Amy Kronenberg

Heavy-ion radiobiology: new approaches to delineate mechanisms underlying enhanced biological effectiveness

Shortly after the discovery of polonium and radium by Marie Curie and her husband and colleague, Pierre Curie, it was learned that exposure to these alpha-particle emitters produced deleterious biological effects. The mechanisms underlying the increased biological effectiveness of densely ionizing radiations, including alpha particles, neutrons and highly energetic heavy charged particles, remain an active area of investigation. In this paper, we review recent advances in several areas of the radiobiology of these densely ionizing radiations, also known as heavy ions. Advances are described in the areas of DNA damage and repair, chromosome aberrations, mutagenesis, neoplastic transformation in vitro, genomic instability, normal tissue radiobiology and carcinogenesis in vivo. We focus on technical innovations, including novel applications of pulsed-field gel electrophoresis, fluorescence in situ hybridization (FISH), linkage analysis, and studies of gene expression and protein expression. We also highlight the use of new cellular and animal systems, including those with defined DNA repair deficiencies, as well as epithelial cell model systems to assess neoplastic transformation both in vitro and in vivo. The studies reviewed herein have had a substantial impact on our understanding of the genotoxic effects of heavy ions as well as their distinct effects on tissue homeostasis. The use of these radiations in cancer therapy is also discussed. The use of both heavy-ion and proton therapy is on the upswing in several centers around the world, due to their unique energy deposition characteristics that enhance the therapeutic effect and help reduce damage to normal tissue.

Homologous Recombination Contributes to the Repair of DNA Double-Strand Breaks Induced by High-Energy Iron Ions

Abstract To test the contribution of homologous recombinational repair (HRR) in repairing DNA damage sites induced by high-energy iron ions, we used (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We found that in response to exposure to iron ions, HRR contributed to cell survival in rodent cells and that HRR deficiency abrogated RAD51 focus formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 focus formation. For human cells irradiated with iron ions, cell survival was decreased, and in p53 mutant cells, the levels of mutagenesis were increased when HRR was impaired. Human cells synchronized in S phase exhibited a more pronounced resistance to iron ions compared with cells in G1 phase, and this increase in radioresistance was diminished by RAD51 knockdown. These results indicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged-particle radiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival after exposure to high-energy high-LET radiation.

Heavy ion mutagenesis: linear energy transfer effects and genetic linkage.

We have characterized a series of 69 independent mutants at the endogenoushprt locus of human TK6 lymphoblasts and over 200 independent S 1-deficient mutants of the humanxhamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/µm). We find that large deletions are common. The entirehprt gene (>44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entirehprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency ofhprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of thehprt locus to mutation is likely due to tight linkage to a gene that is required for viability.

Understanding cancer development processes after HZE-particle exposure: roles of ROS, DNA damage repair and inflammation

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.

Karyotypic Instability and Centrosome Aberrations in the Progeny of Finite Life-Span Human Mammary Epithelial Cells Exposed to Sparsely or Densely Ionizing Radiation

Abstract Sudo, H., Garbe, J., Stampfer, M. R., Barcellos-Hoff, M. H. and Kronenberg, A. Karyotypic Instability and Centrosome Aberrations in the Progeny of Finite Life-Span Human Mammary Epithelial Cells Exposed to Sparsely or Densely Ionizing Radiation. Radiat. Res. 170, 23–32 (2008). The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer.

Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions.

We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

Comparative Analysis of Cell Killing and Autosomal Mutation in Mouse Kidney Epithelium Exposed to 1 GeV/nucleon Iron Ions In Vitro or In Situ

Abstract Astronauts receive exposures to high-energy heavy ions from galactic cosmic radiation. Although high-energy heavy ions are mutagenic and carcinogenic, their mutagenic potency in epithelial cells, where most human cancers develop, is poorly understood. Mutations are a critical component of human cancer, and mutations involving autosomal loci predominate. This study addresses the cytotoxic and mutagenic effects of 1 GeV/nucleon iron ions in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events contributing to human cancer. Results for kidneys irradiated in situ are compared with results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3–4 months postirradiation in situ, but in situ exposures were less likely to result in cell death than in vitro exposures. Prolonged incubation in situ (8–9 months) further attenuated cell killing at lower doses. Iron ions were mutagenic to cells in vitro and for irradiated kidneys. No sparing was seen for mutant frequency with a long incubation period in situ. In addition, the degree of mutation induction (relative increase over background) was similar for cells exposed in vitro or in situ. We speculate that the latent effects of iron-ion exposure contribute to the maintenance of an elevated mutation burden in an epithelial tissue.

Measuring the Spectrum of Mutation Induced by Nitrogen Ions and Protons in the Human–Hamster Hybrid Cell Line ALC

Abstract Kraemer, S. M., Kronenberg, A., Ueno, A. and Waldren, C. A. Measuring the Spectrum of Mutation Induced by Nitrogen Ions and Protons in the Human–Hamster Hybrid Cell Line ALC. Astronauts can be exposed to charged particles, including protons, α particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human–hamster cell lines, AL and ALC. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human–hamster hybrid cell lines ALC and AL. CD59– mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid ALC, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in ALC cells than in AL cells. In the case of nitrogen ions, the mutations observed in ALC cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59– mutant fraction for ALC cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or “skipping” mutations. We demonstrate here that ALC cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.

Galactic cosmic ray simulation at the NASA Space Radiation Laboratory

Most accelerator-based space radiation experiments have been performed with single ion beams at fixed energies. However, the space radiation environment consists of a wide variety of ion species with a continuous range of energies. Due to recent developments in beam switching technology implemented at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), it is now possible to rapidly switch ion species and energies, allowing for the possibility to more realistically simulate the actual radiation environment found in space. The present paper discusses a variety of issues related to implementation of galactic cosmic ray (GCR) simulation at NSRL, especially for experiments in radiobiology. Advantages and disadvantages of different approaches to developing a GCR simulator are presented. In addition, issues common to both GCR simulation and single beam experiments are compared to issues unique to GCR simulation studies. A set of conclusions is presented as well as a discussion of the technical implementation of GCR simulation.

Comparison of Autosomal Mutations in Mouse Kidney Epithelial Cells Exposed to Iron Ions In Situ or in Culture

Abstract Exposure to accelerated iron ions represents a significant health risk in the deep space environment because it induces mutations that can cause cancer. A mutation assay was used to determine the full spectrum of autosomal mutations induced by exposure to 2 Gy of 1 GeV/nucleon iron ions in intact kidney epithelium, and the results were compared with mutations induced in cells of a kidney epithelial cell line exposed in vitro. A molecular analysis for loss of heterozygosity (LOH) for polymorphic loci on chromosome 8, which harbors Aprt, demonstrated iron-ion induction of mitotic recombination, interstitial deletion, and discontinuous LOH events. Iron-ion-induced deletions were detected more readily with the in vitro assay, whereas discontinuous LOH was detected more readily in the intact kidney. The specific induction of discontinuous LOH in vivo suggests that this mutation pattern may serve as an indicator of genomic instability. Interestingly, the frequency of small intragenic events increased as a function of time after exposure, suggesting non-targeted effects. In total, the results demonstrate that 1 GeV/nucleon iron ions can elicit a variety of autosomal mutations and that the cellular microenvironment and the sampling time after exposure can influence the distribution of these mutations in epithelial cell populations.