Britten, R. A., Peters, L. J. and Murray, D. Biological Factors Influencing the RBE of Neutrons: Implications for Their Past, Present and Future Use in Radiotherapy. Radiat. Res. 156, 125–135 (2001).

The recent resurgence of interest in fast-neutron therapy, particularly for the treatment of prostate cancer, warrants a review of the original radiobiological basis for this modality and the evolution of these concepts that resulted from subsequent experimentation with the fast-neutron beams used for randomized clinical trials. It is clear from current radiobiological knowledge that some of the postulates that formed the mechanistic basis for past clinical trials were incorrect. Such discrepancies, along with the inherent physical disadvantages of neutron beams in terms of collimation and intensity modulation, may partially account for the lack of therapeutic benefit observed in many randomized clinical trials. Moreover, it is equally apparent that indiscriminate prescription of fast-neutron therapy is likely to lead to an adverse clinical outcome in a proportion of patients. Hence any renewed efforts to establish a niche for this modality in clinical radiation oncology will necessitate the development of a triage system that can discriminate those patients who might benefit from fast-neutron therapy from those who might be harmed by it. In the future, fast-neutron therapy might be prescribed based upon the relative status of appropriate molecular parameters that have a differential impact upon radiosensitivity to photons compared to fast neutrons.

Travis, L. B., Land, C. E., Andersson, M., Nyberg, U., Goldman, M. B., Knudson Gaul, L., Berger, E., Storm, H. H., Hall, P., Auvinen, A., Janower, M. L., Holm, L-E., Monson, R. R., Schottenfeld D. and Boice, J. D., Jr. Mortality after Cerebral Angiography with or without Radioactive Thorotrast: An International Cohort of 3,143 Two-Year Survivors. Radiat. Res. 156, 136–150 (2001).

There are few studies on the long-term sequelae of radionuclides ingested or injected into the human body. Patients exposed to radioactive Thorotrast in the 1930s through the early 1950s provide a singular opportunity, since the administration of this radiographic contrast agent resulted in continuous exposure to α particles throughout life at a low dose rate. We evaluated cause-specific mortality among an international cohort of 3,143 patients injected during cerebral angiography with either Thorotrast (n = 1,736) or a similar but nonradioactive agent (n = 1,407) and who survived 2 or more years. Standardized mortality ratios (SMRs) for Thorotrast and comparison patients were calculated, and relative risks (RR), adjusted for population, age and sex, were obtained by multivariate statistical modeling. Most patients were followed until death, with only 94 (5.4%) of the Thorotrast patients known to be alive at the closure of the study. All-cause mortality (n = 1,599 deaths) was significantly elevated among Thorotrast subjects [RR 1.7; 95% confidence interval (CI) 1.5–1.8]. Significantly increased relative risks were found for several categories, including cancer (RR 2.8), benign and unspecified tumors (RR 1.5), benign blood diseases (RR 7.1), and benign liver disorders (RR 6.5). Nonsignificant increases were seen for respiratory disease (RR 1.4) and other types of digestive disease (RR 1.6). The relative risk due to all causes increased steadily after angiography to reach a threefold RR at 40 or more years (P < 0.001). Excess cancer deaths were observed for each decade after Thorotrast injection, even after 50 years (SMR 8.6; P < 0.05). Increasing cumulative dose of radiation was directly associated with death due to all causes combined, cancer, respiratory disease, benign liver disease, and other types of digestive disease. Our study confirms the relationship between Thorotrast and increased mortality due to cancer, benign liver disease, and benign hematological disease, and suggests a possible relationship with respiratory disorders and other types of digestive disease. The cumulative excess risk of cancer death remained high up to 50 years after injection with >20 ml Thorotrast and approached 50%.

Abdoul-Carime, H. and Sanche, L. Sequence-Specific Damage Induced by the Impact of 3–30 eV Electrons on Oligonucleotides. Radiat. Res. 156, 151–157 (2001).

The ability of low-energy electrons to induce single- and double-strand breaks in DNA has recently been demonstrated. Here we show the propensity of 3–30 eV electrons to initiate base sequence-dependent damage to a short single DNA strand. Solid monolayer films of homogeneous thymidine (T₉) and deoxycytidine (dCy₉) and heterogeneous oligomers (T₆dCy₃) are bombarded with 1–30 eV electrons in an ultrahigh-vacuum system. CN, OCN and/or H₂NCN are detected by a mass spectrometer as the most intense neutral fragments desorbing in vacuum. A weaker signal of CH₃CCO is also detected, but only from oligonucleotides containing thymine. Below 17 eV, the energy dependence of the yields of CN, OCN and CH₃CCO exhibits resonance-like structures, attributed to dissociative electron attachment (DEA). Above 17 eV, the monotonic increase in the fragment yields indicates that nonresonant processes (i.e. dipolar dissociation) control the fragmentation of these molecules. Within the energy range investigated, comparison of the magnitude of the total fragment yields produced by electron attack on dCy₉, T₆-dCy₃ and T₉ suggests the following order in the sensitivity of single-strand DNA: dCy₉ > T₆-dCy₃ > T₉. At 12 eV, the total fragment yields are found to be 5.8, 5.0 and 3.9 × 10^–3 fragment/electron, respectively. From the yields obtained with the two homo-oligonucleotides, we differentiate between contributions arising from the chemical nature of the base and the effect of environment (i.e. the sequence) when a thymidine unit in T₉ is replaced by dCy. The base sequence-dependent damage is found to vary with incident electron energy. These results reinforce the idea that genomic sensitivity to ionizing radiation depends on local genetic information. Furthermore, they underscore the possible role of low-energy electrons in the pathways responsible for the induction of specific genomic lesions.

Panyutin, I. V., Luu, A. N., Panyutin, I. G. and Neumann, R. D. Strand Breaks in Whole Plasmid DNA Produced by the Decay of ¹²⁵I in a Triplex-Forming Oligonucleotide. Radiat. Res. 156, 158–166 (2001).

DNA strand breaks produced by the decay of ¹²⁵I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the ¹²⁵I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of ¹²⁵I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the ¹²⁵I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.

Bryntesson, F., Regan, J. C., Jeggo, P. A., Taccioli, G. E. and Hubank, M. Analysis of Gene Transcription in Cells Lacking DNA-PK Activity. Radiat. Res. 156, 167–176 (2001).

The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs^–/– and DNA-PKcs ^/ mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

Sawant, S. G., Randers-Pehrson, G., Metting, N. and Hall, E. J. Adaptive Response and the Bystander Effect Induced by Radiation in C3H 10T½ Cells in Culture. Radiat. Res. 156, 177–180 (2001).

This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose–response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T½ cells in culture. When the single-cell microbeam delivered from 1 to 12 α particles through the nuclei of 10% of C3H 10T½ cells, more cells were inactivated than were actually traversed by α particles. The magnitude of this bystander effect increased with the number of particles per cell. An adaptive dose of 2 cGy of γ rays, delivered 6 h beforehand, canceled out about half of the bystander effect produced by the α particles.

Tsoulou, E., Kalfas, C. A. and Sideris E. G. Probing Irradiated DNA with the Perturbed Angular Correlation Method. Radiat. Res. 156, 181–185 (2001).

The technique of perturbed angular correlations of γ rays has been used to study the effects of radiation on DNA molecules. The samples are buffered solutions of calf thymus DNA exposed to various doses (0–80 Gy) of γ rays. Indium-111 is used as a probe. Rotational correlation times, τ_c, a parameter measuring the flexibility of a macromolecule, are obtained that show a dependence on radiation dose.

Evans, H. H., Horng, M-F., Evans, T. E., Jordan, R. and Schwartz, J. L. Genotoxic Effects of High-Energy Iron Particles in Human Lymphoblasts Differing in Radiation Sensitivity. Radiat. Res. 156, 186–194 (2001).

The effects of ⁵⁶Fe particles and ¹³⁷Cs γ radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of ⁵⁶Fe particles than to those of γ rays. However, the mutagenicity of ⁵⁶Fe particles and γ rays at the TK locus was the same per unit dose and was higher for γ rays than for ⁵⁶Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by ⁵⁶Fe particles are repaired less efficiently than γ-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both γ rays and ⁵⁶Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53 ). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.

Nose, M., Wang, B., Itsukaichi, H., Yukawa, O., Hayata, I., Yamada, T. and Ohyama, H. Rescue of Lethally Irradiated Mice from Hematopoietic Death by Pre-exposure to 0.5 Gy X Rays without Recovery from Peripheral Blood Cell Depletion and its Modification by OK432. Radiat. Res. 156, 195–204 (2001).

Exposing mice to 0.5 Gy X rays 2 weeks before lethal irradiation has been reported to induce marked radioresistance and to rescue them from hematopoietic death. Here we examined effects of the 0.5-Gy pre-exposure on hematological changes in C57BL mice that were lethally irradiated with 6.5 Gy X rays. Approximately 77% of pre-exposed mice survived 30 days after this irradiation, whereas 80% of mice that did not receive this pre-exposure died by day 20. However, regardless of the pre-exposure, peripheral blood cell counts decreased markedly by day 3 and reached a nadir at day 20. CFU-S in femur and CFU-GM in spleen had started to recover at day 10 and 14, respectively, but recovery of functional peripheral blood cells occurred later. The effect of pre-exposure on survival was altered by OK432, a bioresponse modifier; the effect depended on the timing of its administration. OK432 given 2 days before 0.5 Gy enhanced the protective effect of pre-exposure, resulting in the survival of 97% of the mice. In contrast, injection of OK432 1 day before or 2 days after pre-exposure led to 100% mortality. Thus the survival-promoting effect of 0.5 Gy could be altered by OK432. The OK432-induced changes in the survival of mice could not be attributed solely to hematological changes, as shown by blood cell counts and progenitor cell contents. These results suggest that radioresistance induced by pre-exposure to 0.5 Gy X rays is not stable, but rather varies with the physiological conditions, and can be modulated by factors such as OK432.

Nagler, R. M., Eichen, Y. and Nagler, A. Redox Metal Chelation Ameliorates Radiation-Induced Bone Marrow Toxicity in a Mouse Model. Radiat. Res. 156, 205–209 (2001).

Since zinc desferrioxamine (Zn-DFO) has been shown to be a very potent protector against injuries induced by redox-active metal ions, we examined its protective effect against radiation-induced toxicity. We found that treatment with Zn-DFO given before TBI increased the survival of mice irradiated with 7.5 and 8.5 Gy. Zn-DFO also protected against radiation-induced myelosuppression and body weight loss, while soluble Il6 levels in serum were normalized in mice pretreated with Zn-DFO. We concluded that administration of Zn-DFO prior to TBI protected BALB/c mice from radiation-induced toxicity, increasing survival rates by up to 75%. The biological effect of Zn-DFO is known to result from its effect on the production of intracellular hydroxyl free radicals mediated by redox-active metal ions, and both metal chelation and zinc delivery appear to be equally likely mechanisms for this outcome. We suggest that radiation-induced toxicity is caused by the deleterious effect of redox-active metal ions, and that compounds which modulate this redox activity may act as radioprotectors.

Randers-Pehrson, G., Geard, C. R., Johnson, G., Elliston, C. D. and Brenner, D. J. The Columbia University Single-Ion Microbeam. Radiat. Res. 156, 210–214 (2001).

A single-ion microbeam facility has been constructed at the Columbia University Radiological Research Accelerator Facility. The system was designed to deliver defined numbers of helium or hydrogen ions produced by a van de Graaff accelerator, covering a range of LET from 30 to 220 keV/μm, into an area smaller than the nuclei of human cells growing in culture on thin plastic films. The beam is collimated by a pair of laser-drilled apertures that form the beam-line exit. An integrated computer control program locates the cells and positions them for irradiation. We present details of the microbeam facility including descriptions of the collimators, hardware, control program, and the various protocols available. Various contributions to targeting and positioning precision are discussed along with our plans for future developments. Beam time for outside users is often available (see www.raraf.org).

Box, H. C., Budzinski, E. E., Dawidzik, J., Patrzyc, H. B. and Freund, H. G. A Novel Double Lesion in X-Irradiated DNA Consists of a Strand Break and a Base Modification. Radiat. Res. 156, 215–219 (2001).

A single free radical-initiating event can produce a pair of base lesions in DNA oligomers exposed to ionizing radiation. Whereas double base lesions have been observed previously, the present study shows that double lesions may sometimes consist of a base lesion and an associated strand break. The mechanism for the formation of double lesions is discussed. A redox process is postulated in which guanine is the source of the electron. It is suggested that double lesions may be formed in DNA either on adjacent nucleotides or, alternatively, on nucleotides separated by one, two or possibly more intervening nucleotides. It is hypothesized that intramolecular electron transfer facilitates the formation of double lesions on nonadjacent nucleotides.