Orthologs of human circulating miRNAs associated with hepatocellular carcinoma are elevated in mouse plasma months before tumour detection.

Research examining the potential for circulating miRNA to serve as markers for preneoplastic lesions or early-stage hepatocellular carcinoma (HCC) is hindered by the difficulties of obtaining samples from asymptomatic individuals. As a surrogate for human samples, we identified hub miRNAs in gene co-expression networks using HCC-bearing C3H mice. We confirmed 38 hub miRNAs as associated with HCC in F2 hybrid mice derived from radiogenic HCC susceptible and resistant founders. When compared to a panel of 12 circulating miRNAs associated with human HCC, two had no mouse ortholog and 7 of the remaining 10 miRNAs overlapped with the 38 mouse HCC hub miRNAs. Using small RNA sequencing data generated from serially collected plasma samples in F2 mice, we examined the temporal levels of these 7 circulating miRNAs and found that the levels of 4 human circulating markers, miR-122-5p, miR-100-5p, miR-34a-5p and miR-365-3p increased linearly as the time approaching HCC detection neared, suggesting a correlation of miRNA levels with oncogenic progression. Estimation of change points in the kinetics of the 4 circulating miRNAs suggested the changes started 17.5 to 6.8 months prior to HCC detection. These data establish these 4 circulating miRNAs as potential sentinels for preneoplastic lesions or early-stage HCC.

Mitochondrial Effects in the Liver of C57BL/6 Mice by Low Dose, High Energy, High Charge Irradiation.

Galactic cosmic rays are primarily composed of protons (85%), helium (14%), and high charge/high energy ions (HZEs) such as 56Fe, 28Si, and 16O. HZE exposure is a major risk factor for astronauts during deep-space travel due to the possibility of HZE-induced cancer. A systems biology integrated omics approach encompassing transcriptomics, proteomics, lipidomics, and functional biochemical assays was used to identify microenvironmental changes induced by HZE exposure. C57BL/6 mice were placed into six treatment groups and received the following irradiation treatments: 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), 350 MeV/n 28Si (0.2 Gy), 137Cs (1.0 Gy) gamma rays, 137Cs (3.0 Gy) gamma rays, and sham irradiation. Left liver lobes were collected at 30, 60, 120, 270, and 360 days post-irradiation. Analysis of transcriptomic and proteomic data utilizing ingenuity pathway analysis identified multiple pathways involved in mitochondrial function that were altered after HZE irradiation. Lipids also exhibited changes that were linked to mitochondrial function. Molecular assays for mitochondrial Complex I activity showed significant decreases in activity after HZE exposure. HZE-induced mitochondrial dysfunction suggests an increased risk for deep space travel. Microenvironmental and pathway analysis as performed in this research identified possible targets for countermeasures to mitigate risk.

Transcriptomic analysis links hepatocellular carcinoma (HCC) in HZE ion irradiated mice to a human HCC subtype with favorable outcomes.

High-charge, high-energy ion particle (HZE) radiations are extraterrestrial in origin and characterized by high linear energy transfer (high-LET), which causes more severe cell damage than low-LET radiations like γ-rays or photons. High-LET radiation poses potential cancer risks for astronauts on deep space missions, but the studies of its carcinogenic effects have relied heavily on animal models. It remains uncertain whether such data are applicable to human disease. Here, we used genomics approaches to directly compare high-LET radiation-induced, low-LET radiation-induced and spontaneous hepatocellular carcinoma (HCC) in mice with a human HCC cohort from The Cancer Genome Atlas (TCGA). We identified common molecular pathways between mouse and human HCC and discovered a subset of orthologous genes (mR-HCC) that associated high-LET radiation-induced mouse HCC with a subgroup (mrHCC2) of the TCGA cohort. The mrHCC2 TCGA cohort was more enriched with tumor-suppressing immune cells and showed a better prognostic outcome than other patient subgroups.

Effects of Low Dose Space Radiation Exposures on the Splenic Metabolome.

Future space missions will include a return to the Moon and long duration deep space roundtrip missions to Mars. Leaving the protection that Low Earth Orbit provides will unavoidably expose astronauts to higher cumulative doses of space radiation, in addition to other stressors, e.g., microgravity. Immune regulation is known to be impacted by both radiation and spaceflight and it remains to be seen whether prolonged effects that will be encountered in deep space can have an adverse impact on health. In this study, we investigated the effects in the overall metabolism of three different low dose radiation exposures (γ-rays, 16O, and 56Fe) in spleens from male C57BL/6 mice at 1, 2, and 4 months after exposure. Forty metabolites were identified with significant enrichment in purine metabolism, tricarboxylic acid cycle, fatty acids, acylcarnitines, and amino acids. Early perturbations were more prominent in the γ irradiated samples, while later responses shifted towards more prominent responses in groups with high energy particle irradiations. Regression analysis showed a positive correlation of the abundance of identified fatty acids with time and a negative association with γ-rays, while the degradation pathway of purines was positively associated with time. Taken together, there is a strong suggestion of mitochondrial implication and the possibility of long-term effects on DNA repair and nucleotide pools following radiation exposure.

137Cs γ Ray and 28Si Irradiation Induced Murine Hepatocellular Carcinoma Lipid Changes in Liver Assessed by MALDI-MSI Combined with Spatial Shrunken Centroid Clustering Algorithm: A Pilot Study.

Characterization of lipids by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is of great interest because not only are lipids important structural molecules in both the cell and internal organelle membranes, but they are also important signaling molecules. MALDI-MSI combined with spatial image segmentation has been previously used to identify tumor heterogeneities within tissues with distinct anatomical regions such as the brain. However, there has been no systematic study utilizing MALDI-MSI combined with spatial image segmentation to assess the tumor microenvironment in the liver. Here, we present that image segmentation can be used to evaluate the tumor microenvironment in the liver. In particular, to better understand the molecular mechanisms of irradiation-induced hepatic carcinogenesis, we used MALDI-MSI in the negative ion mode to identify lipid changes 12 months post exposure to low dose 28Si and 137Cs γ ray irradiation. We report here the changes in the lipid profiles of male C3H/HeNCrl mice liver tissues after exposure to irradiation and analyzed using the spatial shrunken centroid clustering algorithm. These findings provide valuable information as astronauts will be exposed to high-charge high-energy (HZE) particles and low-energy γ-ray irradiation during deep space travel. Even at low doses, exposure to these irradiations can lead to cancer. Previous studies infer that irradiation of mice with low-dose HZE particles induces oxidative damage and microenvironmental changes that are thought to play roles in the pathophysiology of hepatocellular carcinoma.

Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of 56Fe, 16O, and 28Si irradiation on the induction of murine hepatocellular carcinoma.

BACKGROUND: One of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC. RESULTS: We performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes (e.g., Acute Phase Response Signaling, B Cell Receptor Signaling, IL-3 Signaling), and RNA Transcription/Expression. CONCLUSIONS: A large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC.

Efficient identification of multiple pathways: RNA-Seq analysis of livers from 56Fe ion irradiated mice.

BACKGROUND: mRNA interaction with other mRNAs and other signaling molecules determine different biological pathways and functions. Gene co-expression network analysis methods have been widely used to identify correlation patterns between genes in various biological contexts (e.g., cancer, mouse genetics, yeast genetics). A challenge remains to identify an optimal partition of the networks where the individual modules (clusters) are neither too small to make any general inferences, nor too large to be biologically interpretable. Clustering thresholds for identification of modules are not systematically determined and depend on user-settable parameters requiring optimization. The absence of systematic threshold determination may result in suboptimal module identification and a large number of unassigned features. RESULTS: In this study, we propose a new pipeline to perform gene co-expression network analysis. The proposed pipeline employs WGCNA, a software widely used to perform different aspects of gene co-expression network analysis, and Modularity Maximization algorithm, to analyze novel RNA-Seq data to understand the effects of low-dose 56Fe ion irradiation on the formation of hepatocellular carcinoma in mice. The network results, along with experimental validation, show that using WGCNA combined with Modularity Maximization, provides a more biologically interpretable network in our dataset, than that obtainable using WGCNA alone. The proposed pipeline showed better performance than the existing clustering algorithm in WGCNA, and identified a module that was biologically validated by a mitochondrial complex I assay. CONCLUSIONS: We present a pipeline that can reduce the problem of parameter selection that occurs with the existing algorithm in WGCNA, for applicable RNA-Seq datasets. This may assist in the future discovery of novel mRNA interactions, and elucidation of their potential downstream molecular effects.

Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity.

In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.

Space radiation-associated lung injury in a murine model.

Despite considerable progress in identifying health risks to crewmembers related to exposure to galactic/cosmic rays and solar particle events (SPE) during space travel, its long-term effects on the pulmonary system are unknown. We used a murine risk projection model to investigate the impact of exposure to space-relevant radiation (SR) on the lung. C3H mice were exposed to (137)Cs gamma rays, protons (acute, low-dose exposure mimicking the 1972 SPE), 600 MeV/u (56)Fe ions, or 350 MeV/u (28)Si ions at the NASA Space Radiation Laboratory at Brookhaven National Laboratory. Animals were irradiated at the age of 2.5 mo and evaluated 23.5 mo postirradiation, at 26 mo of age. Compared with age-matched nonirradiated mice, SR exposures led to significant air space enlargement and dose-dependent decreased systemic oxygenation levels. These were associated with late mild lung inflammation and prominent cellular injury, with significant oxidative stress and apoptosis (caspase-3 activation) in the lung parenchyma. SR, especially high-energy (56)Fe or (28)Si ions markedly decreased sphingosine-1-phosphate levels and Akt- and p38 MAPK phosphorylation, depleted anti-senescence sirtuin-1 and increased biochemical markers of autophagy. Exposure to SR caused dose-dependent, pronounced late lung pathological sequelae consistent with alveolar simplification and cellular signaling of increased injury and decreased repair. The associated systemic hypoxemia suggested that this previously uncharacterized space radiation-associated lung injury was functionally significant, indicating that further studies are needed to define the risk and to develop appropriate lung-protective countermeasures for manned deep space missions.

Effects of 28Si ions, 56Fe ions, and protons on the induction of murine acute myeloid leukemia and hepatocellular carcinoma.

Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the carcinogenic effects of 300 MeV/n 28Si or 600 MeV/n 56Fe ions in a mouse model for radiation-induced acute myeloid leukemia and hepatocellular carcinoma. C3H/HeNCrl mice were irradiated with 0.1, 0.2, 0.4, or 1 Gy of 300 MeV/n 28Si ions, 600 MeV/n 56Fe ions or 1 or 2 Gy of protons simulating the 1972 solar particle event (1972SPE) at the NASA Space Radiation Laboratory. Additional mice were irradiated with 137Cs gamma rays at doses of 1, 2, or 3 Gy. All groups were followed until they were moribund or reached 800 days of age. We found that 28Si or 56Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia. However, 28Si or 56Fe ion irradiated mice had a much higher incidence of hepatocellular carcinoma than gamma ray irradiated or proton irradiated mice. These data demonstrate a clear difference in the effects of these HZE ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis. Also seen in this study was an increase in metastatic hepatocellular carcinoma in the 28Si and 56Fe ion irradiated mice compared with those exposed to gamma rays or 1972SPE protons, a finding with important implications for setting radiation exposure limits for space-flight crew members.

Leukemogenesis in heterozygous PU.1 knockout mice.

Most murine radiation-induced acute myeloid leukemias involve biallelic inactivation of the PU.1 gene, with one allele being lost through a radiation-induced chromosomal deletion and the other allele affected by a recurrent point mutation in codon 235 that is likely to be spontaneous. The short latencies of acute myeloid leukemias occurring in nonirradiated mice engineered with PU.1 conditional knockout or knockdown alleles suggest that once both copies of PU.1 have been lost any other steps involved in leukemogenesis occur rapidly. Yet, spontaneous acute myeloid leukemias have not been reported in mice heterozygous for a PU.1 knockout allele, an observation that conflicts with the understanding that the PU.1 codon 235 mutation is spontaneous. Here we describe experiments that show that the lack of spontaneous leukemia in PU.1 heterozygous knockout mice is not due to insufficient monitoring times or mouse numbers or the genetic background of the knockout mice. The results reveal that spontaneous leukemias that develop in mice of the mixed 129S2/SvPas and C57BL/6 background of knockout mice arise by a pathway that does not involve biallelic PU.1 mutation. In addition, the latency of radiation-induced leukemia in PU.1 heterozygous mice on a genetic background susceptible to radiation-induced leukemia indicates that the codon 235 mutation is not a rate-limiting step in radiation leukemogenesis driven by PU.1 loss.

Molecular characterisation of murine acute myeloid leukaemia induced by 56Fe ion and 137Cs gamma ray irradiation.

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

Animal studies of charged particle-induced carcinogenesis.

The distribution of energy deposition in cells and tissues by high-charge, high-energy (HZE) nuclei differs considerably from that of low linear energy transfer (LET) radiation, raising concerns that charged particle exposure may be more efficient in inducing radiogenic cancers or may induce a different spectrum of tumors. The authors have performed a review of charged particle carcinogenesis in animals with the following observations. A limited number of animal studies with carcinogenesis endpoints have been performed to evaluate the effectiveness of HZE ions. These include the induction of skin and mammary tumors in the rat and Harderian gland tumors, acute myeloid leukemia (AML), and hepatocellular carcinomas in the mouse. In general, high relative biological effectiveness (RBE) has been reported for solid tumor induction. RBE dependence on HZE radiation quality has been most extensively characterized in studies of mouse Harderian gland tumorigenesis. In this model, the RBE increases with LET and plateaus in the 193-953 keV µm(-1) range. Unlike the results of solid tumor studies, a leukemogenesis study found 1 GeV nucleon(-1) 56Fe ions no more efficient than gamma-rays for AML induction. No novel tumor types have been observed in HZE irradiated animals as compared with those that occur spontaneously or following low-LET radiation exposures. Genetic background of the irradiated animals is critical; the tumor types induced in HZE irradiated mice depend on their strain background, and the incidence of HZE ion-induced mammary carcinogenesis in the rat is also strain dependent.

Radiation-induced carcinogenesis: mechanistically based differences between gamma-rays and neutrons, and interactions with DMBA.

Different types of ionizing radiation produce different dependences of cancer risk on radiation dose/dose rate. Sparsely ionizing radiation (e.g. γ-rays) generally produces linear or upwardly curving dose responses at low doses, and the risk decreases when the dose rate is reduced (direct dose rate effect). Densely ionizing radiation (e.g. neutrons) often produces downwardly curving dose responses, where the risk initially grows with dose, but eventually stabilizes or decreases. When the dose rate is reduced, the risk increases (inverse dose rate effect). These qualitative differences suggest qualitative differences in carcinogenesis mechanisms. We hypothesize that the dominant mechanism for induction of many solid cancers by sparsely ionizing radiation is initiation of stem cells to a pre-malignant state, but for densely ionizing radiation the dominant mechanism is radiation-bystander-effect mediated promotion of already pre-malignant cell clone growth. Here we present a mathematical model based on these assumptions and test it using data on the incidence of dysplastic growths and tumors in the mammary glands of mice exposed to high or low dose rates of γ-rays and neutrons, either with or without pre-treatment with the chemical carcinogen 7,12-dimethylbenz-alpha-anthracene (DMBA). The model provides a mechanistic and quantitative explanation which is consistent with the data and may provide useful insight into human carcinogenesis.

Murine Prkdc polymorphisms impact DNA-PKcs function.

Polymorphic variants of DNA repair genes can increase the carcinogenic potential of exposure to ionizing radiation. Two single nucleotide polymorphisms (SNPs) in Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), have been identified in BALB/c mice and linked to reduced DNA-PKcs activity and mammary cancer susceptibility. We examined three additional mouse strains to better define the roles of the BALB/c Prkdc SNPs (R2140C and M3844V). One is a congenic strain (C.B6) that has the C57BL/6 Prkdc allele on a BALB/c background, and the other is a congenic strain (B6.C) that has the BALB/c variant Prkdc allele on a C57BL/6 background. We also examined the LEWES mouse strain, which possesses only one of the BALB/c Prkdc SNPs (M3844V). Our results demonstrate that both Prkdc SNPs are responsible for deficient DNA-PKcs protein expression, DNA repair and telomere function, while the LEWES SNP affects only DNA-PKcs expression and repair capacity. These studies provide insight into the separation of function between the two BALB/c SNPs as well as direct evidence that SNPs positioned within Prkdc can significantly influence DNA-PKcs function involving DNA repair capacity, telomere end-capping, and potentially cancer susceptibility.

The balance between initiation and promotion in radiation-induced murine carcinogenesis.

Studies of radiation carcinogenesis in animals allow detailed investigation of how the risk depends on age at exposure and time since exposure and of the mechanisms that determine this risk, e.g., induction of new pre-malignant cells (initiation) and enhanced proliferation of already existing pre-malignant cells (promotion). To assist the interpretation of these patterns, we apply a newly developed biologically based mathematical model to data on several types of solid tumors induced by acute whole-body radiation in mice. The model includes both initiation and promotion and analyzes pre-malignant cell dynamics on two different time scales: comparatively short-term during irradiation and long-term during the entire life span. Our results suggest general mechanistic similarities between radiation carcinogenesis in mice and in human atomic bomb survivors. The excess relative risk (ERR) in mice decreases with age at exposure up to an exposure age of 1 year, which corresponds to mid-adulthood in humans; the pattern for older ages at exposure, for which there is some evidence of increasing ERRs in atomic bomb survivors, cannot be evaluated using the data set analyzed here. Also similar to findings in humans, initiation dominates the ERR at young ages in mice, when there are few background pre-malignant cells, and promotion becomes important at older ages.

Incidence of acute myeloid leukemia and hepatocellular carcinoma in mice irradiated with 1 GeV/nucleon (56)Fe ions.

Abstract Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV (56)Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon (56)Fe ions or (137)Cs gamma rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon (56)Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia (AML). However, (56)Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than gamma-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.

Radiation leukemogenesis in mice: loss of PU.1 on chromosome 2 in CBA and C57BL/6 mice after irradiation with 1 GeV/nucleon 56Fe ions, X rays or gamma rays. Part I. Experimental observations.

Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of gamma-, X- and 56Fe-ion-irradiated mice at various times postirradiation. Although 56Fe ions were initially some two to three times more effective than X or gamma rays in inducing PU.1 deletions, by 1 month postirradiation, the proportions of cells with PU.1 deletions were similar for the HZE particles and the sparsely ionizing radiations. These results indicate that while 56Fe ions are more effective in inducing PU.1 deletions, they are also more effective in causing collateral damage that removes hit cells from the bone marrow. After X, gamma or 56Fe-ion irradiation, AML-resistant C57BL/6 mice have fewer cells with PU.1 deletions than CBA mice, and those cells do not persist in the bone marrow of the C57B6/6 mice. Our findings suggest that quantification of PU.1 deleted bone marrow cells 1 month postirradiation can be used as surrogate for the incidence of radiation-induced AML measured in large-scale mouse studies. If so, PU.1 loss could be used to systematically assess the potential leukemogenic effects of other ions and energies in the space radiation environment.