Manganese superoxide dismutase (SOD2) is a nuclear encoded and mitochondria localized antioxidant enzyme known to convert mitochondria generated O2•−
. The present study was designed to determine whether SOD2 activity, and therefore mitochondria generated ROS regulate ionizing radiation (IR) induced normal cell transformation. Mouse embryonic fibroblasts (MEFs) carrying wild type (+/+), heterozygous (+/−) and homozygous knockout (−/−) SOD2 were irradiated with equitoxic doses of IR (dose rate, 0.83 Gy/min) and assayed for cellular transformation by measuring Type II and III foci. The transformation frequency was approximately 5-fold higher in SOD2 (−/−) compared to SOD2 (+/+) MEFs (, ); SOD2 (+/−) showed an intermediate response. These results are consistent with earlier reports in the literature demonstrating SOD2 overexpression protecting cells from carcinogen induced transformation8, 43
. Furthermore, these results also provide a direct evidence for the original hypothesis of SOD2 activity and carcinogenesis proposed by Oberley and Buettner17
Since SOD2 activity is known to convert mitochondria generated O2•−
, results from the transformation assay also suggest that mitochondrial ROS could influence cellular transformation in irradiated cells. Irradiation is a classical generator of ROS that persist for milliseconds and result in oxidative damage to cellular macromolecules12
. It has been hypothesized that ROS mediated covalent modifications of cellular macromolecules could regulate some aspects of the cellular responses to IR exposure. This hypothesis is based on the observations that ROS scavengers suppress many of the biological effects of irradiation. However, it is believed that the amount of ROS generated from the primary ionization events are significantly lower than ROS generated from cellular metabolism12
. Therefore, the initial production of ROS might not be entirely responsible for all cellular effects of irradiation. This observation has led to the hypothesis that ROS imbalance due to metabolic oxidative stress long after the initial radiation exposure could be an additional regulatory mechanism controlling the fate of the irradiated cell population.
A key observation of our study was the late ROS accumulation and DNA damage in SOD2 (−/−) compared to SOD2 (+/+) MEFs, correlating with an increase in transformation frequency (–, and ). Cellular redox environment was assessed by flow cytometry measurements of DHE and DCFH-oxidation in time matched control and irradiated MEFs. Initially, there was no significant difference in cellular ROS levels among all 3 cell types compared to their respective un-irradiated control at 24 h post-IR (). The absence of any significant changes in cellular ROS levels during 24 h post-irradiation is comparable to our previously published results6, 14
. In our previous report, we used electron paramagnetic resonance spectroscopy to measure superoxide steady state levels in 6 Gy irradiated control and SOD2 overexpressing human oral squamous carcinoma cells. These results showed no significant change in ROS levels in irradiated control compared to SOD2 overexpressing cells within hours of the radiation treatment6
. Likewise, measurements of DHE-oxidation in irradiated SOD1 overexpressing human glioma cells were comparable to irradiated SOD1 wild-type cells during 1–5 h post-IR14
. While the earlier results were obtained using cancer cells, results from the present study showed similar effects in normal cells, suggesting that the initial changes in cellular ROS levels immediately following the IR exposure could be independent of cellular transformation state and antioxidant enzyme activities. This notion is further supported by our results (, ) demonstrating that the IR induced DNA damage was essentially similar in all 3 cell types within 24 h post-IR. IR exposure increased the percentage of MNBNCs significantly (~3–4 folds) at 24 h post-IR; however, there was no difference among the 3 cell types. Likewise, IR exposure substantially increased γH2AX within 15 min, peaked at 30 min, and decreased by 60 min post-IR in all 3 cell types (). These results indicate that the IR induced early effects on cellular ROS levels and DNA damage are essentially similar among SOD2 (+/+), SOD2 (+/−), and SOD2 (−/−) MEFs.
However, IR induced late effects on cellular ROS levels and DNA damage could differ depending upon the antioxidant capacity of the cell. This “metabolic redox-response” to radiation exposures could determine the fate of the redox-sensitive cellular processes in irradiated cells. This hypothesis is supported by an earlier report demonstrating an increase in the pentose cycle activity in irradiated cells, which is believed to provide NADPH required for repair and biosynthetic processes44
. Furthermore, an earlier report by Petkau et al
showed that administration of SOD1 2–4 h post-IR protected Swiss mice from radiation induced lethality. Likewise, antioxidant manipulations long after the initial radiation exposures have been shown to suppress radiation induced late effects13
. In these previously published reports, the evidence for IR induced late effects on cellular ROS levels and DNA damage were indirect, and primarily based on the observations of elevated levels of oxidized products including 4-hydroxynonenal, 8-hydroxy-2′-deoxyguanosine, and malondialdehyde13, 45, 46
. Our results showed a significant increase in cellular ROS levels in SOD2 (−/−) compared to SOD2 (+/+) MEFs at 72 h post-IR (). Consistent with this increase in cellular ROS levels, the percentage of micronuclei remained higher in SOD2 (−/−) vs.
SOD2 (+/+) MEFs (, ). Because the late ROS accumulation and percentage of micronuclei (DNA damage) were suppressed in SOD2 (+/+) compared to SOD2 (−/−) MEFs, and SOD2 activity is well known to scavenge mitochondria generated superoxide, our results indicate that mitochondria generated superoxide-signaling could regulate IR induced late effects in MEFs.
SOD2 activity and therefore mitochondria derived superoxide-signaling could influence IR induced cell cycle checkpoint activation. IR exposures are known to cause cell cycle arrest in G1, S, and/or G2 to prevent replication of damaged DNA or to prevent aberrant cell division. The regulatory mechanisms are known as checkpoints, and their primary function is to delay progression until the cells have adequately repaired the damage. While the IR induced G1-checkpoint activation was comparable in all 3 cell types (data not shown), the kinetics of the G2-checkpoint activation differed in SOD2 (−/−) vs. SOD2 (+/+) MEFs (). All three cell types showed approximately 2.5-fold increase in G2-cells at 8 h post-IR demonstrating that the G2-checkpoint is intact in all 3 cell types. However, exit from G2 appears to be faster in SOD2 (+/−) and SOD2 (−/−) compared to SOD2 (+/+) MEFs at 24 h post-IR (). Interestingly, the fold-change in G2 in AdSOD2 infected SOD2 (−/−) MEFs was higher than AdEmpty infected SOD2 (−/−) MEFs at 24 h post-IR (); this observation was comparable to irradiated SOD2 (+/+) MEFs.
In summary, mouse embryonic fibroblasts lacking SOD2 activity demonstrated approximately 5-fold higher IR induced transformation frequency compared to wild type cells. SOD2 activity, therefore mitochondria derived superoxide-signaling, did not affect cellular ROS levels and DNA damage within 24 h of irradiation (early response). However, SOD2 activity significantly suppressed IR induced late ROS accumulation and DNA damage at 72 h post-IR (late response). SOD2 activity appears to influence exit from G2 in irradiated cells. These results suggest that long after the initial irradiation a “metabolic redox-response” regulates IR induced transformation in mouse embryonic fibroblasts. These results support the hypothesis that interventions of late ROS accumulation could be a viable redox-based countermeasure for radiation exposures.