Both natural and man-made sources of ionizing radiation contribute to human exposure and consequently pose a possible risk to human health. Much of this is unavoidable, e.g., natural background radiation, but as the use of radiation increases, so does the potential health risk and the public's concerns. This perspective reflects the authors' view of current issues in low dose radiation biology research, highlights some of the controversies therein, and suggests areas of future research to address both issues in low dose radiation research and the controversies. This is a critical time for the radiation sciences and the implications of future research will have a significant impact on radiation protection, medicine, national security, research and industry. The views expressed here are the authors' own and do not represent any institution, organization or funding body.

A long-standing dogma in the radiation sciences is that energy from radiation must be deposited in the cell nucleus to elicit a biological effect. A number of non-targeted, delayed effects of ionizing radiation have been described that challenge this dogma and pose new challenges to evaluating potential hazards associated with radiation exposure. These effects include induced genomic instability and non-targeted bystander effects. The in vitro evidence for non-targeted effects in radiation biology will be reviewed, but the question as to how one extrapolates from these in vitro observations to the risk of radiation-induced adverse health effects such as cancer remains open.

Studies of health effects in animals after exposure to internally deposited radionuclides were intended to supplement observational studies in humans. Both nuclear workers and Beagle dogs have exhibited plutonium-associated lung fibrosis; however, the dogs' smaller gene pool may limit the applicability of findings to humans. Data on Beagles that inhaled either plutonium-238 dioxide ( 238 PuO 2 ) or plutonium-239 dioxide ( 239 PuO 2 ) were analyzed. Wright's Coefficient of Inbreeding was used to measure genetic or familial susceptibility and was assessed as an explanatory variable when modeling the association between lung fibrosis incidence and plutonium exposure. Lung fibrosis was diagnosed in approximately 80% of the exposed dogs compared with 23.7% of the control dogs. The maximum degree of inbreeding was 9.4%. Regardless of isotope, the addition of inbreeding significantly improved the model in female dogs but not in males. In female dogs, an increased inbreeding coefficient predicted decreased hazard of a lung fibrosis diagnosis. Lung fibrosis was common in these dogs with inbreeding affecting models of lung fibrosis incidence in females but not in males. The apparent protective effect in females predicted by these models of lung fibrosis incidence is likely to be minimal given the small degree of inbreeding in these groups.

Repair of DNA damage through homologous recombination (HR) pathways plays a crucial role in maintaining genome stability. However, overstimulation of HR pathways in response to genotoxic stress may abnormally elevate recombination frequencies, leading to increased mutation rates and delayed genomic instability. Radiation-induced genomic instability has been detected after exposure to both low- and high-linear energy transfer (LET) radiations, but the mechanisms responsible for initiating or propagating genomic instability are not known. We have demonstrated that WR-1065, the active metabolite of amifostine, protects against radiation-induced cell killing and delayed genomic instability. We hypothesize that hyperstimulation of HR pathways plays a mechanistic role in radiation-induced genomic instability and that, in part, WR-1065 exerts it radioprotective effect through suppression of the HR pathway. Results of this study demonstrate that WR-1065 treatment selectively protected against radiation-induced cell killing in HR-proficient cell lines compared to an HR-deficient cell line. Further, WR-1065 treatment decreases HR in response to DNA damage using two different mammalian cell systems. This suppression of hyper-recombination is a previously unrecognized mechanism by which WR-1065 effects radioprotection in mammalian cells.

Straume, T., Amundson, S. A., Blakely, W. F., Burns, F. J., Chen, A., Dainiak, N., Franklin, S., Leary, J. A., Loftus, D. J., Morgan, W. F., Pellmar, T. C., Stolc, V., Turteltaub, K. W., Vaughan, A. T., Vijayakumar, S. and Wyrobek, A. J. NASA Radiation Biomarker Workshop. September 27–28, 2007. Radiat. Res. 170, 393–405 (2008). A summary is provided of presentations and discussions at the NASA Radiation Biomarker Workshop held September 27–28, 2007 at NASA Ames Research Center in Mountain View, CA. Invited speakers were distinguished scientists representing key sectors of the radiation research community. Speakers addressed recent developments in the biomarker and biotechnology fields that may provide new opportunities for health-related assessment of radiation-exposed individuals, including those exposed during long-duration space travel. Topics discussed included the space radiation environment, biomarkers of radiation sensitivity and individual susceptibility, molecular signatures of low-dose responses, multivariate analysis of gene expression, biomarkers in biodefense, biomarkers in radiation oncology, biomarkers and triage after large-scale radiological incidents, integrated and multiple biomarker approaches, advances in whole-genome tiling arrays, advances in mass spectrometry proteomics, radiation biodosimetry for estimation of cancer risk in a rat skin model, and confounding factors. A summary of conclusions is provided at the end of the report.

Murley, J. S., Kataoka, Y., Baker, K. L., Diamond, A. M., Morgan, W. F. and Grdina, D. J. Manganese Superoxide Dismutase ( SOD2 )-Mediated Delayed Radioprotection Induced by the Free Thiol Form of Amifostine and Tumor Necrosis Factor α. Radiat. Res. 167, 465–474 (2007). RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 μ M and 4 m M or the cytokine tumor necrosis factor α (TNFα, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFα used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFα treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFα exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 μ M or 4 m M WR-1065 or 10 ng/ml TNFα or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFα were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 μ M WR-1065 or TNFα. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 m M WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 μ M or 4 m M WR-1065 and 20 h after TNFα treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFα.

Nagar, S. and Morgan, W. F. The Death-Inducing Effect and Chromosomal Instability. Radiat. Res. 163, 316–323 (2005). Exposure to ionizing radiation can induce a heritable change in the unirradiated progeny of irradiated cells. This non-targeted effect of ionizing radiation manifests as genomic instability, and although there is some debate as to the role of genomic instability in the carcinogenic process, it is thought by some to be an early step in radiation carcinogenesis. Although the mechanism of induction of genomic instability is not clearly understood, evidence suggests that secreted factors from irradiated cells may be involved. We have previously identified another non-targeted effect of ionizing radiation, the death-inducing effect. Exposure of unirradiated GM10115 cells to medium from chromosomally unstable clones was generally found to be cytotoxic. However, occasionally cells will survive in medium from unstable clones and can be clonally expanded. The absolute yield of survivors is independent of the initial number of cells plated when cell densities reached 5,000 or more cells/dish. After cytogenetic analysis of the surviving colonies, we found chromosomal instability in three of 40 clones analyzed, while some clones exhibited increased micronucleus frequency and HPRT mutation frequency. These data suggest that our chromosomally unstable GM10115 cells secrete factors that are cytotoxic to the majority of stable, parental cells but are also capable of inducing a heritable change in some of the survivors that can manifest as delayed genomic instability. These results suggest a mechanism whereby instability can be perpetuated through the influences of potentially cytotoxic factors produced by genomically unstable clones.

Nagar, S., Smith, L. E. and Morgan, W. F. Variation in Apoptosis Profiles in Radiation-Induced Genomically Unstable Cell Lines. Radiat. Res. 163, 324–331 (2005). Delayed reproductive cell death or lethal mutations in the survivors of irradiated cells is a well-characterized end point associated with radiation-induced genomic instability. Although the mechanism for this delayed lethality has not been identified, it is thought to be a means of eliminating cells that have sustained extensive damage, thus preventing tissue disruption after radiation exposure. In this study we have tested the hypothesis that delayed reproductive cell death in chromosomally unstable GM10115 clones is due to persistently increased levels of apoptosis. Evidence for differences in apoptosis in two representative genomically unstable clones after irradiation is presented. In addition, one of the unstable clones was found to have abnormal levels of apoptosis after radiation exposure. An understanding of apoptosis in genomically unstable clones may provide insight into the maintenance of genomic instability and the mechanism by which genomically unstable cells evade cell death, potentially contributing to carcinogenesis.

Morgan, W. F. Non-targeted and Delayed Effects of Exposure to Ionizing Radiation: II. Radiation-Induced Genomic Instability and Bystander Effects In Vivo, Clastogenic Factors and Transgenerational Effects. Radiat. Res. 159, 581–596 (2003). The goal of this review is to summarize the evidence for non-targeted and delayed effects of exposure to ionizing radiation in vivo. Currently, human health risks associated with radiation exposures are based primarily on the assumption that the detrimental effects of radiation occur in irradiated cells. Over the years a number of non-targeted effects of radiation exposure in vivo have been described that challenge this concept. These include radiation-induced genomic instability, bystander effects, clastogenic factors produced in plasma from irradiated individuals that can cause chromosomal damage when cultured with nonirradiated cells, and transgenerational effects of parental irradiation that can manifest in the progeny. These effects pose new challenges to evaluating the risk(s) associated with radiation exposure and understanding radiation-induced carcinogenesis.

Morgan, W. Non-targeted and Delayed Effects of Exposure to Ionizing Radiation: I. Radiation-Induced Genomic Instability and Bystander Effects In Vitro. Radiat. Res. 159, 567–580 (2003). A long-standing dogma in the radiation sciences is that energy from radiation must be deposited in the cell nucleus to elicit a biological effect. A number of non-targeted, delayed effects of ionizing radiation have been described that challenge this dogma and pose new challenges to evaluating potential hazards associated with radiation exposure. These effects include induced genomic instability and non-targeted bystander effects. The in vitro evidence for non-targeted effects in radiation biology will be reviewed, but the question as to how one extrapolates from these in vitro observations to the risk of radiation-induced adverse health effects such as cancer remains open.

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min -1 ). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around ∼30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the γ-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min -1 ) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.

We have previously described chromosomal instability in cells of a human-hamster hybrid cell line after exposure to X rays. Chromosomal instability in these cells is characterized by the appearance of novel chromosomal rearrangements multiple generations after exposure to ionizing radiation. To identify the cellular target(s) for radiation-induced chromosomal instability, cells were treated with 125 I-labeled compounds and frozen. Radioactive decays from 125 I cause damage to the cell primarily at the site of their decay, and freezing the cells allows damage to accumulate in the absence of other cellular processes. We found that the decay of <tex-math>${}^{125}{\rm I}\text{-iododeoxyuridine}$</tex-math>, which is incorporated into the DNA, caused chromosomal instability. While cell killing and first-division chromosomal rearrangements increased with increasing numbers of 125 I decays, the frequency of chromosomal instability was independent of dose. Chromosomal instability could also be induced from incorporation of <tex-math>${}^{125}{\rm I}\text{-iododeoxyuridine}$</tex-math> without freezing the cells for accumulation of decays. This indicates that DNA double-strand breaks in frozen cells resulting from 125 I decays failed to lead to instability. Incorporation of an 125 I-labeled protein (<tex-math>${}^{125}{\rm I}\text{-succinyl-concanavalin}$</tex-math> A), which was internalized into the cell and/or bound to the plasma membrane, neither caused chromosomal instability nor potentiated chromosomal instability induced by <tex-math>${}^{125}{\rm I}\text{-iododeoxyuridine}$</tex-math>. These results show that the target for radiation-induced chromosomal instability in these cells is the nucleus.

Genomic instability is characterized by the increased rate of acquisition of alterations in the mammalian genome. These changes encompass a diverse set of biological end points including karyotypic abnormalities, gene mutation and amplification, cellular transformation, clonal heterogeneity and delayed reproductive cell death. The loss of stability of the genome is becoming accepted as one of the most important aspects of carcinogenesis, and the numerous genetic changes associated with the cancer cell implicate genomic stability as contributing to the neoplastic phenotype. Multiple metabolic pathways govern the accurate duplication and distribution of DNA to progeny cells; other pathways maintain the integrity of the information encoded by DNA and regulate the expression of genes during growth and development. For each of these functions, there is a normal baseline frequency at which errors occur, leading to spontaneous mutations and other genomic anomalies. This review summarizes the current status of knowledge about radiation-induced genomic instability. Those events and processes likely to be involved in the initiation and perpetuation of the unstable phenotype, the potential role of epigenetic factors in influencing the onset of genomic instability, and the delayed effects of cellular exposure to ionizing radiation are discussed.

It is proposed that genomic integrity is preserved after DNA damage in a variety of ways. X irradiation induces a p53-dependent G 1 -phase cell cycle checkpoint which putatively allows time for repair of DNA damage. The p53 protein is also involved in the initiation of apoptosis after radiation-induced DNA damage, presumably leading to the elimination of lethally damaged cells from the irradiated population. To test the hypothesis that repair occurs in the additional time provided by the activation of the G 1 -phase checkpoint, we investigated whether the presence of a G 1 -phase arrest modified the frequency and type of chromosomal rearrangements at the first mitosis after irradiation. Isogenic cell lines derived from the same human glioma cell line, but differing in p53 status, were used. Purified G 1 -phase cells, isolated by centrifugal elutriation and X-irradiated, were studied. The wild-type p53 cell line demonstrated a dose-dependent arrest during G 1 phase, as determined by flow cytometry. These cells remained in G 1 phase as long as 48 h after irradiation. Cells expressing a dominant-negative p53 mutation accumulated to a much lesser extent in G 1 phase after irradiation. Cells lacking the G 1 -phase checkpoint showed increased survival at all radiation doses. There were no significant differences in the type or frequency of total chromosomal aberrations in mitotic cells from either cell line after 1, 2, 4, or 6 Gy X rays, as measured by conventional cytogenetic analysis. There was an increase, however, in the number of reciprocal translocations in mitotic cells with mutant p53 (lacking a G 1 -phase checkpoint), as measured by fluorescence in situ hybridization with a chromosome 4-specific DNA library, but only after 6 Gy. The results suggest that the presence of a well-defined p53-dependent G 1 -phase arrest does not reduce chromosomal aberrations caused by low doses of ionizing radiation markedly, but may reduce the overall degree of survival by triggering other G 1 -phase events.

Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3′ cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5′ cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.

Once electroporated into the nucleus of eukaryotic cells, restriction enzymes will bind at specific DNA sequences and cleave DNA to make double-strand breaks. These induced breaks can lead to chromosome aberrations and consequently offer one approach to determining the mechanism(s) of aberration formation. Because the higher-order structure of DNA in eukaryotic cells might influence the ability of restriction enzymes to locate their recognition sequence, bind, and cleave DNA, we have investigated whether enzymes will cut DNA during metaphase when the chromosomes are most condensed. Chinese hamster ovary cells synchronized in mitosis and treated with either AluI or Sau3AI showed few chromosome aberrations when held in mitosis for 1, 2, or 3 h after enzyme treatment. However, some disruption of chromosome morphology was seen, especially after exposure to Sau3AI. When cells were allowed to complete one cell cycle after enzyme treatment in the preceding mitosis, there was extensive chromosome damage, with the most abundant type of lesion being the interstitial deletion. It appears that restriction enzymes will cleave the highly condensed DNA in mitotic cells but that decondensation, DNA replication, and recondensation are required before the aberrations are manifested.

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-β-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases α and δ or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases α and δ, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases α and δ as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.

Neoplastic transformation in vitro of hamster embryo cells and mouse C3H 10T 1/2 cells by X rays and ultraviolet light was suppressed by benzamide or 3-aminobenzamide, agents which inhibit poly(ADP-ribose) polymerization. Suppression was observed under conditions in which the inhibitors reduce poly(ADP-ribose) polymerization by about 75% and increase sister chromatid exchange frequencies, but have no influence on repair of X-ray and uv damage and reportedly have no detectable side effects on nucleotide precursor metabolism. These findings suggest that the mechanisms regulating neoplastic transformation differ from those regulating mutagenesis and sister chromatid exchanges and are mediated via alterations in poly(ADP-ribosylation), causing changes in gene control and expression.

To investigate whether a delay in the rejoining of radiation-induced strand breakage can lead to sister chromatid exchange formation, Chinese hamster ovary cells were prelabeled with 5-bromodeoxyuridine and X-irradiated in the presence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase. The resulting sister chromatid exchange frequencies were consistent with those expected if 3-aminobenzamide and X-ray treatments were independent and additive. A similar but much smaller additive effect was also observed in cells cultured in the presence of 3-aminobenzamide and X-irradiated immediately before the addition of bromodeoxyuridine to the culture medium. These findings support previous studies indicating that X rays are poor inducers of sister chromatid exchanges and suggest that the normally rapid resealing of DNA strand breaks does not account for this inefficiency.