Detection of 7-(2′-Carboxyethyl)guanine but not 7- carboxymethylguanine in human liver DNA

Article abstract of DOI:10.1021/tx100062v

7-Carboxymethylguanine (7-CMGua) and 7-(2′-carboxyethyl)guanine (7-CEGua) are DNA adducts that potentially could be formed upon the metabolism of the carcinogenic nitrosamines N-nitrososarcosine (NSAR) and 3-(methylnitrosamino)propionic acid (MNPA), respectively, or from other sources such as nitrosation of glycine (7-CMGua) or reaction of DNA with acrylic acid (7-CEGua). Since both NSAR and MNPA have been detected in human urine and there are plausible sources of exposure to other precursors to these adducts, we analyzed human liver DNA for 7-CMGua and 7-CEGua, using liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM). Human hepatic DNA was mixed with [ ¹⁵N₅]7-CMGua and [¹⁵N₅]7-CEGua as internal standards and enzymatically hydrolyzed. The hydrolysate was partially purified by solid-phase extraction, and the resulting fraction was treated with acetyl chloride in methanol to convert 7-CMGua and 7-CEGua to their methyl esters. After a second solid-phase extraction, LC-ESI-MS/MS-SRM analysis was carried out using the transitions m/z 224 [M + H]⁺ → m/z 164 [(M + H) - HCOOCH₃]⁺ and m/z 238 [M + H]⁺ → m/z 152 [BH]⁺ for the methyl esters of 7-CMGua and 7-CEGua, respectively. The method was sensitive, accurate, precise, and apparently free from artifact formation. 7-CEGua, as its methyl ester, was detected in all 24 human liver samples analyzed, mean ± SD, 373 ± 320 fmol/μmol Gua (74.6 adducts per 10⁹ nucleotides), range 17-1189 fmol/μmol Gua, but the methyl ester of 7-CMGua was not detected in any sample. These results demonstrate the ubiquitous presence of 7-CEGua in human liver DNA. Acrylic acid may be a likely endogenous precursor to 7-CEGua.

Full text of DOI:10.1021/tx100062v

Chem. Res. Toxicol. 2010, 23, 1089–1096
1089
Detection of 7-(2′-Carboxyethyl)guanine but Not
7-Carboxymethylguanine in Human Liver DNA
Guang Cheng, Mingyao Wang, Peter W. Villalta, and Stephen S. Hecht*
Masonic Cancer Center, UniVersity of Minnesota, MMC 806, 420 Delaware Street SE,
Minneapolis, Minnesota 55455
ReceiVed February 18, 2010
7-Carboxymethylguanine (7-CMGua) and 7-(2′-carboxyethyl)guanine (7-CEGua) are DNA adducts
that potentially could be formed upon the metabolism of the carcinogenic nitrosamines N-nitrososarcosine
(NSAR) and 3-(methylnitrosamino)propionic acid (MNPA), respectively, or from other sources such as
nitrosation of glycine (7-CMGua) or reaction of DNA with acrylic acid (7-CEGua). Since both NSAR
and MNPA have been detected in human urine and there are plausible sources of exposure to other
precursors to these adducts, we analyzed human liver DNA for 7-CMGua and 7-CEGua, using liquid
chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-
ESI-MS/MS-SRM). Human hepatic DNA was mixed with [15N5]7-CMGua and [15N5]7-CEGua as internal
standards and enzymatically hydrolyzed. The hydrolysate was partially purified by solid-phase extraction,
and the resulting fraction was treated with acetyl chloride in methanol to convert 7-CMGua and 7-CEGua
to their methyl esters. After a second solid-phase extraction, LC-ESI-MS/MS-SRM analysis was carried
out using the transitions m/z 224 [M + H]+ f m/z 164 [(M + H) - HCOOCH3]+ and m/z 238 [M +
H]+ f m/z 152 [BH]+ for the methyl esters of 7-CMGua and 7-CEGua, respectively. The method was
sensitive, accurate, precise, and apparently free from artifact formation. 7-CEGua, as its methyl ester,
was detected in all 24 human liver samples analyzed, mean ( SD, 373 ( 320 fmol/µmol Gua (74.6
adducts per 109 nucleotides), range 17-1189 fmol/µmol Gua, but the methyl ester of 7-CMGua was not
detected in any sample. These results demonstrate the ubiquitous presence of 7-CEGua in human liver
DNA. Acrylic acid may be a likely endogenous precursor to 7-CEGua.
Although 88% of NSAR is excreted unchanged in rats (8), its
carcinogenicity strongly indicates that it is metabolized to some
extent. Cytochrome P450 catalyzed metabolism of NSAR by
hydroxylation of its methyl group, a well established and common
metabolic pathway for N-methyl-N-nitroso compounds (9), is
expected to yield the unstable intermediate R-hydroxymethyl
NSAR (2). Intermediate 2 will spontaneously eject formaldehyde
with the formation of the carboxymethyl diazonium ion 3, which
exists in equilibrium with diazotate 4. Multiple studies have
demonstrated that this intermediate, generated from various sources,
is a carboxymethylating agent and reacts with DNA to produce
7-CMGua as well as O6-CMGua (6). Thus, Zurlo et al. demon-
strated that 7-CMGua was formed in pancreatic acinar cells exposed
to azaserine (7) (10). The Shuker group carried out a series of
studies investigating the formation of O6-CMGua. They demon-
strated that the nitrosated bile acid conjugate N-nitrosoglycocholic
acid (8) and related N-nitroso-compounds produced 7-CMGua and
O6-CMGua (as well as O6-methylGua) in vitro (11, 12). They also
showed that O6-CMGua could be formed upon the nitrosation of
glycine (9) in vitro, presumably via intermediate 4, and detected
O6-CMGua in three human blood samples using an immunoslot
blot assay (13). Using immunohistochemistry techniques, they
demonstrated positive staining of exfoliated colonic cells for O6-
CMGua, and this staining was significantly higher in cells from
humans who consumed a high red meat diet (14). Further studies
demonstrated that potassium diazotate, a stable precursor to 4,
produced mutations in the p53 gene in vitro (15).
Introduction
The ability of DNA adducts to induce mutations has been
clearly demonstrated in multiple studies (1). When these
mutations occur in critical regions of important genes such as
ras and p53, the result can be loss of normal cellular growth
control mechanisms, genomic instability, and cancer (2-4). By
characterizing DNA adducts in human tissues and establishing
their miscoding potential, new leads for understanding cancer
etiology can potentially be obtained.
In this study, we focused on the analysis of 7-carboxymeth-
ylguanine (7-CMGua, 5, Scheme 1) and 7-(2′-carboxyethyl)gua-
nine (7-CEGua, 13). These adducts could potentially be formed
from the carcinogenic nitrosamines N-nitrososarcosine (NSAR,
1) and 3-(methylnitrosamino)propionic acid (MNPA, 10). NSAR
causes esophageal tumors in rats, nasal tumors in Swiss mice,
and liver tumors in newborn mice (5). The major sources of
human exposure to NSAR are believed to be consumption of
smoked and cured meats, and endogenous formation (5, 6).
NSAR has been consistently detected in human urine (6). Levels
of NSAR in the urine of healthy individuals averaged 0.42 and
0.94 µg/day in two studies in Germany, 0.36 µg/day in the
United Kingdom, 0.1 µg/day in Poland, 3.4 µg/day in Egypt,
and 0.1-1.5 µg/day in China. Increased levels were found in
regions of high esophageal cancer incidence in China and high
gastric cancer incidence in Poland (6). NSAR is “reasonably
anticipated to be a human carcinogen” according to the U. S.
Department of Health and Human Services (7).
MNPA is a relatively weak inducer of lung adenomas in A/J
mice in the one reported study of its carcinogenicity (16). The
greatest known source of human exposure to MNPA is
* To whom correspondence should be addressed. Phone: (612) 624-7604.
Fax: (612) 626-5135. E-mail: hecht002@umn.edu.
10.1021/tx100062v  2010 American Chemical Society
Published on Web 05/03/2010

1090 Chem. Res. Toxicol., Vol. 23, No. 6, 2010
Scheme 1. Reactions Which Can Produce 7-CMGua (5) and 7-CEGua (13)a
Cheng et al.
a Carboxylic acids are shown in protonated form for consistency with adduct structures 5 and 13 which are detected in this form.
CMGua, 7-CEGua, and [15N5]7-CEG. System 2 used a 4.6 mm ×
25 cm Luna 5 µm C18 column with a gradient from 5 to 40%
CH3OH in H2O over the course of 35 min at a flow rate of 0.7
mL/min. This system was used for the quantitation of dGuo or Gua
in DNA samples. Results from the two methods agreed within 5%.
The Gua values reported here were calculated from measured dGuo.
smokeless tobacco which contains substantial amounts of this
nitrosamine (17). MNPA has been reported in human urine at
levels of 0.1-0.4 µg/day in studies of healthy individuals on
normal diets (18). Its levels were higher in areas of Poland where
inhabitants have a relatively high risk for stomach cancer
compared to low risk areas (18, 19). It is not clear whether the
occurrence of MNPA in human urine is related to tobacco use.
R-Methyl hydroxylation of MNPA would produce intermediate
11, which upon loss of formaldehyde yields the carboxyethyl
diazonium ion 12. This intermediate would be expected to react
with DNA to produce 7-CEGua (13). 7-CEGua has been
previously detected in liver DNA of rats treated with the
hepatocarcinogen 1-nitroso-5,6-dihydrouracil (14) (20). 7-CEGua
is also formed in the reactions of acrylic acid (16) and
ꢀ-propiolactone (17) with DNA (21).
Chemicals, Enzymes, and Chromatography Supplies.
[15N5]dGuo was purchased from Spectra Stable Isotopes (Columbia,
MD). Ethanol was obtained from AAPER Alcohol and Chemical
Co. (Shelbyville, KY). 2-Propanol was procured from Acros
Organics (Morris Plains, NJ). Puregene DNA purification solution
was obtained from Qiagen (Morris Plains, NJ). Oasis MCX 3 cc,
60 mg LP Extraction cartridges were obtained from Waters
(Milford, MA). Strata-X cartridges (33 µm, 30 mg/1 mL) were
obtained from Phenomenex. Alkaline phosphatase was purchased
from Roche Diagnostic Corp. (Indianapolis, IN). N2-(1′-Carboxy-
ethyl)deoxyguanosine was prepared essentially as described (22).
All other chemicals and enzymes were obtained from Sigma-Aldrich
(St. Louis, MO).
Collectively, these data provided a strong rationale for
investigating the presence of 7-CMGua and 7-CEGua in human
DNA. Therefore, we used liquid chromatography-electrospray
ionization-tandem mass spectrometry-selected reaction moni-
toring (LC-ESI-MS/MS-SRM) to analyze human liver DNA for
these adducts.
7-Carboxymethylguanine (7-CMGua, 5) and [15N5]7-CMGua.
7-CMGua was prepared as described by Zurlo et al (10) with minor
modifications. Iodoacetic acid (238.6 mg, 1.3 mmol) and Guo (92.2
mg, 0.33 mmol) were dissolved in 600 µL of H2O containing 16.3
mg of LiOH. The mixture was heated at 100 °C for 70 min. The
color of the solution turned to brownish red. The product was
Experimental Procedures
1
purified using HPLC system 1. H NMR: δ8.25 (bs, 1H, N1H),
HPLC-UV Analysis. This was carried out using Waters As-
sociates (Milford, MA) instruments equipped with a model 996
Photodiode array detector for system 1 and a Shimadzu SPD-10A
UV detector (Shimadzu Scientific Instruments, Columbia, MD) for
system 2. The UV detector was operated at 254 nm. System 1 used
a 10 mm ×25 cm 5 µm Luna C18 column (Phenomenex, Torrance,
CA) with a gradient from 5% CH3OH in 0.35% aq formic acid to
40% CH3OH over the course of 35 min at a flow rate of 3.3 mL/
min. This system was used for collecting 7-CMGua, [15N5]7-
7.66 (s, 1H, C8H), 6.00 (s, 2H, NH2), 4.46 (s, 2H, CH2). Positive
ESI-MS: m/z 210 [M + H]+, MS/MS of m/z 210, m/z 152 [BH]+;
UV λmax 252 nm, λmin 230 nm.
[15N5]7 -CMGua was prepared from [15N5]dGuo using the same
procedure. Positive ESI-MS: m/z 215 [M + H]+, MS/MS of m/z
215, m/z 157 [BH]+.
The concentration of 7-CMGua in standard solutions was
determined by 1H NMR using toluene as internal standard, as

7-(2′-Carboxyethyl)guanine in Human LiVer DNA
Chem. Res. Toxicol., Vol. 23, No. 6, 2010 1091
previously described (23). The concentration of [15N5]7-CMG was
determined by UV at 254 nm.
mM NH4OAc buffer (pH 6.6), and 8 µL aliquots were analyzed
by LC-ESI-MS/MS-SRM.
7-(2′-Carboxyethyl)guanine (7-CEGua, 13) and [15N5]7-
CEGua. 7-CEGua was prepared essentially as described above.
3-Iodopropionic acid (240 mg, 1.2 mmol) and dGuo (80 mg, 0.3
mmol) were dissolved in 1 mL of dimethylacetamide, and the
mixture was stirred at room temperature overnight, then heated at
100 °C for 5 h. The product was purified by HPLC using system
The analysis was carried out with either a Quantum Ultra AM
(with Ion Max ESI source) or a Discovery Max triple quadrupole
mass spectrometer (Thermo Scientific, San Jose, CA) interfaced
with an Agilent Technologies (Palo Alto, CA) 1100 capillary flow
HPLC equipped with a 150 mm × 0.5 mm Zorbax SB C18 column
(Agilent). The column was operated at 50 °C and eluted with a
gradient from 5% to 25% CH3CN in 15 mM NH4OAc buffer (pH
6.6) for 10 min, then a gradient from 25-80% CH3CN in 2 min,
then held at 80% for 6 min, and finally returning to 5% CH3CN in
2 min, at a flow rate of 15 µL/min. The first 2 min of eluant was
directed to waste, and the 2-15 min fractions were diverted to the
ESI source, then the remaining 5 min eluant to waste.
A second HPLC system was used for the confirmation of identity.
A 150 mm × 0.5 mm, 4 µm, Synergi Polar-RP column (Phenom-
enex) was eluted with a gradient from 5 to 35% CH3OH in 15 mM
NH4OAc buffer (pH 6.6) in 35 min at a flow rate of 10 µL/min at
50 °C. The first 15 min of eluant was directed to waste, and the 15
to 35 min fraction was diverted to the ESI source.
The MS parameters were set as follows: spray voltage, 4 kV;
sheath gas pressure, 30; capillary temperature, 250 °C; collision
energy, 22 V; scan width, 0.1 amu; scan time, 0.4 s; Q1 peak width,
0.7; Q3 peak width, 0.7; Q2 pressure, 1.0 mTorr; source CID, 8 V;
and tube lens offset, 95 V. Transitions monitored were as follows,
for the methyl esters of 7-CMGua, [15N5]7-CMGua, 7-CEGua, and
[15N5]7-CEGua, respectively: m/z 224 [M + H]+ f m/z 164
[(M + H) - HCOOCH3]+; m/z 229 [M + H]+ f m/z 169
[(M + H) - HCOOCH3]+; m/z 238 [M + H]+ f m/z 152 [BH]+;
and m/z 243 [M + H]+ f m/z 157 [BH]+.
1
1. H NMR δ 8.23(bs, 1H, N1H), 7.79 (s, 1H, C8H), 6.11 (s, 2H,
NH2), 4.28 (t, 2H, CH2CH2COOH), 2.74 (t, 2H, CH2CH2COOH).
Positive ESI-MS: m/z 224 [M + H]+, MS/MS of m/z of 224, m/z
152 [BH]+; UV λmax 250 nm, λmin 233 nm. [15N5]7-CEG was
prepared from [15N5]dGuo using the same procedure. Positive ESI-
MS: m/z 229 [M + H]+, MS/MS of m/z of 229, m/z 157 [BH]+.
The concentrations of 7-CEGua and [15N5]7-CEGua in standard
solutions were determined by 1H NMR and UV at 254 nm,
respectively.
7-(1′-Carboxyethyl)guanine (18). This was prepared as de-
scribed for 7-CEGua from 2-bromopropionic acid and dGuo
followed by heating at 100 °C for 5 h. Positive ESI-MS: m/z 224
[M + H]+, MS/MS of m/z of 224, m/z 152 [BH]+; UV λmax 250
nm, λmin 233 nm.
Human Liver Samples and DNA Isolation. This study was
approved by the University of Minnesota Research Subjects’
Protection Program Institutional Review Board Human Subjects
Committee. Twenty-four de-identified liver samples were obtained
through The Masonic Cancer Center Tissue Procurement Facility.
The samples were removed during surgery, immediately frozen in
liquid N2, confirmed histopathologically as normal, and stored at
-80 °C.
Calibration curves were constructed before each analysis using
standard solutions of 7-CEGua and [15N5]7-CEGua. A constant amount
of [15N5]7-CEGua (1040 fmol) was mixed with differing amounts of
7-CEGua (20, 40, 80, 160, 320, and 640 fmol), and these were
esterified with CH3COCl and CH3OH and analyzed by LC-ESI-MS/
MS-SRM. Each set of human liver samples contained negative (buffer
blanks) and positive (calf thymus DNA samples) controls.
Accuracy and precision were determined by adding 20, 40, 80,
160, or 320 fmol of 7-CEGua in triplicate to 0.5 mg aliquots of
calf thymus DNA and analyzing each sample in duplicate. The
amount of 7-CEGua which was present in calf thymus DNA (52
fmol/0.5 mg) was subtracted from the amount detected. Recovery
was determined by adding 1040 fmol of [15N5]7-CEGua to 0.5 mg
of calf thymus DNA and processing as described above. The results
were compared to the same analysis without solid-phase extraction.
Effects of N-Acetylcysteine on the Formation of 7-CEGua
in Reactions of Acrylic Acid with dGuo and Calf Thymus
DNA. A mixture of dGuo (30 mg, 110 µmol), acrylic acid (0.32
mg, 4.5 µmol), and N-acetylcysteine (final concentration 0, 1, 2, 5,
or 10 mM) in 15 mL of 0.1 M phosphate buffer, pH 7, was
incubated at 37 °C for 24 h in a shaking water bath. The resulting
mixture was analyzed as described above for 7-CEGua. Reactions
with calf thymus DNA (30 mg) were carried out and analyzed in
a similar fashion.
DNA isolation was performed as previously described with minor
modifications (24). Briefly, the liver samples (0.5 g) were homog-
enized with 15 mL of cell lysis solution. After the samples were
treated with RNase A and protein was precipitated, DNA was
precipitated with 2-propanol. The DNA was dissolved in 4 mL of
10 mM Tris-HCl/1 mM EDTA buffer (pH 7), and the mixture was
extracted twice with CHCl3 containing 4% isoamyl alcohol. The
DNA was precipitated by the addition of 5 M NaCl and ice-cold
ethanol, and washed with 70% and 100% ethanol.
For experiments using N-acetylcysteine to investigate artifactual
formation of 7-CEGua, 10 mM N-acetylcysteine was added to the
cell lysis solution before the tissue was homogenized. The DNA
was isolated as described above.
Analysis of DNA for 7-CMGua and 7-CEGua. The procedure
for enzymatic hydrolysis of DNA was previously described (25).
Briefly, DNA (1 mg) was dissolved in 1 mL of 10 mM Tris-HCl/5
mM MgCl2 buffer (pH 7.0) containing [15N5]7-CMGua (1920 fmol)
and [15N5]7-CEGua (1040 fmol). The DNA was enzymatically
hydrolyzed for 70 min at 37 °C with 1060 units of DNase I (type
II, bovine pancreas), 0.05 units of phosphodiesterase I (type II,
Crotalus adamanteus venom), and 300 units of alkaline phosphatase
(calf intestine). The hydrolysate, after the removal of a 10 µL aliquot
for dGuo quantitation, was purified using a mixed mode cation
exchange (MCX Vac RC, 60 mg, Waters) cartridge. The cartridge
was conditioned with 2 mL of CH3OH and 2 mL of 2% H3PO4.
The sample, which had been acidified with 10 µL of 86% H3PO4,
was loaded, and the column was washed with 2 mL of 0.1% H3PO4
and 2 mL of CH3OH, which were discarded. It was then washed
with 2 mL of 3% NH4OH in CH3OH. This fraction was collected
and evaporated to dryness on a SpeedVac. To the vial was added
1 mL of freshly prepared 10% CH3COCl in CH3OH, and the
mixture was heated for 1 h at 50 °C, then concentrated to dryness.
The residue was dissolved in 1 mL of 15 mM NH4OAc buffer (pH
6.6) and purified using a Strata-X (33 µm, 30 mg/1 mL, Phenom-
enex) solid-phase extraction cartridge. The cartridge was condi-
tioned with 1 mL of CH3OH, 1 mL of H2O, and 1 mL of 15 mM
NH4OAc buffer (pH 6.6). The sample was loaded on the cartridge,
which was washed with 1 mL of 15 mM NH4OAc buffer (pH 6.6),
1 mL of H2O, and 1 mL of 2% aq CH3OH. It was then washed
with 1 mL of 80% CH3OH, and this eluant was collected and
evaporated to dryness. The residue was dissolved in 40 µL of 15
Effects of N-Acetylcysteine on Levels of 7-CEGua in
Human Liver DNA. N-Acetylcysteine (final concentration, 10 mM)
was added to the cell lysis solution before homogenizing the tissue,
and DNA was isolated and analyzed as described above.
Results
The internal standards, [15N5]7-CMGua and [15N5]7-CEGua,
were prepared by reactions of iodoacetic acid and 3-iodopro-
pionic acid, respectively, with [15N5]dGuo, followed by neutral
thermal hydrolysis. We also prepared adducts 18 and 19, which
could have interfered with the analysis of 7-CMGua and
7-CEGua. Adduct 18 was prepared by the reaction of dGuo
with 2-bromopropionic acid followed by neutral thermal hy-
drolysis and adduct 19 by reaction of methylglyoxal with dGuo
(22).

1092 Chem. Res. Toxicol., Vol. 23, No. 6, 2010
Cheng et al.
Since 7-CMGua and 7-CEGua would be released from DNA
by neutral thermal hydrolysis due to the lability of the Gua N9-
deoxyribose 1′ bond, our initial plan was to use neutral thermal
hydrolysis followed by solid-phase extraction to enrich the
adducts. This was not successful because neutral thermal
hydrolysis produced a relatively high background in the LC-
ESI-MS/MS-SRM analysis of these adducts. Experimentation
with calf thymus DNA (which contains 7-CEGua) showed that
enzyme hydrolysis was superior to neutral thermal hydrolysis
or the combination of both methods of hydrolysis in the release
of 7-CEGua from DNA because it produced the highest signal-
to-noise ratio in the LC-ESI-MS/MS-SRM analysis.
Solid-phase extraction of 7-CMGua and 7-CEGua using
mixed mode cation exchange cartridges was successful, but we
were unable to achieve good results with Strata-X reverse-phase
cartridges. Since both enrichment steps were necessary and the
high polarity of the adducts was suspected to be a reason for
their poor retention on Strata-X columns, we converted 7-CM-
Gua and 7-CEGua to their methyl esters by treatment with
CH3COCl and MeOH.
Figure 1. Chromatograms obtained upon LC-ESI-MS/MS-SRM analy-
sis of a hydrolysate of human liver DNA for the methyl esters of
7-CMGua and 7-CEGua. A Zorbax SB C18 column was used for the
analysis. Shaded peaks correspond to the methyl esters of [15N5]7-
CMGua internal standard (panel B); 7-CEGua (panel C); and [15N5]7-
CEGua internal standard (panel D). No peak was observed correspond-
ing to the methyl ester of 7-CMGua (panel A).
The analytical method is summarized in Scheme 2. Human
hepatic DNA and the internal standards were subjected to
enzyme hydrolysis followed by solid-phase extraction using
mixed mode cation exchange cartridges. The adducts were
converted to their methyl esters 20 and 21, which proceeded in
90% yield, and the resulting mixture was purified by solid-phase
extraction on Strata-X cartridges. The appropriate fraction was
collected and analyzed by LC-ESI-MS/MS-SRM at m/z 224
[M + H]+ f m/z 164 [M - HCOOCH3]+ for 20 and m/z 238
[M + H]+ f m/z 152 [BH]+ for 21.
A typical chromatogram obtained upon LC-ESI-MS/MS-SRM
analysis using this method is shown in Figure 1. A clear peak
for 7-CEGua methyl ester 21 was observed in the m/z 238 f
m/z 152 channel, and this peak coeluted with the corresponding
internal standard [15N5]7-CEGua methyl ester, monitored at m/z
243 f m/z 157. In contrast, no peak was observed for 7-CMGua
methyl ester 20, in the m/z 224 f m/z 164 channel. Using a
different LC column (Synergi Polar-RP) to confirm the presence
of 7-CEGua methyl ester 21, a sharp peak corresponding to 21
was observed, and it coeluted with the internal standard (Figure
2). The related standards 18 and 19, as their methyl esters, eluted
at retention times different from those of 21 under the conditions
used in Figure 1: 0.1 min later for 18 and 3.6 min later for 19.
A calibration curve for methyl ester 21 was linear in the range
measured (20-640 fmol); R2 ) 1. The accuracy and precision
of the method were tested by adding various amounts of
7-CEGua to calf thymus DNA and carrying out the analysis of
three aliquots at each added concentration. The results which
are summarized in Table 1 demonstrate outstanding accuracy
Scheme 2. Method of Analysis of Human Liver DNA for 7-CMGua and 7-CEGua

7-(2′-Carboxyethyl)guanine in Human LiVer DNA
Chem. Res. Toxicol., Vol. 23, No. 6, 2010 1093
Table 2. Effects of N-Acetylcysteine on the Formation of
7-CEGua in Reactions of Acrylic Acid (16) with dGuo or DNAa
(A)
reaction of
16 with
N-acetylcysteine
7-CEGua
%
(mM)
(fmol/µmol Gua)b
reduction
dGuo
0
1
2
5
10
5600
4030
3640
1810
1110
28
35
68
80
(B)
N-acetylcysteine
7-CEGua
%
(mM)
(fmol/µmol Gua)c
reduction
DNA
0
5
10
20
1370
308
168
305
77
88
77
a Reactions were carried out with 0.3 mM 16 and dGuo (30 mg) or
calf thymus DNA (30 mg) in 15 mL of 0.1 M phosphate buffer (pH 7)
at 37 °C for 24 h. b dGuo contained 7-CEGua, 947 fmol/µmol Gua. This
amount was subtracted from each value. c Calf thymus DNA contained
7-CEGua, 185 fmol/µmol Gua. This amount was subtracted from each
value.
Figure 2. Chromatograms obtained upon LC-ESI-MS/MS-SRM analy-
sis of a hydrolysate of human liver DNA for the methyl ester of
7-CEGua. A Synergi Polar-RP column was used for the analysis.
Shaded peaks correspond to the methyl esters of (A) 7-CEGua and (B)
[15N5]7-CEGua internal standard.
Table 3. 7-(2′-Carboxyethyl)guanine in Human Liver DNAa
DNA
7-CEGua
sample
gender
age
analyzed (mg)
(fmol/µmol Gua)
Table 1. Relationship between Added and Measured
Amounts of 7-CEGua in Calf Thymus DNA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
F
F
F
M
M
M
F
F
F
F
F
M
F
F
F
F
M
F
F
31
21
61
60
54
54
34
83
30
58
45
18
51
50
51
38
64
43
37
48
64
59
77
38
0.18
0.19
0.84
0.56
0.58
0.16
0.54
0.26
0.12
0.38
0.13
0.11
0.17
0.20
0.95
0.12
0.69
0.06
0.45
0.53
0.42
0.20
0.64
0.22
112
196
246
278
268
296
267
160
177
190
704
1120
769
326
17
accuracy
(%)
precision
(CV, %)
addeda
detectedb,c
20
40
80
160
320
22.3 ( 0.4
40.3 ( 3.5
81.0 ( 5.0
169 ( 3.6
328 ( 4.9
111
100
100
106
103
1.2
8.7
6.2
2.1
1.5
a Added to 0.5 mg calf thymus DNA. b Amount present in calf
thymus DNA was subtracted from each value. 7-CEGua was detected as
its methyl ester 21. c Mean ( SD, N ) 3.
and precision. The on column detection limits for esters 20 and
21 were 10 fmol and 5 fmol, respectively. Recovery was about
50%. The detection limit for 21 in DNA, starting with 1 mg,
was approximately 8 fmol.
1190
39
842
211
128
206
490
311
402
M
M
M
M
M
We considered the possibility that 7-CEGua could be formed
as an artifact by reaction of acrylic acid, a ubiquitous compound,
with DNA during isolation and analysis. We investigated this
problem by first determining the effects of N-acetylcysteine on
the reaction of acrylic acid with dGuo or DNA. As shown in
Table 2, N-acetylcysteine inhibited this reaction, presumably
by scavenging acrylic acid by Michael addition. Maximum
inhibition of about 80% was obtained with 10 mM N-
acetylcysteine. We then analyzed three human liver DNA
samples in the absence and presence of 10 mM N-acetylcysteine,
added at the DNA isolation step. The amounts of 21 detected
in these analyses, with or without N-acetylcysteine addition,
were sample 1, 419 and 414 fmol/µmol Gua; sample 2, 1673
and 1491 fmol/µmol Gua; and sample 3, 177 and 180 fmol/
µmol Gua. These results demonstrate that artifact formation was
not occurring during the isolation of DNA and analysis for
7-CEGua.
a 7-(2′-Carboxyethyl)guanine (7-CEGua) was determined as its methyl
ester 21. Negative controls did not have detectable 21. Positive control
values were 172 ( 42 fmol/µmol Gua in calf thymus DNA samples run
with each set.
detected in any sample. There was no relationship of 7-CEGua
levels to age and gender. No information on tobacco use was
available.
Discussion
We present convincing evidence for the presence of 7-CEGua
in human liver DNA. We monitored the transition m/z 238
[M + H]+ f m/z 152 [BH] +, which is characteristic of our
analyte 21, the methyl ester of 7-CEGua. Our LC-ESI-MS/MS-
SRM chromatograms showed clear peaks which coeluted with
the internal standard [15N5]7-CEGua, as its methyl ester, in two
different LC systems (Figures 1 and 2). We found no evidence
however for the presence of 7-CMGua in these liver DNA
The results of the analyses of 24 human liver DNA samples
are presented in Table 3. 7-CEGua, as its methyl ester 21, was
detected in all of the samples; mean ( SD, 373 ( 320 fmol/
µmol Gua (74.6 adducts per 109 nucleotides) and range 17-1189
fmol/µmol Gua. 7-CMGua, as its methyl ester 20, was not

1094 Chem. Res. Toxicol., Vol. 23, No. 6, 2010
Cheng et al.
samples, as there were no clear peaks eluting at the retention
time of its methyl ester 20.
countries, levels of NSAR reported in human urine are similar
to those of MNPA (6), we might have expected to detect both
7-CMGua and 7-CEGua if these nitrosamines were their sources.
It is possible, however, that NSAR is metabolized to a lesser
extent than MNPA by R-methyl hydroxylation. This requires
further study.
As indicated in Scheme 1, there are several possible sources
of 7-CEGua (13) in human liver DNA. One of the most likely
may be the reaction of acrylic acid (16) with DNA. Acrylic
acid is a metabolite of acrolein in the rat, on the basis of in
vitro studies with rat liver preparations and on in vivo studies
in which the excretion of the mercapturic acid N-acetyl-S-(2-
carboxyethyl)cysteine was observed in the urine of rats treated
with either acrolein or acrylic acid (26-29). Acrolein is a
commonly observed lipid peroxidation product and ubiquitous
environmental pollutant, occurring in various types of engine
exhaust and industrial emissions (30). It is formed in high
temperature cooking and is found in various foods (30, 31).
Acrolein forms exocyclic adducts with dGuo in DNA, and these
have been detected in some human tissues (30, 32-39). Acrylic
acid is widely employed in the production of acrylates which
are used in the manufacturing of polymers, leading potentially
to human exposure (40). Mercapturic acid metabolites of
acrolein and acrylic acid, N-acetyl-S-(3-hydroxypropyl)cysteine
and N-acetyl-S-(2-carboxyethyl)cysteine, are routinely detected
in the urine of nonsmokers (41, 42). Acrylic acid reacts with
DNA in vitro giving 7-CEGua as a product, although the
reaction is slow and has not been reported in vivo (21).
Nevertheless, these data collectively provide a plausible mech-
anism for our finding of 7-CEGua in human hepatic DNA.
A second possible source is 1-nitroso-5,6-dihydrouracil (14).
Favoring this hypothesis is the fact that 7-CEGua has been
detected in the hepatic DNA of rats treated with 14. Therefore,
if there is human exposure to 14, its metabolism to 7-CEGua
in the liver is likely. Human exposure to 14 could potentially
occur by endogenous nitrosation of 5,6-dihydrouracil (22). This
is formed in the metabolism of uracil, catalyzed by dihydro-
pyrimidine dehydrogenase. Subsequent steps in the metabolism
of uracil lead to ꢀ-ureidopropionic acid (23) and finally to
ꢀ-alanine (24). Levels of 22 in the urine of healthy volunteers
averaged 1.41 µmol/mmol creatinine (about 1 mg per day), while
those of 23 and 24 were 2.09 and 0.69 µmol/mmol creatinine,
respectively (43). Levels of 22 in the plasma of healthy subjects
averaged 36 ng/mL plasma (44). Endogenous nitrosation of 22
could potentially occur in situations of high nitrite exposure or
the generation of endogenous NO, but this requires further
investigation (6, 45-47). Nitrosation of 23 and 24 could also
lead to carboxyethyl diazonium ion 12.
In view of the extensive studies on O6-CMGua formation,
we were somewhat surprised to find no evidence for the presence
of 7-CMGua in human liver DNA. In vitro reactions of
N-nitrosoglycocholic acid (8) with DNA produced 7-CMGua
and O6-CMGua in an approximate 10:1 ratio, consistent with
data on other alkylating agents (11, 48). Other studies by Shuker
et al. provide convincing evidence for a number of N-nitroso
compound precursors to O6-CMGua in DNA via diazoacetate
(4), a reaction which also produces O6-methylGua via the
decarboxylation of 4 (12, 13). They also reported the presence
of this adduct in exfoliated colonic cells obtained from
volunteers participating in a diet study to investigate the potential
genotoxic effects of N-nitroso compounds arising from red meat
consumption, an area of research that has been extensively and
convincingly documented by the Bingham group (14, 49, 50).
There are no reports in the literature of O6-CMGua in hepatic
DNA. It is possible that the formation of this adduct is specific
to favorable conditions which exist in the colon. Decarboxylation
of 4 to O6-methylGua might also partially limit the observed
levels of 7-CMGua.
The levels of 7-CEGua found in this study (373 fmol/µmol
Gua) are quite comparable to those of the acetaldehyde DNA
adduct N2-ethylidene-dGuo (534 fmol/µmol dGuo), which we
have previously quantified in human hepatic DNA by LC-ESI-
MS/MS-SRM (51). 7-CEGua levels were substantially higher
than those of 7-ethylGua (42 fmol/µmol Gua) (24), N2-ethyl-
dGuo (12 fmol/µmol dGuo) (51), and the 1,N2-propano-dGuo
adduct of crotonaldehyde and acetaldehyde (ND-15 fmol/µmol
dGuo) (52), all quantified in human liver DNA by LC-ESI-
MS/MS-SRM. The levels reported here are also higher than
those of several endogenous DNA adducts reported in human
hepatic DNA analyzed by various methods (53). These results
suggest that there is a commonly occurring and rich source of
7-CEGua adducts in hepatic DNA.
There are no reports on the mutagenic properties of 7-CEGua.
In general, 7-alkylGua derivatives are not considered to be
mutagenic per se. However, they are prone to depurination,
which can lead to abasic sites in DNA (54). If these are not
repaired, the result can be G f T mutations and other changes
(54, 55).
Our finding of 7-CEGua in human hepatic DNA raises
significant questions regarding its source and the potential role
of its precursors in human carcinogenesis. It will be important
to determine whether acrylic acid, endogenous nitrosation of
5,6-dihydrouracil or glycine, or another mechanism is its main
source. Another consideration, not addressed in this study, is
tobacco use. These questions cannot be answered by assaying
for this adduct in hepatic DNA because liver tissue is not
routinely available. Therefore, we are investigating the detection
of this adduct in human leukocyte DNA or urine.
A limitation of this study is that we do not know to what
extent depurination of 7-CMGua or 7-CEGua may take place
during DNA isolation, before the addition of the internal
standards. If depurination does occur during DNA isolation, the
values reported here would be lower than those that actually
exist in hepatic DNA. A solution to this would be the synthesis
of a DNA strand containing [15N]7-CEdGuo, which could be
added at the beginning of the DNA isolation procedure.
Our original hypothesis for this study pertained to the
nitrosamines NSAR and MNPA. Although MNPA has not been
reported frequently as a constituent of human urine, the levels
reported in the one study carried out in Poland were similar to
those of NSAR and indicate an environmental or endogenous
source of exposure to this nitrosamine in humans (6, 18).
R-Methyl hydroxylation of MNPA, while apparently not
reported in the literature, is certainly a likely route of metabolism
of this nitrosamine. Thus, the generation of carboxyethyldia-
zonium ion 12 in this way is completely reasonable and could
be the source of 7-CEGua in hepatic DNA. Arguing against
this hypothesis, however, is the lack of detection of 7-CMGua
in our samples. Since, in most studies carried out in Western

7-(2′-Carboxyethyl)guanine in Human LiVer DNA
Chem. Res. Toxicol., Vol. 23, No. 6, 2010 1095
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J., Krygier, M., Chmielarczyk, W., and Bartsch, H. (1989) Urinary
excretion of N-nitrosamino acids and nitrate by inhabitants of high-
and low-risk areas for stomach cancer in Poland. Int. J. Cancer 44,
823–827.
(19) Forman, D., Al Dabbagh, S., Knight, T., and Doll, R. (1988) Nitrate
exposure and the carcinogenic process. Ann. N.Y. Acad. Sci. 534, 597–
603.
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and in vivo formation of 7-(2′-carboxyethyl)guanine from the liver
carcinogen 1-nitroso-5,6-dihydrouracil and its reactions with water and
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Kadlubar, F. F., Segerba¨ck, D., and Bartsch, H., Eds.) pp 179-198,
International Agency for Research on Cancer, Lyon, France.
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(23) Sturla, S. J., Scott, J., Lao, Y., Hecht, S. S., and Villalta, P. W. (2005)
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In summary, we report the detection of substantial amounts
of 7-CEGua in human hepatic DNA. The related adduct,
7-CMGua, was not detected. These results provide some new
insights on possible mechanisms of DNA damage in humans.
While our original hypothesis involved the possible production
of these adducts from N-nitroso compounds, our results suggest
that acrylic acid may be a more likely precursor.
Acknowledgment. This study was supported by grant CA-
85702 from the National Cancer Institute. We thank Li Chen
for her contributions to this study and Bob Carlson for editorial
assistance.
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