Louise Honikel

Attenuation of oxidative damage and inflammatory responses by apigenin given to mice after irradiation

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.

No Evidence for the in vivo Induction of Genomic Instability by Low Doses of 137Cs Gamma Rays in Bone Marrow Cells of BALB/cJ and C57BL/6J Mice

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 137Cs 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 137Cs gamma rays in the radiosensitive strain only.

Effects of 100MeV protons delivered at 0.5 or 1cGy/min on the in vivo induction of early and delayed chromosomal damage.

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.

Late-occurring chromosome aberrations and global DNA methylation in hematopoietic stem/progenitor cells of CBA/CaJ mice exposed to silicon (28Si) ions

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.

Protein-expression profiles in mouse blood-plasma following acute whole-body exposure to 137Cs γ rays

Purpose: To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of 137Cs 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 Students 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.

Identification of proteins secreted into the medium by human lymphocytes irradiated in vitro with or without adaptive environments.

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 &ggr; rays 4 h prior to a challenging dose of 1 Gy 137Cs &ggr; 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.

Induction of Chronic Inflammation and Altered Levels of DNA Hydroxymethylation in Somatic and Germinal Tissues of CBA/CaJ Mice Exposed to 48Ti Ions

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 48Ti 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 48Ti-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 48Ti ions in the testis were studied. There were five mice per treatment group at each harvest time. We investigated inflammatory responses after exposure to 48Ti 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 48Ti ions. Overall, our data showed that 48Ti ions may create health risks in both lung and testicular tissues.

Proteomic Profiling of Hematopoietic Stem/Progenitor Cells after a Whole Body Exposure of CBA/CaJ Mice to Titanium (48Ti) Ions

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 (48Ti ions), which are the important component of radiation found in the space environment. Mice exposed to 0 Gy of 48Ti 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 48Ti 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 48Ti 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 48Ti 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 48Ti 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 48Ti 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.

Persistent depletion of plasma gelsolin (pGSN) after exposure of mice to heavy silicon ions

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 (28Si) 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 28Si 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 28Si 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 28Si-ion exposure at both harvest times but also early and late-occurring damage.

Protein expression profiles in hematopoietic stem/progenitor cells after exposure of mice to silicon (28Si) ions

It has been well recognized that exposure to space radiation is a major challenge to space exploration. To protect astronauts in space environments, improvement in our knowledge of radiation-induced changes in specific target cells that may affect the health of astronauts is required. Cancer of blood cells, in particular myeloid leukemia (ML), is one of the major health concerns from exposure to space radiation. However, the predictions of risks for developing ML post-exposure to space radiation are unsatisfactory. To increase the reliability of predicting risk for ML, a much improved understanding of space radiation-induced changes in the target cells, i.e. hematopoietic stem/progenitor cells (HSPCs), is critically important. In vivo studies of humans are not possible. Thus, controlled and randomized animal experiments are critically important. Most proteomic applications mentioned above have used 2-DE or stable isotope-tagged mass spectrometry. Although the 2-DE has several advantages (e.g. the detection of potential post-translational modifications of proteins which can be readily visualized on the gel, although the exact type of modification requires determination by mass spectrometry), such technology is simply not as comprehensive and reliable as desired. To overcome these limitations, we recently developed a unique label-free quantitative mass spectrometry (LFQMS) platform [ 1]. This is an innovative, experimentally based method that accurately determines peptide peak retention-time and uses multiple filters for exclusion of unqualified peptides by peptide frequency, retention time, intensity coefficient of variation and intensity correlation to enhance protein quantification of qualified peptides and proteins. In this study, we used the LFQMS platform to examine protein expression-profiles in the colonies of HSPCs (the best population of cells for studying in vivo biological effects of radiation on hematopoietic stem cells) obtained at 6 months after exposure (at which radiation-induced genomic instability and chronic inflammation have been detected [ 2]) of CBA/CaJ mice whole-body to a total dose of 0, 0.1, 0.25 or 0.5 Gy of 300 MeV/nucleon 28Si ions, using a fractionated schedule (two exposures, 15 days apart that totaled each selected dose). These doses of 300 MeV/nucleon 28Si ions are comparable to what astronauts encounter in space. Mice exposed to 0 Gy of 28Si ions served as non-irradiated sham controls. The colonies of HSPCs were obtained from BM cells of five mice per treatment group, by means of an in vitro colony forming unit assay (CFU-A) using methylcellulose-based medium. Proteins were extracted from HSPC colonies and protein concentrations were determined by the Bradford Protein Assay. The trans-proteomic pipeline (TPP) was used to assign a probability of a particular protein being in the sample. A total of 1344 unique, non-redundant proteins were identified with ≥90% confidence from 3254 peptides, quantified and their abundances were compared statistically. Among the 1344 proteins, differential expression of 198 proteins was found to be statistically significant in HSPC colonies obtained from treated groups, in relation to those found in non-irradiated sham controls. A proof-of-concept-based Ingenuity Pathway Analysis (IPA, www.ingenuity.com) was used to characterize the functions and pathways of these 198 identified proteins. The majority of these proteins are cancer-related (P < 0.0001). Biochemical analyses of the molecular and cellular functions of these proteins reveal association with perturbation in cell survival, free radical scavenging, cell cycle, DNA repair, cellular assembly, hematological system development and inflammatory responses. These proteins are linked to two major molecular networks that are linked to cancer and inflammatory responses (i.e. nuclear factor-κ B and the protein phosphatase 2 A networks). Our results indicated 28Si ion-induced damage in HSPCs.