Radiation Protection by the Antioxidant Alpha-Tocopherol Succinate

Radiological terrorism and use of nuclear weapons are major concerns for national defense and homeland security. At low doses of radiation, the hazards from these scenarios may not be apparent immediately, but may result in late arising pathologies like cancer and pulmonary fibrosis. At high doses, the hazards vary from incapacitation due to nausea and diarrhea to mortality. Free radical species of oxygen, derived from the interaction of ionizing radiation with critical biological targets and with the aqueous cellular milieu, are implicated in these hazards. Scavengers of free radicals have been shown to be effective protectors from radiation damage. However, many of these protectors are either toxic or cannot be administered orally at doses that are effective. The data presented here indicate that α-tocopherol succinate (TS), a free radical scavenger, can be used as a radioprotector with low toxicity. Tocopherol succinate was dispersed in a vehicle containing polyethylene glycol-400 (PEG) and given orally (PO) to male CD2F1 mice. About 22-24 hours later, they were irradiated at different doses of Co radiation at a dose rate of 0.6 Gy/min. To maximize the protection, different formulations of the vehicle were used. Mice were monitored for body weight and survival for 30 days. In vitro experiments were done to study the effects of TS on radiation-induced apoptosis of Jurkat cells (lymphoblastoid cell line) using flow cytometry. Although different formulations for solubilizing TS were used, oral formulations based on PEG were found to provide better protection than those based on oil emulsions. The best protection was obtained with a combination of PEG-400 and an emulsifier consisted of benzyl alcohol and ethyl alcohol. The PEG and emulsifier combination provided 60-70% protection at 9 Gy, a radiation dose at which only 0-13% of the animals treated with the vehicle survived. Other available analogues/derivatives of tocopherol were not protective with any of the formulations. In vitro experiments indicated that TS promoted radiation-induced apoptosis in human lymphocyte cell cultures. In vitro apoptosis experiments were not done with other tocopherol analogues/derivatives. The results with bone marrow cells were not conclusive. Protection of irradiated mice by oral TS but not by other analogues probably indicates the need for amphipathic groups on tocopherol to obtain protection by oral administration. A hemisuccinate moiety on TS may satisfy this requirement. However, the increase in apoptosis in in vitro experiments suggests that the role of the hemisuccinate moiety is only in facilitating the transport of TS across the intestine and that the in vivo protection is probably not due to the whole TS molecule but only the tocopherol released from TS in vivo. Paper presented at the NATO Human Factors and Medicine Panel Research Task Group 099 “Radiation Bioeffects and Countermeasures” meeting, held in Bethesda, Maryland, USA, June 21-23, 2005, and published in AFRRI CD 05-2. Radiation Protection by the Antioxidant Alpha-Tocopherol Succinate 16–2 NATO RTG-099 2005 1.0 INTRODUCTION Military personnel and civilian population are at risk of exposure to radiation from nuclear or radiological attack. Use of improvised nuclear devices (IND), also known as “dirty bombs,” is not a question of if; it is a question of when [Kimery 2003]. Therefore, radioprotectors for use prior to exposure has been identified as one of the highest priority areas for research [Pellmar 2005]. Depending on the dose rate and dose of exposure, the effects of radiation range from nausea and vomiting to immune system compromise to death from either radiation-induced tissue damage or infection. Although it is believed that the effects of exposure to moderate doses (1–8 Gy) of γ-radiation result in hematopoietic dysfunction, the net injury is a result of a dynamic process involving cell killing, altered cell-to-cell communication, inflammatory responses, compensatory tissue hypertrophy and repair [Stone 2003, Stone 2002]. Higher radiation doses compound these effects with gastrointestinal (GI) and neurovascular tissue damage [Weiss 1988, Coleman 2004]. Ionizing radiation triggers the formation of free radicals, which interact among themselves and with critical biological targets with the formation of a plethora of newer free radicals. It is generally believed that production of these free radicals is the main mechanism through which radiation induces biological damage at lower radiation doses [Weiss 1988]. Some of these stable free radicals may be responsible for the genomic instability mediated by the microenvironment [Barcellos-Hoff 2001]. The cells of the immune and blood-forming systems are particularly sensitive to changes in oxidant/antioxidant balance because of a high percentage of polyunsaturated fatty acids in their plasma membranes. The oxidant/antioxidant balance is an important determinant for both immune and blood-forming functions, not only for maintaining the integrity and function of the plasma membrane, cellular proteins, and nucleic acids, but also for control of signal transduction and gene expression [Aw 1999]. A safe compound that would prevent the ablation of immune system function and other radiation-induced oxidative tissue damage would provide an effective medical countermeasure against nuclear or radiological attack. Despite over four decades’ effort in this area, no compound has yet been identified and fielded that has broad-spectrum radioprotective attributes necessary to protect populations from the unwanted radiation exposures associated with catastrophic accidental or intentional (terrorist associated) events. Amifostine (WR2721) is considered a “gold standard” radioprotectant by many investigators, due to its broad spectrum, efficacious nature. Despite the fact that Amifostine is currently FDA (U.S. Food and Drug Administration)approved and commercially marketed as a normal tissue protectant in cancer patients undergoing intense chemoand/or radiotherapy, the drug is quite toxic and debilitating when applied at relatively high doses required for radioprotection. Amifostine is, therefore, suitable for use only in a clinical setting and could not be used in a clinically unsupervised scenario such as to protect military or civilian population that has undergone, or is expected to undergo, radiation exposure. 2.0 ANTIOXIDANTS AS RADIOPROTECTANTS An ideal radioprotectant should offer significant protection against lethality from acute and long-term effects of radiation exposure; be suitable for oral administration and be rapidly absorbed and distributed throughout the body; cause no significant toxicological effects, particularly those on behaviour; be readily available and affordable; and be chemically stable to permit easy handling and storage. Although the radioprotectants studied earlier do not meet these requirements, some of the recently investiRadiation Protection by the Antioxidant Alpha-Tocopherol Succinate NATO RTG-099 2005 16–3 gated agents appear to satisfy many of these criteria. Antioxidants are one such class of agents, which are nontoxic and moderately radioprotective. These antioxidants include tocols (tocopherols and tocotrienols), soyisoflavones, vitamin A, β-carotene, selenium (organic and inorganic), zinc, copper, and the enzyme superoxide dismutase and its mimetics [Kumar 1988, Kumar 2002, Kumar 2003, Weiss 2000, Seifter 1984, Weiss 1990]. Among these compounds, vitamin E has attracted considerable attention. The term “vitamin E” refers to a family of 8 tocols—4 each of α, β, γ, and δ tocopherols and tocotrienols (Figure 1).