Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo.

Article abstract of DOI:10.1021/tx980225b

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.

Full text of DOI:10.1021/tx980225b

Chem. Res. Toxicol. 1999, 12, 335-340
335
3
2
Develop m en t of a P -P ostla belin g Meth od for th e
2
Detection of 1,N -P r op a n od eoxygu a n osin e Ad d u cts of
2
-Hexen a l in Vivo
D. Schuler, Budiawan, and E. Eder*
Department of Toxicology, University of W u¨ rzburg, Versbacher Strasse 9,
D-97078 W u¨ rzburg, Germany
Received October 5, 1998
2
2
-Hexenal is an R,â-unsaturated carbonyl compound which forms cyclic 1,N -propano adducts
in vitro. The adduct formation in vivo was not reported by others to date. Because this type of
adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans
are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in
3
2
carcinogenicity. To improve the cancer risk assessment, we developed a new P-postlabeling
technique for this compound and optimized the different steps of the postlabeling procedure.
The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of
1
0% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 108
nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats,
the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate
in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2
days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts
were detected 8 h after gavage. The respective adducts could not be found as a background in
tissues of untreated rats or in calf thymus DNA at the limit of detection.
In tr od u ction
genesis. Detection and quantitation of the respective
DNA adducts in animals or in humans could lead to a
clear improvement in cancer risk assessment of 2-hex-
enal; however, attempts to detect the respective adducts
in vivo, e.g., after gavage of 2-hexenal to animals, have
not been successful to date (19). Therefore, we developed
2
-Hexenal is a bifunctional R,â-unsaturated carbonyl
compound which reacts with DNA. The in vitro adduct
formation of this compound was investigated in detail
by our group (1). Like other â-alkyl-substituted pro-
2
penals, it forms predominantly cyclic 1,N -propano-
32
2
a new
P-postlabeling technique for 1,N -propano-
deoxyguanosine adducts. A pair of diastereomers of only
that regioisomer were found in which the OH group is
adjacent to the N1 of the guanine moiety (N9 in the new
nomenclature; see Figure 2).The respective regioisomer
possesses a trans configuration; the cis isomer was not
detected in vitro (1). Site-specific studies and investiga-
tions with a plasmid shuttle vector system with this type
of adducts suggest that they constitute promutagenic
lesions (2, 3).
deoxyguanosine adducts of 2-hexenal.
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d Rea gen ts. Ca u tion : 2-Hexenal is irritating
and genotoxic. trans-2-Hexenal (99%) was purchased from
Aldrich (Steinheim, Germany). dG,1 dGp, and NS1 from As-
pergillus oryzae were obtained from Sigma (Deisenhofen, Ger-
many). Nuclease P1 from Penicillium citrinum was purchased
from Boehringer (Mannheim, Germany). [γ- P]ATP (4500 Ci/
mmol, 10 µCi/µL) and T4 PNK (30 units/µL) were obtained from
USB (Amersham, Braunschweig, Germany). PEI-cellulose TLC
plates were purchased from Macherey & Nagel (D u¨ ren, Ger-
many). RP18 TLC plates were obtained from Merck (Darmstadt,
Germany).
In str u m en ta tion . 1H NMR spectra were recorded on a
Bruker DMX 600 instrument, and mass spectra were measured
on a Fisons VG Trio 2000 spectrometer.
Syn th esis of Hex-d Gp Ad d u cts. A sample of 30 mg (0.077
mM) of dGp was reacted with 50 µL (0.43 mM) of 2-hexenal in
3
2
As expected from its interaction with DNA, 2-hexenal
is mutagenic in the Ames test with the Salmonella
typhimurium strain TA 104 (4) as well as with strain TA
1
00 (5). 2-Hexenal induces DNA strand breaks in mouse
L1210 cells (6) and in human Namalva cells (7), and
micro nuclei and sister chromatid exchange in human
lymphocytes (8).
2
-Hexenal is a common constituent of plants and is
therefore also called leave aldehyde. It occurs practically
in all vegetables and fruits in concentrations of 3-40 ppm
1
mL of 0.1 M sodium phosphate buffer (pH 10.7) at room
(9-17). 2-Hexenal evidently acts as a natural fungicide
and prevents fungi from penetrating into plant parts and
fruits after they have been injured (18).
Because of its widespread occurrence in vegetarian food
and its mutagenic and genotoxic activities, 2-hexenal is
considered to play an important role in human carcino-
1
Abbreviations: dA, deoxyadenosine; dAp, deoxyadenosine 3′-
monophosphate; dCp, deoxycytidine 3′-monophosphate; dG, deoxygua-
nosine; dGp, deoxyguanosine 3′-monophosphate; dTp, thymidine 3′-
monophosphate; DTT, dithiothreitol; Hex-dGp, 8-hydroxy-6-propyl-
,N -propano-2′-deoxyguanosine 3′-monophosphate; Hex-pdGp, 8-hy-
droxy-6-propyl-1,N -propano-2′-deoxyguanosine 3′,5′-bisphosphate; MN,
micrococcus nuclease; NP1, nuclease P1; NS1, nuclease S1; PEI,
polyethyleneimine; PNK, polynucleotide kinase; RP, reverse phase;
SPDE, spleen phosphodiesterase.
2
1
2
*
To whom correspondence should be addressed.
1
0.1021/tx980225b CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/19/1999

3
36 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Schuler et al.
temperature. Maximum conversion (40%) and maximum yield
were obtained after 8 days. The Hex-dGp adducts were
separated by HPLC (system 1) performed on a Hewlett-Packard
In the case of DNA hydrolysates, 6 µL of the NP1 mixture
consisting of 1.2 µL (6 µg) of the NP1 solution, 1.8 µL of 0.3 µM
ZnCl , and 3 µL of 250 mM sodium acetate (pH 4.0) was added
2
1
050 pump, a Rheodyne injector, and a Hewlett-Packard photo-
to a total volume of 20 µL of digested DNA. The mixture was
incubated for 1 h at 37 °C, and the reaction was stopped by
adding 2.4 µL of 0.5 M Tris base. The solution was desiccated
to dryness and redissolved in 10 µL of water.
diode array detector. The reaction mixture was separated on a
Nucleosil C18 column (250 mm long, 8 mm inside diameter, and
1
0 µm particle size) with a linear gradient from 10 mM
ammonium formate buffer (pH 4.7) to methanol with a flow rate
of 3.0 mL/min over the course of 80 min (HPLC system 1).
According to HPLC-UV detection at 254 nm, the yield was 25%
and the purity was >98%.
In h ibition of NP 1 by th e Nu cleosid e d A. Amounts from
1 to 10 nmol of dTp in a total volume of 20 µL were incubated
at 37 °C for 10 min with 4 µL (200 nmol) of dA and 6 µL of the
NP1 mixture (see NP1 Incubation). In this case, however,
Hex-d Gp 1: UV (pH 7) λmax ) 259 and 200 nm, λmin ) 230
2
sodium succinate and CaCl were not used. After the addition
of 2.4 µL of 0.5 M Tris base, 10 µL of the solution was analyzed
by HPLC system 2.
Op tim iza tion of th e La belin g Rea ction . To a solution of
300 fmol of Hex-dGp adducts in 10 µL of water was added 7.2
µL of labeling mixture consisting of a mixture of 1 µL of kinase
1
nm; H NMR (Bruker 600 MHz, D
3
2
O) δ 0.92 (t, J 13,12 ) 7.4 Hz,
H, H-13), 1.35-1.48 (m, 2H, H-12), 1.51-1.69 (m, 3H, H-11,
H-7a), 2.24 (pseudo-td, J 7b,7a ) 13.9 Hz, J 7b,6 ) J 7b,8 ) 3.1 Hz,
1
3
J
J
1
)
(
)
4
H, H-7b), 2.61 (ddd, J 2′a,2′b ) 13.9 Hz, J 2′a,1′ ) 6.4 Hz, J 2′a,3′
.2 Hz, 1H, H-2′a), 2.78 (pseudo-td, J 2′b,2′a ) 13.9 Hz, J 2′b,1′
)
)
2′b,3′ ) 7.0 Hz, 1H, H-2′b), 3.64-3.72 (m, 1H, H-6), 3.76 (dd,
5′a,5′b ) 12.5 Hz, J 5′a,4′ ) 4.7 Hz, 1H, H-5′a), 3.77 (dd, J 5′b,5′a
2.5 Hz, J 5′a,4′ ) 3.6 Hz, 1H, H-5′b), 4.20 (pseudo-q; J 4′,3′ ) J 4′,5′a
4′,5′b ) 3.8 Hz, 1H, H-4′), 4.78-4.84 (m, 1H, H-3′), 6.24
buffer [100 mM DTT, 100 mM MgCl , 10 mM spermidine, and
2
)
200 mM Bicine/NaOH (pH 9.5)], 1 µL of 2.2 µM [γ-32P]ATP (167
TBq/mmol, 4500 Ci/mmol; 0.37 MBq, 10 µCi), 1 µL of 20 µM
ATP, and 0.25 µL (7.5 units) of T4 PNK. The mixture was
incubated for 45 min at 37 °C. Subsequently, the solution was
reacted with 4 µL (40 milliunits) of apyrase for 30 min at the
same temperature.
J
pseudo-t, J 1′,2′a ) J 1′,2′b ) 7.0 Hz, 1H, H-1′), 6.29 (pseudo-t, J 8,7a
J
8,7b ) 2.6 Hz, 1H, H-8), 7.92 (s, 1H, H-2); MS (electrospray,
+
kV) m/z 446 (M + H).
Hex-d Gp 2: UV (pH 7) λmax ) 259 and 200 nm, λmin ) 230
Aliquots of 4 µL were applied to two-directional TLC on
prewashed PEI-cellulose sheets (without contact transfer) as
described in Chromatography.
On the basis of this method, single parameters were varied
as described in the Results.
1
nm; H NMR (Bruker 600 MHz, D
3
2
O) δ 0.91 (t, J 13,12 ) 7.4 Hz,
H, H-13), 1.34-1.44 (m, 2H, H-12), 1.51-1.71 (m, 3H, H-11,
H-7a), 2.24 (pseudo-td, J 7b,7a ) 13.9 Hz, J 7b,6 ) J 7b,8 ) 3.1 Hz,
1
3
J
J
1
)
(
)
4
H, H-7b), 2.60 (ddd, J 2′a,2′b ) 14.0 Hz, J 2′a,1′ ) 6.4 Hz, J 2′a,3′
.3 Hz, 1H, H-2′a), 2.77 (pseudo-td, J 2′b,2′a ) 14.0 Hz, J 2′b,1′
)
)
Tr ea tm en t of An im a ls. An oral dose of 500 mg of 2-hexenal/
kg of body weight dissolved in corn oil was administered to
2′b,3′ ) 7.0 Hz, 1H, H-2′b), 3.64-3,72 (m, 1H, H-6), 3.75 (dd,
5′a,5′b ) 12.6 Hz, J 5′a,4′ ) 4.7 Hz, 1H, H-5′a), 3.76 (dd, J 5′b,5′a
2.6 Hz, J 5′a,4′ ) 3.6 Hz, 1H, H-5′b), 4.19 (pseudo-q; J 4′,3′ ) J 4′,5′a
4′,5′b ) 3.8 Hz, 1H, H-4′), 4.80-4.84 (m, 1H, H-3′), 6.23
)
8
-week-old male Fischer 344 rats (190-210 g) by gavage.
Control animals were treated with corn oil only. The rats were
sacrificed at different times from 8 h to 4 days. Two rats were
used for each group, and two postlabeling determinations were
carried out for each rat liver. The liver was perfused with 0.9%
sodium chloride solution, and the DNA was isolated according
to the phenol extraction method as described by Gupta (20). The
concentration of the DNA was quantitated using the absorption
at 260 nm.
J
pseudo-t, J 1′,2′a ) J 1′,2′b ) 7.0 Hz, 1H, H-1′), 6.29 (pseudo-t, J 8,7a
J
8,7b ) 2.7 Hz, 1H, H-8), 7.91 (s, 1H, H-2); MS (electrospray,
+
kV) m/z 446 (M + H).
The concentrations were quantified by UV absorption, on the
basis of the molar absorbance coefficient derived for Hex-dG
259 ) 15 100 (1)].
[A
Sta bility of th e Ad d u cts. A sample of 7.5 nmol of the Hex-
DNA Hyd r olysis. DNA (10 µg) was incubated for 4 h at 37
dGp adducts was incubated in 0.1 M phosphate buffer at 37 °C
for 5 days. The pH value was varied from 2 to 13. Aliquots were
analyzed by the same HPLC setup as described above using a
Nucleosil C18 column (250 mm long, 4 mm inside diameter, and
°
C with 3 µL of the enzyme mixture containing 0.2 unit/µL (1
µg/µL) MN and 0.002 unit/µL (1 µg/µL) SPDE and with 4 µL of
DNA digestion buffer [25 mM CaCl and 50 mM sodium
2
succinate (pH 6.0)] in a total volume of 20 µL. Control experi-
ments were performed to examine whether the DNA was
completely digested to the respective 3′-monophosphates under
these conditions by HPLC analysis of the 3′-monophosphate
5
µm particle size) with a linear gradient from 10 mM am-
monium formate buffer (pH 4.7) to methanol with a flow rate
of 1.0 mL/min over the course of 20 min (system 2).
NS1 In cu ba tion . Samples of 8 nmol of the different nucleo-
side 3′-monophosphates (equivalent to 10 µg of DNA) were
incubated in a total volume of 15 µL at 37 °C for 60 min with
nucleotides using HPLC system 1.
32P -P ostla belin g. To the digested and NP1-enriched solution
was added 6.5 µL of the labeling mixture. It was made as a
mixture of 1 µL of kinase buffer [100 mM DTT, 100 mM MgCl
0
.7 µg (180 units) of NS1 and 3 µL of NS1 mixture which
consisted of 83 mM sodium acetate (pH 4.5), 83 mM sodium
chloride, and 1.67 mM ZnCl . Ten microliters of the solution
2
,
1
0 mM spermidine, and 200 mM Bicine/NaOH (pH 9.5)], 0.5
2
µL of 800 mM Bicine, 5 µL of 2.2 µM [γ- P]ATP (167 TBq/mmol,
500 Ci/mmol; 1.9 MBq, 50 µCi), and 0.25 µL (7.5 units) of T4
32
was analyzed by HPLC system 2.
4
For the examination of the stability of Hex-dGp, a sample
of 7 nmol of the adduct was incubated with 12.5 µg (3300 units)
of NS1 for 120 min.
NP 1 In cu ba tion . For NP1 incubation of the nucleoside 3′-
monophosphates, samples of 8 nmol of the different nucleoside
PNK. The sample was incubated for 45 min at 37 °C. Then, 4
µL (40 milliunits) of apyrase was added, and the solution was
incubated for 30 min at the same temperature. The labeling
efficiency was determined by application of defined amounts of
adduct standards from 1 to 100 fmol. The samples were
measured with the PhosphorImager additionally calibrated
3
′-monophosphates (equivalent to 10 µg of DNA) were incubated
3
2
with 6 µL of the NP1 mixture in a total volume of 18 µL for 10
min at 37 °C. The NP1 mixture consisted of 1.2 µL (0.6 µg, 10%
of the regular amount used for PL) of the NP1 solution, 1.8 µL
using adenosine [ P]triphosphate.
Ch r om a togr a p h y. For the detection of the Hex-dG adducts
in vivo, a contact transfer method was used. The different
samples were applied in portions of about 5 µL to an RP18 TLC
sheet (10 cm long) to which a wick was attached (7 cm,
Whatman #17). The chromatography was performed with 0.4
M ammonium formate/formic acid (pH 6.0) overnight. The
origins were cut out (1 cm × 1 cm, no washing) and attached
with clothes pegs to PEI-cellulose sheets (10 cm × 10 cm). The
transfer to the PEI-cellulose material was carried out by
developing the sheets with n-propanol/water/nonidet P40 (50:
of 0.3 mM ZnCl
2
, and 3.0 µL of buffer (0.25 M sodium acetate,
). After 10 min, the
4
0 mM sodium succinate, and 20 mM CaCl
2
reaction was stopped by adding 2.4 µL of 0.5 M Tris base. Ten
microliters of the mixture was analyzed by HPLC system 2.
The pH value of the sodium acetate buffer was varied for the
determination of the pH optimum.
Using Hex-dGp, the incubation was performed with 6 µg of
NP1 for 180 min.

Development of a 32P-Postlabeling Method
Chem. Res. Toxicol., Vol. 12, No. 4, 1999 337
F igu r e 1. HPLC chromatogram of the reaction of 2-hexenal
with deoxyguanosine 3′-phosphate at pH 10.7 after 8 days.
HPLC conditions are described in Materials and Methods.
5
0:1, v/v/v) at 50 °C. The RP18 chips were removed, and the
TLC plates were washed thoroughly with water. After the plates
were dried, chromatography was performed in the first direction
(D ) with 1.2 M ammonium formate/formic acid (pH 3.5) after
1
attaching wicks (4 cm, Whatman #1). The wicks were removed,
and the TLC sheets were washed in water and air-dried.
Another wick (4 cm, Whatman #1) was attached, and for
chromatography in the second direction, 0.3 M ammonium
sulfate (adjusted to pH 7.5 with sodium phosphate) was used.
The chromatograms were visualized and quantitated by a
PhosphorImager (Molecular Dynamics).
Qu a n tita tion of DNA Ad d u cts. Quantitation of the adduct
levels was based on the recovery for the reference compound
Hex-dGp in an extra sample with DNA of a control animal for
the amount of adducts. The number of nucleotides was calcu-
lated via the UV absorption of the DNA solution.
F igu r e 2. 1H- H COSY NMR spectrum of Hex-dGp1 in D
1
O.
2
Resu lts a n d Discu ssion
Syn th esis of Sta n d a r d s. The synthesis of the 1,N2-
propanodeoxyguanosine 3′-monophosphate adducts of
2
-hexenal as standards is a prerequisite for developing
the postlabeling technique and for optimizing the differ-
ent steps of the technique. Furthermore, the utilization
of the standard to work out the chromatographic meth-
ods, in particular if using the standard for cochromatog-
raphy, can avoid possible artifacts. Therefore, we syn-
thesized the standards by reaction of 2-hexenal with
deoxyguanosine 3′-monophosphate (dGp) as described in
Materials and Methods. Figure 1 shows the HPLC
chromatogram of the reaction mixture after reaction for
F igu r e 3. Dependence of the rate of dephosphorylation of
different nucleoside 3′-monophosphates by NP1 on pH.
the coupling constants (Materials and Methods). The
results are also consistent with our previous publications
(1) (see also the Introduction).
2
8
days. A pair of diastereomers is formed and can be well
Well-characterized and pure (>98%) 1,N -propanode-
separated under our chromatographic conditions (see
Materials and Methods). Besides the two adduct peaks,
those of the starting materials, dGp and 2-hexenal, can
be seen. This means that despite the long reaction time
the reaction is not complete. The relatively long reaction
time is a general feature of the reaction of R,â-unsatu-
rated carbonyl compounds with DNA components (1, 21,
oxyguanosine 3′-monophosphate adducts are now avail-
able for further studies.
Hyd r olysis of DNA. In our control experiments, we
found that DNA was practically completely digested to
the 3′-monophosphates under the described conditions
already after 30 min and measured concentrations of 30%
for dAp, 29% for dGp, and 21% for dCp and dTp were
related to the total amount of DNA digested after
incubation for 30 min. Nevertheless, we used the stand-
ard method and incubated the DNA for 4 h at 37 °C.
Resu lts of th e P ostla belin g Tech n iqu e. (1) En -
r ich m en t P r oced u r e. The lipophilicity of the cyclic
propano adducts Hex-dGp1 and -2 is not sufficient for
the butanol extraction (not shown). Also, the utilization
of HPLC methods as a single enrichment method turned
out not to be practical (not shown). Therefore, we tried
the nuclease P1 and the nuclease S1 enrichment. To
determine optimum conditions, we first investigated the
stability of the standards at different pH values as
described in Materials and Methods and found that the
adducts are stable over a wide pH range of pH 4-11.
Figure 3 shows the dependence of the nuclease P1-
catalyzed dephosphorylation of the normal 3′-nucleotides
on pH. Maximum dephosphorylation rates were obtained
at pH 4. Therefore, the nuclease P1 enrichment was
2
2).
Figure 2 shows the H- H COSY NMR spectrum of
Hex-dGp1 in D O which enables a clearer attribution
1
1
2
1
1
13
of the H signals. Besides H NMR spectra, C NMR
spectra, mass spectra, and UV spectra were recorded to
characterize the standards (Materials and Methods). The
spectroscopic data for Hex-dGp1 and Hex-dGp2 are
nearly identical. The 13C NMR spectra of the Hex-dGp
adducts are practically identical with those of the respec-
tive Hex-dG adducts (1) and are therefore not shown
and discussed here. The spectroscopic data shown here
are in accordance with our in vitro studies with dG
published recently (1). Because of the newly formed
stereocenters at the C-6 and C-8 atoms in the newly
formed ring, four different isomers could be formed
theoretically. We could, however, find only the two
isomers in which the substituents in the newly formed
ring are in the trans position as can be demonstrated by

3
38 Chem. Res. Toxicol., Vol. 12, No. 4, 1999
Schuler et al.
F igu r e 6. Dependence of the labeling efficiency on pH (a) and
the reaction time (b).
F igu r e 4. Michaelis-Menten kinetics for the dephosphoryl-
ation of Hex-dGp by NP1 at pH 4.0.
parameters were examined, e.g., the influence of the pH
value on the labeling efficiency, the influences of the
reaction time, of the ATP concentration, and of the used
radioactivity, the amount of the polynucleotide enzyme
on the labeling efficiency, or the effect of the reaction
temperature on the labeling efficiency. Furthermore, the
influences of the concentration of the components of the
2
labeling buffer, i.e., DTT, spermidine, Bicine, and MgCl ,
were proven. Because metal ions can influence the
substrate specificity of DNA polynucleotides (23) and also
of the enzyme polynucleotide kinase (24), we, in addition,
investigated the dependence of the labeling efficiency on
F igu r e 5. Inhibition of the dephosphorylation of dTp by NP1
in the presence of dA (Michaelis-Menten kinetics and Line-
weaver-Burk plot).
concentrations of the five different metal salts (CaCl
2
,
BeCl , CdCl , CoCl , and MnCl ). The labeling efficiencies
2
2
2
2
under standard conditions were in every case lower after
addition of any of these metal salts in a concentration of
1 mM than without these metal ions. Therefore, addition
of any of these metal salts does not lead to a better
labeling efficiency and is not recommended.
Figure 6 shows a selection of parameters that were
determined so the labeling efficiency could be optimized.
The highest labeling efficiencies were obtained at pH 8.7,
and at a reaction time of 45 min. Furthermore, we found
that the Bicine concentration must be higher than 30 mM
so a decrease in the labeling efficiency could be avoided
(not shown).
There is a remarkably steep decrease in the labeling
efficiency from pH 7.8 to 7.0. To compensate for the acid
buffer of the DNA digest (pH 6.0) and that of the nuclease
P1 enrichment (pH 4.0), we used a pH value of 9.5 for
the kinase buffer. Although a maximum labeling ef-
carried out at pH 4 throughout the studies with the
2
-hexenal adducts.
In contrast to the normal nucleotides, the respective
Hex-dGp adducts are rather stable against nuclease P1
under our conditions (Figure 4). A rate constant k of
-1
0
.00056 min was calculated for these 2-hexenal adducts
-
1
compared with a rate constant k of 3.5 min for dTp,
the most stable normal nucleotide (see Figure 4). The rate
constants indicate that the nuclease P1 enrichment
should be an appropriate method for the 2-hexenal
adducts. When examining these values by measuring the
dTp concentration after nuclease P1 treatment, we
surprisingly found that the decrease in the concentration
of dTp was somewhat lower than that calculated on the
basis of the Michaelis-Menten equation. This discrep-
ancy can be explained by an inhibition of nuclease P1 by
the formed nucleoside thymidine. To verify this hypoth-
esis, we measured the enzyme kinetics of dTp in the
presence of deoxyadenosine as a representative of a
nucleoside as described in Materials and Methods (In-
hibition of NP1 by the Nucleoside dA). The results are
demonstrated in Figure 5. Whereas the addition of
2
ficiency was found at a MgCl concentration of 2.8 mM
and at a spermidine concentration of 0.28 mM (not
shown), we did not change the standard concentrations
of 100 and 10 mM, respectively, since the effects of the
concentrations of MgCl and spermidine on the labeling
2
efficiency were only marginal. With these labeling condi-
tions, we achieved a labeling efficiency of 35%.
(3) Ch r om a togr a p h y a n d Qu a n tita tion . The chro-
matographic analysis of the adducts formed in vivo was
performed on a PEI-cellulose sheet as described in
Materials and Methods. The contact transfer method
performed on an RP18 TLC sheet (25-27) was used, in
addition, to minimize the radioactive background on the
TLC sheet, in particular, in the region where the adduct
spot appears at the PEI-cellulose sheet and to avoid
artifacts. Information about the reproducibility of the
contact transfer method is actually rather scarce in the
literature (25-27). We found a relative standard devia-
tion of 27%.
deoxyadenosine leads to a clear decrease of the maximum
conversion rate from 7.4 to 3.1 nmol min-1 mg enzyme
-1
,
M
the K remains nearly uninfluenced (0.71 vs 0.58 mM).
The performance of the complete labeling technique
showed, however, that the nuclease P1 enrichment
procedure is an excellent tool for the separation of the
normal nucleotides from Hex-dGp adducts, despite the
inhibition of the enzyme by the formed nucleosides.
Besides the nuclease P1, the utilization of the nuclease
S1 was examined using the methods described for nu-
clease P1. The application of nuclease S1 did, however,
not provide any advantages and did not lead to better
results compared with the nuclease P1 method.
(
2) Op tim u m La belin g Con d ition s. To achieve the
During the development of the TLC conditions, we
tried different solvents that were capable of separating
monophosphates from bisphosphates and compared the
highest labeling efficiencies and the highest sensitivity,
we determined optimum labeling conditions. Several

Development of a 32P-Postlabeling Method
Chem. Res. Toxicol., Vol. 12, No. 4, 1999 339
F igu r e 7. Postlabeling analysis of liver DNA in male Fischer
3
2
44 rats 2 days after administering an oral dose of 500 mg of
-hexenal/kg of body weight: (left) control and (right) treated
F igu r e 8. Dependence of Hex-dG levels in the liver of male
Fischer 344 rats on the time after application of an oral dose of
animal.
5
00 mg of 2-hexenal/kg of body weight. The values represent
results with other adducts to be sure that the spot in
Figure 7 is actually the 3′,5′-bisphosphate adduct and not
the 5′-monophosphate which should have different chro-
matographic properties (unpublished results). Further-
more, the labeling efficiency increases from pH 7 to a
maximum at pH 8.8 (see Figure 6a), whereas the 3′-
phosphatase activity of the PNK is increasing with
decreasing pH values. The phosphatase activity of PNK
is extremely low under the conditions at pH 9.5 used here
so that it is unlikely that substantial and detectable
amounts of 5′-monophosphate adducts are formed (this
phenomenon is dealt with in more detail in a forthcoming
paper). The TLC conditions described offer the advantage
that the adduct spot is effectively distinguished from the
normal nucleotides and impurities and that the two
diastereomers appear in one spot (data not shown
because it is practically identical with Figure 7). The
chromatograms were visualized and analyzed via a
PhosphorImager, and the adduct levels were measured
on the basis of the recovery of an added standard to the
DNA of a control animal. Although the recovery was only
means from two different rat livers per group and two deter-
minations per liver.
amounts of 2-hexenal are formed in vivo. This conforms
with our in vitro results. On the other hand, the 2-hex-
enal adducts are repaired rather soon because after 4
days the adduct level is decreased to about one-third of
the maximum level found 2 days after gavage. This
discrepancy can, however, be explained easily by me-
tabolization and elimination of 2-hexenal and by DNA
repair. These considerations could be important for
cancer risk assessment of 2-hexenal.
The 32P-postlabeling technique developed and opti-
mized for 2-hexenal adducts provides the possibility of
sensitively measuring DNA adduct levels of 2-hexenal
in animals after high levels of exposure. Approaches for
estimating a possible cancer risk of 2-hexenal can now
be carried out on the basis of adduct levels and daily
intake of 2-hexenal via food.
Refer en ces
1
0% if the contact transfer method was being used, the
8
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