Terrorist threats and recent accidents in nuclear installations emphasize the urgent need to develop radiation countermeasures [3
]. We reported recently that TS is a promising radiation countermeasure that protects mice against hematopoietic as well as GI doses of irradiation when administered 24 h before radiation exposure [13
]. Administration of TS modulated the expression of antioxidant enzymes and inhibited expression of oncogenes in irradiated mice. It also increased colony-forming unit-spleen numbers and bone marrow cellularity in irradiated mice. We were interested to explore the mechanism of radio-protective efficacy provided by TS. In this study, we demonstrate that TS treatment inhibits CD68-positive cells, radiation-induced DNA damage and apoptosis, and increases mitosis in irradiated mice.
CD68 is particularly useful as a marker for the various cells of the macrophage lineage including monocytes, histocytes, giant cells, Kupffer cells and osteoclasts. There are a large number of studies using CD68 markers to identify inflammatory cells [24
]. We observed lower numbers of CD68-positive cells 2 and 4 h after irradiation in the jejunum of mice receiving TS treatment compared with vehicle control, suggesting that TS treatment is involved in suppressing inflammation in response to irradiation.
Ionizing radiation has been shown to induce apoptotic pathways. Several caspases play essential roles in the execution of apoptosis [26
]. Caspase 3, an effector caspase, is responsible for the proteolytic cleavage of several key proteins including the nuclear enzyme poly(ADP-ribose) polymerase (PARP) [27
]. TS inhibited the expression of caspase 3 in irradiated mice, which further explains the role of TS in the inhibition of radiation-induced apoptosis. Our observation of lower numbers of caspase 3-positive cells in TS-treated mice compared with vehicle-treated mice also explains the lower number of cleaved PARP-positive cells in TS-treated mice.
To further evaluate the molecular mechanism of the inhibition of caspase 3 in the TS-treated mice, we evaluated the expression of BAX protein. BAX is a pro-apoptotic protein of the Bcl2 family containing BH1, BH2 and BH3 domains. Expression of BAX is known to accelerate apoptotic cell death by releasing cytochrome c oxidase from mitochondrion to cytoplasm [28
]. Released cytochrome c from mitochondria facilitates the formation of the apoptosome-containing Apaf-1 and caspase 9, which converts the pro-caspase 3 to active caspase 3 [29
]. TS inhibited the expression of BAX in the jejunal crypts of irradiated mice, which supports our earlier finding of regeneration of crypts in TS-treated and irradiated mice [14
]. Lower numbers of BAX-positive cells in TS-treated and irradiated mice are in correlation with lower numbers of active caspase 3-positive cells in TS-treated mice. Park et al.
have also shown the eckol-mediated inhibition of BAX in the crypt of irradiated mice, supporting our current observations [31
The comet assay, also called single-cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. This is a standard technique for evaluation of DNA damage/repair. Several radio-protective agents such as eckol and dieckol have been shown to reduce radiation-induced DNA damage using comet assays [22
]. Our comet assay results support earlier findings that radio-protective agents can reduce radiation-induced DNA damage/repair. We found similar results in comet assay with samples obtained from spleen, thymus and peripheral blood cells of TS-treated and irradiated mice. We have used a 9.2-Gy radiation dose for experiments presented in Figure since 9.2 Gy is the LD90/30
dose that causes hematopoietic injury and this dose was suitable for analyzing DNA damage in cells of spleen, thymus and peripheral blood.
The effect on mitosis, which is crucial for the regular replacement of intestinal epithelial lining, may be an important mechanism for the protection of gastrointestinal tract, as observed in mice injected with TS. Octamers of histone proteins (two each of H2A, H2B, H3 and H4 histone proteins) function as spools to package eukaryotic DNA into repeating nucleosome units. During the prophase of mitosis, chromosomal condensation starts with phosphorylation of histone H3, which occurs in a step-wise and orderly manner [33
]. Phosphorylation of histone 3 has been considered a hallmark of mitosis [35
]. We evaluated the expression of phospho-histone H3 (pH3) expression as a mitotic marker of intestinal epithelial cells. As expected, we observed significantly higher numbers of pH3-positive cells in mice treated with TS and exposed to 60
Co γ-radiation, compared with vehicle control. Results presented here further confirm our earlier findings with bromodeoxyuridine and Ki-67 markers in tocopherol succinate-treated and irradiated mice [14
]. The phosphorylation of histone H3 at Ser10 (pH3) has been considered an important marker of mitosis [35
], since it only occurs in cells undergoing mitosis. We observed significantly higher numbers of pH3-positive cells per crypt in TS-treated mice exposed to 60
Co γ-radiation compared with vehicle-treated mice. PCNA is a nuclear protein and a co-factor of DNA polymerase δ, which is involved in the control of eukaryotic DNA replication and repair. In response to DNA damage, this protein is ubiquitinated and is involved in the RAD6-dependent DNA repair pathway. Our results show higher numbers of PCNA-positive cells per crypt in TS-injected mice compared with vehicle control. Overall these results demonstrate an increased proliferation of jejunum cells of lethally irradiated mice treated with TS. The effect on mitosis, which is crucial for the regular replacement of intestinal epithelial lining, may be an important mechanism for the protection of the gastrointestinal tract observed in mice treated with TS. Park et al.
have demonstrated a similar radio-protective mechanism with eckol [22
]. They have demonstrated that eckol's protective effects include an improvement in hematopoietic recovery, the repair of damaged DNA in immune cells and an enhancement of their proliferation, which had been severely suppressed by ionizing radiation.
Our results demonstrate that TS has the potential to protect tissue from radiation injury beyond the hematopoietic system by improving structural integrity, inhibiting apoptosis and enhancing cell proliferation in vital gastrointestinal tissue in mice exposed to high doses of 60
Co γ-radiation. TS did not protect mice when administered as a mitigator after irradiation [14
] and it can be used only as a radio-protector. TS will be of use to the military where they are aware of potential incidences of exposure, such as missions where a significant possibility of exposure to radiation exists. Furthermore, TS could also be used in limited situations where the potential for exposure will continue long after a nuclear accident and new workers might be brought in who may benefit from pretreatment with such an agent. TS has been used at a dose of 400 mg/kg to be consistent with recent published results [14
]. The lowest effective dose of TS is 200 mg/kg [36
]. Furthermore, different species require different drug doses based on body surface area [37
]. Protection provided by TS is of great significance because the radiation dose used in this study is equivalent to highly lethal doses for humans. TS appears to be an attractive radiation countermeasure candidate with no known toxicity. It is a candidate for further development as a radiation countermeasure in large animals such as canines, minipigs or nonhuman primates for the ultimate use in humans.