Simultaneous quantitation of N2,3-ethenoguanine and 1,N2-ethenoguanine with an immunoaffinity/gas chromatography/high-resolution mass spectrometry assay

Article abstract of DOI:10.1021/tx0002076

We have previously described an immunoaffinity/gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (IA/GC/ECNCI-HRMS) assay for quantitation of the promutagenic DNA adduct N²,3-ethenoguanine (N²,3-εGua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N²-ethenoguanine (1,N²-εGua), in the same DNA sample. 1,N²-εGua and N²,3-εGua were purified together from hydrolyzed DNA using two immobilized polyclonal antibodies. GC/ECNCI-HRMS was used to quantitate the 3,5-bis(pentafluorobenzyl) (PFB) derivative of each adduct against an isotopically labeled analogue. Selected ion monitoring was used to detect the [M - 181]^- fragments of 3,5-(PFB)₂-N²,3-εGua and 3,5-(PFB)₂-[¹³C₄,¹⁵N ₂]-N²,3-εGua and the [M - 201]^- fragments of 3,5-(PFB)₂-1,N²-εGua and 3,5-(PFB)₂-[¹³C₃]-1,N²-εGua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N²,3-εGua and 15 fmol of 1,N²-εGua in ～250 μg of DNA, which corresponded to 5.0 N²,3-εGua and 8.7 1,N²-εGua adducts/10⁸ unmodified Gua bases, respectively. 1,N²-εGua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N²-εGua to N²,3-εGua were 5:1 and 38:1 when calf thymus DNA was treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions. Only N²,3-εGua was detected in DNA treated with the vinyl chloride (VC) metabolite 2-chloroethylene oxide and in hepatocyte DNA from rats exposed to 1100 ppm VC for 4 weeks (6 h/day for 5 days/week). These data suggest that 1,N²-εGua plays a minor role relative to N²,3-εGua in VC-induced carcinogenesis, but that 1,N²-εGua may be formed to a larger extent from endogenous oxidative processes.

Full text of DOI:10.1021/tx0002076

Chem. Res. Toxicol. 2001, 14, 327-334
327
2
Sim u lta n eou s Qu a n tita tion of N ,3-Eth en ogu a n in e a n d
2
1
,N -Eth en ogu a n in e w ith a n Im m u n oa ffin ity/Ga s
Ch r om a togr a p h y/High -Resolu tion Ma ss Sp ectr om etr y
Assa y
†
‡
§
Eric J . Morinello, Amy-J oan L. Ham, Asoka Ranasinghe,
§
,†,‡,§
Ramiah Sangaiah, and J ames A. Swenberg*
Curriculum in Toxicology and Departments of Pathology and Environmental Sciences and
Engineering, University of North Carolina, Chapel Hill, North Carolina 27599
Received September 27, 2000
We have previously described an immunoaffinity/gas chromatography/electron capture
negative chemical ionization high-resolution mass spectrometry (IA/GC/ECNCI-HRMS) assay
2
2
for quantitation of the promutagenic DNA adduct N ,3-ethenoguanine (N ,3-ꢀGua) in vivo.
Here we present an expanded assay that allows simultaneous quantitation of its structural
2
2
2
2
isomer, 1,N -ethenoguanine (1,N -ꢀGua), in the same DNA sample. 1,N -ꢀGua and N ,3-ꢀGua
were purified together from hydrolyzed DNA using two immobilized polyclonal antibodies. GC/
ECNCI-HRMS was used to quantitate the 3,5-bis(pentafluorobenzyl) (PFB) derivative of each
adduct against an isotopically labeled analogue. Selected ion monitoring was used to detect
-
2
13
15
2
the [M - 181] fragments of 3,5-(PFB)
2
-N ,3-ꢀGua and 3,5-(PFB)
2
-[ C
4
13
, N
2
]-N ,3-ꢀGua and
]-1,N -ꢀGua. The
-
2
2
the [M - 201] fragments of 3,5-(PFB)
2
-1,N -ꢀGua and 3,5-(PFB)
2
-[ C
3
2
demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N ,3-ꢀGua and 15
2
2
2
fmol of 1,N -ꢀGua in ∼250 µg of DNA, which corresponded to 5.0 N ,3-ꢀGua and 8.7 1,N -ꢀGua
8
2
adducts/10 unmodified Gua bases, respectively. 1,N -ꢀGua was found to be the predominant
ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The
2
2
respective ratios of 1,N -ꢀGua to N ,3-ꢀGua were 5:1 and 38:1 when calf thymus DNA was
treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions.
2
Only N ,3-ꢀGua was detected in DNA treated with the vinyl chloride (VC) metabolite
2
-chloroethylene oxide and in hepatocyte DNA from rats exposed to 1100 ppm VC for 4 weeks
2
(6 h/day for 5 days/week). These data suggest that 1,N -ꢀGua plays a minor role relative to
2
2
N ,3-ꢀGua in VC-induced carcinogenesis, but that 1,N -ꢀGua may be formed to a larger extent
from endogenous oxidative processes.
2
In tr od u ction
this laboratory (4, 5). The amount of 1,N -ꢀGua formed
from in vitro reactions with DNA has been measured
using HPLC with UV or fluorescence detection (2, 3), a
competitive immunoassay (6), and LC/MS/MS (7-9).
However, these methods were unable to detect the low
2
2
The exocyclic DNA adducts N ,3-ethenoguanine (N ,3-
1
2
2
ꢀ
Gua) and 1,N -ethenoguanine (1,N -ꢀGua) have been
identified in reactions of the vinyl chloride (VC) metabo-
lites 2-chloroacetaldehyde and 2-chloroethylene oxide
CEO) with DNA (1-3). The formation of N ,3-ꢀGua in
vivo has been characterized in rats exposed to VC and
other chemicals by several methods, including ultrasen-
sitive, highly specific gas chromatography/electron cap-
ture negative chemical ionization high-resolution mass
spectrometry (GC/ECNCI-HRMS) assays developed in
2
concentrations of 1,N -ꢀGua expected to be present in
vivo. A sensitive P-postlabeling assay used for the
detection of 1,N -ꢀGua standard added to DNA (10) has
not been applied to the routine analysis of DNA.
Both 1,N -ꢀGua and N ,3-ꢀGua have been shown to be
highly promutagenic when tested with in vitro and in
vivo systems (11-16). 1,N -ꢀGua has been detected in
reactions of dGuo with the lipid peroxidation products
2
(
32
2
2
2
2
*
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-
139. Fax: (919) 966-6123.
Curriculum in Toxicology.
trans-4-hydroxy-2-nonenal (HNE) or 2,3-epoxy-4-hydroxy-
2
2
2-nonanal (17, 18), and both 1,N -ꢀGua and N ,3-ꢀGua
were demonstrated in reactions of dGuo with HNE or
ethyl linoleate (EtLA) in the presence of tert-butylhydro-
6
†
‡
Department of Pathology.
2
§
peroxide (t-BuOOH) (9). Recently, 1,N -ꢀdGuo was dem-
Department of Environmental Sciences and Engineering.
1
2
2
2
2
Abbreviations: N ,3-ꢀGua, N ,3-ethenoguanine; 1,N -ꢀGua, 1,N -
onstrated from the reaction of calf thymus DNA with the
linoleic acid peroxidation product trans,trans-2,4-deca-
dienal in the presence of peroxides (8). Consequently,
both etheno adducts are suspected to play significant
roles as endogenous mutagens, especially under condi-
tions of oxidative stress (19).
ethenoguanine; VC, vinyl chloride; CEO, 2-chloroethylene oxide;
ECNCI, electron capture negative chemical ionization; HRMS, high-
resolution mass spectrometry; HNE, trans-4-hydroxy-2-nonenal; EtLA,
ethyl linoleate; t-BuOOH, tert-butyl hydroperoxide; IA, immunoaffinity;
KLH, keyhole limpet hemocyanin; PFB, pentafluorobenzyl; ESI, elec-
trospray ionization; EI, electron impact; BHT, butylated hydroxytolu-
ene.
1
0.1021/tx0002076 CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/03/2001

3
28 Chem. Res. Toxicol., Vol. 14, No. 3, 2001
Morinello et al.
In this work, we describe the extension of the existing
mL/min. The high-molecular weight product (0.6-1.2 min) was
2
collected, pooled, and lyophilized.
immunoaffinity (IA)/GC/ECNCI-HRMS assay for N ,3-
2
ꢀ
Gua to allow the simultaneous quantitation of 1,N -ꢀGua
Im m u n iza tion s. Two New Zealand white rabbits (2-3 kg)
in DNA. The formation of both adducts in DNA was
examined to determine the relative role each may play
in carcinogenesis under various conditions.
were purchased from Robinson Services (Clemmons, NC). The
2
KLH-1,N -ꢀGuo conjugate (3.0 mg) was added to 1.5 mL of
sterile 10 mM phosphate buffer and 0.85% saline (pH 7.5) (PBS),
and the suspension was emulsified in 1.5 mL of Freund’s
complete adjuvant. Immunizations (1.0 mL total) were made
at four sc and six id sites on the back and hind of each rabbit.
The rabbits were boosted in the same manner at 2, 4, and 6
weeks with the conjugate similarly prepared in Freund’s
incomplete adjuvant. A test bleed from the ear vein was
conducted at 7 weeks. Blood was harvested 3 days later by
exsanguination from the abdominal aorta after each rabbit was
anesthetized with acepromazine and ketamine by a member of
the veterinary staff. Plasma was obtained by centrifuging the
blood from each rabbit at 1000g for 15 min at 4 °C and collecting
the supernatant. The plasma was stored at -80 °C until it was
used.
Exp er im en ta l P r oced u r es
Ma ter ia ls. Ca u tion : VC, CEO, 2-chloroacetaldehyde, and
chloroform are known or probable carcinogens. Phenol is severely
caustic. Pentafluorobenzyl bromide is a potent lachrymator. Each
of these chemicals should be handled carefully with gloves in
an operating fume hood. VC (99% chemically pure) was pur-
chased from Supelco (Bellefonte, PA). Solubilized keyhole limpet
hemocyanin (KLH), calf thymus DNA, and RNAases A and T
1
were obtained from Sigma (St. Louis, MO). Clostridium his-
tolyticum type 2 collagenase was obtained from Worthington
Biochemical (Freehold, NJ ). Nucleic acid purification grade lysis
buffer, a phenol/chloroform/water mixture, and proteinase K
P r ep a r a tion of Im m u n oa ffin ity Colu m n s. Immunoaffin-
ity columns were prepared as described by Ham et al. with
2
were purchased from PE Biosystems (Foster City, CA). 1,N -
2
modifications (5). To prepare hybrid columns, 4 mL of anti-1,N -
1
3
2
ꢀ
3
dGuo and [2,9,9a- C ]-1,N -ꢀdGuo were generous gifts from
ꢀ
Guo plasma was added to 2.5 mL of protein A-Sepharose, and
F. Peter Guengerich (Vanderbilt University, Nashville, TN) and
were synthesized as described by M u¨ ller et al. (7). CEO was
synthesized as previously described (20). 2-Chloroacetaldehyde
and 2,3,4,5,6-pentafluorobenzyl bromide were purchased from
Aldrich (Milwaukee, WI). Other materials were obtained as
described by Ham et al. (5, 9).
2
1
.5 mL of anti-N ,3-ꢀGuo plasma was added to 1.0 mL of protein
A-Sepharose. Following the coupling procedures, the resins
were pooled and used to construct 20 columns. To prepare 10
2
columns for the purification of 1,N -ꢀGua only, 2 mL of anti-
2
1
,N -ꢀGuo plasma was added to 1.25 mL of protein A-Sepharose.
The columns were washed sequentially with 5 mL of PBS
containing 0.02% sodium azide (w/v), 5 mL of water, 20 mL of
methanol, 5 mL of water, 20 mL of 0.1 M formic acid, 10 mL of
water, and 10 mL of PBS before use.
HP LC. Individual systems and conditions are described in
detail below. Reverse-phase chromatography was performed
with either Beckman Ultrasphere semipreparative (250 mm ×
1
0 mm × 5 µm) or analytical (250 mm × 4.6 mm × 5 µm)
Syn th esis a n d Ch a r a cter iza tion of Bis(p en ta flu or o-
octadecyl silane columns. Strong cation-exchange chromatog-
raphy was performed as described by Ham et al. (5). The eluent
was monitored with either a HP 1040A diode array UV detector
or an Applied Biosystems 757 single-wavelength UV detector.
Injections were made manually with a Rheodyne injector, or
automatically with a Waters 712 WISP autosampler. In each
case, Waters 510 pumps were used.
2
2
ben zyl)-1,N -EGu a Der iva tives. 1,N -ꢀGuo (3.0 mg) was dis-
2
solved in 2 mL of 1 N hydrochloric acid and hydrolyzed to 1,N -
ꢀ
Gua by incubation at 80 °C for 8 h. The solution was
2
neutralized with 1 N sodium hydroxide, and 1,N -ꢀGua was
purified by reverse-phase semipreparative HPLC. The mobile
phase was 20% methanol at 2 mL/min, and A285 was monitored
2
with a diode array detector. 1,N -ꢀGua was collected (10.9 min)
2
2
Syn th esis of 1,N -EGu o. 1,N -ꢀGuo was synthesized by the
reaction of 2-chloroacetaldehyde (45% aqueous solution) with
Guo as described by Sattsangi et al. (21). The product was
purified by semipreparative reverse-phase HPLC, and A285 of
the eluent was monitored. Waters Baseline software was used
for data acquisition and to control a Pharmacia FRAC-200
fraction collector. The methanol concentration was 20% from 0
to 16 min, was linearly increased to 50% at 19 min, and was
and lyophilized. The pentafluorobenzyl (PFB) derivatives were
synthesized as described in the next section, except that 26 µL
of undiluted pentafluorobenzyl bromide was used. A portion of
the product was purified by reverse-phase semipreparative
HPLC. The mobile phase was 50% methanol from 0 to 15 min,
and the methanol concentration was linearly increased to 90%
at 45 min with a flow rate of 2 mL/min. Full scan mass spectra
of the crude product and each major HPLC peak were obtained
by GC/MS in the ECNCI and electron impact (EI) modes at a
2
maintained at 50% until 35 min. The 1,N -ꢀGuo fractions (15.7
2
min) were collected, pooled, and lyophilized. 1,N -ꢀGuo was
3
mass resolving power of 1 × 10 as described below. Electrospray
purified a second time using the same system with an isocratic
ionization (ESI) mass spectra of the analytical standards were
obtained by infusion with a Finnigan (San J ose, CA) TSQ7000
instrument. Each HPLC peak was analyzed by ESI-MS by
2
0% methanol mobile phase. The flow rate was 2 mL/min in
each case.
2
2
DECA
1
P r od u ction of th e KLH-1,N -EGu o Con ju ga te. 1,N -ꢀGuo
infusion with a Finnigan LCQ
instrument. H NMR spectra
was conjugated to KLH by a modified periodate oxidation
procedure (22). 1,N -ꢀGuo (5 mg) was dissolved in 1 mL of 100
were recorded at 500 MHz on a Varian (Palo Alto, CA) Inova500
spectrometer at the University of North Carolina School of
2
2
mM sodium acetate buffer (pH 5.5). Sodium periodate was added
to a concentration of 30 mM and incubated for 15 min at room
temperature before the reaction was stopped by addition of 50
µL of 1 M ethylene glycol. After 5 min at 4 °C, the product was
purified in a single 1 mL injection using a Pharmacia FPLC
system fitted with a HR10/10 gel filtration column. The mobile
phase was 100 mM sodium bicarbonate (pH 9.5) used at a flow
rate of 3 mL/min. The A254 and A280 of the eluent were monitored
with a Pharmacia UV-2 detector coupled to Waters Baseline
software for data acquisition. The eluent corresponding to the
low-molecular weight peak was collected (3.5-7.0 min), and 10
mL of KLH (57 mg) was added. The solution was incubated for
Medicine facility. 1,N -ꢀGua: ESI-MS (assignments in paren-
+
1
6
theses) m/z 176 (MH ); H NMR (DMSO-d ) δ 12.52 (br s, 1H,
N1 or N3-H), 12.22 (br s, 1H, N1 or N3-H), 8.18 and 8.12 (2 s,
1H, N5), 7.82 (s, 1H, H-2), 7.56 (d, 1H, J ) 2.4 Hz, H-7), 7.37
(d, 1H, J ) 2.4 Hz, H-6); UV (20% methanol/H O) λmax ) 223,
2
1
3
2
+
292 nm. [ C
3
]-1,N -ꢀGua: ESI-MS m/z 179 (MH )], [3,5-(PFB)
2
-
2
1,N -ꢀGua: ESI-MS (assignments and relative abundances in
parentheses) m/z 535 (M , 100), 516 (M - F , 18), 354 (M -
PFB , 37); ECNCI-MS (assignments and relative abundances
+
+
+
-
-
in parentheses) m/z 354 (M - PFB , 83), 334 (M - PFB - HF ,
1
100); H NMR (CDCl
Hz, H-7), 7.35 (d, 1H, J ) 1.9 Hz, H-6), 6.15 (s, 2H, CH
5.74 (s, 2H, CH -N5); UV (77% methanol/H O) λmax ) 226, 253,
308 nm. 1,5-(PFB) -1,N -ꢀGua: EI-MS (assignments and rela-
tive abundances in parentheses) m/z 535 (M , 100), 516 (M -
3
) δ 7.79 (s, 1H, H-2), 7.68 (d, 1H, J ) 1.9
2
-N3),
2
4 h at 4 °C, and the product was reduced with 190 µL of 100
2
2
2
2
mM sodium borohydride. After 30 min at 4 °C, the KLH-1,N -
Guo conjugate was purified with multiple 1 mL injections using
the same system with water as the mobile phase at a rate of 4
2
+
ꢀ
+
+
F , 15), 354 (M - PFB , 34); ECNCI-MS (assignments and

Simultaneous Quantitation of Ethenoguanine Adducts
Chem. Res. Toxicol., Vol. 14, No. 3, 2001 329
-
relative abundances in parentheses) m/z 354 (M - PFB , 100),
DNA Hyd r olysis. One hundred microliters of 1 N HCl, 30
-
1
13
15
2
1
1
81 (PFB , 7); H NMR (CDCl
H, J ) 2.5 Hz, H-7), 6.97 (d, 1H, J ) 2.5 Hz, H-6), 5.90 (s, 2H,
-N1), 5.40 (s, 2H, CH -N5); UV (67% methanol/H
3
) δ 8.88 (br s, 1H, H-2), 7.58 (d,
µL of the [ C
4
, N
2
]-N ,3-ꢀGua standard (66.3 fmol), and 50 µL
1
3
2
of the [ C
3
]-1,N -ꢀGua standard (177 fmol) were added to 5-800
CH
27, 308 nm. O ,5-(PFB)
parentheses) m/z 536 (MH ); H NMR (CDCl
H-2), 7.62 (d, 1H, J ) 1.5 Hz, H-7), 6.92 (d, 1H, J ) 1.5 Hz,
H-6), 5.34 (s, 2H, OCH ), 5.31 (s, 2H, CH -N5).
2
2
2
O) λmax
)
µg of aqueous DNA. Water was added to bring the total volume
of each sample to 1000 µL, and the tubes were incubated at 70
°C for 60 min. The samples were cooled, transferred to Centri-
con-10 concentrators (Millipore, Bedford, MA), and centrifuged
at 5000g (60 min at 4 °C). In the case of DNA samples, a 50-
50 µL aliquot of the filtrate was retained for Gua quantitation
see a section below). Each tube was rinsed with 1 mL of 500
mM sodium phosphate buffer (pH 7.2), which was transferred
to the Centricon retentate vial. An additional 1 mL of buffer
was added to each retentate, and the samples were similarly
centrifuged for 90 min.
9
2
2
2
-1,N -ꢀGua: ESI-MS (assignments in
+
1
3
) δ 7.69 (br s, 1H,
2
2
2
(
An im a l Exp osu r es a n d Tissu e Collection . Exposures
were conducted at Huntington Life Sciences (East Millstone,
NJ ). Eleven-week-old male Sprague-Dawley rats (450-550 g)
were purchased from Charles River Laboratories (Portage, MI)
and housed in stainless steel cages with a 12 h light/dark cycle.
Certified Rodent Diet No. 5002 (PMI Feeds, St. Louis, MO) was
provided ad libitum during nonexposure periods, and water was
provided without restriction at all times. Ten animals per group
were acclimated for 1 week and then exposed to nominal
concentrations of 0 or 1100 ppm VC in a whole-body inhalation
apparatus for 1 or 4 weeks (6 h/day for 5 days/week). The
animals were anesthetized ip with pentobarbital, iv heparinized,
and killed by incision of the abdominal aorta. The livers were
perfused in situ with collagenase via the portal vein (23). After
Im m u n oa ffin ity Ch r om a togr a p h y. All steps were per-
formed at 4 °C unless otherwise noted. The IA columns were
preconditioned by the sequential addition of 5 mL of water, 10
mL of methanol, 10 mL of water, and 10 mL of PBS/sodium
azide. Each Centricon filtrate was diluted with 2 mL of water
and transferred to an IA column at room temperature. The
filtrate vials were rinsed with 5 mL of PBS/sodium azide, which
was also transferred to the columns. The samples were washed
with 5 mL of water and 10 mL of 5% methanol. The adducts
were eluted with 5 mL of methanol into silanized test tubes and
were dried in a rotary evaporator. The columns were recondi-
tioned with 5 mL of methanol, 5 mL of water, 10 mL of 0.1 M
formic acid, 10 mL of water, and 10 mL of PBS/sodium azide.
∼
5 min, the livers were removed and sieved through 149 and
05 µm polypropylene meshes to obtain mixed cell suspensions.
Hepatocytes were isolated by centrifugation at 50g for 3 min
1
(24), frozen on dry ice, and stored at -80 °C.
DNA Extr a ction . Cellular DNA was isolated using a modi-
fied phenol/chloroform extraction procedure with 2% butylated
hydroxytoluene (BHT) in 2-propanol (w/v) added to reagents and
samples as an antioxidant. The hepatocyte pellet was suspended
in 5 mL of lysis buffer containing 0.04% BHT (w/v), and an
additional 25 µL of 2% BHT solution was added. RNAases A (5
units) and T (270 units) were added, and the samples were
1
incubated for 2 h at 37 °C. Next, proteinase K (45 units) was
added, and the samples were incubated for an additional 2 h at
Der iva tiza tion . To each sample were added ∼25 mg of oven-
dried, powdered potassium carbonate and 500 µL of acetone.
Next, 35 µL of a 10% pentafluorobenzyl bromide/acetone mixture
(v/v) was added. The tubes were capped and mixed vigorously
at 50 °C for 70 min. The samples were dried in a rotary
evaporator and were then extracted and transferred in two 100
µL portions of dichloromethane to disposable silica gel columns
prepared as previously described (5). The samples were washed
with 4 mL of hexane and 6 mL of a 5% ethyl acetate/hexane
mixture (v/v). The derivatives were eluted in 3 mL of ethyl
acetate and dried again. The samples were redissolved in 15
µL of toluene and transferred to silanized GC vial inserts for
analysis.
3
7 °C. The samples were extracted twice with 5 mL of a phenol/
chloroform/water mixture containing 0.04% BHT (w/v) and once
with 5 mL of alumina-purified chloroform containing 0.04% BHT
(w/v). DNA was precipitated by addition of 0.5 mL of 3 M sodium
acetate and 10 mL of cold 95% ethanol. The pellet was washed
with cold 70% ethanol and redissolved overnight in water at 4
GC/ECNCI-HRMS. The instruments and conditions de-
scribed by Ham et al. (5) were used for the GC/ECNCI-HRMS
°
C. The concentration of each sample was estimated by A260,
and the DNA was stored at -80 °C until analysis was carried
out.
3
analyses. The mass resolving power was 7-10 × 10 . A 2 µL
volume of each sample was injected. Baseline resolution of the
derivatized adducts was achieved without modification of the
GC parameters. Selected ion monitoring at m/z 334.0351 and
2
13
15
2
Sta n d a r d s. N ,3-ꢀGua and [ C
4 2
, N ]-N ,3-ꢀGua standards
were prepared and characterized as described by Ham et al.
2
13
2
-
(
5). 1,N -ꢀdGuo and [ C
3
]-1,N -ꢀdGuo were hydrolyzed to the
337.0453 was used to monitor the [M - 201] fragments of 3,5-
2
13
2
corresponding nucleobases in 2.5% formic acid at 60 °C for 4 h.
The bases were purified by reverse-phase analytical HPLC. The
methanol concentration was increased from 5 to 18% from 0 to
(PFB)
2
-1,N -ꢀGua and 3,5-(PFB)
2
-[ C
3
]-1,N -ꢀGua, respectively.
-
2
The [M - 181] fragments of 3,5-(PFB)
(PFB) -[ C , N ]-N ,3-ꢀGua were monitored at m/z 354.0414
2 4 2
2
-N ,3-ꢀGua and 3,5-
1
3
15
2
1
0 min, and was then increased to 45% from 10 to 15 min. The
and 360.0489, respectively. The absolute retention times of the
peaks decreased over time since the GC column was shortened
by ∼30 cm before each reinstallation.
pooled nucleobase fractions (8.1 min) were dried in a rotary
evaporator, and the concentration of each standard was deter-
mined by UV absorbance in 0.1 N HCl (21). GC/ECNCI-HRMS
was used to calibrate the internal standard by comparison to
the better-characterized analyte standard, and the calculated
concentration was within 1% of that determined by UV. No
Gu a n in e Qu a n tita tion . The Gua content of each sample
was determined with HPLC system 2 described by Ham et al.
(5). In the case of in vitro DNA reactions using less than 50 µg
of DNA, each 200-250 µL aliquot was analyzed undiluted.
Otherwise, 50 µL aliquots were diluted to 200 µL with water
and analyzed. The Gua content of each sample was determined
by comparison to an external standard curve prepared with each
experiment.
2
contamination of the internal standard with unlabeled 1,N -
ꢀGua was detected by GC/ECNCI-HRMS (data not shown).
DNA Rea ction s. The reaction of HNE and EtLA with calf
thymus DNA was performed as described by Ham et al. (9). For
CEO reactions, calf thymus DNA (3 mg/mL) was dissolved in
Ba sic p H In cu ba tion s. Calf thymus DNA (200 µg) was
hydrolyzed, and the first centrifugation was performed as
described above. To stop hydrolysis of the DNA backbone, 2 mL
of 500 mM sodium phosphate buffer (pH 7.2) was added to the
retentate vial. A 50 µL Gua aliquot was removed from the
filtrate vial, and the filtrates were treated with 1 mL of 50 mM
sodium bicarbonate buffer (pH 10.5) and 55 µL of 1 N sodium
hydroxide. The pH of each sample was measured at pH 10.5-
10.6 with a micro Ag/AgCl electrode. The samples were incu-
bated at 4 °C for 1 or 12 h before the filtrates were neutralized
1
00 mM potassium phosphate buffer (pH 7.4) and sheared to
homogeneity. Three 1 mL aliquots were equilibrated at 4, 23,
and 37 °C for 30 min. Next, 15 µL of CEO (53% pure as
determined by GC; 0.1 mmol) was added, and each sample was
incubated at the appropriate temperature for 30 min before
being stored at -80 °C.
Assa y P r oced u r es. Samples were prepared using the mild
acid hydrolysis method described by Ham et al. (5) with minor
modifications as described below.

3
30 Chem. Res. Toxicol., Vol. 14, No. 3, 2001
Morinello et al.
2
2
F igu r e 1. Structures of 1,N -ꢀGua, N ,3-ꢀGua, and their
bispentafluorobenzyl derivatives.
F igu r e 4. Full scan EI (A) and ECNCI (B) mass spectra of
2
2
3
,5-(PFB)
2
-1,N -ꢀGua. The spectra of 1,5-(PFB)
2
-1,N -ꢀGua are
similar.
2
F igu r e 2. Reverse-phase HPLC profile of 1,N -ꢀGua derivati-
9
2
zation products. Peak 1 was identified as O ,5-(PFB)
2
-1,N -ꢀGua.
2
Peaks 2 and 3 correspond to 1,5-(PFB)
2
-1,N -ꢀGua and 3,5-
2
(
PFB)
tuted derivative of HO-ethanoGua that contaminated the 1,N -
Gua synthesis product.
2
-1,N -ꢀGua, respectively. Peak 4 was due to a trisubsti-
2
ꢀ
F igu r e 5. 1H NMR spectra (500 MHz) of 1,5-(PFB)
-1,N -ꢀGua
2
2
2
(
top) and 3,5-(PFB)
2
-1,N -ꢀGua (bottom) in CDCl
3
(δ 7.24 ppm).
Chemical shifts are reported relative to TMS. The assignment
of the fragmented group is arbitrary.
MS in the EI and ECNCI modes (Figure 4A,B). The
identification of the isomers was made with NMR in
2
analogy to the pentafluorobenzyl derivatives of N ,3-ꢀGua
F igu r e 3. GC/ECNCI-MS total ion chromatogram of the crude
(
4), and in accordance with the work of Kjellberg and
2
1
,N -ꢀGua derivatization product. The first peak corresponds
J ohansson (25). The downfield shift of the H-2 proton in
2
2
to 3,5-(PFB) -1,N -ꢀGua, and the second corresponds to 1,5-
2
HPLC peak 3 relative to that in HPLC peak 2 indicates
(
2
PFB) -1,N -ꢀGua.
2
that the former corresponds to the 3,5-(PFB)
2
-1,N -ꢀGua
2
isomer, while the latter corresponds to the 1,5-(PFB) -
1,N -ꢀGua isomer (Figure 5). The 3,5-(PFB) -1,N -ꢀGua
2
isomer was monitored in subsequent quantitative experi-
ments since it produced the strongest signal in the
ECNCI mode (Figure 4B), and displayed superior chro-
by centrifugation at 5000g for 90 min. The samples were purified
with IA chromatography and analyzed as described above.
Sta tistica l An a lysis. All results are reported as the means
2
2
(
SD. Statistical analyses were completed with the aid of InStat
version 3.00 for Windows 95 (GraphPad Software, San Diego,
CA). Two-tailed p values were used in each case.
matographic properties. In the ECNCI mode, there was
2
significant fragmentation of 3,5-(PFB)
2
-1,N -ꢀGua into
Resu lts
-
-
both [M - 201] (m/z 334) and [M - 181] (m/z 354) ions.
-
The major (66%) [M - 201] fragment was chosen for
Ch a r a cter iza tion of P en ta flu or oben zyl Der iva -
2
2
2
quantitative analyses. The minor fragment of 3,5-(PFB) -
tives of 1,N -ꢀGu a . Four products of the 1,N -ꢀGua
2
1
,N -ꢀGua did not interfere with quantitation of the major
fragment of 3,5-(PFB) -N ,3-ꢀGua (and vice versa) since
2
derivatization were detected with HPLC (Figure 2). When
2
analyzed with ESI-MS, HPLC peaks 1-3 demonstrated
2
the two isomers were resolved by GC.
molecular ions that corresponded to (PFB)
2
-1,N -ꢀGua
derivatives. Two of these derivatives, corresponding to
HPLC peaks 2 and 3, were eluted from the GC column
when the crude product was analyzed by EI-MS (Figure
The NMR and ESI-MS spectra of HPLC peak 1 were
9
2
consistent with a novel O ,5-(PFB)
2
-1,N -ꢀGua derivative.
9
This derivative was identified as an O -substituted
isomer on the basis of the large upfield shift of the PFB
3
). Each of the isolated HPLC peaks was analyzed by GC/

Simultaneous Quantitation of Ethenoguanine Adducts
Chem. Res. Toxicol., Vol. 14, No. 3, 2001 331
Ta ble 1. Recover y of Ad d u ct Sta n d a r d s
%
recovered (mean ( SD)
2
2
n
1,N -ꢀGua
N ,3-ꢀGua
a
hydrolysis + IA
5
5
84.4 ( 2.2
85.3 ( 2.7
71.4 ( 2.6
84.3 ( 2.1
b
IA only
a
2
Recovery was determined by processing 177 fmol of 1,N -ꢀGua
2
and 50.0 fmol of N ,3-ꢀGua standards through the entire method
and adding both internal standards to the immunoaffinity eluent.
b
2
Recovery was determined by applying 177 fmol of 1,N -ꢀGua and
2
5
0.0 fmol of N ,3-ꢀGua standards directly to the columns and
adding both internal standards to the immunoaffinity eluent.
2
2
F igu r e 6. Calibration curves for N ,3-ꢀGua (b) and 1,N -ꢀGua
2
(
9) standards processed through the entire method (r > 0.995
in each case). The ratio of each analyte standard (AS) to the
appropriate internal standard (IS) was calculated.
2
F igu r e 7. Detection of 15 fmol of the spiked 1,N -ꢀGua
2
methylene protons relative to those in the 1,5- and 3,5-
standard (A) and 5 fmol of endogenous N ,3-ꢀGua (C) in 250 µg
of calf thymus DNA. The chromatograms corresponding to the
respective internal standards are shown in panels B and D.
2
(PFB) isomers. This substitution was unexpected since
no analogous product was reported from the pentafluoro-
2
benzylation of N ,3-ꢀGua (4). The ESI mass spectrum of
The accuracy of adduct measurements in samples of
HPLC peak 4 was consistent with a trisubstituted PFB
derivative of HO-ethanoGua (3). Although this related
adduct contaminated the product of the large-scale
hydrolyzed DNA was evaluated by spiking 200 µg of calf
2
thymus DNA with 177 fmol of the 1,N -ꢀGua standard
2
and 50.0 fmol of the N ,3-ꢀGua standard. The amount of
synthesis, its presence did not affect the quantitation of
2
2
1,N -ꢀGua detected in four samples was 177 ( 6 fmol.
1
,N -ꢀGua since the analytical standards were obtained
2
The N ,3-ꢀGua determinations were complicated by the
from a different source, and both were demonstrated to
be free of HO-ethanoGua with ESI-MS.
endogenous presence of this adduct in calf thymus DNA.
2
When the amount of N ,3-ꢀGua measured in an equiva-
Im m u n oa ffin ity Ch r om a togr a p h y. The recovery of
lent amount of control calf thymus DNA was subtracted
2
2
1
,N -ꢀGua and N ,3-ꢀGua standards from the IA columns
2
from the amount of N ,3-ꢀGua detected in the spiked
was evaluated by GC/ECNCI-HRMS (Table 1). No car-
ryover of either adduct from previous samples was
observed, and no decrease in the recovery of either adduct
was apparent after the columns were each used seven
times over a 3 month period.
2
DNA, the result was 56.4 ( 8.0 fmol of N ,3-ꢀGua.
Detection Lim its in DNA. The limits of quantitation
2
2
for 1,N -ꢀGua and N ,3-ꢀGua processed through the
entire method were determined in samples containing
2
calf thymus DNA. Known amounts of the 1,N -ꢀGua
Meth od P er for m a n ce. The incubation time for the
.1 N HCl DNA hydrolysis was increased from 30 to 60
standard were added to the DNA prior to hydrolysis.
Since N ,3-ꢀGua is present as an endogenous lesion in
2
0
min to account for the relatively strong glycosidic bond
DNA (5, 26), it was unnecessary to add any standard to
2
2
associated with 1,N -ꢀdGuo (2). 1,N -ꢀdGuo was quanti-
the samples. The demonstrated limits of quantitation for
2
2
2
tatively converted to 1,N -ꢀGua under these conditions
N ,3-ꢀGua and 1,N -ꢀGua in these samples were 7.6 and
15 fmol of adduct/sample, respectively (Figure 7). In the
∼250 µg DNA samples, these values corresponded to 5.0
2
2
(
data not shown). 1,N -ꢀGua and N ,3-ꢀGua standards
were processed together through the entire method to
generate the standard curves shown in Figure 6. When
the detector was operated at maximum sensitivity, its
2
2
8
N ,3-ꢀGua and 8.7 1,N -ꢀGua adducts/10 unmodified
Gua bases. The assay is inherently less sensitive toward
2
2
2
response was linear when analyzing samples containing
1,N -ꢀGua than N ,3-ꢀGua since only 24% of the N ,3-
2
5
-300 fmol of the N ,3-ꢀGua standard and 10-350 fmol
ꢀGua derivative is present as the minor isomer, and since
2
2
of the 1,N -ꢀGua standard. If the derivatization efficiency
3,5-(PFB)
2
-N ,3-ꢀGua fragments almost exclusively to the
2
-
and recovery of 1,N -ꢀGua are assumed to be equal to
[M - 181] ion (4).
2
those of N ,3-ꢀGua at 30% (4), these values correspond
DNA Rea ction s. Both adducts were formed in large
amounts in DNA treated with EtLA in the presence of
t-BuOOH (Figure 8). When 8 µg of DNA was analyzed
2
to quantitation limits of ∼200 amol of N ,3-ꢀGua and
2
∼
400 amol of 1,N -ꢀGua per injection.

3
32 Chem. Res. Toxicol., Vol. 14, No. 3, 2001
Morinello et al.
2
2
be 26.1 ( 3.3 1,N -ꢀGua and 4.75 ( 0.38 N ,3-ꢀGua
6
adducts/10 unmodified Gua bases.
2
1
,N -ꢀGua was also the predominant ethenoGua adduct
formed in reactions of DNA with HNE in the presence of
t-BuOOH (Table 2). When 5 µg of DNA from each of
2
triplicate reaction mixtures was analyzed, the mean 1,N -
2
ꢀ
Gua:N ,3-ꢀGua ratio was 38:1.
When calf thymus DNA was reacted with the VC
2
metabolite CEO at several temperatures, only N ,3-ꢀGua
was detected (Table 3). When 100 µg of CEO-treated calf
thymus DNA (reacted at 23 °C) was analyzed using IA
2
columns constructed using only 1,N -ꢀGua antibodies (to
prevent suppression due to the very large amounts of
2
2
N ,3-ꢀGua present in this DNA), the amount of 1,N -ꢀGua
was still below the limit of quantitation (data not shown).
2
2
Thus, the N ,3-ꢀGua:1,N -ꢀGua ratio observed in this
experiment is clearly much greater than that previously
reported using HPLC/fluorescence (3) and LC/MS/MS (7)
assays.
2
VC-Tr ea ted Ra ts. Only N ,3-ꢀGua, at a concentration
2
7
of 8.06 ( 2.58 N ,3-ꢀGua adducts/10 unmodified Gua
bases, was detected in hepatocyte DNA from three rats
exposed to 1100 ppm VC for 4 weeks (5 days/week) by
inhalation (Table 3). On the basis of the demonstrated
2
detection limit of 15 fmol of 1,N -ꢀGua/DNA sample, and
a mean sample size of 150 µg of DNA, the upper limit
2
for 1,N -ꢀGua in these samples was estimated to be 1.5
2
7
2
1
,N -ꢀGua adducts/10 unmodified Gua bases. 1,N -ꢀGua
was also not detected in brain and whole liver DNA from
similarly exposed rats (data not shown).
Ba sic p H In cu ba tion s. As shown in Figure 9, both
,N -ꢀGua and N ,3-ꢀGua were readily induced in calf
2
2
F igu r e 8. Formation of 1,N -ꢀGua (A) and N ,3-ꢀGua (C) in
2
2
1
2
00 µg of DNA treated with EtLA in the presence of t-BuOOH.
The chromatograms corresponding to the respective internal
standards are shown in panels B and D. The smaller peak in
panels B is due to the minor fragment of 3,5-(PFB)
thymus DNA incubated under basic conditions. Although
2
undetectable in control DNA, 1,N -ꢀGua was apparent
2
2
-1,N -ꢀGua
after a 1 h incubation at pH 10.5. The concentration of
at m/z 354.
2
N ,3-ꢀGua detected following the 1 h incubation was
2
2
similar to the endogenous concentration measured in
untreated DNA. The concentrations of both adducts were
increased significantly following a 12 h incubation under
the same conditions (p < 0.05).
Ta ble 2. F or m a tion of 1,N -EGu a a n d N ,3-EGu a in DNA
Rea cted w ith EtLA or HNE
6
mol of ꢀGua/10 mol of Gua
2
2
treatment
n
1,N -ꢀGua
N ,3-ꢀGua
t-BuOOH (EtLA control)
3
3
3
3
3
NDa
28.1 ( 3.5
28.3 ( 5.2
ND
0.14 ( 0.03c
5.28 ( 0.61
5.22 ( 0.96c
0.20 ( 0.02c
3.87 ( 1.33
Discu ssion
d
b
b
t-BuOOH + EtLA
t-BuOOH + EtLA
e
This IA/GC/ECNCI-HRMS assay is at least as sensitive
as the published LC/MS/MS methods (7, 8) for the
t-BuOOH (HNE control)
t-BuOOH + HNE
146 ( 66
2
quantitation of 1,N -ꢀGua in DNA, and offers the advan-
a
b
ND, not detectable (see the text). Significantly greater than
2
tage of simultaneous N ,3-ꢀGua measurements. It is
2
the amount of N ,3-ꢀGua in the same samples using the paired t
comparable in sensitivity, but is much more specific and
quantitative than the existing 32P-postlabeling method
(10). Furthermore, the potential for contamination or
artifactual adduct formation during sample preparation
is minimized by the mild and rapid immunoaffinity
enrichment procedure.
An unexpected finding was the apparent absence of
1,N -ꢀGua in reactions of CEO with calf thymus DNA.
The discrepancy between this and previous reports (3,
7) may be due to minor differences between the reaction
conditions, the higher specificity of this method for 1,N -
ꢀGua, or the complete avoidance of basic conditions. Here,
it was demonstrated that both ethenoguanine adducts
were readily formed by incubations of otherwise un-
treated DNA at pH 10.5. Although the sources of these
adducts were not identified in this study, it has been
reported that intermediates formed from the reaction of
c
test (p < 0.02). Significantly greater than the matched control
(
p < 0.05) using the unpaired t test with a Welch correction.
d
e
Eight micrograms of DNA analyzed. Sixteen micrograms of
DNA analyzed.
from each of triplicate reaction mixtures, the concentra-
2
tions were measured at 28.1 ( 3.5 1,N -ꢀGua and 5.28 (
2
6
2
0
2
.61 N ,3-ꢀGua adducts/10 unmodified Gua bases (Table
). The linearity of the assay was demonstrated when
similar adduct concentrations were measured after the
amount of DNA analyzed from each reaction was doubled
Table 2). The mean 1,N -ꢀGua:N ,3-ꢀGua ratio in these
2
2
2
(
six samples was 5.4:1. The precision of the assay was
2
demonstrated when 23.8 ( 1.5 1,N -ꢀGua and 4.57 ( 0.21
2
6
N ,3-ꢀGua adducts/10 unmodified Gua bases were found
in quadruplicate samples of 8 µg of DNA from a single
EtLA-DNA reaction. When the same sample was ana-
lyzed on three separate occasions over the course of 5
months, the adduct concentrations were determined to
2
HNE or its epoxide with dGuo can be converted to 1,N -
ꢀGua at this pH (17, 18). Thus, there appears to be a

Simultaneous Quantitation of Ethenoguanine Adducts
Chem. Res. Toxicol., Vol. 14, No. 3, 2001 333
Ta ble 3. Qu a n tita tion of EGu a Ad d u cts in Ca lf Th ym u s a n d Ra t Hep a tocyte DNA
6
mol of ꢀGua/10 mol of Gua
2
2
DNA sample
n
DNA (µg)
1,N -ꢀGua
N ,3-ꢀGua
untreated calf thymus DNA
8
300
5
5
5
150
NDa,b
ND
ND
ND
ND
0.08 ( 0.03
1330 ( 20
1510 ( 50
1730 ( 50
0.81 ( 0.26
c
CEO-treated calf thymus DNA (4 °C)
CEO-treated calf thymus DNA (25 °C)
CEO-treated calf thymus DNA (37 °C)
VC-exposed rat hepatocytes
3
3
3
3
c
c
a
2
b
n ) 3 since the 1,N -ꢀGua standard was added to five other samples for detection limit determinations. Not detectable (see the
c
text). Samples prepared from a single DNA reaction.
2
artifactually. Since 1,N -ꢀGua was not detected with the
GC/ECNCI-HRMS method in untreated calf thymus
DNA at neutral pH, its endogenous concentration ap-
pears to be very low under normal physiological condi-
tions. Thus, despite the additional sensitivity offered by
2
both MS-based methods, the existence of 1,N -ꢀGua in
vivo remains unproven. However, the results of the EtLA
2
and HNE experiments suggest that 1,N -ꢀGua may play
a significant role when tissues are subjected to oxidative
stress. This assay will be used in the future to determine
2
if significant amounts of endogenous 1,N -ꢀGua are
present in animal models, disease states, or dietary
conditions that promote lipid peroxidation.
2
2
F igu r e 9. Formation of 1,N -ꢀGua and N ,3-ꢀGua in untreated
DNA under basic conditions. The results that are shown are
the means ( SD of three reactions. Calf thymus DNA (200 µg)
was incubated in pH 10.5 buffer for the indicated times as
The combination of IA chromatography and MS has
had a significant impact on DNA adduct research in
recent years. These techniques, coupled with the use of
isotopically labeled internal standards, allow highly
specific and precise measurements of adducts that are
present in parts per million concentrations or lower
relative to unmodified bases. To the best of our knowl-
edge, this is the first example of the use of hybrid IA
columns to purify multiple adducts from the same sample
of hydrolyzed DNA. In this case, the similar chemical
properties of these structural isomers allowed knowledge
2
described in detail in Experimental Procedures. 1,N -ꢀGua was
not detected in the control DNA [an asterisk denotes that the
level is significantly greater than that of N ,3-ꢀGua in the same
samples by a paired t test (p < 0.05); the dot denotes that the
level is significantly increased compared to the value at 1 h with
ANOVA followed by the Tukey-Kramer multiple-comparisons
test (p < 0.01)].
2
significant potential to form ethenoguanine adducts
during sample preparation and analysis, and these
results strongly suggest that basic conditions should be
avoided at all times.
2
gained from the study of N ,3-ꢀGua to be applied to the
2
quantitation of 1,N -ꢀGua. The capability to measure
In lieu of positive results from the CEO-DNA samples,
the reactions of EtLA and HNE with DNA served as
positive controls during method development. As reported
levels of both ethenoGua adducts with one procedure will
greatly aid in assessing the potential role of each in
carcinogenesis under various conditions.
2
by Ham et al. using LC/MS/MS (9), both 1,N -ꢀGua and
2
N ,3-ꢀGua were formed from the reaction of either EtLA
Ack n ow led gm en t. We thank Dr. Raymond Schroed-
er (Huntington Life Sciences) for supervising the rat
exposures, Dr. F. P. Guengerich (Vanderbilt University)
or HNE with DNA in the presence of t-BuOOH. Although
2
1
,N -ꢀGua was the predominant ethenoGua adduct in
2
2
each case, the ratio of 1,N -ꢀGua:N ,3-ꢀGua formed from
the reaction of DNA with EtLA was lower than that
formed from the reaction with its product, HNE. This
suggests that products other than HNE are involved in
2
13
2
3
for providing 1,N -ꢀdGuo and [ C ]-1,N -ꢀdGuo for ana-
lytical standards, and Mr. Sherif Ghobrial (University
of North Carolina) for performing the CEO-DNA reac-
tions. Additional support was provided by NIH Training
Grants ES07126 and CA72319, the Superfund Basic
Research Program under NIH Grant ES05948, and the
Chemical Manufacturer’s Association.
2
the formation of N ,3-ꢀGua when DNA is subjected to
peroxidizing conditions.
The results of 1,N -ꢀGua and N ,3-ꢀGua measurements
2
2
in hepatocyte DNA from rats exposed to VC mirrored the
2
findings from the CEO-DNA reactions. While N ,3-ꢀGua
Refer en ces
was formed in relatively large amounts following expo-
sure to VC, no 1,N -ꢀGua was detected in these samples.
Although it may be possible to detect small amounts of
,N -ꢀGua by analyzing greater amounts of DNA, these
data suggest that 1,N -ꢀGua plays a minor role in VC-
induced carcinogenesis relative to N ,3-ꢀGua in this
2
(
1) Oesch, F., and Doerjer, G. (1982) Detection of N ,3-ethenoguanine
in DNA after treatment with chloroacetaldehyde in vitro. Car-
cinogenesis 3, 663-665.
2
2
1
(2) Kusmierek, J . T., and Singer, B. (1992) 1,N -Ethenodeoxygua-
2
2
nosine: Properties and formation in chloroacetaldehyde-treated
polynucleotides and DNA. Chem. Res. Toxicol. 5, 634-638.
2
(
3) Guengerich, F. P., Persmark, M., and Humphreys, W. G. (1993)
model.
2
2
Formation of 1,N - and N ,3-ethenoguanine from 2-haloox-
iranes: Isotopic labeling studies and isolation of a hemiaminal
2
The level of 1,N -ꢀdGuo was recently determined with
LC/MS/MS in calf thymus DNA to be 2.79 adducts/10
unmodified dGuo bases (8), a concentration that would
be easily detected by the GC/MS method. However, the
DNA in that experiment was apparently dissolved at pH
.4, a condition that seems likely to generate 1,N -ꢀGua
6
2
derivative of N -(2-oxoethyl)guanine. Chem. Res. Toxicol. 6, 635-
6
48.
(
4) Fedtke, N., Boucheron, J . A., Turner, M. J ., and Swenberg, J . A.
1990) Vinyl chloride-induced DNA adducts. I: Quantitative
(
2
determination of N ,3-ethenoguanine based on electrophore label-
ing. Carcinogenesis 11, 1279-1285.
2
9

3
34 Chem. Res. Toxicol., Vol. 14, No. 3, 2001
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(
5) Ham, A. J . L., Ranasinghe, A., Morinello, E. J ., Nakamura, J .,
Upton, P. B., J ohnson, F., and Swenberg, J . A. (1999) Immunoaf-
finity/gas chromatography/high-resolution mass spectrometry
of nucleotides opposite five-membered exocyclic ring guanine
derivatives by Escherichia coli polymerases in vitro and in vivo:
2
1,N -ethenoguanine, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]pu-
2
method for the detection of N ,3-ethenoguanine. Chem. Res.
rine, and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine.
Biochemistry 37, 5184-5193.
Toxicol. 12, 1240-1246.
(
6) Foiles, P. G., Miglietta, L. M., Nishikawa, A., Kusmierek, J . T.,
(16) Langou e¨ t, S., M u¨ ller, M., and Guengerich, F. P. (1997) Misincor-
Singer, B., and Chung, F. L. (1993) Development of monoclonal
poration of dNTPs opposite 1,N -ethenoguanine and 5,6,7,9-
2
2
2
antibodies specific for 1,N -ethenodeoxyguanosine and N ,3-
ethenodeoxyguanosine and their use for quantitation of adducts
in G12 cells exposed to chloroacetaldehyde. Carcinogenesis 14,
tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in oligonucle-
otides by Escherichia coli polymerases I exo- and II exo-, T7
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1
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