Antioxidant Activity

An illustration of different targets of radioprotectors. The targets are free radical stress, oxidative stress, cellular response, cell cycle, D N A repair pathway, and cell death.

Fig. 11.5
General therapeutic approaches to develop novel radioprotective agents. IR, directly or indirectly, causes damage to macromolecules such as DNA, lipids, and proteins. As a result, oxidative stress is generated, which either triggers DNA damage repair or induces p53-mediated cell disorders, such as cell cycle arrest and cell apoptosis. When the damage exceeds the cell’s ability to repair itself, the cell appears to follow the death program. The protective activities of potential radioprotectors should target such phases/mechanisms (described in blue dotted box) with the aim to shield the normal cells from harmful insults of irradiation. Inspired from/based on “General principles of developing novel radioprotective agents for nuclear emergency” from Radiation Medicine and Protection (Volume 1, Issue 3, Pages 120–126), by Du et al. 2020, Copyright Elsevier (2022)


An illustration of different targets of radioprotectors. The targets are free radical stress, oxidative stress, cellular response, cell cycle, D N A repair pathway, and cell death.


Radioprotectors should prevent/suppress the formation of radiation-induced free radicals (most of them are produced during radiolysis with water), thereby inhibiting their reactions with biomolecules, reducing the incidence of DNA strand breaks, and preventing the occurrence of cellular malfunction (more detail in Chap. 2). Since free radicals are short-lived (approximately 10−10 s) and interact rapidly with biomolecules, it is necessary that radioprotectors are present in sufficient concentration in the cellular milieu, at the time of radiation exposure.


Molecules or compounds which increase the activity or expression of antioxidant enzymes are also considered radioprotectors. Many antioxidants have the potential to act as radioprotectors; however, not all antioxidants offer radioprotection, and this paradox may be explained by the relative activity of a compound when reacting with radiation-induced reactive species compared with those generated under H2O2 induced oxidative stress. Conventional antioxidants may not be able to scavenge this less reactive secondary species because either they do not accumulate in proximity to the secondary radicals or they may not have enough kinetic reactivity to scavenge them effectively. Thiols (e.g., amifostine), hydrophilic antioxidants (e.g., GSH), and newly developed cyclic nitroxides have adequate reactivity to effectively scavenge •OH and secondary radicals as well.


Molecules or events that play a role late in signaling and IR-induced apoptotic pathways may act as potential targets for post-irradiation interventions.
ATM/ATR is activated by DNA damage and DNA replication stress; however, they often work together to signal DNA damage and trigger apoptotic cell death by upregulating proapoptotic proteins such as apoptotic protease-activating factor-1 (Apaf-1), phorbol-12-myristate-13-acetate-induced protein 1 (Noxa), and Bcl2-associated X (Bax) after IR.

Pifithrin (PFT)-μ (2-phenylethynesulfonamide) directly inhibits p53 binding to mitochondria as well as inactivates the antiapoptotic proteins Bcl-xL and Bcl-2 on the mitochondrial surface, thereby suppressing subsequent release of cytochrome c and apoptosis, whereas PFT-μ reversibly inhibits transcriptionally mediated p53-dependent apoptosis.

Signal transducer and activator of transcription 3 (STAT3) can be activated by various growth factors and protects against IR damage. The protection mediated by STAT3 is attributed to its genomic actions as a transcription factor (such as upregulating genes that are antioxidative, antiapoptotic, and proangiogenic, but suppressing anti-inflammatory and antifibrotic genes) and other nongenomic roles targeting mitochondrial function and autophagy.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a well-characterized ubiquitous master transcription factor, whose activity is tightly controlled by cytoplasmic association along with its redox-sensitive transcriptional inhibitor Kelch-like ECH-associated protein 1 (Keap1). A well-known mechanism of activation of Nrf2 signaling protects cells against radiation-induced oxidative stress and also maintains cellular reduction-oxidation homeostasis. Upon oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus to activate a series of antioxidant response elements, such as GPx, SOD, CAT, and heme oxygenase-1 (HO-1), increasing total cellular antioxidant capacity (TAC), accompanied by suppressed expression of inflammatory-related genes, avoiding oxidative stress and excessive inflammatory response, which is particularly important in radioprotection.

Heat-shock proteins (HSPs), molecular chaperones, are induced in cells during stress conditions. Importantly, HSPs are cytoprotective and can mediate cell and tissue repair after IR-induced deleterious effects. Higher cytosolic levels of HSPs have been shown to induce radioprotective effects by interfering with apoptotic pathways.

Peroxisome proliferator-activated receptor-γ (PPAR-γ), ligand-activated transcription factors, is a part of the nuclear hormone receptor family. It suppresses IR-induced survival signals and DNA damage responses and enhances IR-induced apoptosis signaling in human cells.


ATM/ATR is activated by DNA damage and DNA replication stress; however, they often work together to signal DNA damage and trigger apoptotic cell death by upregulating proapoptotic proteins such as apoptotic protease-activating factor-1 (Apaf-1), phorbol-12-myristate-13-acetate-induced protein 1 (Noxa), and Bcl2-associated X (Bax) after IR.


Pifithrin (PFT)-μ (2-phenylethynesulfonamide) directly inhibits p53 binding to mitochondria as well as inactivates the antiapoptotic proteins Bcl-xL and Bcl-2 on the mitochondrial surface, thereby suppressing subsequent release of cytochrome c and apoptosis, whereas PFT-μ reversibly inhibits transcriptionally mediated p53-dependent apoptosis.


Signal transducer and activator of transcription 3 (STAT3) can be activated by various growth factors and protects against IR damage. The protection mediated by STAT3 is attributed to its genomic actions as a transcription factor (such as upregulating genes that are antioxidative, antiapoptotic, and proangiogenic, but suppressing anti-inflammatory and antifibrotic genes) and other nongenomic roles targeting mitochondrial function and autophagy.


Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a well-characterized ubiquitous master transcription factor, whose activity is tightly controlled by cytoplasmic association along with its redox-sensitive transcriptional inhibitor Kelch-like ECH-associated protein 1 (Keap1). A well-known mechanism of activation of Nrf2 signaling protects cells against radiation-induced oxidative stress and also maintains cellular reduction-oxidation homeostasis. Upon oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus to activate a series of antioxidant response elements, such as GPx, SOD, CAT, and heme oxygenase-1 (HO-1), increasing total cellular antioxidant capacity (TAC), accompanied by suppressed expression of inflammatory-related genes, avoiding oxidative stress and excessive inflammatory response, which is particularly important in radioprotection.


Heat-shock proteins (HSPs), molecular chaperones, are induced in cells during stress conditions. Importantly, HSPs are cytoprotective and can mediate cell and tissue repair after IR-induced deleterious effects. Higher cytosolic levels of HSPs have been shown to induce radioprotective effects by interfering with apoptotic pathways.


Peroxisome proliferator-activated receptor-γ (PPAR-γ), ligand-activated transcription factors, is a part of the nuclear hormone receptor family. It suppresses IR-induced survival signals and DNA damage responses and enhances IR-induced apoptosis signaling in human cells.


In the search for an effective radioprotective agent, the Walter Reed Army Research Institute (USA) screened approximately 4500 compounds from the late 1950s. Cysteine was the first agent to confer radiation protection in mice after total body irradiation (TBI) in 1949. Later, various synthetic compounds with the aminothiol group were developed and proved to be highly effective in preclinical models [4]. Among them, the most effective was WR-2721 or amifostine, a prodrug activated by alkaline phosphatase to an active sulfhydryl compound WR-1065, and at this moment, it is the only cytoprotective agent specifically approved by the FDA as a radioprotector (Fig. 11.6). The efficacy of amifostine is attributed to the free radical scavenging, along with DNA protection and repair, all of which are coupled with the initial induction of cellular hypoxia. At the cellular level, amifostine has significant effects on cell cycle progression and has antimutagenic and anticarcinogenic properties [5]. In fact, amifostine indirectly induces the expression of proteins involved in DNA repair and triggers antiapoptotic pathways [6] and expression of antioxidant enzymes. Some authors have also proposed that it may enhance protective effects by increasing nuclear accumulation and inducing transcription factors related to p53 expression [7].

An illustration of the mechanism of Amifostine. It gives radioprotection to normal cells and no radioprotection to cancer cells. After hydrolysis, Amifostine becomes W R 1065 that binds to reactive nucleophiles, resulting in D N A protection in normal cells and D N A damage in cancer cells.

Fig. 11.6
Mechanisms of radioprotection by amifostine


An illustration of the mechanism of Amifostine. It gives radioprotection to normal cells and no radioprotection to cancer cells. After hydrolysis, Amifostine becomes W R 1065 that binds to reactive nucleophiles, resulting in D N A protection in normal cells and D N A damage in cancer cells.


Moreover, WR-1065 accumulates more rapidly in normal tissues than in malignant cells, because the concentration of membrane-bound alkaline phosphatase tends to be higher on normal cells. Moreover, the lower vascular supply and the acidic environment of many tumors reduce the rate of dephosphorylation of WR-2721 and its uptake. It thus seems to be a really unique molecule that might potentiate radiotherapy (RT) efficacy in two opposite ways at the same time [8]. The US FDA has approved the use of amifostine in preventing/reducing xerostomia (dry mouth) in head and neck cancer patients undergoing RT [5]. It has also been assayed in clinical trials to reduce mucositis, dysphagia, dermatitis, and pneumonitis during radiotherapy of head and neck cancers [9].


However, like other radioprotective aminothiols, the safety profile of amifostine has considerable limitations. Although the side effects such as nausea, vomiting, and hypotension are not life threatening, they can further aggravate the gastrointestinal syndrome. As it will be exposed latter, amifostine has been assessed in combination with other FDA-approved drugs (growth factors, cytokines, vitamin E, metformin, etc) looking for additive or synergistic radioprotective effects to prevent Acute Radiation Syndrome (ARS). Nevertheless, in most of cases none of these novel strategies completely counteracts amifostine’s toxic side effects at the doses needed to be efficacious as radioprotector [5].


Dimethyl sulfoxide (DMSO) has been shown to prevent the loss of proliferative lingual epithelial stem and progenitor cells upon irradiation by facilitating DNA DSB repair, thereby protecting against radiation-induced mucositis without tumor protection. Given its high efficacy and low toxicity, DMSO appears to be a potential treatment option to prevent radiation-induced oral mucositis [10].


GSH (L-γ-glutamyl-L-cysteinyl-glycine) plays a crucial role in the detoxification of reactive oxygen species, H2O2, lipid peroxyl radicals, peroxynitrites through enzymatic reactions, such as those catalyzed by GPxs, glutathione-S-transferases (GSTs), formaldehyde dehydrogenase, maleylacetoacetate isomerase, and glyoxalase I [11]. GSH not only protects DNA and other biomolecules against oxidative stress and radioinduced damages, it is also essential to activate DNA repairment mechanisms, to activate proliferation and to avoid radio-induced cell death [12]. In fact, the selective depletion of GSH in cancer cells has been shown to have potent radiosensitizing effects on tumor cells [13].


N-acetylcysteine (NAC) has a powerful antioxidant capacity, preserves GSH cellular levels, and prevents oxidative stress-induced apoptosis. NAC treatment (300 mg/kg, subcutaneous), starting either 4 h prior to or 2 h after radiation exposure reduced early deaths in abdominally irradiated (X-rays, 20 Gy) C57BL/6 mice, attenuating gastrointestinal syndrome [14]. More recently, preclinical studies have evidenced that NAC can prevent/reduce cardiac, ovarian, renal, and testicular radiation-induced toxicity in rats. Nevertheless, NAC and GSH cannot be used as a radioprotector in cancer patients because they also enhance antioxidant defenses in cancer cells and may increase their metastatic potential [12].


Treatment with erdosteine (a homocysteine derivative) before γ-radiation exposure ameliorated nephrotoxicity and altered kidney function in rats. It is a potent scavenger of free radicals, increases GPx and CAT activity, and reduces oxidized glutathione levels displaying almost normal concentrations with respect to the irradiated group. Moreover, IL-1, IL-6, and TNF-α circulating levels were also significantly improved thus erdosteine provide substantial protection against radiation-induced inflammatory damage as evidenced in the biochemical and histopathological samples [15].


Phosphorothioates and other aminothiols are usually administered shortly before irradiation. They have been hypothesized to act as radioprotectors by one or a combination of the following effects: scavenging radiation-induced free radicals before their reaction with biomolecules; inducing hypoxia; scavenging metals; repairing DNA damage through hydrogen donation to carbon-centered radicals; and stabilizing genome. Moreover, high doses of phosphorothioates administered to mice before radiation have demonstrated anticarcinogenic effects [4]. However, as it happens with other more powerful thiolic radioprotectors (such as amifotine), its use is limited due to undesirable side effects.


NRs, like Tempol, JP4–039, XJB-5-131, TK649.030, or JRS527.084, are stable free radicals containing a nitroxyl group (-NO.) with an unpaired electron. The action of nitroxides to metabolize ROS is ascribed primarily to cyclic one- or two-electron transfer among three oxidation states: the oxoammonium cation, the nitroxide, and the hydroxylamine. Nitroxides undergo a very rapid, one-electron reaction to the corresponding hydroxylamine, which has antioxidant activity. In addition to their ability to neutralize free radicals, NR can easily diffuse through the cell membranes (and have SOD-like activity) (Fig. 11.7), prevent Fenton and Haber-Weiss reactions by oxidation of transition metal ions to a higher oxidation state, confer catalase-like activity on heme proteins, and inhibit lipid peroxidation. NRs are able to mitigate TBI-induced hematopoietic syndrome, when are administered before or as late as 72 h after radiation exposition [16].

An illustration of oxidative and reductive reversible chemical processes. Hydroxylamines form oxoammonium with the loss of two electrons. Hydroxylamines and oxoammonium form nitroxide radicals, an antioxidant, with the loss of one electron.

Fig. 11.7
Radioprotective properties of cyclic nitroxides include scavenger free radical capacity and SOD-like activity. Adapted from “Nitroxides as Antioxidants and Anticancer Drugs,” by Lewandowski M. and Gwozdzinski K. 2017, Licensed under CC BY 4.0


An illustration of oxidative and reductive reversible chemical processes. Hydroxylamines form oxoammonium with the loss of two electrons. Hydroxylamines and oxoammonium form nitroxide radicals, an antioxidant, with the loss of one electron.


Gramicidin S-derived nitroxide (JP4–039) is an effective TBI mitigator when is delivered intravenously up to 72 h after exposure. JP4–039 treatment ameliorated head and neck radiation-induced mucositis and distant marrow suppression in mice [17]. In a comparative study with other four nitroxides, JP4-039 demonstrated the best median survival after radiation exposition [18]. The potential of this type of molecules as radioprotectors and/or mitigators has raised the interest of researchers, and nitroxidic structures has evidenced radioprotective activity. That is the case of nitronyl- nitroxide radical spin-labeled resveratrol [19].


Primary experiments performed in the 1960s reported that antibiotic treatment and a single transfusion of allogeneic platelets significantly reduced mortality among monkeys exposed to TBI X-irradiation. Oral administration of streptomycin, kanamycin, neomycin, or gentamicin with drinking water (4 mg/mL) for 2 weeks before supralethal TBI (28.4 Gy) prolonged mean survival in mice (8.2–8.9 days vs. 6.9 for controls) [20]. The efficacy of antibiotics and other antimicrobials (antifungal and antiviral agents) is best explained as a countermeasure for radiation-induced neutropenia and immunosuppression.


Tetracycline and ciprofloxacin protected human lymphoblastoid cells, reducing radiation-induced DNA double-strand breaks (DSB) by 33% and 21%, respectively. Their radioprotective efficacy was attributed to the activation of the Tip60 histone acetyltransferase and altered chromatin structure [21]. Tetracycline hydrochloride is a free radical scavenger, protects DNA, and increases survival of C57BL/6 mice by 20% upon a lethal radiation dose of 9 Gy [22].


Mucositis is the most common side effect of RT for head and neck cancers. Preventive measures used in clinical medicine include good oral hygiene, dental and periodontal treatment, avoidance of tobacco products and alcohol, and frequent oral rinsing with a bland mouthwash such as povidone-iodine. Nonabsorbable antibiotic lozenges and/or antifungal topical agents (i.e., bicarbonates and amphotericin B) are also recommended [23].


Minocycline prevented radiation-induced apoptosis and promoted radiation-induced autophagy in primary neurons in vitro. Minocycline also increases the counts of splenic macrophages, granulocytes, natural killer cells, and lymphocytes, and accelerates neutrophil recovery in C57BL/6 mice exposed to 1-3 Gy 60Co γ-rays. The mechanisms involved in this radioprotective effect were the suppression of cytokines that could prevent hematopoiesis (e.g. macrophage inflammatory protein-1α, TNF-α and INF-γ) and the increased production of IL-1α and β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) [24].


Furazolidone (FZD) is an antimicrobial agent effective on both Gram+ and Gram− bacteria by interfering with bacterial oxidoreductase activity. In vitro, FZD treatment reduced unstable chromosomal aberrations (CAs) (such as acentric and dicentric chromosomes (DC)), chromosome breaks, and radiosensitivity of intestinal epithelial cells. Ma et al. [25] showed that FZD treatment significantly improved the survival of lethal dose-irradiated mice, decreased the number of micronuclei (MN), increased the number of leukocytes and immune organ indices, and reversed the apoptosis and autophagy in the small intestine, thus restoring intestinal integrity. Their experiments showed that irradiation resulted in villous shortening and crypt dilation accompanied by epithelial atrophy or slough, and even marked edema and inflammatory cell infiltration, and how FZD significantly induced damage recovery. FZD is a clinically used antibiotic with few side effects and has been proposed as an efficacious medical countermeasure (MCM). However, detailed radiation protection activity and clinical applications need to be further studied, because radioprotective efficacy of antibiotics has not yet been tested in clinical trials.


Considerable information from in vivo, ex vivo, and/or in vitro studies suggests that crude extracts, fractionated extracts, isolated phytoconstituents, and plant polysaccharides from various plants such as Alstonia scholaris, Centella asiatica, Hippophae rhamnoides, Ginkgo biloba, Ocimum sanctum, Panax ginseng, Podophyllum hexandrum, Amaranthus paniculatus, Emblica officinalis, Phyllanthus amarus, Piper longum, Tinospora cordifolia, Mentha arvensis, Mentha piperita, Syzygium cumini, Zingiber officinale, Ageratum conyzoides, Aegle marmelos, and Aphanamixis polystachya protect against radiation-induced lethality, lipid peroxidation, and DNA damage [26]. From these extracts, polyphenolic and nonpolyphenolic active principles and a range of secondary metabolites (e.g., carotenoids, alkaloids, sulfur compounds), already known for their anticancer properties, have also demonstrated radioprotective potential. Although many have been tested for brevity, this chapter focuses on those with the most promising results in vivo.


Over the last decades, plant-derived polyphenols have been screened for their potential ability to confer radioprotection. The free radical scavenger potential and antioxidant activity of polyphenols depends, in part, on their ability to delocalize electron distribution, resulting in a more stable phenoxy group. Moreover, intercalation in DNA double helices induces stabilization and condensation of DNA structures making them less susceptible to free radicals’ attack, reducing genotoxic damage induced by IR [27]. They are capable of trapping and neutralizing lipoperoxide radicals and can chelate metal ions (i.e., iron and copper), which play an important role in the initiation of oxidative stress reactions [28, 29]. Polyphenols radioprotective efficacy is mainly attributed to its (Fig. 11.8) antioxidant and antiinflammatory properties, to their capacity to detoxify free radicals, eliciting DNA repair pathways, stimulating the recovery of hematopoietic and immune functions [28, 29].

An illustration of the biological properties of polyphenols. They are neuroprotection, cardiovascular protection, anti-inflammatory, anti-diabetic, anti-microbial, anti-viral, anti-aging, anti-proliferative, anti-tumor, and cytoprotective against radiation with anti-oxidant properties.

Fig. 11.8
Radioprotective and biological properties of polyphenols


An illustration of the biological properties of polyphenols. They are neuroprotection, cardiovascular protection, anti-inflammatory, anti-diabetic, anti-microbial, anti-viral, anti-aging, anti-proliferative, anti-tumor, and cytoprotective against radiation with anti-oxidant properties.


In addition to the biochemical scavenger theory, there is also evidence of another potential mechanism by which polyphenols activate Nrf2, exhibiting cellular protection against excessive ROS production, oxidative stress, and inflammation as well. Since the chemical features of these natural organic compounds are analogous to phenolic substances, their antioxidant and antiradical/scavenging radical (such as H2O2, 2,2-diphenyl-1-picrylhydrazyl) properties may be correlated positively with the number of hydroxyl groups bonded to the aromatic ring. They can exert their protection against environmental stimuli with the aid of remarkable antioxidant power by balancing the organic oxidoreductase enzyme system, regulating antioxidant-responsive signaling pathways, and restoring mitochondrial function.


Although topically administered polyphenols may provide strong antioxidant protection, various challenges still exist and are onerous as well: (1) improving the bioavailability of polyphenols more effectively in order to promote their effectiveness is challenging; (2) if the polyphenols are extracted as the medicine or as health supplements, attention should be paid to the activity loss and degradation of polyphenols during the extraction process; (3) the effects cannot be generalized for all kinds of polyphenols, because each polyphenol has its own unique features; and (4) polyphenols have limited water solubility, and so it is important for polyphenols to be involved in rapid metabolism and also prove its chemical stability and solubility under in vivo conditions. To overcome this limitation, Obrador et al. [30] suggested a few feasible options: structural modifications of natural molecules (e.g., in the form of salts) to increase their hydro-solubility for intravenous administration or oral formulations to increase their bioavailability (e.g., cocrystals, nanoparticles, nanozymes). The promising phytochemical, pharmacodynamic, and toxicological research into the properties of polyphenols may serve as potential candidates for radioprotection in the near future.


Apigenin exhibits anticancer properties associated with its prooxidant activity, inhibiting tumor growth and inducing cell cycle arrest and apoptosis. Apigenin pretreatment displayed efficacy for radioprotection in TBI Swiss albino mice by reducing cytogenetic alterations and biochemical and hematological changes [31]. Further, when apigenin was administered intraperitoneally at a dose level equal to 15 mg/kg body, it was found to ameliorate radiation-induced gastrointestinal (GI) damages and restore intestinal crypt-villus architecture [32]. These attributes could be due to its ability to activate the endogenous antioxidants, suppress lipid peroxidation, and modulate inflammatory (NF-κB) and apoptotic signaling mediator/marker (p53, p21, Bax, caspase-3, caspase-9) expression. The in vivo efficacy of apigenin was also evidenced when it was intraperitoneally administered to mice 3 h after receiving γ-rays [33]. A significant reductions in the level of 8-hydroxy-2-deoxyguanosine (8-OH-dG), suppressed expression of NF-κB and NF-κB-regulated proinflammatory cytokines were observed, thus showing the radioprotective potential of apigenin.


Curcumin, a yellow pigment of turmeric, is naturally found in the rhizome of Curcuma longa and other Curcuma spp. It is an active immunomodulatory agent which has many scientifically proven health benefits, such as the potential to improve symptoms of anxiety, depression, arthritis, and heart health and prevent Alzheimer’s, cancer, and oxidative and inflammatory conditions. Administration of curcumin in patients undergoing RT has demonstrated a dual action: radioprotection to normal cells through its ability to reduce oxidative stress, scavenge free radicals, inhibit transcription of genes related to oxidative stress, and suppress inflammatory response, as well as radiosensitization in tumor cells [34]. Curcumin, administered before or after a single 50 Gy radiation dose, showed protective effect on radiation-induced cutaneous damage in mice by significantly decreasing mRNA expression of early-responding cytokines (IL-1, IL-6, IL-18, TNF-α, and lymphotoxin-beta) and fibrogenic cytokines [35]. Oral administration of curcumin in mouse before irradiation resulted in a significant rise in activities of GPx and SOD enzymes while declining lipid peroxidation significantly, which indicates increased antioxidant status in mouse exposed to different doses of fractionated γ-radiation [36]. These protective qualities of curcumin may be due to free radical scavenging and upregulation of Nrf2 expression.


Ellagic acid (EA), a strong natural antioxidant, has a major protecting role against different diseases associated with oxidative stress and inflammation. It also exerts antiangiogenesis effects via down regulation of vascular endothelial growth factor-2 (VEGF-2) signaling pathways in cancer. The amount and duration of EA used play a significant role in suppressing in vivo and in vitro oxidative stresses. In vitro studies [37] displayed high DPPH radical scavenging and lipid peroxidation inhibition activities of EA. It triggered the actions of antioxidant enzymes such as SOD, CAT, and GPx in V79–4 cells; reduced cell proliferation; and induced apoptosis in human osteogenic sarcoma cells as evidenced by chromosomal DNA degradation and apoptotic body appearance. When the human breast cancer cells (MCF-7) were treated with EA (10μM) and exposed with γ-radiation, the rate of apoptotic cell death in sub-G1 phase of cell cycle was high due to decreased mitochondrial membrane potential, upregulated proapoptotic Bax, and downregulated Bcl2, suggesting EA’s role in tumor toxicity to improve cancer radiotherapy [38].


Epicatechin (EC) is a common flavanol found in tea, cocoa, dark chocolates, and red wine. It has the ability to cross the blood-brain barrier and activate brain-derived neurotrophic factor pathways, suggesting its neuroprotective effects. In addition to general antioxidant activities, it aids with the modulation of metabolism of nitric oxide (NO) and other reactive nitrogen species (RNS). To evaluate the radioprotective effects of EC, Swiss albino mice were administered with EC for three consecutive days before exposing them to 5 Gy 60Co γ-irradiation [39]. EC pretreatment ameliorated γ-radiation-mediated alterations in mice, protected the liver and testis from radiation-induced oxidative stress, prevented systemic and cellular stress, and developed inflammation. It may possibly be due to the influence on the endogenous antioxidant defenses system after TBI in mice [40]. Another study [41] intended to investigate the effectiveness of EC in scavenging mitochondrial ROS and mitigating mitochondrial damage as radiation countermeasure agents by using human and mouse cells. It was observed that preradiation and postradiation treatments with EC mitigated ROS-mediated mitochondrial damage and IR-induced oxidative stress responses in mice. Also, oral administration of EC significantly enhanced the recovery of mouse hematopoietic cells from radiation injury in vivo, suggesting EC as a potentially viable countermeasure agent which is immediately effective against accidental IR exposure.


Epigallocatechin-3-gallate (EGCG) is a natural polyphenolic antioxidant found in a number of plants, predominantly in green tea and black tea and also in small amounts in fruits and nuts. It gets a lot of attention for its potential positive impact on health. It aids weight loss, reduces inflammation, and helps prevent certain chronic conditions, including heart disease, diabetes, and cancers. Pretreatment with EGCG significantly enhanced the viability of human skin cells which were irradiated with X-rays and decreased radiation-induced apoptosis [42]. It was found that EGCG suppressed IR-induced damage to mitochondria via upregulation of SOD2 and induced expression of cytoprotective molecule HO-1 in a dose-dependent manner via transcriptional activation. The therapeutic effects and mechanism of EGCG on radiation-induced intestinal injury (RIII) have not yet been determined; however, Xie et al. recently [43] investigated it both in vitro and in vivo and revealed that treatment with EGCG not only prolonged the survival time of lethally irradiated mice, but also mitigated RIII. Besides, it significantly augmented proliferation and survival of intestinal stem cells and their progeny cells in irradiated mice. Their findings demonstrated that EGCG protected against RIII by reducing the level of IR-induced ROS and DNA damage, inhibiting apoptosis and ferroptosis through activating transcription factor Nrf2-mediated signaling pathway and its downstream targets comprising antioxidant proteins Slc7A11, HO-1, and GPx4, suggesting that EGCG could be a promising medical countermeasure for the alleviation of RIII.


Genistein (GEN), an isoflavonoid compound, is commonly found in soybeans and its products. Mechanistic insights reveal its potential beneficial effects on human diseases such as cancer, by inducing apoptosis and cell cycle arrest. GEN has antiangiogenic, antimetastatic, and anti-inflammatory effects. Besides, various studies of GEN have revealed its radioprotective properties by protecting against radiation-induced DNA damage, scavenging free radicals, and altering cell cycle effects. Davis et al. [44] revealed GEN-induced radioprotection against hematopoietic- acute radiation syndrome (H-ARS) by altering the cell cycle of hematopoietic stem and progenitor cells in a murine model. The extracted GEN displayed protection against IR-induced GI injury and bone marrow toxicity by upregulating the Rassf1a and Ercc1 genes to effectively attenuate DNA damage in a TBI mouse model [45]. Moreover, Song et al. [46] showed that low concentration of GEN (1.5μM) lessened radiation-induced injuries by way of inhibiting apoptosis, alleviating chromosomal and DNA damage, downregulating GRP78, and upregulating HERP, HUS1, and hHR23A. In contrast, high concentration of GEN (20μM) demonstrated radiosensitizing characteristics in cancer cells. The role of genistein as a radiosensitizer will be further discussed in Sect. 11.4.


Naringin, a predominant flavone glycoside, is present in citrus fruits. Manna et al. [47] demonstrated that pretreatment with naringin significantly prevented γ-radiation (6Gy)-induced intracellular ROS-mediated oxidative DNA damage; inhibited radiation-induced G1/S-phase cell cycle arrest by modulating p53-dependent p21/WAF1, cyclin E, and cyclin dependent kinase 2 (CDK2) activation; and reversed the inflammation through downregulating nuclear factor kappa B (NF-kB) signaling pathways and balancing the expression of C-reactive protein, monocyte chemoattractant protein-1 (MCP-1), and iNOS2 at sites of inflammation in murine splenocytes. Besides, naringin pretreatment could effectively deter UVB-mediated DNA damage, alter apoptotic marker expression (Bax, BCl-2, caspase-9, and caspase-3), and potentially modulate NER gene (XPC, TFIIH, XPE, ERCC1, and GAPDH) expression, thereby augmenting DNA repair [48].


Naringenin is present in peppermint and citrus fruits such as oranges, grapefruit, and tangerines. It is endowed with biological effects on human health, which includes a great ability to modulate signaling pathways; efficient impairing of plasma lipid and lipoprotein accumulation; and antiatherogenic and anti-inflammatory effects. To evaluate radioprotective effects of naringenin in vivo, Swiss albino mice were orally administered 50 mg/kg body weight of naringenin prior to radiation exposure [49], and it protected mice against radiation-induced DNA, chromosomal, and membrane damage. Naringenin pretreatment increased antioxidant status and survival chances, inhibited NF-kB pathway, and downregulated radiation-induced apoptotic proteins (p53, Bax, and Bcl-2) in normal cells resulting in radioprotection at the cellular, tissue, and organism levels.


Resveratrol (RV), a natural polyphenol, is produced in several plants in response to stress, injury, and UV radiation. It is present in fruits such as grapes, strawberries, and red wine. It is known for its analgesic, antiviral, cardioprotective, neuroprotective, and antiaging actions. Different doses of RV were administered intraperitoneally to mice prior to total-body γ-irradiation (2 Gy), and it was observed that RV significantly reduced lymphocyte damage in mice caused by γ-radiation due to its ability to scavenge free radicals, restore the levels of intracellular antioxidants (GPx, SOD, CAT activity), and cause cell cycle arrest [50]. RV is also known to have a significant effect in stabilizing p53 and altering proapoptotic and antiapoptotic protein concentration [51]. Zhang et al. [52] treated with RV IR-exposed C57BL/6N mice. RV reduced radioinduced-intestinal injury (upregulating Sirt1 and acetylating p53 expression), improved intestinal morphology, decreased apoptosis of crypt cells, maintained cell regeneration, and ameliorated SOD2 expression, evidencing its radioprotective potential. The role of RV together with pterostilbene as a radiosensitizer will be further discussed in Sect. 11.4.


Pterostilbene (PT), is another stilbenoid compound, structurally similar to RV, present in blueberries, grapes, and other similar fruits. It is an active phytonutrient with many biomedical applications in cancer treatment, insulin sensitivity, cardiovascular diseases, aging, and cognition. Moreover, it has a greater bioavailability, efficacy and lower toxicity than RV [53]. Sirerol et al. [54] evidenced that pterostilbene reduced chronic UVB irradiation-induced skin damage and carcinogenesis in hairless mice through maintaining antioxidant defenses, including GSH, CAT, SOD, and GPx. Recently, a combination of natural polyphenols (PT and silibinin) with a NAD+ precursor and a TLR2/6 ligand was shown to protect mice against lethal γ-radiation, increasing long term survival up to 90% of the treated mice [55].


Caffeic acid/caffeic acid phenethyl ester (CAPE) is found in coffee, tea, chocolate, and colas. It has numerous pharmacological and physiological effects, including cardiovascular, respiratory, renal, and smooth muscle effects, as well as effects on mood, memory, alertness, and physical and cognitive performance. It is essentially regarded as a radiosensitizer by virtue of its inhibition of DNA repair after irradiation. The radioprotective properties of CAPE have also been shown in the bone marrow chromosomes of mice exposed to TBI (1.5 Gy 60Co γ-rays), regardless of its time of administration [56]. Caffeic acid, a known dietary antioxidant, could be used as a supplemental drug which has a dual effect: ameliorating hematopoietic stem cell (HSC) senescence-accompanied long-term BM injury in single (sublethal dose of 5 Gy) TBI and stimulating apoptotic cell death of colon cancer cells in mice [57]. Khayyo et al. [58] intraperitoneally administered CAPE prior to total-head γ-irradiation and observed that the oxidant stress parameters (total oxidant status, oxidative stress index, and lipid hydroperoxide) were significantly reduced, whereas antioxidant parameters (activity of paraoxonase, arylesterase, total GSH levels) were increased in the rat brain tissue, signifying the protective role of CAPE as an important antioxidant against ROS accumulation induced by total-head irradiation. The role of CAPE as a radiosensitizer will be further discussed in Sect. 11.4.1.7.


Sesamol is found in sesame seeds and oil. It has many biological activities and health-promoting benefits such as inducing growth arrest and apoptosis in cancer and cardiovascular cells and enhancing vascular fibrinolytic capacity, antioxidant activity, chemoprevention, antimutagenic, and antihepatotoxic activities. Naturally occurring or synthetic substances of sesamol counteract the damaging effects of oxidation by inhibiting or retarding oxidation reactions. Also, it has the potential to scavenge free radicals and therefore reduces the radiation-induced cytogenetic damage in cells. Kumar et al. [59] investigated its radioprotective potential against radiation-induced genotoxicity in hematopoietic bone marrow of whole-body 𝛾-irradiated (2Gy) mice. Preadministration of 20 mg/kg body weight sesamol reduced the frequency of radiation-induced MN, CAs, and comets (% damaged DNA streak in tail), suggesting its major role in direct scavenging of free radicals to protect bone marrow, spleen, and lymphocytes from radiation-induced cytogenetic damages and genotoxicity. Besides, intraperitoneal pretreatment of sesamol offered protection to hematopoietic and GI systems against γ-radiation-induced injury in C57BL/6 male mice by inhibiting lipid peroxidation; translocating gut bacteria to spleen, liver, and kidney; enhancing regeneration of crypt cells in GI; reducing the expression of p53 and Bax apoptotic proteins in the bone marrow, spleen, and GI; and alleviating the total antioxidant capacity in spleen and GI tissue [60]. Recently, Majdaeen et al. [61] concluded that regular oral consumption of sesamol extract is more effective than consuming it once before irradiation.


3,3′-Diindolylmethane (DIM), a small-molecule compound and a major bioactive metabolite, is formed by acid hydrolysis of indole-3-carbinol (one of the best characterized components in Cruciferae). It can inhibit invasion, angiogenesis, and proliferation and induce apoptosis in tumor cells by modulating signaling pathways involving AKT, NF-kB, and FOXO3 [62]. Chen et al. [63] investigated the radioprotective effects of DIM in normal tissues using a mouse model approach. It was indicated that treatment with DIM increased the expression of some stress-responsive genes without causing DNA damage, delayed radiation-induced cell cycle arrest, and apoptosis. Fan et al. [64] reported that administration of DIM in a multidose schedule protected rodents against lethal doses of TBI up to 13 Gy. Transcriptomic profiling showed that DIM’s mechanism of radioprotection involved regulation of responses to DNA damage and oxidative stress by inducing ataxia-telangiectasia mutated (ATM)-driven DDR-like response, enhancing radiation-induced ATM signaling and NF-κB activation, suggesting its potential role as a MCM in protecting or mitigating adverse effects of RT.


With the understanding that free radicals perpetuate a significant amount of the damage caused by IR, vitamins with antioxidant potential (A, C, and E and its derivatives) have been assayed as radioprotectors (Fig. 11.9). Vitamin A and β-carotenes (lutein, lycopene, phytofluene, phytoene, and others) reduced mortality and morbidity in mice exposed to partial or TBI. Dietary vitamin A offered protection in mice subjected to localized radiation exposure focused on the intestine (13 Gy, TBI) and the esophagus (29 Gy) [30]. A single dose of vitamin A injected intraperitoneally 2 h before 2 Gy of γ-radiation exposition, significantly reduced the number of MN in the bone marrow and the genetic damages, due to its capacity to trap free radicals [65]. Carotenoids such as crocin and crocetin (isolated from the dietary herb saffron) have antioxidant, anti-inflammatory, and antiapoptotic effects. In mice bearing pancreatic tumors, crocin significantly reduced tumor burden, radiation-induced toxicity, and hepatic damage and preserved liver morphology [66] while crocetin also reduced radiation injury in intestinal epithelial cells [67].

An illustration of the vitamins that provide radioprotection with their effects. The vitamins are beta carotene, or provitamin A, ascorbic acid, or vitamin C, retinol or vitamin A, vitamin E, alpha lipoic acid, and vitamin D.

Fig. 11.9
Radioprotective effects of vitamins


An illustration of the vitamins that provide radioprotection with their effects. The vitamins are beta carotene, or provitamin A, ascorbic acid, or vitamin C, retinol or vitamin A, vitamin E, alpha lipoic acid, and vitamin D.


Lutein is a pigment classified as a carotenoid, found in plants such as green leafy vegetables (spinach, kale), fruits, corn, egg yolk, and animal fats. While this pigment plays an important role in eye health, lutein supplements also help to prevent colon and breast cancer, diabetes, and heart disease due to its powerful antioxidant potential. In vitro and in vivo lutein was found to scavenge free radicals and inhibit lipid peroxidation by increasing the activity of CAT, SOD, and glutathione reductase [68]. Lutein showed maximum survival in mice treated with 250 mg/kg body weight against a lethal dose of 10 Gy γ-radiation. Pretreatment of lutein maintained near-normal levels of hematological parameters indicating resistance/recovery from the radiation-induced damages [69]. Furthermore, lycopene has the highest antioxidant activity among carotenoids, and it reduces proinflammatory cytokine expression such as IL-8, IL-6, and NF-κB. Many preclinical studies evidence its radioprotective efficacy, in particular, if it is administered before or as soon as possible after radiation exposure [70].


Vitamin C is the reduced form of ascorbic acid (AA) and a water-soluble vitamin. The intake of vitamin C decreases the risk of getting cataracts after radiation exposition. AA has low toxicity and cost and is easily available, making it an attractive radioprotective agent. Administration of AA before γ-irradiation prevents chromosomal damage in bone marrow cells, mainly due to its scavenging activity of ROS, protecting lipid membranes and proteins from oxidative damage. It has also been reported that AA can prevent the adverse effects of TBI by increasing the antioxidant defense systems in the liver and kidney of irradiated animals [71]. Sato et al. [72] demonstrated the significant radioprotective effect of AA on the ARS in special GI syndrome, especially if it is administered before or not later than 24 h after radiation exposition.


Vitamin E is an essential fat-soluble nutrient with antioxidant, neuroprotective, and anti-inflammatory properties. Vitamin E family includes eight vitamers, four saturated (α, β, γ, and δ) called tocopherols, and four unsaturated analogs (α, β, γ, and δ) referred to as tocotrienols, which are collectively called tocols, with α-tocopherol being the most abundant in human tissues. Tocols administered subcutaneously 1 h prior to or during 15 min postirradiation improved the 30-day survival in mice, and other tocopherol derivatives, such as α-tocopherol-succinate and α-tocopherol-mono-glucoside, have also shown radioprotective effects in vivo. Moreover, subcutaneous injection of γ-tocotrienol (100–200 mg/kg) 24 h prior to 60Co γ-irradiation showed a significant protective effect in mice facing radiation doses as high as 11.5 Gy and increased mice survival rate [73].


Preclinical studies have provided evidence that tocotrienols exert radioprotection at least in part via induction of G-CSF, reducing inflammatory response suppressing the expression of TNFα, inducible NO synthase (iNOS), and IL-6 and 8, as well as inhibiting NF-κB signaling [74]. Endothelial cells activated through IR downregulate the expression of thrombomodulin (TM) and increase endothelial surface expression of adhesion molecules, which allow the attachment of immune cells, and thereby contribute to inflammation and activation of the coagulation cascade. The greater efficacy of tocotrienols is attributed to their higher antioxidant potential and its ability to inhibit HMG-CoA reductase activity (decreasing serum cholesterol levels) and increase TM expression in endothelial cells, which result in antipermeability, anti-inflammatory, and antithrombotic response in order to decrease radiation-induced vascular damages.


Nevertheless, low bioavailability of tocotrienols is an important limiting factor for their use as radioprotectants, and thus a novel water-soluble liposomal formulation of γ-tocotrienol (GT3) has been developed. GT3 has shown to increase the delivery of γ-tocotrienol in the spleen and bone marrow and offered significant radioprotection in vivo [75]. Despite these promising results, the use of vitamin E derivatives as radioprotectants must be evaluated with caution for their potential toxic effects. More recently, several laboratories have assayed the potential synergistic effect of tocols with other radioprotectants, such as pentoxifylline (PTX) (an antioxidant and anti-inflammatory xanthine derivative, approved by the FDA) which increased survival of mice subjected to 12 Gy 60Co γ-irradiation. Efficacy of PTX and α-tocopherol against radiation-induced fibrosis has been observed in animal models and clinical studies, even though the treatment started after radiation-induced fibrosis manifested clinically. Three clinical trials have evaluated if PTX enhances the radiation-protective properties of γ-tocotrienol, but the results of these studies have not yet been published [74]. At least, two randomized controlled trials provided evidence that dietary supplementation of α-tocopherol and β-carotene during radiation therapy could reduce the severity of treatment adverse effects, but these trials also evidenced that the use of high doses of antioxidants might compromise radiation treatment efficacy. Other combinations like α-tocopherol, acetate and AA showed radioprotective effects and enhanced apoptosis in irradiated cancer cells [76].


Cholecalciferol (D3) and ergocalciferol (D2) are the two forms of vitamin D provided by the food. Exposure to UV radiation of the skin also induces the endogenous synthesis of D3, and for that reason, it is also called the “sunshine vitamin.” D3 and D2 have to undergo a double hydroxylation (in the liver and in the kidney) to form the biologically active derivative, that is, calcitriol (1,25-(OH)2-vitamin D), an essential hormone in the regulation of phosphocalcic metabolism. In vitro and in vivo studies evidenced the radioprotective efficacy of calcitriol enhancing the expression of genes coding for antioxidant enzymes (such as SODs and GPxs) and metallothioneins which are ROS scavengers [77]. Jain and Micinski [78] showed a positive link between vitamin D and GSH concentrations, as well as reduction in levels of ROS and proinflammatory cytokines, which is undoubtedly beneficial in protecting against IR. Populations of radiologically contaminated areas close to the Chernobyl accident had lower vitamin D blood levels compared to those in the uncontaminated Ukrainian regions [79]. Therefore, oral supplementation with vitamin D during RT or in medical professionals chronically exposed to low IR doses should be taken into consideration also because radiation toxicity can reduce mineral bone density. Recent studies evidence that calcitriol also radiosensitizes cancer cells by activating the NADPH/ROS pathway, which can makes it a promising adjuvant in RT [80].


Many antioxidant/defense enzymes, like SOD and metalloproteins, require trace elements as cofactors. The main oligoelements showing protective effects against radiation-induced DNA damage are zinc (Zn), manganese (Mn), and selenium (Se) [81] (Fig. 11.10). Se is an essential component of selenoenzymes such as GPx, thioredoxin reductase-1 (TR1), and ribonucleotide reductase (RNR). Se compounds and their metabolites possess a wide range of biological functions including anticancer and cytoprotection effects and modulation of hormetic genes and antioxidant enzyme activities. Exposure to radiation has been associated with a decrease in Se blood levels, and thus administration of seleno-compounds has emerged as a radioprotective strategy to reduce radiation toxicity. Mechanisms underlying the radioprotection effects include Nrf2 transcription factor activation and the consequent upregulation of the antioxidant-adaptive response in bone marrow stem cells and hematopoietic precursors [82]. 3,3-Diselenopropionic acid (at an IP dose of 2 mg/kg for 5 days prior to γ-TBI) showed radioprotection in mice by decreasing DNA damage and apoptosis [83]. Another recent formulations, poly-vinylpyrrolidone and selenocysteine-modified Bi2Se3 nanoparticles, improved the RT efficacy against tumors while exerting radioprotection in normal tissues [84]. Cancer patients, treated orally with Selenium Selenite, experienced a a lower incidence of diarrhoea compared to the placebo group [85]. Selenomethionine also reduces mucositis in patients with advanced head and neck cancer who are receiving cisplatin and radiation therapy (NCT01682031, www.clinicaltrials.gov).

An illustration of copper, zinc, manganese, selenium, or metal complexes causing different types D N A damage.

Fig. 11.10
Radioprotection by oligoelements


An illustration of copper, zinc, manganese, selenium, or metal complexes causing different types D N A damage.


Radiation-induced lung pneumonitis is a major dose-limiting side effect of thoracic RT, and the therapeutic options for its prevention are limited. 3,3′-Diselenodipropionic acid (DSePA), a synthetic organoselenium compound, shows moderate GPx-like activity and is an excellent scavenger of ROS. DSePA reduced the radiation-mediated infiltration of polymorphonuclear neutrophils (PMN) and suppressed NF-kB/IL-17/G-CSF/neutrophil axis as well as elevation in levels of proinflammatory cytokines such as IL1-β, ICAM-1 (intercellular adhesion molecule-1), E-selectin, IL-17, and TGF-β in the bronchoalveolar fluid of irradiated mice, thus ameliorating inflammatory responses. Administration of DSePA has shown a survival advantage against TBI and a significant protection to lung tissue against thoracic irradiation [86]. Wang et al. [87, 88] developed a highly efficient radioprotection strategy using a selenium-containing polymeric drug, with low toxicity and long-term bioavailability, The radioprotection activity of (VSe) and N-(2-hydroxyethyl) acrylamide shows more remarkable effects both in cell culture and mice models compared to the commercially available ebselen (organoselenium compound) and also exhibits a much longer retention time in blood (half-life ∼10 h).


Crescenti et al. [89] evaluated in vivo the tolerance induced by the combination of Se, Zn, and Mn (4 microg/mL each) plus Lachesis muta venom (O-LM) (4 ng/mL) to high doses of TBI (10 Gy, 137Cs source) IR in mice. Mice who received daily O-LM subcutaneous injections, starting 30 days before irradiation, showed a higher number of crypts, enhanced villous conservation, and lack of edema or vascular damage compared to the untreated and irradiated group. O-LM treatment also decreased vascular damage and grade of aplasia of mice bone marrow. O-LM treatment safety and efficacy were tested in a phase I clinical trial, and results indicated that it is an attractive candidate as a radioprotective agent for patients undergoing RT. Other clinical evidence indicates that Zn supplementation may act as an effective radioprotector in patients during RT. In a randomized clinical study, patients treated with Zn sulfate suffered a lower degree of mucositis compared to the placebo group [90]. Orally administered Zn-carnosine reduced oral mucositis and xerostomia in head and neck cancer patients [91].


SODs are a group of metalloenzymes that catalyze the dismutation of superoxide radicals (O2˙-) to H2O2 and O2, thus are first line of defense to prevent IR damages. In the event of a radio-nuclear attack or nuclear accident, the skin damage used to be severe. A synthetic SOD/CAT mimetic (EUK-207) administered 48 h after irradiation significatively mitigated radiation dermatitis, suppressed indicators of tissue oxidative stress, and enhanced wound healing [92].


Clinical applications of SODs mimetics are limited by their structural instability deficient availability and high cost. Compared with natural enzymes, nanozymes (nanomaterials with enzyme-like activity) are more stable, are economically affordable, and can be easily modified. Due to these characteristics, nanozymes are expected to become effective substitutes for natural enzymes for medical applications. Nanozymes with SOD-like activity have been developed and proved to have a mitigating effect on diseases involving oxidative stress [93]. As shown in Fig. 11.11, after administration, they are internalized by the cells and imitate SOD2 activity in order to inhibit ROS-induced cell damage.

An illustration of the actions of antioxidants when a nanozyme with S O D like activity attacks a cell. The antioxidants that respond against free radicals are superoxide dismutase, S O D, catalase, C A T, and glutathione peroxidase, G P x. These inhibit various R O S induced cell damage.

Fig. 11.11
Nanozymes with SOD-like activities


An illustration of the actions of antioxidants when a nanozyme with S O D like activity attacks a cell. The antioxidants that respond against free radicals are superoxide dismutase, S O D, catalase, C A T, and glutathione peroxidase, G P x. These inhibit various R O S induced cell damage.


Patients treated with RT for cancers of the head, neck, or lung suffer damage to the mucosa of the upper aerodigestive tract. Most of them develop ulcerative forms of mucositis, and severe forms lead to inability to eat solid foods, and in some cases, they cannot drink liquids. Results of clinical trials (now in phase III, e.g., NCT03689712) demonstrated the efficacy of the SOD mimetic, avasopasem manganese (GC4419) [94].


Mn porphyrin-based SOD mimics (MnPs) are reactive with superoxide and with other reactive oxygen, nitrogen, and sulfur species (Fig. 11.12). MnPs have CAT and GPx-like activities and peroxynitrite-reducing activity [93]. MnPs administered before and continued after radiation exposure protect from γ-ray, X-ray, and proton beam irradiation damages in different animal models, and a few studies indicate that beginning treatment with MnPs after radiation exposure is also effective. In normal tissues, MnPs treatment reduces oxidative stress, NF-κB, and TGF-β signaling pathways and activates Nrf2-dependent pathways. On the contrary, MnPs administration in combination with cancer therapy results in more oxidative stress in cancer cells, which leads to the reduction of NF-κB and HIF-1α and their downstream signaling pathways (Fig. 11.12). These changes are associated with increasing apoptosis and reducing overall cancer growth [95].

An illustration of the effect of M n porphyrin based S O D mimics, M n Ps, on normal cells and cancer cells Normal cells: Protection against radiation induced toxicity. Cancer cell: Enhancing cancer cell radiation therapy efficacy.

Fig. 11.12
Effects of Mn porphyrin-based SOD mimics in normal and cancer cells


An illustration of the effect of M n porphyrin based S O D mimics, M n Ps, on normal cells and cancer cells Normal cells: Protection against radiation induced toxicity. Cancer cell: Enhancing cancer cell radiation therapy efficacy.


BMX-001 is a porphyrin mimetic of the human mitochondrial manganese SOD, with the capacity to cross the blood-brain barrier and protect the brain against IR while acting as a tumor radiosensitizer [96]. It has been assayed as a radioprotector in different clinical trials, e.g., NCT03386500 (patients with recently diagnosed anal cancer), NCT03608020 (cancer patients with multiple brain metastases), NCT02990468 (head and neck cancer), and NCT02655601 (high-grade glioma treated with radiation therapy and temozolomide) [30].


All previous SOD mimetics suppress oxidative stress-mediated injuries, supporting the survival of the normal tissue, while promoting apoptotic processes in tumor tissues. The results from the clinical trials will provide us invaluable information on their real clinical utility as radioprotectors.


Radiation dermatitis is a common side effect of irradiation that limits cancer RT courses. It has already been described how the induction of hypoxia limits the damage associated with radiation, and consequently the option of using vasoconstrictor substances as radioprotectors has been proposed. Topical application of adrenergic vasoconstrictors (epinephrine or norepinephrine) to rat skin before radiation exposition (17.2 Gy) confers 100% protection against radiation dermatitis [97], and similar results were obtained when phenylephrine was topically administered to prevent radiation mucositis.


Indralin is an α1-adrenoceptor agonist with vasoconstrictor effects similar to those of epinephrine. Indralin (120 mg/kg)-treated rhesus monkeys survived better (five of six) after being exposed to a lethal TBI 60Co ɣ-irradiation of 6.8 Gy, than nontreated ones (all died). Moreover, less pronounced manifestations of hemorrhagic syndrome, leukopenia, and anemia were also noted [98]. Norepinephrine and α1-adrenoceptor agonists accelerate differentiation of hematopoietic stem cells by blocking their proliferation, thus avoiding, at least, earlier manifestation of radiation injury. A common feature of the radioprotective action of biogenic amines like indralin and aminothiols is the induction of hypoxia, although their mechanisms of action differ significatively. Norepinephrine and indralin exert their effect through the neurohormonal α1-adrenoceptors, but sulfur-containing radioprotectors act directly on tissues. Nevertheless, the use of α-catecholaminergic agonists entails a high risk of increased blood pressure or pressure decompensation in hypertensive patients, which would compromise their widespread use in an accidental emergency radiation exposure.


GI radiation vulnerability to a certain extent can be caused by release of potent pancreatic enzymes into the intestinal lumen after radiation exposure. Therefore, reducing intraluminal proteolytic activity may help attenuate intestinal radiation toxicity.


Somatostatin and its analogs (octreotide and pasireotide) inhibit exocrine pancreatic secretions. Octreotide reduces both acute and delayed intestinal radiation injury and diarrhea [99], as it has also been evidenced in a randomized controlled trial in patients who were undergoing radiation therapy to the pelvis (NCT00033605, www.clinicaltrials.gov). Nevertheless, some common side effects such as allergy, nausea, rash, and light-headedness may limit the routine use of octreotide. Moreover, it could also induce hypoglycemia [99] and reduce secretion of GH and IGF1, which could be highly counterproductive for the recovery of damaged tissues.


SOM230 (pasireotide) is another somatostatin analog under preclinical evaluation as a radioprotector. SOM230 reduced intestinal mucosa injury and increased mouse survival after TBI by inhibiting exocrine pancreatic secretion. Moreover, SOM230 has a 40-fold improved affinity to somatostatin receptor 5 than other somatostatin analogs, and it proved to be beneficial when administered prior to radiation exposure, and also when the treatment started up to 48 h following the exposure [100].


Several hormones are known to exhibit radioprotective characteristics, and melatonin, N-acetyl-5-methoxytryptamine, is one of them. It is the main secretory product of the pineal gland. Its radioprotective properties are outlined in Figs. 11.13 and 11.14.

An illustration of the properties of melatonin. Melatonin enhances endogenous D N A repair activity, directly neutralized R O S and R N S and acts as free radical scavenger, ameliorates inflammatory response, and increases expression of antioxidant levels, reducing oxidative stress.

Fig. 11.13
Radioprotective properties of melatonin

An illustration of the different radioprotective and radio mitigative effects of melatonin in the extracellular and intracellular regions and in the nucleus.

Fig. 11.14
Melatonin can exert radioprotective and radiomitigative effects


An illustration of the properties of melatonin. Melatonin enhances endogenous D N A repair activity, directly neutralized R O S and R N S and acts as free radical scavenger, ameliorates inflammatory response, and increases expression of antioxidant levels, reducing oxidative stress.


An illustration of the different radioprotective and radio mitigative effects of melatonin in the extracellular and intracellular regions and in the nucleus.


Melatonin has the ability to neutralize both ROS and NO directly leading to the production of less/nontoxic agents or indirectly increasing the activity of antioxidant enzymes such as SODs, GPx, GR, and CAT at the same time suppressing prooxidant enzymes like xanthine oxidase (XO) [101]. In addition, melatonin induces DNA repair mechanisms, which reduce mutagenic damage and also induction of DNA DSBs, which are lethal events for the cell. Melatonin administration before irradiation with a lethal dose of 60Co γ-rays reversed the upregulation of Bax and p53 proapoptotic genes and elevated Bcl-2, which led to 100% survival and preservation of hematopoietic and GI systems in mice [102]. Inflammation and fibrosis are two degenerative phenomena that are typical pathophysiological processes following RT. Melatonin via inhibition of NF-kB, COX-2, and iNOS enzymes has the ability to reduce the release of inflammatory cytokines and chemokines. Attenuation of these enzymes’ activities is associated with reduced level of oxidative stress, infiltration of macrophages and lymphocytes, as well as suppression of fibrosis, which prevents radio-induced pneumonitis and lung fibrosis [103], and also heart [104] and brain [105] damage associated with radiation exposition.


The physiological concentrations of melatonin in the human blood are approximately much lower during the day than during the night. Therefore, it seems that radiation therapy with supplementary melatonin leads to more beneficial effects during the nighttime. Melatonin exhibits multiple neutralizing actions to reduce radio-induced damage. Together with its low toxicity and its ability to cross biological barriers, these are all significant properties to consider it for clinical RT applications as well as for mitigation of radiation injury in a possible radiation accident scenario; however, its short half-life in vivo (<1 h) and the need of high doses to achieve radioprotective effects could limit its use in practice.


At this moment, just a few clinical trials have studied the therapeutic usefulness of melatonin as a radiosensitizer. Many preclinical studies evidence that it increases ROS production, inhibits telomerase activity and DNA repair mechanisms in cancer cells, reduces tumor angiogenesis and inflammatory response associated with high doses of radiation exposure, and enhances anticancer immunity. All these oncostatic properties make melatonin an interesting molecule to increase the efficacy of RT on cancer cells [106].


Apart from being a common antidiabetic drug, MTF has demonstrated potential antioxidant, radioprotective, and anticarcinogenic properties [107]. It is a hydrogen-rich agent able to neutralize free radicals, increase GSH, and upregulate the activity of SOD and CAT enzymes [108], which all favors the antioxidant defense of normal cells. MTF has been reported to reduce the generation of ROS at the complex 1 and to prevent mitochondrial mediated apoptosis [109]. It also decreases production of the inflammatory cytokine IL-1β in response to lipopolysaccharide (LPS) in macrophages [110] and inhibits NADPH oxidase, COX-2, and inducible NO synthase, thereby limiting macrophage recruitment and inflammatory responses. MTF stimulates the DNA repair pathways of nonhomologous end joining (NHEJ) or homologous recombination (HR), and nucleotide excision repair (NER) pathways [111].


In contrast to other radioprotectors, MTF has shown modulatory effects through induction of several redox-related genes, such as the PRKAA2 gene (which encodes the AMPK), thereby suppressing redox reactions, protecting cells from accumulation of unrepaired DNA, and attenuating initiation of inflammation as well as fibrotic pathways [108] involved in lung fibrosis [104]. Cardiovascular disease is one of the most pivotal disorders after RT. The administration of MTF to γ-irradiated (5 Gy) rats significantly ameliorated the changes in cardiac disease biomarkers (LDH and CK-MB) and in NF-κB, IL-6, and TNF-α levels compared to the control group. MTF also reduced E-selectin as well as ICAM and VCAM-1. These results demonstrate that concomitant administration of MTF during RT can act as an efficient heart protector from oxidative stress, inflammatory mediators, and endothelial dysfunction-induced damages [112–114]. MTF does not have significant adverse effects at the normal clinical level, but it may cause severe lactic acidosis and increase the risk of hypoglycemia. In animal models, MTF has demonstrated synergistic effects with melatonin mitigating the radiation-associated damages, and both of them radiosensitize cancer cells increasing RT efficacy (this will be later exposed in Sect. 11.4).


Radiomitigators are those agents/compounds which can be administered during or shortly after radiation therapy or IR exposure to reduce the effects of radiation on normal tissues before the onset of symptoms. These compounds are capable of minimizing the toxicity even after radiation has been delivered, which differentiates them from radioprotectors (reducing direct damage caused by radiation in normal tissues). At this moment, all FDA-approved radiation countermeasures (filgrastim, a recombinant DNA form of the naturally occurring G-CSF; pegfilgrastim, a PEGylated form of the recombinant human G-CSF; sargramostim, a recombinant GM-CSF) are classified as radiomitigators [30].


In some cases, these agents have protective properties that are similar to the action of “classic” radioprotectors, even if they are administered after radiation exposure. However, these agents are most effective not only at administration shortly after irradiation, but also during the irradiation. For radiologic terrorism and space research, much of the focus of radiomitigators has been in the field of developing chemopreventives strategies in order to reduce carcinogenesis of TBI.


An ideal radiomitigator should (a) offer the possibility of easy administration, (b) protect normal sensitive tissues which are associated with dose-limiting toxicity and significant reduction in quality of life, (c) be stable and easily available, and (d) have no relevant toxicity.


Postradiation changes in normal tissue such as constant mitotic cell death and perpetually active cytokine cascades can sooner or later lead to vascular damage, tissue hypoxia, and excessive extracellular matrix deposition [115]. Radiation mitigators should aim to interrupt these cascades prior to the manifestation of toxicity or intervene to prevent the prolongation of molecular and cellular damage, and therefore reduce the expression of radiation-induced tissue toxicity or prevent the acute toxicity.


Potential radiation mitigators are described in this chapter; their possible mechanism of action is represented in Fig. 11.15.

An illustration of the mechanism of action of radiomitigators. The mechanisms are mitigation D N A damage, accelerate D N A damage repair, gene expression, signal transduction, repopulation or proliferation, physiological effects, inflammation, differentiation, and activation of host cells.

Fig. 11.15
Radiomitigators: mechanism of action


An illustration of the mechanism of action of radiomitigators. The mechanisms are mitigation D N A damage, accelerate D N A damage repair, gene expression, signal transduction, repopulation or proliferation, physiological effects, inflammation, differentiation, and activation of host cells.


Radiomitigators can modulate the radiation-induced molecular, cellular, and tissue toxicity/injuries even when they are administered after radiation exposure. Gene and stem cell therapies as therapeutic radiation countermeasures are being developed and may be applied in the near future to minimize the side effects of radiation exposure through tissue regeneration.


Several studies have suggested that cellular recovery and repair processes can be enhanced by radiomitigators. Double-strand breaks (DSBs) are the most common form of DNA damage associated with IR. After DSBs are generated, a cascade of enzymatic processes, such as, HR and NHEJ (mediated by BRCA 1 and BRCA 2 enzymes), activation of p53, and induction of cell cycle arrest triggered to allow DNA repair or to induce apoptosis. The pharmacological improvement of these mechanism can contribute to mitigate IR damages. However, this must be done with care, because failure of these processes can lead to carcinogenic transformation [116]. Future studies should focus on compounds that have the potential to enhance the process of DNA repair after radiation exposure. In that sense, higher cellular pools of DNA precursors can create a radioprotective cellular environment, and drugs and chemicals that stimulate the activity of precursor-synthesizing enzymes can function as radiomitigators [117].


Anti-inflammatory Activity

An illustration of anti-inflammatory activity. I R activates the immune response, causing inflammation. Inflammation occurs in the presence of pro inflammatory cytokines and chemokines, such as I L 1, I L 6, T N F alpha, and T G F beta.


An illustration of anti-inflammatory activity. I R activates the immune response, causing inflammation. Inflammation occurs in the presence of pro inflammatory cytokines and chemokines, such as I L 1, I L 6, T N F alpha, and T G F beta.


IR is indirectly toxic by activating immune response, and patients undergoing radiation therapy may occasionally suffer from widespread inflammation. Various proinflammatory cytokines and chemokines are generated after radiation exposure, which particularly mediate inflammation, fibrosis, and other serious injuries in tissues and organs. Some natural products and their bioactive components can reduce the expression of these small cell signaling protein molecules and relieve the inflammation-associated side effects through their healing properties. Some phytochemicals, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoid, and other molecules reduce inflammatory response, reducing long-term side effects like fibrosis.

An illustration of I R causing death. I R causes dose-depended cascade or hematopoietic syndrome, resulting in injury to lymphoid and hematopoietic system. The injury causes septicemia, anemia, and hemorrhages, leading to death.


An illustration of I R causing death. I R causes dose-depended cascade or hematopoietic syndrome, resulting in injury to lymphoid and hematopoietic system. The injury causes septicemia, anemia, and hemorrhages, leading to death.


Hematopoietic stem cell injury is the primary cause of death after accidental or intentional exposure to a moderate or high dose of IR. Hence, compounds which can stimulate the regeneration of hematopoietic cells and immune system by mechanisms such as increasing spleen colony-forming units in synergy with interleukins may have good ability to protect cells and tissues against radiation exposure. A range of endogenous compounds like IL-1, TNFα, G-CSF, stem cell factor (SCF), erythropoietin (EPO), and GM-CSF stimulate stem cell progenitors and promote hematopoietic bone marrow repopulation and thus have been further investigated as potential radiomitigators. So, agents that upregulate endogenous radioprotective factors can also act as radioprotectors.


A variety of agents (such as vitamins, TLR ligands, and β-glucan) and many natural antioxidants are classified as immunomodulators as they regulate different cytokines (cell growth factors, colony-stimulating factors, etc.) in order to facilitate patient recovery from IR-induced injuries. These regulators inhibit cell apoptosis, promote differentiation and development of gastrointestinal or hematopoietic stem cells, and have radiomitigator effects (Box 11.3).


Radiomitigators (cytokines, growth factors, hormones, synthetic analogs, immunological adjuvants, immune regulatory peptides, etc.) accelerate the postradiation restoration of radiosensitive tissues.

At times, these agents can exert protective effects in a similar way to the action of “classic” radioprotectors; therefore, radiomitigators reduce oxidative stress and inflammatory damages, activate enzymes involved in repair mechanisms, and/or stimulate the replenishment of damaged tissues.

Compared to radioprotectors, they have the advantage of being effective despite being administered after exposure to IR. They are usually administered during the early period of postradiation and prior to the development of acute radiation syndrome (ARS).


Radiomitigators (cytokines, growth factors, hormones, synthetic analogs, immunological adjuvants, immune regulatory peptides, etc.) accelerate the postradiation restoration of radiosensitive tissues.


At times, these agents can exert protective effects in a similar way to the action of “classic” radioprotectors; therefore, radiomitigators reduce oxidative stress and inflammatory damages, activate enzymes involved in repair mechanisms, and/or stimulate the replenishment of damaged tissues.


Compared to radioprotectors, they have the advantage of being effective despite being administered after exposure to IR. They are usually administered during the early period of postradiation and prior to the development of acute radiation syndrome (ARS).


Four radiomitigators have been approved by the US Food and Drug Administration (US FDA) for the management of hematopoietic acute radiation syndrome (H-ARS). They include human recombinant G-CSF (filgrastim/Neupogen®), long-acting PEGylated form of G-CSF (filgrastim/Neulasta®), GM-CSF (sargramostim/Leukine®), and romiplostim (Nplate®), a fusion protein containing a peptide region that binds to the thrombopoietin receptor (c-Mpl) and an Fc carrier domain that increases its circulating half-life. Romiplostim was approved for use in radiation injury by the FDA based on the Animal Rule (United States Food and Drug Administration, 2021, Highlights of prescribing information. Nplate® (romiplostim) for injection, for subcutaneous use). Except for romiplostim, they have all been used to treat radiation accident victims with beneficial results [118–121].


G-CSF and PEGylated G-CSF promote the differentiation and proliferation of myeloid progenitor cells and their progeny. These effects promote neutrophil recovery after radiation-induced neutropenia. In addition, they enhance the function of neutrophils and improve survival. A World Health Organization Consultancy recommended that Neupogen and Neulasta should be administered subcutaneously, as soon as possible to individuals who have been exposed to radiation doses of >2 Gy [118]. Neulasta has the advantage that it is administered weekly, compared to daily administrations that is required for Neupogen treatment. GM-CSF increases the differentiation and proliferation of macrophage and granulocyte progenitor cells. When administered as late as 48 h after radiation exposure, GM-CSF reduced the recovery time for neutropenia and thrombocytopenia and decreased the rate of infection [5]. In addition, GM-CSF appears to exhibit an antifibrotic effect in the setting of radiation-induced lung injury (RILI) in experimental animals and humans [122, 123].


Keratinocyte growth factor (KGF), a factor that is produced by mesenchymal cells, protects and repairs epithelial tissues. Early studies suggested that KGF promotes the recovery of the oral mucosa after radiation-induced injury, improves gastrointestinal barrier function, and limits bacterial translocation and subsequent sepsis after irradiation. In clinical studies, Palifermin®, a human recombinant KGF product, reduced the incidence, duration, and severity of oral mucositis and esophagitis in patients treated with chemoradiotherapy and stimulated immune reconstitution following hematopoietic stem cell transplantation [124].


Many cell types release epidermal growth factor (EGF), which promotes the regeneration of hematopoietic stem cells in vivo. EGF was reported to have an additive effect on overall survival with G-CSF (survival of 20% for controls, 67% for EGF, 86% for EGF plus G-CSF) [125]. Fibroblast growth factor (FGF) is found in many tissues throughout the body, and its levels decrease after irradiation. FGF-P is a human recombinant derivative that is capable of activating FGF receptor-1, resulting in protection of the crypts located in the duodenum and improved survival in a GI-ARS mouse model. In addition, platelet counts were found to be higher in FGF-P-treated animals, resulting in decreased hemorrhages and cutaneous ulcerations postirradiation. It has been suggested that FGF-P has the potential to treat radiation-induced skin ulcerations and thermal burns and that it holds potential promise in the management of ischemic wounds and the promotion of tissue engineering and stem cell regeneration [125].


Interleukin-12 (IL-12) has pleiotropic effects on the innate and adaptive immune cells, including stimulation of hematopoiesis. Treatment with HemaMax® (human recombinant IL-12) restored all cell types in bone marrow when administered at 24 and 48 h post-TBI in non-human primates (HNPs) and mice, respectively. Compared to Neupogen, Neulasta, and Leukine, the single administration of HemaMax® is another advantage in the event of a mass casualty incident [126]. A novel, PEGylated IL-11 (Neumega®) is approved to treat thrombocytopenia in cancer patients, but must be injected daily, making its use inconvenient as a radiomitigator. To circumvent this problem, another mono-PEGylated IL-11 analog (BBT-059) was designed and demonstrated higher bioavailability and potency in vivo. In mouse model exposed to high TBI doses, BBT-059 leads to bone marrow cell reconstitution, leading to an accelerated recovery of platelets, erythrocytes, and neutrophils and an increase of survival higher than that obtained with treatment with the PEGylated derivatives of G-CSF and GM-CSF [125].


Erythropoietin is prescribed for the treatment of severe anemia arising from intense chemo- and/or radiation therapies. Erythropoietin and thrombopoietin (TPO) have been used for the victims of radiation exposure in the Tokaimura accident. Romiplostim (Nplate) is a synthetic TPO receptor agonist that preferentially increases platelet generation in bone marrow; contributes to mitigation of radiation-induced thrombocytopenia, anemia, and leukopenia; gives protection; and enhances regeneration of vascular endothelium. Romiplostim has recently received FDA approval to treat acutely irradiated and severely myelosuppressed adult and pediatric patients. More recently, ALXN4100TPO (a TPO receptor agonist) has been shown to stimulate megakaryopoiesis, reduce bone marrow atrophy and radiation-induced mortality in acutely irradiated mice, with the advantage of being less immunogenic than Nplate.


Combinations of hematopoietic growth factors and cytokines (G-CSF, GM-CSF, EPO, SCF, and IL-3) have already been used in the treatment of radiological accident victims, but the relative efficacy of this combined treatment is difficult to evaluate due to differing radiation sources, exposure doses, and other circumstances [127].


As explained in detail in Chap. 2, irradiation directly causes ROS overproduction, apoptosis, and/or necrosis, which activate the inflammatory response. In the short term, proinflammatory cytokines, such as IL-1, IL-6, IL-8, IL-33, TNF-α, and TGF-β, help to activate the immune response and bone marrow cellular recovery, but if it is excessive or is maintained for a long time, it can contribute to bystander/nontargeted effect (damages in tissues that have not been directly exposed), in special autoimmune diseases, fibrosis, and/or cancer initiation and progression. Therefore, the use of cytokines or growth factors capable of increasing the inflammatory response should be carefully evaluated. Moreover, the use of substances that inhibit its release or antagonize its proinflammatory effects has been shown to have mitigating effects on the damage caused by IR.


Fibrogenic cytokines like TGF-β, vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) are involved in radiation-induced fibrosis. TGF-β is able to stimulate ROS and NO production by the immune system, involved in the initiation and progression of chronic oxidative damage after exposure to a high dose of radiation. It is therefore not surprising that combined inhibition of TGF-β and PDGF signaling attenuates radiation-induced pulmonary fibrosis associated with decreased pneumonitis and leading to prolonged survival. Inhibition of TGF-β also reduced radiation-induced endothelial vascular damages [113, 114]. Moreover, different phase I/II clinical trials have shown more successful RT response with the combined use of TGF-β inhibitor in metastatic breast cancer patients (LY2157299, NCT02538471). This is of special interest, because the reduction in plasma levels of TGF-β is associated with greater efficacy of RT on different types of cancer and some studies have proposed that attenuation of cytokines by genistein or quercetin ameliorates late effects of IR such as pneumonitis and fibrosis [128].


The necrosis of central nervous system (CNS) tissue is one of brain irradiation’s main risk factors. The same is true for radiation-induced increase of capillary permeability resulting from cytokine release, causing extracellular edema. A recombinant human monoclonal antibody (bevacizumab), which prevents the VEGF from binding to its receptors, reduced brain necrosis in a patient subjected to cranial irradiation and further experiments evidenced its efficacy for the management of edema associated with radiation necrosis [129].


Toll-like receptors (TLRs) play critical roles in basal resistance to IR in animals and multiple radiosensitive tissues. Several TLR ligands had been proved to exert protective roles against IR both in vitro and in vivo, downstream effectors including NF-κB (controller of inflammation, and immune response), interferon regulatory factors, and stress-activated protein kinase (Jnk), which in turn results in inhibition of apoptosis, promotion of cell proliferation, regulation of cell cycle, and secretion of cytokines. In cultured cells, TLR2, TLR5, or TLR9 agonists inhibit radiation-induced apoptosis and increase cell survival. CBLB502 (a TLR5 agonist) was reported to alleviate bone marrow and intestinal injuries in mice and rhesus monkeys. Activation of TLR4 by its agonist LPS can protect bone marrow damage and lower mice mortality after irradiation. Moreover, some kinds of TLR agonists, such as TLR2/6 coagonist CBLB613, were reported to be more effective in radiomitigation than single-TLR agonists. In conclusion, TLRs and their ligands provide novel strategies for radiation protection in nuclear accidents [28, 29, 55].


IR is known to be especially damaging on highly proliferative tissues. Cellular sensitivities in approximate descending order from most to least sensitive are lymphocytes, germ cells, proliferating bone marrow and intestinal epithelial cells, and epithelial stem cells. Hematopoietic syndrome (HS) is the dominant manifestation after whole-body doses of about 1–6 Gy and consists of a generalized pancytopenia, due to bone marrow stem cell depletion, although, excepting lymphocytes, mature blood cells in circulation are largely unaffected. Patients remain asymptomatic during a latent period as the impediment to hematopoiesis progresses. Risk of infection and sepsis is increased as a result of neutropenia (most prominent at 2–4 weeks) and decreased antibody production. Petechiae and bleeding result from thrombocytopenia, which develops within 3–4 weeks and can persist for months. Anemia develops more slowly because circulating erythrocytes have a longer life span. Clinical management of the HS with risk of sepsis, hemorrhage, and/or acute anemia is related to the standard clinical protocols. Therapy would certainly encompass, but not limited to, the use of antibiotics, blood, and platelet transfusion, although the latter is limited by the recipient’s own immune response. Moreover, aseptic protocols must be rigidly employed. Allogeneic hematopoietic stem cell transplantation can restore bone marrow and immune functions. In the past, stem cells were harvested directly from donor bone marrow in the operating room, but at present, peripheral blood is most used as a source of stem cells for both autologous and allogeneic grafts [130]. Bone marrow stromal cell transplantation has also been shown to renew the irradiated intestinal stem and alleviate radiation-induced GIS [131]. To date, about 50 patients with acute radiation sickness have been treated with allogeneic hematopoietic stem cell transplants, but the median survival time has not yet exceeded 1 month. Despite these results, the efficacy of bone marrow transplantation in patients undergoing RT treatments highlights the need to have mechanisms in place to implement this procedure for patients exposed during a nuclear emergency [132].


Mesenchymal stem cells (MSCs) are nonhematopoietic adult stem cells with self-renewal and multilineage differentiation potential, low immunogenicity, and capacity to restore cell loss in damaged microenvironments. Moreover, MSCs secrete different interleukins, which help in the repair and recovery of cells. Although MSCs were traditionally isolated from bone marrow, cells with MSC-like characteristics are much easier to isolate from a variety of neonatal and adult tissues, including amniotic fluid, umbilical cord, peripheral blood, fat tissue, etc. Treatment with MSCs has shown efficacy in protecting the liver against radiation-induced injury; healing irradiated skin in mice; mitigating radiation-induced GIS, HS, brain injury, and neurological complications of RT; and increasing survival in irradiated mice [102]. Moreover, MSCs have successfully been assayed against radiation-induced pulmonary fibrosis (NCT02277145) and xerostomia (NCT03876197) (www.clinicaltrials.gov) [30]. Nevertheless, despite the extensive use of MSCs in preclinical and ongoing clinical trials, there is a lack of long-term safety in humans. During recent years, it has been demonstrated in animal models that MSC-derived extracellular (EVs). EVs can exert the same therapeutic effect of MSC; therefore, EVs can be used as an alternative MSC-based therapy [133]. To cite some examples, EVs inhibit DNA damage and cell death and preserve intestinal function [134] and bone marrow activity providing long-term survival in mice exposed to TBI [135].


Radiation initiates many enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) to produce ROS or NO, involved in the activation of inflammatory response. Most NSAIDs, such as aspirin, ibuprofen, indomethacin, diclofenac, and flurbiprofen, are able to inhibit COX-1 and COX-2 enzymes. The protective action COX inhibitors (COXi) is ascertained to the inhibition of the prostaglandin synthesis and directly or indirectly linked with the ability of NSAIDs to arrest cells in the G0 or G1 phase where cells are less sensitive to radiation damage and/or stimulation of the hematopoietic recovery [136]. Both pre- and post-irradiation treatments with sodium diclofenac reduced radiation-induced formation of DC and MN formation in human peripheral blood lymphocytes [137]. Flurbiprofen also showed radioprotection in clinical studies, e.g., delaying the onset of mucositis and reducing its severity after RT in 12 head and neck cancer patients, although the overall severity or duration of mucositis was not improved [138]. A recent meta-analysis of randomized controlled trials indicates that aspirin reduces the overall risk of recurrence and mortality of colorectal cancer and/or colorectal adenomas, which increases the interest in its possible use as a radiomitigator [24, 25]. However, nonselective COXi are known to cause undesirable side effects including GI ulcers and bleeding when taken for continued periods of time.


Increase of COX-2 gene expression is associated with decreased survival in patients receiving RT [139]. COX-2 selective inhibitors (COXi, as celecoxib, meloxicam, indomethacin) lack the GI toxicity of classical NSAIDs, and therefore, the use of COX-2i like meloxicam has been extensively assayed. Meloxicam administered either before or repeatedly after irradiation exposure has enhanced the recovery of hematopoietic progenitor cells committed to granulocyte-macrophage and erythroid development in sublethally irradiated mice [140], but the increase in survival was only observed when meloxicam was applied before lethal TBI. Sequential administration of PGE2 and meloxicam was shown to increase hematopoiesis and survival in irradiated mice [141], and meloxicam combined with ibuprofen treatment reduced bone loss after radiation exposure [142]. Radiation pneumonitis is a severe and dose-limiting side effect in lung cancer treatment. In this regard, celecoxib was tested in rats after single-dose X-ray irradiation of the right hemithorax and mediastinal region with 20 Gy revealing a dose-dependent protective effect on lipid peroxidation (MDA levels) and histopathological parameters. Celecoxib treatment induced a decrease in severe skin reactions after a high single dose of 50 Gy [136]. Moreover, celecoxib was also found to alleviate radiation-induced brain injury by maintaining the integrity of the BBB (blood-brain barrier) and reducing the inflammation in the rat brain tissues by inhibition of apoptosis in vascular endothelial cells [143]. RIVAD018 is another selective COX-2i which adds to its anti-inflammatory effects the ability to exert antioxidant activity, preventing oxidation of low-density lipoproteins, showing protection on both cellular and vascular models [144].


Several studies have also described that overexpression of COX-2 in cancer cells results in increased tumor angiogenesis, growth, and metastasis; thus, several COX-2 inhibitors have been described as radiosensitizers [136]. Celecoxib restricts neoangiogenesis, leading to a reduction in the survival of hepatocarcinoma and lung and skin cancer cells. In glioblastoma cells, the combined effect of radiation and celecoxib increased tumor cell necrosis, showing a significant reduction in tumor microvascular density compared to irradiation alone [139].


Radiation exposure of skin with high doses (>20 Gy) results in erythema, blistering, and necrosis in sequence. The necrosis generally occurs 10–30 days after exposure, although it may appear earlier in the most severe cases. The earliest administration of systemic and topical anti-inflammatory agents reduces the need for surgical excision of the affected tissue. Current therapy might make use of transplanted autologous keratinocytes combined with allogeneic stem cells. Advances in the knowledge of the radiomitigating properties of these compounds may prove to be very useful, particularly for the relatively low cost and toxicity, and specially for their analgesic effects [139].


Steroidal anti-inflammatory drugs such as dexamethasone can be administered after radiation exposure to attenuate fever and inflammatory or pain symptoms or to treat acute pathologies such as pneumonitis. Some authors reported that dexamethasone administration prior or immediately after radiation exposure reduced the risk of cardiac and other tissue fibrosis. Moreover, dexamethasone is often used to manage the inflammatory response in the brain during RT treatment of glioblastoma and other intracranial tumors. The effects of dexamethasone on patient survival however remain controversial because several clinical studies suggest that dexamethasone could potentially restrict effective RT [145].


Pathologically, acute intestinal epithelium damage is described as dilatation or destruction of crypt cells, decrease in villous height and number, ulceration, severe mucosal and submucosal inflammation, and sepsis associated with a pathogen bacterial translocation. Because of the rapid turnover of intestinal mucosa, the acute-phase symptoms (nausea, vomiting, diarrhea, abdominal pain, and acute mucositis) persist for hours to several months, while other intestinal complications such as obliterative vasculitis, mucosal ulceration, bowel wall thickening or progressive interstitial fibrosis, bowel obstruction, and fistulae formation, with or without fecal incontinence, are late events, often associated with chronic radiation exposition [146]. The reported incidence of severe late chronic radiation enteritis varies between 5 and 15% of patients treated with pelvic RT.


Probiotics, prebiotics, and FMT target intestinal microbiota by inhibiting colonization of pathogenic bacteria and restoring microbiome normobiosis. They increase production of mucin in the intestinal epithelial cells and expression of tight junction protein and occludin, thereby enhancing mucus layer function and improving survival of intestinal crypts (Fig. 11.16).

An illustration of the effect of probiotics, prebiotics, and fecal microbiota transplantation. The effects include inhibition, restoration, enhancement, reduction, increase, decrease, release, and recruitment.

Fig. 11.16
Effect of probiotics, prebiotics, and FMT on the function of the intestinal epithelium and gut microbiome


An illustration of the effect of probiotics, prebiotics, and fecal microbiota transplantation. The effects include inhibition, restoration, enhancement, reduction, increase, decrease, release, and recruitment.


A diverse and healthy commensal intestinal microbiota plays an essential role in GI homeostasis. It has been found that postirradiation enteropathy is associated with low mucosal microbiota diversity, in particular, a decrease of Lactobacillus and Bifidobacterium spp. and an increase in the relative abundance of opportunistic pathogens. Gut microbiota dysbiosis aggravates radiation enteritis, reduces the absorbing surface of intestinal epithelial cells, weakens intestinal epithelial barrier function, promotes intestinal inflammation, and contributes to the development of mucositis, leading to a persistent diarrhea and bacteremia [147]. Correction of the microbiome by application of probiotics, prebiotics, FMT, and/or antibiotics helps to prevent and treat radiation-induced enteritis [148].