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Radiation-induced DNA damage and the relative biological effectiveness of 18F-FDG in wild-type mice

Radiation-induced DNA damage and the relative biological effectiveness of 18F-FDG in wild-type mice Abstract Clinically, the most commonly used positron emission tomography (PET) radiotracer is the glucose analog 2-[18F] fluoro-2-deoxy-d-glucose (18F-FDG), however little research has been conducted on the biological effects of 18F-FDG injections. The induction and repair of DNA damage and the relative biological effectiveness (RBE) of radiation from 18F-FDG relative to 662 keV γ-rays were investigated. The study also assessed whether low-dose radiation exposure from 18F-FDG was capable of inducing an adaptive response. DNA damage to the bone marrow erythroblast population was measured using micronucleus formation and lymphocyte γH2A.X levels. To test the RBE of 18F-FDG, mice were injected with a range of activities of 18F-FDG (0–14.80 MBq) or irradiated with Cs-137 γ-rays (0–100 mGy). The adaptive response was investigated 24h after the 18F-FDG injection by 1 Gy in vivo challenge doses for micronucleated reticulocyte (MN-RET) formation or 1, 2 and 4 Gy in vitro challenges doses for γH2A.X formation. A significant increase in MN-RET formation above controls occurred following injection activities of 3.70, 7.40 or 14.80 MBq (P < 0.001) which correspond to bone marrow doses of ~35, 75 and 150 mGy, respectively. Per unit dose, the Cs-137 radiation exposure induced significantly more damage than the 18F-FDG injections (RBE = 0.79±0.04). A 20% reduction in γH2A.X fluorescence was observed in mice injected with a prior adapting low dose of 14.80 MBq 18F-FDG relative to controls (P < 0.019). A 0.74 MBq 18F-FDG injection, which gives mice a dose approximately equal to a typical human PET scan, did not cause a significant increase in DNA damage nor did it generate an adaptive response. Typical 18F-FDG injection activities used in small animal imaging (14.80 MBq) resulted in a decrease in DNA damage, as measured by γH2A.X formation, below spontaneous levels observed in control mice. The 18F-FDG RBE was <1.0, indicating that the mixed radiation quality and/or low dose rate from PET scans is less damaging than equivalent doses of gamma radiation. Introduction The use of diagnostic imaging in health care has seen a dramatic increase over the last two decades. In the USA, nuclear medicine procedures have increased >7% per annum, with positron emission tomography (PET) showing the greatest increases at 57% per year with expected increases due to its unprecedented sensitivity for the detection of biological processes (1, 2). Clinically, the most commonly used PET radiotracer is the glucose analog 2-[18F] fluoro-2-deoxy-d-glucose (18F-FDG). PET scans with 18F-FDG are used to image disease states characterised by alterations in metabolism: epilepsy (3), Alzheimer’s disease (4), infection (5), heart disease (6, 7) but most often, malignancy (8, 9). 18F-FDG is transported into the cytoplasm of metabolically active cells by glucose transport membrane proteins (GLUT) and undergoes phosphorylation to form 18F-FDG-6-phosphate by hexokinase (10). At this point, 18F-FDG becomes trapped in the cell because of the substitution at the hydroxyl group (11). The absorbed radiation dose in tissue depends on the glucose requirements of that tissue. The radiation exposure is a result of positrons (β+, Emax = 634 keV) emitted by 18F-FDG and subsequent annihilation photons (γ-rays, 511 keV). During a typical clinical protocol involving the administration of 350–750 MBq 18F-FDG (12), most tissues will be irradiated throughout the patient’s body (4–9 mGy). However, in organs with high energy requirements, doses can be much higher, i.e. the brain (10–36 mGy) and heart (16–51 mGy) or organs within the excretory system including kidneys (7–23 mGy) and bladder (13–233 mGy) through which the radiopharmaceutical is voided (13–17). Dose is delivered at a low decaying dose rate reflective of the physical (109.7min) and biological half-life of 18F-FDG. The biological half-life depends on the residence times of the radiopharmaceutical within different tissues. The MIRD dose estimate report for 18F-FDG provides a whole body residence time of 2.38±0.12h (15). It is important to note that regardless of the site being imaged, an injection of the radiopharmaceutical 18F-FDG will result in systemic uptake and radiation exposure (18). It has been reported that, compared to all other nuclear medicine procedures, PET scans with 18F-FDG deliver one of the highest effective doses to patients (14.1 mSv) (18). The effective dose is a value which allows the summation of dose contributions from radionuclides non-uniformly distributed throughout the body to evaluate the associated stochastic effects (cancer & hereditary) relative to other radiation exposures. It requires a calculation using radiation weighting factors (wR) and tissue weighting factors (wT) published by the International Commission on Radiological Protection (ICRP) (19). The increase in nuclear medicine procedures has stimulated interest in whether a rise in radiation-induced cancers should be expected. Certain studies, utilising the linear no threshold (LNT) model of risk estimation, assert that PET scans will result in a measurable increase in the lifetime risk of cancer among patients (20). These types of studies use dose as a surrogate for risk. While accurate dosimetry in nuclear medicine is important, it should only be used as a starting point in evaluating the possible health effects associated with nuclear medicine procedures. Nuclear medicine examinations involve protracted dose rates which have been shown to significantly reduce the biological risk associated with a given dose of ionising radiation (21–24). In fact, many studies have shown that low dose-rate exposures may be protective to an organism by generating an adaptive response. A number of experiments using cell lines have shown that low dose-rate radiation exposures reduced the level of transformation or damage below that of controls (25–27). In addition, both low dose (28, 29) and lose dose-rate (26, 30, 31) exposures have been shown to reduce the full detriment of a subsequent high-dose radiation exposure. In contrast, there are certain elements of nuclear medicine procedures that may augment the associated risk. Certain groups have identified that decaying dose rates generate more damage than fixed low dose rates (32) or that damage delivered at a very low dose rate (9.4 cGy/h) evades DNA damage surveillance mechanisms (33). Current investigations into medical isotope health effects have been focused almost exclusively on radioiodine (I-131, I-125) (34–38). There are few investigations that address the possible health effects of radiation exposure from newer imaging agents (39) and none that are focused on PET imaging. Moreover, many radiobiological investigations into the characteristics associated with nuclear medicine procedures (low dose rates, changing dose rates) have been investigated in cell lines. In this study, the response to 18F-FDG was investigated in mice to gain a better understanding of the in vivo radiation-induced health effects associated with PET scans. Inbred mice were chosen as the model system to eliminate the high degree of interindividual variability observed in patient studies (35, 40). DNA double-strand breaks (DSBs) following 18F-FDG injections were measured by γH2A.X fluorescence at 24-h post-injection and micronucleated reticulocyte (MN-RET) formation at 43-h post-injection. Additionally, the potential of this injection to induce an adaptive response was evaluated using these same endpoints by irradiating mice or cells at 24-h post-injection with high doses of radiation (1, 2 and 4 Gy). We hypothesise that the low dose rate associated with PET scans employing 18F-FDG reduces the detrimental effects of the radiation exposure and that this exposure may have the capacity to generate an adaptive response. Materials and methods Mice In these experiments, 7- to 9-week-old wild-type female mice (B6.129S2-Trp53tm1Tyj/1x 129X1/SvJ, Jackson Laboratory; Bar Harbor, ME, USA) were used. Mice were housed five per cage in specific pathogen-free conditions. The housing room was maintained on a 12-h light/dark cycle at a temperature of 24±1°C. Protocols were approved by the Animal Research Ethics Board at McMaster University and carried out as per the Canadian Council on Animal Care. 18F-FDG production and injection Mice were injected with 18F-FDG prepared at Hamilton Health Sciences (Hamilton, Ontario, Canada). The 18F was produced by 18O(p,n)18F reaction using a Siemens RDS112 11MeV Proton-Cyclotron and the FDG was prepared as per the procedure listed in (41), meeting all USP and Health Canada regulatory requirements. All mice were fasted overnight prior to the isotope injection (12–14h) and water was provided ad libitum. Mice were housed individually for the duration of the experiment to minimise inter-mouse irradiation following the isotope injection. Individual isotope doses were measured using a CRC-12 radioisotope calibrator (Capintec). Pre- and post-injection activity of the syringe in addition to the time of each measurement was recorded. This information was used to accurately calculate the activity administered to each mouse by correcting for decay. The weight of each mouse was also recorded prior to the injection. The average weight of mice used in these experiments was 20±0.08g. The 18F-FDG was diluted into a total volume of 200 µl with saline and administered by tail vein injection. Control mice were injected with 200 µl saline only. Anaesthesia was not used since image acquisition was not the objective of this work and may interfere with the biological processes under investigation. Food was returned to the mice 2h after the injection to maximise uptake of the 18F-FDG and to simulate the conditions of a typical PET procedure. Calculation of absorbed doses from 18F-FDG Previously published absorbed dose estimates for 18F-FDG in mice (42, 43) were used to convert the injected activities used into whole body doses and bone marrow doses (Table I). The whole body dose estimates using both approaches are in good agreement. Small samples of mice were imaged at 7–8 weeks of age to verify the distribution of 18F-FDG in the mouse model (Figure 1). Table I. Injection activities and corresponding doses for 20g mice injected with 18F-FDG n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 Activities are expressed in MBq (µCi). Absorbed dose estimates are expressed in mGy. Data are expressed mean ± SEM. n = number of mice per group. aObtained using absorbed dose values (14 mGy/MBq) from Taschereau et al. (43). bObtained using an approximate dose calculation and S value (22.1×10−13 Gy/Bq/s) from Funk et al. (42). cObtained using the absorbed dose in marrow (10 mGy/MBq) from Taschereau et al. (43). Open in new tab Table I. Injection activities and corresponding doses for 20g mice injected with 18F-FDG n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 Activities are expressed in MBq (µCi). Absorbed dose estimates are expressed in mGy. Data are expressed mean ± SEM. n = number of mice per group. aObtained using absorbed dose values (14 mGy/MBq) from Taschereau et al. (43). bObtained using an approximate dose calculation and S value (22.1×10−13 Gy/Bq/s) from Funk et al. (42). cObtained using the absorbed dose in marrow (10 mGy/MBq) from Taschereau et al. (43). Open in new tab Fig. 1. Open in new tabDownload slide Representative PET scan of an 8-week-old female healthy wild-type mouse from this study acquired with a Philips MOSIAC animal PET scanner at the McMaster Center for Pre-Clinical and Translational Imaging. Mouse (fasted) was injected with 18.5 MBq 18F-FDG. Static scan (15min in duration) was acquired 45min post-injection.(A) Coronal view: increased 18F-FDG uptake in the bladder (4), heart (3), brain (1) and active skeletal muscle/brown fat (2) is evident. (B) Sagittal view: reveals 18F-FDG uptake in kidneys (5). The pattern of uptake is consistent with the predicted distribution of absorbed doses. Fig. 1. Open in new tabDownload slide Representative PET scan of an 8-week-old female healthy wild-type mouse from this study acquired with a Philips MOSIAC animal PET scanner at the McMaster Center for Pre-Clinical and Translational Imaging. Mouse (fasted) was injected with 18.5 MBq 18F-FDG. Static scan (15min in duration) was acquired 45min post-injection.(A) Coronal view: increased 18F-FDG uptake in the bladder (4), heart (3), brain (1) and active skeletal muscle/brown fat (2) is evident. (B) Sagittal view: reveals 18F-FDG uptake in kidneys (5). The pattern of uptake is consistent with the predicted distribution of absorbed doses. Biological experiments Two flow cytometry-based assays were used to evaluate the biological effects of 18F-FDG injections in mice: MN-RET formation and γH2A.X mean fluorescence. All samples were kept at 0°C during preparation and analyzed using an EPICS XL (Beckman Coulter, Miami, FL, USA) flow cytometer. MN-RET assay All reagents used for the MN-RET assay were included the Mouse MicroFlowPLUS® kit (Litron Laboratories, Rochester, NY, USA). Cells were fixed in absolute methanol (Sigma Aldrich, Mississauga, Ontario, Canada) at −80°C and stored at −80°C for a minimum of 24h before staining and flow cytometric analysis. The fixed blood samples were washed and labelled for flow cytometric analysis according to the Mouse MicroFlowPLUS Kit manufacture’s procedure and previously described by Dertinger et al. (44). Briefly, fixed blood cells were washed with 12ml buffer solution and cell pellets were maintained at 0°C until staining. Following the wash, 80 µl of reagent mixture containing anti-CD71-FITC, anti-CD61-PE, RNase and buffer solution was added to 20 µl aliquot of each fixed blood sample in duplicate. The cells were incubated on ice for 30min followed by 30min at room temperature, and then returned to ice. Immediately prior to analysis by flow cytometry, 1ml of 4°C propidium iodide (1.25 µg/ml in buffer solution) was added to each tube. Data acquisition was performed using the EPICS XL flow cytometer (Beckman Coulter, Brea, CA, USA) equipped with a 488nm argon laser. The gating logic used to quantitatively analyse the erythrocyte subpopulations has been described previously (44). Analysis windows were set to quantify the number of reticulocytes (RETs) and MN-RETs for each sample. Representative bivariate graphs illustrating the resolution of the various erythrocyte populations have been previously published (44). The number of RETs was measured in 2×105 erythrocytes. The number of MN-RETs was determined based on a total of 2×104 total RETs per sample. MN-RET formation kinetics An experiment was first performed to evaluate if the kinetics of MN-RET formation induced by a single injection of 18F-FDG corresponded to previously published values induced by gamma radiation (44). Mice were injected with saline (n = 1–3 induplicate per time point) or 18.5 MBq 18F-FDG (n = 3 in duplicate per time point). Blood samples were collected at 30-, 43-, 47-, 51- and 72-h post-injection. MN-RET dose response For the activity–response experiments, mice were injected with a range of 18F-FDG doses (0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq) and the MN-RET frequency was evaluated at 43-h post-injection in a blood sample obtained from each mouse in duplicate. For all samples, the percentage of MN-RETs within a population of 20000 RETs was determined. Relative biological effectiveness In order to compare the effectiveness of the radiation exposure from PET scans employing 18F-FDG to that of a reference dose of radiation (662 keV γ-rays), the bone marrow dose was determined for the injection activities listed in Table I using conversion values determined by Taschereau and Chatziioannou (43). The gamma-irradiated mice received acute doses of 0 (n = 3), 10 (n = 5), 20 (n = 5), 50 (n = 5) or 100 mGy (n = 5) at a dose rate of 0.018 Gy/min. Gamma doses were delivered using a Cs-137 source (662 keV γ-rays) at the McMaster Taylor Radiobiology Source Facility and mice were placed in a customised sectioned polycarbonate restraint tube for the γ-ray irradiation. These doses were corrected for attenuation through the mouse to the site of bone marrow using previously determined experimental values from our laboratory. Adaptive response For the in vivo adaptive response experiments, mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 9–11, duplicate samples) and irradiated with 1 Gy whole body (662 keV γ-rays, 0.35 Gy/min) 24-h post-injection. The MN-RET frequency was evaluated 43h after the 1 Gy dose. For the in vitro adaptive response experiments, mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 6–10, duplicate samples). The bone marrow was extracted at 24-h post-injection and irradiated with 0, 1, 2 or 4 Gy in vitro (662 keV γ-rays, 0.19 Gy/min, 0°C). Bone marrow cultures (1×106 cells/ml) were incubated at 37°C for 30min following irradiation, then fixed with 70% EtOH. For analysis, the fixed bone marrow samples were centrifuged at 5°C (250 × g, 8min) and the supernatant was discarded. Cells were then washed in 3ml of Tris-buffered saline (1× TBS; Trizma base + NaCl; Sigma Aldrich), centrifuged (250 × g, 8min), resuspended in 1ml of Tris–saline–triton [TST; TBS + 4% fetal bovine serum + 0.1% Triton X-100 (Sigma Aldrich)] and incubated on ice for 10min to permeabilise cells. The cells were again centrifuged (250 × g, 8min), the supernatant discarded and cells were resuspended in 200 µl of a 1:400 dilution of anti-phospho-H2A.X (ser139) antibody (Upstate Cell Signaling, Charlottesville, VA, USA). Samples were incubated at room temperature for 2h in the dark. The cells were then washed with 3ml of TST, resuspended in 200 µl of a 1:500 dilution of Alexa Fluor™ 488-conjugated goat anti-rabbit IgG F(ab′)2 antibody (Invitrogen Canada, Burlington, Ontario, Canada) and incubated at room temperature for 1h in the dark. The cells were then washed once in TBS and resuspended in 300 µl TBS + 5 µl propidium iodide (1mg/ml; Sigma Aldrich). Samples were placed on ice and promptly analysed by flow cytometry. Mean γH2A.X fluorescence was measured in 5000 bone marrow cells gated on the lymphocyte-rich population based on forward and side-scatter characteristics. Only G0/G1 cells were analyzed for mean γH2A.X fluorescence, due to confounding factors associated with γH2A.X in S and G2/M cells (45). Statistics Statistical analysis of the biological data was performed using Sigma Plot version 11.0 (Systat Software, Germany). Data points represent the mean ± SEM. Two sided P values ≤0.05 were considered statistically significant. One-way analysis of variance (ANOVA) was performed on ranks (Kruskal–Wallis). Two-way ANOVA with Bonferroni was also used. T-tests, when appropriate, were performed to examine differences between two groups in particular. Dose–response curves were analyzed using linear regression analysis and multiple linear regression analysis was used for comparisons. Results Validation of isotope distribution The pattern of 18F-FDG uptake is consistent with the predicted distribution of absorbed doses from (42) listed in Table I. Two separate approaches for whole body dose calculation were used to assess consistency in methodology. Although the estimation methods were divergent, either using phantoms and PET images (43) or calculations based on theoretical ellipsoids (42), the results of both approaches were reasonably consistent. Due to the tissue-specific calculations, dose estimates based on Taschereau and Chatziioannou (43) were used throughout this study. A representative image is provided in Figure 1. The image shows that as expected, the isotope preferentially accumulated in more metabolically active tissues, consequently increasing the absorbed dose to that organ relative to the whole body average. Areas of high uptake were observed in the brain, heart, brown fat, kidneys and bladder, which is consistent with the established pattern of uptake observed in 18F-FDG PET scans. Kinetics of MN-RET formation following an 18F-FDG injection Emphasis was placed on time points after 43h, the published MN-RET peak following acute gamma irradiation, as it was suspected that the protracted exposure associated with the isotope decay may lead to a persistent elevation in MN-RET levels. The injection caused a significant increase in MN-RETS relative to controls (P < 0.001; Figure 2). The level of MN-RETS in the saline injected mice (controls) did not change significantly with time. Following the injection, the maximum level of MN-RETs occurred at 43h and subsequently decreased to control levels by 72-h post-injection. These data are consistent with published MN-RET kinetics by acute gamma radiation exposure. The MN-RET level at 43h was not significantly different from that at 47h (P = 1.000), but decreased significantly at 49h (P < 0.001; Figure 3). At 72h, the average level of MN-RETS in the 18F-FDG injected mice (0.19±0.01) decreased below control levels (0.22±0.05) although this decrease was not significant (P = 0.600). The 43-h time point was subsequently used to further characterise the in vivo response to 18F-FDG in mice. Fig. 2. Open in new tabDownload slide MN-RET kinetics in vivo formation of MN-RETs following injection with 18.5 MBq 18F-FDG (n = 3, duplicate samples) or saline (n = 1–3, duplicate samples). Blood samples were collected at 30-, 43-, 47-, 51- and 72-h post-injection. Results are depicted as mean ± SEM. Fig. 2. Open in new tabDownload slide MN-RET kinetics in vivo formation of MN-RETs following injection with 18.5 MBq 18F-FDG (n = 3, duplicate samples) or saline (n = 1–3, duplicate samples). Blood samples were collected at 30-, 43-, 47-, 51- and 72-h post-injection. Results are depicted as mean ± SEM. Fig. 3. Open in new tabDownload slide Activity–response curve in vivo formation of MN-RETs following injection with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples). Each point (x, y) represents one mouse (duplicates averaged) with corresponding injected activity. Fig. 3. Open in new tabDownload slide Activity–response curve in vivo formation of MN-RETs following injection with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples). Each point (x, y) represents one mouse (duplicates averaged) with corresponding injected activity. 18F-FDG dose response (MN-RET) The induction of micronuclei in bone marrow by 18F-FDG was found to vary non-linearly with injected activity, corresponding with a second-order polynomial (R2 = 0.9997; Figure 3). All injection activities caused an increase in micronuclei relative to controls although this increase was only significant following injections of 3.70, 7.40 and 14.80 MBq (P < 0.001). The injection activities corresponded to bone marrow doses of 35.89±0.62, 74.30±1.59 and 150.32±4.12 mGy, respectively (Table I). Relative biological effectiveness of 18F-FDG measured (MN-RET) Similar to 18F-FDG, the gamma dose–response curve was also found to be non-linear (R2 = 0.9945), however the gamma curve differed significantly from the 18F-FDG (Figure 4). Multiple linear regression analysis determined that the type of radiation exposure (18F-FDG or 662 keV γ-rays) was a significant determinant of the MN-RET dose response (P < 0.004) with 662 keV γ-rays generating significantly more damage per unit dose. The relative biological effectiveness (RBE) for 18F-FDG was calculated by taking the ratio of the linear regression coefficients from the two dose–response curves (18F-FDG: 662 keV γ-rays). Using this approach, the RBE for 18F-FDG was determined to be 0.79±0.04. Fig. 4. Open in new tabDownload slide RBE comparison of the MN-RET frequency per unit dose between 18F-FDG (internal) and 662 keV γ-rays (external). For the 18F-FDG curve, mice were injected with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples) and converted to bone marrow doses. For the 662 keV γ-rays, mice were irradiated whole body with 0, 10, 20, 50 or 100 mGy (n = 3–5, duplicate samples) at a dose rate of 18 mGy/min which were also converted to bone marrow doses using an attenuation factor. Radiation-induced values are displayed; MN-RET levels were normalised from background MN-RET values. Results displayed are mean ± SEM. Fig. 4. Open in new tabDownload slide RBE comparison of the MN-RET frequency per unit dose between 18F-FDG (internal) and 662 keV γ-rays (external). For the 18F-FDG curve, mice were injected with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples) and converted to bone marrow doses. For the 662 keV γ-rays, mice were irradiated whole body with 0, 10, 20, 50 or 100 mGy (n = 3–5, duplicate samples) at a dose rate of 18 mGy/min which were also converted to bone marrow doses using an attenuation factor. Radiation-induced values are displayed; MN-RET levels were normalised from background MN-RET values. Results displayed are mean ± SEM. In vivo induction of an adaptive response by 18F-FDG (MN-RET) Four independent experiments were performed and pooled to examine whether injections of 0 (0), 0.74 (20), 3.70 (100) or 14.80 (400) MBq (µCi) could modify the effect of a 1 Gy (662 keV γ-rays) in vivo challenge dose given 24-h post-injection. The PET injection did not significantly alter the damage induced by the 1 Gy challenge dose in vivo as measured by the MN-RET assay (P = 0.959; Figure 5). Fig. 5. Open in new tabDownload slide In vivo adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 9–11, duplicate samples) and irradiated 24-h post-injection with 1 Gy whole body (662 keV γ-rays, 0.35 Gy/min). The MN-RET frequency was evaluated 43h after the 1 Gy dose. Results displayed are mean ± SEM. Fig. 5. Open in new tabDownload slide In vivo adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 9–11, duplicate samples) and irradiated 24-h post-injection with 1 Gy whole body (662 keV γ-rays, 0.35 Gy/min). The MN-RET frequency was evaluated 43h after the 1 Gy dose. Results displayed are mean ± SEM. In vitro induction of an adaptive response by 18F-FDG injections (γH2A.X) The lower limit of detection of the flow cytometry-based γH2A.X assay is 100 mGy for acute radiation exposures (46); consequently, this assay would consequently not be useful in measuring the direct effects of the standard dose range of 18F-FDG injections. It was used instead to measure the potential modification of an acute radiation exposure by 18F-FDG by the induction of an adaptive response. Mice were injected with 0 (0), 0.74 (20), 3.70 (100) or 14.80 (400) MBq (µCi). At 24-h post-injection, bone marrow from these mice were extracted and irradiated in culture with 0, 1, 2 or 4 Gy (662 keV γ-rays). The mean γH2A.X fluorescence in the lymphocyte-rich population was measured 30min after the challenge irradiation as this has been shown previously to be the optimal time for measurement (45). The level of γH2A.X increased significantly with the magnitude of the challenge dose (P < 0.001; Figure 6A). None of the injection activities (0.74, 3.70 or 14.80 MBq) significantly modified the effect of any of the challenge doses (1, 2, 4 Gy), relative to the saline injected controls (P = 0.322; Figure 6A). It was observed in the non-challenged mice (0 Gy) that γH2A.X levels decreased with increasing injection activity (P = 0.019; Figure 6B) at 24h. The 14.80 MBq injection of 18F-FDG, in particular, caused a significant decrease in γH2A.X below controls (P < 0.014). Fig. 6. Open in new tabDownload slide In vitro adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 6–10, duplicate samples) and irradiated at 24-h post-injection with 0, 1, 2 or 4 Gy in vitro. Mean γH2A.X fluorescence was measured 30min after the challenge dose (A). Sham-irradiated samples only. (B) Results displayed are mean ± SEM. Fig. 6. Open in new tabDownload slide In vitro adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 6–10, duplicate samples) and irradiated at 24-h post-injection with 0, 1, 2 or 4 Gy in vitro. Mean γH2A.X fluorescence was measured 30min after the challenge dose (A). Sham-irradiated samples only. (B) Results displayed are mean ± SEM. Discussion The short-term DNA damage response to 18F-FDG PET scans in mice was evaluated using two well-established radiation biology assays: MN-RET formation and γH2A.X fluorescence. Micronuclei occur in newly formed RETs in the blood stream of mice following DNA damage to precursors in bone marrow. This process generally takes between 43 and 55h and varies with mouse strain (44). The MN-RET technique has been adopted by genetic toxicology and product safety research programs (47, 48) because of its exceptional sensitivity for measuring the mutagenic potential of low level exposures. It has been shown to be a reliable biodosimeter for acute radiation exposures and various clastogenic/aneugenic chemicals (44, 49, 50), however, to our knowledge, this assay has never been used to measure the DNA damage associated with radioisotopes. Previous studies demonstrated that a 1 Gy acute γ-radiation exposure induces a maximum MN-RET frequency at 43h and returns to spontaneous levels by 137h (44). The formation of γH2A.X foci occurs at the site of new DSBs within minutes. The phosphorylation of histone H2A.X to form γH2A.X is a key component in sensing DNA damage and recruiting repair proteins. It has been shown that the majority of radiation-induced foci disappear by 8-h post-irradiation to doses below 1 Gy (51), and disappeared in bone marrow cells by 24h following 4 Gy (52). Residual foci have been associated with on-going genetic damage typical of genomic instability (53, 54), so measuring γH2A.X foci 24-h post-irradiation consequently provides information about residual cytogenetic damage. We examined the biological response to a range of injection activities of 18F-FDG between 0.74 (20) and 14.80 (400) MBq (µCi), the lowest activity used (0.74 MBq) produces absorbed doses in a mouse similar to the average patient absorbed dose during clinical PET scans. The highest dose (14.80 MBq) represents a typical injection activity used in small animal imaging investigations. A proportionally larger amount of activity is needed to generate diagnostically useful images in mice, related to the higher spatial resolution requirements in small animal imaging (55). The 43-h peak of MN-RET expression was reduced in magnitude compared to that seen with acute gamma exposures, but the following pattern of decay was similar. A prolonged elevation in MN-RETS compared to the acute gamma exposure was expected based on the characteristics of isotope decay but was not observed. This suggests the DSB repair mechanisms induced by the low dose rates associated with 18F-FDG were effective despite the protracted exposure. By 72h, the level of MN-RETS in 18F-FDG-injected mice had decreased to ~14% below control levels. This decrease was not significant but suggests that higher doses of 18F-FDG may be required to fully induce adaptive mechanisms. We demonstrated that a single 10 mGy acute whole body exposure significantly increased MN-RET frequency above controls. In contrast, a 3.70 MBq 18F-FDG injection, corresponding to a whole body dose of 50–57 mGy and a bone marrow dose of 36 mGy, was required to significantly elevate MN-RET frequency. This highlights the increased resistance to DNA damage in vivo when low dose rates protract the radiation exposure. As the dose rate decreases, the temporal abundance of damage in the cell is decreased and the ability to accurately repair DNA damage increases (56, 57). The RBE for 18F-FDG, a combined exposure from 634 keV positrons and 511 keV γ-rays, was calculated to be 0.79±0.04 in mice for the MN-RET endpoint. RBE is used to derive radiation weighting factors for use in radiation protection, with a factor of 1 used for all low linear energy transfer radiations (58). We have shown that the low dose rate and radiation quality associated with 18F-FDG reduces its effectiveness in generating micronuclei in bone marrow. RBE has been shown to vary substantially based on the radiation quality, biological endpoint, organism and dose rate. It has been shown previously that single low dose or dose-rate exposures have the capacity to modify the subsequent response of a biological system to a large dose of radiation (26, 28–31, 59). We investigated the potential for 18F-FDG to generate an adaptive response at 24-h post-injection using the MN-RET and γH2A.X endpoints. The 24-h interval was chosen to allow for complete decay of the isotope in order to capture the full extent of biological changes induced by the injection. A single injection of 0.74, 3.70 or 14.80 MBq 18F-FDG did not modify the magnitude of the response to a 1 Gy acute dose of radiation in vivo as measured with the MN-RET endpoint. Injections of 0.74, 3.70 or 14.80 MBq correspond to approximate whole body doses of 10, 50 and 200 mGy and approximate bone marrow doses of 7, 35 and 150 mGy (Table I). Similarly, 0.74, 3.70 or 14.80 MBq 18F-FDG injections did not modify the response of bone marrow lymphocytes to 1, 2 or 4 Gy in vitro as measured by γH2A.X foci formation. While the 18F-FDG doses in this study did not induce an adaptive response, there are factors characteristic of this radioisotope that require modifications to the classic adaptive response experimental design. The low dose rate associated with 18F decay appears to require significantly greater doses than those used in this study to induce protective mechanisms. This is supported by the reduction in baseline MN-RET levels following injection of the largest doses of 18F-FDG. Additionally, the dose-dependent decrease in γH2A.X following injections of 18F-FDG at 24h in the sham-irradiated mice (0 Gy), and more specifically, a significant 20% reduction in γH2A.X fluorescence following the 14.80 MBq injection indicates the induction protective mechanisms in this study. The heterogeneity of 18F-FDG uptake into tissues provides another confounding factor for adaptation responses. Bone marrow may not have sufficient uptake to obtain the dose necessary for induction of protective mechanisms, however tissues with higher absorbed doses may in fact undergo adaptation. Further studies in tissues including the heart, brain and kidneys of these mice are required to clarify this issue. Timing between the adapting and challenge doses is also crucial, however determining optimal timing with radioisotope decay can be problematic given that the timing is dependent on individual tissue characteristics (30, 60). We have studied the DNA damage response to radiation exposure from 18F-FDG and found a dose-dependent increase in radiation-induced damage. This initial damage response, as measured by MN-RET formation, offers insight into the biophysical properties of the energy deposition in bone marrow associated with 18F-FDG. By studying later time points, we were able to understand the biological impact of this initial response. For example, the highest injection activities used (14.80–18.5 MBq) did not lead to residual DNA damage, but in fact, decreased the level of damage below controls. This illustrates the importance of examining later time points in these types of investigations as subsequent biological responses may alter dose–response dynamics. The LNT model of risk would predict that the total damage from this combination of treatments would be equal to the damage due to the isotope injection plus that induced by the high-dose radiation exposure. Consistent with the other observations in this study, the damage induced by the radiation exposure from the 0.74, 3.70 or 14.80 MBq 18F-FDG injections was effectively repaired and did not add to damage induced by the high-dose challenge. The LNT model represents a practical approach to radiation protection and extrapolates epidemiological high dose-effects into the low dose range using a linear relationship. In doing so, certain assumptions are made: that dose is a surrogate for risk, that there is no threshold for radiation effects and that risk is additive and can only increase. Our results indicate that in the context of 18F-FDG exposure from PET scans, this does not hold true. We have shown that there is threshold for DNA damage to bone marrow following injections of 18F-FDG and the dose–response curve for the radioisotope is non-linear. We have also shown that the added radiation exposure (low dose isotope + high-dose challenge) does not result in additive damage. We have examined the radiobiological changes induced by 18F-FDG to improve the current understanding of the health effects associated with radiation exposure from PET scans. We tested injection activities relevant to both human imaging and small animal studies, demonstrating that the low dose rate of 18F-FDG exposure substantially reduces the DNA damage generated for a given dose. Later sampling times revealed that the larger injection activities may actually reduce the level of DNA damage below that of controls. Together, these results provide evidence against the use of LNT to predict the risk to patients from nuclear medicine procedures such as PET. Funding US Department of Energy Low Dose Radiation Program (DE-FG02-07ER64343) and the National Sciences and Engineering Council of Canada (238495). Acknowledgements We sincerely thank Mary Ellen Cybulski, Lisa Laframboise and Nicole McFarlane for their important technical contributions to this study. 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Radiation-induced DNA damage and the relative biological effectiveness of 18F-FDG in wild-type mice

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Oxford University Press
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© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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Abstract

Abstract Clinically, the most commonly used positron emission tomography (PET) radiotracer is the glucose analog 2-[18F] fluoro-2-deoxy-d-glucose (18F-FDG), however little research has been conducted on the biological effects of 18F-FDG injections. The induction and repair of DNA damage and the relative biological effectiveness (RBE) of radiation from 18F-FDG relative to 662 keV γ-rays were investigated. The study also assessed whether low-dose radiation exposure from 18F-FDG was capable of inducing an adaptive response. DNA damage to the bone marrow erythroblast population was measured using micronucleus formation and lymphocyte γH2A.X levels. To test the RBE of 18F-FDG, mice were injected with a range of activities of 18F-FDG (0–14.80 MBq) or irradiated with Cs-137 γ-rays (0–100 mGy). The adaptive response was investigated 24h after the 18F-FDG injection by 1 Gy in vivo challenge doses for micronucleated reticulocyte (MN-RET) formation or 1, 2 and 4 Gy in vitro challenges doses for γH2A.X formation. A significant increase in MN-RET formation above controls occurred following injection activities of 3.70, 7.40 or 14.80 MBq (P < 0.001) which correspond to bone marrow doses of ~35, 75 and 150 mGy, respectively. Per unit dose, the Cs-137 radiation exposure induced significantly more damage than the 18F-FDG injections (RBE = 0.79±0.04). A 20% reduction in γH2A.X fluorescence was observed in mice injected with a prior adapting low dose of 14.80 MBq 18F-FDG relative to controls (P < 0.019). A 0.74 MBq 18F-FDG injection, which gives mice a dose approximately equal to a typical human PET scan, did not cause a significant increase in DNA damage nor did it generate an adaptive response. Typical 18F-FDG injection activities used in small animal imaging (14.80 MBq) resulted in a decrease in DNA damage, as measured by γH2A.X formation, below spontaneous levels observed in control mice. The 18F-FDG RBE was <1.0, indicating that the mixed radiation quality and/or low dose rate from PET scans is less damaging than equivalent doses of gamma radiation. Introduction The use of diagnostic imaging in health care has seen a dramatic increase over the last two decades. In the USA, nuclear medicine procedures have increased >7% per annum, with positron emission tomography (PET) showing the greatest increases at 57% per year with expected increases due to its unprecedented sensitivity for the detection of biological processes (1, 2). Clinically, the most commonly used PET radiotracer is the glucose analog 2-[18F] fluoro-2-deoxy-d-glucose (18F-FDG). PET scans with 18F-FDG are used to image disease states characterised by alterations in metabolism: epilepsy (3), Alzheimer’s disease (4), infection (5), heart disease (6, 7) but most often, malignancy (8, 9). 18F-FDG is transported into the cytoplasm of metabolically active cells by glucose transport membrane proteins (GLUT) and undergoes phosphorylation to form 18F-FDG-6-phosphate by hexokinase (10). At this point, 18F-FDG becomes trapped in the cell because of the substitution at the hydroxyl group (11). The absorbed radiation dose in tissue depends on the glucose requirements of that tissue. The radiation exposure is a result of positrons (β+, Emax = 634 keV) emitted by 18F-FDG and subsequent annihilation photons (γ-rays, 511 keV). During a typical clinical protocol involving the administration of 350–750 MBq 18F-FDG (12), most tissues will be irradiated throughout the patient’s body (4–9 mGy). However, in organs with high energy requirements, doses can be much higher, i.e. the brain (10–36 mGy) and heart (16–51 mGy) or organs within the excretory system including kidneys (7–23 mGy) and bladder (13–233 mGy) through which the radiopharmaceutical is voided (13–17). Dose is delivered at a low decaying dose rate reflective of the physical (109.7min) and biological half-life of 18F-FDG. The biological half-life depends on the residence times of the radiopharmaceutical within different tissues. The MIRD dose estimate report for 18F-FDG provides a whole body residence time of 2.38±0.12h (15). It is important to note that regardless of the site being imaged, an injection of the radiopharmaceutical 18F-FDG will result in systemic uptake and radiation exposure (18). It has been reported that, compared to all other nuclear medicine procedures, PET scans with 18F-FDG deliver one of the highest effective doses to patients (14.1 mSv) (18). The effective dose is a value which allows the summation of dose contributions from radionuclides non-uniformly distributed throughout the body to evaluate the associated stochastic effects (cancer & hereditary) relative to other radiation exposures. It requires a calculation using radiation weighting factors (wR) and tissue weighting factors (wT) published by the International Commission on Radiological Protection (ICRP) (19). The increase in nuclear medicine procedures has stimulated interest in whether a rise in radiation-induced cancers should be expected. Certain studies, utilising the linear no threshold (LNT) model of risk estimation, assert that PET scans will result in a measurable increase in the lifetime risk of cancer among patients (20). These types of studies use dose as a surrogate for risk. While accurate dosimetry in nuclear medicine is important, it should only be used as a starting point in evaluating the possible health effects associated with nuclear medicine procedures. Nuclear medicine examinations involve protracted dose rates which have been shown to significantly reduce the biological risk associated with a given dose of ionising radiation (21–24). In fact, many studies have shown that low dose-rate exposures may be protective to an organism by generating an adaptive response. A number of experiments using cell lines have shown that low dose-rate radiation exposures reduced the level of transformation or damage below that of controls (25–27). In addition, both low dose (28, 29) and lose dose-rate (26, 30, 31) exposures have been shown to reduce the full detriment of a subsequent high-dose radiation exposure. In contrast, there are certain elements of nuclear medicine procedures that may augment the associated risk. Certain groups have identified that decaying dose rates generate more damage than fixed low dose rates (32) or that damage delivered at a very low dose rate (9.4 cGy/h) evades DNA damage surveillance mechanisms (33). Current investigations into medical isotope health effects have been focused almost exclusively on radioiodine (I-131, I-125) (34–38). There are few investigations that address the possible health effects of radiation exposure from newer imaging agents (39) and none that are focused on PET imaging. Moreover, many radiobiological investigations into the characteristics associated with nuclear medicine procedures (low dose rates, changing dose rates) have been investigated in cell lines. In this study, the response to 18F-FDG was investigated in mice to gain a better understanding of the in vivo radiation-induced health effects associated with PET scans. Inbred mice were chosen as the model system to eliminate the high degree of interindividual variability observed in patient studies (35, 40). DNA double-strand breaks (DSBs) following 18F-FDG injections were measured by γH2A.X fluorescence at 24-h post-injection and micronucleated reticulocyte (MN-RET) formation at 43-h post-injection. Additionally, the potential of this injection to induce an adaptive response was evaluated using these same endpoints by irradiating mice or cells at 24-h post-injection with high doses of radiation (1, 2 and 4 Gy). We hypothesise that the low dose rate associated with PET scans employing 18F-FDG reduces the detrimental effects of the radiation exposure and that this exposure may have the capacity to generate an adaptive response. Materials and methods Mice In these experiments, 7- to 9-week-old wild-type female mice (B6.129S2-Trp53tm1Tyj/1x 129X1/SvJ, Jackson Laboratory; Bar Harbor, ME, USA) were used. Mice were housed five per cage in specific pathogen-free conditions. The housing room was maintained on a 12-h light/dark cycle at a temperature of 24±1°C. Protocols were approved by the Animal Research Ethics Board at McMaster University and carried out as per the Canadian Council on Animal Care. 18F-FDG production and injection Mice were injected with 18F-FDG prepared at Hamilton Health Sciences (Hamilton, Ontario, Canada). The 18F was produced by 18O(p,n)18F reaction using a Siemens RDS112 11MeV Proton-Cyclotron and the FDG was prepared as per the procedure listed in (41), meeting all USP and Health Canada regulatory requirements. All mice were fasted overnight prior to the isotope injection (12–14h) and water was provided ad libitum. Mice were housed individually for the duration of the experiment to minimise inter-mouse irradiation following the isotope injection. Individual isotope doses were measured using a CRC-12 radioisotope calibrator (Capintec). Pre- and post-injection activity of the syringe in addition to the time of each measurement was recorded. This information was used to accurately calculate the activity administered to each mouse by correcting for decay. The weight of each mouse was also recorded prior to the injection. The average weight of mice used in these experiments was 20±0.08g. The 18F-FDG was diluted into a total volume of 200 µl with saline and administered by tail vein injection. Control mice were injected with 200 µl saline only. Anaesthesia was not used since image acquisition was not the objective of this work and may interfere with the biological processes under investigation. Food was returned to the mice 2h after the injection to maximise uptake of the 18F-FDG and to simulate the conditions of a typical PET procedure. Calculation of absorbed doses from 18F-FDG Previously published absorbed dose estimates for 18F-FDG in mice (42, 43) were used to convert the injected activities used into whole body doses and bone marrow doses (Table I). The whole body dose estimates using both approaches are in good agreement. Small samples of mice were imaged at 7–8 weeks of age to verify the distribution of 18F-FDG in the mouse model (Figure 1). Table I. Injection activities and corresponding doses for 20g mice injected with 18F-FDG n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 Activities are expressed in MBq (µCi). Absorbed dose estimates are expressed in mGy. Data are expressed mean ± SEM. n = number of mice per group. aObtained using absorbed dose values (14 mGy/MBq) from Taschereau et al. (43). bObtained using an approximate dose calculation and S value (22.1×10−13 Gy/Bq/s) from Funk et al. (42). cObtained using the absorbed dose in marrow (10 mGy/MBq) from Taschereau et al. (43). Open in new tab Table I. Injection activities and corresponding doses for 20g mice injected with 18F-FDG n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 n . Nominal activity . Injected activity . Whole body dosea . Whole body doseb . Bone marrow dosec . 10 0.00 (0.0) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 10 0.74 (20.0) 0.66±0.03 9.21±0.41 10.36±0.47 6.58±0.30 10 1.48 (40.0) 1.42±0.04 19.88±0.56 22.37±0.64 14.20±0.40 9 3.70 (100.0) 3.59±0.06 50.25±0.86 56.55±0.97 35.89±0.62 10 7.40 (200.0) 7.43±0.15 104.02±2.33 117.05±2.40 74.30±1.59 8 14.80 (400.0) 15.03±0.41 210.44±5.77 236.80±6.49 150.32±4.12 Activities are expressed in MBq (µCi). Absorbed dose estimates are expressed in mGy. Data are expressed mean ± SEM. n = number of mice per group. aObtained using absorbed dose values (14 mGy/MBq) from Taschereau et al. (43). bObtained using an approximate dose calculation and S value (22.1×10−13 Gy/Bq/s) from Funk et al. (42). cObtained using the absorbed dose in marrow (10 mGy/MBq) from Taschereau et al. (43). Open in new tab Fig. 1. Open in new tabDownload slide Representative PET scan of an 8-week-old female healthy wild-type mouse from this study acquired with a Philips MOSIAC animal PET scanner at the McMaster Center for Pre-Clinical and Translational Imaging. Mouse (fasted) was injected with 18.5 MBq 18F-FDG. Static scan (15min in duration) was acquired 45min post-injection.(A) Coronal view: increased 18F-FDG uptake in the bladder (4), heart (3), brain (1) and active skeletal muscle/brown fat (2) is evident. (B) Sagittal view: reveals 18F-FDG uptake in kidneys (5). The pattern of uptake is consistent with the predicted distribution of absorbed doses. Fig. 1. Open in new tabDownload slide Representative PET scan of an 8-week-old female healthy wild-type mouse from this study acquired with a Philips MOSIAC animal PET scanner at the McMaster Center for Pre-Clinical and Translational Imaging. Mouse (fasted) was injected with 18.5 MBq 18F-FDG. Static scan (15min in duration) was acquired 45min post-injection.(A) Coronal view: increased 18F-FDG uptake in the bladder (4), heart (3), brain (1) and active skeletal muscle/brown fat (2) is evident. (B) Sagittal view: reveals 18F-FDG uptake in kidneys (5). The pattern of uptake is consistent with the predicted distribution of absorbed doses. Biological experiments Two flow cytometry-based assays were used to evaluate the biological effects of 18F-FDG injections in mice: MN-RET formation and γH2A.X mean fluorescence. All samples were kept at 0°C during preparation and analyzed using an EPICS XL (Beckman Coulter, Miami, FL, USA) flow cytometer. MN-RET assay All reagents used for the MN-RET assay were included the Mouse MicroFlowPLUS® kit (Litron Laboratories, Rochester, NY, USA). Cells were fixed in absolute methanol (Sigma Aldrich, Mississauga, Ontario, Canada) at −80°C and stored at −80°C for a minimum of 24h before staining and flow cytometric analysis. The fixed blood samples were washed and labelled for flow cytometric analysis according to the Mouse MicroFlowPLUS Kit manufacture’s procedure and previously described by Dertinger et al. (44). Briefly, fixed blood cells were washed with 12ml buffer solution and cell pellets were maintained at 0°C until staining. Following the wash, 80 µl of reagent mixture containing anti-CD71-FITC, anti-CD61-PE, RNase and buffer solution was added to 20 µl aliquot of each fixed blood sample in duplicate. The cells were incubated on ice for 30min followed by 30min at room temperature, and then returned to ice. Immediately prior to analysis by flow cytometry, 1ml of 4°C propidium iodide (1.25 µg/ml in buffer solution) was added to each tube. Data acquisition was performed using the EPICS XL flow cytometer (Beckman Coulter, Brea, CA, USA) equipped with a 488nm argon laser. The gating logic used to quantitatively analyse the erythrocyte subpopulations has been described previously (44). Analysis windows were set to quantify the number of reticulocytes (RETs) and MN-RETs for each sample. Representative bivariate graphs illustrating the resolution of the various erythrocyte populations have been previously published (44). The number of RETs was measured in 2×105 erythrocytes. The number of MN-RETs was determined based on a total of 2×104 total RETs per sample. MN-RET formation kinetics An experiment was first performed to evaluate if the kinetics of MN-RET formation induced by a single injection of 18F-FDG corresponded to previously published values induced by gamma radiation (44). Mice were injected with saline (n = 1–3 induplicate per time point) or 18.5 MBq 18F-FDG (n = 3 in duplicate per time point). Blood samples were collected at 30-, 43-, 47-, 51- and 72-h post-injection. MN-RET dose response For the activity–response experiments, mice were injected with a range of 18F-FDG doses (0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq) and the MN-RET frequency was evaluated at 43-h post-injection in a blood sample obtained from each mouse in duplicate. For all samples, the percentage of MN-RETs within a population of 20000 RETs was determined. Relative biological effectiveness In order to compare the effectiveness of the radiation exposure from PET scans employing 18F-FDG to that of a reference dose of radiation (662 keV γ-rays), the bone marrow dose was determined for the injection activities listed in Table I using conversion values determined by Taschereau and Chatziioannou (43). The gamma-irradiated mice received acute doses of 0 (n = 3), 10 (n = 5), 20 (n = 5), 50 (n = 5) or 100 mGy (n = 5) at a dose rate of 0.018 Gy/min. Gamma doses were delivered using a Cs-137 source (662 keV γ-rays) at the McMaster Taylor Radiobiology Source Facility and mice were placed in a customised sectioned polycarbonate restraint tube for the γ-ray irradiation. These doses were corrected for attenuation through the mouse to the site of bone marrow using previously determined experimental values from our laboratory. Adaptive response For the in vivo adaptive response experiments, mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 9–11, duplicate samples) and irradiated with 1 Gy whole body (662 keV γ-rays, 0.35 Gy/min) 24-h post-injection. The MN-RET frequency was evaluated 43h after the 1 Gy dose. For the in vitro adaptive response experiments, mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 6–10, duplicate samples). The bone marrow was extracted at 24-h post-injection and irradiated with 0, 1, 2 or 4 Gy in vitro (662 keV γ-rays, 0.19 Gy/min, 0°C). Bone marrow cultures (1×106 cells/ml) were incubated at 37°C for 30min following irradiation, then fixed with 70% EtOH. For analysis, the fixed bone marrow samples were centrifuged at 5°C (250 × g, 8min) and the supernatant was discarded. Cells were then washed in 3ml of Tris-buffered saline (1× TBS; Trizma base + NaCl; Sigma Aldrich), centrifuged (250 × g, 8min), resuspended in 1ml of Tris–saline–triton [TST; TBS + 4% fetal bovine serum + 0.1% Triton X-100 (Sigma Aldrich)] and incubated on ice for 10min to permeabilise cells. The cells were again centrifuged (250 × g, 8min), the supernatant discarded and cells were resuspended in 200 µl of a 1:400 dilution of anti-phospho-H2A.X (ser139) antibody (Upstate Cell Signaling, Charlottesville, VA, USA). Samples were incubated at room temperature for 2h in the dark. The cells were then washed with 3ml of TST, resuspended in 200 µl of a 1:500 dilution of Alexa Fluor™ 488-conjugated goat anti-rabbit IgG F(ab′)2 antibody (Invitrogen Canada, Burlington, Ontario, Canada) and incubated at room temperature for 1h in the dark. The cells were then washed once in TBS and resuspended in 300 µl TBS + 5 µl propidium iodide (1mg/ml; Sigma Aldrich). Samples were placed on ice and promptly analysed by flow cytometry. Mean γH2A.X fluorescence was measured in 5000 bone marrow cells gated on the lymphocyte-rich population based on forward and side-scatter characteristics. Only G0/G1 cells were analyzed for mean γH2A.X fluorescence, due to confounding factors associated with γH2A.X in S and G2/M cells (45). Statistics Statistical analysis of the biological data was performed using Sigma Plot version 11.0 (Systat Software, Germany). Data points represent the mean ± SEM. Two sided P values ≤0.05 were considered statistically significant. One-way analysis of variance (ANOVA) was performed on ranks (Kruskal–Wallis). Two-way ANOVA with Bonferroni was also used. T-tests, when appropriate, were performed to examine differences between two groups in particular. Dose–response curves were analyzed using linear regression analysis and multiple linear regression analysis was used for comparisons. Results Validation of isotope distribution The pattern of 18F-FDG uptake is consistent with the predicted distribution of absorbed doses from (42) listed in Table I. Two separate approaches for whole body dose calculation were used to assess consistency in methodology. Although the estimation methods were divergent, either using phantoms and PET images (43) or calculations based on theoretical ellipsoids (42), the results of both approaches were reasonably consistent. Due to the tissue-specific calculations, dose estimates based on Taschereau and Chatziioannou (43) were used throughout this study. A representative image is provided in Figure 1. The image shows that as expected, the isotope preferentially accumulated in more metabolically active tissues, consequently increasing the absorbed dose to that organ relative to the whole body average. Areas of high uptake were observed in the brain, heart, brown fat, kidneys and bladder, which is consistent with the established pattern of uptake observed in 18F-FDG PET scans. Kinetics of MN-RET formation following an 18F-FDG injection Emphasis was placed on time points after 43h, the published MN-RET peak following acute gamma irradiation, as it was suspected that the protracted exposure associated with the isotope decay may lead to a persistent elevation in MN-RET levels. The injection caused a significant increase in MN-RETS relative to controls (P < 0.001; Figure 2). The level of MN-RETS in the saline injected mice (controls) did not change significantly with time. Following the injection, the maximum level of MN-RETs occurred at 43h and subsequently decreased to control levels by 72-h post-injection. These data are consistent with published MN-RET kinetics by acute gamma radiation exposure. The MN-RET level at 43h was not significantly different from that at 47h (P = 1.000), but decreased significantly at 49h (P < 0.001; Figure 3). At 72h, the average level of MN-RETS in the 18F-FDG injected mice (0.19±0.01) decreased below control levels (0.22±0.05) although this decrease was not significant (P = 0.600). The 43-h time point was subsequently used to further characterise the in vivo response to 18F-FDG in mice. Fig. 2. Open in new tabDownload slide MN-RET kinetics in vivo formation of MN-RETs following injection with 18.5 MBq 18F-FDG (n = 3, duplicate samples) or saline (n = 1–3, duplicate samples). Blood samples were collected at 30-, 43-, 47-, 51- and 72-h post-injection. Results are depicted as mean ± SEM. Fig. 2. Open in new tabDownload slide MN-RET kinetics in vivo formation of MN-RETs following injection with 18.5 MBq 18F-FDG (n = 3, duplicate samples) or saline (n = 1–3, duplicate samples). Blood samples were collected at 30-, 43-, 47-, 51- and 72-h post-injection. Results are depicted as mean ± SEM. Fig. 3. Open in new tabDownload slide Activity–response curve in vivo formation of MN-RETs following injection with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples). Each point (x, y) represents one mouse (duplicates averaged) with corresponding injected activity. Fig. 3. Open in new tabDownload slide Activity–response curve in vivo formation of MN-RETs following injection with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples). Each point (x, y) represents one mouse (duplicates averaged) with corresponding injected activity. 18F-FDG dose response (MN-RET) The induction of micronuclei in bone marrow by 18F-FDG was found to vary non-linearly with injected activity, corresponding with a second-order polynomial (R2 = 0.9997; Figure 3). All injection activities caused an increase in micronuclei relative to controls although this increase was only significant following injections of 3.70, 7.40 and 14.80 MBq (P < 0.001). The injection activities corresponded to bone marrow doses of 35.89±0.62, 74.30±1.59 and 150.32±4.12 mGy, respectively (Table I). Relative biological effectiveness of 18F-FDG measured (MN-RET) Similar to 18F-FDG, the gamma dose–response curve was also found to be non-linear (R2 = 0.9945), however the gamma curve differed significantly from the 18F-FDG (Figure 4). Multiple linear regression analysis determined that the type of radiation exposure (18F-FDG or 662 keV γ-rays) was a significant determinant of the MN-RET dose response (P < 0.004) with 662 keV γ-rays generating significantly more damage per unit dose. The relative biological effectiveness (RBE) for 18F-FDG was calculated by taking the ratio of the linear regression coefficients from the two dose–response curves (18F-FDG: 662 keV γ-rays). Using this approach, the RBE for 18F-FDG was determined to be 0.79±0.04. Fig. 4. Open in new tabDownload slide RBE comparison of the MN-RET frequency per unit dose between 18F-FDG (internal) and 662 keV γ-rays (external). For the 18F-FDG curve, mice were injected with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples) and converted to bone marrow doses. For the 662 keV γ-rays, mice were irradiated whole body with 0, 10, 20, 50 or 100 mGy (n = 3–5, duplicate samples) at a dose rate of 18 mGy/min which were also converted to bone marrow doses using an attenuation factor. Radiation-induced values are displayed; MN-RET levels were normalised from background MN-RET values. Results displayed are mean ± SEM. Fig. 4. Open in new tabDownload slide RBE comparison of the MN-RET frequency per unit dose between 18F-FDG (internal) and 662 keV γ-rays (external). For the 18F-FDG curve, mice were injected with 0, 0.74, 1.48, 3.70, 7.40 or 14.80 MBq 18F-FDG (n = 8–10, duplicate samples) and converted to bone marrow doses. For the 662 keV γ-rays, mice were irradiated whole body with 0, 10, 20, 50 or 100 mGy (n = 3–5, duplicate samples) at a dose rate of 18 mGy/min which were also converted to bone marrow doses using an attenuation factor. Radiation-induced values are displayed; MN-RET levels were normalised from background MN-RET values. Results displayed are mean ± SEM. In vivo induction of an adaptive response by 18F-FDG (MN-RET) Four independent experiments were performed and pooled to examine whether injections of 0 (0), 0.74 (20), 3.70 (100) or 14.80 (400) MBq (µCi) could modify the effect of a 1 Gy (662 keV γ-rays) in vivo challenge dose given 24-h post-injection. The PET injection did not significantly alter the damage induced by the 1 Gy challenge dose in vivo as measured by the MN-RET assay (P = 0.959; Figure 5). Fig. 5. Open in new tabDownload slide In vivo adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 9–11, duplicate samples) and irradiated 24-h post-injection with 1 Gy whole body (662 keV γ-rays, 0.35 Gy/min). The MN-RET frequency was evaluated 43h after the 1 Gy dose. Results displayed are mean ± SEM. Fig. 5. Open in new tabDownload slide In vivo adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 9–11, duplicate samples) and irradiated 24-h post-injection with 1 Gy whole body (662 keV γ-rays, 0.35 Gy/min). The MN-RET frequency was evaluated 43h after the 1 Gy dose. Results displayed are mean ± SEM. In vitro induction of an adaptive response by 18F-FDG injections (γH2A.X) The lower limit of detection of the flow cytometry-based γH2A.X assay is 100 mGy for acute radiation exposures (46); consequently, this assay would consequently not be useful in measuring the direct effects of the standard dose range of 18F-FDG injections. It was used instead to measure the potential modification of an acute radiation exposure by 18F-FDG by the induction of an adaptive response. Mice were injected with 0 (0), 0.74 (20), 3.70 (100) or 14.80 (400) MBq (µCi). At 24-h post-injection, bone marrow from these mice were extracted and irradiated in culture with 0, 1, 2 or 4 Gy (662 keV γ-rays). The mean γH2A.X fluorescence in the lymphocyte-rich population was measured 30min after the challenge irradiation as this has been shown previously to be the optimal time for measurement (45). The level of γH2A.X increased significantly with the magnitude of the challenge dose (P < 0.001; Figure 6A). None of the injection activities (0.74, 3.70 or 14.80 MBq) significantly modified the effect of any of the challenge doses (1, 2, 4 Gy), relative to the saline injected controls (P = 0.322; Figure 6A). It was observed in the non-challenged mice (0 Gy) that γH2A.X levels decreased with increasing injection activity (P = 0.019; Figure 6B) at 24h. The 14.80 MBq injection of 18F-FDG, in particular, caused a significant decrease in γH2A.X below controls (P < 0.014). Fig. 6. Open in new tabDownload slide In vitro adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 6–10, duplicate samples) and irradiated at 24-h post-injection with 0, 1, 2 or 4 Gy in vitro. Mean γH2A.X fluorescence was measured 30min after the challenge dose (A). Sham-irradiated samples only. (B) Results displayed are mean ± SEM. Fig. 6. Open in new tabDownload slide In vitro adaptive response mice were injected with 0, 0.74, 1.48 or 3.70 MBq of 18F-FDG (n = 6–10, duplicate samples) and irradiated at 24-h post-injection with 0, 1, 2 or 4 Gy in vitro. Mean γH2A.X fluorescence was measured 30min after the challenge dose (A). Sham-irradiated samples only. (B) Results displayed are mean ± SEM. Discussion The short-term DNA damage response to 18F-FDG PET scans in mice was evaluated using two well-established radiation biology assays: MN-RET formation and γH2A.X fluorescence. Micronuclei occur in newly formed RETs in the blood stream of mice following DNA damage to precursors in bone marrow. This process generally takes between 43 and 55h and varies with mouse strain (44). The MN-RET technique has been adopted by genetic toxicology and product safety research programs (47, 48) because of its exceptional sensitivity for measuring the mutagenic potential of low level exposures. It has been shown to be a reliable biodosimeter for acute radiation exposures and various clastogenic/aneugenic chemicals (44, 49, 50), however, to our knowledge, this assay has never been used to measure the DNA damage associated with radioisotopes. Previous studies demonstrated that a 1 Gy acute γ-radiation exposure induces a maximum MN-RET frequency at 43h and returns to spontaneous levels by 137h (44). The formation of γH2A.X foci occurs at the site of new DSBs within minutes. The phosphorylation of histone H2A.X to form γH2A.X is a key component in sensing DNA damage and recruiting repair proteins. It has been shown that the majority of radiation-induced foci disappear by 8-h post-irradiation to doses below 1 Gy (51), and disappeared in bone marrow cells by 24h following 4 Gy (52). Residual foci have been associated with on-going genetic damage typical of genomic instability (53, 54), so measuring γH2A.X foci 24-h post-irradiation consequently provides information about residual cytogenetic damage. We examined the biological response to a range of injection activities of 18F-FDG between 0.74 (20) and 14.80 (400) MBq (µCi), the lowest activity used (0.74 MBq) produces absorbed doses in a mouse similar to the average patient absorbed dose during clinical PET scans. The highest dose (14.80 MBq) represents a typical injection activity used in small animal imaging investigations. A proportionally larger amount of activity is needed to generate diagnostically useful images in mice, related to the higher spatial resolution requirements in small animal imaging (55). The 43-h peak of MN-RET expression was reduced in magnitude compared to that seen with acute gamma exposures, but the following pattern of decay was similar. A prolonged elevation in MN-RETS compared to the acute gamma exposure was expected based on the characteristics of isotope decay but was not observed. This suggests the DSB repair mechanisms induced by the low dose rates associated with 18F-FDG were effective despite the protracted exposure. By 72h, the level of MN-RETS in 18F-FDG-injected mice had decreased to ~14% below control levels. This decrease was not significant but suggests that higher doses of 18F-FDG may be required to fully induce adaptive mechanisms. We demonstrated that a single 10 mGy acute whole body exposure significantly increased MN-RET frequency above controls. In contrast, a 3.70 MBq 18F-FDG injection, corresponding to a whole body dose of 50–57 mGy and a bone marrow dose of 36 mGy, was required to significantly elevate MN-RET frequency. This highlights the increased resistance to DNA damage in vivo when low dose rates protract the radiation exposure. As the dose rate decreases, the temporal abundance of damage in the cell is decreased and the ability to accurately repair DNA damage increases (56, 57). The RBE for 18F-FDG, a combined exposure from 634 keV positrons and 511 keV γ-rays, was calculated to be 0.79±0.04 in mice for the MN-RET endpoint. RBE is used to derive radiation weighting factors for use in radiation protection, with a factor of 1 used for all low linear energy transfer radiations (58). We have shown that the low dose rate and radiation quality associated with 18F-FDG reduces its effectiveness in generating micronuclei in bone marrow. RBE has been shown to vary substantially based on the radiation quality, biological endpoint, organism and dose rate. It has been shown previously that single low dose or dose-rate exposures have the capacity to modify the subsequent response of a biological system to a large dose of radiation (26, 28–31, 59). We investigated the potential for 18F-FDG to generate an adaptive response at 24-h post-injection using the MN-RET and γH2A.X endpoints. The 24-h interval was chosen to allow for complete decay of the isotope in order to capture the full extent of biological changes induced by the injection. A single injection of 0.74, 3.70 or 14.80 MBq 18F-FDG did not modify the magnitude of the response to a 1 Gy acute dose of radiation in vivo as measured with the MN-RET endpoint. Injections of 0.74, 3.70 or 14.80 MBq correspond to approximate whole body doses of 10, 50 and 200 mGy and approximate bone marrow doses of 7, 35 and 150 mGy (Table I). Similarly, 0.74, 3.70 or 14.80 MBq 18F-FDG injections did not modify the response of bone marrow lymphocytes to 1, 2 or 4 Gy in vitro as measured by γH2A.X foci formation. While the 18F-FDG doses in this study did not induce an adaptive response, there are factors characteristic of this radioisotope that require modifications to the classic adaptive response experimental design. The low dose rate associated with 18F decay appears to require significantly greater doses than those used in this study to induce protective mechanisms. This is supported by the reduction in baseline MN-RET levels following injection of the largest doses of 18F-FDG. Additionally, the dose-dependent decrease in γH2A.X following injections of 18F-FDG at 24h in the sham-irradiated mice (0 Gy), and more specifically, a significant 20% reduction in γH2A.X fluorescence following the 14.80 MBq injection indicates the induction protective mechanisms in this study. The heterogeneity of 18F-FDG uptake into tissues provides another confounding factor for adaptation responses. Bone marrow may not have sufficient uptake to obtain the dose necessary for induction of protective mechanisms, however tissues with higher absorbed doses may in fact undergo adaptation. Further studies in tissues including the heart, brain and kidneys of these mice are required to clarify this issue. Timing between the adapting and challenge doses is also crucial, however determining optimal timing with radioisotope decay can be problematic given that the timing is dependent on individual tissue characteristics (30, 60). We have studied the DNA damage response to radiation exposure from 18F-FDG and found a dose-dependent increase in radiation-induced damage. This initial damage response, as measured by MN-RET formation, offers insight into the biophysical properties of the energy deposition in bone marrow associated with 18F-FDG. By studying later time points, we were able to understand the biological impact of this initial response. For example, the highest injection activities used (14.80–18.5 MBq) did not lead to residual DNA damage, but in fact, decreased the level of damage below controls. This illustrates the importance of examining later time points in these types of investigations as subsequent biological responses may alter dose–response dynamics. The LNT model of risk would predict that the total damage from this combination of treatments would be equal to the damage due to the isotope injection plus that induced by the high-dose radiation exposure. Consistent with the other observations in this study, the damage induced by the radiation exposure from the 0.74, 3.70 or 14.80 MBq 18F-FDG injections was effectively repaired and did not add to damage induced by the high-dose challenge. The LNT model represents a practical approach to radiation protection and extrapolates epidemiological high dose-effects into the low dose range using a linear relationship. In doing so, certain assumptions are made: that dose is a surrogate for risk, that there is no threshold for radiation effects and that risk is additive and can only increase. Our results indicate that in the context of 18F-FDG exposure from PET scans, this does not hold true. We have shown that there is threshold for DNA damage to bone marrow following injections of 18F-FDG and the dose–response curve for the radioisotope is non-linear. We have also shown that the added radiation exposure (low dose isotope + high-dose challenge) does not result in additive damage. We have examined the radiobiological changes induced by 18F-FDG to improve the current understanding of the health effects associated with radiation exposure from PET scans. We tested injection activities relevant to both human imaging and small animal studies, demonstrating that the low dose rate of 18F-FDG exposure substantially reduces the DNA damage generated for a given dose. Later sampling times revealed that the larger injection activities may actually reduce the level of DNA damage below that of controls. Together, these results provide evidence against the use of LNT to predict the risk to patients from nuclear medicine procedures such as PET. Funding US Department of Energy Low Dose Radiation Program (DE-FG02-07ER64343) and the National Sciences and Engineering Council of Canada (238495). Acknowledgements We sincerely thank Mary Ellen Cybulski, Lisa Laframboise and Nicole McFarlane for their important technical contributions to this study. 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