Formation of a substituted 1,N6-etheno-2'-deoxyadenosine adduct by lipid hydroperoxide-mediated generation of 4-oxo-2-nonenal

Article abstract of DOI:10.1021/tx0000771

Analysis of the reaction between 2'-deoxyadenosine and 13-hydroperoxylinoleic acid by liquid chromatography/constant neutral loss mass spectrometry revealed the presence of two major products (adducts A and B). Adduct A was shown to be a mixture of two isomers (A₁ and A₂) that each decomposed with the loss of water to form adduct B. The mass spectral characteristics of adduct B were consistent with the substituted 1,N⁶-etheno-2'-deoxyadensoine adduct 1'-[3-(2'-deoxy-β-D-erythro-pentafuranosyl)-3H-imidazo[2,1-i]purin-7-yl]hept an-2'-one. Adducts A₁, A₂, and B were formed when 2'-deoxyadenosine was treated with synthetic 4-oxo-2-nonenal, which suggested that it was formed by the breakdown of 13-hydroperoxylinoleic acid. A substantial increase in the rate of formation of adducts A₁, A₂, and B was observed when 13-hydroperoxylinoleic acid and 2'-deoxyadenosine were incubated in the presence of Fe(II). Thus, 4-oxo-2-nonenal was most likely formed by a homolytic process. Although adducts A₁, A₂, and B were formed in the reaction between 4-hydroxy-2-nonenal and 2'-deoxyadenosine, a number of additional products were observed. This suggested that 4-hydroxy-2-nonenal was not a precursor in the formation of 4-oxo-2-nonenal from 13-hydroperoxylinoleic acid. This study has provided additional evidence which shows that 4-oxo-2-nonenal is a major product of lipid peroxidation and that it reacts efficiently with DNA to form substituted etheno adducts.

Full text of DOI:10.1021/tx0000771

846
Chem. Res. Toxicol. 2000, 13, 846-852
F or m a tion of a Su bstitu ted
1,N6-Eth en o-2′-d eoxya d en osin e Ad d u ct by Lip id
Hyd r op er oxid e-Med ia ted Gen er a tion of 4-Oxo-2-n on en a l
Diane Rindgen,† Seon Hwa Lee, Masaharu Nakajima,‡ and Ian A. Blair*
Center for Cancer Pharmacology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104-6160
Received April 7, 2000
Analysis of the reaction between 2′-deoxyadenosine and 13-hydroperoxylinoleic acid by liquid
chromatography/constant neutral loss mass spectrometry revealed the presence of two major
products (adducts A and B). Adduct A was shown to be a mixture of two isomers (A1 and A2)
that each decomposed with the loss of water to form adduct B. The mass spectral characteristics
of adduct B were consistent with the substituted 1,N6-etheno-2′-deoxyadensoine adduct 1′′-
[3-(2′-deoxy-â-D-erythro-pentafuranosyl)-3H-imidazo[2,1-i]purin-7-yl]heptan-2′′-one. Adducts A1,
A2, and B were formed when 2′-deoxyadenosine was treated with synthetic 4-oxo-2-nonenal,
which suggested that it was formed by the breakdown of 13-hydroperoxylinoleic acid. A
substantial increase in the rate of formation of adducts A1, A2, and B was observed when
13-hydroperoxylinoleic acid and 2′-deoxyadenosine were incubated in the presence of FeII. Thus,
4-oxo-2-nonenal was most likely formed by a homolytic process. Although adducts A1, A2, and
B were formed in the reaction between 4-hydroxy-2-nonenal and 2′-deoxyadenosine, a number
of additional products were observed. This suggested that 4-hydroxy-2-nonenal was not a
precursor in the formation of 4-oxo-2-nonenal from 13-hydroperoxylinoleic acid. This study
has provided additional evidence which shows that 4-oxo-2-nonenal is a major product of lipid
peroxidation and that it reacts efficiently with DNA to form substituted etheno adducts.
deoxyguanosine (dGuo) to produce a tricyclic substituted
propano adduct (19, 20). It is also readily oxidized to the
epoxide derivative which can covalently modify both
dGuo and dAdo to form etheno adducts (21, 22).
We have suggested that 4-oxo-2-nonenal is a novel
product of lipid peroxides, which covalently modifies
dGuo either as the free nucleoside or when present in
DNA (23). The adducts that arise from the reaction of
4-oxo-2-nonenal with dAdo have recently been character-
ized (24). A detailed analysis of the reaction between
dAdo and linoleic acid hydroperoxide {13-hydroperoxy-
[S-(Z,E)]-9,11-octadecadienoic acid (13-HPODE1)}, a rep-
resentative ω - 6 lipid hydroperoxide has now been
performed. The decomposition product of 13-HPODE
responsible for covalent modifications to dAdo was un-
equivocally identified as 4-oxo-2-nonenal. Therefore, this
study provides additional insight into the mechanism by
which lipid hydroperoxides can initiate the formation of
DNA adducts (25).
In tr od u ction
Free radical-mediated lipid peroxidation results in the
formation of lipid hydroperoxides that readily decompose
to the corresponding alkoxy radicals (1). The alkoxy
radicals can either chain terminate by the abstraction
of a hydrogen atom from a suitable donor or undergo
various secondary reactions which lead to the formation
of highly reactive unsaturated aldehydes (2, 3). Lipid
peroxidation has been implicated as a contributing factor
in the etiology of a number of disease states (4-7).
Several lipid peroxidation end products have been shown
to covalently modify nucleic acids to form mutagenic
lesions in DNA (8). Malondialdehyde (MDA, â-hydroxy-
acrolein) is a well-characterized R,â-unsaturated alde-
hyde, which arises from both free radical (9-11) and
enzymatic (12, 13) peroxidation of polyunsaturated fatty
acids. MDA modifies both purine bases to generate a
tricyclic adduct with guanine (14), which has been
detected in rat (15) and human liver DNA (16). It also
forms an acyclic adduct with adenine (17, 18). The
degradation of lipid hydroperoxides results in the produc-
tion of 4-hydroxy-2-nonenal, another well-studied lipid
peroxidation product (2, 3). 4-Hydroxy-2-nonenal is a
bifunctional electrophile, which reacts directly with 2′-
Ma ter ia ls a n d Meth od s
Ma ter ia ls. 2′-Deoxyadenosine, soybean lipoxidase (type V),
and calf thymus DNA were purchased from Sigma Chemical
Co. (St. Louis, MO). 9,11-Octadecadienoic acid, 13-hydroperoxy-
[S-(Z,E)], was obtained from Cayman Chemical Co. (Ann Arbor,
* To whom correspondence should be addressed: Center for Cancer
Pharmacology, University of Pennsylvania School of Medicine, 1254
BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160. Fax: (215)
573-9889. E-mail: ian@spirit.gcrc.upenn.edu.
1
Abbreviations: CID, collision-induced dissociation; CNL, constant
neutral loss; ESI, electrospray ionization; 13-HPODE, 13-hydroperoxy-
[S-(Z,E)]-9,11-octadecadienoic acid; LC/MS, liquid chromatography/
mass spectrometry; LOX, lipoxygenase; MH+, protonated molecular
ion; MSn, multiple tandem mass spectrometry; PBS, phosphate-
buffered saline; SRM, selected reaction monitoring; TIC, total ion
current.
† Present address: Drug Metabolism and Pharmacokinetics, Scher-
ing-Plough Research Institute, K-15-3700, 2015 Galloping Hill Rd.,
Kenilworth, NJ 07033-0539.
‡ Present address: Niigata Prefectural Food Research Institute,
2-25, Shin-eicho, Kamo-shi, Niigata 959-1381, J apan.
10.1021/tx0000771 CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/04/2000

Etheno-dAdo Adduct from Lipid Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 847
MI). 4-Hydroxy-2-nonenal was purchased from Sigma. HPLC
grade water was obtained from Fisher Scientific Co. (Fair Lawn,
NJ ). HPLC grade methanol was purchased from Burdick and
J ackson (Muskegon, MI). Gases were supplied by BOC Gases
(Lebanon, NJ ).
Ma ss Sp ectr om etr y. The data presented were acquired on
either a Finnigan TSQ 7000 or a Finnigan LCQ ion trap mass
spectrometer (ThermoQuest, San J ose, CA) equipped with a
Finnigan electrospray source. The mass spectrometers were
operated in the positive ion mode with a potential of 4.25 kV
applied to the electrospray needle. Nitrogen was used as the
sheath and auxiliary gas to assist with nebulization. The
capillary temperature was held at 200 °C. Full scanning
analyses were performed in the range of m/z 100-900. Helium
was used as the collision gas.
Liqu id Ch r om a togr a p h y. Chromatography was performed
using a Waters Alliance 2690 HPLC system (Waters Corp.,
Milford, MA). Gradient elutions were all performed in the linear
mode. Gradient systems 1 and 2 employed a C18 ODS-AQ
column (250 mm × 2.0 mm i.d., 3 µm; YMC, Inc., Wilmington,
NC) at a flow rate of 170 µL/min. Solvent A was 5 mM aqueous
ammonium acetate and 0.01% trifluoroacetic acid, and solvent
B was 5 mM methanolic ammonium acetate and 0.01% trifluo-
roacetic acid. The gradient conditions for system 1 were as
follows: 0% B at 0 min, 100% B at 15 min, and 0% B at 20 min,
followed by an equilibration time of 20 min. The gradient
conditions for system 2 were as follows: 30% B at 0 min, 30%
B at 5 min, 100% B at 16 min, 100% B at 26 min, and 30% B at
31 min, followed by an equilibration time of 19 min. Gradient
system 3 employed a YMC C18 ODS-AQ column (250 mm × 4.6
mm i.d., 5 µm; YMC, Inc.) at a flow rate of 1 mL/min. The
gradient conditions were as follows: 30% B at 0 min, 30% B at
5 min, 100% B at 16 min, 100% B at 24 min, and 30% B at 26
min, followed by an equilibration time of 4 min.
F igu r e 1. Analysis of the reaction between dAdo and 13-
HPODE after 36 h at 37 °C by LC/MS with CNL analysis using
gradient system 1. (A) Selected ion chromatogram for the MH+
of adduct A (m/z 406). (B) Selected ion chromatogram for the
MH+ of adduct B (m/z 388).
stirred for 30 min in an ice/water bath followed by 3 h at room
temperature. The reaction mixture was then extracted with
chloroform (3 × 1 mL); the chloroform extracts were dried over
sodium sulfate and filtered, and the chloroform was evaporated
under nitrogen. The 2,3-epoxy-4-hydroxynonanal was redis-
solved in ethanol (800 µL). dAdo (43 mg, 0.17 mmol) in a 1:1
PBS/H2O solution (3 mL of PBS/3 mL of H2O) was added to the
stirred solution of 2,3-epoxy-4-hydroxynonanal in ethanol (400
µL) and incubated at 37 °C for 60 h. The reaction mixture was
placed in an ice bath prior to analysis by LC/MS using gradient
system 2.
Syn th esis of 4-Oxo-2-n on en a l. 4-Hydroxy-2-nonenal di-
ethyl acetal was oxidized with activated MnO2 as described by
Esterbauer and Weger (28). The resulting 4-oxo-2-nonenal
diethyl acetal was then hydrolyzed by citric acid/HCl as
described previously (23).
Rea ction of d Ad o w ith 13-HP ODE. dAdo (44 µg, 0.18 µmol)
in H2O (40 µL) was added together with 13-HPODE (500 µg,
1.6 µmol) in ethanol (120 µL) to PBS (210 µL). The final pH
was 7.0. Reaction mixtures were incubated at 37 °C for 36 h,
after which they were placed on ice prior to LC/MS analysis
using gradient system 1 or 2.
Rea ction of d Ad o w ith 4-Oxo-2-n on en a l. A solution of
4-oxo-2-nonenal (1300 µg, 8.4 µmol) in ethanol (15 µL) was added
to dAdo (1200 µg, 4.8 µmol) in water (250 µL). The reaction
mixture was sonicated for 15 min at room temperature and
incubated at 37 or 60 °C for 24 h, after which it was placed on
ice. An aliquot of the sample (20 µL) was diluted to 100 µL with
a water/methanol mixture (7:3 v/v) and filtered through a 0.2
µm CoStar cartridge prior to analysis of a portion of the sample
(10 µL) by LC/MS using gradient system 2.
Rea ction of d Ad o w ith 13-HP ODE/F eII. 13-HPODE (100
µg, 0.32 µmol) in ethanol (20 µL) was added together with 50
µL of an aqueous solution of FeSO4‚7H2O (0.89 mg/mL, 0.16
µmol of FeII) and dAdo (40 µg, 0.16 µmol) in water (45 µL) to
water (135 µL). The reaction mixture (total volume of 250 µL;
640 µM FeII) was incubated at 37 °C for 30 min. Samples were
placed on ice prior to LC/MS analysis using gradient system 2.
P r ep a r a tion of [13C5]d Ad o. [13C5]Ade was prepared as
described previously (26) and was used to generate [13C5]dAdo
via the procedure described by Chapeau and Marnett (27).
Rea ction of d Ad o w ith 13-HP ODE fr om Lip oxygen a se
(LOX)/Lin oleic Acid . Linoleic acid (450 µg, 1.6 µmol) or [13C18]-
linoleic acid (475 µg, 1.6 µmol) in ethanol (2 µL) was added to
soybean LOX (1100 units, type V) in PBS (300 µL, pH 7.4). The
samples were incubated at 37 °C for 24 h. They were then cooled
to room temperature, and dAdo (40 µg, 0.16 µmol) or [13C5]dAdo
(42 µg, 0.16 µmol) in H2O (40 µL) was added. The incubations
were continued for a further 12 h at 37 °C. After being cooled
on ice, the samples were filtered through 0.2 µm CoStar filter
cartridges prior to LC/MS analysis using gradient system 3.
Rea ction of d Ad o w ith 4-Hyd r oxy-2-n on en a l. dAdo (45
µg, 0.18 µmol) in H2O (35 µL) was added together with
4-hydroxy-2-nonenal (280 µg, 1.8 µmol) in hexane (26 µL) to PBS
(289 µL, pH 7.4) or 25 mM KH2PO4 (289 µL, pH 7.4). The slightly
turbid solutions were incubated at 37 °C for 37 h and then
placed on ice prior to analysis by LC/MS using gradient system
2.
Resu lts
Rea ction of d Ad o w ith 13-HP ODE. LC/MS analysis
was conducted using gradient system 1 coupled with
constant neutral loss (CNL) scanning to screen the
reaction mixture for compounds undergoing the loss of
116 amu under collision-induced dissociation (CID) con-
ditions (29). This fragmentation (which is characteristic
of 2′-deoxynucleosides) revealed the presence of adduct
A and adduct B with MH+ ions at m/z 406 and 388,
respectively (Figure 1). Adduct B appeared to be a
dehydration product of adduct A because of its mass
spectral characteristics and its longer retention time
under reversed-phase conditions. The response in the m/z
406 channel suggested the presence of two components,
and this was confirmed when the reaction mixture was
analyzed using modified gradient system 2 (Figure 2).
The reconstructed mass chromatogram for m/z 406
showed the presence of two isomeric compounds (adduct
A1 and adduct A2) which both possessed major aglycone
product ions at m/z 290. A single response was observed
Rea ction of d Ad o w ith 2,3-Ep oxy-4-h yd r oxyn on a n a l. A
stirred aqueous solution of tert-butylhydroperoxide (246 µL of
a 70% solution) was adjusted to pH 8 with 0.01 N NaOH. This
was followed by the addition of 4-hydroxy-2-nonenal (100 mg,
0.64 mmol) in methanol (500 µL). The reaction mixture was

848 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Rindgen et al.
F igu r e 2. Analysis of the reaction between dAdo and 13-
HPODE after 36 h at 37 °C by LC/MS2 using gradient system
2. Product ion spectra are shown in the insets. The spectrum
for adduct A1 was identical with that for adduct A2. (A) TIC
chromatogram for adducts A1 and A2 (m/z 406 f). (B) TIC
chromatogram for adduct B (m/z 388 f).
F igu r e 3. Analysis of the reaction between dAdo and 13-
HPODE after 36 h at 37 °C by LC/MS3 using gradient system
2. Product ion spectra are shown in the insets. The spectrum
for adduct A1 was identical with that for adduct A2. (A) TIC
chromatograms for adducts A1 and A2 (m/z 406 f 290 f). (B)
TIC chromatogram for adduct B (m/z 388 f 272 f).
for the m/z 388 product (adduct B) with the expected
aglycone ion detected at m/z 272.
The LCQ instrument was used to further study the
fragmentation behavior of the aglycone ions of adducts
A1 and A2 (m/z 406) and adduct B (m/z 388). Each ion,
namely, m/z 290 (adducts A1 and A2) and 272 (adduct
B), was isolated and CID conducted. The aglycone ions
at m/z 290 from adducts A1 and A2 each generated two
identical product ions at m/z 272 and 136 (Figure 3A).
This confirmed that they were isomeric dAdo adducts.
The ion at m/z 272 arose through the loss of a water
molecule, while the ion at m/z 136 was protonated
adenine. The aglycone ion at m/z 272 from adduct B
generated one major product ion at m/z 174 (-C5H10CO)
(Figure 3B).
(Figure 5). The reaction was repeated using [13C18]linoleic
acid as substrate, and the products were compared with
those obtained with the unlabeled fatty acid. Adducts A1,
A2, and B each displayed a 9 Da increase in MH+, which
confirmed the incorporation of a nine-carbon fragment
from linoleic acid into the three adducts. These experi-
ments were also conducted using [13C5]dAdo, and the
expected 5 Da mass shifts in MH+ were observed.
Rea ction of d Ad o w ith 4-Hyd r oxy-2-n on en a l. A
targeted CID analysis was conducted on the reaction
mixture focusing on the product ions derived from the
aglycone ion (m/z 290) of m/z 406 from adducts A1 and
A2. A number of compounds were formed in this reaction
(Figure 6). It should be noted that there was no clear
evidence for the formation of the Michael addition
product, which would generate an MH+ ion at m/z 408
(data not shown). The mass spectrometric behavior of the
two earliest eluting compounds at 17.2 and 17.6 min was
identical to that of adducts A1 and A2 generated in the
dAdo/13-HPODE incubation; i.e., major product ions were
observed at m/z 272 (loss of water) and 136 (Ade + H).
Each compound eluting between 18.0 and 20.0 min
exhibited a major product ion at m/z 160. This product
ion is known to arise by CID of the MH+ ion (m/z 276) of
1,N6-etheno-dAdo, which suggested that these compounds
were substituted etheno adducts such as those described
by Sodum and Chung (21). The later two eluting com-
pounds (20.3 and 20.8 min) did not fragment readily
under the conditions of the analysis, although the
elimination of water was observed.
Rea ction of d Ad o w ith 13-HP ODE in th e P r es-
en ce of F eII. Small amounts of adducts A1, A2, and B
could be observed in the LC/MS chromatogram after
reaction for only 30 min between 13-HPODE and dAdo
(Figure 4A,C). However, there was a large increase in
the signal intensity for A1, A2, and B when FeII (640 µM)
was included in the incubation mixture. After incubation
for 30 min, the magnitude of the m/z 406 signals (adducts
A1 and A2) exhibited an increase of more than 2 orders
of magnitude (8.4 × 106; Figure 4B) when compared with
the magnitude of the same signals in the absence of FeII
(1.9 × 104; Figure 4A). The signal corresponding to adduct
B in the m/z 388 channel increased almost 20-fold (1.7 ×
105; Figure 4D) when compared to the signal in the
absence of FeII (9.2 × 103; Figure 4C).
Rea ction of d Ad o w ith LOX/Lin oleic Acid -De-
r ived 13-HP ODE. Linoleic acid is converted primarily
to 13-HPODE by soybean LOX (30). When 13-HPODE
was generated enzymatically by soybean LOX, the reac-
tion products observed with dAdo were identical to those
formed in the reaction of synthetic 13-HPODE with dAdo
Rea ction of d Ad o w ith 2,3-Ep oxy-4-h yd r oxyn on a -
n a l. Two major products were observed with retention
times of 20.3 and 20.8 min (data not shown). Their mass

Etheno-dAdo Adduct from Lipid Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 849
F igu r e 6. Analysis of the reaction between dAdo and 4-hy-
droxy-2-nonenal for 36 h at 37 °C by LC/MS3 using gradient
system 2. (A) TIC. (B) SRM chromatogram for adducts A1 and
A2 (m/z 406 f 290 f 272). (C) SRM chromatogram for m/z 406
f 290 f 160. (D) SRM chromatogram for m/z 406 f 290 f
136.
F igu r e 4. Analysis of the reaction between dAdo and 13-
HPODE after 30 min at 37 °C by LC/MS2 using gradient system
2. (A) SRM chromatogram for adducts A1 and A2 (m/z 406 f
290) in the absence of FeII. (B) SRM chromatogram for adducts
A1 and A2 (m/z 406 f 290) in the presence of FeII (640 µM). (C)
SRM chromatogram for adduct B (m/z 388 f 272) in the absence
of FeII. (D) SRM chromatogram for adduct B (m/z 388 f 272)
in the presence of FeII (640 µM).
F igu r e 7. Analysis of the reaction between 4-oxo-2-nonenal
and dAdo for 36 h at 37 °C by LC/MS3 using gradient system 2.
The product ion spectra are shown in the insets. The spectrum
for adduct A1 was identical with that for adduct A2. (A) TIC
chromatogram for adducts A1 and A2 (m/z 406 f 290 f). (B)
TIC chromatogram for adduct B (m/z 388 f 272 f).
F igu r e 5. Analysis of the reaction between dAdo and LOX/
linoleic acid-derived 13-HPODE after 12 h at 37 °C by LC/MS
using gradient system 3. (A) TIC chromatogram. Asterisks
denote signals observed in a control incubation with no dAdo
added. (B) Selected ion chromatogram for adducts A1 and A2
(m/z 406). (C) Selected ion chromatogram for adduct B (m/z 388).
dAdo with 4-oxo-2-nonenal revealed the presence of three
major compounds (Figure 7). Their retention times and
mass spectrometric behavior were identical when com-
pared to those of adducts A1, A2, and B observed in the
reaction of 13-HPODE and dAdo. None of the later
eluting m/z 406 isomers (18.0-21.0 min) observed in the
chromatograms from reactions of dAdo with 4-hydroxy-
2-nonenal or 2,3-epoxy-4-hydroxynonanal were detected.
spectral properties were identical with those of the later
eluting compounds observed at 20.3 and 20.8 min in the
chromatogram from the dAdo/4-hydroxy-2-nonenal reac-
tion (Figure 6).
Rea ction of d Ad o w ith 4-Oxo-2-n on en a l. A tar-
geted CID analysis of the products from the reaction of

850 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Rindgen et al.
Sch em e 1. P r op osed Mech a n ism for th e Rea ction
betw een 4-Oxo-2-n on en a l a n d DAd o
Discu ssion
Previous studies have investigated the covalent modi-
fications that can occur to DNA bases from electrophilic
breakdown products of peroxidized methyl linoleate (31,
32). Characteristic fluorescent adducts were observed
only in reactions with adenine-containing compounds.
Structures for the reactive secondary lipid oxidation
products responsible for adduct formation were inferred
from the adduct structures. Thus, one of the adducts was
suggested to contain a substituted 1,N6-propano adduct
from the reaction between 3-nonenal and adenine. The
adduct structure was only tentatively assigned, and it is
difficult to envisage a reasonable pathway for the genera-
tion of this adduction product.
MDA, a well-studied lipid peroxidation breakdown
product, generates an acyclic N6-oxopropenyl adduct
when reacted with dAdo (17, 18). This adduct is thought
to result from initial 1,4-addition to the â-hydroxyacrolein
form of MDA followed by dehydration. 4-Hydroxy-2-
nonenal, another characteristic lipid breakdown product,
does not appear to react very efficiently with dAdo.
However, the epoxide derivative, 2,3-epoxy-4-hydroxy-
nonanal, has been shown to form substituted and un-
substituted etheno adducts with dAdo (21, 25, 33). The
proposed mechanism for formation of the etheno adducts
involves nucleophilic attack by the exocyclic N6 amino
group of dAdo at the C1 aldehyde of 2,3-epoxy-4-hydroxy-
nonanal followed by addition of N1 to C2 of the epoxide
to generate an intermediate ethano adduct.
We have proposed that 4-oxo-2-nonenal is a previously
unrecognized product of lipid peroxidation (23). Further-
more, using NMR and LC/MS, it was demonstrated that
4-oxo-2-nonenal reacts with dAdo to give two isomeric
substituted ethano adducts (adducts A1 and A2), which
dehydrate to give the etheno adduct B, 1′′-[3-(2′-deoxy-
â-D-erythro-pentafuranosyl)-3H-imidazo[2,1-i]purin-7-yl]-
heptan-2′′-one (24). In the study presented here, we have
compared the products of the reaction between dAdo and
13-HPODE with those derived from synthetic 4-oxo-2-
nonenal. The reaction was conducted at a higher con-
centration of dAdo than the reaction between 13-HPODE
and dAdo, and the concentration of ethanol was lower.
However, the three dAdo adducts were identical, and no
other adducts were detected. This suggested that the 13-
HPODE had decomposed to 4-oxo-2-nonenal and that this
had then reacted with the dAdo to form adducts A1, A2,
and B. Formation of these adducts is thought to arise by
a mechanism similar to that described above for 2,3-
epoxy-4-hydroxynonanal (21). Thus, initial nucleophilic
addition of N6 to the C1 aldehyde of 4-oxo-2-nonenal
occurs. This is followed by reaction of N1 at C2 of the
resulting R,â-unsaturated ketone to generate adducts A1
and A2 as a mixture of diastereomers. Subsequent
dehydration of adducts A1 and A2 results in the formation
of adduct B (Scheme 1).
Three major products (adducts A1, A2, and B) were
detected in the reaction between dAdo and 13-HPODE
after 36 h (Figures 2 and 3). The reaction was then
performed for 30 min using a 2-fold excess of 13-HPODE
and 8% ethanol rather than a 10-fold excess of 13-
HPODE and 32% ethanol. The same three products were
observed, although in much smaller amounts (Figure
4A,C). A dramatic increase in the extent of adduct
formation was observed when the reaction was performed
for 30 min in the presence of FeII (Figure 4B,D). This
indicated that the adducts were formed through a ho-
molytic process (34). The MS fragmentation patterns of
these adducts were not consistent with the direct in-
volvement of either 4-hydroxy-2-nonenal or 2,3-epoxy-4-
hydroxynonanal in their formation. For example, the
product of direct reaction between 4-hydroxy-2-nonenal
and dAdo would generate an MH+ ion at m/z 408. This
ion was not observed in any reaction mixture containing
dAdo and 13-HPODE. The substituted 1,N6-etheno-dAdo
adduct derived from the reaction of 2,3-epoxy-4-hydroxy-
nonanal and dAdo would exhibit an MH+ ion at m/z 406
(21). However, LC/MSn analysis of the MH+ ions at m/z
406 derived from adducts A1 and A2 shown in Figures 2
and 3 did not support this structure. LC/MS2 analysis
revealed that both adduct A1 and adduct A2 formed
intense aglycone product ions at m/z 290. CID analysis
of these aglycone ions showed two major product ions at
m/z 272 (-H2O) and 136 (Ade + H) (Figure 3A). The
aglycone ion (m/z 272) of adduct B generated a product
ion at m/z 174 (-C5H10CO) (Figure 3B). Although these
data were consistent with the addition of a nine-carbon
unit from 13-HPODE to dAdo, the fragmentation path-
ways could not be reconciled with any of the known dAdo
adducts derived from C9 products of lipid peroxidation.
We examined the reaction of 4-hydroxy-2-nonenal
using conditions similar to those of the reaction between
13-HPODE and dAdo except that 7% hexane was used
to dissolve the lipid instead of 32% ethanol. As noted in
the Results, LC/MS analysis of the reaction between
4-hydroxy-2-nonenal and dAdo revealed the absence of
a signal from the direct Michael addition product (m/z
408). However, there was a series of signals arising from
MH+ ions at m/z 406. Adducts of dAdo with a molecular
weight of 405 are most likely derived from a two-electron
oxidation (-2H) of 4-hydroxy-2-nonenal or initial epoxi-
dation (+O) followed by dehydration (-H2O). Using
targeted LC/MS3 analysis (406 f 290 f), the early
eluting peaks (17.2 and 17.6 min) were shown to be
identical with adducts A1 and A2, respectively (Figure
6). The later eluting peaks (20.3 and 20.8 min) were
identical with the products derived from the reaction
between 2,3-epoxy-4-hydroxynonanal and dAdo (Figure

Etheno-dAdo Adduct from Lipid Hydroperoxides
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 851
Sch em e 2. P r op osed Mech a n ism for th e
F or m a tion of 4-Oxo-2-n on en a l fr om 13-HP ODE
the concerted loss of water and a C9 aldehyde (Scheme
2). However, the possibility that 4-oxo-2-nonenal is
formed from 4-hydroxy-2-nonenal as described by Xu and
Sayre or by some alternative pathway cannot be ruled
out (35).
Interestingly, 4-oxo-2-nonenal has been observed in the
oxidation of 3(Z)-nonenal by soybean LOX when hydro-
peroxide concentrations are low (41). Therefore, it is
conceivable that 4-oxo-2-nonenal-derived dAdo adducts
observed in the soybean LOX reaction may have been a
consequence of the hydroperoxide lyase activity that is
also present. The hydroperoxide lyase activity could have
converted 13-HPODE to 3(Z)-nonenal (42). LOX-medi-
ated oxidation of the 3(Z)-nonenal to 4-hydroperoxy-2-
nonenal (43) followed by FeII-mediated generation of the
4-alkoxy radical and a â-scission (similar to that postu-
lated for intermediate III) would have resulted in the
formation of 4-oxo-2-nonenal (41). However, the observa-
tion that 4-oxo-2-nonenal-derived dAdo adducts were also
formed from 13-HPODE in the absence of soybean LOX
suggests that this pathway is not of major importance.
In summary, we have provided further evidence that
4-oxo-2-nonenal is a novel product of lipid peroxidation
and that it can form substituted etheno adducts with
DNA bases. The role of etheno DNA adducts in mutagen-
esis and carcinogenesis is currently under intense study
(8). The ability to quantify 4-oxo-2-nonenal-derived DNA
adducts will help to further our understanding of the role
that this novel bifunctional electrophile plays in lipid
hydroperoxide-mediated carcinogenesis. Therefore, our
current studies are focused on the development of LC/
MS assay methodology for the quantitation of 4-oxo-2-
nonenal-derived etheno-DNA adducts in tissues and
biological fluids.
6). Adduct B was also observed (data not shown). It is
unlikely that adducts A1 and A2 observed in the reaction
between 4-hydroxy-2-nonenal and dAdo are different
from those observed with 4-oxo-2-nonenal because they
have the same retention time, the same parent ion, and
the same product ions. These data suggested that both
2,3-epoxy-4-hydroxynonanal and 4-oxo-2-nonenal were
formed by oxidation of 4-hydroxy-2-nonenal under the
reaction conditions. It is noteworthy that 4-oxo-2-non-
enal-derived adducts were observed when 4-hydroxy-2-
nonenal was reacted with n-butylamine in the presence
of oxygen (35).
There are two possible explanations for the formation
of 4-oxo-2-nonenal from 13-HPODE. The 13-HPODE
could have decomposed to 4-hydroxy-2-nonenal, which
was then oxidized by excess 13-HPODE to 4-oxo-2-
nonenal. Alternatively, the 13-HPODE could have de-
composed directly to 4-oxo-2-nonenal without the inter-
mediate formation of 4-hydroxy-2-nonenal (Scheme 2).
The former possibility was considered less likely for three
reasons. First, Chen and Chung (36) have demonstrated
that 4-hydroxy-2-nonenal is oxidized to 2,3-epoxy-4-
hydroxynonanal rather than 4-oxo-2-nonenal by lipid
hydroperoxides. Second, we were unable to detect any
products of reaction between dAdo and 2,3-epoxy-4-
hydroxynonanal (21, 22) in the reaction between 13-
HPODE and dAdo (Figures 3 and 5). Third, LC/MS
analysis of products from the reaction of 13-HPODE and
dAdo (Figure 3) revealed a different profile when com-
pared to the profile of the products of the reaction
between 4-hydroxy-2-nonenal and dAdo (Figure 6).
Pryor and Porter have proposed a mechanism to
account for the formation of 4-hydroxy-2-nonenal from
13-HPODE (3). Initial FeII-mediated one-electron reduc-
tion of the hydroperoxide to generate alkoxy radical I (37)
is followed by epoxide formation and concomitant addi-
tion of oxygen to give 12,13-epoxy-9-hydroperoxy-10-
octadecenoic acid (II) (38, 39). Another FeII-mediated one-
electron reduction of the hydroperoxide results in the
formation of alkoxy radical intermediate III (39). â-Scis-
sion of this alkoxy radical would lead to the formation of
the known lipid peroxidation end product 4,5-epoxy-2-
decenal (39, 40). Alkoxy radical III was proposed to form
a bis-epoxy carbon-centered radical (intermediate IV)
that rearranges to intermediate V with the concomitant
addition of oxygen (3). Loss of a C9 aldehyde moiety then
provides intermediate VI, which rearranges to 4-hydroxy-
2-nonenal. Hydroperoxide-mediated oxidation of 4-hy-
droxy-2-nonenal would provide 2,3-epoxy-4-hydroxynona-
nal (22) (Scheme 2). As we were unable to detect any
dAdo adducts derived from either 4-hydroxy-2-nonenal
or 2,3-epoxy-4-hydroxynonanal, we propose that 4-oxo-
2-nonenal is formed directly from intermediate V through
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial support from the National Institutes of Health
in the form of an RO1 grant to I.A.B. (CA65878) and an
NRSA fellowship to D.R. (GM19388-02).
Refer en ces
(1) Loidl-Stahlhofen, A., Hannemann, K., and Spiteller, G. (1994)
Generation of R-hydroxyaldehydic compounds in the course of
lipid peroxidation. Biochim. Biophys. Acta 1213, 140-148.
(2) Esterbauer, H., Schaur, R. J ., and Zollner, H. (1991) Chemistry
and biochemistry of 4-hydroxynonenal, malonaldehyde and re-
lated aldehydes. Free Radical Biol. Med. 11, 81-128.
(3) Pryor, W. A., and Porter, N. A. (1990) Suggested mechanisms for
the production of 4-hydroxy-2-nonenal from the autoxidation of
polyunsaturated fatty acids. Free Radical Biol. Med. 8, 541-543.
(4) Horton, A. A., and Fairhurst, S. (1987) Lipid peroxidation and
mechanisms of toxicity. Crit. Rev. Toxicol. 18, 27-79.
(5) Halliwell, B., and Chirico, S. (1993) Lipid peroxidation: its
mechanism, measurement, and significance. Am. J . Clin. Nutr.
57, 715-725.
(6) Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Oxidants,
antioxidants, and the degenerative diseases of aging. Proc. Natl.
Acad. Sci. U.S.A. 90, 7915-7922.
(7) Stadtman, E. R., and Berlett, B. S. (1997) Reactive oxygen-
mediated protein oxidation in aging and disease. Chem. Res.
Toxicol. 10, 485-494.
(8) Burcham, P. C. (1998) Genotoxic lipid peroxidation products:
Their DNA damaging properties and role in formation of endog-
enous DNA adducts. Mutagenesis 13, 287-305.
(9) Basu, A. K., and Marnett, L. J . (1983) Unequivocal demonstration
that malondialdehyde is a mutagen. Carcinogenesis 4, 331-333.
(10) Esterbauer, H. (1982) Aldehydic products of lipid peroxidation.
In Free Radicals, Lipid Peroxidation and Cancer (McBrien, D. C.
H., and Slater, T. F., Eds.) pp 101-128, Academic Press, London.
(11) Dedon, P. C., Plastaras, J . P., Rouzer, C. A., and Marnett, L. J .
(1998) Indirect mutagenesis by oxidative DNA damage: formation
of the pyrimidopurinone adduct of deoxyguanosine by base
propenal. Proc. Natl. Acad. Sci. U.S.A. 95, 11113-11116.

852 Chem. Res. Toxicol., Vol. 13, No. 9, 2000
Rindgen et al.
(12) Hamberg, M., and Samuelsson, B. (1967) On the mechanism of
the biosynthesis of prostaglandins E1 and F1R. J . Biol. Chem. 242,
5336-5343.
(13) Hecker, M., and Ullrich, V. (1989) On the mechanism of prosta-
cyclin and thromboxane A2 biosynthesis. J . Biol. Chem. 264, 141-
150.
(14) Marnett, L. J ., Basu, A. K., O’Hara, S. M., Weller, P. E., Rahman,
A. F. M. M., and Oliver, J . P. (1986) Reaction of malondialdehyde
with guanine nucleosides: formation of adducts containing oxa-
diazabicyclononene residues in the base-pairing region. J . Am.
Chem. Soc. 108, 1348-1350.
(15) Chaudhary, A. K., Nokubo, M., Marnett, L. J ., and Blair, I. A.
(1994) Analysis of malondialdehyde-2′-deoxyguanosine adduct in
rat liver DNA by gas chromatography/electron capture negative
chemical ionization mass spectrometry. Biol. Mass Spectrom. 23,
457-464.
(16) Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow,
J . D., Blair, I. A., and Marnett, L. J . (1994) Detection of
endogenous malondialdehyde-deoxyguanosine adducts in human
liver. Science 265, 1580-1582.
(17) Stone, K., Ksebati, M. B., and Marnett, L. J . (1990) Investigation
of the adducts formed by reaction of malondialdehyde with
adenosine. Chem. Res. Toxicol. 3, 33-38.
(18) Chaudhary, A. K., Reddy, R. G., Blair, I. A., and Marnett, L. J .
(1996) Characterization of an N6-oxopropenyl-2′-deoxyadenosine
adduct in malondialdehyde-modified DNA using liquid chroma-
tography/electrospray ionization tandem mass spectrometry. Car-
cinogenesis 17, 1167-1170.
(19) Winter, C. K., Segall, H. J ., and Haddon, W. F. (1986) Formation
of cyclic adducts of deoxyguanosine with the aldehydes trans-4-
hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro. Cancer
Res. 46, 5682-5686.
(20) Yi, P., Zhan, D. J ., Samokyszyn, V. M., Doerge, D. R., and Fu, P.
P. (1997) Synthesis and 32P-postlabeling/high-performance liquid
chromatography separation of diastereomeric 1,N2-(1,3-propano)-
2′-deoxyguanosine 3′-phosphate adducts formed from 4-hydroxy-
2-nonenal. Chem. Res. Toxicol. 10, 1259-1265.
(21) Sodum, R. S., and Chung, F.-L. (1991) Stereoselective formation
of in vitro nucleic acid adducts by 2,3-epoxy-4-hydroxynonanal.
Cancer Res. 51, 137-143.
(22) Chen, H.-J . C., and Chung, F.-Y. (1996) Epoxidation of trans-4-
hydroxy-2-nonenal by fatty acid hydroperoxides and hydrogen
peroxide. Chem. Res. Toxicol. 9, 306-312.
(23) Rindgen, D., Nakajima, M., Wehrli, S., Xu, K., and Blair, I. A.
(1999) Covalent modifications to 2′-deoxyguanosine by 4-oxo-2-
nonenal a novel product of lipid peroxidation. Chem. Res. Toxicol.
12, 1195-1204.
(24) Lee, S. H., Rindgen, D., Bible, R. A., Hajdu, E., and Blair, I. A.
(2000) Characterization of 2′-deoxyadenosine adducts derived
from 4-oxo-2-nonenal, a novel product of lipid peroxidation. Chem.
Res. Toxicol. 13, 565-574.
(25) Chung, F.-L., Chen, H.-J . C., and Nath, R. G. (1996) Lipid
peroxidation as a potential endogenous source for the formation
of exocyclic DNA adducts. Carcinogenesis 17, 2105-2111.
(26) Yeola, S. N., Chaudhary, A. K., Marnett, L. J ., and Blair, I. A.
(1994) Preparation of isotope labeled [4,5,6,8-13C4; 9-15N1] guanine
for the analysis of endogenous DNA products. Proc. 42nd ASMS
Conf. Mass Spectrom. All. Topics, p 367.
(28) Esterbauer, H., and Weger, W. (1967) U¨ ber die wirkungen von
aldehyden auf gesunde und maligne zellen, 3. mitt.: synthese von
homologen 4-hydroxy-2-alkenalen, II. Monatsh. Chem. 98, 1994-
2000.
(29) Chaudhary, A. K., Nokubo, M., Oglesby, T. D., Marnett, L. J .,
and Blair, I. A. (1995) Characterization of endogenous DNA
adducts by liquid chromatography/electrospray ionization tandem
mass spectrometry. J . Mass Spectrom. 30, 1157-1166.
(30) Verhagen, J ., Veldink, G. A., Egmond, M. R., Vliegenthart, J . F.
G., Boldingh, J ., and Van Der Star, J . (1978) Steady-state kinetics
of the anaerobic reaction of soybean lipoxygenase-1 with linoleic
acid and 13-L-hydroperoxylinoleic acid. Biochim. Biophys. Acta
529, 369-379.
(31) Hasegawa, K., Fujimoto, K., Kaneda, T., Neff, W. E., and Frankel,
E. N. (1989) Formation of fluorescent products in the reaction of
adenine and other bases with secondary oxidation products of
methyl linoleate hydroperoxides. Agric. Biol. Chem. 53, 1575-
1581.
(32) Hasegawa, K., Fujimoto, K., Kaneda, T., and Frankel, E. N. (1988)
Characterization of fluorescent products from reaction of methyl
linoleate hydroperoxides with adenine in the presence of Fe2+ and
ascorbic acid. Biochim. Biophys. Acta 962, 371-376.
(33) Chen, H.-J . C., and Chung, F.-L. (1994) Formation of etheno
adducts in reactions of enals via autoxidation. Chem. Res. Toxicol.
7, 857-860.
(34) Spiteller, P., and Spiteller, G. (1998) Strong dependence of the
lipid peroxidation product spectrum whether Fe2+/O2 or Fe3+/O2
is used as oxidant. Biochim. Biophys. Acta 1392, 23-40.
(35) Xu, G., and Sayre, L. M. (1998) Structural characterization of a
4-hydroxy-2-alkenal-derived fluorophore that contributes to li-
poperoxidation-dependent protein cross-linking in aging and
degenerative disease. Chem. Res. Toxicol. 11, 247-251.
(36) Chen, H.-J . C., and Chung, F.-L. (1996) Epoxidation of trans-4-
hydroxy-2-nonenal by fatty acid hydroperoxides and hydrogen
peroxide. Chem. Res. Toxicol. 9, 306-312.
(37) Rota, C., Barr, D. P., Martin, M. V., Guengerich, F. P., Tomasi,
A., and Mason, R. P. (1997) Detection of free radicals produced
from the reaction of cytochrome P-450 with linoleic acid hydro-
peroxide. Biochem. J . 328, 565-571.
(38) Reeder, B. J ., and Wilson, M. T. (1998) Mechanism of reaction of
myoglobin with the lipid hydroperoxide hydroperoxyoctadecadi-
enoic acid. Biochem. J . 330, 1317-1323.
(39) Iwahashi, H. (2000) Some polyphenols inhibit the formation of
pentyl radical and octanoic acid radical in the reaction mixture
of linoleic acid hydroperoxide with ferrous irons. Biochem. J . 346,
265-273.
(40) Zamora, R., Alaiz, M., and Hidalgo, F. J . (1999) Modification of
histidine residues by 4,5-epoxy-2-alkenals. Chem. Res. Toxicol.
12, 654-660.
(41) Gardner, H. W., and Grove, M. J . (1998) Soybean lipoxygenase-1
oxidizes 3Z-nonenal. A route to 4S-hydroperoxy-2E-nonenal and
related products. Plant Physiol. 116, 1359-1366.
(42) Gardner, H. W. (1999) Biosynthesis of hydroxyalkenals by plants.
Recent Res. Dev. Lipids Res. 3, 15-21.
(43) Gardner, H. W., and Hamberg, M. (1993) Oxygenation of (3Z)-
Nonenal to (2E)-4-hydroxy-2-nonenal in the broad bean (Vicia
faba L.). J . Biol. Chem. 268, 6971-6977.
(27) Chapeau, M. C., and Marnett, L. J . (1991) Enzymatic synthesis
of purine deoxynucleoside adducts. Chem. Res. Toxicol. 4, 636-
638.
TX0000771