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Carcinogenesis Advance Access originally published online on March 14, 2007
Carcinogenesis 2007 28(11):2363-2366; doi:10.1093/carcin/bgm057
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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 Tomokuni1 and Masayoshi Ichiba1

Graduate School of Global Environmental Studies, Kyoto University, Kyoto 606-8501, Japan
1 Department of Social and Environmental Medicine, Saga Medical School, Saga 849-8501, Japan
2 Department of Environmental Biosciences, Yokohama City University, Yokohama, Kanagawa 236-0027, Japan
3 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA
4 Department of Environmental Health, University of Occupational and Environmental Health, Kitakyusyu, Fukuoka 807-8555, Japan
5 First Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan
6 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyusyu University, Fukuoka 812-8582, Japan

* To whom correspondence should be addressed. Tel: +75 753 5052 Fax: +81 75 753 3335; Email: matsuda{at}eden.env.kyoto-u.ac.jp

Correspondence may also be addressed to Masayoshi Ichiba. Fax: +81 952 34 2065; Email: ichiba{at}cc.saga-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 {alpha}-methyl-{gamma}-hydroxy-1,N2-propano-2'-deoxyguanosine ({alpha}-Me-{gamma}-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 Aldh2+/+ mice, the level of N2-ethylidene-dG adduct in liver DNA was 1.9 ± 0.7 adducts per 107 bases and was not significantly different than that of Aldh2+/– and –/– mice. In alcohol-fed mice (20% ethanol for 5 weeks), the adduct levels of Aldh2+/+, +/– and –/– mice were 7.9 ± 1.8, 23.3 ± 4.0 and 79.9 ± 14.2 adducts per 107 bases, 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 {alpha}-Me-{gamma}-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.

Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; {epsilon}dA, 1,N6-etheno-2'-deoxyadenosine; LC/MS/MS, liquid chromatography tandem mass spectrometry; {alpha}-Me-{gamma}-OH-PdG, {alpha}-methyl-{gamma}-hydroxy-1,N2-propano-2'-deoxyguanosine; N2-Et-dG, N2-ethyl-2'-deoxyguanosine; N2-ethylidene-dG, N2-ethylidene-2'-deoxyguanosine


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Alcohol consumption is a risk factor for hepatocellular carcinoma and acetaldehyde, a carcinogenic intermediate of ethanol, has been suggested to be involved in the occurrence of hepatocellular carcinoma. Two large-scale epidemiological studies revealed that habitual alcohol drinking was probably lead to an increased risk of hepatocellular carcinoma and that a lack of acetaldehyde-metabolizing enzyme activity [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 {alpha}, ß and {gamma} 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 determined by their Michaelis constant values for acetaldehyde: the low Km ALDH (ALDH1 and ALDH2) and high Km ALDH (ALDH3 and ALDH4). The Km values of ALDH3 and ALDH4 are on the order of millimolar (5–83 mM) (3), cytosolic ALDH1 is on the order of micromolar (180 µM) 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 homotetrameric 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 containing 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 acetaldehyde-derived stable DNA adducts, N2-ethyl-2'-deoxyguanosine (N2-Et-dG), {alpha}-S- and {alpha}-R-methyl-{gamma}-hydroxy-1,N2-propano-2'-deoxyguanosine ({alpha}-S-Me-{gamma}-OH-PdG and {alpha}-R-Me-{gamma}-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 N2 position 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 confirmed by detection of N2-Et-dG after reduction of DNA during isolation 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.


Figure 1
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  		Fig. 1. Formation of acetaldehyde–deoxyguanosine adducts. 1, N2-ethylidene-dG; 2, N2-Et-dG and 3, {alpha}-Me-{gamma}-OH-PdG.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Aldh2-knockout mice
Aldh2-knockout mice, which had been backcrossed with C57BL6, were obtained from the Department of Environmental Health, University of Occupational 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).

Alcohol feeding
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.

DNA isolation from mouse liver
For quantification of N2-Et-dG, {alpha}-methyl-{gamma}-hydroxy-1,N2-propano-2'-deoxyguanosine ({alpha}-Me-{gamma}-OH-PdG) and 1,N6-etheno-2'-deoxyadenosine ({epsilon}dA), DNA was purified from mouse liver (~50 mg amounts) by using PuregeneTM DNA Purification System Cell and Tissue kit. The protocol was performed basically as described according to the manufacturer except that desferroxamine (final concentration: 0.1 mM) was added to all solutions to avoid formation of oxidative adducts during the purification step.

For quantification of N2-ethylidene-dG, DNA was isolated from mouse liver (~50 mg amounts) as described by Wang et al. (15). The PuregeneTM DNA Purification System Cell and Tissue kit was used. The protocol was basically 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, {alpha}-Me-{gamma}-OH-PdG and their [U-15N5]-labeled standards were synthesized as described previously (14). {epsilon}dA was purchased from Sigma-Aldrich Japan, Tokyo, Japan and [U-15N5] {epsilon}dA was prepared from [U-15N5] dA (Cambridge Isotope Laboratory, Andover, MA, USA) following a method as described previously (17).

DNA digestion
DNA samples (20 µg) were digested to their corresponding 2'-deoxyribonucleoside-3'-monophosphates by the addition of 15 µl of 17 mM citrate plus 8 mM CaCl2 buffer 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 µl of 0.5 M Tris–HCl (pH 8.5), 5 µl of 20 mM ZnSO4 and 67 µl of distilled water were added and incubated further for 3 h at 37°C. The digested sample was extracted twice with methanol. The methanol fractions were evaporated to dryness, re-suspended in 100 µl of distilled water and subjected to LC/MS/MS.

Instrumentation
LC/MS/MS analyses were performed using a Shimadzu LC system (Shimadzu, Kyoto, Japan) interfaced with a Quattro Ultima triple stage quadrupole MS (Waters–Micromass, Manchester, UK). The LC column was eluted over a gradient 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 injection volumes of 50 µl each were separated on a Shim-pack FC-ODS column (4.6 x 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 temperature was 130°C, the desolvation gas temperature was 380°C and the cone voltage was 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 x 10–3 mbar. Positive ions were acquired in multiple reaction monitoring (MRM) mode. The MRM transitions monitored were as follows: [15N5]-{alpha}-Me-{gamma}-OH-PdG, m/z 343 -> 227; {alpha}-Me-{gamma}-OH-PdG, m/z 338 -> 222; [15N5]-N2-Et-dG, m/z 301 -> 185; N2-Et-dG, m/z 296 -> 180; [15N5]-{epsilon}dA, m/z 281 -> 165 and {epsilon}dA, 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 adduct concentrations. The amount of deoxyguanosine was monitored by a Shimadzu SPD-10A UV-Visible detector that was in place before the tandem MS.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
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 genotypes. 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 {alpha}-Me-{gamma}-OH-PdG, were analyzed and {epsilon}dA, a DNA adduct induced by lipid peroxidation, was also analyzed for comparative purposes. The LC/MS/MS instrument employed for analyzing these adducts was sensitive enough to detect at least one adduct per 108 bases in this experimental protocol (14). However, N2-Et-dG was not detected in any liver DNA samples for both alcohol-treated and non-treated mice for any Aldh2 genotype. {alpha}-Me-{gamma}-OH-PdG and {epsilon}dA were detected in all the samples analyzed but neither alcohol-dependent nor Aldh2 genotype-dependent increases in adduct levels were observed (Figure 2).


Figure 2
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  		Fig. 2. DNA adduct levels in control and alcohol-treated mice having different Aldh2 genotypes. Mice were fed with water (Aldh2+/+: n = 5, +/–: n = 7 and –/–: n = 5) or 20% ethanol (Aldh2+/+: n = 6, +/–: n = 5 and –/–: n = 2) for 5 weeks. Liver DNA samples were purified without addition of reducing agent NaBH3CN. (A) The levels of {alpha}-Me-{gamma}-OH-PdG (open bar: {alpha}-S-Me-{gamma}-OH-PdG and closed bar: {alpha}-R-Me-{gamma}-OH-PdG). (B) The levels of {epsilon}dA. The error bars represent the standard deviation.

 
Detection of hepatic N2-ethylidene-dG adduct in Aldh2-knockout mice
To measure N2-ethylidene-dG in DNA, liver samples were homogenized 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 Aldh2+/+ mice was 1.9 ± 0.7 adducts per 107 bases. 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 Aldh2+/+ mice (7.9 ± 1.8 adducts per 107 bases) and Aldh2+/– and Aldh2–/– mice were observed; levels were 23.3 ± 4.0 and 79.9 ± 14.2 adducts per 107 bases in Aldh2+/– 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.


Figure 3
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  		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 (Aldh2+/+: n = 5, +/–: n = 7 and –/–: n = 5) or 20% ethanol (Aldh2+/+: n = 6, +/–: n = 5 and –/–: n = 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 Aldh2+/+ (B), +/– (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 control (+/+); **significantly increased from water control (+/–) or ethanol-treated Aldh2+/+ mice and ***significantly increased from water control (–/–) or ethanol-treated Aldh2+/+ and +/– mice (P < 0.01).

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The ALDH2-knockout mouse developed by Kitagawa et al. (16) has a portion of the phosphoglycerate kinase (PGK) gene promoter containing 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 oxidation 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 Aldh2+/– mice liver, half of the activity for metabolizing acetaldehyde remains compared with the Aldh2+/+ mouse. On the other hand, human ALDH2*1/2*2 heterozygotes have only 13% of the native activity because the heterotetramers of the ALDH2*1 and ALDH2*2 subunits do not function properly (8). Thus, ALDH2 activity in a human ALDH2*2/2*1 heterozygote corresponds with that of the homozygous knockout (Aldh2–/–) mouse rather than the heterozygous (Aldh2+/–) 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 µM and that was 9.3 times higher than that of Aldh2+/+ 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 Aldh2+/+ mice, and these data are consistent with data of acetaldehyde burden. Human alcohol challenge tests have shown that after drinking a moderate amount of ethanol (0.8 g/kg body wt), the average peak in blood acetaldehyde concentrations in ALDH2*1/2*2 individuals was 23 µM and that was 7.5 times greater than that of active ALDH2*1/2*1 homozygotes (19). Thus, it is possible that higher 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. {alpha}-Me-{gamma}-OH-PdG, another acetaldehyde-induced DNA adduct, was detected at the level of 4.5–8.1 adducts per 108 nucleotides, however, neither significant alcohol-dependent nor Aldh2 genotype-dependent increases 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 {alpha}-Me-{gamma}-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 differences 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 {alpha}-Me-{gamma}-OH-PdG, is a sensitive biomarker for acetaldehyde exposure in vivo.

There have been several studies in regard to the mutagenicity of N2-Et-dG and {alpha}-Me-{gamma}-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 {alpha} and {delta} (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 containing a single diastereoisomer of {alpha}-Me-{gamma}-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, {alpha}-Me-{gamma}-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 {alpha}-Me-{gamma}-OH-PdG adducts are mutagenic DNA lesions that may cause human cancers, 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 properties of this sensitive and abundant alcohol- and Aldh2 genotype-dependent biomarker in the near future.


   Acknowledgments
 
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.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 13, 2006; revised February 22, 2007; accepted March 2, 2007.


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R. Ogusucu, D. Rettori, L. E. S. Netto, and O. Augusto
Superoxide Dismutase 1-mediated Production of Ethanol- and DNA-derived Radicals in Yeasts Challenged with Hydrogen Peroxide: MOLECULAR INSIGHTS INTO THE GENOME INSTABILITY OF PEROXIREDOXIN-NULL STRAINS
J. Biol. Chem., February 27, 2009; 284(9): 5546 - 5556.
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