Lactols in hydrolysates of DNA treated with α-acetoxy-n- nitrosopyrrolidine or crotonaldehyde

Article abstract of DOI:10.1021/tx980165+

α-Acetoxy-N-nitrosopyrrolidine (α-acetoxyNPYR) is a stable precursor to α-hydroxyNPYR, the initial product of metabolism and proposed proximate carcinogen of N-nitrosopyrrolidine (NPYR). Crotonaldehyde (2-butenal) is a metabolite of NPYR and also a mutagen and carcinogen. Both α-acetoxyNPYR and crotonaldehyde form DNA adducts, but these reactions have not been completely characterized. In previous studies, we detected substantial amounts of unidentified radioactivity in hydrolysates of DNA that had been treated with radiolabeled α-acetoxyNPYR. In this study, we have characterized these products as 2-hydroxytetrahydrofuran, the cyclic form of 4-hydroxybutanal, and paraldol, the dimer of 3-hydroxybutanal. These products were identified by comparison to standards and by conversion to 2,4-dinitrophenylhydrazones. 2-Hydroxytetrahydrofuran is the major product in neutral thermal hydrolysates of α-acetoxyNPYR-treated DNA and is derived predominantly from N²- (tetrahydrofuran-2-yl)deoxyguanosine 8. Paraldol is present to a lesser extent than 2-hydroxytetrahydrofuran in these reactions and is formed from paraldol-releasing adducts, which in turn are produced in the reaction of crotonaldehyde, a solvolysis product of α-acetoxyNPYR, with DNA. Other products in hydrolysates of α-acetoxyNPYR-treated DNA are N⁷-substituted guanines 5 and 6, cyclic N⁷-C8 guanines 4, 11, and 12, and 1,N²- propanodeoxyguanosines 9 and 10. Paraldol is a major product in hydrolysates of crotonaldehyde-treated DNA, being present in amounts 100 times greater than those of previously identified adducts 9 and 10. The results of this study provide a more complete picture of the reactions of α-acetoxyNPYR with DNA and yield some new insights about possible endogenous DNA adducts formed from crotonaldehyde.

Full text of DOI:10.1021/tx980165+

Chem. Res. Toxicol. 1998, 11, 1567-1573
1567
La ctols in Hyd r olysa tes of DNA Tr ea ted w ith
r-Acetoxy-N-n itr osop yr r olid in e or Cr oton a ld eh yd e
Mingyao Wang, Pramod Upadhyaya, Trieu T. Dinh, Leo E. Bonilla, and
Stephen S. Hecht*
University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware Street SE,
Minneapolis, Minnesota 55455
Received J uly 13, 1998
R-Acetoxy-N-nitrosopyrrolidine (R-acetoxyNPYR) is a stable precursor to R-hydroxyNPYR,
the initial product of metabolism and proposed proximate carcinogen of N-nitrosopyrrolidine
(NPYR). Crotonaldehyde (2-butenal) is a metabolite of NPYR and also a mutagen and
carcinogen. Both R-acetoxyNPYR and crotonaldehyde form DNA adducts, but these reactions
have not been completely characterized. In previous studies, we detected substantial amounts
of unidentified radioactivity in hydrolysates of DNA that had been treated with radiolabeled
R-acetoxyNPYR. In this study, we have characterized these products as 2-hydroxytetra-
hydrofuran, the cyclic form of 4-hydroxybutanal, and paraldol, the dimer of 3-hydroxybutanal.
These products were identified by comparison to standards and by conversion to 2,4-
dinitrophenylhydrazones. 2-Hydroxytetrahydrofuran is the major product in neutral thermal
hydrolysates of R-acetoxyNPYR-treated DNA and is derived predominantly from N2-(tetra-
hydrofuran-2-yl)deoxyguanosine 8. Paraldol is present to a lesser extent than 2-hydroxy-
tetrahydrofuran in these reactions and is formed from paraldol-releasing adducts, which in
turn are produced in the reaction of crotonaldehyde, a solvolysis product of R-acetoxyNPYR,
with DNA. Other products in hydrolysates of R-acetoxyNPYR-treated DNA are N7-substituted
guanines 5 and 6, cyclic N7-C8 guanines 4, 11, and 12, and 1,N2-propanodeoxyguanosines 9
and 10. Paraldol is a major product in hydrolysates of crotonaldehyde-treated DNA, being
present in amounts 100 times greater than those of previously identified adducts 9 and 10.
The results of this study provide a more complete picture of the reactions of R-acetoxyNPYR
with DNA and yield some new insights about possible endogenous DNA adducts formed from
crotonaldehyde.
acyclic nitrosamines such as N-nitrosodimethylamine and
N-nitrosodiethylamine is well-characterized and results
from predictable reactions of alkanediazonium ions with
DNA bases (3), adduct formation from cyclic nitrosamines
such as NPYR is more complex, presumably due to the
intermediacy of aldehydic diazonium ions and related
intermediates such as 2, 3, and 7 (Figure 1), which are
formed by spontaneous decomposition of R-hydroxyNPYR
(1). An understanding of DNA adduct formation from
NPYR will provide insights about these pathways for
other carcinogenic cyclic nitrosamines such as N-nitro-
sopiperidine, N-nitrosomorpholine, and N′-nitrosonorni-
cotine, for which the spectrum of DNA adducts is largely
uncharacterized (14-18). Moreover, DNA adducts and
related products of DNA hydrolysis can be used as
biomarkers for the uptake and metabolic activation of
carcinogens. This is particularly important in the case
of nitrosamines such as NPYR which are extensively
metabolized in vivo. Thus, only small amounts of NPYR
and 2-hydroxyTHF, a major metabolite resulting from
R-hydroxylation in vitro, are detected in the urine of
NPYR-treated animals (2, 6).
In tr od u ction
N-Nitrosopyrrolidine (NPYR)1 is a representative car-
cinogenic cyclic nitrosamine. R-AcetoxyNPYR (Figure 1)
is a stable precursor to R-hydroxyNPYR (1), the major
initial metabolite and proximate carcinogen of NPYR (1,
2). NPYR is a well-established hepatocarcinogen in the
rat and a respiratory carcinogen in the Syrian golden
hamster (3). NPYR occurs in the human diet and in
tobacco smoke (4, 5). It is a likely product of nitric oxide-
mediated endogenous nitrosation in humans and has
been detected in human urine (6-9). Crotonaldehyde (2-
butenal) is a mutagen and a hepatocarinogen in rats (10,
11). It occurs in the human diet, tobacco smoke, and
mobile source emissions as well as being a product of lipid
peroxidation (11, 12). It is also a solvolysis product of
R-acetoxyNPYR and a metabolite of NPYR (Figure 1)
(13). In this study, we identified lactols as significant
products in hydrolysates of DNA treated with R-acetoxy-
NPYR or crotonaldehyde.
Our goal is to understand the metabolism and DNA
adduct formation by NPYR. DNA adducts are important
in carcinogenesis. While adduct formation from simple
In previous studies, we have characterized several
DNA adducts as products of the reaction of R-acetoxyN-
PYR with DNA. These adducts are illustrated in Figure
1. The N7-C8 guanine adduct 4, the open chain N7-
substituted adduct 5, and the N2-tetrahydrofuranyl ad-
duct 8 are the most prevalent (15, 19). Adducts 9-12
1
Abbreviations: R-acetoxyNPYR, R-acetoxy-N-nitrosopyrrolidine;
2,4-DNP, 2,4-dinitrophenylhydrazone; 2,4-DNP reagent, 2,4-dinitro-
phenylhydrazine reagent; 3-HOBA, 3-hydroxybutanal; 4-HOBA, 4-hy-
droxybutanal; R-hydroxyNPYR, R-hydroxy-N-nitrosopyrrolidine; 2-hy-
droxyTHF, 2-hydroxytetrahydrofuran; NPYR, N-nitrosopyrrolidine;
NTH, neutral thermal hydrolysis; PCI, positive ion chemical ionization.
10.1021/tx980165+ CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/12/1998

1568 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Wang et al.
F igu r e 1. Products formed in the solvolysis of R-acetoxyNPYR and in its reactions with DNA. See the text for details. NTH is
neutral thermal hydrolysis.
Gilson model 116 UV detector (Gilson Medical Electronics,
Middletown, WI), an RF-10AXL spectrofluorometric detector
(Shimadzu Scientific Instruments, Columbia, MA), or a â-RAM
radioactivity HPLC flow-through detector (IN/US Systems,
Tampa, FL). The following systems were used: (1) two 4.6 mm
× 25 cm Partisil-10 SCX strong cation exchange columns
(Whatman, Clifton, NJ ) and an elution medium of 40 mM
ammonium phosphate buffer (pH 2.0) at a flow rate of 1 mL/
min with fluorescence (excitation at 290 nm and emission at
380 nm) or radio-flow detection; (2) two 4.6 mm × 25 cm
Supelcosil LC 18-DB columns (Supelco, Bellefonte, PA), the
eluant being a gradient from 0 to 20% CH3CN in H2O over the
course of 20 min at a flow rate of 1 mL/min with UV detection
(280 nm); (3) the same columns as in system 2 with isocratic
elution by 5% CH3CN in 10 mM sodium phosphate buffer (pH
7.0) for 10 min and then a gradient from 5 to 25% CH3CN over
the course of 60 min at a flow rate of 1 mL/min with UV
detection (254 nm); (4) a 9.4 mm × 50 cm Partisil 10 ODS-3
Magnum 9 column (Whatman) with elution by a gradient from
0 to 80% CH3CN in H2O over the course of 20 min at a flow
rate of 4 mL/min with UV detection (280 nm); (5) a 4.6 mm ×
25 cm 5µ OD5 octadecyl column (Burdick and J ackson, Baxter,
McGaw Park, IL) with elution by a gradient from 40 to 60%
CH3CN in H2O over the course of 40 min at a flow rate of 1
mL/min with UV detection (365 nm); and (6) the same column
as in system 5, the eluant in this case being a gradient of 0 to
are formed via crotonaldehyde, a product of the solvolysis
of R-acetoxyNPYR (13, 20-22). Adducts 4, 5, 9, and 10
have been detected in rat liver DNA following treatment
with NPYR (20, 23, 24). Despite what appears to be a
relatively complete characterization of DNA adduct
formation from R-acetoxyNPYR, chromatograms of neu-
tral thermal hydrolysates of DNA that had been treated
with radiolabeled R-acetoxyNPYR showed substantial
amounts of unidentified material. One of these chro-
matograms is illustrated in Figure 2. This chromatogram
had large unidentified early eluting peaks, labeled P9 and
P12. Characterization of this early eluting radioactivity
was the focus of this study.
Exp er im en ta l Section
Ca u tion : R-AcetoxyNPYR is a mutagen and chemically
activated form of the carcinogen NPYR. Crotonaldehyde is a
mutagen and carcinogen. These compounds should be handled
with extreme care, using appropriate safety wear and ventilation
at all times.
Ap p a r a tu 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, a

Lactols in DNA Hydrolysates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1569
thymus DNA in 300 mL of 0.1 M phosphate buffer (pH 7). The
mixture was incubated at 37 °C for 20 h while it was being
shaken. The reaction mixture was diluted with 300 mL of H2O
and then extracted three times with 300 mL of CHCl3/isoamyl
alcohol (24:1). After each extraction, the mixture was centri-
fuged at 10000g for 20 min. The supernatants were removed,
combined, and mixed with 30 mL of 5 M NaCl and 450 mL of
ethanol to precipitate the DNA. The DNA was washed with
450 mL of 70% ethanol and then twice with 100 mL of ethanol.
It was dried under a stream of N2. The DNA was hydrolyzed
and analyzed as described below.
(2) Cr oton a ld eh yd e a n d DNA. Crotonaldehyde (56 mg,
0.76 mmol) was added to a solution of 100 mg of calf thymus
DNA in 6 mL of 0.1 M phosphate buffer (pH 7). The mixture
was incubated at 37 °C for 20 h while it was being shaken. The
reaction mixture was diluted with 22 mL of H2O and then
extracted with 12 mL of CHCl3/isoamyl alcohol (24:1). The
mixture was centrifuged at 10000g for 20 min. The supernatant
was removed and mixed with 2.8 mL of 5 M NaCl and 30 mL of
ethanol to precipitate the DNA. The DNA was washed with 5
mL of 70% ethanol and then twice with 10 mL of ethanol. It
was dried under a stream of N2.
For neutral thermal and acid hydrolysis, DNA (10 mg) was
dissolved in 1 mL of 10 mM sodium cacodylate buffer (pH 7.0)
and the mixture was heated at 100 °C for 1 h. To the resulting
mixture was added 0.1 mL of 1 N HCl to precipitate the partially
apurinic DNA. An aliquot of the supernatant was analyzed for
adducts 4, 11, and 12 using HPLC system 1. Another aliquot
(0.8 mL) was mixed with 50 µL of 2,4-DNP reagent and the
mixture allowed to stand overnight at room temperature. The
mixture was extracted twice with 3 mL of CH2Cl2. The extracts
were concentrated to dryness, redissolved in methanol, and
analyzed by HPLC system 5. The precipitated, partially apu-
rinic DNA was dissolved in 1 mL of 0.1 N HCl and the mixture
heated at 85 °C for 1 h to release adducts 9 and 10, as the
guanine base, which were analyzed using HPLC system 1.
For enzyme hydrolysis, DNA (15 mg) was dissolved in 1.5
mL of 10 mM Tris-HCl/5 mM MgCl2 buffer (pH 7.0). The
mixture was incubated with 7500 units of DNaseI at 37 °C for
10 min and then with 0.38 unit of phophodiesterase I and 2300
units of alkaline phosphatase for an additional 60 min. En-
zymes were removed by centrifugation using a centrifree MPS
device (MW cutoff of 30 000; Amicon, Inc., Beverly, MA). The
hydrolysate (0.4 mL) was analyzed for adducts 8-10 by HPLC
system 3. Another portion of the hydrolysate (0.8 mL) was
treated with 50 µL of 2,4-DNP reagent, extracted with CH2Cl2,
and analyzed by HPLC system 5.
Collection a n d An a lysis of P 9 a n d P 12. The retention
times of paraldol and 2-hydroxyTHF were established by HPLC
system 1. Neutral thermal hydrolysates of DNA reacted with
R-acetoxyNPYR were analyzed in this system. Material eluting
at the retention times of 2-hydroxyTHF and paraldol (1.5-1.7
mL) was collected and then concentrated to 1 mL. 2,4-DNP
reagent (50 µL) was added to each concentrated solution. After
standing overnight at room temperature, the reaction mixture
was extracted twice with 3 mL of CH2Cl2. The extracts were
combined and concentrated in vacuo to dryness. The residue
was dissolved in methanol and analyzed by HPLC system 5.
The retention times of paraldol and 2-hydroxyTHF were also
established by HPLC system 2. Neutral thermal hydrolysates
of DNA reacted with R-acetoxyNPYR were analyzed, and the
material eluting at the correct retention times was collected as
described above. The collected material was concentrated and
repurified in the same HPLC system. This material was
concentrated and analyzed by GC/MS.
F igu r e 2. HPLC analysis of a neutral thermal hydrolysate of
[2,3,4,5-3H]-R-acetoxyNPYR-treated DNA. The analysis was
performed in a previous study (15). The traces were obtained
using HPLC system 1. The upper trace illustrates the retention
times of standard adducts 4, 5, 11, and 12 (Figure 1) and
guanine (G), as detected by fluorescence. The lower panel
illustrates radio-flow detection of products in the neutral
thermal hydrolysate.
30% CH3CN in H2O over the course of 30 min at a flow rate of
1 mL/min with UV (254 nm) or radio-flow detection.
GC/MS was carried out on a Finnigan MAT 90 double-
focusing mass spectrometer (Finnigan MAT, Bremen, Germany)
interfaced with a Hewlett-Packard model 5890 gas chromato-
graph equipped with split-splitless injection and a 0.25 mm ×
60 m DB-5 column (J & W Scientific, Folsom, CA). Samples
were introduced at 50 °C, with no oven temperature ramp.
Mass spectra were obtained using PCI with CH4 or NH3 as the
reagent gas. Electron ionization mass spectra were obtained
by direct insertion. The probe was heated at 25 °C/min to a
final temperature of 150 °C.
1H NMR data were obtained on a GE Omega 300 spectrom-
eter.
Ch em ica ls a n d En zym es. R-AcetoxyNPYR and 2-hydroxy-
THF were synthesized (25, 26). 2,4-DNP reagent was prepared
by dissolving 250 mg of 2,4-dinitrophenylhydrazine (Eastman
Organic Chemicals, Rochester, NY) in 100 mL of 6 N HCI.
Crotonaldehyde-2,4-DNP and 4-HOBA-2,4-DNP were pre-
pared from crotonaldehyde and 2-hydroxyTHF (13). Paraldol
was allowed to react with 2,4-DNP reagent, and the products
were separated by HPLC system 5. Their electron ionization
mass spectra were determined. [3H]H2O was purchased from
Amersham Life Sciences, Inc. (Arlington Heights, IL). All other
reagents and enzymes were obtained from Aldrich Chemical Co.
(Milwaukee, WI) and Sigma Chemical Co. (St. Louis, MO).
2-(2-Hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paral-
dol) was prepared essentially as described previously (27). To
50 g of cold acetaldehyde was added 1.25 mL of 10% aqueous
NaOH dropwise while the mixture was being stirred at 5 °C.
The mixture was kept at 4-5 °C for 30 min, then stirred for 1
h at 5 °C, and then adjusted to pH 3-4 with tartaric acid. After
filtration, the product was distilled in vacuo at an oil bath
temperature of 140 °C. The distillate [bp 57 °C (1 mmHg), 5.4
g] was further purified using HPLC system 5, with a retention
time of 10 min: 1H NMR for the major conformer of paraldol
(Figure 1) (D2O) δ 5.03 (m, 1H, ring 4-CHOH), 4.83 (m, 1H, ring
2-CH), 3.92 (m, ring 6-CHCH3 + 2-CHOHCH3), 1.69 (m, 4H,
ring 5-H2 + CH2CHOH), 1.2 (m, ring 6-CH3 + CHOHCH3),
essentially as described in ref 28; MS PCI (CH4) m/z (relative
intensity) 161 (M - 15, 2), 89 (CHOHCH2CHOHCH3, 9), 73 (22),
71 (OdCHCH2CHCH3, 100).
Resu lts
Preliminary data indicated that P9 and P12 had the
same retention times and formed the same 2,4-DNPs as
two products of the solvolysis of R-acetoxyNPYR-paral-
dol and 2-hydroxyTHF, respectively (Figure 1). Paraldol
R ea ct ion s. (1) r-Acet oxyNP YR a n d DNA. R-Acetoxy-
NPYR (6.0 g, 38 mmol) was added to a solution of 5 g of calf

1570 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Wang et al.
Ta ble 1. HP LC Reten tion Tim es of P a r a ld ol,
2-Hyd r oxyTHF , a n d Der ived 2,4-DNP s
hydrolysis. The hydrolysates were analyzed using strong
cation exchange HPLC. Material which eluted at the
same retention times as those of paraldol and 2-hydroxy-
THF was collected, derivatized with 2,4-DNP reagent,
and analyzed by HPLC. Panels A and B of Figure 4 are
the chromatograms obtained upon derivatization of the
material which coeluted with paraldol and 2-hydroxy-
THF, respectively. The two peaks indicated by arrows
in Figure 4A had the same retention times as 3-HOBA-
2,4-DNP and crotonaldehyde-2,4-DNP, the products of
derivatization of paraldol with 2,4-DNP reagent. Simi-
larly, the indicated peak in Figure 4B had the same
retention time as that of 4-HOBA-2,4-DNP, which is
formed upon derivatization of 2-hydroxyTHF. The UV
spectra of the standard and isolated 2,4-DNPs were
identical (Figure 3A-F).
The same DNA hydrolysates were analyzed by reverse-
phase HPLC. The material which was eluted at the
retention times of paraldol and 2-hydroxyTHF was
collected and analyzed by GC/MS. The mass spectra of
standard paraldol and 2-hydroxyTHF and the corre-
sponding isolated products were essentially identical
(Figure 5A,B). These results demonstrate that paraldol
and 2-hydroxyTHF are products of the neutral thermal
hydrolysis of R-acetoxyNPYR-treated DNA.
DNA treated with R-acetoxyNPYR was also hydrolyzed
enzymatically, and these hydrolysates were treated with
2,4-DNP reagent. The results of this analysis demon-
strated the presence of 3-HOBA-2,4-DNP, crotonalde-
hyde-2,4-DNP, and 4-HOBA-2,4-DNP, as in the neutral
thermal hydrolysates.
It seemed likely that the source of the paraldol-
releasing adducts in R-acetoxyNPYR-treated DNA was
crotonaldehyde, which is known to be a product of the
solvolysis of R-acetoxyNPYR and is known to react with
DNA. Therefore, we investigated the reaction of cro-
tonaldehyde with DNA. This DNA was extensively
purified and then subjected to either neutral thermal or
enzyme hydrolysis, followed by derivatization with 2,4-
DNP reagent. The results of this analysis demonstrated
the presence of 3-HOBA-2,4-DNP and crotonaldehyde-
retention time (min)
HPLC
HPLC
HPLC
system 1
system 2
system 5
paraldol
8.39
9.56
-
12.6
23.8
-
-
2-hydroxyTHF
-
3-HOBA-2,4-DNP
crotonaldehyde-2,4-DNP
4-HOBA-2,4-DNP
11.7
35.9
11.2
-
-
-
-
is the dimer of 3-HOBA, while 2-hydroxyTHF is the cyclic
form of 4-HOBA. Therefore, these standards were pre-
pared and were used to establish the retention times of
P9 and P12 with the strong cation exchange and reverse-
phase HPLC systems employed in this study (Table 1).
In previous studies, we have found that 2,4-DNP
formation is an effective way to isolate and quantify
lactols (2, 13, 29). Published data on 3-HOBA-2,4-DNP,
the expected product of derivatization of paraldol, are
ambiguous with respect to the possible formation of
crotonaldehyde-2,4-DNP (30). Therefore, we reacted
paraldol with 2,4-DNP reagent. The products were
separated by HPLC (Table 1) and identified by their
electron ionization mass and UV spectra. The electron
ionization mass spectrum of the product which was eluted
at 11.7 min had a molecular ion at m/z 268 and a base
peak at m/z 45 ([CH3CHOH]+). Its UV spectrum was
similar to that of 4-HOBA-2,4-DNP (Figure 3). These
results demonstrate that the 11.7 min peak is 3-HOBA-
2,4-DNP. The product which was eluted at 35.9 min had
a molecular ion and base peak of m/z 250, expected for
crotonaldehyde-2,4-DNP; its retention time and UV
spectrum were identical to those of crotonaldehyde-2,4-
DNP. Crotonaldehyde-2,4-DNP presumably formed from
dehydration of either 3-HOBA or 3-HOBA-2,4-DNP
under the reaction conditions. Thus, reaction of paraldol
with 2,4-DNP reagent gives a mixture of 3-HOBA-2,4-
DNP and crotonaldehyde-2,4-DNP.
DNA that had been treated with R-acetoxyNPYR was
precipitated, washed once with 70% ethanol and twice
with ethanol, dried, and subjected to neutral thermal
F igu r e 3. UV spectra of standard 2,4-DNPs (A, C, and E) and 2,4-DNPs isolated from the traces shown in Figure 4 (B, D, and F).

Lactols in DNA Hydrolysates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1571
Figure 2 is a chromatogram of a neutral thermal
hydrolysate of [2,3,4,5-3H]-R-acetoxyNPYR-treated DNA
from an earlier study (15). P9 and P12 in this chromato-
gram are about the same size. But the data in Table 2
demonstrate that levels of paraldol are lower than those
of 2-hydroxyTHF in neutral thermal hydrolysates of
R-acetoxyNPYR-treated DNA. Therefore, P9 of Figure
2 must contain something in addition to paraldol. Our
data indicate that this is [3H]H2O. Thus, when hydroly-
sates of [2,3,4,5-3H]-R-acetoxyNPYR-treated DNA were
stored at -20 °C for 1 year, the P9 level increased at the
expense of the P12 level ([3H]-2-hydroxyTHF). The newly
formed material did not form a 2,4-DNP derivative and
had the same retention time as [3H]H2O in HPLC
systems 1 (retention time of 7.11 min) and 6 (retention
time of 2.55 min). However, in samples of neutral
thermal hydrolysates of [U-14C]-R-acetoxyNPYR-treated
DNA, the ratios of P9 and P12 were similar to those
shown in Table 2, and did not change after storage for 1
year. These data indicate that P9 of Figure 2 is a mixture
of [3H]H2O and paraldol.
F igu r e 4. HPLC trace obtained upon analysis (system 5) of
(A) P9 and (B) P12 of Figure 2 after treatment with 2,4-DNP
reagent. The indicated peaks were identified by their retention
times and UV spectra (Figure 3).
2,4-DNP, as in the studies described above.
Products of the neutral thermal or enzymatic hydroly-
sis of DNA treated with either R-acetoxyNPYR or cro-
tonaldehyde are summarized in Table 2. 2-HydroxyTHF
is the major product of both neutral thermal and enzyme
hydrolysis of DNA treated with R-acetoxyNPYR. The
total amount of 2-hydroxyTHF and the N2-tetrahydro-
furanyl adduct 8 in the enzyme hydrolysates is similar
to the amount of 2-hydroxyTHF in the neutral thermal
hydrolysate, consistent with the known conversion of 8
to 2-hydroxyTHF under neutral thermal hydrolysis con-
ditions (14). Levels of paraldol are significantly lower
than those of 2-hydroxyTHF in hydrolysates of R-acetoxy-
NPYR-treated DNA.
In hydrolysates of DNA treated with crotonaldehyde,
paraldol is present in amounts more than 100 times
greater than the amounts of each of the adducts 9-12,
previously characterized in these reactions (Table 2).
Paraldol levels are greater in neutral thermal hydroly-
sates than in enzyme hydrolysates of both types of DNA,
suggesting the presence of a thermally unstable paraldol-
releasing adducts.
Discu ssion
In an earlier study, we noted the presence of substan-
tial amounts of unidentified early eluted radioactivity in
neutral thermal hydrolysates of [2,3,4,5-3H]-R-acetoxy-
NPYR-treated DNA (Figure 2) (15). This was in contrast
to chromatograms obtained using fluorescence detection
in which adduct 4 predominated (20). In this paper, our
major objective was to characterize this unknown radio-
activity. This goal was accomplished. The main early
eluted products derived from R-acetoxyNPYR-treated
DNA are paraldol, the dimer of 3-HOBA, and 2-hydroxy-
THF, the cyclic and predominant form of 4-HOBA.
[3H]H2O was also identified. Thus, P9 of Figure 2 is
comprised of paraldol and [3H]H2O, while P12 is 2-hy-
droxyTHF.
The N2-tetrahydrofuranyl adduct 8 is the source of
most of the 2-hydroxyTHF observed in these experiments.
F igu r e 5. PCI mass spectra of standard and isolated (HPLC system 2) paraldol (A) and 2-hydroxyTHF (B).

1572 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Wang et al.
Ta ble 2. Qu a n tita tion of P r od u ct F or m a tion in th e Rea ction s of r-AcetoxyNP YR or Cr oton a ld eh yd e w ith DNAa
product (mmol/mol of G or dG)b
reactant
hydrolysis conditions paraldolc 2-hydroxyTHFc
4d
8
9 and 10e
11
12
R-acetoxyNPYR
neutral thermal
or acidf
9.9 ( 2.1f
53.6 ( 3.5
24.3 ( 4.4 NDg
1.8 ( 0.2
0.07 ( 0.01
0.08 ( 0.01
enzyme
4.1 ( 0.8
9.6 ( 3.0
32.7 ( 0.6
ND
NQ
ND
27.3 ( 2.3 NQ
NQh
NQ
crotonaldehyde
neutral thermal
ND
0.07 ( 0.04 0.033 ( 0.006 0.043 ( 0.006
or acidf
enzyme
4.3 ( 0.5
ND
ND
ND
NQ
NQ
NQ
a
R-AcetoxyNPYR (38 mmol) was allowed to react with calf thymus DNA (5.0 g) for 20 h at 37 °C in 0.1 M phosphate buffer (pH 7.0).
b
Crotonaldehyde (0.76 mmol) was similarly reacted with DNA (100 mg). Neutral thermal and acid hydrolysis products expressed per
mole of G and enzyme hydrolysis products per mole of dG. Values are mean ( SD (n ) 3). c Quantified as their 2,4-DNPs: 3-HOBA-
d
2,4-DNP and crotonaldehyde-2,4-DNP (6:1 ratio) from paraldol and 4-HOBA-2,4-DNP from 2-hydroxyTHF. Numbers refer to structures
in Figure 1. Adducts 5 and 6 were not quantified. e Quantified as guanine base. f Acid hydrolysis for adducts 9 and 10 only. ND means
g
h
not detected. NQ means not quantified.
We have previously shown that this adduct is unstable
at the nucleoside level, decomposing to 2-hydroxyTHF
and deoxyguanosine with a t1/2 of 26 min at pH 7 and 37
°C (6, 14). Thus, the formation of 2-hydroxyTHF upon
neutral thermal and enzyme hydrolysis of R-acetoxy-
NPYR-treated DNA is completely consistent with previ-
ous data. Comparison of the amounts of 2-hydroxyTHF
present in neutral thermal and enzyme hydrolysates
indicates that adduct 8 is the main source of 2-hydroxy-
THF. There may also be some minor 2-hydroxyTHF-
releasing adducts in this DNA, since in some experiments
we analyzed each portion of the HPLC chromatogram of
the enzyme hydrolysate for material which gives 4-HO-
BA-2,4-DNP upon derivatization and obtained evidence
for an additional 2-hydroxyTHF-releasing adduct (data
not shown).
The most likely source of the paraldol released upon
hydrolysis of R-acetoxyNPYR-treated DNA is an adduct
or adducts formed in the reaction of crotonaldehyde with
DNA. Crotonaldehyde is known to be a solvolysis product
of R-acetoxyNPYR (13). In 1983, our group demonstrated
that both crotonaldehyde and R-acetoxyNPYR react with
deoxyguanosine, producing the diastereomeric 1,N2-pro-
panodeoxyguanosine adducts 9 and 10 (21). We subse-
quently demonstrated that crotonaldehyde and acrolein
produce 1,N2-propanodeoxyguanosine adducts in DNA
(22). Others have confirmed these results (31). In
several recent studies, Chung and Nath have demon-
strated that crotonaldehyde adducts 9 and 10 are present
in human and laboratory animal DNA, apparently aris-
ing partly from lipid peroxidation and perhaps also from
exogenous sources (32-35). These studies suggest that
crotonaldehyde may be an endogenous carcinogen. The
results of the present study suggest that DNA damage
by crotonaldehyde may be more extensive than previously
realized, since the levels of paraldol-releasing adducts are
about 100 times greater than those of the 1,N2-propano-
deoxyguanosine adducts 9 and 10. It will be important
to determine whether similar ratios of paraldol-releasing
adducts to 1,N2-propanodeoxyguanosine adducts are
found in human DNA. Chung and Nath have shown that
levels of adducts 9 and 10 range from approximately
0.003 to 1 µmol/mol of guanine in human DNA. If the
ratio of paraldol-releasing adducts to 9 and 10 is the same
as observed here, one would expect levels of paraldol-
releasing adducts in human DNA to range from 0.3 to
100 µmol/mol of guanine. For comparison, levels in
human lung DNA of 8-oxodeoxyguanosine, a common
indicator of oxidative DNA damage, are reported to range
approximately from 2 to 12 µmol/mol of guanine (36).
Although our results support the concept that cro-
tonaldehyde is the main source of the paraldol-releasing
adducts in R-acetoxyNPYR-treated DNA, they could also
be formed by reaction of a 3-oxobutyl carbocation with
DNA. This would be produced by rearrangement of a
carbocation derived from intermediate 3 of Figure 1. In
a previous study, we did not find evidence for the
production of 3-HOBA or paraldol in the solvolysis of
R-acetoxyNPYR or in incubations of NPYR with rat liver
microsomes and cofactors (13). However, in this study,
we did identify paraldol as a solvolysis product of
R-acetoxyNPYR (data not shown), supporting the inter-
mediacy of a 3-oxobutyl carbocation. In this study, our
detection limits were better and the reactions were run
on a larger scale.
We do not know the structures of the paraldol-releasing
adducts in crotonaldehyde- or R-acetoxyNPYR-treated
DNA. Crotonaldehyde is known to undergo Michael
additions with deoxyguanosine at both the N2- and
7-positions (20, 21, 37). This is followed by ring closure
to give adducts 9-12. Bis-adducts have also been
observed (37). The paraldol-releasing adducts may be
adducts in which Michael addition, but no ring closure,
has occurred. Cross-links could also be formed. These
adducts could produce 3-HOBA upon hydrolysis, and
3-HOBA is known to dimerize to paraldol. Alternatively,
crotonaldehyde could react with H2O, producing paradol,
which could then react with DNA. We are currently
exploring these hypotheses by developing methods for
stabilizing these adducts in DNA.
In Figure 1, our current understanding of DNA adduct
formation from R-acetoxyNPYR is summarized. Ring
opening of R-hydroxyNPYR (1) gives 4-oxobutanediazo-
hydroxide (2) which most likely is converted to diazonium
ion 3 (2). This may react directly with DNA to produce
adducts 4 and 5, or adduct 6 after oxidation (20). We
refer to these as type 1 adducts (6). They are reasonably
stable in DNA, with half-lives of 33-54 h at 37 °C, but
depurinate upon neutral thermal hydrolysis (19, 20, 23).
Diazonium ion 3 or a derived carbocation can cyclize to
give cyclic oxonium ion 7 which either may react directly
with DNA to produce 8 or may give 8 by way of a reactive
2-tetrahydrofuranyl derivative (14). Adducts formed by
this pathway are called type 2 adducts. Intermediates
2, 3, and 7 react with H2O to produce three solvolysis
products: 4-HOBA, which exists mainly as the lactol
2-hydroxyTHF; 3-HOBA, which exists mainly as the
dimer paraldol; and crotonaldehyde (2, 13, 28). Crotonal-
dehyde reacts with DNA to produce adducts 9-12 as well
as the paraldol-releasing adducts (20, 21, 37). Adducts
9 and 10 may also be produced from intermediate 3 or
its decomposition products since the ratio of 9 and 10 to
11 and 12 is substantially greater in R-acetoxyNPYR-
treated DNA than in DNA reacted with crotonaldehyde

Lactols in DNA Hydrolysates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1573
adducts in the reactions of R-acetoxy-N-nitrosopyrrolidine and
R-acetoxy-N-nitrosopiperidine with DNA. Chem. Res. Toxicol. 8,
617-624.
(Table 2). Adducts formed by reaction with DNA of stable
solvolysis products of R-acetoxyNPYR such as croton-
aldehyde are referred to as type 3 adducts. Overall, the
major DNA adducts produced from R-acetoxyNPYR are
the N7-C8 cyclic adduct 4 and the open chain N7 adduct
5 (type 1 adducts), the N2-tetrahydrofuranyl adduct 8 (a
type 2 adduct), and the paraldol-releasing adducts (type
3 adducts).
In summary, 2-hydroxyTHF and paraldol have been
identified as substantial products in neutral thermal and
enzyme hydrolysates of DNA treated with R-acetoxy-
NPYR. They are formed from the N2-tetrahydrofuranyl
adduct 8 and crotonaldehyde-derived paraldol-releasing
adducts, respectively. Paraldol-releasing adducts appear
to be major products in the reaction of crotonaldehyde
with DNA. These results define more completely the
reactions of R-acetoxyNPYR and crotonaldehyde with
DNA. Moreover, 2-hydroxyTHF and paraldol may be
useful as biomarkers of DNA modification by NPYR and
crotonaldehyde.
(16) Chung, F.-L., and Hecht, S. S. (1985) Formation of the cyclic 1,N2-
glyoxal-deoxyguanosine adduct upon reaction of N-nitroso-2-
hydroxymorpholine with deoxyguanosine. Carcinogenesis 6, 1671-
1673.
(17) Stewart, B. N., Swann, P. F., Holsman, J . W., and Magee, P. N.
(1974) Cellular injury and carcinogenesis. Evidence for alkylation
of rat liver nucleic acids in vivo by N-nitrosomorpholine. Z.
Krebsforsch. 82, 1-12.
(18) Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of
tobacco-specific nitrosamines. Chem. Res. Toxicol. 11, 559-603.
(19) Wang, M., Chung, F.-L., and Hecht, S. S. (1989) Formation of
acyclic and cyclic guanine adducts in DNA reacted with R-acetoxy-
N-nitrosopyrrolidine. Chem. Res. Toxicol. 2, 423-428.
(20) 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.
(21) Chung, F.-L., and Hecht, S. S. (1983) Formation of cyclic 1,N2-
adducts by reaction of deoxyguanosine with R-acetoxy-N-nitros-
opyrrolidine, 4-(carbethoxynitrosamino)butanal, or crotonalde-
hyde. Cancer Res. 43, 1230-1235.
(22) Chung, F.-L., Young, R., and Hecht, S. S. (1984) Formation of
cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction
with acrolein or crotonaldehyde. Cancer Res. 44, 990-995.
(23) Wang, M., Chung, F.-L., and Hecht, S. S. (1992) Formation of
7-(4-oxobutyl)guanine in hepatic DNA of rats treated with N-
nitrosopyrrolidine. Carcinogenesis 13, 1909-1911.
(24) Chung, F.-L., Young, R., and Hecht, S. S. (1989) Detection of cyclic
1,N2-propanodeoxyguanosine adducts in DNA of rats treated with
N-nitrosopyrrolidine and mice treated with crotonaldehyde. Car-
cinogenesis 10, 1291-1297.
(25) Saavedra, J . E. (1979) Oxidation of nitrosamines. 1. Formation
of N-nitrosoimminium ions through the oxidative decarboxylation
of N-nitrosoproline, N-nitrosopipecolic acid, and N-nitrososar-
cosine. J . Org. Chem. 44, 4511-4515.
(26) Paul, R., and Tchelitcheff, S. (1948) Sur l’alde´hyde gamma-
hydroxybutyrique et ses de´rive´s. Bull. Soc. Chim. Fr. 15, 197-
203.
(27) Kyriakides, L. P. (1914) Some organic preparations. J . Am. Chem.
Soc. 36, 530-537.
(28) Vogel, M., and Rhum, D. (1966) The structure of paraldol. J . Org.
Chem. 31, 1775-1780.
(29) Chen, C. B., Hecht, S. S., and Hoffmann, D. (1978) Metabolic
R-hydroxylation of the tobacco specific carcinogen, N′-nitrosonor-
nicotine. Cancer Res. 38, 3639-3645.
Ack n ow led gm en t. This study was supported by
Grant CA-44377 from the National Cancer Institute. We
thank Dr. Edward J . McIntee for obtaining 1H NMR
spectra.
Refer en ces
(1) Baldwin, J . E., and Branz, S. E. (1976) Chemical activation of
nitrosamines into mutagenic agents. Tetrahedron Lett., 333-336.
(2) Hecht, S. S., Chen, C. B., and Hoffmann, D. (1978) Evidence for
metabolic R-hydroxylation of N-nitrosopyrrolidine. Cancer Res.
38, 215-218.
(3) Preussmann, R., and Stewart, B. W. (1984) N-Nitroso carcinogens.
In Chemical Carcinogenesis (Searle, C. E., Ed.) pp 643-828,
American Chemical Society, Washington, DC.
(4) Tricker, A. R., Pfundstein, B., Theobald, E., Preussmann, R., and
Spiegelhalder, B. (1991) Mean daily intake of volatile N-nitro-
samines from foods and beverages in West Germany in 1989-
1990. Food Chem. Toxicol. 29, 729-732.
(5) Hoffmann, D., and Hoffmann, I. (1997) The changing cigarette,
1950-1995. J . Toxicol. Environ. Health 50, 307-364.
(6) Wang, M., and Hecht, S. S. (1997) A cyclic N7, C-8 guanine adduct
of N-nitrosopyrrolidine (NPYR): formation in nucleic acids and
excretion in the urine of NPYR-treated rats. Chem. Res. Toxicol.
10, 772-778.
(30) Grosjean, D. (1983) Chemical ionization mass spectra of 2,4-
dinitrophenylhydrazones of carbonyl and hydroxycarbonyl atmo-
spheric pollutants. Anal. Chem. 55, 2436-2439.
(31) Boerth, D. W., Eder, E., Hussain, S., and Hoffman, C. (1998)
Structures of acrolein-guanine adducts: a semi-empirical self-
consistent field and nuclear magnetic resonance spectral study.
Chem. Res. Toxicol. 11, 284-294.
(32) Nath, R. G., and Chung, F.-L. (1994) Detection of exocyclic 1,N2-
propanodeoxyguanosine adducts as common DNA lesions in
rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495.
(33) Nath, R. G., Ocando, J . E., and Chung, F.-L. (1996) Detection of
1,N2-propanodeoxyguanosine adducts as potential endogenous
DNA lesions in rodent and human tissues. Cancer Res. 56, 452-
456.
(34) Chung, F.-L., Chen, H.-J . C., and Nath, R. G. (1996) Lipid
peroxidation as a potential source for the formation of exocyclic
DNA adducts. Carcinogenesis 17, 2105-2111.
(35) Nath, R. G., Ocando, J . E., Guttenplan, J . B., and Chung, F.-L.
(1998) 1,N2-Propanodeoxyguanosine adducts: Potential new biom-
arkers of smoking-induced DNA damage in human oral tissue.
Cancer Res. 58, 581-584.
(7) Tricker, A. R., Stickler, D. J ., Chawla, J . C., and Preussmann, R.
(1991) Increased urinary nitrosamine excretion in paraplegic
patients. Carcinogenesis 12, 943-946.
(8) Mostafa, M. H., Helmi, S., Badawi, A. F., Tricker, A. R., Spiegel-
halder, B., and Preussmann, R. (1994) Nitrate, nitrite and volatile
N-nitroso compounds in the urine of Schistosoma haematobium
and Schistosoma mansoni infected patients. Carcinogenesis 15,
619-625.
(9) van Maanen, J . M. S., Welle, I. J ., Hageman, G., Dallinga, J . W.,
Mertens, P. L. J . M., and Kleinjans, J . C. S. (1996) Nitrate
contamination of drinking water: relationship with HPRT variant
frequency in lymphocyte DNA and urinary excretion of N-
nitrosamines. Environ. Health Perspect. 104, 522-528.
(10) Chung, F.-L., Tanaka, T., and Hecht, S. S. (1986) Induction of
liver tumors in F344 rats by crotonaldehyde. Cancer Res. 46,
1285-1289.
(11) International Agency for Research on Cancer (IARC) (1995) IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans,
Vol. 63, pp 373-391, IARC, Lyon, France.
(12) Wu, H.-Y., and Lin, J .-K. (1995) Determination of aldehydic lipid
peroxidation products with dabsylhydrazine by high-performance
lipid chromatography. Anal. Chem. 67, 1603-1612.
(13) Wang, M., Chung, F.-L., and Hecht, S. S. (1988) Identification of
crotonaldehyde as a hepatic microsomal metabolite formed by
R-hydroxylation of the carcinogen N-nitrosopyrrolidine. Chem.
Res. Toxicol. 1, 28-31.
(36) Asami, S., Manabe, H., Miyake, J ., Tsurudome, Y., Hirano, T.,
Yamaguchi, R., Itoh, H., and Kasai, H. (1997) Cigarette smoking
induces an increase in oxidative DNA damage, 8-hydroxydeox-
yguanosine, in a central site of the human lung. Carcinogenesis
18, 1763-1766.
(37) Eder, E., and Hoffman, C. (1992) Identification and characteriza-
tion of deoxyguanosine-crotonaldehyde adducts. Formation of
cyclic adducts and 1,N2,7,8 bis-cyclic adducts. Chem. Res. Toxicol.
5, 802-808.
(14) Young-Sciame, R., Wang, M., Chung, F.-L., and Hecht, S. S. (1995)
Reactions of R-acetoxy-N-nitrosopyrrolidine and R-acetoxy-N-
nitrosopiperidine with deoxyguanosine: formation of N2-tetrahy-
drofuranyl or N2-tetrahydropyranyl adducts. Chem. Res. Toxicol.
8, 607-616.
(15) Wang, M., Young-Sciame, R., Chung, F.-L., and Hecht, S. S. (1995)
Formation of N2-tetrahydrofuranyl and N2-tetrahydropyranyl
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