Total body irradiation causes a chronic decrease in antioxidant levels

Ionizing radiation exposure may not only cause acute radiation syndrome, but also an increased risk of late effects. It has been hypothesized that induction of chronic oxidative stress mediates the late effects of ionizing radiation. However, only a few reports have analyzed changes in long-term antioxidant capacity after irradiation in vivo. Our previous study demonstrated changes in whole-blood antioxidant capacity and red blood cell (RBC) glutathione levels within 50 days after total body irradiation (TBI). In this study, seven-week-old, male, C57BL/6J mice exposed to total body irradiation by X-ray and changes in whole-blood antioxidant capacity and RBC glutathione levels at ≥ 100 days after TBI were investigated. Whole-blood antioxidant capacity was chronically decreased in the 5-Gy group. The RBC reduced glutathione (GSH) level and the GSH/oxidative glutathione (GSSG) ratio were chronically decreased after ≥ 1 Gy of TBI. Interestingly, the complete blood counts (CBC) changed less with 1-Gy exposure, suggesting that GSH and the GSH/GSSG ratio were more sensitive radiation exposure markers than whole-blood antioxidant capacity and CBC counts. It has been reported that GSH depletion is one of the triggers leading to cataracts, hypertension, and atherosclerosis, and these diseases are also known as radiation-induced late effects. The present findings further suggest that chronic antioxidant reduction may contribute to the pathogenesis of late radiation effects.

Ionizing radiation exposure may not only cause acute radiation syndrome, but there is also an increased risk of late effects. It has been reported that the mortality or morbidity of cancer 1 , cataracts 2 , hypertension 3 , heart diseases 4 , atherosclerosis 5 , and strokes 4 was increased in atomic bomb survivors a few decades after exposure. Most of these diseases are correlated with oxidative stress and persistent inflammation. Indeed, plasma reactive oxygen species (ROS), C-reactive protein (CRP), and interleukin-6 (IL-6) levels were increased in a dosedependent manner in atomic bomb survivors 6 .
Antioxidants maintain redox homeostasis to maintain health by scavenging ROS and reducing oxidative metabolites and cytokines. We have analyzed changes in whole-blood antioxidant capacity from 30 min to 50 days after total body irradiation (TBI) in mice, finding that whole-blood antioxidant capacity decreased in a dose-dependent manner 2-24 days after TBI at 0.5-3 Gy 7 . Importantly, we also found that low antioxidant capacity persisted for at least 50 days after irradiation with TBI at 2 and 3 Gy 7 .
Glutathione is the most prevalent antioxidant in mammals, and the reduced glutathione (GSH)/oxidative glutathione (GSSG) ratio is considered a marker of redox homeostasis 8 . GSH also has anti-inflammatory activity. Depletion of GSH promotes or enhances oxidative stress 9 , inflammatory cytokine levels 10 , and radiation-induced cell damage 11 . Previously, we analyzed changes in red blood cell (RBC) glutathione levels from 2 to 24 days after TBI in mice, finding that radiation decreased the GSH/GSSG ratio through an increase of GSSG levels and a decrease of GSH levels 7 .
However, there is still a lack of information about whether whole-blood antioxidant capacity and RBC glutathione levels change 100 or more days after ionizing radiation exposure. This study extends our previous work and shows long-term changes in antioxidant levels following irradiation in vivo. Mice were exposed to TBI, and www.nature.com/scientificreports/ whole-blood antioxidant capacity, RBC glutathione levels, and complete blood counts (CBC) were examined every 100 days. It was found that whole-blood antioxidant capacity was chronically decreased in the 5-Gy group, and the RBC GSH level and the GSH/GSSG ratio were chronically decreased after ≥ 1 Gy of irradiation. These results suggest that radiation caused a lasting decline of antioxidant levels that may contribute to the pathogenesis of late effects.

5-Gy TBI chronically decreased whole-blood antioxidant capacity.
The antioxidant capacity of whole blood was examined every 100 days after TBI with 1, 3, and 5 Gy. The 1-Gy group did not show significant changes in antioxidant capacity compared with the 0-Gy group (Fig. 1). The antioxidant capacity was significantly decreased at 100 days after 3-Gy, and 200-600 and 800 days after 5-Gy irradiation compared with the 0-Gy group (Fig. 1).

Changes in CBC after TBI.
To evaluate radiation-induced blood injury, changes in the CBC later than 100 days after irradiation were evaluated. The white blood cell (WBC) count was significantly decreased at 600 days after 1-Gy, at 300, 400, and 600 days after 3-Gy, and at 100-700 days after 5-Gy irradiation compared with the 0-Gy group (Fig. 3a). The RBC count was significantly decreased at 400-700 days after 3-Gy and at 200-700 days after 5-Gy irradiation compared with the 0-Gy group (Fig. 3b). Hemoglobin (HGB) was significantly decreased at 500-800 days after 3-Gy and at 100 and 300-700 days after 5-Gy irradiation compared with the 0-Gy group (Fig. 3c). Hematocrit (HCT) was significantly decreased at 400-800 days after 3-Gy and 100-700 days after 5-Gy irradiation compared with the 0-Gy group (Fig. 3d). There were no significant radiationrelated changes in mean corpuscular volume (MCV) at any time point (Fig. 4a). Mean corpuscular hemoglobin (MCH) was significantly increased at 600 and 800 days after 3-Gy irradiation compared with the 0-Gy group (Fig. 4b). There were no significant radiation-related changes in mean corpuscular hemoglobin concentration (MCHC) at any time point (Fig. 4c). Platelets (PLT) were significantly increased at 500 and 800 days after 3-Gy irradiation compared with the 0-Gy group (Fig. 4d).

Discussion
The purpose of this study was to investigate long-term (over a year) changes in antioxidant levels following irradiation in vivo. Mice were exposed to acute TBI by X-ray at 1, 3, and 5 Gy. Acute exposure was assumed in atomic bombings or serious nuclear power plant accidents. Exposure to 1 Gy significantly increased the risk of non-cancer disease in atomic bomb survivors 12,13 . The half lethal dose within 30 days in humans was presumed to be ~ 4 Gy for acute TBI 14 . Furthermore, our preliminary experiment showed that most mice will be dead within 14 days after 7-Gy or higher irradiation. Thus, we analyzed this dose range in the present study. The antioxidant levels were analyzed in blood, because hematopoiesis is one of the main vital processes in the body of mammals and is one of the most radiosensitive systems 15 . It was found that whole-blood antioxidant capacity decreased chronically in the 5-Gy group (Fig. 1). RBC GSH levels and the GSH/GSSG ratio were chronically decreased after ≥ 1-Gy irradiation (Fig. 2). Furthermore, ≥ 3-Gy irradiation decreased WBC and RBC counts, HGB, and HCT levels (Fig. 3). These results suggested that RBC glutathione levels may be the most sensitive long-term biomarker of radiation among these parameters.
It has been reported that buthionine sulfoximine (BSO), a GSH synthesis inhibitor, treatment induced or worsened radiation-related disease and health risk in mice, such as cataracts 16 , brain inflammation 10 , hypertension 17 , atherosclerosis 18 , decreased HDL levels 19 , increased oxidative DNA damage 20 , and tumorigenesis 21 . Thus, induction of radiation-related disease and health risk may occur through decreased GSH levels. It has been shown that BSO treatment decreased viability and proliferation of tumor cells in vitro and in vivo, suggesting that GSH is also essential in tumor growth 22,23 . Indeed, the present study showed that 1-Gy irradiation decreased GSH levels, but did not shorten the lifespan of C57BL/6J mice (Supplementary Fig. S1b). It has been reported that > 90% of irradiated C57BL/6J mice developed thymic lymphoma 24 . Richie et al. also reported that BSO treatment increased colon tumorigenesis, but it did not shorten mouse survival 21 . Thus, further studies should carefully analyze whether decreased GSH level increase cancer mortality in humans.
Radiation-induced long-term changes in oxidative stress levels and hematology findings have been studied in atomic bomb survivors. They received ~ < 3-Gy acute TBI 12 . Thus, the exposure situation is similar to that of the present study. However, no studies analyzed antioxidant levels in atomic bomb survivors. Hayashi et al. reported that plasma ROS levels were increased in a dose-dependent manner in atomic bomb survivors at ~ 50 years after exposure 6 . This result was concordant with the present results and suggested that chronic oxidative stress was increased after exposure. It has been reported that WBC counts were increased 25 , and HGB levels 26 and RBC counts 27 were decreased in atomic bomb survivors. WBC counts were inconsistent, but RBC counts and HGB levels were consistent with the present study. Chua et al. reported that WBC, RBC, and PLT counts were chronically decreased in surviving mice after ~ 7.8-Gy TBI 28 . These results are consistent with the present study. They also suggested that reduced CBC were induced by hematopoietic stem and progenitor cell dysfunction after TBI 28 .
It has been suggested that radiotherapy induced late tissue damage associated with oxidative stress 29 . Robbins et al. reported that 8-hydroxy-2′-deoxyguanosine levels were continuously increased in kidneys over the 24-week experimental period in 10 Gy or higher acute partial-body irradiated rats 30  reported that 18-Gy irradiation to dog lung increased ROS levels in lung tissue, but serum malondialdehyde (MDA) levels and reductase (superoxide dismutase and glutathione peroxidase) activities were not associated with radiation 32 . These reports are consistent with the present study and suggest that radiation affected the longterm redox state. However, these reports performed local irradiation as for radiotherapy, and doses were higher than in the present study. Further studies should examine whether progression of diseases or tissue damage is associated with imbalance of the redox state in our experimental situation.
Several papers have analyzed the redox state in chronic exposure. Volkova et al. analyzed the Scots pine that is widespread in the area contaminated by the Chernobyl accident, finding increases in the GSH/GSSG ratio and in MDA levels in the exposed group 33 . Urushihara et al. analyzed cattle within the ex-evacuation zone of the Fukushima Daiichi nuclear plant accident, finding increased glutathione peroxidase activity and MDA levels in the exposed group 34 . Malekirad et al. analyzed radiology staff, finding increased total antioxidant capacity and lipid peroxidation levels in the exposed group 35 . Thus, these reports are inconsistent with the present study and showed that chronic radiation exposure enhances both oxidative stress and antioxidants. These results suggest that dose rate and total dose are important factors in radiation-induced antioxidant level modification.
The present results leave several open questions. First, antioxidant levels were analyzed after acute TBI. However, induction of biological radiation effects varies with the exposure conditions (e.g. type of radiation, dose rate, irradiation volume, linear energy transfer, and total dose). Furthermore, TBI models do not completely mimic the nuclear disaster scenario or other uncontrolled nuclear events 28 . Future studies should analyze changes in the blood redox state in partial-body or chronic irradiation and determine whether the blood redox state is related to the progression of diseases. Second, young adult (7-week-old) male mice were used in the present study. It has been reported that biological radiation effects vary with age and sex 36,37 . Further studies should examine whether age and sex affect radiation-induced antioxidant changes. Third, the number of individual samples was decreased at ≥ 700 days, especially in the 3-and 5-Gy groups, which reduced statistical power. This was because most of the mice that received 3-and 5-Gy irradiation were dead before 700 days, and only limited breeding space was available. More importantly, it is necessary to consider survivorship bias in the ≥ 700-day groups.

Conclusions
In conclusion, the present study showed that radiation has lasting effects on the antioxidant homeostasis of blood. Whole-blood antioxidant capacity and the RBC GSH/GSSG ratio were chronically decreased after TBI. Considering the present findings and those of previous studies, chronic antioxidant reduction may contribute www.nature.com/scientificreports/ to the pathogenesis of late radiation effects. Furthermore, the present results also support the hypothesis that the redox state could be a marker for estimating the risk of late radiation effects.

Materials and methods
Mice, irradiation, and blood sampling. Six-week-old, male, C57BL/6J mice were obtained from Japan SLC (Shizuoka, Japan). Their diet and drinking water were sterilized by autoclaving 7 . After at least 1 week of acclimation, the mice received TBI (0, 1, 3, and 5 Gy) using an X-ray generator (150 kV; 20 mA; filter: 0.2 mm Cu and 0.5 mm Al; MBR-1520R-3; Hitachi Power Solutions, Ibaraki, Japan. The dose rate was 0.69 Gy/min). Whole blood was collected by a 0.5-mm Goldenrod Animal Lancet (MEDIpoint, New York, NY, USA) puncture of the submandibular vein at 100, 200, 300, 400, 500, 600, 700, and 800 days after irradiation. Whole blood was collected into heparin-containing tubes and centrifuged at 3000×g and 4 °C for 10 min to separate plasma and red blood cells 7 .

Measurement of whole-blood antioxidant capacity (i-STrap).
Whole-blood antioxidant capacity was measured using the i-STrap technique (Dojindo/Dojin Glocal, Kumamoto, Japan), according to the manufacturer's protocol 7 . Briefly, 100 μL of whole blood, 100 μL of saline, 10 mM of 2-diphenylphosphinoyl-2-methyl-3,4-dihy-dro-2H-pyrrole N-oxide (DPhPMPO), and 10 mM tert-butyl hydroperoxide (tBuOOH) were mixed and incubated at room temperature for 30 min 7 . Then, 1 mL of chloroform/methanol (2:1) solution (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) was added and mixed for 10 min. The samples were centrifuged at 8000×g and 4 °C for 10 min, and the organic layer was collected into a new tube and stored at − 80 °C until electron spin resonance (ESR) measurement 7 . The samples were measured by X-band ESR spectroscopy (JES-TE200; JEOL, Tokyo, Japan). The ESR conditions were as follows: microwave frequency, 9.423 GHz; microwave power, 2 mW; field center, 332.0 mT; sweep width, 20 mT; sweep time, 4 min; and time constant, 0.3 s. The signal of DPhPMPO spin adduct intensity was corrected by marker manganese oxide intensity 7 . The number of mice in each group is shown in Supplementary Table S1.
Measurement of red blood cell glutathione levels. RBC glutathione levels were measured using a GSSG/GSH Quantification Kit (Dojindo) according to the manufacturer's protocol 38 . Briefly, RBCs were hemolyzed with 10 times the amount of 5% 5-sulfosalicylic acid solution (FUJIFILM Wako), and the samples were centrifuged at 8000 × g for 10 min to remove proteins 7 . The samples and buffer solution were mixed and incubated for 1 h at 37 °C. Then Substrate and Enzyme/coenzyme working solution was added. After 10 min of incubation, absorbance was measured at 412 nm using a Varioskan LUX plate reader (Thermo Fisher Scientific, Kanagawa, Japan). The number of mice in each group is shown in Supplementary Table S1.
Complete blood counts. Whole blood was collected into heparin-containing tubes. The samples were analyzed using a pocH-100iV instrument (Sysmex, Hyogo, Japan) 7 . The number of mice in each group is shown in Supplementary Table S1. Statistical analysis. The median survival time and 95% CI were calculated, and the significance of differences in overall survival was determined using the log-rank test. The mean and standard deviation (SD) values were calculated for the data of whole-blood antioxidant capacity, RBC glutathione levels, and CBC. Two-way ANOVA and the post hoc Dunnett's test were used to analyze significant differences with the 0-Gy group. A P-value of less than 0.05 was considered significant.