A new LC-MS/MS method for the quantification of endogenous and vinyl chloride-induced 7-(2-oxoethyl)guanine in Sprague-Dawley rats

Article abstract of DOI:10.1021/tx200447w

Vinyl chloride (VC) is an industrial chemical that is known to be carcinogenic to animals and humans. VC primarily induces hepatic angiosarcomas following high exposures (≥50 ppm). VC is also found in Superfund sites at ppb concentrations as a result of microbial metabolism of trichloroethylene and perchloroethylene. Here, we report a new sensitive LC-MS/MS method to analyze the major DNA adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We used this method to analyze tissue DNA from both adult and weanling rats exposed to 1100 ppm [¹³C₂]-VC for 5 days. After neutral thermal hydrolysis, 7-OEG was derivatized with O-t-butyl hydroxylamine to an oxime adduct, followed by (Figure presented) LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [¹³C₂]-7-OEG from the reaction of calf thymus DNA with [¹³C₁₈]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to 10.0 adducts per 10⁶ guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [¹³C₂]-VC for 5 days was 104.0 ± 23.0 adducts per 10⁶ guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 10 ⁶ guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats, exogenous [ ¹³C₂]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 10⁶ guanine in liver. Studies on the persistence of [¹³C₂]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [¹³C₂]-VC demonstrated a half-life of 7-OEG of 4 days in both liver and lung.

Full text of DOI:10.1021/tx200447w

Article
pubs.acs.org/crt
A New LC-MS/MS Method for the Quantification of Endogenous and
Vinyl Chloride-Induced 7-(2-Oxoethyl)Guanine in Sprague−Dawley
Rats
Esra Mutlu,†,‡ Yo-Chan Jeong,† Leonard B. Collins,† Amy-Joan L. Ham,§ Patricia B. Upton,† Gary Hatch,∥
Darrell Winsett,∥ Paul Evansky,∥ and James A. Swenberg*,†,‡
‡
†Department of Environmental Sciences and Engineering and the Curriculum in Toxicology, Gillings School of Global Public
Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
§Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States
∥NHEERL, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27709, United States
ABSTRACT: Vinyl chloride (VC) is an industrial chemical
that is known to be carcinogenic to animals and humans.
VC primarily induces hepatic angiosarcomas following high
exposures (≥50 ppm). VC is also found in Superfund sites at
ppb concentrations as a result of microbial metabolism of
trichloroethylene and perchloroethylene. Here, we report a
new sensitive LC-MS/MS method to analyze the major DNA
adduct formed by VC, 7-(2-oxoethylguanine) (7-OEG). We
used this method to analyze tissue DNA from both adult
and weanling rats exposed to 1100 ppm [13C2]-VC for 5 days.
After neutral thermal hydrolysis, 7-OEG was derivatized with
O-t-butyl hydroxylamine to an oxime adduct, followed by
LC-MS/MS analysis. The limit of detection was 1 fmol, and the limit of quantitation was 1.5 fmol on the column. The use of
stable isotope VC allowed us to demonstrate for the first time that endogenous 7-OEG was present in tissue DNA. We
hypothesized that endogenous 7-OEG was formed from lipid peroxidation and demonstrated the formation of [13C2]-7-OEG
from the reaction of calf thymus DNA with [13C18]-ethyl linoleate (EtLa) under peroxidizing conditions. The concentrations of
endogenous 7-OEG in liver, lung, kidney, spleen, testis, and brain DNA from adult and weanling rats typically ranged from 1.0 to
10.0 adducts per 106 guanine. The exogenous 7-OEG in liver DNA from adult rats exposed to 1100 ppm [13C2]-VC for 5 days
was 104.0 23.0 adducts per 106 guanine (n = 4), while concentrations in other tissues ranged from 1.0 to 39.0 adducts per 106
guanine (n = 4). Although endogenous concentrations of 7-OEG in tissues in weanling rats were similar to those of adult rats,
exogenous [13C2]-7-OEG concentrations were higher in weanlings, averaging 300 adducts per 106 guanine in liver. Studies on the
persistence of [13C2]-7-OEG in adult rats sacrificed 2, 4, and 8 weeks postexposure to [13C2]-VC demonstrated a half-life of
7-OEG of 4 days in both liver and lung.
proteins.8 Although 7-(2-oxoethyl)guanine (7-OEG) is the
INTRODUCTION
Vinyl chloride (VC) is an industrial chemical that is known
■
major DNA adduct (∼98%)9−13 that arises from the reaction
of CEO with DNA, it has been reported to be devoid of
to be a human and rodent carcinogen that also is found in over
miscoding properties,14 and it is lost primarily by chemical de-
100 Superfund sites as a result of microbial metabolism of
purination. Conversely, exocyclic etheno adducts of VC, N2,
trichloroethylene (TCE) and perchloroethylene (PCE). VC
had been regarded as a relatively nontoxic industrial chemical
until in 1974, the first epidemiology studies of occupationally
3-ethenoguanine (εG), 1,N6-ethenodeoxyadenosine (εdA), and
3,N4-ethenodeoxycytidine (εdC), display clear promutagenic
activity during DNA synthesis.15−17 Because promutagenic
exposed workers identified hepatic angiosarcomas, a rare neo-
plasm in humans.1−3 VC is still widely used in industry for
properties of etheno adducts are known, they have been studied
in more detail than 7-OEG to further characterize their molec-
ular dosimetry, promutagenic properties, and repair.16−18
Low concentrations of endogenous etheno adducts have been
detected in tissues19−25 and are believed to result from lipid
peroxidation and oxidative stress.26−29 In contrast, 7-OEG
the preparation of polyvinyl chloride. While prolonged expo-
sure to high concentrations of VC is known to cause liver angio-
sarcoma,4 it remains unclear why carcinogenesis is primarily
associated with high exposures (≥50 ppm VC). VC is meta-
bolized by CYP450 2E1 to chloroethylene oxide (CEO).5,6
CEO, the ultimate carcinogenic metabolite of VC,7 covalently
binds to DNA and induces four DNA adducts, whereas chloro-
acetaldehyde, a secondary metabolite, reacts primarily with
Received: October 14, 2011
Published: January 2, 2012
© 2012 American Chemical Society
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a
Scheme 1. VC-Induced Major DNA Adduct, 7-OEG
a
In the case of [13C2]-VC exposures, * indicates positions of labeled atoms.
Figure 1. Chromatograms of (A) brain DNA from an adult rat exposed to 1100 ppm [13C2]-VC for 5 days, 6 h per day, and (B) brain DNA from an
unexposed adult rat.
adducts were only known to result from the reaction of CEO
(Scheme 1) or the epoxide of urethane with the nucleophilic
N7 position on dG.
of hydrogen from unsaturated fatty acids to generate a fatty
acid radical. This is followed by a reaction with O2 to give a
peroxy radical (propagation), which continues a chain reaction
until production is stopped by the formation of a nonradical
species. Following oxidation, α,β-unsaturated aldehydes, such as
4-oxo-2-nonenal (ONE), 4-hydroxy-2-nonenal (HNE), malon-
dialdehyde (MDA), and acrolein, form by the decomposition
of hydroperoxides.36,37 Such aldehydes react with DNA and
proteins to induce cell proliferation and apoptosis.36−38 Some
of the LPO-induced DNA adducts are εdA, εG, and pyrimido-
[1,2-a]-purin-10(3H)-one (M1G).39,40
Previously, VC has been shown to be more carcinogenic
in young animals.41−44 Maltoni et al. showed that 1 day old
Sprague−Dawley rats exposed to 6000 or 12000 ppm VC
(4 h/day, 5 day/week, 5 weeks) had about a 50% incidence of
angiosarcoma, whereas 13 week old rats exhibited less than a
10% incidence.41 Drew et al. also reported that rats, mice, and
hamsters exposed to 50−200 ppm VC by inhalation (6 h/day,
5 days/week), regardless of duration of exposure, had a higher
incidence of neoplasms when exposures are started early in
life.42 It was also shown that rat fetuses and neonates have
higher susceptibility to angiosarcomas and hepatocellular
carcinomas.44,45
Reactive oxygen species (ROS), such as super oxide anion
radical (O2−), hydrogen peroxide (H2O2), and hydroxy radical
(·OH), are generated during oxidative stress by biochemical
reactions and cellular functions.30 They are highly reactive with
DNA, protein, lipids, and glycation products and are well-
known to be cytotoxic.31 While ROS are produced as a product
of normal cellular function (i.e., mitochondrial metabolism),
oxidative stress occurs when an imbalance arises between
ROS production and antioxidant capacity, either through excess
ROS production and/or antioxidant depletion. Oxidative stress
can induce oxidative damage in cellular DNA, which if left
unrepaired may induce mutations and, thus, play an important
role in multistage carcinogenesis and progression of cancer.32
Excess free radical formation and oxidative stress are asso-
ciated with various human diseases including a variety of can-
cers, neurodegenerative diseases, aging, and cardiovascular
diseases.33−35
Oxidative degeneration of lipids, also known as “lipid per-
oxidation” (LPO), consists of three major steps: initiation, pro-
pagation, and termination. LPO is first initiated by abstraction
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Figure 2. Chromatograms of (A) liver DNA from an adult rat exposed to 1100 ppm [13C2]-VC for 5 days, 6 h per day, and (B) liver DNA from an
unexposed adult rat.
methanol, and acetic acid were purchased from Thermo Fisher Sci-
entific (Raleigh, NC), ethylene oxide, O-t-butyl hydroxylamine, RNase A,
CtDNA, and dG were purchased from Sigma Aldrich (St. Louis, MO),
and t-butyl hypochlorite was purchased from TCI America (Portland,
OR). Nucleic acid purification grade lysis buffer, protein precipita-
tion solution, and proteinase K were purchased from Qiagen
(Valencia, CA).
Animal studies also indicated that young animals are more
susceptible than adults to the formation of DNA adducts by VC
exposures.46−48 It was previously reported that following VC
exposure via inhalation (600 ppm, 4 h/day, 5 days), 7-OEG and
εG concentrations in 10 day old rats were ∼4-fold higher than
lactating rats.46 Age-dependent differences in the formation of
εG by VC exposure were also studied by Morinello et al.47 The
concentration of εG in hepatocytes from weanling rats exposed
to 1100 ppm VC (6 h/day, 5 days/week, 1 week) was reported
to be ∼1.8-fold greater than that found in adult rats.47
Several methods were developed to measure 7-OEG induced
by VC in mice and rats.12,13,49,50 Fedtke et al. used HPLC to
determine the formation and persistence of 7-OEG in female
rats exposed to 600 ppm VC.9,10,51 The half-life of 7-OEG in
these animals was reported as 62 h, which was shorter than
found for the etheno adducts. The concentration of 7-OEG was
also measured by LC-MS/MS after the reduction of 7-OEG to
7-(2-hydroxyethyl)guanine (7-HEG) with NaCNBH3.47 In that
study, 7-OEG was detectable in hepatocytes but not adjoining
nonparenchymal cells.
In the present study, we developed a new highly sensitive and
specific LC-MS/MS method to analyze 7-OEG in tissues from
both adult and weanling male rats exposed to 1100 ppm [13C2]-
VC for 5 days and 1100 ppm VC for 1 day. Because of the
increased sensitivity of the new assay and the use of stable
isotope-labeled VC, we were able to detect and quantify endo-
genous 7-OEG in vivo in tissues of control rats and [13C2]-VC-
exposed rats (Figures 1 and 2). We hypothesized that the
source leading to the formation of 7-OEG was LPO and pre-
sent data to support our hypothesis. The reaction of calf thy-
mus DNA (CtDNA) with [13C18]-ethyl linoleate (EtLa) under
peroxidizing conditions resulted in the formation of [13C2]-
7-OEG,52 which could be quantified by LC/MS-MS (Figure 3).
In addition to quantitating endogenous and exogenous 7-OEG
after [13C2]-VC exposures, the half-life of [13C2]-7-OEG in liver
and lung was determined.
Synthesis of 7-OEG Analyte and Internal Standards. 7-OEG
was synthesized from the reaction of CEO with dG. CEO was syn-
thesized as previously described by Holt et al.53 by photochlorination
of ethylene oxide with t-butyl hypochlorite. After distillation of the
crude product, the exact composition of the collected product was
verified by 1H NMR. A solution of dG (20 mM) in water was reacted
with excess CEO in a sealed tube at room temperature for 15 min and
then at 37 °C for an additional 15 min. After hydrolysis with 0.1 N HCl at
70 °C for 40 min, 7-OEG standard was purified by HPLC. Chroma-
tography was performed on a RP-18 (15 mm × 3.2 mm) precolumn
(Brownlee Laboratories) attached to RP-SCX column (ES Industries,
Chromega column, 250 mm × 4.6 mm, 5 μm) with a flow rate of
1.5 mL/min using a linear gradient program from 20 to 100% B in
20 min [A, 75 mM ammonium formate/10% acetonitrile (ACN), pH 2.8,
and B, 250 mM ammonium formate/10% ACN, pH 2.8).49
7-OEG analyte standard (AST) was collected at ∼7 min and quan-
tified by fluorescence measurement with excitation wavelength of 255 nm
and a 340 nm emission cutoff filter. Isotopically labeled 7-OEG internal
standard (IST) also was synthesized from the reaction of [15N5]-dG with
CEO as described and purified by the same procedure as for AST.
Animals, Exposure, and Tissue Collection. Inhalation expo-
sures were conducted at the U.S. EPA NHEERL facility in Research
Triangle Park, NC. All procedures that involved the use of animals
were approved by the U.S. EPA Institutional Animal Care and
Use Committee. Ten week old (300−325 g) adult and 21 day old
(50−60 g) weanling male Sprague−Dawley rats were purchased from
Charles River Laboratories (Raleigh, NC). The rats were acclimated
for 1 week and housed in stainless steel cages with a 12 h light/dark
cycle. Four adult and eight weanling animals per group were ex-
posed to 1100 ppm VC in a nose-only inhalation apparatus for 1 day
(6 h/day) or to 1100 ppm [13C2]-VC (6 h/day) for 5 days. The VC
vapor was generated by metering pure VC or [13C2]-VC vapor into a
mixing chamber with medical grade air using mass flow controllers
(Tylan Instruments, Torrance, CA). VC vapor was delivered to the
inlet of the nose-only chamber (CH Technologies, Westwood, NJ)
after the concentration was determined using a Miran 1A infrared
gas analyzer (Foxboro, MA), which was calibrated with VC (Scott Gas,
99.96% purity). Temperature and relative humidity were monitored
during the exposures using an Omega model RH411 Thermo-
Hygrograph (Stamford, CT) and were within the acceptable limits for
nose-only exposures.
MATERIALS AND METHODS
■
Caution: VC is a known carcinogen and should be handled carefully in
an operating fume hood with protective equipment (i.e., gloves and
laboratory coat).
Chemicals. [13C2]-VC (≥98% chemical purity; 99% isotopic
purity) and [15N5]-dG (98% isotopic purity) were obtained from
Cambridge Isotope Laboratories (Andover, MA). HPLC grade water,
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Figure 3. Chromatograms of (A) CtDNA reacted with [13C18]-EtLa for 89 h and (B) control CtDNA.
At the end of exposure, the rats were anesthetized with Euthasol
by intraperitonial injection and euthanized by exsanguination via the
vena cava. The liver, lung, kidney, spleen, testis, and brain were
removed, frozen on dry ice, and stored at −80 °C. Following 5 days of
[13C2]-VC exposure, four adults and all eight weanling rats were
euthanized within 2 h of the completion of exposure. Three additional
groups of four adults were euthanized 2, 4, or 8 weeks later to deter-
mine the half-life of [13C2]-VC DNA adducts.
DNA Isolation. DNA isolation from tissues was performed as pre-
viously described22 with minor modifications using the Gentra Systems
DNA extraction kit. The homogenized tissues were incubated in lysis
buffer with the addition of 20 mM 2,2,6,6-tetramethyl-1-piperidinyloxy
(TEMPO), followed by protein precipitation solution. After protein
precipitation, the DNA/RNA mixture was precipitated by isopropanol.
The DNA/RNA pellet then was resuspended in lysis buffer containing
TEMPO and incubated with RNase A at 37 °C for 30 min, which was
then followed by protein and DNA precipitation. The DNA pellet was
resuspended in distilled water containing 1 mM TEMPO and stored
at −80 °C until analysis. DNA was quantified by a Biomate UV
spectrophotometer (Thermo Scientific) at A260 and calculated as
1 AU = 50 μg/mL.
DNA Hydrolysis. DNA solutions (50 μg) were spiked with the IST
(640 fmol, [15N5]-7-OEG) and diluted to 500 μL with HPLC water.
Samples were incubated at 100 °C for 30 min. Immediately after
incubation, samples were cooled on ice. The DNA backbone was
separated by Microcon 10 filtration (11500 rpm, 4 °C, 40 min),
after which the filtrate was placed in a separate tube, and 400 μL of
HPLC water was added to the retentate to wash the DNA back-
bone (11500 rpm, 4 °C, 40 min), followed by combining both filtrates.
Four mM O-t-butyl hydroxylamine (O-tBHA) (50 μL) and methanol
(100 μL) were added to each DNA filtrate, incubated at 45 °C for
30 min, and dried by vacuum evaporation in a SpeedVac concentrator.
The dried fractions were transferred to glass autosampler vials by
rehydration with HPLC water (3 × 100 μL), dried under vacuum, and
then diluted in 50 μL of HPLC water to be analyzed by LC-MS/MS.
Positive CtDNA controls (with and without O-tBHA) and negative
reagent-only controls (with and without O-tBHA) were also prepared
as described above. These samples proved no artificial contribution to
the exogenous mass transitions, and endogenous 7-OEG was present
only in samples containing DNA.
volume of 5 μL. Samples were kept at 4 °C in the autosampler during
analysis. Separation was performed on a Thermo Scientific Aquasil
C18 column (150 mm × 1 mm, 5 μ) with a flow rate of
0.120 mL/min using the following gradient: (A) 0.1% acetic acid in
water and (B) acetonitrile; 0−1.5 min, 5% B; 1.5−3 min, 30% B;
3−6 min, 90% B; 6−7 min, 5% B; and 7−12 min, 5% B.
Instrument conditions were optimized for maximum signal of
7-OEG by direct infusion of AST and IST. MS settings were as
follows: electrospray voltage, 3000 V; capillary temperature, 285 °C;
HESI temperature, 150 °C; sheath and auxiliary gas pressures, 30 and
20 arbitrary units; collision energy, 20 V; and Q2 collision gas pres-
sure, 1.5 mTorr. Calibration curves using O-tBHA derivatized 7-OEG
AST and [15N5]-7-OEG IST in HPLC water were generated by
diluting from derivatized stock standards with each sample set. Stan-
dard curves were calculated using the peak area ratio of 7-OEG-tBHA
to IST versus fmol of 7-OEG-tBHA injected (Figure 5). The amounts
of both 7-OEG and exogenously derived [13C2]-7-OEG in each sample
were calculated from the ratio of the AST and IST peak areas.
Injection of a high amount of [15N5]-7-OEG-tBHA IST (>3 pmol)
showed no contribution to either the endogenous or the exogenous
mass transitions. The assay performance was evaluated by adding
known amounts of 7-OEG to freshly isolated CtDNA. DNA was
quantified by UV before spiking 50 μg aliquots with 3.2, 32, and
64 fmol of 7-OEG and 640 fmol of IST. Sample sets were assayed on
two different days by separate analysts, and results are summarized in
Table 1. The endogenous 7-OEG concentration found in CtDNA was
determined to be 2.4 0.8 fmol (n = 5) per 50 μg of DNA.
Reaction of [13C18]-EtLa with CtDNA. The reaction of [13C18]-
EtLa with CtDNA was carried out according to a previously published
method29 with minor modifications. Ten milligrams of [13C18]-EtLa
(100 μL, 100 mg/mL in methanol) was added to 300 μg of CtDNA in
1 mL of 50 mM sodium phosphate buffer (pH 7.4). After the addition
of 10 μL of t-butylhydroperoxide (70% solution, 70 μmol), the mixture
was incubated at 37 °C for 24 h. After the addition of 1 mL of water,
10 μL of 2% butylated hydroxytoluene in isopropanol (w/v), and
0.5 mL of methanol to the reaction mixture, the sample was extracted
two times with 2 mL of chloroform containing 0.5% t-butylhydroper-
oxide (w/v); the aqueous layer was dried under vacuum, and the
sample was resuspended in 1 mL of water. Samples were left at 4 °C
overnight to rehydrate and stored at −80 °C until analysis.
LC-MS/MS Analysis. Quantitative LC-MS/MS data were obtained
using a Thermo Finnigan TSQ Quantum Ultra triple-quadrupole mass
spectrometer equipped with Waters Acquity UPLC. A heated electro-
spray ionization (HESI) interface was operated in positive selected
reaction monitoring (SRM) mode. The transitions monitored were
m/z 265 → 152 for 7-OEG-tBHA (Figure 4), m/z 267 → 152 for
13C2-7-OEG-tBHA, and m/z 270 → 157 for [15N5]-7-OEG-tBHA. The
method gave a detection limit of 1 fmol on the column using an injection
RESULTS AND DISCUSSION
■
Although 7-OEG lacks miscoding properties, it is the major
DNA adduct formed by VC and is therefore an important
biomarker for VC exposure. This paper details a new approach
to determine the formation and persistence of 7-OEG. We
developed a sensitive LC-MS/MS analysis to determine 7-OEG
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Table 1. Intraday and Interday Assay Variability Summarized
for 50 μg Aliquots of CtDNA Spiked with Increasing
a
Amounts of 7-OEG
fmol of 7-OEG fmol of 7-OEG
fmol of 7-OEG combined interday
spiked
found day 1 (n = 5) found day 2 (n = 5) results (n = 10)
3.2
32
7.9 3.3
34.8 7.3
70.4 10.3
7.4 3.3
38.3 7.7
69.0 9.2
7.6 3.1
36.6 7.1
70.0 9.2
64
a
Data are presented as the mean fmol found per sample standard
deviation. Amounts measured in this standard deviation are the com-
bination of the endogenous concentration and spiked concentration
7-OEG.
concentration of [13C2]-7-OEG detected in DNA from adult
rats exposed to 1100 ppm [13C2]-VC for 5 days was found in
liver, with 104.0 23.0 adducts per 106 guanine. Lung, kidney,
and spleen had the next highest concentrations of [13C2]-7-
OEG with 39.0
2.0, 28.0
7.0, and 10.0
2.0 adducts
0.3) and testis
per 106 guanine, respectively. Brain (3.0
(1.0 0.1) had the lowest number of [13C2]-7-OEG adducts
per 106 guanine.
Figure 4. Product ion spectrum of 7-OEG-tBHA (precursor ion
m/z 265). The spectrum was obtained by injecting 6.4 pmol of
7-OEG-tBHA on the LC system, selecting m/z 265 in Q1, and
scanning Q3 from m/z 50 to 300. The argon collision gas pressure was
1.5 mTorr, and the collision energy was 20 eV.
We found that the concentrations of exogenous 7-OEG
adducts were ∼50-fold higher than endogenous 7-OEG in adult
livers and ∼300-fold higher in weanling rat livers. The endog-
enous 7-OEG adduct concentrations in target tissues in adult
and weanling rats showed no significant difference. Therefore,
we conclude that there is no effect of age on the endogenous
7-OEG formation. The ratio of exogenous/endogenous 7-OEG
in adult rats was ∼200-fold in lung, ∼40-fold in kidney, ∼4-fold
in brain, and ∼1.7-fold higher in spleen, while in testis it was
∼0.1-fold.
The formation of [13C2]-7-OEG in liver of weanling rats
was ∼3-fold greater than adult rats, with 299.0 129.0 adducts
per 106 guanine. This is thought to be due to age-related dif-
ferences in metabolism due to greater CYP2E1 activity in wean-
ling rats. A recent study has shown up to 2-fold higher CYP2E1
activity in 3 week old rat liver.54 In comparison to the adult
rats, weanling rats had [13C2]-7-OEG concentrations in lung,
kidney, and spleen that were approximately 2-fold higher than
adults (Tables 2 and 3). Three-fold higher [13C2]-7-OEG
was also detected in brains of weanling rats exposed to 1100 ppm
[13C2]-VC for 5 days, as compared to the exogenous
concentrations found in similarly exposed adult rats, but was
still 30 times lower than weanling liver. The lowest exogenous
7-OEG was detected in spleen for both weanling and adult rats,
which might suggest that P450 metabolism of VC in this tissue
is minimal. In contrast, endogenous 7-OEG formation was
highest in spleen and testis for both adult and weanling rats as
compared to other tissues.
The persistence of 7-OEG was also determined in adults rats
euthanized 2, 4, and 8 weeks after exposure to [13C2]-VC for
5 days (Table 2). The half-life of [13C2]-7-OEG was calculated
in liver and lung DNA by using the plot of log (adducts) versus
days (Figure 6). The half-life of [13C2]-7-OEG in liver and
lung was ∼4 days, which is consistent with other studies on
N7-guanine adducts. This is thought to primarily represent
chemical depurination.
Figure 5. Calibration curve for 7-OEG-tBHA obtained under SRM
conditions using the present LC-MS/MS method. The plot shows the
peak area ratio of 7-OEG-tBHA to IST vs increasing fmol of 7-OEG-
tBHA. Data points correspond to standard mixtures that give 1.6, 3.2,
6.4, 16, 32, 64, 160, 320, and 640 fmol of 7-OEG-tBHA and 640 fmol
of [15N5]-7-OEG-tBHA on column.
concentrations in tissue DNA of male Sprague−Dawley rats
exposed to 1100 ppm VC for 1 day or [13C2]-VC for 5 days,
including postexposure periods of 2, 4, or 8 weeks. The new
7-OEG assay utilized an oximation reaction with O-tBHA,
which allowed us to measure 7-OEG as a more stable oxime
adduct. Using this new highly sensitive assay, we determined
that 7-OEG concentrations were ∼200-fold higher than εG in
liver of adult rats similarly exposed to VC.48 Laib et al. reported
similar results for adduct formation in liver of rats exposed to
VC (1:100 ratio).12
The exposure of adult and weanling rats to 1100 ppm [13C2]-
VC for 1 week induced [13C2]-7-OEG in liver, lung, kidney,
spleen, brain, and testis as summarized in Table 2. Stable
isotope-labeled VC exposures allowed us to distinguish 7-OEG
from [13C2]-7-OEG and demonstrated that 7-OEG is formed
endogenously. This is clearly shown in Figures 1 and 2, which
contrast chromatograms from an unexposed control animal to
one subjected to 5 days of [13C2]-VC exposure. The greatest
Previously, 7-OEG formation was determined by Fedtke et al.
in rats exposed to 600 ppm VC (1 week).10 DNA (1−2 mg)
was depurinated by acid hydrolysis and analyzed by HPLC
using fluorescence detection with a limit of detection 10 pmol
7-OEG per μmol guanine. 7-OEG was shown to be 144 times
higher than εG. While the data obtained from these studies
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Table 2. 7-OEG (Endogenous) and [13C2]-7-OEG (Exogenous) Adduct Concentrations Determined from Adult (n = 4) Rats
Exposed to 1100 ppm [13C2]-VC for 5 Days and Cage Controls
adult postexposure time
adult control
2 h
2 weeks
4 weeks
8 weeks
7-OEG 13C2-7-OEG
7-OEG
13C2-7-OEG
7-OEG 13C2-7-OEG 7-OEG 13C2-7-OEG 7-OEG 13C2-7-OEG
liver (add/106 gua)
lung (add/106 gua)
kidney (add/106 gua) 1.2 0.4
brain (add/106 gua) 3.0 2.0
spleen (add/106 gua) 3.2 1.5
testis (add/106 gua) 2.3 1.5
1.3 0.5
0.6 0.1
−
−
−
−
−
−
2.0 1.0
0.2 0.1
0.7 0.4
0.8 0.5
6.0 4.0
9.0 4.0
104.0 23.0 1.0 0.3 4.0 3.0 1.0 0.4
1.0 0.6
0.3 0.1
ND
1.0 0.7
0.8 0.4
1.5 0.6
NA
ND
ND
ND
NA
NA
NA
39.0 2.0
28.0 7.0
3.0 0.3
10.0 2.0
1.0 0.1
0.4 0.1 2.0 0.1 0.2 0.1
0.4 0.2 1.0 0.6 0.2 0.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Table 3. 7-OEG (Endogenous) and [13C2]-7-OEG
was only detectable in hepatocyte DNA at 4
0.9 adducts
per 106 guanine.47 7-OEG was measured as 7-HEG after Na-
CNBH3 reduction and analyzed by LC-MS/MS with a limit of
detection 0.3 adducts per 106 guanine. Because only DNA of
unlabeled VC exposed rats was analyzed, no distinction was
made between endogenous and exogenous 7-OEG formation.
7-OEG concentrations reported by Morinello et al.47 were
∼25-fold lower than exogenous 7-OEG concentrations that
we report in this study (Table 2). Because the sufficiency of
NaCNBH3 reduction of 7-OEG is not known, we suggest that
incompletion of the reduction of 7-OEG in DNA might be an
important factor for the lower 7-OEG concentration found in
that study. For comparison reasons, we also analyzed lung,
kidney, and brain DNA of rats exposed to [13C2]-VC from
Morinello et al. study.47 Endogenous and exogenous 7-OEG
concentrations in those samples were very similar to the con-
centrations reported in this study (Table 4).
(Exogenous) Adduct Concentrations Determined from
Weanling (n = 8) Rats Exposed to 1100 ppm [13C2]-VC for
5 Days and Cage Controls
weanling control
7-OEG 13C2-7-OEG 7-OEG
weanling 2 h postexposure
13C2-7-OEG
1.0 0.5 299.0 129.0
liver (add/106 gua)
lung (add/106 gua)
0.8 0.1
0.5 0.1
kidney (add/106 gua) 2.7 1.0
brain (add/106 gua) 5.0 2.0
spleen (add/106 gua) 3.8 1.1
testis (add/106 gua) 1.0 0.6
−
−
−
−
−
−
0.4 0.1
0.8 0.3
1.0 0.3
7.0 5.0
6.0 0.2
83.0 19.0
66.0 12.0
10.0 0.8
16.0 2.0
1.2 0.1
Table 4. 7-OEG (Endogenous) and [13C2]-7-OEG
(Exogenous) Adduct Concentrations Determined from
a
Adult Rats Exposed to 1100 ppm [13C2]-VC for 5 Days
adult control
7-OEG
13C2-7-OEG
NA
adult 2 h postexposure
7-OEG
13C2-7-OEG
lung (n = 8)
−
−
−
0.6 0.2
31.0 11.0
(add/106 gua)
kidney (n = 7)
NA
0.8 0.4
1.0
22.0 3.0
2.0
(add/106 gua)
brain (n = 3, 1)* 5.0 1.0
Figure 6. Plot of log (7-OEG/108 gua) vs days was used to determine
the half-life of 7-OEG in the liver and lung of adult rats exposed to
[13C2]-VC (1100 ppm, 5 days, 6 h per day) and euthanized at the end
of exposure or 2, 4, or 8 weeks later. The half-life of 7-OEG in liver
and lung is 4 days on the basis of the equations.
(add/106 gua)
a
Tissues were analyzed from Morinello et al.47 *Brain control, n = 3;
brain exposed, n = 1.
The data obtained from CtDNA, control, and [13C2]-VC-
exposed animals demonstrated that 7-OEG is formed endog-
enously in relatively high concentrations in comparison to other
endogenous N7-guanine adducts. While endogenous con-
centrations of 7-HEG in tissues from control rats and mice
were reported to range from 1 to 4 adducts per 108 nucleo-
tides,55,56 endogenous N7-methylguanine ranged from 2 to
2.5 adducts per 107 nucleotides.57
On the basis of previous data showing the formation of
etheno DNA adducts by LPO, we hypothesized that endogenous
7-OEG was also formed as a result of LPO.52 Previously, the
key LPO intermediates that have been identified for the for-
mation of DNA adducts19,58−60 are the result of reactions of
HNE, ONE, and MDA with dG and are associated with DNA
adducts formed by VC exposure.58
gave important information on DNA adduct formation from
VC exposures, it lacked the sensitivity of the present LC-MS/
MS assay. Using our mass spectrometry method, we were able
to quantify DNA adducts by using a stable isotope-labeled
7-OEG IST, while in the previous HPLC method, adducts were
quantified by external calibration using CtDNA spiked with
7-OEG. In the previous studies, neither endogenous 7-OEG in
control samples, nor exogenous 7-OEG in brain and spleen of
exposed weanling rats could be detected. In comparison, the
adduct ratio of 7-OEG was 1:1 in lung of adult and weanling
rats, whereas in liver it was 4-fold higher in weanlings. The half-
life of 7-OEG was also calculated as 63 h, while in the present
study, it was ∼96 h.10
Morinello et al. also examined 7-OEG concentrations in
hepatocyte and brain DNA of rats exposed to 1100 ppm VC for
1 week (6 h/day, 5 days/week) and demonstrated that 7-OEG
Previously, the lack of evidence for endogenous 7-OEG for-
mation did not suggest a rationale for studying its formation
396
dx.doi.org/10.1021/tx200447w | Chem. Res. Toxicol. 2012, 25, 391−399

Chemical Research in Toxicology
Article
background amounts will always be present. The new data sup-
port the carcinogenesis and epidemiology findings that VC is
causally associated with cancer following relatively high expo-
sures. In contrast, the number of endogenous adducts is greater
than identical exogenous adducts formed at low exposures.
In summary, we developed a new sensitive, selective, and effi-
cient LC-MS/MS method for quantification of 7-OEG in DNA.
For the first time, endogenous 7-OEG was detected in control
rats and [13C2]-VC exposed rats, and its endogenous formation
from LPO was confirmed in vitro in CtDNA. The half-life of
7-OEG in liver and lung was determined to be ∼4 days.
Table 5. 7-OEG (Endogenous + Exogenous) Adduct
Concentrations Determined from Weanling (n = 8) and
Adult (n = 4) Rats Exposed to 1100 ppm VC for 1 Day
7-OEG
liver (add/ lung (add/ kidney (add/ brain (add/
106 Gua)
106 Gua)
106 Gua)
106 Gua)
1.5 0.6
weanling 1 day
exposure
84.0 47.0 37.0 11.0 28.0 12.0
adult 1 day
exposure
64.0 49.0 28.0 6.0 19.0 3.0
1.6 0.5
in vitro and/or in vivo. The detection of endogenous 7-OEG in
the present study (Figures 1 and 2) prompted us to examine
its route of formation. We tested the reaction of CtDNA with
[13C18]-EtLa under peroxidizing conditions.52 The formation
of 7-OEG by direct alkylation was evaluated by monitoring
the incorporation of the 13C-labeled stable isotope from
EtLa into 7-OEG (Figure 3). Previously, Chung et al.61 and
Ham et al.52 had demonstrated the formation of etheno adducts
1,N2-ethenoguanine, 1,N6-ethenodeoxyadenine, and εG as a re-
sult of the reaction between dG with HNE, CtDNA with HNE
and EtLa under peroxidation conditions.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 919-966-6139. Fax: 919-966-6123. E-mail: jswenber@
email.unc.edu.
Funding
We acknowledge financial support from NIH Grants R42-
ES0011746, T32-ES07126, and P30-ES10126.
ACKNOWLEDGMENTS
■
We thank the American Chemistry Council for providing the
[13C2]-VC.
7-OEG formation in adult and weanling rats exposed to
1100 ppm VC for 1 day was also determined (Table 5). In adult
and weanling rats exposed to unlabeled VC, the concentration of
7-OEG adduct was 64.0 49.0 and 84.0 47.0 in liver, 28.0
6.0 and 37.0 11.0 in lung, 19.0 3.0 and 28.0 12.0 in kidney,
ABBREVIATIONS
■
VC, vinyl chloride; CEO, chloroethylene oxide; 7-OEG, 7-(2-
oxoethyl)guanine; εG, N2,3-ethenoguanine; εdA, 1,N6-etheno-
deoxyadenosine; εdC, 3,N4-ethenodeoxycytidine; ROS, reactive
oxygen species; LPO, lipid peroxidation; ONE, 4-oxo-2-nonenal;
HNE, 4-hydroxy-2-nonenal; MDA, malondialdehyde; M1G,
pyrimido[1,2-a]-purin-10(3H)-one; 7-HEG, 7-(2-hydroxyethyl)-
guanine; CtDNA, calf thymus DNA; EtLa, ethyl linoleate;
AST, analyte standard; IST, internal standard; TEMPO, 2,2,6,6-
tetramethyl-1-piperidinyloxy; O-tBHA, O-t-butyl hydroxylamine;
HESI, heated electrospray ionization; SRM, selected reaction
monitoring
1.6
0.5 and 1.5
0.6 adducts per 106 guanine in brain,
respectively. These animals were exposed to VC to test the nose-
only inhalation apparatus before starting [13C2]-VC exposures but
provided additional information about the ef-
fect of age, tissue, and duration of exposure on 7-OEG for-
mation. The age-dependent formation of 7-OEG in liver from
weanling rats exposed to 1100 ppm VC (1 day) was ∼1.3-fold
greater than the concentrations in liver of adult rats exposed
to 1100 ppm VC (1 day). The concentration of 7-OEG in
lung, kidney, and brain from weanling rats was similar to adult
rats exposed for the same duration. While 7-OEG adducts in liver,
lung, and kidney were higher than control animals for both adult
and weanling rats, in brain, it was similar to that of control
animals. These data, however, reflect a mixture of endogenous
and exogenous adducts, as no distinction between the two could
be made.
In this study, we determined the concentration of 7-OEG in
liver, lung, kidney, spleen, testis, and brain DNA from animals
exposed to VC and [13C2]-VC and demonstrated the presence
of endogenous 7-OEG in brain, liver, lung, kidney, spleen,
and testis. The persistence of 7-OEG in liver, lung, kidney,
and spleen was also demonstrated in this study. The molecular
dosimetry data suggest that not only liver but also other organs
such as lung and kidney might be considered target organs
for VC-induced carcinogenesis following inhalation exposure.
Because of the reactivity and short half-life of CEO, 1.6 min in
aqueous solution at neutral pH,62 we hypothesize that VC is
metabolized in each organ by P450 rather than arising via the
circulation of CEO in the body. The lower exogenous adduct
level in brain, spleen, and testis supports this.
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