proteins. Apart from the induced transcription of antioxidant defence genes, the activation of signaling pathways takes place, as well as mitophagy, due to which the dysfunctional ‘mitochondria are degraded (reviewed in Novak 2012). When the balance between the oxidants and the antioxidants is discurbed leading to oxidative stress, the adverse effects of ROS increase follow, including damage to the molecules impoztant for the proper functioning of the cell. The result is cell death or epigenetic changes enabling adaptation in the surviving cells Prolonged oxidative stress in the irradiated cell (Oxidative stress is induced by a variety of factors, such as ionizing and UV radiations, chemicals present in the envi: ronment or food, and pathogens. The connections between oxidative stress and carcinogenesis as well as tumor pro: gression have been characterized in numerous reviews (Jackson and Loeb 2001, Valko et al. 2006, Goetz and Luch 2008, Lawless et al. 2009, Reuter et al. 2010, Klaunig et al. 211, Kryston et al. 2011, Georgakilas 2012, Kundu and Suth 2012, Nowsheen et al, 2012, Hasselbalch 2013, Luo and Lee 2013). IR induces oxidative stress that is transmit. ted to the progeny ofthe irradiated cells (Clutton et al. 1996, Limoli et al. 1998, Rugo and Schiest! 2004), Ic is paralleled by genomic instability, a pre-requisite of cancer (Hanahan and Weinberg 2011). Hence, it has been of considerable Interest to examine the post-exposure oxidative stress at the cellular level. The mitochondrial origin of ROS in the iradiated cell Upon cell exposure to TR, the above-mentioned (Figure 1) ‘mutual interactions between the cellular ROS-producing systems are joined by ROS produced by water radiolysis. One of the earliest comprehensive descriptions of post inradiation oxidative stress was given by Leach et al. (2001). “These authors used dihydrodichlorofluorescein (DCF) for measuring reactive oxygen and nitrogen species (RONS) during the frst 25 min after exposure of the A431 squamous carcinoma cells to 7 (*Sr source) radiation (1-10 Gy). The amount of the reactive species per cell was constant in the whole dose range, but the percentage of cells produc: ing RONS increased from 20-80. A reversible calcium ion: dependent mitochondrial permeability transition was noted that coincided with the depolarization of the mito chondrial membrane potential and could be inhibited by pre-treatment with cyclosporine A. Generation of RONS was absent in osteosarcoma cells deficient inthe mitochondrial DNA (mtDNA) termed tho(0) cells, pointing to mitochon: drial ETC as the source of the reactive species in these cells after exposure to y radiation. Experiments with the use of rotenone, inhibitor of the mitochondrial ETC complex 1 have further supported this conclusion, Dysfunction of complex Il (succinate: ubiquinone ox: doreductase) was the source of the oxidative stress found in an unstable clone derived from the GM10115 cells exposed to 10 Gy X-rays (Dayal etal. 2008). Aykin-Bums etal (2011) reported a higher steady-state levels of O,+~ and 1,0, as compared tothe parental celllinein hamster B9 cells withthe lonizing radiation-induced oxidative stress 5 ‘mutated mitochondrial succinate dehydrogenase subunit C. ‘The cells were exposed to y (""Cs) radiation (5 eGy-1 Gy). In the dose range 5-50 eGy a survival decrease (as related to the parental cells) was noted in the mutated cell line, but the difference was lost at Gy. ‘Another study was carried out on the effect of X-rays on the heart mitochondria of the C57BL/6N mice (Barjaktarovie et al. 2011) Irradiation (targeted on the heart) with 2 or 0.2 ‘Gy preceded by 4 weeks the sacrifice ofthe animals and the isolation of the mitochondria, The succinate stimulated respiration (state 2 respiration) decreased by 13% in the mitochondria isolated from the mouse hearts irradiated with a dose of 2 Gy, as compared with those from the sham: irradiated mice. Both doses decreased the pyruvate metabo. lism and modified the mitochondrial proteome, but only the higher dose caused a partial deactivation of complexes and IIL, by 32 and 11%, respectively, whereas cytochrome was reduced by 34%, Also, RONS production was mea: sured in heart mitochondria from the sham-irradiated and X-inradiated mice. The '2 Gy mitochondria’ (incubated with DCP in the presence of succinate alone or with added ‘tres sors, 50 HM mercury oF 100 uM ter-butyl-COOH) produced significantly more RONS than the controls only in the pres: fence of stressors. Interestingly, the reduced cytochrome ¢ levels were also found in the chromosomally unstable LS12 cell line, where oxidative stress and genomic instability were contelated with BTC dysfunction (Thomas etal. 2012) Itshould be noted that the disturbed function of ETC was not found in the human lung adenocarcinoma A549 cells after X-irradiation with 10 Gy (Yamamori et al. 2012); this, however, may be explained by the constitutive nuclear factor (cxythroid-derived 2)-like 2 (NRF2) overexpression in these cells and the resulting resistance to oxidants (Kweon et al 2006). The lack of the typical mitochondrial ETC response to X-irradiation in A549 cells shows the importance of the antioxidant defence system in the prevention of the mito chondrial damage. ETC funetions in an array of respiratory complexes that form higher-order complexes known as respirasomes. They consist of varying numbers of copies ofthe complexes I, I, and IV; the position of complex If has not yet been resolved (Schafer et al. 2006, Dudkina et al. 2010, Lenaz et al. 2010, Winge 2012). A plausible assumption is thatthe disruption of the respirasome organization may lead to an increase in ROS generation (Dencher et al. 2007, Lenaz and Genova 20082, 200%, 2012, Lenaz et al. 2010). Relevant to this assumption, as wells othe previously mentioned effects of X-irradiation on the function of mitochondria (Leach etal 2001, Barjaktarovic et al. 2011) isthe conclusion formulated byByrme et al. (2013) on the relative proportions of the num: ber ofonizations and the total energy deposit in the nucleus ‘and cytoplasm in an average mammalian cel: for a macroscopic dose of no more than 1 Gy only a {few hundred ionisations occur in the nucleus volume ‘whereas the number of jonisations in the eytoplasm is over a magnitude larger... the eyroplasm and organelles residing therein can be important targets for low-dose radiation. (p.1251)4. |.Szumiel ‘he results of experiments carried out by Leach et al (2001) are in agreement with this statement and support the notion thatthe primary cause of oxidative stress and of the nhon-targeted effects of irradiation are the mitochondria with the respirasome function directly damaged by IR Extra-mitochondrial ROS sources in the irradiated cell NOX have been implicated in ROS generation as a result of exposure to ionizing radiation both in vitro and in vivo (Pazhanisamy etal. 2011, Datta etal.2012, Kang etal. 2012, ‘Weyemi et al. 2013) as well as in carcinogenesis (Collins Underwood et al. 2008, Pazhanisamy et al. 2011). The par ticipation ofthis group of oxidases in the radiation-induced oxidative stress depends on the cell type. For example, in hematopoietic stem cells (HSC) about half of oxygen con: sumption is extra-mitochondrial (Piccoli etal. 2005). In the ‘murine HSC isolated after total body (Cs) irradiation (6.5 Gy, a sublethal dose) oxidative stress was induced that could be mitigated by postirradiation treatment of mice with diphenyleneiodonium, a pan-NOX inbibitor, but not by apocynin, an inhibitor ofall NOX except NOX4. Neither rotenone, nor inhibitors of cyclooxygenases and lipoxyge nnases significantly decreased ROS generation. This pointed to NOX as the main source of ROS in the oxidative stress induced by IR in HSC (Wang et al. 2010). Both diphenyle neiodonium and N-acetyleysteine increased clonogenic: lty and decreased the nuclear DNA damage and genomic instability (Pazhanisamy et al. 2011). ERstress was shown to beinduced by IR (Zhangetal. 2010, Lim etal. 2012); however, so far there were no experiments to show that this could also conteibute to post irradiation ox: dative stress Antioxidant defence a radiological safety measure? ‘The apprehension of IR-induced malignant transformation is one reason for the conservative guidelines of radiological protection. The key factor that prevents the oxidative stress that would result from the IR impact and lead to carcino genesis isthe antioxidant defence (Zhao and Robbins 2008). “The essential component is mitophagy, an autophagic ‘mechanism of clearance of the dysfunctional mitochonéria. ROS participate in its induction (Lee et al. 2012). So, once IR exposure has occurred, the best safety measure is the anti oxidant defence level that prevents oxidative stress and yet supports mitophagy. Paradoxically, ROS play a role in the induction of apoptosis and senescence - two mechanisms of cancer prevention in vivo (Vurusaner etal. 2012, Kitagishi and Matsuda 2013). The increase in ROS level changes its function inthe cell ‘from an indispensable signaling messen. ger atthe low level toa deadly damaging species atthe high level’ (Hamanaka and Chandel 2010). Taking into account the diversity of cell types and their requirements, the deter rmination of an optimal ROS level seems an impossible task. Nevertheless, some therapeutic strategies have recently been proposed (Zhao and Robbins 2009). 0D?2 as a radioprotecting factor In most cel types examined after exposure to IR, mitochon. dria were the main site of ROS generation. The prolonged oxidative stress contributed both to survival decrease (Hosokietal.2012) and an increase in chromosomal damage seen as a higher frequency of micronuclei (Choi et al. 2007). Being a mitochondrial antioxidant, SOD2 not only counter acts the oxidative stress but also acts as a radioprotecting agent Thisis strongly supported bythe observation thatSOD2 considerably increased cell survival (Epperly et al. 2003), reduced post-radiation oxidative stress (Belikova et al. 2008, Dhar and St Clair 2012) and was active in radioadaptation (Eldridge etal. 2012). its silencing by microRNA interference considerably decreased cell survival after irradiation with 2 Gy (from 0.403-0.021; D, decreased from 1.923-0.617 Gy) (Qu et al. 2010). The high SOD2 expression obtained by the retrovirus-mediated gene transfer had a radioprotecting effect (Southgate et al. 2006). It is noteworthy that overex- pression of the cytosolic SODI did not increase radioresis. tance, again stressing the importance of mitochonérial ROS in the cellular response to IR (Hosoki etal. 2012) Interestingly, the cyclin B1/cyclin-dependent kinase 1 complexlocalizedinthemitochondriauponirradiation with 10 cGy and phosphorylated the SOD2 molecule at serine 106. ts activity and phosphorylation signficantiy increased in the irradiated human breast epithelial MCF1OA cells and ‘mouse tissues, as determined at 8 h postirradiation. This S0D2 modification also resulted in an increased cellular resistance to the subsequent IR (10 Gy)-indueed apopto sis, thus confirming its role in the radioadaptive response (Candas etal. 2013). A radioprotective effect could also be obtained by cell treatment with thiol compounds followed by irradiation (2Gy) 24hhlater The survival increase was 1.40 (for WR-1065) ‘and 1.25 times (for N-acetyleysteine) and the radioprotee: tive effect correlated withthe elevated level of SOD2 protein due tothe thiol-induced NF-xB (nuclear factor kappa-light chain-enhancer of activated B cells) activation (Murley etal 2004, 2006). A similar effect was described for cells with the constitutively active AKT (protein kinase B) which resulted in activation of NF-xB and a radioadaptive response (Park etal. 2013). Also in that case itwas mostlikely the result of an increased NF-xB-mediated SOD2 expression, as observed earlier (Ozeki et al. 2004, Fan etal 2007) ‘An important modulator of SOD2 activity is SIRT3, a NAD~-dependent deacetylase thats linked to aging, inflam: mation and cancer (reviews in Finley and Haigis 2012, Gilt and Villarroya 2012, Bruzzone et al. 2013). Deacetylation by SIRTS increased SOD2 activity and resulted in a lowered ROS generation. Consistently, the lack or down-regulation of SIRTS expression had a radiosensitizing effect (Tao etal 2010, Alhazzazi et al. 2011) and enhanced the sensitivity to ‘oxidative stress, DNA damage and cancer incidence. “These examples of the antioxidative properties of SOD2 permit he expectation thatitshigh acivityis the best defence factor in the case of exposure to IR. Interactions between the DNA and p53 ochondrial “The cellular generation of ROS is of central importance to the cellular redox state. The ROS homeostasis to a considerableextent relies on the ROS-p63 interactions. The expression profile ofp53-transactivated genes depends on the ROS con centration. On the other hand, p53 regulates the cellular ROS generation. This complicated interplay has been reviewed in Liu etal, 2008, Malet and Pervaiz 2012). Among others, 153 controls the mitochondrial ROS generation by transac: tivation of the SOD2 gene. There is also an opposite effect: “That of the mitochondrial function on the expression of p53. Compton etal. (2011) examined the link between mitochon: drial dysfunction and p53 expression in several respiration: deficient Chinese hamster mutant cell lines. The p53 protein expression level was found to be differentially regulated depending on the type of ETC deficiency. Independently of the p53 protein expression profile, upon radiation expo sure the p53 activity was compromised in all the respiration: deficient cells “The mutual dependence between p53 and mitochondrial genes was also described in a pair of human lymphoblas toma cells lines TK6 and WTK1 (expressing a functional p53 and mutated p53, respectively). A connection was found between p53 function and the mitochondrial gene expres sion. In X-irradiated (2 Gy) TK6 cells there was enhanced expression of some of the mitochondrial NADH dehydro genases, cytochrome c oxidase subunits, cytochrome by and an ATP synthase coded by the mitochondrial genes. “his effect lasted for at least 24 h, The transcription of the :ajority of these mitochondrial genes was not enhanced or decreased in the irradiated WTK1 cells expressing mutated 153 (Chaudhry and Omaruddin 2012). It should be added that the mtDNA copy numbers in the irradiated cells of both cell lines were similar “Thus, the mutated m1DNA (or its absence) may affect the expression level of p53 and, in consequence, the expression profile of genes that are under the transcriptional control of 153. Furthermore, the key element ofthe cellular response to IR induction of p53 activityis missingiin the absence of mito chondrial respiration. The detailed molecular mechanisms remain tobe discovered, but the most likely clue seems to be the cellular redox state Radiation carcinogene: ‘The most important studies demonstrating that cancer Induction results from exposure to IR are those published about 20 years ago and later re-evaluated (Doll 1995, Pierce etal. 1996, 2007). In particular, the detailed studies of atomic bomb survivors provided a wealth of information. The established risk of secondary cancer induction from cancer radiotherapy, its dependence on the irradiation technique and genetic factors were recently analyzed (eg, Brenner 2012, Travis et al. 2012) Early and delayed effects of “The early expressed consequences of IR exposure are DNA and chromosomal damage, due to unrepaired or misre paired lesions causing growth delay and cell death. Part of the IR effect in vivo may be non-targeted, as demon strated in a three-dimensional reconstructed system of the normal human skin (Belyakov et al. 2005); the induetion lonizing radiation induced oxidative st ss 5 of micronuclei (1.7-fold increase over background fre quency) and apoptosis (2.8-fold increase) were observed up to 1 mm away from the cells radiated with a micro: beam of 7.2:MeV a-partiles. The lethally damaged cells are eliminated from the exposed cell population by various cell death mechanisms depending on the cell type and the ‘extent of damage inflicted by the radiation exposure. Fault lly repaired DNA isthe cause of both non-lethal mutations and non-lethal chromosomal rearrangements and dele tions which represent @ potentially cancerogenous factor in the surviving cells. Such chromosomal anomalies are 2 tat often found in eancer cells (review in Janssen and Medema 2013), Radiation-induced damage may be expressed after a considerable delay: Zhao and Robbins (2009) analyzed late radiation effects in various normal tissues, including ato: phy, fibrosis, necrosis and vascular injury, and presented evidence supporting the role of induced chronic oxidative suess and inflammation in their origin. In the animal models studied, te tissue injury could be mitigated or prevented by, among other things, antioxidants. Another trait of sustained oxidative stess that characterizes the surviving cell popu lation is that in vivo it may damage the surrounding, non inradiated eels. This is due to the bystander effect, which induces genomic instability transmissible to the progeny ‘over many cell generations (Kadbim eta. 2013). Genomic instability Genomic instability is a late (Litle et al. 1997), carcino: genesis-related (Steffer 2010), non-targeted effect. It is linked with the chronic oxidative stress in cells surviving IR exposure and their progeny and is recognized as the cel lular mechanism that enables pre-caneer cells to acquire the typical traits of malignancy (Hanahan and Weinberg 2011), Genomic instability is expressed as increased levels of chromosomal aberrations, mutations, cell death, and mitotic failure. Its characteristic features enumerated by Kadhim et al. (2013) are high expression rate, non-clonal propagation, and non-linear IR dose dependency. As early as in 1996 it was proposed that an altered DNA methylation pattern (Murnane 1986) and a persistent oxidative stress (Clutton et al. 1996) were involved in the manifestation of the instability. As discussed below, both explanations turn out to be true. IR-induced carcinogenesis - targeted or non-targeted effect? ‘The discovery and gradual identification of mutations in ‘oncogenes (e.g., Der 1987, Steen 2000, Weinstein and Joe 2008) were consistent with the increasing knowledge of radiation-induced DNA damage, its faulty repair and the resulting mutations. Thus, these findings were also consis tent with target theory, nDNA being the radiation-damaged target. Also, the later discovered radiation-induced oxida tive stress and the link between inflammation and cancer (Biswas and Rahman 2009, Kamp et al. 2011, Vendramini Costa and Carvalho 2012) could be explained by the DNA damage caused by ROS, Nevertheless, the next-generation methods of DNA sequencing enabling the determination