Sweasy, J. B., Lauper, J. M. and Eckert, K. A. DNA Polymerases and Human Diseases. Radiat. Res. 166, 693–714 (2006).

DNA polymerases function in DNA replication, repair, recombination and translesion synthesis. Currently, 15 DNA polymerase genes have been identified in human cells, belonging to four distinct families. In this review, we briefly describe the biochemical activities and known cellular roles of each DNA polymerase. Our major focus is on the phenotypic consequences of mutation or ablation of individual DNA polymerase genes. We discuss phenotypes of current mouse models and altered polymerase functions and the relationship of DNA polymerase gene mutations to human cell phenotypes. Interestingly, over 120 single nucleotide polymorphisms (SNPs) have been identified in human populations that are predicted to result in nonsynonymous amino acid substitutions of DNA polymerases. We discuss the putative functional consequences of these SNPs in relation to human disease.

Mushkacheva, G., Rabinovich, E., Privalov, V., Povolotskaya, S., Shorokhova, V., Sokolova, S., Turdakova, V., Ryzhova, E., Hall, P., Schneider, A. B., Preston, D. L. and Ron, E. Thyroid Abnormalities Associated with Protracted Childhood Exposure to ¹³¹I from Atmospheric Emissions from the Mayak Weapons Facility in Russia. Radiat. Res. 166, 715–722 (2006).

Between 1948 and 1960, the Mayak nuclear weapons facility in Ozyorsk, Russia discharged relatively high levels of radionuclides, primarily ¹³¹I, into the atmosphere, resulting in appreciable exposure to the residents of Ozyorsk. To evaluate the association between thyroid diseases and childhood exposure to radioiodines, we screened 894 Ozyorsk residents born between 1952 and 1953. The study population was comprised of 581 exposed individuals living in Ozyorsk during the years of heaviest exposure and 313 nonexposed individuals who moved to Ozyorsk when radiation exposure from Mayak largely had ended. The screening protocol included a patient interview, palpation of the thyroid, cervical lymph nodes and salivary glands, an ultrasound examination, and measurement of fT4, TSH and TPOAb. Twenty-eight percent of the study group was diagnosed with a thyroid abnormality. The prevalence of nodular disease was significantly higher in the exposed group (20.7%) compared with the nonexposed (14.4%) group (relative risk = 1.4, 95% CI = 1.1; 1.9). Risks were larger for solitary nodules and for nodules ≥10 mm in diameter. Expansion of the study to increase the number of persons screened as well as detailed dose estimation would offer an unique opportunity to evaluate thyroid disease in relation to chronic exposure to radioiodines during childhood.

Muto, M., Fujimori, A., Nenoi, M., Daino, K., Matsuda, Y., Kuroiwa, A., Kubo, E., Kanari, Y., Utsuno, M., Tsuji, H., Ukai, H., Mita, K., Takahagi, M. and Tatsumi, K. Isolation and Characterization of a Novel Human Radiosusceptibility Gene, NP95. Radiat. Res. 166, 723–733 (2006).

The murine nuclear protein Np95 has been shown to underlie resistance to ionizing radiation and other DNA insults or replication arrests in embryonic stem (ES) cells. Using the databases for expressed sequenced tags and a two-step PCR procedure, we isolated human NP95, the full-length human homologue of the murine Np95 cDNA, which consists of 4,327 bp with a single open reading frame (ORF) encoding a polypeptide of 793 amino acids and 73.3% homology to Np95. The ORF of human NP95 cDNA is identical to the UHRF1 (ubiquitin-like protein containing PHD and RING domain 1). The NP95 gene, assigned to 19p13.3, consists of 18 exons, spanning 60 kb. Several stable transformants from HEK293 and WI-38 cells that had been transfected with the antisense NP95 cDNA were, like the murine Np95-knockout ES cells, more sensitive to X rays, UV light and hydroxyurea than the corresponding parental cells. In HEK293 cells, the lack of NP95 did not affect the activities of topoisomerase IIα, whose expression had been demonstrated to be regulated by the inverted CCAAT box binding protein of 90 kDa (ICBP90) that closely resembles NP95 in amino acid sequence and in cDNA but differs greatly in genomic organization. These findings collectively indicate that the human NP95 gene is the functional orthologue of the murine Np95 gene.

Santini, M. T., Romano, R., Rainaldi, G., Indovina, P., Ferrante, A., Motta, A. and Indovina, P. L. Temporal Dynamics of ¹H-NMR-Visible Metabolites during Radiation-Induced Apoptosis in MG-63 Human Osteosarcoma Spheroids. Radiat. Res. 166, 734–745 (2006).

The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance (¹H-NMR) spectroscopy. Specifically, the ¹H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50–80 μm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH₂ and CH₃ mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH₂ and CH₃ lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.

Gruel, G., Lucchesi, C., Pawlik, A., Frouin, V., Alibert, O., Kortulewski, T., Zarour, A., Jacquelin, B., Gidrol, X. and Tronik-Le Roux, D. Novel Microarray-Based Method for Estimating Exposure to Ionizing Radiation. Radiat. Res. 166, 746–756 (2006).

Accurate estimation of the dose of ionizing radiation to which individuals have been exposed is critical for therapeutic treatment. We investigated whether gene expression profiles could be used to evaluate the dose received, thereby serving as a biological dosimeter. We used cDNA microarrays to monitor changes in gene expression profiles induced by ionizing radiation in mouse total blood. The subsets of genes best characterizing each dose were identified by resampling the original data set and calculating the intersection of the dose signatures. This analytical strategy minimizes the impact of potential genetic/epigenetic variation between mice and overcomes the bias in gene selection inherent to microarray technology. The significance of the identified signatures was evaluated by monitoring the type I error rate by in silico negative control simulation. Based on the distribution of the mean ratios of the selected probes, we were able to identify transcription profiles giving 83% to 100% correct estimation of the dose received by test mice, demonstrating that the selected probes could be used to determine the dose of radiation to which the animals had been exposed. This method could potentially be generalized to determine the level of exposure to other toxins and could be used to develop new related clinical applications.

Day, T. K., Zeng, G., Hooker, A. M., Bhat, M., Scott, B. R., Turner, D. R. and Sykes, P. J. Extremely Low Priming Doses of X Radiation Induce an Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate. Radiat. Res. 166, 757– 766 (2006).

An adaptive response is a response to a stress such as radiation exposure that results in a lower than expected biological response. We describe an adaptive response to X radiation in mouse prostate using the pKZ1 chromosomal inversion assay. pKZ1 mice were treated with a priming dose of 0.001, 0.01, 1 or 10 mGy followed 4 h later by a 1000-mGy challenge dose. All priming doses caused a similar reduction in inversions compared to the 1000-mGy group, supporting the hypothesis that the adaptive response is the result of an on/off mechanism. The adaptive response was induced by a priming dose of 0.001 mGy, which is three orders of magnitude lower than has been reported previously. The adaptive responses completely protected against the inversions that would have been induced by a single 1000-mGy dose as well as against a proportion of spontaneous background inversions. The distribution of inversions across prostate gland cross sections after priming plus challenge irradiation suggested that adaptive responses were predominantly due to reduced low-dose radiation-induced inversions rather than to reduced high-dose radiation-induced inversions. This study used radiation doses relevant to human exposure.

Datta, K., Jaruga, P., Dizdaroglu, M., Neumann, R. D. and Winters, T. A. Molecular Analysis of Base Damage Clustering Associated with a Site-Specific Radiation-Induced DNA Double-Strand Break. Radiat. Res. 166, 767–781 (2006).

Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted ¹²⁵I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the ¹²⁵I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the ¹²⁵I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the ∼32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the ¹²⁵I-decay site that may form via a charge migration mechanism.

Priest, N. D., Hoel, D. G. and Brooks, P. N. Relative Toxicity of Chronic Irradiation by ⁴⁵Ca β Particles and ²⁴²Cm α Particles with Respect to the Production of Lung Tumors in CBA/Ca Mice. Radiat. Res. 166, 782–793 (2006).

Approximately 1800 female CBA/Ca mice were exposed by inhalation at three dose levels to β particles from ⁴⁵Ca-labeled fused aluminosilicate particles (FAP), to α particles from ²⁴²Cm-labeled FAP, or to carrier control FAP. Another group of mice inhaled no FAP and were designated as untreated cage controls. The FAP in combination with these radionuclides was used to achieve the same spatial and temporal distribution of α- and β-particle dose within the irradiated mice. Some mice were killed to determine the clearance of radiolabeled FAP from their lungs, and the remainder were allocated to a life-span study. All animals were subjected to a detailed necropsy. To facilitate the identification of small tumors, the lungs were rendered transparent in methyl salicylate and examined under back illumination for the presence of lesions. Lung nodules and other microscopic lesions were excised for histological examination. The median survival of mice in all groups was approximately 910 days. The control animals lived longer than those that were irradiated, but it was difficult to determine a dose–response relationship for survival among the exposed mice. Benign adenomas and, less frequently, malignant adenocarcinomas were identified in all animal groups. The prevalence of these tumors was ∼28.8% in the control mice, which is consistent with the results of other studies using the same strain of mouse. After exposure to radionuclide-labeled FAP, there was a significant dose-related increase in the prevalence of lung tumors in ²⁴²Cm- (peak prevalence 55%) and ⁴⁵Ca-exposed (peak prevalence 48.6%) mice. The prevalence of tumors in the mice that received ²⁴²Cm-labeled FAP was approximately twice that in the mice that inhaled ⁴⁵Ca-labeled FAP within the range of doses employed (0.55–4.69 Gy). Using the ratio of the slope of the linear component of the dose–response curves, the toxicity of the α particles relative to the β particles was 1.5 (90% CI: 0.7, 9.0) for all adenomas and 9.4 (90% CI: 5.0, 23.0) for the less frequent adenocarcinomas. The relative toxicity for adenocarcinomas was found to decrease with increasing dose.

Heidenreich, W. F., Carnes, B. A. and Paretzke, H. G. Lung Cancer Risk in Mice: Analysis of Fractionation Effects and Neutron RBE with a Biologically Motivated Model. Radiat. Res. 166, 794–801 (2006).

Data from Argonne National Laboratory on lung cancer in 15,975 mice with acute and fractionated exposures to γ rays and neutrons are analyzed with a biologically motivated model with two rate-limiting steps and clonal expansion. Fractionation effects and effects of radiation quality can be explained well by the estimated kinetic parameters. Both an initiating and a promoting action of neutrons and γ rays are suggested. While for γ rays the initiating event is described well with a linear dose-rate dependence, for neutrons a nonlinear term is needed, with less effectiveness at higher dose rates. For the initiating event, the neutron RBE compared to γ rays is about 10 when the dose rate during each fraction is low. For higher dose rates this RBE decreases strongly. The estimated lifetime relative risk for radiation-induced lung cancers from 1 Gy of acute γ-ray exposure at an age of 110 days is 1.27 for male mice and 1.53 for female mice. For doses less than 1 Gy, the effectiveness of fractionated exposure to γ rays compared to acute exposure is between 0.4 and 0.7 in both sexes. For lifetime relative risk, the RBE from acute neutrons at low doses is estimated at about 10 relative to acute γ-ray exposure. It decreases strongly with dose. For fractionated neutrons, it is lower, down to about 4 for male mice.

Marrale, M., Brai, M., Triolo, A., Bartolotta, A. and D'Oca, M. C. Power Saturation of ESR Signal in Ammonium Tartrate Exposed to ⁶⁰Co γ-Ray Photons, Electrons and Protons. Radiat. Res. 166, 802–809 (2006).

In this paper we present an investigation of the electron spin resonance (ESR) line shape of ammonium tartrate (AT) dosimeters exposed to radiation with different linear energy transfer (LET). We exposed our dosimeters to γ-ray photons (⁶⁰Co), 7 MeV and 14 MeV initial energy electrons, and 19.3 MeV initial energy protons. The differences in the power saturation behavior of ESR spectra of AT irradiated with photons, electrons and protons could be correlated to the effective LET of the radiation beams. We analyzed the behavior of peak-to-peak amplitude as a function of microwave power, and we developed a fitting procedure that permits us to obtain the dependence of the homogeneity parameter of the line shape on the LET of the radiation using the Castner saturation theory. This simple procedure allows us to distinguish the LET of the radiation beam.

Wu, J., Daino, K., Ichimura, S. and Nenoi, M. The Initiator Motif is Preferentially Used as the Core Promoter Element in Ionizing Radiation-Responsive Genes. Radiat. Res. 166, 810– 813 (2006).

Recent improvements in DNA microarray technologies and bioinformatics have made it possible to look for common features of ionizing radiation-responsive genes and their regulatory regions. We analyzed the promoters of 217 radiation-responsive human genes, compiled from microarray databases available in the literature. Using the DBTSS database, the transcriptional start sites were determined, and the core promoter elements, such as the TATA-box, initiator (Inr), GC-box and CCAAT-box, were searched for in the −1000 bp/ 200 bp region of each gene by using MATCH. It was found that the frequency of Inr in radiation-responsive genes was higher than that in general genes, and the frequencies of the GC-box and CCAAT-box were significantly lower than those in general genes. Use of the GC-box and the CCAAT-box in radiation-responsive genes was found to be dependent on the TATA-box status; that is, GC-box frequency was low in TATA box-containing genes, and CCAAT-box frequency was also low in TATA-less genes. When correlations between gene functions and frequencies of core promoter elements were examined, no apparent biased use of the core promoter element in association with a specific gene function was observed. It may be speculated that use of Inr in the core promoter correlates with appearance of radiation-responsive enhancer (silencer) elements in the upstream (downstream) regulatory region.