4-Hydroxy-2-nonenal and ethyl linoleate form N2,3-ethenoguanine under peroxidizing conditions

Article abstract of DOI:10.1021/tx0001124

In these studies, we demonstrate that N²,3-ethenoguanine (N²,3-∈Gua) is formed from lipid peroxidation as well as other oxidative reactions. Ethyl linoleate (EtLA) or 4-hydroxy-2-nonenal (HNE) was reacted with dGuo in the presence of tert-butyl hydroperoxide (t-BuOOH) for 72 h at 50 °C. The resulting N²,3-∈Gua was characterized by liquid chromatography/electrospray mass spectroscopy and by gas chromatography/high-resolution mass spectral (GC/HRMS) analysis of its pentafluorobenzyl derivative following immunoaffinity chromatography purification. The amounts of N²,3-∈Gua formed were 825 ± 20 and 1720 ± 50 N²,3-∈Gua adducts/10⁶ normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases in the amount of N²,3-∈Gua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in a > 1000-fold increase in the level of N²,3-∈Gua over dGuo that was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reacted with calf thymus DNA at 37 °C for 89 h. The amounts of N²,3-∈Gua formed in intact ctDNA were 114 ± 32 and 52.9 ± 16.7 N²,3-∈Gua adducts/10⁶ normal dGuo bases for EtLA and HNE, respectively. These compared to 2.02 ± 0.17 and 2.05 ± 0.47 N²,3-∈Gua adducts/10⁶ normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid [¹³C₁₈]EtLA was reacted with dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. These reactions resulted in a 89-93% level of incorporation of the ¹³C label into N²,3-∈Gua when EtLA and dGuo were in equimolar concentrations, when EtLA was in 10-fold molar excess, and when deoxyribose (thymidine) was in 10-fold molar excess. Similar reactions with ctDNA resulted in an 86% level of incorporation of the ¹³C label. These data demonstrate that N²,3-∈Gua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N²,3-∈Gua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.

Full text of DOI:10.1021/tx0001124

Chem. Res. Toxicol. 2000, 13, 1243-1250
1243
4-Hyd r oxy-2-n on en a l a n d Eth yl Lin olea te F or m
N2,3-Eth en ogu a n in e u n d er P er oxid izin g Con d ition s
Amy-J oan L. Ham,† Asoka Ranasinghe,‡ Hasan Koc,‡ and J ames A. Swenberg*,†,‡
Departments of Pathology and Environmental Sciences and Engineering, University of North
Carolina, Chapel Hill, North Carolina 27599
Received May 18, 2000
In these studies, we demonstrate that N2,3-ethenoguanine (N2,3-ꢀGua) is formed from lipid
peroxidation as well as other oxidative reactions. Ethyl linoleate (EtLA) or 4-hydroxy-2-nonenal
(HNE) was reacted with dGuo in the presence of tert-butyl hydroperoxide (t-BuOOH) for 72 h
at 50 °C. The resulting N2,3-ꢀGua was characterized by liquid chromatography/electrospray
mass spectroscopy and by gas chromatography/high-resolution mass spectral (GC/HRMS)
analysis of its pentafluorobenzyl derivative following immunoaffinity chromatography purifica-
tion. The amounts of N2,3-ꢀGua formed were 825 ( 20 and 1720 ( 50 N2,3-ꢀGua adducts/106
normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases
in the amount of N2,3-ꢀGua compared to controls containing only t-BuOOH. Controls containing
t-BuOOH but no lipid resulted in a >1000-fold increase in the level of N2,3-ꢀGua over dGuo
that was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reacted
with calf thymus DNA at 37 °C for 89 h. The amounts of N2,3-ꢀGua formed in intact ctDNA
were 114 ( 32 and 52.9 ( 16.7 N2,3-ꢀGua adducts/106 normal dGuo bases for EtLA and HNE,
respectively. These compared to 2.02 ( 0.17 and 2.05 ( 0.47 N2,3-ꢀGua adducts/106 normal
dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [13C18]EtLA was reacted
with dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. These
reactions resulted in a 89-93% level of incorporation of the 13C label into N2,3-ꢀGua when
EtLA and dGuo were in equimolar concentrations, when EtLA was in 10-fold molar excess,
and when deoxyribose (thymidine) was in 10-fold molar excess. Similar reactions with ctDNA
resulted in an 86% level of incorporation of the 13C label. These data demonstrate that N2,3-
ꢀGua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The
data also suggest, however, that N2,3-ꢀGua is also formed by an alternative mechanism that
involves some other oxidative reaction which remains unclear.
In tr od u ction
N2,3-Ethenoguanine (N2,3-ꢀGua,1 Figure 1) is a highly
mutagenic adduct formed from a number of carcinogens,
including vinyl chloride (1, 2). More recently, N2,3-ꢀGua
has been discovered in unexposed tissues of mice, rats,
and humans (3). Fedtke et al. (4) originally reported that
N2,3-ꢀGua appeared to have a very long half-life in
hepatic DNA following vinyl chloride exposure (>30
days). On the basis of the discovery of endogenous N2,3-
ꢀGua, it is now believed that the half-life of N2,3-ꢀGua
could not be calculated accurately after exposure to vinyl
chloride because baseline levels of endogenous N2,3-ꢀGua
(3) were being measured.
The importance of etheno adducts (Figure 1) in car-
cinogenesis stems from both their formation by a number
* To whom correspondence should be addressed: Department of
Environmental Sciences and Engineering, CB #7400, University of
North Carolina, Chapel Hill, NC 27599-7400. Telephone: (919) 966-
6139. Fax: (919) 966-6123.
F igu r e 1. Structures of etheno adducts.
† Department of Pathology.
‡ Department of Environmental Sciences and Engineering.
1
Abbreviations: N2,3-ꢀGua, N2,3-ethenoguanine; N2,3-ꢀGuo, N2,3-
of carcinogens and their demonstrated mutagenicity. All
four etheno adducts have demonstrated miscoding po-
tential in mutagenicity studies (2, 5-10). These studies
demonstrated that ꢀAde caused A to G transitions, ꢀCyt
caused C to A tranversions and C to T transitions, N2,3-
ꢀGua caused G to A transitions, and 1,N2-ꢀGua caused
ethenoguanosine; N2,3-ꢀdGuo, N2,3-ethenodeoxyguanosine; ꢀAde, 1,N6-
ethenoadenine; ꢀCyt, 3,N4-ethenocytosine; dGuo, deoxyguanosine;
EtLA, ethyl linoleate; HNE, 4-hydroxy-2-nonenal; BHT, butylated
hydroxytoluene; IA, immunoaffinity; EI, electron ionization; CI, chemi-
cal ionization; ctDNA, calf thymus DNA; t-BuOOH, tert-butyl hydro-
peroxide; SIM, selected ion monitoring; PFB, pentafluorobenzyl; PFK,
perfluorokerosene; M1G, pyrimido[1,2-a]purine-10(3H)-one.
10.1021/tx0001124 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/14/2000

1244 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Ham et al.
Reaction of tr a n s-4-Hydr oxy-2-n on en al, Eth yl Lin oleate,
a n d [13C18]Eth yl Lin olea te w ith d Gu o a n d ctDNA. The
reaction of 4-hydroxy-2-nonenal and ethyl linoleate with dGuo
was carried out according to the methods of Sodum and Chung
(22) with minor modifications. One hundred micrograms (0.64
µmol) of trans-4-hydroxy-2-nonenal (HNE, 10 µL, 10 mg/mL in
hexane) or 10 mg (32.4 µmol) of ethyl linoleate (EtLA) (100 µL,
100 mg/mL in methanol) was added to 0.2 µmol of dGuo in 0.5
mL of 50 mM sodium phosphate buffer (pH 7.4). After the
addition of 10 µL of t-BuOOH (70% solution, 70 µmol), the
mixture was incubated with constant mixing at 50 °C for 72 h.
After the addition of 1 mL of water and 10 µL of 2% butylated
hydroxytoluene (BHT) in 2-propanol (w/v) to both reaction
mixtures and 0.5 mL of methanol to the EtLA reaction mixture,
the samples were extracted with 1 mL of chloroform. The
aqueous layer was dried under vacuum and resuspended in 3
mL of phosphate-buffered saline (pH 7.6) and stored at -80 °C
until analysis was carried out. The reaction of [13C18]EtLA with
dGuo was the same as described above except 10-fold less (10-
fold excess lipid) or 100-fold less (equimolar and dGuo) EtLA
was used and the reaction was carried out for 24 h instead of
72 h. The reaction of ctDNA with HNE and EtLA was essentially
both G to A transitions and G to T transversions. These
mutation studies were consistent with the mutations
observed in tumors from vinyl chloride-exposed humans
and rats. In five of six human liver angiosarcomas
associated with vinyl chloride exposure, GC to AT transi-
tions were observed at codon 13 of c-Ki-ras-2 gene (11).
Tumors from vinyl chloride-exposed rats showed G to A
transitions, A to C transversions, and C to T transitions
in N-ras genes (12). Additionally, angiosarcomas from
rats exposed to vinyl chloride demonstrated p53 muta-
tions which resulted in G to A transitions as well as A to
T transversions (13). All of these mutations observed in
vinyl chloride-induced tumors are consistent with muta-
tions caused by etheno adducts, suggesting an important
role for these adducts in carcinogenesis.
The occurrence of DNA adducts from endogenous
sources has become increasingly apparent. Many of these
adducts have been postulated to occur from the reaction
of DNA with products of lipid peroxidation (14-20). The
etheno adducts, 1,N2-ethenoguanine (1,N2-ꢀGua) and
1,N6-ethenoadenine (ꢀAde) (Figure 1), are formed from
2,3-epoxy-4-hydroxynonanal, the epoxide of the lipid
peroxidation product trans-4-hydroxy-2-nonenal (21-24).
Although these etheno adducts are formed from products
of lipid peroxidation, there has been no direct evidence
to show that N2,3-ꢀGua is formed from lipid peroxidation
as well. In this study, we have used a recently developed
GC/HRMS method for N2,3-ꢀGua using enrichment by
immunoaffinity chromatography to determine if N2,3-
ꢀGua is formed from the reaction of 4-hydroxy-2-nonenal
(HNE) or ethyl linoleate (EtLA) with either deoxygua-
nosine (dGuo) or calf thymus DNA (ctDNA) under per-
oxidizing conditions. In addition, we have reacted dGuo
and ctDNA with [13C18]ethyl linoleate under peroxidizing
conditions to determine the extent that lipid peroxidation
products form N2,3-ꢀGua by direct alkylation by monitor-
ing the incorporation of the 13C-labeled stable isotope into
N2,3-ꢀGua.
the same as for dGuo (using 10-fold molar excess lipid for the
13
[
C ]EtLA experiments) except that 300 µg of DNA in 1 mL of
18
buffer was used (roughly equivalent to 0.2 µmol of dGuo) and
the reaction was carried out at 37 °C for 89 h (24 h for the [13C18]-
EtLA experiments). The samples were then extracted two times
with 2 mL of chloroform containing 0.5% BHT (w/v); the aqueous
layer was dried under vacuum, and samples were resuspended
in 1 mL of water. The EtLA samples were then extracted two
additional times with a 2:1 chloroform (with BHT)/methanol
mixture, and the aqueous layer was dried under vacuum and
resuspended in 1 mL of water. Samples were left at 4 °C
overnight to rehydrate DNA and stored at -80 °C until analysis
was carried out. The resulting ctDNA was separated from
depurinated product prior to analysis by Centricon 10 filtration.
LC/MS of a Rea ction Mixtu r e fr om th e Rea ction of
EtLA w ith d Gu o. A Magic 2002 HPLC system (Michrom,
Auburn, CA), equipped with a 718AL autosampler (Alcott
Chromatography, Inc., Norcross, GA) and a Rheodyne injector,
was connected to a TSQ 7000 triple mass spectrometer (Ther-
moQuest, San J ose, CA) with a diversion valve placed between
the liquid chromatograph and the electrospray source. An
Alltech (Alltech Associates, Inc., Deerfield, IL) Platinum EPS
C18, 5 µm, 100 Å, 4.6 mm × 250 mm column was run at a flow
rate of 0.7 mL/min with the following gradient: from 0 to 20
min, isocratic 90:10 water/methanol; from 20 to 40 min, linear
gradient from 90:10 to 50:50 water/methanol; and from 40 to
65 min, isocratic 50:50 water/methanol. By using the diversion
valve, only the 25-40 min portion of the LC effluent was allowed
into the ESI source. The rest of the effluent was directed to the
waste. Nitrogen was used as the auxiliary (40 psi) and sheath
gas (80 psi). Electrospray and SRM parameters were optimized
for maximum sensitivity using 5 µL loop injections of the N2,3-
ꢀdGuo standard (24 µM). The MS/MS detection was carried out
in the selected reaction monitoring (SRM) mode. SRM transi-
Exp er im en ta l P r oced u r es
Ma ter ia ls. Spleen phosphodiesterase, alkaline phosphatase,
and DNase I were purchased from Sigma Chemical Co. (St.
Louis, MO). Snake venom phosphodiesterase was purchased
from Worthington Biochemical (Freehold, NJ ). Protein
A
Sepharose was purchased from Pharmacia Biotech (Piscataway,
NJ ). Disposable polystyrene columns were purchased from
Pierce (Rockford, IL). Pentafluorobenzyl bromide was obtained
from Aldrich Chemical (Milwaukee, WI). Calf thymus DNA, 2′-
deoxyguanosine, 4-hydroxy-2-nonenal, and tert-butyl hydro-
peroxide (t-BuOOH) were purchased from Sigma Chemical Co.
Ethyl linoleate was obtained from NuChek Prep, Inc. (Elysian,
MN). [13C18]Ethyl linoleate (>98%) was purchased from Cam-
bridge Isotope Laboratories, Inc. (Andover, MA). All other
chemicals were obtained commercially and were of the highest
purity available. Centricon 10 filters were obtained from Ami-
con, Inc. (Beverly, MA). N2,3-ꢀGua was synthesized by the
method of Sattsangi et al. (25). Stable isotope internal standards
of N2,3-ꢀGua were synthesized by first preparing isotopically
labeled guanine which was synthesized by the method of
Scheller et al. (26) with the modification that for [13C4,15N2]-
N2,3-ꢀGua, K13C15N and Na15NO2 were used to label the N-7
and N-9 nitrogens. Isotopically labeled N2,3-ꢀGua was then
synthesized by first protecting labeled Gua with a benzyl group
at the O6 position using a two-step procedure involving acety-
lation at N2 and N9, according to the method of Zou and Robins
(27), followed by a Mitsunobu reaction, according to the method
of Khazanchi et al. (28). The etheno bridge of N2,3-ꢀGua was
then formed according to the method of Sattsangi et al. (25).
tions for N2,3-ꢀdGuo, N2,3-ꢀGua, and
[
13C4,15N2]-N2,3-ꢀGua
(internal standard) were from m/z 292 to 176, 176 to 81, and
182 to 84, respectively. SRM transitions for 1,N2-ꢀdGuo, 1,N2-
ꢀGua, and [13C3]-1,N2-ꢀGua (internal standard) were from m/z
292 to 176, 176 to 148, and 182 to 150, respectively. Collision-
induced dissociation was performed in the presence of argon at
about 2.5 × 10-3 mbar. The collision energy was set to 25 V.
Data acquisition and processing were performed using Xcalibur
version 1.0 software running under the Microsoft NT 4.0
operating system.
Qu a n tita tion a n d GC/HRMS of th e P en ta flu or oben zyl
Der iva tive of N2,3-EGu a . The resulting product from the dGuo
or ctDNA reactions described above was quantitated by immu-
noaffinity chromatography (IA) followed by GC/HRMS (IA/GC/
HRMS) by the method of Ham et al. (29), using enzymatic
digestion for ctDNA. The quantitative analyses and full scan

Lipid Peroxidation Forms N2,3-Ethenoguanine
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1245
F igu r e 3. Chromatogram resulting from the LC/ESI-MS/MS
analysis of the reaction between EtLA and dGuo using selected
reaction monitoring. Stable isotopically labeled internal stan-
dards for N2,3-ꢀGua ([13C4,15N2]-N2,3-ꢀGua) and 1,N2-ꢀGua
([13C3]-1,N2-ꢀGua) were added to the reaction mixture prior to
analysis.
F igu r e 2. Full scan mass spectral analysis of the pentafluo-
robenzyl derivative of the product from the reaction of HNE with
deoxyguanosine. The product was isolated by immunoaffinity
chromatography prior to derivatization and mass spectral
analysis. The top panel is the mass spectrum using positive
electron impact mode, and the bottom panel is the mass
spectrum using negative chemical ionization mode.
that corresponded with the molecular ion (M+), a molec-
ular ion minus a fluorine (M - F), and a PFB fragment,
respectively, consistent with that of the PFB2-N2,3-ꢀGua
standard. Additionally, negative CI analysis resulted in
a mass spectral peak at m/z 354, consistent with a (M -
PFB) fragment. These data matched those of a synthetic
standard for PFB2-N2,3-ꢀGua as well as previously
published data (30).
mass analyses of the resulting pentafluorobenzyl derivatives
were performed on an HP 5890 gas chromatograph interfaced
with a VG70-250SEQ mass spectrometer at mass resolving
powers of 10 000 and 1000 (full scan), respectively. A DB-5MS
fused silica capillary column (30 m × 0.32 mm, 0.1 µm thickness;
J &W Scientific) with a helium (Sunox, 99.999% pure) head
pressure of 10 psi was used in all experiments. The samples
were introduced using the direct injection mode utilizing Restek
Uniliner 20355. The injection port temperature was 290 °C, and
the initial column temperature was 100 °C for 30 s, increasing
at a rate of 25 °C/min to 150 °C and at a rate of 15 °C/min to
300 °C, and held for 3 min. Selected ion monitoring (SIM)
experiments at a mass resolving power of 10 000 were performed
by monitoring the fragment ions, (M - PFB)- [m/z 354.0413
(N2,3-ꢀGua), 356.0481 ([13C2]-N2,3-ꢀGua), 358.0549 (13C4-labeled
internal standard), and 360.0489 (13C4- and 15N2-labeled internal
standard)]. The perfluorokerosene (PFK) lock mass and the
dwell time were 354.9792 amu and 50 ms/ion, respectively. The
methane (Airco, 99.99% pure) pressure was adjusted to give an
ion gauge reading of 3 × 10-5 mbar. The CI slit in the EI/CI
source was selected, and the instrument was tuned using the
PFK mass (m/z 293) under CI negative ion mode. The electron
energy and emission current were also optimized such that the
highest ion abundance of m/z 293 from PFK (PCR Inc.) was
displayed on the oscilloscope. After the tuning, the electron
energy and emission current ranged between 60 and 100 eV and
between 0.5 and 1.0 mA, respectively. EI full scan mass spectra
were obtained using the EI slit and with an electron energy of
70 eV. The ion source temperature was set at 250 °C during all
the experiments.
The reaction mixture dGuo with EtLA was also ana-
lyzed by LC/ESI-MS using selected reaction monitoring
(SRM), to confirm the GC/MS results. The resulting
chromatogram is pictured in Figure 3. From this experi-
ment, we were able to confirm that N2,3-ꢀGua, 1,N2-ꢀGua,
and 1,N2-ethenodeoxyguanosine (1,N2-ꢀdGuo) were formed.
1,N2-ꢀdGuo was analyzed by monitoring the transition
from m/z 292 to 176. The respective nucleobases, N2,3-
ꢀGua and 1,N2-ꢀGua, were analyzed by monitoring the
transitions from m/z 176 to 81 and from m/z 176 to 148,
respectively. The presence of N2,3-ꢀGua and 1,N2-ꢀGua
was confirmed by the addition of authentic internal
standards ([13C4,15N2]-N2,3-ꢀGua and [13C3]-1,N2-ꢀGua)
that coeluted with the identified N2,3-ꢀGua and 1,N2-
ꢀGua peaks, respectively (Figure 3). The amounts of N2,3-
ꢀGua formed were approximately 10-fold lower than the
amount of 1,N2-ꢀGua and 1,N2-ꢀdGuo combined (esti-
mated on the basis of the relative response to the internal
standards [13C4,15N2]-N2,3-ꢀGua and [13C3]-1,N2-ꢀGua).
Further analysis of the reactions of dGuo with HNE
and EtLA was achieved by IA/GC/HRMS of the pen-
tafluorobenzyl derivative of N2,3-ꢀGua, which utilized IA
chromatography and selected ion monitoring mass spec-
troscopy with a resolving power of 10 000 based on a
previously described method (29). These methods allow
for a very high selectivity, thereby increasing the confi-
dence in the formation of N2,3-ꢀGua. It is also important
to note that the antibodies used for the IA chromatog-
raphy do not cross react with 1,N2-ꢀGua and that the
pentafluorobenzyl derivative of 1,N2-ꢀGua can be sepa-
rated from N2,3-ꢀGua by GC under the conditions used
for analysis (data not shown). A representative chro-
matogram after the reaction of dGuo with HNE is shown
in Figure 4.
Resu lts
The reaction mixtures resulting from the reaction of
dGuo with EtLA or HNE were purified by IA chroma-
tography, hydrolyzed to the nucleobase, and derivatized
with pentafluorobenzyl bromide. The resulting derivative
(PFB2-N2,3-ꢀGua) was subjected to positive EI and
negative CI full scan mass spectral analysis. The result-
ing full scan spectra are shown in Figure 2. The positive
EI analysis resulted in peaks at m/z 535, 516, and 181
In addition to confirmation of the presence of N2,3-
ꢀGua, the IA/GC/HRMS method allowed us to quantitate

1246 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Ham et al.
Ta ble 1. N2,3-Eth en ogu a n in e F or m ed fr om th e Rea ction
of EtLA a n d HNE w ith d Gu o or ctDNA u n d er
P er oxid izin g Con d ition s
EtLA reaction
conditionsb
(N2,3-ꢀGua
adducts/
HNE reaction
conditionsb
(N2,3-ꢀGua
adducts/
106 dGuo bases) 106 dGuo bases)
deoxyguanosinea
lipid and t-BuOOH
t-BuOOH only (no lipid)
control (no incubation)
ctDNAa
825 ( 20c
21 ( 5
<0.02
1720 ( 50
21 ( 6
<0.02
lipid and t-BuOOH
t-BuOOH only (no lipid)
control (incubation only)
control (no incubation)
114 ( 32
52 ( 17
2.02 ( 0.17
0.62 ( 0.13
0.26 ( 0.05
2.05 ( 0.47
1.00 ( 0.86
0.26 ( 0.05
F igu r e 4. Representative GC/HRMS chromatogram of N2,3-
ꢀGua resulting from the reaction of HNE with deoxyguanosine.
a
The reactions with deoxyguanosine were carried out at 50 °C
for 72 h. The reactions with calf thymus DNA were carried out at
37 °C for 89 h. b Reaction conditions are described in Experimental
Procedures. The amounts of EtLA and HNE that were used were
32.4 and 0.64 µmol, respectively. c Values are expressed as means
( the standard deviation (n ) 3 for all samples).
the amount of N2,3-ꢀGua formed in these reaction mix-
tures. The results for the formation of N2,3-ꢀGua from
reactions of dGuo with either HNE or EtLA are shown
in Table 1. The amount of EtLA used in these reactions
was greater than the amount of HNE since it was
hypothesized that the epoxide of HNE was the ultimate
reactant and this would have to be formed from EtLA in
a low yield. The reaction of 200 nmol of dGuo with EtLA
in the presence of t-BuOOH resulted in the generation
of 825 ( 20 N2,3-ꢀGua adducts/106 normal dGuo bases.
The same reaction with HNE resulted in the generation
of 1720 ( 50 N2,3-ꢀGua adducts/106 normal dGuo bases.
This corresponded to yields of 0.08 and 0.17%, based on
deoxyguanonsine, for EtLA and HNE, respectively. Ad-
ditionally, the control reaction of dGuo with t-BuOOH
but no lipid resulted in the formation of 21 ( 5 and 21 (
6 N2,3-ꢀGua adducts/106 normal dGuo bases for the
reaction conditions for EtLA and HNE, respectively.
These numbers for control incubations with t-BuOOH
corresponded to a 0.002% yield. Unreacted dGuo was also
analyzed for the presence of N2,3-ꢀGua, and although a
small amount was detected, this number was below the
limit of quantitation and it was at least 1000-fold lower
than that in the control incubations containing t-BuOOH.
comparison to endogenous levels of N2,3-ꢀGua in rat liver
[∼0.07 N2,3-ꢀGua adduct/106 normal dGuo bases (29)].
This may be an artifact induced during commercial
isolation.
Since the incubation times for the reactions of EtLA
and HNE with ctDNA were long (89 h at 37 °C) and N2,3-
ꢀGua is a N3 guanine adduct, there was a possibility that
the total amount of N2,3-ꢀGua measured in ctDNA was
an underestimation of the total amount of N2,3-ꢀGua
formed due to depurination of the adduct. In fact, in
reaction mixtures with dGuo at higher temperatures (50
°C for 72 h), no N2,3-ꢀdGuo was identified via LC/ESI-
MS analysis of the reaction mixture, and only N2,3-ꢀGua
was detected, suggesting that N2,3-ꢀdGuo was completely
hydrolyzed to N2,3-ꢀGua at 50 °C. It is important to note
that the amount of N2,3-ꢀGua measured in ctDNA
reaction mixtures was in intact ctDNA since any depu-
rinated product was separated from the DNA during
Centricon 10 filtration. In addition to measuring the
amount of N2,3-ꢀGua formed in ctDNA exposed to EtLA
and HNE, we also measured the amount of N2,3-ꢀGua
that was released from the DNA by measuring the
amount of N2,3-ꢀGua in the filtrate after the DNA was
subjected to Centricon 10 filtration. The amount of N2,3-
ꢀGua depurinated from the ctDNA reacted with EtLA
and HNE was <0.8%, showing that the amount of N2,3-
ꢀGua measured in ctDNA was an accurate representation
of the total amount of N2,3-ꢀGua formed.
Since N2,3-ꢀGua was formed in dGuo and ctDNA in
the absence of lipid, a mechanism other than direct
alkylation by the lipid or its byproducts was suggested.
Because of this, we could not conclude whether N2,3-ꢀGua
was formed from direct alkylation by the lipid or its
byproducts or if N2,3-ꢀGua was formed from an alterna-
tive mechanism that was more prevalent in the presence
of lipid peroxidation. Reactions were carried out using
[13C18]EtLA to determine the amount of N2,3-ꢀGua formed
from direct alkylation by the lipid or its byproducts
versus some alternative oxidative reaction. By using
stable isotopically labeled EtLA, the N2,3-ꢀGua that is
formed as a result of direct alkylation by the lipid or its
byproducts is 2 mass units greater than that formed from
other reactions, and this difference can be visualized and
quantitated by mass spectral analysis. Representative
Similar reactions were performed using ctDNA instead
of dGuo. The results of these experiments are also shown
in Table 1. The amounts of N2,3-ꢀGua in ctDNA after
reaction with EtLA and HNE were 114 ( 32 and 52 (
17 N2,3-ꢀGua adducts/106 normal dGuo bases, from EtLA
and HNE, respectively. These numbers corresponded to
0.01 and 0.005% yields, based on deoxyguanosine, from
the EtLA and HNE reactions, respectively. The reactions
with EtLA and HNE resulted in 56- and 25-fold increases
in the level of N2,3-ꢀGua, respectively, over the control
reactions of dGuo with t-BuOOH but no lipid. These
control incubations corresponded to 2.02 ( 0.17 and 2.05
( 0.47 N2,3-ꢀGua adducts/106 normal dGuo bases using
the reaction conditions for EtLA and HNE, respectively.
Additionally, N2,3-ꢀGua was detected in ctDNA that was
either not incubated or incubated but not exposed to
either lipid or t-BuOOH. The amount of N2,3-ꢀGua in
control DNA that was incubated but not exposed to
t-BuOOH was 2-4-fold lower than the amount of DNA
incubated with t-BuOOH only. The amount of N2,3-ꢀGua
in control DNA that was not incubated (0.26 ( 0.05 N2,3-
ꢀGua adduct/106 normal dGuo bases) was approximately
20-fold lower than the amount of N2,3-ꢀGua detected in
control incubations in the presence of t-BuOOH. These
control levels of N2,3-ꢀGua in ctDNA were very high in

Lipid Peroxidation Forms N2,3-Ethenoguanine
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1247
Ta ble 2. N2,3-Eth en ogu a n in e F or m ed fr om th e Rea ction
of [13C18]EtLA w ith d Gu o u n d er P er oxid izin g Con d ition sa
unlabeled
13C2-labeled
N2,3-ꢀGua
N2,3-ꢀGua
(N2,3-ꢀGua adducts/ (N2,3-ꢀGua adducts/
106 dGuo bases)
106 dGuo bases)
10-fold molar excess of lipid 75.8 ( 33.9b (7%)c
1086 ( 518 (93%)
191 ( 57 (89%)
162 ( 50 (89%)
equimolar lipid and dGuo
24.4 ( 2.7 (11%)
21.5 ( 5.7 (11%)
10-fold molar excess of
nucleoside (thymidine)
t-BuOOH only (no lipid)
control (incubation only)
11.7 ( 2.1
0.2 ( 0.1
NDd
ND
a
b
Reactions were carried out at 50 °C for 24 h. Values are
expressed as means ( the standard deviation (n ) 3 for all
d
samples). c Percentage of total N2,3-ꢀGua formed. ND, not de-
tected.
gesting that the unlabeled N2,3-ꢀGua resulted from some
oxidation of dGuo other than simple oxidation of the
deoxyribose. The total amount of N2,3-ꢀGua formed
when EtLA and dGuo were equimolar represented an
18-fold increase as compared to controls containing only
t-BuOOH and a 1100-fold increase as compared to
controls which were not subjected to any t-BuOOH or
incubation. These results indicated that lipid peroxida-
tion was the major factor in the formation of this adduct.
The amount of N2,3-ꢀGua in the presence of t-BuOOH
(12 ( 2 N2,3-ꢀGua adducts/106 normal dGuo bases),
however, was still 60-fold greater than the amount
of N2,3-ꢀGua in controls that were not subjected to
t-BuOOH exposure or incubation (0.19 ( 0.05 N2,3-ꢀGua
adduct/106 normal dGuo bases). The amount of unlabeled
N2,3-ꢀGua in these reactions could not be attributed to
isotopic impurity of the [13C18]ethyl linoleate since there
was an only 3.6% total 12C isotopic contamination of the
lipid (data not shown).
In addition to the reaction with dGuo, [13C18]EtLA was
reacted with ctDNA. The results of these experiments
are shown in Table 3. The total amount of N2,3-ꢀGua
formed in the presence of a 10-fold molar excess EtLA
(with respect to the amount of dGuo in the DNA used)
was 21 ( 10 N2,3-ꢀGua adducts/106 normal dGuo bases.
This represented a 20-fold increase in the level of N2,3-
ꢀGua over control DNA that was incubated with
t-BuOOH (1.1 ( 0.2 N2,3-ꢀGua adducts/106 normal dGuo
bases). Of the ꢀGua present following this reaction, 86%
resulted from direct alkylation by the lipid or its byprod-
ucts ([13C2]-N2,3-ꢀGua), whereas 14% was unlabeled N2,3-
ꢀGua, similar to the results from the reactions with dGuo.
Interestingly, unlike the reactions with dGuo and previ-
ous reactions with ctDNA, there was no increase in the
level of N2,3-ꢀGua in controls with t-BuOOH as compared
to controls that were not subjected to t-BuOOH exposure
or incubation. The levels in controls that were not
subjected to t-BuOOH exposure or incubation, however,
were 4-fold higher than the same controls seen in the
previous experiment (Tables 1 and 3). It is unclear why
the level of N2,3-ꢀGua in the control DNA (1.1 ( 0.1 N2,3-
ꢀGua adducts/106 normal dGuo bases) in this experiment
was higher than it was in previous experiments (0.26 (
0.05 N2,3-ꢀGua adduct/106 normal dGuo bases). One
possible explanation is that the ctDNA was stored in
water at -20 °C for an additional 15 months prior to the
[13C18]EtLA reactions. The additional storage of the
ctDNA may have allowed for increases in the level of
N2,3-ꢀGua from reactions similar to those observed when
t-BuOOH was reacted dGuo in the absence of lipid
(Tables 1 and 2). The amounts of N2,3-ꢀGua in this ctDNA
F igu r e 5. Representative GC/HRMS chromatogram of N2,3-
ꢀGua resulting from the reaction of [13C18]EtLA with dGuo.
chromatograms from the reaction of [13C18]EtLA with
dGuo are shown in Figure 5, demonstrating the formation
of 13C2-labeled and unlabeled N2,3-ꢀGua. The results of
these experiments are shown in Table 2. Upon the
reaction of [13C18]EtLA with dGuo in which the lipid was
in 10-fold molar excess, a total of 1160 ( 552 adducts/
106 normal dGuo bases was formed. Of the N2,3-ꢀGua
formed in this reaction, 93% of the N2,3-ꢀGua resulted
from direct alkylation by the lipid or its byproducts (1086
( 518 [13C2]-N2,3-ꢀGua adducts/106 normal dGuo bases),
whereas only 7% resulted from an alternative oxidative
mechanism (75.8 ( 33.9 unlabeled N2,3-ꢀGua adducts/
106 normal dGuo bases). Additionally, the amount of
N2,3-ꢀGua formed with a 10-fold molar excess of EtLA
(1160 ( 550 N2,3-ꢀGua adducts/106 normal dGuo bases)
was greater than when EtLA was equimolar with respect
to dGuo (215 ( 58 N2,3-ꢀGua adducts/106 normal dGuo
bases) for both the unlabeled and labeled N2,3-ꢀGua. This
represented a 3-fold increase in the amount of unlabeled
ꢀGua and a 6-fold increase in the amount of [13C2]-N2,3-
ꢀGua. The presence of the lipid, therefore, increased the
amount of unlabeled N2,3-ꢀGua as well as [13C2]-N2,3-
ꢀGua. Even more striking was the fact that when the
reactants (EtLA and dGuo) were equimolar, the N2,3-
ꢀGua resulting from direct reaction by the lipid or its
byproducts ([13C2]-N2,3-ꢀGua) was still 89% of the total
N2,3-ꢀGua formed. Only 11% of the unlabeled N2,3-ꢀGua
was formed from an alternative oxidative mechanism
when EtLA and dGuo were equimolar, similar to the
relative amounts of N2,3-ꢀGua formed when EtLA was
in excess. We had originally hypothesized that the N2,3-
ꢀGua formed from the alternative oxidative mechanism
may be due to oxidation of the deoxyribose on the
nucleoside, leading to a reactive hydroxyacetaldehyde
that may then react with deoxyguanosine to form N2,3-
ꢀGua. To test this hypothesis, we used thymidine, as a
source of deoxyribose separate from the deoxyguanosine,
in 10-fold molar excess of both EtLA and dGuo. Although
deoxyadenosine had been used previously as a source of
deoxyribose oxidation products (31, 32), we chose to use
thymidine in these experiments since etheno adducts of
dAdo can be formed, but no such adduct for thymidine
exists. By using thymidine instead of dAdo, therefore, we
wanted to eliminate any competitive reactions for the
formation of etheno adducts. The reaction with a 10-fold
excess of thymidine, however, did not lead to any
increases in the level of N2,3-ꢀGua over the reaction in
which EtLA and dGuo were equimolar (Table 2), sug-

1248 Chem. Res. Toxicol., Vol. 13, No. 12, 2000
Ham et al.
Ta ble 3. N2,3-Eth en ogu a n in e F or m ed fr om th e Rea ction
of [13C18]EtLA w ith ctDNA u n d er P er oxid izin g
Con d ition sa
observed (data not shown). Similarly, we subjected 1,N2-
ꢀdGuo to the conditions of the reactions described here
(without the presence of lipid), and no formation of either
N2,3-ꢀGua or 1,N2-ꢀGua was observed (data not shown).
The presence of N2,3-ꢀGua in these reactions and no N2,3-
ꢀdGuo, therefore, could not be explained by simple
depurination of the nucleobase or by conversion of 1,N2-
ꢀdGuo to N2,3-ꢀGua. There are two possible explanations,
therefore, for the formation of N2,3-ꢀGua and not N2,3-
ꢀdGuo. First, the intermediate adducts in the formation
of 1,N2-ꢀdGuo from 2,3-epoxyhydroxynonenal are bulky
adducts containing two exocyclic rings and nine carbons
(22). It is likely that in the reactions of dGuo with EtLA
and HNE the formation of similar intermediates at the
N3 position of deoxyguanosine weakens the glycosidic
bond between the nucleobase and the deoxyribose due
to steric hindrance leading to depurination. This, how-
ever, does not seem likely since the rate of depurination
of this adduct in DNA was insignificant (0.8%). Alterna-
tively, there may be additional radicals present in the
reactions with lipid present that may lead to the cleavage
of this adduct or to the cleavage of dGuo and formation
of guanine and that N2,3-ꢀGua was formed directly from
guanine. In fact, we did observe during HPLC analysis
of the dGuo reaction products using diode array detection
that there was formation of guanine in the reactions at
a yield of approximately 15% (data not shown). In the
reactions of dGuo with EtLA and HNE, therefore, the
formation of N2,3-ꢀGua may have been primarily due to
formation directly from guanine, not dGuo, and would
therefore result in the formation of N2,3-ꢀGua instead of
N2,3-ꢀdGuo. This would also better explain the presence
of 1,N2-ꢀGua, as well, since it is not likely that the bulky
intermediates formed at the 1,N2-position would weaken
the glycosidic bond. We did, however, see the formation
of N2,3-ꢀGua in intact ctDNA and showed very little
depurination of the adduct (<0.8%) from ctDNA, sug-
gesting that an intact adduct is indeed formed in DNA.
Therefore, while no N2,3-ꢀdGuo was formed in the reac-
tions with dGuo, clearly, it was formed in DNA. The yield
in DNA, however, was much lower than it was in the
dGuo reactions. This could easily be explained by the
steric hindrance of the formation of the adduct in DNA
that was not present in the dGuo reactions when guanine
was the reactant.
A surprising result in these studies was that N2,3-ꢀGua
was formed in incubations without any lipid peroxidation
(Tables 1-3). Not only was N2,3-ꢀGua formed in the
absence of lipid, but the amount of adduct formation was
highly significant, at least 1000-fold greater than the
amount of N2,3-ꢀGua detected in dGuo that was not
subjected to incubation. These results suggest that an
alternative mechanism for the formation of N2,3-ꢀGua
exists. It has recently been demonstrated that base
propenals, byproducts of H-4′ hydrogen abstraction from
deoxyribose, form a related exocyclic adduct, pyrimido-
[1,2-R]purin-10(3H)-one (M1G) (31). M1G had previously
been shown to be the most abundant pyrimidopurinone
adduct of deoxyguanosine formed from the lipid peroxi-
dation byproduct malondialdehyde (33). Dedon and col-
leagues demonstrated that M1G was formed not only from
the reaction of ctDNA with adenine propenal formed but
also with greater efficiency with adenine propenal than
with malondiadehyde. Alternatively, the H-3′ hydrogen
abstraction from deoxyribose results in the formation of
a phosphoglycoaldehyde in DNA, or hydroxyacetaldehyde
unlabeled
13C2-labeled
N2,3-ꢀGua
N2,3-ꢀGua
(N2,3-ꢀGua adducts/ (N2,3-ꢀGua adducts/
106 dGuo bases)
106 dGuo bases)
lipid and t-BuOOH
3.0 ( 1.2b (14%)c
1.1 ( 0.2
18.4 ( 9.0 (86%)
t-BuOOH only (no lipid)
control (incubation only)
control (no incubation)
NDd
ND
ND
0.7 ( 0.1
1.1 ( 0.1
a
b
Reactions were carried out at 37 °C for 24 h. Values are
expressed as means ( the standard deviation (n ) 3 for all
d
samples). c Percent of total N2,3-ꢀGua formed. Not detected.
were 1.1 ( 0.1, 0.7 ( 0.1, and 1.1 ( 0.2 N2,3-ꢀGua
adducts/106 normal dGuo bases for ctDNA that was not
subjected to incubation, ctDNA that was incubated but
not exposed to t-BuOOH, and ctDNA that was incubated
with t-BuOOH, respectively.
Discu ssion
The results of this paper clearly demonstrate the
formation of N2,3-ꢀGua from the reaction of dGuo or
ctDNA with either HNE or EtLA under peroxidizing
conditions. We demonstrated that N2,3-ꢀGua is formed
from these reactions using both GC/MS and LC/MS.
Additional support was provided by showing the incor-
poration of two 13C isotopes into the N2,3-ꢀGua product
after the reaction of dGuo or ctDNA with [13C18]ethyl
linoleate. Previously, Chung and colleagues had demon-
strated that the related etheno adducts, 1,N2-ꢀGua and
1,N6-ꢀAde, were formed from the autoxidation product
of HNE, 2,3-epoxy-4-hydroxynonanal (14, 19, 21-24).
Additionally, 1,N6-ꢀAde and 3,N4-ꢀCyt have been detected
by the 32P-labeling method in lipid peroxidation reactions
with nucleic acid bases (17). It is not surprising, there-
fore, that the related adduct, N2,3-ꢀGua, would also be
formed from similar reactions as we have demonstrated
here.
Sodum and Chung (21, 22) reported that 1,N2-ꢀGua
was formed from the reaction of dGuo with HNE under
peroxidizing conditions, similar to the reactions we report
here. As a result of our LC/ESI-MS studies (Figure 3),
we showed the formation of 1,N2-ꢀGua and 1,N2-ꢀdGuo,
consistent with the results of Sodum and Chung (21, 22).
In addition, we observed peaks late in the chromatograms
that had chromatographic characteristics similar to those
of other adducts that Sodum and Chung observed from
these reactions (data not shown) (21-23). On the basis
of LC/ESI-MS analysis, N2,3-ꢀGua was formed after
reaction with EtLA in a yield that was about 20-fold
lower than the total amount of 1,N2-ꢀGua formed (com-
bined total of 1,N2-ꢀGua and 1,N2-ꢀdGuo). This result was
anticipated since the formation of N2,3-ꢀGua should be
affected more by the steric hindrance of the deoxyribose
than it would in the formation of 1,N2-ꢀGua. When the
yield of N2,3-ꢀGua was more accurately quantified using
IA/GC/MS analysis, it was found that N2,3-ꢀGua was
formed at levels of 0.08 and 0.17%, based on deoxygua-
nonsine, for EtLA and HNE, respectively.
We did not observe the formation of N2,3-ꢀdGuo in the
reaction of EtLA with dGuo. Additionally, we observed
the formation of not only 1,N2-ꢀdGuo but also 1,N2-ꢀGua.
We subjected N2,3-ꢀdGuo to the conditions of the reac-
tions described here (without the presence of lipid), but
no release of nucleobase N2,3-ꢀGua or 1,N2-ꢀdGuo was

Lipid Peroxidation Forms N2,3-Ethenoguanine
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1249
ꢀGua after the reaction of dGuo or ctDNA with [13C18]-
EtLA provides concrete evidence for these reactions. The
level of formation of [13C2]-N2,3-ꢀGua was 10-fold greater
than the level of formation of unlabeled N2,3-ꢀGua when
EtLA and dGuo were equimolar. These yields did not
change even in the presence of a 10-fold molar excess of
thymidine, as a source of additional deoxyribose (Table
2). Although the formation of [13C2]-N2,3-ꢀGua predomi-
nated in all of these reactions, clearly there were large
increases in the amounts of N2,3-ꢀGua also seen in the
absence of EtLA and HNE. The mechanism for the
formation of N2,3-ꢀGua not involving lipid peroxidation
warrants further investigation and may prove to be a
significant source of N2,3-ꢀGua resulting from endog-
enous reactions.
Sch em e 1. P r op osed F or m a tion of
P h osp h oglycoa ld eh yd e fr om H-3′ Hyd r ogen
Abstr a ction of Deoxyr ibose
Ack n ow led gm en t. We thank Dr. R. Sangaiah for
the synthesis of chemical standards and Dr. F. Peter
Guengerich of Vanderbilt University for providing the
internal standard for 1,N2-ethenoguanine, [13C3]-1,N2-
ꢀGua. This work was supported by NIEHS Grants
ES05779, ES07017, and ES05948.
in the case of the nucleoside, and the base propenoate
(Scheme 1; see ref 34 for review). This aldehyde is
structurally analogous to chloroacetaldehyde, a metabo-
lite of vinyl chloride that has been shown to form N2,3-
ꢀGua (35-37). It is possible, therefore, that N2,3-ꢀGua
may be formed from the reaction of dGuo with phospho-
glycoaldehyde, resulting from sugar oxidation in DNA,
or hydroxyacetaldehyde, resulting from sugar oxidation
in deoxyguanosine. In fact, J ones and Dedon (32) have
demonstrated that ꢀAde is formed from the reaction of
dAdo with 2-phosphoglycoaldehyde. They were unable to
reproduce, however, the formation of ꢀAde in γ-irradiated
DNA, which should form phosphoglycoaldehyde in DNA.
Our results suggest that this is not the mechanism by
which N2,3-ꢀGua is formed in the absence of lipid in our
studies. Although we showed that there is an alternative
mechanism for the direct alkylation by the lipid, experi-
ments in which an excess of the deoxynucleoside (thy-
midine) was added failed to yield an increase in the level
of N2,3-ꢀGua (Table 2). Although Dedon and colleagues
showed that ꢀAde is formed from the reaction of dAdo
with phosphoglycoaldehyde, the mechanism for the for-
mation of N2,3-ꢀGua remains unknown. In the reactions
described here, dGuo appears to be the limiting reagent
for this alternative mechanism. As discussed above,
guanine was a product of these reactions. There may be
radical reactions that lead to the cleavage of dGuo to
guanine that may form a radical specific to dGuo, which
is not formed with thymidine. Additionally, while the
formation of N2,3-ꢀGua in DNA from this alternative
mechanism was present, the amount was only ∼10-fold
greater than the amount of N2,3-ꢀGua in control DNA
(Table 1). This compares to a 1000-fold greater amount
of N2,3-ꢀGua from this alternative mechanism, compared
to controls, when the reaction mixtures contained dGuo
instead of DNA (Table 1). This further supports the
hypothesis that radical reactions that lead to the cleavage
of dGuo to guanine may form a radical specific to dGuo.
These same reactions most likely do not occur at the same
rate in DNA since <0.8% “depurination” of N2,3-ꢀGua
was seen, yet all of the product from the dGuo reactions
was N2,3-ꢀGua. This alternative mechanism, therefore,
may not be as significant a pathway in DNA as compared
to direct alkylation. Experiments with 13C-labeled de-
oxyribose will be needed to further examine this alterna-
tive mechanism of N2,3-ꢀGua formation.
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