Ultrasensitive simultaneous quantification of 1, N 2-etheno-2'- deoxyguanosine and 1, N 2-propano-2'-deoxyguanosine in DNA by an online liquid chromatography-electrospray tandem mass spectrometry assay

Article abstract of DOI:10.1021/tx1001018

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N²-etheno-2'-deoxyguanosine (1,N ²-εdGuo) and 1,N²-propano-2'-deoxyguanosine (1,N ²-propanodGuo) in DNA. This methodology permits direct online quantification of 2'-deoxyguanosine and ca. 500 amol of adducts in 100 μg of hydrolyzed DNA in the same analysis. Using the newly developed technique, accurate determinations of 1,N²-etheno-2'-deoxyguanosine and 1,N ²-propano-2'-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 ± 0.32 1,N²-εdGuo/10⁸ dGuo and 3.43 ± 0.33 1,N²-propanodGuo/10⁸ dGuo) and rat tissue (liver, 2.47 ± 0.61 1,N²-εdGuo/10⁸ dGuo and 4.61 ± 0.69 1,N²-propanodGuo/10⁸ dGuo; brain, 2.96 ± 1.43 1,N²-εdGuo/10⁸ dGuo and 5.66 ± 3.70 1,N²-propanodGuo/10⁸ dGuo; and lung, 0.87 ± 0.34 1,N²-εdGuo/10⁸ dGuo and 2.25 ± 1.72 1,N²-propanodGuo/10⁸ dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals with pathological conditions and after air pollution exposure.

Full text of DOI:10.1021/tx1001018

Chem. Res. Toxicol. 2010, 23, 1245–1255
1245
Ultrasensitive Simultaneous Quantification of
2
2
1
,N -Etheno-2′-deoxyguanosine and 1,N -Propano-2′-deoxyguanosine
in DNA by an Online Liquid Chromatography-Electrospray
Tandem Mass Spectrometry Assay
Camila C. M. Garcia, Flor eˆ ncio P. Freitas, Paolo Di Mascio, and Marisa H. G. Medeiros*
Departamento de Bioqu ´ı mica, Instituto de Qu ´ı mica, UniVersidade de S a˜ o Paulo,
CEP 05508-000, S a˜ o Paulo, Brazil
ReceiVed March 15, 2010
Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential
tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving
online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry
detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards
2
2
was developed for the simultaneous quantification of 1,N -etheno-2′-deoxyguanosine (1,N -εdGuo) and
2
2
1
,N -propano-2′-deoxyguanosine (1,N -propanodGuo) in DNA. This methodology permits direct online
quantification of 2′-deoxyguanosine and ca. 500 amol of adducts in 100 µg of hydrolyzed DNA in the
2
same analysis. Using the newly developed technique, accurate determinations of 1,N -etheno-2′-
2
deoxyguanosine and 1,N -propano-2′-deoxyguanosine levels in DNA extracts of human cultured cells
2
8
2
8
(
4.01 ( 0.32 1,N -εdGuo/10 dGuo and 3.43 ( 0.33 1,N -propanodGuo/10 dGuo) and rat tissue (liver,
2 8 2 8
2
1
1
.47 ( 0.61 1,N -εdGuo/10 dGuo and 4.61 ( 0.69 1,N -propanodGuo/10 dGuo; brain, 2.96 ( 1.43
2 8 2 8 2
,N -εdGuo/10 dGuo and 5.66 ( 3.70 1,N -propanodGuo/10 dGuo; and lung, 0.87 ( 0.34 1,N -εdGuo/
8
2
8
0 dGuo and 2.25 ( 1.72 1,N -propanodGuo/10 dGuo) were performed. The method described herein
can be used to study the biological significance of exocyclic DNA adducts through the quantification of
different adducts in humans and experimental animals with pathological conditions and after air pollution
exposure.
Introduction
deoxyguanosine adducts (15, 16). These adducts can also be
formed by the reaction of acetaldehyde (AA) with dGuo
Exocyclic DNA adducts arise from the reaction of DNA
with lipid peroxidation products, such as malonaldehyde,
-hydroxy-2(E)-nonenal, 4-oxo-2(E)-nonenal, 2,4-decadienal,
acrolein, and crotonaldehyde (CRO ) (1, 2). The direct
addition of R,ꢀ-unsaturated aldehydes to DNA bases yields
cyclic substituted propano adducts, such as 1,N -propano-
(
17, 18). AA is present in tobacco smoke, vehicle exhaust
due to alcohol fuel combustion, and some beverages and
foods. It is endogenously formed by the metabolic oxidation
of ethanol through hepatic NAD-dependent alcohol dehy-
drogenases in the liver and during threonine catabolism
4
1
2
(
19-21). The production of significant amounts of acetal-
2
′-deoxyguanosine (3, 4). Alternatively, R,ꢀ-unsaturated
dehyde in saliva was observed after the ingestion of moderate
aldehydes can be oxidized to reactive epoxides, giving ethano
or etheno derivatives upon reaction with DNA (5, 6). Some
of these DNA lesions have been shown to be highly
mutagenic (7-9) and are considered to be possible pathways
leading to the carcinogenic effects involved in the lipid
peroxidation process (10).
amounts of ethanol (22). AA reacts with dGuo in DNA, first
2
forming N -ethylidene-dGuo (18). The subsequent reaction
2
of another molecule of AA with N -ethylidene-dGuo gives
2
rise to 1,N -propano-2′-deoxyguanosine (Scheme 1) in both
the (6S,8S) and (6R,8R) configurations. The reaction between
acetaldehyde and dGuo favors the formation of the (8R,6R)
CRO is an important industrial chemical and an environ-
mental pollutant formed by the combustion of plant materials,
including tobacco, and mobile source emissions. Additionally,
it is generated endogenously by the oxidative degradation
of unsaturated lipids and as a metabolite of N-nitrosopyrro-
lidine (11, 12). CRO is mutagenic and carcinogenic (13, 14);
diastereomer, while the reaction involving CRO promotes
2
(
6S,8S) formation (18). In addition, AA-1,N -propano-2′-
deoxyguanosine adduct formation is stimulated by polyamines
and histones (23, 24). In DNA, the 1,N -propano-2′-deoxy-
2
guanosine adduct exists in an equilibrium between the closed
and opened forms because the opened form is more favored
in double-stranded DNA, and the closed-form is predominant
in single-stranded DNA (25).
its reaction with dGuo in DNA leads to the formation of the
6S,8S) and (6R,8R) diastereomers of 1,N -propano-2′-
2
(
2
1
,N -PropanodGuo promotes DNA miscoding in human cells,
*
To whon correspondence should be addressed. Tel: ++ (55) 11
principally through G f T transversions, and can inhibit DNA
synthesis (8, 26). The ring-opened form can lead to an
interstrand cross-link when the adduct is formed in a 5′ CpG
sequence (27). Cross-links involving DNA and proteins can also
occur as shown by Kurtz and Lloyd after the reaction between
3
1
0912153. Fax: ++ (55) 11 30912186. E-mail: mhgdmede@iq.usp.br.
1
2
Abbreviations: CRO, crotonaldehyde; AA, acetaldehyde; 1,N -εdGuo,
2
2
2
,N -etheno-2′-deoxyguanosine; 1,N -propanodGuo, 1,N -propano-2′-deoxy-
guanosine; dGuo, 2′-deoxyguanosine; HPLC/ESI/MS-MS, high performance
liquid chromatography/electrospray ionization tandem mass spectrometry;
MRM, multiple reaction monitoring.
1
0.1021/tx1001018  2010 American Chemical Society
Published on Web 06/15/2010

1
246 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Garcia et al.
2
2
Scheme 1. Formation of 1,N -PropanodGuo in the Reaction
ogy enabled us to determine the basal levels of 1,N -εdGuo
2
of Acetaldehyde or Crotonaldehyde with dGuo
and, 1,N -propanodGuo in cultured human lung fibroblasts
(
IMR-90 cells) and in liver, lung, and brain tissues from male
rats.
Experimental Procedures
Chemicals. All of the chemicals employed were of the highest
1
5
purity grade commercially available. [ N ]-2′-Deoxyguanosine
5
was provided by Cambridge Isotope Laboratories (Andover,
MA). Chromatography grade acetonitrile, 2-propanol, and etha-
nol were obtained from Merck (Darmstadt, Germany). Fetal calf
serum was acquired from Atena Biotecnologia (Campinas, SP,
Brazil). 2′-Deoxyguanosine (dGuo), formic acid, sodium hy-
droxide, potassium phosphate, magnesium sulfate, saccharose,
magnesium chloride, acetaldehyde, 2-chloroacetaldehyde, and
all the other chemicals used were from Sigma (St. Louis, MO).
Water was purified using a Milli-Q system (Millipore, Bedford,
MA).
DNA Modified in ViWo. Twenty-week-old untreated male Wistar
rats (n ) 21) were sacrificed by using a guillotine. Their livers,
lungs, and brains were removed, snap-frozen in liquid nitrogen,
and stored at -80 °C.
Cell Culture. Human lung fibroblasts (line IMR-90) were
routinely grown in Dulbecco’s modified Eagle’s medium (DMEM,
pH 7.4) supplemented with 10% (v/v) fetal calf serum, 3.7 g/L
sodium bicarbonate, 0.04 g/L penicillin, and 0.1 g/L streptomycin
2
sulfate. The cells were incubated in a CO /air atmosphere (1:
1
9) at 37 °C and were allowed to grow until confluence. DNA
was extracted, hydrolyzed, and analyzed as described below.
DNA Extraction. DNA was isolated by the modified chao-
tropic NaI method, as previously described (34). Briefly, the
8
tissues (500 mg) or the cellular pellets (3 × 10 cells) were
the free aldehyde group of the adduct ring-opened form and
peptides (28).
homogenized in 10 mL of a lysis solution (320 mM sucrose, 5
mM MgCl
2
, 10 mM Tris-HCl, 0.1 mM desferroxamine, and 1%
Etheno adducts have been used as biomarkers for DNA
damage resulting from the reactions of endogenous lipid
(
1
v/v) Triton X-100 at pH 7.5). After centrifugation at 1500g for
0 min, the pellets were resuspended in 10 mL of the lysis
2
peroxidation end products (29). 1,N -Etheno-2′-deoxygua-
solution and centrifuged one more time at 1500g for 10 min.
The pellets were then suspended in 6 mL of 10 mM Tris-HCl
buffer (pH 8.0) containing 5 mM EDTA, 0.15 mM desferrox-
amine, and 350 µL of 10% SDS. The enzymes RNase A (30
µL, 10 mg/mL) and RNase T1 (4 µL, 20 U/µL) in 10 mM Tris-
HCl buffer (pH 7.4) containing 1 mM EDTA and 2.5 mM
desferroxamine were added, and the reaction mixture was
incubated at 37 °C. After 1 h, 300 µL of proteinase K (20 mg/
mL) was added, and the reaction was incubated at 37 °C for
2
nosine (1,N -εdGuo) can be formed by the reaction between
2
′-deoxyguanosine and reactive aldehydes, such as chloro-
acetaldehyde, 2,3-epoxyaldehydes and vinyl chloride, that are
derived from oxidized lipids, sugars, or amino acids
2
(
6, 30, 31).The biological relevance of the occurrence of 1,N -
εdGuo has been revealed by studies showing the mutagenicity
of this etheno adduct both in Escherichia coli and in human
2
cells (7, 9, 32, 33). The presence of 1,N -εdGuo leads to
1
h. After centrifugation at 5000g for 15 min, the liquid phase
misincorporation and mutations in in Vitro systems involving
DNA polymerases in bacteria and mammalian cells (7, 32, 33).
Recently, it was reported that the human DNA polymerase
was collected, 1 mL of a solution containing 7.6 M NaI, 40
mM Tris-HCl (pH 8), 20 mM EDTA, and 0.3 mM desferrox-
amine was added, and then 5 mL of isopropanol was added.
The contents of the tube were mixed well by inversion until a
white precipitate appeared. The precipitate was collected by
centrifugation at 5000g for 15 min, washed with 5 mL of 60%
isopropanol, centrifuged at 5000g for 15 min, washed with an
additional 5 mL of 70% ethanol, and centrifuged at 5000g for
2
κ produces frame shift deletions when bypassing a 1,N -
εdGuo adduct in DNA (9).
Today, exocyclic DNA adducts are emerging as new tools
in the study of oxidative stress-related diseases and cancer
etiology. Several methods, including 3 P-postlabeling, competi-
tive immunoassay, gas chromatography/electron capture nega-
tive chemical ionization high-resolution mass spectrometry (GC/
ECNCI-HRMS), and high performance liquid chromatography/
tandem mass spectrometry (HPLC/ESI/MS-MS), have been
employed to quantify DNA adducts and have been previously
reviewed (1, 2, 10).
2
1
5 min. The DNA pellet was solubilized in 500 µL of
desferroxamine (0.1 mM). The DNA concentration was measured
spectrophotometrically at 260 nm.
2
Scheme 2. Structure of DNA Adducts, 1,N -PropanodGuo
2
and 1,N -εdGuo
This work describes the development and validation of a
sensitive method for the simultaneous quantitative determi-
2
2
nation of 1,N -εdGuo and 1,N -propanodGuo from in Vitro
and in ViVo samples using online reverse-phase HPLC
separation with tandem mass spectrometry detection by
multiple reaction monitoring (MRM). In addition, the tech-
nique permits the detection and quantification of dGuo from
crude or extracted DNA in the same analysis. This methodol-

HPLC-MS/MS Quantification of Exocyclic DNA Adducts
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1247
2
Figure 1. Detection of a 500 amol of DNA adducts standard by HPLC/ESI/MS-MS analysis in the MRM mode. (A) 1,N -εdGuo: m/z 292 f 176.
1
5
2
2
15
2
(
B) [ N
5
]-1,N -εdGuo: m/z 297 f 181. (C) 1,N -propanodGuo: m/z 338 f 222. (D) [ N
5
]-1,N -propanodGuo: m/z 343 f 227.
1
5
2
Enzymatic Hydrolysis of DNA. Sodium acetate buffer (1 M,
Synthesis of the [ N
5
]-1,N -Etheno-2′-deoxyguanosine Inter-
15
2
15
2
15
pH 5, 4 µL) and 33 fmol of [ N
5
]-1,N -εdGuo were added to an
nal Standard. [ N
5
5
]-1,N -εdGuo was obtained by reacting [ N ]-
dGuo with chloroacetaldehyde with subsequent purification by
HPLC, as described by Loureiro et al. (36). The identity of the
5
[ N ]-adduct was confirmed by mass spectrometry analysis.
aliquot of a 0.1 mM desferroxamine solution containing 200 µg of
DNA. The DNA was then digested with 2 units of nuclease P1 at
1
5
3
7 °C for 30 min. Tris-HCl buffer (1 M, pH 7.4, 8 µL), 8 µL of
2
phosphatase buffer, and 6 units of alkaline phosphatase were then
added for an additional 1 h of incubation at 37 °C. The final volume
of the solution was adjusted to 200 µL with water before the second
incubation. The enzymes were precipitated by centrifugation at
Synthesis of the 1,N -Propano-2′-deoxyguanosine (6R,8R)
and (6S,8S) Unlabeled Standards. dGuo (25 µmol) was dissolved
in 2 mL of phosphate buffer (50 mM, pH 7.5) containing 1 mmol
of AA and 0.05 mmol of lysine (as a catalyst for adduct
formation). The solution was mixed at 500 rpm at 37 °C for
12 h. The adducts were purified by HPLC (Shimadzu, Kyoto,
Japan) with a Luna C18(2) analytical column (250 mm ×4.6
mm i.d., 5 µm, Phenomenex, Torrance, CA). The following
water/acetonitrile gradient method was used: from 0 to 30 min,
0 to 8% acetonitrile and 0.8 to 0.5 mL/min; from 30 to 50 min,
8 to 15% acetonitrile and 0.5 to 0.6 mL/min; from 50 to
60 min, 15 to 50% acetonitrile, 0.6 to 0.8 mL/min; from 60
to 65 min, 50 to 0% acetonitrile and 0.8 mL/min; and from 65
to 70 min, 0% acetonitrile and 0.8 mL/min.
5
000g for 3 min, and the resulting aqueous layer was subjected to
HPLC/ESI/MS-MS analysis (100 µL of the DNA solution/injec-
tion). The amounts of the reagents and labeled internal standards
were proportionally adjusted for hydrolysis and analysis of other
DNA quantities.
2
Synthesis of the 1,N -Etheno-2′-deoxyguanosine Unlabeled
2
Standard. The 1,N -εdGuo unlabeled standard was obtained by
reacting dGuo with chloroacetaldehyde with subsequent purification
by HPLC, as described by Loureiro et al. (35). The identity of the
1
compound was confirmed by ESI/MS and H NMR.

1
248 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Garcia et al.
Figure 2. (A) Enzymatically hydrolyzed DNA; λ ) 260 nm. Conditions were as described in the Experimental Procedures section. (B) Positions
of the automated switching valve during the HPLC/ESI/MS-MS analysis, cleaning and mass spectrometer positions.
The identities of the two diastereomeric products were confirmed
through the second column to the mass spectrometer. The total time
spent on this analysis was 32 min.
by the following spectroscopic features: UV λmax 260 nm, ε ) 15600
-
1
-1
-1
-1
15
2
M
cm (6S,8S) and ε ) 15700 M cm (6R,8R) (SPD-E10A/
5
The DNA hydrolysates containing 33 fmol of the [ N ]-1,N -
1
5
2
VP Shimadzu). ESI/MS: m/z 338 ([M + H - 2-D-erythro-
pentose] ), 222 ([M + H] ). H, H- H COSY NMR (D
εdGuo and [ N ]-1,N -propanodGuo internal standards were
5
+
+
1
1
1
+
2
O) (Bruker
injected into the system described above. The [M + H] ions
2
DRX 500 MHz). Circular dichroism was recorded in water on a
JASCO J-720 spectropolarimeter (Figures 1 and 2, Supporting
Information).
corresponding to the m/z values 292/176 (1,N -εdGuo), 297/181
1
5
2
2
([ N ]-1,N -εdGuo) and 338/222 (1,N -propanodGuo), 343/
5
1
5
2
227([ N ]-1,N -propanodGuo) were monitored with a dwell time
5
1
5
2
Synthesis of the [ N
5
]-1,N -Propano-2′-deoxyguanosine Internal
of 200 ms.
15
2
15
Standard. [ N
5
]-1,N -propanodGuo was obtained by reacting [ N
5
]-
All of the parameters of the mass spectrometer were adjusted
+
dGuo with AA with subsequent purification by HPLC, as described
for acquisition of the best [M + H] /[M + H - 2-D-erythro-
1
5
pentose]+ transition. The curtain gas was adjusted to 25 psi, the
source temperature was held at 450 °C, the nebulizer and auxiliary
gas were maintained at 60 psi, the Turbo Ion Spray voltage was
above. The identity of the [ N
5
]-adduct was confirmed by mass
15
2
spectrometry analysis. ESI/MS for [ N
43 ([M + H - 2-D-erythro-pentose] ), 227 ([M + H] ).
5
]-1,N -propanodGuo: m/z
+
+
3
5
500 V, the collision gas was set on high, the interface heater was
held at 100 °C, and the entrance potential was set to 10 V. For the
38/222 and 343/227 transitions, the following were selected:
High-Performance Liquid Chromatography/Electrospray Ion-
ization Tandem Mass Spectrometry (HPLC/ESI/MS-MS). Online
HPLC/ESI/MS-MS analyses were carried out in the positive
mode using an API-4000 QTRAP mass spectrometer (Applied
3
collision energy, 19 V; collision cell exit, 20 V; and declastering
potential, 51 V. For the 292/176 and 297/181 transitions were
selected 17 V of collision energy, 16 V of collision cell exit, and
2
2
Biosystems, Foster City, CA). The 1,N -εdGuo and 1,N -
propanodGuo adducts in the DNA samples were detected by
multiple reaction monitoring (MRM). An Agilent HPLC system
4
1 V of declastering potential. The data were processed using
Analyst 1.4.2 software.
(
Kyoto, Japan) consisting of an autosampler (1200 High
performance), a column oven at 18 °C (1200 G1216B), an
automated switching valve, a 1200 Binary Pump SL, a 1200
Isocratic Pump SL, and a UV detector (1200 DAD G1315C)
were used for sample injection and cleanup of the analytical
column (Luna C18(2), 250 mm ×4.6 mm i.d., 5 µm, Phenom-
enex, Torrance, CA). The adduct was eluted from this column
with a gradient of water and acetonitrile containing 0.1% formic
acid with the following method: from 0 to 10 min, 10 to 40%
acetonitrile and 0.65 to 0.2 mL/min; from 10 to 16 min, 40 to
Results
The online HPLC/ESI/MS-MS methodology reported here
permits direct quantification of two DNA adducts, 1,N -
propanodGuo and 1,N -εdGuo (Scheme 2), formed from the
reaction of dGuo with exogenous or endogenous reactive
aldehydes. Figure 1 shows a representative chromatogram
2
2
2
2
3
0% acetonitrile and 0.2 mL/min; from 16 to 20 min, 30 to 60%
of 1,N -εdGuo and 1,N -propanodGuo (Figure 1A and C).
15 2
acetonitrile and 0.2 mL/min; from 20 to 21 min, 60 to 40%
acetonitrile and 0.2 mL/min; from 21 to 22 min, 40 to
5
The use of the stable isotopic internal standards [ N ]-1,N -
15
2
εdGuo and [ N ]-1,N -propanodGuo, five units of mass larger
than the native adducts, ensures accurate quantification of
5
9
9
1
0% acetonitrile and 0.2 to 0.65 mL/min; from 22 to 26 min,
0% acetonitrile and 0.65 mL/min; from 26 to 27 min, 90 to
0% acetonitrile and 0.65 mL/min; from 27 to 32 min, 10%
the adducts (Figure 1B and D). The internal standards were
synthesized as described in the Experimental Procedures
section, characterized by UV and ESI-MS/MS and quantified
with the UV extinction coefficients of the unlabeled adducts.
The stable isotopic internal standards (33 fmol) were added
to the DNA samples prior to enzymatic hydrolysis. The HPLC
program developed herein allowed for adequate separation
of the adducts from the last eluted nucleoside (dThd), as
shown in Figure 2A. The method also allowed the simulta-
neous quantification of dGuo from hydrolyzed DNA samples
acetonitrile and 0.65 mL/min.
An isocratic pump was used to simultaneously load a second
column (Eclipse XDB-C18, 150 mm × 4.6 mm i.d., 5 µm, Agilent)
at 0.2 mL/min with a solution of 60:40 water/acetonitrile with 0.1%
formic acid and maintained a constant flow of the mobile phase to
the mass spectrometer during the analysis. The position of the
switching valve was changed twice: at 13 min to allow the eluent
from the first column to enter the second column and at 25 min to
permit the first column to be washed while the adduct was eluted

HPLC-MS/MS Quantification of Exocyclic DNA Adducts
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1249
2
15
2
)
0.993) and 1,N -propanodGuo to [ N
5
]-1,N -propanodGuo
Figure 3B, r ) 0.999) from each injection were plotted against
the amount (in fmol) of the unlabeled adduct injected (0.01-100
2
(
15
5
fmol/injection). The amount of the [ N ]-labeled internal
standard that was injected was kept constant at 33 fmol. Blank
injections (water) under the same conditions revealed that no
carryover of adducts from previous analyses occurred (data not
shown).
The limit of detection and the possible suppressor effect
of the excess amount of normal DNA bases on the response
of the adduct were evaluated using hydrolyzed calf thymus
DNA spiked with different quantities (0 to 100 fmol) of the
1
5
5
unlabeled DNA adduct standards and 33 fmol of the [ N ]
DNA adducts. All of the standards were added prior to DNA
hydrolysis. The limit of detection of both adducts (S/N ) 3
(
0.2) was 150 amol on the column, and the limit of
quantification (S/N ) 10 ( 0.3) was 500 amol on the column,
9
corresponding to approximately 9.5 adducts/10 dGuo in 100
µg of hydrolyzed DNA. The basal levels present in the calf
thymus DNA were minimized from all DNA samples before
quantification and validation. Figure 4 shows a representative
chromatogram of calf thymus DNA contaminated with 500
amol of adduct standards, corresponding to an S/N value
of approximately 10, the limit of quantification for each
adduct.
The accuracy and precision of the method were determined
by analysis of nine different amounts of hydrolyzed calf
2
thymus DNA contaminated with 1 fmol of 1,N -εdGuo, 1
2
fmol of each diastereomer of the 1,N -propanodGuo stan-
dards, and 33 fmol of each internal standard. These injections
were performed on three consecutive days with three injec-
tions per day. Table 1 summarizes the quantification of the
adducts and the coefficients of variance of the analysis within
the same day and on subsequent days. The values accurately
reflect the known additional amounts of adduct. On the first
2
Figure 3. HPLC/ESI/MS-MS calibration curve for 1,N -etheno-2′-
2
deoxyguanosine (A) and 1,N -propano-2′-deoxyguanosine (B), ob-
tained by plotting the relative area ratios of unlabeled adducts to
1
5
2
the isotopically
N
5
-labeled adduct vs increasing amounts of 1,N -
2
etheno-2′-deoxyguanosine and 1,N -propano-2′-deoxyguanosine.
Conditions were as described in the Experimental Procedures
section.
2
2
occasion, the 1,N -εdGuo and 1,N -propanodGuo levels were
8
found to be 0.95 ( 0.07 fmol or 0.52 ( 0.03 adducts/10
8
dGuo and 2.06 ( 0.10 fmol or 1.14 ( 0.05 adducts/10 dGuo,
respectively. On the second occasion, the measured levels
through absorbance measurements (λ ) 260 nm) in the UV
detector (Figure 2A). An automated switching valve (Figure
B) was programmed to change its position in the interval
between the end of the elution of the last DNA base and the
8
were 0.99 ( 0.04 fmol or 0.52 ( 0.04 adducts/10 dGuo
8
and 2.07 ( 0.11 fmol or 1.08 ( 0.07 adducts/10 dGuo,
2
respectively, and on the third occasion, they were 1.04 (
8
2
0.02 fmol or 0.58 ( 0.01 adducts/10 dGuo and 2.08 ( 0.1
beginning of the elution of the first DNA adduct (1,N -
8
fmol or 1.14 ( 0.03 adducts/10 dGuo, respectively (Table
εdGuo). The automated switching valve directed the unmodi-
fied nucleotides, after elution through column 1 and UV
detection, to the waste (cleaning time 13 min), preventing
the loss of mass spectrometer sensitivity. The adducts were
then eluted through a second narrow bore column at a low
flow rate to concentrate the adduct into a sharp peak,
increasing the sensitivity of mass spectrometer detection in
MRM mode. The m/z transition chosen for the MRM
detection experiments corresponds to the [M + H - 2-D-
erythro-pentose]+ ion of the adducts, a predominant frag-
mentation product characterized by the loss of 116 mass units
1
). The chromatograms (Figure 5) show one of the DNA
samples that contained 1 fmol of unlabeled adduct. The S/N
ratio of the unlabeled adducts shown in Figure 5 is ap-
proximately two times higher than the S/N ratio obtained
with 500 amol of the unlabeled standards (Figure 4),
confirming the linearity in adduct quantification and the fact
that larger hydrolyzed DNA samples did not cause suppres-
sion of the adduct response.
The newly developed methodology was applied to the
2
2
quantification of the basal levels of 1,N -εdGuo and 1,N -
propanodGuo in cultured IMR-90 cells and in the liver, brain,
and lung tissues from untreated male Wistar rats. Analysis
of DNA extracted from the cultured cells (n ) 7 trials)
+
from MH . Representative ion chromatograms obtained by
MRM detection using the m/z transitions from 292 to 176
]-1,N -εdGuo), from 338
to 222 (1,N -propanodGuo), and from 343 to 227 ([ N
2
15
2
(
1,N -εdGuo), from 297 to 181 ([ N
5
2
15
2
5
]-
confirmed the presence of basal levels of 4.01 ( 0.32 1,N -
2
8
2
8
1
,N -propanodGuo) with samples containing 500 amol of the
εdGuo/10 dGuo and 3.43 ( 0.33 1,N -propanodGuo/10
unlabeled adducts and 33 fmol of the stable isotopic internal
standards are shown in Figure 1A-D.
The calibration curves shown in Figure 3 were obtained from
dGuo (data not shown).
Table 2 reports the quantification and SD of basal levels
2
2
of 1,N -εdGuo and 1,N -propanodGuo in the liver (2.47 (
2 8 2
1
4 different concentration points. The chromatographic peak
0.61 1,N -εdGuo/10 dGuo and 4.61 ( 0.69 1,N -propanod-
8 2 8
2
15
2
2
area ratios of 1,N -εdGuo to [ N
5
]-1,N -εdGuo (Figure 3A, r
Guo/10 dGuo), brain (2.96 ( 1.43 1,N -εdGuo/10 dGuo

1
250 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Garcia et al.
2
Figure 4. HPLC/ESI/MS-MS detection of 500 amol of DNA adducts standard contaminated in 100 µg of calf thymus DNA. (A) 1,N -εdGuo: m/z
1
5
2
2
15
2
2
92 f 176. (B) [ N
5 5
]-1,N -εdGuo: m/z 297 f 181. (C) 1,N -propanodGuo: m/z 338 f 222. (D) [ N ]-1,N -propanodGuo: m/z 343 f 227. (E)
Enzymatically hydrolyzed DNA at 260 nm. Conditions were as described in the Experimental Procedures section.

HPLC-MS/MS Quantification of Exocyclic DNA Adducts
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1251
2
2
2
Table 1. Levels of of 1,N -PropanodGuo and 1,N -εdGuo in
Table 2. Levels of 1,N -Propano-2′-deoxyguanosine and
2
Hydrolyzed DNA Calf Thymus Spiked with 1 fmol of Each
1,N - Etheno-2′-deoxyguanosine in DNA from Wistar Rat
a
Adduct
Tissues (Lung, Liver, and Brain) (n ) 21)
1
,N2-
1,N2-
1,N -εdGuo/10 dGuo
2
8
2
2
1
,N -εdGuo- 1,N -εdGuo/ propanodGuo- propanodGuo/
8
8
min. value
max. value
mean
SD
fmol
10 dGuo
fmol
10 dGuo
liver
lung
brain
1.77
0.39
1.83
3.41
2.14
4.19
2.47
0.87
2.96
0.61
0.34
1.43
day 1 DNA A
DNA B
DNA C
mean
0.93
1.02
0.94
0.95
0.07
1.01
1.03
0.95
0.10
0.04
1.02
1.07
1.05
1.04
0.02
0.96
0.05
1.04
0.03
0.98
0.06
1.00
0.06
0.49
0.56
0.52
0.52
0.03
0.56
0.48
0.53
0.52
0.04
0.58
0.59
0.58
0.58
0.01
0.54
0.05
0.54
0.07
0.54
0.03
0.55
0.05
2.16
1.98
2.04
2.06
0.1
1.20
1.10
1.13
1.14
0.05
1.01
1.08
1.16
1.08
0.07
1.18
1.12
1.13
1.14
0.03
1.13
0.10
1.10
0.02
1.14
0.02
1.15
0.11
2
8
1
,N -propanodGuo/10 dGuo
SD
day 2 DNA A
DNA B
DNA C
mean
2.17
1.95
2.09
2.07
0.11
2.20
2.02
2.03
2.08
0.10
2.17
0.02
1.98
0.03
2.05
0.03
2.11
0.17
min. value
max. value
mean
SD
liver
lung
brain
3.02
1.47
1.86
6.93
3.42
10.19
4.61
2.25
5.66
0.69
1.72
3.70
SD
day 3 DNA A
DNA B
DNA C
mean
2
The endogenous formation of 1,N -propanodGuo and its
quantification in animals models are still controversial in the
literature. Previously, Nath and Chung found 0.18-0.94 adducts/
SD
6
1
0
dGuo in the liver DNA of Fisher 344 rats using a
DNA A mean
SD
DNA B mean
SD
DNA C mean
SD
3
2
P-postlabeling methodology (37). In another study, they
6
analyzed DNA from the brain (0.397-0.691 adducts/10 dGuo),
6
lung (0.117-0.469 adducts/10 dGuo), and other tissues from
the same rat model (38). However, Schuler and Eder did not
2
total
mean
SD
detect basal levels of 1,N -propanodGuo in the liver DNA of
3
2
Fisher 344 rats using P-postlabeling coupled with TLC
(39, 40). Later, Pan et al., using a methodology based on solid-
phase extraction/high-performance liquid chromatography and
P-postlabeling, reported basal levels of 1,N -propanodGuo
(25.9 ( 7.8 adducts/10 dGuo) in the liver DNA of Long-Evans
rats (41). This level is comparable with that in liver samples of
Wistar rats reported here (4.61 ( 0.69 1,N -propanodGuo/10
dGuo).
The presence of 1,N -propanodGuo has previously been
reported in human tissue (liver and lung) and blood using two
different methodologies, P-postlabeling combined with HPLC
37) and HPLC/MS/MS analysis (42). Different quantities and
a
The analysis were performed in 3 subsequent days by HPLC-MS/
MS (n ) 3).
32
2
9
2
8
and 5.66 ( 3.70 1,N -propanodGuo/10 dGuo), and lung (0.87
2
8
2
(
0.34 1,N -εdGuo/10 dGuo and 2.25 ( 1.72 1,N -
8
2
8
propanodGuo/10 dGuo) samples from 21 untreated male
Wistar rats. A representative chromatogram of one of these
samples is shown in Figure 6.
2
3
2
Discussion
(
The ultrasensitive methodology described herein permits
us to evaluate and compare the deleterious effects of reactive
aldehydes, such as CRO and AA, from endogenous or
exogenous exposure through the direct and simultaneous
frequencies of the adducts were reported, probably due to
differences in the methodologies and in the levels of aldehyde
exposure and individual repair efficiencies, as discussed before
(38, 42, 43).
2
6
quantification of two resulting DNA adducts, 1,N -propan-
Additionally, etheno adducts, such as 1,N -etheno-2′-
2
6
4
odGuo and 1,N -εdGuo. Advantages of the method include
deoxyadenosine (1,N -εdAdo), 3,N -etheno-2′-deoxycytidine
4 2 2
the online separation of adducts from normal nucleosides
coupled with the accuracy provided by tandem mass spec-
trometry detection. The methodology also excludes the
necessity for a second method for the quantification of
unmodified dGuo, thereby increasing precision and efficiency.
The addition of an isotopically labeled adduct prior to DNA
hydrolysis improves the specificity of the method on adduct
quantification because it allows for the correction of any
possible loss of adducts during the procedure.
(3,N -εdCyd), 3,N -etheno-2′-deoxyguanosine (3,N -εdGuo),
2 2
and 1,N -etheno-2′-deoxyguanosine (1,N -εdGuo), are well-
validated biomarkers for DNA damage arising from the
reaction of endogenous lipid peroxidation end products (29).
2
The in ViVo basal levels of 1,N -εdGuo was first reported by
our group using a methodology based on HPLC/MS-MS. This
adduct was quantified in the liver DNA of untreated female
7
Wistar rats (5.22 ( 1.37 adducts/10 dGuo) (36). These levels
are 10 times higher than those reported here. This difference
can be attributed to improvement in the present methodology,
simultaneous dGuo quantification, and rat gender. More
The methodology enables us to determine the basal levels
2
8
2
of 1,N -εdGuo (4.01 ( 0.32 adducts/10 dGuo) and 1,N -
8
2
propanodGuo (3.43 ( 0.33 adducts/10 dGuo) in the DNA
of human lung fibroblasts (IMR-90). Our group had previ-
ously reported basal levels of 1,N -εdGuo in the DNA of
recently, 1,N -εdGuo was quantified by Pang et al. (44) in
tissue DNA of SJL mice. The basal levels reported in the
2
9
9
liver (39 ( 38/10 dGuo), kidney (20 ( 7/10 dGuo), and
9
green monkey kidney fibroblasts (CV1-P cells) (4.5 ( 0.4
spleen (24 ( 9/10 dGuo) are comparable with those reported
7
adducts/10 dGuo) (35).
here.
Additionally, we measured here the adduct levels in
The quantification of the basal levels of DNA adducts in
different tissues and animals is extremely important for the
unequivocal use of these adducts as biomarkers of disease
development and environmental exposure. Additionally, the
accurate measurement of adducts can be useful for mechanistic
elucidation in pathological situations associated with oxidative
stress.
2
different Wistar rat tissues (liver, 2.47 ( 0.61 1,N -εdGuo/
8
2
8
1
0 dGuo and 4.61 ( 0.69 1,N -propanodGuo/10 dGuo;
2 8
brain, 2.96 ( 1.43 1,N -εdGuo/10 dGuo and 5.66 ( 3.70
2
8
2
1
,N -propanodGuo/10 dGuo; and lung, 0.87 ( 0.34 1,N -
8 2 8
εdGuo/10 dGuo and 2.25 ( 1.72 1,N -propanodGuo/10
dGuo).

1
252 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Garcia et al.
2
15
Figure 5. HPLC/ESI/MS-MS detection of DNA adducts in 100 µg of calf thymus DNA. (A) 1 fmol of 1,N -εdGuo: m/z 292 f 176. (B) [ N
5
]-
2
2
15
2
1
,N -εdGuo: m/z 297 f 181. (C) 1 fmol of each isomer of 1,N -propanodGuo: m/z 338 f 222. (D) [ N
5
]-1,N -propanodGuo: m/z 343 f 227. (E)
Enzymatically hydrolyzed DNA at 260 nm. Conditions were as described in the Experimental Procedures section.
The methodology described herein provides a highly precise
addition, this article demonstrates the unequivocal quantification
2
2
and specific approach to quantify 1,N -εdGuo and 1,N -
propanodGuo adducts in DNA and allows for the future
evaluation of their levels when tissues are subjected to factors
that promote or decrease the lipid peroxidation process. In
of the basal levels of the two DNA adducts in the lung, liver,
and brain tissue of male Wistar rats and in live cells (IMR-90).
This quantification is fundamental for the establishment of these
molecules as biomarkers involving redox processes.

HPLC-MS/MS Quantification of Exocyclic DNA Adducts
Chem. Res. Toxicol., Vol. 23, No. 7, 2010 1253
Figure 6. HPLC/ESI/MS-MS detection of DNA adducts in 100 µg of DNA extracted from the liver of an untreated male Wistar rat. The MRM
2
15
2
2
15
mode was used. (A) 1,N -εdGuo: m/z 292 f 176. (B) [ N
5
]-1,N -εdGuo: m/z 297 f 181. (C) 1,N -propanodGuo: m/z 338 f 222. (D) [ N
,N -propanodGuo: m/z 343 f 227. (E) Enzymatically hydrolyzed DNA at 260 nm. Conditions were as described in the Experimental Procedures
section.
5
]-
2
1

1
254 Chem. Res. Toxicol., Vol. 23, No. 7, 2010
Garcia et al.
Acknowledgment. This work was supported by the Fun-
(19) International Agency for Research on Cancer (1985) Allyl Compounds,
Aldehydes, Epoxides and Peroxides. In Monographs on the EValuation
of the Carcinogenic Risk of Chemicals to Humans,Vol. 36, pp 101-
da c¸ a˜ o de Amparo a` Pesquisa do Estado de S a˜ o Paulo,
FAPESP (Brazil), the Conselho Nacional para o Desenvolvi-
mento Cient ´ı fico e Tecnol o´ gico, CNPq (Brazil), Coordena c¸ a˜ o
de Aperfei c¸ oamento de Pessoal de N ´ı vel Superior, CAPES,
and Instituto Nacional de Ci eˆ ncia e Tecnologia de Processos
Redox em Biomedicina-Redoxoma (INCT - Redoxoma).
1
32, International Agency for Research on Cancer, Lyon, France.
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Supporting Information Available: H NMR spectrum and
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