Kreisheimer, M., Koshurnikova, N. A., Nekolla, E., Khokhryakov, V. F., Romanow, S. A., Sokolnikov, M. E., Shilnikova, N. S., Okatenko, P. V. and Kellerer, A. M. Lung Cancer Mortality among Male Nuclear Workers of the Mayak Facilities in the Former Soviet Union.

An analysis of lung cancer mortality in a cohort of 1,669 Mayak workers who started their employment in the plutonium and reprocessing plants between 1948 and 1958 has been carried out in terms of a relative risk model. Particular emphasis has been given to a discrimination of the effects of external γ-ray exposure and internal α-particle exposure due to incorporated plutonium. This study has also used the information from a cohort of 2,172 Mayak reactor workers who were exposed only to external γ rays. The baseline lung cancer mortality rate has not been taken from national statistics but has been derived from the cohort itself. For both α particles and γ rays, the results of the analysis are consistent with linear dose dependences. The estimated excess relative risk per unit organ dose equivalent in the lung due to the plutonium α particles at age 60 equals, according to the present study, 0.6/Sv, with a radiation weighting factor of 20 for α particles. The 95% confidence range is 0.39/Sv to 1.0/Sv. For the γ-ray component, the present analysis suggests an excess relative risk for lung cancer mortality at age 60 of 0.20/Sv, with, however, a large 95% confidence range of−0.04/Sv to 0.69/Sv.

Fujiwara, S., Kusumi, S., Cologne, J., Akahoshi, M., Kodama, K. and Yoshizawa, H. Prevalence of Anti-hepatitis C Virus Antibody and Chronic Liver Disease among Atomic Bomb Survivors.

To investigate whether exposure to atomic bomb radiation altered the prevalence of hepatitis C virus (HCV) infection or accelerated the progress toward chronic hepatitis after HCV infection, the seropositivity of antibody to hepatitis C virus (anti-HCV) was determined for 6,121 participants in the Adult Health Study of atomic bomb survivors in Hiroshima and Nagasaki. The seropositivity of anti-HCV antibody was 2.5 times higher among those with a history of blood transfusion and 1.2 times higher among those with a family history of liver disease, whereas acupuncture showed no association with anti-HCV. Although the prevalence of anti-HCV was lower for survivors with positive dose estimates than for those with 0 dose (relative prevalence 0.84, P = 0.022), there was no evidence of a smooth dose–response relationship. However, these data suggested that the radiation dose response for chronic liver disease among HCV antibody-positive survivors may be greater than that among HCV antibody-negative survivors (slope ratio 20). In conclusion, no dose–response relationship was found between anti-HCV positivity and radiation dose; a possible increase in the radiation dose response of chronic liver disease among anti-HCV-positive individuals was found. Thus radiation exposure may accelerate the progress of chronic liver disease associated with hepatitis C virus infection.

Wang, L.-E., Bondy, M. L., de Andrade, M., Strom, S. S., Wang, X., Sigurdson, A., Spitz, M. R. and Wei, Q. Gender Difference and Smoking Effect in Chromosome Sensitivity to Gamma Radiation in a Healthy Population.

In the general population, there is variation in radiosensitivity associated with cancer risk. However, data on the role of epigenetic factors in the variation of radiosensitivity are scarce. Thus we investigated the effects of smoking and age on the radiosensitivity of human lymphocytes by measuring the frequency of chromosome aberrations after in vitro exposure to γ rays in peripheral lymphocytes from 441 healthy subjects (18–95 years old). We analyzed the frequency of both spontaneous (baseline) and in vitro γ-ray-induced (1.5 Gy) chromatid breaks in 50 well-spread metaphases per subject. The overall mean frequencies of spontaneous and induced breaks were 0.02 and 0.45 per cell, respectively. The mean frequency of induced breaks was significantly higher in men than in women (P = 0.03) but did not differ by age or ethnicity. Donors who had ever smoked showed a small but significantly increased frequency of induced breaks (mean = 0.47) compared to nonsmokers (mean = 0.41; P = 0.005). Further stratification and multivariate analyses revealed that the smoking effect was more pronounced in men than in women. These findings support a smoking effect on radiosensitivity in a healthy population, particularly in men. Therefore, when evaluating the association between radiosensitivity and susceptibility to smoking-related cancers, the effect of smoking should be taken into account.

Shukitt-Hale, B., Casadesus, G., McEwen, J. J., Rabin, B. M. and Joseph, J. A. Spatial Learning and Memory Deficits Induced by Exposure to Iron-56-Particle Radiation.

It has previously been shown that exposing rats to particles of high energy and charge (HZE) disrupts the functioning of the dopaminergic system and behaviors mediated by this system, such as motor performance and an amphetamine-induced conditioned taste aversion; these adverse behavioral and neuronal effects are similar to those seen in aged animals. Because cognition declines with age, spatial learning and memory were assessed in the Morris water maze 1 month after whole-body irradiation with 1.5 Gy of 1 GeV/nucleon high-energy ⁵⁶Fe particles, to test the cognitive behavioral consequences of radiation exposure. Irradiated rats demonstrated cognitive impairment compared to the control group as seen in their increased latencies to find the hidden platform, particularly on the reversal day when the platform was moved to the opposite quadrant. Also, the irradiated group used nonspatial strategies during the probe trials (swim with no platform), i.e. less time spent in the platform quadrant, fewer crossings of and less time spent in the previous platform location, and longer latencies to the previous platform location. These findings are similar to those seen in aged rats, suggesting that an increased release of reactive oxygen species may be responsible for the induction of radiation- and age-related cognitive deficits. If these decrements in behavior also occur in humans, they may impair the ability of astronauts to perform critical tasks during long-term space travel beyond the magnetosphere.

Hiranuma, H., Jikko, A., Maeda, T., Abe, M. and Fuchihata, H. Effect of X Irradiation on Secondary Palate Development in Mice.

The mechanisms whereby X irradiation induces palatal clefting were investigated in vivo and in an in vitro organ culture system. When pregnant mice at day 12.5 of gestation were exposed to a 4-Gy dose of whole-body X radiation, the incidence of palatal clefting in their offspring was 91%. The volume of the irradiated palatal shelves was too low for them to make contact with each other. On gestational day 13.5 after labeling, bromodeoxyuridine-positive cells were sparse and apoptotic cells were abundant in the irradiated shelves. To prevent secondary effects of irradiation from the injured maternal body, fetal palatal explants were immediately transferred to an organ culture system after X irradiation in utero. The incidence of palatal clefting was 24%, much lower than the incidence in vivo. The addition of 10^–4 M of dexamethasone to the culture medium increased the incidence of palatal clefting to 56%. These findings indicated that X irradiation inhibited cell proliferation and induced apoptosis, resulting in small-volume palatal shelves that could not fuse with each other. The organ culture data also indicated that 4 Gy of irradiation appears to produce its effects both by a direct action on the fetus and indirectly by affecting the metabolism of the pregnant dam.

Kandasamy, S. B. Possible Involvement of L-Type Voltage-Gated Calcium Channels in Release of Dopamine in the Striatum of Irradiated Rats.

The object of this study was to determine the effect of exposure to γ radiation on potassium chloride (KCl)-stimulated release of dopamine (DA) in the striatum of the rat. In addition, the effect of some calcium channel blockers [nicardipine, a blocker of the L-type voltage-gated N-type VGCC; Ω-agatoxin TK, a selective blocker of P-type VGCC; and nickel chloride (NiCl₂), which preferentially blocks the T-type VGCC] on KCl-stimulated release of DA in the striatum in sham-irradiated and irradiated rats was determined. Exposure of rats to 1–10 Gy ⁶⁰Co γ rays had no significant effect on KCl-stimulated release of DA in the striatum in comparison to sham-irradiated animals. Administering 100, 300 and 500 nM of Ω-agatoxin TK or 50, 100 and 200 nM of Ω-conotoxin GVIA significantly decreased the release of DA stimulated by KCl in both irradiated and sham-irradiated animals in a dose-dependent manner. However, 10, 30 and 50 μM of nicardipine decreased the release of DA in irradiated animals but not in sham-irradiated animals. It is unknown why doses of 5–20 μM NiCl₂ had no effect on the release of DA in sham-irradiated and irradiated animals. The results demonstrate that the doses of radiation used in this study had no effect on release of DA in the striatum. Multiple calcium channel types coexist to regulate release of DA. P- and N-type VGCCs are involved in release of DA in sham-irradiated and irradiated animals, whereas only L-type VGCCs are involved in release of DA in irradiated animals.

Philippo, H., Huiskamp, R., Winter, E. A. M., Gharbaran, B. and van der Kogel, A. J. Age Dependence of the Radiosensitivity of Glial Progenitors for In Vivo Fission-Neutron and X Irradiation.

O-2A progenitor cells are the stem cells of the myelin-forming oligodendrocytes in the central nervous system. In the epithermal reactor beams used for boron neutron capture therapy (BNCT) for treatment of brain tumors, fission neutrons are a contaminating component. To estimate the radiosensitivity of the O-2A progenitors for fission neutrons, an in vivo–in vitro clonogenic assay was used. Radiosensitivity of progenitors obtained from the spinal cord of 1- or 5-day-old rats or the optic nerve of 2- or 12-week-old rats for 1 MeV fission neutrons was compared to that for 300 kVp X rays. Dose–survival curves were fitted according to the linear-quadratic model. The resulting β component was very small to negligible. Progenitor cells obtained from rats of different ages show differences in radiosensitivity, characterized by different α values. RBE values for fission neutrons were 3.5 for 1-day-old spinal cord, 3.2 for 5-day-old spinal cord, 3.0 for 2-week-old optic nerve, and 4.3 for 12-week-old optic nerve. These high RBE values indicate the importance of minimizing the fast-neutron component in the epithermal neutron beams used for BNCT.

Warenius, H. M., White, R., Peacock, J. H., Hanson, J., Britten, R. A. and Murray, D. The Influence of Hypoxia on the Relative Sensitivity of Human Tumor Cells to 62.5 MeV (p→Be) Fast Neutrons and 4 MeV Photons.

Fast neutrons have been used in the clinical radiation therapy of tumors largely because of experimental evidence that their cytotoxic effects are much less dependent on oxygen levels than those of low-LET photons. The potential therapeutic advantage of fast neutrons based on hypoxia alone can be calculated as the “hypoxic gain factor”, which is the ratio of the OERs for the fast-neutron compared to the photon beams. The hypoxic gain factor that is generally anticipated based on studies with established mammalian cell lines is about 1.6. However, surprisingly few studies have examined the influence of hypoxia on the fast-neutron radiosensitivity of human tumor cells of different histological types. For this reason, we have determined the OERs of five human tumor cell lines exposed to 62.5 MeV (p→Be) cyclotron-generated fast neutrons or 4 MeV photons from a clinical linear accelerator. The OERs for four chemotherapy-naive cell lines, HT29/5, Hep2, HeLa and RT112, were invariably greater for photons than for neutrons, but all of these values were lower than expected on the basis of the previous literature. Despite their low OERs, these cell lines showed hypoxic gain factors that were within the range of 1.31−1.63, indicating that such effects cannot entirely explain the disappointing clinical results obtained with fast neutrons. In contrast, comparison of the surviving fractions at clinically relevant doses (1.6 Gy of neutrons and 2.0 Gy of photons) for these four tumor cell lines suggested that little benefit should result from neutron treatment. Only the cisplatin-resistant OAW42-CP line showed a significant hypoxic gain factor by this method of analysis. We conclude that, at the dose fractions used in clinical radiation therapy, there may not be a radiobiological precedent for higher local control rates after fast-neutron irradiation of hypoxic tumor cells.

Gupta, A. K., Bernhard, E. J., Bakanauskas, V. J., Wu, J., Muschel, R. J. and McKenna, W. G. RAS-Mediated Radiation Resistance is not Linked to MAP Kinase Activation.

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.

Gorbunov, N. V., Pogue-Geile, K. L., Epperly, M. W., Bigbee, W. L., Draviam, R., Day, B. W., Wald, N., Watkins, S. C. and Greenberger, J. S. Activation of the Nitric Oxide Synthase 2 Pathway in the Response of Bone Marrow Stromal Cells to High Doses of Ionizing Radiation.

Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that γ irradiation with doses of 2–50 Gy from ¹³⁷Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.

Greinert, R., Detzler, E. and Harder, D. The Kinetics of Postirradiation Chromatin Restitution as Revealed by Chromosome Aberrations Detected by Premature Chromosome Condensation and Fluorescence In Situ Hybridization.

In a study of X-ray-induced chromosome aberrations in human G₀ lymphocytes irradiated with 4 Gy using premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH), the time-dependent pattern of chromosome fragments and interchromosomal exchanges involving chromosome 4 was recorded after postirradiation incubation times varying from 0.5 to 46.5 h. Unattached acentric fragments and incomplete interchromosomal exchanges have high initial yields, followed by an exponential decrease, while complete interchromosomal exchanges have almost zero initial yield with a subsequent increase in their number. Plateau values of all yields are reached after about 25 h. This temporal variation of aberration yields can consistently be explained by the competition of disruptive PCC stress with the progress of postirradiation structural restitution at the sites of radiation-induced chromatin instabilities. Details of the temporal pattern of incomplete exchanges reflect the different kinetics of the α and β components of the yield of aberrations. The observed large difference between late-PCC and metaphase yields of unattached acentric fragments and the almost perfect conversion from incomplete prematurely condensed chromosomes into complete metaphase exchanges are explained by a difference in the magnitude of chromosome condensation stress between PCC and mitotic conditions. Chromatin sites prone to fragmentation and incompleteness under conditions of PCC can therefore persist as genetic instabilities hidden during mitosis.

Srivastava, M. and Kale, R. K. Radiomodification of Xanthine Oxidoreductase System in the Liver of Mice by Phenylmethylsulfonyl Fluoride and Dithiothreitol.

The widely distributed xanthine oxidoreductase (XOR) system has been shown to be modulated upon exposure of animals to ionizing radiation through the conversion of xanthine dehydrogenase (XDH) into xanthine oxidase (XO). In the present work, radiomodification of the XOR system by phenylmethylsulfonyl fluoride (PMSF) and dithiothreitol (DTT) was examined using female Swiss albino mice which were irradiated with γ rays at a dose rate 0.023 Gy s^–1. PMSF, a serine protease inhibitor, and DTT, the sulfhydryl reagent, were administered intraperitoneally prior to irradiation. The specific activities of XDH and XO as well as the XDH/XO ratio and the total activity (XDH XO) were determined in the liver of the mice. The inhibition of XO activity, restoration of XDH activity, and increase in the XDH/XO ratio upon administration of PMSF were suggestive of irreversible conversion of XDH into XO mediated through serine proteases. The biochemical events required for the conversion were probably initiated during the early phase of irradiation, as the treatment with PMSF immediately after irradiation did not have a modulatory effect. Interestingly, DTT was not effective in modulating radiation-induced changes in the XOR system or oxidative damage in the liver of mice. The DTT treatment resulted in inhibition of the release of lactate dehydrogenase. However, the protection appears to be unrelated to the formation of TBARS. On the other hand, the presence of PMSF during irradiation inhibited radiation-induced oxidative damage and radiation-induced increases in the specific activity of lactate dehydrogenase. These findings suggest that a major effect of ionizing radiation is irreversible conversion of xanthine to xanthine oxidase.

Panov, V., Salomon, Y., Kabalka, G. W. and Bendel, P. Uptake and Washout of Borocaptate Sodium and Borono-phenylalanine in Cultured Melanoma Cells: A Multi-nuclear NMR Study.

The cellular uptake and washout of the two principal boron neutron capture therapy (BNCT) agents, borocaptate sodium (BSH) and borono-phenylalanine (BPA), were monitored on-line, noninvasively, using nuclear magnetic resonance (NMR) spectroscopy. The uptake and washout of inorganic borate (B_i) was also followed for comparison. M2R mouse melanoma cells grown on polystyrene microspheres were perfused inside the NMR sample tube. ¹¹B NMR was used to detect the presence of B_i, BSH and BPA, and ¹⁹F NMR was applied to detect fluorinated BPA (¹⁹F-BPA). The results revealed chemical modifications of BSH due to spontaneous formation of the borocaptate dimer, BSSB, in the culture medium. BPA readily formed a complex with glucose contained in the culture medium but was also converted in the cells to a yet unidentified compound in a reaction that probably involves the hydrolysis of BPA and the release of B_i. The cellular accumulation ratio for BPA was significantly higher than 1 and was also significantly higher than that for BSH. On the other hand, the cellular retention time observed for BSH was much longer than for BPA, indicating a strong trapping of BSH in cells.

Ogiu, T., Nishimura, M., Watanabe, F., Ukai, H., Ishii-Ohba, H., Shimada, Y., Tsuji, H., Sakurai, J. and Hino, O. Absence of Linkage between Radiosensitivity and the Predisposing Atp7b Gene Mutation for Heritable Hepatitis in the LEC Rat.

The LEC rat is known to be a mutant strain that spontaneously develops heritable hepatitis due to copper accumulation, caused by mutation of the copper-transporting ATPase gene (Atp7b). Immunodeficiency and radiosensitivity have also been observed. Hayashi et al. extensively examined the radiosensitivity of the LEC rat and concluded that its hypersensitivity is controlled by a single autosomal gene. Furthermore, they suggested the possibility that it correlates to copper accumulation due to the Atp7b gene mutation, because ionizing radiation-induced hydroxyl radicals might act in concert with copper-induced hydroxyl radicals. In the present experiment, we analyzed linkage between radiosensitivity and the mutation responsible for hepatitis in F₁ animals of a cross with the F344 rat. Our results clearly demonstrated an absence of any significant association. In addition, partial dominance for radiosensitivity was observed, and radiosensitive (F₁ × LEC) backcross rats were twice as numerous as their radioresistant counterparts, suggesting the possibility of control by two or more recessive genes.