Delta-Tocotrienol Protects Mice from Radiation-Induced Gastrointestinal Injury.

Li XH, Ghosh SP, Ha CT, Fu D, Elliott TB, Bolduc DL, Villa V, Whitnall MH, Landauer MR, Xiao M.

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 60Co 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-1β 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.

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Gamma tocotrienol, a potent radioprotector, preferentially upregulates expression of anti-apoptotic genes to promote intestinal cell survival

Suman S, Datta K, Chakraborty K, Kulkarni SS, Doiron K, Fornace AJ Jr Sree Kumar K, Hauer-Jensen M, Ghosh SP.

Food Chem Toxicol. 2013 Aug 10

Summary

Gamma tocotrienol (GT3) has been reported as a potent ameliorator of radiation-induced gastrointestinal (GI) toxicity when administered prophylactically. This study aimed to evaluate the role of GT3 mediated pro- and anti-apoptotic gene regulation in protecting mice from radiation-induced GI damage.

Male 10- to 12-weeks-old CD2F1 mice were administered with a single dose of 200 mg/kg of GT3 or equal volume of vehicle (5% Tween-80) 24 h before exposure to 11 Gy of whole-body γ-radiation. Mouse jejunum was surgically removed 4 and 24 h after radiation exposure, and was used for PCR array, histology, immunohistochemistry, and immunoblot analysis.

Results were compared among vehicle pre-treated no radiation, vehicle pre-treated irradiated, and GT3 pre-treated irradiated groups. GT3 pretreated irradiated groups, both 4 h and 24 h after radiation, showed greater upregulation of anti-apoptotic gene expression than vehicle pretreated irradiated groups. TUNEL staining and intestinal crypt analysis showed protection of jejunum after GT3 pre-treatment and immunoblot results were supportive of PCR data.

Our study demonstrated that GT3-mediated protection of intestinal cells from a GI-toxic dose of radiation occurred via upregulation of antiapoptotic and downregulation of pro-apoptotic factors, both at the transcript as well as at the protein levels.

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Vitamin E: tocopherols and tocotrienols as potential radiation countermeasures.

Singh VK, Beattie LA, Seed TM.

J Radiat Res, 2013 May 8. [Epub ahead of print]

Summary

Despite the potential devastating health consequences of intense total-body irradiation, and the decades of research, there still remains a dearth of safe and effective radiation countermeasures for emergency, radiological/nuclear contingencies that have been fully approved and sanctioned for use by the US FDA. Vitamin E is a well-known antioxidant, effective in scavenging free radicals generated by radiation exposure. Vitamin E analogs, collectively known as tocols, have been subject to active investigation for a long time as radioprotectors in patients undergoing radiotherapy and in the context of possible radiation accidents or terrorism scenarios. Eight major isoforms comprise the tocol group: four tocopherols and four tocotrienols. A number of these agents and their derivatives are being investigated actively as radiation countermeasures using animal models, and several appear promising. Although the tocols are well recognized as potent antioxidants and are generally thought to mediate radioprotection through ‘free radical quenching’, recent studies have suggested several alternative mechanisms: most notably, an ‘indirect effect’ of tocols in eliciting specific species of radioprotective growth factors/cytokines such as granulocyte colony-stimulating factor (G-CSF). The radioprotective efficacy of at least two tocols has been abrogated using a neutralizing antibody of G-CSF. Based on encouraging results of radioprotective efficacy, laboratory testing of γ-tocotrienolhas moved from a small rodent model to a large nonhuman primate model for preclinical evaluation. In this brief review we identify and discuss selected tocols and their derivatives currently under development as radiation countermeasures, and attempt to describe in some detail their in vivo efficacy.

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Granulocyte colony-stimulating factor antibody abrogates radioprotective efficacy of gamma-tocotrienol, a promising radiation countermeasure.

Kulkarni S, Singh PK, Ghosh SP, Posarac A, Singh VK.

Cytokine. 2013 May;62(2):278-85. doi: 10.1016/j.cyto.2013.03.009. Epub 2013 Apr 3

This study aimed to determine the role of granulocyte colony-stimulating factor (G-CSF), induced by a promising radiation countermeasure, gamma tocotrienol (GT3), in protecting mice from lethal doses of ionizing radiation. CD2F1 mice were injected with an optimal dose of GT3 and a G-CSF antibody, and their 30-d survival was monitored. An appropriate antibody isotype was used as a control. Multiplex Luminex was used to analyze GT3-induced cytokines. G-CSF neutralization by exogenous administration of a G-CSF antibody was confirmed by analyzing serum cytokine levels. Our results demonstrate that GT3 significantly protected mice against ionizing radiation, and induced high levels of G-CSF in peripheral blood 24h after administration. Injection of a G-CSF neutralizing antibody to the GT3-treated mice resulted in complete neutralization of G-CSF and abrogation of its protective efficacy. Administration of a G-CSF antibody did not affect levels of other cytokines induced by GT3. Histopathology of bone marrow from GT3-treated and -irradiated mice demonstrated protection of the hematopoietic tissue, and also that such protection was abrogated by administering a G-CSF antibody. Our results suggest that induction of high levels of G-CSF by GT3 administration is responsible for its protective efficacy against radiation injury.

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

Ray, S.,Kulkarni, S. S., Chakraborty, K.,Pessu, R.,Hauer-Jensen, M.,Kumar, K. S.,Ghosh, S. P.

Int Immunopharmacol 2013;15(3):557-64

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 72h 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.

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

Kulkarni SS, Cary LH, Gambles K, Hauer-Jensen M, Kumar KS, Ghosh SP.

Int Immunopharmacol. 2012 Sep 20;14(4):495-503. 2012.09.001. [Epub ahead of print]

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-1β, IL-6, IL-12p70, IL-17, MIP-1α, and KC levels. G-CSF levels increased markedly within 12-24h after administration (5441pg/ml in GT3-treated groups compared to 17pg/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 48h 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.

Mechanism of radioprotection by δ-tocotrienol: Pharmacokinetics, pharmacodynamics and modulation of signalling pathways

Satyamitra M, Ney P, Graves J, Mullaney C, Srinivasan V.

Br J Radiol. 2012 Jun 6.

Objective: To investigate the correlation between in vivo δ-tocotrienol (DT3) pharmacokinetcs, pharmacodynamics and radiation protection, and to evaluate the effect of DT3 pre-treatment on radiation-induced alterations in apoptotic and autophagic pathways.Methods: We evaluated pharmacokinetics (plasma, 0.5 to 12 h) and pharmacodynamics (peripheral blood indices, day 3, 7, 10 and 14) after a single subcutaneous injection of 300 mg kg DT3 in unirradiated CD2F1 mice. Next, we monitored 30-day post-irradiation survival (9.25 Gy) and haematopoietic recovery of DT3-treated mice (7 Gy) exposed to cobalt-60 γ irradiation. The effects of DT3 on irradiated bone marrow apoptosis and autophagy were determined by analyses of key caspases (3, 7, 9 and 8), beclin-1 and LC3 conversion.Results: Plasma concentration of DT3 reached ∼195 µM (Cmax) 1 h after injection (Tmax), and DT3 was eliminated from plasma 12 h later. In unirradiated mice, DT3 significantly increased white blood cells (WBCs), neutrophils, lymphocytes (day 3 post-DT3 injection) and platelets (day 7) by 1.5-2-fold, over vehicle-treated control. DT3 pre-treatment improved 30-day survival to 100% (∼15% in control) and accelerated recovery of reticulocytes, platelets, WBCs, neutrophils, lymphocytes and monocytes in peripheral blood. DT3 reduced activation of caspase-8, caspases-3 and -7, inherent to apoptosis, while increasing autophagy-related beclin-1 expression in irradiated bone marrow.Conclusion: These data indicate that DT3 stimulates multilineage haematopoiesis, protects against radiation-induced apoptosis downstream of the mitochondria and stimulates cytoprotective autophagy. Apart from a potent antioxidant activity, DT3 may elicit survival advantage following irradiation by enhancing haematopoiesis and modulating signalling pathways.

Novel drugs to ameliorate gastrointestinal normal tissue radiation toxicity in clinical practice: What is emerging from the laboratory?

Berbée M, Hauer-Jensen M.

Curr Opin Support Palliat Care. 2012 Mar;6(1):54-9.

Purpose of Review: To give an overview of promising novel agents under development for the prevention and reduction of gastrointestinal radiation injury.

Recent Findings: Currently, several novel agents are being tested as drugs to prevent or reduce gastrointestinal radiation injury. These drugs may not only prevent injury, but also mitigate toxicity, that is, reduce injury after radiation exposure has occurred. Promising novel agents include the somatostatin analogue SOM230, growth factors, agents acting on the toll-like receptor 5 pathway, endothelial protectants, and the vitamin E analogue γ-tocotrienol.

Summary: Gastrointestinal radiation injury is the most important dose-limiting factor during radiotherapy of the abdomen or pelvis. It may severely affect the quality of life both during radiotherapy treatment and in cancer survivors. To date, there are no agents that can prevent or reduce intestinal radiation injury. Hence, there is an urgent need for the development of novel drugs to ameliorate intestinal toxicity during and after radiotherapy. This review summarizes the several agents that have been shown to reduce intestinal radiation injury in animals. Further research is needed to investigate their safety and efficacy in patients receiving radiotherapy for abdominal or pelvic tumours.

Mechanisms underlying the radioprotective properties of γ-tocotrienol: Comparative gene expression profiling in tocol-treated endothelial cells

Berbée M, Fu Q, Boerma M, Sree Kumar K, Loose DS, Hauer-Jensen M.

Genes Nutr. 2012 Jan;7(1):75-81. Epub 2011 Apr 24.

Among the eight naturally occurring vitamin E analogs, γ-tocotrienol (GT3) is a particularly potent radioprophylactic agent in vivo. Moreover, GT3 protects endothelial cells from radiation injury not only by virtue of its antioxidant properties but also by inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and by improving the availability of the nitric oxide synthase cofactor tetrahydrobiopterin. Nevertheless, the precise mechanisms underlying the superior radioprotective properties of GT3 compared with other tocols are not known. This study, therefore, examined the differences in gene expression profiles between GT3 and its tocopherol counterpart, γ-tocopherol, as well as between GT3 and α-tocopherol in human endothelial cells. Cells were treated with vehicle or the appropriate tocol for 24 h, after which total RNA was isolated and genome-wide gene expression profiles were obtained using the Illumina platform. GT3 was far more potent in inducing gene-expression changes than α-tocopherol or γ-tocopherol. In particular, GT3 induced multiple changes in pathways known to be of importance in the cellular response to radiation exposure. Affected GO functional clusters included response to oxidative stress, response to DNA damage stimuli, cell cycle phase, regulation of cell death, regulation of cell proliferation, hematopoiesis, and blood vessel development. These results form the basis for further studies to determine the exact importance of differentially affected GO functional clusters in endothelial radioprotection by GT3.

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Clinical evaluation of photoprotective effect by a topical antioxidants combination (tocopherols and tocotrienols)

Pedrelli VF, Lauriola MM, Pigatto PD.

J Eur Acad Dermatol Venereol. 2011 Sep 14.

Background: Vitamin E is among the earliest recognized antioxidants. Recent findings suggested that tocotrienols have superior activity than tocopherols. Moreover, vitamin A is well-known in dermatology for its actions, including the ultraviolet radiation absorbing property.

Objectives: In view of experimental evidence for the photoprotective properties of these antioxidants, we evaluated in 30 patients with photosensitivity, the prophylactic efficacy of a new topical agent, containing tocopherols 10% and tocotrienols 0.3%, compared with retinol, simple vehicle and untreated areas.

Methods: After determination of the minimal UVB erythema dose (MED), two areas of 2 × 2 cm were selected on the buttocks of each subject, one of which was treated with the antioxidant formulation whereas the other field did not undergo any treatment. Therefore, both areas were irradiated with a twofold MED. As further controls, other two similar areas, selected on the forearm of 15 patients, were photo-irradiated similarly, 30 min after application of the simple vehicle to a field and of vitamin A in the same vehicle to the other. Reactions (erythema/oedema/itch/vesciculation) assessment was carried out assigning scores indicative of their intensity; then, mean values +DS of scores were calculated. Results  The pre-treatment with the vitamin E formulation highly protects against photosensitivity, and all reactions to irradiation were significantly lower in the areas treated with the topical vitamin E formulation compared to those treated with the simple vehicle or vitamin A.

Conclusions: The use of a new topical formulation containing significant concentrations of tocotrienols and tocopherols represents a promising strategy to reduce the photo-induced skin damage.