From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
The study of DNA oxidation has progressed from
an exploratory phase, during which its basic biochemistry was
established, into a field branching out into numerous areas. Early on,
radiation biologists discovered that radiolysis of water generates
oxygen free radicals, which are responsible for many of the
consequences of irradiating living things. The characterization of
radiation-induced oxidative DNA lesions, and the connection between
radiation and cancer, caused a surge of interest in DNA oxidation
per se and raised the possibility of DNA damage from
biological oxidants. Nucleic acid biochemists, cancer biologists, and
toxicologists then set out to ask key questions: "how much oxidative
DNA damage is there, how does it get there, how and when is it removed,
and what are the consequences?" A proliferation of techniques has resulted in the confirmation of the early hypotheses and also delivered
some surprises. In this minireview, we have outlined some of the most
interesting recent results. Extensive reviews on DNA oxidation
published elsewhere have discussed the earlier work in detail (1-5). A
companion minireview by Henle and Linn (6) covers in depth the
biochemistry of DNA oxidation.
Methods for Measuring Oxidative DNA Damage The steady-state amount of DNA oxidation appears to be massive,
with oxidative adducts occurring at a frequency that is 1 or more
orders of magnitude higher than non-oxidative adducts (1, 7). Despite
their abundance, oxidative DNA adducts exist in a large background
(105-106) of unaltered nucleotides, which may
be prone to oxidation during sample preparation and analysis. Concerns
about artifactual oxidation, combined with the different values that
have been generated by alternative methods, have fueled an ongoing
debate over the most appropriate techniques for studying DNA
oxidation.
Gas chromatography coupled with mass spectroscopy
(GC-MS),1 initially used in characterizing
oxidative adducts, is also a quantitative tool whose principal
advantage is the simultaneous analysis of a number of different adducts
(5). DNA is chemically hydrolyzed, derivatized, and injected onto
GC-MS. In the absence of a mass spectrometer, an alternative approach
is the enzymatic hydrolysis of DNA to nucleosides and chromatography of
the hydrolysate by HPLC (8). The adducts
8-oxo-7,8-dihydro-2 Generally speaking, GC-MS estimates of DNA oxidation have been higher
than HPLC-EC estimates, by about a factor of 10 (9). The debate about
the cause of the difference (an overestimate due to artifactual
oxidation with GC-MS versus an underestimate due to
inefficient enzymatic digestion with HPLC-EC) has now been settled;
artifactual oxidation occurs during GC-MS derivatization, in the case
of guanine/oxo8dG (10) and in the case of adducts formed
from adenine, cytosine, thymine, and thymidine (9). The HPLC-EC method
itself, however, has also been criticized on the grounds that the
variability of the assay is unacceptable (11-15). Estimates of the
ratio of oxo8dG to dG (for example, in rat tissues) have
ranged from approximately 0.25 × 10 Artifactual oxidation poses problems of accuracy and precision. For
example, the total cellular burden of oxidative adducts has been
estimated from HPLC-EC measurements of oxo8dG (1), on the
assumption that oxo8dG represents 5% of all adducts (it is
one of about 20 major radiation adducts characterized by GC-MS (16)).
This estimate, about a million oxidative adducts per rat cell (1), is a
number which argues forcibly that oxidative mutagenesis must be
important in vivo (2, 3, 7). To the extent that the initial
measurement of oxo8dG may be artificially elevated, this
estimate may also be inaccurate. Worse, perhaps, is the effect that
artifactual oxidation has on the ability to detect an elevation of
oxo8dG. If the DNA damage "signal" is obscured by
background artifact "noise," then real increases in DNA oxidation
may be obscured or fail to achieve statistical significance.
Fortunately, a number of incremental improvements have recently been
introduced (12, 13), driving down the estimate of steady-state
oxo8dG 2-5-fold from previous estimates. The current
lowest estimates of the ratio of oxo8dG/dG (in rat
hepatocytes and human lymphocytes) cluster around 0.25 × 10 150,000 oxidative adducts per cell represents a huge load of damage: is
there solid evidence that this number is accurate? Although it is
difficult to rule out some contribution by artifactual oxidation to these values (or indeed to values derived from any of the
techniques that have been devised), emerging features of experiments
with oxo8dG lend them credibility. Namely, recent studies
show low sample-to-sample variance, as well as dramatic
patterns of appearance and disappearance of
oxo8dG following oxidant challenges, occurring in parallel
with the induction of oxo8dG repair activity (13, 17-21).
Together with the fact that radically different techniques (discussed
below) have demonstrated similar degrees and patterns of induced DNA
damage, these results suggest that the problem of artifactual noise has
been tamed, if not eliminated. Elsewhere, we have discussed in detail
how to avoid the artifacts that can occur with
HPLC-EC.2
The cloning and overexpression of repair enzymes continue to provide
new ways to detect oxidative adducts. Enzymes such as Escherichia
coli endonuclease III and formamidopyrimidine glycosylase (Fapy
glycosylase), which recognize and excise oxidized pyrimidines and
purines, respectively, possess associated lyase activities that result
in strand cleavage (23). Treating DNA with these enzymes introduces
nicks, which may then be measured by alkaline elution (24), nick
translation (25), or ring opening of supercoiled molecules (26).
The polymerase chain reaction (PCR) has provided an approach called
"quantitative PCR" (Q-PCR), which takes advantage of the fact that
many DNA lesions block thermostable DNA polymerases, thereby decreasing
the efficiency of amplification (27). As the length of the desired
amplicon increases, the probability that a strand-terminating adduct
will occur also increases, as does the sensitivity of the method. With
appropriate controls and calculations, the technique is quantitative,
although the types of DNA lesions resulting in decreased amplification
are only known in general. There is potential that Q-PCR may be coupled with repair endonucleases, enabling the quantification of specific adducts.
All of the techniques discussed so far, from GC-MS to Q-PCR, require
purified DNA. A technique for estimating the rate of oxo8dG formation, which does not require the isolation of
DNA and its associated problems, is the measurement of its repair
products excreted into urine or tissue culture medium (3, 28, 29). The
daily flux of repaired adducts should reflect the intracellular rate of
DNA damage, if not in a direct way. Although there is a different set
of concerns surrounding the accuracy of these methods (such as
uncertainty about repair pathways and products, oxidation of free
nucleotide pools, and the contribution of cell and mitochondrial
turnover), an overwhelming advantage of these methods is that they are
non-invasive and integrative. As a consequence, the measurement of
repaired adducts in urine is one of the few techniques that has been
routinely applied to humans. Recently, an elegant and related approach
has been reported: the measurement of oxo8dG in small
amounts of heart muscle interstitial fluid, collected with a
microdialysis probe. During a period of reperfusion following ischemia
(a well established model of oxidative stress) a rapid increase in
intercellular oxo8dG was observed (30).
Last, there exist two techniques that preserve cellular integrity:
single-cell gel electrophoresis and immunohistochemistry with
anti-DNA-adduct monoclonal antibodies (mAbs). Single-cell gel
electrophoresis, also descriptively termed the "comet" assay, involves casting cells in a thin agarose gel on a microscope slide and
running the DNA out of the nuclei by electrophoresis. The more
fragmented the chromatin, the more it migrates, assuming (upon
staining) the appearance of a comet's tail streaking away from the
nucleus in the direction of the anode. The analysis of the length and
intensity of the tail (its "moment") is achieved with the help of
software (31). Modifications of the assay permit the analysis of
specific lesions; alkaline conditions are used to study single-strand
nicks, and by treating cells in agarose in situ with an
enzyme such as Fapy glycosylase prior to single-cell gel
electrophoresis, the method has been used to detect the substrate oxo8Gua (32, 33). The specificity of mAbs has also been
utilized; mAbs to thymine glycol, for instance, are used in
enzyme-linked immunosorbent assays of purified DNA (34). The ultimate
power of mAbs, however, may lie in their ability to detect DNA damage in fixed cells and tissues in situ, as was recently reported
for a mAb to oxo8Gua (35).
There is no single ideal method for measuring oxidative lesions, as all
have their strengths and weaknesses. For instance, GC-MS and HPLC-EC
are rigorously quantitative but require relatively large quantities of
pure nucleic acids. Molecular biological methods like Q-PCR require
less DNA but are not as specific in their detection. Cellular assays
are ideal for analyzing tiny samples (hundreds of cells) and avoiding
cellular disruption but are semi-quantitative. What is encouraging
about recent results is the growing congruence between studies using
different approaches.
Mechanisms and Location of DNA Oxidation Until the last 2 years, it had become almost accepted wisdom that
the role of the superoxide anion radical (O Reactive nitrogen intermediates such as peroxynitrite
(ONOO A fascinating subtlety of DNA oxidation involves the possibility that
oxidation may be mediated by long distance electron transport along the
Oxidative Mutagenesis: GOing, GOing, GOne In E. coli, oxidative DNA damage is removed by pathways
involving both nucleotide and base excision pathways. The latter
include endonuclease III, which recognizes and removes oxidized
pyrimidines, and Fapy glycosylase (23), which performs a similar role
on oxidized purines. The latter enzyme is one of three gene products in
the coordinated "GO system" (mutM, mutT,
mutY), a set of three repair enzymes that suppress
mutagenesis by Guanine Oxidation, by removing
oxo8Gua paired with cytosine (mutM), adenine
paired with oxo8Gua (mutY), and by hydrolyzing
the oxidized nucleotide oxo8dGTP to the nucleoside
monophosphate (mutT), thereby preventing its incorporation
into DNA. Although space constraints preclude a full discussion of
oxidative repair enzymes here, it is important to stress the fact that
losses of the E. coli activities may result in 10-1000-fold
increases in the rate of spontaneous mutagenesis and that homologous
genes or activities have been identified in humans, including the
cloning of human homologs of endonuclease III (52), mutY
(53), and mutT (54) and the recent identification of a
MutM-like activity (55). As would be expected of a fundamental type of
DNA damage, repair of oxidative lesions appears widely conserved. The
cloning of mouse genes involved in repair of oxidative damage and the
subsequent generation of transgenic knockout mice by recombination will
permit a powerful test of the "oxidative mutagenesis" hypotheses of
cancer and aging (53).
Eukaryotes likely possess unique systems in addition to their homologs
of prokaryotic enzymes. The Drosophila ribosomal S3 protein,
which possesses an associated oxo8Gua glycosylase/AP lyase
activity and is able to restore the wild-type phenotype to
mutM mutants of E. coli, may be one such example (56). In addition to its involvement in protein synthesis, the S3
protein possesses a nuclear localization signal, hinting at communication between translation and DNA repair. Moreover, aberrant levels of the human ribosomal S3 protein have been reported in xeroderma pigmentosum and Fanconi's anemia, a disease associated with
elevated levels of the adduct oxo8dG. Mammalian cells also
face the additional burden of delivering DNA repair capacity to their
mitochondria, an organelle which, despite early reports to the
contrary, is able to process oxidative DNA damage (57). Therefore, in
addition to phenotypes resulting from loss of nuclear repair, there may
be collateral or independent syndromes associated with inefficient
repair of mtDNA. In xeroderma pigmentosum complementation group A, for
instance, a deficiency in the repair of oxidative damage of both
nuclear and mtDNA is observed (58).
The removal of oxidative adducts in human cells appears to be very
rapid. In lymphoblasts, the half-lives of
H2O2-induced adducts ranged from 8.5 to 62 min
(59). In human respiratory tract epithelial cells, repair of some
H2O2-induced adducts (for example,
oxo8dG) is so rapid that a narrow window of opportunity
(approximately 30 min) exists for their detection (60). The
heterogeneity of DNA adducts should be noted; whereas
oxo8dG may rapidly appear and disappear with an hour, in
the same cells thymine glycol and single-strand breaks (themselves a
result of repair) may increase (60).
Hormesis or the "beneficial effect of a low level exposure to an
agent that is harmful at high levels" (61) may be relevant for some
oxidative stresses, as has been argued to be the case for low level
radiation exposure (62). Hyperbaric oxygen therapy of humans (100%
O2 at 2.5 atm), for instance, induces significant oxidative
DNA damage to peripheral blood cells on the first day of therapy but
fails to cause damage on subsequent days (33); in fact, it results in a
lower base-line level of total and oxidative DNA damage. Similarly,
Experimental and epidemiological evidence suggests that DNA
oxidation is mutagenic and is a major contributor to human cancer through three major sources: smoking, chronic inflammation, and endogenous oxidants such as leakage from mitochondria (1, 3, 7, 64,
65). Cigarette smoke contains high levels of NOx and depletes the body's antioxidants, and phagocytic cells
recruited to sites of chronic infection abundantly generate reactive
oxidants such as NOx and HOCl. Oxidative
stresses such as these may contribute to as much as half of all human
cancers, and evidence of oxidative damage during experimental
carcinogenesis is accumulating. Merely to cite the most recent results,
elevated DNA oxidation has been measured during early
Helicobacter pylori infection (stomach cancer (66)), ferric
nitrilotriacetate administration (experimental rodent renal cancer
(67)), smoking (lung cancer (17, 68)), and exposure to diesel exhaust
particles (lung cancer (69)), asbestos (lung cancer (70)), benzene
(leukemia (71, 72)), and aflatoxin (liver cancer (73)). Some studies
have provided more than a simple association between carcinogenic
agents and oxidative DNA damage by measuring the specific induction of
repair enzymes by oxidative carcinogens (74) and by demonstrating the suppression of carcinogenesis by administration of antioxidants (21,
75). The latter results are consistent with the strong correlation
between a high intake of fruits and vegetables, which are the principal
source of dietary antioxidants, and reduction in cancer risk by as much
as half (1, 7). We have reported elevated oxidative damage to sperm DNA
in smokers and in men on low serum antioxidants (vitamin C) (76) and
have hypothesized that oxidative damage to male germ cells contributes
to cancer and birth defects in the children of male smokers (77).
Indeed, new epidemiological evidence indicates that all types of
childhood cancer studied are increased in offspring of male smokers;
for example, the risks of acute lymphoblastic leukemia, lymphoma, and
brain tumors are increased three to four times (78).
A transgenic mouse model, in which somatic mutations occurring in
vivo can be measured ex vivo with the use of a shuttle
vector incorporated into the mouse genome, has recently been used to quantify a 5-fold increase in mutant frequency in vivo in
response to short term ischemia-reperfusion, an oxidative stress (79). Transgenic in vivo mutagenesis models, the use of which is
becoming routine (80), should soon permit the degree and spectrum of mutagenesis to be measured in tandem with increases in oxidative damage. Already, the spectrum of alterations in the growing data base
of oncogenic human mutations provides some evidence of relevant oxidative mutagenesis, as shown by the high frequency of G-to-T transversions (a signature mutation resulting from oxo8Gua)
in human p53 and ras (81).
Stress and Damage from Cradle to Grave The role of DNA oxidation in diseases of aging and in
developmental abnormalities is less well established but ripe for
investigation. A fundamental unanswered question is whether or not DNA
oxidation is able to adversely affect quiescent and postmitotic cells
in diseases in which uncontrolled proliferation is not an issue. There
is evidence that the frequency of oxidative DNA adducts increases by as
much as 2-fold with age in a number of species and tissues (19, 82,
83). We have recently found that mitochondria from senescent animals
produce a greater flux of oxidants than young mitochondria, which is
consistent with these results and suggests a mechanism for the
observation (65). Even potent exogenous oxidants result in a limited
(several-fold at most) and rather short term increase in adduct
frequency (60). Therefore, it may be that an age-related, persistent
50-100% increase in the steady-state level of adducts is
physiologically relevant, representing an inability to prevent or
repair oxidative damage. Unfortunately, the detection of such a change
in the steady-state frequency of adducts requires the virtual absence
of artifactual background noise. Two recent and independent studies, in
which the frequency of oxo8dG in a variety of organs of
Fisher 344 rats was studied, illustrate this point. In the first, a
clear increase in the ratio of oxo8dG/dG was noted (18),
whereas in the second, the lack of a significant increase in the ratio
of oxo8dG/dG was associated with higher base-line values in
young animals (19).
So far, we have not made a distinction between the oxidation of nDNA
and that of mtDNA, but there is a body of work based on the HPLC-EC
detection of oxo8dG which suggests that the latter is
higher by more than 10-fold (48) and that age-related mtDNA oxidation
is particularly dramatic (84). These empirical observations have been
attributed to a number of properties of mtDNA, including its proximity
to metabolic oxidant generation and its established sensitivity to
mutagens in general. It is worth stressing that measuring mitochondrial oxo8dG by HPLC-EC represents a great methodological
challenge, due to the difficulty in purifying it in quantity. The
correspondingly greater potential for artifactual oxidation during the
preparation and analysis of mtDNA may explain the large disparity
between published values (85). The technique of Q-PCR, which requires neither DNA purification nor large amounts of DNA, has recently been
used in studies of mtDNA oxidation and has confirmed the greater
sensitivity of mtDNA than nDNA to exogenous oxidants (27, 68, 81).
However, the small number of studies, pitfalls of analyzing mtDNA, and
large range of credible values (85) indicate that more work on mtDNA
oxidation is needed, particularly in light of the potential role of
mitochondria in age-related human diseases (65).
Last (or perhaps first), there is evidence from the other end of the
human lifespan that oxidative damage may interfere with development.
The elevation of oxo8dG by teratogens has been observed
(86), and the phenotype of Cockayne's syndrome, which includes mental
retardation, developmental defects, and (often) premature death in
childhood, has been tightly associated with the specific lack of
transcription-coupled repair of oxidative damage (34). Even the process
of birth itself, which represents, among other things, the first direct
oxidative stress encountered by newborn mammals, induces a measurable
degree of genotoxic oxidative stress (87).
In this minireview, we have attempted to highlight the recent
results in this fast moving field. A principal outstanding question is:
"what proportion of carcinogenic and spontaneous mutations are caused
by metabolic oxidants?" Although there is evidence that specific
oxidative injury, such as that associated with reperfusion injury,
chronic infection, or smoking, may result in mutagenesis, it is less
clear that spontaneous mutations are oxidative. The coordinated use of
three different types of transgenic mouse models should soon make these
tractable problems. Transgenic mice have been established that possess
altered antioxidant activities (22) and permit the in vivo
quantification of mutagenesis (80). Combined with transgenic mice
possessing deleted or overexpressed oxidative repair endonucleases,
they may provide ideal models (53) for studies in which analytical
measurements of oxidative adducts are combined with direct measurements
of mutagenesis.
-deoxyguanosine (oxo8dG) and its
corresponding base 8-oxo-guanine (oxo8Gua) are especially
useful in this regard, since they are electrochemically active, lending
themselves to sensitive electrochemical (EC) detection. The relative
simplicity and high sensitivity of HPLC-EC detection of
oxo8dG have made it the most popular method for monitoring
DNA oxidation in vivo.
5 to higher than
10
4, and it has been suggested that artifactual oxidation
is to blame.
5, equivalent to approximately 7,500 oxo8dG
or about 1.5 × 105 oxidative adducts per human cell
(if oxo8dG represents 5% of all such adducts) (12,
13).
2) in DNA oxidation was its ability to reduce ferric iron (Fe3+) to ferrous
iron (Fe2+); Fe2+ catalyzes the formation of
the hydroxyl radical ·OH (from H2O2)
which, according to the scheme (referred to as Fenton chemistry), is
the ultimate reactive species in DNA oxidation (6). Support for the
roles of all three components of this model (O
2, iron, and
H2O2) continues to accumulate (36, 37). However, as is discussed in the companion minireview by Henle and Linn
(6), the nuances of DNA oxidation have turned out to be more complex
and interesting. For one, the nature of the ultimate oxidant
responsible for DNA damage by H2O2 is unclear. Detailed experiments have illustrated that a model of freely diffusible ·OH fails to account for the strikingly parallel dynamics of DNA strand scission in vitro and cytotoxicity of
H2O2 to E. coli. Rather, multiple
classes of oxidant appear to exist, associated with the DNA double
helix to different extents (6). Moreover, the role of O
2 in
reducing free ferric iron has been challenged by experiments suggesting
that its principal role is to release iron from protein-bound
iron-sulfur clusters (38). Besides O
2, there are other
reductants (such as NADH) that effectively reduce Fe3+ to
Fe2+ and that may be more relevant as reductants of free or
DNA-bound iron than is O
2 (39). Copper (40) and less well
studied transition metals such as chromium (41) also take part in
Fenton-like chemistry in DNA oxidation.
) also react with DNA, forming (among other lesions)
the adduct oxo8dG (42-44). Interestingly,
oxo8dG itself is far more susceptible to peroxynitrite than
dG, which emphasizes the fact that more stable oxidative end products
than oxo8dG exist (45, 46). Also, oxidative DNA adducts may
be formed indirectly; the peroxidation of membrane lipids results in
various aldehyde breakdown products that are able to form covalent
mutagenic adducts (47). Recently, we have reported that the
concentration of protein-bound aldehyde accumulates with age in rats
(48), suggesting that aldehyde-DNA adducts may also increase with age. A final complication is the evidence that DNA is not a homogeneous target of oxidative damage and repair. Internucleosomal DNA appears at
least 3.5 times more susceptible than nucleosomal DNA to oxidation by
physiological iron chelates (49), and repair of a number of adducts is
more rapid in DNA in the nuclear matrix than in total chromatin
(50).
stack of the DNA double helix. Experiments with synthetic
double-stranded oligonucleotides have shown that long range oxidative
damage may occur, resulting in the formation of oxo8dG in
susceptible 5-GG-3
at a distance from a covalently attached terminal
oxidant (51). If such a phenomenon is important in vivo, it
may mean that the topology of DNA serves to channel or trap oxidation
in zones.
-irradiation of rats, which significantly elevates oxidative adducts
in hepatic chromatin, results in lower base-line levels of some
oxidative DNA adducts 24 h after an acute exposure (50), and other
observations of the lowering of base-line oxidative damage by oxidants
have appeared (27). These results are not surprising, since defense
systems are often induced in response to oxidative stress, a
generalization that has recently been extended to oxo8Gua
glycosylase activity in E. coli (63) and rats (20). This implies that there is a degree of slack in oxidative defense and repair
under "normal" circumstances and that cells may ordinarily tolerate
a burden of oxidative adducts that contributes to the spontaneous rate
of mutation.
We thank Hal Helbock, Mark Shigenaga, and Stu Linn for critical reading of the manuscript.