A potent antioxidative compound in crude canola oil, canolol, was recently identified, and reported herein are studies of its scavenging capacity against the endogenous mutagen peroxynitrite (ONOO(-)). ONOO(-) is generated by the reaction between superoxide anion radical and nitric oxide, both of which are produced by inflammatory leukocytes. Among various antioxidative substances of natural or synthetic origin, canolol was one of the most potent antimutagenic compounds when Salmonella typhimurium TA102 was used in the modified Ames test. Its potency was higher than that of flavonoids (e.g., rutin) and alpha-tocopherol and was equivalent to that of ebselen. Canolol suppressed ONOO(-)-induced bactericidal action. It also reduced intracellular oxidative stress and apoptosis in human cancer SW480 cells when used at a concentration below 20 microM under H(2)O(2)-induced oxidative stress. In addition, canolol suppressed plasmid DNA (pUC19) strand breakage induced by ONOO(-), as revealed by agarose gel electrophoresis.

Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO ؊ ), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO 2 and NH 3 from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 M ONOO ؊ , i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO ؊ against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO ؊ -induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 M flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO ؊ was also suppressed by the addition of 20 mM NaHCO 3 but not by the addition of 20 mM NH 3 . In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO ؊ was significantly reduced by the addition of urea or NaHCO 3 , as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO ؊ scavenger, CO 2 /HCO 3 ؊ , that defends the bacteria from ONOO ؊ cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.

In the present study it has been shown that the reactive nitrogen species, peroxynitrite, can cause at least a 7.1-fold increase in the frequency of occurrence of drug-resistant mutants of Helicobacter pylori at a pathophysiological concentration (e.g. 1.0 μM) and in the presence of CLR. Furthermore, the CLR MIC of these resistant H. pylori strains increased by at least 250 times or higher in CLR susceptibility. In the 45 resistant strains, the modification of 23S rRNA A2142G was the predominant mutation (22/45), followed by A2143G (17/45) within the sequences of 23S rRNA. The other mutants were one each (1/45) in A2142T, and T2269G, and two each (2/45) in C2695G and T1944C, respectively. These results show that the inflammatory host reaction involving induction of reactive oxygen species (e.g. O ·−2 ), and the inducible form of nitric oxide synthase, is a significant cause of mutation via peroxynitrite formation, particularly in drug-resistant bacterial strains. Key words drug resistant mutant, free radical, peroxynitrite, 23S rRNAGeneration of drug-resistant mutant pathogenic bacteria is a serious problem. In previous studies (1, 2), we have demonstrated greatly increased mutation frequency in a mouse model of influenza viral infection. During one 10-14 day disease-span of a mouse, a 6-to 7-fold increase in the occurrence of mutant virus compared with the non-diseased state was noted. This mutation is caused primarily by ONOO − (1-4), a product of an endogenous reaction between O ·− 2 and NO, which are generated by inflammatory leukocytes at sites of infection (1-7). ONOO − is a highly oxidative chemical which functions in the rapid oxidation or cleavage of nucleic acids, proteins and lipids (8). It is also, however, a strong nitrating agent, and thus 3-nitrotyrosine (9) and concomitant formation 2 in the presence of cytochrome P450 NADPH reductase, nitric oxide synthases, or cytochrome b5 reductase in a non-stoichiometric manner (10,11), that is, similar to a propagation reaction of lipid peroxidation.This result means that an inflammatory site is a "hot spot" of free radical generation. Also, we have published a more recent report showing that ONOO − in pathophysiological concentration (1-8 μM) greatly increases the mutation frequency in Salmonella typhimurium, as revealed by a modified Ames test in which no cytosolic cytochrome

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