What's Changed in 75 Years of RadRes? - An Australian Perspective on Selected Topics.

Several scientific themes are reviewed in the context of the 75-year period relevant to this special platinum issue of Radiation Research. Two criteria have been considered in selecting the scientific themes. One is the exposure of the associated research activity in the annual meetings of the Radiation Research Society (RRS) and in the publications of the Society's Journal, thus reflecting the interest of members of RRS. The second criteria is a focus on contributions from Australian members of RRS. The first theme is the contribution of radiobiology to radiation oncology, featuring two prominent Australian radiation oncologists, the late Rod Withers and his younger colleague, Lester Peters. Two other themes are also linked to radiation oncology; preclinical research aimed at developing experimental radiotherapy modalities, namely microbeam radiotherapy (MRT) and Auger endoradiotherapy. The latter has a long history, in contrast to MRT, especially in Australia, given that the associated medical beamline at the Australian Synchrotron in Melbourne only opened in 2011. Another theme is DNA repair, which has a trajectory parallel to the 75-year period of interest, given the birth of molecular biology in the 1950s. The low-dose radiobiology theme has a similar timeline, predominantly prompted by the nuclear era, which is also connected to the radioprotector theme, although radioprotectors also have a long-established potential utility in cancer radiotherapy. Finally, two themes are associated with biodosimetry. One is the micronucleus assay, highlighting the pioneering contribution from Michael Fenech in Adelaide, South Australia, and the other is the γ-H2AX assay and its widespread clinical applications.

Mutant p53 Mediates Sensitivity to Cancer Treatment Agents in Oesophageal Adenocarcinoma Associated with MicroRNA and SLC7A11 Expression.

TP53 gene mutations occur in 70% of oesophageal adenocarcinomas (OACs). Given the central role of p53 in controlling cellular response to therapy we investigated the role of mutant (mut-) p53 and SLC7A11 in a CRISPR-mediated JH-EsoAd1 TP53 knockout model. Response to 2 Gy irradiation, cisplatin, 5-FU, 4-hydroxytamoxifen, and endoxifen was assessed, followed by a TaqMan OpenArray qPCR screening for differences in miRNA expression. Knockout of mut-p53 resulted in increased chemo- and radioresistance (2 Gy survival fraction: 38% vs. 56%, p < 0.0001) and in altered miRNA expression levels. Target mRNA pathways analyses indicated several potential mechanisms of treatment resistance. SLC7A11 knockdown restored radiosensitivity (2 Gy SF: 46% vs. 73%; p = 0.0239), possibly via enhanced sensitivity to oxidative stress. Pathway analysis of the mRNA targets of differentially expressed miRNAs indicated potential involvement in several pathways associated with apoptosis, ribosomes, and p53 signaling pathways. The data suggest that mut-p53 in JH-EsoAd1, despite being classified as non-functional, has some function related to radio- and chemoresistance. The results also highlight the important role of SLC7A11 in cancer metabolism and redox balance and the influence of p53 on these processes. Inhibition of the SLC7A11-glutathione axis may represent a promising approach to overcome resistance associated with mut-p53.

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance.

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.

Until There Is a Resolution of the Pro-LNT/Anti-LNT Debate, We Should Head Toward a More Sensible Graded Approach for Protection From Low-Dose Ionizing Radiation.

Current regulation of ionizing radiation is based on the linear no-threshold (LNT) model where any radiation dose increases cancer risk and is independent of dose rate, resulting in large amounts of time and money being spent protecting from extremely small radiation exposures and hence extremely small risk. There are animal studies which demonstrate that LNT is incorrect at low doses, supporting a threshold or hormesis model and thus indicating that there is no need to protect from very low doses. This has led to a sometimes bitter debate between pro-LNT and anti-LNT camps, and the debate has been at a stalemate for some time. This commentary is not aimed at taking either side of the debate. It is likely that the public, workers, and the environment are adequately protected under current regulation, which is the most important outcome. Until those on one side of the debate can convince the other, it would be sensible to move forward toward a graded (risk-based) approach to regulation, where the stringency of control is commensurate with the risk, resulting hopefully in more sensible practical thresholds. This approach is gradually being put forward by international radiation protection advisory bodies.

Low Radiation Environment Switches the Overgrowth-Induced Cell Apoptosis Toward Autophagy.

Low radiation doses can affect and modulate cell responses to various stress stimuli, resulting in perturbations leading to resistance or sensitivity to damage. To explore possible mechanisms taking place at an environmental radiation exposure, we set-up twin biological models, one growing in a low radiation environment (LRE) laboratory at the Gran Sasso National Laboratory, and one growing in a reference radiation environment (RRE) laboratory at the Italian National Health Institute (Istituto Superiore di Sanità, ISS). Studies were performed on pKZ1 A11 mouse hybridoma cells, which are derived from the pKZ1 transgenic mouse model used to study the effects of low dose radiation, and focused on the analysis of cellular/molecular end-points, such as proliferation and expression of key proteins involved in stress response, apoptosis, and autophagy. Cells cultured up to 4 weeks in LRE showed no significant differences in proliferation rate compared to cells cultured in RRE. However, caspase-3 activation and PARP1 cleavage were observed in cells entering to an overgrowth state in RRE, indicating a triggering of apoptosis due to growth-stress conditions. Notably, in LRE conditions, cells responded to growth stress by switching toward autophagy. Interestingly, autophagic signaling induced by overgrowth in LRE correlated with activation of p53. Finally, the gamma component of environmental radiation did not significantly influence these biological responses since cells grown in LRE either in incubators with or without an iron shield did not modify their responses. Overall, in vitro data presented here suggest the hypothesis that environmental radiation contributes to the development and maintenance of balance and defense response in organisms.

DMAPT is an Effective Radioprotector from Long-Term Radiation-Induced Damage to Normal Mouse Tissues In Vivo.

While radiotherapy is widely used in cancer treatment, the benefits can be limited by radiation-induced damage to neighboring healthy tissues. We previously demonstrated in mice that the anti-inflammatory compound dimethylaminoparthenolide (DMAPT) selectively induces radiosensitivity in prostate tumor tissue from transgenic adenocarcinoma of mouse prostate (TRAMP) mice, while simultaneously protecting healthy tissues from 6 Gy whole-body radiation-induced apoptosis. Here, we examined the radioprotective effect of DMAPT on fibrosis in normal tissues after a partial-body fractionated radiation protocol that more closely mimics the image-guided fractionated radiotherapy protocols used clinically. Male C57BL/6J mice, 16 weeks old, received 20 Gy fractionated doses of X rays (2 Gy daily fractions, five days/week for two weeks) or sham irradiation to the lower abdomen, with or without a prior 20 mGy dose to mimic an image dose. In addition, mice received thrice weekly DMAPT (100 mg/kg by oral gavage) or vehicle control from 15 weeks of age until time of analysis at 6 weeks postirradiation. In the absence of exposure to radiation, there were no significant differences observed in the tissues of DMAPT and vehicle-treated mice (P > 0.05). DMAPT treatment significantly reduced radiation-induced testis weight loss by 60.9% (P < 0.0001), protected against a decrease in the seminiferous tubule diameter by 42.1% (P < 0.0001) and largely preserved testis morphology. Inclusion of the image dose had no significant effect on testis mass, seminiferous tubule diameter or testis morphology. DMAPT reduced radiation-induced fibrosis in the corpus cavernous region of the penis (98.1% reduction, P = 0.009) and in the muscle layer around the bladder (80.1% reduction, P = 0.0001). There was also a trend towards reduced collagen infiltration into the submucosal and muscle layers in the rectum. These results suggest that DMAPT could be useful in providing protection from the radiation-induced side effects of impotence and infertility, urinary incontinence and fecal urgency resulting from prostate cancer radiotherapy. DMAPT is a very well-tolerated drug and can conveniently be delivered orally without strict time windows relative to radiation exposure. Protection of normal tissues by DMAPT could potentially be useful in radiotherapy of other cancer types as well.

The Combination of Metformin and Valproic Acid Has a Greater Anti-tumoral Effect on Prostate Cancer Growth In Vivo than Either Drug Alone.

BACKGROUND/AIM: The hypoglycemic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA) have individually shown anti-tumor effects in prostate cancer in vitro. The present study intended to investigate the efficacy of the combination of MET and VPA in prostate cancer treatment in a pre-clinical xenograft model. MATERIALS AND METHODS: Prostate cancer cell lines (LNCaP and PC-3) were inoculated under the skin of BALB/c nude mice. The mice were treated with 200 µl/ml MET and/or 0.4% (w/v) VPA diluted in drinking water, or with vehicle control, and were monitored until the tumor volume reached 2,000 mm3 Evaluation of toxicity of the drug combination was determined in liver and kidney by histology. RESULTS: In both LNCaP and PC-3 xenografts, MET combined with VPA significantly reduced tumor growth during the first 4 weeks following treatment, and delayed the time-to-tumor volume of 2,000 mm3 by 90 days, as compared to MET or to VPA alone, and to vehicle control. There was no significant difference in total mouse weight, liver or kidney morphology in response to combination treatment (MET+VPA) compared to MET or VPA alone and vehicle control. CONCLUSION: The combination treatment of MET with VPA is more effective at slowing prostate tumor growth in vivo compared to either drug alone, in mouse xenografts. These pre-clinical results support previous in vitro data and also demonstrate the low toxicity of the combination of these drugs, suggesting that this may be a potential new therapy to be investigated in clinical trials for prostate cancer.

Combination Therapies Using Metformin and/or Valproic Acid in Prostate Cancer: Possible Mechanistic Interactions.

Prostate cancer (PCa) is the most frequent cancer in men. The evolution from local PCa to castration-resistant PCa, an end-stage of disease, is often associated with changes in genes such as p53, androgen receptor, PTEN, and ETS gene fusion products. Evidence is accumulating that repurposing of metformin (MET) and valproic acid (VPA) either when used alone, or in combination, with another therapy, could potentially play a role in slowing down PCa progression. This review provides an overview of the application of MET and VPA, both alone and in combination with other drugs for PCa treatment, correlates the responses to these drugs with common molecular changes in PCa, and then describes the potential for combined MET and VPA as a systemic therapy for prostate cancer, based on potential interacting mechanisms.

Mealtime Assistance ... From Chaos to Calm: A Collaborative Best Practice Implementation Project.

BACKGROUND: Providing appropriate and timely mealtime assistance to hospitalized patients should be part of a multifaceted and multidisciplinary approach to optimizing a patient's nutritional care plan. PROBLEM: There was anecdotal evidence at the study hospital that patients did not receive adequate and/or timely assistance at mealtimes. APPROACH: A best practice implementation project, using a proven strategy of audit, feedback, and reaudit, was used to effect practice change on an acute general medical ward. OUTCOMES: The combined interventions of staff engagement, redesigning the model of care to reprioritize activities at mealtimes, clarifying nutritional care roles and responsibilities, introducing a protected mealtime and a novel 2-tiered colored tray system, and implementing an awareness and education program have resulted in significant improvements in mealtime assistance. CONCLUSIONS: Success of the project is considered to be replicable and sustainable hospitalwide and more broadly.

Chronic low dose ethanol induces an aggressive metastatic phenotype in TRAMP mice, which is counteracted by parthenolide.

Despite advances in prostate cancer therapy, dissemination and growth of metastases results in shortened survival. Here we examined the potential anti-cancer effect of the NF-κB inhibitor parthenolide (PTL) and its water soluble analogue dimethylaminoparthenolide (DMAPT) on tumour progression and metastasis in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer. Six-week-old male TRAMP mice received PTL (40 mg/kg in 10% ethanol/saline), DMAPT (100 mg/kg in sterile water), or vehicle controls by oral gavage thrice weekly until palpable tumour formation. DMAPT treatment slowed normal tumour development in TRAMP mice, extending the time-to-palpable prostate tumour by 20%. PTL did not slow overall tumour development, while the ethanol/saline vehicle used to administer PTL unexpectedly induced an aggressive metastatic tumour phenotype. Chronic ethanol/saline vehicle upregulated expression of NF-κB, MMP2, integrin ß1, collagen IV, and laminin, and induced vascular basement membrane degradation in primary prostate tumours, as well as increased metastatic spread to the lung and liver. All of these changes were largely prevented by co-administration with PTL. DMAPT (in water) reduced metastasis to below that of water-control. These data suggest that DMAPT has the potential to be used as a cancer preventive and anti-metastatic therapy for prostate cancer. Although low levels of ethanol consumption have not been shown to strongly correlate with prostate cancer epidemiology, these results would support a potential effect of chronic low dose ethanol on metastasis and the TRAMP model provides a useful system in which to further explore the mechanisms involved.

The Combination of Metformin and Valproic Acid Induces Synergistic Apoptosis in the Presence of p53 and Androgen Signaling in Prostate Cancer.

We investigated the potential of combining the hypoglycemic drug metformin (MET) and the antiepileptic drug valproic acid (VPA), which act via different biochemical pathways, to provide enhanced antitumor responses in prostate cancer. Prostate cancer cell lines (LNCaP and PC-3), normal prostate epithelial cells (PrEC), and patient-derived prostate tumor explants were treated with MET and/or VPA. Proliferation and apoptosis were assessed. The role of p53 in response to MET + VPA was assessed in cell lines using RNAi in LNCaP (p53+) and ectopic expression of p53 in PC-3 (p53-). The role of the androgen receptor (AR) was investigated using the AR antagonist enzalutamide. The combination of MET and VPA synergistically inhibited proliferation in LNCaP and PC-3, with no significant effect in PrEC. LNCaP, but not PC-3, demonstrated synergistic intrinsic apoptosis in response to MET + VPA. Knockdown of p53 in LNCaP (p53+, AR+) reduced the synergistic apoptotic response as did inhibition of AR. Ectopic expression of p53 in PC-3 (p53-, AR-) increased apoptosis in response to MET + VPA. In patient-derived prostate tumor explants, MET + VPA also induced a significant decrease in proliferation and an increase in apoptosis in tumor cells. In conclusion, we demonstrate that MET + VPA can synergistically kill more prostate cancer cells than either drug alone. The response is dependent on the presence of p53 and AR signaling, which have critical roles in prostate carcinogenesis. Further in vivo/ex vivo preclinical studies are required to determine the relative efficacy of MET + VPA as a potential treatment for prostate cancer. Mol Cancer Ther; 16(12); 2689-700. ©2017 AACR.

Parthenolide Selectively Sensitizes Prostate Tumor Tissue to Radiotherapy while Protecting Healthy Tissues In Vivo.

Radiotherapy is widely used in cancer treatment, however the benefits can be limited by radiation-induced damage to neighboring normal tissues. Parthenolide (PTL) exhibits anti-inflammatory and anti-tumor properties and selectively induces radiosensitivity in prostate cancer cell lines, while protecting primary prostate epithelial cell lines from radiation-induced damage. Low doses of radiation have also been shown to protect from subsequent high-dose-radiation-induced apoptosis as well as DNA damage. These properties of PTL and low-dose radiation could be used to improve radiotherapy by killing more tumor cells and less normal cells. Sixteen-week-old male Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) and C57BL/6J mice were treated with PTL (40 mg/kg), dimethylaminoparthenolide (DMAPT, a PTL analogue with increased bioavailability) (100 mg/kg), or vehicle control three times over one week prior to combinations of low (10 mGy) and high (6 Gy) doses of whole-body X-irradiation. Tissues were analyzed for apoptosis at a range of time points up to 72 h postirradiation. Both PTL and DMAPT protected normal tissues, but not prostate tumor tissues, from a significant proportion of high-dose-radiation-induced apoptosis. DMAPT provided superior protection compared to PTL in normal dorsolateral prostate (71.7% reduction, P = 0.026), spleen (48.2% reduction, P = 0.0001) and colorectal tissue (38.0% reduction, P = 0.0002), and doubled radiation-induced apoptosis in TRAMP prostate tumor tissue (101.3% increase, P = 0.039). Both drugs induced the greatest radiosensitivity in TRAMP prostate tissue in areas with higher grade prostatic intraepithelial neoplasia (PIN) lesions. A 10 mGy dose delivered 3 h prior to a 6 Gy dose induced a radioadaptive apoptosis response in normal C57Bl/6J prostate (28.4% reduction, P = 0.045) and normal TRAMP spleen (13.6% reduction, P = 0.047), however the low-dose-adaptive radioprotection did not significantly add to the PTL/DMAPT-induced protection in normal tissues, nor did it affect tumor kill. These results support the use of the more bioavailable DMAPT and low-dose radiation, alone or in combination as useful radioprotectors of normal tissues to alleviate radiotherapy-induced side-effects in patients. The enhanced radiosensitisation in prostate tissues displaying high-grade PIN suggests that DMAPT also holds promise for targeted therapy of advanced prostate cancer, which may go on to become metastatic. The redox mechanisms involved in the differential radioprotection observed here suggest that increased radiotherapy efficacy by DMAPT is more broadly applicable to a range of cancer types.

Environmentally Relevant Chronic Low-Dose Tritium and Gamma Exposures do not Increase Somatic Intrachromosomal Recombination in pKZ1 Mouse Spleen.

The toxicity of tritium is a public health concern given its presence and mobility in the environment. For risk predictions using radiological protection models, it is essential to allocate an appropriate radiation weighting factor (WR). This in turn should be consistent with the observed relative biological effectiveness (RBE) of tritium beta radiation. Although the International Commission on Radiological Protection (ICRP) currently recommends a WR of 1 for the calculation of committed effective dose for X rays, gamma rays and electrons of all energies, including tritium energies, there are concerns that tritium health risks are underestimated and that current regulatory tritium drinking water standards need revision. In this study, we investigated potential cytotoxic and genotoxic effects in mouse spleen after one month and eight months of chronic exposure to low-dose tritiated water (HTO). The dose regimes studied were designed to mimic human chronic consumption of HTO at levels of 10 kBq/l, 1 MBq/l and 20 MBq/l. The total doses from these radiation exposures ranged from 0.01 to 180 mGy. We also compared the biological effects of exposure to HTO with equivalent exposure to external whole-body 60Co gamma rays. Changes in spleen weight and somatic intrachromosomal recombination (DNA inversions) in spleen tissue of pKZ1Tg/+ mice were monitored. Our results showed no overall changes in either spleen organ weights and no increase mouse splenic intrachromosomal recombination frequencies, indicating that current drinking water standards for tritium exposure in the form of HTO are likely to be adequately protective against cytotoxic and genotoxic damage in spleen. These results demonstrate no evidence for cytotoxicity or genotoxicity in mouse spleen following chronic exposures to HTO activities (or equivalent gamma doses) up to 20 MBq/L.

Prevention of venous thromboembolism amongst patients in an acute tertiary referral teaching public hospital: a best practice implementation project.

BACKGROUND: Deep vein thrombosis and pulmonary embolism are known collectively as venous thromboembolism (VTE). These conditions are possible complications in hospitalized patients that can extend hospital stay, result in unplanned readmission, and are associated with long-term disability and death. Despite strong evidence, many patients do not receive optimal thromboprophylaxis. VTE prevention is a top priority in healthcare systems worldwide. AIM: The aim of the project was to establish a standardized hospital-wide VTE prevention program and to improve awareness of, and compliance with, best practice standards in the prevention of VTE. METHODS: A multidisciplinary team utilized the Joanna Briggs Institute Practical Application of Clinical Evidence System program to facilitate the collection of pre and post implementation audit data. The Getting Research into Practice program was also used to conduct a situational analysis to identify barriers, enablers, and implementation strategies while taking into account the context in which the changes were to occur. Hospital-acquired VTE data were collected to monitor the impact, if any, on patient outcomes. The project was conducted in three different phases over a 2.5-year period in an acute care public hospital. RESULTS: A comprehensive suite of professionally crafted guidelines, tools, and resources were developed to facilitate clinician acceptance of evidence-based practices. Comparison of compliance results showed variable improvements with four audit criteria. Formalized patient risk assessment improved to 7.5% with the introduction of a new form. High-risk patients receiving appropriate prophylaxis improved to 81% in medical and 83% in surgical patients, on an existing high background compliance rate. A total of 59% of staff attended a VTE update education in-service. No patients received information about adverse VTE events prior to discharge. The hospital-acquired VTE rate decreased slightly from 0.65 to 0.52 events per 1000 overnight bed days. CONCLUSION: Overall the project achieved improvements in compliance with best practice standards. A number of delays and barriers contributed to some of the planned interventions not being fully implemented at the time of the follow-up audit. Contributing factors included the lack of electronic capabilities, some processes not being fully embedded into routine clinical workflows, lack of staff time, and identification of an additional organizational barrier relating to practical issues in providing patient education at discharge. A second action cycle is recommended in an attempt to further improve compliance, ensure intervention fidelity, and embed practices into routine daily workflows to positively impact patient and organizational outcomes.

Temporal Responses to X-Radiation Exposure in Spleen in the pKZ1 Mouse Recombination Assay.

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 ß-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.

A single whole-body low dose X-irradiation does not affect L1, B1 and IAP repeat element DNA methylation longitudinally.

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 methylation of DNA repeat elements is sex-dependent and temporally different in response to X radiation in radiosensitive and radioresistant mouse strains.

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.

Protection from radiation-induced apoptosis by the radioprotector amifostine (WR-2721) is radiation dose dependent.

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.