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Mechanisms of cardiac radiation injury and potential preventive approaches

Jan Slezak*, Branislav Kura, Táňa Ravingerová, Narcisa Tribulova, Ludmila Okruhlicova, Miroslav Barancik Institute for heart research, Slovak Academy of Sciences, Dúbravská cesta 9, 842 33 Bratislava, Slovak Republic *Corresponding author. Present address: Institute for heart research, Slovak Academy of Sciences, Dúbravská cesta 9, 842 33 Bratislava, Slovak Republic. Tel.:+421 903 620 181, Fax.: +421 2 5477 6637, e-mail address: [email protected]

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Abstract The most common cancer treatment in addition to the cytostatic and surgical is using gamma radiation. Despite sophisticated radiological techniques, however, in addition to irradiation of the tumor, irradiation of the surrounding healthy tissue also takes place, which results in various side effects depending on the absorbed dose of radiation. Radiation either directly affects the DNA, or indirectly via the formation of oxygen radicals, which in addition to the DNA damage react with all cell organelles, and interfere with their molecular mechanisms. The main features of radiation injury beside DNA is the inflammation, increased expression of pro-inflammatory genes and cytokines. Endothelial damage and dysfunction of capillaries and small blood vessels plays a particularly important role in radiation injury. Review is focused on summarizing currently available data concerning the mechanisms of radiation injury, and the effectiveness of the use of various antioxidants, anti-inflammatory cytokines and cytoprtective substances that may be utilized in preventing, mitigating or treatment of the toxic effects of ionizing radiation on the heart. Key words: radiation, cardiac toxicity, free oxygen radicals, mechanisms of radiation injury, mitigation, prevention, treatment, antioxidants

Introduction Before 1950, generators of therapeutic radiation were not so powerful, and had only limited depth-dose capacity (Lacher 1990). The advent of megavoltage techniques resulted in higher tumor doses and also adjacent tissues, with a subsequent increase in side effects (Stewart and Fajardo 1984). The clinical importance of radiation-induced heart disease has been recognized for many years. The first indication of the relative radiosensitivity of the heart came from long-term follow-up studies of patients treated for Hodgkin’s disease (Andratschke et al. 2011). Today chronic injury of the myocardium is increasingly recognized as an undesired side effect of irradiation after thoracic/mediastinal radiation therapy of malignancies. Currently, the use of technology to reduce normal tissue toxicity includes radiation techniques such

as

conformal

radiotherapy,

intensity-modulated

radiotherapy,

image-guided

radiotherapy, and proton radiotherapy. Each of these techniques helps to reduce the volume of normal tissue exposed to high doses of radiation, thus reducing the risk of injury to normal -2

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tissue. Despite modern radiotherapy techniques in some cases it inevitably involves exposure of adjacent tissues causing undesired side effect. Radiation-induced heart disease (RIHD) has attracted much interest in the recent 10 years and pointed out to the mechanisms involved in the development of radiation-induced cardiovascular injury. The mechanism of radiation injury is a complex and multifactorial issue and involves endothelial damage of the microvasculature and coronary arteries, DNA damage and release of multiple inflammatory and profibrotic cytokines (Sencus-Konefka and Jassem 2007; Boerma and Hauer-Jensen 2010; Lee et al. 2013). The mechanisms whereby these cardiac effects occur are not fully understood and different factors are probably involved after high therapeutic doses. These various mechanisms probably result in different cardiac pathologies, e.g. coronary artery atherosclerosis leading to myocardial infarction, versus microvascular damage and fibrosis leading to congestive heart failure (Stewart et al. 2013). The inflammatory cell infiltrate disturbs the filtration properties of the endothelium, and the basement membrane of the capillary wall thickens as a result of collagen deposition and fibrosis (Gaya and Ashford 2005). Better understanding of molecular pathways of injury might help to unravel basic mechanisms of RIHD, with the ultimate goal to identify potential targets for intervention (Andratschke et al. 2011). Studies might also provide knowledge of how to modify the

progression of

radiation damage in the heart by drugs or biological molecules (Citrin et al. 2010; Mège et al. 2011). RIHD can be modulated by therapies directed at mitigating the cascade of events resulting from normal tissue injury. This review is focused on the mechanisms of radiation-induced cardiovascular toxicity and prevention of injury of healthy tissues in the areas at risk.

Mechamisms and factors of radiation injury

Mechanisms by which radiation causes tissue injury of both malignant and normal tissues involves induction of apoptosis due to free radical–mediated DNA damage, and the sequence of overlapping events that include activation of the coagulation system, inflammation, and

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tissue remodeling. This complex process is orchestrated by a large number of interacting molecular signals including cytokines, chemokines, and growth factors. There are several crucial factors determining the intensity of radiation tissue damage. These can be shortly summarized as dose- (the higher dose the greater injury), speed of dose delivered- (the faster delivery results in more injurious effect), size of exposed body- (the bigger part of body the more severe injury), sensitivity of tissue to radiation, age, health status and genetic abnormalities (Fig. 1). Dependence of damage from radiation dose targeted on thoracic region is very prominent. Results showed that irradiation with 5 Gy resulted only in a modest increase in right ventricular weight and a reduction in lung angiotensin converting enzyme activity. Rats receiving 10 Gy exhibited pulmonary vascular dropout, right ventricular hypertrophy, increased pulmonary vascular resistance, increased dry lung weight, and decreases in total lung angiotensin converting enzyme activity, as well as pulmonary artery distensibility after one month (Ghosh et al. 2009; Slezak et al. 2011; Slezak et al. 2012; Slezak et al. 2013). Activation of protein kinase C was involved in radiation-induced adaptive responses, and the intracellular signal transduction pathway induced by protein phosphorylation with protein kinase C was a key step in the signal transduction pathways induced by low-dose irradiation (Matsumoto et al. 2004). Radiation at doses of 14 and 25Gy increased cGMP, increased iNOS activity and nitrite content. Both doses of radiation significantly decreased the L-arginine transport and increased iNOS gene expression significantly. It was proposed that radiation induces the NO generation by up-regulating the iNOS activity (Zhong et al. 2004). Radiation damage to vasculature can be demonstrated by the fact that breast cancer patients exposed to post-operative radiotherapy showed in later stages a significant increase in mortality from ischaemic heart disease (Rutqvist et al. 1992). About 50% of the patients had new scintigraphic defects which could be related to radiation damage to the micro-circulation (Gyenes et al. 1996) resulting in reduced myocardial capillary density (Baker et al. 2009), focal loss of endothelial alkaline phosphatase (Schultz-Hector and Balz 1994), and increased expression of von Willebrand factor (Boerma et al. 2004). It was estimated that 1 Gy added to the mean dose would increase the cardiotoxic risk by 4% (Mège et al. 2011). Shortly after 20 Gy dose, cardiac function was slightly reduced then maintained in a steady state for several

weeks, probably due to a compensatory up-regulation of cardiac β-

adrenergic receptors. In denervated working heart preparations (in vitro), however, these -4

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compensatory mechanisms are not effective and stroke volume as well as cardiac contractility show a rapid and steady deterioration (Schultz-Hector 1992). The state after six weeks of 25 Gy irradiation of mediastinal area is characterized by general alteration of the animals, e.g. body and heart weight retardation, presence of exudate in the chest and abdominal cavity. In isolated Langendorff-perfused hearts, the effect of irradiation on the heart function was manifested

by mild bradycardia and surprisingly enhanced

coronary flow, but by no changes in heart contractile parameters. Under conditions of ischaemia/reperfusion (I/R), the incidence of reperfusion arrhythmias was higher than in the control hearts. Interestingly, the size of infarction in the irradiated hearts was smaller than in the intact hearts (Ravingerova et al. 2011; Carnicka et al. 2013a,b; Slezak et al. 2011; Slezak et al. 2012; Slezak et al. 2014). At around 70 days after 20 Gy, a marked reduction in capillary density as well as ultrastructural endothelial cell degeneration can be observed. Simultaneously to structural capillary damage, a focal loss of the endothelial marker enzyme alkaline phosphatase was observed in rats in the areas with subsequent myocardial degeneration (Schultz-Hector 1992). Radiation which is absorbed in a cell, has the potential to influence a variety of critical targets in the cell, the most important of which is the DNA. Evidence indicates that damage to the DNA is what causes cell death, mutation, and carcinogenesis (Schultz-Hector 1992). Radiation further causes alteration in cell membrane and nuclear membrane permeability, functional aberrations in chromosomes and cellular organelles and can change its function (Fig. 2). After exposure to ionizing radiation, atoms or molecules became ionized or excited and these can produce free radicals from other molecules, they can break many of chemical bonds in other chemical compounds or they can produce a new chemical bonds from existing chemical compounds. Ionized/excited form of atoms/molecules can damage some important molecules, for example DNA, RNA or proteins (Fig. 3).

Direct and indirect effects of radiation

Direct Action DNA is the principle target for biological effects of radiation. Radiation may damage the DNA directly, causing ionization of the atoms in the DNA molecule (Fig. 4). The misrepaired -5

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or unrepaired DNA damages, in particular DNA double strand breaks, induce chromosomal aberrations and gene mutations. Radiation-induced DNA double strand breaks play an important role in the induction of apoptosis and cell cycle arrest (Han and Yu 2009). Radiation produces a variety of DNA and other cellular lesions that elicit a stress response. Altered gene profiles are one characteristic feature of this response. Increased expression of pro-inflammatory and other genes has been demonstrated within hours following irradiation (Hong et al. 1995; Kyrkanides et al. 2002). These include genes of transcription factors such as nuclear factor–kappa B (NF-κB), cytokines such as tumor necrosis factor–α (TNFα), interleukin-1β (IL-1β), and basic fibroblast growth factor (bFGF) involved in inflammatory processes. Biological effects of ionizing radiation have long been considered a consequence of DNA damage in the irradiated cells. Unrepaired or misrepaired DNA damage in the irradiated cells are responsible for the genetic effects. At the same time, no effects are expected in cells in the population that have not been exposed to radiation. This conventional dogma was, however, challenged by the occurrence of the radiation-induced bystander effect (RIBE). RIBE was reported back in 1954, when cells exposed to doses of low Linear energy transfer (LET) radiation were found to have an indirect effect in producing a plasma-borne factor, which led to chromosome breakage and cytogenetic disorders (Mothersill and Seymour 2001). From the early 1990s, development in single-cell irradiation has led to an immense interest in the bystander effects. Generally, RIBE can be defined as the phenomenon whereby the irradiated cells can release some signaling molecule, which is transferred via the medium or gap-junctions, so that the same cytotoxicity or genotoxicity can be observed in the nonirradiated cells (Han and Yu 2009).

Indirect action via production of oxygen free radicals

Radiation interacts with non-critical target atoms or molecules, usually water. This results in the production of free radicals. Free radicals can then attack critical targets such as the DNA, because they are able to diffuse some distance in the cell. The initial ionization event does not have to occur so close to the DNA in order to cause damage. Radiation treatment causes direct damage to blood vessels by the generation of reactive oxygen species (ROS) that disrupt DNA strands and leading to an inflammatory cascade (Hatoum et al. 2006). -6

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Mechanisms of free radicals action

Free radiacals are molecules containing one or more unpaired electrons in atomic or molecular orbitals (Gutteridge and Halliwell 2000). Unpaired electrons, result in high chemical reactivity. Most of the energy deposited in cells is absorbed initially in water, which is the main component of cells, leading to a rapid production of oxidizing and reducing reactive hydroxyl radicals. Reactive free radicals play a crucial part in different physiological processes ranging from cell signaling, inflammation and the immune defense (Elahi and Matata 2006). Hydroxyl radicals (·OH) may diffuse over distances to interact with DNA to cause damage. Fortunately, some defensive systems or responses in cells can protect the cells from the damage (Han and Yu 2009). Formation of ROS is originating from a variety of sources such as nitric oxide (NO) synthase (NOS), xanthine oxidases (XO), the cyclooxygenases, nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase isoforms and metal-catalyzed reactions (Elahi et al. 2009). Abnormal production of free radicals leads to changes in molecular pathways resulting in pathogenesis of several important pathological states including heart disease, neurological disease and cancer and is involved in the process of physiological aging. Reduction and oxidation can render the reduced molecule unstable and make it free to react with other molecules to cause damage to cellular and sub-cellular components. This includes free radicals such as superoxide anion (O2·−), hydroxyl radical (HO·), lipid radicals (ROO−) and nitric oxide (NO). Although other reactive oxygen species, hydrogen peroxide (H2O2), peroxynitrite (ONOO−) and hypochlorous acid (HOCl), are not free radicals, they have oxidizing effects that contribute to oxidative stress. ROS have been implicated in cell damage, necrosis and cell apoptosis due to their direct oxidizing effects on macromolecules such as lipids, proteins and DNA (Valko et al. 2005; Valko et al. 2006). Reaction between radicals and polyunsaturated fatty acids within the cell membrane can result in fatty acid peroxyl radicals, which accumulate in the cell membrane and alter protein function and signal transduction. ROS can also induce the opening of the mitochondrial membrane permeability transition pore and cause a release of cytochrome c and other factors that can lead to -7

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apoptosis-mediated cell death (Tatton et al. 2003; Tsutsui et al. 2009). O2 .- radicals can further interact with the signaling molecule nitric oxide (NO) resulting in the formation of reactive nitrogen species (RNS), which further reduce NO bioavailability and cause NO toxicity known as “nitrosative stress” (Elahi et al. 2007). Under the physiological situation, defences such as specialized enzymes and antioxidants can cope with the situation and maintain reduction-oxidation (redox) balance. However, during excessive production of ROS, enzymes and antioxidants can get exhausted resulting in oxidative/nitrosative disbalance, a process that is an important mediator of cell damage (Pacher and Szabo 2008; Vassalle et al. 2008; Elahi et al. 2009). Excessive production of RNS results in nitrosylation reactions that change the structure of proteins (Ridnour et al. 2004) leading to the loss or change of protein function. The oxidation and nitration of cellular proteins, lipids and nucleic acids, and formation of aggregates of oxidized molecules underlie the loss of cellular function, cellular aging and the inability of cells to withstand physiological stresses. ROS modulate signal transduction processes and energy metabolism in response to conditions of oxidative/nitrosative stress. This suggests that radiation causes an inflammatory response and in later phase, oxidative damage in large vessels that in combination with high cholesterol, increases oxidation of lowdensity lipoproteins and allows them to be ingested by macrophages, thus triggering the start of the atherosclerotic process. Once the atherosclerotic process is initiated, the lipid cells secrete further inflammatory cytokines and growth factors, which stimulate proliferation and migration of the smooth muscle cells (Stewart et al. 2010). Ionizing radiation is associated with induction of inflammatory markers including cytokine expression. An increase in cyclooxygenase-2 (COX-2) expression and COX-2-mediated prostanoid production was observed in the irradiated mouse brain. COX-2 is one of two isoforms of the obligate enzyme in prostanoid synthesis and a principal target of non-steroidal anti-inflammatory drugs (NSAIDs). Inhibition of COX-2 attenuates prostanoid induction and cerebral edema in mice after XRT (Moore et al. 2004). Sources of ROS, physiological and pathophysiological conditions, and cellular oxidant targets determine the characteristic feature of a disease process and resultant outcomes (Elahi et al. 2009).

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In this context, cytokines and growth factors probably play a central role in this process and in particular, TGF-β1, TGF-β2, and TGF-β3 are highly pleiotropic cytokines secreted by all cell types; TGF-β molecules are proposed to act as cellular switches that regulate processes such as immune function, proliferation, and epithelial-mesenchymal transition. TGF-β1 is the isoform most frequently implicated in the fibro-proliferative process, and it appears to be a key-molecule and a master switch for the general fibrotic program (Lawrence 1996; Hendry et al. 2008). ROS signaling can progress via NFκB activation. NFκB belongs to a family of inducible transcription factors (Baeuerle and Henkel 1994), and is one of the most commonly studied transcriptional factors influenced by cellular redox state (Imbert et al. 1996). ROS is an important intermediate second messenger of NFκB activation by upstream stimuli such as TNF and IL-1. It is involved in the regulation of inflammation, stress responses, expression of cytokines and cell adhesion molecules, regulation of immune response and programmed cell death. NF-κB targets multiple genes involved in inflammation including ICAM, VCAM, and IL-1, the production of cytokines, upregulation of prothrombotic markers, and pathogenesis of atherosclerosis. (Wilson et al. 2000; Kim et al. 2001). Postirradiation activation of NF-κB was prevented by NO, and thus a reduction in the bioavailability of NO may result in epigenetic changes that promote vascular inflammation and atherosclerosis (Peng et al. 1995). NFκB is found to be upregulated in atherosclerotic vessels and its nuclear translocation has been detected in the intima and media of atherosclerotic lesions and in smooth muscle cells, endothelial cells, macrophages and T cells of atherosclerotic plaques. It has also been reported that NFκB plays a role in mediating of T-cell signaling in atheromatous plaques (Brand et al. 1997; Landry et al. 1997; Mach et al. 1998; Kawano et al. 2006; Barlic et al. 2007).

Peroxisome proliferators activated receptors (PPAR) and ROS signaling

PPARs are expressed in vascular cells where they exert antiatherogenic, anti-inflammatory and vasculoprotective actions. Activators of PPARalpha (fibrates) and PPARgamma (thiazolidinediones or glitazones) antagonize angiotensin II effects in vivo and in vitro and have cardiovascular antioxidant and anti-inflammatory actions. (Touyz and Schriffrin 2006). PPAR transcription factor has been implicated in the inflammatory processes involved in pathogenesis of atherosclerosis. Oxidized LDL has been shown to increase expression of PPAR in foam cells of atherosclerotic lesions. However, PPAR does not have a sole role in -9

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the mediation of inflammation. Activation of PPARalpha and PPARgamma isoforms results in anti-inflammatory responses in blood vessel wall. Specific agonists of PPARgamma have been shown to suppress pro-inflammatory gene expression in monocytes. Activators of PPAR alpha have also been shown to block inflammatory responses in aortic smooth muscle cells and PPARgamma activation has been shown to mitigate the inflammation associated with chronic and acute neurological insults (Rieber and Baeuerle 1991; Slezak et al. 2013).

Pivotal role of endothelium in radiation-induced injury

The endothelium plays an essential role in maintaining of cardiovascular function, and early changes in endothelial function are indicators of cardiovascular morbidity and mortality (Okruhlicova et al. 2012; Triggle et al. 2012). Important functional role of the endothelium is represented by the control of blood flow angiogenesis, inflammation, platelet aggregation, and vascular remodeling, as well as by control of metabolism. Radiation damage to the myocardium is caused primarily by inflammatory changes in the microvasculature, leading to microthrombi and occlusion of vessels, later reduced vascular density, perfusion defects and focal ischemia. This is followed by progressive myocardial cell death and fibrosis. Irradiation of endothelial cells lining large vessels also increases expression of inflammatory molecules, leading to adhesion and transmigration of circulating monocytes that transform into activated macrophages (Stewart et al. 2010). Endothelium is not only an important source of nitric oxide (NO), but also of numerous other signaling molecules, including the putative endothelium-derived hyperpolarizing factor (EDHF), prostacyclin (PGI2), and hydrogen peroxide (H2O2), which have both vasodilating and vasoconstricting properties. It modulates flow-mediated vasodilatation as well as influences mitogenic activity, platelet aggregation, and neutrophil adhesion. These early effects are followed by endothelial cells proliferation and obstruction of myocardial capillary lumen (Gyenes 1998). Radiation injury of endothelial cells results in a reduction in the bioavailability of NO, increases the recruitment of leukocytes to the endothelium indicating that endothelial cellderived NO plays an important anti-inflammatory role that, in part, is mediated by the inhibition of adhesion molecule expression (Kubes et al. 1991; Niu et al. 1994). Reduced bioavailability of NO promotes endothelial and vascular dysfunction not only via profound -10

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effects on vascular tone and blood flow, but also via promotion of cell proliferation and enhanced expression of adhesion molecules. In addition, a reduced bioavailability of NO and (or) PGI2 will also enhance the potential for platelet aggregation (Triggle et al. 2012). It has been postulated that damage to the microvascular components begins with the injury of endothelial cells within heart blood capillaries. Endothelial damage leads to an acute inflammatory reaction and to activation of the coagulation mechanisms with consequent fibrin deposition. The activation of macrophages and monocytes during the inflammatory process results in the continuous secretion of cytokines and growth factors, including Tumor Necrosis Factor (TNF), Interleukin (IL)-1, IL-6, IL-18, monocytes chemotactic factor. Besides induction of adhesion molecules, up-regulation of some cytokines (namely IL-6 and IL-8) has been observed after endothelial cell irradiation in a time- and dose-related fashion manner (Burger et al. 1998; Van der Meeren et al. 1999). Although microvascular injury is a major underlying cause of radiation-induced myocardial damage, radiation could also damage the major arteries leading to an accelerated development of age-related atherosclerosis. The initial event in radiation-induced atherosclerosis is endothelial cell damage and transmigration of monocytes into the intima, with subsequent ingestion of low-density lipoproteins and formation of fatty streaks (Konings et al. 1978; Vos et al. 1983). Schultz-Hector and Trott (2007) concluded that in rodents, radiation-induced heart disease was caused by radiation damage to the micro-vasculature leading to focal ischemia. Atherosclerosis of the coronary arteries has been never observed in rodent hearts except in constitutionally hypertensive rats (Lauk and Trott 1988). Radiation may cause micro-vascular disease which is characterised by a decrease in capillary density causing chronic ischemic heart disease and focal myocardial degeneration, and macrovascular disease through the faster development of age-related atherosclerosis in the coronary arteries (Schultz-Hector and Trott 2007). Progressive decrease of capillary density occurrs later (after two months) both as a random rarefication by disappearance of individual capillaries and as a focal loss of groups of capillaries which gradually lead to ischemic necrosis. Before the focal loss of capillaries, focal disappearance of alkaline phosphatase activity was observed (Lauk 1987; Schultz-Hector and Balz 1994; Seddon et al. 2002). This focal functional injury of endothelial cells is detectable -11

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within few weeks after irradiation. Focal loss of capillaries is preceded by increased endothelial proliferation but in the enzyme-negative areas only (Schultz-Hector et al. 1993). Radiation-induced vascular injury and endothelial dysfunction are mediated also in part by Transforming Growth Factor- ߚ (TGF- ߚ ) (Kruse et al. 2009), a pluripotent growth factor. Also, there is evidence of prothrombotic effects of radiation (Verheij et al. 1994; van Kleef et al. 1998; Boerma et al. 2004) which may be the cause of the increased platelet adherence and thrombus formation observed in irradiated capillaries and arteries (Schultz-Hector et al. 1992; Darby et al. 2005; Ivanov et al. 2006; Hussein et al. 2008). Experimental evidence suggests that RIHD is the result of indirect myocytes secondary effect caused by microvascular and macrovascular damage (Corn et al. 1990; Gagliardi et al. 2001; Jaworski et al. 2013). Endothelial dysfunction is believed to be a precipitating factor in the development of cardiac sequelae (Paris et al. 2001) and is most likely a combination of impaired endothelial function, stimulation of growth factors, and eventual fibrosis (Darby et al. 2010). Interestingly, according to animal studies, the pathophysiology of RIHD seems to be fundamentally different from non-radiation-related chronic heart failure. In the latter, the reduction of cardiac output induces a sustained activation of the sympathetic nervous system and, subsequently, a down-regulation of cardiac β-receptors. In RIHD, the adrenal catecholamine synthesis is unchanged and cardiac catecholamine content is reduced, leading to an increase of β-receptor density (Schultz-Hector et al. 1992; Gyenes 1998). Other strategies thus may include modulating the effects of VEGF on tight junction proteins. VEGF-mediated increases in vascular permeability are associated with endothelial nitric oxide synthase (eNOS) activity and release of nitric oxide (NO) (Bates 2010). This process is probably related to microcirculatory damage, as therapeutic doses of irradiation do not seem to cause direct damage to myocytes. Endothelial damage leads to an acute inflammatory reaction (due to acute swelling of the endothelial cells). The activation of the coagulation mechanisms leads to fibrin deposition. The activation of macrophages and monocytes during the inflammatory process results in the secretion of cytokines, including TNF, IL-1, IL-6, IL-8, monocyte chemotactic factor and later PDGF and TGF-β. These early effects are followed by organized fibrin formation, endothelial proliferation and collagen deposition (Slezak et al. 2013; Slezak et al. 2014), and, in the late phase, fibroblastic proliferation and enhanced atherosclerosis. Microscopy revealed an increased amount of -12

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collagen and a higher proportion of type I collagen (relative to type III) amount (morphometry) of collagen 6 w after irradiation. Significant increase of collagen I, enhances the rigidity of myocardium (Schultz-Hector et al. 1992; Chello et al. 1996; Gyenes 1998, Slezak et al. 2014.). Early and late side-effects of radiation limit dose escalation, and affect the patient's quality of life. Irradiated endothelial cells acquire a proinflammatory, procoagulant and prothrombotic phenotype. Reduced myocardial capillary density in later stages (Baker et al. 2009), focal loss of endothelial alkaline phosphatase (Schultz-Hector and Balz 1994), and increased expression of von Willebrand factor (Boerma et al. 2004) are leading hallmarks of irradiation damage. Von Willebrand factor (vWf), a glycoprotein involved in blood coagulation, is synthesized by endothelial cells. VWF mediates the adherence of platelets to one another and to the sites of vascular damage. Increased amounts of vWf in blood plasma or tissue samples are indicative of damaged endothelium. It is important in modulation of platelets and leukocyte recruitment and formation of blood clots (Gabriels et al. 2012). Six weeks after irradiation, vessels had increased von Willebrand Factor expression, indicative of endothelial cell damage (Slezak et al. 2013). Radiation is altering functional properties of cardiac sarcolemmal Na, K-ATPase (Mezesova et al. 2014). These various mechanisms probably result in different cardiac pathologies, e.g. coronary artery atherosclerosis leading to myocardial infarction, versus microvascular damage and fibrosis leading to congestive heart failure (Stewart et al. 2013).

The role of mononuclears and mastocytes in irradiated myocardium

Early radiation damage to the myocardium was represented primarily by chronic inflammatory cells infiltration in the ventricular myocardium at 6 weeks after 25 Gy irradiation. Microvascular density was not decreased at this time period (after 6 weeks and 25 Gy (Slezak et al 2014)). Myocardial and microvascular inflammatory changes were leading to extravasation of blood cells, creation of microthrombi and signs of fibrosis. Monocytes mononuclear leukocytes infiltrate the myocardium in response to radiation-induced inflammatory signals and undergo activation. Activated monocytes produce and secrete -13

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several pro-inflammatory cytokines (TNF, IL-1, and IL-12) that amplify the inflammatory response (Okunieff et al. 2008). Postirradiation induced mast cells degranulation and interaction with many cellular and molecular systems in the heart. For instance, mast cell-derived proteinases, have been shown to contribute to both the formation and degradation of endothelin-1 (ET-1) (Metsärinne et al. 2002; Maurer et al. 2004). Long-term up-regulation of the endothelin system may have detrimental effects due to the vasopressor, pro-hypertrophic, and pro-fibrotic properties of ET-1 (Giannessi et al. 2001). Mast cells express the receptor endothelin A receptor (ET-A), which, upon activation by ET-1, induces mast cell degranulation (Yamamura et al. 1994), a pathway by which ET-1 may enhance the activity of matrix metalloproteinases (MMPs) in the heart (Janicki et al. 2006; Lundequist et al. 2006). In recent years, microRNAs (miRNAs) have emerged as novel regulators of gene expression. miRNAs participate in many cellular processes, such as apoptosis, fat metabolism, cell differentiation, tumorigenesis and cardiogenesis. MiRNAs are also critically involved in the pathological process of cardiac hypertrophy, angiogenesis, arrhythmogenesis, radiation injury and heart failure. Suppression of injurious genes may lead to upregulation of protective proteins including eNOS and HSP70. Nevertheless, the protection observed clearly suggests that concerted action of one or perhaps several miRNAs, may have been responsible for the increased expression of cardioprotective substances (Yin et al. 2009). MiR15b is proapoptotic and is linked with injury caused by ischemia. Six weeks after 25 Gy mediastinal irradiation, miR-15b was down-regulated in the hearts almost by 42 % which is indicating that these hearts are probably protected or there is an adaptive compensatory mechanism triggered upon irradiation (Slezak et al. 2013). Expression of microRNA-21 in these hearts was increased nearly 10-fold which points to compensatory/protective effect in the myocardium 6 weeks after radiation (Sayed et al. 2010; Qin et al. 2012; Slezak et al. 2013; Skommer et al. 2014). Postischemia/reperfusion levels of mRNA of PPAR alpha were significantly lower in the hearts of irradiated animals than in the hearts of their control counterparts indicating a shift in substrate preferences from fatty acids to glucose. Myocardial Cx43 was upregulated via reduced miRNA-1 that coud be interpreted as is an adaptive/protective mechanism triggered 6 weeks upon irradiation (Radosinska et al. 2011; Viczenczova et al. 2013; Viczenczova et al. 2014). Connexin-43 (Cx43) cardiac gap junction channels play the crucial role in synchronizing myocardium allowing impulse propagation -14

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from pacemaker cells along the conduction system and throughout the atria and ventricles. The channels, in addition, are permeable to ions and small molecules (up to 1 kD) that is important for direct cell-to-cell communication. Cx43 channels are opened and closed (gated) by various treatments. Likewise, Cx43 expression and distribution can be modulated by various physiological and pathophysiological stimuli (Salameh and Dhein 2005). Impaired intercellular communication due to disease-related alterations in myocardial Cx43 distribution and/or expression promotes development of life-threatening arrhythmias and contractile dysfunction (Severs et al. 2004; Tribulova et al. 2008). Barancik et al. (2013) demonstrated tissue-specific alterations in activation of cardiac MMP-2 six weeks after exposure to irradiation. The stimulatory effects of irradiation on circulating MMP-2 in rats suggest that this enzyme plays a significant role in the progression of the effects induced by irradiation and may be responsible for the development of pathological changes induced by cardiac irradiation. Circulating MMPs have been proposed to be a prognostic factor for survival in patients with heart failure, and recent study demonstrated a strong positive correlation between plasma levels of MMP-2, myocardial infarction size, and left ventricular dysfunction. These findings suggest that the observed activation of MMP-2 in circulation may have a negative impact on the progression of pathological changes induced as a consequence of mediastinal irradiation. It was found that irradiation of rats significantly increased the activities of circulating 72 kDa MMP-2. Importantly, application of acetylsalicylic acid (ASA) or statin markedly reduced the effect of irradiation on circulating MMP-2 (Barancik et al. 2013). Induction of heat shock proteins was also reported to be involved in the adaptive response (Kang et al. 2002). Ceramides are a family of waxy lipid molecules found in high concentrations within the cell membranes. A ceramide is composed of sphingosine and a fatty acid. They are one of the structural elements and component that make up sphingomyelin, one of the major lipids in the lipid bilayer that can participate in a variety of cellular signaling including regulation of differentiation, proliferation, and programmed cell death of cells. Apoptotic signaling can be also facilitated by interaction of ionizing radiation with cellular membranes suggesting that direct DNA damage mediates radiation-induced cell death. It is noteworthy that the substances that can cause ceramide to be generated, tend to be stress signals that can cause the cells to go into programmed cell death. Ceramide thus acts as an intermediatory signal that -15

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links the external signal with the internal metabolism of the cells (Haimovitz-Friedman et al. 1994; Hallahan 1996). Recent evidence suggests that ceramide regulates stress signaling via reorganization of the plasma membrane (Stancevic and Kolesnick 2010). Following thoracic irradiation, ICAM-1 and inflammation contribute to pulmonary fibrosis and injury. Expression of vascular cell adhesion molecule-1 VCAM-1 and intracellular adhesion molecule-1 (ICAM-1) in the irradiated mouse lung was decreased by manganese superoxide dismutase– plasmid/liposome gene therapy (Epperly et al. 2002). Experimental findings suggest that radiation injury to the myocardial capillary network is the underlying cause of myocardial degeneration and heart failure after heart irradiation. A number of pro-inflammatory molecules have been reported to be upregulated by endothelial cell irradiation in vitro and in vivo. These adhesion molecules include E-selectin (a mediator of leukocyte rolling), ICAM (a mediator of leukocyte arrest), and PECAM-1 (involved in leukocyte transmigration). These pro-inflammatory factors may be the molecular correlate of early radiation-induced ultrastructural changes observed in the microvessels of the myocardium (Fajardo and Stewart 1971; Schultz-Hector and Balz 1994). The selectins are a family of cell adhesion molecules (or CAMs). All selectins are singlechain transmembrane glycoproteins. Selectins bind to sugar moieties and thus are considered to be a type of lectin, cell adhesion proteins that binds sugar polymers. There are three subsets of selectins: E-selectin (in endothelial cells), L-selectin (in lymphocytes), P-selectin (in platelets and endothelial cells) (Fajardo 1970; Corn et al. 1990; Somers et al. 2000; Ley 2003; van Luijk et a. 2005). The p53 protein plays a key role in the adaptive response. It is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor, preventing cancer and supporting cell and gene stability. The role of p53 in the response to radiation damage is complex since it affects some aspects of DNA repair, controls checkpoint cell cycle arrest and initiates apoptosis, etc. (Fei and El-Deiry 2003). Low doses of radiation can modulate the expression of a variety of genes (Sasaki et al. 2002). Exposure to high-dose radiation could suppress p53-dependent apoptosis (Takahashi 2001). The morphological alterations in the early phase, about 6 h after the exposure, include acute inflammation of small/medium size arteries and a neutrophils infiltrate affecting all layers of the heart. During the following latent phase, only slight progressive fibrosis can be detected -16

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by light microscopy. Electron microscopy studies demonstrate, however, progressive damage of myocardial capillary endothelial cells leading to obstruction of the lumen and the formation of thrombi of fibrin and platelets. Eventually, the decreased patency of capillaries results in ischemic damage and subsequent myocardial cell death. As myocytes have no capability do divide, these processes lead to their replacement by fibrosis. Damage may also affect myocardial cells involved in conduction processes leading to arrhythmias (Stewart et al. 1995; Gagliardi et al. 2001). Early radiation damage to the myocardium was represented primarily by chronic inflammatory cells infiltration in the ventricular myocardium at 6 weeks after 25 Gy irradiation. Suprisingly, microvascular density was not decreased at this time. Presence of monocytes and mast cells represent the important feature of this phase. Myocardial and microvascular inflammatory changes were leading to extravasation of blood cells, creation of microthrombi and signes of fibrosis. A number of more or less acute effects including endothelial damage (seen in EM), inflammatory cell infiltration, and lysosomal activation, were observed (Slezak et al. 2013). Early structural alterations in irradiated endothelial cells are reversible and later are followed by a persistent decrease in capillary density (Schultz-Hector and Balz 1994). Systematic morphometric studies in rats show that capillary volume and length density begin to decline about 20 days after heart irradiation, and the decline continues in a dose-dependent manner. A capillary rarefication is also accompanied by a simultaneous focal loss of the endothelial cell marker enzyme, alkaline phosphatase, which increase is time-dependent. Some evidence shows that this enzyme is involved in regulating endothelial cell proliferation and microvascular blood flow by dephosphorylating extracellular nucleoside phosphates. When foci of myocardial degeneration developed, they were invariably situated in the areas of enzyme deficiency. Ultrastructural studies showed that the enzyme loss was not caused by a loss of endothelial cells but was associated with the signs of endothelial cell activation, such as swelling, lymphocyte adhesion, and extravasation (Schultz-Hector and Balz 1994), as well as with increased endothelial cell proliferation (Lauk and Trott 1990; Slezak et al. 2013; Slezak et al. 2014). In the late phase, vessel lumen progressive obstruction and formation of fibrin thrombi and platelets, like those shown in electron microscopy studies, result in ischemia and subsequent myocardial cell death. This process leads to replacement of cardiac tissue by fibrotic tissueand to chronic heart failure (Stewart et al. 1995; Gyenes 1998; Seddon et al. 2002; Adams et al. 2003; Schultz-Hector 2007).

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Damage may also affect myocardial cells involved in the process of conduction leading to arrhythmias (Stewart et al. 1995). Injury of the pericardium may be present as excessive pericardial fluid effusion, extensive fibrotic thickening, pericardial adhesions, and finally pericarditis (Stewart et al. 1995). Cardiac output did not decrease progressively in the early phase. There was a modest, early decrease of cardiac output, however, after this drop, cardiac output remained stable until the final heart failure (Schultz-Hector et al. 1992; Slezak et al. 2013). The concentration of betaadrenoceptors in the irradiated heart increased by 50%, already 2 months after the irradiation, before any evidence of myocardial damage was apparent (Schultz-Hector et al. 1992). This suggests that the initial radiation damage stimulated an up-regulation of cardiac contractility to a stable level via adrenergic mechanisms until the failure of compensatory mechanisms was evident. At the time of beginning congestive heart failure, a sudden drop of left ventricular ejection fraction and of cardiac output has been measured (Kitahara et al. 1993). This points out that cardiac output is not a safe criterion of sub-clinical radiation damage to the heart, neither in experimental animals nor in the patients. Summary of some of effects on the heart after irradiation is given in the Fig. 5. Radiation protection and treatment

Antioxidants have been studied for their capacity to reduce the cytotoxic effects of radiation in normal tissues for at least 50 years. Early research identified sulfur-containing antioxidants as those with the most beneficial therapeutic ratio. These compounds have substantial toxicity when given in vivo. Radiation protectors that are not primarily antioxidants, including those that act through acceleration of cell proliferation (e.g. growth factors), prevention of apoptosis, other cellular signaling effects (e.g. cytokine signal modifiers), or augmentation of DNA repair, all have direct or indirect effects on cellular redox state and levels of endogenous antioxidants (Okunieff et al. 2008). Differences in the management of radiation-induced oxidative stress between tumors and normal tissues can provide a fundamental basis to design new cancer therapeutic agents which can exploit differences between normal tissue and tumor mechanisms of handling the oxidative stress of ionizing irradiation damage (Epperly et al. 2007; Greenberger and Epperly

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2007). Unfortunately, some of known radioprotectors are toxic at doses required for radioprotection. Treatments that reduce the risk or severity of damage to normal tissue or that facilitate the healing of radiation injury are being developed. These could greatly improve the quality of life of patients treated for cancer (Stone et al. 2003). Although prevention of radiation toxicity may provide the best opportunity to minimize impact on quality of life, few radioprotectors are in clinical use and the treatment of radiation injury remains an important target to deal with radiation-induced side effects. Novel technologies, such as gene therapy, may offer the ability to reverse radiation-induced toxicities (Citrin et al. 2010). In general, chemical/biological agents used to combat tissue toxicity from radiation can be broadly divided into three categories based on the timing of delivery in relation to radiation: chemical radioprotectors, mitigators, and treatment (Stone et al. 2004). Pharmacological agents can be used before or after radiation to reduce side effects and are classified based on the timing of radiation delivery. "Radioprotectors," used as a molecular prophylactic strategy before radiation, are mostly based on antioxidant properties. Currently, amifostine is the only radioprotector approved for the use in the clinic. "Mitigators," given during or shortly after irradiation, reduce the action of cellular ionizing radiation on normal tissues before the appearance of symptoms. Lastly, a "treatment" is the administration of an agent once symptoms have developed in order to reverse those that are mostly due to fibrosis (Bentzen 2006; Citrin et al. 2010; Bourgier et al. 2012). There is a large amount of data on the radioprotective effects of antioxidants at the cellular level, especially at the level of nuclear DNA, where the radical scavenging by the antioxidant protects this and other sensitive cellular targets. Many antioxidants have been shown to protect the cell by increasing cellular antioxidant capacity through their ability to elevate the levels of natural antioxidants and antioxidant enzymes. There are a number of hypotheses that have been suggested to explain the enhanced radioprotective effect of combined antioxidant treatments related to the regulation and response to ROS including the regeneration of vitamin E and other antioxidants by vitamin C, induction of cellular antioxidant systems, and interaction with inflammatory mediators (Okunieff et al. 2008).

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Antioxidants may interfere with the initial mediation of apoptosis by ROS (Salganik 2001), as well as later membrane lipid peroxidation, which is characteristic of radiation-induced apoptosis (McClain et al. 1995). The impact of radiation on the mitochondrial DNA and thus long-term reproductive health of the mitochondria, reproduction of the cell, and on cellular redox and energy state has not been studied in details. The long-term consequences of radiation may be very dependent on this mechanism of radiation toxicity and may be greatly alleviated by properly designed antioxidant treatment (Okunieff et al. 2008). Regarding the oxidative stress in physiological conditions, cells could increase the activities of antioxidant enzymes and other antioxidant defences to counteract the effects of oxidative stress. These include manganese dependent superoxide dismutase (Mn-SOD), Copper/Zinc superoxide dismutase (Cu/Zn SOD), glutathione peroxidase, glutathione reductase and catalase (CAT). MnSOD and Cu/ZnSOD convert O2·− to hydrogen peroxide, which is then transformed to water by glutathione peroxidase or catalase. When discussing antioxidants as radioprotectors, it is worth mentioning the use of SOD as a method to prevent radiotherapy-induced toxicity. Ionizing radiation results in the formation of superoxide radicals that are highly reactive and potentially damaging to cells. SOD is an enzyme that is naturally present in human cells. It catalyzes the conversion of superoxide to oxygen and hydrogen peroxide and functions as an antioxidant during normal conditions and after radiation. Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral vectors (Tribble et al. 1999; Dröge 2002; Ferreira et al. 2004; Vassalle et al. 2008; Veldwijk et al. 2009; Citrin et al. 2010). Administration of manganese superoxide dismutase-plasmid liposomes (MnSOD-PL) has been demonstrated to provide local radiation protection to the lung, esophagus, oral cavity, urinary bladder and intestine. Radiation protection has been shown to be mediated in part by MnSOD stabilization of the antioxidant pool including glutathione and total thiols within cells and in normal tissues. General antioxidant defense is also provided by low molecular weight antioxidants, which are hydrogen atom–donating reducing agents such as ascorbic acid, tocopherols, polyphenols, and thiols such as glutathione. In this situation, the oxidants are neutralized by hydrogen atom donation, resulting in a less reactive or nonreactive product from the original oxidant and a

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radical product from the antioxidant, which no longer can exert detrimental effects (Citrin et al. 2010).

Radioprotective effect of antioxidants

Prevention of immediate radiation-induced genotoxicity requires that an antioxidant be present at the time of irradiation.To be maximally effective, the antioxidant must be present in the vicinity of DNA and thus must have an access to the nucleus. It must be able to either, 1) react with all the oxygen-related free radicals and detoxify them to radicals that are not themselves genotoxic and/or 2) effectively compete with oxygen to repair damage to the DNA chemically through reactions with free radicals on the DNA. Thiol-based compounds are especially good antioxidants because these compounds are capable of both scavenging oxygen radicals and affecting chemical repair of some forms of DNA damage with the subsequent formation of sulfur-based radicals, which are not reactive with DNA (Held 1988). Sulfhydryl compounds such as cysteine and cysteamine have long been known to act as radioprotectors via free radical scavenging and H atom donation (Patt et al. 1949; Bacq 1954). Aminothiols could function as both radical scavengers and polyamine mimetics that influence DNA protection, repair, and synthetic processes. Thiols such as amifostine and the newly developed nitroxides have sufficient reactivity to efficiently scavenge secondary radicals, in fact, amifostine is the only synthetic antioxidant used clinically (Seed 2005). The application of antioxidant radioprotectors in various human radiation exposure situations has not been extensive, although it is generally accepted that endogenous antioxidants, such as cellular non-protein thiols and antioxidant enzymes, provide some degree of protection. Pathohistology examinations revealed better radioprotective effects of fullerenol compared to those of amifostine on the spleen, small intestine and lung, while amifostine had better radioprotective effects than fullerenol in protection of the heart, liver and kidney (Trajković et al. 2007). Captopril (Yarom et al. 1993), amifostine (Kruse et al. 2003) and a combination of pentoxifylline and alpha-tocopherol (Boerma et al. 2008) showed some potential advantage in the prevention of myocardial damage, but their clinical usefulness has to be yet demonstrated. A number of nonthiol radioprotective agents including protease inhibitors, vitamins, metalloelements, and calcium antagonists, are also used. There has been a virtual explosion of -21

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interest in biological, as opposed to chemical, modifiers of radiation injury. These biologic compounds include cytokines, such as interleukin 1 and granulocyte colony-stimulating factor, eicosanoids, such as prostaglandins and leukotrienes, and steroids/glucocorticoids, such as dexamethasone and methylprednisolone. In addition, an array of immunomodulatory agents, such as glucan and endotoxin have been described, which act nonspecifically by enhancing immunological and hemopoietic responses. Although some of these biologic agents act best when given prior to irradiation, many of them can modulate radiation injury when given after irradiation, presumably by affecting the recovery and repopulation of critical tissue elements (Murray and McBride 2000). Activation of proinflammatory and proliferative pathways is an integral part of the inflammatory response and involves activation of nuclear factor kappa B (NF‐kB) and TNF. The involvement of NFκB was demonstrated

in the induced expression of antioxidant

enzyme MnSOD (Murley et al. 2004; Murley et al. 2006; Murley et al. 2007). Antioxidants including vitamin E and N-acetylcysteine have been shown to reverse activation of NFκB by TNF and IL-1 however, it is thought that a non-antioxidant action on NFκB activity may also be responsible (Allport et al. 2000; Yeung et al. 2004). The data indicate that atorvastatin exerts protective effects on irradiated heart by reducing apoptosis by up-regulating thrombomodulin expression , a thrombin membrane receptor and enhancing protein C activation in irradiated tissues (Ran et al. 2010). The new effective prevention of radiation induced inflammation could be based upon using of NF‐kB inhibitors, anti TNF, statins, matrix metalloproteinases inhibitors , free‐radical scavengers, and inhibitors of mast cell degranulation (Rainsford 2007; Slezak et al. 2013; Slezak et al. 2014). Metalloporphyrin antioxidants ameliorate normal tissue radiation damage and are serving as SOD mimetics to combat oxidative stress against acute radiation-induced apoptosis and ameliorated delayed damage to the blood-brain barrier without producing a discernible increase in tissue superoxide disumtase (SOD) activity (Pearlstein et al. 2010). Antioxidants derived from natural sources also exhibit dose-modifying effects on DNA damage and cell survival when present at the time of irradiation. This immediate protection is mediated by the scavenging of radicals. For example, there is a number of antioxidants, including caffeine, melatonin, flavonoids, polyphenols, and other phytochemicals, which are -22

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shown to decrease radiation-induced damage in either plasmid or cellular DNA through the scavenging of oxygen radicals and/or peroxides (Kumar et al. 2001; Frei and Higdon 2003). Antioxidants are also phytochemicals, vitamins and other nutrients that protect cells from damage caused by free radicals. Phytochemicals are nonnutritive bioactive plant substances, such as a flavonoids or carotenoids that are recognized to have a beneficial effect on human health and also called phytonutrients. From antioxidant nutrients only vitamins E and C and the mineral selenium are considered to be dietary antioxidants and are defined as “a substance in foods that significantly decreases the adverse effects of reactive species, such as reactive oxygen and nitrogen species (Weiss and Landauer 2003). Also radioprotection by dietary vitamin A and β-carotene in mice exposed to partial-body irradiation or total-body irradiation has been reported (Seifter et al. 1988). With the understanding that free radicals perpetuate a significant amount of the damage caused by ionizing radiation, multiple vitamin antioxidants have been tested as a method to reduce the toxicity of radiotherapy. Antioxidant compounds such as glutathione, lipoic acid, and the antioxidant vitamins A, C, and E have been evaluated in this context. Conversely, well-known antioxidants such as vitamin C and vitamin E do not act as classic radioprotectors (Citrin et al. 2010). The vitamin E analog γ-tocotrienol (GT3) is regarded to be a potent radioprotector and mitigator. The efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX). (Berbée et al. 2011). Antifibrotic treatments, such as PTX and vitamin E, have shown promise in clinical trials. Oral administration of PTX alone from 3 months to 6 months after irradiation did not significantly reverse myocardial remodeling or intracellular signaling, but instead, had an adverse effect on cardiac rhythm after local heart irradiation. While addition of a tocotrienolenriched oral formulation could not correct the adverse effects of PTX on cardiac function, it reduced myocardial inflammatory infiltration (Sridharan et al. 2013). Treatment with PTX and α-tocopherol may have beneficial effects on radiation-induced myocardial fibrosis and left ventricular ex vivo function, both when started before irradiation and when started later during the process of RIHD (Boerma et al. 2008). This approach should

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be expanded and new promising concepts of delaying the clinical manifestations of progressive ischemic heart disease should also be tested in RIHD (particularly in rats). The ability to treat chronic inflammation by targeting NF-kappaB has been suggested for a number of available drugs, but there is a paucity of therapeutical adjuncts suggested to cope with the adverse effects of radiotherapy. It is, therefore, of utmost interest that the effects of PTX

have been studied even beyond NF-kappaB in the context of radiation-induced

inflammation (Halle and Tornvall 2011). Tocotrienols reduced a number of cardiac mast cells and macrophages. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation (Sridharan et al. 2013). The comment that PTX is suggested to prevent radiation-induced cardiotoxicity is indeed very interesting. Unfortunately, in our review, we were only able to mention a few potential targets of treatment to ameliorate radiation-induced tissue damage. A large number of selenium (Se) derivatives and selenium and selenium/vitamin E combinations have been studied for their radioprotective effects (reviewed by Weiss et al. 1994). Se compounds are found in a variety of foods; for example, Se-methylselenocysteine is found in garlic and broccoli. Selenomethionine is a naturally occurring derivative of low toxicity and is found in soy, grains, legumes, and selenium-enriched yeast (Whanger 2002). Antioxidants derived from natural sources also exhibit dose-modifying effects on DNA damage and cell survival when present at the time of irradiation. This immediate protection is mediated by the scavenging of radicals. For example, there is a number of antioxidants, including caffeine, melatonin, flavonoids, polyphenols, and other phytochemicals (e.g., albana), which are shown to attenuate radiation-induced damage in either plasmid or cellular DNA through the scavenging of oxygen radicals and/or peroxides (Kumar et al. 2001; Frei and Higdon 2003). Finally, a number of phytochemicals including caffeine, genistein, and melatonin, have multiple physiological effects, as well as antioxidant activity, which result in radioprotection in vivo. Many antioxidant nutrients and phytochemicals have antimutagenic properties, and their modulation of long-term radiation effects have been demonstrated. A number of -24

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flavonoids (genistein, quercetin, luteolin and other tea components) reduce the frequency of micronucleated reticulocytes in the peripheral blood of irradiated mice (Shimoi et al. 1994). Procyanadins (flavan-3-ols) from grape seed extract, including rutin, were radioprotective as confirmed by a decrease in number of micronucleated erythrocytes from bone marrow of irradiated mice (Castillo et al. 2000). Melatonin is thought to act as an antioxidant itself (Lopez-Burillo et al. 2003; Reiter et al. 2003) but also is capable to increase the expression of antioxidant enzymes such as SOD and glutathione peroxidase (Okatani et al. 2001). Radioprotection with melatonin and melatonin analogs has been documented in a number of animal models (Manda et al. 2007; Hussein et al. 2008). Melatonin (N-acetyl-5-methoxytryptamine) has been shown to augment the activity of glutathione peroxidase in addition to stimulating the activity of glutathione reductase and increasing the synthesis of glutathione (GSH); all of which are important in reducing levels of oxygen radicals and peroxides in the cells (Reiter et al. 2003; El-Missiry et al. 2007). Melatoninedible plants, such as cherries, may be a significant source of melatonin, an endogenous antioxidant found in high concentrations in the pineal gland (Reiter and Tan 2002). A large number of studies has demonstrated the protective effects of melatonin against oxidative stress caused by radiation. Resveratrol (RSV), a natural polyphenol, produced in many plants. It is present in human diet, e.g. in fruits and in wine. RSV is known for its antioxidant, anti-inflammatory, analgetic, antiviral, cardioprotective, neuroprotective and antiaging effects. Depending on the dose, RSV may act as an antioxidant or as a pro-oxidant. RSV is able to modulate the behavior of the cells in response to radiation-induced damage (Dobrzyńska 2013). Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that modulate responses to radiation. This protective effect was related to the demonstrated antioxidant properties of caffeine in vitro including scavenging of primary and secondary ROS. Dietary flaxseed (FS) displays antioxidant and anti-inflammatory properties in preclinical models of lung disease including radiation-induced pneumonopathy. FS induced significant changes in lung miRNA profile suggesting that modulation of small RNA by dietary supplements may represent a novel strategy to prevent adverse side-effects of thoracic -25

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radiotherapy (Christofidou-Solomidou et al. 2014). Dietary FS given early post irradiation mitigated radiation effects by decreasing inflammation, lung injury and eventual fibrosis while improving survival . FS may be a useful agent mitigating adverse effects of radiation in individuals exposed to radiation, inhaled radioisotopes or even after the initiation of radiation therapy to treat the malignancy (Pietrofesa et al. 2013). A number of new strategies and drugs including certain growth factors, compounds involved in signal transduction and apoptosis, synthetic aminothiols, cytokines and gene therapies are under development to protect the cardiovascular system from the side effects of radiation (Basavaraju and Easterly 2002). Important potential neuroprotector that has a pleiotropic function is erythropoietin (EPO). It is a cytokine (protein signaling molecule) for erythrocyte (Wong and Van der Kogel 2004), which plays an important role in the brain response to neural injury. Studies in experimental animals have shown protective effects of carnitine against exposure to ionizing radiation (Khan and Alhomida 2011). Treatments that reduce the risk or severity of damage to normal tissue or that facilitate the healing of radiation injury are being developed. These could greatly improve the quality of life of patients treated for cancer (Stone et al. 2003). Although prevention of radiation toxicity may provide the best opportunity to minimize the impact on quality of life, few radioprotectors are in clinical use and the treatment of radiation injury remains an important mechanism to deal with radiation-induced toxicity. Novel technologies, such as gene therapy, may offer the capability to reverse radiation-induced toxicities (Citrin et al. 2010). Many cytokines and growth factors are radiation mitigators when used near the time of radiation. These agents stimulate the differentiation of stem cells in bone marrow or the intestine, thus preventing bone marrow failure or gastrointestinal syndrome after total body exposure.

A number of cytokines and growth

factors has been explored as

radioprotectors/mitigators. Keratinocyte-Growth-Factor KGF is a growth factor that stimulates a number of cellular processes such as differentiation, proliferation, DNA repair, and detoxification of ROS (Finch and Rubin 2004). These properties make KGF an attractive method to stimulate the recovery of mucosa after ionizing radiation. -26

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A variety of agents that protect against fibrosis has been evaluated as mitigators of radiation fibrosis. Transforming growth factor (TGF)-β plays a critical role in the development of radiation-induced fibrosis. It is, therefore, not surprising that many of the agents that have been used to prevent the development of radiation fibrosis directly or indirectly inhibit the TGF-β signaling pathway (Citrin et al. 2010). Moreover, some studies used animal models to test potential therapeutic strategies to utilize before and after heart irradiation. It was shown that administration of Methylprednisolon or Ibuprofen retarded the development of myocardial fibrosis, pericarditis and pericardial effusion, and improved survival of rabits in experimental model of radiation-induced heart disease (Reeves et al. 1982). Proinflammatory molecules COX‐2 and mPGES‐1 are involved in irradiation injury. Postirradiation alterations in both COX-1 and COX-2 vasoactive products contribute to endothelial and vascular dysfunction. Stewart et al. (2006) tested the efficacy of acetylsalicylic acid (ASA) and demonstrated protective effect in radiation induced endothelial dysfunction. Therefore, acetylosalicilic acid treatment may attenuate the inflammatory response in the rat heart 6 weeks after irradiation (Slezak et al. 2013; Slezak et al. 2014). Inhibitor of COX-2 regulates production of PGs involved in inflammation, pain and fever. The roles of COX-2 and COX-1 in cardiovascular diseases may bring more light and facilitate developing newer agents to control

conditions of

inflammation and anti‐inflammatory

effects of ASA (Hasan et al. 2013). Protection by ASA against irradiation injury may be mediated in part by inhibition of COX‐2 ASA also increases plaque stability but did not reduce the number or size of endothelial lesions of some ateries in irradiated mice (Hoving et al. 2008). However, anti-inflammatory and anti-coagulant therapies were less effective in inhibiting radiation-induced atherosclerosis than age-related atherosclerosis suggesting more complex underlying mechanistic pathways leading to the development of the irradiation induced lesions. Antibiotics, tetracyclines and fluoroquinolones, which share a common planar ring moiety, were also shown to be

radioprotective. Furthermore, tetracycline protected murine

hematopoietic stem/progenitor cell populations from radiation damage. The choice of antibiotics in such emergencies, as well as in cancer patients receiving radiotherapy, could benefit from consideration of more than purely microbiological criteria since not all classes of antibiotics are active. Although tetracyline and ciprofloxacin have long been utilized in the -27

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clinic and there is no evidence of long-term deleterious effects, their ability to inhibit or enhance radiation carcinogenesis should be investigated (Kim et al. 2009). High dose of Astragalus polysaccharide (APS) pretreatment led to remarkably less morphologic features of IR-induced hepatic and pulmonary injury. Thus, APS exerts protective effects against IR-induced injury in liver in mice, and the related molecular mechanism may involve suppressing the radiation-induced oxidative stress reaction (Liu et al. 2014). Septilin exhibited potential antioxidant activity and showed radioprotective effect against gamma-radiation by preventing oxidative stress and scavenging free radicals (Mansour et al. 2014). The effect of septilin an ayurvedic poly-herbal formulation

exhibited potential

antioxidant activity and showed radioprotective effect against γ-radiation by preventing oxidative stress and scavenging free radicals. Administration of septilin for 5 days (100 mg/kg) prior to radiation resulted in a significant increase in both superoxide dismutase (SOD) activity and total glutathione (GSH) level in hepatic and brain tissues (Mansour et al. 2014). Hydrogen (H2) has a potential as an antioxidant in preventive and therapeutic applications. H2 selectively reduced the hydroxyl radical, the most cytotoxic of reactive oxygen species (ROS), and effectively protected the cells; however, H2 did not react with other ROS, which mediates physiological roles. H2 can be used as an effective antioxidant therapy; due to its ability to rapidly diffuse across membranes, it can reach and react with cytotoxic ROS and thus protect against oxidative damage (Ohsawa et al. 2007). Consumption of hydrogen-rich water reduces the biological reaction to radiation-induced oxidative stress without compromising anti-tumor effects. These encouraging results suggested that H2 represents a potentially novel preventive strategy for radiation-induced oxidative injuries (Chuai et al. 2012; Qian et al. 2013). Chuai et al. (2012) found that pre-treatment with H2 prior to IR (ionizing radiation) significantly suppressed the reaction of •OH and the cellular macromolecules which caused lipid peroxidation, protein carbonyl and oxidatively damaged DNA. The role of the renin angiotensin system (RAS) in normal tissue radiation injury has been well defined (Robbins and Diz 2006). Inhibitors of angiotensin-converting enzyme (ACE) and antagonists of angiotensin type 1 receptors reduce injury in some animal models of irradiation (Robbins et al. 2009). -28

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Inhibition of ACE is considered to be cardioprotective in part by suppressing the breakdown of bradykinin by ACE (Fleming 2006). New treatments for IHD are being developed. Various types of stem cells are being studied to determine whether and how they diferentiate into new cardiomyocytes (Bearzi et al. 2007; Wang and Li 2007). Another approach is to inject mesenchymal stem cells overexpressing hepatocyte growth factor (HGF) (Duan et al. 2003), or application-deffcient adenovirus carrying the HGF gene to stimulate the regeneration of cardiomyocytes. Preliminary data show that HGF gene transfer and expression can improve myocardial perfusion of locally irradiated rat hearts. SPK1 (sphingosine kinase 1, a downstream signal molecule involved in HGF signal cascades) gene transfer also induces neovascularisation, inhibits fibrosis, and improves ischemic heart function (Duan et al. 2007). Stem cell and gene therapy both need further research to establish whether they have the potential to be safe and effective treatments for radiation-induced heart disease. Normal tissue injury remains important limiting factor in the treatment of malignancies by radiotherapy. In order to deliver a radiation dose sufficient to eradicate a localised tumour, the normal tissues need to be protected. A number of pharmacological agents has been used experimentally, and some clinically, to minimize radiation damage to normal tissues. The limited evidence available suggests that radiation insult, like many other tissue injuries, is amenable to pharmacological intervention (Rezvani 2008; Guha and Kavanagh 2011). The knowledge of molecular mechanisms involved in endothelium dysfunction following radiation is needed to identify therapeutic targets and develop strategies to prevent and /or reduce side-effects of radiation therapy (Milliat et al. 2008).

Conclusions While modern sofisticated radio-therapeutic techniques have reduced radiation exposure of the heart, they have not eliminated side effect of radiation. Radiation effects may occur as direct ionizations damage of organic molecules or indirectly via free radical processes, events that occur after radiation and that are responsible for the injury to the normal tissue. This review briefly summarizes the multiple damaging effects of free radicals and reactive oxygen and nitrogen species to the heart resulting in radiation induced heart disease (RIHD). -29

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Despite studies aimed to elucidate molecular and cellular mechanisms of RIHD, the pathogenesis of RIHD is largely unknown, and treatment is not available. Antioxidants are commonly employed to combat molecular damage mediated by oxygen and nitrogen-based reactants. Agents used to minimize toxicity as radioprotectors, mitigators and therapeutics of radiation-induced normal tissue injury are reviewed in this article. Further work is needed to study detailed molecular mechanisms of radiation-induced cardiac injury and develop methods attenuating adverse effects of radiation on normal healthy tissue and to help to improve the quality of life of oncological patients.

Acknowledgements This work was supported by the grant APVV-0241-11.

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Figures caption

Fig. 1.: Determinants of biological effects of radiation. Biological effect of radiation is influenced by many factors, including dose and rate of absorption, exposed area, cell sensitivity and individual sensitivity.

Fig. 2.: Effects of radiation on the cell. Ionizing radiation can damage several cell components. It causes alteration in cell membrane and nuclear membrane permeability, functional aberrations in chromosomes and cellular organelles and can change its function.

Fig. 3.: Effect of radiation on molecules. After exposure to ionizing radiation, atoms or molecules became ionized or excited and these can produce free radicals from other molecules, they can break many of chemical bonds in other chemical compounds or they can produce a new chemical bonds -49

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from existing chemical compounds. Ionized/excited form of atoms/molecules can damage some important molecules, for example DNA, RNA or proteins.

Fig. 4.: Mechanisms of DNA damage after irradiation. The most important molecules which may be affected by radiation are DNA molecules. Radiation can damage DNA in direct or in indirect action. In the first case radiation affects DNA in direct way by breaking chemical bonds in DNA structure. This lead to the change of DNA sequence and to mutations. In the second case DNA molecule is affected by ROS produced from disrupting of water molecules.

Fig 5.: Effect of radiation on the heart. Changes in the irradiated heart were observed at molecular and sturctural levels. Radiation of the heart induces increased levels of ROS which leads to DNA damage, increased levels of inflammatory cells, cytokines, macrophages, monocytes or NFκB, TNFα levels. Changes such as decreasing levels of capillary alkaline phosphatase or increasing von Willebrand factor levels. vascular leakage, increased fibrosis, existance of compensatory mechanisms and final decrease in function of the heart and heart failure are characteristic features of the heart exposed to radiation.

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117x72mm (300 x 300 DPI)

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Effects of radiation on the cell. Ionizing radiation can damage several cell components. It causes alteration in cell membrane and nuclear membrane permeability, functional aberrations in chromosomes and cellular organelles and can change its function. 101x64mm (300 x 300 DPI)

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170x145mm (300 x 300 DPI)

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Mechanisms of DNA damage after irradiation. The most important molecules which may be affected by radiation are DNA molecules. Radiation can damage DNA in direct or in indirect action. In the first case radiation affects DNA in direct way by breaking chemical bonds in DNA structure. This lead to the change of DNA sequence and to mutations. In the second case DNA molecule is affected by ROS produced from disrupting of water molecules. 90x72mm (300 x 300 DPI)

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Effect of radiation on the heart. Changes in the irradiated heart were observed at molecular and sturctural levels. Radiation of the heart induces increased levels of ROS which leads to DNA damage, increased levels of inflammatory cells, cytokines, macrophages, monocytes or NFκB, TNFα levels. Changes such as decreasing levels of capillary alkaline phosphatase or increasing von Willebrand factor levels. vascular leakage, increased fibrosis, existance of compensatory mechanisms and final decrease in function of the heart and heart failure are characteristic features of the heart exposed to radiation. 75x68mm (300 x 300 DPI)

Mechanisms of cardiac radiation injury and potential preventive approaches.

In addition to cytostatic treatment and surgery, the most common cancer treatment is gamma radiation. Despite sophisticated radiological techniques ho...
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