In vitro DNA deamination by α-nitrosaminoaldehydes determined by GC/MS- SIM quantitation

Article abstract of DOI:10.1021/tx990126d

The deamination of DNA bases by three α-nitrosaminoaldehydes, butylethanalnitrosamine, methylethanalnitrosamine, and N-nitroso-2- hydroxymorpholine (NHMOR), the direct metabolite of potent animal carcinogen N-nitrosodiethanolamine, was demonstrated by a set of in vitro experiments. The deamination of guanine, adenine, and cytosine bases in nucleotides, oligonucleotides, and calf thymus DNA gave xanthine, hypoxanthine, and uracil, respectively. The order of relative reactivities of the bases was as listed above. Deamination of cytosine to uracil was detected by the reaction of ³²P-labeled oligonucleotide ([5'-³²P]CGAT) followed by enzymatic hydrolysis. Quantitative analysis of deamination of guanine and adenine in calf thymus DNA was performed by a gas chromatography/mass spectrometry- selected ion monitoring method. Both the extent and the rate of the deamination reactions which occur by transnitrosation from the α- nitrosaminoaldehyde to the base were determined for formation of xanthine and hypoxanthine. The deamination of guanine by NHMOR remained significant at low substrate levels.

Full text of DOI:10.1021/tx990126d

72
Chem. Res. Toxicol. 2000, 13, 72-81
In Vitr o DNA Dea m in a tion by r-Nitr osa m in oa ld eh yd es
Deter m in ed by GC/MS-SIM Qu a n tita tion
Misun Park and Richard N. Loeppky*
Department of Chemistry, University of Missouri, Columbia, Missouri 65211
Received J uly 9, 1999
The deamination of DNA bases by three R-nitrosaminoaldehydes, butylethanalnitrosamine,
methylethanalnitrosamine, and N-nitroso-2-hydroxymorpholine (NHMOR), the direct metabo-
lite of potent animal carcinogen N-nitrosodiethanolamine, was demonstrated by a set of in
vitro experiments. The deamination of guanine, adenine, and cytosine bases in nucleotides,
oligonucleotides, and calf thymus DNA gave xanthine, hypoxanthine, and uracil, respectively.
The order of relative reactivities of the bases was as listed above. Deamination of cytosine to
uracil was detected by the reaction of 32P-labeled oligonucleotide ([5′-32P]CGAT) followed by
enzymatic hydrolysis. Quantitative analysis of deamination of guanine and adenine in calf
thymus DNA was performed by a gas chromatography/mass spectrometry-selected ion
monitoring method. Both the extent and the rate of the deamination reactions which occur by
transnitrosation from the R-nitrosaminoaldehyde to the base were determined for formation
of xanthine and hypoxanthine. The deamination of guanine by NHMOR remained significant
at low substrate levels.
Sch em e 1
In tr od u ction
N-Nitrosodiethanolamine 1 (NDELA),1 a potent liver
carcinogen in experimental animals, is one of the most
prevalent and abundant of the environmentally occurring
nitrosamines (1, 2) because it can be readily formed by
the nitrosation of ubiquitous compounds such as di- or
triethanolamine and their derivatives. It is a common
trace contaminant in cosmetics, personal care items,
tobacco products, metalworking fluids, and pesticides
(3-5). The chemistry and mode of biochemical activation
of this compound have been the subject of considerable
investigation, yet while becoming clearer, an understand-
ing of the initial events in its carcinogenic activation is
lacking (6, 7). Nitrosamines are known to require meta-
bolic activation to exert their carcinogenic effect (8). While
the mode of biotransformation has not been elucidated
for all nitrosamines, many of them are activated through
the R-hydroxylation process (8-10). The biochemistry of
enzymatic R-hydroxylation mediated by various sub-
groups of cytochrome P450 has been studied extensively
and is reasonably well-understood (10). However, only
recently has there been any evidence that the carcino-
genic activation of NDELA and similar â-hydroxynitro-
samines involves R-hydroxylation (6, 7). All prior experi-
mentation has pointed to an activation step involving the
oxidation of the 2-hydroxyethyl group to an aldehyde, a
process known to be mediated by mammalian liver
alcohol dehydrogenase (ADH) as shown in Scheme 1. The
R-nitrosaminoaldehyde generated by the oxidation of
NDELA, N-(2-hydroxyethyl)-N-nitrosoethanal 2 (NHEE),
preferentially exists as a more stable hemiacetal N-ni-
troso-2-hydroxymorpholine 3 (NHMOR), and further
oxidation leads to the formation of N-(2-hydroxyethyl)-
N-nitrosoglycine 4 (NHEG), the isolated urinary metabo-
lite of NDELA (11, 12). It has been shown that â-hydrox-
ynitrosamines are substrates for liver alcohol dehydro-
genase in vitro, being reversibly converted to the corre-
sponding aldehydes (7, 13, 14). The resulting R-nitro-
saminoaldehydes are highly reactive compounds possess-
ing the unique ability of transferring their nitroso groups
to other amines, namely, transnitrosation (12-15), re-
sulting in nitrosamine formation from secondary amines
and diazonium ion formation and thence deamination of
primary amines. This reaction occurs rapidly in organic
solvents and is slower in aqueous buffer to some extent
due to the equilibrium formation of gem-diol by hydration
of the aldehyde group. In organic solvents, the transni-
trosation reaction is often accompanied by the formation
of imines of glyoxal. In aqueous media, both the imines
and their hydrolysis products, primary amines and
glyoxal, are found (13, 14), but it is not yet known if or
how these processes are linked. As shown in Scheme 2
for adenine 6, transnitrosation from the R-nitrosaminoal-
dehyde to one of the primary amino groups in DNA bases
is expected to result in deamination (15) through the
formation of diazonium ion 8, its hydrolysis to 9, and the
generation of a carbonyl 10 through tautomerism.
* Corresponding author. E-mail: LoeppkyR@missouri.edu.
1
Abbreviations: NDELA, N-nitrosodiethanolamine; NHEE, N-
nitrosohydroxyethylethanalnitrosamine; NHMOR, N-nitroso-2-hy-
droxymorpholine; NHEG, N-nitroso(2-hydroxyethyl)glycine; BuNE,
butylethanalnitrosamine; MeNE, methylethanlanitrosamine; ADH,
alcohol dehydrogenase; gG, N1-2-NH2-glyoxal-guanine adduct; SIM,
selective ion monitoring; T4 PNK, T4 polynucleotide kinase; SVP,
snake venom phosphodiesterase; DMP, Dess-Martin periodinane; TeC,
5′-CGAT DNA oligotetramer; TeA, 5′-ATCG DNA oligotetramer; TeG,
5′-GTAC DNA oligotetramer; MSTFA, N-methyl-N-(trimethylsilyl)-
trifluoroacetamide; dUMP, deoxyuridine phosphate; dIMP, deoxyi-
nosine phosphate.
10.1021/tx990126d CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/28/1999

DNA Deamination by R-Nitrosaminoaldehydes
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 73
otide kinase (T4 PNK) was obtained from United States
Biochemicals (Cleveland, OH), and SVP (snake venom phos-
phodiesterase) was from Worthington Biochemical Co. (Free-
hold, NJ ).
Sch em e 2
1H and 13C NMR spectra were recorded on either a Bruker
AMX-500 (500 MHz for 1H and 125.8 MHz for 13C) or a Bruker
ARX-250 (250 MHz for 1H and 62.9 MHz for 13C) spectrometer.
HPLC was performed with a Waters instrument consisting of
a model 510 solvent delivery system, a model 710B WISP
autosampler, a model 490 multiwavelength UV detector, and
HPLC control software of Maxima 820 or Millenium. An A-200
Flo-one/beta radioactive flow detector (Radiomatic Instruments
& Chemical Co., Tampa, FL) was coupled to the HPLC system
for radiochromatography. A programmable FC 203 Gilson
fraction collector (Gilson Medical Electronics, Inc., Middleton,
WI) was utilized for collecting eluted samples from the HPLC
system. Gas chromatography/mass spectrometry (GC/MS) analy-
ses were performed on a Hewlett-Packard 5890 gas chromato-
graph equipped with a Hewlett-Packard 5970 Series mass
selective detector at an ionization voltage of 70 eV and controlled
with Hewlett-Packard 59970 Chemstation software.
A number of the chemical and biological properties of
NHMOR have been investigated after it was recognized
as a probable important intermediate in the activation
of NDELA (11, 12, 15). Chung and Hecht showed that
deoxyguanosine reacts with NHMOR to form the cyclic
1,N2-glyoxal-deoxyguanosine 11 (gG) adduct (16).
Ca u tion : Nitrosamines are potent chemical carcinogens and
should be handled with extreme caution. A solution of anhydrous
HBr in glacial acetic acid (10%) was used to decontaminate
glassware and other washable items exposed to nitrosamines.
Syn th esis of r-Nitr osa m in oa ld eh yd es. Dess-Martin pe-
riodinane (DMP) was prepared as described in the literature
(21). A dried 50 mL flask equipped with a magnetic stir bar, a
septum, and a nitrogen balloon was charged with the starting
nitrosamino alcohol (2.24 mmol, 1 equiv) (NDELA, methyletha-
nolnitrosamine, or butylethanolnitrosamine) and dry CH2Cl2 (10
mL) to give a 0.22 M solution. DMP (1.05 g, 2.47 mmol, 1.1
equiv) dissolved in dry CH2Cl2 (10 mL) was added dropwise via
a syringe. The reaction mixture was allowed to stir at room
temperature for 1 h. The reaction was monitored by TLC. The
white precipitate was removed by filtration, and the filtrate was
diluted with diethyl ether (20 mL). A solution of Na2S2O3 (2.73
g, 17.29 mmol, 7 equiv of DMP) in 20 mL of saturated NaHCO3
was added, and the mixture was stirred for 10 min. The organic
layer was separated, and the aqueous layer was extracted with
ethyl acetate (3 × 20 mL). The combined organic layers were
concentrated on a rotary evaporator to give the desired product
which was purified by flash chromatography. The spectral data
of the resulting R-nitrosaminoaldehydes 5, N-nitroso-2-hydroxy-
morpholine (NHMOR, 3), N-methylnitrosaminoethanal (MeNE),
or N-butylnitrosaminoethanal (BuNE), were in a good agree-
ment with the literature (13).
As mentioned above, because NHMOR is a “masked”
R-nitrosaminoaldehyde, it could deaminate the primary
amino groups in DNA. The deamination process would
change base-pairing characteristics or result in cross-
linking, which could cause critical damage in DNA
without leaving carbon fragments in the DNA backbone.
The delineation of the role that NHMOR plays in NDELA
activation requires that its ability to deaminate the NH2-
containing bases in DNA be defined since the level of
radio-carbon incorporation from NDELA into DNA has
been reported to be very low (17, 18). We have previously
shown the deamination of guanosine by NHMOR and
N-butylnitrosaminoethanal 5 (R ) nBu) (BuNE), as well
as the formation of glyoxal-guanosine adducts 11 (14,
19, 20).
In this paper, we demonstrate the deamination of DNA
bases in oligonucleotides and calf thymus DNA by
NHMOR and other R-nitrosaminoaldehydes. Quantita-
tive analysis of deaminated bases performed by the GC/
MS-selective ion monitoring (SIM) method revealed that
these nitrosamines deaminate guanine, adenine, and
cytosine and that the deamination of guanine by NHMOR
can be considered significant at the level of human
environmental exposure to NDELA, its metabolic pre-
cursor.
Rea ction of DNA Oligotetr a m er s w ith r-Nitr osa m in oa l-
d eh yd es. Stock solutions (2.0 M) of NHMOR, MeNE, and BuNE
were prepared by dissolving the R-nitrosaminoaldehydes (26.4,
28.0, and 32.2 mg, respectively, 200 µmol each) in dimethylfor-
mamide (DMF, 100 µL). The 5′-GTAC tetramer (TeG, 990 µg,
0.79 µmol) was dissolved in sterilized water (100 µL) to give
7.9 nmol/µL of stock solution. The TeG solution (20 µL, 160
nmol) was mixed with the NHMOR or BuNE stock solution (6
µL, 12 µmol) in 100 µL of borate (0.15 M, pH 9.0) or phosphate
(50 mM KH2PO4, pH 7.4) buffer. The mixture was incubated at
50 or 37 °C for 24 h and then hydrolyzed with diluted HCl (1.0
M, 100 µL) at 70 °C for 1 h. The hydrolysate was concentrated
to dryness on a speedvac at room temperature. The residue was
extracted with CHCl3 (4 × 200 µL) to remove excess nitro-
samines and injected into the HPLC system with a µBondapak
C-18 column (10 µm, 30 cm × 3.9 mm, Waters) using a gradient
program (I) as follows: solvent A, 0.1 M NaH2PO4 (pH 5.5);
solvent B, methanol; 100% A from 0 to 5 min, gradient to 8% B
over the course of 20 min and then to 30% B over the course of
5 min; flow rate, 1.2 mL/min.
Ma ter ia ls a n d Meth od s
Ch em ica ls a n d In str u m en ts. Chemicals were purchased
from Aldrich Chemical Co. (Milwaukee, WI) or other commercial
sources and were purified by conventional procedures, if neces-
sary. DNA oligotetramers were prepared with an automatic
DNA synthesizer by L. J . Forrest of the Department of Micro-
biology, University of Missouri. Calf thymus DNA, DNA mono-
nucleotides, DNA bases, and other related biochemicals were
purchased from Sigma Chemical Co. (St. Louis, MO). [1,3-15N2]-
Xanthine was kindly provided by J . Wishnok and S. R. Tan-
nenbaum of the Department of Chemistry, Massachusetts
Institute of Technology (Cambridge, MA). [γ-32P]ATP (3000 Ci/
mmol) was purchased from Dupont (Boston, MA). T4 polynucle-
P r ep a r a tion of 32P -La beled Oligon u cleotid es. To a 1.5
mL microcentrifuge tube containing 10× phosphorylation buffer
[0.5 M Tris-HCl (pH 8.0)/0.1 M MgCl2 and 0.015 M spermidine,
20 µL] was added the DNA oligotetramer solution (20 pmol/µL,
2 µL, 40 pmol), 5′-CGAT (TeC), 5′-ATCG (TeA), or 5′-GTAC

74 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Park and Loeppky
(TeG), followed by 3000 Ci/mmol [γ-32P]ATP (6 µL, 60 µCi, 20
pmol). T4 PNK (30 units/µL) was diluted to a concentration of
3 units/µL with Tris-HCl buffer (50 mM, pH 8.0) immediately
prior to use. The diluted T4 PNK solution (6 µL, 18 units) was
added to the reaction tube, and the final volume was adjusted
to 100 µL with autoclaved water. The mixture was incubated
at 37 °C for 30 min. The labeling reaction was terminated by
adding EDTA solution (0.5 M, 1 µL) followed by heating at 65
°C for 5 min. The labeled tetramer was purified by HPLC
employing a Radiomatic detector. A Zorbax ODS column (4.6
mm × 25 cm) was used, and the following gradient program
(II) was employed: solvent A, 50 mM KH2PO4 (pH 5.2); solvent
B, methanol; gradient from 15 to 20% B over the course of 15
min, then to 30% B over the course of 10 min, and 30% B for 2
min; flow rate, 1.4 mL/min. The 32P-labeled tetramer peak was
collected on a fraction collector and then loaded on a C-18 Sep-
Pak cartridge (Waters) for desalting. The cartridge was washed
with distilled water (10 mL) to remove buffer salts, and the
labeled tetramer was eluted with 75% acetonitrile in water (4
mL). The resulting tetramer solution was divided into six
microcentrifuge tubes and concentrated to dryness on a speedvac
to give about 2 pmol of 32P-labeled tetramer in each tube.
dissolved in dry acetonitrile (100 µL), and then dry pyridine (20
µL) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA,
80 µL, 0.37 mmol) were added. The reaction vial was tightly
sealed and heated at 100 °C for 1 h. The sample (1 µL) was
analyzed by GC/MS under the following conditions. A fused-
silica capillary SPB-1 column, 30 m × 0.25 mm (Supelco), was
used. The injector and transfer lines were kept at 280 °C. The
temperature program was as follows; the initial temperature
was 150 °C with an initial time of 2 min, and the temperature
was increased to 220 °C at a rate of 10 °C/min, and then to 280
°C at a rate of 25 °C/min with a final time of 5 min. The samples
were injected into the GC/MS apparatus with a low split rate.
The base peaks of M-15 of the derivatized bases were observed:
m/z 264 for di-TMS-adenine, m/z 352 for tri-TMS-guanine, m/z
265 for di-TMS-hypoxanthine, m/z 353 for tri-TMS-xanthine,
and m/z 355 for tri-TMS-[15N2]xanthine. A mixture of standards
was derivatized, and only the base peaks were monitored in the
SIM mode which provides a more accurate measurement of the
abundance of ions of interest. Dwell times were 20 ms per ion.
The retention times on the GC/MS apparatus were 8.2 min for
hypoxanthine, 8.9 min for adenine, 10.3 min for xanthine and
[
15N2]xanthine, and 11.1 min for guanine.
Rea ction of th e 32P -La beled Tetr a m er w ith HNO2. The
32P-labeled tetramer (about 2 pmol) in a 1.5 mL microcentrifuge
tube was mixed with NaNO2 solution (5.0 M, 20 µL) and KH2-
PO4 buffer (50 mM, pH 3.0, 200 µL). The reaction mixture was
shaken at 50 °C for 24 h, and then concentrated to dryness on
a speedvac. The residue was dissolved in SVP buffer (100 µL).
A SVP stock solution (1.52 units/µL) was prepared by dissolving
the enzyme (152 units) in SVP buffer [Tris-HCl buffer (50 mM,
pH 8.8) containing 3 mM MgCl2 (90 µL)] and BSA solution
(bovine serum albumin, 10 µg/µL, 10 µL). The stock solution
was stored at -28 °C. Total enzymatic hydrolysis was performed
by adding SVP stock solution (2 µL, 3 units) followed by shaking
at room temperature for 20 h. Partial hydrolysis was carried
out for comparison with the following procedure. The 32P-labeled
tetramer (about 2 pmol) was dissolved in SVP buffer (100 µL)
and mixed with SVP stock solution (0.4 µL) for 5 s at room
temperature. The reaction was stopped by adding 0.1 M EDTA
(3 µL) followed by heating at 65 °C for 1 min. The hydrolysates
were analyzed by HPLC with a Zorbax ODS column employing
the following gradient program (III): solvent A, 50 mM KH2-
PO4 (pH 5.2); solvent B, acetonitrile; gradient from 2 to 30% B
over the course of 30 min and 30% B for 10 min; flow rate, 1.2
mL/min.
Rea ction of th e 32P -La beled Tetr a m er w ith r-Nitr o-
sa m in oa ld eh yd es. The 32P-labeled tetramer in a 1.5 mL
microcentrifuge tube was mixed with R-nitrosaminoaldehyde
stock solution (2.0 M, 5 µL) and borate buffer (0.15 M, pH 9.0,
100 µL). The reaction mixture was shaken at 50 °C for 24 h
and then extracted with CHCl3 (4 × 200 µL) to remove
unreacted nitrosamines. The mixture was concentrated on a
speedvac to dryness, and the resulting residue was dissolved
in SVP buffer (100 µL) and hydrolyzed with SVP (2 µL, 3 units)
at room temperature for 20 h. Control experiments were
performed without nitrosamine. The hydrolysate was analyzed
by HPLC using gradient program III.
Calf thymus DNA (2.5 mg) was dissolved in KH2PO4 buffer
(50 mM, pH 7.4, 0.5 mL) and the solution mixed with R-nitro-
saminoaldehydes (20 mg in 10 µL of DMF). A control DNA
sample was prepared with buffer solution and DMF. The
mixture was shaken at 50 °C for 48 h. A part of the reaction
mixture (100 µL) was taken and dried on a speedvac. This step
differs for the method of Nguyen et al. (22), who utilized ethanol
to precipitate the DNA. Control experiments consistently showed
that smaller amounts of xanthine were obtained when ethanol
precipitation was used compared to the direct speedvac drying
of the sample. The residue was extracted with CHCl3 (4 × 200
µL) and then dissolved in diluted HCl (1.0 M, 100 µL). The
mixture was hydrolyzed at 70 °C for 1 h, and the hydrolysate
was directly injected into the HPLC system with a Synchropak
RP-P-100 column, 4.6 mm × 25 cm (Alltech), using the following
gradient program (IV): solvent A, 0.1 M ammonium acetate (pH
5.2); solvent B, methanol; 100% A from 0 to 15 min, gradient to
30% B over the course of 5 min, and 30% B for 5 min; flow rate,
1.0 mL/min. The eluted fractions corresponding to adenine,
guanine, hypoxanthine, and xanthine were collected on
a
fraction collector. The combined fractions were neutralized to
pH 7 with ammonium hydroxide and concentrated to a volume
of 150-200 µL on a cold trap (dry ice/acetone) rotary evaporator
in a vacuum. The resulting liquid was transferred to a conical
glass vial and dried on a speedvac at 50 °C. The residue was
dissolved in dry acetonitrile (100 µL) and dry pyridine (20 µL),
and then derivatized with MSTFA (80 µL, 0.37 mmol) by heating
at 100 °C for 1 h. The derivatized sample (1 µL) was injected
into the GC/MS system under the same conditions as described
above. Base peaks at M-15, m/z 264 for di-TMS-adenine, m/z
265 for di-TMS-hypoxanthine, m/z 352 for tri-TMS-guanine, and
m/z 353 for tri-TMS-xanthine, were monitored. Dwell times were
20 ms per ion.
Qu a n tita tion of Dea m in a tion by GC/MS-SIM (22). DNA
samples and controls were prepared as described above with
an R-nitrosaminoaldehyde concentration ratio of 64 µmol/mg of
DNA. After acidic hydrolysis, the hydrolysate was combined
with a [15N2]xanthine standard solution (2 µL, 30.3 nmol) and
injected onto the HPLC system (program IV). The modified and
unmodified base fractions were collected, neutralized, and dried
for derivatization. The dried sample was dissolved in dry
acetonitrile (60 µL) and dry pyridine (40 µL), and then deriva-
tized with MSTFA (100 µL, 0.46 mmol) at 100 °C for 1 h. The
derivatized samples were analyzed by the GC/MS-selected ion
monitoring (SIM) process. Base peaks at M-15 were monitored
with a dwell time of 20 ms per ion. The quantities of modified
and unmodified bases were calculated by comparing the abun-
dances of M-15 ions of the derivatized bases with that of the
internal standard (m/z 355 for tri-TMS-[15N2]xanthine). The
method was validated by using a standard curve and by
Dea m in a tion in Ca lf Th ym u s DNA Deter m in ed by GC/
MS-SIM (22). Although the analytical methodology used for the
accurate measurement of DNA deamination, except for note
variations, is essentially the same as that published by Nguyen
et al., the proper analytical conditions were carefully worked
out in our laboratory by the optimization of each step with
respect to precision, reproducibility, and accuracy through the
use of standards. The standard solutions of DNA bases and
modified bases were prepared by dissolving adenine, guanine,
hypoxanthine, and xanthine (2.8, 3.1, 2.8, and 3.1 mg, respec-
tively, 20 µmol) in DMSO (600 µL). The standard solution of
[
15N2]xanthine was prepared by dissolving the sample (1.4 mg,
9.1 µmol) in DMSO (600 µL) to give a concentration of 15.15
nmol/µL. The standard solution (10 µL) was added into a conical
glass vial and dried on a speedvac at 50 °C. The residue was

DNA Deamination by R-Nitrosaminoaldehydes
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 75
determining the ratio of bases (unmodified) in the DNA control
sample in comparison with that reported in the literature (23),
and excellent agreement was observed.
Sch em e 3
A calf thymus DNA solution (5.0 mg/mL) was prepared in
KH2PO4 buffer (50 mM, pH 7.4) and sonicated for 2 min to break
the DNA into small pieces. Stock solutions (16.0 M) of R-nitro-
saminoaldehydes were prepared by dissolving NHMOR, BuNE,
and MeNE (0.21, 0.23, and 0.16 g, respectively, 1.6 mmol) in
DMF (100 µL).
(1) Tim e Dep en d en ce. The nitrosamine stock solution (2
µL, 32 µmol) was mixed with a DNA solution (100 µL, 0.5 mg)
and shaken at 37 °C for the desired length of time (1, 2, 3, 4, 6,
8, 10, 12, 24, and 48 h). Control experiments were performed
via 4, 8, 12, 24, and 48 h incubations of DNA solutions without
nitrosamine.
(2) Con cen tr a tion Dep en d en ce. Various amounts of the
respective nitrosamine stock solutions (2, 4, 6, and 8 µL) were
mixed with a DNA solution (500 µL) and incubated for 24 h at
37 °C.
(3) Gu a n in e Dea m in a tion by NHMOR a t Low er Con -
cen tr a tion s. Reaction mixtures were prepared with same
procedure described above except for the use of a diluted
NHMOR solution (1.0 or 0.1 M) to make the concentration ratios
of nitrosamine 0.1, 0.2, 0.4, 0.6, 1.0, and 2.0 µmol/mg DNA,
respectively. The mixtures were incubated for 4 h at 37 °C.
The reaction mixtures were dried on a speedvac and then
extracted with CHCl3 (5 × 200 µL). The residues were hydro-
lyzed at 70 °C with dilute HCl (2.0 M, 100 µL) for 1 h. The
hydrolysates were combined with the [15N2]xanthine standard
solution (2 µL, 30.3 nmol) and injected into the the HPLC system
using elution program IV. The fractions containing xanthine and
hypoxanthine were collected and derivatized according to the
procedure described above. The derivatized samples were
analyzed with the GC/MS-SIM method.
LA (R ) CH2CH2OH), methylethanol nitrosamine (R )
CH3), and butylethanolnitrosamine (R ) nBu), were
prepared by the nitrosation of the corresponding amines
12 with sodium nitrite and glacial acetic acid in high
yield. The oxidation of nitrosamino alcohols by DMP was
carried out at room temperature in 0.02 M acetic acid.
The reaction was complete within 1 h, providing quan-
titative conversion in all cases as monitored by TLC. In
the case of NDELA, the formation of dialdehyde as a side
product is considered to be prevented under acidic
conditions by facilitating the ring closure of the resulting
monoaldehyde to its hemiacetal, NHMOR 3. A neutral
workup procedure using Na2S2O3 was employed to pre-
vent the decomposition of base-sensitive R-nitrosami-
noaldehydes. The yields of the R-nitrosaminoaldehydes
after chromatographic purification are given in Scheme
3. The application of Dess-Martin oxidation to the
synthesis of R-nitrosaminoaldehydes provides great ad-
vantages. The procedure is simplified, involving only
nitrosation of amino alcohols and oxidation of the result-
ing nitrosamino alcohols to aldehydes. The overall yields
are about 85% from the starting amine, which is a great
improvement especially for highly water-soluble products,
NHMOR and MeNE 5 (R ) CH3). These compounds have
been of great interest in the study of nitrosamine car-
cinogenesis, and a large number of investigations are still
underway. With this new method, the synthesis of
R-nitrosaminoaldehydes has become more convenient and
efficient.
Ch em istr y of Dea m in a tion by r-Nitr osa m in oa l-
d eh yd es. The prior research and model studies on the
properties of R-nitrosaminoaldehydes, and, in particular,
their reactions with guanosine and deoxyguanosine,
clearly lead to the expectation that these reactive N-
nitroso compounds should react with DNA. The two
expected transformations are deamination and glyoxal-
guanine (gG) adduct formation, but the reactions of
NHMOR and other R-nitrosaminoaldehydes with DNA
have not been reported. The focus of this work, therefore,
was to determine whether these reactive aldehydes,
NHMOR in particular, can deaminate DNA bases through
transnitrosation.
Resu lts a n d Discu ssion
Syn th esis of r-Nitr osa m in oa ld eh yd es by Dess-
Ma r tin Oxid a tion . R-Nitrosaminoaldehydes have been
commonly synthesized from the corresponding amine and
R-chloroaldehyde acetal to give aminoacetal followed by
nitrosation with isopropyl nitrite (13). The yield of the
aminoacetal in the first step varies from 42 to 58%. In
this synthetic procedure, isopropyl nitrite was used to
nitrosate the aminoacetals, but the yields of the nitro-
sation range from 65 to 88%, which often requires a long
reaction time, more than 48 h, to obtain a reasonable
amount of the product. The resulting nitrosaminoacetals
were treated with 6 M HCl to generate the corresponding
nitrosaminoaldehydes which were extracted after neu-
tralization with a saturated NaHCO3 solution. The yields
of this final step were about 75% for relatively nonpolar
aldehydes possessing bulky alkyl groups, but were only
about 30% for highly water-soluble compounds such as
N-methylnitrosaminoethanal 5 (R ) CH3) (MeNE) and
NHMOR. In an attempt to overcome difficulties such as
the low overall yield and time-consuming procedure, a
direct oxidation of nitrosamino alcohols to their corre-
sponding aldehydes was taken into consideration. The
oxidation of alcohols to aldehydes or ketones has been
carried out with a variety of methods, and one of the most
effective and convenient methods is the Dess-Martin
reaction which uses an organo-iodine compound (21, 24).
The synthesis of the oxidizing agent, Dess-Martin pe-
riodinane (DMP), was carried out according to the known
procedure (21) with 87% overall yield from the starting
iodobenzoic acid. Application of this effective yet mild
oxidation method to the preparation of R-nitrosaminoal-
dehydes reduces the synthesis procedure to two steps
(Scheme 3). The starting nitrosamino alcohols 13, NDE-
In a preliminary report or our work (19), we presented
evidence which showed that R-nitrosaminoaldehydes
deaminate DNA mononucleotides, dGMP, dAMP, and
dCMP. The mononucleotides were reacted with either
n
NHMOR 3 or BuNE (5, R ) Bu) in buffer (pH 7.4 or 9)
and a small amount of DMF to facilitate dissolution of
the mixture. After reaction for 48 h at 50 °C, the bases
were released by heating in acid and subjected to HPLC.
Appropriate controls were performed to test for deami-
nation under conditions of hydrolysis. Both NHMOR and
BuNE deaminated dGMP 14 and dAMP 6 to give xan-
thine 10 and hypoxanthine 16, respectively. The forma-
tion of glyoxal-guanine (gG) adduct 11 from dGMP was
also observed in high yield (see Scheme 4). In all cases,
BuNE produced more deamination than NHMOR. How-

76 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Park and Loeppky
Sch em e 4
enzymatic hydrolysis. Many of these problems can be
overcome by the use of 5′-32P-labeled oligotetramers
which employ a different base at the 5′-end of each
oligomer, and total enzymatic hydrolysis of the 3′-
phosphodiester bonds is performed (26).
Use of 5′-32P -La beled DNA Oligotetr a m er s. The
tetramers 5′-CGAT (TeC), 5′-ATCG (TeA), and 5′-GTAC
(TeG) were labeled with [γ-32P]ATP according to the
standard procedure and purified by HPLC. These labeled
tetramers were reacted with R-nitrosaminoaldehydes,
NHMOR, BuNE, and MeNE, in borate buffer (pH 9) for
24 h at 50 °C or with HNO2 at pH 3.5 for 2 h (NO2- does
not react with DNA under neutral or basic conditions).
Total enzymatic hydrolysis followed by HPLC coupled
using a radioflow detector permits the detection of
modifications on the 5′-base. The chromatograms of the
nucleotides from the reactions were compared with
standards, and those from reactions with nitrous acid as
well as the hydrolysate of the unreacted tetramer (con-
trol). In each case (data not shown), a partial enzymatic
hydrolysis was performed as a control to ensure that
peaks seen in the total hydrolyses were not due to
oligomers of various sizes.
The radiochromatograms for the reactions of TeC are
shown in Figure 1. All radiochromatograms of the reac-
tions of TeC with the R-nitrosaminoaldehydes showed the
presence of dUMP, the deamination product of cytosine
(after hydrolysis). It was observed in high yield in the
reaction with nitrous acid. The deamination was not
observed in the control experiment. Among the R-nitro-
saminoaldehydes, BuNE exhibited higher reactivity than
NHMOR and MeNE (data not shown). From the reactions
of TeA with R-nitrosaminoaldehydes, deoxyinosine 5′-
monophosphate (dIMP), the deamination product of
adenine, was also observed (data not shown). The inten-
sity of dIMP peaks was very low, but clearly, no dIMP
was detected in the control experiment. The reaction with
nitrous acid exhibited a much higher yield of dIMP. The
reactions of TeG with R-nitrosaminoaldehydes did not
show any common adducts or modifications in the radio-
chromatogram compared to those observed in the nitrous
acid reaction of TeG. For each R-nitrosaminoaldehyde,
the radiochromatograms exhibited a large peak due to
the gG adduct (comparison with standard) (20) which
eluted before dGMP. The same peak was also observed
in high yield from the reaction of glyoxal with TeG
followed by enzymatic hydrolysis. The failure to observe
[5′-32P]deoxyxanthosine monophosphate, the deamination
product of G in TeG, in this case is attributed to either
cleavage of the nucleosidic linkage under the workup and
enzymatic hydrolysis conditions or, more probably, its
being obscured by the gG adduct peak. With the use of
32P-labeled DNA oligotetramers, it was possible to detect
the deamination of cytosine and adenine which were not
well-characterized by UV detection.
ever, the deamination of dCMP 15 to produce uracil 17
was not well-characterized by this method, which was
due to the difficulty in hydrolyzing the glycosidic bond
of pyrimidine nucleotides. The detection of dUMP, the
deamination product of dCMP, from the reaction mixture
was not successful by UV monitoring. It is known that
the reactivity of cytidine toward deamination by nitro-
sating agents is much lower than that of guanosine and
adenosine (25). To detect the deamination of dCMP, a
more sensitive method was required.
Rea ction of DNA Oligotetr a m er s w ith r-Nitr o-
sa m in oa ld eh yd es. The use of individual mononucle-
otides clearly demonstrated that R-nitrosaminoaldehydes
deaminate DNA bases and form the glyoxal-guanine
adduct (19). These reactions were conducted under the
conditions adjusted to provide the maximum rate of
transnitrosation, which were different from physiological
conditions. Since our interest lies in the carcinogenesis
of the reactive aldehydes in biological systems, it is
important to know whether the chemical transformations
observed in the previous experiments would take place
under conditions mimicking more natural biological
conditions. To test this, R-nitrosaminoaldehydes were
reacted with 5′-GTAC (TeG) under both physiological
conditions (pH 7.4, 37 °C) and the reaction conditions
used previously (pH 9, 50 °C) for comparison. The results
showed no major difference in these two reaction condi-
tions. The chromatogram of the reaction of NHMOR with
TeG shows that xanthine and the glyoxal-guanine
adduct are formed (data not shown). Similar observations
were made for BuNE which appeared to produce more
of the glyoxal-guanine adduct than xanthine. The deam-
ination of adenine could not be observed under the
chromatographic conditions used, since hypoxanthine
overlaps with guanine. Attempts to separate the two
bases within an appropriate pH range (4.5-7.5) for this
analysis were not successful. Since the amount of TeG
used and the yield of the products were very low, the
HPLC/UV chromatograms were unable to provide clear
results. Therefore, 32P-labeled DNA oligotetramers were
used for more sensitive detection. Originally (26), we
attempted to use oligotetramers to detect reactions at
each kind of base by employing 5′-32P-labeled oligotet-
ramers, containing one base of each type, followed by
partial enzymatic hydrolysis after the separation of the
modified oligomers. The initial separations are difficult,
however, when reactive substrates produce a myriad of
modifications (adducts). Moreover, adduction can impede
Qu a n tita tion of th e Dea m in a tion in Ca lf Th ym u s
DNA by GC/MS-SIM Meth od . Studies on the reactions
of R-nitrosaminoaldehydes with DNA have shown that
these reactive aldehydes are capable of deaminating DNA
bases. The possible biological significance of these trans-
formations obviously depends on the relative rate of DNA
reactions compared to those of other processes, particu-
larly those which act to detoxify the R-nitrosaminoalde-
hydes by oxidation to unreactive, readily excreted car-
boxylic acids. To elucidate biological consequences of
deamination in relation with mutagenicity of the R-nit-

DNA Deamination by R-Nitrosaminoaldehydes
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 77
Ta ble 1. Yield s a n d Ra tes of P u r in e Dea m in a tion by Nitr osa m in oa ld eh yd es
xanthinea
hypoxanthineb
e
e
aldehyde
nmol/mgc
% deaminatedd
Vo
nmol/mgc
% deaminatedd
Vo
NHMOR
BuNE
MeNE
64.5 ( 0.8
121.2 ( 3
49.1 ( 3
10.4 ( 0.1
19.6 ( 0.5
5.6 ( 0.4
181 ( 23
125 ( 2
73 ( 4
-
7.9 ( 0.3
22.9 ( 0.4
5.6 ( 0.5
0.9 ( 0.03
2.6 ( 0.05
0.10 ( 0.01
0.012 ( 0.003
5 ( 0.6
30 ( 0.7
7 ( 0.2
-
control
1.4 ( 0.09
0.23 ( 0.01
0.10 ( 0.02
a
b
d
From guanine. From adenine. c Yield in nanomoles per milligram of DNA at 48 h, pH 7.4, and 37 °C. The percentage of deamination
was estimated using the amounts of adenine and guanine in calf thymus DNA reported in the literature (23), which are 891.3 and 620.0
nmol/mg of DNA, respectively. e Picomoles of product per milligram of DNA per minute.
To test the method and determine whether we could
observe sufficient R-nitrosaminoaldehyde-mediated deam-
ination of DNA under our experimental conditions, calf
thymus DNA was reacted with NHMOR, BuNE, or
MeNE (64 µmol/mg of DNA) under physiological condi-
tions (pH 7.4, 37 °C) for 48 h. The reaction mixtures were
prepared for HPLC after extracting the remaining nit-
rosamines with chloroform. The fractions containing
adenine, guanine, hypoxanthine, and xanthine were
collected and dried for derivatization. Adenine and
guanine were collected from the control DNA sample to
test the reliability of the method. The derivatized samples
were analyzed by the GC/MS-SIM method. The relative
abundancies of the base peaks of the deaminated bases
compared to those of unmodified bases were calculated,
which provided the following initial results: hypoxan-
thine/adenine, 0.2 for NHMOR, 0.1 for BuNE, and 0.1
for MeNE; xanthine/guanine, 0.5 for NHMOR, 0.7 for
BuNE, and 0.3 for MeNE. The relative yields of deami-
nated bases from this experiment are in a good ac-
cordance with those we obtained from the deamination
of mononucleotides. The ratio of adenine and guanine in
control calf thymus DNA was observed to be 1:0.7, which
agrees with the published data, 1:0.69 (23).
Encouraged by these results, we carefully optimized
our procedures to accurately determine the extent of
deamination as a function of nitrosamine concentration
and time. Each run involved the utilization of three
samples at each time point or concentration. In every
case, a determination of the purine base ratios in a
standard DNA control sample was made, and we mea-
sured the individual concentrations of the hypoxanthine
and xanthine in the control sample. As might be expected,
no hypoxanthine was observed in the control samples at
long run times, although low levels of xanthine were
observed. This “natural” guanine deamination was very
F igu r e 1. Radiochromatograms from the reactions of [5′-32P]-
CGAT (TeC) with the indicated compound, after enzymatic
hydrolysis. (A) NHMOR; peaks at retention times > 19 min are
partial hydrolysis products. (B) BuNE. (C) HNO2. (D) Control;
5′-32P-CGAT after total hydrolysis.
small compared to that produced by the nitrosamine at
the same time point. By this means, the percent error
as measured by the standard deviation of the means for
all runs was in the range of 0.9-1%. Specific values are
given in Table 1 where we report the initial rates of
deamination and the yield of deamination product after
incubation for 25 h at 37 °C and pH 7.4. The results
provided by this GC/MS-SIM analysis clearly show that
the deamination of bases in DNA, including that of
adenine to form hypoxanthine which was not clearly
visible in double-stranded DNA by the HPLC/UV method,
by R-nitrosaminoaldehydes certainly occurs. The highest
yield of deamination was observed in the reaction with
BuNE. NHMOR was moderately reactive among the
three compounds, and MeNE was the least reactive. A
significant amount of deamination occurred in the reac-
tion with NHMOR, a process which could occur in vivo.
To obtain a better understanding of the biological im-
portance of the deamination reaction induced by R-nit-
rosaminoaldehydes, a detailed quantitative investigation
of R-nitrosaminoaldehyde-induced DNA deamination re-
actions was necessary. The quantitation of xanthine and
hypoxanthine produced by the reaction of nitric oxide
with DNA, RNA, or intact human cells has been reported
by Nguyen and co-workers (22). The analysis was carried
out using the GC/MS-SIM method after derivatizing the
dried residue of xanthine and hypoxanthine collected
from HPLC. Quantitation was carried out by comparing
the abundances of the M-15 ions of the TMS-derivatized
bases with that of the internal standard, [1,3-15N2]-
xanthine. This same analytical method was utilized by
us for the quantitative determination of DNA deamina-
tion by R-nitrosaminoaldehydes.

78 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Park and Loeppky
F igu r e 3. Xanthine formation from the deamination of guanine
in calf thymus DNA by NHMOR at low concentrations after 4
h at 37 °C. The inset shows the regression line for the double-
reciprocal plot.
F igu r e 2. Effect of nitrosamine concentration upon the quan-
tity of xanthine produced from DNA within 24 h under physi-
ological conditions. The plots are for the reciprocals of the
variables as indicated by the axes labels at the left and bottom.
The axes labels at the top and right give the corresponding
concentrations of substrate and xanthine, respectively.
cal of eq 1 gives eq 2, which predicts that a plot of the
reciprocal of the xanthine yield, at any time, versus the
reciprocal of the initial aldehyde concentration should be
linear. In fact, we found double-reciprocal plots of the
yields of deaminated bases are linear as is shown in
Figures 2 and 3. Because we do not know the mechanism
of the deamination transformation at this time, we
cannot know with certainty why the double-reciprocal
plots are linear. There are, however, several reasonable
explanations. For example, if we assume that the forma-
tion of a deaminated base is represented simply by eqs 3
and 4 of Scheme 5, and if it is assumed that changes in
the R-nitrosaminoaldehyde and DNA are negligible with
respect to the relatively low yield of the deaminated base
(e.g., xanthine), conditions which certainly hold in our
work, then it can be shown that eq 1 (and eq 2) holds
where a ) k1k2t, b ) (k-1 + k2), and c ) k1. However,
equations of this type can also be derived when the
substrate enters into another process competitively which
does not produce a deaminated base. This could involve
either the formation of the gG adduct in a transformation
which is independent of the deamination reaction or
another reaction of the nitrosaminoaldehyde with the
DNA, e.g., formation of Schiff base adducts or another
undetermined transformation. While we have previously
shown that glyoxal equivalents are generated in the
transnitrosation reactions of R-nitrosaminoaldehydes
(13-15, 19, 20), we have not shown that the transforma-
tions are actually coupled, although current research in
our laboratory is directed at this point.
To further establish the possible biological relevance
of the deamination reaction, we have examined the
deamination of guanine bases in DNA by NHMOR at
concentrations lower than those utilized above. Prior
work in other laboratories led us to conclude that adenine
and cytosine deamination levels would be very low at real
exposure levels due to the low reactivity (22, 27). Since
guanine is more reactive than the other bases, and
NHMOR is a known metabolite of NDELA, a carcinogen
to which humans are exposed, the deamination of gua-
nine was studied with lower NHMOR concentrations.
Concentration-related increases in the level of xanthine
formation after incubation for 4 h with varying concen-
trations of NHMOR, from 0.1 to 2.0 µmol/mg of DNA,
measured by the GC/MS-SIM method are illustrated in
Figure 3. Here again, a double-reciprocal plot was linear
and the curve in Figure 3 was generated by transforma-
Sch em e 5
rosaminoaldehydes, the extent of deamination as a
function of nitrosamine concentration and time was
determined.
Con cen tr a tion Dep en d en ce of Dea m in a tion . The
dependence of the deamination on the R-nitrosaminoal-
dehyde concentration was studied by varying the amount
of the aldehyde from 12.8 to 64.0 µmol/mg of DNA in 100
µL. The reaction mixtures were incubated for 24 h under
physiological conditions. The results are shown in Figure
2 for the production of xanthine from the deamination
of guanine. Similar data were observed for the deami-
nation of adenine to give hypoxanthine, although the
formation of hypoxanthine was 6-10 times slower than
that of xanthine. While the yield of the deaminated base
at a fixed time point increases with the concentration of
the R-nitrosaminoaldehyde, the increase is not linear over
the concentration range. Rather, a saturation phenom-
enon is observed where an increase in the concentration
of the R-nitrosaminoaldehyde does not produce a propor-
tional increase in the concentration of the deaminated
base. This phenomenon was very reproducible and ob-
served over several concentration ranges. These observa-
tions suggested that the yield of deaminated base at any
time point could be represented by a function such as
that shown for xanthine in eq 1 of Scheme 5, where a, b,
and c are constants. Equations of this type are, for
example, typical for adsorption processes where sub-
strates are reversibly competing for sites, such as those
in DNA where a transformation could occur. The recipro-

DNA Deamination by R-Nitrosaminoaldehydes
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 79
tion. The linear regression analysis of the data, while
exhibiting excellent linearity as measured by R2, does not
give slopes (rate constants) which are of value in deter-
mining relative reactivity, because the slopes of such
plots are a function of X∞. Accordingly, we have cal-
culated initial rates (Vo) from the derivative d[xanthine]/
dt ) -X∞me(mt+b) at time zero, where the derivative was
determined from the equation X ) X∞[1 - e(mt+b)] of the
exponential plot resulting from the regression analysis
(above) where m is the slope of that plot and b is the
intercept. The Vo values for xanthine formation from each
of the nitrosamino aldehydes are given in Table 1 for both
xanthine and hypoxanthine. Because of the lower levels
of hypoxanthine formation, there is greater uncertainty
in these values and at best the Vo values can be regarded
as an indicator of relative reactivity. The data for
xanthine show the reactivity order: NHMOR > BuNE
. MeNE. In the case of hypoxanthine formation, there
is a reversal in reactivity between BuNE and NHMOR,
but this may be due to experimental error. In each case,
the methyl compound is significantly less reactive. As
was seen in the yield data, guanine deaminated more
readily than adenine, and this is consistent with other
observations on the relative rates of nitrosative deami-
nation of DNA bases.
We have considered various mechanisms for the tran-
snitrosation reactions of R-nitrosamino aldehydes (6),
which, in the case considered here, leads to deamination
of DNA bases, but we have at present no definitive data
which permit anything but pure speculation about how
this very interesting transformation occurs. It is impor-
tant to emphasize first that common dialkylnitrosamines
do not enter into transnitrosation transformations except
in very strong acid and second that under the neutral to
basic conditions employed here, nitrite cannot enter into
nitrosation processes which result in deamination. To
deaminate nitrosamine and amines, nitrite must be
converted into nitrous acid or oxides of nitrogen, a process
which occurs only at pH values below 5. We do not yet
know the transnitrosation mechanism of R-nitrosami-
noaldehydes. Some of the N-nitroso groups may be
converted into nitrite at physiological pH through “tran-
snitrosation” to H2O, but it is unlikely that these ions
are involved in the deamination chemistry. It is more
probable that the nitroso group is transferred directly
from the R-nitrosamino aldehyde or some derivative
thereof to one of the purine or pyrimidine NH2 groups of
DNA. The chemistry of R-nitrosaminoaldehydes is highly
unusual in this context. Mechanism studies are under-
way.
F igu r e 4. Time course of guanine deamination in calf thymus
DNA by the three R-nitrosamino aldehydes. The inset shows
the data for NHMOR-mediated deamination in the form of a
plot of ln(1 - X/X∞) vs t (hours).
tion of the linear regression of the data in that form
(R2 ) 0.98). A xanthine yield of 1.8 nmol/mg of DNA was
formed from 0.1 µmol of NHMOR. The deaminated bases
in human cells treated with nitric oxide in vitro have been
detected by GC/MS-SIM quantitation at the level of 10-1
nmol/mg of DNA (22). Extrapolation of our data suggests
that the same level of guanine deamination produced by
NO could be achieved by a concentration ratio of
approximately 10-2 µmol of NHMOR/mg of DNA. These
results suggest that detection of DNA deamination by
NHMOR in biological samples where the level of envi-
ronmental exposure to its metabolic precursor, NDELA,
has been high may be possible by the GC/MS-SIM
method. However, as mentioned earlier, a significant
amount of xanthine depurinates from the DNA backbone
and this unknown quantity is excluded when DNA
samples are prepared by the precipitation method.
Because of this fact, accurate quantitative determination
of xanthine in vivo could only be achieved after establish-
ing appropriate procedures for DNA separation from
biological samples.
Tim e Dep en d en ce of Dea m in a tion . The effect of
incubation time on the deamination reaction was exam-
ined by GC/MS-SIM quantitation. A calf thymus DNA
solution (5.0 mg/mL) was prepared in phosphate buffer
and sonicated to break the DNA into small pieces to make
the solution homogeneous. The DNA solution was reacted
with NHMOR, BuNE, or MeNE under physiological
conditions. The concentration of the R-nitrosaminoalde-
hydes was 64.0 µmol/mg of DNA. The samples taken after
incubation for 1, 2, 3, 4, 6, 8, 10, 12, 24, and 48 h were
analyzed for the amount of xanthine and hypoxanthine.
Control samples were prepared by incubation of DNA
solution without nitrosamine for 4, 8, 12, 24, and 48 h.
Figure 4 shows the transformation time course for
xanthine formation as a result of guanine deamination
by the nitrosaminoaldehydes. It is evident from the
inspection of Figure 4 that the reaction exhibits a
saturation phenomenon. The greatest yield was obtained
from BuNE. Plots of ln(1 - X/X∞) versus t, where X is
the xanthine concentration at time t and X∞ is the
xanthine concentration at 48 h, were linear. This form
of plotting is characteristic of a first-order (or pseudo-
first-order) transformation which does not go to comple-
Through this investigation, it has become evident that
R-nitrosaminoaldehydes, including NHMOR, the metabo-
lite of NDELA, can deaminate DNA bases through
transnitrosation. The transformations occur at concen-
tration levels and on time scales which could be biologi-
cally significant. Deamination does not incorporate car-
bon fragments into the DNA backbone but can clearly
produce mutations. This may explain why NDELA does
not incorporate significant levels of its carbon fragments
into DNA yet exerts strong carcinogenicity. In other work,
we have shown that the in vivo administration of NDELA
to rats results in the formation of gG and O6-(hydroxy-
ethyl)guanine adducts (6, 20, 28).2 The former adducts
2
R. N. Loeppky and P. Goelzer, manuscript to be submitted for
publication.

80 Chem. Res. Toxicol., Vol. 13, No. 2, 2000
Park and Loeppky
Michejda, C. J ., Eds.) pp 20-33, American Chemical Society,
Washington, DC.
are hydrolytically unstable and may not have survived
the analyses used to determine the extent of radiocarbon
incorporation from NDELA into DNA. Our current data
are consistent with bioactivation processes for NDELA
which involve both its R- and â-oxidation (6, 7, 20),
despite the fact that NHMOR has been reported to be
only weakly carcinogenic (29). The latter experiments
were carried out at very low doses, however. Clearly,
NHMOR deaminates the three different types of bases
in DNA. In vitro experiments have demonstrated that
the â-oxidation of NDELA can be catalyzed not only by
ADH but also by microsomes (6).3 Therefore, base deami-
nation by NHMOR, in addition to adduct formation, must
be considered as a possible mechanism of carcinogenesis
for NDELA.
(6) Loeppky, R. N. (1999) The mechanism of bioactivation of N-
nitrosodiethanolamine. Drug Metab. Rev. 31, 175-193.
(7) Loeppky, R. N., Fuchs, A., J anzowski, C., Humberd, C., Goelzer,
P., Schneider, H., and Eisenbrand, G. (1998) Probing the mech-
anism of the carcinogenic activation of N-nitrosodiethanolamine
with deuterium isotope effects: In vivo induction of DNA single
strand breaks and related in vitro assays. Chem. Res. Toxicol.
11, 1556-1566.
(8) 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.
(9) Mochizuki, M., Anjo, T., and Okada, M. (1980) Isolation and
characterization of N-alkyl-N-(hydroxymethyl)nitrosamines from
N-alkyl-N-(hydroperoxymethyl) nitrosamines by deoxygenation.
Tetrahedron Lett. 21, 3693-3696.
(10) Yang, C. S., Yoo, J . S. H., Ishizaki, H., and Hong, J . (1990)
Cytochrome P450IIE1: roles in nitrosamine metabolism and
mechanisms of regulation. Drug Metab. Rev. 22, 147-159.
(11) Airoldi, L., Bonfanti, M., Benfenati, E., Tavecchia, P., and Fanelli,
R. (1983) Identification of an acidic metabolite of N-nitrosodi-
ethanolamine isolated from rat urine. Biomed. Mass Spectrom.
10, 334-337.
(12) Airoldi, L., Bonfanti, M., Fanelli, R., Bove, B., Benfenati, E., and
Gariboldi, P. (1984) Identification of a nitrosamino aldehyde and
a nitrosamino acid resulting from â-oxidation of N-nitrosodietha-
nolamine. Chem.-Biol. Interact. 51, 103-113.
(13) Loeppky, R. N., Tomasik, W., and Kerrick, B. E. (1987) Nitroso
transfer from R-nitrosamino aldehydes: Implications for carcino-
genesis. Carcinogenesis 8, 941-946.
(14) Loeppky, R. N., Tomasik, W., Denkel, E., and Eisenbrand, G.
(1987) R-Nitrosaminoaldehydes: Highly reactive metabolites.
IARC Sci. Publ. 84, 94-99.
(15) Loeppky, R. N., Tomasik, W., Kovacs, D. A., Outram, J . R., and
Byington, K. H. (1984) Alternative bioactivation routes for â-hy-
droxynitrosamines. Biochemical and chemical model studies. In
N-Nitroso Compounds: Occurrence, Biological Effects and Rel-
evance to Human. Cancer (Bartsch, H., O’Neill, I., and Von
Borstel, R. C., Eds.) pp 429-436, International Agency for
Research on Cancer, Lyon, France.
(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) Farrelly, J . G., Stewart, M. L., and Lijinsky, W. (1984) The
metabolism of nitrosodi-n-propylamine, nitrosodiallylamine and
nitrosodiethanolamine. Carcinogenesis 5, 1015-1019.
(18) Farrelly, J . G., Thomas, B. J ., and Lijinsky, W. (1987) Metabolism
and cellular interactions of N-nitrosodiethanolamine. IARC Sci.
Publ. 84, 87-90.
(19) Loeppky, R. N., Lee, M. P., and Mueller, S. (1993) DNA modifica-
tion by R-nitrosamino aldehydes. In The Chemistry of DNA
Adducts (Hemminki, K., and Bartsch, H., Eds.) pp 429-432,
International Agency for Research on Cancer, Lyon, France.
(20) Loeppky, R. N., Cui, W., Goelzer, P., and Ye, Q. (1999) Glyoxal-
guanine DNA adducts: Detection, stability and formation from
nitrosamines. IARC Sci. Publ. 150, 155-168.
(21) Dess, D. B., and Martin, J . C. (1991) A useful 12-I-5 triacetoxy-
periodinane (the Dess-Martin periodinane) for the selective
oxidation of primary or secondary alcohols and a variety of related
12-I-5 species. J . Am. Chem. Soc. 113, 7277-7287.
(22) Nguyen, T., Brusnson, D., Crespi, C. L., Penman, B. W., Wishnok,
J . S., and Tannenbaum, S. R. (1992) DNA damage and mutation
in human cells exposed to nitric oxide in vitro. Proc. Natl. Acad.
Sci. U.S.A. 89, 3030-3034.
(23) Gehrke, C. W., McCune, R. A., Gama-Sousa, M., Erlich, M., and
Kuo, K. (1984) Quantitative reversed-phase high-performance
liquid chromatography of major and modified nucleosides in DNA.
J . Chromatogr. 301, 199-219.
(24) Dess, D. B., and Martin, J . C. (1983) Readily accessible 12-I-5
oxidant for the conversion of primary and secondary alcohols to
aldehydes and ketones. J . Org. Chem. 48, 4255-4256.
(25) Shapiro, R., and Pohl, S. H. (1968) Reaction of ribonucleosides
with nitrous acid. Side products and kinetics. Biochemistry 7,
448-455.
Su m m a r y a n d Con clu sion s
We have developed a convenient and simple method
for the production of the reactive R-nitrosaminoaldehydes
by the Dess-Martin oxidation of the corresponding
alcohol. The deamination of the NH2-containing bases in
nucleotides and DNA through the transnitrosation reac-
tion from three different R-nitrosaminoaldehydes has
been demonstrated. The methodology has utilized 32P-
labeled nucleotides and quantitative analysis by means
of the GC/MS-SIM method, employing a 15N-labeled
xanthine. As expected, the extent of deamination depends
on the structure and concentration of the R-nitrosami-
noaldehyde, which are time dependent. The order of base
deamination rates is consistent with other observations:
guanine > adenine . cytosine. High concentrations of
NHMOR deaminate guanine and adenine in calf thymus
DNA up to 10.4 and 7.9%, respectively. Lower concentra-
tions of NHMOR, closer to those generated in the
metabolism of NDELA, to which humans are exposed,
produced a significant amount of deamination in a short
period of time. These results support a role for NHMOR-
mediated base deamination in the mechanism of carci-
nogenicity of NDELA.
Ack n ow led gm en t. The support of this research by
a grant (ES03953) from the National Institutes of Envi-
ronmental Health Sciences is gratefully acknowledged.
We also express our profound appreciation to the re-
search group of Prof. Steven Tannenbaum for the sample
of the 15N-labeled xanthine standard and discussion of
the related analytical methodology.
Refer en ces
(1) Preussmann, R., Habs, M., Habs, H., and Schmaehl, D. (1982)
Carcinogenicity of N-nitrosodiethanolamine in rats at five differ-
ent dose levels. Cancer Res. 42, 5167-5171.
(2) Lijinsky, W., Reuber, M. D., and Manning, W. B. (1980) Potent
carcinogenicity of nitrosodiethanolamine in rats. Nature 288,
589-590.
(3) Fan, T. Y., Morrison, J ., Rounbehler, D. P., Ross, R., Fine, D. H.,
Miles, W., and Sen, N. P. (1977) N-Nitrosodiethanolamine in
synthetic cutting fluids: a part-per-hundred impurity. Science
196, 70-71.
(4) Fan, T. Y., Goff, U., Song, L., Fine, D. H., Arsenault, G. P., and
Biemann, K. (1977) N-Nitrosodiethanolamine in cosmetics, lotions
and shampoos. Food Cosmet. Toxicol. 15, 423-430.
(5) Havery, D. C., and Chou, H. J . (1994) Nitrosamines in sunscreens
and cosmetic products. In Nitrosamines and Related N-Nitroso
Compounds: Chemistry and Biochemistry (Loeppky, R. N., and
(26) Loeppky, R. N., Yu, L., Gu, F., and Ye, Q. (1996) DNA Guanine
Adducts from 3-methyl-1,2,3-oxadiazolinium ions. J . Am. Chem.
Soc. 118, 10995-11005.
(27) Wink, D. A., Kasprzak, K. S., Margos, C. M., Elespuru, P. K.,
Misra, M., Dunams, T. M., Cebula, T. A., Koch, W. H., Andrews,
3
R. N. Loeppky, P. Goelzer, and J . Shi, manuscript to be submitted
for publication.

DNA Deamination by R-Nitrosaminoaldehydes
Chem. Res. Toxicol., Vol. 13, No. 2, 2000 81
A. W., Allen, J . S., and Keefer, L. K. (1991) DNA Deaminating
ability and genotoxicity of nitric oxide and its progenitors. Science
254, 1001-1003.
(29) Hecht, S. S., Lijinsky, W., Kovatch, R. M., Chung, F. L., and
Saavedra, J . E. (1989) Comparative tumorigenicity of N-nitroso-
2-hydroxymorpholine, N-nitrosodiethanolamine, and N-nitroso-
morpholine in A/J mice and F344 rats. Carcinogenesis 10,
1475-1477.
(28) Scherer, G., Ludeke, B., Kleihues, P., Loeppky, R. N., and
Eisenbrand, G. (1991) Mutagenicity, DNA damage and DNA
adduct formation by N-nitroso-2-hydroxyalkylamine and corre-
sponding aldehydes. IARC Sci. Publ. 105, 339-342.
TX990126D