Circulating IL-18 Binding Protein (IL-18BP) and IL-18 as Dual Biomarkers of Total-Body Irradiation in Mice.

We have previously reported that circulating interleukin-18 (IL-18) can be used as a radiation biomarker in mice, minipigs and nonhuman primates. In this study, we further determined the serum levels of IL-18 binding protein (IL-18BP), a natural endogenous antagonist of IL-18, in CD2F1 mice 1-13 days after total-body gamma irradiation (TBI) with different doses (5-10 Gy). We compared the changes in blood lymphocyte, neutrophil and platelet counts as well as the activation of the proapoptotic executioner caspase-3 and caspase-7, and the expression of the inflammatory factor cyclooxygenase 2 (COX-2) in spleen cells, with the changes of IL-18BP and IL-18 in mouse serum. We also evaluated the significance, sensitivity and specificity of alterations in radiation-induced IL-18BP. IL-18 increased from day 1-13 after TBI in a dose-dependent manner that was paralleled with an increase in IL-18 receptor alpha (IL-18Rα) in irradiated mouse spleen cells. IL-18BP rapidly increased (25-63 fold) in mouse serum on day 1 after different doses of TBI. However, it returned to baseline within 3 days after 5-7 Gy doses and within 7 days after 8 Gy dose, and was unaltered thereafter. In contrast, high doses of radiation (9 and 10 Gy) significantly sustained a higher level of IL-18BP in mouse serum and later induced a second phase of increase in IL-18BP on day 9-13 after irradiation, which coincided with the onset of animal mortality. Consistent with this observation, highly activated caspase-3 and -7 in 8-10 Gy irradiated mouse spleen cells exhibited reduced or no activity 24 h after 5 Gy, although radiation induced an inflammatory response, as shown by COX-2 expression in all irradiated cells. Our data suggest that the radiation-induced differential elevation of IL-18 and IL-18BP in animal serum is a dynamic and discriminative indicator of the severity of injury after exposure to ionizing radiation. These findings support the inclusion of the dual biomarkers IL-18BP and IL-18 in the development of a multifactorial strategy for radiation dose and injury assessment.

Delta-tocotrienol suppresses radiation-induced microRNA-30 and protects mice and human CD34+ cells from radiation injury.

We reported that microRNA-30c (miR-30c) plays a key role in radiation-induced human cell damage through an apoptotic pathway. Herein we further evaluated radiation-induced miR-30 expression and mechanisms of delta-tocotrienol (DT3), a radiation countermeasure candidate, for regulating miR-30 in a mouse model and human hematopoietic CD34+ cells. CD2F1 mice were exposed to 0 (control) or 7-12.5 Gy total-body gamma-radiation, and CD34+ cells were irradiated with 0, 2 or 4 Gy of radiation. Single doses of DT3 (75 mg/kg, subcutaneous injection for mice or 2 µM for CD34+ cell culture) were administrated 24 h before irradiation and animal survival was monitored for 30 days. Mouse bone marrow (BM), jejunum, kidney, liver and serum as well as CD34+ cells were collected at 1, 4, 8, 24, 48 or 72 h after irradiation to determine apoptotic markers, pro-inflammatory cytokines interleukin (IL)-1ß and IL-6, miR-30, and stress response protein expression. Our results showed that radiation-induced IL-1ß release and cell damage are pathological states that lead to an early expression and secretion of miR-30b and miR-30c in mouse tissues and serum and in human CD34+ cells. DT3 suppressed IL-1ß and miR-30 expression, protected against radiation-induced apoptosis in mouse and human cells, and increased survival of irradiated mice. Furthermore, an anti-IL-1ß antibody downregulated radiation-induced NFκBp65 phosphorylation, inhibited miR-30 expression and protected CD34+ cells from radiation exposure. Knockdown of NFκBp65 by small interfering RNA (siRNA) significantly suppressed radiation-induced miR-30 expression in CD34+ cells. Our data suggest that DT3 protects human and mouse cells from radiation damage may through suppression of IL-1ß-induced NFκB/miR-30 signaling.

Circulating interleukin-18 as a biomarker of total-body radiation exposure in mice, minipigs, and nonhuman primates (NHP).

We aim to develop a rapid, easy-to-use, inexpensive and accurate radiation dose-assessment assay that tests easily obtained samples (e.g., blood) to triage and track radiological casualties, and to evaluate the radioprotective and therapeutic effects of radiation countermeasures. In the present study, we evaluated the interleukin (IL)-1 family of cytokines, IL-1ß, IL-18 and IL-33, as well as their secondary cytokines' expression and secretion in CD2F1 mouse bone marrow (BM), spleen, thymus and serum in response to γ-radiation from sublethal to lethal doses (5, 7, 8, 9, 10, or 12 Gy) at different time points using the enzyme-linked immune sorbent assay (ELISA), immunoblotting, and cytokine antibody array. Our data identified increases of IL-1ß, IL-18, and/or IL-33 in mouse thymus, spleen and BM cells after total-body irradiation (TBI). However, levels of these cytokines varied in different tissues. Interestingly, IL-18 but not IL-1ß or IL-33 increased significantly (2.5-24 fold) and stably in mouse serum from day 1 after TBI up to 13 days in a radiation dose-dependent manner. We further confirmed our finding in total-body γ-irradiated nonhuman primates (NHPs) and minipigs, and demonstrated that radiation significantly enhanced IL-18 in serum from NHPs 2-4 days post-irradiation and in minipig plasma 1-3 days post-irradiation. Finally, we compared circulating IL-18 with the well known hematological radiation biomarkers lymphocyte and neutrophil counts in blood of mouse, minipigs and NHPs and demonstrated close correlations between these biomarkers in response to radiation. Our results suggest that the elevated levels of circulating IL-18 after radiation proportionally reflect radiation dose and severity of radiation injury and may be used both as a potential biomarker for triage and also to track casualties after radiological accidents as well as for therapeutic radiation exposure.

Delta-tocotrienol protects mice from radiation-induced gastrointestinal injury.

We recently demonstrated that natural delta-tocotrienol (DT3) significantly enhanced survival in total-body irradiated (TBI) mice, and protected mouse bone marrow cells from radiation-induced damage through Erk activation-associated mTOR survival pathways. Here, we further evaluated the effects and mechanisms of DT3 on survival of radiation-induced mouse acute gastrointestinal syndrome. DT3 (75-100 mg/kg) or vehicle was administered as a single subcutaneous injection to CD2F1 mice 24 h before 10-12 Gy (60)Co total-body irradiation at a dose rate of 0.6 Gy/min and survival was monitored. In a separate group of mice, jejunum sections were stained with hematoxylin and eosin and the surviving crypts in irradiated mice were counted. Apoptosis in intestinal epithelial cells was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and bacterial translocation from gut to heart, spleen and liver in irradiated mice were evaluated. DT3 (75 mg/kg) significantly enhanced survival in mice that received 10, 10.5, 11 or 12 Gy TBI. Administration of DT3 protected intestinal tissue, decreased apoptotic cells in jejunum and inhibited gut bacterial translocation in irradiated mice. Furthermore, DT3 significantly inhibited radiation-induced production of pro-inflammatory factors interleukin-1ß and -6 and suppressed expression of protein tyrosine kinase 6 (PTK6), a stress-induced kinase that promotes apoptosis in mouse intestinal cells. Our data demonstrate that administration of DT3 protected mice from radiation-induced gastrointestinal system damage.

Genistein nanoparticles protect mouse hematopoietic system and prevent proinflammatory factors after gamma irradiation.

Previous studies demonstrated that genistein protects mice from radiation-induced bone marrow failure. To overcome genistein's extremely low water solubility, a nanoparticle suspension of genistein has been formulated for more rapid dissolution. In the current study, we evaluated the radioprotective effects of a nanoparticle formulation of genistein on survival and hematopoietic recovery in mice exposed to total-body gamma irradiation. A single intramuscular injection of a saline-based genistein nanosuspension (150 mg/kg) administered to CD2F1 mice 24 h before 9.25 Gy (60)Co radiation exposure resulted in a 30-day survival rate of 95% compared to 25% in vehicle-treated animals. In mice irradiated at 7 Gy, the genistein nanosuspension increased mouse bone marrow cellularity from approximately 2.9% (vehicle treated) to 28.3% on day 7 postirradiation. Flow cytometry analysis demonstrated decreased radiation-induced hematopoietic stem and progenitor cell (HSPC, Lineage(-)/cKit(+)) death from 77.0% (vehicle) to 43.9% (genistein nanosuspension) with a significant recovery of clonogenicity 7 days after irradiation. The genistein nanosuspension also attenuated the radiation-induced elevation of proinflammatory factors interleukin 1 beta (IL-1ß), IL-6 and cyclooxygenase-2 (COX-2) in mouse bone marrow and spleen, which may contribute to protecting HSPCs.

Micro-RNA30c negatively regulates REDD1 expression in human hematopoietic and osteoblast cells after gamma-irradiation.

We recently demonstrated that a novel cell stress response gene REDD1 protects human fetal osteoblast cell line (hFOB) cells from γ-radiation-induced premature senescence. Here we show that levels of endogenous REDD1 are very low in human hematopoietic progenitor CD34+ cells regardless of radiation, but highly expressed in differentiated hematopoietic cells (14 day cultured CD34+ cells) in response to radiation, which might be associated with radiation tolerance of the latter cells. To further understand the mechanisms of radiation-induced damage in different cells, microRNA (miRNA)-arrays were performed using purified miRNAs from CD34+ and hFOB cells before and post-irradiation and real-time reverse transcription (RT)-PCR was used to validate the expression profiles of miRNAs in the radiation-damaged cells. The results indicate that γ-radiation downregulated 16 miRNAs in CD34+ cells and 14 in hFOB cells. Radiation-induced upregulation was observed for 15 miRNAs in CD34+ cells and 18 miRNAs in hFOB cells. The profiles of radiation-induced miRNA expression were completely different in CD34+ vs. hFOB cells. Radiation up-regulated miRNA (miR)-30b, miR-30c and miR-30d in CD34+ cells, whereas it inhibited miR-30c expression in hFOB cells. Since miR-30 has potential target sites located in the 3'untranslated region (UTR) of the REDD1 gene and radiation regulated miR-30c expression in both CD34+ and hFOB cells, we further explored the effects of miR-30c on REDD1 expression using miR-30c inhibitor and precursor (pre-miR-30c). The results show that pre-miR-30c transfection suppressed REDD1 expression in 14 day cultured CD34+ cells and hFOB cells and resulted in hFOB cell death. In contrast, inhibition of miR-30c expression significantly enhanced clonogenicity in CD34+ cells. Our data suggest that CD34+ and hFOB cells have different miRNA expression patterns after irradiation and miR-30c plays a key role in radiation-induced cell damage which might be through regulation of REDD1 expression.

REDD1 protects osteoblast cells from gamma radiation-induced premature senescence.

Radiotherapy is commonly used for cancer treatment. However, it often results in side effects due to radiation damage in normal tissue, such as bone marrow (BM) failure. Adult hematopoietic stem and progenitor cells (HSPC) reside in BM next to the endosteal bone surface, which is lined primarily by hematopoietic niche osteoblastic cells. Osteoblasts are relatively more radiation-resistant than HSPCs, but the mechanisms are not well understood. In the present study, we demonstrated that the stress response gene REDD1 (regulated in development and DNA damage responses 1) was highly expressed in human osteoblast cell line (hFOB) cells after γ irradiation. Knockdown of REDD1 with siRNA resulted in a decrease in hFOB cell numbers, whereas transfection of PCMV6-AC-GFP-REDD1 plasmid DNA into hFOB cells inhibited mammalian target of rapamycin (mTOR) and p21 expression and protected these cells from radiation-induced premature senescence (PS). The PS in irradiated hFOB cells were characterized by significant inhibition of clonogenicity, activation of senescence biomarker SA-ß-gal, and the senescence-associated cytokine secretory phenotype (SASP) after 4 or 8 Gy irradiation. Immunoprecipitation assays demonstrated that the stress response proteins p53 and nuclear factor κ B (NFkB) interacted with REDD1 in hFOB cells. Knockdown of NFkB or p53 gene dramatically suppressed REDD1 protein expression in these cells, indicating that REDD1 was regulated by both factors. Our data demonstrated that REDD1 is a protective factor in radiation-induced osteoblast cell premature senescence.

Delta-tocotrienol protects mouse and human hematopoietic progenitors from gamma-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling.

BACKGROUND: Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments. DESIGN AND METHODS: CD2F1 mice and human hematopoietic progenitor CD34(+) cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay. RESULTS: δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage(-)/Sca-1(+)/ckit(+) stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34(+) cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34(+) cells. CONCLUSIONS: Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34(+) cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.

Diazoxide increases liver and kidney HSP25 and HSP70 after shock and stroke.

BACKGROUND: The compound, diazoxide (DZ), is known to induce preconditioning through its effect as a mitochondrial K(ATP) channel opener and succinate dehydrogenase inhibitor. Our team tested the hypothesis that pharmacological induction of ischemic preconditioning with DZ can offer cytoprotection and preserve vital tissues after hemorrhagic shock and stroke. MATERIALS AND METHODS: Sprague-Dawley male rats received an intraperitoneal injection of sterile saline or 5 mg/kg DZ in saline 24 h prior to 1 h of hemorrhagic shock, by approximately 40% total blood loss volume (Shock Study), or a permanent unilateral common carotid ligation just before shock (Stroke + Shock Study). While remaining under isoflurane anesthesia, animals then received 81 mL/kg intravenous sterile saline over the next 45 min for recovery and survived for another 24 h. RESULTS: When DZ was administered 24 h prior to shock, it significantly reduced hyperglycemia, which in vehicle-treated animals persisted after resuscitation. DZ also attenuated hyperlactatemia during the 1-h shock period. With more severe trauma from combined stroke and shock, DZ also decreased hyperlactatemia and hyperglycemia levels but the reduction was only significant for hyperglycemia. The expression levels of heat shock proteins 25 (HSP25) and 70 (HSP70) were used as biomarkers for response of the kidney and liver to DZ and combined stroke and shock. Compared to vehicle-treated animals, DZ-treated rats subjected to shock and stroke exhibited increased HSP25 and HSP70 in kidney and liver tissue. CONCLUSIONS: DZ-attenuated physiological indicators of metabolic stress following shock or combined shock and stroke and enhanced the up-regulation of cytoprotective heat shock protein expression.

Evaluation of gene expression measurements from commercial microarray platforms.

Multiple commercial microarrays for measuring genome-wide gene expression levels are currently available, including oligonucleotide and cDNA, single- and two-channel formats. This study reports on the results of gene expression measurements generated from identical RNA preparations that were obtained using three commercially available microarray platforms. RNA was collected from PANC-1 cells grown in serum-rich medium and at 24 h following the removal of serum. Three biological replicates were prepared for each condition, and three experimental replicates were produced for the first biological replicate. RNA was labeled and hybridized to microarrays from three major suppliers according to manufacturers' protocols, and gene expression measurements were obtained using each platform's standard software. For each platform, gene targets from a subset of 2009 common genes were compared. Correlations in gene expression levels and comparisons for significant gene expression changes in this subset were calculated, and showed considerable divergence across the different platforms, suggesting the need for establishing industrial manufacturing standards, and further independent and thorough validation of the technology.