Sugarman, J. and McKenna, W. G. Ethical Hurdles in Translational Research. Radiat. Res. 160, 1–4 (2003).

The notion of translational research has gained considerable currency over the past few years. While such an approach promises great scientific and clinical advances, the penumbra of translational research tends to incorporate prioritizing scientific projects based upon their potential for translation; tight financial connections between sponsors, scientists and clinical investigators; and sometimes research involving biological approaches for which there is little experience determining safety. It is these aspects of translational research that raise some serious ethical challenges. In this report, we examine three specific areas that raise ethical questions: (1) the potential implications of prioritizing research objectives based on the potential for translation; (2) cautions related to moving from bench to bedside (and back again); and (3) unique questions for translational research initiatives in academic medical centers. Based on this examination, it is clear that the financial and ethical costs as well as benefits of taking a translational approach need to be considered. In the meantime, exquisite attention needs to be paid whenever translational research is likely to affect the traditional fiduciary responsibilities of scientists, clinicians and institutions to research subjects, patients and students. Successful mechanisms that might be developed to address any untoward effects should be shared and evaluated.

Mao, X. W., Archambeau, J. O., Kubínová, L., Boyle, S., Petersen, G. and Grove, R. Quantification of Rat Retinal Growth and Vascular Population Changes after Single and Split Doses of Proton Irradiation: Translational Study Using Stereology Methods. Radiat. Res. 160, 5–13 (2003).

This study quantified architectural and population changes in the rat retinal vasculature after proton irradiation using stereology. A 100 MeV conformal proton beam delivered 8, 14, 20 and 28 Gy as single and split doses to the whole eye. The vascular networks were prepared from retinal digests. Stereological methods were used to obtain the area of the retina and unbiased estimates of microvessel/artery/vein endothelial, pericyte and smooth muscle population, and vessel length. The retinal area increased progressively in the unirradiated, age-matched controls and in the retinas irradiated with 8 and 14 Gy, indicating uniform progressive retinal growth. No growth occurred after 20 and 28 Gy. Regression analysis of total endothelial cell number in all vessels (arteries, veins and capillaries) after irradiation documented a progressive time- and dose-dependent cell loss occurring over 15 to 24 months. The difference from controls was significant (P < 0.01) after 28 Gy given in single and split doses and after 20 Gy given as a split dose (P < 0.05). Total vessel length in microvessel was significantly shortened at 20 and 28 Gy compared to that of controls (P < 0.05). No evident dose recovery was observed in the endothelial populations after split doses. At 10 Gy, the rate of endothelial cell loss, a dose parameter used to characterize the time- and dose-dependent loss of the endothelial population, was doubled.

Game, J. C., Birrell, G. W., Brown, J. A., Shibata, T., Baccari, C., Chu, A. M., Williamson, M. S. and Brown, J. M. Use of a Genome-Wide Approach to Identify New Genes that Control Resistance of Saccharomyces cerevisiae to Ionizing Radiation. Radiat. Res. 160, 14–24 (2003).

We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae, to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.

Nakasono, S., Laramee, C., Saiki, H. and McLeod, K. J. Effect of Power-Frequency Magnetic Fields on Genome-Scale Gene Expression in Saccharomyces cerevisiae. Radiat. Res. 160, 25–37 (2003).

To estimate the effect of 50 Hz magnetic-field exposure on genome-wide gene expression, the yeast Saccharomyces cerevisiae was used as a model for eukaryotes. 2D PAGE (about 1,000 spots) for protein and cDNA microarray (about 5,900 genes) analysis for mRNA were performed. The cells were exposed to 50 Hz vertical magnetic fields at 10, 150 or 300 mT r.m.s. for 24 h. As positive controls, the cells were exposed to aerobic conditions, heat (40°C) or minimal medium. The 2D PAGE and microarray analyses for the positive controls showed high-confidence differential expression of many genes including those for known or unknown proteins and mRNAs. For magnetic-field exposure, no high-confidence changes in expression were observed for proteins or genes that were related to heat-shock response, DNA repair, respiration, protein synthesis and the cell cycle. Principal component analysis showed no statistically significant difference in principal components, with only insignificant differences between the magnetic-field intensities studied. In contrast, the principal components for the positive controls were significantly different. The results indicate that a 50 Hz magnetic field below 300 mT did not act as a general stress factor like heat shock or DNA damage, as had been reported previously by others. This study failed to find a plausible differential gene expression that would point to a possible mechanism of an effect of magnetic fields. The findings provide no evidence that the magnetic-field exposure alters the fundamental mechanism of translation and transcription in eukaryotic cells.

Marples, B., Wouters, B. G. and Joiner, M. C. An Association between the Radiation-Induced Arrest of G₂-Phase Cells and Low-Dose Hyper-Radiosensitivity: A Plausible Underlying Mechanism? Radiat. Res. 160, 38–45 (2003).

The survival of asynchronous and highly enriched G₁-, S- and G₂-phase populations of Chinese hamster V79 cells was measured after irradiation with ⁶⁰Co γ rays (0.1–10 Gy) using a precise flow cytometry-based clonogenic survival assay. The high-dose survival responses demonstrated a conventional relationship, with G₂-phase cells being the most radiosensitive and S-phase cells the most radioresistant. Below 1 Gy, distinct low-dose hyper-radiosensitivity (HRS) responses were observed for the asynchronous and G₂-phase enriched cell populations, with no evidence of HRS in the G₁- and S-phase populations. Modeling supports the conclusion that HRS in asynchronous V79 populations is explained entirely by the HRS response of G₂-phase cells. An association was discovered between the occurrence of HRS and the induction of a novel G₂-phase arrest checkpoint that is specific for cells that are in the G₂ phase of the cell cycle at the time of irradiation. Human T98G cells and hamster V79 cells, which both exhibit HRS in asynchronous cultures, failed to arrest the entry into mitosis of damaged G₂-phase cells at doses less than 30 cGy, as determined by the flow cytometric assessment of the phosphorylation of histone H3, an established indicator of mitosis. In contrast, human U373 cells that do not show HRS induced this G₂-phase checkpoint in a dose-independent manner. These data suggest that HRS may be a consequence of radiation-damaged G₂-phase cells prematurely entering mitosis.

Neronova, E., Slozina, N. and Nikiforov, A. Chromosome Alterations in Cleanup Workers Sampled Years after the Chernobyl Accident. Radiat. Res. 160, 46–51 (2003).

Cytogenetic analysis performed 4–13 years after the Chernobyl accident showed an elevated frequency of acentrics, chromatid exchanges, dicentrics and rings in Chernobyl cleanup workers compared to the control group. Cytogenetic data were analyzed according to the information on exposure to radiation (the year when the cleanup workers worked at the Chernobyl station, doses rates, time elapsed since exposure to radiation, and cytogenetic examination) and some lifestyle factors. The data obtained suggested that some types of chromosome aberrations could be influenced by the action of different environmental factors or lifestyle factors. The frequency of acentrics was correlated with the age of the cleanup workers, and the increased chromatid exchange frequency was attributed to smoking. The numbers of dicentrics and rings suggested a genotoxic effect of ionizing radiation that is still present over 13 years after the exposure.

Mognato, M., Bortoletto, E., Ferraro, P., Baggio, L., Cherubini, R., Canova, S., Russo, A. and Celotti, L. Genetic Damage Induced by In Vitro Irradiation of Human G₀ Lymphocytes with Low-Energy Protons (28 keV/μm): HPRT Mutations and Chromosome Aberrations. Radiat. Res. 160, 52–60 (2003).

Cell survival, mutations and chromosomal effects were studied in primary human lymphocytes exposed in G₀ phase to a proton beam with an incident energy of 0.88 MeV (incident LET of 28 keV/μm) in the dose range 0.125–2 Gy. The curves for survival and mutations at the hypoxanthine-guanine phosphoribosyl transferase locus were obtained by fitting the experimental data to linear and linear-quadratic equations, respectively. In the dose interval 0–1.5 Gy, the α parameters of the curves were 0.42/Gy and 3.6 × 10⁻⁶ mutants/Gy, respectively. The mutation types at the HPRT locus were analyzed by multiplex-PCR in 94 irradiated and 41 nonirradiated clones derived from T lymphocytes from five healthy donors. All clones showed a normal multiplex-PCR pattern and were classified as point mutations. Chromosome aberration data were fitted as a linear function of dose (α = 0.62 aberrations per cell Gy⁻¹). By irradiating G₀ lymphocytes from a single subject with 28 keV/μm protons and γ rays, an RBE of 6.07 was obtained for chromosome aberrations. An overinvolvement of chromosome 9 relative to chromosome 7 was found in chromosome breaks after chromosome painting analysis.

Hawkins, R. B. A Microdosimetric-Kinetic Model for the Effect of Non-Poisson Distribution of Lethal Lesions on the Variation of RBE with LET. Radiat. Res. 160, 61–69 (2003).

The microdosimetric-kinetic (MK) model for cell killing by ionizing radiation is summarized. An equation based on the MK model is presented which gives the dependence of the relative biological effectiveness in the limit of zero dose (RBE₁) on the linear energy transfer (LET). The relationship coincides with the linear relationship of RBE₁ and LET observed for low LET, which is characteristic of a Poisson distribution of lethal lesions among the irradiated cells. It incorporates the effect of deviation from the Poisson distribution at higher LET. This causes RBE₁ to be less than indicated by extrapolation of the linear relationship to higher LET, and to pass through a maximum in the range of LET of 50 to 200 keV per micrometer. The relationship is compared with several experimental studies from the literature. It is shown to approximately fit their results with a reasonable choice for the value of a cross-sectional area related to the morphology and ultrastructure of the cell nucleus. The model and the experiments examined indicate that the more sensitive cells are to radiation at low LET, the lower will be the maximum in RBE they attain as LET increases. An equation that portrays the ratio of the sensitivity of a pair of cell types as a function of LET is presented. Implications for radiotherapy with high-LET radiation are discussed.

Yamamoto, T., Matsumura, A., Yamamoto, K., Kumada, H., Hori, N., Torii, Y., Shibata, Y. and Nose, T. Characterization of Neutron Beams for Boron Neutron Capture Therapy: In-Air Radiobiological Dosimetry. Radiat. Res. 160, 70–76 (2003).

The survival curves and the RBE for the dose components generated in boron neutron capture therapy (BNCT) were determined separately in neutron beams at Japan Research Reactor No. 4. The surviving fractions of V79 Chinese hamster cells with or without ¹⁰B were obtained using an epithermal neutron beam (ENB), a mixed thermal-epithermal neutron beam (TNB-1), and a thermal (TNB-2) neutron beam; these beams were used or are planned for use in BNCT clinical trials. The cell killing effect of the neutron beam in the presence or absence of ¹⁰B was highly dependent on the neutron beam used and depended on the epithermal and fast-neutron content of the beam. The RBEs of the boron capture reaction for ENB, TNB-1 and TNB-2 were 4.07 ± 0.22, 2.98 ± 0.16 and 1.42 ± 0.07, respectively. The RBEs of the high-LET dose components based on the hydrogen recoils and the nitrogen capture reaction were 2.50 ± 0.32, 2.34 ± 0.30 and 2.17 ± 0.28 for ENB, TNB-1 and TNB-2, respectively. The RBEs of the neutron and photon components were 1.22 ± 0.16, 1.23 ± 0.16, and 1.21 ± 0.16 for ENB, TNB-1 and TNB-2, respectively. The approach to the experimental determination of RBEs outlined in this paper allows the RBE-weighted dose calculation for each dose component of the neutron beams and contributes to an accurate inter-beam comparison of the neutron beams at the different facilities employed in ongoing and planned BNCT clinical trials.

Chaudhuri, K., Banerjee, R., Pandit, B., Mukherjee, A., Das, S., Sengupta, S., Roychoudury, S. and Bhattacharyya, N. P. Identification of Two Differentially Expressed Mitochondrial Genes in a Methotrexate-Resistant Chinese Hamster Cell Strain Derived from V79 Cells Using RNA Fingerprinting by Arbitrary Primed Polymerase Chain Reaction. Radiat. Res. 160, 77–85 (2003).

To identify genes that are differentially expressed in a methotrexate (MTX)-resistant cell strain designated as M5 that exhibits resistance to γ radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was about twofold higher in the M5 cell strain compared to V79 cells. Moreover, the expression of both the subunits decreased in γ-irradiated Chinese hamster V79 cells. Cytochrome oxidase, another enzyme of the mitochondrial electron transport chain encoded in the mitochondrial genome, was also found to be overexpressed in M5 cells. All three genes are under the control of the same promoter. However, no amplification of DNA was observed. These data indicate that the alterations in mitochondrial gene expression may be involved in the recovery of irradiated cells, which may arise from transcriptional modulation of the mitochondria from the nucleus.

Abdoul-Carime, H. and Sanche, L. Alteration of Protein Constituents Induced by Low-Energy (<35 eV) Electrons: II. Dissociative Electron Attachment to Amino Acids Containing Cyclic Groups. Radiat. Res. 160, 86–94 (2003).

We report measurements of the desorption of anions from thin condensed films of tryptophan (Trp), histidine (His) and proline (Pro) stimulated by 5–35 eV electron impact. H⁻, O⁻, OH⁻ and CN⁻ desorb from Trp, His and Pro, whereas CH₂⁻ is observed only from Pro fragmentation. Below 12 eV, the anion yield functions exhibit resonant structures indicative of dissociative electron attachment. For all three amino acids, this process is likely to be initiated by the resonant capture of the incident electron at the NH₃ –CH–…. .–COO⁻ and/or NH₂–CH–…. .–COOH group of the molecule. Temporary electron attachment to the ring leads to anion desorption only for tryptophan and proline. The energy-averaged yields measured at the detector of the mass spectrometer are (4.9, 0.3 and 54.0) × 10⁻⁸ H⁻/incident electron and (3.4, 2.9, 1.8) × 10⁻¹¹ O⁻/incident electron, respectively, from Trp, His and Pro dissociation. Fragmentation of amino acids is found to be as intense as that of the nucleic acid bases. These results are discussed within the context of radiobiological damage induced by secondary electrons.

Kumagai, J., Masui, K., Itagaki, Y., Shiotani, M., Kodama, S., Watanabe, M. and Miyazaki, T. Long-Lived Mutagenic Radicals Induced in Mammalian Cells by Ionizing Radiation are Mainly Localized to Proteins. Radiat. Res. 160, 95–102 (2003).

We have provided evidence that long-lived radicals, produced by ionizing radiation, are highly mutagenic and transforming in mammalian cells. Long-lived radicals are scavenged effectively by vitamin C or by epigallocatechin-3-O-gallate (EGCG). Long-lived radicals are not involved in lethality or in the induction of chromosome aberrations. We now report the results of experiments that define the relative amounts of long-lived radicals in DNA and proteins and identify the major protein radicals as sulfinyl radicals (R-CH₂-S-O^·). To make these assignments, yields of long-lived radicals in γ-irradiated salmon sperm DNA and albumin were compared by ESR. ESR spectra of long-lived radicals produced in irradiated Syrian hamster embryo (SHE) cells were analyzed precisely and compared with ESR parameters obtained by density functional theory calculations. Long-lived radicals yields of 99.8% were produced in proteins. We also identified a new type of long-lived radical as H-added phenylalanine radicals. While our evidence does not rule out the possibility of important biological consequences of the low-level long-lived radicals created by radiation, it implicates radicals in proteins as playing a key role in genetic effects of ionizing radiation. We suggest that these novel radicals, wherever they reside, need to be considered in explanations of biological sequela of radiation.

Engalytcheff, A., Deridder, V., Debuyst, R. and Tilquin, B. Determination of Radical Yields in Solid-State Drugs as One Technique to Identify Drugs that will Withstand Radiosterilization: Radioresistance of Beta Blockers. Radiat. Res. 160, 103–109 (2003).

This article describes a simple preliminary test to determine whether a drug is sufficiently radioresistant to withstand radiosterilization. The test is based on the electron spin resonance (ESR) detection of radicals produced after irradiation of a solid-state drug, assuming that these radicals are the precursors of the final products detected after dissolution of the drug. A calibration curve has therefore been established by measuring ESR spectra of l-alanine irradiated at different doses. The response factor to quantify the radicals is the normalized double integration (DI) of the whole first-derivative ESR spectrum. The curve gives the relationship between the normalized DI and the number of radicals. Eight β blockers have been chosen and their radical yield determined. This is the first time that several different drugs of the same pharmacological group have been studied and compared. The results obtained are similar for seven of the eight β blockers; the mean G value (excepted for nadolol) is 3 × 10⁻⁹ mol/J. This means that β blockers are radioresistant. The two most radiosensitive drugs (nadolol and esmolol hydrochloride) were also studied by high-performance liquid chromatography (HPLC). No significant loss of the active compound was detected, which confirms this radioresistant property. Moreover, no change in color or smell was observed. Using ESR and HPLC, β blockers were identified as potential candidates for radiosterilization.

Kubínová, L., Mao, X. W., Janáček, J. and Archambeau, J. O. Stereology Techniques in Radiation Biology. Radiat. Res. 160, 110–119 (2003).

Clinicians involved in conventional radiation therapy are very concerned about the dose–response relationships of normal tissues. Before proceeding to new clinical protocols, radiation biologists involved with conformal proton therapy believe it is necessary to quantify the dose response and tolerance of the organs and tissues that will be irradiated. An important focus is on the vasculature. This presentation reviews the methodology and format of using confocal microscopy and stereological methods to quantify tissue parameters, cell number, tissue volume and surface area, and vessel length using the microvasculature as a model tissue. Stereological methods and their concepts are illustrated using an ongoing study of the dose response of the microvessels in proton-irradiated hemibrain. Methods for estimating the volume of the brain and the brain cortex, the total number of endothelial cells in cortical microvessels, the length of cortical microvessels, and the total surface area of cortical microvessel walls are presented step by step in a way understandable for readers with little mathematical background. It is shown that stereological techniques, based on a sound theoretical basis, are powerful and reliable and have been used successfully.

Archambeau, J. O., Mao, X. W. and Kubínová, L. Stereology Techniques Have—or Should Have—a Role in Preclinical Radiation Therapy. Radiat. Res. 160, 120–123 (2003).

A paper by Kubinova et al. (Radiat. Res. 000, 000–000, 2003) introduced to radiation biology the techniques of stereology required to quantify the dose response of irradiated brain populations. A paper by Mao et al. (Radiat. Res. 000, 000–000, 2003) and earlier papers by Archambeau et al. applied these techniques to quantify the population changes in the vasculature of the retina. This presentation reviews in broad terms the evolution of the need to quantify population and kinetic techniques and how the need has been met. The in vitro and in vivo descriptive and clonogenic techniques used regularly in radiation biology and in clinical therapy will not be replaced by stereology. While stereology is applicable to all tissue, it proves to be an important technique that allows the investigator to quantify cell population parameters in late-responding and non-proliferative populations.