Parsons, J. L. and Townsend, L. W. Interplanetary Crew Dose Rates for the August 1972 Solar Particle Event.

Using the coupled neutron–proton space radiation transport computer code (BRYNTRN), estimates of dose rates of protons in the skin, ocular lens and bone marrow, behind various thicknesses of aluminum shielding, for crews on space missions outside the Earth's magnetosphere, are made for the large solar particle event (SPE) of August 1972. Overall, the August 1972 dose rates are significantly higher than those estimated for any of the events that occurred in August–December 1989. The dose rates in the August 1972 SPE are not low dose rates as specified by the major national and international advisory bodies and committees.

Hacker-Klom, U. B., Köhnlein, W., Kronholz, H. L. and Göhde, W. The Relative Biological Effectiveness of Low Doses of 14 MeV Neutrons in Steady-State Murine Spermatogenesis as Determined by Flow Cytometry.

The relative biological effectiveness of 14 MeV neutrons in the low-dose range ≤1 Gy has been determined in differentiating and differentiated spermatogonia. Male NMRI mice were exposed to single doses of 2 cGy to 3 Gy of ⁶⁰Co γ rays or neutrons. The ratios of testicular S-phase cells, 4c primary spermatocytes, and elongated spermatids were quantified by DNA flow cytometry 2 to 70 days after irradiation and were found to decrease. Histological samples and testis weight were analyzed in parallel. Doses of 2–5 cGy neutrons and 10–50 cGy γ rays significantly (P < 0.05) decreased the proportions of S-phase cells, spermatocytes and elongated spermatids at 4, 14 and 28 days postirradiation. For S-phase cells, the biphasic shape of the cell survival curves was described with a D₅₀ of 5 cGy neutrons. The D₅₀ for ⁶⁰Co γ rays and the relative biological effectiveness could not be determined. The relative biological effectiveness of neutrons at 50% reductions of testis weight, primary spermatocytes, and elongated spermatids were 2.5, 10.0 and 6.1, respectively. This in vivo assay is interesting because of its sensitivity at dose ranges that are relevant for exposures in the environment, the workplace and radiotherapy.

Kraemer, S. M., Kronenberg, A., Ueno, A. and Waldren, C. A. Measuring the Spectrum of Mutation Induced by Nitrogen Ions and Protons in the Human–Hamster Hybrid Cell Line A_LC.

Astronauts can be exposed to charged particles, including protons, α particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human–hamster cell lines, A_L and A_LC. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human–hamster hybrid cell lines A_LC and A_L. CD59^– mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the A_L hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid A_LC, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in A_LC cells than in A_L cells. In the case of nitrogen ions, the mutations observed in A_LC cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59^– mutant fraction for A_LC cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or “skipping” mutations. We demonstrate here that A_LC cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.

Ulsh, B. A., Mühlmann-Díaz, M. C., Whicker, F. W., Hinton, T. G., Congdon, J. D. and Bedford, J. S. Chromosome Translocations in Turtles: A Biomarker in a Sentinel Animal for Ecological Dosimetry.

Nonhuman organisms are being exposed to ionizing radiations at radionuclide-contaminated sites around the world. Direct methods are seldom available for measuring biologically relevant doses received by these organisms. Here we extend biological dosimetry techniques, which are much better developed for humans and a few other mammalian species, to a nonmammalian species. Turtles were chosen because a long-lived animal would best serve the need for low-level, chronic exposure conditions. We chose the yellow-bellied slider turtle (Trachemys scripta), which is known to have a maximum life span of at least 22 years. As reported elsewhere, we first isolated an embryonic fibroblast cell line and constructed whole-chromosome-specific DNA libraries for chromosome 1 by microdissection and PCR. A FISH painting probe was prepared and used to establish a dose–response curve for ionizing radiation-induced chromosome interchange aberrations in turtle fibroblasts. This was compared to the dose response for human fibroblasts treated under similar conditions in our laboratory. With respect to induction of chromosome interchange aberrations, human fibroblasts were approximately 1.7 times more sensitive than the T. scripta fibroblasts. To the extent that symmetrical interchanges are persistent over long periods, this approach could eventually provide a measure of the integrated lifetime dose these organisms receive from radionuclides in their environment and give a measure of the extent of relevant genetic damage over that time.

Vykhovanets, E. V., Chernyshov, V. P., Slukvin, I. I., Antipkin, Y. G., Vasyuk, A. N. and Colos, V. I. Analysis of Blood Lymphocyte Subsets in Children Living around Chernobyl Exposed Long-Term to Low Doses of Cesium-137 and Various Doses of Iodine-131.

Epidemiological studies have found that children living around Chernobyl have rates of respiratory tract illness that are higher than those seen in the area before the Chernobyl accident. The present study investigates the possible effects of radiation exposure on the composition of peripheral blood lymphocyte subsets in children living around Chernobyl. Two hundred nineteen healthy children and children suffering from recurrent respiratory diseases aged 6–14 years who received both low doses of radiation to the whole body from ¹³⁷Cs and various doses of radiation to the thyroid from ¹³¹I as fallout from the accident were assessed 5 (1991) and 8–10 years (1994–1996) after the accident. A total of 148 healthy children and children suffering from recurrent respiratory diseases living in noncontaminated areas were also evaluated as controls. Children with recurrent respiratory diseases who lived around Chernobyl had a significantly lower percentage of T cells and a higher percentage of NK cells compared to control children with recurrent respiratory diseases during the study period. In contrast to the findings in 1991, a significant decrease in the percentage of helper-inducer cells was observed in children with recurrent respiratory diseases in 1994–1996. In contrast to 1991, there is a positive correlation between the percentage of helper-inducer cells, the helper-inducer/cytotoxic-suppressor cell ratio, and the dose of radiation to the thyroid of healthy children from ¹³¹I in 1994–1996. There was a positive correlation between the dose of radiation to the thyroid from ¹³¹I and the percentage of helper-inducer cells in children with recurrent respiratory diseases 5 years (1991) after the accident. Further, the dose of radiation to the thyroid from ¹³¹I correlated negatively with the percentage of T and B cells and positively with the percentage of NK cells in children with recurrent respiratory diseases 8–10 years (1994–1996) after the accident. These results raise the possibility that long-term exposure to low doses of ¹³⁷Cs may have altered the composition of the T-cell subsets and NK cells in children with recurrent respiratory diseases. The differences in the composition of the peripheral blood lymphocyte subsets between healthy children and those with recurrent respiratory diseases may be attributed to long-term low-dose exposure of the whole body to radiation from ¹³⁷Cs and exposure of the thyroid to radiation from ¹³¹I subsequent to the Chernobyl accident.

Miura, M., Watanabe, H., Okochi, K., Sasaki, T. and Shibuya, H. Biological Response to Ionizing Radiation in Mouse Embryo Fibroblasts with a Targeted Disruption of the DNA Polymerase β Gene.

Base excision repair (BER) is carried out by two distinct pathways in mammalian cells, one dependent on DNA polymerase β (Polb) and the other on proliferating cell nuclear antigen (Pcna). We studied whether the Polb-dependent pathway plays an important role in BER in vivo after exposure to ionizing radiation. For this purpose, we used mouse embryo fibroblasts derived from wild-type and Polb gene knockout littermates. Both cell lines had essentially the same clonogenic cell survival and low levels of apoptosis as determined by a colony formation assay and by a change in mitochondrial membrane potential, respectively. No significant cleavage of protein kinase C δ (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and intact Pkcd was retained in both cell lines for at least 72 h after irradiation. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity in vitro were observed in both cell lines after irradiation. Radiation induced cell cycle arrest in the form of a G₂-phase block, and G₂/M-phase fractions reached a peak approximately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were observed immediately after irradiation in non-S-phase cells of both cell lines and disappeared by 4 h after irradiation. We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results strongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo.

Tuttle, S., Stamato, T., Perez, M. L. and Biaglow, J. Glucose-6-phosphate Dehydrogenase and the Oxidative Pentose Phosphate Cycle Protect Cells against Apoptosis Induced by Low Doses of Ionizing Radiation.

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP . Hence, under normal growth conditions, where NADPH levels exceed NADP levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD⁻) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD⁻ cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD⁻ cells exhibited a significant enhancement in the α component of radiation-induced cell killing, while the values obtained for the β component were similar in both the G6PD⁻ and wild-type CHO cell lines. Here we report that the enhanced α component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD⁻ cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD⁻ cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD⁻ cell line

Price, M. E., McKelvey-Martin, V. J., Robson, T., Hirst, D. G. and McKeown, S. R. Induction and Rejoining of DNA Double-Strand Breaks in Bladder Tumor Cells.

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose–response curves (0–60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0–10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.

Fouladi, B., Waldren, C. A., Rydberg, B. and Cooper, P. K. Comparison of Repair of DNA Double-Strand Breaks in Identical Sequences in Primary Human Fibroblast and Immortal Hamster–Human Hybrid Cells Harboring a Single Copy of Human Chromosome 11.

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Löbrich et al., Proc. Natl. Acad. Sci. USA 92, 12050–12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human–hamster hybrid A_L cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G₁ phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A_L cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

Rydberg, B. Radiation-Induced Heat-Labile Sites that Convert into DNA Double-Strand Breaks.

The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50°C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37°C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50°C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50°C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

Booth, V. K., Roberts, J. C., Warters, R. L., Wilmore, B. H. and Lepock, J. R. Radioprotective Thiolamines WR-1065 and WR-33278 Selectively Denature Nonhistone Nuclear Proteins.

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5–10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23°C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37°C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR-1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein–WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca² ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.

Hole, E. O., Sagstuen, E., Nelson, W. H. and Close, D. M. Free Radical Formation in X-Irradiated Crystals of 2′-Deoxycytidine Hydrochloride. Electron Magnetic Resonance Studies at 10 K.

Single crystals of deoxycytidine hydrochloride (CdR·HCl) have been X-irradiated at 10 K with doses up to about 150 kGy and studied using 24 GHz (K-band) EPR, ENDOR and FSE spectroscopy. In this system, the cytosine base is protonated at the N3 position. Nine different radicals were characterized and identified. Three of these are ascribed to three versions of the one-electron reduced species, probably differing in their protonation state. Radicals formed by net hydrogen addition to the cytosine C5 and C6 positions were observed at 10 K. The hydrogen-abstraction radical at the deoxyribose C1′ position most probably results from initial oxidation of the base. The remaining radical species are all localized to the sugar moiety, representing products formed by net hydrogen abstraction from three of the five available carbons of the deoxyribose sugar. The lack of base-centered oxidation products as well as the structures of the one-electron reduced species is rationalized by considering the specific proton donor–acceptor properties of this crystalline lattice in comparison with similar systems.

Nakagawa, K. and Nishio, T. Electron Paramagnetic Resonance Investigation of Sucrose Irradiated with Heavy Ions.

We investigated the potential use of sucrose to estimate linear energy transfer (LET) for heavy-ion irradiation. We also made a quantitative comparison between heavy-ion and γ irradiation in terms of spin concentration. Heavy-ion irradiation of sucrose produces stable free radicals. Based on the electron paramagnetic resonance (EPR) spectra obtained, the stable sucrose radicals are the same among helium ions, carbon ions and γ rays. The EPR spectrum was ∼70 G wide and was composed of several hyperfine structures. The total spin concentration obtained after the heavy-ion irradiation increased linearly as the absorbed dose increased and decreased logarithmically as LET increased. Production of the spin concentration of helium ions was two times more dependent on LET than that for carbon-ion irradiation. The empirical relationships obtained imply that LET at a certain dose can be determined by the spin concentration. Furthermore, the results of γ irradiation of deuterated sucrose suggest that one of the persistent radicals is a carbon-centered radical.

Hawkins, R. B. Survival of a Mixture of Cells of Variable Linear-Quadratic Sensitivity to Radiation.

The experimentally observed survival of a heterogeneous mixture of cells, each component of which obeys a different linear-quadratic survival response to ionizing radiation, is examined. It is shown that the survival relationship for the mixed population approaches a linear-quadratic form for low doses. The linear parameter of the low-dose relationship approached is equal to the average of the distribution of values of the linear parameter (α_i) of the various components of the mixture. The quadratic parameter of the low-dose relationship approached is equal to the average of the distribution of values of the quadratic parameter (β_i) of the various components of the mixture minus one-half the variance of the distribution of the values of α_i. A numerical example of the survival expected for an exponentially growing population of Chinese hamster V79 cells is presented. From this it can be appreciated that the apparent value of the α and β parameters obtained by fitting an experimentally obtained survival curve will depend on the range of doses over which survival is determined. The apparent value of β is decreased at higher doses, producing a straightening of the survival curve to approach the exponential decrease in survival commonly observed for the terminal high-dose portion of survival curves. Apparent exponential survival at high doses is not inconsistent with linear-quadratic survival and may not indicate a multitarget or other mechanism of cell killing.

Stevens, R. G., Morris, J. E. and Anderson, L. E. Hemochromatosis Heterozygotes May Constitute a Radiation-Sensitive Subpopulation.

A primary mechanism of radiation-induced DNA damage is by generation of free radicals. Chronically increased oxidative stress from elevated levels of iron in the body may increase radiation sensitivity by decreasing cellular oxygen radical scavenging capability. Hemochromatosis heterozygotes have elevated body iron. Low-level radiation sensitization by iron may be particularly pertinent for risk of breast cancer. Since 10% of the population appears to be heterozygous for the hemochromatosis gene, a radiosensitizing effect would have pervasive implications.