Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents

Article abstract of DOI:10.1021/tx960102w

Reactive oxygen species, produced endogenously or by exposure to environmental chemicals and ionizing radiation, induce a wide range of DNA lesions. The variety of chemistries associated with different oxidants suggests that each will produce a unique spectrum of DNA damage products. To extend our efforts to relate genotoxin chemistry to DNA damage, we measured both strand breaks and 8-oxoguanine (8-oxoG) in DNA after exposure to γ- radiation, Fe(II)-EDTA/H₂O₂, Cu(II)/H₂O₂, and peroxynitrite at concentrations approaching physiological relevance. We found that the ratio of 8-oxoG to strand breaks varied more than 10-fold depending on the oxidizing agent: ~0.4 for Cu(II)/H₂O₂ and peroxynitrite and ~0.03 for Fe(II)-EDTA/H₂O₂ and γ-radiation. In the case of Cu(II)/H₂O₂, the relative proportion of 8-oxoG and strand breaks was found to vary more than 2-fold (0.14-0.37) for different Cu(II) concentrations, consistent with other studies. We were able to detect 8-oxoG formation by peroxynitrite by using low peroxynitrite concentrations in conjunction with a sensitive immunoaffinity/HPLC-ECD methodology. The level of 8-oxoG relative to strand breaks produced by peroxynitrite was higher than that produced by Fe(II)- EDTA/H₂O₂ and γ-radiation, which is consistent with the altered reactivity or accessibility of a non-hydroxyl radical species produced by peroxynitrite.

Full text of DOI:10.1021/tx960102w

386
Chem. Res. Toxicol. 1997, 10, 386-392
Qu a n tita tion of 8-Oxogu a n in e a n d Str a n d Br ea k s
P r od u ced by F ou r Oxid izin g Agen ts
Laura J . Kennedy,† Kenneth Moore J r.,‡ J ennifer L. Caulfield,‡
Steven R. Tannenbaum,†,‡ and Peter C. Dedon*,†
Division of Toxicology and Department of Chemistry, Massachusetts Institute of Technology,
77 Massachusetts Avenue, 56-787, Cambridge, Massachusetts 02139-4307
Received J une 20, 1996X
Reactive oxygen species, produced endogenously or by exposure to environmental chemicals
and ionizing radiation, induce a wide range of DNA lesions. The variety of chemistries
associated with different oxidants suggests that each will produce a unique spectrum of DNA
damage products. To extend our efforts to relate genotoxin chemistry to DNA damage, we
measured both strand breaks and 8-oxoguanine (8-oxoG) in DNA after exposure to γ-radiation,
Fe(II)-EDTA/H2O2, Cu(II)/H2O2, and peroxynitrite at concentrations approaching physiological
relevance. We found that the ratio of 8-oxoG to strand breaks varied more than 10-fold
depending on the oxidizing agent: ∼0.4 for Cu(II)/H2O2 and peroxynitrite and ∼0.03 for Fe-
(II)-EDTA/H2O2 and γ-radiation. In the case of Cu(II)/H2O2, the relative proportion of 8-oxoG
and strand breaks was found to vary more than 2-fold (0.14-0.37) for different Cu(II)
concentrations, consistent with other studies. We were able to detect 8-oxoG formation by
peroxynitrite by using low peroxynitrite concentrations in conjunction with a sensitive
immunoaffinity/HPLC-ECD methodology. The level of 8-oxoG relative to strand breaks
produced by peroxynitrite was higher than that produced by Fe(II)-EDTA/H2O2 and γ-radiation,
which is consistent with the altered reactivity or accessibility of a non-hydroxyl radical species
produced by peroxynitrite.
oxynitrite (13). However, peroxynitrite appears to have
a different reactivity than hydroxyl radical (9), which may
be evident in the spectrum of DNA damage products it
produces.
Copper is a biologically important metal that appears
to play a role in organization of the nuclear matrix (14).
In the presence of hydrogen peroxide, copper(II) has been
proposed to generate hydroxyl radical-like species through
Fenton-type reactions (15, 16):
In tr od u ction
DNA damage resulting from exposure to reactive
oxygen species plays a role in a variety of biological
processes such as mutagenesis, aging, and carcinogenesis
(1, 2). Reactive oxygen species arise in normal cellular
processes such as metabolism and inflammation and are
generated during exposure to environmental chemicals,
ionizing radiation, and transition metals (1, 3-5). The
variety of chemistries associated with different oxidants
suggests that each will produce a unique spectrum of
DNA lesions consisting of single and double strand
breaks, modified bases, abasic sites, and DNA-protein
cross-links (6). As part of an effort to relate genotoxin
chemistry to DNA damage, we have undertaken studies
to define the relative quantities of two different DNA
lesions, strand breaks and 8-oxoguanine (8-oxoG), pro-
duced by four oxidizing agents: peroxynitrite, Cu(II)/
H2O2, Fe(II)-EDTA/H2O2, and γ-radiation.
Each of these oxidants produces DNA damage by a
different mechanism. Nitric oxide, an important media-
tor of many biological processes (7), reacts with super-
oxide to form peroxynitrite (ONOO-) with a rate constant
near the diffusion-controlled limit (8). Peroxynitrite and
its conjugate acid, peroxynitrous acid (ONOOH), are
highly reactive at physiological pH and are capable of
oxidizing a variety of biomolecules, including thiols, DNA,
and lipids (9). Peroxynitrite and SIN-1, a compound that
simultaneously generates nitric oxide and superoxide
(10), both produce 8-oxoG (10, 11) and strand breaks in
DNA (10, 12); it is possible that the formation of 8-oxoG
in activated macrophages relates to production of per-
Cu(II) + H2O2 f Cu(I) + H2O + H+
Cu(I) + H2O2 f Cu(II) + OH- + •OH
The preferential binding of both Cu(I) and Cu(II) to the
N7 of guanine may account for the high level of 8-oxoG
produced by Cu and hydrogen peroxide (17-19) and for
the occurrence of Cu-mediated damage (20, 21) and
mutation at runs of guanine (22, 23). This binding mode
may also affect the relative proportions of strand breaks
and 8-oxoG produced by Cu.
Iron is another physiologically important metal that
has been proposed to act as a catalyst for the formation
of hydroxyl radical from hydrogen peroxide by the Fenton
reaction (24):
Fe2+ + H2O2 f Fe3+ + OH- + •OH
Of particular interest is the EDTA complex of Fe(II) that,
in the presence of hydrogen peroxide, has been shown to
generate a hydroxyl radical-like species (25, 26). Unlike
free Cu and Fe, the negative charge of the Fe(II)/EDTA
complex likely prevents it from binding to DNA phos-
phates or bases (26, 27). The resulting hydroxyl radical-
like species may exist in the fluid phase, which could
* To whom correspondence should be addressed.
† Division of Toxicology.
‡ Department of Chemistry.
X Abstract published in Advance ACS Abstracts, March 15, 1997.
S0893-228x(96)00102-6 CCC: $14.00 © 1997 American Chemical Society

8-Oxoguanine and Strand Break Formation by Oxidizing Agents
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 387
bromide-activated Sepharose 4B, N2-methyl guanosine, dieth-
ylenetriaminepentaacetic acid (DETAPAC), and phosphate-
buffered saline (PBS) were purchased from Sigma Chemical Co.
Cupric chloride, ethylenediaminetetraacetic acid (EDTA), potas-
sium phosphate (mono- and dibasic), agarose, and acetonitrile
were obtained from Mallinckrodt. Hydrogen peroxide (30%) and
ammonium acetate were purchased from Fisher Scientific.
Dimethyl sulfoxide (DMSO) and 98% formic acid were obtained
from EM Science. Chelex 100 Resin and Poly-prep columns
were purchased from BioRad Laboratories. SpectraPor 7 mem-
branes with a MWCO of 1000 were obtained from Spectrum.
Bromine was purchased from Aldrich Chemical Co. [3H]8-
oxoGua was generously provided by Dr. William Boadi (Division
of Toxicology, Massachusetts Institute of Technology). Plasmid
Giga purification kits were purchased from Qiagen.
Syn th esis of N2-Meth yl-8-oxoG. N2-Methyl guanosine (500
µg) was dissolved in 500 µL of double distilled water, and 40
µL of brominated water (1:200 Br2 in H2O) was added with
mixing by inversion. N2-Methyl-8-bromoguanosine was purified
from unreacted N2-methyl guanosine by HPLC using an Alltech
C18 column (25 cm × 4.6 mm) with 50 mM ammonium acetate
(pH 5.5) as mobile phase and monitoring at 260 nm. N2-Methyl-
8-bromoguanosine eluting at ∼14 min was collected manually
and then dried by centrifugation under vacuum (0.05 mbar).
Formic acid (500 µL) was added, reacted for 16 h at 130 °C,
and then dried by centrifugation under vacuum; this reaction
was repeated a second time. The product was redissolved in
50 mM ammonium acetate (pH 5.5), filtered, and purified by
HPLC using an Alltech C18 column (25 cm × 4.6 mm) with 2%
methanol in water as mobile phase and monitoring at 260 nm.
The fraction that eluted at 14 min was collected and dried by
centrifugation under vacuum. The fraction was then derivatized
by adding 15 µL of acetonitrile, 10 µL of pyridine, and 25 µL of
BSTFA and heating to 130 °C for 20 min. The product was
identified as N2-methyl-8-oxoguanine by GC/MS. The product
was dried by centrifugation under vacuum and stored at -20
°C.
account for the lack of base sequence-selectivity of
strand breaks generated by the complex (26, 27). In this
respect, Fe(II)/EDTA may be similar to ionizing radiation
in its DNA damage spectrum.
In addition to a minor contribution from direct inter-
action with DNA, ionizing radiation reacts with water
to produce hydroxyl radicals and hydrogen atoms by
homolytic fission of oxygen-hydrogen bonds and to
produce hydrated electrons (28). As with Fe(II)/EDTA,
a radiation-induced hydroxyl radical can react with both
deoxyribose and bases in DNA to produce damage that
includes strand breaks (29, 30) and 8-oxoG (31).
While these four agents all produce strand breaks and
8-oxoG, they do so by different mechanisms and in
different locations in DNA. Even the final common
damage products can arise by different mechanisms. This
is most evident with oxidation of deoxyribose, which can
result in both direct strand breaks and oxidized abasic
sites. For example, radiation and the antibiotics neo-
carzinostatin and bleomycin all produce radicals that can
abstract a hydrogen atom from the C4′-position of deoxy-
ribose (6, 28, 32, 33). The resulting C4′ radical can
undergo further reactions to form either a strand break
with a 3′-phosphoglycolate residue or a 4′-keto-1′-alde-
hyde abasic site that leaves the sugar-phosphate back-
bone intact (6, 28, 32, 33). However, formation of the
ketoaldehyde abasic site is oxygen-independent with
bleomycin (32) and oxygen-dependent with neocarzi-
nostatin and radiation (28, 33). Identical products can
thus arise by different mechanisms depending on the
chemistry of the oxidizing agent.
With regard to base damage, our studies focus on
8-oxoG, which was identified in 1984 by Kasai and
Nishimura (34). The development of a sensitive analyti-
cal technique involving HPLC with electrochemical de-
tection (35) has made 8-oxoG an attractive marker for
monitoring DNA damage in studies with various oxidiz-
ing agents (36). The role of 8-oxoG in mutagenesis and
carcinogenesis has been widely investigated (37), and
many studies have shown a correlation between the
formation of 8-oxoG and carcinogenesis (38). Oxygen
radicals appear to mediate the formation of 8-oxoG in
DNA through a reaction involving the addition of hy-
droxyl radical to the C-8 of guanine (38). This is followed
by the subsequent loss of a hydrogen atom or by a one-
electron oxidation of the C-8 position and subsequent
addition of water (37), which may again depend on the
chemistry of the oxidizing agent.
Syn th esis of P er oxyn itr ite. Peroxynitrite solution was
prepared as described by Pryor et al. (39). Briefly, ozone
generated in a Welsbach ozonator was bubbled into 100 mL of
0.1 M sodium azide in water chilled in an ice bath. After 45
min of ozonation, the peroxynitrite was estimated spectropho-
tometrically in 0.1 N NaOH (
) 1670 M-1 cm-1; 39). The
302
peroxynitrite was further characterized for its ability to cause
the nitration of L-tyrosine using a method described by Beckman
et al. (40) as modified by Pryor et al. (39).
Cou p lin g of Mon oclon a l An tibod ies to CNBr -Activa ted
Sep h a r ose 4B. 8-OxoG-specific monoclonal antibodies were
produced as described previously by Ravanat et al. (41). Ascites
fluid was purified by ammonium sulfate precipitation (40% w/v)
and dialyzed against coupling buffer (0.1 M NaHCO3 and 0.5
M NaCl, pH 8.3). The antibodies were then bound to CNBr-
activated Sepharose 4B (2 mg of protein/mL of gel) that had
been swelled in 1 mM HCl as described previously (42, 43). The
gel was stored at 4 °C in PBS with 0.02% sodium azide. Prior
to use in immunoaffinity columns, the antibody-modified gel was
washed with 50% DMSO (25 gel volumes) on a sintered glass
filter to remove the existing bound 8-oxoG. The gel was then
washed with an equal volume of water and collected in PBS.
The binding efficiency of the antibody-modified gel was deter-
mined to be 75-80% using [3H]8-oxoG.
P r ep a r a tion a n d P u r ifica tion of p UC19 P la sm id DNA.
Cultures of DH5R Escherichia coli were transformed with
pUC19 plasmid and grown on LB plates containing 50 µg/mL
ampicillin (44). Plasmid DNA was isolated using the Qiagen
Giga Plasmid/Cosmid Purification kit according to manufactur-
er’s instructions. After washing the DNA pellet with 80%
ethanol, the solvent was evaporated, and the DNA was redis-
solved in 50 mM potassium phosphate, pH 7.4 (treated with
Chelex 100 resin). Plasmid DNA was exhaustively dialyzed
against 50 mM potassium phosphate containing 1 mM DETA-
PAC for 12 h at 4 °C to remove trace metals and then against
50 mM potassium phosphate for an additional 12 h at 4 °C.
In an effort to understand the relationship between the
reactivity of a genotoxin and the DNA damage it pro-
duces, we have quantified strand breaks and 8-oxoG for
peroxynitrite, Cu(II)/H2O2, Fe(II)-EDTA/H2O2, and γ-ra-
diation at exposure levels approaching physiological
relevance. We found that the ratio of 8-oxoG to strand
breaks was not the same for different oxygen radical
generating systems and that the relevant proportions of
8-oxoG and strand breaks varied as a function of con-
centration for certain oxidizing agents. These results are
discussed in light of other investigations of oxidative DNA
damage spectra and the current models for the mecha-
nism of action of the four oxidizing agents.
Exp er im en ta l P r oced u r es
Ch em ica ls. 2-Amino-6,8-dihydroxypurine (8-oxoG) was ob-
tained from Chemical Dynamics Corp. Plasmid pUC19 (2686
base pairs) was obtained from New England Biolabs. Cyanogen

388 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Kennedy et al.
Following dialysis, the DNA concentration was determined by
a Hoechst dye assay (45) using a Sequoia-Turner Model 450
fluorometer. The pUC19 DNA was stored in 200-µg aliquots
at -80 °C.
Tr ea tm en t of p UC19 P la sm id . The ferrous ammonium
sulfate, cupric chloride, EDTA, and hydrogen peroxide solutions
were prepared fresh for every treatment. pUC19 DNA was
brought to a concentration of 30 µg/mL in Chelex 100-treated
50 mM potassium phosphate, pH 7.4, and the DNA was treated
with the four agents for 30 min at ambient temperature.
Reactions with Fe(II)/EDTA were performed at Fe(II) concentra-
tions of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 5, and 10 µM with 2-fold excess
EDTA and 1 mM H2O2. Control studies revealed that the
damage reaction was complete at the end of the 30-min
incubation and that there was no further damage produced
during subsequent dialysis workup for 8-oxoG analysis (vide
infra; data not shown). Reactions with Cu(II) were performed
at CuCl2 concentrations of 0, 5, 7.5, 10, 12.5, 15, 20, 25, and 30
µM with 1 mM H2O2. The reaction was stopped by adding
EDTA to 1 mM; EDTA abolishes Cu-induced oxidative DNA
damage in the presence of H2O2 (e.g., ref 46). The peroxynitrite
reaction was performed at concentrations of 0, 0.25, 0.5, 1.0, 5,
10, 15, 20, 25, and 50 µM. Peroxynitrite has a half-life of ∼1 s
at pH 7.4, and the nitrate breakdown products are inert (47).
DNA (30 µg/mL) in Chelex 100-treated 50 mM potassium
phosphate, pH 7.4, was also exposed to γ-radiation at 0, 0.125,
0.375, 0.5, 1, 5, and 10 Gy in a 60Co γ source at a dose rate of 4
Gy/min and then placed on ice. The radiation experiment was
performed in either air- or N2O-saturated buffer.
F igu r e 1. 8-oxoG (O) and strand breaks (b) produced by
γ-radiation. DNA damage in plasmid pUC19 was determined
as described in Experimental Procedures.
an ESA Model 5100A Coulochem electrochemical detector
(ECD), and an ESA Model 5010 analytical cell. Separation of
nucleobases was achieved using a Supelco LC-18-DB 5 µm
column (25 cm × 4.6 mm) under isocratic conditions. The mobile
phase was 2% methanol in 50 mM ammonium acetate (pH 5.5)
with a flow rate of 1 mL/min. The background level of 8-oxoG
in untreated plasmid was approximately 5 residues per 106
bases. This value was subtracted from 8-oxoG levels induced
by the oxidizing agents.
Qu a n t it a t ion of St r a n d Br ea k s. Quantitation of
peroxynitrite-, Cu(II)-, Fe(II)-EDTA-, and γ-radiation-induced
DNA damage was accomplished by a method similar to one
described previously (48). After treatment, a portion of the DNA
was treated with putrescine (100 mM, pH 7, 1 h, 37 °C) to cleave
abasic sites (49) and another portion was kept on ice as a control.
Both samples of plasmid DNA (300 ng) were loaded on 1%
agarose gels (Tris-borate-EDTA), and topoisomers were re-
solved at 3 V/cm for 3 h. The gel was then dried and probed
with radiolabeled pUC19 prepared by the random primer
technique (50). The plasmid topoisomers were quantified on a
Molecular Dynamics phosphorimager.
Resu lts
γ-Radiation (29-31), Fe(II)-EDTA/H2O2 (26, 27, 51),
Cu(II)/H2O2 (17, 52, 53), and peroxynitrite (10, 12) all
produce strand breaks and 8-oxoG but apparently by
different mechanisms. Therefore, we used these four
oxidizing agents to compare the relative quantities of
strand breaks and 8-oxoG produced during oxidative
DNA damage.
γ-Ra d ia tion -In d u ced Da m a ge. The quantities of
both 8-oxoG and strand breaks were found to increase
with increasing doses of radiation (Figure 1). For all of
the oxidizing agents, the formation of strand breaks was
studied in the range of “single-hit conditions”, which
corresponds to ∼30-40 direct strand breaks in 106 bases
of pUC19 DNA according to a Poisson distribution (49).
Above this level of damage, the number of strand breaks
is underestimated because additional nicks in an already
nicked plasmid cannot be detected by topoisomer analy-
sis. Under single-hit conditions, the increase in strand
breaks and 8-oxoG produced by γ-radiation was nearly
linear. However, the amount of 8-oxoG formed was
significantly less than the number of strand breaks. The
data reported are for air-saturated solutions since only
slightly higher levels of strand breaks were observed
under N2O, and no significant effect was observed for
8-oxoG (data not shown).
Because oxidation of deoxyribose results in both strand
breaks and abasic sites, we sought to quantify abasic site
formation by converting the lesions to strand breaks with
putrescine. This agent has been shown to cleave virtu-
ally all types of induced abasic sites in DNA (49, 54-
56). Interestingly, putrescine treatment did not increase
the number of apparent strand breaks by more than 5%
for radiation and the other oxidizing agents examined
in these studies (data not shown). This observation
suggests that, under our conditions, abasic sites repre-
sent a relatively small component of the total DNA
damage. Since abasic sites can also arise from loss of
oxidized bases, the small effect of putrescine treatment
DNA Hyd r olysis. For analysis of 8-oxoG, the treated DNA
was dialyzed against distilled and deionized water for 16-20 h
at 4 °C. The absence of reducing agents, the low temperature,
and the fact that the reactions were complete or stopped prior
to dialysis rule out significant adventitious DNA damage during
the dialysis. Furthermore, we have observed no change in the
8-oxoG level of calf thymus DNA or the level of strand breaks
in plasmid DNA following extensive dialysis (unpublished
observations). The concentration of DNA in the solution was
determined by UV at 260 nm (1 OD ) 50 µg), and 50 µg was
aliquotted in triplicate into screw-cap vials. The DNA was then
dried by centrifugation under vacuum (0.05 mbar). Distilled
and deionized H2O (190 µL) and 98% formic acid (310 µL) were
added to produce a final concentration of 60% formic acid, and
hydrolysis was performed for 1 h at 100 °C. Formic acid was
then removed by centrifugation under vacuum.
P u r ifica tion of 8-oxoG on Im m u n oa ffin ity Colu m n s.
8-oxoG was purified on immunoaffinity columns as described
by Ravanat et al. (41) with the following modifications. Antibody-
modified gel (0.2 mL) was packed into BioRad Poly-prep
columns. Hydrolyzed DNA in 0.5 mL of PBS was applied to
the column, and the eluent was re-applied twice. The column
was then washed successively under gravity-induced flow with
2.0 mL of PBS, 2.0 mL of H2O, and 1 mL of acetonitrile. The
bound 8-oxoG was eluted with 1.5 mL of methanol. After
incubation at -20 °C for 30 min and the addition of 150 µL of
N2-methyl-8-oxoG solution (10 pg/µL), the eluent was then dried
by centrifugation under vacuum.
Qu a n tifica tion of 8-oxoG by HP LC. Following hydrolysis
of the DNA, the dried residue was redissolved in 50 µL of the
HPLC mobile phase. The sample was injected into an HPLC
system that consisted of a Hewlett Packard Model 1050 pump,

8-Oxoguanine and Strand Break Formation by Oxidizing Agents
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 389
F igu r e 2. 8-oxoG (O) and strand breaks (b) produced by
Fe(II)-EDTA/H2O2. DNA damage in plasmid pUC19 was deter-
mined as described in Experimental Procedures.
F igu r e 4. 8-oxoG (O) and strand breaks (b) produced by
peroxynitrite. DNA damage in plasmid pUC19 was determined
as described in Experimental Procedures.
F igu r e 3. 8-oxoG (O) and strand breaks (b) produced by
Cu(II)/H2O2. DNA damage in plasmid pUC19 was determined
as described in Experimental Procedures.
F igu r e 5. Comparison of the levels of 8-oxoG and strand breaks
for peroxynitrite, Cu(II)/H2O2, Fe(II)-EDTA/H2O2, and γ-radia-
tion.
also indicates that putrescine did not induce significant
abasic site formation by reaction with bases lesions
produced by the four oxidizing agents.
the shape of the strand break curve at lower concentra-
tions of peroxynitrite suggests that the reaction is bi-
phasic in nature.
Ir on (II)-EDTA-In d u ced Da m a ge. As observed with
γ-radiation, there was a linear increase in both strand
break and 8-oxoG formation upon treatment with Fe(II)-
EDTA/H2O2 (Figure 2). The relative levels of 8-oxoG and
strand breaks were similar to those observed with
γ-radiation, which is consistent with a hydroxyl radical-
like DNA-damaging species.
Cop p er (II)-In d u ced Da m a ge. The levels of strand
breaks and 8-oxoG produced by Cu(II)/H2O2 were differ-
ent from those seen with γ-radiation and Fe(II)-EDTA/
H2O2 (Figure 3). Most significantly, the number of strand
breaks rose sharply between 7.5 and 12.5 µM Cu(II) while
the quantity of 8-oxoG increased gradually. The different
shapes of the strand break and 8-oxoG curves is consis-
tent with the observations of Drouin et al. in their studies
of strand breaks and base damage in isolated genomic
DNA (57) and suggests that strand breaks and 8-oxoG
are produced by different mechanisms. The levels of
8-oxoG were higher than those arising from γ-radiation
and Fe(II)-EDTA, which is consistent with a site-specific
reaction involving copper bound to the N7 position of
guanine (18, 19).
P er oxyn itr ite-In d u ced Da m a ge. As shown in Fig-
ure 4, the levels of 8-oxoG were higher than those
observed for γ-radiation and Fe(II)-EDTA, indicating that
the reactive oxygen species produced by peroxynitrite
reacts with a different selectivity than the hydroxyl
radical produced by radiation or the hydroxyl radical-
like species associated with Fe(II)-EDTA. Additionally,
As mentioned earlier, there was little increase in
strand breaks following putrescine treatment of the
peroxynitrite-damaged plasmid DNA. This is important
in light of the observation by Yermilov et al. that
peroxynitrite produces higher levels of 8-nitroguanine
than 8-oxoG and that 8-nitroguanine undergoes depuri-
nation with a half-life of ∼4 h at 20 °C (11). The lack of
putrescine effect indicates that putrescine did not react
with either 8-oxoG or 8-nitroguanine to produce strand
breaks and that depurination did not occur to any
appreciable extent during DNA processing, since pu-
trescine would have converted the abasic sites to strand
breaks.
Discu ssion
Oxidative DNA damage resulting from exposure to
reactive oxygen species is believed to play a significant
role in mutagenesis, aging, and carcinogenesis (1, 2). In
an ongoing effort to relate the chemistry of genotoxins
to the spectrum of DNA lesions they produce, we have
examined the quantities of 8-oxoG and strand breaks
produced by four different oxidizing agents. We have
demonstrated that the relative levels of 8-oxoG and
strand breaks can vary by more than an order of
magnitude and that they can vary between different

390 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Kennedy et al.
concentrations of a single agent as shown most dramati-
cally for Cu(II)/H2O2. These results indicate the impor-
tance of understanding how the chemistry of the oxidiz-
ing agents contributes to the different levels and types
of oxidative damage to DNA.
more selective than the hydroxyl radical (9, 12). Our data
lend further support to this hypothesis.
There are several interesting features of the peroxy-
nitrite concentration-damage profile shown in Figure 4.
First is the plateau and possibly decrease in 8-oxoG
formation at high concentrations of peroxynitrite (Figure
5). Yermilov et al. also observed a plateau effect with
peroxynitrite (11), while Inoue and Kawanishi, using
SIN-1, observed a decrease in 8-oxoG with increasing
peroxynitrite concentrations (10). These observations are
all consistent with the recent studies of Uppu et al., who
observed that 8-oxoG is susceptible to further oxidation
by peroxynitrite (62). Because they were unable to detect
8-oxoG formation at high peroxynitrite concentrations
(0.1-1 mM), Uppu et al. proposed that either 8-oxoG was
not formed or that any 8-oxoG that is produced is rapidly
oxidized by peroxynitrite (62). Our results support the
latter hypothesis. In addition to the use of a more
sensitive 8-oxoG assay, our ability to detect 8-oxoG is
likely due to that fact we performed the DNA damage
reactions at lower peroxynitrite concentrations (0.25-
100 µM vs 100-1000 µM), which presumably reduced the
second-order oxidation of 8-oxoG by peroxynitrite. Our
results cannot be attributed to different preparations of
peroxynitrite since we used the same procedure as Uppu
et al. to prepare the peroxynitrite (62).
The second interesting feature of peroxynitrite-induced
DNA damage is the biphasic nature of strand break
production. We are presently unable to explain this
phenomenon, but it may be due to some type of competing
second-order reaction such as a self-reaction of peroxy-
nitrite.
In conclusion, we have determined that the relative
quantities of sugar and base damage produced by four
different oxidizing agents vary as a function of the
chemistry and concentration of the agent. The results
are consistent with several previous studies and extend
the observations into physiologically relevant concentra-
tions of oxidizing agents. The observations made with
each agent warrant further study.
Both Fe(II)-EDTA/H2O2 and γ-radiation generate simi-
lar levels of 8-oxoG and strand breaks (Figure 5), which
is consistent with the hypothesis that chemical interme-
diates of similar reactivity are involved in each system.
The results are also consistent with previous studies in
which the sequence specificity of DNA damage produced
by γ-radiation was shown to be similar to that of Fe(II)-
EDTA (58, 59). In both cases, the majority of the DNA
damage can be attributed to a hydroxyl radical-like
species generated by the reagent. It is possible that the
higher levels of strand breaks relative to 8-oxoG reflect
the different fates of the initial one-electron oxidation
products, the accessibility of the Fe/EDTA complex to the
DNA grooves, or in the case of radiation, the higher
concentration of water molecules in the solution phase
compared to the grooves.
The levels of 8-oxoG relative to strand breaks were
higher with Cu(II) than with Fe(II)-EDTA or γ-radiation.
This difference could be due to the generation of a
different DNA-damaging species or to the DNA binding
specificity of Cu(II). Preferential binding of Cu(II) to the
N7 of guanine makes the C-8 position susceptible to
attack by hydroxyl radical or some other Cu-induced
oxidizing species.
Unlike the smooth rise in 8-oxoG formation, there was
a sharp increase in strand breaks between 7.5 and 12.5
µM Cu(II) (Figure 3). This sudden transition was also
observed with the Cu(II)/H2O2/ascorbate system, which
indicates that it is not unique to the Cu(II)/H2O2 reagent
(data not shown). A sharp increase in strand breaks and
a more gradual increase in base lesions was also observed
by Drouin et al. in their studies of DNA lesions produced
by Cu(II)/H2O2/ascorbate (57). The results of both studies
are consistent with a model in which Cu-induced strand
breaks and 8-oxoG are formed by different mechanisms
or DNA binding modes.
Ack n ow led gm en t. The expert assistance of Cynthia
Reyes, Samar Burney, and William LaMarr in several
of the DNA strand break assays is gratefully acknowl-
edged. This work was supported by NIH Grants CA
26731 (S.R.T.), CA09112 (J .C.), CA 64524 (P.C.D.), and
CA 57633 (P.C.D./K.M.), the MIT Ida Green and PEEER
Martin Fellowships (L.J .K.), and the Samuel A. Goldblith
Professorship (P.C.D.).
While the model that Cu-induced strand breaks and
base damage occur by different mechanisms is in contrast
to the model proposed recently by Toyokuni and Sagri-
panti (60), data from the two studies is in fairly good
agreement. At similar ratios of Cu to DNA, we observe
a ratio of strand breaks to 8-oxoG of ∼3 vs a ratio of ∼2
determined by Toyokuni and Sagripanti (60). In their
studies, Toyokuni and Sagripanti held the Cu(II) con-
centration constant and varied the level of H2O2 (60).
Since both base damage and strand breaks produced by
Cu have been shown to increase as a function of increas-
ing H2O2 concentration (57, 61), their observation of a
linear relationship between strand breaks and 8-oxoG
under these conditions is consistent with the model that
Drouin et al. (57) and we have proposed. The ratio of
strand breaks to 8-oxoG remains constant at one Cu
concentration with both lesions varying in parallel as a
function of the H2O2 concentration.
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