Ionizing radiation induces rapid production of ROS, including superoxide. Reactive nitrogen species (RNS) are also present at sites of irradiation as a result of activated immune cells (19
). The capacity of cells to neutralize ROS and RNS through maintenance of an antioxidant pool has been shown to be critical for cell-, tissue- and organ-specific radiation tolerance (20
). The two major defenses against ROS and RNS stress are scavengers, such as glutathione, and antioxidant enzymes, such as superoxide dismutase (SOD). Three forms of superoxide dismutase facilitate reduction of the levels of radiation-induced superoxide. One of these, SOD2, or mitochondrial SOD (MnSOD), has been shown to ameliorate radiation-induced injury through targeted localization to the mitochondrial membrane (22
Organ-specific up-regulation of MnSOD through targeted MnSOD-PL gene therapy has been shown to confer tissue- and organ-specific radioprotection in the lung, oral cavity, bladder and esophagus (23
). The MnSOD gene therapy-mediated reduction in histopathology and tissue injury (as seen by reduced functionality) has been shown to correlate with increased levels of antioxidant capacity in individual cells (21
) and decreased lipid peroxidation in irradiated tissues (9
). Whether MnSOD-PL organ-specific gene therapy affects radiation-induced DNA sequence rearrangements in vivo
had not been evaluated previously.
In the present studies, we delivered 29 Gy radiation to the upper body of FYDR mice and measured the levels of HR, which reflects the repair of DNA double-strand breaks, in explanted esophageal epithelial cells. In our previous studies with the same background mouse strain (C57BL/6HNsd), MnSOD-PL protection was observed at 29 Gy (1
). In the present studies, 29 Gy induced a significant increase in HR in esophageal epithelial cells 7 days after irradiation, the time when significant apoptosis and lipid peroxidation are detected (1
). These results confirm and extend previous studies documenting the value of the FYDR mouse strain for quantifying HR in vivo
Radiation both directly ionizes the DNA and reacts with water to create ROS. Many different types of DNA lesions are created by direct ionization, ROS or RNS associated with inflammation, including base damage and single- and double-strand breaks (12
). Among these, double-strand breaks are thought to be the most deleterious lesions, because they can be highly toxic and can lead to large-scale sequence rearrangements through HR events (28
). HR can lead to insertions, deletions, translocations and LOH, all of which have been shown to promote cancer (29
). Therefore, the levels of HR reflect the extent to which cells have increased levels of cancer-promoting sequence rearrangements. Since radiation-induced sequence changes are one of the most serious long-term consequences of radiation exposure, strategies to suppress such rearrangements would be of great value.
Superoxide is formed both as a consequence of ionization of water and also by activated immune cells, which secrete both superoxide and nitric oxide. Breakdown products of superoxide and nitric oxide have been shown to be highly genotoxic (19
). Furthermore, superoxide and nitric oxide can react to form peroxynitrite, which has been shown to be highly recombinogenic (33
). A reduction in the levels of superoxide might therefore reduce the levels of DNA damage. To explore this possibility, we tested MnSOD-PL gene therapy. We found that MnSOD-PL delivered in a concentrated organ-specific fashion significantly reduced the magnitude of radiation-induced HR in the esophagus in vivo
. These data suggest that MnSOD-PL can exert radioprotective effects in the esophagus at the level of DNA damage, which is detectable as a reduction in radiation-induced HR in individual explanted cells. Alternative explanations include the possibility that MnSOD-PL treatment inhibits the HR process or that MnSOD expression suppresses expression of the YFP protein.
The observation that MnSOD-PL suppresses the genotoxic effects of radiation is consistent with published data on the effects of intraesophageal administration of MnSOD-PL including increased mouse survival (1
), preservation of body weight and hydration (1
), protection of self-renewing transplantable esophageal stem cells (6
), decreased radiation-induced apoptosis (10
), and decreased radiation-induced lipid peroxidation (9
We also explored the potential effects of MnSOD in the pancreas (which has been studied previously) using FYDR mice (16
) and in the bone marrow (which had not previously been studied for HR in FYDR mice) after thoracic irradiation. HR was also suppressed in the pancreas of mice receiving thoracic irradiation after intraesophageal administration of MnSOD-PL. MnSOD administered to the esophagus might lead to a reduction in HR in the pancreas as a result of the ability of MnSOD-PL to reduce the levels of radiation-induced cytokines that might otherwise induce HR (3
). Since radiation did not produce a statistically significant increase in the level of HR in the pancreas, the apparent suppression of HR by administration of MnSOD-PL is inconclusive. There was no detectable increase in HR in the bone marrow of the same FYDR mice. The observation that relatively few bone marrow cells from the positive control FYDR-Rec were detected as YFP+
suggests that expression of EYFP in bone marrow limits the detection of rare fluorescent recombinant cells. The observed variation in the spontaneous levels of recombinant fluorescent cells in different mouse tissues confirms and extend previous studies (16
To further explore the potential for MnSOD to suppress HR in the bone marrow, we irradiated the femurs and tibias of other FYDR mice with 8 or 29 Gy and quantified HR 1 to 14 days after irradiation. Earlier times were chosen for bone marrow examination since hematopoietic stem cells and their progeny are significantly more radiosensitive and undergo apoptosis more rapidly after irradiation than esophageal cells (9
). Radiation induced a detectable increase in HR at day 7 in bone marrow irradiated with 8 Gy, but no increase in recombinant fluorescent cells was apparent on day 14.
Given the very low level of EYFP expression in the bone marrow of the positive control mice, the significant increase on day 7 was unexpected.
Intravenous injection of MnSOD-PL 24 h prior to irradiation did not significantly reduce the elevation of HR in the 8 Gy-irradiated bone marrow. This may have been the result of a low concentration of MnSOD-PL reaching the marrow after i.v. injection compared to the relatively high levels achieved in the locally treated esophagus. Systemic delivery of MnSOD-PL by intravenous injection does protect mice from death after 9.5 or 1.0 Gy total-body irradiation, but uptake of plasmid in marrow was not quantified in that study (34
). We administered 100 μg of plasmid DNA in both intraesophageal and i.v. procedures. The local esophageal delivery results in a detectable level of expression of the MnSOD transgene in the esophagus for 24–72 h (1
). In recent studies, at 24 h after i.v. injection of MnSOD-PL, examination of all explanted mouse tissues by PT-PCR did not detect MnSOD transgene-specific sequences in marrow, only in the liver. Moreover, we did not detect epitope-tagged transgene encoded HA-MnSOD protein by histochemistry at 24 h in explanted marrow, only in the liver. In contrast, after local intraesophageal administration, the transgene and its HA-MnSOD product protein were detected in explanted tissue continually for 24–72 h (1
). Further studies testing the effect of higher levels of local intramedullary injected MnSOD-PL on irradiated bone marrow may help to evaluate the role of marrow levels of transgene and protein in radioprotection.
Radiation was delivered locally to the esophagus and marrow of FYDR mice at a dose permitting survival of some stem cells (35
) (29 Gy for esophagus, 8 Gy for marrow) and induced an increase in HR. A marrow dose of 29 Gy was also given, which was expected to leave no surviving hematopoietic progenitors. We did not observe any induction of HR by an increase in the frequency of YFP+
cells at this toxic dose. This is in contrast to the results for the less toxic dose of 8 Gy. It is not surprising that a recent publication (36
) reported no esophageal stem cell killing after a lower, fractionated radiation dose. The present data provide useful information on the effects of radiation on the bone marrow of FYDR mice. In particular, these studies suggest that FYDR mice could be useful for evaluating the potential benefits on hematopoietic stem cells of systemic i.v. and/or transdermal delivery of new small-molecule mitochondrially targeted radioprotectors and radiation damage mitigators.
Large-scale DNA sequence rearrangements that result from HR are known to contribute to cancer and aging, and HR can be induced by DNA damage caused by radiation. Methods for suppressing radiation-induced HR would therefore be of value. Here we explored the potential for gene therapy to protect against radiation-induced HR and showed that MnSOD-PL suppressed radiation-induced HR in both the esophagus and the pancreas. In contrast, suppression of HR was not observed in the bone marrow, possibly as a result of limited delivery to target cells. While prolonged overexpression of the MnSOD transgene has been shown to reduce levels of other antioxidant proteins such as catalase (37
), acute overexpression in the gene therapy model does not. The demonstration that MnSOD-PL suppresses HR in vivo
shows that a reduction in the levels of superoxide helps to prevent radiation-induced genotoxicity and provides evidence of an additional benefit of MnSOD-PL therapy.