Overexpression of Manganese Superoxide Dismutase (MnSOD) Protects Small Intestine from Ionizing Irradiation
Abstracts
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One of the complications from total body irradiation (TBI) for bone marrow transplantion (BMT) is damage to the intestine. To prevent damage to the intestine during TBI, human MnSOD transgene was overexpressed in the small intestine. MnSOD delivered by plasmid/liposomes (PL) has been demonstrated to prevent irradiation-induced damage to the lung and esophagus. Herpes simplex virus (HSV) vectors containing either the human MnSOD transgene and green fluorescent protein (GFP) transgene, or GFP transgene alone, were constructed and injected into a 2cm length of small intestine of a C3H/HeNsd mouse. Mice were sacrificed 24 hrs later, the injected portion of the bowel removed, compared to the uninjected control sections. RNA was isolated, and nested RT-PCR using primers specific for the human MnSOD transgene was carried out. MnSOD biochemical assays were performed on the injected sections, increased MnSOD mRNA and biochemical activity was detected in HSV-MnSOD injected areas. Other injected segments were frozen in OCT, sectioned, and stained with an anti-GFP antibody. GFP+ cells were demonstrated over the length of intestinal villi. TBI of 7.5 or 10 Gy was delivered to control mice or mice injected 24 hrs earlier with either HSV-MnSOD or HSV-GFP. At 24 or 72 hrs after irradiation, the mice were sacrificed and the injected or control segments of small intestine removed, frozen in OCT, sectioned, stained with H&E stain, and the area of villi measured. The irradiated villi area from control HSV-GFP-injected mice were significantly less (p<0.001) at 0.11±0.06 and 0.16±0.03 mm, compared to villi area injected with HSV-MnSOD, which were not significantly decreased from nonirradiated intestine (0.49±0.10 and 0.35±0.03 mm), respectively. In another experiment, mice were injected with HSV-MnSOD or HSV-GFP, irradiated 24 hrs later to 7.5 or 10 Gy, and sacrificed 72 hrs later. The injected portion of intestine was removed, washed, weighed, and dried in an oven for 24 hrs. Dried intestine was weighed, and the mg protein per weight calculated. Intestine injected with HSV-GFP had a significantly decreased protein content of 106.4±15.9 and 68.0±15.5 µg of protein/mg of weight after 7.5 or 10 Gy, respectively, compared to 199.3±31.1 and 163.7±18.9 for mice injected with HSV-MnSOD irradiated to 7.5 or 10 Gy (p=0.012 and 0.005, respectively). The data demonstrate that overexpression of human MnSOD transgene in small bowel protects against ionizing irradiation damage. |
