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.

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 hours by 5-fold (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.

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 radiationinduced 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.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

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.

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

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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.