Intestinal iron bio-accessibility changes by Lignin and the subsequent impact on cell metabolism and intestinal microbiome communities.

The detrimental effects of high concentrations of colonic iron have been linked to intestinal inflammation and microbial dysbiosis. Exploiting chelation against this luminal pool of iron may restore intestinal health and have beneficial impacts on microbial communities. This study aimed to explore whether lignin, a heterogenous polyphenolic dietary component, has iron-binding affinity and can sequester iron within the intestine and thus, potentially modulate the microbiome. Within in vitro cell-culture models, the treatment of RKO and Caco-2 cells with lignin almost abolished intracellular iron import (96% and 99% reduction of iron acquisition respectively) with corresponding changes in iron metabolism proteins (ferritin and transferrin receptor-1) and reductions in the labile-iron pool. In a Fe-59 supplemented murine model, intestinal iron absorption was significantly inhibited by 30% when lignin was co-administered compared to the control group with the residual iron lost in the faeces. The supplementation of lignin into a microbial bioreactor colonic model increased the solubilisation and bio-accessibility of iron present by 4.5-fold despite lignin-iron chelation previously restricting intracellular iron absorption in vitro and in vivo. The supplementation of lignin in the model increased the relative abundance of Bacteroides whilst levels of Proteobacteria decreased which could be attributed to the changes in iron bio-accessibility due to iron chelation. In summary, we demonstrate that lignin is an effective luminal iron chelator. Iron chelation leads to the limitation of intracellular iron import whilst, despite increasing iron solubility, favouring the growth of beneficial bacteria.

Alternate day versus daily oral iron for treatment of iron deficiency anemia: a randomized controlled trial.

Oral iron is the mainstay of treating iron deficiency anemia. Recent studies indicate better fractional iron absorption with alternate day supplementation. However, the optimal supplementation strategy is unclear. We compared effectiveness of daily versus alternate day supplementation of oral iron for treatment of iron deficiency anemia. This double blind, active control, randomized controlled trial was conducted on two hundred adults having hemoglobin 10 g/dL or less with microcytic hypochromic anemia and/or serum ferritin below 50 ng/mL. They were randomized to receive either two Ferrous sulfate tablets containing 60 mg elemental iron (120 mg total) on alternate days or single tablet of 60 mg elemental iron daily for 8 weeks. Primary outcome was mean change in hemoglobin at week 8 from baseline. Mean hemoglobin was 6.53 (± 1.89) and 6.68 (± 1.89) g/dL in the alternate day and daily arms respectively. Mean change in hemoglobin was + 1.05 ± 1.34 g/dL in alternate day arm and + 1.36 ± 1.51 g/dL in daily arm (p = 0.47) at week 8. There were no statistically significant differences between the arms with respect to any secondary outcome. There is no significant difference between alternate day and daily iron administration in improving hemoglobin. Randomized controlled trials enrolling more participants for longer periods of supplementation and evaluating clinically relevant outcomes like change in hemoglobin may be useful in identifying the ideal dosing strategy.Trial Registration: Clinical Trial Registry of India (CTRI/2019/01/017169).

Iron from nanostructured ferric phosphate: absorption and biodistribution in mice and bioavailability in iron deficient anemic women.

Food fortification with iron nanoparticles (NPs) could help prevent iron deficiency anemia, but the absorption pathway and biodistribution of iron-NPs and their bioavailability in humans is unclear. Dietary non-heme iron is physiologically absorbed via the divalent metal transporter-1 (DMT1) pathway. Using radio- iron isotope labelling in mice with a partial knockdown of intestine-specific DMT1, we assessed oral absorption and tissue biodistribution of nanostructured ferric phosphate (FePO4-NP; specific surface area [SSA] 98 m2g-1) compared to to ferrous sulfate (FeSO4), the reference compound. We show that absorption of iron from FePO4-NP appears to be largely DMT1 dependent and that its biodistribution after absorption is similar to that from FeSO4, without abnormal deposition of iron in the reticuloendothelial system. Furthermore, we demonstrate high bioavailability from iron NPs in iron deficient anemic women in a randomized, cross-over study using stable-isotope labelling: absorption and subsequent erythrocyte iron utilization from two 57Fe-labeled FePO4-NP with SSAs of 98 m2g-1 and 188 m2g-1 was 2.8-fold and 5.4-fold higher than from bulk FePO4 with an SSA of 25 m2g-1 (P < 0.001) when added to a rice and vegetable meal consumed by iron deficient anemic women. The FePO4-NP 188 m2g-1 achieved 72% relative bioavailability compared to FeSO4. These data suggest FePO4-NPs may be useful for nutritional applications.

Long-Term Sex- and Genotype-Specific Effects of 56Fe Irradiation on Wild-Type and APPswe/PS1dE9 Transgenic Mice.

Space radiation presents a substantial threat to travel beyond Earth. Relatively low doses of high-energy particle radiation cause physiological and behavioral impairments in rodents and may pose risks to human spaceflight. There is evidence that 56Fe irradiation, a significant component of space radiation, may be more harmful to males than to females and worsen Alzheimer's disease pathology in genetically vulnerable models. Yet, research on the long-term, sex- and genotype-specific effects of 56Fe irradiation is lacking. Here, we irradiated 4-month-old male and female, wild-type and Alzheimer's-like APP/PS1 mice with 0, 0.10, or 0.50 Gy of 56Fe ions (1GeV/u). Mice underwent microPET scans before and 7.5 months after irradiation, a battery of behavioral tests at 11 months of age and were sacrificed for pathological and biochemical analyses at 12 months of age. 56Fe irradiation worsened amyloid-beta (Aß) pathology, gliosis, neuroinflammation and spatial memory, but improved motor coordination, in male transgenic mice and worsened fear memory in wild-type males. Although sham-irradiated female APP/PS1 mice had more cerebral Aß and gliosis than sham-irradiated male transgenics, female mice of both genotypes were relatively spared from radiation effects 8 months later. These results provide evidence for sex-specific, long-term CNS effects of space radiation.

Iron bioavailability from bouillon fortified with a novel ferric phytate compound: a stable iron isotope study in healthy women (part II).

Bouillon cubes are widely consumed and when fortified with iron could contribute in preventing iron deficiency. We report the development (part I) and evaluation (current part II) of a novel ferric phytate compound to be used as iron fortificant in condiments such as bouillon. Ferric pyrophosphate (FePP), is the compound of choice due to its high stability in foods, but has a modest absorption in humans. Our objective was to assess iron bioavailability from a novel iron fortificant consisting of ferric iron complexed with phytic acid and hydrolyzed corn protein (Fe-PA-HCP), used in bouillon with and without an inhibitory food matrix. In a randomised single blind, cross-over study, we measured iron absorption in healthy adult women (n = 22). In vitro iron bioaccessibility was assessed using a Caco-2 cell model. Iron absorption from Fe-PA-HCP was 1.5% and 4.1% in bouillon with and without inhibitory matrix, respectively. Relative iron bioavailability to FeSO4 was 2.4 times higher than from FePP in bouillon (17% vs 7%) and 5.2 times higher when consumed with the inhibitory meal (41% vs 8%). Similar results were found in vitro. Fe-PA-HCP has a higher relative bioavailability versus FePP, especially when bouillon is served with an inhibitory food matrix.

Liver Iron Retention Estimated from Utilization of Oral and Intravenous Radioiron in Various Anemias and Hemochromatosis in Humans.

Patients with hereditary hemochromatosis and non-transfusion-dependent hereditary anemia develop predominantly liver iron-overload. We present a unique method allowing quantification of liver iron retention in humans during first-pass of 59Fe-labeled iron through the portal system, using standard ferrokinetic techniques measuring red cell iron uptake after oral and intravenous 59Fe administration. We present data from patients with iron deficiency (ID; N = 47), hereditary hemochromatosis (HH; N = 121) and non-transfusion-dependent hereditary anemia (HA; N = 40). Mean mucosal iron uptake and mucosal iron transfer (±SD) were elevated in patients with HH (59 ± 18%, 80 ± 15% respectively), HA (65 ± 17%, 74 ± 18%) and ID (84 ± 14%, 94 ± 6%) compared to healthy controls (43 ± 19%, 64 ± 18%) (p < 0.05) resulting in increased iron retention after 14 days compared to healthy controls in all groups (p < 0.01). The fraction of retained iron utilized for red cell production was 0.37 ± 0.17 in untreated HA, 0.55 ± 0.20 in untreated HH and 0.99 ± 0.22 in ID (p < 0.01). Interestingly, compared to red blood cell iron utilization after oral iron administration, red blood cell iron utilization was higher after injection of transferrin-bound iron in HA and HH. Liver iron retention was considerably higher in HH and HA compared to ID. We hypothesize that albumin serves as a scavenger of absorbed Fe(II) for delivering albumin-bound Fe(III) to hepatocytes.

The complex of ferric-enterobactin with its transporter from Pseudomonas aeruginosa suggests a two-site model.

Bacteria use small molecules called siderophores to scavenge iron. Siderophore-Fe3+ complexes are recognised by outer-membrane transporters and imported into the periplasm in a process dependent on the inner-membrane protein TonB. The siderophore enterobactin is secreted by members of the family Enterobacteriaceae, but many other bacteria including Pseudomonas species can use it. Here, we show that the Pseudomonas transporter PfeA recognises enterobactin using extracellular loops distant from the pore. The relevance of this site is supported by in vivo and in vitro analyses. We suggest there is a second binding site deeper inside the structure and propose that correlated changes in hydrogen bonds link binding-induced structural re-arrangements to the structural adjustment of the periplasmic TonB-binding motif.

Space-like 56Fe irradiation manifests mild, early sex-specific behavioral and neuropathological changes in wildtype and Alzheimer's-like transgenic mice.

Space travel will expose people to high-energy, heavy particle radiation, and the cognitive deficits induced by this exposure are not well understood. To investigate the short-term effects of space radiation, we irradiated 4-month-old Alzheimer's disease (AD)-like transgenic (Tg) mice and wildtype (WT) littermates with a single, whole-body dose of 10 or 50 cGy 56Fe ions (1 GeV/u) at Brookhaven National Laboratory. At ~1.5 months post irradiation, behavioural testing showed sex-, genotype-, and dose-dependent changes in locomotor activity, contextual fear conditioning, grip strength, and motor learning, mainly in Tg but not WT mice. There was little change in general health, depression, or anxiety. Two months post irradiation, microPET imaging of the stable binding of a translocator protein ligand suggested no radiation-specific change in neuroinflammation, although initial uptake was reduced in female mice independently of cerebral blood flow. Biochemical and immunohistochemical analyses revealed that radiation reduced cerebral amyloid-ß levels and microglia activation in female Tg mice, modestly increased microhemorrhages in 50 cGy irradiated male WT mice, and did not affect synaptic marker levels compared to sham controls. Taken together, we show specific short-term changes in neuropathology and behaviour induced by 56Fe irradiation, possibly having implications for long-term space travel.

Comparative study of core- and surface-radiolabeling strategies for the assembly of iron oxide nanoparticle-based theranostic nanocomposites.

This work highlights the superiority of the surface-radiolabeling strategy over the core-labeling strategy in the assembly of radioactive iron oxide nanoparticle (IONP)-based nanocomposites for use in multimodal imaging and targeted therapy. It also implies a possible overestimation of the labeling stability in previous studies and points out directions for further optimization.

Ascorbate and ferritin interactions: Consequences for iron release in vitro and in vivo and implications for inflammation.

This review discusses the chemical mechanisms of ascorbate-dependent reduction and solubilization of ferritin's ferric iron core and subsequent release of ferrous iron. The process is accelerated by low concentrations of Fe(II) that increase ferritin's intrinsic ascorbate oxidase activity, hence increasing the rate of ascorbate radical formation. These increased rates of ascorbate oxidation provide reducing equivalents (electrons) to ferritin's core and speed the core reduction rates with subsequent solubilization and release of Fe(II). Ascorbate-dependent solubilization of ferritin's iron core has consequences relating to the interpretation of 59Fe uptake sourced from 59Fe-lebelled holotransferrin into ferritin. Ascorbate-dependent reduction of the ferritin core iron solubility increases the size of ferritin's iron exchangeable pool and hence the rate and amount of exchange uptake of 59Fe into ferritin, whilst simultaneously increasing net iron release rate from ferritin. This may rationalize the inconsistency that ascorbate apparently stabilizes 59Fe ferritin and retards lysosomal ferritinolysis and whole cell 59Fe release, whilst paradoxically increasing the rate of net iron release from ferritin. This capacity of ascorbate and iron to synergise ferritin iron release has pathological significance, as it lowers the concentration at which ascorbate activates ferritin's iron release to within the physiological range (50-250 µM). These effects have relevance to inflammatory pathology and to the pro-oxidant effects of ascorbate in cancer therapy and cell death by ferroptosis.

Detection and quantitation of iron in ferritin, transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging.

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter 55Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox-active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000 Da) in the neonatal cardiomyocytes.

Space radiation triggers persistent stress response, increases senescent signaling, and decreases cell migration in mouse intestine.

Proliferative gastrointestinal (GI) tissue is radiation-sensitive, and heavy-ion space radiation with its high-linear energy transfer (high-LET) and higher damaging potential than low-LET γ-rays is predicted to compromise astronauts' GI function. However, much uncertainty remains in our understanding of how heavy ions affect coordinated epithelial cell migration and extrusion, which are essential for GI homeostasis. Here we show using mouse small intestine as a model and BrdU pulse labeling that cell migration along the crypt-villus axis is persistently decreased after a low dose of heavy-ion 56Fe radiation relative to control and γ-rays. Wnt/ß-catenin and its downstream EphrinB/EphB signaling are key to intestinal epithelial cell (IEC) proliferation and positioning during migration, and both are up-regulated after 56Fe radiation. Conversely, factors involved in cell polarity and adhesion and cell-extracellular matrix interactions were persistently down-regulated after 56Fe irradiation-potentially altering cytoskeletal remodeling and cell extrusion. 56Fe radiation triggered a time-dependent increase in γH2AX foci and senescent cells but without a noticeable increase in apoptosis. Some senescent cells acquired the senescence-associated secretory phenotype, and this was accompanied by increased IEC proliferation, implying a role for progrowth inflammatory factors. Collectively, this study demonstrates a unique phenomenon of heavy-ion radiation-induced persistently delayed IEC migration involving chronic sublethal genotoxic and oncogenic stress-induced altered cytoskeletal dynamics, which were seen even a year later. When considered along with changes in barrier function and nutrient absorption factors as well as increased intestinal tumorigenesis, our in vivo data raise a serious concern for long-duration deep-space manned missions.

Structure-Activity Relationships of Nitro-Substituted Aroylhydrazone Iron Chelators with Antioxidant and Antiproliferative Activities.

Aroylhydrazone iron chelators such as salicylaldehyde isonicotinoyl hydrazone (SIH) protect various cells against oxidative injury and display antineoplastic activities. Previous studies have shown that a nitro-substituted hydrazone, namely, NHAPI, displayed markedly improved plasma stability, selective antitumor activity, and moderate antioxidant properties. In this study, we prepared four series of novel NHAPI derivatives and explored their iron chelation activities, anti- or pro-oxidant effects, protection against model oxidative injury in the H9c2 cell line derived from rat embryonic cardiac myoblasts, cytotoxicities to the corresponding noncancerous H9c2 cells, and antiproliferative activities against the MCF-7 human breast adenocarcinoma and HL-60 human promyelocytic leukemia cell lines. Nitro substitution had both negative and positive effects on the examined properties, and we identified new structure-activity relationships. Naphthyl and biphenyl derivatives showed selective antiproliferative action, particularly in the breast adenocarcinoma MCF-7 cell line, where they exceeded the selectivity of the parent compound NHAPI. Of particular interest is a compound prepared from 2-hydroxy-5-methyl-3-nitroacetophenone and biphenyl-4-carbohydrazide, which protected cardiomyoblasts against oxidative injury at 1.8 ± 1.2 µM with 24-fold higher selectivity than SIH. These compounds will serve as leads for further structural optimization and mechanistic studies.

Heat-induced radiolabeling and fluorescence labeling of Feraheme nanoparticles for PET/SPECT imaging and flow cytometry.

Feraheme (FH) nanoparticles (NPs) have been used extensively for treatment of iron anemia (due to their slow release of ionic iron in acidic environments). In addition, injected FH NPs are internalized by monocytes and function as MRI biomarkers for the pathological accumulation of monocytes in disease. We have recently expanded these applications by radiolabeling FH NPs for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging using a heat-induced radiolabeling (HIR) strategy. Imaging FH NPs using PET/SPECT has important advantages over MRI due to lower iron doses and improved quantitation of tissue NP concentrations. HIR of FH NPs leaves the physical and biological properties of the NPs unchanged and allows researchers to build on the extensive knowledge obtained about the pharmacokinetic and safety aspects of FH NPs. In this protocol, we present the step-by-step procedures for heat (120 °C)-induced bonding of three widely employed radiocations (89Zr4+ or 64Cu2+ for PET, and 111In3+ for SPECT) to FH NPs using a chelateless radiocation surface adsorption (RSA) approach. In addition, we describe the conversion of FH carboxyl groups into amines and their reaction with an N-hydroxysuccinimide (NHS) of a Cy5.5 fluorophore. This yields Cy5.5-FH, a fluorescent FH that enables the cells internalizing Cy5.5-FH to be examined using flow cytometry. Finally, we describe procedures for in vivo and ex vivo uptake of Cy5.5-FH by monocytes and for in vivo microPET/CT imaging of HIR-FH NPs. Synthesis of HIR-FH requires experience with working with radioactive cations and can be completed within <4 h. Synthesis of Cy5.5-FH NPs takes ∼17 h.

A role for sex and a common HFE gene variant in brain iron uptake.

HFE (high iron) is an essential protein for regulating iron transport into cells. Mutations of the HFE gene result in loss of this regulation causing accumulation of iron within the cell. The mutated protein has been found increasingly in numerous neurodegenerative disorders in which increased levels of iron in the brain are reported. Additionally, evidence that these mutations are associated with elevated brain iron challenges the paradigm that the brain is protected by the blood-brain barrier. While much has been studied regarding the role of HFE in cellular iron uptake, it has remained unclear what role the protein plays in the transport of iron into the brain. We investigated regulation of iron transport into the brain using a mouse model with a mutation in the HFE gene. We demonstrated that the rate of radiolabeled iron (59Fe) uptake was similar between the two genotypes despite higher brain iron concentrations in the mutant. However, there were significant differences in iron uptake between males and females regardless of genotype. These data indicate that brain iron status is consistently maintained and tightly regulated at the level of the blood-brain barrier.

Simulated space radiation-induced mutants in the mouse kidney display widespread genomic change.

Exposure to a small number of high-energy heavy charged particles (HZE ions), as found in the deep space environment, could significantly affect astronaut health following prolonged periods of space travel if these ions induce mutations and related cancers. In this study, we used an in vivo mutagenesis assay to define the mutagenic effects of accelerated 56Fe ions (1 GeV/amu, 151 keV/µm) in the mouse kidney epithelium exposed to doses ranging from 0.25 to 2.0 Gy. These doses represent fluences ranging from 1 to 8 particle traversals per cell nucleus. The Aprt locus, located on chromosome 8, was used to select induced and spontaneous mutants. To fully define the mutagenic effects, we used multiple endpoints including mutant frequencies, mutation spectrum for chromosome 8, translocations involving chromosome 8, and mutations affecting non-selected chromosomes. The results demonstrate mutagenic effects that often affect multiple chromosomes for all Fe ion doses tested. For comparison with the most abundant sparsely ionizing particle found in space, we also examined the mutagenic effects of high-energy protons (1 GeV, 0.24 keV/µm) at 0.5 and 1.0 Gy. Similar doses of protons were not as mutagenic as Fe ions for many assays, though genomic effects were detected in Aprt mutants at these doses. Considered as a whole, the data demonstrate that Fe ions are highly mutagenic at the low doses and fluences of relevance to human spaceflight, and that cells with considerable genomic mutations are readily induced by these exposures and persist in the kidney epithelium. The level of genomic change produced by low fluence exposure to heavy ions is reminiscent of the extensive rearrangements seen in tumor genomes suggesting a potential initiation step in radiation carcinogenesis.

Cellular requirements for iron-sulfur cluster insertion into the antiviral radical SAM protein viperin.

Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and 55Fe-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. 55Fe-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for 55Fe/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.

Transport of Non-Transferrin Bound Iron to the Brain: Implications for Alzheimer's Disease.

A direct correlation between brain iron and Alzheimer's disease (AD) raises questions regarding the transport of non-transferrin-bound iron (NTBI), a toxic but less researched pool of circulating iron that is likely to increase due to pathological and/or iatrogenic systemic iron overload. Here, we compared the distribution of radiolabeled-NTBI (59Fe-NTBI) and transferrin-bound iron (59Fe-Tf) in mouse models of iron overload in the absence or presence of inflammation. Following a short pulse, most of the 59Fe-NTBI was taken up by the liver, followed by the kidney, pancreas, and heart. Notably, a strong signal of 59Fe-NTBI was detected in the brain ventricular system after 2 h, and the brain parenchyma after 24 h. 59Fe-Tf accumulated mainly in the femur and spleen, and was transported to the brain at a much slower rate than 59Fe-NTBI. In the kidney, 59Fe-NTBI was detected in the cortex after 2 h, and outer medulla after 24 hours. Most of the 59Fe-NTBI and 59Fe-Tf from the kidney was reabsorbed; negligible amount was excreted in the urine. Acute inflammation increased the uptake of 59Fe-NTBI by the kidney and brain from 2-24 hours. Chronic inflammation, on the other hand, resulted in sequestration of iron in the liver and kidney, reducing its transport to the brain. These observations provide direct evidence for the transport of NTBI to the brain, and reveal a complex interplay between inflammation and brain iron homeostasis. Further studies are necessary to determine whether transient increase in NTBI due to systemic iron overload is a risk factor for AD.

Neurochemical differences in learning and memory paradigms among rats supplemented with anthocyanin-rich blueberry diets and exposed to acute doses of 56Fe particles.

The protective effects of anthocyanin-rich blueberries (BB) on brain health are well documented and are particularly important under conditions of high oxidative stress, which can lead to "accelerated aging." One such scenario is exposure to space radiation, consisting of high-energy and -charge particles (HZE), which are known to cause cognitive dysfunction and deleterious neurochemical alterations. We recently tested the behavioral and neurochemical effects of acute exposure to HZE particles such as 56Fe, within 24-48h after exposure, and found that radiation primarily affects memory and not learning. Importantly, we observed that specific brain regions failed to upregulate antioxidant and anti-inflammatory mechanisms in response to this insult. To further examine these endogenous response mechanisms, we have supplemented young rats with diets rich in BB, which are known to contain high amounts of antioxidant-phytochemicals, prior to irradiation. Exposure to 56Fe caused significant neurochemical changes in hippocampus and frontal cortex, the two critical regions of the brain involved in cognitive function. BB supplementation significantly attenuated protein carbonylation, which was significantly increased by exposure to 56Fe in the hippocampus and frontal cortex. Moreover, BB supplementation significantly reduced radiation-induced elevations in NADPH-oxidoreductase-2 (NOX2) and cyclooxygenase-2 (COX-2), and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) in the hippocampus and frontal cortex. Overall results indicate that 56Fe particles may induce their toxic effects on hippocampus and frontal cortex by reactive oxygen species (ROS) overload, which can cause alterations in the neuronal environment, eventually leading to hippocampal neuronal death and subsequent impairment of cognitive function. Blueberry supplementation provides an effective preventative measure to reduce the ROS load on the CNS in an event of acute HZE exposure.

TonB-Dependent Heme/Hemoglobin Utilization by Caulobacter crescentus HutA.

Siderophore nutrition tests with Caulobacter crescentus strain NA1000 revealed that it utilized a variety of ferric hydroxamate siderophores, including asperchromes, ferrichromes, ferrichrome A, malonichrome, and ferric aerobactin, as well as hemin and hemoglobin. C. crescentus did not transport ferrioxamine B or ferric catecholates. Because it did not use ferric enterobactin, the catecholate aposiderophore was an effective agent for iron deprivation. We determined the kinetics and thermodynamics of [59Fe]apoferrichrome and 59Fe-citrate binding and transport by NA1000. Its affinity and uptake rate for ferrichrome (equilibrium dissociation constant [Kd ], 1 nM; Michaelis-Menten constant [KM ], 0.1 nM; Vmax, 19 pMol/109 cells/min) were similar to those of Escherichia coli FhuA. Transport properties for 59Fe-citrate were similar to those of E. coli FecA (KM , 5.3 nM; Vmax, 29 pMol/109 cells/min). Bioinformatic analyses implicated Fur-regulated loci 00028, 00138, 02277, and 03023 as TonB-dependent transporters (TBDT) that participate in iron acquisition. We resolved TBDT with elevated expression under high- or low-iron conditions by SDS-PAGE of sodium sarcosinate cell envelope extracts, excised bands of interest, and analyzed them by mass spectrometry. These data identified five TBDT: three were overexpressed during iron deficiency (00028, 02277, and 03023), and 2 were overexpressed during iron repletion (00210 and 01196). CLUSTALW analyses revealed homology of putative TBDT 02277 to Escherichia coli FepA and BtuB. A Δ02277 mutant did not transport hemin or hemoglobin in nutrition tests, leading us to designate the 02277 structural gene as hutA (for heme/hemoglobin utilization).IMPORTANCE The physiological roles of the 62 putative TBDT of C. crescentus are mostly unknown, as are their evolutionary relationships to TBDT of other bacteria. We biochemically studied the iron uptake systems of C. crescentus, identified potential iron transporters, and clarified the phylogenetic relationships among its numerous TBDT. Our findings identified the first outer membrane protein involved in iron acquisition by C. crescentus, its heme/hemoglobin transporter (HutA).