DNA interstrand cross-linking activity of (1-chloroethenyl)oxirane, a metabolite of β-chloroprene

Article abstract of DOI:10.1021/tx9003769

With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane (CEO). We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking at deoxyguanosine residues within 5′-GC and 5′-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO ≥ ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role for interstrand cross-links in the genotoxicology of chloroprene.

Full text of DOI:10.1021/tx9003769

Chem. Res. Toxicol. 2010, 23, 235–239
235
DNA Interstrand Cross-Linking Activity of
(1-Chloroethenyl)oxirane, a Metabolite of ꢀ-chloroprene
Brian A. Wadugu,† Christopher Ng,† Bethany L. Bartley,† Rebecca J. Rowe,‡ and
Julie T. Millard*,†
Department of Chemistry, Colby College, WaterVille, Maine 04901, and Department of Chemistry, UniVersity of
Maine, Orono, Maine 04469
ReceiVed October 15, 2009
With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined
the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane
(CEO). We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of
interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking
at deoxyguanosine residues within 5′-GC and 5′-GGC sites, with the rate of cross-linking depending on
pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally
similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO g
ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role
for interstrand cross-links in the genotoxicology of chloroprene.
Chart 1. Cross-Linkers Used in This Study
Introduction
Chloroprene is a petrochemical produced in large scale
worldwide, at about 4.0 × 106 kg/year (1). Its principal use is
for the production of polychloroprene (Neoprene), a solvent-
resistant elastomer used to make adhesives, automotive parts,
wetsuits, and other consumer goods (2). The main source of
exposure to chloroprene is occupational, with exposure of the
general public thought to be very low (2, 3). Although there is
only inconsistent evidence of chloroprene-induced carcinogen-
esis in humans, correlation between occupational exposure and
elevated liver and lung cancer risk has been reported (4, 5).
However, chloroprene is carcinogenic to rats and mice when
given by inhalation, with comparable carcinogenic potency to
butadiene in mice (6). On the basis of sufficient evidence for
carcinogenicity in animals but inadequate evidence in humans,
the International Agency for Research on Cancer has classified
chloroprene as a possible human (Group 2B) carcinogen (7).
chloroprene. We also compared the cross-linking efficiency,
DNA sequence preferences, and cytotoxicity of CEO to two
established cross-linkers, ECH and diepoxybutane (DEB; 3,
Chart 1). Our data support interstrand cross-linking at deoxy-
guanosine residues by CEO at 5′-GC and 5′-GGC sites, a similar
sequence preference to ECH (10). We also found a correlation
between cross-linking efficiency and cytoxicity, with DEB >
CEO > ECH. These findings suggest a role for CEO interstrand
cross-links in the genotoxicity of chloroprene.
Chloroprene is metabolized by microsomes from rats, mice,
and humans primarily to the monoepoxide (1-chloroethenyl)ox-
irane (CEO1; 1, Chart 1) (1, 5). CEO is mutagenic in the Ames
test, implicating this metabolite in the genotoxicity of chloro-
prene (8). Furthermore, CEO reacts with DNA, with major
adducts derived from reaction with N7 of deoxyguanosine and
N3 of deoxycytidine (5, 9). The structurally similar compound
epichlorohydrin (ECH; 2, Chart 1) likewise alkylates the N7
position of deoxyguanosine to form interstrand cross-links (10),
suggesting the likelihood of DNA cross-linking by CEO.
Experimental Procedures
Caution: CEO, ECH, and DEB are suspect carcinogens and
must be handled appropriately.
Preparation of Racemic (1-Chloroethenyl)oxirane. We modi-
fied a simple two-step literature procedure for the synthesis of CEO
as follows. The intermediate product 2-(1,2-dichloroethyl)oxirane
was prepared as described previously (1). In the second step (11),
5.27 g of KOH (93.6 mmol) was added to a two-necked 100-mL
round-bottom flask equipped with an addition funnel and a short-
path distillation head. KOH was melted at 120 °C using an oil bath,
and then 7.5 g of 2-(1,2-dichloroethyl)oxirane (53.2 mmol) was
added dropwise with rapid stirring. After the complete addition of
the oxirane intermediate, the bath temperature was raised to 200
°C. The desired product 1 distilled into the receiving flask. The
distillate was dried using Na2SO4 and then filtered, affording 3.20 g
Our objective was to investigate possible DNA cross-linking
activity of CEO, which could contribute to the genotoxicity of
* To whom correspondence should be addressed. 5757 Mayflower Hill
Drive, Colby College, Waterville, ME 04901. Tel: (207) 859-5757. E-mail:
jtmillar@colby.edu.
2
1
(58% yield; >90% pure ) of CEO as a colorless liquid. HNMR
for the major isomer (500 MHz, CDCl3): δ 2.90-2.95 (m, 2 H),
3.5-3.52 (m, 1 H), 5.49 (d, 1 H, J ) 1.8 Hz), 5.29 d, 1 H, J ) 1.8
† Colby College.
‡ University of Maine.
1 Abbreviations: CEO, (1-chloroethenyl)oxirane; ECH, epichlorohydrin;
DEB, diepoxybutane; TE, 10 mM Tris buffer and 1 mM EDTA [pH 7.0];
MES, 2-(N-morpholino)ethanesulfonic acid; dPAGE, denaturing polyacryl-
amide gel electrophoresis; LD50, median lethal dose.
2 As determined via GC-MS, impurities included 3-4-dichloro-1-butene
(<3%), 2-chlorobenzoic acid (<5%), and 2-(1,2-dichloroethyl)oxirane
(∼2%).
10.1021/tx9003769  2010 American Chemical Society
Published on Web 12/23/2009

236 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Wadugu et al.
Table 1. DNA Oligomers Used in These Studies
Hz). 13CNMR (500 MHz, CDCl3): δ 47.5 (C-3), 52.5 (C-2), 115.0
(C-2′), 138 (C-1′).
Preparation of Cross-Linked DNA Duplexes. Oligonucleotides
(Integrated DNA Technologies, Inc.) were purified via 20%
denaturing polyacrylamide gel electrophoresis (19:1 acrylamide/
bisacrylamide; 40% urea) followed by UV shadowing and the crush-
and-soak procedure (12). Duplexes radiolabeled on the 3′- or 5′-
terminus were prepared as described previously (10). Racemic DEB
and ECH were purchased from Sigma-Aldrich Chemical Co.
(Milwaukee, WI); CEO was prepared as described above. To 50
µg of radiolabeled duplex DNA in buffer, 250 mM (2 µL of DEB,
2 µL of ECH, or 2.3 µL of CEO) cross-linker was added to achieve
a total volume of 100 µL. All reactions were run at 37 °C, and TE
buffer (10 mM Tris-Cl and 1 mM EDTA), 0.1 M MES buffer, and
0.1 M sodium acetate buffer were used for cross-linking reactions
at pH 7.0, pH 6.0, and pH 5.0, respectively. Previously determined
optimal reaction times were used for ECH (6 h) (10) and DEB (45
min) (13). CEO reaction times (typically 4-6 h) were determined
for each duplex via time trials, in which aliquots were removed
and ethanol precipitated during the course of the reaction.
Separation of Cross-Links. Cross-links were separated from
single strands via 20% denaturing polyacrylamide gels (19:1
acrylamide/bisacrylamide, 50% urea) run at 60 W and ambient
temperature. Gels were dried for analysis via phosphorimagery
(Amersham Biosciences STORM 840) or left wet for purification
of cross-links, which have approximately half the mobility of single
strands, via crush-and soak (12) after autoradiography. Percent
cross-linking was determined through volume analysis of the low-
mobility bands in comparison to total DNA (ImageQuant software).
Piperidine Cleavage of Alkylated DNA. Gel-purified cross-
linked DNA was cleaved at sites of guanine N7 alkylation by
heating at 90 °C in 10% aqueous piperidine for 30 min, followed
by lyophilization and water washing (14). Piperidine-cleavage
products were analyzed via 25% sequencing gels (19:1 acrylamide/
bisacrylamide, 50% urea) run at 60 W and 55 °C. After drying and
phosphorimaging, the relative intensities of bands corresponding
to cleavage at guanine residues were determined through volume
analysis.
mobility product forming only after several hours. No cross-
links were detected at pH 7.0 (Figure 2). This combination of
optimal pH (5.0) and reaction time (∼6 h) is similar to the
conditions previously established for ECH (10).
Corroboration of Interstrand Cross-Linking. In order to
confirm that the low-mobility bands seen in Figure 1 are
interstrand cross-links, we prepared Duplex 2 (Table 1), which
is similar to Duplex 1 but has strands that differ in length. Two
sets of cross-linking reactions were run at pH 5.0, one with the
short strand radiolabeled and the other with the longer strand
radiolabeled. The gel mobility of the presumed cross-link was
the same regardless of which strand was radiolabeled, supporting
our assignment of the low-mobility band as a cross-link rather
than a single-stranded product (Figure 3). Furthermore, these
low-mobility bands comigrated with the low-mobility bands of
samples treated with the established cross-linker DEB (17). The
diffuse nature of the low mobility bands produced by CEO
suggests heterogeneity in the location of these cross-links (18).
Relative Cross-linking Efficiencies of CEO, DEB, and
ECH. We compared the cross-linking efficiency of CEO to that
of ECH and DEB using Duplex 3 (Table 1), which has a central
5′-GGC site, the DEB consensus sequence for cross-linking (13).
Cytotoxicity Assays. We used Trypan blue exclusion (15) to
determine the cytotoxicities of DEB, ECH, and CEO in 6C2 chicken
erythro-progenitor cells. Briefly, confluent cells were grown (37
°C, 5% CO2) at a 1:10 dilution with MEM Richter’s modification
with L-glutamine until the cell count reached between 1.00 × 105
and 1.00 × 106 cells per 10 mL of culture. Aliquots (1.0 mL) were
removed into a 6-well plate with 4 mL of fresh media and incubated
overnight (37 °C, 5% CO2). CEO, DEB, or ECH at concentrations
ranging from 0-1.00 mM was added to different wells, and the
cells were incubated for 24 h (37 °C, 5% CO2). Twenty-microliter
aliquots were taken from each well and mixed with 2 µL of Trypan
blue (0.2% stock), dyed samples were monitored for viable cells
with an automated cell counter (Cellometer Auto T4, Nexcelom
Bioscience), and the viable fraction was plotted versus concentra-
tion. Determinations were made in duplicate, with the Solver tool
in Excel used to optimize the values of a and b in the equation y
) 1/[1 + [x/a]b] to achieve the best-fit dose-response curve, with
a being the LD50 (median lethal dose). Standard deviations were
determined with SolverAid (16).
Figure 1. Reaction of CEO and Duplex 1 over time at pH 5.0 (lanes
1-9) and pH 6.0 (lanes 10-18). Reaction aliquots were taken hourly
from time ) 0 h (lanes 1 and 10) to 8 h (lanes 9 and 18). Presumed
interstrand cross-links appear as low-mobility bands (arrow).
Results
Determination of Optimum pH and Time for CEO Cross-
Linking. To optimize reaction conditions for CEO cross-linking,
we used Duplex 1 (Table 1), containing a central 5′-GGC site
previously shown to be cross-linked efficiently by ECH (10).
Radiolabeled Duplex 1 was incubated with CEO at pH 5.0, 6.0,
or 7.0 and aliquots removed hourly for 8 h. Reaction products
were resolved using denaturing polyacrylamide gel electro-
phoresis (dPAGE), with low mobility bands presumed to be
interstrand cross-links. Cross-link production increased with time
at pH 5.0 and 6.0 (Figure 1), with significant amounts of low-
Figure 2. Reaction of CEO and Duplex 1 over time at pH 7.0.
Reaction aliquots were taken hourly from time ) 0 h (Lane 1) to
8 h (Lane 9).

DNA Interstrand Cross-Linking
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 237
Table 4. Mean Relative Percentages of Cross-Linking As a
Function of Nucleotide Position within Duplex 5 (Containing
Both GC and GGC sites)a
CEO cross-linking DEB cross-linking ECH cross-linking
nucleotide
mean%
SD
mean%
SD
mean%
SD
GC
GGC
GGC
34.0
29.3
36.7
6.9
3.8
4.0
5.5
5.7
88.8
5.2
2.9
8.0
25.6
39.4
35.1
13.7
4.6
10.6
a Relative intensities of piperidine cleavage at the bold
deoxyguanosines from separate experiments in which the top strand was
either 5′- or 3′-end-radiolabeled were averaged (three replicate trials of
each).
Figure 3. Cross-linked Duplex 2, with strands of differing lengths.
Lanes 1-3 have the long strand radiolabeled; lanes 4-6 have the short
strand radiolabeled. Lanes 1 and 4 are DEB products; lanes 2 and 5
are controls (no cross-linker); lanes 3 and 6 are CEO products. Presumed
cross-links (arrow) have the same mobility independent of which strand
is labeled.
Table 2. Mean Percentages of Cross-Linking (% XL) for
Duplex 3, which Contains a Central 5′-GGC Site, with CEO,
DEB, and ECHa
CEO
DEB
ECH
mean % XL
SD
7.4
1.5
21.2
4.8
5.3
1.4
a Data for three replicate trials were averaged.
Table 3. Mean Percentages of Cross-Linking (% XL) for
Duplex 4, Which Contains a Central 5′-GC Site, with CEO,
DEB, and ECHa
CEO
DEB
ECH
mean % XL
SD
5.6
0.9
7.7
2.3
4.1
0.7
Figure 4. Survival curves for 6C2 cells treated with CEO (-9-), DEB
(-2-), or ECH (-[-). Viable cells were determined by the Trypan blue
exclusion assay as described in Experimental Procedures. Data are the
averages from two trials per compound with standard deviations
reported as error bars. The following LD50 values were calculated from
the best-fit lines: DEB, 0.025 mM ( 0.064; CEO, 0.14 mM ( 0.03;
ECH, 0.59 mM ( 0.14. Despite the relatively high standard deviation,
the LD50 value for DEB was reproducible to within ∼12% in a separate
set of experiments conducted in triplicate.
a Data for three replicate trials were averaged.
Radiolabeled duplexes were independently incubated with CEO,
DEB, or ECH under optimal reaction conditions (pH 5.0, 37
°C) and analyzed via dPAGE. The phosphorimagery volume
analysis results (Table 2) revealed that DEB had a significantly
higher efficiency than either CEO or ECH. Additionally, CEO
was more efficient than ECH, although this difference was not
significant at the Bonferronin-corrected level of 0.05. Therefore,
the trend for cross-linking follows the order DEB > CEO > ECH,
although the latter difference is not statistically significant.
Sequence Specificity of CEO. In order to establish the
sequence specificity of CEO, we did two separate experiments.
In the first, we compared cross-linking of Duplex 3, with its
central 5′-GGC site, to Duplex 4 (Table 1), which has a central
5′-GC site. The 5′-GC sequence, which has the minimal N7-
to-N7 interstrand distance, was originally proposed to be the
sole target of cross-linkers with chains of five or fewer atoms
(19). The formation of CEO cross-links was comparable for
Duplex 3 and Duplex 4 (Table 3), suggesting that there is no
significant difference in CEO cross-linking at 5′-GC and 5′-
GGC sites. Similar findings were observed for ECH, consistent
with literature reports that it cross-links these sequences about
equally (10). In contrast, DEB, which has a strong preference
for 5′-GGC sites in comparison to 5′-GC sites (13, 17), had an
almost 3-fold preference for Duplex 3 relative to Duplex 4.
For the second experiment, we designed Duplex 5 (Table
1), which contains both 5′-GC and 5′-GGC sites. Presenting
both sites within the same duplex ensured identical reaction
conditions for the two sequences. Duplex 5 was radiolabeled
on either the 5′- or 3′-end of the top strand and then treated
with DEB, CEO, or ECH under optimum reaction conditions.
Cross-links were purified from wet denaturing polyacrylamide
gels for piperidine cleavage at sites of deoxyguanosine alkylation
at N7 positions. Cleavage products were resolved on sequencing
gels and analyzed via phosphorimagery to determine cross-
linking efficiencies at each site. We averaged data for 5′- and
3′-radiolabeling experiments to minimize the effects of over-
alkylation, which inflates the abundance of fragments closest
to the radiolabeled end.
Our data confirmed that CEO, like ECH (10), displays a
reduced sequence preference relative to DEB (Table 4). As
expected from its established consensus sequence for cross-
linking at 5′-GGC sequences (13, 17), DEB showed an 18-fold
preference for the first residue within the 5′-GGC site. In
contrast, CEO and ECH both partitioned about equally between
the 5′-GGC and 5′-GC sites. Although the two deoxyguanosine
residues on the bottom strand were cleaved approximately
equally after piperidine treatment of the purified cross-link
radiolabeled on that strand (data not shown), some cross-link
remained intact. This observation is suggestive of some cross-
linking at sites other than deoxyguanosine residues, such as
terminal residues, which are often hyperreactive (18).
Cytotoxicity Studies. In order to determine a possible
relationship between interstrand cross-linking and cytotoxicity
for CEO, we used the Trypan blue exclusion assay to obtain
cell survival curves for CEO, DEB, and ECH in 6C2 chicken
erythro-progenitor cells (Figure 4). With a 24-h treatment,
cytotoxicity followed the order DEB (LD50 ) 0.025 mM (
0.064) > CEO (LD50 ) 0.14 mM ( 0.03) > ECH (LD50 ) 0.59
mM ( 0.14), supporting a relationship between interstrand
cross-linking and cytotoxicity. This finding is consistent with
previous reports that the interstrand cross-linking ability of

238 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Scheme 1. Possible Mechanism for CEO Cross-Linking at Deoxyguanosine Residues
Wadugu et al.
bifunctional alkylating agents often, although not always,
this step, recent experimental evidence supports the feasibility
correlates with cytotoxicity (20).
of this reaction (25).
For some of the duplexes in these experiments, we observed
multiple low-mobility bands after CEO treatment, suggesting
heterogeneous products (e.g., Figure 3). Terminal residues of
synthetic DNA duplexes are often hyperreactive, leading to
diffuse or multiple cross-linking bands for agents with flexible
sequence specificity (26). In general, low-mobility bands with
the greatest retardation correspond to centrally cross-linked
products (24), a fact that we confirmed for our duplexes through
piperidine cleavage and subsequent analysis of the fragments.
We observed a decrease in low-mobility bands and the appear-
ance of products corresponding to cleavage at deoxyguanosine
residues following treatment with piperidine, which supports
alkylation at N7, consistent with previous reports for calf thymus
DNA treated with CEO (5, 9). However, some low-mobility
products were not cleaved by piperidine, suggesting linkage at
other sites as well.
Discussion
The goal of this study was to investigate the DNA cross-
linking potential of (1-chloroethenyl)oxirane, CEO, a metabolite
of the possible human carcinogen chloroprene. Structurally
similar bifunctional alkylating agents such as DEB, ECH, and
the nitrogen mustards have been demonstrated to form inter-
strand cross-links between deoxyguanosine residues on opposite
strands (17, 10, 21, 22). Initially, we screened for CEO cross-
linking with a duplex containing a central 5′-GGC site previ-
ously established to be efficiently cross-linked by ECH (10).
Low-mobility bands suggestive of interstrand cross-links were
resolved via dPAGE, with the intensity of these bands increasing
with decreasing pH. Although we did not detect cross-links at
pH 7.0, their formation after longer incubation times in ViVo is
likely. Even a small number of interstrand cross-links can be
lethal because of blockage of DNA replication and transcription.
Indeed, it has been proposed that as few as 20 interstrand cross-
links in the mammalian genome can result in cell death (23).
In general, cross-linking is a two-step process with the
formation of monoadducts followed by closure to cross-links
(24). The acid catalysis of CEO cross-linking, similar to that
observed for ECH (10), is consistent with a similar mechanism
in which the initial reaction is at the epoxide. Our data suggest
that CEO monoalkylation proceeds by nucleophilic attack on
the protonated epoxide by N7 of a deoxyguanosine residue,
leading to N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine. In-
deed, this proposed monoadduct is the major product formed
in the reaction of calf thymus DNA with CEO (5). Formation
of a cross-link could then proceed via nucleophilic attack by a
second deoxyguanosine on the opposite strand after protonation
of the terminus of the butenyl group (Scheme 1). While the
presence of a chlorine substituent might be expected to impede
Comparison of the cross-linking efficiencies of CEO, ECH,
and DEB revealed that DEB was several times more efficient
at cross-linking the duplex containing its consensus sequence,
5′-GGC. CEO was modestly more efficient than ECH. DEB
also required much shorter incubation times (45 min) than CEO
(4-6 h) and ECH (6 h). We therefore rank the cross-linking
efficiencies of these compounds as DEB > CEO g ECH.
Reminiscent of ECH, CEO appears to be somewhat flexible
in its sequence requirements for cross-linking, as confirmed by
our findings for Duplexes 3, 4, and 5. Whereas DEB has a strong
preference for the 5′-GNC sequence relative to 5′-GC (17), CEO
and ECH cross-linked distal deoxyguanosine residues at these
sites about equally, despite the relatively short alkyl chain
lengths of these compounds. Interestingly, early reports proposed
that cross-linking between N7 of guanine would be precluded
for agents of less than seven carbon atoms in length (27).

DNA Interstrand Cross-Linking
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 239
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Acknowledgment. We thank Professor Liam O’Brien, Pro-
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assistance. This work was supported by the Donors of the
American Chemical Society Petroleum Research Fund (PRF#
44839-B4) and NIH Grant Number P20 RR-016463 from the
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