Gamma-tocotrienol, a tocol antioxidant as a potent radioprotector

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Int. J. Radiat. Biol., Vol. 85, No. 7, July 2009, pp. 598–606

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Gamma-tocotrienol, a tocol antioxidant as a potent radioprotector


Armed Forces Radiobiology Research Institute, USUHS, Bethesda, 2Department of Preventive Medicine and Biometrics, USUHS, Bethesda, Maryland, and 3University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare Systems, Little Rock, Arkansas, USA (Received 16 December 2008; Revised 1 April 2009; Accepted 7 April 2009) Abstract 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 [LD50/30 (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 survivalprotection 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.

Keywords: Gamma tocotrienol, ionising radiation, anti-oxidant, radioprotectant, mice

Introduction There is a need to provide first responders with radioprotective agents/compounds for use during nuclear/radiological accidents within a radiationexposure field (Kumar et al. 2008) and during a combined injury scenario involving radiation plus other insults such as infection (Hauer-Jensen et al. 2008). Therefore, development of radioprotectants (i.e., drugs capable of preventing radiation induced tissue injuries and lethality) that can be used prior to exposure is a high priority research area (Pellmar and Rockwell 2005). Although several medical protocols have been proposed (Mettler and Voelz 2002, Moulder 2004), a safe and effective radioprotectant

is not yet available. A number of compounds including thiols (Davidson et al. 1980, Srinivasan et al. 2002), cytokines (Waddick et al. 1991, Singh and Yadav 2005), steroids (Whitnall et al. 2001), antioxidants (Kumar et al. 1988, Srinivasan and Weiss 1992, Kumar et al. 2002) have been reported as radioprotectants. Among these, only amifostine is approved for clinical use in conjunction with cisplatin and for patients undergoing radiotherapy for head and neck cancer. However, amifostine has not been approved as a radioprotectant for military personnel or first responders because of its performance-degrading toxicity (Landauer et al. 1992). Some of the naturally occurring tocol isoforms have been reported as potential radioprotectants.

Correspondence: Sanchita P. Ghosh, PhD, Armed Forces Radiobiology Research Institute, USUHS 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA. Tel: þ1 301 295 1945. Fax: þ1 301 295 0292. E-mail: [email protected] ISSN 0955-3002 print/ISSN 1362-3095 online Ó 2009 Informa Healthcare USA, Inc. DOI: 10.1080/09553000902985128

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Gamma-tocotrienol – a potent radioprotector These tocols exist in nature as eight isoforms – four each of tocopherols having a saturated side chain and tocotrienols with an unsaturated side chain (Ghosh et al. 2008). Srinivasan et al. (1983) reported that alpha-tocopherol (AT) protected mice from radiation lethality and inhibited radiation-induced delayed type hypersensitivity. The same authors later showed that the efficacy of S-[2-(3-methylaminopropyl) aminoethyl] phosphorothioate acid (WR-3689), a methylated derivative of amifostine, can be enhanced by combining it with alpha-tocopherol (Srinivasan and Weiss 1992). We have reported that the efficacy of alpha-tocopherol can be increased by using an improved formulation (Kumar et al. 2002). Although extensive studies have been made with AT, comparatively fewer studies have been carried out with the unsaturated tocol isoforms. Among these, gamma-tocotrienol (GT3) has received greater attention in recent years. Preliminary studies reported by us (Kumar et al. 2008) indicated that GT3 is a better radioprotectant than AT in mice, even at supra-lethal radiation doses. Moreover, GT3 is a potent inhibitor of hydroxymethyl glutaryl CoA reductase (Qureshi et al. 1986, Rukmini and Raghuram 1991, Baliarsingh et al. 2005), a pivotal enzyme in the synthesis of mevalonic acid, a key metabolic intermediate in the biosynthesis of cholesterol. Surprisingly, statins, which inhibit the same enzyme, were shown to ameliorate radiation induced enterotoxicity (Wang et al. 2004). In terms of antioxidant activity, GT3 was also shown to be superior to AT. The superior antioxidant activity plus its similarity to statins, prompted us to make an extensive study of GT3’s radioprotectant profile. We report in this paper that GT3, to the extent of our knowledge, is a naturally occurring antioxidant that provides high dose reduction factor (1.29). Our objective is to develop GT3 as a prophylactic agent (i.e., administered before ionising radiation exposure) against the short-term consequences of radiation exposure such as hematopoietic death and/or gastro intestinal (GI) failure and lethality.


quarantine for two weeks and were used after microbiology, serology, and histopathology examination of representative samples, ensured the absence of Pseudomonas aeruginosa and common murine diseases. Mice were provided certified rodent rations (Harlan Teklad Rodent diet #8604 (w), Harlan Teklad, Madison, WI, USA) and acidified water (with HCl, pH 2.5–3.0) ad libitum. All mice were kept in rooms with a 12 h light/dark cycle with lights on from 06:00 to 18:00 h. All animal procedures were performed in accordance with a protocol approved by the AFRRI’s Animal Care and Use Committee. Research was conducted according to the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, National Research Council, U.S. National Academy of Sciences. Irradiation Mice were irradiated bilaterally at the AFRRI’s cobalt-60 gamma radiation facility. The midline tissue dose to the animals was 3–12 Gy at a dose rate of 0.6 Gy/min. An alanine/ESR (electron spin resonance) dosimetry system (American Society for Testing and Material Standard E 1607) was used to measure dose rates (to water) in the cores of acrylic mouse phantoms. Phantoms were 3 inches long and 1 inch in diameter and were located in all empty compartments of the exposure rack. The ESR signals were measured with a calibration curve based on standard calibration dosimeters provided by the National Institute of Standard and Technology (NIST, Gaithersburg, MD, USA). The accuracy of the calibration curve was verified by intercomparison with the National Physical Laboratory (NPL), United Kingdom. The only corrections applied to the dose rates in phantoms were for the decay of cobalt-60 source and for a small difference in mass energy-absorption coefficients for water and soft tissue. The radiation field was uniform within + 2%. Drug formulation and administration

Materials and methods Animals Six- to 8-week-old male CD2F1 mice (Harlan Laboratories, Indianapolis, IN, USA) were housed (eight per cage) in an air-conditioned facility at the Armed Forces Radiobiology Research Institute (AFRRI, Bethesda, MD, USA), accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The holding rooms for mice were maintained at 218C + 28 with 10–15 hourly cycles of fresh air and a relative humidity of 50% + 10%. Mice were held in

Gammatocotrienol (2,7,8-trimethyl-2-(4,8,12-trimethyltrideca-3,7,11-trienyl) chroman-6-ol) formulated in 5% Tween-80 was purchased from Yasoo Health Inc. (Johnson City, TN, USA). Olive oil was used as vehicle control (equivalent to the amount of gamma-tocotrienol) in 5% Tween-80. The final GT3 concentrations (50–400 mg/kg) were adjusted to deliver 0.1 ml of GT3. Control mice received 0.1 ml of vehicle. All subcutaneous (SC) injections of the drug and vehicle in animals were done aseptically at the nape of the neck with a 23G needle before radiation. No infections or local reactions were noted at the site of injection. At least 16 animals


S. P. Ghosh et al

(unless otherwise mentioned) were used per group in all the following experiments and all injections were done SC.

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Dose optimisation Groups of mice were given four doses of GT3 (50, 100, 200, and 400 mg/kg of body weight) and irradiated after 24 h. Radiation doses were 10.5, 11, and 11.5 Gy at a rate 0.6 Gy/min. After irradiation mice were returned to their cages with free access to food and water and monitored (30 days) for weight loss, apparent behavioural deficit, and survival. Time optimisation Dose optimisation studies indicated that the optimum GT3 dose was 200 mg/kg body weight. To determine the optimum time, GT3 at this dose was injected at 48 h, 24 h, 12 h, 8 h, 4 h, and 2 h before radiation at 11 Gy and mice were monitored for 30-day survival protection. Determination of Dose Reduction Factor (DRF) Six groups of mice (n ¼ 16 for each group) were injected with vehicle and another six groups of mice were injected with 200 mg/kg of GT3. The vehicleinjected groups were irradiated at 8.5, 9, 9.5, 10, 10.5, and 11 Gy; the GT3-injected groups were irradiated at 9.5, 10, 10.5, 11, 11.5, and 12 Gy. The range of radiation doses for vehicle or GT3-treated groups were selected based on previous observations so that the lowest radiation dose will result in 100% survival and the highest dose will result in 100% lethality. Survival was monitored for 30 days and from survival data LD50/30 (lethal radiation dose that results in the mortality of 50% mice in 30 days) radiation doses for vehicle- and GT3-treated mice were calculated using probit analysis. DRF was calculated as the ratio of the LD50/30 radiation dose of GT3-treated mice to the LD50/30 radiation dose of vehicle-treated mice (Whitnall et al. 2000).

0 was the day of irradiation). Total white blood cells (WBC), absolute neutrophil counts (ANC), monocytes (MONO), lymphocytes (LYMP), platelets (PLT), and reticulocytes (RETIC) were counted using an Advia 120 cell counter (Bayer Corporation, Terrytown, NY, USA). From these values, we obtained the pattern of declination and recovery graphs drawn with post-irradiation days and cell counts. We evaluated the effect of GT3 in the amelioration of cytopenia from these graphs. Statistical analysis For the survival data, Fisher’s exact test was used to compare survival at 30 days and a log-rank test was used to compare survival curves. Means and standard errors were reported for cytopenia data. Analysis of variance (ANOVA) was used to determine if there was a significant difference among different groups. For a given day, if there was a significant difference among the groups, a pair-wise comparison was done using the Tukey-Kramer method. A significance level was set at 5% for each test. All statistical tests were two-sided. Statistical software, PC SAS, was used for statistical analyses.

Results Comparison of radioprotective efficacy at various doses of GT3 Figure 1 illustrates the radioprotective efficacy of mice treated with various doses of GT3 (50, 100, 200, and 400 mg/kg) 24 h before radiation at 10.5 Gy. There were no survivors in the vehicle group. The percentages of mice surviving after 30 days at GT3 doses of 50, 100, 200, and 400 mg/kg were

Peripheral blood cytopenia Mice (n ¼ 10 per group) were given either vehicle or 200 mg/kg GT3 24 h before radiation at 7 Gy at 0.6 Gy/min or sham irradiated. Mice were anaesthetised with an overdose of isoflurane (Hospira Inc., Lake Forest, IL, USA) and blood (0.6–1.0 ml) was collected from the posterior vena cava using a 23gauge needle. Blood was transferred immediately into ethylenediamine tetraacetic acid (Sigma, St Louis, MO, USA) containing blood collection tubes and mixed gently in a rotary shaker until analysis. Blood was collected at day 0, 1, 4, 8, 16, and 30 (day

Figure 1. Radioprotection provided by GT3 at 10.5 Gy. Thirtyday survival of mice (n ¼ 16 per group) treated 24 h before receiving 10.5 Gy of cobalt-60 gamma radiation, with a single SC injection of vehicle (5% Tween 80) or GT3 at doses of 50–400 mg/kg body weight. Mice that received a GT3 dose of 100, 200, and 400 mg/kg exhibited a significant increase from vehicle control group ( p 5 0.0001, Chi-square ¼ 59.95, df ¼ 4).

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Gamma-tocotrienol – a potent radioprotector 31%, 81%, 94%, and 88%, respectively. There was a significant difference in 30-day survival for groups that received GT3 doses of at least 100 mg/kg (Figure 1). There were no significant differences in survival between GT3 doses of 100, 200, and 400 mg/kg, however they differed significantly (p 5 0.005) from the lowest GT3 dose (50 mg/kg). Survival rates at the higher doses were significantly different ( p 5 0.005) from the vehicle. When the radiation dose was increased to 11 Gy, almost similar survival pattern was observed with various GT3 doses (Figure 2). However, when the radiation dose was increased to 11.5 Gy, the pattern of survival was somewhat different. Survival percentages at 100 mg/ kg and 200 mg/kg were identical (44%), which was significantly higher than vehicle control group (p 5 0.005) (Figure 3). The highest dose of


GT3 (400 mg/kg) did not show significant survival at 11.5 Gy. Survival with a dose of 200 mg/kg GT3 was slightly better at 10.5 Gy and 11 Gy than with 100 mg/kg. At 11.5 Gy, it was higher than 400 mg/kg. Therefore, this dose (200 mg/kg GT3) was judged to be the optimum dose and was used in all further studies. Determination of optimal time for administering GT3 Figure 4 shows survival curves for mice administered 200 mg/kg of GT3 at 48 h, 24 h, and 12 h before radiation at 11.0 Gy. Only 38% of mice that received GT3 12 h before radiation survived compared to 95% of those that received GT3 24 h before radiation ( p 5 0.001 survival at 12 h vs. 24 h). There were no survivors in the group that received GT3 48 h before radiation. Although we tested shorter time points (8, 4, and 2 h) before radiation, there were no survivors at these time points (data not shown). From these two studies, we concluded that radioprotective efficacy of GT3 is optimal at a dose of 200 mg/kg, given SC 24 h before radiation. Determination of dose reduction factor (DRF)

Figure 2. Radioprotection provided by GT3 at 11 Gy. Thirty-day survival of mice (n ¼ 16 per group) treated 24 h before receiving 11 Gy of cobalt-60 gamma radiation, with a single SC injection of vehicle (5% Tween 80) or GT3 at doses of 50 to 400 mg/kg body weight. Mice that received a GT3 dose of 100, 200, and 400 mg/kg exhibited a significant increase from vehicle control group ( p 5 0.0001, Chi-square ¼ 69.74, df ¼ 4).

Figure 3. Radioprotection provided by GT3 at 11.5 Gy. Thirtyday survival of mice (n ¼ 16 per group) treated 24 h before receiving 11.5 Gy of cobalt-60 gamma radiation, with a single SC injection of vehicle (5% Tween 80) or GT3 dose of 100–400 mg/ kg body weight. Mice that received a GT3 at doses of 100 and 200 mg/kg exhibited a significant increase from vehicle control group ( p 5 0.0001, Chi-square ¼ 29.34, df ¼ 3).

A GT3 dose of 200 mg/kg, which resulted in the highest survival rate when administered to mice 24 h before irradiation, was used to evaluate DRF. Mice were exposed to whole body gamma-radiation and received doses between 8.5 and 12 Gy. Mortality of the GT3 and vehicle treatment is plotted in Figure 5. The probit line was shifted to the right in the drugtreated animals. The LD50/30 radiation doses were 8.6 Gy for vehicle and 11.12 Gy for GT3,

Figure 4. Survival studies evaluating the optimum time for administering GT3 for radioprotection. Thirty-day survival of mice (n ¼ 16 per group) treated at various times (748, 724, 712, 78, 74, and 72 h) before receiving 11 Gy of gamma radiation with a single SC injection of Tween-80 vehicle or 200 mg/kg of GT3. Survival was significantly enhanced in the group receiving the drug at 724 h and 712 h, compared to vehicle (*p 5 0.001 for GT3 compared to vehicle control group). No mice survived among groups that received the drug at 748, 78, 74, and 72 h (time points with no survival are not shown in the curve).


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GT3-treated mice had significantly ( p 5 0.05) higher levels of PLT on days 4, 8, 16, and 30 after irradiation. The nadir for RETIC also occurred on day 4 after radiation, and recovery was faster in GT3-treated mice, which showed a significantly (p 5 0.05) higher level on day 8 (Figure 6E). GT3 had no effect on radiation-induced declination and recovery of lymphocytes and erythrocytes (data not shown). Discussion Figure 5. Dose reduction factor (DRF) determination. Mice (n ¼ 16 per group) were treated, 24 h before receiving irradiation, with a single SC injection of Tween-80 vehicle or 200 mg/kg of GT3. Radiation doses for the vehicle group were 8.5, 9, 9.5, 10, 10.5, and 11 Gy; doses for the GT3-treated group were 9.5, 10, 10.5, 11, 11.5, and 12 Gy. Probit mortality curves were generated and a DRF of 1.29 with 95% confidence interval (1.27–1.32) was calculated from the ratio of the LD50/30 for GT3-treated to vehicletreated mice.

respectively. The DRF calculated was 1.29 with 95% confidence interval (1.27–1.32). DRF calculation was done as described by Whitnall et al. (2000) and Davis et al. (2007). Effect of GT3 on radiation-induced peripheral blood cytopenia Figure 6A–E shows the effects of GT3 on cytopenia of WBC, ANC, PLT, MONO, and RETIC. Other than RETIC (Figure 6E), there were significant (p 5 0.05) increases in WBC, ANC, MONO, and PLT when mice were treated with GT3 alone compared to untreated or vehicle-treated groups during days 4–16. For WBC (Figure 6A) and ANC (Figure 6C), the differences were significant during days 1–16. The MONO increases (Figure 6D) were significant at all time points tested from four days after irradiation; PLT (Figure 6B) levels were significantly higher only at 8 and 16 days after irradiation. WBC, ANC, and MONO levels began declining after irradiation and reached their nadir at day 4, resulting in neutropenia and monocytopenia. These cells had recovered to their normal levels by day 30. Recovery was significantly faster in GT3-treated animals after the 4th day of radiation. On day 16 after irradiation, GT3-treated mice exhibited WBC, ANC, and MONO counts that were significantly higher ( p 5 0.05) than in mice treated with vehicle (Figures 6A, C, D). The post-irradiation declination pattern for PLT (Figure 6B) was different from other blood elements. The nadir for platelets was reached 8 days after irradiation but did not result in thrombocytopenia;

Alpha-tocopherol (AT) has long been recognised as a chain-breaking antioxidant, affecting several pathological conditions, from heart disease (Yusuf et al. 2000) to diabetes (Jain et al. 2000). In recent years an unsaturated isoform, GT3, has been receiving attention due to its potent inhibition of hydroxyl methyl glutaryl coenzyme A (HMG-CoA) reductase enzyme in vitro (Rukmini and Raghuram 1991, Qureshi et al. 1986) and in vivo (Baliarsingh et al. 2005). HMG-CoA reductase is a pivotal enzyme in the synthesis of mevalonic acid, a key metabolic intermediate in the biosynthesis of cholesterol. Tocotrienols had been shown to suppress the proliferation of a wide variety of tumour cells in culture, including breast (Guthrie et al. 1997, Yu et al. 1999, Nesaretnam et al. 2000, Shun et al. 2004, Sylvester and Shah 2005), prostate (Galli et al. 2004, Srivastava and Gupta 2006), and colon (Eitsuka et al. 2006). Animal studies have shown that tocotrienols can also suppress the growth of several tumours (Gould et al. 1991, Ngah et al. 1991, Goh et al. 1994, He et al. 1997, Iqbal et al. 2004, Wada et al. 2005, Kumar et al. 2006) and they are neuroprotective agents (Sen et al. 2006). Although several studies have reported on GT3, none dealt with the radiation protection, GT3 provides. Some studied AT alone (Srinivasan et al. 1983, Kumar et al. 2002, Seed et al. 2002) and in combination with other agents. Compared to the number of studies done with AT, radioprotection studies with GT3 are very preliminary (Kumar et al. 2008). We report here on GT3’s optimal drug dose, time of administration, amelioration of pancytopenia, and DRF. A possible mechanism for the protection of GT3 may be due to higher antioxidant activity than has been achieved for tocopherols (Watkins et al. 1999). GT3’s higher level of antioxidant activity will enhance the efficiency of scavenging radiationinduced reactive oxygen species (ROS), providing increased radioprotection. Drug formulation is an important and integral concern, especially for lipophilic drugs such as GT3. An improved formulation of AT has been shown to increase its radioprotective efficacy (Kumar et al. 2002). Our

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Gamma-tocotrienol – a potent radioprotector


Figure 6. GT3 pretreatment enhances recovery of peripheral blood cells in irradiated mice. Effect of GT3 on (A) total white blood cells (WBC), (B) platelets (PLT), (C) absolute neutrophil count (ANC), (D) monocytes (MONO), and (E) reticulocytes (RETIC) in mice (n ¼ 10 per group) subjected to total-body gamma radiation with 7 Gy (0.6 Gy/min). Mice were administered a single SC injection of GT3 (200 mg/kg) or vehicle (5% Tween-80) 24 h prior to gamma radiation. Day 0 represents day of radiation. Data represented are means + standard error of the mean (SEM) for n ¼ 10 mice for each time point. The marked group (* or #) indicated significant difference for irradiated GT3 compared to irradiated vehicle group or unirradiated GT3 compared to unirradiated vehicle group by Tukey-Kramer method. Some points in the Figure do not have error bars that are visible because they are smaller than data points.

studies with another radioprotectant, Ex-Rad (a chlorobenzene sulfone derivative), indicated that use of Tween-80 as an excipient conferred an increase in radiation protection at lethal radiation dose (Ghosh et al. 2009). Using the same excipient in the current studies might have facilitated better permeation of GT3 from the site of injection into the systemic circulation, resulting in enhanced protection. Previous pharmacokinetic study indi-

cates that maximum plasma concentration (Cmax) of GT3 is reached within 5 h of oral intake in human (Yap et al. 2001). In our study, maximum survival in mice after radiation was observed when the drug was given 24 h before radiation, indicating that the antioxidant property of GT3 may or may not contribute to the radioprotection. The DRF is a measure of a drug’s efficacy over a wide range of radiation doses, and is a standard

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S. P. Ghosh et al

measure for comparing radioprotectors. A DRF of 1.16 (based on 30-day survival) was obtained with a single SC administration of genestein, a non-toxic isoflavone from soybeans, at a dose of 200 mg/kg 24 h before radiation (Davis et al. 2007). The DRF of GT3 was found to be 1.29 which indicates that survival enhancing efficacy of GT3 goes beyond hematopoietic protection. GT3 may be a better radioprotectant than AT comes from the fact that AT (400 mg/kg injected SC 24 h before radiation) demonstrated lower DRF (1.23) compared to GT3 (Seed et al. 2002). GT3 also accelerated the regeneration and repletion of peripheral blood cells, particularly WBC, ANC, MONO, and PLT. Radiation-induced peripheral blood neutropenia and thrombocytopenia can result in infectious and hemorrhagic complications that could be lethal. Previous studies show that genestein (a non-toxic isoflavone from soybeans) pretreatment enhances recovery of peripheral blood cells in irradiated mice (Davis et al. 2007). Extrapolating results from studies reported here at 7 Gy, GT3 appears to be an ideal choice to prevent radiation-induced infectious and hemorrhagic complications. That GT3 is effective in stimulating bone marrow to increase white cell populations was evident in the increased numbers of WBC, ANC, MONO, and PLT even in unirradiated mice. In this respect, GT3 appears to mimic hematopoietic cytokines like granulocyte colony stimulating factor (G-CSF) to a limited extent. As we have reported earlier (Singh et al. 2006), the induction of cytokines by GT3 and the subsequent stimulation of the hematopoietic system may be the cause of increased numbers of peripheral blood elements. Thus, restoring the deranged innate immune system may be another mechanism of radioprotection provided by GT3. Stimulating the production of peripheral blood elements even in unirradiated mice has potential applications in therapeutic radiology and chemotherapy. Currently, G-CSF or granulocyte macrophage colony stimulating factor (GM-CSF) is given to restore neutrophils depleted by radiation or chemotherapy. However, these cytokines are also known to cause side effects such as fever. If instead, GT3 is used to induce the production of these cytokines de novo, it may prevent such side effects without compromising the beneficial effects of restoring neutrophils. In conclusion, radioprotection provided by GT3 offers another potent, naturally occurring agent that could be considered for further development as a radioprotectant for first responders. The protection by GT3 assumes greater significance since the radiation doses used in the study are equivalent to lethal human doses. Our studies are carried out at a

drug dose of 200 mg/kg body weight given SC which is equivalent to 16 mg/kg body weight (equivalent to 1120 mg for a 70 kg person) for humans based on the calculation as described by Food and Drug Administration (FDA 2005). However, for actual human use, GT3 will have to go through extensive safety and toxicity studies and efficacy trial. Acknowledgements This work was supported by the U.S. Department of Defense Threat Reduction Agency grant H.10027_07_AR_R (KSK), administered by The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. and HDTRA 1-07-C0028 (MH-J). SPG, SK, KH, and LR are affiliated with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., 1401 Rockville pike, Rockville, MD 20852, USA. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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