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

# **11.2 Radiomitigators**

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

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

#### **Characteristics of an Ideal Radiomitigator**

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

#### **Mechanism of Action**

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

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

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

#### *DNA Repair and Cell Recovery Process*

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

**Fig. 11.15** Radiomitigators: mechanism of action

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

# *Hematopoietic and Immunostimulating Activity (Regeneration)*

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

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

#### **Box 11.3: Radiomitigators**

stress and infammatory damages, activate enzymes involved in repair mechanisms, and/or stimulate the replenishment of damaged tissues.

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

# **11.2.1 Growth Factors and Cytokines**

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

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

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

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

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

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

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

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

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

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

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

# **11.2.2 Cell Therapy Replacement**

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

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

# **11.2.3 Nonsteroidal (NSAIDs) and Steroidal Anti-infammatory Radiomitigators**

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

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

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

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

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

# **11.2.4 Probiotics, Prebiotics, and Fecal Microbiota Transplantation (FMT)**

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

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

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

Probiotics are live microorganisms, added to aliments, that have a benefcial role in reducing pathogenic bacteria multiplying without competitors, promoting intestinal immune barrier function, and preventing translocation of harmful bacteria. Preparations containing Bifdobacterium, Lactobacillus, and Streptococcus ameliorated radiationinduced gut toxicity, reducing the incidence of diarrhea, and delaying the necessity for rescue treatment with loperamide [147]. Randomized controlled trial evidenced that live Lactobacillus acidophilus plus Bifdobacterium bifdum treatment reduced the incidence of radiationinduced diarrhea and the need for antidiarrheal medication and had a signifcant beneft on stool consistency [149]. The anti-infammatory effect of probiotics has been shown in other pathologies such as ulcerative colitis and Crohn's disease. The administration of Lactobacillus spp. decreased levels of different colonic infammatory cytokines such as IL-6, TNF-α, or NF-κB p65 and recruitment of leukocytes to the colonic mucosa. In mice model, administration of *Lactobacillus rhamnosus* increased the crypts survival in radiation-induced enteritis by approximately twofold and reduced epithelial cell apoptosis, which depends on intact TLR2 and COX-2 inhibition in

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

mesenchymal stem cells of crypt [150]. Genetically engineered species of *Lactobacillus plantarum* and *Lactococcus lactis* release SOD inducing antiinfammatory effects and attenuation of enteritis symptoms [151]. Increased production of short-chain fatty acids is one of the most important probiotic protective effects implicated in GI and hematopoietic tissue protection and increased survival of irradiated mice [152]. Several clinical trials seem to indicate that probiotics reduce the incidence of radiotherapy-induced mucositis [148], even though results are diffcult to evaluate, as they vary in the type of cancer patients recruited, radiotherapy modalities used, and type of bacteria used as probiotic [146]. In this regard, choosing the right probiotic can be crucial, and a recently published systematic review concludes that a combination of *Bifdobacterium longum*, *Lactobacillus acidophilus*, *Bifdobacterium breve*, *Bifdobacterium infantis*, and *Saccharomyces boulardii* could be a good combination of probiotics to reduce incident rates of mucositis or ameliorate its symptoms in chemo- or radiotherapy-treated patients [153].

Prebiotics offer a source of enrichment to the microbiome, and dietary interventions have demonstrated to reduce the severity of infammatory intestinal pathologies and thus can potentially serve as a radiomitigative strategy. In fact, a clinical trial (NCT01549782) evidenced that increased consumption of certain prebiotics (fber and plant sugars) was associated with a reduction in days of diarrhea and improved quality of life for irradiated patients [154].

FMT increased the survival rate, elevated peripheral white blood cell counts, and alleviated GI toxicities and intestinal epithelial integrity in irradiated mice [155]. Radiationinduced intestinal edema was strikingly alleviated after 8 weeks of FMT of gut microbes from healthy donors, enhancing benefcial bacteria such as Alistipes, Phascolarctobacterium, Streptococcus, and Bacteroides recovery, whereas the abundance of Faecalibacterium decreased. FMT can reduce the intestinal leakage and enhance the intestinal functions and epithelial integrity in patients with chronic radiation enteritis [156].

Researchers have long known that administering antibiotics to irradiated animals can enhance survival by avoiding opportunistic infections. As previously have been exposed, antibiotics such as fuoroquinolones and ciprofoxacin also have the advantage of reducing radiation damage to hematopoietic progenitor cells. Antibiotic cocktail and metronidazole pretreatment are benefcial to the reconstruction of gut microbes in irradiated mice. Abx pretreatment regulates macrophage polarization in the ileum and downregulates the expression of TGF-β1, thereby preventing intestinal fbrosis and ultimately improving the survival of mice with radiation-induced intestinal injury [157].

# **11.2.5 Angiotensin Axis-Modifying Agents**

Radiation nephropathy has emerged as a signifcant complication in RT and is a potential sequela of radiological terrorism and radiation accidents. The use of a high-salt diet in the immediate post-irradiation period signifcantly decreases renal injury but is deleterious for the treatment of established disease. FDA-approved drugs that modify the reninangiotensin system are habitually used for the treatment of hypertension and cardiac and/or renal insuffciency. ACEIs constrain angiotensin-converting enzyme (ACEs) and reduce the formation of angiotensin II (AII). Angiotensin receptor blockers (ARBs) impede the function of the angiotensin AT1 or AT2 receptors and decrease the actions of AII.

The effcacy of ACEIs and ARBs has also been long studied for their effects in radiation protection, modulation, or mitigation (Fig. 11.17). Clinical trials have evidenced the potential of ACE inhibitors to reduce radiation-induced pneumonitis and fbrosis (enalapril, NCT01754909, www. clinicaltrials.gov).

Results of a recent meta-analysis review evidenced that the use of ACEIs, but not ARBs, effectively reduced the incidence of radiation pneumonitis in most lung cancer patients. That has important clinical implications because lung cancer patients receiving thoracic radiation could take an appropriate dose of ACEIs to prevent radiation-induced pneumonitis, during or after the period of RT, which would greatly improve the quality of life and therapeutic effect. By contrast, even the most expensive ARBs were ineffective [158].

Five different **ACEIs** (captopril, lisinopril, enalapril, ramipril, and fosinopril), at clinically relevant doses, have been examined for effcacy as mitigators of radiation-induced nephropathy. Overall, survival in rats is higher after an 11–12 Gy TBI when treated with any of the ACEIs captopril, enalapril, or fosinopril starting 1 week postirradiation [159]. All, except fosinopril, effectively abrogated radiation nephropathy, with captopril being the most effective [160].

**Captopril** treatment increased survival from thoracic irradiation to 75% compared with 0% survival in vehicletreated animals, and suppression of infammation and senescence markers, combined with an increase of anti-infammatory factors, was part of the mechanism involved in its therapeutic effects [161]. Captopril reduced radiation-induced cytokines EPO, G-CSF, and SAA (Noninvasive serum amyloid A) in the plasma, mitigated brain microhemorrhage at 21 days postirradiation, and increased EPO levels postirradiation if started prior to radiation exposure. These data suggest that captopril may be an ideal countermeasure to mitigate H-ARS following accidental radiation exposure [162]. A trial of captopril in patients receiving TBI demonstrated not only safety, but also effcacy against renal and pulmonary injury [163]. Moreover, prophylactic administration of captopril reduced radiation-induced hypertension

**Fig. 11.17** Role of ACEIs, ARBs, and renin inhibitors in the renin–angiotensin system

and renal failure and mitigated pulmonary endothelial dysfunction and radiation-induced pneumonitis and fbrosis. The isofavone genistein appears to work synergistically with captopril, improving the 30-day survival in mice receiving both drugs from 0 to 95% after 8.25 Gy TBI. The combination therapy reduced anemia and increased the number of circulating hematopoietic cells [164].

In murine models, administration of AT1 receptor antagonist before, during, and after fractionated whole-brain irradiation prevented or reduced cognitive impairment. It is also hypothesized that ARBs may attenuate radiation-induced brain injury by increasing the generation of anti-infammatory peptide, angiotensin (1–7). ACEI or AT1 antagonist treatment in hypertensive patients increases blood levels of angiotensin (1–7); prevents oxidative stress, infammatory cytokine release, and fbrotic events; and also has anticarcinogenic effects, thus having radiomitigating potential as it has been evidenced recently [165].

While other types of antihypertensive drugs are ineffective, ACEIs and AII receptor antagonist type I are effective in the mitigation of radiation damages. Moreover, some of them also exhibit antitumor effects; thus, there is a strong case for the clinical use of these agents in the treatment of radiation-induced late effects.

# **11.2.6 Statins**

The incidence of cardiovascular disease was observed in the atomic bomb survivors, and cardiovascular disease is a known side effect of radiation therapy [166]. Statins (simvastatin, lovastatin, pravastatin, and others) are inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is a rate-limiting enzyme for the synthesis of cholesterol and serves to upregulate low-density lipoprotein (LDL) synthesis. Therefore, statins are clinically used to reduce LDL levels in the blood and, consequently, to treat atherosclerosis and hypercholesterolemia. Statins also strongly induce thrombomodulin (TM) expression, which in turn forms a complex with thrombin. Thrombin-TM complexes activate protein C, which has anti-infammatory, anticoagulant, and antioxidant properties. All these benefcial effects may help to attenuating radiation injuries [167].

Radiation exposure (5 Gy X-rays) increased cholesterol levels, and those were reduced by simvastatin treatment [168]. Simvastatin treatment (20 mg/kg/d over 2 weeks) mitigates, to a limited extent, radiation-induced enteric injury (4–8 Gy), as evidenced by improved structural integrity of the mucosa, reduced neutrophil infltration, decreased thickening of the intestinal wall, and reduced accumulation of collagen I in jejunum and bone marrow in male C57BL/6J mice [169]. Simvastatin also prevented radiation-induced marrow adipogenesis and provided radioprotection to the niche cells [170], and attenuated radiation-induced salivary gland dysfunction in mice [171]. Pathak et al. [167] demonstrated that a single subcutaneous dose of γ-tocotrienol (GT3) rescues mice from lethal radiation doses, and combined treatment (GT3 + simvastatin) provides substantial protection against radiation-induced lethality, hematopoietic injury, and bone marrow damage compared to the single treatment.

A combination of statin and ACEI agents has shown effcacy in reducing GI toxicity in patients receiving pelvic RT [172]. Lovastatin treatment of irradiated mice (15 Gy wholelung irradiation), starting immediately after irradiation or 8 weeks post-irradiation (three times a week), demonstrated a reduction in lung tissue lymphocytes and macrophages, decreased collagen content, prevented lung fbrosis, and improved rates of survival [173].

**Pravastatin** (30 mg/kg body weight given 4 h before irradiation) protected the normal intestine and lung tissues from radiation. The radiomitigating effect of pravastatin was associated with a reduction in the level of radiation-induced DNA DSB. The pravastatin-treated group showed a signifcantly lower apoptotic index of the lung and intestinal epithelial cells and reduced the intestinal expression of ataxiatelangiectasia mutated and γ-H2A histone family member X (H2AX) after irradiation [174]. Statins are generally well tolerated, and their effect was pronounced for delayed radiation injury and for that reason shows potential as radiomitigators.

# **11.2.7 Growth Hormone (GH) and Somatomedin C (IGF1) Analogs**

Long et al. [175] demonstrated that chimeric protein dTMP-GH, a tandem dimer formed by thrombopoietin mimetic peptide and GH treatment, increased survival in mice exposed to 60Co γ-ray photons (6 Gy). Meanwhile, dTMP-GH treatment accelerated the recovery of bone marrow hematopoiesis, promoted skin wound closure, and mitigated ileum injury. Zinc sulfate and GH administration prevented radiation-induced dermatitis in rats [176], and increased GH/

IGF1 levels also reduced radio-induced intestinal epithelial cell apoptosis preserving, in the short term, the effcacy of RT on tumors [177]. GH signifcantly restored follicular development and preserved fertility in female rats exposed to a single TBI of 3.2 Gy [178]. However, in oncology, GH and IGF1 reduce the effectiveness of RT and may frequently cause metastasis and cancer recurrence. Therefore, even if GH/IGF-derived radiomitigative effects are confrmed, further studies of these hormonal treatments would be necessary before translating the results to human clinical trials.

# **11.2.8 Molecular Hydrogen (H2)**

Hydrogen can mitigate IR damages through various mechanisms [122, 123]: (a) directly neutralizes hydroxyl radicals and peroxynitrite [179]; (b) indirectly reduces oxidative stress, by upregulating the expression of different endogenous antioxidant enzymes, i.e., SOD, CAT, and GPx; and (c) shows antiapoptotic and anti-infammatory properties [180]. H2 reduces 8-hydroxy-2′-deoxyguanosine and malondialdehyde levels and increases SOD activity and GSH levels. These fndings suggest that the radioprotective effect of H2 is largely due to the inhibition of oxidative stress. In that sense, H2 has demonstrated in vitro radioprotective effects in cells especially sensitive to IR, such as intestinal epithelial cells, hematopoietic precursors, and spermatogonia [180] these protective effect of H2 are not signifcant when it is administered after radiation [181].

Shin et al. [182] observed that application of H (H2O) to human skin prevented UV-induced erythema and DNA damage, administered even after exposure to RI. Although a lot of in vitro and in vivo research has been done to investigate the potential use of H2 as a radiomitigator, there are scarce clinical data. Kang et al. [183] performed a placebo-controlled, randomized study to evaluate the validity of ingesting hydrogen-rich water in 49 patients with malignant liver tumors, while they were receiving RT at the same time. Patients drinking H2-rich water had considerably higher quality of life (QOL) scores, notably less appetite loss, and much fewer tasting disorders than patients drinking placebo water, and most importantly, no differences were found in tumor response to RT comparing both groups of patients [183]. In cancer patients, H2 has also shown protective effects against brain, lung, and myocardial injury associated with RT, furthermore preventing side effects like anorexia, taste disorders, or bone marrow damage without compromising the antitumor effects of the treatment [180].

The use of H2 is feasible in the clinical practice because it is stable at normal temperatures; it can be easily administered through various routes such as inhalation, drinking, injection, etc. (Fig. 11.18); it can even cross the blood-brain barrier; has a very favorable tolerability profle; and it shows

**Fig. 11.18** Delivery of hydrogen and its protective and therapeutic opportunities in various systems. Adapted from "Molecular hydrogen: A potential radioprotective agent," by Hu et al. [122, 123], Licensed under CC BY 4.0

great effcacy as a potential radioprotective agent [122, 123]. Although the human body does not have the enzymes necessary to produce H2, the colonic microbiota can produce about 12 L of H2 per day under physiological conditions. Many results support the idea that upregulation of H2 gas produced by intestinal bacteria could be used as a valid treatment strategy for various diseases. Since there are several methods to supply external H2, it can be easily administered with little or no adverse effects.

# **11.2.9 Vitamins**

1-Methyl nicotinamide (MNA), a derivative of vitamin B3, signifcantly prolonged survival of mice irradiated at LD30/30 (6.5 Gy), LD50/30 (7.0 Gy), or LD80/30 (7.5 Gy) of γ-rays when the MNA administration started as late as 7 days post-irradiation. Another vitamin B3 derivative, 1-methyl-3-acetylpyridine, was slightly less effcient when it was administered after 7.5 Gy γ-ray exposition. These prosurvival effects might be related to the anti-infammatory and/or antithrombotic properties of the vitamin B3 derivatives and do not seem to be mediated by stimulation of hematopoiesis. These results show that MNA may represent a prototype of a radiomitigator because it reduces the severity and/or progression of radiation-induced injuries when applied several hours or days after exposure to high doses of IR [184].

# **11.3 Internal Contamination by Radionuclides and Treatment**

After various radiological and nuclear incidents, radioactive materials (radionuclides) may be released in the atmosphere where they could be either inhaled as gas, ingested as particulates, or absorbed through intact skin or subcutaneous tissue [185].

The medical consequences of internal contamination are determined primarily by radiation dose and radiation quality. Deleterious effects include dose-dependent deterministic (i.e., predictable) effects; stochastic (i.e., random) effects such as cancer in tissues where radionuclides are retained for prolonged times, and at a suffciently high quantity of contamination; multiorgan failure; and death. The radiation quality or specifc radionuclide(s) has (have) a characteristic emitted energy (alpha, beta, or gamma/Xray), solubility, radioactive half-life, and biological halflife, which is determined by the time required for a compartment, defned by a body organ or tissue or part of an organ or tissue (see Fig. 11.19) to eliminate half of its radionuclide content. The particle size and chemical composition of the radioactive material impact the site of deposition within the body and route of elimination. Finally, comorbidities such as renal insuffciency, hepatic failure, and pulmonary disorders may impair pathways needed for radionuclide elimination from the body, thereby prolonging exposure [186].

The internal contamination with radionuclides involves four metabolic phases:

The excretion of these radionuclides by natural processes can be accelerated using decorporation therapies. This consists of enhancing the action of biological processes through chemical or biological agents, thereby facilitating radionuclide elimination. In the event that radionuclides have been incorporated internally, the objective of the therapy is to reduce the internal dose and thus the risk of biological effects on health. This can be achieved by preventing the incorporation, reducing the absorption and internal deposit of radionuclides, and also promoting their excretion. The decorporation process may have adverse side effects. Therefore, these therapies must be based on risk criteria and applied as soon as possible.

The general procedures are intended to reduce or inhibit the absorption of radionuclides from the GI system, the respiratory tract, or the skin and wounds (Fig. 11.19). Some examples of general procedures are the use of emetics, gastric lavage, laxatives, gastric alkalinization, and irrigation if there are wounds, especially in an emergency scenario. The use of specifc drugs to impede the deposition of radionuclides (decorporation agents) in organs or tissues could avoid accumulation and retention of radionuclides and, obviously, is more effective if treatment is started immediately after internal contamination. Decorporation agents can reduce radionuclide absorption, entry, and deposit in organs and tissues and/or accelerate its excretion, fnally minimizing the absorbed dose.

# **11.3.1 Blockers (Metabolic Blocking)**

Blocking agents work by reducing the absorption of the radionuclide in the body, since they saturate tissues, organs, and metabolic processes using a stable isotope (identical to the nonradioactive element). Among these agents, the best known is potassium iodide (KI), used to prevent the deposit in the thyroid gland of radioactive iodine delivered to the atmosphere as a result of uncontrolled nuclear accident, which can lead to an increased risk of developing thyroid cancers, particularly in infants and young children [187]. KI prevents binding of radioiodine by three mechanisms: a) it will dilute the radioiodine circulating inside the body and available for thyroid uptake; b) it will saturate the active transport mechanism of iodine mediated by the sodium iodine symporter (NIS); and c) it will inhibit the organifcation of iodine, also called *Wolff-Chaikoff* phenomenon, a mechanism that could lead to a decrease in the synthesis of thyroid hormones and a possible hypothyroidism; but this effect is usually of short duration. This measure only protects the thyroid from radioactive iodine, not other parts of the body.

Pharmacologic thyroid blockade by oral KI (50–100 mg in adults) can substantially reduce radioiodine thyroid uptake and was one of the frst and urgent protective actions recommended by the World Health Organization (WHO) (1960–1970s).

The recommendations adopted for iodine prophylaxis, in particular those regarding the administration timing, the iodine quantity to be given, and the possible side effects occurring as a result of this measure, are included in the Guide [188]. Although stable iodine is usually considered as the standard for thyroid protection against radioiodine [189], perchlorate can be considered as an alternative, provided that it is administered at equi-effective dosages (1000 mg perchlorate is as effective as 100 mg stable iodine in the aftermath of an acute radioiodine exposure). Perchlorate also protects the thyroid by competition with radioiodine at the NI-symporter site. Considering its simpler protective mechanism and potential advantages in particularly vulnerable subpopulations and its acceptable adverse effects, it seems promising for future studies to focus more closely on perchlorate as an alternative to stable iodine for thyroid protection against radioiodine [187].

# **11.3.2 Reduced Absorption**

Absorption is defned as a movement of material that reaches the blood regardless of the mechanism. This generally applies to the entrance in the bloodstream of soluble substances and material dissociated from particles (NCRP 161).

Prussian blue, a nonabsorbable resin (approved by the FDA), acts as a laxative agent that promotes the fecal elimination of ingested radiocesium and thallium. The most effective form of this compound is its colloidal soluble form. This compound was used in the Goiânia accident extensively and successfully for the decorporation of 137Cs. Different silicabased materials have also been tested to capture various radionuclides of plutonium, americium, uranium, and thorium [190].

Natural products have also been used to reduce the absorption of radionuclides. An example is that orally administered *Chlorella algae* inhibited the absorption of strontium (90Sr) into the blood and enhanced its fecal elimination [191].

# **11.3.3 Dilution (Isotopic Dilution)**

Increasing the intake of liquids, such as water, milk, and tea, or intravenous administration of isotonic saline solution, is a rapid method to increase the excretion of soluble radionuclides. This would be the case of tritium, where ingestion of suffcient liquids reduces the time of permanence in the body [192].

# **11.3.4 Displacement**

Displacement shares the same principle as dilution and blocking therapies. However, in this specifc case, an element is used that has a different atomic number. Thereby, that element will compete for internal scavenging sites, displacing the radioisotope from a receptor/target. Calcium gluconate, for example, competes with radiostrontium in bone deposition, or stable iodine, which displaces technetium-99m [193].

This method consists of increasing the natural renewal process of the release of radionuclides from organs and tissues, thus reducing deposition and improving the elimination rate by diuresis. As an example, ammonium chloride, which if administered orally, lowers the pH of the blood and increases the elimination of radiostrontium once internalized. Or the use of sodium bicarbonate increases the pH of the blood and favors the removal of uranium [194].

# **11.3.5 Chelators and Functional Sorbents**

Chelating agents are classifed as organic or inorganic agents capable of binding to metal ions and forming complex ring structures, known as "chelates." These agents possess atoms of union or "ligands" that generally form covalent bonds and facilitate the excretion by the kidneys or other organs [186].

Some examples of this method are the one used to facilitate the elimination of plutonium complexes by the kidneys and the GI. DTPA (diethylenetriaminepentaacetic acid with calcium or zinc) is the chelator with the widest range of potential use [186]. Other chelators commonly used are dimercaptosuccinic acid, dimercaprol, and deferoxamine. Different silica-based materials (such as isomers of diphosphonic acid, hydroxypyridinone, acetamide phosphonic acid, DTPA, and glycinyl-urea) have also been tested to capture various radionuclides of plutonium, americium, uranium, and thorium [190]. Importantly, factors that can potentially affect the stability of any chelating agent must always be taken into account, i.e., (but not limited to) acidity and alkalinity, chemical properties of the agent, its selectivity, and concentration of competing metals.

Internal contamination with actinides, whether by inhalation, ingestion, or injuries, represents a serious risk to the health. Some guidelines to assist physicians or other professionals in treating workers or members of the public who may suffer internal contamination with compounds such as plutonium tributyl phosphate, plutonium nitrate, americium oxide, or nitrate can be found in [195].

The use of these types of agents is most effective when administered immediately after exposure to radiation because the radionuclides are still circulating in the body and may not yet have deposited in target organs or cells (liver and bone are examples of preferred targets).

# **11.3.6 Surgical Excision**

This method is used for the elimination of a fxed radionuclide contaminant in the body. The surgery must be evaluated carefully, taking into account risks and benefts, and must be carried out with the support and collaboration of radiation protection staff [196].

Occasionally, debridement and excision of the wound may be necessary in order to remove the fxed contamination. It is important that a well-established evaluation is carried out by specialized personnel to support the medical decision, considering the benefts and risks of the surgical procedure. When surgical exploration is necessary, as well as the removal of tissue/foreign material, it should be performed with the help of a radiation protection professional, a radiophysicist who uses a specifc probe for wounds. Once the surgical material has been removed, it should be saved for subsequent radioanalysis. There are no contraindications regarding the use of local anesthetics or systemic anesthetic agents.

# **11.3.7 Lung Lavage (Mechanical)**

Lung lavage is an invasive medical procedure that involves the same risks as general anesthesia and is only indicated for a limited number of cases. The parameters that are taken into account are the patient's age, clinical status, existence of comorbidities, radiotoxicity of the contaminant, and dose.

This technique will only be used after a meticulous medical and dosimetric evaluation, and in case inhaled and insoluble radioactive particles (plutonium for example) are deposited in the lungs. Other isotopes and focal accumulation are depicted in Fig. 11.20. A fexible bronchoscopy should be performed to enhance bronchoalveolar lavage [197]. This type of bronchoscopy should be performed only if the lung load is high and incorporates a large amount of insoluble inhaled particles, such as alpha particles (α).

The objective of this procedure is to avoid deterministic effects for pulmonary doses above 6 Gy-equivalents (Gy-Eq)

**Fig. 11.20** Isotopes and focal accumulation in the body

and is stochastic when the committed doses are lower in the lung. All this within a period of 30 days and individualized for each case.

The Clinical Decision Guide (CDG) and the IAEA EPR 2018 Guide provide bases that can be used by healthcare providers to treat cases where radionuclides have been deposited internally as explained above. Both guides are useful for medical management of individuals contaminated with radionuclides as a consequence of a nuclear or radiological emergency, or due to an industrial scintigraphy accident, or in patients undergoing treatments with radionuclides.

See Annex 1: Contamination by radionuclides and MCMs table.

# **11.4 Radiosensitizers**

Radiotherapy (RT) is a treatment that uses high doses of radiation to kill cancer cells and shrink tumors. Radiosensitizers are chemicals or pharmaceutical agents that increase the cytotoxic effect of IR on cancer cells by accelerating DNA damage and producing free radicals, suppressing the antioxidant mechanism of defenses, or inhibiting the repair of biomolecules, among others. In most cases, radiosensitizers have less effect on normal cells; however, some can also be administered after radiation exposure to treat or reduce the late side effects to healthy tissue. The effectiveness of potential radiosensitizers is measured in terms of the enhancement ratio (ER) (Box 11.4):

*ER* <sup>=</sup> Radiation dose required toobtain a given biological effect Radiation dose required to obtain thesame effect in the presence of sensitizer

#### **Box 11.4: Radiosensitizers**

#### **Characteristics of an Ideal Radiosensitizer**

For use as an adjunct in RT, an ideal radiosensitizer should not be harmful to healthy tissues and not interfere with other therapies, as well as should be highly effcient on tumor and hypoxic cells. It should also be economically affordable.

A radiosensitizer should be nontoxic and should produce an advantage in enlarging the therapeutic window, increasing tumor control probability, and limiting the normal tissue toxicity. This effective gain could result from a selective uptake or absorption rate or half-life of the radiosensitizing molecule in a tumor with respect to normal tissue.

#### **Mechanism of Action**

Radiosensitizers have been developed to modulate the response that occurs during or after the radiation exposure. At a molecular level, these molecules stimulate the fxing of free radicals generated by radiation. Similarly to the oxygen effect, the biochemical mechanism prevents the repair of damaged molecules. The electron affnity of the radiosensitizers captures independently existing free radicals, rendering the molecules incapable of repair [198]. Although each radiosensitizer has different rationales and limitations, they interact with specifc biological targets, i.e., the signaling pathway/cascade (Table 11.1) at diverse levels (Fig. 11.21) from molecules to cells to tissues to organs to a whole organism. The core mechanisms for radiosensitization include:

#### **Classifcation**

Based on the DNA damage and repair mechanisms, radiosensitizers are divided into fve groups [199, 200]: (1) reduction of thiols or other intracellular radioprotective molecules; (2) radiolysis of the radiosensitizer, which results in the production of cytotoxic chemicals; (3) inhibitors of repair of biomolecules; (4) thymine analogs incorporated into DNA chain; and (5) oxygen mimetics with electrophilic properties.

With the continuous technological innovation, radiosensitizers can be classifed into three categories: (1) molecular

**Table 11.1** Potential biological targets at different levels for developing radiosensitizers

Abbreviations: *DDR* DNA damage response, *DNA-PKcs* DNA-dependent protein kinase, *ATM/ATR* ataxia–telangiectasia mutated and ATM and Rad3 related, *PARP* poly[ADP-ribose] polymerase, *MDC1* mediator of DNA damage checkpoint protein 1, *LIG4* ligase IV, *CDK1* cyclin-dependent kinase 1, *BRCA1* breast cancer gene 1, *CHK1* checkpoint kinase 1, *HIF-1* hypoxia-inducible factor-1, *ECM* extracellular matrix

**Fig. 11.21** Development of potential radiosensitizers at different levels. Potential radiosensitizers can be developed focusing on the molecular, cellular, or organismic levels, which may be useful in modulating the radiation effects on cancer cells as well as on normal cells

#### **Table 11.2** Small molecules as radiosensitizers

#### *Hyperbaric oxygen*

 A potent radiosensitizer, which promotes toxic and relatively stable free radical formation, useful to effectively enhance the radiosensitivity of the tumors which contain numerous hypoxic cancer cells.

#### *NItroxides*

 The most representative are nitro-containing compounds (such as nitrobenzene, nitroimidazoles, and its derivatives) and nitric oxides (NOs). These are "true radiosensitizers," having higher electron affnity and better diffusion properties than molecular oxygen. It can theoretically substitute for oxygen in "repairing/ fxing" radiation-induced DNA damage.

#### *Carbogen*

 A mixture of 95% oxygen and 5% carbon dioxide, which improves tumor oxygenation contrasting with hypoxia.

#### *Hypoxia-specifc cytotoxins*

 Bioreductive agents, such as aromatic N-oxides, transition metal complexes, quinones (mitomycin C, porfromycin, and E09), aliphatic N-oxides, and nitro compounds, that selectively radiosensitize the hypoxic cells by virtue of their preferential cytotoxicity.

#### *Chemical radiosensitizers*

 Chemicals targeting a variety of cell signaling pathways, suppressing radioprotective substances, pseudo-substrates, and targeted delivery systems for radiosensitization. Examples are BKM120 (an oral pan-class I PI3K inhibitor), targets of PI3K-Akt pathway, NVP-BEZ235 (a mTOR inhibitor), AMG 232 (an MDM2-p53 interaction), GSH inhibitors, and radiosensitizing nucleosides (5-fuorouracil (FUra), bromodeoxyuridine (BrdUrd), iododeoxyuridine (IdUrd), hydroxyurea, gemcitabine (dFdCyd), fudarabine).

*Natural radiosensitizers*

 Natural molecules are safer than synthetic compounds and have anti-infammatory and antioxidant properties: curcumin, genistein, resveratrol, zerumbone, ursolic acid, etc.

structures of small molecules (Table 11.2); (2) macromolecules with their mechanism of radiosensitivity (Table 11.3); and (3) nanomaterials (Table 11.4) with low cytotoxicity, good biocompatibility, usability, and functionality (Box 11.5).

# **11.4.1 Nutraceutical Compounds**

Several nutraceutical chemicals have attracted signifcant interest in recent decades due to their possible involvement in the prevention and treatment of various illnesses, as well as their favorable effects in boosting human and animal health. In particular, literature data often report their positive effect in combination with chemotherapy in cancer care. Even while intriguing results have been published on this issue at multiple cellular levels, less is known about their role as radiosensitizers. Presence of these compounds during radiation augments their effect by several mechanisms including the lethal reactions of free radicals.

Among compounds of various origins that showed radiosensitizer potential, numerous studies have revealed the

#### **Table 11.3** Macromolecules as radiosensitizers

#### *Proteins and peptides*

 Antibody conjugates and cell-penetrating peptides selectively deliver a cytotoxic payload to a tumor and spare most healthy cells. Examples are HER3-ADC (targeting HER3), SYM004, and nimotuzumab (targeting EGFR) and cetuximab (inhibitor of EGFR).

#### *miRNAs*

 Endogenous noncoding microRNAs (miRNAs) can be used as RT sensitization targets. These can be regulatory miRNAs of DNA damage response (DDR) and HR repair factors.

#### *siRNAs*

 Exogenous short interfering RNAs or silencing RNAs (siRNAs), which are noncoding RNA molecules, that can selectively target key mRNAs belonging to pathways involved in the response to radiation, such as DDR, cell cycle regulation, and survival/ apoptosis balance.

#### *Oligonucleotides*

 Small DNA or RNA sequences are able to disturb key mRNA translation. Studies have concerned oligonucleotides targeting the telomerase RNA subunit or telomerase reverse transcriptase (hTERT) or cyclic AMP response element (CRE) decoy oligonucleotide.

important role of molecules of natural origin, when administered in combination to IR.

The use of nutraceuticals as sensitizers, in addition to being generally well tolerated, is also easily recovered and less expensive in comparison to synthesized drugs. Their administration reduces the collateral effects frequently associated with medication delivery, and in certain situations, they can help attenuate IR adverse effects through biological processes like those shown in Fig. 11.22. Indeed, in most cases, they show anti-infammatory and antioxidant properties, which are precious arms to counter the RI side effects on healthy tissues.

However, in most cases, they showed direct anticancer activity, as demonstrated by numerous scientifc papers. The most studied natural compounds are exposed in Table 11.5 [203].

#### **<IP_ADDRESS> Curcumin**

Curcumin, the main component in the Indian culinary spice turmeric (*Curcuma longa*), has been shown to have anticancer potential in several studies. The biological mechanism can be ascribed to cell signaling pathway effects, resulting in the inhibition of cell proliferation and induction of apoptosis.

Regarding its radiosensitizing properties evaluated by an in vitro approach, the inhibition of survival and proliferation has been observed on the MCF-7 breast cancer cell line. In addition, the effect of vehicolated curcumin, using solid nanoparticles, combined with X-ray radiation was tested by Minafra and coworkers [204] on the human nontumorigenic breast epithelial MCF10A cell line and the breast adenocar-

#### **Table 11.4** Nanomaterials as radiosensitizers

#### *Noble metal nanomaterials*

 Nanoparticles, such as gold (Au, *Z* = 79), silver (Ag, *Z* = 47), and platinum (Pt, *Z* = 78), can effectively interact with radiation, emitting secondary electrons which amplify the radiation effects.  Examples are gadolinium-based nanoparticles (AGuIX), hafnium oxide (HfO2) nanoparticle (NBTXR3), tantalum pentoxide (Ta2O5) and tantalum oxide (TaOx), bismuth oxide (BiO) nanoparticles, and tungsten oxide nanopowder or nanoparticles (WO3).

#### *Ferrite nanomaterials*

 They can catalyze the reaction of H2O2, generating highly toxic hydroxyl free radicals in the tumor microenvironment with the aim of boosting the radiation therapeutic effcacy. Explored examples are superparamagnetic magnesium ferrite spinel (MgFe2O4) nanoparticles (SPMNPs) and zinc ferrite (ZnFe2O4) nanoparticles

*Semiconductor nanomaterials*

 Semiconductor nanosensitizer materials, such as silicon (Si), germanium (Ge), gallium arsenide (GaAs), and semiconductor quantum dots, have unique properties making them great candidates as photosensitizers and radiosensitizers for tumor treatment ([201]; [202]). Explored examples are WO2.9-WSe2- PEG semiconductor heterojunction nanoparticles (WSP NPs), titanium peroxide (PAA-TiOx) nanomaterial, copper bismuth sulfde (Cu3BiS3,CBS) nanoparticles, and TiO2 nanotubes.

*Nonmetallic nanomaterials*

 Similarly to the metallic nanoparticles' mechanism of action, nonmetallic nanomaterials can increase oxidative damage. Explored examples are ultrasmall uncapped and amino-silanized oxidized silicon nanoparticles; nanocrystals of underivatized fullerene, C60, (nano-C60); nanodiamonds and carbon nanotubes; and selenium (Se) nanoparticles.

*Nanostructured substances and drug delivery systems* Chemicals, oxygen carriers, siRNAs, and other radiosensitizing agents are transported via relatively new nano-based delivery systems. Explored examples are the poly(D,L-lactide-coglycolide) (PLGA) nanoparticles containing paclitaxel (a cell cycle-specifc radiosensitizer) and etanidazole (a hypoxic radiosensitizer).

#### **Box 11.5: Radiation Sensitizers**

cinoma MCF7 and MDA-MB-231 cell lines. The vehicolated curcumin has been shown to be more effective than the free curcumin on MCF7 and MCF10A, whereas the free molecule resulted to be slightly more effective on MDA-MB-231. The dose-modifying factors (DMFs) were calculated to quantify the radiosensitizing effect, which resulted in 1.78 for MCF7 using vehicolated curcumin and 1.38 with free curcumin on MDA-MB-231 cells. Transcriptomic and metabolomics approach supported this study, revealing the double-positive effect of curcumin as an autophagy enhancer for tumor cells and antioxidant agents [204].

Antiapoptotic signals and block in G2/M cell cycle phase mediated by Bcl-2 were demonstrated in human immortalized prostate adenocarcinoma cells (PC-3) after 5 Gy irradiation combined with 2μM curcumin. Instead, an increased radiosensitivity was observed in HCT116 and HT29 human colorectal cancer cell lines treated with 25μM of curcumin and a single dose of X-ray radiation (10 Gy). Curcumin was also able to decrease COX-2 expression by the inhibition of EGFR phosphorylation both in vitro on the human head and neck squamous cell carcinoma (HNSCC) cell line and in two in vivo models of head and neck tumor.

On the human glioblastoma U87MG cell line, the viability was reduced in a dose-dependent manner by 3 Gy of X-ray combined with curcumin at a concentration range of 5–10μM, sustained by the arrest of cell cycle in phase G2/M (which is the most sensitive step to radiation) and the inhibition of two master regulators of tumor progression, the MAP kinases ERK and JNK [203].

However, curcumin is an unstable, nonbioavailable compound due to its poor absorption in the GI system. Hence, its therapeutic application is delimited by its pharmacokinetics. Despite promising preclinical studies, no double-blinded placebo-controlled clinical trial, using curcumin as a radiosensitizer, has been successful. The interaction of curcumin with RT on different cancer types has been reviewed by Verma [205]; however, there is still a lack of solid clinical evidence of radiosensitization. For instance, in vitro and in vivo studies together with clinical bioavailability data do not give evidence for a radiosensitizing effect of curcumin in the treatment of high-grade brain tumors (glioblastoma multiforme). On the other hand, there is limited data on curcumin's radioprotective function, despite the fact that some clinical trials suggest that curcumin is benefcial for the management of radiation toxicities [205].

# **<IP_ADDRESS> Resveratrol (RV) and Pterostilbene (PT)**

The antineoplastic ability of RV encouraged its application also as a radiosensitizer to overcome radioresistance of many cancers.

A dose-dependent reduction in the surviving fraction of a non-small cell lung cancer (NSCLC) cell line after irradiation with 0–8 Gy of γ-rays in combination with 20μM of RV was

**Fig. 11.22** Radiation therapy and nutraceutical substances may infuence signaling pathways involved in migration, infammatory response, autophagy, and formation of reactive oxygen species (ROS). Adapted

from "Nutraceutical Compounds as Sensitizers for Cancer Treatment in Radiation Therapy," by [203], Licensed under CC BY 4.0

**Table 11.5** Natural compounds related to cancer radiation treatments

observed along with accelerated senescence and cell death following enhanced DNA DSB induced by ROS [203].

However, an increased expression of LC3-II for autophagy response after X-ray and RV treatment (75μM) was demonstrated in SU-2 glioblastoma multiforme cell lines [206]. Also, in GBM, RV showed inhibition of the hypoxiainducible factor HIF-1α, which is responsible for a wellknown mechanism of radioresistance. Moreover, the interaction of RV with other agents as iododeoxyuridine (IUdR) was also tested and demonstrated the ability to decrease the formation of cancer colonies [203].

In the HNSCC cancer model, suppression of cell proliferation was obtained on a cell line, treated with 100μM of RV combined with 10 Gy of X-ray, also observing the inhibition of STAT3 phosphorylation, a well-known transcription factor driving infammation and cancer progression. Even the peanut stem extract (PSE), which contains a high amount of RV, has been tested in combination with X-rays, which showed similar radiosensitization effects on radioresistant human prostate cancer cell lines. In this regard, the tumor growth of a prostate cancer xenograft mouse model was reduced with RV and/or PSE (total dose 12 Gy, 5 or 250 mg/ kg, respectively) [203]. RV was also used as a pretreatment (25–150μM) to treat the human NPC CNE-1 cell line with X-ray irradiation (0–6 Gy), revealing the inhibition of the AKT phosphorylated form, a known proproliferative marker. These effects were also confrmed in NPC xenograft models, combining the RV treatment with 4 Gy for 3 days, resulting in a tumor volume reduction.

Nevertheless, a key problem is the short RV half-life and low bioavailability under in vivo conditions. In vivo, pterostilbene was proven to be benefcial in the treatment of melanoma and pancreatic cancer. This study demonstrated that PT can be helpful against melanoma by inhibiting the generation of adrenocorticotropic hormone in the brain of a mouse, which impairs the Nrf2-dependent antioxidant defenses of melanoma and pancreatic tumors. This produces tumor growth restraining and tumor sensitization to oxidative stress. In addition, PT has been shown to increase cancer cell death by the induction of lysosomal membrane permeabilization [53].

#### **<IP_ADDRESS> Withaferin A**

Withaferin A (WA) was the frst withanolide to be isolated and extracted from the plant *Withania somnifera*. WA-induced radiosensitization has been observed in human histiocytic lymphoma, renal carcinoma, and liver, breast, and several other types of cancer. Overall, these studies highlight the effect of combined treatment, mediated by the increase of apoptosis and production of ROS.

WA has been shown to suppress cancer cell growth by targeting the intermediate flament protein vimentin, a structural protein of the cell cytoskeleton. In light of its anticancer capability, WA was also tested in order to investigate its effect in inducing the radiosensitization of cancer cells.

WA's effects were initially investigated in vitro and in combination with γ-irradiation on a lung fbroblast cell line, and WA was found to be well tolerated by cells and to mediate a synergistic impact with γ-rays in terms of cell death. Based on these encouraging results, WA was further tested in vivo to assess its effect as a radiosensitizing agent in several cancer models such as the spontaneous murine mammary adenocarcinoma (Ehrlich ascites carcinoma—EAC), a mouse model of fbrosarcoma, and a mouse model of melanoma. Overall, each of the studies demonstrated that the WA and γ-ray combined treatment inhibits tumor growth, increasing tumor-free survival and median survival time of animals [203].

#### **<IP_ADDRESS> Celastrol**

Celastrol, also known as tripterine, is a triterpenoid derived from the root of the "thunder god wine" plant often found in China and utilized in traditional Chinese medicine for its anti-infammatory qualities in a variety of conditions, including autoimmune diseases. Moreover, anticancer properties have been revealed, due to its proteasome inhibitory activity and antimetastatic ability.

A study has evaluated its radiosensitizer effect on PC-3 cells, both in vitro and in vivo. The in vitro pretreatment with celastrol before irradiation with X-rays resulted in a signifcant dose-dependent enhancement of IR-induced clonogenic cell killing. This effect was explained by (1) a longer gH2AX activation for a longer time in combined treated cells with respect to the only irradiated ones, thus revealing a DNA repair impairment action by celastrol, and (2) a major expression of apoptosis markers (cleaved PARP and caspase-3).

Thus, the same group tested the celastrol radiosensitizer effect on a PC-3 xenograft model. 1 mg/kg of celastrol (5 days/week for 3 weeks) was given to the mice 1 h before irradiation with a single dose of 2 Gy (5 days/week for 2 weeks). The histological analysis showed a signifcantly increased apoptosis and angiogenesis reduction in the combined treated tumors [203].

Similar effects have been found on the NCI-H460 human lung cancer cell line, combining celastrol with 0–4 Gy of X-rays. Indeed, the EGFR, ErbB2, and survivin irradiation markers were found to be reduced, whereas the celastroldependent inhibition of HSP90 was observed. Furthermore, celastrol induces a more pronounced ROS generation after irradiation, thanks to its quinone methide moiety [203].

Finally, the effect of celastrol as a radiosensitizer was evaluated on lung cancer with different approaches. Indeed, one research group has identifed it as one of the most promising sensitizer candidates among 30 drugs, by an in silico study. Thus, they tested its effect in vitro on A549 and H460 cells, subjected to pretreatment with celastrol and 2–10 Gy of dose range. The encouraging results from this in vitro study were the premises for a preclinical study on a A549 xenograft mouse model. The combined treatment using 2 mg/kg/5 each day and 10 Gy of IR for 12 days produced larger intratumoral necrotic areas [203].

#### **<IP_ADDRESS> Ursolic Acid**

Ursolic acid (UA) belongs to the family of the pentacyclic triterpenoids. It is generally obtainable from the peel of many fruits, e.g., apples, blueberries, and prunes, and also in many herbs, such as rosemary and thyme. Recently, the following therapeutic properties of UA have been described: anticancer, anti-infammatory, and antimicrobial, and also its radiosensitization activity in models in in vitro and in vivo studies. For example, in human prostate and colon cancer cells, and in mouse melanoma cells, the UA is able to radiosensitize cells with a signifcant reduction in cell viability associated with an increase of typical signs of apoptosis cascade, such as cell volume reduction, nuclei fragmentation or condensation, caspase-3 activation (one of the key enzymes involved in the apoptotic pathway), increased levels of cleaved PARP (enzyme involved in DNA repair processes), DNA fragmentation, and also increased ROS generation. In melanoma mouse models, the treatment with UA and IR is able to inhibit tumor growth owing to a downregulation of Bcl-2 and survivin, two known key protein regulators of cell survival [203].

Moreover, UA can also exert a differential effect after exposure of normal or cancer cells to UV, acting as a photosensitizer for the latter and as a photoprotector for normal ones. This action was observed in human melanoma cells and in human retinal pigment epithelium control cells, where induced oxidative stress by ROS production, cell cycle arrest, and cell death induction were evaluated following UA and UV treatments. Furthermore, the UA has a signifcant radiosensitizing effect in human gastric adenocarcinoma cells, as evidenced by (i) a decrease in the cell survival fraction and otherwise an increase in the number of apoptotic cells (positive to the propidium iodide and annexin V apoptotic markers); (ii) the arrest of the cell cycle (in the G1 and G2/M phases); and (iii) the increase in ROS amount and a decrease of Ki-67-positive proliferating cells [207].

#### **<IP_ADDRESS> Zerumbone**

Zerumbone (ZER) is a cyclic ketone and a sesquiterpene compound, a cytotoxic component obtained by steam distillation of the *Zingiber zerumbet* Smith. ZER is used in food and herbal medicine, and it also has anti-infammatory, antiproliferative, and antitumor properties, as observed in many tumor types (including breast, pancreas, colon, lung, and skin). In addition, the radiosensitizing effects of ZER on tumors, by means of its regulatory activities on DNA DSB repair, cell cycle, and apoptotic pathways, have been highlighted too [203].

ZER was able to signifcantly increase radiation-induced cell death in human lung adenocarcinoma cells by inhibiting heat-shock proteins (HSP), increasing caspase 3 and PARP cleavage, and inhibiting HSP27 binding to apoptotic molecules such as PKCδ and cytochrome C [203].

In addition, the radiosensitizing effect was also observed in human glioblastoma cells. The same authors showed an IR-induced decrease of cell survival on human prostate cancer cells, associated with a reduced expression of proteins involved in the DNA damage repair pathway, such as γH2AX and ATM [203].

Moreover, in human colon-rectal cancer cells, ZER pretreatment is able to induce apoptosis and enhance radiationinduced G2/M arrest and reduction of activation of the DSB DNA repair machinery.

#### **<IP_ADDRESS> Cafeic Acid Phenethyl Esther**

CAPE is an active component of honeybee propolis, a phenolic compound, and a structural derivative of favonoids. It was described for its antiviral, bactericidal, antiinfammatory, and antioxidant properties. CAPE compound is also able to change the redox state by perturbing the activation of GSH and to induce apoptosis. Furthermore, it has been shown to be more toxic to cancer cells than normal cells, as well as to amplify the action of RT in a variety of cancers.

CAPE has been shown to improve radiation-induced cell cycle arrest and death in human medulloblastoma DAOY cells. In particular, the combined treatment with CAPE and 2 Gy of IR caused an ROS enhancement production, a signifcant inhibition of NF-kB activity, apoptosis activation, and downregulation of cyclin B1 protein expression. In line with these data, a strong reduction of cell survival, in a concentration-dependent manner, was described in the same cell line pretreated with CAPE (0.1–10 M) for 24 h before exposure to γ-ray irradiation at various doses (0–8 Gy), associated with cell cycle progression inhibition, by arresting cells in the S phase [203].

The CAPE pretreatment radiosensitizing effect was also shown in mouse CT26 adenocarcinoma cells, using both in vitro and in vivo approaches showing decreased cell survival rate and reduced NF-kB activation. CAPEinduced decrease of survival rate was also described in breast and lung cancer cell lines. In particular, in MDA-MB-231 and T47D breast cancer cell lines, CAPE and X-ray combined treatments decreased cell growth and delayed the DNA repair process for up to 60 min after exposure [203].

#### **<IP_ADDRESS> Emodin**

Traditional Chinese medicine uses emodin (6-methyl-1,3,8 trihydroxyanthraquinone), a natural phenolic derived from the roots and rhizomes of numerous plants (e.g., *Polygonum cuspidatum* and *Cascara buckthorn*).

Emodin is chemically similar to the mitochondrial ubiquinone named DMNQ (2,3-dimethoxy-1,4 naphthoquinone), an endogenous ROS inductor, as it is able to transfer electrons. It is also known to have antibacterial, antiviral, anti-infammatory, and anticancer effects. The emodin's antitumor effect has been observed in several types of cancer (leukemia, breast, colon, and lung cancer), also in combination with RT schedules, although its mechanism of action still remains unclear.

Under hypoxic conditions, emodin treatment enhanced the radiosensitivity of CNE-1 NPC human nasopharyngeal carcinoma cell line. In particular, treatment with 3.9 and 7.8 g/mL emodin 24 h before 2 Gy IR induced an increase in the apoptosis ratio and cell cycle arrest in the G2/M phase. Moreover, an increase of ROS production in tandem with a downregulation of HIF-1 levels (both mRNA and protein) was also described. These data were also confrmed by using CNE-1 xenograft models where a tumor growth delay was observed after emodin and IR combined treatments [203].

The radiosensitizing effect of emodin has also been observed in the HeLa cervical cancer cell line, where pretreatment with different concentrations of aloe emodin (AE) before X-ray irradiation (0–10 Gy) leads to decrease in the mean lethal dose (D0) in a concentration-dependent manner, as well as an enhancement in the percentage of cells in the G2/M phase and a sub-G1 peak at 24, 48, and 72 h, using 50 M and 4 Gy IR. In addition, an increased expression of cyclin B, γ-H2AX, and alkaline phosphatase (ALP) activity was also described. Similar data regarding a decrease of cell growth and viability were observed also in human HepG2 hepatocellular carcinoma cell line treated with 10 Gy of γ-irradiation and AE, under hypoxic conditions. This combined treatment leads to higher increase in both G2/M and apoptotic populations [203].

#### **<IP_ADDRESS> Flavopiridol**

Flavopiridol is a favone originating from the *Dysoxylum binectariferum* plant commonly used in Indian medicine. This molecule is able to arrest cell cycle by acting on cyclindependent kinases (CDKs) during the G1/S or G2/M phases, which is confrmed in several cancer cell types (chronic lymphocytic leukemia, squamous cancer, breast cancer cells). In addition, favopiridol is able to induce the transcriptional suppression of genes involved in the proliferation pathways, to stimulate apoptosis, to inhibit angiogenesis, and to increase the chemotherapeutic effects [203].

The power of favopiridol to affect cell radiosensitivity, in tandem with docetaxel, was described in H460 human lung carcinoma, by using both in vitro and in vivo approaches. Multiple treatments with docetaxel (10 M), γ-irradiation (0–5 Gy), and favopiridol (120 M) are able to augment radiation effects by inducing cell cycle arrest in the G1 and G2/M phases. On the other hand, in esophageal squamous carcinoma cell lines, cell cycle arrest after irradiation was described with the decrease of cyclin D1 and retinoblastoma protein (Rb) levels. Additionally, in the SEG-1 esophageal cancer cell line, treatment with favopiridol 24 h before γ-radiation (2–6 Gy) increased radiosensitivity compared to the control, due to inhibition of several CDKs, cell cycle redistribution in G1 and G2 phases, and induction of apoptosis [203].

The experimental evidences show that cells containing mutated p53 or overexpressed Bcl-2 are more radioresistant than wild type. However, favopiridol increased the cytotoxic effects of radiation in cells with altered status of p53 and Bcl-2, confrming the hypothesis according to which these two pathways are targeted by radiosensitizer mechanism exerted by favopiridol [203]. Moreover, the radiosensitizing effects of favopiridol were evaluated in vivo on glioma xenograft models using GL261 cells. The interaction of γ-radiation (5 Gy), fractionated for 10 days, with favopiridol (5 mg/kg) resulted in a decrease in cell proliferation, which was mainly mediated by the favopiridol's antiangiogenic activity, which also inhibited the HIF-1 pathway [203].

On the other hand, as described in OCA-I ovarian carcinoma cells, the radiosensitizing action of favopiridol could be sustained also by the downregulation of Ku70 and Ku80 proteins, known to be involved in DNA repair mechanisms after radiation exposure, by the redistribution of the cell cycle with a greater accumulation of cells in the two more radiosensitive G1 and G2 phases [203].

#### **<IP_ADDRESS> Berberine**

Berberine is an alkaloid which can be extracted from the roots of many plants like the barberry, the tree turmeric, and the California poppy. Berberine is used to treat health problems like hypercholesterolemia and type 2 diabetes mellitus.

Berberine works by inhibiting cell cycle progression, thereby exerting, in vitro, an antitumor activity in a large array of tumors, and its radiosensitizing properties were investigated on lung, esophageal, and breast cancer cells. Since berberine interferes with the expression and activity of RAD51, involved in DNA damage repair response, its radiosensitizing mechanism is based on hindering DNA damage recovery after X-ray irradiation. In vitro and in vivo experimental data has revealed the ability of berberine to inhibit HIF-1α and suppress VEGF. For example, in an in vitro nasopharyngeal carcinoma study, berberine when combined with γ-rays demonstrated a reduction of cancer cell proliferation, viability, and Sp1 decreased expression, a protein involved in tumor motility and invasion [203].

#### **<IP_ADDRESS> Genistein**

As expected, genistein also acts as a radiosensitizing agent, if combined with γ-irradiation, as shown in vitro in cervical cancer cells, where the growth inhibition was associated with survivin downregulation, a prosurvival protein. Again, in cervical neoplasms, genistein enhanced RT effects in multiple ways: by inhibiting G2/M phase of cell cycle; by reducing the expression of two prosurvival proteins, Mcl-1 and AKT; and by triggering cell apoptosis via cytochrome c release, cleavage of caspase-3 and -8, inhibition of Bcl-2, and enhancement of Bax expression. Similar results were also shown on breast and non-small cell lung cancers, where the radiosensitizing ability was associated with the inhibition of Bcl-x, ROS production enhancement, and antioxidant molecule downregulation [203].

#### **<IP_ADDRESS> BP-C2**

BP-C2, a lignin-derived polymer containing benzene polycarboxylic acids complexed with ammonium molybdate, is an antioxidant that promotes the release of prorepair cytokines (IL-4 and IL-10) and suppresses the release of proinfammatory cytokines (TNF-α and IL-6). Orally administered BP-C2 was found to have radioprotective and mitigative activity in H-ARS and GI-ARS [208]. Topical BP-C2 was found to have radiomitigative activity in a cutaneous radiation injury model (CRI-ARS) [209].

#### **<IP_ADDRESS> Sodium Selenite**

Several studies have revealed the prooxidant and cytotoxic properties of sodium selenite, with respect to other selenium compounds, recognized for their antioxidant activity. In particular, the effect on natural killer (NK) cell activation is known, as well as the inhibition of the disulfde exchange on cell surface, a remodeling process, which drives cancer to uncontrolled cell division [203].

Schueller et al. [210] tested a 14-day pretreatment of C6 rat glioma cell line with selenite in the range concentration of 2–3.6 mM, before applying 0–20 Gy of γ-rays. The results showed a signifcant difference between the 0 mM and 3 mM survival curves applying 5 Gy (*p* = 0.02) and 10 Gy (*p* = 0.009). Also, the vehiculated sodium selenite nanoparticles (nano-Se) were tested as radiosensitizers, using the 0–3 mg/mL range concentrations pretreatment, before treating with 0–8 Gy of X-rays. In this case, the authors showed the effect on MCF7 breast cancer cells, observing that combined treatment generated a higher mortality rate of the IR or nano-Se single treatments, inducing block at the G2/M phase of cell cycle, autophagy activation, and ROS generation. Moreover, A375 melanoma cells were subjected to 4-h pretreatment with a selenium nanosystem, using 0–15 mM coated hemocompatible erythrocyte membrane combined with bevacizumab (RBCs@Se/Av) and 2–8 Gy of X-rays. This study showed a strong cell survival reduction, an increase in the sub-G1 cell proportion, apoptotic pathway activation, and ROS generation. In addition, as expected by the bevacizumab treatment, decreased VEGF and VEGF2 levels were observed as tumor angiogenesis reduction [203].

# **11.4.2 Corticosteroids**

Corticosteroids are a group of hormones, produced by the cortex of the adrenal glands, having the characteristic steroid nucleus and derived from subsequent degradations of the cholesterol side chain. They include numerous molecules with different actions, including sex hormones. However, they are divided into glucocorticoids, such as cortisol which controls the metabolism, and mineralocorticoids, such as aldosterone which controls the concentration of electrolyte and water in the blood.

Among these molecules, many are used for their potent anti-infammatory and immunosuppressive properties, such as corticosterone (C21H30O4) and cortisone (C21H28O5, 17-hydroxy-11-dehydrocorticosterone).

In the context of clinical RT, corticosteroids are currently used as mitigators of side effects caused by irradiation [211]. However, some researchers have highlighted the radiosensitizing effects of these molecules, used in the pretreatment phase.

Glucocorticoids (GCs), acting on stress pathways, are well known in the treatment of different types of tumors. They have a strong inhibitory action on the proinfammatory cytokine production, although their action mechanisms need deeper investigation, if used in combination with IR.

An in vitro study has investigated the role of dexamethasone (Dex), a synthetic glucocorticoid, in DNA damage response (DDR) pathway, on three astrocytoma cell lines (CT2A, APP.PS1 L.1, and APP.PS1 L.3). The results showed increased basal levels of γ-H2AX foci, keeping them higher 4 h after irradiation (IR) of the cells, while no effect was shown on the 53BP1 foci formation, compared to untreated cells. The high-level expression of γ-H2AX was reversed by ascorbic acid administration, a strong inhibitor of reactive oxygen species, showing that DEXA induces DNA damage by oxidative stress [203].

In addition, in a preclinical study on rat model, the effect of 1 mg Dex was studied alone or in combination with radioprotective molecules turpentine oil (TO), α2-macroglobulin (α2-M), or amifostine, before the administration of 6.7 Gy (LD50/30) of RI, evaluating survival and blood infammatory markers. The results showed that Dex alone was lethal for 45% and 55% of control and irradiated rats, respectively. On the other hand, from the combination of pretreatments, it emerged that 1 mg Dex reduced the radioprotective effcacy of TO and Ami to 30% and 40%, respectively, even if, given together, TO and Ami provided 70% protection to rats receiving Dex. Instead, TO and α2-M enhanced the rate of survival from 50% to 90% and 100%, respectively [203].

# **11.4.3 Nanoparticles**

A crucial question for cancer treatment is how to increase the therapeutic window, enhancing radiation damage in tumors, while preserving the surrounding healthy tissues. One promising strategy is the accumulation of nanoparticles composed of high-Z materials (e.g., gold, palladium, platinum, gadolinium) in the tumor cells.

High-atomic-number (Z) compounds have long been used as image contrast agents due to their high X-ray attenuation properties compared to soft tissues. The higher energy absorption of elements such as iodine and barium can enhance the contrast of the organs and tissues in which they are injected. The concentration of the compounds and the radiation doses used for diagnostic applications are usually so low that radiation effects and risks can be neglected. However, the same differential energy absorption principle can be exploited for therapeutic use. Recent developments in nano-manufacturing have provided reasonable and affordable methods to produce high-Z structures with dimensions smaller than 100 nm, which can be loaded in tumor volumes and in tumor cells. Their small size allows the nanostructures to escape the leaky vasculature system of tumor regions, providing a natural method for passive tumor accumulation. The majority of work has been concentrated on gold thanks to its biocompatibility and easy functionalization. The former means that considerable concentrations of gold nanostructures can be administered without toxicity effects, while the latter allows for the development of bespoke products able to accumulate in specifc tissues/cells (active accumulation). Gold's high atomic number (*Z* = 79) provides excellent radiation absorption contrast as indicated

**Fig. 11.23** Mass energy absorption coeffcient (left-hand-side *Y*-axis) for gold (purple) and soft tissue (blue) as a function of X-ray energy. Right-hand-side *Y*-axis indicates the ratio (black)

in Fig. 11.23. Other materials such as gadolinium (*Z* = 64) and more recently superparamagnetic iron oxide nanoparticles (SPION, *Z*Fe = 26) have also been suggested and explored.

Early work by Hainfeld [212] demonstrated the potential of high-Z nanostructures to enhance the effect of radiation and improve tumor control in mice treated with kilovoltage X-rays minutes after injection of gold nanoparticles (GNP). In vitro work using a wide range of cell lines and radiation qualities confrmed that the presence of GNP can enhance the effect of radiation by 10–100% [213].

Interestingly, the radiation sensitization observed in in vitro and in vivo work is often signifcantly greater than that predicted from simple macroscopic dose models. Furthermore, the size, shape, and surface coating of the nanoparticle as well as the radiation quality and cell line have been shown to affect the radiation response observed. The discrepancy between dosimetric and experimental results regarding the radiosensitization effect emphasizes that complex physical, chemical, and biological interactions are involved in high-Z nanoparticle-mediated radiosensitization, which still need to be fully elucidated in order to extrapolate the nanoparticle radiosensitization concept to patient cancer RT. Physical, chemical, and biological mechanisms of nanoparticle radiosensitization are shown in Fig. 11.24.

#### **<IP_ADDRESS> Physical Radiosensitization**

The physical processes driving the enhancement in radiation effectiveness in the presence of nanoparticles strongly depend on the radiation quality used. For medium-energy X-rays (<300 kVp), the radiation-nanoparticle interaction is dominated by the photoelectric effect, especially for photons with energies around the L- and K-shell excitation edges. This causes the emission of inner shell electrons from the high-*Z* material, resulting in a cascade of low-energy Auger

**Fig. 11.24** Physical, chemical, and biological mechanisms of nanoparticle. Nanoparticles radiosensitization. Reproduced with permission of Dove Medical Press Ltd., from Application of Radiosensitizers in

Cancer Radiotherapy, International Journal of NanoMedicine, 16: 1083–1102, by Gong L et al. 2021

electrons (10–20 electrons per interaction). The majority of these low-energy electrons are reabsorbed by the nanoparticle. Only a few Auger electrons escape the nanoparticle, releasing their energy in a few 10 s of nm around the nanoparticle [214]. As a result, the presence of high-*Z* nanoparticles increases the dose absorbed by the volume in which the nanoparticles are loaded, and it changes the spatial distribution of the ionizations concentrating the energy deposition around the nanoparticles.

At higher photon energies (such as MV which is routinely used for cancer treatment), the dominant process in the interaction between radiation and nanoparticles is Compton scattering. At these energies, the Compton cross sections for high-Z materials are similar to that of soft tissues, and therefore no differences should be expected from the presence of high-*Z* nanoparticles. However, MV photon beams usually contain a considerable fraction of lower keV photons and electrons, which can also increase due to scattering processes (e.g., at 10 cm depth, the fraction of <150 keV photons from a fattening flter-free beam is between 13% and 20%). MV photon beams can therefore produce a considerable amount of Auger electrons from interaction with high-*Z* nanoparticles and alter both the macroscopic and the microscopic dose absorbed.

With protons and charged particles, the probability of interaction between the primary beam particle and the nanoparticles is considerably smaller than that for photon beams due to the lower number of primary tracks required to deliver a given dose. The dominant interaction process is the production of secondary electrons from the nanoparticle via small-angle scattering, which is proportional to the density and therefore higher for high-Z nanoparticles than for soft tissues. In contrast to the photon interactions, these secondary electrons are produced from the outer atomic orbital of the nanoparticle and contribute only a few percentages of the additional absorbed dose.

In all cases, the presence of high-Z nanoparticles causes an increase in the overall macroscopic dose absorbed and high localized energy deposition spikes. The former was initially thought to be the main cause of the observed radiobiological enhancement. However, calculations clearly show that the additional absorbed macroscopic dose by itself is not enough to explain the increased effectiveness observed in experimental studies. The localized energy deposition spikes, caused by the presence of nanoparticles, are similar to those produced by the traversal of charged particles, and their impact on the biological effects can therefore be estimated using radiobiological models based on microscopic dose distributions. The local effect model (LEM) is widely used for charged particles and has been employed to demonstrate that higher radiosensitization enhancement ratios can be expected when local energy distribution is taken into account. Radiosensitization prediction by the LEM-based model strongly depends on the location of the nanoparticles and the radiation quality, with the closer the nanoparticles are to the critical structures of the cell (e.g., DNA), the larger the effect.

#### **<IP_ADDRESS> Chemical Radiosensitization**

A key property of nanoparticles is the increased interaction with the surrounding environment (due to the high surfaceto-volume ratio). The intracellular nanoparticle concentration is an important determinant for radiation sensitization. Numerous studies have investigated the importance of the nanoparticle size on cellular uptake, and an optimum diameter appears to range between 10 nm and 50 nm, showing a strong correlation between radiosensitization and nanoparticle concentration [215]. However, the precise value also depends on the coating and on the cell line. High-Z nanostructures are generally coated with a layer of polyethylene glycol (PEG) to provide a hydrophilic nature, preventing the nanostructures from aggregating and increasing their cellular internalization by macrophage recognition. Moreover, nanoparticles are often also conjugated with other biomolecules to achieve specifc cellular and subcellular targeting. By using such an approach, it is possible to manufacture nanoparticles that dock to specifc cell surface proteins or distinct subcellular compartments such as mitochondria using specifc peptide sequences. The presence of the chemical coating and the high-Z element itself also change the chemical environment of the cell.

Reactive oxygen species (ROS) play an important role in mediating DNA damage produced by radiation. In fact, 50–70% of the DNA lesions in standard RT upon X-ray irradiation are attributed to the hydroxyl radical (OH). By altering the chemical environment of the cell, the nanoparticles can in principle affect both the yield and the spectrum of ROS produced, which in turn has consequences for the DNA damage. Competing mechanisms may be at play depending on the composition of the nanoparticles and their coating. For instance, certain chemical compounds, such as PEG, can act as ROS scavengers and actually detoxify radicals formed by the interaction of radiation with water molecules. Studies aiming at quantifying the G-value (i.e., the number of molecules of a specifc radical produced per 100 eV of energy absorbed) have indicated three possible mechanisms through which nanoparticles can affect the ROS production by radiation and therefore affect the radiation effectiveness (Fig. 11.25). Increased radicals can be produced as a result of direct interaction between radiation and nanoparticles through the production of electrons and low-energy photons emitted by the nanoparticles. As the energy spectrum of the secondary radiation emitted by the nanoparticle is different from the primary radiation beam, a different spectrum of radicals is to be expected and the yield is generally higher

**Fig. 11.25** Schematic representation of the possible pathways through which nanoparticles can affect the yield of radicals following radiation exposure: (**a**) primary water radiolysis, (**b**) secondary water radiolysis, and (**c**) radical scavenging from nanoparticles

due to their lower energy and cascade of secondary electrons. The presence of the nanoparticles, however, can also affect the yield of radicals produced by the direct interaction of radiation with the water molecules. This may occur through scavenging action from the nanoparticle-coating elements or by chemical interaction between the nanoparticles and the radiolysis products.

#### **<IP_ADDRESS> Biological Radiosensitization**

When nanoparticles reach the cell surface, they are usually internalized via endocytosis and end up in intracellular vesicles, also called endosomes. This endosomal pathway is often a barrier hindering biological or therapeutic effects of nanoparticles. Nevertheless, nanoparticles are able to escape the endosomal transport system, accessing the cytoplasm and organelles by destabilizing the endosomal membrane, inducing osmotic swelling and membrane rupture, or passively crossing the plasma membrane or the endosomal membrane [216]. Once internalized, the nanoparticles can affect different cellular and molecular processes. For instance, apart from creating additional DNA damage, nanoparticles directly or indirectly affect the functioning of DNA repair proteins by preventing their synthesis, posttranslational modifcation, or recruitment to the site of damage. For instance, due to the large specifc surface area, nanoparticles are very effcient in capturing a large amount of proteins, including DNA repair proteins, eventually leading to their deprivation and hence reducing the DNA repair effciency [217]. On the other hand, the release of metal ions could imbalance the metal homeostasis in the cells, which is critical for protein folding and could replace the metallic cofactor in active sites of enzymes that are involved in the antioxidant defense system, altering their structure and inhibiting their activity.

Besides affecting the DNA damage repair machinery and causing antioxidant enzyme inhibition, multiple in vitro studies showed that nanoparticles can cause cell cycle disruption. The radiosensitivity of cells can vary depending on their cell cycle phase, with cells in the late G2 and mitosis (G2/M) phases being the most radiosensitive, presumably because the condensed chromatin in mitotic cells is more susceptible to radiation-induced double-strand breaks, which are commonly repaired by the error-prone NHEJ mechanism. On the other hand, cells in the late S phase are the most radioresistant, due to the diffused chromatin regions and the fact that during the S phase, DNA damage is usually repaired by the accurate HR mechanism. Multiple research groups reported an elevated proportion of cells in the G2/M phase and a decreased cell number in the G0/G1 phase after gold nanoparticle exposure [218]. As a result, the radiosensitizing effects of gold nanoparticles can also be attributed to stalling of the cell cycle in the radiosensitive G2/M phase.

Finally, mitochondria, located in the cytoplasm and having their own DNA, are another potential target for nanoparticle-mediated radiosensitization as the same processes as described above for nuclear DNA may affect mitochondrial (mt) DNA. However, nanoparticles are hardly ever detected in mitochondria and are often accumulated in endosomes and lysosomes. In general, although hypothetical, nanoparticles accumulated and irradiated within lysosomes or other organelles may decrease cell survival by directly harming these organelles and their functions [219]. Even milder damage to organelles could potentially lead to altered signaling and eventually increased cell death.

Cytoplasmic organelles might thus represent a parallel or even dominant target to nuclear DNA. It is obvious from the previous paragraphs that nanoparticle-mediated radiosensitization is not fully understood. The research is complicated by extreme complexity and variability of the systems studied, including different materials, sizes, shapes, and modifcations of nanoparticles; different cell types; different types of radiation and irradiation conditions, etc. Under certain experimental conditions, a plethora of biological processes may be induced so that it might not be excluded that different types and sizes of nanoparticles do interact according to specifc mechanisms and their combinations (Box 11.6).

#### **Box 11.6: Nanoparticle-Mediated Radiosensitization**

# **11.4.4 Autophagy Inhibitors**

Autophagy is a basic catabolic mechanism that involves cell degradation of unnecessary or dysfunctional cell components, such as damaged endoplasmic reticulum (ER) and other cytoplasmic constituents through lysosome action.

The autophagy is mediated by protein complexes, such as class III PI3K, autophagy-related gene (Atg) proteins, and others containing microtubule-associated protein 1 lightchain subunit 3 (LC3), recruited to the membrane favoring membrane expansion and phagophore elongation. Particularly, autophagy can be activated by multiple signaling pathways, mainly through energy signals via AMPK. AMPK activation can phosphorylate ULK1, inhibit mTOR signaling, and activate Beclin-1 and Vps34 molecules resulting in the upregulation of autophagy intensity. Thus, the AMPK-mTOR-ULK1 pathway plays an important role in autophagy. Recently, the autophagy-related protein BECN1 has been shown to regulate radiation-induced G2/M arrest. In the context of a disease, autophagy has been described as an adaptive response to survival, a strategy to maintain metabolic homeostasis. In cancer cells, autophagy is a double-edged sword. In early stages, it could limit tumorigenesis. However, it could also provide a prosurvival function for adaptation and detoxifcation in a stressful environment, such as starvation, hypoxia, and chemotherapy/ radiotherapy.

Some studies show that the autophagy preventing is radiosensitive, while the autophagy promoting is radioprotective, suggesting that IR-induced autophagy may represent an adaptive response to maintain tumor growth and survival. In order to improve IR tumor responses, several sensitization agents to radiation-induced autophagy are currently being studied. The molecular machinery involved in IR-induced autophagy is still not clear. Recent studies show that p53 and PARP-1, a DNA repair enzyme triggered by DNA damage, exert essential roles in starting the autophagy process regulating the PI3K/PKB/AKT/mTOR signaling pathway that represents an autophagy key regulator. Reports by investigators have recently shown that autophagy activity increased after IR and chemotherapy. It is an escape mechanism for cell survival in response to cytotoxic agents, including IR and temozolomide (TMZ) in glioblastoma (GBM) treatment [220]. In radioresistant breast cancer (BC) cells, a strong postirradiation autophagy induction has been observed as a protective and prosurvival mechanism of radioresistance after exposure to IR. Studies have also shown that induced autophagy in some radioresistant cancers, such as GBM, causes IR sensitization and increased cell death.

In normal conditions, microautophagy and chaperonemediated autophagy permit the breakdown of abnormal proteins, cellular debris, or damaged organelles, maintaining cellular homeostasis and/or as tools to recycle biological constituents (e.g., amino acid, fatty acid, and energy in the form of ATP). After stress stimuli, such as nutrient starvation, protein aggregation, organelle damage, and oxidative or genotoxic stress, including by IR, the autophagy hyperactivation promotes cell death, via nonapoptotic and caspaseindependent mechanisms, and this case is also called macroautophagy [221].

As described above, AMPK promotes the activation of autophagy. Among factors activated by AMPK, an interesting and not well-described role in the autophagy process, it was supposed to be for glucose transporter GLUT-1, often upregulated in cancer cells. In this sense, the cross talk among GLUT1, curcumin, and AMPK pathway in LC remains vague. Interestingly, it was recently described that the treatment with GLUT1 siRNA alone or in combination with curcumin resulted in profound improvement of the radiosensitivity of LC cells after irradiation. In particular, curcumin and GLUT1 siRNA combined treatment not only promoted apoptosis of LC cells, but also induced autophagyassociated cell death through activation of AMPK/mTOR/ ULK1 signaling-mediated autophagy with or without irradiation treatment [222].

Taking all these observations, we can speculate that the role of autophagy in cancer cells depends upon certain factors, such as cell type, specifc characteristics of tumor cells, microenvironment of the tumor, and type of treatment applied. In addition, a variety of radiation-resistant molecules, such as PI3K/AKT, EGFR, NF-κB, and p53, may play an important role in the regulation of autophagy, thus indicating that the mechanisms that regulate autophagy are very complex. Thus, many questions and contradictory fndings have yet to be clarifed.

# **11.4.5 Metformin (MTF)**

In cancer patients, MTF may affect tumor growth and treatment response in two different ways: directly by inhibiting mitochondrial metabolism and activating downstream cell signaling pathways in cancer cells or indirectly by keeping low the levels of glucose, insulin, and other factors which can activate cancer cell proliferation [223]. MTF has also shown anticancer effects in nondiabetic patient populations (described in Sect. 11.1.9).

With regard to the relationship between MTF and IR, MTF is able to increase intrinsic radiosensitivity, as tested in vitro in many cell lines of different tumors, including lung, head and neck, breast, liver, prostate, and pancreas cancers and murine fbrosarcoma, with specifc cell line-dependent radiosensitizing effect [203, 223].

Several clinical data on the effect of MTF on patient outcome, focused particularly on patient populations undergoing RT, have shown that these patients have better outcomes if they are treated with MTF. These and other data show the signifcant role of MTF in enhancing the RT effcacy and suggest an interaction between MTF and IR [223].

In vitro and in vivo studies, concerning the MTF radiosensitizing effect, have showed an enhanced DNA damage after MTF treatment combined with RT, as tested by phosphorylation of histone γH2AX, a well-known marker for DNA damage [203].

In particular, MTF may increase initial DNA damage or inhibit repair processes, being able to reduce the expression of DNA repair protein Ku70 and hamper radiation-induced activation of EGFR and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Considering that the primary targets of MTF are the mitochondrial complex I and mGPD (glycerol-3-phosphate dehydrogenase), it may induce an increase of ROS generation and oxidative damage to lipids, protein, and DNA that could strengthen the effects of IR. Indeed, several data have demonstrated that MTF can increase ROS in cancer cells when delivered alone or in combination with IR. In general, as regards the main known MTF mechanisms of action when combined with RT, in vitro and in vivo data show a signifcant role of MTF in affecting at least four different parameters at radiobiological level, including intrinsic cell radiosensitivity (SF2, i.e., the fraction of cells surviving after a single IR dose of 2 Gy), cancer stem cell fraction, tumor proliferation rates, and tumor hypoxia [223].

# **11.4.6 PARP Inhibitors**

Poly-(adenosine diphosphate-ribose)-polymerase (PARP) are cellular enzymes that play crucial roles in various cell processes like replication, transcription, cellular repair, and also death [224]. PARP detects single-strand breaks and triggers the activation of cellular machinery involved in the repair of the single-strand break. Studies have shown that PARP inhibitors (PARPi) exhibit enhanced radiosensitivity when combined with IR. Radiation works by damaging the DNA, which can activate the singlestrand break (SSB) repair pathway like base excision repair (BER) or double-strand break (DSB) pathways like the HR and the NHEJ pathway (more details in Chap. 3). SSB, if unrepaired, gets converted to double-stranded breaks, which consequently hinder normal cellular processes. PARPi imparts radiosensitivity through the SSB and base excision pathway, which substantially increases the risk to collapsed replication fork, thereby producing a stable DSB [225]. Due to the crucial role of PARP in the DNA repair pathway, PARPi have proved as effective radiosensitizers, especially in tumors harboring DNA repair defciencies like the BRCA mutation. The replication-reliant operations of PARPi facilitate the establishment of differential outcomes in tumor and healthy tissues. Other mechanisms that are known to induce the radiosensitization effects include inhibition of chromatin remodeling, G2/M arrest, vasodilatory effect induced by PARPi, etc. Characteristically, factors that affect radiosensitivity are the capability of tumors to repair the damage, redistribution of the cell cycle, process of reoxygenation, vascular endothelial damage process, tumor immunity, and repopulation of the tumor tissue. A radiosensitizer should have the potential not only to infuence these processes but also prevent the increase in the toxicity. Since PARPi possess most of these qualities necessary for being a potential radiosensitizer (Fig. 11.26), it has gained interest in the medical community [226].

**Fig. 11.26** The advantages and various modes of action by which PARPi enhance the radiosensitivity of tumor cells. Adapted from "Poly- (ADP-ribose)-polymerase inhibitors as radiosensitizers: a systematic review of preclinical and clinical human studies," by [224], Licensed under CC BY 3.0

# **<IP_ADDRESS> Nitroxides**

Nitroxides are a class of stable free radical compounds that exhibit antioxidant mechanisms, thereby safeguarding the cells from several lethal agents like superoxide and hydrogen peroxide (also described in Sect. 11.1.2). However, the rationale to use nitroxides in cancer RT comes from the role of free radicals in tumor development and capacity of inhibitors of radical reactions to suppress tumorigenesis. The underlying mode of action of nitroxyls exhibiting the radiosensitization effect can be attributed to cell signaling, enhanced blood fow to the tumor, consequences on the cellular respiration, and generation of reactive oxygen and nitrogen species that can operate as metabolite radiosensitizers. The effects of nitroxides with radiation were found conficting, leading to uncertainty about their radiosensitizing nature.

Tempol (TPL) is a piperidine nitroxide that possesses an unpaired electron and goes through swift reversible transfer among the three forms: nitroxide, hydroxylamine, and oxoammonium cation. Based on its concentration in the cell, it can act as an oxidative or reductive agent. In cancer cells, TPL favorably inhibits growth by increasing the generation of cellular ROS [227]. When used with RT and chemotherapy, TPL exhibits a differential effect of protecting the normal healthy cells from RT and cisplatin-mediated damage, whereas in cancer cells, tempol is reduced to its hydroxylamine form that is unable to protect the cells from radiation and cisplatin-mediated damage. This differential or selective acting on cancer cells while sparing the normal cells is particularly of signifcance in cancer radio- as well as chemotherapy [228]. It was observed that the anticancer effects of cisplatin increased due to the prooxidant activity of TPL via the increased ROS-mediated cell apoptosis [227]. In several cancer cell lines, TPL was able to free radical-dependent apoptosis. On 24-h exposure to TPL, human promyelocytic leukemic cell line (HL-60) showed reduced levels of mitochondrial and intracellular glutathione, failure in the oxidative phosphorylation process, and diminished mitochondrial membrane potential. TPL also particularly targeted the respiratory chain complex I and showed some insignifcant effects on the complexes II and IV. This can be attributed to the role of mitochondria in apoptosis and it being a free radical resource and target. In HL-60, TPL works by targeting the mitochondria, which subsequently leads to mitochondria associated with oxidative stress and apoptosis. This in turn can sensitize the tumor cells to the proapoptotic effects of cytotoxic agents [229]. When human breast cancer cells (MDA-MB 231) were treated with tempo, another nitroxide, it exhibited considerable levels of tyrosine phosphorylation of numerous unknown proteins when evaluated with the equimolar concentration, i.e., 10 mm TPL. The compounds tempo and TPL lead to the phosphorylation of tyrosine and trigger the Raf-1 protein kinase (30 min, two- to threefold) [230]. Nevertheless, TPL leads to augmented extracellular signalregulated kinase 1 activity. Tempo also activated the stressassociated protein kinase (2 h, threefold) and induced apoptosis (2 h, >50%). The ceramide levels signifcantly increase (54% over control) at 30 min and (71% over control) at 1 h after treatment, prior to activation of stress-activated protein kinase and cell death via apoptosis [230]. TPL protects normal cells and tissues from oxidative damage and remarkably hampers the proliferation of cancer cells. These clearly imply that the nitroxide TPL shows the potential to be a good prooxidant and can be a potent radiosensitizing agent for cancer treatment.

# **11.5 Exercises and Self-Assessment**

# **11.6 Exercise Solutions**

thus able to improve the effectiveness of radiation therapy.

References for use (from https://remm.hhs.gov/int\_contamination.htm#countermeasures\_refs) **FDA approved**: Countermeasures so marked have been approved as treatment for internal contamination with the listed radioisotope by the US Food and Drug Administration (FDA) **NCRP preferred**: Countermeasures so marked have been listed as preferred treatments for internal contamination with the listed radioisotope by the National Council on Radiation Protection and Measurements [Management of Persons Contaminated with Radionuclides: Handbook (NCRP Report No. 161, Vol. I)]. Except where noted, use of these countermeasures has not been approved by the US Food and Drug Administration (FDA)

**NCRP suggested**: Countermeasures so marked have been listed as suggested treatments for internal contamination with the listed radioisotope by the National Council on Radiation Protection and Measurements [Management of Persons Contaminated with Radionuclides: Handbook (NCRP Report No. 161, Vol. I)]. Use of these countermeasures has not been approved by the US Food and Drug Administration (FDA)

# **References**

ide radical spin-labeled resveratrol derivatives. Fitoterapia. 2021;155:105053.

resulting from exposure to ionizing radiation. Disaster Med Public Health Prep. 2011;5(3):202–12.

of thyroidal protection by iodine and perchlorate against radioiodine exposure in Caucasians and Japanese. Arch Toxicol. 2021;95(7):2335–50.

# **Further Reading**

**Open Access** This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons. org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# **12 Ethical, Legal, Social, and Epistemological Considerations of Radiation Exposure**

Alexandra Dobney, Abel Julio González, Deborah Oughton, Frances Romain, Gaston Meskens, Michel Bourguignon, Tim Wils, Tanja Perko, and Yehoshua Socol

#### **Learning Objectives**

A. Dobney (\*)

A. J. González

Argentine Nuclear Regulatory Authority, Buenos Aires, Argentina

D. Oughton Norwegian University of Life Sciences (NMBU), Ås, Norway e-mail<EMAIL_ADDRESS>
#### F. Romain

University of Manchester, Manchester, United Kingdom

#### G. Meskens

Science and Technology Studies Unit, Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium

Centre for Ethics and Value Inquiry, Ghent University, Ghent, Belgium e-mail<EMAIL_ADDRESS>
M. Bourguignon University of Paris Saclay (UVSQ), Paris, France

T. Wils

Catholic University of Leuven (KU Leuven), Leuven, Belgium

T. Perko Belgian Nuclear Research Centre, SCK CEN, Mol, Belgium

University of Antwerp, Antwerp, Belgium e-mail<EMAIL_ADDRESS>
#### Y. Socol

Jerusalem College of Technology, Jerusalem, Israel e-mail<EMAIL_ADDRESS>

# **12.1 Introduction**

Ionizing radiation and radioactive substances can be natural or human-made. Humans have always been exposed to natural ionizing radiation (background radiation), because of the exposure of the Earth's surface to cosmic rays and the radioactivity contained in rocks that form the continental crust. The use of radiation and radioactive substances in medicine, research, industry, agriculture, and teaching, as well as the generation of nuclear power, have brought important benefts to society. Acceptance by society of the risks associated with radiation depends on the perceived relationship between these risks and the benefts to be gained from the use of radioactive sources. Logically, risks must be limited, and adequate protection provided. This does not mean that individuals or the environment must be protected from any and

Queen Mary University of London, London, United Kingdom

all effects of ionizing radiation, but rather to ensure that the amount of radiation absorbed does not have negative consequences that outweigh the benefts. The need to balance risks and benefts makes radiation a matter of science and values, meaning that in addition to technical assessments, ethical and legal issues also apply in the judgement of the acceptability of radiation risks.

# **12.2 The Radiological Protection System**

After the initial and isolated observations of the frst health effects of ionizing radiation (i.e., skin burns and cancers) by the pioneers at the turn of the nineteenth and twentieth centuries, two decades passed before a clear need for radiation protection was identifed. One of the frst signs was the leukemia epidemic that developed among radiologists after World War I, refecting the use of X-ray radioscopy by military surgeons, without any kind of protection, to localize shrapnel in wounded soldiers. This epidemic lasted until the 1950s and affected approximately 500 radiologists [1].