Base modification and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3- morpholinosydnonimine

Article abstract of DOI:10.1021/tx960084i

Exposure of isolated calf thymus DNA and human skin epidermal keratinocytes to peroxynitrite or the peroxynitrite generator, 3- morpholinosydnonimine (SIN-1), led to extensive DNA base modification. Large increases in xanthine and hypoxanthine, possible deamination products of guanine and adenine, respectively, and in 8-nitroguanine were observed, but only small changes in some oxidized base products were seen. This pattern of damage suggests that hydroxyl radicals were not major contributors to base modification caused by peroxynitrite, as OH· is known to cause multiple oxidative modifications to all four DNA bases. Instead, it seems that reactive nitrogen species play a much greater role in the mechanism of base damage, producing both nitration and deamination of purine bases when DNA or whole cells are exposed to peroxynitrite. If this pattern of damage is unique to peroxynitrite, it might act as a marker of cellular damage by this species in vivo.

Full text of DOI:10.1021/tx960084i

1
152
Chem. Res. Toxicol. 1996, 9, 1152-1158
Ba se Mod ifica tion a n d Str a n d Br ea k a ge in Isola ted Ca lf
Th ym u s DNA a n d in DNA fr om Hu m a n Sk in Ep id er m a l
Ker a tin ocytes Exp osed to P er oxyn itr ite or
3
-Mor p h olin osyd n on im in e
,
†
‡
†
†
J eremy P. E. Spencer,* J on Wong, Andrew J enner, Okezie I. Aruoma,
‡
†,‡
Caroll E. Cross, and Barry Halliwell
Neurodegenerative Disease Research Centre, Pharmacology Group,
University of London King’s College, Manresa Road, London SW3 6LX, U.K., and
Division of Pulmonary/ Critical Care Medicine, Department of Internal Medicine,
University of California Davis Medical Centre, Sacramento, California 95817
Received May 22, 1996X
Exposure of isolated calf thymus DNA and human skin epidermal keratinocytes to
peroxynitrite or the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), led to extensive
DNA base modification. Large increases in xanthine and hypoxanthine, possible deamination
products of guanine and adenine, respectively, and in 8-nitroguanine were observed, but only
small changes in some oxidized base products were seen. This pattern of damage suggests
that hydroxyl radicals were not major contributors to base modification caused by peroxynitrite,
•
as OH is known to cause multiple oxidative modifications to all four DNA bases. Instead, it
seems that reactive nitrogen species play a much greater role in the mechanism of base damage,
producing both nitration and deamination of purine bases when DNA or whole cells are exposed
to peroxynitrite. If this pattern of damage is unique to peroxynitrite, it might act as a marker
of cellular damage by this species in vivo.
In tr od u ction
Many pathological conditions including ischemia/re-
has been obtained by ESR spin trapping (14) and by
aromatic hydroxylation experiments (13, 15). However,
production of OH via simple homolytic fission of per-
•
perfusion, inflammation, and sepsis may induce tissues
to simultaneously produce an excess of both superoxide
oxynitrous acid is thought to be unlikely due to thermo-
dynamic constraints (7). Peroxynitrite also oxidizes
sulfhydryl groups and induces membrane lipid oxida-
tion (16-18). It nitrates tyrosine residues in proteins
to form 3-nitrotyrosine (6, 19), which has been meas-
ured as a putative marker of peroxynitrite-mediated
damage in inflamed tissues (19-23). The postulated
ability of peroxynitrous acid to cross cell membranes (6)
means it may penetrate into the cell nucleus where it
might induce damage to DNA. Yermilov et al. (24, 25)
•
-
•
•
•-
(
O
2
) and nitric oxide (NO ). NO and O
2
produced by
activated phagocytes may play a role in the multistage
carcinogenesis process, triggered by chronic infection and
•
inflammation (1, 2). It has been reported that NO can
induce damage by leading to deamination of DNA bases,
resulting in mutation (3, 4).
•
-
•
Both O
2
and NO contain unpaired electrons and can
react at near diffusion limited rates to form the peroxy-
-
-
nitrite anion (ONOO ). The reaction rate constant has
have shown that ONOO can rapidly react with gua-
9
-1
-1
been determined to be 6.7 ((0.9) × 10
M
s
(5).
nine under physiological conditions to form 8-nitrogua-
nine. It has also been reported that peroxynitrite in-
duces strand breaks in plasmid DNA and oxidative
damage in isolated DNA in vitro (26, 27) as well as being
mutagenic (28).
Peroxynitrite is stable at alkaline pH, but decomposes
to nitrate at physiological pH after protonation to form
peroxynitrous acid (ONOOH) (pK ) 6.8). The half-life
a
of ONOOH is extremely short, but it may be able to
diffuse some distance on a cellular scale (100 µm) and it
may even cross cell membranes (6).
In this study we show that ONOO- not only causes
extensive base modification to isolated DNA but also
produces significant base modification and causes strand
breakage to DNA when human keratinocytes are exposed
It has become clear that peroxynitrite is a powerful,
toxic oxidizing and nitrating agent that can be produced
in vivo (7-11). It may initiate reactions currently
-
-
to ONOO or to the ONOO generator 3-morpholinosyd-
•
nonimine (SIN-1).1 We also show that the pattern of
damage caused by peroxynitrite suggests that free OH
thought to be characteristic of hydroxyl radical (OH ),
+
•
nitryl cation (NO
2
), and the nitrogen dioxide radical
•
(
NO
2
) (7, 9, 12, 13). Many different oxidative pathways
radicals are not involved but that reactive nitrogen
species seem to play the major role in DNA base
modification.
exist due to the chemical nature of the O-O bond of
peroxynitrous acid, which may react as if it was cleaved
•
•
2
either homolytically into OH and NO or heterolytically
-
+
•
into OH and NO
2
. Some evidence for OH formation
1
Abbreviations: GC-MS: gas chromatography-mass spectroscopy;
FAPy-adenine: 4,6-diamino-5-formamidopyrimidine; FAPy-guanine:
,6-diamino-5-formamidopyrimidine; 5-OH,Me-uracil: 5-(hydroxy-
2
*
Corresponding author, at telephone: +44 171 333 4760, fac-
methyl)uracil; 5-OH,Me-hydantoin: 5-(hydroxymethyl)hydantoin;
SIN-1: 3-morpholinosydnonimine, BSTFA: bis(trimethylsilyl)trifluo-
racetamide, TMS: trimethylchlorosilane; HBSS: Hank’s balanced salt
solution; CDTA: trans-1,2-diaminocyclohexane-N,N,N′,N′ -tetraacetic
acid; SDS: sodium dodecyl sulfate.
simile: +44 171 333 4949, e.mail: J .Spencer@kcl.ac.uk.
†
University of London King’s College.
University of California Davis Medical Centre.
Abstract published in Advance ACS Abstracts, September 15, 1996.
‡
X
S0893-228x(96)00084-7 CCC: $12.00 © 1996 American Chemical Society

DNA Damage by ONOO- and SIN-1
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1153
Ma ter ia ls a n d Meth od s
sterile, filtered PBS. A Hank’s balanced salt solution (HBSS)
was used for the exposure experiments. HBSS did not affect
cell viability, measured using the AlamarBlue assay (33). Stock
solutions of both peroxynitrite and SIN-1 were made up im-
mediately prior to experiments. Peroxynitrite exposures were
to cells in suspension. Cells were removed from plates using
trypsin and resuspended in HBSS (∼5 million/mL). Peroxyni-
trite (1 mM) was added directly to cells in suspension followed
by constant mixing for 10 s (final pH 7.2). SIN-1 (1 mM) in
HBSS was added to cell plates and a 60 min incubation at 37
Ma ter ia ls. Chemicals were of the highest quality available
from Sigma Chemical Co. (Poole, Dorset, U.K.), from the BDH
Chemical Co. (Gillingham, Dorset, U.K.) and/or Aldrich Chemi-
cal Co. (Milwaukee, WI). Calf thymus DNA was Sigma type I.
SIN-1 was purchased from Molecular Probes Inc. (Eugene,
OR). 8-Azaadenine, 6-azathymine, diaminopurine, 8-bromoad-
enine, 5-hydroxyuracil (isobarbituric acid), 4,6-diamino-5-for-
mamidopyrimidine (FAPy-adenine), 2,5,6-triamino-4-hydroxy-
pyrimidine, and 5-(hydroxymethyl)uracil were purchased from
Sigma. 8-Hydroxyguanine was purchased from Aldrich (Poole,
Dorset, U.K.). 8-Hydroxyadenine and FAPy guanine were
synthesized (courtesy of Dr. H. Kaur, King’s College London)
by, respectively, treatment of 8-bromoadenine with concentrated
formic acid (95%) at 150 °C for 45 min with purification by
crystallization from water, as described in ref 29, and treatment
of 2,5,6-triamino-4-hydroxypyrimidine with concentrated formic
acid with purification by crystallization from water, as described
in ref 30. Thymine glycol was synthesized by reaction of
°
C followed. SIN-1 incubated at 37 °C for 24 h before addition
to cells was the control for SIN-1 experiments. After incubation,
the solutions were removed and cells were washed twice in
filtered PBS.
Ala m a r Blu e Assa y for Cell Via bility. The AlamarBlue
assay (33) was performed as previously published (34) and is a
measure of cell viability based on mitochondrial dehydrogenase
activity.
Isola tion of DNA fr om Cu ltu r ed Ma m m a lia n Cells.
DNA isolation from cells and assessment of RNA contamination
were carried out as previously described (34).
5
-methyluracil with OsO
4 4
for 1 h at 60 °C, and excess OsO was
removed by freeze-drying (31). 8-Nitroguanine was synthesized
by reaction of peroxynitrite (5 mM) with guanine (3 mg) under
constant stirring and was purified by preparative HPLC (24).
Purity of synthesized standards was assessed by mass spec-
trometry, and all were found to be >99% pure. 2-Hydroxyad-
enine, 5-hydroxycytosine, and 5-(hydroxymethyl)hydantoin were
gifts from Dr. M. Dizdaroglu (National Institute of Standards
and Technology, Gaithersburg, MD).
Cellu.Sep dialysis membranes with a relative molecular mass
cut off of 3500, silylation grade acetonitile, and bis(trimethyl-
silyl)trifluoracetamide (BSTFA) (containing 1% trimethylchlo-
rosilane, TMS) were obtained from Pierce Chemical Co. (Rock-
ford, IL). Distilled water passed through a purification system
An a lysis of DNA Ba se Mod ifica t ion b y Ga s
Ch r om atogr aph y-Mass Spectr oscopy. Derivatized samples
were analyzed by GC-MS (Hewlett-Packard 5890II gas chro-
matograph interfaced with a Hewlett-Packard 5917A mass
selective detector). Preparation, hydrolysis, derivatization, and
analysis of samples were performed as described previously
(35, 36) with the following modifications. The injection port
and the GC-MS interface were kept at 250 and 290 °C,
respectively. Separations were carried out on a fused silica
capillary column (12 m × 0.2 mm i.d.) coated with cross-linked
5% phenylmethylsiloxane (film thickness 0.33 µm) (Hewlett-
Packard). Helium was the carrier gas with a flow rate of 0.93
mL/min. Derivatized samples (1.0 µL) were injected into the
GC injection port using a split ratio of 8:1. Column temperature
was increased from 125 to 175 °C at 8 °C/min after 2 min at
125 °C, then from 175 to 220 °C at 30 °C/min and held at 220
°C for 1 min, and finally from 220 to 290 °C at 40 °C/min and
held at 290 °C for 2 min. Selected-ion monitoring was performed
using the electron-ionization mode at 70 eV with the ion source
maintained at 185 °C.
(
Elga, High Wycome, Bucks, U.K.) was used for all purposes.
Alamar blue was from Alamar Biosciences (Sacramento, CA).
Dulbecco’s modified Eagle’s medium was from Flow Laboratories
Mclean, VA), and fetal calf serum was from Hyclone Labora-
tories (Logan, UT).
(
DNA Da m a ge In d u ced by P er oxyn itr ite. Peroxynitrite
was synthesized in a quenched flow reaction system, by mix-
ing H
2
O
2
(0.6 M) with NaNO
2
(0.6 M) under acidic conditions.
-
The ONOO was trapped in a NaOH solution (1.2 M), in which
it was stable for several weeks. Peroxynitrite was used from
the liquid layer formed by freeze fractionation, and excess H
Quantitation of modified bases was achieved by relating the
peak area of the compound with the internal standard peak area
and applying the following formula:
2 2
O
was destoyed by granular MnO to eliminate possible base
2
modification by species derived from this oxidant. Prior to
concn (nmol/mg of DNA) ) A/AIST × [IST] × (1/K)
-
each experiment, ONOO was quantified spectrophotometrically
-
1
at 302 nm using a molar extinction coefficient of 1670 M
-
1
where K ) relative molar response factor” for each damaged
base, A ) peak area of product, AIST ) the peak area of internal
standard, and [IST] ) concentration of the internal standard
5 nmol/mg of DNA). K constants were calculated from the
slopes of the calibration curves constructed using known
concentrations of internal standards and authentic compounds.
cm (typical concentrations were 200-300 mM), and thawed
ONOO solutions were kept on ice (6). The reactions were
-
carried out at room temperature (∼22 °C). Peroxynitrite at
various concentrations was added to a reaction mixture (final
volume 1.0 mL) containing sodium phosphate buffer (0.1 M; pH
(
7
.0) and calf thymus DNA (0.2 mg). Solutions were mixed
-
rapidly during addition of ONOO for approximately 10 s. The
maximum volume of the ONOO solution added was 10 µL
Mea su r em en t of 8-Nitr ogu a n in e. DNA samples were
hydrolyzed with 60% (v/v) formic acid for 45 min at 150 °C.
Hydrolysates were then analyzed by UV-HPLC. The HPLC
system consisted of a pump (ACS isocratic pump 351, Applied
Chemical Systems, Macclesfield, Cheshire, U.K.), Rheodyne
injection valve, Model 7125 (Fisons, Loughborough, Leicester-
shire, U.K.), and a stainless steel column (250 × 4.6 mm) packed
with Nucleosil RP-C18, 5 µm (Fisons, Loughborough, Leices-
tershire, U.K.). The detection was carried out by means of a
UV detector (ACS, Macclesfield, Cheshire, U.K.) at 396 nm, as
previously reported by Yermilov et al. (25). An integrator (Data
system 450 PCIP version 3.94, Kontron Instruments, Watford,
U.K.) linked to a PC was used to monitor the current signal at
a chart speed of 0.25 cm/min. The mobile phase (pH 6.0)
consisted of ammonium formate buffer (20 mM) which was
pumped through the system at a flow rate of 1.0 mL/min. Levels
were quantified using a standard curve generated using the
synthesized standard which was checked for purity using mass
spectroscopy.
-
which gave a final pH of 7.2. Experiments were repeated in
reaction mixtures of pH 6.0 and 8.0 to determine the pH
dependence of reactivity. Control experiments were performed
using the same concentrations of decomposed peroxynitrite
(dilution of peroxynitrite in phosphate buffer at pH 7.4 results
in rapid decomposition and generated a solution which was used
as the control).
Cell Cu ltu r e. A spontaneously immortalized line of human
skin epidermal keratinocytes (HaCaT), derived from adult trunk
skin and maintaining features of normal epidermal growth and
differentiation, was used (32). The cells were routinely cultured
in Dulbecco’s modified Eagle’s medium supplemented with 10%
(
9
v/v) fetal calf serum at 37 °C in a humidified atmosphere of
5% air and 5% CO
Exp osu r e of Hu m a n Ker a tin ocytes to P er oxyn itr ite
2
.
a n d SIN-1. Cells of approximately 90% confluency were used.
Growth medium was removed and cells were washed twice with

1
154 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Spencer et al.
Ta ble 1. Levels of DNA Ba se Mod ifica tion P r od u cts in
Con tr ol DNA a n d in DNA Exp osed to P er oxyn itr ite (1
m M) a t p H 7.4a
levels in control
DNA (nmol/mg
of DNA)
levels in DNA exposed
to ONOO (1 mM)
(nmol/mg of DNA)
-
base product
5
5
5
-OH-hydantoin
-OH-uracil
-OH,Me-uracil
0.36 ( 0.20
0.11 ( 0.02
0.03 ( 0.01
0.77 ( 0.07
1.33 ( 0.11
1.83 ( 0.21
0.21 ( 0.02
0.49 ( 0.03
0.45 ( 0.03
0.21 ( 0.01
0.87 ( 0.07
1.06 ( 0.07*
0.14 ( 0.01
0.15 ( 0.06*
1.86 ( 0.11*
5.96 ( 0.56*
303.85 ( 26*
0.38 ( 0.04*
0.52 ( 0.06
thymine glycol
hypoxanthine
xanthine
FAPy-adenine
8
FAPy-guanine
8
8
-OH-adenine
0.68 ( 0.05*
1.23 ( 0.07*
123.52 ( 7.90*
-OH-guanine
-Nitro-guanine
a
Control DNA was commercial calf thymus DNA. Data are
plotted as the mean (SD, n ) 4. *Significant increase over
-
F igu r e 1. The generation of peroxynitrite (ONOO ) from SIN-
. SIN-1 is stable at acidic pH but breaks down at neutral pH.
In the process, several intermediates are formed including both
control, p e 0.05).
1
•
-
•
-
superoxide (O
2
) and nitric oxide ( NO), resulting in ONOO
formation. At physiological pH, SIN-1 decomposition has been
physiological pH. Small but significant increases in
8
5
-hydroxyguanine, 5-OH-hydantoin, thymine glycol (trans),
-OH,Me-uracil, and the ring opened products FAPy-
-
shown to generate about 1 µM/min ONOO .
adenine and FAPy-guanine were observed. No signifi-
cant changes in 5-OH-uracil, 5-OH-cytosine, 5-OH,Me-
hydantoin, 2-hydroxyadenine, and 8-hydroxyadenine were
apparent. By contrast, there were very large rises in
xanthine, hypoxanthine, and 8-nitroguanine (Table 1;
F lu or om etr ic Detection of DNA Str a n d Br ea k s. DNA
strand breaks were detected using the alkaline unwinding/
ethidium bromide assay in which the fluorescent dye binds
selectively to double-stranded DNA (37, 38).
Cells after exposure were washed with PBS and then pelleted
and resuspended to achieve a 5-10 million cell/mL concen-
tration. Aliquots of the cell suspension (0.2 mL) were placed
in 12 tubes labelled T (total), P (test), or B (blank) in groups of
-
Figure 3) when DNA was exposed to ONOO . Figure 3
also shows the pH dependence of xanthine, hypoxanthine,
-
and 8-nitroguanine generation by ONOO . Product
4
. To each was added 0.2 mL of cell lysis and chromatin
-
generation on exposure to ONOO (1 mM) appears to be
disruption buffer (urea, 9.0 M; NaOH, 10 mM; CDTA, 2.5 mM;
SDS, 0.1%), and tubes were incubated at 0 °C for 10 min to
allow total disruption of chromatin. To tubes P and B, 0.1 mL
of alkaline unwinding solution A (0.45 volume of cell lysis buf-
fer in 0.2 M NaOH) and 0.1 mL of alkaline unwinding solu-
tion B (0.4 volume of cell lysis buffer in 0.2 M NaOH) were
added without mixing. During the subsequent incubation
at 0 °C for 30 min, the alkali diffuses into the viscous lysate
to give a final pH of about 12.8. Before the alkali solutions
were added to tube T, 0.4 mL of denaturation prevention
solution was added (glucose, 1 M; mercaptoethanol, 14 mM),
ensuring that the DNA is never exposed to a denaturing pH
dependent on pH. 8-Hydroxyguanine formation is opti-
mal at pH 6.0, while xanthine and hypoxanthine forma-
tion is favored at pH 7.4, and 8-nitroguanine yields are
maximal at pH 8.0 (Figure 4). No changes in base
damage were observed when DNA was treated with
decomposed peroxynitrite, indicating that changes in
products were dependent on peroxynitite and not due to
other constituents of the solution.
St u d ies w it h Ker a t in ocyt es. Exposure of human
-
keratinocytes to ONOO (1 mM) or SIN-1 (1 mM, 60 min)
(
∼11.0). All tubes were then incubated for 60 min at 15 °C,
led to small but significant increases in DNA strand
breakage. SIN-1 produced a slightly greater extent of
and denaturation was stopped by addition of 0.4 mL of dena-
turation prevention solution and chilling to 0 °C. To each tube
is added 1.5 mL of ethidium bromide solution (ethidium
bromide, 6.7 µg/mL; NaOH, 13.3 mM) and their fluorescence
is read at room temperature (excitation: 520 nm; emission:
-
-
strand breakage than ONOO (ONOO : 21.0 ( 2.6%;
SIN-1: 29.0 ( 3.5% strand breakage). These levels of
the compounds did not affect cell viability measured just
before DNA extraction, but further incubation for 24 h
led to small reductions in viability which were most
5
90 nm).
The extent of DNA unwinding after a given time of exposure
-
-
apparent in ONOO exposed cells (ONOO : 81% viable;
SIN-1: 92% viable compared to control cells: 100%
of cell extracts to alkali is calculated from the formula:
-
%
DS DNA ) (P - B)/(T - B) × 100
viable). Thus, neither ONOO nor SIN-1 appears lethal
at the concentrations used in our experiments.
Resu lts
Levels of oxidative DNA base modification in the
control (see Materials and Methods) cells were low, as
Stu d ies w ith Isola ted DNA. The use of gas chro-
matography-mass spectroscopy enabled us to demon-
-
expected. Exposure of cells to ONOO and SIN-1 pro-
-
-
strate that both ONOO and the ONOO generator SIN-1
Figure 1) cause damage to all four DNA bases. However,
duced small but significant rises in 8-OH-guanine, FAPy-
guanine, and FAPy-adenine and in 5-OH-hydantoin
(Figure 5). However, as in the experiments with calf
thymus DNA, the most striking increases were in xan-
thine, hypoxanthine, and 8-nitroguanine (Figure 5). No
significant changes in 5-OH,Me-hydantoin, 5-OH-uracil,
5-OH,Me-uracil, 5-OH-cytosine, thymine glycol, or 8-OH-
adenine were observed. Levels of 8-nitroguanine of about
8 nmol/mg of DNA correspond to 2-3 8-nitroguanines
(
oxidized base products did not increase very much in
amount, whereas, levels of xanthine, hypoxanthine, and
nitrated guanine increased dramatically (Table 1). Fur-
thermore, generation of damaged products appeared to
be dependent on the exposure environment, in particular
pH.
Panels A and B of Figure 2 show the increases in
modified pyrimidine and modified purine bases, respec-
tively, when isolated DNA is exposed to ONOO at
3
per 10 DNA bases, about the same as the level observed
-
-
when DNA is exposed to 100 µM ONOO in vitro (25).

DNA Damage by ONOO- and SIN-1
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1155
the C-8 position requires either an amino or a hydroxyl
group at the C-2 position and only hydrogens at the C-8
carbon and N-7 nitrogen. This would explain why no
nitrated adenine product was observed. Our results
agree with previous studies (24) that the formation of
8
-nitroguanine is optimal at a pH close to 8, suggesting
that either a heterolytic cleavage of peroxynitrite to form
+
a nitryl cation (NO
2
) or a high energy intermediate
) 7.9) could be
derived from trans peroxynitrite (pK
a
involved (7). This would be similar to the mechanism of
tyrosine nitration and other phenolic compounds by
peroxynitrite, which is also seen to be optimum at pH
∼
8 (12, 19, 21). Another possibility is that the nitrogen
•
2
dioxide radical (NO ) is involved.
The question as to whether OH is a significant
•
-
mediator of ONOO -dependent damage has been repeat-
edly debated (6-10, 12-15). Kinetic and thermodynamic
calculations suggest that the hydroxyl radical-like reac-
tivity of peroxynitrous acid is mediated by a vibrationally
excited intermediate that does not physically separate
•
into free OH and nitrogen dioxide (7). Our data show
•
that only low levels of products characteristic of OH
attack upon DNA were observed when DNA was exposed
to peroxynitrite (Figure 2). This would suggest that that
•
OH radicals are not major contributors to the DNA
•
damage, since OH is known to produce a wide range of
base modifications which results in generation of signifi-
cant amounts of all the products measured here (36, 39-
4
3). The low level of hydroxylated (and ring opened)
products observed in our data might result from a
mechanism by which a high energy intermediate form
of peroxynitrous acid reacts with the target molecule (i.e.,
guanine) to promote homolytic cleavage of the attacking
species (7) (Figure 6). Since our paper was submitted,
Douki and Cadet (44) have reported their inability to find
-
8
-OH-guanine in DNA exposed to 30 mM ONOO . A
possible explanation of this discrepancy is that, at such
-
high levels, ONOO leads to oxidative loss of 8-OH-
guanine (45). In any case, we are all agreed that 8-OH-
guanine, like the other oxidized bases, is at most a minor
product (44, 45).
The most striking increases in base damage products
were in xanthine and hypoxanthine, which can arise as
deamination products of guanine and adenine, respec-
-
tively (46, 47). Deamination could involve ONOO itself
or other species derived from it, and deamination has
been observed in NO-stressed cells (47).
When human skin epidermal keratinocytes are exposed
to either peroxynitrite or SIN-1, the percentage of double
stranded DNA measured after alkali treatment decreases
and so the number of strand breaks in the DNA must
have risen (37, 38). The extent of DNA strand breakage
F igu r e 2. Effect of increasing peroxynitrite concentrations on
the generation of oxidized base products. The increase in
modified bases derived from pyrimidine bases is shown in (A)
and purine bases in (B). Experiments were conducted as in the
Materials and Methods section. Data points are means of three
separate experiments plotted (SD (n ) 4).
-
is greater in the cells exposed to the ONOO generator,
SIN-1. This may be due to the fact that the majority of
ONOO added as such reacts with other cell components,
Discu ssion
-
In this study we have demonstrated that the exposure
of DNA to peroxynitrite in vitro yields increases in
products derived from all four DNA bases and leads to
DNA strand breakage. The use of GC-MS allowed us to
identify rises in hydroxylated products, for example,
for example, lipid membranes and cytosolic constituents,
before it can enter the nucleus. From the amount of
8-nitroguanine formed, it seems that only about 10% of
-
the added ONOO gave rise to nitrating species close to
-
DNA. SIN-1, in contrast, generates ONOO at a of rate
-
1
8
-OH-guanine, and in the possible deamination products
1 µM min over the 1 h incubation period, perhaps
-
of guanine and adenine, xanthine and hypoxanthine
respectively. Large rises in 8-nitroguanine were also
observed in agreement with ealier studies (24, 25) and
quantified by the use of HPLC. Although the reaction
mechanism for the formation of 8-nitroguanine is un-
known, studies have suggested that purine nitration at
allowing a greater chance of ONOO reaching the DNA.
Strand breakage or base modification do not appear to
be lethal either directly after the experiment or after 24
h. Strand breakage was accompanied by increases in
many modified base products, suggesting that both are
due to the same mechanism. The pattern of base damage

1
156 Chem. Res. Toxicol., Vol. 9, No. 7, 1996
Spencer et al.
F igu r e 3. (A) Effect of increasing peroxynitrite concentrations on the generation of xanthine at different pH’s. Experiments were
conducted as in the Materials and Methods section. Data points are means of three separate experiments plotted (SD (n ) 4). (B)
Effect of increasing peroxynitrite concentrations on the generation of hypoxanthine at different pH’s. (C) Effect of increasing
peroxynitrite concentrations on the generation of 8-nitroguanine at different pH’s.
F igu r e 4. pH dependence of hypoxanthine, xanthine, 8-OH-guanine, and 8-nitroguanine formation by peroxynitrite (1 mM). Data
points are mean (SD (n ) 4).
is similar to that seen when isolated DNA was exposed
to ONOO . The levels of only a few oxidized bases
thine, hypoxanthine, and 8-nitroguanine, which is similar
to that seen in isolated DNA exposed to ONOO . Deami-
-
-
increased and these changes were small. This pattern
of damage is again not suggestive of OH attack upon
nation of guanine and adenine could cause DNA strand
breakage due to the relative instability of xanthine and
hypoxanthine. Changes in DNA resulting from deami-
nation reactions are consistant with the types of muta-
tions observed in many cancers, e.g., in the p53 gene (1,
46). Deamination of unmethylated cytosine residues in
vivo produces uracil and its removal by uracil glycosylase
(48) can lead to abasic site formation (49). Misrepair
would produce a G:C f A:T transition, which has been
implicated in familial Alzheimer’s disease (50).
•
DNA (see above). Increases in the putative deamination
products xanthine and hypoxanthine and in 8-nitrogua-
nine indicate attack by reactive nitrogen species. All the
products identified and quantified were observed in DNA
isolated from untreated cells (including some 8-nitrogua-
nine) and probably arose due to physiological levels of
oxidative stress in these cells. The largest increases over
background levels were observed in the products xan-

DNA Damage by ONOO- and SIN-1
Chem. Res. Toxicol., Vol. 9, No. 7, 1996 1157
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2) Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Oxidants,
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29-240.
F igu r e 5. Effect of peroxynitrite (1 mM) and SIN-1 (1 mM; 60
min) on the generation of modified DNA bases in human skin
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F igu r e 6. Proposed mechanism of 8-OH-guanine by peroxyni-
trite. Transfer of the hydroxyl group proceeds via a vibrationally
excited intermediate which results from vibrations of the
N-O-O bond angle and lengthening of the O-O bond. When
this intermediate oxidizes a target molecule, it promotes ho-
(
molytic cleavage of peroxynitrous acid to leave 8-OH-guanine
•
and NO
2
.
(
14) Augusto, O., Gatti, R. M., and Radi, R. (1994) Spin-trapping
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Overall, we have shown that the pattern of DNA
damage produced by peroxynitrite in isolated DNA and
in keratinocyte cells is both complex and extensive.
Deamination and nitration reactions appear to be the
(
15) van der Vliet, A., O’Neill, C. A., Halliwell, B., Cross, C. E., and
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potential of superoxide annd nitric oxide. Arch. Biochem. Biophys.
-
dominant mechanisms of damage inflicted by ONOO .
However, we have shown that it is also capable of
producing a small number of products similar to those
(
(
•
produced by OH attack on DNA. The pattern of damage
observed suggests that reactive nitrogen species rather
than reactive oxygen species are responsible for by far
the greater amount of damage to DNA in cells generating
2
88, 481-487.
-
ONOO . It is possible that the pattern of damage is
(18) van der Vliet, A., Smith, D., O’Neill, C. A., Kaur, H., Darley-
Usmar, V., and Cross, C. E. (1994) Interaction of peroxynitrite
with human plasma and its constituents: Oxidative damage and
antioxidant depletion. Biochem. J . 303, 295-301.
unique to peroxynitrite and could be used as a “finger-
print” of peroxynitrite-induced DNA damage in vivo. If
this is true, it would not only provide vital information
on whether peroxynitrite is involved in pathogenic pro-
cesses but would also go some way to help devise possible
(
19) Beckman, J . S., Ischiropoulos, H., Zhu, L., van der Woerd, M.,
Smith, C., Chen, J ., Harrison, J ., Martin, J . C., and Tsai, M. (1992)
Kinetics of superoxide dismutase- and iron-catalyzed nitration
of phenolics by peroxynitrite. Arch. Biochem. Biophys. 298, 438-
-
ways of protecting cells against damage by ONOO .
4
45.
Another such “fingerprint” may be the formation of a
peroxynitrite-guanine adduct in DNA (51).
(20) Beckman, J . S, Ye, Y. Z., Anderson, P. G., Chen, J ., Accavitti, M.
A., Tarpey, M. M., and White, C. R. (1994) Extensive nitration of
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Biophys. 298, 431-437.
(
(
Ack n ow led gm en t. We are grateful to Asta Medica
Pharmaceuticals, the National Institutes of Health (U.S.),
the U.K. Ministry of Agriculture Fisheries and Food, the
World Cancer Research Fund, and Nestec SA, Switzer-
land, for research support.
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Invest. 94, 2407-2413.
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mediated oxidative damage in chronic inflammation. Nitroty-
rosine in serum and synovial fluid from rheumatoid patients.
FEBS Lett. 350, 9-12.
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