The Influence of Computed Tomography Contrast Agent on Radiation-Induced Gene Expression and Double-Strand Breaks.

After nuclear scenarios, combined injuries of acute radiation syndrome (ARS) with, e.g., abdominal trauma, will occur and may require contrast-enhanced computed tomography (CT) scans for diagnostic purposes. Here, we investigated the effect of iodinated contrast agents on radiation-induced gene expression (GE) changes used for biodosimetry (AEN, BAX, CDKN1A, EDA2R, APOBEC3H) and for hematologic ARS severity prediction (FDXR, DDB2, WNT3, POU2AF1), and on the induction of double-strand breaks (DSBs) used for biodosimetry. Whole blood samples from 10 healthy donors (5 males, 5 females, mean age: 28 ± 2 years) were irradiated with X rays (0, 1 and 4 Gy) with and without the addition of iodinated contrast agent (0.016 ml contrast agent/ml blood) to the blood prior to the exposure. The amount of contrast agent was set to be equivalent to the blood concentration of an average patient (80 kg) during a contrast-enhanced CT scan. After irradiation, blood samples were incubated at 37°C for 20 min (DSB) and 8 h (GE, DSB). GE was measured employing quantitative real-time polymerase chain reaction. DSB foci were revealed by γH2AX + 53BP1 immunostaining and quantified automatically in >927 cells/sample. Radiation-induced differential gene expression (DGE) and DSB foci were calculated using the respective unexposed sample without supplementation of contrast agent as the reference. Neither the GE nor the number of DSB foci was significantly (P = 0.07-0.94) altered by the contrast agent application. However, for some GE and DSB comparisons with/without contrast agent, there were weakly significant differences (P = 0.03-0.04) without an inherent logic and thus are likely due to inter-individual variation. In nuclear events, the diagnostics of combined injuries can require the use of an iodinated contrast agent, which, according to our results, does not alter or influence radiation-induced GE changes and the quantity of DSB foci. Therefore, the gene expression and γH2AX focus assay can still be applied for biodosimetry and/or hematologic ARS severity prediction in such scenarios.

Short-term Effects of Non-invasive Physical Plasma Treatment on Genomic Stability.

BACKGROUND/AIM: The application of non-invasive physical plasma (NIPP) generates reactive oxygen species. These can lead to chemical oxidation of cellular molecules including DNA. On the other hand, NIPP can induce therapeutically intended apoptosis, which also leads to DNA fragmentation in the late phase. Therefore, to assess unwanted genotoxic effects, the formation of DNA damage was investigated in this study in discrimination from apoptotic processes. MATERIALS AND METHODS: Mutation events after NIPP application were analyzed in CCL-93 fibroblast cells using the hypoxanthine phosphoribosyl transferase assay. Additionally, DNA single-strand breaks (SSB) and double-strand breaks (DSB) were quantified by performing the alkaline comet assay, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. DSBs were quantified by phospho-histone 2AX-p53-binding protein 1 co-localization DSB focus assay. The data were compared with cell death quantification by the caspase-3/7 apoptosis assay. RESULTS: Treatment with NIPP led to exceedingly rapid damage to genomic DNA and the appearance of DNA SSBs and DSBs in the initial 4 h. However, damage decreased again within the first 4-8 h, then the late phase began, characterized by DNA DSB and increasing caspase-3/7 activation. CONCLUSION: Although NIPP treatment leads to extremely rapid damage to genomic DNA, this damage is reversed very quickly by efficient DNA-repair processes. As a consequence, only those cells whose genome damage can be repaired actually survive and proliferate. Persistent genotoxic effects were not observed in the cell system used.

Nano-Architecture of Persistent Focal DNA Damage Regions in the Minipig Epidermis Weeks after Acute γ-Irradiation.

Exposure to high acute doses of ionizing radiation (IR) can induce cutaneous radiation syndrome. Weeks after such radiation insults, keratinocyte nuclei of the epidermis exhibit persisting genomic lesions that present as focal accumulations of DNA double-strand break (DSB) damage marker proteins. Knowledge about the nanostructure of these genomic lesions is scarce. Here, we compared the chromatin nano-architecture with respect to DNA damage response (DDR) factors in persistent genomic DNA damage regions and healthy chromatin in epidermis sections of two minipigs 28 days after lumbar irradiation with ~50 Gy γ-rays, using single-molecule localization microscopy (SMLM) combined with geometric and topological mathematical analyses. SMLM analysis of fluorochrome-stained paraffin sections revealed, within keratinocyte nuclei with perisitent DNA damage, the nano-arrangements of pATM, 53BP1 and Mre11 DDR proteins in γ-H2AX-positive focal chromatin areas (termed macro-foci). It was found that persistent macro-foci contained on average ~70% of 53BP1, ~23% of MRE11 and ~25% of pATM single molecule signals of a nucleus. MRE11 and pATM fluorescent tags were organized in focal nanoclusters peaking at about 40 nm diameter, while 53BP1 tags formed nanoclusters that made up super-foci of about 300 nm in size. Relative to undamaged nuclear chromatin, the enrichment of DDR protein signal tags in γ-H2AX macro-foci was on average 8.7-fold (±3) for 53BP1, 3.4-fold (±1.3) for MRE11 and 3.6-fold (±1.8) for pATM. The persistent macro-foci of minipig epidermis displayed a ~2-fold enrichment of DDR proteins, relative to DSB foci of lymphoblastoid control cells 30 min after 0.5 Gy X-ray exposure. A lasting accumulation of damage signaling and sensing molecules such as pATM and 53BP1, as well as the DSB end-processing protein MRE11 in the persistent macro-foci suggests the presence of diverse DNA damages which pose an insurmountable problem for DSB repair.

Postirradiation temperature influences DSB repair and dicentric chromosome formation-potential impact for dicentric chromosome analysis in interlaboratory comparisons.

The objective was to investigate the influence of different pre-storage temperatures in the dicentric chromosome analysis (DCA) protocol (22°C vs. 37°C) by using γ-H2AX + 53BP1 foci as a marker for deoxyribonucleic acid (DNA) double-strand break (DSB) damage induction and repair and the formation of dicentric chromosomes as a result of mis-repair. Repair of γ-H2AX + 53BP1 DSB foci was absent in samples that were incubated for 2 h at 22°C after exposure of 0.5 and 1.2 Gy. When 0.5- and 1.2-Gy-exposed samples were incubated at 37°C for 2 h, there was an average decline of 31 and 52% of DSB foci, respectively. This indicated that DNA repair occurred. There was a 27% decrease in dicentric chromosome yield at 1.2 Gy and a 15% decrease at 3.5 Gy after post-irradiation incubation for 2 h at 37°C relative to the observed dicentric frequencies at 22°C. Recommended to re-phase: our data suggested that there were more open DSBs after a 2-h incubation at 22°C, which contributed to more mis-repair and dicentric formation from the start of culture. Our findings are corroborated by publications showing that lesion interaction based on enzymatic activity is suppressed below 21°C. As such temperature variations can be a source of variation in DCA during interlaboratory comparison studies, we propose to establish a common guide for the standardisation of pre-culture conditions in cytogenetic dosimetry proficiency testing.

Radiation-induced double-strand breaks by internal ex vivo irradiation of lymphocytes: Validation of a Monte Carlo simulation model using GATE and Geant4-DNA.

This study describes a method to validate a radiation transport model that quantifies the number of DNA double-strand breaks (DSB) produced in the lymphocyte nucleus by internal ex vivo irradiation of whole blood with the radionuclides 90Y, 99mTc, 123I, 131I, 177Lu, 223Ra, and 225Ac in a test vial using the GATE/Geant4 code at the macroscopic level and the Geant4-DNA code at the microscopic level. METHODS: The simulation at the macroscopic level reproduces an 8 mL cylindrical water-equivalent medium contained in a vial that mimics the geometry for internal ex vivo blood irradiation. The lymphocytes were simulated as spheres of 3.75 µm radius randomly distributed, with a concentration of 125 spheres/mL. A phase-space actor was attached to each sphere to register all the entering particles. The simulation at the microscopic level for each radionuclide was performed using the Geant4-DNA tool kit, which includes the clustering example centered on a density-based spatial clustering of applications with noise (DBSCAN) algorithm. The irradiation source was constructed by generating a single phase space from the sum of all phase spaces. The lymphocyte nucleus was defined as a water sphere of a 3.1 µm radius. The absorbed dose coefficients for lymphocyte nuclei (dLymph) were calculated and compared with macroscopic whole blood absorbed dose coefficients (dBlood). The DBSCAN algorithm was used to calculate the number of DSBs. Lastly, the number of DSB∙cell-1∙mGy-1 (simulation) was compared with the number of radiation-induced foci per cell and absorbed dose (RIF∙cell-1∙mGy-1) provided by experimental data for gamma and beta emitting radionuclides. For alpha emitters, dLymph and the number of α-tracks∙100 cell-1∙mGy-1 and DBSs∙µm-1 were calculated using experiment-based thresholds for the α-track lengths and DBSs/track values. The results were compared with the results of an ex vivo study with 223Ra. RESULTS: The dLymph values differed from the dBlood values by -1.0% (90Y), -5.2% (99mTc), -22.3% (123I), 0.35% (131I), 2.4% (177Lu), -5.6% (223Ra) and -6.1% (225Ac). The number of DSB∙cell-1∙mGy-1 for each radionuclide was 0.015 DSB∙cell-1∙mGy-1 (90Y), 0.012 DSB∙cell-1∙mGy-1 (99mTc), 0.014DSB∙cell-1∙mGy-1 (123I), 0.012 DSB∙cell-1∙mGy-1 (131I), and 0.016 DSB∙cell-1∙mGy-1 (177Lu). These values agree very well with experimental data. The number of α-tracks∙100 cells-1∙mGy-1 for 223Ra and 225Ac where 0.144 α-tracks∙100 cells-1∙mGy-1 and 0.151 α-tracks∙100 cells-1∙mGy-1, respectively. These values agree very well with experimental data. Moreover, the linear density of DSBs per micrometer α-track length were 11.13 ±â€¯0.04 DSB/µm and 10.86 ±â€¯0.06 DSB/µm for 223Ra and 225Ac, respectively. CONCLUSION: This study describes a model to simulate the DNA DSB damage in lymphocyte nuclei validated by experimental data obtained from internal ex vivo blood irradiation with radionuclides frequently used in diagnostic and therapeutic procedures in nuclear medicine.

The order of sequential exposure of U2OS cells to gamma and alpha radiation influences the formation and decay dynamics of NBS1 foci.

DNA double strand breaks (DSBs) are a deleterious form of DNA damage. Densely ionising alpha radiation predominantly induces complex DSBs and sparsely ionising gamma radiation-simple DSBs. We have shown that alphas and gammas, when applied simultaneously, interact in producing a higher DNA damage response (DDR) than predicted by additivity. The mechanisms of the interaction remain obscure. The present study aimed at testing whether the sequence of exposure to alphas and gammas has an impact on the DDR, visualised by live NBS1-GFP (green fluorescent protein) focus dynamics in U2OS cells. Focus formation, decay, intensity and mobility were analysed up to 5 h post exposure. Focus frequencies directly after sequential alpha → gamma and gamma → alpha exposure were similar to gamma alone, but gamma → alpha foci quickly declined below the expected values. Focus intensities and areas following alpha alone and alpha → gamma were larger than after gamma alone and gamma → alpha. Focus movement was most strongly attenuated by alpha → gamma. Overall, sequential alpha → gamma exposure induced the strongest change in characteristics and dynamics of NBS1-GFP foci. Possible explanation is that activation of the DDR is stronger when alpha-induced DNA damage precedes gamma-induced DNA damage.

Analysis of Single- and Double-Stranded DNA Damage in Osteoblastic Cells after Hyperbaric Oxygen Exposure.

(1) Background: Hyperbaric oxygen (HBO) exposure induces oxidative stress that may lead to DNA damage, which has been observed in human peripheral blood lymphocytes or non-human cells. Here, we investigated the impact of hyperbaric conditions on two human osteoblastic cell lines: primary human osteoblasts, HOBs, and the osteogenic tumor cell line SAOS-2. (2) Methods: Cells were exposed to HBO in an experimental hyperbaric chamber (4 ATA, 100% oxygen, 37 °C, and 4 h) or sham-exposed (1 ATA, air, 37 °C, and 4 h). DNA damage was examined before, directly after, and 24 h after exposure with an alkaline comet assay and detection of γH2AX+53BP1 colocalizing double-strand break (DSB) foci and apoptosis. The gene expression of TGFß-1, HO-1, and NQO1, involved in antioxidative functions, was measured with qRT-PCR. (3) Results: The alkaline comet assay showed significantly elevated levels of DNA damage in both cell lines after 4 h of HBO, while the DSB foci were similar to sham. γH2AX analysis indicated a slight increase in apoptosis in both cell lines. The increased expression of HO-1 in HOB and SAOS-2 directly after exposure suggested the induction of an antioxidative response in these cells. Additionally, the expression of TGF-ß1 was negatively affected in HOB cells 4 h after exposure. (4) Conclusions: in summary, this study indicates that osteoblastic cells are sensitive to the DNA-damaging effects of hyperbaric hyperoxia, with the HBO-induced DNA damage consisting largely of single-strand DNA breaks that are rapidly repaired.

RENEB Inter-Laboratory Comparison 2021: The Gamma-H2AX Foci Assay.

The Running the European Network of biological and retrospective dosimetry (RENEB) network of laboratories has a range of biological and physical dosimetry assays that can be deployed in the event of a radiation incident to provide exposure assessment. To maintain operational capability and provide training, RENEB runs regular inter-laboratory comparison (ILC) exercises. The RENEB ILC2021 was carried out with all the biological and physical dosimetry assays employed in the network. The focus of this paper is to evaluate the results from 6 laboratories that took part using the gamma-H2AX radiation-induced foci assay. For two laboratories this was their first RENEB ILC. Blood samples were homogenously exposed to 240 kVp X rays (1 Gy/min) to provide calibration data, (0-4 Gy), and a few weeks later three blind coded test samples, (0, 1.2 and 3.5 Gy) were prepared. All samples were allowed a 2 h repair time at 37°C before being transported, on ice packs, to the participating laboratories. On arrival, the samples were processed, scored either manually or automatically for gamma-H2AX foci and dose estimates for the 3 blind coded samples sent to the organizing laboratory. The temperature of samples during transit and the time taken to report the dose estimates were recorded. Subsequent examination of the data from each laboratory used the doses estimates to assign triage categories to the samples. After receipt of the samples, the quickest report of dose estimates was 4.6 h. Analysis of variance revealed that the laboratory carrying out the assay had a significant effect on the foci yield (P < 0.001) for the calibration data, but not on the dose estimates of the blind coded samples (P = 0.101). All laboratories correctly identified the unirradiated and irradiated samples, although the dose estimates for the latter tended to under-estimate the dose. Two participants seriously under-estimated the dose for the highly exposed sample, which resulted in the sample being placed in the lowest triage category not the highest. However, this under-estimation resulted from the samples not remaining cold during shipment, due to a delay in transit and was not related to the experience of the participating laboratory. Overall, the RENEB network laboratories have demonstrated it is possible to quickly identify a recent whole-body acute exposure using the gamma-H2AX assay within the conditions of the ILC. In addition, an ILC provides a useful training and harmonization exercise for laboratories.

Ex-vivo dose response characterization of the recently identified EDA2R gene after low level radiation exposures and comparison with FDXR gene expression and the γH2AX focus assay.

OBJECTIVE: Recently, promising radiation-induced EDA2R gene expression (GE) changes after low level radiation could be shown. Stimulated by that, in this study, we intended to independently validate these findings and to further characterize dose-response relationships in comparison to FDXR and the γH2AX-DNA double-strand break (DSB) focus assay, since both assays are already widely used for biodosimetry purposes. MATERIALS AND METHODS: Peripheral blood samples from six healthy human donors were irradiated ex vivo (dose: ranging from 2.6 to 49.7 mGy). Subsequently, the fold-differences relative to the sham irradiated reference group were calculated. Radiation-induced changes in GE of FDXR and EDA2R were examined using the quantitative real-time polymerase-chain-reaction (qRT-PCR). DSB foci were quantified in 100 γH2AX + 53BP1 immunostained cells employing fluorescence microscopy. Examinations were performed at single time points enabling sufficient detection of both endpoints. RESULTS: A significant increase in EDA2R GE relative to the unexposed control was observed in the range of 2.6 mGy (1.6-fold, p = .045) to 5.4 mGy (2.2-fold, p = .0002), whereas the copy numbers increased linearly up to 13.1-fold at 49.7 mGy. On the contrary, FDXR upregulation (2.2-fold) became significant after a 22.6 mGy exposure (p ≤ .02) and increased linearly up to 4-fold at 49.7 mGy. A significant increase in radiation-induced foci (relative to unexposed, RIF-fd) was observed after 11.3 mGy (RIF-fd: 1.5 ± 0.5, p ≤ .03), while the foci increased linearly up to 3-fold at 49.7 mGy. From this, the FDXR and RIF-fd slopes have shown comparability, while the EDA2R slope was five times higher. Nevertheless, the coefficient of variation (CV) of EDA2R was about 30% higher than for RIF-fd. CONCLUSION: Higher radiation-induced EDA2R GE changes and a lower radiation detection level compared to RIF-fd and FDXR GE changes examined under optimal conditions ex vivo on human samples appear promising. Yet, our results represent just the beginning of further studies to be conducted in animal models for further time- and dose-dependent evaluation and additional examinations on radiologically examined patients to evaluate the impact of confounder, such as age, sex, social behavior, or diseases.

Initial experience on abdominal photon-counting computed tomography in clinical routine: general image quality and dose exposure.

OBJECTIVES: Photon-counting computed tomography has lately found its way into clinical routine. The new technique could offer substantial improvements regarding general image quality, image noise, and radiation dose reduction. This study evaluated the first abdominal examinations in clinical routine and compared the results to conventional computed tomography. METHODS: In this single-center retrospective study, 66 patients underwent photon-counting and conventional abdominal CT. Four radiologists assessed general image quality, image noise, and image artifacts. Signal-to-noise ratio and dose properties of both techniques within the clinical application were compared. An ex vivo phantom study revealed the radiobiological impact by means of DNA double-strand break foci in peripheral blood cells by enumerating γ-H2AX+53BP1 foci. RESULTS: General image quality in accordance with the Likert scale was found superior for photon-counting CT (4.74 ± 0.46 vs. 4.25 ± 0.54; p < 0.001). Signal-to-noise ratio (p < 0.001) and also dose exposure were higher for photon-counting CT (DLP: 419.2 ± 162.2 vs. 372.3 ± 236.6 mGy*cm; p = 0.0435). CT exposure resulted in significantly increased DNA damage in comparison to sham control (p < 0.001). Investigation of the average foci per cell and radiation-induced foci numbers revealed significantly elevated numbers (p = 0.004 and p < 0.0001, respectively) after photon-counting CT. CONCLUSION: Photon-counting CT in abdominal examinations showed superior results regarding general image quality and signal-to-noise ratio in clinical routine. However, this seems to be traded for a significantly higher dose exposure and corresponding double-strand break frequency. Optimization of standard protocols in further clinical applications is required to find a compromise regarding picture quality and dose exposure. KEY POINTS: • Photon-counting computed tomography promises to enhance the diagnostic potential of medical imaging in clinical routine. • Retrospective single-center study showed superior general image quality accompanied by higher dose exposure in initial abdominal PCCT protocols compared to state-of-the-art conventional CT. • A simultaneous ex vivo phantom study revealed correspondingly increased frequencies of DNA double-strand breaks after PCCT.

The microRNA-202 as a Diagnostic Biomarker and a Potential Tumor Suppressor.

MicroRNA-202 (miR-202) is a member of the highly conserved let-7 family that was discovered in Caenorhabditis elegans and recently reported to be involved in cell differentiation and tumor biology. In humans, miR-202 was initially identified in the testis where it was suggested to play a role in spermatogenesis. Subsequent research showed that miR-202 is one of the micro-RNAs that are dysregulated in different types of cancer. During the last decade, a large number of investigations has fortified a role for miR-202 in cancer. However, its functions can be double-edged, depending on context they may be tumor suppressive or oncogenic. In this review, we highlight miR-202 as a potential diagnostic biomarker and as a suppressor of tumorigenesis and metastasis in several types of tumors. We link miR-202 expression levels in tumor types to its involved upstream and downstream signaling molecules and highlight its potential roles in carcinogenesis. Three well-known upstream long non-coding-RNAs (lncRNAs); MALAT1, NORAD, and NEAT1 target miR-202 and inhibit its tumor suppressive function thus fueling cancer progression. Studies on the downstream targets of miR-202 revealed PTEN, AKT, and various oncogenes such as metadherin (MTDH), MYCN, Forkhead box protein R2 (FOXR2) and Kirsten rat sarcoma virus (KRAS). Interestingly, an upregulated level of miR-202 was shown by most of the studies that estimated its expression level in blood or serum of cancer patients, especially in breast cancer. Reduced expression levels of miR-202 in tumor tissues were found to be associated with progression of different types of cancer. It seems likely that miR-202 is embedded in a complex regulatory network related to the nature and the sensitivity of the tumor type and therapeutic (pre)treatments. Its variable roles in tumorigenesis are mediated in part thought its oncogene effectors. However, the currently available data suggest that the involved signaling pathways determine the anti- or pro-tumorigenic outcomes of miR-202's dysregulation and its value as a diagnostic biomarker.

Repair of α-particle-induced DNA damage in peripheral blood mononuclear cells after internal ex vivo irradiation with 223Ra.

PURPOSE: As α-emitters for radiopharmaceutical therapies are administered systemically by intravenous injection, blood will be irradiated by α-particles that induce clustered DNA double-strand breaks (DSBs). Here, we investigated the induction and repair of DSB damage in peripheral blood mononuclear cells (PBMCs) as a function of the absorbed dose to the blood following internal ex vivo irradiation with [223Ra]RaCl2. METHODS: Blood samples of ten volunteers were irradiated by adding [223Ra]RaCl2 solution with different activity concentrations resulting in absorbed doses to the blood of 3 mGy, 25 mGy, 50 mGy and 100 mGy. PBMCs were isolated, divided in three parts and either fixed directly (d-samples) or after 4 h or 24 h culture. After immunostaining, the induced γ-H2AX α-tracks were counted. The time-dependent decrease in α-track frequency was described with a model assuming a repair rate R and a fraction of non-repairable damage Q. RESULTS: For 25 mGy, 50 mGy and 100 mGy, the numbers of α-tracks were significantly increased compared to baseline at all time points. Compared to the corresponding d-samples, the α-track frequency decreased significantly after 4 h and after 24 h. The repair rates R were (0.24 ± 0.05) h-1 for 25 mGy, (0.16 ± 0.04) h-1 for 50 mGy and (0.13 ± 0.02) h-1 for 100 mGy, suggesting faster repair at lower absorbed doses, while Q-values were similar. CONCLUSION: The results obtained suggest that induction and repair of the DSB damage depend on the absorbed dose to the blood. Repair rates were similar to what has been observed for irradiation with low linear energy transfer.

Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks.

Hyperthermia inhibits DNA double-strand break (DSB) repair that utilizes homologous recombination (HR) pathway by a poorly defined mechanism(s); however, the mechanisms for this inhibition remain unclear. Here we report that hyperthermia decreases H4K16 acetylation (H4K16ac), an epigenetic modification essential for genome stability and transcription. Heat-induced reduction in H4K16ac was detected in humans, Drosophila, and yeast, indicating that this is a highly conserved response. The examination of histone deacetylase recruitment to chromatin after heat-shock identified SIRT1 as the major deacetylase subsequently enriched at gene-rich regions. Heat-induced SIRT1 recruitment was antagonized by chromatin remodeler SMARCAD1 depletion and, like hyperthermia, the depletion of the SMARCAD1 or combination of the two impaired DNA end resection and increased replication stress. Altered repair protein recruitment was associated with heat-shock-induced γ-H2AX chromatin changes and DSB repair processing. These results support a novel mechanism whereby hyperthermia impacts chromatin organization owing to H4K16ac deacetylation, negatively affecting the HR-dependent DSB repair.

Planar Proton Minibeam Irradiation Elicits Spatially Confined DNA Damage in a Human Epidermis Model.

Purpose: High doses of ionizing radiation in radiotherapy can elicit undesirable side effects to the skin. Proton minibeam radiotherapy (pMBRT) may circumvent such limitations due to tissue-sparing effects observed at the macro scale. Here, we mapped DNA damage dynamics in a 3D tissue context at the sub-cellular level. Methods: Epidermis models were irradiated with planar proton minibeams of 66 µm, 408 µm and 920 µm widths and inter-beam-distances of 2.5 mm at an average dose of 2 Gy using the scanning-ion-microscope SNAKE in Garching, GER. γ-H2AX + 53BP1 and cleaved-caspase-3 immunostaining revealed dsDNA damage and cell death, respectively, in time courses from 0.5 to 72 h after irradiation. Results: Focused 66 µm pMBRT induced sharply localized severe DNA damage (pan-γ-H2AX) in cells at the dose peaks, while damage in the dose valleys was similar to sham control. pMBRT with 408 µm and 920 µm minibeams induced DSB foci in all cells. At 72 h after irradiation, DNA damage had reached sham levels, indicating successful DNA repair. Increased frequencies of active-caspase-3 and pan-γ-H2AX-positive cells revealed incipient cell death at late time points. Conclusions: The spatially confined distribution of DNA damage appears to underlie the tissue-sparing effect after focused pMBRT. Thus, pMBRT may be the method of choice in radiotherapy to reduce side effects to the skin.

Ascorbic acid-2 glucoside mitigates intestinal damage during pelvic radiotherapy in a rat bladder tumor model.

PURPOSE: Ascorbic acid is a strong antioxidant and has potent radioprotective effects on radiation injuries. Ascorbic acid 2-glucoside (AA2G) is a stabilized derivative of ascorbic acid and rapidly hydrolyzed into ascorbic acid and glucose. Since there is the possibility that AA2G treatment interferes with the antitumor activity of radiotherapy, we investigated the effect of AA2G treatment during radiotherapy on acute radiation enteritis and antitumor activity of radiotherapy in rats. MATERIALS AND METHODS: AY-27 rat bladder tumor cells were used to induce bladder tumors in rats. Two weeks after inoculation rats received fractionated pelvic radiotherapy in eight fractions for 4 weeks totaling 40 Gy. During radiotherapy, one group of rats received per os AA2G (ascorbic acid: 250 mg/kg/day) and its bolus engulfment (ascorbic acid: 250 mg/kg) 8 h before each X-irradiation fraction. Seven days after the last X-irradiation, we studied histology, DNA double strand break (DSB) damage (by 53BP1 foci staining), and the M1/M2 macrophage response by immunohistochemistry of paraffin-fixed bladder and intestinal tissues. RESULTS: AA2G treatment reduced the intestinal damage (shortening of villi) but did not reduce antitumor effectiveness of radiotherapy against bladder tumors. Like the controls, AA2G-treated rats showed no residual tumor lesions in the bladder after X-irradiation. Both AA2G-treated and control groups showed similar persistent DSB damage (53BP1 foci) both in bladders and ilea seven days after radiotherapy. Radiotherapy tended to reduce CD163+ M2 macrophages, which are considered as an anti-inflammatory subtype favoring tissue repair, in the bladders. X-irradiation also reduced the occurrence of M2 macrophages in the ilea. AA2G treatment significantly increased CD163+/CD68+ macrophage ratio in the ilea of rats after pelvic irradiation in comparison to the sham irradiated control rats. AA2G treatment increased, albeit not significantly, the CD163+/CD68+ macrophage ratio in the irradiated bladders relative to the control irradiated rats. On the other hand, bladders and ilea of the irradiated rats with and without AA2G treatment showed similar frequencies of CD68+ macrophages. CONCLUSIONS: AA2G treatment mitigated radiation-induced intestinal damage without reducing antitumor activity after fractionated pelvic radiotherapy against bladder tumors in rats. The beneficial effect of AA2G treatment seems to promote a restoration of the M2 answer as well as tissue remodeling and wound healing. Similar residual DNA damage in bladders and ilea seven days post-irradiation is consistent with tumor control in both groups.

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra.

Dual-energy CT provides enhanced diagnostic power with similar or even reduced radiation dose as compared to single-energy CT. Its principle is based on the distinct physical properties of low and high energetic photons, which, however, may also affect the biological effectiveness and hence the extent of CT-induced cellular damage. Therefore, a comparative analysis of biological effectiveness of dual- and single-energy CT scans with focus on early gene regulation and frequency of radiation-induced DNA double strand breaks (DSBs) was performed. Blood samples from three healthy individuals were irradiated ex vivo with single-energy (80 kV and 150 kV) and dual-energy tube voltages (80 kV/Sn150kV) employing a modern dual source CT scanner resulting in Volume Computed Tomography Dose Index (CTDIvol) of 15.79-18.26 mGy and dose length product (DLP) of 606.7-613.8 mGy*cm. Non-irradiated samples served as a control. Differential gene expression in peripheral blood mononuclear cells was analyzed 6 h after irradiation using whole transcriptome sequencing. DSB frequency was studied by 53BP1 + γH2AX co-immunostaining and microscopic evaluation of their focal accumulation at DSBs. Neither the analysis of gene expression nor DSB frequency provided any evidence for significantly increased biological effectiveness of dual-energy CT in comparison to samples irradiated with particular single-energy CT spectra. Relative to control, irradiated samples were characterized by a significantly higher rate of DSBs (p < 0.001) and the shared upregulation of five genes, AEN, BAX, DDB2, FDXR and EDA2R, which have already been suggested as radiation-induced biomarkers in previous studies. Despite steadily decreasing doses, CT diagnostics remain a genotoxic stressor with impact on gene regulation and DNA integrity. However, no evidence was found that varying X-ray spectra of CT impact the extent of cellular damage.

Human skin aging is associated with increased expression of the histone variant H2A.J in the epidermis.

Cellular senescence is an irreversible growth arrest that occurs as a result of damaging stimuli, including DNA damage and/or telomere shortening. Here, we investigate histone variant H2A.J as a new biomarker to detect senescent cells during human skin aging. Skin biopsies from healthy volunteers of different ages (18-90 years) were analyzed for H2A.J expression and other parameters involved in triggering and/or maintaining cellular senescence. In the epidermis, the proportions of H2A.J-expressing keratinocytes increased from ≈20% in young to ≈60% in aged skin. Inverse correlations between Ki67- and H2A.J staining in germinative layers may reflect that H2A.J-expressing cells having lost their capacity to divide. As cellular senescence is triggered by DNA-damage signals, persistent 53BP1-foci, telomere lengths, and telomere-associated damage foci were analyzed in epidermal keratinocytes. Only slight age-related telomere attrition and few persistent nuclear 53BP1-foci, occasionally colocalizing with telomeres, suggest that unprotected telomeres are not a significant cause of senescence during skin aging. Quantification of integrin-α6+ basal cells suggests that the number and function of stem/progenitor cells decreased during aging and their altered proliferation capacities resulted in diminished tissue renewal with epidermal thinning. Collectively, our findings suggest that H2A.J is a sensitive marker of epidermal aging in human skin.

Genomic Adaption and Mutational Patterns in a HaCaT Subline Resistant to Alkylating Agents and Ionizing Radiation.

Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.

Parp1-Dependent DNA Double-Strand Break Repair in Irradiated Late Pachytene Spermatocytes.

Poly (ADP-ribose) polymerase-1 (Parp1) is a member of nuclear enzymes family involved in to the response to genotoxic stresses, DNA repair, and is critical for the maintenance of genome stability. During gametogenesis, genome stability is essential for inheritance and formation of healthy gametes. The latter involves DNA double-strand break (DSB)-driven pairing of homologous chromosomes in first meiotic prophase. By analysis of DSB repair kinetics in male meiotic prophase cells of homologous recombination (HR) and nonhomologous end joining (NHEJ)-deficient mouse models, we previously demonstrated an interplay between HR and the conventional NHEJ repair pathway. In the current work, we evaluate the relative contribution of Parp1-dependent NHEJ to the repair of ectopic ionizing radiation (IR)-induced DSBs in control and Parp1-inhibited mouse pachytene spermatocytes before and after the completion of meiotic recombination in stages VI-XI. The disappearance of large, exogenous DSB-related γ-H2AX foci was quantified 1 and 8 h after 1 Gy γ-irradiation of control and 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)quinolinone (DPQ) Parp1-inhibited mice. Late pachytene control spermatocytes obtained 8 h after IR had repaired >80% of DSBs observed at 1 h after IR. However, only 64% of DSBs were repaired in late spermatocytes of DPQ-treated (Parp1-inhibited) mice. Thus, it appears that Parp1 contributes to the repair of a fraction of DSBs in late prophase I, providing further insights in DNA repair pathway choreography during spermatogenic differentiation.

Catalase T-Deficient Fission Yeast Meiocytes Show Resistance to Ionizing Radiation.

Environmental stress, reactive oxygen species (ROS), or ionizing radiation (IR) can induce adverse effects in organisms and their cells, including mutations and premature aging. DNA damage and its faulty repair can lead to cell death or promote cancer through the accumulation of mutations. Misrepair in germ cells is particularly dangerous as it may lead to alterations in developmental programs and genetic disease in the offspring. DNA damage pathways and radical defense mechanisms mediate resistance to genotoxic stresses. Here, we investigated, in the fission yeast Schizosaccharomyces pombe, the role of the H2O2-detoxifying enzyme cytosolic catalase T (Ctt1) and the Fe2+/Mn2+ symporter Pcl1 in protecting meiotic chromosome dynamics and gamete formation from radicals generated by ROS and IR. We found that wild-type and pcl1-deficient cells respond similarly to X ray doses of up to 300 Gy, while ctt1∆ meiocytes showed a moderate sensitivity to IR but a hypersensitivity to hydrogen peroxide with cells dying at >0.4 mM H2O2. Meiocytes deficient for pcl1, on the other hand, showed a resistance to hydrogen peroxide similar to that of the wild type, surviving doses >40 mM. In all, it appears that in the absence of the main H2O2-detoxifying pathway S. pombe meiocytes are able to survive significant doses of IR-induced radicals.