Intercellular communications-redox interactions in radiation toxicity; potential targets for radiation mitigation

Abstract

Nowadays, using ionizing radiation (IR) is necessary for clinical, agricultural, nuclear energy or industrial applications. Accidental exposure to IR after a radiation terror or disaster poses a threat to human. In contrast to the old dogma of radiation toxicity, several experiments during the last two recent decades have revealed that intercellular signaling and communications play a key role in this procedure. Elevated level of cytokines and other intercellular signals increase oxidative damage and inflammatory responses via reduction/oxidation interactions (redox system). Intercellular signals induce production of free radicals and inflammatory mediators by some intermediate enzymes such as cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS), NADPH oxidase, and also via triggering mitochondrial ROS. Furthermore, these signals facilitate cell to cell contact and increasing cell toxicity via cohort effect. Nitric oxide is a free radical with ability to act as an intercellular signal that induce DNA damage and changes in some signaling pathways in irradiated as well as non-irradiated adjacent cells. Targeting of these mediators by some anti-inflammatory agents or via antioxidants such as mitochondrial ROS scavengers opens a window to mitigate radiation toxicity after an accidental exposure. Experiments which have been done so far suggests that some cytokines such as IL-1β, TNF-α, TGF-β, IL-4 and IL-13 are some interesting targets that depend on irradiated organs and may help mitigate radiation toxicity. Moreover, animal experiments in recent years indicated that targeting of toll like receptors (TLRs) may be more useful for radioprotection and mitigation. In this review, we aimed to describe the role of intercellular interactions in oxidative injury, inflammation, cell death and killing effects of IR. Moreover, we described evidence on potential mitigation of radiation injury via targeting of these mediators.

Introduction

Nowadays, human exposure to ionizing radiation (IR) is an unavoidable phenomenon. The main source of human exposure in developed countries is medical uses. Also, the use of medical IR sources for diagnostic or therapeutic aims is growing. In addition to clinical applications, exposure to high doses of IR is one of the main threats for human space mission as well as accidental or terrorism related radiological events (Moulder 2004; Valentin 2005). A Knowledge about the mechanisms of IR-induced normal tissues toxicity is one of the main goals in radiation biology. Enhancements in this knowledge can lead to better management of radiation toxicity, improve clinical outcomes of radiotherapy, and also in mitigation of human radiation injury after a nuclear disaster. The major concerns for radiotherapy patients or irradiated people in an accidental disaster are carcinogenesis and organ failure that threaten the lives of irradiated individuals (Moulder 2003).

A large body of experiments have shown that intracellular signaling plays a significant role in radiation toxicity in irradiated cells (Prise et al. 2005). Moreover, intercellular communications between irradiated and non-irradiated cells, induce various damages via a phenomenon known as bystander effect (Azzam et al. 1998). Interestingly, in vitro studies showed that the mechanisms involved in bystander effect are very similar to directly irradiated cells (Najafi et al. 2014; Yahyapour et al. 2018c). Furthermore, inhibition of bystander effect can lead to alleviation of oxidative cellular damage in both directly irradiated and bystander cells (Zhou et al. 2005). This shows that after exposure to radiation, cells release some clastogenic factors that amplify radiation toxicity in irradiated cells and also cause damage to non-irradiated cells (Yang et al. 2005). The radiation toxicity by released signals involves two distinct processes which include the production of danger signals and response of other irradiated or non-irradiated cells to these signals (Yahyapour et al. 2018a). Based on these conclusions, it has been proposed that identification of intercellular signals can help better management of radiation toxicity in radiotherapy patients, deep space missions, as well as nuclear/radiological disasters. Although, several studies have been conducted to define the mechanisms of radiation-induced normal tissue toxicity, radiobiologists still agree that the complete involvement of signaling pathways remain to be known.

Radiation toxicity in radiotherapy

Response of normal tissues to radiotherapy is so dependent on the type of irradiated organs, as well as radiation dose. In conventional radiotherapy, 2Gy of Gamma or X-rays is a typical radiation dose in each fraction. However, in some new radiotherapy techniques such as stereotactic, higher radiation dose in each fraction is available. On the other hand, in some hyper-fractionation techniques, doses lower than 1Gy per fraction may be used. For every radiation dose, DNA is the most critical target within cells, whose interaction with IR may lead to cell death. Following exposure to a radiation dose lower than 1Gy, apoptosis which is caused by DNA damage is the most common type of cell death in irradiated organs. However, in higher doses such as in conventional or stereotactic radiotherapy, necrosis is more common compared to apoptosis (Najafi et al. 2018a). Necrosis is as a result of damage to other organelles in cells such as the membrane. The response of immune cells to each of these cell death types are different. Each of these responses have toxic effects on survived cells. Although, most of the late effects of radiotherapy such as bleeding, fibrosis, pneumonitis, heart disorders, myelopathy etc., are as a result of inflammatory responses following higher doses of IR, anti-inflammatory responses which are common after exposure to lower doses of IR are involved in some other side effects such as fibrosis (Najafi et al. 2017b). Each of these side effects may limit the required radiation dose for tumor, leading to higher possibility of tumor recurrence and reduction of therapeutic ratio.

Radiation toxicity in a nuclear/radiological disaster

Nuclear or radiological disasters are threats to human. Results of studies on an atomic bomb explosion in Hiroshima and Nagasaki showed that exposure to a high dose of IR following nuclear weapon explosion led to deaths some hours to weeks after exposure (Barnaby 1995). Moreover, exposure to a sub-lethal dose of IR can cause appearance of various disorders after some decades (Douple et al. 2011; Kamiya et al. 2015). The most sensitive organs to external exposure to gamma rays or neutron particles includes the bone marrow and gastrointestinal system (Grammaticos et al. 2013). While, in Chernobyl disaster, it has been shown that exposure to beta-emitter particles including radioactive iodine isotopes can cause severe skin burning (Peter et al. 1994). Detrimental effects of IR on these organs may lead to death after some weeks. Some sensitive and late responding organs to IR includes lung, heart, liver and kidney (Peter et al. 1994; Dorr and Meineke 2011). Some evidences proposed that exposure of these organs to a high dose of IR cause death some months to years after exposure (Yahyapour et al. 2017a). Exposure of lung tissue to a high dose of radionuclides received through inhalation, leads to pneumonitis and fibrosis that cause death after some years (Medhora et al. 2012; Mahmood et al. 2013). Although, heart fibrosis may appear 30 years after exposure. By contrast to the lung, heart disorders can be seen following exposure to a low dose of IR (Eldabaje et al. 2015; Boerma et al. 2016).

Autocrine, paracrine and endocrine signals in radiation injury

Damage to a single cell by IR can trigger some signals that amplify further free radical production in irradiated cell. Depending on the irradiated cell type, this can continue for some hours, days or months after exposure (Pazhanisamy et al. 2011; Chang et al. 2015). Hence, exposure of irradiated cells to these signals play a key role in detrimental effects of IR on normal tissues. In addition, secreted signals can migrate to other irradiated cells. Interactions between irradiated cells that further increase oxidative stress and cell death is known as cohort effect (Blyth and Sykes 2011). It plays a key role in amplification of radiation injury by the extent of time that cells are exposed to free radicals, chemokines, cytokines and others (Lorimore et al. 2001). These changes are associated with long-term change in reduction/oxidation (redox) system and cell metabolism (Yahyapour et al. 2018b). Released signals from irradiated cells can also migrate to adjacent non-irradiated cells. This phenomenon is known as bystander effect, which causes activation of redox metabolism in non-irradiated cells (Mothersill et al. 2000). Some secreted signals from irradiated cells may be released to the circulatory system and then stimulate ROS/NO production in distant organs. This phenomenon is known as non-targeted/out-of-field effect (Najafi et al. 2014).

The origin of intercellular signaling after exposure to radiation is DNA damage and cell death. Massive DNA damage and free radical production by IR that are seen following exposure to an accidental nuclear/radiological disaster or radiotherapy lead to cellular death through mitotic catastrophe, apoptosis, necrosis, senescence and autophagy (Maier et al. 2016). Among these cell death mechanisms, mitotic catastrophe cannot trigger secretion of danger signals. By contrast, other types of cell death stimulate immune system responses including both inflammatory and anti-inflammatory responses (Golden et al. 2012). Released danger signals from damaged cells are known as damage-associated molecular patterns (DAMPs). DAMPs are detected by some receptors on cell surface that are known as pattern recognition receptors (PRRs) (Krysko et al. 2012; Multhoff and Radons 2012). Toll like receptors (TLRs) are the most common PRRs in cell surface that detect DAMPs. Some experiments have shown that after an exposure, some TLRs such as TLR2, TLR4, TLR5 and TLR9 are upregulated (Piccinini and Midwood 2010). These TLRs stimulate the expression of some transcription factors such as mitogen activated protein kinases (MAPKs), nuclear factor of kappa B (NF-KB), signal transducer and activator of transcriptions (STATs), and some others (Piccinini and Midwood 2010; Karki and Igwe 2013). Therefore, depending on the TLRs-transcription factor pathway, various types of immune system mediators and cytokines are released into cells and intracellular space. As some of immune mediators are able to produce ROS/NO, these signaling pathways are associated with oxidative damage, chromosomal aberrations and cell death. Thus, secretion of danger alarms can stimulate redox system in targeted, bystander and distant non-targeted cells. Upregulation of transcription factors, immune mediators and free radicals have been reported in all these cells, while the expression of genes profile may be different (Ghandhi et al. 2008; Chaudhry and Omaruddin 2012).

Intercellular mediators in radiation toxicity

As mentioned above, after exposure to IR, DNA damage and cell death lead to the release of some danger alarm into intracellular space. Oxidized DNA in both nucleus and mitochondria is a danger alarm that is secreted to outer space of cells after damage and deletion of a part of DNA. High mobility group box 1 (HMGB1) is a key molecule that is secreted by necrosis cells. This molecule triggers inflammatory cytokines via binding to TLR4. Sulfonyl HMGB1 is another type of this molecule that is secreted following apoptosis and has no stimulatory effect on inflammatory responses (Yang et al. 2015). HMGB1 through interaction with TLR4 upregulates NF-κB and MAPKs, leading to filtration of macrophages and production of inflammatory cytokines such as IL-1, IL-6, IL-8, TNF (Park et al. 2006; Yang et al. 2010). As inflammatory cytokines stimulate ROS and NO generation via redox system, this is associated with oxidative damage in cells that are exposed to HMGB1 (Li et al. 2013). Moreover, HMGB1 is able to provoke ROS/NO production by NADPH oxidase enzymes (Klune et al. 2008). By contrast to inflammatory pathways of cell death by necrosis, removal of apoptotic bodies by macrophages cause release of anti-inflammatory cytokines such as TGF-β and IL-10 in autocrine and paracrine cells (Szondy et al. 2017; Fadok et al. 1998). On the other hand, TGF-β stimulates apoptosis through some signaling pathways such as SMAD and Rho/ROCK pathways (Jang et al. 2002; Schuster and Krieglstein 2002). TGF-β also triggers macrophages and lymphocytes to generate free radicals through some signaling pathway such as iNOS, NADPH oxidase and cyclooxygenase-2 (COX-2). Production of free radicals stimulate further release of HMGB1 by macrophages and monocytes (Tang et al. 2007).

Various studies have shown that after exposure of cells to radiation, some of these mediators are released to intracellular spaces as well as blood circulation. For example, Wang et al. showed that exposure to 4 or 12 Gy X-rays causes dramatic intracellular release of HMGB1 in human skin fibroblast and bronchial epithelial cells, as well as in irradiated rats (Wang et al. 2016). Elevated level of both inflammatory and anti-inflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, IL-33, TGF-β and TNF-α, as well as some siRNAs, miRNAs and vesicles after exposure to IR have been confirmed by several studies (Yahyapour et al. 2017b; Prise and O'Sullivan 2009). Cell-free chromatin, which may be produced after interaction of IR with DNA is another intracellular mediator that can trigger inflammation and oxidative damage in other cells (Mittra et al. 2015). Studies confirmed that oxidized DNA which originates from the nucleus or mitochondrial DNA is able to stimulate inflammation and oxidative stress in adjacent cells (Havaki et al. 2015; Kostyuk et al. 2012). Similar to HMGB1, oxidized DNA can bind to TLR9 and then upregulate inflammatory mediators and cytokines, leading to free radical production in a positive feedback loop (Yahyapour et al. 2017b). An experiment by Mittra et al. revealed that co-culture of dying cells along with live cells cause induction of bystander effect via transition of cell-free chromatin. They showed a rapid entrance of cell free chromatins into live cells and induction of DNA damage. Moreover, they showed that injection of dead Jurkat cells to mice cause stimulation of inflammation and upregulation of inflammatory mediators such as NF-κB, IL-6, TNFα and IFN-γ in different tissues. However, injection of live cells had less effect (Mittra et al. 2017).

These studies show that there is a potent link between radiation-induced DNA damage, cell death, inflammation and oxidative damage. Cytokines, siRNAs, miRNAs, vesicles, HMGB1 and cell-free DNA fragments are intracellular mediators. These mediators amplify cellular damages by chronic ROS/NO production in irradiated cells. Furthermore, these mediators can affect other non-irradiated cells, leading to bystander effect. Although the severity of oxidative damage in bystander cells is lower than directly irradiated cells, this effect increases the risk of carcinogenesis. Inhibition of these mediators can be proposed to ameliorate oxidative stress in directly irradiated, bystander cells and non-targeted tissues (Ghobadi et al. 2017; Fardid et al. 2017; Najafi et al. 2017a).

Intercellular connections

For several years, it has been known that normal activity of cells in tissues depends on communication between cells. After exposure to IR and production of toxic agents or clastogenic factors, intercellular communications which link adjacent cells to each other facilitate the propagation of toxic products between irradiated cells (Autsavapromporn et al. 2011). Also, intercellular communications can transfer clastogenic factors to non-irradiated/bystander cells. Connexin channels including connexin26 (Cx26) and Cx43, are the most important junctions that amplify radiation toxicity or induce bystander effect in non-irradiated cells (de Toledo et al. 2017; Azzam et al. 2001).

Zhao et al. in an in-vitro study showed that exposure of adenocarcinoma HeLa cells to IR upregulates Cx26 in both irradiated and bystander cells. This was associated with increased micronuclei formation. They showed that there is a crosstalk between COX-2 and Cx26 in irradiated and bystander cells. COX-2 upregulation in irradiated cells induces Cx26 in bystander cells, leading to COX-2 expression and mutation in these cells (Zhao et al. 2014). Although, the role of connexins and gap junctions in mediating radiation injury has been confirmed, it has also shown cell specific and radiation quality effects (Autsavapromporn et al. 2013a, 2013b). However, this may not be seen in all cell types (Mothersill and Seymour 1998).

Cytokines

Cytokines are products of immune system cells including macrophages and lymphocytes T. These molecules are the most important mediators for interrelation between immune cells, and also response to DAMPs. In response to IR, cytokines play a central role in promoting various side effects such as inflammation, fibrosis and carcinogenesis. Increased level of cytokines is a marker for radiation toxicity and its modulation can be proposed for alleviating side effects of radiotherapy. Depending on the type of cell death, exposure to IR stimulates the production of inflammatory cytokines such as IL-1, IL-2, IL-6, IL-8, IL-18, IL-33, TNF-α and IFN-γ, or anti-inflammatory cytokines which include TGF-β and IL-10. These cytokines stimulate several signaling pathways that induce inflammation and oxidative damage in irradiated as well as bystander cells. Upregulation of TGF-β in non-targeted tissues can trigger COX-2 and ROS production (Cheki et al. 2018).

Epigenetic modulators

In the last two decades, several studies have shown that miRNAs and siRNAs play a key role in response of normal tissues to IR. Although there has not been any clear understanding of how IR upregulates or downregulates the expression of some miRNAs and siRNAs, it has been confirmed that after exposure to IR, these mediators are released from irradiated cells to intracellular space (Xu et al. 2015). Also, miRNAs and siRNAs can transfer to other cells through vesicles. The role of epigenetic modulators in radiation-induced free radical production in irradiated and bystander cells have been studied. Similar studies on the role of these mediators in radiation induced DNA damage and genomic instability in distant non-targeted tissues have been conducted (Mothersill and Seymour 2012).

Let-7 subfamily of miRNAs that upregulates after exposure of cells to IR are involved in free radical production (He and Jiang 2016). A well-known effect of miRNAs in ROS production and oxidative stress is increasing super oxidase level via suppression of antioxidant enzymes. A good example is upregulation of mir-21 in both targeted and bystander cells. mir-21, which is itself triggered by TGF-β inhibits SOD2 gene expression, leading to decreasing SOD2 activity and damage to irradiated and bystander cells by superoxide (Jiang et al. 2014; Tian et al. 2015; Xu et al. 2014). Suppression of SOD activity and GSH level have been revealed in non-targeted lung tissues (Ghobadi et al. 2017; Najafi et al. 2016).

Cohort/Bystander/Non-targeted signals induce Redox system and amplify radiation injury in irradiated and non-irradiated cells

As previously mentioned, interaction between cells amplify radiation toxicity. The most critical products of intracellular signals involved in radiation toxicity are free radicals. In irradiated organs, intracellular signals are involved in early and late side effects of IR. There are some immune mediators such as COX-2, NADPH oxidase and iNOS that produce ROS and NO after stimulation by cytokines or others. Moreover, some organelles including mitochondria, Endoplasmic reticulum and lysosomes amplify DNA damage after interaction with free radicals.

COX-2

COX-2 is an isoenzyme that plays a central role in inflammation. It also has a key role in radiation injury and continuous ROS production after exposure to IR. Thus, targeting COX-2 has been proposed for protection and mitigation of radiation injury (Cheki et al. 2018). It has been confirmed that COX-2 is involved in radiation toxicity in irradiated and bystander cells, as well as in non-targeted tissues. In-vitro studies have shown that COX-2 can be stimulated by IGF-1–COX-2 and TNF-α–PKC–COX-2 pathways (Zhou et al. 2005; Fang et al. 2016). While in-vivo studies have shown that COX-2 in the lung and bronchia tissues of mice is upregulated up to 30 fold via TGF-βR-COX-2 pathway (Chai et al. 2012, 2013). However, these studies showed that TNF-α is not involved in non-targeted effect. Based on these studies, so far, it has been shown that IGF-1 and TNF-α are the most important mediators in radiation toxicity by COX-2 pathway in targeted cells.

Nitric oxide synthase (NOS)

NO is an important mediator within and between cells in the immune system, cardiovascular, and nervous systems (Calabrese et al. 2007). It is generated from arginine by metabolism of some enzymes known as nitric oxide synthase (NOS). NO is one of the most important mediators that play a key role in oxidative damage in targeted and bystander cells (Tomita et al. 2015; Han et al. 2007). NO has a low weight and higher half-life compared to other free radicals. Hence, enabling it to migrate to distant cells. These properties make it to reach to bystander cells without the need of gap junctions (Cali et al. 2015). NO plays an important role in various signaling pathways of the immune system. In addition, it can be produced by some enzymes such as inducible form of NO synthetize (iNOS). Although, other forms of NO synthesis such as constitutive NOS (cNOS) may be involved in early effects of IR, it seems that iNOS is the main source of NO production for long time following exposure to IR (Hong et al. 2013).

Upregulation of iNOS and subsequent NO production in irradiated and bystander cells are due to inflammatory signaling pathways. In hematopoietic system, which has the most critical organs in radiation disasters, the activation of macrophages can lead to NO production by iNOS for days or weeks after exposure (Hildebrandt et al. 2003). A study showed that irradiation of macrophages upregulates iNOS expression and increases NO production, leading to chromosome damage in bystander cells. Suppression of this enzyme by its selective inhibitor causes a significant reduction in DNA damage (Ghosh et al. 2008). Although, exact signaling pathways of NO production by iNOS in targeted and bystander cells remain to be elucidated, some studies proposed that IL-1β, TNF-α and TGF-β are the main activators (Wu et al. 2017). The role of NO in non-targeted effect is not yet studied.

NADPH oxidase

NADPH oxidase includes five NOX subfamilies (NOX1-5) and two dual oxidases (DUOX1-2). These enzymes are producers of H₂O₂, which help to kill foreign bodies in cells. Moreover, in responses to a wide range of cytokines and growth factors. Studies have revealed that both NOX and DUOX subfamilies have involvement in radiation toxicity in irradiated cells. Also, some limited studies proposed that NOX system may be involved in radiation toxicity in bystander cells (Azzam et al. 2002). After activation, these enzymes have high stability, leading to continuous free radical generation after exposure to IR (Battino et al. 1999). Studies proposed that inflammatory cytokines such as IL-1β, TNF-α and TGF-β are stimulators of NOX1-5, while DUOX1-2 is activated by IFN-γ, IL-4 and IL-13 (Battino et al. 1999; Cho et al. 2017).

A study showed that NOX2 and NOX4 are the main sources of free radical production in bone marrow cells. This study revealed that exposure to IR can upregulate NOX4 for 2 months, leading to the death of bone marrow stem cells and genomic instability (Pazhanisamy et al. 2011). As hematopoietic system failure is a main reason of death after an accidental radiation disaster, modulation of this enzyme may help mitigate death and possible radiation diseases in these organs. In addition to hematopoietic syndrome, suppression of NOX1-5 and DUOX1-2 system pathways have been proposed for mitigation of radiation-induced lung injury, mucositis, enteritis, fibrosis, and etc. (Ameziane-El-Hassani et al. 2015; Piwkowska et al. 2013; Wu and Doroshow 2014a; Najafi et al. 2018b).

Mitochondria

Mitochondria is an ATP source for the energy of cells. This procedure is a result of oxidative phosphorylation that occurs in the mitochondrial inner membrane. The mitochondrial DNA (mtDNA) is located close to the mitochondrial inner membrane and produces superoxide by oxidative phosphorylation after exposure. Experiments have shown that mutation in mtDNA can increase superoxide production by oxidative phosphorylation (Brand et al. 2004; Yoshida et al. 2012). After irradiation, an Increase in Ca²⁺ ions also increases ROS production by the mitochondria (Leach et al. 2001). Under normal conditions, ROS produced by the mitochondria are neutralized by mitochondria antioxidant enzymes such as catalase, SOD2, glutathione (GSH) and glutathione peroxidase (GPx). Oxidative stress occurs when the production of ROS exceeds the levels of these enzymes. Excessive free radical production after damage to mtDNA can react with DNA and intracellular organelles, leading to lipid peroxidation, protein hydroxylation and chromosomal aberrations. Studies have confirmed the key role of the mitochondria in radiation-induced inflammation and chronic oxidative stress in targeted and bystander cells (Tartier et al. 2007; Zhou et al. 2008a, 2008b, 2011; Zorov et al. 2006). In addition, targeting of mitochondrial ROS has been proposed for mitigation of radiation injury (Rwigema et al. 2011) (Fig. 1).

Fig. 1.

Mechanisms of radiation toxicity via activation of redox system by intra and intercellular mediators. a; a single irradiated cell induces ROS production which amplify radiation toxicity, leading to more production of ROS. Necrosis and apoptosis cause stimulation macrophages and lymphocytes T to produce several inflammatory or anti-inflammatory cytokines. This is associated with increased redox activity in irradiated cells, as well as in other adjacent non-irradiated cells.

Suppression of Intercellular-Redox signaling for mitigation of radiation injury

Suppression of cytokines

Suppression of cytokines is an interesting strategy for the amelioration of radiation toxicity. Elevated levels of cytokines including inflammatory and pro-fibrotic cytokines are a result of immunologic cell death (apoptosis and necrosis). Hence, inhibition of these cytokines with their antagonists or via reduction of cell death can ameliorate inflammation and fibrosis induced by IR (Yahyapour et al. 2017a). Using selective inhibitors or other agents have been studied for evaluating possible mitigatory effects of cytokines targeting. Among various cytokines that are increased by IR, IL-4, IL-13 and TGF-β have been proposed for mitigation of radiation fibrosis (Gauter-Fleckenstein et al. 2010b). These cytokines can upregulate some months after exposure to IR, leading to stimulation of fibrosis mediators and collagen deposition in intercellular space (Groves et al. 2016; Wu and Doroshow 2014b). This suggests that chronic stimulation of ROS production by Redox interactions play a key role in the development of fibrosis. IL-4 induces expression of DUOX2, while IL-13 stimulates DUOX1 gene expression. These changes are associated with long term ROS production by these enzymes (Ameziane-El-Hassani et al. 2015). In addition, upregulation of these enzymes can stimulate upregulation of TGF-β. TGF-β is one of the most important players of radiation toxicity in directly irradiated as well as non-irradiated bystander cells (Anscher 2010). TGF-β regulates several signaling pathways that are involved in fibrosis. Among these pathways, TGF-β-Rho/ROCK, TGF-β-Smad2, and TGF-β-NOX4 promote fibrosis after exposure to IR (Dancea et al. 2009; Martin et al. 2000). In addition to these pathways, various studies have proposed that inflammatory cytokines amplify fibrosis induced by IR (Medhora et al. 2012; Oikonomou et al. 2006; Robbins and Zhao 2004).

An in-vivo study by Chung et al. have shown that IL-13 is a potential target for mitigation of radiation-induced lung fibrosis. They showed that exposure to 5 daily fractions of 6 Gy lead to an increase in IL-13, but not for IL-4 in the lung 16 weeks after irradiation. Moreover, their results showed that IL-13 upregulation stimulates TGF-β, leading to phosphorylation and activation of smad2/smad3 signaling and other pro-fibrosis factors such as fibrillin-1, matrix metalloproteinase-2 (MMP-2), and MMP-3. Furthermore, they revealed that targeting of IL-13 can mitigate radiation death induced by lung fibrosis via downregulation of these pathways (Chung et al. 2016). Although, this study proposed no role for IL-4 in lung injury, some other studies have proposed a role for this cytokine (Huaux et al. 2003). Groves et al. showed that IL-4 stimulates macrophage accumulation and activity in irradiated lung mice. They revealed that while inhibition of IL-4 does not prevent fibrosis, it can promote the maintenance of macrophages in lung tissue following irradiation (Groves et al. 2016).

Targeting of TGF-β is the most studied approach for amelioration of radiation injury in normal tissues, as well as increasing therapeutic ratio through inhibition of angiogenesis and tumor growth. In addition to fibrosis progression, TGF-β stimulates oxidative damage via upregulation of ROS producing enzymes such as NOX2, NOX4 and COX-2, and also via stimulation of NO producing enzymes like iNOS. These changes may continue for some days to months after exposure to IR. In the bone marrow, which is the most critical organ in a radiation disaster, ROS generation by activation of NOX2 and NOX4 can continue for some months and involves hematopoietic syndrome and genomic instability. Inhibition of TGF-β has shown reduced oxidative damage and cell death in irradiated bone marrow of mice. Zhang et al. have shown that treatment of mice with SB431542 (a selective TGF-β inhibitor) reduces expression of NOX2 and NOX4, and also free radical production in bone marrow mononuclear cells following exposure to 2-4 Gy of IR. Results also showed an increase in NOX1, but it was independent of TGF-β. The most effective concentration was 1 μM SB431542. This increases the ability of CFU-GM by 60% (Zhang et al. 2013).

In addition to stimulation of ROS/NO production and DNA damage, TGF-β is a potent player in late effects of IR, including fibrosis (Boerma et al. 2013). As earlier mentioned, TGF-β can induce fibrosis via some different signaling pathways. Expression of TGF-β and lung fibrosis is increased after exposure (Rube et al. 2000). Rabender et al. investigated selective targeting of TGF-β for mitigation of radiation-induced fibrosis in the lung and heart after exposure to 11.5 Gy. IPW-5371 as a selective inhibitor of TGF-β was administered 24 h after irradiation at a dose of 10 mg/kg or 30 mg/kg per day. This process continued for 6 or 20 weeks in irradiated groups. Results showed a significant increase in survival and reducing fibrosis. Meanwhile, results indicated that higher dose of TGF-β inhibitor for longer periods of time was more effective. The median survival of mice was increased from 135 days in irradiated mice without TGF-β inhibitor to 180 days in irradiated plus treated mice. Several analyses showed a significant improvement in respiratory function, reduced heart injury, decreased collagen deposition and attenuation of TGF-β/Smad3 signaling (Rabender et al. 2016). In another study by Flechsig et al., they showed that targeting of TGF-β receptor I by LY2109761 produced similar results. Results of these studies was associated with decreased pneumonitis and lung fibrosis, leading to prolonged survival (Flechsig et al. 2012). Other studies confirmed the inhibition of TGF-β via different agents such as activin receptor-like kinase-5 inhibitor, adenoviral vectors, TGF-β antibody, halofuginone (Koh et al. 2015; Nishioka et al. 2004, 2015; Anscher et al. 2006; Xavier et al. 2004).

Some other studies proposed that IL-17 has a role in the development of radiation fibrosis. Wang et al. showed that after irradiation of mice lung with 15 Gy the level of this cytokine elevates after 1 week and reaches its peak after 4 weeks. They showed that attenuation of this cytokine by dexamethasone leads to reducing macrophage infiltration and lung injury (Wang et al. 2014b). In another study, they evaluated targeting of IL-17 on radiation-induced fibrosis and pneumonitis. They used an IL-17 antibody (4 μg, 4 days/month) before exposure for 4 months after chest irradiation with 15 Gy (Wang et al. 2014a). Moreover, inhibition of IL-17 can reduce expression of TGF-β and IL-13 (pro-fibrotic cytokines) and augment that of IFN-γ (Mi et al. 2012).

In addition to selective inhibitors, some studies proposed that attenuation of cytokines by other agents ameliorate late effects of IR such as pneumonitis and fibrosis. Genistein or quercetin as an isoflavonoid, flaxseed as a lignan and EUK-207 as an SOD-catalase mimetic, have shown ability to mitigate thorax radiation injury and reduce TGF-β signaling (Mahmood et al. 2013; Horton et al. 2013; Pietrofesa et al. 2013; Lee et al. 2009). This was confirmed for other SOD mimetic, MnTE-2-PyP(5+) (Yakovlev et al. 2010; Gauter-Fleckenstein et al. 2010a; Archambeau et al. 2013). Due to the synergic effect on tumor therapy after targeting some of these cytokines, agents such as LY2109761 and ZnSO₄ can be proposed as an adjuvant in radiotherapy (Zhang et al. 2011; Melisi et al. 2008; Hardee et al. 2012; Reeves et al. 2007).

Targeting of TLRs

TLRs are mediators between DNA/cell damage and inflammation. Among the known TLRs; TLR2, TLR4, TLR5 and TLR9 are involved in inflammation signaling pathways and redox activation by IR (Yahyapour et al. 2017a). Targeting of these receptors have been proposed for radiation mitigation and protection of normal tissues in some studies. Shakhov et al. examined an agonist of TLR2 for mitigation of radiation injury in mice after whole body irradiation with 9 or 10 Gy gamma rays. Results showed that pretreatment with TLR2 agonist before exposure to 10 Gy gamma rays increases survival by more than 80%, while using placebo does not lead to survival. Moreover, they showed that agonist administration 24 to 48 h after irradiation increases survival more potently. The best time for administering TLR2 agonist was 1 h after irradiation. Their results showed an increase in survival (73% against 7%). More analyses showed an amelioration of inflammatory responses and recovery of hematopoietic system following irradiation (Shakhov et al. 2012). Similar results have been obtained for TLR2/6 agonist (Kurkjian et al. 2017). Targeting of TLR4 has shown ability to mitigate radiation toxicity in spleen, testis, intestine and bone marrow. This was associated with increased survival, reducing apoptosis and protection of CD34+ hematopoietic stem cells (HSC), and suppression of inflammatory and pro-fibrotic cytokines such as IL-1, IL-4, IL-6, IL-13 and TNF-α (Guo et al. 2017).

TLR5 is another target that has shown both radioprotection and mitigation effects. A single administration of CBLB502 (a TLR5 agonist) has shown ability to protect against both hematopoietic and gastrointestinal syndrome following irradiation of mice with 10 or 13 Gy. Results showed that while exposure to 13 Gy caused no survival in irradiated mice, treatment with CBLB502 gave more than 80% survival. Interestingly, survival for mice treated with 150 mg/kg amifostine was lower than CBLB502 (87% against 54%) (Burdelya et al. 2008). Treatment with TLR5 agonist has shown increased survival by 2-3 fold via amelioration of hematopoietic and gastrointestinal syndrome (Krivokrysenko et al. 2015). In addition to early radiosensitive organs in hematopoietic and gastrointestinal systems, targeting of TLR5 has been proposed for mitigation of radiodermatitis and mucositis (Toshkov et al. 2017).

Targeting of ROS/NO producing enzymes

ROS/NO producing enzymes are some immune enzymes that are involved in defense against pathogens as well as inflammatory responses. Moreover, NO and ROS play key roles in various signaling pathways. Overproduction of these enzymes after exposure to IR, and subsequent products induce the production of ROS and NO over a long period of time. Hence, attenuation of these enzymes may be proposed for mitigation of radiation injury. Among these enzymes, COX-2 inhibition has been studied. However, its protective effect may be due to inhibition of inflammatory mediators and cytokines (Cheki et al. 2018). Inhibition of COX-2 has shown ability to mitigate pneumonitis, arthritis, and inflammatory cytokines such as TNF and IL-1 (Khayyal et al. 2009). In addition, COX-2 suppression by celecoxib may reduce oxidative damage in bone marrow cells (Hosseinimehr et al. 2017). Claude et al. evaluated the mitigatory effect of targeting nitric oxide synthase enzymes in C57BL/6 NHsd mice after exposure to 9.5 Gy X-rays. They showed that targeting of NOS enzymes by MCF201-89 with a dose of 10 mg/kg can mitigate lethal dose of radiation. They showed that treatment of mice with MCF201-89 increases survival from 20% up to 80% (Rwigema et al. 2011). It has been shown that suppression of COX-2 and iNOS by melatonin is associated with mitigation of oxidative damage in non-targeted lung tissues of rats (Ghobadi et al. 2017; Fardid et al. 2017).

NADPH oxidase are ROS producing enzymes whose role in IR-induced injury have been widely studied in recent years. However, studies for mitigation of normal tissues injury by suppression of these enzymes are limited. Inhibition of NOX-2 in mice brain has shown ability to ameliorate oxidative injury and inflammatory markers such as TNF-α and MCP-1 (Cho et al. 2017). Another study by Collins-Underwood showed that upregulation of NADPH oxidase in brain cells results from angiotensin II stimulation. In addition, they proposed that targeting of these enzymes can ameliorate neurovascular syndrome and brain injury following exposure to IR (Collins-Underwood et al. 2007). Saka et al. showed that among different subfamilies of NADPH oxidase, NOX4 is upregulated following irradiation of mouse embryonic fibroblasts. They showed that inhibition of NOX4 can reduce ROS production and recruitment of inflammatory cells (Sakai et al. 2018). Targeting of NOX4 has also been proposed for the mitigation of radiation injury in fibroblast cells (Weyemi et al. 2015). These studies indicated that overproduction of these enzymes are associated with increased risk of carcinogenesis, even though it seems that targeting of ROS/NO producing enzymes can reduce the risk of carcinogenesis following an accidental disaster, or second primary cancers after radiotherapy.

Targeting of mitochondria

As mentioned earlier, mtDNA mutation leads to an excessive ROS production by mitochondria. ROS produced by the mitochondria can interact with NO, resulting in nitroxide production. Mutations in complex II in mitochondria resulting from superoxide production can lead to genomic instability by IR (Dayal et al. 2009). Hence, targeting of mitochondrial ROS and nitroxide free radicals can mitigate radiation injury. An in-vitro study by Jiang et al. showed that targeting mitochondrial nitroxide free radicals by [2-(1-oxyl-2,2,6,6-tetramethyl-piperidin-4-ylimino)-ethyl] -triphenyl-phosphonium (TPEY-Tempo) reduces mitochondrial apoptosis and increases cell survival. Mouse embryonic cells were irradiated with 10 Gy gamma rays and then incubated by 10 μM TPEY-Tempo. Results showed that TPEY-Tempo neutralize nitroxide and prevent the release of cytochrome C, leading to decreasing apoptosis. Furthermore, they showed that the major mitigatory effect of TPEY-Tempo is not a result of direct scavenging of free radicals and is mediated by mitochondria-targeted nitroxides (Jiang et al. 2009). Similar results have been shown for S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) on same cells (Jiang et al. 2008). The mitochondria-targeted nitroxide (JP4-039) is another agent that has been shown to mitigate murine hematopoietic progenitor cells (Rajagopalan et al. 2009).

The mitigatory effect of mitochondria targeting has also been shown in in-vivo studies. Results of a study by Claude et al. revealed the mitigatory effect of JP4-039 in a C57BL/6 NHsd mice model. Total body of mice was irradiated with 9.5 Gy and 24 h after exposure received 5 mg/kg JP4-039. This method increased survival from 20% to 70% (Rwigema et al. 2011). Another study showed that treatment with 10 mg/kg JP4-039 after exposure to 9-9.25 Gy total body irradiation mitigates hematopoietic syndrome by increasing bone marrow progenitor cells (Goff et al. 2011). JP4-039 has shown ability to ameliorate radiation-induced distant bone marrow suppression in mice, which gives an indication of its potential to suppress non-targeted signals (Willis et al. 2018; Berhane et al. 2014). JP4-039 can be proposed for clinical radioprotection of normal tissues in patients with cancer since it has shown no protective effect on some tumor cell types in mice (Shinde et al. 2016).

Summary and conclusion

The aim of this review was to clarify the mechanisms by which IR secrete intracellular mediators that cause cellular damage and tissue failure following exposure to radiation. The most important final product of these mediators that amplify radiation toxicity are free radicals such as ROS and NO. Knowledge of these mediators which are involved in radiation toxicity can help mitigate death and other organ damages in radiation disaster. This can also help to alleviate normal tissue damage in radiotherapy, thus improve cancer therapy outcome. Among various type of intracellular mediators, inflammatory cytokines such as IL-1β, TNF-α and TGF-β, pro-fibrotic cytokines such as IL-4 and IL-13, microRNAs including mir21, HMGB1 and oxidized DNA are known. These mediators induce free radical production and some other signaling pathways that extend the acute reaction of normal tissues.

The long term generation of intracellular mediators that can be seen during chronic inflammation has a central role in late effects of radiotherapy such as fibrosis, pneumonitis, enteritis, mucositis, bleeding as well as carcinogenesis. Furthermore, modulation of these mediators is a potential strategy for mitigation of possible radiological/nuclear disasters. In this situation, based on the injured organ, each of these mediators can be proposed for alleviation of radiation damage that may threaten the life of exposed people. Therefore, targeting these mediators or signaling pathways reduces risk of carcinogenesis.

The simplest strategy for the mitigation of radiation injury is administration of a high dose of antioxidants such as selenium, ascorbic acid, melatonin, resveratrol etc. Similarly, treatment with some mitochondrial antioxidants have shown promising results. Among various agents, mitochondrial ROS antioxidants have shown promising results. The application of these antioxidants in radiotherapy is associated with doubt, hence this strategy is not recommended in clinical use of IR. However, the targeting of other mediators is a more useful strategy. For example, targeting of TGF-β, IL-4 and IL-13 has been proposed for alleviation of fibrosis and bone marrow toxicity, while the inhibition of IL-1β and TNF-α can reduce inflammatory reactions such as pneumonitis, arthritis, mucositis, dermatitis and others.

Targeting of some TLRs including TLR2, TLR4, TLR5 and TLR9 is other strategy, which has shown interesting results. Inhibition of these mediators, in addition to amelioration of inflammation in irradiated organs, reduce activity of ROS producing enzymes, including COX-2, NADPH oxidase subfamilies, iNOS, and others. Moreover, superoxide production by mitochondria can be reduced. In a study, it has shown more protective effect and survival compared to amifostine. Hence, their administration can be proposed as a potent antagonist.

Studies conducted to define the role of gap junctions in radiation toxicity in cohort and bystander effect proposed that inflammation facilitates migration of clastogenic factors to adjacent cells. Therefore, inhibition of these mediators can reduce tissue toxicity through amelioration of inflammation, reduction of redox system and suppression of cell to cell contact.

So far, it has been confirmed that mir21 is a key player in oxidative damage by suppression of SOD2 activity. Stimulation of antioxidant enzymes by some agents such as melatonin and selenium can reverse inhibition of SOD activity. For some complicated situations such as in whole body exposure in a radiological or nuclear accident, using a combination of agents to neutralize free radicals and ameliorate inflammatory responses is a more useful strategy.

Compliance with ethical standards

Conflict of interest

No