FAILLA MEMORIAL LECTUREHow We Got Here: One Laboratory's Odyssey in the Field of Radiation-Inducible Genes.

This review focuses on early discoveries that contributed to our understanding and the scope of transcriptional responses after radiation damage. Before the development of modern approaches to assess overall global transcriptomic responses, the idea that mammalian cells could respond to DNA-damaging agents in a manner analogous to bacteria was not generally accepted. To investigate this possibility, the development of technology to identify differentially expressed low-abundance transcripts substantially facilitated our appreciation that DNA damaging agents like UV radiation and subsequently ionizing radiation did in fact produce robust transcriptional responses. Here we focus on our identification and characterization of radiation-inducible genes, and how even early studies on stress gene signaling highlighted the broad scope of transcriptional responses to radiation damage. Since then, the central role of transcriptional responses to radiation injury in maintaining genome integrity has been highlighted in many processes, including cell cycle checkpoint control, resistance to cancer by p53 and other key factors, cell senescence, and metabolism.

Expression of Stem Cell Markers in High-LET Space Radiation-Induced Intestinal Tumors in Apc1638N/+ Mouse Intestine.

Estimation of cancer risk among astronauts planning to undertake future deep-space missions requires understanding the quantitative and qualitative differences in radiogenic cancers after low- and high-LET radiation exposures. Previously, we reported a multifold higher RBE for high-LET radiation-induced gastrointestinal (GI) tumorigenesis in Apc1638N/+ mice. Using the same model system, i.e., Apc1638N/+ mice, here, we report qualitative differences in the cellular phenotype of low- and high-LET radiation-induced GI tumors. Stem cell (SC) phenotypes were identified using BMI1, ALDH1, CD133, DCLK1, MSI1, and LGR5 markers in low (γ-rays)- and high (56Fe)-LET radiation-induced and spontaneous tumors. We also assessed the expression of these markers in the adjacent normal mucosa. All six of these putative SC markers were shown to be overexpressed in tumors compared to the adjacent normal intestinal tissue. A differential SC phenotype for spontaneous and radiogenic intestinal tumors in Apc1638N/+ mice was observed, where the ALDH1, BMI1, CD133, MSI1, and DCLK1 expressing cells were increased, while LGR5 expressing cells were decreased in 56Fe-induced tumors compared to γ-ray-induced and spontaneous tumors. Furthermore, higher ß-catenin activation (marked by nuclear localization) was observed in 56Fe-induced tumors compared to γ and spontaneous tumors. Since differential tumor cell phenotype along with activated ß-catenin may very well affect malignant progression, our findings are relevant to understanding the higher carcinogenic risk of high-LET radiation. This study has implications for the assessment of GI-cancer risk among astronauts, as well as for the estimation of secondary cancer risk among patients receiving hadron therapy, considering that our results indicate increased stemness properties after radiation.

Biomarker integration for improved biodosimetry of mixed neutron + photon exposures.

There is a persistent risk of a large-scale malicious or accidental exposure to ionizing radiation that may affect a large number of people. Exposure will consist of both a photon and neutron component, which will vary in magnitude between individuals and is likely to have profound impacts on radiation-induced diseases. To mitigate these potential disasters, there exists a need for novel biodosimetry approaches that can estimate the radiation dose absorbed by each person based on biofluid samples, and predict delayed effects. Integration of several radiation-responsive biomarker types (transcripts, metabolites, blood cell counts) by machine learning (ML) can improve biodosimetry. Here we integrated data from mice exposed to various neutron + photon mixtures, total 3 Gy dose, using multiple ML algorithms to select the strongest biomarker combinations and reconstruct radiation exposure magnitude and composition. We obtained promising results, such as receiver operating characteristic curve area of 0.904 (95% CI: 0.821, 0.969) for classifying samples exposed to ≥ 10% neutrons vs. < 10% neutrons, and R2 of 0.964 for reconstructing photon-equivalent dose (weighted by neutron relative biological effectiveness) for neutron + photon mixtures. These findings demonstrate the potential of combining various -omic biomarkers for novel biodosimetry.

Impact of GADD45A on Radiation Biodosimetry Using Mouse Peripheral Blood.

High-dose-radiation exposure in a short period of time leads to radiation syndromes characterized by severe acute and delayed organ-specific injury accompanied by elevated organismal morbidity and mortality. Radiation biodosimetry based on gene expression analysis of peripheral blood is a valuable tool to detect exposure to radiation after a radiological/nuclear incident and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation, can potentially obscure the predictive power of the method. GADD45A (Growth arrest and DNA damage-inducible gene a) plays important roles in cell growth control, differentiation, DNA repair, and apoptosis. GADD45A-deficient mice develop an autoimmune disease, similar to human systemic lupus erythematosus, characterized by severe hematological disorders, kidney disease, and premature death. The goal of this study was to elucidate how pre-existing inflammation in mice, induced by GADD45A ablation, can affect radiation biodosimetry. We exposed wild-type and GADD45A knockout male C57BL/6J mice to 7 Gy of X rays and 24 h later RNA was isolated from whole blood and subjected to whole genome microarray and gene ontology analyses. Dose reconstruction analysis using a gene signature trained on gene expression data from irradiated wild-type male mice showed accurate reconstruction of either a 0 Gy or 7 Gy dose with root mean square error of ± 1.05 Gy (R^2 = 1.00) in GADD45A knockout mice. Gene ontology analysis revealed that irradiation of both wild-type and GADD45A-null mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. However, based on their z-score, these pathways were predicted to be more significantly overrepresented in GADD45A-null mice, implying that GADD45A deletion may exacerbate the deleterious effects of radiation on blood cells. Numerous immune cell functions and quantities were predicted to be underrepresented in both genotypes; however, differentially expressed genes from irradiated GADD45A knockout mice predicted an increased deterioration in the numbers of T lymphocytes, as well as myeloid cells, compared with wild-type mice. Furthermore, an overrepresentation of genes associated with radiation-induced hematological malignancies was associated with GADD45A knockout mice, whereas hematopoietic and progenitor cell functions were predicted to be downregulated in irradiated GADD45A knockout mice. In conclusion, despite the significant differences in gene expression between wild-type and GADD45A knockout mice, it is still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of pre-existing inflammation status.

High-LET-Radiation-Induced Persistent DNA Damage Response Signaling and Gastrointestinal Cancer Development.

Ionizing radiation (IR) dose, dose rate, and linear energy transfer (LET) determine cellular DNA damage quality and quantity. High-LET heavy ions are prevalent in the deep space environment and can deposit a much greater fraction of total energy in a shorter distance within a cell, causing extensive DNA damage relative to the same dose of low-LET photon radiation. Based on the DNA damage tolerance of a cell, cellular responses are initiated for recovery, cell death, senescence, or proliferation, which are determined through a concerted action of signaling networks classified as DNA damage response (DDR) signaling. The IR-induced DDR initiates cell cycle arrest to repair damaged DNA. When DNA damage is beyond the cellular repair capacity, the DDR for cell death is initiated. An alternative DDR-associated anti-proliferative pathway is the onset of cellular senescence with persistent cell cycle arrest, which is primarily a defense mechanism against oncogenesis. Ongoing DNA damage accumulation below the cell death threshold but above the senescence threshold, along with persistent SASP signaling after chronic exposure to space radiation, pose an increased risk of tumorigenesis in the proliferative gastrointestinal (GI) epithelium, where a subset of IR-induced senescent cells can acquire a senescence-associated secretory phenotype (SASP) and potentially drive oncogenic signaling in nearby bystander cells. Moreover, DDR alterations could result in both somatic gene mutations as well as activation of the pro-inflammatory, pro-oncogenic SASP signaling known to accelerate adenoma-to-carcinoma progression during radiation-induced GI cancer development. In this review, we describe the complex interplay between persistent DNA damage, DDR, cellular senescence, and SASP-associated pro-inflammatory oncogenic signaling in the context of GI carcinogenesis.

Variable Dose Rates in Realistic Radiation Exposures: Effects on Small Molecule Markers of Ionizing Radiation in the Murine Model.

Novel biodosimetry assays for use in preparedness and response to potential malicious attacks or nuclear accidents would ideally provide accurate dose reconstruction independent of the idiosyncrasies of a complex exposure to ionizing radiation. Complex exposures will consist of dose rates spanning the low dose rates (LDR) to very high-dose rates (VHDR) that need to be tested for assay validation. Here, we investigate how a range of relevant dose rates affect metabolomic dose reconstruction at potentially lethal radiation exposures (8 Gy in mice) from an initial blast or subsequent fallout exposures compared to zero or sublethal exposures (0 or 3 Gy in mice) in the first 2 days, which corresponds to an integral time individuals will reach medical facilities after a radiological emergency. Biofluids (urine and serum) were collected from both male and female 9-10-week-old C57BL/6 mice at 1 and 2 days postirradiation (total doses of 0, 3 or 8 Gy) after a VHDR of 7 Gy/s. Additionally, samples were collected after a 2-day exposure consisting of a declining dose rate (1 to 0.004 Gy/min) recapitulating the 7:10 rule-of-thumb time dependency of nuclear fallout. Overall similar perturbations were observed in both urine and serum metabolite concentrations irrespective of sex or dose rate, with the exception of xanthurenic acid in urine (female specific) and taurine in serum (VHDR specific). In urine, we developed identical multiplex metabolite panels (N6, N6,N6-trimethyllysine, carnitine, propionylcarnitine, hexosamine-valine-isoleucine, and taurine) that could identify individuals receiving potentially lethal levels of radiation from the zero or sublethal cohorts with excellent sensitivity and specificity, with creatine increasing model performance at day 1. In serum, individuals receiving a 3 or 8 Gy exposure could be identified from their pre-irradiation samples with excellent sensitivity and specificity, however, due to a lower dose response the 3 vs. 8 Gy groups could not be distinguished from each other. Together with previous results, these data indicate that dose-rate-independent small molecule fingerprints have potential in novel biodosimetry assays.

Simulated galactic cosmic radiation (GCR)-induced expression of Spp1 coincide with mammary ductal cell proliferation and preneoplastic changes in ApcMin/+ mouse.

Female astronauts inevitably exposed to galactic cosmic radiation (GCR) are considered at a greater risk for mammary cancer development. The purpose of this study is to assess the status of mammary cancer-associated preneoplasia markers after GCR and γ-ray irradiation using a mouse model of human mammary cancer. Female ApcMin/+ mice were irradiated to 50 cGy of either γ-ray (137Cs) or full-spectrum simulated galactic cosmic radiation (GCR) (33-beam), and at 110 - 120 days post-irradiation mice were euthanized, and normal-appearing mammary tissues were analyzed for histological and molecular markers of preneoplasia. Whole-mount staining, hematoxylin and eosin-based histological assessment, and Cyclin D1 immunohistochemistry (IHC) were performed to analyze ductal outgrowth and cell proliferation. Additionally, mRNA expression of known mammary preneoplasia markers (Muc1, Exo1, Foxm1, Depdc1a, Nusap1, Spp1, and Rrm2) was analyzed using qPCR, and their respective protein expression was validated using immunohistochemistry. A significant increase in ductal outgrowth and cell proliferation in mammary tissues of GCR-irradiated mice was noted which indicates a higher risk of mammary cancer, relative to γ-rays. Increased mRNA and protein expression of Spp1 was observed in the GCR group, relative to γ-rays. This study demonstrates the plausibility of Spp1 as a preneoplasia marker in the early detection of mammary cancer after space radiation exposure.

Galactic cosmic ray simulation at the NASA space radiation laboratory - Progress, challenges and recommendations on mixed-field effects.

For missions beyond low Earth orbit to the moon or Mars, space explorers will encounter a complex radiation field composed of various ion species with a broad range of energies. Such missions pose significant radiation protection challenges that need to be solved in order to minimize exposures and associated health risks. An innovative galactic cosmic ray simulator (GCRsim) was recently developed at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). The GCRsim technology is intended to represent major components of the space radiation environment in a ground analog laboratory setting where it can be used to improve understanding of biological risks and serve as a testbed for countermeasure development and validation. The current GCRsim consists of 33 energetic ion beams that collectively simulate the primary and secondary GCR field encountered by humans in space over the broad range of particle types, energies, and linear energy transfer (LET) of interest to health effects. A virtual workshop was held in December 2020 to assess the status of the NASA baseline GCRsim. Workshop attendees examined various aspects of simulator design, with a particular emphasis on beam selection strategies. Experimental results, modeling approaches, areas of consensus, and questions of concern were also discussed in detail. This report includes a summary of the GCRsim workshop and a description of the current status of the GCRsim. This information is important for future advancements and applications in space radiobiology.

Low dose radiation upregulates Ras/p38 and NADPH oxidase in mouse colon two months after exposure.

BACKGROUND: Exposure to ionizing is known to cause persistent cellular oxidative stress and NADPH oxidase (Nox) is a major source of cellular oxidant production. Chronic oxidative stress is associated with a myriad of human diseases including gastrointestinal cancer. However, the roles of NADPH oxidase in relation of long-term oxidative stress in colonic epithelial cells after radiation exposure are yet to be clearly established. METHODS AND RESULTS: Mice were exposed either to sham or to 0.5 Gy γ radiation, and NADPH oxidase, oxidative stress, and related signaling pathways were assessed in colon samples 60 days after exposure. Radiation exposure led to increased expression of colon-specific NADPH oxidase isoform, Nox1, as well as upregulation of its modifiers such as Noxa1 and Noxo1 at the mRNA and protein level. Co-immunoprecipitation experiments showed enhanced binding of Rac1, an activator of NADPH oxidase, to Nox1. Increased 4-hydroxynonenal, 8-oxo-dG, and γH2AX along with higher protein carbonylation levels suggest increased oxidative stress after radiation exposure. Immunoblot analysis demonstrates upregulation of Ras/p38 pathway, and Gata6 and Hif1α after irradiation. Increased staining of ß-catenin, cyclinD1, and Ki67 after radiation was also observed. CONCLUSIONS: In summary, data show that exposure to a low dose of radiation was associated with upregulation of NADPH oxidase and its modifiers along with increased Ras/p38/Gata6 signaling in colon. When considered along with oxidative damage and proliferative markers, our observations suggest that the NADPH oxidase pathway could be playing a critical role in propagating long-term oxidative stress after radiation with implications for colon carcinogenesis.

White-Nose Syndrome Disrupts the Splenic Lipidome of Little Brown Bats (Myotis lucifugus) at Early Disease Stages.

White-nose syndrome (WNS)-positive little brown bats (Myotis lucifugus) may exhibit immune responses including increased cytokine and pro-inflammatory mediator gene levels. Bioactive lipid mediators (oxylipins) formed by enzymatic oxidation of polyunsaturated fatty acids can contribute to these immune responses, but have not been investigated in WNS pathophysiology. Nonenzymatic conversion of polyunsaturated fatty acids can also occur due to reactive oxygen species, however, these enantiomeric isomers will lack the same signaling properties. In this study, we performed a series of targeted lipidomic approaches on laboratory Pseudogymnoascus destructans-inoculated bats to assess changes in their splenic lipidome, including the formation of lipid mediators at early stages of WNS. Hepatic lipids previously identified were also resolved to a higher structural detail. We compared WNS-susceptible M. lucifugus to a WNS-resistant species, the big brown bat (Eptesicus fuscus). Altered splenic lipid levels were only observed in M. lucifugus. Differences in splenic free fatty acids included both omega-3 and omega-6 compounds. Increased levels of an enantiomeric monohydroxy DHA mixture were found, suggesting nonenzymatic formation. Changes in previously identified hepatic lipids were confined to omega-3 constituents. Together, these results suggest that increased oxidative stress, but not an inflammatory response, is occurring in bats at early stages of WNS that precedes fat depletion. These data have been submitted to metabolomics workbench and assigned a study number ST002304.

Heavy-ion radiation-induced colitis and colorectal carcinogenesis in Il10-/- mice display co-activation of ß-catenin and NF-κB signaling.

Space radiation-induced gastrointestinal (GI) cancer risk models for future interplanetary astronauts are being developed that primarily rely on quantitative animal model studies to assess radiation-quality effects of heavy-ion space radiation exposure in relation to γ-rays. While current GI-cancer risk estimation efforts are focused on sporadic GI-cancer mouse models, emerging in-vivo data on heavy-ion radiation-induced long-term GI-inflammation are indicative of a higher but undetermined risk of GI-inflammation associated cancers, such as colitis-associated cancer (CAC). Therefore, we aimed to assess radiation quality effects on colonic inflammation, colon cancer incidence, and associated signaling events using an in-vivo CAC model i.e., Il10-/- mice. Male Il10-/- mice (8-10 weeks, n = 12/group) were irradiated with either sham, γ-rays or heavy-ions (28Si or 56Fe), and histopathological assessments for colitis and CAC were conducted at 2.5 months post-exposure. qPCR analysis for inflammation associated gene transcripts (Ptges and Tgfb1), and in-situ staining for markers of cell-proliferation (phospho-histone H3), oncogenesis (active-ß-catenin, and cyclin D1), and inflammation (phospho-p65NF-κB, iNOS, and COX2) were performed. Significantly higher colitis and CAC frequency were noted after heavy-ion exposure, relative to γ and control mice. Higher CAC incidence after heavy-ion exposure was associated with greater activation of ß-catenin and NF-κB signaling marked by induced expression of common downstream inflammatory (iNOS and COX2) and pro-proliferative (Cyclin D1) targets. In summary, IR-induced colitis and CAC incidence in Il10-/- mice depends on radiation quality and display co-activation of ß-catenin and NF-κB signaling.

Countermeasure development against space radiation-induced gastrointestinal carcinogenesis: Current and future perspectives.

A significantly higher probability of space radiation-induced gastrointestinal (GI) cancer incidence and mortality after a Mars mission has been projected using biophysical and statistical modeling approaches, and may exceed the current NASA mandated limit of less than 3% REID (risk of exposure-induced death). Since spacecraft shielding is not fully effective against heavy-ion space radiation, there is an unmet need to develop an effective medical countermeasure (MCM) strategy against heavy-ion space radiation-induced GI carcinogenesis to safeguard astronauts. In the past, we have successfully applied a GI cancer mouse model approach to understand space radiation-induced GI cancer risk and associated molecular signaling events. We have also tested several potential MCMs to safeguard astronauts during and after a prolonged space mission. In this review, we provide an updated summary of MCM testing using the GI cancer mouse model approach, lessons learned, and a perspective on the senescence signaling targeting approach for desirable protection against space radiation-induced GI carcinogenesis. Furthermore, we also discuss some of the advanced senotherapeutic candidates/combinations as a potential MCM for space radiation-induced GI carcinogenesis.

Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells.

Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq®). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT's mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.

Predominant contribution of the dose received from constituent heavy-ions in the induction of gastrointestinal tumorigenesis after simulated space radiation exposure.

Gastrointestinal (GI) cancer risk among astronauts after encountering galactic cosmic radiation (GCR) is predicted to exceed safe permissible limits in long duration deep-space missions. Current predictions are based on relative biological effectiveness (RBE) values derived from in-vivo studies using single-ion beams, while GCR is essentially a mixed radiation field composed of protons (H), helium (He), and heavy ions. Therefore, a sequentially delivered proton (H) → Helium (He) → Oxygen (O) → Silicon (Si) beam was designed to simulate simplified-mixed-field GCR (Smf-GCR), and Apc1638N/+ mice were total-body irradiated to sham or γ (157Cs) or Smf-GCR followed by assessment of GI-tumorigenesis at 150 days post-exposure. Further, GI-tumor data from equivalent doses of heavy-ions (i.e., 0.05 Gy of O and Si) in 0.5 Gy of Smf-GCR were compared to understand the contributions of heavy-ions in GI-tumorigenesis. The Smf-GCR-induced tumor and carcinoma count were significantly greater than γ-rays, and male preponderance for GI-tumorigenesis was consistent with our earlier findings. Comparison of tumor data from Smf-GCR and equivalent doses of heavy ions revealed an association between higher GI-tumorigenesis where dose received from heavy-ions contributed to > 95% of the total GI-tumorigenic effect observed after Smf-GCR. This study provides the first experimental evidence that cancer risk after GCR exposure could largely depend on doses received from constituent heavy-ions.

Crohn's disease in endoscopic remission, obesity, and cases of high genetic risk demonstrates overlapping shifts in the colonic mucosal-luminal interface microbiome.

BACKGROUND: Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype. METHODS: Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry. RESULTS: CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls. CONCLUSIONS: Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.

Biofluid Metabolomics and Lipidomics of Mice Exposed to External Very High-Dose Rate Radiation.

High-throughput biodosimetry methods to determine exposure to ionizing radiation (IR) that can also be easily scaled to multiple testing sites in emergency situations are needed in the event of malicious attacks or nuclear accidents that may involve a substantial number of civilians. In the event of an improvised nuclear device (IND), a complex IR exposure will have a very high-dose rate (VHDR) component from an initial blast. We have previously addressed low-dose rate (LDR, ≤1 Gy/day) exposures from internal emitters on biofluid small molecule signatures, but further research on the VHDR component of the initial blast is required. Here, we exposed 8- to 10-week-old male C57BL/6 mice to an acute dose of 3 Gy using a reference dose rate of 0.7 Gy/min or a VHDR of 7 Gy/s, collected urine and serum at 1 and 7 d, then compared the metabolite signatures using either untargeted (urine) or targeted (serum) approaches with liquid chromatography mass spectrometry platforms. A Random Forest classification approach showed strikingly similar changes in urinary signatures at 1 d post-irradiation with VHDR samples grouping closer to control samples at 7 d. Identical metabolite panels (carnitine, trigonelline, xanthurenic acid, N6,N6,N6-trimethyllysine, spermine, and hexosamine-valine-isoleucine-OH) could differentiate IR exposed individuals with high sensitivity and specificity (area under the receiver operating characteristic (AUROC) curves 0.89-1.00) irrespective of dose rate at both days. For serum, the top 25 significant lipids affected by IR exposure showed slightly higher perturbations at 0.7 Gy/min vs. 7 Gy/s; however, identical panels showed excellent sensitivity and specificity at 1 d (three hexosylceramides (16:0), (18:0), (24:0), sphingomyelin [26:1], lysophosphatidylethanolamine [22:1]). Mice could not be differentiated from control samples at 7 d for a 3 Gy exposure based on serum lipid signatures. As with LDR exposures, we found that identical biofluid small molecule signatures can identify IR exposed individuals irrespective of dose rate, which shows promise for more universal applications of metabolomics for biodosimetry.

GADD45 in Stress Signaling, Cell Cycle Control, and Apoptosis.

GADD45 is a gene family consisting of GADD45A, GADD45B, and GADD45G that is often induced by DNA damage and other stress signals associated with growth arrest and apoptosis. Many of these roles are carried out via signaling mediated by p38 mitogen-activated protein kinases (MAPKs). The GADD45 proteins can contribute to p38 activation either by activation of upstream kinase(s) or by direct interaction, as well as suppression of p38 activity in certain cases. In vivo, there are important tissue and cell type specific differences in the roles for GADD45 in MAPK signaling. In addition to being p53-regulated, GADD45A has also been found to contribute to p53 activation via p38. Like other stress and signaling proteins, GADD45 proteins show complex regulation and numerous effectors. More recently, aberrant GADD45 expression has been found in several human cancers, but the mechanisms behind these findings largely remain to be understood.

Small Molecule Signatures of Mice Lacking T-cell p38 Alternate Activation, a Model for Immunosuppression Conditions, after Total-Body Irradiation.

Several diagnostic biodosimetry tools have been in development that may aid in radiological/nuclear emergency responses. Of these, correlating changes in non-invasive biofluid small-molecule signatures to tissue damage from ionizing radiation exposure show promise for inclusion in predictive biodosimetry models. Integral to dose reconstruction has been determining how genotypic variation in the general population will affect model performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway [p38αßY323F, double knock-in (DKI) mice] to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. We exposed adult male DKI mice (8-10 weeks old) to 2 and 7 Gy in parallel with wild-type mice and assessed perturbations in urine (days 1, 3, 7) and serum (day 1) using a global metabolomics approach. A multidimensional scaling plot showed excellent separation of radiation-exposed groups in wild-type mice with slightly dampened responses in DKI mice. Validated metabolite panels were developed for urine [N6,N6,N6-trimethyllysine (TML), N1-acetylspermidine, spermidine, carnitine, acylcarnitine C21H35NO5, aminohippuric acid] and serum [phenylalanine, glutamine, propionylcarnitine, lysophosphatidylcholine (LysoPC 14:0), LysoPC (22:5)] to determine the area under the receiver operating characteristic curve (AUROC). For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤2 Gy vs. 7 Gy mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes after irradiation. Overall, these results suggest immunosuppression should not compromise small molecule multiplex panels used in dose reconstruction for biodosimetry.

Effect of the p38 Mitogen-Activated Protein Kinase Signaling Cascade on Radiation Biodosimetry.

Radiation biodosimetry based on transcriptomic analysis of peripheral blood is a valuable tool to detect radiation exposure after a radiological/nuclear event and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation or immune suppression, can potentially obscure the predictive power of the method. Members of the p38 mitogen-activated protein kinase (MAPK) family respond to pro-inflammatory signals and environmental stresses, whereas genetic ablation of the p38 signaling pathway in mice leads to reduced susceptibility to collagen-induced arthritis and experimental autoimmune encephalomyelitis that model human rheumatoid arthritis and multiple sclerosis, respectively. p38 is normally regulated by the MAP3K-MAP2K pathway in mammalian cells. However, in T cells there is an alternative pathway for p38 activation that plays an important role in antigen-receptor-activated T cells and participates in immune and inflammatory responses. To examine the role of p38 in response to radiation, we used two mouse models expressing either a p38α dominant negative (DN) mutation that globally suppresses p38 signaling or a p38αß double-knock-in (DKI) mutant, which inhibits specifically T-cell receptor activation. We exposed p38 wild-type (p38WT) and mutant male mice to 7 Gy X rays and 24 h later whole blood was isolated subjected to whole-genome microarray and gene ontology analysis. Irradiation of p38WT mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. In contrast, these pathways were significantly underrepresented in p38DN and p38DKI mutant mice, suggesting that p38 attenuation may protect blood cells from the deleterious effects of radiation. Furthermore, radiation exposure in p38 mutant mice resulted in an enrichment of phagocytosis-related pathways, suggesting a role for p38 signaling in restricting phagocytosis of apoptotic cells after irradiation. Finally, despite the significant changes in gene expression, it was still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of p38 status.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow.

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/µm), or high-LET 56Fe-ions (148 keV/µm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and 56Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, 56Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.