Badhwar, G.D. and Cucinotta, F.A. A Comparison of Depth Dependence of Dose and Linear Energy Transfer Spectra in Aluminum and Polyethylene.

A set of four tissue-equivalent proportional counters (TEPCs), with their detector heads at the centers of 0 (bare), 3, 7 and 9-inch-diameter aluminum spheres, were flown on Shuttle flight STS-89. Five such detectors at the centers of polyethylene spheres were flown 1 year earlier on STS-81. The results of dose–depth dependence for the two materials convincingly show the merits of using material rich in hydrogen to decrease the radiation exposure to the crew. A comparison of the calculated galactic cosmic radiation (GCR) absorbed dose and dose-equivalent rates using the radiation transport code HZETRN with nuclear fragmentation model NUCFRG2 and the measured GCR absorbed dose rates and dose-equivalent rates shows that they agree within root mean square (rms) error of 12.5 and 8.2%, respectively. However, there are significant depth-dependent differences in the linear energy transfer (LET) spectra. A comparison for trapped protons using the proton transport code BRYNTRN and the AP-8 MIN trapped-proton model shows a systematic bias, with the model underpredicting dose and dose-equivalent rates. These results show the need for improvements in the radiation transport and/or fragmentation models.

Søyland, C., Hassfjell, S.P. and Steen, H.B. A New Alpha-Particle Irradiator with Absolute Dosimetric Determination.

A new experimental setup for uniform α-particle irradiation of cells in vitro is described. The α-particle irradiator is based on a radioactive ²¹²Pb/²¹² Bi source. In the experimental setup proposed, cells are grown directly on a polylysine-coated track-etch material that forms the base of custom-made cell dishes. Alpha-particle irradiation is done through the base of the dish. Immediately prior to irradiation, the cell dish is scanned under a microscope, and images of cells with the corresponding coordinates are saved. After irradiation and after the biological end point under study has been determined, the cell dish is etched to develop α-particle tracks in the dish base. A microscope image series of α-particle track images is obtained by accurately revisiting every original (preirradiation) cell position in the track-etched dish. The number of α-particle traversals of each individual cell is scored by mapping images of α-particle tracks onto the images of cells recorded prior to irradiation. The uncertainty of the α-particle hit determination is 0.9 μm. The procedure described thus presents a method for radiobiological experiments with absolute, rather than statistical, cell dosimetry.

Roeske, J.C. and Stinchcomb, T.G. Tumor Control Probability Model for Alpha-Particle-Emitting Radionuclides.

Alpha-particle emitters are currently being evaluated for the treatment of metastatic disease. The dosimetry of α-particle emitters is a challenge, however, because the stochastic patterns of energy deposition within cellular targets must be taken into account. We propose a model for the tumor control probability of α-particle emitters which takes into account these stochastic effects. An expression for cell survival, which is a function of the microdosimetric single-event specific-energy distribution, is multiplied by the number of cells within the tumor cluster. Poisson statistics is used to model the probability of zero surviving cells within the cluster. Based on this analysis, a number of observations have been made: (1) The dose required to eradicate a tumor is nearly a linear function of the cell survival parameter z₀. (2) Cells with smaller nuclei will require more dose to achieve the same level of tumor control probability, relative to cells with larger nuclei, for an identical source–target configuration and cell sensitivity. (3) As the targeting of α-particle emitters becomes more specific, the dose required to achieve a given level of tumor control decreases. (4) Additional secondary effects include cell shape and the initial α-particle energy.

Abdoul-Carime, H., Dugal, P-C. and Sanche, L. Damage Induced by 1–30 eV Electrons on Thymine- and Bromouracil-Substituted Oligonucleotides.

The impact of low-energy (1–30 eV) electrons on self-assembled monolayers of heterogeneous oligonucleotides chemisorbed on a gold surface has been investigated by mass spectrometry of desorbed neutral species in an attempt to understand the consequences of secondary electron damage in a short sequence of a DNA single strand. We demonstrate that the most intense observable neutral species (CN, OCN and/or H₂NCN) desorbed from Cy₆-Th₃ and Cy₆-(BrdU)₃ oligos are related to primary fragmentation of the bases induced by electron impact. The dependence of the neutral species desorption on electron energy shows typical signatures of dissociative electron attachment initiated by the formation of shape- and core-excited resonances (i.e. single-electron and two-electron–one-hole transitory anions, respectively). Substitution of dTh by BrdU increases the production of neutral fragments by as much as a factor of about 3 for the entire electron energy range. When the distribution of secondary electrons along radiation tracks in H₂O is taken into account, we show that the probability for electron damage to heterogeneous oligonucleotides is enhanced by a factor of 2.5–3 for electron energies below 20 eV for both sensitized and unsensitized strands.

Douki, T., Bretonniere, Y. and Cadet., K. Protection against Radiation-Induced Degradation of DNA Bases by Polyamines.

Polyamines have been reported to protect DNA against the formation of radiation-induced strand breaks and crosslinks to proteins. The present study was aimed at investigating the protective effect of spermine, spermidine and putrescine against the degradation of DNA bases upon exposure to γ rays in aerated aqueous solution. The yield of 8-oxo-7,8-dihydroguanine and 5-hydroxycytosine was found to decrease for concentrations of spermine and spermidine greater than 0.1 mM. A protection factor of 10 was observed for a concentration of 1 mM of the latter two polyamines. Putrescine afforded a lower protection. In addition, the formation yield of a series of radiation-induced degradation products of the purine and pyrimidine bases was determined within DNA in the presence or absence of spermine. The protection factor was within the same range for all the lesions measured. The latter observation ruled out the possibility of degradation of DNA by radiation-induced polyamine peroxyl radicals. This was confirmed by studies involving radiolysis of DMSO and decomposition of 2,2′-azobis(2-methyl-propionamidine) as sources of alkylperoxyl radicals. Therefore, it is likely that the polyamine-mediated protection against the radiation-induced degradation of DNA bases is due to the compaction of the DNA structure and the reduction in the accessibility of DNA to ˙OH rather than by scavenging ˙OH in the bulk solution or in the vicinity of the DNA.

Endlich, B., Radford, I.R., Forrester, H.B. and Dewey, W.C. Computerized Video Time-Lapse Microscopy Studies of Ionizing Radiation-Induced Rapid-Interphase and Mitosis-Related Apoptosis in Lymphoid Cells.

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10–20-min burst of membrane blebbing followed by swelling for 2–4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1–4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30–60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G₂-phase delay, and then most of the cells (76–97%) attempted to divide for the first or second time at ∼18–30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation–refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36–60 h postirradiation.

Albanese, J. and Dainiak, N. Ionizing Radiation Alters Fas Antigen Ligand at the Cell Surface and on Exfoliated Plasma Membrane-Derived Vesicles: Implications for Apoptosis and Intercellular Signaling.

Resident proteins that reside on the plasma membrane are continually exfoliated from the cell surface. Exfoliation is a selective, energy-dependent process that mediates intercellular communication. Ionizing radiation modulates the expression of many plasma membrane-bound growth regulators, including the “death” ligand, TNFSF6 (formerly known as FasL, CD95L). Here we report that ionizing radiation induces dose-dependent up-regulation of TNFSF6 on plasma membranes purified from SW620 cells, a TNFSF6-expressing colon cancer cell line. Serum-free medium conditioned by exposed and control cells was collected and exfoliated vesicles were obtained by ultracentrifugation. Western blot analysis of vesicles from unexposed cells and from cells treated with 10 Gy showed increased amounts of TNFSF6 compared to that on vesicles from unexposed cells. Cells treated with 4 Gy released vesicles having a low level of TNFSF6 on their surface relative to that on vesicles exfoliated from unexposed cells. When assayed for bioactivity, vesicles from unexposed cells induced the greatest level of apoptosis in TNFRSF6 (formerly known as FAS) receptor-bearing Jurkat cells (cell surviving fraction of 43.7 ± 6.1; P < 0.05), followed by vesicles collected from cells treated with 4 Gy (79.6 ± 2.6%; P < 0.05). Despite having a high level of TNFSF6 by Western analysis, vesicles collected from cells exposed to 10 Gy display minimal biological activity (77.9 ± 3.2%; P < 0.05), suggesting that modification of the vesicle-associated ligand has occurred. Our results indicate that ionizing radiation increases the level of TNFSF6 exfoliated on extracellular vesicles. The data may provide a mechanism for abscopal and bystander effects after irradiation.

Akudugu, J.M., Slabbert, J.P., Serafin, A. and Bohm, L. Frequency of Radiation-Induced Micronuclei in Neuronal Cells Does Not Correlate with Clonogenic Survival.

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2α, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2α, G28, G120 and G44; SF2 ≥ 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of ⁶⁰Co γ radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 ≤ 0.27) produced 0.08 ± 0.01 and 0.04 ± 0.01 MN per gray, respectively. OP-6, SK-N-BE(2c), G112, G62, G60 and KELLY cells constituted an intermediate group and displayed a micronucleus formation index between 0.10 and 0.24 MN per gray per binucleated cell. SHSY5Y cells showed no detectable formation of MN. In two groups [OP-6, SK-N-BE(2c), G112, G62, N2α and G28 or G120, G60, OP-27 and SK-N-SH], the more resistant cell lines produced more MN per unit dose. Another group [OP-6, SK-N-BE(2c), G112, G62, G44 and G120] showed no correlation between micronucleus formation and radiosensitivity. We conclude that the relationship between cell survival and micronucleus formation is not straightforward and that it would be simplistic to translate micronucleus frequency into radiosensitivity.

Inano, H., Onoda, M., Suzuki, K., Kobayashi, H. and Wakabayashi, K. Inhibitory Effects of WR-2721 and Cysteamine on Tumor Initiation in Mammary Glands of Pregnant Rats by Radiation.

We evaluated the effect of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid] and cysteamine (2-mercaptoethylamine) on the development of radiation-induced mammary tumors in rats. Pregnant rats were treated with WR-2721 or cysteamine 30 min prior to whole-body irradiation with γ rays from a ⁶⁰Co source at a dose of 1.5 or 2.6 Gy. Additional pregnant rats were given saline and then exposed to γ rays at a dose of 0, 1.5 or 2.6 Gy as a control. All rats were implanted with pellets of diethylstilbestrol, a tumor promoter, 1 month after termination of nursing and were observed for 1 year to detect palpable mammary tumors. No mammary tumors developed in the saline-injected nonirradiated rats. However, when rats were irradiated with 1.5 or 2.6 Gy after saline treatment, the incidence of mammary tumors was high (71.4 and 92.3%, respectively). Administration of WR-2721 or cysteamine prior to irradiation with 1.5 Gy significantly decreased the tumor incidence (23.8 and 20.8%, respectively). Tumor prevention by either agent was less effective at the higher dose. The appearance of the first mammary tumor occurred later in rats treated with WR-2721 or cysteamine than in the control rats. An increasing rate of adenocarcinoma in the control group was observed with increasing dose from 1.5 Gy up to 2.6 Gy. However, the development of adenocarcinoma did not increase after pretreatment with WR-2721 or cysteamine in rats irradiated with 2.6 Gy. Many of the mammary tumors that developed in the control rats were of the ER PgR type. Administration of WR-2721 produced no tumors of the ER PgR type. Cysteamine treatment increased the development of ER-negative tumors. The serum concentration of progesterone was significantly higher in rats treated with WR-2721 or cysteamine than in the control rats. On the other hand, the estradiol-17β concentration was reduced by treatment with WR-2721, but not significantly compared to the control. WR-2721 and cysteamine had no effect on the prolactin concentration of the irradiated rats. The results suggest that administration of WR-2721 or cysteamine prior to the irradiation has a potent preventive effect on the initiation phase during mammary tumorigenesis.

Fenton, B.M., Lord, E.M. and Paoni, S.F. Enhancement of Tumor Perfusion and Oxygenation by Carbogen and Nicotinamide during Single- and Multifraction Irradiation.

Numerous experimental and clinical studies have been completed regarding the effects of carbogen and nicotinamide on tumor oxygenation and radiosensitivity. The current study incorporates three physiological measurement techniques to further define spatial variations in oxygen availability and development of hypoxia after single- and multifraction irradiation in KHT murine fibrosarcomas. Distances to anatomical and perfused blood vessels were measured using immunohistochemical and fluorescent staining, intravascular oxygen levels were determined cryospectrophotometrically, and tumor hypoxia was quantified using uptake of EF5, a marker of hypoxia. Carbogen, nicotinamide, and the combination of both all increased intravascular oxygen availability compared to controls. While nicotinamide had no effect on the number of perfused blood vessels in nonirradiated tumors, carbogen produced a substantial closing of vessels. After a single dose of 4 Gy, only the combination of nicotinamide and carbogen produced significant improvements in oxygen availability, while numbers of perfused vessels were significantly increased for nicotinamide, unchanged for the combination of nicotinamide and carbogen, and significantly decreased for carbogen. After 4 × 4-Gy fractions, oxygen availability was increased substantially with the combination of nicotinamide and carbogen, somewhat with carbogen, and not at all with nicotinamide. Tumor oxygenation changes were estimated by EF5/Cy3 intensity distributions, which demonstrated that manipulative agents could produce disparate effects on tumor hypoxia when combined with either single- or multifraction irradiation.

Rasey, J.S., Casciari, J.J., Hofstrand, P.D., Muzi, M., Graham, M.M. and Chin, L.K. Determining the Hypoxic Fraction in a Rat Glioma by Uptake of Radiolabeled Fluoromisonidazole.

The usefulness of radiolabeled nitroimidazoles for measuring hypoxia will be clarified by defining the relationship between tracer uptake and radiobiologically hypoxic fraction. We determined the radiobiologically hypoxic fraction from radiation response data in 36B10 rat gliomas using the paired cell survival curve technique and compared the values to the radiobiologically hypoxic fraction inferred from mathematical modeling of time–activity data acquired by PET imaging of [¹⁸F]FMISO uptake. Rats breathed either air or 10% oxygen during imaging, and timed blood samples were taken. The uptake of [³H]FMISO by 36B10 cells in vitro provided cellular binding characteristics of this radiopharmaceutical as a function of oxygen concentration. The radiobiologically hypoxic fraction determined for tumors in air-breathing rats using the paired survival curve technique was 6.1% (95% CL = 4.3–8.6%), which agreed well with that determined by modeling FMISO time–activity data (7.4%; 95% CL = 2.5–17.3%). These results are consistent with the agreement between the two techniques for measuring radiobiologically hypoxic fraction in Chinese hamster V79 cell spheroids. In contrast, the FMISO-derived radiobiologically hypoxic fraction in rats breathing 10% oxygen was 13.1% (95% CL 7.9–8.3%), much lower than the radiobiologically hypoxic fraction of 43% determined from the radiation response data. This discrepancy may be due to the failure of FMISO to identify hypoxic cells residing at or above an oxygen level of 2–3 mmHg that will still confer substantial protection against radiation. The presence of transiently hypoxic cells in rats breathing reduced oxygen may also be under-reported by nitroimidazole binding, which is strongly dependent on time and concentration.

Nekolla, E.A., Kreisheimer, M., Kellerer, A.M., Kuse-Isingschulte, M., Gössner, W. and Spiess, H. Induction of Malignant Bone Tumors in Radium-224 Patients: Risk Estimates Based on the Improved Dosimetry.

Mainly between 1945 and 1955, several thousand German patients with ankylosing spondylitis, tuberculosis, or—in a few cases—other diseases received multiple injections of the short-lived α-particle emitter radium-224. In the early 1950s, the follow-up of 899 patients was initiated, and the study has continued since then. It includes most of the high-dose patients and nearly all of those treated as children or juveniles, i.e. under the age of 21. In the study cohort, 56 malignant bone tumors occurred in a temporal wave that peaked 8 years after exposure, whereas less than one case would have been expected during the follow-up. Most of the malignant bone tumors were osteosarcomas and fibrous-histiocytic sarcomas. A new analysis has now been performed, primarily because an improved dosimetry resulted in modified bone surface doses, especially for those treated at younger ages. A significant increase in bone tumor risk with decreasing age at exposure is now demonstrated. The earlier finding of an inverse protraction factor is confirmed. In the new formulation, the dependence on dose rate or duration applies only at higher doses; i.e., the initial slope of the dose dependence is unrelated to dose rate or exposure duration, which is in contrast to earlier analyses but is in agreement with microdosimetric considerations and general radiobiological experience.

Rezvani, M., Hopewell, J.W., Wilkinson, J.H., Bray, S., Morris, G.M. and Charles, M.W. Time- and Dose-Related Changes in the Thickness of Skin in the Pig after Irradiation with Single Doses of Thulium-170 Beta Particles.

Time-related changes in skin thickness have been evaluated in the pig using a noninvasive ultrasound technique after exposure to a range of single doses of 0.97 MeV β particles from ¹⁷⁰Tm plaques. The reduction in relative skin thickness developed in two phases; the separation into two phases was statistically justified only after 120 Gy (P = 0.04). The first phase was between 12 weeks and 24 weeks after irradiation. No further changes were seen until 48–60 weeks after irradiation, when a second phase of skin thinning was observed. No further changes in relative skin thickness were seen in the follow-up period of 104 weeks. The timing of these phases of relative skin thinning was totally independent of the radiation dose; however, the severity of each phase of radiation-induced skin thinning was related to the dose. The pattern of changes was similar to that reported previously after irradiation with 2.27 MeV β particles from ⁹⁰Sr/⁹⁰Y, but the degree of dermal thinning was less for a similar skin surface dose. From a comparison of the depth–dose distribution of the β particles from the two radionuclides, it was concluded that the target cell population responsible for both the first and second phase of skin thinning in pig skin after irradiation may be located at approximately 800 μm depth. This corresponds to an area in the reticular dermis in pig skin and may be the appropriate site at which to measure the average dose to the dermal tissue.

Spruill, M. D., Nelson, D. O., Ramsey, M. J., Nath, J. and Tucker, J. D. Lifetime Persistence and Clonality of Chromosome Aberrations in the Peripheral Blood of Mice Acutely Exposed to Ionizing Radiation.

As the measurement of chromosomal translocations increases in popularity for quantifying prior radiation exposure, information on the possible decline of these “stable” aberrations over time is urgently needed. We report here information about the persistence of radiation-induced chromosome aberrations in vivo over the life span of a rodent. Female C57BL/6 mice were given a single whole-body acute exposure of 0, 1, 2, 3 or 4 Gy ¹³⁷Cs γ rays at 8 weeks of age. Chromosome aberrations were analyzed from peripheral blood samples at various intervals between 1 day and 21 months after exposure. Aberrations were detected by painting chromosomes 2 and 8. Translocations decreased dramatically during the first 3 months after irradiation, beyond which time the frequencies remained relatively constant out to 1 year, when the effects of aging and clonal expansion became significant. Both reciprocal and nonreciprocal translocations increased with age in the unexposed control animals and were involved in clones. As expected of unstable aberrations, dicentrics decreased rapidly after exposure and reached baseline levels within 3 months. These results indicate that the persistence of translocations induced by ionizing radiation is complicated by aging and clonal expansion and that these factors must be considered when quantifying translocations at long times after exposure. These results have implications for biological dosimetry in human populations.

Kondrashova, T.V. and Ivanova, T.I. Do Quiescent Human Peripheral Lymphocytes Repair Radiation-Induced Chromosome Damage?

Peripheral blood samples from seven healthy donors were exposed to 2 Gy of ⁶⁰Co γ rays. Lymphocyte cultures were prepared from the samples and stimulated to proliferate either immediately after the exposure or after 1, 2 or 4 h. No significant differences were found between the frequencies of chromosome aberrations (breaks or exchange) observed in lymphocytes stimulated after different postirradiation periods. We conclude that unstimulated lymphocytes do not undergo significant levels of recovery from potentially lethal damage at the chromosomal level.