Modulation of Neuronal Nitric Oxide Synthase (NOS1) Sensitized NOS1 -/- Mice to Total Body Irradiaton.

Abstract 4597

Ionizing irradiation results in increased superoxide and nitric oxide (NO) production. These products combine to form peroxynitrite which, through reactions with DNA, lipids, proteins, and cell membranes, initiates apoptotic cell death. Removal of nitric oxide synthase should reduce NO production and limit peroxynitrite formation thereby limiting irradiation induced apoptosis. To test this hypothesis, bone marrow stromal cell lines were derived from long term bone marrow cultures established from NOS1 -/-, NOS2 -/-, NOS3 -/-, or control background strain C57BL/6NHsd mice. Irradiation survival curves using doses ranging from 0 to 8 Gy, and scoring 7 day colonies of greater than 50 cells revealed that NOS1 -/- bone marrow stromal cells were radioresistant compared to all others showing an increased shoulder with a Ñ = 20.8 ± 5.6 compared to 8.3 ± 2.4 for C57BL/6NHsd mice (p = 0.0356). There were no significant differences between the NOS 2 -/- and NOS3 -/- stromal cells and the C57BL/6NHsd stromal cells. To determine whether inhibition of nitric oxide synthase in vivo protected mice from irradiation, groups of C57BL/6NHsd mice had Alzet osmotic pumps containing either the NOS inhibitor, L-NAME, or control phosphate buffered saline (PBS) placed subcutaneously. Three days later, at a time when NO production had been inhibited, mice were irradiated to the LD 50/30 dose of 9.5 Gy total body irradiation, and followed for expected development of the hematopoietic syndrome. Mice with pumps delivering L-NAME showed significantly increased survival (p = 0.0011), compared to control PBS pump containing mice (50% survival at 14 days compared to 8 days). Since L-NAME inhibits all NOS isoforms, NOS1 -/- cell lines were radioresistant in vitro, and NOS1 -/- mice demonstrate in vivo bladder radioresistance (Kanai, Epperly, Pearce et al, American Journal of Physiology-Heart & Circulatory Physiology, 286:H13-H21, 2004) we sought to establish that NOS1-/- mice would be relatively radioresistant to total body irradiation. Groups of female NOS1 -/-, NOS2 -/-, NOS3 -/-, and C57BL/6NHsd mice were total body irradiated to 9.5 Gy. Unexpectedly, NOS1 -/- mice were significantly more radiosensitive (50% survival at 9 days) (p = 0.0006) compared to other groups (50% survival at 25 days for C57BL/6NHsd, 19 days for NOS2 -/-, and greater than 30 days for NOS3 -/-). To determine the mechanism of rapid death in irradiated NOS1-/- mice, peripheral blood was analyzed before and 6 days after 9.5 Gy irradiation. Freshly removed bone marrow CFU-GEMM was tested for clonagenic radiation survival in vitro. There was no significant difference in radiation response of hematocrit, white cell or platelet counts, or marow CFU-GEMM between strains. While NOS1 -/- mice show reduced density of enteric neurons and associated developmental non-morbid pyloric stenosis and gastoesophageal dilation, there was no significant detectable difference in post irradiation histopathology of the esophagus, duodenum, jejunum, ileum, cecum, or colon. There was also no difference in numbers or density of intestinal crypt cells. Intestinal transit studies in irradiated mice demonstrated no significant difference in transit times. Therefore, NOS1 -/- mice display a novel total body irradiation sensitivity that is independent of hematopoietic or gastrointestinal syndromes.

Supported by NIAID/NIH grant U19AI068021