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Department of Animal Biology-II (Animal Physiology), Faculty of Biology, Complutense University, Madrid 28040, Spain.
1Correspondence: Departamento de Biología Animal-II, Facultad de Biología, Universidad Complutense, Madrid 28040.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: 8-hydroxy-deoxyguanosine • longevity • aging • free radical
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
, especially when
focused on mitochondria (3
, 4)
, is frequently being
tested. Oxygen radicals leaking from the mitochondrial respiratory
chain are thought to continuously damage important cellular
macromolecules in postmitotic tissues. Among these targets, there is
wide agreement that damage to DNA is of utmost importance, since repair
of all the rest of the biologically relevant macromolecules is
ultimately dependent on the information coded in the DNA. Although
studies of variations as a function of age in tissue levels of
oxidative damage in nuclear DNA (nDNA) have yielded controversial
results (5
6
7
8
9
10
11
12)
, almost all the studies performed to date
have found increases in oxidative damage in mitochondrial DNA (mtDNA)
during aging (5
, 6
, 13
14
15
16)
. Furthermore, the few
available studies indicate that oxidative damage is higher in mtDNA
than in nDNA (5
, 6
, 17
, 18)
. This is consistent with the
observation that mitochondrial DNA mutations accumulate during aging
(19
, 20)
.
Different mammalian species can age at very different rates. Rapidly
aging species like rodents have short maximum life spans (MLSPs: 3.5–4
years in mice and rats), whereas slowly aging species like cows and
horses reach MLSPs of 30 and 46 years, respectively. In agreement with
the mitochondrial free radical theory of aging, all comparative studies
performed to date have found that the rate of oxygen radical production
by mitochondria is inversely correlated with MLSP (21
22
23
24
25
26
27
;
see ref 28
for a review). It remains to be demonstrated,
however, if the relatively lower rate of free radical generation of
long-lived animals is accompanied by a lower rate of oxidative damage
to their DNA. If the mitochondrial free radical-DNA damage hypothesis
of aging is correct, at least the oxidative damage to mtDNA, which is
placed near the main oxygen radical generator of healthy tissues,
should be less in slow than in rapidly aging animal species. However,
this important test has never been performed.
In this investigation, oxidative damage to both nDNA and mtDNA,
estimated through the measurement of
8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by high performance
liquid chromatography (HPLC), was studied in the heart of eight and in
the brain of six mammalian species showing more than one order of
magnitude of difference in MLSP (from 3.5 years in mice to 46 years in
horses). Heart and brain were selected because they are vital tissues
containing postmitotic cells, the kind of tissues where aging-related
changes are more readily observed. Previous descriptions of the
existence of higher 8-oxodG levels in mtDNA than in nDNA are restricted
to rodent liver (6
, 17
, 18)
, with a study available for
human postmortem brain tissue (5)
. The simultaneous study
of mtDNA and nDNA in the same individual animals pertaining to the
above-mentioned different species in the present investigation can
answer the questions of whether a higher level of oxidative damage in
mitochondrial than in the nuclear DNA in heart and brain is a general
characteristic of mammals and whether oxidative damage to mtDNA and
nDNA is related to the rate of aging.
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MATERIALS AND METHODS |
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Animals and tissue samples
Male mice, rats, guinea pigs, and rabbits were killed at the
laboratory by decapitation. Male sheep, pigs, cows, and horses were
killed at the abattoir. The mean age of the animals was 8 months
(mice), 11 months (rat), 1.4 years (guinea pig), 1.5 years (rabbit), 1
year (pig), and 1.5–2.5 years (sheep, cow, and horse). The selection
of these ages allows a comparison among young adults of all the species
while maintaining a compromise between the choice of similar
chronological or biological ages for interspecies comparison. The
maximum longevities of the selected species are well known
(29)
and vary progressively from 3.5 to 46 years: mouse
(Mus musculus, MLSP=3.5 years), rat (Rattus
norvegicus, MLSP=4 years), guinea pig (Cavia porcellus,
MLSP=8 years), rabbit (Oryctolagus caniculus, MLSP=13
years), sheep (Ovis aries, MLSP=20 years), pig (Sus
scrofa, MLSP=27 years), cow (Bos taurus, MLSP=30
years), and horse (Equus caballus, MLSP=46 years). All the
animals were in good health according to routine veterinary controls at
the abattoir and no animal was obese or scraggy. Heart samples were
taken from ventricles and brain samples were taken from occipital
cortex (cow and horse) or were whole brains (rodents). The samples were
obtained in all animals at the same time after death (5 min), they were
cut in small pieces, immediately frozen in liquid nitrogen, and
transferred before 2 h to a -80°C freezer for storage to be
used later for isolation of nuclear and mitochondrial DNA and analysis
of 8-oxodG and deoxyguanosine (dG).
Isolation of DNA
Nuclear DNA was isolated, after sodium dodecyl sulfate treatment
of heart samples, by chloroform extraction and ethanol precipitation
following the method of Loft and Poulsen (30)
as described
previously (31)
. Mitochondrial DNA was isolated by the
method of Latorre et al. (32)
, following the procedure of
Asunción et al. (15)
exactly. The mtDNA preparations
were free of nuclear DNA as tested by agarose gel electrophoresis and
staining with ethidium bromide. Before digestion, the amount of mtDNA
was determined in a small aliquot with high sensitivity by measuring
the fluorescence after binding of the Hoechst 33258 dye (Pharmacia;
Piscataway, N.J.).
DNA digestion
The isolated nuclear and mitochondrial DNAs were digested to
deoxynucleoside level by incubation at 37°C with 5 U of nuclease P1
(in 20 µl of 20 mM sodium acetate, 10 mM ZnCl2,
15% glycerol, pH 4.8) for 30 min and 1 U alkaline phosphatase (in 20
µl of 1 M Tris-HCl, pH 8.0) for 1 h (30)
.
8-oxodG and dG assays
The concentrations of 8-oxodG and deoxyguanosine (dG) were
measured by HPLC with on-line electrochemical and ultraviolet
detection, respectively. For analysis, the deoxynucleoside mixture was
injected into a reverse-phase Beckman Ultrasphere ODS column (5 µM,
4.6 mm x 25 cm) eluted with 2.5% acetonitrile in 50 mM phosphate
buffer pH 5.5. The amount of deoxynucleosides injected in the HPLC was
similar in all species. The mean amounts of heart mtDNA (determined by
the Hoechst method) used for injection in the HPLC after digestion were
7.5 µg (mouse), 16.5 µg (rat), 16.5 µg (guinea pig); 17.4 µg
(rabbit), 15.0 µg (sheep), 12.0 µg (pig), 13.3 µg (cow), and 14.4
µg (horse). In the case of brain mtDNA, the mean amounts used for
injection were 12.0 µg (mouse), 20.1 µg (rat), 26.1 µg (guinea
pig), 16.6 µg (rabbit), 14.0 µg (cow), and 15.2 µg (horse). These
amounts were not significantly correlated with the MLSP of the donor
species according to the criteria stated in the Statistical analysis
section (see below). A Waters 590 pump at 1 ml/min was used. 8-oxodG
and dG were detected with an ESA Coulochem II electrochemical
coulometric detector (ESA, Inc., Bedford, Mass.) with a high
sensitivity 5011 analytical cell run in the oxidative mode (E1=0 mV,
E2=200 mV) and a Bio-Rad model 1806 UV detector at 254 nm. For
quantification, peak areas of dG standards and three level calibrations
with pure 8-oxodG standards (Sigma) were analyzed during each HPLC run.
Statistical analyses
The statistical relationships between MLSP and 8-oxodG in nDNA
or mtDNA were studied first by linear regression (equation y=a+b.x), where y is 8-oxodG and x is MLSP. When
the correlation was found to be significant, adjustment by nonlinear
regression to the power function y = a .
xb (where y is 8-oxodG and x is MLSP) was
performed with Table-Curve software (Jandel Scientific). The
correlations were analyzed using the Pearson correlation coefficient
(r) selecting 0.05 as the point of minimum statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, this trend did not reach statistical significance (r=-0.68,
P<0.059). A different situation was observed for heart
mtDNA. In this case, a significant inverse correlation between 8-oxodG
values and MLSP was found (Fig. 2
; r=-0.92, P<0.001). This relationship followed the power
function y = a.xb,
where y is 8-oxodG in mtDNA and x is MLSP.
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The results obtained in the brain were qualitatively similar to those
obtained in the heart for both kinds of DNA, although lower correlation
coefficients and statistical significances were observed in this case.
Brain 8-oxodG in nDNA did not show a correlation with MLSP (Fig. 3
; r = 0.53, P<0.27). However, similar to what happened in
the heart, brain 8-oxodG levels in mtDNA were inversely correlated with
the species-specific MLSP in the mammals studied (Fig. 4
; power equation y= a.xb;
r=-0.88; P<0.016).
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8-oxodG values in mtDNA were also compared with those in nDNA in both
tissues when both values were available in the same individual animal
(Table 1
). In all species and tissues, 8-oxodG values were significantly higher
in mtDNA than in nDNA. Since heart 8-oxodG values tended to decrease
with increases in MLSP in both nDNA and mtDNA (Figs. 1
and 2)
, the
ratio ‘8-oxodG in mtDNA/8-oxodG in nDNA’ tended to be similar in all
the species, ranging from 3.2-fold in pigs and rabbits to 5.5-fold in
mouse, with a mean value of 4.1-fold for all the mammals studied (Table 1)
. Since brain 8-oxodG was inversely correlated with MLSP in mtDNA
(Fig. 4)
whereas the nonsignificant trend of the correlation with MLSP
was positive in the case of brain nDNA (Fig. 3)
, the ratio ‘8-oxodG in
mtDNA/8-oxodG in nDNA’ in the brain was inversely correlated with MLSP
(r==-0.84; P<0.025), i.e., the difference in 8-oxodG
between mtDNA and nDNA (always higher in mtDNA) tended to be lower in
long-lived than in short-lived mammals. This difference was around
9-fold in mice and rats, 5- to 6-fold in guinea pigs, rabbits and cows,
and 3-fold in horses, with a mean value of 6.6-fold for all the species
(Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 34)
. This disparity with the nature
of our nuclear DNA results could be due to methodological differences,
especially since the values reported in those investigations were high
for rodent liver (8.5 per 105 dG) in relation to
present day values, suggesting artifactual 8-oxodG formation; however,
this cannot be ascertained since those data were included in review
studies (33
, 34)
without stating the methodological
details used and were not published as original contributions. Tissue
differences could also be involved, although in principle we would not
expect to see aging-related differences in a nonpostmitotic tissue
(liver) or in partially or totally postmitotic ones (brain and heart).
The main oxygen radical generator of the intensely respiring heart and
brain cells, the inner mitochondrial membrane, is situated near mtDNA
(not near nDNA), and the most reactive free radicals (like
OH.) cannot diffuse far away and will react
unspecifically with macromolecules situated near their sites of
generation. Therefore, it is logical to think that oxygen radicals
generated by mitochondria can be a main cause of the
8-oxodG(mtDNA)-MLSP inverse relationship found in this investigation.
In strong agreement with a such a causal relationship, all studies
performed to date (including those carried out in heart and brain) have
consistently shown that the rate of mitochondrial oxygen radical
generation is also inversely related to MLSP (21
22
23
24
25
26
27
28)
. The
higher mtDNA 8-oxodG levels of short-lived animals in comparison with
long-lived ones would thus be caused, at least in part, by their higher
rates of mitochondrial oxygen radical generation—the reverse being
true in long-lived species.
Oxidative attack due to free radicals of mitochondrial origin are among
the primary possible causes of mtDNA damage. Recent measurements using
total detection polymerase chain reaction-based assays show that
multiple deleterious modifications (fragmentations, deletions, point
mutations, etc.) accumulate in the mtDNA with age to such an extent
that the percentage of full-length wild-type mtDNA decreases even down
to 11% (19)
or to undetectable or trace amounts
(20)
in muscle or heart of old individuals. Others,
however, have recently questioned (35)
the results
describing accumulations of mutations amounting to 90% or more of
mtDNA during aging without ruling out the possibility that a low
overall level of mtDNA damage could have a strong physiological
significance due to possible phenomena like clonal expansion of
defective mtDNA molecules (36
, 37)
. This would explain the
presence of mosaics in postmitotic tissues containing cells heavily
loaded with mutant mtDNA molecules (38)
. With one
exception(14)
, almost all studies of 8-oxodG in mtDNA
(although scarce) have found (5
, 6
, 13
, 15
, 16)
higher
values in old than in young animals of the same species; in the case of
nDNA, controversial results have been reported for age-related
variations of 8-oxodG in heart (6
7
8)
, brain
(5
6
7
8
9
10
11
12)
, and other tissues. However, those age-related
increases cannot be the result of a lifetime accumulation of oxidative
damage in mtDNA. The levels of 8-oxodG found in the mtDNA of old
individuals, which are between 2- and 15-fold higher than those of
young ones according to the different reports (5
, 6
, 13
, 15
, 16)
, as well as those found in the mtDNA of short-lived species
in this investigation, would be quickly reached in the absence of
repair systems. Mitochondria lack repair systems for some forms of DNA
damage like nucleotide excision repair or repair of pyrimidine dimers
(39)
. But contrary to previous beliefs, recent
investigations show that, analogous to what happens for nDNA, repair of
oxidative damage is very active in mtDNA (39
, 40)
. Since
mitochondrial free radical generation is known to be higher in
short-lived than in long-lived species (21
22
23
24
25
26
27
28)
, we expect
the same for the repair of endogenous oxidative damage to mtDNA
(although this still has not been investigated); otherwise, the
damage would soon rise to unbearable levels. Nevertheless, a higher
level of oxidative attack to the mtDNA of short-lived animals compared
with long-lived ones also suggested that higher 8-oxodG levels can help
to explain the quicker rate of accumulation of other multiple mtDNA
deleterious modifications during aging (19
, 20)
, which are
repaired with more difficulty or not at all.
In agreement with the existence of a higher turnover of oxidatively
damaged DNA bases in short-lived than in long-lived species, the rate
of urinary excretion of 8-oxodG and thymine and thymidine glycol is
known to be higher in rats and mice than in monkeys and humans
(41
, 42)
. On the other hand, the data obtained in this
investigation show that the equilibrium between oxidative attack and
repair in mtDNA is reached at higher 8-oxodG steady-state levels in
short-lived than in old animals. The same is true for old when compared
to young individuals (5
, 6
, 13
, 15
, 16)
. Since 8-oxodG is
mutagenic (43
, 44)
, the relatively higher steady-state
8-oxodG levels observed in short-lived species and in old individuals
would constantly impose a higher risk of mutations in their mtDNA. The
presence of a higher oxidative attack and higher 8-oxodG levels in the
mtDNA of short-lived in comparison with long-lived animals can help to
explain why mtDNA mutations accumulate faster in rodents (in 2–3
years) than in slow-aging animals (90 or more years in humans).
Our results should be considered within the context of current
gerontological facts and theories. The maximum difference in mtDNA
8-oxodG levels between short- and long-lived animals (4-fold) was
smaller than their difference in longevity (13-fold between mouse and
horse). The same occurred in all of our previous studies comparing
rates of mitochondrial oxygen radical generation among animals showing
different longevities (24
25
26
27)
. These observations do not
contradict the free radical theory of aging. On the contrary, they are
consistent with the widely accepted concept that aging is due to more
than one single major cause. Aging is thought to be due to relatively
few causes with major effects (the most interesting ones to study) plus
many causes with minor effects. Besides, even if oxidative stress were
the main cause of endogenous DNA damage, it would not be its only
basis. Other causes that are of minor importance when considered
individually could nevertheless have a relevant role collectively,
taking into account all their effects. A single mechanism cannot
explain all the variance of longevity between species, whereas the
quantitative differences in oxidative damage to mtDNA are consistent
with a multicausal origin of aging.
Previous investigations have consistently found that 8-oxodG
concentration is higher in mtDNA than in nDNA. Very few studies are
available, however, and those that are are limited to rodent liver
(6
, 17
, 18)
, with a single study available in postmortem
human brain (5)
. Other reports describing high 8-oxodG
levels in mtDNA (13
, 15
, 16)
are not useful for this kind
of discussion because 8-oxodG in nDNA was not simultaneously measured
in those investigations, and 8-oxodG values are known to vary depending
on the laboratories and methods of measurement used. This scarcity of
data and other considerations have led to doubts that oxidative damage
is in fact higher in mtDNA than in nDNA (2
, 39)
. In the
present investigation we have found that this difference between the
two types of DNA exists in the heart and brain of all the mammals
studied, using exactly the same method for mtDNA and nDNA in all the
steps in which it is possible to do it: DNA digestion and
chromatographic separation of 8-oxodG and dG. 8-oxodG levels were
around fourfold higher in mtDNA than in nDNA in the heart of all
mammals and between three- and ninefold higher in the case of the
brain, the highest difference being observed in the brain of
short-lived mammals. Thus, our results confirm earlier reports from
rodent liver (6
, 17
, 18)
and human brain after autopsy
(5)
, extending them to the heart and brain of mammals in
general. Since it is now known that the rate of repair of 8-oxodG is
rapid in mitochondria, similar to what happens in the nucleus
(39)
, the higher steady-state 8-oxodG levels observed in
mtDNA in relation to nDNA must be due to the higher oxidative attack
caused by the higher rate of oxygen radical generation present in the
organelles. This higher oxidative attack suffered by mtDNA can help
explain the accumulation of mtDNA mutations in postmitotic tissues
during aging of all kinds of animal species (short-lived or long-lived)
at the end of their life span (19
, 20)
.
In summary, the results obtained here show that levels of the oxidative
damage marker 8-oxodG are higher in mitochondrial than in nuclear DNA
in the heart and brain of a wide range of mammalian species.
Furthermore, the steady-state 8-oxodG concentration in mtDNA is higher
in rapidly aging mammalian species than in those that age slowly,
whereas 8-oxodG levels in nuclear DNA do not correlate with mammalian
longevity. Taken together, these results are consistent with the
predictions of a free radical theory of aging focused on mitochondria
(3
, 4
, 28
, 45
, 46)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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