( )
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.
Ž
.
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.
Ž
.
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
Ž
.
two major products were formed Fig. 1 . The two
Ž
.
Ž
adducts peaks a and b had
at 259 nm pH
.
Ž
.
Žmax
.
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
Ž
.
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
Ž
in both BD-exposed rats and humans Licea Perez
´
Ž
.
6
.
et al., 1997 . Therefore, N -adenine adducts of
epoxybutanediol might be a useful biomarker of
BD exposure.
Ž
.
Ž
mrz 434.6, deriving from 331 dAMP q105 tri-
6
Acknowledgements
.
Ž
hydroxybutyl group . This N - 2,3,4-trihydroxy-
.
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
Ž
Dr Kimmo Peltonen and Pertti Koivisto are
thanked for rat liver samples. The study was
supported by the Swedish Work-Environment
Fund.
.
Zhao, Chunyan
