Carcinogenesis vol.17 no.6 pp.1297-1303, 1996
32
P-Postlabelling of diastereomeric 7-alkylguanine adducts of
butadiene monoepoxide
Rajiv Kumar, Pavel Vodicka1, Pertti Koivisto2,
Kimmo Peltonen2 and Kari Hemminki
Center for Nutrition and Toxicology, Karolinska Institute, Novum,
141 57 Huddinge, Sweden, 'Department of Developmental Genotoxicology,
Institute of Experimental Medicine, Czech Academy of Science, Videnska
1083, 14020 Prague 4, Czech Republic and 2Finnish Institute of
Occupational Health, Molecular Dosimetry Group, Topeliuksenkatu 41 aA,
FIN-00250 Helsinki, Finland
The reaction of 3,4-epoxy-l-butene (BMO) with deoxyguanosine-3'-monophosphate (3'-dGMP) resulted in the
formation of two pairs of diastereomeric 7-alkyl-3'-dGMP
derivatives corresponding to two isomers C"-l and C"-2.
The T4 polynucleotide kinase-mediated phosphorylation
with [y-32P]-ATP showed preferential labelling of diastereomers of the C"-l isomer. The diastereomers 1 and 2 of the
C"-l isomer had labelling efficiencies of 42%. However,
the labelling efficiencies of diastereomers 3 and 4 of the
C"-2 isomer were 11 and 10%, respectively. The 32Ppostlabelling of BMO-modified DNA yielded four isomers
in the ratio of 4:4:1:1 with overall recoveries being 14%.
The two isomers had a half-life of 270 min (C"-l isomer)
and 300 min (C"-2 isomer) which is in accordance with the
stability predicted by other similar adduct experiments. The
molecular modelling experiments showed more pronounced
restricted rotation of butadiene residue in C"-2 isomers
due to steric interaction between butadiene residue at N-7
and O6 atom of guanine than in C"-l isomer. The butadiene
residue also leads to steric overcrowding at 3'-phosphate
in C"-2 isomer which probably restricts the access to the
active site of T4 polynucleotide kinase.
Introduction
32
P-Postlabelling is by far the most sensitive technique for the
detection of DNA adducts (1,2). The technique involves
transfer of the terminal phosphate group from [y-32P]-ATP to
the 5'-hydroxyl group of a 3'-nucleotide, mediated by T4
polynucleotide kinase enzyme and chromatographic separation
by thin-layer chromatography (TLC*) or high-pressure liquid
chromatography (HPLC) of 32P-postlabelled adducted nucleotides (3,4). Over the years this technique has evolved to the
extent that it is now possible to detect a variety of adducts
using variations of this technique (3,5-8). We had earlier
described the detection of 7-alkylguanine adducts formed by
the reaction of different 1,2-epoxides with DNA using a
modified 32P-postlabelling method (9,10). In this communication we describe the extension of that method for the detection
of 7-alkylguanine adduct formed by the reaction of butadiene
monoepoxide with 2'-deoxyguanosine-3'-monophosphate (3'dGMP) and DNA. Butadiene monoepoxide (BMO) is a major
•Abbreviations: BMO, butadiene monoepoxide; BD, 1,3-butadiene; HPLC,
high-pressure liquid chromatography; 3'-dGMP, 2'-deoxyguanosine-3'-monophosphate; TLC, thin-layer chromatography; ATP, adenosine triphosphate;
dGMP, 2'-deoxyguanosine-5'-monophosphate.
© Oxford University Press
metabolite of 1,3-butadiene which is an important industrial
chemical (11).
Butadiene monoepoxide reacts with the N-7 position of
guanine to form two pairs of diastereomers corresponding to
C"-l and C"-2 isomers (12,13). The results of polynucleotide
kinase mediated reaction showed that diastereomers 1 and 2
of C"-l isomer undergo 5'-phosphorylation more efficiently
than diastereomers 3 and 4 of C"-2 isomer. Molecular modelling
experiments show the intrinsic differences in the three dimensional structures between the two isomers which probably
result in restricted access of C"-2 isomers to the active site of T4
polynucleotide kinase enzyme and consequently less efficient
phosphorylation than C"-l isomers.
Materials and methods
Salmon testis DNA and micrococcal nuclease were obtained from Sigma
Chemical Co., St Louis, USA; nuclease PI and spleen phosphodiesterase
from Boehringer Mannheim, Germany. [y-32P]-adenosine triphosphate (ATP)
(sp. act. 3000 Ci/mmol) was purchased from Amersham, UK and cloned T-4
polynucleotide kinase from US Biochemical, USA. BMO was from AldrichChemie, Germany. HPLC analysis was performed on a Beckman Gold system
coupled with a module 168 diode array detector. The analysis of 32 Ppostlabelled samples was done on a Beckman model coupled with Beckman
166 UV and 171 radioisotope detectors in a series. In the radioisotope detector
a 50 H.1 Teflon tube was used as a flow cell.
Reaction of 5'-dGMP and 3'-dGMP with butadiene monoepoxide
2'-Deoxyguanosine-5'-monophosphate (5'-dGMP) and 3'-dGMP (2 mg each)
were treated with butadiene monoepoxide (100 mM) in 50 mM Tris-HCl
buffer, pH 8.0 and 30% methanol. The reactions were incubated at 37°C for
5 h. Thereafter, the reaction mixtures were extracted with ethylacetate and
the ethylacetate extracts were discarded. The aqueous phases were freeze
dried and then reconstituted in 100 \i\ 5 mM ammonium formate buffer,
pH 5.3. These reconstituted samples were loaded on the strong anion exchange
cartridges (1 ml, J.T.Baker, Holland) and eluted with 5 mM ammonium
formate buffer (4 ml), pH 5.3. The eluents were injected into HPLC columns
to separate isomers of 7-alkyl-dGMP derivatives of butadiene monoepoxide.
The HPLC separation was carried out on a C-18 reverse phase column by
elution (flow rate 0.7 ml/min) with 100% 50 mM ammonium formate buffer,
pH 4.6, for an initial 10 min followed by linear gradient to 10% methanol
over the next 10 min which was maintained for 5 min. The concentration of
methanol was increased to 30% in 10 min and then further to 100% in the
next 10 min. The peaks were detected by UV absorption at 254 nm.
Aliquots of the HPLC fractions collected were brought to pH 7 with 0.1 M
NaOH and heated at 100°C for 30 min in a water bath. The depurinated
samples were analysed on HPLC using the solvent system described above.
The depurinated isomers were identified by HPLC after spiking separately
with the authentic samples of 7-(2-hydroxy-3-buten-l-yl)guanine and 7-(lhydroxy-3-buten-2-yl)guanine.
Determination of half-lives {tln) °f isomers of butadiene monoepoxide 7alkyl-3'-dGMP adducts
The two separated isomers [ 1 and 4] corresponding to C"-1 and C"-2 derivatives
were incubated in 20 nl IX polynucleotide kinase buffer (40 mM bicine
buffer, 20 mM MgCl2 and 2 mM spermidine), pH 9.6 at 37°C. The aliquots
were taken at time points of 0, 30, 60, 120, 240, 300 and 360 min and
analysed on HPLC under similar conditions as described above. The halflives of these isomers were calculated from the slope of the straight line. The
other two isomers [2 and 3] which were collected as a single peak were
collectively incubated as described and analysed on HPLC at different
time points.
Reaction of salmon testis DNA with butadiene monoepoxide
Salmon testis DNA (4 |ig/|il) was reacted with 100 mM butadiene monoepoxide in 50 mM Tris-HCl, pH 7.4. The reaction mixture was incubated at
1297
R.Kumar et al.
0,3
0,25
g
c
0,2
•I 0,15
<
0,1
0.05
0
20
25
Retention time (minutes)
0.35
b
0,3
0.25
b
0.2
0.15
0.1
IA x
0.05
0
10
15
20
25
Retention times (minutes)
Fig. 1. HPLC analysis of diastereomers of 7-alkyl-dGMP formed by reaction of butadiene monoepoxide with (A) 5'-dGMP and (B) 3'-dGMP. The peaks a
and b correspond to C"-l and C"-2 depurinated products formed spontaneously during the reaction.
37°C for 20 h. The reaction mixture was extracted with ethylacetate and the
modified DNA was ethanol precipitated. The DNA precipitate was washed
with 70% ethanol and dissolved in water. The concentration of DNA was
measured by a spectrophotometer.
An aliquot of modified DNA equivalent to 800 u.g was placed in a boiling
water bath for 10 min. The DNA was ethanol precipitated and the 7-alkylguanine adducts released were measured by injecting the supernatant into
HPLC. The peaks corresponding to 7-(2-hydroxy-3-buten-l-yl)guanine and 7(l-hydroxy-3-buten-2-yl)guanine eluted at 35.1 and 38.0 min, respectively.
32
P-postlabelling of separated diastereomers of 7-alkyl-3'-dCMP adducts
formed by the reaction of 3'-dGMP and butadiene monoepoxide
The three fractions separated by HPLC from the reaction of 3'-dGMP with
BMO were phosphorylated with [y-32P]-ATP in kinase mediated reaction as
described earlier (9). Briefly, 500 fmol of isomers 1 and 4 were labelled
separately, whereas isomers 2 and 3 (500 fmol each) were labelled together.
The reaction was carried out in a total volume of 2.0 nl containing 2 pmol
of [y-32P]-ATP, 40 mM bicine buffer, pH 9.6, 20 mM MgCI2, 2 mM spermidine
and 6 U T4 polynucleotide kinase. The mixture was incubated at 37°C for
1 h followed by addition of nuclease PI (2.5 ng) and IM ammonium formate
buffer (0.5 |il) pH 4.0 for the adjustment of the pH of the reaction mixture
to 5.5. The incubation was carried on for another 15 min.
The postlabelled samples were diluted to 100 nl with water and 20 |il
aliquots were mixed with synthesized 7-alkyl-5'-dGMP isomers as UV
markers. These samples were injected into HPLC with on-line UV and
radioisotope detectors. The separation was carried out on a Kromasil CI8,
250 X 2.0 mm, 5 \ua column. The initial elution was carried out with 0.2 M
ammonium formate/ 20 mM o-phosphoric acid, pH 4.6 for 10 min. This was
followed by a linear gradient of methanol up to 100% in the next 50 min.
The flow rate was maintained at 0.2 ml per min and the UV absorption was
detected at 254 nm. The postlabelled adducts were also analysed by HPLC
as nucleoside bisphosphates without carrying out nuclease PI treatment after
kinase mediated reaction.
s2
P-Postlabelling assay of DNA modified with BMO
The butadiene monoepoxide modified salmon testis DNA (5 ng) samples
were incubated at 37°C with micrococcal nuclease (80 mU/Hg DNA) in 3 mM
bicine, pH 9.0 and 0.2 mM CaCl2 for 2 h followed by addition of spleen
phosphodiesterase (1.6 mU/ng DNA) and 20 mM ammonium acetate, pH 5.0.
The incubation was continued for another 2 h. The digested DNA samples
were applied to strong anion-exchange cartridges equilibrated with 5 mM
ammonium formate, pH 5.3. Samples of 7-alkyl dGMP adducts were eluted
with 5mM ammonium formate by centrifugation at 1600 r.p.m. Then 10 |il
aliquots from the eluent equivalent to 500 fmol adduct were freeze dried and
taken for postlabelling. The postlabelling and subsequent analysis on HPLC
1298
were carried out as described. The adducts were quantified by conversion of
peak areas into c.p.m. units using diluted [y-32P]-ATP as reference.
Molecular modelling
All the computations were done by using HyperChem software package
(Hypercube Inc., Waterloo, Ontario, Canada), visualization utilized ChemPlus
software package also from Hypercube. The computer used was an IBM
compatible PC with 90 MHz Pentium processor.
The butadiene monoepoxide residues were first optimized as to local energy
minima using molecular mechanic computations. The computations were
performed with MM+ force field, the first optimization used was steepest
descent algorithm subsequently followed by the Polak—Ribiere conjugate
gradient optimization.
After docking to 3'dGMP at the position of N-7 nitrogen atom the butadiene
residues were frozen and the adducted 3'dGMPs were computed by using
Amber force field and the steepest descent algorithm. A molecular dynamics
simulation was used for a conformational search to find a low energy
conformer. The simulation temperature was 300 K for 50 ps.
Results
Synthesis of 7-alkylguanine derivatives of 3'- and 5'-dGMP
by the reaction of butadiene monoepoxide with 3'- and 5'dGMP, respectively
The reaction of butadiene monoepoxide with 5'-dGMP under
aqueous conditions formed two diastereomeric pairs corresponding to C"-l and C"-2 isomers. The four diastereomeric 7alkylguanine derivatives were separated by HPLC which
migrated at the retention times of 25.9, 28.5, 30.4 and
32.4 min respectively (Figure 1A). The depurinated C"-l
and C"-2 derivatives migrated at the retention times of
35.1 and 38.0 min, respectively. The depurination of peaks at
25.9 and 28.5 min (diastereomers 1 and 2) under neutral
conditions yielded a 7-alkylguanine derivative corresponding
to C"-l isomer migrating at the retention time of 35.1 min.
Similarly peaks at 30.4 and 32.4 min (diastereomers 3 and 4)
after depurination under neutral conditions produced C"-2
depurinated derivative with the retention time of 38.0 min.
The identity of the depurinated derivatives was confirmed as
7-(2-hydroxy-3-buten-l-yl)guanine and 7-(l-hydroxy-3-buten-
32
c
A
80000
E
ATP
70000
iPO
6OO00
ATP
120000
70000
I
100000
20000
20000
II
L
40000
| diastereomer 3
I
uv.
0
20
0
40
10
B
. .nI
20
V
30
40
0
10
.
A_ A
20
30
40
minutes
F
D
A T P
180000
iPO
300000
ATP
160000
ATP
30000
diastereomer 4
minutes
minutes
35000
60000
20000
10000
II
c
I
I
30000
n
10000
diastereomer 2
1 40000
I
I
30000
iPO
80000
' PO
50000
diastereomer 1
40000
ATP
80000
60000
50000
1
P-postlabelling of butadiene monoepoxide adducts
250000
140000
25000
E
120000
20000
E
diastereomer 1
u
15000
»
I
200000
iPO
100000
t 150000
80000
1OO00
5000
,_
Q
0
\J\ 20
minutes
l
20000
Q
40
0
10
20
ipo
100000
I
ft
40000
n
u
diastereomers 2 & 3
60000
i
50000
diastereomer 4
Q
30
40
0
minutes
10
20
30
40
minutes
Fig. 2. HPLC analysis of 32P-postlabelled isomeric 7-alkyl-dGMP derivative formed by the reaction of BMO with 3'-dGMP. Diastereomer 1 of C"-l isomer
analysed as (A) monophosphate and (B) bisphosphate. Diastereomers 2 and 3 of C"-l and C"-2 isomers as (C) monophosphates and (D) bisphosphates.
Diastereomer 4 of C"-2 isomer as (E) monophosphate and (F) as bisphosphate.
2-yl)guanine by their co-migration in HPLC with authentic
samples characterized by 'H and I3C nuclear magnetic resonance in an earlier study (12).
The reaction of 3'-dGMP with butadiene monoepoxide under
similar conditions yielded four corresponding diastereomers.
These isomers gave three peaks on separation by HPLC which
migrated at the retention times 26.8, 29.5 and 33.6 min (Figure
IB). The peaks at 26.8 and 33.6 min corresponded to one
diastereomer each [1 and 4] of C"-l and C"-2 derivatives
respectively. These two peaks on depurination at neutral pH
gave 7-alkylguanine derivatives which migrated at 35.1 and
38.0 min, respectively, corresponding to the C"-l and C"-2
isomers. However, the peak at the retention time 29.5 min
corresponded to two diastereomers [2 and 3] of the C"-l and
C"-2 derivatives which unlike 5'-dGMP derivatives did not
separate. This peak on depurination at neutral pH gave two
peaks corresponding to the C"-l and C"-2 depurinated derivatives.
Reaction of BMO with DNA
The reaction of BMO with salmon testis DNA resulted in the
formation of 7-alkylguanine adducts with a total level of 6.3
adducts per 103 normal nucleotides. This total adduct level
was determined by depurination of BMO modified DNA at
neutral pH (9) which resulted in the release of two isomeric
7-alkylguanine adducts. The two isomers (C"-l and C"-2) were
formed in the ratio of 1.3:1, respectively.
32
P-postlabelling of separated 7-alkyl-3'-dGMP isomers
The 32P-postlabelling of the isolated isomers of 7-alkyl3'-dGMP derivative of BMO was carried out in the T4
polynucleotide kinase mediated reaction. The postlabelled
isomers were analysed both as bisphosphate nucleosides as
well as monophosphate nucleosides. The diastereomer 1 of
C"-l isomer had the retention time of 24.0 min as monophosphate (Figure 2A) and 15.6 min as bisphosphate (Figure 2B).
60
50
40
0)
.Q
JO
8
S.
30
20
I
10
1
2
3
4
Diastereomers
Fig. 3. Relative efficiencies of isomeric 7-alkyl-3'-dGMP derivatives in
T4 PNK mediated 32P-postlabelling reaction. Numbers 1 and 2 are
diastereomeric forms of C"-l isomer and 3 and 4 are diastereomers of
isomer C"-2.
The diastereomers 2 (of C"-l isomer) and 3 (of C"-2 isomer)
which could not be separated as 3'-monophosphates did
not separate as bisphosphates also and eluted at a retention
time of 20.4 min (Figure 2D). However, after treatment with
nuclease PI these were separated as 5'-monophosphates with
retention times of 26.4 and 27.5 min (Figure 2C). The
diastereomer 4 (of C"-2 isomer) had a retention time of
29.6 min as a monophosphate (Figure 2E) and 22.9 min as
a bisphosphate (Figure 2F). The labelling efficiencies of
isomers determined by the analysis of both mono- and bisphosphates were 42 ± 9.1, n = 5 (isomer 1), 42 ± 6.6, n =
5 (isomer 2), 11 ± 2.5, n = 5 (isomer 3) and 10 ± 2.4,
n = 5 (isomer 4) per cent. In all the experiments, isomers
corresponding to the C"-1 derivative showed about a four-fold
1299
R.Kumar el at.
20
Retention time (minutes)
0,05
0,04
0,03
0,02:
0,01
10
20
Retention time (minutes)
Fig. 4. HPLC chromatogram of 32P-postlabelled BMO modified DNA analysed with UV and radioisotope detectors. (A) shows radioactive peaks. The peaks
marked 1 and 2 are diastereomers of C"-l isomer and peaks marked 3 and 4 are diastereomers of isomer C"-2 of 7-alkyl-dGMP adducts analysed as
nucleoside-5'-monophosphates. The identity of isomers was confirmed by their co-migration with synthesized standards used as UV markers shown in (B).
higher labelling efficiency than the isomers corresponding to
C"-2 derivatives (Figure 3).
32
P-Postlabelling of BMO modified DNA
The digested DNA was passed through a strong anion-exchange
cartridge for adduct enrichment. 32P-Postlabelled samples were
analysed by HPLC after nuclease PI treatment. The adduct
peaks corresponding to diastereomers of C"-l and diastereomers of C"-2 were identified by their co-migration with
corresponding 7-alkyl-5'-dGMP isomers used as UV markers
(Figure 4A and B). The total recovery of labelled butadiene
7-alkyl-dGMP adducts from DNA by 32P-postlabelling method
was 14 ± 4.2%. The recovery of diastereomers 1-4 was in
the ratio of 4: 4 : 1 : 1 indicating the preferential labelling of
isomers corresponding to C"-l butadiene monoepoxide 7-alkyl3'-dGMP derivative.
Half-lives of diastereomers of butadiene monoepoxide 7-alkyl3'-dGMP derivative
Since the 32P-postlabelling reaction was carried out at pH 9.6
the stability of the individual diastereomers was ascertained
by determining their half-lives of disappearance at that pH.
The diastereomer 1 of C"-l derivative showed a half-life of
270 min whereas the diastereomer 4 of C"-2 derivative had a
half-life of 300 min. The diastereomers 2 (of C"-l isomer)
and 3 (of C"-2 isomer) had a half-life of 250 min, determined
together as these two could not be separated on HPLC.
Molecular modelling
After molecular dynamic simulation and conformation optimization the C"-l and C"-2 adducts showed a 4.5% difference
in potential energies. The potential energy of the C"-l isomer
was 104.2 kcal/mol and in the similar simulation condition
the C-2 isomer gave a potential energy of 109.6 kcal/mol.
The results of calculations are in good agreement with the
experimental observations that the products do not show
1300
different stability/lability in solutions. The non-modified 3'dGMP showed a lower potential energy—only 31.2 kcal/mol.
The torsion angles measured from C-l(sugar)-N9-C8-N7
showed a minor difference between the two isomers revealing
torsion angles of 143.8 and 135.7 degrees of C"-l and C"-2
isomers, respectively. Also the torsion angles from O6-C5-N7C"-l/C"-2 showed only a slight difference between the isomers,
and 5.9 and 3.3 degrees were measured for C"-l and C"-2
isomers, respectively.
The major difference between the isomers shown by the
modelling was that in all cases the C"-2 isomer had arms of
the butadiene residue pointing out perpendicularly to both
sides of the purine ring system (Figure 5). The butadiene
residue could not lie along the purine plane because of the
steric conflict between the residue and O6. The O 6 atom also
caused a restricted rotation of the butadiene residue in isomer
C"-2 allowing the residue to rotate only 63 degrees measured
from one extreme to another. The butadiene residue in the
C"-l isomer has freedom to rotate, because there was no steric
conflict between C"-l hydrogens and O 6 atom (van der Waals
radii do not overlap). This property leaves one side of the
purine plane not crowded by the butadiene residue. The key
structure in the nucleotides that kinase enzyme recognizes is
the 3'-phosphate group in the sugar moiety. If steric crowding
exists in the vicinity of this phosphate group, this may result
in low labelling efficiency. In the case of the C"-2 isomer both
sides of the purine plane are occupied with butadiene residues,
but in the C"-1 isomer one side of the plane is always nonoccupied. These properties of the two isomers revealed in
silico (reactions simulated on computer) may partially explain
the difference observed in the labelling efficiency in vitro.
Discussion
Butadiene is an important chemical used in the petrochemical
and rubber industry. The occupational exposure to butadiene
32
P-postlabelllng of butadiene monoepoxide adducts
06
C"4
Fig. 5. Molecular models of (left) C - l isomer, (centre) C-2 isomer of 7-alkyl-3'-dGMP formed by the reaction of BMO with 3'-dGMP and (right)
unmodified 3'-dGMP.
ranges from a few to hundreds of p.p.m. (11,14). Butadiene
has also been identified as one of the volatile chemicals emitted
during the heating of Chinese rape seed oil in a study to
determine genotoxic and lung cancer risk factors in Chinese
women (15,16). In carcinogenecity studies in animals 1,3butadiene (BD) has been shown to be a potent carcinogen in
mice (17,18). 1,3-Butadiene has been found to cause mutations in K-ras and p53 genes in studies on mice (19) and
increased frequency of mutations at A:T base pairs in lacl
transgenic mice (20). Butadiene is metabolized in vivo by
cytochrome P450-dependent mono-oxygenases and also by
human liver microsomes to butadiene monoepoxide and
butadiene diepoxide (21-23).
Butadiene monoepoxide, like other 1,2-epoxides, is a direct
acting mutagen (24,25) which reacts with different DNA bases
to form adducts (26). The major lesion formed during the
reaction of BMO with DNA is probably the 7-alkylguanine
adduct. However, adenine adducts formed by butadiene monoepoxide and diepoxide by 32P-postlabelling have been detected
(27-31).
In this study we have used a previously developed technique
to measure 7-alkylguanine adducts by the 32P-postlabelling
method formed by the reaction of BMO with 3'-dGMP and
DNA. T4 polynucleotide kinase mediated phosphorylation
reaction of the isomeric 7-alkyl-3'-dGMP derivatives with
[y-32P]-ATP did result in the labelling of all four diastereomers
that were detected both as 5', 3'- bisphosphates as well as 5'monophosphates by HPLC coupled with on-line radioisotope
detector. However, the labelling efficiencies of diastereomers
of C-1 isomer were 42% each whereas the labelling efficiencies
of diastereomers of C - 2 isomer were 11 and 10%, respectively.
These results were consistent in all postlabelling assays when
the labelled adducts were measured both as 3',5'-bisphosphates
as well as 5'-phosphates, which rules out selectivity by nuclease
PI, that was used for the conversion of labelled bisphosphate
adducts to monophosphate nucleotides. Neither can the apparent four-fold difference in labelling efficiencies between the
two isomers be explained on the basis of their relative
stabilities. Both isomers showed similar stabilities as determined by their half-lives at the pH of the labelling reaction.
In 32P-postlabelling experiments with BMO-modified DNA,
the recovery of diastereoisomers corresponding to C - l isomers
was four times higher than diastereomers corresponding to
C-2 isomers, with overall recovery being 14%.
The similar stabilities of isomers were further shown by
calculation of potential energies by molecular modelling
studies. The difference in the potential energies between the
two isomers of 5.4 kcal/mol was consistent with experimental
results obtained from stability experiments. However, the
potential energy differences between the parent compound 3'dGMP and the 7-alkyl-dGMP derivatives were large. The
relative spatial arrangement of the butadiene residue on the
N-7 position of guanine does however show a difference in
isomers. In the case of the C-2 isomers, the rotation of
butadiene residue is restricted due to steric interaction between
the C-1 hydrogens and the O 6 of guanine residue. However,
in the case of C - l isomers no such restriction of rotation
seems to exist. Moreover, in the case C - 2 isomers, butadiene
residue which is perpendicular to the plane of the purine ring
system is extended both above and below the plane of the
ring. This arrangement creates an overcrowding in the vicinity
of 3'-phosphate residue which is a key recognition unit for
polynucleotide kinase enzyme. Additionally, the imidazole ring
is slightly pushed out from planarity. In the case of C - l
isomers the presence of butadiene residue on the N-7 position
seems to have no or little effect on the planarity of purine
systems and the 3'-phosphate residue is far from overcrowded.
Presumably these are the major factors responsible for the
four-fold difference in the labelling efficiencies between the
two isomers.
T4 polynucleotide-kinase mediated 5'-phosphorylation of
nucleic acids and 3'-nucleotides has been shown in different
studies to be substrate specific, as different substrates have been
shown to undergo phosphorylation with different efficiencies
1301
R.Kumar el al.
(4,9,32-34). The basic requirements for this enzymatic reaction
include the presence of a 3'-phosphate group and proceeds
with the inversion of configuration at the phosphorus atom (35).
However, there are a few reports where the phosphorylation of
non-nucleotide substrates or altered fidelity of PNK has been
shown (36-38). The difference in labelling efficiencies between
the diastereomers has been reported (39) however, the difference in regioisomers is not known. The molecular modelling
experiments carried out in this study provide an insight into
the probable inhibition mechanism. Despite the widespread
use of T4 PNK in molecular biology, which has both 5'-kinase
and 3'-phosphatase activities (40), not much information
about its three-dimensional structure is known. Moreover, the
mammalian homologs (41) of T4 PNK enzyme expressed by
PseT gene on T4 phage (42) with complex in vivo functions
are also known. Therefore, further investigation is required to
study the substrate selectivity exhibited by T4 PNK and its
possible in vivo implications.
In this study we have shown that the differences in efficiency
of kinase-mediated phosphorylation between the individual
isomers of the same adduct necessitate the determination
of labelling efficiencies of individual isomers for accurate
quantification of isomeric adducts. We also report the extension
of a modified postlabelling method for the detection of 7alkylguanine adducts formed by BMO with DNA. The method
was sensitive enough to detect these adducts at femtomolar
level and has potential of increased sensitivity by a combination
of HPLC and TLC (43), thus, making it useful for monitoring
human exposure to butadiene.
Acknowledgements
The project was supported by the EU Environment Programme, partly through
the Swedish Medical Research Council, the National Environment Protection
Board, the Swedish Cancer Fund, the EU PECO programme, the Swedish
Work-Environment Fund, the OK Environment Fund and by grant
no. 3157-3 IGA, Czech Ministry of Health.
References
l.Randerath.K., Reddy,M.V. and Gupta,R.C. (1981) 32P-labelling test for
DNA damage. Proc. Nail Acad. Sci. USA, 78, 6126-6129.
2. Randerath.K. and Randerath.E. (1990) Detection of human DNA adducts
by 32P-postlabelling. Basic Life Sci., 53, 13-32.
3.Beach,A.C. and Gupta,R.C. (1992) Human biomonitoring and the 32 Ppostlabelling assay. Carcinogenesis, 13, 1053-1074.
4. Szyfter.K., Kriiger,J., Ericson.P., Vaca.C, F6rsti,A. and Hemminki,K.
(1994) 32P-postlabelling analysis of DNA adducts in humans: adduct
distribution and method improvement. Mutat. Res., 313, 269-276.
5.Mustonen,R., Forsti.A., Hietanen.P. and Hemminki.K. (1991) Measurement
by 32P-postlabelling of 7-methylguanine levels in white blood cell DNA
of healthy individuals and cancer patients treated with dacarbazine. Human
data and method development for 7-alkylguanines. Carcinogenesis, 12,
1423-1431.
6. Vodicka.P., Vodickova.L., Trejbalova.K., Sram.RJ. and Hemminki.K.
(1994) Persistence of 06-guanine adducts in styrene-exposed lamination
workers determined by 32P-postlabelling. Carcinogenesis, 15, 1949-1953.
7. Hemminki.K., Widlak,P. and Hou,S.-M. (1995) DNA adducts caused by
tamoxifene and toremifene in human microsomal system and lymphocytes
in vitro. Carcinogenesis, 16, 1661-1664.
8.Nath,R.G. and Chung,F.-L. (1994) Detection of exocyclic 1,N2propanodeoxyguanosine adducts as DNA lesions in rodents and humans.
Proc. Nail Acad. Sci. USA, 91, 7491-7495.
9.Kumar,R., StaffasJ., Forsti.A. and Hemminki,K. (1995) 32P-postlabelling
method for the detection of 7-alkylguanine adducts formed by the reaction
of different 1,2-alkyl epoxides with DNA. Carcinogenesis, 16, 483^89.
lO.Eide.I., Hagemann.R., Zahlsen.K., Tareke,E., Tbrnqvist.M., Kumar.R.,
Vodicka.P. and Hemminki,K. (1995) Uptake, distribution and formation
of hemoglobin and DNA adducts after inhalation of C2-C8 1-alkenes
(olefins) in the rat. Carcinogenesis, 16, 1603-1609.
1302
ll.Fajen.J., Lundsford,R. and Roberts.D. (1993) Industrial exposure to 1,3butadiene monomer, polymer and end-user industries. In Sorsa,M.,
Peltonen.K., Vainio,H. and Hemminki,K. (eds), Butadiene and Styrene:
Assessments of Health Hazards, I ARC Scientific Publications no. 127,
IARC, Lyon, pp. 3-14.
12. Neagu.I., Koivisto.P., Neagu.C, Kostiainen.R., Stenby.K. and Peltonen.K.
(1995) Butadiene monoepoxide and deoxyguanosine alkylation products
at the N7-position. Carcinogenesis, 16, 1809-1813.
13.Citti,L., Gervasi.G., Turchi.G., Bellucci.G. and Bianchini.R. (1984) The
reaction of 3,4-epoxy-1 -butene with deoxyguanosine and DNA in vitro:
synthesis and characterization of the main adducts. Carcinogenesis, 5,
47-52.
14. Peltonen.K. and Vaaranrinta.R. (1995) Sampling and analysis of 1,3butadiene in air by gas chromatography on a porous-layer open tubular
fused-silca column. J. Chromatogr., 710, 237-241.
15.Pellizzari,E.D., Michael,L.C, Thomas.K.W., Shields.P.G. and Harris.C.
(1995) Identification of 1,3-butadiene, benzene and other volatile organics
from wok oil emissions. J. Exposure Anal. Environ. Epidemiology, 5,77-87.
16. Shields.P.G., Xu.G.X., Blot.W.J.,
FraumeniJ.F.Jr,
Trivers.G.E.,
Pellizzari.E.D., Qu,Y.H., Gao.Y.T. and Harris.C. (1995) Mutagens from
heated Chinese and US cooking oils. J. Natl Cancer Inst., 87, 836-841
17.Huff,J., Melnick.R., Solleveld.H., HazemanJ., Powers.M. and Miller.R.
(1985) Multiple organ carcinogenecity of 1,3-butadiene in B6C3F1 mice
after 60 weeks of inhalation exposure. Science, 227, 548-549.
18.Melnick.R., Huff,J.,Chan,B. and Miller.R. (1990) Carcinogenecity of 1,3butadiene in C57BL/6 X C3HF1 mice at low exposure concentrations.
Cancer Res., 50, 6592-6599.
19.Jacobson-Kram,D. and Rosenthal.S.L. (1995) Molecular and genetic
toxicology of 1,3-butadiene. Mutat. Res., 339, 121-30.
20. Recio.L. and Meyer,K.G. (1995) Increased frequency of mutations at A:T
base pairs in the bone marrow of B6C3F1 lad transgenic mice exposed
to 1,3-butadiene. Environ. Mol. Mutagenesis, 26, 1-8.
21.Duescher,R.J. and Elfarra,A.A. (1994) Human liver microsomes are
efficient catalysts of 1,3-butadiene oxidation: evidence for major roles by
cytochromes P450 2A6 and 2E1. Arch. Biochim. Biophys., 311, 342-349.
22.Thornton-Manning,J.R., Dahl.A.R., Bechtold.W.E., Griffith.W.C.Jr and
Henderson,R.F. (1995) Disposition of butadiene monoepoxide and
butadiene diepoxide in various tissues of rats and mice following a low
level inhalation exposure to 1,3-butadiene. Carcinogenesis, 16, 1723-31.
23.Maniglier-Poulet,C, Cheng,X., Ruth.J.A. and Ross,D. (1995) Metabolism
of 1,3-butadiene to butadiene monoepoxide in mouse and human bone
marrow cells. Chem. -Biol. Interactions, 97, 119-129.
24.Cochrane,J.E. and Skopek.T.R. (1994) Mutagenicity of butadiene and its
epoxide metabolites: I. Mutagenicity potential of 1,2-epoxybutene, 1,2,3,4diepoxybutane and 3,4-epoxy-1,2-butanediol in cultured human
Iymphoblasts. Carcinogenesis, 15, 713-717.
25.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 hprt locus in splenic T-cells from exposed B6C3F1
mice. Carcinogenesis, 15, 719-723.
26.Neagu,I., Koivisto.P., Neagu.C, Kaltia,S., Kostiainen,R. and Peltonen,K.
(1994) Alkylation of purine bases with 3,4-epoxy-1-butene, a reactive
metabolite of 1,3-butadiene. In Hemminki.K., Dipple,A., Shuker,D.E.G.,
Kadlubar.F.F, Segerback.D. and Bartsch,H. (eds), DNA AdductsIdentification and Biological Significance, IARC Scientific Publications
no. 125, IARC, Lyon, pp. 419-422.
27. Leuratti.C, Jones.N.J., Marafante.E., Kostiainen.R., Peltonen.K. and
Waters,R. (1994) DNA damage induced by the environmental carcinogen
butadiene: identification of a diepoxybutane-adenine adduct and its
detection by 32P-postlabelling. Carcinogenesis, 15, 1903-10.
28. Leuratti.C., Jones.N.J., Marafante.E., Peltonen.K., Kostiainen.R. and
Waters,R. (1993) Biomonitoring of exposure to 1,3-butadiene: detection
by high-performance liquid chromatography and 32P-postlabelling of an
adenine adduct formed by diepoxybutane. In Sorsa.M., Peltonen.K.,
Vainio,H. and Hemminki,K. (eds), Butadiene and Styrene: Assessments of
Health Hazards, IARC Scientific Publications no. 127, IARC, Lyon,
pp. 143-150.
29. Leuratti.C., Marafante,E., Jones,N.J. and Waters,R. (1994) Detection by
HPLC 32P-postlabelling of DNA adducts formed after exposure to
epoxybutene and diepoxybutane. In Phillips.D.H., Castegnaro.M. and
Bartsch.H. (eds), Postlabelling Methods for Detection of DNA Adducts,
IARC Scientific Publications no. 124, IARC, Lyon, pp. 211-282.
30. Koivisto.P. Kostiainen.R., Kilpetainen.A., Steinly.K. and Peltonen.K.
(1995) Preparation, characterization and 32P-postlabelling of butadiene
monoepoxide N6-adenine adducts. Carcinogenesis, 16, 2999-3007.
3I.Koivisto,P, Peltonen.K., Adler,I.-D. and Sorsa.M. (1996) Inhalation
exposure of rats and mice to 1,3-butadiene induces N6-adenine adducts of
32
P-postlabelling of butadiene monoepoxide adducts
butadiene monoepoxide detected by 32P-postlabelling and HPLC. Environ.
Health Perspect., in press.
32.Hemminki.K., SzyfterX, Vodicka,P, Koivisto,P., Mustonen.R. and
Reunanen.A. (1991) Quantitative aspects of 32P-postlabelling. In
Gledhill,B.L. and Mauro.F. (eds). New Horizons in Biological
Dosimetry, Wiley-Liss, New York, pp. 219-228.
33.Segerback,D. and Vodicka,P. (1993) Recoveries of DNA adducts of
polycyclic aromatic hydrocarbons in 32P-postlabelling assay.
Carcinogenesis, 14, 2463-2469.
34.Bykov,V., Kumar.R., F6rsti,A. and Hemminki,K. (1995) Analysis of UV induced DNA photoproducts by 32P-postlabelling. Carcinogenesis, 16,
113-116.
35.Jarvest,R.L. and Lowe.G. (1981) The stereochemical course of phosphoryl
transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected
Escherichia coli B). Biochemical J., 199, 273-276.
36.Fontanel,M.-L., Bazin.H. and Teoule.R. (1994) Sterical recognition by
T4 polynucleotide kinase of non nucleosidic moieties 5'-attached to
oligonucleotides. Nucleic Acids Res., 22, 2022-2027.
37. Masento,M.S., Hewer,A., Grover,P.L. and Phillips.D.H. (1989) Enzymemediated phosphorylation of polycyclic aromatic hydrocarbon metabolites:
detection of non-adduct compounds in the 32P-postlabelling assay.
Carcinogenesis, 10, 1557-1559.
38. Randerath,K., Gupta.K.P. and van Golen.K.L. (1993) Altered fidelity of a
nucleic acid modifying enzyme, T4 polynucleotide kinase, by safrole
induced DNA damage. Carcinogenesis, 14, 1523-1529.
39.Hemminki,K., Szyfter.K. and Kadlubar,F.F. (1991) Quantitation of the 32 Ppostlabeling reaction using Nl, N2 and C8 modified deoxyguanosine 3'monophosphates as substrates. Chem. -Biol. Interactions, 77, 51-61.
40Soltis,D.A. and Uhlenbeck,O.C. (1982) Independent locations of kinase
and 3'-phosphatase activities on T4 polynucleotide kinase. J. Biol. Chem.,
257, 11340-11345.
41.Sprinkle,T.J., Tippins.R.B. and Kestler,D.P. (1987) Inhibition of bovine
and human brain 2',3'-cyclic nucleotide-3'-phosphodiesterase by heparin
and polyribonucleotides and evidence for an associated 5'-polynucleotide
kinase activity. Biochem. Biophys. Res. Commun., 145, 686-691.
42.Midgley,C.A. and Murray.N.E. (1985) T4 polynucleotide kinase; cloning
of the gene (pseT) and amplification of its product. EMBO J., 4, 2695-2703.
43.Kumar,R. and Hemminki.K. (1996) Separation of 7-methyl- and 7-(2hydroxyethyl)-guanine adducts in human DNA samples using a
combination of TLC and HPLC. Carcinogenesis, 17, 485-492.
Received on December 12, 1995; revised on February 13, 1996; accepted on
March 13, 1996
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