Pretreatment with ascorbic acid is believed to be effective for attenuating radiation-induced GI damage because of its potent antioxidative effect [8
]. It is considered that the production of reactive oxygen species (ROS) is evoked immediately after radiation exposure, and that ascorbic acid may scavenge these ROS. In line with this theory, post-treatment with ascorbic acid alone (without pretreatment) was ineffective (0% survival, Figure 5
), likely because it could not scavenge the ROS generated immediately after irradiation. However, even when we performed pretreatment with ascorbic acid for three days and engulfment eight hours before the irradiation, the irradiated mice showed only 20% survival (Figure 5
), despite the fact that this boosting pretreatment significantly increased the tissue ascorbic acid levels just before irradiation (Table 1
Nevertheless, the addition of post-treatment ascorbic acid to the pretreatments increased the survival of the irradiated mice to 100%. It is possible that scavenging the ROS generated immediately after radiation by the boosting pretreatment with ascorbic acid may be necessary to improve the survival of irradiated mice. However, additional post-treatment with ascorbic acid also may be indispensable for further improving the survival due to late or ongoing damage.
In our previous study [4
], we examined the efficacy of ascorbic acid therapy in irradiated mice at a dose of 150 mg/kg/day. However, most clinical studies of high-dose ascorbic acid therapy, which were performed to examine the effects of ascorbic acid on the prognoses in cancer patients, were evaluated by p.o. supplementation of ascorbic acid at 10 g/day [9
]. Therefore, we based our dosing on this level (200–250 mg/kg/day).
It is known that plants and most animals, including mice, can synthesize ascorbic acid from glucose. Primitive fish, amphibians and reptiles synthesize ascorbic acid in the kidneys, whereas most mammals, such as mice, synthesize it in the liver. In contrast, humans, other primates, guinea-pigs and a few species of fruit-eating bats cannot synthesize ascorbic acid because the gene encoding l
-gulonolactone oxidase (GLO)—the enzyme required for the last step in ascorbate synthesis—is not functional [11
]. The ascorbic acid concentrations in such mammals appear to be affected by three mechanisms, particularly in the human; intestinal absorption of the ingested ascorbic acid, tissue accumulation and renal reabsorption and excretion [9
]. We think that the mice receiving high-dose ascorbic acid treatment likely have an ascorbic acid metabolism which is similar to that in humans. The gastrointestinal absorption of ascorbic acid usually occurs through an active transport process, as well as through passive diffusion. Although the active transport of ascorbic acid predominates at low intra-intestinal tract concentrations, when the hosts receives a high oral dose of ascorbic acid and develops high intra-tract concentrations (as would be expected in the present model), active transport becomes saturated, leaving only passive diffusion. In such a case, the intestinal tissue levels of ascorbic acid are important, and this is the case for the hosts receiving p.o. therapy with high-dose ascorbic acid.
The half-life of the radicals evoked by radiation is considered to be extremely short (nanoseconds), and these radicals interact with various biological molecules, with some breaking DNA chains [13
]. It has been believed that these short-lived radicals play crucial roles in modulating the radiation-induced biological effects on the tissue and cells, such as apoptotic cell death [14
]. When the radiation hits the water molecules in cells, which are composed of more than 80% water, it leads to the prompt generation of radicals of water origin, such as H and OH, resulting in ROS-induced tissue damage [14
]. However, a cell is not a homogenous water solution of organic matter, and almost half of the water in cells exists as bound water [15
]. Therefore, it is doubtful that the active radicals derived from the ionization water molecules directly participate in cell death [16
Miyazaki et al.
have identified long-lived radicals, which have a half-life of more than 20 h, in γ-irradiated cells, using electron spin resonance (ESR) spectroscopy and highly sensitive measurement techniques [17
]. Interestingly, ESR spectroscopy showed that when ascorbic acid is added after irradiation, it removes the long-lived radicals from irradiated cells [17
]. In another in vitro
study, they demonstrated that the long-lived radicals induced by ionizing radiation are highly mutagenic and transforming in mammalian cells, although they are not involved in the lethality or in the induction of chromosome aberrations in cultured cells [16
]. When ascorbic acid was added 20 h after irradiation, it reduced the frequency of radiation-induced hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutation in human embryonic (HE17) cells. However, ascorbic acid did not change the survival of the cultured cells. The cell damage caused by the long-lived radicals may be limited, because suppressing the short-lived radicals by pretreatment with ascorbic acid clearly increased the cell survival [16
]. In the present study, there is a possibility that the additional treatment with ascorbic acid following the pretreatment period may have effectively reduced the levels of long-lived radicals in the irradiated mice. However, we could not detect the long-lived radicals induced by irradiation in mice, and therefore, did not examine whether the long-lived radicals are scavenged by post-treatment with ascorbic acid. There is plenty of room for further research on other possible mechanisms by which the combination therapy with ascorbic acid induces a beneficial effect on the survival of irradiated mice.
Proinflammatory cytokines, as well as ROS, may play important roles in the occurrence of radiation-induced injury [18
]. The levels of proinflammatory cytokines, e.g., TNF in the intestinal tissue (but not plasma) and IL-6 in the plasma (but not intestinal tissue), are reportedly increased in mice three to six days after abdominal radiation [18
]. Mice receiving repeated abdominal radiation also reportedly exhibit a positive expression of TNF (but not IL-1β or IL-6) in the intestinal mucosa on immunohistochemistry [19
]. Interestingly, in the present study, pre/post-treatment and engulfment of ascorbic acid significantly reduced the tissue TNF levels and free radical metabolite levels in the murine small intestine (but not plasma) seven days after abdominal radiation, although the treatment affected neither the IL-1β nor IL-6 levels in the intestinal tissue or plasma (Table 2
). Treatment with ascorbic acid may reduce ROS and TNF-induced/involved tissue inflammatory responses, especially in the intestinal tissue, resulting in the attenuation of radiation-induced tissue injury. Further studies are required to determine how ascorbic acid therapy affects radiation-induced inflammation and cytokine responses.
The antioxidant N
-acetyl-cysteine (NAC) reportedly protected mice from GI damage when the administration was started not only 4 h before, but also 2 h after, a lethal level of abdominal radiation [21
]. Although half of the irradiated mice receiving post-treatment with NAC still died (50% survival), its pharmaceutical efficacy when administered after exposure is attractive. On the one hand, the intravenous or oral administration of ascorbic acid, including high-dose administration, is widely used in humans and is believed to be harmless because ascorbic acid is a hydrosoluble vitamin [9
]. On the other hand, NAC has also been used intravenously and orally in humans [22
]. However, clinicians must pay attention to the possibility of anaphylactoid reactions when intravenously infusing NAC [22
]. Kerr et al.
reported that approximately 15% of patients treated with intravenous NAC develop an anaphylactoid reaction within two hours after the initial infusion [25
]. AEOL 10150, a small-molecule antioxidant analogous to the catalytic site of superoxide dismutase (SOD), reportedly protects cells against radiation-induced lung injury, even when administered after radiation exposure to the right hemithorax [26
]. Although AEOL 10150 is reportedly undergoing evaluations in various clinical trials [27
], it is not yet available in the clinical setting. Antioxidative agents can be potent therapeutic tools for radiation-induced tissue/organ damage.
Although abdominal irradiation is performed in patients as an effective therapy for several kinds of abdominal malignancies, radiation-induced GI damage sometimes occurs, which decreases the quality of life for the patients and treatment may need to be discontinued. The present pre/post-irradiation combination therapy with ascorbic acid may provide an effective therapeutic tool for the patients who develop radiation-induced GI damage. In addition, monitoring the plasma citrulline levels may be useful to evaluate the radiation-induced GI damage, especially after abdominal irradiation [28
], because decreases in the plasma citrulline levels were associated with the intestinal mucosal damage after abdominal radiation.
Considering the potential use of ascorbic acid therapy in cancer patients, we were concerned about the possibility that the radioprotective effect induced by the ascorbic acid might reduce the radiation-induced antitumor cytotoxicity. Of note, it is known that ascorbic acid has some anticancer effects [9
], although this was not confirmed by the clinical trial performed at the Mayo Clinic [10
]. Interestingly, ascorbic acid therapy in mice reportedly exerted radioprotective effects on their skin and bone marrow cells, but did not protect the tumor cells, although the mechanism underlying this differential radiomodification of the normal tissue sensitivity and tumor tissue response is unclear [31
]. A recent paper has also demonstrated that ascorbic acid enhances the radiation-induced apoptosis in tumor cell lines via the activation of caspases-3, 8 and 9 [32
]. Although the precise mechanisms underlying this effect have yet to be elucidated, there is a possibility that ascorbic acid may not reduce the radiation-induced antitumor activity. However, further studies are needed to elucidate this important issue.
When a radiation accident unfortunately occurs, it is quite difficult to treat the victims with acute radiation syndrome. However, rescue team members can be deployed in radiation-contaminated areas to rescue the victims. In such situations, preventing radiation-induced damage, including GI damage, is of vital importance. As described in our previous paper [4
], when rescuing victims from radiation-contaminated areas immediately after a radiation accident or terrorist attack, it is important for the rescue team members to promptly take ascorbic acid orally. The Fukushima nuclear disaster occurred in 2011, one year after the publication of our paper. This was an opportunity for our rescue team members to administer the appropriate mitigating treatment based on our scenario. Some of the volunteers in the rescue team orally took ascorbic acid as a trial, but it showed no significant side effects; however, they were fortunately considered to have received very little radiation exposure.