Skip Navigation


Mutagenesis Advance Access originally published online on July 21, 2008
Mutagenesis 2008 23(6):473-482; doi:10.1093/mutage/gen036
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/6/473    most recent
gen036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lemon, J. A.
Right arrow Articles by Boreham, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lemon, J. A.
Right arrow Articles by Boreham, D. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Elevated DNA damage in a mouse model of oxidative stress: impacts of ionizing radiation and a protective dietary supplement

J. A. Lemon*, C. D. Rollo1 and D. R. Boreham

Medical Physics and Applied Radiation Sciences 1Department of Biology, McMaster University, Nuclear Research Building, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada

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 {gamma}H2AX 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 {gamma}H2AX foci and double the levels of chromosomal aberrations. In unirradiated mice, the dietary supplement significantly reduced constitutive {gamma}H2AX and 8-OHdG in both Nr and Tg mice (normalizing both {gamma}H2AX and 8-OHdG in Tg), with little difference in {gamma}H2AX 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.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
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 (1Go–4Go). Free radicals damage lipids, proteins, DNA and cellular structures. Oxidative DNA base damage, chromosome aberrations, genomic instability and telomere alterations are among the most deleterious consequences of oxidative processes consequently making high doses potentially mutagenic (1Go–13Go).

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 (5Go,8Go–10Go) and are considered valid markers of ageing (7Go). Genomic aberrations increase the risk of carcinogenesis and may actually accelerate the ageing process by contributing to cell dysfunction and death (14Go–17Go). Chronic oxidative stress such as that associated with ageing is a significant contributor to the accumulation of damaged DNA and chromosome rearrangements (12Go,18Go). 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 (19Go–23Go). 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 (16Go,17Go,24Go–27Go). Cellular responses mediating defence, repair and replacement also appear to attenuate with age and contribute to ageing (7Go,17Go,28Go).

Transgenic growth hormone (Tg) mice express elevated free radical processes (29Go) and accelerated ageing (30Go–34Go). Tg mice live only half as long as normal mice (29Go,30Go,35Go,36Go) and experience elevated superoxide radical production and lipid peroxidation in several tissues (29Go,37Go,38Go). They have age related, early onset arthritis, increased incidence of cataracts, reduced motor activity and muscle wasting (30Go,35Go,36Go,39Go,40Go). Brown-Borg et al. (31Go) found elevation of hepatic carbonyls at 12 months. Tg mice also express reduced levels of hepatic antioxidants (38Go,41Go,42Go). Young Tg mice display greatly enhanced cognitive abilities (43Go,44Go), but experience a rapid age-related decline in learning abilities (43Go).

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 supplement would reduce DNA damage in Tg mice. Limoli et al. (45Go) 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 {gamma}H2AX 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 (46Go–49Go). 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 (46Go–57Go).

Multiple cellular mechanisms defend against oxidative stress (58Go–61Go). Antioxidants typically spare, synergize or recycle one another, and if administered singly, they may become pro-oxidants. 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 (62Go). Mechanisms include cell membrane NADPH oxidases and (associated) potassium channels, mitochondrial dysfunction and inflammatory processes (3Go,12Go,63Go–65Go). NADPH oxidases both produce and are activated by free radicals. This can perpetuate chronic free radical production (64Go). Mitochondrial dysfunction induced by irradiation (Ca2+ 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 (65Go,66Go). Leach et al. (66Go) highlighted Ca2+ 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 {gamma}H2AX foci formation, DNA repair fidelity using SKY and assessing oxidative base damage by measuring 8-OhdG levels.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Animals
Our Tg mice (C57BL/6J-SJL background) have transgenes with metallothionein promoters fused to rat growth hormone (GH) structural regions (67Go). 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 (68Go), 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 {gamma}H2AX 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.

Dietary supplement
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. (36Go, 43Go). 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 (69Go). 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.


View this table:
[in this window]
[in a new window]

  		Table I. Components of the dietary supplement and associated cellular targets

 
In vivo irradiation
During irradiation, mice were placed in a polyvinyl chloride (PVC) tube (5 x 12.5 cm) with PVC mesh endcaps. Each mouse was given a 2-Gy whole-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.

SKY
Sample collection and preparation. Four hours after irradiation, mice were anesthetized with IsofluraneTM 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.

Slide preparation. 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.

Hybridization. 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 4x 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 40 min. 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 air-dry. 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.

Fluorescence microscopy. 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 EXPOTM, the image analysis software. A minimum of 50 suitable metaphases were scored for each mouse.

{gamma}H2AX and 8-OHdG
Cell collection. 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 {gamma}H2AX assay. A 3 ml ice cold 70% ethanol was immediately added to each tube and all tubes were place on ice.

Antibody staining. The remaining bone marrow samples were incubated in a 37°C water bath. For the {gamma}H2AX 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, 500-µl 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 Tris-buffered saline (TBS; Trizma base + NaCl, Sigma Aldrich, Mississauga, Ontario) for 10 min to rehydrate cells. The tubes were centrifuged and re-suspended 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 re-suspended in 200 µl either anti-phospho-H2A.X (ser139) antibody ({gamma}H2AX; 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 AlexaFluorTM 488-conjugated goat anti-rabbit IgG F(ab')2 antibody ({gamma}H2AX) or AlexaFluorTM 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).

Statistics
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 between groups.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Chromosome aberrations
Constitutive. 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.


Figure 1
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. The average number of constitutive (0 Gy) and radiation-induced (2 Gy) chromosome aberrations in bone marrow of unsupplemented and diet-supplemented mice. Asterisks indicate values significantly different from unirradiated normal mice (*P < 0.009).

 
Radiation induced. 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 radiation-induced 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.


View this table:
[in this window]
[in a new window]

  		Table II. The types and percentages of constitutive (0 Gy) and radiation-induced (2 Gy) chromosome aberrations in the bone marrow of each group of mice

 


View this table:
[in this window]
[in a new window]

  		Table III. Correlation (R2 value) of chromosome aberrations versus chromosome length, number of genes or gene density

 


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Comparison of the number of breaks in each chromosome after a 2-Gy whole-body dose. (A) Unsupplemented Nr mice versus unsupplemented Tg. (B) Diet-supplemented Nr mice versus diet-supplemented Tg mice. Chromosome breaks from each group of mice have been pooled.

 
DSB repair
Kinetics. There was a reduction in constitutive {gamma}H2AX 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 {gamma}H2AX was the same for all groups of mice, with supplemented mice demonstrating significantly reduced {gamma}H2AX levels (P < 0.045) at all time points (Figure 3B). The level of {gamma}H2AX 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 {gamma}H2AX fluorescence returned to baseline levels. The level of {gamma}H2AX 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.


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Comparison of {gamma}H2A.X levels in the bone marrow of all groups of mice over the 4-h incubation period. (A) Constitutive levels of {gamma}H2A.X. (B) Radiation-induced levels of {gamma}H2A.X.

 
Constitutive foci formation. The background level of {gamma}H2AX 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 {gamma}H2AX 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 {gamma}H2AX 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 {gamma}H2AX levels between supplemented Nr and supplemented Tg (Figure 3A).

Radiation-induced foci formation. Radiation-induced {gamma}H2AX 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). Unsupplemented Nr had significantly lower radiation-induced {gamma}H2AX 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 {gamma}H2AX fluorescence, so that radiation-induced {gamma}H2AX fluorescence did not differ significantly in supplemented mice from constitutive levels. The level of {gamma}H2AX 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).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Comparison of constitutive (0 Gy) and radiation-induced (2 Gy) 8-OHdG levels in the bone marrow of all four groups of mice. Asterisks indicate values significantly different from unirradiated normal mice (*P < 0.005, **P < 0.0001).

 
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 {gamma}H2A.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.


   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Our Tg mice have significantly elevated free radical production and lipid peroxidation in several tissues (29Go). 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 {gamma}H2AX (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 (70Go). In contrast, Brown-Borg et al. (31Go) 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 (33Go,71Go–73Go). As in many models of extended longevity such as birds and dietary restricted rodents (74Go), dwarf (GH deficient) mice were shown to generate lower amounts of mitochondrial free radicals (31Go). In this case, reduced metabolic rate rather than changes in mitochondrial coupling may be the cause. Sanz et al. (75Go) showed that mitochondrial DNA damage was also reduced in dwarfs, especially in brain.

Antioxidant defences may also be reduced in Tg mice (38Go,41Go,42Go). GH receptor knockout mice show strongly elevated hepatic superoxide dismutase that likely reflects down-regulated phoshoinositide-3 kinase (PI3K)/Akt signaling and elevated forkhead box transcription factors (FOXO) activity (76Go). GH administration also up-regulates enzymes involved in glutathione degradation and down-regulates those involved in synthesis (76Go). 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 (33Go,77Go,78Go). 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 (33Go,73Go). 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 (18Go). Persistently elevated free radical processes associated with genomic instability may be associated with dysregulated mitochondria and reduced antioxidant activity (18Go,65Go). 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 (79Go).

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 error-prone 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 (80Go,81Go). It is interesting to note that constitutive levels of {gamma}H2AX 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 (82Go,83Go).

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 (84Go,85Go).

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 {gamma}H2AX 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 {gamma}H2AX (30-min incubation), it was readily apparent that unsupplemented Tg mice had significantly higher levels of {gamma}H2AX fluorescence. Although absolute levels of {gamma}H2AX 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 (86Go–89Go). 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 phenotype.

Antioxidants can protect against chronic oxidative stress associated with ageing or disease (3Go,90Go) and acute oxidative insult such as those induced by irradiation or xenobiotics (56Go,91Go,92Go). Limoli et al. (93Go) found that antioxidants reduced radiation-induced cell mortality, the degree of DNA damage and genomic instability. Antioxidants, including {alpha}-lipoic acid, offset free radical elevations and stem cell dysfunctioning (94Go,95Go). Many individual ingredients in our supplement have been tested as radioprotective agents (56Go), including most of the components that have been postulated as potentially useful in protecting astronauts from high-level radiation exposure (55Go). Single antioxidants (i.e. ascorbate) administered after irradiation have been shown to suppress long-lived radicals that can otherwise cause mutations contributing to genomic instability (57Go).

Gaziev et al. (50Go,51Go) found that a multiple-ingredient supplement was radioprotective for mice and human lymphocytes (vitamins E, C, β-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/β-carotene (52Go,53Go). A vitamin E and C combination reduced bleeding and diarrhoea in patients subjected to pelvic irradiation (54Go). Given that the free radical theory of ageing was born in radiation science (24Go), 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 {gamma}H2AX 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 ROS.

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 radiation-induced 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 (57Go). 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 radioprotectant.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Science and Engineering Research Council (238495), Candu Owners Group (03.014) and the Chemical Biological Radiological and Nuclear Research and Technology Initiative (015ssH1021-021608).


   Acknowledgments
 
The authors acknowledge the expert technical assistance of Sarah Laronde, Ashley Hodgins and Lisa Laframboise.


   Notes
 
* To whom correspondence should be addressed. Tel: +1 905 525 9140; Fax: +1 905 522 5982; Email: lemonja{at}mcmaster.ca


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

    1. Spruill MD, Nelson DO, Ramsey MJ, Nath J, Tucker JD. Lifetime persistence and clonality of chromosome aberrations in the peripheral blood of mice acutely exposed to ionizing radiation. Radiat. Res. (2000) 153:110–121.[CrossRef][Web of Science][Medline]

    2. Tunca B, Egeli U, Aydemir N, Cecener G, Bilaloglu R. Investigation of the genotoxic effect in bone marrow of swiss albino mice exposed long-term to pyrimethamine. Teratog. Carcinog. Mutag. (2002) 22:393–402.[CrossRef]

    3. Parihar MS, Hemnani T. Alzheimer's disease pathologies and therapeutic interventions. J. Clin. Neurosci. (2004) 11:456–467.[CrossRef][Web of Science][Medline]

    4. Singh P, Jain A, Kaur G. Impact of hypoglycaemia and diabetes on CNS: correlation of mitochondrial oxidative stress with DNA damage. Mol Cell. Biochem. (2004) 260:153–159.[CrossRef][Web of Science][Medline]

    5. Hastie N, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with aging. Nature (1994) 346:866–868.[CrossRef]

    6. Crompton NEA. Telomeres, senescence and cellular radiation response. Cell. Mol. Life Sci. (1997) 53:568–575.[CrossRef][Web of Science][Medline]

    7. Bolognesi C, Lando C, Forni A, Landini E, Scarpato R, Migliore L, Bonassi S. Chromosomal damage and ageing: effect on micronuclei frequency in peripheral blood lymphocytes. Age Ageing (1999) 28:393–397.[Abstract/Free Full Text]

    8. Bukvic N, Gentile M, Susca F, Fanelli M, Serio G, Buonadonna L, Capurso A, Guanti G. Sex chromosome loss, micronuclei, sister chromatid exchange and aging: a study including 16 centenarians. Mutat. Res. (2001) 498:159–167.[Web of Science][Medline]

    9. Lezhava T. Chromosome and aging: genetic conception of aging. Biogerontology (2001) 2:253–260.[CrossRef][Web of Science][Medline]

    10. Dolle ME, Vijg J. Genome dynamics in aging mice. Genome Res. (2002) 12:1732–1738.[Abstract/Free Full Text]

    11. Bresgen N, Karlhuber G, Krizbai I, Bauer H, Bauer HC, Eckl PM. Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei and apoptosis. J. Neurosci. Res. (2003) 72:327–333.[CrossRef][Web of Science][Medline]

    12. Samper E, Nicholls DG, Melov S. Mitochondrial oxidative stress causes chromosomal instability of mouse embryonic fibroblasts. Aging Cell (2003) 2:277–285.[CrossRef][Web of Science][Medline]

    13. Haddad JJ. Redox and oxidant-mediated regulation of apoptosis signaling pathways: immuno-pharmaco-redox conception of oxidative siege versus cell death commitment. Int. Immunopharmacol. (2004) 4:475–493.[CrossRef][Web of Science][Medline]

    14. Aigner A, Wolf S, Gassen G. Transport and detoxification principles, approaches and perspectives for research on the blood-brain barrier integrity. Angew. Chem. Int. Ed. Engl. (1997) 36:2441.[CrossRef][Web of Science]

    15. Ames BN, Gold LS, Willett WC. The causes and prevention of cancer. Proc. Natl Acad. Sci. USA (1995) 92:5258–5265.[Abstract/Free Full Text]

    16. Vijg J, Dolle ME. Large genome rearrangements as a primary cause of aging. Mech. Ageing Dev. (2002) 123:907–915.[CrossRef][Web of Science][Medline]

    17. Weirich-Schaiger H, Weirich HG, Gruber B, Schweiger M, Hirsch-Kauffmann M. Correlation between senescence and DNA repair in cells from young and old individuals and in premature aging syndromes. Mutat. Res. (1994) 316:37–48.[CrossRef][Web of Science][Medline]

    18. Limoli CL, Giedzinski E. Induction of chromosome instability by chronic oxidative stress. Neoplasia (2003) 5:339–346.[Web of Science][Medline]

    19. Ziegler D, Hanefeld M, Ruhnau KJ, Meibner HP, Lobisch M, Schutte K, Gries FA. Treatment of symptomatic diabetic peripheral neuropathy with the anti-oxidant {alpha}-lipoic acid. A 3-week multicentre randomized controlled trial (ALADIN study). Diabetologia (1995) 38:1425–1433.[CrossRef][Web of Science][Medline]

    20. Akiyama H, Barger S, Bradt B, et al. Inflammation and Alzheimer's disease. Neurobiol. Aging (2000) 21:383–421.[CrossRef][Web of Science][Medline]

    21. Berr C, Balansard B, Arnaud J, Roussel AM, Alperovitch A. Cognitive decline is associated with systemic oxidative stress: the EVA study. J. Am. Genetics Soc. (2000) 48:1285–1291.

    22. Liu J, Atamna H, Kuratsune H, Ames BN. Delayed brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites. Ann. N. Y. Acad. Sci. (2002) 959:133–166.[Web of Science][Medline]

    23. Tchirkov A, Lansdorp PM. Pole of oxidative stress in telomere shortening in cultured fibroblasts from normal individuals and patients with ataxia-telangiectasia. Hum. Mol. Genetics (2003) 12:227–232.[Abstract/Free Full Text]

    24. Harman D. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. (1956) 11:298–300.[Free Full Text]

    25. Beckman KB, Ames BN. The free radical theory of aging matures. Physiol. Rev. (1998) 78:547–581.[Abstract/Free Full Text]

    26. Hagen TM, Ingersoll RT, Lykkesfeld J, Liu J, Wehr CM, Vinarsky V, Bartholomew JC, Ames BN. R-{alpha}-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate. FASEB J. (1999) 13:411–418.[Abstract/Free Full Text]

    27. Hagen TM, Liu J, Lykkesfeldt J, Wehr CM, Ingersoll RT, Vinarsky V, Bartholomew JC, Ames BN. Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress. Proc. Natl Acad. Sci. (2002) 99:1870–1875.[Abstract/Free Full Text]

    28. Miura Y. Oxidative stress, radiation-adaptive responses, and aging. J. Radiat. Res. (2004) 45:357–372.[CrossRef][Medline]

    29. Rollo CD, Carlson J, Sawada M. Accelerated aging of giant transgenic mice is associated with elevated free radical processes. Can. J. Zool. (1996) 74:606–620.[CrossRef]

    30. Steger RW, Bartke A, Cecim M. Premature ageing in transgenic mice expressing growth hormone genes. J. Reprod. and Fertil. (1993) 46(Suppl.):61–75.

    31. Brown-Borg HM, Johnson WT, Rakoczy S, Romanick M. Mitochondrial oxidant generation and oxidative stress in Ames dwarf and GH transgenic mice. J. Am. Aging Assoc. (2001) 24:85–100.

    32. Rollo CD. Growth negatively impacts the life span of mammals. Evol. Dev. (2002) 4:55–61.[CrossRef][Web of Science][Medline]

    33. Rollo CD. Overview of research on giant transgenic mice with emphasis on the brain and aging. In: Human Body Size and the Laws of Scaling—Samaras T, ed. (2007) New York, NY: Nova Science Publishers. 235–260.

    34. Bartke A. Can growth hormone (GH) accelerate aging? Evidence from GH-transgenic mice. Neuroendocrinology (2003) 78:210–216.[CrossRef][Web of Science][Medline]

    35. Wolf E, Kahnt E, Ehrlein J, Hermanns W, Brem G, Wanke R. Effects of long-term elevated serum levels of growth hormone on life expectancy of mice: lessons from transgenic animal models. Mech. Ageing Dev. (1993) 68:71–87.[CrossRef][Web of Science][Medline]

    36. Lemon JA, Boreham DR, Rollo CD. A complex dietary supplement extends longevity in mice. J. Gerontol. Biol. Med. Sci. (2005) 60A:275–279.

    37. Carlson JC, Bharadwaj R, Bartke A. Oxidative stress in hypopituitary dwarf mice and in transgenic mice overexpressing human and bovine GH. Age (1999) 22:181–186.[CrossRef][Web of Science]

    38. Hauck SJ, Bartke A. Free radical defenses in the liver and kidney of human growth hormone transgenic mice: possible mechanisms of early mortality. J. Gerontol. Biol. Sci. (2001) 56:B153–B162.[Abstract/Free Full Text]

    39. Meliska CJ, Burke PA, Bartke A, Jensen RA. Inhibitory avoidance and appetitive learning in aged normal mice: comparison with transgenic mice having elevated plasma growth hormone levels. Neurobiol. Learn. Mem. (1997) 68:1–12.[CrossRef][Web of Science][Medline]

    40. Ogueta S, Olazabal I, Santos I, Delgado-Baeza E, Garcia-Ruiz JP. Transgenic mice expressing bovine GH develop arthritic disorder and self-antibodies. J. Endocrinol. (2000) 165:321–328.[Abstract]

    41. Hauck SJ, Bartke A. Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase. Free Radic. Biol. Med. (2000) 28:970–978.[CrossRef][Web of Science][Medline]

    42. Brown-Borg HM, Rakoczy SG, Romanick MA, Kennedy MA. Effects of growth hormone and insulin-like growth factor-1 on hepatocyte antioxidative enzymes. Exp. Biol. Med. (2002) 227:94–104.[Abstract/Free Full Text]

    43. Lemon JA, Boreham DR, Rollo CD. A dietary supplement abolishes age-related cognitive decline in transgenic mice expressing elevated free radical processes. Exp. Biol. Med. (2003) 228:800–810.[Abstract/Free Full Text]

    44. Rollo CD, Ko CV, Tyerman JGA, Kajiura L. The growth hormone axis and cognition: empirical results and integrated theory derived from giant transgenic mice. Can. J. Zool. (1999) 77:1874–1890.[CrossRef]

    45. Limoli CL, Giedzinski E, Baure J, Rola R, Fike JR. Altered growth and radiosensitivity in neural precursor cells subjected to oxidative stress. Int. J. Radiat. Biol. (2006) 82:640– 647.[CrossRef][Web of Science][Medline]

    46. Tavares CD, Cecchi AO, Antunes LMG, Takahashi CS. Protective effects of the amino acid glutamine and of ascorbic acid against chromosomal damage induced by doxorubicin in mammalian cells. Teratog. Carcinog. Mutag. (1998) 18:153–161.[CrossRef]

    47. Antunes LMG, Takahashi CS. Protection and induction of chromosomal damage by vitamin C in human lymphocyte cultures. Teratog. Carcinog. Mutag. (1999) 19:53–59.[CrossRef]

    48. Antunes LMG, Francescato HDC, Darin JD, Delourdes P, Bianchi M. Effects of selenium pre-treatment on cisplatin-induced chromosome aberrations in wistar rats. Teratog. Carginog. Mutag. (2000) 20:341–348.[CrossRef]

    49. Jagetia GC, Venkatesha VA, Reddy TK. Naringin, a citrus flavonone, protects against radiation-induced chromosome damage in mouse bone marrow. Mutagenesis (2003) 18:337–343.[Abstract/Free Full Text]

    50. Gaziev AI, Podlutsky AJ, Panfilov BM, Bradbury R. Dietary supplements of antioxidants reduce hprt mutant frequency in splenocytes of aging mice. Mutat. Res. (1995) 338:77–86.[CrossRef][Web of Science][Medline]

    51. Gaziev AI, Sologub GR, Fomenko LA, Zaichkina SI, Kosyakova NI, Bradbury RJ. Effect of vitamin-antioxidant micronutrients on the frequency of spontaneous and in vitro {gamma}-ray-induced micronuclei in lymphocytes of donors: the age factor. Carcinogenesis (1996) 17:493–499.[Abstract/Free Full Text]

    52. Konopacka M, Widel M, Rzeszowska-Wolny J. Modifying effect of vitamin C, E, and beta-carotene against gamma-ray-induced DNA damage in mouse cells. Mutat. Res. (1998) 417:85–94.[Web of Science][Medline]

    53. Blumenthal RD, Lew W, Reising A, Soyne D, Osorio L, Ying Z, Goldberg DM. Anti-oxidant vitamins reduce normal tissue toxicity induced by radio-immunotherapy. Int. J. Cancer (2000) 86:276–280.[CrossRef][Web of Science][Medline]

    54. Kennedy M, Bruninga K, Mutlu EA, Losurdo J, Choudhary S, Keshavarzian A. Successful and sustained treatment of chronic radiation proctitis with antioxidant vitamins E and C. Am. J. Gastroenterol. (2001) 96:1080–1084.[CrossRef][Web of Science][Medline]

    55. Turner ND, Braby LA, Ford J, Lupton JR. Opportunities for nutritional amelioration of radiation-induced cellular damage. Nutrition (2002) 18:904–912.[CrossRef][Web of Science][Medline]

    56. Weiss JF, Landauer MR. Protection against ionizing radiation by antioxidant nutrients and phytochemicals. Toxicology (2003) 189:1–20.[CrossRef][Web of Science][Medline]

    57. Waldren CA, Vannais DB, Ueno AM. A role for long-lived radicals (LLR) in radiation-induced mutation and persistent chromosomal instability: counteraction by ascorbate and RibCys but not DMSO. Mutat. Res. (2004) 551:255–265.[Web of Science][Medline]

    58. Fenech M, Rinaldi J. The relationship between micronuclei in human lymphocytes and plasma levels of vitamin C, vitamin E, vitamin B12 and folic acid. Carcinogenesis (1994) 15:1405–1411.[Abstract/Free Full Text]

    59. Fenech M, Dreositi IE, Aitken C. Vitamin-E supplements and their effects on vitamin-E status in blood and genetic damage rate in peripheral blood lymphocytes. Carcinogenesis (1997a) 18:359–364.[Abstract/Free Full Text]

    60. Fenech M, Dreositi IE, Rinaldi J. Folate, vitamin B12, homocysteine status and chromosomal damage rate in lymphocytes of older men. Carcinogenesis (1997b) 18:1329–1336.[Abstract/Free Full Text]

    61. Fenech M, Stockley C, Aitken C. Moderate wine consumption protects against hydrogen peroxide-induced DNA damage. Mutagenesis (1997c) 12:289–296.[Abstract/Free Full Text]

    62. Spitz DR, Azzam EI, Jian LJ, Gius D. Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology. Cancer Metastasis Rev. (2004) 23:311–322.[CrossRef][Web of Science][Medline]

    63. Saad AH, Zhou Y, Lambe EK, Hahn GM. Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels. Mutat. Res. (1994) 324:171–176.[CrossRef][Web of Science][Medline]

    64. Cai H. NAD(P)H oxidase-dependent self-propagation of hydrogen peroxide and vascular disease. Circ. Res. (2005) 96:818–822.[Abstract/Free Full Text]

    65. Kim GJ, Fiskum GM, Morgan WF. A role for mitochondrial dysfunction in perpetuating radiation-induced genomic instability. Cancer Res. (2006) 66:10377–10383.[Abstract/Free Full Text]

    66. Leach JK, Van Tuyle G, Lin PS, Schmidt-Ullrich R, Mikkelsen RB. Radiation-induced, mitochondrial-dependent generation of reactive oxygen/nitrogen. Cancer Res. (2001) 61:3894–3901.[Abstract/Free Full Text]

    67. Palmiter RD, Brinster RL, Hammer RE. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature (1982) 300:611–615.[CrossRef][Web of Science][Medline]

    68. Clutter AC, Pomp D, Murray JD. Quantitative genetics of transgenic mice: components of phenotypic variation in body weights and weight gains. Genetics (1996) 143:1753–1760.[Abstract]

    69. Calder WA. Size, Function and Life History (1984) Cambridge: Harvard University Press. 431.

    70. Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A. H2AX: the histone guardian of the genome. DNA Repair (2004) 3:959–967.[CrossRef][Medline]

    71. Lai HC, Liu TJ, Ting CT, Sharma PM, Wang PH. Insulin-like growth factor-1 prevents loss of electrochemical gradient in cardiac muscle mitochondria via activation of PI 3 kinase/Akt pathway. Mol. Cell. Endocrinol. (2003) 205:99–106.[CrossRef][Web of Science][Medline]

    72. O'Rourke B, Cortassa S, Aon AA. Mitochondrial ion channels: gatekeepers of life and death. Physiology (2005) 20:303–315.[Abstract/Free Full Text]

    73. Rollo CD. Multidisciplinary aspects of regulatory systems relevant to multiple stressors: aging, xenobiotics, and radiation. In: Multiple Stressors: a Challenge for the Future—Mothersill C, ed. (2007b) New York, NY: Springer. 185–224.

    74. Ogburn CE, Carlberg K, Ottinger MA, Holmes DJ, Martin GM, Austad SN. Exceptional cellular resistance to oxidative damage in long-lived birds requires active gene expression. J. Gerontol. (2001) 56:B468–B474.[Web of Science]

    75. Sanz A, Bartke A, Barja G. Long-lived Ames dwarf mice: oxidative damage to mitochondrial DNA in heart and brain. Age (2002) 25:119–122.[CrossRef]

    76. Brown-Borg HM. Longevity in mice: is stress resistance a common factor? AGE (2006) 28:145–162.[CrossRef][Web of Science]

    77. Droge W. Free radicals in the physiological control of cell function. Physiol. Rev. (2002) 82:47–95.[Abstract/Free Full Text]

    78. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. (2007) 39:44–84.[CrossRef][Web of Science][Medline]

    79. Rollo CD. Radiation and the regulatory landscape of neo2-Darwinism. Mutat. Res. (2006) 597:18–31.[Web of Science][Medline]

    80. Pfeiffer P, Goedecke W, Obe G. Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations. Mutagenesis (2000) 15:289–302.[Abstract/Free Full Text]

    81. Burma S, Chen BP, Chen DJ. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (2006) 5:1042–1048.[Medline]

    82. Mei N, Tamae K, Kunugita N, Hirano T, Kasai H. Analysis of 8-hydroxydeoxyquanosine 5’monophosphate (8-OH-dGMP) as a reliable marker of cellular oxidative DNA damage after gamma-irradiation. Environ. Mol. Mutagen. (2003) 41:332–338.[CrossRef][Web of Science][Medline]

    83. Ogawa Y, Kobayashi T, Nishoka A, Kariya S, Seguchi H, Yoshida S. Radiation-induced oxidative DNA damage, 8-oxoguanosine, in human peripheral T cells. Int. J. Mol. Med. (2003) 11:27–32.[Web of Science][Medline]

    84. Walker JA, Boreham DR, Unrau P, Duncan AM. Chromosome content and ultrastructure of radiation-induced micronuclei. Mutagenesis (1996) 11:419–424.[Abstract/Free Full Text]

    85. Broome EJ, Brown DL, Mitchel REJ. Adaption of human fibroblasts to radiation alters biases in DNA repair at the chromosome level. Int. J. Radiat. Biol. (1999) 75:681–690.[CrossRef][Web of Science][Medline]

    86. Nisitani S, Hosokawa M, Sasaki MS, Yasuoka K, Naiki H, Matsushita T, Takeda T. Acceleration of chromosome aberrations in senescence-accelerated strains of mice. Mutat. Res. (1990) 237:221–228.[CrossRef][Web of Science][Medline]

    87. Mukherjee AB, Costello C. Aneuploidy analysis in fibroblasts of human premature aging syndromes by FISH during in vitro cellular aging. Mech. Ageing Dev. (1998) 103:209–222.[CrossRef][Web of Science][Medline]

    88. Grigorova M, Balajee AS, Natarajan AT. Spontaneous and X-ray-induced chromosomal aberrations in Werner syndrome cells detected by FISH using chromosome-specific painting probes. Mutagenesis (2000) 15:303–310.[Abstract/Free Full Text]

    89. Rosenfeld SV, Togo EF, Mikheev VS, Popovich IG, Zabezhinskii MA, Khavinson VKH, Anisimov VN. Effect of Epithalon on the incidence of chromosome aberrations in senescence-accelerated mice. Bull. Exp. Biol. Med. (2002) 133:274–276.[CrossRef][Web of Science][Medline]

    90. Dusinska M, Kazimirova A, Barancokova M, Beno M, Smolkova B, Horska A, Raslova K, Wsolova L, Collins AR. Nutritional supplementation with antioxidants decreases chromosomal damage in humans. Mutagenesis (2003) 18:371–376.[Abstract/Free Full Text]

    91. Sener G, Jahovic N, Tosun O, Atasoy BM, Yegen BC. Melatonin ameliorates ionizing radiation-induced oxidative organ damage in rats. Life Sci. (2003) 74:563–572.[CrossRef][Web of Science][Medline]

    92. Lazarova M, Slamenova D. Genotoxic effects of a complex mixture adsorbed onto ambient air particles on human cells in vitro; the effects of vitamins E and C. Mutat. Res. (2004) 557:167–175.[Web of Science][Medline]

    93. Limoli CL, Kaplan MI, Giedzinski E, Morgan WF. Attenuation of radiation-induced genomic instability by free radical scavengers and cellular proliferation. Free Radic. Biol. Med. (2001) 31:10–19.[CrossRef][Web of Science][Medline]

    94. Limoli CL, Rola R, Giedzinski E, Mantha S, Huang TT, Fike JR. Cell-density-dependent regulation of neural precursor cell function. Proc. Natl Acad. Sci. (2004) 101:16052–16057.[Abstract/Free Full Text]

    95. Limoli CL, Giedzinski E, Baure J, Doctrow SR, Rola R, Fike JR. Using superoxide dismutase/catalase mimetics to manipulate the redox environment of neural precursor cells. Radiat. Prot. Dosimetry (2006a) 122:228–236.[Abstract/Free Full Text]

Received on December 4, 2007; revised on June 13, 2008; accepted on June 15, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/6/473    most recent
gen036v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lemon, J. A.
Right arrow Articles by Boreham, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lemon, J. A.
Right arrow Articles by Boreham, D. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?