Pajonk, F. and McBride, W. H. The Proteasome in Cancer Biology and Treatment. Radiat. Res. 156, 447–459 (2001).

During the last 30 years, investigation of the transcriptional and translational mechanisms of gene regulation has been a major focus of molecular cancer biology. More recently, it has become evident that cancer-related mutations and cancer-related therapies also can affect post-translational processing of cellular proteins and that control exerted at this level can be critical in defining both the cancer phenotype and the response to therapeutic intervention. One post-translational mechanism that is receiving considerable attention is degradation of intracellular proteins through the multicatalytic 26S proteasome. This follows growing recognition of the fact that protein degradation is a well-regulated and selective process that can differentially control intracellular protein expression levels. The proteasome is responsible for the degradation of all short-lived proteins and 70–90% of all long-lived proteins, thereby regulating signal transduction through pathways involving factors such as AP1 and NFKB, and processes such as cell cycle progression and arrest, DNA transcription, DNA repair/misrepair, angiogenesis, apoptosis/survival, growth and development, and inflammation and immunity, as well as muscle wasting (e.g. in cachexia and sepsis). In this review, we discuss the potential involvement of the proteasome in both cancer biology and cancer treatment.

Cucinotta, F. A., Manuel, F. K., Jones, J., Iszard, G., Murrey, J., Djojonegro, B. and Wear, M. Space Radiation and Cataracts in Astronauts. Radiat. Res. 156, 460–466 (2001).

For over 30 years, astronauts in Earth orbit or on missions to the moon have been exposed to space radiation comprised of high-energy protons and heavy ions and secondary particles produced in collisions with spacecraft and tissue. Large uncertainties exist in the projection of risks of late effects from space radiation such as cancer and cataracts due to the absence of epidemiological data. Here we present the first epidemiological data linking an increased risk of cataracts for astronauts with higher lens doses (>8 mSv) of space radiation relative to other astronauts with lower lens doses (<8 mSv). Our study uses historical data for cataract incidence in the 295 astronauts participating in NASA's Longitudinal Study of Astronaut Health (LSAH) and individual occupational radiation exposure data. These results, while preliminary because of the use of subjective scoring methods, suggest that relatively low doses of space radiation are causative of an increased incidence and early appearance of cataracts.

Whitehouse, C. A. and Tawn, E. J. No Evidence for Chromosomal Instability in Radiation Workers with In Vivo Exposure to Plutonium. Radiat. Res. 156, 467–475 (2001).

The availability of cultured lymphocyte preparations from radiation workers with internal deposits of plutonium provided the opportunity to examine whether irradiation of bone marrow cells had induced a transmissible genomic instability manifesting as an increase in de novo chromosome aberrations in descendant cells in the peripheral blood. The men were originally classified as having more than 20% of the maximum permissible body burdens of plutonium, and recent red bone marrow dose calculations provided individual cumulative estimates at the time of sampling ranging up to 1.8 Sv. The initial sampling occurred approximately 10 years after the main major intake, and samples were subsequently taken during three further periods over the following 20 years. Control samples were available from three of the four sampling times. Chromosome analysis of solid Giemsa-stained material revealed no significant differences either in comparisons between the total group of plutonium workers and controls for comparable periods or when the comparisons were restricted to a group of plutonium workers with initial red bone marrow plutonium doses greater than 0.25 Sv. However, the frequencies of cells containing chromatid exchanges, chromatid breaks, and chromosome and chromatid gaps decreased significantly over the study period for both the plutonium workers as a whole and the controls, and a similar fluctuating pattern was seen when sequential samples from groups of the same individuals were examined. Cells with dicentrics, centric rings and excess acentric fragments remained at similar frequencies throughout the study period. There was therefore no evidence from the study of blood lymphocytes for the induction of persistent transmissible genomic instability in the bone marrow of radiation workers with internal deposits of plutonium. The work has, however, confirmed the need for appropriate controls when conducting studies of cytogenetic end points of instability.

Chumak, A., Thevenon, C., Gulaya, N., Guichardant, M., Margitich, V., Bazyka, D., Kovalenko, A., Lagarde, M. and Prigent, A. F. Monohydroxylated Fatty Acid Content in Peripheral Blood Mononuclear Cells and Immune Status of People at Long Times after the Chernobyl Accident. Radiat. Res. 156, 476–487 (2001).

The monohydroxylated fatty acid content of peripheral blood mononuclear cells from 23 cleanup workers and 16 unexposed individuals was studied in relation to their immune status after the Chernobyl accident. Men with absorbed doses below 0.32 Gy showed higher levels of free and esterified 12-hydroxyeicosatetraenoic acid (12-HETE) than unexposed men, whereas 15-HETE and the 17-hydroxy derivative of C22 fatty acid (17-OH 22), either free or esterified in phospholipids, were increased in a dose-dependent manner. The percentage of CD4-positive cells was also increased significantly in heavily irradiated men, whereas the percentage of CD8-positive cells tended to decrease with dose. Furthermore, the absolute count of CD4-positive cells was correlated positively with the amount of esterified 15-HETE in the phospholipid fraction of the mononuclear cells and with the total 15-HETE. These results show for the first time that the accumulation of autoxidized/lipoxygenase products of polyunsaturated fatty acids in the mononuclear cells of irradiated individuals was associated with immune imbalance. This may be the basis for certain late effects of radiation such as autoimmune disorders, somatic and neoplastic diseases, and early aging.

Hoyes, K. P., Lord, B. I., McCann, C., Hendry, J. H. and Morris I. D. Transgenerational Effects of Preconception Paternal Contamination with ⁵⁵Fe. Radiat. Res. 156, 488–494 (2001).

The conjecture that germline mutations induced by radiation exposure before conception may predispose subsequent offspring to cancer remains contentious. Previous experimental studies have shown that preconception paternal irradiation with ²³⁹Pu induces perturbations in the hemopoietic systems of offspring and influences sensitivity to a secondary carcinogen. In the present study, male DBA2 mice were injected intravenously with the Auger electron emitter ⁵⁵Fe (4 kBq g^–1) 18 or 84 days before mating with normal females. Comet analysis showed an increased incidence of DNA strand breaks in sperm from contaminated animals after 84 days, but not after 18 days, indicating spermatogonial rather than spermatid damage. Offspring were either assayed for changes in bone marrow stem cells and committed progenitors or challenged with the chemical carcinogen methyl nitrosourea (MNU, 50 mg/kg) at 10 weeks of age and monitored for the onset of malignancy. Offspring from irradiated fathers had normal peripheral blood profiles, although the stem cell population was amplified in offspring arising from those exposed to ⁵⁵Fe at 84 days before conception. Exposure to MNU significantly increased the incidence of lympho-hemopoietic malignancies in offspring from the 84-day group, but not in those from the 18-day group. These findings support the hypothesis that aberrations that are potentially leukemogenic may be transmitted to offspring after radiation damage to the paternal germline.

Sykes, P. J., McCallum, B. D., Bangay, M. J., Hooker, A. M. and Morley, A. A. Effect of Exposure to 900 MHz Radiofrequency Radiation on Intrachromosomal Recombination in pKZ1 Mice. Radiat. Res. 156, 495–502 (2001).

Radiofrequency (RF) radiation emitted from mobile phones is not considered to be directly genotoxic, but it may have downstream effects on cellular DNA. We studied the effect of 4 W/kg pulsed 900 MHz RF radiation on somatic intrachromosomal recombination in the spleen in the pKZ1 recombination mutagenesis model. Somatic intrachromosomal recombination inversion events were detected in spleen tissue of pKZ1 mice by histochemical staining for E. coli β-galactosidase protein in cells in which the lacZ transgene has undergone an inversion event. pKZ1 mice were exposed daily for 30 min to plane-wave fields of 900 MHz with a pulse repetition frequency of 217 Hz and a pulse width of 0.6 ms for 1, 5 or 25 days. Three days after the last exposure, spleen sections were screened for DNA inversion events. There was no significant difference between the control and treated groups in the 1- and 5-day exposure groups, but there was a significant reduction in inversions below the spontaneous frequency in the 25-day exposure group. This observation suggests that exposure to RF radiation can lead to a perturbation in recombination frequency which may have implications for recombination repair of DNA. The biological significance of a reduction below the spontaneous frequency is not known. The number of mice in each treatment group in this study was small (n = 10 or n = 20). Therefore, repetition of this study with a larger number of animals is required to confirm these observations.

Murata, R., Siemann, D. W., Overgaard, J. and Horsman, M. R. Improved Tumor Response by Combining Radiation and the Vascular-Damaging Drug 5,6-Dimethylxanthenone-4-acetic Acid. Radiat. Res. 156, 503–509 (2001).

The interaction between 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and radiation was investigated in two different mouse tumor models and a normal mouse tissue. C3H mouse mammary carcinomas transplanted in the feet of CDF1 mice and KHT mouse sarcomas growing in the leg muscles of C3H/HeJ mice were used. DMXAA was dissolved in saline and injected intraperitoneally. Tumors were irradiated locally in nonanesthetized mice, and response was assessed using tumor growth for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. DMXAA alone had an antitumor effect in both tumor types, but only at doses above 15 mg/kg. DMXAA also enhanced radiation damage, and again there was a threshold dose. No enhancement was seen in the C3H mammary carcinoma at 10 mg/kg and below, while in the KHT sarcoma, doses above 15 mg/kg were necessary. This enhancement of radiation damage was also dependent on the sequence of and interval between the treatments with DMXAA and radiation. Combining radiation with DMXAA at the maximum tolerated dose (i.e., the highest dose that could be injected without causing any lethality) of either 20 mg/kg (CDF1 mice) or 17.5 mg/kg (C3H/HeJ mice) gave an additive response when the two agents were administered simultaneously. Even greater antitumor effects were achieved when DMXAA was administered 1–3 h after irradiation. However, when administration of DMXAA preceded irradiation, the effect was similar to that seen for radiation alone, suggesting that appropriate timing is essential to maximize the utility of this agent. When such conditions were met, DMXAA was found to increase the tumor response significantly in the absence of an enhancement of radiation damage in normal skin, thus giving rise to therapeutic gain.

Zhao, D., Constantinescu, A., Hahn, E. W. and Mason, R. P. Tumor Oxygen Dynamics with Respect to Growth and Respiratory Challenge: Investigation of the Dunning Prostate R3327-HI Tumor. Radiat. Res. 156, 510–520 (2001).

We recently described a novel approach to measuring regional tumor oxygen tension using ¹⁹F nuclear magnetic resonance echo planar imaging relaxometry (FREDOM) of hexafluorobenzene. We have now applied this technique to evaluate in detail the oxygen tension dynamics in the relatively slowly growing, moderately well-differentiated Dunning prostate R3327 HI rat tumor with respect to tumor growth and respiratory challenge. Seven individual tumors were assessed repeatedly over a period of 5 weeks (∼4 volume doubling times). For small tumors (<1 cm³), the mean pO₂ ranged from 28 to 44 Torr under baseline conditions, decreasing to less than 10 Torr when the tumors reached 5 to 6 cm³, with a strong inverse correlation between the baseline tumor oxygen tension and the tumor size. The hypoxic fraction (defined as the percentage of the voxels with pO₂ <10 Torr) increased significantly with tumor growth. Administration of oxygen or carbogen produced a significant increase (P < 0.0001) in tumor oxygenation at all stages of tumor growth. Most interestingly, even regions of these tumors that were initially poorly oxygenated responded rapidly, and significantly, to respiratory intervention, in contrast to the behavior of the faster-growing rat prostate tumors investigated previously.

Huo, L., Nagasawa, H. and Little, J. B. HPRT Mutants Induced in Bystander Cells by Very Low Fluences of Alpha Particles Result Primarily from Point Mutations. Radiat. Res. 156, 521–525 (2001).

We have shown previously that damage signals may be transmitted from irradiated cells to nonirradiated cells in monolayer cultures, leading to changes in gene expression and an enhanced frequency of mutations in these “bystander” cells. The present study was designed to test the hypothesis that mutations occurring in bystander cells result from a different mechanism than those occurring in irradiated cells, and thus show differences in molecular structure. Structural changes in the HPRT gene of Chinese hamster ovary (CHO) cells were determined by multiplex PCR analysis. A total of 790 mutant clones derived from monolayer cultures exposed to mean doses of 0, 0.5 or 10 cGy of α-particle radiation (0, 3% or 44%, respectively, of nuclei traversed by one or more α particles) were examined. Whereas mutations induced by 10 cGy included a high frequency of deletions, nearly all mutations occurring in bystander cells in cultures irradiated with 0.5 cGy involved point mutations, confirming our hypothesis that they are induced by a different mechanism.

Schettino, G., Folkard, M., Prise, K. M., Vojnovic, B., Bowey, A. G. and Michael, B. D. Low-Dose Hypersensitivity in Chinese Hamster V79 Cells Targeted with Counted Protons Using a Charged-Particle Microbeam. Radiat. Res. 156, 526–534 (2001).

The Gray Laboratory charged-particle microbeam has been used to assess the clonogenic ability of Chinese hamster V79 cells after irradiation of their nuclei with a precisely defined number of protons with energies of 1.0 and 3.2 MeV. The microbeam uses a 1-μm silica capillary collimator to deliver protons to subcellular targets with high accuracy. The detection system is based on a miniature photomultiplier tube positioned above the cell dish, which detects the photons generated by the passage of the charged particles through an 18-μm-thick scintillator placed below the cells. With this system, a detection efficiency of greater than 99% is achieved. The cells are plated on specially designed dishes (3-μm-thick Mylar base), and the nuclei are identified by fluorescence microscopy. After an incubation period of 3 days, the cells are revisited individually to assess the formation of colonies from the surviving cells. For each energy investigated, the survival curve obtained for the microbeam shows a significant deviation below 1 Gy from a response extrapolated using the LQ model for the survival data above 1 Gy. The data are well fitted by a model that supports the hypothesis that radioresistance is induced by low-dose hypersensitivity. These studies demonstrate the potential of the microbeam for performing studies of the effects of single charged particles on cells in vitro. The hypersensitive responses observed are comparable with those reported by others using different radiations and techniques.

Masson, C., Menaa, F., Pinon-Lataillade, G., Frobert, Y., Radicella, J. P. and Angulo, J. F. Identification of KIN (KIN17), a Human Gene Encoding a Nuclear DNA-Binding Protein, as a Novel Component of the TP53-Independent Response to Ionizing Radiation. Radiat. Res. 156, 535–544 (2001).

Ionizing radiation elicits a genetic response in human cells that allows cell survival. The human KIN (also known as KIN17) gene encodes a 45-kDa nuclear DNA-binding protein that participates in the response to UVC radiation and is immunologically related to the bacterial RecA protein. We report for the first time that ionizing radiation and bleomycin, a radiomimetic drug, which produce single- and double-strand breaks, increased expression of KIN in human cells established from tumors, including MeWo melanoma, MCF7 breast adenocarcinoma, and ATM( )GM3657 lymphoblast cells. KIN expression increased rapidly in a dose-dependent manner after irradiation. Under the same conditions, several genes controlled by TP53 were induced with kinetics similar to that of KIN. Using the CDKN1A gene as a marker of TP53 responsiveness, we analyzed the up-regulation of KIN and showed that is independent of the status of TP53 and ATM. In contrast, the presence of a dominant mutant for activating transcription factor 2 (ATF2) completely abolished the up-regulation of KIN. Our results suggest a role for ATF2 in the TP53-independent increase in KIN expression after γ irradiation.

Costes, S., Sachs, R., Hlatky, L., Vannais, D., Waldren, C. and Fouladi, B. Large-Mutation Spectra Induced at Hemizygous Loci by Low-LET Radiation: Evidence for Intrachromosomal Proximity Effects. Radiat. Res. 156, 545–557 (2001).

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59⁻ mutants, mainly very large-scale deletions (>3 Mbp), in human–hamster hybrid cells, and data from the literature on those HPRT⁻ mutants which involve at least deletion of the whole gene, and often of additional flanking markers (∼50-kbp to ∼4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a “proximity effect”. For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Scholz, M., Jakob, B. and Taucher-Scholz, G. Direct Evidence for the Spatial Correlation between Individual Particle Traversals and Localized CDKN1A (p21) Response Induced by High-LET Radiation. Radiat. Res. 156, 558–563 (2001).

The spatial correlation between individual particle traversals and the nuclear CDKN1A (p21) response after high-LET irradiation of human fibroblasts was investigated. The experiments were based on a technique for the retrospective detection of particle traversals by means of nuclear track detectors, which were used as the cell substratum. This technique requires the precise repositioning of a sample at different steps of the experimental procedure and uses a computerized microscope stage control. The precision of the spatial correlation is further enhanced by means of reference marks in the track etch material that are produced by preirradiation of the plates with charged-particle beams at low fluences. The pattern of the CDKN1A foci that were induced by charged-particle traversals at 1 h postirradiation was found to coincide extremely well with the pattern of particle tracks. This represents direct evidence that CDKN1A foci are located at the sites of particle traversals and thus provides further evidence that the radiation-induced accumulation of the CDKN1A protein takes place at the sites of the primary damage.

Prise, K. M., Pinto, M., Newman, H. C. and Michael, B. D. A Review of Studies of Ionizing Radiation-Induced Double-Strand Break Clustering. Radiat. Res. 156, 572–576 (2001).

Underpinning current models of the mechanisms of the action of radiation is a central role for DNA damage and in particular double-strand breaks (DSBs). For radiations of different LET, there is a need to know the exact yields and distributions of DSBs in human cells. Most measurements of DSB yields within cells now rely on pulsed-field gel electrophoresis as the technique of choice. Previous measurements of DSB yields have suggested that the yields are remarkably similar for different types of radiation with RBE values ≤1.0. More recent studies in mammalian cells, however, have suggested that both the yield and the spatial distribution of DSBs are influenced by radiation quality. RBE values for DSBs induced by high-LET radiations are greater than 1.0, and the distributions are nonrandom. Underlying this is the interaction of particle tracks with the higher-order chromosomal structures within cell nuclei. Further studies are needed to relate nonrandom distributions of DSBs to their rejoining kinetics. At the molecular level, we need to determine the involvement of clustering of damaged bases with strand breakage, and the relationship between higher-order clustering over sizes of kilobase pairs and above to localized clustering at the DNA level. Overall, these studies will allow us to elucidate whether the nonrandom distributions of breaks produced by high-LET particle tracks have any consequences for their repair and biological effectiveness.

Nikjoo, H., O'Neill, P., Wilson, W. E. and Goodhead, D. T. Computational Approach for Determining the Spectrum of DNA Damage Induced by Ionizing Radiation. Radiat. Res. 156, 577–583 (2001).

To study the characteristics of molecular damage induced by ionizing radiation at the DNA level, Monte Carlo track simulation of energetic electrons and ions in liquid water, a canonical model of B-DNA, and a comprehensive classification of DNA damage in terms of the origin and complexity of damage were used to calculate the frequencies of simple and complex strand breaks. A threshold energy of 17.5 eV was used to model the damage by direct energy deposition, and a probability of 0.13 was applied to model the induction of a single-strand break produced in DNA by OH radical reactions. For preliminary estimates, base damage was assumed to be induced by the same direct energy threshold deposition or by the reaction of an OH radical with the base, with a probability of 0.8. Computational data are given on the complexity of damage, including base damage by electrons with energies of 100–4500 eV and ions with energies of 0.3–4.0 MeV/nucleon (59–9 keV μm⁻¹ protons and 170–55 keV μm⁻¹ α particles). Computational data are presented on the frequencies of single- and double-strand breaks induced as a function of the LET of the particles, and on the relative frequencies of complex single- and double-strand breaks for electrons. The modeling and calculations of strand breaks show that: (1) The yield of strand breaks per unit absorbed dose is nearly constant over a wide range of LET. (2) The majority of DNA damage is of a simple type, but the majority of the simple single-strand breaks are accompanied by at least one base damage. (3) For low-energy electrons, nearly 20–30% of the double-strand breaks are of a complex type by virtue of additional breaks. The proportion of this locally clustered damage increases with LET, reaching about 70% for the highest-LET α particles modeled, with the complexity of damage increasing further, to about 90%, when base damage is considered. (4) The extent of damage in the local hit region of the DNA duplex is mostly limited to a length of a few base pairs. (5) The frequency of base damage when no strand breaks are present in the hit segment of DNA varies between 20–40% as a function of LET for protons and α particles.

Weinfeld, M., Rasouli-Nia, A., Chaudhry, M. A. and Britten, R. A. Response of Repair Enzymes to Complex DNA Lesions. Radiat. Res. 156, 584–589 (2001).

There is now increasing evidence that ionizing radiation generates complex DNA damage, i.e. two or more lesions—single-strand breaks or modified nucleosides—located within one to two helical turns on the same strand or on opposite strands. Double-strand breaks are the most readily recognizable clustered lesions, but they may constitute a relatively minor fraction of the total. It is anticipated that clustered lesions may play a significant role in cellular response to ionizing radiation since they may present a major challenge to the DNA repair machinery. The degree of lesion complexity increases with increasing LET. This has potential implications for space travel because of exposure to high-LET cosmic radiation. It is therefore critical that we begin to understand the consequences of such damaged sites, including their influence on DNA repair enzymes. This paper presents a short review of our current knowledge of the action of purified DNA repair enzymes belonging to the base excision repair pathway, including DNA glycosylases and apurinic/apyrimidinic endonucleases, on model complex lesions.

Jenner, T. J., Fulford, J. and O'Neill, P. Contribution of Base Lesions to Radiation-Induced Clustered DNA Damage: Implication for Models of Radiation Response. Radiat. Res. 156, 590–593 (2001).

Biophysical modeling of radiation-induced DNA damage shows that significant yields of clustered DNA damage are formed after energy deposition by a single radiation track. To date, the majority of studies on radiation-induced DNA damage in cells have concentrated on determination of the yields of single- and double-strand breaks (DSBs), the latter representing one type of clustered DNA damage. It was recognized, however, that clustered DNA damage, which does not contain a DSB, might contain a combination of DNA base lesions and single-strand breaks in proximity. This mini-review discusses some of the recent experimental data confirming the induction of non-DSB, clustered DNA damage by radiation.

Ponomarev, A. L., Cucinotta, F. A., Sachs, R. K., Brenner, D. J. and Peterson, L. E. Extrapolation of the DNA Fragment-Size Distribution after High-Dose Irradiation to Predict Effects at Low Doses. Radiat. Res. 156, 594–597 (2001).

The patterns of DSBs induced in the genome are different for sparsely and densely ionizing radiations: In the former case, the patterns are well described by a random-breakage model; in the latter, a more sophisticated tool is needed. We used a Monte Carlo algorithm with a random-walk geometry of chromatin, and a track structure defined by the radial distribution of energy deposition from an incident ion, to fit the PFGE data for fragment-size distribution after high-dose irradiation. These fits determined the unknown parameters of the model, enabling the extrapolation of data for high-dose irradiation to the low doses that are relevant for NASA space radiation research. The randomly-located-clusters formalism was used to speed the simulations. It was shown that only one adjustable parameter, Q, the track efficiency parameter, was necessary to predict DNA fragment sizes for wide ranges of doses. This parameter was determined for a variety of radiations and LETs and was used to predict the DSB patterns at the HPRT locus of the human X chromosome after low-dose irradiation. It was found that high-LET radiation would be more likely than low-LET radiation to induce additional DSBs within the HPRT gene if this gene already contained one DSB.

Kawata, T., Durante, M., Furusawa, Y., George, K., Ito, H., Wu, H. and Cucinotta, F. A. Rejoining of Isochromatid Breaks Induced by Heavy Ions in G₂-Phase Normal Human Fibroblasts. Radiat. Res. 156, 598–602 (2001).

We reported previously that exposure of normal human fibroblasts in G₂ phase of the cell cycle to high-LET radiation produces a much higher frequency of isochromatid breaks than exposure to γ rays. We concluded that an increase in the production of isochromatid breaks is a signature of initial high-LET radiation-induced G₂-phase damage. In this paper, we report the repair kinetics of isochromatid breaks induced by high-LET radiation in normal G₂-phase human fibroblasts. Exponentially growing human fibroblasts (AG1522) were irradiated with γ rays or energetic carbon (290 MeV/nucleon), silicon (490 MeV/nucleon), or iron (200 MeV/nucleon) ions. Prematurely condensed chromosomes were induced by calyculin A after different postirradiation incubation times ranging from 0 to 600 min. Chromosomes were stained with Giemsa, and aberrations were scored in cells at G₂ phase. G₂-phase fragments, the result of the induction of isochromatid breaks, decreased quickly with incubation time. The curve for the kinetics of the rejoining of chromatid-type breaks showed a slight upward curvature with time after exposure to 440 keV/μm iron particles, probably due to isochromatid–isochromatid break rejoining. The formation of chromatid exchanges after exposure to high-LET radiation therefore appears to be underestimated, because isochromatid–isochromatid exchanges cannot be detected. Increased induction of isochromatid breaks and rejoining of isochromatid breaks affect the overall kinetics of chromatid-type break rejoining after exposure to high-LET radiation.

Wu, H. Probabilities of Radiation-Induced Inter- and Intrachromosomal Exchanges and their Dependence on the DNA Content of the Chromosome. Radiat. Res. 156, 603–606 (2001).

A biophysical model has been developed that is based on the assumptions that an interphase chromosome occupies a spherical territory and that chromosome exchanges are formed by the misrejoining of two DNA double-strand breaks induced within a defined interaction distance. The model is used to explain the relative frequencies of inter- and intrachromosomal exchanges and the relationship between radiation-induced aberrations in individual chromosomes and the DNA content of the chromosome. Although this simple model predicts a higher ratio of inter- to intrachromosomal exchanges for low-LET radiation than for high-LET radiation, as has been suggested by others, we argue that the comparison of the prediction of the model with experimental results is not straightforward. With the model, we also show that the probability of the formation of interchromosomal exchanges is proportional to the “surface area” of the chromosome domain plus a correction term. The correction term is small if the interaction distance is less than 1 μm for both low- and high-LET radiations.

Kiefer, J., Schmidt, P. and Koch, S. Mutations in Mammalian Cells Induced by Heavy Charged Particles: An Indicator for Risk Assessment in Space. Radiat. Res. 156, 607–611 (2001).

Induction of mutations at the HPRT locus in V79 Chinese hamster cells by heavy charged particles is reviewed. A unique dependence of mutation induction cross sections on LET or any other physical parameter of the particles cannot be found. There is, however, a general trend showing an increase up to about 100 keV/μm and an indication of a saturation with higher LET. An empirical expression is given approximating the data which may be useful for practical purposes. On the basis of this expression, it is suggested that the formula used by the ICRP for the quality factor overestimates the risk for very heavy particles. This may have implications for the assessment of the risk of exposure to iron ions in space.

Brenner, D. J. and Elliston, C. D. The Potential Impact of Bystander Effects on Radiation Risks in a Mars Mission. Radiat. Res. 156, 612–617 (2001).

Densely ionizing (high-LET) galactic cosmic rays (GCR) contribute a significant component of the radiation risk in free space. Over a period of a few months—sufficient for the early stages of radiation carcinogenesis to occur—a significant proportion of cell nuclei will not be traversed. There is convincing evidence, at least in vitro, that irradiated cells can send out signals that can result in damage to nearby unirradiated cells. This observation can hold even when the unirradiated cells have been exposed to low doses of low-LET radiation. We discuss here a quantitative model based on the a formalism, an approach that incorporates radiobiological damage both from a bystander response to signals emitted by irradiated cells, and also from direct traversal of high-LET radiations through cell nuclei. The model produces results that are consistent with those of a series of studies of the bystander phenomenon using a high-LET microbeam, with the end point of in vitro oncogenic transformation. According to this picture, for exposure to high-LET particles such as galactic cosmic rays other than protons, the bystander effect is significant primarily at low fluences, i.e., exposures where there are significant numbers of untraversed cells. If the mechanisms postulated here were applicable in vivo, using a linear extrapolation of risks derived from studies using intermediate doses of high-LET radiation (where the contribution of the bystander effect may be negligible) to estimate risks at very low doses (where the bystander effect may be dominant) could underestimate the true risk from low doses of high-LET radiation. It would be highly premature simply to abandon current risk projections for high-LET, low-dose radiation; however, these considerations would suggest caution in applying results derived from experiments using high-LET radiation at fluences above ∼1 particle per nucleus to risk estimation for a Mars mission.

Barcellos-Hoff, M. H. and Brooks, A. L. Extracellular Signaling through the Microenvironment: A Hypothesis Relating Carcinogenesis, Bystander Effects, and Genomic Instability. Radiat. Res. 156, 618–627 (2001).

Cell growth, differentiation and death are directed in large part by extracellular signaling through the interactions of cells with other cells and with the extracellular matrix; these interactions are in turn modulated by cytokines and growth factors, i.e. the microenvironment. Here we discuss the idea that extracellular signaling integrates multicellular damage responses that are important deterrents to the development of cancer through mechanisms that eliminate abnormal cells and inhibit neoplastic behavior. As an example, we discuss the action of transforming growth factor β (TGFB1) as an extracellular sensor of damage. We propose that radiation-induced bystander effects and genomic instability are, respectively, positive and negative manifestations of this homeostatic process. Bystander effects exhibited predominantly after a low-dose or a nonhomogeneous radiation exposure are extracellular signaling pathways that modulate cellular repair and death programs. Persistent disruption of extracellular signaling after exposure to relatively high doses of ionizing radiation may lead to the accumulation of aberrant cells that are genomically unstable. Understanding radiation effects in terms of coordinated multicellular responses that affect decisions regarding the fate of a cell may necessitate re-evaluation of radiation dose and risk concepts and provide avenues for intervention.

Carnes, B. A. and Gavrilova, N. Risk Analysis: Divergent Models and Convergent Interpretations. Radiat. Res. 156, 628–630 (2001).

Material presented at a NASA-sponsored workshop on risk models for exposure conditions relevant to prolonged space flight are described in this paper. Analyses used mortality data from experiments conducted at Argonne National Laboratory on the long-term effects of external whole-body irradiation on B6CF₁ mice by ⁶⁰Co γ rays and fission neutrons delivered as a single exposure or protracted over either 24 or 60 once-weekly exposures. The maximum dose considered was restricted to 1 Gy for neutrons and 10 Gy for γ rays. Proportional hazard models were used to investigate the shape of the dose response at these lower doses for deaths caused by solid-tissue tumors and tumors of either connective or epithelial tissue origin. For protracted exposures, a significant mortality effect was detected at a neutron dose of 14 cGy and a γ-ray dose of 3 Gy. For single exposures, radiation-induced mortality for neutrons also occurred within the range of 10–20 cGy, but dropped to 86 cGy for γ rays. Plots of risk relative to control estimated for each observed dose gave a visual impression of nonlinearity for both neutrons and γ rays. At least for solid-tissue tumors, male and female mortality was nearly identical for γ-ray exposures, but mortality risks for females were higher than for males for neutron exposures. As expected, protracting the γ-ray dose reduced mortality risks. Although curvature consistent with that observed visually could be detected by a model parameterized to detect curvature, a relative risk term containing only a simple term for total dose was usually sufficient to describe the dose response. Although detectable mortality for the three pathology end points considered typically occurred at the same level of dose, the highest risks were almost always associated with deaths caused by tumors of epithelial tissue origin.

Bridges, B. A. Radiation and Germline Mutation at Repeat Sequences: Are We in the Middle of a Paradigm Shift? Radiat. Res. 156, 631–641 (2001).

Two assumptions are commonly made in the estimation of genetic risk: (1) that the seven specific loci in the mouse constitute a suitable basis for extrapolation to genetic disease in humans, and (2) that mutations are induced by radiation damage (energy-loss events leading to double-stranded damage) occurring within the gene and are induced linearly with dose, at least at low doses. Recent evidence on the mutability of repeat sequences is reviewed that suggests that neither of these assumptions is as well founded as we like to think. Repeat sequences are common in the human genome, and alterations in them may have health consequences. Many of them are unstable, both spontaneously and after irradiation. The fact that changes in DNA repeat sequences can clearly arise as a result of radiation damage outside the sequence concerned and the likely involvement of some sort of signal transduction process mean that the nature of the radiation dose response cannot be assumed. While the time has not come to abandon the current paradigms, it would seem sensible to invest more effort in exploring the induction of changes in repeat sequences after irradiation and the consequences of such changes for health.

Pandita, T. K. The Role of ATM in Telomere Structure and Function. Radiat. Res. 156, 642–647 (2001).

Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with a wide variety of phenotypic manifestations. AT patients are cancer prone and hypersensitive to ionizing radiation. Cells derived from AT patients require higher levels of serum factors, exhibit cytoskeletal defects, and undergo premature senescence in culture. The gene responsible for AT is ATM (ataxia-telangiectasia mutated), and its product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control. Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance. The ATM gene product influences chromosome end associations, telomere length, and telomere clustering. The defective telomere metabolism in AT cells could be due to altered interactions between the telomeres and the nuclear matrix. These interactions were studied in nuclear matrix halos before and after irradiation. Altered telomere–nuclear matrix interactions were observed in cells derived from individuals with AT. AT cells also had different nucleosomal periodicity in their telomeres from normal cells. Both telomere–nuclear matrix interactions and nucleosomal periodicity were altered by treatment of primary AT fibroblasts with ionizing radiation. This effect was not observed in cells derived from normal individuals. A link was also found between altered telomere–nuclear matrix interactions, aberrant telomere clustering, and gonadal atrophy. The telomere defect was not corrected by the ectopic expression of the catalytic subunit of telomerase (TERT). Since alteration of the yeast telomere chromatin structure is known to influence gene expression, we compared expressed sequence tags (ESTs) of Atm-null mouse cells and normal mouse cells. Several ESTs were found to be aberrantly expressed in Atm-null mouse cells. This paper summarizes our recent publications and presents some new data on the influence of ATM on telomere metabolism.

Sankaranarayanan, K. and Chakraborty, R. Impact of Cancer Predisposition and Radiosensitivity on the Population Risk of Radiation-Induced Cancers. Radiat. Res. 156, 648–656 (2001).

This paper provides a brief overview of the current evidence for cancer predisposition and for an increased sensitivity of individuals carrying such predisposing mutations to cancers induced by ionizing radiations. We also discuss the use of a Mendelian one-locus, two-allele autosomal dominant model for predicting the impact of cancer predisposition and increased radiosensitivity on the risk of radiation-induced cancers in the population and in relatives of affected individuals using breast cancer due to BRCA1 mutations as an example. The main conclusions are the following: (1) The relative risk ratio of the risks of radiation-induced cancer in a heterogeneous population which has subgroups of normal and cancer-predisposed individuals to the risks in a homogeneous population (i.e., one which does not have these subgroups) increases with increasing dose; however, the dose dependence of the RR decreases at higher doses because of the fact that at high doses, the radiation risk to a homogeneous population will already be high. (2) The attributable risk (the proportion of cancers attributable to increased cancer susceptibility and increased radiosensitivity) follows a similar pattern. (3) When the proportion of cancers due to the susceptible genotypes is small (<10%), as is likely to be the case for breast cancers in non-Ashkenazi Jewish women, the increases in risk ratios and attributable risks are small, and become marked only when there are very large increases in cancer susceptibility (>1000-fold) and radiosensitivity (>100-fold) in the susceptible group. (4) When the proportion of cancers due to the susceptible genotypes is appreciable (≥10%), as may be the case for breast cancers in Ashkenazi Jewish women, there may be significant increases in the risk ratios and attributable risk for comparatively moderate increases in cancer susceptibility (>10-fold) and radiosensitivity (>100-fold) in the susceptible subpopulation. (5) The ratio of the risk of radiation-induced cancer in relatives to that in unrelated individuals in the population increases with the biological relatedness of the relative, being higher for close than for distant relatives; however, even when the mutant BRCA1 gene frequency and the proportion of breast cancers due to these mutations are high, as in Ashkenazi Jewish women, for values of predisposition strength and radiosensitivity differential <10, the increase in breast cancer risks is only marginal, even for first-degree relatives.

Amundson, S. A., Bittner, M., Meltzer, P., Trent, J. and Fornace, A. J., Jr. Induction of Gene Expression as a Monitor of Exposure to Ionizing Radiation. Radiat. Res. 156, 657–661 (2001).

The complex molecular responses to genotoxic stress are mediated by a variety of regulatory pathways. The transcription factor TP53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation, but other pathways also play important roles. In addition, differences in radiation quality, such as the exposure to high-LET radiation that occurs during space travel, may influence the pattern of responses. The premise is developed that stress gene responses can be employed as molecular markers for radiation exposure using a combination of informatics and functional genomics approaches. Published studies from our laboratory have already demonstrated such transcriptional responses with doses of γ rays as low as 2 cGy, and in peripheral blood lymphocytes (PBLs) irradiated ex vivo with doses as low as 20 cGy. We have also found several genes elevated in vivo 24 h after whole-body irradiation of mice with 20 cGy. Such studies should provide insight into the molecular responses to physiologically relevant doses, which cannot necessarily be extrapolated from high-dose studies. In addition, ongoing experiments are identifying large numbers of potential biomarkers using microarray hybridization and various irradiation protocols including expression at different times after exposure to low- and high-LET radiation. Computation-intensive informatics analysis methods are also being developed for management of the complex gene expression profiles resulting from these experiments. With further development of these approaches, it may be feasible to monitor changes in gene expression after low-dose radiation exposure and other physiological stresses that may be encountered during manned space flight, such as the planned mission to Mars.

Durante, M., Bonassi, S., George, K. and Cucinotta, F. A. Risk Estimation based on Chromosomal Aberrations Induced by Radiation. Radiat. Res. 156, 662–667 (2001).

The presence of a causal association between the frequency of chromosomal aberrations in peripheral blood lymphocytes and the risk of cancer has been substantiated recently by epidemiological studies. Cytogenetic analyses of crew members of the Mir Space Station have shown that a significant increase in the frequency of chromosomal aberrations can be detected after flight, and that such an increase is likely to be attributed to the radiation exposure. The risk of cancer can be estimated directly from the yields of chromosomal aberrations, taking into account some aspects of individual susceptibility and other factors unrelated to radiation. However, the use of an appropriate technique for the collection and analysis of chromosomes and the choice of the structural aberrations to be measured are crucial in providing sound results. Based on the fraction of aberrant lymphocytes detected before and after flight, the relative risk after a long-term Mir mission is estimated to be about 1.2–1.3. The new technique of mFISH can provide useful insights into the quantification of risk on an individual basis.

Ford, B. N., Wilkinson, D., Thorleifson, E. M. and Tracy, B. L. Gene Expression Responses in Lymphoblastoid Cells after Radiation Exposure. Radiat. Res. 156, 668–671 (2001).

Individual differences in response to radiation are well known, but the molecular basis for these differences is not well understood, and molecular indicators that are useful in assessing individual variation are lacking. Cells from patients developing unexpected radiation responses have occasionally been analyzed for rare genetic anomalies (such as alleles of the ATM gene), but few data exist on the long-term effects of genetic variation on radiation response. We hypothesize that much of the variation in the response to radiation is due to differences in the genes that respond to radiation exposure, and that changes in gene expression may serve as surrogate markers of individual response. As a first step in developing a selection of suitable markers of gene expression, we used cDNA microarrays to identify genes that were altered in expression in lymphoblastoid cells 4 h after exposure to 1 Gy X rays. We found changes in gene expression ranging from a 10-fold repression to a 12-fold induction. Some of the responsive genes have been noted previously in other cell types, whereas others are reported for the first time. Using these data, we are beginning to characterize the range of structural, temporal and functional variations in the responsive genes. The results of this work will assist in developing response markers both for prescreening for sensitive individuals and for risk assessment.

Ohtaki, M. and Niwa, O. A Mathematical Model of Radiation Carcinogenesis with Induction of Genomic Instability and Cell Death. Radiat. Res. 156, 672–677 (2001).

We developed a mathematical model of carcinogenesis that incorporates genomic instability, a feature characterized by long-term destabilization of the genome in irradiated cells that leads to an increase in cancer risk in the exposed individuals at the cancer-prone age. This model also considers the induction of cell death, another important effect of radiation on cells. It is assumed that cell killing by radiation may occur at all stages of the carcinogenic process. The resulting model can explain not only the paradoxical relationship between low mutation rates and high cancer incidence but also the low-order dose–response relationship of cancer risk.

Heidenreich, W. F. and Paretzke, H. G. The Two-Stage Clonal Expansion Model as an Example of a Biologically Based Model of Radiation-Induced Cancer. Radiat. Res. 156, 678–681 (2001).

A model with two stages and clonal expansion (TSCE) is reviewed as a prototype for biologically based models of cancer development. Applications of the TSCE model to data sets for animals and humans for particle radiation (α particles) are presented. The results suggest that the radiation not only influences the initiating mutation, but may also act as a promoter. A possible mechanism for the promoting action is described. The consequences of these results for the shapes of the radiation dose–response curves at low doses and dose rates are discussed.

Cucinotta, F. A., Schimmerling, W., Wilson, J. W., Peterson, L. E., Badhwar, G. D., Saganti, P. B. and Dicello, J. F. Space Radiation Cancer Risks and Uncertainties for Mars Missions. Radiat. Res. 156, 682–688 (2001).

Projecting cancer risks from exposure to space radiation is highly uncertain because of the absence of data for humans and because of the limited radiobiology data available for estimating late effects from the high-energy and charge (HZE) ions present in the galactic cosmic rays (GCR). Cancer risk projections involve many biological and physical factors, each of which has a differential range of uncertainty due to the lack of data and knowledge. We discuss an uncertainty assessment within the linear-additivity model using the approach of Monte Carlo sampling from subjective error distributions that represent the lack of knowledge in each factor to quantify the overall uncertainty in risk projections. Calculations are performed using the space radiation environment and transport codes for several Mars mission scenarios. This approach leads to estimates of the uncertainties in cancer risk projections of 400–600% for a Mars mission. The uncertainties in the quality factors are dominant. Using safety standards developed for low-Earth orbit, long-term space missions (>90 days) outside the Earth's magnetic field are currently unacceptable if the confidence levels in risk projections are considered. Because GCR exposures involve multiple particle or δ-ray tracks per cellular array, our results suggest that the shape of the dose response at low dose rates may be an additional uncertainty for estimating space radiation risks.

De Angelis, G., Caldora, M., Santaquilani, M., Scipione, R. and Verdecchia, A. Health Risks from Radiation Exposure for Civilian Aviation Flight Personnel: A Study of Italian Airline Crew Members. Radiat. Res. 156, 689–694 (2001).

A study of the effects of exposures of civilian aviation flight personnel to atmospheric ionizing radiation (including high-energy neutrons) is being performed. The results of previous studies and of the criteria required for a more satisfactory outcome in future studies are presented, along with a description of the protocol for the Italian national study. A description of the cohort is given in terms of its size, composition and member eligibility. The various ways of determining the exposure and the health status of past and current aircrew members are discussed, and follow-up procedures are described. An overview of the data management and processing philosophy adopted in the Italian study is given with regard to flight routes, radiation dose evaluation along the flight paths, and construction of exposure matrices. Other studies of potential interest are also discussed. The study is still in progress, so the results are preliminary.