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Abstract: Omeprazole is a mainstay of therapy for gastroesophageal reflux disease (GERD) and gastritis, and is increasingly used as an over-the-counter remedy for dyspepsia. Omeprazole acts by selectively oxidizing thiol targets in the gastric proton pump, but it also appears to be toxic to the gastric mucosa. We hypothesized that omeprazole toxicity is due to non-specific oxidation of cell structures other than the proton pump, and tested the efficacy of antioxidants to prevent omeprazole-induced toxicity in isolated rabbit gastric glands. Toxicity was measured by uptake and converstion of calcein-AM, following three hours of exposure to omeprazole and a non-selective thiol-oxidant, monochloramine. Intracellular concentration of Zn2+ and the capacity to maintain luminal acidity were monitored using the fluorescent reporters fluozin-3 and Lysosensor DND-160, respectively. Both omeprazole and monochloramine caused marked reduction in cell viability. The toxicity of omeprazole was independent of monochloramine toxicity. The thiol reducing agent dithiothreitol protected gastric glands from injury. The oxidant scavenger Vitamin C also protected, and did not impair the anti-secretory effects of omeprazole. Thus, omeprazole toxicity appears to be oxidative and preventable with antioxidant therapy, including Vitamin C. Vitamin C may be a safe and efficacious addition to treatments requiring the use of PPIs. [Copyright &y& Elsevier]

Monochloramine (NH2Cl) is a potent, thiol-directed oxidant capable of oxidizing thiol (S-H) residues in a wide variety of proteins. Generated in the stomach by the interaction of bacterial and host products, monochloramine has been shown to dysregulate Ca2+ homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([Zn2+]i) in the gastric gland of the rabbit and that the increased Zn2+ within the cell causes an independent decrease in cell viability. Changes in [Zn2+]i were measured by using the fluorescent reporter FluoZin-3, whereas cell viability was assessed by measuring the conversion of calcein-AM to fluorescent calcein, an assay that is not affected by intracellular oxidation state. Cell death was confirmed using propidium iodide and YO-PRO-1 dye uptake measurements. Our experiments demonstrate that [Zn2+]i is increased in gastric glands exposed to NH2Cl and that elevated [Zn2+]i decreases cell viability. Chelation of Zn2+ with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of NH2Cl, but only when administered concurrently. These findings suggest that the toxic effect of thiol oxidants present during chronic gastritis is partially due to dysregulation of [Zn2+]i early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of NH2Cl. [ABSTRACT FROM AUTHOR]

Background: Caspase-3, a pro-apoptotic enzyme, represents a class of proteins in which the active site contains reduced thiol (S-H) groups and is modulated by heavy metal cations, such as Zn2+. We explored the effects of the thiol oxidant monochloramine (NH2Cl) on caspase-3 activity within cells of isolated rabbit gastric glands. In addition, we tested the hypothesis that NH2Cl-induced alterations of caspase-3 activity are modulated by oxidant-induced accumulation of Zn2+ within the cytoplasm. Materials and methods: Isolated gastric glands were prepared from rabbit mucosa by collagenase digestion. Caspase-3 activity was measured colorimetrically in suspensions of healthy rabbit gastric glands, following exposure to various concentrations of NH2Cl with or without the zinc chelator TPEN [tetrakis-(2-pyridylmethyl)ethylene diamine] for 1 h, and re-equilibration in Ringer''s solution for 5 h. Conversion of procaspase-3 to active caspase-3 was monitored by Western blot. Results: Monochloramine inhibited caspase-3 activity in a dose-dependent fashion. At concentrations of NH2Cl up to 100 μm, these effects were prevented if TPEN was given concurrently and were partly reversed if TPEN was given 1 h later. Caspase-3 activity was preserved by concurrent treatment with a thiol-reducing agent, dithiothreitol. Conclusions: At pathologically relevant concentrations, NH2Cl impairs caspase-3 activity through oxidation of its thiol groups. Independently from its thiol oxidant effects on the enzyme, NH2Cl-induced accumulation of Zn2+ in the cytoplasm is sufficient to restrain endogenous caspase-3 activity. Our studies suggest that some bacterially generated oxidants, such as NH2Cl, impair host pathways of apoptosis through release of Zn2+ from endogenous pools. [Copyright &y& Elsevier]

In Helicobacter pylori-induced gastritis, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the gastric mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH2Cl), to serve as agonists of Ca2+ accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca2+ in the cytoplasm (fura-2 AM) or intracellular stores (mag-fura-2 AM). Conditions were adjusted to screen out contributions from metal cations such as Zn2+, for which these reporters have affinity. Exposure to NH2CI (up to 200 µM) led to dose-dependent increases in intracellular Ca2+ concentration ([Ca2+]i), in the range of 200-400 nM above baseline levels. These alterations were prevented by pretreatment with the oxidant scavenger vitamin C or a thiol-reducing agent, dithiothreitol (DTT), which shields intracellular thiol groups from oxidation by chlorinated oxidants. Introduction of vitamin C during ongoing exposure to NH2CI arrested but did not reverse accumulation of Ca2+ in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of NH2CI. Accumulation of Ca2+ in the cytoplasm induced by NH2CI is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca2+-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca2+]i. Under certain conditions, NH2CI may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH2Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents. [ABSTRACT FROM AUTHOR]