Life Sciences 77 (2005) 2840 – 2854
www.elsevier.com/locate/lifescie
Susceptibility of DNA to oxidative stressors in young
and aging mice
Norma E. López-Diazguerrero a,1, Armando Luna-López a, Marı́a C. Gutiérrez-Ruiz a,
Alejandro Zentellab, Mina Königsberg a,T
a
Departamento de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud, UAM-Iztapalapa, A.P. 55-535,
México, D.F. 09340, México
b
Departamento de Bioquı́mica, Instituto Nacional de Ciencias Médicas y de la Nutrición Salvador Zubirán e Instituto de
Investigaciones Biomédicas, UNAM. México, D.F. 04510, México
Received 19 November 2004; accepted 11 May 2005
Abstract
The changes that accompany aging may be a result of oxidative damage to DNA that accumulates as a
result of aging and age-related illnesses. Furthermore, a higher susceptibility is thought to be more common
among elderly than young individuals. In the present study, we examined the severity of DNA damage caused
by carbon tetrachloride (CCl4) and H2O2 in cells from young (2 month old) and older (14 month old) mice
using both in vivo and in vitro exposures. CCl4 is known to generate radical oxidative species (ROS)
throughout its biotransformation in the liver. Therefore, 8-oxo-7,8-dihydro-2V-deoxyguanosine (8-oxdGuo) was
quantified in liver DNA obtained from young and older mice treated with CCl4. In addition, DNA singlestrand breaks were measured by the Comet assay in primary lung fibroblasts cultured from young and older
mice and treated in vitro with H2O2. Intracellular ROS production and mitochondrial enzyme activity were
determined in parallel. 8-oxodGuo levels were significantly higher in older mouse liver DNA than younger,
and increased significantly with CCl4 treatment. When the basal DNA damage was subtracted, the net damage
was almost equal for both. In addition, untreated cells cultured from older mice had significantly greater levels
of strand breaks than cells derived from young mice. H2O2 increased the level of damage in both cell cultures.
Our findings indicate that the DNA damage observed in older animals probably results from the accumulation
T Corresponding author. Tel.: +52 55 5804 4732; fax: +52 55 5804 4727.
E-mail address: [email protected] (M. Königsberg).
1
Programa de Doctorado en Biologı́a Experimental, UAMI.
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.05.034
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2841
of endogenous damage with age, perhaps due to insufficient repair, which enhances the injury caused by
exposure to the toxic agents.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Aging; DNA damage; Oxidative stress; H2O2; Carbon tetrachloride
Introduction
Aging has been described as the large array of alterations in structure and function that slowly and
inevitably emerge during the post-maturational phases of the life cycle (Martin et al., 1996). These
alterations have been attributed to a wide variety of extrinsic and intrinsic agents to which life forms are
exposed, and which have the potential to cause cellular damage at the molecular level. In recent years,
reactive oxygen species (ROS), which damage lipids, proteins and DNA, have been associated with agerelated deterioration (Barnett and King, 1995; Martinez et al., 2003). This association is supported by the
beneficial effects of antioxidant supplementation, known to provide protection from oxidative damage,
and, which may lead to diminished physiological injury with concurrent life span extension (Mockett et
al., 2001; Melov et al., 2001; Zimmerman et al., 2003).
DNA damage is of special importance to aging because its accumulation and inheritance by subsequent
cellular generations results in altered genotypes and functionally deficient cells (Bohr et al., 1998;
Hamilton et al., 2001). Many reports have found a strong correlation between mutations caused by
oxidative stress during aging and age-related processes (Ames et al., 1990; Wiseman and Halliwell, 1996).
Among the most common age-related illnesses are the neurodegenerative disorders (Emerit et al., 2004),
neoplasias (Halliwell and Gutteridge, 1984; Campisi, 2000) and autoimmune diseases like rheumatoid
arthritis (Weyand et al., 2003). Therapeutic interventions for these diseases in both elderly animals and
humans are extremely challenging due to the unique handling and metabolism of pharmacological agents
in older individuals. Adverse drug reactions are estimated to be 2–3 times higher in elderly patients than in
young adults (Turnheim, 2003; Wright and Warpula, 2004). Therefore, the medical treatment of multiple
illnesses in the elderly are complicated by decreased physiological parameters, adverse events to which the
elderly are especially vulnerable, increased risk for side effects and toxicity, and drug–drug interactions
due to the use of multiple agents (Ramsay et al., 2004; Zhang et al., 2003).
Carbon tetrachloride (CCl4) causes hepatotoxicity following acute exposure and liver cirrhosis as a
result of chronic exposure (Beddowes et al., 2003). Liver cell injury induced by CCl4 is initially due to
its metabolic transformation into a trichloromethyl free-radical (CCl3S), the possible formation of
trichloromethylperoxy (d OOCCl3), chlorine (d Cl) free radicals, and to phosgene and aldehydic products
from lipid peroxidation. CCl4 is metabolized, as are the majority of drugs and xenobiotics, by the mixedfunction oxidase system of the liver endoplasmic reticulum that belongs to the P450 family. It is
postulated that the widespread disturbances in hepatocyte function could involve the generation of toxic
products arising directly from CCl4 metabolism or from peroxidative degeneration of membrane lipids
(Boll et al., 2001). Therefore CCl4 is frequently used as a model of a drug whose detoxifying metabolism
causes oxidative stress (Ahmad et al., 1987).
Nevertheless, it is known that many tissues and most cells lines and primary cell cultures have either
diminished P450 activity or completely lose their P450 systems (Guillouzo, 1998), rendering them
unable to metabolize the CCl4. Hence, P450-independent oxidative stressors are frequently used in cell
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cultures. For instance, hydrogen peroxide (H2O2) has been used to generate oxidative stress and DNA
damage in lung and skin fibroblasts (Lueken et al., 2004), and white blood cells (Giovannelli et al.,
2003). H2O2 readily permeates to cell membranes, and once within the cytoplasm, it can be reduced to
the highly reactive hydroxyl radical by metal ion through the Fenton reaction (McCord, 1993; Halliwell
and Gutteridge, 1999).
Of the several different techniques that can be used to evaluate oxidative DNA damage, two are of
special interest. Alkaline single cell gel electrophoresis (SCGE), or the Comet assay, is a sensitive
method for detecting double-and single-strand DNA breaks caused by multiple toxicants (Singh et al.,
1988; Tice et al., 2000). Also, 8-oxo-7,8-dihydro-2V-deoxyguanosine (8-oxodGuo) is a selective marker
of oxidative DNA damage (Helbock et al., 1998). Quantification of 8-oxodGuo has gained in popularity
because it is one of the most abundant oxidative DNA lesions and its formation correlates with
mutagenesis, carcinogenesis, and aging (Chen et al., 1995). Hence, these methods clearly complement
each other, and both have been used extensively in order to evaluate DNA damage (Pouget et al., 2000;
Wolf et al., 2002).
The present study was designed to examine whether the excessive DNA injury produced by two
different chemicals (H2O2 and CCl4) in the cells derived from old animals, was mostly due to increased
susceptibility to toxicants or as a result of the accumulation of previous DNA damage. Toxicant induced
damage was measured independently in two different models, one in vivo and one in vitro (tissue and
cells). In the first set of experiments, 8-oxodGuo was quantified in liver DNA obtained from young (2month-old) and older (14-month-old) mice treated in vivo with CCl4. In a second set of experiments,
DNA single-strand break induction by H2O2 was measured in vitro using primary lung fibroblast
cultures obtained from young and older mice. The intracellular ROS production was determined in
parallel. Our results indicate that the cells from old mice do not produce more endogenous ROS than the
cells from the young mice. The increased DNA damage found in the cells from older animals might
result from a combination of the damage caused by the toxicant and the pre-existing cellular damage due
to age. These results are of importance when treating elderly patients with pharmacological agents that
could induce oxidative stress.
Materials and methods
Chemicals
All chemicals and reagents were of the highest analytical grade available; most were purchased from
Sigma (St. Louis MO). Those obtained from other sources are detailed in the text.
Animals
Female CD-1 mice were obtained from the closed breeding colony at the Universidad Autónoma
Metropolitana-Iztapalapa (UAM-I). It has been reported (Altamirano, 1994) that vaginal opening for
CD-1 females occurs between 50 and 60 days of age. Thus, CD-1 mice of this age are considered young
adults, completely developed and capable of reproduction. The reproductive span of female breeders is
8 to 9 months, resulting in a total of 6–8 litters (Zurcher et al., 1982). After that period of time, female
mice deteriorate physically and physiologically, and have a classical doldT phenotype, with an increase in
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benign tumors, loss of hair, and muscle weakness. In 14 of the 15 inbred mouse strains that were
evaluated (all descended from the foundation stocks of the Jackson Laboratory), virgin females survived
significantly longer (20–25%) than did their bred sisters (Russel, 1964). It was previously demonstrated
that breeder female mice showed not only phenotypical and morphological characteristics of aging, but
cellular and molecular features of senescence as well (Königsberg et al., 2004). Therefore, 2-month-old
animals with vaginal openings were used as young, fully-developed adults and 14-month-old retired
breeders where used as older or aging mice. Mice were cared for according to the principles of the
Mexican official ethics standard 062-ZOO-1999.
In vivo experiments
Animal treatments
Female CD-1 mice were divided into four groups of three animals each. Two groups (a and b) were
young animals (2-months of age) and two groups (c and d) were older animals (14-months of age).
Groups a and c received single intraperitoneal (ip) injections of a sublethal dose of 0.16 ml/Kg body
weight of 1 part CCl4: 5 parts mineral oil for 3 consecutive days. Groups b and d received only ip
injections of mineral oil for the 3 days. The mice were sacrificed on the fourth day, and the liver was
used for DNA extraction.
DNA extraction and enzymatic hydrolysis
DNA was isolated using the chaotropic-NaI method (Wang et al., 1994), as modified by Matos et al.
(2001). Tissue (1 g) was suspended in 5 ml of a lysis solution (0.32 M sucrose, 5 mM EDTA, 1% (w/v)
Triton X-100, 5 mM MgCl2, 10 mM Tris–HCl, 0.1 mM desferroxamine, pH7.5). After centrifugation at
1500 g for 10 min, the pellets were resuspended in 6 ml of 10 mM Tris–HCl buffer, pH 8.0, containing
5 mM EDTA, 10% SDS and 0.15 mM desferroxamine. 30 Al of 10 mg/ml RNase A and 20 Al of 1000 U/
ml RNase T1 (both in 10 mM Tris–HCl buffer, pH 7.4, containing 1 mM EDTA and 2.5 mM
desferroxamine) were added to the reaction mixture. After a 1 h incubation at 37 8C, 300 Al of a 20 mg/
ml proteinase K solution were added, and the incubation continued at 37 8C for 1 h. After centrifugation
at 5000 g at 4 8C for 15 min, the liquid phase was collected and 4 ml of 7.6 M NaI solution containing
40 mM Tris–HCl, 20 mM EDTA-Na2, 0.3 mM desferroxamine, pH 8.0, were added, followed by the
addition of 8 Al of isopropanol. The tube was mixed by inversion and refrigerated at 70 8C overnight.
The precipitate formed was collected by centrifugation at 9000 g for 15 min and washed with 1 ml of
60% (w/v) isopropanol, followed by washing with 1 ml of 70% (w/v) ethanol. After additional
centrifugation at 9000 g for 15 min, the DNA pellet was dissolved in 500 Al of 0.1 mM
desferroxamine. The DNA concentration was determined spectrophotometrically at 260 nm and its
purity was assessed by ensuring that the A 260 / A 280 ratio was N 1.75. 100 Ag of DNA for each injection
were diluted in 100 Al of deionized water, followed by the addition of 4 Al 1 M sodium acetate with 5
units of nuclease P1; the solution was incubated at 37 8C for 30 min. 150 Al of 1 M Tris–HCl (pH 7.4)
and 3 units of E. coli acid phosphatase were added, followed by incubation at 37 8C for 1 h. The sample
was centrifuged and the aqueous layer was then analyzed by HPLC.
Analysis of 8-oxodGuo by HPLC/electrochemical (EC) detection
100 Ag samples of digested DNA were injected into an HPLC/EC system consisting of a Waters 600 C
pump (Waters, MA) connected to a supelcosil LC-18 (Supelco, Bellefonte, PA) reverse-phase column
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(250 4.6 mm i.d., particle size 5 Am). The isocratic eluent was 50 mM potassium phosphate buffer, pH
5.5, and 8% methanol at a 1 ml/min flow rate. EC detection was performed by a INTRO detector (Antec
Leyden, Leiden, The Netherlands) operated at 290 mV. Elution of unmodified nucleosides was
simultaneously monitored by a 486 Waters UV spectrometer set at 254 nm. The molar ratio of 8oxodGuo to deoxy-guanosine (dGuo) in each DNA sample was determined based on EC detection at
290 mV for 8-oxodGuo and absorbance at 254 nm for dGuo (Matos et al., 2001).
In vitro experiments
Cell culture
Primary cultures of normal lung fibroblasts were obtained from young or old female CD-1 mice
according to the protocol described by Doyle et al. (1998). The cells were maintained in Dulbecco’smodified minimal essential medium (DMEM; GIBCO-Invitrogen, Carlsbad, CA) supplemented with
15% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT), 1% non-essential amino acids, 100
U/ml penicillin and 100 Ag/ml streptomycin (GIBCO-Invitrogen). Cells were grown at 37 8C in 60 mmdiameter culture dishes (Corning, Acton, MA) in an atmosphere of 95% air and 5% CO2. The medium
was replaced every 2–3 days and the cells were subcultured by trypsinization upon reaching confluence.
Cell treatments and Comet assay
Semi-confluent cultures of lung fibroblasts from young and old animals at an early cumulative
number of population doublings (V 10) and in logarithmic growth phase were treated with a sublethal
oxidative stress of 0.3 mM H2O2 in DMEM containing 10% FBS at 37 8C for 30 min. At this
concentration, alterations in DNA structure occur without any accompanying cell death for at least 24 h,
as confirmed by 97% viability assessed by trypan blue dye exclusion. Control cultures free of H2O2 were
incubated in parallel. After incubation the cells were dislodged with trypsin, and centrifuged at 1000 rpm
for 4 min.
The Comet assay was performed essentially as described by Singh et al. (1988), as modified by Tice
et al. (1991) and Betancourt et al. (1995). Briefly, the cell suspension was embedded in a three layer
agarose gel on frosted slides and immersed in a lysis buffer (2.5 M NaCl, 100 mM EDTA, 1% Na
sarcosinate and 10 mM Tris, pH 10.0, with Triton X-100 and 10% (v/v) DMSO added fresh) and
maintained at 4 8C for at least 2 h. The slides were removed and placed in a horizontal electrophoresis
chamber (BioRad, Hercules, CA). The reservoirs were filled with fresh electrophoresis buffer consisting
of 300 mM NaOH and 1 mM EDTA. The slides were left in the alkaline buffer for 20 min to allow DNA
unwinding and the expression of alkali–labile damage. Electrophoresis then was performed in the same
buffer at 25 V and 300 mA for 20 min. Following electrophoresis, the slides were rinsed twice with
neutralization buffer (0.4 M Tris, pH7.5) before staining the DNA with 50 Al of 2 mg/ml ethidium
bromide. Slide analysis was performed using an epifluorescence microscope (Olympus, Tokyo, Japan)
equipped with a 40 objective lens, a 515–560 nm excitation filter and a 590 nm barrier filter. Tail
lengths were measured from the trailing edge of the comet tail. 50 cells per treatment (25 cells on each of
two replicate slides) from three independent experiments were analyzed for DNA migration.
Measurement of ROS production
Intracellular ROS production was measured using the fluorescent dichlorofluorescein (DCF) assay
(Cathcart et al., 1983; Bass et al., 1983). Semi-confluent cultures of lung fibroblasts from young and
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old animals at an early cumulative number of population doublings (V 10) in logarithmic growth phase
were trypsinized and then loaded with 4 Ag/ml of 2V,7V-dichlorodihydrofluorescein diacetate
(H2DCFDA; Molecular Probes, Eugene, OR) for 15 min in the dark to allow the intracellular deesterificaton of H2DCFDA to H2DCF. The cells were centrifuged in order to eliminate the
unincorporated H2DCFDA and the subsequent oxidation of this substrate by the intracellular ROS into
the fluorescent DCF, was quantified with a flow cytometer (FACScan; Becton-Dickinson, San Jose,
CA), using an excitation wavelength of 480 nm, an emission wavelength of 520 nm, and CELLQuest
software for data analysis.
MTT assay
Mitochondrial respiratory function was determined indirectly using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983), which quantifies mitochondrial
succinate dehydrogenase activity. This assay was chosen as an indicator of cellular energetic and
metabolic status. Briefly, treated and control cells were incubated with complete medium containing 0.5
mg/ml MTT for 4 h at 37 8C. The medium was discarded and 800 Al of extracting solution (0.04 M HCl
in isopropanol) were added. After 15 min of incubation at room temperature, the absorbance of the
colored formazan salt was measured at 570 nm using a Beckman DU 640 spectrophotometer.
Data analysis
Data are reported as the means F SD for at least three independent experiments using cells from
different donor animals. The Kruskal–Wallis non-parametric test followed by the Tamhane variance
analysis were used to compare the Comet assay data. The two-tailed Student t-test was used for all the
other comparisons. A 0.05 level of probability was used as a minimum criterion of significance.
Results
8-OxodGuo formation in liver DNA from CCl4 treated mice
The basal level of liver 8-oxodGuo in young control mice was low, averaging 0.49 F 0.10 residues /
10 dGuo, while it was 2.3-fold higher in old control mice, 2.61 F 0.52 residues / 106 dGuo (Fig. 1).
Treatment of young and old mice with CCl4 increased the level of 8-oxodGuo in DNA significantly, to
7.39 F 2.96 residues / 106 dGuo in young animals and 10.14 F 4.16 residues / 106 dGuo in old mice (both
p b 0.05; Fig 1). These data indicate that there was 27% more 8-oxodGuo in the liver DNA of CCl4treated old mice than similarly treated young mice. The insert in Fig. 1 represents the subtraction of the
basal levels of 8-oxodGuo from the levels of damage found in treated mice. The net increase in 8oxodGuo levels in old vs. young mice due to the treatment was only 16%, which was not statistically
significant.
6
In vitro experiments
Properties of cultured cells
Primary lung fibroblasts cultures from young and old mice displayed a characteristic early logarithmic
growth phase followed by decline in the rate of cell proliferation. At day 19, the cells reached their
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Fig. 1. Effect of CCl4 on 8-oxodGuo levels in DNA from young and old mice. Young and old mice were subjected to oxidative
stress with a sublethal treatment of 0.16 ml/kg body weight CCl4 (1 : 5 w/v in mineral oil) for 3 consecutive days. Liver DNA
was isolated using the chaotropic-NaI method and the amount of 8-oxodGuo was quantified by HPLC/EC. Results are the
mean F S.D. of at last three independent experiments. The statistical comparisons were carried out using Student’s t test: *
p b 0.05 for comparisons between DNA from old and young mice; # p b 0.05 for comparisons between DNA from CCl4-treated
and control mice. The insert represents the subtraction of the basal levels of 8-oxodGuo (hatched) from the levels of damage
found in CCl4 treated mice.
capacity to divide and became senescent (Hayflik’s limit). The cells from young animals, however, had a
4.6-fold higher proliferation rate than the cells derived from old animals (data not shown) (Königsberg et
al., 2004). All the experiments described below were performed with semi-confluent logarithmic phase
cultures and normalized per cell number.
DNA single strand breaks in H2O2 treated cells
Cells derived from young and old donor animals were exposed to a sublethal concentration (0.3 mM)
of H2O2 and DNA single-strand breaks were measured using the Comet assay. Fig. 2A shows the
percentage of damaged cells observed in control and treated primary fibroblasts. More than 50% of
untreated control cells from both young and old animals displayed damaged DNA in the form of comets.
This observation is consistent with the observations of Parrinello et al. (2003) indicating that murine
cells have a higher steady-state level of DNA damage than that commonly detected in human cells. Since
tail length or DNA migration can be used as a direct measure of the extent of DNA damage (Tice et al.,
2000), comet tail length was determined in treated and control cultures (Fig. 2B). 96% of control cells
derived from young mice had a tail length between 1–20 Am, which we defined as low damage, and only
4% had comet tails of between 21–40 Am, which we classified as mildly damaged. In contrast, 65% of
cells derived from old mice had low damage and 21% were mildly damaged (tail length 21–40 Am),
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2847
A
100
% of damaged cells
80
60
40
20
0
Young
control
Young
H2O2
Old
control
Old
H2O2
B
% of damaged cells
100
80
60
40
20
0
1-20 21-40 -60 -80 00
41
61 81-1 -120 40
1
101 121-
OH2O2
YH2O2
OC
YC
DNA migration, tail length (µm)
Fig. 2. A. Percentage of damaged cells in control and H2O2-treated primary lung fibroblasts. Cells cultured from young and old
mice were given a sublethal oxidative stress with 0.3 mM H2O2 for 30 min. The Comet assay was performed as described in
Materials and Methods, and the percentage of damaged cells (cells with comets) was determined. Results are the mean of three
independent experiments; n = 150 for each treatment. B. Tail length magnitude. The magnitude of the tail length observed in
H2O2-damaged cells was analyzed. Definitions of tail length and damage are as follows: Tail length between 1–20 Am was
defined as low damage, 21–40 Am as mild damage, 41–60 Am as moderate damage, 61–80 Am as severe damage, and N 81 nm
as very severe damage. Yc: untreated cells derived from young mice (young control); OC: untreated cells derived from old mice
(old control); YH2O2: H2O2-treated cells derived from young mice; OH2O2: H2O2-treated cells derived from old mice. Results
are the mean of three independent experiments.
while 14% had comet tails of 41–60 Am (moderately damaged) and 1.5% had tails of 61–80 Am
(severely damaged).
Treatment with H2O2 resulted in measurable damage in all cells derived from old animals and 79% of
the cells cultured from young animals. In terms of the magnitude of the injury, 46% of cells derived from
young animals had low damage, 38.1% mild damage, and 15.1% moderate damage. By comparison,
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only 10.6% of treated cells from old mice had low damage, 16.3% mild damage, 26.2% moderate
damage, and 23% severe damage, while 24% of the cells had damage in excess of the severe category
(tail length N 81 Am) (Fig. 2B).
The mean tail length for H2O2-treated cells from young animals was 2.3-fold that of the mean tail
length of young control cells. A similar ratio was obtained for cells derived from old mice, where the
mean tail length of treated cells was 2.17-fold greater than the mean tail length for the controls (Fig. 3).
Also, even though control cells from older and younger mice had similar percentages of damaged cells
(Fig. 2A), the control cells from old mice had an average tail length that was 2.2-fold greater than the
control cells from younger mice, which resulted in a 4.84-fold total increase in DNA migration in treated
cells from older mice compared to control cells derived from young mice (Fig. 3). Statistical analysis of
this data reveled a significant difference in the mean tail length between the young and old cells control
groups and among H2O2 treated and untreated cells (Kruskal–Wallis non-parametric method/Tamhane
variance analysis, p b 0.05). The insert in Fig. 3 represents the subtraction of the basal mean tail length
found in control cells from the mean tail length determined for the H2O2-treated mice. The net increase
in DNA damage in old vs. young mice cells due to the treatment was 52%, which was a statistically
significant difference. To be consistent with the previous analysis, damaged cells were grouped into two
Fig. 3. Average tail lengths of damaged cells in control and H2O2-treated primary lung fibroblasts. Cells cultured from young
and old mice were given a sublethal oxidative stress with 0.3 mM H2O2 for 30 min. The mean tail length (Am) of injured cells in
control and H2O2-treated primary fibroblasts was determined. The Comet assay was performed as described in Materials and
Methods. Results are the mean of the damaged cells of three independent experiments. The statistical comparisons were carried
out using the Kruskal–Wallis test followed by the Tamhane variance analysis: * p b 0.05 for comparisons between damaged
cells from old and young control mice; # p b 0.05 for comparisons between the damaged cells from H2O2-treated and control
mice. The insert represents the subtraction of the basal mean tail length (hatched) from the mean tail length determined in H2O2treated mice.
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Table 1
Damaged cells grouped by severity of injury
Tail length (Am)
% of Cells
Control
1–60
61–120
H2O2-treated
Young
Old
Young
Old
100.0
0.0
98.79
1.21
100.0
0.0
48.24
51.76 F 1.66
Damaged cells derived from young and old mice described in Fig. 1. are grouped in two categories: minor damage (tails of 1–60
Am) and severe damage (tails of 61–120 Am). The Comet assay was performed as described in Materials and Methods. Results
are the means of data from three independent experiments.
categories: cells with minor damage (comet tails of 1–60 Am) and cells with severe damage (tails of 60–
120 Am). Table 1 shows that treated and control cells derived from young mice all fell into the minor
damage category, along with 98% of control cells from old animals. In contrast, 48.2% of the H2O2treated cells derived from old animals fell into the severe damage category indicating that approximately
half of the cell population was susceptible to H2O2 treatment.
ROS generation in cells from young and old mice
The ROS content in lung fibroblasts was determined by flow cytometry using the DCF assay
(Cathcart et al., 1983; Bass et al., 1983). The data were normalized as fluorescence intensity (FI) = (mean
fluorescent intensity) (% gated events). Cells derived from young mice had a significantly higher level of
FI (1598.25 F 431.01) than the cells derived from old mice (881.85 F 347.96; p b 0.02).
Mitochondrial enzyme activity in cells from young and old mice
The MTT assay was performed in order to determine if the higher levels of ROS found in control
cells from young animals correlated with a more active mitochondrial respiration (Mosmann, 1983;
Madesh and Balasubramanian, 1998). MTT reduction was determined at three stages of the culture
cycle: at the end of the establishment phase (day 13), during the logarithmic growth phase (day 15),
and at Hayflik’s limit (day 19). MTT reduction was found to be significantly ( p b 0.05) higher for
the lung fibroblasts derived from young animals, independent of the proliferation phase of the
culture (Table 2).
Table 2
Mitochondrial respiratory function in lung fibroblasts from young and old mice
Day of culture
Cells from young mice, OD/cell (normalized)
Cells from old mice, OD/cell (normalized)
13
15
19
From day 13 to 19
1.40 F 0.20
2.10 F 0.21
2.17 F 0.42
1.89 F 0.45
0.96 F 0.21*
1.19 F 0.43*
1.38 F 0.49*
1.18 F 0.42*
Mitochondrial respiratory function was indirectly determined using the MTT assay during the proliferation phase of culture.
MTT reduction was significantly higher (*p b 0.05 for comparisons between damaged cells from old and young control mice) in
the cells derived from young animals, regardless of the proliferation phase. Results are the mean F S.D. of data from at least
four independent experiments (n = 12 for each data set). Statistical comparisons are shown using the Student’s t test.
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Discussion
One of the current hypotheses proposed to explain the molecular basis for aging is that endogenous
or exogenous free radicals result in the accumulation of oxidative damage in various biomolecules
over the course of organism’s lifetime (Harman, 1956). Increased lipid peroxidation, protein carbonyl
levels (Mutlu-Turkoglu et al., 2003) and oxidative DNA damage (Hamilton et al., 2001; Wolf et al.,
2002) have been associated with advanced age. These lesions, on their own or in combination with
other changes, are believed to contribute to age-related pathologies. It is therefore not surprising that
old animals and elderly people are more prone to infections (Effros, 2003), autoimmune and
neuroedegenerative diseases (Weyand et al., 2003), and cancer (Campisi, 2000). Recently, a decline in
stress tolerance has been associated with a considerably higher level of oxidative damage in older
organisms (Zhang et al., 2003).
Our experiments where aimed at determining if the major DNA damage observed in the cells derived
from older female mice when exposed to H2O2 and CCl4, is as a result of an acquired susceptibility to
the toxicant or attributable to the DNA alterations accumulated through age. Old retired breeders were
considered as older or aging animals because they are known to survive significantly less (20–25%) than
their bred sisters; they also presented a classical doldT phenotype, with an increase in benign tumors, loss
of hair, and muscle weakness, as well as cellular deterioration as reported earlier (Russel, 1964;
Königsberg et al., 2004).
The liver and the lung were selected because they are completely different systems in terms of
function and antioxidant response, and the similarity of the results obtained for both set of experiments
support the previous idea.
Oxidative DNA damage is perhaps the most detrimental consequence of oxidative stress, causing
irreversible dysfunctions that eventually can lead to cell death (Yoneda et al., 1995). The level of
oxidative DNA damage has also been used as a marker of aging (Wolf et al., 2002), and was therefore
used as an endpoint in this study.
Previously, Hamilton et al. (2001) reported age related increases in 8-oxodGuo in nuclear and
mitochondrial DNA in mice, however, they found that these increases were not the result of a reduction
in the rate of removal this lesion, but rather to an age related increase in the sensitivity of tissues to
oxidative stress. Our results indicated that liver DNA extracted from control old mice had 2.3-fold higher
levels of 8-oxodGuo than DNA from control young adults. CCl4 treatment increased the levels the 8oxodGuo in both groups of animals, with liver DNA from the old mice containing more 8-oxodGuo than
liver DNA from young mice. However, the differences between young and old mice were not significant
This result suggests that the amount of oxidative damage generated by CCl4 in the DNA obtained from
old and young animals is almost the same. The major difference between young and old animals, and the
decrease in stress tolerance, appears to be due to the accumulation of damage during aging or the lack of
reparation.
In the in vitro experiments, the primary cultures from young and old animals displayed a marked
difference in cell kinetics, even though both reached Hayflik’s limit at day 19. We hypothesize that this
dissimilarity can be explained by the presence of senescent cells that increase as the cultures age in vitro,
especially in the cultures derived from old animals. Hence, both cultures are composed of a mixed
population of senescent and pre-senescent cells, as confirmed by the SA-h-Gal assay (Königsberg et al.,
2004). It is likely that the differences between cell cultures from young and old mice, do not result from
differences between individual cells (i.e., a young cell vs. an old cell), but reflect differences among the
N.E. López-Diazguerrero et al. / Life Sciences 77 (2005) 2840–2854
2851
populations. This possibility has to be taken into consideration when comparing the two cultures, since
the in vitro experiments were performed with semi-confluent logarithmic-phase cultures and normalized
per cell number.
The Comet assay experiments which quantified the DNA strand breaks in vitro, showed that
35% of the control cells derived from old animals, but only 4% of control cells from young
animals, had tail lengths z20 Am (defined as mild to moderate damage). This observation supports
the notion that the level of cellular DNA damage increases with age (Hamilton et al., 2001) (Fig.
2B). When treated with a sub-lethal dose of H2O2, the tail length of cells from the old animals
was considerably greater than the comet tails of cells from young mice. Almost 50% of the cells
derived from old animals showed tail lengths z60 Am (defined as severe damage), while none of
the cells derived from young animals had comets exceeding 60 Am, indicating that 52% had
increased susceptibility to the toxicant (determined by the subtraction of the basal damage, as in
the CCl4 experiments). This can hardly be explained by DNA single strand breaks only, but is
more likely related to epigenetic changes and chromatin structure disorders, as suggested by
Collins and others (Collins et al., 1997; Fairban et al., 1995). It may be significant to note that
chromatin alterations have been related to aging (Lezhava, 2001; Issa, 2002; Ikura and Ogryzko,
2003). The results obtained with the Comet assay in the present study are consistent with this
proposal, suggesting that the decreased stress tolerance observed in cells from old animals results,
in a substantial part, from the dbasalT damage (oxidative and epigenetic) accumulated as a result of
aging and not necessarily from a decrease in the primary antioxidant defense system. Indeed, the
flow cytometry experiments that measured endogenous ROS levels indicated that primary cells
derived from old mice had lower steady-state ROS levels than cells derived from young mice,
which probably is explained by the higher metabolic rates in the cells from the young mice. This
higher rate of metabolism was reflected in the relatively high rates of proliferation in the cells
from young mice and in the results of the MTT reduction assay indicating that cells from young
mice had relatively high levels of energetic activity independent of the proliferation phase of the
culture (Table 2).
One of the reasons used to explain the increase in DNA damage in cells from older organisms
was related to a diminished anti-oxidant capacity, but recent data indicate that cellular defense
systems vary with age in an irregular manner. No significant differences were found in the blood
levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, caeruloplasmin, uric
acid and bilirubin in old and young humans (Barnett and King, 1995). In contrast, SOD activity
was higher in the liver and brain of old rats, while GPx was lower, and glutathione (GSH) content
did not change with age in these organs (Kim et al., 2003). Mosoni et al. (2004) found that,
although GSH levels decreased in rat muscle, GPx and GSSG reductase activities changed little
with age in muscle and liver. Therefore, it has been suggested (Barnett and King, 1995), that agerelated increases in DNA damage and in the frequency of mutations are not due to age-related
alterations of in vivo antioxidant status, but by a decrease in the efficacy of DNA-repairing systems
(Bohr, 2002; Hashiguchi et al., 2004; Park and Gerson, 2005; Takahashi et al., 2005). It has also
been proposed that ROS are not only a cause of structural damage, but are also of physiological
importance as mediators in biological signaling processes. ROS-induced altered signaling might be
responsible for age-dependent changes in post-translational modifications and import of DNA repair
enzymes into nuclei and mitochondria, which in turn might affect genome repair (Szczesny et al.,
2004).
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N.E. López-Diazguerrero et al. / Life Sciences 77 (2005) 2840–2854
Conclusion
In summary, the results of this study indicate that when old cells are treated with a toxic agent, the
age-related increase in the level of DNA damage is not only a result of an inappropriate response of the
cells to the immediate damage. In agreement with Harman’s theory of aging (Harman, 1956), the
apparently higher susceptibility of DNA from old animals is in large part explained by the basal level of
DNA damage that may have accumulated with age.
Acknowledgments
The authors would like to thank Dr. Rocı́o González Vieira MVZ who cared for the animals; Dr.
Manuel Castillo for his advice on statistical analysis; Dr. Marisa HG Medeiros and Osmar Gomes
from the Universidade de Sao Paulo for their guidance in 8-oxodGuo quantification; and Dr. Ma de
los Angeles Aguilar and Dr. Victor Fainstein for their critical reading of the manuscript. This work
was supported by CONACyT’s grant No. 400200-5-J34194-M. Norma E. López-Dı́azguerrero and
Armando Luna-López are scholarship holders from CONACYT with respective numbers: 137897
and 185567.
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