Radiation therapy (RT) plays a vital role in managing thoracic cancers, though it can lead to adverse effects, including significant cardiotoxicity. Understanding the risk factors like hypertension in RT is important for patient prognosis and management. A Dahl salt-sensitive (SS) female rat model was used to study hypertension effect on RT-induced cardiotoxicity. Rats were fed a high-salt diet to induce hypertension and then divided into RT and sham groups. The RT group received 24 Gy of whole-heart irradiation. Cardiac function was evaluated using MRI and blood pressure measurements at baseline, 8 weeks and 12 weeks post-RT. Histological examination was performed after the last timepoint or animal death. The hypertensive RT rats demonstrated significant decreases in left-ventricular ejection fraction (EF) (45 ± 7.2%) compared to sham (68 ± 7.3%). Furthermore, circumferential (Ecc) and radial (Err) myocardial strains were significantly reduced (Ecc: -7.4 ± 2.0% RT rats vs. -11 ± 2.4% sham; Err: 15 ± 6.5% RT rats vs. 23 ± 8.9% sham). Histological analysis revealed significant pathophysiological remodeling post-RT, including nuclear size, interstitial fibrosis, necrosis, and the presence of inflammatory cells. This study provides valuable insights into the cardiotoxic effects of RT in the context of hypertension, highlighting the potential of using MRI for improved risk assessment with potential for future clinical translation.

Exposure to diagnostic and therapeutic radiation introduces risks for development of diseases later in life by causing DNA damage in cells. Currently, there is no clinical method for determining exposure risk caused by radiation toxicity to DNA. Cell-free DNA (cfDNA), a marker of DNA damage, is currently used to assess risk for long-term effects following organ transplantation, surgery and inflammation. The goal of our proposed study is to develop cfDNA as an early biomarker for assessing risk for cardiovascular disease and cancer from radiation exposure so that strategies to mitigate the damaging effects of medical radiation can be assessed. Hearts from male and female WAG/RijCmcr rats (n = 6-10/group) were exposed to increasing doses of X-radiation (50 mGy and 3.5 Gy). Blood was collected prior to and after (15 minutes-96 hours) irradiation, and cell-free plasma was prepared. Primers and probes were designed for quantitative analysis of sequences of mitochondria (12S rRNA) and nuclear (Gapdh) cfDNA present in rat plasma using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Exposure of hearts to radiation increased nuclear and mitochondrial cfDNA in a dose-dependent manner. Three point five grays from X-radiation increase cfDNA for Gapdh in plasma after 1 hour with a 15.8-fold increase (P < 0.001) after 6 hours. The earliest time nuclear and mitochondrial cfDNA increases were detected in plasma was at 60 minutes following exposure to 3.5 Gy. cfDNA has potential to advance as a biomarker of exposure to medical doses of radiation in patients.

Introduction: The space environment astronauts experience during space missions consists of multiple environmental challenges, including microgravity. In this study, we assessed the behavioral and cognitive performances of male Fisher rats 2 months after sham irradiation or total body irradiation with photons in the absence or presence of simulated microgravity. We analyzed the plasma collected 9 months after sham irradiation or total body irradiation for distinct alterations in metabolic pathways and to determine whether changes to metabolic measures were associated with specific behavioral and cognitive measures.Methods: A total of 344 male Fischer rats were irradiated with photons (6 MeV; 3, 8, or 10 Gy) in the absence or presence of simulated weightlessness achieved using hindlimb unloading (HU). To identify potential plasma biomarkers of photon radiation exposure or the HU condition for behavioral or cognitive performance, we performed regression analyses.Results: The behavioral effects of HU on activity levels in an open field, measures of anxiety in an elevated plus maze, and anhedonia in the M&M consumption test were more pronounced than those of photon irradiation. Phenylalanine, tyrosine, and tryptophan metabolism, and phenylalanine metabolism and biosynthesis showed very strong pathway changes, following photon irradiation and HU in animals irradiated with 3 Gy. Here, 29 out of 101 plasma metabolites were associated with 1 out of 13 behavioral measures. In the absence of HU, 22 metabolites were related to behavioral and cognitive measures. In HU animals that were sham-irradiated or irradiated with 8 Gy, one metabolite was related to behavioral and cognitive measures. In HU animals irradiated with 3 Gy, six metabolites were related to behavioral and cognitive measures.Discussion: These data suggest that it will be possible to develop stable plasma biomarkers of behavioral and cognitive performance, following environmental challenges like HU and radiation exposure.

IntroductionEffects and interactions between different spaceflight stressors are expected to be experienced by crew on missions when exposed to microgravity and galactic cosmic rays (GCRs). One of the limitations of previous studies on simulated weightlessness using hindlimb unloading (HU) is that a control HU condition was not included.MethodsWe characterized the behavioral performance of male Fischer rats 2 months after sham or total body irradiation with a simplified 5-ion 6-mixed-beam exposure representative of GCRs in the absence or presence of HU. Six months later, the plasma, hippocampus, and cortex were processed to determine whether the behavioral effects were associated with long-term alterations in the metabolic pathways.ResultsIn the open field without and with objects, interactions were observed for radiation × HU. In the plasma of animals that were not under the HU or control HU condition, the riboflavin metabolic pathway was affected most for sham irradiation vs. 0.75 Gy exposure. Analysis of the effects of control HU on plasma in the sham-irradiated animals showed that the alanine, aspartate, glutamate, riboflavin, and glutamine metabolisms as well as arginine biosynthesis were affected. The effects of control HU on the hippocampus in the sham-irradiated animals showed that the phenylalanine, tyrosine, and tryptophan pathway was affected the most. Analysis of effects of 0.75 Gy irradiation on the cortex of control HU animals showed that the glutamine and glutamate metabolic pathway was affected similar to the hippocampus, while the riboflavin pathway was affected in animals that were not under the control HU condition. The effects of control HU on the cortex in sham-irradiated animals showed that the riboflavin metabolic pathway was affected. Animals receiving 0.75 Gy of irradiation showed impaired glutamine and glutamate metabolic pathway, whereas animals receiving 1.5 Gy of irradiation showed impaired riboflavin metabolic pathways. A total of 21 plasma metabolites were correlated with the behavioral measures, indicating that plasma and brain biomarkers associated with behavioral performance are dependent on the environmental conditions experienced.DiscussionPhenylalanine, tyrosine, and tryptophan metabolism as well as phenylalanine and tryptophan as plasma metabolites are biomarkers that can be considered for spaceflight as they were revealed in both Fischer and WAG/Rij rats exposed to simGCRsim and/or HU.

Heart disease is a significant adverse event caused by radiotherapy for some cancers. Identifying the origins of radiogenic heart disease will allow therapies to be developed. Previous studies showed non-targeted effects manifest as fibrosis in the non-irradiated heart after 120 days following targeted X-irradiation of the kidneys with 10 Gy in WAG/RijCmcr rats. To demonstrate the involvement of T cells in driving pathophysiological responses in the out-of-field heart, and to characterize the timing of immune cell engagement, we created and validated a T cell knock downrat on the WAG genetic backgrou nd. Irradiation of the kidneys with 10 Gy of X-rays in wild-type rats resulted in infiltration of T cells, natural killer cells, and macrophages after 120 days, and none of these after 40 days, suggesting immune cell engagement is a late response. The radiation nephropathy and cardiac fibrosis that resulted in these animals after 120 days was significantly decreased in irradiated T cell depleted rats. We conclude that T cells function as an effector cell in communicating signals from the irradiated kidneys which cause pathologic remodeling of non-targeted heart.

Long-duration space exploratory missions to the Earth's moon and the planet Mars are actively being planned. Such missions will require humans to live for prolonged periods beyond low earth orbit where astronauts will be continuously exposed to high energy galactic cosmic rays (GCRs). A major unknown is the potential impact of GCRs on the risks of developing degenerative cardiovascular disease, which is a concern to NASA. A ground-based rat model has been used to provide a detailed characterization of the risk of long-term cardiovascular disease from components of GCRs at radiation doses relevant to future human missions beyond low earth orbit. Six month old male WAG/RijCmcr rats were irradiated at a ground-based charged particle accelerator facility with high energy ion beams broadly representative of GCRs: protons, silicon and iron. Irradiation was given either as a single ion beam or as a combination of three ion beams. For the doses used, the single ion beam studies did not show any significant changes in the known cardiac risk factors and no evidence of cardiovascular disease could be demonstrated. In the three ion beam study, the total cholesterol levels in the circulation increased modestly over the 270 day follow up period, and inflammatory cytokines were also increased, transiently, 30 days after irradiation. Perivascular cardiac collagen content, systolic blood pressure and the number of macrophages found in the kidney and in the heart were each increased 270 days after irradiation with 1.5 Gy of the three ion beam grouping. These findings provide evidence for a cardiac vascular pathology and indicate a possible threshold dose for perivascular cardiac fibrosis and increased systemic systolic blood pressure for complex radiation fields during the 9 month follow up period. The development of perivascular cardiac fibrosis and increased systemic systolic blood pressure occurred at a physical dose of the three ion beam grouping (1.5 Gy) that was much lower than that required to show similar outcomes in earlier studies with the same rat strain exposed to photons. Further studies with longer follow up periods may help determine whether humans exposed to lower, mission-relevant doses of GCRs will develop radiation-induced heart disease.

In the original publication [...].

A limitation of simulated space radiation studies is that radiation exposure is not the only environmental challenge astronauts face during missions. Therefore, we characterized behavioral and cognitive performance of male WAG/Rij rats 3 months after sham-irradiation or total body irradiation with a simplified 5-ion mixed beam exposure in the absence or presence of simulated weightlessness using hindlimb unloading (HU) alone. Six months following behavioral and cognitive testing or 9 months following sham-irradiation or total body irradiation, plasma and brain tissues (hippocampus and cortex) were processed to determine whether the behavioral and cognitive effects were associated with long-term alterations in metabolic pathways in plasma and brain. Sham HU, but not irradiated HU, rats were impaired in spatial habituation learning. Rats irradiated with 1.5 Gy showed increased depressive-like behaviors. This was seen in the absence but not presence of HU. Thus, HU has differential effects in sham-irradiated and irradiated animals and specific behavioral measures are associated with plasma levels of distinct metabolites 6 months later. The combined effects of HU and radiation on metabolic pathways in plasma and brain illustrate the complex interaction of environmental stressors and highlights the importance of assessing these interactions.

Cardiac disease is a frequent and significant adverse event associated with radiotherapy for cancer. Identifying the underlying mechanism responsible for radiation injury to the heart will allow interventions to be developed. In the present study, we tested if local kidney irradiation results in remodeling of the shielded, nontargeted heart. One kidney, two kidneys, or the total body of male WAG and Dahl SS rats were irradiated with 10 Gy of X-rays. Local kidney irradiation resulted in systemic hypertension, increased BUN, infiltration of T lymphocytes, natural killer cells, and macrophages into the renal cortex and medulla, and renal fibrosis. Local irradiation of kidneys in WAG rats resulted in remodeling in the nontargeted heart after 120 days, manifested by perivascular fibrosis and increased interventricular septal thickness, but was not seen in Dahl SS rats due to a high baseline level of fibrosis in the sham-irradiated animals. Genetic depletion of T cells mitigated the nephropathy after local kidney irradiation, indicating a role for the immune system in mediating this outcome. Local kidney irradiation resulted in a cascade of pro-inflammatory cytokines and low-molecular weight metabolites into the circulation associated with transmission of signals resulting in pathologic remodeling in the nontargeted heart. A new model is proposed whereby radiation-induced cardiac remodeling in susceptible animals is indirect, with lower hemi body organs such as the kidney exporting factors into the circulation that cause remodeling outside of the irradiated field in the shielded, nontargeted heart. This nontargeted effect appears to be mediated, in part, by the immune system.

Radiotherapy with sparsely ionizing photons is a cornerstone of successful cancer treatment. Age at time of exposure to radiation is known to influence biological outcomes for many end points. The effect of dose and age at exposure upon the occurrence of radiogenic cardiovascular disease is poorly understood. The goal of this work was to determine the response of maleWAG/RijCmcr rats at 6 months of age to gamma rays, and at 6 months or 6 weeks of age to X rays, using clinically relevant biomarkers of cardiovascular disease and kidney injury. Overall, there were significant radiation-induced effects on the levels of bicarbonate (P=0.0016), creatinine (P=0.0002), calcium (P = 0.0009), triglycerides (P = 0.0269) and blood urea nitrogen, albumin, protein, AST, alkaline phosphatase, total cholesterol and HDL (all P < 0.0001). Of those variables with a significant radiation-dose effect, there were significant modifications by age at time of exposure for bicarbonate (P = 0.0033), creatinine (P = 0.0015), AST (P = 0.0040), total cholesterol (P = 0.0006) and blood urea nitrogen, calcium, albumin, protein, alkaline phosphatase and HDL (all P < 0.0001). Cardiac perivascular collagen content was significantly increased in rats that were 8.0 Gy X-ray irradiated at 6 weeks of age (P < 0.047) but not at 6 months of age. While systemic blood pressure was elevated in both cohorts after 8.0 Gy X-ray irradiation (compared to agematched sham-irradiated controls), the magnitude of the increase above baseline was greater in the younger rats (P < 0.05). These findings indicate that dose and age at time of irradiation determine the timeline and severity of cardiac and renal injury.

Radiation is a cornerstone of successful cancer treatment, with one-half to two-thirds of all patients receiving radiotherapy. Survivors of cancer treated with radiation are at increased risk for cardiovascular disease (CVD). Understanding how radiation causes CVD will allow development of novel therapies. Irradiation of the lower hemi body, but not the upper hemi body, with 10 Gy in rats increases risk factors for CVD and results in cardiac fibrosis quantitatively similar to total body irradiation, suggesting radiation injury to the heart can be indirect. Simvastatin, an inhibitor of liver cholesterol synthesis administered continuously to rats (10 mg/kg/day) after 10 Gy lower hemi body irradiation mitigated against increased blood cholesterol and cardiac fibrosis. These findings indicate simvastatin limits transmission of a signal from the lower hemi body that decreases risk for and occurrence of CVD independent of any direct exposure of the heart to radiation. Bivariate examination of 3,607 patients following therapeutic lower hemi body irradiation using Chi-square, Wilcoxon rank-sum and t-tests was used to examine risk factors for CVD in patients diagnosed with congestive heart failure, myocardial infarction, atrial fibrillation, and cardiomyopathy before 80 years of age. We found that 47.4% of patients age 70-80 developed CVD compared to 29.7% who received simvastatin (p= < 0.001, n = 293 and 361, respectively). Patients who were male, overweight, smokers, and had a diagnosis of chronic kidney disease and diabetes also had significantly higher risk of CVD. Race and hypertension were not indicative of increased risk for CVD. These clinical findings, taken together with the results from our animal studies, support a new research paradigm where radiation-induced heart disease can be indirect, with abdominal organs exporting factors that cause CVD. Simvastatin can be developed to mitigate and treat CVD after therapeutic radiation.

Activators of G-protein Signaling (AGS) are a family of accessory proteins that were discovered as modulators of heterotrimeric G-protein subunits. The primary aim of the present study was to localize Group I and II AGS proteins and determine the renal expression profile using immunohistochemistry and quantitative RT-PCR, respectively, during normal and injured states of the kidney. Group I AGS1 was found to be predominantly localized to the proximal tubule, Group II AGS3 and AGS5 were exclusively localized to the distal tubular segments, and Group II AGS6 was ubiquitously expressed in every nephron segment of the rodent kidney. In rat kidneys following ischemia–reperfusion injury (IRI), Group I AGS1 mRNA was dramatically increased after 24 h by fivefold ( P < 0.05), whereas Group II AGS3 and AGS4 mRNA was significantly decreased at the same time point ( P < 0.05). No significant change in the transcript levels were detected at other time points for any of the AGS genes between control and IRI groups. In polycystic diseased kidneys, mRNA levels for AGS3, AGS4 and AGS6 was significantly increased ( P < 0.05) by 75–80 % in PCK rat kidneys. The identification of Group I and II AGS mRNA and protein in the kidney may provide insight into the potential mechanism of action during normal and varying states of renal disease or injury.

The ability of simvastatin to mitigate the increases in risk factors for and the occurrence of cardiac disease after 10 Gy total body irradiation (TBI) was determined. This radiation dose is relevant to conditioning for stem cell transplantation and threats from radiological terrorism. Male rats received single dose TBI of 10 Gy. Age-matched, sham-irradiated rats served as controls. Lipid profile, heart and liver morphology and cardiac mechanical function were determined for up to 120 days after irradiation. TBI resulted in a sustained increase in total-and LDL-cholesterol (low-density lipoprotein-cholesterol), and triglycerides. Simvastatin (10 mg/kg body weight/day) administered continuously from 9 days after irradiation mitigated TBI-induced increases in totaland LDL-cholesterol and triglycerides, as well as liver injury. TBI resulted in cellular peri-arterial fibrosis, whereas control hearts had less collagen and fibrosis. Simvastatin mitigated these morphological injuries. TBI resulted in cardiac mechanical dysfunction. Simvastatin mitigated cardiac mechanical dysfunction 20-120 days following TBI. To determine whether simvastatin affects the ability of the heart to withstand stress after TBI, injury from myocardial ischemia/reperfusion was determined in vitro. TBI increased the severity of an induced myocardial infarction at 20 and 80 days after irradiation. Simvastatin mitigated the severity of this myocardial infarction at 20 and 80 days following TBI. It is concluded simvastatin mitigated the increases in risk factors for cardiac disease and the extent of cardiac disease following TBI. This statin may be developed as a medical countermeasure for the mitigation of radiation-induced cardiac disease.

Activator of G‐protein signaling (AGS) are a family of accessory proteins that regulate heterotrimeric G‐protein function in a receptor‐independent mechanism. Recent studies in our lab have identified a crucial role for AGS3/GPSM1 in the recovery of the renal epithelial cells following acute kidney injury. Under normal conditions, AGS3/GPSM1 is exclusively expressed in the distal and collecting ducts. Following ischemia‐reperfusion injury, AGS3/GPSM1 is induced in the proximal tubules in the outer medulla, which is a highly sensitive site of injury due to ischemia. To determine whether other AGS proteins are expressed and may demonstrate biological activity during acute kidney injury, this study was designed to localize AGS proteins within the kidney using immunohistochemistry, and also determine the renal expression profile using quantitative RT‐PCR of all 13 known AGS proteins during quiescence and following an injury response. AGS1 was found to be predominantly localized to the proximal tubule in the renal cortex and outer medulla from mice, whereas AGS3, 5, and 8 were exclusively localized to the distal and collecting ducts in rat kidneys. In rat kidneys following ischemia‐reperfusion injury, AGS1 and AGS7 were both dramatically increased after 24 hours by 6‐7 fold, and slowly decreased towards normal (sham) levels at 72 and 168 hours after reperfusion. Messenger RNA levels of AGS4, 8, 9, 10, 11 and 12 were decreased by 40‐90% in the ischemia‐reperfusion injured kidneys compared to their sham (time‐control) kidneys at 24 hours, which tended to return towards normal (sham) levels by 168 hours after reperfusion. This study provides new information as to the site of synthesis of AGS protein, and could shed new light on their potential roles during normal and pathologic conditions in the kidney.Grant Funding Source: Supported by National Institutes of Health

BACKGROUND:Chronic persistent oxidative stress has been proposed as a mechanism for late radiation injury to normal tissue. Using biochemical, histological, and pharmacological techniques, we have not been able to confirm this hypothesis for late renal radiation injury. Gene expression may be more revealing, especially since the initial effects of radiation are to damage DNA.METHODS:Gene array studies were done using kidney tissue from irradiated rats, with particular attention to genes pertinent to oxidative stress. The time points were from 1 to 49 days after irradiation. Cellular RNA and mitochondrial DNA were isolated, for gene expression analysis and common deletion testing, respectively.RESULTS:For the gene expression studies, and from over 30,000 transcripts, only nine related to oxidative stress had 1.4 fold or greater changes in expression. Mitochondrial DNA showed no changes in the common deletion.CONCLUSION:These studies do not support the hypothesis of chronic oxidative stress as a mechanism for radiation nephropathy.

The objective of this study was to determine whether radiation-induced injury to the heart after 10 Gy total body irradiation (TBI) is direct or indirect. Young male WAG/RijCmcr rats received a 10 Gy single dose using TBI, upper hemi-body (UHB) irradiation, lower hemi-body (LHB) irradiation, TBI with the kidneys shielded or LHB irradiation with the intestines shielded. Age-matched, sham-irradiated rats served as controls. The lipid profile, kidney injury, heart and liver morphology and cardiac function were determined up to 120 days after irradiation. LHB, but not UHB irradiation, increased the risk factors for cardiac disease as well as the occurrence of cardiac and kidney injury in a way that was quantitatively and qualitatively similar to that observed after TBI. Shielding of the kidneys prevented the increases in risk factors for cardiac disease. Shielding of the intestines did not prevent the increases in risk factors for cardiac disease. There was no histological evidence of liver injury 120 days after irradiation. Injury to the heart from irradiation appears to be indirect, supporting the notion that injury to abdominal organs, principally the kidneys, is responsible for the increased risk factors for and the occurrence of cardiac disease after TBI and LHB irradiation.

Abstract Lenarczyk, M., Cohen, E. P., Fish, B. L., Irving, A. A., Sharma, M., Driscoll, C. D. and Moulder, J. E. Chronic Oxidative Stress as a Mechanism for Radiation Nephropathy. Radiat. Res. 171, 164–172 (2009). Suppression of the renin-angiotensin system has proven efficacy for mitigation and treatment of radiation nephropathy, and it has been hypothesized that this efficacy is due to suppression of radiation-induced chronic oxidative stress. It is known that radiation exposure leads to acute oxidative stress, but direct evidence for radiation-induced chronic renal oxidative stress is sparse. We looked for evidence of oxidative stress after total-body irradiation in a rat model, focusing on the period before there is physiologically significant renal damage. No statistically significant increase in urinary 8-isoprostane (a marker of lipid peroxidation) or carbonylated proteins (a marker of protein oxidation) was found over the first 42 days after irradiation, while a small but statistically significant increase in urinary 8-hydroxydeoxy-guanosine (a marker of DNA oxidation) was detected at 35–55 days. When we examined renal tissue from these animals, we found no significant increase in either DNA or protein oxidation products over the first 89 days after irradiation. Using five different standard methods for detecting oxidative stress in vivo, we found no definitive evidence for radiation-induced renal chronic oxidative stress. If chronic oxidative stress is part of the pathogenesis of radiation nephropathy, it does not leave widespread or easily detectable evidence behind.

Induced genomic instability in the human B lymphoblastoid cell line TK6 manifests itself as increases in end-to-end chromosome fusions and non-reciprocal chromosome translocations. It is not associated with elevated frequencies of specific locus mutations or other cytogenetic alterations. Previous studies on a limited number of cells and end-points suggested that induced instability in TK6 mirrors spontaneous instability in terms of the types of alterations observed. In the present study we expanded on our previous analysis to include more cells and more end-points in order to derive a more precise measure of spontaneous instability in TK6 cells. The frequency of normal growth rate thymidine kinase mutants (TK(-/-)), measured in 44 independently isolated clones, was 2.73 +/- 0.78 x 10(-6)/cell, while that for slow growth mutants was 2.39 +/- 0.52 x 10(-6)/cell. These are similar to the frequencies observed for HPRT mutants in primary human cells. There was wide variation in chromatid break frequencies, but the average break frequency, at 0.04+/-0.01 breaks/cell, was only slightly higher than that reported for primary human cells. In contrast, the dicentric frequency of 0.006/cell was more than 10-fold higher for TK6 cells than that reported for normal primary human cells. Furthermore, the dicentrics in TK6 cells are unusual in that they are the result of end-to-end chromosome fusions. TK6 cells also show much higher levels of non-reciprocal chromosome translocations than are usually observed in primary human cells. The results suggest an inherent instability in TK6 cells that differs from what is observed in primary cells in that it affects the frequency of end-to-end chromosome fusions and non-reciprocal chromosome translocations, but not TK gene mutations or other cytogenetic alterations.