Radiation therapy (RT) plays an important role in cancer treatment. The clinical efficacy of radiation therapy is, however, limited by normal tissue toxicity in areas surrounding the irradiated tumor. Compared to conventional radiation therapy (CONV-RT) in which doses are typically delivered at dose rates between 0.03–0.05 Gy/s, there is evidence that radiation delivered at dose rates of orders of magnitude higher (known as FLASH-RT), dramatically reduces the adverse side effects in normal tissues while achieving similar tumor control. The present study focused on normal cell response and tested the hypothesis that proton-FLASH irradiation preserves mitochondria function of normal cells through the induction of phosphorylated Drp1. Normal human lung fibroblasts (IMR90) were irradiated under ambient oxygen concentration (21%) with protons (LET = 10 keV/µm) delivered at dose rates of either 0.33 Gy/s or 100 Gy/s. Mitochondrial dynamics, functions, cell growth and changes in protein expression levels were investigated. Compared to lower dose-rate proton irradiation, FLASH-RT prevented mitochondria damage characterized by morphological changes, functional changes (membrane potential, mtDNA copy number and oxidative enzyme levels) and oxyradical production. After CONV-RT, the phosphorylated form of Dynamin-1-like protein (p-Drp1) underwent dephosphorylation and aggregated into the mitochondria resulting in mitochondria fission and subsequent cell death. In contrast, p-Drp1 protein level did not significantly change after delivery of similar FLASH doses. Compared with CONV irradiation, FLASH irradiation using protons induces minimal mitochondria damage; our results highlight a possible contribution of Drp1-mediated mitochondrial homeostasis in this potential novel cancer treatment modality.

An effective method that can protect radiation-damaged tissues from apoptosis and promote tissue repair has not been reported to date. Hypoxanthine (Hx) is an intermediate metabolite in the purine degradation system that serves as a substrate for ATP synthesis via the salvage pathway. In this study, we focused on the transient decrease in intracellular ATP concentration after radiation exposure and examined the protective effect of Hx against radiation-induced tissue damage. Human umbilical vein endothelial cells were X irradiated, and the cell viability and incidence of apoptosis and DNA double-strand breaks (DSBs) were evaluated at different Hx concentrations. We found that in the presence of 2–100 µM Hx, the percentages of DSBs and apoptotic cells after 2, 6 and 10 Gy dose of radiation significantly decreased, whereas cell viability increased in a concentration-dependent manner. Moreover, the addition of Hx increased the levels of AMP, ADP, and ATP in the cells at 2 h postirradiation, suggesting that Hx was used for adenine nucleotide synthesis through the salvage pathway. Administration of a xanthine oxidoreductase inhibitor to a mouse model of radiation dermatitis resulted in increased blood Hx levels that inhibited severe dermatitis and accelerated recovery. In conclusion, the findings provide evidence that increasing the levels of Hx to replenish ATP could be an effective strategy to reduce radiation-induced tissue damage and elucidating the detailed mechanisms underlying the protective effects of Hx could help develop new protective strategies against radiation.

In this study, an improved method using scavenger-free plasmid DNA was established to accurately determine yields of DNA damage induced by direct and indirect actions of ionizing radiation. The scavenger-free plasmid DNA was obtained by dialysis over 5–7 days, and the DNA solvent was replaced with phosphate buffer to completely remove impurities, which could be scavengers of radicals produced as a result of water radiolysis. DNA samples of films and dilute aqueous solutions were used to separately evaluate contributions of the direct and indirect actions of X rays (150–160 kVp). The irradiated DNA was analyzed by agarose gel electrophoresis to quantify strand-break yields. The yields of single-strand breaks (SSBs), n(SSB), were determined to be (6.5 ± 2.0) × 10^–10 and (3.1 ± 0.9) × 10^–7 SSBs/Gy/Da for the film and solution samples, respectively, showing a significant contribution of hydroxyl radicals (^•OH) compared with direct energy depositions from ionizing radiation to DNA. As observed in SSBs, the yields of double-strand breaks (DSBs), n(DSB), were (5.6 ± 1.1) × 10^–11 and (1.3 ± 0.2) × 10^–8 DSBs/Gy/Da for the film and solution samples, respectively. The yield ratio of DSBs to SSBs, that is, n(DSB)/n(SSB), was 0.091 ± 0.026 for the film samples, while it was much lower for the solution samples (0.045 ± 0.010), indicating that direct actions result in more localized strand breaks relative to indirect actions. Base excision repair enzymes, namely, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), were utilized after irradiations to convert base lesions and apurinic/apyrimidinic (AP) sites into strand breaks. The amounts of Nth and Fpg for the conversion were optimized to a few units per µg of DNA, although the optimal concentrations can differ among conditions.

Medical imaging plays a major role in coronavirus disease-2019 (COVID-19) patient diagnosis and management. However, the radiation dose received from medical procedures by these patients has been poorly investigated. We aimed to estimate the cumulative effective dose (CED) related to medical exposure in COVID-19 patients admitted to the intensive care unit (ICU) in comparison to the usual critically ill patients. We designed a descriptive cohort study including 90 successive ICU COVID-19 patients admitted between March and May 2020 and 90 successive non-COVID-19 patients admitted between March and May 2019. In this study, the CED resulting from all radiological examinations was calculated and clinical characteristics predictive of higher exposure risk identified. The number of radiological examinations was 12.0 (5.0–26.0) [median (interquartile range) in COVID-19 vs. 4.0 (2.0–8.0) in non-COVID-19 patient (P < 0.001)]. The CED during a four-month period was 4.2 mSv (1.9–11.2) in the COVID-19 vs. 1.2 mSv (0.13–6.19) in the non-COVID-19 patients (P < 0.001). In the survivors, the CED in COVID-19 vs. non-COVID-19 patients was ≥100 mSv in 3% vs. 0%, 10–100 mSv in 23% vs. 15%, 1–10 mSv in 56% vs. 30% and <1 mSv in 18% vs. 55%. The CED (P < 0.001) and CED per ICU hospitalization day (P = 0.004) were significantly higher in COVID-19 than non-COVID-19 patients. The CED correlated significantly with the hospitalization duration (r = 0.45, P < 0.001) and the number of conventional radiological examinations (r = 0.8, P < 0.001). To conclude, more radiological examinations were performed in critically ill COVID-19 patients than non-COVID-19 patients resulting in higher CED. In COVID-19 patients, contribution of strategies to limit CED should be investigated in the future.

Several diagnostic biodosimetry tools have been in development that may aid in radiological/nuclear emergency responses. Of these, correlating changes in non-invasive biofluid small-molecule signatures to tissue damage from ionizing radiation exposure show promise for inclusion in predictive biodosimetry models. Integral to dose reconstruction has been determining how genotypic variation in the general population will affect model performance. Here, we used a mouse model that lacks the T-cell receptor specific alternative p38 pathway [p38αβ^Y323F, double knock-in (DKI) mice] to determine how attenuated autoimmune and inflammatory responses may affect dose reconstruction. We exposed adult male DKI mice (8–10 weeks old) to 2 and 7 Gy in parallel with wild-type mice and assessed perturbations in urine (days 1, 3, 7) and serum (day 1) using a global metabolomics approach. A multidimensional scaling plot showed excellent separation of radiation-exposed groups in wild-type mice with slightly dampened responses in DKI mice. Validated metabolite panels were developed for urine [N6,N6,N6-trimethyllysine (TML), N1-acetylspermidine, spermidine, carnitine, acylcarnitine C₂₁H₃₅NO₅, aminohippuric acid] and serum [phenylalanine, glutamine, propionylcarnitine, lysophosphatidylcholine (LysoPC 14:0), LysoPC (22:5)] to determine the area under the receiver operating characteristic curve (AUROC). For both urine and serum, excellent sensitivity and specificity (AUROC > 0.90) was observed for 0 Gy vs. 7 Gy groups irrespective of genotype using identical metabolite panels. Similarly, excellent to fair classification (AUROC > 0.75) was observed for ≤2 Gy vs. 7 Gy mice for both genotypes, however, model performance declined (AUROC < 0.75) between genotypes after irradiation. Overall, these results suggest immunosuppression should not compromise small molecule multiplex panels used in dose reconstruction for biodosimetry.

Stereotactic body radiation therapy (SBRT) has shown promising results in the treatment of pancreatic cancer and other solid tumors. However, wide adoption of SBRT remains limited largely due to uncertainty about the treatment's optimal fractionation schedules to elicit maximal tumor response while limiting the dose to adjacent structures. A small animal irradiator in combination with a clinically relevant oncological animal model could address these questions. Accurate delivery of X rays to animal tumors may be hampered by suboptimal image-guided targeting of the X-ray beam in vivo. Integration of bioluminescence imaging (BLI) into small animal irradiators in addition to standard cone-beam computed tomography (CBCT) imaging improves target identification and high-precision therapy delivery to deep tumors with poor soft tissue contrast, such as pancreatic tumors. Using bioluminescent BxPC3 pancreatic adenocarcinoma human cells grown orthotopically in mice, we examined the performance of a small animal irradiator equipped with both CBCT and BLI in delivering targeted, hypo-fractionated, multi-beam SBRT. Its targeting accuracy was compared with magnetic resonance imaging (MRI)-guided targeting based on co-registration between CBCT and corresponding sequential magnetic resonance scans, which offer greater soft tissue contrast compared with CT alone. Evaluation of our platform's BLI-guided targeting accuracy was performed by quantifying in vivo changes in bioluminescence signal after treatment as well as staining of ex vivo tissues with γH2AX, Ki67, TUNEL, CD31 and CD11b to assess SBRT treatment effects. Using our platform, we found that BLI-guided SBRT enabled more accurate delivery of X rays to the tumor resulting in greater cancer cell DNA damage and proliferation inhibition compared with MRI-guided SBRT. Furthermore, BLI-guided SBRT allowed higher animal throughput and was more cost effective to use in the preclinical setting than MRI-guided SBRT. Taken together, our preclinical platform could be employed in translational research of SBRT of pancreatic cancer.

Radiation-induced muscle fibrosis is a long-term side effect of radiotherapy that significantly affects the quality of life and even reduces the survival of cancer patients. We have demonstrated that radiation induces satellite cell (SC) activation at the molecular level; however, cellular evidence in a rat model of radiation-induced muscle fibrosis was lacking. In this study, we evaluated SC activation in vivo and investigated whether radiation affects the proliferation and differentiation potential of SCs in vitro. For in vivo studies, Sprague-Dawley rats were randomly divided into six groups (n = 6 per group): non-irradiated controls, 90 Gy/1 week-, 90 Gy/2 weeks-, 90 Gy/4 weeks-, 90 Gy/12 weeks- and 90 Gy/24 weeks-postirradiation groups. Rats received a single dose of radiation in the left groin area and rectus femoris tissues were collected in the indicated weeks. Fibrosis, apoptosis, and autophagy were evaluated by Masson's trichrome staining, TUNEL staining, and electron microscopy, respectively. SC activation and central nuclear muscle fibers were evaluated by immunofluorescence staining and hematoxylin and eosin staining. IL-1β concentrations in serum and irradiated muscle tissue samples were determined by ELISA. For in vitro studies, SCs were isolated from rats with radiation-induced muscle fibrosis and their proliferation and differentiation were evaluated by immunofluorescence staining. In vivo, fibrosis increased over time postirradiation. Apoptosis and autophagy levels, IL-1β concentrations in serum and irradiated skin tissues, and the numbers of SCs and central nuclear muscle fibers were increased in the irradiated groups when compared with the control group. In vitro, cultured SCs from irradiated muscle were positive for the proliferation marker Pax7, and differentiated SCs were positive for the myogenic differentiation marker MyHC. This study provided cellular evidence of SC activation and proliferation in rats with radiation-induced muscle fibrosis.

Irradiation protocols for murine experiments often use standardized dose rate estimates for calculating dose delivered, regardless of physical variations between mouse subjects. This work sought to determine the significance of mouse size on absorbed dose. Five mouse-like phantoms of various sizes based on the mouse whole-body (MOBY) model were 3D printed. The phantoms were placed in an X-Rad320 biological irradiator and a standard irradiation protocol was used to deliver dose. Dose was measured using thermoluminescent dosimeter (TLD) microcubes inside each phantom, and the relative readings were used to calculate output factors (OFs), normalized to the phantom of median volume. Additionally, the OF for each mouse was simulated in Monte Carlo N-Particle (MCNP) code. For both the TLD measurements and MCNP simulations, the OF for each mouse was determined by both experiments and calculations to be unity within the relative standard uncertainties (k = 1). This work supports comparing results across various studies using the X-Rad320 irradiator without need for corrections based on mouse size.

The microbeam radiation therapy (MRT), a spatially micro-fractionated synchrotron radiotherapy, leads to better control of incurable high-grade glioma than that obtained upon homogeneous radiotherapy. We evaluated the effect of meloxicam, a non-steroidal anti-inflammatory drug (NSAID), to increase the MRT response. Survival of rats bearing intracranial 9L gliosarcoma treated with meloxicam and/or MRT (400 Gy, 50 µm-wide microbeams, 200 µm spacing) was monitored. Tumor growth was assessed on histological tissue sections and COX-2 transcriptomic expression was studied 1 to 25 days after radiotherapy. Meloxicam significantly extended the median survival of microbeam-irradiated rats (from +10.5 to +20 days). Dual treatment led to last survivors until D₉₀ (D₃₉ for the MRT group) and to tumor 9.5 times smaller than MRT alone. No significant modification of COX-2 expression was induced by MRT in normal and tumor tissues. The meloxicam reinforced the anti-tumor effect of MRT for glioma treatment. Although the mechanisms of interaction between meloxicam and MRT remain to be elucidated, the addition of this NSAID, easily implemented as a supplement to water for example, is a very favorable therapeutic regimen since it doubled the survival benefit compared to MRT alone.

The biological effects of ultrasound may be classified into thermal and nonthermal mechanisms. The nonthermal effects may be further classified into cavitational and noncavitational mechanisms. DNA damage induced by ultrasound is considered to be related to nonthermal cavitations. For this aspect, many in vitro studies on DNA have been conducted for evaluating the safety of diagnostic ultrasound, particularly in fetal imaging. Technological advancement in detecting DNA damage both in vitro and in vivo have elucidated the mechanism of DNA damage formation and their cellular response. Damage to DNA, and the residual damages after DNA repair are implicated in the biological effects. Here, we discuss the historical evidence of ultrasound on DNA damage and the mechanism of DNA damage formation both in vitro and in vivo, compared with those induced by ionizing radiation. We also offer a commentary on the safety of ultrasound over X-ray-based imaging. Also, understanding the various mechanisms involved in the bioeffects of ultrasound will lead us to alternative strategies for use of ultrasound for therapy.