Reactions of 2,6-dimethyl-1,3-dioxane-4-ol (aldoxane) with deoxyguanosine and DNA

Article abstract of DOI:10.1021/tx0100385

In a recent study, we identified several new DNA adducts of the carcinogen acetaldehyde, including N²-(2,6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (N²-aldoxane-dG, 2). Our goal in this study was to investigate further the formation of 2 by allowing 2,6-dimethyl-1,3-dioxane-4-ol (aldoxane, 5) to react with dG and DNA. Aldoxane is readily formed by trimerization of acetaldehyde. The reaction of aldoxane with dG and DNA produced diastereomers of N²-aldoxane-dG (2) as observed in the reactions of acetaldehyde with dG and DNA, supporting the intermediacy of aldoxane in their formation. Unexpectedly, however, an array of other adducts was formed in these reactions, including 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2- a]purine-10(3H)one (3), 2-amino-7,8-dihydro-8-hydroxy-6-methyl-3H-pyrrolo[2,1-f] purine-4(6H)one (13), N²-(3-hydroxybutylidene)dG (9), N²-[(2-hydroxypropyl)-6-methyl-1,3-dioxane-4-yl]dG (14), and N²-ethylidene-dG (1). Adduct 1 was the major product and was found to be quite stable in DNA. The adducts result from a cascade of aldehydes, e.g., 2-butenal (crotonaldehyde, 12), 3-hydroxybutanal (7) and its dimer (2-hydroxypropyl)-6-methyl-1,3-dioxane-4-ol (paraldol, 6), and acetaldehyde, produced from aldoxane under the reaction conditions. The reactions of aldoxane with dG and DNA were compared with those of paraldol. The paraldol reactions gave products resulting from reactions of dG and DNA with paraldol, 3-hydroxybutanal, and crotonaldehyde (adducts 3, 13, and 9) but the products of the aldoxane and acetaldehyde reactions (adducts 1 and 2) were not observed, indicating that paraldol is more stable under the reaction conditions than is aldoxane. The results of this study provide new insights about the formation of DNA adducts from aldehydes via condensation products of the latter.

Full text of DOI:10.1021/tx0100385

Chem. Res. Toxicol. 2001, 14, 1025-1032
1025
Rea ction s of 2,6-Dim eth yl-1,3-d ioxa n e-4-ol (Ald oxa n e)
w ith Deoxygu a n osin e a n d DNA
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 February 22, 2001
In a recent study, we identified several new DNA adducts of the carcinogen acetaldehyde,
2
2
including N -(2,6-dimethyl-1,3-dioxan-4-yl)deoxyguanosine (N -aldoxane-dG, 2). Our goal in
this study was to investigate further the formation of 2 by allowing 2,6-dimethyl-1,3-dioxane-
4
-ol (aldoxane, 5) to react with dG and DNA. Aldoxane is readily formed by trimerization of
acetaldehyde. The reaction of aldoxane with dG and DNA produced diastereomers of
2
N -aldoxane-dG (2) as observed in the reactions of acetaldehyde with dG and DNA, supporting
the intermediacy of aldoxane in their formation. Unexpectedly, however, an array of other
adducts was formed in these reactions, including 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-
hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)one (3), 2-amino-7,8-dihydro-8-hydroxy-6-meth-
2
2
yl-3H-pyrrolo[2,1-f]purine-4(6H)one (13), N -(3-hydroxybutylidene)dG (9), N -[(2-hydroxypropyl)-
6
2
-methyl-1,3-dioxane-4-yl]dG (14), and N -ethylidene-dG (1). Adduct 1 was the major product
and was found to be quite stable in DNA. The adducts result from a cascade of aldehydes, e.g.,
2
1
-butenal (crotonaldehyde, 12), 3-hydroxybutanal (7) and its dimer (2-hydroxypropyl)-6-methyl-
,3-dioxane-4-ol (paraldol, 6), and acetaldehyde, produced from aldoxane under the reaction
conditions. The reactions of aldoxane with dG and DNA were compared with those of paraldol.
The paraldol reactions gave products resulting from reactions of dG and DNA with paraldol,
3
-hydroxybutanal, and crotonaldehyde (adducts 3, 13, and 9) but the products of the aldoxane
and acetaldehyde reactions (adducts 1 and 2) were not observed, indicating that paraldol is
more stable under the reaction conditions than is aldoxane. The results of this study provide
new insights about the formation of DNA adducts from aldehydes via condensation products
of the latter.
2
In tr od u ction
base, N -ethylidene-dG (1) (9, 10). The reduced form of
Acetaldehyde is mutagenic and carcinogenic (1, 2). It
causes mutations, sister chromatid exchanges, micronu-
clei formation, and aneuploidy in cultured mammalian
cells, and gene mutations in bacteria. It induces nasal
tumors in rats and laryngeal carcinomas in hamsters
when administered by inhalation (1, 2).
Human exposure to acetaldehyde is extensive. It is the
primary metabolite of ethanol and is an intermediate in
the metabolism of sugars (1-3). Several recent studies
have implicated acetaldehyde as a cause of cancer in
individuals who consume alcohol and are carriers of a
mutant aldehyde dehydrogenase gene leading to enzyme
inactivity (4-7). The concentration of acetaldehyde in
tobacco smokes920 µg/cigarettesis far greater than the
levels of polycyclic aromatic hydrocarbons or tobacco-
specific nitrosamines (8). Acetaldehyde is also an inter-
mediate in chemical production, with considerable po-
tential for worker exposure (3). Acetaldehyde is classified
by the International Agency for Research on Cancer as
2
this adduct, N -ethyl-dG, has been detected in the DNA
of peripheral white blood cells of alcohol abusers, in
human buccal cell DNA following exposure to acetalde-
hyde, and in human urine (11-13). We recently identified
additional products of acetaldehyde’s reaction with
DNA: diastereomers of N -(2,6-dimethyl-1,3-dioxan-4-yl)-
dG (N -aldoxane-dG, 2), the 1,N -propano-dG adducts
3, as well as the interstrand cross-link 4 (10).
2
2
1
2
“possibly carcinogenic in humans” and by the U.S.
Department of Health and Human Services as “reason-
ably anticipated to be carcinogenic in humans” (1-3).
Acetaldehyde reacts with DNA forming a variety of
adducts. The major adduct identified to date is the Schiff
In the present study, we investigated the reactions of
,6-dimethyl-1,3-dioxane-4-ol (aldoxane, 5) with dG and
2
1
2
Abbreviations: aldoxane, 2,6-dimethyl-1,3-dioxane-4-ol; N -aldox-
ane-dG, N -(2,6-dimethyl-1,3-dioxan-4-yl)dG; paraldol, 2-(2-hydroxy-
propyl)-6-methyl-1,3-dioxane-4-ol; N -paraldol-dG, N -[2-(2-hydroxy-
2
2
2
*
To whom correspondence should be addressed. Phone: (612) 624-
7
604. Fax: (612) 626-5135. E-mail: hecht002@umn.edu.
propyl)-6-methyl-1,3-dioxane-4-yl]dG.
1
0.1021/tx0100385 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/29/2001

1
026 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Wang et al.
°
C. After an additional 30 min of stirring at 5 °C, the temper-
ature was increased to 30 °C, and the reaction mixture was
allowed to stand for an additional 30 min. The pH was adjusted
to 3 with tartaric acid. After filtration, the filtrate was subjected
to column (5 × 40 cm) chromatography on silica gel, with elution
by a gradient of 0 to 100% ethyl acetate in hexane. Aliquots of
fractions were treated with 2,4-dinitrophenylhydrazine reagent
to identify aldoxane, which gives the 2,4-dinitrophenylhydra-
zones of acetaldehyde, 3-hydroxybutanal, and crotonaldehyde
DNA. Aldoxane is readily formed from acetaldehyde and
appeared to play a central role in producing adducts 2.
We also wanted to compare the reactions of aldoxane with
dG and DNA to those of 2-(2-hydroxypropyl)-6-methyl-
(10). These fractions were combined and concentrated in vacuo
to give a mixture of crude aldoxane and paraldol. Aldoxane was
collected and purified using HPLC system 4: UV, λmax 282 nm;
1
yield, 1.3% from acetaldehyde. H NMR data for aldoxane are
summarized in Table 1.
1
3
,3-dioxane-4-ol (paraldol, 6). Paraldol is the dimer of
-hydroxybutanal (7), the aldol condensation product of
Aldoxane was also prepared from paraldol. To 500 mg of
paraldol was added chilled acetaldehyde (3.5 mL) with stirring,
until the paraldol dissolved. The temperature of the mixture
was increased to room temperature. After 30 min, the temper-
ature of the reaction mixture was raised slowly to 60 °C, then
stored at this temperature overnight. Aldoxane was collected
and purified as above.
acetaldehyde, and thus could potentially play some role
in adduct formation between acetaldehyde and DNA.
Exp er im en ta l Section
HP LC An a lysis. HPLC analysis was carried out with a
Waters Corporation (Milford, MA) system equipped with a
model 991 or 996 photodiode array detector. Flow rates were 1
mL/min, unless noted otherwise. The following columns and
solvent elution systems were used: (1) two 4.6 mm × 25 cm
Supelcosil LC 18-BD columns (Supelco, Bellefonte, PA) with
Rea ction s. (1) Ald oxa n e or P a r a ld ol a n d d G. Aldoxane
(0.8 mmol), freshly collected using HPLC system 4 or 5, was
allowed to react with dG (2.7 mg) in 1 mL of 0.1 M phosphate
buffer (pH 7.0) at 37 °C for 45 h. Paraldol (0.5 mmol) was
similarly allowed to react with dG.
(
2) Ald oxa n e or P a r a ld ol a n d DNA. Aldoxane or paraldol
isocratic elution by 5% CH
buffer (pH 7) for 10 min, then a gradient from 5 to 25% CH
3
CN in 10 mM sodium phosphate
CN
(0.6 mmol) was allowed to react with calf thymus DNA (20 mg)
3
in 1.5 mL of 0.1 M phosphate buffer (pH 7.0) at 37 °C for 40 h.
The modified DNA was precipitated by addition of ethanol, then
washed with 70% ethanol, and ethanol. DNA was hydrolyzed
enzymatically as previously described (10, 14). In some cases,
the DNA was treated with NaBH CN before hydrolysis, using
3
previously described conditions (10).
over the course of 60 min, with UV detection (254 nm). This
system was used for all adduct analyses; (2) The same column
as in system 1, with isocratic elution by 5% CH
ammonium acetate buffer (pH 6.6) for 10 min, then a gradient
from 5 to 35% CH OH in 60 min, and finally a gradient from
5 to 50% CH OH in 10 min, at a flow rate of 0.5 mL/min with
3
OH in 40 mM
3
3
3
detection by UV (254 nm) for analysis of all adducts; (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 0 to
Resu lts
8
0% CH
analysis of aldoxane and paraldol, and from 40 to 60% CH
in H O over the course of 40 min with UV detection (365 nm)
3
OH in H
2
O in 20 min, with UV detection (280 nm) for
We prepared aldoxane by base treatment of acetalde-
hyde. The reaction mixture was purified by silica gel
chromatography. Fractions containing aldoxane, as iden-
tified by reaction with 2,4-dinitrophenylhydrazine re-
agent, were combined. HPLC analysis of these combined
fractions produced the chromatogram illustrated in Fig-
ure 1. Peaks 1 and 3 are apparently decomposition
products of aldoxane, which in our hands was somewhat
unstable. Peak 2 was confirmed as aldoxane by H NMR
Table 1), and was used for all studies described here.
Although aldoxane is said to be one of the major products
of the aldol condensation (18), we could find no H NMR
3
CN
2
for analyzing the 2,4-dinitrophenylhydrazones of acetaldehyde,
crotonaldehyde, and 3-hydroxybutanal; (4) a 9.4 mm × 50 cm
Whatman Partisil 10 Magnum 9 column (Whatman, Clifton, NJ )
with elution by a gradient from 0 to 50% ethyl acetate in hexane
over the course of 25 min, and then from 50 to 65% ethyl acetate
in 15 min, at a flow rate of 3 mL/min, with UV detection (280
nm) for collection of aldoxane; (5) a 4.6 mm × 25 cm Phenom-
enex Luna 5 µm silica column (Phenomenex, Torrance, CA) with
the same program as used in system 4, except the flow rate was
1
(
1
1
mL/min. This system was used to assess formation of aldoxane
1
from acetaldehyde.
data for this compound in the literature. The H NMR
MS An a lysis. LC-MS analysis was carried out on a Finnigan
MAT LCQ Deca instrument (Thermoquest LC/MS Division, San
J ose, CA) interfaced with an Alliance 2690 HPLC multisolvent
delivery system (Waters) equipped with an SPD-10A UV-Vis
detector (Shimadzu Scientific Instruments, Columbia, MD). The
column was the same as in system 2, except that the flow rate
was 0.3 mL/min. APCI source parameters were as follows:
voltage, 4.5 kV; current, 5.0 µA; capillary temperature, 200 °C,
vaporizer temperature, 400 °C.
NMR An a lysis. Spectra were obtained on an 800 MHz
instrument (Varian, Inc., Palo Alto, CA).
Ch em ica ls a n d En zym es. 2,4-Dinitrophenylhydrazine re-
agent was purchased from Eastman Organic Chemicals (Roch-
data in Table 1 are consistent with the presence of two
isomers in a ratio of approximately 60:40. Isomer a has
all equatorially oriented substituents whereas isomer b
has an axial hydroxyl group. All assignments were
confirmed by COSY and NOESY experiments.
Aldoxane was allowed to react with dG. HPLC analysis
of the reaction mixture produced the chromatogram
illustrated in Figure 2. Peaks were identified by com-
parison of their retention times, UV, and MS to those of
standards (Table 2). Structures corresponding to each
peak are shown in Figure 2.
An HPLC chromatogram of an enzymatic hydrolysate
of DNA that had been allowed to react with aldoxane is
shown in Figure 3. Peaks were identified by comparison
of retention times, UV, and MS data to those of standards
3
ester, NY). Acetaldehyde and NaBH CN were obtained from
Aldrich Chemical Co. (Milwaukee, WI). Alkaline phosphatase
was purchased from Boehringer Mannheim Co. (Indianapolis,
IN). Paraldol and all adduct standards were prepared as
described (10, 14-17). All other chemicals and enzymes were
obtained from Sigma Chemical Co. (St. Louis, MO).
(Table 3). Structures of the compounds corresponding to
the HPLC peaks are illustrated in Figure 3. Most of the
larger peaks without numbers were also present in the
HPLC traces of hydrolysates of DNA that had not been
Aldoxane was prepared by adding 2.5 mL of 10% aqueous
NaOH dropwise to 100 g of cold acetaldehyde with stirring at 5

Aldoxane-DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1027
Ta ble 1. H NMR Da ta for Ald oxa n ea
1
2
-H
4-OH
4-Heq
4-Hax
4.74(dd)
J ) 9.6, 1.6 Hz
5-Heq
1.61(m) 1.12-1.07(m) 3.64(m) 1.17(d)
J ) 5.6 Hz
3.96(m) 1.10(d)
J ) 4.8 Hz
5-Hax
6-H
2-CH3
6-CH3
1.08(d)
J ) 4.8 Hz
1.03(d)
J ) 5.6 Hz
isomer a 4.67(q)
J ) 4.8, 10 Hz
isomer b 5.20(q)
J ) 4.8, 11.2 Hz
6.57(bs)
6.29(bs) 5.13(d)
J ) 4 Hz
1.42(m) 1.47(m)
a
Values are chemical shifts (δ) for samples dissolved in DMSO-d6.
Ta ble 2. Sp ectr a l Da ta for P r od u cts of th e Rea ction of
Ald oxa n e a n d d G (F igu r e 2)a
UV
peak
LC-APCI-MS (m/z)
(λmax, nm)
1
2
3
4
5
6
7
8
9
ndb
250, 285
254
254
250, 285
254
254
260
260
254
+
338 (M + 1); 204 (BH -H2O)
+
338 (M + 1); 204 (BH -H2O)
nd
+
338 (M + 1); 204 (BH -H2O)
294 (M + 1)
+
338 (M + 1); 222 (BH )
+
338 (M + 1); 222 (BH )
+
382 (M + 1); 266 (BH );
+
2
04 (BH -CH3CHO-H2O)
+
1
1
1
1
1
0
1
2
3
4
382 (M + 1); 266 (BH );
2
382 (M + 1); 266 (BH );
2
426 (M + 1); 310 (BH );
2
426 (M + 1); 310 (BH );
2
426 (M + 1); 310 (BH );
2
426 (M + 1); 310 (BH );
204 (BH -CH3CHO-H2O)
254
254
254
254
254
254
+
04 (BH -CH3CHO-H2O)
+
+
04 (BH -CH3CHO-H2O)
+
+
04 (BH -CH3CHO-H2O)
+
+
04 (BH -CH3CHO-H2O)
+
+
04 (BH -CH3CHO-H2O)
+
F igu r e 1. Chromatogram obtained upon HPLC analysis
system 4) of a silica gel column chromatography fraction
15
+
(
containing aldoxane, peak 2. Peak 2 was collected and used for
the studies reported in this paper.
a
All peaks had the same retention time as standards. b nd )
not determined.
treated with aldoxane. The major difference between the
chromatograms shown in Figures 2 and 3 is the absence
of peaks corresponding to the Schiff base adducts (peaks
Ta ble 3. Sp ectr a l Da ta for P r od u cts of th e Rea ction of
Ald oxa n e a n d DNA (F igu r e 3)a
UV
peak
LC-APCI-MS (m/z)
(λmax, nm)
2
, 3, 5, and 6 of Figure 2) in the HPLC traces of the DNA
1
2
3
ndb
259
259
253
hydrolysates. This is due to their instability at the
nucleoside level. However, the formation of these prod-
ucts was confirmed by treatment of the aldoxane-reacted
+
338 (M + 1); 222 (BH )
382 (M + 1); 266 (BH );
+
+
2
04 (BH -CH3CHO-H2O)
DNA with NaBH
3
CN prior to enzyme hydrolysis. HPLC
+
4
5
382 (M + 1); 266 (BH );
253
253
+
analysis of this mixture gave the chromatogram il-
204 (BH -CH3CHO-H2O)
+
2
382 (M + 1); 266 (BH );
lustrated in Figure 4. The structures of N -(3-hydroxy-
+
2
04 (BH -CH3CHO-H2O)
2
butyl)dG (peaks 1 and 2) and N -ethyl-dG (peak 3),
6
7
8
426 (M + 1)
426 (M + 1)
nd
253
253
253
2
resulting from reduction of Schiff base adducts N -(3-
hydroxybutylidene)dG (peaks 2, 3, and 5 of Figure 2) and
2
N -ethylidene-dG (peak 6 of Figure 2) were confirmed by
a
All peaks had the same retention time as standards. b nd )
LC-APCI-MS analysis and by comparison to standards.
not determined.
Figure 5 illustrates an HPLC chromatogram obtained
upon analysis of the reaction of paraldol with dG. Each
peak was identified by comparison of its HPLC retention
time and UV with those of standards. The results are
similar to those of our published studies on paraldol-dG
reactions (14, 17). The major difference between the
products of this reaction and those formed in the reaction
Figure 6 shows a chromatogram obtained by HPLC
analysis of an enzymatic hydrolysate of DNA that had
been allowed to react with paraldol. Peaks were identified
by comparison of their HPLC retention times and UV
spectra to those of standards. When the DNA was treated
2
with NaBH
3
CN prior to hydrolysis, N -(3-hydroxybutyl)-
2
dG was also detected, resulting from reduction of N -(3-
hydroxybutylidene)dG. Table 4 summarizes for compari-
son the differences in adduct formation between aldoxane
and paraldol in their reactions with DNA.
2
of aldoxane with dG was the absence of N -ethylidene-
dG (peak 6 of Figure 2) and the diastereomers of
2
N -aldoxane-dG (peaks 9-11 of Figure 2).

1
028 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Wang et al.
F igu r e 2. Chromatogram obtained upon HPLC analysis (system 1) of the products of reaction between aldoxane and dG. Structures
of the numbered peaks are indicated. Peaks 1 and 4 are cis- and trans-isomers, respectively. Partial information on the structures
of diastereomers of peaks 2, 3, and 5; 7 and 8; and 12-15 is available in refs 14, 15, and 17. A small amount of another isomer of
peaks 9-11 may coelute with peak 12.
F igu r e 3. Chromatogram obtained upon HPLC analysis (system 1) of an enzymatic hydrolysate of DNA that had been allowed to
react with aldoxane. Structures of the numbered peaks are indicated.
2
Since N -ethylidene-dG (1) is the major DNA adduct
incubated at 37 °C. Aliquots were withdrawn at various
intervals, treated with NaBH CN, enzymatically hydro-
lyzed, and analyzed for N -ethyl-dG. The results were:
of aldoxane, we investigated its stability in DNA. DNA
containing N -ethylidene-dG (14 mmol/mol dG) was
3
2
2

Aldoxane-DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1029
F igu r e 4. Chromatogram obtained upon HPLC analysis (system 1) of an enzymatic hydrolysate of DNA that had been allowed to
react with aldoxane, and then was treated with NaBH CN. Structures of the numbered peaks are indicated.
3
F igu r e 5. Chromatogram obtained upon HPLC analysis (system 1) of the products of the reaction between paraldol and dG. Structures
of the numbered peaks are indicated.
time (h), (% of the amount at t ) 0); 0 (100); 24 (55); 72
41); 96 (35). Thus, in contrast to its instability at the
nucleoside level (t1/2 < 5 min at 37 °C), N -ethylidene-
dG (1) is quite stable in DNA.
Discu ssion
(
2
One goal of this study was to assess the role of
aldoxane (5) in the formation of the N -aldoxane-dG
2

1
030 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Wang et al.
F igu r e 6. Chromatogram obtained upon HPLC analysis (system 1) of an enzymatic hydrolysate of DNA that had been allowed to
react with paraldol. Structures of the numbered peaks are indicated.
Ta ble 4. Com p a r a tive Ad d u ct F or m a tion in th e Rea ction s of DNA w ith Ald oxa n e (5) a n d P a r a ld ol (6)
adducts (mmol/mol dG)
2
a
2
N -aldoxane-dGb
2
2
N -(3-hydroxybutylidene)dGc
2
N -ethylidene-dG
1,N -propano-dG
N -paraldol-dG
reactant
(1)
(2)
3
(9)
(14)
aldoxane (5)
paraldol (6)
20.4
nd
1.1
nd
0.6
1.5
3.0
6.7
0.1
1.8
a
As N -ethyl-dG after NaBH3CN treatment of the DNA. b Using N -paraldol-dG as a UV standard for quantitation. As N -(3-
2
2
c
2
hydroxybutyl)dG after NaBH3CN treatment of the DNA.
adducts 2 which were also observed as products of the
reaction of acetaldehyde with dG and DNA. This would
provide additional support for the structures of 2, which
were previously assigned based only on spectral data (10).
A second goal was to gain insight into the mechanisms
by which these adducts are formed. Our results confirm
mechanism in which 3-hydroxybutanal reacts with dG
to form Schiff base 9, which then reacts with another
molecule of acetaldehyde to give 10 and ultimately 2.
An unexpected observation in this study was the rich
variety of adducts produced in the reactions of aldoxane
with dG and DNA (Scheme 2). In addition to the
2
2
that the structures of adducts 2 are diastereomers of N -
aforementioned diastereomeric N -aldoxane-dG adducts
aldoxane-dG (2). Two major diastereomers, corresponding
to peaks 10 and 11 of Figure 2, are formed in the dG
reactions, and three major diastereomers, represented by
peaks 3-5 of Figure 3, are produced in the DNA reaction.
These products are apparently identical to those formed
in the reaction of acetaldehyde with dG and DNA (10).
The stereochemistry of these adducts requires further
investigation. A plausible mechanism for the formation
2, we also observed adducts characteristic of reactions
of crotonaldehyde (12), 3-hydroxybutanal (7), and acetal-
dehyde with dG. These results are rationalized in Scheme
2. Under our conditions, aldoxane is in equilibrium with
its open chain tautomer 8, which in turn is in equilibrium
with 3-hydroxybutanal (7) and its dimer paraldol (6).
Additionally, aldoxane is in equilibrium with crotonal-
dehyde (12) and acetaldehyde via 3-hydroxybutanal (7).
Each of these aldehydes reacts with dG. Adducts 2 are
formed by the reaction of open chain tautomer 8 with
dG as discussed above. Adducts 9 are formed by the
2
of N -aldoxane-dG in reactions of acetaldehyde with dG
and DNA is shown in Scheme 1. Aldoxane (5) is produced
by trimerization of acetaldehyde, which occurs under the
conditions of the present study (data not shown). Aldox-
ane is also in equilibrium with its tautomer 8. The latter
reacts with dG producing intermediate 10, which cyclizes
2
reaction of 3-hydroxybutanal with dG while N -[2-(2-
2
hydroxypropyl)-6-methyl-1,3-dioxane-4-yl]dG (N -paral-
dol-dG, 14) is produced by reaction of paraldol (6) with
dG via its open chain tautomer 11 (14). Reaction of
crotonaldehyde with dG produces the N7,C8 adducts 13
2
to N -aldoxane-dG (2). Support for this mechanism comes
from the apparent similarity of adduct 2 diastereomers
independent of whether acetaldehyde or aldoxane is the
starting material, consistent with the equilibria shown
in Scheme 1. However, we cannot exclude a second
2
as well as the 1,N -propano-dG adducts 3 (15, 16). The
latter can also be formed from acetaldehyde, but the
approximately 1:1 diastereomeric ratio of adducts 3

Aldoxane-DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 8, 2001 1031
2
Sch em e 1. P r op osed Mech a n ism s for F or m a tion of N -a ld oxa n e-d G (2)
Sch em e 2. F or m a tion of d G a n d DNA Ad d u cts of Ald oxa n e (5)
Ta ble 5. Over view of Ad d u ct F or m a tion in Rea ction s w ith d G
adduct detected
2
N -(3-hydroxy-
butylidene)dG
2
2
2
2
N -ethylidene-
N -aldoxane-
1,N -propano-
crosslink
N7-C8-dG
N -paraldol-
reactant
dG (1)
dG (2)
dG 3
4
(9)
13
dG (14)
acetaldehyde
crotonaldehyde
aldoxane
yes
no
yes
no
yes
no
yes
no
yes
yes
yes
yes
yes
yes
no
no
no
noa
yes
yes
yes
yes
yes
yes
yes
yes
yes
paraldol
no
a
2
Small amounts of N -paraldol-dG may be formed, but not confirmed.
indicates that their major source is crotonaldehyde (10).
Finally, acetaldehyde reacts with dG and DNA to produce
Schiff base adduct 1, which is the major product of the
reaction of aldoxane with DNA. The stability of adduct
1 in DNA suggests that it may contribute to the biological
effects of acetaldehyde and aldoxane.
The reactions of paraldol (6) with dG and DNA were
not as complex as those of aldoxane (14). Paraldol reacts

1
032 Chem. Res. Toxicol., Vol. 14, No. 8, 2001
Wang et al.
2
via its open chain tautomer 11 to produce N -paraldol-
dG (14, Scheme 2). Paraldol is also in equilibrium with
its monomer, 3-hydroxybutanal (7), which reacts with dG
Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol.
3
6, pp 101-132, IARC, Lyon, FR.
(
3) U.S. Department of Health and Human Services (2000) Ninth
Report on Carcinogens, III-65-III-66.
2
to produce N -(3-hydroxybutylidene)dG (9)(17). 3-Hy-
(
4) Yokoyama, A., Muramatsu, T., Omori, T., Matsushita, S., Yoshimi-
zu, H., Higuchi, S., Yokoyama, T., Maruyama, K., and Ishii, H.
1999) Alcohol and aldehyde dehydrogenase gene polymorphisms
droxybutanal in turn is in equilibrium with crotonalde-
hyde (12), which gives rise to adducts 3 and 13 (15, 16).
Paraldol is more stable under our conditions than aldox-
ane, and apparently is not in equilibrium with ap-
preciable amounts of acetaldehyde or aldoxane, because
we did not detect adducts 1 or 2 in these reactions. We
(
influence susceptibility to esophageal cancer in J apanese alcohol-
ics. Alcohol.: Clin. Exp. Res. 23, 1705-1710.
(
5) Yokoyama, A., Muramatsu, T., Ohmori, T., Yokoyama, T., Okuya-
ma, K., Takahashi, H., Hasegawa, Y., Higuchi, S., Maruyama,
K., Shirakura, K., and Ishii, H. (1998) Alcohol-related cancers and
aldehyde dehydrogenase-2 in J apanese alcoholics. Carcinogenesis,
2
also did not detect significant amounts of N -paraldol-
1
9, 1383-1387.
dG (14) in reactions of acetaldehyde with dG or DNA,
although we cannot completely exclude their formation.
Qualitative comparisons of product formation in the
reactions with dG of acetaldehyde, crotonaldehyde, al-
doxane, and paraldol are summarized in Table 5. We did
not investigate reactions with other DNA bases. A
previous study indicates some reactivity of acetaldehyde
with dAdo (19).
The reactions with dG and DNA examined here were
carried out at nonphysiological concentrationss400-800
mMsof aldoxane and paraldol. These concentrations
were 10-20 times as great as those of dG. It was not
our purpose to assess the relevance of these reactions
under conditions which may exist in vivo, but rather to
investigate the reactions of aldoxane and paraldol with
dG and DNA under conditions which allow product
analysis by HPLC with UV detection. In the aldoxane-
DNA reactions, yields of adducts other than Schiff base
(
6) Yokoyama, A., Muramatsu, T., Ohmori, T., Higuchi, S., Ha-
yashida, M., and Ishii, H. (1996) Esophageal cancer and aldehyde
dehydrogenase-2 genotypes in J apanese males. Cancer Epide-
miol., Biomarkers Prev. 5, 99-102.
(
7) Yokoyama, A., Muramatsu, T., Ohmori, T., Makuuchi, H., Higu-
chi, S., Matsushita. S., Yoshino, K., Nakano, M., and Ishii, H.
(1996) Multiple primary esophageal and concurrent upper aero-
digestive tract cancer and the aldehyde dehydrogenase-2 genotype
of J apanese alcoholics. Cancer 77, 1987-1990.
(
8) Chepiga, T. A., Morton, M. J ., Murphy, P. A., Avalos, J . T.,
Bombick, B. R., Doolittle, D. J ., Borgerding, M. F., and Swauger,
J . E. (2000) A comparison of the mainstream smoke chemistry
and mutagenicity of a representative sample of the U.S. cigarette
market with two Kentucky reference cigarettes (K1R4F and
K1R5F). Food Chem. Toxicol. 38, 949-962.
9) Fang, J . L., and Vaca, C. E. (1995) Development of a 32P-post-
(
labeling method for the analysis of adducts arising through the
reaction of acetaldehyde with 2′-deoxyguanosine-3′-monophos-
phate and DNA. Carcinogenesis 16, 2177-2185.
(
10) Wang, M., McIntee, E. J ., Cheng, G., Shi, X., Villalta, P. W., and
Hecht, S. S. (2000) Identification of DNA adducts of acetaldehyde.
Chem. Res. Toxicol. 13, 1149-1157.
1
were low (Table 4). DNA adduct yields from paraldol
(11) Vaca, C. E., Nilsson, J . A., Fang, J . L., and Grafstrom, R. C. (1998)
Formation of DNA adducts in human buccal epithelial cells
exposed to acetaldehyde and methylglyoxal in vitro. Chem.-Biol.
Interact. 108, 197-208.
(12) Fang, J . L., and Vaca, C. E. (1997) Detection of DNA adducts of
acetaldehyde in peripheral white blood cells of alcohol abusers.
Carcinogenesis 18, 627-632.
were also low under these conditions. Further studies will
be required to examine product formation in the micro-
molar or nanomolar range more likely to be encountered
in vivo. It is not known whether aldoxane and paraldol
are produced in vivo from acetaldehyde or crotonalde-
hyde.
(
13) Matsuda, T., Terashima, I., Matsumoto, Y., Yabushita, H., Matsui,
2
S., and Shibutani, S. (1999) Effective utilization of N -ethyl-2′-
In summary, the results of this study provide further
evidence supporting the role of aldoxane as an intermedi-
ate in the reactions of dG and DNA with acetaldehyde.
Moreover, aldoxane is in equilibrium with a variety of
closely related aldehydes resulting in the formation of a
cascade of dG and DNA adducts. These results provide
new insights into the potential DNA damaging properties
of aldehydes in vivo.
deoxyguanosine triphosphate during DNA synthesis catalyzed by
mammalian replicative DNA polymerases. Biochemistry 38, 929-
9
35.
(
14) Wang, M., McIntee, E. J ., Cheng, G., Shi, Y., Villalta, P. W., and
Hecht, S. S. (2000) Identification of paraldol-deoxyguanosine
adducts in DNA reacted with crotonaldehyde. Chem. Res. Toxicol.
13, 1065-1074.
2
(
15) Chung, F. L., and Hecht, S. S. (1983) Formation of cyclic 1,N -
adducts by reaction of deoxyguanosine with R-acetoxy-N-nitroso-
pyrrolidine, 4-(carbethoxynitrosamino)butanal, or crotonaldehyde.
Cancer Res. 43, 1230-1235.
Ack n ow led gm en t. This study was supported by
grant CA-85702 from the National Cancer Institute.
Stephen S. Hecht is an American Cancer Society Re-
search Professor, supported by ACS grant RP-00-138.
(16) Chung, F. L., Wang, M., and Hecht, S. S. (1989) Detection of
exocyclic guanine adducts in hydrolysates of hepatic DNA of rats
treated with N-nitrosopyrrolidine and in calf thymus DNA reacted
with R-acetoxy-N-nitrosopyrrolidine. Cancer Res. 49, 2034-2041.
(
17) Wang, M., McIntee, E. J ., Cheng, G., Shi, Y., Villalta, P. W., and
Hecht, S. S. (2001) A Schiff base is a major DNA adduct of
crotonaldehyde. Chem. Res. Toxicol. 14, 423-430.
18) Nielson, A. T., and Houlihan, W. J . (1963) The aldol condensation.
Org. React. 16, 1-87.
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(
(
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(
TX0100385