32P-postlabelling of N6-adenine adducts of epoxybutanediol in vivo after 1,3-butadiene exposure

Article abstract of DOI:10.1016/S0378-4274(98)00253-7

Epoxybutanediol is one of the epoxide metabolites of butadiene (BD). A pair of diastereomeric N-1-adenine adducts were formed by reacting epoxybutanediol with deoxyadenosine 5'-monophosphate (5'-dAMP). These two N-1-adenine adducts rearranged in a base-catalysed reaction to an N⁶-trihydroxybutyl-adenine adduct, which was characterized by UV and mass spectroscopy. Using the ³²P-postlabelling/HPLC assay the same adducts were detected in diepoxybutane (DEB)-treated DNA in vitro and in liver DNA samples from rats exposed to BD by inhalation. Adenine adducts of epoxybutanediol are probably suitable for monitoring BD exposure. Copyright (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Full text of DOI:10.1016/S0378-4274(98)00253-7

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Toxicology Letters 102᎐103 1998 591᎐594
32 P-postlabelling of N 6-adenine adducts of
epoxybutanediol in vivo after 1,3-butadiene exposure
Chunyan Zhao, Mikko Koskinen, Kari HemminkiU
Center for Nutrition and Toxicology, Department of Biosciences, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden
Abstract
Epoxybutanediol is one of the epoxide metabolites of butadiene BD . A pair of diastereomeric N-1-adenine
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adducts were formed by reacting epoxybutanediol with deoxyadenosine 5Ј-monophosphate 5Ј-dAMP . These two
N-1-adenine adducts rearranged in a base-catalysed reaction to an N 6-trihydroxybutyl-adenine adduct, which was
characterized by UV and mass spectroscopy. Using the 32P-postlabellingrHPLC assay the same adducts were
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detected in diepoxybutane DEB -treated DNA in vitro and in liver DNA samples from rats exposed to BD by
inhalation. Adenine adducts of epoxybutanediol are probably suitable for monitoring BD exposure.
Elsevier Science Ireland Ltd. All rights reserved.
ᮊ 1998
Keywords: Epoxybutanediol; 32P-postlabelling; DNA adducts; HPLC
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1. Introduction
DEB Malvoisin and Roberfroid, 1982 . The epox-
ide metabolites of BD are DNA alkylating agents.
In vitro studies have shown the formation of
several N7-guanine and N 6-adenine adducts from
EB and DEB. The observation of a high fre-
quency of AªG transitions in mice exposed to
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1,3-Butadiene BD is mainly used in the man-
ufacture of polymers and copolymers. Rats and
mice exposed to BD develop tumours at multiple
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sites Melnick et al., 1987; Owen et al., 1987 . BD
is metabolized by cytochrome P450 to 1,2-epoxy-
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BD Recio et al., 1993 suggests that adenine
adducts might play an important role in the toxic-
ity of BD.
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3-butene EB Malvoisin et al., 1979 , which may
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also be further oxidized to diepoxybutane DEB .
Epoxybutanediol may be formed via hydrolysis of
EB followed by oxidation, or via hydrolysis of
In this paper N-1- and N 6-adenine adducts of
epoxybutanediol were identified with the aid of
rearrangement experiments, UV and mass spec-
troscopy. Using the synthesized standard adducts
as UV markers and a 32 P-postlabellingrHPLC
assay, these adducts were measured in DEB-ex-
U Corresponding author. Tel.: q46 8 6089243; fax: q46 8
6081501; e-mail: kari.hemminki@cnt.ki.se
0378-4274r98r$ - see front matter ᮊ 1998 Elsevier Science Ireland Ltd. All rights reserved.
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P I I S 0 3 7 8 - 4 2 7 4 9 8 0 0 2 5 3 - 7

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C. Zhao et al. rToxicology Letters 102᎐103 1998 591᎐594
592
posed DNA in vitro and, for the first time, in liver
DNA of rats exposed to BD by inhalation.
2. Materials and methods
Epoxybutanediol was prepared by hydrolysis of
DEB in water. The remaining DEB was extracted
with toluene. N-1-adenine adducts were formed
by incubation of 5Ј-dAMP with the hydrolysate.
Part of the mixture was treated with base at 37ЊC
overnight to convert the N-1-adenine adducts into
the corresponding N 6-adenine adducts by Dim-
roth rearrangement. Salmon testis DNA was re-
acted with DEB at 37ЊC for 24 h.
Fig. 1. HPLC separation of products formed by 5Ј-dAMP
treated with epoxybutanediol. Peaks a and b are N-1-adenine
adducts and c is an N 6-adenine adduct. UV detection at 260
nm.
Rats were exposed to 300 ppm BD for 5 days, 6
hrday. DNA adducts were analyzed by the post-
labelling procedure described by Randerath et al.
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1989 . The postlabelled sample mixed with the
and rat liver DNA samples. The synthesized N-
1-alkyl- and N 6-alkyl-5Ј-dAMP were used as UV
markers in HPLC. As shown in Fig. 2A, two
N-1-adenine adducts of epoxybutanediol were de-
tected in DEB-treated DNA. N 6-adenine adducts
in DEB-treated DNA and rat liver DNA samples
synthesized UV-marker, N-1-epoxybutanediol-
dAMP, was injected into a Beckman HPLC.
HPLC fractions containing 32 P-labelled N-1-
adenine adducts were collected and treated with
base at 37ЊC overnight. The corresponding N 6-
adenine adducts were further analyzed by HPLC.
Radioactivity was measured on-line with a Beck-
man 171 Radioisotope detector.
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Fig. 2B᎐D were analyzed by HPLC after rear-
rangement of the collected labelled N-1-adenine
adduct fractions. Since much less radioactivity
was injected into HPLC and since N 6-adenine
adducts eluted at a later retention time than
N-1-adenine adducts, the sensitivity was in-
creased. The approximate detection limit was 1
adductr109 nucleotides using 10 ␮g DNA. The
N 6-adenine adduct level in rat liver was on aver-
age 4.5r109 nucleotides, and nil in control rat
liver.
3. Results and discussion
In the reaction of epoxybutanediol with dAMP
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two major products were formed Fig. 1 . The two
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adducts peaks a and b had ␭
at 259 nm pH
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1 , 270 nm pH 7 and 270 nm pH 13 . These UV
spectra at different pH were similar to those of
other reported N-1-adenine adducts. The later
N 6-adenine adducts are chemically stable and
efficiently labelled. In addition, recent studies
have found substantially higher levels of epoxybu-
tanediol᎐haemoglobin adducts than those of EB
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eluting adduct peak c had UV spectra at differ-
ent pH identical to those of authentic N 6-adenine
adducts. After incubation of the purified peaks a
and b at pH 13 and 37ЊC overnight, they were
fully converted to a new product peak c . This
suggests that peaks a and b are diastereomeric
N-1-adenine adducts. The mass spectroscopic
analysis of peak c showed a molecular ion at
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in both BD-exposed rats and humans Licea Perez
´
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et al., 1997 . Therefore, N -adenine adducts of
epoxybutanediol might be a useful biomarker of
BD exposure.
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mrz 434.6, deriving from 331 dAMP q105 tri-
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Acknowledgements
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hydroxybutyl group . This N - 2,3,4-trihydroxy-
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but-1-yl adenine adduct has previously been iden-
tified in DEB-treated DNA Tretyakova et al.,
1997 .
Using the 32 P-postlabellingrHPLC method, the
same adducts were analyzed in DEB-treated DNA
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Dr Kimmo Peltonen and Pertti Koivisto are
thanked for rat liver samples. The study was
supported by the Swedish Work-Environment
Fund.
.

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C. Zhao et al. rToxicology Letters 102᎐103 1998 591᎐594
593
Fig. 2. HPLC chromatograms of 32 P-postlabelled DEB-treated DNA and rat liver DNA analysed with radioisotope and UV
6
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.
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detectors. A N-1-adenine adducts in DEB-treated DNA, B N -adenine adducts in DEB-treated DNA, C an exposed rat liver
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sample, D a control rat liver sample. The positions of the adducts are indicated with arrows.ᎏᎏᎏ radioactivity; . . . UV. Note that
the UV detector is installed 0.6 min after the radioactivity detector.

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C. Zhao et al. rToxicology Letters 102᎐103 1998 591᎐594
Inhalation toxicity studies with 1,3-butadiene. 3. Two-year
toxicityrcarcinogenicity study in rats. Am. Ind. Hyg. Assoc.
J. 48, 407᎐413.
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