Sex as a Factor in Murine Radiation Research: Implications for Countermeasure Development.

There is an increased threat of exposure to ionizing radiation; in the event of such exposure, the availability of medical countermeasures will be vital to ensure the protection of the population. Effective countermeasures should be efficacious across a varied population and most importantly amongst both males and females. Radiation research must be conducted in animal models which act as a surrogate for the human response. Here, we identify differences in survival in male and female C57BL/6 in both a total body irradiation (TBI) model using the Armed Forces Radiobiology Research Institute (AFRRI) 60Co source and a partial body irradiation (PBI) model using the AFRRI Linear Accelerator (LINAC) with 4 MV photons and 2.5% bone marrow shielding. In both models, we observed a higher degree of radioresistance in female animals and a corresponding radiosensitivity in males. One striking difference in male and female rodents is body size/weight and we investigated the role of pre-irradiation body weight on survivability for animals irradiated at the same dose of irradiation (8 Gy TBI, 14 Gy PBI). We found that weight does not influence survival in the TBI model and that heavier males but lighter females have increased survival in the PBI model. This incongruence in survival amongst the sexes should be taken into consideration in the course of developing radiation countermeasures for response to a mass casualty incident.

Impairment of IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity and Mortality.

Following exposure to high doses of ionizing radiation, diverse strains of vertebrate species will manifest varying levels of radiation sensitivity. To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, two mouse strains with varying radiosensitivity (C3H/HeN, and CD2F1), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling pathway is associated with radiosensitivity, we investigated the link between systemic or tissue-specific IGF-1 signaling and radiosensitivity. Adult male C3H/HeN and CD2F1 mice were irradiated using gamma photons at Lethal Dose-70/30 (LD70/30), 7.8 and 9.35 Gy doses, respectively. Those mice that survived up to 30 days post-irradiation, were termed the survivors. Mice that were euthanized prior to 30 days post-irradiation due to deteriorated health were termed decedents. The analysis of non-irradiated and irradiated survivor and decedent mice showed that inter-strain radiosensitivity and post-irradiation survival outcomes are associated with activation status of tissue and systemic IGF-1 signaling, nuclear factor erythroid 2-related factor 2 (Nrf2) activation, and the gene expression profile of cardiac mitochondrial energy metabolism pathways. Our findings link radiosensitivity with dysregulation of IGF-1 signaling, and highlight the role of antioxidant gene response and mitochondrial function in radiation sensitivity.

Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling.

Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9-2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries.

Dysregulated Cardiac IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity.

Acute exposure to ionizing radiation leads to Hematopoietic Acute Radiation Syndrome (H-ARS). To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, adult males of two strains of minipig, one with higher radiosensitivity, the Gottingen minipig (GMP), and another strain with comparatively lower radiosensitivity, the Sinclair minipig (SMP), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling is associated with radiation sensitivity and regulation of cardiovascular homeostasis, we investigated the link between dysregulation of cardiac IGF-1 signaling and radiosensitivity. The adult male GMP; n = 48, and SMP; n = 24, were irradiated using gamma photons at 1.7-2.3 Gy doses. The animals that survived to day 45 after irradiation were euthanized and termed the survivors. Those animals that were euthanized prior to day 45 post-irradiation due to severe illness or health deterioration were termed the decedents. Cardiac tissue analysis of unirradiated and irradiated animals showed that inter-strain radiosensitivity and survival outcomes in H-ARS are associated with activation status of the cardiac IGF-1 signaling and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated induction of antioxidant gene expression. Our data link H-ARS with dysregulation of cardiac IGF-1 signaling, and highlight the role of oxidative stress and cardiac antioxidant response in radiation sensitivity.

Use of antisense oligonucleotides to correct the splicing error in ISCU myopathy patient cell lines.

ISCU myopathy is an inherited disease that primarily affects individuals of northern Swedish descent who share a single point mutation in the fourth intron of the ISCU gene. The current study shows correction of specific phenotypes associated with disease following treatment with an antisense oligonucleotide (ASO) targeted to the site of the mutation. We have shown that ASO treatment diminished aberrant splicing and increased ISCU protein levels in both patient fibroblasts and patient myotubes in a concentration dependent fashion. Upon ASO treatment, levels of SDHB in patient myotubular cell lines increased to levels observed in control myotubular cell lines. Additionally, we have shown that both patient fibroblast and myotubular cell lines displayed an increase in complex II activity with a concomitant decrease in succinate levels in patient myotubular cell lines after ASO treatment. Mitochondrial and cytosolic aconitase activities increased significantly following ASO treatment in patient myotubes. The current study suggests that ASO treatment may serve as a viable approach to correcting ISCU myopathy in patients.

FSL-1: A Synthetic Peptide Increases Survival in a Murine Model of Hematopoietic Acute Radiation Syndrome.

In the current geopolitical climate there is an unmet need to identify and develop prophylactic radiation countermeasures, particularly to ensure the well-being of warfighters and first responders that may be required to perform on radiation-contaminated fields for operational or rescue missions. Currently, no countermeasures have been approved by the U.S. FDA for prophylactic administration. Here we report on the efficacious nature of FSL-1 (toll-like receptor 2/6 agonist) and the protection from acute radiation syndrome (ARS) in a murine total-body irradiation (TBI) model. A single dose of FSL-1 was administered subcutaneously in mice. The safety of the compound was assessed in non-irradiated animals, the efficacy of the compound was assessed in animals exposed to TBI in the AFRRI Co-60 facility, the dose of FSL-1 was optimized, and common hematological parameters [complete blood cell (CBC), cytokines, and bone marrow progenitor cells] were assessed. Animals were monitored up to 60 days after exposure and radiation-induced damage was evaluated. FSL-1 was shown to be non-toxic when administered to non-irradiated mice at doses up to 3 mg/kg. The window of efficacy was determined to be 24 h prior to 24 h after TBI. FSL-1 administration resulted in significantly increased survival when administered either 24 h prior to or 24 h after exposure to supralethal doses of TBI. The optimal dose of FSL-1 administration was determined to be 1.5 mg/kg when administered prior to irradiation. Finally, FSL-1 protected the hematopoietic system (recovery of CBC and bone marrow CFU). Taken together, the effects of increased survival and accelerated recovery of hematological parameters suggests that FSL-1 should be developed as a novel radiation countermeasure for soldiers and civilians, which can be used either before or after irradiation in the aftermath of a radiological or nuclear event.

Development of a Multi-Organ Radiation Injury Model with Precise Dosimetry with Focus on GI-ARS.

The goal of this study was to establish a model of partial-body irradiation (PBI) sparing 2.5% of the bone marrow (BM2.5-PBI) that accurately recapitulates radiological/nuclear exposure scenarios. Here we have reported a model which produces gastrointestinal (GI) damage utilizing a clinical linear accelerator (LINAC) with precise dosimetry, which can be used to develop medical countermeasures (MCM) for GI acute radiation syndrome (ARS) under the FDA animal rule. The PBI model (1 hind leg spared) was developed in male and female C57BL/6 mice that received radiation doses ranging from 12-17 Gy with no supportive care. GI pathophysiology was assessed by crypt cell loss and correlated with peak lethality between days 4 and 10 after PBI. The radiation dose resulting in 50% mortality in 30 days (LD50/30) was determined by probit analysis. Differential blood cell counts in peripheral blood, colony forming units (CFU) in bone marrow, and sternal megakaryocytes were analyzed between days 1-30, to assess the extent of hematopoietic ARS (H-ARS) injury. Radiation-induced GI damage was also assessed by measuring: 1. bacterial load (16S rRNA) by RT-PCR on days 4 and 7 after PBI in liver, spleen and jejunum, 2. liposaccharide binding protein (LBP) levels in liver, and 3. fluorescein isothiocyanate (FITC)-dextran, E-selectin, sP-selectin, VEGF, FGF-2, MMP-9, citrulline, and serum amyloid A (SAA) levels in serum. The LD50/30 of male mice was 14.3 Gy (95% confidence interval 14.1-14.7 Gy) and of female mice was 14.5 Gy (95% confidence interval 14.3-14.7 Gy). Secondary endpoints included loss of viable crypts, higher bacterial loads in spleen and liver, higher LBP in liver, increased FITC-dextran and SAA levels, and decreased levels of citrulline and endothelial biomarkers in serum. The BM2.5-PBI model, developed for the first time with precise dosimetry, showed acute radiation-induced GI damage that is correlated with lethality, as well as a response to various markers of inflammation and vascular damage. Sex-specific differences were observed with respect to radiation dose response. Currently, no MCM is available as a mitigator for GI-ARS. This BM2.5-PBI mouse model can be regarded as the first high-throughput PBI model with precise dosimetry for developing MCMs for GI-ARS under the FDA animal rule.

Time- and sex-dependent delayed effects of acute radiation exposure manifest via miRNA dysregulation.

The detrimental effects of high-dose ionizing radiation on human health are well-known, but the influence of sex differences on the delayed effects of acute radiation exposure (DEARE) remains unclear. Here, we conducted six-month animal experiments using escalating radiation doses (7-9 Gy) on male and female C57BL/6 mice. The results show that female mice exhibited greater resistance to radiation, showing increased survival at six months post-total body irradiation. LD50/30 (lethal dose expected to cause 50% lethality in 30 days) for female mice is 8.08 Gy, while for male mice it is 7.76 Gy. DEARE causes time- and sex-dependent dysregulation of microRNA expression, processing enzymes, and the HOTAIR regulatory pathway. Differential regulation of molecular patterns associated with growth, development, apoptosis, and cancer is also observed in male and female mice. These findings shed light on the molecular basis of age and sex differences in DEARE response and emphasize the importance of personalized medicine for mitigating radiation-induced injuries and diseases.

Iron content of Saccharomyces cerevisiae cells grown under iron-deficient and iron-overload conditions.

Fermenting cells were grown under Fe-deficient and Fe-overload conditions, and their Fe contents were examined using biophysical spectroscopies. The high-affinity Fe import pathway was active only in Fe-deficient cells. Such cells contained ~150 µM Fe, distributed primarily into nonheme high-spin (NHHS) Fe(II) species and mitochondrial Fe. Most NHHS Fe(II) was not located in mitochondria, and its function is unknown. Mitochondria isolated from Fe-deficient cells contained [Fe(4)S(4)](2+) clusters, low- and high-spin hemes, S = (1)/(2) [Fe(2)S(2)](+) clusters, NHHS Fe(II) species, and [Fe(2)S(2)](2+) clusters. The presence of [Fe(2)S(2)](2+) clusters was unprecedented; their presence in previous samples was obscured by the spectroscopic signature of Fe(III) nanoparticles, which are absent in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. The majority of Mn ions in wild-type yeast cells was electron paramagnetic resonance-active Mn(II) and not located in mitochondria or vacuoles. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400-450 µM Fe. In these cells, the concentration of nonmitochondrial NHHS Fe(II) declined 3-fold, relative to that in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe(III) increased to a limiting cellular concentration of ~300 µM. Isolated mitochondria contained more NHHS Fe(II) ions and substantial amounts of Fe(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change in nutrient Fe levels. The ability to limit Fe import prevents cells from becoming overloaded with Fe.

PrC-210 Protects against Radiation-Induced Hematopoietic and Intestinal Injury in Mice and Reduces Oxidative Stress.

The development of safe, orally available, and effective prophylactic countermeasures to protect our warfighters is an unmet need because there is no such FDA-approved countermeasure available for use. Th 1-Propanethiol, 3-(methylamino)-2-((methylamino)methyl) (PrC-210), a synthetic small molecule, is a member of a new family of aminothiols designed to reduce toxicity while scavenging reactive oxygen species (ROS). Our study investigated the protective role of a single oral administration of PrC-210 against radiation-induced hematopoietic and intestinal injury in mice. Pre-treatment with PrC-210 significantly improved the survival of mice exposed to a lethal dose of radiation. Our findings indicated that the radioprotective properties of PrC-210 are achieved by accelerating the recovery of the hematopoietic system, stimulating bone marrow progenitor cells, and ameliorating additional biomarkers of hematopoietic injury. PrC-210 pre-treatment reduced intestinal injury in mice exposed to a lethal dose of radiation by restoring jejunal crypts and villi, reducing translocation of bacteria to the spleen, maintaining citrulline levels, and reducing the sepsis marker serum amyloid A (SAA) in serum. Finally, PrC-210 pre-treatment led to a significant reduction (~10 fold) of Nos2 expression (inducible nitric oxide) in the spleen and decreased oxidative stress by enhancing the antioxidant defense system. These data support the further development of PrC-210 to receive approval from the FDA to protect warfighters and first responders from exposure to the harmful effects of ionizing radiation.

Early to sustained impacts of lethal radiation on circulating miRNAs in a minipig model.

Early diagnosis of lethal radiation is imperative since its intervention time windows are considerably short. Hence, ideal diagnostic candidates of radiation should be easily accessible, enable to inform about the stress history and objectively triage subjects in a time-efficient manner. Therefore, the small molecules such as metabolites and microRNAs (miRNAs) from plasma are legitimate biomarker candidate for lethal radiation. Our objectives were to comprehend the radiation-driven molecular pathogenesis and thereby determine biomarkers of translational potential. We investigated an established minipig model of LD70/45 total body irradiation (TBI). In this pilot study, plasma was collected pre-TBI and at multiple time points post-TBI. The majority of differentially expressed miRNAs and metabolites were perturbed immediately after TBI that potentially underlined the severity of its acute impact. The integrative network analysis of miRNA and metabolites showed a cohesive response; the early and consistent perturbations of networks were linked to cancer and the shift in musculoskeletal atrophy synchronized with the comorbidity-networks associated with inflammation and bioenergy synthesis. Subsequent comparative pipeline delivered 92 miRNAs, which demonstrated sequential homology between human and minipig, and potentially similar responses to lethal radiation across these two species. This panel promised to retrospectively inform the time since the radiation occurred; thereby could facilitate knowledge-driven interventions.

PEGylated thrombopoietin mimetic, JNJ­26366821 a novel prophylactic radiation countermeasure for acute radiation injury.

Thrombopoietin (TPO) is the primary regulator of platelet generation and a stimulator of multilineage hematopoietic recovery following exposure to total body irradiation (TBI). JNJ­26366821, a novel PEGylated TPO mimetic peptide, stimulates platelet production without developing neutralizing antibodies or causing any adverse effects. Administration of a single dose of JNJ­26366821 demonstrated its efficacy as a prophylactic countermeasure in various mouse strains (males CD2F1, C3H/HeN, and male and female C57BL/6J) exposed to Co-60 gamma TBI. A dose dependent survival efficacy of JNJ­26366821 (- 24 h) was identified in male CD2F1 mice exposed to a supralethal dose of radiation. A single dose of JNJ­26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of TBI with a dose reduction factor of 1.36. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pre-treated with JNJ­26366821. The drug also increased bone marrow cellularity and megakaryocytes, accelerated multi-lineage hematopoietic recovery, and alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage. These results indicate that JNJ­26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Biophysical investigation of the ironome of human jurkat cells and mitochondria.

The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with (57)Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K Mössbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to S = 0 [Fe(4)S(4)](2+) clusters and low-spin (LS) Fe(II) heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be Fe(III) oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) Fe(II) species. A portion of these species may constitute the "labile iron pool" (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. Electron paramagnetic resonance spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS Fe(III) heme and non-heme species. The basal heme redox state of mitochondria within cells was reduced; this redox poise was unaltered during the anaerobic isolation of the organelle. Contributions from heme a, b, and c centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using inductively coupled plasma mass spectrometry. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells - the first "ironome" profile of a human cell.

CDX-301 prevents radiation-induced dysregulation of miRNA expression and biogenesis.

Risks of radiation exposure necessitate the development of radioprophylactic drugs. We have reported the efficacy of CDX-301, a recombinantly developed human protein form of Fms-related tyrosine kinase 3 ligand (Flt3L), as a radioprophylactic and radiomitigatory agent. Here, we performed global microRNA profiling to further understand the mechanism of action of CDX-301. We find that CDX-301 administration 24 h prior to total body irradiation prevents radiation-induced dysregulation of microRNA biogenesis and expression in murine serum and spleen samples in a time- and tissue-dependent manner. Further analysis shows that activation of the HOTAIR regulatory pathway has a prominent function in radiation-induced injury responses, which is inhibited by pre-treatment with CDX-301. Moreover, CDX-301 attenuates radiation-induced dysregulation of several cellular functions such as inflammatory and immune responses. In corroboration, we also find that pre-treatment with CDX-301 restores the expression of bone marrow aplasia markers and inflammatory cytokines and growth factors, as well as the expression of genes associated with MAP kinase and TGF-ß pathways that are altered by radiation. Our findings provide new insights into CDX-301-mediated molecular and cellular mechanisms and point to a possible novel radioprotective drug for the prevention of irradiation-induced injury and hematopoietic acute radiation syndrome.

Pre-Administration of PLX-R18 Cells Protects Mice from Radiation-Induced Hematopoietic Failure and Lethality.

Acute Radiation Syndrome (ARS) is a syndrome involving damage to multiple organs caused by exposure to a high dose of ionizing radiation over a short period of time; even low doses of radiation damage the radiosensitive hematopoietic system and causes H-ARS. PLacenta eXpanded (PLX)-R18 is a 3D-expanded placenta-derived stromal cell product designated for the treatment of hematological disorders. These cells have been shown in vitro to secrete hematopoietic proteins, to stimulate colony formation, and to induce bone marrow migration. Previous studies in mice showed that PLX-R18 cells responded to radiation-induced hematopoietic failure by transiently secreting hematopoiesis related proteins to enhance reconstitution of the hematopoietic system. We assessed the potential effect of prophylactic PLX-R18 treatment on H-ARS. PLX-R18 cells were administered intramuscularly to C57BL/6 mice, −1 and 3 days after (LD70/30) total body irradiation. PLX R18 treatment significantly increased survival after irradiation (p < 0.0005). In addition, peripheral blood and bone marrow (BM) cellularity were monitored at several time points up to 30 days. PLX-R18 treatment significantly increased the number of colony-forming hematopoietic progenitors in the femoral BM and significantly raised peripheral blood cellularity. PLX-R18 administration attenuated biomarkers of bone marrow aplasia (EPO, FLT3L), sepsis (SAA), and systemic inflammation (sP-selectin and E-selectin) and attenuated radiation-induced inflammatory cytokines/chemokines and growth factors, including G-CSF, MIP-1a, MIP-1b, IL-2, IL-6 and MCP-1, In addition, PLX-R18 also ameliorated radiation-induced upregulation of pAKT. Taken together, prophylactic PLX-R18 administration may serve as a protection measure, mitigating bone marrow failure symptoms and systemic inflammation in the H-ARS model.

Mitigation of total body irradiation-induced mortality and hematopoietic injury of mice by a thrombopoietin mimetic (JNJ-26366821).

The threat of a nuclear attack has increased in recent years highlighting the benefit of developing additional therapies for the treatment of victims suffering from Acute Radiation Syndrome (ARS). In this work, we evaluated the impact of a PEGylated thrombopoietin mimetic peptide, JNJ-26366821, on the mortality and hematopoietic effects associated with ARS in mice exposed to lethal doses of total body irradiation (TBI). JNJ-26366821 was efficacious as a mitigator of mortality and thrombocytopenia associated with ARS in both CD2F1 and C57BL/6 mice exposed to TBI from a cobalt-60 gamma-ray source. Single administration of doses ranging from 0.3 to 1 mg/kg, given 4, 8, 12 or 24 h post-TBI (LD70 dose) increased survival by 30-90% as compared to saline control treatment. At the conclusion of the 30-day study, significant increases in bone marrow colony forming units and megakaryocytes were observed in animals administered JNJ-26366821 compared to those administered saline. In addition, enhanced recovery of FLT3-L levels was observed in JNJ-26366821-treated animals. Probit analysis of survival in the JNJ-26366821- and saline-treated cohorts revealed a dose reduction factor of 1.113 and significant increases in survival for up to 6 months following irradiation. These results support the potential use of JNJ-26366821 as a medical countermeasure for treatment of acute TBI exposure in case of a radiological/nuclear event when administered from 4 to 24 h post-TBI.

Biophysical investigation of the iron in Aft1-1(up) and Gal-YAH1 Saccharomyces cerevisiae.

Aft1p is a major iron regulator in budding yeast Saccharomyces cerevisiae. It indirectly senses cytosolic Fe status and responds by activating or repressing iron regulon genes. Aft1p within the Aft1-1(up) strain has a single amino acid mutation which causes it to constitutively activate iron regulon genes regardless of cellular Fe status. This leads to elevated Fe uptake under both low and high Fe growth conditions. Ferredoxin Yah1p is involved in Fe/S cluster assembly, and Aft1p-targeted iron regulon genes are also upregulated in Yah1p-depleted cells. In this study Mössbauer, EPR, and UV-vis spectroscopies were used to characterize the Fe distribution in Aft1-1(up) and Yah1p-depleted cells. Aft1-1(up) cells grown in low Fe medium contained more Fe than did WT cells. A basal level of Fe in both WT and Aft1-1(up) cells was located in mitochondria, primarily in the form of Fe/S clusters and heme centers. The additional Fe in Aft1-1(up) cells was present as mononuclear HS Fe(III) species. These species are in a nonmitochondrial location, assumed here to be vacuolar. Aft1-1(up) cells grown in high Fe medium contained far more Fe than found in WT cells. The extra Fe was present as HS Fe(III) ions, probably stored in vacuoles, and as Fe(III) phosphate nanoparticles, located in mitochondria. Yah1p-deficent cells also accumulated nanoparticles in their mitochondria, but they did not contain HS Fe(III) species. Results are interpreted by a proposed model involving three homeostatic regulatory systems, including the Aft1 system, a vacuolar iron regulatory system, and a mitochondrial Fe regulatory system.

Mössbauer and EPR study of iron in vacuoles from fermenting Saccharomyces cerevisiae.

Vacuoles were isolated from fermenting yeast cells grown on minimal medium supplemented with 40 µM (57)Fe. Absolute concentrations of Fe, Cu, Zn, Mn, Ca, and P in isolated vacuoles were determined by ICP-MS. Mössbauer spectra of isolated vacuoles were dominated by two spectral features: a mononuclear magnetically isolated high-spin (HS) Fe(III) species coordinated primarily by hard/ionic (mostly or exclusively oxygen) ligands and superparamagnetic Fe(III) oxyhydroxo nanoparticles. EPR spectra of isolated vacuoles exhibited a g(ave) ~ 4.3 signal typical of HS Fe(III) with E/D ~ 1/3. Chemical reduction of the HS Fe(III) species was possible, affording a Mössbauer quadrupole doublet with parameters consistent with O/N ligation. Vacuolar spectral features were present in whole fermenting yeast cells; however, quantitative comparisons indicated that Fe leaches out of vacuoles during isolation. The in vivo vacuolar Fe concentration was estimated to be ~1.2 mM while the Fe concentration of isolated vacuoles was ~220 µM. Mössbauer analysis of Fe(III) polyphosphate exhibited properties similar to those of vacuolar Fe. At the vacuolar pH of 5, Fe(III) polyphosphate was magnetically isolated, while at pH 7, it formed nanoparticles. This pH-dependent conversion was reversible. Fe(III) polyphosphate could also be reduced to the Fe(II) state, affording similar Mössbauer parameters to that of reduced vacuolar Fe. These results are insufficient to identify the exact coordination environment of the Fe(III) species in vacuoles, but they suggest a complex closely related to Fe(III) polyphosphate. A model for Fe trafficking into/out of yeast vacuoles is proposed.

Catalytic mechanism and three-dimensional structure of adenine deaminase.

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k(cat) = 2.0 s(-1); k(cat)/K(m) = 2.5 × 10(3) M(-1) s(-1)). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn(2+) prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k(cat) and k(cat)/K(m) values of 200 s(-1) and 5 × 10(5) M(-1) s(-1), respectively. The apoenzyme was prepared and reconstituted with Fe(2+), Zn(2+), or Mn(2+). In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe(II)/Fe(II)]-ADE was oxidized to [Fe(III)/Fe(III)]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe(III)/Fe(III)]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Mössbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 Å resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps.

Investigating the Multifaceted Nature of Radiation-Induced Coagulopathies in a Göttingen Minipig Model of Hematopoietic Acute Radiation Syndrome.

Coagulopathies are well documented after acute radiation exposure at hematopoietic doses, and radiation-induced bleeding is notably one of the two main causes of mortality in the hematopoietic acute radiation syndrome. Despite this, understanding of the mechanisms by which radiation alters hemostasis and induces bleeding is still lacking. Here, male Göttingen minipigs received hematopoietic doses of 60Co gamma irradiation (total body) and coagulopathies were characterized by assessing bleeding, blood cytopenia, fibrin deposition, changes in hemostatic properties, coagulant/anticoagulant enzyme levels, and markers of inflammation, endothelial dysfunction, and barrier integrity to understand if a relationship exists between bleeding, hemostatic defects, bone marrow aplasia, inflammation, endothelial dysfunction and loss of barrier integrity. Acute radiation exposure induced coagulopathies in the Göttingen minipig model of hematopoietic acute radiation syndrome; instances of bleeding were not dependent upon thrombocytopenia. Neutropenia, alterations in hemostatic parameters and damage to the glycocalyx occurred in all animals irrespective of occurrence of bleeding. Radiation-induced bleeding was concurrent with simultaneous thrombocytopenia, anemia, neutropenia, inflammation, increased heart rate, decreased nitric oxide bioavailability and endothelial dysfunction; bleeding was not observed with the sole occurrence of a single aforementioned parameter in the absence of the others. Alteration of barrier function or clotting proteins was not observed in all cases of bleeding. Additionally, fibrin deposition was observed in the heart and lungs of decedent animals but no evidence of DIC was noted, suggesting a unique pathophysiology of radiation-induced coagulopathies. These findings suggest radiation-induced coagulopathies are the result of simultaneous damage to several key organs and biological functions, including the immune system, the inflammatory response, the bone marrow and the cardiovasculature.