Inducible nitric oxide synthase (iNOS) expression and NO production increase after radiation exposure. We showed previously that inhibiting iNOS expression prevents hemorrhage injury; we therefore investigated whether inhibiting iNOS expression also limits radiation injury. Human Jurkat T cells were exposed to gamma radiation (2, 4, 6 or 8 Gy), and cell lysates were collected for analysis at selected times afterward. Radiation exposure increased iNOS expression within 4 h postirradiation by increasing the levels of the iNOS transcription factors NF-kappaB and KLF6. By 24 h postirradiation cell viability was reduced. In these cells, NO production, lipid peroxidation, protein nitration, apoptosomes (formed by cytochrome c, caspase 9 and Apaf-1), and caspase 3 activity were significantly elevated, suggesting that the iNOS pathway had been activated. Treatment with the iNOS inhibitors 17-DMAG or L-NIL-6 24 h prior to irradiation limited these changes, as did treatment with iNOS siRNA to silence the iNOS gene. These results suggest radiation injury involves the iNOS pathway, and iNOS-mediated NO produced endogenously in the T cell alters overall T-cell function and results in apoptosis and cell lethality. Control of iNOS expression may represent a useful approach for protecting T cells from radiation injury.

BackgroundHemorrhage increases inducible nitric oxide synthase (iNOS) and depletes ATP levels in various tissues. Previous studies have shown that geldanamycin, an inducer of heat shock protein 70kDa (HSP-70) and inhibitor of iNOS, limits both processes. Reduction in NO production limits lipid peroxidation, apoptosome formation, and caspase-3 activation, thereby increasing cellular survival and reducing the sequelae of hemorrhage. The poor solubility of geldanamycin in aqueous solutions, however, limits its effectiveness as a drug. 17-DMAG is a water-soluble analog of geldanamycin that might have greater therapeutic utility. This study investigated the effectiveness of 17-DMAG at reducing hemorrhagic injury in mouse small intestine.ResultsIn mice, the hemorrhage-induced iNOS increase correlated with increases in Kruppel-like factor 6 (KLF6) and NF-kB and a decrease in KLF4. As a result, increases in NO production and lipid peroxidation occurred. Moreover, hemorrhage also resulted in decreased Bcl-2 and increased TNF-α, IL-6, and IL-10 concentrations, p53 protein, caspase-3 activation, and cellular ATP depletion. A shortening and widening of villi in the small intestine was also observed. Treatment with 17-DMAG significantly reduced the hemorrhage-induced increases in iNOS protein, jejunal alteration, and TNF-α and IL-10 concentrations, but 17-DMAG did not affect the hemorrhage-induced increases in p53 and IL-6 concentration. 17-DMAG treatment by itself upregulated HSP-70, Bcl-2, and p53.ConclusionSince 17-DMAG is water soluble, bioactive, and not toxic, 17-DMAG may prove useful as a prophylactic drug for hemorrhage.