Bedford, J. S. and Dewey, W. C. Historical and Current Highlights in Radiation Biology: Has Anything Important Been Learned by Irradiating Cells? Radiat. Res. 158, 251–291 (2002).

Around 30 years ago, a very prominent molecular biologist confidently proclaimed that nothing of fundamental importance has ever been learned by irradiating cells! The poor man obviously did not know about discoveries such as DNA repair, mutagenesis, connections between mutagenesis and carcinogenesis, genomic instability, transposable genetic elements, cell cycle checkpoints, or lines of evidence historically linking the genetic material with nucleic acids, or origins of the subject of oxidative stress in organisms, to name a few things of fundamental importance learned by irradiating cells that were well known even at that time. Early radiation studies were, quite naturally, phenomenological. They led to the realization that radiations could cause pronounced biological effects. This was followed by an accelerating expansion of investigations of the nature of these radiobiological phenomena, the beginnings of studies aimed toward better understanding the underlying mechanisms, and a better appreciation of the far-reaching implications for biology, and for society in general. Areas of principal importance included acute tissue and tumor responses for applications in medicine, whole-body radiation effects in plants and animals, radiation genetics and cytogenetics, mutagenesis, carcinogenesis, cellular radiation responses including cell reproductive death, cell cycle effects and checkpoint responses, underlying molecular targets leading to biological effects, DNA repair, and the genetic control of radiosensitivity. This review summarizes some of the highlights in these areas, and points to numerous examples where indeed, many things of considerable fundamental importance have been learned by irradiating cells.

Sèche, E., Sabattier, R., Bajard, J-C., Blondiaux, G., Breteau, N., Spotheim-Maurizot, M. and Charlier, M. Direct Effect in DNA Radiolysis. Boron Neutron Capture Enhancement of Radiolysis in a Medical Fast-Neutron Beam. Radiat. Res. 158, 292–301 (2002).

This paper is devoted to the study of the molecular basis of the boron neutron capture enhancement of fast-neutron radiotherapy. Plasmid DNA was irradiated with a medical fast-neutron beam in the presence of either ¹⁰B or ¹¹B. The number of induced SSBs and DSBs was much higher in samples containing ¹⁰B compared to ¹¹B. The additional breaks are attributed to the nuclear reaction ¹⁰B(n, α)⁷Li induced by the capture by ¹⁰B of thermal neutrons produced in the medium by scattering and slowing down of neutrons. Irradiation in the presence of DMSO (OH radical scavenger) allows the number of nonscavengeable breaks to be determined. The ratio DSB/SSB is within the range of those observed with heavy ions, in good agreement with the hypothesis that the additional breaks are due to α particles and recoil lithium nuclei. The simulation of the energy deposition along the paths of the α and ⁷Li particles allows the calculation of core and penumbra track volumes. Further, the number of plasmids encountered by the core and the penumbra was evaluated. Their number was compared to the nonscavengeable additional breaks. Since the two sets of values are of the same order of magnitude, we conclude that the nonscavengeable additional SSBs and DSBs could be due to direct effects.

Buchko, G. W. and Weinfeld, M. DNA-Targeted 2-Nitroimidazoles: Studies of the Influence of the Phenanthridine-Linked Nitroimidazoles, 2-NLP-3 and 2-NLP-4, on DNA Damage Induced by Ionizing Radiation. Radiat. Res. 158, 302–310 (2002).

The nitroimidazole-linked phenanthridines 2-NLP-3 (5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide) and 2-NLP-4 (5-[3-(2-nitro-1-imidazoyl)-butyl]-phenanthridinium bromide) are composed of the radiosensitizer, 2-nitroimidazole, attached to the DNA intercalator phenanthridine by a 3- and 4-carbon linker, respectively. Previous in vitro assays showed both compounds to be 10–100 times more efficient as hypoxic cell radiosensitizers (based on external drug concentrations) than the untargeted 2-nitroimidazole radiosensitizer, misonidazole (Cowan et al., Radiat. Res. 127, 81–89, 1991). Here we have used a ³²P postlabeling assay and 5′-end-labeled oligonucleotide assay to compare the radiation-induced DNA damage generated in the presence of 2-NLP-3, 2-NLP-4, phenanthridine and misonidazole. After irradiation of the DNA under anoxic conditions, we observed a significantly greater level of 3′-phosphoglycolate DNA damage in the presence of 2-NLP-3 or 2-NLP-4 compared to irradiation of the DNA in the presence of misonidazole. This may account at least in part for the greater cellular radiosensitization shown by the nitroimidazole-linked phenanthridines over misonidazole. Of the two nitroimidazole-linked phenanthridines, the better in vitro radiosensitizer, 2-NLP-4, generated more 3′-phosphoglycolate in DNA than did 2-NLP-3. At all concentrations, phenanthridine had little effect on the levels of DNA damage, suggesting that the enhanced radiosensitization displayed by 2-NLP-3 and 2-NLP-4 is due to the localization of the 2-nitroimidazole to the DNA by the phenanthridine substituent and not to radiosensitization by the phenanthridine moiety itself.

Mitchell, C. R., Folkard, M. and Joiner M. C. Effects of Exposure to Low-Dose-Rate ⁶⁰Co Gamma Rays on Human Tumor Cells In Vitro. Radiat. Res. 158, 311–318 (2002).

Cells of three asynchronously growing human tumor cell lines, PC3 (human prostate carcinoma), T98G and A7 (human glioblastomas), which have been shown previously to demonstrate low-dose hyper-radiosensitivity to low acute single doses, were irradiated with ⁶⁰Co γ rays at low dose rates (2 cGy–1 Gy h⁻¹). Instead of a dose-rate sparing response, these cell lines demonstrated an inverse dose-rate effect on cell survival at dose rates below 1 Gy h⁻¹, whereby a decrease in dose rate resulted in an increase in cell killing per unit dose. A hyper-radiosensitivity-negative cell line, U373MG, did not demonstrate an inverse dose-rate effect. Analysis of the cell cycle indicated that this inverse dose-rate effect was not due to accumulation of cells in G₂/M phase or to other cell cycle perturbations. T98G cells in reversible G₁-phase arrest also showed an inverse dose-rate effect at dose rates below 30 cGy h⁻¹ but a sparing effect as the dose rate was reduced from 60 to 30 cGy h⁻¹. We conclude that this inverse dose-rate effect in continuous exposures reflects the hyper-radiosensitivity seen in the same cell lines in response to very small acute single doses.

Little, J. B., Nagasawa, H., Dahlberg, W. K., Zdzienicka, M. Z., Burma, S. and Chen, D. J. Differing Responses of Nijmegen Breakage Syndrome and Ataxia Telangiectasia Cells to Ionizing Radiation. Radiat. Res. 158, 319–326 (2002).

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G₀/G₁ phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G₁-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G₁-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21^Waf1) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.

Yu, Y., Inamdar, K. V., Turner, K., Jackson-Cook, C. K. and Povirk, L. F. Base Substitutions, Targeted Single-Base Deletions, and Chromosomal Translocations Induced by Bleomycin in Plateau-Phase Mammary Epithelial Cells. Radiat. Res. 158, 327–338 (2002).

Previous work showed that treatment of plateau-phase Chinese hamster ovary cells with the radiomimetic double-strand cleaving agent bleomycin induced very small deletions as well as interchromosomal reciprocal translocations, both of which could be ascribed to errors in end joining of DNA double-strand breaks. In an attempt to assess the possible role of TP53 in suppressing such repair errors, bleomycin-induced mutagenesis at the HPRT locus was examined in immortalized 184B5 human mammary epithelial cells (TP53 ), and in a TP53-defective derivative, 184B5-E6tfxc6. For both cell lines, the most frequent bleomycin-induced mutations were base substitutions, with no apparent targeting to major bleomycin lesions. However, both lines also sustained single-base deletions that were targeted to expected sites of double-strand breaks, suggesting that they arose by end-joining repair of the breaks. Surprisingly, only a few large deletions or rearrangements, and no interchromosomal events involving the HPRT locus were detected among the mutants. The results suggest that in both cell lines, errors in double-strand break repair resulting in heritable large deletions and rearrangements are rare. Spectral karyotyping of bleomycin-treated 184B5 cells showed that a significant number of translocations were present shortly after bleomycin exposure, but their frequency decreased upon continued culture of the cells. Thus, for these cells, the lack of induced interchromosomal rearrangements can be explained in part by selection against such events as the cells proliferate.

Coutier, S., Bezdetnaya, L., Foster, T. H., Parache, R-M. and Guillemin, F. Effect of Irradiation Fluence Rate on the Efficacy of Photodynamic Therapy and Tumor Oxygenation in Meta-Tetra (Hydroxyphenyl) Chlorin (mTHPC)-Sensitized HT29 Xenografts in Nude Mice. Radiat. Res. 158, 339–345 (2002).

We present direct experimental evidence of the fluence-rate-dependent, radiation-induced variations in intratumor oxygen partial pressure (pO₂) in HT29 human colon adenocarcinoma xenografts subjected to meta-tetra(hydroxyphenyl)chlorin (mTHPC)-based photodynamic therapy (PDT). The data establish a correlation between tumor oxygenation and treatment outcome. Tumor-bearing mice were injected with 0.3 mg/kg photosensitizer and subjected 72 h later to a 12 J/cm² red light dose administered at fluence rates of 5, 30, 90 and 160 mW/cm². A significant decrease in mean and median pO₂ was registered at approximately half of the total radiation fluence was delivered in tumors treated at rates of 160 and 90 mW/cm². Conversely, with the two lower fluence rates, intratumor pO₂ was maintained at levels comparable to those measured before illumination. Tumor oxygenation values registered shortly after every treatment protocol were at least equal to baseline levels, thus excluding the possibility of significant acute vessel damage during illumination. The tumor regrowth profile correlated with the pO₂ values monitored during irradiation. Tumors treated with fluence rates of 5 and 30 mW/cm² exhibited significantly longer tumor quadrupling times than those treated at 160 and 90 mW/cm². Improved tumor destruction could be expected by reducing the rate and the extent of oxygen depletion during meta-tetra(hydroxyphenyl)chlorin photodynamic therapy using low fluence rates.

Nakashima, E., Fujiwara, S. and Funamoto, S. Effect of Radiation Dose on the Height of Atomic Bomb Survivors: A Longitudinal Study. Radiat. Res. 158, 346–351 (2002).

We conducted a longitudinal analysis of height after age 20 for atomic bomb survivors in the Adult Health Study (AHS) cohort. The measurements we used were made from July 1958 to June 1998 (AHS examination cycles 1–20). We analyzed only the subjects with known atomic bomb radiation doses, excluding those who were not in the city at the time of bombing (ATB) and those exposed in utero. We also excluded from the analysis measurements made after the occurrence of vertebral fracture. The total number of subjects was 11,862, and the total number of measurements was 109,770; the mean number of measurements per subject was 9.25. Assuming that stature after age 20 is approximately constant, a simple mixed-effects model was fitted to stature after age 20, and linear dose effects for young ATB subjects were modeled for both sexes. The estimated mean heights for subjects born in 1945 in Hiroshima were 166.0 cm for men and 155.4 cm for women. The sex difference in height was 10.6 cm, with men significantly taller than women (P < 0.001). The difference between the cities was not significant (P = 0.162). The birth cohort effects per decade were −1.7 cm for men (P < 0.001) and −2.1 cm for women (P < 0.001). A reduction of stature due to radiation exposure was observed for individuals of both sexes who were below 19 years of age ATB (95% confidence interval, 17–21 years), and the dose effect was larger for women than for men (P = 0.028). The estimated effects per gray for those who were age 0 ATB were −1.2 cm for men and −2.0 cm for women and for those who were age 10 ATB were−0.57 cm for men and −0.96 cm for women.

Braune, S., Riedel, A., Schulte-Mönting, J. and Raczek, J. Influence of a Radiofrequency Electromagnetic Field on Cardiovascular and Hormonal Parameters of the Autonomic Nervous System in Healthy Individuals. Radiat. Res. 158, 352–356 (2002).

The potential health risks of radiofrequency electromagnetic fields (EMFs) emitted by mobile phones are of considerable public interest. The present study investigated the hypothesis, based on the results of our previous study, that exposure to EMFs can increase sympathetic vasoconstrictor activity. Forty healthy young males and females underwent a single-blind, placebo-controlled protocol once on each of two different days. Each investigation included successive periods of placebo and EMF exposure, given in a randomized order. The exposure was implemented by a GSM-like signal (900 MHz, pulsed with 217 Hz, 2 W) using a mobile phone mounted on the right-hand side of the head in a typical telephoning position. Each period of placebo exposure and of EMF exposure consisted of 20 min of supine rest, 10 min of 70° upright tilt on a tilt table, and another 20 min of supine rest. Blood pressure, heart rate and cutaneous capillary perfusion were measured continuously. In addition, serum levels of norepinephrine, epinephrine, cortisol and endothelin were analyzed in venous blood samples taken every 10 min. Similar to the previous study, systolic and diastolic blood pressure each showed slow, continuous, statistically significant increases of about 5 mmHg during the course of the protocol. All other parameters either decreased in parallel or remained constant. However, analysis of variance showed that the changes in blood pressure and in all other parameters were independent of the EMF exposure. These findings do not support the assumption of a nonthermal influence of EMFs emitted by mobile phones on the cardiovascular autonomic nervous system in healthy humans.

Utteridge, T. D., Gebski, V., Finnie, J. W., Vernon-Roberts, B. and Kuchel, T. R. Long-Term Exposure of Eμ-Pim1 Transgenic Mice to 898.4 MHz Microwaves does not Increase Lymphoma Incidence. Radiat. Res. 158, 357–364 (2002).

A total of 120 Eμ-Pim1 heterozygous mice and 120 wild-type mice were exposed for 1 h/day 5 days/week at each of the four exposure levels in “Ferris-wheel” exposure systems for up to 104 weeks to GSM-modulated 898.4 MHz radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg. In addition, 120 heterozygous and 120 wild-type mice were sham-exposed; there was also an unrestrained negative control group. Four exposure levels were used to investigate whether a dose–response effect could be detected. Independent verification confirmed that the exposures in the current study were nonthermal. There was no significant difference in the incidence of lymphomas between exposed and sham-exposed groups at any of the exposure levels. A dose–response effect was not detected. The findings showed that long-term exposures of lymphoma-prone mice to 898.4 MHz GSM radiofrequency (RF) radiation at SARs of 0.25, 1.0, 2.0 and 4.0 W/kg had no significant effects when compared to sham-irradiated animals. A previous study (Repacholi et al., Radiat. Res. 147, 631–640, 1997) reported that long-term exposure of lymphoma-prone mice to one exposure level of 900 MHz RF radiation significantly increased the incidence of non-lymphoblastic lymphomas when compared to sham-irradiated animals.