Induction of SOS response in Salmonella typhimurium TA4107/pSK1002 by peroxynitrite-generating agent, N-morpholino sydnonimine.
Salmonella typhimurium TA4107/pSK1002 strain was used to measure the SOS response induced by peroxynitrite. The parent strain TA4107 (oxydelta1[oxydelta(oxyR argH)1]) is sensitive to oxidative stress and the plasmid of pSK1002 carries a fused gene umuC'-'lacZ, in which umu and lacZ genes are involved in the induction of mutagenesis and beta-galactosidase activity, respectively. Therefore, the level of SOS response was monitored via beta-galactosidase activity. A bolus addition of authentic peroxynitrite (0.3-0.6 mM) increased about eight times the enzyme activity. In N-morpholino sydnonimine (SIN-1), which produces peroxynitrite from superoxide and nitric oxide generated through hydrolysis, addition of over 1mM SIN-1 induced four-five-fold activity. The SIN-1-induced SOS response was scarcely influenced by superoxide dismutase (SOD), catalase or a combination of both, removing the possibility of induction by superoxide, hydrogen peroxide and hydroxyl radical. Two types of peroxynitrite scavengers, mannitol (type I) and glutathione (type II), decreased the response. Mannitol showed a constant inhibition (70%) at a concentration up to 20 mM, exhibiting kinetics that are zero-order in mannitol and first-order in peroxynitrite. On the other hand, glutathione sharply reduced the response dependent on concentration up to 2 mM (90%), indicating second-order kinetics, first-order in both glutathione and peroxynitrite. Dihydrorhodamine (DHR)123, which traps peroxynitrite in a molar ratio of 1:1, efficiently inhibited the SOS response. These effects suggest that peroxynitrite, generated gradually from SIN-1, penetrates through the cell membrane, damages the DNA and induces the SOS response. This strain can thus, be used in screening of antioxidants against peroxynitrite-induced DNA damage in cells.
Motohashi
Noriko
N
Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe, Japan. motohasi@kobepharma-u.ac.jp
Saito
Yutaka
Y
D007700:Kinetics; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology*; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000096:biosynthesis*; D010957:Plasmids; D013014:SOS Response, Genetics; D012486:Salmonella typhimurium / Q000235:genetics*
D030421:Peroxynitrous Acid; C002385:linsidomine; D008981:Molsidomine