Review of the evidence of radioprotective potential of creatine and arginine as dietary supplements

Abstract

Purpose

Creatine (Cr) and l-arginine are naturally occurring guanidino compounds, commonly used as ergogenic dietary supplements. Creatine and l-arginine exhibit also a number of non-energy-related features, such as antioxidant, anti-apoptotic, and anti-inflammatory properties, which contribute to their protective action against oxidative stress (OS). In this regard, there are a number of studies emphasizing the protective effect of Cr against OS, which develops in the process of aging, increased physical loads as part of athletes’ workouts, as well as a number of neurological diseases and toxic effects associated with xenobiotics and UV irradiation. Against this backdrop, and since ionizing radiation causes OS in cells, leading to radiotoxicity, there is an increasing interest to understand whether Cr has the full potential to serve as an effective radioprotective agent. The extensive literature search did not provide any data on this issue. In this narrative review, we have summarized some of our own experimental data published over the last years addressing the respective radioprotective effects of Cr. Next, we have additionally reviewed the existing data on the radiomodifying effects of l-arginine presented earlier by other research groups.

Conclusions

Creatine possesses significant radioprotective potential including: (1) radioprotective effect on the survival rate of rats subjected to acute whole-body X-ray irradiation in a LD_70/30 dose of 6.5 Gy, (2) radioprotective effect on the population composition of peripheral blood cells, (3) radioprotective effect on the DNA damage of peripheral blood mononuclear cells, (4) radioprotective effect on the hepatocyte nucleus-nucleolar apparatus, and (5) radioprotective effect on the brain and liver Cr–Cr kinase systems of the respective animals. Taking into account these cytoprotective, gene-protective, hepatoprotective and energy-stimulating features of Cr, as well as its significant radioprotective effect on the survival rate of rats, it can be considered as a potentially promising radioprotector for further preclinical and clinical studies. The review of the currently available data on radiomodifying effects of l-arginine has indicated its significant potential as a radioprotector, radiomitigator, and radiosensitizer. However, to prove the effectiveness of arginine (Arg) as a radioprotective agent, it appears necessary to expand and deepen the relevant preclinical studies, and, most importantly, increase the number of proof-of-concept clinical trials, which are evidently lacking as of now.

Keywords:

• Creatine
• l-arginine
• radioprotection

Introduction

The growing application of nuclear technologies for military and peaceful purposes raises a need to ensure radiation safety to a humankind. Radiation therapy, while one of the most effective treatment modalities in clinical radiology, is simultaneously toxic to normal cells and induces adverse effects. To restrain toxicity to normal tissues, patients are usually treated with smaller radiation doses (1.8–2 Gy in a period from 4 to 8 weeks). At the same time, the recent advances in clinical radiology allowed to harmlessly use a limited number of high doses (up to 15–20 Gy) in the case of patients with certain types of cancer (Moding et al. 2013).

It is a well-known fact that biological molecules are exposed to both direct and indirect effects of ionizing radiation (IR). The direct effects of IR emerge through its direct interaction with individual DNA moieties. The indirect effect is generated through interactions with water radiolysis products, such as hydrogen atoms, hydroxyl radicals, and solvated electrons perform with DNA and other cell constituents, such as lipids, proteins, etc. (Desouky et al. 2015; Suzuki et al. 2021). It is shown that direct radiation induces predominantly DNA damage in cells, along with some of its most prominent consequences, such as apoptosis, necrosis (Sia et al. 2020), mitotic catastrophe (Galluzzi et al. 2018), autophagy (Levy et al. 2017), cell cycle arrest, and/or senescence (Li et al. 2018). Moreover, IR causes disturbances in the DNA synthesis, which may result in altered or lost genetic material (Painter 1985; Jaspers and Zdzienicka 2006).

Another type of cell damage may include the damage of the cell membrane, as well as membranes of cellular organelles (Somosy 2000). In addition, due to the impaired protein synthesis, disruption of the cellular metabolism and/or cellular structure may occur. Consequently, all these processes lead to the development of oxidative stress (OS) in the cell, which in turn may lead to the radiation induced toxicity (Azzam et al. 2012; Reisz et al. 2014; Akbari et al. 2019). At the same time, it has been noted that maintaining basal levels of reactive oxygen species (ROS) in the cells is essential for cell viability (Turpaev 2002; Starkov 2008; Morgan and Liu 2011; Villalpando-Rodriguez and Gibson 2021). Cells can neutralize toxic effects of ROS when the latter is directly reduced with the help of antioxidant enzymes, as well as through maintaining a healthy mitochondrial pool (Starkov 2008).

Currently, there is an accepted approach to grouping various radioprotective agents, depending on the timing of their administration, which assumes one of the following: protection, mitigation, or therapy (Mishra and Alsbeih 2017; Mun et al. 2018; Liu et al. 2023). Radioprotectors are used as a preventive measure and are normally meant to be applied before radiotherapy or IR exposure. They protect organisms from cellular and molecular damage, predominantly by enhancing antioxidant defense mechanisms through the scavenging of free radicals (Singh and Hauer-Jensen 2016).

Mitigators are usually administered shortly after the given exposure to IR. Thus, they lessen the general effect that IR causes to tissues before the onset of symptoms by enhancing DNA repair, cellular signaling, and modulating thiols redox system of the cells (Citrin et al. 2010). Therapeutic agents may be administered following IR and/or after the symptoms appear, and thus serve as a form of medication to regenerate tissues by stimulating division of functionally undamaged cells (Citrin et al. 2010; Bourgier et al. 2012). The disadvantages of synthetic chemical radioprotectors, including high toxicity and side effects, as well as the limited duration of the positive effect explain the fact as to why nowadays only Amifostine is approved by the US Food and Drug Administration (FDA) (Mun et al. 2018).

Against this backdrop, the study of radioprotective properties of low-toxic natural compounds is becoming particularly relevant. The FDA-approved radioprotective agents, such as Filgrastim (a recombinant form of the naturally occurring granulocyte colony-stimulating factor, G-CSF), Pegfilgrastim (a PEGylated form of the recombinant human G-CSF, PEGylated G-CSF), and Sargramostim (a recombinant granulocyte-macrophage colony-stimulating factor, GM-CSF) were considered as radiomitigators (Singh et al. 2017; Mun et al. 2018). However, to date, no radioprotector has been approved for acute radiation syndrome (Obrador et al. 2020).

It is well known that the whole variety of animals, as well as humans, own a common system of storing and transporting chemical energy. This system includes phosphagens, which are guanidinо substances having an energy-rich, phospho-amido bond, and phosphagen kinases (guanidine kinases), which are isofunctional enzymes catalyzing the reversible reaction of the transfer of the macroergic phosphoryl residue from ATP to guanidine-containing bases (Ellington 2001). To date, eight various phosphagens with their own phosphagen kinases have been documented. In fact, among them, most widely known are the creatine (Cr) phosphate-creatine kinase (PCr-CK) and arginine phosphate (AP)-arginine kinase (AK). Moreover, it was shown that creatine kinase (CK) superseded AK during the transition from invertebrates to vertebrates (Lyzlova and Stefanov 1991; Ellington 2001). In the meantime, the other six phosphagens (guanidine acetate phosphate, taurocyamine phosphate, lombricine phosphate, opheline phosphate, hypotaurocyamine phosphate and thalassemine phosphate) can be seen extremely rarely. CK is the only phosphagen kinase that vertebrates have, unlike invertebrates featuring also other phosphagen kinases, alongside AK (Lyzlova and Stefanov 1991).

In this narrative review, we first provide a brief background on the physiological roles and some applications of Cr and arginine (Arg), the substrates of the CK and AK, respectively. Further, the part devoted to Cr, is followed by a summary of our own experimental data on the radioprotective effects of Cr, presented in the number of our recent publications (Nersesova et al. 2013; Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021). Concurrently, we summarized the existing findings in the literature on the radioprotective effects of l-arginine. The respective Cr and Arg data indicate significant radioprotective potential for both compounds as dietary supplements.

Radioprotective potential of creatine as dietary supplement

Brief overview of creatine

Cr is a naturally occurring guanidino compound (methyl-guanidine-acetic acid) that plays a key role in the maintenance of the cellular energy and calcium homeostasis (Walker 1979; Brosnan et al. 2007; Wallimann et al. 2011). The human body gets the bigger share of Cr necessary for normal physiological functioning of the its organs through the meat and fish uptake, while the remaining required part of Cr is synthesized in liver, spleen, and kidneys. The average diet provides 1 g of Cr per day, and about the same amount of Cr is synthesized (Brosnan et al. 2007). The latter is then exported to the blood to reach the tissues where it is absorbed by cells with the help of specialized Na-dependent Cr transporter (Nash et al. 1994).

In conjunction with CK, Cr and phosphocreatine (PCr) as substrates establish the Cr–PCr–CK system. This system, in addition to its key role in providing energy and maintaining calcium homeostasis of the cell, also performs a number of other functions. One is the special role which refers to the maintenance of the structural/functional status of mitochondria, closely related to the antioxidant and anti-apoptotic properties of Cr (Schlattner et al. 2006; Wallimann et al. 2011). CK catalyzes the conversion of excessive ATP produced by the cell into PCr to refill the pool of this reserve macroerg until its next use. Still, PCr not only serves as a temporary energy buffer but also participates in transporting energy from the places of its production to the places of its consumption (Figure 1). Figure 1 is a classical graphical representation of the role played by Cr–PCr–CK system, whether as an energy buffer or an energy shuttle (Wallimann et al. 2011). On top of everything else, there are a few non-energy-related functions of Cr, including: (1) Cr being one of the main osmolytes of the central nervous system; (2) Cr serving as a neurotransmitter (Marques and Wyse 2019).

Figure 1. Graphical presentation of the role of the Cr–PCr–CK system as an energy buffer and as an energy shuttle. (1) Cr enters the cells through the Cr-transporter (CRT). Within the cell, the PCr/Cr and ATP/ADP ratios are regulated by the soluble fraction of cytoplasmic CK (CK-c; see 3). The cytoplasmic CK specifically associated with glycolytic enzymes (G; CK-g; see 2), takes on glycolytic ATP regenerating PCr, whereas mitochondrial CK (mt-CK; see 1) associated with the adenine nucleotide transporter (ANT) accepts ATP generated through oxidative phosphorylation (OP), releasing PCr into the cytoplasm. The cytoplasmic CK, specifically associated with subcellular sites of ATP consumption (CK-a; see 4), forms microcompartments that regenerate in place with an excess of PCr ATP consumed by ATP-ase reactions. Thus, the Cr–PCr energy shuttle connects subcellular energy production sites with subcellular energy consumption sites (Wallimann et al. 2011 adapted).

The cytoplasmic isoenzymes of CK (cyt-CK), which have a dimer structure, include muscle (CK-MM), brain (CK-BB), and cardiac (CK-MB) isoenzymes (Wallimann et al. 2011). Further, the mitochondrial isoenzymes of CK (mit-CK), which have an octamer/dimer structure, are represented in tissues by the sarcomeric isoenzyme (striated muscle tissue), as well as the ubiquitous isoenzyme (most other tissues). Мit-CK structure is critical to its ability to interact with other mitochondrial proteins, as well as perform its functions. For example, the octameric mit-CK provides a functional link between two adjacent proteins, namely porin and adenine nucleotide transporter, located on the outer and inner mitochondrial membranes, respectively, thereby inhibiting the opening of the mitochondrial permeability transition pores (Schlattner et al. 2006; Wallimann et al. 2011).

Why creatine may have radioprotective potential

The antioxidant and antiapoptotic properties of Cr and its protective effect against oxidative stress

The antioxidant and antiapoptotic properties of Cr are important components for its protective action against OS (Wallimann et al. 2011). Within the cell, Cr delivers both direct and indirect antioxidant action. For example, Cr has been shown to be effective in direct scavenging of a number of free radicals, such as superoxide anion, peroxynitrite, and several others (Lawler et al. 2002; Sestili et al. 2006; Sestili et al. 2011). With regard to the indirect antioxidant effect of Cr, it is worth noting that the regulation of mitochondrial respiration is closely related to the Cr–PCr–CK system. By stimulating mitochondrial respiration, Cr promotes the efficient circulation of ADP inside mitochondria via the mit-CK reaction. The latter provides more efficient connection of mitochondrial respiration with the ATP synthesis, which, in turn, contributes to the suppression of ROS production (Kay et al. 2000). One of the best examples of the antioxidant activity of Cr extends to the protective effect of Cr against the UV radiation induced OS in skin keratinocytes (Lenz et al. 2005). Another example is about its cytoprotective effect against free oxygen radicals, as detected using a range of cell lines (Brewer and Wallimann 2000; Sestili et al. 2006; Qasim and Mahmood 2015).

As for the anti-apoptotic effect of Cr, it has been shown that Cr, together with the octameric mit-CK, prevents or stops the opening of mitochondrial permeability transition pores, through which apoptotic factors, such as cytochrome c and caspase substrates, as well as Ca²⁺, are released from mitochondria (Schlattner et al. 2006). It should be noted here that in the case of Cr supplementation, liver cells of transgenic mice, which feature high levels of BB-CK expression, appear to be resilient to hypoxia-induced apoptosis (Miller et al. 1993). The opposite, subjects with Cr deficiency syndrome demonstrate increased OS and apoptosis induced by ROS (Brewer and Wallimann 2000; Alcaide et al. 2011; Wallimann et al. 2011; Clarke et al. 2020).

The protective effect of creatine as a dietary supplement against oxidative stress associated with several diseases and conditions of the body

Numerous in vivo and in vitro studies reported the protective effect of Cr against OS, which develops in the process of aging, increasing physical loads in athletes’ workouts as well as a number of neurological diseases and toxic effects associated with xenobiotics and UV irradiation (Lenz et al. 2005; Fimognari et al. 2009; Sestili et al. 2011; Gonzales et al. 2014; Kreider and Stout 2021). One of the advantages of Cr is that in relatively high doses, such as 2–20 g per day, Cr itself is still safe (Kim et al. 2011). As a dietary supplement, it increases the energy status of the organism, fuels its activity and endurance, as well as prevents muscle atrophy. This is why it is widely used as an ergogenic agent in sports (Antonio et al. 2021).

Even today, elderly people use Cr to prevent sarcopenia (Dolan, Artioli, et al. 2019). A study looking at the effects of a commercial Cr–folic acid formulation designed to fight the clinical signs of premature aging, induced by the UV exposure, found that the respective formulation provided an effective treatment option for photoaged skin (Knott et al. 2008). Oral Cr supplementation was shown to improve memory in healthy individuals, especially older adults and stressed-out individuals (Avgerinos et al. 2018; Dolan, Gualano, et al. 2019; Forbes et al. 2022; Prokopidis et al. 2023).

Of special interest are the data reflecting on the considerable gene-protective effect of Cr on the mitochondrial DNA of human cells exposed to OS (Guidi et al. 2008). The protective effect of Cr intake was also demonstrated against oxidative DNA damage and lipid peroxidation caused by the critically increasing physical loads in athletes’ workouts (Mirzaei et al. 2013).

The use of Cr as a form of adjuvant therapy for various neurodegenerative diseases associated with OS, possible mechanisms of the neuroprotective effect of Cr, as well as reasons for the controversial data in some of the clinical trials, have been studied in detail and subsequently discussed in a number of reviews (Gualano et al. 2012; Gonzales et al. 2014; Hersch et al. 2017; Mo et al. 2017; Clarke et al. 2020; Forbes et al. 2022; Marshall et al. 2022; Nersesova et al. 2022). There is also some solid evidence that the dietary supplementation with Cr may have therapeutic value in the treatment of cancer in experiments with rodents (Pal et al. 2016; Cella et al. 2020). Comparable benefits of Cr supplementation were also registered among humans suffering from various types of cancer (Fairman et al. 2019). In addition, when used against renal dysfunction due to cytotoxicity of the anticancer drug cisplatin, Cr exhibits considerable therapeutic effect, which could be of solid clinical interest (Genc et al. 2014). Figure 2 demonstrates generalized data about the relative efficiency of the use of Cr as a dietary supplement in case of various diseases and conditions of the body, based on the studies cited above (Hersch et al. 2006; Adhihetty and Beal 2008; Gualano et al. 2012; Gonzales et al. 2014; Smith et al. 2014; Hersch et al. 2017; Mo et al. 2017; Clarke et al. 2020; Harmon et al. 2021; Forbes et al. 2022; Marshall et al. 2022; Nersesova et al. 2022) and the following systematic reviews (Avgerinos et al. 2018; Bonilla et al. 2021; Kreider and Stout 2021; Prokopidis et al. 2023). The ‘probable’ rating denotes the case of strongly established efficacy of Cr in preclinical and double-blind, placebo-controlled clinical trials. Further, the ‘possible’ rating is used in the case of some reliably established efficacy of Cr in preclinical studies with controversial results and/or insufficient number of participants in clinical trials or other insufficient conditions in terms of a clinical trial protocol (Gualano et al. 2012).

Figure 2. The relative effectiveness of the therapeutic and preventive use of creatine in various diseases, metabolic disorders and body conditions.

And finally, the traditional target theory of radiobiology considers the damage to the nuclear DNA to be the primary cause of the biological effects of IR (Desouky et al. 2015; Shibata and Jeggo 2019). However, the data available in literature and accumulated to date point out to an equally important role of mitochondria in the development of intracellular radiation effects (Kim et al. 2019). In this regard, it should be noted that in the case of low doses irradiation, the biological effects induced by ROS are relatively more significant than those caused through DNA breaks (Yang et al. 2014; Kawamura et al. 2018; Kim et al. 2019; Patten et al. 2019). Cr is one of the most popular adaptogens, and as part of the Cr–PCr–CK system, it is considered to be one of the most effective mitochondrial protectors against OS (Schlattner et al. 2006; Wallimann et al. 2011).

Thus, considering the protective effects of Cr against OS induced by various factors described above (Lenz et al. 2005; Gonzales et al. 2014; Qasim and Mahmood 2015), and especially by UV radiation (Lenz et al. 2005), as well as considering the importance of the Cr–CK system in providing cellular energy and Ca homeostasis, along with its role in the provision of the structural and functional stability of mitochondria (Schlattner et al. 2006; Wallimann et al. 2011), we attached high importance to the issue of assessing the radioprotective potential of Cr. This gains even more importance because the extensive literature search did not provide any data on this issue.

Our group has studied the CK radiosensitivity and its adaptive plasticity by conducting a comparative assessment of the dynamics of post-radiation changes of the brain and liver CK activity, serving as an indicator of the respective cell energy status (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021). Further, we have studied the radioprotective activity of dietary Cr against the acute total-body X-ray irradiation of rats based on the comparative assessment of the post-radiation changes in the (1) status of Cr–CK system in the brain and liver, (2) morphofunctional status of the nucleus-nucleolar apparatus of hepatocytes, (3) population composition of the peripheral blood cells, as well as radioprotective effects of Cr on (4) the DNA damage of the mononuclear cells of the peripheral blood and (5) the rats’ survival (Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019).

Creatine kinase radiosensitivity and its adaptive plasticity

A course of radiation therapy, various radiation accidents, as well as nuclear/radiological terrorism, may lead to an exposure to radiation doses in the range from 1 to 10 Gy (Coleman et al. 2003). Apparently, the effect of radiation induced OS depends on the strength of the respective irradiation. Cells can return to normal physiological state in case of light disorders, while a more serious OS may cause their death (Reisz et al. 2014).

It was shown that the final manifestation of the damage caused to the enzymes in vivo may be delayed for several days (Azzam et al. 2012). The evaluation of the post-radiation changes in the enzyme activity levels on the 1st, 6/7th, and 14/15th days following irradiation showed that in the case of a single whole-body X-ray irradiation, both in the 4.5 Gy and 6.5 Gy doses, the brain and liver CK, as well as the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (both studied for comparative purposes), demonstrate different radiosensitivity and adaptive capabilities. This finding may be corroborated based on the differences in the magnitude and direction of the changes in the activity levels of these enzymes, as well as based on the oscillatory changes in the activity levels of each of the respective enzymes (Nersesova et al. 2013; Nersesova Petrosyan, Babayan, et al. 2019; Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021). It should be noted that the relatively stronger radiosensitivity of CK revealed by our group is consistent with the data in literature on high sensitivity of CK to OS, at both the post-transcriptional and genetic levels (Malone and Ullrich 2007; Sersa et al. 2010).

Concerning the oscillatory nature of post-radiation changes in the activity levels of these enzymes, it has been found that changes of this kind are characteristic of both the levels of activity and expression of enzymes, as well as of other post-radiation biological effects (Morgan 2003; Malone and Ullrich 2007; Sersa et al. 2010). This phenomenon is associated with the development of oxidative processes over time and, apparently, inclusion of various adaptation mechanisms of the cell (Morgan 2003; Sersa et al. 2010). Moreover, the post-radiation changes in the enzymes studied over time indicate certain stabilization of their activity levels up to the control, or increased compensatory level by the 13–15th days after the irradiation (Nersesova, Petrosyan, Babayan, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021), which more or less coincides with the end of the peak of acute radiation sickness in rats irradiated in sub-lethal doses (Shevtsov et al. 2004). Thus, the changes in the CK activity after X-ray irradiation have an adaptive nature aimed at restoring the energy homeostasis of the cell. At the same time, it appears that the adaptive properties of CK are tissue-specific and apparently depend on the CK content in the tissue (Hatano et al. 1996; Auricchio et al. 2001).

Radioprotective effects of creatine on DNA damage and Cr–CK system

Based on the abovementioned studies as well as generally accepted approaches to assessing the radiomodifying effects of potential radioprotectors, our group has analyzed the effect of Cr monohydrate on some of the X-ray irradiation induced biological effects described below (Nersesova, Petrosyan, Babayan, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021). During the respective studies, the rats received Cr for two weeks before and two weeks after irradiation in a dose of 1 g per kg of BW per day as part of a 0.9% solution of glucose. The latter, as argued by several authors, increases the bioavailability of Cr (Green et al. 1996). In the first series of experiments, the radiomodifying activity of Cr was assessed for DNA damage in the peripheral blood mononuclear cells, as well as for the CK activity and Cr content in the brain and liver of the rats on the 1st, 7th, and 15th days after irradiation in a dose of 4.5 Gy (Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova et al. 2021). In the second series of experiments, described in the following subsection, the radiomodifying activity of Cr was assessed for the following: (1) survival of rats, (2) activity of CK and content of Cr in the brain and liver, (3) status of the nuclear-nucleolar apparatus of hepatocytes, and (4) population composition of the peripheral blood cells on the 30th day after the irradiation in a dose of 6.5 Gy (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021).

Genoprotective effect of creatine in peripheral blood mononuclear cells

The DNA damage model is widely used in both in vitro and in vivo studies to evaluate the effectiveness of new radioprotectors (Dahle and Kvam 2003; Rodriguez-Rocha et al. 2011; Cinkilic et al. 2013; Maurya and Devasagayam 2013; Reisz et al. 2014; Kumar et al. 2018; Shibata and Jeggo 2019). A comparative analysis of the dynamics of post-radiation changes in the DNA damage level in peripheral blood mononuclear cells of rats exposed to radiation in a dose of 4.5 Gy on the 1st, 7th, and 15th days after irradiation has shown a significant protective effect of Cr on DNA damage. Moreover, the type of the dynamics of these post-radiation changes appears to be indicative of the systemic nature of this radioprotective effect of Cr (Nersesova, Petrosyan, Babayan, et al. 2019). In this regard, the data on the protective effect of Cr on the damage induced by H₂O₂, including DNA breaks, in isolated human blood lymphocytes may be of interest (Qasim and Mahmood 2015). Particularly, these authors (Qasim and Mahmood 2015) demonstrated that the respective cytoprotective effect could be associated with the ability of Cr to neutralize ROS and prevent suppression of the cellular enzymatic and non-enzymatic antioxidant systems. This study unequivocally supports our conclusion about the radioprotective feature of Cr in relation to the genotoxic effect of IR.

Radioprotective effect of creatine on the rat brain and liver Cr–CK systems

According to the in vivo and in vitro studies, the enrichment with Cr has direct effect on increasing the bioenergetic balance of cells through the increase in both CK activity and content of PCr and ATP within a cell (Greenhaff et al. 1994; Dechent et al. 1999; Brewer and Wallimann 2000). It should be noted that Cr from the bloodstream easily passes through the blood–brain barrier with the help of a special Na-dependent transport protein-carrier, after which it is actively extracted from the extracellular fluid of the brain by neurons and oligodendrocytes. Moreover, astrocytes lacking this transport protein are able to synthesize Cr themselves (Braissant et al. 2007; Roschel et al. 2021).

The studies conducted by Nersesova et al. found that Cr demonstrates a protective effect on the brain and liver CK of rats exposed to a dose of 4.5 Gy. This was evidenced by a decrease in the damaging effect of radiation on the CK activity induced on the first day after irradiation on the one hand, and the stimulation of the adaptive properties of CK through the compensatory increase of the enzyme activity level on the 7th and 15th days after irradiation on the other hand (Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova et al. 2021). At the same time, the brain, which is characterized by a rather pronounced energy metabolism and contains higher amounts of CK and Cr than the ones found in the liver (Wallimann et al. 2011), shows greater radiosensitivity, as well as greater plasticity in its adaptation to IR than what is indicative of the liver (Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova et al. 2021). The dynamics of the noted compensatory changes in the CK activity in the presence and absence of Cr as a dietary supplement, largely coincided with the timing of the development of acute radiation sickness in rats (Shevtsov et al. 2004), which signals about significant radioprotective properties of Cr (Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019; Nersesova et al. 2021).

As for the post-radiation changes in the content of Cr in the brain, as well as liver, several bursts of the respective levels were registered throughout the entire study. However, they did not significantly differ from the control levels, with a single exception concerning a significant increase in the content of Cr (by 27%) on the first post-radiation day in the brains of rats receiving Cr (Nersesova, Petrosyan, Babayan, et al. 2019; Nersesova et al. 2021). It can be assumed that the increased content of Cr is associated with the enrichment of the organism with a Cr supplement or stimulation of endogenous synthesis of Cr by radiostress.

Radioprotective effects of creatine on the population composition of peripheral blood cells, brain and liver Cr–CK systems, hepatocyte nucleus-nucleolar apparatus and survival of rats

As noted above, we evaluated the protective effect of Cr on the survival of rats exposed to acute whole-body irradiation in a dose of LD_70/30 equal to 6.5 Gy. Тhe rats in the experimental group received Cr in a dose of 1 g per 1 kg of BW per day as part of 0.9% glucose solution, while the rats in the two control groups received just the 0.9% glucose solution or simply water, respectively. It should be noted that, according to the results of this experiment, glucose did not show a significant radiomodifying effect on the parameters studied (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019).

In our experiments, we used Cr monohydrate, since this form of Cr is the best studied and most widely used both for scientific purposes and as a dietary supplement (LeBaron 2011; Hall and Trojian 2013). Cr is absorbed by cells only in a dissolved state, and the solubility of Cr in water does not exceed 1 g per 100 ml of water; therefore, we did not look at higher concentrations of Cr (LeBaron 2011). According to our preliminary experiments, the most effective dose of the Cr supplement is 1 g per 1 kg of BW, and the most effective administration regimen is two weeks before and two weeks after irradiation (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). The latter is consistent with the data reported in the literature on the need for accumulation of Cr in the body to ensure its protective effect (Green et al. 1996; Cooke and Cribb 2015).

Cytoprotective effect of creatine on the population composition of peripheral blood cells

During X-ray irradiation of animals, and particularly rats, in a dose of 6.5–7.0 Gy, it is mainly the hematopoietic tissue that is affected. That normally occurs along with the bone marrow form of the acute radiation sickness (of moderate severity) that develops soon and implies long-lasting (up to several months) recovery (Shevtsov et al. 2004).

The data obtained by our group showed that on the 30th day following irradiation, the signs of the classical form of acute radiation sickness characteristic of the population composition of peripheral blood cells were still persisting (Nersesova, Petrosyan, Karalova, et al. 2019). For example, while the control group of intact rats lacked early forms of blood cells, the control group of irradiated rats featured monoblasts, lymphoblasts, metamyelocytes, as well as basophilic and polychromatophilic erythroblasts, all together making up approximately 15% of the total population of peripheral blood cells. Further, the share of reticulocytes reached more than half of the population of peripheral blood cells (more precisely, 60% vs. 15% in the native control). At the same time, the number of lymphocytes in the blood of the same group of the irradiated rats decreased sharply, more than four times, which is the first and most straightforward sign of radiation sickness. The comparative analysis of the population composition of blood cells in these irradiated rats and that of the irradiated rats treated with Cr showed that under the latter’s influence, the severity of pathological changes in the blood composition significantly decreased (Nersesova, Petrosyan, Karalova, et al. 2019). Thus, in the rats treated with Cr-supplement, the content of lymphocytes in the blood was almost three times higher compared to the irradiated control, which demonstrates the effect of restoring the respective indicator to the normative level, which may well be associated with the radioprotective effect of Cr. Concurrently, the enrichment with Cr leads to the elevation of the number of monocytes, bringing their content closer to that in the blood of intact rats, as well as inducing a decrease in the number of nuclear forms of erythroblasts as well as pathological and destroyed cells. Under the influence of Cr, it was easy to notice the leveling of other post-radiation effects in the population composition of peripheral blood, which by approximating the blood picture to a native one, may further indicate the cytoprotective effect of Cr (Nersesova, Petrosyan, Karalova, et al. 2019).

Hepatoprotective effect of creatine

The adaptive capabilities of functionally active cells of an organ can be manifested through cell proliferation for the purpose of regeneration, as well as activation of surviving cells, in order to replenish their function (Gentric and Desdouets 2014; Gentric et al. 2015). Before evaluating the hepatoprotective effect of Cr in the case of acute X-ray irradiation of rats in a dose of 6.5 Gy on the 30th day after irradiation, we preliminarily studied the post-radiation changes in the hepatocyte nuclear-nucleolar apparatus induced by acute X-ray irradiation of rats in a dose of 4.5 Gy. On the 6th day after irradiation, we noticed the development of destructive processes in rat hepatocytes, which was characterized by a shrinking nuclei area and perimeter, as well as decreasing the average number of nucleoli per nucleus, on the one hand, and increasing proportion of aneuploid and, in particular, hypodiploid nuclei without nucleoli, on the other hand. The latter most likely indicates cells, have died, possibly through apoptosis. This assumption also comes from the detection of cells with ‘apoptotic bodies’ (Nersesova et al 2013).

Further, in the hepatocyte population of the irradiated rats, compared with the intact control, the number of euploid nuclei significantly decreases, mainly due to the decrease in the number of diploid cells. At the same time, the proportion of tetraploid cells increases significantly, along with hyperoctaploid cells also emerging. Here, it must be noted that in the samples studied, cells almost do not divide (Nersesova et al. 2013), which indicates that in this case compensatory mechanisms are switched on, aiming at changing the functional activity of surviving hepatocytes, but not replacing dead cells with the new ones (Gentric and Desdouets 2014; Gentric et al. 2015).

It turns out that, irradiation in a dose of 6.5 Gy causes changes in the distribution of nuclear DNA according to ploidy, almost similar to those that happen during the irradiation in a dose of 4.5 Gy. On the 30th day after the exposure, the latter manifests itself in the form of an increase in the proportion of aneuploid and especially hypodiploid hepatocytes, as well as an increase in nuclear polyploidization. The enrichment of the rats’ diet with Cr brings the pattern of distribution of hepatocytes by ploidy closer to that of intact rats of the control group, which further indicates the hepatoprotective effect of Cr (Nersesova, Petrosyan, Karalova, et al. 2019). Statistically reliable data on the postradiation changes of average content of DNA in the nucleus, its average area and perimeter, as well as the average number of nuclei per nucleus on the 30th day after irradiation, have not been detected.

Radioprotective effect of creatine on the brain and liver Cr–CK systems

The radiomodifying effect of Cr on the cerebral and hepatic Cr–CK systems of rats in the experimental groups on the 30th day after irradiation in a dose of 6.5 Gy manifests itself in the form of increased, compensatory levels of both the CK activity and content of Cr in these organs, potentially indicating the presence of delayed effects of radiation (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). Of particular interest are the data reflecting on the changes in the level of cerebral CK activity, which is induced by the same irradiation in rats having survived in the control irradiated group (the one that received water instead of Cr). For these animals, featuring a normal level of brain Cr, a significantly higher level of cerebral CK activity (compared to that for intact animals in the control group) was determined, thus clearly indicating that the increased natural resistance of the respective animals to the IR effects engages the brain Cr–CK system (Petrosyan et al. 2019; Nersesova et al. 2021). It may be assumed that the mechanisms of long-term adaptation associated with the additional biosynthesis of enzymatic proteins become activated specifically during the post-radiation period. A somewhat different effect was observed for the hepatic Cr–CK system of the rats examined (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). With a statistically unreliable increase in the CK activity, some significant increase in the content of Cr in the liver can still be found, which is possibly associated with a certain compensatory increase in its endogenous synthesis, quite typical to this organ (Hatano et al. 1996; Auricchio et al. 2001).

Radioprotective effect of creatine on the survival of rats

And finally, the gold standard for evaluating the effectiveness of potential radioprotectors, known as the animal survival model, fully confirms the radioprotective effect of Cr (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). Various criteria are used to evaluate the anti-radiation agents’ radioprotective ability, including, for instance, the impact they have on DNA breaks in in vivo and in vitro models, activity and expression of metabolically important enzymes, indicators of the OS and DNA repair. However, the evaluation of the 30-day survival rate of rodents is considered as a benchmark in radioprotective activity assessments (Orsolić et al. 2007).

The comparative analysis conducted by our group shows that the death of animals in the experimental group of rats treated with Cr in glucose solution begins on the 7th day after irradiation, while in the control group of irradiated animals that did not receive Cr, this occurs two days earlier, indicating somewhat greater resistance of the former group to the effects of IR. Moreover, in the same control group, the death of animals continues for up to 28 days, while in the experimental group, the incidence of death stops already on the 21st day after irradiation. The mortality rate of animals in the control group was 72.2%, which is significantly higher than the respective indicator for the rats in the experimental group, equal to only 33.3% (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). Thus, Cr reduces the mortality of rats that received X-ray radiation in a dose of 6.5 Gy equal to LD_70/30 by 39%. The rat survival rate calculated according to the Kaplan–Meier method (Rich et al. 2010) was 67%, and the increase in their average life expectancy was 39% (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). Thus, the data presented explicitly testify to the anti-radiation activity of Cr, which, as it appears, is capable of increasing the resistance of rats to IR.

Despite the fact that the comparison of the survival data that we obtained with similar data on other natural radioprotectors may be complicated by the fact that the key parameters largely depend on the combination of existing experimental conditions (e.g. types and doses of IR, animal species, methods and time of administration of preparations, as well as their doses), it seems necessary to provide some distinct data on the most actively studied natural radioprotective agents. First of all, we need to name the recombinant GM-CSF, which, when administered 48 hours after total irradiation of primates, reduces the level of mortality by 36% in the LD_50-60/60 group and 44% in the LD_70-80/60 group (Clayton et al. 2016). When flavanols (such as rutin and quercetin) are used in the doses of 10 mg per 1 kg of BW and 20 mg per 1 kg of BW, respectively, for five days before γ-irradiation in the dose of 10 Gy, the survival rate of mice in the experimental groups appears to be 70% and 69%, respectively (Patil et al. 2012). The survival rate of mice treated one hour before X-ray exposure with synthetic genistein in a dose of 200 mg per 1 kg of BW depends on the radiation dose. Thus, in the doses of 7 Gy, 8 Gy and 9 Gy, the survival rate increased by 30%, 44% and 37%, respectively. When the drug is administered 24 hours before irradiation, it loses its effectiveness (Grebeniuk et al. 2013).

In general, the data presented in this review, testify to the radioprotective efficacy of Cr, which can apparently increase the adaptability and resistance of rats to acute X-ray irradiation. It must be noted that, to the best of the knowledge we have, our study is the first of its kind in this area; hence, it is possible that the radioprotective efficacy of Cr (measured while using new combinations of experimental conditions) may turn out to be higher than what we observe based on the indicators rendered by our experiments (Nersesova, Petrosyan, Karalova, et al. 2019; Petrosyan et al. 2019). Therefore, the main goal of this review is to trigger interest in the topic under discussion in order to fuel research in this area.

Thus, the radioprotective effect of Cr on the survival rate of rats subjected to acute whole-body X-irradiation, as well as its cytoprotective, gene-protective, hepatoprotective and energy-stimulating activities, provide a solid background to consider Cr as a potential radioprotector (Figure 3). In line with its broad-based prerequisites, an ideal radioprotector, while nontoxic, should be easily dispensed, as well as let tumors be directly affected by IR. At the same time, it should be able to exclude any negative effect on a normal tissue (Mun et al. 2018). In the context of the above, it is important to note the following advantages of Cr: it is readily available; does not cause toxic effects, even in high doses; does not accumulate in a human body, even with repeated doses; has the ability to act through a number of mechanisms; can be administered orally; has a protective effect that extends to a number of organ systems; remains stable while stored; and, finally, no less important, is cost effective (Jäger et al. 2011; Kreider et al. 2017).

Figure 3. A generalized illustration representing the radioprotective effects of creatine.

Radioprotective potential of l-arginine as a dietary supplement

Brief overview of l-arginine

l-Arginine (Arg) is a conditionally essential amino acid when referring to adult population and an essential amino acid when it comes to children’s optimal growth and development. Similar to the vertebrates’ PCr-CK shuttle, the AP-AK shuttle has an important role in cell bioenergetics (Voncken et al. 2013).

Arg was found in many foods of both animal and plant origin, namely meat, dairy products, nuts, seeds, wheat, algae, and other products (Wu et al. 2007; King et al. 2008). A person with an average weight of 70 kg is able to tolerate intake of 10 g of Arg daily for a long period of time, which indicates its essential safety (Closs et al. 2004; Böger 2007; Wu et al. 2007).

For an adult, the average daily intake of Arg with food ranges from 3 to 6 g (Mirmiran et al. 2017). Arg taken with food is absorbed in the small bowel from where it enters the liver. In the latter, its main amount is utilized in the ornithine cycle. As a result of Arg degradation, the metabolites with huge biological importance, such as glutamate, NO, Cr, polyamines are produced (Wu and Morris 1998; Mirmiran et al. 2017). An important function of Arg is the detoxification of ammonia toxic to the central nervous system (Wu et al. 2009).

The structure of the Arg molecule suggests its participation in a large number of cellular processes, which are discussed in detail in the respective reviews (Tapiero et al. 2002; Wu et al. 2009; Morris 2016; Wu et al. 2021). Consider, for instance, the following cellular processes: Arg structure is favorable to the binding of the phosphate anion and, therefore, it is involved in the catalysis of phosphorylation reactions (O'Brien et al. 2008). Further, Arg participates in maintaining the charge of many proteins (Harms et al. 2011). It also promotes the secretion of hormones (Oh et al. 2017). In mammals, Arg activates cellular signaling pathways, thereby stimulating protein synthesis, enhancing cell migration, as well as increasing the production of milk proteins (Wu et al. 2004). It appears that Arg maintains the vascular tone and hemodynamics and can also have some complex effect on platelets, coagulation, and fibrinolytic systems (Wu and Meininger 2009; Heffernan et al. 2010). Finally, it has been shown that Arg is critical for embryonic survival, as well as fetal and neonatal growth (Rosselli et al. 1998; Wu et al. 2004).

The metabolite of Arg, namely NO, while a vasodilator, allows more blood to pass through the tissues, thus ensuring more nutrients to the body. The latter, in turn, stimulates the synthesis of muscle tissue and also helps to improve performance and increase endurance, which is very important, for example, in bodybuilding (Stefani et al. 2018). Since NO plays a major role in vasodilation, Arg may combat erectile dysfunction (Koolwal et al. 2019) and stimulate sperm production (Tanimura 1967). As a signaling molecule, physiological levels of NO enhance glucose uptake, glucose and fatty acid oxidation, as well as inhibit the synthesis of glucose, glycogen, and fat in target tissues, inhibit almost all enzymatic reactions (Jobgen et al. 2006), modulate the vascular system, regulate immune processes and control neuronal functions (Wu and Morris 1998; Wu et al. 2021). Thus, the great essentiality of Arg is attributed to its role as a precursor for the synthesis of NO.

The antioxidant and antiapoptotic properties of l-arginine

In the context of assessing the radioprotective action of Arg, the antioxidant and anti-apoptotic features of Arg are of special interest. NO, by itself, demonstrates an ability to function as a potent inhibitor of lipid peroxidation, mostly by scavenging lipid peroxyl radicals (Violi et al. 1999; Lass et al. 2002). Thus, NO has a well-established antioxidant effect (Hogg and Kalyanaraman 1999; Violi et al. 1999). At the same time, NO can perform as a pro-oxidant (Rubbo et al. 1994; Hogg and Kalyanaraman 1999).

Arg, like Cr, has both direct and indirect antioxidant effects. For example, it has been shown that Arg can develop good radical scavenging activity (Ahmad et al. 2015). The indirect antioxidant effects of Arg have been well documented in several reports (Liang et al. 2018; Wu et al. 2021). Dietary Arg supplementation of rabbits suffering from hypercholesterolemia may help form a certain capacity to limit the vascular release of superoxide anions and thus bring back NO production (Böger et al. 1995). Enrichment with Arg leads to lower level of the lipid peroxidation product malondialdehyde in patients suffering from diabetes mellitus (Lubec et al. 1997). Furthermore, it appears that Arg can activate the Nrf2 pathway and glutathione synthesis and, as a result, induce antioxidant response. The latter allows the authors to view Arg as a key element to induce an antioxidant response and defeat OS (Liang et al. 2018).

With regard to antiapoptotic activity of NO, Trachtman et al. (2000) showed that Arg was able to directly modulate apoptosis in mesangial cells in response to a variety of stimuli. In a different study, it was shown that Arg could help in maintaining and stimulating the skin fibroblast proliferative, as well as antiapoptotic and immune defense functions (Kocic et al. 2017).

l-arginine as a dietary supplement

As a dietary supplement, Arg exhibits a number of beneficial pharmacological effects, such as a reduced risk of vascular and heart diseases (Wu and Meininger 2000; Fayh et al. 2013), an improved immune response (Popovic et al. 2007) and enhanced male fertility (Tanimura 1967), inhibition of gastric hyperacidity (Nagahama et al. 2012), as well as a lower incidence of erectile dysfunction (Rhim et al. 2019), to name just a few. Of considerable interest are the promising anti-aging benefits of Arg, which are superior to those of any other similar agents (Gad 2010).

To date, some strong experimental basis for the use of Arg to prevent and treat intrauterine growth retardation in humans and animals has been provided (Wu et al. 2004; Mateo et al. 2008). Arg intake in women with gestational hypertension reduced blood pressure and decreased the frequency of babies with low-birth weights (Facchinetti et al. 1996). Considering the great importance for Arg in conceptus survival and growth, Wu et al. have evolved Arg treatment protocols, thus enhancing pregnancy outcomes for various species (Wu et al. 2004).

Another well-studied scope of dietary supplementation with Arg is endothelial dysfunction. It has been established that the NO deficiency is the basic cause of endothelial dysfunction, occurring in a number of metabolic diseases, such as hypercholesterolemia, hypertension, diabetes, and several others (Wu and Meininger 2000; Wu et al. 2009). Multiple lines of evidence show that Arg supplementation promotes reversing endothelial dysfunction happening under these conditions (Fayh et al. 2013). Furthermore, Arg administration may avert a major dysfunction of the cardiovascular system, which is done via several mechanisms. One of them refers to bringing back endothelial NO synthesis and diminishing superoxide production (Wu and Meininger 2000; Wu et al. 2007; Wu and Meininger 2009). However, as in the case of Cr, varying sources of Arg or populations under consideration, as well as different treatment regimens may distort the overall picture of the effectiveness of Arg supplementation. For instance, the results of the study evaluating the effects of dietary supplementation with Arg in patients who are obese or suffer from cardiovascular disease, or diabetes in contrast to the above, have shown that oral Arg supplementation had no connection to improvements on select variables in examined patients (Rodrigues-Krause et al. 2018).

The effects of dietary supplementation with Arg, noted above, as well as those identified for diseases, such as chronic metabolic disease, tissue injury and wound healing, immune status disorders, skeletal muscle disorders, cancer and others have been discussed in detail in the respective reviews (Tapiero et al. 2002; Wu and Meininger 2009; Gad 2010; Dong et al. 2011; Rodrigues-Krause et al. 2018; Rhim et al. 2019; Wu et al. 2021). Figure 4, compiled based on these studies, as well as the conclusions made respectively in several systematic reviews (Hadi et al. 2019; Khosroshahi et al. 2020; Viribay et al. 2020; Shiraseb et al. 2022; Sadeghi et al. 2023) demonstrates the comparative effectiveness of the use of Arg in case of several diseases and conditions of the body. The ‘probable’ rating is given in the case of a strongly established efficacy of Arg in preclinical and double-blind, placebo-controlled clinical trials. The ‘possible’ rating then is given in the case of a reliably established efficacy of Arg in preclinical studies with controversial results and/or insufficient number of participants in clinical trials or other insufficient conditions for the clinical trial protocol. It should be noted that in some cases similar protective effects have been recorded for both Cr and Arg. This phenomenon may assume that Arg, serving as a precursor for the synthesis of Cr, may have its protective effect indirectly, that is through Cr.

Figure 4. The relative effectiveness of the therapeutic and preventive use of Arg in various diseases, metabolic disorders and conditions of the body.

Radioprotective effects of l-arginine

It is fair to note that, the amount of data referring to the radioprotective effects of Arg as discussed in preclinical in vivo and in vitro studies and clinical studies with patients who tested Arg as a radioprotective agent is rather limited. For instance, a group of Brazilian scientists studied the protective effect of Arg on the prostate (non-neoplastic) of rats with radiation-induced injury (Pinto et al. 2016). The rats, which were supplemented with Arg in a dose of 0.65 g per kg of BW over 22 days, received a single-dose pelvic irradiation on the 8th day of the experiment. Arg was shown to have reduced radiation-induced diarrhea, maintained body weight in rats, and increased weight gain, as well as regenerated the acinar epithelium, thus suggesting systemic protective effects of supplementation with Arg (Pinto et al. 2016).

In another study, the effects of nutritional supplementation with Arg were examined on the rat bladder wall injury caused by pelvic radiation (Costa et al. 2013). The one-time irradiation of the rat pelvic-abdominal region was done in a dose of 10 Gy, and Arg was administered in a dose of 0.65 g per kg of BW per day, starting seven days before the irradiation and up until the euthanasia occurred on the 16th day after irradiation. It was concluded that pelvic radiation led to significant modifications in the morphology of blood vessels and vascular endothelial growth, as well as fibroblast growth factors expression in the wall of the rat bladder. Treatment based on the intake of Arg was effective in preventing all the negative changes. Thus, the use of Arg in its capacity of a radioprotective drug was strongly highlighted (Costa et al. 2013).

Yavas et al. described the effects of l-glutamine, Arg, and β-hydroxy-β-methylbutyrate on the acute inflammation, as well as mucosal atrophy in the oral mucosa induced by irradiation of rats (Yavas et al. 2013). The extent of surface epithelium smoothing, villous atrophy, lamina propria inflammation, cryptitis, crypt distortion, regenerative atypia, vascular dilatation and congestion, as well as fibrosis was quantified for histological sections of intestinal mucosa. Concurrent use of these supplements appears to mitigate acute inflammation, induced by irradiation (Yavas et al. 2013). However, according to the authors, this finding needs to be clarified in further clinical trials.

Radiation therapy also induces mucosal injury to the small bowel that can cause enteritis, complications of which may require a surgery (Stacey and Green 2014). Ersin et al. (2000) performed histopathological and microbiological evaluation of the preventive and therapeutic effects of Arg and glutamine enriched diets in a modeled case of radiation enteritis in rats exposed to acute abdomen-focused irradiation in a dose of 11 Gy. Seven-day designated diets were administered in pre- and post-irradiation patterns, respectively. With the post-irradiation scenario, both diets demonstrated protective effects on gut mucosa. At the same time, the simultaneous pre- and post-irradiation administration mode for the two diets does not provide any extra protective effect (Ersin et al. 2000).

Studies, where an attempt was made to reveal the molecular-cellular mechanisms of the protective action of Arg against IR, deserve some special attention. Shukla et al. (2008) show that Arg, administered two hours after 2 Gy total-body gamma irradiation, helps mitigate inflammatory processes, prevent apoptosis, and restore the spleen immune functions of the exposed mice. The spleen arginase activity recorded a considerable increase among the mice subjected to total-body irradiation, with Arg intake following it. This is in sharp contrast to the mice that have either received total-body irradiation (with no Arg following it) or been supplemented with Arg, having the total-body irradiation coming next (Shukla et al. 2008).

Another work by the same authors (Shukla et al. 2011) was devoted to the study of the cardiac dysfunction up to 24 hours after 2 Gy total-body irradiation and its mitigation by Arg. Total-body irradiation also triggered inflammatory responses in the cardiac tissue and spleen. Arg administered two hours after total-body irradiation of mice mitigated the entire inflammatory response and skewed electrocardiographic profile in favor of normal indicators. Arg administered just before total-body irradiation was unable to reverse the negative impact of irradiation. Radiation-induced inflammatory responses in cardiac tissue and spleen (measured at time intervals of 4 and 24 hours following total-body irradiation) appeared in direct correlation with the changes detected through electrocardiographic checkups, per the same time periods respectively. Thus, this study indicates the ability of Arg, administered at 4- and 24-hour intervals after total-body irradiation, to mitigate cardiac dysfunction induced by radiation (Shukla et al. 2011).

As Pearce et al. (2012) have demonstrated, Arg protects hematopoietic progenitor (32D cl 3) cells exposed to gamma irradiation from death. The addition of Arg to the culture media (to 5 mM) one hour prior to irradiation grants significant protection for these cells from the IR damaging activity, corresponding to a dose-modifying factor of 1.5 at 2 Gy and 1.2 at >5 Gy. No better survival has been observed in the post-irradiation scenario. Thus, in this case, Arg manifests itself more as a protector rather than a mitigator (Pearce et al. 2012).

It is well known that the lactogenic metabolism characteristic of brain metastases and radio-resistance are interlinked (Marullo et al. 2021). A bigger group of researchers representing various scientific and medical centers in the United States and Argentina has recently demonstrated that oral Arg results in radiosensitization in patients with brain metastases (a placebo-controlled trial). It has been demonstrated that brain metastases express NO synthase 2 and that the intake of Arg, being a substrate for the synthesis of NO, decreases tumor lactate in patients’ brain metastases. At the same time, preclinical studies have showed that Arg radio-sensitization is a NO-mediated mechanism (Marullo et al. 2021).

Oral mucositis and esophagitis are common acute toxicities of radiotherapy for head and neck cancer. In a different clinical study, authors (Yuce Sari et al. 2016) compared the quality of life for patients suffering from head and neck cancer who either did not or, alternatively, did receive a glutamine and Arg-enriched solution during radiotherapy in order to curb the rates of the respective toxicities. The global health status, functional and symptom scale scores were similar in both groups on the 1st day of observation. However, on the 15th and last days of observation, the scores of social functions, pain, appetite, dry mouth, sticky saliva, trouble with taste and swallowing problems were significantly worse in the control group. Authors concluded, that use of glutamine and Arg-enriched solution might have mediated the negative effect of radiotherapy (Yuce Sari et al. 2016).

To conclude, the review of the currently available data on the radiomodifying effects of Arg indicates its significant potential: Arg may work as both a radioprotector and radiomitigator, as well as a radiosensitizer. To prove the effectiveness of Arg as a radioprotective agent, it seems necessary to expand and deepen relevant preclinical studies as well as conduct proof-of-concept clinical trials with the inclusion of a sufficient number of patients and the use of scientifically based protocols.

Conclusions

A dietary supplement, Cr, as part of a 0.9% glucose solution, is capable of considerably stimulating the tissue-specific adaptive capabilities of the rat brain and liver Cr–CK systems against an X-ray exposure, hence making these organs more radio-resistant. Cr/glucose supplementation also significantly decreases genomic instability of hepatocytes, caused by acute total-body X-ray irradiation in a dose of 6.5 Gy (LD_70/30). The list of the benefits of Cr/glucose supplementation also includes: an increased (up to 67%) rat survival rate and greater (by at least 39%) lifespan; genoprotective effect due to a lower post-radiation incidence of DNA breaks in the peripheral blood mononuclear cells; a cytoprotective effect due to the leveling of pathological post-radiation changes in the population composition of the peripheral blood cells of the rats studied.

Considering the radioprotective effect of Cr on the survival rate of rats, as well as its energy-stimulating, hepatoprotective, genoprotective, and cytoprotective activities, this natural adaptogen can be considered as a potentially promising radioprotector. In this regard, it is important to note the following advantages of Cr: it is readily available; does not cause toxic effects, even in high doses; does not accumulate in the human body, even with repeated doses; has the ability to act through a number of mechanisms; can be administered orally; has a protective effect that extends to a number of organ systems; remains stable while stored; and, importantly, is cost effective (Jäger et al. 2011; Wallimann et al. 2011; Kreider et al. 2017). All of the above forms a good basis for further active research on Cr as a promising radioprotective agent.

The review of the currently available data on the radiomodifying effects of l-arginine indicates its significant potential as a radioprotector and radiomitigator, as well as a radiosensitizer. To prove the effectiveness of Arg as a radioprotective agent, it seems necessary to expand and deepen relevant preclinical studies, and, most importantly, increase the number of proof-of-concept clinical trials, which are clearly lacking as of now.

Acknowledgements

We thank Dr. Elina Arakelova (Institute of Molecular Biology NAS RA) for her valuable and helpful comments on the manuscript. We are also grateful to Mikayel Karapetyan for his technical help in the design of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this review article as no new data were created or analyzed in this study.

Additional information

Funding

This work has been supported by the Science Committee of the Ministry of Education, Science, Culture and Sports of the Republic of Armenia in the frames of the 20TTCG-1F003 and 21T-1F066 research projects.

Notes on contributors

Lyudmila Nersesova

Lyudmila Nersesova, PhD, is a Senior Scientist at the Laboratory of Experimental Biology of the Institute of Molecular Biology NAS RA, Yerevan, Armenia. Her main interest focuses on studying the role of creatine kinase as a biomarker of the impact ionizing and non-ionizing electromagnetic radiation as well as xenobiotics may cause to organism. Particularly under the spotlight comes the identification of new natural radioprotectors.

Mariam Petrosyan

Mariam Petrosyan, PhD, is a Junior Scientist at the Laboratory of Experimental Biology of the Institute of Molecular Biology NAS RA, Yerevan, Armenia. Her main interest is the identification and study of different radioprotective agents.

Gohar Tsakanova

Gohar Tsakanova, PhD, is the Deputy Director, a Leading Scientist and Head of the Laboratory of Experimental Biology at the Institute of Molecular Biology NAS RA, Yerevan, Armenia, as well as a Leading Scientist and Head of Laboratory of Experimental Biology at the CANDLE Synchrotron Research Institute. Her scientific interests include study of potential radioprotectors and radioenhancers, as well the studies of the biomedical effect of ultrashort pulsed electron beam irradiation.