We have previously shown significant pathology in the heart and kidney of murine hematopoietic-acute radiation syndrome (H-ARS) survivors of 8.7–9.0 Gy total-body irradiation (TBI). The goal of this study was to determine temporal relationships in the development of vasculopathy and the progression of renal and cardiovascular delayed effects of acute radiation exposure (DEARE) at TBI doses less than 9 Gy and to elucidate the potential roles of senescence, inflammation and oxidative stress. Our results show significant loss of endothelial cells in coronary arteries by 4 months post-TBI (8.53 or 8.72 Gy of gamma radiation). This loss precedes renal dysfunction and interstitial fibrosis and progresses to abnormalities in the arterial media and adventitia and loss of coronary arterioles. Major differences in radiation-induced pathobiology exist between the heart and kidney in terms of vasculopathy progression and also in indices of inflammation, senescence and oxidative imbalance. The results of this work suggest a need for different medical countermeasures for multiple targets in different organs and at various times after acute radiation injury to prevent the progression of DEARE.

Unthank, J. L, Ortiz, M., Trivedi, H., Pelus, L. M., Sampson, C. H., Sellamuthu, R., Fisher, A., Chua, H. L., Plett, A., Orschell, C. M., Cohen, E. P. and Miller, S. J. Cardiac and Renal Delayed Effects of Acute Radiation Exposure: Organ Differences in Vasculopathy, Inflammation, Senescence and Oxidative Balance. Radiat. Res. 191, 383–397 (2019).

Nguyen, P., Shukla, S., Liu, R., Abbineni, G. and Smart, D. K. Sirt2 Regulates Radiation-Induced Injury. Radiat. Res. 191, 398–412 (2019).

Sirtuin 2 (SIRT2) plays a major role in aging, carcinogenesis and neurodegeneration. While it has been shown that SIRT2 is a mediator of stress-induced cell death, the mechanism remains unclear. In this study, we report the role of SIRT2 in mediating radiation-induced cell death and DNA damage using mouse embryonic fibroblasts (MEFs), progenitor cells and tissues from Sirt2 wild-type and genomic knockout mice, and human tumor and primary cell lines as models. The presence of Sirt2 in cells and tissues significantly enhanced the cell's sensitivity to radiation-induced cytotoxicity by delaying the dispersion of radiation-induced γ-H2AX and 53BP1 foci. This enhanced cellular radiosensitivity correlated with reduced expression of pro-survival and DNA repair proteins, and decreased DNA repair capacities involving both homologous repair and non-homologous end joining DNA repair mechanisms compared to those in Sirt2 knockout (KO) and knockdown (KD) phenotypes. Together, these data suggest SIRT2 plays a critical role in mediating the radiation-induced DNA damage response, thus regulating radiation-induced cell death and survival.

Deyhle Jr., R. T., Wong, C. P., Martin, S. A., McDougall, M. Q., Olson, D. A., Branscum, A. J., Menn, S. A., Iwaniec, U. T., Hamby, D. M and Turner, R. T. Maintenance of Near Normal Bone Mass and Architecture in Lethally Irradiated Female Mice following Adoptive Transfer with as few as 750 Purified Hematopoietic Stem Cells. Radiat. Res. 191, 413–427 (2019).

Total-body irradiation (TBI) followed by transfer of bone marrow cells from donors is routinely performed in immunology research and can be used to manipulate differentiation and/or function of bone cells. However, exposure to high-dose radiation can result in irreversible osteopenia, and transfer of heterogeneous cell populations can complicate interpretation of results. The goal of this research was to establish an approach for reconstituting bone marrow using small numbers of purified donor-derived hematopoietic stem cells (HSCs) without negatively affecting bone metabolism. Gamma-irradiated (9 Gy) WBB6F1 mice were engrafted with bone marrow cells (5 × 10⁶ cells) or purified HSCs (3,000 cells) obtained from GFP transgenic mice. In vivo analysis and in vitro differentiation assays performed two months later established that both methods were effective in reconstituting the hematopoietic compartment with donor-derived cells. We confirmed these findings by engrafting C57Bl/6 (B6) mice with bone marrow cells or purified HSCs from CD45.1 B6 congenic mice. We next performed adoptive transfer of purified HSCs (750 cells) into WBB6F1 and radiosensitive Kit^W/W-v mice and evaluated the skeleton two months later. Minimal differences were observed between controls and WBB6F1-engrafted mice that received fractionated doses of 2 × 5 Gy. Kit^w/wv mice lost weight and became osteopenic after 2 × 5 Gy irradiations but these abnormalities were negligible after 5 Gy irradiation. Importantly, adoptive transfer of wild-type cells into Kit^w/wv mice restored normal Kit expression in bone marrow. Together, these findings provide strong evidence for efficient engraftment with purified HSCs after lethal TBI with minimal collateral damage to bone. This approach will be useful for investigating mechanisms by which hematopoietic lineage cells regulate bone metabolism.

Jackson,. I. L., Gibbs, A., Poirier, Y., Wathen, L., Eley, J., Draeeger, E., Gopalakrishnan, M., Benjamin, B. and Vujaskovic, Z. Hematological Effects of Non-Homogenous Ionizing Radiation Exposure in a Non-Human Primate Model. Radiat. Res. 191, 428–438 (2019).

Detonation of a radiological or nuclear device in a major urban area will result in heterogenous radiation exposure, given to the significant shielding of the exposed population due to surrounding structures. Development of biodosimetry assays for triage and treatment requires knowledge of the radiation dose-volume effect for the bone marrow (BM). This proof-of-concept study was designed to quantify BM damage in the non-human primate (NHP) after exposure to one of four radiation patterns likely to occur in a radiological/nuclear attack with varying levels of BM sparing. Rhesus macaques (11 males, 12 females; 5.30–8.50 kg) were randomized by weight to one of four arms: 1. bilateral total-body irradiation (TBI); 2. unilateral TBI; 3. bilateral upper half-body irradiation (UHBI); and 4. bilateral lower half-body irradiation (LHBI). The match-point for UHBI vs. LHBI was set at 1 cm above the iliac crest. Animals were exposed to 4 Gy of 6 MV X rays. Peripheral blood samples were drawn 14 days preirradiation and at days 1, 3, 5, 7 and 14 postirradiation. Dosimetric measurements after irradiation indicated that dose to the mid-depth xiphoid was within 6% of the prescribed dose. No high-grade fever, weight loss >10%, dehydration or respiratory distress was observed. Animals in the bilateral- and unilateral TBI arms presented with hematologic changes [e.g., absolute neutrophil count (ANC) <500/ll; platelets <50,000/ll] and clinical signs/symptoms (e.g., petechiae, ecchymosis) characteristic of the acute radiation syndrome. Animals in the bilateral UHBI arm presented with myelosuppression; however, none of the animals developed severe neutropenia or thrombocytopenia (ANC remained >500/µl; platelets >50,000/µl during 14-day follow-up). In contrast, animals in the LHBI arm (1 cm above the ilieac crest to the toes) were protected against BM toxicity with no marked changes in hematological parameters and only minor gross pathology [petechiae (1/5), splenomegaly (1/5) and mild pulmonary hemorrhage (1/ 5)]. The model performed as expected with respect to the dose-volume effect of total versus partial-BM irradiation, e.g., increased shielding resulted in reduced BM toxicity. Shielding of the major blood-forming organs (e.g., skull, ribs, sternum, thoracic and lumbar spine) spared animals from bone marrow toxicity. These data suggest that the biological consequences of the absorbed dose are dependent on the total volume and pattern of radiation exposure.

After a planned or unplanned radiation exposure, determination of absorbed dose has great clinical importance, informing treatment and triage decisions in the exposed individuals. Biodosimetry approaches allow for determination of dose in the absence of physical measurement apparatus. The current state-of-the-art biodosimetry method is based on the frequency of induced dicentric chromosomes in peripheral blood T cells, which is proportional to the absorbed radiation dose. Since dose-response curves used for obtaining absorbed dose for humans are based on data sourced from in vitro studies, a concerning discrepancy may be present in the reported dose. Specifically, T-cell survival after in vitro irradiation is much higher than that measured in humans in vivo and, in addition, is not dose dependent over some dose ranges. We hypothesized that these differences may lead to inappropriately inflated dicentric frequencies after in vitro irradiation when compared with in vivo irradiation of the same samples. This may lead to underestimation of the in vivo dose. To test this hypothesis, we employed the humanized mouse model, which allowed direct comparison of cell depletion and dicentric frequencies in human T cells irradiated in vivo and in vitro. The results showed similar dicentric chromosome induction frequencies measured in vivo and in vitro when assessed 24 h postirradiation despite the differences in cell survival. These results appear to validate the use of in vitro data for the estimation of the absorbed dose in human radiation biodosimetry.

Pujol-Canadell, M., Young, E. and Smilenov, L. Use of a Humanized Mouse Model System in the Validation of Human Radiation Biodosimetry Standards. Radiat. Res. 191, 439–446 (2019).

To identify modifications to amino acids that are directly induced by ionizing radiation, free amino acids and 3-residue peptides were irradiated using a linear accelerator (Linac) radiotherapy device. Mass spectrometry was performed to detail the relative sensitivity to radiation as well as identify covalent, radiation-dependent adducts. The order of reactivity of the 20 common amino acids was generally in agreement with published literature except for His (most reactive of the 20) and Cys (less reactive). Novel and previously identified modifications on the free amino acids were detected. Amino acids were far less reactive when flanked by glycine residues in a tripeptide. Order of reactivity, with GVG most and GEG least, was substantially altered, as were patterns of modification. Radiation reactivity of amino acids is clearly and strongly affected by conversion of the α-amino and α-carboxyl groups to peptide bonds, and the presence of neighboring amino acid residues.

Minkoff, B. B., Bruckbauer, S. T., Sabat, G., Cox, M. M. and Sussman, M. R. Covalent Modification of Amino Acids and Peptides Induced by Ionizing Radiation from an Electron Beam Linear Accelerator Used in Radiotherapy. Radiat. Res. 191, 447–459 (2019).

Evaluation of the characteristics of accelerator-based thermal neutron fields is recognized as an important issue when discussing the effectiveness of boron neutron capture therapy (BNCT). In this study, we propose that the radiation chemical yield (G value) of hydroxyl radicals (G_oh^•) can be considered a universal parameter for the description of the accelerator-based thermal neutron field. The G_oh^• of the ¹⁰B(n,α)⁷Li reaction was quantitatively evaluated using an aqueous coumarin-3-carboxylic acid (3CCA) solution, and was discriminated from that of contaminations (i.e., γ rays and fast neutrons). The G_oh^• of the ¹⁰B(n,α)⁷Li reaction was 0.107 ± 0.004 OH^•/100 eV, which is almost equivalent to that exposed to α particles with an energy of 6.0 MeV. Since the G_oh^• of γ rays from a ⁶⁰Co source is 2.03 ± 0.05 OH^•/100 eV, this lower value suggests that indirect action by the ¹⁰B(n,α)⁷Li reaction is not dominant in BNCT. However, our results indicate that one can assess the ⁶⁰Co equivalent dose of the ¹⁰B(n,α)⁷Li reaction in water from the G_oh^• derived using aqueous 3CCA solution in the accelerator-based thermal neutron field.

Kusumoto, T. and Ogawara, R. Radiation Chemical Yield of Hydroxyl Radicals for Accelerator-based Boron Neutron Capture Therapy: Dose Assessment of ¹⁰B(n,α)⁷Li Reaction Using Coumarin-3-Carboxilic Solution. Radiat. Res. 191, 460–465 (2019).

In this study, the behavior dynamics of Zirconium-89 (⁸⁹Zr)-labeled monoclonal antibodies (MAbs) after injection into the human body were modeled. This modified biokinetic model can be used for dose assessment, not only for ⁸⁹Zr-labeled MAb tumor visualization, but also for diagnostic and therapeutic radiation with MAb labeled with other radionuclides. A modified biokinetic model was created based on experimental data from previously published studies. Cumulative ⁸⁹Zr activity in organs and tissues per Bq of administered activity was calculated using the WinAct program. For most organs receiving the highest radiation dose, average absorbed doses were estimated using IDAC version 2.1 software. The results from the modeled calculations were compared with recently published results from studies of real patients. The calculations revealed that organs with the highest dose were the spleen, liver, kidneys and red bone marrow, at 1.54, 1.33, 0.81 and 0.82 mGy/MBq, respectively. In the modified biokinetic model, with MAb injection, organs exhibiting the highest dose were liver, heart wall, spleen, red bone marrow and pancreas, at 1.05, 0.93, 0.79, 0.69 and 0.67 mGy/MBq, respectively.

Zakaly, H. M. H., Mostafa, M. Y. A. and Zhukovsky, M. Dosimetry Assessment of Injected 89Zr-Labeled Monoclonal Antibodies in Humans. Radiat. Res. 191, 466–474 (2019).

One highly promising approach to cancer treatment, especially for tumors that have undergone micrometastasis, is targeted alpha-particle therapy (TAT). However, the development of a TAT drug has been impeded due to numerous unsuccessful attempts to establish effective in vitro screening methods. The goal of this study was to construct a model to predict and optimize in vitro screening of potential TAT drugs. Based on mean field hypothesis, microdosimetry and the classic linear-quadratic equation, a novel model was built, which can predict our own in vitro experiments and replicate published data from others. Interestingly, this model can also be used to quickly optimize several key parameters in in vitro screening of potential TAT drugs, instructing the optimal combinations of the expression level of antigen, the binding affinity of antibody and drug antibody ratio, as well as others. In addition, to conveniently evaluate the therapeutic benefit of different drugs, a simple but universal parameter, the death ratio, is proposed. To our knowledge, this is the first model that can predict and guide the optimization of in vitro potential targeted alpha-particle therapy drug screening, which may then accelerate the development of potential targeted alpha-particle therapy drugs dramatically.

Ma, W., Wang, X., Liu, W., Ma, H., Su, Y., Yang, Y., Liu, N., Wang, Y. and Yang, G. A Theoretical Model for Predicting and Optimizing In Vitro Screening of Potential Targeted Alpha-Particle Therapy Drugs. Radiat. Res. 191, 475–482 (2019).

Jamsranjav, E., Ito, A., Kato, Y., Tatebe, Y., Takase, N. and Yoshida, S. DNA Strand Breaks Induced by Fast and Thermal Neutrons from YAYOI Research Reactor in the Presence and Absence of Boric Acid. Radiat. Res. 191, 483–489 (2019).

It is well known that neutrons have more damaging effects, with high relative biological effectiveness or a radiation weighting factor depending on neutron energy, compared to low-LET ionizing radiations. In the current work, we evaluated the contribution of the indirect effect induced by radicals for the purpose of studying the mechanisms of fast neutron effects or mechanisms of boron neutron capture therapy (BNCT) using relatively lower energy neutrons. Plasmid pBR322 DNA with a supercoiled structure was irradiated with fast neutrons (1–10 MeV) in the reactor core of the YAYOI research reactor at the University of Tokyo, and with thermalized neutrons passing through a phantom made of acrylic resin to simulate a human body. The single- and double-strand breaks (SSBs and DSBs) of the plasmid were evaluated from the numbers of its open circular and linear forms, respectively, detected using agarose gel electrophoresis. The number of supercoiled forms decreased exponentially with the absorbed dose of fast neutrons. The decrease was inhibited by dimethyl sulfoxide (DMSO) in a concentration-dependent manner. The contributions of the indirect effect to DNA strand breaks by fast neutrons and thermalized neutrons were calculated to be 50–65% which, while lower than the value for X rays, still accounts for the majority of the DNA strand breaks. In the case of thermalized neutrons, SSB and DSB yields were increased by the addition of boric acid. Moreover, an increased ¹⁰B isotope concentration was found to increase the DSB/SSB ratio.