Pluripotent stem cells (PSCs) hold great promise in regenerative medicine, disease modeling, functional genomics, toxicological studies and cell-based therapeutics due to their unique characteristics of self-renewal and pluripotency. Novel methods for generation of pluripotent stem cells and their differentiation to the specialized cell types such as neuronal cells, myocardial cells, hepatocytes and beta cells of the pancreas and many other cells of the body are constantly being refined. Pluripotent stem cell derived differentiated cells, including neuronal cells or cardiac cells, are ideal for stem cell transplantation as autologous or allogeneic cells from healthy donors due to their minimal risk of rejection. Radiation-induced DNA damage, ultraviolet light, genotoxic stress and other intrinsic and extrinsic factors triggers a series of biochemical reactions known as DNA damage response. To maintain genomic stability and avoid transmission of mutations into progenitors cells, stem cells have robust DNA damage response signaling, a contrast to somatic cells. Stem cell transplantation may protect against radiation-induced late effects. In particular, this review focuses on differential DNA damage response between stem cells and derived differentiated cells and the possible pathways that determine such differences.

Estimating cancer risk from space radiation has been an ongoing challenge for decades primarily because most of the reported epidemiological data on radiation-induced risks are derived from studies of atomic bomb survivors who were exposed to an acute dose of gamma rays instead of chronic high-LET cosmic radiation. In this study, we introduce a formalism using cellular automata to model the long-term effects of ionizing radiation in human breast for different radiation qualities. We first validated and tuned parameters for an automata-based two-stage clonal expansion model simulating the age dependence of spontaneous breast cancer incidence in an unexposed U.S. population. We then tested the impact of radiation perturbation in the model by modifying parameters to reflect both targeted and nontargeted radiation effects. Targeted effects (TE) reflect the immediate impact of radiation on a cell's DNA with classic end points being gene mutations and cell death. They are well known and are directly derived from experimental data. In contrast, nontargeted effects (NTE) are persistent and affect both damaged and undamaged cells, are nonlinear with dose and are not well characterized in the literature. In this study, we introduced TE in our model and compared predictions against epidemiologic data of the atomic bomb survivor cohort. TE alone are not sufficient for inducing enough cancer. NTE independent of dose and lasting ∼100 days postirradiation need to be added to accurately predict dose dependence of breast cancer induced by gamma rays. Finally, by integrating experimental relative biological effectiveness (RBE) for TE and keeping NTE (i.e., radiation-induced genomic instability) constant with dose and LET, the model predicts that RBE for breast cancer induced by cosmic radiation would be maximum at 220 keV/μm. This approach lays the groundwork for further investigation into the impact of chronic low-dose exposure, inter-individual variation and more complex space radiation scenarios.

We implemented a two-stage study to predict late occurring hematologic acute radiation syndrome (HARS) in a baboon model based on gene expression changes measured in peripheral blood within the first two days after irradiation. Eighteen baboons were irradiated to simulate different patterns of partial-body and total-body exposure, which corresponded to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts the surviving baboons (n = 17) exhibited mild (H1–2, n = 4) or more severe (H2–3, n = 13) HARS. Blood samples taken before irradiation served as unexposed control (H0, n = 17). For stage I of this study, a whole genome screen (mRNA microarrays) was performed using a portion of the samples (H0, n = 5; H1–2, n = 4; H2–3, n = 5). For stage II, using the remaining samples and the more sensitive methodology, qRT-PCR, validation was performed on candidate genes that were differentially up- or down-regulated during the first two days after irradiation. Differential gene expression was defined as significant (P < 0.05) and greater than or equal to a twofold difference above a H0 classification. From approximately 20,000 genes, on average 46% appeared to be expressed. On day 1 postirradiation for H2–3, approximately 2–3 times more genes appeared up-regulated (1,418 vs. 550) or down-regulated (1,603 vs. 735) compared to H1–2. This pattern became more pronounced at day 2 while the number of differentially expressed genes decreased. The specific genes showed an enrichment of biological processes coding for immune system processes, natural killer cell activation and immune response (P = 1 × E-06 up to 9 × E-14). Based on the P values, magnitude and sustained differential gene expression over time, we selected 89 candidate genes for validation using qRT-PCR. Ultimately, 22 genes were confirmed for identification of H1–3 classifications and seven genes for identification of H2–3 classifications using qRT-PCR. For H1–3 classifications, most genes were constantly three to fivefold down-regulated relative to H0 over both days, but some genes appeared 10.3-fold (VSIG4) or even 30.7-fold up-regulated (CD177) over H0. For H2–3, some genes appeared four to sevenfold up-regulated relative to H0 (RNASE3, DAGLA, ARG2), but other genes showed a strong 14- to 33-fold down-regulation relative to H0 (WNT3, POU2AF1, CCR7). All of these genes allowed an almost completely identifiable separation among each of the HARS categories. In summary, clinically relevant HARS can be independently predicted with all 29 irradiated genes examined in the peripheral blood of baboons within the first two days postirradiation. While further studies are needed to confirm these findings, this model shows potential relevance in the prediction of clinical outcomes in exposed humans and as an aid in the prioritizing of medical treatment.

Heart disease is an increasingly recognized, serious late effect of radiation exposure, most notably among breast cancer and Hodgkin's disease survivors, as well as the Hiroshima and Nagasaki atomic bomb survivors. The purpose of this study was to evaluate the late effects of total-body irradiation (TBI) on cardiac morphology, function and selected circulating biomarkers in a well-established nonhuman primate model. For this study we used male rhesus macaques that were exposed to a single total-body dose of ionizing gamma radiation (6.5–8.4 Gy) 5.6–9.7 years earlier at ages ranging from ∼3–10 years old and a cohort of nonirradiated controls. Transthoracic echocardiography was performed annually for 3 years on 20 irradiated and 11 control animals. Myocardium was examined grossly and histologically, and myocardial fibrosis/collagen was assessed microscopically and by morphometric analysis of Masson's trichrome-stained sections. Serum/plasma from 27 irradiated and 13 control animals was evaluated for circulating biomarkers of cardiac damage [N-terminal pro B-type natriuretic protein (nt-proBNP) and troponin-I], inflammation (CRP, IL-6, MCP-1, sICAM) and microbial translocation [LPS-binding protein (LBP) and sCD14]. A higher prevalence of histological myocardial fibrosis was observed in the hearts obtained from the irradiated animals (9/14) relative to controls (0/3) (P = 0.04, χ²). Echocardiographically determined left ventricular end diastolic and systolic diameters were significantly smaller in irradiated animals (repeated measures ANOVA, P < 0.001 and P < 0.008, respectively). Histomorphometric analysis of trichrome-stained sections of heart tissue demonstrated ∼14.9 ± 1.4% (mean ± SEM) of myocardial area staining for collagen in irradiated animals compared to 9.1 ± 0.9 % in control animals. Circulating levels of MCP-1 and LBP were significantly higher in irradiated animals (P < 0.05). A high incidence of diabetes in the irradiated animals was associated with higher plasma triglyceride and lower HDLc but did not appear to be associated with cardiovascular phenotypes. These results demonstrate that single total-body doses of 6.5–8.4 Gy produced long-term effects including a high incidence of myocardial fibrosis, reduced left ventricular diameter and elevated systemic inflammation. Additional prospective studies are required to define the time course and mechanisms underlying radiation-induced heart disease in this model.

The goal of this study was to determine whether in vivo X irradiation induces nontargeted effects, such as delayed effects and bystander effects in ICR mouse lymphocytes. We first examined the generation of DNA double-strand breaks (DSBs) in lymphocytes, isolated from ICR mice exposed to 1 Gy X irradiation, by enumeration of p53 binding protein 1 (53BP1) foci, and observed that the number of 53BP1 foci reached their maximum 3 days postirradiation and decreased to background level 30 days postirradiation. However, the number of 53BP1 foci was significantly increased in lymphocytes isolated from ICR mice 90–365 days postirradiation. This result indicates that in vivo X irradiation induced delayed DSBs in ICR mouse lymphocytes. We next counted the number of 53BP1 foci in lymphocytes isolated from sham-irradiated ICR mice that had been co-cultured with lymphocytes isolated from 1 Gy X-irradiated ICR mice, and observed a significant increase in the number of 53BP1 foci 1–7 days postirradiation. This result indicates that in vivo X irradiation induced bystander effects in ICR mouse lymphocytes. These findings suggest that in vivo X irradiation induces early and delayed nontargeted effects in ICR mouse lymphocytes.

Animal models of hematopoietic and gastrointestinal acute radiation syndromes (ARS) have been characterized to develop medical countermeasures. Acute radiation-induced decrease of intestinal absorptive function has been correlated to a decrease in the number of intestinal crypt cells resulting from apoptosis and enterocyte mass reduction. Citrulline, a noncoded amino acid, is produced almost exclusively by the enterocytes of the small intestine. Citrullinemia has been identified as a simple, sensitive and suitable biomarker for radiation-induced injury associated with gastrointestinal ARS (GI-ARS). Here we discuss the effect of radiation on plasma citrulline levels in three different species, C57BL/6 mice, Göttingen minipigs and rhesus nonhuman primates (NHPs), measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of experimental study conditions such as feeding and anesthesia were also examined on plasma citrulline levels in the NHPs. Both the mice and Göttingen minipigs were partial-body irradiated (PBI) with doses from 13–17 Gy and 8–16 Gy, respectively, whereas NHPs were total-body irradiated (TBI) with doses from 6.72–13 Gy. Blood samples were taken at different time points and plasma citrulline levels were measured in the three species at baseline and after irradiation. Basal plasma citrulline concentrations (mean ± SEM) in mice and minipigs were 57.8 ± 2.8 μM and 63.1 ± 2.1 μM, respectively. NHPs showed a basal plasma citrulline concentration of 32.6 ± 0.7 μM, very similar to that of humans (∼40 μM). Plasma citrulline progressively decreased after irradiation, reaching nadir values between day 3.5 and 7. The onset of citrulline recovery was observed earlier at lower radiation doses, while only partial citrulline recovery was noted at higher radiation doses in minipigs and NHPs, complete recovery was noted in mice at all doses. Plasma citrulline levels in NHPs anesthetized with ketamine and acepromazine significantly decreased by 35.5% (P = 0.0017), compared to unanesthetized NHPs. In the postprandial state, citrulline concentrations in NHPs were slightly but significantly decreased by 12.2% (P = 0.0287). These results suggest that plasma citrulline is affected by experimental conditions such as anesthesia and feeding.

The extracellular microenvironment affects cellular responses to various stressors including radiation. Annexin A2, which was initially identified as an intracellular molecule, is also released into the extracellular environment and is known to regulate diverse cell surface events, however, the molecular mechanisms underlying its release are not well known. In this study, we found that in cultured human cancer and non-cancerous cells an extracellular release of annexin A2 was greatly enhanced 1–4 h after a single 20 cGy X-ray dose, but not after exposure to ultraviolet C (UVC) radiation. Extracellular release of annexin A2 was also enhanced after H₂O₂ and nicotine treatments, which was suppressed by pretreatment with the antioxidant, N-acetyl cysteine. Among the oxidative stress pathway molecules examined in HeLa cells, AMP-activated protein kinase α (AMPKα) and p38 mitogen-activated protein kinase (MAPK) were mostly activated by low-dose X-ray radiation, and the p38 MAPK inhibitor, SB203580, but not compound C (an AMPKα inhibitor), suppressed the enhancement of the annexin A2 extracellular release after low-dose X irradiation. In addition, the enhancement was suppressed in the cells in which p38α MAPK was downregulated by siRNA. HeLa cells and human cultured cells preirradiated with 20 cGy or precultured in media from low-dose X-irradiated cells showed an increase in resistance to radiation-induced cell death, and the increase was suppressed by treatment of the irradiated cell-derived media with anti-annexin A2 antibodies. In addition, extracellularly added recombinant annexin A2 conferred cellular radiation resistance. These results indicate that an oxidative stress-activated pathway via p38 MAPK was involved in the extracellular release of annexin A2, and this pathway was stimulated by low-dose X-ray irradiation. Furthermore, released annexin A2 may function in low-dose ionizing radiation-induced responses, such as radioresistance.

Metabolomic analysis of easily accessible biofluids has provided numerous biomarkers in urine and blood for biodosimetric purposes. In this pilot study we assessed saliva for its utility in biodosimetry using a mouse model. Mice were exposed to 0.5, 3 and 8 Gy total-body gamma irradiation and saliva was collected on day 1 and 7 postirradiation. Global metabolomic profiling was conducted through liquid chromatography mass spectrometry and metabolites were positively identified using tandem mass spectrometry. Multivariate data analysis revealed distinct metabolic profiles for all groups at day 1, whereas at day 7 the two lower dose profiles appeared to have minimal differences. Metabolites that were identified include amino acids and fatty acids, and intermediates of the nicotinate and nicotinamide metabolism. The specificity and sensitivity of the radiation signature, as expected, was higher for the 8 Gy dose at both time points, as determined through generation of receiver operating characteristic curves. To the best of our knowledge, this is the first metabolomics study in saliva of irradiated mice to demonstrate the utility of this biofluid as a potential matrix for identification of radiation and dose-specific biomarkers.