The Biodistribution of the Spike Protein after Ad26.COV2.S Vaccination Is Unlikely to Play a Role in Vaccine-Induced Immune Thrombotic Thrombocytopenia.

Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation.

Sustained Liver HBsAg Loss and Clonal T- and B-Cell Expansion upon Therapeutic DNA Vaccination Require Low HBsAg Levels.

BACKGROUND: Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. METHODS: AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. RESULTS: GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. CONCLUSIONS: This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

DNA damage and repair: underlying mechanisms leading to microcephaly.

DNA-damaging agents and endogenous DNA damage constantly harm genome integrity. Under genotoxic stress conditions, the DNA damage response (DDR) machinery is crucial in repairing lesions and preventing mutations in the basic structure of the DNA. Different repair pathways are implicated in the resolution of such lesions. For instance, the non-homologous DNA end joining and homologous recombination pathways are central cellular mechanisms by which eukaryotic cells maintain genome integrity. However, defects in these pathways are often associated with neurological disorders, indicating the pivotal role of DDR in normal brain development. Moreover, the brain is the most sensitive organ affected by DNA-damaging agents compared to other tissues during the prenatal period. The accumulation of lesions is believed to induce cell death, reduce proliferation and premature differentiation of neural stem and progenitor cells, and reduce brain size (microcephaly). Microcephaly is mainly caused by genetic mutations, especially genes encoding proteins involved in centrosomes and DNA repair pathways. However, it can also be induced by exposure to ionizing radiation and intrauterine infections such as the Zika virus. This review explains mammalian cortical development and the major DNA repair pathways that may lead to microcephaly when impaired. Next, we discuss the mechanisms and possible exposures leading to DNA damage and p53 hyperactivation culminating in microcephaly.

Gravitational effects on fibroblasts' function in relation to wound healing.

The spaceflight environment imposes risks for maintaining a healthy skin function as the observed delayed wound healing can contribute to increased risks of infection. To counteract delayed wound healing in space, a better understanding of the fibroblasts' reaction to altered gravity levels is needed. In this paper, we describe experiments that were carried out at the Large Diameter Centrifuge located in ESA-ESTEC as part of the ESA Academy 2021 Spin Your Thesis! Campaign. We exposed dermal fibroblasts to a set of altered gravity levels, including transitions between simulated microgravity and hypergravity. The addition of the stress hormone cortisol to the cell culture medium was done to account for possible interaction effects of gravity and cortisol exposure. Results show a main impact of cortisol on the secretion of pro-inflammatory cytokines as well as extracellular matrix proteins. Altered gravity mostly induced a delay in cellular migration and changes in mechanosensitive cell structures. Furthermore, 20 × g hypergravity transitions induced changes in nuclear morphology. These findings provide insights into the effect of gravity transitions on the fibroblasts' function related to wound healing, which may be useful for the development of countermeasures.

Limnospira indica PCC 8005 Supplementation Prevents Pelvic Irradiation-Induced Dysbiosis but Not Acute Inflammation in Mice.

Pelvic irradiation-induced mucositis secondarily leads to dysbiosis, which seriously affects patients' quality of life after treatment. No safe and effective radioprotector or mitigator has yet been approved for clinical therapy. Here, we investigated the potential protective effects of fresh biomass of Limnospira indica PCC 8005 against ionizing irradiation-induced mucositis and dysbiosis in respect to benchmark probiotic Lacticaseibacillus rhamnosus GG ATCC 53103. For this, mice were supplemented daily before and after 12 Gy X-irradiation of the pelvis. Upon sacrifice, food supplements' efficacy was assessed for intestinal barrier protection, immunomodulation and changes in the microbiota composition. While both could not confer barrier protection or significant immunomodulatory effects, 16S microbial profiling revealed that L. indica PCC 8005 and L. rhamnosus GG could prevent pelvic irradiation-induced dysbiosis. Altogether, our data show that-besides benchmarked L. rhamnosus GG-L. indica PCC 8005 is an interesting candidate to further explore as a radiomitigator counteracting pelvic irradiation-induced dysbiosis in the presented in vivo irradiation-gut-microbiota platform.

The Effects of Combined Exposure to Simulated Microgravity, Ionizing Radiation, and Cortisol on the In Vitro Wound Healing Process.

Human spaceflight is associated with several health-related issues as a result of long-term exposure to microgravity, ionizing radiation, and higher levels of psychological stress. Frequent reported skin problems in space include rashes, itches, and a delayed wound healing. Access to space is restricted by financial and logistical issues; as a consequence, experimental sample sizes are often small, which limits the generalization of the results. Earth-based simulation models can be used to investigate cellular responses as a result of exposure to certain spaceflight stressors. Here, we describe the development of an in vitro model of the simulated spaceflight environment, which we used to investigate the combined effect of simulated microgravity using the random positioning machine (RPM), ionizing radiation, and stress hormones on the wound-healing capacity of human dermal fibroblasts. Fibroblasts were exposed to cortisol, after which they were irradiated with different radiation qualities (including X-rays, protons, carbon ions, and iron ions) followed by exposure to simulated microgravity using a random positioning machine (RPM). Data related to the inflammatory, proliferation, and remodeling phase of wound healing has been collected. Results show that spaceflight stressors can interfere with the wound healing process at any phase. Moreover, several interactions between the different spaceflight stressors were found. This highlights the complexity that needs to be taken into account when studying the effect of spaceflight stressors on certain biological processes and for the aim of countermeasures development.

Comparison of in vitro scratch wound assay experimental procedures.

Fibroblast migration is an important aspect of wound healing. Different factors can influence migration and as such proper wound healing. In vitro scratch wound assays are used to examine cellular migration. However, the wide array of techniques available reduces reproducibility of findings. In this paper, we compare two techniques for wound creation; i.e. the exclusion method or scratching of cell monolayers. Furthermore, we investigate if analysis software influences experimental outcome by comparing both commercially and freely available analysis software. Besides, we examine the effect of cortisol on migration behavior of fibroblasts and identify possible caveats in experimental design. Results show a significantly reduced migration of fibroblasts when wounds are created using a cell exclusion method. Furthermore, addition of cortisol to the cell culture media only reduced migration of fibroblast monolayers that had been scratched but not in those where wounds were created using the exclusion method. A possible explanation related to cytokine expression is discussed.

Spaceflight Stressors and Skin Health.

Traveling to space puts astronauts at risk of developing serious health problems. Of particular interest is the skin, which is vitally important in protecting the body from harmful environmental factors. Although data obtained from long-duration spaceflight studies are inconsistent, there have been indications of increased skin sensitivity and signs of dermal atrophy in astronauts. To better understand the effects of spaceflight stressors including microgravity, ionizing radiation and psychological stress on the skin, researchers have turned to in vitro and in vivo simulation models mimicking certain aspects of the spaceflight environment. In this review, we provide an overview of these simulation models and highlight studies that have improved our understanding on the effect of simulation spaceflight stressors on skin function. Data show that all aforementioned spaceflight stressors can affect skin health. Nevertheless, there remains a knowledge gap regarding how different spaceflight stressors in combination may interact and affect skin health. In future, efforts should be made to better simulate the spaceflight environment and reduce uncertainties related to long-duration spaceflight health effects.

Prenatal Radiation Exposure Leads to Higher-Order Telencephalic Dysfunctions in Adult Mice That Coincide with Reduced Synaptic Plasticity and Cerebral Hypersynchrony.

Higher-order telencephalic circuitry has been suggested to be especially vulnerable to irradiation or other developmentally toxic impact. This report details the adult effects of prenatal irradiation at a sensitive time point on clinically relevant brain functions controlled by telencephalic regions, hippocampus (HPC), and prefrontal cortex (PFC). Pregnant C57Bl6/J mice were whole-body irradiated at embryonic day 11 (start of neurogenesis) with X-ray intensities of 0.0, 0.5, or 1.0 Gy. Female offspring completed a broad test battery of HPC-/PFC-controlled tasks that included cognitive performance, fear extinction, exploratory, and depression-like behaviors. We examined neural functions that are mechanistically related to these behavioral and cognitive changes, such as hippocampal field potentials and long-term potentiation, functional brain connectivity (by resting-state functional magnetic resonance imaging), and expression of HPC vesicular neurotransmitter transporters (by immunohistochemical quantification). Prenatally exposed mice displayed several higher-order dysfunctions, such as decreased nychthemeral activity, working memory defects, delayed extinction of threat-evoked response suppression as well as indications of perseverative behavior. Electrophysiological examination indicated impaired hippocampal synaptic plasticity. Prenatal irradiation also induced cerebral hypersynchrony and increased the number of glutamatergic HPC terminals. These changes in brain connectivity and plasticity could mechanistically underlie the irradiation-induced defects in higher telencephalic functions.

Irradiation at Ultra-High (FLASH) Dose Rates Reduces Acute Normal Tissue Toxicity in the Mouse Gastrointestinal System.

PURPOSE: Preclinical studies using ultra-high dose rate (FLASH) irradiation have demonstrated reduced normal tissue toxicity compared with conventional dose rate (CONV) irradiation, although this finding is not universal. We investigated the effect of temporal pulse structure and average dose rate of FLASH compared with CONV irradiation on acute intestinal toxicity. MATERIALS AND METHODS: Whole abdomens of C3H mice were irradiated with a single fraction to various doses, using a 6 MeV electron linear accelerator with single pulse FLASH (dose rate = 2-6 × 106 Gy/s) or conventional (CONV; 0.25 Gy/s) irradiation. At 3.75 days postirradiation, fresh feces were collected for 16S rRNA sequencing to assess changes in the gut microbiota. A Swiss roll-based crypt assay was used to quantify acute damage to the intestinal crypts to determine how tissue toxicity was affected by the different temporal pulse structures of FLASH delivery. RESULTS: We found statistically significant improvements in crypt survival for mice irradiated with FLASH at doses between 7.5 and 12.5 Gy, with a dose modifying factor of 1.1 for FLASH (7.5 Gy, P < .01; 10 Gy, P < .05; 12.5 Gy, P < .01). This sparing effect was lost when the delivery time was increased, either by increasing the number of irradiation pulses or by prolonging the time between 2 successive pulses. Sparing was observed for average dose rates of ≥280 Gy/s. Fecal microbiome analysis showed that FLASH irradiation caused fewer changes to the microbiota than CONV irradiation. CONCLUSIONS: This study demonstrates that FLASH irradiation can spare mouse small intestinal crypts and reduce changes in gut microbiome composition compared with CONV irradiation. The higher the average dose rate, the larger the FLASH effect, which is also influenced by temporal pulse structure of the delivery.

Intestinal mucositis precedes dysbiosis in a mouse model for pelvic irradiation.

Pelvic radiotherapy is known to evoke intestinal mucositis and dysbiosis. Currently, there are no effective therapies available to mitigate these injuries, which is partly due to a lack of insight into the events causing mucositis and dysbiosis. Here, the complex interplay between the murine host and its microbiome following pelvic irradiation was mapped by characterizing intestinal mucositis along with extensive 16S microbial profiling. We demonstrated important morphological and inflammatory implications within one day after exposure, thereby impairing intestinal functionality and inducing translocation of intraluminal bacteria into mesenteric lymph nodes as innovatively quantified by flow cytometry. Concurrent 16S microbial profiling revealed a delayed impact of pelvic irradiation on beta diversity. Analysis of composition of microbiomes identified biomarkers for pelvic irradiation. Among them, members of the families Ruminococcaceae, Lachnospiraceae and Porphyromonadaceae were differentially affected. Altogether, our unprecedented findings showed how pelvic irradiation evoked structural and functional changes in the intestine, which secondarily resulted in a microbiome shift. Therefore, the presented in vivo irradiation-gut-microbiome platform allows further research into the pathobiology of pelvic irradiation-induced intestinal mucositis and resultant dysbiosis, as well as the exploration of mitigating treatments including drugs and food supplements.

Hippocampal and cortical tissue-specific epigenetic clocks indicate an increased epigenetic age in a mouse model for Alzheimer's disease.

Epigenetic clocks are based on age-associated changes in DNA methylation of CpG-sites, which can accurately measure chronological age in different species. Recently, several studies have indicated that the difference between chronological and epigenetic age, defined as the age acceleration, could reflect biological age indicating functional decline and age-associated diseases. In humans, an epigenetic clock associated Alzheimer's disease (AD) pathology with an acceleration of the epigenetic age. In this study, we developed and validated two mouse brain region-specific epigenetic clocks from the C57BL/6J hippocampus and cerebral cortex. Both clocks, which could successfully estimate chronological age, were further validated in a widely used mouse model for AD, the triple transgenic AD (3xTg-AD) mouse. We observed an epigenetic age acceleration indicating an increased biological age for the 3xTg-AD mice compared to non-pathological C57BL/6J mice, which was more pronounced in the cortex as compared to the hippocampus. Genomic region enrichment analysis revealed that age-dependent CpGs were enriched in genes related to developmental, aging-related, neuronal and neurodegenerative functions. Due to the limited access of human brain tissues, these epigenetic clocks specific for mouse cortex and hippocampus might be important in further unravelling the role of epigenetic mechanisms underlying AD pathology or brain aging in general.

An appraisal of folates as key factors in cognition and ageing-related diseases.

Folic acid (FA) is often consumed as a food supplement and can be found in fortified staple foods in various western countries. Even though FA supplementation during pregnancy is known to prevent severe congenital anomalies in the developing child (e.g., neural tube defects), much less is known about its influence on cognition and neurological functioning. In this review, we address the advances in this field and situate how folate intake during pregnancy, postnatal life, adulthood and in the elderly affects cognition. In addition, an association between folate status and ageing, dementia and other neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis is discussed. While its role in the incidence and severity of these diseases is becoming apparent, the underlying action of folates and related metabolites remains elusive. Finally, the potential of FA as a nutraceutical has been proposed, although the efficacy will highly depend on the interplay with other micronutrients, the disease stage and the duration of supplementation. Hence, the lack of consistent data urges for more animal studies and (pre)clinical trials in humans to ascertain a potential beneficial role for folates in the treatment or amelioration of cognitive decline and ageing-related disorders.

Abnormal retinal pigment epithelium melanogenesis as a major determinant for radiation-induced congenital eye defects.

Recent studies highlighted a link between ionizing radiation exposure during neurulation and birth defects such as microphthalmos and anophthalmos. Because the mechanisms underlying these defects remain largely unexplored, we irradiated pregnant C57BL/6J mice (1.0 Gy, X-rays) at embryonic day (E)7.5, followed by histological and gene/protein expression analyses at defined days. Irradiation impaired embryonic development at E9 and we observed a delayed pigmentation of the retinal pigment epithelium (RPE) at E11. In addition, a reduced RNA expression and protein abundance of critical eye-development genes (e.g. Pax6 and Lhx2) was observed. Furthermore, a decreased expression of Mitf, Tyr and Tyrp1 supported the radiation-induced perturbation in RPE pigmentation. Finally, via immunostainings for proliferation (Ki67) and mitosis (phosphorylated histone 3), a decreased mitotic index was observed in the E18 retina after exposure at E7.5. Overall, we propose a plausible etiological model for radiation-induced eye-size defects, with RPE melanogenesis as a major determining factor.

Folic Acid Fortification Prevents Morphological and Behavioral Consequences of X-Ray Exposure During Neurulation.

Previous studies suggested a causal link between pre-natal exposure to ionizing radiation and birth defects such as microphthalmos and exencephaly. In mice, these defects arise primarily after high-dose X-irradiation during early neurulation. However, the impact of sublethal (low) X-ray doses during this early developmental time window on adult behavior and morphology of central nervous system structures is not known. In addition, the efficacy of folic acid (FA) in preventing radiation-induced birth defects and persistent radiation-induced anomalies has remained unexplored. To assess the efficacy of FA in preventing radiation-induced defects, pregnant C57BL6/J mice were X-irradiated at embryonic day (E)7.5 and were fed FA-fortified food. FA partially prevented radiation-induced (1.0 Gy) anophthalmos, exencephaly and gastroschisis at E18, and reduced the number of pre-natal deaths, fetal weight loss and defects in the cervical vertebrae resulting from irradiation. Furthermore, FA food fortification counteracted radiation-induced impairments in vision and olfaction, which were evidenced after exposure to doses ≥0.1 Gy. These findings coincided with the observation of a reduction in thickness of the retinal ganglion cell and nerve fiber layer, and a decreased axial length of the eye following exposure to 0.5 Gy. Finally, MRI studies revealed a volumetric decrease of the hippocampus, striatum, thalamus, midbrain and pons following 0.5 Gy irradiation, which could be partially ameliorated after FA food fortification. Altogether, our study is the first to offer detailed insights into the long-term consequences of X-ray exposure during neurulation, and supports the use of FA as a radioprotectant and antiteratogen to counter the detrimental effects of X-ray exposure during this crucial period of gestation.

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity.

Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity.

Food Supplements to Mitigate Detrimental Effects of Pelvic Radiotherapy.

Pelvic radiotherapy has been frequently reported to cause acute and late onset gastrointestinal (GI) toxicities associated with significant morbidity and mortality. Although the underlying mechanisms of pelvic radiation-induced GI toxicity are poorly understood, they are known to involve a complex interplay between all cell types comprising the intestinal wall. Furthermore, increasing evidence states that the human gut microbiome plays a role in the development of radiation-induced health damaging effects. Gut microbial dysbiosis leads to diarrhea and fatigue in half of the patients. As a result, reinforcement of the microbiome has become a hot topic in various medical disciplines. To counteract GI radiotoxicities, apart from traditional pharmacological compounds, adjuvant therapies are being developed including food supplements like vitamins, prebiotics, and probiotics. Despite the easy, cheap, safe, and feasible approach to protect patients against acute radiation-induced toxicity, clinical trials have yielded contradictory results. In this review, a detailed overview is given of the various clinical, intestinal manifestations after pelvic irradiation as well as the role of the gut microbiome herein. Furthermore, whilst discussing possible strategies to prevent these symptoms, food supplements are presented as auspicious, prophylactic, and therapeutic options to mitigate acute pelvic radiation-induced GI injury by exploring their molecular mechanisms of action.

A detailed characterization of congenital defects and mortality following moderate X-ray doses during neurulation.

BACKGROUND: Both epidemiological and animal studies have previously indicated a link between in utero radiation exposure and birth defects such as microphthalmos, anophthalmos, and exencephaly. However, detailed knowledge on embryonic radiosensitivity during different stages of neurulation is limited, especially in terms of neural tube defect and eye defect development. METHODS: To assess the most radiosensitive stage during neurulation, pregnant C57BL6/J mice were X-irradiated (0.5 Gy or 1.0 Gy) at embryonic days (E)7, E7.5, E8, E8.5, or E9. Next, the fetuses were scored macroscopically for various defects and prenatal resorptions/deaths were counted. In addition, cranial skeletal development was ascertained using the alcian-alizarin method. Furthermore, postnatal/young adult survival was followed until 5 weeks (W5) of age, after X-irradiation at E7.5 (0.1 Gy, 0.5 Gy, or 1.0 Gy). In addition, body and brain weights were registered at adult age (W10) following X-ray exposure at E7.5 (0.1 Gy, 0.5 Gy). RESULTS: Several malformations, including microphthalmos and exencephaly, were most evident after irradiation at E7.5, with significance starting respectively at 0.5 Gy and 1.0 Gy. Prenatal mortality and weight were significantly affected in all irradiated groups. Long-term follow-up of E7.5 irradiated animals revealed a reduction in survival at 5 weeks of age after high dose exposure (1.0 Gy), while lower doses (0.5 Gy, 0.1 Gy) did not affect brain and body weight at postnatal week 10. CONCLUSIONS: With this study, we gained more insight in radiosensitivity throughout neurulation, and offered a better defined model to further study radiation-induced malformations and the underlying mechanisms.

Current Evidence for Developmental, Structural, and Functional Brain Defects following Prenatal Radiation Exposure.

Ionizing radiation is omnipresent. We are continuously exposed to natural (e.g., radon and cosmic) and man-made radiation sources, including those from industry but especially from the medical sector. The increasing use of medical radiation modalities, in particular those employing low-dose radiation such as CT scans, raises concerns regarding the effects of cumulative exposure doses and the inappropriate utilization of these imaging techniques. One of the major goals in the radioprotection field is to better understand the potential health risk posed to the unborn child after radiation exposure to the pregnant mother, of which the first convincing evidence came from epidemiological studies on in utero exposed atomic bomb survivors. In the following years, animal models have proven to be an essential tool to further characterize brain developmental defects and consequent functional deficits. However, the identification of a possible dose threshold is far from complete and a sound link between early defects and persistent anomalies has not yet been established. This review provides an overview of the current knowledge on brain developmental and persistent defects resulting from in utero radiation exposure and addresses the many questions that still remain to be answered.

Higher Initial DNA Damage and Persistent Cell Cycle Arrest after Carbon Ion Irradiation Compared to X-irradiation in Prostate and Colon Cancer Cells.

The use of charged-particle beams, such as carbon ions, is becoming a more and more attractive treatment option for cancer therapy. Given the precise absorbed dose-localization and an increased biological effectiveness, this form of therapy is much more advantageous compared to conventional radiotherapy, and is currently being used for treatment of specific cancer types. The high ballistic accuracy of particle beams deposits the maximal dose to the tumor, while damage to the surrounding healthy tissue is limited. In order to better understand the underlying mechanisms responsible for the increased biological effectiveness, we investigated the DNA damage and repair kinetics and cell cycle progression in two p53 mutant cell lines, more specifically a prostate (PC3) and colon (Caco-2) cancer cell line, after exposure to different radiation qualities. Cells were irradiated with various absorbed doses (0, 0.5, and 2 Gy) of accelerated (13)C-ions at the Grand Accélérateur National d'Ions Lourds facility (Caen, France) or with X-rays (0, 0.1, 0.5, 1, 2, and 5 Gy). Microscopic analysis of DNA double-strand breaks showed dose-dependent increases in γ-H2AX foci numbers and foci occupancy after exposure to both types of irradiation, in both cell lines. However, 24 h after exposure, residual damage was more pronounced after lower doses of carbon ion irradiation compared to X-irradiation. Flow cytometric analysis showed that carbon ion irradiation induced a permanent G2/M arrest in PC3 cells at lower doses (2 Gy) compared to X-rays (5 Gy), while in Caco-2 cells the G2/M arrest was transient after irradiation with X-rays (2 and 5 Gy) but persistent after exposure to carbon ions (2 Gy).