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Therapies that enhance antitumor immunity have altered the natural history of many cancers. Consequently, leveraging nonoverlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyl-transferase METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA (dsRNA) formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signaling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that although METTL3 inhibition is equally efficacious to anti-PD-1 therapy, the combination has far greater preclinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD-1 therapy and METTL3 inhibition target distinct malignant clones, and the combination of these therapies overcomes clones insensitive to the single agents. These data provide the mole-cular and preclinical rationale for employing METTL3 inhibitors to promote antitumor immunity in the clinic., Significance: This work demonstrates that METTL3 inhibition stimulates a cell-intrinsic interferon response through dsRNA formation. This immunomodulatory mechanism is distinct from current immunotherapeutic agents and provides the molecular rationale for combination with anti-PD-1 immune-checkpoint blockade to augment antitumor immunity. This article is featured in Selected Articles from This Issue, p. 2109., (©2023 American Association for Cancer Research.)

Calorie restriction (CR) suppresses not only spontaneous but also chemical- and radiation-induced carcinogenesis. Our previous study revealed that the cancer-preventive effect of CR is tissue dependent and that CR does not effectively prevent the development of thymic lymphoma (TL). We investigated the association between CR and the genomic alterations of resulting TLs to clarify the underlying resistance mechanism. TLs were obtained from previous and new experiments, in which B6C3F1 mice were exposed to radiation at 1 week of age and fed with a CR or standard (non-CR) diet from 7 weeks throughout their lifetimes. All available TLs were used for analysis of genomic DNA. In contrast to the TLs of the non-CR group, those of the CR group displayed suppression of copy-neutral loss of heterozygosity (LOH) involving relevant tumor suppressor genes (Cdkn2a, Ikzf1, Trp53, Pten), an event regarded as cell division-associated. However, CR did not affect interstitial deletions of those genes, which were observed in both groups. In addition, CR affected the mechanism of Ikzf1 inactivation in TLs: the non-CR group exhibited copy-neutral LOH with duplicated inactive alleles, whereas the CR group showed expression of dominant-negative isoforms accompanying a point mutation or an intragenic deletion. These results suggest that, even though CR reduces cell division-related genomic rearrangements by suppressing cell proliferation, tumors arise via diverse carcinogenic pathways including inactivation of tumor suppressors via interstitial deletions and other mutations. These findings provide a molecular basis for improved prevention strategies that overcome the CR resistance of lymphomagenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nakayama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The development of in vivo lung cancer models that faithfully mimic the human disease is a crucial research tool for understanding the molecular mechanisms driving tumorigenesis. Subcutaneous transplantation assays are commonly employed, likely due to their amenability to easily monitor tumor growth and the simplistic nature of the technique to deliver tumor cells. Importantly however, subcutaneous tumors grow in a microenvironment that differs from that resident within the lung. To circumvent this limitation, here we describe the development of an intrapulmonary (iPUL) orthotopic transplantation method that enables the delivery of lung cancer cells, with precision, to the left lung lobe of recipient mice. Critically, this allows for the growth of lung cancer cells within their native microenvironment. The coupling of iPUL transplantation with position emission tomography (PET) imaging permits the serial detection of tumors in vivo and serves as a powerful tool to trace lung tumor growth and dissemination over time in mouse disease models., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The gastrin-releasing peptide receptor (GRPR) is a promising molecular target for imaging and therapy of prostate cancer using bombesin peptides that bind to the receptor with high affinity. Targeted copper theranostics (TCTs) using copper radionuclides, 64 Cu for imaging and 67 Cu for therapy, offer significant advantages in the development of next-generation theranostics. [ 64 Cu]Cu-SAR-BBN is in clinical development for PET imaging of GRPR-expressing cancers. This study explores the therapeutic efficacy of [ 67 Cu]Cu-SAR-BBN in a pre-clinical mouse model. The peptide was radiolabeled with 67 Cu, and specific binding of the radiolabeled peptide towards GRPR-positive PC-3 prostate cancer cells was confirmed with 52.2 ± 1.4% total bound compared to 5.8 ± 0.1% with blocking. A therapy study with [ 67 Cu]Cu-SAR-BBN was conducted in mice bearing PC-3 tumors by injecting 24 MBq doses a total of six times. Tumor growth was inhibited by 93.3% compared to the control group on day 19, and median survival increased from 34.5 days for the control group to greater than 54 days for the treatment group. The ease and stability of the radiochemistry, favorable biodistribution, and the positive tumor inhibition demonstrate the suitability of this copper-based theranostic agent for clinical assessment in the treatment of cancers expressing GRPR.

The risk of radiation-induced carcinogenesis depends on age at exposure. We previously reported principal causative genes in lymphomas arising after infant or adult exposure to 4-fractionated irradiation as Pten or Ikzf1 , respectively, suggesting that cells with mutation in these genes might be the origin of lymphomas arising after irradiation depending on age at exposure. Here, we clarified the age-dependent differences in thymus-cell dynamics in mice during the initial post-irradiation period. The thymocyte number initially decreased, followed by two regeneration phases. During the first regeneration, the proportion of phosphorylated-AKT-positive (p-AKT + ) cells in cell-cycle phases S+G2/M of immature CD4 - CD8 - and CD4 + CD8 + thymocytes and in phases G0/G1 of mature CD4 + CD8 - and CD4 - CD8 + thymocytes was significantly greater in irradiated infants than in irradiated adults. During the second regeneration, the proportion of p-AKT + thymocytes in phases G0/G1 increased in each of the three populations other than CD4 - CD8 - thymocytes more so than during the first regeneration. Finally, PI3K-AKT-mTOR signaling in infants contributed, at least in part, to biphasic thymic regeneration through the modification of cell proliferation and survival after irradiation, which may be associated with the risk of Pten mutation-associated thymic lymphoma.

Radionuclide therapy targeting prostate-specific membrane antigen (PSMA) is promising for prostate cancer. We previously reported a ligand, 64 Cu-CuSarbisPSMA, featuring 2 lysine-ureido-glutamate groups. Here, we report the therapeutic potential of 67 Cu-CuSarbisPSMA. Methods: Growth of PSMA-positive xenografts was evaluated after treatment with 67 Cu-CuSarbisPSMA or 177 Lu-LuPSMA imaging and therapy (I&T). Results: At 13 d after injection, tumor growth was similarly inhibited by the 2 tracers in a dose-dependent manner. Survival was comparable after single (30 MBq) or fractionated (2 × 15 MBq, 2 wk apart) administrations. Conclusion: 67 Cu-CuSarbisPSMA is efficacious in a PSMA-expressing model of prostate cancer., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Background: Muscle wasting (Sarcopenia) is associated with poor outcomes in cancer patients. Early identification of sarcopenia can facilitate nutritional and exercise intervention. Cross-sectional skeletal muscle (SM) area at the third lumbar vertebra (L3) slice of a computed tomography (CT) image is increasingly used to assess body composition and calculate SM index (SMI), a validated surrogate marker for sarcopenia in cancer. Manual segmentation of SM requires multiple steps, which limits use in routine clinical practice. This project aims to develop an automatic method to segment L3 muscle in CT scans., Methods: Attenuation correction CTs from full body PET-CT scans from patients enrolled in two prospective trials were used. The training set consisted of 66 non-small cell lung cancer (NSCLC) patients who underwent curative intent radiotherapy. An additional 42 NSCLC patients prescribed curative intent chemo-radiotherapy from a second trial were used for testing. Each patient had multiple CT scans taken at different time points prior to and post- treatment (147 CTs in the training and validation set and 116 CTs in the independent testing set). Skeletal muscle at L3 vertebra was manually segmented by two observers, according to the Alberta protocol to serve as ground truth labels. This included 40 images segmented by both observers to measure inter-observer variation. An ensemble of 2.5D fully convolutional neural networks (U-Nets) was used to perform the segmentation. The final layer of U-Net produced the binary classification of the pixels into muscle and non-muscle area. The model performance was calculated using Dice score and absolute percentage error (APE) in skeletal muscle area between manual and automated contours., Results: We trained five 2.5D U-Nets using 5-fold cross validation and used them to predict the contours in the testing set. The model achieved a mean Dice score of 0.92 and an APE of 3.1% on the independent testing set. This was similar to inter-observer variation of 0.96 and 2.9% for mean Dice and APE respectively. We further quantified the performance of sarcopenia classification using computer generated skeletal muscle area. To meet a clinical diagnosis of sarcopenia based on Alberta protocol the model achieved a sensitivity of 84% and a specificity of 95%., Conclusions: This work demonstrates an automated method for accurate and reproducible segmentation of skeletal muscle area at L3. This is an efficient tool for large scale or routine computation of skeletal muscle area in cancer patients which may have applications on low quality CTs acquired as part of PET/CT studies for staging and surveillance of patients with cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amarasinghe, Lopes, Beraldo, Kiss, Bucknell, Everitt, Jackson, Litchfield, Denehy, Blyth, Siva, MacManus, Ball, Li and Hardcastle.)

Purpose: Projects evaluating the effects of radiation, within the National Institutes of Quantum and Radiological Science and Technology (QST), National Institute of Radiological Sciences (NIRS), have focused on risk analyses for life shortening and cancer prevalence using laboratory animals. Genetic and epigenetic alterations in radiation-induced tumors have been also analyzed with the aim of better understanding mechanisms of radiation carcinogenesis. As well as the economic and practical limitations of repeating such large-scale experiments, ethical considerations make it vital that we store and share the pathological data and samples of the animal experiments for future use. We are now constructing such an archive called the Japan-Storehouse of Animal Radiobiology Experiments (J-SHARE). Methods: J-SHARE records include information such as detailed experimental protocols, necropsy records and photographs of organs at necropsy. For each animal organs and tumor tissues are dissected, and parts are stored as frozen samples at -80 °C. Samples fixed with formalin are also embedded in paraffin blocks for histopathological analyses. Digital copies of stained tissues are being systematically saved using a virtual slide system linked to original records by barcodes. Embedded and frozen tissues are available for molecular analysis. Conclusion: Similar archive systems for radiation biology have also been under construction in the USA and Europe, the Northwestern University Radiation Archive (NURA), and STORE at the BfS, respectively. The J-SHARE will be linked with the sister-archives and made available for collaborative research to institutions and universities all over the world.

Radiation exposure during the peri-pubertal period is a proven risk factor for breast cancer, whereas parity is an established protective factor. The present study investigated whether parity imposes differential protective effects against radiation-induced rat mammary carcinoma depending on the age at exposure. Pre- and post-pubertal female rats, irradiated or left unirradiated, were mated and allowed to nurse until weaning or left unmated. Appearance of mammary tumors was monitored, and serum concentrations of estradiol and progesterone were measured following weaning. Carcinomas were evaluated by immunohistochemistry for estrogen receptor, progesterone receptor, and the cell proliferation marker Ki-67. Parity reduced the risk of carcinoma in unirradiated and pre-pubertally irradiated rats but not post-pubertally irradiated rats. Although radiation exposure increased serum progesterone level, parity after pre-pubertal exposure significantly decreased the elevated progesterone to a normal level, reflecting a protective effect. Moreover, parity significantly decreased the proportion of hormone receptor-positive carcinomas after pre-pubertal exposure. Parity was also related to the observed positive association between progesterone receptor and Ki-67 indices in cancer tissue, implying progesterone receptor-dependent cell proliferation. Thus, parity protects against radiation-induced rat mammary carcinogenesis depending on the age at exposure; the mechanisms may involve changes in hormone levels and cancer tissue.

Purpose: Despite decades of research into radiation-induced adaptive responses, where prior irradiation changes the response to subsequent irradiations, the field of radiation oncology relies upon models of tumor control that assume that each radiation therapy fraction reproduces the same effect, known as iso-effect per fraction. Can these radiobiology principles both be true, forming a paradox or is only one of them right? Here, the apparent coexistence of these two contradictory observations is considered, examining how adaptive responses might apply in radiotherapy scenarios that are inconsistent with the majority of adaptive response experimental designs., Conclusion: While the iso-effect per fraction assumption would preclude the observation of adaptive responses for cells survival after radiotherapy fractions, this does not preclude the observation of adaptive responses for other endpoints. Adaptive responses for cell survival might also manifest without invalidating the iso-effect principle in practical terms. It may also be the case that instances of both phenomena can be observed under different conditions, but not at the same time.

Radiation therapy is an effective means of achieving local control in a wide range of primary tumours, with the reduction in the size of the tumour(s) thought to mediate the observed reductions in metastatic spread in clinical trials. However, there is evidence to suggest that the complex changes induced by radiation in the tumour environment can also present metastatic risks that may counteract the long-term efficacy of the treatment. More than 25 years ago, several largely theoretical mechanisms by which radiation exposure might increase metastatic risk were postulated. These include the direct release of tumour cells into the circulation, systemic effects of tumour and normal tissue irradiation and radiation-induced changes in tumour cell phenotype. Here, we review the data that has since emerged to either support or refute these putative mechanisms focusing on how the unique radiobiology underlying modern radiotherapy modalities might alter these risks.

Whilst the near instantaneous physical interaction of radiation energy with living cells leaves little opportunity for inter-individual variation in the initial yield of DNA damage, all the downstream processes in how damage is recognized, repaired or resolved and therefore the ultimate fate of cells can vary across the population. In the clinic, this variability is observed most readily as rare extreme sensitivity to radiotherapy with acute and late tissue toxic reactions. Though some radiosensitivity can be anticipated in individuals with known genetic predispositions manifest through recognizable phenotypes and clinical presentations, others exhibit unexpected radiosensitivity which nevertheless has an underlying genetic cause. Currently, functional assays for cellular radiosensitivity represent a strategy to identify patients with potential radiosensitivity before radiotherapy begins, without needing to discover or evaluate the impact of the precise genetic determinants. Yet, some of the genes responsible for extreme radiosensitivity would also be expected to confer susceptibility to radiation-induced cancer, which can be considered another late adverse event associated with radiotherapy. Here, the utility of functional assays of radiosensitivity for identifying individuals susceptible to radiotherapy-induced second cancer is discussed, considering both the common mechanisms and important differences between stochastic radiation carcinogenesis and the range of deterministic acute and late toxic effects of radiotherapy., Competing Interests: The authors declare no conflict of interest.

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

There is a natural tendency to expect that irradiation of an infant organ prior to development-related expansion will result in a higher risk of developing cancer than that of fully-developed adult tissue, and this has generally been observed. However, if tissues also vary in their initial responses to radiation depending on age, the interplay between tissue- and age-dependent risk would potentially be quite complex. We have previously shown opposing age-dependent induction of apoptosis for the intestinal epithelium and hematopoietic cells in mice, but such data are not yet available for the liver. Here, we have examined markers of DNA damage, initiation of DNA damage responses, cell cycle arrest, apoptosis and proliferation, as well as gene expression, in the B6C3F1 mouse liver over the hours and days after irradiation of mice at 1 or 7 weeks of age. We found that induction and resolution of radiation-induced DNA damage is not accompanied by significant changes in these cellular end points in the adult liver, while in infant hepatocytes modest induction of p53 accumulation and p21-mediated cell cycle arrest in a small fraction of damaged cells was overshadowed by a further stimulation of proliferation over the relatively high levels already found in the neonatal liver. We observed distinct expression of genes that regulate cell division between the ages, which may contribute to the differential responses. These data suggest that the growth factor signaling environment of the infant liver may mediate radiation-induced proliferation and increased liver cancer risk after irradiation during early life.

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis., (© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Epidemiology studies have shown that children are at greater overall risk of radiation-induced cancer, but the modifying effect of age at exposure in different tissues is heterogeneous. Early epidemiology findings of increased lung cancer risk with increasing age at the time of exposure have been dismissed, with suggestions that the trend is an artefact from a failure to adequately correct for the effects of tobacco smoking. Yet, differing models used in subsequent analyses have shown that the increased susceptibility with age, counter to the overall solid tumor trend, can either be confirmed or discounted depending on the model parameters used. In this study, we analyzed the induction of tumors in female Wistar rats exposed to increasing thoracic doses of X-ray as neonates, juveniles or young adults, to allow the effect of age at exposure in this early period to be observed in the absence of any interactions with smoking. Histology was used to compare tumor subtypes among groups, and genomic DNA copy number alterations in a number of tumors arising after irradiation at different ages were examined. Induction of lung cancers increased with radiation dose, with the frequency of early occurring lung adenomas greater in rats irradiated at older ages. At the highest dose, the rats irradiated at 5 or 15 weeks of age showed increased age-specific rates of lung adenocarcinomas in later life compared to those irradiated at 1 week of age. However, thoracic mammary gland tumors induced by the highest dose at the later ages significantly decreased the lifespan in these groups, reducing the number of rats at risk of radiation-induced lung adenocarcinoma. There was no induction of mammary tumors outside of the irradiated field. Lung adenocarcinomas showed widespread DNA copy number aberrations at the chromosome level, but the only recurrent lesions were intragenic Fhit deletions and losses on chromosome 4. The results presented here suggest that the risk of radiation-induced lung cancer after irradiation may not monotonically decrease with age, and demonstrate that increasing lung cancer risk with exposure age can be observed independent of corrections for smoking, and that mammary tumors may show a similar relationship with age.

Several lines of evidence indicate one's age at exposure to radiation strongly modifies the risk of radiation-induced breast cancer. We previously reported that rat mammary carcinomas induced by pre- and post-pubertal irradiation have distinct gene expression patterns, but the changes underlying these differences have not yet been characterized. The aim of this investigation was to see if differences in CpG DNA methylation were responsible for the differences in gene expression between age at exposure groups observed in our previous study. DNA was obtained from the mammary carcinomas arising in female Sprague-Dawley rats that were either untreated or irradiated (γ-rays, 2 Gy) during the pre- or post-pubertal period (3 or 7 weeks old). The DNA methylation was analyzed using CpG island microarrays and the results compared to the gene expression data from the original study. Global DNA hypomethylation in tumors was accompanied by gene-specific hypermethylation, and occasionally, by unique tumor-specific patterns. We identified methylation-regulated gene expression candidates that distinguished the pre- and post-pubertal irradiation tumors, but these represented only 2 percent of the differentially expressed genes, suggesting that methylation is not a major or primary mechanism underlying the phenotypes. Functional analysis revealed that the candidate methylation-regulated genes were enriched for stem cell differentiation roles, which may be important in mammary cancer development and worth further investigation. However, the heterogeneity of human breast cancer means that the interpretation of molecular and phenotypic differences should be cautious, and take into account the co-variates such as hormone receptor status and cell-of-origin that may influence the associations., Competing Interests: The authors have declared that no competing interests exist.

Recently reported studies have led to a heightened awareness of the risks of cancer induced by diagnostic radiological imaging, and in particular, the risk of brain cancer after childhood CT scans. One feature of Ptch1 +/- mice is their sensitivity to radiation-induced medulloblastomas (an embryonic cerebellar tumor) during a narrow window of time centered on the days around birth. Little is known about the dynamics of how dose protraction interacts with such narrow windows of sensitivity in individual tissues. Using medulloblastomas from irradiated Ptch1 +/- mice with a hybrid C3H × C57BL/6 F1 genetic background, we previously showed that the alleles retained on chromosome 13 (which harbors the Ptch1 gene) reveal two major mechanisms of loss of the wild-type allele. The loss of parental alleles from the telomere extending up to or past the Ptch1 locus by recombination (spontaneous type) accounts for almost all medulloblastomas in nonirradiated mice, while tumors in irradiated mice often exhibited interstitial deletions, which start downstream of the wild-type Ptch1 and extend up varying lengths towards the centromere (radiation type). In this study, Ptch1 +/- mice were exposed to an acute dose of either 100 or 500 mGy gamma rays in utero or postnatally, or the same radiation doses protracted over a four-day period, and were monitored for medulloblastoma development. The results showed dose- and age-dependent radiation-induced type tumors. Furthermore, the size of the radiation-induced deletion differed with the dose rate. The results of this work suggest that tumor latency may be related to the size of the deletion. In this study, 500 mGy exposure produced radiation-induced type tumors at all ages and dose rates, while 100 mGy exposure did not significantly produce radiation-induced type tumors. The radiation signature allows for unique mechanistic insight into the action of radiation to induce DNA lesions with known causal relationship to a specific tumor type, particularly for doses and dose rates that are relevant to both diagnostic and accidental radiological exposures.

The risk of radiation-induced cancer adds to anxiety in low-dose exposed populations. Safe and effective lifestyle changes which can help mitigate excess cancer risk might provide exposed individuals the opportunity to pro-actively reduce their cancer risk, and improve mental health and well-being. Here, we applied a mathematical multi-stage carcinogenesis model to the mouse lifespan data using adult-onset caloric restriction following irradiation in early life. We re-evaluated autopsy records with a veterinary pathologist to determine which tumors were the probable causes of death in order to calculate age-specific mortality. The model revealed that in both irradiated and unirradiated mice, caloric restriction reduced the age-specific mortality of all solid tumors and hepatocellular carcinomas across most of the lifespan, with the mortality rate dependent more on age owing to an increase in the number of predicted rate-limiting steps. Conversely, irradiation did not significantly alter the number of steps, but did increase the overall transition rate between the steps. We show that the extent of the protective effect of caloric restriction is independent of the induction of cancer from radiation exposure, and discuss future avenues of research to explore the utility of caloric restriction as an example of a potential post-irradiation mitigation strategy.

The in vivo mouse transgenic pKZ1 chromosomal inversion assay is a sensitive assay that responds to very low doses of DNA-damaging agents. pKZ1 inversions are measured as the frequency of cells expressing E. coli β-galactosidase protein, which can only be produced from an inverted pKZ1 transgene. In previous studies we reported that a single whole-body low dose of 0.01 mGy X rays alone caused an increase in pKZ1 chromosomal inversions in spleen when analyzed 3 days postirradiation, and yet this same dose could protect from high-dose-induced inversions when delivered as a conditioning dose 4 h before or after a 1 Gy challenge dose. In an attempt to explain these results, we performed temporal studies over a wide radiation dose range to determine if the inversion response was temporally different at different doses. pKZ1 mice were irradiated with a single whole-body X-ray dose of 0.01 mGy, 1 mGy or 1 Gy, and spleen sections were then analyzed for pKZ1 inversions at 7 h, 1 day or 7 days after exposure. No change in inversion frequency was observed at the 7 h time point at any dose. At day 1, an increase in inversions was observed in response to the 0.01 mGy dose, whereas a decrease in inversions below sham-treated frequency was observed for the 1 mGy dose. Inversion frequency for both doses returned to sham-treated inversion frequency by day 7. To our knowledge, this is the first reported study to examine the temporal nature of a radiation response spanning a wide dose range, including doses relevant to occupational exposure, and the results are dynamic and dose specific. The results suggest that inversions induced after low-dose irradiation are removed by homeostatic mechanisms within a short time frame, and underscore the importance of studying responses over a period of time when interpreting radiation effects.

Since diagnostic reference levels (DRLs) for children are not currently established in Japan, the authors determined local DRLs for the full range of paediatric CT examinations in a single tertiary care children's hospital. A retrospective review of 4801 CT performance records for paediatric patients (<15 y old) who had undergone CT examinations from 2008 to 2011 was conducted. The most frequent examinations were of the head (52 %), followed by cardiac (15 %), temporal bone (9 %), abdomen (7 %), chest (6 %) and others (11 %). Approximately one-third of children received two or more CT scans. The authors' investigation showed that mean CTDIvol and DLP for head, chest and abdomen increased as a function of age. Benchmarking of the results showed that CTDIvol, DLP and effective dose for chest and abdomen examinations in this hospital were below average, whereas those for the head tended to be at or slightly above average of established DRL values from five countries. The results suggest that CT examinations as performed in a tertiary children's hospital in Japan are well optimised., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Renal toxicity is a hallmark of uranium exposure, with uranium accumulating specifically in the S3 segment of the proximal tubules causing tubular damage. As the distribution, concentration and dynamics of accumulated uranium at the cellular level is not well understood, here, we report on high-resolution quantitative in situ measurements by high-energy synchrotron radiation X-ray fluorescence analysis in renal sections from a rat model of uranium-induced acute renal toxicity. One day after subcutaneous administration of uranium acetate to male Wistar rats at a dose of 0.5 mg uranium kg(-1) body weight, uranium concentration in the S3 segment of the proximal tubules was 64.9 ± 18.2 µg g(-1) , sevenfold higher than the mean renal uranium concentration (9.7 ± 2.4 µg g(-1) ). Uranium distributed into the epithelium of the S3 segment of the proximal tubules and highly concentrated uranium (50-fold above mean renal concentration) in micro-regions was found near the nuclei. These uranium levels were maintained up to 8 days post-administration, despite more rapid reductions in mean renal concentration. Two weeks after uranium administration, damaged areas were filled with regenerating tubules and morphological signs of tissue recovery, but areas of high uranium concentration (100-fold above mean renal concentration) were still found in the epithelium of regenerating tubules. These data indicate that site-specific accumulation of uranium in micro-regions of the S3 segment of the proximal tubules and retention of uranium in concentrated areas during recovery are characteristics of uranium behavior in the kidney., (Copyright © 2015 John Wiley & Sons, Ltd.)

Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult-irradiation group, with Ikaros frequently (50%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc-finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult-irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2a and Ikaros are a prominent feature of young adult irradiation-induced T-cell lymphoma, tumors arising after irradiation from infancy suffer a second hit in Pten by mis-segregation or recombination. This is the first report showing an influence of age-at-exposure on genomic alterations of tumor suppressor genes and their relative involvement in radiation-induced T-cell lymphoma. These data are important for considering the risks associated with childhood exposure to radiation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Monitoring mice exposed to carbon ion radiotherapy provides an indirect method to evaluate the potential for second cancer induction in normal tissues outside the radiotherapy target volume, since such estimates are not yet possible from historical patient data. Here, male and female B6C3F1 mice were given single or fractionated whole-body exposure(s) to a monoenergetic carbon ion radiotherapy beam at the Heavy Ion Medical Accelerator in Chiba, Japan, matching the radiation quality delivered to the normal tissue ahead of the tumour volume (average linear energy transfer = 13 keV x μm(-1)) during patient radiotherapy protocols. The mice were monitored for the remainder of their lifespan, and a large number of T cell lymphomas that arose in these mice were analysed alongside those arising following an equivalent dose of 137Cs gamma ray-irradiation. Using genome-wide DNA copy number analysis to identify genomic loci involved in radiation-induced lymphomagenesis and subsequent detailed analysis of Notch1, Ikzf1, Pten, Trp53 and Bcl11b genes, we compared the genetic profile of the carbon ion- and gamma ray-induced tumours. The canonical set of genes previously associated with radiation-induced T cell lymphoma was identified in both radiation groups. While the pattern of disruption of the various pathways was somewhat different between the radiation types, most notably Pten mutation frequency and loss of heterozygosity flanking Bcl11b, the most striking finding was the observation of large interstitial deletions at various sites across the genome in carbon ion-induced tumours, which were only seen infrequently in the gamma ray-induced tumours analysed. If such large interstitial chromosomal deletions are a characteristic lesion of carbon ion irradiation, even when using the low linear energy transfer radiation to which normal tissues are exposed in radiotherapy patients, understanding the dose-response and tissue specificity of such DNA damage could prove key to assessing second cancer risk in carbon ion radiotherapy patients.

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and β-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1), B1 and Intracisternal-A-Particle (IAP) repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons. By 420 days post-irradiation, neither radiation- or aging-related changes in the methylation of peripheral blood, spleen or liver L1, B1 and IAP elements were observed. Analysis of the spleen and liver tissues of cohorts of untreated aging mice showed that the 17-19 month age group exhibited higher repeat element methylation than younger or older mice, with no overall decline in methylation detected with age. This is the first temporal analysis of the effect of low dose radiation on repeat element methylation in mouse peripheral blood and the first to examine the long term effect of this dose on repeat element methylation in a radiosensitive tissue (spleen) and a tissue fundamental to the aging process (liver). Our data indicate that the methylation of murine DNA repeat elements can fluctuate with age, but unlike human studies, do not demonstrate an overall aging-related decline. Furthermore, our results indicate that a low dose of ionising radiation does not induce detectable changes to murine repeat element DNA methylation in the tissues and at the time-points examined in this study. This radiation dose is relevant to human diagnostic radiation exposures and suggests that a dose of 10 mGy X-rays, unlike high dose radiation, does not cause significant short or long term changes to repeat element or global DNA methylation.

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure. We chose to study a relatively low dose which is the maximum radiation dose for radiation emergency workers (0.25 Gy) and a high dose relevant to radiotherapy exposures (6 Gy). Mice were administered 400 mg/kg amifostine 30 min before, or 3 h after, whole-body irradiation with 0.25 or 6 Gy X-rays and apoptosis was analysed 3 or 7 h later in spleen and bone marrow. We observed a significant increase in radiation-induced apoptosis in the spleen of mice when amifostine was administered before or after 0.25 Gy X-rays. In contrast, when a high dose of radiation was used (6 Gy), amifostine caused a reduction in radiation-induced apoptosis 3 h post-irradiation in spleen and bone marrow similar to previously published studies. This is the first study to investigate the effect of amifostine on radiation-induced apoptosis at a relatively low radiation dose and the first to demonstrate that while amifostine can reduce apoptosis from high doses of radiation, it does not mediate the same effect in response to low-dose exposures. These results suggest that there may be a dose threshold at which amifostine protects from radiation-induced apoptosis and highlight the importance of examining a range of radiation doses and timepoints.

The effects of ionizing radiation on DNA methylation are of importance due to the role that DNA methylation plays in maintaining genome stability, and the presence of aberrant DNA methylation in many cancers. There is limited evidence that radiation-sensitivity may influence the modulation of DNA methylation by ionizing radiation, resulting in a loss of methylation. The BALB/c, CBA and C57Bl/6 strains are the most commonly utilized mouse strains in radiation research and are classified as radiation sensitive (BALB/c and CBA) or radiation resistant (C57Bl/6). We present here the first direct comparison of changes in repeat element DNA methylation (L1, B1 and Intracisternal A Particle; IAP) over time in these three mouse strains after high-dose radiation exposure. Using a high-resolution melt assay, methylation of the spleen repeat elements was investigated between 1 and 14 days after whole-body irradiation with 1 Gy X rays. Our study demonstrated that rather than a loss of methylation at the elements, all strains exhibited an early increase in L1 methylation one day after irradiation. In the most radiosensitive strain (BALB/c) the increase was also detected at 6 days postirradiation. The radioresistant C57Bl/6 strain exhibited a loss of L1 methylation at 14 days postirradiation. Less extensive changes to the B1 and IAP elements were detected at various time points, and pyrosequencing revealed that the responses of the strains were influenced by sex, with the male BALB/c and CBA mice exhibiting a greater response to the irradiation. The results of our study do not support the hypothesis that the most radiosensitive strains exhibit the greatest loss of repeat element DNA methylation after exposure to high-dose radiation. While the exact mechanism and biological outcome of the changes in DNA methylation observed here are still to be elucidated, this study provides the first evidence that radiation exposure elicits time-dependent changes in the methylation of repeat elements that are influenced by the genetic background, gender and the type of repeat element investigated. Furthermore, it suggest that any induced changes may not be persistent.

The objective of the current study was to determine the classification accuracy of serum S100B and apolipoprotein (apoA-I) for mild traumatic brain injury (mTBI) and abnormal initial head computed tomography (CT) scan, and to identify ethnic, racial, age, and sex variation in classification accuracy. We performed a prospective, multi-centered study of 787 patients with mTBI who presented to the emergency department within 6 h of injury and 467 controls who presented to the outpatient laboratory for routine blood work. Serum was analyzed for S100B and apoA-I. The outcomes were disease status (mTBI or control) and initial head CT scan. At cutoff values defined by 90% of controls, the specificity for mTBI using S100B (0.899 [95% confidence interval (CI): 0.78-0.92]) was similar to that using apoA-I (0.902 [0.87-0.93]), and the sensitivity using S100B (0.252 [0.22-0.28]) was similar to that using apoA-I (0.249 [0.22-0.28]). The area under the receiver operating characteristic curve (AUC) for the combination of S100B and apoA-I (0.738, 95% CI: 0.71, 0.77), however, was significantly higher than the AUC for S100B alone (0.709, 95% CI: 0.68, 0.74, p=0.001) and higher than the AUC for apoA-I alone (0.645, 95% CI: 0.61, 0.68, p<0.0001). The AUC for prediction of abnormal initial head CT scan using S100B was 0.694 (95%CI: 0.62, 0.77) and not significant for apoA-I. At a S100B cutoff of <0.060 μg/L, the sensitivity for abnormal head CT was 98%, and 22.9% of CT scans could have been avoided. There was significant age and race-related variation in the accuracy of S100B for the diagnosis of mTBI. The combined use of serum S100B and apoA-I maximizes classification accuracy for mTBI, but only S100B is needed to classify abnormal head CT scan. Because of significant subgroup variation in classification accuracy, age and race need to be considered when using S100B to classify subjects for mTBI.

The TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) and LADY (Probasin-large T antigen transgenic mouse) mice are widely used autochthonous models of prostate cancer. Both models utilise probasin promoters to direct androgen-regulated expression of oncogenic SV40 specifically to epithelial cells of the mouse prostate. The oncogenic processes and phenotypes which result mimic many features of human prostate cancer, making these transgenic mouse models useful experimental systems. The terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP in situ nick end labelling (TUNEL) assay is a commonly used method for the detection of cells undergoing apoptosis. In this study, we demonstrate false-positive TUNEL staining in frozen prostate tissue from TRAMP and LADY mice, which was not observed in non-transgenic control animals and is not due to non-specific binding of labelled-dUTP substrate. The false-positive signal co-localised with large SV40 T-antigen expression. False-positive signal was apparent using multiple commercial TUNEL kits with different detection systems. These results caution against the use of the TUNEL assay for detection of apoptosis in frozen prostate tissue of large T-antigen based autochthonous transgenic models of prostate cancer.

Cancer of the prostate is a highly prevalent disease with a heterogeneous aetiology and prognosis. Current understanding of the biological mechanisms underlying the responses of prostate tissue to ionizing radiation exposure, including cancer induction, is surprisingly limited for both high- and low-dose exposures. As population exposure to radiation increases, largely through medical imaging, a better understanding of the response of the prostate to radiation exposure is required. Low-dose radiation-induced adaptive responses for increased cancer latency and decreased cancer frequency have been demonstrated in mouse models, largely for hematological cancers. This study examines the effects of high- and low-dose whole-body radiation exposure on prostate cancer development using an autochthonous mouse model of prostate cancer: TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP). TRAMP mice were exposed to single acute high (2 Gy), low (50 mGy) and repeated low (5 × 50 mGy) doses of X rays to evaluate both the potential prostate cancer promoting effects of high-dose radiation and low-dose adaptive response phenomena in this prostate cancer model. Prostate weights and histopathology were examined to evaluate gross changes in cancer development and, in mice exposed to a single 2 Gy dose, time to palpable tumor was examined. Proliferation (Ki-67), apoptosis, DNA damage (γ-H2AX) and transgene expression (large T-antigen) were examined within TRAMP prostate sections. Neither high- nor low-dose radiation-induced effects on prostate cancer progression were observed for any of the endpoints studied. Lack of observable effects of high- or low-dose radiation exposure suggests that modulation of tumorigenesis in the TRAMP model is largely resistant to such exposures. However, further study is required to better assess the effects of radiation exposure using alternative prostate cancer models that incorporate normal prostate and in those that are not driven by SV40 large T antigen.

Renal toxicity is the principal health concern after uranium exposure. Children are particularly vulnerable to uranium exposure; with contact with depleted uranium in war zones or groundwater contamination the most likely exposure scenarios. To investigate renal sensitivity to uranium exposure during development, we examined uranium distribution and uranium-induced apoptosis in the kidneys of neonate (7-day-old), prepubertal (25-day-old) and adult (70-day-old) male Wistar rats. Mean renal uranium concentrations increased with both age-at-exposure and exposure level after subcutaneous administration of uranium acetate (UA) (0.1-2 mg kg(-1) body weight). Although less of the injected uranium was deposited in the kidneys of the two younger rat groups, the proportion of the peak uranium content remaining in the kidneys after 2 weeks declined with age-at-exposure, suggesting reduced clearance in younger animals. In situ high-energy synchrotron radiation X-ray fluorescence analysis revealed site-specific accumulation of uranium in the S3 segment of the proximal tubules, distributed in the inner cortex and outer stripe of the outer medulla. Apoptosis and cell loss in the proximal tubules increased with age-at-exposure to 0.5 mg kg(-1) UA. Surprisingly, prepubertal rats were uniquely sensitive to uranium-induced lethality from the higher exposure levels. Observations of increased apoptosis in generating/re-generating tubules particularly in prepubertal rats could help to explain their high mortality rate. Together, our findings suggest that age-at-exposure and exposure level are important parameters for uranium toxicity; uranium tends to persist in developing kidneys after low-level exposures, although renal toxicity is more pronounced in adults., (Copyright © 2013 John Wiley & Sons, Ltd.)

Assessment of risks associated with childhood exposure to ionizing radiation when combined with chemical carcinogens is of great importance. We studied the age-dependence of the effect of combined exposure to ionizing radiation (IR) and a chemical carcinogen on lung carcinogenesis. Female 1-, 5-, and 22-week-old Wistar rats were locally irradiated on the thorax with X-rays (3.18 Gy) and/or were injected intraperitoneally with N-nitrosobis(2-hydroxypropyl)amine (BHP) (1g/kg body weight) 1 week after X-ray exposure or at 23 weeks of age. Rats were terminated at 90 weeks of age. We found that: (i) the incidence of lung tumors (adenoma and adenocarcinoma) increased slightly as a function of age at X-ray exposure, although this was not statistically significant, while the incidence induced by BHP decreased with increasing age at administration; (ii) combined exposure to X-rays at 5 or 22 weeks with BHP 1 week later enhanced the tumor incidence, and the effect at early-life stage (5 weeks irradiation) was more effective than that at late-life stage (22 weeks irradiation); (iii) combined exposure preferentially enhanced malignant transformation; (iv) although a longer interval between the X-ray and BHP treatments reduced the combined effect, risks of early-life irradiation at 1 or 5 weeks of age lasted into adulthood; (v) adenomas and adenocarcinomas induced by X-ray and/or BHP originated from surfactant apoprotein A-positive alveolar type II cells; and (vi), extracellular signal-regulated kinase pathway activation was observed in half the adenocarcinomas, regardless of the exposure schedule. In conclusion, combined exposure may enhance lung tumorigenesis more synergistically at early-life stage (5 weeks of age) than later-life stage., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2'-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.

Serum S100B elevations accurately reflect blood-brain barrier (BBB) damage. Because S100B is also present in peripheral tissues, release of this protein may not be specific to central nervous system (CNS) injury. Ubiquitin C-terminal hydrolase 1 (UCHL1), and phosphorylated neurofilament heavy chain (pNF-H) are found exclusively in neurons, but their relationship to BBB dysfunction has not been determined. The objective of this study was to determine the accuracy of serum UCHL1 and pNF-H as measures of BBB integrity after traumatic brain injury (TBI), to and compare them to S100B. We performed a prospective study of 16 patients with moderate to severe TBI (Glasgow Coma Scale [GCS] score ≤12) and 6 patients with non-traumatic headache who had cerebrospinal fluid (CSF) collected by ventriculostomy or lumbar puncture (LP). Serum and CSF were collected at the time of LP for headache patients and at 12, 24, and 48 h after injury for TBI patients. BBB function was determined by calculating albumin quotients (Q(A)), where Q(A)=[albumin(CSF)]/[albumin(serum)]. S100B, UCHL1, and pNF-H were measured by enzyme-linked immunosorbent assay (ELISA). Pearson's correlation coefficient and area under the receiver operator characteristic (ROC) curve were used to determine relationships between serum markers and Q(A). At 12 hours after TBI, a significant relationship was found between Q(A) and serum UCHL1 concentrations (AUC=0.76; 95% CI 0.55,1.00), and between Q(A) and serum S100B concentrations (AUC=0.794; 95% CI 0.57,1.02). There was no significant relationship found between these markers and Q(A) at other time points, or between pNF-H and Q(A) at any time point. We conclude that serum concentrations of UCHL1 are associated with abnormal BBB status 12 h after moderate to severe TBI. This relationship is similar to that observed between serum S100B and Q(A,) despite the fact that S100B may be released from peripheral tissues after multi-trauma. We conclude that peripheral release of S100B after multi-trauma is probably negligible and that UCHL1 may have some utility to monitor BBB disruption following TBI.

The term radiation-induced bystander effect is used to describe radiation-induced biological changes that manifest in unirradiated cells remaining within an irradiated cell population. Despite their failure to fit into the framework of classical radiobiology, radiation-induced bystander effects have entered the mainstream and have become established in the radiobiology vocabulary as a bona fide radiation response. However, there is still no consensus on a precise definition of radiation-induced bystander effects, which currently encompasses a number of distinct signal-mediated effects. These effects are classified here into three classes: bystander effects, abscopal effects and cohort effects. In this review, the data have been evaluated to define, where possible, various features specific to radiation-induced bystander effects, including their timing, range, potency and dependence on dose, dose rate, radiation quality and cell type. The weight of evidence supporting these defining features is discussed in the context of bystander experimental systems that closely replicate realistic human exposure scenarios. Whether the manifestation of bystander effects in vivo is intrinsically limited to particular radiation exposure scenarios is considered. The conditions under which radiation-induced bystander effects are induced in vivo will ultimately determine their impact on radiation-induced carcinogenic risk.

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by five- to sevenfold 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.

Mild traumatic brain injury (mTBI) refers to the clinical condition of transient alteration of consciousness as a result of traumatic injury to the brain. The priority of emergency care is to identify and facilitate the treatment of rare but potentially life-threatening intracranial injuries associated with mTBI through the judicious application of appropriate imaging studies and neurosurgical consultation. Although post-mTBI symptoms quickly and completely resolve in the vast majority of cases, a significant number of patients will complain of lasting problems that may cause significant disability. Simple and early interventions such as patient education and appropriate referral can reduce the likelihood of chronic symptoms. Although definitive evidence is lacking, mTBI is likely to be related to significant long-term sequelae such as Alzheimer disease and other neurodegenerative processes., (Copyright 2010 Elsevier Inc. All rights reserved.)

The potential for irradiated cells to induce biological effects in their unirradiated neighbors (known as the bystander effect) has been observed repeatedly in vitro. However, whether bystander effects occur in vivo under the specific conditions relevant to low-dose radiation protection is still unclear. To test this, the fate of bystander cells in the mouse spleen was examined using an adoptive transfer method designed to replicate the rare, irradiated cells in an organ that might be expected after a low-dose-rate, low-LET radiation exposure. Splenic lymphocytes radiolabeled with low activities of (3)H-thymidine were introduced into the spleens of unirradiated recipient mice. In this study, the apoptotic and proliferative response of the neighboring bystander spleen cells was compared to the response of spleen cells in parallel control recipients that received sham-irradiated cells. Neither the local area surrounding lodged radiolabeled cells nor the spleen as a whole showed a change in apoptosis or proliferation either 1 or 3 days after adoptive transfer. Increasing the irradiated cell numbers, increasing the mean (3)H-thymidine activity per cell, or exposing cells ex vivo to an acute X-ray dose also had no effect. Possible reasons for the absence of a bystander effect in the spleen under these conditions are discussed.