Synthesis and mutagenesis of the butadiene-derived N3 2′-deoxyuridine adducts

Article abstract of DOI:10.1021/tx060016o

1,3-Butadiene is a known carcinogen and mutagen that acts through a variety of metabolic intermediates that react with DNA, forming stable and unstable lesions on dG, dA, dC, and dT. The N3 2′-deoxyuridine adducts are a highly stable, stereoisomeric mixture of adducts derived from the reaction of cytosine with the monoepoxide metabolite of butadiene, followed by spontaneous deamination. In this study, the phosphoramidites and subsequent oligodeoxynucleotides containing the N3 2′-deoxyuridine adducts have been constructed and characterized. Using a single-stranded shuttle vector DNA, the mutagenic potential of these adducts has been tested following replication in mammalian cells. Replication past the N3 2′-deoxyuridine adducts was found to be highly mutagenic with an overall mutation yield of ～97%. The major mutations that were observed were C to T transitions and C to A transversions. In vitro, these adducts posed a complete block to both the Klenow fragment of Escherichia coli polymerase I and polymerase ε, while these lesions significantly blocked polymerase δ. These data suggested a possible involvement of bypass polymerases in the in vivo replication of these lesions. Overall, these findings indicate that the N3 2′-deoxyuridine adducts are highly mutagenic lesions that may contribute to butadiene-mediated carcinogenesis.

Full text of DOI:10.1021/tx060016o

968
Chem. Res. Toxicol. 2006, 19, 968-976
Synthesis and Mutagenesis of the Butadiene-Derived N3
2′-Deoxyuridine Adducts
Priscilla H. Fernandes,† Linda C. Hackfeld,‡ Ivan D. Kozekov,§ Richard P. Hodge,‡ and
R. Stephen Lloyd*,†
Center for Research on Occupational and EnVironmental Toxicology, and Department of Molecular and Medical
Genetics, Oregon Health and Science UniVersity, Portland, Oregon 97239-3098, National Institute of
EnVironmental Health Sciences Center, and Sealy Center for Molecular Science, UniVersity of Texas Medical
Branch, GalVeston, Texas 77555, and Department of Chemistry, Vanderbilt UniVersity,
NashVille, Tennessee 37235
ReceiVed January 24, 2006
1,3-Butadiene is a known carcinogen and mutagen that acts through a variety of metabolic intermediates
that react with DNA, forming stable and unstable lesions on dG, dA, dC, and dT. The N3 2′-deoxyuridine
adducts are a highly stable, stereoisomeric mixture of adducts derived from the reaction of cytosine with
the monoepoxide metabolite of butadiene, followed by spontaneous deamination. In this study, the
phosphoramidites and subsequent oligodeoxynucleotides containing the N3 2′-deoxyuridine adducts have
been constructed and characterized. Using a single-stranded shuttle vector DNA, the mutagenic potential
of these adducts has been tested following replication in mammalian cells. Replication past the N3 2′-
deoxyuridine adducts was found to be highly mutagenic with an overall mutation yield of ∼97%. The
major mutations that were observed were C to T transitions and C to A transversions. In vitro, these
adducts posed a complete block to both the Klenow fragment of Escherichia coli polymerase I and
polymerase ꢀ, while these lesions significantly blocked polymerase δ. These data suggested a possible
involvement of bypass polymerases in the in vivo replication of these lesions. Overall, these findings
indicate that the N3 2′-deoxyuridine adducts are highly mutagenic lesions that may contribute to butadiene-
mediated carcinogenesis.
BD toxicity is mediated by its metabolic activation to reactive
epoxides (reviewed in 15). BD is primarily oxidized to 1,2-
epoxy-3-butene (EB) by cytochrome P450 2E1 and 2A6. EB
can be further oxidized to 1,2,3,4-diepoxybutane (DEB). EB
can also be hydrolyzed by epoxide hydrolase to 1,2-dihydroxy-
3-butene that can be metabolized by cytochrome P450 to
hydroxymethylvinyl ketone (HMVK) (16). 3,4-epoxy-1,2-bu-
tanediol (EBD) can be produced through partial hydrolysis of
DEB and/or epoxidation of hydrolyzed EB. EB, DEB, EBD,
and HMVK can react with nucleophilic sites in DNA to form
their corresponding adducts (17-19). DEB is more genotoxic
than EB, which in turn is more genotoxic than EBD (17, 20,
21). The high genotoxicity of DEB has been attributed to its
ability to form DNA-DNA cross-links (22-24). The ability
of EB, DEB, and EBD to induce toxicity is also affected by
polymorphisms in the enzymes that metabolize them. For
instance, an increase in hprt mutant frequency was observed in
humans with polymorphic variants that confer reduced microso-
mal epoxide hydrolase activity that is responsible for the
detoxification of EB and DEB (25).
The mechanisms underlying BD-induced carcinogenesis are
not clearly understood. Because covalent modification of DNA
represents the initial step to carcinogenesis, one may gain a
better understanding of this process through the study of DNA
adducts of BD. To date, many BD-derived adducts have been
detected following treatment of DNA in vitro, as well as in DNA
isolated from exposed organisms. These include N1, N2, and
N7 adducts of guanine (26-29), N1, N3, and N6 adducts of
adenine (30-33), and the N3 adducts of cytidine and thymidine
(34, 35). Some of these adducts (e.g., N7 guanine and N3
adenine) are unstable, giving rise to apurinic sites. Site-specific
Introduction
1,3-Butadiene (BD)1 is a high volume, industrial chemical
used in the manufacture of synthetic rubber, plastics, and resins.
BD is also a constituent of cigarette smoke, automobile exhaust,
and gasoline vapor, thus making it a relevant environmental
hazard (1, 2). It is an established rodent carcinogen with its
effect being more pronounced in mice than in rats (3, 4). BD-
induced specific genetic alterations lead to the development of
lymphomas, brain tumors, and mammary adenocarcinomas in
mice (5-7). Occupational exposure to BD is correlated with
an increased risk of developing leukemia (8-11). BD has been
classified by the International Agency for Research on Cancer
as a probable human carcinogen (12). More recently, it has been
classified by the National Toxicology Program as a known
human carcinogen (13) and by the U.S. Environmental Protec-
tion Agency as carcinogenic to humans by inhalation (14).
* To whom correspondence should be addressed. Tel: 503-494-9957.
Fax: 503-494-6831. E-mail: lloydst@ohsu.edu.
† Oregon Health and Science University.
‡ University of Texas Medical Branch.
§ Vanderbilt University.
1 Abbreviations: BD, 1,3-butadiene; EB, 1,2-epoxy-3-butene; DEB,
1,2,3,4-diepoxybutane; EBD, 3,4-epoxy-1,2-butanediol; HMVK, hydroxym-
ethylvinyl ketone; BD N3-dU, N3 2′-deoxyuridine; Kf, Klenow fragment
of Escherichia coli polymerase I; pol δ, polymerase δ; PCNA, proliferating
cell nuclear antigen; pol ꢀ, polymerase ꢀ; ss, single-stranded; DMT-dU,
5′-O-dimethoxytrityl-2′-deoxyuridine; DMT-Cl, dimethoxytrityl chloride;
TLC, thin-layer chromatography; Rf, relative front; DMT-dU-Tol, 5′-O-
DMT-3′-O-toluoyl-2′-deoxyuridine; DMT-dU-(Bu-OSi)-Tol, 5′-O-DMT-
3′-O-toluoyl-N3-(2-O-tBDMSi-3-butene)-2′-deoxyuridine; DMT-dU-Bu-
OSi, 5′-O-DMT-2′-deoxyuridine-N3-(2-O-tBDMSi-3-butene); DMT-dU-
BuOSi-CE, 5′-O-DMT-2′-deoxyuridine-N3-(2-O-tBDMSi-3-butene)-3′-O-
(O-cyanoethyl-N,N-diisopropyl)phosphoramidite; tBDMSi, tert-butyl-
dimethylsilyl; CGE, capillary gel electrophoresis.
10.1021/tx060016o CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/01/2006

Synthesis and Mutagenesis of the BD N3-dU Adducts
Chem. Res. Toxicol., Vol. 19, No. 7, 2006 969
Scheme 1. Synthesis of 5′-DMT-3′ p-Tol-2′-deoxyuridine
mutagenesis studies have made it possible to examine the role
of some of the other adducts. These include previous studies in
our lab that have demonstrated that N6 deoxyadenosine and N2
deoxyguanosine adducts are weakly mutagenic in Escherichia
coli (36, 37), while the adducts of N1 deoxyinosine and the
N6,N6 -deoxyadenosine intrastrand cross-link were considerably
mutagenic in E. coli and mammalian cells (38).
The role of BD-derived pyrimidine adducts has not yet been
examined, particularly the N3 cytosine adducts formed from
the reaction of EB with deoxycytidine or calf thymus DNA (31,
35). These adducts were unstable due to epoxidation, and they
subsequently deaminated to form the corresponding N3 2′-
deoxyuridine (BD N3-dU) adducts. These BD N3-dU adducts
were stable toward hydrolysis in DNA and, as such, represent
a potential source of mutagenesis, since this type of alkylation
not only changes the base pairing of the parent nucleoside, but
also has the potential to block DNA replication enzymes.
Mutations at GC base pairs are critical events in BD mutage-
nicity as indicated by studies of mutations in lacI and lacZ mice
and hprt mutational analyses in rodents and humans (reviewed
in 39), thus suggesting modification of cytosine by BD
metabolites and formation of the N3-dU adducts in vivo.
Therefore, the current study focuses on the synthesis of these
BD N3-dU adducts, their incorporation into oligodeoxynucle-
otides, and their subsequent use in cellular and in vitro studies
to examine their effects on replication.
Dimethoxytrityl chloride (DMT-Cl) (4.45 g, 13.14 mmol) was added
at 0 °C (ice bath) and stirred under argon for 30 min. A second
addition of DMT-Cl (4.45 g, 13.14 mmol) was then introduced at
0 °C, and after 10 min, the ice bath was removed and the reaction
was stirred for 2 h. The reaction was monitored by thin-layer
chromatography (TLC) using 100% ethyl acetate as the eluant
[starting material relative front (Rf) ) 0.01, product Rf ) 0.40].
The reaction was quenched by cooling to 0 °C, followed by addition
of 40 mL of methanol and stirring for 15 min. The reaction mixture
was then evaporated to a minimum under reduced pressure and
subsequently redissolved in 100 mL of methylene chloride. The
mixture was then neutralized with 5% aqueous sodium bicarbonate
while stirring until the aqueous layer remained at pH 8. The layers
were then separated, and the aqueous layer was extracted a second
time with fresh methylene chloride. The organic layers were then
combined, dried over sodium sulfate, filtered, and evaporated to
give 14.2 g of crude DMT-dU. This was purified by silica gel
chromatography using a step gradient from 49.9% ethyl acetate:
49.9% hexane:0.2% triethylamine to 99.8% ethyl acetate:0.2%
triethylamine. Fractions containing the product were combined and
evaporated to give 10.2 g (87.7%) of DMT-dU or product 1
Materials and Methods
Materials. All solvents and reagents for the synthesis of the BD
N3-dU were obtained from Fisher-Acros or Aldrich Chemicals
except for bis-N,N,N′,N′-tetraisopropyl-cyanoethyl phosphine, which
was obtained from Chemgenes (Cambridge, MA). MALDI-TOF
mass spectra of modified oligodeoxynucleotides were performed
on an ABI Voyager 4211 system using R-cyano-4-hydroxycinnamic
acid as the matrix and calibrated against 10 and 4 kDa oligode-
oxynucleotide standards from Genosys. T4 DNA ligase, T4
polynucleotide kinase, EcoRV, and Klenow fragment of E. coli
polymerase I (Kf) were obtained from New England BioLabs
(Ipswich, MA). S1 nuclease and proteinase K were purchased from
Invitrogen (Rockville, MD). Calf thymus DNA polymerase δ (pol
δ) and proliferating cell nuclear antigen (PCNA) were generous
gifts from Dr. K. M. Downey (University of Miami, Miami, FL).
DNA polymerase ꢀ (pol ꢀ) from HeLa cells was kindly provided
by Dr. S. Linn (University of California, Berkeley, CA), and yeast
DNA pol δ was a generous gift from Dr. P. M. Burgers (Washington
University School of Medicine, St. Louis, MO). Single-stranded
(ss) pMS2 DNA was a generous gift from Dr. M. Moriya (State
University of New York, Stony Brook, NY). [γ32P] Ribo-ATP was
purchased from PerkinElmer Life Science (Boston, MA). Bio-Spin
columns were purchased from Bio-Rad (Hercules, CA). Centricon
100 concentrators were obtained from Amicon Inc. (Beverly, MA).
Dulbecco’s modified Eagle’s medium, fetal bovine serum, Opti-
MEM (reduced serum medium), L-glutamine, antibiotic-antimycotic,
and Lipofectin reagent for tissue culture were obtained from
Invitrogen. Trypsin-EDTA and HEPES buffer were purchased from
Cellgro Mediatech (Herndon, VA). COS-7 cells were purchased
from American Type Culture Collection (Rockville, MD). Non-
damaged 12-mer and 32-mer with a deoxycytosine (dC) in place
of BD N3-dU were purchased from Midland Reagent Co. (Midland,
TX).
1
(Scheme 1). H NMR (d6-DMSO): δ 11.3 (s, 1H, NH), 7.63 (d,
1H, J ) 7.53, H-6), 7.37 (d, 2H, J ) 7.87, DMT), 7.31 (m, 2H,
DMT), 7.24 (m, 5H, DMT), 6.90 (m, 4H, DMT), 6.14 (m, 1H,
H-1′), 5.37 (d, 1H, J ) 7.87, H-5), 5.32 (d, 1H, J ) 4.79, 3′-OH),
4.27 (m, 1H, H-4′), 3.87 (m, 1H, H-3′), 3.74 (s, 6H, DMT-OCH3s),
3.23 (m, 1H, H-5′), 3.17 (m, 1H, H-5′′), 2.18 (m, 2H, H-2′, H-2′′).
2. Synthesis of 5′-O-DMT-3′-O-toluoyl-2′-deoxyuridine (DMT-
dU-Tol). DMT-dU (500 mg, 0.942 mmol) was added to a 100 mL
flask with 5 mL of dry pyridine under argon with stirring. Once
dissolved, 150 µL of p-toluoyl chloride (1.13 mmol) was added
and the mixture was stirred for 3 h at room temperature under argon.
The reaction was monitored by TLC using 100% ethyl acetate as
the eluant (starting material Rf ) 0.40, product Rf ) 0.75). The
product was recovered by diluting the mixture with 20 mL of
methylene chloride and carefully washing with 20 mL of saturated
sodium bicarbonate. The organic layer was then separated, dried
over sodium sulfate, filtered, and evaporated to yield 720 mg of
crude product that was then purified by silica gel chromatography
using 49.9% ethyl acetate:49.9% hexane:0.2% triethylamine as
eluant. The pure product fractions were pooled and evaporated to
yield 460 mg (75%) of product 2 (Scheme 1). 1H NMR (d6-
DMSO): δ 11.3 (br s, 1H, N3-H), 7.89 (d, 2H, J ) 6.84,
p-toluoyl), 7.69 (d, 1H, J ) 7.87, H-6), 7.36 (m, 4H, DMT +
p-toluoyl), 7.29 (m, 2H, DMT), 7.24 (m, 5H, DMT), 6.87 (m, 4H,
DMT), 6.23 (m, 1H, H-1′), 5.50 (m, 1H, H-3′), 5.47 (d, 1H, J )
7.87, H-5), 4.23 (m, 1H, H-4′), 3.72 (s, 6H, DMT-OCH3s), 3.41
(m, 2H, H-5′, H-5′′), 2.59 (m, 2H, H-2′, H-2′′), 2.40 (s, 3Hs,
p-toluoyl CH3s).
Synthesis of the BD N3-dU Adducts and Their Incorporation
into Oligodeoxynucleotides.
1. Synthesis of 5′-O-Dimethoxytrityl-2′-deoxyuridine (DMT-
dU). Synthesis of DMT-dU was performed based on methods
described by Beaucage et al. (40). 2′-dU (5 g, 21.9 mmol) was
coevaporated twice with anhydrous pyridine in a 250 mL flask and
redissolved with 100 mL of fresh anhydrous pyridine under argon.
3. Synthesis of 2-Hydroxy-3-butene-1-(p-toluenesulfonate).
This synthesis was based on methods used by Martinelli et al. (41).
Dibutyltin oxide (2.82 g, 11.35 mmol) was added to 1,2-dihydroxy-
3-butene (R,S mixture) (10 g, 113.5 mmol) in methylene chloride

970 Chem. Res. Toxicol., Vol. 19, No. 7, 2006
Fernandes et al.
Scheme 3. Synthesis of 5′-DMT-N3-(3-buten-2-O-
Scheme 2. Synthesis of
2-O-t-Butyldimethylsilyl-3-butene-1-(p-toluenesulfonate)
t-butyldimethylsilyl-1yl)-2′-deoxyuridine-3′-O-
(N,N-diisopropyl-O-cyanoethyl)phosphoramidite
(200 mL). Toluenesulfonyl chloride (21.6 g, 113.5 mmol) and
triethylamine (16 mL, 113.5 mmol) were added, and the mixture
was stirred at room temperature overnight. Water (50 mL) was
added, and the layers were separated. The aqueous layer was
extracted twice with methylene chloride (50 mL each), and the
combined organic layers were washed with water and saturated
NaCl solution (100 mL each), dried over sodium sulfate, filtered,
and evaporated to give 3 (Scheme 2) as an oil that crystallized
upon cooling (26.8 g, 97% yield). 1H NMR (CDCl3): δ 7.78 (d, J
) 8.27, 2H, Tos-ortho Hs), 7.33 (d, J ) 8.07, 2H, Tos-meta Hs),
5.73 (m, 1H, H-3), 5.34 (d, J ) 17.15, 1H, H-4a), 5.21 (d, J )
10.49, 1H, H-4b), 4.37 (m, 1H, H-2), 4.03 (dd, J ) 3.43, 1H, H-1b),
3.89 (dd, J ) 7.26, 1H, H-a), 2.50 (s, 1H, OH), 2.41 (s, 3H, Tos-
CH3).
4. Synthesis of 2-O-tert-butyldimethylsilyl-3-butene-1-(p-tolu-
enesulfonate). 2-Hydroxy-3-butene-1-(p-toluenesulfonate) (130 mg,
0.537 mmol) was added to 500 µL of anhydrous dimethylformamide
under argon. Anhydrous potassium carbonate (74.2 mg, 0.537
mmol) was added, and the mixture was stirred under argon for 5
min. tert-Butyldimethylsilyl chloride (89 mg, 0.59 mmol) was then
added, and the mixture was stirred under argon at room temperature
overnight. The reaction was assayed by TLC (20% ethyl acetate/
hexane). The reaction products showed over an 80% yield (Rf )
0.85). The mixture was then filtered of solids, washed with
methylene chloride, evaporated to a minimum, and purified by flash
chromatography column (10-20% gradient ethyl acetate/hexane)
to give 4 (86.7 mg, 45.7%) along with 25 mg of recovered 3
(19.2%) (Scheme 2). 1H NMR (CDCl3): δ 7.78 (d, 2H, J ) 7.76,
Tos-ortho Hs), 7.34 (d, 2H, J ) 7.76, Tos-meta Hs), 5.71 (m, 1H,
H-3), 5.31 (d, 1H, J ) 17.22, H-4 trans), 5.17 (d, 1H, J ) 10.13,
H-4 cis), 4.34 (m, 1H, H-2), 3.91 (m, 1H, H-1a), 3.82 (m, 1H,
H-1b), 2.45 (s, 3H, Tos-CH3), 0.86 (s, 9H, tert-Butyl-Si-CH3s), 0.04
(s, 3H, Si-CH3), 0.03 (s, 3H, Si-CH3).
5. Synthesis of 5′-O-DMT-3′-O-toluoyl-N3-[2-O-tert-butyldim-
ethylsilyl (tBDMSi)-3-butene]-2′-deoxyuridine [DMT-dU-(Bu-
OSi)-Tol]. DMT-dU-Tol (350 mg, 0.54 mmol) was dissolved in 5
mL of anhydrous dimethyl sulfoxide in a 20 mL flask under argon
with stirring. Anhydrous potassium carbonate (100 mg) was added,
and the mixture was stirred under argon for 10 min before adding
2-O-tert-butyldimethylsilyl-3-butene-1-(p-toluenesulfonate) (200
mg, 0.562 mmol). The mixture was sealed and stirred under argon
for 8 days. The crude mixture was added directly to a silica gel
column packed in 25% ethyl acetate/hexane and eluted by step
gradient to 33% ethyl acetate/hexane resulting in a yield of 215
mg of product 5 (47%) (Scheme 3). 1H NMR (CDCl3): δ 7.94 (d,
2H, J ) 8.06, p-toluoyl-ortho Hs), 7.69 (m, 1H, H-6), 7.30 (d, 2H,
J ) 8.06, p-toluoyl-meta Hs), 7.27 (m, 5H, DMT-Bz), 7.17 (d,
4H, J ) 8.79, DMT- ortho Hs), 6.84 (d, 4H, J ) 8.42, DMT-meta
Hs), 6.34 (m, 1H, H-1′), 5.86 (m, 1H, butenol H-3), 5.81 (d, 1H,
J ) 8.06, H-5), 5.57 (m, 1H, H-3′), 5.24 (d, 1H, J ) 17.58, butenol
H-4 trans), 5.12 (d, 1H, J ) 9.16, butenol H-4 cis), 4.55 (m, 1H,
butenol H-2), 4.26 (s, 1H, H-4′), 4.17 (m, 1H, butenol H-1a), 4.01
(s, 2H, H-5′, 5′′), 3.83 (m, 1H, butenol H-1b), 3.81 (s, 3H, DMT-
OCH3), 3.80 (s, 3H, DMT-OCH3), 2.59 (m, 1H, H-2′), 2.50 (m,
1H, H-2′′), 2.44 (s, 3H, p-toluoyl-CH3), 0.85 (s, 9H, silyl tert-butyl
CH3s), -0.01 (s, 3H, silyl CH3), -0.04 (s, 3H, silyl CH3).
6. Synthesis of 5′-O-DMT-2′-deoxyuridine-N3-(2-O-tBDMSi-
3-butene) (DMT-dU-Bu-OSi). DMT-dU-(Bu-OSi)-Tol (190 mg,
0.228 mmol) was dissolved in 10 mL of anhydrous methanol in a
sealed 50 mL flask under argon. To this was added 276 mg of
anhydrous potassium carbonate, and the suspension was stirred
under argon for 3 h. TLC (49.9% ethyl acetate:49.9% hexane:0.2%
triethylamine) showed complete deprotection. The reaction was
quenched with 40 mL of 0.5 M sodium phosphate buffer (pH 5.5)
and quickly extracted with 100 mL of methylene chloride. The
methylene chloride fraction was dried over sodium sulfate, filtered,
and evaporated. The crude product was purified by preparative
CycloGraph TLC using a 25% ethyl acetate to 50% ethyl acetate
(in 1% ethylamine/hexane) step gradient. The product was moni-
tored by UV 254 nm and collected to yield 160 mg of 6 (98%)
(Scheme 3). HPLC analysis showed the product to be greater than
1
99% pure. H NMR (CDCl3): δ 7.53 (m, 1H, H-6), 7.27 (m, 5H,
DMT-Bz), 7.18 (d, 4H, J ) 7.69, DMT- ortho Hs), 6.83 (d, 4H, J
) 7.69, DMT-meta Hs), 6.17 (m, 1H, H-1′), 5.85 (m, 1H, butenol
H-3), 5.77 (d, 1H, J ) 7.69, H-5), 5.23 (d, 1H, J ) 17.21, butenol
H-4 trans), 5.11 (d, 1H, J ) 10.26, butenol H-4 cis), 4.59 (m, 1H,
H-3′), 4.53 (m, 1H, butenol H-2), 4.15 (m, 1H, butenol H-1a), 4.03
(s, 1H, H-4′), 3.94 (m, 1H, butenol H-1b), 3.84 (m, 2H, H-5′, 5′′),
3.81 (s, 6H, DMT-OCH3s), 2.40 (m, 2H, H-2′, 2′′), 0.84 (s, 9H,
silyl tert-butyl CH3s), -0.01 (s, 3H, silyl CH3), -0.06 (s, 3H, silyl
CH3).
7. Synthesis of 5′-O-DMT-2′-deoxyuridine-N3-(2-O-tBDMSi-
3-butene)-3′-O-(O-cyanoethyl-N,N-diisopropyl) (DMT-dU-BuOSi-
CE) Phosphoramidite. DMT-dU-Bu-OSi (112 mg, 0.16 mmol) 6
was dissolved with 1 mL of dry methylene chloride in a 20 mL
flask under argon. To this was added simultaneously via syringe,
while stirring under argon, 71 µL (0.224 mmol) of neat bis-
N,N,N′,N′-tetra-isopropyl-O-cyanoethyl-phosphine and 448 µL
(0.192 mmol) of tetrazole solution in tetrahydrofuran (30 µg/µL).
The solution was stirred for 1 h under argon at room temperature.
TLC (29.9% ethyl acetate:69.9% hexane:0.2% triethylamine, prerun
TLC plates) showed that the reaction had proceeded to completion.
The mixture was extracted with ethyl acetate and 5% sodium
bicarbonate, and the ethyl acetate fraction was further extracted
with saturated aqueous NaCl. The ethyl acetate fraction was then
dried over sodium sulfate, filtered, and evaporated. The crude
product was purified using a preparative TLC CycloGraph system
prewetted and run with the same TLC solvent above. Pure product
fractions were pooled to yield 95 mg of pure product 7 (67%)
1
(Scheme 3). H NMR (300 MHz, CD3CN): δ 7.63 (m, 1H, H-6),
7.42 (m, 2H, DMT-ortho Hs), 7.25 (m, 7H, DMT- ortho Hs and
Bz), 6.85 (m, 4H, DMT-meta Hs), 6.19 (m, 1H, H-1′), 5.84 (m,
1H, butenol H-3), 5.41 (m, 1H, H-5), 5.23 (d, 1H, J ) 17.3, butenol

Synthesis and Mutagenesis of the BD N3-dU Adducts
Chem. Res. Toxicol., Vol. 19, No. 7, 2006 971
H-4 trans), 5.10 (d, 1H, J ) 10.5, butenol H-4 cis), 4.60 (m, 1H,
H-3′), 4.53 (m, 1H, butenol H-2), 4.05 (m, 2H, butenol H-1a and
H-4′), 3.76, (s, 3H, DMT-OCH3), 3.75 (s, 3H, DMT-OCH3), 3.62
(m, 1H, butenol H-1b), 3.60 (m, 2H, H-5′, 5′′), 3.33 (m, 2H,
isopropyl CH3s) 2.63 (t, 1H, OCH, cyanoethyl), 2.52 (t, 1H, OCH,
cyanoethyl), 2.42 (m, 1H, H-2′), 2.27 (m, 1H, H-2′′), 1.93 (m, 2H,
CH2CN), 1.16 (m, 6H, isopropyl CH3s), 0.82 (s, 9H, silyl tert-
butyl CH3s), -0.03 (s, 3H, silyl CH3), -0.10 (s, 3H, silyl CH3).
31P NMR (121.5 MHz, CD3CN): δ 147.3, 147.2.
2. Enzymatic Hydrolysis. The 12-mer oligodeoxynucleotide 5′-
GCTAGCXAGTCC-3′ (0.3 A260 units) was dissolved in 30 µL of
10 mM Tris-HCl buffer (pH 7.0) containing 10 mM MgCl2 and
incubated with DNase I (8 units, Promega), snake venom phos-
phodiesterase I (0.02 units, Sigma), and alkaline phosphatase (1.7
units, E. coli; Sigma) at 37 °C for 24 h. The mixture was analyzed
by reversed phase HPLC. The adducted nucleosides were identified
by comparison with authentic samples based on retention times,
coinjection, and ultraviolet spectra. HPLC analyses of the enzymatic
digestion were performed on a Beckman HPLC system (32 Karat
software version 3.1, pump module 125) with a diode array UV
detector (module 168) monitoring at 260 nm using a Waters YMC
ODS-AQ columns (250 mm × 4.6 mm i.d., 1.5 mL/min) with H2O
and CH3CN using the following gradient: 1-10% acetonitrile over
15 min, 10-20% acetonitrile over 5 min, hold for 5 min, 100%
acetonitrile over 3 min, hold for 2 min, and then to 1% acetonitrile
over 3 min.
3. DNA Sequencing. The 12-mer oligodeoxynucleotide 5′-
GCTAGCXAGTCC-3′ (0.03 A260 units) in 24 µL of ammonium
hydrogen citrate buffer (pH 9.4) containing 20 mM MgSO4 was
incubated at 37 °C with 2 milliunits of snake venom phosphodi-
esterase I. Aliquots (4 µL) were taken before enzyme addition and
at 1, 8, 18, 28, and 38 min time points after enzyme addition. The
aliquots were combined and kept frozen until the analysis. The
combined aliquots were desalted using a Millipore C18 Ziptips and
eluted directly onto a MALDI plate in 3-hydroxypicolinic acid
containing ammonium hydrogen citrate (7 mg/mL). MALDI-TOF
analysis gave a sequencing ladder from the 3′ f 5′ direction. An
identical analysis was performed using 12 milliunits of bovine
spleen phosphodiesterase II in 24 µL of 20 mM ammonium acetate
buffer (pH 6.6) that provided a complementary sequencing ladder
from the 5′ f 3′ direction.
Construction of Circular ss pMS2 DNAs Containing the BD
N3-dU Adducts. The methodologies for introducing site-specific
DNA lesions into the ss pMS2 vector (42) were performed as
described by Kanuri et al. (43) and Fernandes et al., (44). In brief,
ss pMS2 DNA (59 pmol) was annealed to a 58-mer scaffold (295
pmol), and the annealed product was digested with EcoRV to release
the hairpin loop structure of ss pMS2 DNA. This procedure results
in the creation of a precise gap in the DNA. The scaffolding DNA
was designed such that 22 nucleotides on the 5′-end and 24
nucleotides on the 3′-end were complementary to the two termini
of the digested ss pMS2. The 58-mer also had a central sequence
that was complementary to the 12-mer oligodeoxynucleotide
sequence 5′-GCTAGCXAGTCC-3′ where X is control dC or BD
N3-dU. To ensure that scaffolding DNA was not used as a substrate
for replication in COS-7 cells, all thymines were substituted by
uracil in the 58-mer scaffold. The BD N3-dU 12-mer and the control
12-mer were phosphorylated at the 5′-end using T4 DNA kinase
(5 units/pmol) and then ligated (325 units of T4 DNA ligase/pmol)
into the partially duplex pMS2 vector. The ligated DNAs were then
purified using Centricon 100 concentrators (to remove unligated
12-mers) and recovered by ethanol precipitation. The products
obtained were treated with uracil DNA glycosylase for 1 h to
severely damage the scaffold DNA and prevent it from being used
as a primer for (-)-strand replication.
Site-Specific Mutagenesis in COS-7 Cells. The overall strategy
for replication and mutagenic analyses was as previously described
(43, 44). COS-7 cells were grown to approximately 70% confluency
and transfected with 1 µg of control or BD N3-dU-modified DNAs
using lipofectin. The transfection medium was replaced after 24 h
with DMEM medium containing 10% fetal bovine serum. Following
replication for 48 h, the COS-7 cells were harvested and their
progeny plasmid was recovered as a Hirt supernatant (45). To ensure
removal of any input ss DNA and progeny associated with the
original ss pMS2 vector, the DNAs so obtained were treated with
1 unit of S1 nuclease in a 60 µL reaction mixture and with 20
units of EcoRV in a 70 µL reaction mixture.
8. Oligodeoxynucleotide Synthesis and Purification. Two
oligodeoxynucleotides (12-mer 5′-GCTAGCXAGTCC-3′ and 32-
mer 5′-ACCATGCCTGCAAGAAXTAAGCAATGATCGCC-3′)
were synthesized incorporating N3-(3-butene-2-O-tert-butyldim-
ethylsilyl)-2′-deoxyuridine in place of a 2′-deoxycytidine at site X
using phosphoramidite chemistry and standard oligodeoxynucleotide
synthesis procedures. Oligodeoxynucleotides were synthesized at
the 1.0 µM scale using a PerSeptive Biosystems Expedite 8909
DNA synthesizer with off-line coupling of the DMT-dU-BuOSi-
CE phosphoramidite by placing a hold in the sequence program at
the coupling step and introducing the modified nucleoside by mixing
of the phosphoramidite (10 mg/100 µL anhydrous acetonitrile) and
tetrazole (3 mg/100 µL anhydrous acetonitrile) solutions across the
synthesis cassette via syringes. The coupling was carried out for
20 min with occasional mixing, followed by placement of the
cassette back on the synthesizer, washing with acetonitrile, and
continuation of the sequence synthesis program. Standard phos-
phoramidites were used for all other coupled bases, and the final
5′-dimethoxytrityl group was left on. Oligodeoxynucleotide cassettes
were treated using standard cleavage from the support (1 h
concentrated ammonia at room temperature) and deprotection of
the resulting oligodeoxynucleotide/ammonia solution (60 °C for 16
h). Following deprotection, an aliquot of the crude sample was
subjected to analytical HPLC analysis [Waters Delta Pak C-18,
0-20% acetonitrile gradient in 0.1 M ammonium formate (pH 7.2)
for 20 min, at 1 mL/min flow rate], and the bulk of the product
was evaporated to a minimum on a Savant SpeedVac system. The
5′-dimethoxytrityl containing full-length oligodeoxynucleotides was
then purified on C-18 Sep Pak cartridges. A sample of this material
was also analyzed by HPLC using the same system with a more
shallow gradient (4-10% acetonitrile, 20 min). Purification of the
base-protected oligodeoxynucleotides after DMT removal was
conducted on a preparative Hamilton PRP column using a 5 mL/
min flow rate and gradient of 2-10% acetonitrile in 0.1 M
ammonium formate (pH 7.2).
9. Deprotection of t-BDMSi from the Oligodeoxynucleotides.
The remaining tert-butyldimethylsilyl protecting groups on the
butenol side chain on N3-adducted deoxyuridine oligodeoxynucle-
otides were removed using 200 µL of a 67/33 mixture of anhydrous
1.0 M tetrabutylammonium fluoride/neat triethylammonium hy-
drofluoride solution overnight at 0-4 °C, followed by Nap-10
purification against 0.1 M sodium phosphate buffer (pH 7.2) to
remove residual tert-butyldimethylsilyl fluoride and reagents. This
was necessary since triethylammonium hydrofluoride alone was too
acidic, causing noticeable degradation of the oligodeoxynucleotides,
whereas tetrabutylammonium fluoride deprotection alone failed to
achieve greater than 60% deprotection. All products were analyzed
by HPLC and MALDI-TOF mass spectrometry. The observed pre-
and postdesilylation mass spectrometric values (vs calculated values)
were 3789.4 (3789.7) and 3675.4 (3675.7) for the 12-mer and
9951.6 (9951.8) and 9836.5 (9837.7) for the 32-mer, respectively,
within 0.012% of instrument accuracy.
Characterization of the 12-mer Oligodeoxynucleotide Con-
taining the BD N3-dU Adduct.
1. Capillary Gel Electrophoresis (CGE). Electrophoretic
analyses were carried out using a Beckman P/ACE MDQ instrument
system (using 32 Karat software, version 5.0) monitored at 260
nm on a 31.2 cm × 100 µm eCAP capillary, with samples applied
at 10 kV and run at 9 kV. The capillary was packed with 100-R
gel (for ss DNA) using the Tris-borate buffer system containing 7
M urea.
The Hirt supernatant DNAs were used to transform E. coli DH5R
cells via electroporation, and the resultant transformants were
selected on LB plates containing 100 µg/mL ampicillin. To detect

972 Chem. Res. Toxicol., Vol. 19, No. 7, 2006
Fernandes et al.
the presence of mutants, the transformant colonies were individually
picked and grown overnight at 37 °C in 96 well plates containing
ampicillin-supplemented LB medium. These cultures were then
transferred, via a 48 pin replicator, onto Whatman 541 filter papers
on ampicillin-containing LB plates in four replicates (one for each
probe), for both the adducts and the control. The E. coli on filter
papers were then incubated overnight at 37 °C to form distinct
colonies that were then lysed and differentially hybridized using
the four probes, 5′-GATGCTAGCNAGTCCATC-3′, where N refers
to G, C, A, or T.
In Vitro Replication Using BD N3-dU Containing 32-mer
Template. In vitro polymerase reactions were carried out using
14-mer oligodeoxynucleotide primers annealed to the template, 5′-
ACCATGCCTGCAAGAAXTAAGCAATGATCGCC-3′, where X
represents the site of the BD N3-dU adducts or control dC. Primer
oligodeoxynucleotides were first phosphorylated with T4 poly-
nucleotide kinase using [γ-32P] ATP and purified using P-6 Bio-
Spin columns supplied with Tris-HCl buffer (pH 7.4). The 32P-
labeled primers were then mixed with either adducted or nonadducted
template oligodeoxynucleotides at a molar ratio of 1:2 in 25 mM
Tris HCl buffer (pH 7.6), 50 mM NaCl, heated at 90 °C for 2 min,
and cooled to room temperature overnight. To confirm the
completion of primer annealing, aliquots were assayed on a 7.5%
native PAGE. The Kf reaction mixture contained 5 nM primer/
template DNA in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM
MgCl2, 1 mM dithiothreitol, and dNTPs at a final concentration of
100 µM and were incubated for 15 min at room temperature with
increasing concentrations (0.0005-0.5 units) of the polymerase
(where one unit is defined as the amount of enzyme required to
convert 10 nmol of dNTPs to an acid-insoluble material in 30 min
at 37 °C). The pol δ reaction mixture contained 5 nM primer/
template DNA in 40 mM HEPES-KOH (pH 6.8), 10% glycerol,
200 µg/mL BSA, 6 mM MgCl2, 1 mM dithiothreitol, 70 ng of calf
thymus PCNA, and dNTPs at a final concentration of 100 µM and
was incubated for 15 min at room temperature with increasing
concentrations of pol δ from 0.008 to 0.8 units of polymerase. The
pol ꢀ reaction mixture contained 5 nM primer/template DNA in 50
mM HEPES-KOH (pH 7.5), 100 mM potassium glutamate, 20%
glycerol, 200 µg/mL BSA, 15 mM MgCl2, 10 mM dithiothreitol,
0.03% Triton X-100, 100 µM each of the dNTPs, and 0.1 unit of
the polymerase. These reactions were carried out for increasing
time intervals (0, 1, 5, 10, 15, 20, and 30 min) at room temperature.
All reactions were terminated by the addition of stop solution
consisting of 95% (v/v) formamide, 20 mM EDTA, 0.02% (w/v)
xylene cyanol, and 0.02% (w/v) bromphenol blue. Reaction products
were resolved on a 15% denaturing PAGE and visualized by
autoradiography. Quantitative analyses were performed using a
PhosphorImager screen and Image-Quant software.
Figure 1. Analysis of 12-mer oligodeoxynucleotide 5′-GCTAGCX-
AGTCC-3′. (A) CGE profile. (B) HPLC profile of the enzymatic
hydrolysate. The HPLC trace of an authentic sample of the adducted
nucleoside is superimposed.
for DNA synthesis. HPLC analysis of the 12-mer dU-BuOSi
oligodeoxynucleotide showed two distinct overlapping species;
however, they could not be independently isolated upon further
purification. Subsequently, the silyl protecting group was
removed by fluoride treatment and Nap-10 purification to give
the BD N3-dU 12-mer oligodeoxynucleotide that was used as
the R, S mixture in all further experiments. HPLC analysis of
the 32-mer dU-BuOSi oligodeoxynucleotide also showed two
distinct overlapping species; however, these could not be
independently isolated upon further purification. Subsequently,
this product was treated and purified as above to remove the
silyl protecting group and used as the R, S mixture in all further
experiments.
The 12-mer adducted oligodeoxynucleotide used for the
construction within the pMS2 shuttle vector was analyzed by
CGE and judged to be >96% pure (Figure 1A). Under the CGE
conditions used, the diastereomers could be partially resolved.
Enzymatic digestion of the oligodeoxynucleotide using a mixture
of DNase I, snake venom phosphodiesterase I, and alkaline
phosphatase, followed by HPLC analysis, showed the appropri-
ate nucleosides in the correct molar ratios (Figure 1B). The
modified nucleoside was identified by comparison of the
retention time with an authentic sample and by coinjection.
The oligodeoxynucleotide was then sequenced by partial
enzymatic digestion with snake venom phosphodiesterase I.
Aliquots were taken at various time points, combined, and then
analyzed by MALDI-TOF mass spectrometry. A sequencing
ladder was obtained as a result of sequential loss of the
nucleotides from the 3′ f 5′ direction (46) (Figure 2A). For
phosphodiesterase I, the digestion was arrested at the adducted
nucleotide. A complementary analysis was performed using
bovine spleen phosphodiesterase II to determine the sequence
from the 5′ f 3′ direction (Figure 2B). In this analysis, the
progress of the phosphodiesterase II was halted one base before
the adducted nucleotide. Although the nucleases were unable
to digest through the adducted nucleotide on the time scale of
these analyses, the results are consistent with the sequence noted.
Replication and Mutagenesis of DNA Containing the Site-
Specific N3-dU Adducts of Butadiene. The ss pMS2 shuttle
vector was annealed to a scaffold and digested with EcoRV to
create a gap into which the BD N3-dU adducted or control dC
Results
Synthesis and Characterization of the BD N3-dU Contain-
ing Oligodeoxynucleotides. Standard protecting group chem-
istries were utilized in the formation of the 2′-deoxyuridine
precursor 2 for alkylation (Scheme 1). For the synthesis of the
buten-ol alkylating reagent 4 (Scheme 2), the C1 alcohol was
selectively protected in high yield as the p-toluenesulfonate ester
3 using the R-chelating method of Martinelli et al. (41). The
toluenesulfonate group served as both protections for subsequent
silylation of the C2 alcohol to give 4 and as the leaving group
for the alkylation reaction at N3 of 2′-deoxyuridine. However,
the alkylation reaction of 2 under aprotic conditions was very
slow and did not go to completion (47%) (Scheme 3). Even
after 8 days, unreacted starting materials were still recoverable.
The p-toluoyl group of the C-3′ alcohol of alkylated product 5
was readily removed by a protic solution of KOH, and the
resulting product 6 was then phosphitylated using the bis-
N,N,N′N′-tetraisopropyl-2-O-cyanoethyl-phosphine procedure to
give the required phosphoramidite 7 in good yield and purity

Synthesis and Mutagenesis of the BD N3-dU Adducts
Chem. Res. Toxicol., Vol. 19, No. 7, 2006 973
Figure 2. MALDI-TOF analysis of partial exonuclease digestion of 12-mer oligodeoxynucleotide 5′-GCTAGCXAGTCC-3′. (A) 3′f5′ sequencing
using phosphodiesterase I. (B) 5′f3′ sequencing using phosphodiesterase II.
Table 1. Mutation Yield of the BD N3-dU Adducts in COS-7
Mammalian Cells
%
C
C
C
total
ss vector pMS2
containing
no. of
to to
to mutation
colonies nonmutagenic
G
A
T
0
yield
control dC
BD N3-dU adducts
199
191
100
3.1
0
0
0
11 32.5 53.4
96.9
C to G transversions (11%). To verify the correct identification
of mutant sequences derived by the differential hybridization
method, random colonies that hybridized to specific probes were
sequenced using a primer located upstream from the original
adducted site. The sequencing data confirmed that the point
mutations detected by autoradiography were properly identified,
and these data did not reveal any alternate replication bypass
events such as deletions or frameshifts. These analyses indicate
that these lesions primarily (if not exclusively) induce point
mutations.
In Vitro Replication. To assess the effect of the BD N3-dU
lesions on in vitro replication, a labeled 14-mer primer was
annealed to the 32-mer template containing the BD N3-dU
adducts or control dC and several polymerases assayed for their
ability to replicate past these lesions. The location of the 3′-
OH was such that the polymerases would incorporate one
nucleotide prior to incorporation opposite the lesion. Replication
past the adducts was first tested with Kf. On the nondamaged
DNA substrate, an efficient extension of the primers was
observed (Figure 4, lanes 2-5). In contrast, on the BD N3-dU-
containing substrate, after one nucleotide was incorporated,
indicating efficient loading and polymerization, further synthesis
was greatly reduced, suggesting that the BD N3-dU adducts
posed a near complete block to further replication. No formation
of the full-length size products was detected (Figure 4, lanes
7-10).
Mammalian replicative polymerases δ and ꢀ were also
examined for their ability to bypass these lesions. In the presence
of pol δ and PCNA, the primer was extended by one nucleotide
but further polymerization opposite the BD N3-dU adducts was
greatly reduced (Figure 5A, lanes 6-8). However, partial bypass
of the lesions was observed (Figure 5A, lane 8) under conditions
that allowed nearly complete replication of the nondamaged
DNA template (Figure 5A, lane 4). PCNA was added to this
reaction since it is a pol δ processivity factor known to stimulate
the bypass of certain DNA lesions [γ-hydroxypropano deox-
yguanosine (43) and thymine dimers (47)], even on DNA
templates that are not capped at their termini to restrict the
Figure 3. Autoradiographs displaying the result of mutagenic replica-
tion past the BD N3-dU adducts in COS-7 mammalian cells. ss pMS2
vector containing the BD N3-dU adducts or control dC was transfected
into COS-7 cells and replicated for 48 h. The progeny plasmids were
then recovered from the COS-7 cells and used to transform DH5R E.
coli cells. Individual transformants were picked and grown in 96-well
plates containing LB with ampicillin. The resultant colonies were
transferred in four replicates onto Whatman 541 filter papers, one for
each of the four probes G, C, A, and T, and were differentially
hybridized.
oligodeoxynucleotide was ligated, followed by digestion of the
scaffold. These modified vectors were replicated in COS-7 cells,
and the resultant progeny DNAs were transformed into DH5R
E. coli cells. There was no appreciable difference in the absolute
number of transformants between the adducted and the control
DNAs, implying that these lesions may not pose total blocks to
replication within cells. These transformants were subjected to
differential hybridization (43, 44) using one of the four probes
(5′-GATGCTAGCNAGTCCATC-3′ where N refers to G, C,
A, or T). Typical autoradiographic results are shown in Figure
3. The intensely hybridizing cells were scored as positive
colonies that contained the progeny DNAs derived from the
replication of adduct-containing or control sequences. All
experimental procedures were repeated in triplicate, and ∼190
colonies were analyzed for both adduct-containing and control
oligodeoxynucleotides. The overall mutation yield for the BD
N3-dU adducts was around 97%, while no mutational events
were detected with the control dC (Table 1). The predominant
mutations were C to T transitions (53.4%) and C to A
transversions (32.5%), followed by a much lower frequency of

974 Chem. Res. Toxicol., Vol. 19, No. 7, 2006
Fernandes et al.
tions of pol ꢀ that enabled complete polymerization on the
unmodified templates.2
Discussion
Research in BD-mediated carcinogenesis has included the
identification of those DNA adducts that may induce mutagen-
esis. Site-specific methodologies have made it possible to
evaluate the effect of individual DNA adducts on replication
as compared to general BD exposure studies. Of the various
BD-derived DNA monoadducts analyzed through these methods,
the two most highly mutagenic adducts found were the N1-
inosine adducts (38) and the BD N3-dU adducts as shown in
this study. The N1-inosine adducts, like the N3-dU adducts,
are deamination products following its epoxidation by EB (30).
Interestingly, the N1 inosine adducts exhibited a stereospecific
difference in their overall mutation yield. While the mutagenic
frequencies for the R N1-deoxyinosine adducts were 59%, those
of the S N1-deoxyinosine adducts were 90% (38). Because the
BD N3-DU adducts are overall highly mutagenic, both isomers
should exhibit similar mutation yields, even though the pattern
of mutations may be different with each isomer. Separate
syntheses of the R and S diastereoisomers of the adducts are
currently underway to test this hypothesis.
Figure 4. Primer extension reactions catalyzed by Kf on the BD N3-
dU adducted template. Control dC or BD N3-dU adducted 32-mer DNA
templates were annealed to a -2 primer. The DNA substrates (5 nM)
were incubated in the presence of all four dNTPs (100 µM) without or
with increasing concentrations (0.0005, 0.005, 0.05, and 0.5 units) of
Kf (lanes 2-5 and 7-10) for 15 min at room temperature. The position
of the 14-nucleotide primer is indicated. U* indicates the position of
the modified uracil on the template.
Nucleotide incorporation studies using exonuclease-deficient
Kf with N3-hydroxyethyl uracil have revealed this adduct to
be mutagenic, implicating it in the induction of mainly C to T
transitions and C to A and C to G transversions to a minor
extent (48). This is similar to the trend of mutations measured
with the BD N3-dU adducts (Table 1). These data suggest that
adduction at the N3 position of uracil renders it mutagenic
irrespective of the exact identity of the parent epoxide. Structural
data on the N3 BD-dU adducts are not yet available but would
give a better understanding of the relationship of the positioning
of this adduct within DNA to explain the incorporation of the
incorrect nucleotides opposite these lesions during replication
in cells. This has been demonstrated in the case of other adducts
of BD like the N1 deoxyinosine adducts (49, 50) in which the
syn conformation of the adducts at the glycosyl bond could
explain the induction of A to G mutations.
Assays to detect the formation of BD N3-dU adducts
following in vivo exposure to butadiene gas or activated
metabolites have not yet been carried out; thus, it is an open
question whether these lesions form at an appreciable rate in
humans. It is reasonable to speculate that they may be present
at low levels as was seen in the case of 3-hydroxypropyl uracil,
the only uracil DNA lesion to be detected in vivo. This adduct
was found at 0.02 lesions/106 nucleotides and at 0.02% of the
corresponding 7-hydroxypropyl guanine (51). However, the data
presented herein on the extremely high mutagenic frequency
associated with the replication of these lesions suggest that such
an investigation is well-warranted.
In addition to the BD N3-dU adducts that are the C1 adducts
formed from the reaction of deoxycytidine with EB, the C2 BD
N3-dU adducts were also detected (35). However, the deoxy-
cytidine form of their precursors was not detected apparently
due to more rapid rates of deamination as compared to the C1
BD N3-dU adducts. Both regioisomers, i.e., the C1 and C2
adducts, are formed from reactions of EB with deoxyguanosine
(26, 31, 52). This also holds true for reactions of EB with N1
deoxyadenosine that can give rise to both C1 and C2 N1
deoxyadenosine adducts, C2 N1 inosine adduct, or can undergo
Dimroth rearrangement to give rise to C1 and C2 N6 deoxy-
adenosine regioisomers (30). Similarly, both C1 and C2 N3
deoxyadenosine adducts are formed from in vitro reactions of
Figure 5. Primer extension reactions catalyzed by calf thymus pol δ
and human pol ꢀ on the BD N3-dU adducted template. Control dC or
BD N3-dU adducted 32-mer DNA templates were annealed to a -2
primer. (A) The DNA substrates (5 nM) were incubated in the presence
of all four dNTPs (100 µM) without or with increasing concentrations
(0.008, 0.08, and 0.8 units) of pol δ, in the presence of 70 ng of calf
thymus PCNA for 30 min at room temperature. (B) Time course
experiments (at time intervals of 0, 1, 5, 10, 15, 20, and 30 min) were
carried out with the DNA substrates (5 nM) at room temperature in
the presence of all four dNTPs (100 µM) with 0.1 units of pol ꢀ. The
positions of the 14-nucleotide primers and the 32-nucleotide full-length
products are indicated. U* indicates the position of the modified uracil
on the template.
release of the PCNA. Interestingly, the lesions posed an extreme
block for replication by yeast pol δ.2 In kinetic experiments
using pol ꢀ, primer extension was completely blocked one
nucleotide prior to and at the site of the lesions under conditions
that supported polymerization on unmodified templates (Figure
5B). The same results were observed with increasing concentra-
2 Unpublished data.

Synthesis and Mutagenesis of the BD N3-dU Adducts
Chem. Res. Toxicol., Vol. 19, No. 7, 2006 975
(3) 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.
(4) 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.
(5) Zhuang, S. M., Cochran, C., Goodrow, T., Wiseman, R. W., and
Soderkvist, P. (1997) Genetic alterations of p53 and ras genes in 1,3-
butadiene- and 2′,3′-dideoxycytidine-induced lymphomas. Cancer Res.
57, 2710-2714.
(6) Zhuang, S. M., Wiseman, R. W., and Soderkvist, P. (2002) Frequent
mutations of the Trp53, Hras1 and beta-catenin (Catnb) genes in 1,3-
butadiene-induced mammary adenocarcinomas in B6C3F1 mice.
Oncogene 21, 5643-5648.
(7) Kim, Y., Hong, H. H., Lachat, Y., Clayton, N. P., Devereux, T. R.,
Melnick, R. L., Hegi, M. E., and Sills, R. C. (2005) Genetic alterations
in brain tumors following 1,3-butadiene exposure in B6C3F1 mice.
Toxicol. Pathol. 33, 307-312.
(8) Santos-Burgoa, C., Matanoski, G. M., Zeger, S., and Schwartz, L.
(1992) Lymphohematopoietic cancer in styrene-butadiene polymeri-
zation workers. Am. J. Epidemiol. 136, 843-854.
(9) Delzell, E., Sathiakumar, N., Hovinga, M., Macaluso, M., Julian, J.,
Larson, R., Cole, P., and Muir, D. C. (1996) A follow-up study of
synthetic rubber workers. Toxicology 113, 182-189.
(10) Macaluso, M., Larson, R., Delzell, E., Sathiakumar, N., Hovinga, M.,
Julian, J., Muir, D., and Cole, P. (1996) Leukemia and cumulative
exposure to butadiene, styrene and benzene among workers in the
synthetic rubber industry. Toxicology 113, 190-202.
(11) Graff, J. J., Sathiakumar, N., Macaluso, M., Maldonado, G., Matthews,
R., and Delzell, E. (2005) Chemical exposures in the synthetic rubber
industry and lymphohematopoietic cancer mortality. J. Occup. EnViron.
Med. 47, 916-932.
EB with adenine (31). Inhalation exposure studies revealed that
the C2 N7 guanine adduct derived from the R enantiomer was
the major adduct found in the rat liver (53). Both C1 and C2
adducts were also detected in the testis and lung of mice (54).
Thus, the C2 adducts are as important as the C1 adducts.
Because these adducts are formed to both carbons associated
with the epoxy group, the chemistry of EB seems to more
resemble that of styrene oxide than that of saturated aliphatic
epoxides that react mainly with the terminal carbon of the
epoxide (55).
The BD N3-dU adducts are formed from the N3 (2-hydroxy-
3-buten-1-yl) deoxycytidine adducts that are unstable under
physiological conditions. The deoxycytidine adducts have a high
deamination rate with short half-lives of 2.3 and 2.5 h, whereas
their deaminated products are stable for up to 168 h (35).
Similarly, 3-(2-hydroxyethyl)-2′-deoxycytidine derived from bis-
(2-chloroethyl)nitrosourea is unstable under physiological condi-
tions and undergoes deamination to 3-(2-hydroxyethyl)-2′-
deoxyuridine with a half-life of approximately 5 h (56). Thus,
these uracil adducts are readily formed and may remain
persistent in DNA. Other epoxides, which yield a uracil product
on reaction with cytidine or calf thymus DNA, include propylene
oxide (57), ethylene oxide (58), 2-cyanoethyleneoxide (59),
acrylonitrile, (60) epichlorohydrin (61), and glycidol (62). It is
suggested that once the epoxide attacks the N3 of deoxycytidine,
the hydroxyalkyl side chain intramolecularly catalyzes the
hydrolysis of the adjacent imine CdNH bond. As a result, an
unstable carbinolamine intermediate is formed that loses am-
monia to form the uracil adduct (60).
(12) IARC (1999) IARC Monographs on the Evaluation of Carcinogenic
Risks to Humans, International Agency for Research on Cancer, Lyon.
IARC Sci. Publ. 71, 109-225.
(13) National Toxicology Program (2001) The Ninth Report on Carcino-
gens, U.S. Department of Health and Human Services, Washington,
DC.
(14) U.S. Environmental Protection Agency (2002) Publication 600/P-98/
001F, United States Environmental Protection Agency National Center
for Environmental Assessment, Washington, DC.
(15) Anderson, D. (1998) Butadiene: Species comparison for metabolism
and genetic toxicology. Mutat. Res. 405, 247-258.
Our studies indicate that mammalian pol ꢀ and bacterial Kf
are severely blocked upon encountering these adducts. They
also block pol δ, although some bypass activity was observed.
In vivo, these adducts may be recognized and bypassed more
efficiently by specialized translesion polymerases such as
polymerases η, ι, ú, and κ that are implicated in the bypass of
certain DNA adducts (63-66). Studies are currently underway
to test this hypothesis.
(16) Kemper, R. A., Elfarra, A. A., and Myers, S. R. (1998) Metabolism
of 3-butene-1,2-diol in B6C3F1 mice. Evidence for involvement of
alcohol dehydrogenase and cytochrome p450. Drug Metab. Dispos.
26, 914-920.
In summary, we have synthesized and established the
mutagenic effect of the BD N3-dU adducts. They are stable
adducts that are blocking to replicative and repair polymerases
and mutagenic in mammalian cells. These data thus suggest the
importance of the BD N3-dU adducts as crucial lesions
contributing to BD carcinogenesis.
(17) 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.
(18) Cochrane, J. E., and Skopek, T. R. (1994) Mutagenicity of butadiene
and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-
epoxybutene and diepoxybutane at the hprt locus in splenic T cells
from exposed B6C3F1 mice. Carcinogenesis 15, 719-723.
(19) Powley, M. W., Jayaraj, K., Gold, A., Ball, L. M., and Swenberg, J.
A. (2003) 1,N2-propanodeoxyguanosine adducts of the 1,3-butadiene
metabolite, hydroxymethylvinyl ketone. Chem. Res. Toxicol. 16,
1448-1454.
(20) Bernardini, S., Pelin, K., Peltonen, K., Jarventaus, H., Hirvonen, A.,
Neagu, C., Sorsa, M., and Norppa, H. (1996) Induction of sister
chromatid exchange by 3,4-epoxybutane-1,2-diol in cultured human
lymphocytes of different GSTT1 and GSTM1 genotypes. Mutat. Res.
361, 121-127.
(21) Adler, I. D., Kliesch, U., Nylund, L., and Peltonen, K. (1997) In vitro
and in vivo mutagenicity of the butadiene metabolites butadiene
diolepoxide, butadiene monoepoxide and diepoxybutane. Mutagenesis
12, 339-345.
(22) Lawley, P. D., and Brookes, P. (1967) Interstrand cross-linking of
DNA by difunctional alkylating agents. J. Mol. Biol. 25, 143-160.
(23) Park, S., Hodge, J., Anderson, C., and Tretyakova, N. (2004) Guanine-
adenine DNA cross-linking by 1,2,3,4-diepoxybutane: Potential basis
for biological activity. Chem. Res. Toxicol. 17, 1638-1651.
(24) Park, S., and Tretyakova, N. (2004) Structural characterization of the
major DNA-DNA cross-link of 1,2,3,4-diepoxybutane. Chem. Res.
Toxicol. 17, 129-136.
(25) Abdel-Rahman, S. Z., El-Zein, R. A., Ammenheuser, M. M., Yang,
Z., Stock, T. H., Morandi, M., and Ward, J. B., Jr. (2003) Variability
in human sensitivity to 1,3-butadiene: Influence of the allelic variants
of the microsomal epoxide hydrolase gene. EnViron. Mol. Mutagen.
41, 140-146.
Acknowledgment. We acknowledge Dr. Masaaki Moriya,
Dr. Kathleen M. Downey, Dr. Hitomi Asahara, Dr. Stuart Linn,
and Dr. Peter M. Burgers for the generous gifts of ss pMS2,
pol δ, pol ꢀ, and yeast DNA pol δ, respectively. We also
acknowledge continued support from the UTMB Sealy Center
of Molecular Science and the Sealy Center for Environmental
Health and Medicine. We thank Dr. Irina G. Minko for helpful
discussions and Dr. Carmelo Rizzo for his efforts in facilitating
the characterization of the modified oligodeoxynucleotides. This
work was supported by NIH Grant P01-ES05355 and NIEHS
Center Grants P30-ES06676 and P30-ES00267.
References
(1) 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.
(2) Pelz, N., Dempster, N. M., and Shore, P. R. (1990) Analysis of low
molecular weight hydrocarbons including 1,3-butadiene in engine
exhaust gases using an aluminum oxide porous-layer open-tubular
fused-silica column. J. Chromatogr. Sci. 28, 230-235.

976 Chem. Res. Toxicol., Vol. 19, No. 7, 2006
Fernandes et al.
(26) Selzer, R. R., and Elfarra, A. A. (1996) Synthesis and biochemical
characterization of N1-, N2-, and N7-guanosine adducts of butadiene
monoxide. Chem. Res. Toxicol. 9, 126-132.
(27) Boogaard, P. J., van Sittert, N. J., Watson, W. P., and de Kloe, K. P.
(2001) A novel DNA adduct, originating from 1,2-epoxy-3,4-butane-
diol, is the major DNA adduct after exposure to [2,3-(14)C]-1,3-
butadiene,[4-(14)C]-1,2-epoxy-3-butane. Chem.-Biol. Interact. 135-
136, 687-693.
(28) Zhang, X. Y., and Elfarra, A. A. (2004) Characterization of the reaction
products of 2′-deoxyguanosine and 1,2,3,4-diepoxybutane after acid
hydrolysis: Formation of novel guanine and pyrimidine adducts. Chem.
Res. Toxicol. 17, 521-528.
(29) Powley, M. W., Li, Y., Upton, P. B., Walker, V. E., and Swenberg,
J. A. (2005) Quantification of DNA and hemoglobin adducts of 3,4-
epoxy-1,2-butanediol in rodents exposed to 3-butene-1,2-diol. Car-
cinogenesis 26, 1573-1580.
(30) Selzer, R. R., and Elfarra, A. A. (1996) Characterization of N1- and
N6-adenosine adducts and N1-inosine adducts formed by the reaction
of butadiene monoxide with adenosine: Evidence for the N1-adenosine
adducts as major initial products. Chem. Res. Toxicol. 9, 875-881.
(31) Selzer, R. R., and Elfarra, A. A. (1999) In vitro reactions of butadiene
monoxide with single- and double-stranded DNA: Characterization
and quantitation of several purine and pyrimidine adducts. Carcino-
genesis 20, 285-292.
(47) O’Day, C. L., Burgers, P. M., and Taylor, J. S. (1992) PCNA-induced
DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf
thymus DNA polymerase delta in vitro. Nucleic Acids Res. 20, 5403-
5406.
(48) Zhang, W., Johnson, F., Grollman, A. P., and Shibutani, S. (1995)
Miscoding by the exocyclic and related DNA adducts 3,N4-etheno-
2′-deoxycytidine, 3,N4-ethano-2′-deoxycytidine, and 3-(2-hydroxy-
ethyl)-2′-deoxyuridine. Chem. Res. Toxicol. 8, 157-163.
(49) Scholdberg, T. A., Merritt, W. K., Dean, S. M., Kowalcyzk, A., Harris,
C. M., Harris, T. M., Rizzo, C. J., Lloyd, R. S., and Stone, M. P.
(2005) Structure of an oligodeoxynucleotide containing a butadiene
oxide-derived N1 beta-hydroxyalkyl deoxyinosine adduct in the human
N-ras codon 61 sequence. Biochemistry 44, 3327-3337.
(50) Merritt, W. K., Kowalczyk, A., Scholdberg, T. A., Dean, S. M., Harris,
T. M., Harris, C. M., Lloyd, R. S., and Stone, M. P. (2005) Dual
roles of glycosyl torsion angle conformation and stereochemical
configuration in butadiene oxide-derived N1 beta-hydroxyalkyl deox-
yinosine adducts: A structural perspective. Chem. Res. Toxicol. 18,
1098-1107.
(51) Plna, K., Nilsson, R., Koskinen, M., and Segerback, D. (1999) 32P-
postlabeling of propylene oxide 1- and N6-substituted adenine and
3-substituted cytosine/uracil: Formation and persistence in vitro and
in vivo. Carcinogenesis 20, 2025-2032.
(32) 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.
(33) Zhao, C., Vodicka, P., Sram, R. J., and Hemminki, K. (2000) Human
DNA adducts of 1,3-butadiene, an important environmental carcinogen.
Carcinogenesis 21, 107-111.
(52) Neagu, I., Koivisto, P., Neagu, C., Kostiainen, R., Stenby, K., and
Peltonen, K. (1995) Butadiene monoxide and deoxyguanosine alky-
lation products at the N7-position. Carcinogenesis 16, 1809-1813.
(53) Koivisto, P., Sorsa, M., Pacchierotti, F., and Peltonen, K. (1997) 32P-
postlabeling/HPLC assay reveals an enantioselective adduct formation
in N7 guanine residues in vivo after 1,3-butadiene inhalation exposure.
Carcinogenesis 18, 439-443.
(54) Koivisto, P., Adler, I. D., Pacchierotti, F., and Peltonen, K. (1998)
DNA adducts in mouse testis and lung after inhalation exposure to
1,3-butadiene. Mutat. Res. 397, 3-10.
(55) Latif, F., Moschel, R. C., Hemminki, K., and Dipple, A. (1988) Styrene
oxide as a stereochemical probe for the mechanism of aralkylation at
different sites on guanosine. Chem. Res. Toxicol. 1, 364-369.
(56) Zhang, W., Rieger, R., Iden, C., and Johnson, F. (1995) Synthesis of
3,N4-etheno, 3,N4-ethano, and 3-(2-hydroxyethyl) derivatives of 2′-
deoxycytidine and their incorporation into oligomeric DNA. Chem.
Res. Toxicol. 8, 148-156.
(57) Solomon, J. J., Mukai, F., Fedyk, J., and Segal, A. (1988) Reactions
of propylene oxide with 2′-deoxynucleosides and in vitro with calf
thymus DNA. Chem.-Biol. Interact. 67, 275-294.
(58) Li, F., Segal, A., and Solomon, J. J. (1992) In vitro reaction of ethylene
oxide with DNA and characterization of DNA adducts. Chem.-Biol.
Interact. 83, 35-54.
(59) Solomon, J. J., Singh, U. S., and Segal, A. (1993) In vitro reactions
of 2-cyanoethylene oxide with calf thymus DNA. Chem.-Biol. Interact.
88, 115-135.
(60) Solomon, J. J., and Segal, A. (1989) DNA adducts of propylene oxide
and acrylonitrile epoxide: Hydrolytic deamination of 3-alkyl-dCyd
to 3-alkyl-dUrd. EnViron. Health Perspect. 81, 19-22.
(61) Singh, U. S., Decker-Samuelian, K., and Solomon, J. J. (1996) Reaction
of epichlorohydrin with 2′-deoxynucleosides: Characterization of
adducts. Chem.-Biol. Interact. 99, 109-128.
(62) Segal, A., Solomon, J. J., and Mukai, F. (1990) In vitro reactions of
glycidol with pyrimidine bases in calf thymus DNA. Cancer Biochem.
Biophys. 11, 59-67.
(63) Masutani, C., Kusumoto, R., Iwai, S., and Hanaoka, F. (2000)
Mechanisms of accurate translesion synthesis by human DNA poly-
merase eta. EMBO J. 19, 3100-3109.
(64) Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and
Prakash, L. (2000) Eukaryotic polymerases iota and zeta act sequen-
tially to bypass DNA lesions. Nature 406, 1015-1019.
(65) Zhang, Y., Yuan, F., Wu, X., Wang, M., Rechkoblit, O., Taylor, J.
S., Geacintov, N. E., and Wang, Z. (2000) Error-free and error-prone
lesion bypass by human DNA polymerase kappa in vitro. Nucleic Acids
Res. 28, 4138-4146.
(66) Washington, M. T., Minko, I. G., Johnson, R. E., Wolfle, W. T., Harris,
T. M., Lloyd, R. S., Prakash, S., and Prakash, L. (2004) Efficient and
error-free replication past a minor-groove DNA adduct by the
sequential action of human DNA polymerases iota and kappa. Mol.
Cell. Biol. 24, 5687-5693.
(34) Selzer, R. R., and Elfarra, A. A. (1997) Characterization of four N-3-
thymidine adducts formed in vitro by the reaction of thymidine and
butadiene monoxide. Carcinogenesis 18, 1993-1998.
(35) Selzer, R. R., and Elfarra, A. A. (1997) Chemical modification of
deoxycytidine at different sites yields adducts of different stabilities:
Characterization of N3- and O2-deoxycytidine and N3-deoxyuridine
adducts of butadiene monoxide. Arch. Biochem. Biophys. 343, 63-
72.
(36) Carmical, J. R., Zhang, M., Nechev, L., Harris, C. M., Harris, T. M.,
and Lloyd, R. S. (2000) Mutagenic potential of guanine N2 adducts
of butadiene mono- and diolepoxide. Chem. Res. Toxicol. 13, 18-25.
(37) Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd,
R. S. (2000) Mutagenic potential of adenine N6 adducts of monoep-
oxide and diolepoxide derivatives of butadiene. EnViron. Mol. Mu-
tagen. 35, 48-56.
(38) Kanuri, M., Nechev, L. V., Tamura, P. J., Harris, C. M., Harris, T.
M., and Lloyd, R. S. (2002) Mutagenic spectrum of butadiene-derived
N1-deoxyinosine adducts and N6,N6-deoxyadenosine intrastrand cross-
links in mammalian cells. Chem. Res. Toxicol. 15, 1572-1580.
(39) Jackson, M. A., Stack, H. F., Rice, J. M., and Waters, M. D. (2000)
A review of the genetic and related effects of 1,3-butadiene in rodents
and humans. Mutat. Res. 463, 181-213.
(40) Beaucage, S. L., and Iyer, R. P. (1992) Advances in the synthesis of
oligonucleotides by the phosphoramidite approach. Tetrahedron 48,
2223-2311.
(41) Martinelli, M. J., Vaidyanathan, R., Pawlak, J. M., Nayyar, N. K.,
Dhokte, U. P., Doecke, C. W., Zollars, L. M., Moher, E. D., Khau,
V. V., and Kosmrlj, B. (2002) Catalytic regioselective sulfonylation
of alpha-chelatable alcohols: Scope and mechanistic insight. J. Am.
Chem. Soc. 124, 3578-3585.
(42) Moriya, M. (1993) Single-stranded shuttle phagemid for mutagenesis
studies in mammalian cells: 8-Oxoguanine in DNA induces targeted
G.CfT.A transversions in simian kidney cells. Proc. Natl. Acad. Sci.
U.S.A. 90, 1122-1126.
(43) Kanuri, M., Minko, I. G., Nechev, L. V., Harris, T. M., Harris, C. M.,
and Lloyd, R. S. (2002) Error prone translesion synthesis past gamma-
hydroxypropano deoxyguanosine, the primary acrolein-derived adduct
in mammalian cells. J. Biol. Chem. 277, 18257-18265.
(44) Fernandes, P. H., Wang, H., Rizzo, C. J., and Lloyd, R. S. (2003)
Site-specific mutagenicity of stereochemically defined 1,N2-deox-
yguanosine adducts of trans-4-hydroxynonenal in mammalian cells.
EnViron. Mol. Mutagen. 42, 68-74.
(45) Hirt, B. (1967) Selective extraction of polyoma DNA from infected
mouse cell cultures. J. Mol. Biol. 26, 365-369.
(46) Tretyakova, N., Matter, B., Ogdie, A., Wishnok, J. S., and Tannen-
baum, S. R. (2001) Locating nucleobase lesions within DNA sequences
by MALDI-TOF mass spectral analysis of exonuclease ladders. Chem.
Res. Toxicol. 14, 1058-1070.
TX060016O