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

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.

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