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The search for medical treatments to prevent radiation-induced damage to gastrointestinal tissue is crucial as such injuries can be fatal. This study aimed to investigate the effects of apigenin (AP) on the gut microbiome of irradiated mice, as it is a promising radiation countermeasure. Male C57BL/6J mice were divided into four groups, with six mice in each group. Two groups were given food with apigenin (20 mg/kg body weight or AP 20) before and after exposure to 0 or 50 cGy of silicon (28Si) ions, while another two groups of mice received regular diet without apigenin (0 mg/kg body weight or AP 0) before and after irradiation. The duodenum, the primary site for oral AP absorption, was collected from each mouse seven days after radiation exposure. Using 16S rRNA amplicon sequencing, we found significant differences in microbial diversity among groups. Firmicutes and Bacteroidetes were the major phyla for all groups, while actinobacterial and proteobacterial sequences represented only a small percentage. Mice not given dietary apigenin had a higher Firmicutes and Bacteroidetes (F/B) ratio and an imbalanced duodenal microbiota after exposure to radiation, while irradiated mice given apigenin had maintained homeostasis of the microbiota. Additionally, irradiated mice not given apigenin had decreased probiotic bacteria abundance and increased inflammation, while apigenin-supplemented mice had reduced inflammation and restored normal histological structure. In conclusion, our results demonstrate the potential of dietary apigenin as a countermeasure against radiation-induced gut injuries due to its anti-inflammatory activity, reduction of gut microbiota dysbiosis, and increase in probiotic bacteria (e.g., Lachnospiraceae, Muribaculaceae and Bifidobacteriaceae)., (©2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Purpose: To determine the early- and late-occurring damage in the bone marrow (BM) and peripheral blood cells of male CBA/Ca mice after exposure to 0, 0.1, 0.25, or 0.5 Gy of 1 GeV/n titanium ( 48 Ti) ions (one type of space radiation)., Method: We used the mouse in vivo blood-erythrocyte micronucleus (MN) assay for evaluating the cytogenetic effects of various doses of 1 GeV/n 48 Ti ions. The MN assay was coupled with the characterization of epigenetic alterations (the levels of global 5-methylcytosine and 5-hydroxymethylcytosine) in DNA samples isolated from BM cells. These analyses were performed in samples collected at an early time-point (1 week) and a late time-point (6 months) post-irradiation., Results: Our results showed that 48 Ti ions induced genomic instability in exposed mice. Significant dose-dependent loss of global 5-hydroxymethylcytosine was found but there were no changes in global 5-methylcytosine levels., Conclusion: Since persistent genomic instability and loss of global 5-hydroxymethylcytosine are linked to cancer, our findings suggest that exposure to 48 Ti ions may pose health risks.

We used 3 biological metrics highly relevant to health risks, that is, cell death, inflammation, and global DNA methylation, to determine the late effects of low doses (0.05 or 0.1 Gy) of 137 Cs γ rays on the bone marrow, lung, and testis collected at 6 months post-irradiation from the same exposed BALB/cJ mouse. This integrative approach has not been used for such a purpose. Mice exposed to 0 or 1 Gy of radiation served as a sham or positive control group, respectively. The results could deliver information for better health risk assessment across tissues, including better scientific basis for radiation protection and clinical application. We found no changes in the levels of all studied biological metrics (except a significant increase in the levels of an anti-inflammatory cytokine, ie, interleukin 10) in tissues of 0.05-Gy exposed mice, when compared to those in sham controls. In contrast, significantly increased levels of cell death and inflammation, including a significant loss of global 5-hydroxymethylcytosine, were found in all tissues of the same mice exposed to 0.1 or 1.0 Gy. Our data demonstrated not only no harm but also hormesis in the 0.05-Gy exposed mice. However, the hormetic effect appears to be dependent on biological metrics and tissue., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Little is known about plasma proteins that can be used as biomarkers for early and late responses to radiation. The purpose of this study was to determine a link between depletion of plasma gelsolin (pGSN) and cell-death as well as inflammatory responses in the lung (one of the tissues known to be radiosensitive) of the same exposed CBA/CaJ mice after exposure to heavy silicon ( 28 Si) ions. To prevent the development of multiple organ dysfunctions, pGSN (an important component of the extracellular actin-scavenging system) is responsible for the removal of actin that is released into the circulation during inflammation and from dying cells. We evaluated the levels of pGSN in plasma collected from groups of mice (5 mice in each) at 1 week (wk) and 1 month (1 mo) after exposure whole body to different doses of 28 Si ions, i.e. 0, 0.1, 0.25, or 0.5 Gy (2 fractionated exposures, 15 days apart that totaled each selected dose). In the same mouse, the measurements of pGSN levels were coupled with the quantitation of injuries in the lung, determined by (a) the levels of cleaved poly (ADP-ribose) polymerase (cleaved-PARP), a marker of apoptotic cell-death, (b) the levels of activated nuclear factor-kappa B (NF-κB) and selected cytokines, i.e. tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6, from tissue-lysates of the lung. Further, the ratio of neutrophils and lymphocytes (N/L) was determined in the same mouse. Our data indicated: (i) the magnitude of pGSN depletion was dependent to radiation dose at both harvest times, (ii) a persistent depletion of pGSN up to 1 mo post-exposure to 0.25 or 0.5 Gy of 28 Si ions, (iii) an inverse-correlation between pGSN depletion and increased levels of cleaved-PARP, including activated NF-κB/pro-inflammatory cytokines in the lung, and (iv) at both harvest times, statistically significant increases in the N/L ratio in groups of mice exposed to 0.5 Gy only. Our findings suggested that depletion in pGSN levels reflects not only the responses to 28 Si-ion exposure at both harvest times but also early and late-occurring damage., (Copyright © 2018. Published by Elsevier Ltd.)

Low-dose radiation is widely used across the world for the diagnosis of many diseases by means of a variety of imaging technologies. However, the harmful effects of exposure to low-dose radiation during medical examination remain controversial. The authors studied the effects of medical diagnostic low-dose x rays (i.e., 0.03, 0.05, or 0.1 mGy) after an in vitro exposure of human lymphocytes. Cells with no irradiation served as the non-irradiated control group. Three biological indicators were used to determine the effects of medical diagnostic low-dose x rays at 4, 8, 24, 48, and 72 h post-irradiation. These biological endpoints were mitochondrial membrane potential (ΔΨm), cell cycle, and apoptosis. Results indicated no changes in the ΔΨm, number of apoptotic cells, and cell cycle in lymphocytes exposed to these low doses of radiation, as compared to the corresponding non-irradiated lymphocytes at all harvest time-points. These results suggested that there were no harmful effects of the diagnostic low-dose x rays when human lymphocytes were exposed in an in vitro condition.

Although the lung is one of the target organs at risk for cancer induction from exposure to heavy ions found in space, information is insufficient on cellular/molecular responses linked to increased cancer risk. Knowledge of such events may aid in the development of new preventive measures. Furthermore, although it is known that germinal cells are sensitive to X- or γ-rays, there is little information on the effects of heavy ions on germinal cells. Our goal was to investigate in vivo effects of 1 GeV/n (48)Ti ions (one of the important heavy ions found in the space environment) on somatic (lung) and germinal (testis) tissues collected at various times after a whole body irradiation of CBA/CaJ mice (0, 0.1, 0.25, or 0.5 Gy, delivered at 1 cGy/min). We hypothesized that (48)Ti-ion-exposure induced damage in both tissues. Lung tissue was collected from each mouse from each treatment group at 1 week, 1 month, and 6 months postirradiation. For the testis, we collected samples at 6 months postirradiation. Hence, only late-occurring effects of (48)Ti ions in the testis were studied. There were five mice per treatment group at each harvest time. We investigated inflammatory responses after exposure to (48)Ti ions by measuring the levels of activated nuclear factor kappa B and selected pro-inflammatory cytokines in both tissues of the same mouse. These measurements were coupled with the quantitation of the levels of global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Our data clearly showed the induction of chronic inflammation in both tissues of exposed mice. A dose-dependent reduction in global 5hmC was found in the lung at all time-points and in testes collected at 6 months postirradiation. In contrast, significant increases in global 5mC were found only in lung and testes collected at 6 months postirradiation from mice exposed to 0.5 Gy of 1 GeV/n (48)Ti ions. Overall, our data showed that (48)Ti ions may create health risks in both lung and testicular tissues.

Although there is no doubt that exposure to high doses of radiation (delivered at a high dose-rate) induces harmful effects, the health risks and benefits of exposure to low levels (delivered at a low dose-rate) of toxic agents is still a challenging public health issue. There has been a considerable amount of published data against the linear no-threshold (LNT) model for assessing risk of cancers induced by radiation. The LNT model for risk assessment creates "radiophobia," which is a serious public health issue. It is now time to move forward to a paradigm shift in health risk assessment of low-dose exposure by taking the differences between responses to low and high doses into consideration. Moreover, future research directed toward the identification of mechanisms associated with responses to low-dose radiation is critically needed to fully understand their beneficial effects.

Although myeloid leukemia (ML) is one of the major health concerns from exposure to space radiation, the risk prediction for developing ML is unsatisfactory. To increase the reliability of predicting ML risk, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is important. We focused on the in vivo induction of late-occurring damage in HSPCs of mice exposed to (28)Si ions since such damage is associated with radiation-induced genomic instability (a key event of carcinogenesis). We gave adult male CBA/CaJ mice, known to be sensitive to radiation-induced ML, a whole-body exposure (2 fractionated exposures, 15 days apart, that totaled each selected dose, delivered at the dose-rate of 1 cGy/min) to various doses of 300 MeV/n (28)Si ions, i.e. 0 (sham controls), 0.1, 0.25, or 0.5 Gy. At 6 months post-irradiation, we collected bone marrow cells from each mouse (five mice per treatment-group) for obtaining the myeloid-lineage of HSPC-derived clones for analyses. We measured the frequencies of late-occurring chromosome aberrations (CAs), using the genome-wide multicolor fluorescence in situ hybridization method. The measurement of CAs was coupled with the characterization of the global DNA methylation patterns, i.e. 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC). A dose-dependent increase in the frequencies of CAs was detected (Analysis of Variance or ANOVA, p<0.01), indicating the induction of genomic instability after exposure of mice to 300 MeV/n (28)Si ions. Slight increases in the levels of 5 mC were observed in all treatment groups, as compared to the sham-control level. In contrast, there was a significant reduction in levels of 5 hmC (ANOVA, p<0.01). Since these endpoints were evaluated in the same mouse, our data suggested for the first time a link between a reduction in 5 hmC and genomic instability in HSPC-derived myeloid colonies of CBA/CaJ mice exposed to 300 MeV/n (28)Si ions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Myeloid leukemia (ML) is one of the major health concerns from exposure to radiation. However, the risk assessment for developing ML after exposure to space radiation remains uncertain. To reduce the uncertainty in risk prediction for ML, a much increased understanding of space radiation-induced changes in the target cells, i.e. , hematopoietic stem/progenitor cells (HSPCs), is critically important. We used the label-free quantitative mass spectrometry (LFQMS) proteomic approach to determine the expression of protein in HSPC-derived myeloid colonies obtained at an early time-point (one week) and a late time-point (six months) after an acute whole body exposure of CBA/CaJ mice to a total dose of 0, 0.1, 0.25, or 0.5 Gy of heavy-ion titanium ( 48 Ti ions), which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48 Ti ions served as non-irradiated sham controls. There were five mice per treatment groups at each harvest time. The Trans-Proteomic Pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A proof-of-concept based Ingenuity Pathway Analysis (IPA) was used to characterize the functions, pathways, and networks of the identified proteins. Alterations of expression levels of proteins detected in samples collected at one week (wk) post-irradiation reflects acute effects of exposure to 48 Ti ions, while those detected in samples collected at six months (mos) post-irradiation represent protein expression profiles involved in the induction of late-occurring damage (normally referred to as genomic instability). Our results obtained by using the IPA analyses indicate a wide array of signaling pathways involved in response to 1 GeV/n 48 Ti ions at both harvest times. Our data also demonstrate that the patterns of protein expression profiles are dose and time dependent. The majority of proteins with altered expression levels are involved in cell cycle control, cellular growth and proliferation, cell death and survival, cell-to-cell signaling and interaction. The IPA analyses indicate several important processes involved in responses to exposure to 48 Ti ions. These include the proteosme/ubiquination, protein synthesis, post-translation modification, and lipid metabolism. The IPA analyses also indicate that exposure to 1 GeV/n 48 Ti ions affects the development and function of hematological system, immune cell trafficking, including the cytoskeleton. Further, the IPA analyses strongly demonstrate that the NF-κB and MAPKs (ERKs, JNKs, and p38MAPK) pathways play an essential role in signal transduction after exposure to 1 GeV/n 48 Ti ions. At an early time-point (1 week), the top networks identified by the IPA analyses are related to metabolic disease, lipid metabolism, small molecule biochemistry, and development disorder. In contrast, the top networks identified in samples collected at a late time-point (6 mos post-irradiation) by the IPA analyses are related to cancer, hematological disorders, and immunological diseases. In summary, the proteomic findings from our study provide a foundation to uncover compounds potentially be highly effective in radiation countermeasures.

Purpose: To investigate the biological effects of titanium ((48)Ti, one of the important heavy ions found in space) in the liver of exposed-mice., Materials and Methods: We gave adult male CBA/CaJ mice a whole-body exposure to a total dose of 0, 0.1, 0.25 or 0.5 Gy of (48)Ti ions. The liver was collected at 1 week, 1 month, and 6 months post-irradiation (five mice per treatment-group at each harvest-time). Three biological endpoints were used for evaluating the effects of (48)Ti ions: Oxidative-stress, inflammatory responses, and DNA-methylation (5-methylcytosine and 5-hydroxymethylcytosine)., Results: Our data clearly demonstrated dose-dependent increases in oxidative stress and inflammatory responses in the liver of exposed mice at all time-points (Analysis of Variance or ANOVA, p < 0.05). Significant dose-dependent increases in the levels of 5-methylcytosine were detected at 1 week and 1 month (ANOVA, p < 0.05). At 6 months post-irradiation, a significant increase in the level of 5-methylcytosine was found only in 0.5-Gy-(48)Ti-ion-exposed mice. In contrast, dose-dependent decreases in 5-hydroxymethylcytosine levels were found in the liver of exposed mice (ANOVA, p < 0.05) at all time-points., Conclusions: Chronic oxidative-stress, chronic inflammation, and persistent aberrant DNA-methylation occurred in the liver of (48)Ti-exposed mice. Hence, exposure to (48)Ti ions in space may pose health risks.

During space travel astronauts are exposed to a variety of radiations, including galactic cosmic rays composed of high-energy protons and high-energy charged (HZE) nuclei, and solar particle events containing low- to medium-energy protons. Risks from these exposures include carcinogenesis, central nervous system damage and degenerative tissue effects. Currently, career radiation limits are based on estimates of fatal cancer risks calculated using a model that incorporates human epidemiological data from exposed populations, estimates of relative biological effectiveness and dose-response data from relevant mammalian experimental models. A major goal of space radiation risk assessment is to link mechanistic data from biological studies at NASA Space Radiation Laboratory and other particle accelerators with risk models. Early phenotypes of HZE exposure, such as the induction of reactive oxygen species, DNA damage signaling and inflammation, are sensitive to HZE damage complexity. This review summarizes our current understanding of critical areas within the DNA damage and oxidative stress arena and provides insight into their mechanistic interdependence and their usefulness in accurately modeling cancer and other risks in astronauts exposed to space radiation. Our ultimate goals are to examine potential links and crosstalk between early response modules activated by charged particle exposure, to identify critical areas that require further research and to use these data to reduced uncertainties in modeling cancer risk for astronauts. A clearer understanding of the links between early mechanistic aspects of high-LET response and later surrogate cancer end points could reveal key nodes that can be therapeutically targeted to mitigate the health effects from charged particle exposures.

Little is known about in vivo cytogenetic effects of protons delivered at the dose and dose rates encountered in space. We determined the effects of 100MeV protons, one of the most abundant type of protons produced during solar particle events (SPE), on the induction of chromosome aberrations (CAs) in bone marrow (BM) cells collected at early (3 and 24h) and late (6 months) time-points from groups of BALB/cJ mice (a known radiosensitive strain) exposed whole-body to 0 (sham-controls), 0.5, or 1.0Gy of 100MeV protons, delivered at 0.5 or 1.0cGy/min. These doses and dose-rates are comparable to those produced during SPE events. Additionally, groups of mice were exposed to 0 or 1Gy of (137)Cs γ rays (delivered at 1cGy/min) as a reference radiation. The kinetics of formation/reduction of gamma-histone 2-AX (γH2AX) were determined in BM cells collected at 1.5, 3, and 24h post-irradiation to assess the early-response. There were five mice per treatment-group per harvest-time. Our data indicated that the kinetics of γH2AX formation/reduction differed, depending on the dose and dose rate of protons. Highly significant numbers of abnormal cells and chromatid breaks (p<0.01), related to those in sham-control groups, were detected in BM cells collected at each time-point, regardless of dose or dose-rate. The finding of significant increases in the frequencies of delayed non-clonal and clonal CAs in BM cells collected at a late time-point from exposed mice suggested that 0.5 or 1Gy of 100MeV protons is capable of inducing genomic instability in BM cells. However, the extent of effects induced by these two low dose rates was comparable. Further, the results showed that the in vivo cytogenetic effects induced by 1Gy of 100MeV protons or (137)Cs γ rays (delivered at 1cGy/min) were similar., (Copyright © 2013 Elsevier B.V. All rights reserved.)

It has been well established that the bone marrow (BM) is a radiosensitive tissue, but the radiosensitivity of the heart is poorly understood. In this study, we investigated the comparative effects of ²⁸Silicon (²⁸Si) ions (one type of heavy ion found in space) on tissue from the heart and the BM of exposed mice. We gave adult male CBA/CaJ mice a whole-body exposure to a total dose of 0, 0.1, 0.25, or 0.5 Gy of 300 MeV/nucleon (n) ²⁸Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). The heart and BM were collected from 5 mice per treatment group at various times up to 6 months post-irradiation. In each mouse, we obtained tissue lysates from the heart and from the total population of BM cells for measuring the levels of cleaved poly (ADP-ribose) polymerase (cleaved PARP, a marker of apoptotic cell death) and the levels of activated nuclear factor-kappa B (NF-κB) and selected NF-κB-regulated cytokines known to be involved in inflammatory responses. Our data showed that, up to 6 months post-irradiation, the levels of apoptotic cell death and inflammatory responses in tissues from the heart and BM collected from exposed mice were statistically higher than those in sham controls. Hence, these findings are suggestive of chronic apoptotic cell death and inflammation in both tissues after exposure to ²⁸Si ions. In summary, our data are indicative of a possible association between exposure to ²⁸Si ions during space flight and long-term health risk.

We thank Dr. Baverstock [1] for his interest in reading our article and his time in writing his comments for our work [2]. We, however, respectfully disagree with his statement that we made "two category errors" associated with the assessment of the occurrence of "genomic instability" by determining the frequencies of delayed- or late-occurring chromosomal damage. Our disagreement is based upon the well-known fact that radiation-induced genomic instability (or delayed/late-occurring damage) can be manifested in many ways. These include late-occurring chromosomal damage, or mutations, or gene expression, or gene amplifications, or transformation, or microsatellite instability, or cell killing [3-9]. Such phenomena have been detected many cell generations after irradiation. We agree that genomic instability may well be the consequence of epigenetic changes. Another mechanism mentioned by Dr. Bavertock as being probably unlikely is the reversibility of damage. This potential may not be discarded off-hand, as Dr. Baverstock prefers to do. There is much reproducible evidence of adaptive protection that depending on absorbed dose precisely may reverse early damage, and damage appearing late may be due to some form of residual damage letting the cell become genetically unstable. In other words, the argument by Dr. Baverstock regarding upward or downward causation appears to be rather speculative and far from being settled.

It is clear that high-dose radiation is harmful. However, despite extensive research, assessment of potential health-risks associated with exposure to low-dose radiation (at doses below or equal to 0.1 Gy) is still challenging. Recently, we reported that 0.05 Gy of 137Cs gamma rays (the existing limit for radiation-exposure in the workplace) was incapable of inducing significant in vivo genomic instability (measured by the presence of late-occurring chromosomal damage at 6 months post-irradiation) in bone marrow (BM) cells of two mouse strains, one with constitutively high and one with intermediate levels of the repair enzyme DNA-dependent protein-kinase catalytic-subunit (DNA-PKcs). In this study, we present evidence for a lack of genomic instability in BM cells of the severely combined-immunodeficiency (SCID/J) mouse (which has an extremely low-level of DNA-PKcs activity) exposed whole-body to low-dose radiation (0.05 Gy). Together with our previous report, the data indicate that low-dose radiation (0.05 Gy) is incapable of inducing genomic instability in vivo (regardless of the levels of DNA-PKcs activity of the exposed mice), yet higher doses of radiation (0.1 and 1 Gy) do induce genomic instability in mice with intermediate and extremely low-levels of DNA-PKcs activity (indicating an important role of DNA-PKcs in DNA repair).

We determined the in vivo efficacy of apigenin, as an anti-oxidant and anti-inflammatory agent, given to mice after irradiation. Various concentrations of apigenin (0, 10, 20, and 40mg/kg body weight) were administered to mice by a single intraperitoneal injection 3hr after receiving 0 or 3Gy of (137)Cs gamma rays. Mice receiving vehicle only (no radiation and no apigenin) served as sham controls. We assessed the anti-oxidative activity of apigenin in vivo by measuring levels of 8-hydroxy-2-deoxy guanosine (8-OH-dG) in bone marrow (BM) cells, collected at days 3 and 10 after irradiation, from groups of mice (5 mice per treatment group) with or without apigenin treatment. Simultaneously, we evaluated the ability of apigenin to diminish radiation-induced inflammatory responses in bone-marrow-derived macrophages (BMDMs) from the same individual mice used for measuring the level of 8-OH-dG. To do this, the levels of activated nuclear factor-kappa B (NF-kappa B) and NF-kappa B-regulated pro-inflammatory cytokines [i.e. interleukin 1-beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha)] were measured in BMDMs. Our results indicated significant reductions (p<0.01 or <0.05) in the levels of 8-OH-dG in BM cells collected at both harvest times from irradiated mice receiving apigenin treatment, at all apigenin concentrations tested. Likewise, activation of NF-kappa B in BMDMs collected from gamma-irradiated mice that received apigenin was suppressed at both harvest times. Further, the levels of pro-inflammatory cytokines in gamma-irradiated mice treated with 20 or 40mg/kg body weight apigenin were significantly lower than those in mice receiving radiation only (p<0.01 or <0.05) even at day 10 post-irradiation. Additionally, the ratio of neutrophils to lymphocytes indicated that apigenin ameliorated radiation-induced hematological toxicity. Our study is the first to demonstrate the mitigative/therapeutic effects of apigenin given to mice after irradiation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

There is increasing evidence to support the hypothesis of adaptive response, a phenomenon in which protection arises from a low-dose radiation (<0.1 Gy) against damage induced by subsequent exposure to high-dose radiation. The molecular mechanisms underlying such protection are poorly understood. The goal of this study was to fill this knowledge gap. Mass spectrometry-based proteomics was used to characterize global protein expression profiles in the medium collected from human lymphocyte cultures given sham irradiation (0 Gy) or a priming low dose of 0.03 Gy 137Cs γ rays 4 h prior to a challenging dose of 1 Gy 137Cs γ rays. Adaptive response was determined by decreased micronucleus frequencies in lymphocytes receiving low dose irradiation prior to high dose irradiation compared to those receiving only high dose irradiation. Adaptive response was found in these experiments. Proteomic analysis of media revealed: (a) 55 proteins with similar abundance in both groups; (b) 23 proteins in both groups, but 7 of them were high abundance in medium with adaptive environment, while 16 high abundance proteins were in medium without adaptive environment; (c) 17 proteins in medium with adaptive environment only; and (d) 8 proteins in medium without adaptive environment only. The results provide a foundation for improving understanding of the molecular mechanisms associated with the beneficial effects of low dose radiation that, in turn, will have an important impact on radiation risk estimation. Hence, these studies are highly relevant to radiation protection due to an increased use of low dose radiation in daily life (e.g., medical diagnosis or airport safety) or an unavoidable exposure to low level background radiation.

In spite of extensive research, assessment of potential health risks associated with exposure to low-dose (≤ 0.1 Gy) radiation is still challenging. We evaluated the in vivo induction of genomic instability, expressed as late-occurring chromosome aberrations, in bone-marrow cells of two strains of mouse with different genetic background, i.e. the radiosensitive BALB/cJ and the radioresistant C57BL/6J strains following a whole-body exposure to varying doses of (137)Cs gamma rays (0, 0.05, 0.1, and 1.0 Gy). A total of five mice per dose per strain were sacrificed at various times post-irradiation up to 6 months for sample collections. Three-color fluorescence in situ hybridization for mouse chromosomes 1, 2, and 3 was used for the analysis of stable-aberrations in metaphase-cells. All other visible gross structural-abnormalities involving non-painted-chromosomes were also evaluated on the same metaphase-cells used for scoring the stable-aberrations of painted-chromosomes. Our new data demonstrated in bone-marrow cells from both strains that low doses of low LET-radiation (as low as 0.05 Gy) are incapable of inducing genomic instability but are capable of reducing specific aberration-types below the spontaneous rate with time post-irradiation. However, the results showed the induction of genomic instability by 1.0 Gy of (137)Cs gamma rays in the radiosensitive strain only.

The objective of this study was to determine the kinetics of nuclear factor-kappa B (NF-kappaB) activation and cytokine expression in bone marrow (BM) cells of exposed mice as a function of the dose rate of protons. The cytokines included in this study are pro-inflammatory [i.e., tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and IL-10). We gave male BALB/cJ mice a whole-body exposure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons, delivered at 5 or 10 mGy min(-1), the dose and dose rates found during solar particle events in space. As a reference radiation, groups of mice were exposed to 0 (sham-controls) or 1 Gy of (137)Cs gamma rays (10 mGy min(-1)). After irradiation, BM cells were collected at 1.5, 3, 24 h, and 1 month for analyses (five mice per treatment group per harvest time). The results indicated that the in vivo time course of effects induced by a single dose of 1 Gy of 100 MeV protons or (137)Cs gamma rays, delivered at 10 mGy min(-1), was similar. Although statistically significant levels of NF-kappaB activation and pro-inflammatory cytokines in BM cells of exposed mice when compared to those in the corresponding sham controls (Student's t-test, p < 0.05 or <0.01) were induced by either dose rate, these levels varied over time for each protein. Further, only a dose rate of 5 mGy min(-1) induced significant levels of anti-inflammatory cytokines. The results indicate dose-rate effects of protons.

Purpose: To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of (137)Cs gamma rays., Materials and Methods: Two-dimensional electrophoresis gel coupled with mass spectrometry was used to analyze blood-samples collected at days 2 and 7 post-irradiation. At each sacrifice-time, alterations in expression-level of protein spots between control- and exposed-groups were analyzed statistically by the PDQuest software using Student's t-test (at the significance level of p < 0.05). Mass spectrometry was used to identify the identity of protein-spots with significantly altered expression-level., Results: At day 2, 18 proteins were significantly up-regulated in exposed-mice. These included: alpha-2-Heremans-Schmid (HS)-glycoprotein, apolipoprotein (Apo)-AII-precursor, Apo-E, beta-2-glycoprotein-I, clusterin, fibrinogen-alpha-chain, fibrinogen-gamma-polypeptide, fetuin-B, haptoglobin, high-molecular-weight (HMW)-kininogen (Kng), low-MW-Kng, Kng1-precursor, liver-carboxylesterase-I-precursor, major-urinary-protein-6-precursor, mannose-binding-protein-C-precursor, mannose-binding-lectin-C, and prothrombin-precursor. Gelsolin was detected in control-mice only. At day 7, high expression-levels of 14 proteins were detected in control-mice (i.e., alpha-1-antitrypsin-precursor, carboxylesterase-N, cholesterol-7-alpha-hydroxylase, contraspin, coagulation-factor-II, coagulation-factor-XIII, gelsolin, immunoglobulin-G-heavy-chain, neurexin, prothrombin-precursor, protein-phosphatase, putative-calcium-influx-channel, vitamin-D-binding-protein, and 1110018G07Rik); while 15 proteins were highly expressed in exposed-mice. These included: alpha-1-acid-glycoprotein, alpha-2-HS-glycoprotein, alpha-1-protease-inhibitor-2, ApoA-IV, ApoC-I, ApoH, beta-1-globin, clusterin, complement-component-3, fibrinogen-beta-chain, HMW-Kng, major-histocompatibility-complex-class-Ia-H2-K, serine-(cysteine)-proteinase-inhibitor, retinoblastoma-associated-protein-140, and vascular-cell-adhesion-molecule-1., Conclusion: Although different proteins (mostly involved in inflammatory responses) were detected in exposed-mice, alterations in expression-levels of clusterin, gelsolin, kininogen, and alpha-2-HS-glycoproteins were found at both times. Despite the need for validation, the results suggested that alterations in expression-levels of specific proteins may be indicative of radiation-exposure. The results also provided the important step in an eventual establishment of blood-based biomarkers of radiation-exposure in vivo.

The diagnosis of pediatric multiple sclerosis (MS) is challenging due to its low frequency and the overlap with other acquired childhood demyelinating disorders of the central nervous system. To identify potential protein biomarkers which could facilitate the diagnosis, we used two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry to identify proteins associated with pediatric MS. Plasma samples from nine children with MS and nine healthy subjects, matched in aggregate by age and gender, were analyzed for differences in their patterns of protein expression. We found 12 proteins that were significantly up regulated in the pediatric MS group: alpha-1-acid-glycoprotein 1, alpha-1-B-glycoprotein, transthyretin, apoliprotein-C-III, serum amyloid P component, complement factor-I, clusterin, gelsolin, hemopexin, kininogen-1, hCG1993037-isoform, and vitamin D-binding protein. These results show that 2-DE in combination with mass spectrometry is a highly sensitive technique for the identification of blood-based biomarkers. This proteomic approach could lead to a new panel of diagnostic and prognostic markers in pediatric MS.

A pilot study was conducted to examine the magnitude of cell-cycle delay and apoptosis in bone marrow (BM) cells collected at 18, 42 and 66 hr from radiosensitive CBA/CaJ mice and radioresistant C57BL/6J mice following a whole-body in vivo exposure to 1 GeV/amu (56)Fe ions or (137)Cs gamma rays. At each sacrifice, BM cells were collected from three mice of each strain per dose of (56)Fe ions (0, 10 and 100 cGy) and two mice of each strain per dose of (137)Cs gamma rays (0, 100 and 300 cGy). A significant G1-arrest (ANOVA, p < 0.05) was observed at 18 hr after exposure of mice to 100 cGy of (56)Fe ions or 300 cGy of (137)Cs gamma rays, relative to their corresponding sham-controls, resulting in a significant decrease in the percentage of cells cycling into S-phase in both strains. The percentage of S-phase cells subsequently increased and persisted up to 66 hr post-irradiation. Significant numbers of G2/M cells were found at 18 and 66 (but not at 42) hr post-irradiation, regardless of radiation-type or mouse-strain. It is likely that BM cells have undergone at least one cell cycle at 66 hr after exposure of mice to either 100 cGy (56)Fe ions or 300 cGy (137)Cs gamma rays. Our study is the first to investigate the in vivo effects of (56)Fe ions (1 GeV/amu) on the cell cycle of mouse BM cells using flow cytometry. The cell-cycle distribution (but not the number of apoptotic cells) was dependent on radiation-dose and harvest-time.

Previous research has demonstrated that adding a very small gamma-ray dose to a small alpha radiation dose can completely suppress lung cancer induction by alpha radiation (a gamma-ray hormetic effect). Here we investigated the possibility of gamma-ray hormesis during low-dose neutron irradiation, since a small contribution to the total radiation dose from neutrons involves gamma rays. Using binucleated cells with micronuclei (micronucleated cells) among in vitro monoenergetic-neutron-irradiated human lymphocytes as a measure of residual damage, we investigated the influence of the small gamma-ray contribution to the dose on suppressing residual damage. We used residual damage data from previous experiments that involved neutrons with five different energies (0.22-, 0.44-, 1.5-, 5.9-, and 13.7-million electron volts [MeV]). Corresponding gamma-ray contributions to the dose were approximately 1%, 1%, 2%, 6%, and 6%, respectively. Total absorbed radiation doses were 0, 10, 50, and 100 mGy for each neutron source. We demonstrate for the first time a protective effect (reduced residual damage) of the small gamma-ray contribution to the neutron dose. Using similar data for exposure to gamma rays only, we also demonstrate a protective effect of 10 mGy (but not 50 or 100 mGy) related to reducing the frequency of micronucleated cells to below the spontaneous level.

To date, there is scant information on in vivo induction of chromosomal damage by heavy ions found in space (i.e. 56Fe ions). For radiation-induced response to be useful for risk assessment, it must be established in in vivo systems especially in cells that are known to be at risk for health problems associated with radiation exposure (such as hematopoietic cells, the known target tissue for radiation-induced leukemia). In this study, the whole genome multicolor fluorescence in situ hybridization (mFISH) technique was used to examine the in vivo induction of chromosomal damage in hematopoietic tissues, i.e. bone marrow cells. These cells were collected from CBA/CaJ mice at day 7 following whole-body exposure to different doses of 1 GeV/amu 56Fe ions (0, 0.1, 0.5 and 1.0 Gy) or (137)Cs gamma rays as the reference radiation (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min using a GammaCell40). These radiation doses were the average total-body doses. For each radiation type, there were four mice per dose. Several types of aberrations in bone marrow cells collected from mice exposed to either type of radiation were found. These were exchanges and breaks (both chromatid- and chromosome-types). Chromosomal exchanges included translocations (Robertsonian or centric fusion, reciprocal and incomplete types), and dicentrics. No evidence of a non-random involvement of specific chromosomes in any type of aberrations observed in mice exposed to 56Fe ions or 137Cs gamma rays was found. At the radiation dose range used in our in vivo study, the majority of exchanges were simple. Complex exchanges were detected in bone marrow cells collected from mice exposed to 1 Gy of 56Fe ions or 3 Gy of 137Cs gamma rays only, but their frequencies were low. Overall, our in vivo data indicate that the frequency of complex chromosome exchanges was not significantly different between bone marrow cells collected from mice exposed to 56Fe ions or 137Cs gamma rays. Each type of radiation induced significant dose-dependent increases (ANOVA, P < 0.01) in the frequencies of chromosomal damage, including the numbers of abnormal cells. Based upon the linear-terms of dose-response curves, 56Fe ions were 1.6 (all types of exchanges), 4.3 (abnormal cells) and 4.2 (breaks, both chromatid- and chromosome-types) times more effective than 137Cs gamma rays in inducing chromosomal damage.

Objective: The objectives of this study were to identify protein biomarkers of radiation-induced acute myeloid leukemia (rAML) in CBA/CaJ mice, and to examine the similarities or differences in the patterns of protein-expression profiles among AMLs induced by low linear energy transfer (LET) radiation (e.g., gamma- or x-rays), and high LET radiation (i.e., neutrons)., Materials and Methods: We used two-dimensional electrophoresis gel in combination with mass spectrometry (MS), i.e., matrix-assisted laser desorption ionization/time-of-flight MS and electrospray ionization-liquid chromatography/tandem mass spectrometry, to identify protein signatures in blood-plasma samples collected from control and rAML mice. There were nine cases of rAML (three cases induced by high LET radiation; six induced by low LET radiation) and eight control mice at similar ages., Results: The results showed differences in the patterns of protein profiles from blood-plasma samples collected from rAML vs control mice. Moreover, our data demonstrated, both qualitatively and quantitatively, differences between the plasma protein profiles obtained from mice with AML induced by low vs high LET radiation. Most of the proteins that were present at greater levels in normal samples than in rAML samples were associated with normal metabolism and growth. Several acute-phase proteins were upregulated in rAML samples., Conclusion: The data present, for the first time, evidence for increased expression of clusterin and a loss of gelsolin expression in blood plasma as potential biomarkers of rAML in the CBA/CaJ mouse. Results also indicate that two-dimensional electrophoresis, in combination with MS, is a highly sensitive technique for identification of blood-based biomarkers of rAML.

We measured levels of NF-kappaB activation in bone marrow (BM) cells collected at 1 and 4 h from male BALB/cJ mice (10-12 weeks old) given a whole body dose of 0, 0.05, 0.1 and 1 Gy of (137)Cs gamma-rays (at the dose rate of 0.75 Gy/min). At each harvest time-point, BM cells were collected from five mice per dose of radiation. We used two methods for detecting NF-kappaB activation (1) the NF-kappaB/p65 transcription factor enzyme-linked immunosorbance assay (ELISA) and (2) immunofluorescence staining with NF-kappaB/p65 antibody. Results from ELISA indicated 2.0 and 2.8-fold increases in NF-kappaB activation in BM cells isolated at 1 h post-exposure of mice to 0.1 or 1.0 Gy. The immunofluorescence staining method showed similar results. In samples isolated 4 h post-irradiation, however, no activated NF-kappaB signal was found, regardless of the method of detection. The data also demonstrated that NF-kappaB was not activated in bone marrow cells collected either at 1 or 4 h from BALB/cJ mice exposed to a single dose of 0.05 Gy (137)Cs gamma-rays. Taken together, the results from our in vivo study indicate the involvement of NF-kappaB activation in early response to 0.1 and 1.0 Gy (but not 0.05 Gy) of (137)Cs gamma-rays.

The potential use of flavonoids as a radioprotector is of increasing interest because of their high antioxidant activity and abundance in the diet. The aim of this study is to examine genotoxic and radioprotective effects of one of the most common flavonoids, apigenin, on radiation-induced chromosome aberrations in human lymphocytes. The cytokinesis-block micronucleus (CBMN) assay was used to evaluate such effects of apigenin. Blood samples were collected from two non-smoking healthy male volunteers who had no history of previous exposure to other clastogenic agents. Isolated lymphocytes were cultured. There were two tubes per concentration for all treatments. To evaluate the genotoxicity of apigenin, cells were first treated with different concentrations of apigenin (0, 2.5, 5, 10 and 25 microg/mL) at 24 h after culture initiation, followed by cytochalasin-B (Cyt-B) treatment (3 microg/mL) and cell harvest at 44 and 72 h, respectively. Secondly, to investigate the radioprotective effect, cell cultures were exposed to different concentrations of apigenin as described above for 30 min before being irradiated to 2 Gy of 137Cs gamma rays (at a dose rate of 0.75 Gy/min). In all instances, the frequency of MN was scored in binucleated (BN) cells. The nuclear proliferation index also was calculated. We did not detect an increase in the frequency of MN in non-irradiated human lymphocyte cultures treated with 2.5, 5.0 or 10 microg/mL apigenin; although, we did observe an increase in cultures treated with 25 microg/mL apigenin (the highest concentration of apigenin used in our study). We also observed a significant increase in the frequency of MN in irradiated cells overall; however, the frequency was decreased as the concentration of apigenin increased, suggesting a radioprotective effect. These findings provide a basis for additional studies to help clarify the potential use and benefit of apigenin as a radioprotector.

The objectives of this review are to: (a) demonstrate that the male CBA/Ca mouse has several characteristics that make it an excellent animal for the study of leukemogenesis, (b) show that several of the genetic abnormalities observed in the male CBA/Ca mouse during the development of radiation induced acute myeloid leukemia (AML) are syntenic with those frequently detected in patients with myeloid disorders such as myelodysplastic syndrome and AML, (c) illustrate that leukemia-related chromosomal lesions are the indicators for high risk individuals.

Although microsatellite instability (MSI), usually detected by DNA length polymorphisms, has been implicated in the induction of solid tumors in both humans and animals, its role in leukemogenesis is unclear. The goal of this study was to investigate whether there is an association between MSI and radiation leukemogenesis in CBA/Ca mice. Microsatellite lengths at 55 loci, mapped to eight different mouse chromosomes, were examined in two groups of DNA samples: 1) 10 normal DNA samples collected from the bone marrow cells of control male CBA/Ca mice, and 2) 17 DNA samples isolated from the spleens of mice that developed myeloid leukemia (ML) after exposure to neutrons, or X rays, or gamma rays. Microsatellite markers were amplified using the non-radioisotopic multiplex-touchdown PCR protocols developed in our laboratory, and the sizes of amplicons were examined on 6% non-denaturing polyacrylamide gels. Although no correlation between microsatellite length polymorphisms and radiation leukemogenesis was observed at the 55 CBA/Ca mouse loci tested in this study, an uncommon MSI, manifested as the absence of DNA bands after PCR amplification at 2 loci (D2MIT140 and D4MIT104), was observed in both control and ML samples. However, the frequency of ML samples showing this type of MSI is statistically significant (p<0.05). Although there is no direct evidence that this type of MSI predisposes mice to the development of leukemia, the results suggests that genes flanking the D2MIT140 and D4MIT104 are susceptible to spontaneous mutation and perhaps to damage caused by ionizing radiation.

N-ras mutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon- (3 gamma-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of the N-ras gene: (i) a 120 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of the N-ras gene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GAC-->GAT), 62 (CAA-->CAC), or 70 (CAG-->CAA) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CAA-->CAC) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency of N-ras mutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at the N-ras region of the genome. More importantly, N-ras mutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding that N-ras mutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence of N-ras mutations. Alternatively, N-ras may be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.

G-banded metaphase chromosomes prepared from 14 male CBA/Ca mice with histologically confirmed myeloid leukemia (ML) were studied in an effort to identify specific chromosomal changes associated with radiation leukemogenesis. The chromosome studies were undertaken as part of a larger investigation of radiation carcinogenesis, in which mice were exposed to radiation of several different qualities, i.e., x-rays, gamma-rays and "monoenergetic" fast neutrons of 5 mean energies ranging from 0.2 to 14 MeV. The 14 ML cases showed no histologically phenotypic differences and they were transplantable in syngeneic mice. We detected a specific chromosomal deletion in 1 copy of mouse chromosome 2 at regions D-E in all radiation-induced ML cells, regardless of radiation quality. Our results strongly implicate the involvement of genes within or close to regions D-E of chromosome 2 in radiation leukemogenesis. In addition to the specific deletion in chromosome 2, loss or gain of the Y chromosome was also detected in some cells from 6 ML cases. Because this hypo- or hyperploidy occurred in only a small fraction of leukemic cells, a causative role in radiation leukemogenesis appears unlikely.