Received in revised form16 July 2013Accepted 16 July 2013

iobiology posits that damage to the DNA in the cell nucleus is the primaryeffects of radiation. However, emerging experimental evidence suggests that

this theoretical framework is insufcient for describing extranuclear radiation effects, particularly theresponse of the mitochondria, an important site of extranuclear, coding DNA. Here, we discuss

discussed. In this review, we summarize the current understanding of targets for ionizing radiationoutside the cell nucleus. Available experimental data suggest that an increase in the tumoricidal efcacyof radiation therapy might be achievable by targeting mitochondria. Likewise, more specic protection ofmitochondria and its coding DNA should reduce damage to healthy cells exposed to ionizing radiation.

Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.

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. . . . . . . . . . . . 610. . . . . . . . . . . . 611. . . . . . . . . . . . 611. . . . . . . . . . . . 611. . . . . . . . . . . . 612. . . . . . . . . . . . 612

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/freeradbiomed

Free Radical Biology and Medicine

Free Radical Biology and Medicine 65 (2013) 607619E-mail addresses: wik@ansto.gov.au, winikam@gmail.com (W.W.-Y. Kam).0891-5849/$ - see front matter Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.freeradbiomed.2013.07.024

Abbreviations: OXPHOS, Oxidative phosphorylation; PGC-1, Peroxisome-proliferator-activated receptor- coactivator 1; ROS, Reactive oxygen species

n Corresponding author at: Australian Nuclear Science and TechnologyOrganisation, Lucas Heights, Menai, Sydney, NSW 2234, Australia. Fax: +612 9717 9262.The source of radiation-induced oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612The role of manganese superoxide dismutase in radioprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612The localization of antioxidant enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613Common deletionmtDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A change in the mitochondrial DNA copy number after ionizing irradiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .p0 cells in radiation research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mitochondrial function after ionizing radiation stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .The effects of radiation on the electron transport chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .The effects of radiation on oxidative phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A change in the mitochondrial mass after ionizing irradiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Radiation-induced oxidative stress and antioxidant enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Susceptibility of nuclear and mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6094977 . . . . . . . . . . . . 609Available online 26 July 2013

Keywords:MitochondriaNucleusIonizing radiationDNARNACopy numberMammalian cellsRadiation responseManganese superoxide dismutaseReactive oxygen species (ROS)SuperoxideCommon deletionOxidative phosphorylation (OXPHOS)Electron transport chainp0 cells

Contents

Introduction. . . . . . . . . . . . . . . . . . . . .Mitochondrial DNA after ionizing radi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608ress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608experimental observations of the effects of ionizing radiation on the mitochondria at (1) the DNA and(2) functional levels. The roles of mitochondria in (3) oxidative stress and (4) late radiation effects areArticle history:Received 28 February 2013

The current concept of radcause for the detrimentalReview Article

Effects of ionizing radiation on mitochondria

Winnie Wai-Ying Kam a,b,n, Richard B. Banati a,b,c

a Australian Nuclear Science and Technology Organisation, Lucas Heights, Sydney, New South Wales 2234, Australiab Medical Radiation Sciences, Faculty of Health Sciences, University of Sydney, Cumberland, Sydney, New South Wales 2141, Australiac National Imaging Facility at Brain and Mind Research Institute (BMRI), University of Sydney, Camperdown, Sydney, New South Wales 2050, Australia

a r t i c l e i n f o a b s t r a c t

Mitochondria, the cell nucleus, and late ionizing radiation effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613Signal propagation between mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613Signal propagation between mitochondria and the cell nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614Delayed increase in radiation-induced mitochondrial oxidative stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614Mitochondrial dysfunction and late radiation effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614

Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616

Introduction

The main target of ionizing radiation damage is believed to be

The number of studies investigating the effects of ionizingradiation on the mitochondria is far less than that on the cell nucleus.Specically, a citation analysis using Web of Science on the published

mitochondrial disease screening [50,51], mitochondrial dysfunction

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619608Fig. 1. Citation analysis of published literature. A citation analysis performed on 22 May 2013, through Web of Science/Thomas Reuters, using keywords (1) nucle* ANDionizing radiation AND DNA damage NOT nuclear energy (black bars) versus (2) mitochondr* AND ionizing radiation AND DNA damage NOT nuclear energy(white bars) as Topic for All Years. Only Articles (a document type with original scientic ndings) were included into the analysis. The number of papers published inthe DNA in the cell nucleus [1]. However, the validity of thecurrent radiation damage models has been challenged [2]. Thediscovery of nontargeted phenomena, such as radiation-inducedgenomic instability in the progeny of cells that survive afterirradiation [3,4] and bystander effects on cells that have notdirectly been exposed to radiation [5], call this central dogma ofradiation biology further into question [1].

There are reports of the effects of radiation on cell organellesother than the nucleus [615]. It has been suggested that theseextranuclear effects are not subsequent to nuclear responses toradiation but are instead due to the direct effect of radiation onother organelles [12,13,16]. Mitochondria may account for up to 30%of the total cell volume (e.g., in lymphocytes [17]). Notably, mito-chondria are the only sites where extranuclear DNA resides [1820].Ionizing radiation can induce various lesions in the circular mito-chondrial DNA, such as strand breaks [21], base mismatches [22], andlarge deletions [23], which are also observed in nuclear DNA[10,24,25]. Therefore, mitochondria are likely to be a major targetof ionizing radiation in addition to the cell nucleus [26,27].

Ionizing radiation alters mitochondrial functions [28,29], increasesmitochondrial oxidative stress [3033], and induces apoptosis [3437].Radiation causes specic mitochondrial gene expression changes thatare related to cell survival [38], and mitochondria have been reportedas the primary target for radiation-induced apoptosis [39]. Theseorganelles may also have a role in radiation-induced intra- [40] andintercell [8,4143] signaling. Furthermore, a subcellular proteomicanalysis revealed that proteins involved in processes such as energymetabolism and antioxidant response are regulated after ionizingradiation exposure in vivo [44]. The known involvement of mitochon-dria in these responses/processes, therefore, suggests a role ofmitochondria in the radiation response. Notably, the aforementionedmitochondrial responses cannot be fully accounted for in the currentmodel of radiation effect estimation, which predominantly focuses onthe cell nucleus and its genetic material [17,45].each year is shown.[52], and fetal developmental assessment [53]. Furthermore, mito-chondrial DNA is commonly used for evolutionary analysis because itevolves 10 times faster than the nuclear DNA of the same organism[54]. There is an unequivocal link between mitochondrial geneticvariations and the onset of disease [5563]. The level of mitochon-drial DNA mutation is thus suggested as a marker for cancermalignancy [60,6467].

Mitochondrial DNA genetic variation has also been employed forassessing interindividual radiosensitivity. Higher levels ofliterature from 1990 to 2013 shows that there are 9 times moreoriginal Articles with the keywords nuclen AND ionizing radia-tion AND DNA damage NOT nuclear energy (2214 results), thanwith the keywords mitochondrn AND ionizing radiation ANDDNA damage NOT nuclear energy (246 results) (Fig. 1). In thisreview, we summarize some of the reported mitochondrial responsesto ionizing radiation and point to some avenues for future investiga-tion of the role of mitochondria in the induction of the overall cellresponse to ionizing radiation.

Mitochondrial DNA after ionizing radiation stress

The genetic information of an organism is mostly encoded by theDNA inside the cell nucleus [46]. Some other vital genes are encodedby DNA stored inside the mitochondria. Mitochondrial DNA contains13 genes which code for the subunits of the electron transport chainenzyme complexes (Complexes I, II, III, and IV) and the ATP synthase[1820]. These enzymes are important for respiration, adenosinetriphosphate (ATP) synthesis, and the regulation of many cellularpathways within the cell [47].

Changes in the mitochondrial DNA content, measured as themitochondrial to nuclear DNA (or protein) ratio, can be used as areadout to measure drug [48] or radiation [49] response, for

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 609mitochondrial DNA point mutations and deletions were seen inpatients who received whole body radiation and chemotherapycompared to the control subjects [68]. A high level of mitochondrialDNA sequence variation was observed in nasopharyngeal carcinomapatients who later developed moderate to severe deep tissue brosisas a late complication of radiation therapy. In contrast, the patientswith no or minimal brotic reactions were those with low levels ofmitochondrial DNA sequence variations [69].

Thus, mitochondrial DNA changes can be used in the assess-ment of evolutionary and pathological changes, as well as theanalysis of the effects of radiation.

Susceptibility of nuclear and mitochondrial DNA

The susceptibility of nuclear and mitochondrial DNA to variousstresses has been investigated. Backer and Weinstein showed thatmitochondrial DNA developed more covalent modications thannuclear DNA when treated with a carcinogenic derivative of benzo[a]pyrene [70]. Yakes and Van Houten also observed that themitochondrial genome was more prone to oxidative damage andwas characterized by a reduced ability for repair compared tonuclear DNA. They concluded that mitochondrial DNA is a criticaltarget for reactive oxygen species (ROS) [71].

In addition to its greater sensitivity to chemically inducedoxidative stress, mitochondrial DNA has also been shown to bemore susceptible than its nuclear counterpart to ionizing radiationdamage. Richter et al. directly irradiated mitochondria isolatedfrom rat livers using gamma radiation (150 Gy). They observed a6-fold higher amount of 8-hydroxydeoxyguanosine (oxidizedbase) per unit mass in mitochondrial DNA than nuclear DNA fromthe same liver [72]. May and Bohr found that approximately2 times more gamma radiation-induced (560 Gy) strand breakscould be detected in mitochondrial compared to nuclear DNA.In addition, the amounts of lesions repaired in the nuclearand mitochondrial DNA within the study period (2 h) were 80and 25%, respectively [73]. Furthermore, Yoshida et al. reporteda delayed (24 to 72 h postirradiation) increase in 8-hydroxy-deoxyguanosine lesions in the mitochondrial, but not the nuclear,DNA when A7r5 cells (rat smooth muscle cells) were irradiatedwith 5 Gy of gamma radiation [74].

Although mitochondrial DNA only accounts for 0.25% of thetotal cellular DNA [20], the whole mitochondrial DNA (exceptD-loop) consists of genes for protein synthesis [18,19]. In contrast,the protein coding portion of nuclear DNA is only about 1% of theremaining 99.75% of the total cellular DNA [46]. Therefore, agenetic defect leading to direct biological effects is more likely tohappen within the coding regions of the mitochondrial DNA. Inaddition, mitochondrial DNA lacks histone protection [72] and anefcient DNA repair system [7578]; hence more unrepairedlesions are likely to accumulate.

Common deletionmtDNA4977

A region of the mitochondrial DNA known as mtDNA4977 orthe common deletion, i.e., from nucleotide position 8470 to13446, is a region prone to deletion and is associated with anumber of pathologies [51,7982] and with aging [68,83,84].

The relationship between the common deletion and the radia-tion dose has been examined. Schilling-Toth et al. used humanbroblast cell lines and primary broblast cultures to show thatthe common deletion level increased with doses from 0.1 to 10 Gy(after 72 h postirradiation). Interestingly, they observed hypersen-sitivity at a low dose (0.05 Gy) [85]. A nonlinear relationshipbetween the common deletion level and the radiation dose wasalso observed by Murphy et al. in HPV-G cells (derived from

human neonatal foreskin transfected with the HPV-16 virus);these cells showed a higher frequency of the common deletionat low (0.005 Gy) rather than high (5 Gy) radiation doses whenassayed 96 h after gamma irradiation [86].

The temporal prole of the occurrence of the common deletionafter radiation exposure (5 Gy; X-radiation) was examined by Wanget al. using Hep G2 cells (human liver hepatocellular cells) and subcelllines generated by long-term X-ray treatment (0.5 Gy, twice daily, >4years). The frequency of the common deletion peaked at 24 to 48 h,followed by a sharp decrease; the common deletionwas undetectableafter 10 days in their tested cell lines. In addition, they reported thatthe common deletion was only detectable in the apoptotic/dead cells,but not the viable ones. They also discovered a novel mitochondrialDNA deletion region between nucleotide position 8435 and 13,368and named this region the 4934del; this deletion was specic toionizing radiation exposure in their cell system [87].

Furthermore, the relationship between the frequency of thecommon deletion and the radiosensitivity in vitro was explored.Kubota et al. reported that the frequency of the common deletion (0.5to 10 Gy of X-radiation; 72 h postirradiation) depended on theinherent cellular radiosensitivity among the tested SQ-20B, SCC-61(human squamous carcinoma cells) and AT5BIVA cells (derived fromSV-40 transformed broblasts of an ataxia-telangiectasia patient)[88]. Prithivirajsingh et al. tested their normal broblast cell lines,dermal broblasts from human subjects, ataxia telangiectasia lines,Kearns Sayre syndrome lines, glioblastoma lines with or withoutDNA-PK deciency, and colon carcinoma lines. Although they didobserve an increase in the common deletion after administering aradiation dose (2 to 20 Gy of gamma radiation; 72 h postirradiation),the level of the increase varied and showed no correlation withradiosensitivity among their tested samples [89].

In vivo, Rogounovitch et al. reported a greater frequency ofmitochondrial deletion in radiation-associated post-Chernobyl papil-lary thyroid carcinoma than in sporadic cases [23]. This conclusionwas based on a comparison of Russian (residents of radionuclide-contaminated regions after the Chernobyl fallout) and Japanese(control) subjects, who might have different baseline levels ofmitochondrial deletion due to differences in the environmentalultraviolet background in different geographical locations [84].

Wen et al. used human peripheral blood lymphocytes and found adose-dependent decrease in the common deletion in subjects withtotal body irradiation (4.5 or 9 Gy; X-radiation) compared to theunirradiated controls. As previously suggested [87], the authorsexplained that radiation might cause lymphocytes to differentiateinto two populations, one with a high frequency of the commondeletion, which would be more prone to apoptosis, one with a lowfrequency of the common deletion, which would be more radio-resistant. A sample mixture with a high percentage of apoptotic cellsmight have led to the seemingly high frequency of the commondeletion measured in other studies [90].

A change in the mitochondrial DNA copy number after ionizingirradiation

Yoneda et al. pointed out that polymerase chain reaction (PCR) ismore sensitive than conventional Southern blotting in detectingmitochondrial DNA damage because the latter involves washingand transfer steps that could elute the lesions from the analysis[91]. Moreover, with specic primers to amplify mitochondrial genes,a reduction in PCR product yield indicates a loss in mitochondrialDNA integrity [9295], and this principle can be used to assess thedamage induced by ionizing radiation [25]. In contrast, an increase inthe PCR product yield suggests an increase in the mitochondrial DNAcopy number. An increase in the mitochondrial DNA copy numberwas observed in response to oxidative stress in a yeast model [96].Similar observations have been reported in in vitro and in vivo

mammalian systems after ionizing irradiation.

In vitro, Murphy et al. showed an up to 2-fold increase in themitochondrial DNA copy number after 0.5 Gy of gamma irradiation inHPV-G cells followed by 96 h of recovery [86]. They later used qPCRand again showed a 1.3-fold increase in the mitochondrial DNAcopy number in the same cell system under the same experimentalprocedure [97]. Wang et al. reported a postponed increase in themitochondrial DNA copy number (24 h to 45 days) in their Hep G2cell line and its derivatives after being exposed to 5 Gy of X-radiation.The level of the mitochondrial DNA increase varied among thedifferent cell lines and uctuated during the examination period[87]. Kulkarni et al. exposed normal B-lymphoblastoid cells andcell lines with either Leigh's syndrome or Leber's optic atrophy toX-radiation between 0.5 and 4 Gy and observed an increase in themitochondrial DNA copy number (up to 30%) 24 h after the exposure[38]. Zhou et al. also reported an increase in the mitochondrial DNAcopy number (up to 70%) in MCF-7 cells after the administration of0.05 to 4 Gy of X-radiation followed by 4 to 72 h of recovery [98].

In vivo, the mouse brain and spleen tissues showed an increase inthe mitochondrial DNA copy number after 24 to 96 h of recoverysubsequent to a 3 Gy gamma-irradiation [99]. The mouse small boweland bone marrow tissues also showed a mitochondrial DNA copynumber increase after gamma-irradiation. The effect appears to betissue-independent and increases with the dose (2, 4, 7 Gy) andrecovery time (24, 48 h) [100]. Using 10 Gy of X-radiation, an increasein the mitochondrial DNA copy number was again observed in themurine liver, skeletal muscle, and brain when assayed at 1 to 72 hpostirradiation [101]. One day after 10 Gy of total body X-radiation, a3-fold increase in the mitochondrial DNA copy number in mice spleencells compared to the control was observed. The authors suggestedthat mitochondrial DNA replication was activated [102]. Using periph-eral blood lymphocytes from acute lymphoblastic leukemia patients,

Wen et al. reported that there was an average 2-fold increase in themitochondrial DNA copy number 24 h after the patients received totalbody irradiation (4.5 or 9 Gy; X-radiation) [90].

A mitochondrion contains multiple copies of its genome [103]. Theproper control of the mitochondrial DNA copy number is believed tobe important for normal cell function [104]. An increase in themitochondrial DNA copy number after radiation stimulation, termedmitochondrial polyploidization [99], is believed to be a compensa-tory mechanism [105] or an adaptive response of mitochondria tomaintain function in postirradiated cells [23,97] and malignantlytransformed progeny that survive after radiation exposure [106].

The benet of such an increase in the mitochondrial DNA copynumber postirradiation is under debate. It has been proposed thatDNAprotein complexes might shield some mitochondrial DNAmolecules from direct radiation-induced ROS damage [99]. Suchintact or partially damaged mitochondrial DNA might then replicateto compensate for the loss and to maintain mitochondrial function[49,51,90,99,100]. However, the increase in the mitochondrial DNAcopy number was found to be correlated with oxidative stress levelsin human leukocytes [107]. Thus, an increase in the mitochondrialcontent could be a transient gain that later might strain the cells asmore resources are needed to support this high proliferation andcope with the subsequent increase in ROS [108].

p0 cells in radiation research

The role of mitochondrial DNA in the cellular response toradiation has been extrapolated by investigating p0 cells, i.e., cellswithout mitochondrial DNA derived from a cell line containingmitochondria. p0 cells can be generated by long-term, low dosetreatment of ethidium bromide [109], or enzymatically using

Table 1

rent

hecdecrover

tethmir ceincr

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619610Radiation responses of p0 cells oFN>mitochondrial membrane potential.

Reference Cell type Cell type Dose(Gy)

Type ofradiation

Changes relative to pa

Cellsurvival

Cell cycle

[175] 701.2.8C Humanbroblast cells

2 to 8 Gamma No change

[117] 701.2.8C Humanbroblast cells

2 to 8 Gamma Nochange

n/a

[176] 701.2.8C& 143.TK-

Humanbroblast cells& humanosteosarcomacells

2 to 10 Gamma Nochange

n/a

[115] 143.TK- Humanosteosarcomacells

2 to 8 Gamma Nochange

n/a

[116] 143.TK- Humanosteosarcomacells

2 to 8 Gamma Nochange

n/a

[34] 143.TK- Humanosteosarcomacells

1 to 10 Gamma n/a n/a

[177] Hela Humancervicalcancer cells

2 to 8,20

Gamma Delayed G2 carrest and aability to recG2 arrest

[8] Hela Humancervicalcancer cells

N/a Helium ionmicrobeam

n/a n/a

[111] Miapaca Humanpancreatictumor cells

2 to 6 X ray n/a G2 checkpoinactivation togcyclin B1 (forand CDK1 (foprogression)al cells or controls after radiation exposure

Cellgrowth

Micronucleusformation

DNA damage ROSproduction

ATPcontent

n/a n/a n/a n/a n/a n/a

n/a n/a n/a n/a

n/a No change(DNAfragmentation)

n/a n/a n/a

Nochange(celldoublingtime)

n/a n/a n/a n/a

n/a n/a n/a n/a

n/a n/a n/a No change Nochange

n/a

kpointeasedfrom

n/a n/a n/a n/a n/a

n/a n/a (53BP1 fociformation)

n/a n/a n/a

er withtosis)ll cycleease

minimalchange

n/a n/a n/a n/a n/a

p cells was used (generated from 701.2.8C cells; studies by

The effects of radiation on oxidative phosphorylation

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 611Yoshioka et al. (2004) and Tang et al. (1999)).The observed discrepancies in the radiation responses of p0 cells

could be due to varying levels of remnant mitochondrial DNA ormitochondrial function. One study found no evidence of mitochondrialDNA in their p0 cells (Fig. 1 of Kawamura et al.), while others studiesdeemed the level of mitochondrial DNA nonnegligible(Fig. 1A of Clooset al. (2009) and Fig. 5A of Tartier et al. (2007)). Moreover, most of themitochondrial proteins in all enzyme complexes are encoded bynuclear DNA (77 subunits) [112]. p0 cells, therefore, may not be thebest model to assess the role of mitochondria in cellular radiationresponse. Specically, p0 cells could still retain Complex II (solelyencoded by nuclear genes) activity [113,114]. Indo et al. observed thatp0 cells responded to electron transport chain inhibitors; this observa-tion suggested that the function and electron ow of the electrontransport chain may not have been completely abolished [114].Furthermore, a shift in the energy metabolism from OXPHOS (OX,i.e., oxidation/respiration; PHOS, i.e., phosphorylation/ATP synthesis)to glycolysis (anaerobic respiration) in p0 cells [109,113] could likelyreduce the proliferation rate of these cells [115117] and, therefore,may indirectly lead to differences in their responses to radiation.

Notably, the absence of functional mitochondria in p0 cells mayinduce other nonmitochondrial changes, which could in turn affecttheir radiation responses [111] and complicate the response analysis[118]. Park et al. showed that p0 cells generated from a wide range ofcell lines were characterized by a 21-fold higher expression of themanganese superoxide dismutase gene and were more resistant toROS when compared to their parental cells. This increase in themitochondrial antioxidant activity in p0 cells may allow them to adaptand establish a new equilibrium of the superoxide levels, thus makingp0 cells less suitable as models for ROS investigations [119]. In addition,Ivanov et al. showed that p0 cells generated from human skinbroblasts differ from their parental cells in terms of their mitochon-drial function, oxygen consumption, mitochondrial membrane poten-tial, nuclear-encoded mitochondrial protein expression, and NF-B/STAT3 signaling pathways (for cellular response); the parent cells andthe p0 cells also differed in the expression of 2100 genes [120].

Mitochondrial DNA is more vulnerable to ionizing radiationdamages than its nuclear counterpart. Ionizing radiation inducesnonlinear and cell-type-specic damages directly to mitochondrialDNA, which may increase its copy number as a compensatoryresponse to counteract a loss in mitochondrial function.

The role of mitochondrial DNA in the radiation response hasbeen studied using p0 cells. However, these cells are intrinsicallydifferent from their parental cells. In addition, the use of pseudo p0

cells, which are dened as cells with a reduced but nonzeroamount of mitochondrial DNA, in some studies limits the applic-ability of p0 cells in elucidating the role of mitochondrial DNA inthe radiation response.

Mitochondrial function after ionizing radiation stress

The effects of radiation on the electron transport chain

Pearce et al. irradiated isolated bovine heart mitochondria with50 Gy of gamma radiation and showed an activity inhibition inComplexes I and III [121]. Barjaktarovic et al. locally irradiatedmitochondrial-targeted restriction endonuclease to avoid themutagenic effect of ethidium bromide [110]. Although p0 cellsappear to be a useful tool to assess the mitochondrial response toradiation, conicting results have been reported (Table 1); thesedisparities are likely due to differences in the cell origin orwhether the parental cells have been virally transduced [111].Yet, the results were still conicting even when the same kind of0(2 Gy; X-radiation) the heart of C57BL/6 N mice and observed aHall et al. performed a whole body irradiation (8.4 Gy; X-radia-tion) in rats and reported that phosphorylation was decreased by20% 1 h postirradiation and remained low for the next 12 h. Thefunctionality of OXPHOS appeared to return to baseline after 24 hof recovery [125].

In a similar but modied experiment, Hwang et al. directlyirradiated (5 to 20 Gy; gamma radiation) the murine liver whichwas squeezed out through an upper abdomen opening andassayed for mitochondrial respiratory function immediately afterirradiation. They noted a signicant decrease in both state 3 andstate 4 respiratory rates as the dose of radiation increased.However, they did not nd a signicant change in the adenosine5-diphosphate/oxygen ratio after irradiation [28]; this nding isin contrast to the ndings of Hall's report. The authors suggestedthat the measured effect of radiation on energy metabolism mightvary at different sampling times [28]. In addition, rats weresubjected to whole body irradiation in Hall's study [125], but theliver was directly irradiated in Hwang's experiment [28]. Thedifference in the irradiated region and hence the overall systemicinvolvement might lead to differences in the measured radiationresponse.

Yoshida et al. showed that human osteosarcoma cells underwenta 20% reduction in ATP content 1 h after 8 Gy of gamma radiation,followed by a full recovery in approximately 3 h [116]. Arguably, theeffect of radiation on OXPHOS could be a cell-specic event, assuggested by Nugent et al. who showed that the change in cellularoxygen consumption after 5 Gy of gamma irradiation varied betweentheir CHO-K1 and HPV-G cells upon recovery (4 to 24 h) [108].

These studies show that ionizing radiation, to some extent,alters oxidation and/or phosphorylation; however, these changesdelayed (4 weeks postirradiation) and partial deactivation ofComplex I (32%) and Complex III (11%), a decreased succinate-driven respiratory capacity (13%), and an increased level of ROS[122]. Yoshida et al. also reported a 50% reduction in Complex Iactivity in their rat A7r5 cells 12 h after 5 Gy of gamma irradiation.The observed changes were associated with the delayed ( 24 hpostirradiation) increase in mitochondrial ROS and the subsequentmitochondrial DNA oxidation and growth inhibition [74]. Nugentet al. showed a cell-specic change (CHO-K1: derived from ovaryof the Chinese hamster; HPV-G cells), either a decrease or anincrease, in OXPHOS enzyme activities soon (4 h) after irradiation.More importantly, the activity of some of the examined enzymes(Complexes II, III, IV, and ATP synthase) did recover upon 12 to96 h of recovery [97] (see section 3.2).

Chinese hamster lung broblast cells with Complex II mutated(single-base mutation that truncates 33 amino acids from the COOH-terminal integral membrane portion of succinate dehydrogenasesubunit C protein) are found to have a higher level of superoxideand hydrogen peroxide [123]. Later, Aykin-Burns et al. reported thatthese cells were more radiosensitive and affected by poorer survivalwhen compared to their parental cells after low dose irradiation(5 cGy to 50 cGy; gamma radiation). The above phenomenon couldbe reversed when the mutant cells were made to overexpress wild-type human succinate dehydrogenase subunit C [124].

These results show that the electron transport chain complexes(including the nuclear-encoded Complex II) and ATP synthasedirectly respond to ionizing radiation. Because these complexesare involved in electron transport within the electron transportchain and ATP synthesis, processes associated with OXPHOS arelikely to be affected.could later be recovered.

HPV-G cells) after 4 to 96 h of recovery [108]. Wang et al. also

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619612found a 3.2-fold increase in the mitochondrial mass in their HepG2 cells 24 h after being exposed to 5 Gy of X-radiation [87].

Using Mitotracker Green and uorescence-activated cell sorting(FACS) analysis, Zhou et al. reported a 1.4- to 1.5-fold increase inthe mitochondrial mass in MCF-7 cells (human breast adenocarci-noma cell line) 4 h after 0.05 and 0.1 Gy of X-radiation exposure[98]. Using the same technique, Dayal et al. noted a 1.5- to 2-foldincrease in the mitochondrial mass in both genetically stable andunstable clones isolated from irradiated CHO cells (10 Gy; X-radia-tion). More importantly, they also showed a decrease in themitochondrial membrane potential and an increase in oxygenconsumption, while the ATP levels were unchanged. These authorssuggested that radiation causes mitochondrial dysfunction, and anincrease in the mitochondrial mass is needed to maintain cellularenergy status [126].

The increase in mitochondrial mass is likely to be associatedwith an increase in the total amount of mitochondrial DNA.However, this assumption has been challenged [51]. MitochondrialDNA replication is independent of and seems to be uncoupledfrom mitochondrial ssion [103]. Because mitochondria containproteins synthesized from both nuclear and mitochondrial DNA[61,112], the nuclear-encoded proteins can still be produced eventhough the mitochondrial DNA is defective. This disproportionalsynthesis of nuclear-encoded proteins may contribute to theincrease in mitochondrial mass, even when the mitochondriamay lack mitochondrial DNA [51]. However, it was later shownthat the temporal [98] and relative [87] increase in the mitochon-drial mass, at least when stimulated by X-radiation, was associatedwith the increase in mitochondrial DNA in vitro.

Ionizing radiation causes mitochondrial dysfunction by alteringthe activity of the electron transport chain complexes and ATPsynthase, hence OXPHOS. Mitochondria seem to respond to suchchanges by increasing their DNA copy number (see section 2.3).Whether the benet of such an increase is the restoration ofmitochondrial function is yet to be conrmed. Interestingly, wehave observed an increase in the expression of 18 mitochondrialgenes as well as peroxisome-proliferator-activated receptor-coactivator 1 (PGC-1; a regulator of mitochondrial gene expres-sion) in mammalian cells after gamma irradiation (unpublisheddata). The increase in mitochondrial content (DNA and RNA)postirradiation may thus lead to an overproduction of mitochond-rially encoded subunits. This could cause an imbalance in thecomposition of the electron transport chain complexes and theATP synthase, therefore, impairing the mitochondrial function andleading to an increase in oxidative stress. Notably, PGC-1 isalso associated with mitochondrial proliferation/number [127].The increase in PGC-1 postirradiation might relate to the increasein mitochondrial mass/number (see section 3.3) which furtherstresses the cell due to an accompanying increase in mitochondrialoxidative stress.

Radiation-induced oxidative stress and antioxidant enzyme

The source of radiation-induced oxidative stress

Electrons may leak during transport within the electron trans-port chain. The partial reduction of an oxygen molecule by a freeA change in the mitochondrial mass after ionizing irradiation

Using MitoTracker Green (marker for mitochondria) and uor-escent microscopy, it was shown that 0.005 to 5 Gy of gammairradiation could lead to a 1.5- to 3.8-fold increase in the mito-chondrial mass in two cell lines of different origin (CHO-K1 andelectron generates a superoxide anion, which is the precursor ofmost ROS [128130]. Slane et al. have thoroughly characterized thetype of ROS released in mitochondrially impaired cells (Chinesehamster lung broblast cells with mutated Complex II subunit C).Their ow cytometry and high-performance liquid chromatogra-phy (HPLC) results demonstrated the oxidation of dihydroethi-dium (nonuorescent) to 2-OH-ethidium (uorescent) in thesemutated cells, suggesting the increase in mitochondrial superoxidelevel [123]. Mitochondria, therefore, produce most of the ROS(likely superoxide) under physiological and abnormal conditions,making them constantly under high oxidative stress [71]. Notably,the superoxide anion was found to be the major cause of radiation-induced apoptosis, at least in the peritoneal resident macrophagesof C3H mice [131]. The role of mitochondria in radiation-inducedoxidative stress, thus, is one of the major study areas in radiationresearch.

2,7-Dichlorouorescein diacetate and hydroethidine, as wellas their derivatives, are the dyes commonly used to detectoxidative products, including those induced by ionizing radiation[25,31,33,34,74,116,132136]. However, the application of thesedyes is limited because they cannot differentiate the source of theoxidative stress.

2,7-Dichlorouorescein and its diacetate form are found toproduce ROS when oxidized and generate more ROS during thedetection process [137]. This assay may also register ROS gener-ated in the extracellular medium as those formed intracellularly byionizing radiation and is reported to be dependent on theconcentration of serum in the medium during irradiation [138].When hydroethidine is oxidized by superoxide, it does not changeto ethidium, which was thought to bind to DNA and lead to anenhancement of uorescence [139].

A number of dyes are used for radiation-induced oxidativestress detection at the subcellular level. Motoori et al. useddihydrohodamine 123 to detect mitochondrial ROS and showeda radiation-induced (15 Gy; X-radiation) elevation in mitochon-drial ROS in their control HLE cells (human hepatoma cells), butnot in cells overexpressing an enzyme that breaks down mito-chondrial superoxide radicals. These authors suggested that mito-chondria are one of the major sites of ROS production and thatsuperoxide radicals are the primary radicals that give rise to theelevation in ROS levels upon irradiation [30].

Indo et al. used confocal microscopy to observe an instant,gradual increase in ROS levels, which peaked and then declined atthe 2nd hour, in their HLE cells after 18.8 Gy of X-radiation. Theyalso demonstrated the colocalization of hydroxyphenyl uorescein(marker for hydroxyl radicals) and MitoTracker in the irradiatedcells, thus conrming that the ROS were generated by themitochondria. Moreover, their electron spin resonance spectro-scopy results veried that the ROS were hydroxyl radicals andsuperoxide anions [140].

The role of manganese superoxide dismutase in radioprotection

Manganese superoxide dismutase is an enzyme that resideswithin the mitochondria; this enzyme is responsible for thedismutation of highly reactive superoxides to less toxic forms,i.e., water and hydrogen peroxide [129,130]. Oberley et al. usedmouse heart tissues and showed that radiation (absorbed dose of7.7 Gy to the heart; X-radiation) only induced the activity andprotein expression of manganese superoxide dismutase, but not itscytosol counterpart, the Cu/Zn superoxide dismutase [141]. Thetime- and dose-dependent increase in the mRNA expression ofmanganese superoxide dismutase was also observed by Akashiet al. in irradiated human embryonic lung broblasts [142]. Theseresults suggest the likely involvement of manganese superoxidedismutase in modulating the cellular response to daily and

radiation-induced oxidative stress both in vivo and in vitro.

crucial role of mitochondrial ROS (likely superoxide [71]), rather than

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 613Cells overexpressing manganese superoxide dismutase wereused to further examine the role of this enzyme in the radiationresponse. Hirose et al. overexpressed manganese superoxidedismutase in CHO cells (Chinese hamster ovary cells) and reportedthe positive effect of this enzyme on cell survival after gammairradiation (1 to 11 Gy). These authors hypothesized that theradioprotective role of manganese superoxide dismutase is prob-ably tied to its ROS scavenging capability [143]. Later, Motoori et al.reported that HLE cells transfected with manganese superoxidedismutase were more radioresistant (15 Gy; X-radiation), asshown by the lower level of apoptosis, mitochondrial ROS produc-tion, and membrane lipid peroxidation [30]. Hosoki et al. per-formed a microarray experiment to compare gamma-irradiated orunirradiated HeLa cells (human cervical cancer cells) overexpres-sing manganese superoxide dismutase or a control plasmid. Cellsoverexpressing manganese superoxide dismutase had a highendogenous level or a greater radiation-stimulated upregulationof genes associated with the cell cycle and stress response [33].These reports provide insights for future investigations of thepotential use of manganese superoxide dismutase in radiationprotection.

Epperly et al. delivered an adenovirus-carried manganesesuperoxide dismutase transgene into their athymic nude micevia intratracheal injection. After X-radiation treatment (850 to950 cGy), these animals were found to have a lower expression ofcytokine mRNA (IL-1, TGF-, TNF-), as well as organizing alveo-litis/brosis development, when compared to the controls [144].They also conrmed that the IL-1 mRNA level correlates with theacute pneumonitis phase and that the late elevations of TNF-,TGF-1, and TGF-2 are associated with the onset of organizingalveolitis/brosis and mortality [145]. They later applied differentmodes of transgene delivery, such as plasmid/liposomes [146] anda minicircle plasmid [147], to further explore the potential ofmanganese superoxide dismutase in radioprotective gene therapy.

The localization of antioxidant enzymes

The importance of the mitochondrial localization of manganesesuperoxide dismutase in modulating the stress response has beenexamined by a number of groups. Hirai et al. found that themitochondrial ROS level and membrane lipid peroxidation, whichwas induced by hypoxia and reoxygenation treatment, weresuppressed only if the manganese superoxide dismutase wasproperly expressed within the mitochondria (i.e., a transgene witha mitochondrial targeting signal) [148].

Other than hypoxia and reoxygenation stress, Epperly et al. usedgenetic approaches to demonstrate the importance of the mitochon-drial localization of superoxide dismutases in protecting cells fromradiation-induced oxidative stress. In vitro, they reported that whensuperoxide dismutases, including Cu/Zn superoxide dismutase, whichis normally expressed within the cytosol, were expressed within themitochondria, their 32Dcl3 cells (murine myeloid cell line) showedbetter survival and less apoptosis relative to the control cell lines aftergamma irradiation. However, their in vivo results showed that onlysome of the mice injected with mitochondrially targeted superoxidedismutase, manganese or Cu/Zu type, were protected from radiation-induced esophagitis (35 Gy; X-radiation). The authors explained thisobservation by suggesting the presence of cell phenotype-specictransgenic effects for manganese superoxide dismutase in vivo [149].Furthermore, this group also demonstrated the ability of mitochond-rially targeted catalase to confer radioprotection. In vitro (32Dcl3 cells)and in vivo (development of alveolitis in mouse lungs by 20 Gy ofX-radiation) radioprotection could be seenwhen catalase was directedinto the mitochondria. These authors suggested that mitochondriallytargeted catalase could be an additional radioprotector alongside with

manganese superoxide dismutase, at least in vitro [150].intracellular ROS, in mediating cellular damages after ionizing radia-tion exposure.

Mitochondria, the cell nucleus, and late ionizing radiationeffects

Signal propagation between mitochondria

In addition to the studies that have identied mitochondria asthe major source of radiation-induced oxidative stress, there arereports of signal propagation between mitochondria, which canamplify the effects of radiation.

Leach et al. reported that about 50% of the wild-type or controlplasmid-transfected CHO cells showed a signicant increase inROS after irradiation (4 Gy; gamma radiation). However, less than10% of the cells overexpressing calbindin 28 K, a Ca2+-bindingprotein, showed an increase in radiation-induced ROS. They thenproposed that mitochondria directly hit by ionizing radiation aresubjected to a transition in their permeability. Ca2+ ions arereleased and then taken up by adjacent mitochondria, which thendepolarize, leading to a subsequent increase in ROS production.Thus, the Ca2+ ion could act as a signaling molecule in a chainreaction among the mitochondria after irradiation, leading to thepropagation and amplication of the ROS signal; this chainreaction would then impact the nucleus [34].

Communication between mitochondria for damage signal propa-gation was also investigated by Brady et al. using rodent cardiomyo-cytes, which are cells with a regular array of mitochondria. Theauthors noted that laser-beam induced local ROS development andthe subsequent mitochondrial permeability transition could spread toadjacent mitochondria. This change was a mitochondrial-potentialdriven process, mediated via the mitochondrial permeability transi-tion pore, and ROS, as opposed to Ca2+ ions, appeared to participate inSimilar results were obtained by other groups. Hosoki et al.showed that radioresistance was conferred only by cells overexpres-sing mitochondrially targeted superoxide dismutase [33]. Indo et al.also noted that authentic manganese superoxide dismutase, but notthe one without the mitochondrial targeting sequence, was needed toreduce the level of radiation-induced ROS generation and hence lipidperoxidation and eventual apoptotic cell death [140]. Furthermore,treatment using mitochondrially targeted ROS scavenging enzymeshas also been reported to reduce cytotoxicity or to enhance radiationresistance in cells [124,136].

Ionizing radiation induces both intracellular and mitochondrialoxidative stress [32]. Manganese superoxide dismutase and Cu/Zusuperoxide dismutase are important for antioxidant defense withinthe mitochondria and cytosol, respectively. These superoxide dismu-tases (and other antioxidant enzymes) can be genetically modied tohave their subcellular localization altered. The change in the level ofradiation-induced oxidative stress in different subcellular compart-ment can thus be differentially investigated in cells after expressingsuch genetically modied antioxidant enzymes. Such enzymes,targeting different organelles, would help to examine the role ofdifferent subcellular compartments to the increase in oxidative stressafter ionizing radiation exposure. Collectively, these studies establishthe importance of the mitochondrial localization of antioxidantenzymes in protecting cells from oxidative stress induced by ionizingradiation.

Fluorescent assays have revealed that mitochondria are the majorsubcellular site of radiation-induced oxidative stress. Transgenicexperiments have further conrmed that antioxidant enzymes needto reside within the mitochondria to alleviate the oxidative stresscaused by ionizing radiation. Collectively, these ndings suggest thethis signal propagation [151]. As discussed above, ionizing radiation

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619614impairs the electron transport chain leading to a persistent elevationin mitochondrial oxidative stress. This imbalance in mitochondrialROS might form a positive feedback mechanism for more depolariza-tion and greater ROS production, leading to further cell damages, assuggested by the ROS-induced ROS release [152] and mitochon-drial ROS-induced ROS [153] phenomena originally established byZorov and colleagues.

Of note, cardiomyocytes are terminally differentiated, whileCHO cells are immortal cells with continuous cell divisions. Thedifference in material might, in part, contribute to the discrepancyobserved between the results from Brady's [151] and Leach'steams [34] associated with the role of Ca2+ ions in signalpropagation between irradiated cells.

Signal propagation between mitochondria and the cell nucleus

Changes in the functional state of mitochondria can be com-municated to the cell nucleus via mitochondrial retrograde signal-ing to achieve an adaptive cell response to stimulations/stresses[154]. It is, therefore, possible that radiation damage, too, maytrigger a mitochondrial-nuclear communication.

Tartier et al. showed that a signicant increase in nuclear 53BP1foci, which is a marker for DNA damage, could be observed in 3 hwhen the cytoplasm of their HeLa cells was irradiated; such foci couldbe seen in just 5 min when the nucleus was targeted. They proposedthat an additional step is needed to produce a biological response inthe cell nucleus from cytoplasmic irradiation [8].

Fisher and Goswami further suggested that mitochondrial ROScould have a role in the regulation of radiation-induced cell cyclecheckpoints and overall cellular radiosensitivity, at least in MiaPaCa-2 cells (human pancreatic cancer cells). In their study, theyhypothesized that communication between the mitochondria andthe nucleus, via radiation-induced mitochondrial ROS signaling,could inuence the cellular response to radiation exposure [136].

Delayed increase in radiation-induced mitochondrial oxidative stress

Radiation causes an acute, transient increase in intracellularoxidative stress [32]. The increase in oxidative stress is most likelydue to water radiolysis because water constitutes a substantialportion of a cell. Free radicals generated from water usually have avery short life span of only 10-9 s [155]; yet, a number of studieshave reported the persistent increase in oxidative stress afterradiation exposure.

For example, Tulard et al. used dihydrohodamine 123 andrecorded a dose-dependent (1.5 to 7 Gy; gamma radiation),delayed (24 h onward), and persistent (for days) increase inmitochondrial ROS in their SW620 (human colon cells) andSW620IR1 (radiosensitive clone derived from SW620 cells) cellsafter gamma irradiation. Their results suggest a role for mitochon-dria in the radiation-induced late production of ROS [31]. Thisstudy also used 2,7-dichlorouorescein diacetate for intracellularROS detection. However, this part of their study is not discussed inthis review due to the aforementioned uncertainties associatedwith this dye (see section 4.1).

Ogura et al. showed that 10 Gy of X-radiation caused anincrease in intracellular ROS, which was associated with therelease of cytochrome c from the mitochondria and the inductionof apoptosis in A549 cells (adenocarcinomic human alveolar basalepithelial cells). They further used MitoAR, which selectivelylocalizes within mitochondria and reacts with ROS, to show thatmitochondria are the source for the late (tested at the 6th hourpostirradiation) increase in ROS in their tumor cell model [156].

A more detailed study was performed by Kobashigawa et al. usingamino-uorescein and MitoSOX, which are markers for intracellular

and mitochondrial ROS, respectively. Using normal human broblast-Mitochondria may be permanently impaired after ionizingirradiation, leading to the observed persistent mitochondrialoxidative stress long after the initial exposure.

At the mitochondrial DNA level, Schilling-Toth et al. reportedthat their radiosensitive human broblast cells showed a two-wave pattern of the common deletion in the mitochondrial DNA:the frequency of the common deletion peaked around Day 14 andthen returned to the baseline level. This decrease was followed byanother peak around Day 49 that persisted until Day 63 after 0.1 or2 Gy of gamma irradiation. This nding shows that damage to themitochondrial DNA, which manifests as the common deletion, canbe a long-term, persistent event, which the authors dened asradiation-induced instability of the mitochondrial genome [85].

At the functional level, a number of studies have shown achange in mitochondrial function in the progenies of the cells thathave survived from ionizing radiation exposure. Kim et al. usedgenetically unstable clones LS-12 and Fe10-3, which were isolatedfrom GM101115 cells (contain 1 copy of human chromosome 4 in abackground of 20 to 24 CHO chromosomes) after 10 Gy of X-ray oriron ions irradiation, respectively. They reported that both celllines were characterized by higher intracellular ROS levels andlower manganese superoxide dismutase activity as well as areduced Complex IV activity and hence a defective respiratorypathway [135].

Miller et al. later reported that unstable clones (LS-12, CS-9, 115cell lines) were characterized by an altered expression of mito-chondrial proteins and an elevated ROS level. Their results suggesta casual association between mitochondrial dysfunction andradiation-induced genome instability [158].

Dayal et al. reported that the relationship between genomicinstability and persistent oxidative stress was mediated by hydrogenlike cells, they reported that radiation caused a dose-dependentincrease in ROS and a difference in the temporal prole of theintracellular and mitochondrial ROS levels upon radiation stimulation(2 to 6 Gy; gamma radiation). They observed an acute increase inintracellular ROS, which subsided in about 24 h, and the levels thenrose again and remained high from the 72nd hour onward. Themitochondrial ROS level showed a gentle increase, peaked at the72nd hour postirradiation, and remained steadily high until the endof the study period (7 days postirradiation) [32].

A recent report by Hosoki et al. also used MitoSOX anddemonstrated a delayed elevation of mitochondrial superoxide inHeLa cells upon radiation stimulation (5 Gy gamma radiation; 72 hpostirradiation) [33]. A postponed, persistent increase in themitochondrial ROS level was also observed by Yoshida et al. whenirradiated A7r5 cells (embryonic rat thoracic aorta cells) wereexamined using the MitoSOX assay (5 Gy gamma radiation; from24 to 72 h postirradiation) [74].

Yamamori et al. exposed A549 cells to 10 Gy X-radiation andsimilarly observed a delayed (peak at 12th hour postirradiation)increase in oxidative stress; this change was associated with anincrease in the mitochondrial membrane potential, mitochondrialrespiration, and ATP production. More importantly, they demon-strated a radiation-induced G2/M arrest and showed that cells in thatphase featured an increased mitochondrial content (mitochondrialmass and DNA) and a higher intracellular ROS level [157].

Different cell lines were used in the aforementioned studiesand may contribute to the discrepancies in the reported ROSproduction proles. Nonetheless, these reports show that radia-tion causes a late increase in ROS, which are likely to be generatedby the mitochondria.

Mitochondrial dysfunction and late radiation effectsperoxide [159]. They later observed that their genetically unstable

clones had increased oxygen consumption (CS-9 cells) and elevatedComplex II activity, but reduced stability (LS-12 cells) [126].

A comprehensive mitochondrial subproteomic study, togetherwith mRNA and microRNA investigations, was performed byThomas et al. Their ndings suggested that unstable clones (LS-12 cells) were likely to have a compromised electron transportchain function. In addition, these cells were characterized by anelevated response to oxidative stress and an upregulated tricar-boxylic acid cycle, which was necessary to maintain cell survival inthe case of mitochondrial dysfunction. These responses are likelyto be epigenetically regulated and thus constitute a feedback loopleading to genomic instability [160].

Furthermore, it is evident that mitochondrial dysfunction couldlead to nuclear damage or tumor development. Choi et al. reportedthat the mitochondria, Nox1 (gene coding for NAPDH oxidase 1),and JNK (for stress response) are likely to be involved in radiation-induced (2.5 Gy; gamma-radiation) ROS production and hencemicronucleus formation [161]. St Clair et al. overexpressed man-ganese superoxide dismutase in C3H10T1/2 cells (mouse cell linewith broblastic morphology and functionally similar to mesench-ymal stem cells) and found that these cells were protected fromradiation-induced neoplastic transformation [162]. Du et al. usedmouse embryonic broblasts and showed that the increase inmanganese superoxide dismutase activity was associated with thedecrease in ROS production and, more importantly, the suppres-sion of the late (72 h post gamma irradiation) increase in ROS,

micronuclei formation, and the subsequent cellular transformation[134]. Furthermore, Zhang et al. showed that malignant trans-formed human small airway epithelial cells, induced by 0.2 to 1 Gyof 4He ion irradiation, had an increased mitochondrial content anda greater frequency of the common deletion; however, these cellswere characterized by a reduction in oxygen consumption, themitochondrial membrane potential, and Complex II activity [106].

These studies suggest that mitochondrial dysfunction is a likelycause of radiation-induced genomic instability [163], which couldpropagate to cell progenies/offspring, leading to long-term radia-tion effects including nuclear damage [164].

Ionizing radiation permanently impairs mitochondria, leading toa persistent production of mitochondrial ROS [165]. MitochondrialROS are likely to act as signaling molecules for intermitochondrialand mitochondrial-nuclear communication, which might promotesubsequent long-term radiation effects.

Outlook

Here, based on the available experimental data, we infer theeffects of ionizing radiation on mitochondria:

Mitochondria are more susceptible to ionizing radiation thanthe nucleus (Fig. 2A). Mitochondrial DNA can be directly damaged,e.g., through mitochondrial DNA deletion. Interestingly, the mito-chondria can cope with such damages, which might lead to

) Wr genfounspontitide mhondages

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 615Fig. 2. Radiation-induced mitochondrial superoxide mediated nuclear damage. (Acontaining sites) are likely to be affected. The mitochondrial genome is of higheradiation-induced lesions that could lead to functional changes are more likely to beupon ionizing radiation exposure, therefore, altering the function of the electron tranpostirradiation. Mitochondria may respond to such damages by increasing the quafunction necessary for cell survival. (C) Nevertheless, the radiation-induced superoxdiffuse to the nearby mitochondrion, both steps similarly cause further mitoc(D) Furthermore, mitochondrial superoxide may diffuse intracellularly causing dam

nuclear DNA.hen a cell is exposed to ionizing radiation, its nucleus and mitochondria (DNA-etic information density as compared to that of the nuclear genome; therefore,d in mitochondrial DNA. (B) Regions within the mitochondrial DNA could be deletedrt chain (ETC) and leading to a persistent mitochondrial superoxide (O2-) productiony or synthesizing new mitochondrial DNA in order to maintain the mitochondrialay continue to damage the mitochondrial DNA of that impaired mitochondrion orrial dysfunction and hence amplify the amount of mitochondrial superoxide.to other parts of the cells including the cell nucleus (micronuclei formation) and

production could alter the activity of the electron transport chaincomplexes/OXPHOS, leading to an elevated superoxide production.

W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619616The increase in superoxide in one mitochondrion might diffuse(via an unknown active pathway or passively) to another mito-chondrion (Fig. 2C). This intermitochondrial communicationamplies the damage signal and permanently damages the restof the mitochondria within a cell, resulting in further superoxideproduction. Impaired mitochondria, after the initial radiationexposure, continue to produce superoxide, which eventuallydiffuse into the cell nucleus and cause nuclear DNA damage(Fig. 2D). Furthermore, the nuclear lesion produced by thisradiation-induced mitochondrial superoxide-mediated nucleardamage, if not properly repaired, might propagate to the cellprogenies resulting in secondary cancer development in cells longafter the initial radiation exposure.

A further theoretical consideration is that radiation-induceddamage to mitochondrial DNA may cause mutations or deletionswithin the mitochondrial genome which could be inherited by theoffspring through the maternal germline. Though ndings from arecent report [166] appear to contradict with this concept, a largerstudy cohort is warranted for a clearer examination on thepossibility of transgenerational increase in cancer risk due tomitochondrial abnormality subsequent to ionizing radiation expo-sure to the mother. Likewise, it has been suggested that mitochon-drial superoxide radicals have been a driving factor in theevolutionary move of mitochondrial genetic information into thenucleus [167]. However, it is still a theoretical speculation whetherionizing radiation [168] and the associated oxidative stress mayindeed lead to the insertion of mitochondrial DNA fragments intothe nuclear genome, and whether such nuclear mitochondrialpseudogenes could disrupt the nuclear genome and contribute toan increase in cancer risk within an exposed individual or even intheir offsprings.

The reported observations summarized in this review suggest aneed for further investigations into the effects of ionizing radiationon mitochondria, including (a) the mechanisms of radiation-induced alterations in mitochondrial electron transport chainstoichiometry, residence time, and accessibility of electrons tomediate increased levels of one-electron reductions of oxygen forthe late increase in oxidative stress; and (b) the mechanisms andbiological signicance of the change in mitochondrial mass andcontent in cells after being exposed to ionizing radiation.

Echoing a recent review [26], the interactions between themitochondria and the cell nucleus in response to ionizing irradia-tion are likely to determine short- and long-term radiation effects.For example, how exactly the different nuclear and mitochond-rially encoded subunits of the electron transport chain interact toalter mitochondrial superoxide production, and thus contributegenomic instability, requires further systematic investigation.Furthermore, it is likely that mitochondria are potential targetsfor radiation protection [169174]. The latter has broad relevancefor clinical applications, in the mitigation of environmental orindustrial exposure and in space biology.

Acknowledgments

We thank Mrs. Geetanjali Dhand for her generous assistance incollecting the literature for this review; Dr. Aimee McNarama, fordiscussing and proofreading the manuscript; and A/Prof Zdenkamitochondrial dysfunction, by increasing their quantity and DNAcopy number (Fig. 2B).

However, more mitochondria might lead to more superoxideproduction. In addition, the increase in mitochondrial DNA mightresult in an overproduction of mitochondrial protein. This over-Kuncic, for discussing the manuscript.References

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