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Laboratory of Molecular Genetics, National Institute on Aging, Baltimore, Maryland 21224-6825, USA
3Correspondence: Laboratory of Molecular Genetics, National Institute on Aging, GRC, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224-6825, USA. E-mail: vbohr{at}nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: 8-hydroxy-2'-deoxyguanosine • 8-hydroxydeoxyguanosine • 8-oxoguanine • 8-oxodG • aging • mitochondria
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and numbers as high as
163 per genome (4840 per 106 bases) have been
reported (2)
. This damage was suggested to cause the
eventual breakdown of proper mitochondrial function and to be a primary
cause of aging (3
, 4)
. However, the reported levels of
8-oxo-dG in mtDNA vary by five orders of magnitude, ranging from 0.08
per 106 bases in mtDNA isolated from cultured
HeLa cells (5)
to 4840 per 106 bases
in mtDNA isolated from the hearts of 100-wk-old rats (2)
.
Even within a single system, the values cover a wide range. Levels
reported for mtDNA isolated from rat liver range from 4 per
106 bases (6)
to 110 per
106 bases (2)
(reviewed in refs
1
, 7
). At least two factors have contributed to this
diversity. First, several methods are commonly used to measure the
level of 8-oxo-dG in DNA, and very little information is available
concerning their relative sensitivity. Second, in order to measure the
level of damage in mtDNA it has been necessary to isolate the
mitochondria. Mitochondrial isolation provides opportunity for the
induction of oxidative damage. Also, since mitochondria are able to
repair 8-oxo-dG (8
9
10)
and a mitochondrial enzyme
responsible for excision of 8-oxo-dG from double-stranded DNA
(11)
has been isolated, the possibility of post-isolation
repair is an issue. Thus, measurement of oxidative damage to mtDNA
after mitochondrial isolation may in part reflect the ability of the
isolated mitochondria to resist or remove damage during the isolation
procedure rather than the in vivo steady-state levels.
To address these issues, we measured 8-oxo-dG in DNA using two methods:
high-pressure liquid chromatography with electrochemical detection
(HPLC/ECD) (12
, 13)
and an enzymatic/Southern blot assay
(14)
using the bacterial repair enzyme formamidopyrimidine
glycosylase (Fpg). The former method is the most widely used to detect
endogenous 8-oxo-dG in DNA. The latter is used to measure the removal
of exogenously induced 8-oxo-dG from DNA in studies of DNA repair. It
has two major advantages over chemical methods such as HPLC/ECD: only a
few micrograms of total DNA are required, as opposed to 50 or more
micrograms of purified mtDNA; with the enzymatic/Southern blot method,
isolation of mitochondria and mtDNA is not required. However, its
sensitivity relative to HPLC/ECD had not been established.
In the present paper we used photoactivated methylene blue (MB) to create a series of DNA standards that contain linearly increasing amounts of 8-oxo-dG. This technique permits determination of the sensitivity and precision of a measurement system across a range of DNA damage levels. These standards were used to compare the HPLC/ECD and enzymatic/Southern blot methods for measurement of 8-oxo-dG in DNA. The results were linear and there was close agreement between the two methods. This showed that both measurement systems provide precise determinations of the level of damage, and the agreement between the two methods argues that the actual levels detected are accurate. We then applied the enzymatic/Southern blot method to the measurement of oxidative damage in mtDNA both with and without isolation of mitochondria. The level of damage seen in the two samples, from the same animals, was threefold higher in mtDNA when the mitochondria had been subjected to isolation. In the absence of organelle isolation, most genomes did not contain enzyme-sensitive sites (that is, they were lesion free). The implications of this finding, as well as the advantages and disadvantages of each method and questions that can best be addressed by the application of both methods simultaneously, will be discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was used as a DNA source for in vitro
work. Six- to 7-month-old outbred male Wistar rats (Gerontology Research Center Colony) were used in this study. They were maintained on a 12/12 L-D cycle with lights on at 07:00 h and food (NIH Open Formula 079) and water available ad libitum. Animals were killed by cervical dislocation between 8:30 and 9:30 a.m.).
Mitochondrial isolation
Mannitol, EGTA, spermine, and spermidine were obtained from
Sigma Chemical Company (St. Louis, Mo.). EDTA was from Quality
Biological, Inc. (Gaithersburg, Md.). HEPES was obtained from Advanced
Biotechnologies, Inc. (Columbia, Md.) and sucrose from ICN Biomedicals,
Inc. (Aurora, Ohio).
Livers were removed and placed immediately in ice-cold MSHE buffer (0.21 M mannitol, 0.07 M sucrose, 10 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.15 mM spermine, 0.75 mM spermidine), where they were minced. The MSHE was replaced with fresh buffer and the livers were homogenized with a glass and Teflon homogenizer. The unbroken cells and nuclei were pelleted by centrifugation at 500 x g for 7 min. This pellet was resuspended in cold MSHE buffer and an aliquot was taken for isolation of nuclear DNA. The supernatant, which contained the mitochondria, was centrifuged again for 7 min at 9500 x g. The mitochondrial pellet was washed twice by resuspension in MSHE buffer, followed by centrifugation for 7 min at 9500 x g before the final resuspension in MSHE buffer.
DNA isolation
Total DNA for use in experiments involving in vitro
damage was isolated from BL-2 human transformed lymphoblasts
(15)
by salt extraction (16)
.
Rat liver total and mtDNA was isolated as follows: sodium dodecyl sulfate (SDS) (Quality Biological, Inc.) was added to the mitochondria or homogenate to 1% and the lysate incubated for 1 h at 37°C to promote complete lysis. After this treatment, 1/4 volume of 55°C saturated NaCl was added and the mixture was cooled to 4°C. The resulting protein and SDS precipitate was removed by centrifugation at 10,000 x g for 5 min and the supernatant was purified using freshly prepared phenol (Life Technologies, Gaithersburg, Md.), phenol/chloroform, and chloroform (Mallinckrodt, Phillipsburg, N.J.). DNA was precipitated overnight and resuspended at ~500 µg/ml in TE, pH 8.0, prior to treatment with 100 µg/ml RNAse for 1 h at 37°C. The RNAase was removed by addition of SDS to 1%, followed by precipitation with NaCl as described above. The precipitate was removed by centrifugation at 10,000 x g for 5 min and the DNA in the supernatant was precipitated with 2 volumes of ethanol.
Fpg digestion
Reaction conditions for digestion with Fpg (which for this study
was obtained from Dr. Arthur Grollman, State University of New York,
Stony Brook, N.Y.) were as follows: concentrated reaction buffer was
mixed with DNA dissolved in TE (final 1x concentrations: 0.5 mg/ml
bovine serum albumin, 50 mM Tris-Cl, pH 7.5, 50 mM KCl, 1 mM EDTA). The
Fpg:DNA ratio was held between 0.01 to 0.1 µg Fpg:µg DNA. All
incubations were at 37°C.
The Fpg/Southern blot assay has been described previously
(10)
. In brief, a master mix of DNA was prepared in 1x
reaction buffer for each sample. One aliquot of this mix was then
treated with Fpg and a second with 1x reaction buffer. Between 0.5
µg and 2 µg of DNA was used for each reaction, using digestion
conditions as described above. After Fpg treatment, the DNA was fully
denatured by a 37°C incubation in 0.06 M NaOH for 15 min, then the
entire reaction run on an alkaline gel and analyzed by Southern blot
hybridization. The principle behind the assay is that Fpg generates a
single-strand nick at the site of damage in a restricted DNA fragment.
In a denaturing gel, the cleaved strand migrates faster than the
full-length strand and thus moves forward, away from the main band.
After blotting and probing, this loss of damaged DNA from the main band
allows quantitation of the undamaged DNA.
Southern blotting and probing
Southern blotting was performed essentially as described
previously (10)
. Membranes were hybridized to
32P-labeled probes generated as riboprobes
(SP6/T7 Transcription Kit, Boehringer Mannheim Corporation,
Indianapolis, Ind.) or by random primed labeling (Random Primed DNA
Labeling Kit, Boehringer Mannheim). Two probes were used, one for the
mitochondrial genome and one for nuclear DNA. The mitochondrial probe
was directed against the heavy strand of the mitochondrial ribosomal
sequence and the nuclear probe was directed against the nuclear
ribosomal sequence. Both probes have been described previously
(10)
. Radioactivity in the bands was quantitated using a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, Calif.) and
ImageQuant software (Molecular Dynamics).
Damaging DNA in vitro
After treatment with Fpg and restriction with PvuII
(Boehringer Mannheim), BL-2 DNA was diluted in 10 mM Tris-HCl, 1 mM
EDTA, pH 8 (TE) to 150 µg/ml. One volume of 6 µM MB (Ricca Chemical
Company, Arlington, Tex.: 
max = 655 nm, 
=89125) was added to
the DNA in the dark, mixed rapidly, and the mixture was aliquoted into
60 mm plastic tissue culture plates. The solution depth was ~2 mm.
The dish was exposed to visible light [0.187
kW/m2, measured with an IL1400A
Radiometer/Photometer (International Light, Inc., Newburyport, Mass.)]
from a 300 W tungsten bulb situated beneath the plate. Heat buildup was
prevented by use of a fan beneath the Plexiglas surface on which the
dish rested. To remove MB, SDS was added to 1%, the solution was
extracted three times with butanol, and the DNA was then ethanol
precipitated, washed with 70% ethanol, and resuspended in TE. One-half
of each sample was digested with Fpg at this stage. All samples were
repurified with SDS and salt (16)
and the DNA was stored
as an ethanol precipitate until ready for analysis. All work was done
under a dim blue light while MB was present in solution with DNA.
HPLC/ECD
Nucleosides were prepared enzymatically from DNA using nuclease
P1 and alkaline phosphatase (Boehringer Mannheim Corporation), then
filtered through a 0.22-micron filter and a 30 kDa cutoff spin column
(12)
. Desferal (1 mM) was included to chelate iron and
reduce the risk of artifactual oxidative damage.
The nucleosides were separated isocratically using a C-8 column (YMC,
Inc., Wilmington, N.C.) at a flow rate of 1 ml/min for 30 min to avoid
spillover to the successive run. The mobile phase was composed of 100
mM sodium acetate (Sigma Chemical Company), pH 5.15, and 5% methanol
(Fisher Scientific International Inc., Hampton, N.H.). Both 8-oxo-dG
and dG were detected electrochemically using a four-channel coularray
(ESA, Inc., Chelmsford, Mass.). Two channels were set at low potentials
(285, 400 mV) for detection of 8-oxodG and two channels at higher
potentials for detection of 2-deoxyguanosine (800–900 mV). The peaks
were identified in the samples according to the retention time and
ratio accuracy. All samples were analyzed twice. A 2'-deoxyguanosine
standard was purchased from Sigma Chemical Company and an 8-oxo-dG
standard from ESA, Inc. The concentrations of the standards were
determined spectrophotometrically. For 8-oxo-dG, we used = 11300 at
295 nm in mobile phase and for 2'-deoxyguanosine, = 13000 at 254
nm.
Data analysis
Damage, as measured by HPLC/ECD, is normalized to unmodified dG.
For conversion to standard units (lesions per 106
dN), 8-oxo-dG per 105 dG was multiplied by 2.2 based on
22% dG for mammalian DNA (for a list of nucleic acid compositions by
species, see ref 17
).
To calculate the amount of damage in standard units using the Fpg/Southern blot assay, the number of incisions per fragment was calculated using the Poisson distribution: Incisions = -ln(Fpg treated/untreated). This number was divided by the number of dGs present in the fragment and multiplied by 2.2 x 105. For the ribosomal fragment, for which the exact sequence was not known, an approximate conversion was obtained by assuming that the fragment has an average dG content and multiplying the number of lesions in the fragment by 106/fragment length. (In actuality, the ribosomal region is known to be dG-rich, and so this procedure probably leads to a slight overestimation of the true level in the bulk DNA.)
A single-factor ANOVA was used to determine whether the probability there was a difference between the levels of 8-oxo-dG measured in mtDNA from isolated mitochondria, mtDNA from the same animals measured without mitochondrial isolation, and nuclear ribosomal DNA from the same animals. When a significant difference was observed, a T test assuming unequal variance was used to determine the differences between pairs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, filled circles). Aliquots of DNA from each time point were
treated with Fpg after damage, and residual 8-oxo-dG was measured by
HPLC/ECD. Less than 10% of the induced 8-oxo-dG remained after Fpg
treatment (Fig. 1A
, open circles). This percentage was not
dependent on the amount of damage present prior to Fpg digestion within
the range measured, suggesting that 10% of the induced 8-oxo-dG may
have been inaccessible to the Fpg enzyme.
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DNA damaged in vitro with MB was analyzed by the
Fpg/Southern blot method. Both nuclear ribosomal and mitochondrial
sequences were probed, and the number of Fpg-sensitive sites was
determined using the Poisson equation. This number was converted to
lesions/106 dN based on the approximate fragment
length (for nuclear ribosomal DNA) or the number of guanines present in
the sequence (for mtDNA). The results were compared with the results of
an HPLC/ECD analysis of the same DNA (Fig. 1B
). The methods
gave virtually identical answers. This was true regardless of which
sequence was probed (data not shown).
In addition to 8-oxo-dG, photoactivation of MB also causes DNA strand
breaks. This can be seen as a shift in the molecular weight of the DNA
that has not been treated with Fpg, as viewed on a denaturing agarose
gel stained with Sybrgreen (Molecular Probes) (data not shown), as well
as a weakened signal in the lanes not treated with Fpg on the Southern
blot (Fig. 1C
).
DNA from mitochondria isolated from rat liver or from the crude
homogenate were subjected to Fpg/Southern blot analysis (Fig. 2
). The endogenous levels based on DNA from the crude homogenate were
between 5 and 10, 8-oxo-dG per 106 dN for both
nuclear and mtDNA, and were not significantly different
(P=0.36). (This is equivalent to 0.16, 8-oxo-dG per 32 kdN,
which is the number of nucleotides in a double-stranded mitochondrial
genome. Thus, on average, only 16 of every 100 mitochondrial genomes
would contain the lesion.) The endogenous levels based on DNA from
isolated mitochondria from the same animals were approximately
threefold higher (P<0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Prior to treatment of DNA with photoactivated MB, endogenous 8-oxo-dG
was removed from the DNA enzymatically (18)
. This was done
for two reasons: 1) to generate samples that had less damage
than would be found in vivo, and 2) to correct
for the possibility that endogenous damage would be more extensive in
some sequences than in others. This is necessary because the HPLC/ECD
and Fpg/Southern blot methods measure different parameters to determine
the amount of damage present in a sample. The HPLC/ECD system measures
damage directly, and a random distribution is assumed when the number
of lesions detected is divided by the amount of DNA assayed. The
Fpg/Southern blot method, in contrast, measures undamaged DNA. A random
distribution is assumed when the number of lesion-free DNA strands is
used in the Poisson equation (14)
to calculate the average
number of lesions in a given amount of DNA. If damage is distributed
randomly, the distinction is not important. If it is not, however,
HPLC/ECD will overestimate the number of DNA strands that actually
contained damage, whereas the Fpg/Southern blot method will
underestimate the number of lesions present in the sample. Although
there may be some minor effect of sequence, DNA in vitro is
much more homogenous than it is in situ, where protein
binding and cellular differences make it likely that some cells and
sequences will contain more damage than others.
The efficacy of this removal of endogenous damage and the
enzymatic/Southern blot method both depend on the efficiency of the
enzymatic reaction. To determine this efficiency, we treated aliquots
of damaged DNA from each time point with Fpg and measured residual
8-oxo-dG using HPLC/ECD. Greater than 90% of the induced 8-oxo-dG was
removed by Fpg treatment (Fig. 1A
). This percentage was not
dependent on the amount of damage present prior to Fpg digestion within
the range measured.
Next we analyzed the damaged samples using the Fpg/Southern blot method
and compared the results with those of the HPLC/ECD analysis of the
same DNA. The methods give virtually identical answers (Fig. 1B
). However, at least one FapyGua is produced by treatment
of DNA with photoactivated MB for every 5 to 10 8-oxo-dGs
(19)
. FapyGua is not alkali sensitive and is excised by
Fpg, and so contributes to the damage measured by the Fpg/Southern blot
method. Since the two assays agree exactly, and yet roughly 10% of the
damage detected by the Fpg/Southern blot assay is not the lesion of
interest, then by subtraction the Fpg assay is detecting only ~90%
of the 8-oxo-dG detected by HPLC/ECD. This agrees with the 90%
enzymatic efficiency calculated above based on HPLC/ECD detection of
residual 8-oxo-dG in Fpg treated DNA (Fig. 1A
). The
linearity of the results shows that both measurement systems provide
precise determinations of the level of damage, and close agreement
between the two methods argues strongly for the precision and accuracy
of both assays.
To address the issue of mitochondrial isolation, we applied the
Fpg/Southern blot analysis (Fig. 2)
to the measurement of oxidative
damage in mtDNA both with and without isolation of mitochondria.
Nonisolated mtDNA contained ~5 lesions per 106
bases, whereas levels of damage were approximately threefold higher in
the isolated mtDNA from the same animals (P<0.01). Since
the DNA isolation protocol was the same for all samples, it is likely
that the damage is occurring during the mitochondrial isolation.
Published values for the ratio of mitochondrial to nuclear levels of
8-oxo-dG range from 2.0 (20)
to 16.0 (21)
. To
calculate such a ratio in the present study, it must be assumed that
the nuclear sequence probed is representative of the remainder of the
genome. This may not be the case: as noted above, there are theoretical
reasons to suspect that nuclear ribosomal DNA may contain higher levels
of damage than other nuclear sequences. However, this assumption does
allow a rough comparison with earlier work in the field. Basing the
ratio on mtDNA damage present without isolation of organelles, the
ratio of mitochondrial to nuclear levels of 8-oxo-dG is less than 1.
Basing the calculation on the level of damage in mtDNA from isolated
mitochondria, the ratio is 2.5.
Many previous studies have shown that oxidative DNA damage in isolated
mtDNA increases with age (22)
and in certain disease
states, such as Alzheimer’s Disease (23)
and
atherosclerosis (24)
. Although the present work suggests
that the absolute numbers reported in earlier studies may have been too
high, the changes attributed to age or disease remain valid, since both
young and old (or diseased and healthy) mitochondria were subjected to
the same analysis. The reason for the observed changes must now be
revisited: did the change in the DNA truly occur in vivo, or
was there a change in other mitochondrial components caused by age or
disease that led the mtDNA to be more readily oxidized during
isolation? There are many opportunities to oxidize DNA as it is
prepared for analysis. This is especially true for mtDNA, which cannot
be isolated from the organelles until the mitochondria themselves have
been isolated and manipulated extensively. Since an increase in
vulnerability during isolation is likely to reflect an increased
vulnerability to challenge in vivo, the underlying reason
for the vulnerability would be of great importance. If the increase in
damage is not due to sensitivity during isolation but instead occurs
in vivo, questions concerning the cause of the increased
damage, and the cellular and subcellular distribution of the damage,
become even more pressing.
In summary, using an enzymatic/Southern blot method that does not
require mitochondrial isolation, the present study shows that damage in
mtDNA is not extensive in rat liver from young rats. Indeed, in the
absence of organelle isolation, most genomes did not contain
enzyme-sensitive sites (were lesion-free). Rather, low levels of
oxidative damage are induced during mitochondrial isolation. It has
been proposed in the past that it is high steady-state levels of damage
that lead to functional consequences, and that the increase seen with
age is due to a vicious cycle of damage causing dysfunction, which in
turn causes more damage (25)
. In view of the present
results, this idea must be reevaluated. Steady-state levels of damage
in young, normal mtDNA are maintained at low levels, and the cause for
increases commonly seen with age and in some disease states must be
investigated. The increases may be due to an age-related failure of
anti-oxidative pathways or of mtDNA repair, to increased susceptibility
to oxidation during isolation, or to some other factor as yet not
considered.
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FOOTNOTES |
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2 Current affiliation: IGEN International, Gaithersburg, MD 20877, USA.
Received for publication July 2, 1999. Revised for publication September 15, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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