Identification of an N′-Nitrosonornicotine-Specific Deoxyadenosine Adduct in Rat Liver and Lung DNA

Article abstract of DOI:10.1021/acs.chemrestox.1c00013

The tobacco-specific nitrosamines N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be two of the most important carcinogens in unburned tobacco and its smoke. They readily cause tumors in laboratory animals and are classified as "carcinogenic to humans"by the International Agency for Research on Cancer. DNA adduct formation by these two carcinogens is believed to play a critical role in tobacco carcinogenesis. Among all the DNA adducts formed by NNN and NNK, 2′-deoxyadenosine (dAdo)-derived adducts have not been fully characterized. In the study reported here, we characterized the formation of N6-[4-(3-pyridyl)-4-oxo-1-butyl]-2′-deoxyadenosine (N6-POB-dAdo) and its reduced form N6-PHB-dAdo formed by NNN 2′-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyadenosine (N6-HPB-dAdo) formed after NaBH3CN or NaBH4 reduction both in vitro in calf thymus DNA reacted with 5′-acetoxy-N′-nitrosonornicotine and in vivo in rat liver and lung upon treatment with NNN. This adduct was specifically formed by NNN 5′-hydroxylation. Chemical standards of N6-HPB-dAdo and the corresponding isotopically labeled internal standard [pyridine-d4]N6-HPB-dAdo were synthesized using a four-step method. Both NMR and high-resolution mass spectrometry data agreed well with the proposed structure of N6-HPB-dAdo. The new adduct coeluted with the synthesized internal standard under various LC conditions. Its product ion patterns of MS2 and MS3 transitions were also consistent with the proposed fragmentation patterns. Chromatographic resolution of the two diastereomers of N6-HPB-dAdo was successfully achieved. Quantitation suggested a dose-dependent response of the levels of this new adduct in the liver and lung of rats treated with NNN. However, its level was lower than that of 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-deoxyinosine, a previously reported dGuo adduct that is also formed from NNN 5′-hydroxylation. The identification of N6-HPB-dAdo in this study leads to new insights pertinent to the mechanism of carcinogenesis by NNN and to the development of biomarkers of NNN metabolic activation.

Full text of DOI:10.1021/acs.chemrestox.1c00013

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Article
Identification of an N′-Nitrosonornicotine-Specific Deoxyadenosine
Adduct in Rat Liver and Lung DNA
Yupeng Li and Stephen S. Hecht*
Cite This: Chem. Res. Toxicol. 2021, 34, 992−1003
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ABSTRACT: The tobacco-specific nitrosamines N′-nitrosonorni-
cotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta-
none (NNK) are considered to be two of the most important
carcinogens in unburned tobacco and its smoke. They readily cause
tumors in laboratory animals and are classified as “carcinogenic to
humans” by the International Agency for Research on Cancer. DNA
adduct formation by these two carcinogens is believed to play a
critical role in tobacco carcinogenesis. Among all the DNA adducts
formed by NNN and NNK, 2′-deoxyadenosine (dAdo)-derived adducts have not been fully characterized. In the study reported
here, we characterized the formation of N6-[4-(3-pyridyl)-4-oxo-1-butyl]-2′-deoxyadenosine (N6-POB-dAdo) and its reduced form
N6-PHB-dAdo formed by NNN 2′-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct
N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyadenosine (N6-HPB-dAdo) formed after NaBH3CN or NaBH4 reduction both in
vitro in calf thymus DNA reacted with 5′-acetoxy-N′-nitrosonornicotine and in vivo in rat liver and lung upon treatment with NNN.
This adduct was specifically formed by NNN 5′-hydroxylation. Chemical standards of N6-HPB-dAdo and the corresponding
isotopically labeled internal standard [pyridine-d4]N6-HPB-dAdo were synthesized using a four-step method. Both NMR and high-
resolution mass spectrometry data agreed well with the proposed structure of N6-HPB-dAdo. The new adduct coeluted with the
synthesized internal standard under various LC conditions. Its product ion patterns of MS2 and MS3 transitions were also consistent
with the proposed fragmentation patterns. Chromatographic resolution of the two diastereomers of N6-HPB-dAdo was successfully
achieved. Quantitation suggested a dose-dependent response of the levels of this new adduct in the liver and lung of rats treated with
NNN. However, its level was lower than that of 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-deoxyinosine, a previously reported dGuo
adduct that is also formed from NNN 5′-hydroxylation. The identification of N6-HPB-dAdo in this study leads to new insights
pertinent to the mechanism of carcinogenesis by NNN and to the development of biomarkers of NNN metabolic activation.
the nitroso group of NNN forms 2′-hydroxyNNN (5) and 5′-
hydroxyNNN (6). They spontaneously yield the reactive
intermediate diazonium ions 9 and 10 as a consequence of
two-step reactions including pyrrolidine ring opening and loss
of H2O. Similarly, the diazonium ion 9 can also be formed
from α-methyl hydroxylation of NNK via the intermediate α-
hydroxymethylNNK (4).
DNA is alkylated by these diazonium ions to form a panel of
adducted products on both the nucleobases10−19 and
phosphate backbone.20−23 Figure 1 summarizes recent
progress toward identifying new pyridyl DNA adducts derived
from NNN and NNK metabolism upon reaction with four
nucleobases in vitro and in vivo. Among these, 2′-
deoxyadenosine (dAdo)-derived adducts represent a small
INTRODUCTION
■
Cigarette smoking remains the strongest single risk factor for
cancer in the United States, accounting for 19% of all cancer
cases and 29% of all cancer deaths.1 Among the multiple
carcinogens identified in both tobacco and tobacco smoke, the
tobacco-specific nitrosamines N′-nitrosonornicotine (NNN,
Scheme 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta-
none (NNK) are among the most abundant.2 In laboratory
animals including rats, Syrian golden hamsters, and mink,
target tissues of NNN treatment are predominantly esophagus,
oral mucosa, trachea, and nasal mucosa.3 NNN is the most
abundant oral carcinogen in smokeless tobacco, an established
cause of oral cavity cancer in smokeless tobacco users.4
Epidemiological studies have provided strong evidence that
urinary levels of NNN in cigarette smokers predict future
esophageal cancer incidence.5,6 The International Agency for
Research on Cancer has classified NNN and NNK as group 1
carcinogens (“carcinogenic to humans”).7
Received: January 8, 2021
Published: March 11, 2021
DNA adduct formation plays a critical role in the process of
tobacco carcinogenesis.8 To exert their carcinogenicity, NNN
and NNK require metabolic activation by cytochrome P450s
(Scheme 1).3,9 Initial hydroxylation of the carbons adjacent to
https://dx.doi.org/10.1021/acs.chemrestox.1c00013
© 2021 American Chemical Society
Chem. Res. Toxicol. 2021, 34, 992−1003
992

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Article
Scheme 1. Mechanisms of NNN and NNK Metabolic Activation Leading to dAdo-Derived Adducts in DNA
Figure 1. Summary of previously investigated pyridyl DNA base adducts formed by NNN and NNK metabolism in vitro and in vivo. dR = 2′-
deoxyribose.
portion and are reported in quite low abundance in rat tissues.
Three dAdo-derived adducts (11−13, Scheme 1) have been
identified in tissues of rats treated with NNN, NNK, or the
NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta-
nol (NNAL).
However, the levels of 7-POB-Gua and O2-POB-Thd in the
same tissue were 688 65 fmol/mg DNA and 4409 320
fmol/mg DNA, respectively. Similarly, the level of N6-PHB-
dAdo was 35 5 fmol/mg DNA, while the levels of 7-PHB-
Gua and O2-PHB-Thd were 129
34 fmol/mg DNA and
N6-[4-(3-Pyridyl)-4-oxo-1-butyl]-2′-deoxyadenosine (11,
N6-POB-dAdo) formed by NNK and N6-[4-(3-pyridyl)-4-
hydroxy-1-butyl]-2′-deoxyadenosine (12, N6-PHB-dAdo)
formed by NNK and NNAL were characterized and quantified
in rat liver and rat lung.18 Their levels were significantly lower
than 7-POB-Gua/7-PHB-Gua and O2-POB-Thd/O2-PHB-Thd
(structures shown in Figure 1). For example, in the lung DNA
of rats treated with 5 ppm of NNK in the drinking water for 50
weeks, the level of N6-POB-dAdo was 99 3 fmol/mg DNA.
1259 66 fmol/mg DNA, respectively, from the same rat lung
tissue. The other adduct 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2′-
deoxynebularine (13, Py-Py-dN) formed through NNN 5′-
hydroxylation followed by NaBH3CN reduction was identified
in rat lung and rat nasal mucosa but in levels too low to
quantify.15 Collectively, these data seemed to indicate the
unfavorable formation of dAdo-derived adducts by NNN and
NNK. However, studies with other chemical carcinogens such
as aristolochic acid24 and colibactin25 provided good evidence
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a
Scheme 2. Synthetic Routes for N6-HPB-dAdo and [pyridine-d4]N6-HPB-dAdo
a
Reagents and conditions: (i) γ-Butyrolactone, n-BuLi, Et2O, −78 °C to rt, 2 h; (ii) methoxyamine hydrochloride, Na2CO3, EtOH, 80 °C, 1 h; (iii)
borane-THF complex, THF, 0 °C to rt, 1 h, then reflux for 2.5 h; H2O; 1 N NaOH; 65 °C, 1 h; (iv) Et3N, DMF, 45 °C, 12 d; (v) compound 19,
Et3N, DMF, 60 °C, 3 d.
dAdo was synthesized as described before.27 The isotopically labeled
of the formation of dAdo-derived adducts. These data
internal standards [pyridine-d4]N6-POB-dAdo and [15N5]N6-PHB-
encouraged us to examine dAdo-derived adduct formation
more thoroughly both in vitro and in vivo.
Considering the convergence of the same alkylating
dAdo were available from our previous study.18 N6-HPB-dAdo and
[pyridine-d4]N6-HPB-dAdo were synthesized as described below. 3-
Bromopyridine-d4 (catalog number D-6066, lot number EF-132,
purity of 99.1%-d4) was purchased from CDN Isotopes Inc. (Quebec,
Canada). 6-Chloropurine-2-deoxyriboside (CAS number 4594-45-0)
diazonium ion 9 formed by NNK α-methyl hydroxylation
and NNN 2′-hydroxylation, the dAdo adducts N6-POB-dAdo
(11) and N6-PHB-dAdo (12, after reduction) were expected to
was procured from Alfa Aesar (Tewksbury, MA). DNA hydrolysis
be similarly formed in the tissues of rats treated with NNN.
However, our previous study using a method with relatively
low sensitivity suggested only the presence of low levels of Py-
Py-dN (13) in rat tissues after NaBH3CN reduction.15 The
apparent absence of N6-PHB-dAdo (12) formed by NNN 2′-
hydroxylation and the possible formation of the open-chain
adduct N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyadeno-
sine (N6-HPB-dAdo, 14) formed by NNN 5′-hydroxylation
(both after reduction) encouraged us to investigate these
adducts using a new highly sensitive and specific liquid
chromatography-nanoelectrospray ionization-high-resolution
tandem mass spectrometry (LC-NSI-HRMS/MS) method.
We found that N6-PHB-dAdo (12) was formed after NaBH4
reduction in the liver and lung of rats treated with racemic
NNN in the drinking water for 3 weeks. Its precursor adduct
N6-POB-dAdo (11) was similarly observed in the rat tissues.
However, both of these adducts occurred in very low
abundance. Surprisingly, a new DNA adduct nearly coeluting
with N6-PHB-dAdo was observed both in vitro and in vivo in
relatively high abundance. It was characterized as the open-
chain adduct N6-HPB-dAdo (14) using the authentic
synthesized standard, and its levels in the liver and lung of
rats treated with NNN were quantified. This structurally new
NNN-specific adduct N6-HPB-dAdo leads to new insights for
understanding mechanisms of carcinogenesis by tobacco-
specific nitrosamines.
enzymes including deoxyribonuclease I (catalog number P4527-
40KU), phosphodiesterase I (catalog number P3243-1VL, lot number
SLCD8995, ≥100 units/vial), and alkaline phosphatase (Roche,
catalog number 10567752001) were purchased from Sigma-Aldrich
and purified before use.23 Porcine liver esterase was obtained from
Sigma-Aldrich (catalog number E3019-20KU, lot number
SLBM1761V) and purified in the same way as the DNA hydrolysis
enzymes before use. All other chemicals and supplies were purchased
from Sigma-Aldrich or Fisher Scientific. Milli-Q water (Millipore) was
routinely used unless otherwise mentioned.
General Synthetic Procedures. All commercial reagents were
used without further purification except where noted. Reactions were
monitored routinely by TLC-UV or reverse phase HPLC. Polygram
precoated silica gel TLC plates (40 × 80 mm, 0.2 mm thick) with 254
nm fluorescence indicator were utilized. Products were purified
through silica gel column chromatography or reverse phase HPLC.
NMR spectra were recorded on a Bruker AC 500 MHz spectrometer.
Chemical shifts are reported as parts per million (ppm) using residual
solvent peaks as the internal references. The first-order peak patterns
are indicated as s (singlet), d (doublet), t (triplet), q (quadruplet),
and p (pentuplet). Complex nonfirst-order signals are indicated as m
(multiplet).
Synthesis of 5′-AcetoxyNNN (3). 5′-AcetoxyNNN was synthe-
sized using the method described before.15,26 The reaction details and
1H NMR data can be found in the Supporting Information (Scheme
S1 and Figure S1). NMR data were consistent with the reported
values.
Synthesis of N6-HPB-dAdo (14). The synthetic route is
illustrated in Scheme 2. The reaction conditions are detailed below.
4-Hydroxy-1-(pyridin-3-yl)butan-1-one (16, HPB). This com-
pound was synthesized using the method reported before with slight
modifications.20,28 To a solution of 3-bromopyridine (2.5 mmol, 241
μL) in dry Et2O (15 mL) was slowly added a solution of n-
butyllithium (∼1.6 M in hexanes) (3.75 mmol, 2.34 mL) at −78 °C
under a nitrogen atmosphere. The solution was stirred for an
additional 15 min at −78 °C, and then a solution of γ-butyrolactone
MATERIALS AND METHODS
■
Caution! NNN and 5′-acetoxyNNN are strongly carcinogenic. They
should be handled in a well-ventilated fume hood with extreme caution
and with appropriate protective equipment.
Chemicals and Supplies. 5′-Acetoxy-N′-nitrosonornicotine (5′-
acetoxyNNN) was synthesized as reported previously.15,26 N6-PHB-
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(2.5 mmol, 192 μL) in dry Et2O (5 mL) was added dropwise. The
reaction mixture was allowed to warm to room temperature and
stirred for 2 h. After completion, saturated NaHCO3 solution was
added to quench the reaction. The mixture was extracted 3 times with
EtOAc. The organic layers were combined and dried over anhydrous
Na2SO4. The solvent was evaporated, and the resulting crude product
16 was purified by flash silica gel column chromatography (EtOAc) as
a yellow oil (227 mg, 55%). 1H NMR (500 MHz, CDCl3) δ 9.20 (d, J
= 2.3 Hz, 1H, pyr-H2), 8.78 (dd, J = 4.9, 1.7 Hz, 1H, pyr-H6), 8.25
(dt, J = 8.0, 2.0 Hz, 1H, pyr-H4), 7.43 (dd, J = 8.0, 4.8 Hz, 1H, pyr-
H5), 3.77 (t, J = 6.1 Hz, 2H, −CH2OH), 3.16 (t, J = 6.9 Hz, 2H,
COCH2−), 2.05 (p, J = 6.5 Hz, 2H, −CH2CH2CH2−), 1.62 (s, 1H,
OH).
(Z/E)-4-Hydroxy-1-(pyridin-3-yl)butan-1-one O-methyl oxime
(17). A solution of compound 16 (1.35 mmol, 223 mg), methoxy-
amine hydrochloride (1.62 mmol, 135 mg), and Na2CO3 (1.62 mmol,
172 mg) in EtOH (2 mL) was stirred at 80 °C for 1 h. The reaction
mixture was cooled to room temperature before filtration. The filtrate
was concentrated and purified by flash silica gel column
chromatography (hexanes/EtOAc) to afford the desired product 17
as a light yellow oil, mixture of (Z)- and (E)-isomers (171 mg, 65%).
1H NMR (500 MHz, CDCl3) δ 8.89 (dd, J = 2.3, 0.9 Hz, 1H, pyr-H2),
−CH2CH2OH), 4.40 (s, 1H, 3′-H), 3.86 (d, J = 5.1 Hz, 1H, 4′-H),
3.60 (dd, J = 11.6, 4.8 Hz, 1H, 5′-Ha), 3.56−3.47 (m, 1H, 5′-Hb),
3.41 (p, J = 5.7 Hz, 2H, −CH(NH)CH2-), 2.77−2.66 (m, 1H, 2′-Ha),
2.24 (ddd, J = 13.3, 6.1, 2.9 Hz, 1H, 2′-Hb), 2.01 (s, 1H,
−CH2CH2aOH), 1.87 (s, 1H, −CH2CH2bOH), 1.55 (s, 1H,
−CH2aCH2OH), 1.47−1.36 (m, 1H, −CH2bCH2OH). 13C NMR
(126 MHz, DMSO-d6) δ 154.0 (ade-C6), 152.2 (ade-C2), 148.5
(overlapped, pyr-C2 and ade-C4), 147.9 (pyr-C6), 139.6 (overlapped,
pyr-C3 and ade-C8), 134.2 (pyr-C4), 123.4 (pyr-C5), 119.6 (ade-C5),
88.0 (C4′), 83.9 (C1′), 70.9 (C3′), 61.9 (C5′), 60.3 (−CH(NH)
CH2−), 51.2 (−CH(NH)CH2−), 39.8 (C2′), 32.2 (−CH2OH), 29.7
(−CH2CH2OH). HRMS (Orbitrap Fusion): [M + H]+ calc’d
401.1932; found 401.1930.
Synthesis of [Pyridine-d4]N6-HPB-dAdo (24). This compound
was synthesized similarly as N6-HPB-dAdo, except starting with 3-
bromopyridine-d4 (Scheme 2).
[Pyridine-d4]4-hydroxy-1-(pyridin-3-yl)butan-1-one (21, [Pyri-
dine-d4]HPB). Under an atmosphere of N2, to a stirred solution of
3-bromopyridine-d4 (20) (1.54 mmol, 0.25 g) in anhydrous Et2O (3.0
mL) was added a solution of n-butyllithium (1.6 M in hexanes, 3.09
mmol, 1.93 mL) dropwise over 15 min at −78 °C. Then a solution of
γ-butyrolactone (1.85 mmol, 142 μL) in Et2O (2.0 mL) was added
dropwise. The reaction was allowed to warm to room temperature
and stirred for 2 h. After completion, the reaction mixture was
quenched with saturated NaHCO3 solution. The mixture was
extracted with EtOAc 3 times. The combined organic layers were
evaporated to dryness. The residue was purified by flash silica gel
column chromatography (EtOAc) to afford the desired product 21 as
8.60 (dd, J = 4.8, 1.6 Hz, 1H, pyr-H6), 7.98 (dt, J = 8.1, 2.0 Hz, 1H,
pyr-H4), 7.30 (ddd, J = 8.1, 4.8, 0.9 Hz, 1H, pyr-H5), 4.02 (s, 3H,
−OCH3), 3.62 (t, J = 6.3 Hz, 2H, −CH2OH), 2.89 (t, J = 7.3 Hz, 2H,
−CN(OMe)-CH2), 1.96 (s, 1H, −OH), 1.80 (ddd, J = 13.2, 7.3,
6.0 Hz, 2H, −CH2CH2OH).
4-Amino-4-(pyridin-3-yl)butan-1-ol (18). A solution of the oxime
17 (0.6 mmol, 116 mg) in THF (1.2 mL) was cooled in an ice bath.
Borane-THF complex (1 M in THF) (1.8 mmol, 1.8 mL) was added
dropwise. The resulting solution was allowed to warm to room
temperature, stirred for 1 h, and then heated at reflux for 2.5 h. After
cooling in an ice bath, the mixture was treated with H2O (1 mL) and
1 N NaOH (2 mL) sequentially. The mixture was again warmed to 65
°C for 1 h. After reaction, all the solvents were evaporated. The
resulting residue was purified by flash silica gel column chroma-
tography (DCM/MeOH) to afford the desired product 18 as a
1
a yellow oil (170 mg, 65%). H NMR (500 MHz, DMSO-d6) δ 4.51
(t, J = 5.2 Hz, 1H, −OH), 3.46 (q, J = 6.3 Hz, 2H, −CH2OH), 3.09
(t, J = 7.2 Hz, 2H, −COCH2−), 1.78 (p, J = 6.7 Hz, 2H,
−CH2CH2OH). 13C NMR (126 MHz, DMSO-d6) δ 199.5 (CO),
59.9 (−CH2OH), 34.9 (COCH2−), 26.9 (−CH2CH2OH).
[Pyridine-d4](Z/E)-4-Hydroxy-1-(pyridin-3-yl)butan-1-one O-
methyl oxime (22). A solution of [pyridine-d4]HPB (21) (0.2
mmol, 34 mg), methoxyamine hydrochloride (0.24 mmol, 20 mg),
and sodium carbonate (0.24 mmol, 25.4 mg) in EtOH (1.0 mL) was
stirred at 80 °C for 1 h. The mixture was concentrated and purified by
flash silica gel column chromatography (hexanes/EtOAc). Pure
fractions were concentrated to afford the desired product 22 as a
1
colorless oil (60 mg, 60%). H NMR (500 MHz, CDCl3) δ 8.58 (s,
1H, pyr-H2), 8.51 (d, J = 5.7 Hz, 1H, pyr-H6), 7.95 (d, J = 7.9 Hz, 1H,
pyr-H4), 7.48 (dd, J = 8.0, 5.7 Hz, 1H, pyr-H5), 4.10 (dd, J = 7.6, 5.6
Hz, 1H, −CH(NH2)−), 3.67 (dt, J = 6.6, 5.0 Hz, 2H, −CH2OH),
1.89−1.73 (m, 2H, −CH(NH2)CH2−), 1.62 (ddt, J = 26.2, 13.6, 7.1
Hz, 2H, −CH2CH2OH).
1
colorless oil (16 mg, 40%). H NMR (500 MHz, CDCl3) δ 4.02 (s,
3H, −OCH3), 3.62 (s, 2H, −CH2OH), 2.89 (t, J = 7.3 Hz, 2H, −C
N(OMe)-CH2), 2.04 (s, 1H, −OH), 1.86−1.75 (m, 2H,
−CH2CH2OH). The other geometric isomer was also observed in
the spectrum, accounting for 28% of the product mixture.
[Pyridine-d4]4-amino-4-(pyridin-3-yl)butan-1-ol (23). The oxime
22 (0.08 mmol, 16 mg) in THF (1.5 mL) was cooled in an ice water
bath. Borane-THF complex (1 M in THF, 0.5 mmol, 0.5 mL) was
added dropwise. Under the same workup condition as described
above, the desired product 23 was obtained as a colorless oil (11 mg,
81%).
N6-[4-Hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyadenosine (14,
N6-HPB-dAdo). A solution of 6-chloropurine-2′-deoxyribose (19)
(0.35 mmol, 95 mg), compound 18 (0.35 mmol, 58 mg) and
triethylamine (0.52 mmol, 73 μL) in DMF (1.0 mL) was stirred at 45
°C for 12 days. The reaction mixture was diluted with H2O (0.5 mL)
before subjecting it to reverse phase HPLC purification. HPLC was
carried out with Waters Associates (Milford, MA) systems equipped
with a Shimadzu SPD-10A 0.2 mm Prep UV−vis detector set to 254
nm. Separation was performed using a Luna 5 μm C18(2) 100A 250
× 10 mm column purchased from Phenomenex (Torrance, CA). A 50
min program was used, with a flow rate of 4 mL/min and a gradient
starting from 10% MeOH in H2O for 10 min, then ramping up to
90% MeOH in H2O over 25 min. After holding at 90% MeOH in
H2O for 3 min, the gradient was returned to 10% MeOH in H2O over
5 min. The instrument was equilibrated for 7 min before the next
injection. The crude product was collected at the retention time of
∼25 min as a colorless oil after evaporation of solvents. This crude
product (containing 2 peaks) was further purified under isocratic
conditions (45% MeOH in H2O). The first peak eluting at 5.8 min
was collected; it contained the desired product 14 (16 mg, 11%).
One-dimensional and two-dimensional NMR spectra of compound
[Pyridine-d4]N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2′-deoxyade-
nosine (24, [pyridine-d4]N6-HPB-dAdo). A solution of compound 19
(0.02 mmol, 5.4 mg), compound 23 (0.02 mmol, 3.4 mg), and
triethylamine (0.03 mmol, 4.2 μL) in DMF (0.5 mL) was stirred at 60
°C for 3 days. Using the same HPLC conditions as described above,
the desired product 24 was collected at the retention time of 25.3 min
as a colorless oil after evaporation (1.3 mg, 16%). See Figure S3 for
1
the NMR of compound 24. H NMR (500 MHz, DMSO-d6)δ 8.41
(s, 1H, −NHCH(pyridine)−), 8.37 (d, J = 1.3 Hz, 1H, ade-H8), 8.16
(s, 1H, ade-H2), 6.39−6.22 (m, 1H, 1′-H), 5.38 (s, 1H, −NHCH-
(pyridine)−), 5.29 (d, J = 3.8 Hz, 1H, 3′-OH), 5.21−5.11 (m, 1H, 5′-
OH), 4.45−4.41 (t, J = 5.1 Hz, 0.5H, 3′-Ha), 4.40 (s, 1H,
−CH2CH2OH), 4.35 (t, J = 5.1 Hz, 0.5H, 3′-Hb), 3.87 (s, 1H, 4′-
H), 3.65−3.55 (m, 1H, 5′-Ha), 3.55−3.47 (m, 1H, 5′-Hb), 3.45−3.36
(m, 2H, −CH2CH2CH2OH), 2.79−2.66 (m, 1H, 2′-Ha), 2.29−2.20
(m, 1H, 2′-Hb), 2.01 (s, 1H, −CH2aOH), 1.87 (s, 1H, −CH2bOH),
1.55 (s, 1H, −CH2aCH2OH), 1.42 (s, 1H, −CH2bCH2OH). HRMS
(Orbitrap Fusion): [M + H]+ calc’d 405.2183; found 405.2182. MS
1
14 are presented in Figure S2. H NMR (500 MHz, DMSO-d6) δ
8.65 (s, 1H, pyr-H2), 8.40 (d, J = 4.9 Hz, 1H, pyr-H6), 8.37 (d, J = 1.3
Hz, 1H, ade-H8), 8.16 (s, 1H, ade-H2), 7.86 (dd, J = 7.7, 2.2 Hz, 1H,
pyr-H4), 7.32 (dd, J = 7.9, 4.8 Hz, 1H, pyr-H5), 6.33 (t, J = 6.9 Hz,
1H, 1′-H), 5.39 (s, 1H, pyr-CH(NH)−), 5.28 (d, J = 4.0 Hz, 1H, 3′-
OH), 5.21−5.10 (m, 1H, 5′-OH), 4.42 (t, J = 5.2 Hz, 1H,
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Table 1. Exact Masses of Target Analytes and Their Most Abundant First-Generation and Second-Generation Product Ions
parent ion [M + H]+
first-generation product ions [MS2 transition + H]+ second-generation product ions [MS3 transition + H]+
analyte
(m/z)
(m/z)
(m/z)
N6-HPB-dAdo
401.1932
405.2183
285.1458
289.1709
150.0913; 136.0618
154.1164; 136.0618
[pyridine-d4]N6-HPB-
dAdo
N6-PHB-dAdo
401.1932
406.1784
399.1775
403.2026
285.1458; 267.1353
290.1310; 272.1204
283.1302; 265.1196
287.1553; 269.1447
136.0618; 132.0808
141.0469; 132.0808
148.0757; 136.0618
152.1008; 136.0618
[15N5]N6-PHB-dAdo
N6-POB-dAdo
[pyridine-d4]N6-POB-
dAdo
data suggested compound 24 contains ∼0.5% unlabeled N6-HPB-
dAdo.
increased to 25% MeOH in H2O over 10 min. The gradient ramped
to 95% MeOH in H2O within 3 min and was held at 95% MeOH in
H2O for 5 min, and then returned to 5% MeOH in H2O over 2 min.
The system was equilibrated at 5% MeOH in H2O for 8 min before
the next injection. The injection volume was 2 μL and the flow rate
was 15 μL/min. The amount of dGuo was determined by a standard
calibration curve, and the amount of DNA was calculated by
considering that dGuo comprises 22% of all nucleotides in rat DNA.23
The remaining filtrate was purified using a 30 mg Strata-X
polymeric reversed-phase SPE cartridge (1 mL, Phenomenex, catalog
number 8B-S100-TAK, Torrance, CA). The cartridge was activated
with MeOH (2 mL) and preconditioned with H2O (2 mL) before
loading the filtrate. The cartridge was washed with H2O (2 mL) and
30% MeOH solution (2 mL) sequentially. Then 2 mL of 75% MeOH
was applied to elute the analytes. The 75% MeOH fractions were
concentrated by SpeedVac and reconstituted in 100 μL 10% CH3CN
solution (Fisher, Optima H2O and Optima CH3CN). The sample was
transferred to a glass vial with a 300 μL insert and concentrated by
SpeedVac to dryness. Ten μL of H2O (Optima H2O) was added to
reconstitute the analytes prior to mass spectrometric analysis.
Calf thymus DNA incubated with a precursor of hydroxyme-
thylNNK, 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone
(NNKOAc), was also used for the analysis of dAdo-derived adducts.
The isolated DNA was obtained from our previous study.20 The
procedures of DNA enzymatic hydrolysis, dGuo quantitation, analyte
enrichment, and sample preparation prior to mass spectrometric
analysis were essentially the same as described above.
Sample Preparation for Identifying dAdo-Derived Adducts
In Vivo. The animal studies were approved by the University of
Minnesota Institutional Animal Care and Use Committee and
reported in detail previously.15 In brief, liver and lung DNA from
rats treated with 50, 100, or 500 ppm of racemic NNN in the drinking
water for 3 weeks were used for the analysis of dAdo-derived adducts.
The control group rats were treated under the same conditions except
for drinking only plain tap water. Total NNN exposures to the rats
from the 50, 100, or 500 ppm groups over the 3 weeks were estimated
as 21 mg, 41 mg, and 128 mg per rat, respectively. All of these DNA
samples were obtained from our previous study. DNA hydrolysis,
dGuo quantitation, analyte enrichment through solid-phase extraction
(SPE), and sample preparation prior to mass spectrometric analysis
were performed using the protocol detailed above.
Alternate Synthesis of N6-PHB-dAdo (12). Two diastereomers
can be formed during the synthesis of 12 using the method reported
by us before.18,27 However, the standard compound synthesized
previously showed only one single peak upon HPLC purification and
LC-MS analysis. To exclude the possibility that we missed one of the
two isomers when performing the HPLC fraction collection, N6-PHB-
dAdo was resynthesized using the method illustrated in Scheme S2.27
The reaction details are described in Scheme S2. NMR spectra of N6-
PHB-dAdo and its precursor compound N6-POB-dAdo are presented
in Figure S4. HPLC traces of both N6-POB-dAdo and N6-PHB-dAdo
are shown in Figure S5. The newly synthesized N6-PHB-dAdo was
essentially identical to the one synthesized before, with no
chromatographic separation of the two diastereomers under any
tested conditions.
Sample Preparation for Identifying dAdo-Derived Adducts
In Vitro. Calf thymus DNA (2 mg) was dissolved in 1.0 mL of 0.1 M
phosphate buffer (pH = 7.4). To the solution were added 5′-
acetoxyNNN (2 mg, 8.5 μmol) and purified porcine liver esterase (4
units). The mixture was incubated at 37 °C overnight (20 h). The
resulting mixture (1.0 mL) was washed 3 times with CHCl3/isoamyl
alcohol (24:1). Each time, 2 mL of the mixed solvent was added, and
the mixture was vortexed thoroughly for 10 min before centrifuging at
4000 g for 15 min. The upper layer was then transferred to a 5 mL
centrifuge tube prefilled with 0.2 mL of 5 M NaCl solution. To the
centrifuge tube was then added 1.5 mL of ice-cold isopropanol with
gentle swirling to precipitate the DNA. The DNA was transferred to a
4 mL silanized glass vial and washed 2 times with 0.5 mL of ice-cold
75% EtOH and 100% EtOH sequentially. The DNA was dried under
a stream of N2 yielding a white solid (crude weight: 1.9 mg) and
stored at −20 °C until hydrolysis.
The isolated DNA was dissolved in 500 μL of 10 mM sodium
succinate buffer containing 5 mM CaCl2 (pH = 7.3). For the samples
reduced with NaBH3CN, 100 μL of 20 mg/mL NaBH3CN in the
succinate buffer was added to the DNA solution. The mixture was
incubated at 37 °C for 1 h before adding the isotopically labeled
standard [pyridine-d4]N6-HPB-dAdo (and [15N5]N6-PHB-dAdo and
[pyridine-d4]N6-POB-dAdo when necessary). For the unreduced
samples, no reducing agent was added. Three purified DNA
hydrolysis enzymes were added as follows: deoxyribonuclease I (0.4
units), phosphodiesterase I (0.1 units), and alkaline phosphatase (0.8
units). After incubating at 37 °C overnight, the hydrolysate was
filtered through a Microcon-10 kDa centrifugal filter unit (Millipore
Sigma, MRCPRT010) at 14,000 g for 60 min at 4 °C. For the samples
reduced with NaBH4, 100 μL of 20 mg/mL NaBH4 in the succinate
buffer was added to the DNA hydrolysate and incubated at 37 °C for
1 h. The resulting solution was adjusted to pH ∼ 8.0 with 50 μL of 1
N HCl before centrifugal filtration.
Liver and lung DNA of rats treated with 5 ppm of NNK in the
drinking water for 50 weeks were also used for the analysis of dAdo-
derived adducts in this study. The isolated NNK-treated rat DNA
samples were obtained from a previous study.20 The protocol to
prepare the samples for MS analysis was the same as described above.
Qualitative Analysis of dAdo-Derived Adducts by LC-NSI-
HRMS/MS. The hydrolysates of DNA from in vitro samples (5′-
acetoxyNNN- or NNKOAc-incubated calf thymus DNA) or rat livers
and lungs (NNN treatment, NNK treatment, or control group) were
analyzed by a new LC-NSI-HRMS/MS method. An aliquot of 2 μL of
DNA hydrolysate was injected into a Dionex UltiMate 3000
RSLCnano UPLC system equipped with a 5 μL autosampler injection
loop. The UPLC system was coupled to the nanospray ion source.
Chromatographic separation was achieved using a capillary column
(75 μm i.d., 15−22 cm length, 10 μm orifice) created by hand-
packing a fused-silica emitter (New Objective, Woburn, MA) with 5
Ten μL of the filtrate was taken for dGuo quantitation by HPLC as
described before.23 The HPLC method was modified to achieve a
better separation of the four nucleotide peaks. Quantitation of dGuo
was carried out with a Dionex UltiMate 3000 RSLCnano System
(Thermo Scientific, Waltham, MA) with a UV detector set at 254 nm.
A Luna 5 μm C18(2) 100 Å capillary column (250 × 0.5 mm)
(Phenomenex, Torrance, CA) was used with H2O and MeOH as
mobile phases. Starting from 5% MeOH in H2O, a linear gradient was
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μm particle size XBridge BEH Luna C18 stationary phase (Waters,
Milford, MA). The LC conditions are described in Table S1.
determining the limit of detection to be 6.5 amol (on column). For
the method validation of accuracy and precision, two amounts of N6-
HPB-dAdo (1 and 5 fmol) were spiked into 40 μg of calf thymus
DNA in succinate buffer with 1 fmol of [pyridine-d4]N6-HPB-dAdo
added as the internal standard. These two amounts represent the low
and high levels of N6-HPB-dAdo in rat tissue samples. All the samples
were prepared using the same method as described above. The
recovery was 75%. The limit of quantitation was 200 amol (on
column) as assessed by the lowest spiked level of N6-HPB-dAdo in
calf thymus DNA that produced a coefficient of variation (CV) of
<10%. The determined levels of N6-HPB-dAdo in the rat liver and
lung DNA samples were calculated by the calibration curve, using the
ratio of the peak areas of MS2 quantitative transitions of N6-HPB-
dAdo and [pyridine-d4]N6-HPB-dAdo. All the MS data were
processed using Thermo Xcalibur Qual Browser (version 4.3.73.11)
software.
The samples were analyzed using a Thermo Scientific Orbitrap
Fusion Tribrid Mass Spectrometer (Thermo Scientific, San Jose, CA)
in the positive ion profile mode. Four experiments were included for
each scan cycle for the qualitative analysis. They are full scan, targeted
selected-ion monitoring (SIM) scan, targeted MS2 scan, and targeted
MS3 scan. Exact masses of all the analytes and their characteristic
transitions are summarized in Table 1. For the full scan experiment,
the resolution of the Orbitrap detector was 7500; the scan range was
m/z 100−1000; and the maximum injection time was 20 ms. For the
targeted SIM scan experiment, precursor ions of the analytes
including m/z 401.2 for N6-HPB-dAdo or N6-PHB-dAdo, m/z
405.2 for [pyridine-d4]N6-HPB-dAdo; m/z 406.2 for [15N5]N6-PHB-
dAdo (only included for the qualitative analysis of the reduced
samples); m/z 399.2 for N6-POB-dAdo and (N6-[5-(3-pyridyl)-
tetrahydrofuran-2-yl]-2′-deoxyadenosine (N6-Py-THF-dAdo)),27 m/z
403.2 for [pyridine-d4]N6-POB-dAdo (only included for the
qualitative analysis of the unreduced samples) were isolated with a
quadrupole isolation window of m/z 1.5. The resolution of the
Orbitrap detector was 15,000. The maximum injection time was 22
ms. For the targeted MS2 scan experiment, the quadrupole isolation
window for isolating the precursor ions listed above was 1.5, and 30%
of the higher-energy collisional dissociation (HCD) collision energy
was used in the ion routing multipole. The Orbitrap detector was set
up with a resolution of 60,000 and a scan range of product ions of m/
z 100−500. The maximum injection time was 118 ms. For the
targeted MS3 scan experiment, the first-generation precursor ions, as
listed above and in Table 1, were isolated with a quadrupole isolation
window of m/z 2.0, and the HCD collision energy setting was 30%.
The second-generation precursor ions were m/z 285.1 for N6-HPB-
Ade, m/z 289.2 for [pyridine-d4]N6-HPB-Ade; m/z 267.1 for N6-
PHB-Ade, and m/z 272.1 for [15N5]N6-PHB-Ade (only included for
the qualitative analysis of the reduced samples); m/z 265.1 for N6-
POB-Ade with a H2O loss, and m/z 269.1 for [pyridine-d4]N6-POB-
Ade with a H2O loss (only included for the qualitative analysis of the
unreduced samples). They were isolated with a quadruple isolation
window of m/z 1.5, and the HCD collision energy setting was 50%.
The resolution of the Orbitrap detector was 30,000. The scan range of
the product ions was m/z 100−500. The maximum injection time was
54 ms. For all four experiments, the normalized automatic gain
control (AGC) target (%) setting was chosen as “standard” (5 × 104)
and the RF lens (%) setting was 60. The number of microscans was
set as 1. The spray voltage was 2.2 kV. The ion-transfer tube
temperature was 300 °C. The EASY-IC internal mass calibration
feature was used to ensure maximum mass accuracy. Extraction of
precursor ion signals and product ion signals was performed with an
accurate mass tolerance of 5 ppm. Thermo Xcalibur Qual Browser
(version 4.3.73.11) was used to process all the MS data.
RESULTS
■
Three types of pyridyl dAdo-derived DNA adducts formed by
NNN or NNK metabolism in vivo have been previously
reported (Scheme 1 and Figure 1), but our understanding of
adduct formation from these carcinogens is incomplete. In this
study, we hypothesized that N6-POB-dAdo (11), that has been
observed in tissues of NNK-treated rats, and results from the
intermediate 9 (Scheme 1), would be similarly formed by the
same metabolite produced by NNN 2′-hydroxylation.
Preliminary Study of dAdo-Derived Adduct forma-
tion In Vitro and In Vivo. Hydrolysates of liver and lung
DNA of rats treated with 500 ppm racemic NNN in the
drinking water for 3 weeks were first analyzed by MS using the
LC method described in Table S2. As shown in Figure S6B,
N6-PHB-dAdo was barely observed in the NaBH3CN-reduced
sample from liver DNA of rats treated with 500 ppm racemic
NNN. This was consistent with our previous observation when
using a method with relatively low sensitivity.15
However, it was surprising to observe the formation of N6-
PHB-dAdo in calf thymus DNA incubated with 5′-acetoxy-
NNN, followed by NaBH3CN reduction, as confirmed by
coelution with its isotopically labeled internal standard [15N5]
N6-PHB-dAdo (Figure S6A). The presence of N6-PHB-dAdo
in calf thymus DNA incubated with 5′-acetoxyNNN, followed
by reduction with NaBH3CN, raised some questions regarding
its precursor adduct, since N6-POB-dAdo was not supposed to
be formed by 5′-acetoxyNNN (Scheme 1) and was not
observed in the same but unreduced sample (Figure S9C).
Further study suggested that N6-PHB-dAdo was formed by the
reduction of N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2′-deoxy-
adenosine (N6-Py-THF-dAdo) (Scheme S3), which had been
previously observed only at the nucleoside level, for example,
in the reaction of 5′-acetoxyNNN with dAdo.27 However, our
present results described in the accompanying manuscript
suggested that it is also present in calf thymus DNA incubated
with 5′-acetoxyNNN but not in liver and lung DNA of rats
treated with NNN.29
More surprisingly, a peak eluting only slightly earlier, at 22.3
min, than N6-PHB-dAdo was observed both in vitro and in vivo
after reduction with NaBH3CN (Figure S6). Fragmentation
ions of m/z 136.0618 [Ade + H]+ and m/z 150.0913 (pyridyl
fragment) of MS3 transitions of this new peak suggested one
unknown type of pyridyl dAdo adduct (Figure S7). Based on
the abundance of the first-generation and second-generation
product ions as well as our knowledge of NNN metabolism
mechanisms, the chemical structure of this new peak was
proposed to be N6-HPB-dAdo (14, Scheme 1). It is
Quantitative Analysis of N6-HPB-dAdo in Rat Liver and Rat
Lung. The hydrolysates of DNA from the rat livers and lungs were
analyzed by the LC-NSI-HRMS/MS method similarly as described
above. Modifications of MS parameters were made to further increase
the method sensitivity and specificity. Only two experiments were
included for each scan cycle of the quantitation method: full scan and
targeted MS2 scan. For the full scan, the maximum injection time was
increased to 50 ms; the scan range was set as m/z 100−800. For the
targeted MS2 scan, the Orbitrap detector was set up with a resolution
of 240,000; the normalized AGC target (%) was chosen as 1000 (5 ×
105); and the maximum injection time was 500 ms. Only the
precursor ions of m/z 401.2 for N6-HPB-dAdo and m/z 405.2 for
[pyridine-d4]N6-HPB-dAdo were included for the MS2 scan.
Quantitation was performed using the MS2 transition of [M + H]+
→ [M + H-dR]+.
For the calibration curve, the ratios of N6-HPB-dAdo/[pyridine-d4]
N6-HPB-dAdo were 0.5, 1, 2, 5, 10, with [pyridine-d4]N6-HPB-dAdo
at a constant concentration of 0.1 fmol/μL in H2O. The injection
volume was 2 μL for each injection. The concentration range chosen
was to cover the range of N6-HPB-dAdo levels in rat liver and lung
DNA samples. Low concentrations of N6-HPB-dAdo were used for
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Figure 2. Representative chromatograms and MS2 product ion spectra of N6-HPB-dAdo and [pyridine-d4]N6-HPB-dAdo versus N6-PHB-dAdo and
[15N5]N6-PHB-dAdo. All the results agreed well with the proposed fragmentation patterns of the four chemical standards. The ion m/z 173.53 was
a coeluting interfering ion.
structurally close to N6-PHB-dAdo, consistent with their very
close retention times and similar fragmentation patterns. To
confirm the proposed structure, N6-HPB-dAdo and its
isotopically labeled internal standard [pyridine-d4]N6-HPB-
dAdo were synthesized.
Synthesis and Characterization of N6-HPB-dAdo and
[pyridine-d4]N6-HPB-dAdo. N6-HPB-dAdo and [pyridine-
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Figure 3. Representative extracted product ion chromatograms of MS2 transitions for the analysis of N6-HPB-dAdo and N6-PHB-dAdo formation
in DNA samples upon NaBH4 reduction. (A) Blank calf thymus DNA, (B) calf thymus DNA incubated with 5′-acetoxyNNN, (C) liver DNA of rat
from the control group, (D) liver DNA of rats treated with 500 ppm racemic NNN for 3 weeks, (E) lung DNA of rat from the control group, (F)
lung DNA of rats treated with 500 ppm racemic NNN for 3 weeks, (G) calf thymus DNA incubated with NNKOAc, and (H) lung DNA of rats
treated with 5 ppm of NNK for 70 weeks.
d4]N6-HPB-dAdo were synthesized by a four-step procedure
(Scheme 2). Conversion of the carbonyl group of 16 to the
amino group of 18 was accomplished in two steps via the
oxime intermediate. The final SNAr reaction with 19 yielded
the desired product N6-HPB-dAdo (14). Its structure was
confirmed by one- and two-dimensional NMR experiments
(Figure S2) and HRMS. [Pyridine-d4]N6-HPB-dAdo was
synthesized using the same method, and the structure was
similarly confirmed (Figure S3).
acetoxyNNN and treated with a reducing agent (NaBH4 or
NaBH3CN) was analyzed by the new LC-NSI-HRMS/MS
method. As shown in Figure 3B, N6-PHB-dAdo was clearly
formed in vitro upon NaBH4 reduction. No such peak was
observed in the NaBH4-reduced untreated calf thymus DNA
(Figure 3A). The same peak was also observed in the calf
thymus DNA incubated with NNKOAc (Figure 3G) as
previously reported.18 The precursor compound of N6-PHB-
dAdo is considered to be N6-Py-THF-dAdo (vide supra). No
N6-POB-dAdo was observed in vitro without reduction (Figure
S9C), which agreed well with the metabolic mechanism of 5′-
acetoxyNNN as depicted in Scheme S3.
In the liver and lung DNA of rats treated with racemic
NNN, N6-PHB-dAdo was formed upon NaBH4 reduction
(Figure 3D,F). No such peaks were observed in the control
samples (Figure 3C,E). Consistent with our previous
observations, N6-PHB-dAdo was also formed in the lung
DNA of rats chronically treated with 5 ppm of NNK in the
drinking water for 50 weeks (Figure 3H).18 The precursor
compound of N6-PHB-dAdo in vivo is different from in vitro; it
is N6-POB-dAdo. As shown in Figure S9D,E, N6-POB-dAdo
coeluted with its isotope-labeled internal standard [pyridine-
d4]N6-POB-dAdo in the liver and lung DNA of rats treated
with NNN. The formation of N6-POB-dAdo in NNKOAc-
treated calf thymus DNA and the liver DNA of rats treated
with NNK was also reaffirmed as reported before (Figure
S9A,B).18
Representative LC chromatograms of chemical standards
N6-HPB-dAdo and N6-PHB-dAdo with their corresponding
isotopically labeled internal standards are shown in Figure 2.
Under the LC conditions described in Table S1, the peaks of
N6-HPB-dAdo elute slightly later than N6-PHB-dAdo, with
clear separation of the two diastereomers, in ratios near unity.
It is noteworthy that the separation was easily compromised by
changes of hand-packed column quality (Figure S8). Even
though N6-PHB-dAdo also has two diastereomers, no such
separation was achieved under the conditions we have tried.
The pattern of the product ions of N6-HPB-dAdo and
[pyridine-d4]N6-HPB-dAdo agreed well with the proposed
fragmentation pattern (Figure 2). The most abundant product
ions in the MS2 transitions were m/z 285.1459 [N6-HPB-Ade
+ H]+ and 289.1710 [[pyridine-d4]N6-HPB-Ade + H]+. The
product ion pattern of the MS3 transition of each standard was
also consistent with the proposed fragmentation pattern. The
subtle difference of product ion patterns of both MS2 and MS3
transitions between N6-HPB-dAdo and N6-PHB-dAdo is also
noted in Figure 2. The unique features of the MS2 fragments
m/z 267.1353 and 272.1204 of N6-PHB-dAdo and [15N5]N6-
PHB-dAdo respectively were helpful to distinguish this adduct
from N6-HPB-dAdo in the rat DNA samples.
It is noteworthy that N6-PHB-dAdo was formed in very low
levels upon NaBH3CN reduction of DNA from rats treated
with NNN. The effect is clearly observed when comparing the
levels of N6-PHB-dAdo in the same rat liver and lung DNA
samples, upon reduction of NaBH4 (Figure 3D,F) versus
NaBH3CN (Figure S10D,F). Since NaBH3CN was used for
the previous study15 which failed to observe the formation of
Investigation of N6-PHB-dAdo In Vitro and In Vivo
after Reduction. Calf thymus DNA incubated with 5′-
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N6-PHB-dAdo, the insufficient conversion of N6-POB-dAdo to
N6-PHB-dAdo by NaBH3CN reduction is likely to be one
potential explanation.
Table 2. Interday Precision and Accuracy for the Mass
Spectrometry Quantitation of N6-HPB-dAdo
spiked
(fmol)
measured
(fmol)
precision
(CV)
Investigation of N6-HPB-dAdo In Vitro and In Vivo
after Reduction. The formation of the new adduct N6-HPB-
dAdo was confirmed using its authentic synthesized internal
standard [pyridine-d4]N6-HPB-dAdo. As shown in Figure 3B,
this adduct was unambiguously formed in vitro upon NaBH4
reduction of calf thymus DNA incubated with 5′-acetoxyNNN,
coeluting with [pyridine-d4]N6-HPB-dAdo. The MS2 and MS3
fragmentation patterns of the two peaks were also similar to
the synthesized standard. The precursor compound of N6-
HPB-dAdo is considered to be N6-[4-oxo-1-(pyridine-3-
yl)butyl]-2′-deoxyadenosine (N6-OPB-dAdo), in equilibration
with 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2′-deoxynebu-
larine (Py-Py(OH)-dN), which has been observed in vitro and
in vivo (Scheme S3).29 No such peak was observed in the
untreated (Figure 3A) or NNKOAc-treated calf thymus DNA
(Figure 3G). This strongly indicates that N6-HPB-dAdo is
specifically formed by NNN metabolism. Both NaBH3CN and
NaBH4 were able to form N6-HPB-dAdo by reduction.
However, higher abundance of N6-HPB-dAdo formed by the
reduction of NaBH4 than NaBH3CN was clearly observed
(Figure 3 versus Figure S10). Thus, NaBH4 reduction was
chosen for the following quantitation study.
experiment
accuracy
1
5
day 1
day 2
day 3
mean SD
day 1
day 2
day 3
mean SD
0.99
1.14
1.11
1.08 0.08
6.36
5.63
5.75
5.92 0.39
108%
118%
7.2%
6.6%
Table 3. Levels of N6-HPB-dAdo and Py-Py-dI (fmol/μmol
dGuo) in the liver and lung DNA of rats (N = 3 per Dose)
Treated with Different Doses of Racemic NNN in the
Drinking Water for 3 Weeks
dose of racemic NNN
DNA adduct
N6-HPB-dAdo
rat tissue
50 ppm
74
110 18
56 14
580 41
100 ppm
500 ppm
liver
lung
liver
lung
4
84 20
176 34
157 42
1045 69
233 84
328 44
412 53
2018 200
a
Py-Py-dI
a
Data for Py-Py-dI levels in the same rat tissues were adapted from
our previous study.15
In the reduced DNA hydrolysates of rat liver and lung, N6-
HPB-dAdo was formed in relatively high abundance (Figure
3D,F). This adduct was not observed in the liver and lung
DNA of rats from the control group (Figure 3C,E) or the lung
DNA of rats treated with NNK (Figure 3H). The levels of N6-
HPB-dAdo in the liver and lung DNA of rats treated with
NNN appeared to be 71- and 486-fold higher than N6-PHB-
dAdo, respectively, by estimating the MS responses and
assuming similar ionization efficiencies of the two adducts
(Figure 3D,F). Thus, it is of great interest to quantify the levels
of N6-HPB-dAdo in rat tissues such as liver and lung and
compare to other DNA adducts specially formed by NNN
metabolism.
DISCUSSION
■
This is the first study to characterize an NNN-specific dAdo
adduct N6-HPB-dAdo formed by this tobacco-specific
carcinogen. Its structure was confirmed with authentic
synthesized standards. This new adduct was observed both in
vitro (calf thymus DNA treated with 5′-acetoxyNNN) and in
vivo (liver and lung DNA from rats treated with NNN).
Quantitation suggested a rough dose-dependent response of
NNN treatment and the adduct level in rat liver and lung. N6-
HPB-dAdo is a new and structurally unique type of DNA
adduct formed only by NNN 5′-hydroxylation.
The retention times of N6-HPB-dAdo and the structurally
similar adduct N6-PHB-dAdo differed to a very small extent.
Baseline separation was achieved under two different HPLC
conditions. Using the preliminary HPLC method with 2 mM
NH4OAc and CH3CN as the mobile phase (Table S2), N6-
HPB-dAdo eluted ∼1.5 min earlier than N6-PHB-dAdo
(Figure S6). An optimized HPLC method using 0.05% formic
acid in H2O and CH3CN as the mobile phase (Table S1)
eluted N6-HPB-dAdo ∼0.1 min later than N6-PHB-dAdo. This
method also achieved a good separation of the two
diastereomers of N6-HPB-dAdo (Figure 2). The liquid
chromatographic baseline separation of these two adducts,
together with the unique MS2 transition of m/z [M + H]+
401.1932 → [M + H-dR-H2O]+ 267.1353 of N6-PHB-dAdo,
provided unambiguous evidence for the formation of the new
adduct N6-HPB-dAdo in rat liver and lung. The difference of
formation and persistence of the two diastereomers of N6-
HPB-dAdo was considered minor, as shown in Figure 3. Thus,
the absolute configuration of the two isomers and assignment
of each peak were not pursued in this study.
Mass Spectrometry Quantitation of N6-HPB-dAdo in
Liver and Lung of Rats Treated with NNN. Our previous
animal study provided the liver and lung tissues of rats treated
with different doses of racemic NNN, which allowed us to
investigate the dose-dependent response of N6-HPB-dAdo to
NNN exposure in vivo.15 DNA hydrolysates were quantita-
tively analyzed for N6-HPB-dAdo using the MS2 transition of
m/z 401.2 [M + H]+ → 285.1458 [M + H-dR]+. The
quantitative MS method was validated with good accuracy and
precision (Table 2). The calibration curve for N6-HPB-dAdo
showed good linearity with an R2 value of 0.9989 (Figure S11).
As depicted in Table 3, levels of N6-HPB-dAdo in the liver
DNA of rats ranged from 74 4 to 233 84 fmol/μmol dGuo
(or from 49
2 to 154
55 fmol/mg DNA), and were
roughly dose dependent. The levels of N6-HPB-dAdo in rat
liver DNA were comparable to the levels of 2-[2-(3-pyridyl)-N-
pyrrolidinyl]-2′-deoxyinosine (Py-Py-dI, Figure 1), the only
NNN-specific DNA adduct quantified before,15 in the same rat
liver tissues. However, N6-HPB-dAdo occurred in substantially
lower abundance (∼6-fold) in the rat lung DNA when
compared with Py-Py-dI, ranging from 110 18 to 328 44
In agreement with our hypothesis that the formation of N6-
POB-dAdo and N6-PHB-dAdo (after reduction) should occur
in the liver and lung DNA of rats by the treatment of NNN,
these adducts were observed but in very low abundance. Levels
fmol/μmol dGuo (or from 73
12 to 217
29 fmol/mg
DNA).
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of N6-PHB-dAdo were estimated to be 71- and 486-fold lower
than N6-HPB-dAdo in the rat liver and rat lung, respectively
(Figure 3D,F). This was likely the reason for not observing
these adducts in our previous study15 and highlighted the
advantage of using the highly sensitive and specific LC-NSI-
HRMS/MS method for investigating trace levels of DNA
adducts in in vivo samples.
applied to reveal a strong correlation between NNN exposure
in human smokers’ urine and future esophageal cancer
incidence in the Shanghai Cohort study.5 However, urinary
total NNN does not necessarily reflect the metabolic fate of
NNN. A previous study of NNN metabolism in the patas
monkey suggested only <1% of intravenously dosed NNN was
excreted unchanged in the urine.37 This finding suggested that
the majority of NNN injected into the patas monkey was
biotransformed to its metabolites. Most major NNN
metabolites are identical to minor metabolites of nicotine,
thus precluding their use as specific biomarkers of NNN
exposure in people who use tobacco products.39,40 Thus, it will
potentially be beneficial to identify NNN-specific metabolites
as activation biomarkers to evaluate NNN exposure and uptake
for clinical trials. The formation of N6-HPB-dAdo in rat liver
and lung DNA indicates that adducted products can also be
formed when the diazonium ion 10 from NNN 5′-
hydroxylation reacts with various nucleophiles that are widely
distributed in the body, for example, glutathione, amino acids,
or taurine. The adductive products, if formed and stable in vivo,
may have the potential to serve as the surrogate biomarkers for
monitoring exposure to and bioactivation of NNN.
In summary, we have shown for the first time the formation
of N6-HPB-dAdo in the liver and lung DNA of rats treated
with NNN using a highly sensitive and specific LC-NSI-
HRMS/MS method. This represents a new type of DNA
adduct specifically formed by NNN 5′-hydroxylation. The
levels of this adduct in the liver and lung DNA of rats were
roughly dose dependent to the NNN treatment. The reported
findings here lead to new insights for identifying tobacco-
specific DNA adducts as well as investigating new activation
biomarkers of NNN metabolism.
Levels of N6-HPB-dAdo in the liver and lung DNA of rats
treated with different doses of racemic NNN quantified by MS
suggested a rough dose-dependent response to the NNN
exposure. This result agreed with our previous study of Py-Py-
dI.15 Even though N6-HPB-dAdo levels were comparable to
Py-Py-dI in rat liver DNA, it occurred in substantially lower
levels in rat lung DNA ranging from 110 18 to 328 44
fmol/μmol dGuo, when compared to Py-Py-dI which occurred
in the levels of 580 41 to 2018 200 fmol/μmol dGuo. The
low levels of N6-HPB-dAdo may be due to the lower formation
of this adduct by NNN. It is also possible that N6-HPB-dAdo
might be more prone to be repaired than Py-Py-dI, leading to
relatively low levels of persistence in the lung. Considering that
target tissues of NNN treatment in rats are mainly esophagus,
oral mucosa, and nasal mucosa,11−13,23 quantitation of N6-
HPB-dAdo in those tissues will provide a better understanding
of the persistence and distribution of this newly identified
dAdo adduct. Levels of N6-HPB-dAdo in comparison to those
of Py-Py-dI in the target tissues of rats will be more important.
A study is currently in progress to provide these tissues for
analysis.
The formation of DNA adducts by the metabolic activation
of the tobacco carcinogens NNN and NNK has been studied
intensively.30,31 However, only a few pyridyl dAdo-derived
adducts have been observed in vivo. They were N6-POB-dAdo
and N6-PHB-dAdo observed in rat lung and liver and Py-Py-
dN observed in rat lung and nasal cavity (structures shown in
Scheme 1).15,18 The new adduct N6-HPB-dAdo reported in
this study enlarges the repertoire of dAdo-derived adducts.
More importantly, N6-HPB-dAdo represents a new type of
DNA adduct that is structurally specific to NNN treatment. As
illustrated in Scheme 1, metabolic activation of NNN through
2′-hydroxylation forms the diazonium ion 9 which can also be
formed by NNK bioactivation. Thus, the resulting dAdo
adducts such as N6-POB-dAdo and N6-PHB-dAdo are not
structurally specific to NNN or NNK metabolism. However,
the new adduct N6-HPB-dAdo characterized in this study is
formed by the diazonium ion 10, which is a specific metabolite
of NNN 5′-hydroxylation. Considering previous findings that
5′-hydroxylation predominates in the metabolic activation of
NNN in some cultured human tissues32−36 and in the patas
monkey,37 the new adduct may be present in relatively higher
abundance in human DNA samples. Similarly, we might expect
dGuo and Thd to form analogous NNN-specific adducts such
as N2-HPB-dGuo/O6-HPB-dGuo and O2-HPB-Thd when
treated with 5′-acetoxyNNN in vitro or NNN in vivo. These
adducts could comprise a new group of DNA lesions that have
not been biologically studied. They may also shed light on the
current challenging studies directed toward identifying
tobacco-specific DNA adducts in samples from human
smokers and smokeless tobacco users.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.chemrestox.1c00013.
■
sı
Synthetic details for 5′-acetoxyNNN and N6-PHB-dAdo
(Schemes S1−S2); proposed mechanisms of formation
of Py-Py-dN, N6-HPB-dAdo, and N6-HPB-dAdo upon
reduction of treated calf thymus DNA (Scheme S3);
NMR spectra of 5′-acetoxyNNN, N6-HPB-dAdo,
[pyridine-d4]N6-HPB-dAdo, N6-POB-dAdo, and N6-
PHB-dAdo (Figures S1−S4); HPLC traces of N6-
POB-dAdo and N6-PHB-dAdo (Figure S5); preliminary
results of investigating new dAdo adducts in rat liver
after reduction (Figure S6); fragmentation pattern
comparison between N6-PHB-dAdo and the new peak
(Figure S7); compromised HPLC separation results as
column quality decreased (Figure S8); formation of N6-
POB-dAdo in unreduced DNA samples (Figure S9);
formation of N6-PHB-dAdo and N6-HPB-dAdo in DNA
samples reduced by NaBH3CN (Figure S10); calibration
curve for N6-HPB-dAdo (Figure S11); HPLC flow rate
and gradient composition at different time points in the
LC-NSI-HRMS/MS method and the preliminary LC
method (Tables S1−S2) (PDF)
The structural feature of N6-HPB-dAdo also provides new
insights for the investigation of activation biomarkers of NNN
metabolism. The current biomarker used for evaluating NNN
exposure in humans is urinary total NNN (free NNN plus its
glucuronide derivative NNN-N-Gluc).5,38 It was successfully
AUTHOR INFORMATION
Corresponding Author
■
Stephen S. Hecht − Masonic Cancer Center, University of
Minnesota, Minneapolis, Minnesota 55455, United States;
1001
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Article
(6) Stepanov, I., Sebero, E., Wang, R., Gao, Y. T., Hecht, S. S., and
Yuan, J. M. (2014) Tobacco-specific N-nitrosamine exposures and
cancer risk in the Shanghai Cohort Study: remarkable coherence with
rat tumor sites. Int. J. Cancer 134, 2278−2283.
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nitrosamines, In IARC Monographs on the Evaluation of Carcinogenic
Risks to Humans, Vol 89, pp 1−592, IARC, Lyon, France.
(8) Wiencke, J. K. (2002) DNA adduct burden and tobacco
carcinogenesis. Oncogene 21, 7376−7391.
(9) Jalas, J. R., Hecht, S. S., and Murphy, S. E. (2005) Cytochrome
P450 enzymes as catalysts of metabolism of 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Chem. Res.
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Reactions with deoxyguanosine of 4-(carbethoxynitrosamino)-1-(3-
pyridyl)-1-butanone, a model-compound for α-hydroxylation of
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chronically treated with (R)- and (S)-N’-nitrosonornicotine. Chem.
Res. Toxicol. 20, 246−256.
orcid.org/0000-0001-7228-1356; Phone: (612) 624-
7604; Email: hecht002@umn.edu; Fax: (612) 624-3869
Author
Yupeng Li − Masonic Cancer Center, University of Minnesota,
Minneapolis, Minnesota 55455, United States; orcid.org/
0000-0001-5403-5880
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.1c00013
Funding
This study was supported by grant CA-81301 from the
National Cancer Institute. Mass spectrometry was carried out
in the Analytical Biochemistry Shared Resource of the Masonic
Cancer Center, University of Minnesota, supported in part by
Cancer Center Support grant CA-077598.
Notes
The authors declare no competing financial interest.
(12) Zhang, S., Wang, M., Villalta, P. W., Lindgren, B. R., Lao, Y.,
and Hecht, S. S. (2009) Quantitation of pyridyloxobutyl DNA
adducts in nasal and oral mucosa of rats treated chronically with
enantiomers of N’-nitrosonornicotine. Chem. Res. Toxicol. 22, 949−
956.
(13) Zhao, L., Balbo, S., Wang, M., Upadhyaya, P., Khariwala, S. S.,
Villalta, P. W., and Hecht, S. S. (2013) Quantitation of
pyridyloxobutyl-DNA adducts in tissues of rats treated chronically
with (R)- or (S)-N’-nitrosonornicotine (NNN) in a carcinogenicity
study. Chem. Res. Toxicol. 26, 1526−1535.
(14) Balbo, S., Johnson, C. S., Kovi, R. C., James-Yi, S. A.,
O’Sullivan, M. G., Wang, M., Le, C. T., Khariwala, S. S., Upadhyaya,
P., et al. (2014) Carcinogenicity and DNA adduct formation of 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its
metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F-344
rats. Carcinogenesis 35, 2798−2806.
(15) Zarth, A. T., Upadhyaya, P., Yang, J., and Hecht, S. S. (2016)
DNA adduct formation from metabolic 5′-hydroxylation of the
tobacco-specific carcinogen N’-nitrosonornicotine in human enzyme
systems and in rats. Chem. Res. Toxicol. 29, 380−389.
(16) Yang, J., Villalta, P. W., Upadhyaya, P., and Hecht, S. S. (2016)
Analysis of O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine and
other DNA adducts in rats treated with enantiomeric or racemic N’-
nitrosonornicotine. Chem. Res. Toxicol. 29, 87−95.
(17) Leng, J., and Wang, Y. (2017) Liquid chromatography-tandem
mass spectrometry for the quantification of tobacco-specific nitros-
amine-induced DNA adducts in mammalian cells. Anal. Chem. 89,
9124−9130.
(18) Carlson, E. S., Upadhyaya, P., Villalta, P. W., Ma, B., and Hecht,
S. S. (2018) Analysis and identification of 2′-deoxyadenosine-derived
adducts in lung and liver DNA of F-344 rats treated with the tobacco-
specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
and enantiomers of its metabolite 4-(methylnitrosamino)-1-(3-
pyridyl)-1-butanol. Chem. Res. Toxicol. 31, 358−370.
(19) Guo, S., Leng, J., Tan, Y., Price, N. E., and Wang, Y. (2019)
Quantification of DNA lesions induced by 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanol in mammalian cells. Chem. Res. Toxicol. 32,
708−717.
ACKNOWLEDGMENTS
■
The authors thank Drs. Erik S. Carlson, Adam T. Zarth, and
Pramod Upadhyaya for their input and preliminary work on
this NNN project. We thank Drs. Peter W. Villalta and
Yingchun Zhao for help with the operation of the mass
spectrometer. We also thank Bob Carlson for his editorial
assistance. Y.L. would like to thank Valeria Guidolin (Silvia
Balbo lab) for her help with method validation and dGuo
quantitation. Y.L. also appreciates Dr. Bin Ma for his valuable
suggestions about mass spectrometric analysis.
ABBREVIATIONS
■
NNN, N′-nitrosonornicotine; NNK, 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone; dAdo, 2′-deoxyadenosine; NNAL, 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanol; POB, pyridylox-
obutyl; PHB, pyridylhydroxybutyl; Gua, guanine; Thd,
thymidine; NaBH3CN, sodium cyanoborohydride; LC-NSI-
HRMS/MS, liquid chromatography-nanoelectrospray ioniza-
tion-high-resolution tandem mass spectrometry; HPLC, high-
performance liquid chromatography; SPE, solid-phase extrac-
tion; i.d., inner diameter; SIM, selected-ion monitoring; HCD,
higher-energy collisional dissociation; AGC, automatic gain
control; CV, coefficient of variation
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