Radiation sensitivity of biological systems, its modification by chemical modifiers and its quantitative evaluation

Abstract

Large number of sensitizers and protectors of cells, tissues, organs and organisms are being discussed in this review. Attention is paid to metals, nanoparticles or organic compounds as modifiers. They can modify the radiation sensitivity by 20–60%. In most cases, sensitization is caused by an increase in DNA damage or a decrease in the repair of radiation damage. The protection can be caused in many cases by repair of damaged DNA molecules, proteins or enzymes, or by reducing the ROS concentration. The most current research tasks in this area are formulated. A new quantitative and reproducible method of evaluating modifiers is discussed.

Introduction

Since the turn of the nineteenth and twentieth centuries, two different but related fields have developed, both focused on the interaction of ionizing radiation with matter (since the discovery of X-rays by Wilhelm Conrad Röntgen in 1895, followed by Hugo Fricke´s recommendations to study the chemical effects of radiation in order to understand the reaction of living organisms to radiation (1928), or the first radiotherapy (breast cancer) performed by E. H. Grubbé in 1933). The first of them is radiation chemistry, the second is radiation biology (more often called radiobiology). A huge number of papers (monographs or original papers) have been published in both these fields. Over such a long period of time, it has been shown that the chemical aspects of interaction of ionizing radiation with biological subjects are very important for understanding the nature of reactions of biological materials both with radiation itself and primary radiolytic products [1]. Currently, there is a large amount of papers published, dealing with the bordering areas between radiation chemistry and radiation biology (this field can be referred as bioradiation (or radiobiological) chemistry—similar to already established biophysical, bioinorganic, bioorganic chemistry or bioradiation physics). The purpose of this paper can be summarized as follows: First, to give critical overview of papers dealing with the modification (positive or negative) of the radiation sensitivity of biologically interesting objects by chemical modifiers and try to make some generalizations, since majority of the original work published in this field is just a description of the experiments and gathered data and there is a lack of critical comparison and evaluation of the obtained results. Therefore, the presented work is not oriented specifically to clinical practice, although it touches this area. Some review articles summarizing the state of the art are focused on a specific area of the research (focusing on specialty chemicals, protection against low doses or clinical applications), and therefore a general overview is needed. The second goal of the paper is to present a description of an objective and quantitative method for evaluating the effects of chemical modifiers. Practical applicability of the method was repeatedly verified [2,3,4].

Influencing radiation sensitivity with chemical agents

Ionizing radiation (X-rays, γ-radiation, electron beams or beams of other charged particles originated from radionuclides or accelerators) or non-ionizing UV- radiation or neutrons can act on biological objects at different levels, from biomolecules, organelles, cells, tissues, organs up to whole organisms. The overall effect of irradiation depends on the type of radiation, parameters (dose, dose rate) and method of irradiation and, last but not least, on the irradiated subject. The same parameters also determine the ways of influencing the radiation sensitivity of biomaterials with modifiers which can be applied before, during or after irradiation.

A positive influencing the sensitivity of biological material to radiation leads to increase in its radiation sensitivity. This increase may be caused by various factors such as the formation of new radicals or other reactive species in the presence of sensitizers (e. g. lipid peroxides [5,6,7,8,9,10]), inhibition of reparative processes or stimulating the occurrence of DNA breaks caused by radiation. On the other hand, a negative influencing leading to a decrease in radiation sensitivity can also be caused by various factors. The direct effect of ionizing radiation can be significantly reduced via chemical shielding by added chemicals (e.g. adsorbed on cell membranes) or by stimulating the rate of repair processes. One notable mechanism involves the reduction of single-strand breaks (SSBs) or double-strand breaks (DSBs) of DNA in cells. Mechanisms for reducing radiosensitivity to the indirect action of ionizing radiation include (but are not limited to) steric reasons, scavenging radicals, or reducing the concentration of oxygen through its chemical reactions with chemical agents. Regardless of the above, the detailed mechanisms of influencing sensitivity to radiation by chemical agents are still not fully understood. But in general, chemical agents improve the quality of radiation therapy either by increasing the killing of tumor cells or protecting normal tissues [11].

Changes in biological materials can also be caused by UV radiation. At present, however, one can generally observe a disappearing difference between the action of ionizing and non-ionizing radiation (i.e. between radiation chemistry and photochemistry). Among other things, because UV radiation up to a wavelength of at least 254 nm can also produce in water or diluted aqueous solutions some radicals (e. g. OH radicals) by two-photon processes [12].

The chemical nature of the modifiers can significantly vary. It is worth noting that the same chemicals (such as melatonin or nitric oxide) can act as sensitizers or protectors depending on the conditions under which they are used. The study of the action of chemical modifiers (a part of bioradiation chemistry) is useful in the radiotherapy of cancer, where the highest sensitivity of malignant cells is required and, on the other hand, the lowest possible radiation sensitivity for surrounding healthy cells is desired. It is possible that the right combination of suitable chemical sensitizers and protectors would be able to meet these opposing requirements.

Effective protectors are also important for the protection of the human population moving in an environment with an increased level of radiation or for the sterilization of drugs. The basics of this complex issue were treated from a chemical point of view by von Sontag in the book "The Chemical Bases of Radiation Biology" [1]. The subject of the book is, in the words of the author, "radiation chemistry of biological systems”.

In the next chapters, the results of recent works (especially since around 2013) dealing with influencing the radiation sensitivity of various biological objects with chemical modifiers are discussed. An irradiated biological subject was chosen as the general criterion according to which the annotated original works were processed. This approach makes it possible to create a certain picture of the current state of optimization of radiation treatment of various biological subjects. An overview of the discussed papers is given in Table 1 (sensitizers) and Table 2 (protectors). The tables can be considered as a guide to the literature in the field. In most cases, gamma radiation or X-ray radiation has been used in published works (even where not explicitly stated). The use of other types of radiation (UV, protons, ions or neutrons) is indicated.

Table 1 Sensitizers

Table 2 Protectors

Increase in radiation sensitivity

Several review papers devoted to this topic have been published (radiosensitizers published in Int. J. of Radiation Biology is listed in an overview article by J. Moulder [13]). Each of them prefers a certain area. Some of original works use the dose enhancement factor (DEF) to quantitatively and uniformly evaluate the results (DEF is defined as the ratio Ds / D₀, where Ds and D₀ are the doses absorbed by the irradiated object in the presence and absence of the sensitizer, respectively, for the same biological effect).

The oldest classical sensitizer is probably oxygen. The well-known oxygen effect occurring even at low concentrations was observed around 1900. Since 1990, great progress has been made in its research [14]. Therefore, some authors of overview articles paid attention to the effect of oxygen on the radiation sensitivity of the cells. For example, Moulder has devoted much attention in the last two decades to studies of the radiation sensitivity of hypoxic cells [13]. An interpretation of oxygen-dependent molecular modifications and their effect on tumor response in mammalian cells to therapy was offered by Liu et al. [14]. The high resistance of cells under anoxia conditions can be according to Liu et al. caused by the fact that DNA free radicals can be relatively easily regenerated by chemical reduction with sulfhydryl compounds and/or others reducing molecules, while DNA radicals converted to peroxides in the presence of oxygen cannot be structurally restored. On the other hand, according to Wardman, oxygen modifies the chemical pathways to DNA strand breaks, and peroxides are just accidental byproducts of those pathways. It is now clear that the oxygen effect is mainly realized inside the cells at the moment of irradiation [15] and the rate of the ongoing reactions is relatively high. It is also clear that the generation of oxygen radicals plays a significant role in cell sensitization. However, we still do not fully understand the nature of the oxygen effect at the molecular level.

However, knowledge of the processes and mechanisms underlying the effects of sensitizers is also of utmost importance from a clinical point of view to ensure that the sensitizer sensitizes cancer cells more than normal ones. However, differentiated cell sensitization is still, according to J. E. Moulder, “the holy grail" of clinically oriented radiobiology [13]. Finding a sensitizer (or a combination of several modifiers) with a maximum effect on malignant cells and at the same time with a minimum impact on normal cells is therefore one of the most important tasks of bioradiation chemistry also in this case.

To achieve this goal, it is important to focus current research of sensitizers in three areas: First, search for the most effective nanosensitizers with a high atomic number and with high cell penetration and low biological incompatibility including cytotoxicity, where a synergistic effect can be expected. With sizes ranging from tens to hundreds of nm, nanoparticles have high cell penetration and mostly low incompatibility. Many different sensitizers have been intensively studied such as gold, silver, gadolinium, iron oxides, zirconia and others, which have a high atomic number and consequently a high mass absorption coefficient. Gold nanoparticles (AuNPs) with a high photon absorption cross-section are the preferred nanoscale radiosensitizers and are therefore used for clinical purposes. To make their effectiveness even greater, they are modified with hydrophilic polymers directed to the targeting ligands [16]. This multimodal approach is the second promising area of current sensitizer research. The third area is the combination of radiotherapy and photodynamic therapy [17,18,19] and the combination of several different sensitizers or sensitizers with protectors. In all these areas, it is necessary to carry out research both in vitro and in vivo. The results of both these methods may be different. This was shown for example in the irradiation of mouse tumor cells (P 53 SA-NH, P 53 FSa lines) in the presence of metformin (C₄H₁₁N₅) where the applied concentration for in vitro use was 5 mM and the dose for in vivo application was 250 mg kg^–1 body weight [20].

In the following subsections, the results of the study of the action of sensitizers according to the irradiated objects will be discussed.

Deoxyribonucleic acid

In the area of the basic study, many works are dedicated to the increase in radiation sensitivity of DNA. Nevertheless, also increasing radiation damage to many other biomolecules (e. g. proteins, lipids, carbohydrates, enzymes, vitamins, hormones) leads to significantly increased radiation sensitivity of cells.

One of the most recently studied sensitizers acting in the sensitization of DNA plasmids is cis-platinum (PtCl₂(NH₃)₂). Cis-platinum appears to increase DNA damage through increased formation of both single-strand (SSB) and double-strand (DSB) breaks [21]. The cause of this effect is apparently the formation of adducts of DNA molecules with cis-platinum. The presence of an average of two cisplatin per plasmid increased single- and double-strand break yields by factors of 1.9 and 2.2, respectively, compared to irradiated unmodified DNA samples [22]. However, the effects caused by the action of cis-Pt are complex in nature. Furthermore, in addition to the increased formation of DNA DSB breaks in the presence of cis-platinum the promotion of formation of DNA clusters, which are known to be difficult to repair and which are toxic, is also known. Such lesions present in significant concentration indicate loss of DNA functionality [23].

It seems that the mechanism of sensitizing effect of cis-Pt on DNA is closely related to the interaction of electrons released in ionization processes with cis Pt. It turns out that low-energy electrons (from 10 to 10⁴ eV) play a decisive role in this process [24]. Even with the application of ultra-low-energy (about 0.5 eV) electrons, it is possible to predict the formation of DSB lesions in the platinum-modified DNA structure [25]. Electrons in this energy state are already hydrated and react as relatively stable particles with oligonucleotides, leading to the formation of both single- and double-strand breaks in platinized DNA, but not in unmodified DNA. This reaction is obvious controlled by diffusion [26, 27]. The participation of slow (hydrated) electrons in the radiosensitization of DNA seems indisputable [28]. More detailed mechanism leads to reactions of hydrated electrons with platinum-thymine or platinum-guanine adducts [26].

Increased attention is also paid to the study of changes in the genetic apparatus of cells under the influence of irradiation with accelerated ions [29, 30]. The study of this process in the presence of suitable sensitizers (or modifiers in general) can contribute to the clarification of the complexities of the processes taking place here.

Lung cancer cells

Lung cancer is one of the most common cause of cancer death. Currently, its incidence is increasing especially among women. It is necessary to state critically that the results of the treatment are still unsatisfactory. It is a diagnosis with the worst prognosis, as statistically only 10% of patients survive for more than 5 years. One method of treatment is radiotherapy, either as maintenance treatment after surgery or radical radiotherapy of circumscribed findings.

The study of the effect of various sensitizers on the radiation sensitivity of cancerous lung cells has received a lot of attention in recent years. The results so far are rather qualitative, without the ambition to search for the causes of the observed effects of sensitizers. Nevertheless, it seems that the delay in the repair processes of radiation-damaged DNA molecules plays a significant role here. This was observed when non-small cell lung cancer was treated with cis-Pt prior to irradiation [31]. The detailed mechanism of delayed repair is not yet sufficiently clarified. However, the conclusions about the action of cis-Pt on DNA presented in chap. 2. 1. 1 are valid even in this particular case. Some progress was made when cofilin 1 (intercellular actin modulating protein) for sensitization of human non-small cell lung cancer H1299 was used. Two repair systems of radiation-induced DSBs were considered [32].

In this context, it is worth mentioning the situation when a substance with sensitizing effects is used as a contrast agent for diagnostic examination. Such a case is the use of dotarem as a contrast agent. Sensitizing effect is undesirable here. Examinations with dotarem (2 and 20 mM) were performed with lung (H460) and five other organs (pancreas (MiaPaCa2), prostate (DU145), breast (MCF7), brain (U87) and liver (HEPG2) irradiated with225 kV X-ray (dose of 1 Gy). The active substance of dotarem is gadoteric acid (C₁₆H₂₅GdN₄O₈) (gadolinium compounds are commonly used as contrast agents for magnetic resonance imaging). Although a statistically significant increase in DNA damage was observed in all cell lines, no statistically significant decrease in cell survival was observed in the clonogenic assay due to the high level of repair (observed after 24 h) of damaged DNA structures [33].

In radiotherapy practice, it is generally important that the effect of the sensitizer on malignant cells is more pronounced compared to the healthy cells. Such a different effect was found with human lung cancer cells H1299 compared to normal HFL-III cells (or mouse fibroblasts C3H/MCA clone 15 compared to C3H/10T1/2 cells) irradiated with X-rays using syringetin (a natural substance contained in lilac-like plants) as a sensitizer [34]. Overall, it can be sad that syringetin can be considered as a promising effective radiosensitizer in suppressing the growth of lung cancer cells.

Another important condition for the successful application of radiosensitizer is its ability to penetrate into the cells. This also applies to the treatment of malignant lung lesions. Therefore, in this case too, due attention was given to the application of various sensitizers in the form of nanoparticles. Thus, for example, the radiosensitive effect of selenium nanoparticles (about 89 nm) under the influence of X-rays against lung cancer was described. SeNPs themselves are relatively less toxic in normal healthy cells than in cancer cells. These findings revealed the cytotoxic potential of the SeNP + X-ray combination in lung cancer cell lines [35]. In combination with thioglucose, 16 nm AuNPs (up to a non-toxic concentration of 100 µM) after intercellular uptake inhibit the proliferation of human lung cancer cells QU-DB (similarly to breast cancer cells MCF 7 – chap. 2. 1. 3.) by 64 and 32 percent after irradiation with 100 kV and 6 MV X-ray, respectively [36].

In addition to the most common application of wave radiation (X or γ), the use of corpuscular radiation, especially neutron radiation, accelerated protons or carbon ions, is also being developed in lung cancer therapy. Various sensitizers are used successfully in these cases.

In the boron neutron capture therapy (BNCT), various “sensitizers” (boron compounds) are tested. In fact, it is not about sensitizers in the true sense of the word. The nuclei of boron atoms in compounds located directly in tumor cells after capturing neutrons produce energetic alpha particles with a destructive effect on the surrounding cells (¹⁰B + n → α + ⁷Li). In this way, irradiation of cells with neutrons in the presence of various borate compounds leads to inhibition of DNA replication and arrest of the cell cycle. However, considering the relatively simple process of alpha particle genesis by the aforementioned nuclear reaction, it is interesting that the chemical structure of such "sensitizers" can affect the effectiveness of BNCT. For example, human A 549 cell lung cancer lines were incubated with boron reagent solutions (2.3 mM) for 5 h and then irradiated with a neutron dose of 0.33 Sv. It was found that borate esters derived from the adenosine triphosphate, adenosine diphosphate, adenosine monophosphate nucleosides showed significantly higher sensitization values than those derived from glycerol, inosine (nucleoside C₁₀H₁₂N₄O₅) or triethanolamine (N(CH₂CH₂OH)₃) [37].

In the case of accelerated particles, it is mainly about precisely targeted irradiation of tumor lesions with accelerated protons or heavy ions with a high linear energy transfer (LET). Proton irradiation can be carried out in the presence of compounds labeled with ¹¹B isotopes, where, similar to the BNCT therapy, the resulting alpha particles (¹¹B + p → 3α) have a destructive effect on the immediate surroundings. In the case of accelerated ions (mostly multiply charged carbon ions Cⁿ⁺) the use of sensitizers AZD7762 ((S)-5-(3-fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide-C₁₇H₁₉FN₄O₂S), honokiol (5,3′-diallyl-2,4′ -dihydroxybiphenyl—C₁₈H₁₈O₂) or tunicamycin (a mixture of homologous antibiotics containing uracil, N-acetylglycosamine and 11-carbon aminodialdose) leads to higher sensitivity of non-small cell lung cancer lines A549 and H1299 after their irradiation. These agents cause an increased concentration of unrepairable DSB defects on cellular DNA. Therefore, the use of high-LET carbon ions in combination with these compounds may be a potentially effective therapeutic strategy for locally advanced non-small cell lung cancer lines A549 and H1299 [38].

Breast cancer cells

Much attention was also paid to increasing the radiation sensitivity of breast cancer cells. This disease is one of the most common malignant diseases. According to a statistical survey, it is, for example, in the second place in the USA, right after skin cancer.

Promising results were obtained with the cellular response to combined treatment with an anti-miR-21 (oligonucleotide) sensitizer and radiation when MDA-MB-361 breast cancer cells and the non-transformed breast epithelial cell line MCF-10A were irradiated with doses of 0.25 and 2.5 Gy [39]. The observations made showed that the cellular response to the combined treatment is different in cancerous and non-cancerous cells. This is a promising result for therapeutic purposes. Using nitric oxide (NO) as a sensitizer, formation of SSBs and DSBs on DNA was observed when MCF7 and UT-SCC-14 breast cell lines were irradiated. In addition, a longer repair time (except for the formation of SSBs and DSBs) was found. The authors assume the formation of stable adducts of nitric oxide with uracil radicals. The process was consistent with a nearly diffusion-controlled reaction. The efficiency of NO oxide (70 nM) was found to be several times higher than that of oxygen [40].

Similar to lung tumor cells, the sensitizing effects of various sensitizers in the form of nanoparticles are successfully used in breast tissue tumor cell therapy. The radiosensitization by AuNPs may also involve inhibition of DNA-DSB repair. Experiments performed with an AuNPs concentration of 0.5 mg/ml led to the dose enhancement factor lying in the interval from 1.09 to 1.39. The effect was most pronounced under the oxic conditions [41]. In combination with thioglucose, AuNPs were found to inhibit the proliferation of MCF 7 breast cancer cells by 64 and 38% after their irradiation with 100 kV and 6 MV X-rays, respectively, similar to lung cells (chap. 2.1.2) [36]. Positive results were also achieved by the application of iron oxide nanoparticles coated with dextran (80 μg/ml), when MCF 7 cells were irradiated with 6 MV photons at a dose rate of 370 cGy/min [42]. The radiation sensitivity was shown to increase with increasing dose rate and the dose enhancement factor was found to be 1.21. Another sensitizer applied to breast tumor cells is melatonin (5-methoxy-N-acetyltryptamine—a hormone produced by the pineal gland—the so-called "sleep hormone"). Its application before radiation was found to increase the radiation sensitivity of breast cancer cells [43]. This is mediated by a reduction in cell proliferation, an increase in p53 mRNA expression, and may be related to the action of melatonin on estrogen regulation. Melatonin can be considered promising, non-toxic and inexpensive drug for adjuvant radiotherapy. Similarly, manganese porphyrins could also serve as promising radiosensitizers, as they have been shown in mammary tumor radiotherapy to sensitize tumor cells and simultaneously protect normal tissues from damage through modulation of the redox status of various tissue types [44]. Also, the PKI 402 inhibitor (C₂₉H₃₄N₁₀O₃) in combination with ionizing radiation causes more apoptotic cell death than IR alone in the MCF-7 cells [45].

It is clear from the above examples that the mechanism of action of different sensitizers on breast cancer cells is different. Mostly it involves stimulation of the formation of DSB or SSB DNA defects (NO, Dotarem). In some cases, it can affect the formation of enzymes (melatonin) or prolong the repair time of damaged DNA (AuNPs). It is not excluded that with some sensitizers it may be a combination of several different mechanisms, e.g. with nitric oxide, where in addition to increased DNA damage, there is probably also an increase in the repair time of these damages. On the contrary, with some sensitizers, such as Dotarem, the increased intensity of repair processes can work against the sensitizing effect (see chap. 2. 1. 2.). Some examples give the possibility to assume that many sensitizers investigated so far have a real hope for practical use in the treatment of breast cancer. This is for example the case of some oligonucleotides (such as anti-miR-21), which can be characterized by a significant difference in their action on cancer and non-cancer cells. Manganese porphyrins have a similar perspective. Similarly, some hormonal preparations (e.g. melatonin) with favorably low side effects can be considered perspective. Preparations of a nanoparticle nature can also have a great perspective in this direction. Of them, gold nanoparticles modified in various ways currently appear to be the most promising.

Cervical cancer cells

Cervical cancer is the fourth most common type of cancer and the fourth leading cause of cancer death in women. Every year, this diagnosis is indicated in approximately 500,000 women around the world. In addition to surgical treatment and chemotherapy, radiation therapy is one of the standard methods of therapy for this disease. The classic chemotherapeutic agent is a cis-platinum that can lead to a synergistic effect in combination with radiotherapy. The classic research object is HeLa tumor cervical cells. The possible mechanism of the effect of the cis-platinum is based on the delay or inhibition of DNA damage repair (see also chaps. 2. 1. 1.–2. 1. 3.). However, it should be noted that the sensitizing effect of platinum compounds (and many other sensitizers) may be complicated by the fact that virtually all the sensitizers behave as scavengers of free radicals. That´s why a combination of betamethasone (corticosteroid C₂₂H₂₉FO₅) with platinum salt led to an increase in cell mortality, but less than the platinum salt itself. This effect was observed when the cancer cervical cell line HeLa was irradiated with accelerated positive carbon ions [46]. Today, this method of irradiation is one of the progressive promising methods of radiotherapy due to the possibility of relatively accurate targeting of the corpuscular beam to an irradiated object. Nevertheless, most of the irradiation is performed by electromagnetic radiation of different wavelengths (from UVC radiation to hard gamma radiation) using sensitizers which significantly increase the efficacy of radiotherapy. However, their effect depends on many factors, including the nature of malignant cells of cervical tissue. This has been proven when rosiglitazone (ROSI) (thiazolidined) was used before irradiation. In some of the cells (HeLa and SiHa) a growing level of antioxidant enzymes (catalase, superoxiddismutase) was found and the number of surviving cells remained unchanged after irradiation. On the other hand, no increase in antioxidant enzymes and a decreased number of surviving cells was observed in the Me-180 cells [47].

As with lung or breast cells, sensitizers in the form of differently modified nanoparticles are also used as effective sensitizers in radiotherapy of cervical tumor cells. The AuNPs (47 nm) used in radiotherapy can be combined with some additives such as folic acid (vitamin B type) where dose enhancement factors of 1.64 and 1.35 were observed when HeLa cells were irradiated in the presence of a folate-conjugated sensitizer compared to unconjugated (pegylated) sensitizer [48]. It is remarkable that in the case of irradiation of breast cells in presence of AuNPs, a similar DEF (from 1.09 to 1.39) was achieved [41] (chap. 2. 1. 3). A similar conclusion can be reached by comparison of results when HeLa cells were irradiated with X-rays in the presence of iron oxide nanoparticles covered with dextran, where the DEF of 1.19 was found, with the results when MCF 7 breast tumor cells were irradiated (DEF = 1.21—chap. 2. 1. 3.). This finding is all the more interesting because, as mentioned above, the changes induced by radiation in the presence of ROSI-sensitizer differ for different cells. All of this shows that the response of the cells to irradiation itself is a complex process additionally complicated by the presence of sensitizer. Nevertheless, the resulting sensitizer effect expressed by DEF may be similar for different cells and sensitizers.

Cervical tumor cells are sensitive not only to relatively high-energy radiation but they can be sensitive also to a low-energy radiation in the presence of sensitizer. This is the case of radio-photothermal therapy of cervical cancer with low concentration of Fe3O4@AuNPs. Time-dependent death of cervical cancer cells was observed after a short period of near-infrared radiation [49]. It represents a promising method of combined radiotherapy with low radiation exposure of the patient.

Glioma

Gliomas are one of the most common types of primary brain tumors. It accounts for about 30% of all brain and central nervous system tumors. In addition to surgical therapy and chemotherapy, radiotherapy is used for treatment either as a primary procedure (when surgery is impossible or too complicated) or as a secondary treatment after surgery. In both cases, it is necessary to ensure the highest possible efficiency of radiation treatment with the lowest possible radiation load of the surrounding healthy cells. These requirements are particularly urgent in the treatment of brain cancer to avoid damage to vital centers in the nervous system. Both mentioned requirements can be achieved on the one hand by maximally increasing the radiation sensitivity of tumor cells to the sensitivity of healthy tissue and on the other hand by hitting the malignant focus as precisely as possible with a beam of radiation from an external radiation source. The first condition can be met by the application of suitable sensitizers, the second by the application of targeted radiotherapy (e.g. irradiation with corpuscular radiation (protons, accelerated ions, neutrons).

Similar to many other cases, in radiotherapy of glioma, the effort is to increase the effectiveness of the treatment by using modified sensitizers (mostly gold nanoparticles (GNPs)) or by combining radiotherapy with some other suitable methods. For example, gold-coated iron oxide nanoparticles can be considered as a good thermo-radio-sensitizer without toxicity below 10 µg/ml. The best results were obtained when AuNPs, hyperthermia and radiation were simultaneously applied to U87-MG human glioma cells [50]. Ultra-small gold nanoparticles (with a hydrodynamic diameter < 3 nm) coated with polyethylene glycol and conjugated with a cyclic (Arg-Gly-Asp-D-Phe-Lys) peptide (cRGDfK) showed negligible toxicity in ALTS1C1 murine glioma at a moderate concentration and can be considered a suitable radiosensitizer for proton and kV and MV photon radiotherapy applications. The dose enhancement factor moves in the interval from 1.14 to 1.33 [51]. Even in this case, the increase in radiation sensitivity expressed by DEF is practically the same as in the previously cited studies. For practical applications, increased attention will need to be paid to developing delivery methods to increase AuNPs uptake in tumor cells and reduce uptake by unwanted cells.

The work based on targeted chemo-proton therapy of C6 brain cancer cell line with modified superparamagnetic iron oxide nanoparticles conjugated with folate and paclitaxel (with a size of 5.4 nm and a non-toxic concentration of 0.2 µg/ml) demonstrated an improvement in therapeutic efficacy due to the synergistic effect of drug administration [52]. DEF values from 1.16 to 1.52 were achieved using this method. This work shows that modern proton therapy can find applications in the radiotherapy of malignant brain tumors and the method can be optimized using sensitizers based on modified nanoparticles.

Boron neutron capture therapy (BNCT) is another modern radiotherapy method in the treatment of brain cancer which has already been mentioned in connection with lung radiotherapy (chap. 2.1.2). The decisive factor of this treatment seems to be the ability of boron compounds to penetrate the cells of the tumor tissue. This ability can be quite different for different compounds. For example, this was clearly shown in the U87 heterotopic glioblastoma model in SCID mice. The concentration of borophenylalanine (BPA) in glioblastoma was the highest, followed by liposomal sodium borocaptate (BSH) and non-liposomal BSH. However, the effect of boron compounds is not an unequivocal function of their concentration in cells. It was found that liposomal BSH showed better long-term results compared to BPA or non-liposomal BSH. These differences became significant from day 50 after BNTC [53]. In this context, it is interesting that a low dose of γ-radiation (0.1 Gy) increased the accumulation of BPA (used at a concentration of 400 mg/kg) in tumors by 52.2%. In addition, both tumor/normal tissue boron ratio and tumor/blood boron ratio were increased from 3.77 to 5.31 and from 3.47 to 4.46, respectively. Since no BPA toxicity was detected according to histopathological findings [54], such pre-irradiation leads to an improvement in the efficacy of BNCT.

The importance of the transport of boron compounds into cancer cells during the application of BNCT is also confirmed by works in which this transport was directly affected by the introduction of the relevant transporter. Modification of the expression level of L-amino acid transporter 1 of human cancer cells (glioblastoma cell line T98G), which transports BPA into cells, leads to greater intracellular uptake of BPA. Intracellular absorption of BPA was 1.5–5.0 times higher in the treated clones than in the control clone. This effect results in enhanced anticancer effects of boron neutron capture therapy [55]. Recently, plasmids that induced overexpression of L-type amino acid transporter 1 (LAT1) were developed for this purpose to increase the efficacy of BNCT therapy [56].

Based on the results obtained during the BNCT study of the brain (and lungs), it can be stated that the effect of the sensitizer largely depends on its ability to penetrate the cells. It is highly likely that this applies in general to the BNCT of all biological objects.

For the sake of completeness, it is appropriate to add that dotarem can also be used as a contrast agent for the examination of glioma (as in lung or breast, as mentioned earlier) without the risk of radiation damage due to rapid recombination processes [33] (see chap. 2. 1. 2.).

Fibroblasts and melanoma

Similar to lung cancer cells (chap. 2.1.2), a higher sensitization effect of the syringetin was found in fibroblastic cancer cells compared to healthy ones [34]. Experiments with early-studied mouse embryo fibroblasts and later studied nine human cancer cell lines (lung adenocarcinoma (A549 and H358), glioblastoma (LN229), cervical cancer (HeLa), colorectal carcinoma (SW480 and HCT116) and nasopharyngeal carcinoma (5–8 Cell lines F, 6–10B and HK1)) showed that another sensitizer, an ATM inhibitor, significantly sensitizes cancer cells to ionizing radiation as a result of which it inhibits cancer cell growth. These findings allow the use of the ATM inhibitor for radiation therapy of resistant tumors [57]. These findings document that one and the same sensitizer can significantly increase the radiation sensitivity of cells not only of different lines of a given organ, but also of cells of different organs. This also documents the action of the nitric oxide which increases the radiation sensitivity of breast tumor cells (chap. 3. 2. 3.) and also effectively increases the radiation sensitivity of fibroblast cells [40].

While the sensitization of fibroblastic cells is mainly motivated by research interests, papers on radiation sensitization of skin tumor cells are usually focused more practically. About 73% of skin cancer deaths are melanomas of different species. In addition to surgical therapy, radiotherapy for this disease may be one of the promising adjuvant treatments (except for chemotherapy or biological treatment). However, its efficiency must be increased by suitable sensitizers. As in other areas, research is also focused here on nanoparticle sensitizers for the reasons stated in the chap. 2. 1. An example is a study in which 50 nm commercial spherical AuNPs allows the dose to be reduced from 4.3 to 2 Gy in the treatment of highly resistant melanoma with 150 and 450 kV X-radiation [58]. AuNPs (50 nm) and Au-particles (320 µm) reduce a part of the melanoma cells in the radiosensitive G2/M phase. The progression of melanoma cells treated with AuNPs after 150 kV X-rays irradiation (2–4 Gy) was faster than without AuNPs [59]. It is remarkable that the DEF of 2.15 is in this case remarkably high compared to the breast cancer radiotherapy (from 1.09 to 1.39–chap. 2. 1. 3.). In any case, even in this area of radiotherapy, it would be promising to investigate the effect of modified AuNPs, for example with thioglucose, as in the case of lung cancer (chap. 2. 1. 2.) or malignant breast tumors (chap. 2. 1. 3.) or AuNPs modified with folate or combination with photothermal therapy, as in the case of cervical cancer treatment (chap. 2. 1. 4.). This is only a proposal of other possibilities motivated by analogies in other areas of radiotherapy.

Very interesting modification of the combined radiotherapeutic method represents dual irradiation with laser light (1 and 1.5 W/cm²) and X-ray (2, 4 and 6 Gy) in the presence of Pt-nanoparticles (PtNPs). Dual radiation has significantly improved the treatment of mice malignant melanoma cell line B16/F10 by producing reactive oxygen species (ROS) [60].

In addition to nanoparticles, in the treatment of melanomas, as in many other cases, various bioorganic molecules can play the role of sensitizers. An example is the use of cold inducible RNA binding protein, which is a critical protein suitable for sensitization of A375 and M624 melanoma cancer cells. It causes increased DNA damage and reduced DNA repair [61]. Many other promising sensitizers are studied in this area, facilitating the treatment of often difficult-to-manage melanomas.

Colon and prostate cancer

If colon cancer develops, there are many treatment procedures to help control it (surgery, chemotherapy, targeted therapy or immunotherapy). One of them is also radiation therapy often supported by sensitizers.

AuNPs can also here increase the efficiency of the absorbed dose in the human colorectal carcinoma HT-29 cells irradiated with 9 mV X-ray (similar to lung or breast cells (chaps. 2. 1. 2 and 2. 1. 3)). In doing so, it was proven that Au-particles (10–100 µm) seem to be a biologically inert material and do not affect the cell cycle [62]. All cases of AuNPs application discussed so far (lungs, breasts, cervix, glioma, melanoma, colon) clearly prove that AuNPs are effective and almost universal sensitizer. Possibly, its operation is based on a general principle. Most likely, the accumulation of radiation energy in Au nanoparticles plays an essential role.

Other promising sensitizer is aurapetene (natural bioactive monoterpene coumarin ether C₁₉H₂₂O₃), which can be used as a supplement to the ionizing radiation in the treatment of colon cancer. Auraptene was applied in vitro before irradiation of CT26 cells and intraperitoneally in vivo before irradiation colon cancer in mouse. The conducted experiments revealed an increased number of apoptotic cells and a regression in the size of the tumor [63].

Prostate cancer is one of the most common types of cancer. Therefore, it is also necessary here to look for suitable sensitizers to increase the effectiveness of radiation treatment of this disease. It was shown that iron oxide nanoparticles surface modified with dextran (a polymer of anhydroglucose) or dextran with an amino group can enter DU145 human prostate cancer cells. Similar to MCF7 breast cancer cells (chap. 2. 1. 3.) or HeLa cervical cells (chap. 2. 1. 4.), it increases their radiosensitivity to 6 MV-X-rays. The detected DEF value was approximately 1.2 at different radiation doses in the range of 2–8 Gy [64]. Like AuNPs, the overall sensitizing effect of FeONPs in cells of different organs is practically the same. So, it seems that the character of the irradiated cells is not decisive for the final effect of nanoparticle sensitizers.

Similar to lungs (chap. 2. 1. 2.), breast (chap. 2. 1. 3.) or brain (chap. 2. 1. 5.), the use of dotarem as a contrast agent does not lead to a decrease in the number of surviving cells even in the examination of the prostate, although the sensitizer causes DNA damage [33].

For the treatment of castration-resistant prostate cancer, the use of docetaxel (C₄₃H₅₃NO₁₄) is suggested as a suitable sensitizer [65].

Some other irradiated biological objects

The sensitizing effect of dotarem is manifested by a decrease in the number of surviving cells also in pancreatic cancer cells (MiaPaCa2) or liver cancer cells (HEPG2) due to significant recombination of radiation-induced DNA damage [33]. An increase in DSBs and significant simultaneous decrease in DNA repair efficiency was observed after stimulation of X-ray exposure (1 Gy) with germanium oxide GeO₂ applied at concentrations from 0 to 22 mM 12 h before irradiation of Chinese hamster ovary cells (CHOK1). The application after irradiation was also effective. Initiated processes lead to cell death, without increased ROS production [66].

The effect of p-boronophenylalanine applied 2 h before neutron therapy on the kidneys of female C57BL6 mice was studied. This application has been reported to lead to the accumulation of fat in the liver and to fibrosis [67].

Sensitizer made of gold nanoparticles modified by bovine serum albumin and folic acid and core-loaded with paclitaxel and curcumin is shown to be suitable for the treatment of esophageal cancer. The sensitizer shows inhibitory effect on proliferation, with no obvious toxicity on healthy cells Het-1A. In such a way, the sensitizer acts as a safe and effective radiotherapy sensitizer to improve the radiotherapy efficacy [68].

Summary

In general, it can be stated that the effects of sensitizers on lung cancer cells result in a decreased rate of proliferation (gold nanoparticles + thioglucose). It may be caused by reduced or even blocked DNA DSB repairs (cofilin 1, AZD inhibitors, honokiol, tunicamycin). On the other hand, the increase in DNA damage observed in the presence of some sensitizers (e. g. dotarem) may be partially compensated by a higher level of repair. Such contradictory processes can also occur in the cells of other organs (pancreas, prostate, breast, brain, liver). Until now, the influence of sensitizers on the radiation sensitivity of brain tumor cells (glioma, glioblastoma) has been mostly concentrated on BNCT or proton therapy. The difference in the effectivity of “scavengers” can be largely assigned to different levels of their concentration in tumor cells. Various suitably modified nanomaterials enabling a synergy were found useful in proton therapy of brain tumors. Extensive application of heavy accelerated ions in the presence of sensitizers may be expected in near future. Research on modified radiotherapy of cervical-HeLa cells showed that the effectiveness of radiotherapy increases with pretreatment or with the simultaneous use of a combined sensitizer composed of two or more components (Pt-salt + metasone, AuNP + folic acid). This trend can be considered as part of the development of modern modified radiation therapy. More attention should be focused on dose rate effects, as it strongly influenced the sensitivity of cervical cells as well as breast tumor cells to radiation applied in the presence of a suitable sensitizer (such as dextran-coated FeO nanoparticles). Irradiation of fibroblasts showed that there are suitable sensitizers strongly affecting sensitivity of tumor cells and at the same time only weakly affecting sensitivity of normal cells (ATMi, syringe). Negative side effects on normal cells can also be suppressed e.g. by melatonin or Mn-porphyrin, as shown in the case of radiotherapy of breast tumor cells. Similarly, positive results were obtained with modified radiotherapy of colon tumors in the presence of various sensitizers (aurapten, metformin, gold or platinum nanoparticles, FeO-Pt core nanoparticles). Radiotherapy modified with a sensitizer has also been successfully tested on human malignant melanoma cells, human prostate cancer cells and some others.

In majority of cases, the sensitizers were applied before irradiation (e. g. melatonin—breast cancer cells, aurapten—CT 26 cells, rosiglitazone—colon and cervical cancer cells). In some cases, however, the effects were also observed when sensitizers were applied during irradiation (various combined sensitizers – HeLa cells) or after irradiation (germanium oxide – DNA).

Great attention has been focused on gold nanoparticles due to their high photon absorption cross section. Typically, various combinations of gold nanoparticles with other components (thioglucose, folic acid, PEG + c RGDfKs or iron oxides) have been studied in proton therapy. The reason was to achieve maximum damage to malignant tumor cells with minimal damage to normal cells. Nanoparticle sensitizers based on iron or selenium oxides also seem promising. A rapidly developing neutron capture therapy uses boron compounds necessary for the application of this method (borate esters, borocaptates, boron phenylalanine, etc.). Even in this case, attempts have been made to combine boron compounds with other suitable components (e.g. transport amino acids).

As it follows from Table 3, the effectiveness of radiotherapy in the presence of selected sensitizers evaluated using the dose enhancement factor ranges from 1.19 ± 0.1 to 1.64 ± 0.2. AuNPs modified with folic acid applied in the treatment of the cervix appear to be the most effective from this point of view (DEF = 1.64) while iron oxide nanoparticles modified with dextran show the lowest efficiency in this treatment (DEF = 1.19). As can be further seen from Table 3, the same sensitizer modified in the same way (FeONPs + dextran) has practically the same DEF (from 1.21 to 1.19) when applied to different organs (breast, prostate or HeLa cells). On the other hand, FeONPs modified in another way (folate and paclitaxel) lead to the same significant increase in the effectiveness of the sensitizer (DEF = 1.52).

Table 3 Dose enhancement factors (DEF)

In liquid and gas phases, the primary interactions of ionizing radiation with matter consist of only three simple processes: excitations, ionizations of atoms and molecules, and bond cleavage. These processes occur regardless of the type of irradiated object and in principle also regardless of the type of ionizing radiation. Differentiation occurs only in the later stages due to the high variability of subsequent chemical reactions. This differentiation leads to a great variety of the resulting effects. The situation is even more complicated in the presence of a sensitizer (generally modifier). Among the many final consequences of the influence of the sensitizer on radiation sensitivity, one of the most important can be considered the increased formation of DSBs and SSBs in DNA molecules and inhibition of repair processes. The first of them is probably caused by slow electrons resulting from primary ionization and subsequent thermalization. Their reactions with biomolecules may be stimulated by various sensitizers. However, the detailed mechanism of such sensitization process is still unknown in most cases. The second process (inhibition of the repair of radiation damage) can be caused by a direct reaction of the sensitizer with fragments produced by irradiation (e.g. reaction of nitrous oxide with purine or pyrimidine radicals). Some sensitizers (GeO₂) act through both of these mechanisms. An important step in the action of a sensitizer is its ability to penetrate inside the cell. Sensitizers in the form of nanoparticles have a great advantage from this point of view.

Some research results have already been translated into practical applications. BNCT is used to treat brain tumors. Except for neutron therapy, rapid development of nanoparticles as sensitizers in proton therapy of brain tumors (e.g. FeONPs or AuNPs applied to target chemoproton therapy) is perspective. The application of nanoparticles in conventional radiotherapy leads to a significant increase in the effectiveness of multimodal treatment consisting of radiotherapy and photodynamic therapy. In such a combined approach, gold nanoparticles, sometimes with other additives, are used in clinical practice (e.g. melanoma radiotherapy). This allows the applied dose to be reduced while maintaining the desired therapeutic effect. There are many other sensitizers that are promising for practical use, such as Mn-porphyrins in mammary tumor therapy, aurapten in colon cancer therapy, various inhibitors in high-LET Cⁿ⁺ therapy (non-small cell lung cancer). The application of cold-inducible RNA binding proteins or cis-Pt to sensitize cancer cells in radiotherapy or photon dynamic therapy also appears to be quite promising.

Based on the summarization of the results of the study of the influence of sensitizers on the radiation sensitivity of biological material, the following specific conclusions can be drawn. The facts that can be considered proven can be formulated as follows:

The most likely underlying mechanism of action of the sensitizer is the promotion of DSB and SSB formation in DNA.

Another frequent mechanism is reduction or complete blocking the repair of radiation-damaged biomolecules.

An important role in sensitization is played by slow electrons arising from subsequent thermalization after primary ionization.

The effect of sensitizers can be manifested by their application before, during or even after irradiation.

The effectiveness of boron compounds in BNTC applications increases proportionally with the concentration of these "sensitizers" in the irradiated tumor.

In the future, it would be advisable to pay attention to the following areas of sensitizer research:

A systematic basic study of the reactions of slow electrons in presence of different kinds of sensitizers.

Suppression of the negative effect of repair processes on the increased radiation sensitivity of biological objects.

Multimedia therapy (e.g. sensitized radiation therapy combined with photodynamic therapy) with a high synergistic effect.

Possibilities of using other types of radiation with high LET (except for p + and Cⁿ⁺) in sensitized radiotherapy and the use of other boron compounds in BNCT.

It would be convenient to standardly compare the effectiveness of sensitizers using the DEF value.

Reduction of radiation sensitivity

While tumor cells should be maximally sensitive to ionizing radiation, the radio sensitivity of healthy cells should be kept to a minimum. This is important not only for malignant lesions irradiation but the protection of healthy human cells must also be ensured in cases where there may be an increased level of radiation in the environment, e.g. in nuclear power plants other nuclear facilities, in the cabins of jet aircrafts or in the case of space flights or potentially in the event of a nuclear accident. Therefore, radiation protection research and in particular research of various radioprotectors is an important part of radiobiology or bioradiation chemistry. Cysteine (NH₂ CH(COOH)CH₂SH) or thiourea (S = C(NH₂)₂) were used as one of the first protectors. Later, it was shown that various sulfur or phosphorus compounds, such as aminothiols, disulfides, phosphorothioates, thiazolidines, aminoethylisothiourea or dithiolanes, exhibit significant radioprotective activities [69]. After World War II, and especially since 1957, when radiodiagnostic methods were expanded, research on low-dose protection has been developing. A review dedicated to this issue was recently published [70]. An overview article dedicated to protectors containing sulfur, phosphorus or organometallic protectors containing silicon, germanium or selenium was published in the monograph "Radiation Chemistry. From fundamentals to applications in materials and life sciences [69]. A comprehensive recent work discusses the clinical applications of many different protectors used in medical practice [71]. At present, the attention is given to a variety of chemically different protectors. Unfortunately, the vast majority of chemicals with protective effects against radiation are more or less toxic to the living cells. For practical use, it is therefore necessary to search for substances and conditions of their application under which their protective effect is maximal, but their toxicity is as minimal as possible. The potential toxicity may be associated with impurities present in the administered drug, for example in the case of some fullerenes [72].

The ratio of indirect and direct effects of ionizing radiation on living cells reaches more than 90% [73]. Indirect effect is applied in living cells containing 50–60% water mainly through its radiolysis. Various intermediate products of water radiolysis are created by the indirect action of ionizing radiation. The most important of them are OH^., e⁻_aq and H^. radicals and various ROS arising as secondary products. They attack the main structures of living cells, including the structure of DNA. It can be considered proven that the oxidative effects of OH radicals are to a great extent responsible for the DNA damage [74]. Therefore, the removal of radiolytic products, especially OH radicals, can lead to cell protection. Consequently, scavengers of OH radicals can contribute to effective cell protection. However, according to Ewing, their protective effect may not be based solely on the scavenging OH radicals [75].

Indeed, many different OH radical scavengers such as oxides, acids, thiophosphates, nitrites, alcohols, sugars, amines, amides, thiols, ketones, vitamins, phenolic compounds, various extracts with antioxidant effects and many other compounds have been investigated as radioprotectors. Their effect is complex and it may depend on the character of protector, conditions of irradiation [76] and the type of irradiated object. Accordingly, the protection mechanisms can be variable. In addition to radical scavenging, other processes may apply in the protective mechanism, such as DNA protection and its repair, caloric restriction, anti-inflammatory or antioxidant processes and many others [77].

DNA molecules

Deoxyribonucleic acid (DNA) has received the most attention in the study of radiation desensitization at the molecular level due to its dominant importance in radiation biology and bioradiation chemistry. In the DNA radiation sensitivity reduction, different protectors were studied. One of the tasks of protectors is to reduce the yield of DNA SSB and DSB defects. This has been achieved with various protectors. For example, after irradiation of DNA with high-energy ions (Cⁿ⁺- 290 MeV/u, Feⁿ⁺- 500 MeV/u) with doses up to 25 Gy in the presence of coumarin-3-carboxylic acid, or dimethyl sulfoxide (DMSO) with concentrations from 10^–4 up to 10^–2 M or glycylglycine in the concentration range from 10^–3 to 10^–1 M the concentration of DSB sites decreased linearly with increasing scavenger efficiency of protectors. A protective effect based on reducing the occurrence of non-DSB clusters was also observed [78].

DNA plasmids modified with organometallic compounds such as RAPTA C (Ru(η6-p-cymene)Cl₂(1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane-phosphine)) also showed greater resistance to plasmid damage induced by OH radicals than unmodified ones [21]. The scavenging of OH radicals by protectors is the basis of the protective effect of many scavengers. This applies to the irradiation of DNA plasmid with accelerated carbon ions 290 MeV/u in the presence of D- or L-methionine (butanoic acid—(2S)-2-amino -4-(methylsulfanyl)) [79]. It is worth noting that no difference in the action of the two isomers was found. This means that the three-dimensional structure of the scavenger has no essential effect on the protection mechanism in this case.

The reaction of water radiolysis products, presumably OH radicals, with sugar phosphate groups in the DNA backbone is also known to induce clustered abasic sites (AP) (with similar efficiency to SSB). The scavenging of OH radicals can therefore lead to protection also against this type of DNA damage. It was observed when DNA-plasmids were irradiated with X-rays or accelerated Cⁿ⁺-ions in the presence of Tris (hydroxymethylaminomethane—C₄H₁₁NO₃) scavenger (concentration ranging from 0.66 to 200 mM). At a higher concentration, the yields of AP sites were significantly lower than the yields of basal lesions [80].

The DNA protection can also be mediated by agents protecting the functional activity of the DNA-ligase enzyme involved in DNA replication and repair. Such an effect was observed when DNA-ligase was irradiated with gamma radiation (3–5 kGy) in the presence of radioprotectors such as amifostine (organothiophosphate), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid—soluble analog of vitamin E), melatonin, semiquinone glucoside derivative (derivatives isolated from Bacillus sp. INM‐1) or gallic acid (trihydroxybenzoic acid) showing antioxidant effect [81]. Complete DNA ligation was observed in the presence of protectors, while a functional impairment in ligation was detected in their absence.

In the proposed mechanisms of DNA protection there are still many uncertainties. An example is the following observation. When the two scavengers arginine (amino acid) and Tris (buffer) were compared to each other, both in concentrations from 1 to 200 mM, surprisingly a higher rate of DNA chain cleavage was found in the case of arginine with a higher scavenging efficiency (the systems were irradiated with ultra soft K_α Al—X-ray radiation up to a dose of 88 Gy) [73]. According to the authors, the observation may be related to specific binding modes of arginine to DNA. It is important to realize that the situation can be complicated by the fact that many scavenging processes lead to various radical products, which can subsequently enter into a reaction with cellular structures. In the following example, propan-2-ol reacts with OH/H radicals via water or hydrogen abstraction [82]:

$$ \left( {{\text{CH}}_{{3}} } \right)_{{2}} {\text{CHOH}}\, + \,{\text{OH}}/{\text{H}}\, \to \,\left( {{\text{CH}}_{{3}} } \right)_{{2}} {\text{COH}}\, + \,{\text{H}}_{{2}} {\text{O}}/{\text{H}}_{{2}} $$

where 2-hydroxyprop-2-yl radical (CH₃)₂COH is a reducing agent [83]. Subsequent reduction processes can complicate the protective effects of the original protector.

Cells

Many works deal with the protection of cells. A reduction in the number of DNA double-strand breaks was observed when human MCF-7 breast or HTB 177 lung cancer cells were irradiated after DMSO or glycerol application [84]. Different DNA damage repair mechanisms between the two cell lines have been hypothesized. Therefore, the type of cells can be decisive in this case even when using the same protector. Sodium selenite, acting as an antioxidant, is used in the treatment of breast cancer cells to protect normal tissue, while physiological concentrations of the protector (70; 140 µg/L) do not impair efficacy of radiotherapy with X-ray doses up to 10 Gy [85]. DMA protector (3, 4-dimethoxyphenyl bis-benzimidazole) also has a protective effect. Based on a multifactorial analysis, its protective effect is believed to be based on the repair of proteins essential for the normal function of mammalian HEK cells or U 87 cell lines [86]. Irradiation of IEC-6 normal rat small intestine epithelial cells with a dose of 9 Gy in presence of ferrostatin (C₁₅H₂₂N₂O₂) led to a decrease in ROS production and inhibition of both ferroptosis and apoptosis [87]. Similarly, connexin 43, a gap junction protein responsible for intercellular communication prevents radiation-induced intestinal damage via the Ca²⁺ concentration in IEC-6 irradiated intestinal epithelial cells [88]. Promising results were found in human colon cancer cells exposed to DMSO (0.28 M) at low doses (1 Gy) of X-rays [89]. DMSO significantly increased clonogenic cell survival, but DSBs produced by irradiation were slower repaired (DMSO alone had no effect on apoptosis). This indicates the complex action of DMSO. Similarly, when human colon cancer cells were pretreated with some endogenous metabolites such as phenylacetate or tauroursodeoxycholate, the radioprotective properties of the protectors were noted, but no mitigation effects were observed [90].

As already mentioned, the protective effects can be based, besides others mechanisms, on the scavenging of various oxidants (not only OH radicals). This is apparently also the case with curcumin (diferulylmethane) applied 30 min. before γ-irradiation with a dose of 4 Gy in a concentration of 0.5 µg L^–1. The scavenger acts as a cell-permeable and irreversible anti-tumor and anti-inflammatory agent reducing the formation of micronuclei in human peripheral blood lymphocytes by inhibiting the formation of radiation-induced free radicals (in addition to the hydroxyl radical, these are 2,2-azinobis (3-ethylbenzothiazoline-6-sulfone) acid, 2,2′-diphenyl-1-picrylhydrazyl and also nitric oxide) [91]. On this occasion, it is appropriate to point out that the NO radical increases the radiation sensitivity of cells by reducing DNA repair capacity, as already mentioned in chap. 2. 1. [40]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid—a component of lignin) acts as a natural antioxidant and significantly reduces endothelial cell damage of the human umbilical vein and thus increases cell viability. Ferulic acid applied 12 h before ⁶⁰Co γ -irradiation (dose 4 Gy) has been shown to act through the thrombomodulin (Thbd) pathway [92]. Yokota and co-workers observed that the decreased survival rate of surrounding normal human fibroblast WI-38 cells caused by gamma radiation or accelerated carbon C^n + ions can be alleviated by special nitric oxide radical scavengers [93]. In addition, the authors claim that, surprisingly, the effect of radiation dose is more important than the quality of radiation. Indeed, the discussed results indicate that the effects of protectors on biological objects (plasmids, fibroblasts) exposed to accelerated heavy charged particles (C^n +) may be generally like those observed in gamma/photon irradiation (when applying the same dose). This is true despite fundamental differences in the secondary mechanisms of interaction of these two types of radiation with the irradiated matter. Therefore, the last cited article states that the applied dose is an important parameter influencing the effect of protectors. All these observations again show the complexity of the processes taking place in living organisms irradiated in the presence of the modifier. Nevertheless, it can be assumed that even at the cellular level, the protective effect of the protector is primarily based on the protection of the DNA of the cell. This was proven, for example, when human spermatozoa were irradiated with doses from 22.62 to 67.88 mGy in the presence of e-aminocaproic acid (50 ng/mL). A decrease in sperm DNA fragmentation, an increase in total DNA integrity and, as a result, an increase in sperm motility and viability were observed [94].

A special area in the study of radiation protection of cells is the use of microbial cultures. This is an area in which it is possible to apply the exact and quantitative approach to evaluation of protectors. From the obtained results it is then possible to better understand the mechanism of action of individual protectors.

A study of large number of different scavengers of OH radicals led to the conclusion that scavengers affect both the fraction of surviving cells and the shoulders of the survival curves [95]. Systematic studies have shown [2, 3] that the protective effect of each scavenger is unique and specific. It was found that alcoholic scavengers (methanol, ethanol) are more effective than non-alcoholic ones (e.g. potassium formate). It has been shown that bacterial cultures are more sensitive to the presence of scavengers than yeast cultures [2,3,4]. In addition, it was found that the protective effect is a non-monotonic function of gamma radiation dose rate [96] and the effect was not affected by hypothermia [97]. However, unlike hypothermia, hypoxia is a factor affecting the protective effect, which not only reduces the radiosensitivity of cells [14] but also reduces the protective effect of scavengers such as methanol, ethanol or potassium formate [4]. The exact method of evaluating the effectiveness of protectors and, in particular, the specific conditions under which they can be used are described in chap. 2. 3.

Tissues/organs

Much attention was paid to the protection of the glands, especially the thyroid gland. Melatonin appears to be a promising protector in this area. It helps to protect the structure of the thyroid gland from the unwanted cytotoxic effects of radiotherapy. This was confirmed in rats when melatonin was administered (50 mg/kg) intraperitoneally 15 min before irradiation with 6 MV photon beams with a total dose of 18 Gy (dose rate 2 Gy/min.). The percentage of apoptosis during radiotherapy alone was significantly higher (p < 0.05) than in the melatonin and radiotherapy group [98]. No difference was found between low and high dose rates at the same dose [99]. It is possible that the active substance in melatonin affects DNA-ligase (chap. 2. 2. 1). Similarly, intraperitoneal injection (1 mg/kg) of cimetidine (H₂—blocker; C₁₀H₁₆N₆S) administered 1 h before irradiation (gamma radiation dose 10 Gy) of mice minimized thyroid damage. At the same time, the scavenger prevents significant changes in weight loss and causes a reduction in serum levels of thyroid hormones [100]. The process is obviously associated with a number of secondary changes. A protective effect was also detected during local pretreatment of the salivary gland of rats with the dose of 20 Gy and 5 mg/kg phenylephrine (aromatic amine C₉H₁₃NO₂). According to the authors [101], the protective mechanism of phenylephrine may be related to improved expression of aquaporin 5 and reduced activation of c-Jun N-terminal kinase. A similar radioprotective effect on the salivary gland was noted with the intraperitoneal application of kaempferol (a flavonoid from fruits and vegetables) at a dose of 10 mg/kg before fractionated irradiation of male Wistar rats with a total dose of 20 Gy [102]. Kaempferol is a known antioxidant agent and this may be related to its protective effect. Thanks to this, the structure of the salivary gland was preserved in previously protected rats and almost no further degradation, except for some biochemical changes, occurred. Infliximab (monoclonal antibody) also shows a protective effect on the salivary gland of rats irradiated with a dose of 20 Gy by reducing inflammatory processes [103].

The application of radioprotectors leads to numerous changes in radiation-induced nephrotoxicity. Atorvastatin (a fluorocyclic hydrocarbon C₃₃H₃₅FN₂O₅) administered in doses of 10, 20 and 50 mg/kg by oral gavage for 7 days was found to have a significant protective effect against radiation-induced nephrotoxicity in mice. Decreased levels of oxidative stress, urea and creatinine concentrations, tubular degeneration, decreased atrophy, as well as caspase-3 immunoreactivity were observed on the eighth day of the experiment in animals irradiated with X-ray. Based on these results, atorvastatin may be considered a promising drug to protect patients during radiotherapy [104]. Selenium nanoparticles or sodium selenite can also be used against radiation-induced nephropathy. Intraperitoneal doses (0.1 mg kg^–1) applied for 14 days during intermittent gamma radiation (0, 2, 8 Gy) led to the normalization of all monitored parameters. The first modifier, acting as a strong antioxidant, was more effective than the second one [105]. The protective effect of sodium selenite is also related to its antioxidant effects, similar to the treatment of breast cancer, where a similar dose of X-rays (10 Gy) was applied (chap. 2. 2. 2.). The antioxidant effects of silymarin (extract from the seeds of Silybum marianum) can also be used to protect the kidneys. Silymarin has been successfully applied to human embryonic kidneys (HEK). Preincubation of HEK in the extract reduced the concentration of ROS induced by gamma radiation by 21%. As a result, a decrease in apoptosis (about 76%), radiation-induced micronuclei and DNA damage concentration (> 69%) and an increase in mitochondrial membrane potential were observed [106].

It was shown that for kidney cancer radiotherapy, as for many other cases, the agents with strong antioxidant effects are effective protectors. This was also confirmed by whole-body irradiation of rats with gamma radiation with a total dose of 10 Gy in the presence of the antioxidant quercetin (natural polyphenol substance of plant origin) administered orally before and after irradiation at a dose of 50 mg/kg day, where after examination of the kidneys and liver it was stated that quercetin is thought to have a protective potential against radiation induced liver and kidney damage [107]. Mouse livers can also be protected by miR-146a-5p protector (small microRNA molecules), which inhibits the progression of hepatic fibrosis [108]. Melatonin reduces lung radiation damage caused by X-rays (12 Gy) [109]. Here too, however, it is possible, similar to when melatonin is applied to the thyroid gland (see above), that part of the mechanism of melatonin's activity is the influence of DNA-ligase. In addition to antioxidants, immunostimulators are also often used as protectors. They help reduce the risk of carcinogenesis induced by radiation (including low-dose exposure e.g. during CT diagnostics). An example can be CpG-oligodeoxynucleotide (short single-stranded synthetic DNA molecules) used in CT diagnosis of mouse lungs [110]. Similarly, the alkaloid endogenous metabolite sepiapterin (C₉H₁₁N₅O₃) has an ameliorative effect on mouse lung (or heart) after post-radiation oral administration [111].

As in many other cases, nanoparticles are used as protectors to protect the lungs. For example, cerium oxide nanoparticles were applied 2 h after 15 Gy irradiation protected lungs in irradiated mice. Significantly reduced collagen deposition and reduction in structural damage was observed. In addition, a reduction in the inflammatory response was found after applying the protector. As a result, an increase in percent survival of 30–90% was observed for protector doses of 100 nM and 10 µM [112]. It is obvious that the protective effect of cerium oxide nanoparticles manifests itself comprehensively.

The lungs are particularly sensitive to the occurrence of delayed reactions to acute radiation syndrome. So far, it's hard to find ways to effectively mitigate them. Even so, it was demonstrated that the preparation BIO 300 (200 mg kg^–1) used in C57L/J mice whole-body subsequently irradiated with a dose of 7.75 Gy, reduces the occurrence of interstitial lung inflammation and pulmonary fibrosis after 180 days [113]. Therefore, according to the authors, BIO 300 seems to be a promising prophylactic lung protection agent.

Melatonin administered intraperitoneally (10 mg/kg) 15 min before low-dose rate (LDR-RT) or high-dose rate (HDR-RT) radiotherapy (at a total dose of 16 Gy) protected against induced brain damage in the early period of radiotherapy. Significant differences in histopathological and biochemical parameters were found in the experimental rat model [114].

Similarly, intraperitoneal administration of melatonin (50 mg/kg) 15 min before 6 MV X-ray irradiation of rats with a single dose of 8 Gy at dose rates of 4 and 14 Gy/min. found that melatonin can be effective in the preventive protection of rat testes [115]. At the same time, the dose rate does not have a fundamental influence on the effect of the protector.

From the research to date, it is clear that melatonin is a promising protector applied in the therapy of various organs (thyroid gland, lungs). Its action apparently depends on the conditions of its use. This is evident from the fact that in certain cases, melatonin can, on the other hand, increase radiation sensitivity, as it was mentioned in connection with breast cancer radiotherapy (chap. 2.1.3.) (a similar situation as with nitric oxide – chap. 2.1).

Alleviation of histopathological changes in the rat brain was observed also with the use of wogonin (5,7-dihydroxy-8-methoxyflavone, an active ingredient isolated from the root of Scutellaria baicalensis Georgi) with antioxidant and anti-inflammatory properties at a concentration of 30 mg kg^–1 and applied before or after irradiation [116].

Amifostine also has protective effect on irradiated brain tissue. This was confirmed in zebrafish larval brain irradiated with ¹³⁷ Cs gamma radiation at a total-body dose of 2 Gy. Application of amifostine (4 mM) resulted in suppression of apoptosis [117]. It may be related to the protective effect on the functional activity of the DNA-ligase enzyme involved in DNA (chap. 2.2.1.).

Effective intervention against brain damage caused by radiation can be ensured by the application of ginkgolide B (terpenic lactone C₂₀H₂₄O₁₀) extracted from ginkgo biloba leaves. This was demonstrated by irradiating C57BL/6 mice and SH-SY5Y cells with X-rays at a dose of 20 Gy, where a reduction in ROS production and a change in the concentration of some proteins were demonstrated [118].

In terms of radiation protection, attention was paid to some other organs. Radiation damage to the heart could be alleviated by injections of the Chinese preparation shenmai (Chinese patent-protected injection, which was mainly made from Red Ginseng and Radix Ophiopogonis). The study was performed on Sprague–Dawley rats and H9c2 cell line. In vivo, the shenmai reduced the serum biomarkers of cardiac damage, ameliorated cardiac dysfunction after irradiation, mitigated cardiac structural damage, fibrosis, and macrophage infiltration. In addition, the inhibition of oxidative stress was found. In vitro, increased cell viability and decreased excess ROS production were found [118]. Ethanol extract of Alocasia indica tuber applied for 7 days at doses of 200 and 400 mg kg^–1 before γ-irradiation (2.9 Gy) showed a radioprotective effect against radiation damage to the ovaries and uterus of female Swiss albino mice. Reducing oxidative stress and ROS level led to the restoration of ovarian and uterine function and many other improvements [120]. The mouse uterus can be protected by L-carnitine (a quaternary ammonium compound and a derivative of the amino acids lysine and methionine). After irradiation with a dose of 8.3 Gy the oxidative stress decreased in the L-quarantine applied groups and the tumor was not protected from radiation damage to the same extent as normal tissue [121]. Litchi chinensis leaf extract showed photo-chemo-protection of human skin L 929 or red blood cells against UVB radiation through reduction of ROS production and cytoprotective activity against hydrogen peroxide. In this way, the protector (which is not cytotoxic) acts against DNA damage of cells [122]. In the radiotherapy of AY-27 rat bladder tumor using multistage X-ray irradiation with a total dose of 40 Gy using ascorbic acid-2 glucoside (a natural vitamin C) as a protector applied in an amount of 250 mg kg^–1 before each irradiation was proved that the intestinal damage (shortening of villi) was reduced without reducing the effectiveness of radiotherapy against bladder tumors [123]. Effective mitigation of radiation damage to the intestinal tract of mice irradiated with the dose of 15 Gy has been observed with the use of trichostatin A (C₁₇H₂₂N₂O₃ – antifungal antibiotic). The protector promoted survival and structural restoration of the irradiated intestine [124]. Ferrostatin 1 (C₁₅H₂₂N₂O₂) also has protective effects on the intestinal system, which acts via a reductive mechanism to prevent damage to membrane lipids and thereby inhibits cell death [125].

Large body of papers exist on dealing with the substances protecting living cells from UV radiation. The articles presented here are only examples from such a wide field. They indicate that the mechanism of action of UV protectors consists primarily in a decrease in ROS production as a result of the antioxidative properties of the protectors. Obviously, the same mechanism also applies in many cases of irradiation with ionizing radiation.

Organisms

Great attention is paid to radiation protection of the organism against acute radiation syndrome. One of the effective protectors is human umbilical cord blood, a rich source of stem cells. Its use results in improved survival, accelerated hematopoietic recovery, reduced gastrointestinal tract damage, and mitigation of radiation-induced pneumonitis and pulmonary fibrosis [126].

Much of the testing has been done on rats. For example, α-tocopherol (vitamin E—10 mg kg^–1) in combination with ascorbic acid (vitamin C—20 mg/kg) administered before irradiation protected rats irradiated with total body doses from 2 to 100 Gy, while individual vitamins showed no protection. The protective effect was attributed to the reduction of chromosomal aberrations [127]. As another example can be given a mixture of green tea (GTE) and grape seed extracts (GSE) applied to rats. Application of the mixture (GTE:GSE = 1:2) after gamma irradiation with a dose from 5 to 10 Gy increases the concentration of immune cells and all hematological parameters. In addition, it leads to a decrease in the pro-inflammatory cytokines tumor necrosis factor α, C-reactive protein, cholesterol and triglycerides [128]. Similarly, ethanol extract of mulberry (Morus alba) leaves administered orally (100 and 200 mg kg^–1) before exposure to gamma radiation (7 Gy) had a radiation protective effect against irradiation of male rats by improving many biochemical parameters [129]. The protective effect of silymarin applied (70 mg kg^–1) before irradiation (4 Gy) in combination with mesenchymal stem cells (introduced intravenously after irradiation) in male rats is probably also due to hematological and biochemical changes [130]. According to the authors, the action of this protector is based on antioxidant effects, similar to the radiation protection of the kidneys (chap. 2. 2. 3.). Effective protection of male Wistar rats irradiated with whole-body X-rays at a dose of 6 Gy can be ensured by oral application of green barley juice. The conducted experiments confirm that this natural protector exhibited a potential prophylactic radioprotective effect in rats, particularly in testes [131]. Rats were also pretreated with 240 mg kg^–1 N-acetylcysteine (NAC). The results of electrocardiograms and many biochemical tests led to the conclusion that NAC counteracts against cardiac damage induced by radiotherapy [132]. Therefore, the authors believe that NAC may be a potential radioprotector able to prevent heart damage. Overall, it can be stated that a large number of protectors used in whole-body irradiation and in particular protectors obtained from natural substances (extracts from green tea and grape seeds, ethanol extract from mulberry leaves, extract from milk thistle seeds) lead to various changes in hematological and biochemical parameters, acting against various radiation damages inclusive inflammation processes.

Many experiments have been done with mice. Polyaminothiol structures with acceptable systemic toxicity were found to protect γ-irradiated (8.6 Gy) mice. The presence of an aminothiol topically applied to the skin of mice served as a prevention of induced dermatitis after irradiation with the dose of 17.3 Gy [133]. 6,7,3′,4′-tetrahydroxyisoflavone applied subcutaneously before irradiation of mice led to various changes, including a reduction in apoptosis. All observed changes resulted in protection of mice from γ-irradiation-induced lethal damage presumably by restoration of hematopoietic function [134]. The protective effect can also be induced by prophylactic nitric oxide synthase, compound T 1023 (a vasoactive enzyme creating nitric oxide with a specific hypoxic mechanism of action in tissues), applicated 5–120 min. before whole-body irradiation [135]. When intraperitoneal administration (75 mg/kg) was used, the dose reduction factors (ratio of doses with and without modifying agents producing the same level of biological effect) of 1.6 to 1.9 were achieved. According to the authors, nitric oxide synthase inhibitors are sufficiently safe compounds and represent new tools for the prevention of acute radiation syndrome. It was recently demonstrated that ferrostatin applied intraperitoneally to mice 1 h before and 24 h after total body irradiation (9.0 Gy) restored intestinal structure and its physiological function [87]. A newly developed polyphenolic composition derived from lignin with ammonium molybdate is recommended for relief of acute radiation syndrome. Its effect was verified on mice exposed to total body irradiation (4–8 Gy). Attenuator (81 mg kg^–1) was applied 24 h after irradiation. The achieved dose reduction factor was 1.1 and improved survival. The mitigating effect was mediated by an increase in extramedullary hematopoiesis in the spleen and a protective effect on the intestinal epithelium [136].

It is clear that, similar to the case of rats, also in the case of irradiation of mice, it is proven that suitable protectors of a chemically different nature (e. g. thiol, flavone, phenolic structures) can show protective effects against the consequences of whole-body irradiation. Some of them are applicable in clinical practice. Under certain conditions, they can lead to the prevention of radiation damage, decrease in apoptosis, recovery processes or mitigation of acute radiation syndrome.

Summary

Many protectors have already been successfully applied to protect normal cells during irradiation of cell systems (nitrous oxide during irradiation of fibroblasts), without significantly reducing the effectiveness of curative processes (NaSe during irradiation of breast cancer cells, phenylacetates, oxycholates or DMSO during irradiation of human colon cancer cells). Protecting effect often occurs as a result of a reduction in oxidative stress due to the blocking of free radicals produced during irradiation. DNA protection and protein repair play an important role in mammalian or breast cancer cells irradiated in the presence of benzimidazole, DMSO or glycerol. However, the data on the effectiveness of protectors need to be presented in a quantitative and unified way. The presence of oxygen is very important for the protective effects and the effect of oxygen depends non-monotonically on the dose rate. It was clearly shown by the results of the study of microbial cell cultures. A similar study would need to be done with somatic cells in the future.

Whole organ irradiation studies have confirmed that many organic substances reduce radiation damage of various organs (e. g. thyroid gland, submandibular gland). The mechanism of such a complex action remains unknown, but it is quite evident that the protective effect may also depend on the dose rate (observed in the case of glands and brain). Renal radiotherapy can be successfully performed in the presence of some organic protectors or selenium compounds in the form of nanoparticles. Positive results were also obtained for lung irradiation in the presence of CeO₂-nanoparticles. The use of various extracts during ovaries and uterus irradiation or for mitigating the adverse effects of UV radiation on the skin or red blood cells seems promising. These protective effects appear to be often related to a decrease in ROS concentration in irradiated organs.

Research on the effect of protectors on irradiated organisms focuses especially on the irradiation of mice or rats. Interestingly, combination of different protective substances (vitamin E + vitamin C, green tea + grapefruit seed extract, silymarin + mesenchymal stem cells, polyphenolic composition derived from lignin + ammonium molybdate) have been found to be effective means to alleviate acute radiation syndrome. Many protectors show a protective effect when applied prior to radiation (vitamin E + vitamin C, N-acetylcysteine, cimetidine, phenylephrine, silymarin, ethanol extract of mulberry leaves applied to rats, 6, 7, 3′, 4′ tetrahydroxyisoflavone applied to mice, ferulic acid extract applied to human umbilical vein endothelial cells). In the case of wogonin, a protective effect was observed before and after irradiation of rats. Applications after irradiation were successfully tested on various objects (cerium oxide applied to lungs, green tea + grape seed extracts applied to rats, polyphenolic composition + ammonium molybdate applied to mice).

Similar to sensitizers, no specific group of chemicals has yet been preferred. However, nanoparticle protectors have been studied quite extensively (e.g., Se-nanoparticles, CeO₂ -nanoparticles). Again, similar to sensitizers, attention is focused on the study of multi-component protectors (vitamin E + vitamin C, silymarin + stem cells, polyphenolic composition derived from lignin + ammonium molybdate). Most research papers focus on maximum protection of normal cells while maintaining maximum radiation damage to malignant cells.

As mentioned earlier, melatonin, one of the frequently used modifiers, protects against radiation damage of thyroid glands through affection of DNA-ligase, lungs through reduction of oxidative stress or brain where differences in histopathological and biochemical parameters were observed. On the other hand, however, it is remarkable that melatonin increases the radiation sensitivity of breast cancer cells via estrogen regulation leading to the reduction in cell proliferation. These diametrically different activities can be caused by the fact that, on the one hand, melatonin affects the formation of certain enzymes and thus increases the sensitivity of the irradiated biomaterial. On the other hand, it protects the functional activity of DNA-ligase and has antioxidant effects, as a result of which it acts as a protector under certain conditions. These effects are, of course, accompanied by other histopathological and biochemical changes. Similar contradictory effects were observed when nitrous oxide was applied: mitigates the radiation-induced decrease in fibroblast survival and at the same time prolongs the repair time of radiation-induced damage caused by the formation of purine and pyrimidine radical adducts. In these cases, the modifier initiates different reactions depending on the conditions of its application, and the resulting effect is given by their superposition.

The protective effect can be determined by a certain functional group of the protector. For example, phenylacetate (salt) shows an optimal protective effect in the radiotherapy of human colon cancer cells, and phenylephrine (an aromatic amine) shows also a protective effect in the irradiated submandibular gland. Therefore, it can be assumed that the phenyl functional group plays an important role in this protective mechanism.

Regardless of the variety of processes caused by the interaction of ionizing radiation and the protectors, it seems that the action of protectors is caused by two main final processes. The first one is the reduction of DSB and SSB damage, the second is the scavenging of radiation-induced radicals and other species with high redox potential towards the chemical structures present in the irradiated objects. In addition to these mechanisms, the protector can act through a variety of other interactions. Some protectors act through enzyme protection (DNA-ligase) or through the repair of damaged proteins (DMA-protector). In some special cases, protection is realized by reducing the deposition of collagen (CeO₂-nanoparticles in protection of lung cells). The protective effect can also be realized by changes in hematological and biochemical parameters or restoration of hematopoietic functions. In general, it can be stated that the effect of protectors is often attributed to reduction in the concentration of ROS in cells and tissue and consequently to the antioxidant and anti-inflammatory effects and, therefore, to anti-oxidative stress. In addition to these processes, a number of other mechanisms are involved in cell protection. In total, about ten types of radiation protection processes for biological objects can be distinguished [71].

Many protectors are currently in clinical use. They mainly serve to protect the normal cells during the irradiation of a lesion or during the examination of various organs using radiation techniques (X-ray, CT, etc.). They are used, for example, in CT diagnosis of breast cancer cells (sodium selenite), radiotherapy of thyroid or brain tumors (melatonin). The application of atorvastatin acting against radiation-induced nephrotoxicity appears to be highly promising. N-acetylcysteine has been identified as another promising protector counteracting the damage of the heart caused by radiotherapy. It can be assumed that around fifty different radioprotectors, mainly of organic origin, are currently used (or can be potentially used) in clinical practice [69].

Regarding the amount of protector applied, it can be stated that this is limited, among others, by the chemical toxicity of the protector. On average, this amount ranges from tens to hundreds mg kg^–1. The applied doses are on average around 10 to 20 Gy. In general, the clinical application of protectors before, during or after radiation results in a better condition of patients.

Summarizing the results achieved in the field of radiation protection of cells by chemical means leads to the following conclusions:

The following facts can be considered proven:

The main basal mechanism of the protective effect of protectors is the reduction of the number of DSB and SSB DNA damage.

An equally important mechanism is support for the repair of radiation-damaged DNA molecules, proteins and enzymes.

The protective effect is in many cases related to the presence of oxygen during irradiation and can be influenced by the dose rate.

From an overall point of view, the action of protectors leads to a reduction in the concentration of ROS, inclusive OH radicals, which results in the elimination of oxidative stress.

In the future, it will be interesting and useful to focus, among other things, on:

Development and application of standard methods for evaluating the effectiveness of protectors.

Systematic research on the influence of different functional groups in the molecules of protectors.

Detailed examination of the controversial effect of some protectors (melatonin, nitrous oxide) on various biological objects.

Compare the effectiveness of protectors in a standard way, for example using the DRF (dose reduction factor) value.

General notes on the method of quantitative assessment of the effect of chemical substances on the radiation sensitivity of cells

The last chapters showed that a relatively large number of works deal with the influence of various chemical substances on the radiation sensitivity of cells, their components, organelles or whole organisms. However, in many works, mainly qualitative or semi-quantitative methods of evaluating the obtained results have been used so far. Their repeatability and reproducibility are therefore often problematic and makes their mutual comparison difficult. Perhaps the most suitable system for the basic studies are cell cultures or microbial cultures. A common method of evaluating the growth of a microbial culture (unirradiated, irradiated, irradiated in the presence of a modifier) is to measure the slopes of the linear parts of survival curves presented in semi-logarithmic coordinates [137]. Various mathematical methods are also developed to describe survival curves in the presence of chemical modifiers. An example is the linear-quadratic model used for description of the influence of halogenated pyrimidines on growth curves of microorganisms at both low and high doses of radiation [138].

One of the objective methods was developed to evaluate the effect of OH radical scavenging on the radiation sensitivity of yeast and bacteria [2, 3] or Chinese hamster V79 lung fibroblast cells [96]. This method can be extended to other cells irradiated in the presence of sensitizers or protectors. The method is based on the repeatedly proven fact that the modification effects Ϭ or α, defined by the relations Ϭ = ln S₀ × (ln S_M)^–1 and α = (S_M – S₀) × S₀^–1, are linearly proportional to the concentration c_M or scavenging efficiency Q_M of modifiers in a wide range of concentrations or modification efficiencies [2,3,4]. The symbols S₀ and S_M represent the fractions of surviving cells without and in the presence of the modifier. The scavenging efficiency Q_M is defined by the relation Q_M = k_M × c_M, where k_M is the rate constant of the reaction between scavenger and OH radicals. In this way, the scavenging efficiency represents the reaction rate of the scavenger with OH radicals. The extent of modification is defined by the slopes k_Ϭc, k_ϬQ or k_αc, k_αQ of the linear parts of the dependence Ϭ = f (c_M) or α = f (c_M) and analogously Ϭ = f (Q_M) or α = f (Q_M). The slopes k_Ϭc = dϬ/dc and k_αc = dα /dc (specific modification effects) represent the modifying effects at unit concentration of the modifier, and analogously the slopes k_ϬQ = dϬ/dQ and k_αQ = dα/dQ represent the modifying effects at unit reaction rate of the modifier with OH radicals. To compare the modification effects of different modifiers, it is convenient to use the modification efficiency Q_M instead of the c_M concentration and describe the modifier effect using constants k_ϬQ or k_αQ. The meaning of all symbols is clearly shown in Table 4. The appropriateness of using the quantity Ϭ or α depends on the nature of the irradiated materials, the applied radiation dose or the effectiveness of the modifier. In general, in most cases the Ϭ quantity appears to be more advantageous [3]. Therefore, the following discussion will be focused only on the Ϭ-quantity (the α-quantity can be discussed similarly).

Table 4 Quantities for the evaluation of modification of radiation sensitivity of cells by chemical agents

Due to the definition of fractions S₀ and S_M (Table 4), both quantities can be generated only from the open interval (0, 1) (we do not consider the hormesis effect). Therefore, the Ϭ quantity has only positive values. The openness of the above interval is obvious because the limit values of S₀ and S_M (0 or 1) lead to two extreme effects (both without the possibility of modification): the first is the complete killing of irradiated cells (generally total inactivation of irradiated objects)—value 0, the second is zero effect of radiation—value 1. These extreme values of S₀ and S_M therefore lead to indefinite expressions for the Ϭ quantity (Table 5). The following relations apply to protectors: S₀ < S_M = >|ln S₀ | >|ln S_M |=>  Ϭ > 1 and k_Ϭ > 0. Analogously in the case of sensitizers Ϭ < 1 and k_Ϭ < 0. For an inert modifier (S₀ = S_M) Ϭ = 1 and k_Ϭ = 0. These basic criteria are summarized in Table 6. For both cases (Ϭ > 1 (protector), Ϭ < 1 (sensitizer)), the linear part of the dependence Ϭ = f (c_M) is described by the equation Ϭ = k_Ϭc × c_M + 1. The constant k_Ϭc represents the sensitivity of the modifying effect Ϭ to changes in the concentration of the modifier c_M. An analogous relationship and physical interpretation apply to the quantities k_ϬQ and Q_M. As an example of the application of the above criteria, Fig. 1 shows the dependence of Ϭ values on the scavenging efficiency of OH radicals (Q_M) taken from our previously cited works. In this case, Escherichia coli was irradiated with ⁶⁰Co gamma radiation at a dose of 90 Gy and a dose rate of 30 Gy h⁻¹ in the presence of methanol, ethanol or potassium formate. It is obvious that the basic condition of the method is fulfilled for all scavengers, thanks to the linear increase of Ϭ values with increasing scavenging efficiency Q_M in the entire interval of measured Q_M-values. Therefore, k_Ϭ > 0. Furthermore, it is clear that Ϭ > 1. This means that all three scavengers act as protectors. It is also clear that the values of k_Ϭ are significantly different (and consequently characteristic and unique) for each protector. The effect of the modification Ϭ is therefore different for each scavenger, even with the same scavenging efficiency (at the same rate of scavenger reaction with OH radicals). It follows from this finding that the protection of bacteria is not determined solely by the rate of scavenging of OH radicals by the investigated scavenger. It is worth noting that this phenomenon has been predicted previously [73] but has not yet been explained. Similar conclusions were made for the yeast Saccharomyces cerevisiae [2, 4].

Table 5 Description of the effects of irradiation in the case of extreme (0 or 1) values of the S₀ and S_M quantities and the indeterminate expressions for the Ϭ–quantity

Table 6 Relations between the S₀ and S_M quantities and intervals for the Ϭ and k _Ϭ quantities for sensitizers, protectors and inert modifiers

Fig. 1

Dependence of the modifying effect Ϭ on the modification efficiency Q_M of methanol (CH₃OH), ethanol (C₂H₅OH) and potassium formate (HCOOK) for E. coli bacteria irradiated with gamma irradiation at the dose of 90 Gy and a dose rate of 30 Gy h⁻¹ and the slopes k_S of these dependences

Conclusions

An overview of both the increase and the decrease in the radiation sensitivity of cells or other biological systems by chemical agents applied in vitro and in vivo was presented.

It was possible to draw some general conclusions from the annotated results of the selected papers although most of the works published so far represent only a simple description of the observed facts. In addition to the conclusions formulated separately for sensitizers and protectors, several general conclusions for modifiers (sensitizers and protectors) can be made:

The fact that a large number of modifiers have already found application in medical practice can be considered a significant result of the study of modifiers.

In the future, it will be useful to pay attention to the following facts:

• The search for new systems of modifiers formed by an optimal combination of various sensitizers or protectors, or even a combination of sensitizers with protectors, in order to achieve a sufficient degradation effect on malignant cells with maximum protection of healthy cells.

• Studying the reactions of modifiers with ionizing radiation and with fragments arising from interactions of ionizing radiation with biological material in order to suppress side effects.

• Study of the kinetics and mechanism of penetration of modifiers into the cytoplasm of cells and their components.

• Pay increased attention to the effect of dose rate on the effectiveness of modifiers.

The overview and conclusions will contribute to creating a picture of the current development of knowledge in the field of modifying the radiation sensitivity of biological material and to the further development of this current topic. They will also help prospectively to increase the efficiency of radiation degradation of tumors and the protection of the organism against ionizing radiation.

The general characteristics of the universal and objective method of evaluating effectiveness of modifiers (sensitizers and protectors) was presented. It will be useful to extend this method to other microbial cultures and to verify its applicability to other biological objects. This method gives the possibility of a deeper understanding of the processes of its sensitization and protection.