Accidental radiation exposure or a terrorist attack with a radioactive dirty bomb poses a serious threat to public health. Management of radiation injuries is a complex medical challenge, requiring a careful encounter as well as therapeutic agents administered at the appropriate time following the radiation exposure [1, 23, 32]. HSCs and hematopoiesis are among the tissues/organs most vulnerable to radiation injury. Radiation-induced damage to HSCs leads to HSC cell senescence and defects in HSC self-renewal capacity, and contributes to several manifestations of acute radiation sickness. Currently, there are very few agents that can effectively rescue HSCs from radiation injury when given after radiation exposure . Antioxidant and HSC growth factors have limitations, and the major drawback of these agents is that they should be administrated before radiation or immediately after radiation [33–35]. There is an unmet medical need for identifying and developing effective agents that can be used to rescue lethal-dose radiation injury and enhance all-lineage hematopoietic cell recovery when given after irradiation.
Previously, we reported that TXN mitigates mice from radiation-induced death and enhances HSC recovery when given at 2 h after radiation exposure in a mouse model of radiation injury . Our current study has important clinical relevance. Individuals exposed to radiation may not be aware of the exposure until a few hours later. Therefore, agents that are effective in rescuing victims from radiation injury when given 24 h after radiation exposure have great potential for clinical use. The remarkable ability of TXN to mitigate radiation-induced damage when administered intravenously 24 h after lethal TBI makes it an attractive radiation countermeasure agent for further development in the use against radiation injury. Further supporting this proposition is that TXN is highly competent in ameliorating the radiation-induced hematopoietic injury by facilitating HSC recovery. We have shown that TXN enhanced the recovery of multi-lineages of peripheral blood cells such as WBCs and platelets. TXN-treated mice demonstrated more cellular bone marrow. Importantly, TXN treatment had a higher number of CFU-GEMM, KSL cells, and primitive SLAM + KSL HSCs.
Radiation causes HSC damage through several mechanisms: increased production of ROS and induction of oxidative stress [36, 37]; increased oxidative DNA damage [38, 39]; activation of apoptotic cell death ; enhanced cell senescence [41, 42]; and promotion of HSC differentiation . Cell senescence, an irreversible proliferative arrest, plays a critical role in radiation-induced HSC injury. HSC senescence impairs HSC replication and self-renewal, and thus reduces the HSC long-term repopulating capacity . Originally described as an antioxidant, TXN also plays numerous roles as a transcription factor and signaling molecule [20, 21]. The levels of TXN were found to correlate with organismal lifespan [45, 46]. Pharmacological and genetic inhibition of TXN induced premature senescence in skin fibroblasts and hepatic cancer cells, suggesting a role for TXN in the regulation of cell senescence [47, 48]. Our results demonstrated that TXN-treated mice had lower SA-gal-positive cells compared to saline-treated mice. The effects of TXN in reducing cell senescence after radiation injury were further validated in primary fibroblasts. p16 is one of the vital biomarkers and an important mediator for cell senescence [27, 49]. TXN treatment suppressed the p16 expression level, further supporting the effects of TXN on cell senescence.
Activation of the p38 pathway contributes to the induction of p16 and HSC senescence following exposure to irradiation [43, 50]. It has been shown that radiation causes HSC cell senescence through the activation of the p38 pathway, and the inhibition of p38 activity with a specific inhibitor (SB203580) attenuated radiation-induced hematopoietic cell injury. Inhibition of p38 activity appears to be a promising strategy for HSC proliferation. We have shown that TXN is able to downregulate phosphorylated p38. TXN serves as a potential mediator of redox signaling by ROS-dependent and -independent pathways [20, 51] and is involved in the regulation of multiple biological processes such as antiapoptotic, anti-inflammatory, and mitogenic activities . The major target of TXN in the cytosol is apoptosis signal-regulating kinase 1 (ASK1). ASK1 is a member of MAP3 kinase family, which activates both the c-Jun N-terminal kinase (JNK) and p38 MAPK pathways . TXN binds to ASK1 and prevents ASK1 from full activation, thus downregulating the p38 pathway .
Radiation-induced DNA damage can occur due to the direct effect of radiation on DNA molecules, which accounts for 30–40% of lesions, or by free radicals, which accounts for 60–70% of lesions [39, 53]. Irradiation induces a variety of DNA lesions, including oxidized base damage, abasic sites, single-strand breaks (SSBs), double-strand breaks (DSBs), and DNA protein crosslinks . DSBs are thought to be the most lethal lesion induced by irradiation, as one unrepaired DSB can be sufficient to trigger apoptosis [8, 17, 22]. γH2AX is a vital marker for DSBs. We have found that TXN reduces γH2AX expression in both murine Lin– bone marrow cells and in primary fibroblasts. The reduction in γH2AX expression could be due to less double-strand DNA breaks from its antioxidant function and/or enhanced DNA repair by TXN. Our preliminary data indicate that TXN could upregulate the gene expression of the Fanconi anemia/BRCA DNA repair pathway (data not shown).
Radiation damage is complex and there are many mechanisms underlying radiation damage such as iNOS and cytokines , miRNA regulation , NF-kB activation , caspase-dependent apoptosis , and LC-II-induced autophagy . In addition to reducing cell senescence and downregulating p38 and γH2AX as shown in the current study, TXN likely acts on other signaling pathways and affects various cellular events. TXN can act as a cellular growth factor and promotes the proliferation of B cells and various transformed cells [58, 59]. Since ERK1/2 and JNK are members of the MAPK family, TXN modulation on ERK1/2 and JNK after radiation cannot be excluded. Further studies with these two enzymatic molecules should be explored. Recent studies have implicated TXN in the regulation of cell cycle progression through G2/M  and in the p53-mediated base excision repair pathway . TXN activates the MEKK1-JNK signaling pathway, leading to IkB degradation and NF-kB activation . It has been shown that TXN directly interacts with PTEN, inhibits phosphatase activity and membrane binding of PTEN, and activates the Akt pathway . TXN can translocate to the nucleus and regulates the functions of several transcription factors including Ref-1, GR, HSF1, HDAC4, HIF1a, NFkB, Nrf2, PPARg, RUNX2, and SP1 [63, 64].
In the current study, TXN was given intravenously every other day for five doses. We are currently optimizing thioredoxin administration regimens and testing different administration routes, including intramuscular or subcutaneous injection. Intramuscular or subcutaneous injection will offer a simpler and more practical route of administration, particularly in a mass casualty scenario. Importantly, TXN has several important features that make it an attractive candidate for further development as a radiation mitigator. TXN promotes the recovery of hematopoietic stem cells and enhances the recovery of multiple lineages of hematopoietic cells. This is significant as G-CSF only works on myeloid progenitors and only enhances the recovery of neutrophils. TXN affects and modulates diverse cellular events, including cell senescence, apoptosis, and double-strand DNA breaks. TXN is a ubiquitously expressed endogenous protein, eliminating the concerns of developing immune response following administration. TXN can cross the cell membrane and enter cells efficiently. Therefore, TXN can be simply added into an HSC culture or administered systematically.