Variable low-dose irradiator.
A recently developed 125
I-based low dose-rate irradiator provides an effective method to continuously expose mice to low dose-rate radiation (Olipitz et al. 2010
). While 125
I is not a radionuclide found in nature, its photon emissions are a reasonable surrogate for both background radiation [the majority of background radiation tracks through our bodies are photon tracks] and environmental contamination [the radionuclide of most concern for long-term contamination after nuclear reactor accidents or nuclear weapons explosions is cesium-137 (137
Cs), a photon emitter].
DNA base-lesion levels in splenic tissue.
Radiation-induced reactive oxygen species (ROS), such as hydroxyl radical (OH•
), superoxide radical (O2•–
), and hydrogen peroxide (H2
), can create mutagenic and cytotoxic DNA base lesions (Halliwell and Aruoma 1991
). In addition, the cellular damage caused by ionizing radiation can potentially cause inflammation, with local generation of high levels of reactive nitrogen species (RNS), including nitric oxide (NO), nitrous anhydride (N2
), and peroxynitrite (ONOO–
) (Dedon and Tannenbaum 2004
). While ONOO–
causes DNA oxidation, N2
can cause nitrosative deamination of DNA nucleobases (Dedon and Tannenbaum 2004
). We therefore set out to determine the extent to which continuous low dose-rate radiation affects DNA damage levels by direct or indirect mechanisms that potentially modulate the formation or clearance of DNA damage.
LC-MS/MS is highly sensitive and can be used to measure the steady-state levels of DNA lesions (Dedon et al. 2007
). In the present study, we quantified mutagenic and cytotoxic base lesions, including 8-oxodG (a DNA oxidation product), dI (a nucleobase deamination product), and εdA and εdC (two lesions derived from reactions of DNA with lipid peroxidation products). The spleen was chosen for analysis given its radiosensitivity. After exposure to approximately 400-fold background radiation for 5 weeks, we did not detect any significant changes in the levels of base lesions in spleen tissue from irradiated mice ().
Figure 1 Exposure to 10.5 Gy acute (7.1 cGy/min) and chronic irradiation (0.0002 cGy/min) does not change steady-state base lesion levels. Effects of chronic, low dose-rate and acute irradiation on DNA base lesion levels of (A) 8-oxodG, (B) dI, (C) εdA, (more ...)
One possible reason that base damage might not accumulate is that radiation-induced DNA damage may be rapidly repaired. We therefore asked if the same total dose of radiation induces base damage when delivered acutely, at a dose-rate that was approximataely four orders-of-magnitude higher (7.1 cGy/min). Even under acute conditions, we did not detect any significant difference in the levels of base lesions (). Together these results show that exposure to 10.5 cGy does not significantly affect the levels of several key DNA base lesions that are known to be formed in response to radiation and inflammation, regardless of the dose-rate (ranging from 0.0002 to 7.1 cGy/min).
Micronuclei analysis in RBCs.
Although far less frequent than radiation-induced base lesions, radiation-induced double strand breaks (DSBs) are severely cytotoxic and mutagenic (Helleday et al. 2007
). The micronucleus assay is an exquisitely sensitive approach for detecting DSBs (Hayashi et al. 2000
). Using the in vivo
RBC micronucleus assay, small chromosomal fragments can be detected in enucleated RBCs () (Kirsch-Volders et al. 2000
). To explore the impact of dose-rate on susceptibility to DSBs, we compared the extent to which 10.5 cGy radiation induces micronuclei when delivered acutely versus chronically. Consistent with previous studies, exposure to 10.5 cGy delivered acutely (7.1 cGy/min) resulted in a significant increase in micronuclei in mice in vivo
< 0.005) () (Abramsson-Zetterberg et al. 1996
; Bhilwade et al. 2004
; Uma Devi and Sharma 1990
). In contrast, no significant increase in micronuclei was observed in continuously irradiated mice (). These data reveal that dose-rate can significantly affect radiation-induced DNA damage levels.
Figure 2 Representative image of a PCE-containing micronuclei (MN-PCE; arrowheads) and of a normal RBC (arrow) isolated from bone marrow; bar = 20 µm (A). Low dose-rate (0.0002 cGy/min) irradiation (B) does not induce micronuclei in PCEs, whereas acute (more ...) Frequency of HR events in the pancreas.
An alternative approach for studying DSBs is to assess DSB repair activity. We have recently developed FYDR mice that allow investigation of mitotic HR, one of the major DSB repair pathways in mammals (Wiktor-Brown et al. 2006a
). FYDR mice carry a direct repeat recombination substrate for which an HR event can restore the full length Eyfp
coding sequence () (Hendricks et al. 2003
). The frequency of fluorescent yellow recombinant cells can be assessed using in situ
imaging or flow cytometry (). Recombinant cells can continue to fluoresce for their lifespan, making it possible to monitor the accumulation of recombinant cells over time (Wiktor-Brown et al. 2006b
). Thus, although induction of recombination can potentially be detected by an increase in the frequency of recombinant cell foci (compare ), no difference was observed in the frequency of HR among low dose-rate irradiated and non-irradiated animals ().
Figure 3 FYDR mice carry a recombination substrate (A) that results in expression of Eyfp upon recombination repair. The Eyfp signal can be detected by in situ imaging. The frequency of Eyfp+ cells increases with age [(B) 4-week-old (young) mouse; (C) 24-week-old (more ...)
Although these data suggest that low dose-rate radiation did not affect the frequency of HR, it remained formally possible that radiation caused silencing of the Eyfp
gene (Suzuki et al. 2011
), which could lead to a false negative result. We therefore exploited FYDR-Rec positive-control mice to test for radiosuppression of Eyfp
expression; however, no suppression was detected (). Therefore, we conclude that low dose-rate radiation does not significantly affect HR.
To explore the possibility that acute exposure might induce HR, animals were exposed to 10.5 cGy at a dose-rate of 7.1 cGy/min. Although there appears to be a slight increase in HR frequency by in situ imaging, the difference is not statistically significant (). Taken together, our analysis of DSB repair indicates that long-term low dose-rate irradiation at approximately 400-fold background for 5 weeks does not lead to a detectable increase in the frequency of either micronuclei or HR.
Gene expression analysis of DNA damage response genes.
Gene expression changes have been observed in response to acute irradiation delivered at doses as low as 1 cGy (Alvarez et al. 2006
; Amundson et al. 2000
; Fujimori et al. 2005
). Several genes found to be consistently affected by radiation are part of the transformation related protein 53 [p53
)] DNA damage response: cyclin-dependent kinase inhibitor 1A (Cdkn1a
), growth arrest and DNA-damage-inducible 45 alpha (Gadd45a
), transformed mouse 3T3 cell double minute 2 (Mdm2
), ataxia telangiectasia mutated homolog (human) (Atm
), and damage specific DNA binding protein 2 (Ddb2
) (Gruel et al. 2008
). As WBCs are particularly responsive to radiation exposure (Amundson et al. 2000
), we assessed gene expression levels for Cdkn1a
, and Ddb2
in primary WBCs after exposure to low dose-rate radiation (0.0002 cGy/min). We found that there was no significant difference in gene expression between irradiated and non-irradiated animals for any of the five genes (). To explore the impact of dose-rate, we exposed mice to 10.5 cGy irradiation delivered acutely (7.1 cGy/min). At this higher dose-rate, Cdkn1a
was significantly up-regulated (), indicating that DNA damage responses are dose-rate dependent, which is consistent with previous studies (Amundson et al. 2003
Figure 4 Effects of chronic, low dose-rate (0.0002 cGy/min; A,B) and acute (7.1 cGy/min; C,D) ionizing radiation on gene expression in WBCs. Gene expression changes were compared between control and treated groups after irradiation (A,C) and in irradiated animals (more ...)
A significant challenge for all animal studies is variability due to interindividual differences. We therefore developed an approach for a paired analysis, wherein blood samples were collected from the same animals both before and after radiation exposure. Regardless of whether the data was paired or pooled, Cdkn1a was significantly induced by acute irradiation, though we detected a greater induction using the paired experimental design (compare ). Furthermore, using paired analysis conditions, we also detected a significant increase in expression of Mdm2 (). These studies suggest that longitudinal assessment increases the sensitivity of the assay to subtle changes in gene expression. Nevertheless, under the conditions of low dose-rate exposure (0.0002 cGy/min), there were no significant changes in gene expression, even with a paired analysis ().
Taken together, studies of animals that live under conditions of prolonged continuous exposure to radiation at approximately 400-fold background do not show any evidence of increased levels of base damage (for 8-oxodG, dI, εdA, εdC) nor DSBs (micronuclei and HR), nor induction of a DNA damage response (at the level of p53-inducible gene expression). Importantly, when delivered acutely, the same total dose induced micronuclei and induced key genes involved in the DNA damage response.