Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N′-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.

While thyroid cancer risks from exposure to ionizing radiation early in life are well characterized quantitatively, the association of radiation with nonmalignant, functional thyroid disorders has been less studied. Here, we report on a risk analysis study of hypothyroidism with radiation dose to the thyroid gland and the hypothalamic-pituitary axis among survivors of childhood cancer. Utilizing data from the Childhood Cancer Survivor Study, a cohort of 14,364 five-year survivors of childhood cancer diagnosed at 26 hospitals in the U.S. and Canada between 1970 and 1986 and followed through 2009, the occurrence of hypothyroidism was ascertained among 12,015 survivors through serial questionnaires. Radiation doses to the thyroid gland and pituitary gland were estimated from radiotherapy records. Binary outcome regression was used to estimate prevalence odds ratios for hypothyroidism at five years from diagnosis of childhood cancer and Poisson regression to model incidence rate ratios (RR) after the first five years. A total of 1,193 cases of hypothyroidism were observed, 777 (65%) of which occurred five or more years after cancer diagnosis. The cumulative proportion affected with hypothyroidism (prevalence at five years after cancer diagnosis plus incidence through 30 years after cancer diagnosis) was highest among five-year survivors of Hodgkin lymphoma (32.3%; 95% CI: 29.5–34.9) and cancers of the central nervous system (17.7%; 95% CI: 15.2–20.4). The incidence rate was significantly associated with radiation dose to the thyroid and pituitary. The joint association of hypothyroidism with thyroid and pituitary dose was sub-additive for pituitary doses greater than 16 Gy. In particular, a very strong thyroid radiation dose dependence at low-to-moderate pituitary/hypothalamic doses was diminished at high pituitary doses. Radiation-related risks were higher in males than females and inversely associated with age at exposure and time since exposure but remained elevated more than 25 years after exposure. Our findings indicated that hypothyroidism was significantly associated with treatment with bleomycin (RR = 3.4; 95% CI: 1.6–7.3) and the alkylating agents cyclohexyl-chloroethyl-nitrosourea (CCNU) (RR = 3.0; 95% CI: 1.5–5.3) and cyclophosphamide (RR = 1.3; 95% CI: 1.0–1.8), with a significant dose response for CCNU (P < 0.01). The risk of hypothyroidism among childhood cancer survivors treated with radiation depends both on direct, dose-dependent radiation-induced damage to the thyroid gland and on dose-dependent indirect effects secondary to irradiation of the hypothalamic-pituitary axis. The dose-response relationship for each site depends on dose to the other. Radiation-related risk persists for more than 25 years after treatment. Treatment with certain chemotherapy agents may increase the risk of hypothyroidism.

While computed tomography (CT) is now commonly used and considered to be clinically valuable, significant DNA double-strand breaks (γ-H2AX foci) in white blood cells from adult and pediatric CT patients have been frequently reported. In this study to determine whether γ-H2AX foci and X-ray-induced naked DNA damage are suppressed by administration of the PrC-210 radioprotector, human blood samples were irradiated in a CT scanner at 50–150 mGy with or without PrC-210, and γ-H2AX foci were scored. X-ray-induced naked DNA damage was also studied, and the DNA protective efficacy of PrC-210 was compared against 12 other common “antioxidants.” PrC-210 reduced CT radiation-induced γ-H2AX foci in white blood cells to near background (P < 0.0001) at radiation doses of 50–150 mGy. PrC-210 was most effective among the 13 “antioxidants” in reducing naked DNA X-ray damage, and its addition at 30 s before an ^•OH pulse reduced to background the ^•OH insult that otherwise induced >95% DNA damage. A systemic PrC-210 dose known to confer 100% survival in irradiated mice had no discernible effect on micro-CT image signal-to-noise ratio and CT image integrity. PrC-210 suppressed DNA damage to background or near background in each of these assay systems, thus supporting its development as a radioprotector for humans in multiple radiation exposure settings.

Astronauts traveling in deep space are exposed to high-charge and energy (HZE) particles from galactic cosmic rays. We have previously determined that irradiation of adult female mice with iron HZE particles induces DNA double-strand breaks, oxidative damage and apoptosis in ovarian follicles, causing premature ovarian failure. These effects occur at lower doses than with conventional photon irradiation. Ovarian failure with resultant loss of negative feedback and elevated levels of gonadotropin hormones is thought to play a role in the pathophysiology of ovarian cancer. Therefore, we hypothesized that charged-iron-particle irradiation induces ovarian tumorigenesis in mice. In this study, three-month-old female mice were exposed to 0 cGy (sham) or 50 cGy iron ions and aged to 18 months. The 50 cGy irradiated mice had increased weight gain with age and lack of estrous cycling, consistent with ovarian failure. A total of 47% and 7% of mice irradiated with 50 cGy had unilateral and bilateral ovarian tumors, respectively, whereas 14% of mice in the 0 cGy group had unilateral tumors. The tumors contained multiple tubular structures, which were lined with cells positive for the epithelial marker cytokeratin, and had few proliferating cells. In some tumors, packets of cells between the tubular structures were immunopositive for the granulosa cell marker FOXL2. Based on these findings, tumors were diagnosed as tubular adenomas or mixed tubular adenoma/granulosa cell tumors. In conclusion, charged-iron-particle-radiation induces ovarian tumors in mice, raising concerns about ovarian tumors as late sequelae of deep space travel in female astronauts.

To better understand the spatiotemporal course of radiation-induced central nervous system (CNS) vascular necrosis and assess the therapeutic potential of approaches for protecting against radiation-induced necrosis, adult female Sprague Dawley rats received 40 Gy surface dose centered on the T9 thoracic spinal cord segment. Locomotor function, blood-spinal cord barrier (BSCB) integrity and histology were evaluated throughout the study. No functional symptoms were observed for several months postirradiation. However, a sudden onset of paralysis was observed at approximately 5.5 months postirradiation. The progression rapidly led to total paralysis and death within less than 48 h of symptom onset. Open-field locomotor scores and rotarod motor coordination testing showed no evidence of neurological impairment prior to the onset of overt paralysis. Histological examination revealed minimal changes to the vasculature prior to symptom onset. However, Evans blue dye (EvB) extravasation revealed a progressive deterioration of BSCB integrity, beginning at one week postirradiation, affecting regions well outside of the irradiated area. Minocycline treatment significantly delayed the onset of paralysis. The results of this study indicate that extensive asymptomatic disruption of the blood-CNS barrier may precede onset of vascular breakdown by several months and suggests that minocycline treatment has a therapeutic effect by delaying radiation-induced necrosis after CNS irradiation.

Although bone marrow aplasia has been considered for the past decades as the major contributor of radiation-induced blood disorders, cytopenias alone are insufficient to explain differences in the prevalence of bleeding. In this study, the minipig was used as a novel preclinical model of hematopoietic acute radiation syndrome to assess if factors other than platelet counts correlated with bleeding and survival. We sought to determine whether radiation affected the insulin-like growth factor-1 (IGF-1) pathway, a growth hormone with cardiovascular and radioprotective features. Gottingen and Sinclair minipigs were exposed to ionizing radiation at hematopoietic doses. The smaller Gottingen minipig strain was more sensitive to radiation; differences in IGF-1 levels were minimal, suggesting that increased sensitivity could depend on weak response to the hormone. Radiation caused IGF-1 selective resistance by inhibiting the anti-inflammatory anti-oxidative stress IRS/PI3K/Akt but not the pro-inflammatory MAPK kinase pathway, shifting IGF-1 signaling towards a pro-oxidant, pro-inflammatory environment. Selective IGF-1 resistance associated with hemorrhages in the heart, poor prognosis, increase in C-reactive protein and NADPH oxidase 2, uncoupling of endothelial nitric oxide synthase, inhibition of nitric oxide (NO) synthesis and imbalance between the vasodilator NO and the vasoconstrictor endothelin-1 molecules. Selective IGF-1 resistance is a novel mechanism of radiation injury, associated with a vicious cycle amplifying reactive oxygen species-induced damage, inflammation and endothelial dysfunction. In the presence of thrombocytopenia, selective inhibition of IGF-1 cardioprotective function may contribute to the development of hemostatic disorders. This finding may be particularly relevant for individuals with low IGF-1 activity, such as the elderly or those with cardiometabolic dysfunctions.

DosiKit is a new field-radiation biodosimetry immunoassay for rapid triage of individuals exposed to external total-body irradiation. Here, we report on the validation of this immunoassay in human blood cell extracts 0.5 h after in vitro exposure to ¹³⁷Cs gamma rays, using γ-H2AX analysis. First, calibration curves were established for five donors at doses ranging from 0 to 10 Gy and dose rates ranging from ∼0.8 to ∼3 Gy/min. The calibration curves, together with a γ-H2AX peptide scale, enabled the definition of inter-experimental correction factors. Using previously calculated correction factors, blind dose estimations were performed at 0.5 h postirradiation, and DosiKit performance was compared against concomitant dicentric chromosome assay (DCA), the current gold standard for external irradiation biodosimetry. A prototype was then assembled and field tested. We show that, despite significant inter-individual variations, DosiKit can estimate total-body irradiation doses from 0.5 to 10 Gy with a strong linear dose-dependent signal and can be used to classify potentially exposed individuals into three dose ranges: below 2 Gy, between 2 and 5 Gy and above 5 Gy. The entire protocol can be performed in 45 min, from sampling to dose estimation, with a new patient triaged every 10 min. While DCA enables precise measurement of doses below 5 Gy, it is a long and difficult method. In contrast, DosiKit is a quick test that can be performed directly in the field by operational staff with minimal training, and is relevant for early field triage and identification of individuals most likely to experience acute radiation syndrome. These findings suggest that DosiKit and DCA are complementary and should be combined for triage in a mass scale event. While the proof-of-concept reported here validates the use of DosiKit at 0.5 h postirradiation, further studies are needed to calibrate and evaluate the performance of the DosiKit assay at longer times after irradiation.

Exposure to galactic cosmic rays (GCR) poses an obstacle to successful deep space missions, including missions to the Moon or Mars. Previously, we and others have identified chronic cognitive impairments associated with GCR in rodent model systems. The persistent cognitive loss previously reported is indicative of global changes in different regions of the brain, including the prefrontal cortex and the hippocampus. It has been shown that both of these brain regions are involved in social functions. Here we demonstrate that four months after a single exposure to oxygen ionizing radiation, which is a component of GCR, adult male mice have social memory deficits. Importantly, we identified circulating levels of CD8 T cells as predictors of social behavioral changes. Thus, CD8 T cells could be used as a potential peripheral biomarker. To the best of our knowledge we demonstrate for the first time that GCR-induced impairments in social behavior are directly linked to peripheral immune changes. These results further advance our understanding of the challenges encountered during space exploration.

As one of the most crucial epidermal growth factor receptor (EGFR) variants, EGFRvIII can be detected in various tumors but rarely in normal tissues, making it an ideal target for prognosis, diagnosis or immune therapy. The recently developed anti-EGFRvIII monoclonal antibody (mAb), 4G1, has been validated as a promising molecular probe to detect EGFRvIII expression in tumors by single-photon emission computed tomography/computed tomography imaging. To overcome shortcomings associated with the whole antibody, including long-term retention, circulation and enhanced permeability and retention effects, the Fab fragment of 4G1 (Fab-4G1) was generated, labeled with ¹³¹I and evaluated in vitro and in vivo to test its potential application in molecular imaging. Whole mAb 4G1 was first digested by immobilized ficin and then purified through a protein A column to generate the Fab fragment, Fab-4G1. Next, SDS-PAGE, Western blot, indirect fluorescence assay, flow cytometry and enzyme-linked immunosorbent assay were performed to verify molecular weight, specificity and affinity of Fab-4G1. Finally, biodistribution planar gamma imaging was performed by injection of ¹³¹I-labeled Fab-4G1 into xenografted EGFRvIII-overexpressed tumors in nude mice. Parallel studies were also performed with intact 4G1. The molecular weight of Fab was determined to be 35–40 kDa by SDS-PAGE. In vitro tests confirmed both intact 4G1 and Fab-4G1 specifically bound EGFRvIII but not wild-type EGFR, and Fab-4G1 showed decreased affinity. Compared to ¹³¹I-4G1, biodistribution studies showed lower tumor uptake of ¹³¹I-Fab-4G1 at all time points, but much faster elimination in all normal organs. As for planar gamma imaging, ¹³¹I-Fab-4G1 and ³¹I-4G1 showed similar imaging effect at 2 h after injection of tracer, while ¹³¹I-Fab-4G1 was eliminated more quickly with time, suggesting radiolabeled Fab-4G1 could be potentially used for imaging of EGFRvIII-positive tumors at early time points. Radiolabeled Fab-4G1 would be a promising nuclear probe for future clinical EGFRvIII tumor detection.

We investigated the relationship between significantly different genes of the mitochondria-to-nucleus retrograde signaling pathway (RTG) in H1299 ρ⁰ cells (mtDNA depleted cell) and compared their radiosensitivity to that of parental ρ cells, to determine the possible intervention targets of radiosensitization. ρ⁰ cells were depleted of mitochondrial DNA by chronic culturing in ethidium bromide at low concentration. Radiosensitivity was analyzed using clonogenic assay. Western blot was used to analyze the cell cycle-related proteins, serine/threonine kinase ataxia telangiectasia mutant (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and cyclin B1 (CCNB1). The γ-H2AX foci were detected using confocal fluorescence microscopy. RNA samples were hybridized using the Agilent human genome expression microarray. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for Gene Ontology (GO) Consortium and pathway annotations of differentially expressed genes, respectively. The H1299 ρ⁰ cells were found to be more radioresistant than ρ cells. The ATP production of H1299 ρ⁰ cells was lower than that of the ρ cells before or after irradiation. Both H1299 ρ⁰ and ρ cells had higher ROS levels after irradiation, however, the radiation-induced ROS production in ρ⁰ cells was significantly lower than in ρ cells. In addition, the percentage of apoptosis in H1299 ρ⁰ cells was lower than in ρ cells after 6 Gy irradiation. As for the cell cycle and DNA damage response-related proteins ATM, ATR and CCNB1, the expression levels in ρ⁰ cells were significantly higher than in ρ cells, and there were less γ-H2AX foci in the ρ⁰ than ρ cells after irradiation. Furthermore, the results of the human genome expression microarray demonstrated that the phosphorylated protein levels of the NF-κB/PI3K/AKT2/mTOR signaling pathway were increased after 6 Gy irradiation and were decreased after treatment with the AKT2-specific inhibitor MK-2206 combined with radiation in H1299 ρ⁰ cells. MK-2206 treatment also led to an increase in pro-apoptotic proteins. In conclusion, these results demonstrate that mtDNA depletion might activate the mitochondria-to-nucleus retrograde signaling pathway of NF-κB/PI3K/AKT2/mTOR and induce radioresistance in H1299 ρ⁰ cells by evoking mitochondrial dysfunctions.

The transcription factor Nrf2 is an important modulator of antioxidant and drug metabolism, carbohydrate and lipid metabolism, as well as heme and iron metabolism. Regulation of Nrf2 expression occurs transcriptionally and post-transcriptionally. Post-transcriptional regulation entails ubiquitination followed by proteasome-dependent degradation. Additionally, Nrf2-mediated gene expression is subject to negative regulation by ATF3, Bach1 and cMyc. Nrf2-mediated gene expression is an important regulator of a cell's response to radiation. Although a majority of studies have shown that Nrf2 deficient cells are radiosensitized and Nrf2 over expression confers radioresistance, Nrf2's role in mediating the radiation response of crypt cells is controversial. The Nrf2 activator CDDO attenuates radiation-mediated crypt injury, whereas intestinal crypts in Nrf2 null mice are radiation resistant. Further investigation is needed in order to define the relationship between Nrf2 and radiation sensitivity in Lgr5 and Bmi1 cells that regulate regeneration of crypt stem cells. In hematopoietic compartments Nrf2 promotes the survival of irradiated osteoblasts that support long-term hematopoietic stem cell (LT-HSC) niches. Loss of Nrf2 in LT-HSCs increases stem cell intrinsic radiosensitivity, with the consequence of lowering the LD50₃₀. An Nrf2 deficiency drives LT-HSCs from a quiescent to a proliferative state. This results in hematopoietic exhaustion and reduced engraftment after myoablative irradiation. The question of whether induction of Nrf2 in LT-HSC enhances hematopoietic reconstitution after bone marrow transplantation is not yet resolved. Irradiation of the lung induces pulmonary pneumonitis and fibrosis. Loss of Nrf2 promotes TGF-β/Smad signaling that induces ATF3 suppression of Nrf2-mediated target gene expression. This, in turn, results in elevated reactive oxygen species (ROS) and isolevuglandin adduction of protein that impairs collagen degradation, and may contribute to radiation-induced chronic cell injury. Loss of Nrf2 impairs ΔNp63 stem/progenitor cell mobilization after irradiation, while promoting alveolar type 2 cell epithelial-mesenchymal transitions into myofibroblasts. These studies identify Nrf2 as an important factor in the radiation response of normal tissue.