Acute and chronic stress-induced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate.

Reactive oxygen species (ROS) are implicated in the pathogenesis of stress-induced gastrointestinal mucosal injury. In the present study, we have investigated the effects of acute and chronic stress on the enhanced production of ROS including superoxide anion [SA; as determined by cytochrome c reduction (CCR)] and hydroxyl radicals (OH), and correlated the enhanced production of these free radicals with increased lipid peroxidation, membrane microviscosity and DNA fragmentation, indices of oxidative tissue damage, in the gastric and intestinal mucosa of female Sprague-Dawley rats. Furthermore, the protective ability of bismuth subsalicylate (BSS) against the gastrointestinal mucosal injury induced by acute and chronic stress was determined. Acute stress was induced for a period of 90 min, while chronic stress was induced for 15 min/day for 15 consecutive days. Half of the animals exposed to acute stress were pretreated orally with 15 mg BSS/kg 30 min prior to the exposure to acute stress. Similarly, half of the animals exposed to water-immersion restraint chronic stress were pretreated orally with 7.5 mg BSS/kg/day for 15 consecutive days 30 min prior to the exposure to chronic stress. Acute stress produced greater injury to both gastric and intestinal mucosa as compared to chronic stress. Acute stress increased CCR and OH production by 10.0- and 14.3-fold, respectively, in the gastric mucosa, and 10.4- and 17.0-fold, respectively, in the intestinal mucosa. Pretreatment with BSS prevented the acute stress-induced increase in CCR and OH production. Acute stress increased lipid peroxidation, DNA fragmentation and membrane microviscosity by 3.6-, 4.0- and 11.6-fold, respectively, in gastric mucosa, and 4.1-, 5.0- and 16.2-fold, respectively, in intestinal mucosa. BSS decreased acute stress-induced lipid peroxidation, DNA fragmentation and membrane microviscosity by approximately 26, 35 and 30%, respectively, in gastric mucosa, and by 20, 36 and 30%, respectively, in the intestinal mucosa. Chronic stress increased CCR and OH production by 4.8- and 6.3-fold, respectively, in gastric mucosa, and 4.6- and 6.9-fold, respectively, in intestinal mucosa. Chronic stress increased lipid peroxidation and DNA fragmentation by 2.9- and 3.3-fold, respectively, in gastric mucosa, and 3.3- and 4.2-fold, respectively, in intestinal mucosa. BSS decreased chronic stress-induced lipid peroxidation, DNA fragmentation and membrane microviscosity by approximately 41, 44 and 45%, respectively, in gastric mucosa, and by 39, 52 and 51%, respectively, in the intestinal mucosa. Daily administration of BSS provided greater protection against chronic stress-induced oxidative gastrointestinal injury as compared to the acute stress. These results demonstrate that both acute and chronic stress can induce gastrointestinal mucosal injury through enhanced production of ROS, and that BSS can significantly protect against gastrointestinal mucosal injury.

Mechanism of gastroprotection by bismuth subsalicylate against chemically induced oxidative stress in cultured human gastric mucosal cells.

Reactive oxygen species (ROS) are implicated in the pathogenesis of chemically induced gastric mucosal injury. We have investigated the effects of ethanol, hydrochloric acid (HCl), and sodium hydroxide (NaOH) on: (1) enhanced production of ROS including superoxide anion and hydroxyl radicals, (2) modulation of intracellular oxidized states by laser scanning confocal microscopy, and (3) DNA fragmentation, indices of oxidative tissue, and DNA damage in a primary culture of normal human gastric mucosal cells (GC), which were isolated and cultured from Helicobacter pylori-negative endoscopic biopsies from human subjects. The induction of ROS and DNA damage in these cells following exposure to ethanol (15%), HCl (150 mM) and NaOH (150 mM) were assessed by cytochrome c reduction (superoxide anion production), HPLC detection for enhanced production of hydroxyl radicals, changes in intracellular oxidized states by laser scanning confocal microscopy, and DNA damage by quantitating DNA fragmentation. Furthermore, the protective ability of bismuth subsalicylate (BSS) was assessed at concentrations of 25, 50, and 100 mg/liter. Incubation of GC with ethanol, HCI, and NaOH increased superoxide anion production by approximately 8.0-, 6.1-and 7.1-fold and increased hydroxyl radical production by 13.3-, 9.6-, and 8.9-fold, respectively, compared to the untreated gastric cells. Incubation of GC with ethanol, HCl, and NaOH increased DNA fragmentation by approximately 6.7-, 4.3-, and 4.8-fold, respectively. Approximately 20.3-, 17.5-, and 13.1-fold increases in fluorescence intensities were observed following incubation of gastric cells with ethanol, HCl, and NaOH, respectively, demonstrating dramatic changes in the intracellular oxidized states of GC following exposure to these necrotizing agents. Preincubation of GC with 25, 50, and 100 mg/liter of BSS decreased ethanol-induced increases in intracellular oxidized states in these cells by 36%, 56%, and 66%, respectively, demonstrating a concentration-dependent protective ability by BSS. Similar results were observed with respect to BSS in terms of superoxide anion and hydroxyl radical production, and DNA damage. The present study demonstrates that ethanol, HCl, and NaOH induce oxidative stress and DNA damage in GC and that BSS can significantly attenuate gastric injury by scavenging these ROS.

Effect of pepper and bismuth subsalicylate on gastric pain and surface hydrophobicity in the rat.

BACKGROUND: The mechanism by which dietary pepper causes dyspepsia and epigastric pain is poorly understood, as is the ability of bismuth subsalicylate (BSS) to relieve these symptoms. AIM: To investigate the ability of black pepper, red pepper and BSS to affect gastric surface hydrophobicity and induce/relieve visceral pain in rat model systems. METHODS: Fasted rats were administered intragastrically Vivonex containing varying concentrations of either black or red pepper (0-200 mg/mL) and gastric contact angles were read after 1-24 h. Some rats were post-treated with BSS (2.0-17.5 mg/mL) and contact angles were read after 2-18 h. To study pain sensitivity in rats treated with pepper/BSS, we compared tail-flick latencies after the application of radiant heat. RESULTS: Both black and red pepper rapidly (< 1 h) induced a decrease in gastric surface hydrophobicity in a dose-dependent fashion. This spice-induced increase in surface wettability was long-lasting, could be enhanced in the presence of ethanol and reversed by post-treating the rats with BSS. Both black and red pepper induced an increase in pain sensitivity, consistent with the presence of gastric pain, which could also be reversed by post-treating the rats with BSS. CONCLUSION: Both black and red pepper may induce epigastric pain by removing the stomach's hydrophobic lining and activating intramucosal pain receptors. BSS may provide relief from postprandial dyspepsia by restoring the stomach's non-wettable properties.

Stress, diet and alcohol-induced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate.

Oxygen free radicals are implicated in the pathogenesis of stress and food/alcohol-induced gastrointestinal injury. We have investigated the effects of restraint stress, spicy food diet, high-fat diet and 40% ethanol on the enhanced production of reactive oxygen species, including superoxide anion and hydroxyl radicals, and on DNA fragmentation, lipid peroxidation and membrane microviscosity (indices of oxidative tissue damage) in gastric and intestinal mucosa of Sprague-Dawley rats. Furthermore, the protective ability of bismuth subsalicylate (BSS; 15 mg kg(-1) was determined against the gastrointestinal mucosal injury induced by these stressors. Animals on the high-fat diet consumed 31% more food as compared to other animals. Animals on the spicy food diet consumed ca. 23% more water as compared to control animals, and the high-fat diet animals consumed 17% less water. Restraint stress provided greater injury to both gastric and intestinal mucosa as compared to other stressors. Restraint stress, spicy food diet, high-fat diet and ethanol increased superoxide anion production by 10.0-, 4.3-, 5.7- and 4.8-fold, respectively, in the gastric mucosa, and by 10.4-, 5.3-, 7.0- and 5.5-fold in the intestinal mucosa. Exposure to restraint stress, spicy food diet, high-fat diet and 40% ethanol also increased hydroxyl radical production by ca. 14.3-, 4.5-, 3.5- and 4.8-fold, respectively, in the gastric mucosa, and by 17.0-, 4.8-, 3.5- and 4.7-fold in the intestinal mucosa. Bismuth subsalicylate administration to the animals provided significant protection against superoxide anion and hydroxyl radical production. Restraint stress, spicy food diet, high-fat diet and ethanol increased lipid peroxidation by 3.6-, 2.4-, 2.6- and 2.0-fold, respectively, in the gastric mucosa, and by 4.1-, 3.5-, 3.6- and 2.7-fold in intestinal mucosa. Administration of BSS decreased restraint stress, spicy food diet, high-fat diet and ethanol-induced gastric mucosal lipid peroxidation by ca. 26%, 36%, 45% and 18%, and intestinal mucosa lipid peroxidation by 20%, 21%, 46% and 42%, respectively. Approximately 4.0-, 2.0-, 2.4- and 2.0-fold increases in DNA fragmentation were observed in the gastric mucosa of rats exposed to restraint stress, spicy food diet, high-fat diet and 40% ethanol, respectively, and similar increases in the intestinal mucosa. These same four stressors increased membrane microviscosity by 11.6-, 6.1-, 7.3- and 5.4-fold, respectively, in the gastric mucosa, and by 16.2-, 7.9-, 9.5- and 7.8-fold in the intestinal mucosa. Bismuth subsalicylate exerted significant protection against DNA damage and changes in membrane microviscosity induced by the four stressors. Excellent correlations existed between the production of reactive oxygen species and the tissue damaging effects in both gastric and intestinal mucosa. In summary, the results demonstrate that physical and chemical stressors can induce gastrointestinal oxidative stress and mucosal injury through enhanced production of reactive oxygen species, and that BSS can significantly attenuate gastrointestinal injury by scavenging these reactive oxygen species.

Protection against chemically-induced oxidative gastrointestinal tissue injury in rats by bismuth salts.

Oxygen free radicals (OFR) are implicated in the pathogenesis of stress, chemically induced gastric lesions, and gastrointestinal injury. The concentration-dependent scavenging abilities of bismuth subsalicylate (SBS), colloidal bismuth subcitrate (CBS), and selected OFR scavengers, including superoxide dismutase (SOD), catalase, mannitol, and allopurinol were examined against biochemically or chemically generated superoxide anion, hydroxyl radical, and hypochlorite radical plus hypochlorous acid based on a chemiluminescence assay. Furthermore, both gastric (GM) and intestinal mucosa (IM) were individually exposed in vitro to these free radical generating systems, and the concentration-dependent protective abilities of SBS and CBS against lipid peroxidation (LP) were compared with selected OFR scavengers. In addition, 24-hr fasted rats were orally treated with the necrotizing agents 0.6 M HCl, 0.2 M NaOH, 80% ethanol, and aspirin (200 mg/kg). The extent of tissue injury in the GM and IM was determined by assessing LP, DNA fragmentation, and membrane microviscosity. Dose- and time-dependent in vivo protective abilities of CBS (100 mg/kg) and SBS (15 mg/kg) were also assessed. Following incubations with superoxide anion and hydroxyl radical generating systems in the presence of 125 mg SBS/liter, approximately 47% and 61% inhibitions were observed in the chemiluminescence response, respectively, while 48% and 46% inhibitions were observed with 125 mg CBS/liter. SBS and CBS exerted similar abilities towards hypochlorite radical plus hypochlorous acid. Approx. 3.1- and 3.7-fold increases in LP were observed in the GM and IM of rats following oral administration of 0.6 M HCl. Pretreatment of the rats with SBS and CBS decreased 0.6 M HCl-induced LP in the GM by approx. 39% and 27%, respectively, with similar decreases in LP in the IM. SBS exhibited better protective abilities towards 0.6 M HCl and 0.2 m NaOH-induced GM and IM injury as compared to CBS. SBS and CBS provided similar protection towards 80% ethanol-induced gastric injury, while CBS exerted a superior protective ability towards aspirin-induced gastric injury. The results demonstrate that both SBS and CBS can scavenge reactive oxygen species and prevent tissue damage produced by OFR.

Cytoprotective effect of bismuth subsalicylate in indomethacin-treated rats is associated with enhanced mucus bismuth concentration.

BACKGROUND: Bismuth compounds prevent gastric injury from the short-term administration of nonsteroidal anti-inflammatory drugs. We studied the mechanisms underlying the gastroprotective actions of bismuth subsalicylate against indomethacin-induced injury in rats. METHODS: An in vivo microscopic technique was used in which acid output, surface cell intracellular pH (pHi), gastric mucus gell thickness and mucosal blood flow were measured simultaneously. Concentrations of bismuth in mucus were measured by atomic absorption. RESULTS: Indomethacin (60 mg/kg) significantly thinned the mucus gel layer and augmented the decrease of pHi during luminal acid superfusion, consistent with a weakened gastric mucosal barrier to acid. Bismuth subsalicylate partially reversed this effect of indomethacin on pHi, consistent with gastroprotection. Neither a prostaglandin-inhibiting but non-injurious dose of indomethacin (5 mg/kg), bismuth subsalicylate, or their combination affected mucus gel thickness or pHi homeostasis. In separate experiments, indomethacin (60 mg/kg) significantly increased gastric mucus bismuth concentration in rats given bismuth subsalicylate. CONCLUSION: Bismuth accumulation in the gastric mucus during the evolution of mucosal injury may play an important role in the gastroprotective effect of bismuth subsalicylate against indomethacin injury.

Comparative effects of nabumetone, naproxen, piroxicam, and diclofenac on rat gastric irritancy following acute exposure to OTC non-steroidal anti-inflammatory agents and other gastric irritants.

This study examined the relative effects of equally-effective anti-inflammatory doses of nabumetone, naproxen, piroxicam and diclofenac on gastric irritancy induced by over-the-counter (OTC) non-steroidal anti-inflammatory drugs (NSAIDs) aspirin and ibuprofen and a variety of necrotizing agents (0.6 N HCl, 0.2 N NaOH and 25% NaCl). Within one hour, aspirin 100 and 200 mg/kg and ibuprofen up to 15 mg/kg produced significant gastric mucosal injury. Aspirin 50 mg/kg produced only minimal damage that was enhanced by 5 x ID25 piroxicam and naproxen, but not by nabumetone or diclofenac. 5 x ID25 naproxen, piroxicam, and diclofenac significantly enhanced mucosal damage produced by ibuprofen 2.5 mg/kg. An equivalent anti-inflammatory dose of nabumetone failed to enhance the gastric irritancy produced by ibuprofen 2.5 mg/kg. Similarly, naproxen, piroxicam, and diclofenac enhanced the susceptibility of the gastric mucosa to the necrotizing actions of 0.6 N HCl, 0.2 N NaOH or 25% NaCl. Naproxen, piroxicam, or diclofenac are more likely than nabumetone to enhance gastric mucosal injury produced by OTC NSAIDs (aspirin and ibuprofen) or other gastric irritants.

Effects of nicotine and ethanol on rat atrial membrane potentials.

The purpose of this research was to study the effects of nicotine and ethanol, alone and in combination, on cardiac membrane potentials (MP). Rat atrial preparations driven at 5 Hz were superfused with Tyrode's solution (37 degrees C) while recording MP with intracellular microelectrodes. Nicotine concentrations below and including 6.2 x 10(-5) M did not affect MP. Within 15 s, nicotine 3.1 x 10(-3) M shortened the action potential duration (APD) and depressed the overshoot of the action potential (OS). This action was blocked by atropine. After 3 min, nicotine prolonged the APD and depressed Vmax of phase O, OS and the amplitude of the action potential (AAP), without affecting the resting membrane potential (RMP). Nifedipine blocked the depression of the OS while tetraethylammonium chloride blocked the prolongation of the APD. Acute exposure to ethanol depressed OS and AAP and shortened APD, but it did not affect RMP or Vmax of phase O. When nicotine and ethanol were administered simultaneously, the APD-prolonging effects of nicotine prevailed. The influence of chronic ethanol ingestion on the acute action of nicotine and/or ethanol was studied in rats pair-fed a liquid diet with (ER) or without (NR) ethanol (35% of total caloric intake) for 24 weeks. Chronic ethanol ingestion accentuated the depressant effect of nicotine 3.1 x 10(-3) M on OS and AAP, but it did not modify the APD-prolonging action of nicotine. The same results were observed when ER and NR were exposed to nicotine and ethanol simultaneously.

Cardiac chronotropic effects of nicotine and ethanol in the rat.

The purpose of this research was to study the chronotropic effects of ethanol (ETOH) and nicotine (NIC), alone and in combination, on the heart. Rat sinoatrial preparations superfused with Tyrode's solution (37 degrees C) were used. The sinoatrial rate (SAR) was monitored using intracellular microelectrodes. NIC concentrations below and including 6.2 x 10(-5) M did not affect the SAR. NIC 6.2 x 10(-4) M and above depressed the SAR. This chronotropic effect of NIC was in part muscarinic. Acute in vitro exposure to ETOH diminished the chronotropic effect of NIC. Chronic ingestion of ETOH (35% of total caloric intake) for 24 weeks did not modify the effect of NIC on the SAR. In summary, there is no positive component in the chronotropic effect of NIC on the rat heart, which is probably due to absence of NIC receptors for the release of norepinephrine. Acute in vitro exposure to ETOH, but not chronic ingestion of ETOH, diminished the negative chronotropic action of NIC.

Actions and interactions of dihydropyridines and ethanol on the rat sinus node.

The actions and interactions of dihydropyridines (DHPs) and ethanol on sinus node (SN) automaticity were studied using rat sinoatrial preparations superfused with Tyrode's solution at 37 degrees C. Intracellular microelectrodes were used to monitor SN rate (SNR). The automaticity of the SN was not affected by nimodipine 10(-10) M, but it was depressed by a higher concentration of this calcium antagonist (10(-8) M). The racemic compound (+/-)Bay K 8644 had a dose-dependent biphasic chronotropic action: At 10(-8) M it decreased the SNR, while at 10(-7) M it increased the SNR. The negative component was blocked by atropine. A small concentration of ethanol (1.8 x 10(-2) M) had a positive chronotropic effect on the SN, and this action was blocked by nimodipine 10(-10) M. This small concentration of ethanol did not modify the chronotropic effects of either of the DHPs. A higher concentration of ethanol (5.3 x 10(-2) M) did not by itself affect the SNR, but it antagonized the chronotropic actions of the two DHPs.