Helicobacter pyloriGenerates Superoxide Radicals and Modulates Nitric Oxide Metabolism

Abstract

During studies of the bactericidal action of nitric oxide (NO), we found that it reversibly inhibited the respiration of Escherichia coli and irreversibly inhibited the respiration of Helicobacter pylori. Peroxynitrite, a reaction product of NO and superoxide, irreversibly inhibited the respiration of both H. pylori and E. coli. H. pylori, but not E. coli, generated substantial amounts of superoxide radicals. These results suggest that NO directly inhibits the respiration of E. coli whereas it rapidly reacts with endogenously generated superoxide radicals in H. pylori. The resulting peroxynitrite inactivates the respiration of H. pylori. Nitric oxide (NO)1 is a multifunctional gaseous free radical produced by NO synthase in various types of cells, such as endothelial cells, neurons, neutrophils, and macrophages (1). Nitric oxide is also generated from nitrite in saliva and from food by microorganisms in the oral cavity as well as by nonenzymatic mechanisms under acidic conditions, such as in gastric juice (12). Because NO synthase is also present in gastric mucosa (13,14), physiological concentrations of NO in gastric juice are fairly high.We have shown that the biological activity of NO is augmented significantly by physiologically low levels of oxygen tension (2-5). Although NO plays important roles in defense mechanisms against enteric bacteria (6, 7), few studies have explored the mechanism of its bactericidal action at physiological levels of oxygen tension.Helicobacter pylori is a Gram-negative and microaerophilic bacterium that resides in the mucus layer overlying the gastric epithelium of the human stomach. This organism is thought to play essential roles in the pathogenesis of gastric inflammation, ulceration, and carcinogenesis (8 -11). Although H. pylori is exposed to fairly high concentrations of NO in gastric juice, which has low oxygen tension, the effect of NO on the metabolism of H. pylori remains to be defined. We therefore studied the effects of NO on the respiration of H. pylori and Escherichia coli under physiologically low levels of oxygen tension. MATERIALS AND METHODSReagents-Peroxynitrite solution and carboxy-2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (cPTIO) were obtained from Dojin Co. (Kumamoto, Japan). Mn-type superoxide dismutase (SOD) from E. coli and 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazol[1,2-␣]pyra zin-3-one (MCLA) were obtained from Sigma and Tokyo Kasei Kogyo Co. (Tokyo, Japan), respectively. Nitric oxide solution was prepared as described previously (5).Bacterial Strains and Their Culture-Two types of enteric bacteria, E. coli K-12 JM109 and H. pylori NCTC-11637, were used in all experiments. E. coli was cultured at 37°C with shaking in nutrient broth (Difco) containing 0.5% NaCl. H. pylori was cultured in Brucella broth containing 5% horse serum under a microaerophilic atmosphere, produced with the use of a gas pack BBL CampyPak (Becton, MD), at 37°C for 20 h as described previously (15). The cultured E. coli and H. pylori were harvested at the logarithmic...

Interleukin-8 Induction by Helicobacter pylori in Gastric Epithelial Cells is Dependent on Apurinic/Apyrimidinic Endonuclease-1/Redox Factor-1

“…ROS enhance APE-1/Ref-1 expression and activity in fibroblasts, macrophages, B cells, and other cell types (25)(26)(27)(28). Studies have shown that infection with H. pylori generates ROS (29), resulting in the accumulation of ROS within the gastric mucosa (18) including the epithelial cells (30), which, in turn, leads to oxidative DNA damage (31) in infected patients. Previously, we reported that treatment with hydrogen peroxide as well as infection with H. pylori, but not Campylobacter jejuni, enhanced APE-1/Ref-1 expression in human gastric epithelial cells (32).…”

“…Studies have shown that AP-1 and NF-B are redox-responsive elements (12) and that the generation of ROS by H. pylori (29) may trigger their activation in gastric epithelial cells. Furthermore, APE-1/Ref-1 itself is regulated by ROS and other environmental stimuli (25,26,28,61), and it has been suggested that the induction of IL-8 and other chemokines by H. pylori is redox sensitive and may depend on ROS-mediated activation of NF-B and AP-1 in gastric epithelial cells (17).…”

Molecular mechanisms ofH. pyloriassociated gastric carcinogenesis

“…Many studies have indicated that ROMs can directly interact with genomic DNA and cause damage in specific genes that control cell growth and differentiation [46][47][48] . Furthermore, it has been reported that intact H. pylori, as well as isolated cellular components, stimulate nitric oxide (NO) synthesis [49][50][51] . High concentrations of NO induce wild-type p53 protein accumulation [52,53] , and the NO-related deamination of DNA has been reported to cause GC-AT transitions, which are frequently found in p53 mutations in gastric cancer [23,54] .…”

Helicobacter pylori and Migraine: Is Eradication of Helicobacter pylori Effective in Relief of Migraine Headache?

“…This cascade is associated with the host immune response and alterations of vascular permeability, as a result of released vasoactive substances (15). Superoxide radicals and nitric oxide are also produced (16). Therefore, the prolonged oxidative injury caused by the persistent infection and the release of vasoactive substances might be involved in regional cerebral blood flow changes during migraine (4).…”