Appraisal of biochemical classes of radioprotectors: evidence, current status and guidelines for future development

Abstract

The search for efficient radioprotective agents to protect from radiation-induced toxicity, due to planned or accidental radiation exposure, is still ongoing worldwide. Despite decades of research and development of widely different biochemical classes of natural and derivative compounds, a safe and effective radioprotector is largely unmet. In this comprehensive review, we evaluated the evidence for the radioprotective performance of classical thiols, vitamins, minerals, dietary antioxidants, phytochemicals, botanical and bacterial preparations, DNA-binding agents, cytokines, and chelators including adaptogens. Where radioprotection was demonstrated, the compounds have shown moderate dose modifying factors ranging from 1.1 to 2.7. To date, only few compounds found way to clinic with limited margin of dose prescription due to side effects. Most of these compounds (amifostine, filgratism, pegfilgrastim, sargramostim, palifermin, recombinant salmonella flagellin, Prussian blue, potassium iodide) act primarily via scavenging of free radicals, modulation of oxidative stress, signal transduction, cell proliferation or enhance radionuclide elimination. However, the gain in radioprotection remains hampered with low margin of tolerance. Future development of more effective radioprotectors requires an appropriate nontoxic compound, a model system and biomarkers of radiation exposure. These are important to test the effectiveness of radioprotection on physiological tissues during radiotherapy and field application in cases of nuclear eventualities.

Background

The quest for agents that can protect organisms from radiation-induced damage began shortly aftershock of the nuclear detonation in Hiroshima and Nagasaki (Japan, 1945) during the Second World War. This was followed by several major radiological accidents, such as Three-Miles island (USA, 1979), Chernobyl (Russia, 1986), Fukushima (Japan, 2011) in addition to many isolated incidents (Singh et al. 2015; Gupta et al. 2013; Coeytaux et al. 2015). Furthermore, radiation preparedness plan anticipates disposing of low toxicity radioprotectors as countermeasures following threats of nuclear detonation, radiological terrorisms, and accidental radiation contamination. Radioprotectors may also be useful for staff working at different facilities of radiation sources, radiographers, patients undergoing lengthy radiological procedures, naval cadets in nuclear submarine, armed forces, flight pilots and astronauts (Stone et al. 2004). In addition, increasing number of cancer patients undergoing radiotherapy can benefit from such applications, to protect normal tissues from radiation-induced side effects, which otherwise may compromise the quality of life of cancer survivors (Hall and Giaccia 2006; Nair et al. 2001; Zelefsky et al. 2002; Kry et al. 2005).

There are two distinct mechanisms by which ionizing radiation damages DNA and causes cell death. One involves ionization of atoms in the DNA (direct effect) while the other involves attacks by free radicals produced by the radiolysis of surrounding water molecules (indirect effect). The total biological effects result in damaging cellular and tissue structures that lead to dysfunction of organs. In case of planned or inadvertent, whole or large body radiation exposure, the physiologic failures of organ systems are known as acute radiation syndromes (hematopoietic, gastrointestinal, neuro-vascular), which may generally develop after doses of as low as 1–2 Gy. The lethal dose that will result in the death of 50% of exposed individuals (LD50) is 3.5–4 Gy. With optimal supportive care and bone marrow transplantation, this dose can be increased by two-fold. Interestingly, saving only 10% of bone marrow during whole body exposure may shun the effects of LD50 dose (Kumar et al. 2012). Thus, the protection of hematopoietic system is important to rescue individuals where radioprotectors with ability to protect hematopoietic system can come into play as major radiation countermeasures. The desired properties of a radioprotector include: low toxicity in the therapeutic concentrations, ability to reduce damage to numerous organs with high-dose reduction factor, economical, abundant, and orally administered.

Radioprotective agents are broadly classified into three groups: radioprotectors, radiation mitigators and therapeutic agents. Historically, radioprotectors are referred to the agents that protect organisms from cellular and molecular damage during irradiation, predominantly by enhancing antioxidant defense mechanism through scavenging of free radicals (Singh and Hauer-Jensen 2016). Mitigators are agents administered after radiation exposure but before the appearance of symptoms and generally protect organism by enhancing DNA repair, cellular signaling and modulating thiols redox system of cells. Radiation therapeutic agents are administered after appearance of symptoms to regenerate tissues by stimulating division of functional undamaged cells (Citrin et al. 2010). Alternatively, radioprotective agents are also categorized as radioprotectors, adaptogens and absorbents (Nair et al. 2001). Radioprotectors are composed of antioxidant and sulphahydryl compounds. Adaptogens are compounds that enhance radioresistance by acting as stimulator of defense system via boosting antioxidants and repair. Lastly, absorbents are compounds that likely to perform action as chelating agents to protect individual from ingested radionuclides.

Appraisal of various classes of radioprotective agents

Although radioprotective agents can be grouped into radioprotectors, radiation mitigators and therapeutic agents or alternatively into radioprotectors, adaptogens and absorbents, there is no unified system that can unambiguously classify various compounds. There is no doubt that the number of agents studied for their radioprotective potential is overwhelmingly large. Here we sought to render a comprehensive, yet brief, current account of these diverse compounds. We evaluated the evidence for their radioprotective efficacy and the future developments in this field. Therefore, in this review we have grouped known radioprotective agents according to their biochemical classes along with the potential clinical applications as illustrated in the schematic presentation (Fig. 1). To assess the radioprotective ability, the efficacy of different radioprotectors is expressed in terms of dose modifying factor (DMF). The DMF or reduction factor is defined as the ratio of radiation dosage producing similar effects in the presence or absence of the compound. Since human experiment is not feasible, investigations were carried out using in vitro cell cultures and in vivo animal models. The 30 days survival of irradiated mice is considered the gold standard endpoint to test the efficiency of a radioprotector. The various biochemical classes of radioprotectors and derivatives were discussed below and agents with quantified level of radioprotection (DMF or % survival) are listed in Table 1 along with the mechanisms of action (Table 1).

Fig. 1.

Schematic representation of various classes of radioprotectors citing most studied compounds. Asterisks indicate agents approved or under considerations for clinical use as follows: Amifostine (Ethyol) is specifically approved by FDA as a radioprotector that prevents cumulative normal tissues toxicity associated with cancer treatments, and offers significant reduction of radiation-induced xerostomia in head and neck radiotherapy patients. Genistein (BIO300) has currently an investigational new drug (IND) status as radioprotector of normal tissues to prevent acute radiation syndromes. While FDA has approved Neupogen (filgratism), Neulasta (pegfilgrastim) and Leukine (sargramostim) to ameliorate neutropenia induced by cancer treatment, these compounds are currently under investigation as radiation countermeasure agents. Palifermin (Kepivance) is an FDA-approved recombinant derivative of human keratinocyte growth factor (KGF) that is used to treat oral mucositis in patient undergoing hematopoietic stem cell transplantation. Recombinant salmonella flagellin CBLB502 (Entilimod) is FDA approved as off-label drug that can be used during nuclear or radiological accidents to protect against acute radiation syndromes. Prussian blue (Radiogardase) and potassium iodide (KI) are FDA-approved decorporating agents to increase the rate of elimination of radionuclides in internal contamination

Table 1.

List of main radioprotective compounds studied with their dose modifying factor (DMF) or the percentage (%) of survival after 30 days following its application along with the probable mechanism of action and chemical structure where available

Group and name of compounds	DMF or % survival	Mechanism of action	Chemical structure (pubchem)	References
Sulfhydryl compounds
Amifostine (WR-2721)	2.7	Free radical scavenging, repair		Hall and Giaccia (2006)
Cystaphos (WR-638)	2.1	Free radical scavenging, repair		Hall and Giaccia (2006)
N-Acetylcysteine	1.1	Free radical scavenging		Landauer et al. (1988)
Vitamins and hormones
Vitamin E (tocopherol)	1.1	Antioxidant, free radical scavenging		Srinivasan et al. (1997)
Tocopherol monoglucoside	1.23	Antioxidant, free radical scavenging		Satyamitra et al. (2003)
Gamma-tocotrienol (GT3)	1.29	Antioxidant, free radical scavenging, stimulation of G-CSF		Ghosh et al. (2009)
Tocopherol succinate	1.28	Free radical scavenging, modulation of antioxidant enzymes		Landauer et al. (1988)
Vitamin A	1.11	Free radical scavenging		Seifter et al. (1984)
Vitamin C	No protection	Free radical scavenging		Harapanhalli et al. (1996)
Lipoic acid	1.26	Free radical scavenging		Ramakrishnan et al. (1992)
l-Selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetylcysteine (mixed)	1.6	Free radical scavenging		Wambi et al. (2008)
Melatonin	86	Free radical scavenging		Vijayalaxmi et al. (1999b)
Phytochemicals and dietary antioxidants
Orientin and vicenin	1.3–1.37	Free radical scavenging		Uma Devi et al. (1999)
Genistein	1.16	Free radical scavenging		Landauer et al. (2003)
Sesamol	100	Free radical scavenging		Khan et al. (2015)
Botanical and herbal extracts
Podophyllum hexadendrum	1.62	Free radical scavenging	N/A	Lata et al. (2009), Gupta et al. (2007)
Ageratum conyzoides	1.3	Free radical scavenging	N/A	Jagetia et al. (2003b)
Aegle marmelos	1.2	Inhibition of lipid peroxidation	N/A	Jagetia et al. (2004b)
Zingiber officinale	1.15	Free radical scavenging	N/A	Jagetia et al. (2003a, 2004a)
Hippophae rhamnoides	80	Free radical scavenging	N/A	Prakash et al. (2005)
Radiola Imbricata	90	Free radical scavenging	N/A	Goel et al. (2006)
Bacterial extracts
CBLB502 (recombinant salmonella flagellin)	1.6	Binding to toll-like receptor 5 and activation of nuclear factor-kB	N/A	Burdelya et al. (2008)
CBLB613 (lipopeptide from Mycoplasma arginini)	1.25	Induction of interleukins and chemokines	N/A	Singh et al. (2012)
Cytokines
IL-1	88–100 1.05–1.12	Regulates the proliferation of hematopoietic cells	N/A	Neta et al. (1986), Dorie et al. (1989)

N/A not available

Sulphahydryl compounds

The marathon search for agents that can protect humans from radiation was initiated by U.S. Army due to vivid memory of victims of Hiroshima and Nagasaki nuclear detonation. Cysteine, a thiol compound, was the first agent to confer radiation protection of mice from whole body exposure in 1949 (Patt et al. 1949). However, cysteine and its derivatives N-acetylcysteine (DMF = 1.1) had shown toxicity (nausea and vomiting) that limited its usefulness (Landauer et al. 1988). To reduce toxicity, a series of chemical compounds were synthesized and tested in Walter Reed Institute of Research (USA). The discovery of cysteamine (β-mercaptoethyamine, MEA) further accelerated the research and an analog compound named amifostine (WR-2721) was synthesized with a DMF of 2.7 (Hall and Giaccia 2006). Marketed under the trade name of Ethyol, it is the only cytoprotective agent specifically approved by the FDA as a radioprotector. Amifostine selectively protects normal tissues in multiple organs against the toxic effects of radiation and various cytotoxic drugs in cancer patients with advanced stages (Brizel et al. 2000). The compound offers significant reduction of radiation-induced xerostomia and is currently being used during radiotherapy of head and neck cancers. A related phosphocysteamine compound (WR-638, cystaphos, DMF = 2.1) was carried by Russians army for infield radioprotection. Amisfostine is an injectable non-reactive phosphorothioate prodrug that does not readily permeate cells because of phosphorothioic acid group. It is believed that dephosphorylation by alkaline phosphatase enhances the uptake of amifostine and its conversion to the active metabolite WR-1065. The free radical scavenging (hydrogen donation), DNA protection and enhanced repair properties of thiols are believed to be the mechanisms of protection. However, similar to other thiols drugs, amifostine also exhibits toxicity and causes nausea, vomiting and hypotension (Kouvaris et al. 2007). This limits the use of amifostine in fields for mass radiation casualties.

The significant toxicity and short half-life (>90% clearance in 6 min) of sulphahydryl compounds fueled the search for non-thiols-based compounds (van der Vijgh and Korst 1996). Several chemicals such as alcohols, glycerol, and glycol were also tested but failed again due to toxicity. Different antioxidant mimetic agents, nitroxide, superoxide dismutase (SOD) were also studied and boosted the exploration of natural nontoxic compounds for their radioprotective properties (Weiss and Landauer 2003).

Vitamins, minerals and hormones

Varieties of vitamins and minerals are present in the nature. Most of them are natural antioxidants owing to their abilities to neutralize free radicals, and act as cofactors in different metabolic processes. The radioprotective properties of most studied vitamins, hormones, and minerals are described here.

Vitamin E

The study on vitamin E (tocopherol, C29H50O2) and its isoforms started in early 1980, when it had been shown that either injected (100 IU/kg) or fed as diet enhances the survival of CD2F1 mice exposed to lethal dose of radiation (Srinivasan et al. 1997). A DMF of 1.06–1.1 was obtained when vitamin E was injected 1 h before irradiation. In addition, vitamin E has been shown to reduce oral mucositis in cancer patients during radiotherapy (Srinivasan and Weiss 1992). Furthermore, tocopherol monoglucosides (a vitamin E derivative) protected plasmid from radiation-induced DNA strand breaks (Rajagopalan et al. 2002). It also provided relative radiation protection of animals from lethal dose when injected intraperitoneally (600 mg/kg), with a DMF of 1.09 (Satyamitra et al. 2003). However, low DMF and requirement of high concentration limit the application of these molecules in clinics.

A series of studies on gamma-tocotrienol (GT3), a member of vitamin E family and inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA), suggested a protective effect in mice, with a DMF of 1.29, at 100 and 200 mg/kg body weight when injected 24 h before irradiation (Ghosh et al. 2009). GT3 significantly increased granulocyte colony-stimulating factor (G-CSF), interleukin-6 (IL-6) and reduced radiation-induced cytopenia (Kulkarni et al. 2010, 2012). Tocopherol succinate (TS) inhibited apoptosis in various cells and had protected mice from lethal radiation dose when it was administered (400 mg/kg) 24 h before irradiation with a DMF of 1.28 (Singh et al. 2009). The mechanism of protection is believed to be due to the stimulation of G-CSF, a glycoprotein that stimulates bone marrow to produce granulocytes and stem cells (Singh et al. 2013). Recently, TS has been suggested to mobilize progenitor cells (Singh et al. 2014). Although TS and tocotrienols had higher DMF compared to classical tocopherol (alpha), recent studies suggest that injection of high dose (75 mg/kg) of these drugs causes adverse effects in non- human primates and could increase mortality with no significant protection compared to irradiated control (Singh et al. 2016). These results suggest that vitamin E and its derivatives at high concentrations might be toxic to humans.

Ascorbic acid (Vitamin C)

Many studies have been carried out to investigate the radioprotective effect of vitamin C (C₆H₈O₆). Although a role as antioxidant and radical scavenger is established, conflicting results envelop its effect in improving the overall survival after lethal radiation dose. Ascorbic acid was shown to reduce the formation of micronuclei and dicentrics in mice (Sarma and Kesavan 1993), and protect radionuclides-mediated DNA damage in mice sperm with a DMF of 2.2 (Narra et al. 1993). Monoglucosides’ derivatives of ascorbic acid have been shown to scavenge different free radicals and protect plasmid from radiation-induced DNA strand breaks (Mathew et al. 2007). Although supplemental vitamin C improved the survival of mice’ nucleated bone marrow cells following whole body sublethal irradiation (3.5 Gy, DMF = 1.7), it had no significant effect after lethal irradiation (9 Gy) on 30-day survival outcome (Harapanhalli et al. 1996). Nonetheless, pre-treatment of ascorbic acid (150 mg/kg/day) orally for 3 days before a lethal radiation dose (14 Gy) followed by bone marrow transplantation had reduced gastrointestinal syndrome and resulted in an enhancement of mice survival (Yamamoto et al. 2010). Post-treatment with ascorbic acid at high dose (3 g/kg) immediately after exposure (7–8 Gy) resulted in a recovery of hematopoietic system, reduced apoptosis in bone marrow cells and effectively improved survival of mice (Sato et al. 2015). It appears that ascorbic acid at high dosage could provide protection to mice after whole body irradiation. However, a high dose of 3 g/kg for 60 kg humans will be impracticable. Therefore, it has been concluded that the requirement of high pharmacologic effective doses limits the application of this molecules in clinics (Du et al. 2015).

The reason for failure of such classic antioxidants and vitamins as radioprotectors is due to their poor efficacy in whole body irradiation. Low reduction factors (DMF 1.11–1.26) were also observed for vitamin A and lipoic acid (Seifter et al. 1984; Harapanhalli et al. 1996; Ramakrishnan et al. 1992). However, a mixture of vitamins (l-selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetyl cysteine) provided improved protection with a DMF of 1.6 (Wambi et al. 2008). Clearly, vitamins and minerals act as cofactors and regulate many physiological systems in addition to their anti-radical activities. These essential nutrients are required in small amounts by living organisms for normal function. Thus, they may harm rather than help at high concentration needed a priory for radiation protection purposes.

Melatonin

The pineal hormone melatonin (C₁₃H₁₆N₂O₂) had attracted much attention when it was shown to reduce radiation-induced genetic damage and protect mice from a lethal radiation dose (8.15 Gy) in a series of work by Vijyalaxmi et al. (1999a, b). The increase in survival was dose dependent and improved from 45% without treatment to 60% (DMF = 1.33) and 85% (DMF = 1.78) for mice pretreated with 125 and 250 mg/kg melatonin, respectively. Furthermore, clinical reports indicated that melatonin administration results in a favorable ratio of radiotherapy efficacy versus toxicity during the treatment of human cancers (Vijayalaxmi et al. 2004). The mechanism of radiation protection by melatonin has been attributed to scavenging of varieties of oxygen-based radicals and DNA protective properties (Reiter et al. 2010). It has also been reported that melatonin possesses the unique property of selectively sensitizing tumor (Um et al. 2011) while protecting normal cells (Mishra et al. 2011). Further, it was suggested that melatonin could protect antioxidative enzymes including catalase (CAT), glutathione peroxidase (GPx), and SOD from depletion following UV radiation (Fischer et al. 2013).

Melatonin is highly tolerated over a wide range of doses and recent studies suggest that up to 8000 mg/kg of body weight is not toxic in mice (Ali et al. 2012). Assessing the efficacy and safety of melatonin as adjuvant therapy in concurrent chemo-radiotherapy for solid tumors, a meta-analysis of 8 randomized controlled trials concluded that 20 mg orally, once a day, led to substantial improvements in tumor remission, 1-year survival, and alleviation of therapy-related side effects (Wang et al. 2012). Although melatonin has shown some characteristics of a promising candidate radioprotector, its implication in regulating physiological functions is still inadequately understood. In addition, its short active half-life (20–50 min) and the requirement of rather high doses for radiation protection are still challenging for developing melatonin as radiation countermeasures.

Selenium

Selenium (Se) is an important chemical element that exists as cofactor for many enzymes such as GPx, thioredoxin reductase and ribonucleotide reductase in different prokaryotes and eukaryotes. Early report using selenium and its derivatives had described radioprotective effect in mice (Weiss et al. 1992). Intraperitoneal injection either before (≤1 h) or shortly after (15 min) 9 Gy radiation has enhanced the 30-day survival. Selenomethionine (SLM) has lower lethal and behavioral toxicity (locomotor activity depression) compared to sodium selenite (Weiss et al. 1992). A derivative of selenium, 3, 3, di-selenopropionic acid had also protected mice by reducing DNA damage and apoptosis (Kunwar et al. 2010). Furthermore, dietary selenium (100 µg) had mitigated the radiation-induced nephropathy in mice (Sieber et al. 2011). The combination of selenium with vitamin E has also been reported to protect gastrointestinal system from ionizing radiation (Mutlu-Turkoglu et al. 2000). A recent review of clinical trials has concluded that selenium supplementation (200–500 µg/day) improves the general conditions and the quality of life of patients and reduces the side effects without reducing the effectiveness of radiotherapy (Puspitasari et al. 2014). The authors, however; warned that high-dose and long-term supplementation may be unsafe due to selenium toxicity and that more evidence-based information and research are needed to ensure its therapeutic benefits. In addition, in a more recent randomized phase II clinical trial on 18 radiotherapy patients, selenomethionine did not lower the incidence of severe mucositis or improve quality of life or survival outcomes (Mix et al. 2015).

Phytochemicals and dietary antioxidants

Many dietary compounds from a wide range of foods and beverages have been screened for their potential modulatory effects in various human ailments on the belief that they do not only provide us with nutrients, but also enhance our capacity to fight against oxidative stress and diseases. Different natural compounds possess hepatoprotective, cardioprotective, neuroprotective, and anti-tumorigenic properties (Omar 2010; Alqasoumi 2012; Shareef et al. 2016) and these are desired traits for effective medicinal agents. The radioprotective properties of the most common natural dietary compounds are discussed here.

Curcumin

The phenolic compound curcumin (C₂₁H₂₀O₆) present in spices (curcuma longa) was shown to protect radiation-induced genotoxicity (micronuclei and dicentrics) in cultured rat lymphocytes and hepatocytes and decreased lipid peroxidation, particularly at the highest dose (10 µg/ml) used (Srinivasan et al. 2006, 2007). An in vivo study, on the lens of rats fed with 100 mg/kg curcumin for 28 days, had reported significant decrease (from 100 to 40%) in radiation-induced (15 Gy) cataract (Ozgen et al. 2012). In addition, pre-treatment with curcumin (25–200 mg/kg) led to a dose-dependent increase in wound healing following 6 Gy whole body irradiation of mice (Jagetia and Rajanikant 2004).

In contrast to these radioprotective effects in normal cells and tissues, curcumin was reported to radiosensitize variety of tumor cells in vitro, especially by blocking NF-kB pathways (Chendil et al. 2004; Qiao et al. 2012), modulating p53, thioredoxin reductase-1, enhancing reactive oxygen species (ROS) and MAP kinase pathways (Javvadi et al. 2008, 2010; Veeraraghavan et al. 2010). The apparent differential effect of curcumin between normal (radioprotective) and tumor (radiosensitizer) cells is difficult to interpret in radiobiological terms. Curcumin is traditionally known for its anti-inflammatory effects. It has been shown to be a potent immunomodulatory agent that can regulate the activation of T cells, B cells, macrophages, neutrophils, natural killer cells, dendritic cells and can enhance antibody responses (Jagetia and Aggarwal 2007). It remains to be demonstrated whether this dissimilarity in effects pertains to a potential metamorphic ability of curcumin to modulate the immune system in such a way that it distinguishes between good (normal) and bad (cancer) cells or simply by harnessing the differences in cellular homeostasis? Thus, the studies on the radioprotective effects of curcumin are inconclusive. In addition, its poor aqueous solubility, hydrolytic degradation, poor bioavailability and metabolism may introduce technical bias in various studies (Chidambaram and Krishnasamy 2014). Although these limitations preclude the use of curcumin as radioprotector or radiosensitizer in clinic, its immunomodulatory roles merit in depth exploration for a well-defined use in medicine (Srivastava et al. 2011).

Genistein

Genistein (C₁₅H₁₀O₅) is a natural isoflavone from soybeans that has drawn wide attention for its bioactivity and potential role in modulating radiation damage (Song et al. 2015). According to in vivo studies, genistein was reported to have a protective effect on radiation-induced intestinal damage in tumor-bearing mice and to also delay tumor growth (Son et al. 2013). A study by Calveley and colleagues on rat lung tissue had shown that although genistein treatment decreased the levels of many inflammatory cytokines and protected from DNA damage, it provided partial protection against pneumonitis and fibrosis (Calveley et al. 2010). Subcutaneous pre-administration of genistein (25–400 mg/kg), 24 h before irradiation (9.5 Gy) enhanced the 30-day survival of mice (DMF = 1.16), whereas survival was not different from controls when it was administered one hour before irradiation (Landauer et al. 2003). In addition, enhanced hematopoietic cell recovery and 30-day survival of mice (97% compared to 31%) were described following subcutaneous administration of genistein (200 mg/kg) 24 h prior to lethal radiation (8.75 Gy) dose (Davis et al. 2007).

As for in vitro studies, it has been shown that low concentration of genistein (1.5 µM) could protect L-02 cells from radiation-induced injury via inhibition of apoptosis, DNA damage and chromosome aberration; meanwhile, high concentration (20 µM) demonstrated radiosensitizing effect via opposite increase in apoptosis, chromosome aberration and impaired DNA repair along with up-regulation and down-regulation of certain genes (Song et al. 2015). Genistein at 10 µM had also radiosensitized breast cancer cell strains via G2/M cell cycle arrest, increased DNA damage and apoptosis (Liu et al. 2013). In addition, genistein had potentiated radiation-induced cell killing in vitro and in vivo orthotopic model of PC-3 prostate carcinoma (Hillman et al. 2004). Again, these apparent differential effects of genistein on normal and tumor cells are difficult to interpret. Currently genistein has an investigational new drug (IND) status as radioprotector of normal tissues for acute radiation syndrome under name BIO300 (Singh et al. 2016). However, its ability to inhibit many enzymes (tyrosine kinase) and modulate different signal transduction pathways infers a complex role in biological systems and call for further investigation to establish genistein as a radioprotective agent (Grabowski et al. 2015; Qian et al. 2015).

Sesamol

Sesamol (C₇H₆O₃) is a natural compound found in sesame (sesamum indicum) oil. Although sesame seeds are popular nutritional food, it is a relatively new antioxidant molecule having potential radioprotective effect with strong anti-radical properties (Mishra 2012; Kumar et al. 2015). Sesamol exhibited more free radicals scavenging capacity compared to melatonin and demonstrated greater radioprotective efficacy in plasmid and calf thymus DNA (DMF = 10 at 100 µM), human lymphocytes, and V79 cell survival (DMF = 2) in vitro (Prasad et al. 2005; Mishra et al. 2011). Sesamol has been shown to protect C57BL/6 male mice (100 mg/kg) in vivo following 7.5 Gy radiation dose by protecting hematopoietic and gastrointestinal systems (Khan et al. 2015). Radioprotection was also described in the jejunum of Swiss albino mice irradiated with 15 Gy, where it showed increased crypt cells, maintained villus height, and prevented mucosal erosion (Parihar et al. 2006). In addition, sesamol was also reported to reduce radiation-induced cytogenetic damage in bone marrow cells of mice (Kumar et al. 2015). These studies are encouraging and put forth to consider sesamol as a potent candidate radioprotector.

Botanical and herbal extracts

Worldwide, different botanical, herbal, and Ayurvedic preparations have been traditionally used to treat different ailments including stress-induced disorders. Therefore, it was assumed that these preparations might be effective against radiation-induced mortality (Jagetia 2007). Many botanical and herbal extracts have been studied to determine their capability to demonstrate radioprotective effect. The crude extracts of different parts of plants such as root, leaf, bark, seeds, flower were examined. The radioprotective properties were revealed for some botanical or herbal plants, such as Tinospora cardifolia, Panax ginseng, Rosemarinus officinalis, Aloe vera, Embilica officinalis, Alstonia scholaris and Mentha piperata. Extracts of these plants have showed significant protection against radiation-induced syndromes (hematopoietic, gastrointestinal), as well as cellular and molecular damages in mice (Goyal and Gehlot 2009; Jindal et al. 2010; Samarth et al. 2004).

The high-altitude plant Hippophae rhamnoides (seabuckthorn) was studied in different model systems for potential radioprotective properties. It was shown to protect hematopoietic and gastrointestinal systems when administered 30 min before irradiation (Goel et al. 2002; Bala et al. 2015). A 30 mg/kg of this botanical extracts (RH-3) rendered a more than 80% survival after whole body lethal irradiation (10 Gy) in mice (Prakash et al. 2005). The hippophae extracts had also protected mitochondrial and genomic DNA from radiation damage (Shukla et al. 2006). In contrast, it showed enhanced apoptosis in thymocytes (Goel et al. 2004). Another high-altitude plant that has been studied is Podophyllum hexadendrum. The crude extracts are rich in podophylotoxins, such as 4-O-α-(d)-6-acetylglucopyranoside and podophyllotoxin-4-O-β-(d)-6-acetylglucopyranosid (Puri et al. 2006). A single dose (10–15 mg/kg body weight) of a semi-purified extract (REC-2001) conferred greater than 90% of survival when administered before whole body irradiation (10 Gy) with a DMF of 1.6 (Lata et al. 2009). Conversely, high dose (115 mg/kg body weight) was toxic and killed 100% of the animals. At pharmacological doses, the extract protected different organs such as liver, testis, gastrointestinal, brain and reduced genotoxicity in peripheral blood lymphocytes (Dutta and Gupta 2014; Dutta et al. 2015). The extract had also protected HePG2 cells from radiation-induced cell death (Gupta et al. 2003). Another extract of P. hexadendrum (REC-2006) had protected 90% of the mice from lethal whole body irradiation (10 Gy) at significantly lower dose of 6–8 mg/kg (Gupta et al. 2007). The names of these extracts were based on the years they were isolated and the active constituents provided protection even after years of extraction from the plants. The mechanism of radioprotection was attributed to scavenging of free radicals and immunomodulation (Rajesh et al. 2007).

The radioprotective properties of many traditional medicinal plants were widely studied by various groups of investigators (Table 1). The overall outcome indicates that dietary supplements of Ageratum conyzoides (DMF = 1.3), Aegle marmelos (DMF = 1.2), Zingiber (Ginger) officinalis (DMF = 1.15), Radiola Imbricata (90% survival), magiferin (Mangifera indica), LiV52, naringin (citrus flavonoids), triphala, chavanyaprash, mint (Mentha arvensis), Syzygium cumini (Jamun), Coleus aromaticus can confer protection to mice from radiation-induced cell death (Jagetia et al. 2003a, 2004b; Goel et al. 2006; Jagetia 2007). Active flavonoids Orientin and Vicenin from basil plants (Ocimum sanctum) have also demonstrated potential radioprotective properties (Uma Devi et al. 1999). The results also showed that most of these herbal extracts possess strong antioxidants and immunomodulatory properties.

Plant products have great potentials as pharmacological agents. However; these herbal preparations are mixtures of compounds. The scientific communities believe that it is very difficult in such situation to predict the effects of a single constituent alone (Ramana et al. 2014). In addition, the limited knowledge regarding dosing of different herbals and botanicals preparations during treatment of various pathological conditions restricts their universal applications and carries the risk of inefficiency or toxicity due to miss-dosing. Also, the relative availability of specific constituents in plants at specific geographical areas further complicates their study as radioprotective drugs.

Bacterial extracts

It has been observed that many bacterial species may survive at extreme conditions of heat and/or radiation (thermophiles and radiophiles). These species must be adapted to these environmental conditions by changing their physiological or genetic constitution (Satoh et al. 2016). It is assumed that understanding the mechanism of radioresistance and mimicking the condition may provide protection to humans against ionizing radiation. A few bacterial species are radioresistant (such as Deinococcus radiodurans, radiophilus, and grandis), and evidence suggests that extracts of those bacteria could confer radioprotective properties in different experimental models (Fedorocko and Mackova 1996; Daly et al. 2010).

Various other bacterial extracts were also tested for radioprotective potential. Intraperitoneal administration of 250 and 500 µg endotoxin-free extract of bronchovaxom (bacteria responsible for respiratory infection, available as drug) at 24 h before whole body irradiation (9.5 Gy) protected mice from death with DMFs of 1.12 and 1.25; respectively (Fedorocko et al. 1992). A single subcutaneous injection of extract prepared from heat killed Lactobacillus casei protected mice from a lethal radiation dose of 8.5 Gy (Tsuneoka et al. 1994). The effect was attributed to enhanced protection of hematopoietic tissues due to activation of macrophage stimulating factor. Also, a single injection of recombinant polypeptide derived from salmonella flagellin (CBLB502) 30 min before lethal total body irradiation had protected mice from hematopoietic and gastrointestinal syndromes and brought about two-fold (DMF = 1.6) improvement in mice survival and mortality in rhesus macaques (Burdelya et al. 2008). CBLB502 protected animals by binding to toll-like receptor 5 and activation of nuclear factor-kB, which presents a landmark work that changed the way of earlier thinking considering only radical scavengers as good radioprotectors. CBLB502 (marketed as Entilimod) is FDA approved as off-label drug that can be used during nuclear or radiological incidents for acute radiation syndrome (Singh et al. 2015). In addition, CBLB613, a toll-like receptor 2/6 (TLR 2/6) agonist and natural lipopeptide obtained from Mycoplasma arginini, had also shown significant radioprotective capacity in CD2F1 mice against hematopoietic syndrome (DMF = 1.25), and stimulated induction of various interleukins and chemokines (Singh et al. 2012). Intraperitoneal administration of aqueous solution of semi-quinone glucosides (SQGD) obtained from bacillus species INM-1 showed antioxidant and radioprotective properties in vitro and enhanced the expression of immuno-stimulatory cytokines such as IL-12p40, IL-12p35, IL-23p19, and Rel A genes in human peripheral blood mononuclear cells (Kumar et al. 2011). Alteration of host immunomodulatory activity seems to be the primary mode of action of many bacterial products. However, immune responses in humans are very complex in nature and the mechanisms of different bacterial products as drug are not well understood.

Cytokines

Cytokines, which include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, are proteins of small molecular weight that regulate the interaction among lymphoid, inflammatory and hematopoietic cells (Schaue et al. 2012). Interleukins were the first to demonstrate radioprotective effect. Intraperitoneal injection of 2000 units of synthetic interlukin-1 (IL-1) showed to protect 88 and 100% of C57BL/6 mice following LD100/17 and LD50/30 radiation doses, respectively (Neta et al. 1986). Other study, however, showed rather modest DMFs (1.05–1.12) for 100 ng/mouse (3 Âµg/kg) in C57BL/Ka and C3H/Km mice (Dorie et al. 1989). In addition, the administration of antibodies against interleukins and tumor necrosis factor (TNFα) reduced survival of CD2F1 mice following whole body irradiation due to myeloid suppression in exposed animals (Neta et al. 1991). Furthermore, many cytokines, such as IL-3, IL-6, IL-12, IL-11, G-CSF, granulocytes-macrophages colony-stimulating factors (GM-CSF), erythropoietin, and thrombopoietin that regulate the proliferation of hematopoietic cells at various stages, were clinically tested for their ability to restore bone marrow after chemotherapy or radiotherapy of cancer patients. However, only IL-1, IL-12, TNF-α, basic fibroblast growth factor (bFGF), and G-CSF had protected animals when given prior to irradiation [(Singh and Yadav 2005) and references therein]. Although cytokines can potentially be used as candidate radioprotectors due to decreased toxicity, few cytokines such as TNF-α sensitize cells and, therefore, their mechanism and role in radiation-induced cell death need to be explored in more detail before being used as radioprotectors. Furthermore, the natural compound curcumin downregulates the expression of pro-inflammatory cytokines such as TNF, IL-1, IL-2, IL-6, IL-8 and IL-12, mostly through an activation of Nf-kB transcription factor (Jagetia 2007).

Interleukins such as G-CSF and GM-CSF, and their pharmaceutical analogs, filgratism, lenogratism sargramostim, regulate maturation and differentiation of progenitor cells in the bone marrow to granulocytes, macrophages and T cells. Interleukins G-CSF and GM-CSF are also shown to ameliorate cancer therapy-induced neutropenia for which FDA has approved Neupogen (filgratism), Neulasta (pegfilgrastim) and Leukine (sargramostim) and currently under investigation as radiation countermeasure agents (Singh et al. 2015). In this context, palifermin (Kepivance), a recombinant derivative of human keratinocyte growth factor (KGF), is also an FDA-approved drug (Fig. 1) that is used to treat oral mucositis in patient undergoing hematopoietic stem cell transplantation (Vadhan-Raj et al. 2013). In contrast to amifostine, the mechanism of action of palifermin is related to epithelial cell proliferation leading to significant reduction in oral mucositis in head and neck cancer patients. Further studies are required to explore its usefulness as radiation countermeasure agent after radiological events (Johnke et al. 2014).

Thus, cytokines are diversified in their nature and functions. The intricate actions of interleukins and their targets during irradiation of animals are poorly understood in the scenarios of whole body exposure. Since these signaling molecules are produced during different physiological processes and each interleukin is involved in more than one pathway, it will be difficult to demonstrate the mechanism of action responsible for the desired radioprotective effect. On the other side, it is a fact that these interleukins promote also tumor progression and are responsible for the poor prognosis in many cases of cancers (Rastogi et al. 2015).

DNA-binding agents

DNA-binding or damaging agents are broadly anticancer drugs. In general, there are three modes of binding of small-molecule with double-stranded DNA: intercalation, groove binding and covalent binding. The minor groove of DNA is of particular interest due to sequence-specific interactions with a large number of small molecules. These minor groove ligands bind typically to AT-rich sequences and either induce permanent DNA damage or cause only reversible inhibition of DNA-dependent functions (Baraldi et al. 2004). Although most of these small molecules have cytotoxic activities, a few modulates cellular pathways that promote cell survival and exhibit radioprotective properties (Gurova 2009). The mechanism of radioprotection seems to be hydrogen or electron transfer from the ligands to the DNA radical at binding sites leading to DNA structural stabilization.

The Hoechst minor groove ligands H33258 (C₂₅H₂₄N₆O·3HCl), H33342 (C₂₇H₂₈N₆O·3HCl) and amino derivatives methylproamine had protected V79 cells from radiation-induced DNA damage and death with a DMF of 2.1 (Martin et al. 2004). Also, dioxomolybdenum and dioxotungusten hydroxamato had protected DNA from radiation-induced strand breaks (Paul et al. 2014). Netropsin (C₁₈H₂₆N₁₀O₃·2HCl), which is another minor groove binding ligand, had radioprotective properties through DNA structural stability mechanism (Mishra et al. 2009). In contrast with H33258, the netropsin protection was at specific DNA site. H33342 derivatives such as tris (TBZ), and dimethoxty (DMA) benzimidazole were also studied for their radioprotective efficacy in vivo. DMA showed an effective radioprotection in mice at single nontoxic oral dose with a DMF of 1.28 (Nimesh et al. 2015). DMA-treated mice showed delayed radiation sickness, such as weight loss, irritability and lethargy.

DNA-binding drugs raise, however, a serious concern due to potential mutagenic effects. Many classes of DNA minor groove binding ligands induce aneuploidy, polyploidy, chromosome decondensation at heterochromatic regions rich in AT content, fragile sites, which may have a propensity to develop particular cancers (Turner and Denny 1996). Nonetheless, pentamidine (a minor groove binder used in the treatment of AIDS-related pneumocystis pneumonia) did not show so far mutagenic effects in nuclear DNA. Thus, minor groove binding does not necessarily lead to mutagenesis and further studies are required to ascertain their role as safe radioprotectors.

Absorbents and chelators

In general, absorbent is defined as a substance endowed with the property of attaching other substances to its surface without covalent bonding, while chelator is a chemical compound that bonds with a metal ion to form a chelate. Yet, the exact distinction between the two terms is not unanimous and often used interchangeably. In radiation protection, absorbents do often include chelating and are the compounds that can sequester radionuclides after internal contamination and nullify toxic effects to protect biological systems (Nair et al. 2001). During a nuclear explosion, distinctive radioactive materials may be produced and released to the atmosphere where they could be inhaled either as gases, ingested as particulates by mouth, or through skin contamination. Hence, suitable decorporating agents are required to remove internal contamination of different radionuclides (Rump et al. 2016). Radionuclides have unstable nucleus and in the process of stabilization, they emit gamma radiation, alpha or beta particles which are ionizing radiations causing a high risk of stochastic and sometimes deterministic health effects. Radioactive nuclides, such as strontium 90, cesium 137, plutonium 239, cobalt 60, radium 226, polonium 210, iridium 192, iodine 131 and americium 241, might be also a component of potential radioactive dirty bombs (Rump et al. 2016).

Radioprotective agents can be used as decorporating agents to remove radionuclides from the whole body. Fast and effective removal of radionuclides from biological system are the properties required from these agents. Prussian blue (FDA approved decorporating agents) has been used as radioprotector to eliminate cesium and thallium ingested radionuclides (Bhardwaj et al. 2006). Prussian blue, a non-absorbable resin dye, acts as laxative agent that promotes the elimination of radionuclides from the digestive system. The absorption of certain divalent radionuclides, such as strontium, may be inhibited using calcium chloride in large quantity. Iodine radionuclides accumulate in the thyroid gland and potassium iodide is used to eliminate the incorporated isotopes. Chelating agents, such as calcium-DTPA and zinc-DTPA, may be administered to chelate a number of radioactive materials and promote their elimination via urinary system. Changes in the alkalinity by sodium bicarbonate can be used to eliminate uranium poisoning (Dominguez-Gadea and Cerezo 2011).

A variety of synthetic and natural products have been studied for decorporation of radionuclides from the body. Orally administered dietary recombinant Chlorella algae were shown to inhibit the absorption of strontium (90Sr) into the blood and enhance its elimination from mice body through adsorption in intestine (Ogawa et al. 2016). The transdermal delivery of synthetic DTPA di- and tri-ethyl ester has been reported to enhance decorporation in a dose-dependent manner (Zhang et al. 2013). Different silica-based materials have also been tested to capture various radionuclides of Plutonium, Americium, Uranium, and Thorium (Yantasee et al. 2010). The mechanisms of radioprotection in case of radionuclide’s toxicity might be chelating, inhibiting uptake by changing chemical state, diluting, and flooding of active compounds (Dominguez-Gadea and Cerezo 2011). However, most of these absorbents are not well tolerated due to their side effects. Research is still in progress for more suitable agents with less toxicity to various organs.

Conclusions

An overwhelming number of natural agents and derivatives were studied for their radioprotective potentials. These compounds belong to a broad range of unrelated chemical groups. Although the mechanisms of radioprotection differ widely among groups, most compounds act primarily via scavenging of radiation-induced free radicals or by quenching of secondary biomolecular reactive species. Where radioprotection was demonstrated, the compounds showed modest dose reduction factor (DMF) ranging from 1.1 to 2.7. However, the resulting number of useful radioprotectors that can be used in human remains surprisingly limited. To date, not a single drug is ready to be used with acceptable degree of confidence except amifostine. Several drugs obtained an Investigational New Drug (IND) status; however, their use in clinical setting is still to be confirmed. Hence, the ideal radioprotector remedy has not been achieved yet despite the extensive research for last five decades.

Future development of ideal radioprotectors requires taking into consideration the following points. The interaction of radiation with biological systems involves physico-chemical and molecular processes and, hence, these are complex in nature. A radioprotector comes into play as an intermediate neutralizing the produced free radicals, thus preventing their consequences on the biological system. Whether the intent of their use is for initial radiation protection, mitigation or therapeutic, they are often required to be present in effective concentrations during or shortly after the radiation exposure. The development of suitable radiation countermeasures that could possibly be used as prophylactic and/or therapeutic agents is in nascent stage. The quest is still ongoing to discover less toxic compounds that could be used under minimal medical supervision during radiotherapy and field applications.

Varieties of molecules have shown their radioprotective properties and potential (Fig. 1; Table 1), but their detailed molecular, physiological and pharmacological mechanisms need to be fully studied and understood before being applied. Attempts to repurpose suitable nontoxic drug for the management of radiation victims are also an attractive approach and that can save time, money, and risk associated with new drug development. It is evident that natural compounds are currently the most attractive source due to their potential lower toxicity than synthetic compounds. The major concerns of high dosage and limited bioavailability and efficacy can be improved by designing more efficient derivatives of selected potential compounds.

One of the important challenges of radioprotector development is the model system where these promising molecules could be tested. Therefore, the identification of biomarkers that can be used to test putative agents is also needed. Although the ability to reduce radiation-induced xerostomia was tested, many more test systems are needed for different category of radioprotective agents. The risk of radiation-induced secondary malignancy and cardiovascular diseases due to the use of radiotherapy for cancer patients is also legitimate targets for radiation protection. The risk of cardiovascular diseases during breast cancer therapy is of growing concern and has attracted more attention recently. Therefore, suitable agents with the ability to protect cardiovascular system will be an achievement of great importance.

In summary, the future development of ideal radioprotector requires an appropriate nontoxic drug, a model system and biomarkers of radiation exposure to test and verify the effects on physiological systems. Multifaceted mechanistic understanding is essential in this development. Such approach is essential to ensure the efficacy and safety of customized agents for use during radiotherapy and field application in cases of nuclear eventualities.

Abbreviations

bFGF

Beta fibroblast growth factors

CD2F1

Cluster of differentiations 2F1

DMA

(5-2-[2′-(3, 4-Dimethoxyphenyl)-5′-benzimidazolyl]

DMF

Dose modifying factor

DTPA

Diethylenetriaminepentaacetic acid

FDA

Food and Drug Administration

G-CSF

Granulocyte colony-stimulating factor

GM-CSF

Granulocyte macrophage colony-stimulating factor

GPx

Glutathione peroxidase

GT3

Gamma tocotrienol

Gy

Gray (dose of ionizing radiation)

HMG-CoA

3-Hydroxy-3-methyl-glutaryl-coenzyme A

IL

Interleukin

MAP kinases

Mitogen activated protein kinases

MEA

β-Mercaptoethyamine

NF-kB

Nuclear factor kappa beta

ROS

Reactive oxygen species

SOD

Superoxide dismutase

SQGD

Semiquinone glucoside

TBZ

2-(4-Thiazolyl) benzimidazole

TS

Tocopherol succinate

LD50

Lethal dose for 50% killing

TLR 2/6

Toll-like receptor 2/6

TNF-α

Tumor necrosis factor-α

Author contributions

KM collected information and drafted the manuscript and GA conceived the review and critically revised and edited the content.

Compliance with ethical standards

Funding

This work was partially supported the National Science, Technology and Innovation Plan (NSTIP, KACST) Grants 12-MED2945-20 and 15-MED4114-20 (KFSHRC, RAC 2130-025 and 2170 005).

Conflict of interest

The authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Availability of data and material

Data sharing is not applicable to this review manuscript as no datasets were generated or analyzed during the current study.