Cancer Letters 327 (2012) 48–60

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Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

Mini-review

Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury

Edouard I. Azzam a,⇑, Jean-Paul Jay-Gerin b, Debkumar Pain c
a
Department of Radiology, UMDNJ – New Jersey Medical School Cancer Center, Newark, NJ 07103, United States
b
Département de Médecine Nucléaire et de Radiobiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke (Québec), Canada J1H 5N4
c
Department of Pharmacology and Physiology, UMDNJ – New Jersey Medical School, Newark, NJ 07101, United States

a r t i c l e i n f o a b s t r a c t
Keywords: Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through direct
Ionizing radiation
interactions of radiation with target macromolecules or via products of water radiolysis. Further, the oxi-
Reactive oxygen/nitrogen species
dative damage may spread from the targeted to neighboring, non-targeted bystander cells through
Oxidative metabolism
Mitochondria redox-modulated intercellular communication mechanisms. To cope with the induced stress and the
Genomic instability, adaptive responses, changes in the redox environment, organisms elicit transient responses at the molecular, cellular and tis-
bystander effects sue levels to counteract toxic effects of radiation. Metabolic pathways are induced during and shortly
after the exposure. Depending on radiation dose, dose-rate and quality, these protective mechanisms
may or may not be sufficient to cope with the stress. When the harmful effects exceed those of homeo-
static biochemical processes, induced biological changes persist and may be propagated to progeny cells.
Physiological levels of reactive oxygen and nitrogen species play critical roles in many cellular functions.
In irradiated cells, levels of these reactive species may be increased due to perturbations in oxidative
metabolism and chronic inflammatory responses, thereby contributing to the long-term effects of expo-
sure to ionizing radiation on genomic stability. Here, in addition to immediate biological effects of water
radiolysis on DNA damage, we also discuss the role of mitochondria in the delayed outcomes of ionization
radiation. Defects in mitochondrial functions lead to accelerated aging and numerous pathological con-
ditions. Different types of radiation vary in their linear energy transfer (LET) properties, and we discuss
their effects on various aspects of mitochondrial physiology. These include short and long-term in vitro
and in vivo effects on mitochondrial DNA, mitochondrial protein import and metabolic and antioxidant
enzymes.
Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction of continuous generation of reactive oxygen (ROS) and nitrogen

(RNS) species [3]. Remarkably, these processes occur not only in
The absorption of ionizing radiation by living cells can directly the irradiated cells but also in their progeny [2,4,5]. Furthermore,
disrupt atomic structures, producing chemical and biological radiation-induced oxidative stress may spread from targeted cells
changes. It can also act indirectly through radiolysis of water, to non-targeted bystander cells through intercellular communica-
thereby generating reactive chemical species that may damage nu- tion mechanisms [6–9]. The progeny of these bystander cells also
cleic acids, proteins and lipids [1] (Fig. 1). Together, the direct and experience perturbations in oxidative metabolism and exhibit a
indirect effects of radiation initiate a series of biochemical and wide range of oxidative damages, including protein carbonylation,
molecular signaling events that may repair the damage or culmi- lipid peroxidation, and enhanced rates of spontaneous gene muta-
nate in permanent physiological changes or cell death [2]. tions and neoplastic transformation [10,11] (Fig. 3). The persis-
Interestingly, the early biochemical modifications, which occur tence of such stressful effects in progeny cells has profound
during or shortly after the radiation exposure, were thought to be implications for long-term health risks, including emergence of a
responsible for most of the effects of ionizing radiation in mamma- second malignancy following radiotherapy treatments [12–15].
lian cells. However, oxidative changes may continue to arise for Oxidative DNA damages in key genes such as the tumor suppres-
days and months after the initial exposure presumably because sors P53 and RETINOBLASTOMA may be responsible for the induc-
tion of such malignancies [16,17]. Increasing evidence also
supports the role of chronic oxidative stress in the progression of
⇑ Corresponding author. Tel.: +1 973 972 5323; fax: +1 973 972 1865.
degenerative diseases and radiation-induced late tissue injury
E-mail addresses: azzamei@umdnj.edu (E.I. Azzam), jean-paul.jay-gerin@
[2,18,19]. Therefore, understanding the molecular and biochemical
USherbrooke.ca (J.-P. Jay-Gerin), painde@umdnj.edu (D. Pain).

0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2011.12.012
 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60 49

Fig. 1. The direct and indirect cellular effects of ionizing radiation on macromolecules. Absorption of ionizing radiation by living cells directly disrupts atomic structures,
producing chemical and biological changes and indirectly through radiolysis of cellular water and generation of reactive chemical species by stimulation of oxidases and nitric
oxide synthases. Ionizing radiation may also disrupt mitochondrial functions significantly contributing to persistent alterations in lipids, proteins, nuclear DNA (nDNA) and
mitochondrial DNA (mtDNA).

events that promote early and late oxidative stress in irradiated superoxide/perhydroxyl ðO  
2 =HO2 Þ radicals, where the O2 radical
cells/tissues will be informative for counteracting adverse health exists in a pH-dependent equilibrium with its conjugate acid
effects of ionizing radiation. (pKa = 4.8) [23]. Thus, in an aerobic cellular environment at physi-
ological pH, the major reactive species at homogeneity (106 s)
include O 
2 , OH, and H2O2 (Fig. 2). H2 plays only a limited role in
2. Primary effects of ionizing radiation the radiolysis of aqueous solutions, and most of it escapes from
solution.
2.1. Water radiolysis and generation of reactive oxygen species In biological systems, organic radicals (R) are also formed, most
often by H-abstraction reactions (initiated by OH radicals for
Water is the major (80%) constituent of cells. A thorough example). These carbon-centered radicals usually react rapidly
knowledge of water radiolysis is therefore critical for understand- with O2 to give peroxyl radicals ðRO2 Þ, which are stronger oxidizing
ing radiobiological effects. The absorption of energetic radiations agents than their parent radicals [24]. The RO2 radicals can abstract
by water results in both excitations and ionizations leading to pro- H from other molecules to form hydroperoxides (ROOH), a reac-
duction of free radicals that in turn can attack other critical mole- tion known to be involved in lipid peroxidation. In this context,
cules (indirect effect) (Fig. 1). For brevity, the complex events that the progression of radiation damage in cells likely involves persis-
accompany the absorption of high-energy photons or the passage tent lipid peroxidation reactions that are intertwined with protein
of fast charged particles can be divided into four, more or less inactivation [3,25].
clearly demarcated, consecutive, temporal stages [20]. During the
first or ‘‘physical’’ stage, the energy deposition is caused by the
incident radiation and secondary electrons are generated. The 2.2. Generation of reactive nitrogen species
resulting species are extremely unstable and undergo fast reorga-
nization in the second or ‘‘physicochemical’’ stage. These processes Ionizing radiation can also stimulate inducible nitric oxide syn-
produce radical and molecular products of radiolysis that are dis- thase (NOS) activity in hit cells [26], thereby generating large
tributed in a highly non-homogeneous track structure. Secondary amounts of nitric oxide (NO). Although NO is chemically inert to-
electrons slow down to sub-excitation energies and following ther- ward most cellular constituents (except for heme), it reacts with

malization, they become trapped (e 
tr ) and hydrated (eaq ). The initial O
2 to form the peroxynitrite anion (ONOO ) with a rate constant
(1012 s) spatial distribution of reactants is then directly used as that is larger than that for the superoxide dismutase (SOD)-cata-

the starting point for the so-called stage of ‘‘non-homogeneous lyzed dismutation of O2 [27]. Like hydroxyl radicals, ONOO is also
chemistry’’. During this third stage, the various chemically reactive highly reactive and capable of attacking a wide range of cellular
species diffuse and react with one another or with the environ- targets, including lipids, thiols, proteins and DNA bases. This high
ment, until all intra-track reactions are complete (106 s). Finally, reactivity of ONOO implies low selectivity, confined reactivity
in a physiologic system, there follows a ‘‘biological’’ stage in which with molecules in immediate vicinity, and inability to act as a cel-
the cells respond to the damage resulting from the products lular messenger. By contrast, the much lower reactivity of H2O2
formed in the preceding stages. During this stage (103 s or long- and O 
2 (or HO2 , depending on pH) allows them to diffuse a longer
er, depending very much upon the medium), the biological re- distance away from the originating site (the diffusion coefficients
9
sponses affecting the long-term consequences of radiation of H2O2, O 
2 , and HO2 being 2.3  10 , 1.75  109, and
exposure are induced. Fig. 2 illustrates the time scale of the stages 2.3  109 m2 s1, respectively [28]). In the presence of catalytic
of water radiolysis by sparsely ionizing types (e.g. cobalt-60 or ce- redox metal ions (principally iron and copper ions), these species
sium-137 c rays). It also shows the time scale of chemical reactions lead to the production of OH radicals via Fenton and Haber–Weiss
leading to generation of specific radiolytic products. chemistry [29], which can enhance damage [30].
On a quantitative basis, the species produced in the radiolysis of In summary, the radiolysis of water and early activation of nitric
pure deaerated water are e  
aq , OH, H , H2, and H2O2 [21,22] (Fig. 2). oxide synthases is a major source of ROS/RNS in irradiated cells un-
 
In the presence of oxygen, eaq and H atoms are rapidly converted to der ambient oxygen. Interestingly, the yield of these species is
50 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60

Event Approximate
time scale

H2O

H2O + H2O•+ + e− + H2O H2O*elec ---------- ~10-16 s

−
H2O•
−
H2O*vib e th
(< 10-40 fs) H• + •OH H2 + O(1D)
Geminate (~10-40 fs)
recombination 2H• + O(3P)
(< 10 fs)
H− + •OH
−
•
OH + H3O+ e tr H2O
(~50-300 fs) H2O Nonradiative decay
Proton transfer
(~10 fs) back to ground-state water
−
H2 + OH
Dissociative electron
e−aq attachment
Electron
hydration ---------- ~10-12 s
(~240 fs-1 ps)

Formation of free radicals and molecular products in the

tracks and diffusion of species out of the tracks

−
e aq , H•, •OH, H2, H2O2, H+, OH−, O2•− (or HO2•),… ---------- ~10-6 s

Fig. 2. Time scale of events in the radiolysis of water by low linear energy transfer radiations.

Progeny

IR Survival - Oxidative stress

- Genetic/epigenetic changes
(chromosomal rearrangements
enhanced mutations, aneuploidy
Death
gene amplification, change in
gene expression, etc.)

Bystander
Stresses similar to those in
cells
progeny of irradiated cells

Fig. 3. Ionizing radiation (IR) induces targeted and non-targeted (bystander) effects. Communication of stress-inducing molecules from cells exposed to IR propagates
stressful effects, including oxidative stress, to the bystander cells and their progeny. The induced effects may be similar in nature to those observed in progeny of irradiated
cells.

strongly modulated by different types of radiation. With increasing crosslinks and telomere dysfunction [35–37]. If unrepaired or
linear energy transfer (LET) of the irradiating particles, an increase mis-repaired, these damages may lead to mutations and promote
in the yield of molecular products (such as H2O2) is accompanied neoplastic transformation or cell death [4].
by a corresponding decrease in the yield of radicals (such as

OH). In contrast, O 
2 (or HO2 ) is the most abundant radical species 2.3. Ionizing radiation track structure and the nature of induced
produced by radiations with high LET character [31,32]. Evidently, biological effects
the yield of these products and their concentrations along the
tracks of irradiating particles has important consequences to the Ionizing radiation is classified as either electromagnetic or par-
extent and nature of induced DNA damages [33,34]. Reactive oxy- ticulate. Whereas X and c rays belong to electromagnetic radiation,
gen and nitrogen species can attack DNA resulting in several alter- energetic electrons, protons, neutrons, a particles and heavy
ations, including DNA breaks, base damage, destruction of sugars, charged particles are different forms of particulate radiation [1].
 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60 51

Many of the damaging effects of water radiolysis are due to the neighboring cells. Whereas, certain genetic and epigenetic changes
geometry of the physical energy deposition of the impacting radi- in targeted and non-targeted cells may be observed shortly after
ation, referred to as the track structure or LET effects [38]. In irra- exposure [5,6,54,77], others require several generations to be ex-
diated cells, such energy deposition causes endogenous bursts of pressed [10,78–80]. Certain effects (e.g. microRNA expression) were
ROS in and around the radiation track as well as in the intercellular detected as early as several hours after whole body irradiation of
matrix. The track structure determines the relative potency of dif- mice, and persisted for days, weeks, and even months after irradia-
ferent types of radiation in causing biological effects [39,40]. Fol- tion [79]. Together, the changes due to radiation-induced chronic
lowing exposure to high LET radiations (e.g. a particles, high inflammation and perturbations in oxidative metabolism can lead
charge and high energy (HZE) particles), the yield of locally multi- to genomic instability in targeted and non-targeted cells [81], caus-
ply damaged (LMDS) sites in DNA is greatly increased [4,41,42]. ing serious health effects, including neurodegeneration, cardiovas-
The clustering of lesions induced by ionizing radiation is thought cular diseases and cancer [82–84].
to play a central role in long-term biological effects [43]. However,
the concept that low doses/low fluences of ionizing radiation gen- 2.4. Endogenous and radiation-induced DNA alterations
erate LMDS is not universally accepted [44].
Whereas 60 ROS per nanogram of tissue are generated within A strong emphasis has been on the effect of exogenous agents
less than a microsecond from a hit caused by 137Cs c rays, 2000 such as radiation on DNA damage. However, improvements in
ROS are generated from a 3.2 MeV a particle traversal, which cor- the sensitivity of analytic methods to measure oxidative damage
responds to a ROS concentration of 19 nM in the nucleus [45]. [68] have revealed altered bases and nucleotides in the DNA of nor-
Such a nuclear ROS concentration can obviously cause extensive mal cells that have not been exposed to ionizing radiation or other
oxidative injury and modify normal biochemical reactions mutagens [85]. The analyses have shown that endogenous bio-
[46,47]. As a result, different signaling cascades responding to chemical processes greatly contribute to genome mutations. The
these stress conditions are triggered. For example, adaptive re- ROS produced during normal cellular metabolic processes (mainly
sponses encompassing DNA repair and antioxidation reactions O
2 and H2O2) cause extensive depurinations and to lesser extent
may be triggered following exposures to low doses of low LET radi- depyrimidinations. In addition, ROS can oxidize bases in DNA, such
ations (X and c rays) [48–50]. The protective mechanisms may as the oxidation of deoxyguanosine (dG) to 8-hydroxyguanine (8-
over-compensate, resulting in stimulatory responses that enhance oxodG), with 100–500 of such lesion being formed per day in a
the well-being of the organism long after the exposure [51,52]. In human cell [86]. The rate of occurrence of these alterations has
contrast, basal and induced signaling cascades do not seem to com- been closely linked to the rate of oxidative metabolism: higher
pletely alleviate the complex damages induced by low fluences of oxygen consumption in different species correlated with an in-
high LET radiations (e.g. a and HZE particles) [53]. Damaging ef- creased rate of base oxidation in DNA [87]. A failure to repair oxi-
fects endure and may spread to neighboring bystander cells [54] dized bases creates a risk of mutation during DNA replication. For
and persist in their progeny [11,55] (Fig. 3). Since low-dose radia- example, 8-oxodG mispairs with deoxyadenosine (dA) rather than
tion-induced bystander effects and adaptive responses appear to deoxycytosine (dC) resulting in a C–A point mutation.
involve ROS and RNS [2,56], the track structure is crucial for dictat- In addition to base oxidation, depurination and depyrmidin-
ing the size and precise location of the initial radiation-induced ation, other spontaneous damages can also occur in DNA. The mod-
ROS bursts and their subsequent signaling or damaging effects ification of cytosine to 5-methylcytosine (5mC) caused by reaction
[57–60]. As discussed below, the track structure of the impacting with S-adenosylmethionine, mainly in CpG doublets in the mam-
radiation is also critical in determining the nature of long-term ef- malian genome [88], creates a mutable site that generates thy-
fects on oxidative metabolism [10,11]. The bursts of ROS and RNS mine. The latter is a component of normal DNA, and the T:G
may affect directly or indirectly proteins/genes that participate in basepair may escape detection and serves as template in a subse-
oxidative metabolism [26,61]. The persistence of such perturba- quent cycle of DNA replication, leading to a C- to T point mutation.
tions in the normal oxidative metabolism is associated with Several defenses act to restore DNA integrity. In response to
chronic inflammatory responses [4,62,63]. The latter is a field of DNA damage, cells activate cell cycle checkpoints that provide time
growing interest from radiotherapy [19] and radiation protection for DNA repair machinery to mend the damage. Base excision re-
[18,64] perspectives; it is the subject of intense investigations (re- pair (BER) recognizes and restores spontaneous base modifications,
viewed in [4,63,65]). Levels of the inflammatory markers C-reac- abasic sites and single strand breaks [89,90]. Other modes of DNA
tive protein (CRP), interleukin-6 (IL-6), white cell blood counts, repair, including nucleotide excision repair (NER), mismatch repair
and sialic acid levels were found to be increased in survivors of and double strand break repair restitute other types of DNA dam-
the A-bomb long after the event [64,66]. age [91].
Chronic inflammation is a dynamic and progressive process. The spectrum of ROS generated during and shortly after irradi-
When recruited to sites of inflammation, macrophages and neutro- ation is similar to that produced by metabolic processes. However,
phils generate diffusible and reactive species, including ROS and differences exist in microdistribution (single molecules and clus-
RNS. These species can cause a large spectrum of oxidative DNA ters of ROS produced by radiation vs. single molecules produced
damage that can lead to mutations as well as DNA cross-links in by endogenous processes), relative yield of specific products
neighboring cells (reviewed in [67,68]). Further, the processing of (mainly O2 and H2O2 produced by endogenous processes vs. OH


closely spaced oxidative lesions (oxidatively-induced clustered in highest yield in irradiated cells), and timing of production
DNA lesions or OCDLs) can induce DNA double strand breaks, a seri- (chronic release of endogenous ROS vs. instantaneous production
ous type of DNA lesion that leads to cell death or long-term stressful during irradiation) [92]. As a result, while damage from metabolic
effects in surviving cells [69–71]. Increasing evidence indicates that ROS is randomly distributed in the DNA, radiation-induced DNA
inflammatory cells in circulating blood of patients that received damage frequently occurs in clusters [93]. Whereas 1/3 of DNA
partial body irradiation may also induce DNA damage at sites that damage from ionizing radiation (low LET type) emanates from di-
are distant from the irradiated target [4,72], hence contributing to rect interaction of DNA with the irradiating particle and 2/3 due to
‘out-of-field’ or abscopal effects [5,73–75]. Macrophages also se- indirect effects (Fig. 1), DNA damage from endogenous sources is
crete cytokines [76] that may perturb physiological functions in due primarily to indirect effect.
normal surrounding cells. Hence, the effects of localized energy Techniques such as the ‘comet assay’ [93,94] and newer
deposition events in a cell may not be assessed independently of techniques (e.g. gas chromatography, high pressure liquid
52 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60

chromatography, immunoassays) [4,68] can distinguish gross DNA cell-cycle functions [98]. In addition to mediating the anti-micro-
damage produced by ionizing radiation and damage from oxidative bial activity of phagocytes [114], the multicomponent NAD(P)H
attacks. For low doses of radiation, the total number of induced oxidase affects signal transduction by growth factor receptors
DNA alterations is probably small when compared with the total [115] and promotes proliferation in a variety of cell types [116].
number of equivalent alterations from endogenous sources [95]. In turn, mitogenic signaling elicited by cytokines or growth factors
induces intracellular ROS production via activation of the PI3K
3. Reactive oxygen species and cellular homeostasis pathway resulting in stimulation of Rac1, which up-regulates
NAD(P)H-oxidase activity [117]. However, the exact molecular
Although excess ROS produced by ionizing radiation is toxic, in- and biochemical events by which non-phagocytic NAD(P)H-oxi-
tra-cellular ROS produced under physiologic conditions serve as dase (and/or other oxidases) mediate cellular proliferation under
essential signaling molecules that regulate numerous cellular pro- physiological oxidative conditions are not clear. Nevertheless, the
cesses [96–98]. Homeostatic cellular functions therefore require levels and activity of cyclins that drive cellular proliferation (e.g.
tight control of the redox environment [2,99]. At low levels, ROS cyclin D) are redox sensitive [112,118]. Therefore, the cell cycle ar-
participate in signaling pathways that maintain normal cellular rest in G1 phase through modulation of the cellular redox environ-
functions by regulating the expression of specific genes [100– ment offers a therapeutic potential to control tumor growth.
103], modulating ion channel activities [104], and mimicking or Notably, many cancer cells show increased production of hydrogen
affecting intermediates (e.g. second messengers) in signal trans- peroxide and other oxidizing species, which are associated with
duction [105]. NAD(P)H-oxidases, lipoxygenases, nitric oxide syn- cellular proliferation [119]. Hence, the approach of inhibiting basal
thases, xanthine oxidase, microsomal cytochrome P-450, and oxidant production by flavin-oxidases could be an attractive mode
mitochondrial electron transport chain are the major sources of of cancer therapy [111,118,120–122]. Importantly, most tumors
cellular ROS [106]. The levels of these ROS greatly depend on the harbor proliferating cells in different phases of the growth cycle.
availability of cellular oxygen [30]. Most of the oxidants and their Blocking cells in G1 phase, by inhibiting ROS generation by fla-
byproducts are metabolized by various enzymes and small mole- vin-containing oxidases, could result in depletion of tumor cells
cule antioxidants [97]. Most likely, it is the ROS that escape antiox- from the relatively radioresistant S-phase compartment [123],
idant defense participate in homeostatic regulation of redox thereby enhancing radiosensitivity and therapeutic response. This
signaling [107]. Certain ROS (e.g. HO2 , H2O2) can permeate lipid approach may complement radiotherapy of cancer cells, which
bilayers [108] and traverse membranes [109], thus contributing are normally not arrested in G1 phase after irradiation [124]. Aber-
to the propagation of signaling events among cells in a confluent rant cell proliferation associated with oxidative stress also contrib-
cell culture. utes to other pathological conditions such as neurodegenerative
Although a major emphasis in the literature has been on phys- and cardiovascular diseases and diabetes [125]. Reestablishing cell
iological functions at homeostatic or higher than normal levels of cycle regulation by manipulating the cellular redox environment
ROS [110], lower ROS levels also result in important changes in cel- may help ameliorate some of these disorders [98].
lular functions [111]. For example, inhibition of superoxide pro-
duction by NAD(P)H-oxidases (but not nitric oxide generated by 4. Mitochondria and delayed effects of ionizing radiation
nitric oxide synthases) leads to strong arrest of cells in G1 phase
of the growth cycle [112] (Fig. 4). The effect occurs in the absence Upon cellular exposure to ionizing radiation, ROS generating-
of induced DNA damage and is dependent on the Ataxia Telangiec- oxidases may be activated, antioxidants modulated, and metabolic
tasia Mutated (ATM) gene, a critical player in the early detection activity altered in response to the oxidative insult. Among the mul-
and repair of ionizing radiation-induced DNA damage [113]. titude of induced effects, ionizing radiation may disrupt mitochon-
Importantly, the induced G1-delay is attenuated when cells treated drial functions because of several reasons. For example,
with inhibitors of NAD(P)H-oxidases are simultaneously exposed mitochondria occupy a fairly substantial fraction of cell volume
to low dose-rate c rays. These results suggest that ROS generated (4–25% depending on the cell) [126], which renders them a likely
by c rays substitute for effects of ROS generated by NAD(P)H-oxi- target of radiation traversal through the cell. More importantly,
dases [112]. It is often asked whether the chemical species and/or mitochondria consume about 90% of the body’s oxygen and are
genetic lesions induced by ionizing radiation and those induced by the richest source of ROS [127–130]. They divert about 1–5% of
normal oxidative metabolism are distinct or similar [92]. There- electrons from the respiratory chain to the formation of superoxide
fore, the ability of radiation-induced ROS to replace ROS generated radicals by ubiquinone-dependent reduction [131]. Mitochondrial
by flavin-oxidases (e.g. NAD(P)H-oxidase) for restoration of normal dysfunction in irradiated cells may thus significantly contribute
cell cycle progression suggests that they are similar. to perturbation in oxidation–reduction reactions that determine
The findings as described above emphasize the multi-function- the cellular redox environment. The mitochondrion serves as the
ality of oxidases in modulating normal cellular responses and high- ‘‘real power plant of the cell’’ through aerobic respiration [132].
light a regulatory link between oxidative metabolic processes and It is the cell’s principal source of energy that includes the

ROS Ionizing Radiation

Oxidizing agents

S
ROS Homeostasis G1

ROS NADP(H) oxidase

M G2
inhibition

Fig. 4. Reactive oxygen species (ROS) and the regulation of cell proliferation. Higher or lower than normal levels of ROS can induce cell cycle delays in different phases of the
cell cycle.
 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60 53

tricarboxylic acid (TCA) cycle, electron transport chain (ETC), and Recent evidence has indicated that base excision repair [172]
oxidative phosphorylation. and mismatch repair [173] may be induced in mitochondria during
The TCA cycle occurs in the mitochondrial matrix, and oxidative oxidative insult. However, excess oxidative stress appears to target
metabolism of carbohydrates, lipids, and amino acids integrate the mitochondrial DNA polymerase c (pol c) activity required for
into the TCA cycle to produce NADH and FADH2. These essential replication and repair of mtDNA [174], thereby reducing the over-
intermediates are electron-rich donors that enter the ETC on the all repair capacity [175–177]. In addition, cells from knockout mice
mitochondrial inner membrane for use in ATP production. The for 8-oxoguanine DNA glycosylase (ogg1) spontaneously accumu-
ETC is composed of five protein complexes (I–V) that perform a late mtDNA damage [178]. Therefore, subsequent to radiation
series of oxidation-reduction reactions in which O2 serves as the fi- exposure, mtDNA might be preferentially damaged or lost due to
nal electron acceptor and is reduced to H2O. Electron transfer is oxidative stress with an ensuing decrease in respiratory chain
coupled with the ejection of H+ out of the matrix into the inter- activity and decrease of mitochondrial function. Mutations in
membrane space, creating a proton gradient that drives the pro- mtDNA causing disruptions in the proper assembly and/or function
duction of ATP through oxidative phosphorylation [133]. A conse- of mitochondrial ETCs could lead to an increase in accessibility of
quence of this energy production is the generation of ROS reduced components of the ETCs to O2, which may result in an in-
byproducts. crease in prooxidant formation [2]. The net consequence being a
The premature leakage of electrons (mainly from complexes I condition of persistent metabolic oxidative stress that continues
and III, and complex II to a minor extent) results in the reduction to cause de-novo oxidative damage to critical biological structures
of O2 to create superoxide (O2 ) [134–136]. Apart from this normal long after the radiation exposure. Radiation-induced mtDNA muta-
basal level of ROS production, radiation causes further leakage of tions may segregate in post-mitotic cells [179] and become herita-
electrons from the ETC, and therefore results in excess O 2 genera- ble traits that contribute to genomic instability [180]. Changes in
tion [97]. This occurs in addition to the ROS produced during water the structure and/or function of genes coding for mitochondrial
radiolysis. ROS production by mitochondria plays multiple roles in ETC proteins in progeny cells can give rise to increases in mito-
signaling cascades [137–139] and mediates apoptosis [140]. Excess chondria-derived oxidants that contribute to nuDNA damage
ROS may cause mutations in mitochondrial DNA, and damage or [162].
alter the expression of proteins required for critical mitochondrial The ‘common deletion’ in the mitochondrial genome is also in-
and cellular functions. Several endpoints have been tested to duced following cellular exposure to ionizing radiation [181]. This
examine mitochondrial dysfunction in irradiated cells and their deletion involves the loss of 4977 base pairs coding for genes that
progeny. For example, early and late generation of radiation-in- include subunits of the mitochondrial ATPase, NADH dehydroge-
duced mitochondrial ROS/RNS mediates changes in mitochondrial nase complex I and cytochorome c oxidase. Once formed, the ‘com-
DNA copy number [141], mutations [142] and gene expression mon deletion’ becomes stable [182]. It has been proposed that the
[143,144], autophagy [145,146], apoptosis [147–149], propagation ‘common deletion’ leads to inefficient mitochondrial metabolism
of non-targeted responses [73,150–155], the induction of nuclear and thus increased ROS production [183]. Whereas irradiated nor-
DNA damage [156], genomic instability [157], neoplastic transfor- mal cells accumulate significant levels of the 4977 base-deletion,
mation [158], and degenerative conditions [37,63,159] among tumor cells show only a modest induction [181]. Surprisingly, a
other outcomes [2,37]. We investigated effects of ionizing radiation relationship between radiation-induced deletions and sensitivity
on mitochondrial protein import, assembly of large protein com- to cell killing by radiation was not observed in the same study
plexes, protein oxidation and activity of metabolic and antioxidant among the various cell strains/lines tested [181].
enzymes [10,11,45,48,160,161]. Significantly, the persistence of The traffic of DNA from mitochondria to the nucleus has been
radiation effects that lead to mitochondrial dysfunction in progeny demonstrated in Saccharomyces cerevisiae [184], and mtDNA se-
cells has serious consequences to health risks [2,162]. quences are found in at least 27 sites in human chromosomes
[185]. It is therefore attractive to speculate that migration of
4.1. Mitochondrial DNA mtDNA fragments to the nucleus following exposure to ionizing
radiation may lead to their insertion in nuclear genome, particu-
Human mitochondrial DNA (mtDNA) is a circular molecule; it is larly during repair of DNA double strand breaks [186], which
among the smallest known mtDNAs and contains 16,659 bases. A would contribute to alterations in genomic DNA and the induction
mammalian cell contains several copies of mtDNA that code for of genomic instability. Irradiation of mice stimulated the appear-
rRNA, tRNA and proteins (13 in humans). The close proximity of ance of mtDNA fragments in the cytosolic fractions of the brain
mtDNA to sites of ROS production renders it particularly suscepti- as early as 1 h after exposure to 5 Gy of 137CS c rays [187]. By tar-
ble to damage, which led Harman to propose that damage to geting regulatory sequences, insertion of mtDNA in nuDNA may re-
mtDNA contributes to the aging process [163]. Various pathologies sult in permanent changes in gene expression [180]. Therefore, a
have been attributed to mutations in mtDNA; their severity de- persistent increase in mitochondrial oxidative stress following
pends on the nature of the mutation and on the proportion of mu- exposure to ionizing radiation may lead to a mutagenic phenome-
tant and wild-type mtDNA present in a particular cell type [164– non that could contribute to cancer and accelerated aging
166]. Tissues that have a high requirement for ATP are most af- [188,189]. Consistent with a role of oxidative stress in the translo-
fected by mutations in mtDNA [167], and express a variety of cation of mtDNA to the nucleus, experiments in yeast have shown
pathologies as well as age associated disease [168,169]. that the migration rate increases during chronological aging [189],
Short- and long-term radiation-induced ROS/RNS could result in which suggests that a similar phenomenon may occur following
damage to mtDNA and/or nuclear DNA (nuDNA) coding for mito- exposure to moderate doses of radiation.
chondrial electron transport chain (ETC) subunits as well as the
biochemical machinery necessary for their proper expression and 4.2. Effects of ionizing radiation on mitochondrial functions
assembly [170]. Interestingly, cells deficient in mitochondrial elec-
tron transport (rho(o) cells) do not experience radiation-induced 4.2.1. Mitochondrial protein import
ROS/RNS production [126]. Rho(o) cells are more resistant to radia- Although mitochondria contain their own DNA and complete
tion-induced cell killing than rho(+) cells; they show delayed G2 ar- systems for replication, transcription and translation, they synthe-
rest and decreased ability to recover from the G2 checkpoint than size only a few (13 in humans) proteins [190]. All other mitochon-
rho(+) cells [171]. drial proteins are nuclear-encoded and are synthesized on
54 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60

cytoplasmic ribosomes. These proteins must be transported from expression [199]. Mammalian cells contain two aconitases that
cytosol to the correct mitochondrial sub-compartment – outer contain Fe–S clusters: the mitochondrial enzyme (ACO2) involved
membrane, intermembrane space, inner membrane or matrix in the TCA cycle, and a cytosolic enzyme referred to as the iron reg-
[191] (Fig. 5). Protein import into mitochondria is therefore a fun- ulatory protein-1 (IRP1).
damental mechanism of mitochondrial biogenesis – maintenance The TCA cycle in the mitochondrial matrix is a central pathway
and regeneration of mitochondria. Defects in mitochondrial pro- of oxidative metabolism. This pathway is critical for the oxidation
tein import resulting from exposure to ionizing radiation may am- of acetyl-CoA and for the production of reducing equivalents that
plify the oxidative stress and lead to late health effects, including are used by the respiratory complexes to produce ATP. Aconitase
degenerative diseases and metabolic disorders [192–195]. (ACO2) is one of the eight enzymes that participate in the TCA cy-
We have demonstrated that mitochondrial protein import could cle. It reversibly catalyzes the conversion of citrate to isocitrate.
be used as a sensitive marker for early detection of long-term ef- The cubane [4Fe–4S]2+ cluster in the active site of aconitase is
fects of ionizing radiation [161]. Specifically, irradiated normal hu- essential for this catalytic activity, but it also renders the enzyme
man diploid cells show differential effects of high and low doses of highly vulnerable to oxidative stress. The cytosolic IRP1 in its
c rays on mitochondrial membrane potential (Dw) and protein im- [4Fe–4S] cluster form exhibits aconitase activity. As in the case
port [161]. Protein import into the mitochondrial matrix requires for the mitochondrial aconitase (ACO2), the [4Fe–4S] cluster of
Dw across the inner membrane, and our data show that ionizing cytosolic IRP1 is also highly susceptible to oxidative damage.
radiation-induced defects in import are not solely due to changes The data presented in Fig. 6 illustrate in-gel measurement of
in Dw. These import studies were performed using the frataxin aconitase activity 24 h after exposure of Chinese hamster lung
precursor protein as a model substrate, and it remains to be deter- fibroblasts to a moderate dose of c rays (50 cGy). The assay differ-
mined if radiation affects mitochondrial import of other proteins as entiates between mitochondrial and cytoplasmic aconitase activi-
well. For example, cellular exposure to ionizing radiation may ties, and shows that both are decreased in irradiated cells (Fig. 6).
cause damages to key components of the protein translocation We have also shown that ACO2 activity is decreased in tissues
machinery, and this in turn may result in a global defect in protein of irradiated mice or in cultured cells after exposure to doses as
targeting/import. low as 1 cGy from sparsely ionizing 137Cs c rays. The decreases
We also found that exposure to ionizing radiation causes a de- were transient and the activity returned to basal levels within
crease in the levels of some of the components of the protein im- 48–120 h after irradiation. This is an important finding because
port machinery in vivo. These observations were made with such a low dose is often used for certain diagnostic procedures.
tissues of rats exposed to mean moderate doses (50 cGy) of high In confluent cell cultures exposed to low fluences of densely ioniz-
LET radiations (HZE particles). The effects persisted for months ing radiations (e.g. HZE particles such as Fe or Ti ions found in deep
after the exposure, occurred in both the targeted and non-targeted space), significant decreases in ACO2 were detected in protein ly-
tissues of irradiated animals, and correlated with greatly reduced sates even when only 1–5% of the cells in the culture were irradi-
mitochondrial protein import. These novel findings highlight the ated. These data suggest that oxidative stress in irradiated cells is
physiological relevance of radiation dose and quality (LET) with re- propagated to neighboring bystander cells, and this notion has
spect to long-term effects on mitochondria. The results also show been confirmed in co-culture studies of HZE-particle-irradiated
that radiation dose-rate is an important factor modulating mito- cells and bystander cells [10,11]. Strikingly, the effect on aconitase
chondrial biogenesis. Compared to cultured cells or rodents ex- activity in the progeny of bystander cells persisted and was de-
posed to acute radiation doses, protraction of delivery of the low tected after 20 population doublings [11]. These results strongly
LET radiation dose attenuated reduction in mitochondrial protein suggest that perturbations in oxidative metabolism persist long
import. A reduction in import efficiency is normally indicative of after the radiation exposure. The effect was associated with en-
stress conditions. However, it may also represent an adaptive re- hanced levels of micronuclei (a form of DNA damage that arises
sponse to minimize ROS-mediated metabolic changes. In fact, pro- mainly from DNA double strand breaks [200]), protein carbonyl-
teomic analyses of mitochondria from tissues of irradiated rodents ation, lipid peroxidation and decreases in cloning efficiency [11].
[73] reveal both stressful effects (e.g. decrease in proteins involved One possibility is that the decreases in aconitase activity leads
in fatty acid elongation, a process required for energy storage and to reduced NADH supply for electron transport, thereby limiting
synthesis of lipids [196]), and protective responses (e.g. upregula- the production of free radical species [201]. Thus, in the case of
tion of Nrf2 signaling implicated in important antioxidant func- sparsely ionizing radiations (i.e. c rays), the transient decreases
tions [197,198]). in ACO2 activity in cells and tissues exposed to low doses represent
an adaptive response to reduce further generation of ROS until the
4.2.2. Metabolic enzymes: Aconitases redox environment is restored to homeostatic levels. However, the
Iron-sulfur (Fe–S) clusters are ancient modular co-factors of continual reduction in aconitase activity in distant progeny of by-
proteins that are involved in many cellular processes, including stander cells that were in co-culture with cells exposed to densely
enzymatic catalysis, electron transport, and regulation of gene ionizing radiations is indicative of persistent oxidative stress

Matrix
IM OM
OM: Outer membrane
IMS IMS: Intermembrane space
IM: Inner membrane
TOM: Translocase outer membrane
TIM: Translocase inner membrane
TIM TIM

Precursor TOM
protein Cytosol

Fig. 5. Schematic of protein import into mitochondria.

 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60 55

C 50 cGy A B
Protein carbonylation Lipid peroxidation
MW [0] [10] [200] [0] [10] [200] cGy
ACO2 (kDa)

97

68
IRP1
43
100 30 % Relative intensity of ACO2
100 43 % Relative intensity of IRP1
29
Fig. 6. Native in-gel assay for the mitochondrial (ACO2) and cytoplasmic (IRP1)
aconitase activities in control and c-ray-exposed Chinese hamster lung fibroblasts.
21

[10,11]. These radiations include a particles emitted from radon

gas and HZE particles encountered during deep space travel, and Fig. 7. Long-term effects of ionizing radiation on protein oxidation. Immunoblots
are likely to be associated with significant health risks [202,203]. showing protein carbonylation (panel A) and proteins with 4-hydroxynonenal
Measurement of aconitase activity may be used as another sen- (HNE) adducts (panel B) in progeny of bystander cells that were cultured for 5 h
sitive marker of oxidative stress in irradiated cells and tissues. It with 1 GeV/nucleon 56Fe-irradiated cells. Note that the radiation dose described in
the figure refers to the absorbed dose by the irradiated cells.
may be particularly informative of the level of stress induced in sit-
uations of mixed exposures to both sparsely and densely ionizing
quences for the maintenance of genomic integrity [210]. It alters
radiations. The oxidized form of aconitase (ACO2) appears to be
chromatin compactness with significant loss of DNA repair. There-
selectively degraded by the Lon protease in mitochondria
fore, persistent oxidative damage in irradiated and non-targeted
[204,205] Thus, studies of mitochondrial aconitase activity/protein
organs may be a factor in late tissue injury following radiotherapy
levels and degradation of the aconitase protein by Lon protease
[63,106].
may provide critical understanding of radiation-induced biochem-
ical events in mitochondria.
4.2.4. Modulation of antioxidants
Central to antioxidant defense are superoxide dismutases
4.2.3. Large protein complexes and oxidative damage to mitochondrial
(SOD), glutathione, glutathione peroxidases, catalase as well as
proteins
nutrient-derived antioxidant compounds such as vitamin E and
Proteins that participate in critical mitochondrial functions of-
selenium [211,212]. The use of antioxidants in the treatment of
ten exist in large complexes (e.g. respiratory complexes I–V). These
radiation injury and other diseases associated with oxidative stress
complexes may be susceptible to oxidative stress from exposure to
(e.g. Crohn’s disease, rheumatoid arthritis, osteoarthritis) contin-
ionizing radiation. Consistent with this concept, dysfunction in
ues to be advocated [3,106,213]. For example, SOD has been shown
mitochondrial complex II was shown to contribute to genomic
to protect other enzymes, mitochondria, membranes, microsomes,
instability in progeny of cells exposed to ionizing radiation [206].
DNA and normal mammalian cells [106]; its anti-inflammatory
In these cells, aberrant expression of subunit B protein of complex
properties were discovered well before the protein was identified
II led to increased steady-state levels of O
2 and H2O2.
as an enzyme [214]. Indeed, the protective effects of antioxidants
Radiation induced ROS may also result in oxidative damage to
support the role of ROS in the progression of diseases [215]. How-
other proteins in mitochondria. Significant increases in protein car-
ever, the dose, timing and mode of delivery of antioxidants should
bonylation and also in levels of proteins with 4-hydroxynonenal
be carefully evaluated. For example, the superoxide radical serve as
adducts were observed in high LET-irradiated cells within hours
both an initiator and a terminator of the free radical-mediated
after exposure [45]. 4-hydroxynonenal is a notoriously reactive
chain reaction that results in lipid peroxidation [216]. Thus,
molecule that covalently modifies proteins and DNA, thereby caus-
depending on dose, in some circumstances SOD may not alleviate
ing dysregulation of their function [207]. As shown in Fig. 7, these
radiation injury; in contrast, it may exacerbate the toxic effects.
modifications were also detected in the distant progeny of bystan-
Similar to mitochondrial protein import and aconitase activity
der cells that were in co-culture with cells exposed to high LET
described above, radiation also modulates antioxidant enzyme
radiations [11]. These findings are highly relevant to understand-
activity in a dose, dose-rate and LET-dependent manner. Low doses
ing of long-term health effects of radiation. Such a notion is consis-
of low LET radiation (137Cs c rays) delivered at low dose-rate up-
tent with our in vivo work showing extensive protein oxidation in
regulate antioxidant defense (e.g. increase in the level of glutathi-
targeted and non targeted tissues of mice and rats even 2 years
one together with up-regulation of c-glutamylcysteine synthetase
after exposure to radiations with high LET character. These effects
expression [48,217,218]). By contrast, high LET radiation propa-
appear to be tissue specific and are associated with chronic inflam-
gates oxidative stress in the irradiated cells and their neighboring
mation, a dynamic and progressive process that begins with ROS
bystanders [219–221]. In progeny of bystander cells, oxidative
and RNS generation and is closely associated with radiation-in-
stress is associated with decreases in activity of MnSOD, CuZnSOD,
duced oxidative stress [19].
catalase and glutathione peroxidase [11]. With relevance to radia-
Oxidative damage to proteins renders them prone to segrega-
tion protection and radiotherapy, similar effects in progeny cells
tion and degradation. Further, carbonylation damage is unrepair-
were not induced when bystander cells were co-cultured with
able, which may impair the activity of key proteins essential for
low LET-irradiated cells [11].
healthy survival [208]. It may disrupt protein structures with con-
sequent loss of functions. It may also change the transport proper-
ties of lipid bilayers and the transmembrane potential of both 5. Perspective
plasma and nuclear membranes, and cause the accumulation of
cytotoxic products [209]. Carbonylation of histones, the essential Reactive oxygen and nitrogen species have multiple roles that
components of eukaryotic chromatin, has potentially severe conse- greatly depend on their concentrations. At normal physiologic
56 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60

levels, ROS/RNS participate in signal transduction functions that that determines radiation sensitivity. In sum, both genetic and
are essential for healthy survival [107]. However, at aberrant lev- metabolic susceptibility to ionizing radiation greatly affect the out-
els, they function as toxic agents and are associated with abnormal come of radiation exposure. At the organelle level, mitochondria
cell proliferation [215,222]. Ionizing radiation is a strong inducer of being the major site of oxidative metabolism are almost certainly
ROS and RNS [35]. Depending on concentration, reactivity, spatial going to be affected. Whereas many studies have addressed the ef-
and temporal distribution, these species may mediate either adap- fects of ionizing radiation on biochemical changes in mitochondria,
tive/protective responses or genomic instability in progeny of irra- studies of the radiation-induced morphological changes of the
diated cells and their neighboring bystanders [223–226]. organelle are lacking. This is particularly important since mito-
Against a background of 109 ROS/cell/day derived from chondrial structure and bioenergetics may be tightly linked; mito-
metabolized oxygen [227], a few hundred ROS are generated from chondrial size, shape and fusion/fission may significantly vary
cellular exposure to low doses/low fluences of ionizing radiation depending on cell type, the metabolic energy status, radiation dose
mainly due to water radiolysis [95]. Yet, cells sense this increase and quality [244–246].
and up-regulate signaling cascades to counteract the effects of
additional reactive species. Metabolic protections are mobilized
during and soon after the insult at molecular, cellular, tissue and Acknowledgments
organism levels [228]. In addition to up-regulation of DNA repair
mechanisms, antioxidants scavenge excess ROS and proteases re- We are grateful to the members of our laboratories for their dil-
move oxidized proteins. Further, cells/tissues may downregulate igent input into the research. This work was supported by grants
oxidative metabolism to lower the concentrations of reactive from the National Aeronautical Space Radiation Administration
chemical species and may mobilize immune responses as well (Grant NNJ06HD91G), the US Department of Energy Low Dose
[11,161,229]. Whereas the induced mechanisms adequately pro- Radiation Research Program (Grant FG02-02ER63447) and the Na-
tect against oxidative stress from low doses of low LET radiations tional Institute of Health (Grant CA049062) to E.I.A., the Natural
delivered at low dose rates [48,230–235], they are inefficient at Sciences and Engineering Research Council of Canada to J.-P.J.-G.,
low fluences of high LET radiations. In fact, the latter radiations ap- and the National Institute of Aging (Grant AG030504) and Ameri-
pear more effective at inducing long-term clastogenic/toxic effects can Heart Association (Grant 09GRNT2260364) to D.P.
[236,237] than previously thought [238,239]. The concentration of
ROS/RNS in confined space along the track of densely ionizing radi-
ations is an important factor that determines the damaging effects References
of high LET radiations [33,47,59,240,241]. Mounting evidence indi-
[1] E.J. Hall, A.J. Giaccia, Radiobiology for the Radiologist, 6th ed., Lippincott
cates that perturbations in oxidative metabolism may persist long Williams & Wilkins, Philadelphia, PA, 2006.
after the decay of primary and secondary chemical species, and [2] D.R. Spitz, E.I. Azzam, J.J. Li, D. Gius, Metabolic oxidation/reduction reactions
these conditions likely disrupt stability and activity of DNA repair and cellular responses to ionizing radiation: a unifying concept in stress
response biology, Cancer Metastasis Rev. 23 (2004) 311–322.
proteins. Consequently, de novo DNA damages continue to occur [3] A. Petkau, Role of superoxide dismutase in modification of radiation injury, Br.
[10,242], thereby affecting gene expression. Therefore, under- J. Cancer (Suppl. 8) (1987) 87–95.
standing of the processes that lead to perturbations in oxidative [4] T.B. Kryston, A.B. Georgiev, P. Pissis, A.G. Georgakilas, Role of oxidative stress
and DNA damage in human carcinogenesis, Mutat. Res. 711 (2011) 193–201.
metabolism would contribute to devising strategies to counteract [5] J. Tamminga, O. Kovalchuk, Role of DNA damage and epigenetic DNA
oxidative stress (e.g. by administration of antioxidants, inhibitors methylation changes in radiation-induced genomic instability and
of mitochondrial ATP-dependent proteases that target metabolic bystander effects in germline in vivo, Current Mol. Pharmacol. 4 (2011)
115–125.
enzymes, etc.) to control the progression of cancer and other path- [6] E.I. Azzam, S.M. de Toledo, J.B. Little, Oxidative metabolism, gap junctions and
ological states [243]. On the other hand, enhancing oxidative stress the ionizing radiation-induced bystander effect, Oncogene 22 (2003) 7050–
selectively in cancer cells to induce lethal effects is also desirable. 7057.
[7] T.K. Hei, H. Zhou, Y. Chai, B. Ponnaiya, V.N. Ivanov, Radiation induced non-
Coupling radiotherapy with strategies to deplete cancer cells of targeted response: mechanism and potential clinical implications, Current
antioxidants may enhance lethal effects in cancer cells. Further, Mol. Pharmacol. 4 (2011) 96–105.
enhancing the propagation of death-inducing events among irradi- [8] C. Mothersill, C.B. Seymour, Radiation-induced bystander effects–
implications for cancer, Nat. Rev. Cancer 4 (2004) 158–164.
ated cancer cells and between irradiated and bystander cancer
[9] K.M. Prise, J.M. O’Sullivan, Radiation-induced bystander signalling in cancer
cells through redox-modulated events would also contribute to tu- therapy, Nat. Rev. Cancer 9 (2009) 351–360.
mor control [45,75]. [10] M. Buonanno, S.M. de Toledo, E.I. Azzam, Increased frequency of spontaneous
Although targets of ROS/RNS have been identified, many others neoplastic transformation in progeny of bystander cells from cultures
exposed to densely-ionizing radiation, PloS One 6 (2011). art. no. e21540.
remain to be explored [110]. For this purpose, proteomic analyses [11] M. Buonanno, S.M. De Toledo, D. Pain, E.I. Azzam, Long-term consequences of
of covalent modifications (oxidative/nitrosative changes) in anti- radiation-induced bystander effects depend on radiation quality and dose
oxidant enzymes and DNA repair proteins combined with studies and correlate with oxidative stress, Radiat. Res. 175 (2011) 405–415.
[12] F.A. Cucinotta, L.J. Chappell, Non-targeted effects and the dose response for
of mitochondrial protein content [244] and functions may be infor- heavy ion tumor induction, Mutat. Res. 687 (2010) 49–53.
mative. Revealing the association of these changes with radiation- [13] E.J. Hall, Henry S. Kaplan, Distinguished Scientist Award 2003. The crooked
induced protective or detrimental effects may identify regulated shall be made straight; dose-response relationships for carcinogenesis, Int. J.
Radiat. Biol. 80 (2004) 327–337.
pathways in the context of metabolic changes. Such an approach [14] M. Tubiana, Can we reduce the incidence of second primary malignancies
may also uncover different mechanisms for early and late cellular occurring after radiotherapy? A critical review, Radiother. Oncol. 91 (2009)
responses to ionizing radiation. However care must be taken to 4–15. discussion 11-13.
[15] BEIR-VII, Health Risks from Exposure to Low Levels of Ionizing Radiation, in,
precisely define the effects of dose, dose rate and radiation quality. National Research Council of the National Academies, Washington, D.C., 2005.
For example, molecular and biochemical effects that underlie high [16] J.H. Hendry, Genomic instability: potential contributions to tumour and
dose radiations may not necessarily mediate low dose radiations. normal tissue response, and second tumours, after radiotherapy, Radiother.
Oncol. 59 (2001) 117–126.
Further, when extrapolating results across species, the metabolic
[17] A.I. Robles, S.P. Linke, C.C. Harris, The p53 network in lung carcinogenesis,
rate of the system being investigated may need to be integrated. Oncogene 21 (2002) 6898–6907.
Metabolic rates greatly vary, with that of humans being an order [18] S.J. Schonfeld, P. Bhatti, E.E. Brown, M.S. Linet, S.L. Simon, R.M. Weinstock,
of magnitude lower than mice [87], which may be a factor in deter- A.A. Hutchinson, M. Stovall, D.L. Preston, B.H. Alexander, M.M. Doody, A.J.
Sigurdson, Polymorphisms in oxidative stress and inflammation pathway
mining species-specific long-term effects. Likewise, basal meta- genes, low-dose ionizing radiation, and the risk of breast cancer among US
bolic activity in specific tissues is likely to be an important factor radiologic technologists, Cancer Causes Control 21 (2010) 1857–1866.
 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60 57

[19] W. Zhao, M.E. Robbins, Inflammation and chronic oxidative stress in human fibroblasts: the role of growth architecture and oxidative metabolism,
radiation-induced late normal tissue injury: therapeutic implications, Curr. Radiat. Res. 166 (2006) 849–857.
Med. Chem. 16 (2009) 130–143. [49] L.E. Feinendegen, M. Pollycove, R.D. Neumann, Whole-body responses to low-
[20] R. Platzman, The physical and chemical basis of mechanisms in radiation level radiation exposure: new concepts in mammalian radiobiology, Exp.
biology, in: W. Claus (Ed.), Radiation Biology and Medicine. Selected Reviews Hematol. 35 (2007) 37–46.
in the Life Sciences, Addison-Wesley, Reading, MA, 1958, pp. 15–72. [50] S. Wolff, The adaptive response in radiobiology: evolving insights and
[21] C. Ferradini, J.P. Jay-Gerin, Radiolysis of water and aqueous solutions: histrory implications, Environ. Health Perspect. 106 (Suppl 1) (1998) 277–283.
and present state of the science, Can. J. Chem. 77 (1999) 1542–1575. [51] T. Nomura, X.H. Li, H. Ogata, K. Sakai, T. Kondo, Y. Takano, J. Magae,
[22] J.W.T. Spinks, R.J. Wodds, An Introduction to Radiation Chemistry, 3rd ed., Suppressive Effects of Continuous Low-Dose-Rate gamma Irradiation on
Wiley, New York, 1990. Diabetic Nephropathy in Type II Diabetes Mellitus Model Mice, Radiat. Res.
[23] B.H.J. Bielski, D.E. Cabelli, R.L. Arudi, A.B. Ross, Reactivity of HO2 /O2 - radicals 176 (2011) 356–365.
in aqueous solution, J. Phys. Chem. Ref. Data 14 (1985) 1041–1100. [52] E.I. Azzam, Exposure to low level environmental agents: The induction of
[24] Z.B. Alfassi (Ed.), Peroxyl Radicals, Springer, Berlin, 1997. hormesis, Mutat. Res. 726 (2011) 89–90.
[25] B.S. Berlett, E.R. Stadtman, Protein oxidation in aging, disease, and oxidative [53] M.A. Kadhim, D.A. Macdonald, D.T. Goodhead, S.A. Lorimore, S.J. Marsden, E.G.
stress, J. Biol. Chem. 272 (1997) 20313–20316. Wright, Transmission of chromosomal instability after plutonium a-particle
[26] R.B. Mikkelsen, P. Wardman, Biological chemistry of reactive oxygen and irradiation [see comments], Nature 355 (1992) 738–740.
nitrogen and radiation-induced signal transduction mechanisms, Oncogene [54] H. Nagasawa, J.B. Little, Induction of sister chromatid exchanges by extremely
22 (2003) 5734–5754. low doses of a-particles, Cancer Res. 52 (1992) 6394–6396.
[27] J.-P. Jay-Gerin, C. Ferradini, Are there protective enzymatic pathways to [55] B. Ponnaiya, M. Suzuki, C. Tsuruoka, Y. Uchihori, Y. Wei, T.K. Hei, Detection of
regulate high local nitric oxide (NO) concentrations in cells under stress chromosomal instability in bystander cells after Si490-ion irradiation, Radiat.
conditions?, Biochimie 82 (2000) 161–166. Res. 176 (2011) 280–290.
[28] Y. Frongillo, T. Goulet, M.J. Fraser, V. Cobut, J.P. Patau, J.P. Jay-Gerin, Monte [56] H. Matsumoto, M. Tomita, K. Otsuka, M. Hatashita, N. Hamada, Nitric oxide is
Carlo simulation of fast electron and proton tracks in liquid water. II. a key molecule serving as a bridge between radiation-induced bystander and
Nonhomogeneous chemistry, Radiat. Phys. Chem. 51 (1998) 245–254. adaptive responses, Curr. Mol. Pharmacol. 4 (2011) 126–134.
[29] B. Halliwell, J.M. Gutteridge, Biologically relevant metal ion-dependent [57] F.A. Cucinotta, R. Katz, J.W. Wilson, Radial distribution of electron spectra
hydroxyl radical generation: an update, FEBS Lett. 307 (1992) 108–112. from high-energy ions, Radiat. Environ. Biophys. 37 (1998) 259–265.
[30] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 4th ed., [58] D.T. Goodhead, J. Thacker, R. Cox, Effects of radiations of different qualities on
Oxford University Press, Oxford, 2007. cells: molecular mechanisms of damage and repair (Weiss Lecture), Int. J.
[31] J.A. La Verne, Radiation chemical effects of heavy ions, in: A. Mozumder, Y. Radiat. Biol. 63 (1993) 543–556.
Hatano (Eds.), Charged Particle and Photon Interactions with Matter. [59] Y. Muroya, I. Plante, E.I. Azzam, J. Meesungnoen, Y. Katsumura, J.-P. Jay-Gerin,
Chemical, Physicochemical, and Biological Consequences with Applications, High-LET ion radiolysis of water: visualization of the formation and evolution
Marcel Dekker, New York, 2004, pp. 403–429. of ion tracks and relevance to the radiation-induced bystander effect, Radiat.
[32] J. Meesungnoen, J.-P. Jay-Gerin, Radiation chemistry of liquid water with Res. 165 (2006) 485–491.
heavy ions: Monte Carlo simulation studies, in: Y. Hatano, Y. Katsumura, A. [60] I. Plante, A. Ponomarev, F.A. Cucinotta, 3D visualisation of the stochastic
Mozumder (Eds.), Charged Particle and Photon Interactions with Matter. patterns of the radial dose in nano-volumes by a Monte Carlo simulation of
Recent Advances, Applications, and Interfaces, Taylor and Francis, Boca Raton, HZE ion track structure, Radiat. Prot. Dosimetry 143 (2011) 156–161.
FL, 2011, pp. 355–400. [61] C. Wu, A.M. Parrott, C. Fu, T. Liu, S.M. Marino, V.N. Gladyshev, M.R. Jain, A.T.
[33] D.T. Goodhead, The initial physical damage produced by ionizing radiations, Baykal, Q. Li, S. Oka, J. Sadoshima, A. Beuve, W.J. Simmons, H. Li, Thioredoxin
Int. J. Radiat. Biol. 56 (1989) 623–634. 1-mediated post-translational modifications: reduction, transnitrosylation,
[34] A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W. Friedland, denitrosylation, and related proteomics methodologies, Antioxid. Redox
S. Gerardi, S. Molinelli, A. Ottolenghi, H. Paretzke, G. Simone, M.A. Tabocchini, Signal 15 (2011) 2565–2604.
DNA DSB induced in human cells by charged particles and gamma rays: [62] S.C. Formenti, S. Demaria, Local control by radiotherapy: is that all there is?,
experimental results and theoretical approaches, Int. J. Radiat. Biol. 81 (2005) Breast Cancer Res 10 (2008) 215.
841–854. [63] M.E. Robbins, W. Zhao, Chronic oxidative stress and radiation-induced late
[35] P. O’Neill, P. Wardman, Radiation chemistry comes before radiation biology, normal tissue injury: a review, Int. J. Radiat. Biol. 80 (2004) 251–259.
Int. J. Radiat. Biol. 85 (2009) 9–25. [64] T. Hayashi, Y. Kusunoki, M. Hakoda, Y. Morishita, Y. Kubo, M. Maki, F. Kasagi,
[36] E. Sahin, S. Colla, M. Liesa, J. Moslehi, F.L. Muller, M. Guo, M. Cooper, D. Kotton, K. Kodama, D.G. Macphee, S. Kyoizumi, Radiation dose-dependent increases
A.J. Fabian, C. Walkey, R.S. Maser, G. Tonon, F. Foerster, R. Xiong, Y.A. Wang, in inflammatory response markers in A-bomb survivors, Int. J. Radiat. Biol. 79
S.A. Shukla, M. Jaskelioff, E.S. Martin, T.P. Heffernan, A. Protopopov, E. (2003) 129–136.
Ivanova, J.E. Mahoney, M. Kost-Alimova, S.R. Perry, R. Bronson, R. Liao, R. [65] S. Demaria, S.C. Formenti, Sensors of ionizing radiation effects on the
Mulligan, O.S. Shirihai, L. Chin, R.A. DePinho, Telomere dysfunction induces immunological microenvironment of cancer, Int. J. Radiat. Biol. 83 (2007)
metabolic and mitochondrial compromise, Nature 470 (2011) 359–365. 819–825.
[37] K. Valerie, A. Yacoub, M.P. Hagan, D.T. Curiel, P.B. Fisher, S. Grant, P. Dent, [66] K. Neriishi, E. Nakashima, R.R. Delongchamp, Persistent subclinical
Radiation-induced cell signaling: inside-out and outside-in, Mol. Can. Ther. 6 inflammation among A-bomb survivors, Int. J. Radiat. Biol. 77 (2001) 475–482.
(2007) 789–801. [67] P.C. Dedon, S.R. Tannenbaum, Reactive nitrogen species in the chemical
[38] R.A. Roth, S.C. Sharma, R. Katz, Systematic evaluation of cellular biology of inflammation, Arch. Biochem. Biophys. 423 (2004) 12–22.
radiosensitivity parameters, Phys. Med. Biol. 21 (1976) 491–503. [68] J. Cadet, T. Douki, J.L. Ravanat, Measurement of oxidatively generated base
[39] A. Ottolenghi, M. Merzagora, H.G. Paretzke, DNA complex lesions induced by damage in cellular, DNA, Mutat. Res. 711 (2011) 3–12.
protons and alpha-particles: track structure characteristics determining [69] C.E. Redon, J.S. Dickey, A.J. Nakamura, I.G. Kareva, D. Naf, S. Nowsheen, T.B.
linear energy transfer and particle type dependence, Radiat. Environ. Kryston, W.M. Bonner, A.G. Georgakilas, O.A. Sedelnikova, Tumors induce
Biophys. 36 (1997) 97–103. complex DNA damage in distant proliferative tissues in vivo, Proc. Natl. Acad.
[40] D.T. Goodhead, Energy deposition stochastics and track structure: what about Sci. USA 107 (2010) 17992–17997.
the target?, Radiat Prot. Dosimetry 122 (2006) 3–15. [70] J.M. Hair, G.I. Terzoudi, V.I. Hatzi, K.A. Lehockey, D. Srivastava, W. Wang, G.E.
[41] J.F. Ward, Biochemistry of DNA lesions, Radiat. Res. (Suppl. 8) (1985) S103– Pantelias, A.G. Georgakilas, BRCA1 role in the mitigation of radiotoxicity and
111. chromosomal instability through repair of clustered DNA lesions, Chem. Biol.
[42] A.G. Georgakilas, From chemistry of DNA damage to repair and biological Interact. 188 (2010) 350–358.
significance Comprehending the future, Mutat. Res. 711 (2011) 1–2. [71] O.A. Sedelnikova, C.E. Redon, J.S. Dickey, A.J. Nakamura, A.G. Georgakilas,
[43] E. Gollapalle, R. Wang, R. Adetolu, D. Tsao, D. Francisco, G. Sigounas, A.G. W.M. Bonner, Role of oxidatively induced DNA lesions in human
Georgakilas, Detection of oxidative clustered DNA lesions in X-irradiated pathogenesis, Mutat. Res. 704 (2010) 152–159.
mouse skin tissues and human MCF-7 breast cancer cells, Radiat. Res. 167 [72] O.A. Martin, C.E. Redon, A.J. Nakamura, J.S. Dickey, A.G. Georgakilas, W.M.
(2007) 207–216. Bonner, Systemic DNA damage related to cancer, Cancer Res 71 (2011) 3437–
[44] D. Boucher, I. Testard, D. Averbeck, Low levels of clustered oxidative DNA 3441.
damage induced at low and high LET irradiation in mammalian cells, Radiat. [73] M.R. Jain, M. Li, W. Chen, T. Liu, S.M. De Toledo, B.N. Pandey, H. Li, B.M. Rabin,
Environ. Biophys. 45 (2006) 267–276. E.I. Azzam, In vivo space radiation-induced non-targeted responses: late
[45] N. Autsavapromporn, S.M. de Toledo, J.B. Little, J.-P. Jay-Gerin, A.L. Harris, E.I. effects on molecular signaling in mitochondria, Current Mol. Pharmacol. 4
Azzam, The role of gap junction communication and oxidative stress in the (2011) 106–114.
propagation of toxic effects among high-dose a-particle-irradiated human [74] R.H. Mole, Whole body irradiation; radiobiology or medicine?, Br J. Radiol. 26
cells, Radiat. Res. 175 (2011) 347–357. (1953) 234–241.
[46] B.M. Sutherland, P.V. Bennett, O. Sidorkina, J. Laval, Clustered DNA damages [75] K. Ohba, K. Omagari, T. Nakamura, N. Ikuno, S. Saeki, I. Matsuo, H. Kinoshita, J.
induced in isolated DNA and in human cells by low doses of ionizing Masuda, H. Hazama, I. Sakamoto, S. Kohno, Abscopal regression of
radiation, Proc Natl Acad Sci U S A 97 (2000) 103–108. hepatocellular carcinoma after radiotherapy for bone metastasis, Gut 43
[47] H. Nikjoo, D.T. Goodhead, D.E. Charlton, H.G. Paretzke, Energy deposition in (1998) 575–577.
small cylindrical targets by monoenergetic electrons, Int. J. Radiat. Biol. 60 [76] S. Gordon, Alternative activation of macrophages, Nat. Rev. Immunol. 3
(1991) 739–756. (2003) 23–35.
[48] S.M. de Toledo, N. Asaad, P. Venkatachalam, L. Li, R.W. Howell, D.R. Spitz, E.I. [77] J.B. Little, Failla Memorial Lecture. Changing views of cellular radiosensitivity,
Azzam, Adaptive responses to low-dose/low-dose-rate c rays in normal Radiat. Res. 140 (1994) 299–311.
58 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60

[78] U. Aypar, W.F. Morgan, J.E. Baulch, Radiation-induced genomic instability: are [113] Y. Shiloh, ATM and related protein kinases: safeguarding genome integrity,
epigenetic mechanisms the missing link?, Int J. Radiat. Biol. 87 (2011) 179– Nat. Rev. Cancer 3 (2003) 155–168.
191. [114] M. Ushio-Fukai, A.M. Zafari, T. Fukui, N. Ishizaka, K.K. Griendling, p22phox is
[79] Y. Ilnytskyy, O. Kovalchuk, Non-targeted radiation effects-an epigenetic a critical component of the superoxide-generating NADH/NADPH oxidase
connection, Mutat. Res. 714 (2011) 113–125. system and regulates angiotensin II-induced hypertrophy in vascular smooth
[80] Y.E. Dubrova, A.J. Jeffreys, A.M. Malashenko, Mouse minisatellite mutations muscle cells, J. Biol. Chem. 271 (1996) 23317–23321.
induced by ionizing radiation, Nat Genet 5 (1993) 92–94. [115] R. Ammendola, M.R. Ruocchio, G. Chirico, L. Russo, C. De Felice, F. Esposito, T.
[81] S.A. Lorimore, D. Mukherjee, J.I. Robinson, J.A. Chrystal, E.G. Wright, Long- Russo, F. Cimino, Inhibition of NADH/NADPH oxidase affects signal
lived inflammatory signaling in irradiated bone marrow is genome transduction by growth factor receptors in normal fibroblasts, Arch.
dependent, Cancer Res 71 (2011) 6485–6491. Biochem. Biophys. 397 (2002) 253–257.
[82] P.A. Cerutti, Prooxidant states and tumor promotion, Science 227 (1985) 375– [116] K. Irani, P.J. Goldschmidt-Clermont, Ras, superoxide and signal transduction,
381. Biochem. Pharmacol. 55 (1998) 1339–1346.
[83] W.F. Morgan, J.P. Day, M.I. Kaplan, E.M. McGhee, C.L. Limoli, Genomic [117] Y.S. Bae, S.W. Kang, M.S. Seo, I.C. Baines, E. Tekle, P.B. Chock, S.G. Rhee,
instability induced by ionizing radiation, Radiat. Res. 146 (1996) 247–258. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide.
[84] F. Weinberg, N.S. Chandel, Reactive oxygen species-dependent signaling Role in EGF receptor-mediated tyrosine phosphorylation, J. Biol. Chem. 272
regulates cancer, Cell Mol Life Sci 66 (2009) 3663–3673. (1997) 217–221.
[85] R.A. Weinberg, The biology of cancer, Garland Science, Taylor & Francis [118] S.G. Menon, E.H. Sarsour, A.L. Kalen, S. Venkataraman, M.J. Hitchler, F.E.
Group, LLC, New York, 2007. Domann, L.W. Oberley, P.C. Goswami, Superoxide signaling mediates N-
[86] T. Lindahl, Instability and decay of the primary structure of DNA, Nature 362 acetyl-L-cysteine-induced G1 arrest: regulatory role of cyclin D1 and
(1993) 709–715. manganese superoxide dismutase, Cancer Res 67 (2007) 6392–6399.
[87] B.N. Ames, Endogenous oxidative DNA damage, aging, and cancer, Free Radic. [119] T.P. Szatrowski, C.F. Nathan, Production of large amounts of hydrogen
Res. Commun. 7 (1989) 121–128. peroxide by human tumor cells, Cancer Res 51 (1991) 794–798.
[88] R. Holliday, G.W. Grigg, DNA methylation and mutation, Mutat. Res. 285 [120] K.Y. Kim, T. Rhim, I. Choi, S.S. Kim, N-acetylcysteine induces cell cycle arrest
(1993) 61–67. in hepatic stellate cells through its reducing activity, J. Biol. Chem. 276 (2001)
[89] V.A. Bohr, Repair of oxidative DNA damage in nuclear and mitochondrial 40591–40598.
DNA, and some changes with aging in mammalian cells, Free Radic Biol Med [121] M. Kyaw, M. Yoshizumi, K. Tsuchiya, Y. Izawa, Y. Kanematsu, Y. Fujita, N. Ali,
32 (2002) 804–812. K. Ishizawa, A. Yamauchi, T. Tamaki, Antioxidant effects of stereoisomers of
[90] D.M. Wilson 3rd, D. Kim, B.R. Berquist, A.J. Sigurdson, Variation in base N-acetylcysteine (NAC), L-NAC and D-NAC, on angiotensin II-stimulated MAP
excision repair capacity, Mutat. Res. 711 (2011) 100–112. kinase activation and vascular smooth muscle cell proliferation, J. Pharmacol.
[91] E.C. Friedberg, L.D. McDaniel, R.A. Schultz, The role of endogenous and Sci. 95 (2004) 483–486.
exogenous DNA damage and mutagenesis, Curr Opin Genet Dev 14 (2004) 5– [122] P. Venkatachalam, S.M. de Toledo, E.I. Azzam, Flavin-containing oxidases
10. regulate progression from G1 to S phase of the cell cycle in normal human
[92] L.E. Feinendegen, R.D. Neumann, (Eds.), Workshop Report: Cellular Responses fibroblasts, Radiat. Phys. Chem. 72 (2004) 315–321.
to Low Doses of Ionizing Radiation, United States Department of Energy and [123] R. Terasima, L.J. Tolmach, X-ray sensitivity and DNA synthesis in synchronous
the National Institutes of Health, Bethesda, 1999. populations of Hela cells, Science 140 (1963) 490–492.
[93] A.G. Georgakilas, S.M. Holt, J.M. Hair, C.W. Loftin, Measurement of [124] H. Nagasawa, C.Y. Li, C.G. Maki, A.C. Imrich, J.B. Little, Relationship between
oxidatively-induced clustered DNA lesions using a novel adaptation of radiation-induced G1 phase arrest and p53 function in human tumor cells,
single cell gel electrophoresis (comet assay), Curr. Protoc. Cell Biol. (2010). Cancer Res 55 (1995) 1842–1846.
Chapter 6. [125] S. Kar, S. Subbaram, P.M. Carrico, J.A. Melendez, Redox-control of matrix
[94] P.L. Olive, Impact of the comet assay in radiobiology, Mutat. Res. 681 (2009) metalloproteinase-1: a critical link between free radicals, matrix remodeling
13–23. and degenerative disease, Respir. Physiol. Neurobiol.iochem J 174 (2010)
[95] L.E. Feinendegen, Reactive oxygen species in cell responses to toxic agents, 299–306.
Hum. Exp. Toxicol. 21 (2002) 85–90. [126] J.K. Leach, G. Van Tuyle, P.S. Lin, R. Schmidt-Ullrich, R.B. Mikkelsen, Ionizing
[96] R.H. Burdon, V. Gill, C. Rice-Evans, Cell proliferation and oxidative stress, Free radiation-induced, mitochondria-dependent generation of reactive oxygen/
Radic. Res. Commun. 7 (1989) 149–159. nitrogen, Cancer Res 61 (2001) 3894–3901.
[97] W. Droge, Free radicals in the physiological control of cell function, Physiol. [127] A. Boveris, B. Chance, The mitochondrial generation of hydrogen peroxide.
Rev. 82 (2002) 47–95. General properties and effect of hyperbaric oxygen, Biochem. J. 134 (1973)
[98] E.H. Sarsour, M.G. Kumar, L. Chaudhuri, A.L. Kalen, P.C. Goswami, Redox 707–716.
control of the cell cycle in health and disease, Antioxid. Redox Signal 11 [128] E. Cadenas, K.J. Davies, Mitochondrial free radical generation, oxidative stress,
(2009) 2985–3011. and aging, Free Radic Biol Med 29 (2000) 222–230.
[99] F.Q. Schafer, G.R. Buettner, Redox environment of the cell as viewed through [129] B.M. Babior, NADPH oxidase: an update, Blood 93 (1999) 1464–1476.
the redox state of the glutathione disulfide/glutathione couple, Free Radic [130] M. Los, H. Schenk, K. Hexel, P.A. Baeuerle, W. Droge, K. Schulze-Osthoff, IL-2
Biol Med 30 (2001) 1191–1212. gene expression and NF-kappa B activation through CD28 requires reactive
[100] R.G. Allen, M. Tresini, Oxidative stress and gene regulation, Free Radic Biol oxygen production by 5-lipoxygenase, Embo J. 14 (1995) 3731–3740.
Med 28 (2000) 463–499. [131] A. Boveris, E. Cadenas, A.O. Stoppani, Role of ubiquinone in the mitochondrial
[101] P. Herrlich, F.D. Bohmer, Redox regulation of signal transduction in generation of hydrogen peroxide, Biochem. J. 156 (1976) 435–444.
mammalian cells, Biochem. Pharmacol. 59 (2000) 35–41. [132] A. Claude, The coming of age of the cell, Science 189 (1975) 433–435.
[102] C. Meplan, M.J. Richard, P. Hainaut, Redox signalling and transition metals in [133] P.C. Champe, R.A. Harvey, D.R. Ferrier, Biochemistry, 4th ed., Wolters Kluwer
the control of the p53 pathway, Biochem. Pharmacol. 59 (2000) 25–33. Health/Lippincott Williams & Wilkins, Philadelphia, 2008.
[103] B.D. Price, S.K. Calderwood, Gadd45 and Gadd153 messenger RNA levels are [134] J.F. Turrens, A. Alexandre, A.L. Lehninger, Ubisemiquinone is the electron
increased during hypoxia and after exposure of cells to agents which elevate donor for superoxide formation by complex III of heart mitochondria, Arch.
the levels of the glucose-regulated proteins, Cancer Res 52 (1992) 3814– Biochem. Biophys. 237 (1985) 408–414.
3817. [135] H. Nohl, W. Jordan, The mitochondrial site of superoxide formation, Biochem.
[104] J. Lopez-Barneo, J.R. Lopez-Lopez, J. Urena, C. Gonzalez, Chemotransduction in Biophys. Res. Commun. 138 (1986) 533–539.
the carotid body: K+ current modulated by PO2 in type I chemoreceptor cells, [136] A. Boveris, Mitochondrial production of superoxide radical and hydrogen
Science 241 (1988) 580–582. peroxide, Adv. Exp. Med. Biol. 78 (1977) 67–82.
[105] K. Schulze-Osthoff, M. Bauer, M. Vogt, S. Wesselborg, P.A. Baeuerle, Reactive [137] V. Goossens, K. De Vos, D. Vercammen, M. Steemans, K. Vancompernolle, W.
Oxygen Intermediates as Primary Signals and Second Mesengers in the Fiers, P. Vandenabeele, J. Grooten, Redox regulation of TNF signaling,
Activation of transcription Factors, in: H.J. Forman, E. Cadenas (Eds.), Biofactors 10 (1999) 145–156.
Oxidative Stress and Signal Transduction, Chapman & Hall, New York, 1997, [138] T.L. Vanden Hoek, L.B. Becker, Z. Shao, C. Li, P.T. Schumacker, Reactive oxygen
pp. 239–259. species released from mitochondria during brief hypoxia induce
[106] A. Petkau, Scientific basis for the clinical use of superoxide dismutase, Cancer preconditioning in cardiomyocytes, J. Biol. Chem. 273 (1998) 18092–18098.
Treat Rev. 13 (1986) 17–44. [139] E. Werner, Z. Werb, Integrins engage mitochondrial function for signal
[107] T. Finkel, Redox-dependent signal transduction, FEBS Lett. 476 (2000) 52–54. transduction by a mechanism dependent on Rho GTPases, J Cell Biol 158
[108] G.V. Rumyantseva, L.M. Weiner, Y.N. Molin, V.G. Budker, Permeation of (2002) 357–368.
liposome membrane by superoxide radical, FEBS Lett. 108 (1979) 477–480. [140] C. Sidoti-de Fraisse, V. Rincheval, Y. Risler, B. Mignotte, J.L. Vayssiere, TNF-
[109] R.E. Lynch, I. Fridovich, Permeation of the erythrocyte stroma by superoxide alpha activates at least two apoptotic signaling cascades, Oncogene 17 (1998)
radical, J. Biol. Chem. 253 (1978) 4697–4699. 1639–1651.
[110] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology of ageing, [141] L. Malakhova, V.G. Bezlepkin, V. Antipova, T. Ushakova, L. Fomenko, N. Sirota,
Nature 408 (2000) 239–247. A.I. Gaziev, The increase in mitochondrial DNA copy number in the tissues of
[111] S.G. Menon, E.H. Sarsour, D.R. Spitz, R. Higashikubo, M. Sturm, H. Zhang, P.C. gamma-irradiated mice, Cell Mol Biol Lett 10 (2005) 721–732.
Goswami, Redox regulation of the G1 to S phase transition in the mouse [142] V.N. Antipova, L.V. Malakhova, V.G. Bezlepkin, [Detection of large deletions of
embryo fibroblast cell cycle, Cancer Res 63 (2003) 2109–2117. mitochondrial DNA in tissues of mice exposed to X-rays], Biofizika 56 (2011)
[112] P. Venkatachalam, S.M. de Toledo, B.N. Pandey, L.A. Tephly, A.B. Carter, J.B. 439–445.
Little, D.R. Spitz, E.I. Azzam, Regulation of normal cell cycle progression by [143] Q. Chen, Y.C. Chai, S. Mazumder, C. Jiang, R.M. Macklis, G.M. Chisolm, A.
flavin-containing oxidaseso, Oncogene 27 (2008) 20–31. Almasan, The late increase in intracellular free radical oxygen species during
 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60 59

apoptosis is associated with cytochrome c release, caspase activation, and [170] J.M. Berg, J.L. Tymoczko, L. Stryer, Biochemistry, 6th ed., W. H. Freeman and
mitochondrial dysfunction, Cell Death Differ. 10 (2003) 323–334. Company, New York, NY, 2006.
[144] M.A. Chaudhry, R.A. Omaruddin, Mitochondrial Gene Expression in Directly [171] S. Kawamura, D. Takada, K. Watanabe, J. Hayashi, K. Hayakawa, M. Akashi,
Irradiated and Nonirradiated Bystander Cells, Cancer Biother. Radiopharm. Role of mitochondrial DNA in cells exposed to irradiation: generation of
(2011). reactive oxygen species (ROS) is required for G2 checkpoint upon irradiation,
[145] H.W. Chiu, W. Lin, S.Y. Ho, Y.J. Wang, Synergistic effects of arsenic trioxide J. Health Sci. 51 (2005) 385–393.
and radiation in osteosarcoma cells through the induction of both autophagy [172] V.I. Grishko, L.I. Rachek, D.R. Spitz, G.L. Wilson, S.P. LeDoux, Contribution of
and apoptosis, Radiat. Res. 175 (2011) 547–560. mitochondrial DNA repair to cell resistance from oxidative stress, J. Biol.
[146] S.L. Lomonaco, S. Finniss, C. Xiang, A. Decarvalho, F. Umansky, S.N. Kalkanis, T. Chem. 280 (2005) 8901–8905.
Mikkelsen, C. Brodie, The induction of autophagy by gamma-radiation [173] P.A. Mason, E.C. Matheson, A.G. Hall, R.N. Lightowlers, Mismatch repair
contributes to the radioresistance of glioma stem cells, Int J Cancer 125 activity in mammalian mitochondria, Nucl. Acids Res. 31 (2003) 1052–1058.
(2009) 717–722. [174] M.A. Graziewicz, B.J. Day, W.C. Copeland, The mitochondrial DNA polymerase
[147] O. Cuisnier, R. Serduc, J.P. Lavieille, M. Longuet, E. Reyt, C. Riva, Chronic as a target of oxidative damage, Nucl. Acids Res. 30 (2002) 2817–2824.
hypoxia protects against gamma-irradiation-induced apoptosis by inducing [175] T.H. Zastawny, M. Dabrowska, T. Jaskolski, M. Klimarczyk, L. Kulinski, A.
bcl-2 up-regulation and inhibiting mitochondrial translocation and Koszela, M. Szczesniewicz, M. Sliwinska, P. Witkowski, R. Olinski, Comparison
conformational change of bax protein, Int. J. Oncol. 23 (2003) 1033–1041. of oxidative base damage in mitochondrial and nuclear DNA, Free Radic Biol
[148] A. Ogura, S. Oowada, Y. Kon, A. Hirayama, H. Yasui, S. Meike, S. Kobayashi, M. Med 24 (1998) 722–725.
Kuwabara, O. Inanami, Redox regulation in radiation-induced cytochrome c [176] D.C. Wallace, Diseases of the mitochondrial DNA, Annu. Rev. Biochem. 61
release from mitochondria of human lung carcinoma A549 cells, Cancer Lett (1992) 1175–1212.
277 (2009) 64–71. [177] C. Richter, J.W. Park, B.N. Ames, Normal oxidative damage to mitochondrial
[149] C. Cherbonnel-Lasserre, S. Gauny, A. Kronenberg, Suppression of apoptosis by and nuclear DNA is extensive, Proc. Natl. Acad. Sci. USA 85 (1988) 6465–
Bcl-2 or Bcl-xL promotes susceptibility to mutagenesis, Oncogene 13 (1996) 6467.
1489–1497. [178] W. Wang, Y. Esbensen, D. Kunke, R. Suganthan, L. Rachek, M. Bjoras, L. Eide,
[150] S. Chen, Y. Zhao, G. Zhao, W. Han, L. Bao, K.N. Yu, L. Wu, Up-regulation of ROS Mitochondrial DNA damage level determines neural stem cell differentiation
by mitochondria-dependent bystander signaling contributes to genotoxicity fate, J. Neurosci. 31 (2011) 9746–9751.
of bystander effects, Mutat. Res. 666 (2009) 68–73. [179] D. Bogenhagen, D.A. Clayton, Mouse L cell mitochondrial DNA molecules are
[151] P. Maguire, C. Mothersill, C. Seymour, F.M. Lyng, Medium from irradiated selected randomly for replication throughout the cell cycle, Cell 11 (1977)
cells induces dose-dependent mitochondrial changes and BCL2 responses in 719–727.
unirradiated human keratinocytes, Radiat. Res. 163 (2005) 384–390. [180] A.I. Gaziev, G.O. Shaikhaev, [Ionizing radiation can activate the insertion of
[152] S. Rajendran, S.H. Harrison, R.A. Thomas, J.D. Tucker, The role of mitochondria mitochondrial DNA fragments in the nuclear genome] Radiatsionnaia
in the radiation-induced bystander effect in human lymphoblastoid cells, biologiia, radioecologiia/Rossiiskaia akademiia nauk 47 (2007) 673–683.
Radiat. Res. 175 (2011) 159–171. [181] S. Prithivirajsingh, M.D. Story, S.A. Bergh, F.B. Geara, K.K. Ang, S.M. Ismail,
[153] L. Tartier, S. Gilchrist, S. Burdak-Rothkamm, M. Folkard, K.M. Prise, C.W. Stevens, T.A. Buchholz, W.A. Brock, Accumulation of the common
Cytoplasmic irradiation induces mitochondrial-dependent 53BP1 protein mitochondrial DNA deletion induced by ionizing radiation, FEBS Lett. 571
relocalization in irradiated and bystander cells, Cancer Res 67 (2007) 5872– (2004) 227–232.
5879. [182] B. Chen, Y. Zhong, W. Peng, Y. Sun, Y.J. Hu, Y. Yang, W.J. Kong, Increased
[154] H. Zhou, V.N. Ivanov, Y.C. Lien, M. Davidson, T.K. Hei, Mitochondrial function mitochondrial DNA damage and decreased base excision repair in the
and nuclear factor-kappaB-mediated signaling in radiation-induced auditory cortex of D-galactose-induced aging rats, Mol. Biol. Rep. 38 (2011)
bystander effects, Cancer Res 68 (2008) 2233–2240. 3635–3642.
[155] M. He, M. Zhao, B. Shen, K.M. Prise, C. Shao, Radiation-induced intercellular [183] S. Biskup, D.J. Moore, Detrimental deletions: mitochondria, aging and
signaling mediated by cytochrome-c via a p53-dependent pathway in Parkinson’s disease, Bioessays 28 (2006) 963–967.
hepatoma cells, Oncogene 30 (2011) 1947–1955. [184] P.E. Thorsness, T.D. Fox, Escape of DNA from mitochondria to the nucleus in
[156] K.M. Choi, C.M. Kang, E.S. Cho, S.M. Kang, S.B. Lee, H.D. Um, Ionizing Saccharomyces cerevisiae, Nature 346 (1990) 376–379.
radiation-induced micronucleus formation is mediated by reactive oxygen [185] M. Ricchetti, F. Tekaia, B. Dujon, Continued colonization of the human
species that are produced in a manner dependent on mitochondria, Nox1, genome by mitochondrial DNA, PLoS Biol. 2 (2004) E273.
and JNK, Oncol. Rep. 17 (2007) 1183–1188. [186] M. Ricchetti, C. Fairhead, B. Dujon, Mitochondrial DNA repairs double-strand
[157] C.L. Limoli, E. Giedzinski, W.F. Morgan, S.G. Swarts, G.D. Jones, W. Hyun, breaks in yeast chromosomes, Nature 402 (1999) 96–100.
Persistent oxidative stress in chromosomally unstable cells, Cancer Res 63 [187] M. Patrushev, V. Kasymov, V. Patrusheva, T. Ushakova, V. Gogvadze, A.I.
(2003) 3107–3111. Gaziev, Release of mitochondrial DNA fragments from brain mitochondria of
[158] C. Du, Z. Gao, V.A. Venkatesha, A.L. Kalen, L. Chaudhuri, D.R. Spitz, J.J. Cullen, irradiated mice, Mitochondrion 6 (2006) 43–47.
L.W. Oberley, P.C. Goswami, Mitochondrial ROS and radiation induced [188] J.W. Shay, H. Werbin, New evidence for the insertion of mitochondrial DNA
transformation in mouse embryonic fibroblasts, Cancer Biol. Ther. 8 (2009) into the human genome: significance for cancer and aging, Mutat. Res. 275
1962–1971. (1992) 227–235.
[159] J.R. Fike, S. Rosi, C.L. Limoli, Neural precursor cells and central nervous system [189] X. Cheng, A.S. Ivessa, The migration of mitochondrial DNA fragments to the
radiation sensitivity, Semin. Radiat. Oncol. 19 (2009) 122–132. nucleus affects the chronological aging process of Saccharomyces cerevisiae,
[160] N. Autsavapromporn, S.M. de Toledo, M. Buonanno, J.P. Jay-Gerin, A.L. Harris, Aging Cell 9 (2010) 919–923.
E.I. Azzam, Intercellular communication amplifies stressful effects in high- [190] I.E. Scheffler, Mitochondria, 2nd ed., Wiley-Liss, Hoboken, NJ, 2008.
charge, high-energy (HZE) particle-irradiated human cells, J. Radiat. Res. [191] D. Pain, H. Murakami, G. Blobel, Identification of a receptor for protein import
(Tokyo). 52 (2011) 408–414. into mitochondria, Nature 347 (1990) 444–449.
[161] B.N. Pandey, D.A. Gordon, S.M. de Toledo, D. Pain, E.I. Azzam, Normal human [192] K.M. Davey, J.S. Parboosingh, D.R. McLeod, A. Chan, R. Casey, P. Ferreira, F.F.
fibroblasts exposed to high or low dose ionizing radiation: differential effects Snyder, P.J. Bridge, F.P. Bernier, Mutation of DNAJC19, a human homologue of
on mitochondrial protein import and membrane potential, Antioxid. Redox yeast inner mitochondrial membrane co-chaperones, causes DCMA
Signal 8 (2006) 1253–1261. syndrome, a novel autosomal recessive Barth syndrome-like condition, J.
[162] G.J. Kim, G.M. Fiskum, W.F. Morgan, A role for mitochondrial dysfunction in Med. Genet. 43 (2006) 385–393.
perpetuating radiation-induced genomic instability, Cancer Res 66 (2006) [193] A. Huckriede, A. Heikema, K. Sjollema, P. Briones, E. Agsteribbe, Morphology
10377–10383. of the mitochondria in heat shock protein 60 deficient fibroblasts from
[163] D. Harman, The biologic clock: the mitochondria?, J Am. Geriatr. Soc. 20 mitochondrial myopathy patients. Effects of stress conditions, Virchows.
(1972) 145–147. Arch. 427 (1995) 159–165.
[164] I.J. Holt, A.E. Harding, J.A. Morgan-Hughes, Deletions of muscle mitochondrial [194] U. Rothbauer, S. Hofmann, N. Muhlenbein, S.A. Paschen, K.D. Gerbitz, W.
DNA in patients with mitochondrial myopathies, Nature 331 (1988) 717– Neupert, M. Brunner, M.F. Bauer, Role of the deafness dystonia peptide 1
719. (DDP1) in import of human Tim23 into the inner membrane of mitochondria,
[165] D.C. Wallace, G. Singh, M.T. Lott, J.A. Hodge, T.G. Schurr, A.M. Lezza, L.J. Elsas J. Biol. Chem. 276 (2001) 37327–37334.
2nd, E.K. Nikoskelainen, Mitochondrial DNA mutation associated with Leber’s [195] F. Takakubo, P. Cartwright, N. Hoogenraad, D.R. Thorburn, F. Collins, T.
hereditary optic neuropathy, Science 242 (1988) 1427–1430. Lithgow, H.H. Dahl, An amino acid substitution in the pyruvate
[166] D.C. Wallace, X.X. Zheng, M.T. Lott, J.M. Shoffner, J.A. Hodge, R.I. Kelley, C.M. dehydrogenase E1 alpha gene, affecting mitochondrial import of the
Epstein, L.C. Hopkins, Familial mitochondrial encephalomyopathy (MERRF): precursor protein, Am. J. Hum. Genet. 57 (1995) 772–780.
genetic, pathophysiological, and biochemical characterization of a [196] C. Wang, S. Rajput, K. Watabe, D.F. Liao, D. Cao, Acetyl-CoA carboxylase-a as a
mitochondrial DNA disease, Cell 55 (1988) 601–610. novel target for cancer therapy, Frontiers in bioscience (Scholar edition), vol.
[167] S. DiMauro, M. Hirano, Pathogenesis and treatment of mitochondrial 2, pp. 515–526.
disorders, Adv. Exp. Med. Biol. 652 (2009) 139–170. [197] M.K. Kwak, K. Itoh, M. Yamamoto, T.W. Kensler, Enhanced expression of the
[168] K.B. Beckman, B.N. Ames, The free radical theory of aging matures, Physiol. transcription factor Nrf2 by cancer chemopreventive agents: role of
Rev. 78 (1998) 547–581. antioxidant response element-like sequences in the nrf2 promoter, Mol.
[169] C.Y. Lu, H.C. Lee, H.J. Fahn, Y.H. Wei, Oxidative damage elicited by imbalance Cell Biol. 22 (2002) 2883–2892.
of free radical scavenging enzymes is associated with large-scale mtDNA [198] M.K. Kwak, N. Wakabayashi, K. Itoh, H. Motohashi, M. Yamamoto, T.W.
deletions in aging human skin, Mutat. Res. 423 (1999) 11–21. Kensler, Modulation of gene expression by cancer chemopreventive
60 E.I. Azzam et al. / Cancer Letters 327 (2012) 48–60

dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene [224] S.M. de Toledo, E.I. Azzam, Adaptive and bystander responses in human and
clusters for cell survival, J. Biol. Chem. 278 (2003) 8135–8145. rodent cell cultures exposed to low level ionizing radiation: the impact of
[199] T.A. Rouault, W.H. Tong, Iron-sulphur cluster biogenesis and mitochondrial linear energy transfer, Dose Response 4 (2006) 291–301.
iron homeostasis, Nature Rev. 6 (2005) 345–351. [225] A.L. Brooks, Paradigm shifts in radiation biology: their impact on intervention
[200] M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat. for radiation-induced disease, Radiat. Res. 164 (2005) 454–461.
Res. 147 (1985) 29–36. [226] W.F. Morgan, Non-targeted and delayed effects of exposure to ionizing
[201] A.L. Bulteau, M. Ikeda-Saito, L.I. Szweda, Redox-dependent modulation of radiation: II. Radiation-induced genomic instability and bystander effects
aconitase activity in intact mitochondria, Biochemistry 42 (2003) 14846– in vivo, clastogenic factors and transgenerational effects, Radiat. Res. 159
14855. (2003) 581–596.
[202] Managing Space Radiation Risk in the New Era of Space Exploration, in: [227] T. Lindahl, DNA repair enzymes acting on spontaneous lesions in DNA, in:
National Research Council of the National Academies (Ed.), Springer, berlin, W.W. Nichols, D.G. Murphy, (Eds.), DNA Repair Processes, Symposia
2008. Specialists, Miami, 1977, pp. 225–240.
[203] BEIR-IV, Health Risks of Radon and Other Internally Deposited Alpha- [228] L.E. Feinendegen, A.L. Brooks, W.F. Morgan, Biological consequences and
Emitters (BEIR IV), Springer, Berlin, 1988. health risks of low-level exposure to ionizing radiation: commentary on the
[204] D.A. Bota, K.J. Davies, Lon protease preferentially degrades oxidized workshop, Health Phys 100 (2011) 247–259.
mitochondrial aconitase by an ATP-stimulated mechanism, Nat. Cell Biol. 4 [229] M. Ina, K. Sakai, Activation of immunological network by chronic low-dose-
(2002) 674–680. rate irradiation in wild-type mouse strains: analysis of immune cell
[205] C.K. Suzuki, K. Suda, N. Wang, G. Schatz, Requirement for the yeast gene LON populations and surface molecules, Int. J. Radiat. Biol. 81 (2005) 721–729.
in intramitochondrial proteolysis and maintenance of respiration, Science [230] E.I. Azzam, S.M. de Toledo, G.P. Raaphorst, R.E. Mitchel, Low-dose ionizing
264 (1994) 891. radiation decreases the frequency of neoplastic transformation to a level
[206] D. Dayal, S.M. Martin, K.M. Owens, N. Aykin-Burns, Y. Zhu, A. Boominathan, D. below the spontaneous rate in C3H 10T1/2 cells, Radiat. Res. 146 (1996) 369–
Pain, C.L. Limoli, P.C. Goswami, F.E. Domann, D.R. Spitz, Mitochondrial 373.
complex II dysfunction can contribute significantly to genomic instability [231] E.I. Azzam, G.P. Raaphorst, R.E. Mitchel, Radiation-induced adaptive response
after exposure to ionizing radiation, Radiat. Res. 172 (2009) 737–745. for protection against micronucleus formation and neoplastic transformation
[207] J. Fang, A. Holmgren, Inhibition of thioredoxin and thioredoxin reductase by in C3H 10T1/2 mouse embryo cells, Radiat. Res. 138 (1994) S28–31.
4-hydroxy-2-nonenal in vitro and in vivo, J Am Chem Soc 128 (2006) 1879– [232] R.E. Mitchel, J.S. Jackson, R.A. McCann, D.R. Boreham, The adaptive response
1885. modifies latency for radiation-induced myeloid leukemia in CBA/H mice,
[208] T. Nystrom, Role of oxidative carbonylation in protein quality control and Radiat. Res. 152 (1999) 273–279.
senescence, Embo J. 24 (2005) 1311–1317. [233] G. Olivieri, J. Bodycote, S. Wolff, Adaptive response of human lymphocytes to
[209] C. Ziegler, J.M. Wessels, Investigation of lipid peroxidation in liposomes low concentrations of radioactive thymidine, Science 223 (1984) 594–597.
induced by heavy ion irradiation, Radiat. Environ. Biophys. 37 (1998) 95–100. [234] J.L. Redpath, R.J. Antoniono, Induction of an adaptive response against
[210] G.T. Wondrak, D. Cervantes-Laurean, E.L. Jacobson, M.K. Jacobson, Histone spontaneous neoplastic transformation in vitro by low-dose gamma
carbonylation in vivo and in vitro, Biochem. J. 351 Pt 3 (2000) 769–777. radiation, Radiat. Res. 149 (1998) 517–520.
[211] S.C. Lu, Regulation of glutathione synthesis, Curr. Top Cell Regul. 36 (2000) [235] X.C. Le, J.Z. Xing, J. Lee, S.A. Leadon, M. Weinfeld, Inducible repair of thymine
95–116. glycol detected by an ultrasensitive assay for DNA damage, Science 280
[212] B. Chance, H. Sies, A. Boveris, Hydroperoxide metabolism in mammalian (1998) 1066–1069.
organs, Physiol. Rev. 59 (1979) 527–605. [236] D.J. Brenner, R. Doll, D.T. Goodhead, E.J. Hall, C.E. Land, J.B. Little, J.H. Lubin,
[213] M.W. Epperly, H. Wang, J.A. Jones, T. Dixon, C.A. Montesinos, J.S. Greenberger, D.L. Preston, R.J. Preston, J.S. Puskin, E. Ron, R.K. Sachs, J.M. Samet, R.B. Setlow,
Antioxidant-chemoprevention diet ameliorates late effects of total-body M. Zaider, Cancer risks attributable to low doses of ionizing radiation:
irradiation and supplements radioprotection by MnSOD-plasmid liposome Assessing what we really know, Proc. Natl. Acad. Sci. USA 100 (2003) 13761–
administration, Radiat. Res. 175 (2011) 759–765. 13766.
[214] J.M. McCord, I. Fridovich, Superoxide dismutase. An enzymic function for [237] D.J. Brenner, C.D. Elliston, The potential impact of bystander effects on
erythrocuprein (hemocuprein), J. Biol. Chem. 244 (1969) 6049–6055. radiation risks in a Mars mission, Radiat. Res. 156 (2001) 612–617.
[215] C.J. Weydert, T.A. Waugh, J.M. Ritchie, K.S. Iyer, J.L. Smith, L. Li, D.R. Spitz, L.W. [238] G.W. Barendsen, Differences between biological effects of high LET and low
Oberley, Overexpression of manganese or copper-zinc superoxide dismutase LET radiations in relation to their application in radiotherapy, Radiol Clin
inhibits breast cancer growth, Free Radic Biol Med 41 (2006) 226–237. (Basel) 46 (1977) 380–389.
[216] J.M. McCord, Superoxide dismutase, lipid peroxidation, and bell-shaped dose [239] A.L. Brooks, Chromosome damage in liver cells from low dose rate alpha, beta,
response curves, Dose Response 6 (2008) 223–238. and gamma irradiation: Derivation of RBE, Science 190 (1975) 1090–1092.
[217] S. Kojima, H. Ishida, M. Takahashi, K. Yamaoka, Elevation of glutathione [240] F.A. Cucinotta, H. Wu, M.R. Shavers, K. George, Radiation dosimetry and
induced by low-dose gamma rays and its involvement in increased natural biophysical models of space radiation effects, Gravit. Space Biol. Bull. 16
killer activity, Radiat. Res. 157 (2002) 275–280. (2003) 11–18.
[218] A. Bravard, C. Luccioni, E. Moustacchi, O. Rigaud, Contribution of antioxidant [241] J. Meesungnoen, M. Benrahmoune, A. Filali-Mouhim, S. Mankhetkorn, J.P. Jay-
enzymes to the adaptive response to ionizing radiation of human Gerin, Monte Carlo calculation of the primary radical and molecular yields of
lymphoblasts, Int. J. Radiat. Biol. 75 (1999) 639–645. liquid water radiolysis in the linear energy transfer range 0.3-6.5 keV/um:
[219] E.I. Azzam, S.M. de Toledo, D.R. Spitz, J.B. Little, Oxidative metabolism application to 137Cs gamma rays, Radiat. Res. 155 (2001) 269–278 (Erratum
modulates signal transduction and micronucleus formation in bystander cells Published in Radiat. Res. 155 (2001) 2873).
from alpha-particle-irradiated normal human fibroblast cultures, Cancer Res. [242] S.M. Poulose, D.F. Bielinski, K. Carrihill-Knoll, B.M. Rabin, B. Shukitt-Hale,
62 (2002) 5436–5442. Exposure to 16O-particle radiation causes aging-like decrements in rats
[220] P.K. Narayanan, E.H. Goodwin, B.E. Lehnert, Alpha particles initiate biological through increased oxidative stress, inflammation and loss of autophagy,
production of superoxide anions and hydrogen peroxide in human cells, Radiat. Res. 176 (2011) 761–769.
Cancer Res. 57 (1997) 3963–3971. [243] P.T. Schumacker, Reactive oxygen species in cancer cells: live by the sword,
[221] P.K. Narayanan, K.E.A. LaRue, E.H. Goodwin, B.E. Lehnert, Alpha particles die by the sword, Cancer Cell 10 (2006) 175–176.
induce the production of interleukin-8 by human cells, Radiat. Res. 152 [244] S.E. Calvo, V.K. Mootha, The mitochondrial proteome and human disease,
(1999) 57–63. Annu. Rev. Genomics. Hum. Genet. 11 (2010) 25–44.
[222] L.W. Oberley, T.D. Oberley, Role of antioxidant enzymes in cell [245] J.B. Brocard, G.L. Rintoul, I.J. Reynolds, New perspectives on mitochondrial
immortalization and transformation, Mol Cell Biochem 84 (1988) 147–153. morphology in cell function, Biol Cell 95 (2003) 239–242.
[223] E.I. Azzam, J.B. Little, The radiation-induced bystander effect: evidence and [246] L. Griparic, A.M. van der Bliek, The many shapes of mitochondrial
significance, Hum. Exp. Toxicol. 23 (2004) 61–65. membranes, Traffic 2 (2001) 235–244.