham’s F-12 medium, and foetal bovine serum were purchased from Gibco (Grand Island, NY). Hyaluronidase (type 1-S), antibiotic-antimycotic, N-[2-hydroxyethyl] piperazine-N′-[2-ethanesulphonic acid] (HEPES), cytochrome c, superoxide dismutase (SOD) (from bovine liver) and Triton X-100 were all obtained from Sigma (St Louis, MO). H2O2 (30%), EtOH (99.5%) and formalin (60%) were purchased from Wako Pure Chemicals (Osaka, Japan). L-carnosine and zinc sulphate heptahydrate (ZnSO4·7H2O) were obtained from Hamari Chemicals Ltd. (Osaka, Japan) and Nacalai Tesque Inc. (Kyoto, Japan), respectively. Polaprezinc was a generous gift from Zeria Pharmaceutical Co. (Saitama, Japan). Polaprezinc was dissolved in 100 m M HCl solution, and the stock solution of polaprezinc (20 m M) in 100 m M HCl was diluted with Earle’s balanced salt solution (EBSS) (pH 6.0) to obtain a final concentration of 1 m M. The solution was adjusted to pH 6.0 by the addition of small amounts of 5 N NaOH. Control EBSS solution was prepared similarly, and the pH was adjusted to pH 6.0. Because polaprezinc precipitates at a pH above 6.5, all incubations in the cytotoxicity and cytochrome c reduction assay, described below, were carried out under experimental conditions of pH 6.0. We have demonstrated previously that the cells used in the present study were resistant to the acidic culture conditions at pH 6.0; 51Cr release from cells incubated at pH 6.0 for 2 h was not significantly different from that from cells incubated at pH 7.4.23 Hanks’ balanced salt solution (HBSS) and EBSS supplemented with 15 m M HEPES were obtained from Gibco, and adjusted to pH 7.4 and pH 6.0, respectively. 51Cr (sodium chromate, 200–900 Ci/g chromium) was obtained from NEN, Boston, MA. Tissue culture plates and dishes were purchased from Corning Glass Works (Corning, NY).
Primary monolayer cultures of rat gastric fundic mucosa were prepared as previously described,24 and studied as a representative of normal gastric cells with functional integrity and cellular polarity.25 Over 90% of the cells have been identified previously as mucus-producing epithelial cells, and shown to be capable of synthesizing DNA as well as cyclic nucleotides, producing and secreting mucous glycoprotein, and generating prostaglandins.25 Confluent monolayers were studied 3 days after seeding.
Cytotoxicity was quantified by measuring 51Cr release from pre-labelled cells.21, 23 Confluent monolayers of epithelial cells in 24-well culture plates were labelled overnight with 2 μCi 51Cr/well/0.5 mL of culture medium. After pre-labeling, the cells were washed three times with HBSS to remove unincorporated 51Cr, and then incubated with either EBSS alone (as a control) or EBSS containing H2O2 (0.1–1 m M) for up to 3 h, or with either EBSS alone or EBSS containing ethanol (12–18 %) for 1 h. After designated periods of time, aspirated buffer was centrifuged at 1000 g for 2 min, and 100 μL-aliquots of cell-free supernatant buffer were removed for the determination of specific 51Cr release (A–B/C – B) × 100% as follows: where A represents the mean test 51Cr c.p.m. released, B represents the mean spontaneous 51Cr c.p.m. released, and C represents the mean maximum 51Cr c.p.m. released. Maximum 51Cr release was determined by incubating the cells in 0.2% Triton X-100. Spontaneous 51Cr release was determined in control monolayers incubated in EBSS only and was 5–7% and 8–12% of maximum 51Cr release after 1 h and 3 h of incubation, respectively. 51Cr radioactivity was counted with a JDC-751 Auto Well Gamma Counting System (Aloca, Tokyo, Japan).
Cytotoxicity was also qualitatively assessed under phase contrast microscopy. After the incubation of cells with either H2O2 (1 m M) for 3 h or with EtOH (18%) for 1 h in the absence or presence of polaprezinc (1 m M), formalin was added slowly to the monolayers to achieve a final concentration of 10%, and microscope pictures of the monolayers were taken under phase contrast microscopy (IMT-2-21, Olympus Optical, Tokyo, Japan) with a camera (OM-4Ti, Olympus).
Detection of superoxide anion (O2–·) was based on its ability to reduce cytochrome c.26 Monolayer-cultured cells in 35-mm culture dishes were incubated at 37 °C for 1 h in EBSS (without phenol red) containing graded concentrations of ethanol (0–18%) and cytochrome c (10 nmol), with or without polaprezinc (1 m M). Part of the buffer was incubated at 37 °C without cultured cells for use as a blank control. After incubation, the buffer was cleared by centrifugation at 3000 g for 5 min. In some experiments, to examine the SOD inhibition of cytochrome c reduction, monolayers in 35-mm dishes were incubated with ethanol (18%), cytochrome c (10 nmol), and SOD (500 U/mL) for 1 h. Cytochrome c reduction in an aliquot of the supernatant buffer was determined by measuring the absorbance at 550 nm on a Shimadzu UV-210 Spectrophotometer (Shimadzu, Kyoto, Japan), using the blank as reference. ΔEmM (ferrocytochrome c minus ferricytochrome c) at 550 nm was taken as 18.5.27
Data were expressed as means ± s.e. Analysis of variance ( ANOVA) was performed when more than two groups were compared, and when the differences were significant (P < 0.05), Scheffe’s multiple comparison test was applied to test for the differences between individual groups. The significance of the differences between two groups was determined by an unpaired Student’s t-test; P-values of 0.05 or less were considered significant.
Whether or not polaprezinc exerts an antioxidant property in vitro was investigated by determining the effect of this agent on H2O2-induced injury.21 H2O2 (1 m M) caused a time-related increase in specific 51Cr release for up to 3 h (Figure 2 ). H2 O2 (0.1–1 m M) also caused a dose-dependent increase in specific 51Cr release during a 3 h incubation (Figure 3 ). The dose-response curve of specific 51Cr release brought about by H2O2 shifted to the right in the presence of polaprezinc (1 m M) (Figure 3 ). Polaprezinc, at submillimolar concentrations, prevented H2O2-induced specific 51Cr release in a dose-dependent manner (Figure 4 4).
EtOH (12–18%) caused a dose-dependent increase in specific 51Cr release during a 1 h incubation. The presence of polaprezinc (1 m M) caused a right-shift of the dose-response curve for EtOH (Figure 5 ). Furthermore, polaprezinc (0.1–1 m M) prevented EtOH-induced specific 51Cr release in a dose-dependent manner (Figure 6 6).
Cells were incubated with either EBSS alone, H2O2 (1 m M), polaprezinc (1 m M), or H2O2 plus polaprezinc for 3 h, or with either EBSS alone, EtOH (18%), polaprezinc, or EtOH plus polaprezinc for 1 h. The incubation of monolayers with H2O2 or EtOH induced extensive cytolysis and cell detachment, whereas incubation with polaprezinc alone did not cause any structural change in the monolayers compared with the control incubation. The protection of cells against H2O2 or EtOH by polaprezinc, as assessed by the inhibition of increased 51Cr release (Figures 3–6), was associated with the prevention of microscopic damage (cell detachment and cytolysis) (Figures 7 and 8).
The incubation of cells with EtOH (12–18%) caused an increase in cytochrome c reduction during a 1 h incubation, as the concentrations of EtOH increased (Table 1 ). An increased reduction of cytochrome c was prevented almost completely by the presence of superoxide dismutase (500 U/mL), indicating that reduction of cytochrome c was largely attributable to O2–· production. The addition of polaprezinc (1 m M) to the reaction mixture inhibited EtOH (18%)-induced cytochrome c reduction from 6.05 ± 0.03 nmol/106 cells in the control to 2.05 ± 0.04 nmol/106 cells (Table 1 1).
In order to better understand how polaprezinc can exert the putative antioxidant action, we have determined whether either zinc sulphate or L-carnosine alone is capable of exerting the beneficial effect against EtOH. Zinc sulphate (1 m M), but not L-carnosine (1 m M), offered a significant protection against EtOH (18%), which was similar to polaprezinc (1 m M) in its degree of protection (Table 2 2).
The major findings of the present study are that: (i) polaprezinc directly protects cultured gastric mucosal cells from oxidant stress (H2O2) or EtOH; (ii) EtOH-induced cytotoxicity is associated with O2–· production from cells; and (iii) polaprezinc-induced protection from EtOH damage seems to be attributed in part to the capability of the agent for scavenging ROS. Therefore, it is suggested that polaprezinc is antioxidative and protects gastric mucosal cells from noxious agents through its antioxidant properties in vitro, independently of microcirculatory and neural or hormonal factors.
Reactive oxygen species may be pathophysiologically involved in a variety of gastroduodenal disorders such as those induced by ischaemia/reperfusion,10 EtOH,11, 12 or NSAIDs.13, 14 Polaprezinc is a novel antiulcer agent recently developed in Japan1 and has been used widely for the treatment of gastric ulceration.7 It has been demonstrated that this agent exerts an antioxidant property in an in vitro experiment,9 is protective against a variety of experimental ulcerations in rats1–6 and accelerates the healing of gastric ulceration in humans.7 Peptic ulcerations in humans are highly associated with H. pylori infection. Moreover, the proposed mechanisms by which H. pylori infection causes gastric disorders include oxidant stress induced by the generation of reactive oxygen species from activated inflammatory cells.19, 20 This is possibly supported by the concomitant association of lowered levels of antioxidants in the stomach; a reduction in vitamin E-values of the corpus mucosa28 and a substantially lower concentration of vitamin C in the gastric juice.29 Therefore, it is of great interest to determine whether an antiulcer agent with antioxidant action, such as polaprezinc, has the ability to protect gastric mucosal cells from oxidant injury. It is possible to directly assess a possible protective effect of such an agent on oxidant-induced cellular injury in vitro.
First, we examined the effect of polaprezinc on injury brought about by H2O2, which has been shown to induce cytolysis partly through lipid peroxidation,30 as an oxidant stress in cultured gastric monolayers. H2O2-induced damage was associated with extensive disruption of the monolayers (cytolysis and cell detachment) (Figure 7 ) as well as increased 51Cr release (Figures 2 and 3 ). Polaprezinc not only significantly inhibited the increase in 51Cr release brought about by H2O2, but also prevented the disruption of the monolayers (Figure 7 7). Thus, these findings clearly show that this agent has the ability to protect gastric cells from oxidant stress.
Second, we employed EtOH as an agent that is noxious to gastric cells. EtOH (12–18%) induced a dose-dependent injury of these cells, which was prevented significantly by the presence of polaprezinc (1 m M) (Figure 5 ). Furthermore, polaprezinc, in the 0.033–1 m M concentration range, significantly prevented EtOH (18%)-induced damage (Figure 6 6). Microscope observations also demonstrated that the protection of polaprezinc against EtOH was associated with the prevention of monolayer disruption (Figure 8 ). It has been suggested that the pathogenesis of ethanol-induced gastric cellular and mucosal damage includes oxidant stress in vivo.11, 12 Earlier studies on isolated or cultured gastric epithelial cells from rats have shown that EtOH induces dose-dependent injury in vitro.22, 31 In the present study where O2–· generation was detected by measuring the reduction of an electron acceptor, i.e. cytochrome c, we have found that these cells are capable of producing O2–· in response to exposure to ethanol without the involvement of blood flow or polymorphonuclear cells (Table 1 ), which is in agreement with the results of our previous study.22 These findings indicate that gastric epithelial cells, when exposed to ethanol, generate O2–· as a function of ethanol concentration. Other researchers have also shown that primary cultures of gastric surface epithelial cells from guinea-pigs are capable of releasing considerable amounts of O2–· in response to a variety of stimuli.32
With respect to the source of O2–· generation by gastric cells, one possibility may be the oxidative metabolism of ethanol by microsomes involving cytochrome P450.33 However, this explanation is less likely because P450IIE1, a microsomal P450 enzyme inducible by ethanol, is not detected in the rat gastric mucosa, either constitutively or after ethanol treatment.34 Another possibility may be the cellular xanthine oxidase system. Gastric mucosae of the rat and human are shown to possess alcohol dehydrogenase activity,35 which can convert ethanol to acetaldehyde; acetaldehyde can serve as a substrate of xanthine oxidase to produce reactive oxygen species including O2–·.36 Indeed, two inhibitors of xanthine oxidase, allopurinol and oxypurinol, have been shown to provide protection against ethanol-induced injury in the rat stomach.11 Alternatively, a membrane-bound NADPH oxidase-like system may be the source of O2–· production.37 These issues are currently under investigation in our laboratory.
Polaprezinc is a chelate compound consisting of zinc and L-carnosine (Figure 1 ).2L-carnosine and its related compounds are present in high concentrations (1–20 m M) in the muscle and brain of mammals including humans,38 and these compounds have been shown to have antioxidant activities both in vivo and in vitro.38, 39 Earlier studies have also shown that the zinc ion exerts an antioxidant effect through the protection of sulfhydryl groups against oxidation and through the inhibition of the production of reactive oxygen species by transition metals such as iron.40 Thus, both zinc ions and L-carnosine may contribute to the antioxidant property of polaprezinc. Indeed, it has been shown that polaprezinc has the ability to scavenge O2–·, to inhibit the Fenton reaction (in which most toxic hydroxyl radical is produced from H2O2 by the catalytic action of transition metals), and to inhibit lipid peroxidation in an in vitro experiment using the electron spin resonance trapping method.9 However, the present in vitro study has shown that zinc sulphate (1 m M), but not L-carnosine (1 m M), significantly protects gastric cells from EtOH (Table 2 2 ). Therefore, it appears that the protection of gastric cells against oxidants by polaprezinc is mainly attributable to the effect of zinc under the conditions used in the present study.
The clinical dosage of polaprezinc that is used to treat gastric ulceration in humans is 150 mg/day (75 mg given twice daily).7 Although the concentration of polaprezinc in the human gastric mucosa after polaprezinc ingestion has not been determined, it has been estimated that the concentration of zinc in the gastric mucosa around acetic acid-induced ulceration rises from 16.7 μg/g tissue (equivalent to 255 μM) in controls to 27.1 μg/g tissue (equivalent to 414 μM) 1 h after the oral administration of polaprezinc (3 mg/kg) in the rat.41 The present study has demonstrated that the concentrations of polaprezinc needed to exert an antioxidative effect as well as to offer protection against noxious agents, are of submillimolar orders (Figures 4 and 6), suggesting that these polaprezinc concentrations fall within the ranges observed in the human gastric mucosa in clinical use. Therefore, protection against oxidant injury by polaprezinc may provide a theoretical basis for the usage of an antiulcer drug with antioxidant properties for the treatment of gastric inflammatory diseases induced by ethanol and also perhaps those induced by NSAIDs or H. pylori, where the generation of reactive oxygen and/or nitrogen species is proposed to be pathophysiologically involved. 13, 14, 19, 20 Moreover, our preliminary study found that polaprezinc suppresses proinflammatory cytokine-induced NF-κB activation and interleukin-8 expression in gastric epithelial cells in vitro, which may indicate an additional anti-inflammatory effect of this agent.42
In summary, the present study demonstrated that (i) polaprezinc protects gastric cells from H2O2 and EtOH in vitro; (ii) EtOH-induced cytotoxicity is linked with O2–· production from cells; and (iii) polaprezinc-induced protection against EtOH seems to be attributed, at least in part to scavenging reactive oxygen species. These observations lead to the conclusion that polaprezinc is antioxidative, and directly protects gastric mucosal cells from noxious agents through its antioxidant properties in vitro, independently of microcirculatory and neural or hormonal factors. This report may provide the theoretical basis for the use of an antiulcer drug with antioxidant properties for the treatment of gastric inflammatory diseases such as gastric ulceration with H. pylori infection.
This research was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.