4,5-Epoxy-2(E)-decenal-induced formation of 1,N6-etheno-2′-deoxyadenosine and 1,N2-etheno-2′-deoxyguanosine adducts

Article abstract of DOI:10.1021/tx010147j

Trans-4,5-Epoxy-2(E)-decenal reacted with 2′-deoxyadenosine to give 1,N⁶-etheno-2′-deoxyadenosine as well as other 2′-deoxyadenosine adducts. It also reacted with 2′-deoxyguanosine to give 1,N²-etheno-2′-deoxyguanosine and other 2′-deoxyguanosine adducts. Synthetic trans-4,5-epoxy-2(E)-decenal was quite stable under the reaction conditions that were used. It was not contaminated with 2,3-epoxyoctanal, a potential precursor to the formation of unsubstituted etheno adducts. Furthermore, using a sensitive LC/MS assay, it was possible to show that no 2,3-epoxyoctanal was formed during prolonged incubations of trans-4,5-epoxy-2(E)-decenal. Therefore, trans-4,5-epoxy-2(E)-decenal, a primary product of lipid peroxidation, is a precursor to the formation of 1,N⁶-etheno-2′-deoxyadenosine and 1,N²-etheno-2′-deoxyguanosine. There is no need for an additional oxidation step such as would be required if trans,trans-2,4-decadienal or 4-hydroxy-2-nonenal were the lipid hydroperoxide decomposition products that initiated the formation of unsubstituted etheno adducts. These findings provide an important link between a primary product of lipid peroxidation and a mutagenic DNA lesion that has been detected in human tissues.

Full text of DOI:10.1021/tx010147j

300
Chem. Res. Toxicol. 2002, 15, 300-304
Communications
4,5-Ep oxy-2(E)-d ecen a l-In d u ced F or m a tion of
1,N6-Eth en o-2′-d eoxya d en osin e a n d
1,N2-Eth en o-2′-d eoxygu a n osin e Ad d u cts
Seon Hwa Lee, Tomoyuki Oe, and Ian A. Blair*
Center for Cancer Pharmacology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104-6160
Received September 6, 2001
trans-4,5-Epoxy-2(E)-decenal reacted with 2′-deoxyadenosine to give 1,N6-etheno-2′-deoxy-
adenosine as well as other 2′-deoxyadenosine adducts. It also reacted with 2′-deoxyguanosine
to give 1,N2-etheno-2′-deoxyguanosine and other 2′-deoxyguanosine adducts. Synthetic trans-
4,5-epoxy-2(E)-decenal was quite stable under the reaction conditions that were used. It was
not contaminated with 2,3-epoxyoctanal, a potential precursor to the formation of unsubstituted
etheno adducts. Furthermore, using a sensitive LC/MS assay, it was possible to show that no
2,3-epoxyoctanal was formed during prolonged incubations of trans-4,5-epoxy-2(E)-decenal.
Therefore, trans-4,5-epoxy-2(E)-decenal, a primary product of lipid peroxidation, is a precursor
to the formation of 1,N6-etheno-2′-deoxyadenosine and 1,N2-etheno-2′-deoxyguanosine. There
is no need for an additional oxidation step such as would be required if trans,trans-2,4-
decadienal or 4-hydroxy-2-nonenal were the lipid hydroperoxide decomposition products that
initiated the formation of unsubstituted etheno adducts. These findings provide an important
link between a primary product of lipid peroxidation and a mutagenic DNA lesion that has
been detected in human tissues.
In tr od u ction
Lipid peroxidation of polyunsaturated fatty acids
(PUFAs)1 is thought to play an important role the
degenerative diseases of aging such as cancer (1, 2). The
formation of lipid hydroperoxides from PUFAs by free
radical processes is a complex process, which leads to a
number of different regioisomers and stereoisomers (3).
LOX-mediated oxidation of PUFAs results in the forma-
tion of lipid hydroperoxides with much greater stereo-
selectivity (4). Lipid hydroperoxides undergo homolytic
decomposition to bifunctional electrophiles (5) that can
F igu r e 1. Formation of 4-oxo-2-nonenal (4-ONE), 4-hydrop-
eroxy-2-nonenal (4-HPNE), 4-hydroxy-2-nonenal (4-HNE), and
4,5-epoxy-2(E)-decenal (4,5-EDE) from 13-HPODE.
react with DNA (2). In previous studies, we examined
the homolytic decomposition of 13-[S-(Z,E)]-9,11-hydro-
peroxyoctadecadienoic acid (13-HPODE; a prototypic ω-6
PUFA lipid hydroperoxide) in the presence of the DNA
bases 2′-deoxyadenosine (dAdo) and 2′-deoxyguanosine
(dGuo). From structures of the resulting DNA adducts,
we proposed that the major covalent modifications arose
through generation of 4-oxo-2-nonenal from 13-HPODE.
The same adducts were formed when DNA bases were
treated with synthetic 4-oxo-2-nonenal (6, 7).
Subsequently, 4-oxo-2-nonenal was confirmed as a
major breakdown product of homolytic lipid hydroper-
oxide decomposition (Figure 1) (8), a finding that was
recently confirmed by Spiteller et al. (9). Surprisingly,
4-hydroperoxy-2-nonenal was also characterized as a
product of 13-HPODE decomposition by our laboratory
and by Schneider et al. (8, 10). 4-Hydroperoxy-2-nonenal
was subsequently shown to be a precursor in the forma-
tion of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal (11)
(Figure 1). The other major bifunctional electrophile iden-
tified in homolytic 13-HPODE decomposition was 4,5-
epoxy-2(E)-decenal (11) (Figure 1), a recently identified
product from the autoxidation of arachidonic acid (12).
trans,trans-2,4-Decadienal is an environmental con-
taminant in water and food (see ref 13 for discussion). It
is also a product of lipid peroxidation through R-cleavage
* Correspondence should be addressed to this author at the 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.
1Abbreviations: APCI, atmospheric pressure chemical ionization;
brs, broad singlet; CID, collision-induced dissociation; dAdo, 2′-
deoxyadenosine; d, doublet; dd, doublet of doublets; dGuo, 2′-deox-
yguanosine; Gua, guanine; 4-HNE, 4-hydroxy-2-nonenal; 4-HPNE,
4-hydroperoxy-2-nonenal; 13-HPODE, 13-[S-(Z,E)]-9,11-hydroperoxy-
octadecadienoic acid; LC/MS, liquid chromatography/mass spectrom-
etry; LOX, lipoxygenase; MH+, protonated molecular ion; MSn, multiple
tandem mass spectrometry; 4-ONE, 4-oxo-2-nonenal; PUFA, polyun-
saturated fatty acid; s, singlet; td, triplet of doublets; SIM, selected
ion monitoring; TIC, total ion current.
10.1021/tx010147j CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/12/2002

Communications
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 301
roacetic acid, and solvent B was 5 mM ammonium acetate in
methanol containing 0.01% (v/v) trifluoroacetic acid. The linear
gradient was as follows: 30% B at 0 min, 30% B at 3 min, 55%
B at 13 min, 55% B at 16 min, 70% B at 24 min, 100% B at 26
min, 100% B at 30 min with a flow rate of 1.0 mL/min. For
system 7, solvent A was 5 mM ammonium acetate in water,
and solvent B was 5 mM ammonium acetate in acetonitrile. The
linear gradient was as follows: 6% B at 0 min, 6% B at 3 min,
20% B at 9 min, 20% B at 13 min, 60% B at 21 min, 80% B at
22 min, 80% B at 24 min with a flow rate of 1.0 mL/min. All
separations were performed at ambient temperature.
1H NMR. Spectra (500 MHz) were determined at 25 °C using
a Varian UNITY 500 instrument. Samples were dissolved in
750 µL of CDCl3 [trans-4,5-epoxy-2(E)-decenal] or 750 µL of
DMSO-d6 (1,N2-etheno-Gua and N2,3-etheno-Gua) and intro-
duced into a Kontes (Vineland, NJ ) NMR tube (200 mm × 5
mm o.d.). The CHCl3 (7.25 ppm) or DMSO (2.50 ppm) residual
signal was used as the reference signal. Coupling constants are
shown as hertz values. Acquisition conditions were as follows:
spectral width of 6000 Hz [trans-4,5-epoxy-2(E)-decenal] or 7000
Hz (1,N2-etheno-Gua and N2,3-etheno-Gua), 30° pulse flip angle,
32K data points, and 32 transients.
of the alkoxy radicals derived from 9-hydroperoxy-(E,E)-
10,12-octadecadienoic acid or 11-hydroperoxy-(Z,Z,E,E)-
eicosa-5,8,12,14-tetraenoic acid (9, 12). Recent studies
have shown that the reaction of peroxide-treated
trans,trans-2,4-decadienal with dAdo or dGuo results in
the formation of 1,N6-etheno-dAdo (13) and 1,N2-etheno-
dGuo (14), respectively. We reasoned that 4,5-epoxy-2(E)-
decenal could have been formed when trans,trans-2,4-
decadienal was treated with peroxides (12, 15) and that
this bifunctional electrophile was in fact the precursor
to the formation of etheno adducts from lipid hydroper-
oxides.
Ma ter ia ls a n d Meth od s
Ma t er ia ls. Ammonium acetate, trifluoroacetic acid, dAdo,
dGuo, 1,N6-etheno-dAdo, O6-benzylguanine, chloroacetaldehyde,
(triphenylphosphoranylidene)acetaldehyde, deuterated NMR
solvents, and hydrogen peroxide were purchased from Sigma-
Aldrich. (St. Louis, MO). 3-Morpholinopropanesulfonic acid
(MOPS) was obtained from Fluka BioChemika (Milwaukee, WI).
Chelex-100 chelating ion-exchange resin (100-200 mesh size)
was purchased from Bio-Rad Laboratories (Hercules, CA). HPLC
grade water, methanol, acetonitrile, hexane, and 2-propanol
were obtained from Fisher Scientific Co. (Fair Lawn, NJ ). Gases
were supplied by BOC Gases (Lebanon, NJ ).
Ma ss Sp ectr om etr y. Mass spectrometry was conducted with
a Thermo Finnigan LCQ ion trap mass spectrometer (Thermo
Finnigan, San J ose, CA) equipped with an APCI source. The
mass spectrometer was operated in the positive ion mode with
a discharge current of 5 µA applied to the corona needle.
Nitrogen was used as the sheath (80 units) and auxiliary (10
units) gas to assist with nebulization. The vaporizer and heated
capillary temperatures were set at 450 and 150 °C, respectively.
Full scanning analyses were performed in the range of m/z 50
to m/z 800. Collision-induced dissociation (CID) experiments
coupled with multiple tandem mass spectrometry (MSn) em-
ployed helium as the collision gas. The relative collision energy
was set at 20% of maximum (1 V).
Liqu id Ch r om a togr a p h y. Analysis and purification of
synthetic DNA adducts was conducted on a Hitachi L7100
module (Hitachi, San J ose, CA) equipped with a Waters 996
photodiode array detector. System 1 employed a Beckman
Ultrasphere-Si column (250 × 10 mm i.d., 5 µm; Alltech,
Deerfield, IL). Systems 2 and 3 employed a YMC basic column
(150 × 2.0 mm i.d., 5 µm); system 4 employed a YMC-Pack ODS-
AQ column (250 × 10 mm i.d., 5 µm). For normal phase system
1, solvent A was hexane/2-propanol (197:3, v/v), and solvent B
was hexane/2-propanol (7:3, v/v). The linear gradient for system
1 was as follows: 1% B at 0 min, 2% B at 18 min, 100% B at 20
min, 100% B at 24 min with a flow rate of 2 mL/min.
For systems 2 and 3, solvent A was 5 mM aqueous ammonium
acetate containing 0.01% (v/v) trifluoroacetic acid, and solvent
B was 5 mM ammonium acetate containing 0.01% (v/v) trifluo-
roacetic acid in methanol. The linear gradient for system 2 was
as follows: 1% B at 0 min, 91% B at 18 min, 91% B at 20 min
with a flow rate of 0.25 mL/min. The linear gradient for system
3 was as follows: 10% B at 0 min, 100% B at 18 min, 100% B
at 20 min with a flow rate of 0.25 mL/min. System 4 employed
an isocratic mobile phase of methanol/water (1:1, v/v; 2 mL/min).
All separations were performed at ambient temperature.
Liqu id Ch r om a togr a p h y/Ma ss Sp ectr om etr y. Chroma-
tography for LC/MS studies was performed using a Waters
Alliance 2690 HPLC system (Waters Corp., Milford, MA).
System 5 employed two silica columns in series (250 × 4.6 mm
i.d., 5 µm; Regis, Morton Grove, IL). Systems 6 and 7 employed
a YMC C18 ODS-AQ column (250 × 4.6 mm i.d., 5 µm; Waters,
Milford, MA). System 5 used normal phase solvents. Solvent A
was hexane/2-propanol (197:3, v/v), and solvent B was hexane/
2-propanol (7:3, v/v). Isocratic elution was performed with 50%
B at a flow rate of 0.8 mL/min. For system 6, solvent A was 5
mM aqueous ammonium acetate containing 0.01% (v/v) trifluo-
Syn t h esis of tr a n s-4,5-E p oxy-2(E)-d ecen a l. trans-2-
Octenal was subjected to epoxidation with hydrogen peroxide
under alkaline conditions as described by Lin et al. (16). The
crude trans-2,3-epoxyoctanal was recrystallized from ethyl ace-
tate to remove 5% of contamination by cis-2,3-epoxyoctanal, and
then subjected to a Wittig reaction with (triphenylphosphor-
anylidene)acetaldehyde. After silica gel column chromatography
with hexane/diethyl ether/triethylamine (350:150:0.2; v/v/v), the
crude trans-4,5-epoxy-2(E)-decenal was isolated and purified by
distillation under vacuum (60 °C, 2 mmHg) as a colorless oil
(UV λmax ) 229 nm; hexane/2-propanol) with a purity of >98%
as determined by HPLC analysis using system 1 [tR ) 13.5 min;
1
cis-4,5-epoxy-2(E)-decenal < 0.1%]. The H NMR (CDCl3) spec-
trum was identical with that reported by Lin et al. (16): δ 0.90
[3H, t, C(10)-H3], 1.33 [4H, m, C(8)-H2 and C(9)-H2], 1.47 [2H,
m, C(7)-H2], 1.63 [2H, m, C(6)-H2], 2.96 [1H, td, J 56 ) 5.5 Hz,
J 45 ) 2 Hz, C(5)-H], 3.32 [1H, dd, J 34 ) 7 Hz, J 45 ) 2 Hz, C(4)-
H], 6.54 [1H, dd, J 23 ) 16 Hz, J 34 ) 7 Hz, C(3)-H], 6.38 [1H,
dd, J 23 ) 16 Hz, J 12 ) 7.5 Hz, C(2)-H], 9.56 [1H, d, J 12 ) 7.5
Hz, C(1)HO]. APCI/MS: m/z 169 (MH+), m/z 151 (MH+-H2O).
APCI/MS/MS of m/z 169 (MH+): m/z 151 [MH-H2O]+, m/z 133
[MH-2H2O]+, m/z 123 [MH-H2O-CO]+, m/z 113 [MH-C4H8]+,
m/z 109 [MH-H2O-C3H6]+, m/z 99 [MH-C5H10]+, m/z 95 [MH-
H2O-C4H8]+, m/z 85 [MH-CO-C4H8]+, m/z 81 [MH-H2O-
CO-C3H6]+, m/z 67, [MH-H2O-CO-C4H8]+.
Syn th esis of 1,N2-Eth en ogu a n in e (Gu a ). 1,N2-Etheno-Gua
was prepared by a modification of the method of Sattsangi et
al. (17) from the reaction of dGuo with chloroacetaldehyde at
37 °C at pH 6.4 for 5 days in water. After purification by
reversed-phase HPLC using system 2 (tR ) 12 min) followed by
system 4 (tR ) 8 min), 1,N2-etheno-dGuo was obtained in poor
yield (5%) as a white solid (UV λmax ) 226, 285 nm in methanol/
water containing 0.1% trifluoroacetic acid) with >97% purity
by HPLC system 2 (tR ) 12 min). APCI/MS: m/z 292 (MH+),
m/z 176 (BH2+). MS2 (m/z 292 f): m/z 176 [BH2]+. MS3 (m/z
292 f m/z 176 f): m/z 148 [MH-CO]+, m/z 121 [MH-CO-
CNH]+. 1,N2-Etheno-dGuo was depurinated by dissolving in
aqueous 1 N HCl and allowing to stand for 4 h at 60 °C. The
aqueous HCl was evaporated under nitrogen and the resulting
solid dried under vacuum at 60 °C. 1,N2-Etheno-Gua was
obtained in essentially quantitative yield from 1,N2-etheno-dGuo
as a white amorphous solid (UV λmax ) 221, 268, 295 nm; 0.1 N
HCl) with >97% purity as determined by reversed-phase HPLC
1
analysis on system 2 (tR ) 10 min). The H NMR spectrum was
essentially identical with that we reported previously for [13C3]-
etheno-Gua (18) (DMSO-d6): δ 7.38 (1H, d, J olefinic ) 2.5 Hz,
etheno), 7.56 (1H, d, J olefinic ) 2.5 Hz, etheno), 7.82 [1H, s, C(8)-
H]. The assignment of C-8 as δ 9.37 by Sattsangi et al. (17)
appears to be incorrect. APCI/MS: m/z 176 (MH+). MS2 (m/z
176 f): m/z 148 [MH-CO]+, m/z 121 [MH-CO-CNH]+.

302 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Communications
Syn th esis of N2,3-Eth en o-Gu a . N2,3-Ethenoguanine was
also prepared by a modification of the method of Sattsangi et
al. (17). O6-Benzylguanine was reacted with chloroacetaldehyde
in 75% aqueous ethanol at 37 °C for 5 days at pH 3.5. The
solvent was evaporated under vacuum, and the residue was
crystallized from ethanol/water (1:1, v/v). After drying under
vacuum, O6-benzyl-N2,3-etheno-Gua was isolated as a white
solid with >98% purity (system 4, tR ) 17 min) in 80% yield.
The benzyl moiety was removed by heating at 100 °C in aqueous
1 N HCl for 4 h. The aqueous solution was neutralized with
aqueous sodium hydroxide and washed with ether, the water
was evaporated, and the resulting white solid was dried under
vacuum. After crystallization from ethanol/water, N2,3-etheno-
Gua was obtained in quantitative yield as a white amorphous
powder with >99% purity (UV λmax ) 215, 256 nm; 0.1 M HCl)
on HPLC system 3 (tR ) 6 min). The 1H NMR (DMSO-d6)
spectrum was similar to that reported previously (17): δ 7.09
(1H, d, J olefinic ) 2 Hz, etheno), 7.60 (1H, d, J olefinic ) 2 Hz,
etheno), 8.14 [1H, s, C(8)-H], 12.3 (1H, brs, NH or OH), 13.8
(1H, brs, NH or OH). However, reported chemical shifts for the
etheno protons were at lower field (δ 7.61 and 8.02) than these
values, although the coupling constants were identical (2 Hz).
APCI/MS: m/z 176 (MH+). MS2: no product ions were detected.
LC/MS An a lysis of Ep oxid es. LC/APCI/selected ion moni-
toring (SIM)/MS analysis of the two epoxides employed MH+
for 2,3-epoxyoctanal and trans-4,5-epoxy-2(E)-decenal at m/z 143
and m/z 169, respectively. 2,3-Epoxyoctanal and trans-4,5-epoxy-
2(E)-decenal eluted with retention times of 8.34 and 9.04 min,
respectively, on HPLC system 5. Under these conditions, there
was a clear separation of the two epoxides with no interfering
signals at the retention times of either analyte.
Qu a n tita tion of 2,3-Ep oxyocta n a l in Syn th etic tr a n s-
4,5-Ep oxy-2(E)-d ecen a l. Calibration standards containing
known amounts of 2,3-epoxyoctanal (0.02, 0.10, 0.20, 1, 2, 10,
and 20 µg) were prepared as an ether solution (100 µL)
containing trans-4,5-epoxy-2(E)-decenal (20 µg). A portion of the
standard solution (10 µL) was subjected to LC/APCI/SIM/MS
analysis using normal phase system 5 by monitoring m/z 143
(MH+) for 2,3-epoxyoctanal and m/z 169 (MH+) for trans-4,5-
epoxy-2(E)-decenal. A typical regression line was y ) 6E+06x
+ 194 385, r2 ) 0.9995. The amount of 2,3-epoxyoctanal in the
authentic trans-4,5-epoxy-2(E)-decenal (20 µg, 2 µg on column)
was then determined. The 2,3-epoxyoctanal was below the
detection limit of the assay (<2 ng on column, <0.1%).
LC/MS An a lysis of tr a n s-4,5-Ep oxy-2(E)-d ecen a l u p on
P r olon ged Hea tin g. A solution of trans-4,5-epoxy-2(E)-decenal
(1680 µg, 10 µmol) in ethanol (16.8 µL) was added to Chelex-
treated 100 mM MOPS containing 150 mM NaCl (pH 7.4, 233.2
µL). The mixture was incubated for 24, 48, or 72 h with
continuous shaking at 37 °C after sonication for 15 min at room
temperature. At the end of the incubation, the sample was
cooled in an ice bath and extracted with diethyl ether (250 µL).
Samples were filtered through a 0.2 µm Costar cartridge prior
to analysis of a portion of the sample (10 µL, 67.2 µg on column)
by LC/APCI//MS using normal phase system 5 as described
above.
F igu r e 2. Analysis of 1,N6-etheno-dAdo from the reaction of
trans-4,5-epoxy-2(E)-decenal with dAdo. (A) LC/MS chromato-
grams showing the total ion current chromatogram (TIC; upper),
the ion chromatogram for MH+ (m/z 276; middle), and the SRM
chromatogram for MH+ (m/z 276) f BH2+ (m/z 160; lower). (B)
Product ion spectrum of MH+ (m/z 276).
48, or 72 h at pH 7.4 was below the limit of detection
using LC/MS system 5. This corresponded to 2 ng on
column or <0.003% conversion to 2,3-epoxyoctanal. The
LC/MS response for trans-4,5-epoxy-2(E)-decenal at each
time point was identical with that observed before the
sample was placed in the incubator.
LC/MS An a lysis of Au th en tic 1,N6-Eth en o-d Ad o.
The APCI/MS spectrum of 1,N6-etheno-dAdo showed an
intense protonated molecular ion (MH+) at m/z 276
together with a fragment ion at m/z 160 corresponding
to the loss of 2′-deoxyribose with the addition of hydrogen
atom (BH2+). MS2 analysis of m/z 276 resulted in the
+
exclusive formation of the BH2 product ion at m/z 160.
No product ion was observed in the MS3 analysis (m/z
276f m/z 160f) up to a collision energy of 3V.
LC/MS An a lysis of Au th en tic 1,N2-Eth en o-Gu a
a n d N2,3-Eth en o-Gu a . The APCI/MS spectra of 1,N2-
etheno-Gua and N2,3-etheno-Gua were identical. Each
spectrum exhibited an intense MH+ ion at m/z 176. MS2
analysis of 1,N2-etheno-Gua (m/z 176f) gave rise to
product ions at m/z 148 (MH+-CO) and m/z 121 (MH+-
CO-CNH). For N2,3-etheno-Gua, no product ions were
observed when MS2 analysis of m/z 176 was conducted
at collision energies below 3 V. At higher collision
energies, the product ion yield was very low, and spectra
were lost in background noise.
Rea ction of tr a n s-4,5-Ep oxy-2(E)-d ecen a l w ith DNA
Ba ses. A solution of trans-4,5-epoxy-2(E)-decenal (1680 µg, 10
µmol) in ethanol (16.8 µL) was added to dAdo (251 µg, 1.0 µmol)
or dGuo (267 µg, 1.0 µmol) in Chelex-treated 100 mM MOPS
containing 150 mM NaCl (pH 7.4, 233.2 µL). The reaction
mixture was sonicated for 15 min at room temperature, incu-
bated at 37 °C for 48 h, and then placed on ice. Each sample
was filtered through a 0.2 µm Costar cartridge prior to analysis
of a portion of the sample (20 µL) by LC/MS using gradient
system 6 (for the dAdo reaction mixture) or system 7 (for the
dGuo reaction mixture).
An a lysis of Rea ction betw een tr a n s-4,5-Ep oxy-
2(E)-d ecen a l a n d d Ad o. LC/APCI/MS analysis of the
reaction mixture after 48 h using gradient system 6
revealed the presence of six dAdo adducts together with
residual dAdo. LC/MS characteristics (Figure 2A) of the
earliest eluting adduct were identical to authentic 1,N6-
etheno-dAdo. Its mass spectrum exhibited an intense
MH+ at m/z 276, together with a BH2 ion at m/z 160.
+
MS2 analysis of MH+ (m/z 276) gave rise to exclusive
formation of the BH2+ product ion at m/z 160 (Figure 2B).
The later eluting dAdo adducts had LC/MS properties
that were similar to those reported by Carvalho et al.
(13).
Resu lts
LC/MS An a lysis of tr a n s-4,5-Ep oxy-2(E)-d ecen a l
on P r olon ged Hea tin g. The amount of 2,3-epoxyoctanal
in the trans-4,5-epoxy-2(E)-decenal after heating for 24,

Communications
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 303
F igu r e 4. Formation of unsubstituted etheno adducts from
trans-4,5-epoxy-2(E)-decenal.
Discu ssion
1,N6-Etheno-dAdo has been detected in the liver of rats
treated with vinyl chloride (19). Its persistence in rat liver
suggested that it may also be an endogenous DNA
adduct. 1,N6-Etheno-dAdo was formed in vitro from
peroxide-treated 4-hydroxy-2-nonenal (20) and peroxide-
treated trans,trans-2,4-decadienal (13). A highly specific
LC/MS assay for 1,N6-etheno-dAdo was able to detect the
adduct in human tissues (21). 1,N6-Etheno-dAdo is highly
mutagenic in mammalian cells and much more mu-
tagenic than lesions that arise from oxidative damage
such as 7,8-dihydro-8-oxo-dGuo (22). This most likely
stems from the ability of atypical mammalian DNA
polymerases to perform translesional synthesis, which
results in A to T transversions (23).
N2,3-Etheno-Gua was also found in the liver DNA of
rats treated with vinyl chloride (24). 1,N2-Etheno-Gua
was identified in chloroacetaldehyde-treated DNA (25),
and both N2,3-etheno-Gua and 1,N2-etheno-Gua were
isolated from 2-halooxirane-treated DNA (26). 1,N2-
Etheno-dGuo was isolated from the reaction of dGuo with
peroxide-treated 4-hydroxy-2-nonenal (20) and peroxide-
treated trans,trans-2,4-decadienal (14). It was shown to
be mutagenic in mammalian cells (27), although the
mutation profile was much more complex than for 1,N6-
etheno-dAdo (22).
In vitro studies with DNA bases have demonstrated
that peroxide treatment of 4-hydroxy-2-nonenal and
trans,trans-2,4-decadienal results in the formation of
etheno DNA adducts (13, 14, 20). However, it is not clear
that such reactions could occur in vivo because of the
competition between detoxication by glutathione-S-trans-
ferases and aldo-keto reductases (28) and activation by
peroxidation. The reaction of dAdo and dGuo with trans-
4,5-epoxy-2(E)-decenal resulted in the formation of un-
substituted etheno adducts. The structure of 1,N6-etheno-
dAdo was confirmed by LC/MS analysis (Figure 2A) and
by MSn analyses (Figure 2B). The structure of 1,N2-
etheno-dGuo was established by hydrolysis to the corre-
sponding Gua adduct and comparison with authentic
etheno-Gua isomers. N2,3-Etheno-Gua and 1,N2-etheno-
Gua were readily separated by LC/MS (Figure 3A). They
were also distinguished by the ease with which 1,N2-
etheno-Gua underwent CID compared with N2,3-etheno-
Gua. At normal collision energies, N2,3-etheno-Gua gave
F igu r e 3. LC/MS analysis of 1,N2-etheno-Gua and N2,3-etheno-
Gua. (A) Authentic standards showing the TIC (upper), ion
chromatograms for MH+ (m/z 176; middle), and TIC for MS/
MS analysis of MH+ (m/z 176 f; lower). (B) Etheno-Gua from
the reaction of trans-4,5-epoxy-2(E)-decenal with dGuo showing
the TIC (upper), the ion chromatogram for MH+ (m/z 176;
middle), and TIC for MS/MS analysis of MH+ (m/z 176 f; lower).
(C) Product ion spectrum of MH+ (m/z 176) for etheno-Gua from
reaction of trans-4,5-epoxy-2(E)-decenal with dGuo.
An a lysis of Rea ction betw een tr a n s-4,5-Ep oxy-
2(E)-d ecen a l a n d d Gu o. LC/APCI/MS analysis of the
reaction mixture after 48 h revealed three major adducts.
The most polar adduct had a retention time of 10.4 min.
Its APCI mass spectrum revealed an intense MH+ at m/z
292, together with a BH2 ion at m/z 176. MS2 analysis
+
of MH+ (m/z 292) resulted in a BH2 product ion at m/z
+
176. MS3 analysis of m/z 176 revealed product ions at
m/z 148 and m/z 121. These LC/MS characteristics were
identical to those for 1,N2-etheno-dGuo. The adduct was
isolated by preparative HPLC using system 7 and sub-
jected to acid hydrolysis (1 N HCl, 100 °C, 1 h). After
depurination, the retention time of the adduct was 8.7
min (Figure 3B), and the MS2 spectrum showed two
major product ions at m/z 148 (MH+-CO) and m/z 121
(MH+-CO-CHN) (Figure 3C). This was identical with
the LC/MS characteristics of authentic 1,N2-etheno-Gua
(Figure 3A). The later eluting adducts had similar LC/
MS characteristics to those reported by Loureiro et al.
(14).

304 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Communications
virtually no product ions. Therefore, LC/MS2 analysis of
MH+ (m/z 176f) gave a very weak signal in the LC/MS2
chromatogram for N2,3-etheno-Gua (Figure 3A, lower
scan). Etheno-Gua from trans-4,5-epoxy-2(E)-decenal had
identical LC (Figure 3B) and MS (Figure 3C) character-
istics with authentic 1,N2-etheno-Gua.
(9) Spiteller, P., Kern, W., Reiner, J ., and Spiteller, G. (2001)
Aldehydic lipid peroxidation products derived from linoleic acid.
Biochim. Biophys. Acta 1531, 188-208.
(10) Schneider, C., Tallman, K. A., Porter, N. A., and Brash, A. R.
(2001) Two distinct pathways of formation of 4-hydroxynonenal.
Mechanisms of nonenzymatic transformation of the 9- and 13-
hydroperoxides of linoleic acid to 4-hydroxyalkenals. J . Biol.
Chem. 276, 20831-20838.
Transition metal ion-free buffers were used in the
reactions of dAdo and dGuo with trans-4,5-epoxy-2(E)-
decenal, and the pH was maintained at 7.4. Under these
conditions, trans-4,5-epoxy-2(E)-decenal was quite stable,
and so the unsubstituted etheno adducts could not have
been formed from further breakdown products of the
epoxide. 2,3-Epoxyoctanal, used in the synthesis of trans-
4,5-epoxy-2(E)-decenal, is much more efficient at convert-
ing both dAdo and dGuo to unsubstituted etheno adducts
(data not shown). Therefore, we considered the possibility
that the trans-4,5-epoxy-2(E)-decenal was contaminated
with 2,3-epoxyoctanal. A normal phase LC/MS assay was
developed that would detect trace amounts of 2,3-epoxy-
octanal. Using this assay, we demonstrated that there
was <0.1% contamination of 2,3-epoxyoctanal in the
synthetic trans-4,5-epoxy-2(E)-decenal. At this level of
contamination, there would have been no significant
contribution from 2,3-epoxyoctanal to the formation of
unsubstituted etheno adducts. Furthermore, 2,3-epoxy-
octanal was not formed during prolonged incubations of
trans-4,5-epoxy-2(E)-decenal at pH 7.4.
In summary, 4,5-epoxy-2(E)-decenal, a primary product
of lipid hydroperoxide decomposition (11, 12), was un-
equivocally shown to be a precursor in the formation of
1,N6-etheno-dAdo and 1,N2-etheno-dGuo. Previous stud-
ies have suggested that an additional oxidative biotrans-
formation step is required before primary products of
lipid hydroperoxide decomposition such as trans,trans-
2,4-decadienal (13, 14) and 4-hydroxy-2-nonenal (20) can
form unsubstituted etheno adducts. Therefore, our study
provides an important link between a primary product
of lipid peroxidation and a mutagenic DNA lesion (22,
27) that has been detected in human tissues (21).
(11) Lee, S. H., Oe, T., and Blair, I. A. (2001) Vitamin C-induced
decomposition of lipid hydroperoxides to endogenous genotoxins.
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of key odorants formed by autoxidation of arachidonic acid using
isotope dilution assay. Lipids 36, 749-756.
(13) Carvalho, V. M., Asahara, F., Di Mascio, P., de Arruda Campos,
I. P., Cadet, J ., and Medeiros, M. H. G. (2000) Novel 1,N6-etheno-
2′-deoxyadenosine adducts from lipid peroxidation products.
Chem. Res. Toxicol. 13, 397-405.
(14) Loureiro, A. P. M., Di Mascio, P., Gomes, O. F., and Medeiros,
M. H. G. (2000) trans,trans-2,4-Decadienal-induced 1,N2-etheno-
2′-deoxyguanosine adduct formation. Chem. Res. Toxicol. 13, 601-
609.
(15) Zamora, R., and Hidalgo, F. J . (1995) Linoleic acid oxidation in
the presence of amino compounds produces pyrroles by carbonyl-
amine reactions. Biochim. Biophys. Acta 1258, 319-327.
(16) Lin, J ., Fay, L. B., Welti, D. H., and Blank, I. (1999) Synthesis of
trans-4,5-epoxy-(E)-2-decenal and its deuterated analogue used
for the development of a sensitive and selective quantification
method based on isotope dilution assay with negative chemical
ionization. Lipids 34, 1117-1126.
(17) Sattsangi, P. D., Leonard, N. J ., and Frihart, C. R. (1977) 1,N2-
Ethenoguanine and N2,3-ethenoguanine. Synthesis and compari-
son of the electronic spectral properties of these linear and
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3292-3296.
(18) Mu¨ller, M., Belas, F. J ., Blair, I. A., and Guengerich, F. P. (1997)
Analysis of 1,N2-ethenoguanine and 5,6,7,9-tetrahydro-7-hydroxy-
9-oxoimidazo[1,2-a]purine in DNA treated with 2-chlorooxirane
by high performance liquid chromatography/electrospray mass
spectrometry and comparison of amounts to other DNA adducts.
Chem. Res. Toxicol. 10, 242-247.
(19) Swenberg, J . A., Fedtke, N., Ciroussel, F., Barbin, A., and Bartsch,
H. (1992) Etheno adducts formed in DNA of vinyl chloride-exposed
rats are highly persistent in liver. Carcinogenesis 13, 727-729.
(20) Chen, H.-J . C., and Chung, F.-L. (1994) Formation of etheno
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(21) Doerge, D. R., Churchwell, M. I., Fang, J . L., and Beland, F. A.
(2000) Quantification of etheno-DNA adducts using liquid chro-
matography, on-line sample processing, and electrospray tandem
mass spectrometry. Chem. Res. Toxicol. 13, 1259-1264.
(22) Levine, R. L., Yang, I.-Y., Hossain, M., Pandya, G. A., Grollman,
A. P., and Moriya, M. (2000) Mutagenesis induced by a single
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60, 4098-4104.
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. (CA91016).
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