Interindividual Differences in DNA Adduct Formation and Detoxification of 1,3-Butadiene-Derived Epoxide in Human HapMap Cell Lines

Article abstract of DOI:10.1021/acs.chemrestox.9b00517

Smoking-induced lung cancer is a major cause of cancer mortality in the US and worldwide. While 11-24% of smokers will develop lung cancer, risk varies among individuals and ethnic/racial groups. Specifically, African American and Native Hawaiian cigarette smokers are more likely to get lung cancer as compared to Caucasians, Japanese Americans, and Latinos. It is important to identify smokers who are at the greatest risk of developing lung cancer as they should be candidates for smoking cessation and chemopreventive intervention programs. Among 60+ tobacco smoke carcinogens, 1,3-butadiene (BD) is one of the most potent and abundant (20-75 μg per cigarette in mainstream smoke and 205-361 μg per cigarette in side stream smoke). BD is metabolically activated to 3,4-epoxy-1-butene (EB), which can be detoxified by glutathione S-transferase theta 1 (GSTT1)-mediated conjugation with glutathione, or can react with DNA to form 7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) adducts. In the present study, we employed EBV-transformed human lymphoblastoid cell lines (HapMap cells) with known GSTT1 genotypes to examine the influence of the GSTT1 gene on interindividual variability in butadiene metabolism, DNA adduct formation/repair, and biological outcomes (apoptosis). We found that GSTT1- HapMap cells treated with EB in culture produced lower levels of glutathione conjugates and were more susceptible to apoptosis but had similar numbers of EB-GII adducts as GSTT1+ cells. Our results suggest that GSTT1 can influence an individual's susceptibility to butadiene-derived epoxides.

Full text of DOI:10.1021/acs.chemrestox.9b00517

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Article
Interindividual Differences in DNA Adduct Formation and
Detoxification of 1,3-Butadiene-Derived Epoxide in Human HapMap
Cell Lines
Amanda Degner,⊥ Rashi Arora,⊥ Luke Erber, Christopher Chao, Lisa A. Peterson,
and Natalia Y. Tretyakova*
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ABSTRACT: Smoking-induced lung cancer is a major cause of
cancer mortality in the US and worldwide. While 11−24% of
smokers will develop lung cancer, risk varies among individuals and
ethnic/racial groups. Specifically, African American and Native
Hawaiian cigarette smokers are more likely to get lung cancer as
compared to Caucasians, Japanese Americans, and Latinos. It is
important to identify smokers who are at the greatest risk of
developing lung cancer as they should be candidates for smoking
cessation and chemopreventive intervention programs. Among 60+
tobacco smoke carcinogens, 1,3-butadiene (BD) is one of the most
potent and abundant (20−75 μg per cigarette in mainstream
smoke and 205−361 μg per cigarette in side stream smoke). BD is metabolically activated to 3,4-epoxy-1-butene (EB), which can be
detoxified by glutathione S-transferase theta 1 (GSTT1)-mediated conjugation with glutathione, or can react with DNA to form 7-
(1-hydroxy-3-buten-2-yl)guanine (EB-GII) adducts. In the present study, we employed EBV-transformed human lymphoblastoid
cell lines (HapMap cells) with known GSTT1 genotypes to examine the influence of the GSTT1 gene on interindividual variability in
butadiene metabolism, DNA adduct formation/repair, and biological outcomes (apoptosis). We found that GSTT1− HapMap cells
treated with EB in culture produced lower levels of glutathione conjugates and were more susceptible to apoptosis but had similar
numbers of EB-GII adducts as GSTT1+ cells. Our results suggest that GSTT1 can influence an individual’s susceptibility to
butadiene-derived epoxides.
compared to other cigarette smoke components.9 In inhalation
studies in laboratory mice and rats, BD exposure led to the
INTRODUCTION
■
Cigarette smoking is a leading cause of cancer worldwide,
accounting for 81% of lung cancer incidence.1,2 On average,
development of tumors in multiple organs including lympho-
mas, hemangiosarcomas of the heart, and lung tumors.10,11
people who smoke cigarettes are 15 to 30 times more likely to
Epidemiological studies in workers occupationally exposed to
BD showed an increased risk of developing leukemia and
lymphoma.12,13
The genotoxic effects of BD are attributed to its epoxide
metabolites 3,4-epoxy-1-butene (EB), 3,4-epoxy-1,2-butanediol
(EBD), and 1,2,3,4-diepoxybutane (DEB), which are formed via
metabolic activation by cytochrome P450 monooxygenases,
namely CYP 2E1 and CYP 2A6 (Scheme 1).14−16 EB and other
BD-derived epoxides are direct genotoxic agents that induce
mutations in cells and animals.16 BD-derived epoxides are
get lung cancer or die from lung cancer than people who do not
smoke; however, the risk varies widely between individuals and
ethnic groups.3 After adjusting for smoking history, African
American and Native Hawaiian smokers are at the highest risk of
developing lung cancer, while Japanese American and Latino
smokers have much lower risks, and Caucasian smokers have
intermediate risk.3,4 While the source of these ethnic differences
is not known, frequencies of genetic polymorphisms in
metabolic enzymes differ between ethnic groups, potentially
influencing the ability of smokers to metabolize tobacco
carcinogens. This, in turn, could lead to increased carcinogen
bioactivation and/or decreased detoxification, elevated levels of
DNA-reactive metabolites, greater DNA adduct load, and a
higher chance for cancer causing mutations in affected
individuals.
Received: December 20, 2019
Published: April 2, 2020
1,3-Butadiene (BD) is a known human carcinogen5 and is
among the most abundant carcinogens in cigarette smoke.6−8
BD has a high cancer risk index per cigarette per day as
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Scheme 1. Bioactivation/Detoxification Pathways of BD and the Formation of EB-DNA Adducts
detoxified through enzyme catalyzed hydrolysis by epoxide
hydrolases (EPHX1) or through glutathione (GSH) conjuga-
tion by glutathione S-transferase theta 1 (GSTT1). Glutathione
conjugates metabolized through the mercapturic acid pathway,
resulting in N-acetylcysteine conjugates (mercapturic acids)
which are excreted in urine (Scheme 1). For EB, the major
routes of detoxification are hydrolysis by EPHX1 to 1-butene-
3,4-diol (EB-diol) and GSH conjugation to form EB-GSH,
which is further metabolized through the mercapturic acid
pathway to (2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene
and 1-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene)
(MHBMA) (Scheme 1).
abundant in Caucasian smokers as compared to African
American smokers (0.048 0.09 vs 0.12 0.02 pg/mg of
creatinine, p = 3.1 × 10−7),28 and their levels did not correlate
with the GSTT1 copy number. However, urinary EB-GII adduct
levels measured in large human studies can be affected by many
other factors such as diet, disease, and polymorphisms in DNA
repair genes. Therefore, although the GSTT1 gene appears to
play a key role in BD metabolism,29,30 it is less clear whether
GSTT1 polymorphisms influence BD-DNA adduct load,
genotoxicity, and susceptibility toward the development of
cancer.
In the present study, we employed human B-lymphocytes
from the International HapMap project31−33 to examine the
differences in individual response to BD-derived epoxide, 3,4-
epoxy-1-butene (EB), due to variations in GSTT1 expression/
activity. The HapMap project aims to make a haplotype map of
the human genome to observe common patterns of human
genetic variation.34 A subset of HapMap cells was selected based
on the presence or the absence of the GSTT1 gene in their
genome. Following treatment of GSTT1+ and GSTT1− cells
with EB, we quantified the levels of EB-GSH conjugates in the
media, genomic EB-DNA adduct levels, and biological response
(apoptosis) in order to characterize the influence of the GSTT1
gene on interindividual response to BD.
EB forms covalent adducts with DNA including N6-(2-
hydroxy-3-buten-1-yl)-2′-deoxyadenosine, 1-(1-hydroxy-3-
buten-2-yl)-adenine, 1-(2-hydroxy-3-buten-1-yl)-adenine, 3-
(1-hydroxy-3-buten-2-yl)-adenine, 3-(2-hydroxy-3-buten-1-yl)-
adenine, 7-(2-hydroxy-3-buten-1-yl)-adenine (EB-GI), and 7-
(1-hydroxy-3-buten-2-yl)guanine (EB-GII).17−19 These ad-
ducts can cause DNA polymerase stalling and misreading
during DNA replication, leading to toxicity and mutations.17,20 If
present in a tumor suppressor gene or a proto-oncogene, such
mutations can lead to unrestricted cell growth and the
development of cancer.21,22
BD-derived metabolites and DNA adducts are established
biomarkers of BD exposure and cancer risk.23,24 Urinary levels of
MHBMA (2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene and
1-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene), a mercapturic
acid ultimately resulting from BD detoxification (Scheme 1), are
associated with cigarette smoking.25,26 MHBMA levels were
measured in urine samples of Caucasian, Japanese American,
and African American smokers, where they differed significantly
based on ethnicity (p = 4.0 × 10−25).27 African Americans
excreted the highest levels of MHBMA, followed by Caucasians
and Japanese Americans.27 A genome-wide association study
revealed a significant association between urinary MHBMA
levels and the GSTT1 copy number, with GSTT1+ individuals
excreting significantly higher levels of MHBMA (p < 0.0001).27
In contrast, urinary BD-DNA adducts (EB-GII) were more
MATERIALS AND METHODS
■
Note: EB is a known carcinogen35 and must be handled with adequate
safety precautions in a well-ventilated fume hood strictly following its
material safety data sheet.
LC-MS grade water and acetonitrile and HPLC grade methanol were
obtained from Fisher Scientific (Pittsburgh, PA). 15N1,13C2-glutathione
was procured from Toronto Research Chemicals (North York, ON,
Canada). EB-GII and 15N5-EB-GII were synthesized in our laboratory
as previously described.36,37 All other chemicals and solvents were
obtained from Sigma-Aldrich (St. Louis, MO).
Synthesis of EB-GSH and 15N1,13C2-EB-GSH Standards. EB-
GSH (Scheme 1) was prepared using a variation of the previously
published methodology.38 Glutathione (3.07 mg, 10 μmol, 1 equiv,
Chem-Impex, Wood Dale, IL) was dissolved in 900 μL of 100 mM
B
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sodium phosphate buffer (pH 7.4) in a suitable Eppendorf tube. The
recombinant human GSTT1 enzyme (20 μg, Sigma-Aldrich, St. Louis,
MO) was added to the mixture, which was then warmed to 37 °C in an
oil bath. Racemic EB (16.1 μL, 200 μmol, 20 equiv, Alfa Aesar, Ward
Hill, MA) diluted in 80 μL of 100 mM sodium phosphate buffer (pH
7.4) was added, and this mixture was incubated at 37 °C without stirring
for 2 h (total reaction volume: 980 μL). The reaction was quenched
with one-half volume (500 μL) of 15% (v/v) dichloroacetic acid
solution. The GSTT1 protein was removed by ultrafiltration through
Nanosep 10K filters (5000g for 10 min, Pall Life Sciences, Ann Arbor,
MI).
This filtered reaction mixture was subjected to SPE through Oasis
MCX cartridges (Waters Corporation, Milford, MA) (30 mg/mL)
before HPLC purification. Cartridges were first conditioned with 4%
(v/v) formic acid in water (1 mL) followed by a rinse of water (1 mL).
After conditioning, 500 μL portions of the filtered reaction mixture
diluted by an equal volume of additional 4% (v/v) formic acid in water
were then loaded onto each cartridge. Cartridges were washed once
with 1 mL of water followed by two 1 mL washes of HPLC-grade
methanol. Finally, the conjugate was eluted using a mixture of (30:70)
methanol:4% aqueous NH4OH (1 mL). The SPE eluates were
combined and dried in vacuo to yield a crude foam (1.2 mg) that was
reconstituted in 1.8 mL of 0.01% acetic acid in water.
Cells were counted using the Countess automated cell counter
(Thermo Fisher Scientific, Waltham, MA), seeded in T-25 flasks, and
grown at 37 °C for 24 h before EB exposure. In our preliminary
experiments, HapMap cell lines (∼5 million cells) were treated 2 mM
EB for 0, 2, 4, 8, or 24 h to determine the ideal EB treatment length.
Based on these results, 6 and 24 h time points were selected for
apoptosis measurements.
In order to compare the cellular response to EB in GSTT1+ and
GSTT1‑ lymphoblasts, six GSTT1−/− cell lines (GM12874, GM18508,
GM18912, GM19128, GM19139, and GM18517), one GSTT11/− line
(GM12145), and four GSTT1+/+ cell lines (GM19130, GM12717,
GM19200 and GM12155) (∼3 million cells, in triplicate) were treated
with 2 mM EB for 6 h before being harvested. The cells were then
centrifuged at 300g for 5 min, and the supernatants were saved for EB-
GSH measurement. The pellet was resuspended in fresh media, and the
cells were divided for DNA adduct measurement (2.5 million) and
apoptosis measurement (0.5 million).
In a separate experiment, three GSTT1−/− cell lines (GM12874,
GM18912, GM19128), one GST (GM12145), and two GSTT1+/+ cell
lines (GM19130 and GM19200) were treated with 10 μM EB using the
same procedure described above. Cells were centrifuged at 300g for 5
min. Cell media was saved for EB-GSH measurements, while the cell
pellet was used for DNA adduct measurements as described below.
Confirmation of GSTT1 Transcript Expression in HapMap
Cell Lines. GSTT1 expression levels in HapMap cell lines were
confirmed at the transcript level by quantitative RT PCR. RNA was
isolated from the cell lines using a RNeasy plus kit (Qiagen, Hilden,
Germany) as per manufacturer’s protocol. cDNA was synthesized from
1 μg of RNA using a RevertAid First Strand cDNA Synthesis Kit
(Thermo Fisher Scientific, Waltham, MA) as per manufacturer’s
protocol. A real time PCR reaction was carried out using Prime PCR
assay (Bio-Rad Laboratories, Hercules, CA) for GSTT1 and the
housekeeping control GAPDH on 250 ng of cDNA as the template as
per manufacturer’s protocol in ABI step one plus real time PCR system
(Applied Biosystems, Foster City, CA). The relative expression was
thus noted using the standard fold change method using GAPDH as the
housekeeping control and cell line GM12717 as the expression control.
Confirmation of GSTT1 Protein Expression in HapMap Cell
Lines by Western Blotting. Total cellular protein was extracted from
10 million untreated cells. Cells were pelleted at 300g for 5 min and
washed twice with PBS. Cell pellets were resuspended in 500 μL of
RIPA buffer (Thermo Fisher Scientific, Waltham, MA) and incubated
on ice for 45 min, followed by 5 cycles of sonication of 2 min each on
ice. The lysed cells were centrifuged at 13000g for 15 min at 4 °C, and
the supernatant, containing total cellular protein, was collected. Protein
concentrations were measured using the Pierce BCA protein assay kit
(Thermo Fisher Scientific, Waltham, MA).
EB-GSH was purified by HPLC using an Agilent 1100 series HPLC
system (Santa Clara, CA) with a UV−vis variable wavelength detector.
The reconstituted solution of the crude product was loaded (in 100 μL
injections) on a Luna C18(2) column (250 × 4.6 mm, 5 μm,
Phenomenex, Torrance, CA) and eluted with 0.01% acetic acid in water
(solvent A) and 0.01% acetic acid in (95:5) acetonitrile:water (solvent
B) at a flow rate of 1.00 mL/min. Solvent composition started at 2% B
and was increased to 10% B over 5 min. The gradient was further
changed to 30% B over 5 min and held for 5 min before reducing back to
2% B over 10 min, followed by re-equilibration of the column at 2% B
for an additional 5 min. The UV absorbance at 215 nm was monitored.
EB-GSH eluted over 2 min starting at 11.5 min. Fractions containing
EB-GSH were combined, concentrated, and dried in vacuo. EB-GSH
1
was quantified via H NMR in 150 μL of D2O and adding a known
amount of methanol (0.55 mg, 17 μmol). The sample was determined
to contain 0.70 μmol or 0.26 mg of the conjugate, representing 7% yield
of pure compound.
EB-GSH: 1H NMR (D2O, 600 MHz): δ 5.91 (ddd, J = 17.2, 10.5, 6.2
Hz, 1H, CHCH2), 5.32 (d, J = 17.2 Hz, 1H, trans CHCH2), 5.23
(d, J = 10.5 Hz, 1H, cis CHCH2), 4.59 (dd, J = 8.9, 4.8 Hz, 1H, Cys α
H), 4.32 (q, J = 6.2 Hz, 1H, CHOH), 4.04 (m, 1H, Glu α H), 3.78 (s,
2H, Gly CH2), 3.13 (dd, J = 14.2, 4.9 Hz, 1H, Cys CH2), 2.92 (dd, J =
14.0, 8.9 Hz, 1H, Cys CH2), 2.83 (dd, J = 13.6, 5.0 Hz, 1H,
CH2CHOH), 2.73 (dd, J = 13.6, 7.3 Hz, 1H, CH2CHOH), 2.55 (m,
2H, Glu γ H), 2.16 (q, J = 7.44 Hz, 2H, Glu β H) ppm.
An equal amount of proteins (100 μg) was boiled in Laemmli buffer
(Bio-Rad Laboratories, Hercules, CA), separated on 10% SDS-
polyacrylamide gels, and transferred onto a polyvinylidene fluoride
(PVDF) membrane (MilliporeSigma, Burlington, MA). Bovine Serum
Albumin (BSA), 5%, was used to block the PVDF membrane. Blocking
was followed by incubation with a primary antibody (Sigma ABS1653
for GSTT1 and Sigma SAB 1404522 for vinculin) for 3 h and
horseradish peroxidase (HRP) conjugated secondary antibody for 45
min at room temperature. The membrane was then washed 3 times with
0.05% tween-20 in PBS (v/v) at room temperature for 15 min each.
Immunoreactive bands were probed with the enhanced chemilumi-
nescence (ECL) Western blot detection system (Bio-Rad Laboratories,
Hercules, CA) and viewed in Image studio. The signal intensity of
GSTT1 was noted relative to the housekeeping control (vinculin).
Measurement of Apoptosis Following Exposure to EB. For
apoptosis assessment, EB-treated cell pellets were transferred to fresh
media and subjected to 24 h staining with allophycocyanin (APC)
labeled annexin-V and PI as per the manufacturer’s guidelines
(eBiosciences, San Diego, CA). The cell population was then analyzed
for the percentage of cells in a healthy and apoptotic phase on a LSR II
4760 flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using
FACSDiva and Flowjo software (Becton Dickinson, Franklin Lakes,
NJ). The increase in the % of Annexin V positive cells caused by EB was
The 15N1,13C2-EB-GSH (internal standard for mass spectrometry)
was prepared analogously starting with 1.6 μmol of 15N1,13C2-GSH
(Toronto Research Chemicals, North York, ON, Canada), and the
amounts of the GSTT1 enzyme, GSH, EB, and total reaction volume
were scaled down accordingly to accommodate the smaller amounts of
starting material. 15N1,13C2-EB-GSH amounts were determined from
HPLC-ESI-MS/MS peak areas after spiking with known amounts of
EB-GSH.
Cell Culture Experiments. Human-derived B-lymphocyte cells
from the HapMap project were obtained from the NIGMS Human
Genetic Cell Repository at the Coriell Institute for Medical Research
(Camden, NJ). Based on previous reports on GSTT1 polymorphisms in
HapMap cell lines,39 11 human HapMap cell lines were selected: six
with the GSTT1 null genotype (GM12874, GM18508, GM18912,
GM19128, GM19139, and GM18517) and five with at least one copy of
the GSTT1 gene present (GM19130, GM12145, GM12717,
GM19200, and GM12155).39 Cells were cultured in Gibco RPMI
1640 Medium (Thermo Fisher Scientific, Waltham, MA) supple-
mented with 15% heat inactivated fetal bovine serum (GIBCO,
Thermo Fisher Scientific, Waltham, MA) at 37 °C in 5% CO2
humidified atmosphere. All experiments were performed using cells
at a passage number below 10.
C
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determined by subtracting the % of Annexin V positive cells in the EB-
treated cells from that observed in the control cells for each cell line.
HPLC-ESI+-MS/MS Analysis of EB-GSH. EB-GSH was quantified
in cell media using isotope dilution HPLC-ESI+-MS/MS. Cell media
from EB-treated and control cells (1 mL) was spiked with the 15N1,13C2-
EB-GSH internal standard (126 pmol). Samples were purified by a two-
stage solid phase extraction (SPE) method incorporating Oasis MCX
cartridges (30 mg/mL) and Oasis MAX cartridges (30 mg/mL). MCX
cartridges were conditioned with 1 mL of 2% formic acid in water,
followed by 1 mL of water. Samples were acidified by adding 20 μL of
formic acid before being loaded onto the prepared cartridges and
washed with 1 mL of water, followed by 2 mL of MeOH. EB-GSH and
its internal standard were eluted with 0.5 mL of 30% MeOH in 2%
NH4OH, dried under vacuum, and reconstituted in 100 μL of 2%
NH4OH in preparation for the next SPE cartridge. MAX cartridges
were conditioned with 1 mL of 2% NH4OH followed by 1 mL of water.
Samples were then loaded onto the prepared cartridges and washed
with 1 mL of water followed by 2 mL of MeOH. EB-GSH and its
internal standard were eluted with 0.5 mL of 2% formic acid in water
and dried under vacuum. Samples were reconstituted in 20 μL of 5 mM
ammonium formate (pH 5) in water in preparation for mass
spectrometry analysis.
DNA concentrations were estimated using a nanodrop UV
spectrophotometer (Thermo Scientific, Waltham, MA) based on UV
absorbance at 260 nm. DNA purity was assessed from A260/A280
absorbance ratios, which were between 1.8 and 1.9. Accurate DNA
amounts were confirmed by HPLC-UV quantitation of dG after
enzymatic hydrolysis as described previously.40
EB-GII adducts were quantified by isotope dilution HPLC-ESI-MS/
MS as described previously.28,41 Briefly, DNA samples (10−25 μg)
were spiked with 50 fmol of the 15N5-EB-GII internal standard and
heated at 70 °C for 1 h to release EB-GII adducts from the DNA
backbone. The DNA backbone was then removed by ultrafiltration with
Nanosep 10K filters (Pall Life Sciences, Ann Arbor, MI, USA) at 5000g
for 10 min. The filtrate was subjected to SPE using Strata-X polymeric
reverse phase cartridges (30 mg/mL). Cartridges were conditioned
with 2 mL of HPLC grade methanol, followed by 2 mL of water. After
conditioning, samples were loaded. Cartridges were washed with 1 mL
of water followed by 1 mL of 10% methanol in water. Finally, EB-GII
and its internal standard were eluted in 1 mL of 60% methanol in water.
Samples were dried under vacuum, reconstituted in 200 μL of water,
and filtered using Nanosep 10K filters at 5000g for 10 min in order to
remove any SPE particles. Filtered samples were then dried under
vacuum and reconstituted in 15 μL of 0.01% acetic acid in water in
preparation for nanoLC-nanoESI+-MS/MS analysis.
HPLC-ESI+-MS/MS analyses of EB-GSH were conducted using a
Dionex LC system interfaced with a TSQ Quantiva instrument
(Thermo Fisher Scientific, Waltham, MA, USA). Solvent A was 5 mM
ammonium formate (pH 5) in LCMS grade water, and solvent B was
LCMS grade acetonitrile. Samples (1 μL) were injected onto an
Acquity UPLC HSS T3 column (1 × 100 mm, 1.8 μm, Waters Corp.,
Milford, MA, USA) and eluted at 20 μL/min. A valve switch was set up
after the column to direct the eluent to waste for the first 3 min, before
switching to the MS. The solvent gradient started at 2% B, increased to
10% B in 10 min, and increased further up to 70% B in 1 min. The
solvent composition was held at 70% B for 4 min, and the column was
then re-equilibrated for 4 min at 2% B. EB-GSH and its internal
standard eluted as a sharp peak at ∼5.6 min.
A TSQ Quantiva triple quadrupole mass spectrometer (Thermo
Scientific, Waltham, MA) was operated in the positive ion mode using
Ar as a collision gas (1.5 mTorr). The MS parameters were optimized
upon infusion of the authentic EB-GSH solution to achieve maximum
sensitivity. Quantitative analyses were conducted using a selected
reaction monitoring (SRM) mode, following the MS/MS transitions
corresponding to the loss of glutamate from the analyte (m/z 378.1 [M
+ H]+ → m/z 249.1 [M + H − Glu]+) and the loss of both glutamate
and water (m/z 378.1 [M + H]+ → m/z 231.1 [M + H − Glu − H2O]+).
Typical instrument settings included a spray voltage of 2.9 kV, capillary
temperature of 400 °C, and collision energy of 10.25 V for both
transitions. The corresponding transitions for the 15N1,13C2 isotopically
labeled internal standard were m/z 381.1 [15N1,13C2-M + H]+ → m/z
252.1 [15N1,13C2-M + H − Glu]+ and m/z 381.11 [15N1,13C2-M + H]+
→ m/z 234.1 [15N1,13C2-M + H − Glu − H2O]+. The peak width for
both Q1 and Q3 was 0.7 amu. HPLC-ESI+-MS/MS quantitation was
based on the areas of the peak for the first transition in the extracted ion
chromatograms corresponding to the analyte and the internal standard.
Solvent blanks were injected every 4−6 samples to monitor for
potential analyte carryover.
The method used for nanoLC-nanoESI+-MS/MS analysis of EB-GII
adducts was based on our previously published method,41 which was
adapted for a TSQ Quantiva triple quadrupole MS. NanoLC-nanoESI+-
MS/MS analysis was conducted using a Dionex LC system with a 5 μL
loop interfaced with a TSQ Quantiva instrument (Thermo Fisher
Scientific, Waltham, MA, USA). Solvent A was 0.01% acetic acid in
LCMS grade water, and solvent B was 0.02% acetic acid in LCMS grade
acetonitrile. Samples (1 μL) were injected onto a nano-LC column with
a fused-silica tipped emitter (75 μm ID, from New Objective, Woburn,
MA) that was manually packed with Synergi Hydro-RP, 80 Å, 4 μm
chromatographic packing (Phenomenex, Torrance, CA). The solvent
composition started at 2% B at 800 nL/min, which was held for 7.5 min
to allow the sample to reach the nano-LC column. The flow rate was
reduced to 300 nL/min and maintained at 2% B for 0.5 min. The
gradient was then increased linearly to 25% B in 9 min and then up to
50% B in 10 min. The column was re-equilibrated at 800 nL/min at 2%
B for 7 min. EB-GII and its internal standard eluted as a sharp peak at
∼16.5 min.
A TSQ Quantiva triple quadrupole mass spectrometer was operated
in the positive ESI mode using argon as a collision gas (1.5 mTorr). The
mass spectrometer parameters were optimized upon infusion of
authentic EB-GII solution to achieve maximum sensitivity. Quantitative
analyses were conducted using the selected reaction monitoring (SRM)
mode by fragmenting the [M + H]+ ions of EB-GII (m/z 222.2) to [Gua
+ H]+ ions (m/z 151.8) via collision induced dissociation (CID) in Q2
using a collision energy of 13.8 V. Typical instrument settings included
a spray voltage of 2.5 kV, capillary temperature of 375 °C, RF lens of 64
V, and a dwell time of 100 ms. The peak width for both Q1 and Q3 was
0.4 and 0.7 amu, respectively. EB-GII was quantified using the extracted
ion chromatogram for m/z 222.2 [M + H]+ → m/z 151.8 [Gua + H]+.
The corresponding transitions for the 15N5 isotopically labeled internal
standard were m/z 227.1 [15N5-M + H]+ → m/z 156.8 [15N5-Gua +
H]+. NanoLC-nanoESI+-MS/MS quantitation was done by comparing
the areas of the peaks for the analyte and the internal standard. Solvent
blanks were injected every 4−6 samples to monitor for potential analyte
carryover.
NanoLC-NSI+-MS/MS Analysis of EB-GII Adducts. DNA was
extracted from the control and EB-treated cells using a Qiagen
Puregene DNA extraction solution set (Qiagen, Hilden, Germany).
Briefly, 1 mL of cell lysis buffer and 3.4 μL of the proteinase K solution
were added to ∼3 million cells. Following overnight incubation at room
temperature with slow inversion mixing, RNA was degraded by
incubation with RNase A (5 μL) at room temperature for 3 h. Proteins
were precipitated out by adding 0.5 mL of the Qiagen Puregene DNA
protein precipitation solution, vortexing samples for ∼20 s, and
centrifuging at 2000g for 15 min. DNA was precipitated by adding 34
μL of 5 M NH4OAc and 1.5 mL of ice-cold isopropyl alcohol (IPA) and
storing overnight in a −20 °C freezer. Extracted DNA was subsequently
washed twice with 1 mL of 70% EtOH in water and sheered using 19
and 22 gauge needles.
RESULTS
■
Experimental Approach. The present study was designed
to elucidate the effects of the GSTT1 genotype on cellular
responses to active metabolite of BD, EB (Scheme 1). Our
ultimate goal was to investigate the role of the GSTT1 gene in
cellular protection against EB toxicity and carcinogenesis. For
this reason, we compared the extent of GSH conjugation, DNA
adduct formation, and apoptosis following exposure to EB in a
range of human HapMap cells with different GSTT1 genotypes
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Scheme 2. Experimental Procedure for HPLC-ESI-MS/MS Analysis of EB-GSH Conjugates, EB-GII Adducts, and Apoptosis in
Cells Exposed to EB
(Scheme 2). As discussed above, cell lines from the HapMap
project were derived from human populations from all over the
world and have been extensively genotyped, allowing for cell
culture studies that mimic the genetic diversity of real human
populations.34
For our study, we selected cell lines from the HapMap project
with GSTT1− and GSTT1+ genotypes, and their phenotypes
were confirmed by RT PCR and Western blotting (Figure 1).
Following treatment with EB, a flow cytometry assay utilizing
the Annexin V marker was used to detect EB-induced
apoptosis.42 The levels of EB-GSH conjugates in the media
were quantified to examine the role of GSTT1 in detoxification
of EB. EB-GII (Scheme 1) was chosen as a representative EB-
DNA adduct. EB-GII, along with its structural isomer EB-GI, is
the most prevalent DNA adduct resulting from exposure to
EB.43
Confirmation of GSTT1 Expression at Transcript and
Protein Levels. GSTT1 gene expression levels in 11 HapMap
cell lines were confirmed by RT-PCR, and GSTT1 protein levels
were determined by Western blot. GSTT1 expression levels
correlated with the published genotype39 at both transcript and
protein levels in all 11 cell lines selected for this study. Cell lines
with the GSTT1 deletion contained no GSTT1 transcripts or the
corresponding protein, whereas the cell lines with at least one
copy of the gene had detectable levels of transcript and protein
(Figure 1).
Apoptosis in Cells Treated with EB. The ability of EB to
Figure 1. GSTT1 expression levels in HapMap cells. A. Expression of
the GSTT1 transcript in selected HapMap cell lines, confirmed by
qualitative RT PCR. *Fold change was noted with respect to cell line
GM12717. B. Expression of the GSTT1 protein in selected HapMap
induce apoptosis in HapMap cells was determined using a
commercially available annexin V flow assay. Preliminary studies
established that there was no change in the % apoptotic cells
below 1 mM EB (data not shown). As shown in Figure 2,
HapMap cell lines lacking GSTT1 were more sensitive to the
toxic effects of EB. The average increase in the % of Annexin V
positive cells in EB-treated GSTT1− cell lines (9.4% 0.3%) was
higher than in cell lines containing at least one copy of GSTT1
(4.1% 0.3%; p < 0.001).
cell lines, confirmed by Western blot. *Signal intensity was normalized
to the vinculin signal. Values given represent average
deviation.
standard
human cells, a quantitative HPLC-ESI-MS/MS method for EB-
GSH was developed (Scheme 1). Authentic standards of EB-
GSH and 15N1,13C2-EB-GSH were prepared in our laboratory
and fully characterized by NMR (Figures S1−S3) and HR MS/
MS (Figure 3). In animals and humans that have been exposed
to EB, EB-GSH is further metabolized through the mercapturic
acid pathway to form N-acetylcysteine conjugate (MHBMA),
HPLC-ESI+-MS/MS Method Development for EB-GSH
in Cellular Media. In order to compare the amounts of EB that
undergoes conjugation with GSH in GSTT1+ and GSTT1−
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the analyte (m/z 378.1 → 249.1) and a loss of both glutamate
and water from the analyte (m/z 378.1 → 231.1). The first
transition showed better reproducibility and sensitivity, and this
was selected for quantitative analyses; while the second
transition was used for confirmation purposes. Similar MS/MS
transitions (m/z 381.1 → 252.1 and m/z 378.1 → 234.1) were
used for 15N1,13C2-EB-GSH (internal standard for quantifica-
tion) (Figure 4).
Quantification of EB-GSH in Cellular Media of HapMap
Cells Treated with 2 mM EB. To examine the effects of the
GSTT1 genotype on EB-GSH conjugate formation after EB
exposure, 6 GSTT1−/− HapMap cell lines (GM12874,
GM18508, GM18912, GM19128, GM19139, and GM18517),
1 GSTT1 line (GM12145), and 4 GSTT1+/+ cell lines
(GM19130, GM12717, GM19200 and GM12155) were
exposed to EB (2 mM) for 6 h. EB concentration was selected
in order to match the treatment levels that resulted in detectable
apoptosis (see above). Culture media (1 mL) from each cell line
was removed and spiked with the 15N1,13C2-EB-GSH internal
standard. The resulting samples were processed by two-step SPE
and analyzed by LC-ESI+-MS/MS methods as described above.
EB-GSH amounts in treated cells varied between 1.0 and 5.9
nmol of EB-GSH per mL of media. No EB-GSH was observed in
media from control cells. While interindividual differences in
EB-GSH levels between cell lines were seen, these differences
did not correlate with GSTT1 expression (Figure 5A), probably
due to nonenzymatic GSH conjugation reactions predominating
at these high EB exposure levels. Indeed, when the treatment was
repeated using physiologically relevant concentrations of EB (10
μM), EB-GSH conjugate formation was significantly higher in
GSTT1+ cells (8.5 4.8 vs 22.9 10.7 pmol EB-GSH per mL
media, p < 0.001, Figure 5B). Our results support a potentially
important role of the GSTT1 enzyme in detoxification of BD-
derived epoxides under physiological exposure levels.
Quantification of EB-GII in Genomic DNA of HapMap
Cells Treated with EB. To examine the effects of the GSTT1
genotype on DNA adduct formation after exposure to EB, 6
GSTT1‑ and 5 GSTT1+ HapMap cell lines were exposed to 2
mM EB for 6 h. Genomic DNA was extracted and analyzed by
nanoLC-ESI+-MS/MS methods as described above. No EB-GII
adducts were observed in genomic DNA isolated from control
cells (data not shown). DNA adduct numbers in treated cells
varied between 35.0 and 70.9 adducts per 107 nucleotides
depending on the cell line. However, differences in EB-GII
adduct formation in GSTT1− cell lines (48.3 7.6 adducts per
107 nucleotides) and GSTT1+ cell lines (52.7 8.6 adducts per
107 nucleotides) were not statistically significant (Figure 6A, p =
0.096).
Figure 2. EB causes an increase in the number of apoptotic cells.
HapMap human lymphoblastoid cells (in triplicate) were exposed to 2
mM EB for 6 h. The cells were then incubated in fresh media for an
additional 24 h prior to measuring apoptosis using Annexin V marker
expression on the cell surface. The average increase in the % of Annexin
V positive cells induced by EB in GSTT1− cells is significantly higher
than that observed in GSTT+ cells, p < 0.001. Values represent average
standard deviation.
which was quantified in our previous studies.27,44,45 However,
the enzymes responsible for further metabolism of GSH
conjugates to mercapturic acids are mostly found in the liver
and kidney tissues and not in B-lymphocytes like the HapMap
cell lines.46 Thus, EB-GSH is less likely to be further metabolized
in this cell line model.
EB-GSH and 15N1,13C2-EB-GSH were prepared by incubating
GSH and 15N1,13C2- GSH with excess EB in the presence of
recombinant GSTT1 protein. Both standards were isolated by
HPLC. EB-GSH was quantified by proton NMR in D2O using
methanol as an internal standard (Figures S1−S3), and
15N1,13C2-EB-GSH was quantified by mass spectrometry by
spiking with known amounts of EB-GSH. These authentic
standards were used for quantitative HPLC-ESI-MS/MS
method development.
Early experiments revealed that EB-GSH was rapidly excreted
from cells into the media. A two-step SPE method was devised to
purify the conjugate. Samples were first acidified and subjected
to mixed-mode strong cation exchange SPE (Oasis MCX
cartridges, 30 mg/mL, Waters Corporation, Milford, MA) to
remove salts, neutrals, and negatively charged compounds. The
resulting eluent was dried down, reconstituted in a basic
solution, and subjected to mixed-mode strong anion exchange
SPE (Oasis MAX cartridges, 30 mg/mL, Waters Corporation,
Milford, MA), to remove any contaminants that cannot hold a
negative charge. The resulting eluent containing EB-GSH and its
internal standard was subjected to capillary HPLC-ESI-MS/MS
analysis. A valve switch was added to the beginning of the LC-
MS run, to divert the first 2 min of the gradient to be sent to
waste instead of the MS, as an added precaution to keep the MS
system clean.
Similar results were observed in a subset of 6 cell lines (3
GSTT1−, 3 GSTT1+) treated with 10 μM EB for 6 h. EB-GII
adduct levels in cells treated with 10 μM EB were approximately
100-fold lower than those in cells treated with 2 mM EB (0.05
0.01 vs 50.5 8.3 adducts per 107 nucleotides) (Figure 6). The
difference in EB-GII amounts in genomic DNA of GSTT1− (5.2
0.6 adducts per 108 nucleotides) and GSTT1+ cell lines (4.7
0.5 adducts per 108 nucleotides) was not statistically significant
(Figure 6B, p = 0.102).
EB-GSH is a very polar molecule and has poor retention on
most reverse phase HPLC columns. An Acquity UPLC HSS T3
(Waters Corporation, Milford, MA) column was chosen as it
retained EB-GSH longer than 3 min. Next, several different
mobile phases and gradients were tested. A gradient of 5 mM
ammonium formate in water (pH 5) and acetonitrile afforded
the best chromatography and ionization efficiency. The
optimized chromatography conditions use a gradient of 5 mM
ammonium formate in water (pH 5) and acetonitrile, where EB-
GSH elutes at ∼6.4 min (Figure 4).
DISCUSSION
■
GSTT1 is a key enzyme in the detoxification pathway of BD-
derived epoxides. It catalyzes the conjugation of these
electrophilic species with glutathione, leading to their
detoxification and excretion in urine following further
Two SRM transitions for HPLC-ESI-MS/MS analysis of EB-
GSH were selected corresponding to the loss of glutamate from
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Figure 3. HR MS/MS spectra of EB-GSH (A) and 15N1,13C2-EB-GSH (B) obtained on an Orbitrap Q Exactive mass spectrometer.
processing by the mercapturic acid pathway.47−49 Previous
studies have shown that urinary levels of MHBMA, the ultimate
product of EB-GSH conjugation excreted in urine, was
associated with cigarette smoking and the GSTT1 copy
number.25,26 Specifically, smokers carrying one copy of the
GSTT1 gene excreted more MHBMA than GSTT1 null
individuals, and subjects with two copies of the gene exhibited
proportionally higher levels of the conjugate.25,26 Genome-wide
association analyses of the data indicated that the GSTT1 copy
number largely accounted for ethnic differences in MHBMA
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Figure 4. Isotope dilution HPLC-ESI+-MS/MS quantification of EB-
GSH in cellular media from the GM12874 cell line treated with 2 mM
EB for 6 h.
Figure 6. Quantitation of EB-GII adducts in GSTT1− and GSTT1+ cell
lines. HapMap cells (3 million) were treated with 2 mM EB (A) and 10
μM EB (B) for 6 h in triplicate. DNA was extracted, and EB-GII adduct
levels were quantified by isotope dilution HPLC-ESI-MS/MS. Values
represent averages standard deviation.
metabolic detoxification of EB via glutathione conjugation,
potentially increasing their concentrations in cells and leading to
higher levels of DNA adducts and increased toxicity. Human
EBV-transformed lymphoblastoid cell lines were selected from
the HapMap project designed to describe the common patterns
of human DNA sequence variation and to represent the genetic
diversity of human populations.34 Six GSTT1−/− cell lines and
five cell lines containing at least one copy of the GSTT1 gene
were chosen for this investigation.
GSTT1− cells exhibited a higher sensitivity toward EB-
induced apoptosis as determined by Annexin V flow cytometry
assay (Figure 2) and produced higher levels of EB-GSH
conjugates when treated with 10 μM EB (Figure 5B). These
results support the hypothesis that GSTT1 has a protective
effect against EB toxicity. However, GSTT1 expression had no
effect on EB-GSH conjugate levels in lymphoblasts treated with
high concentrations of EB (2 mM) (Figure 5A). This can be
explained by nonenzymatic conjugation of GSH with EB and/or
contributions of other GST isoforms under these conditions.
While previous studies found that GSTT1 has the highest
activity in detoxification of BD-derived epoxides, GST mu 1
(GSTM1) and GST theta 2 (GSTT2) could also be
involved.27,50,51 However, when EB-GSH levels in EB-treated
cells were stratified by GSTM1 status, no significant difference in
adduct formation was observed (Figure S4).
Figure 5. HPLC-ESI-MS/MS quantitation of EB-GSH conjugates in
HapMap cell lines treated with 2 mM (A) or 10 μM EB (B) for 6 h in
triplicate. Values represent averages standard deviation.
Although HapMap cells are a valuable biological resource for
characterizing human variability in response to environmental
chemicals,52 these cells have relatively low enzymatic activity in
regard to xenobiotic metabolism. In humans, liver hepatocytes
are the primary source of GSH conjugation.46 While the
expression of GSTT1 was confirmed in all five GSTT1+ cell lines
(Figure 1), expression levels in HapMap cells were low as
compared to its amounts in hepatocytes (data not shown).
levels between Caucasian, Japanese American, and African
American smokers.27
In the present study, we investigated the effects of the GSTT1
gene on BD metabolism, EB-DNA adduct formation, and
apoptotic cell death in human HapMap cells after exposure to
EB. Reduced GSTT1 activity was anticipated to impair
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Authors
These inherently low levels of GSTT1 activity in lymphoblastoid
cell lines may result in saturation of enzymatic detoxification
pathways when cells are exposed to mM concentrations of EB,
with nonenzymatic GSH conjugation contributing to the
majority of EB-GSH formation.
Amanda Degner − Department of Medicinal Chemistry and
Masonic Cancer Center, University of Minnesota, Minneapolis,
Minnesota 55455, United States
Rashi Arora − Masonic Cancer Center, University of Minnesota,
Minneapolis, Minnesota 55455, United States
Another gene critically important for detoxification of BD-
derived epoxides is microsomal epoxide hydrolase (mEH coded
by the EPHX gene). The mEH enzyme catalyzes epoxide
hydrolysis to the corresponding diols, which are typically
unreactive toward DNA and therefore show reduced genotox-
icity. Wickliffe et al. have shown that the frequency of DEB-
induced SCE in human lymphocytes was dependent on EPHX1
polymorphisms.53 Several polymorphic sites within the 5′-
flanking region of the EPXH gene have been identified, which
are likely to lead to altered levels of gene expression.54 The
frequency of low mEH activity in WH, AA, and JA individuals is
28, 21, and 44%, respectively.55,56 These genetic variations are
likely to affect an individual’s sensitivity to butadiene-induced
mutagenesis and carcinogenesis.57 Unfortunately, our Western
blotting and RT PCR experiments revealed that EPHX1
expression levels in HapMap cells were below the limit of
detection. Therefore, studies in a different set of cell lines are
needed to elucidate the role of EPHX1 in detoxification of
butadiene epoxides.
The differences in EB-Gua DNA adduct levels in EB-treated
HapMap cells were not statistically different between GSTT1+
and GSTT1− cells (Figure 6), despite significant variation in
formation of GSH conjugates (Figure 5B). Our earlier studies
revealed that the excretion of urinary EB-Gua adducts by current
smokers was influenced by smoking status and ethnicity but did
not correlate with urinary levels of BD-mercapturic acids.28,58 It
is possible that other factors such as polymorphisms in DNA
repair genes have a greater effect on BD-DNA adduct levels in
treated cells.
Luke Erber − Department of Medicinal Chemistry and Masonic
Cancer Center, University of Minnesota, Minneapolis, Minnesota
55455, United States
Christopher Chao − Department of Medicinal Chemistry and
Masonic Cancer Center, University of Minnesota, Minneapolis,
Minnesota 55455, United States
Lisa A. Peterson − Masonic Cancer Center and Division of
Environmental Health Sciences, University of Minnesota,
Minneapolis, Minnesota 55455, United States; orcid.org/
0000-0001-8715-4480
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrestox.9b00517
Author Contributions
⊥These authors contributed equally to the manuscript.
Funding
This study was supported by a Program Project grant from the
National Cancer Institute (5P01CA138338). NMR experiments
were supported by Grant 1S10OD021536 (G. Veglia) from the
National Institute of General Medical Sciences.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Todd Rappe for his help with NMR analyses, Karin
Vevang for technical assistance and data analysis, Gunnar
Boysen for extensive manuscript edits, and Bob Carlson for his
help with the figures for this manuscript.
In summary, our results indicate that GSTT1 protects
HapMap cells against EB-mediated apoptosis and plays an
important role in metabolic detoxification of EB under
physiological exposure concentrations. In addition, our findings
suggest that another GST enzyme may catalyze the same
conjugation reaction in the absence of GSTT1. Future studies in
human cells are needed to elucidate the role of other metabolic
enzymes such as GSTT2, EPHX1, and DNA repair genes in
defining human sensitivity to butadiene.
REFERENCES
■
(1) Siegel, R. L., Miller, K. D., and Jemal, A. (2019) Cancer statistics,
2019. Ca-Cancer J. Clin. 69, 7−34.
(2) American Cancer Society. (2019) Cancer Facts & Figures 2019,
ACS, Atlanta, GA.
(3) Stram, D. O., Park, S. L., Haiman, C. A., Murphy, S. E., Patel, Y.,
Hecht, S. S., and Le Marchand, L. (2019) Racial/ethnic differences in
lung cancer incidence in the multiethnic cohort study: An update. J.
Natl. Cancer Inst 111, 811−819.
(4) Haiman, C. A., Stram, D. O., Wilkens, L. R., Pike, M. C., Kolonel,
L. N., Henderson, B. E., and Le Marchand, L. (2006) Ethnic and racial
differences in the smoking-related risk of lung cancer. N. Engl. J. Med.
354, 333−342.
(5) International Agency for Research on Cancer. (2012) Chemical
Agents and Related Occupations, IARC Monographs on the Evaluation of
Carcinogenic Risk to Humans, Vol. 100F, IARC, Lyon, France.
(6) White, W. C. (2007) Butadiene production process overview.
Chem.-Biol. Interact. 166, 10−14.
(7) Kagawa, J. (2002) Health effects of diesel exhaust emissions–a
mixture of air pollutants of worldwide concern. Toxicology 181−182,
349−353.
(8) Brunnemann, K. D., Kagan, M. R., Cox, J. E., and Hoffmann, D.
(1990) Analysis of 1,3-butadiene and other selected gas-phase
components in cigarette mainstream and sidestream smoke by gas
chromatography-mass selective detection. Carcinogenesis 11, 1863−
1868.
(9) Fowles, J., and Dybing, E. (2003) Application of toxicological risk
assessment principles to the chemical constituents of cigarette smoke.
Tob Control 12, 424−430.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.chemrestox.9b00517.
1H, HSQC, and HMBC NMR spectra of synthetic EB-
GSH (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Natalia Y. Tretyakova − Department of Medicinal Chemistry and
Masonic Cancer Center, University of Minnesota, Minneapolis,
Minnesota 55455, United States; orcid.org/0000-0002-
0621-6860; Phone: 612-626-3432; Email: trety001@
umn.edu; Fax: 612-624-3869
I
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(10) Melnick, R. L., Huff, J., Chou, B. J., and Miller, R. A. (1990)
Carcinogenicity of 1,3-butadiene in C57BL/6 x C3H F1 mice at low
exposure concentrations. Cancer Res. 50, 6592−6599.
(11) Owen, P. E., Glaister, J. R., Gaunt, I. F., and Pullinger, D. H.
(1987) Inhalation toxicity studies with 1,3-butadiene. 3. Two year
toxicity/carcinogenicity study in rats. Am. Ind. Hyg. Assoc. J. 48, 407−
413.
(12) Delzell, E., Sathiakumar, N., Hovinga, M., Macaluso, M., Julian, J.,
Larson, R., et al. (1996) A follow-up study of synthetic rubber workers.
Toxicology 113, 182−189.
(13) Sathiakumar, N., Graff, J., Macaluso, M., Maldonado, G.,
Matthews, R., and Delzell, E. (2005) An updated study of mortality
among North American synthetic rubber industry workers. Occup.
Environ. Med. 62, 822−829.
in humans and their use as biomarkers of exposure to 1,3-butadiene.
Chem. Res. Toxicol. 30, 678−688.
(29) Norppa, H. (2004) Cytogenetic biomarkers and genetic
polymorphisms. Toxicol. Lett. 149, 309−334.
(30) Norppa, H. (2003) Genetic susceptibility, biomarker respones,
and cancer. Mutat. Res., Rev. Mutat. Res. 544, 339−348.
(31) de Vries, P. S., Sabater-Lleal, M., Chasman, D. I., Trompet, S.,
Ahluwalia, T. S., Teumer, A., et al. (2017) Comparison of HapMap and
1000 genomes reference panels in a large-scale genome-wide
association study. PLoS One 12, e0167742.
(32) International HapMap Consortium (2003) The International
HapMap Project. Nature 426, 789−796.
(33) Yamamura, T., Hikita, J., Bleakley, M., Hirosawa, T., Sato-
Otsubo, A., Torikai, H., et al. (2012) HapMap SNP Scanner: an online
program to mine SNPs responsible for cell phenotype. Tissue Antigens
80, 119−125.
(14) Duescher, R. J., and Elfarra, A. A. (1994) Human liver
microsomes are efficient catalysts of 1,3-butadiene oxidation: evidence
for major roles by cytochromes P450 2A6 and 2E1. Arch. Biochem.
Biophys. 311, 342−349.
(34) Consortium, I. H. (2005) A haplotype map of the human
genome. Nature 437, 1299−1320.
(15) Csanady, G. A., Guengerich, F. P., and Bond, J. A. (1992)
Comparison of the biotransformation of 1,3-butadiene and its
metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues
from humans, rats and mice. Carcinogenesis 13, 1143−1153.
(16) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of
butadiene and its epoxide metabolites: I. Mutagenic potential of 1,2-
epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butanediol in
cultured human lymphoblasts. Carcinogenesis 15, 713−717.
(17) Kotapati, S., Wickramaratne, S., Esades, A., Boldry, E. J., Quirk
Dorr, D., Pence, M. G., et al. (2015) Polymerase bypass of N6-
deoxyadenosine adducts derived from epoxide metabolites of 1,3-
butadiene. Chem. Res. Toxicol. 28, 1496−1507.
(18) Kowal, E. A., Wickramaratne, S., Kotapati, S., Turo, M.,
Tretyakova, N., and Stone, M. P. (2014) Major groove orientation of
the (2S)-N6-(2-hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA ad-
duct induced by 1,2-epoxy-3-butene. Chem. Res. Toxicol. 27, 1675−
1686.
(19) Wickramaratne, S., Banda, D. M., Ji, S., Manlove, A. H.,
Malayappan, B., Nunez, N. N., et al. (2016) Base excision repair of N6-
deoxyadenosine adducts of 1,3-butadiene. Biochemistry 55, 6070−6081.
(20) Minca, E. C., and Kowalski, D. (2011) Replication fork stalling by
bulky DNA damage: localization at active origins and checkpoint
modulation. Nucleic Acids Res. 39, 2610−2623.
(21) Hemminki, K. (1993) DNA adducts, mutations and cancer.
Carcinogenesis 14, 2007−2012.
(22) Basu, A. K. (2018) DNA damage, mutagenesis and cancer. Int. J.
Mol. Sci. 19, 970.
(23) Swenberg, J. A., Boysen, G., Georgieva, N., Bird, M. G., and
Lewis, R. J. (2007) Future directions in butadiene risk assessment and
the role of cross-species internal dosimetry. Chem.-Biol. Interact. 166,
78−83.
(24) Swenberg, J. A., Koc, H., Upton, P. B., Georguieva, N.,
Ranasinghe, A., Walker, V. E., and Henderson, R. (2001) Using DNA
and hemoglobin adducts to improve the risk assessment of butadiene.
Chem.-Biol. Interact. 135−136, 387−403.
(25) Roethig, H. J., Munjal, S., Feng, S., Liang, Q., Sarkar, M., Walk, R.
A., and Mendes, P. E. (2009) Population estimates for biomarkers of
exposure to cigarette smoke in adult U.S. cigarette smokers. Nicotine
Tob. Res. 11, 1216−1225.
(26) Carmella, S. G., Chen, M., Han, S., Briggs, A., Jensen, J.,
Hatsukami, D. K., and Hecht, S. S. (2009) Effects of smoking cessation
on eight urinary tobacco carcinogen and toxicant biomarkers. Chem.
Res. Toxicol. 22, 734−741.
(27) Boldry, E. J., Patel, Y. M., Kotapati, S., Esades, A., Park, S. L.,
Tiirikainen, M., et al. (2017) Genetic determinants of 1,3-butadiene
metabolism and detoxification in three populations of smokers with
different risks of lung cancer. Cancer Epidemiol., Biomarkers Prev. 26,
1034−1042.
(28) Sangaraju, D., Boldry, E. J., Patel, Y. M., Walker, V., Stepanov, I.,
Stram, D., et al. (2017) Isotope dilution nanoLC/ESI+-HRMS3
quantitation of urinary N7-(1-hydroxy-3-buten-2-yl) guanine adducts
(35) International Agency for Research on Cancer. (2008) in 1,3-
butadiene, ethylene oxide, and vinyl halides (vinyl flouride, vinyl chloride
and vinyl bromide), IARC Monographs on the Evaluation of
Carcinogenic Risks to Humans, Vol. 97, Lyon, France.
(36) Tretyakova, N., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg,
J. A. (1997) Identification and quantitation of DNA adducts from calf
thymus DNA exposed to 3,4-epoxy-1-butene. Carcinogenesis 18, 137−
147.
(37) Citti, L., Gervasi, P. G., Turchi, G., Bellucci, G., and Bianchini, R.
(1984) The reaction of 3,4-epoxy-1-butene with deoxyguanosine and
DNA in vitro: synthesis and characterization of the main adducts.
Carcinogenesis 5, 47−52.
(38) Cho, S. H., Loecken, E. M., and Guengerich, F. P. (2010)
Mutagenicity of a glutathione conjugate of butadiene diepoxide. Chem.
Res. Toxicol. 23, 1544−1546.
(39) McCarroll, S. A., Hadnott, T. N., Perry, G. H., Sabeti, P. C., Zody,
M. C., Barrett, J. C., et al. (2006) Common deletion polymorphisms in
the human genome. Nat. Genet. 38, 86−92.
(40) Michaelson-Richie, E. D., Ming, X., Codreanu, S. G., Loeber, R.
L., Liebler, D. C., Campbell, C., and Tretyakova, N. Y. (2011)
Mechlorethamine-induced DNA-protein cross-linking in human
fibrosarcoma (HT1080) cells. J. Proteome Res. 10, 2785−2796.
(41) Sangaraju, D., Villalta, P. W., Wickramaratne, S., Swenberg, J.,
and Tretyakova, N. (2014) NanoLC/ESI+ HRMS3 quantitation of
DNA adducts induced by 1,3-butadiene. J. Am. Soc. Mass Spectrom. 25,
1124−1135.
(42) Vermes, I., Haanen, C., Steffens-Nakken, H., and
Reutelingsperger, C. (1995) A novel assay for apoptosis. Flow
cytometric detection of phosphatidylserine expression on early
apoptotic cells using fluorescein labelled Annexin V. J. Immunol.
Methods 184, 39−51.
(43) Tretyakova, N., Chiang, S. Y., Walker, V. E., and Swenberg, J. A.
(1998) Quantitative analysis of 1,3-butadiene-induced DNA adducts in
vivo and in vitro using liquid chromatography electrospray ionization
tandem mass spectrometry. J. Mass Spectrom. 33, 363−376.
(44) Park, S. L., Kotapati, S., Wilkens, L. R., Tiirikainen, M., Murphy,
S. E., Tretyakova, N., and Le Marchand, L. (2014) 1,3-Butadiene
exposure and metabolism among Japanese American, Native Hawaiian,
and White smokers. Cancer Epidemiol., Biomarkers Prev. 23, 2240−
2249.
(45) Kotapati, S., Esades, A., Matter, B., Le, C., and Tretyakova, N.
(2015) High throughput HPLC-ESI−-MS/MS methodology for
mercapturic acid metabolites of 1,3-butadiene: Biomarkers of exposure
and bioactivation. Chem.-Biol. Interact. 241, 23−31.
(46) Hinchman, C. A., and Ballatori, N. (1994) Glutathione
conjugation and conversion to mercapturic acids can occur as an
intrahepatic process. J. Toxicol. Environ. Health 41, 387−409.
(47) Kasparkova, J., Novakova, O., Vrana, O., Intini, F., Natile, G., and
Brabec, V. (2006) Molecular aspects of antitumor effects of a new
platinum (IV) drug. Mol. Pharmacol. 70, 1708−1719.
J
https://dx.doi.org/10.1021/acs.chemrestox.9b00517
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology
pubs.acs.org/crt
Article
(48) Dolan, M. E., Roy, S. K., Fasanmade, A. A., Paras, P. R., Schilsky,
R. L., and Ratain, M. J. (1998) O6-Benzylguanine in humans: metabolic,
pharmacokinetic, and pharmacodynamic findings. J. Clin. Oncol. 16,
1803−1810.
(49) Hoes, I., Van Dongen, W., Lemiere, F., Esmans, E. L., Van
Bockstaele, D., and Berneman, Z. N. (2000) Comparison between
capillary and nano liquid chromatography-electrospray mass spectrom-
etry for the analysis of minor DNA-melphalan adducts. J. Chromatogr.,
Biomed. Appl. 748, 197−212.
(50) Wiencke, J. K., Pemble, S., Ketterer, B., and Kelsey, K. T. (1995)
Gene deletion of glutathione S-transferase theta: correlation with
induced genetic damage and potential role in endogenous mutagenesis.
Cancer Epidemiol., Biomarkers Prev. 4, 253−259.
(51) Fustinoni, S., Soleo, L., Warholm, M., Begemann, P., Rannug, A.,
Neumann, H. G., et al. (2002) Influence of metabolic genotypes on
biomarkers of exposure to 1,3-butadiene in humans. Cancer Epidemiol.,
Biomarkers Prev. 11, 1082−1090.
(52) O’Shea, S. H., Schwarz, J., Kosyk, O., Ross, P. K., Ha, M. J.,
Wright, F. A., and Rusyn, I. (2011) In vitro screening for population
variability in chemical toxicity. Toxicol. Sci. 119, 398−407.
(53) Schilsky, R. L., Dolan, M. E., Bertucci, D., Ewesuedo, R. B.,
Vogelzang, N. J., Mani, S., et al. (2000) Phase I clinical and
pharmacological study of O6-benzylguanine followed by carmustine
in patients with advanced cancer. Clin. Cancer Res. 6, 3025−3031.
(54) Goldenberg, G. J., Lam, H. Y., and Begleiter, A. (1979) Active
carrier-mediated transport of melphalan by two separate amino acid
transport systems in LPC-1 plasmacytoma cells in vitro. J. Biol. Chem.
254, 1057−1064.
(55) London, S. J., Smart, J., and Daly, A. K. (2000) Lung cancer risk in
relation to genetic polymorphisms of microsomal epoxide hydrolase
among African-Americans and Caucasians in Los Angeles County. Lung
Cancer 28, 147−155.
(56) Yoshikawa, M., Hiyama, K., Ishioka, S., Maeda, H., Maeda, A.,
and Yamakido, M. (2000) Microsomal epoxide hydrolase genotypes
and chronic obstructive pulmonary disease in Japanese. Int. J. Mol. Med.
5, 49−53.
(57) Dunnick, J. K., Elwell, M. R., Radovsky, A. E., Benson, J. M.,
Hahn, F. F., Nikula, K. J., et al. (1995) Comparative carcinogenic effects
of nickel subsulfide, nickel oxide, or nickel sulfate hexahydrate chronic
exposures in the lung. Cancer Res. 55, 5251−5256.
(58) Jokipii Krueger, C. C., Madugundu, G., Degner, A., Patel, Y.,
Stram, D. O., Church, T. R., and Tretyakova, N. (2020) Urinary N7-(1-
hydroxy-3-buten-2-yl) guanine adducts in humans: temporal stability
and association with smoking. Mutagenesis 35, 19−26.
K
https://dx.doi.org/10.1021/acs.chemrestox.9b00517
Chem. Res. Toxicol. XXXX, XXX, XXX−XXX