Determination of sample sizes for demonstrating efficacy of radiation countermeasures.

In response to the ever increasing threat of radiological and nuclear terrorism, active development of nontoxic new drugs and other countermeasures to protect against and/or mitigate adverse health effects of radiation is ongoing. Although the classical LD(50) study used for many decades as a first step in preclinical toxicity testing of new drugs has been largely replaced by experiments that use fewer animals, the need to evaluate the radioprotective efficacy of new drugs necessitates the conduct of traditional LD(50) comparative studies (FDA, 2002, Federal Register 67, 37988-37998). There is, however, no readily available method to determine the number of animals needed for establishing efficacy in these comparative potency studies. This article presents a sample-size formula based on Student's t for comparative potency testing. It is motivated by the U.S. Food and Drug Administration's (FDA's) requirements for robust efficacy data in the testing of response modifiers in total body irradiation experiments where human studies are not ethical or feasible. Monte Carlo simulation demonstrated the formula's performance for Student's t, Wald, and likelihood ratio tests in both logistic and probit models. Importantly, the results showed clear potential for justifying the use of substantially fewer animals than are customarily used in these studies. The present article may thus initiate a dialogue among researchers who use animals for radioprotection survival studies, institutional animal care and use committees, and drug regulatory bodies to reach a consensus on the number of animals needed to achieve statistically robust results for demonstrating efficacy of radioprotective drugs.

gamma-Tocotrienol ameliorates intestinal radiation injury and reduces vascular oxidative stress after total-body irradiation by an HMG-CoA reductase-dependent mechanism.

Analogs of vitamin E (tocols) are under development as radioprophylactic agents because of their high efficacy and lack of toxicity. Gamma-tocotrienol (GT3) is of particular interest because, in addition to being an antioxidant, it also inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and accumulates to greater extent in endothelial cells than other tocols. We addressed in vivo whether HMG-CoA reductase inhibition contributes to the radioprotection conferred by GT3. Groups of mice were treated with vehicle, mevalonate (the product of the reaction catalyzed by HMG-CoA reductase), GT3 alone or GT3 in combination with mevalonate. Lethality and standard parameters of injury to the hematopoietic, intestinal and vascular/endothelial systems were assessed after exposure to total-body irradiation. GT3 improved postirradiation survival and decreased radiation-induced vascular oxidative stress, an effect that was reversible by mevalonate. GT3 also enhanced hematopoietic recovery, reduced intestinal radiation injury, and accelerated the recovery of soluble markers of endothelial function. These parameters were not reversed by mevalonate co-administration. Our data confirm GT3's radioprophylactic properties against hematopoietic injury and, for the first time, demonstrate benefits in terms of protection against gastrointestinal and vascular injury. The radioprotective efficacy of GT3 against vascular injury is related to its properties as an HMG-CoA reductase inhibitor.

Radiation protection by a new chemical entity, Ex-Rad: efficacy and mechanisms.

Ex-Rad is among a series of small molecule kinase inhibitors developed for modifying cell cycle distribution patterns in cancer cells subjected to radiation therapy, and it has been identified as a potential candidate for radiation protection studies. We have investigated its radioprotective efficacy using mouse and in vitro models. Thirty-day survival studies with C3H/HeN male mice revealed 88% survival when 500 mg/kg of Ex-Rad was injected subcutaneously 24 h and 15 min before gamma irradiation with 8.0 Gy. To understand Ex-Rad's mechanism of action, we also studied its radioprotective efficacy in lung fibroblast (HFL-1), skin fibroblast (AG1522) and human umbilical vein endothelial cells (HUVECs). Colony-forming assays indicated that Ex-Rad protected cells from radiation damage after exposure to (60)Co gamma radiation. A study using single-cell gel electrophoresis (SCGE; also known as the alkaline comet assay) showed that Ex-Rad protected cells from radiation-induced DNA damage. Western blot analyses indicated that the radiation protection provided by Ex-Rad resulted in reduced levels of pro-apoptosis proteins such as p53 as well as its downstream regulators p21, Bax, c-Abl and p73, indicating that Ex-Rad could rescue cells from ionizing radiation-induced p53-dependent apoptosis. In conclusion, it appears that Ex-Rad's radioprotective mechanisms involve prevention of p53-dependent and independent radiation-induced apoptosis.

Gamma-tocotrienol, a tocol antioxidant as a potent radioprotector.

PURPOSE: To assess the radioprotective potential of gamma-tocotrienol. MATERIALS AND METHODS: To optimise its dose and time regimen, gamma-tocotrienol (GT3) was injected subcutaneously (SC) at different doses into male CD2F1 mice [LD(50/30) (lethal radiation dose that results in the mortality of 50% mice in 30 days) radiation dose of 8.6 Gy with vehicle]. The mice were given 10.5, 11 and 11.5 Gy cobalt-60 radiation, and 30-day survival-protection was determined. Time optimisation was done by SC administration of GT3 at different intervals before irradiation. Dose reduction factor (DRF) was determined by probit analysis using mortality as the end point at six radiation doses. Protection from radiation induced pancytopenia was determined by enumerating peripheral blood cells from mice given GT3 and irradiated at 7 Gy. RESULTS: At an optimal dose of 200 mg/kg given SC 24 h before irradiation, GT3 had a DRF of 1.29. GT3 accelerated the recovery of total white blood cells, neutrophils, monocytes, platelets, and reticulocytes in irradiated mice, compared to vehicle-injected, irradiated controls. CONCLUSION: GT3 is a radioprotectant having a higher DRF than any other tocols. The protection it provides close to the gastro-intestinal range indicate that GT3 can be considered as an ideal radioprotectant meriting further drug development stages for the ultimate use in humans.

Preferential radiation sensitization of prostate cancer in nude mice by nutraceutical antioxidant gamma-tocotrienol.

Gamma-tocotrienol (GT) is a member of the vitamin E family. Our preliminary studies indicated that it protected mice from lethal irradiation, so we hypothesized that GT might be a radiation sensitizing agent for tumors. To test this, we induced prostate tumors by injecting PC3 cells into nude BALB/c mice. When the tumors were about 5 mm in diameter, mice were injected subcutaneously with 400 mg/kg gamma-tocotrienol and irradiated 24 h later at the site of the tumor with a dose of 12 Gy (60)Cobalt. Tumor size was monitored for 24 days after radiation. Tumor tissues as well as normal tissues like rectum, kidney, and liver were monitored for lipid peroxidation on day 4 and day 24 after radiation. The results indicated that the size of the tumors was reduced by almost 40%, but only in GT-treated and irradiated mice. In unstimulated and Fe-stimulated lipid peroxidation groups, lipid peroxidation in the tumors from irradiated mice increased to 135% and 150%, respectively, four days after irradiation and 33% and 66% in the same groups, respectively, 24 days after irradiation. In general, lipid peroxidation in the rectum did not increase in GT-treated and irradiated mice, although there was a slight increase in Fe-stimulated lipid peroxidation (29%) four days after irradiation. Unexpectedly, the kidneys were as equally sensitized to lipid peroxidation as the tumors. Liver tissue was protected in the short-term from radiation-induced lipid peroxidation. These studies indicate that the radiotherapy efficacy of prostate cancer can be increased with GT and a pro-oxidant if the kidneys can be shielded.

Radioprotective Efficacy of Gamma-Tocotrienol in Nonhuman Primates.

The search for treatments to counter potentially lethal radiation-induced injury over the past several decades has led to the development of multiple classes of radiation countermeasures. However, to date only granulocyte colony-stimulating factor (G-CSF; filgrastim, Neupogen)and pegylated G-CSF (pegfilgrastim, Neulasta) have been approved by the United States Food and Drug Administration (FDA) for the treatment of hematopoietic acute radiation syndrome (ARS). Gamma-tocotrienol (GT3) has demonstrated strong radioprotective efficacy in the mouse model, indicating the need for further evaluation in a large animal model. In this study, we evaluated GT3 pharmacokinetics (PK) and efficacy at different doses of cobalt-60 gamma radiation (0.6 Gy/min) using the nonhuman primate (NHP) model. The PK results demonstrated increased area under the curve with increasing drug dose and half-life of GT3. GT3 treatment resulted in reduced group mean neutropenia by 3-5 days and thrombocytopenia by 1-5 days. At 5.8 and 6.5 Gy total-body irradiation, GT3 treatment completely prevented thrombocytopenia. The capability of GT3 to reduce severity and duration of neutropenia and thrombocytopenia was dose dependent; 75 mg/kg treatment was more effective than 37.5 mg/kg treatment after a 5.8 Gy dose. However, the higher GT3 dose (75 mg/kg) was associated with higher frequency of adverse skin effects (small abscess) at the injection site. GT3 treatment of irradiated NHPs caused no significant difference in animal survival at 60 days postirradiation, however, low mortality was observed in irradiated, vehicle-treated groups as well. The data from this pilot study further elucidate the role and pharmacokinetics of GT3 in hematopoietic recovery after irradiation in a NHP model, and demonstrate the potential of GT3 as a promising radioprotector.

High-dose antibiotic therapy is superior to a 3-drug combination of prostanoids and lipid A derivative in protecting irradiated canines.

There is an urgent need to develop non-toxic radioprotectors. We tested the efficacy of a 3-drug combination (3-DC) of iloprost, misoprostol, and 3D-MPL (3-deacylated monophosphoryl lipid A) and the effects of postirradiation clinical support with high doses of antibiotics and blood transfusion. Canines were given 3-DC or the vehicle and exposed to 3.4 Gy or 4.1 Gy of 60Co radiation. Canines irradiated at 4.1 Gy were also given clinical support, which consisted of blood transfusion and antibiotics (gentamicin, and cefoxitin or cephalexin). Peripheral blood cell profile and 60-day survival were used as indices of protection. At 3.4 Gy, 3-DC- or vehicle-treated canines without postirradiation clinical support survived only for 10 to 12 days. Fifty percent of the canines treated with 3-DC or vehicle and provided postirradiation clinical support survived 4.1-Gy irradiation. Survival of canines treated with vehicle before irradiation significantly correlated with postirradiation antibiotic treatments, but not with blood transfusion. The recovery profile of peripheral blood cells in 4.1 Gy-irradiated canines treated with vehicle and antibiotics was better than drug-treated canines. These results indicate that therapy with high doses of intramuscular aminoglycoside antibiotic (gentamicin) and an oral cephalosporin (cephalexin) enhanced survival of irradiated canines. Although blood transfusion correlated with survival of 3-DC treated canines, there were no additional survivors with 3-DC treated canines than the controls.

Determination of WR-1065 and WR-33278 by liquid chromatography with electrochemical detection.

A liquid chromatographic method using electrochemical detection is presented for measuring the thiol WR-1065 12-(3-aminopropylamino) ethanethiol] and its symmetrical disulfide WR-33278 [NH2(CH2)3NHCH2CH2S]2. WR- 1065 is the active, radioprotective drug derived from the phosphorothioate pro-drug WR-2721 (amifostine). External standard curves for both compounds were linear over the range of 40-200 pmol injected (r2 = 0.999 and 0.996 for the thiol and disulfide, respectively). The detection and quantitation limits for WR-1065 were 9 and 18 pmol, respectively, whereas the corresponding values for WR-33278 were 30 and 59 pmol, respectively. Within- and between-day determinations of measurement Vision and accuracy for both compounds validated the suitability of this assay method.

Nutritional approaches to radioprotection: vitamin E.

Low-level radiation injury is dependent on the radiation dose and dose rate. The major military use of any potential radioprotectant is to prevent the short-term effects of lethality and the long-term effects of cancer and other pathologies from radiation exposure that may occur in a nuclear battlefield or in a nuclear material contaminated field of operation. Therefore, a radioprotectant should not affect the ability of military personnel to perform tasks. Because exposure to ionizing radiation induces free radical species, effective antioxidants, either alone or in combination with other agents, can be used as potential radioprotectors. To test this hypothesis, we studied vitamin E for its radioprotective efficacy. Using CD2F1 male mice as the model system, we observed that vitamin E at a dose of 400 IU/kg acts as a good radioprotectant against lethal doses of cobalt-60 radiation. Vitamin E was more efficacious when given subcutaneously than when given orally.

Protection against gamma-irradiation with 5-androstenediol.

We showed previously that treatment of gamma-irradiated female B6D2F1 mice with 5-androstenediol (AED) enhanced survival, stimulated myelopoiesis, and ameliorated radiation-induced decreases in circulating neutrophils and platelets. We have now tested survival in male CD2F1 mice, and we have investigated molecular and functional effects on neutrophils and bone marrow stromal cells and screened for toxicity in female B6D2F1 mice. AED (160 mg/kg, subcutaneously, 24 hours before irradiation) enhanced survival in male CD2F1 mice with a dose-reduction factor of 1.23, similar to the dose-reduction factor of 1.26 found previously for female B6D2F1 mice. Expression of CD11b, a developmental marker, was reduced on circulating neutrophils after either in vivo AED administration or whole-body gamma-irradiation (3 Gy), but neutrophil peroxidase activity was unchanged. Stromal cell progenitors (fibroblastoid colony-forming units) were reduced in marrow 5 days after AED injection in nonirradiated mice. Clinical chemistry, histopathology, and behavioral assays showed no evidence of toxicity. We conclude that AED and related steroids are attractive candidates to explore as countermeasures to high- and low-level ionizing radiation.

Mobilization of progenitor cells into peripheral blood by gamma-tocotrienol: a promising radiation countermeasure.

Gamma-tocotrienol (GT3), a vitamin E isoform, is shown to induce high levels of granulocyte colony stimulating factor (G-CSF) in mice. G-CSF is a key cytokine used for stimulation of hematopoiesis, and mobilization of hematopoietic stem and progenitor cells into peripheral blood. GT3 is also shown to induce vascular endothelial growth factor (VEGF), another important cytokine necessary for vasculogenesis and endothelial progenitor mobilization. Since GT3 induces both these cytokines, we tested whether GT3 mobilizes hematopoietic and endothelial progenitors in mice. GT3 (200mg/kg) was injected in 10-week-old CD2F1 mice and mobilization of progenitors in peripheral blood was analyzed at 24, 48, and 72 h post-administration. Circulating hematopoietic progenitor cells (HPCs, Lin(-), cKit(+)), endothelial progenitor cells (EPCs, Lin(-), CD34(+), Flk(+)), and stromal progenitor cells (SPCs, Lin(-), CD29(+), CD105(+)) in peripheral blood mononuclear cells (PBMCs) were analyzed simultaneously by flow cytometry. Mobilized HPCs, EPCs and SPCs in PBMC were also measured by colony-forming unit (CFU) assay in progenitor-specific media. Three groups of mice received vehicle, GT3 and GT3 plus AMD3100, a receptor antagonist used to enhance mobilization. GT3 induced significant mobilization of all three progenitor cell types compared to vehicle in peripheral blood; AMD3100 enhanced GT3-induced mobilization even further. Mobilization of progenitor cells in peripheral blood by GT3 indicates that GT3 can be used as an alternative to G-CSF and VGEF to mobilize HPCs and EPCs.

Synergistic radioprotection by gamma-tocotrienol and pentoxifylline: role of cAMP signaling.

Purpose. This study was designed to determine the efficacy and mechanisms of radioprotection by the combination of gamma-tocotrienol (GT3) and pentoxifylline (PTX) against acute radiation injury. Materials and Methods. Post-irradiation survival was monitored to determine the most efficacious dose and time of administration of PTX. Dose reduction factor (DRF) was calculated to compare the radioprotective efficacy of the combination. To determine the mechanism of synergistic radioprotection by the combination, mevalonate or calmodulin were coadministered with the GT3-PTX combination. Mevalonate was used to reverse the inhibitory effect of GT3 on 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and calmodulin was used to reverse the inhibition of phosphodiesterase (PDE) by PTX. Results. The combination was most effective when 200 mg/kg of PTX was administered 15 min before irradiation along with 200 mg/kg of GT3 (-24 h) and resulted in a DRF of 1.5. White blood cells and neutrophil counts showed accelerated recovery in GT3-PTX-treated groups compared to GT3. Mevalonate had no effect on the radioprotection of GT3-PTX; calmodulin abrogated the synergistic radioprotection by GT3-PTX. Conclusion. The mechanism of radioprotection by GT3-PTX may involve PDE inhibition.

Metabolomic changes in gastrointestinal tissues after whole body radiation in a murine model.

Exposure to ionizing radiation (IR) elicits a set of complex biological responses involving gene expression and protein turnover that ultimately manifest as dysregulation of metabolic processes representing the cellular phenotype. Although radiation biomarkers have been reported in urine and serum, they are not informative about IR mediated tissue or organ specific injury. In the present study we report IR induced metabolic changes in gastrointestinal (GI) tissue of CD2F1 mice using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry. Post-radiation GI injury is a critical determinant of survival after exposure to IR. Our results show a distinct dose and time dependent response to GI tissue injury.

Gamma-tocotrienol, a radiation prophylaxis agent, induces high levels of granulocyte colony-stimulating factor.

Gamma-tocotrienol (GT3), a promising radioprotectant, is shown to protect CD2F1 mice from radiation-induced neutropenia and thrombocytopenia when given 24h prior to total-body irradiation. GT3 also is shown to increase white blood cells (WBC) and absolute neutrophil counts (ANC) transiently in peripheral blood. We hypothesized that increases in WBC and ANC may involve stimulation of hematopoiesis possibly by cytokines and growth factors. To evaluate the effects of GT3 on hematopoietic system, we measured various cytokines, chemokines and growth factors by cytokine array and Bio-Plex assays. Both showed strong induction of various cytokines and chemokines. GT3 treatment resulted in significant increases in G-CSF, IL-1α, IL-1ß, IL-6, IL-12p70, IL-17, MIP-1α, and KC levels. G-CSF levels increased markedly within 12-24h after administration (5441 pg/ml in GT3-treated groups compared to 17 pg/ml in vehicle control). Most of these cytokine levels were elevated in the presence or absence of radiation. Time-course analysis of G-CSF and IL-6 induction showed that both cytokines were induced transiently after GT3 administration, and returned to normal levels by 48 h post-administration. For G-CSF, the peak was observed between 12 and 24h post-administration of GT3; however, the highest levels of IL-6 were obtained between 6 and 12h. These results demonstrate that GT3 induced high levels of G-CSF and other inflammatory cytokines and chemokines within 24h after administration. Survival studies reported showed that the most efficacious time for administering GT3 was 24h prior to irradiation, possibly because it induced key hematopoietic cytokines in that time window. These results also suggest a possible role of GT3-induced G-CSF stimulation in protecting mice from radiation-induced neutropenia and thrombocytopenia.

Amelioration of radiation-induced hematopoietic and gastrointestinal damage by Ex-RAD(R) in mice.

The aim of the present study was to assess recovery from hematopoietic and gastrointestinal damage by Ex-RAD(®), also known as ON01210.Na (4-carboxystyryl-4-chlorobenzylsulfone, sodium salt), after total body radiation. In our previous study, we reported that Ex-RAD, a small-molecule radioprotectant, enhances survival of mice exposed to gamma radiation, and prevents radiation-induced apoptosis as measured by the inhibition of radiation-induced protein 53 (p53) expression in cultured cells. We have expanded this study to determine best effective dose, dose-reduction factor (DRF), hematological and gastrointestinal protection, and in vivo inhibition of p53 signaling. A total of 500 mg/kg of Ex-RAD administered at 24 h and 15 min before radiation resulted in a DRF of 1.16. Ex-RAD ameliorated radiation-induced hematopoietic damage as monitored by the accelerated recovery of peripheral blood cells, and protection of granulocyte macrophage colony-forming units (GM-CFU) in bone marrow. Western blot analysis on spleen indicated that Ex-RAD treatment inhibited p53 phosphorylation. Ex-RAD treatment reduces terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay (TUNEL)-positive cells in jejunum compared with vehicle-treated mice after radiation injury. Finally, Ex-RAD preserved intestinal crypt cells compared with the vehicle control at 13 and 14 Gy. The results demonstrated that Ex-RAD ameliorates radiation-induced peripheral blood cell depletion, promotes bone marrow recovery, reduces p53 signaling in spleen and protects intestine from radiation injury.

Hemangiopericytoma - the need for a protocol-based treatment plan.

Hemangiopericytoma is a vascular tumor which comprises only 1% of all vascular tumors. The frequency of occurrence in the head and neck accounts for about 16-33% of all hemangiopericytomas. In this paper we discuss the surgical management, the difficulties in decision-making and treatment-planning in a case of a maxillary tumor in a five-year-old boy with a two-year follow-up. A five-year-old boy presented with a large unilateral maxillary tumor with nasal obstruction. Computed tomography revealed a heterogeneous mass completely occupying the right maxillary sinus and displacing the lateral wall of the nose and nasal septum. The lesion was diagnosed as hemangiopericytoma after histopathological confirmation. The option of surgical resection (total maxillectomy) was carried out after evaluating the available literature. Various treatment modalities like surgery, chemotherapy and radiotherapy were taken into consideration as the tumor has an aggressive nature. Due to the inadequate literature on definitive treatment options for these types of tumors, there was difficulty in arriving at a protocol-based treatment plan.

Reduction of radiation-induced vascular nitrosative stress by the vitamin E analog γ-tocotrienol: evidence of a role for tetrahydrobiopterin.

PURPOSE: The vitamin E analog γ-tocotrienol (GT3) is a powerful radioprotector. GT3 reduces postradiation vascular peroxynitrite production, an effect dependent on inhibition of hydroxy-methylglutaryl-coenzyme A reductase. Hydroxy-methylglutaryl-coenzyme A reductase inhibitors mediate their pleiotropic effects via endothelial nitric oxide synthase that requires the cofactor tetrahydrobiopterin (BH4). This study investigated the effects of radiation on BH4 bioavailability and of GT3 on BH4 metabolism. METHODS AND MATERIALS: Mice were exposed to 8.5 Gy of total body irradiation (TBI). Lung BH4 and total biopterin concentrations were measured 0, 3.5, 7, 14, and 21 days after TBI by use of differential oxidation followed by high-performance liquid chromatography. The effect of exogenous GT3 and BH4 treatment on postradiation vascular oxidative stress and bone marrow colony-forming units were assessed in vivo. The effect of GT3 on endothelial cell apoptosis and endothelial expression of guanosine triphosphate (GTP) cyclohydrolase 1 (GTPCH), GTPCH feedback regulatory protein (GFRP), GFRP transcription, GFRP protein levels, and GFRP-GTPCH protein binding was determined in vitro. RESULTS: Compared with baseline levels, lung BH4 concentrations decreased by 24% at 3.5 days after TBI, an effect that was reversed by GT3. At 14 and 21 days after TBI, compensatory increases in BH4 (58% and 80%, respectively) were observed. Relative to vehicle-treated controls, both GT3 and BH4 supplementation reduced postirradiation vascular peroxynitrite production at 3.5 days (by 66% and 33%, respectively), and BH4 resulted in a 68% increase in bone marrow colony-forming units. GT3 ameliorated endothelial cell apoptosis and reduced endothelial GFRP protein levels and GFRP-GTPCH binding by decreasing transcription of the GFRP gene. CONCLUSIONS: BH4 bioavailability is reduced in the early postradiation phase. Exogenous administration of BH4 reduces postirradiation vascular oxidative stress. GT3 potently reduces the expression of GFRP, one of the key regulatory proteins in the BH4 pathway, and may thus exert some of its beneficial effects on postradiation free radical production partly by counteracting the decrease in BH4.

Pentoxifylline enhances the radioprotective properties of γ-tocotrienol: differential effects on the hematopoietic, gastrointestinal and vascular systems.

The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3+PTX before being exposed to 8.5-13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3+PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3+PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3+PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3+PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.

Hematological targets of radiation damage.

Radiation-induced myelosuppression remains a rate-limiting factor of radiotherapy and chemotherapy. Therefore, hematological targets of radiation damage are of great significance for radiation oncology and normal tissue injury and protection. Protection of hematopoietic stem and progenitor cells is pivotal. In order to develop therapeutic targets, it is necessary to understand the mechanisms of stem cell renewal and differentiation. Recent advances in the molecular pathology of hematopoietic stem cells indicate a fine balance between various extrinsic and intrinsic signaling pathways in preserving the self-renewal and proliferative capacity of stem cells. Extrinsic signaling involves a microenvironment niche factors such as neighboring stromal cells, osteoblasts, and adipocytes secreting cytokines, chemokines, and metalloproteinases; intrinsic regulation involves Wnt/hedgehog/Notch signaling, DNA damage-induced epigenetic alterations, telomere shortening, and early senescence. Various drugs including synthetic cytokine mimetics, cytokine stimulators, and DNA repair modulators are being tested as radioprotectants. Colony-stimulating factors are routinely used in clinics to treat neutropenia induced by chemotherapy and radiotherapy as well as to mobilize and expand progenitors used in autologous transplantation. However, toxicity has limited their use. The vitamin E isoforms gamma tocotrienol, a potent free radical scavenger that has displayed promising anticarcinogenic properties, was recently shown to protect bone marrow (BM) from radiation injury and to stimulate hematopoiesis in a murine model. This chapter focuses on the potential targets of radiation damage in BM and speculates on the mechanisms of protection by γ-tocotrienol and how these mechanisms can contribute to radioprotection in general and to protection of BM during chemotherapy and radiotherapy in particular.

Novel strategies to ameliorate radiation injury: a possible role for tetrahydrobiopterin.

Novel pharmacological strategies are urgently needed to prevent or reduce radiation-induced tissue injury. Microvascular injury is a prominent feature of both early and delayed radiation injury. Radiation-induced endothelial dysfunction is believed to play a key role in the pathogenesis of post-irradiation tissue injury. Hence, strategies that could prevent or improve endothelial malfunction are expected to ameliorate the severity of radiation injury. This review focuses on the therapeutic potential of the nitric oxide synthase (NOS) cofactor 5,6,7,8-tetrahydrobiopterin (BH4) as an agent to reduce radiation toxicity. BH4 is an essential cofactor for all NOS enzymes and a critical determinant of NOS function. Inadequate availability of BH4 leads to uncoupling of the NOS enzyme. In an uncoupled state, NOS produces the highly oxidative radicals superoxide and peroxynitrite at the cost of NO. Under conditions of oxidative stress, such as after radiation exposure, BH4 availability might be reduced due to the rapid oxidation of BH4 to 7,8-dihydrobiopterin (7,8-BH2). As a result, free radical-induced BH4 insufficiency may increase the oxidative burden and hamper NO-dependent endothelial function. Given the growing evidence that BH4 depletion and subsequent endothelial NOS uncoupling play a major role in the pathogenesis of endothelial dysfunction in various diseases, there is substantial reason to believe that improving post-irradiation BH4 availability, by either supplementation with it or modulation of its metabolism, might be a novel strategy to reduce radiation-induced endothelial dysfunction and subsequent tissue injury.