Carcinogenesis vol.28 no.11 pp.2363–2366, 2007
Advance Access publication March 14, 2007
Increased formation of hepatic N2-ethylidene-2#-deoxyguanosine DNA adducts in
aldehyde dehydrogenase 2-knockout mice treated with ethanol
Tomonari Matsuda?, Akiko Matsumoto1, Mitsuhiro
Uchida, Robert A. Kanaly2, Kentaro Misaki, Shinya
Shibutani3, Toshihiro Kawamoto4, Kyoko Kitagawa5,
Keiichi I.Nakayama6, Katsumaro Tomokuni1and
Graduate School of Global Environmental Studies, Kyoto University, Kyoto
606-8501, Japan,1Department of Social and Environmental Medicine, Saga
Medical School, Saga 849-8501, Japan,2Department of Environmental
Biosciences, Yokohama City University, Yokohama, Kanagawa 236-0027,
Japan,3Department of Pharmacological Sciences, State University of New
York at Stony Brook, Stony Brook, NY 11794-8651, USA,4Department of
Environmental Health, University of Occupational and Environmental Health,
Kitakyusyu, Fukuoka 807-8555, Japan,5First Department of Biochemistry,
Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192,
Japan and6Department of Molecular and Cellular Biology, Medical Institute
of Bioregulation, Kyusyu University, Fukuoka 812-8582, Japan
?To whom correspondence should be addressed. Tel: þ75 753 5052
Fax: þ81 75 753 3335;
Correspondence may also be addressed to Masayoshi Ichiba.
Fax: þ81 952 34 2065;
N2-ethylidene-2#-deoxyguanosine (N2-ethylidene-dG) is a major
DNA adduct induced by acetaldehyde. Although it is unstable in
the nucleoside form, it is relatively stable when present in DNA. In
this study, we analyzed three acetaldehyde-derived DNA adducts,
N2-ethylidene-dG, N2-ethyl-2#-deoxyguanosine (N2-Et-dG) and
OH-PdG) in the liver DNA of aldehyde dehydrogenase (Aldh)-2-
knockout mice to determine the influence of alcohol consumption
and the Aldh2 genotype on the levels of DNA damage. In control
Aldh2þ/þ mice, the level of N2-ethylidene-dG adduct in liver
DNAwas 1.9 ± 0.7 adducts per 107bases and was not significantly
different than that of Aldh2þ/? and ?/? mice. In alcohol-fed
mice (20% ethanol for 5 weeks), the adduct levels of Aldh2þ/þ,
þ/? and ?/? mice were 7.9 ± 1.8, 23.3 ± 4.0 and 79.9 ± 14.2
adducts per 107bases, respectively, and indicated that adduct
level was alcohol and Aldh2 genotype dependent. In contrast, an
alcohol- or Aldh2 genotype-dependent increase was not observed
for a-Me-g-OH-PdG, and N2-Et-dG was not detected in any of
the analyzed samples. In conclusion, the risk of formation of N2-
ethylidene-dG in model animal liver in vivo is significantly higher
in the Aldh2-deficient population and these results may contribute
to our understanding of in vivo adduct formation in humans.
Alcohol consumption is a risk factor for hepatocellular carcinoma and
acetaldehyde, a carcinogenic intermediate of ethanol, has been sug-
gested to be involved in the occurrence of hepatocellular carcinoma.
Two large-scale epidemiological studies revealed that habitual alco-
hol drinking was probably lead to an increased risk of hepatocellular
carcinoma and that a lack of acetaldehyde-metabolizing enzyme ac-
tivity [aldehyde dehydrogenase (ALDH)-2] was associated with this
increased risk (1,2).
There are several enzymes responsible for metabolizing alcohol in
the liver. The first step is oxidization of ethanol to acetaldehyde by
alcohol dehydrogenase (ADH) and the ADH holoenzyme may exist as
either a homodimer or heterodimer of a, b and c subunits, encoded by
ADH1, ADH2 and ADH3, respectively. The second step is oxidation
of acetaldehyde to acetic acid by ALDH or inducible cytochrome
P450 2E1. Human ALDH isozymes are divided into two groups de-
termined by their Michaelis constant values for acetaldehyde: the low
KmALDH (ALDH1 and ALDH2) and high KmALDH (ALDH3 and
ALDH4). The Kmvalues of ALDH3 and ALDH4 are on the order of
millimolar (5–83 mM) (3), cytosolic ALDH1 is on the order of mi-
cromolar (180 lM) and mitochondrial ALDH2 is on the order of
nanomolar (200 nM) (4), suggesting that ALDH2 is a key enzyme
responsible for catalyzing oxidation acetaldehyde in human liver.
Approximately 40% of Japanese have a mutation in the ALDH2 gene
whereas most Caucasians and Africans do not (5). ALDH2 is a homo-
tetrameric enzyme and the mutant ALDH2?2 allele (Glu487Lys)
encodes for a catalytically inactive subunit (6). It is predicted that
individuals who possess the ALDH2?1/2?2 genotype will have only
6.25% of the normal ALDH2 protein and that other tetramers con-
taining one or more of the ALDH2?2 subunits are mostly inactive.
However, when taken together, the overall measured activity of the
five possible tetramer combinations of the ALDH2?1/2?2 genotype is
?13% (7,8). Lastly, individuals who are ALDH2?2/2?2 homozygous
have little ALDH2 activity.
Acetaldehyde itself is a carcinogen that induced nasal tumors in
experimental animals by inhalation (9), and is thought to be a tumor
initiator because of its mutagenic and DNA-damaging properties
(10–13). Recently, we developed an analytical method for acetalde-
hyde-derived stable DNA adducts, N2-ethyl-2#-deoxyguanosine
(N2-Et-dG), a-S- and a-R-methyl-c-hydroxy-1,N2-propano-2#-deoxy-
guanosine (a-S-Me-c-OH-PdG and a-R-Me-c-OH-PdG) by using
sensitive liquid chromatography tandem mass spectrometry (LC/
MS/MS) (14). Other than these stable DNA adducts, the reaction of
acetaldehyde with deoxyguanosine results in the formation of
an unstable Schiff base at the N2position of deoxyguanosine [N2-
ethylidene-2#-deoxyguanosine (N2-ethylidene-dG)] (Figure 1). Wang
et al. (15) showed that N2-ethylidene-dG in human liver DNA is
relatively stable and that the presence of this adduct could be con-
firmed by detection of N2-Et-dG after reduction of DNA during iso-
lation and enzymatic hydrolysis. They showed that when the
reduction step was included during these steps that approximately
a few 100 times more N2-Et-dG was detected in some cases. In this
study, we analyzed these acetaldehyde-derived DNA adducts in the
liver DNA of Aldh2-knockout mice that were exposed to alcohol to
determine the effects of alcohol consumption and the Aldh2 genotype
on the levels of DNA damage in the target organ.
Materials and methods
Aldh2-knockout mice, which had been backcrossed with C57BL6, were ob-
tained from the Department of Environmental Health, University of Occupa-
tional and Environmental Health, Japan. Male mice, aged 10–11 weeks old,
were used in conformity with the regulations of the committee on animal
experiments of Saga University, Japan. The genomic DNA of all subjects
was extracted twice—from a small part of the ear and the lung—and the
genotype of Aldh2 was determined by polymerase chain reaction according
to the method of Kitagawa et al. (16).
Male mice were fed an ethanol solution (20%) and standard hard feed CR-LPF
(348 kcal/100 g) (Charles River Japan, Yokohama, Japan) for 5 weeks. The
number of mice ranged from four to six per group. After 5 weeks, the mice
were killed and liver tissue specimens were removed immediately after blood
collection, and then parts of the tissue specimens were frozen in liquid nitrogen
and stored at ?80?C until they were analyzed.
Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydroge-
nase; edA, 1,N6-etheno-2#-deoxyadenosine; LC/MS/MS, liquid chromatogra-
phy tandem mass spectrometry; a-Me-c-OH-PdG, a-methyl-c-hydroxy-1,
N2-propano-2#-deoxyguanosine; N2-Et-dG, N2-ethyl-2#-deoxyguanosine; N2-
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DNA isolation from mouse liver
For quantification of N2-Et-dG, a-methyl-c-hydroxy-1,N2-propano-2#-deoxy-
guanosine (a-Me-c-OH-PdG) and 1,N6-etheno-2#-deoxyadenosine (edA),
DNA was purified from mouse liver (?50 mg amounts) by using Puregene?
DNA Purification System Cell and Tissue kit. The protocol was performed
basically as described according to the manufacturer except that desferrox-
amine (final concentration: 0.1 mM) was added to all solutions to avoid for-
mation of oxidative adducts during the purification step.
For quantification of N2-ethylidene-dG, DNAwas isolated from mouse liver
(?50 mg amounts) as described by Wang et al. (15). The Puregene? DNA
PurificationSystemCell and Tissue kit wasused. The protocol wasbasically as
described according to the manufacturer except that NaBH3CN was added to
the Puregene cell lysis solution (final concentration was 150 mM) and other
solutions (2-propanol, Tris–ethylenediaminetetraacetic acid, ethanol and 70%
ethanol; final concentration was 100 mM). After the purification step, DNA
was dissolved in 10 mM Tris–HCl/5 mM ethylenediaminetetraacetic acid
buffer (pH 7), extracted with chloroform and precipitated with ethanol also
as described by Wang et al. (15).
DNA adduct standards and their stable isotopes
N2-Et-dG, a-Me-c-OH-PdG and their [U-15N5]-labeled standards were synthe-
sized as described previously (14). edA was purchased from Sigma-Aldrich
Japan, Tokyo, Japan and [U-15N5] edAwas prepared from [U-15N5] dA (Cam-
bridge Isotope Laboratory, Andover, MA, USA) following a method as de-
scribed previously (17).
DNA samples (20 lg) were digested to their corresponding 2#-deoxyribo-
nucleoside-3#-monophosphates by the addition of 15 ll of 17 mM citrate plus
8 mM CaCl2buffer that contained micrococcal nuclease (22.5 U) and spleen
phosphodiesterase (0.075 U) plus internal standards. Solutions were mixed and
incubated for 3 h at 37?C, after which alkaline phosphatase (3 U), 10 ll of 0.5
M Tris–HCl (pH 8.5), 5 ll of 20 mM ZnSO4and 67 ll of distilled water were
added and incubated further for 3 h at 37?C. The digested samplewas extracted
twice with methanol. The methanol fractions were evaporated to dryness, re-
suspended in 100 ll of distilled water and subjected to LC/MS/MS.
LC/MS/MSanalyses were performedusing a ShimadzuLC system (Shimadzu,
Kyoto, Japan) interfaced with a Quattro Ultima triple stage quadrupole MS
(Waters–Micromass, Manchester, UK). The LC column was eluted over a gra-
dient that began at a ratio of 2% methanol to 98% water and was changed to
40% methanol over a period of 40 min, changed to 80% methanol from 40 to
45 min and finally returned to the original starting conditions, 2:98, for the
remaining 15 min. The total run time was 60 min. Sample injectionvolumes of
50 ll each were separated on a Shim-pack FC-ODS column (4.6 ? 150 mm;
Shimadzu) and eluted at a flow rate of 0.4 ml/min. Mass spectral analyses were
carried out in positive ion mode with nitrogen as the nebulizing gas. The ion
source temperaturewas 130?C, the desolvation gas temperaturewas 380?C and
the conevoltagewas operated at a constant 35 V. Nitrogen gas was also used as
the desolvation gas (700 l/h) and cone gas (35 l/h) and argon was used as the
collision gas at a collision cell pressure of 1.5 ? 10?3mbar. Positive ions were
acquired in multiple reaction monitoring (MRM) mode. The MRM transitions
monitored were as follows: [15N5]-a-Me-c-OH-PdG, m/z 343 / 227; a-Me-c-
OH-PdG, m/z 338 / 222; [15N5]-N2-Et-dG, m/z 301 / 185; N2-Et-dG, m/z
296 / 180; [15N5]-edA, m/z 281 / 165 and edA, m/z 276 / 160. The
amount of each DNA adduct was quantified by the ratio of the peak area of
the target adducts and of its stable isotope. QuanLynx (version 4.0) software
(Waters–Micromass) was used to create standard curves and to calculate the
madzu SPD-10AUV-Visible detector that was in place before the tandem MS.
Ethanol and food intake by male mice
The male mice were fed with water or 20% ethanol and standard hard
feed for 5 weeks. Feed intake was slightly decreased in the 20%
ethanol group, but not significantly different between Aldh2 geno-
types. The average ethanol intake in the case of the 20% ethanol group
was not significantly different between Aldh2 genotypes (?23 g/day/
kg body wt) whereas significant losses in body weight were observed
only in the Aldh2?/? mice (data not shown).
DNA adduct levels in the liver of control and alcohol-treated mice
After 5 weeks, mice were killed and their liver DNA was purified to
detect DNA adduct levels. The acetaldehyde-inducible stable DNA
adducts, N2-Et-dG and a-Me-c-OH-PdG, were analyzed and edA,
a DNA adduct induced by lipid peroxidation, was also analyzed for
comparative purposes. The LC/MS/MS instrument employed for an-
alyzing these adducts was sensitive enough to detect at least one
adduct per 108bases in this experimental protocol (14). How-
ever, N2-Et-dG was not detected in any liver DNA samples for both
alcohol-treated and non-treated mice for any Aldh2 genotype.
a-Me-c-OH-PdG and edA were detected in all the samples analyzed
but neither alcohol-dependent nor Aldh2 genotype-dependent in-
creases in adduct levels were observed (Figure 2).
Detection of hepatic N2-ethylidene-dG adduct in Aldh2-knockout
To measure N2-ethylidene-dG in DNA, liver samples were homoge-
nized in lysis buffer containing the strong reducing agent NaBH3CN,
followed by DNA purification in the presence of NaBH3CN. During
the purification step, it was expected that N2-ethylidene-dG would be
converted to stable N2-Et-dG. The average N2-ethylidene-dG level in
liver DNA from untreated Aldh2þ/þ mice was 1.9 ± 0.7 adducts per
107bases. Both Aldh2 hetero- and homo-deficient genotypes did not
affect N2-ethylidene-dG levels in untreated mice. However, in the
20% ethanol-consuming mice, significant increases in the levels of
N2-ethylidene-dG in the liver DNA of Aldh2þ/þ mice (7.9 ± 1.8
adducts per 107bases) and Aldh2þ/? and Aldh2?/? mice were ob-
served;levels were 23.3 ± 4.0 and 79.9 ± 14.2 adducts per 107bases in
Aldh2þ/? and Aldh2?/? mouse liver DNA, respectively (Figure 3).
These data indicated an Aldh2 genotype-dependent increase in the
levels of N2-ethylidene-dG in liver DNA.
Fig. 1. Formation of acetaldehyde–deoxyguanosine adducts.
1, N2-ethylidene-dG; 2, N2-Et-dG and 3, a-Me-c-OH-PdG.
T.Matsuda et al.
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The ALDH2-knockout mouse developed by Kitagawa et al. (16) has a
portion of the phosphoglycerate kinase (PGK) gene promoter contain-
ing an in frame termination codon inserted immediately downstream
of exon 3. The Aldh2?/? mouse has null mRNA of Aldh2, null
ALDH2 protein and null mitochondrial aldehyde oxidation activity
in the liver, but maintains a normal level of cytosolic aldehyde oxi-
dation activity. In the mouse model, no ALDH2 protein is expressed
from the Aldh2-knockout gene due to the stop codon present in the
inserted PGK promoter gene. In the Aldh2þ/? mice liver, half of the
activity for metabolizing acetaldehyde remains compared with the
Aldh2þ/þ mouse. On the other hand, human ALDH2?1/2?2 hetero-
zygotes have only 13% of the native activity because the heterote-
tramers of the ALDH2?1 and ALDH2?2 subunits do not function
properly (8). Thus, ALDH2 activity in a human ALDH2?2/2?1 het-
erozygote corresponds with that of the homozygous knockout
(Aldh2?/?) mouse rather than the heterozygous (Aldh2þ/?) mice.
Isse et al. (18) reported that the blood acetaldehyde concentration
after gavage of ethanol (1 g/kg body wt) of Aldh2?/? mice was ?18
lM and that was 9.3 times higher than that of Aldh2þ/þ mice. Our
observations show that the N2-ethylidene-dG levels in the liver DNA
of the ethanol-fed Aldh2?/? mice was 10 times higher than that of
Aldh2þ/þ mice, and these data are consistent with data of acetalde-
hyde burden. Human alcohol challenge tests have shown that after
drinking a moderate amount of ethanol (0.8 g/kg body wt), the aver-
age peak in blood acetaldehyde concentrations in ALDH2?1/2?2 in-
dividuals was 23 lM and that was 7.5 times greater than that of active
ALDH2?1/2?1 homozygotes (19). Thus, it is possible that higher
Fig. 2. DNAadductlevelsincontrolandalcohol-treatedmicehavingdifferent
Aldh2 genotypes. Mice were fed with water (Aldh2þ/þ: n 5 5, þ/?: n 5 7
and ?/?: n 5 5) or 20% ethanol (Aldh2þ/þ: n 5 6, þ/?: n 5 5 and ?/?:
n 5 2) for 5 weeks. Liver DNA samples were purified without addition of
reducing agent NaBH3CN. (A) The levels of a-Me-c-OH-PdG (open bar:
a-S-Me-c-OH-PdG and closed bar: a-R-Me-c-OH-PdG). (B) The levels of
edA. The error bars represent the standard deviation.
Fig. 3. Alcohol- and Aldh2 genotype-dependent increases in N2-ethylidene-
dG levels in mice liver DNA. Mice with various Aldh2 genotypes were fed
with water (Aldh2þ/þ: n 5 5, þ/?: n 5 7 and ?/?: n 5 5) or 20% ethanol
(Aldh2þ/þ: n 5 6, þ/?: n 5 5 and ?/?: n 5 4) for 5 weeks and the liver
DNA was purified under the presence of NaBH3CN to reduce unstable
N2-ethylidene-dG to stable N2-Et-dG. N2-ethylidene-dG was detected as
N2-Et-dG by using LC/MS/MS. (A) A representative LC/MS/MS
chromatogram of transition m/z 301 / 185 for [U-15N5] N2-Et-dG as an
internal standard. (B–D) Representative LC/MS/MS chromatograms of
transition m/z 296 / 180 for N2-Et-dG in Aldh2þ/þ (B), þ/? (C) and ?/?
(D) mice. (E) The levels of N2-ethylidene-dG in mice liver DNA. The error
bars represent the standard deviation.?Significantly increased from water
treated Aldh2þ/þ mice and???significantly increased from water control
(?/?) or ethanol-treated Aldh2þ/þ and þ/? mice (P , 0.01).
DNA damage in ALDH2-knockout mice
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N2-ethylidene-dG levels in liver DNA exist in drinkers having
ALDH2?1/2?2 genotypes more than in ALDH2?1/2?1 genotypes.
On the other hand, N2-Et-dG, a reduced product of N2-ethylidene-
dG, was not detected in any of the liver DNA samples analyzed. Since
our LC/MS/MS method can detect at least one N2-Et-dG adduct in 108
nucleotides, the adduct level should be at least 18–800 times lower
than in the case of N2-ethylidene-dG in mouse liver DNA. a-Me-c-
OH-PdG, another acetaldehyde-induced DNA adduct, was detected at
the level of 4.5–8.1 adducts per 108nucleotides, however, neither
significant alcohol-dependent nor Aldh2 genotype-dependent in-
creases in adduct levels were observed. Previously, we determined
the DNA adducts in the blood of 44 DNA samples from Japanese
alcoholic patients who consumed an average of 116 g of ethanol every
day for 25 years, and the levels of N2-Et-dG and a-Me-c-OH-PdG
were significantly higher in alcoholics with the ALDH2?1/2?2
genotype as compared with those with the ALDH2?1/2?1 genotype
(14). Since many lymphoid cells are long-lived and may persist as
memory cells for several years (20), N2-Et-dG may accumulate in the
lymphoid cells of such subjects. In this study, mice were fed alcohol
for only 5 weeks and that may not have been enough time for these
adducts to accumulate to detectable levels in the liver, although we
should consider species-specific differences and tissue-specific differ-
ences with respect to endogenous reduction of N2-ethylidene-dG and
DNA repair activity. From our data in this study at least, we can
clearly say that N2-ethylidene-dG, rather than N2-Et-dG and a-Me-
c-OH-PdG, is a sensitive biomarker for acetaldehyde exposure
There have been several studies in regard to the mutagenicity of
N2-Et-dG and a-Me-c-OH-PdG. N2-Et-dG adducts induce G to C
mutations during DNA synthesis catalyzed by the Escherichia coli
DNA polymerase I Klenow fragment (21) and G to T mutations
during gap-filling DNA synthesis in E.coli cells (22). N2-ethyl-2#-
deoxyguanosine triphosphate (N2-Et-dGTP) was effectively utilized
during DNA synthesis catalyzed by mammalian DNA polymerases
a and d (23). Additionally, it has been shown that N2-Et-dG strongly
blocks replicative DNA polymerization, which leads to frameshift
deletion mutations (24,25). When a single-strand shuttle vector con-
taining a single diastereoisomer of a-Me-c-OH-PdG was propagated
in a mammalian cell line, the mutational frequency was 5–6%; G to T
transversions were detected as the dominant form of damage (26). In
addition, a-Me-c-OH-PdG adducts are thought to be the precursor
lesions to DNA–DNA or DNA–protein cross-links (27,28). Taken
together, these observations suggest that N2-Et-dG and a-Me-c-OH-
PdG adducts are mutagenic DNA lesions that may cause human can-
cers, however, in regard to N2-ethylidene-dG, little information is yet
available about its biological significance.
In closing, although the biological significance of N2-ethylidene-
dG is not clear, it was clearly shown that the adduct levels in liver
DNA were relatively high and significantly increased after alcohol
uptake. It will be essential to study the mutagenicity and repair prop-
erties of this sensitive and abundant alcohol- and Aldh2 genotype-
dependent biomarker in the near future.
This research was supported in part by Grants-in-aid for Cancer Research from
the Ministry of Health, Labor and Welfare of Japan and Grants-in-aid for
Scientific Research from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
Conflict of Interest Statement: None declared.
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Received November 13, 2006; revised February 22, 2007;
accepted March 2, 2007
T.Matsuda et al.
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