A schiff base is a major DNA adduct of crotonaldehyde

Article abstract of DOI:10.1021/tx000234w

Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in human tissues as well as tissues of untreated laboratory animals. A second class of crotonaldehyde - DNA ad

Full text of DOI:10.1021/tx000234w

Chem. Res. Toxicol. 2001, 14, 423-430
423
A Sch iff Ba se Is a Ma jor DNA Ad d u ct of Cr oton a ld eh yd e
Mingyao Wang, Edward J . McIntee, Guang Cheng, Yongli Shi,
Peter W. Villalta, and Stephen S. Hecht*
University of Minnesota Cancer Center, Minneapolis, Minnesota 55455
Received November 9, 2000
Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces
Michael addition products, and these have been detected in human tissues as well as tissues
of untreated laboratory animals. A second class of crotonaldehyde-DNA adducts releases 2-(2-
hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these
adducts are quantitatively more significant than the Michael addition adducts in vitro. In
this study, we demonstrate that the major source of the paraldol-releasing DNA adducts of
crotonaldehyde is a Schiff base. Reaction of crotonaldehyde with DNA, followed by treatment
with NaBH3CN and enzyme hydrolysis, resulted in the formation of N2-(3-hydroxybutyl)dG
(10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with
dG demonstrated that the Schiff base precursor to N2-(3-hydroxybutyl)dG is N2-(3-hydroxy-
butylidene)dG (7), identified by UV, LC-APCI-MS, and MS/MS. Four isomers of N2-(3-
hydroxybutylidene)dG were observed. The (R)- and (S)-isomers were identified by reactions of
chiral paraldol with dG; each existed as a pair of interconverting (E)- and (Z)-isomers. These
data indicate that the structure of the major Schiff base DNA adduct in crotonaldehyde-treated
DNA is N2-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and
accounts for more than 90% of the paraldol released from crotonaldehyde-treated DNA.
However, the adduct is stable in DNA and therefore is a likely companion to the Michael
addition adducts in human DNA.
tonaldehyde-treated DNA is subjected to enzymatic hy-
drolysis (EH), neutral thermal hydrolysis (NTH),1 or acid
hydrolysis, significant amounts of 2-(2-hydroxypropyl)-
4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12, Scheme 1)
are released. Paraldol is the dimer of 3-hydroxybutanal
(6). Paraldol-releasing DNA adducts were 13-75 times
more prevalent than Michael addition adduct 2 in cro-
tonaldehyde-treated DNA (11). This suggests that these
adducts may also be found in significant quantities in
human DNA. Therefore, we investigated the structures
of the paraldol-releasing DNA adducts. We identified
several diastereomers of N2-[2-(2-hydroxypropyl)-6-meth-
yl-1,3-dioxan-4-yl]deoxyguanosine (N2-paraldol-dG, 11)
and some related adducts resulting from incomplete
hydrolysis of the adducted DNA (11). However, N2-
paraldol-dG and related adducts accounted for less than
10% of the paraldol released from EH or NTH of cro-
tonaldehyde-treated DNA. At present, we have charac-
terized the major paraldol-releasing DNA adduct of
crotonaldehyde as N2-(3-hydroxybutylidene)dG (7, Scheme
1).
In tr od u ction
Crotonaldehyde (1, Scheme 1) is a mutagen and
carcinogen (1, 2). This simple R,â-unsaturated aldehyde
is a common environmental contaminant, found in mobile
source emissions, the atmosphere, tobacco smoke, and
other thermal degradation mixtures (1). Crotonaldehyde
is also a product of lipid peroxidation and a metabolite
of the hepatocarcinogen, N-nitrosopyrrolidine (3, 4). In
a shuttle vector system, crotonaldehyde induces a variety
of mutations, including dG-dThd transversions, dG-
dAdo transitions, and tandem base substitutions (5).
These mutations result from crotonaldehyde-DNA ad-
ducts.
Initial investigations of crotonaldehyde’s reactions with
dG and DNA led to the discovery of the Michael addition
pathway, resulting in 1,N2-propano-dG adducts 2 as well
as adducts 3-5 (Scheme 1) (6-9). Diastereomers of each
of these products have been characterized. Chung and
co-workers have detected two diastereomers of 2 (with
methyl and hydroxy trans to each other) in DNA of
various tissues from untreated laboratory animals as well
as humans (8, 10). They propose that these adducts arise
from endogenous lipid peroxidation as well as exogenous
exposures such as cigarette smoke. The presence of DNA
adducts of crotonaldehyde in human tissues suggests that
they may play some role in cancer development.
We have recently discovered a second pathway of
crotonaldehyde-DNA adduct formation (11). When cro-
Exp er im en ta l Section
Ap p a r a tu s a n d Assa y Con d ition s. HPLC analyses were
carried out with Waters Associates (Waters Division, Millipore,
Milford, MA) systems equipped with
a model 991 or 996
photodiode array detector. Flow rates were 1 mL/min unless
noted otherwise. The following solvent elution systems were
* To whom correspondence should be addressed: University of
Minnesota Cancer Center, Mayo Mail Code 806, 420 Delaware St. SE,
Minneapolis, MN 55455. Telephone: (612) 624-7604. Fax: (612) 626-
5135. E-mail: hecht002@umn.edu.
1 Abbreviations: 2,4-DNP, 2,4-dinitrophenylhydrazine; N2-paraldol-
dG, N2-[2-(2-hydroxypropyl)-6-methyl-1,3-dioxan-4-yl]deoxyguanosine;
NTH, neutral thermal hydrolysis; paraldol, 2-(2-hydroxypropyl)-4-
hydroxy-6-methyl-1,3-dioxane.
10.1021/tx000234w CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/17/2001

424 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Sch em e 1. Ad d u ct F or m a tion in th e Rea ction of Cr oton a ld eh yd e (1) w ith d G or DNAa
Wang et al.
a
Several stereoisomers of Michael addition products 2-5 as well as N2-paraldol-dG (11) have been characterized (4, 7-9, 11). EH,
enzyme hydrolysis; NTH, neutral thermal hydrolysis.
used: (1) two 4.6 mm × 25 cm, 5 µm Supelcosil LC 18-BD
columns (Supelco, Bellefonte, PA) with isocratic elution by 5%
CH3CN in 10 mM sodium phosphate buffer (pH 7) for 10 min
and then a gradient from 5 to 25% CH3CN over the course of
60 min and detection by UV (254 nm), for analysis of reactions
of crotonaldehyde or racemic paraldol with dG, and hydrolysates
of crotonaldehyde-treated DNA; (2) one of the columns used in
system 1 with isocratic elution by 5% CH3OH in 40 mM
ammonium acetate buffer (pH 6.6) for 10 min and then a
gradient from 5 to 35% CH3OH over the course of 60 min at a
flow rate of 0.5 mL/min, with detection by UV (254 nm), for
collection of Schiff bases for LC-APCI-MS analysis and analysis
of reactions of chiral paraldol with dG; and (3) a 4.6 mm × 25
cm, 5 µm OD5 octadecyl column (Burdick & J ackson, Baxter,
McGaw Park, IL) with elution by a gradient from 40 to 60%
CH3CN in H2O over the course of 40 min with UV detection
(365 nm), for analysis of 2,4-dinitrophenylhydrazones of cro-
tonaldehyde and 3-hydroxybutanal.
NMR data were obtained on an 800 MHz instrument (Varian,
Inc., Palo Alto, CA) using Shigemi 3 mm tubes (Shigemi, Inc.,
Allison Park, PA).
Ch em ica ls a n d En zym es. 2,4-Dinitrophenylhydrazine (2,4-
DNP) reagent was purchased from Eastman Organic Chemicals
(Rochester, NY). Crotonaldehyde and NaBH3CN were obtained
from Aldrich Chemical Co. (Milwaukee, WI). Freshly opened
bottles of crotonaldehyde had no detectable impurities, as
determined by GC analysis (11). Alkaline phosphatase was
purchased from Boehringer Mannheim Co. (Indianapolis, IN).
Paraldol and chiral paraldol were prepared as described previ-
ously (11, 12). All other chemicals and enzymes were obtained
from Sigma Chemical Co. (St. Louis, MO).
Rea ction s. (1) Cr oton a ld eh yd e a n d DNA. Crotonaldehyde
(0.4 mmol) from a freshly opened bottle was allowed to react
with calf thymus DNA (20 mg) in 2 mL of 0.1 M phosphate
buffer (pH 7) at 37 °C for 96 h. The modified DNA was
precipitated by addition of ethanol and then washed with 70%
ethanol and ethanol. This DNA sample was used for the
analyses presented in Figures 1 and 2.
LC-MS and LC-MS/MS analyses were carried out on a
Finnigan MAT LCQ Deca instrument (Thermoquest LC/MS
Division, San J ose, CA) interfaced with a Waters Alliance 2690
HPLC multisolvent delivery system and equipped with an SPD-
10A UV-vis detector (Shimadzu Scientific Instruments, Co-
lumbia, MD). For LC-APCI-MS, the same column as in HPLC
system 3 was used, with elution by a gradient from 0 to 60%
CH3CN in H2O over the course of 60 min at a flow rate of 0.5
mL/min. The APCI source was set as follows: voltage, 3.5 kV;
current, 4.8 µA; capillary temperature, 200 °C; and vaporizer
temperature, 400 °C. For LC-ESI-MS, the same column as in
HPLC system 3 was used, with elution by a gradient from 0 to
30% CH3CN in 1% acetic acid over the course of 60 min at a
flow rate of 0.5 mL/min. The ESI source was set as follows:
voltage, 2.0 kV; current, 11 µA; and capillary temperature, 275
°C. MS/MS data were acquired with the following parameters:
parent mass, 338 amu; isolation width, 1.5 amu; activation
amplitude, 25%; activation Q, 0.25; and activation time, 30 ms.
(2) Cr oton a ld eh yd e or P a r a ld ol a n d d G. Crotonaldehyde
(0.18 mmol) was allowed to react with dG (20 mg) in 10 mL of
0.1 M phosphate buffer (pH 7) at 37 °C for 22 h. Paraldol or
chiral paraldol (0.13 mmol) was allowed to react with dG (6.7
mg) in 2.5 mL of 0.1 M phosphate buffer at 37 °C for 22 (racemic)
or 96 h (chiral).
Hyd r olysis of DNA. EH and NTH were carried out as
previously described (11, 13). Treatment of DNA with NaBH3-
CN followed by enzymatic hydrolysis was also performed as
described previously (9). Paraldol was also treated in this way.
Sta bility of th e Sch iff Ba se Ad d u ct in DNA. Aliquots of
DNA (2.5 mg) that had been allowed to react with crotonalde-
hyde for 24 h were washed with ethanol, then were dissolved
in 1 mL of Tris-HCl buffer (pH 7), and allowed to stand at
37 °C for 4, 8, 24, or 70 h. The DNA was then treated with

Crotonaldehyde-DNA Adduct
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 425
F igu r e 1. DNA was allowed to react with crotonaldehyde and then subjected to NTH (A) or treated with NaBH3CN prior to NTH
(B). The resulting mixtures were treated with 2,4-DNP reagent and then analyzed by HPLC (system 3). Chromatograms are illustrated.
The arrows in panel B represent the retention times of the 2,4-dinitrophenylhydrazones of 3-hydroxybutanal and crotonaldehyde,
which were detected only in panel A.
F igu r e 2. DNA was allowed to react with crotonaldehyde and then subjected to enzyme hydrolysis (A) or treated with NaBH3CN
prior to enzyme hydrolysis (B). The resulting mixtures were analyzed by HPLC (system 1), and the chromatograms are illustrated.
Numbers above the peaks refer to the structures in Scheme 1.
from DNA adducts such as N2-paraldol-dG which release
paraldol when treated with the acidic 2,4-DNP reagent.
NaBH3CN and enzymatically hydrolyzed. The hydrolysates were
analyzed for 10 by HPLC system 2.
On the basis of these results, we speculated that the
major unstable paraldol-releasing DNA adduct of cro-
tonaldehyde was a Schiff base such as 7 or 8.
Resu lts
In a previous study, we carried out HPLC analysis of
enzymatic hydrolysates of crotonaldehyde-treated DNA
(11). Fractions were collected from each portion of the
chromatogram, and each fraction was assayed for paral-
dol (12) by reaction with 2,4-DNP reagent. When paraldol
itself or a paraldol-releasing DNA adduct is present, the
2,4-dinitrophenylhydrazones of 3-hydroxybutanal and
crotonaldehyde are detected. The results of this study
demonstrated that, during enzymatic hydrolysis of this
crotonaldehyde-treated DNA, large amounts of paraldol
itself are released, presumably from an unstable DNA
adduct. This elutes in the 5-15 min retention time
fraction and accounts for at least 90% of the paraldol
detected at various points in the chromatogram by the
2,4-DNP reagent method. The remaining 10% is produced
In our study of acetaldehyde-DNA reactions, we
developed conditions for analyzing the Schiff base N2-
ethylidene-dG, which is the major DNA adduct of acetal-
dehyde (9). Reaction of acetaldehyde-treated DNA with
NaBH3CN results in near-quantitative conversion of N2-
ethylidene-dG to N2-ethyl-dG. If a Schiff base were
responsible for release of paraldol during hydrolysis of
crotonaldehyde-treated DNA, then reaction of this DNA
with NaBH3CN prior to hydrolysis should suppress
release of paraldol. As shown in Figure 1A, NTH of
crotonaldehyde-treated DNA followed by treatment with
2,4-DNP reagent resulted in formation of the 2,4-dini-
trophenylhydrazones of 3-hydroxybutanal and crotonal-
dehyde, as expected. However, these products were not
detected when the DNA was treated with NaBH3CN prior

426 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Wang et al.
F igu r e 3. UV spectra of (A) N2-ethylidene-dG, (B) N2-ethyl-dG, (C) peak C of Figure 5A, and (D) peak A of Figure 2B. All spectra
were obtained in 0.1 M phosphate buffer (pH 7.0).
to NTH (Figure 1B). These results suggested the presence
of a Schiff base.
In the next step, crotonaldehyde-treated DNA was
either hydrolyzed enzymatically or allowed to react with
NaBH3CN before enzyme hydrolysis. The hydrolysates
were analyzed by HPLC. As shown in Figure 2A, the
1,N2-propano-dG adducts 2 as well as N2-paraldol-dG
adducts 11 were detected in hydrolysates of DNA that
had not been treated with NaBH3CN, consistent with
previous results. However, hydrolysates of the NaBH3-
CN-treated DNA showed two major new peaks (A and
B) eluting at 37.8 min, just prior to the 1,N2-propano-dG
diastereomers 2 (Figure 2B). It is evident from Figure
2B that the levels of these peaks far exceed those of 1,N2-
propano-dG adducts 2 or N2-paraldol-dG (11). Peaks A
and B were also formed upon NaBH3CN treatment of
reaction mixtures of crotonaldehyde with dG, or paraldol
with dG. The UV spectra of peaks A and B were identical;
that of peak A is shown in Figure 3D. It is identical to
F igu r e 4. Chromatogram obtained upon HPLC analysis
that of N2-ethyl-dG (Figure 3B). ESI-MS of peaks A and
(system 1) of products formed in the reaction of crotonaldehyde
B each give a base peak at m/z 340, which is M + 1 of a
with dG. Numbers by the peaks refer to the structures in
Scheme 1. Peaks C-F are isomers of N2-(3-hydroxybutylidene)-
dG (see the text).
1
hydroxybutyl-substituted dG. H NMR data for peaks A
and B, collected together from HPLC chromatograms of
the NaBH3CN-treated paraldol-dG reaction mixture, are
summarized in Table 1. All assignments were confirmed
by COSY. The 1H NMR data are similar to those
previously reported for N2-(4-hydroxybutyl)dG (14). Col-
lectively, these spectral data conclusively establish the
structure of peaks A and B as two diastereomers of N2-
(3-hydroxybutyl)dG (10).
examined the reaction of crotonaldehyde with dG. This
produced the chromatogram illustrated in Figure 4.
Peaks C-F are the hypothesized Schiff base precursors
to 10. Peaks C-F were also formed but in greater relative
yield in the reaction of paraldol (12) with dG as il-
lustrated in Figure 5A. The UV spectra and retention
times of peaks C-F in Figures 4 and 5A are identical.
When the paraldol-dG reaction mixture was treated
with NaBH3CN, peaks C-F disappeared with appear-
Our next goal was to isolate and characterize the
postulated Schiff base precursor to 10. As the Schiff base
would be unstable to conditions of DNA hydrolysis, we

Crotonaldehyde-DNA Adduct
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 427
Ta ble 1. 1H NMR Da ta (800 MHz) for N2-(3-Hyd r oxybu tyl)d Ga
deoxyguanosyl protons
C-8H
N2-H
1′-H
2′-Ha,b
3′-H
4′-H
5′-Ha,b
3′-OH
5′-OH
7.79 (s)
7.60 (bs)
6.13 (dd)
J ) 7.2, 7.2 Hz
2.17 (m)
2.62 (m)
4.34 (m)
3.79 (m)
3.49 (m)
3.55 (m)
5.25 (bs)
4.91 (bs)
butyl protons
1-Ha,b
2-Ha,b
3-H
4-CH3
3.29 (t)b
J ) 6.8 Hz
1.53 (m)
1.59 (m)
3.69 (m)
1.07 (d)
J ) 6.4 Hz
a
b
Collected via HPLC; see Figure 5B. Obscured in the DMSO spectrum. Measured with DMSO and D2O.
F igu r e 5. Chromatogram obtained upon HPLC analysis (system 1) of products formed in the reaction of paraldol with dG (A) or of
paraldol with dG followed by treatment with NaBH3CN (B). Numbers above the peaks refer to the structures in Scheme 1. Peaks
C-F are isomers of N2-(3-hydroxybutylidene)dG (see the text).
ance of N2-(3-hydroxybutyl)dG (Figure 5B). Treatment
with NaBH3CN of each of peaks C-F of Figure 4A gave
N2-(3-hydroxybutyl)dG, identified by retention time, UV,
and MS. Furthermore, treatment of each of peaks C-F
of Figure 5A with 2,4-DNP reagent gave a mixture of the
2,4-dinitrophenylhydrazones of 3-hydroxybutanal and
crotonaldehyde. These data demonstrate that peaks C-F
in Figures 4 and 5A are the Schiff base precursors to N2-
(3-hydroxybutyl)dG.
We sought further information about the structures of
Schiff base peaks C-F. The UV spectra of peaks C-F
were identical; that of peak C of Figure 5A is shown in
Figure 3C. A shoulder appears at 275 nm compared to
280 nm in the spectrum of N2-(3-hydroxybutyl)dG (Figure
3D). An identical relationship exists between N2-eth-
ylidene-dG, the Schiff base of acetaldehyde and dG
(Figure 3A), and N2-ethyl-dG [Figure 3B (9)]. These UV
data, together with the chemical studies described above,
provide convincing evidence that peaks C-F of Figures
4 and 5A are Schiff bases.
and then analyzing by LC-APCI-MS and MS/MS. These
data are summarized in Table 2. MS data for peaks C
and D (together) and for each of peaks E and F were
similar. Each exhibited an M + 1 ion at m/z 338, as well
as ions corresponding to loss of H2O, deoxyribose, deoxy-
ribose with H2O, and deoxyribose with CH3CHO. MS/MS
analysis of m/z 338 gave similar fragments for each peak,
C-F. These results are consistent with N2-(3-hydroxy-
butylidene)dG (7), Scheme 1, as the structure of the Schiff
base.
When peaks E and F were collected without cooling,
and then immediately re-injected under the same condi-
tions, the chromatogram illustrated in Figure 6 was
obtained. These results demonstrate that peaks E and F
are unstable, giving dG as well as smaller amounts of
peaks C and D. After the mixture stood for 3 h at room
temperature, peaks C-F had completely reverted to dG
plus paraldol, as determined by HPLC. Due to the
instability of peaks C-F, we were not able to obtain
interpretable NMR data.
MS data on peaks C-F were obtained by collecting
them in HPLC system 2, cooling immediately to -80 °C,
Further information about the structures of peaks C-F
was obtained by examining the reactions with dG of

428 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Ta ble 2. AP CI-MS a n d MS/MS Da ta for P ea k s C-F of F igu r es 4 a n d 5Aa
(A) APCI-MS
Wang et al.
m/z 204 (M -
deoxyribose -
H2O + 1)
m/z 178 (M -
deoxyribose -
CH3CHO + 1)
m/z 338
(M + 1)
m/z 320
(M + 1 - H2O)
m/z 294 (M +
1 - CH3CHO)
m/z 222 (M -
deoxyribose + 1)
peak
C + Db
E
F
10
33
36
8
18
25
NDc
ND
3
27
100
100
20
50
62
9
22
28
(B) MS/MS of m/z 338
peak
m/z 294
m/z 222
m/z 204
C
D
E
F
26
24
16
22
100
100
100
100
13
11
7
12
a
b
Values are relative intensities. Peaks for dG + 1 (m/z 268, relative intensity of 35%) and G + 1 (m/z 152, relative intensity of 100%)
were also observed, presumably due to dG in the sample. c ND, not detected.
the hemiacetal linkage of 9 was stable. Although these
products were not identified, the results indicate that the
hemiacetal linkage of 8 would be stable during NaBH3-
CN reduction. Therefore, 10 would not be the expected
major product of NaBH3CN treatment of 8. The LC-
APCI-MS data which we obtained on the Schiff base
products C-F are also not consistent with 8, as we could
not detect any ions at m/z 426, which is M + 1 of 8.
Furthermore, the presence of two pairs of interconverting
isomers (C-F), as discussed above, is consistent with 7,
while there are 16 stereoisomers of 8.
We investigated the stability of the Schiff base adduct
in DNA by incubating crotonaldehyde-treated DNA at pH
7.0 and 37 °C, treating with NaBH3CN, hydrolyzing
enzymatically, and analyzing for N2-(3-hydroxybutyl)dG
(10). The Schiff base adduct was stable in DNA for at
least 70 h under these conditions.
F igu r e 6. Chromatogram obtained upon HPLC analysis
(system 1) of peaks E and F, which had been collected from the
mixture shown in Figure 5A, and re-injected using the same
HPLC system.
chiral 3-hydroxybutanal. (S)- and (R)-3-hydroxybutanal
(as the corresponding paraldol dimers 12) were prepared
as described previously by reduction of either ethyl (S)-
or (R)-3-hydroxybutyrate with diisobutylaluminum hy-
dride. Reaction of (S)-3-hydroxybutanal (as paraldol
dimer 12) with dG gave the chromatogram illustrated
in Figure 7A in which peaks D and F prevailed, while
the corresponding (R)-isomer gave the chromatogram
shown in Figure 7B in which peaks C and E prevailed.
Co-injection of these two mixtures of products gave four
peaks, C-F. These results demonstrate that peaks D and
F have an (S)-3-hydroxybutylidene group, whereas the
corresponding configuration in peaks C and E is (R).
Therefore, peaks D and F must be (E)- or (Z)-isomers of
N2-(S)-(3-hydroxybutylidene)dG, whereas peaks C and E
are (E)- or (Z)-isomers of N2-(R)-(3-hydroxybutylidene)-
dG.
These data are all consistent with our assigned struc-
ture, N2-(3-hydroxybutylidene)dG (7). However, we could
not fully exclude Schiff base 8 in the absence of NMR
data. It was possible that treatment of 8 with NaBH3-
CN could yield N2-(3-hydroxybutyl)dG (10), if the hemi-
acetal linkage in 8 dissociated under the reaction condi-
tions. To test this possibility, we treated paraldol (12)
with NaBH3CN under the same conditions used for
reduction of paraldol-dG reaction mixtures. If the hemi-
acetal linkage of 9 were stable under these conditions,
we should obtain products with a molecular weight of 178
resulting from reduction of the aldehyde carbonyl group
in 9, whereas if it were unstable, the major product would
be 1,3-butanediol, with a molecular weight of 90. LC-
APCI-MS analysis of the reaction mixture showed the
presence of several peaks having ions at m/z 179, which
is consistent with M + 1 of a reduction product in which
Discu ssion
The results of this study clearly demonstrate that a
Schiff base is a major DNA adduct of crotonaldehyde. All
of our data are consistent with N2-(3-hydroxybutylidene)-
dG (7, Scheme 1) as the structure of this Schiff base.
Thus, reaction of crotonaldehyde with DNA followed by
treatment with NaBH3CN produces N2-(3-hydroxybutyl)-
dG. UV and MS data obtained from reactions of crotonal-
dehyde or paraldol with dG also support 7 as the
structure of the Schiff base. However, the instability of
7 at the nucleoside level, leading to its decomposition
during enzyme hydrolysis, prevented us from obtaining
spectral data directly on the DNA adduct. Therefore, it
is possible that the Schiff base in DNA is not identical
to that formed in the dG reactions. We do not consider
this likely on the basis of previous studies of crotonal-
dehyde-DNA reactions which indicate that adducts in
DNA are structurally the same as those formed in
reactions with dG (6, 9, 11, 15, 16). In contrast to the
instability of 7 at the nucleoside level, our data show that
the Schiff base is stable in DNA, where it is presumably
protected from hydrolysis.
The concentration of crotonaldehyde used in the DNA
reactions in this study was high, 200 mM, to facilitate
adduct identification. However, we have previously de-
tected substantial amounts of paraldol-releasing adducts
in DNA reacted with 0.2 mM crotonaldehyde, and it is
very likely that these adducts would also be detected at
lower concentrations (11). As shown in Figure 2B, levels
of the Schiff base adduct far exceed those of Michael

Crotonaldehyde-DNA Adduct
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 429
F igu r e 7. Chromatograms obtained upon HPLC analysis (system 2) of products formed in the reaction of paraldol with dG: (A)
paraldol from (S)-3-hydroxybutanal and (B) paraldol from (R)-3-hydroxybutanal. Peaks D and F are (E)- or (Z)-isomers of N2-(S)-
(3-hydroxybutylidene)dG, and peaks C and E are (E)- or (Z)-isomers of N2-(R)-(3-hydroxybutylidene)dG. Numbers above the peaks
refer to the structures in Scheme 1. The major isomer of 11 formed from (S)-3-hydroxybutanal under these conditions is the first
eluting of the four peaks observed with racemic paraldol, and the major isomer from (R)-3-hydroxybutanal is the third eluting peak.
[note that in Figure 7 and in the corresponding text of ref 11 describing the formation of 11, the (R)- and (S)-labels were inadvertently
switched, and in Figure 8 and the corresponding text of ref 11, the A and B peak labels were switched].
addition adducts 2 in DNA reacted with crotonaldehyde,
consistent with our previous finding that paraldol-
releasing DNA adducts were 13-75 times more prevalent
than 2.
dG or DNA followed by Michael addition of H2O, and
tautomerization. We consider this less likely than the
mechanism shown in Scheme 1 because NaBH3CN treat-
ment did not appear to produce major products resulting
from reduction of a crotonaldehyde Schiff base. However,
further studies would be necessary to exclude this pos-
sibility.
The relatively high concentration of the Schiff base
compared to that of 2 in crotonaldehyde-treated DNA
could have important implications. Chung and co-workers
have shown that adducts 2 are present in human tissues,
ranging in concentration from 0.003 to 6 µmol/mol of G
(8, 10, 17-19). While the highest levels were found in
the oral mucosa of smokers, considerable amounts of
these adducts have also been detected in various tissues
of apparently unexposed individuals. These adducts,
which have also been detected in tissues of laboratory
animals, most likely arise from endogenous lipid peroxi-
dation reactions. Since the Schiff base adduct is stable
in DNA and is formed in vitro in amounts considerably
greater than the amount of adducts 2, it is likely that
significant amounts of this adduct are present in human
DNA. This could have important implications with
respect to cancer etiology. Therefore, we are developing
MS methods for quantifying this adduct in human DNA.
While the concentration of Schiff base adduct 7 in DNA
reacted with crotonaldehyde exceeds those of adducts 2,
the opposite is seen in reactions with dG. Inspection of
Figure 4 indicates that levels of adducts 2 are greater
than those of 7 in dG reacted with crotonaldehyde.
Formation of the Schiff base can occur by addition of H2O
to crotonaldehyde, producing 3-hydroxybutanal (6). The
amount of 6 in these reaction mixtures will be far smaller
than that of crotonaldehyde. This indicates that 6 reacts
considerably faster with DNA than does crotonaldehyde.
Apparently, Schiff base formation occurs more rapidly
than Michael addition in the DNA reactions, but not in
the dG reaction. Alternatively, hydrolysis of the Schiff
base back to dG and 6 may compete significantly with
the forward reaction, whereas this is not the case in DNA.
An alternate pathway leading to Schiff base adduct 7
is initial formation of a crotonaldehyde Schiff base with
As shown in Figure 6, collection of Schiff base peaks
E and F, followed by re-injection in the same system,
results in appearance of peaks C and D as well as dG.
This indicates that peaks E and F, and C and D,
interconvert and are therefore pairs of (E)- and (Z)-
isomers. We could not assign the stereochemistry. How-
ever, facile (E)-(Z) interconversion of Schiff bases has
been previously demonstrated, particularly in cases
where the group attached to nitrogen is aromatic (20, 21).
Our data are consistent with these studies.
In summary, this study demonstrates that a Schiff
base is a major DNA adduct of crotonaldehyde, being
responsible for most of the paraldol released upon hy-
drolysis of crotonaldehyde-treated DNA. This Schiff base
is stable in DNA and is present in substantially greater
quantities than the Michael adducts 2, suggesting that
the Schiff base adduct may be present in significant
quantities in human DNA.
Ack n ow led gm en t. This study was supported by
Grant CA-85702 from the National Cancer Institute.
S.S.H. is an American Cancer Society Research Professor,
supported by ACS grant RP-00-138.
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