Mutagenesis. 2008 (Nov); 23 (6): 465–472 ~ FULL TEXT
Lemon JA, Rollo CD, McFarlane NM, Boreham DR.
Department of Medical Physics and Applied Radiation Sciences,
1280 Main Street West,
Hamilton, Ontario L8S 4K1,
This study examined whether radiation sensitivity measured by lymphocyte apoptosis could be ameliorated by a complex anti-oxidant/anti-ageing dietary supplement. We also examined lymphocytes from both genders of normal (Nr) mice as well as transgenic growth hormone (Tg) mice that express strongly elevated reactive oxygen species processes and a progeroid syndrome of accelerated ageing. We introduce Tg mice as a potentially valuable new model to study radiation sensitivity. Isolated lymphocytes from all experimental groups were exposed to gamma radiation and the time course of apoptosis was measured in vitro. Kinetics of radiation-induced apoptosis was similar among groups, which peaked at 8 h, but maximal levels differed significantly between groups. Nr male mice had 60% lower levels of radiation-induced apoptosis than Tg males, supporting our hypothesis that Tg mice would be radiation sensitive. The dietary supplement protected lymphocytes in male mice of both strains, with proportionally greater reductions in Tg mice. Lymphocytes from female mice (both Nr and Tg) were highly radiation resistant compared to males and the supplement provided no additional benefit at the doses used in this study. These results highlight that radiation-induced apoptosis is complex and is modified by genotype, dietary supplements and gender.
From the FULL TEXT Article:
Oxidative stress, predominantly from increased reactive
oxygen species (ROS), is a potent inducer of apoptosis in
a wide variety of cells. Increased production of ROS (i.e.
through exposure to ionizing radiation) contributes to the
initiation of apoptosis in several ways. Key processes include
altering the redox status of cells  and contributing significant
damage to cellular macromolecules (lipids, DNA and proteins)  and subcellular organelles of which mitochondria are
particularly susceptible. [3, 4] These perturbations can result in
decreased cellular efficiency and functionality, as well as
a further increase in ROS production , creating an escalating
cycle of oxidative stress-induced apoptosis.
Cells have extensive protective mechanisms against oxidative
damage. Enzymatic and non-enzymatic antioxidants
remove free radicals before they cause damage; while recycling
and repair mechanisms remove or repair ROS-damaged
macromolecules and organelles. However, if the damage is
too extensive, cells are removed through apoptotic or necrotic
processes. Several strains of mice with compromised antioxidant
systems have illustrated that increases in oxidative stress
exacerbates apoptotic cell loss, reduces lifespan and increases
the prevalence of age-associated diseases such as cancer and
neuropathologies. [5, 6]
The use of antioxidants and other compounds to ameliorate
the physiological signs of oxidative stress have been investigated
for decades. However, with few exceptions [7, 8],
most studies, which have tested materials alone or a few in
combination, yielded poor or inconsistent results when looking
at higher-order end points such as cognitive ability and
longevity. [9–11] We developed a complex dietary supplement
comprised of 31 ingredients with well-documented effects
known to reduce oxidative stress and inflammation, promote
membrane and mitochondrial integrity and/or increase insulin
sensitivity. [12, 13]
Transgenic growth hormone (Tg) mice have highly elevated
free radical processes in all tissues examined and express
a progeroid syndrome resembling accelerated ageing. Characteristically,
Tg mice have a shortened lifespan (50% that of Nr
mice) and early onset of symptoms associated with senescence
in mice including arthritis, reduced activity, cognitive decline,
cataracts, sarcopenia and poor fur quality. [12, 14–20] Younger
Tg mice possess remarkable learning and memory abilities,
learning an eight-choice radial maze roughly twice as quickly
as normal controls. [12,21] Within 11 months, however, 95%
of Tg mice were unable to learn, whereas Nr mice showed no
cognitive changes at that age.  The complex dietary
supplement completely abolished the age-related cognitive
decline of Tg mice. 
The dietary supplement significantly extended lifespan in
both Nr and Tg mice. Mean longevity of Nr mice is extended
11 ± 2% [mean ± standard error (SE)], whereas there was
a much larger and significant increase of 28 ± 1% for Tg mice.  Comparison of the physical condition of 12-month-old
Tg with age-matched Tg mice on the supplement showed
amelioration of most symptoms of ageing including cataracts,
sarcopenia and arthritis, coat quality and locomotor activity.  The superior physical condition of supplemented Tg mice
continued for several months after most unsupplemented Tg
mice had died.  Given the powerful effects of the diet
supplement on cognition and longevity and the fact that most
of the five targets of the supplement directly or indirectly relate
to free radical processes, we postulated that the dietary
supplement was altering the effects of elevated ROS and
associated processes, as such there may be a systemic effect of
the supplement which could be radioprotective.
We also considered gender in radiation-induced apoptosis of
lymphocytes. There have been numerous reports showing that
there is a significant gender difference in apoptosis in several
species including mice and humans. [22–25] Oestrogen and
progesterone have anti-apoptotic actions on several tissues
including brain [25, 26], thymus , heart [28, 29] and
lymphocytes. [22, 24, 30] Alternatively, testosterone is generally
pro-apoptotic. [22, 27, 31, 32] These actions are mediated
through several receptor-independent and -dependent effectors,
including regulation of the Bcl-2 proto-oncogene family  (22,25) and other proto-oncogenes and oncosuppressor genes , tumour necrosis factor-a [24, 27], modification of signal
transduction pathways [34–38] and modifying oxidative stress
and inflammation processes. [39–42] The specific actions of
sex hormones and their signalling are tissue specific and remain
largely unexplored with respect to radiation.
Lymphocyte apoptosis is an established biomarker of
oxidative stress. Numerous methods are available to measure
this end point and advances in technology have made it a fast
and reliable test used in a broad range of applications from
markers of age-related diseases to biodosimetry and indicators
of radiation risk. [43–46] In this study we have compared
radiation-induced lymphocyte apoptosis in Nr mice and
oxidatively stressed Tg mice. We have tested the postulate
that a complex dietary supplement can alter radiation-induced
apoptosis in lymphocytes and that gender affects the frequency
of apoptotic death.
Materials and methods
Experimental animals were Nr and Tg male and female C57Bl-6J/SJL mice. Tg
mice have metallothionein promoters fused to rat growth hormone (GH)
structural genes.  The rat GH genes are incorporated into the mouse
genome thereby chronically elevating plasma GH levels .100-fold. Tg mice
can be identified morphologically by their significantly larger size by 28 days of
age; this technique has been shown to be extremely reliable, negating the
necessity for continuous molecular testing. [47, 48] The kinetics of radiationinduced
apoptosis in lymphocytes used 48 mice aged 11–12 months, divided
into eight experimental groups (six mice per group; Figure 1). The dose–
response experiments consisted of 12 mice in four experimental groups (three
mice per group). A maximum of four mice were maintained per cage (27 x 12 x 15.5 cm) containing woodchip bedding (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 were approved by the Animal
Research Ethics Board at McMaster University and carried out according to the
Canadian Council on Animal Care regulations.
The anti-ageing supplement was designed to simultaneously ameliorate five
major processes implicated in ageing (oxidative stress, inflammatory processes,
insulin resistance, membrane deterioration and mitochondrial dysfunction).
Criteria for selecting materials for the supplement were as follows:
(i) scientifically documented evidence as effective for one or more of the targeted features,
(ii) can be taken orally,
(iii) ‘over the counter’ products and
(iv) safe for mouse (and human) consumption.
Dosages for the mice were reformulated based on amounts commonly
prescribed to humans. Values were adjusted for the difference in body size and
then increased by a factor of 10 in consideration of the higher gram-specific
metabolic rate (and faster nutrient utilization) of mice.  Details of the
ingredients and associated targets are outlined in Table I. Dosages and
preparation of the diet have been previously described. [12, 13] The supplement
was prepared in liquid form and a 0.4 ml volume was absorbed into a 1 x 1.5 x 1 cm piece of bagel and allowed to air-dry. Each mouse received one piece of
dried bagel daily either with or without the supplement, depending on the
treatment group. The mice were given the bagel pieces midway through the
photoperiod. The bagel pieces were always immediately and completely
ingested by the mice, ensuring mice obtained full and equivalent doses. Mice
were fed the supplement for a minimum of 3 months prior to testing to allow
the compounds within the supplement to reach equilibrium at maximal
Sample collection and preparation
Mice were anesthetized with Isoflurane™ and blood obtained via cardiac
puncture. Samples were kept on ice during preparation unless otherwise stated.
The whole blood was diluted 1:1 in complete RPMI 1640 growth media (10%
foetal bovine serum, 1% L-glutamine and 1% penicillin–streptomycin; all
components from Invitrogen, Mississauga, Ontario, Canada). Half of the blood
from each mouse held at 0°C was irradiated with gamma radiation from a 137Cs
source (0.25 Gy/min dose rate). The remaining volume of blood under the same
conditions was used as the sham-irradiated control. After irradiation, the red
blood cells were removed by lysis using ammonium chloride solution [154 nM
ammonium chloride + 1.5 mM potassium bicarbonate 0.1 mM ethylenediaminetetraacetic
acid (EDTA); at room temperature for 15 min]. The
white blood cells were centrifuged at 200 x g for 10 min at room temperature,
washed once with phosphate-buffered saline (PBS; 137 mM NaCl + 2.7 mM
KCl + 4.3 mM Na2HPO4 + 1.4 mM KH2PO4 + 0.1 mM EDTA), resuspended
in 2.5 ml complete RPMI 1640 and incubated at 37°C (5% CO2,
98% humidity) for designated time periods (see below).
Sample staining and flow cytometry
Annexin V was used as the indicator of apoptosis for this assay with 7-amino
actinomycin D (7-AAD) as the counterstain. The reagents were purchased as
a commercial kit (Annexin V–FITC/7-AAD; IM3614, Beckman Coulter,
Miami, FL). Annexin V is a molecule with a high affinity for phosphatidylserine
residues that become exposed on the external plasma membrane of
apoptotic lymphocytes.  In later stages of apoptosis, plasma membrane
integrity is lost, allowing 7-AAD to enter the cell and bind to DNA. The
combination of the Annexin V and 7-AAD allowed the enumeration of the
various stages of apoptosis. In all groups of mice, apoptotic cells were
identified as Annexin V-positive and Annexin V–7-AAD-positive lymphocytes.
The relative levels of Annexin V-positive to Annexin V + 7-AADpositive
cells did not differ based on genotype or gender and were consistent
between all groups of mice. Loss of Annexin V positivity requires degradation
and loss of the cell membrane, as such, events identified as Annexin V negative
and 7-AAD positive were not included in the analysis.
Since apoptosis is the predominant mode of death (.99%) for lymphocytes,
it was not necessary to further differentiate between apoptotic and necrotic
Cell suspensions were incubated for 0, 1, 2, 4, 6 and 8 h and analysed for
apoptosis as described above. A cell suspension of 4 x 105 cells was washed
with 4 ml PBS + 0.1 mM EDTA and re-suspended in 100 µl of 1x Binding
Buffer (supplied in kit). To each 5-ml polypropylene assay tube, 10 µl Annexin
V–FITC and 20 ll 7-AAD were added and incubated for 15 min on ice as per
the manufacturer’s instructions. An additional 400 µl of 1x Binding Buffer was
then added to each assay tube and analysed within 30 min on a Beckman-
Coulter Epics XL flow cytometer. A minimum of 1.5 x 104 lymphocytes were
analysed from each sample.
All values were represented as the mean and SE of the mean. Student’s t-tests
were performed to determine if significant differences existed between groups.
Analysis of variance (ANOVA) was carried out to clarify which (if any) of the
independent variables (i.e. dietary supplement, gender and genotype) associated
with the groups of mice contributed significantly to the differences in mean
apoptosis and whether those factors had any interactive effects.
Kinetics of apoptosis
Spontaneous and radiation-induced apoptosis was monitored in
all groups of mice (Figure 1, Groups A–P,) over 12-h postirradiation
to determine if differences in kinetics existed
between experimental groups (Figure 2A and B). In all groups,
both spontaneous and radiation-induced apoptotic levels from
in vitro samples peaked at 8 h. Levels plateaued out to 12 h,
after which a continuous decline in levels of apoptotic cells was
observed out to 16 h (data not shown). Since maximal
differences between groups were observed after an incubation
period of 8 h, this incubation time was used for all remaining
analyses. Although the number of apoptotic lymphocytes varied
between groups, all groups demonstrated similar kinetic patterns
during the 8-h incubation period (Figure 2A and B). There was
no difference between spontaneous and radiation-induced
apoptosis for any group of mice between 0 and 2 h incubation
and overall levels of apoptosis did not increase during this time
period. The proportion of apoptotic lymphocytes then began to
increase substantially at 4 h and continued to increase up to 8 h
(Figure 2A and B). At 8 h, male Tg mice (Figure 1, group L)
had the highest level of apoptosis after a 2 Gy dose (Figures 2A
and 4). Nr male mice were the next most sensitive (Figure 1,
Group D). The female mouse lymphocytes were much more
resistant than males and did not differ significantly from each
other (Nr versus Tg) (Figure 1, Groups B and J).
The dose of radiation was proportional to the apoptotic
response for all unsupplemented mice (Groups B, D, J and
L) (Figure 3). Lymphocytes from male Tg mice had
significantly higher levels of apoptosis compared to Nr male
mice and both groups of female mice at doses of ≥2 Gy and
above (2 Gy: P < 0.005, 4 Gy: P < 0.01 and 6 Gy: P < 0.04).
Female Nr and Tg mice had apoptosis levels that were not
significantly different from each other at any dose (P > 0.05),
although female Tg mice demonstrate moderately greater
radiosensitivity >4 Gy. Female mice had significantly lower
levels of lymphocyte apoptosis than Nr and Tg males at doses
of ≥2 Gy (P < 0.01; Figure 3).
Unirradiated lymphocytes in (Figure 1, Groups A, C, E, G, I,
K, M and O) exhibited spontaneous induction of apoptosis
in vitro. The frequency of spontaneous apoptosis was subtracted
from the respective irradiated group to distinguish radiationinduced
levels. After 8-h incubation at 37°C, there was no
significant difference in spontaneous levels (0 Gy dose groups)
of lymphocyte apoptosis between unsupplemented Nr and Tg
males (26.87 ± 0.96 and 27.67 ± 4.08%, respectively, P < 0.85; Figure 3). Spontaneous apoptosis was also similar in
unsupplemented Nr females (23.70 ± 2.16%) and Tg females
(17.73 ± 3.30%; P < 0.16). Spontaneous lymphocyte
apoptosis in diet-supplemented male Nr mice was 34.98 ± 3.17% which was not statistically significantly different (P < 0.11) from diet-supplemented male Tg mice at 22.47 ± 6.46%.
Spontaneous lymphocyte apoptosis in diet-supplemented
female Nr mice (25.23 ± 4.49%) was similar to supplemented
female Tg mice (19.87 ± 2.50%; P < 0.32). When
spontaneous lymphocyte apoptosis was compared within
genotypes, unsupplemented male Nr mice had a similar level
of apoptotic lymphocytes compared to diet-supplemented male
Nr mice (P <0.73). Unsupplemented and diet-supplemented
male Tg mice did not differ significantly (P <0.11). There was
also no significant difference between unsupplemented and
diet-supplemented female Nr mice (P <0.50) or unsupplemented
and diet-supplemented female Tg mice (P < 0.62).
There was no significant difference in spontaneous apoptosis
between male and female mice in any experimental group (P > 0.09 in all cases).
Diet and radiation-induced apoptosis: dependence on gender and genotype
After an exposure of 2 Gy, there was no significant difference
in the levels of apoptosis in lymphocytes from female mice in
any group (Groups B, F, J and N) (Figure 4). Diet, genotype
and gender did not appear to affect the radiation response of
lymphocytes from females (P < 0.17). However, there were
large differences in radiation sensitivity in lymphocytes from
male mice (Groups D, H, L and P) depending on the genotype
and if the diet supplement was given (Figure 4). In Nr male
mice on the standard diet, lymphocyte sensitivity was
significantly higher than any female group (24.11 ± 0.54%;
P < 0.010), but Tg males were nearly twice as sensitive as the
Nr males (40.39 ± 4.19%; P < 0.00023). Lymphocytes from
diet-supplemented Nr mice had lower levels of apoptosis, but
the decrease was not statistically significant (17.97 ± 3.91%;
P < 0.15). However, diet-supplemented Tg mice had a 68%
reduction in radiation-induced apoptosis levels (13.14 ± 3.49%; P < 0.00054) compared to unsupplemented Tg mice (Figure 4).
ANOVA (Table II) determined that individually, the variables
‘gender’ and ‘supplementation’, but not ‘genotype’ significantly
influenced the level of radiation-induced apoptosis. The
interactive effect of variables genotype and ‘diet supplement’
also significantly influenced radiation-induced apoptosis.
Significant interactions were also found for genotype, gender
and diet supplement (Table II).
Lymphocytes are frequently used for studying the physiological
impacts of radiation since immunocytes are relatively
radiosensitive, readily accessible and relatively long lived.
Lymphocyte apoptosis is a well-established and relatively
specific biomarker for radiation-induced damage and free
radical impacts. There is great interest in developing
interventions to prevent radiation damage to biological tissues
(particularly ingestible substrates) but to date, there are few
materials with exceptional efficacy. [8–11]
Apoptosis was examined in Nr mice, which are assumed to
have normal metabolism and free radical production representative
of a typical mouse population, and Tg mice, which have
substantially elevated levels of endogenous free radicals. 
We hypothesized that the apoptosis levels in Tg mice would be
higher under normal growth conditions because of increased
oxidative stress. However, although Tg mice had significantly
higher endogenous ROS production, spontaneous levels of
lymphocyte apoptosis were not altered and were essentially
identical for all groups of mice; these results did not support
our postulate that spontaneous apoptosis levels might be
different in these two mouse strains. There may be several
reasons for this, including the possibility that Tg cells were
able to adapt to the effects of the chronically elevated ROS
through up-regulation in cellular recycling and repair mechanisms
and/or antioxidant systems  and cope with tissue
culture stress like normal cells. The increased radiosensitivity
of Tg mice indicates that these systems may be saturated and
are unable to cope with the increased radiation-induced ROS.
Alternatively, the elevated endogenous ROS production could
suppress the ability of the Tg lymphocytes to undergo
apoptosis since chronic low doses of ionizing radiation have
been shown to suppress leukocyte apoptosis.  Although the
treatment used by Joksic and Pretrovic  involves radiationinduced
generation of ROS, the outcome is likely to be similar
to that in Tg cells with endogenous ROS. Either postulate
could also be exacerbated by the known anti-apoptotic actions
of GH and its downstream effector, insulin-like growth factor-I
(IGF-I), both strongly up-regulated in Tg mice, which could
increase the apoptotic threshold of Tg cells. [53, 54] Interestingly,
all groups of female mice had similar levels of
spontaneous apoptosis, which were lower than that of the male
mice (data not shown), indicating that, regardless of the level of
cellular oxidative damage, the anti-apoptotic actions of
oestrogen and progesterone may play an important role in
reducing lymphocyte apoptosis in these mice.
Radiation-induced apoptosis differed significantly between
groups of male mice. Unsupplemented Nr and Tg males had
highly elevated apoptosis following irradiation, with unsupplemented
Tg showing the greatest radiation sensitivity >1 Gy
compared to any other group of mice (Figure 3). Consequently,
our hypothesis of increased radiation sensitivity was confirmed,
despite the lack of spontaneous/basal response observed in
unirradiated cells. Although chronic low doses of ionizing
radiation/free radicals can ameliorate leukocyte apoptosis ,
the mechanism probably involves adaptive up-regulation of
stress-response systems against a background of otherwise
normal endogenous ROS generation. This mechanism may be
otherwise engaged in Tg mice, thus up-regulation of
endogenous repair, replacement and defensive systems to
offset elevated ROS processes in Tg mice is consistent with
increased radiosensitivity if these systems are stressed to
limited capacity. In that case, further ROS generated by
radiation would overwhelm these protective capabilities. It is
also likely that general levels of oxidative damage in Tg mice
are higher (see above) before radiation exposure [18–20], so
that further damage triggers apoptosis, even though the
threshold is set higher by anti-apoptotic regulatory impacts of
GH and IGF-1.
As might be expected from their low spontaneous levels of
apoptosis in vitro, female mice demonstrated greater radioresistance
than male mice at doses >2 Gy (Figure 3). There
was also no significant difference in radiation-induced
apoptosis between any groups of female mice. These results
are supported by other studies which indicate that females of
other mammalian species also show greater radioresistance
than their male counterparts. [53, 54] Perhaps in females, the
apoptotic threshold is higher such that radiation impacts are
ameliorated or absorbed and are not significant enough to elicit
the response. This might explain why the diet had no apparent
benefit in females. We are currently assessing other biomarkers
of ROS damage since it is possible that females do derive
benefit from the diet, despite the fact that apoptosis does not
It is possible that the anti-apoptotic actions of
oestrogen and progesterone [22, 24, 25, 28, 36] are reducing
lymphocyte apoptosis in Nr females and could be interacting
either additively or synergistically with the anti-apoptotic
effects of GH/IGF-I [55–58] in female Tg mice, completely
masking the effects of increased oxidative damage caused
by radiation. This would likely work in combination with
the antioxidant activity of oestrogen [40, 41] which of itself
could reduce oxidative damage in female mice, thereby
reducing both spontaneous and radiation-induced apoptosis.
Impact of the dietary supplement
There is likely an imbalance in the two major subsets of
antioxidants (endogenous versus nutritional) in cells experiencing
chronic oxidative stress in Tg mice. Typically, there is
an up-regulation of enzymatic antioxidants in response to the
redox status of the cells (as long as sufficient substrates are
available) , with a concomitant depletion of non-enzymatic
antioxidants unless replenished from some external source (i.e.
through diet). [60, 61] The results for supplemented mice
suggest that chronic oxidative stress in Tg cells has depleted
non-enzymatic antioxidants, and possibly enzymatic antioxidants,
although further study is required for confirmation.
Radiation-induced apoptosis is significantly reduced in supplemented
male mice and there was a trend (while not significant)
for a reduction in lymphocyte apoptosis in supplemented
female mice that were already resistant.
We speculate that the reduction in radiation-induced
apoptosis could be due to two aspects of the dietary
(i) The first may be higher inter- and intracellular concentrations
of enzymatic and non-enzymatic antioxidants that
scavenge ROS before they can damage cellular macromolecules.
The baseline level of damage to the cellular
components of male Nr and Tg mice on the dietary supplement
is likely to be reduced compared to unsupplemented
mice since the increase in antioxidants provides a protective
effect from on-going cellular ROS production as well as for
acute radiation insult.  The supplement was designed to
provide antioxidant protection to all critical cell components,
including nuclear material, membranes, the cytosol and
associated proteins and subcellular organelles (particularly
the mitochondria), using compounds with documented
specific protective effects for each of these subcellular regions.  Reduction in the quantity of oxidatively modified
cellular components allows recycling and repair processes to
function more effectively, an issue particularly important in
older organisms since these processes appear to be compromised
in senescent animals. [62–65]
(ii) The second effect may be an amelioration of processes
associated with oxidative stress, which can further increase
ROS production and cellular oxidative stress. These include
disruption of mitochondrial metabolism and reduced electron
transport chain substrate availability, resulting in
reductions in ATP production [64, 66], impaired glucose
metabolism [67–69] and inflammatory processes typically
associated with oxidative damage [70, 71], which are
normally exacerbated in senescent animals [64, 72]. The
lower radiation-induced apoptosis in diet-supplemented Nr
and Tg mice lend support to the idea that a broad-spectrum
dietary supplement can provide a significant protective
effect, even to cells near limiting capacity (Tg mice) or
exposed to an acute increase in ROS damage due to radiation
exposure. While it is unknown if some factors provide
greater protective effects than others, it has been established
that several of the components of the supplement act
additively or synergistically. [73–75] It is also likely that the
immediate free radical scavenging effects of some of the
components and the factors that provide additional support
for the processes associated with oxidative stress act synergistically
to enhance the supplement’s overall protective
Time course studies showed that the kinetics of lymphocyte
apoptosis among all groups was similar despite GH levels,
gender or diet supplementation. Only the magnitude of the
response varied; we postulate that this indicates that overall
initial damage levels must be altered in the various groups due
to enhanced free radical scavenging and/or DNA repair
This study emphasizes the importance of genotype, gender
and diet on the modulation of radiation response. The data
support the contention that male Tg mice provide an excellent
model to study the effects of long-term oxidative stress and its
effects on radiation response in male mice only. There is
a relative paucity of studies addressing gender differences in
radiation biology. The striking sexual dichotomy in apoptotic
response indicates female mice may prove to be an interesting
model for radiation risk modification associated with apoptotic
mechanisms. Although results vary, males and females generally
acquire oxidative damage at the same rate. [76–78]
Unsupplemented Tg mice (both genders) accumulate greater
oxidative damage to cellular components than age-matched
unsupplemented normals , which would imply that Tg
mice are less able to adequately respond to additional oxidative
stress from external sources (i.e. ionizing radiation) and more
importantly, from the escalating ROS production normally
associated with ageing [2, 4, 10, 72]. However, cells from female
mice (Nr and Tg) appear to have the capacity to cope with
greater amounts of oxidative damage before apoptotic processes
are triggered. The fact that females of both genotypes
are similar suggests that the regulation of the threshold in
females differs from males, and was not affected by GH
transgenesis or background ROS processes implying that
oestrogen may have a priority role in determining an apoptotic
threshold over other factors.
The greater overall resistance to radiation-induced apoptosis
demonstrated by female mice requires further study to
determine what processes (i.e. involving sex hormones or
other factors) are contributing to this increased resistance. This
might be extended to considerations of dwarf (GH deficient)
mice that were recently shown to express increased resistance
to free radical stress than normal animals. [79, 80]
Regardless of genetic factors that influence radiation-induced
apoptosis (i.e. Nr versus Tg mice), we have shown that diet can
change the probability of a cell undergoing radiation-induced
apoptosis. Since apoptosis is regarded as an essential
mechanism associated with genomic instability, dietary supplements
could have important ramifications for human diseases
associated with apoptosis, such as ageing and cancer, and
health risks from exposure to environmental mutagens and
carcinogens. We have also demonstrated that gender plays
a significant role in the modulation of apoptosis, indicating that
the use of apoptosis for issues such as biodosimetry and risk
assessment may be problematic if gender is not taken into
CANDU Owners Group (03.014), the Chemical Biological
Radiological Nuclear Research and Technology Initiative
(015ssH1021-021608) and the National Science and Engineering
Research Council (238495).
Conflict of interest statement: None declared.
Haddad, J. J. (2004) Redox and oxidant-mediated regulation of apoptosis
signaling pathways: immuno-pharmaco-redox conception of oxidative
siege versus cell death commitment. Int. Immunopharmacol., 4, 475–493.
Beckman, K. B. and Ames, B. N. (1998) The free radical theory of aging
matures. Physiol. Rev., 78, 547–581.
Genova, M. L., Pich, M. M., Bernacchia, A. et al. (2004) The
mitochondrial production of reactive oxygen species in relation to aging
and pathology. Ann. N. Y. Acad. Sci., 1011, 86–100.
Rafique, R., Schapira, A. H. and Coper, J. M. (2004) Mitochondrial
respiratory chain dysfunction in ageing; influence of vitamin E deficiency.
Free Radic. Res., 38, 157–165.
Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P. P.,
Lafrancone, L. and Pelicci, P. G. (1999) The p66shc adaptor protein controls
oxidative stress response and life span in mammals. Nature, 402, 309–313.
Kokoszka, J. E., Coskun, P., Esposito, L. A. and Wallace, D. C. (2001)
Increased mitochondrial oxidative stress in the Sod2(þ/) mouse results in
the age-related decline of mitochondrial function culminating in increased
apoptosis. Proc. Natl Acad. Sci., 98, 2278–2283.
Liu, J., Head, E., Gharib, A. M., Yuan, W., Ingersoll, R. T., Hagen, T. M.,
Cotman, C. W. and Ames, B. N. (2002) Memory loss in old rats is
associated with brain mitochondrial decay and RNA/DNA oxidation:
partial reversal by feeding acetyl-L-carnitine and/or R-a-lipoic acid. Proc.
Natl Acad. Sci., 99, 2356–2361.
Qin, X.-J., He, W., Hai, C.-X., Liang, X. and Liu, R. (2008) Protection of
multiple antioxidants Chinese herbal medicine on the oxidative stress
induced by adriamycin chemotherapy. J. Appl. Toxicol., 28, 271–282.
Blacker, D. (2005) Mild cognitive impairment—no benefit from vitamin E,
little from donepezil. N. Engl. J. Med., 352, 2439–2441.
Lee, C. K., Pugh, T. D., Klopp, R. G., Edwards, J., Allison, D. B.,
Weindruch, R. and Prolla, T. A. (2004) The impact of alpha-lipoic acid,
coenzyme Q10 and caloric restriction on life span and gene expression
patterns in mice. Free Radic. Biol. Med., 36, 1043–1057.
Yaffe, K., Clemons, T. E., McBee, W. L. and Lindblad, A. S. (2004)
Impact of antioxidants, zinc, and copper on cognition in the elderly:
a randomized, controlled trial. Neurology, 63, 1705–1707.
Lemon, J. A., Boreham, D. R. and Rollo, C. D. (2003) A dietary
supplement abolishes age-related cognitive decline in transgenic mice
expressing elevated free radical processes. Exp. Biol. Med., 228, 800–810.
Lemon, J. A., Boreham, D. R. and Rollo, C. D. (2005) A complex dietary
supplement extends longevity in mice. J. Gerontol. Biol. Med. Sci., 60A,
Steger, R. W., Bartke, A. and Cecim, M. (1993) Premature ageing in
transgenic mice expressing growth hormone genes. J. Reprod. Fertil.
46 (Suppl), 61–75.
Wolf, E., Kahnt, E., Ehrlein, J., Hermanns, W., Brem, G. and Wanke, R.
(1993) Effects of long-term elevated serum levels of growth hormone on
life expectancy of mice: lessons from transgenic animal models. Mech.
Ageing Dev., 68, 71–87.
Meliska, C. J., Burke, P. A., Bartke, A. and Jensen, R. A. (1997) Inhibitory
avoidance and appetitive learning in aged normal mice: comparison with
transgenic mice having elevated plasma growth hormone levels. Neurobiol.
Learn. Mem., 68, 1–12.
Ogueta, S., Olazabal, I., Santos, I., Delgado-Baeza, E. and Garcia-
Ruiz, J. P. (2000) Transgenic mice expressing bovine GH develop arthritic
disorder and self-antibodies. J. Endocrinol., 165, 321–328.
Carlson, J. C., Bharadwaj, R. and Bartke, A. (1999) Oxidative stress in
hypopituitary dwarf mice and in transgenic mice overexpressing human and
bovine GH. Age Ageing, 22, 181–186.
Rollo, C. D., Carlson, J. and Sawada, M. (1996) Accelerated aging of giant
transgenic mice is associated with elevated free radical processes. Can. J.
Zool., 74, 606–620.
Hauke, S. J. and Bartke, A. (2001) Free radical defenses in the liver and
kidney of human growth hormone transgenic mice: possible mechanisms of
early mortality. J. Gerontol. Biol. Sci., 56, B153–B162.
Rollo, C. D., Ko, C. V., Tyerman, J. G. A. and Kajiura, L. (1999) The
growth hormone axis and cognition: empirical results and integrated theory
derived from giant transgenic mice. Can. J. Zool., 77, 1874–1890.
Huber, S. A., Kupperman, J. and Newell, M. K. (1999) Estradiol prevents
and testosterone promotes Fas-dependent apoptosis in CD4þ Th2 cells by
altering Bcl 2 expression. Lupus, 8, 384–387.
Hofmann-Lehmann, R., Holznagel, E. and Lutz, H. (1998) Female cats
have lower rates of apoptosis in peripheral blood lymphocytes than male
cats: correlation with estradiol-17b, but not with progesterone blood levels.
Vet. Immunol. Immunopathol., 65, 151–160.
Evans, M. J., MacLaughlin, S., Marvin, R. D. and Abdou, N. I. (1997)
Estrogen decreases in vitro apoptosis of peripheral blood mononuclear cells
from women with normal menstrual cycles and decreases TNF-alpha
production in SLE but not in normal cultures. Clin. Immunol. Immunopathol.,
Yao, X.-L., Liu, J., Lee, E., Ling, G. S. F. and McCabe, J. T. (2005)
Progesterone differentially regulates pro- and anti-apoptotic gene expression
in the cerebral cortex following traumatic brain injury in rats. J.
Neurotrauma, 22, 656–668.
Bramlett, H. M. (2005) Sex differences and the effect of hormonal therapy
on ischemic brain injury. Pathophysiology, 12, 17–27.
Guevara Patino, J. A., Marino, M. W., Ivanov, V. N. and Nikolich-
Zugich, J. (2000) Sex steroids induce apoptosis of CD8þCD4þ doublepositive
thymocytes via TNF-a. Eur. J. Immunol., 30, 2586–2592.
Boddaert, J., Mallat, Z., Fornes, P., Esposito, B., Lecomte, D., Verny, M.,
Tedgui, A. and Belmin, J. (2005) Age and gender effects on apoptosis in
the human coronary arterial wall. Mech. Ageing Dev., 126, 678–684.
Patten, R. D., Pourati, I., Aronovitz, M. J. et al. (2004) 17b-Estradiol
reduces cardiomyocyte apoptosis in vivo and in vitro via activation of
phospho-inositide-3 kinase/Akt signaling. Circ. Res., 95, 692–699.
Shirshev, S. V., Kuklina, E. M. and Yarilin, A. A. (2003) Role of
reproductive hormones in control of apoptosis of t-lymphocytes. Biochemistry
(Mosc.), 68, 577–583.
Verzola, D., Gandolfo, M. T., Salvatore, F., Villaggio, B., Gianiorio, F.,
Traverso, P., Deferrari, G. and Garibotto, G. (2004) Testosterone
promotes apoptotic damage in human renal tubular cells. Kidney Int., 65,
Dulos, G. J. and Bagchus, W. M. (2001) Androgens indirectly accelerate
thymocyte apoptosis. Int. Immunopharmacol., 1, 321–328.
Cutolo, M., Sulli, A., Seriolo, B., Accardo, S. and Masi, A. T. (1995)
Estrogens, the immune response and autoimmunity. Clin. Exp. Rheumatol.,
Vilatoba, M., Eckstein, C., Bilbao, G., Frennete, L., Eckhoff, D. E. and
Contreras, J. L. (2005) 17b-Estradiol differentially activates mitogenactivated
protein-kinases and improves survival following reperfusion
injury of reduced-size liver in mice. Transplant. Proc., 37, 399–403.
Lu, A., Ran, R. Q., Clark, J., Reilly, M., Nee, A. and Sharp, F. R. (2002)
Estradiol induces heat shock proteins in brain arteries and potentiates
ischemic heat shock induction in glia and neurons. J. Cereb. Blood Flow
Metab., 22, 183–195.
Singh, M., Setalo, G., Jr, Guan, X., Frail, D. E. and Toran-Allerand, C. D.
(2000) Estrogen-induced activation of the mitogen-activated protein kinase
cascade in the cerebral cortex of estrogen receptor-a knock-out mice.
J. Neurosci., 20, 1694–1700.
Palmon, S. C., Williams, M. J., Littleton-Kearney, M. T., Traystman, R. J.,
Kosk-Kosicka, D. and Hurn, P. D. (1998) Estrogen increases cGMP in
selected brain regions and in cerebral microvessels. J. Cereb. Blood Flow
Metab., 18, 1248–1252.
Satoh, M., Matter, C. M., Ogita, H., Takeshita, K., Wang, C.-Y.,
Dorn, G. W. and Liao, J. K. (2007) Inhibition of apoptosis-regulated
signaling kinase-1 and prevention of congestive heart failure by estrogen.
Mol. Cardiol., 115, 3197–3204.
Vedder, H., Anthes, N., Stumm, G., Wurz, C., Behl, C. and Krieg, J.-C.
(1999) Estrogen hormones reduce lipid peroxidation in cells and tissues of
the central nervous system. J. Neurochem., 72, 2531–2538.
Moosmann, B. and Behl, C. (1999) The antioxidant neuroprotective effects
of estrogens and phenolic compounds are independent from their estrogenic
properties. Proc. Natl Acad. Sci., 96, 8867–8872.
Behl, C., Skutella, T., Lezoualc’h, F., Post, A., Widmann, M.,
Newton, C. J. and Holsboer, F. (1997) Neuroprotection against oxidative
stress by estrogens: structure-activity relationship. Mol. Pharmacol., 51,
Roof, R. L., Hoffman, S. W. and Stein, D. G. (1997) Progesterone protects
against lipid peroxidation following traumatic brain injury. Mol. Chem.
Neuropathol., 31, 1–11.
Cui, Y. F., Gao, Y. B., Yang, H., Xiong, C. Q., Xia, G. W. and
Wang, W. F. (1999) Apoptosis of circulating lymphocytes induced by
whole body gamma-irradiation and its mechanism. J. Environ. Pathol.
Toxicol. Oncol., 18, 185–189.
Pollack, M., Phaneuf, S., Dirks, A. and Leeuwenburgh, C. (2002) The role
of apoptosis in the normal aging brain, skeletal muscle, and heart. Ann. N.
Y. Acad. Sci., 959, 93–107.
Bridger, J. M. and Kill, I. R. (2004) Aging of Hutchinson-Gilford progeria
syndrome fibroblasts is characterised by hyperproliferation and increased
apoptosis. Exp. Gerontol., 39, 717–724.
Ginaldi, L., De Martinis, M., Monti, D. and Franceschi, C. (2004) The
immune system in the elderly: activation-induced and damage-induced
apoptosis. Immunol. Res., 30, 81–94.
Palmiter, R. D., Brinster, R. L. and Hammer, R. E. (1982) Dramatic growth
of mice that develop from eggs microinjected with metallothionein-growth
hormone fusion genes. Nature, 300, 611–615.
Clutter, A. C., Pomp, D. and Murray, J. D. (1996) Quantitative genetics of
transgenic mice: components of phenotypic variation in body weights and
weight gains. Genetics, 143, 1753–1760.
Calder, W. A. (1984) Size, Function and Life History. Harvard University
Press, Cambridge, pp. 431.
Lang, F., Gulbin, E., Szabo, I., Lepple-Wienhues, A., Huber, S. M.,
Duranton, C., Lang, K. S., Lang, P. A. and Wieder, T. (2004) Cell
volume and the regulation of apoptotic cell death. J. Mol. Recognit., 17,
Geracitano, L. A., Bocchetti, R., Monserrat, J. M., Regoli, F. and
Bianchini, A. (2004) Oxidative stress responses in two populations of
Laeonereis acuta (Polychaeta, Nereididae) after acute and chronic exposure
to copper. Mar. Environ. Res., 58, 1–17.
Joksic, G. and Petrovic, S. (2000) Lack of adaptive response of human
lymphocytes exposed in vivo to low doses of ionizing radiation. J. Environ.
Pathol. Toxicol. Oncol., 23, 195–206.
Joksic, G., Petrovic, S. and Ilic, Z. (2004) Age-related changes in radiationinduced
micronuclei among healthy adults. Braz. J. Med. Biol. Res., 37,
Pieter, R. S., Niemierko, A., Fullerton, B. C. and Munzenrider, J. E. (2006)
Cauda equine tolerance to high-dose fractionated irradiation. Int. J. Radiat.
Oncol. Biol. Phys., 64, 251–257.
Matsuda, T., Saito, H., Inoue, T., Fukatsu, K., Han, I., Furukawa, S.,
Ikeda, S. and Muto, T. (1998) Growth hormone inhibits apoptosis and
up-regulates reactive oxygen intermediates production by human polymorphonuclear
neutrophils. J. Parenter. Enteral Nutr., 22, 368–374.
Gonzalez-Juanatey, J. R., Pineiro, R., Iglesias, M. J., Gualillo, O.,
Kelly, P. A., Deiguez, C. and Lago, F. (2004) GH prevents apoptosis in
cardiomyocytes cultured in vitro through a calcineurin-dependent mechanism.
J. Endocrinol., 180, 325–335.
Bogazzi, F., Ultimieri, F., Raggi, F. et al. (2004) Growth hormone inhibits
apoptosis in human colonic cancer cell lines: antagonistic effects of
peroxisome proliferators activated receptor-c ligands. Endocrinology, 145,
Shin, D. H., Lee, E., Kim, J.-W., Kwon, B.-S., Jung, M. K., Jee, Y. H.,
Kim, J., Bae, S.-R. and Chang, Y. P. (2004) Protective effect of growth
hormone on neuronal apoptosis after hypoxia-ischemia in the neonatal rat
brain. Neurosci. Lett., 354, 64–68.
Cuzzocrea, S., Thiemermann, C. and Salvemini, D. (2004) Potential
therapeutic effect of antioxidant therapy in shock and inflammation. Curr.
Med. Chem., 11, 1147–1162.
Monget, A. L., Richard, M. J., Cournot, M. P., Arnaud, J., Galan, P.,
Preziosi, P., Herbeth, B., Favier, A. and Hercberg, S. (1996) Effect of 6
month supplementation with different combinations of an associated of
antioxidant nutrients on biochemical parameters and markers of the
antioxidant defence system in the elderly. Eur. J. Clin. Nutr., 50, 443–449.
Rinaldi, P., Polidori, M. C., Metastasio, A. et al. (2003) Plasma
antioxidants are similarly depleted in mild cognitive impairment and in
Alzheimer’s disease. Neurobiol. Aging, 24, 915–919.
Agarwal, S. and Sohal, R. S. (1994) Aging and proteolysis of oxidized
proteins. Arch. Biochem. Biophys., 309, 24–28.
Starke-Reed, P. E. and Oliver, C. N. (1989) Protein oxidation and
proteolysis during aging and oxidative stress. Arch. Biochem. Biophys.,
Van Remmen, H. and Richardson, A. (2001) Oxidative damage to
mitochondria and aging. Exp. Gerontol., 36, 957–968.
Chen, D., Cao, G., Hastings, T., Feng, Y., Pei, W., O’Horo, C. and Chen, J.
(2002) Age-dependent decline of DNA repair activity for oxidative lesions
in rat brain mitochondria. J. Neurochem., 81, 1273–1284.
Esposito, L. A., Melov, S., Panov, A., Cottrell, B. A. and Wallace, D. C.
(1999) Mitochondrial disease in mouse results in increased oxidative stress.
Proc. Natl Acad. Sci., 96, 4820–4825.
Kumari, M., Brunner, E. and Fuhrer, R. (2000) Minireview: mechanisms by
which the metabolic syndrome and diabetes impair memory. J. Gerontol.
Biol. Sci., 55A, B228–B232.
Ludwig, D. S. (2003) Diet and development of the insulin resistance
syndrome. Asia Pac. J. Clin. Nutr., 12, S4.
Sonta, T., Inoguchi, T., Tsubouchi, H., Sekiguchi, N., Matsumoto, S.,
Utsumi, H. and Nawata, H. (2004) Evidence for contribution of vascular
NAD(P)H oxidase to increased oxidative stress in animal models of
diabetes and obesity. Free Radic. Biol. Med., 37, 115–123.
Murata, M. and Kawanishi, S. (2004) Oxidative DNA damage induced by
nitrotyrosine, a biomarker of inflammation. Biochem. Biophys. Res.
Wu, L., Ashraf, H. M., Facci, M., Wang, R., Paterson, P. G., Ferrie, A. and
Juurlink, B. H. (2004) Dietary approach to attenuate oxidative stress,
hypertension, and inflammation in the cardiovascular system. Proc. Natl
Acad. Sci., 101, 7094–7099.
Sastre, J., Pallardo, F. V. and Vina, J. (2003) The role of mitochondrial
oxidative stress in aging. Free Radic. Biol. Med., 35, 1–8.
Hagen, T. M., Liu, J., Lykkesfeldt, J., Wehr, C. M., Ingersoll, R. T.,
Vinarsky, V., Bartholomew, J. C. and Ames, B. N. (2002) Feeding acetyl-
L-carnitine and lipoic acid to old rats significantly improves metabolic
function while decreasing oxidative stress. Proc. Natl Acad. Sci., 99,
Dhitavat, S., Ortiz, D., Rogers, E., Rivera, E. and Shea, T. B. (2005) Folate,
vitamin E, and acetyl-L-carnitine provide synergistic protection against
oxidative stress resulting from exposure of human neuroblastoma cells to
amyloid-beta. Brain Res., 1061, 114–117.
Sener, G., Tosun, O., Sehirli, A. O., Kacmaz, A., Arbak, S., Ersoy, Y. and
Ayanoglu-Dulger, G. (2003) Melatonin and N-acetylcysteine have
beneficial effects during hepatic ischemia and reperfusion. Life Sci., 72,
Kuo, H. W., Chang, S. F., Wu, K. Y. and Wu, F. Y. (2005) Chromium (VI)
induced oxidative damage to DNA: increase of urinary 8-hydroxydeoxyguanosine
concentrations (8-OHdG) among electroplating workers.
Occup. Environ. Med., 60, 590–594.
Garaj-Vrhovac, V. and Kopjar, N. (2003) The alkaline comet assay as
biomarker in assessment of DNA damage in medical personnel
occupationally exposed to ionizing radiation. Mutagenesis, 18, 265–271.
Bolognesi, C., Abbondandolo, A., Barale, R. et al. (1997) Age-related
increase of baseline frequencies of sister chromatid exchanges, chromosome
aberrations and micronuclei in human lymphocytes. Cancer
Epidemiol. Biomarkers Prev., 6, 249–256.
Hauck, S. J., Aaron, J. M., Wright, C., Kopchick, J. J. and Bartke, A.
(2002) Antioxidant enzymes, free-radical damage and response to paraquat
in liver and kidney of long-living growth hormone receptor/binding protein
gene-disrupted mice. Horm. Metab. Res., 34, 481–486.
Bartke, A. and Brown-Borg, H. (2004) Life extension in the dwarf mouse.
Curr. Top. Dev. Biol., 63, 189–225.
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