Exposure to ionising radiation causes a series of physiological changes known as acute radiation syndrome (ARS), the severity of which is related to the exposure dose and dose rate [1
]. The haematopoietic tissue is a rapidly proliferating, self-renewing organ, which is highly vulnerable to radiation injury, making it a major dose-limiting organ for triage following radiological accidents [15
], and the status of bone marrow determines the course of treatment with radiation and/or chemotherapy [16
]. Therefore, a major research goal in radiation biology and oncology has been the identification of protectants that can ameliorate radiation-induced ARS.
We have previously demonstrated significant protective and mitigatory effects of DT3 against radiation-induced oxidative stress and lethality [5
]. The present study assessed the relationship between pharmacokinetics of DT3 and its pharmacodynamic properties, to support the development of DT3 as a radiation countermeasure. We examined pharmacokinetics using the dose and route of DT3 that showed optimum efficacy as a radiation protectant; thus 300 mg kg−1
DT3 administered sc has a half-life (t1/2
) of 1.8 h, and is cleared from plasma within 12 h after a single dose. This is comparable with a mouse study wherein an oral dose of DT3 (100 mg kg−1
) had a t1/2
of 3.5 h [18
], and a clinical study with a t1/2
of 2.3 h for humans [19
]. The slightly longer half-lives reported in these studies could be because orally administered DT3 requires a longer time interval for absorption by the intestine and entry into systemic circulation, as opposed to the sc route. The plasma clearance time for DT3 is 12 h after sc administration; this finding is similar to previous reports on the pharmacokinetics of DT3 [18
] and confirms the rapid clearance of DT3 from plasma.
The pharmacodynamic effects of a single dose of DT3 was seen 3–7 days after drug administration in the haematopoietic tissue as increased end-cell counts of lymphoid, myeloid and megakaryocytic lineages. Previous studies have shown that treatment with vitamin E increased the number of colony forming units of erythroid precursors (CFU-E), enhanced erythropoiesis and haemoglobin levels, and corrected experimentally induced anaemia in laboratory animals [20
]. Our data indicate that the stimulatory effect of DT3 is not limited to erythropoiesis, but extends to leukopoiesis and megakaryopoiesis. Indeed, in related studies, DT3 treatment increased progenitor cells and clonogenicity (CFU-E, CFU-GM and CFU-GEMM) in bone marrow of non-irradiated mice [6
]. Several reports have shown that pharmacokinetics of DT3 vary in different tissues; in the pancreatic tissue, which contains high levels of adipocytes, DT3 level peaked at 8 h [23
]. Adipocytes are the most abundant cells in bone marrow [24
], and we hypothesise that uptake of DT3 by bone marrow tissue might result in a longer half-life and clearance, prolonging the time apportioned for DT3 to modulate cell survival pathways and haematopoiesis. HPLC analysis of DT3 content in bone marrow cells is necessary to test this theory.
Earlier, we reported that a single dose of DT3 protected against post-irradiation lethality in mice, with a DRF of 1.27 [5
]. The survival studies presented here corroborate our earlier findings and, further, suggest that tissue-specific DT3 concentrations, rather than plasma DT3 concentration, may be responsible for the pronounced radioprotection observed following DT3 pre-treatment. The cause of death in mice exposed to TBI can be deduced from the time course of mouse survival; death from haematopoietic syndrome peaks between day 10 and 15 after exposure, and therefore 30-day survival studies following exposure are used to evaluate protection against bone marrow death [25
]. In the present study, ~80% of vehicle-treated animals died between days 10 and 14 following exposure to 9.25 Gy. This indicated that the primary cause of death was haematopoietic toxicity and that DT3 prevented lethality by protecting bone marrow. The data presented here on radiation-induced haematological deficits confirm the marked effect of DT3 on blood-forming tissue.
Significant reduction in haematological indices following irradiation reported here is consistent with earlier reports on radiation-induced ablation of bone marrow [26
]. Irradiation severely depleted reticulocytes, WBCs, neutrophils, lymphocytes, platelets and haematocrit counts, an effect that persisted 21–28 days after exposure. This outcome may be attributed to the clastogenic effects of ionising radiation, which include destruction of mature circulating cells, loss of cells from the circulation by haemorrhage or leakage through capillary walls, and reduced reconstitution due to destruction of the stem/progenitor pool [28
]. Lacking DNA, RBCs are highly radioresistant; therefore, the effects of irradiation on RBC counts are not as pronounced. Further, the long circulating lifespan of the RBCs makes anaemia less of an issue in the early period after radiation exposure [29
]. However, radiation-induced neutropaenia and thrombocytopenia can result in opportunistic infections and aberrant haemorrhagic complications within 10 days of exposure, leading to lethality [30
] or interruption of radiotherapy [31
]. DT3 pre-treatment accelerated the reconstitution of peripheral blood cells, including WBCs, neutrophils, platelets and lymphocytes, indicative of a multilineage recovery.
Initially, we attributed this recovery to a potent antioxidant activity of DT3 [5
], together with stimulation of haematopoietic progenitors and activation of pro-survival pathways [6
]. Here, we posed the question: does DT3 treatment also modulate apoptotic and autophagic signal transduction pathways in mouse bone marrow? To this end, we evaluated several key proteins involved in the apoptotic and autophagic pathways.
DT3 pre-treatment without subsequent irradiation did not activate caspase-8, caspase-3 or caspase-7, although cytochrome c
release and caspase-9 expression were increased. This finding supports earlier studies, where tocotrienols (including DT3) did not injure normal, non-neoplastic cells [10
], but were pro-apoptotic in a variety of malignant cells, such as liver [8
], breast [9
], colon [34
], prostate [10
] and pancreas [17
]. In the present study, we report that irradiation of the whole animal induced a concomitant initiation of the extrinsic and intrinsic apoptotic pathways in CD2F1 mouse bone marrow. Increased activation of caspase-8 and caspase-9, accompanied by cytochrome c
release from mitochondria, is a signature event of apoptosis, and compatible with radiation-induced triggering of the death receptors and mitochondrial perturbation. Cytochrome c
associates with procaspase-9 and Apaf-1 (apoptotic protease activating factor 1), and ATP, resulting in the cleavage of caspase-9. Cleaved caspase-8 and caspase-9 converge on the executor caspase-3, triggering its activation and committing the cell to apoptotic death by further cleaving caspase-6 and caspase-7, and poly(ADP-ribose) polymerase [35
]. DT3 pre-treatment reduced activation of caspase-8, and hence the extrinsic pathway, but we did not see any reduction of radiation-induced cytochrome c
release and caspase-9 levels. However, downstream apoptotic executor molecules caspase-3 and caspase-7 were effectively suppressed by DT3.
Intriguingly, we observed that in DT3-treated and irradiated bone marrow, the expression of caspase-9 increased substantially over both normal and irradiated controls. Because a recent publication suggested that inhibition of caspase-9 could prevent the autophagic flux and enhanced cell death due to blockage of cytoprotective autophagy [36
], we asked what, if any, was the correlation between caspase-9 overexpression and the autophagic process?
Along with apoptosis, autophagy represents an alternative type of programmed cell death (PCD-II) [37
]. Nevertheless, recent insights into autophagy indicate that this may be a response that promotes cell survival [38
]. Some evidence even suggests that autophagy might be a protective mechanism for cells exposed to ionising radiation [40
]. Recently, an autophagy-inducing drug (carbamazepine) was reported to be a radiation protectant and mitigator [41
]. Our immunoblot data demonstrate upregulation of beclin-1 by DT3, and a complex effect on LC3-I and LC3-II in irradiated bone marrow, indicative of a possible increase in autophagy. These data suggest that DT3-induced autophagy could function by improving cell survival, and a causal role of caspase-9 is indicated; on the other hand, the precise role of autophagy in radiation protection is not known. Further experiments, including techniques such as electron microscopy and green fluorescent protein-LC3, are required to further examine this possibility.
In summary, the current paper reports several novel findings. First, plasma pharmacokinetic peak of DT3 was 1 h, with a t1/2 of 1.8 h; however, the increase in peripheral blood counts was observed 3–7 days after the initial drug administration in non-irradiated mice. In irradiated animals, the potent radioprotection observed can be linked to DT3-induced accelerated recovery of haematopoiesis. Finally, DT3 suppressed apoptotic death pathways and modulated autophagic markers, indicating that cytoprotection may have spared haematopoietic stem and progenitor cells from radiation clastogenicity.