Review Article

Effects of ionizing radiation on mitochondria

Winnie Wai-Ying Kam
a,b,n
, Richard B. Banati
a,b,c
a
Australian Nuclear Science and Technology Organisation, Lucas Heights, Sydney, New South Wales 2234, Australia
b
Medical Radiation Sciences, Faculty of Health Sciences, University of Sydney, Cumberland, Sydney, New South Wales 2141, Australia
c
National Imaging Facility at Brain and Mind Research Institute (BMRI), University of Sydney, Camperdown, Sydney, New South Wales 2050, Australia
a r t i c l e i n f o
Article history:
Received 28 February 2013
Received in revised form
16 July 2013
Accepted 16 July 2013
Available online 26 July 2013
Keywords:
Mitochondria
Nucleus
Ionizing radiation
DNA
RNA
Copy number
Mammalian cells
Radiation response
Manganese superoxide dismutase
Reactive oxygen species (ROS)
Superoxide
Common deletion
Oxidative phosphorylation (OXPHOS)
Electron transport chain
p
0
cells
a b s t r a c t
The current concept of radiobiology posits that damage to the DNA in the cell nucleus is the primary
cause for the detrimental effects of radiation. However, emerging experimental evidence suggests that
this theoretical framework is insufcient for describing extranuclear radiation effects, particularly the
response of the mitochondria, an important site of extranuclear, coding DNA. Here, we discuss
experimental observations of the effects of ionizing radiation on the mitochondria at (1) the DNA and
(2) functional levels. The roles of mitochondria in (3) oxidative stress and (4) late radiation effects are
discussed. In this review, we summarize the current understanding of targets for ionizing radiation
outside the cell nucleus. Available experimental data suggest that an increase in the tumoricidal efcacy
of radiation therapy might be achievable by targeting mitochondria. Likewise, more specic protection of
mitochondria and its coding DNA should reduce damage to healthy cells exposed to ionizing radiation.
Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.
Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Mitochondrial DNA after ionizing radiation stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608
Susceptibility of nuclear and mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
Common deletionmtDNA
4977
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
A change in the mitochondrial DNA copy number after ionizing irradiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
p
0
cells in radiation research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
Mitochondrial function after ionizing radiation stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
The effects of radiation on the electron transport chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
The effects of radiation on oxidative phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
A change in the mitochondrial mass after ionizing irradiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
Radiation-induced oxidative stress and antioxidant enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
The source of radiation-induced oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
The role of manganese superoxide dismutase in radioprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
The localization of antioxidant enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/freeradbiomed
Free Radical Biology and Medicine
0891-5849/$ - see front matter Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.freeradbiomed.2013.07.024
Abbreviations: OXPHOS, Oxidative phosphorylation; PGC-1, Peroxisome-
proliferator-activated receptor- coactivator 1; ROS, Reactive oxygen species
n
Corresponding author at: Australian Nuclear Science and Technology
Organisation, Lucas Heights, Menai, Sydney, NSW 2234, Australia. Fax: +612 9717 9262.
E-mail addresses: wik@ansto.gov.au, winikam@gmail.com (W.W.-Y. Kam).
Free Radical Biology and Medicine 65 (2013) 607619
Mitochondria, the cell nucleus, and late ionizing radiation effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
Signal propagation between mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613
Signal propagation between mitochondria and the cell nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
Delayed increase in radiation-induced mitochondrial oxidative stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
Mitochondrial dysfunction and late radiation effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
Introduction
The main target of ionizing radiation damage is believed to be
the DNA in the cell nucleus [1]. However, the validity of the
current radiation damage models has been challenged [2]. The
discovery of nontargeted phenomena, such as radiation-induced
genomic instability in the progeny of cells that survive after
irradiation [3,4] and bystander effects on cells that have not
directly been exposed to radiation [5], call this central dogma of
radiation biology further into question [1].
There are reports of the effects of radiation on cell organelles
other than the nucleus [615]. It has been suggested that these
extranuclear effects are not subsequent to nuclear responses to
radiation but are instead due to the direct effect of radiation on
other organelles [12,13,16]. Mitochondria may account for up to 30%
of the total cell volume (e.g., in lymphocytes [17]). Notably, mito-
chondria are the only sites where extranuclear DNA resides [1820].
Ionizing radiation can induce various lesions in the circular mito-
chondrial DNA, such as strand breaks [21], base mismatches [22], and
large deletions [23], which are also observed in nuclear DNA
[10,24,25]. Therefore, mitochondria are likely to be a major target
of ionizing radiation in addition to the cell nucleus [26,27].
Ionizing radiation alters mitochondrial functions [28,29], increases
mitochondrial oxidative stress [3033], and induces apoptosis [3437].
Radiation causes specic mitochondrial gene expression changes that
are related to cell survival [38], and mitochondria have been reported
as the primary target for radiation-induced apoptosis [39]. These
organelles may also have a role in radiation-induced intra- [40] and
intercell [8,4143] signaling. Furthermore, a subcellular proteomic
analysis revealed that proteins involved in processes such as energy
metabolism and antioxidant response are regulated after ionizing
radiation exposure in vivo [44]. The known involvement of mitochon-
dria in these responses/processes, therefore, suggests a role of
mitochondria in the radiation response. Notably, the aforementioned
mitochondrial responses cannot be fully accounted for in the current
model of radiation effect estimation, which predominantly focuses on
the cell nucleus and its genetic material [17,45].
The number of studies investigating the effects of ionizing
radiation on the mitochondria is far less than that on the cell nucleus.
Specically, a citation analysis using Web of Science on the published
literature from 1990 to 2013 shows that there are 9 times more
original Articles with the keywords nuclen AND ionizing radia-
tion AND DNA damage NOT nuclear energy (2214 results), than
with the keywords mitochondrn AND ionizing radiation AND
DNA damage NOT nuclear energy (246 results) (Fig. 1). In this
review, we summarize some of the reported mitochondrial responses
to ionizing radiation and point to some avenues for future investiga-
tion of the role of mitochondria in the induction of the overall cell
response to ionizing radiation.
Mitochondrial DNA after ionizing radiation stress
The genetic information of an organism is mostly encoded by the
DNA inside the cell nucleus [46]. Some other vital genes are encoded
by DNA stored inside the mitochondria. Mitochondrial DNA contains
13 genes which code for the subunits of the electron transport chain
enzyme complexes (Complexes I, II, III, and IV) and the ATP synthase
[1820]. These enzymes are important for respiration, adenosine
triphosphate (ATP) synthesis, and the regulation of many cellular
pathways within the cell [47].
Changes in the mitochondrial DNA content, measured as the
mitochondrial to nuclear DNA (or protein) ratio, can be used as a
readout to measure drug [48] or radiation [49] response, for
mitochondrial disease screening [50,51], mitochondrial dysfunction
[52], and fetal developmental assessment [53]. Furthermore, mito-
chondrial DNA is commonly used for evolutionary analysis because it
evolves 10 times faster than the nuclear DNA of the same organism
[54]. There is an unequivocal link between mitochondrial genetic
variations and the onset of disease [5563]. The level of mitochon-
drial DNA mutation is thus suggested as a marker for cancer
malignancy [60,6467].
Mitochondrial DNA genetic variation has also been employed for
assessing interindividual radiosensitivity. Higher levels of
Fig. 1. Citation analysis of published literature. A citation analysis performed on 22 May 2013, through Web of Science/Thomas Reuters, using keywords (1) nucle* AND
ionizing radiation AND DNA damage NOT nuclear energy (black bars) versus (2) mitochondr* AND ionizing radiation AND DNA damage NOT nuclear energy
(white bars) as Topic for All Years. Only Articles (a document type with original scientic ndings) were included into the analysis. The number of papers published in
each year is shown.
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 608
mitochondrial DNA point mutations and deletions were seen in
patients who received whole body radiation and chemotherapy
compared to the control subjects [68]. A high level of mitochondrial
DNA sequence variation was observed in nasopharyngeal carcinoma
patients who later developed moderate to severe deep tissue brosis
as a late complication of radiation therapy. In contrast, the patients
with no or minimal brotic reactions were those with low levels of
mitochondrial DNA sequence variations [69].
Thus, mitochondrial DNA changes can be used in the assess-
ment of evolutionary and pathological changes, as well as the
analysis of the effects of radiation.
Susceptibility of nuclear and mitochondrial DNA
The susceptibility of nuclear and mitochondrial DNA to various
stresses has been investigated. Backer and Weinstein showed that
mitochondrial DNA developed more covalent modications than
nuclear DNA when treated with a carcinogenic derivative of benzo
[a]pyrene [70]. Yakes and Van Houten also observed that the
mitochondrial genome was more prone to oxidative damage and
was characterized by a reduced ability for repair compared to
nuclear DNA. They concluded that mitochondrial DNA is a critical
target for reactive oxygen species (ROS) [71].
In addition to its greater sensitivity to chemically induced
oxidative stress, mitochondrial DNA has also been shown to be
more susceptible than its nuclear counterpart to ionizing radiation
damage. Richter et al. directly irradiated mitochondria isolated
from rat livers using gamma radiation (150 Gy). They observed a
6-fold higher amount of 8-hydroxydeoxyguanosine (oxidized
base) per unit mass in mitochondrial DNA than nuclear DNA from
the same liver [72]. May and Bohr found that approximately
2 times more gamma radiation-induced (560 Gy) strand breaks
could be detected in mitochondrial compared to nuclear DNA.
In addition, the amounts of lesions repaired in the nuclear
and mitochondrial DNA within the study period (2 h) were 80
and 25%, respectively [73]. Furthermore, Yoshida et al. reported
a delayed (24 to 72 h postirradiation) increase in 8-hydroxy-
deoxyguanosine lesions in the mitochondrial, but not the nuclear,
DNA when A7r5 cells (rat smooth muscle cells) were irradiated
with 5 Gy of gamma radiation [74].
Although mitochondrial DNA only accounts for 0.25% of the
total cellular DNA [20], the whole mitochondrial DNA (except
D-loop) consists of genes for protein synthesis [18,19]. In contrast,
the protein coding portion of nuclear DNA is only about 1% of the
remaining 99.75% of the total cellular DNA [46]. Therefore, a
genetic defect leading to direct biological effects is more likely to
happen within the coding regions of the mitochondrial DNA. In
addition, mitochondrial DNA lacks histone protection [72] and an
efcient DNA repair system [7578]; hence more unrepaired
lesions are likely to accumulate.
Common deletionmtDNA
4977
A region of the mitochondrial DNA known as mtDNA
4977
or
the common deletion, i.e., from nucleotide position 8470 to
13446, is a region prone to deletion and is associated with a
number of pathologies [51,7982] and with aging [68,83,84].
The relationship between the common deletion and the radia-
tion dose has been examined. Schilling-Toth et al. used human
broblast cell lines and primary broblast cultures to show that
the common deletion level increased with doses from 0.1 to 10 Gy
(after 72 h postirradiation). Interestingly, they observed hypersen-
sitivity at a low dose (0.05 Gy) [85]. A nonlinear relationship
between the common deletion level and the radiation dose was
also observed by Murphy et al. in HPV-G cells (derived from
human neonatal foreskin transfected with the HPV-16 virus);
these cells showed a higher frequency of the common deletion
at low (0.005 Gy) rather than high (5 Gy) radiation doses when
assayed 96 h after gamma irradiation [86].
The temporal prole of the occurrence of the common deletion
after radiation exposure (5 Gy; X-radiation) was examined by Wang
et al. using Hep G2 cells (human liver hepatocellular cells) and subcell
lines generated by long-term X-ray treatment (0.5 Gy, twice daily, >4
years). The frequency of the common deletion peaked at 24 to 48 h,
followed by a sharp decrease; the common deletion was undetectable
after 10 days in their tested cell lines. In addition, they reported that
the common deletion was only detectable in the apoptotic/dead cells,
but not the viable ones. They also discovered a novel mitochondrial
DNA deletion region between nucleotide position 8435 and 13,368
and named this region the 4934del; this deletion was specic to
ionizing radiation exposure in their cell system [87].
Furthermore, the relationship between the frequency of the
common deletion and the radiosensitivity in vitro was explored.
Kubota et al. reported that the frequency of the common deletion (0.5
to 10 Gy of X-radiation; 72 h postirradiation) depended on the
inherent cellular radiosensitivity among the tested SQ-20B, SCC-61
(human squamous carcinoma cells) and AT5BIVA cells (derived from
SV-40 transformed broblasts of an ataxia-telangiectasia patient)
[88]. Prithivirajsingh et al. tested their normal broblast cell lines,
dermal broblasts from human subjects, ataxia telangiectasia lines,
Kearns Sayre syndrome lines, glioblastoma lines with or without
DNA-PK deciency, and colon carcinoma lines. Although they did
observe an increase in the common deletion after administering a
radiation dose (2 to 20 Gy of gamma radiation; 72 h postirradiation),
the level of the increase varied and showed no correlation with
radiosensitivity among their tested samples [89].
In vivo, Rogounovitch et al. reported a greater frequency of
mitochondrial deletion in radiation-associated post-Chernobyl papil-
lary thyroid carcinoma than in sporadic cases [23]. This conclusion
was based on a comparison of Russian (residents of radionuclide-
contaminated regions after the Chernobyl fallout) and Japanese
(control) subjects, who might have different baseline levels of
mitochondrial deletion due to differences in the environmental
ultraviolet background in different geographical locations [84].
Wen et al. used human peripheral blood lymphocytes and found a
dose-dependent decrease in the common deletion in subjects with
total body irradiation (4.5 or 9 Gy; X-radiation) compared to the
unirradiated controls. As previously suggested [87], the authors
explained that radiation might cause lymphocytes to differentiate
into two populations, one with a high frequency of the common
deletion, which would be more prone to apoptosis, one with a low
frequency of the common deletion, which would be more radio-
resistant. A sample mixture with a high percentage of apoptotic cells
might have led to the seemingly high frequency of the common
deletion measured in other studies [90].
A change in the mitochondrial DNA copy number after ionizing
irradiation
Yoneda et al. pointed out that polymerase chain reaction (PCR) is
more sensitive than conventional Southern blotting in detecting
mitochondrial DNA damage because the latter involves washing
and transfer steps that could elute the lesions from the analysis
[91]. Moreover, with specic primers to amplify mitochondrial genes,
a reduction in PCR product yield indicates a loss in mitochondrial
DNA integrity [9295], and this principle can be used to assess the
damage induced by ionizing radiation [25]. In contrast, an increase in
the PCR product yield suggests an increase in the mitochondrial DNA
copy number. An increase in the mitochondrial DNA copy number
was observed in response to oxidative stress in a yeast model [96].
Similar observations have been reported in in vitro and in vivo
mammalian systems after ionizing irradiation.
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 609
In vitro, Murphy et al. showed an up to 2-fold increase in the
mitochondrial DNA copy number after 0.5 Gy of gamma irradiation in
HPV-G cells followed by 96 h of recovery [86]. They later used qPCR
and again showed a 1.3-fold increase in the mitochondrial DNA
copy number in the same cell system under the same experimental
procedure [97]. Wang et al. reported a postponed increase in the
mitochondrial DNA copy number (24 h to 45 days) in their Hep G2
cell line and its derivatives after being exposed to 5 Gy of X-radiation.
The level of the mitochondrial DNA increase varied among the
different cell lines and uctuated during the examination period
[87]. Kulkarni et al. exposed normal B-lymphoblastoid cells and
cell lines with either Leigh's syndrome or Leber's optic atrophy to
X-radiation between 0.5 and 4 Gy and observed an increase in the
mitochondrial DNA copy number (up to 30%) 24 h after the exposure
[38]. Zhou et al. also reported an increase in the mitochondrial DNA
copy number (up to 70%) in MCF-7 cells after the administration of
0.05 to 4 Gy of X-radiation followed by 4 to 72 h of recovery [98].
In vivo, the mouse brain and spleen tissues showed an increase in
the mitochondrial DNA copy number after 24 to 96 h of recovery
subsequent to a 3 Gy gamma-irradiation [99]. The mouse small bowel
and bone marrow tissues also showed a mitochondrial DNA copy
number increase after gamma-irradiation. The effect appears to be
tissue-independent and increases with the dose (2, 4, 7 Gy) and
recovery time (24, 48 h) [100]. Using 10 Gy of X-radiation, an increase
in the mitochondrial DNA copy number was again observed in the
murine liver, skeletal muscle, and brain when assayed at 1 to 72 h
postirradiation [101]. One day after 10 Gy of total body X-radiation, a
3-fold increase in the mitochondrial DNA copy number in mice spleen
cells compared to the control was observed. The authors suggested
that mitochondrial DNA replication was activated [102]. Using periph-
eral blood lymphocytes from acute lymphoblastic leukemia patients,
Wen et al. reported that there was an average 2-fold increase in the
mitochondrial DNA copy number 24 h after the patients received total
body irradiation (4.5 or 9 Gy; X-radiation) [90].
A mitochondrion contains multiple copies of its genome [103]. The
proper control of the mitochondrial DNA copy number is believed to
be important for normal cell function [104]. An increase in the
mitochondrial DNA copy number after radiation stimulation, termed
mitochondrial polyploidization [99], is believed to be a compensa-
tory mechanism [105] or an adaptive response of mitochondria to
maintain function in postirradiated cells [23,97] and malignantly
transformed progeny that survive after radiation exposure [106].
The benet of such an increase in the mitochondrial DNA copy
number postirradiation is under debate. It has been proposed that
DNAprotein complexes might shield some mitochondrial DNA
molecules from direct radiation-induced ROS damage [99]. Such
intact or partially damaged mitochondrial DNA might then replicate
to compensate for the loss and to maintain mitochondrial function
[49,51,90,99,100]. However, the increase in the mitochondrial DNA
copy number was found to be correlated with oxidative stress levels
in human leukocytes [107]. Thus, an increase in the mitochondrial
content could be a transient gain that later might strain the cells as
more resources are needed to support this high proliferation and
cope with the subsequent increase in ROS [108].
p
0
cells in radiation research
The role of mitochondrial DNA in the cellular response to
radiation has been extrapolated by investigating p
0
cells, i.e., cells
without mitochondrial DNA derived from a cell line containing
mitochondria. p
0
cells can be generated by long-term, low dose
treatment of ethidium bromide [109], or enzymatically using
Table 1
Radiation responses of p
0
cells oFN>mitochondrial membrane potential.
Reference Cell type Cell type Dose
(Gy)
Type of
radiation
Changes relative to parental cells or controls after radiation exposure
Cell
survival
Cell cycle Cell
growth
Micronucleus
formation
DNA damage ROS
production
ATP
content
[175] 701.2.8C Human
broblast cells
2 to 8 Gamma No change n/a n/a n/a n/a n/a n/a
[117] 701.2.8C Human
broblast cells
2 to 8 Gamma No
change
n/a n/a n/a n/a n/a
[176] 701.2.8C
& 143.
TK-
Human
broblast cells
& human
osteosarcoma
cells
2 to 10 Gamma No
change
n/a n/a No change
(DNA
fragmentation)
n/a n/a n/a
[115] 143.TK- Human
osteosarcoma
cells
2 to 8 Gamma No
change
n/a No
change
(cell
doubling
time)
n/a n/a n/a n/a
[116] 143.TK- Human
osteosarcoma
cells
2 to 8 Gamma No
change
n/a n/a n/a n/a n/a
[34] 143.TK- Human
osteosarcoma
cells
1 to 10 Gamma n/a n/a n/a n/a n/a No change No
change
n/a
[177] Hela Human
cervical
cancer cells
2 to 8,
20
Gamma Delayed G2 checkpoint
arrest and a decreased
ability to recover from
G2 arrest
n/a n/a n/a n/a n/a
[8] Hela Human
cervical
cancer cells
N/a Helium ion
microbeam
n/a n/a n/a n/a (53BP1 foci
formation)
n/a n/a n/a
[111] Miapaca Human
pancreatic
tumor cells
2 to 6 X ray n/a G2 checkpoint
activation together with
cyclin B1 (for mitosis)
and CDK1 (for cell cycle
progression) increase
minimal
change
n/a n/a n/a n/a n/a
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 610
mitochondrial-targeted restriction endonuclease to avoid the
mutagenic effect of ethidium bromide [110]. Although p
0
cells
appear to be a useful tool to assess the mitochondrial response to
radiation, conicting results have been reported (Table 1); these
disparities are likely due to differences in the cell origin or
whether the parental cells have been virally transduced [111].
Yet, the results were still conicting even when the same kind of
p
0
cells was used (generated from 701.2.8C cells; studies by
Yoshioka et al. (2004) and Tang et al. (1999)).
The observed discrepancies in the radiation responses of p
0
cells
could be due to varying levels of remnant mitochondrial DNA or
mitochondrial function. One study found no evidence of mitochondrial
DNA in their p
0
cells (Fig. 1 of Kawamura et al.), while others studies
deemed the level of mitochondrial DNA nonnegligible(Fig. 1A of Cloos
et al. (2009) and Fig. 5A of Tartier et al. (2007)). Moreover, most of the
mitochondrial proteins in all enzyme complexes are encoded by
nuclear DNA (77 subunits) [112]. p
0
cells, therefore, may not be the
best model to assess the role of mitochondria in cellular radiation
response. Specically, p
0
cells could still retain Complex II (solely
encoded by nuclear genes) activity [113,114]. Indo et al. observed that
p
0
cells responded to electron transport chain inhibitors; this observa-
tion suggested that the function and electron ow of the electron
transport chain may not have been completely abolished [114].
Furthermore, a shift in the energy metabolism from OXPHOS (OX,
i.e., oxidation/respiration; PHOS, i.e., phosphorylation/ATP synthesis)
to glycolysis (anaerobic respiration) in p
0
cells [109,113] could likely
reduce the proliferation rate of these cells [115117] and, therefore,
may indirectly lead to differences in their responses to radiation.
Notably, the absence of functional mitochondria in p
0
cells may
induce other nonmitochondrial changes, which could in turn affect
their radiation responses [111] and complicate the response analysis
[118]. Park et al. showed that p
0
cells generated from a wide range of
cell lines were characterized by a 21-fold higher expression of the
manganese superoxide dismutase gene and were more resistant to
ROS when compared to their parental cells. This increase in the
mitochondrial antioxidant activity in p
0
cells may allow them to adapt
and establish a new equilibrium of the superoxide levels, thus making
p
0
cells less suitable as models for ROS investigations [119]. In addition,
Ivanov et al. showed that p
0
cells generated from human skin
broblasts differ from their parental cells in terms of their mitochon-
drial function, oxygen consumption, mitochondrial membrane poten-
tial, nuclear-encoded mitochondrial protein expression, and NF-B/
STAT3 signaling pathways (for cellular response); the parent cells and
the p
0
cells also differed in the expression of 2100 genes [120].
Mitochondrial DNA is more vulnerable to ionizing radiation
damages than its nuclear counterpart. Ionizing radiation induces
nonlinear and cell-type-specic damages directly to mitochondrial
DNA, which may increase its copy number as a compensatory
response to counteract a loss in mitochondrial function.
The role of mitochondrial DNA in the radiation response has
been studied using p
0
cells. However, these cells are intrinsically
different from their parental cells. In addition, the use of pseudo p
0
cells, which are dened as cells with a reduced but nonzero
amount of mitochondrial DNA, in some studies limits the applic-
ability of p
0
cells in elucidating the role of mitochondrial DNA in
the radiation response.
Mitochondrial function after ionizing radiation stress
The effects of radiation on the electron transport chain
Pearce et al. irradiated isolated bovine heart mitochondria with
50 Gy of gamma radiation and showed an activity inhibition in
Complexes I and III [121]. Barjaktarovic et al. locally irradiated
(2 Gy; X-radiation) the heart of C57BL/6 N mice and observed a
delayed (4 weeks postirradiation) and partial deactivation of
Complex I (32%) and Complex III (11%), a decreased succinate-
driven respiratory capacity (13%), and an increased level of ROS
[122]. Yoshida et al. also reported a 50% reduction in Complex I
activity in their rat A7r5 cells 12 h after 5 Gy of gamma irradiation.
The observed changes were associated with the delayed ( 24 h
postirradiation) increase in mitochondrial ROS and the subsequent
mitochondrial DNA oxidation and growth inhibition [74]. Nugent
et al. showed a cell-specic change (CHO-K1: derived from ovary
of the Chinese hamster; HPV-G cells), either a decrease or an
increase, in OXPHOS enzyme activities soon (4 h) after irradiation.
More importantly, the activity of some of the examined enzymes
(Complexes II, III, IV, and ATP synthase) did recover upon 12 to
96 h of recovery [97] (see section 3.2).
Chinese hamster lung broblast cells with Complex II mutated
(single-base mutation that truncates 33 amino acids from the COOH-
terminal integral membrane portion of succinate dehydrogenase
subunit C protein) are found to have a higher level of superoxide
and hydrogen peroxide [123]. Later, Aykin-Burns et al. reported that
these cells were more radiosensitive and affected by poorer survival
when compared to their parental cells after low dose irradiation
(5 cGy to 50 cGy; gamma radiation). The above phenomenon could
be reversed when the mutant cells were made to overexpress wild-
type human succinate dehydrogenase subunit C [124].
These results show that the electron transport chain complexes
(including the nuclear-encoded Complex II) and ATP synthase
directly respond to ionizing radiation. Because these complexes
are involved in electron transport within the electron transport
chain and ATP synthesis, processes associated with OXPHOS are
likely to be affected.
The effects of radiation on oxidative phosphorylation
Hall et al. performed a whole body irradiation (8.4 Gy; X-radia-
tion) in rats and reported that phosphorylation was decreased by
20% 1 h postirradiation and remained low for the next 12 h. The
functionality of OXPHOS appeared to return to baseline after 24 h
of recovery [125].
In a similar but modied experiment, Hwang et al. directly
irradiated (5 to 20 Gy; gamma radiation) the murine liver which
was squeezed out through an upper abdomen opening and
assayed for mitochondrial respiratory function immediately after
irradiation. They noted a signicant decrease in both state 3 and
state 4 respiratory rates as the dose of radiation increased.
However, they did not nd a signicant change in the adenosine
5-diphosphate/oxygen ratio after irradiation [28]; this nding is
in contrast to the ndings of Hall's report. The authors suggested
that the measured effect of radiation on energy metabolism might
vary at different sampling times [28]. In addition, rats were
subjected to whole body irradiation in Hall's study [125], but the
liver was directly irradiated in Hwang's experiment [28]. The
difference in the irradiated region and hence the overall systemic
involvement might lead to differences in the measured radiation
response.
Yoshida et al. showed that human osteosarcoma cells underwent
a 20% reduction in ATP content 1 h after 8 Gy of gamma radiation,
followed by a full recovery in approximately 3 h [116]. Arguably, the
effect of radiation on OXPHOS could be a cell-specic event, as
suggested by Nugent et al. who showed that the change in cellular
oxygen consumption after 5 Gy of gamma irradiation varied between
their CHO-K1 and HPV-G cells upon recovery (4 to 24 h) [108].
These studies show that ionizing radiation, to some extent,
alters oxidation and/or phosphorylation; however, these changes
could later be recovered.
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 611
A change in the mitochondrial mass after ionizing irradiation
Using MitoTracker Green (marker for mitochondria) and uor-
escent microscopy, it was shown that 0.005 to 5 Gy of gamma
irradiation could lead to a 1.5- to 3.8-fold increase in the mito-
chondrial mass in two cell lines of different origin (CHO-K1 and
HPV-G cells) after 4 to 96 h of recovery [108]. Wang et al. also
found a 3.2-fold increase in the mitochondrial mass in their Hep
G2 cells 24 h after being exposed to 5 Gy of X-radiation [87].
Using Mitotracker Green and uorescence-activated cell sorting
(FACS) analysis, Zhou et al. reported a 1.4- to 1.5-fold increase in
the mitochondrial mass in MCF-7 cells (human breast adenocarci-
noma cell line) 4 h after 0.05 and 0.1 Gy of X-radiation exposure
[98]. Using the same technique, Dayal et al. noted a 1.5- to 2-fold
increase in the mitochondrial mass in both genetically stable and
unstable clones isolated from irradiated CHO cells (10 Gy; X-radia-
tion). More importantly, they also showed a decrease in the
mitochondrial membrane potential and an increase in oxygen
consumption, while the ATP levels were unchanged. These authors
suggested that radiation causes mitochondrial dysfunction, and an
increase in the mitochondrial mass is needed to maintain cellular
energy status [126].
The increase in mitochondrial mass is likely to be associated
with an increase in the total amount of mitochondrial DNA.
However, this assumption has been challenged [51]. Mitochondrial
DNA replication is independent of and seems to be uncoupled
from mitochondrial ssion [103]. Because mitochondria contain
proteins synthesized from both nuclear and mitochondrial DNA
[61,112], the nuclear-encoded proteins can still be produced even
though the mitochondrial DNA is defective. This disproportional
synthesis of nuclear-encoded proteins may contribute to the
increase in mitochondrial mass, even when the mitochondria
may lack mitochondrial DNA [51]. However, it was later shown
that the temporal [98] and relative [87] increase in the mitochon-
drial mass, at least when stimulated by X-radiation, was associated
with the increase in mitochondrial DNA in vitro.
Ionizing radiation causes mitochondrial dysfunction by altering
the activity of the electron transport chain complexes and ATP
synthase, hence OXPHOS. Mitochondria seem to respond to such
changes by increasing their DNA copy number (see section 2.3).
Whether the benet of such an increase is the restoration of
mitochondrial function is yet to be conrmed. Interestingly, we
have observed an increase in the expression of 18 mitochondrial
genes as well as peroxisome-proliferator-activated receptor-
coactivator 1 (PGC-1; a regulator of mitochondrial gene expres-
sion) in mammalian cells after gamma irradiation (unpublished
data). The increase in mitochondrial content (DNA and RNA)
postirradiation may thus lead to an overproduction of mitochond-
rially encoded subunits. This could cause an imbalance in the
composition of the electron transport chain complexes and the
ATP synthase, therefore, impairing the mitochondrial function and
leading to an increase in oxidative stress. Notably, PGC-1 is
also associated with mitochondrial proliferation/number [127].
The increase in PGC-1 postirradiation might relate to the increase
in mitochondrial mass/number (see section 3.3) which further
stresses the cell due to an accompanying increase in mitochondrial
oxidative stress.
Radiation-induced oxidative stress and antioxidant enzyme
The source of radiation-induced oxidative stress
Electrons may leak during transport within the electron trans-
port chain. The partial reduction of an oxygen molecule by a free
electron generates a superoxide anion, which is the precursor of
most ROS [128130]. Slane et al. have thoroughly characterized the
type of ROS released in mitochondrially impaired cells (Chinese
hamster lung broblast cells with mutated Complex II subunit C).
Their ow cytometry and high-performance liquid chromatogra-
phy (HPLC) results demonstrated the oxidation of dihydroethi-
dium (nonuorescent) to 2-OH-ethidium (uorescent) in these
mutated cells, suggesting the increase in mitochondrial superoxide
level [123]. Mitochondria, therefore, produce most of the ROS
(likely superoxide) under physiological and abnormal conditions,
making them constantly under high oxidative stress [71]. Notably,
the superoxide anion was found to be the major cause of radiation-
induced apoptosis, at least in the peritoneal resident macrophages
of C3H mice [131]. The role of mitochondria in radiation-induced
oxidative stress, thus, is one of the major study areas in radiation
research.
2,7-Dichlorouorescein diacetate and hydroethidine, as well
as their derivatives, are the dyes commonly used to detect
oxidative products, including those induced by ionizing radiation
[25,31,33,34,74,116,132136]. However, the application of these
dyes is limited because they cannot differentiate the source of the
oxidative stress.
2,7-Dichlorouorescein and its diacetate form are found to
produce ROS when oxidized and generate more ROS during the
detection process [137]. This assay may also register ROS gener-
ated in the extracellular medium as those formed intracellularly by
ionizing radiation and is reported to be dependent on the
concentration of serum in the medium during irradiation [138].
When hydroethidine is oxidized by superoxide, it does not change
to ethidium, which was thought to bind to DNA and lead to an
enhancement of uorescence [139].
A number of dyes are used for radiation-induced oxidative
stress detection at the subcellular level. Motoori et al. used
dihydrohodamine 123 to detect mitochondrial ROS and showed
a radiation-induced (15 Gy; X-radiation) elevation in mitochon-
drial ROS in their control HLE cells (human hepatoma cells), but
not in cells overexpressing an enzyme that breaks down mito-
chondrial superoxide radicals. These authors suggested that mito-
chondria are one of the major sites of ROS production and that
superoxide radicals are the primary radicals that give rise to the
elevation in ROS levels upon irradiation [30].
Indo et al. used confocal microscopy to observe an instant,
gradual increase in ROS levels, which peaked and then declined at
the 2nd hour, in their HLE cells after 18.8 Gy of X-radiation. They
also demonstrated the colocalization of hydroxyphenyl uorescein
(marker for hydroxyl radicals) and MitoTracker in the irradiated
cells, thus conrming that the ROS were generated by the
mitochondria. Moreover, their electron spin resonance spectro-
scopy results veried that the ROS were hydroxyl radicals and
superoxide anions [140].
The role of manganese superoxide dismutase in radioprotection
Manganese superoxide dismutase is an enzyme that resides
within the mitochondria; this enzyme is responsible for the
dismutation of highly reactive superoxides to less toxic forms,
i.e., water and hydrogen peroxide [129,130]. Oberley et al. used
mouse heart tissues and showed that radiation (absorbed dose of
7.7 Gy to the heart; X-radiation) only induced the activity and
protein expression of manganese superoxide dismutase, but not its
cytosol counterpart, the Cu/Zn superoxide dismutase [141]. The
time- and dose-dependent increase in the mRNA expression of
manganese superoxide dismutase was also observed by Akashi
et al. in irradiated human embryonic lung broblasts [142]. These
results suggest the likely involvement of manganese superoxide
dismutase in modulating the cellular response to daily and
radiation-induced oxidative stress both in vivo and in vitro.
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 612
Cells overexpressing manganese superoxide dismutase were
used to further examine the role of this enzyme in the radiation
response. Hirose et al. overexpressed manganese superoxide
dismutase in CHO cells (Chinese hamster ovary cells) and reported
the positive effect of this enzyme on cell survival after gamma
irradiation (1 to 11 Gy). These authors hypothesized that the
radioprotective role of manganese superoxide dismutase is prob-
ably tied to its ROS scavenging capability [143]. Later, Motoori et al.
reported that HLE cells transfected with manganese superoxide
dismutase were more radioresistant (15 Gy; X-radiation), as
shown by the lower level of apoptosis, mitochondrial ROS produc-
tion, and membrane lipid peroxidation [30]. Hosoki et al. per-
formed a microarray experiment to compare gamma-irradiated or
unirradiated HeLa cells (human cervical cancer cells) overexpres-
sing manganese superoxide dismutase or a control plasmid. Cells
overexpressing manganese superoxide dismutase had a high
endogenous level or a greater radiation-stimulated upregulation
of genes associated with the cell cycle and stress response [33].
These reports provide insights for future investigations of the
potential use of manganese superoxide dismutase in radiation
protection.
Epperly et al. delivered an adenovirus-carried manganese
superoxide dismutase transgene into their athymic nude mice
via intratracheal injection. After X-radiation treatment (850 to
950 cGy), these animals were found to have a lower expression of
cytokine mRNA (IL-1, TGF-, TNF-), as well as organizing alveo-
litis/brosis development, when compared to the controls [144].
They also conrmed that the IL-1 mRNA level correlates with the
acute pneumonitis phase and that the late elevations of TNF-,
TGF-1, and TGF-2 are associated with the onset of organizing
alveolitis/brosis and mortality [145]. They later applied different
modes of transgene delivery, such as plasmid/liposomes [146] and
a minicircle plasmid [147], to further explore the potential of
manganese superoxide dismutase in radioprotective gene therapy.
The localization of antioxidant enzymes
The importance of the mitochondrial localization of manganese
superoxide dismutase in modulating the stress response has been
examined by a number of groups. Hirai et al. found that the
mitochondrial ROS level and membrane lipid peroxidation, which
was induced by hypoxia and reoxygenation treatment, were
suppressed only if the manganese superoxide dismutase was
properly expressed within the mitochondria (i.e., a transgene with
a mitochondrial targeting signal) [148].
Other than hypoxia and reoxygenation stress, Epperly et al. used
genetic approaches to demonstrate the importance of the mitochon-
drial localization of superoxide dismutases in protecting cells from
radiation-induced oxidative stress. In vitro, they reported that when
superoxide dismutases, including Cu/Zn superoxide dismutase, which
is normally expressed within the cytosol, were expressed within the
mitochondria, their 32Dcl3 cells (murine myeloid cell line) showed
better survival and less apoptosis relative to the control cell lines after
gamma irradiation. However, their in vivo results showed that only
some of the mice injected with mitochondrially targeted superoxide
dismutase, manganese or Cu/Zu type, were protected from radiation-
induced esophagitis (35 Gy; X-radiation). The authors explained this
observation by suggesting the presence of cell phenotype-specic
transgenic effects for manganese superoxide dismutase in vivo [149].
Furthermore, this group also demonstrated the ability of mitochond-
rially targeted catalase to confer radioprotection. In vitro (32Dcl3 cells)
and in vivo (development of alveolitis in mouse lungs by 20 Gy of
X-radiation) radioprotection could be seen when catalase was directed
into the mitochondria. These authors suggested that mitochondrially
targeted catalase could be an additional radioprotector alongside with
manganese superoxide dismutase, at least in vitro [150].
Similar results were obtained by other groups. Hosoki et al.
showed that radioresistance was conferred only by cells overexpres-
sing mitochondrially targeted superoxide dismutase [33]. Indo et al.
also noted that authentic manganese superoxide dismutase, but not
the one without the mitochondrial targeting sequence, was needed to
reduce the level of radiation-induced ROS generation and hence lipid
peroxidation and eventual apoptotic cell death [140]. Furthermore,
treatment using mitochondrially targeted ROS scavenging enzymes
has also been reported to reduce cytotoxicity or to enhance radiation
resistance in cells [124,136].
Ionizing radiation induces both intracellular and mitochondrial
oxidative stress [32]. Manganese superoxide dismutase and Cu/Zu
superoxide dismutase are important for antioxidant defense within
the mitochondria and cytosol, respectively. These superoxide dismu-
tases (and other antioxidant enzymes) can be genetically modied to
have their subcellular localization altered. The change in the level of
radiation-induced oxidative stress in different subcellular compart-
ment can thus be differentially investigated in cells after expressing
such genetically modied antioxidant enzymes. Such enzymes,
targeting different organelles, would help to examine the role of
different subcellular compartments to the increase in oxidative stress
after ionizing radiation exposure. Collectively, these studies establish
the importance of the mitochondrial localization of antioxidant
enzymes in protecting cells from oxidative stress induced by ionizing
radiation.
Fluorescent assays have revealed that mitochondria are the major
subcellular site of radiation-induced oxidative stress. Transgenic
experiments have further conrmed that antioxidant enzymes need
to reside within the mitochondria to alleviate the oxidative stress
caused by ionizing radiation. Collectively, these ndings suggest the
crucial role of mitochondrial ROS (likely superoxide [71]), rather than
intracellular ROS, in mediating cellular damages after ionizing radia-
tion exposure.
Mitochondria, the cell nucleus, and late ionizing radiation
effects
Signal propagation between mitochondria
In addition to the studies that have identied mitochondria as
the major source of radiation-induced oxidative stress, there are
reports of signal propagation between mitochondria, which can
amplify the effects of radiation.
Leach et al. reported that about 50% of the wild-type or control
plasmid-transfected CHO cells showed a signicant increase in
ROS after irradiation (4 Gy; gamma radiation). However, less than
10% of the cells overexpressing calbindin 28 K, a Ca
2+
-binding
protein, showed an increase in radiation-induced ROS. They then
proposed that mitochondria directly hit by ionizing radiation are
subjected to a transition in their permeability. Ca
2+
ions are
released and then taken up by adjacent mitochondria, which then
depolarize, leading to a subsequent increase in ROS production.
Thus, the Ca
2+
ion could act as a signaling molecule in a chain
reaction among the mitochondria after irradiation, leading to the
propagation and amplication of the ROS signal; this chain
reaction would then impact the nucleus [34].
Communication between mitochondria for damage signal propa-
gation was also investigated by Brady et al. using rodent cardiomyo-
cytes, which are cells with a regular array of mitochondria. The
authors noted that laser-beam induced local ROS development and
the subsequent mitochondrial permeability transition could spread to
adjacent mitochondria. This change was a mitochondrial-potential
driven process, mediated via the mitochondrial permeability transi-
tion pore, and ROS, as opposed to Ca
2+
ions, appeared to participate in
this signal propagation [151]. As discussed above, ionizing radiation
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 613
impairs the electron transport chain leading to a persistent elevation
in mitochondrial oxidative stress. This imbalance in mitochondrial
ROS might form a positive feedback mechanism for more depolariza-
tion and greater ROS production, leading to further cell damages, as
suggested by the ROS-induced ROS release [152] and mitochon-
drial ROS-induced ROS [153] phenomena originally established by
Zorov and colleagues.
Of note, cardiomyocytes are terminally differentiated, while
CHO cells are immortal cells with continuous cell divisions. The
difference in material might, in part, contribute to the discrepancy
observed between the results from Brady's [151] and Leach's
teams [34] associated with the role of Ca
2+
ions in signal
propagation between irradiated cells.
Signal propagation between mitochondria and the cell nucleus
Changes in the functional state of mitochondria can be com-
municated to the cell nucleus via mitochondrial retrograde signal-
ing to achieve an adaptive cell response to stimulations/stresses
[154]. It is, therefore, possible that radiation damage, too, may
trigger a mitochondrial-nuclear communication.
Tartier et al. showed that a signicant increase in nuclear 53BP1
foci, which is a marker for DNA damage, could be observed in 3 h
when the cytoplasmof their HeLa cells was irradiated; such foci could
be seen in just 5 min when the nucleus was targeted. They proposed
that an additional step is needed to produce a biological response in
the cell nucleus from cytoplasmic irradiation [8].
Fisher and Goswami further suggested that mitochondrial ROS
could have a role in the regulation of radiation-induced cell cycle
checkpoints and overall cellular radiosensitivity, at least in Mia
PaCa-2 cells (human pancreatic cancer cells). In their study, they
hypothesized that communication between the mitochondria and
the nucleus, via radiation-induced mitochondrial ROS signaling,
could inuence the cellular response to radiation exposure [136].
Delayed increase in radiation-induced mitochondrial oxidative stress
Radiation causes an acute, transient increase in intracellular
oxidative stress [32]. The increase in oxidative stress is most likely
due to water radiolysis because water constitutes a substantial
portion of a cell. Free radicals generated from water usually have a
very short life span of only 10
-9
s [155]; yet, a number of studies
have reported the persistent increase in oxidative stress after
radiation exposure.
For example, Tulard et al. used dihydrohodamine 123 and
recorded a dose-dependent (1.5 to 7 Gy; gamma radiation),
delayed (24 h onward), and persistent (for days) increase in
mitochondrial ROS in their SW620 (human colon cells) and
SW620IR1 (radiosensitive clone derived from SW620 cells) cells
after gamma irradiation. Their results suggest a role for mitochon-
dria in the radiation-induced late production of ROS [31]. This
study also used 2,7-dichlorouorescein diacetate for intracellular
ROS detection. However, this part of their study is not discussed in
this review due to the aforementioned uncertainties associated
with this dye (see section 4.1).
Ogura et al. showed that 10 Gy of X-radiation caused an
increase in intracellular ROS, which was associated with the
release of cytochrome c from the mitochondria and the induction
of apoptosis in A549 cells (adenocarcinomic human alveolar basal
epithelial cells). They further used MitoAR, which selectively
localizes within mitochondria and reacts with ROS, to show that
mitochondria are the source for the late (tested at the 6th hour
postirradiation) increase in ROS in their tumor cell model [156].
A more detailed study was performed by Kobashigawa et al. using
amino-uorescein and MitoSOX, which are markers for intracellular
and mitochondrial ROS, respectively. Using normal human broblast-
like cells, they reported that radiation caused a dose-dependent
increase in ROS and a difference in the temporal prole of the
intracellular and mitochondrial ROS levels upon radiation stimulation
(2 to 6 Gy; gamma radiation). They observed an acute increase in
intracellular ROS, which subsided in about 24 h, and the levels then
rose again and remained high from the 72nd hour onward. The
mitochondrial ROS level showed a gentle increase, peaked at the
72nd hour postirradiation, and remained steadily high until the end
of the study period (7 days postirradiation) [32].
A recent report by Hosoki et al. also used MitoSOX and
demonstrated a delayed elevation of mitochondrial superoxide in
HeLa cells upon radiation stimulation (5 Gy gamma radiation; 72 h
postirradiation) [33]. A postponed, persistent increase in the
mitochondrial ROS level was also observed by Yoshida et al. when
irradiated A7r5 cells (embryonic rat thoracic aorta cells) were
examined using the MitoSOX assay (5 Gy gamma radiation; from
24 to 72 h postirradiation) [74].
Yamamori et al. exposed A549 cells to 10 Gy X-radiation and
similarly observed a delayed (peak at 12th hour postirradiation)
increase in oxidative stress; this change was associated with an
increase in the mitochondrial membrane potential, mitochondrial
respiration, and ATP production. More importantly, they demon-
strated a radiation-induced G2/M arrest and showed that cells in that
phase featured an increased mitochondrial content (mitochondrial
mass and DNA) and a higher intracellular ROS level [157].
Different cell lines were used in the aforementioned studies
and may contribute to the discrepancies in the reported ROS
production proles. Nonetheless, these reports show that radia-
tion causes a late increase in ROS, which are likely to be generated
by the mitochondria.
Mitochondrial dysfunction and late radiation effects
Mitochondria may be permanently impaired after ionizing
irradiation, leading to the observed persistent mitochondrial
oxidative stress long after the initial exposure.
At the mitochondrial DNA level, Schilling-Toth et al. reported
that their radiosensitive human broblast cells showed a two-
wave pattern of the common deletion in the mitochondrial DNA:
the frequency of the common deletion peaked around Day 14 and
then returned to the baseline level. This decrease was followed by
another peak around Day 49 that persisted until Day 63 after 0.1 or
2 Gy of gamma irradiation. This nding shows that damage to the
mitochondrial DNA, which manifests as the common deletion, can
be a long-term, persistent event, which the authors dened as
radiation-induced instability of the mitochondrial genome [85].
At the functional level, a number of studies have shown a
change in mitochondrial function in the progenies of the cells that
have survived from ionizing radiation exposure. Kim et al. used
genetically unstable clones LS-12 and Fe10-3, which were isolated
from GM101115 cells (contain 1 copy of human chromosome 4 in a
background of 20 to 24 CHO chromosomes) after 10 Gy of X-ray or
iron ions irradiation, respectively. They reported that both cell
lines were characterized by higher intracellular ROS levels and
lower manganese superoxide dismutase activity as well as a
reduced Complex IV activity and hence a defective respiratory
pathway [135].
Miller et al. later reported that unstable clones (LS-12, CS-9, 115
cell lines) were characterized by an altered expression of mito-
chondrial proteins and an elevated ROS level. Their results suggest
a casual association between mitochondrial dysfunction and
radiation-induced genome instability [158].
Dayal et al. reported that the relationship between genomic
instability and persistent oxidative stress was mediated by hydrogen
peroxide [159]. They later observed that their genetically unstable
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 614
clones had increased oxygen consumption (CS-9 cells) and elevated
Complex II activity, but reduced stability (LS-12 cells) [126].
A comprehensive mitochondrial subproteomic study, together
with mRNA and microRNA investigations, was performed by
Thomas et al. Their ndings suggested that unstable clones (LS-
12 cells) were likely to have a compromised electron transport
chain function. In addition, these cells were characterized by an
elevated response to oxidative stress and an upregulated tricar-
boxylic acid cycle, which was necessary to maintain cell survival in
the case of mitochondrial dysfunction. These responses are likely
to be epigenetically regulated and thus constitute a feedback loop
leading to genomic instability [160].
Furthermore, it is evident that mitochondrial dysfunction could
lead to nuclear damage or tumor development. Choi et al. reported
that the mitochondria, Nox1 (gene coding for NAPDH oxidase 1),
and JNK (for stress response) are likely to be involved in radiation-
induced (2.5 Gy; gamma-radiation) ROS production and hence
micronucleus formation [161]. St Clair et al. overexpressed man-
ganese superoxide dismutase in C3H10T1/2 cells (mouse cell line
with broblastic morphology and functionally similar to mesench-
ymal stem cells) and found that these cells were protected from
radiation-induced neoplastic transformation [162]. Du et al. used
mouse embryonic broblasts and showed that the increase in
manganese superoxide dismutase activity was associated with the
decrease in ROS production and, more importantly, the suppres-
sion of the late (72 h post gamma irradiation) increase in ROS,
micronuclei formation, and the subsequent cellular transformation
[134]. Furthermore, Zhang et al. showed that malignant trans-
formed human small airway epithelial cells, induced by 0.2 to 1 Gy
of
4
He ion irradiation, had an increased mitochondrial content and
a greater frequency of the common deletion; however, these cells
were characterized by a reduction in oxygen consumption, the
mitochondrial membrane potential, and Complex II activity [106].
These studies suggest that mitochondrial dysfunction is a likely
cause of radiation-induced genomic instability [163], which could
propagate to cell progenies/offspring, leading to long-term radia-
tion effects including nuclear damage [164].
Ionizing radiation permanently impairs mitochondria, leading to
a persistent production of mitochondrial ROS [165]. Mitochondrial
ROS are likely to act as signaling molecules for intermitochondrial
and mitochondrial-nuclear communication, which might promote
subsequent long-term radiation effects.
Outlook
Here, based on the available experimental data, we infer the
effects of ionizing radiation on mitochondria:
Mitochondria are more susceptible to ionizing radiation than
the nucleus (Fig. 2A). Mitochondrial DNA can be directly damaged,
e.g., through mitochondrial DNA deletion. Interestingly, the mito-
chondria can cope with such damages, which might lead to
Fig. 2. Radiation-induced mitochondrial superoxide mediated nuclear damage. (A) When a cell is exposed to ionizing radiation, its nucleus and mitochondria (DNA-
containing sites) are likely to be affected. The mitochondrial genome is of higher genetic information density as compared to that of the nuclear genome; therefore,
radiation-induced lesions that could lead to functional changes are more likely to be found in mitochondrial DNA. (B) Regions within the mitochondrial DNA could be deleted
upon ionizing radiation exposure, therefore, altering the function of the electron transport chain (ETC) and leading to a persistent mitochondrial superoxide (O
2
-
) production
postirradiation. Mitochondria may respond to such damages by increasing the quantity or synthesizing new mitochondrial DNA in order to maintain the mitochondrial
function necessary for cell survival. (C) Nevertheless, the radiation-induced superoxide may continue to damage the mitochondrial DNA of that impaired mitochondrion or
diffuse to the nearby mitochondrion, both steps similarly cause further mitochondrial dysfunction and hence amplify the amount of mitochondrial superoxide.
(D) Furthermore, mitochondrial superoxide may diffuse intracellularly causing damages to other parts of the cells including the cell nucleus (micronuclei formation) and
nuclear DNA.
W.W.-Y. Kam, R.B. Banati / Free Radical Biology and Medicine 65 (2013) 607619 615
mitochondrial dysfunction, by increasing their quantity and DNA
copy number (Fig. 2B).
However, more mitochondria might lead to more superoxide
production. In addition, the increase in mitochondrial DNA might
result in an overproduction of mitochondrial protein. This over-
production could alter the activity of the electron transport chain
complexes/OXPHOS, leading to an elevated superoxide production.
The increase in superoxide in one mitochondrion might diffuse
(via an unknown active pathway or passively) to another mito-
chondrion (Fig. 2C). This intermitochondrial communication
amplies the damage signal and permanently damages the rest
of the mitochondria within a cell, resulting in further superoxide
production. Impaired mitochondria, after the initial radiation
exposure, continue to produce superoxide, which eventually
diffuse into the cell nucleus and cause nuclear DNA damage
(Fig. 2D). Furthermore, the nuclear lesion produced by this
radiation-induced mitochondrial superoxide-mediated nuclear
damage, if not properly repaired, might propagate to the cell
progenies resulting in secondary cancer development in cells long
after the initial radiation exposure.
A further theoretical consideration is that radiation-induced
damage to mitochondrial DNA may cause mutations or deletions
within the mitochondrial genome which could be inherited by the
offspring through the maternal germline. Though ndings from a
recent report [166] appear to contradict with this concept, a larger
study cohort is warranted for a clearer examination on the
possibility of transgenerational increase in cancer risk due to
mitochondrial abnormality subsequent to ionizing radiation expo-
sure to the mother. Likewise, it has been suggested that mitochon-
drial superoxide radicals have been a driving factor in the
evolutionary move of mitochondrial genetic information into the
nucleus [167]. However, it is still a theoretical speculation whether
ionizing radiation [168] and the associated oxidative stress may
indeed lead to the insertion of mitochondrial DNA fragments into
the nuclear genome, and whether such nuclear mitochondrial
pseudogenes could disrupt the nuclear genome and contribute to
an increase in cancer risk within an exposed individual or even in
their offsprings.
The reported observations summarized in this review suggest a
need for further investigations into the effects of ionizing radiation
on mitochondria, including (a) the mechanisms of radiation-
induced alterations in mitochondrial electron transport chain
stoichiometry, residence time, and accessibility of electrons to
mediate increased levels of one-electron reductions of oxygen for
the late increase in oxidative stress; and (b) the mechanisms and
biological signicance of the change in mitochondrial mass and
content in cells after being exposed to ionizing radiation.
Echoing a recent review [26], the interactions between the
mitochondria and the cell nucleus in response to ionizing irradia-
tion are likely to determine short- and long-term radiation effects.
For example, how exactly the different nuclear and mitochond-
rially encoded subunits of the electron transport chain interact to
alter mitochondrial superoxide production, and thus contribute
genomic instability, requires further systematic investigation.
Furthermore, it is likely that mitochondria are potential targets
for radiation protection [169174]. The latter has broad relevance
for clinical applications, in the mitigation of environmental or
industrial exposure and in space biology.
Acknowledgments
We thank Mrs. Geetanjali Dhand for her generous assistance in
collecting the literature for this review; Dr. Aimee McNarama, for
discussing and proofreading the manuscript; and A/Prof Zdenka
Kuncic, for discussing the manuscript.
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