Mutagenicity of a glutathione conjugate of butadiene diepoxide

Article abstract of DOI:10.1021/tx100304f

The mutagenicity and carcinogenicity of the important commodity chemical 1,3-butadiene are attributed to the epoxide products. We confirmed our previous work showing that expression of rat glutathione (GSH) transferase 5-5 enhances the mutagenicity of butadiene diepoxide in Salmonella typhimurium TA1535. A GSH-butadiene diepoxide conjugate was isolated and fully characterized by mass spectrometry and nuclear magnetic resonance as S-(2-hydroxy-3,4-epoxybutyl)GSH. The conjugate had a t_1/2 of 2.6 h (pH 7.4, 37 °C) and was considerably more mutagenic than butadiene diepoxide or monoepoxide in S. typhimurium. We propose that the GSH conjugate may be a major species involved in butadiene genotoxicity, not a detoxication product.

Full text of DOI:10.1021/tx100304f

1
544
Chem. Res. Toxicol. 2010, 23, 1544–1546
Mutagenicity of a Glutathione Conjugate of Butadiene Diepoxide
Sung-Hee Cho, Elisabeth M. Loecken, and F. Peter Guengerich*
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine,
NashVille, Tennessee 37232-0146, United States
ReceiVed September 6, 2010
The mutagenicity and carcinogenicity of the important commodity chemical 1,3-butadiene are attributed
to the epoxide products. We confirmed our previous work showing that expression of rat glutathione
(
GSH) transferase 5-5 enhances the mutagenicity of butadiene diepoxide in Salmonella typhimurium
TA1535. A GSH-butadiene diepoxide conjugate was isolated and fully characterized by mass spectrometry
and nuclear magnetic resonance as S-(2-hydroxy-3,4-epoxybutyl)GSH. The conjugate had a t1/2 of 2.6 h
(
pH 7.4, 37 °C) and was considerably more mutagenic than butadiene diepoxide or monoepoxide in S.
typhimurium. We propose that the GSH conjugate may be a major species involved in butadiene
genotoxicity, not a detoxication product.
1,3-Butadiene is an important commodity chemical in industry,
particularly in the production of synthetic rubber and other
7
polymers. In 2008, 1.3 × 10 kg was produced in the United
States alone (1). Butadiene is of concern in toxicology because
the compound is carcinogenic in mice and rats, particularly the
former species (2). In addition, some epidemiology studies
suggest that butadiene is a human carcinogen (1, 3). The
metabolism of butadiene is understood in the pathways shown
in Scheme 1, and it is highly likely that one or more of the
epoxide products of butadiene are responsible for its carcino-
genicity, probably because of genotoxic mechanisms (4, 5). At
least 23 adducts of DNA bases have been reported, with the
number being even higher when stereochemistry is considered
Figure 1. Determination of the t1/2 of the purified GSH-butadiene
diepoxide conjugate using a p-nitrobenzylpyridine assay (18, 19). The
results shown are from three separate experiments (separate symbols),
with the mean t1/2 ( standard deviation indicated.
(
6-13).
We previously reported that the base pair mutagenicity of
butadiene diepoxide in Salmonella typhimurium TA1535 is
considerably increased by the cellular expression of rat glu-
tathione (GSH) transferase 5-5 (14). That result, unexpected at
the time, has been repeatable (Figure S1 of the Supporting
Information) and implies that a GSH conjugate is genotoxic.
GSH conjugation has been reported, but the exact structure of
the conjugate was not determined (15, 16). However, GSH
conjugation has usually been considered to be a detoxication
process in the metabolism of butadiene diepoxide (17).
the HPLC peaks (Figure S3 of the Supporting Information), only
one gave a positive colorimetric assay with p-nitrobenzylpyri-
dine reagent (19), indicating the presence of an electrophile.
The conjugate was isolated in preparative incubations, frozen,
and lyophilized. The t1/2 was determined in 0.10 M potassium
phosphate buffer (pH 7.4) at 37 °C and was 2.6 ( 0.1 h (Figure
1
). The structure was established by a combination of NMR
1
13
1
methods [ H, C, total correlation spectroscopy (TOCSY),
heteronuclear multiple-bond correlation (HBMC), and hetero-
nuclear single-quantum correlation (HSQC) (Figures S4-S8 of
the Supporting Information)] and is shown as 1 in Scheme 1.
A GSH-butadiene diepoxide conjugate (1) was prepared by
either (i) reacting butadiene diepoxide with GSH in CH
3
OH in
0
the presence of 3 equiv of Na or NaOCH
3
(18) or (ii) enzymatic
In particular, GSH attack at C-2 is ruled out (2), as is structure
1
reaction of butadiene diepoxide (2 mM) with GSH (5 mM) in
the presence of a commercial mixture of 0.1 mg/mL rat liver
GSH transferase (Sigma, St. Louis, MO) in 0.10 M Tris-HCl
buffer (pH 7.7) for 1 h at 37 °C. The latter approach yielded
less complex products and was used in the work presented here.
Liquid chromatography-mass spectrometry (LC-MS) analysis
showed the formation of a peak with the features expected for
a GSH conjugate (Figure S2 of the Supporting Information),
and high-resolution analysis yielded an MH+ ion at m/z
3
. The product was quantified using H NMR and an internal
-
1
-1
standard (benzene). An ε560 value of 6.57 mM cm was
determined for the p-nitrobenzylpyridine assay based on the H
1
NMR signals (which can be used for convenient determination
of stock concentrations).
The conjugate, S-(2-hydroxy-3,4-epoxybutyl)GSH, was con-
siderably more mutagenic than several other related epoxides
in S. typhimurium TA1535, a tester strain that reports base pair
mutations at a GC site (Figure 2). Other compounds tested
included butadiene monoepoxide, butadiene diepoxide, and
butadiene diol epoxide [prepared by reaction of 3,4-dihydroxy-
3
24 3 8
94.1276 (calcd for C14H N O S, 394.1284, -2.0 ppm). Of
*
To whom correspondence should be addressed: Department of
Biochemistry and Center in Molecular Toxicology, Vanderbilt University
School of Medicine, 638 Robinson Research Building, 2200 Pierce Ave.,
Nashville, TN 37232-0146. Telephone: (615) 322-2261. Fax: (615) 322-
1
Abbreviations: HBMC, heteronuclear multiple-bond correlation; HSQC,
heteronuclear single-quantum correlation; TOCSY, total correlation
spectroscopy.
3
141. E-mail: f.guengerich@vanderbilt.edu.
1
0.1021/tx100304f  2010 American Chemical Society
Published on Web 09/29/2010

Rapid Report
Chem. Res. Toxicol., Vol. 23, No. 10, 2010 1545
a
Scheme 1. Metabolism of Butadiene
a
Three possible GSH adducts formed from butadiene diepoxide are shown. All compounds in dotted boxes can potentially react with DNA.
Figure 2. Mutagenicity of the GSH-butadiene diepoxide conjugate in S. typhimurium TA1535. Direct comparisons were made with the three other
indicated epoxides, using the methods described in ref 14. Results are shown as means and ranges obtained for duplicate assays.
butene with m-chloroperbenzoic acid (Figures S9 and S10 of
the Supporting Information)]. These results, along with the
enhancement of butadiene diepoxide by GSH transferase
expression, argue for a prominent role of the characterized
GSH-butadiene diepoxide conjugate in butadiene genotoxicity.
of the Supporting Information), but a mixture of other rat GSH
transferases could be used, as shown in the preparative work.
Acknowledgment. This work was supported in part by U.S.
Public Health Service Grants R01 ES010546, T32 ES007028,
and P30 ES00267. We thank K. D. Hardy for performing the
confirming experiments presented in Figure S1 of the Supporting
Information and D. A. Hachey, M. W. Calcutt, and D. F. Stec
for assistance with mass spectrometry and NMR.
Although many butadiene-derived adducts of DNA bases have
been prepared (6-13), limited information about their occur-
rence in biological systems and/or their biological activities is
available. We show a role for a defined GSH conjugate of
butadiene diepoxide in (base pair) mutagenicity (Figure 2),
congruent with previous results for the role of GSH conjugation
in butadiene bioactivation (14, 20) (Figure S1 of the Supporting
Information). We have not yet evaluated the mutagenicity and
other properties of the S-(2-hydroxy-3,4-epoxybutyl)GSH con-
jugate in mammalian systems, which will be of interest.
Preliminary LC-MS studies have shown the formation of Gua
and Ade adducts when the GSH-butadiene diepoxide conjugate
was incubated with DNA or DNA bases (results not presented).
Future studies are focused on characterization of the DNA
adducts formed from the GSH-butadiene diepoxide conjugate,
identification of which of these are miscoding, and analysis of
these adducts in vivo. Three stereoisomers of butadiene diep-
oxide (R,R, S,S, and meso) are known, and some differences
have been reported for their rates of formation and reactivity
Supporting Information Available: GSH transferase-de-
pendent mutagenicity of butadiene diepoxide in S. typhimurium
TA1535, LC-MS characterization of the GSH-butadiene diep-
oxide conjugate, NMR spectra of the GSH-butadiene diepoxide
conjugate, and MS and NMR spectra of butadiene diol epoxide.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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