1
150 Chem. Res. Toxicol., Vol. 13, No. 11, 2000
Wang et al.
2
yl)-dG and N -(4-hydroxybut-2-yl)-dG (6). Cooperative
reactions of acetaldehyde and ethanol with deoxyribo-
nucleosides and acetaldehyde and malondialdehyde with
dAdo have also been reported (6, 10-12).
J ose, CA) interfaced with a Waters 600 HPLC multisolvent
delivery system. The API source of the MS was set as follows:
voltage, 4.5 kV; current, 7.0 µA; and capillary interface tem-
perature, 350 °C. The analyses were performed in the positive
ion electrospray ionization (ESI) mode. HPLC system 4 was
used, except that the eluting solvent was a gradient from 25 to
In ongoing studies of crotonaldehyde-DNA adducts,
we have characterized products resulting from the reac-
tion with DNA of 2-(2-hydroxypropyl)-4-hydroxy-6-meth-
yl-1,3-dioxane (paraldol), the dimer of 3-hydroxybutanal,
which is present in aqueous solutions of crotonaldehyde
6
2
5% methanol in H O containing 1% acetic acid. This system
was used for analysis of all adducts except the guanine analogue
of 12, which was analyzed using HPLC system 3.
1H NMR. Spectra were acquired on an 800 MHz instrument
(
13). Since 3-hydroxybutanal is also readily formed by
(Varian, Inc., Palo Alto, CA) using standard 5 mm tubes or 3
aldol condensation of acetaldehyde, we hypothesized that
paraldol-derived adducts may be produced in the reac-
tions of acetaldehyde with DNA. In this study, we
identified three new types of acetaldehyde-DNA adducts.
These were distinct from the paraldol-derived adducts
which we observed in the crotonaldehyde-DNA reac-
tions.
mm Shigemi tubes (Shigemi, Inc., Allison Park, PA).
Ch em ica ls a n d En zym es. Crotonaldehyde, acetaldehyde,
and NaBH CN were purchased from Aldrich Chemical Co.
3
(Milwaukee, WI). Calf thymus DNA was obtained from Sigma
Chemical Co. (St. Louis, MO). Alkaline phosphatase was
procured from Boehringer Mannheim Co. (Indianapolis, IN).
2
Paraldol, diastereomers of adduct 9, and N -ethyl-dG were
synthesized as described previously (14-16). All other chemicals
and enzymes were obtained from Sigma.
Exp er im en ta l Section
2
N -(2,6-Dim eth yl-1,3-dioxan -4-yl)-dG (11, Sch em e 1, peaks
4
-6, F igu r es 1 a n d 3). This was prepared in reactions with
HP LC An a lysis. HPLC was carried out with Waters Associ-
ates (Milford, MA) systems equipped with a model 991 or 996
photodiode array detector and an RF-10 AXL spectrofluoromet-
ric detector (Shimadzu Scientific Instruments, Columbia, MD).
Columns and solvent elution systems were as follows. For
system 1, we used two 4.6 mm × 25 cm Supelcosil LC 18-BD
columns (Supelco, Bellefonte, PA) with isocratic elution by 5%
DNA or dG. For the DNA reactions, acetaldehyde (12 mmol)
was allowed to react with calf thymus DNA (100 mg) in 6 mL
of 0.1 M phosphate buffer (pH 7.0) for 96 h at 37 °C. The DNA
was precipitated by addition of ethanol and then hydrolyzed
enzymatically as described below. For the dG reactions, acetal-
dehyde (22.5 mmol) was allowed to react with dG (0.17 mmol)
in 10 mL of 0.1 M phosphate buffer (pH 7.0) for 55 h at 37 °C.
Peaks 4-6 were collected from HPLC system 1 followed by
desalting using system 4 and were obtained in 2% yield based
on dG. Peak 4: 1H NMR (DMSO-d
7
7
4
CH
and then a gradient from 5 to 25% CH
0 min at a rate of 1 mL/min and UV detection (254 nm). This
3
CN in 10 mM sodium phosphate buffer (pH 7) for 10 min
3
CN over the course of
6
) δ 10.7 (bs, 1H, dG-N1-H),
.93 (s, 1H, dG-C8-H), 7.24 (bs, 1H, dG-N -H), 6.14 (dd, J )
system was used for analysis of all acetaldehyde adducts. For
system 2, we used the same columns as system 1, with elution
by a gradient from 0 to 30% CH CN in 10 mM sodium phosphate
3
buffer (pH 7) over the course of 40 min at a rate of 1 mL/min
and UV detection (254 nm). This system was used for quanti-
tation of dG. For system 3, we used one of the columns employed
6
2
.2, 7.2 Hz, 1H, 1′-H), 5.40 (dd, J ) 9.6, 10.4 Hz, 1H, dioxane
-H), 5.25 (s, 1H, 3′-OH), 4.87 (bs, 1H, 5′-OH), 4.83 (m, 1H,
dioxane 2-H), 4.36 (m, 1H, 3′-H), 3.80 (m, 2H, 4′-H and dioxane
-H), 3.57 (m, 1H, 5′-H ), 3.50 (m, 1H, 5′-H ), 2.61 (m, 1H, 2′-
), 2.18 (m, 1H, 2′-H ), 1.75 (m, 1H, dioxane 5eq-H), 1.30 (m,
H, dioxane 5ax-H), 1.18 (d, J ) 4.8 Hz, dioxane 2-CH ), 1.15
d, J ) 4.8 Hz, dioxane 6-CH ); MS (positive ion LC-ESI) m/z
6
H
1
(
a
b
in system 1, with elution by a gradient from 10 to 40% CH
in 20 mM ammonium acetate buffer (pH 3) over the course of
0 min at a flow rate of 0.5 mL/min with UV detection (254
3
CN
a
b
3
6
3
+
+
+
(relative intensity) 382 (MH , 100), 266 (BH , 33), 248 (BH
-
nm). This system was used for purification of adduct 10 as the
guanine base. For system 4, we employed a 4.6 mm × 25 cm, 5
µm OD5 octadecyl column (Burdick and J ackson, Baxter,
McGaw Park, IL) with elution by a gradient from 20 to 80%
+
+
H
H
2
O, 8), 222 (BH - CH
3
CHO, 54), 204 (BH - CH
CHO, 49), 152 (15); MS/MS of m/z
O) λmax 254, 275 (sh) nm. Peak 5:
) δ 10.8 (bs, 1H, dG-N1-H), 7.90 (s, 1H, dG-
3
CHO -
+
2
O, 20), 178 (BH - 2CH
3
3
82; 266 (65), 222 (4); UV (H
2
1
H NMR (DMSO-d
C8-H), 7.25 (bs, 1H, dG-N -H), 6.12 (dd, J ) 6.4, 6.4 Hz, 1H,
6
3 2
CH CN in H O over the course of 40 min at a rate of 1 mL/min
2
with UV detection at 254 nm. This system was used for desalting
of adducts collected in system 1. For system 5, we used the same
column and flow rate as in system 4, with a gradient from 40
1
3
′-H), 5.39 (dd, J ) 9.4, 9.4 Hz, 1H, dioxane 4-H), 5.26 (s, 1H,
′-OH), 4.84 (bs, 1H, 5′-OH), 4.83 (bs, 1H, dioxane 2-H), 4.37
(
m, 1H, 3′-H), 3.82 (m, 1H, dioxane 6-H), 3.77 (dt, J ) 4.2, 4.2
Hz, 1H, 4′-H), 3.53 (m, 1H, 5′-H ), 3.44 (m, 1H, 5′-H ), 2.70 (m,
H, 2′-H ), 2.18 (m, 1H, 2′-H ), 1.75 (m, 1H, dioxane 5eq-H), 1.31
),
); MS (positive ion LC-
to 60% CH
detection (365 nm). This system was employed for analysis of
,4-dinitrophenylhydrazones. For system 6, we used two 4.6 mm
25 cm Partisil-10 SCX strong cation exchange columns
Whatman, Clifton, NJ ) and an elution medium of 100 mM
3 2
CN in H O over the course of 40 min with UV
a
b
1
a
b
2
(
1
m, 1H, dioxane 5ax-H), 1.18 (d, J ) 4 Hz, 3H, dioxane 2-CH
3
×
.14 (d, J ) 6.4 Hz, 3H, dioxane 6-CH
3
(
+
+
ESI) m/z (relative intensity) 382 (MH , 100), 266 (BH , 38), 248
ammonium phosphate buffer (pH 2) at a rate of 1 mL/min with
fluorescence detection (excitation at 290 nm and emission at
+
+
+
(
BH - H
CHO - H
UV (H
2
O, 8), 222 (BH - CH
3
CHO, 54), 204 (BH - CH
3
3
-
+
O, 20), 178 (BH - 2CH
CHO, 48), 152 (15), 117 (9);
3
80 nm). This system was used for quantitation of adduct 9 (as
2
O) λmax 254, 275 (sh) nm.
the guanine base) and guanine.
2
2
GC An a lysis. GC with flame ionization detection was
performed with a HP 6890 series gas chromatograph (Hewlett-
Packard, Palo Alto, CA) with a 30 m × 0.32 mm i.d., 3.0 µm
film thickness, DB-1 column (J &W Scientific, Folsom, CA). One
microliter of sample was injected in the split mode (1:100). The
injector temperature was 200 °C, and He was used as a carrier
gas (32 cm/s at 40 °C). The initial temperature of the oven was
3-(2-De oxyr ib os-1-yl)-5,6,7,8-t e t r a h yd r o-8-(N -d e oxy-
gu a n osyl)-6-m eth ylp yr im id o[1,2-a ]p u r in e-10(3H)on e (12,
Sch em e 1, p ea k 3, F igu r es 1 a n d 3). Acetaldehyde was
allowed to react with DNA as described above for the prepara-
tion of adduct 11. The DNA was enzymatically hydrolyzed, and
adduct 12 (peak 3) was collected as described above: H NMR
2
(D O) δ 8.01 (s, 1H, C2-H or C8′-H), 8.00 (s, 1H, C2-H or C8′-
1
4
0 °C, which was maintained for 4 min, and followed by a
programmed rate of 10 °C/min to 210 °C. The flame ionization
detector was set at 25 °C with flow rates of H (40 mL/min), air
400 mL/min), and He (25 mL/min). The retention times of
acetaldehyde, crotonaldehyde, and paraldol were 2.52, 9.10, and
H), 6.87 (m, 1H, C8-H), 6.44 (dd, J ) 7.3, 6.7 Hz, 1H, 1′-H),
6.35 (dd, J ) 6.7, 6.7 Hz, 1H, 1′-H), 4.68 (m, 1H, 3′-H), 4.17 (m,
2H, 4′-H), 3.86 (m, 5H, 5′-Ha,b and C6-H), 3.12 (m, 1H, 2′-H),
2.86 (m, 1H, 2′-H), 2.56 (m, 3H, 2′-Ha,b and C7-H), 1.87 (td, J )
2
(
13.1, 3.1 Hz, 1H, C7-H), 1.41 (d, J ) 6.7 Hz, 3H, C6-CH
3
); MS
+
1
1.97 min, respectively.
MS An a lysis. LC/MS analysis was performed on a Finnigan-
(positive ion LC-ESI) m/z (relative intensity) 587 (MH , 100),
+
+
471 (MH - 116, 23), 355 (MH - 232, 18), (294, 48); UV (H
2
O)
MAT LCQ Deca instrument (Thermoquest LC/MS Division, San
λmax 258, 280 (sh) nm. Hydrolysis of 12 to the guanine base was