*Review* **Nuclear and Radiological Emergencies: Biological Effects, Countermeasures and Biodosimetry**

**Elena Obrador 1,\*, Rosario Salvador-Palmer 1, Juan I. Villaescusa 2,3, Eduardo Gallego 4, Blanca Pellicer 1, José M. Estrela <sup>1</sup> and Alegría Montoro 2,3**


**Abstract:** Atomic and radiological crises can be caused by accidents, military activities, terrorist assaults involving atomic installations, the explosion of nuclear devices, or the utilization of concealed radiation exposure devices. Direct damage is caused when radiation interacts directly with cellular components. Indirect effects are mainly caused by the generation of reactive oxygen species due to radiolysis of water molecules. Acute and persistent oxidative stress associates to radiation-induced biological damages. Biological impacts of atomic radiation exposure can be deterministic (in a period range a posteriori of the event and because of destructive tissue/organ harm) or stochastic (irregular, for example cell mutation related pathologies and heritable infections). Potential countermeasures according to a specific scenario require considering basic issues, e.g., the type of radiation, people directly affected and first responders, range of doses received and whether the exposure or contamination has affected the total body or is partial. This review focuses on available medical countermeasures (radioprotectors, radiomitigators, radionuclide scavengers), biodosimetry (biological and biophysical techniques that can be quantitatively correlated with the magnitude of the radiation dose received), and strategies to implement the response to an accidental radiation exposure. In the case of large-scale atomic or radiological events, the most ideal choice for triage, dose assessment and victim classification, is the utilization of global biodosimetry networks, in combination with the automation of strategies based on modular platforms.

**Keywords:** nuclear and radiological emergencies; radioprotectors; radiomitigators; radionuclide scavengers; radiation biodosimetry

#### **1. Introduction**

Nuclear and radiological accidents can cause huge harm to individuals, the environment, and the economy. Chernobyl (USSR, 1986), Goiania (Brazil, 1987), and Fukushima Daiichi (2011, Japan) were awful catastrophes demonstrating how wrecking these mishaps can be. Moreover, since 11 September 2001, the danger of terrorism has become a public security concern in numerous nations. The number of known terrorist associations with worldwide reach, just like the expanded multiplication and transfer of technical data through the web, raises the chance of shocking assaults with chemical, biological, radiological, or even atomic weapons [1–3] (http://www.dni.gov/index.php/nctc-home, accessed on 15 December 2021; https://www.europol.europa.eu/about-europol/european-counterterrorism-centre-ectc, accessed on 15 December 2021).

**Citation:** Obrador, E.;

Salvador-Palmer, R.; Villaescusa, J.I.; Gallego, E.; Pellicer, B.; Estrela, J.M.; Montoro, A. Nuclear and Radiological Emergencies: Biological Effects, Countermeasures and Biodosimetry. *Antioxidants* **2022**, *11*, 1098. https://doi.org/10.3390/ antiox11061098

Academic Editor: Stanley Omaye

Received: 8 May 2022 Accepted: 27 May 2022 Published: 31 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Radiation exposure is a danger from both potential "dirty bomb" terrorist events and industrial mishaps including problems with atomic reactors or misplaced radioactive sources. Calamities including exposure to radiological materials require technical planning and readiness to guarantee the health of first responders, the evacuation and clinical therapy of possibly contaminated casualties, and the management of the process of triage. Significant advances have been made throughout the most recent decade in public health and clinical planning intended to improve the response to an atomic explosion or a radiological episode [4–6].

A mass victim event would surpass the reaction capacity of the local responders and, subsequently, its methodology would require the mediation of exceptionally well prepared personnel and extensive public activity, based on a fast intervention plan arranged ahead of time. The best model (even though the most improbable) would be the explosion of an improvised nuclear device (IND), which would produce a fireball and a bright glimmer of irradiation followed by an impact wave and thermal pulse. That scenario would make it very hard to get supplies and personnel into the harmed areas, as well as the clearing of the injured to clinics. Mass screening of the affected people would be important to isolate those exposed from non-exposed and to take decisions based on the estimated dose received [7].

Exposure would result from irradiation close to the site of the explosion, which emits radiation at a high dose rate for a brief timeframe; and from deposited radioactivity (also known as aftermath), which has a lower dose rate. The absolute ingested dose would be reliant on the location of the people and the term of their exposure.

The number of individuals exposed, and the dosages received would likewise rely upon a number of factors, e.g., geological characteristics of the area (metropolitan or countryside, protection against radiation provided by buildings), environmental conditions, and the protection set up during the first hours.

Independent of the type of atomic or radiological crisis, explicit (pre-events) plans and reactions should incorporate innovative work in comprehending the pathophysiology of radiation injury, improvement of clinical countermeasures (MCM) (i.e., radioprotectors, radiomitigators, and radionuclide scavengers), and investigating a range of analytic tests to help the clinical decision-makers [8]. Ideally, planning should include energy, health, human management, security, work, transportation, ecology, aeronautics, and atomic guidelines.

#### **2. Nuclear and Radiological Accidents**

The scenario of the Chernobyl and Fukushima-Daiichi accidents comprised release of large amounts of radionuclides. In water reactors, vaporous and unpredictable splitting of items, specifically isotopes of iodine and cesium, would be determinant for the radiological issues off-site [9], as occurred in Fukushima (https://www.iaea.org, accessed on 15 December 2021). Less unpredictable splitting items or actinides would be critical in case of extreme reactivity accidents (like Chernobyl) in which fuel "hot particles" were delivered [10]. In the primary time frame during the crisis period of an atomic mishap, large amounts of iodine isotopes can reach individuals, with the thyroid being a basic target organ. Triage is critical to distinguish between individuals who need care because of their degree of exposure and those who need health observation. The characterization of the radiological circumstances of individuals and the environment is key to setting up protecting activities (https://www.icrp.org, accessed on 15 December 2021). In the more extended term, contamination of the environment with cesium and other seemingly perpetual radionuclides will influence life in the affected areas, where the external and interior exposure of people ought to be checked to implement effective countermeasures.

The scenario after a huge radiological accident, similar to that which occurred in Goiania (1987) with an enormous 137Cs source left in a closed oncologic facility, can likewise be difficult to oversee. In the Goiania accident, four deaths were recorded, 250 people suffered contamination, 62 of them were administered a radionuclide scavenger (Prussian blue), whereas more than 112,000 individuals were radiologically observed, and 3000 m<sup>3</sup>

of radioactive wastes was generated (https://www.iaea.org, accessed on 15 December 2021). Another significant radiological event was that of the 210Po poisoning of Aleksandr Litvinenko in 2006 [11], which required follow up of the polonium pollution and screening of more than 750 individuals for their likely internal contamination, thus requiring a huge coordinated effort [12].

#### **3. Main Radiations Associated to Nuclear and Radiological Emergencies**

Injury from an atomic explosion will fluctuate contingent upon the exposure to various sorts of energy: heat, representing around 35% of total energy; blast, representing roughly half of total energy; and radiation, representing the leftover 15% of energy [13]. Here, the brief acute exposure would be promptly caused by emitted gamma rays combined with a subordinate dose of fast-moving neutrons. Neutrons can represent comprise 25 to 50% of the absolute radiation dose at a distance of approx. 1 km. This is important because, due to its high radiation biological effectiveness (RBE) and radiation weighting factors (WR) (www.icrp.org, accessed on 15 December 2021), the neutron dose can increase multiple times the harm of an equal photon absorbed dose.

The radiation dose received from an atomic blast will be prompt (that delivered with the impact wave), plus an additional relevant component due to fallout of fission and activation products that can be extended (from the aftermath) for a long time as polluted materials tumble to the earth [14]. The mean deadly dose of radiation that would kill half of the people in 60 days (LD50/60), after a total-body irradiation (TBI), is of approx. 3.25–4 Gy in individuals without supportive care; and 6–7 Gy when anti-infection agents and additional support are given [15,16].

In an IND-related event, gamma and neutron radiations will be released, and then gamma and beta radiations from items delivered by the blast [17–19].

In a radiological dispersal device (RDD or dirty bombs)-related event, the radiation exposure would be limited, as most likely just one sort of isotope would be utilized. In most RDD scenarios, even with the utilization of solid gamma-discharging radionuclides, huge radiation wounds should not to be normal. The dispersal impacts of the weapon would dissipate the radioactive source [20,21].

An individual exposed to radiation is not radioactive, while an individual contaminated with radionuclides (internal or remotely) may emit radioactivity that is perceptible with hand-held Geiger counters or whole-body scanners. Contamination results when a radioisotope (as gas, fluid, or solid) is delivered to the environment, and afterward ingested, inhaled, or deposited on the body's surface [22]. A prominent exemption is a neutron radiation exposure, where the cycle of neutron actuation can create biological radioactive material [23].

#### **4. Triage and Categorization**

The kind of triage varies with the type of radiological or atomic event. For instance, in the case of an atomic explosion, an enormous number of individuals should be assessed, including those affected by a high dose and those having negligible or no actual injury. The dose will be a critical boundary for a clinical triage. As of now, the most productive and available triage technique is the utilization of consecutive complete blood counts to evaluate lymphocyte exhaustion that is associated with assessed whole-body dose radiation exposure. If fast blood testing would not be conceivable, dose assessment can be at first evaluated dependent on basic boundaries, i.e., correlations between the extent of the body exposed to the radiation and the % of the radiation levels estimated in the environment; victim's shielding activities after the explosion; and signs and side effects from exposure to radiation or early radioactive particles' aftermath [24]. The radiation dose classes allude to dosages affecting the whole body or a large portion of the body (partial exposure). Notwithstanding the straightforward boundaries referenced above, the dose can be additionally be assessed dependent on: (a) the period until onset of early signs, (b) the seriousness of the signs (i.e., the acute radiation syndrome, ARS), and

(c) the biodosimetry methods [25–27]. Even though vomiting is a serious basic symptom after whole or huge partial body radiation exposure, it cannot be utilized to anticipate the radiation dose received. Vomiting can likewise be brought about by head injury, uneasiness, or other pathology [28].

It is key to point out that viable clinical triage can save numerous lives. In this, a fast reaction, sufficient coordination, and the accessibility of innovative biodosimetry is required. Clinical triage following an atomic explosion ought to be a stepwise cycle, where the principal point is abbreviated as "SALT"- Sort, Assess, Lifesaving Interventions, Treatment/Transport [29]. In the military, operational organizers use 'parts' to characterize the four levels where military clinical help is coordinated on a reformist premise to lead triage evaluation, quick treatment, evacuation, resupply, and capacities basic to the upkeep of health [30]. Stepwise triage should incorporate the point of care (POC) evaluation (blood counts, see above), followed by secondary evaluation, perhaps with high throughput screening to additionally characterize a person's dose (so that individuals considered in danger of showing ARS throughout the following weeks are identified). Also, assays which could be utilized for assessing long-haul malignant growth hazards (for example quality screening) ought to likewise be incorporated [31]. It is also imperative to consider that amid a radiological or atomic crisis, where the coordination of numerous individuals and management is fundamental, an unmistakable and agile command chain is vital.

#### **5. Biological Effects in Nuclear and Radiological Accidents**

#### *5.1. Oxidative Stress and Inflammation at the Core of Ionizing Radiation-Induced Damage*

Ionizing radiation (IR) can break covalent bonds and cause oxidative harm to DNA, lipids, proteins, and numerous metabolites [32]. In experimental processes it is shown that the DNA molecule is more radiosensitive when it is irradiated in solution than in a dry environment [33]. The effects of IR on the DNA molecule are single and double chain fractures, structural alterations and elimination of the bases generating apurinic and apyrimidinic sites (AP sites), sugar damage, cross-links between DNA-DNA or between DNA-protein, and breaking of hydrogen bonds [34,35]. Moreover, overproduced reactive oxygen species (ROS) can react with cell membrane fatty acids and proteins impairing their function [36]. The primary event for the formation of a free radical in the radiolysis of water is the release of an electron in the interaction of low linear energy transfer (LET) ionizing radiation with the water molecule [37]. While the physicochemical events are a quick result of radiation exposure, the damage propagates the reaction by producing repeating waves of ROS, reactive nitrogen species (RNS), cytokines, chemokines, and other factors with related incendiary penetration [38].

During the radiolysis of water, ROS like superoxide anion (O2 •−), hydroxyl radical ( •OH), hydrated electron and hydrogen peroxide (H2O2) are produced [39]. The release of nitric oxide (NO•) and its metabolites such as peroxynitrite (ONOO−) and nitrogen dioxide (NO2 •) are also involved in IR genomic damage [5]. Overproduction of ROS and RNS is a harmful process that can cause damage to cellular biomolecules (DNA, proteins, and lipids), and affect the cell membrane, cellular signaling and genome integrity. These effects can influence numerous cellular processes linked to cell death, carcinogenesis, and cancer progression [40–42]. Indeed, oxidative stress, and the associated redox status shifts, can cause cell transition from quiescent to proliferative status, growth arrest or cell death activation according to the duration and extent of the redox imbalance [43]. In turn, cells injured by IR are responsible for inducing radiation bystander effects (RIBEs) in non-irradiated cell recipients, manifested by changes including (but not limited to) gene expression, protein synthesis, chromosomal aberrations, micronuclei formation, secretion of exosomes and miRNAs, and cell death/proliferation or transformation [44–46]. ROS are considered initiators, and NO, the transforming growth factor beta (TGF-β) and other inflammatory cytokines effectors are involved in RIBE [47,48]. Moreover, the inflammatory response generates recurring waves of ROS, cytokines, chemokines and growth factors with associated inflammatory infiltrates [49,50]. This represents a vicious circle where

both oxidative stress and inflammation induce each other. These concepts are supported because non-steroidal anti-inflammatory drugs and antioxidants decrease some of that latent damage, as well as the inflammation-associated mutations. This is a crucial point that determines that MCM to reduce the damage induced by IR is based on free radical scavengers, antioxidants, and anti-inflammatory agents [51–54].

#### *5.2. Acute and Chronic Radiation Syndromes*

Biological impacts will fluctuate contingent upon the type and dose of radiation, and the time and recurrence (single or serial) of exposure [55]. The impacts of radiation on the body may show up rapidly (acute radiation syndrome, ARS) or require several years after exposure (deferred impacts, for example, fibrosis, sterility, genetic impacts, or malignancies). By and large, exposure to higher doses of radiation produces symptoms more quickly [54]. In the case of an atomic impact, radiation-derived wounds will be of different types, for example, injuries or thermal burns [56]. Heat and light cause thermal injury, including flash burns, fire burns, flash blindness (because of transitory loss of photopigments from retinal receptors), and retinal burns. The impact wave can cause breaks, slashes and cracks of the viscera, and aspiratory drain and edema [56].

Non-deadly harm (mis/unrepaired) may prompt genomic unsteadiness, for example, chromosomal variations, DNA mutations, and cell senescence. According to radiation assurance measures, radiation-prompted impacts are classified as (a) deterministic (tissue responses which require a threshold dose to exceed) which result from cell execution or the deficiency of cell capacity; and (b) stochastic or irregular (not relying upon such a limit, although its likelihood increases as the radiation dose expands) which are brought about by hereditary deviations and mutations setting off long term inherited impacts and malignancies [57].

The Life Span Study (LSS, https://www.rerf.or.jp, accessed on 7 January 2022) is an exploratory program examining deep-rooted health impacts dependent on epidemiologic (accomplice and case-control) considerations. Its most significant target is to explore the longer term impacts of bomb-derived radiation on reasons for death and the occurrence of malignancy. The examination has indicated that the danger of solid malignancy and leukemia among atomic specialists is steady with the dosage assessed, even if they get the radiation at low dose rates over numerous years [58]. The global INWORKS study has shown that in any event, when the combined dose of atomic industry laborers was under 100 mSv and the dose rate was under 10 mGy every year, the danger of solid malignancy is steady based on the dosage assessment [59]. A recent review [60] identified a large body of epidemiological data (published between 2006–2017) that assesses the evidence of an increase in solid cancer risks and/or leukemia, following low-dose IR exposure (<100 mGy).

ARS involves different phases of biological injury that may follow exposure (of the whole body or its majority) to a high dose of radiation (ordinarily in a brief timeframe). Its seriousness relies upon the radiation dose and normally includes syndromes whose term is directly correlated with the total dose received (and, ultimately, with the pace of exposure) [61–64]. Initially, a prodromal phase may show up with side effects, for example, sickness, spewing, and torpidity. This is continued (in hours to weeks) by various conceivable subsyndromes (related to various dose limits) for example the hematological (at doses of 1–2 Gy), gastrointestinal (GI) (dosages of 4–6 Gy), cutaneous (approx. 6 Gy), cerebrovascular (approx. 10 Gy) [65,66]. Lung wounds (approx. 8 Gy) may likewise show up half a month after exposure. An idle period of hematological ARS may infer a time of 1-3 weeks after getting a total dose of 2–4 Gy. Higher dosages may abbreviate or eliminate the inert phase [66].

Chronic radiation syndrome (CRS) results from long-term repeated exposure (external and/or radionuclide intake) to rather low doses [from 0.7–1.5 Gy (at rates > 0.1 Gy/year) to 2–3 Gy] and has a long-term intermittent course. It is worthwhile to point out that cancer induction can also be found at lower doses (<0.7 Gy). In the beginning, it was considered that CRS manifestations could also include the chronic ARS damages, but as tissue reaction mechanisms of ARS and CRS differ, such association was recognized as incorrect [67]. The CRS term does not refer to the duration of disease (ARS manifestations can also remain for a long time, and develop chronic pathologies), but characterizes the result of protracted (chronic) radiation exposure [67–69].

Initial CRS symptoms are nonspecific, and can be reversible, if there is a decrease or a break in radiation exposure. If exposure continues, the initial symptoms grow progressively worse, and others may appear. The earliest manifestations of CRS are a dose-dependent inhibition of hematopoiesis and neurologic dysfunctions. Moderate but persistent leukopenia induced by neutropenia is one of the typical changes in peripheral blood, although in certain patients lymphopenia was also noted [68,70]. A severe degree of CRS is characterized by the development of bone marrow hypoplasia, persistent and marked granulocytopenia, profound thrombocytopenia, and moderate anemia. In these cases, hematopoiesis recovery is quite difficult or even impossible, even though the radiation exposure is discontinued [69]. Three sequential neurologic syndromes have been identified: vegetative dysfunction with impairment of neuro-visceral regulation, asthenic syndrome, and encephalomyelitis-type lesions of the central nervous system. Neurosensitive dysfunctions (olfactory and vestibular excitability decline, taste fatigue, etc.) sometimes precede the neuro-vegetative syndrome which is considered the earliest manifestation of the CRS [70]. Signs of vegetative dysfunction include: decrease in capillary tone (especially in skin vessels), an intense histamineinduced skin reaction, instability of the pulse with a tendency to hypotension, changes in the secretory and motor activity of the GI tract, etc. [70]. Some women develop changes in the sex hormone ratios (total estrogen levels were found at the lowest limit), in most cases accompanied by menstrual cycle disorders [69]; in animal models, a reduced number of follicles have been evidenced [71]. The rate of spontaneous abortions was five times higher than that without exposure [72]. The asthenic syndrome has a gradual progression, i.e., fatigue, headaches, dizziness, general weakness, hypersomnia, decreased working capacity and considerable memory deterioration [69,73]. At this stage patients can suffer cataracts, skin disorders such as a decrease in elasticity, dermatitis, xeroderma or hair loss [74]. Vascular dysfunction and thrombocytopenia play a key role in predisposition to hemorrhagic events like cutaneous petechial, mucosal, and visceral hemorrhages. Functional activity of organs and tissues, as well as structure, can undergo considerable changes (fibrosis, hypoplasia, malignant transformation, etc.). Radiation-related risk of cardiovascular disease is increased and can be associated with lung and heart fibrosis and atherosclerotic disorders [75]. Quite often, the CRS of medium severity is complicated by infections of respiratory and digestive systems [69,73]. When a demyelinating encephalomyelitis is developed, the patients' health status deteriorates dramatically, accompanied by general weakness and adynamy [69]. Although the brain has been classically regarded as a radioresistant organ, vascular lesions (edema, thrombosis, hemorrhage) and Blood-Brain Barrier (BBB) disruption are considered to be a precipitating factor for white matter necrosis [76]. Causes of death in the late period of CRS are sepsis and hemorrhages resulting from inhibition of hematopoiesis and immunity, malignant solid tumors and especially leukemia and chronic myeloleukemia [73]. There is evidence that relative risks are generally higher after radiation exposures in utero or during childhood [77].

Hereditary harm brought about by radiation is behind the expansion in the recurrence of malignant growths and can show both in the early phases and throughout the long term. As a reasonable model, notwithstanding acute ailments, numerous survivors of Chernobyl, Nagasaki, and Hiroshima additionally endured leukemia, and thyroid, stomach, and skin malignant growths (https://www.unscear.org, accessed on 7 January 2022) [74]. Studies on the nuclear bomb survivors in Japan revealed that the danger of mortality of solid malignant growth became apparent approximately ten years after detonation and expanded by half when the dose to which the colon was exposed arrived at 1 Gy; the danger of mortality from leukemia was quadrupled when the dose to which the red bone marrow was exposed reached 1 Gy [78–80].

#### **6. Medical Countermeasures**

It is critical to develop effective radioprotectors as a preventive measure for their application in planned radiation usage, such as radiation therapy, as well as unplanned exposure, such as natural background radiation, space travel, nuclear disasters, and nuclear warfare. The IR research program of the US National Cancer Institute proposed the following pharmacological classification of agents with IR protection properties according to the timing of administration: (a) protection, (b) mitigation, and (c) therapeutic agents [81]. In general, radioprotectors are used before IR exposure to protect cells and tissues from being damaged; radiomitigators are administered during or shortly after IR exposure, and attenuate damage and/or contribute to tissue recovery. Lastly, therapeutic agents are administered after symptoms have presented, acting as palliation or support [82]. As we will explain below, due to the capacity to scavenge free radicals, some antioxidants can be considered as radioprotectors, and many of them act also as radiomitigators for their capacity to enhance cellular antioxidant and repair mechanisms, during and after IR exposure. Finally, only a few can also be considered as therapeutic agents by reducing or palliating the clinical symptoms induced by exposure to IR.

The improvement of viable MCM to shield individuals from the unsafe impacts of normal radiation constitutes a neglected need [54]. Considering explicitly the radiological or atomic crises where earnest assistance is required, it is critical to plan separately for first responders, and for those directly presented to radiation during the mishap. First responders' vulnerability may be reduced by radioprotectors and radiomitigators, while those exposed to radiation may require radiomitigators, and of course, therapeutic support.

#### *6.1. Radioprotectors*

An ideal radioprotective agent should fulfil several criteria, i.e., provide significant protection, be stable, offer the chance of a simple formulation, have an easy route of administration, and have no significant toxicity (mainly in particularly sensitive tissues, in which acute or late toxicity would be dose restricting). No single molecule so far has every one of these properties, and at this moment, radiation MCMs for ARS and other exposure-related injuries are assigned FDA orphan drug status [83].

Many different molecules have been assayed as potential radioprotectors. Some show promising properties but, considering pharmacokinetic properties and ease of in vivo administration, we might suggest the following for a potential radioprotective formula:

#### 6.1.1. Thiol-Containing Compounds

Since the detonation of the Hiroshima and Nagasaki bombs, the Walter Reed Army Research Institute (USA) enhanced its research program on radioprotective countermeasures and screened more than 4000 compounds [84]. Cysteine was the first one to confer radiation protection in mice subjected to total body radiation (TBI) [85], and since then many synthetic aminothiols have been developed and proved. Undoubtedly, the most effective was WR-2721 or amifostine, a sulfhydryl prodrug activated by alkaline phosphatase to the active WR-1065. Salivary glands and the epithelial cells of intestine are highly enriched in this activating enzyme, and thus oral administration of WR-2721 just before radiation results in localized high production of the bioactive derivate, preventing radio-induced mucositis and GI damage without significant systemic side effects [86–89]. The underlying mechanisms of action are free radical scavenging and hydrogen atom donation, along with DNA protection and repair; all coupled to an initial induction of cellular hypoxia [90–92]. WR-1065 has anti-mutagenic and anti-carcinogenic properties evidenced using in vitro testing systems [91], induces G1 cycle-arrest and p53 dependent-cytoprotection [52], upregulates the expression of mitochondrial Mn-SOD2 and proteins responsible for DNA repair, and inhibits apoptosis through Bcl-2 and hypoxia-inducible factor-1α (HIF-1α) [87]. Amifostine was the first Food and Drug Administration (FDA)-approved clinical radiation protector intended to reduce the impact of radiation on normal tissue, and more specifically, to decrease xerostomia in patients undergoing radiotherapy for head and neck cancers [92]. WR-1065 accumulates more rapidly in normal tissues than in malignant cells, due to the relative lower activity of alkaline phosphatase in cancer cells and acidic pH in the environment of many tumors. Amifostine is clinically used to prevent xerostomia, mucositis, dysphagia, dermatitis, and pneumonitis during radiotherapy of head and neck cancers, and a meta-analysis carried out in 2014 pointed out its beneficial effects [88]. However, a more recent randomized double-blind trial [89] does not support any benefit. Despite the heterogeneity, results appear to show some benefit to its use as radioprotector [87].

The glutathione redox status (GSH/GSSG) decreases after irradiation, mainly due to an increase in glutathione disulfide (GSSG) levels. Two reasons may explain the radiationinduced increase in blood GSSG: (a) GSH reacts with radiation-induced free radicals forming thiol radicals that react to produce GSSG; and (b) GSSG is released from different organs (e.g., the liver) into the blood. In fact, GSH is essential to prevent radiation damage and the glutathione redox ratio in the blood can be used as an index of radiation-induced oxidative stress [93]. The DNA single-strand breaks repair system is absent in GSH-deficient cells, and GSH is also essential to activate proliferation and repair of damaged tissues and to prevent cell death [94]. In fact, the main mechanism of action of most radioprotectors is to maintain intracellular levels of GSH. An illustrative example is *N*-acetylcysteine (NAC), a potent antioxidant and GSH precursor. NAC treatments (300 mg/kg, sc), starting either 4 h prior to or 2 h after radiation exposure, and with six subsequent daily injections over 7 days, reduced early deaths in abdominally irradiated (X-rays, 20 Gy) C57BL/6 mice [95]. More recently, radioprotective effects of NAC have been demonstrated in multiple studies [96,97], but the use of GSH or NAC with oncoradiotherapy cannot be supported because it may also favor cancer cell metastasis and radioresistance. Erdosteine (a homocysteine derivative) is a potent free radical scavenger, increases GPx and catalase (CAT) activities and GSH intracellular levels. Erdosteine treatment before γ-radiation ameliorated nephrotoxicity, and decreased IL-1, IL-6, and tumor necrosis factor alpha (TNF-α) blood levels, thus suggesting substantial protection against radiation-induced inflammatory damage [98].

Aminothiols and their phosphothioate derivatives, administered shortly before irradiation, exert radioprotection by one or a combination of effects: scavenging of radiationinduced free radicals; induction of hypoxia; formation of mixed disulfides; quenching of metals; repair of DNA and genome stabilization. However, radioprotectors of this type, including amifostine, have important side effects and a short pre-exposure time window of radioprotectiveness, which limit their use as radiation countermeasures [92,99]. Any strategy aimed at reducing toxicity, without reducing their radioprotective efficacy, would be a great advance. Rather novel approaches include: (a) slow-release delivery of drugs, (b) combined treatments with other radioprotectors/radiomitigators such as cytokines (G-CSF), selenium, metformin, antioxidants, etc.; (c) re-engineering better tolerated analogs like HL-003 or combining with antiemetic drugs; and (d) molecular conjugates and nanoparticle formulation designed to extend amifostine or WR1065 circulating half-life or to avoid intravenous administration. As reviewed by Singh y Seed [92], these approaches have proven to be useful but without a complete elimination of the toxicity or just increasing the radioprotection to a limited extent.

#### 6.1.2. Natural Phytochemicals

Over the last decades many phytochemicals, and especially polyphenols, have been broadly considered as radioprotectors and/or radiomitigators. The antioxidant activity of polyphenols depends, in part, on their ability to delocalize electron distribution, resulting in a more stable phenoxy group. Thereby, differences in ROS scavenging potential can be attributed to the different functional groups attached to the main nucleus [100]. Intercalation in DNA double helices induces stabilization and condensation of DNA structures making them less susceptible to free radicals' attack [100], reducing genotoxic damage induced by IR [101]. Xanthine oxidase and lipoxygenase are inhibited by many polyphenols, thus reducing the generation of free radicals. Finally, many polyphenols decrease the activation of NF-κB and MAPK, thus reducing the release of inflammatory cytokines which play a role in the radiation-induced inflammatory response [102–104].

Genistein nanoparticles increase the expression of metallothionein genes and suppress the post-irradiation increase of cytokine production (IL-1-beta, IL-6) and cyclo-oxygenase-2 (COX-2) activity, thus preserving bone marrow progenitors and increasing survival on day 7 post-irradiation (9.25 Gy 60Co) [105]. The radioprotective effects of genistein are due to its ability to inhibit NF-κB, MMPs, and Bax/Bcl-2 signaling pathways and attenuate the inflammatory response induced by IR. In rodents, genistein has been shown to mitigate the effect of radiation on the lungs [106] and the intestinal tract [107]; used in combination with radiotherapy in prostate cancer patients, it can reduce intestinal, urinary, and sexual adverse effects.

The positive effects of curcumin as a radioprotector involve its free radical scavenging activity, antioxidant properties targeting the Nrf2 pathway [108], and its anti-inflammatory effects mediated by modulation of COX-2, IL-1, IL-6 [109], tumor necrosis factor alpha (TNF-α), TGF-β expression, release and/or activity [110,111]. Curcumin ameliorated radiation-induced pneumonitis and pulmonary fibrosis [112,113] and cognitive deficits (including learning and memory defects), exerted cardioprotective, neuroprotective, hepatoprotective, and renoprotective activities [108,110], and decreased pain severity [114]. Additionally, curcumin has antitumor effects [115] that can synergize with radiotherapy [116–118]; it should thus be considered a good option to increase the efficacy of radiotherapy on cancer cells, as well as to prevent the radiotherapy-induced adverse effects in normal tissues [112,114]. A few human studies have confirmed its efficacy for the management of radiotherapy induced dermatitis [119] and mucositis [120,121]. To modify the pharmacokinetic profile of curcumin and increase its bioefficacy, new formulations have been introduced [122].

Epigallocatechin-3-gallate (EGCG) and other flavonoids from green tea inhibited radiation-induced damage [123]. EGCG scavenges free radicals, increases the levels of several antioxidant enzymes, i.e., glutamate-cysteine ligase, SOD, and heme oxygenase-1 [124,125] and induces Nrf2 activation which, in turn, represses radiation-induced apoptosis and attenuates TBI-induced intestinal injury [126]. The inhibition of the proteasome, a regulator of inflammation, has been reported as well and, consequently, extracts of green tea decreased the release of pro-inflammatory cytokines, i.e., TNF-α, PGE2, IL-1β, IL-6 and IL-8 in vivo [127]. Epicatechin blocked ROS production and radiation-induced apoptosis via down-regulation of JNK and p-38, which ameliorated oral mucositis and survival rates [128], inhibited radiation-induced auditory cell death of rats [129], and enhanced the recovery of hematopoietic cells in mice [130].

Resveratrol (RES) has demonstrated potential anti-cancer, antioxidant, neuroprotective, anti-inflammatory and cardioprotective effects. It is noteworthy that RES serves as a scavenger of O2 •−, •OH and metal-induced radicals, and increases the activity of many antioxidant enzymes [131]. RES significantly reduced radiation-induced chromosome aberration [132], DNA damage [133] and apoptosis, supported cell regeneration, and induced repression of the NLRP-3 inflammasome subset [134]. In mice, administration of RES attenuates radiation-induced intestinal damage via activation of sirtuin-1 [135], supporting lymphocyte [136] and intestinal functions recovery [137]. Under oxidative stress, RES promotes tyrosyl-tRNA synthetase acetylation, regulates relevant signaling proteins, and reduces apoptosis and DNA damage [138]. Clinical studies on RES as a normal tissue protector and potential tumor sensitizer are limited [139], mainly because RES possesses unfavorable bioavailability and pharmacokinetic properties. Synergistic effects with other polyphenols such as curcumin have also been evidenced and new formulations (hybrid molecules or nanoparticles) are being tested to increase its bioavailability and efficacy [140]. The use of derivatives, such as pterostilbene, with similar properties and a longer biological half-life, can significantly contribute to improve the radioprotective effects in vivo, as we have evidenced in our laboratory [141].

Oral silibinin treatment (100 mg/kg/day) reduced late-phase pulmonary inflammation and fibrosis in C57BL/6 mice after 13 Gy thoracic irradiation, via downregulation of NF-κB [142]. We have reported synergic radioprotective effects of silibinin with pterostilbene, resulting in 100% of the mice surviving, 30 days after TBI g-irradiation of 7.6 Gy (LD50/30) [141]. Silibinin can chelate thorium radionuclides (232Th) preventing hemolysis and enhancing liver cells decorporation, which is important because those cells are the major targets of internalized 232Th [143].

Quercetin minimizes radio-induced oxidative damages and genotoxicity, preventing hematopoietic genomic instability and dysfunction [144] and skin fibrosis [145]. Quercetin pre-treatment attenuated ROS generation, downregulated NF-κB and reduced expression of proinflammatory cytokines (PGE2, IL-1β, IL6, IL-8 and TGF-β) [146]; it also reduced DNA double-strand breaks and cellular senescence in C57BL/6 mice exposed to a single-dose (25 Gy) or fractionated IR doses [147]. The anti-inflammatory effects of quercetin are also favored by its ability to reduce recruitment of neutrophils, myeloperoxidase and COX-2 activity, MAP kinases signaling and NLRP3 inflammasome activation in macrophages [148].

Recently, Faramarzi et al. [103] reviewed the radioprotective potential of natural polyphenols and, based on their dose-dependent antioxidant/pro-oxidant efficacy, concluded that they could represent a valuable alternative to synthetic compounds. Polyphenols provide protection to normal cells, with little or no protection to cancer cells, and in some cases, have the additional advantage of increasing cancer radiosensitivity. The potential use of polyphenols as radioprotectors is based on their low toxicity, the suitability of oral administration, and the possibility of combining several of them. Nevertheless, their low bioavailability due to poor absorption, rapid metabolism, and/or rapid systemic elimination, can compromise their efficacy. Thus, new pharmaceutical formulations (nanoparticles, vesicles, cocrystals ... ) are being implemented and tested to facilitate oral administration and/or increase their effectiveness (see, e.g., https://www.circecrystal.com, accessed on 21 January 2022) [149–151].

The most promising non-polyphenolic phytochemicals with radioprotective effects are sesamol, gallic acid and caffeic acid derivates. The strong antioxidant activity of sesamol has been reported in comparison to standard antioxidants like vitamin C, curcumin, etc. Sesamol pre-treatment at 50 mg/kg (oral) was found to be the most effective dose in reducing mortality in irradiated Swiss albino mice exposed to 9.5 or 15 Gy γ-TBI [152]. The radiation-induced increase of apoptotic biomarkers and decrease in endogenous antioxidants (GSH, GST, CAT) was reduced by sesamol treatment, preserving crypt cells, villus height, and intestinal [152] and hematopoietic functions [153]. A recent study evidenced that daily oral consumption of sesamol is more effective than administration of a single dose before irradiation [154]. Similar results were observed using 100 mg/kg of gallic acid 1h prior to 10 Gy radiation exposure [155]. The cytoprotective effects of gallic acid are also due to its ability to enhance DNA repair, chelate metal ions, through the attenuation of MAPK and NF-κB/AP-1 signaling pathways, and reduce the release of inflammatory cytokines and adhesion molecules involved in leukocyte infiltration [156]. Caffeic acid (CA) and caffeic acid phenethyl ester (CAPE) act as free radical scavengers, compete with oxygen for IR-induced electrons, have antioxidant effects [151,157,158], decrease lipid peroxidation and increase antioxidant defenses in the heart and lung tissue of irradiated mice [159]. Treatment with CAPE prior to irradiation of rats effectively ameliorated intestinal [160], and hepatic [161] injuries. CA and CAPE inhibit activation of NF-kB, VEGF secretion and COX-2 activity, being considered potent anti-inflammatory agents [159,162]. In addition, CA stimulates cell cycle arrest and increases cell death in tongue, neck, and mouth cancer cells [158] and both molecules have anticarcinogenic properties attributed to their capacity to reduce tumoral angiogenesis, cancer growth and metastasis progression [158,162,163]. CAPE is a lipophilic agent, but incorporation into nanoparticles facilitates its administration. Moreover, nanoparticles can be modified to respond to different stimuli, such as pH, temperature, magnetic fields, oxidative stress, irradiation etc., thus facilitating the sustained release of drugs in selected tissues. That is the reason why even though CAPE-nanoparticles

showed a similar protective activity compared to CAPE under in vitro conditions, mice treated with nanoparticles had a longer survival after being exposed to IR [151].

Dietary sources of phytochemicals mentioned in this article and their radioprotective properties are detailed and reviewed in [102–104,164].

#### 6.1.3. Vitamins

With the understanding that free radicals perpetuate a significant amount of the damage caused by IR, vitamins with antioxidant potential (A, C, and E and its derivates) have been assayed as radioprotectors. Vitamin A and carotenes have antioxidant activity and capacity to enhance DNA repair, and in vivo reduced mortality and morbidity in mice exposed to partial or TBI [165]. Carotenoids such as crocin and crocetin (isolated from saffron) have antioxidant, anti-inflammatory and antiapoptotic effects [166]. In mice bearing pancreatic tumors, crocin significantly reduced tumor burden and radiation-induced hepatic damage [167], while crocetin reduced in vitro radiation injury in intestinal epithelial cells [168] and testis injury in pubertal mice exposed to 2 Gy X-rays [169]. Lycopene is the carotene isomer with the highest antioxidant potential and capacity to reduce proinflammatory cytokines expression such as IL-8 and IL-6 or NF-κB. Pre-clinical studies evidenced its radioprotective efficacy, particularly, if it is administered previously to or as soon as possible after radiation exposure [170,171] which is very interesting because lycopene has also anti-cancer activity, as recently reviewed in [172].

Administration of vitamin C (ascorbic acid, AA) before g-irradiation prevents chromosomal damage in bone marrow cells, mainly due to its antioxidant activity [173], reduces the GIS severity [174] and the adverse effects of TBI in the liver and kidney [175]. Moreover, intraperitoneal administration of 3 g AA/kg, up to 24 h after TBI (7.5 Gy), significantly increased survival in mice, reduced radiation-induced apoptosis in bone marrow cells, and restored hematopoietic function [176]. Nevertheless, administration of less than 3 g AA/kg was ineffective, and doses of 4 or more g/kg were harmful to mice. Moreover, treatments beyond 36 h were ineffective [176]. These facts highlight the limited efficacy margins of the treatment and compromise its use as a radioprotective measure.

Vitamin E is an essential fat-soluble nutrient with antioxidant, anti-inflammatory and neuroprotective properties. Eight vitamers are included in the vitamin E family, four saturated (α, β, γ, and δ) called tocopherols, and four unsaturated analogs (α, β, γ, and δ) referred as tocotrienols [177]. All of them are collectively known as tocols, and α-tocopherol is the most abundant in human tissues. Tocols are free radical scavengers, potent antioxidants and anti-inflammatory agents with capacity to attenuate fibrosis in tissues exposed to IR [177–179]. α-tocopherol succinate inhibited radiation-induced apoptosis and DNA damage, increased antioxidant enzymes activity, protected active mitotic tissues, and inhibited the expression of oncogenes in irradiated mice [180]. Moreover, when *α*-tocopherol was administered 24 h before 60Co *γ*-radiation, there was a significatively increase in the survival rate of mice, attributed to the capacity to restore crypt cellularity and inhibit bacterial translocation from the gut to the bloodstream [181]. Further studies revealed that α-tocopherol succinate significantly reduced thrombocytopenia, neutropenia, and monocytopenia, an effect mediated through induction of high levels of granulocyte colony-stimulating factor (G-CSF) [182]. Moreover, pre-clinical studies provided evidence that tocotrienols radioprotection is exerted, in part, via induction of G-CSF [183,184], suppressing expression of TNF-α, IL-6, IL-8, inducible nitric oxide synthase (iNOS), and NF-κB signaling [179]. IR downregulates the expression of thrombomodulin (TM) and increases endothelial surface expression of adhesion molecules which allow the attachment of immune cells and, thereby, contribute to inflammation and activation of the coagulation cascade. In this regard, the efficacy of tocotrienols is attributed to their higher antioxidant potential, their ability to inhibit HMG-CoA reductase activity [185], and increase TM expression in endothelial cells [186], which result in anti-permeability, anti-inflammatory and anti-thrombotic response [179]. Promising radioprotective results of γ-tocotrienol (GT3) have been demonstrated in mice [187] and primate models, by preserving the hematopoietic stem and progenitor cells, and recovery from γ-irradiation (5.8 or 6.5 Gy)-induced neutropenia and thrombocytopenia [188,189]. Recent preclinical studies evidenced that GT3 may be a potential countermeasure against late degenerative tissue effects of high-LET radiation in the heart [190] and lung radiation injury [191]. Tocotrienols accumulate in the intestine to a greater level than tocopherols, and this can be involved in its greater ability to attenuate GIS [192]. γ-tocotrienols seem to have a greater efficacy as radioprotectors attributed [189] to their: (a) higher antioxidant potential [191], (b) capacity to downregulate proapoptotic/antiapoptotic ratio [193], (c) ability to accumulate in endothelial cells and intestinal epithelium which facilitates the recovery of mesenchymal immune cells [192], and (d) ability to inhibit HMG-CoA reductase, helping to avoid chronic inflammatory responses associated to radio-induced vascular and intestinal damage [185]. Moreover, recent studies have also evidenced the anti-cancer properties of γ-tocotrienols [179] and, although their low bioavailability is an important limiting factor [177], new formulations may help to overcome this pitfall. In this sense, a novel water-soluble liposomal formulation of γ-tocotrienol selectively targets the spleen and bone marrow with high efficiency, and facilitates rapid recovery of hematopoietic components after lethal TBI radiation in mouse models [194]. High doses of tocols are required to exert radioprotective effects, which increase the risk of toxic accumulative side effects. To ameliorate this risk, several trials have assayed and evidenced additive/synergistic effects with other radioprotectants such us aminofostine [195], simvastin [196], and others. For instance, pentoxifylline (a xanthine derivative approved by the FDA as a phosphodiesterase inhibitor, with antioxidant and anti-inflammatory effects) improved survival and enhanced the radioprotective properties of γ-tocotrienol on the hematopoietic, GI and vascular systems in mice subjected to 12 Gy 60Co γ-irradiation [197]. A Phase II clinical trial also demonstrated the radioprotective efficacy of the combination pentoxifylline+vitamin E to attenuate radiation-induced fibrosis [198]. Two randomized controlled trials provided evidence that dietary supplementation of alpha-tocopherol and beta-carotene during radiation therapy could reduce the severe adverse effects of treatment, but also warned that high doses might compromise radiation treatment efficacy [199,200]. Other radioprotective combinations, such as α-tocopherol acetate and AA, had the additional advantage of enhancing apoptosis in irradiated cancer cells [201,202].

Calcitriol upregulates the expression of SirT1, SODs and GPxs and induces the synthesis of metallothioneins in vitro [203,204]. Jain et al. (2013) showed a positive link between vitamin D and GSH concentrations, as well as a reduction in the levels of pro-inflammatory cytokines [205]. Inhabitants of contaminated regions near Chernobyl had lower vitamin D blood levels compared to those living in uncontaminated regions [206]. Therefore, oral supplementation with vitamin D during radiotherapy or in professionals chronically exposed to low IR doses could be doubly useful, preventing radioinduced oxidative stress and osteoporosis [207]. Recent studies evidence that calcitriol selectively radiosensitizes cancer cells by activating the NADPH/ROS pathway [208].

#### 6.1.4. Antioxidant Enzyme Activities and Oligoelements

Many antioxidant/defense enzymes, such as SODs, GPxs, and metalloproteins require trace elements as cofactors (e.g., Cu, Mn, or Se), thus, their dietary supplementation has been widely evaluated as a radioprotective strategy [54,209]. As cofactor for selenoenzymes, i.e., GPxs, thioredoxin reductase-1 and ribonucleotide reductase, Se supplementation enhances GPxs activity, thus reducing intracellular H2O2 and organic peroxide levels. Both sodium selenite and selenomethionine, i.p. injected before or shortly after (+15 min) radiation exposure (60Co, 9 Gy), enhance the survival of irradiated mice, but selenomethionine had lower toxicity [210]. Se treatment enhances Nrf2 transcription and upregulates the adaptive response to IR in bone marrow and hematopoietic precursors [211]. 3,3 -diselenodipropionic acid (DSePA) had maximum absorption in the lung, suppressed NF-kB/IL-17/G-CSF/neutrophil axis and significantly reduced infiltration of neutrophils and levels of IL1-β, ICAM-1, E-selectin, IL-17 and TGF-β in the bronchoalveolar fluid, prevented pneumonitis and increased survival of irradiated mice without affecting radiation sensitivity of tumors [212]. During the reaction with oxidizing free radicals DSePA generates intermediates with GPx like activity that reduce lipid peroxidation, apoptosis and excessive inflammatory response in radiosensitive tissues such as lung, liver, spleen, and GI tract, increasing survival against supra-lethal doses of γ-radiation [213]. Se compounds are less effective than aminofostine as radioprotectors, but have also lower toxicity and can be used in combined treatments [214].

Two consecutive systematic reviews, carried out between 1987 and 2012 [215] and 2013 and 2019 [216] evidenced that cancer patients tend to have low Se blood levels, which is aggravated by radiotherapy and/or its side effects (vomiting, etc.), and associates to a decrease in the activity of different antioxidant enzymes. Based on the results from clinical trials in patients who underwent radiotherapy, it was concluded that Se supplementation prevented or reduced the side effects of radiotherapy without compromising its anticancer efficacy; and consequently, authors highly recommend sodium selenite (200–500 μg/daily) oral supplementation [216]. On the other hand, it is paradoxical that several studies have demonstrated Se can act as prooxidant in a dose dependent fashion and can attenuate DNA repair mechanisms as well as antiapoptotic genes in some cancer cells, being nowadays assayed as a radiosensitizer in oncoradiotherapy. In vivo, the variability in redox potential gradients, the lower pH and the redox imbalance existing in the cancer microenvironment can facilitate the conversion of Se nanoparticles (SeNPs) into a pro-oxidant agent causing mitochondrial dysfunction, cell cycle arrest, and ultimately cancer cell death [217]. Organic Se compounds and especially SeNPs are better candidates as radioprotectors and radiosentitizers for their lower toxicity and higher cancer cell selectivity compared to sodium selenite [217,218].

SODs exist as CuZnSOD (cytosolic and nuclear fraction) and mitochondrial MnSOD, and both scavenge O2 •− by accelerating its conversion to H2O2. Attempts to supplement the activity of endogenous SOD include the induction of in vivo gene expression using adenovirus or plasmid liposomes, and administration of nanozymes with SOD-like activity [219]. A porphyrin-mimetic of the human MnSOD (BMX-001), which crosses the BBB, protected the brain's white matter at the same time that it increased the sensitivity of the cancer cells to IR [220]. BMX-001 can potentially interact with numerous redox-sensitive pathways, such as those involving NF-κB and Nrf2, thus having an impact on their transcriptional activity [219]. The ability of BMX-001 to reduce the toxic effects of radiotherapy in cancer patients is being evaluated in phase II clinical trials (www.clinicaltrials.gov, accessed on 3 February 2022), e.g., NCT05254327 (rectal Cancer), NCT03608020 (brain metastases), NCT02655601 (high-grade glioma) and NCT02990468 (head and neck cancer) [54], and initial results seem to indicate that BMX-001 reduces side effects of radiotherapy.

#### 6.1.5. Cyclic Nitroxides

Synthetic cyclic stable nitroxide radicals (NRs), such as Tempo, Tempol, XJB-5-131, TK649.030, JRS527.084 or JP4-039, contain a nitroxyl group with an unpaired electron (-NO) and are stabilized by methyl groups, which prevent radical-radical dismutation. In vivo, NRs undergo a very rapid, one-electron reaction to the corresponding hydroxylamine, which has also antioxidant activity. NRs stabilize free radicals, easily diffuse through the cell membranes, have SOD and CAT-like activity, prevent the Fenton and Haber–Weiss reactions and are capable of protecting cells from radical induced damage [54,221].

Gramicidin *S*-nitroxide JP4-039 is a free radical scavenger and antioxidant targeting mitochondria through a segment of a cyclopeptide gramicidin that abrogates mitochondrial oxidative stress and cardiolipin oxidation, playing a pivotal role in the execution of apoptosis. JP4-039 effectively protects and mitigates TBI-induced hematopoietic, GI syndrome and skin damage even when it is delivered intravenously up to 72 h after exposure [222,223]. JP4-039 treatment ameliorated head and neck radiation-induced mucositis and marrow suppression in mice [224]. In a comparative study with other four nitroxides, JP4-039 demonstrated the best median survival after radiation exposition [225]. Based on these

properties, Luo et al. have synthesized and analyzed a series of nitronyl nitroxide radical spin-labeled RES derivatives that have also shown important radioprotective effects [226].

#### 6.1.6. Melatonin

*N*-acetyl-5-methoxytryptamine (melatonin), the main secretory product of the pineal gland, is a free radical scavenger with strong antioxidant properties, related to its chemical structure (specifically, the aromatic ring indole rich in delocalized electrons). Melatonin indirectly affects the oxidative–antioxidant balance, stimulating the expression of genes encoding for SODs, GPxs and GR, and ameliorates inflammatory responses. Such protection is evidenced by the capacity of melatonin to reduce 8-hydroxy-2 -deoxyguanosine levels and associated DNA lesions [227,228]. Moreover, animal studies confirmed that melatonin is able to alleviate radiation-induced cell death via inhibiting proapoptotic genes (e.g., Bax) and upregulating antiapoptotic genes (e.g., Bcl-2) [229]. Its radioprotective efficacy in pre-clinical models has been recently reviewed in [230]. Melatonin has some characteristics of an ideal radioprotector (multiple ways of action, low toxicity, and ability to cross biological barriers), and also has anti-cancer properties, i.e., apoptotic, antiangiogenic, antiproliferative, and metastasis-inhibitory effects reviewed in [231]. A meta-analysis of eight randomized controlled trials concluded that melatonin (20 mg, orally administered, once a day) led to substantial improvements regarding tumor remission, 1-year survival, and alleviation of therapy-related side effects [232].

#### *6.2. Radiomitigators*

Radiomitigators minimize the toxicity of IR even when they are administered after radiation exposure, which differentiates them from radioprotectors that almost prevent/reduce the direct damages. Since most radiological and atomic mishaps are unexpected events, decision-making specialists should consider the use of radiomitigators that can most assist with limiting the destructive impacts of radiation exposure in those already affected. In this technical sense, ideal radiomitigators ought to be anti-inflammatory, enhance antioxidant defenses, have antimutagenic properties, upregulate the DNA repair mechanisms, activate mitotic processes, cell growth and differentiation to promote the regeneration of damaged tissues, and forestall or reduce ARS and CRS. At present, no molecule under study meets all these prerequisites, but there are a large number of choices [54,233,234], which may be combined, for quick administration to affected individuals. For such situations, we may recommend the following:

#### 6.2.1. Antiemetic Drugs, Probiotics, Prebiotics, and Toll-like Receptor Agonists

The pathophysiology of radioinduced GI toxicity is mediated by enterocyte loss, vascular injury, and bacterial translocation. The symptoms involve nausea, vomiting and diarrhea that aggravate electrolyte and fluid loss and lead to morbidity/mortality. Anti-emetics are useful for the stabilization of affected patients, with 5-hydroxytryptamine-3 receptor antagonists (granisetron and ondansetron) often being the first choice of treatment, whereas the addition of dexamethasone provides a modest improvement in prophylaxis [235]. Higher half-life and effectivity make granisetron a better option. The disadvantage of the preventive antiemetic treatment is that prodromal symptoms will be masked and they are useful bioindicators of ARS [235,236].

Gut microbiota dysbiosis aggravates radiation enteritis, reduces the absorbing surface of intestinal epithelial cells, weakens the intestinal epithelial barrier function, and promotes inflammatory factor expression, thus leading to a persistent mucositis, diarrhea and bacteremia [237]. Cancer patients exposed to radiation therapy exhibit marked alterations in gut microbiota composition, with a decrease in protecting *Bifidobacterium* and *Lactobacillus* spp. together with an excessive growth of Gram-negative pathogen bacilli [238]. Maintenance of normal microbiota using probiotics exerts nutrient competition and avoids binding of intestinal pathogens to host mucosa, thus preventing bacterial translocation. Gut microbiota produces short-chain fatty acids (SCFAs), mainly composed of acetate, propionate and

butyrate, that are the main energy source of colon cells and prevent intestinal inflammation by reducing the production of chemokines or adhesion molecules. Butyrate, in particular, is reported to stimulate a variety of colonic mucosal functions and to induce the expansion of Treg lymphocytes [239]. SCFAs play an important role in relieving intestinal injury induced by radiotherapy, whereas propionate [240] and valeric acid [241] have shown long-term radiomitigation of hematopoietic and GI syndromes by reducing the release of ROS, DNA damage and proinflammatory responses.

Prebiotics, fecal microbiota transplantation and, especially, probiotics prevent and improve radiation-induced enteritis [242,243]. In preclinical and clinical studies, probiotic interventions with *Lactobacilli* and/or *Bifidobacteria* ameliorate micro-intestinal atrophy and diarrheal symptoms [244], and exert cancer protection [245]. Commensal bacteria and probiotics interaction with Toll-like receptors (TLRs) activate the NF-κB, ensuring the development of innate immune responses, maintaining the barrier function, and promoting wound repair and tissue regeneration [237]. Several TLR2 and TLR4 agonists reduce radiation-induced apoptosis in epithelial stem cells, alleviating intestinal damage [246,247]. In clinical trials, probiotics reduce the incidence of diarrhea [242,243,248] and mucositis in cancer patients treated with radiotherapy [238], even though results are difficult to evaluate as they vary with the type of cancer, radiotherapy modality used, and type of probiotic used [246]. A recently published systematic review concludes that *Bifidobacterium longum*, *Lactobacillus acidophilus*, *Bifidobacterium breve*, *Bifidobacterium infantis* and *Saccharomyces boulardii* could be a good combination to prevent mucositis or ameliorate side effects of radiotherapy [249].

β-glucans (constituents of the cell wall in bacteria and plants) administered prior to and after irradiation exposition, prevent intestinal pathogen bacterial translocation, stimulate hematopoiesis and enhance survival in radiation-exposed animals [233,250]. Urolithin A (UroA), a metabolite generated from the transformation of ellagitannins by the gut, shows immunomodulatory and anti-inflammatory activities, and markedly upregulated the survival of irradiated mice. UroA improved the intestine's morphology architecture and the regeneration of enterocytes, and significantly decreased radiation-induced p53 mediated apoptotic cell death [251].

#### 6.2.2. Cytokines and Growth Factors

Any radiation dose >2 Gy results in bone marrow depletion, decreased blood cell counts, hemorrhage, and immunosuppression, leading to secondary infections. In the absence of treatment, death may occur in 2–8 weeks post-irradiation. Clinical therapy can help, and should not be limited to the use of antibiotics, blood, and platelet transfusions [236].

Cytokines like IL-1, IL-6, or TNFα promote inflammation, recruit leukocytes into damaged tissues and have restorative effects on the bone marrow. For that reason, earlier studies considered them as radioprotectors [252,253]. Nowadays, this hypothesis has changed since the proinflammatory states exacerbate IR toxicity.

The bone marrow recovery has been highlighted by the FDA, and in fact, some radioprotectants have been approved act in this sense, i.e., Filgrastim (a recombinant DNA type of the physiological G-CSF), Pegfilgrastim (a PEGlylated type of the previous), Sargramostim (a recombinant granulocyte-macrophage colony-stimulating factor, GM-CSF) and recently (2021) romiplostim (a Fc-peptide fusion protein that activates the thrombopoietin receptor) [54,234,254]. G-CSF and pegylated G-CSF promote proliferation, differentiation and maturation, and enhance blood neutrophil recovery and the survival rate. In 2009, The World Health Organization convened a panel of experts to develop recommendations for MCM in the management of H-ARS in a hypothetical scenario involving the hospitalization of 100–200 patients exposed to IR. According to this First Global Consensus, WHO strongly endorsed cytokine therapy (G-CSF or GM-CSF) within 24 h of exposure, above 2 Gy, for affected individuals with significant lymphopenia or when neutropenia (<500 cells/mm3) persists for more than 7 days [236,255]. Pegylated G-CSF can be used as an alternative to G-CSF, with the advantage that it can be administered weekly (daily in the case of G-CSF), but it appears to be less efficacious in treating injuries combined with skin burns. Treatment should be maintained until the neutrophil count maintains over 1000 cells/mm3 in the absence of infection. Individuals with prolonged anemia can be treated with erythropoietin to avoid transfusions, considering the option of iron supplementation.

GM-CSF, administered as late as 48 h after radiation exposure, accelerates recovery from neutropenia and thrombocytopenia and decreases infection rates [256]. Lung injury (RILI) is a common complication of thoracic cancer radiotherapy, and currently, it has no effective treatment. GM-CSF reduced the occurrence of both pneumonia and pulmonary fibrosis. Moreover, an analysis of the clinicopathological characteristics of 41 patients, undergoing radiotherapy, evidenced that RILI remission was significantly correlated with GM-CSF treatment [257].

Keratinocyte growth factor (KGF) produced by mesenchymal cells protects and repairs epithelial tissues. KGF promotes the recovery of the mucosa, improves intestinal barrier functions and limits bacterial translocation and subsequent sepsis after irradiation. In clinical studies Palifermin®, a human recombinant KFG with analogous activity and higher stability, reduced the incidence, duration and severity of oral mucositis and esophagitis in cancer patients, and stimulated immune recovery following hematopoietic stem cell transplantation [258].

Epidermal growth factor (EGF) promotes epithelial and hematopoietic stem cells regeneration [259]. Bone marrow-derived hematopoietic stem cells (HSCs) express the EGF receptor in response to radiation and, in turn, EGF promotes HSCs regeneration in vivo. Mechanistically, EGF reduced radiation-induced apoptosis through repression of PUMA proapoptotic protein, and EGF receptor signaling was needed for DNA repair and for HSCs regeneration [259,260]. rhNRG-1β is an EGF-like protein that maintained mitochondrial integrity and ATP production in irradiated cardiomyocytes and preserves cardiac function via the ErbB2-ERK-SIRT1 signaling pathway [261]. Cotreatment with G-CSF led to a further increase in survival (20% in controls, 67% in EGF, 86% in EGF+G-CSF) [260].

A decrease in fibroblast growth factor (FGF) blood levels is found after irradiation, and a human recombinant derivative (FGF-P) improved duodenal functions and increased survival in GI-ARS mouse models. After been exposed to IR, FGF-P treated animals showed less hemorrhages and cutaneous ulcerations. FGF-P also holds promise for the treatment of burns, wounds and stem-cell regeneration [262].

It must be pointed out that the increased activity of many of these cytokines can be associated with prolonged ROS and RNS generation, a fact that favors the development of chronic inflammatory problems, and thereby the development of fibrosis and/or carcinogenesis [50]. Moreover, many cancer cells (glioblastoma, lung cancer, etc.) increase expression of EGF and other cytokine receptors, which makes the use of these radioprotectors unfeasible in cancer patients undergoing radiotherapy.

Bleeding due to thrombocytopenia is a common cause of death in ARS patients. Several agents have been assessed, including recombinant human thrombopoietin (TPO) and TPO mimetics like romiplostim (Nplate®) and eltrombopag [263]. Unfortunately, alloimmunization was developed after TPO administration, and it is no longer manufactured [264]. Nplate® (injectable) activates the TPO receptor on megakaryocyte precursors promoting cell proliferation and platelet production. It has been clinically assayed successfully for the treatment of thrombocytopenia and is approved by the FDA and European Medicine Agency for the treatment of idiopathic purpura and immune thrombocytopenia [265]. Romiplostim (administered for 3 consecutive days) increases survival to 100% in C57BL/6J mice exposed to a γ-TBI (7 Gy) and, at day 30, blood cells, hematopoietic progenitors and the histological appearance of the intestine were similar to non-irradiated controls [266]. Furthermore, a single dose of Nplate® (30 μg/kg) enhanced the survival to 40% [267]; combined with G-CSF and EPO, it increased survival to 100% (0% survival in controls 30 days after exposure), recovering hematological parameters to the levels of non-irradiated mice [268]. In non-human primates, Nplate® and pegfilgrastim combined treatment had much greater effects on platelet and neutrophil recovery following γ-irradiation compared

to single agents alone [269]. HemaMax® (human recombinant IL-12) restored all cell progenitor types in the bone marrow, decreased thrombocytopenia, leukopenia and infection rates and preserved GI functions, induced recovery of body weight and increased survival, when administered 24 h post-TBI (8.0 Gy) to mice and rhesus monkeys [270]. Pegylated IL-11 (Neumega®) is FDA approved to treat thrombocytopenia in cancer patients, although it has limited use as a radiomitigator, due to the need to be administered daily. To circumvent this problem, another mono-PEGylated IL-11 analog (BBT-059) was designed and showed higher bioavailability and potency in vivo. In a mouse model, BBT-059 led to multi-lineage hematopoietic reconstitution and appears to increase survival more than PEG-G-CSF and PEG-GM-CSF at high TBI doses [259,271].

HSCs and mesenchymal stem cells (MSCs) have also been proven to be effective in treating ARS in preclinical models. Hematopoietic stem cell therapy is recommended for patients with complete aplasia assessed by bone marrow biopsies [255], but in Chernobyl and other accident scenarios, survival was more likely among individuals that did not received bone marrow transplant [272]. Most recipients died shortly after transplantation due to the rapidly progressing insults to skin, lung and gut, complicated by serious bacterial, fungal and/or viral infections [264]. For that reason, Radiation Emergency Assistance Center/Training Site provides recommendations for the administration of antibiotic and/or other antimicrobial agents [264]. The WHO expert group (2011) recommend "wait and see" for a spontaneous or cytokine induction of hematopoiesis recovery, and to consider the administration of hematopoietic stem cells only after 2–3 weeks, and only in the absence of non-hematopoietic organ failure [255]. This recommendation has not changed as a result of the analysis of more recent studies [264]. Mesenchymal stem cells (MSC) are abundant resources (umbilical cord, bone marrow, blood, adipose tissue, and placental tissue), can differentiate into cells of the mesodermal lineage [273], and have demonstrated capacity to regenerate damaged tissues [274]. Despite this promise, translating the potential into actual clinical practice needs to solve many barriers, including immune-rejection, teratogenesis, and others [275]. A clinical trial is evaluating the efficacy of MSC injections for the treatment of chronic radiotherapy-induced complications (PRISME, NCT02814864) [273].

#### 6.2.3. Inhibitors of the Inflammatory Response

Excess of intracellular ROS, hypoxia and microvascular injury induced early activation of HIF-1α is a powerful stimulator of various pro-fibrotic mediators such as TGF-β, chemokines (e.g., MCP-1 and MIP-1beta), vascular endothelial growth factor (VEGF), and platelet-derived growth factor [276,277]. TGF-β stimulates apoptosis through Smad and Rho/Rock pathways, upregulates enzymes such as NOX2, NOX4, COX-2 and iNOS, inducing oxidative stress and proinflammatory responses that may persist and are associated with vascular damages and fibrosis RIBE [278–280]. Consequently, it is not surprising that halofuginone (an inhibitor of the TGF-β signaling pathway) and bevacizumab (an anti-VEGF antibody) have been shown to prevent or reduce radiation-induced fibrosis [281,282], with the additional advantage of inhibiting tumor angiogenesis and consequently tumor growth and metastasis formation. Different phase I/II clinical trials in women with metastatic breast cancer have shown more successful radiotherapy response if combined with a TGF-β inhibitor (LY2157299, NCT02538471). In fact, reduction of the plasma levels of TGF-β is associated with greater efficacy of radiotherapy on different types of cancer [283]. Some inflammatory polyphenols (genistein, curcumin, resveratrol or quercetin) downregulate TGF-β expression or signaling pathways attenuating radio-induced skin, pulmonary and/or myocardial fibrosis [102,112,113,145,277].

Radiation exposure enhances COX and iNOS activity, increasing the production of PGE2 and NO (respectively), both involved in the activation of the inflammatory response [284,285]. NSAIDs assayed as radiomitigators include non-selective COX inhibitors, e.g., acetylsalicylic acid (aspirin), ibuprofen, indomethacin, diclofenac, and flurbiprofen. Aspirin ameliorates radiation-induced kidney and lung damage and reduces post-irradiation chromosomal aberrations [286]. A recent meta-analysis of randomized controlled trials

indicates that acetylsalicylic acid reduces the overall risk of recurrence and mortality of colorectal cancer and/or colorectal adenomas, which increases the interest in its possible use as a radioprotector/radiosensitizer [287]. Flurbiprofen showed radioprotection in clinical studies, e.g., delaying the onset of mucositis and reducing its severity after radiotherapy in head and neck cancer patients, although the overall severity or duration of mucositis was not improved [288]. Benzydamine (a prostaglandin synthetase inhibitor) decreased the incidence and severity of oral mucositis associated to radiotherapy exposure [289].

Selective COX-2 inhibitors have the advantage of having less undesirable side effects, whereas promote myelopoiesis, thus avoiding the negative feedback control exerted by PGE2 [285]. Meloxicam alone, and in combination with IB-MECA (an adenosine A3 receptor agonist), has been reported to stimulate endogenous production of G-CSF and hematopoiesis, increasing the survival of mice exposed to lethal doses of radiation [290]. Celecoxib (a selective COX-2 inhibitor) attenuated severe skin reactions after a high single dose of 50 Gy and, in rats, reduced brain injury maintaining the integrity of the BBB and reducing inflammation [291]. In a glioblastoma model, the combined effect of radiation and celecoxib increased tumor cell necrosis, showing a significant reduction in tumor microvascular density and prolonged survival compared to irradiation alone [292]. It should be added that the analgesic effects of COX inhibitors can contribute to the wellbeing of of people affected by exposure to IR.

The mainstay of treatment in acute radiation pneumonitis consists of the systemic administration of glucocorticoids at high doses, aiming to reduce inflammation and inhibit TNFα-induced nitric oxide-mediated endothelial cell and lymphocyte toxicity. The use of inhaled corticosteroids ensures the highest dose deposition in the airway, thus decreasing side effects and ameliorating pulmonary fibrosis [293]. Nevertheless, systematic prophylactic use of corticosteroids to prevent toxic pulmonary edema is not recommended in China or Germany [294] and there is no evidence of a significant long-term benefit based on the use of corticosteroids.

#### 6.2.4. Statins

These 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors are commonly used to treat hypercholesterolemia and atherosclerosis. Statins also possess other biological effects, i.e., improving endothelial function, decreasing oxidative stress and inflammation, and regulating the immune system. Statins lessen the mRNA expression of pro-inflammatory and pro-fibrotic cytokines, accelerate the repair of DNA double-strand breaks and mitigate DNA damage [295]. Simvastatin, in particular, has been shown to mitigate radiation-induced enteric injury [296], to prevent radiation-induced marrow adipogenesis [297], to attenuate radiation-induced salivary gland dysfunction in mice [298], and to reduce cardiac dysfunction and capsular fibrosis [299]. GT3 and simvastin provide synergic protection against radiation-induced lethality, hematopoietic and bone marrow injury compared to the single treatments [196]. Pravastatin [300] and atorvastatin [301] have also shown radiomitigative efficacy.

#### 6.2.5. Angiotensin Axis Modifying Agents

There is some evidence that IR upregulates angiotensin II (AngII) expression in a dose-dependent manner, and AngII can increase ROS production through activation of the NADPH oxidase, upregulating inflammatory and profibrogenic pathways involved in long-term radiation injury [302,303]. Moreover, local synthesis of Ang II has been observed in fibrotic plaques and lung myofibroblasts, whereas apoptosis of alveolar epithelial was completely abrogated by an AngII receptor antagonist or by anti-AngII antibodies [304]. In pre-clinical models ACEi (angiotensin-converting enzyme inhibitors) and AngII antagonists, widely used as antihypertensive agents, have been shown to mitigate nephrotoxicity [305], pneumonitis [306,307] and other hematopoietic radio-inducted toxicities [308]. ACEi increases Ang-(1–7) levels which seems to have radioprotective [309] and antitumoral effects [310]. Several retrospective studies reported that ACEi decreased

the risk of radiation pneumonitis in lung cancer patients [311]. In a randomized controlled trial in patients exposed to 14 Gy TBI (9 equal fractions for 3 days) captopril mitigated renal nephropathy—increasing survival but not significantly [312], although a subsequent study by the same authors indicated significant differences in survival were attributable to radiomitigating effects on the respiratory system [313]. Captopril has been shown to be a better mitigator than lisinopril, enalapril, or ramipril [234], but a large prospective study in lung cancer patients treated with captopril and radiotherapy was halted due to insufficient accrual [302]. However, a recent meta-analysis seems to evidence that the use of ACEi decreased the incidence of symptomatic radiation-induced pneumonitis in lung cancer patients, especially in those older than 70 years, while those treated with angiotensin receptor blockers had a slight (non-significant) trend towards developing pneumonitis [314]. In addition, recent studies using ramipril and lorsartan showed reduced neuronal apoptosis, enhanced BBB integrity, and improved cognitive and motor function after TBI [315,316], major side effects of cranial radiotherapy in adult and pediatric cancer survivors.

#### 6.2.6. Molecular Hydrogen (H2)

The antioxidant advantages of H2 gas include [317,318]: (a) selectively scavenging the deleterious ONOO− and •OH radicals, preserving other important ROS and NIS for normal signaling regulation, (b) stronger reductive activity than other dietetic antioxidants as C or E vitamins, (c) enhanced Nrf2 transcription and SOD, CAT and GPx expression [319] and (d) reduced NADPH oxidase activity [320]. In addition to reducing oxidative stress, H2 increases the expression of antiapoptotic proteins (Bcl-xL and Bcl-2) [321], which could be helpful to attenuate damage induced by IR. In addition, H2 downregulates the expression of adhesion molecules, reduces the infiltration of neutrophils and macrophages [322], inhibits NF-κB and reduces serum IL-1β, IL-6, and TNF-α levels, which could prevent RIBE and alleviate inflammatory response [323]. Most of these properties have been evidenced in a recent human clinical trial [324].

Hirano et al. [325] have recently published an interesting review on the potential radioprotective effects of H2 on cognitive function, testis, lungs, heart, skin, cartilage, GI system, hematopoietic organs and the immune system. A randomized placebo-controlled study showed that consumption of H2-supplemented water improved the quality of life of patients treated with radiotherapy for liver tumors [326]. H2 mitigated radio-induced bone marrow damage in cancer patients without compromising the anti-tumor effects of radiotherapy according to a retrospective observational study [327]. In vitro and in vivo, H2-rich water promoted tritium elimination (Table 1), reducing serum levels and tissuebound tritium, and attenuated the genetic damage [328]. In addition to its antioxidant, anti-inflammatory and antiapoptotic effects, H2 can be easily administered through various routes with little adverse effects and great efficacy [317,318,325]. These results show promising potential for the use of H2 as a potential radiomitigator that should be studied in more depth.

#### 6.2.7. Metformin

Metformin is one of the most commonly used anti-diabetic drugs and has shown potential antioxidant, radioprotective, and anticarcinogenic properties [329,330]. It is a hydrogen-rich agent able to neutralize free radicals, increase GSH, and upregulate the activity of SOD and CAT [331], which all favor the antioxidant cell defense. Metformin stimulates DNA repair via non-homologous end joining or homologous recombination, and nucleotide excision repair [50]. Some studies showed that metformin exhibited a radioprotective effect only when administered to mice after radiation exposure; and others evidenced that it can also be considered a radiomitigator, because it reduced chronic production of ROS and pro-fibrotic cytokines such as TGF-β, and attenuated fibrosis through modulation of pro-oxidant genes such as NOX4, if administered after radiation exposure [332]. Metformin can also induce several redox-related genes, such as the PRKAA2 (which encodes the AMPK), a mechanism that helps in protecting cells from the accumulation of unrepaired DNA and attenuates inflammation and pro-fibrotic pathways [329]. Metformin also ameliorates IR hematopoietic stem cell injury in mice [333]. Cardiovascular disease is a pivotal disorder after radiotherapy and the administration of metformin to γ-irradiated (5 Gy) rats significantly ameliorated the increase in plasma of cardiac diseaserelated biomarkers such as, LDH and CK-MB, NF-κB, IL-6 and TNF-α levels compared to the control group, which suggests that concomitant administration of metformin during radiotherapy can act as an efficient heart protector from oxidative stress and inflammatory damages, and endothelial dysfunction-derived damage [334]. Furthermore, several studies have evidenced the synergistic action of metformin when it is administered with sulfhydryl containing drugs [335] or with melatonin [332,336], although in others the synergy was not evidenced and melatonin was shown to be a better radioprotector [337]. It is also worth mentioning that metformin improves tumor oxygenation and the response to radiotherapy in tumor xenograft models. Thus, it can be considered a potential radiosensitizer to improve the outcome of radiotherapy [338,339]. In this regard, the use of metformin in patients with hepatocellular carcinoma and receiving radiotherapy has been associated with a higher overall survival [340]. Metformin's anticancer effects are well documented in preclinical studies, along with early phase clinical trials, but there is a significant lack of late phase clinical trials [341].

As we noted previously, we do not have any molecule that meets the requirements of an ideal radioprotector or radiomitigator. However, a combination of molecules may accomplish summation of protective/mitigating mechanisms and, in the end, synergies. Moreover, it is basic to have MCM that can be effective and that can also be immediately administered to the people in need. Such combinations should be assayed in standard clinical trials. Fortunately, most of the alternatives referenced above have had such preclinical and clinical examinations performed for various indications (see e.g., https://www.clinicaltrials.gov, accessed on 3 February 2022).

#### *6.3. Radionuclide Scavengers*

Table 1 summarizes the diverse radionuclides that might be encountered in contamination events. Clinical countermeasures and treatments, their route of administration and main mechanisms of action, as well as the organs or tissues where those agents may accumulate are also indicated. Radiation exposure brought about by radionuclide contamination only stops if the radionuclide is completely disposed from the body, with or without therapy.


**Table 1.**

Contamination

 by

radionuclides

 and medical

countermeasures.




MCM as listed have been suggested as treatments for internal contamination with radioisotopes by: Hickman, D. P. Management of persons contaminated with radionuclides: NCRP Report No. 161 (Volume 1) [374]. *Medical Management of Persons Internally Contaminated with Radionuclides in a Nuclear or Radiological Emergency*; Emergency Preparedness and Response; International Atomic Energy Agency: Vienna, 2018 [375]. Hübner, K.F.; Watson, E.E. Management of Persons Accidentally Contaminated with Radionuclides: NCRP Report No. 65. Washington, D.C. 1980 [376]. Managing Internal Radiation Contamination—Radiation Emergency Medical Management Available online: https://remm.hhs.gov/int\_contamination.htm (accessed on 5 May 2022) [377]. Ammerich, M.; Giraud, J.M.; Helfer, N.; Menetrier, F.; Schoulz, D.; Blanc, J.; Vilain, D.; Boll, H.; Bourguignon, M.; Chappe, P.; et al. Medical Intervention in Case of a Nuclear or Radiological Event—National Guide, Release V36, 2008 [378].

#### *6.4. Biological Dosimetry*

Despite the fact that there are many approved and expected biomarkers to survey the harmful impacts of IR [54], which biomarkers ought to be suggested in the instance of a radiological or atomic crisis? In these scenarios, speed, reliability, and traceability should be the prevailing criteria. These needs rouse us to choose, as the most suitable, those presented below.

#### 6.4.1. Lymphocyte Depletion Kinetic (LDK) Assay

This measure is utilized to gauge the dose following whole or incomplete body outer radiation exposure which was absorbed over minutes to hours. Methodologically, serial complete blood counts are acquired, and the outright lymphocyte check is determined and followed over time. The typical reach for outright lymphocyte count can be impacted by numerous variables, including the hardware utilized, and the ethnicity, age, health, and sex of the examined reference population. In addition, lymphocyte counts can be decreased or expanded by medications, contamination, and numerous clinical problems disconnected from radiation. What is key is that the lymphocyte exhaustion rate is directly identified with radiation assimilated dose (dose range 0.5–14 Gy). For example, a dose of 2–4 Gy associates with lymphocyte atrophy happening over ~4–6 days, while for a dose of 4–6 Gy the lymphocyte decrease requires ~2–4 days [379]. The US Armed Forces Radiobiology Research Institute (AFRRI) BAT (biodosimetry assessment tool) program (https://www.remm.nlm.gov, accessed on 15 February 2022) proposes acquiring a blood cell count as soon as possible after radiation exposure, and suggests that if the absorbed dose is known or suspected to be ≥5 Gy, blood counts ought to be obtained each 9–12 h for 2 to 3 days after irradiation and afterward at regular intervals of 24 h for 3 to 9 days. Other than this, a >5 Gy gamma-ray equivalent dose can be estimated based on biological end points like initial vomiting <2 h after exposure, or other dosimetric end points like physical dosimetry. If the retained dose is known or suspected to be <5 Gy, blood counts ought to be acquired at regular intervals of 24 h for 9 days. A straightforward dose-prediction algorithm based on lymphocyte kinetics as documented in prior radiation accidents was proposed by Goans et al. [380], where results are determined in gamma dose (Gy) whole body counterparts. Notwithstanding, this technique has impediments since it is not appropriate for surveying fractional body exposures or internally deposited radioisotopes.

In developed nations confronting a large-scale radiation crisis, biodosimetry dependent on LDK, clinical signs and indications, and dose estimated from geographic data are probably going to be accessible more quickly than biodosimetry dependent on cytogenetics [31].

#### 6.4.2. Neutrophils-to-Lymphocytes (NLR) Ratio

The neutrophil-to-lymphocyte ratio (NLR) is a valuable marker of host inflammation, which mirrors the connection between circulating neutrophils and lymphocyte counts (dose rage 0.5–10 Gy). It may conveniently be determined from routine complete blood counts (CBCs) with differentiation. It has been indicated that an increase in NLR throughout radiotherapy has a negative impact on survival in breast cancer patients, putting these patients with radiotherapy-susceptible host immunity at a higher risk of tumor recurrence [381]. An essential investigation of the prognostic estimation of the NLR compared with human whole-body irradiation was published after the mishap at the Chernobyl Nuclear Power Station [382].

#### 6.4.3. Cytogenetics

Cytogenetic dosimetry is a significant dose evaluation strategy, especially when there are challenges in deciphering the information, in scenarios where there is reason to believe that people not wearing dosimeters have been exposed to radiation, in instances of cases claiming for compensation after suffering radiation harms that are not upheld by unequivocal dosimetric proof, or in instances of exposure over a person's working lifetime

(https://www.iaea.org, accessed on 24 February 2022). This incorporates [383,384] the examination of:


In this way, thinking about the technical conditions around a radiological or atomic crisis, it appears reasonable to suggest DSB, DC, and CBMN tests as the most ideal choices. For example, Nakamura et al. [388] considered the causal connection between DNA harm acceptance in bovine lymphocytes and the Fukushima mishap. DNA harm was assessed by evaluating the degrees of DNA DSB in peripheral blood lymphocytes by immunocytofluorescence-based measurement of γ-H2AX foci (dose range 0.5–5 Gy (microscopy) or 0.5–10 Gy (cytometry)). A more than two-fold increment in the fraction of harmed lymphocytes was seen in all animal cohorts within the evacuation zone. These outcomes set up a clear relationship between exposure and elevated levels of harm to DNA in animals living in the area of the atomic power plant mishap.

#### 6.4.4. Other Options

Other likely biomarkers of IR-induced harm incorporate (but are not restricted to) oxidative stress markers (e.g., 8-hydroxy-2 -deoxyguanosine, isoprostanes and protein carbonyls), immune and inflammatory mediators (various cytokines and chemokines), altered gene expression and mutations (e.g., NF-κB activation, GADD45, CDKN1A, genes related with the nucleotide excision repair mechanism, TP53, PPP1R14C, TNFAIP8L1, DNAJC1, PRTFDC1, KLF10, TNFAIP8L1, Slfn4, Itgb5, Smim3, Tmem40, Litaf, Gp1bb, Cxx1c, FDXR), epigenetic markers (gene methylation and repetitive components), metabolomics-related markers (e.g., urine glyoxylate, threonate, thymine, uracil, citrate,2-oxoglutarate, thymidine, 2 -deoxyuridine, 2 -deoxyxanthosine; blood serum inositol, serine, lysine, glycine, threonine, glycerol, isocitrate, gluconic acid, stearic acid, methylglutarylcarnitine), proteomicsrelated markers (e.g., plasma ferredoxin reductase, α-2-macroglobulin, chromogranin-A, GPx-3, lipidomics-related markers (e.g., blood serum linoleic acid, palmitic acid, phosphatidylcholines, glycerolipids, glycerophospholipids and esterified sterols), and miRNAs (e.g., miR-150, miR-30a, miR-30c, miR-34a, miR-200b, miR-29a, miR-29b, miR-144-5p, miR-144-3p). As of now, the hardware required, the need for automation (see for example [54]), and the absence of explicit investigations of this topic, do not prompt us to recommend any of these choices as satisfactory in a radiological or nuclear mishap scenario.

#### 6.4.5. Networks

The WHO set up in 1987 the REMPAN organization (Radiation Emergency Medical Preparedness and Assistance Network) in light of the tasks assigned to it in the conventions on early notice and help with the event of atomic mishaps, for which the International Atomic Energy Agency (IAEA, https://www.iaea.org, accessed on 1 March 2022) is the responsible association. In 2007, WHO directed an overview of biological dosimetry laboratories and their crisis reaction abilities in chosen districts. The outcomes demonstrated a robust capacity, although there were not many local or public organizations set up. WHO BioDoseNet was then implemented as a worldwide organization of biodosimetry laboratories whose job is to help in management and decision-making in instances of big radiation crisis events where the capacity of individual labs is likely to be overwhelmed. Global biodosimetry networks have been set up, such as the Latin American Biological Dosimetry Network (LBDNet), the organization from Canada and The United States of America (North American BD Network), the Chromosome Network Council coordinated by Japan, the Asian Network of Biological Dosimetry (ARADOS), the Biological Dose Network in China and the European Network for Biological and Retrospective Physical Dosimetry (RENEB). At global level, worldwide organizations have been set up by the WHO (BioDoseNet), the IAEA (within RANET), EURADOS, and the Global Health Security Initiative (GHSI). This speaks to coordinated worldwide action to organise dose assessment. As an illustration of systems management efficacy, the MULTIBIODOSE project (multi-disciplinary biodosimetric instruments to oversee high scale radiological victims), established as a feature of the FP7 Euratom program in May 2010, showed genuinely similar outcomes among various research centers, with reliable dose estimates [389].

Systems management should give: (1) agility, by offering permanent support, every day/365 days a year, (2) prompt admittance to a facilitated server with stand-up capacity for integration, and (3) use of same equipment and supplies, standard operating procedures (SOPs), alignment curves and scoring rules for validated tests. Well built-up coordination can give the upgraded capacity to react to either demand for help from entities without dose assessment capability, or those who may be overwhelmed due to an abrupt surge of patients with suspected or known exposures. Consequently, in the event that one research center gets overwhelmed, tests can be shipped off to different labs within the network with certainty that the dose assessments will be reliable and equivalent.

#### 6.4.6. Advances in Automation

An ideal reaction to radiological or nuclear crises (large-scale specifically) suggests the need for multi-parametric examination combined with a quality assurance/control, speed in collecting samples, high-throughput technology for test planning and analysis, right linkage with clinical triage and treatment surge, and proficient data management frameworks.

The US Biomedical Advanced Research and Development Authority (BARDA) program (https://www.phe.gov, accessed on 7 March 2022) is currently financing two correlative classifications of biodosimetry innovations: (1) A point of care test for a brisk triage of an exposed populace, categorized as having a biological dose above or under 2-Gy to determine the danger of suffering ARS. This test is intended to be managed by an individual with next to zero clinical preparation in a field hospital or triage station. Preferably, results would be accessible in less than 15 min after getting the sample with minimal sample handling. The point of this test is to isolate the people with radiationrelated clinical necessities from the individuals who may not need explicit therapy. (2) A lab based high-throughput assay to quantitatively evaluate the dose retained by a person. The framework being created is fit for evaluating the assimilated dose in the range of 0.5–10 Gy with a superior exactness contrasted with the point-of-care devices [390]. This framework is required to be capable of processing up to 400,000 samples per week with a high level of lab computerization. Automation in obtaining results after sample assortment would in a perfect world be under 8 h. Over time, the utilization of robotized platforms and the improvement of research facility surge capacity networks can help customary cytogenetic

evaluation techniques. In this sense, enhancements incorporate the utilization of barcoded test compartments, mechanical fluid handlers, and computerized metaphase cell collectors, metaphase cell spreaders, slide stainers, and coverslippers [385]. Two primary methodologies have been utilized to diminish the time expected to gauge a dose—first, the work of robotized metaphase finders, and second, decreases in the number of metaphases scored. A few programming platforms have been created for triage management utilizing existing biodosimetry procedures; for example, time to emesis, LDK, and DCA (dicentric chromosome assay) have been implemented for use in the point-of-care setting. The BAT program (see above) is a model. The DCA QuickScan strategy further speeds up scoring [391]. Additionally, the RABIT-II-DCA is a completely computerized DCA in multiwell plates. All activities, from test stacking to chromosome scoring, are performed, without human mediation, by the second generation Rapid Automated Biodosimetry Tool II (RABiT-II) mechanical framework, a plate imager, and custom programming, FluorQuantDic. The framework requires small volumes of blood (30 μL per individual) to appraise the radiation dose received because of a radiation mishap or terrorist assault [392].

The CBMN is a biodosimetric instrument to measure chromosomal harm in mitogenstimulated human lymphocytes. A scanning and image processing system with a robotized micronucleus scoring, the Radometer MN-Series (RS-MN) microscopic system designed by Radosys (Budapest, Hungary, https://www.radosys.com, accessed on 15 March 2022), has been presented for triage [393]. A similar model is the CytoRadx Assay (https://asell.com, accessed on 15 March 2022).

The FAST-DOSE (Fluorescent Automated Screening Tool for Dosimetry) is an immunofluorescent, biomarker-based framework intended to reproduce assimilated radiation dose in blood tests from possibly exposed people. This framework is intended to evaluate intracellular protein changes in blood leukocytes, and has been shown to effectively differentiate beneath or over 2 Gy as long as 8 days after complete body exposure in non-human primates [394].

The G0-PCC (G0-Phase Premature Chromosome Condensation) permits chromosome aberration analysis within the space of hours after blood assortment. Among all deviations, the examination of chromosomal fragments is the fastest [386]. Significantly, this approach holds potential for multi-parametric dosimetry in combination with FISH.

Mechanization of sample analysis by flow cytometry may overcome the time restrictions connected to the utilization of magnifying instrument-based examination (e.g., γ-H2AX identification [395]). This a phosphorylated type of the H2A histone family member X forming when twofold strand breaks show up, and it has been proposed for screening radiation-initiated DNA harm [396].

The DosiKit is a field-radiation biodosimetry immunoassay for fast triage of people exposed to outer TBI, which was validated in human blood cell extracts 0.5 h after in vitro exposure to 137Cs γ rays, utilizing γ-H2AX analysis. DosiKit can appraise absolute body irradiation doses from 0.5 to 10 Gy with a solid linear dose-dependent signal and can be utilized to differentiate possibly exposed people into three dose ranges: under 2 Gy, in the range of 2 to 5 Gy, and above 5 Gy (DCA permits exact estimation of dosages under 5 Gy). The fundamental preferred position is a brisk test that can be performed directly in the field by operational personnel with minimal preparation [397]. The DosiKit framework was completely integrated into a deployable radiological crisis research lab, and the reaction to operational necessities was exceptionally favorable [398].

The REDI-Dx Biodosimetry Test System (https://www.redidx.com, accessed on 15 March 2022) has been created as an in vitro analytic test, which uses blood collected into DxCollect® Blood Collection Tubes (BCT) for the quantitative assessment of the absorbed IR dose. Test outcomes are analyzed with the ABI 3500xL Dx Genetic Analyzer (Rancho Dominguez, CA, USA) and the REDI-Dx Interpretive Analysis Software (Rancho Dominguez, CA, USA), for the gene expression of a set of radiation responsive genes based on the DxDirect® genomic platform. REDI-Dx has been demonstrated to be a good indicator of dosage, for deciding treatment classification dependent on either 2.0 or 6.0 Gy [399].

The HemoDose is a software device, which estimates absorbed doses based on blood counts (https://www.remm.nlm.gov, accessed on 21 March 2022) [400]. The dose assessed by HemoDose dependent on lymphocyte counts and DC chromosome indicated an equivalent correlation with hematological ARS degrees 1 and 4 (in light of the clinical therapy protocols for radiation mishap casualties, METREPOL) [401,402].

#### *6.5. Biophysical Dosimetry* 6.5.1. External Exposure

#### Personal Dosimetry

These devices should be utilized when individuals are in danger of exposure to IR. The chest or stomach locations are predominant. Dosimeters can be additionally positioned in a limb for the scenario where the assessed dose received could be higher in the limb than in the rest of the body [403]. A standard individual dosimeter ought to be fit for giving data on ingested dosages from photons of at least 10 Gy (https://www.iaea.org, accessed on 21 March 2022). Naito et al. [404] estimated individual external doses during the recovery phase in a town after the Fukushima Daiichi atomic power plant mishap. They utilized an individual dosimeter (D-Shuttle) combined with a global positioning system gadget to quantify, and subsequently comprehend, individual outer doses relying upon the resident's area. At the point when a mishap happens outside the controlled territory, exposed people not wearing dosimeters cannot be checked for radiation exposure.

#### Area Monitoring

This is not expected to survey individual doses, but to give a rough estimate of the dose rate at the mishap site [405]. The requirement for exact and fair-minded radiation monitoring is exemplified by the disarray with respect to radiation levels around the Fukushima nuclear site [406]. Examinations may include different likely areas and hotspots for the evaluation of the presence and power of radioactivity in the environment and for the affected people. Region monitors ought to work dependent on explicit measures for the required level of precision, considering the reliance on radiation energy, direction of incidence, temperature, radiofrequency interference, as well as other expected factors.

Instruments used in screening a territory can include: (a) instruments for photons; (b) instruments to detect β particles and low energy photons; (c) instruments for neutrons; (d) passive γ monitors; (e) passive neutron survey meters; and (f) spectrometers (https://www.iaea.org, accessed on 21 March 2022). For example, after the Fukushima fiasco, most information with respect to the diminishing of biological radioactivity was estimated utilizing a NaI (Tl) scintillation meter (Hitachi Aloca Medical, Ltd., TCS-172B), aligned according to the International Electrotechnical Commission's norms (IEC 60846- 1:2009) [407].

#### Dose Reconstruction

Dose reproduction is a review assessment of radiation dose(s) received by recognizable or representative people from a specific exposure. Much of the time, it is the only strategy to assess γ radiation or low dose exposure. Numerous factors, e.g., distance from the source, exposure length, irradiation geometry, and shielding, should be considered in dose assurance, making this technique a tedious system (https://www.icrp.org, accessed on 21 March 2022).

As depicted by [408], scientific issues in radiation dose recreation can be assembled into three distinct classifications: (a) information issues, for example, demographic data, changes in site tasks over the long run, characterization of intermittent versus persistent exposures, and the utilization of colleagues' information; (b) dosimetry issues, for example, strategies for evaluation of exposures, missed dose, unmonitored dose, and clinical radiation dose brought about as a condition of the workplace; (c) explicit issues identified with outer dose, such as affectability, precision and energy reliance of individual monitors, exposure geometries, and ongoing uncertainties. Issues identified with inner dose incorporate sensitivity of bioassay techniques, uncertainties in biokinetic models, suitable dose coefficients, and modelling uncertainties.

As instances of this strategy, Ivanova et al. [409] built up a system for the reproduction of individualized exposure doses for people dwelling at Chernobyl at the hour of the mishap. The strategy depends on the information of radio-biological (ground, meal) and dosimetric (whole body estimations) checking held in Ukraine during the period 1986–2013. Related to the Fukushima atomic mishap, Technical Report No. 162 (2012) gives a thorough record of the estimations and studies undertaken by the Australian Radiation Protection and Nuclear Health Agency (https://www.arpansa.gov.au, accessed on 1 April 2022) to survey the effect on the health of people and the environment in Australia. This report incorporates radiation observations of the environment and seas and testing of imported food and merchandise.

#### Electron Paramagnetic Resonance (EPR)

EPR dosimetry depends on the evaluation by EPR spectroscopy of dose subordinate changes in the concentration of free radicals, defects, or any species with paramagnetic properties that is shaped in a given material under exposure to IR [410]. The capacity of electron paramagnetic reverberation to quantify radiation-derived paramagnetic species, which persist in specific tissues (e.g., teeth, fingernails, toenails, bone, and hair), has made this procedure a noteworthy technique for screening significantly exposed people [411] for dose range 1–30 Gy.

#### Optically Stimulated Luminescence (OSL)

Luminescence signals utilized in dosimetry comprise light emitted under stimulation by a material able to store energy from radiation. Such materials incorporate insulators and semi-conductors [410]. The standard method for estimating OSL is to illuminate the sample with a steady power incitement source and measure the resulting luminiscence (identified as CW-OSL). The OSL signal arrives at the greatest outflow very quickly after the irradiation is turned on, and from that point decays dramatically as the snares are discharged [412]. The essential points of interest when contrasted with EPR are that there is no spectral deconvolution required and the equipment needed is considered less complex and more suited to field events. The applicable dose range is 0.03–10 Gy.

#### Thermoluminescent (TL) Material

TL-detectors were used for cosmic radiation dosimetry in early 1960s, and since then they have been applied in numerous space missions for personal dosimetry, for biological experiments and for medical applications [413].

The TL material has the capacity of storing energy when presented to IR. This energy is re-discharged as visible light when the material is heated to a suitable temperature. The unadulterated materials with ideal grid structure are not considered as TL materials; however, when certain materials are added (which serve as activators), they display thermoluminescence [414]. TL materials can demonstrate the environment radiation dose at the site of a mishap but not the assimilated dose of a victim.

Dose computation utilizing luminescence of solid-state dosimeter has become a significant field of innovative work, and has been effectively applied in territories affected by the Hiroshima and Nagasaki bombs [415], at the Nevada test site [416], and the Semipalatinsk test site [417]. The strategy was additionally utilized for areas affected by the Chernobyl mishap [418] and in contaminated settlements of the upper Techa River in the Southern Urals [419]. The applicable dose range is 0.01 mSv–10 Sv.

#### 6.5.2. Internal Exposure Whole-Body Counters (WBC)

A WBC is a gadget to measure principally γ-rays emitted by radioactive material present in the body, which may vary contingent upon the radionuclide. Alpha particle decaysa can likewise be identified by their coincident γ radiation [420]. Detection can be accomplished by utilizing either a scintillation indicator or a semiconductor locator set in proximity to the body. A fundamental constraint of this strategy is that WBC might not be able to distinguish radioisotopes that have comparable γ energies.

#### Thyroid Monitoring

After the Chernobyl mishap, thyroid malignancy expanded, a problem that was particularly evident among children who had internal exposure to radioiodine through milk consumption [365,421]. This was expected due to the inclination of radioactive iodine to collect in the thyroid and children's thyroids having a higher susceptability to radiation than adults. For that reason, radiological prophylaxis must be dedicated primarily to ensuring that the measure reaches, in optimal conditions, children and young people under 18 years of age, pregnant and lactating women [365].

131I has a short half-life of around 8 days; however, once it enters the body, a high percentage will accumulate in the thyroid, and the gland will thus be directly exposed to β-particles and γ-beams. A reduction of radioiodine uptake into the thyroid can be achieved by administering a large dose of IK (130 mg, in adolescents older than 12 years and adults) shortly before and up to 2 h after, the expected onset of radiation exposure [365,366]. Saturation of the sodium/iodide symporter (NIS) and the Wolff–Chaikoff effect are the main mechanisms involved in transient blockage of 131I uptake in the thyroid [422]. As indicated in Table 1, stable iodine blocks about 98% of 131I thyroid uptake if it is given several minutes before incorporation. If the administration is simultaneous, the efficacy drops to 90%, being of the order of 50% when the iodine is administered 4–6 h later. Administering IK 24 h after exposure can even be counterproductive, because it prolongs the biological half-life of 131I that is already accumulated in the thyroid [365]. A single administration of IK is normally enough, although repetitive administrations might be required in the case of prolonged or repeated exposures. The latter is not recommended in neonates orpregnant and breastfeeding women due to the risk of adverse effects [365,366].

NIS transports other monovalent anions, with the following decreasing activity: TcO4 − > ClO4 − > I− > Br−. Perchlorate has a higher affinity to the NIS than iodide, thus it can be a good alternative in case of iodine sensitivity [365,366]. The Japanese have a delayed responsiveness to iodine transport saturation; thus, potassium perchlorate confers a much better protection in acute 131I exposure. In case of longer or repeated exposures, preference should be given to perchlorate in both Caucasians and Japanese [366].

#### Lung Monitoring

Lung checking is desirable not long after the intake, as it gives a more precise estimation of lung deposition and retention than whole-body estimation [423]. Suggestions have been made for computation of radiation dosages to the respiratory tract of laborers exposed to airborne radionuclides (Human Respiratory Tract Model for Radiological Protection, https://www.icrp.org, accessed on 1 April 2022). The retention is given for the complete body (for example, activity in all compartments of the biokinetic model, including respiratory tract and the thoracic lymph nodes). These capacities are determined several times after an acute intake (for example, inhalation or ingestion).

Lung counting is the sort of in vivo estimation suggested for radionuclides with long residence times in the lung, for example, uranium oxides, plutonium, and 241Am oxides. In vivo estimations of radionuclides in the lung commonly include the detection of X rays as well as photons with energies < 200 keV (https://www.iaea.org, accessed on 1 April 2022).

#### Bioassays

Concerning contamination, a bioassay is characterized as the assurance of amounts of radioactive material in the human body, regardless of whether by direct estimation, in vivo checking, or by measuring materials discharged or eliminated from the human body (for example the utilization of nasal swipes and swabs to evaluate for inhalation; stools and urine to survey for ingestion; and excisional biopsies of cutaneous scraped spots, slashes, and soft tissue wounds to evaluate for transdermal retention or absorption through injuries) [424].

After the Fukushima mishap in 2011, it was decided at the National Radiation Triage Medical Center (NREMC) of the Korea Institute of Radiological and Medical Sciences that mobile units for inward contamination checking would be more effective than the utilization of fixed-type WBCs to screen individuals. Accordingly, the NREMC developed a Mobile Radiobioassay Laboratory (MRL) for fast field-based checking of internal contamination following atomic or radiological crises [425].

#### 6.5.3. Body Surface Contamination

In the early phases of an atomic mishap, it is important to screen the surface radioactive contamination of everyone living and working around the accident site (https://www. irpa.net, accessed on 1 April 2022). Proper survey meters ought to be utilized. In such a situation, radiation type, size of contaminated territory, and strength and compactness of the detector itself are key determinants. Surface contamination is estimated by surface review meters, for example, a Geiger–Müller (G-M) counter, which is valuable for the concentration level, adequacy of the disinfecting method, and skin dose [426]. During evacuation following the Fukushima mishap, evacuees were screened for body surface contamination utilizing a G-M meter [201]. Body surface contamination levels should be related to inhaled thyroid dosages.

#### 6.5.4. Neutron Exposure

The neutron activation strategy is based on the measurement of radiation released by the decay of radioactive nuclei formed by neutron irradiation of the samples (body organs or tissues). At the point when radioactive atoms decay in the sample, γ rays with traceable energies are emitted by each nuclide. Utilizing a γ-ray spectroscope, the amount and energy of radiated γ rays can be estimated. For a mishap associated with neutron emission, neutron activation is the best technique to assessing the dose [204]. Ekendahl et al. [427] assessed neutron exposure to radioactivity in body tissues utilizing samples of human blood and hair dependent on neutron-spectrum calculations.

#### **7. Conclusions**

Biological and biophysical dosimetry is fundamental in distinguishing the individuals who need prompt clinical mediation from those with a possibility for postponed therapy, those who only require long follow up, and those potentially requiring no medical care. However, as of now, biomarkers and procedures do not appropriately fit a triage scenario. As no single biodosimetry method is adequate for dose prediction, time points, and exposure conditions, use of a combination of various methodologies is essential. Biomarker research likewise faces constraints, notably, on experimentation with humans, albeit sometimes the clinical exposure to therapeutic radiation has created informative outcomes. Moreover, most investigations have been performed utilizing a single type of radiation. Nonetheless, e.g., photon and neutron dosages in mixed exposure scenarios ought to be evaluated independently, since this may be key to assessing the danger of radiation-induced clinical syndromes. Lab information is basic to help decision-making, yet biomarker data are not sufficient for a health evaluation, triage, therapy, and clinical management. Research on biomarkers for dose assessment ought to incorporate exposure to mixed field radiation (synchronous exposure to various sorts of radiation), internal exposures from inhalation or ingestion of radionuclides, accumulated serial exposures, and combined injury (as the inflammatory reaction will occur in organs/tissues affected by injuries, which will confound the assessment). Finally, the utilization of radiation biomarkers to anticipate levels of exposure should address the characteristic differences in radiosensitivities across a population. As a supplement to the biodosimetry procedures, we additionally recommend the option of utilizing biobanks to safeguard samples that do not need prompt examination. Consequently, a multiparametric approach based on physical, biological, and clinical strategies appears the most proper decision. For instance, Figure 1 lays outs the dosimetry/assessment measures that, with the available technology, ought to be performed in the event of a nuclear explosion.

**Figure 1.** Stepwise dosimetry and evaluation after a nuclear detonation. (1) Results can be obtained more quickly if automation systems are available. GIS, gastrointestinal syndrome; HS, hematopoietic syndrome; CNS, central nervous system syndrome; mod, moderate; (pb), personal belongings; CM, construction materials. (2) Samples of human blood and hair. These cytogenetic techniques require 48–72 h to process the samples (not indicated). We have focused on the specific case of samples which are processed right after the accident, in a scenario where triage is rapid to avoid deterministic effects. If there is no option, samples can be processed later, and the results used in a retrospective manner. A recognized drawback of the dicentric (DC) and cytokinesis-block micronucleus (CBMN) assays is that the damage is unstable and therefore can be eliminated from the peripheral blood lymphocyte pool. Nevertheless, some lymphocytes containing aberrations continue to exist in the peripheral circulation for many years after an irradiation, although a high dose exposure or a long delay between irradiation and sampling can reduce the aberration yield. Sevan'kaev et al. remark that "the pattern of decline was biphasic with a more rapid first phase, with a half-life of 4 months, followed by a slower decline with half-lives around 2–4 years. It is usually assumed that for biological dosimetry purposes, where delayed sampling requires an extrapolation to zero time, the yield of DIC decreases with a half-life of about 3 years" [428]. Therefore, DIC and CBMN are useful for dose assessment from 2–3 days to 3 years after the accident, whereas FISH, as a stable alteration, has a time window of more years. It is recommended to run these assays within the first year after harmful radiation exposure.

During an accidental radiation exposure, MCM should separate first responders and individuals directly exposed to radiation. Ideal radioprotectors or radiomitigators for such scenarios have not been found. Based on recent advances and studied mechanisms, in this review we have discussed those that in our opinion appear most promising. Radioprotectors include derivatives of aminothiols and cyclic nitroxides (which, despite having toxic effects, have been shown to have greater radioprotective efficacy), natural products (vitamin antioxidants, trace elements such as Se, phytochemicals, etc.), and antioxidant enzyme mimetics, among others. Much more has been achieved in terms of increasing the knowledge about the mechanisms related to RIBE and in reducing side effects of radiotherapy in cancer patients. These advances will help in the development of new radiomitigating strategies. Among the most promising therapies are those aimed at activating recovery of the tissues (cytokines and growth factors), preventing side effects (probiotics, prebiotics, etc.), reducing the inflammatory response (bevacizumab, COXi, angiotensin axis modifying agents, statins) and, thereby, radio-induced chronic side effects such as fibrosis or others. The combination of antioxidant and anti-inflammatory effects of many of them prevents DNA damage and reduces the risk of developing tumors and/or cancers. Some molecules (e.g., melatonin, metformin, curcumin, caffeic acid, bevacizumab, etc.) offer the additional advantage of increasing the efficacy of radiotherapy on cancer cells, and thus can be used as radiosensitizers in cancer cells. Given that none of the tested molecules have total radioprotective/radiomitigating effects, it is evident that more work is needed to implement combined strategies aiming to find synergistic and/or additive effects. The development of new formulations (nanoparticles, nanocrystals, nanovesicles) will facilitate oral administration or the release of said molecules in especially sensitive tissues, thereby contributing to effective radioprotective/mitigating doses and reducing possible toxic effects. In the event of a nuclear emergency, the protocols and time sequence for selecting/caring for the affected exposed, as well as the appropriate treatments (radioprotectants/radiomitigators), including doses and route of administration, need to be implemented. MCM are still assigned FDA orphan drug status, and thus many of these radioprotective strategies could be considered by FDA under "fast track" approval process.

The utilization of complementary tools, preferably shaping parts of automated equipment and established networks, is the future of radioprotection research. Furthermore, in practice, clinical evaluation faces restrictions for large scale screening, i.e., the need of uniquely prepared medical care workers or the low throughput due to the short time available to finish a correct evaluation. Straightforward strategies such as an early lymphocyte count are needed to set up a baseline. However, this may not be conceivable in a large-scale event because of time limitations and the absence of enough technical personnel.

Therefore, despite the many advances discussed in this review, there are many challenges that still need to be addressed to deal effectively with nuclear and radiological accidents.

**Author Contributions:** Conceptualization, E.O. and A.M.; methodology, E.O. and A.M.; software, R.S.-P. and B.P.; validation, E.O., R.S.-P. and A.M.; formal analysis, R.S.-P. and J.I.V.; investigation, all authors; resources, all authors; data curation, R.S.-P.; writing—original draft preparation, E.O., J.M.E. and A.M.; writing—review and editing, E.O. and R.S.-P.; visualization, R.S.-P. and E.G.; supervision, J.M.E.; project administration, E.O.; funding acquisition, E.O. and J.M.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the MINECO (Ministerio de Economía y Competitividad), Spain (grant number SAF2017-83458-R); Elysium Health Inc., NY, USA (grant number OTR2017- 17899INVES); and the Agencia Valenciana de Innovación (grant number INNVA1/2021/22).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

AA, acid ascorbic; ACEi, angiotensin-converting enzyme inhibitors; AFRRI, United States armed powers radiobiology research institute; AngII, angiotensine II; AP sites, apyrimidinic sites; ARADOS, Asian network of biological dosimetry; ARS, acute radiation syndrome; AS, abasic sites; ASA; acetylsalicylic acid; BARDA, United States biomedical advanced research and development authority; BAT, biodosimetry assessment tool; BBB, Blood-Brain Barrier; BBT-059, PEGylated interleukin-11; BCT, Blood Collection Tubes; BD, base damage; CA, caffeic acid; CAPE, caffeic acid phenethyl ester; CAT, catalase; CBCs, complete blood counts; CBMN, cytokinesis-blocked micronucleus assay; COX, cyclooxygenase; CNS, central nervous system syndrome; CRS, chronic radiation syndrome; DC, dicentric chromosome; DPC, DNA-protein cross-links; DSB, double strand breaks; EGCG, Epigallocatechin-3-gallate; EGF, epidermal growth factor; EPR, electron paramagnetic resonance; FAST-DOSE, fluorescent automated screening tool for dosimetry; FDA, Food and Drug Administration; FGF, fibroblast growth factor; FISH, fluorescent in situ hybridization; G-CSF, granulocyte colonystimulating factor; GHSI, global health security initiative; GI, gastrointestinal; GI-ARS, gastrointestinal ARS; GIS, gastrointestinal syndrome; GM-CSF, granulocyte macrophage colony-stimulating factor; G-m, Geiger-Müller; GPx, Glutathione peroxidase; GSH, L-γ-glutamyl-L-cysteinyl-glycine; GSSG, oxidized glutathione; GT3, γ-tocotrienol; H-ARS, hematopoietic acute radiation syndrome; HIF-1α, hypoxia-inducible factor-1α; IAEA, international atomic energy agency; IEC, international electrotechnical commission; IND, improvised nuclear devices; iNOS, inducible nitric oxide synthase; IR, ionizing radiation; JP4-039, Gramicidin *S*-derived nitroxide; KGF, Keratinocyte growth factor; LBDnet, latin American biological dosimetry network; LDK, lymphocyte depletion kinetic assay; LET, linear energy transfer; LSS, Life Span Study; MCM, medical countermeasures; Melatonin, *N*-acetyl-5-methoxytryptamine; MRL, mobile radiobioassay laboratory; NAC, *N*-acetylcysteine; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NIS, sodium/iodide symporter; NREMC, national radiation Triage medical center; Nrf2, Nuclear factor erythroid 2-related factor 2; NRL, neutrophils-to-lymphocytes ratio; OSL, optically stimulated luminescence; PCC, premature chromosome condensation; POC, point of care; PPARs, peroxisome proliferator-activated receptors; RABIT-II, rapid automated biodosimetry tool II; RED, radiation exposure devices; RDD, radiological dispersal devices; REMPAN, radiation Triage medical preparedness and assistance; RENEB, European network for biological and retrospective physical dosimetry; RES, resveratrol; RILI, Radiation-induced lung injury; RNS, reactive nitrogen species; ROS, reactive oxygen species; RS-MN, radiometer MN-series; SALT, Sort Assess Lifesaving Interventions Treatment/Transport; SCFAs, short-chain fatty acids; SOD, superoxide dismutase; SOP, standard operating procedure; SSB, single strand breaks; TNF-α, tumor necrosis factor alpha; TGF-β, transforming growth factor beta; TBI, total body irradiation; TL, thermoluminiscent material; TLRs, Toll-like receptors; TM, thrombomodulin; UroA, urolithin A; VEGF, vascular endothelial growth factor; WBC, whole body counters.

#### **References**


## *Article* **Radioprotective and Radiomitigative Effects of Melatonin in Tissues with Different Proliferative Activity**

**Serazhutdin A. Abdullaev \*, Sergey I. Glukhov and Azhub I. Gaziev**

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russia; s.glukhov@iteb.ru (S.I.G.); gaziev@iteb.ru (A.I.G.)

**\*** Correspondence: abdullaev@iteb.ru; Tel.: +7-(4967)739364

**Abstract:** We used various markers to analyze damage to mouse tissues (spleen and cerebral cortex) which have different proliferative activity and sensitivity to ionizing radiation (IR). We also assessed the degree of modulation of damages that occurs when melatonin is administered to mice prior to and after their X-ray irradiation. The data from this study showed that lesions in nuclear DNA (nDNA) were repaired more actively in the spleen than in the cerebral cortex of mice irradiated and treated with melatonin (N-acetyl-5-methoxytryptamine). Mitochondrial biogenesis involving mitochondrial DNA (mtDNA) synthesis was activated in both tissues of irradiated mice. A significant proportion of the newly synthesized mtDNA molecules were mutant copies that increase oxidative stress. Melatonin reduced the number of mutant mtDNA copies and the level of H2O2 in both tissues of the irradiated mice. Melatonin promoted the restoration of ATP levels in the tissues of irradiated mice. In the mouse tissues after exposure to X-ray, the level of malondialdehyde (MDA) increased and melatonin was able to reduce it. The MDA concentration was higher in the cerebral cortex tissue than that in the spleen tissue of the mouse. In mouse tissues following irradiation, the glutathione (GSH) level was low. The spleen GSH content was more than twice as low as that in the cerebral cortex. Melatonin helped restore the GSH levels in the mouse tissues. Although the spleen and cerebral cortex tissues of mice differ in the baseline values of the analyzed markers, the radioprotective and radiomitigative potential of melatonin was observed in both tissues.

**Keywords:** radiation; melatonin; nDNA-repair; mtDNA-mutations; oxidation stress; protection; mitigation; H2O2; ATP; MDA; GSH

#### **1. Introduction**

Ionizing radiation is often used in the treatment of various tumor diseases. However, healthy tissues may also be damaged by radiation, including the induction of short-term and long-term effects and the appearance of secondary tumors [1]. Medical staff who use IR sources for diagnosis and therapy and professionals involved in the production of nuclear technologies can be exposed to radiation. A significant number of people can be exposed to IR during radiological or nuclear technology incidents or accidents. The impact of cosmic irradiation on astronauts is also a critical factor for space flights outside the Earth's orbit [2]. Therefore, the search for and study of radioprotectors, radiomitigators, and means of treating radiation injuries remain rather topical problems. The development of such drugs has been the focus of attention of radiobiologists and radiologists for decades [3]. Antioxidant compounds account for a significant proportion of preclinical studies of radioprotectors and radiomitigators, since radiation exposure to cells is associated with the induction of prolonged intracellular oxidative stress [4]. Melatonin (N-acetyl-5-methoxytryptamine) was found to be extremely effective among the numerous compounds that passed preclinical tests as radioprotectors as it reduced the in vitro and in vivo effects of IR [5,6]. Currently, melatonin (MEL) is widely used clinically as an adaptogenic drug that normalizes circadian rhythms and is increasingly finding clinical use as an adjuvant in the radiotherapy of tumors [7–9]. According to the analysis of data from a large number of studies, the provisions

**Citation:** Abdullaev, S.A.; Glukhov, S.I.; Gaziev, A.I. Radioprotective and Radiomitigative Effects of Melatonin in Tissues with Different Proliferative Activity. *Antioxidants* **2021**, *10*, 1885. https://doi.org/10.3390/antiox 10121885

Academic Editors: Elena Obrador Pla and Alegria Montoro

Received: 8 October 2021 Accepted: 24 November 2021 Published: 25 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

on the possibility to use melatonin to protect astronauts from hard cosmic irradiation have been substantiated [10]. In this case, it happens that the main risks are mostly associated with the possible consequences of cosmic irradiation's effects on the central nervous system and spleen, which lead to potential neurological disorders, degenerative effects, and decrease in the immune system and affect many aspects of the crew's health [11,12]. As is known, that brain and spleen tissues exhibit different radiosensitivity [3]. Many years ago (in 1906) J. Bergonié and L. Tribondeau proposed a "rule" stating that ionizing radiation is more harmful to cells with a faster turnover. Therefore, there is a relationship between the radiosensitivity and proliferative activity of various tissues [13]. According to this rule, the brain can be considered a radioresistant tissue, and the spleen can be considered a radiosensitive tissue. Today, it is a generally accepted understanding [3]. We can agree with this only based on data on structural disorders and cell death in these tissues, since functional physiological disorders in the brain are observed even under the action of small doses of radiation [14]. It should also be noted that a significant amount of research is devoted to the study of the modulation of radiation damage to the brain under the action of various compounds, including MEL, while similar studies devoted to the modulation of spleen damage are rather limited.

This study is devoted to the comparative assessment on a number of markers, damages in the cerebral cortex and spleen tissues of mice after irradiation of their whole bodies with X-rays, and the modulation of these damages when MEL was administered before and after irradiation. Nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) damage and repair, change in the number of mtDNA copies, H2O2, ATP, reduced glutathione (GSH) as a marker of the antioxidant system, and malondialdehyde (MDA) as a marker of oxidative stress were used as markers.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

All chemicals were of the "high purity" category from the Alamed company, Moscow, Russia and from the Sigma-Aldrich company, St. Louis, MO, USA. All solutions were prepared in deionized water obtained from the Milli-Q system (Millipore, Bedford, MA, USA). Melatonin (MEL) was obtained from Sigma-Aldrich, St. Louis, MO, USA.

#### *2.2. Animals and Their Irradiation*

Male mice C57BL/6 at the age of 2 months weighing 20–22 g were obtained from Stolbovaya nursery (Settlement Stolbovaya; Moscow, Russia). The mice were used in experiments after 7 days of acclimatization in the animal room. All experiments with animals were performed in accordance with the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes, Directive 2010/63/EU. The protocol was approved by the Committee on Biomedical Ethics of the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences/the Physiology Section of the Russian Committee on Bioethics (Protocol N◦ 20 dated 9 February 2021). The animals were fed a special diet for mice and rats and had free access to clean drinking water. The animals were irradiated at the Research Equipment Sharing Center, a group of radiation sources of the Institute of Cell Biophysics of the Russian Academy of Sciences, on a RUT-250-15-1 X-ray machine (280 kVp, 20 mA) with AL and Cu filters of 1 mm with a dose rate of 1 Gy/min. The animals were irradiated in plastic containers at a dose of 5 Gy. The irradiation of mice was carried out for 5 min.

#### *2.3. Administration of Melatonin to Mice and Collection of Tissues for Analysis*

A freshly prepared MEL solution was used for administration. To do this, 250 mg of MEL was dissolved in boiled drinking water (at room temperature) containing 0.1% dimethyl sulfoxide (DMSO). The final concentrations of this solution were 2.5% MEL and 0.1% DMSO. Mice were orally treated with 100 μL of this solution, corresponding to doses of MEL of 125 mg/kg and DMSO of 0.1 mg/kg of a mouse's body weight [15]. A 0.1% DMSO solution was also prepared separately for administration to control groups of mice. The solutions were administered to groups of mice 30 min before irradiation or 20 min after irradiation. Each individual analysis group consisted of 5–6 mice. The preparation was additionally injected into drinking water (0.3 mg/mL) within 24 and 48 h for mice that were treated with MEL after irradiation, given the short clearance of MEL [15]. To isolate the cerebral cortex and spleen tissues, mice were sacrificed by decapitation 15 min and 24 and 48 h after irradiation. Groups of unirradiated and irradiated mice not treated with MEL were used as controls. The spleen and brain tissue (cortex) were separated with a scalpel on ice immediately after decapitation, then were frozen and stored at −80 ◦C until analysis.

#### *2.4. DNA Isolation and Purification*

Tissues were homogenized in a glass homogenizer and DNA was isolated using the QIAGEN Genomic-tip Kit and Genomic DNA Buffer (QIAGEN, Hilden, Germany). The amount of DNA in all cases was determined by its reaction with the PicoGreen reagent according to the manufacturer's protocol (Molecular Probes Inc., Eugene, OR, USA) and fluorescence was registered on an NanoQuant Infinite M200 instrument (Tecan Group Ltd., Grödig/Salzburg, Austria). DNA samples for mitochondrial genome PCR-analysis were incubated within 20 min at 25 ◦C in TE buffer with XhoI restriction endonuclease (New England Biolabs, Ipswich, MA, USA). XhoI endonuclease initiates a break at the site of the CTCGAG hexamer of the supercoiled mtDNA outside the amplified region and leads to relaxation of the mtDNA, making the selected region available for PCR [16].

#### *2.5. Analysis of Damage and Repair of Mitochondrial DNA and Nuclear DNA*

To determine the damage and repair of nDNA and mtDNA, we used the long amplicon quantitative polymerase chain reaction (LA-QPCR) method [17] taking into account our previous experience [18]. In these analyses, we used (2U/μL) KAPA Long Range Hot Start Kit (KAPA Biosystems, Humboldt County, CA, USA). LA-QPCR was used to amplify a 8.7 kb region of nDNA and 10.9 kb of mtDNA. For amplification of a long fragment of mtDNA (10.9 kb), the standard thermocycler program included initial denaturation at 94 ◦C for 5 min, with 18 cycles of 94 ◦C for 30 s and 68 ◦C for 12.5 min, and with a final extension at 72 ◦C for 10 min. To amplify a long fragment of nDNA (8.7 kb), the thermocycler profile included initial denaturation at 94 ◦C for 5 min, and 28 cycles of 94 ◦C for 30 s and 68 ◦C for 12 min, with a final extension at 72 ◦C for 10 min. Preliminary assays were carried out to ensure the linearity of PCR amplification with respect to the number of cycles and DNA concentration. Since the amplification of a small region would be relatively independent of oxidative DNA damage (low probability), a small DNA fragment for nDNA (110 bp) and for mtDNA (117 bp) was also amplified for normalization of the data obtained with the large fragments, as described previously [18,19]. PCR analyses were performed in triplicate for each DNA sample. All of the amplified products were resolved and visualized using agarose gel electrophoresis and quantitated with an Image Quant (Molecular Dynamics, Waukesha, WI, USA) or VarsaDoc (Bio-Rad, Hercules, CA, USA). The data were plotted as histograms with relative amplification, such as the *y*-axis, which was calculated by comparing the values of exposed samples with the control. All primers are presented in Table 1.



#### *2.6. Quantitative Analysis of Mitochondrial DNA Copies Relative to the Nuclear DNA*

Quantitative analysis of mtDNA was carried out by real-time PCR with TaqMan oligonucleotides on a Prism 7500 thermal cycler (Applied Biosystems, Foster City, CA, USA) [20]. The changes in the relative quantity of mtDNA with respect to nDNA were determined as a ratio between the number of copies of the mitochondrial *ND4* gene and that of the *GAPDH* gene of nDNA in the same test tube. The 2−ΔΔCT method was used for analysis. PCR tests were carried out in triplicate for each DNA sample. The following PCR program was used: 5 min at 95 ◦C followed by 40 cycles (95 ◦C for 30 s, annealing and elongation at 60 ◦C for 1 min). The results are presented as a percentage of data compared to unirradiated mice (taken as 100%). The PCR primers used in this study are given in Table 1.

#### *2.7. Surveyor Nuclease Assay of mtDNA Mutant Copies*

To evaluate the relative level of mutant copies of mtDNA isolated from brain tissue, we used the Surveyor® Mutation Detection Kit (Transgenomic, Omaha, NE, USA), as described in [21,22]. To estimate mutations in mtDNA, a region including the *ND3* gene (534 bp) was chosen for amplification. The PCR primers employed in this study are given in Table 1. PCR was carried out by a programmed thermocycler Thermal Cycler 2720 (Applied Biosystems, Foster City, CA, USA). PCR was performed in a 25 μL volume containing 1.0 ng of total DNA, 75 mM of Tris-HCl, a pH of 8.8, 20 mM of (NH4)2SO4, 2.5 mM of MgCl2, 200 μM of each dNTP, 250 nM of each primer, 0.01% tween-20, and 1.0 unit of total mixture of Taq and Pfu polymerases (Thermo Scientific, Pittsburgh, PA, USA). PCR was initiated by a "hot start" after initial denaturation for 4 min at 94 ◦C. The amplification was carried out in 40 cycles under the following conditions: 30 s at 94 ◦C, 30 s at 62 ◦C, and 1 min at 72 ◦C; the final extension step of 4 min was at 72 ◦C. After the PCR was completed, all amplification products were diluted to an equal concentration. To obtain heteroduplex DNA, equal volumes (7 μL) of PCR products of mtDNA amplification from control and exposed mice were mixed. The mixtures were heated at 95 ◦C for 10 min and cooled slowly to 40 ◦C for 70 min at a rate of 0.3 ◦C/min. Then, 1/10 volume of 0.15 M MgCl2 solution, 1 μL of Surveyor Enhancer S, and 1 μL of Surveyor Nuclease S were added to the heteroduplex mixture. The mixture was incubated at 42 ◦C for 60 min. The reaction was stopped by adding 1/10 volume of stop solution. Nuclease digestion products were

analyzed by electrophoresis in a 2.0% agarose gel stained with ethidium bromide. PCR tests of heteroduplexes were carried out in triplicate for each DNA sample. The fluorescence intensity of DNA bands in the gels was registered by the AlphaImager Mini System (Alpha Innotech, Santa Clara, CA, USA). The ratio of the cleavage products' fluorescence to the total intensity of fluorescence of DNA bands in the gel (% of Surveyor Nuclease cleaved DNA) was calculated using the ImageJ software package (Wayne Rasband, Kensington, MD, USA).

#### *2.8. Determination of Hydrogen Peroxide Level*

A Fluorimetric Hydrogen Peroxide Assay Kit 165 (Sigma-Aldrich Co., St. Louis, MO, USA) was used for the quantitative measurement of hydrogen peroxide (H2O2) in mice tissues. This kit uses peroxidase substrate that generates a red fluorescent product that can be analyzed in 96-well black transparent bottom microplates. All analyses were performed in accordance with the recommendations of the manufacturer. The amount of H2O2 was calculated on the basis of a standard curve obtained using a concentration range of an H2O2 solution obtained by diluting a 30% H2O2 solution with ultrapure water. Each test sample was run in triplicate. Data were obtained from 6 mice in each group. The amount of H2O2 was expressed in nmol per mg of protein using a standard curve. Protein was assessed in these and other analyses by the method of Lowry et al. [23] using bovine serum albumin as a standard.

#### *2.9. ATP Analysis*

The ATP content was determined following the recommendations indicated in [24]. ATP was extracted from tissue homogenates after the removal of proteins with TE buffer saturated with phenol. ATP level was measured using a luciferin–luciferase kit with a Glo-Max 96 Microplate Luminometer (Promega, E6521, Madison, WI, USA). ATP concentration was assessed using a standard curve in nmol per mg of protein. Data were normalized to total protein, and tissue ATP levels were expressed in μmol per 100 mg of protein.

#### *2.10. Determination of Lipid Peroxidation*

The lipid peroxidation level was judged by changes in malondialdehyde (MDA) content after reaction with thiobarbituric acid (TBA) by the method of Buget and Aoust [25]. For this purpose, the cerebral cortex and spleen tissues of mice were homogenized in lysis buffer (50 mM Tris-Cl, 1% NP-40, 0.2% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 1 mM EDTA). Then, one volume of tissue lysate was mixed with two volumes of TBA reagent (15% TCA, 0.375% TBA, and 0.25 N HCl), followed by incubation at 90 ◦C for 30 min. After cooling, the reaction mixture was centrifuged at 10,000 rpm for 15 min. The supernatant absorbance was measured at 533 nm with respect to the blank. The amount of lipid peroxidation was calculated from the MDA level in nmol per milligram of protein (nmol/mg of protein).

#### *2.11. Determination of Glutathione Level (GSH)*

Tissues were homogenized in lysis buffer (50 mM Tris-Cl, 1% NP-40, 0.2% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 1 mM EDTA) as indicated in the determination of lipid oxidation [25]. A total of 1.8 mL of 0.05 M EDTA and 3 mL of a precipitator (containing 1.67 g of HPO3, 0.2 g of disodium EDTA salt, and 30 g of NaCl per liter of water) were added to 0.2 mL of tissue homogenate. After thorough mixing, the solution was kept for 5–7 min and then centrifuged. This step promotes the separation of GSH (in the supernatant) from the rest of the proteins and other cellular elements (in the sediment). Then, two volumes of 0.3 M Na2HPO4 solution and 0.5 volumes of 4 mM DTNB (5,5 -dithiobis-2-nitrobenzoic acid) were added to one volume of the supernatant [26]. Absorbance was determined at 412 nm against a mixture of solutions without biomaterial additives (blank). GSH was expressed in nmol per mg of protein using a standard curve.

#### *2.12. Statistical Analysis*

All numerical results are expressed as the mean ± SEM of 5–6 independent experiments and *p* < 0.05 was considered statistically significant. The statistical analyses were performed using GraphPad Prism 8.0 software (San Diego, CA, USA).

#### **3. Results**

#### *3.1. Damage and Repair of Nuclear DNA and Mitochondrial DNA following Irradiation*

**1.** As established in a number of studies, exogenous melatonin is a powerful antioxidant and has *in vitro* and *in vivo* radioprotective and radiomitigator effects [5,6]. Melatonin also exhibits a wide range of antioxidant defense reactions at various cellular levels. It helps to reduce oxidative stress caused by active forms of oxygen and nitrogen (RONS) and acts as an absorber of free radicals [27,28]. Therefore, it is of interest to elucidate changes in the most important markers of radiation damage in tissues with different radiosensitivity and proliferative activity in animals when they are administered with melatonin. In our study on mice, the spleen and cerebral cortex were taken as such tissues. In this study, mice were irradiated on a RUT-250-15-1 X-ray machine (280 kVp, 20 mA) with AL and Cu filters of 1 mm with a dose rate of 1 Gy/min. As is known, the most important marker of radiation exposure to living organisms is DNA damage. To determine nDNA and mtDNA damage, we used the method of quantitative PCR with a long amplicon (LA-QPCR) [17]. The presence of damage such as modified bases, single-strand and double-strand breaks, or DNA–protein crosslinking can block the activity of KAPA Biosystems' DNA polymerase. Thus, this method allowed us to assess the overall level of DNA damage.

**2.** The amplification products of long sections of nDNA and mtDNA from the tissues of unirradiated mice were taken as 100% control. It can be seen that the level of synthesized products of nDNA and mtDNA LA-QPCR from the mice's spleens and cerebral cortexes 15 min after irradiation was significantly lower than that of the unirradiated mice (Figure 1). Such a reduction in LA-QPCR products indicates that these amplifiable areas of nDNA and mtDNA contained damages capable of blocking KAPA Long Range Rapid PCR DNA polymerase (KAPA Biosystems, Wilmington, MA, USA). The preservation of low levels of amplification of nDNA and mtDNA regions indicates the presence of non-repaired damages in them. However, there was an increase in LA-QPCR products by 24 and 48 h post-radiation time, which indicates the functioning of DNA damage repair processes. According to the results obtained, nDNA and mtDNA from the tissues of mice treated with MEL before IR-radiation and after irradiation had significantly less damages capable of blocking KAPA Long Range DNA polymerase. As can be expected, this shows that MEL contributes to the DNA damage reduction (Figure 1). nDNA and mtDNA from the tissues of mice treated with MEL before irradiation and after irradiation had significantly less damages capable of blocking KAPA Long Range DNA polymerase. As can be expected, this also shows that MEL contributes to the DNA damage reduction. The results obtained show that the nDNA repair occurs more actively in the spleen and cerebral cortex tissues of mice treated with MEL after irradiation. When comparing the LA-QPCR amplification data of nDNA from two tissues of mice irradiated and treated with MEL, it seems that the nDNA repair in the spleen tissue for the indicated periods of post-radiation time was more active than in the cerebral cortex (Figure 1).

**3.** According to the experiment results, we can also conclude that mtDNA in the spleen and the cerebral cortex was actively restored, especially in mice that were treated with MEL after irradiation (Figure 1). However, if the increase in the synthesis of the LA-QPCR product of nDNA during the post-irradiation period was due to the repair of nDNA damages that inhibited KAPA Long Range DNA polymerase, this is unlikely to be the reason for the sharp increase in the synthesis of LA-QPCR products of mtDNA from the same tissues of mice. It is known that only base excision repair (BER) effectively functions in mammalian mitochondria [29]. Other pathway of repairing mutagenic mtDNA damage do not function in mammalian mitochondria. Moreover, double-strand breaks (DSBs) of mtDNA in mammalian cells are not repaired [30,31], and damaged mtDNA can undergo degradation [32]. In this experiment, we most likely registered the activation of mitochondrial biogenesis with the mtDNA synthesis (Figure 1). To test this assumption, we decided to continue experiments to elucidate the effect of MEL on the quantitative content of mtDNA relative to nDNA in the spleen and cerebral cortex tissues of mice exposed to radiation.

**Figure 1.** Analysis of damage and repair of nuclear DNA and recovery of mitochondrial DNA. Long fragments of nDNA (8.7 kb) and mtDNA (10.9 kb) were measured. These data were normalized by the measured levels of the short fragment of nDNA (110 bp) and mtDNA (117 bp), obtained using the same DNA sample. (**A**) Quantitative analysis of the LA-QPCR amplicons of nDNA extracted from spleen and cerebral cortex. (**B**) Quantitative analysis of the LA-QPCR amplicons of mtDNA extracted from spleen and cerebral cortex. Data are presented in % to control (C). Here and in other figures: the dose of X-ray irradiation of mice was 5 Gy and MEL was administered to mice before and after irradiation as a single dose of 125 mg/kg. Electropherogram samples of synthesized amplicons are presented above the histograms. The numbers (15 min, 24 h, 48 h) above and below indicate the time after irradiation. I—mice without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05.

#### *3.2. Effect of Melatonin on Mitochondrial Biogenesis in Tissues of X-Irradiated Mice*

A change in the number of mtDNA copies or the ratio of mtDNA/nDNA is the most important criterion for assessing mitochondrial biogenesis in tissues or cells [30,31]. The results of the analyses obtained by the real-time PCR method show that the number of mtDNA copies increased in the spleen and cerebral cortex tissues of mice 24 and 48 h after their irradiation with a dose of 5 Gy in comparison with the data of the control (non-irradiated) animals group (Figure 2). As judged from the number of mtDNA copies, the enhancement of mtDNA synthesis was more pronounced in the spleen tissue than in the cerebral cortex of the irradiated mice. It should also be noted that the content of mtDNA in the tissues of mice, for a 15-min time after irradiation, remained at the level of the data from the control non-irradiated mice. These results indicate that mtDNA synthesis and, accordingly, mitochondrial biogenesis were activated much later; we registered their increase 24 and 48 h after irradiation. At the same time, we can see that when MEL was administered, the synthesis of mtDNA molecules occurred less actively than in the data obtained in irradiated mice without the administration of MEL. This gives the impression that MEL partially suppresses IR-induced mtDNA synthesis in the tissues of the spleen and cerebral cortex. In fact, most likely, this is the result of a decrease in the level of RONS generated by dysfunctional mitochondria under the influence of MEL. At the same time, the inhibition effect of IR-induced mtDNA synthesis upon administration of MEL to animals after irradiation was more pronounced in comparison with the data of the group of mice treated with MEL before irradiation. Based on the data obtained, it can be assumed that upon initiation of replicative synthesis involving a damaged mtDNA template and with the participation of DNA polymerase γ and DNA polymerase θ in mitochondria [32,33], the appearance of new copies of mtDNA with mutations and deletions in the tissues of mice after irradiation with IR can be expected.

**Figure 2.** Ratio of mtDNA/nDNA in the tissues of the spleen and cerebral cortex of mice after their irradiation. The *y*-axis shows the percentage (%) of the change in mtDNA to nDNA ratio relative to control. The numbers (15 min, 24 h, 48 h) on *X*-axis indicate the time after irradiation. I—mice without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05.

#### *3.3. Analysis of Mitochondrial DNA Mutant Copies*

As noted above, with the exception of BER, other DNA repair pathways are not involved in repairing mtDNA damage in mammalian cells [26]. Therefore, the observed increase in the number of mtDNA copies in the tissues of irradiated mice (Figure 2) suggested that it was associated with increased mtDNA mutagenesis. Our subsequent analyses confirmed this assumption. Electropherograms of the Surveyor nuclease digestion products of mtDNA PCR amplicon heteroduplexes and their quantitative analysis are shown in Figure 3. The quantitative analysis of the cleavage products of heteroduplexes showed that the level of mtDNA mutant copies significantly increased in the spleen and cerebral cortex tissues of mice within 24–48 h after irradiation (Figure 3B). The number of mutant copies in the spleen tissue increased to 30% by 48 h post-radiation time, and it also increased to 20% in the cerebral cortex tissue relative to the control. On the other hand, the data from the analysis of the mtDNA mutant copies number in the tissues of mice treated with MEL before and after irradiation were significantly lower than those from mice that were not treated with MEL. It should also be noted that a significant decrease in the mtDNA mutant copies number was recorded, as can be seen, in the cerebral cortex tissue when MEL was administered into mice after irradiation in comparison with data from the spleens of groups of irradiated mice that were treated with MEL.

**Figure 3.** Detection of mtDNA mutant copies of spleen and cerebral cortex tissues in mice 15 min, 24, and 48 h after X-ray irradiation. (**A**) Electrophoresis of cleavage products obtained by Surveyor nuclease digestion of heteroduplexes of mtDNA PCR amplicons from spleen and cerebral cortex tissues. (**B**) Percentage of Surveyor nuclease cleaved heteroduplexes of PCR amplicons of mtDNA (ND3 gene, 534 bp). I—mice without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05, \*\* *p* < 0.01.

#### *3.4. Changes in H2O2 Content in Tissues of X-Irradiated Mice*

As is known, mitochondria and a number of extramitochondrial oxidases generate various reactive oxygen and nitrogen species (RONS). However, not all RONS can diffuse through the membranes of mitochondria or other organelles and reach the cell nucleus, since most of them migrate only over short distances. H2O2 molecules are the most stable and capable of migrating over long distances (1 μm or more) [34,35]. Therefore, we decided to determine changes in oxidative stress in the spleen and cerebral cortex tissues of irradiated mice by the level of hydrogen peroxide. The analysis results are shown in Figure 4. The data show that H2O2 production increased more sharply in the spleen tissue of mice during 24–48 h of the post-radiation period. At the same time, with the introduction of MEL, the level of H2O2 in the spleen significantly decreased. In the tissue of the cerebral cortex, the tendency for changes in the content of H2O2 is the same as in the spleen, but less pronounced. Here (Figure 4) it can be seen that, after the irradiation of mice, the H2O2

level increased immediately after 15 min and this level remained for 24 and 48 h. At the same time, we observed a decrease in the H2O2 level after only 48 h in the cerebral cortex tissue of mice treated with MEL after irradiation.

**Figure 4.** Changes in the H2O2 content in spleen and cerebral cortex tissues of mice 15 min, 24, and 48 h after their exposure to X-rays. I—mice groups without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05; \*\* *p* < 0.01.

#### *3.5. Changes in ATP Content in Tissues of X-Irradiated Mice*

Maximum energy support is required for DNA repair and cell recovery. This can ensure the synthesis of ATP in functionally active mitochondria [36]. Therefore, it is very important to evaluate the change in the ATP content in the tissues of irradiated mice and the effect of MEL on the correction of its synthesis level. The results of our analyses show that the content of ATP in the spleen tissue was approximately two times less per unit mass of tissue compared to its content in the cerebral cortex tissue of control and irradiated mice (Figure 5). The observed difference was obviously due to the unequal content of mitochondria in these tissues. Nevertheless, the post-radiation changes in the ATP content in both tissues were relatively similar. It can be seen that the ATP content in both tissues sharply decreased in the initial period after irradiation, especially after 15 min. However, a tendency towards restoration of the ATP content in both tissues of the irradiated mice was observed after 24 and 48 h of post-radiation time. Moreover, the restoration of the ATP content in the tissues of the mice that were treated with MEL before and after irradiation was more active. This is best seen in the results obtained on the cerebral cortex tissues. Thus, we can conclude that MEL contributes to the maintenance of mitochondrial functions and the synthesis of the required level of ATP in the spleen and cerebral cortex tissues of irradiated mice.

**Figure 5.** Changes in the ATP content in spleen and cerebral cortex tissues of mice 15 min, 24, and 48 h after their irradiation. I—mice groups without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05; \*\* *p* < 0.01.

#### *3.6. Changes in MDA Content in Tissues of X-Irradiated Mice*

In radiation biology, an increase in the level of the lipid oxidation product malondialdehyde (MDA) in cells or tissues is considered as one of the most important markers of radiation damage. This marker indicates the occurrence of oxidative stress.

The results of our analyses gave quite different results of the content of MDA in the tissues of the spleen and cerebral cortex of mice exposed to X-rays (Figure 6).

**Figure 6.** Changes in the MDA content in spleen and cerebral cortex tissues of mice 15 min, 24, and 48 h after their exposure to X-rays. I—mice without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05, \*\* *p* < 0.01.

Administration of MEL to mice before and after irradiation promoted a significant decrease in MDA in the spleen tissue. Similar results were obtained by MDA analyses in the cerebral cortex tissue of the same mice. However, the results of the brain tissue analyses were quantitatively different from those of the spleen tissue analyses. First of all, the MDA level in the cerebral cortex tissue was higher in comparison with the data of the spleen analyses. Moreover, the administration of MEL to mice before and after irradiation in the brain tissue retained an increased content of MDA, although it was significantly lower than that in the analysis data from the tissues of mice that were not treated with MEL. It can also be noted that the data obtained from MDA analyses both in the spleen tissue and in the brain tissue of irradiated mice that were treated with MEL after irradiation were lower than the results obtained in the tissues of mice that were treated with MEL before irradiation.

#### *3.7. Changes in Glutathione Content in Tissues of X-Irradiated Mice*

Reduced glutathione (GSH) is an essential non-enzymatic antioxidant that plays a prominent part in determining cell radiosensitivity. A decrease in the content of GSH in tissues or in the blood is considered as a marker of a decrease in the level of antioxidants in the body as a result of radiation exposure. The results of our analyses show that there was a sharp decrease in glutathione in the spleen and cerebral cortex tissues of mice after irradiation of the whole body with X-rays (Figure 7). These data also show that the content of GSH in the spleen was more than two times less than that in the cerebral cortex tissue. At the same time, reduced levels of GSH were retained in both tissues during the postradiation time (up to 48 h). We observed an active increase in the content of reduced GSH in the tissues of these mice only after oral administration of MEL to mice before or after irradiation. At the same time, the results show that the restoration of GSH level in the cerebral cortex tissue occurred more actively in mice treated with MEL after irradiation.

**Figure 7.** Changes in the GSH in spleen and cerebral cortex tissues of mice 15 min, 24, and 48 h after their irradiation. I—mice without MEL administration; II—MEL administration before irradiation; III—MEL administration after irradiation. The data are presented as mean ± SEM of 5–6 independent experiments. Statistical significance was set at \* *p* < 0.05, \*\* *p* < 0.01.

#### **4. Discussion**

In a normally functioning cell, DNA is constantly subject to oxidation and "spontaneous" hydrolytic degradation [37]. RONS cause a lot of damage in DNA, including base modifications, the destruction of deoxyribose, formation of apurinic/apyrimidinic sites and single-strand breaks (SSBs) [38]. In addition, double-strand breaks (DSBs) can also form in DNA as a result of a close match of SSBs or in the process of repair of closely spaced damaged bases on complementary strands of a double helix [39]. When IR is exposed to cells, DNA damage is induced much more (depending on the dose of IR). Moreover, there is a sharp increase in the production of RONS in irradiated cells, which can last from several minutes to tens of days, depending on the radiation dose [40]. Therefore, supporting the activity of DNA repair systems and the level of antioxidants play a crucial role in the fate of the irradiated organism. It is obvious that in the regulation of these processes, along with other protective systems of the cell, melatonin can play a primary role [41].

The review article by Galano et al. [41] analyzes the data of many studies on the role of MEL in protecting DNA from oxidative damage. It is shown here that MEL provides cleaning of free radicals and other forms of RONS from cells and activates enzymes involved in the BER. MEL activates the expression of genes encoding DNA repair enzymes and antioxidant enzymes, but suppresses the activity of pro oxidant enzymes. Thus, it is clear that MEL provides protection of the nuclear genome in different directions [41].

It has recently been reported that MEL not only protects DNA, to a large extent, from mutagenic damage, but also from the induction of DNA DSBs, which are lethal events for the cell if they are not repaired. Thus, in patients undergoing computed tomography (CT), DNA DSBs induction was recorded in blood lymphocytes. Moreover, in the group who received a single oral dose of 100 mg of MEL 5–10 min before and 30 min after CT examination, these DNA damages was not recorded [42]. These results are confirmed in another study. The authors observed DNA DSBs in lymphocytes when exposed to IR at doses of 10 mGy and 100 mGy. Administration of 100 mg of MEL to patients before irradiation caused a decrease in DNA DSBs levels [43]. In another study, when incubating human blood lymphocytes in an environment with the addition of radioactive iodine I131 for 2 h in the presence of MEL, the number of induced DNA DSBs decreased by 40% relative to the control (lymphocytes incubated with I131 without MEL) [44].

As described above, for a comparative assessment of damage and repair of nDNA and mtDNA in different tissues, we used the method of quantitative PCR with a long amplicon (LA-QPCR) [17]. The presence of damage such as modified bases, SSBs, DSBs, or DNA– protein crosslinking can block the activity of KAPA Biosystems DNA polymerase and, accordingly, reduce the PCR synthesis product. The results of our analyses indicate that the repair of nDNA total damage capable of blocking KAPA Long Range DNA polymerase in the spleen and cerebral cortex of irradiated mice proceeds rather slowly within 48 h after total body irradiation, and occurs more slowly in the brain cells (Figure 1). It is known that in postmitotic cells, different DNA repair pathways are less active than in dividing cells [45]. Recently, it was shown that after irradiation of the rat head with X-rays, DSBs nDNA in the cortical neurons persisted for a long post-radiation time [46]. We also recently reported that DNA damage repair in irradiated rats is slower in cortical tissue than in hippocampal tissue [18].

The spleen is an organ of the reticuloendothelial system with proliferative activity [47]. The nDNA damage repair in the spleen tissue is more active, although it could not be completed by 48 h without the administration of MEL.

It is possible that the observed slow DNA repair within 24–48 h in the tissues of irradiated mice without the introduction of MEL was due to the occurrence of additional damage in the same DNA. These additional damages may occur as a result of the action of RONS, generated in the dysfunctional mitochondria of the same cells. With the introduction of MEL, obviously, there is a significant cleaning of these RONS. Exposure to ionizing radiation can not only cause acute radiation syndrome, but also increase the risk of developing long-term consequences. IR stimulates RONS production by mitochondria for a few hours to a few days after irradiation. This prolonged RONS generation in mitochondria can induce additional damage to nDNA and mtDNA cells after radiation exposure [40]. It has long been established that the cause of increased oxidative stress in the cells of irradiated mammals is mitochondrial dysfunction [48]. At the same time, the antioxidant activity in the tissues and blood of irradiated rodents sharply decreases [49,50].

We found increased mtDNA synthesis in mouse tissues after irradiation, clearly associated with mitochondrial biogenesis. This well-known phenomenon is the induction of biogenesis with the synthesis of mtDNA under radiation exposure to the cells of animals [51–53]. It is caused by the occurrence of mitochondrial dysfunction, increased oxidative stress, and a decrease in ATP synthesis, along with the emergence of increased energy needs in damaged cells.

As noted above, the processes of mtDNA damage repair occur with low efficiency in mammalian mitochondria. In these organelles, only the pathway of the BER functions efficiently [29]. The results of our analyses (Figure 2) showed an increased mtDNA synthesis in the spleen and cerebral cortex cells by 24–48 h after irradiation. As might be expected, the activity of mtDNA synthesis decreased in both tissues when mice were treated with MEL before and after irradiation, which lowered the RONS content. Various mammalian tissue cells may exhibit tissue-specific features in the activation of mitochondrial biogenesis and mtDNA synthesis, associated with their activity in the generation of ATP and RONS [54].

The subsequent results of our studies showed that in the post-radiation mitochondrial biogenesis, some of the synthesized mtDNA molecules were mutant copies. Obviously, mutations in the newly synthesized mtDNA molecules appeared during the replication of damaged mtDNA matrixes with the participation of DNA polymerase γ and DNA polymerase θ in mitochondria [35,36]. The increased levels of mtDNA mutant copies were observed in the spleen and cerebral cortex tissues of irradiated mice; however, their number significantly decreased upon the administration of MEL before and after irradiation (Figure 3). Therefore, it can be assumed that the antimutagenic effect of MEL is due to both the interception of initial RONS (the administration of MEL before irradiation) and the neutralization of RONS generated in the cells of irradiated mice (the administration of MEL after irradiation) [55]. These results are consistent with the previously obtained data of Tan et al. [56], who concluded that MEL protects mitochondria, has a regulatory effect on mitochondrial biogenesis and dynamics, and contributes to the preservation of the functions of these organelles. The increased level of mutant mtDNA copies in mammalian tissues after irradiation is due to both the low efficiency of the mtDNA repair systems and the effect of RONS; their production in the mitochondria of mammalian cells can continue over a long post-radiation period [40].

The results of a number of studies show that the mitochondrial dysfunction detected in human and animal cells after irradiation is largely associated with the induction of mutations in mtDNA genes encoding proteins of electron transport chain complexes, which continue to operate with the overproduction of RONS [57,58]. Obviously, the expression of mtDNA mutation genes leads to the synthesis of aberrant proteins. The latter can lead to perturbation of the oxidative phosphorylation system in mitochondria, with prolongs the increased generation of ROSNS and increased oxidative stress. This causes even more damage to macromolecules in the organelles and the entire cell, including nDNA. A "vicious cycle" is formed for a long period. This cycle operates in the various mammalian tissue cells at different rates and leads to the differential accumulation of mutant mtDNA copies, which, in turn, increase oxidative stress for a long post-radiation period. Thus, it can be assumed that, when IR is exposed to mammalian tissues, mitochondria containing mtDNA mutant copies become dysfunctional with enhanced RONS generation, which supports the induction of additional nDNA damage and genome instability of surviving cells, the development of degenerative diseases, aging, and oncogenesis for a long post-radiation period [59]. The mitochondrial respiratory chain is considered to be the most important cellular source providing most of the RONS in the cells of an aerobic organism [60]. However, there are other sources of RONS in mammalian cells that can be activated by radiation exposure. These include peroxisomes and many oxidases [61]. NADPH oxidases, a family of NOX enzymes that are located in various cellular compartments, can make a significant contribution to the enhancement of oxidative stress [62]. NADPH oxidases catalyze the one-electron reduction of O2 to produce a superoxide anion (O2 •−) followed by the formation of H2O2 and hydroxyl radicals (OH•) [62].

Although all types of RONS are generated in irradiated cells, the greatest contribution to nDNA damage and other macromolecules is made by H2O2, OH•, and ONOO, which can diffuse over long distances. Especially H2O2 molecules capable of diffusing over distances are attainable by nDNA [63]. It has been noted that in physiological conditions, the level of H2O2 can reach 1–10 nM, whereas at "supraphysiological" concentrations, its content will be higher (>100 nM) [62].

In our analyses, the increase in the H2O2 level in the spleen may have been due to the low level of antioxidants compared to the cerebral cortex tissue (Figure 4). It was also reported that with an increase in the frequency of mtDNA mutations, the level of RONS may raise in the spleen [64]. Due to the specificity of this tissue, it can be noted that iron ions are released in the spleen after irradiation, which can increase the level of RONS with the induction of cell ferroptosis [65]. However, the increased H2O2 level in the spleen tissue of irradiated mice can be significantly reduced by the administration of MEL before and after irradiation. It is possible to observe not only an increase in H2O2, but also a decrease in ATP synthesis

with a loss of mitochondrial membrane potential (ΔΨm) in the initial period in the cells' mitochondria after IR exposure [61]. The ATP content decreases unevenly in different tissues of mice. We found that the decrease in ATP was more actively manifested in the spleen (by 80%) than in the brain tissue (by 20%) (Figure 5) [60]. First of all, the reason for this is that the number of mitochondria in the brain tissue of two-month-old mice is three times greater than in the spleen [62]. The content of ATP in the spleen tissue decreases after irradiation of the mice, however, unlike the cerebral cortex, with the introduction of MEL it increases only to the control level. Perhaps this is a manifestation of the tissue specificity of the mitochondrial reaction [63]. After a short-term decrease in the content of ATP in the tissues of irradiated mice in the post-radiation period, its synthesis is restored. MEL has an active effect on the restoration of ATP synthesis, as well as on the biogenesis of mitochondria in the tissues of irradiated mice.

In radiation biology, the levels of MDA (a marker of oxidative stress) and antioxidant enzymes or reduced GSH (a marker of the antioxidant system) are often used to assess changes in the redox status of cells after irradiation. There are a number of publications in the literature which show that the radioprotective effect of MEL, observed in experiments on animals, is associated with the decreased MDA and increased GSH levels in their tissues [66–68]. According to the results of our study, the MDA levels in the spleen and cerebral cortex tissues of mice 48 h after irradiation remain elevated compared to the non-irradiated control tissues (Figure 6). As might be expected, the data of the GSH content analyses show decreased values. The GSH level in the spleen of the control mice was much lower than in the cerebral cortex tissue (Figure 7). There is a tendency to restore the MDA and GSH content to their reference values after the administration of MEL to mice before and after irradiation. There is still an increased MDA level in the cerebral cortex tissue after the administration of MEL, although the GSH level increases more noticeably to the reference values. The high MDA level in the cerebral cortex tissue might be due to the increased content of lipids in this tissue.

#### **5. Conclusions**

Numerous studies show that MEL is a strong antioxidant that exhibits radioprotective and radiomitigative effects. The results of our study on the evaluation of the effect of MEL on tissues with different proliferative activity and radiosensitivity in mice exposed to IR confirm this position. The results showed that, although the tissues of the spleen and cerebral cortex of mice differ in the initial control values of the analyzed markers, the potential of radiation protection of MEL is successfully implemented in both tissues.

It should also be noted that the issue of the expediency of splenectomy in radiotherapy of tumors of intra-abdominal organs or in astronauts during long-term space flights outside the protection of the Earth's magnetosphere is currently being discussed. Of course, the data obtained by exposure to X-rays on the body is difficult to completely extrapolate to damage to normal tissues during hadron therapy of tumors or to the effects of cosmic radiation on astronauts. Nevertheless, since oxidative stresses of different levels occur when cells are exposed to different IR (56Fe, protons, and X-rays) [69], it seems possible to suppress them by MEL and refrain from splenectomy.

**Author Contributions:** Conceptualization, A.I.G.; methodology, S.A.A. and A.I.G.; conducting all analyses, S.A.A., S.I.G. and A.I.G.; writing—review and editing, A.I.G. and S.A.A.; funding acquisition, A.I.G., S.A.A. and S.I.G.; statistical analysis, S.A.A. and A.I.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Russian government contract N◦ 075-00381-21-00 (2021– 2023) of the Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, and with the support of the Russian Foundation for Basic Research under grant N◦ 17-29-01007.

**Institutional Review Board Statement:** The study was approved by the Committee on Biomedical Ethics of the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences/the Physiology Section of the Russian Committee on Bioethics (Protocol N◦ 20/2021 dated 9 February 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors express their gratitude to A.V. Pisakov for carrying out work on the irradiation of mice and to T. Hilscher for technical support during a number of analyses and for working with animals.

**Conflicts of Interest:** All authors declare no conflict of interest.

#### **References**


## *Article* **Peroxiredoxin 6 Applied after Exposure Attenuates Damaging Effects of X-ray Radiation in 3T3 Mouse Fibroblasts**

**Elena G. Novoselova \*, Mars G. Sharapov, Sergey M. Lunin , Svetlana B. Parfenyuk, Maxim O. Khrenov, Elvira K. Mubarakshina, Anna A. Kuzekova, Tatyana V. Novoselova, Ruslan G. Goncharov and Olga V. Glushkova**

> Institute of Cell Biophysics of the Russian Academy of Sciences, 142290 Pushchino, Russia; sharapov.mg@yandex.ru (M.G.S.); lunin@rambler.ru (S.M.L.); lana\_kras2@rambler.ru (S.B.P.); xpehob2004@mail.ru (M.O.K.); mubarakshina\_e@rambler.ru (E.K.M.); 13krevetka@gmail.com (A.A.K.); novossulova\_t@rambler.ru (T.V.N.); ruslangoncharov071@gmail.com (R.G.G.); glushckova@mail.ru (O.V.G.) **\*** Correspondence: elenanov\_06@mail.ru

**Abstract:** Although many different classes of antioxidants have been evaluated as radioprotectors, none of them are in widespread clinical use because of their low efficiency. The goal of our study was to evaluate the potential of the antioxidant protein peroxiredoxin 6 (Prdx6) to increase the radioresistance of 3T3 fibroblasts when Prdx6 was applied after exposure to 6 Gy X-ray. In the present study, we analyzed the mRNA expression profiles of genes associated with proliferation, apoptosis, cellular stress, senescence, and the production of corresponding proteins from biological samples after exposure of 3T3 cells to X-ray radiation and application of Prdx6. Our results suggested that Prdx6 treatment normalized p53 and NF-κB/p65 expression, p21 levels, DNA repair-associated genes (XRCC4, XRCC5, H2AX, Apex1), TLR expression, cytokine production (TNF-α and IL-6), and apoptosis, as evidenced by decreased caspase 3 level in irradiated 3T3 cells. In addition, Prdx6 treatment reduced senescence, as evidenced by the decreased percentage of SA-β-Gal positive cells in cultured 3T3 fibroblasts. Importantly, the activity of the NRF2 gene, an important regulator of the antioxidant cellular machinery, was completely suppressed by irradiation but was restored by post-irradiation Prdx6 treatment. These data support the radioprotective therapeutic efficacy of Prdx6.

**Keywords:** X-ray radiation; 3T3 fibroblasts; proliferation; apoptosis; cellular stress; senescence; peroxiredoxin 6; Prdx6; radioprotector

#### **1. Introduction**

It is well-known that ionizing radiation (IR) leads to the formation of free radicals and reactive oxygen species (ROS). Ionizing radiation induces cellular stress and damage mediated through either direct changes in DNA or indirect effects on DNA via generation of ROS [1]. Exposure of cells to IR may have various consequences, including cell death, mutations, transformation, and cell cycle arrest. Radiation-induced ROS cause singleand double-stranded DNA breaks and extensive base modifications. To evaluate cellular responses to IR, many different approaches have been used, ranging from chromosomal changes visualization to cell viability analysis, assessments of cell activity, and transcription profiling with an expression analysis of a large array of genes. Overall, this allows researchers to gain insight into the molecular mechanisms underlying the response to IR exposure.

The fate of irradiated cells is influenced by the activities of various transcription factors and interactions between them during the cell response to irradiation. Based on the fact that IR induces the active production of ROS in cells, it is reasonable to study effects of antioxidant enzymes, including peroxiredoxins, as radioprotectors. We have previously shown the beneficial effects of recombinant peroxiredoxin 6 (Prdx6, EC:1.11.1.27) in various pathologies associated with oxidative stress, such as mechanical and thermal skin injuries, chemical burns

**Citation:** Novoselova, E.G.; Sharapov, M.G.; Lunin, S.M.; Parfenyuk, S.B.; Khrenov, M.O.; Mubarakshina, E.K.; Kuzekova, A.A.; Novoselova, T.V.; Goncharov, R.G.; Glushkova, O.V. Peroxiredoxin 6 Applied after Exposure Attenuates Damaging Effects of X-ray Radiation in 3T3 Mouse Fibroblasts. *Antioxidants* **2021**, *10*, 1951. https:// doi.org/10.3390/antiox10121951

Academic Editors: Elena Obrador Pla and Alegria Montoro

Received: 28 October 2021 Accepted: 3 December 2021 Published: 5 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of the respiratory tract, ischemia-reperfusion injuries [2–4], and type 1 diabetes mellitus [5]. In the latter study, we demonstrated that Prdx6 protected RIN-m5F (rat insulinoma) beta cells cultured with high glucose levels through a mechanism that leads to a reduction in ROS production and apoptosis. It was shown that peroxiredoxins (Prdxs), an evolutionarily ancient family of peroxidases capable of reducing a wide range of inorganic and organic peroxide substrates, may play an important role in radioprotection [6,7]. It should be noted that we have recently demonstrated penetration of exogenous Prdx6 into the cells using FITC-labeled Prdx6 [8].

We also studied the radioprotective activity of Prdx6 in different models in vivo and in vitro, and these studies were associated with the prophylactic application of Prdx6 before exposure to IR [9–12]. In addition, we recently demonstrated that preliminarily applied Prdx6 protected 3T3 mouse fibroblasts against LD50 X-ray irradiation in vitro. Thus, pretreatment with Prdx6 increased cell survival, stimulated proliferation, normalized the level of ROS in the culture, and suppressed apoptosis and necrosis in 3T3 fibroblasts [8]. We believe that it is equally important to test whether Prdx6 is capable of exerting a radioprotective effect when applied several hours after irradiation.

The effects of IR are the result of the activation of complex signaling pathway networks in response to DNA damage, which may lead either to recovery that is DNA repair and cell cycle arrest or cell death. These pathways are triggered by the activation of transcription factors, such as p53, nuclear erythroid-derived 2-related factor 2 (Nrf2), nuclear factor kappa B (NF-κB), and activating protein 1 (AP-1). Different radiation doses [13,14] and types of radiation produce different effects on gene expression [15]. High radiation doses are associated with increased severity of DNA damage, accompanied by responses to genotoxic stress, including the recognition of DNA damage, as well as altered repair mechanisms and immunological changes [16].

Among the transcription factors, nuclear factor kappa B (NF-κB) has been recognized as a key agent for the protection of cells against apoptosis in most cell types [17]. Both p53 and NF-κB are activated after exposure to IR, whereas activating protein 1 (AP-1) may control proliferation, aging, differentiation, and apoptosis, and Nrf2 may stimulate cellular antioxidant defense systems [18]. In addition, p21 was originally identified as a common inhibitor of cyclin-dependent kinases, transcriptionally modulated by p53, as well as a marker of cellular senescence. Earlier, p21 was considered a tumor suppressor that acted mainly by arresting the cell cycle and leading to the suppression of tumor growth. However, detailed studies of p21 have shown that p21 regulates responses to many cellular processes, including cell cycle arrest, apoptosis, DNA repair, aging, and autophagy [19].

In contrast, NF-κB has been shown to induce the activation of inflammatory and oxidative mediators, thus causing increased oxidative stress in cells [20,21]. Additionally, the transcription factor Nrf2, via the regulation of many antioxidant enzymes, such as glutathione peroxidase, may protect cells and tissues against inflammatory damage, mainly by inhibiting NF-κB signaling and suppressing the expression of several inflammatory and oxidative mediators [22,23].

Thus, the aim of this work was to study the effects of Prdx6 added to 3T3 fibroblasts several hours after irradiation of cells at a dose of 6 Gy. Thereby, the main subject of the work was not to identify the preventive effect of Prdx6, which has been demonstrated, but to investigate the possibility of its therapeutic activity when the enzyme is applied after irradiation. For this purpose, we analyzed mRNA expression profiles for genes associated with proliferation, apoptosis, senescence, and the production of corresponding proteins from biological samples after exposure to high doses of X-ray radiation and application of Prdx6. Finally, the aim of this study was to assess the role of Prdx6 in the regulation of therapeutic targets, such as NF-κB, Nrf2, TLR, p53, and p21, in irradiated fibroblasts to elucidate the therapeutic value of Prdx6 in counteracting X-ray toxicity. In parallel, we studied the responses of 3T3 cells by determining the production of cytokines IL-6 and TNF-α, expression of toll-like receptors (TLR1, TLR2, and TLR4), as well as the JNK

pathway and SA-β-Gal activity. In addition, the stress response of the 3T3 cells was assessed using the heat shock protein system, including Hsp70, Hsp90α, and Hsp90β.

#### **2. Materials and Methods**

#### *2.1. Cell Culture and Evaluation of Cell Proliferation*

Cells of BALB/3T3 lineage (American Type Culture Collection) were seeded into 25 cm2 culture flasks (volume 5 mL), at concentration 1 × <sup>10</sup><sup>6</sup> cells/flask, in DMEM (PanEco, Moscow, Russia) with addition of 10% fetal calf serum (Thermo, Swindon, UK) and an antibiotic/antimycotic solution (Sigma, Ronkonkoma, NY, USA). Cells were cultivated in a CO2 incubator at 37 ◦C and 5% CO2. For the experiments, cells of 5th–8th passages were used. Cells were allowed to attach for 24 h and then exposed to X-ray irradiation or a sham-irradiation (the same manipulations, excluding the X-ray device activation). Then, four hours later, 0.15 mg/mL Prdx6 was added.

Four groups of cells were used: (1) "control", sham-irradiated 3T3 cells; (2) "Prdx6", sham-irradiated 3T3 cells incubated in presence of Prdx6; (3) "6 Gy", 3T3 cells irradiated with X-ray in dosage 6 Gy; (4) "6 Gy + Prdx6", 3T3 cells irradiated with X-ray in dosage 6 Gy incubated in presence of Prdx6.

To assess survival, cells of 4 groups were placed into 96-well plates at concentration of 1 × <sup>10</sup><sup>4</sup> cells/well and maintained at 37 ◦C and 5% CO2 for 24, 48, 72, or 120 h for subsequent survival evaluation, which included staining the cells with 0.05% Crystall Violet and counting using a Crystal Viollet counting (measure OD 595 nm) [5].

#### *2.2. X-ray Treatment*

Irradiation was performed using a RUT-15 therapeutic X-ray device (focal length 8.5 cm, current 20 mA, voltage 200 kV) (Mosrentgen, Moscow, Russia) at a dose rate of 1 Gy/min. 3T3 cells were irradiated in culture flask, or 24, or 96-well plates at ambient temperature, and the accumulated dose was 6Gy. Sham-exposed cells were kept in the same conditions, excluding X-ray irradiation.

#### *2.3. Isolation and Purification of the PRDX6*

Genetic constructions encoding human Prdx6 enzymes were expressed in E. coli, strain BL21 (DE3), as described earlier [24]. The obtained recombinant proteins included a Histag. The proteins were purified by affinity chromatography on Ni-NTA-agarose (Thermo Fisher Scientific, Waltham, MA, USA), as described in the manufacturer's instructions. Isolation of proteins was performed as previously described [10]. The purity of the enzymes as measured by electrophoresis in 12% SDS/PAGE was at least 98%. Prdx6 diluted in phosphate buffer (1.7 mmol/L KH2PO4, 5.2 mmol/L Na2HPO4, 150 mmol/L NaCl, pH 7.4) at a concentration of 10 mg/mL was stored at −20 ◦C. Two months storage time at the above conditions produced no reduction in enzymatic activity. A peroxidase activity of Prdx6 in relation to hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (t-BOOH) was according to Kang, with minor modifications. The peroxidase activity of recombinant Prdx6 was 230 nmol/min/mg of protein (in relation to H2O2) and 100 nmol/min/mg of protein (in relation to t-BOOH).

#### *2.4. Senescence-Associated Beta-Galactosidase Staining*

Cellular senescence of 3T3 cells exposed to X-ray radiation was detected using a senescence-associated β-galactosidase (SA-β-gal) assay. 3T3 cells cultured in 24-well plates at 1 × <sup>10</sup><sup>4</sup> cells/well at 37 ◦C and 5% CO2 were treated as previously described [25]. After 120 h, the cells were washed with PBS and fixed in 2% formaldehyde/0.2% glutaraldehyde solution. The fixed cells were maintained overnight at 37 ◦C (without CO2) with SA-β-Gal staining solution. Finally, green blue–colored cells were counted (at least 100-200 cells per microscopic field in six fields) as a percentage of the total cell number and displayed as a percentage of cell senescence.

#### *2.5. Electrophoresis and Immunoblotting*

3T3 cells were seeded into cell culture flasks (T25) at concentration of 1 × <sup>10</sup><sup>6</sup> cells/flask, allowed to attach for 24 h and then exposed to X-ray irradiation or sham-irradiation. After irradiation (sham exposure) cells maintained at 37 ◦C and 5% CO2 for 120 h. Proteins from 3T3 cells were isolated using a lysis buffer as described previously [26]. The total protein concentration was measured using a spectrophotometer NanoDrop2000c (ThermoFisher Scientific, Wilmington, DE, USA). Equal quantities of total protein from samples were applied onto 10% SDS-PAGE and separated by electrophoresis. Then, a semi-dry transfer onto PVDF Hybond-P membranes (Amersham, Buckinghamshire, UK) was performed. Afterwards, the membranes were blocked using 5% fat milk in Tris-HCl buffer (pH 7.4) with 0.05% Tween 20 for 1 h, and monoclonal primary antibodies (1:1000) were applied, followed by incubation overnight at 4 ◦C. Following three washes with Tris-buffered saline/Tween 20, the membranes were maintained with an HRP-conjugated secondary goat anti-rabbit IgG antibody (P-GAR Iss, IMTEK, Moscow, Russia) (1:1000) for 1 h at ambient conditions. Primary monoclonal rabbit antibodies against GAPDH (14C10, #2118), NF-κB p65(C22B4, #4764), Phospho-NF-kB p65 Ser536 (93H1, #3033), Phospho-NF-kB p65 (Ser276) (93H1, #3037), Phospho-SAPK/JNK (Thr183/Tyr185 (#9251), Phospho-p53 (Ser46, #2521), Phospho-p53 (Ser15, #9284), p21 (#64016), Phospho-H2AX Ser139 (#2577), HSP70 (#4872), HSP90α (D1A7, #8165), HSP90β (#5087), Caspase-3 (#9662) (Cell Signaling Technology, USA) were used. GAPDH was used as a loading control. To develop blots, ECL Plus chemiluminescent cocktail (Amersham/GE) was used according to the manufacturer's instructions. The blots were photographed using WL transilluminator (Vilber Lourmat, Collégien, France). Quantification of the protein bands was performed densitometrically with Image Studio Software ver. 5.2.5 (Li-COR, USA). The averaged results normalized to the corresponding loading control (GAPDH) were expressed in relative units.

#### *2.6. Gene Expression Analysis*

3T3 cells were seeded into cell culture flasks (T25) at concentration of 1 × <sup>10</sup><sup>6</sup> cells/flask, allowed to attach for 24 h, and then exposed to X-ray irradiation or sham-irradiation. After irradiation (sham exposure) cells maintained at 37 ◦C and 5% CO2 for 120 h. The gene expression level was determined by reverse transcription real-time PCR. Total RNA was isolated from 3T3 cells with ExtractRNA reagent (Evrogen, Moscow, Russia). RNA quality was estimated electrophoretically in 1.5% agarose gel. RNA concentration was determined using NanoDrop 1000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Two micrograms of total RNA were used for reverse transcription with MMLV reverse transcriptase and standard dT15 oligonucleotide ("Evrogen", Russia). The synthesized cDNA was used for real-time PCR with qPCRmix-HS SYBR kit ("Evrogen", Russia) and 200 nM gene-specific primers (Table S1). The genes expression related to the cellular antioxidant response system (SOD3, PRDX1, PRDX2, PRDX3, PRDX4, PRDX5, PRDX6), apoptosis (CASP3, p53), DNA repair system (APEX1, XRCC4, XRCC5, and Ogg1), senescence marker (CDKN1), some transcription factors (p65/NF-κB, NRF2, AP-1), Toll-like receptors (TLR1, TLR2, TLR4), heat shock proteins (HSP90 and HSP70), and cytokine IL-6 were analyzed. Real-time PCR was carried out using DNA amplifier DTlite (DNA-Technology, Moscow, Russia) with cycling mode: (1) «hot-start»: 95 ◦C, 5 min; (2) denaturation, 95 ◦C, 15 s; (3) primer annealing and DNA synthesis at 60◦C, 30 s. Stages (2–3) were repeated 40 times. The expression levels of genes studied was normalized to that of the housekeeping gene-βactin (ACTB). The 2−ΔΔCt method was used to calculate differences in genes expression [27].

#### *2.7. Measurement of Cytokine Production*

3T3 cells of all groups were cultured in 24-well plates at 2 × <sup>10</sup><sup>5</sup> cells/well at 37 ◦C and 5% CO2 for 120 h (for ELISA assay). The cytokine concentrations were determined in the cell lysates by ELISA method in 96-well plates. Commercial reagent kits for quantification of murine interleukin-6 (IL-6) or tumor necrosis factor (TNFα) were used (Peprotech, Rocky Hill, NJ, USA) as described earlier [28].

#### *2.8. Statistical Analysis*

Statistical data analysis was carried out using the Sigma Plot 11 software package (Systat Software Inc., San Jose, CA, USA). Statistical significance between experimental groups was determined using two-way ANOVA with Bonferroni post-hoc tests for survival analysis or unpaired Student's *t*-tests for all other analyses. *p* < 0.05 was considered statistically significant. The results are presented as mean value ± standard error (SE).

#### **3. Results**

#### *3.1. Effects of Prdx6 on the Survival, Proliferation, and Antioxidant Status of Irradiated 3T3 Cells*

Prdx6 added to the 3T3 cell culture in vitro 4 h after irradiation of the cells with a dose of 6 Gy significantly reduced the radiation-induced cell's death. A significant increase in cell survival was especially pronounced in the first two days after exposure to X-ray radiation in the group with the application of exogenous Prdx6 (4 h after irradiation). The number of viable cells in the Prdx6-treated irradiated group was 15–20% higher than in the irradiated control group (Figure 1A). The cyclin-dependent kinase inhibitor 1 (mRNA-CDKN1, protein-p21) was also increased after irradiation, while the presence of Prdx6 almost completely abolished this effect. At the same time, the application of exogenous Prdx6 to the culture of unirradiated 3T3 cells did not significantly affect the level of p21 expression (Figure 1B,C).

**Figure 1.** Effects of X-ray irradiation and Prdx6 addition on (**A**) cell proliferation (Ftime(9.105) = 18.569, *p* < 0.001; Fexposure(3.105) = 240.29, *p* < 0.001; Ftime∗exposure(3.105) = 133.92, *p* < 0.001); (**B**) mRNA level of CDKN1 in the 3T3 cells; (**C**) p21 protein level in 3T3 cells measured by Western blot analysis. Equal amounts of total proteins were analyzed with the corresponding antibodies with normalization to a GAPDH loading control (bottom). Blot images show a single representative experiment, while values below the protein bands show protein level in relative units corresponding to the internal GAPDH control calculated from 3 independent experiments, and all mRNA evaluation experiments were performed in 6 repetitions. \* Significantly different from the sham-irradiated control, *p* < 0.05, & significantly different from the irradiated cells, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

Thus, the addition of Prdx6 prevented an increase of the senescence marker in irradiated 3T3 cells. In addition, evidence was obtained that the X-ray irradiation increased the percentage of SA-β-Gal positive cells, confirming the post-radiation oxidative stress and the activation of cell senescence mechanisms (Figure 2). In contrast, the addition of Prdx6

to the culture medium of the irradiated 3T3 cells led to a relative decrease in the percentage of SA-β-Gal-positive cells in comparison with non-treated irradiated cells. Meanwhile, the introduction of Prdx6 into the culture of non-irradiated cells did not affect the number of SA-β-Gal positive cells (Figure 2).

**Figure 2.** Representative images of effects of X-ray irradiation and Prdx6on SA-β-gal staining and the quantification of SA-β-gal-positive 3T3 cells. Data are percentages of total amounts of cells counted in the microscope's field of vision, *n* = 300 or more ± SEM. Scale bars are 100 μm. \* Significantly different from the sham-irradiated control, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

The expression of some antioxidant response system genes, for which the most significant change in expression after irradiation of 3T3 cells was previously shown, was assessed [8]. Interestingly, X-ray irradiation with a dose of 6 Gy resulted in a prolonged activation of the expression of isoforms PRDX2, PRDX3, and PRDX4, while the expression level of PRDX1, PRDX5, and PRDX6 did not differ significantly from the control values (Figure 3A). The addition of Prdx6 to the culture medium of 3T3 cells significantly normalized the expression of endogenous peroxiredoxins. The evaluation of SOD3 gene expression showed significant post-irradiation stimulation of the expression of this gene, whereas, in the presence of Prdx6, the expression of SOD3 in irradiated cells was almost normalized

(Figure 3B). Opposite effects of radiation and Prdx6 were found for the expression of the NRF2 gene (Figure 3C).

**Figure 3.** Effects of X-ray irradiation and Prdx6 addition on expression of genes regulating antioxidant status in 3T3 cells: (**A**) Peroxiredoxins; (**B**) SOD3; (**C**) NRF2. The mRNA evaluation experiments were performed in 6 repetitions. \* Significantly different from the sham-irradiated control, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

#### *3.2. Post-Irradiation Effects of Prdx6 on Cytokine Production, TLR's Expression, Apoptosis, and Cellular Stress in Irradiated 3T3 Cells*

By evaluating the effects of radiation on the 3T3 cell's activity, we observed that X-ray radiation with a dose of 6 Gy led to the activation of the NF-κB pathway, increased the production of pro-inflammatory cytokines IL-6 and TNF-α, and increased the expression of toll-like receptors TLR1, TLR2, and TLR4 (Figure 4). Notably, the administration of Prdx6 after irradiation removed the pro-inflammatory effects of radiation, both at the gene and protein levels. The only exception was a JNK signaling cascade, whose activity did not increase, but decreased by almost 10 times after irradiation, while the addition of Prdx6 did not change the effect of radiation on JNK activity (Figure 4B).

Apoptosis in irradiated 3T3 fibroblasts was assessed by the expression and activity of caspase 3 (Figure 5A,B). The results showed that IR sharply accelerated apoptosis in 3T3 cells, and the addition of Prdx6 to the cell culture medium decreased the expression of the gene regulating the production of caspase 3 below the control level, which indicated the ability of Prdx6 to protect cells from radiation-induced apoptosis. Additionally, the production of the p53 protein was assessed in 3T3 cells, as well as levels of phosphorylated forms of this protein, ph-p53 (S46) and ph-p53 (S15), which have different roles in the cell. It was shown that IR significantly increased the total level of p53 in the cells, as well as the phosphorylation of p53 at Ser 46 and Ser 15 (Figure 5B,C). In addition, the Prdx6 added to the cells 4 h after irradiation demonstrated an obvious protective effect, manifesting in the normalization of the p53 level, as well as in a tendency to restoring of ph-p53 (S46) and ph-p53 (S15) levels, especially the ph-p53 (S15) form, which promotes cell survival [29].

**Figure 4.** Effects of X-ray irradiation and Prdx6 addition on expression of genes and levels of proteins regulating and characterizing the immune status in the 3T3 cells: (**A**) NF-κB mRNA level; (**B**) NF-κB, p-NF-κB (Ser276 and Ser536, were normalized with total p65) and JNK proteins level; (**C**) TLRs mRNA level; (**D**) IL-6 mRNA level and TNF-α and IL-6 production in the 3T3 cells; (**E**) mRNA IL-6 level. The explanations for Western blot and mRNA analysis as in the legend for Figure 1. \* Significantly different from the sham-irradiated control, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

We also evaluated the expression of a panel of DNA repair-associated genes (such as XRCC4, XRCC5, Apex1, and Ogg1) (Figure 6). The results showed that IR stimulates the expression of these genes, indicating DNA damage in the 3T3-irradiated cells. In addition, irradiation led to increase in phosphorylation of H2AX which is a clear indication of DNA damage. Along with that, IR modulated the expression of H2AX that may indicate its effects on global histone regulation. Prdx6 protein added to the culture medium after irradiation exerted a protective effect in cells, which was indicated by a decrease in the expression of these genes associated with DNA damage and cell senescence, as well as a decrease in p-H2AX level, an important marker of IR-induced double-strand DNA breaks [30,31].

The expression of genes regulating the production of heat shock proteins HSP90α, HSP90β, and HSP70 is a direct indicator of cellular stress. We found that the irradiation of 3T3 fibroblasts led to the significant activation of genes that regulate the production of the inducible form of the heat shock proteins HSP90α, HSP90β, and HSP70 (Figure 7). The presence of Prdx6 in the cell culture medium prevented the stress response of 3T3 cells to X-ray irradiation.

**Figure 5.** Effects of X-ray irradiation and Prdx6 addition on expression of genes and levels of proteins regulating and characterizing apoptosis in 3T3 cells: (**A**) mRNA of caspase 3; (**B**) p53, p-p53 (was normalized with total p53), and caspase 3 protein level; (**C**) mRNA of p53. The explanations for Western blot and mRNA analysis as in the legend for Figure 1. \* Significantly different from the sham control cells, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

**Figure 6.** Effects of X-ray irradiation and Prdx6 addition on expression of genes and level of proteins regulating and characterizing DNA reparation in the 3T3 cells: (**A**) mRNA of XRCC4; (**B**) mRNA of XRCC5; (**C**) mRNA of Apex 1; (**D**) mRNA of Apex 1; (**E**) mRNA of H2AX; (**F**) The level of p-H2AX protein. The explanations for Western blot and mRNA analysis as in the legend for Figure 1. \* Significantly different from the sham-irradiated control, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

**Figure 7.** Effects of X-ray irradiation and Prdx6 addition on expression of genes and levels of proteins, regulating and characterizing heat shock proteins in the 3T3 cells: (**A**) HSP90α and HSP90β proteins levels; (**B**) mRNA of HSP70; (**C**) mRNA of HSP90. The explanations for Western blot and mRNA analysis as in the legend for Figure 1. \* Significantly different from the sham-irradiated cells, *p* < 0.05, # significantly different from the irradiated cells, *p* < 0.05.

#### **4. Discussion**

Earlier, we demonstrated that preliminary administration of exogenous Prdx6 before irradiation of cells [8] and animals [10] provided a radioprotective effect. The radioprotective effect of Prdx6 approximately for 80% was due to its peroxidase activity, and, for 20%, due to stimulation of the TLR4 receptor [8]. It should be noted that exposure to ionizing radiation produces a long-term oxidative stress. In particular, after irradiation, a prolonged (hours-days) increase in the level of lipid peroxidation was observed [32], as well as formation of long-lived reactive protein species [33] that were shown to be effectively eliminated by Prdx6 [10]. In addition, after exposure to radiation, a dysfunction of the electron transport chain of mitochondria and the activation of a number of oxidases (NAD (P) H-oxidase, xanthine oxidase, cyclooxygenase etc.) were observed, which also contribute to an increase in the level of intracellular ROS and the progression of oxidative stress [34]. In this regard, it was interesting to test the effects of antioxidant enzyme Prdx6 applied after exposure to X-rays. Therefore, the purpose of this study was to evaluate the radiomitigating properties of recombinant Prdx6 in the culture of 3T3 embryonic fibroblasts.

Prdx6 can neutralize the broadest range of hydroperoxides and, unlike other members of the Prdx family, is able to reduce phospholipid peroxides and peroxynitrite, as well as phospholipase A2 activity (aiPLA2) under certain conditions [35]. Apparently, due to the aiPLA2 activity, Prdx6 may penetrate into cells, thereby directly affecting their redox status [8].

In cells that survive X-ray irradiation, changes in the expression of genes associated with DNA repair, cell cycle, inflammation, and immune responses are usually observed [36]. To study the effects of recombinant Prdx6 protein on irradiated 3T3 cells, we measured the key regulators of cellular processes. Among them, nuclear transcription factor kappa B (NF-κB) is a key factor in the regulation of metabolic pathways in most cell types. NF-κB is a central transcription factor in the immune system and influences cell survival. Moreover, the induction of radioresistance is mediated by several NF-κB regulated genes [17]. The p53 protein plays an important role in the regulation of the cell cycle, DNA repair, and apoptosis and is an attractive therapeutic target for cancer treatment [37]. Surprisingly, we have shown that both p53 and NF-κB are activated in 3T3 cells after exposure to IR, whereas the presence of Prdx6 significantly reduced the effect of X-ray irradiation.

When studying the expression of PRDX1-6, which are considered the most important intracellular hydroperoxidases, it was found that isoform-specific expression of peroxiredoxins was induced on 5th day after irradiation (Figure 3), which may be explained by adaptation to the changing spectrum of hydroperoxides in the cell, because peroxiredoxin isoforms have different efficacy towards different peroxide substrates. However, in general, the induction of the expression of these genes was several times lower than in the first 6 h after irradiation of 3T3 cells [8], which may be due to the suppression of NRF2 (Figure 3), which regulates the expression of genes of many antioxidants' enzymes [38,39]. It should be noted that suppression of NRF2 may be mediated via activation of the transcription factor NF-κB (Figure 4), which is shown to suppress NRF2 activity [40]. The activation of NF-κB may explain the increase in the expression of some genes, which are controlled by NF-κB, related to the antioxidant response (SOD3) [41] and DNA repair (XRCC4, XRCC5, H2AX, Ogg1, and Apex1) [40].

The panel of DNA repair-associated genes in the irradiated 3T3 fibroblasts was markedly activated, whereas, after the Prdx6 addition, the activation of the gene panel was significantly reduced (Figure 6), which may indicate a decrease in ROS-induced oxidative DNA damage in the presence of Prdx6. These results support the radioprotective efficacy of Prdx6.

Moreover, the anti-inflammatory effect of Prdx6 in irradiated 3T3 fibroblasts was demonstrated. Indeed, while irradiation induced the expression of toll-like receptors (TLR1, TLR2, and TLR4), which is consistent to previous data [42], the production of proinflammatory cytokines IL-6 and TNFα, cell senescence (assessed by SA-β-Gal staining), and Prdx6 almost completely removed the pro-inflammatory effects of X-ray irradiation.

One of the important regulators of cellular activity is a low-molecular-weight p21 protein, transcribed from the CDKN1A gene, which was first described as a cyclin-dependent kinase 1 (CDKN1) inhibitor. It plays an important role in cell cycle control [43]. p21 stops cell cycle progression during G1 and S-phases via the binding and inhibition of cyclin-CDKN1,2,4,6 complexes [44]. Indeed, we have shown that X-ray irradiation at a dose of 6 Gy significantly increased CDKN1 gene expression, p21 production, and p21 phosphorylation, while the addition of Prdx6 into the culture medium of the X-ray irradiated fibroblasts normalized the proliferation of the 3T3 cells.

In addition, we have shown that genes regulating the production of heat shock proteins HSP90 and HSP70 were activated in the irradiated cells, with an inducible form of the HSP90 protein, HSP90β, with the most noticeable activation. Since these proteins are markers of cellular stress, we may conclude that IR induces cellular stress. It should be noted that Prdx6 also acts as a radioprotector, reducing the cellular stress caused by irradiation.

Advantages of new radioprotector development are related not only to providing protection in "working spaces" or during incidents of radioactive contamination but also to the use of radiation therapy [45]. Radiation therapy is currently one of the main treatments for cancer; despite the many benefits of this treatment, such as non-invasiveness, preservation of organ integrity, and precision when targeting a tumor, it can lead to complications in the irradiated healthy tissue. Therefore, applying radioprotective means may alleviate radiation-induced complications. Although many studies have aimed to identify radioprotective agents [46], there is still a need for new effective radioprotectors. Previously, we demonstrated that the radioprotective effects of Prdx6 are based on its capability for ROS neutralization and, potentially, on its ability to activate signaling regulatory mechanisms for the restoration of unbalanced redox homeostasis [10]. The summary on the protective effects of exogenous Prdx6 in the irradiated cells is shown in Figure 8. This study additionally supports this conclusion, importantly, using the post-radiation administration of the recombinant antioxidant protein peroxiredoxin 6, which may be a promising radioprotector/ radiomitigator.

**Figure 8.** Schematic representation of the action of exogenous Prdx6 applied after exposure to X-rays. Ionizing radiation directly and indirectly (via ROS) causes single-stranded (SSBs) or double-stranded (DSBs) DNA breaks. The MRN complex (Mre11, Rad50, and Nbs1 proteins) recognizes DNA damage and activates ataxia telangiectasia-mutated (ATM) kinase. ATM phosphorylates histone H2AX at Ser139 (γH2AX), as well as checkpoint kinases CHK1 and CHK2, which phosphorylate p53. Phosphorylation of p53 at Ser15 leads to cell cycle arrest, while phosphorylation at Ser46 promotes apoptosis. In turn, p-p53 (ar Ser15) promotes the expression of p21 (cyclin-dependent kinase inhibitor p21 (CDKN1A)), which inhibits cyclin-dependent kinase 2 (CDK2), thereby inhibiting phosphorylation of retinoblastoma (Rb) protein and causing cell arrest. Exogenous Prdx6 prevents increase in the level of intracellular ROS by their elimination in the extracellular space, as well as directly inside the cell after Prdx6 s penetration into cytoplasm [8]. Thus, the recombinant Prdx6 inhibits an increase of DNA damage and p21 activation, preventing the development of senescence. Prdx6 also prevents apoptosis by suppressing ROS-mediated activation of the ASK-1/JNK/AP-1 signaling pathway and an increase in the level of p-p53 (Ser46). An important role in the suppression of apoptosis is played by NF-κB, which is activated with the participation of NEMO (NF-κB essential modulator), and stimulation of the TLR4 receptor by exogenous Prdx6.

> **Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/antiox10121951/s1, Table S1: Oligonucleotides used for real-time PCR.

> **Author Contributions:** Conceptualization, O.V.G. and E.G.N.; methodology, O.V.G., S.B.P., M.G.S., M.O.K.; formal analysis, O.V.G., M.G.S., E.K.M.; investigation, O.V.G., S.B.P., M.O.K., M.G.S., E.K.M., A.A.K., R.G.G., T.V.N.; data curation, O.V.G., S.M.L., M.O.K., M.G.S., E.K.M.; writing—original draft preparation, E.G.N., O.V.G., S.M.L.; writing—review and editing, E.G.N., S.M.L., O.V.G., M.G.S.; visualization, O.V.G., E.K.M.; supervision, O.V.G., E.G.N.; project administration, E.G.N., O.V.G., M.G.S., S.M.L.; funding acquisition, O.V.G., M.G.S. All authors have read and agreed to the published version of the manuscript.

> **Funding:** This work was supported by Russian Foundation for Basic Research [grants 20-015-00216 and 19-04-00080]. The authors report no conflict of interest.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or Supplementary Materials.

**Acknowledgments:** Part of the experiments was carried out using the technical base of Collective Use Centers of ICB RAS (X-ray system).

**Conflicts of Interest:** Authors report no conflict of interests.

#### **References**

