Ionizing radiation initiates cellular damage directly by ionization and indirectly by producing free radicals. Approximately two-thirds of radiation-induced damage is caused by the free radicals that are generated during exposure. In addition to short-lived free radicals produced during exposure, free radicals are generated after the radiation exposure; ROS and pro-inflammatory cytokines induce a multitude of biological injuries long after the radiation exposure has ended. One of the approaches to counter oxidative stress caused by free radicals and ROS is to use antioxidants such as α-tocopherol succinate, ascorbic acid, β-carotene, vitamin A, α-lipoic acid, N-acetylcysteine, selenium or an SH compound (e.g. amifostine) (9
The rationale for using a combination of antioxidants is based on a number of observations. Individual antioxidants can act as pro-oxidants when they themselves are oxidized; therefore, individual antioxidants could enhance the progression of postirradiation damage to tissues and organs. In addition, humans have a pool of antioxidants, both endogenous antioxidants that are constitutively synthesized by cells and antioxidants that are consumed in the diet. Individual antioxidants function by different mechanisms and have different affinities for various free radicals. For example, α-tocopherol is more effective as a quencher of free radicals in a reduced oxygen environment, vitamin E has little effect on oxidants derived from nitric oxide, and vitamin A is most effective under higher atmospheric pressures. Ascorbic acid is needed to protect cellular components in aqueous environments, whereas carotenoids, vitamins A and E protect cellular components in non-aqueous environments. Vitamin C recycles oxidized vitamin E to an active form (11
). Vitamins E and C combined inhibit apoptosis in human endothelial cells more effectively than each alone, increasing Bcl-2 and down-regulating the pro-apoptotic Bax (12
Other observations affected our choice of antioxidant mixture. The form and type of vitamin E are important in determining its functional abilities. For example, various organs of rats selectively absorb the natural form of vitamin E and α-tocopherol succinate, the most effective form of vitamin E, for inhibiting cancer growth and a potent radioprotector when given prior to TBI (13
). Selenium is a co-factor of glutathione peroxidase, and Se-glutathione peroxidase acts as an antioxidant. Glutathione cannot be used orally to increase intracellular levels of glutathione, because it is completely hydrolyzed in the gut. In contrast, an oral administration of N-acetylcysteine (NAC) and α-lipoic acid, another endogenous antioxidant, can increase the intracellular levels of glutathione by different mechanisms and can be used in place of oral glutathione to reduce the radiation injury. Co-enzyme Q10, a weak endogenous antioxidant, scavenges peroxy radicals at a faster rate than α-tocopherol and, like vitamin C, can regenerate vitamin E in a redox cycle. The foregoing discussion suggests that a combination of antioxidants may be more effective in reducing the radiation-induced injury than any individual antioxidant alone. Guan et al
. showed that diet supplement with a combination of antioxidants completely prevented the reduction in the plasma levels of total antioxidant status in mice and rats exposed to proton or HZE-particle radiation (5
). Recent studies with 225 kVp X rays demonstrated marked protection from radiation injury, but generally it is believed that antioxidants need to be present during the irradiation or up to 2 h after irradiation to have a significant protecting effect (1
The data presented here show that an antioxidant-supplemented diet started 24 h after an otherwise lethal radiation exposure effectively mitigated death () mediated by a sparing of bone marrow cells (), perhaps due to a reduction in reactive oxygen species (). The effect of 8 Gy on the gastrointestinal system warrants discussion. Recent evidence suggests that the mechanisms governing the bone marrow syndrome and the gastrointestinal syndrome after TBI evolve concomitantly (14
). Consequently, the possible implications of radiation damage for the uptake of antioxidants need be considered. One might expect an even greater mitigating effect if the biodistribution of antioxidants were compromised by gastrointestinal injury.
The connection between ROS and hematopoiesis is being elucidated on a molecular level. Growth factors that stimulate hematopoiesis such as IL3 and GM-CSF have been shown to cause an increase in intracellular ROS levels (15
). The generation of ROS in response to hematopoietic growth factors contributes to downstream signaling events involving tyrosine phosphorylation such as cell proliferation (15
) and apoptosis (17
). Iiyama et al
) implicated ROS in hematopoietic cytokine-induced cell cycle progression from G1
to S phase through inducing expression of c-Myc, cyclin D2 and cyclin E and reducing expression of p27. Iiyama et al
) also showed that ROS play a role in cytokine activation of Jak2 with downstream signaling of proapoptosis pathways including MEK/ERK. Treatment with antioxidants inhibits the increase in ROS, reduces tyrosine phosphorylation, reduces proliferation induced by GM-CSF (15
), and reduces apoptosis (1
Our data are the first to show that a delay in antioxidant administration after cellular stress can be beneficial to cell and animal survival. The kinetics of ROS generation by hematopoietic cytokines as well as the mechanisms by which ROS are involved in cytokine receptor signaling to regulate proliferation and apoptosis of hematopoietic cells was studied by Iiyama et al
). They demonstrated that hematopoietic cytokines IL3 and Epo induce a rapid and transient increase in ROS that peaked at 30 min followed by a slow progressive increase in ROS 24 h after the cytokine administration. It would appear that ROS pathways controlling proliferation and apoptosis of hematopoietic cells involve two separate increases in ROS, a transient increase at 30 min and a prolonged increase that continues for at least 24 h.
Mitigation of radiation lethality by antioxidants administered soon after radiation exposure has been attributed to a reduction in apoptosis (1
). Our experience with C57BL/6 mice is not inconsistent with these results, as shown in , which also illustrates the added benefit of waiting to start administering a diet supplemented with antioxidants until 24 h after irradiation. It would appear that the first transient wave of ROS has some beneficial effect on survival since minimizing ROS early has a detrimental effect on bone marrow cell survival.
In addition to inhibiting apoptosis, reducing ROS by antioxidants soon after the radiation exposure inhibits the progression of cells from G1
to S (18
), the phase of the cell cycle in which repair of DNA damage is most efficient (19
). Repair of DNA damage has a half-time of 1 to 2 h (20
). Consequently arresting cells before S phase too soon after a radiation exposure may decrease the ability of the cells to completely repair the damaged DNA. One explanation for the increased animal survival when the antioxidant diet is given starting 24 h after irradiation is that delaying the start of the antioxidant diet allows for the most efficient repair of radiation injury and the largest increase in the survival of bone marrow cells. Further studies are needed to confirm or refute this hypothesis.
In conclusion, our results extend the work of others to show that a diet supplemented with antioxidants is effective at mitigating radiation lethality when it is started 24 h after the radiation exposure and is more effective than if given soon after the exposure. Our results support the value of antioxidants as countermeasures against radiological terrorism, especially in the practical scenario of starting a diet supplemented with antioxidants 24 h after the exposure.