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Cancer Research 68, 4142, June 1, 2008. doi: 10.1158/0008-5472.CAN-08-0796
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

AlkB Homologue 2–Mediated Repair of Ethenoadenine Lesions in Mammalian DNA

Jeanette Ringvoll1, Marivi N. Moen1,3, Line M. Nordstrand1, Lisiane B. Meira4, Bo Pang4, Anders Bekkelund2, Peter C. Dedon4, Svein Bjelland3, Leona D. Samson4, Pål Ø. Falnes1,2 and Arne Klungland1

1 Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Rikshospitalet HF and University of Oslo, 2 Department of Molecular Biosciences, University of Oslo, Blindern, Oslo, Norway; 3 Faculty of Science and Technology, Department of Mathematics and Natural Sciences, University of Stavanger, Stavanger, Norway; and 4 Biological Engineering Department, Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts

Requests for reprints: Arne Klungland, Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Sognsvannsveien 20, Oslo 0027, Norway. Phone: 47-2307-4072; Fax: 47-2307-4061; E-mail: aklungla{at}medisin.uio.no.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Endogenous formation of the mutagenic DNA adduct 1,N6-ethenoadenine ({varepsilon}A) originates from lipid peroxidation. Elevated levels of {varepsilon}A in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for {varepsilon}A lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair {varepsilon}A lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2–/– mice indicates that mABH2 is the principal dioxygenase for {varepsilon}A repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of {varepsilon}A lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of {varepsilon}A, comprise the cellular defense against {varepsilon}A lesions. [Cancer Res 2008;68(11):4142–9]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Etheno adducts are ubiquitous and have been found in genomic DNA from a variety of rodent and human tissues (1, 2). These lesions are formed by oxidative stress through lipid peroxidation, and by the reaction of DNA with vinyl chloride. 1,N6-Ethenoadenine ({varepsilon}A) residues are highly mutagenic in mammalian cells (3–5). Upon transfection of human cells with double-stranded and single-stranded plasmid DNA containing a single {varepsilon}A at a defined position, A -> T, A -> G, and A -> C mutations were observed (3). The efficient induction of A -> T transversions supports the hypothesis that human and rodent tumors induced by vinyl compounds reflect the misincorporation of A opposite {varepsilon}A (6–8). In such studies, A -> T transversions were observed both in the p53 tumor suppressor gene and in the ras oncogene. In addition, a decrease in the activities for repairing {varepsilon}A and 3,N4-ethenocytosine ({varepsilon}C) residues has been observed in patients with lung adenocarcinoma (9).

In mammalian cells, the alkyl-N-adenine-DNA glycosylase (ANPG, also known as MPG, APG, and AAG) efficiently excises {varepsilon}A adducts from DNA, leaving behind an abasic site (10–14). Complete repair requires additional enzymes in the base excision repair pathway (15). Experiments in gene-targeted mice lacking ANPG (16, 17) show that ANPG is the major DNA glycosylase for the removal of {varepsilon}A as well as for the cytotoxic 3-methyladenine (3meA) and the mutagenic hypoxanthine lesions. However, the incidence of carcinoma following treatment with vinyl carbamate (Vcar) was similar in wild-type and ANPG-deficient mice (18). This may be explained by recent results using new immunoaffinity liquid chromatography-tandem mass spectrometric methodology, which has revealed that the lesion is also slowly removed from the genome in the absence of ANPG (18, 19). The recent observation that Escherichia coli AlkB repairs, in addition to deleterious methyl lesions, {varepsilon}A adducts in DNA by direct reversal might explain the slow repair in the absence of ANPG; if a mammalian dioxygenase possesses the same activity (20). Eight mammalian AlkB homologues, ABH1 to ABH8 (denoted hABH1–8 in humans; mABH1–8 in mice), have been identified by bioinformatics (21), two of which—ABH2 and ABH3—have been shown to share the ability of E. coli AlkB to directly reverse damaged nucleic acids in vitro (22, 23). In mice, ABH2 is the major, or possibly only, dioxygenase for the repair of methylated DNA in vivo (24). In previous studies, conditions for in vitro repair of {varepsilon}A were optimized for ANPG. Therefore, reactions did not contain iron and oxoglutarate, two essential cofactors for AlkB-mediated damage reversal, and consequently, AlkB-mediated repair was not discovered (22, 23).

We report here that recombinant human ABH2 exhibits robust activity for direct reversal of {varepsilon}A lesions. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2–/– mice showed that ABH2 is the principal dioxygenase for {varepsilon}A repair in vivo. This is further substantiated by experiments analyzing the ability of human AlkB homologues (hABH2 and hABH3) to complement the E. coli alkB mutant for its defective repair of etheno adducts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Protein purification (hABH2 and hABH3). The construct pET28a-hABH2 (provided by Dr. Geir Slupphaug, the Norwegian University of Science and Technology, Trondheim, Norway) was transferred into the expression host E. coli strain BL21 (DE3) RIL and grown at 37°C in Luria-Bertani medium containing kanamycin (100 µg/mL) until OD600 = 0.8. Expression of recombinant protein was induced by adding isopropyl β-D-thiogalactoside to a final concentration of 1 mmol/L and the cells further incubated at 18°C for 6 h. Pellets were harvested at 4°C by centrifugation at 6,000 rpm for 10 min and resuspended in a buffer containing 50 mmol/L of NaP (pH 7.5), 300 mmol/L of NaCl, 0.01% Tween 20, 1x Complete-Protease inhibitor EDTA-free tablet (Roche), and 2 mmol/L of β-mercaptoethanol. Lysozyme was added to 1 µg/µL and the suspension incubated on ice for 30 min. The protein extract was prepared from the cell suspension by sonication (3 x 30 s, 60 amplitude) and denatured proteins and cell debris were removed by centrifugation (12,000 rpm for 10 min at 4°C). Precipitation of nucleic acid with protamine sulfate (0.4% w/v final concentration; Sigma) was done twice with centrifugation after each precipitation to reduce viscosity. The cell lysate was filtered (0.45 µm; Sarstedt) and directly loaded onto a TALON Superflow resin (Clontech), which was washed and eluted as recommended (Clontech). Fractions were analyzed using SDS-PAGE (NuPAGE gel and 10% SeeBlue Plus 2 prestained 1x standard; both from Invitrogen). Finally, purified protein was dialyzed against a buffer containing 50 mmol/L of Tris (pH 7.0), 100 mmol/L of NaCl, 10 mmol/L of β-mercaptoethanol, and stored at –80°C until use. hABH3 was purified as described previously (24).

Preparation of nuclear extracts. Five livers were collected from wild-type and mABH2–/–mice. Extracts and fractionations were prepared as described previously (24). Fractions 26 to 35 were tested for enzymatic DNA repair activity.

Preparation of oligonucleotide substrate. A 49-nucleotide DNA sequence containing the site-specifically inserted {varepsilon}A residue (underlined; 5'32P-TAGACATTGCCATTCTCGATAGG-{varepsilon}A-TCCGGTCAAACCTAGACGAATTCCG-3') or N-3-methylC (3meC) residue (5'32P-TAGACATTGCCATTCTCGATAGG-3meC-TCCGGTCAAACCTAGACGAATTCCG-3') were provided by Midland Certified Reagent Company, Inc. and ChemGenes, respectively. The oligonucleotides were 5'-end–labeled, where the 3meC substrate was further processed as previously described (24). The {varepsilon}A–containing oligonucleotide substrates were separated on a denaturing polyacrylamide gel and purified by the "Crush-and-Soak" method (25) and ethanol precipitation. Duplex DNA substrates were prepared by annealing labeled oligonucleotides to a 2-fold molar quantity (56 pmol) of unlabeled complementary oligonucleotides containing a thymine residue opposite to {varepsilon}A and a guanine residue opposite to 3meC. Annealing was achieved by incubation for 2 min at 90°C followed by slow cooling to room temperature. The end-labeled duplex oligonucleotide substrates were purified following separation on a nondenaturing 20% polyacrylamide gel. The concentration of unlabeled oligonucleotides was measured using Nanodrop. The specific activity of substrate (in cpm) was determined in triplicate by scintillation counting [1 mL substrate or ({gamma}-32P)ATP placed in a 6 mL scintillation tube]. Substrate (32P-labeled) concentration (fmol/mL) was calculated according to a formula recommended by the manufacturer (Amersham).

DNA repair reactions. Repair reactions were performed by incubating hABH2 or AlkB with {varepsilon}A- and 3meC-containing DNA at 37°C for 30 min in a total volume of 50 µL, containing 50 mmol/L of Tris (pH 8.0), 2 mmol/L of ascorbic acid, 100 mmol/L of 2-oxoglutarate, and 40 mmol/L of FeSO4. In typical experiments, 2 nmol/L of double-stranded {varepsilon}A-DNA substrate was incubated with 12 pmol of AlkB or 10.3 pmol of hABH2 (Fig. 1 ). Incubation times, and substrate and enzyme concentrations varied as indicated in the figures. MgCl2 (10 mmol/L) was added to optimize hABH2 reaction conditions. As a positive control, {varepsilon}A-DNA was incubated with 12 pmol of a 26-kDa truncated human ANPG protein (26) at 37°C for 30 min followed by NaOH-mediated abasic site cleavage (27). AlkB and its homologues oxidize the methyl group, resulting in direct reversal of the damage without cleaving the DNA strand. The restriction enzyme DpnII is methyl-sensitive and ethenoadenine-sensitive and was used to distinguish between damaged and repaired oligonucleotides. In reactions containing ssDNA substrate, the complementary DNA strand was added prior to DpnII cleavage. The site of the damage is within the DpnII recognition sequence (5'-GATC-3'). Repaired product DNA was cleaved by incubating the reaction with 20 units of DpnII for 30 min at 37°C and resolved by 20% denaturing PAGE. Visualization and quantification were performed by phosphorimaging analysis using ImageQuant Software (Molecular Dynamics, Inc.).


Figure 1
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  		Figure 1. DNA substrates for {varepsilon}A and 3meC repair and {varepsilon}A repair activities of AlkB and hABH2. A, to create {varepsilon}A- and 3meC-containing substrates, 49mer oligonucleotides were modified with an {varepsilon}A in position 24, or 3meC in position 26, radioactively labeled at the 5'-end and hybridized to a complementary strand. The modified bases are located in a DpnII restriction site such that this enzyme cleaves the substrate only if the etheno/methyl group is removed. The unrepaired substrate appears as a band of 49 nucleotides, whereas repaired and cleaved substrate will appear as a 22-nucleotide band following denaturing PAGE. B, activities of purified AlkB, hABH2, and ANPG on {varepsilon}A in ssDNA and dsDNA. DNA substrates were incubated with purified enzymes as indicated, digested with DpnII, or treated with 0.1 mol/L of NaOH for 30 min at 90°C if reacted with ANPG, and separated by 20% denaturing PAGE. Labeled DNA was visualized by phosphorimaging. Untreated DNA substrates were incubated with DpnII as a negative control. C, activities of purified hABH3 on {varepsilon}A in ssDNA and dsDNA. Similar reaction conditions were used (as in B). D, prior to incubation, cold DNA substrates were added. Similar reaction conditions were used (as in B). ss, single strand; ds, double strand.

 
Host cell reactivation of methyl methanesulfonate-treated and chloroacetaldehyde-treated M13 ssDNA phage. The experiments were performed as previously described (23). The toluic acid–inducible expression plasmid pJB658 (28) carrying genes encoding hABH2, hABH3, or AlkB (29) was transfected into an F-pilus–expressing, AlkB-deficient E. coli strain, HK82/F'. Overnight cultures of bacteria were diluted 100-fold in selective Luria-Bertani medium and grown at 37°C until A600 = 0.1. Expression of recombinant protein was then induced by the addition of m-toluate (Fluka) to a final concentration of 2 mmol/L, and the cultures were grown further until A600 = 1.0 (~2.5 h). The ssDNA phage M13 was treated at 30°C for 0.5 h with different concentrations of methyl methanesulfonate (Sigma-Aldrich) or chloroacetaldehyde (Sigma-Aldrich) to introduce methyl lesions or etheno adducts, respectively. A volume of 100 µL of various dilutions of the treated phage was mixed with 300 µL of induced bacteria and 3 mL of Luria-Bertani top agar, and plated onto Luria-Bertani plates. Plates were incubated overnight at 37°C, and progeny phages were scored by counting the resulting plaques. M9 minimum salt medium containing 1 mmol/L of MgSO4 was added for all treatments and dilutions of the M13 phage.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed in mouse embryo fibroblast cell lines and cultured as described (24). The cells were seeded in 96-well plates with ~3,000 cells/well for the wild-type, mABH2–/–, and ANPG–/– cell lines. Cells were exposed to medium containing chloroacetaldehyde for 2 h (0–20 µmol/L, 100 µL/well), 22 h after seeding. The cells were then washed twice with PBS and fresh medium was added. Relative cell numbers were measured after 48 h using the MTT assay (Roche).

Preparation of genomic DNA from mouse livers and mass spectrometric analysis. Frozen mouse liver tissue was homogenized in cell lysis buffer (Roche) using a Brinkmann Polytron homogenizer and genomic DNA was isolated using a Roche genomic DNA isolation kit, following the directions of the manufacturer. The following antioxidants and deaminase inhibitors were added to the PBS, cell lysis buffer, and protein precipitation solution prior to use: coformycin (5 µg/mL; Sigma), desferrioxamine (0.6 mg/mL; Sigma), and butylated hydroxytoluene (100 µmol/L; Sigma). {varepsilon}A in DNA was quantified as described previously (30). The GraphPad Prism Software was used to estimate the P value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Direct reversal of {varepsilon}A lesions in dsDNA and ssDNA by AlkB and hABH2. Recent in vitro studies using recombinant protein have shown that recombinant E. coli AlkB corrects 1meA and 3meC lesions in DNA by oxidative demethylation, and that two human homologues, hABH2 and hABH3, share this ability (29, 31). In vitro, hABH3 displays a strong preference for ssDNA and ssRNA, whereas hABH2 is exclusively active on DNA, and displays a moderate preference for dsDNA over ssDNA (24, 29, 31, 32). However, studies conducted in ABH3-targeted mice have not revealed substrates for ABH3 in vivo (24).5 Recently, {varepsilon}A was discovered to be a substrate for E. coli AlkB (20).

The current work was initiated by an in vitro assay in which we show that recombinant hABH2 (Fig. 1B), but not hABH3 (Fig. 1C), produces a DpnII cleavable substrate when incubated with a single-stranded or a double-stranded synthetic oligonucleotide containing an {varepsilon}A lesion in the G-{varepsilon}A-T-C sequence (Fig. 1A). As illustrated in Fig. 1B, the 49-nucleotide 32P 5'-end–labeled oligonucleotides were cleaved into 22-nucleotide fragments only when the DpnII digestion was preceded by incubation with AlkB or hABH2, indicating the high quality of the substrate DNA. As a positive control for {varepsilon}A repair, we used human ANPG protein, which is able to excise {varepsilon}A residues only from dsDNA substrates (12). Minor repair of ssDNA by ANPG might be due to some repair activity on ssDNA or, alternatively, flexible structures of the single-stranded oligonucleotide (Fig. 1B). It is difficult to measure the accurate concentration of the 32P-labeled {varepsilon}A-DNA substrate. Therefore, we added, in excess, an increasing amount of unlabeled {varepsilon}A-DNA to the specific amount of labeled substrate used in the reaction mixture, to decide the concentration of substrate DNA being used in subsequent single-turnover kinetic experiments (Fig. 1D).

Single-turnover kinetics for repair of {varepsilon}A in DNA by AlkB and hABH2. In contrast with the 1meA and 3meC lesions, which are preferentially introduced into single-stranded regions of DNA, the majority of {varepsilon}A lesions is introduced into dsDNA, and so we investigated the kinetic variables of AlkB and hABH2 (0–100 pmol) for {varepsilon}A repair in dsDNA substrates (Fig. 2 ). Enzyme activity was measured under single-turnover conditions as described in detail elsewhere (27). The concentration of repairable substrate was determined to be 0.32 ± 0.02 nmol/L for hABH2 and 0.51 ± 0.01 nmol/L for AlkB (Fig. 2A), indicating that more {varepsilon}As are present in an appropriate conformation to be repaired by AlkB than hABH2. The reaction rate was determined by the slopes of the linear regression curves presented in Fig. 2B (hABH2) and Fig. 2C (AlkB), which, divided by the active substrate concentration, leads to the first-order rate constant k (Fig. 2D), which describes the overall accumulation of product during the reaction. The calculations show that AlkB and hABH2 remove the etheno structure from adenine with essentially identical efficiency (hABH2, k2 = 0.094 ± 0.009 min–1, KD = 210 ± 60 nmol/L; AlkB, k2 = 0.100 ± 0.009 min–1, KD = 260 ± 70; Fig. 2D).


Figure 2
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  		Figure 2. Single-turnover kinetics for repair of {varepsilon}A. Substrate DNA (2 nmol/L) was incubated with three different (2 times to 100 times higher) concentrations of hABH2 and AlkB to measure product formation as a function of time. Points, averages of three independent measurements. A, plots of product formation as a function of time using 1,000 nmol/L enzyme, together with a curve fit (r2 values: hABH2, 0.99651; AlkB, 0.97915) to the experimental data (see ref. 27), to determine the active substrate concentration. B and C, linear regression curves (r2 values hABH2: 20 nmol/L, 0.94361; 200 nmol/L, 0.99617; 1,000 nmol/L, 0.96471; r2 values AlkB: 20 nmol/L, 0.94061; 200 nmol/L, 0.97327; 1,000 nmol/L, 0.92739) showing the initial product formation as a function of time (in which the reaction rate is determined by their slopes) at the different enzyme concentrations. D, calculated k values (determined from the value of the slope of the curves presented in B divided by the active enzyme concentration) as a function of total enzyme concentration together with a curve fit (r2 values: hABH2, 0.99533; AlkB, 0.99631) to the experimental data (27).

 
Repair of {varepsilon}A and 3meC by freshly prepared protein fractions from wild-type and mABH2–/– mice. The activity of hABH2 has previously been characterized in vitro and in vivo, measured by the repair of 1meA, 3meC, 1meG, and 3meT lesions (24, 29, 31, 32). To determine the properties and relevance of ABH2 in the repair of {varepsilon}A, the double-stranded {varepsilon}A-oligonucleotide was incubated with fractionated nuclear extracts from mouse liver because the activity of ABH2 is difficult to measure in whole nuclear extracts (24). The repair activity eluted at ~370 mmol/L NaCl, in good agreement with our previous results (Fig. 3A and B; ref. 24 ). The fractionation of the nuclear extracts increased the repair activities for both {varepsilon}A and 3meC by several fold. Repair of {varepsilon}A and 3meC peaked in fractions 29 to 31, and was dependent on mABH2 activity because no activity was observed in fractions prepared from mABH2–/– mice (Fig. 3C and D). No activity was detected from the eluted fractions nor in the flow-through (Fig. 3). The relative repair capacities for {varepsilon}A and 3meC of individual fractions prepared from wild-type mice were quantified (Fig. 3A and B, right) and matched each other very well.


Figure 3
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  		Figure 3. Repair of {varepsilon}A and 3meC in wild-type and mABH2–/– cell extracts. Repair of {varepsilon}A (A) and 3meC (B, positive control) in fractionated wild-type extract is shown with DpnII cleavage, as described in Fig. 1. Repair of {varepsilon}A and 3meC was measured in freshly prepared protein fractions corresponding to 0.75 µg of total protein. Repair activities peaked in fractions 29 to 31 and were completely dependent on mABH2 as no activity was observed in fractions from mABH2–/– mice (C and D). Right, the proportion of the substrate (from the gels shown) that has been repaired and cleaved by DpnII (A and B). Details and substrate as in Fig. 1. NE, nuclear extract; F, flow-through.

 
hABH2-mediated repair of etheno adducts in M13 ssDNA in E. coli. The E. coli alkB mutant is defective in reactivating single-stranded phage DNA containing methyl or etheno lesions (20, 33, 34). In the case of methyl lesions, this defect has been shown to be suppressed by the expression of functional AlkB homologues (29, 31), and we expected the same to be true for etheno adducts. The alkB mutant carrying an expression plasmid for hABH2 was infected with M13 ssDNA phage treated with different concentrations of chloroacetaldehyde, a vinyl chloride metabolite known to introduce etheno adducts in DNA. The number of surviving phages decreased with increasing concentrations of chloroacetaldehyde (Fig. 4A ). Higher phage survival was observed in the hABH2-expressing bacteria than in the control, indicating that hABH2-mediated removal of etheno adducts did occur. Compared with hABH2, the E. coli AlkB protein mediated a substantially higher degree of M13 phage survival. Despite the almost identical kinetic properties of the two enzymes measured in vitro (Fig. 2), there were two important differences between the experimental conditions. First, although ssDNA was used as a substrate in the phage reactivation experiments, dsDNA was used in the in vitro repair reactions, and AlkB actually prefers ssDNA over dsDNA, whereas the opposite is true for hABH2 (29, 35). Second, in the in vitro reactions we used an {varepsilon}A-containing oligonucleotide as substrate, but {varepsilon}C, and not {varepsilon}A, is the etheno lesion introduced most frequently when DNA is exposed to chloroacetaldehyde (36, 37).


Figure 4
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  		Figure 4. Survival of chloroacetaldehyde- or methyl methanesulfonate–treated M13 ssDNA phage in alkB mutant bacteria producing different AlkB proteins. HK82/F' alkB bacteria carrying expression plasmids for the proteins indicated were infected with ssDNA phage M13 treated with the indicated concentrations of chloroacetaldehyde (A) or methyl methanesulfonate (B). Formation of progeny phage was assessed by counting plaques, and values are expressed relative to untreated M13. Points, mean of samples in triplicate; bars, SD (the apparent nonsymmetry of the error bars is due to the logarithmic scale). CAA, chloroacetaldehyde; MMS, methyl methanesulfonate.

 
In these experiments, hABH3 did not display any ability to remove chloroacetaldehyde-induced etheno adducts, whereas in control experiments, there were appreciable repair of methyl methanesulfonate–induced methyl lesions by hABH3, hABH2, and AlkB (Fig. 4B), in accordance with previous reports (29). In summary, our phage reactivation experiments indicate, in accordance with the in vitro data, that chloroacetaldehyde-induced etheno lesions are substrates for AlkB and hABH2, but not hABH3.

Cellular sensitivity to chloroacetaldehyde. Chloroacetaldehyde introduces mutagenic and toxic etheno adducts into genomic DNA. Mammalian cells can repair etheno adducts via the ANPG DNA glycosylase of the base excision repair pathway (17, 19), and our kinetic analyses indicate that ANPG is six times more efficient than ABH2 for the repair of {varepsilon}A (see Discussion). E. coli cells deficient in either AlkB or AlkA are sensitive to chloroacetaldehyde treatment, although loss of AlkA causes a more severe phenotype than loss of AlkB (20). AlkA is the E. coli counterpart of ANPG. The relative survival of mABH2–/– cells after 48 hours was reduced by ~25%, 30%, and 40% for chloroacetaldehyde concentrations of 2, 5, and 10 µmol/L, respectively, whereas the corresponding numbers for wild-type were 5%, 15%, and 30%, respectively (Fig. 5A ). Similar experiments with ANPG–/– cells also indicated that the mutant cell lines could be slightly more sensitive than the corresponding wild-type cell line. In general, the differences in relative cell survival between wild-type and mABH2–/– cells, and between wild-type and ANPG–/– cells, were reproducibly modest but not significant. Similar data is previously presented for methyl methanesulfonate survival (24). We observed no difference in viability and growth for the two cell lines, at chloroacetaldehyde concentrations >10 µmol/L (concentrations between 20 and 100 µmol/L are not shown).


Figure 5
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  		Figure 5. Effect of chloroacetaldehyde on the growth and viability of wild-type, mABH2–/–, and ANPG–/–MEF cells. Wild-type cells, mABH2–/– cells, and ANPG–/– cells were used at passages 16 to 34. Cells were seeded in 96-well plates with ~3,000 cells/well. After 22 h, the cells were treated with 0 to 20 µmol/L of chloroacetaldehyde for 2 h, washed twice with PBS, before fresh medium was added. Cell survival was measured after 48 h. Results are expressed as cell numbers in chloroacetaldehyde-treated cultures compared with untreated controls. Cell numbers were measured using the tetrazolium cytotoxicity (MTT) assay (Roche). Untreated wild-type, mABH2–/– (A) and ANPG–/– (B) cell lines had a density of ~60% after 48 h. Points, average of 5 to 10 independent experiments; bars, SD (always <10%). CAA, chloroacetaldehyde.

 
Age-related accumulation of {varepsilon}A lesions in mABH2–/– and ANPG–/– mice. To study the role played by ABH2 and ANPG in the repair of spontaneously formed {varepsilon}A lesions, genomic DNA was extracted from the livers of untreated wild-type, mABH2–/–, and ANPG–/– mice at different ages (Fig. 6A and B ). Steady-state levels of {varepsilon}A were determined at 4 and 12 months in mABH2–/– and mABH3–/– mice, and at 2, 9, and 24 months for ANPG–/– mice by liquid chromatography-tandem mass spectrometry as described in Materials and Methods. Two to five mice of each genotype were sacrificed at each time point. No {varepsilon}A accumulation could be detected in wild-type, mABH2–/–, and mABH3–/– null mice up to 12 months of age. By contrast, significant {varepsilon}A accumulation was observed in 9- and 24-month-old ANPG–/– mice. These results suggest that a substantial degree of {varepsilon}A production occurs under normal physiologic conditions. At 24 months of age, a small increase of {varepsilon}A bases was also observed in wild-type mice. Thus, these data are in keeping with the obtained kinetic variables of the ABH2 and ANPG enzymes, and the accumulation seen in ANPG–/– mice may suggest that the activity provided by ABH2 is not sufficient to clear all endogenously generated {varepsilon}A adducts.


Figure 6
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  		Figure 6. Age-dependent accumulation of {varepsilon}A in wild-type, ABH2–/–, ABH3–/–, and ANPG–/– mice. Histogram showing the quantification of {varepsilon}A DNA lesions in liver tissue from wild-type, mABH2–/–, and mABH3–/– mice ranging in age from 4 to 12 mo (A) and wild-type and ANPG–/– mice ranging in age from 2 to 24 mo (B). Lesions were quantified using a highly sensitive high-performance liquid chromatography-tandem mass spectrometric method (30). A minimum of two and a maximum of five mice were used per time point/per genotype. *, P < 0.05, 24-month-old ANPG–/– mice compared with 24-month-old wild-type or 2-month-old ANPG–/– mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Many types of human cancers are linked to oxidative stress conditions resulting from chronic inflammations. Reactive oxygen species can damage DNA, RNA, proteins, and lipids. Lipid peroxidation causes the formation of mutagenic DNA adducts such as {varepsilon}A and {varepsilon}C. Being the predominant lesions formed in response to inflammation, etheno bases outscore oxidized and deaminated bases (30). Mutation spectra obtained from tumors induced by Vcar in mice are compatible with {varepsilon}A being the initiating lesion (38). The removal of {varepsilon}A and {varepsilon}C from genomic DNA has long been attributed to ANPG and the mismatch-specific thymine-DNA glycosylase, respectively (12, 39, 40). Thymine-DNA glycosylase removes {varepsilon}C residues from dsDNA without a strict preference for a certain opposite base, although the {varepsilon}C/G mismatch is the best substrate (39). Because the increased formation and persistence of {varepsilon}A in DNA in ANPG knockout mice treated with Vcar was not reported to be associated with higher susceptibility to cancer, the ABH2 dioxygenase could account for the slow but significant repair of {varepsilon}A observed in the ANPG–/– liver (18). The half-life of {varepsilon}A in nondividing cells increased from 8 hours in wild-type to 56 hours in ANPG–/– livers. The kinetic variables of ANPG and ABH2, together with the repair kinetics of {varepsilon}A in wild-type and ANPG–/– mice [this work and refs. (18, 41)], indicate that ANPG and ABH2 together might account for the complete repair of {varepsilon}A lesions in vivo.

The development of a mABH2–/–/ANPG–/– double mutant is currently in progress. This might well yield results equivalent to those seen with OGG1–/– and MYH–/– (MutY homologue)–deficient mice. OGG1 removes the highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA. 8-oxoG lesions (42), which appear in greatly increased numbers in OGG1–/– mice (43, 44), can mispair with adenine during replication; this 8-oxoG/A mismatch is handled by the MYH glycosylase which removes the mispaired A. Whereas both OGG1–/– and MYH–/– single mutant mice seem normal with no overt phenotype (43–45), simultaneous deletions of both OGG1 and MYH predisposed two-thirds of the mice to tumors, establishing an obvious link between oxidative DNA damage and tumorigenesis (45). The MYH–/–/OGG1–/–-related tumors are characterized by unique G -> T transversions in codon 12, GGT, of the K-ras oncogene (45, 46).

ABH2 is the major (or probably the only), enzyme responsible for the repair of 1meA in the genome. In unexposed mice lacking ABH2, 1meA lesions accumulate roughly at a rate of one per 107 adenines per year (24). A similar value would be obtained by continuous exposure to 20 nmol/L of methyl methanesulfonate alone, in which a possible intracellular source of methylation is S-adenosyl-methionine (47). Endogenous formation of {varepsilon}A adducts, probably through lipid peroxidation, has been shown by the presence of {varepsilon}A in genomic DNA prepared from human and rodent tissue (1, 48). The greatly variable levels of {varepsilon}A indicate variable sources for {varepsilon}A generation and/or variable capacity for repair (1). The recent development of ultrasensitive methods for the detection of {varepsilon}A has made it possible to measure this lesion in vivo and to study its formation and role in experimental carcinogenesis. Nevertheless, the endogenous sources and concentration of {varepsilon}A are not well characterized. In our experiments, {varepsilon}A lesions accumulate four times faster in the liver of ANPG–/– mice as compared with wild-type mice. In line with previous experiments in which significant amounts of this lesion were detected in the genome of unexposed tissues (1, 48, 49), these data confirm that the lesion is produced at significant levels under normal cellular conditions. Based on this study, we conclude that the activity of ABH2 is not sufficient for the removal of spontaneously produced {varepsilon}A lesions in ANPG–/– mouse liver. On the contrary, ANPG activity is sufficient for the removal of spontaneously produced {varepsilon}A lesions in mABH2–/– mouse liver, supporting the notion that ANPG is the most effective enzyme for the repair of this lesion. The significance of the endogenous production of {varepsilon}A, as well as its induction of carcinogenesis following treatment with Vcar, for example, will be further studied in cells and in mice with combined mABH2–/–/ANPG–/– deficiency. Such studies, which are in good progress, should also identify the potential added effect of combined gene deletion.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Norwegian Cancer Society, the National Program in Functional Genomics sponsored by the Research Council of Norway and the University of Oslo, and by NIH grants ES02109, CA75576, and AI37750 (L.D. Samson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the Norwegian Transgenic Center and the Center for Comparative Studies at Rikshospitalet HF for the excellent services provided.


   Footnotes
 
5 Unpublished data. Back

Received 3/ 3/08. Revised 3/10/08. Accepted 3/13/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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