Peroxidase-catalyzed oxidative damage of DNA and 2′-deoxyguanosine by model compounds of lipid hydroperoxides: Involvement of peroxyl radicals

Article abstract of DOI:10.1021/tx0001880

The peroxidase-catalyzed decomposition of 3-hydroperoxy-1-butene (1), 2,3-dimethyl-3-hydroperoxy-1-butene (2), tert-butyl hydroperoxide (3), ethyl oleate hydroperoxide 4, and linoleic acid hydroperoxide 5 was applied as a chemical model system to assess whether lipid hydroperoxides may cause DNA damage under peroxidase catalysis. For this purpose, the Coprinus peroxidase (CIP), horseradish peroxidase (HRP), and the physiologically important lactoperoxidase (LP) were tested. Indeed, hydroperoxides 1-5 induce strand breaks in pBR 322 DNA upon peroxidase catalysis. For the nucleoside dG, the enzymatic decomposition of hydroperoxides 1-4 led to significant amounts of 4,8-dihydro-4-hydroxy-8-oxo-2′deoxyguanosine (4-HO-8-oxo-dG) and guanidine-releasing products (GRP), whereas 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) was not obtained. In isolated calf thymus DNA, the efficient conversion of the guanine base (Gua) was observed. Peroxyl radicals, which are generated in situ from the hydroperoxides by one-electron oxidation with the peroxidases, are proposed as the active oxidants on the basis of the following experimental facts. (i) Radical scavengers strongly inhibit the guanine oxidation in dG and DNA and strand-break formation in the latter. (ii) EPR spectral studies with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap confirmed the formation of peroxyl radicals. (iii) The release of molecular oxygen was demonstrated, produced through the disproportionation of peroxyl radicals. The biological relevance of these findings should be seen in the potential role of the combined action of lipid hydroperoxides and peroxidases in damaging cellular DNA through peroxyl radicals.

Full text of DOI:10.1021/tx0001880

Chem. Res. Toxicol. 2000, 13, 1199-1207
1199
P er oxid a se-Ca ta lyzed Oxid a tive Da m a ge of DNA a n d
2
′-Deoxygu a n osin e by Mod el Com p ou n d s of Lip id
Hyd r op er oxid es: In volvem en t of P er oxyl Ra d ica ls
Waldemar Adam,* Annemarie Kurz, and Chantu R. Saha-M o¨ ller
Institut f u¨ r Organische Chemie, Universit a¨ t W u¨ rzburg, Am Hubland, D-97074 W u¨ rzburg, Germany
Received August 25, 2000
The peroxidase-catalyzed decomposition of 3-hydroperoxy-1-butene (1), 2,3-dimethyl-3-
hydroperoxy-1-butene (2), tert-butyl hydroperoxide (3), ethyl oleate hydroperoxide 4, and linoleic
acid hydroperoxide 5 was applied as a chemical model system to assess whether lipid
hydroperoxides may cause DNA damage under peroxidase catalysis. For this purpose, the
Coprinus peroxidase (CIP), horseradish peroxidase (HRP), and the physiologically important
lactoperoxidase (LP) were tested. Indeed, hydroperoxides 1-5 induce strand breaks in pBR
3
22 DNA upon peroxidase catalysis. For the nucleoside dG, the enzymatic decomposition of
hydroperoxides 1-4 led to significant amounts of 4,8-dihydro-4-hydroxy-8-oxo-2′-deoxygua-
nosine (4-HO-8-oxo-dG) and guanidine-releasing products (GRP), whereas 7,8-dihydro-8-oxo-
2
′-deoxyguanosine (8-oxo-dG) was not obtained. In isolated calf thymus DNA, the efficient
conversion of the guanine base (Gua) was observed. Peroxyl radicals, which are generated in
situ from the hydroperoxides by one-electron oxidation with the peroxidases, are proposed as
the active oxidants on the basis of the following experimental facts. (i) Radical scavengers
strongly inhibit the guanine oxidation in dG and DNA and strand-break formation in the latter.
(
ii) EPR spectral studies with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap confirmed
the formation of peroxyl radicals. (iii) The release of molecular oxygen was demonstrated,
produced through the disproportionation of peroxyl radicals. The biological relevance of these
findings should be seen in the potential role of the combined action of lipid hydroperoxides
and peroxidases in damaging cellular DNA through peroxyl radicals.
In tr od u ction
it was shown that the photosensitized decomposition of
fatty ester hydroperoxides produces oxyl radicals, which
in turn induce strand breaks in DNA (7).
The peroxidation of unsaturated lipids leads to a large
number of oxidation products, which include lipid hy-
droperoxides, as well as peroxyl and alkoxyl radicals (1,
In view of the fact that peroxidases, e.g., chloroperoxi-
dase (8) and horseradish peroxidase (HRP) (9), release
peroxyl radicals from a variety of hydroperoxides by redox
chemistry, it is plausible that in cellular systems the
peroxidase-catalyzed degradation of lipid hydroperoxides
may generate peroxyl radicals, and these in turn cause
oxidative DNA damage. To probe this possibility, we
decided to investigate as a model system the peroxidase-
catalyzed induction of strand breaks in supercoiled pBR
322 DNA in the presence of hydroperoxides, as well as
the oxidation of the guanine base in calf thymus DNA
and in the nucleoside 2′-deoxyguanosine (dG). As model
compounds (Scheme 1) for the lipid hydroperoxides, we
chose 3-hydroperoxy-1-butene (1) (10), ethyl oleate hy-
droperoxide as a 1:1 regioisomeric mixture of ethyl trans-
2
). The lipid hydroperoxides are known to induce a range
of alterations in DNA. For example, it was shown that
incubation of plasmid DNA with autoxidized linoleic,
linolenic, or arachidonic acid resulted in DNA strand
breaks (3-5). When calf thymus DNA was exposed to
lipid hydroperoxides, the guanine oxidation product 7,8-
1
dihydro-8-oxoguanine (8-oxo-Gua) was formed (6). Since
the detected DNA alterations were enhanced by the
presence of transition metals (4, 5), it was concluded that
these oxidations arise from the oxyl radicals generated
in situ in the transition metal-catalyzed decomposition
of the hydroperoxides (Fenton reaction). More recently,
9
-hydroperoxyoctadec-10-enoate (4a ) and trans-10-hy-
*
To whom correspondence should be addressed. Telephone: +49-
9
31-888-5340, ext 339. Fax: +49-931-888-4756. E-mail: adam@
droperoxyoctadec-8-enoate (4b ) (11), and the enantio-
merically pure (S)-13-hydroperoxy-9(Z),11(E)-octadeca-
dienoic acid (5) (12). In addition, we examined tert-
BuOOH (3) and 2,3-dimethyl-3-hydroperoxy-1-butene (2)
chemie.uni-wuerzburg.de.
1
Abbreviations: dG, 2′-deoxyguanosine; 8-oxo-Gua, 7,8-dihydro-8-
oxoguanine; HRP, horseradish peroxidase; CIP, Coprinus peroxidase;
LP, lactoperoxidase; OC, open-circular; BHT, 2,6-di-tert-butyl-4-meth-
ylphenol; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; SOD, superoxide
dismutase; 4-HO-8-oxo-dG, 4,8-dihydro-4-hydroxy-8-oxo-2′-deoxygua-
nosine; GRP, guanidine-releasing products; 8-oxo-dG, 7,8-dihydro-8-
oxo-2′-deoxyguanosine; AAPH, 2,2′-azobis(2-methylpropionamidine)
dihydrochloride; ABTS, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic
acid); oxazolone, 2,2-diamino-4-[(2-deoxy-â-D-erythro-pentafuranosyl)-
amino]-5-(2H)-oxazolone; imidazolone, 2-amino-5-[(2-deoxy-â-D-erythro-
pentofuranosyl)amino]-4H-imidazol-4-one.
(13); the latter has been previously applied as a model
compound for lipid hydroperoxides (14). As enzymes, the
Coprinus peroxidase (CIP) from the basidiomycete Co-
prinus cinereus (15, 16), the horseradish peroxidase
(HRP), and the physiologically important lactoperoxidase
(LP, from bovine milk) were employed.
1
0.1021/tx0001880 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/17/2000

1
200 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Sch em e 1. Str u ctu r es of Hyd r op er oxid es 1-5
Adam et al.
detector (SunChrom, Friedrichsdorf, Germany), and the latter
was connected in series with an ESA Coulchem model 5100A
electrochemical detector, supplied with a model 5011 high-
sensitivity analytical cell. For the 4,8-dihydro-4-hydroxy-8-oxo-
2
′-deoxyguanosine (4-HO-8-oxo-dG) analysis, a Waters 994
photodiode array detector (Waters Millipore Co., Milford, MA)
was used. Alternatively, a Shimadzu model RF-551 spectro-
fluorometric detector (Shimadzu, Kyoto, J apan) was employed,
which allows the detection of guanidine-releasing products
(GRP). The HPLC eluents were passed through a 0.45 µm
Sartorius cellulose filter before use for degassing purposes.
The separation of 7,8-dihydro-8-oxoguanine (8-oxo-Gua) was
achieved on a 250 mm × 4.6 mm (i.d.) Eurospher 100-C18 7 µm
column (Knauer GmbH, Berlin, Germany) by using a mixture
of 50 mM sodium citrate buffer (pH 5.0) and methanol (90:10,
v/v; 1.0 mL/min) as the eluent. For the detection of 8-oxo-Gua,
the oxidation potential of the electrode (Eox) was set at 350 mV
Exp er im en ta l Section
(17). The separation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-
oxo-dG) was achieved with an 80:20 mixture of sodium citrate
buffer and methanol as the eluent and detected at an oxidation
potential of 450 mV. The DNA bases were monitored at 254 nm
by UV spectroscopy.
Ch em ica ls. Coprinus peroxidase (CIP) was obtained from
Novo Nordisk as an aqueous solution (ca. 2.98 mM) with an
3
activity of 643 kPODU/g (1 g of enzyme contains 643 × 10
peroxidase units). Horseradish peroxidase (HRP grade I, 256
units/mg of solid) was supplied by Boehringer Mannheim GmbH
and superoxide dismutase (SOD from bovine erythrocytes) by
Roche Diagnostics GmbH. Lactoperoxidase (LP from bovine
milk, 123 units/mg of solid), bromophenol blue gel-loading
solution, tris(hydroxymethyl)aminomethane (tris base), 2,2′-
azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and so-
dium salt of 1,2-naphthoquinone-4-sulfonic acid (NQS) were
For the analysis of 4-HO-8-oxo-dG, a 10 mm × 4.5 mm (i.d.)
Lichrospher 100-NH
mm (i.d.) Lichrospher 100-NH 5 µm column (Knauer GmbH)
2
5 µm guard column and a 250 mm × 4.5
2
were used by eluting with a mixture of 25 mM ammonium
formate (20%) and acetonitrile (80%) (18, 19). The detection was
performed at 230 nm by UV monitoring.
The guanidine-releasing products (GRP), e.g., oxazolone (for
the structure, see Scheme 4), were detected by an indirect
fluorescence labeling assay, after release of guanidine in alkaline
solution and its condensation with 1,2-naphthoquinone-4-sul-
fonate (NQS). The products were separated on a 250 mm × 4.6
mm (i.d.) Eurospher 100-C18 5 µm column (Knauer GmbH) with
a mixture of 25 mM ammonium formate and methanol (75:25,
v/v; 1.0 mL/min) as the eluent and detected spectrofluorometri-
cally [λex ) 355 nm, λem ) 405 nm (18, 20, 21)]. The oxidation
products were quantified against an external standard, and the
product yields are reported as the average of at least two runs
under identical conditions.
obtained from Sigma-Aldrich Chemie GmbH. Supercoiled pBR
6
3
22 DNA (form I, molecular mass of 2.9 × 10 Da, 4365 base
pairs per molecule) was ordered from Pharmacia Biotech Europe
GmbH. Calf thymus DNA, 2′-deoxyguanosine (dG), ethidium
bromide for biochemical use, and boric acid were purchased from
Merck KGaA. Agarose was acquired from Serva Feinbiochemica
GmbH. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride
(
AAPH) was available from ACROS. Adenine, cytosine, guanine,
thymine, ammonium formate, and 5,5-dimethyl-1-pyrroline
N-oxide (DMPO) were from Fluka Chemie AG. Citric acid, Na
HPO , and KH PO were from Riedel-de Ha e¨ n. Methanol and
2
-
4
2
4
acetonitrile (HPLC grade) were from Fisher Scientific GmbH.
Water was deionized by employing Millipore MilliQ equipment.
F or m a tion of Str a n d Br ea k s in Su p er coiled p BR 322
DNA. The samples were prepared in a final volume of 10 µL
and thermolyzed at 25-37 °C. The concentration of supercoiled
pBR 322 DNA in all experiments was 10 mg/L in normal aerated
Oxid a tion of DNA. The reaction mixture contained 0.125
g/L calf thymus DNA (corresponds to 78.1 µM guanine) in
normal aerated 5.0 mM KH PO /Na HPO buffer (pH 7.0), 42.5
2 4 2 4
µM CIP, and 60 mM hydroperoxide in 10% acetonitrile as the
cosolvent. The total volume of the reaction mixture was 400 µL.
The reactions were initiated by addition of the enzyme solution,
followed by the hydroperoxide solution. All experiments were
performed at 37 °C, and after a selected reaction time, a 100
µL aliquot of the solution was extracted with ethyl acetate (2 ×
5
.0 mM phosphate buffer (pH 7.4). The reactions were initiated
by addition of the enzyme solution, followed by the reactant
final concentration range of 0.88-60 mM) as an acetonitrile
(
solution (20%). Upon single-strand breakage, the supercoiled
DNA is converted to the open-circular (OC) form, while double-
stranded breaks lead to the linear form. After 60-160 min, to
the samples was added 2.50 µL of a buffer solution with 0.05%
bromophenol blue, 40% sucrose, 0.5% sodium lauryl sulfate, and
2
00 µL) and lyophilized (20 °C/0.01 Torr). Treatment with 12
µL of the HF/pyridine mixture (70% HF) for 45 min at 37 °C
22) afforded a brown solution, which was neutralized by
(
addition of 15 mg of calcium carbonate, suspended in 200 µL of
water, and stirred vigorously for 30 min. After centrifugation
0
.1 M EDTA. An aliquot (8 µL) of the resulting mixture was
(15 min at 15 000 revolutions/min) and washing of the residue
transferred to the 1% agarose gel, which contained 0.50 (8.0 µL)
mg/L ethidium bromide. The gel electrophoresis was carried out
in tris buffer (18 mM tris base, 18 mM boric acid, and 10 mM
EDTA) at pH 8.0 by running the gels on a Pharmacia horizontal
apparatus (GNA 100), with a power supply set at 78 V for 3 h
at room temperature (ca. 20 °C). The DNA spots were detected
by ethidium bromide excitation (fluorescence) with a UV trans-
illuminator (366 nm) and recorded by photography with a
Herolab camera EASY 429K, which was connected to a personal
computer, equipped with a Herolab EASY software program.
The ratio of open-circular and linear DNA relative to the total
amount of DNA was determined from the light intensities of
the spots.
with water, the combined aqueous solutions were lyophilized
and dissolved in 100 µL of water prior to the quantitative
determination of 8-oxo-Gua by HPLC/EC and guanine by HPLC/
UV analysis.
Oxid a tion of 2′-Deoxygu a n osin e (d G). To a 0.50 mM
solution of dG in 50 mM KH PO /Na HPO buffer (pH 7.0) were
2 4 2 4
added an aqueous enzyme solution and 10% of a solution of the
hydroperoxide in acetonitrile. The total volume of the reaction
mixture was 400 µL. The samples were thermostated at 25-37
°C. After modification of the dG, either the sample was directly
analyzed by HPLC or, if necessary, a 100 µL aliquot of the
solution was extracted with ethyl acetate (2 × 200 µL), lyoph-
ilized (ca. 20 °C/0.01 Torr), and dissolved in 100 µL of water
prior to the quantitative determination of the dG conversion
and 4-HO-8-oxo-dG yield by HPLC/UV and 8-oxo-dG by HPLC/
EC analysis.
HP LC An a lysis. The HPLC analytical system consisted of
Bischoff model 2200 analytical pumps (Bischoff GmbH, Leon-
berg, Germany), equipped with a Rheodyne model 7125 loop
injector (Berkeley, CA) and a SpectraFlow 600 photodiode array

Peroxidase-Catalyzed DNA Oxidation
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1201
F igu r e 2. Gel electrophoretic analysis of strand breaks in pBR
F igu r e 1. Gel electrophoretic analysis of strand breaks in pBR
332 DNA (10 mg/L in 5.0 mM KH
2
PO
4
buffer at pH 7.4 with
3
32 DNA induced by hydroperoxides 1-4 under Coprinus
20% CH
3
CN at 25 °C for 4 h) induced by ROOH 2 (6.0 mM)
peroxidase (CIP) catalysis (10 mg/L DNA in 5.0 mM KH
buffer at pH 7.4 with 20% CH CN and 0.90 µM CIP at 37 °C):
a) 60 mM ROOH 1-3 for 160 min and (b) 37 mM ROOH 4 for
0 min. As a blank, the DNA was thermolyzed alone (lane 1)
2
PO
4
under lactoperoxidase (LP) catalysis (0.14 µM, lane 5) and by
ROOH 2/LP in the presence of 0.64 µM superoxide dismutase
(SOD, lane 6). As a blank, the DNA was thermolyzed alone (lane
1) and led to 12% open-circular (OC) DNA, which has been
subtracted from the OC values (lanes 1-6) (error ( 5%).
3
(
9
and led to 18% open-circular (OC) DNA, which has been
subtracted from the OC values (lanes 1-10) (error ( 5%); for
lane 4, the linear form was also observed, given in parentheses.
gers iPrOH (23), 2,6-di-tert-butyl-4-methylphenol (BHT)
(
24), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) re-
For the analysis of the guanine moiety of dG, a 10 µL aliquot
of the reaction solution was extracted with ethyl acetate (2 ×
sulted in a significant decrease in the level of strand
breaks (up to 100%, data not shown). The addition of
superoxide dismutase (SOD, 0.6 µM) caused only a minor
effect.
2
1
0 µL), lyophilized (ca. 20 °C/0.01 Torr), and hydrolyzed with
2 µL of the HF/pyridine mixture. The procedure described
above was followed.
Analogous to that for 3-hydroperoxy-1-butene (1), the
DNA-cleaving propensities for hydroperoxides 2-5 were
assessed. Indeed, a relatively strong DNA-cleaving activ-
ity was found for tertiary hydroperoxides 2 and 3 under
CIP catalysis (Figure 1, lanes 6 and 8) and a moderate
activity for ethyl oleate hydroperoxide 4 (lane 10),
whereas linoleic acid hydroperoxide 5 exhibited only a
very weak effect (5% OC, not shown). Accordingly, the
rank order of efficacy of the hydroperoxides in inducing
CIP-catalyzed DNA cleavage is as follows: 1 > 3 > 2 .
4 > 5. Similar results, i.e., a substantial DNA-cleaving
activity, were obtained for hydroperoxides 1-4 (data not
shown) with HRP.
Once it was confirmed that hydroperoxides induce
DNA strand breaks in the presence of Coprinus and
horseradish peroxidases, the physiologically more im-
portant enzyme lactoperoxidase (LP) was tested. The
ability of the hydroperoxide 2/LP oxidant to induce
breaks in pBR 322 DNA is displayed in Figure 2. The
reaction of hydroperoxide 2 (6.0 mM) and LP (0.14 µM)
Assa y for Gu a n id in e-Relea sin g P r od u cts (GRP ). For the
analysis of the guanidine-releasing products (GRP), e.g., ox-
azolone, a 200 µL aliquot of the reaction mixture was extracted
with ethyl acetate (2 × 400 µL) and dried (ca. 20 °C/0.01 Torr),
dissolved in 100 µL of water, and stored for 24 h at room
temperature (ca. 20 °C) to achieve complete hydrolysis of
imidazolone, the oxazolone precursor (for the structures, see
Scheme 4). After addition of 37 µL of aqueous NaOH (1 M) and
2
0 µL of an aqueous solution of NQS (0.02 M), the mixture was
kept at 65 °C for 9 min in the dark. The resulting orange
solution was neutralized with 43 µL of aqueous HCl (1 M) and
analyzed by HPLC by employing a fluorometric detector.
EP R Stu d ies. These studies were carried out on a Bruker
EPR 420 spectrometer in a flat quartz cell (10 mm × 1 mm).
Samples (volume of 800 µL) were prepared by adding, im-
mediately before the EPR spectroscopy, a stock solution of CIP
[final concentration of 57 µM in 40 mM phosphate buffer (pH
7
.0)], 10 µL of the radical trap DMPO, and 80 µL of the
hydroperoxide. The following spectrometer settings were em-
5
ployed: receiver gain, 4 × 10 ; modulation amplitude, 0.52 G;
power, 20 mW; time constant, 163.84 ms; sweep time, 167.77 s;
and sweep width, 80 G.
with pBR 322 DNA [10 mg/L in 5.0 mM KH
2
PO
4
buffer
(
2
pH 7.4) with 20% acetonitrile at 25 °C for 4 h] afforded
6% OC DNA (Figure 2, lane 5) [corrected by subtraction
Resu lts
F or m a tion of Str a n d Br ea k s in th e P er oxid a se-
Ca ta lyzed Oxid a tion of p BR 322 DNA by Hyd r o-
p er oxid es 1-5. To establish whether the peroxidase-
catalyzed degradation of lipid hydroperoxides may effect
the oxidative damage of DNA, the DNA-cleaving proper-
ties of hydroperoxides 1-5 under peroxidase catalysis
were first assessed. Thus, supercoiled pBR 322 DNA (10
of the blank (lane 1), which amounted to 12% OC]. Only
4% OC DNA was obtained with hydroperoxide 2 alone
(lane 2) and 6% OC DNA with LP alone (lane 4). These
results clearly demonstrate the effective LP-catalyzed
oxidation of DNA by the hydroperoxide 2. In contrast,
3-hydroperoxy-1-butene (1) and ethyl oleate hydroper-
oxide 4 exhibited no DNA-cleaving ability under LP
catalysis (not shown).
Oxid a t ive Da m a ge of d G in t h e P er oxid a se-
Ca ta lyzed Degr a d a tion of Hyd r op er oxid es 1-5. In
addition to the DNA-cleaving activity of the hydroper-
oxide 1-5/peroxidase systems, the oxidation of the
nucleoside 2′-deoxyguanosine (dG) was investigated.
Thus, 0.50 mM dG in 50 mM phosphate buffer (pH 7.0,
10% acetonitrile) was treated with 3-hydroperoxy-1-
butene (1, 6.0 mM) in the presence of Coprinus peroxi-
dase (29.8 µM) at 25 °C to establish whether the CIP-
catalyzed decomposition of the hydroperoxides modifies
the nucleoside by oxidation. After 170 min, the reaction
mg/L DNA in 5.0 mM KH
acetonitrile) was incubated with 3-hydroperoxy-1-butene
1, 60 mM), which served as a model compound for lipid
hydroperoxides, in the presence of Coprinus peroxidase
CIP, 0.9 µM) at 37 °C for 160 min. The oxidation resulted
2 4
PO buffer at pH 7.4, with 20%
(
(
in a significant amount of open-circular (OC) DNA [40%,
Figure 1, lane 4; the yields of OC DNA have been
corrected by subtraction of the blank (18%)] and linear
DNA (11%). This is compared in Figure 1 with 3% OC
DNA in the reaction with the enzyme CIP alone (Figure
1
, lane 2) and with 15% OC DNA by hydroperoxide 1
alone (lane 3). Moreover, the addition of radical scaven-

1
202 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Adam et al.
F igu r e 4. Concentration profile for the dG conversion by the
hydroperoxide 1/Coprinus peroxidase (CIP) system (0.50 mM
dG in 50 mM phosphate buffer at pH 7.0 with 10% CH CN and
3
F igu r e 3. dG conversion and formation of 4-HO-8-oxo-dG and
guanidine-releasing products (GRP) for the Coprinus peroxidase
(
H
CIP)-catalyzed aerobic oxidation of dG by hydroperoxide 1 in
2
9.8 µM CIP at 25 °C for 255 min) (error ( 5%) of the stated
2
O and D O compared with that caused by the thermal
2
values. The inset shows the conversion of the guanine in dG.
decomposition of the azoalkane AAPH in the presence of
molecular oxygen. 8-Oxo-dG was not observed.
It was important to assess whether singlet oxygen
participates in the hydroperoxide 1/CIP-mediated dG
oxidation. For this purpose, the dG oxidation by the
hydroperoxide 1/CIP system was conducted in D O [0.50
2
mM dG in 50 mM phosphate buffer (pD 7.0), 10% aceto-
afforded 4,8-dihydro-4-hydroxy-8-oxo-2′-deoxyguanosine
(4-HO-8-oxo-dG) and guanidine-releasing products (GRP)
in 13.3 ( 0.1% and 16.2 ( 0.8% yields at 82.4 ( 0.8%
conversion of dG (Figure 3). No 7,8-dihydro-8-oxo-2′-
deoxyguanosine (8-oxo-dG) was observed in this reaction,
nor was dG consumed; only negligible amounts of the
oxidation products (less than 1.2%) were detected when
either CIP or hydroperoxide 1 was absent. Similar effects
were obtained with the enzymes HRP and LP (data not
shown), but were much less pronounced. Therefore, all
subsequent dG experiments were performed exclusively
with CIP.
nitrile, 6.0 mM 3, and 29.8 µM CIP at 25 °C for 170 min].
Clearly, the dG oxidation is not enhanced in D
); on the contrary, in D O the extent of dG conversion
is slightly reduced, i.e., 74.0 ( 0.2% in D O compared to
O. Also, the yields of 4-HO-8-oxo-dG (11.5 (
.2%) and guanidine-releasing products (13.5 ( 0.1%)
2
O (Figure
3
2
2
8
0
2.4% in H
2
were lower; 8-oxo-dG was not observed. These results
suggest that singlet oxygen is not involved in the dG
oxidation induced by the hydroperoxide 1/CIP oxidant.
To establish the oxidation efficacy of 3-hydroperoxy-
1
-butene (1), concentration-dependent studies were per-
In Figure 3, the dG conversion and formation of 4-HO-
8-oxo-dG and guanidine-releasing products for the CIP-
catalyzed oxidation of dG in the presence of hydroper-
oxide 1 are compared with those caused in the thermal
decomposition of the azoalkane 2,2′-azobis(2-methylpro-
pionamidine) dihydrochloride (AAPH). The latter is
widely used as radical initiator for the examination of in
situ-generated peroxyl radical effects (25). The thermoly-
sis of AAPH [2.5 mM in 50 mM phosphate buffer (pH
7.0)] in the presence of 0.50 mM dG at 40 °C for 61 h
resulted in a level of dG conversion of 55.0 ( 0.5%, and
5.3 ( 0.2% 4-HO-8-oxo-dG and 14 ( 1% guanidine-
releasing products were yielded; again, 8-oxo-dG was not
observed. Evidently, the efficiency of dG oxidation by the
hydroperoxide 1/CIP combination is comparable to that
caused by the peroxyl radical source AAPH.
formed. The concentration profile (Figure 4) clearly shows
that hydroperoxide concentrations of <2.0 mM (4 equiv)
have no effect on the conversion of dG. With increasing
hydroperoxide concentrations, the extent of dG conver-
sion increases; at 6.0 mM hydroperoxide (12 equiv), more
than 80% of dG was consumed. To assess whether the
oxidation occurs on the guanine or on the sugar moiety
of dG, the dG samples were hydrolyzed with the HF/
pyridine mixture after treatment with the hydroperoxide/
peroxidase oxidant and the remaining amount of guanine
was determined. As the inset Figure 4 exhibits, the
guanine conversion correlates exactly with the dG con-
version. These results imply that the hydroperoxide
1
/CIP combination oxidizes exclusively the guanine base
and not the sugar moiety of dG.
The major products in the peroxidase-catalyzed deg-
radation of 3-hydroperoxy-1-butene (1) are 3-hydroxy-1-
butene (6) and 1-buten-3-one (7). To assess whether these
nonperoxidic compounds participate in the observed dG
oxidation, both products were allowed to react with dG
under the same conditions that were applied in the
reaction of hydroperoxide 1/CIP with dG. Neither of the
two products caused any dG oxidation, with or without
CIP.
To provide further evidence for the intervention of
radicals as the oxidizing species in the CIP-catalyzed
oxidation of dG by the hydroperoxide 1, the inhibitory
effect of the well-established radical scavengers DMPO,
tert-BuOH (26), iPrOH (23), BHT (24), and glutathione
(27) was examined (Figure 5). Indeed, a moderate to
strong inhibitory effect was observed on the dG conver-
sion for all of these additives, which confirms the involve-
ment of radicals in the dG oxidation.

Peroxidase-Catalyzed DNA Oxidation
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1203
Ta ble 1. Oxid a tion of d G by th e 2,3-Dim eth yl-3-h yd r op er oxy-1-bu ten e (2)/Copr in u s P er oxid a se (CIP ) Com bin a tion ver su s
th e H2O2/CIP System
entry
reagenta
dG conversion (%)
4-HO-8-oxo-dG (%)
GRPb (%)
1
2
3
4
5
CIP (59.6 µM, 48 h)
0
0
0.35
0
0.47 ( 0.02
0.10 ( 0.01
3.53 ( 0.04
1.22 ( 0.01
0.39 ( 0.07
ROOH 2 (30 mM, 48 h)
ROOH 2 (30 mM)/CIP (59.6 µM, 48 h)
H2O2 (30 mM, 84 h)
H2O2 (30 mM)/CIP (59.6 µM, 84 h)
30 ( 3
12 ( 1
2 ( 2
3.9 ( 0.3
c
nd
nd
c
a
With 0.50 mM dG, 50 mM phosphate buffer (pH 7.0), and 10% CH3CN at 37 °C. b Guanidine-releasing products. c Not determined.
F igu r e 6. Guanine conversion in calf thymus DNA by ROOH
F igu r e 5. Effect of radical scavengers on the dG conversion
for the Coprinus peroxidase (CIP)-catalyzed oxidation of dG in
the presence of hydroperoxide 1 [0.50 mM dG in 50 mM
(
white bars), by ROOH under Coprinus peroxidase (CIP)
catalysis (gray bars), and by the ROOH/CIP combination in the
presence of 10 vol % of the radical scavenger iPrOH (black bars).
The conditions were as follows: 0.125 g/L calf thymus DNA
phosphate buffer at pH 7.0 with 10% CH
3
CN, 30.0 µM CIP, and
5
.0 mM 3-hydroperoxy-1-butene (1) at 37 °C for 3 h].
(
corresponding to 78.1 µM guanine) in 5 mM phosphate buffer
(pH 7.0), 10% CH CN, 42.5 µM CIP, and 60 mM ROOH at 37
3
The remaining hydroperoxides 2-5 were also tested
°C for 23 h.
for their dG-oxidizing properties by CIP catalysis (data
not shown). In general, tertiary hydroperoxides 2 and 3
and sterically hindered ethyl oleate hydroperoxide 4
oxidize dG in the CIP catalysis less effectively than the
smaller secondary 3-hydroperoxy-1-butene (1). To achieve
may not be detected because it is quickly oxidized to
4
-HO-8-oxo-dG.
Oxid a tive Da m a ge of Ca lf Th ym u s DNA in th e
Cop r in u s P er oxid a se (CIP )-Ca ta lyzed Decom p osi-
tion of Hyd r op er oxid es 1-3. After the oxidation of
guanine in dG by the hydroperoxide/peroxidase systems
was established, the guanine oxidation in DNA was
examined. Therefore, calf thymus DNA [0.125 g/L, cor-
responding to 78.1 µM guanine in 5.0 mM phosphate
buffer (pH 7.0), with 10% acetonitrile] was incubated at
3
0% dG conversion by the hydroperoxide 2/CIP combina-
tion, a large excess of hydroperoxide (30 mM, 60 equiv)
and a long reaction time (48 h) were required (Table 1,
entry 3). The oxidation product 4-HO-8-oxo-dG was
formed in 3.9 ( 0.3% yield and guanidine-releasing
products in 3.5 ( 0.4% yield. The order of efficiency of
the hydroperoxides in inducing CIP-catalyzed dG oxida-
tion is as follows: 1 . 3 > 2 > 4 . 5. Application of
HPR instead of CIP produced the same trend.
The observed dG consumption by the hydroperoxide/
CIP system may arise from direct oxidation of dG by the
hydroperoxide-activated Coprinus peroxidase Compound
I (CIP-I). However, no dG oxidation was observed when
3
7 °C with hydroperoxides 1-3 (60 mM) in the presence
of CIP (42.5 µM). The CIP-catalyzed degradation of
hydroperoxide 1 resulted in the significant consumption
of guanine [31%, Figure 6; the guanine conversion values
have been corrected by subtraction of the blank values
(12%)]. For tert-BuOOH (3)/CIP and hydroperoxide 2/CIP
combinations, the percent conversion values of guanine
were 22 and 9, respectively. The order of efficiency of the
hydroperoxides in oxidizing guanine in DNA under CIP
catalysis follows the same order as in the dG case: 1 >
hydroperoxide 2 was replaced with H
). Surprisingly, addition of the enzyme efficiently in-
hibited the low-level dG oxidation caused by H . The
activated CIP-I appears not to participate in the observed
dG consumption by the hydroperoxide/CIP oxidant.
Oxid a tion of 8-Oxo-d G by th e 3-Hyd r op er oxy-1-
bu ten e (1)/CIP Com bin a tion . As shown in the previous
section, none of the hydroperoxide/peroxidase combina-
tions effected the formation of dG oxidation product
2 2
O (Table 1, entry
5
2
O
2
3
> 2. Since the extents of DNA oxidation for the
hydroperoxide 2/CIP and 3/CIP cases were moderate to
low, experiments with hydroperoxides 4 and 5 were not
pursued. Evidently, the hydroperoxide/CIP oxidant is
substantially less effective in oxidizing the guanine in
DNA than in dG.
8
-oxo-dG. To examine whether 8-oxo-dG is further oxi-
dized by the hydroperoxide 1/CIP systems and, therefore,
does not accumulate for detection, the authentic 8-oxo-
dG [89 µM in 45 mM phosphate buffer (pH 7.0), with 11%
acetonitrile] was treated at 25 °C with 5.5 mM 3-hydro-
peroxy-1-butene (1) and CIP (0.9 µM). Within 90 min,
the 8-oxo-dG was completely consumed and the product
Addition of the radical scavenger iPrOH (23) resulted
in a significant decrease in the level of guanine conver-
sion (Figure 6). None of the hydroperoxide/CIP combina-
tions afforded guanine oxidation product 8-oxo-Gua. As
in the case of dG, no guanine conversion in calf thymus
DNA was observed when the hydroperoxides were re-
4
-HO-8-oxo-dG was found in 76% yield. This result
placed with H
of the enzyme inhibited the guanine oxidation caused by
2 2
H O alone.
2 2
O (data not shown), but again the addition
implies that 8-oxo-dG (provided that it is formed in the
reaction of dG with the hydroperoxide 1/CIP combination)

1
204 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Adam et al.
eters are in reasonable agreement with the literature
values (9) (R
N
) 14.48 G, R
) 14.2 G, R â ) 10.5 G, and R
.0060. The dominant nine-line spectrum (R
H
â ) 10.88 G, and R
γ ) 1.3 G and g )
) 7.1 G and
) 4.2 G and g ) 2.0065) is assigned to 5,5-dimethyl-
H
γ ) 1.30
G): R
N
H
H
2
N
R
H
2
-pyrrolidone N-oxyl (DMPOX) (28), which may arise
from decomposition of the DMPO-OOR adduct (29) in a
Kornblum-DeLaMare reaction (30). Indeed, the DMPO-
OOR signal disappears within 15 min. The remaining
signals are ascribed to a DMPO-OH or a DMPO-OR
adduct (R
N
) R ) 14.9 G and g ) 2.0061) (31). The
H
presence of DMPO in the hydroperoxide 2/CIP pair
afforded only a weak DMPO-OOR signal (not shown).
In the case of the hydroperoxide 1/CIP combination, a
complex EPR signal was recorded (not shown), which
presumably derives from ring-opened decomposition
products of DMPO.
Deter m in a tion of th e Rela tive Con ver sion Ra tes
of Hyd r op er oxid es 1-4 in th e P er oxid a se Ca ta lysis.
For this purpose, CIP [1.0 µM in 72 mM phosphate buffer
F igu r e 7. Evolution of oxygen gas during the 0.74 mM
Coprinus peroxidase (CIP)-catalyzed disproportionation of 28
mM 3-hydroperoxy-1-butene (1, in 50 mM phosphate buffer at
pH 7.0 and 20 °C) in the absence (b) and presence (O) of 0.94
mM dG and with 1.50 mg/mL (corresponding to 0.94 mM Gua)
calf thymus DNA (1).
(pH 7.0), with 10% acetonitrile] was added to a 2.0 mM
solution of ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-
sulfonic acid)] and the particular hydroperoxide (1.0 mM)
at 37 °C. The reaction was monitored by the change of
the ABTS absorption at 405 nm for 30 min, as described
in the literature (32). The ratio of the relative rates of
CIP-catalyzed decomposition of hydroperoxides 1, 3, and
2
was determined to be 6:2:1. The extent of conversion
of ethyl oleate hydroperoxide 4 was very low (data not
shown) under these conditions, and therefore, the conver-
sion rate could not be determined reliably. For this
reason, experiments with linoleic acid hydroperoxide 5
were not pursued.
Discu ssion
The enzymatic decomposition of hydroperoxides 1-5
by peroxidases such as CIP, HRP, and the mammalian
lactoperoxidase (LP) induces strand breaks in pBR 322
DNA (Figures 1 and 2) and guanine conversion in both
the nucleoside 2′-deoxyguanosine (dG, Figure 4) and calf
thymus DNA (Figure 6). Detailed product studies have
revealed that dG was oxidized by the hydroperoxide/
peroxidase systems to guanidine-releasing products and
F igu r e 8. EPR spectrum of 5,5-dimethyl-1-pyrroline N-oxide
(
DMPO) radical adducts formed in the incubation of tert-BuOOH
3) with Coprinus peroxidase (CIP). The signals are assigned
(
to DMPO-OOR (b) and DMPO-OH (2) adducts (for clarity,
only the outer peaks are marked) and to the DMPO oxidation
product DMPOX (1); the hyperfine coupling constants of each
species and the spectrometer settings are given in the Experi-
mental Section.
4
-HO-8-oxo-dG (Figure 3), while the formation of 8-oxo-
dG was not observed.
Oxygen Ga s Evolu tion d u r in g th e Cop r in u s P er -
oxid a se (CIP )-Ca ta lyzed Decom p osition of 3-Hy-
d r op er oxy-1-bu ten e (1). When CIP [0.74 mM in 50 mM
phosphate buffer (pH 7.0)] was allowed to react with
hydroperoxide 1 (28 mM) at room temperature, oxygen
gas evolution was observed (upper curve in Figure 7),
which was monitored by means of an oxygen electrode
Which oxidizing species is responsible for the dG and
DNA oxidation by the hydroperoxide/peroxidase oxi-
dants? For this purpose, we will mainly focus on the
3-hydroperoxy-1-butene (1)/CIP system, because this
combination has proven to be the most effective in the
DNA cleavage and guanine oxidation.
(
System Clark, Bachofer GmbH, Reutlingen, Germany).
The DNA cleavage (cf. the Results) and the dG conver-
sion (Figure 5) by the hydroperoxide 1/CIP oxidant were
efficiently inhibited by radical scavengers. These results
indicate that radicals are involved in this enzymatic
process. The superoxide ion may be ruled out, because
the addition of SOD did not significantly reduce the level
of strand break formation by the hydroperoxide 1/CIP
system (data not shown).
Are hydroxyl or alkoxyl radicals, which may be formed
by the catalytic action of transition metals (Fenton-type
reaction) or by homolytic cleavage of the peroxide bond,
involved in the oxidation of dG and DNA? The fact that
no dG oxidation was observed with the hydroperoxides
in the absence of the enzyme (cf. Table 1, entries 2 and
In the presence of dG (0.94 mM), the hydroperoxide 1/CIP
system evolved oxygen gas less efficiently (lower curve
in Figure 7). Likewise, in the presence of calf thymus
DNA (1.50 mg/mL corresponds to 0.94 mM guanine), the
rate of liberation of oxygen gas was reduced (middle curve
in Figure 7).
EP R Stu d ies. To provide spectral evidence for the
generation of peroxyl radicals in the CIP-catalyzed
decomposition of the hydroperoxides, spin trapping ex-
periments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO)
in water were performed (Figure 8). The tert-BuOOH (3)/
CIP combination gave a spectrum which may be assigned
to a DMPO-OOR adduct, and the EPR spectral param-

Peroxidase-Catalyzed DNA Oxidation
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1205
inhibited (Figure 7). The lower rate of O
release in the
presence of dG or DNA may be due to the reaction of
peroxyl radicals with guanine in generating the guanine
radical (addition, H abstraction). The resulting guanine
radicals trap the molecular oxygen generated in the
disproportionation of peroxyl radicals. Additionally, due
to their reaction with guanine, fewer peroxyl radicals are
Sch em e 2. Ru ssell Mech a n ism for th e F or m a tion
of Molecu la r Oxygen fr om P er oxyl Ra d ica ls in th e
CIP -Ca ta lyzed Oxid a tion of
2
3
-Hyd r op er oxy-1-bu ten e (1)
available for dimerization and release of O
Russell mechanism (Scheme 2).
2
through the
Sch em e 3. Gen er a tion of P er oxyl Ra d ica ls fr om
3
-Hyd r op er oxy-1-bu ten e (1) by Copr in u s
The inhibition of oxygen evolution by dG is more
effective than the inhibition by calf thymus DNA. This
experimental fact is consistent with the results depicted
in Figures 4 and 7, in that the oxidation of dG by the
hydroperoxides under CIP catalysis is much more pro-
nounced than the oxidation of calf thymus DNA. Unfor-
tunately, to date no rate constants of the reaction of
peroxyl radicals with DNA or dG are known. For com-
parison, reactive oxygen species such as singlet oxygen
react in water with dG (35) approximately 10 times faster
than with DNA (36, 37); the same is valid for the hydroxyl
radical (38). We assume that the reaction of peroxyl
P er oxid a se (CIP )
•
radical 1 with dG is likewise faster than with the calf
thymus DNA. In the case of DNA, its oxidation by peroxyl
•
radical 1 is supposedly slow, and other reaction path-
ways, namely, the disproportionation of the peroxyl
radicals, may compete with the DNA reaction.
3
) argues against the generation of alkoxyl and hydroxyl
As for the possible participation of singlet oxygen as
an oxidant, previously it was shown that about 12% of
the released molecular oxygen in the peroxidase-cata-
lyzed decomposition of hydroperoxides is electronically
excited singlet oxygen (39). The lifetime of singlet oxygen
radicals by homolytic cleavage of the hydroperoxides
under our conditions. Moreover, no significant guanine
oxidation was observed when H O was employed instead
2 2
of hydroperoxides in the presence of CIP; indeed, CIP
even inhibits the low-level guanine oxidation caused by
is ca. 10-fold higher in D
therefore, if singlet oxygen were involved in the present
case, the reaction in D O would enhance DNA and dG
2 2
O than in H O (40, 41);
H
2
O
2
itself. It has been well established that CIP pos-
sesses catalase activity (33), and thus, this peroxidase
decomposes readily H and protects DNA from possible
oxidative damage by H . Therefore, it is unlikely that
2
O
2
oxidation. As shown in Figure 3, the dG conversion and
2
O
2
formation of the guanidine-releasing products, as well as
2
1
the peroxidase generates hydroxyl and alkoxyl radicals
from the hydroperoxides (Fenton-type reaction). On the
contrary, it has been reported that peroxidases release
peroxyl radicals from hydroperoxides by redox chemistry
the acclaimed characteristic O
(42), are not enhanced in D
2
product 4-HO-8-oxo-dG
O. Clearly, in
2
O versus H
2
the present case, singlet oxygen does not play a signifi-
cant role in the observed DNA and dG oxidation.
To provide additional evidence that peroxyl radicals
are involved in the peroxidase-catalyzed DNA and dG
oxidation by hydroperoxides, studies with the azoalkane
AAPH have been performed. The latter was recently used
for probing peroxyl radical effects in DNA and dG damage
(43-46). As shown in Figure 3, the dG oxidation to
guanidine-releasing products and 4-HO-8-oxo-dG in the
thermolysis of AAPH is comparable to that caused by the
hydroperoxide 1 under CIP catalysis. These AAPH
results confirm that peroxyl radicals are responsible for
the peroxidase-mediated oxidative damage of dG and
DNA by hydroperoxides observed herein.
The order of efficiency of the hydroperoxides in induc-
ing CIP-catalyzed dG oxidation by peroxyl radicals is as
follows: 1 > 3 > 2 > 4 . 5 (cf. the Results). In general,
the acceptance of sterically hindered and, particularly,
tertiary hydroperoxides by HRP (47, 48) and CIP (33) is
poor; thus, this reactivity order of hydroperoxides 1-4
reflects presumably the ease of substrate acceptance by
the enzymes. Indeed, a control experiment (cf. the
Results) corroborates that the reactivity order is consis-
tent with the relative rates of the CIP-catalyzed decom-
position of hydroperoxides 1-4.
(8, 9, 33).
In view of the aforementioned examples, the most
likely reactive species in the peroxidase-catalyzed oxida-
tive damage of DNA and dG by hydroperoxides are
peroxyl radicals. Our EPR experiments provide evidence
for this, in accord with literature reports (8, 9). As shown
in the EPR spectrum (Figure 8) for the spin trapping with
DMPO, a weak signal for the peroxyl radical DMPO-
OOR adduct is observed for the tert-BuOOH (3)/CIP
system; however, the major signal is due to the DMPOX
species (9). The latter is the decomposition product of the
DMPO-OOR adduct (29), formed through base-catalyzed
elimination of ROH in a Kornblum-DeLaMare reaction
(30). Indeed, the DMPO-OOR adduct is a transient
species, and the weak EPR signal is no longer observed
after 15 min.
In the reaction of CIP with hydroperoxide 1, the
evolution of oxygen gas is observed (Figure 7), which
arises from the disproportionation of peroxyl radicals
according to the Russell mechanism (34), as shown in
•
Scheme 2. The generation of the peroxyl radicals 1
(Scheme 3) is rationalized in terms of one-electron
oxidation of hydroperoxide 1 by the hydroperoxide-
activated enzyme (Compound I and II). In the presence
of dG or calf thymus DNA, the evolution of oxygen gas
from the hydroperoxide 1/CIP system was significantly
For the most effective peroxyl radical source, namely,
the hydroperoxide 1/CIP system, the yield of OC DNA
(single strand breaks) was 40%. For a neutral peroxyl

1
206 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Adam et al.
Sch em e 4. P r op osed Mech a n ism for th e Oxid a tion
of d G to th e Gu a n id in e-Relea sin g P r od u ct
Oxa zolon e (p a th A) a n d to th e 4-HO-8-oxo-d G (p a th
B) by P er oxyl Ra d ica ls
catalytic decomposition of hydroperoxides 2 and 3 by CIP.
(ii) In the reaction of CIP with hydroperoxide 1, the
evolution of oxygen gas was observed; the O
from the hydroperoxide, since it has been established that
the CIP/ROOH mixture releases O through the dispro-
2
must come
2
portionation of peroxyl radicals according to the Russell
mechanism. (iii) The dG oxidation in the thermolysis of
the azoalkane AAPH is comparable to that caused by the
hydroperoxide 1/CIP combination. (iv) Control experi-
ments have revealed that other oxidizing species, such
as singlet oxygen, superoxide ion, and hydroxyl and
alkoxyl radicals, and the activated CIP Compound I are
not involved.
The results of this chemical study might have biological
relevance, since 3-hydroperoxy-1-butene (1), which in
combination with CIP proved to be the most effective
oxidant, contains an allylic hydroperoxy group analogous
to that in naturally occurring lipid hydroperoxides.
Ack n ow led gm en t. We thank Prof. P. Schreier (In-
stitute of Food Chemistry, University of W u¨ rzburg) for
a sample of the (S)-13-hydroperoxy-9(Z),11(E)-octadeca-
dienoic acid (5), Novo Nordisk for a donation of Coprinus
peroxidase, and Roche Diagnostics GmbH for the super-
oxide dismutase. This work was supported by the Deut-
sche Forschungsgemeinschaft (Sonderforschungsbereich
1
72 “Molekulare Mechanismen kanzerogener Prim a¨ rv-
er a¨ nderungen”) and Fonds der Chemischen Industrie.
radical, as in this case, this presents a substantial DNA
cleaving activity (46). Since hydroperoxide 1 in combina-
tion with CIP oxidized only the guanine base, and not
the sugar moiety of the isolated nucleoside dG (Figure
Refer en ces
(
1) Porter, N. A. (1986) Mechanisms for the autoxidation of polyun-
saturated lipids. Acc. Chem. Res. 19, 262-268.
4
), it is not likely that the observed DNA strand scission
(
2) Esterbauer, H., Eckl, P., and Ortner, A. (1990) Possible mutagens
derived from lipids and lipid precursors. Mutat. Res. 238, 223-
233.
arises from the direct attack of the peroxyl radicals on
the deoxyribose. We propose that the strand breaks are
formed by oxidative modifications of the nucleobases (49).
Now the following question arises: how are products
(
3) Inoue, S. (1984) Site-specific cleavage of double-strand DNA by
hydroperoxide of linoleic acid. FEBS Lett. 172, 231-234.
4) Ueda, K., Kobayashi, S., Morita, J ., and Komano, T. (1985) Site-
specific DNA damage caused by lipid peroxidation products.
Biochim. Biophys. Acta 824, 341-348.
(
4
-HO-8-oxo-dG and GRP (e.g., oxazolone) formed in the
oxidation of dG by peroxyl radicals? A plausible mecha-
nism is proposed in Scheme 4, based on literature
speculation and the experimental results presented here.
The oxidation of dG to the guanidine-releasing product
oxazolone may proceed through hydrogen abstraction
(
5) Yang, M. H., and Schaich, K. M. (1996) Factors affecting DNA
damage caused by lipid hydroperoxides and aldehydes. Free
Radical Biol. Med. 20, 225-236.
(6) Park, J . W., and Floyd, R. A. (1992) Lipid peroxidation products
mediate the formation of 8-hydroxydeoxyguanosine in DNA. Free
Radical Biol. Med. 12, 245-250.
(Scheme 4, path A) by peroxyl radicals to the guanosine
(
(
(
7) Adam, W., Andler, S., and Saha-M o¨ ller, C. R. (1998) DNA cleavage
induced by oxyl radicals generated in the photosensitized decom-
position of fatty ester hydroperoxides derived from oleic and
linoleic acid. Arch. Biochem. Biophys. 349, 261-266.
8) Chamulitrat, W., Takahashi, N., and Mason, R. P. (1989) Peroxyl,
alkoxyl, and carbon-centered radical formation from organic
hydroperoxides by chloroperoxidase. J . Biol. Chem. 264, 7889-
7899.
radical. The latter may trap molecular oxygen and form
oxazolone in several steps, as described elsewhere (18,
2
0, 21). The oxidation of dG to 4-HO-8-oxo-dG may be
initiated by the addition of the peroxyl radical (Scheme
, path B) at the C8 position of dG (50). The resulting
4
dG radical species may trap molecular oxygen to yield a
peroxyl radical, which in turn abstracts a hydrogen atom
from the employed hydroperoxide. The resulting dG
hydroperoxide eliminates alcohol under base catalysis in
a Kornblum-DeLaMare reaction (30) to yield 4,8-dihy-
dro-4-hydroperoxy-8-oxo-2′-deoxyguanosine (4-HOO-8-
oxo-dG). In the final step, the 4-HOO-8-oxo-dG is reduced
by CIP to 4-HO-8-oxo-dG under generation of the acti-
vated CIP I complex.
9) Davies, M. J . (1988) Detection of peroxyl and alkoxyl radicals
produced by reaction of hydroperoxides with heme proteins by
electron spin resonance spectroscopy. Biochim. Biophys. Acta 964,
2
8-35.
(10) Lubeigt, X., Flies, F., Bourgeois, M. J ., Montaudon, E., and
Maillard, B. (1991) D e´ placements homolytiques intramol e´ culaires.
1
9. St e´ r e´ ochimie de la d e´ composition induite de peroxydes insa-
tur e´ s conduisant a` la formation d′h e´ t e´ rocycles a` trois et quatre
cha ˆı nons. Can. J . Chem. 69, 1320-1325.
(11) Porter, N. A., and Wujek, J . S. (1987) Allylic hydroperoxide
rearrangement: â-scission or concerted pathway? J . Org. Chem.
5
2, 5085-5089.
Con clu sion s
(
12) Hatanaka, A., Kajiwara, T., Sekiya, J ., and Inouye, S. (1982)
Solubilization and properties of the enzyme cleaving 13-L-
hydroperoxy-linolenic-acid in tea thea-sinensis leaves. Phytochem-
istry 21, 13-17.
From our results, we conclude that peroxidases release
peroxyl radicals from hydroperoxides, which in turn
oxidize the guanine in both dG and DNA, and induce
strand breaks in the latter. The following experimental
facts support this conclusion. (i) EPR spectral evidence
was provided for the formation of peroxyl radicals in the
(
13) Gollnick, K., and Griesbeck, A. (1984) Solvent dependence of
singlet oxygen/substrate interactions in ene-reactions, (4+2)- and
(2+2)-cycloaddition reactions. Tetrahedron Lett. 25, 725-728.
(
14) Timmins, G. S., dos Santos, R. E., Whitwood, A. C., Catalani, L.
H., Di Mascio, P., Gilbert, B. C., and Bechara, E. J . H. (1997)

Peroxidase-Catalyzed DNA Oxidation
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1207
Lipid peroxidation-dependent chemiluminescence from the cy-
clization of alkylperoxyl radicals to dioxetane radical intermedi-
ates. Chem. Res. Toxicol. 10, 1090-1096.
(32) P u¨ tter, J ., and Becker, R. (1983) Peroxidases. In Methods of
Enzymatic Analysis (Bergmeyer, H. U., Ed.) Vol. 3, pp 286-293,
VCH, Weinheim, Germany.
(
15) Shinmen, Y., Asami, S., Amachi, T., Shimizu, S., and Yamada,
H. (1986) Crystallization and characterization of an extracellular
fungal peroxidase. Agric. Biol. Chem. 50, 247-249.
16) Morita, Y., Yamashita, H., Mikami, B., Iwamoto, H., Aibara, S.,
Terada, M., and Minami, J . (1988) Purification, crystallization,
and characterization of peroxidase from Coprinus cinereus. J .
Biochem. 103, 693-699.
17) Floyd, R. A., West, M. S., Eneff, K. L., Schneider, J . E., Wong, P.
K., Tingley, D. T., and Hogsett, W. E. (1990) Conditions influenc-
ing yield and analysis of 8-hydroxy-2′-deoxyguanosine in oxida-
tively damaged DNA. Anal. Biochem. 188, 155-158.
(
33) Adam, W., Mock-Knoblauch, C., and Saha-M o¨ ller, C. R. (1999)
Asymmetric synthesis with the enzyme Coprinus peroxidase:
kinetic resolution of chiral hydroperoxides and enantioselective
sulfoxidation. J . Org. Chem. 64, 4834-4839.
(
(
34) Russell, G. A. (1957) Deuterium-isotope effects in the autoxidation
of aralkyl hydrocarbons. Mechanism of the interaction of peroxyl
radicals. J . Am. Chem. Soc. 79, 3871-3877.
(
(
35) Devasagayam, T. P., Steenken, S., Obendorf, M. S., Schulz, W.
A., and Sies, H. (1991) Formation of 8-hydroxy(deoxy)guanosine
and generation of strand breaks at guanine residues in DNA by
singlet oxygen. Biochemistry 30, 6283-6289.
(
18) Ravanat, J .-L., Berger, M., Benard, F., Langlois, R., Ouellet, R.,
van Lier, J . E., and Cadet, J . (1992) Phthalocyanine and naph-
thalocyanine photosensitized oxidation of 2′-deoxyguanosine dis-
tinct type I and type II products. Photochem. Photobiol. 55, 809-
(
36) Lee, P. C. C., and Rodgers, M. A. J . (1987) Laser flash photokinetic
studies of rose bengal sensitized photodynamic interactions of
nucleotides and DNA. Photochem. Photobiol. 45, 79-86.
8
14.
(37) Davila, J ., and Harriman, A. (1989) Photosensitized oxidation of
biomaterials and related model compounds. Photochem. Photobiol.
50, 29-35.
(
19) Ravanat, J .-L., Berger, M., Buschko, G. W., Benard, F., van Lier,
J . E., and Cadet, J . (1991) Photooxydation sensibilis e´ e de la
d e´ soxy-2′-guanosine par des phtalocyanines et naphtalocyanines.
D e´ termination de l’importance des m e´ canismes de type I et de
type II. J . Chim. Phys. Phys. Chim. Biol. 88, 1069-1076.
20) Buschko, G. W., Cadet, J ., Ravanat, J .-L., and Labataille, P. (1993)
Isolation and characterization of a new product produced by
ionizing irradiation and type I photosensitization of 2′-deoxygua-
nosine in oxygen-saturated aqueous solution: (2S)-2,5′-anhydro-
(38) Michaels, H. B., and Hunt, J . W. (1973) Reactions of the hydroxyl
radical with polynucleotides. Radiat. Res. 56, 57-70.
(
39) Hall, R. D., Chamulitrat, W., Takahashi, N., Chignell, C. F., and
(
1
2
Mason, R. P. (1989) Detection of singlet ( O ) oxygen phospho-
rescence during chloroperoxidase-catalyzed decomposition of ethyl
hydroperoxide. J . Biol. Chem. 264, 7900-7906.
(40) Ogilby, P. R., and Foote, C. S. (1983) Chemistry of singlet oxygen.
1
-(2′-deoxy-â-D-erythro-pentofuranosyl)-5-guanidinylidene-2-hy-
4
2. Effect of solvent, solvent isotopic substitution, and tempera-
droxy-4-oxoimidazolidine. Int. J . Radiat. Biol. 63, 669-676.
21) Cadet, J ., Berger, M., Buschko, G. W., J oshi, P. C., Raoul, S., and
Ravanat, J .-L. (1994) 2,2-Diamino-4-[(3,5-di-O-acetyl-2-deoxy-â-
D-erythro-pentafuranosyl)amino]-5-(2H)-oxazolone: a novel and
predominant radical oxidation product of 3′,5′-di-O-acetyl-2′-
deoxyguanosine. J . Am. Chem. Soc. 116, 7403-7404.
1
ture on the lifetime of singlet molecular oxygen ( ∆
Chem. Soc. 105, 3423-3430.
g
). J . Am.
(1∆
)
g
(
(
41) Rodgers, M. A. J ., and Snowden, P. T. (1982) Lifetime of O
2
in liquid water as determined by time-resolved infrared lumines-
cence measurement. J . Am. Chem. Soc. 104, 5541-5543.
(
42) Ravanat, J .-L., and Cadet, J . (1995) Reaction of singlet oxygen
with 2′-deoxyguanosine and DNA. Isolation and characterization
of the main oxidation products. Chem. Res. Toxicol. 8, 379-388.
(
22) Polverelli, M., Berger, M., Mouret, J .-F., Odin, F., and Cadet, J .
(1990) Acidic hydrolysis of the N-glycosidic bonds of deoxyribo-
nucleic acid by hydrogen fluoride stabilized in pyridine. Nucleo-
sides Nucleotides 9, 451-452.
(43) Harkin, L. A., and Burcham, P. C. (1997) Formation of novel C1-
oxidised abasic sites in alkylperoxyl radical-damaged plasmid
DNA. Biochem. Biophys. Res. Commun. 237, 1-5.
(44) Valentine, M. R., Rodriguez, H., and Termini, J . (1998) Mutagen-
esis by peroxy radical is dominated by transversions at deoxy-
guanosine: evidence for the lack of involvement of 8-oxo-dG and/
or abasic site formation. Biochemistry 37, 7030-7038.
(
23) Thomas, J . K. (1965) Rates of reaction of the hydroxyl radical.
Trans. Faraday Soc. 61, 702-707.
(
24) Trotta, R. J ., Sullivan, S. G., and Stern, A. (1983) Lipid peroxi-
dation and haemoglobin degradation in red blood cells exposed
to tert-butyl hydroperoxide. The relative roles of haem- and
glutathione-dependent decomposition of tert-butyl hydroperoxide
and membrane lipid hydroperoxides in lipid peroxidation and
haemolysis. Biochem. J . 212, 759-772 (since phenols serves as
electron donors to peroxidases, BHT may prevent the formation
of peroxyl radicals).
(
45) Simandan, T., Sun, J ., and Dix, T. A. (1998) Oxidation of DNA
bases, deoxyribonucleosides and homopolymers by peroxyl radi-
cals. Biochem. J . 335, 233-240.
(
46) Paul, T., Young, M. J ., Hill, I. E., and Ingold, K. U. (2000) Strand
cleavage of supercoiled DNA by water-soluble peroxyl radicals.
The overlooked importance of peroxyl radical charge. Biochemistry
(
(
(
(
25) Niki, E. (1990) Free radical initiators as source of water-soluble
or lipid-soluble peroxyl radicals. Methods Enzymol. 186, 100-
1
08.
3
9, 4129-4135.
26) Ito, S., Ueno, K., Mitarai, A., and Sasaki, K. (1993) Evidence for
hydroxyl radicals as an active species generated from Uden-
friend’s reagent. J . Chem. Soc., Perkin Trans. 2, 255-259.
27) Spear, N., and Aust, S. D. (1995) Effects of glutathione on Fenton
reagent-dependent radical production and DNA oxidation. Arch.
Biochem. Biophys. 324, 111-116.
28) Floyd, R. A., and Soong, L. M. (1977) Spin trapping in biological
systems. Oxidation of the spin trap 5,5-dimethyl-1-pyrroline-1-
oxide by a hydroperoxide-hematin-system. Biochem. Biophys. Res.
Commun. 74, 79-84.
29) Rosen, G. M., and Rauckman, E. J . (1980) Spin trapping of the
primary radical involved in the activation of the carcinogen
N-hydroxy-2-acetylaminofluorene by cumene hydroperoxide-he-
matin. Mol. Pharmacol. 17, 233-238.
(
47) Adam, W., Hoch, U., Humpf, H. U., Saha-M o¨ ller, C. R., and
Schreier, P. (1996) Horseradish-peroxidase (HRP)-catalyzed enan-
tioselective reduction of racemic hydroperoxy homoallylic alco-
hols: a novel enzymatic method for the preparation of optically
active, unsaturated diols and hydroperoxy alcohols. J . Chem. Soc.,
Chem. Commun., 2701-2702.
(48) Hoch, U., Adam, W., Fell, R. T., Saha-M o¨ ller, C. R., and Schreier,
P. (1997) Horseradish peroxidase: a biocatalyst for the one-pot
synthesis of enantiomerically pure hydroperoxides and alcohols.
J . Mol. Catal. A: Chem. 117, 321-328.
(49) Burrows, C. J ., and Muller, J . G. (1998) Oxidative nucleobase
modifications leading to strand scission. Chem. Rev. 98, 1109-
1151.
(
(
30) Kornblum, N., and DeLaMare, H. E. (1951) The base-catalyzed
decomposition of a dialkyl peroxide. J . Am. Chem. Soc. 73, 880-
(
50) Shi, X., Mao, Y., Ahmed, N., and J iang, H. (1995) HPLC
investigation on Ni(II)-mediated DNA damage in the presence of
tert-butyl hydroperoxide and glutathione. J . Inorg. Biochem. 57,
8
81.
(
31) Finkelstein, E., Rosen, G. M., and Rauckman, E. J . (1980) Spin
trapping of superoxide and hydroxyl radical: practical aspects.
Arch. Biochem. Biophys. 200, 1-16.
9
1-102.
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