Detection and quantification of specific DNA adducts by liquid chromatography-tandem mass spectrometry in the livers of rats given estragole at the carcinogenic dose

Article abstract of DOI:10.1021/tx100410y

Estragole (ES) is a natural constituent of several herbs and spices that acts as a carcinogen in the livers of rodents. Given that the proximal electrophilic form of ES with a reactive carbocation is generated by cytochrome P450 and a sulfotransferase met

Full text of DOI:10.1021/tx100410y

ARTICLE
pubs.acs.org/crt
Detection and Quantification of Specific DNA Adducts by Liquid
Chromatography-Tandem Mass Spectrometry in the Livers of Rats
Given Estragole at the Carcinogenic Dose
Yuji Ishii,*,† Yuta Suzuki,† Daisuke Hibi,† Meilan Jin,† Kiyoshi Fukuhara,‡ Takashi Umemura,† and
Akiyoshi Nishikawa†
†Division of Pathology and ‡Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo
158-8501, Japan
ABSTRACT: Estragole (ES) is a natural constituent of several
herbs and spices that acts as a carcinogen in the livers of rodents.
Given that the proximal electrophilic form of ES with a reactive
carbocation is generated by cytochrome P450 and a sulfotrans-
ferase metabolizing pathway, there is a possibility that the
resultant covalent adducts with DNA bases may play a key role
in carcinogenesis. The existence of ES-specific deoxyguanosine
(dG) and deoxyadenosine (dA) adducts has already been
reported with the precise chemical structures of the dG adducts
being confirmed. In the present study, we examined ES-specific
dA adduct formation using LC-ESI/MS after the reaction of dA
with 10-acetoxy-ES produced by a sulfotransferase metabolic
pathway mimic. Although two peaks were observed in the LC-ESI/MS chromatogram, the identification of ES-30-N6-dA as the
measurable peak was determined by NMR analysis. To confirm ES-specific dG and dA adduct formation in vivo, an isotope dilution
LC-ESI/MS/MS method applicable to in vivo samples for ES-30-N6-dA together with the two major dG adducts, that is, ES-30-C8-
dG and ES-30-N2-dG, was developed using selected ion recording. The limit of quantification was 0.2 fmol on column for ES-30-C8-
dG and ES-30-N2-dG and 0.06 fmol on column for ES-30-N6-dA, respectively. Using the developing analytical method, we attempted
to measure adduct levels in the livers of rats treated with ES at a possible carcinogenic dose (600 mg/kg bw) for 4 weeks. ES-30-C8-
dG, ES-N2-dG, and ES-30-N6-dA were detected at levels of 3.5 ( 0.4, 4.8 ( 0.8, and 20.5 ( 1.6/106 dG or dA in the livers of ES-
treated rats. This quantitative data and newly developed technique for adduct observation in vivo might be helpful for ES
hepatocarcinogenesis investigations.
’ INTRODUCTION
carcinogenic dose of ES still remains unknown. Transient base
modifications of DNA do not always result in gene mutations
because of chemical DNA instability12 and the existence of
specific DNA repair systems.13 Because the specific activity of
repair enzymes is primarily dependent on stereochemical struc-
tures and concentrations of their substrates, confirmation of
DNA base modifications and precise quantification are necessary
for understanding their biological significance.14,15
The dG modifications have generally been reported as the
major adduct,16,17 because dG is the most potent nucleophilic
nucleoside that can efficiently react with the electrophilic carbo-
cation form.18 Furthermore, the findings of Y-family polymerase
κ, which selectively acts on translesion synthesis on N2-dG
adducts, showed the presence of a linkage between dG modifica-
tion and mutation in the chemical carcinogenicity.19-21 How-
ever, several carcinogens including aristolochic acid22,23 and
polyaromatic hydrocarbons24 preferentially form dA adducts,
Estragole (4-allyl-1-methoxybenzene; ES) is a natural consti-
tuent of essential oils of various herbs and spices (including
tarragon, basil, fennel, and anise) present in food.1 Previous
studies have revealed that ES has genotoxicity and carcinogeni-
city in the livers of mice, concluding that ES is a naturally
occurring genotoxic carcinogen.2-4 ES is metabolized to the
proximate carcinogen, 10-hydroxy-ES, by cytochrome P450 en-
zymes (P450), and sulfotransferase (SULT) converts 10-hydor-
oxy-ES to 10-sulfooxy-ES.2-5 The ultimate electrophilic
carbocation structure, which can form covalent adducts with
DNA bases, is formed from 1-sulfooxy-ES through dissociation
of the sulfate group.6,7 Because postlabeling analysis demon-
strated the formation of four different adducts such as ES-30-N2-
dG (deoxyguanosine), ES-30-C8-dG, ES-10-N2-dG, and ES-30-
N6-dA (deoxyadenosine) in the livers of mice administered the
proximate carcinogenic metabolite 10-hydroxy-ES, it has been
accepted that the carcinogenicity of ES is caused by the formation
of these adducts.8-11 However, the precise amount of these
adducts formed in vivo under continuous administration with a
Received: November 27, 2010
Published: March 08, 2011
r
2011 American Chemical Society
532
dx.doi.org/10.1021/tx100410y Chem. Res. Toxicol. 2011, 24, 532–541
|

Chemical Research in Toxicology
ARTICLE
Figure 1. Typical LC-PDA chromatograms (280 nm) in the reaction of (A) 10-acetoxy-ES alone and (B) 10-acetoxy-ES with dA. LC-PDA conditions are
described in the Materials and Methods.
thereby predominantly inducing dA mutations.25,26 Thus, quan-
titative analysis for each ES-modified base is necessary to
investigate the role of specific DNA adducts in ES carcinogenesis.
Although the exact chemical structure of three dG adducts has
already been confirmed by mass spectrometry (MS) techniques,
that of the dA adduct has not been identified. In this study, ES-dA
adduct formation by reaction of dA with 10-acetoxy-ES (used as a
10-sulfooxy-ES mimic) was investigated by liquid chromatography
(LC)-electron spray ionization (ESI)/MS analysis. The precise
chemical structures of the detected adducts were identified using
nuclear magnetic resonance (NMR). Subsequently, a quantitative
analytical method usingLC-ESI/tandem mass spectrometry (MS/
MS) for ES-specific dG and dA adducts was developed. After the
evaluation of applicability to in vivo samples, our LC-ESI/MS/MS
method was applied to quantify ES-specific DNA adducts in the
livers of rats treated with 600 mg/kg bw of ES for 4 weeks.
were purchased from Sigma Chemical Co. (St. Louis, MO). Nuclease P1
was from Yamasa Shoyu Co. (Chiba, Japan). Stable isotope-labeled
[15N5]-dG and [15N5]-dA were obtained from Cambridge Isotope
Laboratories (Cambridge, MA). Acetic anhydride ammonium carbo-
nate, dimethylsulfoxide (DMSO), dichloromethane, N,N-dimethylfor-
mamide (DMF), diethyl ether, isopropyl alcohol, and DNA extractor
TIS kit were purchased from Wako Pure Chemicals (Tokyo, Japan). All
other chemicals used were of specific analytical or HPLC grade.
Synthesis of 10-Hydroxyestragole. 10-Hydroxy-ES was synthe-
sized from p-anisaldehyde as described by Punt et al.7 The synthesis of
10-hydroxy-ES encompassed a Grignard reaction, using vinylmagnesium
bromide as the Grignard reagent (1 M solution in THF). Briefly, p-
anisaldehyde (0.0165 mol) was dissolved in 10 mL of dry THF, and this
solution was added dropwise over a period of 30 min to the Grignard
reagent (0.035 mol) while stirring at 50 °C in anhydrous conditions
under a nitrogen atmosphere. The reaction mixture was further incu-
batedfor 90min, and theresultingsolution wasaddedtoa solution of4.5 g
of ammonium chloride in 200 mL of ice cold water. The emulsion was
stirred for several minutes, and 10-hydroxy-ES was extracted with diethyl
ether. The organic solution was dried with magnesium sulfate, and the
yield was 94%. The identity of the product was confirmed by 1H NMR.
1H NMR spectra were recorded with a Varian 500 MHz NMR system
’ MATERIALS AND METHODS
Chemicals and Reagents. ES, p-anisaldehyde, vinylmagnesium
bromide, tetrahydrofuran (THF), dG, dA, and alkaline phosphatase
533
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
Figure 2. Mass spectra of peaks 1 (A-C) and 2 (D-F) obtained from LC-ESI/MS analysis for the reaction of 10-acetoxy-ES with dA. Mass analysis was
performed in scan mode. The cone voltages were 20 (A and D), 40 (B and E), and 60 V (C and E) in positive ion mode. LC-ESI/MS conditions are
described in the Materials and Methods.
1
(Varian Inc. Corp.). 10-Hydroxy-ES: H NMR (DMSO-d6): δ 7.22-
7.29 (m, 2H, Ar), 6.82-6.89 (m, 2H, Ar), 5.98-6.10 (m, 1H,
-CHCH2), 5.26-5.35 [m, 2H, -C(OH)CH-], 5.10-5.20 (m, 2H,
-CHCH2), 3.79 (s, 1H, -OCH3), 2.24 [s, 1H, -C(OH)-].
-CHCH2), 5.26-5.27 [m, 2H, -CH(OH)CH-], 5.19-5.23 (m,
2H, -CHCH2), 3.75 (s, 1H, -OCH3), 2.05 (s, 1H, -CO-CH3).
Determination of ES-Specific dA Adduct. ES-specific dA
adducts were determined from reactions between 10-acetoxy-ES and
dA based on the protocol of Phillips et al.8 The reaction products
containing 10-acetoxy-ES were diluted 50-fold in DMF from which 200
μL was added to 1.8 mL of 10 mM dA solution in 50 mM sodium
phosphate buffer (pH 7.4). The reaction mixture was stirred for 24 h at
37 °C. The reaction mixture was then passed through an HLC-DISK
syringe filter (Kanto Chemical Co Inc., Tokyo) and was separated on a
LC-photodiode array (PDA)-ESI/MS system. LC-PDA-ESI/MS ana-
lyses were performed using an Alliance HT model 2695 liquid chroma-
tographic system coupled to a 996 PDA detector and Micromass ZQ, a
single quadrupole, MS system (Waters Corp., Miliford, MA) equipped
with an ESI source through a splitter. Twenty microliters of the reaction
mixture was injected directly onto a reverse-phase C18 column
(Mightysil RP-18, 4.6 mm ꢀ 150 mm, 5 μm, Kanto Chemical Co.,
Synthesis of 10-Acetoxyestragole. 10-Acetoxy-ES was synthe-
sized from 10-hydroxy-ES as described by Punt et al.7 and Drinkwater
et al.2 In brief, 10-hydroxy-ES (50 mg) was dissolved in 200 μL of
pyridine. Acetic anhydride (33 μL) was added dropwise to this solution,
and the reaction mixture was stirred for 5 h at room temperature after
which 400 μL of dichloromethane was added. The reaction mixture was
extracted several times with aliquots of 200 μL of 1 N HCl. When the
aqueous phase reached pH 2-3, the organic layer then immediately was
extracted with 400 μL of 1 M sodium carbonate solution (pH 7.6). The
organic solution was dried with magnesium sulfate, and the solvent was
evaporated in a nitrogen atmosphere. The identity of the product was
confirmed by 1H NMR. 10-Hydroxy-ES: 1H NMR (DMSO-d6): δ 7.26-
7.28 (m, 2H, Ar), 6.92-6.95 (m, 2H, Ar), 5.99-6.07 (m, 1H,
534
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
1
Table 1. H NMR Chemical Shifts of Peak 1a
H-10
H-20
H-200
H-30
H-40
H-50
H-500
OH-30
6.34 (1H, m)
2.70 (1H, m)
2.25 (1H, m)
4.40 (1H, m)
3.87 (1H, m)
3.61 (1H, m)
3.51 (1H, m)
5.31 (1H, s)
5.22 (lH,bs)
8.21 (1H, bs)
8.37 (1H, s)
8.07 (lH,bs)
6.42 (1H, m)
6.21 (1H, m)
4.22 (2H, bs)
7.30 (2H, m)
6.85 (2H, m)
3.75 (3H, m)
Figure 3. Chemical structure of ES-30-N6-dA adducts and the stable
isotopically labeled compounds. The asterisk (*) indicates nitrogen
[15N]-labeling.
OH-50
H-2
H-8
N6-H
ESH-10
ESH-20
of ES-30-C8-dG and ES-30-N2-dG were 63.4 (5.2%) and 103.5 mg
(8.56%), respectively, in the reaction. The syntheses of [15N5]-ES-30-8-
dG, [15N5]-ES-30-N2-dG, and [15N5]-ES-30-N6-dA as stable isotopically
labeled surrogate standards were also performed on a small scale by the
same method. 15N5-labeled ES-dG adducts including four isomers and
ES-30-N6-dA were repeatedly purified using LC-UV system until 98%
and over.
ESH-30
ESH-2,6
ESH-3,5
One millimolar solutions of ES-30-C8-dG, ES-30-N2-dG, and ES-30-
N6-dA were prepared in methanol and immediately diluted with
methanol/HPLC grade water (50/50, v/v) to 10 μM (stock solution).
Working solutions for calibration (0.01-10 nM for ES-30-C8-dG, ES-30-
N2-dG, and 0.003-10 nM for ES-30- N6-dA) were prepared by the
addition of an adequate amount of surrogate standard and diluted
with methanol/HPLC grade water (50/50, v/v) to appropriate
concentrations.
LC-ESI/MS/MS Conditions. LC-ESI/MS/MS analyses were per-
formed using a Quattoro Ultima (Micromass) coupled to a Hewlett
Packard 1100 series (G1322A, degasser; G1312A, bin pump; G1316A,
Colcom; G1329A, ALS; Agilent Technologies, Palo Alto, CA). The mass
spectrometer was operated using an ESI source in the positive ion mode
for multiple reaction monitoring (MRM). An aliquot (20 μL) of the
sample was injected into a Mightysil C18-GP (2.0 mm ꢀ 150 mm, 5 μm;
Kanto Chemical Co., Tokyo, Japan) maintained at 40 °C. Solvent A was
0.001% formic acid, and solvent B was 0.001% formic acid containing
acetonitrile. The column was equilibrated with a mixture of solvent A/
solvent B (75/25, v/v). The mobile phase consisted of a mixture of
0.001% formic acid/0.001% formic acid containing acetonitrile at an
initial ratio of 75/25, employing a linear gradient to a final ratio of 30/70
(v/v) over 30 min, at a constant flow rate of 0.2 mL/min.
ESOCH3
a m, multiplet; s, singlet.
Inc.) maintained at 40 °C. Solvent A was water, solvent B was methanol,
and solvent C was 0.1% formic acid. The column was equilibrated with a
mixture of solvent A/solvent B/solvent C (70/10/20, v/v). A linear
gradient was applied from 30 to 90% methanol over 40 min, kept at 90%
for 10 min, lowered to 20% over 2 min, and re-equilibrated at the initial
conditions for 15 min. Mass analysis was performed in scan mode. The
cone voltages were 20, 40, and 60 V in the positive ion mode.
Synthesis of ES-Derived dA Adduct. ES-dA adducts were
synthesized from a reaction between 10-acetoxy-ES and dA. Four
milliliters of 30 mM 10-acetoxy-ES solution in dichloromethane was
diluted 50-fold in DMF from which 200 mL was added to 1800 mL of
10 mM dG or dA solution in 50 mM sodium phosphate buffer (pH 7.4).
The reaction was stirred for 24 h at 50 °C. The yield of peak 1 was 86 mg
(7.1%). The reaction was repeated several times as needed to acquire
enough products for NMR analysis.
Purification was performed using the combined reaction mixtures
that were evaporated and reconstituted in 50% methanol/water. The
concentrated reaction mixture was separated by a LC system equipped
with a UV detector (LC-UV) (PU-2080 Plus Intelligent HPLC Pump,
AS-2057 plus Intelligent Sampler, CO-966 Intelligent Column Thermo-
stat, and UV-970 Intelligent UV/vis Detector; Jasco Co., Tokyo). Two
milliliters of sample was injected directly on to a reverse phase C18
column (Mightysil RP-18 GP, 20 mm ꢀ 250 mm, 5 μm, Kanto Chemical
Co., Inc.) maintained at 40 °C. Solvent A was 0.01% formic acid, and
solvent B was methanol containing 0.01% formic acid. The column was
equilibrated with a mixture of solvent A/solvent B (70/30, v/v). A linear
gradient was applied from 30 to 90% methanol over 30 min, kept at 90%
for 10 min, lowered to 30% over 2 min, and equilibrated at the initial
conditions for 15 min. Products eluting at 22.5 and 31.2 min (UV
absorbance at 280 nm; flow rate, 10 mL/min) were collected. The
fractions were dried in vacuo and weighed. The identity of the product
In the assay for ES-30-N2-dG and ES-30-C8-dG, the precursor ion ([M
þ H]þ) was m/z 414, and the selected product ion [M þ H -
glycoside]þ was m/z 298. Correspondingly, for [15N]-labeled ES-30-
N2-dG and ES-30-C8-dG, the precursor ion was m/z 419, and the
selected product ion was m/z 303. The cone voltage used was 18 V, and
the collision energy was 10 eV. In the assay for ES-30-N6-dA, the
precursor ion ([M þ H]þ) was m/z 398, and the selected product
ion [M þ H - glycoside]þ was m/z 282. Correspondingly, for [15N]-
labeled ES-30-N6-dA, the precursor ion was m/z 403, and the selected
product ion was m/z 287. The cone voltage used was 12 V, and the
collision energy was 18 eV. The source block temperature was 120 °C,
and the desolvation temperature was 400 °C. The flow rate of the cone
gas was set at 150 L/h, while that of the desolvation gas was set at 600 L/
h. Under these conditions, the standard retention times were 11.8, 12.7,
and 15.1 min for ES-30-N2-dG, ES-30-C8-dG, and ES-30-N6-dA,
respectively.
1
1
was confirmed by LC-ESI/MS and H NMR. H NMR spectra were
recorded with a Varian 600 MHz NMR system (Varian Inc. Corp., Palo
Alto, CA). Chemical shifts are expressed in ppm downfield shift from
trimethylsilane (TMS) (δ scale). The synthesis of stable isotopically
labeled surrogate standards was also performed on a small scale by the
same method. The reaction products were purified using the LC-UV
system.
Recovery. The recovery was evaluated by calculating the mean of
the response at each concentration. The spiked concentrations (low,
middle, and high dose) of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA
were determined from the concentrations of each compound in the liver
DNA of control rats, using LC-ESI/MS/MS. A standard sample was
Standard Solutions. ES-30-C8-dG and ES-30-N2-dG were synthe-
sized from 10-acetoxy-ES and dG as described by Punt et al.7 The yields
535
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
Figure 4. Product ion spectra of (A) ES-30-C8-dG, (B) ES-30-N2-dG, and (C) ES-30-N6-dA. The cone voltages and collision energies were set at the
optimum conditions for each compound in negative ion mode. LC-ESI/MS/MS conditions are described in the Materials and Methods.
added together with an adequate amount of surrogate standards to
20 mM sodium acetate buffer (pH 4.2) for DNA digestion so that the
final concentration might be set to 0.05, 0.5, and 5 nM. The extracted
DNA pellets of rat liver were redissolved in this buffer and digested
according to the protocol. The sample was analyzed by LC-ESI/MS/
MS, and the recovery rates were calculated.
Animal and Treatment. The protocol for this study was approved
by the Animal Care and Utilization Committee of the National Institute
of Health Sciences (Tokyo, Japan). Five week old male F344 rats were
obtained from Japan SLC (Shizuoka, Japan). Ten rats were housed in
polycarbonate cages (five rats per cage) with hardwood chips for
bedding in conventional temperature (23 ( 2 °C), humidity (55 (
5%), air change (12 times per hour), and lighting (12 h light/dark cycle)
and were given free access to CRF-1 basal diet (Oriental Yeast Co., Ltd.,
Tokyo, Japan) and tap water. Groups of 10 rats were given ES by gavage
in corn oil at 600 mg/kg bw per day, 5 days/week. All rats were killed at 4
weeks by exsanguination under ether anesthesia, and the livers were
immediately removed and weighed. Samples were frozen with liquid
nitrogen and stored at -80 °C until measurement of ES-specific DNA
adducts.
The dried DNA pellet was dissolved in surrogate standard containing
20 mM sodium acetate buffer, pH 4.8, and was incubated with 4 μL of
nuclease P1 (2000 U/mL) at 70 °C for 15 min. Then, 20 μL of 1.0 M Tris-
HCl buffer, pH 8.2, was added, and the sample was incubated with 4 μL of
alkaline phosphatase (2500 U/mL) at 37 °C for 60 min. After the addition
of 3.0 M sodium acetate buffer, pH 5.1, the digested DNA samples were
used for adduct analysis and base analysis. Then, 50 μL of the digested
sample for dG and dA analysis was passed through 100000 NMWL filter
(Millipore, Bedford, MA) and injected into the LC-UV. One hundred
microliters of digested sample for adduct analysis was diluted with an equal
volume of methanol and injected into the LC-ESI/MS/MS.
LC-UV Analysis for dG and dA. dGanddA were determinedby an
LC-UV system (Jasco Co.: PU-980 Intelligent HPLC Pump, AS-950-10
Intelligent Sampler, CO-1560 Intelligent Column Thermostat, MD-1515
Multiwavelength Detector, Tokyo, Japan). Two milliliters of sample was
injecteddirectly on toareversedphaseC18 column (Ultrasphere ODS, 4.6
mm ꢀ 250 mm, 5 μm, Beckman Coulter, Inc.) maintained at 40 °C.
Solvent A was 0.01% formic acid containing water, and solvent B was 0.01%
formic acid containing methanol. The column was equilibrated with a
mixtureof solvent A/solvent B(98/2, v/v). The compoundswere elutedat
a flow rate of 1.0 mL/min. A linear gradient was applied from 2 to 10%
methanol over 20 min, kept at 10% for 5 min, lowered to 2% over 2 min,
and equilibrated at these initial conditions for 15 min. The wavelength of
the UV detector was set at 280 nm for the detection of dG and dA.
DNA Isolation and Enzymatic Digestion. DNA extraction and
digestion were performed according to the method of Nakae et al.27 and
our previous report.28 The samples were homogenized with lysis buffer
including commercial DNA isolation kit. The mixture was centrifuged at
10000g for 20 s at 4 °C. The deposit was dissolved in 200 μL of enzyme
reaction buffer. After treatment with RNase and protease K, the DNA
pellet was obtained by washing with 2-propanol and ethanol and
centrifugation.
’ RESULTS
Identification of Luc-N6-dA. The reaction mixtures for 10-
acetoxy-ES with/without dA were separated by LC-PDA-ESI/MS.
536
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
Figure 5. MRM chromatograms of LOD (A) and LOQ (B) levels of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA adducts and each surrogate standard
from extracted DNA samples.
Table 2. Recoveries of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA Adducts in Rat Liver Samples
compounds
added (pmol/L)
concentration (pmol/L)
recovery (%)
RSD (%)
ES-30-C8-dG (n = 5)
50
500
5000
50
50.0 ( 2.3
501.1 ( 26.5
4938.8 ( 135.5
50.6 ( 3.7
98.0 ( 4.6
100.3 ( 5.3
98.8 ( 2.7
101.3 ( 7.5
99.4 ( 3.7
99.6 ( 1.6
103.5 ( 1.5
102.0 ( 1.5
99.8 ( 2.2
4.7
5.3
2.7
7.4
3.7
1.6
1.5
1.4
2.2
ES-30-N2-dG (n = 5)
ES-30-N6-dA (n = 5)
500
5000
50
497.2 ( 18.6
4981.2 ( 79.2
51.7 ( 0.77
500
5000
509.9 ( 7.29
4988.0 ( 109.8
Typical LC-PDA chromatograms are shown in Figure 1A,B. Two
unknown peaks were observed in the PDA chromatogram
(280 nm) after the reaction of 10-acetoxy-ES with dA
(Figure1B). Simultaneously, mass spectra and UV-vis absorp-
tion spectra were obtained by ESI/MS (cone voltage 20, 40, and
60 V) and PDA. MS spectra of all unknown peaks were analyzed
according to the ES and dA structure, and then, two peaks were
analyzed in the chromatograms. One of the two peaks (peak 1)
was observed at 22.1 min in the chromatogram of dA reaction
(Figure 1B). The UV-vis absorption spectrum of peak 1 had
λmax at 260 nm. In the mass spectra of peak 1 when the cone
voltage was set at 20 V (Figure 2A), the precursor ion ([M þ
H]þ, m/z 398) and product ion (m/z 282), corresponding to an
ES-adenine adduct following glycoside bond cleavage, were
clearly observed. A product ion (m/z 147) corresponding to
2-allyl-4-mthoxybenzene structure was observed in the mass
spectra at 40 and 60 V (Figure 2B,C). The other peak (peak
2) was observed at 22.4 min in the chromatogram for the reaction
of 10-acetoxy-ES and dA (Figure 1B). The UV-vis absorption
spectrum was virtually identical to peak 1 and also had at λmax at
260. In the mass spectra of peak 2 when the cone voltage was set
at 20 and 40 V (Figure 2D,E), the precursor ion ([M þ H]þ, m/z
398) and product ion (m/z 282), corresponding to ES-adenine
adduct following glycoside bond cleavage, were clearly observed.
A product ion (m/z 147) corresponding to 2-allyl-4-mthoxy-
benzene structure was also observed in the mass spectra of 40 and
60 V (Figure 2E,F).
10-Acetoxy-ES was used as an electrophilic synthon in large
scale synthesis to identify the chemical structures of peaks 1 and 2
from dA, respectively. These adducts were synthesized repeat-
edly by reaction with each base until the quantity was sufficient
for 1H NMR after HPLC-UV chromatography. However, peak 2
could not be collected in sufficient amounts for structural
analysis. 1H NMR chemical sifts of peak 1 redissolved in
DMSO-d6 are shown in Table 1. On the basis of these data, we
judged that peak 1 was ES-30-N6-dA. Chemical structures of ES-
30-N6-dA and stable isotopically labeled compound as a surrogate
standard for LC-ESI/MS/MS analysis are shown in Figure 3.
Optimal Conditions for LC-MS/MS Detection. Figure 4
shows the product ion spectra for ES-30-C8-dG, ES-30-N2-dG,
537
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
Figure 6. MRM chromatograms of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA adducts and each surrogate standard in rat liver DNA samples. (A)
Liver from rat administrated with vehicle as a control group. (B) Liver from rat orally administrated with 600 mg/kg ES for 4 weeks.
Table 3. LC-MS/MS Analysis of Liver DNA Samples Obtained from Rats Treated with 600 mg/kg bw ES for up to 4 Weeks
group
control
sample no.
ES-30-C8-dG/106dG
ES-30-N2-dG/106dG
ES-30-N6-dA/106dA
1
NDa
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2
3
4
5
average
ES (600 mg/kg)
6
3.02
4.16
18.96
7
3.53
4.28
20.05
8
3.42
4.37
21.22
9
4.12
6.18
22.89
10
3.33
5.07
20.47
average
3.48 ( 0.40
4.81 ( 0.84
20.47 ( 1.61
a ND, not detected.
and ES-30-N6-dA. The mass spectrometer equipped with an ESI
source using a crossflow counter electrode was run in positive ion
mode to detect MRM of the transitions 414 > 298, 414 > 298,
and 398 >282, respectively.
the LC-MS interface was affected by the mobile phase; hence, a
mobile phase containing a volatile acid or salt was generally used.
In this study, the responses were measured using 0-0.1% formic
acid in water-acetonitrile (v/v) as the mobile phase. The
responses of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA were
increased by the addition of formic acid to the mobile phase. The
increase in response reached a maximum and leveled off when
0.001% formic acid was added.
Validation of LC-MS/MS. The calculated instrument detection
limit (IDL) of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA of the
standard solutions was 3.0, 3.0, and 1.0 pM, respectively, for LC-
MS/MS detection at the ratio of the compound's signal to the
background signal (S/N) of 3. In addition, the instrument quanti-
fication limit (IQL) was calculated when S/N = 10 was 10, 10, and
3.0 pM, respectively. The limit of detection (LOD) and limit of
quantification (LOQ) in the real sample were the same as IDL and
The crucial parameters affecting LC-ESI/MS/MS, namely,
cone voltage, collision energy, and mobile phase, were investi-
gated. To establish the optimum cone voltage and collision
energy for the detection of these adducts, the signals at m/z
414 and 419 precursor ions versus cone voltage were investi-
gated, respectively. The optimal cone voltages were 18, 18, and
12 V in the negative ion mode for ES-30-C8-dG, ES-30-N2-dG,
and ES-30-N6-dA standard solutions, respectively. Then, the m/z
298 and 282 product ion signals versus collision energy were
investigated, respectively. The optimal collision energies were 10,
10, and 18 eV for ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA
standard solutions, respectively. The ionization of the samples at
538
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
Table 4. Interday Precision for the Determination of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA Adduct Levels in the Rat Liver
DNA Samples on Five Different Days Using LC-MS/MS
repeat analysis of ES-30-8- dG adduct (nM)
sample no.
day 1
day 2
day 3
day 4
day 5
mean (nmol/L)
RSD (%)
6
7
0.510
0.431
0.462
0.474
0.484
0.518
0.443
0.470
0.490
0.483
0.491
0.450
0.482
0.480
0.483
0.506
0.432
0.483
0.458
0.511
0.509
0.459
0.445
0.476
0.447
0.507 ( 0.010
0.443 ( 0.012
0.468 ( 0.016
0.476 ( 0.011
0.482 ( 0.023
1.96
2.68
3.39
2.39
4.74
8
9
10
repeat analysis of ES-30-N2-dG adduct (nM)
sample no.
day 1
day 2
day 3
day 4
day 5
mean (nmol/L)
RSD (%)
6
7
0.701
0.522
0.590
0.711
0.737
0.711
0.597
0.626
0.706
0.676
0.733
0.597
0.608
0.705
0.717
0.703
0.586
0.638
0.678
0.706
0.727
0.546
0.605
0.661
0.718
0.715 ( 0.014
0.569 ( 0.034
0.613 ( 0.019
0.692 ( 0.022
0.711 ( 0.022
1.97
5.91
3.06
3.15
3.13
8
9
10
repeat analysis of ES-30-N6-dA adduct (nM)
sample no.
day 1
day 2
day 3
day 4
day 5
mean (nmol/L)
RSD (%)
6
7
3.100
2.286
2.699
2.444
2.736
2.967
2.333
2.617
2.455
2.735
3.098
2.236
2.738
2.546
2.846
3.044
2.339
2.797
2.506
2.880
2.949
2.250
2.766
2.526
2.830
3.032 ( 0.071
2.289 ( 0.047
2.723 ( 0.069
2.495 ( 0.044
2.805 ( 0.066
2.36
2.04
2.55
1.77
2.36
8
9
10
IQL, respectively. The peak area ratio with respect to each
surrogate standard was plotted, and the response was found to
be linear (ES-30-C8-dG, y = 1.9707x - 0.0096 and y = 2.0318x -
0.0989; ES-30-N2-dG, y = 0.6359x þ 0.0048 and y = 0.6336x -
0.0263; and ES-30-N6-dA, y = 1.8567x þ 0.012 and y = 1.8205x þ
0.065) over the calibration range, from LOQ to 1.0 nM (low range)
and from 0.1 to 10 nM (high range), with a correlation coefficient
(r) of over 0.999. The average retention times of ES-30-C8-dG, ES-
30-N2-dG, and ES-30-N6-dA standards were11.8 (RSD = 0.08%, n =
5), 12.7 (RSD = 0.12%, n = 5), and 15.1 min (RSD = 0.06%, n = 5),
respectively. MRM chromatograms of the mixture of three adduct
and their corresponding surrogate standards in 50% methanol at
LOD and LOQ levels are shown in Figure 5.
respectively. The interday sample variation of ES-30-C8-dG, ES-
30-N2-dG, and ES-30-N6-dA adduct levels analyzed on different
days resulted in an average coefficient of variation (CV) of 3.0,
3.4, and 2.2% for different samples that were analyzed (Table 4).
All of these adducts were not detected in the control group.
’ DISCUSSION
Phillips et al. have suggested the chemical structure of four ES-
specific DNA adducts, ES-30-N2-dG, ES-30-C8-dG, ES-10-N2-dG,
and ES-30-N6-dA, using radio isotope-labeled nucleosides. The
formation of these adducts in mouse liver treated with [14C]-
labeled ES was demonstrated by radioactive detection after
HPLC fractionation.8 Subsequently, LC-MS analysis by Punt
et al. demonstrated the formation of three ES-dG adducts, ES-30-
N2-dG, ES-30-C8-dG, and ES-10-N2-dG, after in vitro nucleoside
reaction.7 However, the existence of the dA adduct reported
previously and its chemical structure have not been confirmed
using MS technique. In the present study, we examine dA adduct
formation using LC-ESI/MS by taking advantage of reactive
carbocation formation generated by acetylation of ES proposed
by Punt et al.7 In the reaction of dA with 10-acetoxy-ES, two peaks
including ions characteristic for the ES-dA adduct were observed
by total ion chromatography. Subsequently, 1H NMR analysis of
large-scale synthesized and HPLC purified samples led to the
conclusion that the major adduct was ES-30-N6-dA, in line with
the previous report.8 Although a minor and unknown ES-dA
adduct was also found, its precise chemical structure could not be
elucidated due to the low yield.
As shown in Table 2, the average recoveries of ES-30-C8-dG,
ES-30-N2-dG, and ES-30-N6-dA from DNA sample in the livers of
nontreated rats were 99.0, 100.1, and 101.7% for each adduct.
Analysis of ES-N2-dG, C8-dG, and N6-dA in the Livers DNA
Extracted from Rats Treated with ES. The potential formation
of ES adducts in livers of rats exposed to ES was assessed using
the isotope dilution LC-ESI/MS/MS method. Typical selected
ion recording (SIR) chromatograms are shown in Figure 6. No
peaks indicative of ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA
were observed in liver DNA extracted from control rats. In
contrast, ES-30-C8-dG, ES-30-N2-dG, and ES-30-N6-dA were
detected at 11.8, 12.7, and 15.1 min in the liver DNA MRM
chromatograms extracted from rats treated with 600 mg/kg ES
for up to 4 weeks, respectively. Quantitative data are summarized
in Table 3. The ES-30-C8-dG, ES-30-N2-dG/106dG, and ES-30-
N6-dA/106dA ratios were 3.5 ( 0.4, 4.8 ( 0.8, and 20.5 ( 1.6,
539
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
ES has been reported to have genotoxicity and carcinogenicity
in the livers of mice of both sexes.2 ES treatment at a dose of 600
mg/kg by gavage for 16 weeks induced significant developments
of glutathione S-transferase placenta form (GST-P) foci in the
liver of rats (unpublished data). To examine the precise quantity
of ES-specific adducts in ES-hepatocarcinogenesis, we used
interim samples (4 weeks) for the carcinogenicity study men-
tioned above. On the basis of previous reports, we attempted to
detect ES-30-N2-dG, ES-30-C8-dG, and ES-30-N6-dA. Because
appropriate sample preparations and a highly sensitive analytical
method are necessary to detect modified DNA bases in genomic
DNA, we developed a new analytical method by an isotope
dilution LC-ESI/MS/MS method using SIR. 32P-postlabeling
analysis without internal standards for identifying the products
neither provides structural characterization of adducts nor has
sufficient selectivities. In addition, this assay requires radioactive
γ-32P-labeled ATP in the analytical process, which raises the
necessity of rigorous handling. The LC-MS/MS technique using
stable isotope adducts as internal standards achieves accurate
quantification of DNA adducts along with structural character-
ization. The LOQs of ES-30-N2-dG, ES-30-C8-dG, and ES-30-N6-
dA in our method were determined to be 2-5 adducts/108
unmodified dG or dA bases from a 150 mg liver sample. In
addition, the high recoveries of these adducts in the wide range
from LOQ level indicated that our new method enables precise
adduct determination with the use of surrogate standards and is
applicable to the detection of these compounds in animal tissue
samples. As a result, our method was able to quantify these three
adducts in liver DNA of rats treated with 600 mg/kg ES for 4
weeks, and we observed that the ES-30-N6-dA adduct is pre-
dominant. Phillips et al. have suggested that ES-30-N2-dG was a
major adduct in the livers of mice treated with single dose of 10-
hydroxy-ES (12 μmol/mouse), a metabolite of ES. Considering
that the present experiment was performed under carcinogenic
conditions, it is highly probable that the status of DNA adduct
formation observed in the present study may be reflected in the
ES carcinogenesis. The modification at the N6-position of dA has
been reported following treatment with other potent mutagens
including aristolochic acid,22,23 polyaromatic hydrocarbon,
benzo[c]phenanthrene,29,30 and 5,6-dimathylchrysene.31 It is
likely that the formation of various types of chemical-specific
base modifications is dependent on accessibility of the chemical
(or the proximate form) to the reactive amino group between dG
and dA in the DNA helical structure.32 Those mutagens pre-
dominantly induce AT-TA transversion and AT-GC transition
mutations.25,33,34 Therefore, our unpublished data that the AT-
GC transition mutation was predominant in ES-treated rat livers
allow us to hypothesize that N6-dA adducts might play a key role
in ES mutagenicity. Thus, information regarding the precise
concentrations of ES-specific DNA adducts discovered in the
present study would be very helpful for further research on ES
hepatocarcinogenesis.
ES far from the benzene ring is predominant both in vivo and in
vitro, it is conceivable that the priority of these reactions is
determined by steric hindrance between DNA bases and ES. The
suggestion that unknown minor dA adducts also might result
from modification at the 10-position of ES is in line with this
hypothesis. Therefore, the fact that genomic DNA possesses
more steric hindrance than nucleosides might explain the lack of
detection of these minor adducts in vivo.
In conclusion, dA modified by ES was determined to be EG-30-
N6-dA as the major adduct. ES-30-N2-dG, ES-30-C8-dG, and ES-
30-N6-dA adducts can be identified and quantified by this new
method, which may prove useful in other related studies.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ81-3-3700-9819. Fax: þ81-3-3700-1425. E-mail: y-ishii@
nihs.go.jp.
’ ABBREVIATIONS
ES, estragole; dG, deoxyguanosine; dA, deoxyadenosine; LC, li-
quid chromatography; ESI, electron spray ionization; MS, mass
spectrometry; NMR, nuclear magnetic resonance; MS/MS, tan-
dem mass spectrometry; SIR, selected ion recording; LOQ, limit
of quantification; SULT, sulfotransferase; THF, tetrahydrofuran;
DMSO, dimethylsulfoxide; DMF, dimethylformamide; TMS, tri-
methylsilane; MRM, multiple reaction monitoring; PDA, photo-
diode array; IDL, instrument detection limit; IQL, instrument
quantification limit; LOD, limit of detection; GST-P, glutathione
S-transferase placenta form.
’ REFERENCES
(1) Smith, R. L., Adams, T. B., Doull, J., Feron, V. J., Goodman, J. I.,
Marnett, L. J., Portoghese, P. S., Waddell, W. J., Wagner, B. M., and
Rogers, A. E. (2002) Safety assessment of allylalkoxybenzene derivatives
used as flavouring substances-Methyl eugenol and estragole. Food Chem.
Toxicol. 40, 851–870.
(2) Drinkwater, N. R., Miller, E. C., Miller, J. A., and Pilot, H. C.
(1976) Hepatocarcinogenicity of estragole (1-allyl-4-methoxybenzene)
and 10-hydroxyestragole in the mouse and mutagenicity of 10-aceotyes-
tragole in bacteria. J. Natl. Cancer Inst. 57, 1323–1331.
(3) Miller, E. C., Swanson, A. B., Phillips, D. H., Fletcher, T. L., Liem,
A., and Miller, J. A. (1983) Structure-activity studies of the carcinogeni-
cities in the mouse and rat of some naturally occurring and synthetic
alkenylbenzene derivatives related to safrole and estragole. Cancer Res.
43, 1124–1134.
(4) Wiseman, R. W., Miller, E. C., Miller, J. A., and Liem, A. (1987)
Structure-activity studies of the hepatocarcinogenicities of alkenylben-
zene derivatives related to estragole and safrole on administration to
preweanling male C57BL/6J x C3H/HeJ F1 mice. Cancer Res.
47, 2275–2283.
(5) Jeurissen, S. M., Punt, A., Boersma, M. G., Bogaards, J. J. P.,
Fiamegos, Y. C., Schilter, B., Van Bladeren, P. J., Cnubben, N. H. P., and
Rietjens, I. M. C. M. (2007) Human cytochrome P450 enzyme
specificity for the bioactivation of estragole and related alkenylbenzenes.
Chem. Res. Toxicol. 20, 798–806.
(6) Fennell, T. R., Wiseman, R. W., Miller, J. A., and Miller, E. C.
(1985) Major role of hepatic sulfotransferase activity in the metabolic
activation, DNA adduct formation, and carcinogenicity of 10-hydroxy-
2030-dehydroestragole in infant male C57BL/6J x C3H/HeJ F1 mice.
Cancer Res. 45, 5310–5320.
Minor adducts of dG (ES-10-N2-dG) and dA (not identified)
were hardly detected in in vivo samples even though such adducts
are detectable in the reaction of 10-acetoxy-ES with deoxy-
nucleoside.7 Dissociation of the ester group from an ester of
1-hydroxy-ES generates an electrophilic ion in which the positive
charge may reside on the double bond between the 2-, 3- and 1-,
2-positions on the allyl chain. Nucleophilic attack by the purine
bases of DNA at the 10-position of ES would account for ES-10-
N2-dG formation, while attack at the 30-position would account
for ES-30-N2-dG. Because dG modification at the 30-position of
(7) Punt, A., Delatour, T., Scholz, G., Schilter, B., van Bladeren, P. J.,
and Rietjens, I. M. (2007) Tandem mass spectrometry analysis of
N2-(trans-isoestragol-30-yl)-20-deoxyguanosine as a strategy to study
540
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541

Chemical Research in Toxicology
ARTICLE
species differences in sulfotransferase conversion of the proximate
carcinogen 10-hydroxyestragole. Chem. Res. Toxicol. 20, 991–998.
(8) Phillips, D. H., Reddy, M. V., Miller, E. C., and Adams, B. (1981)
Structures of the DNA adducts formed in mouse liver after administra-
tion of the proximate hepatocarcinogen 10-hydroxyestragole. Cancer Res.
41, 176–186.
Jakovina, K., Brdar, B., Slade, N., Turesky, R. J., Goodenough, A. K.,
Rieger, R., Vukeliꢀc, M., and Jalakoviꢀc, B. (2007) Aristolochic acid and the
etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci. U.S.A.
104, 12129–12134.
(24) Kim, S. J., Jajoo, H. K., Kim, H. Y., Zhou, L., Horton, P., Harris,
C. M., and Harris, T. M. (1995) An efficient route to N6 deoxyadenosine
adducts of diol epoxides of carcinogenic polycyclic aromatic hydrocar-
bons. Bioorg. Med. Chem. 3, 811–822.
(25) Schmeiser, H. H., Scherf, H. R., and Wiessler, M. (1991)
Activating mutations at codon 61 of the c-Ha-ras gene in thin-tissue
sections of tumors induced by aristolochic acid in rats and mice. Cancer
Lett. 59, 139–143.
(26) Schmeiser, H. H., Janssen, J. W., Lyons, J., Scherf, H. R., Pfau,
W., Buchmann, A., Bartram, C. R., and Wiessler, M. (1990) Aristolochic
acid activates ras genes in rat tumors at deoxyadenosine residues. Cancer
Res. 50, 5464–5469.
(27) Nakae, D., Mizumoto, E., Kobayashi, E., Noguchi, O., and
Konishi, Y. (1995) Improved genomic/nuclear DNA extraction for
8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue.
Cancer Lett. 97, 233–239.
(28) Ishii, Y., Okamura, T., Inoue, T., Fukuhara, K., Umemura, T.,
and Nishikawa, A. (2010) Chemical structure determination of DNA
bases modified by active metabolites of lucidin-3-O-primeveroside.
Chem. Res. Toxicol. 23, 134–141.
(29) Yakovleva, L., Handy, C. J., Sayer, J. M., Pirrung, M., Jerina,
D. M., and Shuman, S. (2004) Benzo[c]phenanthrene adducts and
nogalamycin inhibit DNA transesterification by vaccinia topoisomerase.
J. Biol. Chem. 279, 23335–23342.
(30) Ponten, I., Sayer, J. M., Pilcher, A. S., Yagi, H., Kumar, S., Jerina,
D. M., and Dipple, A. (1999) Sequence context effects on mutational
properties of cis-opened benzo[c]phenanthrene diol epoxide-deoxya-
denosine adducts in site-specific mutation studies. Biochemistry
38, 1144–1152.
(9) Phillips, D. H., Reddy, M. V., and Randerath, K. (1984) 32P-post-
labelling analysis of DNA adducts formed in the livers of animals treated
with safrole, estragole and other naturally-occurring alkenylbenzens II.
Newborn male B6C3F1 mice. Carcinogenesis 5, 1623–1628.
(10) Rnaderathe, K., Haglund, R. E., Phillips, D. H., and Reddy,
M. V. (1984) 32P-post-labelling analysis of DNA adducts formed in the
livers of animals treated with safrole, estragole and other naturally-
occurring alkenylbenzenes. I. Adult female CD-1 mice. Carcinogenesis
5, 1613–1622.
(11) Wiseman, R. W., Fennell, T. R., Miller, J. A., and Miller, E. C.
(1985) Further characterization of the DNA adducts formed by
electrophilic esters of the hepatocarcinogens 10-hydroxysafrole and
10-hydroxyestragole in vitro and in mouse liver in vivo, including new adducts
at C-8 and N-7 of guanine residues. Cancer Res. 45, 3096–3105.
(12) Prakash, A. S., Tran, H. P., Peng, C., Koyalamudi, S. R., and
Dameron, C. T. (2000) Kinetics of DNA alkylation, depurination and
hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of
cadmium on DNA alkylation. Chem.-Biol. Interact. 125, 133–150.
(13) Moustacchi, E. (2000) DNA damage and repair: Consequences
on dose-responses. Mutat. Res. 464, 35–40.
(14) Choi, J. Y., and Guengerich, F. P. (2005) Adduct size limits
efficient and error-free bypass across bulky N2-guanine DNA lesions by
human DNA polymerase eta. J. Mol. Biol. 352, 72–90.
(15) Wei, D., Maher, V. M., and McCormick, J. J. (1996) Site-
specific excision repair of 1-nitrosopyrene-induced DNA adducts at the
nucleotide level in the HPRT gene of human fibroblasts: Effect of adduct
conformation on the pattern of site-specific repair. Mol. Cell. Biol.
16, 3714–3719.
(16) Singh, R., Gaskell, M., Le Pla, R. C., Kaur, B., Azim-Araghi, A.,
Roach, J., Koukouves, G., Souliotis, V. L., Kyrtopoulos, S. A., and Farmer,
P. B. (2006) Detection and quantification of benzo[a]pyrene-derived
DNA adducts in mouse liver by liquid chromatography-tandem mass
spectrometry: comparison with 32P-postlabeling. Chem. Res. Toxicol.
19, 868–878.
(17) Pfau, W., Brockstedt, U., S€ohren, K. D., and Marquardt, H.
(1994) 32P-post-labelling analysis of DNA adducts formed by food-
derived heterocyclic amines: Evidence for incomplete hydrolysis and a
procedure for adduct pattern simplification. Carcinogenesis 15, 877–882.
(18) Lyle, T. A., Royer, R. E., Daub, G. H., and Vander Jagt, D. L.
(1980) Reactivity-selectivity properties of reactions of carcinogenic
electrophiles and nucleosides: Influence of pH on site selectivity.
Chem.-Biol. Interact. 29, 197–207.
(31) Misra, B., Amin, S., and Hecht, S. S. (1992) Dimethylchrysene
diol epoxides: Mutagenicity in Salmonella typhimurium, tumorigenicity
in newborn mice, and reactivity with deoxyadenosine in DNA. Chem.
Res. Toxicol. 5, 248–254.
(32) Blake, R. D. (2005) Informational Biopolymers of Genes and Gene
Expression, p 290, University Science Books, CA.
(33) Page, J. E., Szeliga, J., Amin, S., Hecht, S. S., and Dipple, A.
(1995) Mutational spectra for 5,6-dimethylchrysene 1,2-dihydrodiol
3,4-epoxides in the supF gene of pSP189. Chem. Res. Toxicol. 8, 143–137.
(34) Schmeiser, H. H., Janssen, J. W., Lyons, J., Scherf, H. R., Pfau,
W., Buchmann, A., Bartram, C. R., and Wiessler, M. (1990) Aristolochic
acid activates ras genes in rat tumors at deoxyadenosine residues. Cancer
Res. 50, 5464–5469.
(19) Yuan, B., Cao, H., Jiang, Y., Hong, H., and Wang, Y. (2008)
Efficient and accurate bypass of N2-(1-carboxyethyl)-20-deoxyguano-
sine by DinB DNA polymerase in vitro and in vivo. Proc. Natl. Acad. Sci.
U.S.A. 105, 8679–8684.
(20) Xu, P., Oum, L., Geacintov, N. E., and Broyde, S. (2008)
Nucleotide selectivity opposite a benzo[a]pyrene-derived N2-dG ad-
duct in a Y-family DNA polymerase: A 50-slippage mechanism. Biochem-
istry 47, 2701–2709.
(21) Stover, J. S., Chowdhury, G., Zang, H., Guengerich, F. P., and
Rizzo, C. J. (2006) Translesion synthesis past the C8- and N2-
deoxyguanosine adducts of the dietary mutagen 2-Amino-3-
methylimidazo[4,5-f]quinoline in the Narl recognition sequence by
prokaryotic DNA polymerases. Chem. Res. Toxicol. 19, 1506–1517.
(22) Chan, W., Yue, H., Poon, W. T., Chan, Y. W., Schmitz, O. J.,
Kwong, D. W., Wong, R. N., and Cai, Z. (2008) Quantification of
aristolochic acid-derived DNA adducts in rat kidney and liver by using
liquid chromatography-electrospray ionization mass spectrometry.
Mutat. Res. 646, 17–24.
(23) Grollman, A. P., Shibutani, S., Moriya, M., Miller, F., Wu, L.,
Moll, U., Suzuki, N., Fernandes, A., Rosenquist, T., Medverec, Z.,
541
dx.doi.org/10.1021/tx100410y |Chem. Res. Toxicol. 2011, 24, 532–541