Overexpression of extracellular superoxide dismutase protects mice from radiation-induced lung injury

Abstract

Purpose/Objective: Superoxide dismutase (SOD) is a key enzyme in the metabolism of reactive oxygen species (ROS). Transgenic mice overexpressing extracellular-SOD (EC-SOD) have demonstrated protection of normal tissue from ROS mediated injury in several models, including cerebral ischemia, cardiac ischemia/reperfusion injury, and lung injury from hyperoxia, influenza, and hemorrhage. Therefore, we hypothesized that overexpression of EC-SOD in the mouse lung would be protective against radiation injury by ameliorating damage due to oxidative stress. Materials/Methods: Fourteen B6C3 transgenic (TG) mice that overexpress EC-SOD in the lungs were compared to 9 wild-type (WT) counterparts. Whole lung radiation was delivered to 9 TG mice (XRT-TG) and 6 WT mice (XRT-WT). The remaining mice were not irradiated (Con-TG and Con-WT). Whole lung radiation was given with a single dose of 15 Gy using 4MV photons through a dorsal-ventral field. Pulmonary function was assessed by measuring breathing frequency every two weeks for 10 weeks, then every week up to 24 weeks. Animals displaying evidence of respiratory distress were sacrificed. The remaining animals, including all controls, were sacrificed at the predetermined time of 6 months post-radiation. Upon sacrifice, the entire right lung wet weight was measured as a gross indication of lung consolidation and edema. Histopathology was used to assess tissue fibrosis and immunohistochemistry to assess macrophage activity. ELISA was performed using lung tissue to measure the percentage of the activated fibrogenic cytokine, TGF-β1. Oxidative stress was assessed indirectly by measuring lipid peroxidation in the form of malondialic acid (MDA) using HPLC. Results: Four of 6 XRT-WT mice required euthanasia at 15-20 weeks post-radiation due to respiratory distress. During the same time period, none of the 9 XRT-TG mice displayed evidence of respiratory distress or increased breathing frequency, but 3 mice were randomly sacrificed for pathologic comparison to the WT mice. The mice sacrificed in this time period of 15-20 weeks comprise the pathologic data presented in this study. At the peak respiratory response (14 weeks), the mean breathing frequency (breaths per minute ±SEM) was significantly higher for XRT-WT mice than XRT-TG mice (380 ±34 vs. 286 ±2, p<0.05). Breathing frequency between XRT-TG and Con-TG was not significantly different (286 ±2 vs. 279 ±3, p>0.05). Similarly, right lung weights (mg ±SEM) were higher for XRT-WT mice (228 ±44) than XRT-TG mice (113 ±7) with a trend toward statistical significance, p=0.08. Histologic evaluation of the XRT-WT mice demonstrated greater fibrosis, consolidation, and collagen deposition compared to the XRT-TG and control mice. The XRT-WT mice (48 ±1) had a ten-fold higher number of activated macrophages per 40X field compared to the XRT-TG mice (4.9 ±0.5). The macrophage count for the XRT-TG mice was not significantly different than the control mice, p>0.05. Fibrogenic cytokine activity, measured by percent activated TGF-β1, was higher in the XRT-WT mice than in the XRT-TG mice (37% vs. 11%, p<0.05). Percent of activated TGF-β1 in the XRT-TG mice was not significantly different than the Con-TG mice (11% vs. 12%, p>0.05). Similar results were obtained for lipid oxidation: XRT-WT mice showed a 1.9-fold increase in MDA levels compared to XRT-TG mice (624 ±123 vs. 323 ±24, p<0.05), and a 2.0-fold increase compared to Con-WT mice (264 ±42, p<0.05). Conclusions: Overexpression of EC-SOD in transgenic mice confers significant protection against radiation-induced lung injury. This is supported by several assessments of lung injury, including pulmonary function, gross pathology, histopathology, immunohistochemistry, and TGF-β1 activity. The high level of radioprotection is demonstrated by the fact that the degree of damage in the irradiated transgenic mice was not significantly different than the unirradiated control mice. Finally, as hypothesized, the mechanism appears to be related to a decrease in oxidative stress, as demonstrated by the levels of lipid oxidation.