Mothersill, C. and Seymour, C. Radiation-Induced Bystander Effects: Past History and Future Directions. Radiat. Res. 155, 757–765 (2001).

There has been a recent upsurge of interest in the phenomenon now known as radiation-induced bystander effects. This is largely due to the increased awareness of the contribution of indirect and delayed effects, such as genomic instability, to cellular outcomes after low-dose exposures. It is also due to the availability of tools such as the microbeam and advanced cell culture systems and to the ability to study end points such as gene or protein expression at low doses which were previously difficult to study. This review looks at the history of bystander effects in the earlier literature, in which the clastogenic effect of plasma from irradiated patients was well known. The effect was known to persist for several years and to cause transgenerational effects, making it similar to what we now call genomic instability. The review then examines the current data and controversies which are now beginning to resolve the questions concerning the mechanisms underlying the induction and transmission of both bystander effects and genomic instability. Finally, the possible impact of data concerning radiation-induced bystander effects on radiotherapy and radiation protection is discussed.

Brennan, R. J. and Schiestl, R. H. Persistent Genomic Instability in the Yeast Saccharomyces cerevisiae Induced by Ionizing Radiation and DNA-Damaging Agents. Radiat. Res. 155, 766–775 (2001).

A “hypermutable” genome is a common characteristic of cancer cells, and it may contribute to the progressive accumulation of mutations required for the development of cancer. It has been reported that mammalian cells surviving exposure to γ radiation display several highly persistent genomic instability phenotypes which may reflect a hypermutability similar to that seen in cancer. These phenotypes include an increased mutation frequency and a decreased plating efficiency, and they continue to be observed many generations after the radiation exposure. The underlying causes of this genomic instability have not been fully determined. We show here that exposure to γ radiation and other DNA-damaging treatments induces a similar genomic instability in the yeast Saccharomyces cerevisiae. A dose-dependent increase in intrachromosomal recombination was observed in cultures derived from cells surviving γ irradiation as many as 50 generations after the exposure. Increased forward mutation frequencies and low colony-forming efficiencies were also observed. Persistently elevated recombination frequencies in haploid cells were dominant after these cells were mated to nonirradiated partners, and the elevated recombination phenotype was also observed after treatment with the DNA-damaging agents ultraviolet light, hydrogen peroxide, and ethyl methanesulfonate. Radiation-induced genomic instability in yeast may represent a convenient model for the hypermutability observed in cancer cells.

White, S. M., Held, K. D., Palmer, M. R. and Yanch, J. C. Biological Dosimetry for Epithermal Neutron Beams. Radiat. Res. 155, 776–782 (2001).

The radiobiological effectiveness of an epithermal neutron beam is described using cell survival as the end point. The M67 epithermal neutron beam at the Nuclear Reactor Laboratory, Massachusetts Institute of Technology, that was used for clinical trials of boron neutron capture therapy was used to irradiate Chinese hamster ovary cells at seven depths in a water-filled phantom that simulated healthy tissue. No boron was added to the samples. Therefore, this experiment evaluates the biological effectiveness of the neutron and photon components, which comprise 80–95% of the dose to healthy tissue. Cell survival was dependent upon the depth in the phantom, as a result of moderation and attenuation of the epithermal neutron beam components by the overlying water. The results were compared with 250 kVp X irradiations to determine relative biological effectiveness values. Cell survival as a function of the dose delivered was lowest at the most shallow depth of 0.5 cm, and increased at depths of 1.5, 3, 4, 5.6, 6.6 and 8.1 cm. The gradual increase in cell survival with increasing depth in the phantom is due to the exponential drop of the fast-neutron intensity of the beam. These results are applicable to clinical boron neutron capture therapy Phase I/II trials in which healthy tissue toxicity was an end point.

Kato, K., Antoku, S., Kodama, K., Kawamura, S., Fujita, Y., Komatsu, K. and Awa, A. A. Organ Doses from Radiation Therapy in Atomic Bomb Survivors. Radiat. Res. 155, 783–793 (2001).

Previous surveys of radiation therapy among the Life Span Study (LSS) population at the Radiation Effects Research Foundation (RERF) revealed that 1,670 (1.4%) of the LSS participants received radiation treatments before 1984. The data on therapeutic radiation doses are indispensable for studying the relationship between radiation treatments and subsequent cancer occurrences. In this study, the radiation treatments were reproduced experimentally to determine the scattered radiation doses. The experiments were conducted using a female human phantom and various radiation sources, including a medium-voltage X-ray machine and a ⁶⁰Co γ-ray source. Doses were measured using thermoluminescence dosimetry and ionization chambers. Radiation doses were determined for the salivary glands, thyroid gland, breast, lung, stomach, colon, ovary and active bone marrow. The results have been used for documenting the organ doses received by patients in previous surveys. The contribution of therapeutic irradiation to the occurrence of chromosome aberrations was studied using data on doses to active bone marrow from both radiation treatments and atomic bomb exposures in 26 RERF Adult Health Study participants. The results suggest that radiation treatments contributed to a large part of their frequencies of stable-type chromosome aberrations. The therapeutic radiation doses determined in the present study are available for investigating the effects of therapeutic irradiation on the subsequent primary cancers among atomic bomb survivors who received radiation treatments.

Stephan, G., Pressl, S., Koshpessova, G. and Gusev, B. I. Analysis of FISH-Painted Chromosomes in Individuals Living near the Semipalatinsk Nuclear Test Site. Radiat. Res. 155, 794–798 (2001).

The Semipalatinsk nuclear test site (STS) is located in the Republic of Kazakhstan. A total of 498 nuclear weapons tests were conducted in this area between 1949 and 1989. The radiation exposure to people who lived close to the STS resulted mostly from the above-ground explosions. Blood samples for chromosome analysis were obtained from 10 subjects who were born before the first explosion in August 1949 and lived continuously in the village of Dolon. The individual calculated effective doses were about 3 Sv. Chromosomes 2, 4 and 8 were painted by means of the FISH technique. In total, 22,240 cells were analyzed. The mean frequency of translocations in the subjects who were irradiated during childhood (2.4/1000 cells) did not differ from the control value (3.1 translocations/1000 cells). It is assumed, therefore, that the calculated physical dose is too high. A significantly increased level of complex cells was determined, however, and this was assumed to have been induced in circulating lymphocytes. The reason for this may be the incorporation of radionuclides from fallout which were not distributed homogeneously within the body, but accumulated instead in tissues that were well supplied with peripheral blood.

Ivanov, V. K., Tsyb, A. F., Rastopchin, E. M., Gorsky, A.;thI., Maksyutov, M. A., Vayzer, V. I., Suspitsin, Y. V. and Fedorov, Y. V. Cancer Incidence among Nuclear Workers in Russia Based on Data from the Institute of Physics and Power Engineering: A Preliminary Analysis. Radiat. Res. 155, 799–806 (2001).

One group that has the potential to be exposed to radiation is workers in the nuclear industry. Results of a systematic medical follow-up and dosimetric monitoring of these workers can form the basis for a study of the relationship between cancer incidence and radiation dose. As part of such efforts in Russia, a major institution of the nuclear industry with an established medical care unit, archiving capabilities, and dosimetry department was selected: the Institute of Physics and Power Engineering (IPPE) in Obninsk. In the study, a comparative analysis of cancer incidence rates for the IPPE workers and for the general population of Russia in 1991–1997 was carried out. The subjects were the IPPE workers hired before 1981. This restriction was imposed to reduce the uncertainty associated with the possible latent period in the development of solid cancers. Thus the possibility of including persons who already had the disease at the time when they were hired was minimized. The analysis is based on information about 158 cancer cases, including 24 cancers in persons under individual dosimetric monitoring. A statistically significant excess in cancer incidence was found among the IPPE workers compared with a comparison population (the general population of Russia) for some types of cancers. The SIR values for all cancers (ICD-9: 140–208) is 0.93 (95% CI 0.76, 1.12) for males and 1.42 (95% CI 1.06, 1.87) for females. A statistically significant excess for all cancers was also observed for residents of Obninsk compared to the control comparison population. The corresponding SIR value was 1.20 (95% CI 1.12, 1.28) for males and 1.58 (95% CI 1.49, 1.69) for females. An important reason for the observed excess in cancer incidence compared to the control population may be the higher level of health care in the so-called nuclear cities of Russia which may have resulted in increased diagnosis and registration of cancers. A statistically significant dependence of the cancer incidence on the dose of ionizing radiation was not established. The excess relative risk per gray for all types of cancer was 0.91 (95% CI −2.75, 4.61) for males and 0.40 (95% CI −6.94, 7.83) for females. These estimates should be considered to be preliminary, as the number of cases considered in the analysis of the dose response is small (17 males and 7 females).

Lloyd, D. C., Lucas, J. N., Edwards, A. A., Deng, W., Valente, E., Hone, P. A. and Moquet, J. E. A Study to Verify a Reported Excess of Chromosomal Aberrations in Blood Lymphocytes of Namibian Uranium Miners. Radiat. Res. 155, 807–815 (2001).

This report describes a study to verify an earlier report of excess chromosomal damage in the blood lymphocytes of uranium miners. Coded blood samples from 10 miners and 10 controls were analyzed conventionally for unstable aberrations and by FISH for translocations. Conventional analysis, scoring 1000 metaphases per subject, showed no significant difference between miners and controls in the frequencies of chromosome- and chromatid-type aberrations. Investigators at two laboratories undertook FISH analyses, each scoring 4000 metaphases per subject. When the data from each laboratory were examined separately, one found slightly more translocations in the miners while the other found fewer. In neither case was the difference significant at the 95% level of confidence. Combining the data likewise showed no significant excess of damage in the miners. This applied to simple one- and two-way translocations and to cells with complex exchanges. There was no correlation between levels of translocations and total lifetime doses from occupational and/or background irradiation. A borderline significant excess of rogue cells was found in the miners. This may be a chance observation, as these rare, highly abnormal cells are considered to be unrelated to radiation exposure and are probably due to a virus. The overall conclusion is that the frequency of chromosomal damage in the miners did not exceed that in the controls. Therefore, the result of the earlier study was not confirmed.

Höglund, E. and Stenerlöw, B. Induction and Rejoining of DNA Double-Strand Breaks in Normal Human Skin Fibroblasts after Exposure to Radiation of Different Linear Energy Transfer: Possible Roles of Track Structure and Chromatin Organization. Radiat. Res. 155, 816–823 (2001).

DNA double-strand breaks are nonrandomly induced by high-LET radiation. Differences in the induction and rejoining of DSBs after irradiation with ions having different LET were detected by fragment analysis. The data obtained indicate that the track structure of the traversing particle and its interaction with the different chromatin structures of the cellular DNA influence the yield as well as the distribution of the induced damage. The induction and rejoining of clustered DSBs induced by the same nitrogen ion fluence at LETs of 80–225 keV/μm were investigated by a detailed analysis of the DNA fragmentation patterns in normal human fibroblasts. The DSBs in the cells were allowed to rejoin during incubations for 0–20 h. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of fragments in the size ranges 1–6 Mbp and 5 kbp–1.5 Mbp. A strong influence of LET on the level of DSB induction was evident. The DSB yield increased from 4.5 ± 0.2 to 10.0 ± 0.3 DSBs per particle traversal through the cell nucleus when LET increased from 80 to 225 keV/μm. Further, the size distribution of the DNA fragments showed a significant dependence on radiation quality, with an excess of fragments at 50–200 kbp and around 1 Mbp. Differences in repair kinetics were also evident, with slower rejoining for increasing LET, and the initial nonrandom fragment distributions were still present after 1 h of repair.

Chernikova, S. B., Lindquist, K. L. and Elkind, M. M. Cell Cycle-Dependent Effects of Wortmannin on Radiation Survival and Mutation. Radiat. Res. 155, 824–829 (2001).

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before γ irradiation caused a greater sensitization in G₁-phase cells relative to late S/G₂-phase cells. Preferential radiosensitization of G₁-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G₁- and S/G₂-phase cells. Interestingly, relative increases in radiation-induced mutations in G₁ and S/G₂ phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.

Chen, S-K., Tsai, M-H., Lin, C-H., Hwang, J. J. and Chang, W. P. Determination of 8-Oxoguanine in Individual Cell Nucleus of Gamma-Irradiated Mammalian Cells. Radiat. Res. 155, 830–834 (2001).

8-Oxoguanine, through its ability to mispair bases other than cytosine, is assumed to be one of the most potent premutagenic lesions in nuclear DNA damaged by reactive oxygen radicals. In this study, we examine whether the presence of residual 8-oxoguanine can be detected in mammalian cells after exposure to ionizing radiation. MOLT-4 human leukemia cells and CHO-K1 Chinese hamster cells were acutely irradiated in vitro with 0, 0.2, 0.4, 0.6 and 1.0 Gy γ radiation at room temperature. The amounts of 8-oxoguanine and total DNA in the cell nucleus were detected by fluorescein-isothiocyanate (FITC)-labeled avidin, which binds specifically and directly to 8-oxoguanine, and propidium iodide, respectively. The intensity ratios between these two fluorescent dyes were then taken as indices to measure the content of 8-oxoguanine within individual cells. We found an apparent dose-dependent increase in the amount of 8-oxoguanine accumulated in cells of both lines. Moreover, the content of 8-oxoguanine decreased from 2 to 20 h after irradiation in CHO-K1 cells, which may reflect the time-dependent repair processes at the 8-oxoguanine lesions. This novel approach may provide a sensitive tool for in situ measurement of 8-oxoguanine in cells or even in the human body after exposure to ionizing radiation.

Seddon, B. M., Honess, D. J., Vojnovic, B., Tozer, G. M. and Workman, P. Measurement of Tumor Oxygenation: In Vivo Comparison of a Luminescence Fiber-optic Sensor and a Polarographic Electrode in the P22 Tumor. Radiat. Res. 155, 835–844 (2001).

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO₂ readings lay in a range over twice that of sensor pO_2(5min) values (–3.2 to 80 mmHg and –0.1 to 34.8 mmHg, respectively). However, there was no significant difference between the means ± 2 SE of the median pO₂ values recorded by each instrument (11.0 ± 3.3 and 8.1 ± 1.9 mmHg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means ± 2 SE of the median pO₂ values reported by the electrode and sensor (1.7 ± 0.9 and 2.9 ± 0.7 mmHg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO₂ values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO₂ values of 0.6 to 28.1 mmHg.

Pateder, D. B., Eliseev, R. A., O'Keefe, R. J., Schwarz, E. M., Okunieff, P., Constine, L. S., Puzas, J. E. and Rosier, R. N. The Role of Autocrine Growth Factors in Radiation Damage to the Epiphyseal Growth Plate. Radiat. Res. 155, 845–855 (2001).

Radiation therapy plays an important role as part of the multimodality treatment for a number of childhood malignancies. Dose-limiting complications of radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken to investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth factors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate chondrocytes and epiphyseal chondrocytes is due to differential modulation of autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the comparative effects of radiation on expression of specific mitogenic growth factors in growth plate chondrocytes. The effect of radiation on the expression of autocrine/paracrine growth factors was examined in an established avian model of epiphyseal growth plate maturation. Exposure of growth plate chondrocytes to radiation resulted in a specific pattern of biochemical and morphological alterations that were dependent on dose and were progressive over time. While radiation did not affect the mRNA expression of some of the autocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expression of PTHrP, a critically important mitogen in growth plate chondrocytes, and a dose-dependent decrease in the PTH/PTHrP receptor mRNA. Interestingly, PTHrP mRNA levels were not affected in irradiated epiphyseal chondrocytes, the main source of PTHrP. Given evidence indicating a role for intracellular calcium levels in regulating PTHrP expression, basal calcium levels in irradiated growth plate chondrocytes and epiphyseal chondrocytes were examined 24 h after treatment. While cytosolic calcium levels were significantly higher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium in mediating radiation damage to growth plate chondrocytes was further demonstrated by the finding that the addition of 4.0 mM EGTA (a calcium chelator) to the cell cultures before irradiation prevented the decrease in PTHrP mRNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irradiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in caspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of growth plate chondrocytes in part by causing an increase in cytosolic calcium levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chondrocyte PTHrP receptor mRNA expression is also inhibited by radiation, further decreasing PTHrP signaling. Despite subtle differences between the chick and mammalian growth plates, further studies should provide an enhanced understanding of the mechanism(s) of radiation injury to the growth plate, as well as possibilities for new therapeutic strategies to protect the growing skeleton from the detrimental effects of radiotherapy.

Van der Meeren, A., Monti, P., Lebaron-Jacobs, L., Marquette, C. and Gourmelon, P. Characterization of the Acute Inflammatory Response after Irradiation in Mice and its Regulation by Interleukin 4 (Il4). Radiat. Res. 155, 856–863 (2001).

The aim of this study was to determine the effects of total-body irradiation of mice on the acute release of a panel of several mediators of inflammation and to evaluate the efficacy of Il4 in regulating these radiation-induced modifications. We studied the effects of exposure of C57BL6/J mice to 8 Gy γ rays on the early release of cytokines, chemokines, acute-phase proteins, prostaglandins and corticosterone in either plasma or tissues compared to those observed after intraperitoneal injection of lipopolysaccharide from 1 h to 3 days after stimulation. During the characterization of the acute inflammatory response induced by radiation or lipopolysaccharide, we observed differences both in the type of mediators produced and in the time course of release. We next determined the anti-inflammatory potential of Il4 in this model of total-body irradiation. We found that Il4 was able to down-regulate the radiation-induced production of mediators of inflammation such as Gro1 (also known as KC, N51) in plasma and lung, corticosterone in blood, Il1b in lung, and prostaglandin E₂ in colon, suggesting the anti-inflammatory potential of Il4 in regulating the radiation-induced response.

Evans, S. C., Mack, D. C., Mason, K. A. and Thames, H. D. The Proliferative Response of Mouse Jejunal Crypt Cells to Radiation-Induced Cell Depletion is not Mediated Exclusively by Transforming Growth Factor Alpha. Radiat. Res. 155, 864–867 (2001).

Several lines of correlative evidence link transforming growth factor alpha (Tgfa, also known as TGF-α) to proliferative activity in jejunal crypt cells. It is therefore tempting to hypothesize that, as a ligand of the epidermal growth factor, it mediates the compensatory proliferative burst in the crypts after radiation-induced cell killing. We have tested this hypothesis by comparing the repopulation response of wild-type and Tgfa-null mice, using the microcolony assay. Mice were exposed whole-body to ¹³⁷Cs γ rays at a dose of approximately 1.6 Gy/min. Single doses and equal doses separated by 4 and 54 h were given. The rightward shift of the dose–response curves for 54 h was identical for wild-type and Tgfa-null mice, and there was no indication of a difference in radiosensitivity. This result indicates that Tgfa is not an essential component of the proliferative response of tissue to radiation-induced cell killing.

Heidenreich, W. F., Atkinson, M. and Paretzke, H. G. Radiation-Induced Cell Inactivation can Increase the Cancer Risk. Radiat. Res. 155, 868–870 (2001).

Radiation can inactivate cells that are replaced by dividing neighboring cells. If cells on the way to malignancy can fill the deficit faster than healthy cells, their number increases. A major part of the radon-induced lung cancers in the Colorado miners can be explained by a moderate increase in the replacement probability.