Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol

Article abstract of DOI:10.1021/acs.chemrestox.7b00281

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized

Full text of DOI:10.1021/acs.chemrestox.7b00281

Subscriber access provided by LAURENTIAN UNIV
Article
Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically
with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers
of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol
Bin Ma, Adam T. Zarth, Erik S Carlson, Peter W. Villalta,
Pramod Upadhyaya, Irina Stepanov, and Stephen S. Hecht
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00281 • Publication Date (Web): 13 Nov 2017
Downloaded from http://pubs.acs.org on November 14, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Chemical Research in Toxicology is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-
(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its
Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bin Ma,* Adam T. Zarth, Erik S. Carlson, Peter W. Villalta, Pramod Upadhyaya, Irina
Stepanov, and Stephen S. Hecht
Masonic Cancer Center, University of Minnesota
Running title: Methyl DNA phosphate adducts formed by NNK and NNAL
Keywords: Methyl DNA phosphate adducts; NNK; NNAL; LC-NSI-HR-MS/MS; methylating agents
*To whom correspondence should be addressed:
Masonic Cancer Center, University of Minnesota, 2231 6th Street SE - 2-152 CCRB, Minneapolis, MN 55455, USA.
phone: (612) 625-4925 fax: (612) 624-3869 e-mail: bma@umn.edu
1
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 2 of 38
1
2
3
4
Table of Contents Graphic
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 38
Chemical Research in Toxicology
1
2
3
4
Abstract
5
6
7
8
9
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a
powerful lung carcinogen in animal models and is considered a causative factor for lung cancer
in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo
metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and
pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also
occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide,
which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are
important in their mechanisms of carcinogenesis. In this study, we characterized and quantified
methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL or (R)-
NNAL in drinking water for 10, 30, 50 and 70 weeks, by using a novel liquid chromatography-
nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23,
21 and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues
of rats treated with NNK, (S)-NNAL and (R)-NNAL, respectively. Levels of the methyl DNA
phosphate adducts were 2290–4510, 872–1120, and 763–1430 fmol/mg DNA, accounting for
15–38%, 8%, and 5–9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL,
and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study
further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide
important new data regarding NNK- and NNAL-derived DNA damage, and new insights
pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical
methylating agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 4 of 38
1
2
3
4
Introduction
5
6
7
8
9
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1,
Figure 1) is a potent lung carcinogen in animal models,1 and together with the related
compound N′-nitrosonornicotine, is classified as a human carcinogen (Group 1) by the
International Agency for Research on Cancer.2 NNK is metabolized to 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanol (NNAL, 2, Figure 1), which is also a potent lung carcinogen with activity
similar to NNK.1, 3-4 Both NNK and NNAL are metabolically activated by α-hydroxylation
catalyzed by cytochrome P450s to form a series of intermediates which react with DNA to form
adducts. The formation of these DNA adducts can cause miscoding and mutations in growth
control genes,5-7 which is a critical step in carcinogenesis by NNK and NNAL. Therefore,
identification and measurement of relevant DNA adducts can enhance our understanding of
mechanisms of NNK- and NNAL-associated carcinogenesis and tobacco-related cancers.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Metabolic activation of NNK by α-hydroxylation on its methyl group produces an unstable
intermediate α-hydroxymethylNNK (3) which decomposes to 4-(3-pyridyl)-4-oxobutane-1-
diazohydroxide (7) that reacts with DNA to form adducts (Figure 1). These adducts are formed
by alkylation of nucleobases to produce pyridyloxobutyl (POB) base adducts,8-9 and by
alkylation of the oxygen of the internucleoside phosphodiester linkages in the DNA to produce
pyridyloxobutyl phosphate adducts [B1p(POB)B2],10 where B1 and B2 represent the same or
different nucleobases. Similar to NNK, α-hydroxylation on the methyl group of NNAL produces
intermediate α-hydroxymethylNNAL (6) and then decomposes to 4-(3-pyridyl)-4-
hydroxybutane-1-diazohydroxide (9), which reacts with DNA to form both pyridylhydroxybutyl
(PHB) base adducts11 and pyridylhydroxybutyl phosphate adducts [B1p(PHB)B2] (Figure 1).12 The
4
ACS Paragon Plus Environment

Page 5 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
major POB and PHB base adducts, and B1p(POB)B2 and B1p(PHB)B2 phosphate adducts have
been characterized, and their levels in NNK- and NNAL-treated animals have been quantified in
our laboratory.4, 8, 10-14 α-Hydroxylation also occurs on the α-methylene carbons of NNK and
NNAL, which produces intermediates α-methylenehydroxyNNK (4) and α-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
methylenehydroxyNNAL (5), respectively. Both 4 and 5 decompose to methane diazohydroxide
(8), which reacts with DNA to form methyl DNA base adducts (Figure 1).1 However, the
formation of methyl DNA phosphate adducts and their levels in NNK- and NNAL-treated animals
are still unknown.
The formation of methyl DNA phosphate adducts by other methylating agents has been
reported previously.15-24 The first identified methyl phosphate adduct, thymidylyl(3'-
5')thymidine methyl phosphotriester (TpMeT), was detected in rat liver DNA treated in vitro
with the carcinogen N-methyl-N-nitrosourea (MNU).16 Two other methyl phosphate adducts –
TpMeA and TpMeC – together with TpMeT were detected in salmon sperm DNA treated with
MNU or dimethyl sulphate (DMS).18 TpMeT was also detected and quantified in salmon testis
DNA and mouse lymphoma cells treated with MNU or methyl methanesulfonate (MMS).19-20
The levels of methyl phosphate adducts were also measured in vivo. Following intraperitoneal
treatment of male mice with MNU, DMS, or MMS, methyl phosphate adducts were detected
and their total amounts were measured in liver DNA.21 The levels of methyl phosphate adducts
were measured in tissue DNA of mice treated with MNU by single or multiple intraperitoneal
injections.22 In addition to the aforementioned direct-acting methylating agents, methyl DNA
phosphate adducts were also formed by certain nitrosamines upon metabolic activation. For
example, methyl phosphate adducts were detected in rats treated with N-
5
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 6 of 38
1
2
3
4
5
6
nitrosodimethylamine (NDMA).23-24 Based on these studies, NNK and NNAL also have the
potential to form methyl DNA phosphate adducts, which is the focus of this study.
7
8
9
We developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high
resolution tandem mass spectrometry (HRMS/MS)-based method for analysis of methyl DNA
phosphate adducts, referred to as B1pMeB2 adducts in this study. The B1pMeB2 adducts were
characterized and quantified in lung DNA of rats treated with NNK, (S)-NNAL, or (R)-NNAL in the
drinking water for 10, 30, 50, and 70 weeks.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL PROCEDURES
Caution: NNK, NNAL and MNU are carcinogenic. They should be handled in a well-ventilated
hood with extreme care and with personal protective equipment.
Materials and Chemicals
TpMeT was purchased from MRIGlobal (Kansas City, MO), and its structure was confirmed by
1D and 2D NMR in our laboratory (Figure S1). MNU was obtained from Sigma-Aldrich Chemical
Co. (Milwaukee, WI). [13C1015N2]TpT was prepared in another study.12 Reagents and enzymes for
DNA isolation were obtained from Qiagen Sciences (Germantown, MD). All other chemicals and
solvents were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI) or Fisher Scientific
(Fairlawn, NJ).
Synthesis of [13C1015N2]TpMeT
6
ACS Paragon Plus Environment

Page 7 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
[13C1015N2]TpMeT was synthesized by reacting [13C1015N2]TpT with MNU in 0.1 M Tris/EDTA
buffer (pH 7.4) at 37°C for 2.5 h, and purified using a 30-mg Strata X cartridge (Phenomenex)
activated with 2 mL of MeOH and 2 mL of H2O. The cartridge was washed with 2 mL of H2O and
1 mL of 10% MeOH sequentially and finally eluted with 2 mL of 50% MeOH. The presence of
two diastereomers of [13C1015N2]TpMeT in the 50% MeOH fraction was confirmed by
comparison with their corresponding unlabeled TpMeT standard (Figure S1) by using the
LC−NSI−HRMS/MS method described below (Figure S2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Rat Lung DNA Samples
The DNA samples were from male F-344 rats (n = 3 per group) in the control group and rats
treated chronically with 5 ppm of NNK, (S)-NNAL or (R)-NNAL in their drinking water for 10, 30,
50, and 70 weeks.4 DNA was isolated using our previously developed protocol, 8 and stored at -
20 °C until analysis.
DNA Hydrolysis and Sample Preparation
The DNA samples were dissolved in 0.6 mL of 10 mM sodium succinate (pH 7.4) buffer
containing 5 mM CaCl2 and 5 fmol [13C1015N2]TpMeT as internal standard, followed by the
addition of deoxyribonuclease I (0.75 units), phosphodiesterase I (0.005 units), and alkaline
phosphatase (0.4 units). The solution was then incubated overnight at 37 °C. On the next day,
20 μL of hydrolysate was collected for dGuo analysis by HPLC-UV and DNA quantitation.8 The
remaining hydrolysate was filtered through 10 K centrifugal filters (Ultracel YM-10, Millipore),
and the filtrates were loaded on 30-mg Strata X cartridges (Phenomenex) activated with 2 mL of
MeOH and 2 mL of H2O. The cartridges were washed with 2 mL of H2O and 1 mL of 10% MeOH
7
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 8 of 38
1
2
3
4
5
6
7
8
9
sequentially and finally eluted with 2 mL of 50% MeOH. The 50% MeOH fraction containing
analytes was concentrated to dryness in a centrifugal evaporator. The residue was redissolved
in 15 μL of deionized H2O prior to analysis by LC−NSI−HRMS/MS.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LC−NSI−HRMS/MS Analysis
The analysis was performed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific,
Waltham, MA) with full scan, selected-ion monitoring (SIM) and product ion scan data
acquisition using Orbitrap detection with internal calibration using the EASY-IC feature of the
instrument. A nanoLC column (75 μm i.d., 360 μm o.d., 20 cm length, and 10 μm orifice) packed
with Luna C18 bonded separation media (Phenomenex, Torrance, CA) was used. The mobile
phase consisted of 2 mM NH4OAc and CH3CN. The sample (4 μL) was loaded onto the column
with a 900 nL/min flow rate under the initial conditions for 6.5 min at which point the injection
valve position was switched to take the injection loop out of the flow path and the flow rate
was reduced to 300 nL/min. Separation on the column was performed with increasing CH3CN
from 2 to 10% over 1.5 min and then from 10 to 15% over 28 min, followed by ramping to 90%
CH3CN within 2 min and holding at this composition for an additional 3 min. The gradient was
then returned to 2% CH3CN in 1 min, and the system was re-equilibrated at this mobile phase
composition for 6 min at 900 nL/min before the next injection. The spray voltage of the mass
spectrometer was 2.2 kV. The capillary temperature was 300 °C, and the S-Lens RF Level was
60%. A multiplexed selected-ion monitoring (SIM) analysis was performed whereby each ion
mass of interest was sequentially isolated by the quadrupole and stored in the ion routing
multipole, and upon collection of all masses of interest, all ions were transported into the
Orbitrap for analysis. The [M + H]+ ions of all possible phosphate adducts listed in Table 1 were
8
ACS Paragon Plus Environment

Page 9 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
analyzed in this fashion using an isolation window of 1.5 m/z, automatic gain control (AGC)
target of 50,000, maximum inject time of 100 ms, and a resolution of 60,000. The product ion
scan was performed using higher-energy collisional dissociation (HCD) fragmentation with a
normalized collision energy of 20 units, isolation widths of 1.5 m/z for all the precursor ions,
and product ion analysis performed with a mass range of m/z 100–700 at a resolution of
15,000. The accurate mass tolerance used for extraction of precursor and fragment ion signals
was 5 ppm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The quantitative analysis of TpMeT was performed using accurate mass extracted ion
chromatograms of m/z 225.0870 [C10H13N2O4]+ for TpMeT (parent ion m/z 561.2) and m/z
237.1146 for [13C1015N2]TpMeT (parent ion m/z 573.2). Quantitation was based on the peak
area ratio of TpMeT to [13C1015N2]TpMeT, the constructed calibration curves, and the amount of
internal standard added. A calibration curve was constructed before each analysis using a series
of standard solutions of TpMeT and [13C1015N2]TpMeT. The calibration standard solutions
contained a constant amount of [13C1015N2]TpMeT (3 fmol on-column) and varying amounts of
TpMeT (0.15, 0.3, 0.6, 1.5, 3, and 6 fmol on-column). The levels of other adducts were
estimated on the basis of their MS signal intensities compared to TpMeT under SIM mode. All
data are presented as mean ± standard deviation (SD). One-way ANOVA followed by a
Bonferroni post test was used for multiple comparisons. A p value of < 0.05 was considered
significant.
9
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 10 of 38
1
2
3
4
RESULTS
5
6
7
Characterization and quantitation of methyl phosphate adducts
8
9
Table 1 summarizes the possible numbers of methyl DNA phosphate adduct isomers which
could be formed from intermediate 8 produced in the metabolism of NNK or NNAL. There can
be 10 different combinations of the four nucleobases, and different stereoisomers for each
combination due to the tetrahedral structure of the phosphate group. Thus, there can be 2
possible isomers of B1pMeB2 with the same nucleobase combination, and 4 isomers with
different nucleobase combinations (two diastereomers of B1-3′-pMe-5′-B2 and two
diastereomers of B2-3′-pMe-5′-B1), resulting in a total of 32 different possible B1pMeB2 adducts.
By analyzing the data obtained from both SIM and product ion scans, as described below, we
observed at least one isomer of each combination of B1pMeB2 phosphate adducts, and a total
of 23, 21 and 22 out of 32 possible adducts were detected in the lung tissues of rats treated
with NNK, (S)-NNAL and (R)-NNAL, respectively (Table 1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Under our chromatographic conditions, the two diastereomers of TpMeT were observed
upon analysis of the TpMeT standard (Figure 2A). The product ion spectra of the isomers
showed similar fragmentation patterns, and only the spectrum of the isomer at 22.6 min with
proposed fragmentation pathways is presented in Figure 2B. The identity of TpMeT was further
confirmed by an MS3 scan on the fragment ion m/z 337.0791, which was fragmented to two
major ions m/z 225.0870 and m/z 207.0764 (Figure S1 and Figure 2B). The DNA samples from
rat lung were analyzed using the same method. No TpMeT was detected upon analysis of DNA
samples from control rats (Figure 3A), while both isomers of TpMeT were detected in rats
10
ACS Paragon Plus Environment

Page 11 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
treated with NNK (Figure 3B) or NNAL. The structures of all the other B1pMeB2 adducts were
characterized based on the data obtained from LC−NSI−HRMS/MS analysis using SIM and
product ion data acquisition of lung DNA samples of rats treated with NNK (Figure 4 and Figure
S3). Taking ApMeC as an example, the exact mass of its precursor ion of m/z 555.1711 was
monitored by SIM and four peaks were detected (Figure 4), indicating the presence of four
isomers. The fragmentation patterns of the two early-eluting isomers were similar but with
different abundances, and so were those of the two isomers which eluted later (Figure S4).
However, the fragmentation patterns of the first two isomers were different compared to the
latter two, which could be due to how the two sugar moieties of the nucleosides connect to the
phosphorus atom, i.e. A-3'-pMe-5'-C and C-3'-pMe-5'-A (Figure S4). The standards of ApMeC
are currently unavailable, and further work needs to be done to assign the configuration of
each isomer. The extracted ion chromatograms and fragmentation patterns for characterization
of all the other B1pMeB2 phosphate adducts are available in Supplementary Figure S3. None of
the B1pMeB2 phosphate adducts was detected in control rats.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Using HCD fragmentation, several major fragment ions were generated in the product ion
spectrum of TpMeT (Figure 2B). Because the highest signal intensities in the spectra of TpMeT
and [13C1015N2]TpMeT were the transitions m/z 561.2→225.0870 and m/z 573.2→237.1146,
respectively, they were selected for quantitative analysis. By using this method, a limit of
detection of 0.15 fmol (on-column) was obtained for TpMeT. The instrument response and the
TpMeT/[13C1015N2]TpMeT ratio were linear in the 0.15−6 fmol (on-column) range of TpMeT with
a typical R2 of 0.9982 (Figure 5).
11
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 12 of 38
1
2
3
4
Methyl phosphate adduct levels in rat lung
5
6
7
8
9
In NNK-treated rats, the two major B1pMeB2 phosphate adducts with the highest levels in lung
DNA were ApMeT and GpMeT, accounting for 38–54% and 16–20% of the total B1pMeB2
phosphate adducts, respectively (Figure 6A). For most of the B1pMeB2 adducts, the levels
increased from 10 weeks-treatment to 30 weeks-treatment, and persisted throughout the rest
of the study. For example, the levels of ApMeT increased from 938 ± 124 fmol/mg DNA at 10
weeks of treatment to 1910 ± 95 fmol/mg DNA at 30 weeks of treatment (p < 0.05), and were
not significantly changed afterwards with levels of 1660 ± 172 and 1870 ± 73 fmol/mg DNA at
50 and 70 weeks, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Similar to the NNK treatment group, the two major B1pMeB2 adducts in (S)-NNAL-treated
rats were also ApMeT and GpMeT, which accounted for 37–55% and 13–21% of the total
B1pMeB2 phosphate adducts, respectively (Figure 6B). The levels of most of the B1pMeB2
adducts did not significantly change over the course of the study. For example, the levels of
GpMeT were 202 ± 48 fmol/mg DNA at 10 weeks of treatment, and did not significantly change
afterwards with levels of 190 ± 60, 163 ± 57 and 170 ± 19 fmol/mg DNA at 30, 50 and 70 weeks,
respectively.
In (R)-NNAL-treated rats, three major B1pMeB2 phosphate adducts were observed – ApMeT
GpMeT, and ApMeC – which accounted for 13–37%, 18–25%, and 13–23% of the total B1pMeB2
phosphate adducts, respectively (Figure 6C). For most of the B1pMeB2 adducts, the levels
increased from 10 weeks-treatment to 30 weeks-treatment, and decreased afterwards. For
example, the levels of ApMeC were 114 ± 22 fmol/mg DNA at 10 weeks of treatment, reached a
12
ACS Paragon Plus Environment

Page 13 of 38
Chemical Research in Toxicology
1
2
3
4
peak at 30 weeks (278 ± 14 fmol/mg DNA, p < 0.05), and decreased to 113 ± 15 fmol/mg DNA at
5
6
70 weeks of treatment.
7
8
9
Comparison of DNA phosphate and base adducts
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Six types of NNK-derived DNA adducts have been characterized so far – B1pMeB2 phosphate
adducts, B1p(POB)B2 phosphate adducts, B1p(PHB)B2 phosphate adducts, methyl base adducts,
POB base adducts, and PHB base adducts.1, 4, 8, 10, 25 To determine the relative proportion of
methyl phosphate adducts to the total measured DNA adducts formed in rats treated with NNK
or NNAL, the levels of the other five types of adducts were obtained from our previous studies
(Table 2).4, 10, 12-13 The levels of total B1p(POB)B2 phosphate adducts in the lung of NNK-treated
rats were reported previously.10 The levels of total B1p(POB)B2 and B1p(PHB)B2 phosphate
adducts in the lung of (R)- and (S)-NNAL-treated rats, and total B1p(PHB)B2 phosphate adducts
in the lung of NNK-treated rats were obtained from other studies.12-13 The levels of one methyl
base adduct – O6-methylguanine, three major POB base adducts – O2-[4-(3-pyridyl)-4-oxobut-1-
yl]thymidine, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine, and O6-[4-(3-pyridyl)-4-oxobut-1-
yl]deoxyguanosine, and two major PHB base adducts – O2-[4-(3-pyridyl)-4-hydroxybut-1-
yl]thymidine and 7-[4-(3-pyridyl)-4-hydroxybut-1-yl]guanine – were reported previously.4 Other
NNK-derived methyl and POB base adducts have been previously identified from in vitro and in
vivo studies,1, 9, 26 but only the base adducts mentioned above were measured in the treated
rats and included in this study.
Methyl phosphate adducts accounted for 15-38%, 8%, and 5-9% of the total measured DNA
adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively (Figure 7). In the NNK-
treated rats, methyl phosphate adducts accounted for 15% of the total adducts at 10 weeks of
13
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 14 of 38
1
2
3
4
5
6
7
8
9
treatment, but became a major contributor afterwards with 26%, 25%, and 38% of the total
adducts at 30, 50, and 70 weeks, respectively. In the (S)-NNAL treatment group, B1p(POB)B2
phosphate adducts were the most abundant type at 10 weeks of treatment (34%), but
decreased afterwards (22%, 11%, and 10% at 30, 50, and 70 weeks of treatment, respectively).
The other two abundant types of adducts were B1p(PHB)B2 phosphate adducts and POB base
adducts, accounting for 27-32% and 23-39%, respectively, of the total measured DNA adducts.
In the (R)-NNAL-treated rats, B1p(PHB)B2 phosphate adducts and PHB base adducts were the
most abundant types of adducts, together accounting for 87-91% of the total measured DNA
adducts. The total amount of measured DNA phosphate and base adducts were calculated and
compared among the three treatment groups (Table 2). At 10 and 30 weeks of treatment, the
total adduct levels in the NNK-treated rats were slightly higher than the other two treatment
groups. At 50 weeks of treatment, the adduct levels were comparable between NNK and (R)-
NNAL groups, which were higher than in the (S)-NNAL group (p < 0.05). There was no significant
difference in the total adduct levels among the three groups at 70 weeks of treatment.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DISCUSSION
We investigated methyl DNA phosphate adduct formation by the tobacco-specific carcinogen
NNK and enantiomers of its metabolite NNAL by using a novel, highly sensitive and specific LC-
NSI-HRMS/MS method. A total of 23, 21 and 22 out of 32 possible methyl DNA phosphate
adducts (B1pMeB2) were detected and quantified in the lung tissues of rats treated with NNK,
(S)-NNAL and (R)-NNAL, respectively. Taken together with our previous studies in which we
14
ACS Paragon Plus Environment

Page 15 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
characterized 30 B1p(POB)B2 adducts,10 and 107 B1p(PHB)B2 adducts,12 we have collectively
identified 160 different structurally unique DNA phosphate adducts in tissues of rats treated
with NNK, which establishes NNK as the source of the most structurally diverse panel of DNA
adducts identified to date from any carcinogen.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
With a total of 32 possible methyl DNA phosphate adducts, the development of a suitable
analytical method for simultaneous monitoring of all the possibilities is challenging, which could
be one reason why the research on methyl phosphate adducts has still not been completely
explored. Early attempts to detect methyl DNA phosphate adducts involved analysis of alkali-
induced strand breaks at the sites of modified lesions. This method was based on the principle
that addition of alkali should lead to the opening of imidazole ring of purines and block the
spontaneous loss of methylated purines, and leave the methyl DNA phosphate adducts as the
only alkali-labile groups. Sedimentation coefficients of the hydrolyzed DNA samples were
measured to determine the amount of methyl DNA phosphate adducts.17, 21, 27-28 However, this
method is not specific and the levels may be overestimated if other alkali-labile lesions are
present in the DNA. The 32P-postlabelling technique, which was widely used for analysis of DNA
base adducts, was another early effort applied to methyl phosphate adducts analysis. However,
the traditional 32P-postlabelling method for DNA base adduct analysis cannot be used for
phosphate adducts because the negative charge on the phosphate group is neutralized by
methylation.29 In order to perform labelling, the methyl group had to be removed under
alkaline conditions and the resulting dinucleoside phosphates or 3'-phosphate alkylated
mononucleotides were measured.30-31 The analysis of methyl phosphate adducts was also
conducted by a transalkylation approach, which utilized the weakly alkylating property of the
15
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 16 of 38
1
2
3
4
5
6
7
8
9
methyl phosphate adducts for the transfer of the methyl group to strong nucleophiles, followed
by measurement of methyl-nucleophile products.32-33 All the above-mentioned methods are
indirect and unable to measure individual phosphate adducts. The first direct measurement of
methyl phosphate adducts was analysis of TpMeT by mass spectrometry,20 which was further
optimized to achieve higher detection sensitivity.19 Together with a method analyzing ethyl
DNA phosphate adducts by mass spectrometry34 and our successful characterization of
B1p(POB)B2 phosphate adducts by an LC−NSI−HRMS/MS method,10 the mass spectrometry-
based approach has proved to be an appropriate technique for the analysis of methyl
phosphate adducts. The method developed in this study is the first example of an HRMS/MS-
based method being used for the analysis of methyl DNA phosphate adducts, demonstrating
high sensitivity and selectivity for specific and accurate measurement of these adducts.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The formation patterns of methyl DNA phosphate and base adducts were different among
the three treatment groups. In NNK-treated rats, the level of total methyl DNA phosphate
adducts increased from 10 to 30 weeks of treatment and stayed constant afterwards, while the
methyl base adduct (O6-methylguanine) levels decreased over the course of the study (Table 2).
A decrease over time was observed for both methyl DNA phosphate and base adducts in (S)-
NNAL-treated rats. In (R)-NNAL-treated rats, a decrease was also observed for methyl DNA
phosphate adducts after 30 weeks of treatment, while the levels of O6-methylguanine were low
throughout the study. The greater persistence of methyl phosphate adducts compared to base
adducts in NNK-treated rats was also observed in animals treated with other methylating
agents. Following a single intraperitoneal injection of MNU to C57BL male mice at 80 mg/kg,
the total methyl DNA phosphate adducts were measured in liver, lung and kidney, and the half-
16
ACS Paragon Plus Environment

Page 17 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
life was estimated to be ~7 days in all the three tissues. In contrast, the half-life of O6-
methylguanine was 13 h,22 which was likely due to its repair by O6-alkylguanine DNA
alkyltransferase (AGT).7 In another study, male Wistar rats were intraperitoneally injected with
a single dose of NDMA at 10 mg/kg. TpMeT was detected and quantified in the liver with a half-
life of 7 days, significantly longer than the corresponding O6-methylguanine with a half-life of
25 h.24 The persistence of DNA phosphate adducts was also observed in our studies of
pyridyloxobutyl10 and pyridylhydroxybutyl12-13 phosphate adducts in the NNK- and NNAL-
treated rats. The persistence of these DNA phosphate adducts could be partially due to their
chemical stability. TpMeT is stable at 37°C for more than three days at pH 7.0 and has a half-life
of 2.75 h even at pH 12.5.35 However, depending on the type of alkyl group, not all the
phosphate adducts are chemically stable. Tp(he)T, with the alkyl group being 2-hydroxyethyl
moiety, has a half-life at 37°C of only 60 min at pH 7.0 and <1 min at pH 12.5, possibly due to
the formation of a reactive dioxaphospholane ring intermediate and subsequent rapid
hydrolysis.35 Another plausible explanation for the persistence of methyl DNA phosphate
adducts is lack of repair mechanisms. The Ada protein in E. coli repairs methyl DNA phosphate
adducts by transferring the aberrant methyl group to one of its own cysteine residues.
However, this repair mechanism was only able to remove the (S)-stereoisomers of the methyl
DNA phosphate adduct, while the (R)-stereoisomer was left unrepaired.36-37 An alkyltransferase
that repairs methyl DNA phosphate adducts was also detected in the eukaryote Aspergillus
nidulans.38 However, repair mechanisms for these adducts in higher eukaryotic organisms have
not been discovered.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 18 of 38
1
2
3
4
5
6
7
8
9
Taking into account the persistence of methyl DNA phosphate adducts, their biological
consequences may be significant. An inhibitory effect on RNA synthesis by methyl DNA
phosphate adducts was demonstrated in vitro by inhibiting exogenously added RNA
polymerases, which occurred primarily at the initiation step.39 Formation of methyl DNA
phosphate adducts also neutralizes the negative charge of the internucleoside phosphodiester
moiety, which affects the binding affinity of DNA to other macromolecules. A single charge
difference due to methylation was shown to induce ~100-fold affinity difference of protein–
DNA interactions.40 Studies on other DNA phosphate adducts – ethyl and isopropyl – also
showed their effects on DNA polymerases and DNA chain elongation.41-43 However, the overall
biological significance of DNA phosphate adducts, including methyl DNA phosphate adducts,
remains largely unknown and warrants further investigation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In contrast to structurally unique B1p(POB)B2 and B1p(PHB)B2 DNA phosphate adducts,
which are specific to NNK and NNAL and possibly N'-nitrosonornicotine, methyl DNA phosphate
adducts are also formed by other chemical methylating agents – MNU, DMS, MMS and
NDMA.16, 18-19, 23 Thus, these adducts could also be potential biomarkers for exposure to these
chemicals, or could contribute to mechanistic understanding. For example, NDMA, which is
classified as a Group 2A carcinogen (probably carcinogenic to humans) by the International
Agency for Research on Cancer,44 is widely detected in drinking water, foodstuffs, and alcoholic
beverages.45-47 The possible formation of methyl DNA phosphate adducts due to long-term
exposure to NDMA or other DNA methylating agents could provide useful information on risk
assessment in humans. In addition to environmental exposure, methylating agents are also
used clinically for cancer treatment. Temozolomide, a prodrug for the treatment of
18
ACS Paragon Plus Environment

Page 19 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
glioblastoma multiforme, acts as a DNA methylating agent and forms O6-methylguanine – the
primary cytotoxic lesion – to exert its chemotherapeutic effects.48 Formation of methyl DNA
phosphate adducts by temozolomide and its potential clinical effects have not been studied,
and such investigations could be conducted to provide more in-depth information.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
We developed and applied a novel LC-NSI-HRMS/MS method to characterize and quantify
methyl DNA phosphate adducts in the lungs of rats chronically treated with NNK, (S)-NNAL, or
(R)-NNAL. A total of 23, 21 and 22 out of 32 possible adducts were detected in the three
treatment groups, respectively. Certain methyl DNA phosphate adducts demonstrated
persistence over the 70 weeks of treatment. These adducts could serve as potential biomarkers
of chronic exposure to NNK and NNAL due to tobacco use, and could also be used to study
exposure to other chemical methylating agents.
19
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 20 of 38
1
2
3
4
Funding
5
6
7
8
9
This study was supported by National Cancer Institute (NCI) grant CA-81301. Mass-
spectrometry analyses were carried out in the Analytical Biochemistry Shared Resource of the
Masonic Cancer Center, supported in part by NCI grant CA-77598. Salary support for P.W.V. was
provided by NCI grant CA-211256.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notes
The authors declare no competing financial interest.
Acknowledgments
We thank Xun Ming and Yingchun Zhao for their help with the mass spectrometry analysis, and
Bob Carlson for editorial assistance.
Supporting Information
NMR and MS3 data of TpMeT, chromatograms of TpMeT and [13C1015N2]TpMeT, chromatograms
of B1pMeB2 phosphate adducts in rat lung DNA, product ion spectra and fragmentation pattern
of ApMeC. This material is available free of charge via the Internet at http://pubs.acs.org.
Abbreviations
DMS, dimethyl sulphate; HCD, higher-energy collisional dissociation; LC-NSI-HRMS/MS, liquid
chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry;
MMS, methyl methanesulfonate; MNU, N-methyl-N-nitrosourea; NDMA, N-
nitrosodimethylamine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PHB,
20
ACS Paragon Plus Environment

Page 21 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
pyridylhydroxybutyl; POB, pyridyloxobutyl; SD, standard deviation; SIM, selected-ion
monitoring; TpMeT, thymidylyl(3'-5')thymidine methyl phosphotriester.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 22 of 38
1
2
3
4
References
5
6
7
8
9
(1) Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-
nitrosamines. Chem. Res. Toxicol. 11, 559-603.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(2) International Agency for Research on Cancer. (2012) Personal habits and indoor
combustions. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, v.
100E. IARC, Lyon, FR. 319–331.
(3) Hecht, S. S., Stepanov, I., and Carmella, S. G. (2016) Exposure and metabolic activation
biomarkers of carcinogenic tobacco-specific nitrosamines. Acc. Chem. Res. 49, 106-114.
(4) 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., and Hecht, S. S. (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.
(5) Ronai, Z. A., Gradia, S., Peterson, L. A., and Hecht, S. S. (1993) G to A transitions and G to T
transversions in codon 12 of the Ki-ras oncogene isolated from mouse lung tumors
induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related DNA
methylating and pyridyloxobutylating agents. Carcinogenesis 14, 2419-2422.
(6) Jasti, V. P., Spratt, T. E., and Basu, A. K. (2011) Tobacco-specific nitrosamine-derived O2-
alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli. Chem. Res.
Toxicol. 24, 1833-1835.
22
ACS Paragon Plus Environment

Page 23 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
(7) Peterson, L. A. (2017) Context matters: Contribution of specific DNA adducts to the
genotoxic properties of the tobacco-specific nitrosamine NNK. Chem. Res. Toxicol. 30,
420-433.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(8) Lao, Y., Villalta, P. W., Sturla, S. J., Wang, M., and Hecht, S. S. (2006) Quantitation of
pyridyloxobutyl DNA adducts of tobacco-specific nitrosamines in rat tissue DNA by high-
performance liquid chromatography-electrospray ionization-tandem mass spectrometry.
Chem. Res. Toxicol. 19, 674-682.
(9) Leng, J., and Wang, Y. (2017) Liquid chromatography-tandem mass spectrometry for the
quantification of tobacco-specific nitrosamine-induced DNA adducts in mammalian cells.
Anal. Chem. 89, 9124-9130.
(10) Ma, B., Villalta, P. W., Zarth, A. T., Kotandeniya, D., Upadhyaya, P., Stepanov, I., and Hecht,
S. S. (2015) Comprehensive high-resolution mass spectrometric analysis of DNA
phosphate adducts formed by the tobacco-specific lung carcinogen 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Chem. Res. Toxicol. 28, 2151-2159.
(11) Upadhyaya, P., Kalscheuer, S., Hochalter, J. B., Villalta, P. W., and Hecht, S. S. (2008)
Quantitation of pyridylhydroxybutyl-DNA adducts in liver and lung of F-344 rats treated
with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Chem. Res. Toxicol. 21, 1468-1476.
(12) Ma, B., Zarth, A. T., Carlson, E. S., Villalta, P. W., Upadhyaya, P., Stepanov, I., and Hecht, S.
S. (2017) Identification of more than one hundred structurally unique DNA-phosphate
adducts formed during rat lung carcinogenesis by the tobacco-specific nitrosamine 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis, in press.
23
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 24 of 38
1
2
3
4
5
6
7
8
9
(13) Ma, B., Zarth, A. T., Carlson, E. S., Villalta, P. W., Stepanov, I., and Hecht, S. S. (2017)
Pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adduct formation in rats treated
chronically with enantiomers of the tobacco-specific nitrosamine metabolite 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Mutagenesis, in press.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(14) Zhang, S., Wang, M., Villalta, P. W., Lindgren, B. R., Upadhyaya, P., Lao, Y., and Hecht, S. S.
(2009) Analysis of pyridyloxobutyl and pyridylhydroxybutyl DNA adducts in extrahepatic
tissues of F344 rats treated chronically with 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone and enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Chem. Res.
Toxicol. 22, 926-936.
(15) Jones, G. D., Le Pla, R. C., and Farmer, P. B. (2010) Phosphotriester adducts (PTEs): DNA's
overlooked lesion. Mutagenesis 25, 3-16.
(16) Swenson, D. H., Farmer, P. B., and Lawley, P. D. (1976) Identification of the methyl
phosphotriester of thymidylyl (3',5')thymidine as a product from reaction of DNA with the
carcinogen N-methyl-N-nitrosourea. Chem. Biol. Interact. 15, 91-100.
(17) Shooter, K. V. (1978) DNA phosphotriesters as indicators of cumulative carcinogen-
induced damage. Nature 274, 612-614.
(18) Swenson, D. H., and Lawley, P. D. (1978) Alkylation of deoxyribonucleic acid by
carcinogens dimethyl sulphate, ethyl methanesulphonate, N-ethyl-N-nitrosourea and N-
methyl-N-nitrosourea. Relative reactivity of the phosphodiester site thymidylyl(3'-
5')thymidine. Biochem. J. 171, 575-587.
(19) Zhang, F., Bartels, M. J., Pottenger, L. H., Gollapudi, B. B., and Schisler, M. R. (2007)
Quantitation of lower levels of the DNA adduct of thymidylyl(3'-5')thymidine methyl
24
ACS Paragon Plus Environment

Page 25 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
phosphotriester by liquid chromatography/negative atmospheric pressure chemical
ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 21, 1043-1048.
(20) Zhang, F., Bartels, M. J., Pottenger, L. H., Gollapudi, B. B., and Schisler, M. R. (2005)
Quantitation of DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by
liquid chromatography/negative electrospray tandem mass spectrometry. Rapid Commun.
Mass Spectrom. 19, 2767-2772.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(21) Shooter, K. V., and Merrifield, R. K. (1976) An assay for phosphotriester formation in the
reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo. Chem. Biol.
Interact. 13, 223-236.
(22) Shooter, K. V., and Slade, T. A. (1977) The stability of methyl and ethyl phosphotriesters in
DNA in vivo. Chem. Biol. Interact. 19, 353-361.
(23) Shooter, K. V., Slade, T. A., and O'Connor, P. J. (1977) The formation and stability of
methyl phosphotriesters in the DNA of rat tissues after treatment with the carcinogen
N,N-dimethylnitrosamine. Chem. Biol. Interact. 19, 363-367.
(24) Den Engelse, L., Menkveld, G. J., De Brij, R. J., and Tates, A. D. (1986) Formation and
stability of alkylated pyrimidines and purines (including imidazole ring-opened 7-
alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with
ethylnitrosourea or dimethylnitrosamine. Carcinogenesis 7, 393-403.
(25) Lao, Y., Yu, N., Kassie, F., Villalta, P. W., and Hecht, S. S. (2007) Formation and
accumulation of pyridyloxobutyl DNA adducts in F344 rats chronically treated with 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite, 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Chem. Res. Toxicol. 20, 235-245.
25
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 26 of 38
1
2
3
4
5
6
7
8
9
(26) Michel, A. K., Zarth, A. T., Upadhyaya, P., and Hecht, S. S. (2017) Identification of 4-(3-
pyridyl)-4-oxobutyl-2'-deoxycytidine adducts formed in the reaction of DNA with 4-
(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone: A chemically activated form of
tobacco-specific carcinogens. ACS omega 2, 1180-1190.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) Snyder, R. D., and Regan, J. D. (1981) Quantitative estimation of the extent of alkylation of
DNA following treatment of mammalian cells with non-radioactive alkylating agents.
Mutat. Res. 91, 307-314.
(28) Shooter, K. V. (1976) The kinetics of the alkaline hydrolysis of phosphotriesters in DNA.
Chem. Biol. Interact. 13, 151-163.
(29) Weinfeld, M., and Livingston, D. C. (1986) Synthesis and properties of
oligodeoxyribonucleotides containing an ethylated internucleotide phosphate.
Biochemistry 25, 5083-5091.
(30) Saris, C. P., Damman, S. J., van den Ende, A. M., Westra, J. G., and den Engelse, L. (1995) A
32P-postlabelling assay for the detection of alkylphosphotriesters in DNA. Carcinogenesis
16, 1543-1548.
(31) Guichard, Y., Jones, G. D., and Farmer, P. B. (2000) Detection of DNA alkylphosphotriesters
by 32P postlabeling: evidence for the nonrandom manifestation of phosphotriester
lesions in vivo. Cancer Res. 60, 1276-1282.
(32) Haglund, J., Ehrenberg, L., and Tornqvist, M. (1997) Studies of transalkylation of
phosphotriesters in DNA: reaction conditions and requirements on nucleophiles for
determination of DNA adducts. Chem. Biol. Interact. 108, 119-133.
26
ACS Paragon Plus Environment

Page 27 of 38
Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
(33) Haglund, J., Rafiq, A., Ehrenberg, L., Golding, B. T., and Tornqvist, M. (2000)
Transalkylation of phosphotriesters using Cob(I)alamin: toward specific determination of
DNA-phosphate adducts. Chem. Res. Toxicol. 13, 253-256.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(34) Haglund, J., Van Dongen, W., Lemiere, F., and Esmans, E. L. (2004) Analysis of DNA-
phosphate adducts in vitro using miniaturized LC-ESI-MS/MS and column switching:
Phosphotriesters and alkyl cobalamins. J. Am. Soc. Mass Spectr. 15, 593-606.
(35) Conrad, J., Muller, N., and Eisenbrand, G. (1986) Studies on the stability of trialkyl
phosphates and di-(2'deoxythymidine) phosphotriesters in alkaline and neutral solution. A
model study for hydrolysis of phosphotriesters in DNA and on the influence of a beta
hydroxyethyl ester group. Chem. Biol. Interact. 60, 57-65.
(36) Weinfeld, M., Drake, A. F., Saunders, J. K., and Paterson, M. C. (1985) Stereospecific
removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract. Nucleic
Acids Res. 13, 7067-7077.
(37) McCarthy, T. V., and Lindahl, T. (1985) Methyl phosphotriesters in alkylated DNA are
repaired by the Ada regulatory protein of E. coli. Nucleic Acids Res. 13, 2683-2698.
(38) Baker, S. M., Margison, G. P., and Strike, P. (1992) Inducible alkyltransferase DNA repair
proteins in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 20, 645-651.
(39) Marushige, K., and Marushige, Y. (1983) Template properties of DNA alkylated with N-
methyl-N-nitrosourea and N-ethyl-N-nitrosourea. Chem. Biol. Interact. 46, 179-188.
(40) He, C., Hus, J. C., Sun, L. J., Zhou, P., Norman, D. P., Dotsch, V., Wei, H., Gross, J. D., Lane,
W. S., Wagner, G., and Verdine, G. L. (2005) A methylation-dependent electrostatic switch
controls DNA repair and transcriptional activation by E. coli ada. Mol Cell. 20, 117-129.
27
ACS Paragon Plus Environment

Chemical Research in Toxicology
Page 28 of 38
1
2
3
4
5
6
7
8
9
(41) Miller, P. S., Chandrasegaran, S., Dow, D. L., Pulford, S. M., and Kan, L. S. (1982) Synthesis
and template properties of an ethyl phosphotriester modified decadeoxyribonucleotide.
Biochemistry 21, 5468-5474.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(42) Tsujikawa, L., Weinfield, M., and Reha-Krantz, L. J. (2003) Differences in replication of a
DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia
coli DNA polymerase I. Nucleic Acids Res. 31, 4965-4972.
(43) Yashiki, T., Yamana, K., Nunota, K., and Negishi, K. (1992) Sequence specific block of in
vitro DNA synthesis with isopropyl phosphotriesters in template
oligodeoxyribonucleotides. Nucleic Acids Symp. Ser. 27, 197-198.
(44) International Agency for Research on Cancer. (1987) Overall evaluations of
carcinogenicity: An updating of IARC monographs. Suppl.7. IARC, Lyon, FR. p67.
(45) Krasner, S. W., Mitch, W. A., McCurry, D. L., Hanigan, D., and Westerhoff, P. (2013)
Formation, precursors, control, and occurrence of nitrosamines in drinking water: a
review. Water Res. 47, 4433-4450.
(46) Dybing, E., O'Brien, J., Renwick, A. G, and Sanner, T. (2008) Risk assessment of dietary
exposures to compounds that are genotoxic and carcinogenic--an overview. Toxicol. Lett.
180, 110-117.
(47) Pflaum, T., Hausler, T., Baumung, C., Ackermann, S., Kuballa, T., Rehm, J., and
Lachenmeier, D. W. (2016) Carcinogenic compounds in alcoholic beverages: an update.
Arch. Toxicol. 90, 2349-2367.
(48) Zhang, J., Stevens, M. F., and Bradshaw, T. D. (2012) Temozolomide: mechanisms of
action, repair and resistance. Curr. Mol. Pharmacol. 5, 102-114.
28
ACS Paragon Plus Environment

Page 29 of 38
Chemical Research in Toxicology
1
2
3
4
Tables
5
6
Table 1. NNK and NNAL-derived methyl DNA phosphate adducts and their [M + H]+ masses
7
8
9
number of isomers detected
B1pMeB2
[M+H]+
possible
NNK
(S)-NNAL
(R)-NNAL
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A-A
C-C
G-G
T-T
579.1824
531.1599
611.1722
561.1592
555.1711
595.1773
570.1708
571.1661
546.1596
586.1657
2
2
2
1
2
1
2
1
2
2
2
2
2
2
2
2
A-C
A-G
A-T
C-G
C-T
4
4
3
3
4
2
2
2
4
3
3
3
4
2
1
2
4
2
2
2
G-T
total
4
3
3
3
32
23
21
22
29
ACS Paragon Plus Environment