Quantitation of pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-performance liquid chromatography- electrospray ionization-tandem mass spectrometry

Article abstract of DOI:10.1021/tx050351x

The tobacco-specific nitrosamines N′-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O²-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O²-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1- butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 12) was the most abundant adduct, followed by O⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′- deoxyguanosine (O⁶-POB-dGuo, 8), O²-POB-dThd, and O ²-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O²-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O⁶-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O⁶-POB-dGuo was more abundant in lung than in liver. O²-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB-DNA adducts, support an important role of O⁶-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O²-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.

Full text of DOI:10.1021/tx050351x

6
74
Chem. Res. Toxicol. 2006, 19, 674-682
Quantitation of Pyridyloxobutyl DNA Adducts of Tobacco-Specific
Nitrosamines in Rat Tissue DNA by High-Performance Liquid
Chromatography-Electrospray Ionization-Tandem Mass
Spectrometry
Yanbin Lao,†,‡ Peter W. Villalta, Shana J. Sturla, Mingyao Wang, and
‡
†,‡
‡
,
‡
Stephen S. Hecht*
Department of Medicinal Chemistry and The Cancer Center, UniVersity of Minnesota,
Minneapolis, Minnesota 55455
ReceiVed December 15, 2005
The tobacco-specific nitrosamines N′-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by
cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce
pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and
NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed
a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-
ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The
corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked
with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis.
The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis.
2
The method was accurate and precise. Excellent sensitivity was achieved, especially for O -[4-(3-pyridyl)-
2
4
-oxobut-1-yl]thymidine (O -POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA
samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone
(
NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine
6
(7-POB-Gua, 12) was the most abundant adduct, followed by O -[4-(3-pyridyl)-4-oxobut-1-yl]-2′-
6
2
2
2
deoxyguanosine (O -POB-dGuo, 8), O -POB-dThd, and O -[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O -
POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the
in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O -
POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in
vivo. In contrast to the other three adducts, O -POB-dGuo was more abundant in lung than in liver.
O -POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-
6
6
2
Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive
6
quantitation of four POB-DNA adducts, support an important role of O -POB-dGuo in NNK lung
2
tumorigenicity in rats, and suggest that O -POB-dThd may be a useful tobacco-specific DNA biomarker
for future tobacco carcinogenesis studies.
Introduction
Tobacco-specific nitrosamines are believed to play an im-
rodents independent of the route of administration. It also
induces liver, pancreas, and nasal cavity tumors in rats (1). NNN
causes esophageal and nasal cavity tumors in rats and respiratory
tract tumors in mice, hamsters, and mink. Oral tumors were
induced when NNN mixed with NNK was swabbed in the rat
oral cavity (1). NNN plus NNK have been evaluated as human
carcinogens (group 1) by the International Agency for Research
on Cancer (2).
portant role in tobacco carcinogenesis. Among these compounds,
1
N′-nitrosonornicotine (NNN, 1, Scheme 1) and 4-(methylni-
trosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are the most
carcinogenic (1). NNK is a potent pulmonary carcinogen in
*
To whom correspondence should be addressed. Tel: 612-626-7604.
Fax: 612-626-5135. E-mail: hecht002@umn.edu.
†
Both NNN and NNK require metabolic activation to express
their carcinogenicity, and DNA adduct formation is central to
their mechanisms of carcinogenesis. Metabolism and DNA
adduct formation by NNN and NNK have been reviewed (1)
and are briefly summarized in Scheme 1. Two pathways
catalyzed by cytochrome P450s, R-methylene and R-methyl
hydroxylation, mainly account for NNK bioactivation. R-Methyl
hydroxylation of NNK produces intermediate 6, which reacts
with H2O to give 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 7).
DNA alkylation by 6 produces pyridyloxobutyl (POB)-DNA
adducts. 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-bu-
Department of Medicinal Chemistry.
‡
The Cancer Center.
1
6
Abbreviations: AGT, O -alkylguanine-DNA-alkyltransferase; HPB,
-hydroxy-1-(3-pyridyl)-1-butanone; HPLC-ESI-MS/MS, high-performance
4
liquid chromatography-electrospray ionization-tandem mass spectrometry;
LOD, limit of detection; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone; NNKOAc, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-bu-
2
2
tanone; NNN, N′-nitrosonornicotine; O -POB-dCyd, O -[4-(3-pyridyl)-4-
2
2
oxobut-1-yl]-2′-deoxycytidine; O -POB-Cyt, O -[4-(3-pyridyl)-4-oxobut-
-yl]cytosine; O -POB-dThd, O -[4-(3-pyridyl)-4-oxobut-1-yl]thymidine;
O -POB-dGuo, O -[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine; 7-POB-
dGuo, 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine; 7-POB-Gua, 7-[4-
2
2
1
6
6
(
4
3-pyridyl)-4-oxobut-1-yl]guanine; PCC, pyridinium chlorochromate; POB,
-(3-pyridyl)-4-oxobut-1-yl; SRM, selected reaction monitoring.
1
0.1021/tx050351x CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/22/2006

Quantitation of Pyridyloxobutyl DNA Adducts
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 675
Scheme 1. Metabolism and POB-DNA Adduct Formation by NNN and NNKa
a
dR, 2′-deoxyribosyl; NTH, neutral thermal hydrolysis.
tanone (NNKOAc, 3), a regiochemically activated form of NNK,
has been extensively used in studies of POB-DNA adduct
formation. Upon hydrolysis by esterase, NNKOAc releases 6,
which alkylates DNA. Similarly, NNN can be metabolically
activated by cytochrome P450-catalyzed 5′- and 2′-hydroxyla-
tion (Scheme 1) (1). The same intermediate 6 produced by NNN
from these adducts with the formation of dGuo and dCyd,
respectively (Scheme 1). Under the same conditions, the stable
adducts, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua,
12) and O -[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O -POB-Cyt,
13), are produced with the loss of 2′-deoxyribose (Scheme 1)
(6, 7). The formation of POB-DNA adducts is believed to be
an important mechanism for NNN and NNK carcinogenesis in
rodents (8-12) and likely in smokers (13, 14).
2
2
2
′-hydroxylation gives rise to POB-DNA adducts. The proper-
6
ties of one adduct, O -[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxy-
guanosine (O -POB-dGuo, 8), have been studied previously (3-
5
6
To further explore the mechanistic role of this bioactivation
pathway, a sensitive analytical method is needed to monitor the
formation, persistence, and repair of POB-DNA adducts in
vivo. Thomson et al. developed a liquid chromatography-
electrospray ionization-tandem mass spectrometry (LC-ESI-
). More recently, three new POB-DNA adducts, 7-[4-(3-
pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (7-POB-dGuo, 9)
2
2
(6), O -[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (O -POB-
2
dCyd, 10) (7), and O -[4-(3-pyridyl)-4-oxobut-1-yl]thymidine
2
6
(O -POB-dThd, 11) (7), have been identified. Upon neutral
MS/MS) method for quantitation of O -POB-dGuo (15). The
thermal hydrolysis (heating at 100 °C for 1 h) of DNA
containing 7-POB-dGuo and O -POB-dCyd, HPB (7) is released
limit of detection (LOD) was 1 fmol of standard. Recently, we
reported the relative levels of 7-POB-Gua, O -POB-dGuo, O -
2
6
2

676 Chem. Res. Toxicol., Vol. 19, No. 5, 2006
Lao et al.
2
POB-dThd, and O -POB-Cyt in reactions of calf thymus DNA
with NNKOAc (16). However, the lack of stable isotope-labeled
internal standards limited the application of that method. In this
study, using all four deuterated internal standards, we developed
and validated a high-performance liquid chromatography-
electrospray ionization-tandem mass spectrometry (HPLC-ESI-
MS/MS) method for quantitative analysis of the above four
adducts. The current method is highly sensitive, accurate, and
precise. It has been applied to quantify POB-DNA adducts in
liver and lung of NNK-treated rats.
0.7 mL/min. This system was used for quantitation of dGuo in
enzymatic hydrolysates of DNA samples.
6
Synthesis of Deuterated Standards. [Pyridine-D
pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine ([pyridine-D
dGuo, [pyridine-D ]8) was synthesized based on a previously
published method for O -POB-dGuo (3, 17). [Pyridine-D
pyridine carboxaldehyde (14) was prepared from commercially
available [pyridine-D ]ethyl nicotinate (15) (18). Compound 15 (2
g, 13 mmol) in MeOH (10 mL) was added dropwise to a solution
of NaBH (4.9 g, 130 mmol) in MeOH (20 mL). The reaction
4
]O -[4-(3-
4
6
]O -POB-
4
6
4
]3-
4
4
mixture was heated under reflux for 2 h. Water (30 mL) was added,
and the MeOH was evaporated. The mixture was extracted with
EtOAc (3 × 30 mL). The organic layers were washed with 1 M
HCl (2 × 50 mL). Then, the aqueous solution was adjusted to pH
Experimental Procedures
7
with 10 N NaOH and extracted with EtOAc (5 × 100 mL). The
2 4
Caution: NNK and NNKOAc are carcinogenic. They should
be handled in a well-Ventilated hood with extreme care and with
personal protectiVe equipment.
Chemicals. O -POB-dGuo, O -POB-dThd, 7-POB-Gua, O -
POB-Cyt, and [pyridine-D ]7-POB-Gua ([pyridine-D ]12) were
prepared as described (16). All standards had purities of at least
9% (HPLC-UV). The isotopic purity of [pyridine-D ]7-POB-Gua
was estimated as 98 at. %D based on the isotopic purity of the
starting material, [pyridine-D ]ethyl nicotinate, which was purchased
from Cambridge Isotope Laboratories (Andover, MA). Micrococcal
nuclease (LS004797, 15 kU) and phosphodiesterase II (LS003603,
0 U) were obtained from Worthington Biochemical Corp. (Lake-
wood, NJ). Alkaline phosphatase (567752, 30 U/µL) was purchased
from Roche Molecular Biochemicals (Indianapolis, IN). Porcine
liver esterase (E-2884, 1.84 U/µL) was obtained from Sigma-
Aldrich Chemical Co. (Milwaukee, WI). All other chemicals and
solvents were acquired from Sigma-Aldrich or Fisher Scientific
EtOAc layers were combined, dried (Na
dryness to give [pyridine-D ]3-hydroxymethylpyridine (16, 1.0 g,
.85 mmol, 68%). A solution of 16 (500 mg, 4.42 mmol) in 10
mL of anhydrous CH Cl was added to a mixture of pyridinium
chlorochromate (PCC, 1.9 g, 8.82 mmol) in 10 mL of CH Cl . The
mixture was stirred under N at room temperature for 1 h. Ether
50 mL) was added, and the mixture was stirred for an additional
SO ), and evaporated to
4
8
6
2
2
2
2
4
4
2
2
2
9
4
(
30 min at room temperature and then loaded onto a short column
4
4
of Florisil. [Pyridine-D ]3-pyridine carboxaldehyde (14, 110 mg,
1
D
mmol, 22%) was eluted from the column with EtOAc. [Pyridine-
6
4
]O -POB-dGuo (6.96 mg, 16.65 µmol) was then synthesized,
1
6
according to the published procedure for O -POB-dGuo (3, 17).
Purity, at least 99% (HPLC-UV); isotopic purity, 98 at. %D. H
1
NMR (CDCl
′H), 4.89 (br, 2H, 2-NH
), 4.18 (br, 1H, 4′H), 3.94 (d, J ) 11.4 Hz, 1H,
′Ha), 3.74 (d, J ) 12.0 Hz, 1H, 5′Hb), 3.22 (t, J ) 7.2 Hz, 2H,
3
): δ 7.61 (s, 1H, 8H), 6.21 (dd, J ) 5.4, 9.6 Hz, 1H,
1
2
), 4.74 (d, J ) 4.8 Hz, 1H, 3′H), 4.59 (t,
J ) 6.0 Hz, 2H, H
A
5
(Fairlawn, NJ).
C B
H ), 3.00 (m, 1H, 2′Ha), 2.29 (m, 2H, H ), 2.23 (m, 1H, 2′Hb).
NMR and MS. Qualitative MS was performed on a Finnigan
UV (MeOH/pH 6.8 buffer): λ (nm) 277.0, 235.8, 210.1. Positive
max
+
LCQ Deca instrument (Finnigan MAT/Thermoquest, San Jose, CA)
or on an Agilent 1100 LC/MSD ion trap instrument (Agilent
Technologies, Inc., Wilmington, DE) in positive-ion mode for
characterization of deuterated standards. The development, valida-
tion, and application of the quantitative method were conducted
on either a Finnigan TSQ Quantum Ultra AM or a TSQ Quantum
Discovery Max (Thermo Electron, San Jose, CA) instrument
coupled with an Agilent 1100 series capillary HPLC system. NMR
spectra were acquired on a Varian Inova 500 or 600 MHz
instrument (Varian, Inc., Palo Alto, CA).
ESI-MS m/z 419: [M + H] , MS/MS of m/z 419, m/z 303 [M -
+
+
116 + H] , m/z 268 [M - [pyridine-D ]POB + H] , m/z 152 [Gua
4
+
+
+ H] and [[pyridine-D ]POB] .
4
2
[
Pyridine-D
4
]O -[4-(3-pyridyl)-4-oxobut-1-yl]thymidine ([Py-
2
ridine-D
4
]O -POB-dThd, [Pyridine-D ]11). Thymidine-5′-tolu-
4
enesulfonate ester (62 mg, 0.15 mmol) (16) was allowed to react
with [pyridine-D ]HPB (464 mg, 2.74 mmol) (16) in the presence
of 1,8-diazobicyclo[5.4.0]undec-7-ene (332 mg, 2.18 mmol), fol-
4
2
lowed by HPLC purification (system 1) to give [pyridine-D
4
]O -
POB-dThd (597 µg, 1.52 µmol, 1%). Purity, at least 99% (HPLC-
1
HPLC Analysis. HPLC was carried out with Waters Associates
Medford, MA) systems equipped with a Shimadzu SPD-10A UV-
vis detector (Shimadzu Scientific Instruments, Columbia, MD) for
systems 1 and 4 or a model 996 photodiode array detector for
systems 2 and 3. UV detection was at 254 nm. Columns and solvent
elution systems were as follows. System 1 was a 250 mm × 21.2
mm Luna 5 µm C18 (2) column (Phenomenex, Torrance, CA) with
isocratic elution by 5% MeOH in H
from 5 to 35% MeOH over 20 min, followed by 35-50% MeOH
over a period of 20 min at a rate of 7.0 mL/min. This system was
UV); isotopic purity, 98 at. %D. H NMR (DMSO-d
1H, 6H), 6.21 (t, J ) 6.0 Hz, 1H, 1′H), 4.35 (t, J ) 6.0 Hz, 2H,
), 4.23 (br, 1H, 3′H), 3.77 (br, 1H, 4′H), 3.60 (dd, J ) 3.0, 12.0
Hz, 1H, 5′Ha), 3.54 (dd, J ) 3.0, 12.0 Hz, 1H, 5′Hb), 3.16 (br,
2H, H ), 2.16 (m, 2H, 2′H), 2.07 (m, 2H, H ), 1.78 (s, 3H, 5-CH ).
UV (acetonitrile/H O): λmax (nm) 258.1, 231.1. Positive ESI-MS
6
): δ 7.82 (s,
(
H
A
C
B
3
2
+
m/z 394 [M + H] , MS/MS of m/z 394: m/z 278 [M - 116 +
+
+
2
O for 5 min and then a gradient
H] , m/z 152 [[pyridine-D
4
]POB] .
2
[Pyridine-D
4
]O -[4-(3-pyridyl)-4-oxobut-1-yl]cytosine ([Pyri-
2
dine-D
35.7 mmol), [pyridine-D
prepared using the synthetic method for NNKOAc (3) (19).
[Pyridine-D ]NNKOAc (138 mg, 0.51 mmol) was then incubated
4
]O -POB-Cyt, [Pyridine-D
4
]13). Starting with 15 (5.54 g,
2
used for purification of [pyridine-D
4
]O -POB-dThd ([pyridine-D
4
]-
4
]NNKOAc (485 mg, 1.80 mmol) was
1
(
1). System 2 was a 250 mm × 3.0 mm Luna 5 µm C18 (2) column
Phenomenex) with isocratic elution by 5% MeOH in 40 mM
ammonium acetate buffer (pH 6.8) for 10 min, then a gradient from
to 25% MeOH over the course of 60 min, followed by 25-50%
4
with dCyd (26 mg, 0.115 mmol) in the presence of porcine liver
5
esterase (170 µL) at 37 °C for 1 h, followed by heating at 100 °C
2
MeOH for an additional 10 min, and then to 5% MeOH in 5 min
at a flow rate of 0.5 mL/min. In system 3, we used the same column
and flow rate as in system 2. However, the gradient was changed
to initial isocratic elution by 5% acetonitrile in 40 mM ammonium
acetate (pH 6.8) for 10 min, then 5-25% acetonitrile in 40 min,
for 1 h. [Pyridine-D
4
]O -POB-Cyt was purified by HPLC using
systems 2 and 3 sequentially. The same procedure was repeated
four times to give 17.8 µg (0.068 µmol, 0.01%) of [pyridine-D ]-
O -POB-Cyt. Purity, at least 99% (HPLC-UV); isotopic purity, 98
4
2
1
at. %D. H NMR (DMSO-d
6.79 (br, 2H, 4-NH ), 6.04 (d, J ) 6.0 Hz, 1H, 5H), 4.20 (t, J )
6.6 Hz, 2H, H ), 2.01 (m, 2H, H ).
6
): δ 7.81 (d, J ) 5.4 Hz, 1H, 6H),
followed by a return to 5% acetonitrile in 2 min. Systems 2 and 3
2
2
were used for the first and second purification of [pyridine-D
POB-Cyt ([pyridine-D
50 mm × 4.6 mm Luna 5 µm C18 (2) column with a gradient
from 5 to 40% MeOH in H O over a period of 35 min, followed
by 40-100% MeOH in 5 min, then held at 100% MeOH for 10
min before going back to 5% MeOH in 10 min at a flow rate of
4
]O -
A
), 3.19 (t, J ) 6.6 Hz, 2H, H
C
B
UV (acetonitrile/pH 6.8 buffer): λmax (nm) 268.8, 227.6. Positive
4
]13), respectively. System 4 consisted of a
+
2
ESI-MS m/z 263 [M + H] , MS/MS of m/z 263: m/z 152
+
+
4
[[pyridine-D ]POB] , m/z 112 [Cyt + H] .
2
6
A stock solution of [pyridine-D
was prepared gravimetrically, and stock solutions of [pyridine-D
4
]O -POB-dGuo (1.67 nmol/µL)
]-
4

Quantitation of Pyridyloxobutyl DNA Adducts
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 677
dThd, m/z 394.1 [M + 1] + to m/z 152.1 ([pyridine-D ]POB])+;
2
2
O -POB-dThd (304 pmol/µL) and [pyridine-D
4
]O -POB-Cyt (13.6
4
+
+
pmol/µL) were prepared by NMR standardization with toluene as
an internal standard, as previously described (16).
7-POB-Gua, m/z 299.1 [M + 1] to m/z 148.1 [POB] ; [pyridine-
+
D
4
4
]7-POB-Gua, m/z 303.1 [M + 1] to m/z 152.1 ([pyridine-D ]-
+
+
2
+
In Vitro DNA Samples. Two NNKOAc-treated calf thymus
DNA samples, ctDNA-A and ctDNA-B, were obtained from the
previous study (16) and used for development and validation of
the method. Briefly, calf thymus DNA (20 mg, 70 µmol) was
incubated with NNKOAc (for ctDNA-A: 10 mg, 40 µmol; for
ctDNA-B: 20 mg, 80 µmol) in the presence of porcine liver esterase
POB]) and [Gua + H] ; O -POB-Cyt, m/z 259.1 [M + 1] to
+
2
+
m/z 148.1 [POB] ; [pyridine-D
to m/z 152.1 ([pyridine-D
4
]O -POB-Cyt, m/z 263.1 [M + 1]
+
4
]POB]) . When the HPLC-ESI-MS/MS
method was being validated for each POB-DNA adduct, the MS
parameters were optimized to achieve the maximum signal for the
adduct by tuning the instrument with the corresponding deuterated
standard. For analysis of in vitro and in vivo DNA samples, the
MS parameters were set as follows: spray voltage, 4 kV; sheath
gas pressure, 30; capillary temperature, 200 °C; collision energy,
20 V; scan width, 0.5 amu; scan time, 0.2 s, Q1 peak width, 0.7
amu; Q3 peak width, 0.7 amu; Q2 gas pressure, 1.0 mTorr; source
CID, 10 V; and tube lens offset, 94 V. MS/MS data were acquired
and processed by Xcaliber software version 1.4 (Thermo Electron).
The amount of each POB-DNA adduct was determined by
comparing the MS peak area ratio of each adduct to its deuterated
standard with a calibration curve. Calibration standards were
prepared by spiking different amounts of each adduct with a
(
0.5-1.0 µL per mg NNKOAc) in 0.1 M phosphate buffer (10 mL,
pH 7.0) at 37 °C for 1.5 h. The incubation mixture was washed
with equal volumes of CHCl /isoamyl alcohol (24:1) and ethyl
3
acetate sequentially. The DNA was precipitated by adding cold
ethanol, washed with 70% ethanol and ethanol sequentially, dried
under a stream of N , and stored at -20 °C until analysis.
2
In Vivo DNA Samples. These were isolated from liver and lung
of NNK-treated male F344 rats. The animals were purchased at
age 5-6 weeks from Charles River (Wilmington, MA). They were
housed under standard conditions and maintained on NIH-07 diet
(Harlan, Madison, WI) in the Research Animal Resources facility
of the University of Minnesota. Groups of five rats (225-275 g)
were treated with saline (0.4 mL) (control group), 5.2 mg (0.025
mmol) NNK/kg body weight in 0.4 mL saline (low dose group),
or 20.7 mg (0.1 mmol) NNK/kg body weight in 0.4 mL saline (high
dose group) once daily for four consecutive days by s.c. injection.
2
constant amount of the corresponding internal standard in H O and
then analyzed by LC-MS/MS without undergoing the sample
preparation procedure described above. The calibration curves were
constructed by plotting concentration ratio vs MS peak area ratio
of each adduct to its deuterated standard. The adduct levels were
expressed as pmol (for in vitro samples) or fmol (for in vivo
samples) per µmol dGuo.
2
Four hours after the final dose, the rats were killed by CO overdose.
Liver and lung were harvested, and DNA was isolated by following
the “DNA Purification from 1 g Animal Tissue” protocol from
Gentra Systems (Minneapolis, MN) with modifications (20) and
stored at -20 °C until analysis.
Results
HPLC-ESI-MS/MS Analysis. The procedure for enzymatic
hydrolysis of DNA was previously described (15). In brief, DNA
samples (about 1 mg for in vitro and 0.5 mg for in vivo) were
We report here the preparation of three deuterated internal
6
2
standards, [pyridine-D4]O -POB-dGuo, [pyridine-D4]O -POB-
2
dThd, and [pyridine-D4]O -POB-Cyt, as well as the development
2
dissolved in 10 mM sodium succinate/5 mM CaCl buffer (1 mL,
and validation of a sensitive HPLC-ESI-MS/MS method for
quantitative analysis of four stable POB-DNA adducts. The
method was applied for analysis of lung and liver DNA isolated
from NNK-treated F344 rats.
pH 7.0), to which the four deuterated internal standards had been
added. The mixture was heated at 100 °C for 30 min and then
cooled to room temperature. Micrococcal nuclease (75 U) and
phosphodiesterase II (450 mU) were added, and the mixture was
incubated at 37 °C. After 6 h, alkaline phosphatase (150 U) was
added, followed by incubation at 37 °C overnight. An aliquot (10
µL) of the enzymatic hydrolysate was saved for quantitation of
dGuo (HPLC system 4), and the remainder was loaded on a Strata-X
cartridge (33 µm, 30 mg/mL, 1 mL, Phenomenex). The cartridge
6
Synthesis and Calibration. [Pyridine-D4]O -POB-dGuo was
6
prepared as reported previously for O -POB-dGuo (17, 18).
2
2
[
Pyridine-D4]O -POB-dThd and [pyridine-D4]O -POB-Cyt were
2
2
synthesized as described for O -POB-dThd and O -POB-Cyt
16). The structures of these deuterated standards were estab-
lished by UV, MS, and H NMR. The deuterated analogues
had the same UV spectra as the nondeuterated adducts, the
expected four unit increases in mass spectra, and lack of pyridyl
(
was sequentially eluted with 2 × 1 mL H
2
O, 1 mL of 10% aqueous
1
MeOH, and 2 × 1 mL MeOH. The MeOH fraction was collected
in a 4 mL silanized vial (ChromTech, Minneapolis, MN) and dried
under reduced pressure using a centrifugal vacuum evaporator
1
protons in the H NMR spectra. Calibration curves were
(Savant SpeedVac, Thermo Electron). The residue was then
transferred with 2 × 100 µL MeOH to a silanized autosampler vial
with an infused 300 µL inset (ChromTech) and dried again under
vacuum. Water (15 µL) was added to the MS vial, and the solution
was sonicated for 10 min and then vortexed and centrifuged.
Eight microliters of the sample per injection was introduced with
an autosampler into the ESI source using a 150 mm × 0.5 mm
Zorbax SB C18 column (Agilent) with a gradient from 0 to 25%
acetonitrile in 15 mM ammonium acetate over a period of 29 min,
then 25-75% acetonitrile for 5 min, then 75% acetonitrile for 5
min, then returning to 0% acetonitrile in 5 min, at a flow rate of
constructed by analysis of calibration standards prepared by
adding a constant amount of each deuterated standard to
increasing concentrations of the nondeuterated standards and
plotting concentration ratios vs MS peak area ratios of analyte/
internal standard. Each adduct was detected by SRM for [M +
+
+
1
]
f [POB] . The plots showed excellent linearity (see
Supporting Information Figure 1A-D).
Method Development. The HPLC-ESI-MS/MS method was
developed on the basis of our previous study, with modifications
2
1
5 µL/min. The column was operated at 25 °C. The first 10 min
(16). In this method, the base adducts 7-POB-Gua and O -POB-
eluant was directed to waste, and the 10 to 40 min fraction was
Cyt, which resulted from unstable adducts bearing quaternary
nitrogens (Scheme 1), were released from the DNA backbone
upon neutral thermal hydrolysis. Further enzymatic hydrolysis
diverted to the ESI source. The retention times of the POB-DNA
6
adducts in this HPLC system were as follows: O -POB-dGuo, 27.5
2
2
min; O -POB-dThd, 24.0 min; 7-POB-Gua, 20.7 min; and O -POB-
Cyt, 27.3 min.
2
6
produced the nucleoside adducts O -POB-dThd and O -POB-
dGuo. The DNA enzymatic hydrolysis procedure was further
investigated. When calf thymus DNA was digested by a
combination of DNAse I, phosphodiesterase I, and alkaline
phosphatase, two significant interfering peaks eluting at the same
The ESI source was operated in the positive ion mode. The
POB-DNA adducts, along with their deuterated standards, were
monitored by selected reaction monitoring (SRM). The ion transi-
6
+
tions were as follows: O -POB-dGuo, m/z 415.1 [M + 1] to m/z
6
2
+
6
+
retention times as O -POB-dGuo and O -POB-Cyt were ob-
1
48.1 [POB] ; [pyridine-D
to m/z 152.1 ([pyridine-D ]POB]) and [Gua + H] ; O -POB-dThd,
m/z 390.1 [M + 1] + to m/z 148.1 [POB] ; [pyridine-D
4
]O -POB-dGuo, m/z 419.1 [M + 1]
+
+
2
2
served, but there were no interferences for O -POB-dThd and
4
+
2
4
]O -POB-
7-POB-Gua (data not shown). When a different procedure was

6
78 Chem. Res. Toxicol., Vol. 19, No. 5, 2006
Lao et al.
2
2
dGuo, 25 and 29; O -POB-dThd, 16 and 16; and O -POB-Cyt,
3 and 12. For each POB-DNA adduct, the ratio of adduct
1
level in ctDNA-B to that in ctDNA-A was 3, which was
consistent with the higher amount of NNKOAc used in the
reaction to form ctDNA-B than ctDNA-A.
Characteristics of the Method. ctDNA-A and ctDNA-B
were diluted with untreated calf thymus DNA and analyzed by
the HPLC-ESI-MS/MS method. The results are shown in Figure
2
2
1
A-D. All R values were 0.99, and the slopes ranged from
.05 to 1.20, supporting the accuracy of this method for
quantitation of each POB-DNA adduct. The precision of the
method was tested by analyzing seven aliquots at each of two
adduct concentrations. Coefficients of variation were (%,
concentration in fmol/mg DNA) 7-POB-Gua (10, 35.5; 11,
Figure 1. Levels of POB-DNA adducts in NNKOAc-treated calf
thymus DNA. Open bars, ctDNA-A; solid bars, ctDNA-B. Each value
is the mean ( SD of at least three measurements.
6
2
8
1
.90); O -POB-dGuo (7, 10.5; 14, 2.62); O -POB-dThd (4, 10.4;
1, 2.08); and O -POB-Cyt (5, 43.6; 6, 10.9). Sensitivity was
used, consisting of micrococcal nuclease, phosphodiesterase II,
and alkaline phosphatase, the levels of both interfering peaks
2
6
determined by estimation of the LOD of each adduct in 1 mg
of DNA matrix with a signal-to-noise ratio of 3. LODs were 3
were lowered to background such that quantitation of O -POB-
2
dGuo and O -POB-Cyt was feasible. Another modification was
6
fmol for 7-POB-Gua, 1 fmol for O -POB-dGuo, 100 amol for
the use of a different ion transition for SRM. In previous studies
2
2
6
O -POB-dThd, and 2 fmol for O -POB-Cyt. At least a 3-fold
improvement in sensitivity was achieved for pure standards in
the absence of the DNA matrix. This implies matrix suppression
of ionization in the MS analysis. Recoveries were determined
based on recoveries of deuterated internal standards (N ) 24-
(
[
15, 16), O -POB-dGuo was quantified by SRM for m/z 415.1
+
+
M + 1] f m/z 299.1 [BH] . However, the MS response
increased five times when the ion transition m/z 415.1
M + 1] f m/z 148.1 [POB] was used. Similarly, the same
modification achieved six times higher MS response for O -
POB-dThd.
+
+
[
2
26). The peak areas of the deuterated standards after the analysis
procedure were compared to the standard curves constructed
with the deuterated standards without undergoing the analysis
procedure. Recoveries were 91 ( 10% for 7-POB-Gua, 84 (
Quantitation of POB-DNA Adducts in Calf Thymus DNA
Treated with NNKOAc. Levels of POB-DNA adducts in
ctDNA-A and ctDNA-B were quantified by the HPLC-ESI-
MS/MS method (Figure 1; also see Table 1 in the Supporting
Information). In each of these NNKOAc-treated DNA samples,
6
2
8
% for O -POB-dGuo, 59 ( 11% for O -POB-dThd, and 61 (
2
4
% for O -POB-Cyt.
6
levels of adducts followed the same order: 7-POB-Gua > O -
Quantitation of POB-DNA Adducts in NNK-Treated
2
2
POB-dGuo > O -POB-dThd > O -POB-Cyt. 7-POB-Gua
accounted for 46% of total POB adducts in ctDNA-A and 43%
in ctDNA-B. The corresponding values for the other adducts
Rats. About 0.5 mg of each DNA sample was subjected to
analysis, and each measurement was carried out in duplicate.
The amounts of internal standards were adjusted to ensure that
peak area ratios of analyte/internal standard fell within the ranges
6
were (% in ctDNA-A and % in ctDNA-B) as follows: O -POB-
6
Figure 2. Correlations between expected and measured levels of POB-DNA adducts in NNKOAc-treated calf thymus DNA (A) O -POB-dGuo,
2
2
(
B) O -POB-dThd, (C) 7-POB-Gua, and (D) O -POB-Cyt. Each value is the mean of duplicate measurements of diluted ctDNA-A or ctDNA-B.
6
2
The ranges of expected levels of POB-DNA adducts were as follows: 1.31-83.83 fmol for O -POB-dGuo, 0.26-51.95 fmol for O -POB-dThd,
2
4
[
.45-142 fmol for 7-POB-Gua, and 10.91-349 fmol for O -POB-Cyt. The amounts of added internal standards were as follows: 49.95 fmol of
6 2 2
4 4 4 4
pyridine-D ]O -POB-dGuo, 45.57 fmol of [pyridine-D ]O -POB-dThd, 80 fmol of [pyridine-D ]7-POB-Gua, and 136.1 fmol of [pyridine-D ]O -
POB-Cyt.

Quantitation of Pyridyloxobutyl DNA Adducts
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 679
Figure 3. SRM chromatograms obtained upon analysis of rat lung DNA isolated from (A) control group and (B) low dose NNK-treated group.
Individual POB-DNA adducts or internal standards were monitored as indicated on each channel. In Figure 3A, the peak eluting at the position
2
2
4
of O -POB-dThd, the area of which is 0.2% of [pyridine-D ]O -POB-dThd, originates from the internal standard.
Figure 4. SRM chromatograms obtained upon analysis of rat liver DNA isolated from (A) control group and (B) low dose NNK-treated group.
Individual POB-DNA adducts or internal standards were monitored as indicated on each channel. In Figure 4A, The peak eluting at the position
2
2
4
of O -POB-dThd, the area of which is 0.3% of [pyridine-D ]O -POB-dThd, originates from the internal standard.
of the calibration curves. POB-DNA adducts were not detected
DNA and 90-130-fold lower in liver DNA. The percentages
2
in lung and liver DNA isolated from control rats (Figures 3A
of O -POB-dThd of total POB adduct levels were 39-40% in
2
2
and 4A). The peaks eluting at the position of O -POB-dThd,
lung DNA and 35.1-38% in liver DNA, and those of O -POB-
2
the areas of which were 0.2 and 0.3% of [pyridine-D4]O -POB-
Cyt were 16-17% in lung DNA and 24-29.2% in liver DNA.
The dose ratio between the two NNK treatments was 4, and
the ratios of individual adduct levels between the high dose and
the low dose groups were 2 for lung DNA and 3-5 for liver
DNA, which was consistent with dose-dependent formation of
these POB-DNA adducts from NNK in vivo.
dThd, originated from the internal standard, as determined by
control experiments in which the deuterated standards were
added to calf thymus DNA. Adducts were readily detected and
quantified in the NNK-treated rats (Figures 3B and 4B). At both
doses, relative adduct levels in lung and liver DNA (Figure
2
2
5
A,B) had the same trend: 7-POB-Gua ≈ O -POB-dThd > O -
Total average levels of POB-DNA adducts formed in the
liver were higher than those in the lung (15100 fmol/µmol dGuo
vs 4180 fmol/µmol dGuo for low dose; 48600 fmol/µmol dGuo
vs 7730 fmol/µmol dGuo for high dose). At the low dose, the
6
POB-Cyt > O -POB-dGuo (see also Tables 2 and 3 in the
Supporting Information). Consistent with the in vitro data
(Figure 1), 7-POB-Gua was a major adduct detected in vivo. It
accounted for 34% of total POB adduct levels in lung DNA in
the low dose group and 35% in the high dose group. In liver
DNA, the percentages of 7-POB-Gua were 35.4% of total POB
adducts in the low dose group and 37.6% in the high dose group.
In contrast to comprising about 25-29% of total POB adducts
in the NNKOAc-treated calf thymus DNA (Table 1 in the
levels of three adducts were 3-6 times higher in the liver than
lung (Figure 6A-C). However, the amount of O6-POB-dGuo
was 9-fold higher in the lung than liver (Figure 6D). Similar
results were seen in the high dose group (Figure 7A-D).
Discussion
6
Supporting Information), the percentages of O -POB-dGuo were
only 9% in lung DNA and 0.3-0.4% in liver DNA. The levels
NNN and NNK, upon metabolic activation, generate the DNA
alkylating agent 6 with consequent formation of POB-DNA
6
of O -POB-dGuo were 4-fold lower than 7-POB-Gua in lung

6
80 Chem. Res. Toxicol., Vol. 19, No. 5, 2006
Lao et al.
Figure 7. Comparisons of POB-DNA adduct levels in liver and lung
DNA of NNK-treated rats in the high dose group (0.1 mmol/kg bw
2
2
per day for 4 days). (A) 7-POB-Gua, (B) O -POB-dThd, (C) O -POB-
6
Cyt, and (D) O -POB-dGuo. Open bars, adduct levels in lung DNA;
solid bars, adduct levels in liver DNA. Each value is the mean ( SD
of duplicate measurements of individual DNA sample isolated from
each of five rats in the high dose group.
Figure 5. Levels of POB-DNA adducts in (A) lung and (B) liver
6
DNA of NNK-treated rats. Inset: plot of O -POB-dGuo on a different
were found in human pulmonary DNA from lung cancer patients
as compared to controls (14). However, the assay of released
HPB measured total levels of POB-DNA adducts, which may
include various nucleobase and phosphotriester adducts (22).
Recently, four POB-DNA adducts were structurally character-
scale. Open bars, low dose; solid bars, high dose. Each value is the
mean ( SD of duplicate measurements of each DNA sample isolated
from five rats per group.
6
2
ized as follows: O -POB-dGuo (3), 7-POB-dGuo (6), O -POB-
2
2
dThd (7), and O -POB-dCyd (7). 7-POB-dGuo and O -POB-
dCyd were shown to release HPB upon neutral thermal
hydrolysis, with the formation of the stable adducts 7-POB-
2
Gua and O -POB-Cyt, respectively (6, 7). The availability of
this structural information allows us to develop an assay to
quantify these specific adducts rather than measuring released
HPB as in previous studies and thus further evaluate the role
of each specific adduct in NNN and NNK carcinogenicity. There
have been no previous reports of individual POB-DNA adduct
levels in rats treated with NNN or NNK. The POB-DNA
adducts may also have potential as biomarkers in studies of
tobacco carcinogenesis and chemoprevention in humans.
The sensitive HPLC-ESI-MS/MS method described here was
developed with modifications from our previous study (16). The
enzymatic hydrolysis procedure was improved to decrease
interferences resulting from incomplete DNA digestion. The
procedure, which uses micrococcal nuclease, phosphodiesterase
II, and alkaline phosphatase, has been shown previously to
completely digest DNA treated with radiolabeled NNKOAc
Figure 6. Comparisons of POB-DNA adduct levels in liver and lung
DNA of NNK-treated rats in the low dose group (0.025 mmol/kg bw
2
2
per day for 4 days). (A) 7-POB-Gua, (B) O -POB-dThd, (C) O -POB-
6
Cyt, and (D) O -POB-dGuo. Open bars, adduct levels in lung DNA;
(15). The HPLC-ESI-MS/MS method was further improved by
solid bars, adduct levels in liver DNA. Each value is the mean ( SD
of duplicate measurements of individual DNA samples isolated from
each of five rats in the low dose group.
monitoring the formation of the POB ion from the parent ion,
6
resulting in 5- and 6-fold increases in sensitivity for O -POB-
2
dGuo and O -POB-dThd, respectively, as compared to our
adducts (1). This pathway was established over 15 years ago
previous study (16). The accuracy and precision of the method
were investigated using NNKOAc-modified DNA containing
known amounts of adducts. Unlike pure POB-DNA adduct
standards, modified DNA more closely mimics real DNA
samples. The method was both accurate and precise. The method
was also quite sensitive with detection limits of 0.1-3 fmol
adduct per mg DNA.
(21) and has been quantified by measuring HPB-releasing DNA
adducts (13). DNA samples were hydrolyzed with acid, and
the released HPB was quantified by GC-MS after derivatization
with pentafluorobenzoyl chloride (13). Using this assay, POB-
DNA adduct formation was shown to be an important mecha-
nism in NNN and NNK carcinogenesis in rodents (8-12).
POB-DNA adducts very likely play an important role in
tobacco carcinogenesis in smokers. Levels of HPB-releasing
adducts were higher in lung DNA from smokers than nonsmok-
ers (13), and significantly higher levels of HPB-releasing adducts
As shown in Figure 1, levels of 7-POB-Gua were the highest
6
followed by O -POB-dGuo among the POB adducts in calf
thymus DNA modified with NNKOAc. These results indicate
that Gua in DNA has the highest reactivity toward pyridylox-

Quantitation of Pyridyloxobutyl DNA Adducts
Chem. Res. Toxicol., Vol. 19, No. 5, 2006 681
obutylating agent 6 generated upon esterase activation of
NNKOAc. Because 7-POB-Gua and O -POB-Cyt are partially
POB-dGuo were 3-9 times higher in lung DNA than in liver
2
6
DNA. The ratios of O -POB-dGuo levels between lung and liver
released by neutral thermal hydrolysis during sample preparation
DNA decreased with increasing dose, which may be due to AGT
saturation. O -POB-dGuo is highly mutagenic in bacteria and
human cells, causing G to A transitions (4). The apparent
2
6
from the unstable adducts, 7-POB-dGuo and O -POB-dCyd,
2
respectively, levels of the initially formed 7-POB-dGuo and O -
POB-dCyd are likely higher than those measured here. As shown
in Scheme 1, neutral thermal hydrolysis of 7-POB-dGuo and
O -POB-dCyd also produces HPB and deoxyribonucleosides,
persistence of this mutagenic DNA adduct in rat lung, the main
target tissue for NNK tumorigenicity, suggests that O -POB-
dGuo plays an important role in NNK carcinogenicity in rats.
6
2
which were not measured here. The absolute levels are unknown
since the absolute yields of 7-POB-Gua from 7-POB-dGuo and
O -POB-Cyt from O -POB-dCyd are difficult to determine.
This result is different from that reported in mice (27). A/J mice
were treated with [5- H]NNK, and O -POB-dGuo was detected
3
6
2
2
in liver DNA but not in lung DNA. Formation and persistence
6
of O -methylguanine are a critical pathway for NNK-induced
The in vitro data obtained in this study (Figure 1) were
lung carcinogenesis in A/J mice (27, 32).
slightly different from our previous study (16), in which 7-POB-
2
6
The in vivo results with respect to O -POB-dThd are also of
Gua accounted for 66% of all POB adducts, O -POB-dGuo for
2
2
considerable interest. It is relatively abundant in both lung and
liver DNA of NNK-treated rats. Its levels are comparable to
those of 7-POB-Gua. It accounts for 35-38% of all adducts in
liver DNA and 39-40% in lung DNA, which may correspond
to poor repair in vivo. In Escherichia coli, 3-methyladenine
5
%, O -POB-dThd for 10%, and O -POB-Cyt for 19%. There
are at least two explanations for these differences. First, a
different enzymatic hydrolysis procedure was used in the
previous study. The DNA may have been digested incompletely
6
due to resistance of O -POB-dGuo to the enzymatic activity of
2
DNA glycosylase II catalyzes the excision of O -alkylthymidines
snake venom phosphodiesterase I (23). Second, the previous
study did not use deuterated internal standards for each adduct.
2
(
33-35), whereas the repair of O -alkylthymidines in mam-
6
malian cells is not clearly understood. Besides O -POB-dGuo,
O -POB-dThd is the only stable deoxyribonucleoside adduct
among the four known POB-DNA adducts. While O -ethyl-
dThd was shown to be mutagenic, causing T to A transversions
The formation of POB-DNA adducts in rats has been
extensively studied by the measurement of HPB released upon
acidic hydrolysis of DNA. Murphy et al. compared levels of
HPB-releasing adducts in rat lung and liver after four daily i.p.
injections of different doses of radiolabeled NNK (24). At low
doses of 15-150 µg/kg/day, levels of HPB released from lung
DNA were higher than those from liver DNA, whereas at high
doses of 600-5000 µg/kg/day, HPB-releasing DNA adduct
levels in liver exceeded those in lung. Consistent with these
results, Boysen et al. found that HPB-releasing DNA adducts
were significantly higher in lung than liver after treatment of
rats with about 150 µg/kg/day of NNK (12). Morse et al. found
higher levels of HPB released from liver DNA than lung DNA
after treatment of rats with four consecutive daily s.c. doses of
2
2
2
(
36, 37), the mutagenicity of O -POB-dThd is unknown. The
2
HPLC-ESI-MS/MS method achieved high sensitivity for O -
POB-dThd with a LOD of about 30 amol for standard and 100
amol in 1 mg of DNA. Current efforts are underway for
2
detection and quantitation of O -POB-dThd and other POB-
DNA adducts in smokers’ DNA.
In summary, a sensitive HPLC-ESI-MS/MS method for
quantitation of four POB-DNA adducts was developed and
validated in this study. The method was applied to the analysis
of DNA samples obtained from both in vitro and in vivo studies.
In vitro, Gua was the most reactive nucleobase to the pyridyl-
6
00 µg/kg/day of NNK (9). Similar results were obtained by
6
oxobutylating agent. In vivo, O -POB-dGuo appeared to be
Peterson et al. in rats treated with 0.8 mg/kg of radiolabeled
NNK (25). In the current study, rats were given NNK at 5.2 or
repaired, especially in liver. The results support the hypothesis
6
that the mutagenic O -POB-dGuo is critical for NNK-induced
20.7 mg/kg/day for 4 days by s.c. injection. Total levels of
2
lung tumorigenesis in rats. O -POB-dThd could be developed
POB-DNA adducts were 4-6 times higher in liver DNA than
in lung DNA, which is fully consistent with previous studies
of HPB-releasing DNA adducts at high doses (9, 24, 25). The
dose-dependent tissue differences in total adduct levels may be
due to different affinities of P450 enzymes in lung and liver
for R-methyl hydroxylation of NNK, or to other factors.
as a biomarker for tobacco carcinogenesis studies, on the basis
of its relatively high abundance in vivo and its low LOD. The
method described here is currently under evaluation for quan-
titation of POB-DNA adducts in human tissues.
Acknowledgment. We acknowledge Steven Carmella and
Jon Bradley Hochalter for their valuable thoughts on method
development. We also thank Patrick M. J. Kenney and Dr.
Pramod Upadhyaya for their assistance in animal experiments
and preparation of NNK, Guang Cheng and Yongli Shi for DNA
isolation, Jana Scott for her help with synthesis of standards,
and Bob Carlson for manuscript preparation. S.S.H. is an
American Cancer Society Research Professor, supported in part
by ACS Grant RP-00-138. This work was supported by Grant
CA-81301 from the National Cancer Institute.
Consistent with the in vitro data, 7-POB-Gua levels were
relatively high in vivo, accounting for about 34-38% of all
6
POB-DNA adducts. However, the levels of O -POB-dGuo
changed dramatically from in vitro to in vivo. It accounted for
2
5-29% of all adducts in vitro, whereas the corresponding
amounts were 9% in lung DNA and 0.3-0.4% in liver DNA.
These results indicate that there is efficient cellular repair of
6
6
O -POB-dGuo in vivo, especially in the liver. O -Alkylguanine-
6
DNA-alkyltransferase (AGT) repairs O -POB-dGuo efficiently
both in vitro and in vivo (5, 26-28). AGT is widely distributed
in rat tissues such as liver, lung, esophagus, colon, and kidney
6
Supporting Information Available: Calibration curves for O -
2
2
(
29-31). AGT activity in liver is about three times higher than
POB-dGuo, O -POB-dThd, 7-POB-Gua, and O -POB-Cyt; levels
of POB-DNA adducts in NNKOAc-treated calf thymus DNA and
in liver and lung DNA of NNK-treated rats. This material is
available free of charge via the Internet at http://pubs.acs.org.
in lung. Different tissue distribution of AGT may explain the
6
apparently more efficient repair of O -POB-dGuo in liver than
lung.
Comparison of adduct levels in the liver and lung DNA was
very interesting. Except for O -POB-dGuo, levels of POB
6
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TX050351X