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NASA NTRS1998

The potential influence of radiation-induced microenvironments in neoplastic progression

Ionizing radiation is a complete carcinogen, able both to initiate and promote neoplastic progression and is a known carcinogen of human and murine mammary gland. Tissue response to radiation is a composite of genetic damage, cell death and induction of new gene expression patterns. Although DNA damage is believed to initiate carcinogenesis, the contribution of these other aspects of radiation response are beginning to be explored. Our studies demonstrate that radiation elicits rapid and persistent global alterations in the mammary gland microenvironment. We postulate that radiation-induced microenvironments may affect epithelial cells neoplastic transformation by altering their number or susceptibility. Alternatively, radiation induced microenvironments may exert a selective force on initiated cells and/or be conducive to progression. A key impetus for these studies is the possibility that blocking these events could be a strategy to interrupt neoplastic progression.

Review, Academic↗

NASA NTRS2006

NASA Radiation Track Image GUI for Assessing Space Radiation Biological Effects

The high-charge high-energy (HZE) ion components of the galactic cosmic rays when compared to terrestrial forms of radiations present unique challenges to biological systems. In this paper we present a deoxyribonucleic acid (DNA) breakage model to visualize and analyze the impact of chromatin domains and DNA loops on clustering of DNA damage from X rays, protons, and HZE ions. Our model of DNA breakage is based on a stochastic process of DNA double-strand break (DSB) formulation that includes the amorphous model of the radiation track and a polymer model of DNA packed in the cell nucleus. Our model is a Monte-Carlo simulation based on a randomly located DSB cluster formulation that accomodates both high- and low-linear energy transfer radiations. We demonstrate that HZE ions have a strong impact on DSB clustering, both along the chromosome length and in the nucleus volume. The effects of chromosomal domains and DNA loops on the DSB fragment-size distribution and the spatial distribution of DSB in the nucleus were studied. We compare our model predictions with the spatial distribution of DSB obtained from experiments. The implications of our model predictions for radiation protection are discussed.

Ponomarev, Artem L.↗

DOE OSTI2024

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in Δnei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of Δlhr cells by mutant lhr alleles depended on Lhr’s ATPase/helicase activities and its tetrameric quaternary structure. The lhr–nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2007

Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

Hu, Shaowen↗

NASA NTRS2007

Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

Hu, Shaowen↗

NASA NTRS2018

Coalescence of DNA Double Strand Breaks Induced by Galactic Cosmic Radiation is Modulated by Genetics in 15 Inbred Strains of Mice

In this manuscript we address the challenges associated with the ability to predict radiation sensitivity associated with exposure to either cosmic radiation or X-rays in a population study, by monitoring DNA damage sensing protein 53BP1 forming small nuclear radiation-induced foci (RIF) as a surrogate biomarker of DNA double strand breaks (DSB). 76 primary skin fibroblasts were isolated from 10 collaborative cross strains and five reference inbred mice (C57Bl/6, BALB/CByJ, B6C3, C3H and CBA/CaJ) and exposed to three different charged nuclei of increasing LET (350 MeV/n Si, 350 MeV/n Ar and 600 MeV/n Fe) and X-ray. Our data brings strong evidence against the classic "contact-first" model where DSBs are assumed to be immobile and repaired at the lesion site. In contrast, our model suggests nearby DSBs move into single repair unit characterized by large RIF before the repair machinery kicks in. Such model has the advantage of being much more efficient molecularly but is poorly suited to deal with cosmic radiation, where energy is concentrated along the particle trajectory, inducing a large density of DSBs along each particle track. In accordance with this model, RIF quantification after X-ray exposition showed a saturated dose response for early time points post-irradiation for all strains. Similarly, the high-LET response showed that RIF number matched the number of track per cell, not the number of expected DSB per cell (1). At the temporal level, we noted that the percentage of unrepaired high-LET tracks over a 48 hour time-course increased with LET, confirming that the DNA repair process becomes more difficult as more DSB coalesce into single RIF. There was also good agreement between persistent RIF levels measured in-vitro in the primary skin cultures and survival levels of T-cells and B-cells collected in blood samples from 10 CC strains 24 hours after 0.1 Gy whole-body dose of X-ray. This suggests that persistent RIF 24 hour post-IR is a good surrogate in-vitro biomarker for in-vivo radiation toxicity. Finally, at the genomic level, large differences in repair rates between strains for high-LET allowed us to identify suggestive genetic loci associated with radiation sensitivity. Interestingly, the two highest LETs provided the most strain variation with a common locus on Chromosome 10 highly enriched for DNA repair associated genes we discussed in detail.

Radiation-Induced Foci↗

NASA NTRS2003

Detection of Strand Cleavage And Oxidation Damage Using Model DNA Molecules Captured in a Nanoscale Pore

We use a biological nano-scale pore to distinguish among individual DNA hairpins that differ by a single site of oxidation or a nick in the sugar-phosphate backbone. In earlier work we showed that the protein ion channel alpha-hemolysin can be used as a detector to distinguish single-stranded from double-stranded DNA, single base pair and single nucleotide differences. This resolution is in part a result of sensitivity to structural changes that influence the molecular dynamics of nucleotides within DNA. The strand cleavage products we examined here included a 5-base-pair (5-bp) hairpin with a 5-prime five-nucleotide overhang, and a complementary five-nucleotide oligomer. These produced predictable shoulder-spike and rapid near-full blockade signatures, respectively. When combined, strand annealing was monitored in real time. The residual current level dropped to a lower discrete level in the shoulder-spike blockade signatures, and the duration lengthened. However, these blockade signatures had a shorter duration than the unmodified l0bp hairpin. To test the pore sensitivity to nucleotide oxidation, we examined a 9-bp hairpin with a terminal 8-oxo-deoxyguanosine (8-oxo-dG), or a penultimate 8-oxo-dG. Each produced blockade signatures that differed from the otherwise identical control 9bp hairpins. This study showed that DNA structure is modified sufficiently by strand cleavage or oxidation damage at a single site to alter in a predictable manner the ionic current blockade signatures produced. This technique improves the ability to assess damage to DNA, and can provide a simple means to help characterize the risks of radiation exposure. It may also provide a method to test radiation protection.

Vercoutere, W.↗

NASA NTRS2004

The Effects of Modeled Microgravity on Nucleocytoplasmic Localization of Human Apurinic/Apyrimidinic

Exposure to space radiation and microgravity occurs to humans during space flight. In order to have accurate risk estimations, answering questions to whether increased DNA damage seen during space flight in modified by microgravity are important. Several studies have examined whether intercellular repair of radiation-induced DNA lesions are modified by microgravity. Results from these studies show no modification of the repair processes due to microgravity. However, it is known that in studies not involving radiation that microgravity interferes with normal development. Interestingly, there is no data that attempts to analyze the possible effects of microgravity on the trafficking of DNA repair proteins. In this study, we analyze the effects of modeled microgravity on nucleocytoplasmic shuttling of the human DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1/Ref1) which is involved in base excision repair. We examined nuclear translocation of APE1 using enhanced green fluorescent protein (EGFP) fused to APE1 as a reporter. While APE1 under normal gravity showed normal nuclear localization, APE1 nuclear localization under modeled microgravity was decreased. These results suggest that nucleocytoplasmic translocation of APE1 is modified under modeled microgravity.

Gonda, Steve↗

NASA NTRS2010

Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

Hu, Shaowen↗

NASA NTRS2007

Involvement of DNA-PK(sub cs) in DSB Repair Following Fe-56 Ion Irradiation

When cells are exposed to radiation, cellular lesions are induced in the DNA including double strand breaks (DSBs), single strand breaks and clustered DNA damage, which if not repaired with high fidelity may lead to detrimental biological consequences. Complex DSBs are induced by ionizing radiation and characterized by the presence of base lesions close to the break termini. They are believed to be one of the major causes of the biological effects of IR. The complexity of DSBs increases with the ionization density of the radiation and these complex DSBs are distinct from the damage induced by sparsely ionizing gamma-radiation. It has been hypothesized that complex DSBs produced by heavy ions in space pose problems to the DNA repair machinery. We have used imm uno-cyto-chemical staining of phosphorylated histone H2AX (gamma-H2AX) foci, as a marker of DSBs. We have investigated the formation and loss of gamma-H2AX foci and RAD51 foci (a protein involved in the homologous recombination pathway) in mammalian cells induced by low fluences of low-LET gamma-radiation and high-LET Fe-56 ions (1GeV/n, 151 keV/micron LET). M059J and M059K cells, which are deficient and proficient in DNA-PK(sub cs) activity respectively, were used to examine the role of DNA-PK(sub cs), a key protein in the non-homologous end joining (NHEJ) pathway of DSB repair, along with HF19 human fibroblasts. Followi ng irradiation with Fe-56 ions the rate of repair was slower in M059J cells compared with that in M059K, indicating a role for DNA-PK(sub cs) in the repair of DSB induced by Fe-56 ions. However a small percentage of DSBs induced are rejoined within 5 h although many DSBs still persist up to 24 h. When RAD51 was examined in M059J/K cells, RAD51 foci are visible 24 hours after irradiation in approximately 40% of M059J cells compared with <5% of M059K cells indicating that persistent DSBs or those formed at stalled replication forks recruit RAD51 in DNA-PK(sub cs) deficient cells. Following 1 Gy gamma-radiation the induction of gamma-H2AX foci is similar in M059J and M059K cells. However, the repair rate of DSBs is slower in M059J cells than in M059K as shown previously but faster than seen with DSB induced by 56Fe ions. Vanillin, an inhibitor of DNA-PK(sub cs), reduces significantly the rate of DSB repair in HF19 cells following 1 Gy gamma-radiation but at 0.25 Gy gamma-irradiation the rate of DSB repair is similar in the presence or absence vanillin, thus suggesting the repair of a sub-set of DSBs induced by low dose, low-LET radiation does not require DNA-PK(sub cs). This sub-set of DSBs is formed in lower yield with high LET radiation. T he complexity of DNA DSBs induced by HZE radiation will be discussed in terms of reduced repair efficiency and provide scope to model different sub-classes of DSBs as precursors that may lead to the detrimental health effects of HZE radiation.

O'Neill, Peter↗

NASA NTRS2022

Agent-Based Modeling of Microbes in Space

Space is tough on organisms. Microorganisms traveling to space experience stress from environmental features such as ionizing radiation and lack of normal gravity, and much remains unknown about the mechanisms by which those environmental features affect microbial physiology. Microbes experience changes in gravity not directly but rather through changes in their fluid environment, and deep-space particle radiation causes cell damage that is complex but rare. Computational modeling at the single-cell level (agent-based modeling) can allow us to probe the spatially heterogeneous processes that characterize space stresses, to gain insight into the relationships of microbial cells with their environments and with each other. Here we present two software packages for simulating microbial population dynamics in space conditions: CAMDLES and AMMPER. Microbes growing in liquid culture medium in the microgravity of an orbital space station experience a quiescent, poorly-mixed fluid environment. CAMDLES (CFD-DEM Artificial Microgravity Developments for Living Ecosystem Simulation) simultaneously simulates biological, chemical, and mechanical processes to predict microbial ecological dynamics in microgravity, and in the rotating culture vessels used to create an artificial microgravity environment in the lab. Initial results demonstrate that the growth of a cross-feeding microbial consortium, dependent on the exchange of soluble metabolites, is sensitive to the initial spatial distribution of cells, and grows differently in real versus artificial microgravity. Microbial populations exposed to deep-space radiation experience spatially and temporally heterogeneous damage from the traversal of high-energy particles. AMMPER (Agent-Based Model for Microbial Populations Exposed to Radiation) pairs a 3d model of energy deposition along a radiation particle track with a microbial population growth and damage model to predict the effects of localized radiation damage on population-level responses. It includes a user-friendly graphical interface. AMMPER growth curves recapitulate experimental results, and allow comparison between direct effects (DNA damage) and indirect effects (reactive oxygen species generation, metabolic impairment) of radiation.

microbiology↗

NASA NTRS2018

How to Kill a Tardigrade - Without Even Trying

Tardigrades are small aquatic animals that are known for their ability to tolerate extreme dessication as well as ionizing radiation. The extent to which different tardigrade species are able to survive extreme doses of radiation has been previously defined, yet the molecular mechanisms underlying such radiation resistance has not been fully characterized. In Ramazzottius varieornatus, high dose radiation resistance been attributed to the presence of a tardigrade-unique DNA-associated protein Dsup, a protein that facilitates in the reduction of DNA fragmentation immediately after radiation exposure. This suggests that tardigrades possess a unique set of proteins that confer enhanced DNA protection as opposed to DNA repair. Previous studies have suggested that tolerance to radiation resistance in the tardigrade Hypsibius dujardini is inversely correlated with cellular division and mitotic activity, yet the molecular mechanisms and identities of such radiation resistance are poorly understood. In the current study, we plan to examine DNA damage by X-ray irradiation of metabolically active Hypsibius dujardini at three different developmental stages (egg, juvenile and adult) to quantitate the relative amount of double-strand breaks per unit DNA. These values will be compared to quantitation using Deinococcus radiodurans and Saccharomyces cerevisiae, at similar X-ray doses. X-ray exposure of D. radiodurans induces many double-stranded DNA breaks from which recovers by efficient repair. S. cerevisiae is not inherently radiation tolerant. Protection of DNA would be evidenced by reduced numbers of DNA double strand breaks in H. dujardini per unit DNA relative to the other two species.

Tardigrades↗

NASA NTRS2011

What Threats to Human Health Does Space Radiation Pose in Orbit

The Space Shuttle program spanned more than the entire length of a solar cycle. Investigations aimed towards understanding the health risks of the astronauts from exposures to space radiation involved mostly physical measurements of the dose and the linear energy transfer (LET) spectrum. Measurement of the dose rate on the Shuttle provided invariable new data for different periods of the solar cycle, whereas measurement of the LET spectrum using the tissue equivalent proportional counter (TEPC) produced the most complete mapping of the radiation environment of the low Earth orbits (LEO). Exposures to the Shuttle astronauts were measured by the personal dosimeter worn by the crewmembers. Analysis of over 300 personal dosimeter readings indicated a dependence on the mission duration, the altitude and inclination of the orbit, and the solar cycle, with the crewmembers on the launch and repair of the Hubble telescope receiving the highest doses due to the altitude of the mission. Secondary neutrons inside the Shuttle were determined by recoil protons or with Bonner spheres, and may contribute significantly to the risks of the crewmembers. In addition, the skin dose and the doses received at different organs were compared using a human phantom onboard a Shuttle mission. A number of radiobiology investigations wer e also performed. The biological doses were determined on six astronauts/cosmonauts on long-duration Shuttle/Mir missions and on two crewmembers on a Hubble repair mission by analyzing the damages in the chromosomes of the crewmembers? white blood cells. Several experiments were also conducted to address the question of possible synergistic effects of spaceflight, microgravity in particular, on the repair of radiation-induced DNA damages. The experimental design included exposure of cells before launch, during flight, or after landing. These physical and biological studies were invaluable in predicting the health risks for astronauts on ISS and future exploration missions. Educational Objectives: A group of high school students flew color negative films on tw o Shuttle missions to detect the radiation environment in orbit. This and other experiments onboard of the Shuttle were aimed at educating the general public of the space program.

Wu, Honglu↗

DOE OSTI2024

Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain

Abstract We lack the fundamental information needed to understand how DNA damage in the brain is generated and how it is controlled over a lifetime in the absence of replication check points. To address these questions, here, we integrate cell-type and region-specific features of DNA repair activity in the normal brain. The brain has the same repair proteins as other tissues, but normal, canonical repair activity is unequal and is characterized by high base excision repair (BER) and low double strand break repair (DSBR). The natural imbalance creates conditions where single strand breaks (SSBs) can convert to double strand breaks (DSBs) and reversibly switch between states in response to oxidation both in vivo and in vitro. Our data suggest that, in a normal background of repair, SSBs and DSBs are in an equilibrium which is pushed or pulled by metabolic state. Interconversion of SSB to DSBs provides a physiological check point, which would allow the formation of unrepaired DSBs for productive functions, but would also restrict them from exceeding tolerable limits.

Science & Technology - Other Topics↗

NASA NTRSDate not supplied

Chimeric Mouse Models for Space Radiation Risk Investigations

Assessment of human health risks associated with space radiation exposure is based largely on the knowledge learned from studies in which animals, mostly rodents, are exposed to high-LET radiation on the ground. It has been recognized that translation of animal results to meaningful implications for human disease can be challenging, particularly for certain risk categories such as the high-LET radiation effects in the central nervous system (CNS). Considering limitations in utilizing non-human primates and clinical studies in humans, chimeric animals can potentially bridge the knowledge gap between rodents and humans. In a chimeric animal, a specific organ or a cell type is replaced with respective human cells that are functional. A number of chimeric mouse models have been developed in the medical research community to study human diseases, and some of the models can potentially be used for NASA applications. Here we investigate use of mice engrafted with human hepatocytes, which have been used in studies of genotoxicity from carcinogen exposures, for assessment of space radiation damage. In a pilot study, we use PXB mice whose livers contain >90% human cells and have been found to function nearly identically to human liver tissues. These mice were exposed to gamma rays for investigations of DNA damage, transcriptomics, and histopathological changes in the humanized livers. Results obtained from PXB mice were compared non-engrafted control animals from the same background strain that are exposed to identical conditions. Preliminary data from histopathological analysis suggest chimeric mouse models are suitable for investigations of space radiation risks, as the humanized liver tissue exhibited changes induced by radiation and was markedly distinct from the liver tissue from the control animals (Fox Chase SCID mice).

Radiation effects↗

NASA NTRSDate not supplied

Chimeric Mouse Models for Space Radiation Risk Investigations

Radiation effects↗

NASA NTRSDate not supplied

Agent-based modeling of microbes in space

Space is tough on organisms. Microorganisms traveling to space experience stress from environmental features such as ionizing radiation and lack of normal microgravity; however, much remains unknown about the mechanisms by which those environmental features affect microbial physiology. Microbes experience changes in gravity not directly but rather through changes in their fluid environment, and deep-space particle radiation causes cell damage that is complex but rare. Computational modeling at the single-cell level (agent-based modeling) can allow us to probe the spatially heterogeneous processes that characterize space stresses, to gain insight into the relationships of microbial cells with their environments and with each other. Here we present two software packages for simulating microbial population dynamics in space conditions: CAMDLES and AMMPER. Microbes growing in liquid culture medium in the microgravity of an orbital space station experience a quiescent, poorly-mixed fluid environment. CAMDLES (CFD-DEM Artificial Microgravity Developments for Living Ecosystem Simulation) simultaneously simulates biological, chemical, and mechanical processes to predict microbial ecological dynamics in microgravity, and in the rotating culture vessels used to create an artificial microgravity environment in the lab. Initial results demonstrate that the growth of a cross-feeding microbial consortium, dependent on the exchange of soluble metabolites, is sensitive to the initial spatial distribution of cells, and grows differently in real versus artificial microgravity. Microbial populations exposed to deep-space radiation experience spatially and temporally heterogeneous damage from the traversal of high-energy particles. AMMPER (Agent-Based Model for Microbial Populations Exposed to Radiation) pairs a 3d model of energy deposition along a radiation particle track with a microbial population growth and damage model to predict the effects of localized radiation damage on population-level responses. It includes a user-friendly graphical interface. AMMPER results agree with experimental data indicating that indirect effects of radiation (reactive oxygen species generation, metabolic impairment) have a greater impact on microorganisms than direct effects (DNA damage).

Jessica A Lee↗

NASA NTRS2004

Induction and quantification of gammma-H2AX foci following cx- and gamma-irradiaton

Following DNA damage the histone H2AX becomes phosphorylated and can be visualised by immunofluorescence as an indicator of DSBs in individual cells. Using a wild type hamster cell line (V79-4) exposed to either a-particles or to Co-60 gamma-rays to induce DNA DSBs at different doses (20-200OmGy), the dose dependent induction of gamma-H2AX foci were scored both manually (by eye) and using image analysis. A linearly dependence on dose was found for both radiations. The number of DSBs determined by image analysis after a post-irradiation period of 30 minutes at 37 C, is 16.6 foci/cell/Gy for alpha-irradiation and 12.2 foci/cell/Gy for gamma-irradiation; the latter being 3-4 times the levels observed by eye and comparable to gamma-radiation-induced levels of prompt DSBs more recently reported using pulse field gel electrophoresis (approx. 16 DSBs/Gy). The average size of the gamma-H2AX foci induced by alpha-irradiation (0.30 square micrometers) is approximately 1.5 times larger than those induced by gamma-irradiation (0.19 square micrometers). The timescale of induction and removal of DSBs up to 24 hours post-irradiation, was investigated with gamma-H2AX foci levels found to remain significantly higher than controls for 4 or 6 hours in gamma-irradiated samples or alpha irradiated samples, respectively. These results demonstrate that not only gamma radiation but also alpha-radiation induce phosphorylation of the H2AX histone in response to DSBs even at low doses (20mGy for gamma-rays, 1 track/cell on average for alpha-particles) and the variation in size and dephosphorylation of the induced foci is dependent on radiation quality (LET).

Leatherbarrow, E. L.↗