NOTE: Changed Division and Discipline/Program to HRP as of FY2006, per program changes at that time, per JSC/A. Chu-ARC (jvp 4/2009)

Aim 1. To test the hypothesis that shielded heavy ions may result in more complex DNA damage to the cells as compared to unshielded heavy ions,

Aim 2. To test the hypothesis that the molecular response to shielded radiation is different from that induced by unshielded radiation, and

Aim 3. To test the hypothesis that shielded radiation may have more deleterious effects on the cell as compared to unshielded radiation and to elucidate the mechanisms involved in repair of DNA damage.

Brief summary of progress: In experiments carried out during the first two years of the project we were able to establish the methods that would be required for successful completion of the project. We were also able to obtain preliminary results that allowed us to estimate the feasibility of the proposed objectives. In the third and fourth years, significant progress was made in most of the proposed aims of the project, resulting in a manuscript that was accepted for publication in DNA Repair. In the fifth and final year we expanded upon the results obtained in the first four years to ask questions pertaining to the long-term consequences of particle radiation, i.e., cellular transformation and carcinogenesis. This has resulted in an additional publication in Carcinogenesis. In sum, we have successfully carried out most of experiments proposed in this project and have also carried out additional experiments to further extend our understanding of the long-term consequences of irradiation by heavy ions and its modulation by shielding. These results have been published in relevant journals.

Detailed summary of progress. We have used pertinent responses to DNA double-strand breaks (DSBs) to understand the consequences of energy loss versus nuclear fragmentation of Fe ions during passage through shielding or tissue-equivalent materials. Phosphorylation of histone H2AX and recruitment of 53BP1 were used to generate 3D reconstructions of DNA damage in human cells and to follow its repair. Human cells are unable to repair a significant portion of DNA damage induced by Fe ions. DNA-PK and ATM are required, to different extents, for the partial repair of Fe-induced DNA damage. Aluminum shielding has little effect on DNA damage or its repair, confirming that the hulls of the Space Shuttle and the International Space Station afford scant protection against these particles. Lead shielding, on the other hand, exacerbates the effects of Fe ions due to energy loss during particle traversal. In sharp contrast, polyethylene (PE), a favored hydrogenous shield, results in DNA damage that is more amenable to repair presumably due to Fe ion fragmentation. Human cells are indeed able to efficiently repair DSBs induced by chlorine ions and protons that represent fragmentation products of Fe. Interestingly, activation of the tumor suppressor p53 in Fe-irradiated cells is uniquely biphasic and culminates in the induction of high levels of p21(Waf1/Cip1), p16(INK4a) and senescence-associated beta-galactosidase activity. Surprisingly, these events occur even in the absence of ATM kinase implying that ATR may be a major responder to the complex DNA damage inflicted by Fe ions. Significantly, fragmentation of the Fe beam through PE attenuates these responses and this, in turn, results in better long-term survival in a colony forming assay. Our results help us to understand the biological consequences of ion fragmentation through materials and provide us with a biological basis for the use of hydrogenous materials like PE as effective space shields. However, it is important to point out the caveat that even after extensive fragmentation of the Fe beam through PE, a small portion of (presumably complex) DSBs are still not repaired by human cells and result in persistent (though attenuated) DNA damage signaling. The improved survival of cells irradiated through PE coupled with the presence of persistent DNA lesions raises the specter of genomic instability in these surviving cells which could eventually trigger cellular transformation. Therefore, it is very important to understand the long-term consequences of such unrepaired DNA lesions (i.e. carcinogenesis) using pertinent model systems. In preliminary studies carried out in the final year of this project, we developed a sensitive in vitro model system (“pre-sensitized” cells with targeted deletions of the Ink4a/Arf tumor suppressor locus). Using this model system and in vitro assays for cellular transformation (growth in soft agar), we find that these cells can indeed be transformed by Fe ions even after fragmentation through PE shielding. In the future, it would be important to evaluate the contribution of Fe particles with or without shielding to carcinogenesis in vivo using mouse models of cancer. In sum, our results show that fragmentation of heavy ions through shielding can significantly alter the biological responses of human cells to the ensuing DNA damage.

Peer reviewed publications from the current NASA funding period (2005-2010):

Research Articles

1. Cell cycle dependence of DNA-PK phosphorylation in response to DNA double-strand breaks. B. Chen, D.W. Chan, J. Kobayashi, S. Burma, A. Asaithamby, K. Morotomi-Yano, E. Botvinick, J. Qin, and D.J. Chen Journal of Biological Chemistry 280:14709-14715 (2005)

2. Gene expression profiles of normal human fibroblasts after ionizing radiation: a comparative study with low and high doses. L.-H. Ding, M. Shingyoji, F. Chen, J.-J. Hwang, S. Burma, J.-F. Cheng, and D. J. Chen Radiation Research 164:17-26 (2005)

3. Effect of Ku proteins on IRES-mediated translation. D. Siilvera, N. Koloteva-Levine, S. Burma, and O. Elroy-Stein Biology of the Cell 98:353-361 (2006)

4. DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells. B. Mukherjee, C. Kessinger, J. Kobayashi, B.P. Chen, D.J. Chen, A. Chatterje, and S. Burma DNA Repair 5:575-590 (2006)

5. Nucleophosmin suppresses oncogene-induced apoptosis and senescence and enhances oncogenic cooperation in cells with genomic instability. J. Li, D.P. Sejas, S. Burma, D.J. Chen, and Q. Pang Carcinogenesis 28:1163-1170 (2007)

6. Ku70/80 modulates ATM and ATR signaling pathways in response to DNA double-strand breaks.N. Tomimatsu, G.G. Tahimic, A. Otsuki, S. Burma, A. Fukuhara, K. Sato, G. Shiota, M. Oshimura, D.J. Chen, and A. Kurimasa Journal of Biological Chemistry 282:10138-10145 (2007)

7. Repair of HZE-partcle-induced DNA double-strand breaks in normal human fibroblasts. A. Asaithamby, N. Uematsu, A. Chatterjee, M.D. Story, S. Burma, and D.J. Chen Radiation Research 169:437-446 (2008)

8. Modulation of the DNA-damage response to HZE particles by shielding. B. Mukherjee, C.V. Camcho, N. Tomimatsu, J. Miller, and S. Burma DNA Repair 7:1717-1730 (2008)

9. Phosphorylation of Exo1 modlates homolgous recombination repair of DNA double-strand breaks. E. Bolderson, N. Tomimatsu, D.J. Richards, D. Boucher, R. Kumar, T.K. Pandita, S. Burma, K.K. Khanna Nucleic Acids Res. 38:1821-1831(2009)

10. Histone H2AX participates in the DNA damage-induced ATM activation through interaction with Nbs1. J. Kobayashi, H. Tauchi, B. Chen, S. Burma, S. Tashiro, S. Matsuura, K. Tanimoto, D.J. Chen, K. Komatsu Biochem. Biphys. Res. Commun. 380:752-757 (2009)

11. RIP-1 activates PI3K-Akt via a dual mechanism involving NF-kB-mediated inhibition of mTOR-S6K-IRS1 negative feedback loop and down-regulation of PTEN. S. Park, D. Zhao, K.J. Hatanpaa, B.E. Mickey, D. Saha, D.A. Boothman, M.D. Story, E.T. Wong, S. Burma, M-M. Georgescu, V.M. Rangnekar, S.S. Chauncey, and A.A. Habib Cancer Research 69:4107-4111 (2009)

12. Distinct roles of ATR and DNA-PKcs in triggering DNA damage responses in ATM-deficient cells. N. Tomimatsu, B. Mukherjee, and S. Burma EMBO Reports 10:629-635 (2009)

13. EGFRvIII and DNA Double-Strand Break Repair: A Molecular Mechanism for Radioresistance in Glioblastoma. B. Mukherjee, B. McEllin, C.V. Camacho, N. Tomimatsu, S. Sirasanagandala, S. Nannepaga, K.J. Hatanpaa, B. Mickey, C. Madden, E. Maher, D.A. Boothman, F. Furnari, W.K. Cavenee, R.M. Bachoo, and S. Burma Cancer Research 69:4252-4259 (2009)

14. Down-regulation of human DAB2IP gene expression in prostate cancer cells results in resistance to ionizing radiation. Z. Kong, D. Xie, T. Boike, P. Raghavan, S. Burma, D.J. Chen, A.A. Habib, A. Chakraborty, J-T. Hsieh and D. Saha Cancer Research 70:2829-2839 (2010)

15. WRN participates in translesion synthesis pathway through interaction with NBS1. J. Kobayashi, M. Okui, A. Asaithamby, S. Burma, B. Chen, K. Tanimoto, S. Matsuura, K. Komatsu, D.J. Chen Mech Ageing Dev. 131:436-444 (2010)

16. Epothilone B confers radiation dose enhancement in DAB2IP gene knock-down radioresistant prostate cancer cells. Z. Kong, P. Raghavan, D. Xie, T. Boike, S. Burma, D.J. Chen, A. Chakraborty, J-T. Hsieh and D. Saha Int. J. Radiation Oncology Biol. Phys. 20:250-257 (2010)

17. Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks. C. V. Camacho, B. Mukherjee, B. McEllin, L-H. Ding, B. Hu, A. Habib, X-J. Xie, C. Nirodi, D. Saha, M. Story, A. Balajee, R. M. Bachoo, D. A. Boothman, and S. Burma Carcinogenesis 31:1889-1896 (2010)

18. PTEN loss compromises homologous recombination repair in astrocytes: implications for glioblastoma therapy with temozolomide or PARP inhibitors. B. McEllin, C.V. Camacho, B. Mukherjee, B. Hahm, N. Tomimatsu, R.M. Bachoo, and S. Burma Cancer Research 70:5457-5464 (2010)

Invited Reviews and Book Chapters

1. Role of non-homologous end joining (NHEJ) in maintaining genomic stability in mammalian cells. S. Burma, B.P. Chen, and D.J. Chen DNA Repair 5:1042-1048 (2006)

2. Role of non-homologous end joining in the repair of DNA double-strand breaks. S. Burma, B. Chen, D.J. Chen in DNA Repair Genetic Instability and Cancer (Q. Wei, L. Li, and D.J. Chen, eds), World Scientific Publishing Co., p157-175 (2007)

3. Epidermal growth factor receptor in glioma: signal transduction, neuropathology, imaging, and radioresistance. K.J. Hatanpaa, S. Burma, D. Zhao, A.A. Habib Neoplasia 12:675-684 (2010)

4. Targeting Non-Homologous End-Joining Through Epidermal Growth Factor Receptor Inhibition: Rationale and Strategies for Radiosensitization. B. Mukherjee, H. Choy, C. Nirodi, and S. Burma Seminars in Radiation Oncology 20:250-257 (2010)

NOTE: Changed Division and Discipline/Program to HRP as of FY2006, per program changes at that time, per JSC/A. Chu-ARC (jvp 4/2009)

Specific Aims: 1) To test the hypothesis that shielded heavy ions may result in more complex DNA damage to the cells as compared to unshielded heavy ions, 2) To test the hypothesis that the molecular response to shielded radiation is different from that induced by unshielded radiation, and 3) To test the hypothesis that shielded radiation may have more deleterious effects on the cell as compared to unshielded radiation and to elucidate the mechanisms involved in repair of DNA damage. Brief summary of progress. In experiments carried out during the first two years of the project we were able to establish the methods that would be required for successful completion of the project. We were also able to obtain preliminary results that allowed us to estimate the feasibility of the proposed objectives. In the third year (2007-2008), significant progress was made in most of the proposed aims of the project, resulting in a manuscript that was accepted for publication in DNA Repair. These results are detailed below along with plans for the coming year.

Detailed summary of progress. Ions of high atomic number and energy (HZE particles) pose a significant cancer risk to astronauts on prolonged space missions. The properties of these particles can be drastically altered during passage through spacecraft shielding, therapy beam modulators, or the human body. In this project, we have used pertinent responses to DNA double-strand breaks (DSBs) to understand the consequences of energy loss versus nuclear fragmentation of Fe ions during passage through shielding or tissue-equivalent materials. Phosphorylation of histone H2AX and recruitment of 53BP1 were used to generate 3D reconstructions of DNA damage in human cells and to follow its repair. Human cells are unable to repair a significant portion of DNA damage induced by Fe ions. DNA-PK and ATM are required, to different extents, for the partial repair of Fe-induced DNA damage. Aluminum shielding has little effect on DNA damage or its repair, confirming that the hulls of the Space Shuttle and the International Space Station afford scant protection against these particles. Lead shielding, on the other hand, exacerbates the effects of Fe ions due to energy loss during particle traversal. In sharp contrast, polyethylene (PE), a favored hydrogenous shield, results in DNA damage that is more amenable to repair presumably due to Fe ion fragmentation. Human cells are indeed able to efficiently repair DSBs induced by chlorine ions and protons that represent fragmentation products of Fe. Interestingly, activation of the tumor suppressor p53 in these cells is uniquely biphasic and culminates in the induction of high levels of p21(Waf1/Cip1), p16(INK4a) and senescence-associated beta-galactosidase activity. Surprisingly, these events occur even in the absence of ATM kinase implying that ATR may be a major responder to the complex DNA damage inflicted by Fe ions.

Significantly, fragmentation of the Fe beam through PE attenuates these responses and this, in turn, results in better long-term survival in a colony forming assay. Our results help us to understand the biological consequences of ion fragmentation through materials and provide us with a biological basis for the use of hydrogenous materials like PE as effective space shields.

Future plans. The long-term goal would be evaluate the contribution of Fe particles with or without shielding to carcinogenesis using models currently being developed in my laboratory. As a model system, we have used “primed” astrocytes bearing some (but not all) of the mutations that would lead to the development of glioblastomas (aggressive brain tumors). These “pre-initiated” cells normally do not form tumors in nude mice. We find, however, that irradiation of these cells with Fe ions results in tumor formation. We can, therefore, use this model system to evaluate the effectiveness of relevant shielding materials.

NOTE: Changed Division and Discipline/Program to HRP as of FY2006, per program changes at that time, per JSC/A. Chu-ARC (jvp 4/2009)

Summary of preliminary results obtained.

Preliminary studies carried out during NSRL6A and 6B runs indicated that significant differences might exist between DNA damage caused by unshielded Fe particles versus particles that have passed through different shielding materials. These preliminary results were reconfirmed during the NSRL 6C, 7A and 7B runs. In addition, studies were initiated to elucidate the molecular and cellular consequences of DNA damage by the fragmentation products of Fe ions that have traversed shielding materials.

General Experimental Strategy: Cells, Irradiation, Dosimetry and Shielding

In this study, we used Fe (56) ions with a kinetic energy of approximately 1 GeV/nucleon at the NSRL (LET 150 keV/micrometers) to irradiate human skin fibroblasts (HSFs) after traversal through specific shielding materials. Two end points were used to visualize DSBs and to quantify its repair: 1) phosphorylation of histone H2AX and 2) recruitment of 53BP1. To understand the long-term consequences of irradiation of cells with fragmented Fe ions, endpoints such as activation of cell cycle checkpoints, senescence and cell death were quantified. For this study, we chose three different materials – 3 cm of aluminum (Al, Z=13), 3 cm of lead (Pb, Z=82), and 19 cm of polyethylene (CH2; Z of C=6; Z of H=1). 3 cm Al is representative of hull material on the Space Shuttle and the International Space Station (ISS) and does not substantially fragment or change the LET of HZE particles traversing it. 3 cm Pb, increases the LET of 1 GeV/nucleon Fe from 150 to almost 200 keV/micrometers with modest fragmentation. 19 cm of polyethylene (PE), a favored shielding material due to its high hydrogen content and a tissue surrogate for the same reason, was calculated to fragment most of the Fe beam; however, the surviving beam particles have approximately the same LET as beam particles after passing through 3 cm Pb. The materials were placed 3 cm upstream of the biological sample and surviving beam ions and charged fragmentation products were measured by solid state detectors at the same time as the biological experiments.

Aim 1. To test the hypothesis that fragmented heavy ions may result in more complex DNA damage to the cells as compared to unfragmented heavy ions.

1. In order to establish a baseline with which to compare the effect of shielding on Fe ions, we visualized the extent of DNA damage induced by 1 GeV Fe and compared this with damage induced by gamma rays. Immunofluorescence staining of irradiated cells in combination with confocal microscopy and modeling of Z-stacks with Imaris software was utilized to generate 3D reconstructions of DNA damage areas.

2. We irradiated cells through the different shielding materials and generated 3D reconstructions of the ensuing DNA damage. Cells irradiated through Al, i.e. by radiation only slightly modified compared to the beam, typically exhibit dense tracks of DNA damage indistinguishable from those produced by the beam alone. Irradiation behind Pb results in denser patterns of DNA damage, presumably due to the higher LET of the surviving beam particles. By contrast, the majority of cells irradiated behind PE do not exhibit discrete DNA-damage tracks, even though the LET of the surviving beam particles is almost the same as with Pb; diffuse areas of DNA damage, heterogeneous in their volume and distribution, are observed instead.

3. We find that cells irradiated with 1 Gy of gamma rays are mostly able to complete DSB repair by 12 h as has been demonstrated before. In contrast, cells irradiated with 1 Gy of Fe ions are unable to repair approximately 30 percent of the initial DNA damage incurred by 12 h; no evidence of further repair is seen after 12 h. We next tried to answer the question of whether fragmentation of a high LET ion into multiple particles, albeit of lower LETs, affects the ability of a cell to repair the ensuing DNA damage. We find that Al has little effect on DNA repair kinetics, which suggests that, by this criterion, the ISS hull affords little protection against HZE. Pb results in even slower kinetics of DNA repair, consistent with the observed increase in LET. The heterogeneous mix of DNA damage induced downstream of PE are repaired with somewhat faster kinetics; we assume that the faster repair is due to the generation of particles with lower LET due to fragmentation through PE; however, the DNA damage is not repaired to completion. Aim 2. To test the hypothesis that the molecular response to mixed field radiation due to nuclear fragmentation is different from that induced by homogeneous heavy ion radiation.

1. In pilot experiments, we have been able successfully study the localization and activation of DNA-PK at Fe particle induced damage with/without shielding. Studies involving ATM are planned for future runs.

2. We have been able to study the co-localization of 53BP1 with H2AX in pilot studies involving beam modification through the three different shielding materials. Detailed experiments on other modulators are being planned for the NSRL8 runs.

3. We examined the activation of p53 as a first step towards understanding the long-term consequences of the failure to repair Fe-induced DNA damage especially those caused by particle fragmentation. HSFs irradiated with gamma rays display rapid and transient p53 accumulation and phosphorylation at ser 15 and transient induction of the cyclin-dependent kinase (CDK) inhibitor p21 (Waf1/Cip1), a downstream target of p53. In contrast, cells irradiated with the unmodified Fe beam display a biphasic response with an initial transient response similar to that seen with gamma rays and a second sustained response starting at about 2 days post-irradiation and lasting for at least 10 days. A high level of the CDK inhibitor p16 (INK4a), that independently arrests cell proliferation in response to stress, is also induced during this stage. The p53 response pattern is largely unaltered after irradiation by the relatively unfragmented beams produced by Al or Pb. Interestingly, irradiation through PE shielding also results in a biphasic response; however, the second phase is modestly attenuated.

Aim 3. To test the hypothesis that mixed field radiation may have more deleterious effects on the cell as compared to unfragmented radiation and to elucidate the mechanisms involved in repair of DNA damage.

1. We have initiated studies with cell lines deficient in specific DNA repair pathways to understand the contribution of these pathways to the repair of fragmented radaition. Specifically, we are utilizing knockout mouse cells with the following genotypes: wild type, DNA-PKcs-/-, and Atm-/-. As a first step, we have characterized the DNA repair response of these lines to proton irradiation to establish the inherent repair capabilities of the lines. As expected, repair of proton-induced DNA damage follows slower kinetics in DNA-PK-/- cells. In future runs we plan to use these lines to delineate the contribution of specific pathways in the repair of fragmented radiation.

2. Detailed colony survival assays were carried out with HSF cells irradiated with gamma rays, 1 GeV protons, 1 GeV Chlorine, and 1 GeV Fe (with no shielding or with Al, Pb or PE shielding) without any shielding or with Al, Pb, or PE shielding. HSFs are extremely sensitive to Fe ions as compared to gamma rays or protons while Cl ions result in intermediate radiation sensitivity which is in keeping with the LETs of these individual ions. As would be expected from our results, modifying the incident particle by passage through the various shields does not result in major differences in survival over unshielded ions.

3. We have characterized the induction of senescence in HSFs as a consequence of radiation using the Senescence-associated beta-galactosidase activity assay. We find that Fe-irradiated cells display a very high percentage of intensely-staining SA beta-Gal-positive cells at 10 d post-irradiation (~84%). This is clearly a consequence of the sustained p53 activation and p21/p16 induction observed in these cells. Induction of senescence is largely unaltered after irradiation by the relatively unfragmented beams produced by Al or Pb (data not shown). Consequently, irradiation by a highly fragmented beam (through PE) results in a modest decrease in the percentage of SA beta-Gal-positive cells at 10 day post-irradiation (~67%) thereby validating the importance, from a radiation protection standpoint, of shielding material that can fragment the incident heavy ion.

Detailed progress:

1. DNA damage induced by Fe particles in comparison to gamma-rays. As a first step we have sought to establish the extent of DNA damage induced by 1 GeV Fe and to compare this with damage induced by gamma rays. We chose to irradiate primary, early passage human skin fibroblasts (HSFs) as these cells exhibit very low levels of background DNA double-strand breaks (DSBs). To examine the induction of DSBs and its repair we used two early DNA damage response proteins as end points: gamma-H2AX (the phosphorylated version of histone H2AX, modified specifically at the sites of DSBs) and p53BP1 (the DNA-damage signaling protein that is recruited specifically to DSBs). We immunofluorescence (IF) stained HSF cells irradiated with gamma-rays or with 1 GeV Fe ions. The damage induced was visualized by laser confocal scanning microscopy of irradiated cells co-immunostained with antibodies to H2AX (red) and BP1 (green). While X-rays resulted in discrete but diffuse DSBs, Fe particles resulted in tracks of DNA damage presumably corresponding to the path of particle traversal.

2. 3D reconstruction of DNA damage induced by Fe particles compared to gamma-rays. Optical slices (Z-stacks) taken of these images were reconstructed to generate 3D images of the DNA damage-tracks. The extensive nature of DNA damage induced by Fe particles, as compared to X-rays, is clearly evident from these reconstructions.

3. Detection of fragmentation of Fe particles through shielding material. In collaboration with Dr. Jack Miller (Lawrence Berkeley National Laboratory), we are comparing the two consequences of traversal of Fe particles through shielding material – 1) fragmentation into smaller particles with generally less deleterious effects than the parent ion and 2) retardation of an unfragmented particle through matter resulting in more deleterious particles with higher LET (linear energy tranfer). In preliminary studies, we used two shielding materials: 1) 3 cm Pb (which retarded the incident Fe ion increasing its LET from 150 keV/micron to approximately 200 keV/micron and 2) 19 cm polyethylene (PE) (which fragmented the majority of incident ions into smaller particles; the unfragmented minority had an LET of 200 keV/micron).

4. Visualization of the effects of traversal of Fe particles through shielding material. We sought to visualize DNA damage induced by Fe particles that have traversed through these shielding materials and to use these end points to estimate the efficacy of space shielding materials. 3D images clearly indicate that while Pb exacerbates the DNA damage induced by Fe, PE results in the fragmentation of the incident ion into smaller particles as evident by the replacement of discrete DNA-damage tracks with diffuse DNA damage. 5. Repair of DNA damage induced by Fe particles after traversal through shielding materials. Based upon our 3D models of DNA damage and beam fragmentation studies, we concluded that beam fragmentation through PE may alleviate the effects of Fe particle irradiation. Thus, we hypothesized that PE may be a better shielding material as compared to Aluminum (the existing shielding material in space crafts and space stations). We attempted to verify this by quantifying the kinetics of DNA damage induced by Fe particles after traversal through shielding materials (using the dissolution of H2AX and 53BP1 foci as end points). Indeed, we find that DNA damage induced by Fe particles is more deleterious to the cell as a large fraction of this damage is unrepaired (in contrast to X-ray induced damage) even at 24h. While 3 cm Al shielding has no beneficial effects, PE shielding results in apparently less complex DNA damage taht is repaired with faster kinetics by the cells with less residual DNA damage at 24hrs. These results confirm the efficacy of PE shielding and affirm that these end points can be successfully used to evaluate the efficacy of futuristic shielding materials.

Future plans. The immediate future goal is to examine if the observed (beneficial) effects of PE shielding is due to fragmentation into smaller particles with lower Z and into protons. For this purpose we will quantify and model DNA damage induced by particles with lower Z than Fe (such as chlorine) and by protons to see if the damage induced is less complex and easily repairable. Data from protons obtained during the last run are currently being evaluated and future experiments will involve chlorine and other ions with lower Z. The long term goal would be evaluate the contribution of Fe particles with or without shielding to carcinogenesis using models currently being developed in my laboratory.