Mutagenesis. 2008 (Nov); 23 (6): 473–482 ~ FULL TEXT
Lemon JA, Rollo CD, Boreham DR.
Medical Physics and Applied Radiation Sciences,
Hamilton, Ontario L8S 4K1,
Transgenic growth hormone (Tg) mice express elevated free radical processes and a progeroid syndrome of accelerated ageing. We examined bone marrow cells of Tg mice and their normal (Nr) siblings for three markers of DNA damage and assessed the impact of free radical stress using ionizing radiation. We also evaluated the radiation protection afforded by a dietary supplement that we previously demonstrated to extend longevity and reduce cognitive ageing of Nr and Tg mice. Spectral karyotyping revealed few spontaneous chromosomal aberrations in Nr or Tg. Tg mice, however, had significantly greater constitutive levels of both gammaH2AX and 8-hydroxy-deoxyguanosine (8-OHdG) compared to Nr. When exposed to a 2-Gy whole-body dose of ionizing radiation, both Nr and Tg mice showed significant increases in DNA damage.
Compared to Nr mice, irradiated Tg mice had dramatically higher levels of gammaH2AX foci and double the levels of chromosomal aberrations. In unirradiated mice, the dietary supplement significantly reduced constitutive gammaH2AX and 8-OHdG in both Nr and Tg mice (normalizing both gammaH2AX and 8-OHdG in Tg), with little difference in gammaH2AX and 8-OHdG over constitutive levels. Induced chromosomal aberrations were also reduced, and in Nr mice, virtually absent. Remarkably, supplemented mice expressed 6-fold lower levels of radiation-induced chromosomal aberrations compared to unsupplemented Nr or Tg mice. Based on our data, the dietary supplement appeared to scavenge free radicals before they could cause damage. This study validates Tg mice as an exemplary model of oxidative stress and radiation hypersensitivity and documents unprecedented radioprotection by a dietary supplement comprised of ingredients available to the general public.
From the FULL TEXT Article:
Oxidative stress arises from diverse causes such as xenobiotics,
nicotinamide adenine dinucleotide phosphate (NADPH) oxidases,
hypoxia, ageing, disease processes, mitochondrial dysfunction
and exposure to ionizing radiation. [1–4] Free radicals damage
lipids, proteins,DNAand cellular structures. OxidativeDNAbase
damage, chromosome aberrations, genomic instability and
telomere alterations are among the most deleterious consequences
of oxidative processes consequently making high doses potentially
Several studies investigating various indicators of DNA
damage, including oxidative base damage, telomere shortening,
chromosome fragments, chromosome loss and aneuploidy have
consistently shown that DNA damage increases proportionately
with age [5, 8–10] and are considered valid markers of
ageing.  Genomic aberrations increase the risk of carcinogenesis
and may actually accelerate the ageing process by
contributing to cell dysfunction and death. [14–17] Chronic
oxidative stress such as that associated with ageing is a
significant contributor to the accumulation of damaged DNA
and chromosome rearrangements. [12, 18] The processes associated
with ageing are the most common causes of chronic
oxidative stress; however, many disease states including neurodegenerative
diseases, cancer, progerias, atherosclerosis, autoimmunity
and diabetes include chronic oxidative stress as a
significant factor in the progression of the disease. [19–23] The
age-related increase in DNA damage is also associated with
progressive mitochondrial dysfunction and rising free radical
production, which may contribute to progressive physiological
dysfunction, deterioration and mortality. [16, 17, 24–27] Cellular
responses mediating defence, repair and replacement also appear
to attenuate with age and contribute to ageing. [7, 17, 28]
Transgenic growth hormone (Tg) mice express elevated free
radical processes  and accelerated ageing. [30–34] Tg mice
live only half as long as normal mice [29, 30, 35, 36] and experience
elevated superoxide radical production and lipid peroxidation
in several tissues. [29, 37, 38] They have age related, early
onset arthritis, increased incidence of cataracts, reduced motor
activity and muscle wasting. [30, 35, 36, 39, 40] Brown-Borg et al.
(31) found elevation of hepatic carbonyls at 12 months. Tg mice
also express reduced levels of hepatic antioxidants. [38, 41, 42]
Young Tg mice display greatly enhanced cognitive abilities [43, 44], but experience a rapid age-related decline in learning abilities. 
Chronically elevated free radical production in Tg mice
would imply that they may display more constitutive chromosomal
aberrations and oxidative base damage. We further hypothesized
that Tg mice would show greater radiosensitivity to
DNA damage than age-matched Nr mice and that the (anti-ageing)
dietary supplementwould reduce DNA damage in Tg mice. Limoli
et al.  found that neuronal precursor cells subjected to an
oxidative environment showed increased radiosensitivity. To
assess these ideas, we examined metaphase chromosomes from
bone marrow cells using spectral karyotyping (SKY) and
γH2AX to examine double-strand breaks (DSBs) and repair
kinetics and 8-OHdG as an indicator of oxidative base damage.
It is established that antioxidants can reduce the severity of
DNA damage and diminish chromosome aberrations. [46–49]
Development of radioprotective diets is a high priority for
individuals exposed to ionizing radiation. For example, diets could
protect astronauts from cosmic radiation and normal tissue in
patients undergoing radiation therapy. Numerous materials show
potential value, but effective protection remains elusive. [46–57]
Multiple cellular mechanisms defend against oxidative stress. [58–61] Antioxidants typically spare, synergize or recycle one
another, and if administered singly, they may become prooxidants.
Various antioxidants occupy different cellular compartments,
so general protection requires multiple components.
For example, lipophilic antioxidants like vitamin E protect cell
membranes but are less effective for hydrophilic components
of the cytosol. Antioxidants that intercept free radicals generated
by the mitochondria or other cytosolic sources may
prevent DNA damage, but those that closely associate with
nuclear DNA may be most effective.
Materials that activate cellular defence systems or provide
support or substrates for enzymatic antioxidant systems and
DNA repair processes are also of significant importance.
Multiple pharmaceuticals are required to serve all these criteria,
even though some may have multiple functions. Since no one
antioxidant can fill all these roles, the ideal supplement must
combine compounds that possess all these characteristics.
Following oxidative insults like radiation, cells may express
elevated free radical production for weeks or even months. 
Mechanisms include cell membrane NADPH oxidases and
(associated) potassium channels, mitochondrial dysfunction
and inflammatory processes. [3, 12, 63–65] NADPH oxidases
both produce and are activated by free radicals. This can
perpetuate chronic free radical production.  Mitochondrial
dysfunction induced by irradiation (Ca2þ elevation, depolarization
of membrane potential, low respiration rate and reduced
activity of manganese superoxide dismutase (MnSOD)) likely
contribute to sustained free radical production generation, even
among clonal cell descendants. [65, 66] Leach et al. 
highlighted Ca2þ inter-mitochondrial signalling in amplifying
radiation-induced free radical generation.
The chronically elevated free radical production characteristic
of Tg mice indicates that older Tg mice may experience
increased constitutive structural chromosome aberrations and
oxidative base damage. Consequently, Tg mice should have
reduced ability cope with acute free radical insult from
radiation exposure, resulting in greater radiation-induced
genomic aberrations compared to age-matched normal mice.
If these hypotheses are supported, then supplementation with
our anti-ageing/antioxidant supplement should reduce the level
of DNA damage in Tg mice. This study tested these hypotheses
by examining metaphase chromosomes from bone marrow to
look at DSB levels using γH2AX foci formation, DNA repair
fidelity using SKY and assessing oxidative base damage by
measuring 8-OhdG levels.
Materials and methods
Our Tg mice (C57BL/6J-SJL background) have transgenes with metallothionein
promoters fused to rat growth hormone (GH) structural regions.  This results in a .100-fold elevation of plasma GH. Tg mice were
reliably identified by their significantly larger size by 28 days of age , as
well as morphological differences (increased body length, significantly broader
nose). All mice used in this study were derived from a closed breeding colony
where Tg sires were bred to Nr dams providing equal numbers of Nr and Tg
mice of similar genetic background. The chromosomal aberration assessment
used 24 female mice aged 11–12 months, evenly divided into four experimental
groups (see below). The γH2AX and 8-OHdG experiments also used 24 female
mice in evenly divided into four experimental groups (11–12 months). For all
assays, experimental groups included unsupplemented Nr mice, unsupplemented
Tg mice, diet-supplemented Nr mice and diet-supplemented Tg mice.
A maximum of four mice were housed in cages (27 x 12 x 15.5 cm) bedded
with wood-chip (Harlan Sani-Chips, 7090). A stainless steel hopper provided food
ad libitum (Harlan Teklad 8640 22/5 rodent chow) and supported a water bottle.
The housing room maintained a 12:12 h light:dark photoperiod at 22 ± 2°C.
All protocols met the standards of McMaster University’s Animal Research
Ethics Board and the Canadian Council on Animal Care.
The dietary supplement was designed to simultaneously ameliorate several
processes implicated in ageing (oxidative stress, inflammatory processes,
insulin resistance and mitochondrial dysfunction). Criteria for selecting specific
ingredients for the supplement were as follows:
(i) scientifically documented evidence as effective for one or more of the targets,
(ii) oral administration and
(iii) approved for human use.
Dosages and preparation of the supplement were described by Lemon et al.. [36, 43] Dosages for mice were based on amounts prescribed to humans. Values were
adjusted for the smaller body size of the mice and then increased by a factor of
10 based on the higher gram-specific metabolic and utilization rates of mice
compared to humans.  Ingredients and associated targets are outlined in Table I.
The supplement was prepared in liquid form and 0.4 ml was allowed to absorb
into a 1 x 1.5 x 1 cm piece of bagel (bread product). This was air-dried and then
refrigerated for immediate use. The mice were given the bagel pieces midway
through the photoperiod. The bagel pieces were immediately and completely
ingested by the mice, ensuring mice obtained full and equivalent doses. All
supplemented mice began receiving the supplement prior to 3 months of age.
In vivo irradiation
During irradiation, mice were placed in a polyvinyl chloride (PVC) tube (5 x 12.5 cm) withPVCmesh endcaps. Eachmousewas given a 2-Gywhole-body dose
of gamma radiation from a 137Cs source (dose rate: 0.5 Gy/min). After irradiation,
each mouse was returned to its housing cage. Non-irradiated sham control
mice were otherwise exposed to exactly the same conditions as irradiated mice.
Sample collection and preparation.
Four hours after irradiation, mice were
anesthetized with Isoflurane~ and euthanized via cervical dislocation. Both
femurs of each mouse were excised and a 3-ml syringe equipped with a 23-g
needle containing 1 ml heparinized complete RPMI 1640 [10% foetal bovine
serum (FBS), 1% L-glutamine, 1% penicillin–streptomycin] was used to flush
the bone marrow from the femurs. Each femur was flushed three times with the
same 1 ml of complete RPMI to ensure complete removal of bone marrow. The
cell suspension was then repeatedly drawn through the syringe gently, 10 times,
to break up any remaining cell clumps. The bone marrow cell suspension was
immediately placed in a 15-ml centrifuge tube containing 4 ml complete RPMI,
2 ml FBS and 0.1 µg/ml colcemide. Cells were incubated at 37°C (5% CO2,
98% humidity) for 4 h. The cells were centrifuged at 200x g for 8 min, the
supernatant aspirated and the cells were re-suspended in ~100 µl of remaining
supernatant by gentle vortexing.
A total of 10 ml of 0.075 M KCl was added to each sample and incubated at
37°C for 20 min. The samples were then centrifuged at 200x g for 8 min, the
supernatant was aspirated and the cells were re-suspended. A total of 10 ml of
3:1 methanol:acetic acid fix was added to each sample and incubated at room
temperature for 15 min. The samples were centrifuged at 300x g for 8 min, the
supernatant was aspirated and the cells were re-suspended. A total of 10 ml of
3:1 methanol:acetic acid fix was added to each tube and samples were stored at
–20°C until analysis.
Samples were allowed to warm to room temperature, then
the assay tubes were centrifuged at 300x g for 7 min and the supernatant was
aspirated to 0.5 ml above the cell pellet. The cells were re-suspended and 5 ml
of freshly prepared 3:1 methanol:acetic acid fix was added to each assay tube.
Slides were dropped in a humidity- and temperature-controlled chamber
(Thermotron®, Holland, MI) onto acid cleaned slides. Prepared slides were
examined under a phase contrast microscope to determine quality of the
chromosome spreads and the mitotic index of each sample. Slides were aged
overnight at 37°C in a dry oven prior to preparation.
The slides were prepared for SKY with a slightly modified
version of the instructions supplied by manufacturer, Applied Spectral
Imaging® (ASI, Vista, CA). The slides were treated as follows: the slides
were treated with pepsin solution for 6 min at 37°C and then washed twice in
phosphate-buffered saline (PBS) at room temperature. The slides were washed
in 1x PBS/MgCl2 at room temperature, then placed in a 1% formaldehyde
solution and incubated for 10 min at room temperature and were washed once
in 1x PBS. At this point, 10 µl of the SkyPaint® mixture (ASI) was denatured
by incubating the mixture at 80°C for 7 min and then placed at 37°C for 90 min.
The slides were incubated in 2x sodium chloride/sodium citrate (SSC) at
70°C for 30 min and then allowed to cool to room temperature (~20 min). The
slides were washed once in 0.1x SSC and denatured in 0.07 M NaOH at room
temperature for 1 min. The slides were then washed once in 0.1x SSC at 4°C,
followed by one wash in 2x SSC at 4°C. The slides were dehydrated in an
ethanol series (70, 80, then 100%) at –20°C and allowed to air-dry.
The denatured SkyPaint® was added to the denatured chromosome
preparation and a 22 x 22-mm glass cover slip was placed over the probe
mixture. The edges of the cover slip are sealed with rubber cement and the
slides were incubated in a humidified chamber at 37°C for 60–64 h. The slides
were washed three times in 50% formamide/2x SSC at 45°C, followed by two
washes in 1x SSC at 45°C. Slides were then washed once in 4x SSC/0.1%
Tween 20 at room temperature. Blocking reagent (80 µl) was applied to each
slide and covered with a plastic cover slip. The slides were incubated for
30 min in a humidified chamber at 37°C. The slides are then drained and 80 µl
of Cy5 buffer (CAD 03 buffer; ASI) was added to each slide. The slides were
incubated at 37°C for 45 min. The slides were then washed three times in 4 SSC/0.1% Tween 20 at 45°C. Slides were drained and 80 µl of Cy5.5 buffer
(CAD 04 buffer; ASI) was added to each slide and incubated at 37°C for 40min.
The slides were washed three times in 4x SSC/0.1% Tween 20. The slides were
finally washed in distilled water to remove detergent residue and allowed to airdry.
Once the slides were fully dry, 15 µl 4,6-diamidino-2-phenylindole (DAPI)/
Antifade solution was applied to each slide and a 22 x 22-mm glass cover slip
was placed over the cell spreads. The slides were allowed to incubate for 5 min,
the slides were gently pressed between two Kimwipes to remove excess solution
and the edges of the cover slips were sealed with clear nail polish. The slides
were then ready for analysis.
The SKY slides were scored using the Spectracube® system (ASI). Suitable metaphases (i.e. those with good chromosome
quality and sufficient chromosome separation) were assessed with a DAPI
(excitation: 310nm, emission: 450nm) filter. Once such a metaphase was found,
a black and white band image was captured, followed by a spectral image with
the SKY filter (proprietary filter). Image acquisition is based on a spectral
imaging system using an interferometer and a CCD camera. Both the band and
spectral images were then used to determine the karyotype of each cell, with
SkyView EXPO™, the image analysis software. A minimum of 50 suitable
metaphases were scored for each mouse.
γH2AX and 8-OHdG
Immediately after irradiation, mice were euthanized via
cervical dislocation and bone marrow cells were obtained from both femurs
as described above. The cell suspension was added to 6 ml of 0°C complete
RPMI 1640 and placed on ice for the duration of sample preparation. Cell
concentrations were determined using the Z2 Coulter particle count and size
analyser (Beckman-Coulter, Miami FL) and adjusted to 1 x 106 cells/ml with
0°C complete RPMI 1640. A 500-µl aliquot of cell suspension was removed for
the 0 h time point from each sample and placed in 5 ml polypropylene tube for
the γH2AX assay. A 3 ml ice cold 70% ethanol was immediately added to each
tube and all tubes were place on ice.
The remaining bone marrow samples were incubated in
a 37°C water bath. For the γH2AX assay, 500-µl aliquots were removed at 15,
30, 60, 120 and 240 min and placed in 5-ml tubes. For the 8-OHdG assay, 500µl aliquots were removed at 240 min and placed in 5-ml tubes. A 3 ml of 0°C
70% ethanol was immediately added to each sample. All tubes were incubated
on ice for 1 h. Samples were stored at –20°C until analysis.
Bone marrow cells fixed in 70% ethanol were allowed to warm to room
temperature prior to the start of antibody staining. Cells were incubated in Trisbuffered
saline (TBS; Trizma base + NaCl, Sigma Aldrich, Mississauga,
Ontario) for 10 min to rehydrate cells. The tubes were centrifuged and resuspended
in Tris–saline–triton [TST; TBS + 4% FBS (VWR International,
Mississauga, Ontario) + 0.1% Triton X-100 (Sigma Aldrich)] and incubated
on ice for 10 min to permeabilize cells. The cells were centrifuged and resuspended
in 200 µl either anti-phospho-H2A.X (ser139) antibody (γH2AX;
Upstate Cell Signaling, Charlottesville, VA) or anti-8-OHdG antibody
(Chemicon International, Temecula, CA).
Both primary antibodies were diluted 1:400 in TST and incubated at room
temperature for 2 h. The cells were washed with TST and re-suspended in
200 µl of either AlexaFluor™ 488-conjugated goat anti-rabbit IgG F(ab')2
antibody (γH2AX) or AlexaFluor™ 488-conjugated rabbit anti-goat IgG
F(ab')2 antibody (8-OHdG). Both secondary antibodies were diluted 1:500 in
TST (Invitrogen Canada, Burlington, Ontario) and incubated at room
temperature for 1 h. The cells were then washed in TBS and re-suspended in
300 µl TBS + 5 µl propidium iodide (1 mg/ml; Sigma Aldrich). Samples were
put on ice and immediately run on the Epics XL flow cytometer (Beckman
Coulter; Mississauga, Ontario).
All values were represented as the mean and standard error of the mean.
Student’s t-tests were performed to determine if significant differences existed
Few spontaneous structural chromosome aberrations
were found in any of the experimental groups (Figure 1).
There were no significant differences in number of chromosome
aberrations between unsupplemented Nr (2.33 ± 0.48%)
and unsupplemented Tg (1.76 ± 0.99%). The number of
constitutive chromosome aberrations in supplemented Nr
(0.56 ± 0.56%) did not differ from supplemented Tg (0.63 ± 0.63%). Supplemented mice had the same number of spontaneous
aberrations as unsupplemented mice. Despite a trend,
levels of spontaneous aberrations between supplemented and
unsupplemented mice were not statistically resolved.
The percentage of cells containing aberrations
increased significantly in unsupplemented Nr exposed to
2 Gy (27.53 ± 5.29%; Figure 1) compared to spontaneous
aberrations in unsupplemented Nr (P < 0.009). Unsupplemented
Tg also had a significant elevation in radiation-induced
chromosome aberrations (46.60 ± 5.33%) compared to constitutive
levels (P < 0.0012). Also, unsupplemented Tg mice
were more sensitive to radiation and had significantly more
cells with radiation-induced chromosome aberrations compared
to unsupplemented Nr mice (P , 0.024; compare in Figure 1).
Diet-supplemented Nr had a significant reduction in radiationinduced
chromosome aberrations (5.27 ± 4.33%) and became
resistant compared to unsupplemented Nr (P < 0.031).
Supplemented Tg also had significantly fewer radiation-induced
aberrations compared to both unsupplemented Tg (P < 0.0031)
and unsupplemented normals (P , 0.033). There was no difference
in radiation-induced chromosome aberrations between
diet-supplemented Nr and diet-supplemented Tg. Supplemented
animals exposed to 2 Gy dose had similar levels of
chromosome aberrations as unirradiated mice, suggesting nearly
complete protection from the effects of a large dose of radiation.
Chromosome-type structural aberrations predominate in
both constitutive and radiation induced for all groups of mice
(Table II). Unrepaired chromatid arm breaks and the corresponding
acentric fragments comprise 100% of constitutive
aberrations in unsupplemented and supplemented mice. The
variety and complexity of radiation-induced aberrations was
higher than constitutive aberrations (Table II), chromatid arm
breaks and corresponding acentric fragments were still the
dominant type of aberration, followed by translocations.
Although it was apparent that some chromosomes were involved
in structural aberrations more frequently than others (Figure 2),
R2 values (Table III) indicated that the frequency with which
each chromosome was involved in chromosome aberrations
does not correlate to chromosome length, number of genes per
chromosome or gene density (genes/Mbp) of the chromosome.
There was a reduction in constitutive γH2AX foci
formation and corresponding fluorescence observed in both
groups of unsupplemented mice during the incubation period
(Figure 3A); however, this trend was only significant in
unsupplemented Tg mice (P < 0.025). The kinetics of
radiation-induced γH2AX was the same for all groups of
mice, with supplemented mice demonstrating significantly
reduced γH2AX levels (P < 0.045) at all time points (Figure 3B). The level of γH2AX fluorescence increased from 0 to
30 min, where it peaked, followed by a decrease in fluorescence
at each of the following time points out to 240 min, when
γH2AX fluorescence returned to baseline levels. The level of
γH2AX fluorescence peaked at 30 min after irradiation in all
groups of mice; to simplify analysis, statistics in the following
sections was based only on the 30-min incubation data.
Constitutive foci formation.
The background level of γH2AX
foci was significantly lower in unsupplemented Nr (1.27 ± 0.13) compared to unsupplemented Tg (1.60 ± 0.14;
P < 0.047). Constitutive expression of γH2AX in supplemented
Nr (1.10 ± 0.12) was lower than unsupplemented Nr; however,
this reduction was also not significant. There was a reduction of
background γH2AX levels in supplemented Tg (0.93 ± 0.09),
significantly lower than both unsupplemented Nr (P < 0.0001)
and unsupplemented Tg (P , 0.0001). However, there was no
difference in γH2AX levels between supplemented Nr and
supplemented Tg (Figure 3A).
Radiation-induced foci formation.
foci was significantly higher in unsupplemented Nr (2.37 ± 0.016; P < 0.0052) and unsupplemented Tg (2.95 ± 0.19;
P , 0.0018) compared to their respective background levels
(Figure 3B). UnsupplementedNr had significantly lower radiationinduced
γH2AX levels compared to unsupplemented Tg (P ,
0.038). Both supplemented Nr (1.77 ± 0.080; P < 0.0066) and
supplemented Tg (1.28 ± 0.10; P < 0.0001) demonstrated
significant reductions in radiation-induced γH2AX fluorescence,
so that radiation-induced γH2AX fluorescence did not
differ significantly in supplemented mice from constitutive
levels. The level of γH2AX in the supplemented groups of
mice did not differ from each other.
Oxidative base damage
Constitutive levels of 8-OHdG.
The constitutive level of
8-OHdG was significantly lower in unsupplemented Nr (1.92 ± 0.08) compared to unsupplemented Tg (3.82 ± 0.10; P < 0.0001). There was no significant difference in constitutive
8-OHdG levels between unsupplemented and supplemented Nr
(1.63 ± 0.12). Supplemented Tg (1.83 ± 0.09) had a dramatic
reduction in 8-OHdG levels compared to unsupplemented Tg
(P < 0.0001); however, there was no difference in 8-OHdG
levels between supplemented Nr and supplemented Tg (Figure 4).
Radiation-induced levels of 8-OHdG.
There was a significant
increase in radiation-induced 8-OHdG levels in unsupplemented
Nr (2.53 ± 0.19) and Tg (4.87 ± 0.27) compared to
constitutive 8-OHdG expression (P < 0.0052 and P < 0.0018,
respectively). Unlike with radiation-induced chromosome
aberrations or cH2A.X levels, the amount of radiation-induced
8-OHdG increased by the same proportion in unsupplemented
Nr and Tg (Figure 4). The level of radiation-induced 8-OHdG
in supplemented Nr (1.85 ± 0.23) was significantly lower than
unsupplemented Nr (P < 0.028). Supplemented Tg also had
a significant reduction in radiation-induced 8-OHdG (2.13 ± 0.21) compared to unsupplemented Tg (P < 0.0001). Levels
of 8-OHdG in supplemented mice were actually slightly lower
than constitutive levels observed in unirradiated unsupplemented
mice. There was also no significant difference in
radiation-induced 8-OHdG fluorescence between supplemented
Nr and supplemented Tg.
Our Tg mice have significantly elevated free radical production
and lipid peroxidation in several tissues (29). These biomarkers
strongly increase with age, correlate with longevity and
indicate that Tg mice are under chronic oxidative stress and
have accumulated significantly greater cellular oxidative damage
compared to Nr. Although spontaneous structural chromosome
aberrations in Tg were no greater than in Nr, unsupplemented Tg
expressed dramatically elevated levels of γH2AX (DSB) and
8-OHdG (oxidative base damage). These results indicate that
there is DNA damage produced by elevated free radicals in Tg
mice, but this does not manifest into genomic DNA damage
seen at the chromosome level.
H2AX has been termed the
‘histone guardian of the genome’ and may function in
chromatin structure alterations at sites of DNA damage. 
In contrast, Brown-Borg et al.  found elevated oxidative
stress to proteins in liver and brain of Tg, but DNA base
damage (8-OHdG) did not differ from controls. However,
inter-individual variation in oxidative biomarkers was very
large. Moreover, oxidized proteins increased with age in liver,
but appeared to decline with age in brain. There are a number
of possible explanations for oxidative stress observed in Tg
mice. GH axis distortion could increase oxidative stress via
alterations in mitochondria, especially via increased coupling
or altered ion channels. [33, 71–73] As in many models of
extended longevity such as birds and dietary restricted rodents , dwarf (GH deficient) mice were shown to generate lower
amounts of mitochondrial free radicals.  In this case,
reduced metabolic rate rather than changes in mitochondrial
coupling may be the cause. Sanz et al.  showed that
mitochondrial DNA damage was also reduced in dwarfs,
especially in brain.
Antioxidant defences may also be reduced in Tg mice. [38, 41, 42] GH receptor knockout mice show strongly elevated
hepatic superoxide dismutase that likely reflects downregulated
phoshoinositide-3 kinase (PI3K)/Akt signaling and
elevated forkhead box transcription factors (FOXO) activity.  GH administration also up-regulates enzymes involved in
glutathione degradation and down-regulates those involved in
synthesis.  Since the glutathione system is crucial for
cellular redox control, redox-regulated signalling, xenobiotic
and stress resistance, impacts on radiosensitivity might be
expected in Tg mice.
Membrane- and receptor-associated NAD(P)H oxidases
generate considerable free radicals that are essential for
mitogenic signalling. [33, 77, 78] There is also a linkage
between NAD(P)H oxidase activity and mitochondrial function.
Thus, growth itself may be associated with free radical
generation and this would likely be greatly accentuated in Tg
mice. [33, 73] Free radical-induced DNA damage can also
derive a state of genomic instability (increased rate of acquiring
DNA modifications) which may well occur in Tg mice (18).
Persistently elevated free radical processes associated with
genomic instability may be associated with dysregulated
mitochondria and reduced antioxidant activity. [18, 65]
NAD(P)H oxidase is also a candidate for the sustained
elevation of free radicals and genomic stability associated with
radiation exposure and the bystander effect. 
The absence of increased spontaneous chromosome aberrations
would suggest that high-fidelity repair processes
dominate in the bone marrow cells of our mice; however,
non-homologous end joining (NHEJ), an inherently errorprone
mechanism, is known to predominate in G0/G1 cells.
SKY is unable to discriminate the small deletions that typically
occur during NHEJ. Further examination, using techniques that
can detect small-scale deletions will be necessary to verify this
hypothesis. Depending on the location of DSBs, alterations or
loss of genetic material can increase cell dysfunction, genomic
instability and carcinogenesis. [80 ,81] It is interesting to note
that constitutive levels of γH2AX in Tg mice are initially higher
than other groups, indicating some residual damage, but decrease
significantly over the incubation time, no other group shows this
trend (Figure 3A). We suggest that increased numbers of DSBs
are being generated in Tg mice with endogenously elevated
reactive oxygen species (ROS), but when cells are removed from
the organismal milieu, DSB production declines and existing
DSBs are repaired. Also, the significant increase in constitutive
8-OHdG levels indicates unsupplemented Tg mice have
dramatically elevated oxidative base damage compared to Nr
mice (see Figure 4). This supports the contention that chronic
oxidative stress in Tg mice results in the accumulation of
considerable DNA damage with ageing. [82, 83]
We considered two possible outcomes of increasing oxidative
stress in Tg mice using ionizing gamma radiation. Adjustments
to offset endogenous free radical elevation could pre-adapt Tg
mice, so they might better cope with additional stress (the radiobiology
adaptive response). Alternatively, endogenous oxidative
stress in Tg mice may overtax defensive and repair abilities
making the mice hypersensitive to radiation-induced damage.
The dramatic increase in structural chromosome aberrations,
oxidative base damage and DSBs in unsupplemented irradiated
Tg versus comparable controls indicates that Tg may function
close to upper levels of manageable oxidative stress, indicating
Tg mice are a good model of elevated free radical processes.
All the spontaneous structural aberrations on both Tg and Nr
mice were chromosome-type aberrations with the apparent
absence in chromatid-type alterations. In radiation-induced
aberrations, unrepaired arm breaks with corresponding acentric
fragments comprised between 69.5 and 85.7% of all aberrations
depending on the experimental group. Translocations
were the next most frequent, followed by dicentric and ring
chromosomes. A small number of chromatid-type aberrations
were present in cells from irradiated mice; however, they only
occurred in unsupplemented animals. Since chromosome-type
aberrations occur in G1 phase of the cell cycle and the majority
of cells in exponentially growing stem cells (i.e. bone marrow)
are in G1, these results correspond well with what is currently
known in stem cell cycle processes.
The frequency of breaks in each chromosome does not
appear to be correlated to chromosome length, gene density or
total genes per chromosome (Table III); however, it must be
emphasized that given the elapsed time after irradiation, we
were only looking at misrepaired breaks. The total number of
breaks induced per chromosome by the radiation exposure
would be proportional to size. When total breaks per
chromosome are correlated to the number of chromosome
aberrations remaining after repair, a relationship to the effect of
Tg or diet may become apparent. Gene activity must be altered
in the different groups and one could argue this changing repair
bias on different chromosomes. [84, 85]
Untreated Nr and Tg mice had significantly higher 8-OHdG
levels after 2-Gy irradiation; however, both groups increased
by the same relative amount over constitutive levels (131 and
129% for Nr and Tg, respectively). This is not surprising given
both groups of mice should have approximately the same
number of free radicals generated within their tissue when given
the same dose of radiation. This finding also indicates that while
Tg mice have increased endogenous ROS production, it does not
appear to exacerbate the radiation-induced oxidative base
damage (i.e. is an additive effect, not synergistic).
The kinetics of radiation-induced γH2AX fluorescence are
the same in Nr and Tg mice, which indicates the rate of DSB
repair are similar in Nr and Tg mice. At the peak fluorescence
for γH2AX (30-min incubation), it was readily apparent that
unsupplemented Tg mice had significantly higher levels of
γH2AX fluorescence. Although absolute levels of γH2AX
were greater in Tg mice, the relative change from constitutive
levels did not differ from unsupplemented Nr mice (increases
of 54 and 56%, respectively). This suggests that the chronically
elevated free radicals in Tg mice did not exacerbate the
radiation-induced DBS production.
Similar to Tg mice, diseases that manifest as accelerated
ageing (i.e. Werner’s, Hutchinson–Gilford progeria syndromes)
and ‘senescence-accelerated mice’ exhibit elevated free radical
production and/or increased chromosome aberrations. [86–89]
These syndromes also display hypersensitivity to mutagens and
ionizing radiation, which cause significant increases in chromosome
instability compared to controls. Although Tg mice
show radiation-induced increases in all parameters of DNA
damage, but relatively minor constitutive DNA damage, they
demonstrate the hypersensitivity to ionizing radiation that is
characteristic of other accelerated ageing phenotypes. Therefore,
a critical attribute of the dietary supplement is that it not
only protects from direct effects of radiation but also over-rides
the ROS processes that influence the accelerated ageing
Antioxidants can protect against chronic oxidative stress
associated with ageing or disease [3, 90] and acute oxidative
insult such as those induced by irradiation or xenobiotics. [56, 91, 92] Limoli et al.  found that antioxidants reduced
radiation-induced cell mortality, the degree of DNA damage
and genomic instability. Antioxidants, including a-lipoic acid,
offset free radical elevations and stem cell dysfunctioning. [94, 95] Many individual ingredients in our supplement have
been tested as radioprotective agents , including most of
the components that have been postulated as potentially useful
in protecting astronauts from high-level radiation exposure.  Single antioxidants (i.e. ascorbate) administered after
irradiation have been shown to suppress long-lived radicals that
can otherwise cause mutations contributing to genomic
Gaziev et al. [50, 51] found that a multiple-ingredient
supplement was radioprotective for mice and human lymphocytes
(vitamins E, C, b-carotene, rutin, selenium and zinc).
Significant radioprotection of BALB/c mice and cells from
bone marrow and bladder were obtained with a combination of
vitamins E, C and vitamin A/b-carotene. [52, 53] A vitamin E
and C combination reduced bleeding and diarrhoea in patients
subjected to pelvic irradiation.  Given that the free radical
theory of ageing was born in radiation science , it is not
surprising that the 31 ingredients in our supplement include
most materials considered to be radioprotective (Table I). Our
results show that a combinatorial approach with multiple
targets and ingredients greatly enhances the radioprotection
afforded by dietary supplements.
Although a reduction in constitutive 8-OHdG and γH2AX
levels was evident in all diet-supplemented mice, it was only
significant in Tg mice. This shows that the dietary supplement
can provide protection from DNA damage resulting from
constitutive cellular processes in normal mice but is particularly
effective in systems that generate excessive amounts of
The dietary supplement provided considerable protection in
all parameters of radiation-induced DNA damage examined in
both Nr and Tg mice. Radiation-induced chromosome aberrations
and oxidative base damage did not differ from constitutive
levels in both groups of diet-supplemented animals,
indicating that even after considerable exposure to radiation the
dietary supplement was able to protect cells from radiationinduced
damage. Since the majority of cellular damage produced
by gamma radiation is indirect, through the production of free
radicals via the hydrolysis of water, rather than by the energy
tract itself, it is likely that the levels of cellular antioxidants are
high enough in supplemented mice to effectively scavenge the
excess ROS produced by the radiation dose.  The small
number of radiation-induced chromosome aberrations in supplemented
mice was likely produced by unscavenged free radicals
or could result directly from the radiation tract.
In conclusion, Tg mice have significantly increased
constitutive DNA damage compared to normal mice and based
on chromosome aberration induction are more vulnerable to
radiation-induced exposure compared to age-matched normal
mice. Our complex dietary supplement provided substantial
protection from constitutive DSBs and oxidative base damage
in both groups of supplemented mice, a strong indication that
the supplement provides meaningful protection from damage
induced by endogenous ROS production.
Even after a substantial dose of ionizing radiation, the
dietary supplement provided significant protection to DNA,
preventing DSBs, chromosome aberrations and oxidative base
damage in both supplemented Nr and supplemented Tg mice.
The defence against ROS-associated damage provided by the
dietary supplement has clearly demonstrated that a holistic
approach provides far greater protection and support for the
maintenance of normal cell functioning than supplementation
with single or a small number of compounds. This diet could
therefore have important applications for protecting individuals
exposed to high levels of occupational radiation such as
astronauts. This protective effect would also apply to individuals
with genetic predisposition to elevated free radicals. Furthermore,
protection of normal tissue during radiation therapy
could be another important use for this dietary supplement. We
conclude that this diet formulation is a safe and potent
National Science and Engineering Research Council (238495),
Candu Owners Group (03.014) and the Chemical Biological
Radiological and Nuclear Research and Technology Initiative
The authors acknowledge the expert technical assistance of Sarah Laronde,
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