Chem. Res. Toxicol. 2000, 13, 1235-1242
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Reactivity and Mutagenicity of Endogenous DNA Oxopropenylating Agents: Base Propenals, Malondialdehyde, and NE-Oxopropenyllysine John P. Plastaras, James N. Riggins, Michael Otteneder, and Lawrence J. Marnett* A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Departments of Biochemistry and Chemistry, Center in Molecular Toxicology and The Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received August 17, 2000
Malondialdehyde (MDA), a mutagenic product of lipid peroxidation, reacts with DNA to form the premutagenic lesion, pyrimido[1,2-a]purin-10(3H)-one (M1G). M1G is present in normal human tissues, but the contribution of other endogenously produced MDA analogues is poorly understood. Oxidation of the DNA backbone can cause strand breaks and release base propenals, and MDA condensation with proteins yields N-oxopropenyllysine. Here we compare the M1G-forming ability and Salmonella typhimurium mutagenicity of MDA with adenine, thymine, and cytosine propenals and NR-acetyl-N-oxopropenyllysine methyl ester. Base propenals are 30-150 times more potent than MDA in M1G formation and are 30-60 times more mutagenic than MDA. In addition, the Fe-bleomycin complex, which generates base propenals, induced M1G, but γ-radiation, which generates mostly MDA, did not. M1G formation by MDA and base propenals was concentration-dependent, time-dependent, and enhanced by acidic conditions. NR-Acetyl-N-oxopropenyllysine methyl ester was less reactive and less mutagenic than MDA. These differences in potency are consistent with differences in leaving group ability. This work supports a role for other MDA analogues, especially base propenals, in the formation of endogenous M1G adducts.
Introduction Oxidizing agents can cause peroxidation of polyunsaturated fatty acids, producing a range of electrophilic products, the most abundant of which is malondialdehyde (MDA)1 (Scheme 1). MDA is also formed enzymatically. Cyclooxygenases convert arachidonic acid to the endoperoxide, prostaglandin H2, which is subsequently converted to a variety of prostaglandins and thromboxanes. Prostaglandin H2 can degrade to MDA in the presence of ferrous ion, and this conversion is dramatically enhanced by a variety of P450 enzymes, especially thromboxane synthase (1-5). MDA is carcinogenic in rats and mutagenic in bacterial and mammalian cells (6-9). MDA forms adducts with adenine and cytosine, but it reacts most readily with guanine to form a 1:1 adduct, pyrimido[1,2-a]purin-10(3H)-one (M1G) (Scheme 2) (7, 10-13). NMR studies of M1G opposite cytosine in oligonucleotides suggest that it exists as the ring-opened form, N2-oxopropenylguanine (Scheme 2) (14, 15). Therefore, when MDA reacts with duplex DNA, it actually results in the formation of N2oxopropenylguanine adducts, which upon heat denaturation cyclize to form M1G. Consequently, M1G is the form * To whom correspondence should be addressed: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-7329. Fax: (615) 343-7534. E-mail: marnett@ toxicology.mc.vanderbilt.edu. 1 Abbreviations: ECL, enhanced chemiluminescence; MDA, malondialdehyde; M1G, pyrimido[1,2-a]purin-10(3H)-one; PBS, 10 mM phosphate-buffered saline (pH 7.4); PBS-T, 0.1% Tween-20 in phosphatebuffered saline; PI, propidium iodide.
that is quantified by analytical methodologies currently in use. M1G itself is mutagenic. When site-specifically incorporated into viral genomes and replicated in bacteria, it induces both base pair substitutions and frameshift mutations (16, 17). M1G has been detected in human leukocyte and liver DNA at levels of 6 and 90 adducts/ 108 bases, respectively (18, 19). This background presence of M1G has been attributed to endogenous production of MDA, but there are other potential pathways for oxopropenylation of DNA. Oxidation of DNA bases can lead to a variety of genotoxic DNA adducts (20), and oxidation of the DNA backbone leads to variety of products and strand breaks (21-23). One of these products is base propenal, which results from oxidation of the C4′ hydrogen of the deoxyribose ring (Scheme 3). Base propenals are generated when DNA is treated with bleomycin, peroxynitrite, or chromium(V) (24-27), but, curiously, not when DNA is oxidized by γ-radiation (28). Base propenals are structurally analogous to the enol tautomer of MDA (β-hydroxyacrolein) and can undergo acid-catalyzed hydrolysis to MDA and the corresponding free base (Scheme 1) (24). Base propenals are reactive electrophiles that are cytotoxic to human cells (29, 30). Dedon et al. (31) have shown that base propenals react with deoxyguanosine residues in DNA to form M1G. Preliminary analysis indicated that base propenals were significantly more reactive than MDA toward DNA. This raises the possibility that agents capable of generating base propenal may be mutagenic by virtue of their ability
10.1021/tx0001631 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/18/2000
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Scheme 1. Tautomerization of MDA to β-Hydroxyacrolein and Structures of Related Compounds
Plastaras et al.
the mutagenicity and reactivity of N-oxopropenyllysine, a product of the reaction of MDA with -amino groups of lysine residues in protein (Scheme 1). Our results indicate substantial variation in mutagenicity and reactivity in this series of oxopropenyl donors that parallels the trend in leaving group ability.
Experimental Procedures
Scheme 2. Interconversion of M1G and N2-Oxopropenylguanine
Scheme 3. Base Propenal Formation Following Deoxyribose 4′-Hydrogen Abstraction under Normoxic Conditions
to produce M1G. We tested this hypothesis by evaluating the mutagenicity of base propenals in strains of Salmonella typhimurium that are sensitive to mutation by MDA. We also used a high-throughput immunoslot blot technique to directly compare the ability of base propenal and MDA to produce M1G. In addition, we determined
Reagents. Calf thymus DNA, 10 mM phosphate-buffered saline (PBS) (pH 7.4), K2HPO4, KH2PO4, Tween-20, ammonium acetate, propidium iodide (PI), sodium acetate, bleomycin sulfate, and ferrous ammonium sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). The goat anti-mouse IgG horseradish peroxidase conjugate was purchased from Dako (Glostrup, Denmark). Super Signal West Dura was purchased from Pierce (Rockford, IL). Sodium MDA, adenine propenal, cytosine propenal, thymine propenal, and NR-acetyl-N-oxopropenyllysine methyl ester were synthesized according to published methods (30, 32, 33). Products were characterized by 1H NMR using (CH3)2SO-d6 as the solvent. Preparation and Analysis of the Modified DNA Standard. Calf thymus DNA (270 ng/µL) in 100 mM KH2PO4 (pH 4.5) was treated with 1 mM sodium-MDA for 4 days at 37 °C. DNA was precipitated by adding 0.1 volume of 3 M sodium acetate and 2.5 volumes of cold ethanol. The DNA pellet was washed twice with cold 70% ethanol and redissolved in water. The M1G content was determined by enzymatic hydrolysis, immunoaffinity column purification, and liquid chromatography/ electrospray ionization/selected reaction monitoring mass spectrometry as described previously (18). The modified calf thymus DNA standard was found to contain 2.03 adducts/104 bases (594 fmol of M1G/µg of DNA). This standard was aliquoted and stored at -80 °C. Immunoslot Blot Assay for M1G. The M1G content of DNA samples was routinely measured by the immunoslot blot assay previously described by Leuratti et al. with minor modifications (34). DNA concentrations were determined with the absorbance at 260 nm using a Hewlett-Packard 8452A diode array spectrophotometer using 50 µg mL-1 (absorbance unit)-1. To construct standard curves, MDA-modified DNA was diluted with unmodified control calf thymus DNA to give 7 µg of DNA in 300 µL of PBS with a range of modification levels. DNA samples to be analyzed were diluted to 3.5 µg in 150 µL of PBS. To generate single-stranded DNA, the tubes were sonicated in a water bath for 15 min. Samples were boiled for 10 min and incubated on ice for 10 min, and 1 volume of 2 M ammonium acetate was added. The Minifold II blotting apparatus (Schleicher and Schuell, Keene, NH) was assembled with a BA79 nitrocellulose membrane (Schleicher and Schuell) and two pieces of blotting paper soaked in 1 M ammonium acetate. Each sample was blotted onto the membrane in triplicate by adding 86 µL (1 µg of DNA) per well and allowed to drain through the membrane under vacuum. The wells were washed with 200 µL of 1 M ammonium acetate. The membrane was baked for 90 min at 80 °C and blocked for 1 h in 0.1% Tween-20 in PBS (PBS-T) with 5% nonfat dry milk (Marvel, Premier Beverages, Stafford, U.K.). The membrane was washed twice for 5 min in PBS-T and then incubated with a 1:50000 dilution of D10A1 anti-M1G monoclonal antibody (35) (0.3 mg/mL of stock) in 0.5% milk PBS-T sealed in 4.5 mil (0.0045 in.) thick polyester plastic bags (Kapak, Minneapolis, MN). Membranes were agitated on an orbital shaker for 1 h at room temperature followed by 15 h at 4 °C. Membranes were washed with PBS-T for 1 min and then twice for 5 min and incubated at room temperature for 2 h with a 1:3000 dilution of goat anti-mouse IgG horseradish peroxidase conjugate in 0.5% milk PBS-T. The membrane was washed with PBS-T for 15 min and then twice for 5 min and incubated in Super Signal West Dura for 5 min. The membrane was sealed in plastic wrap, and the enhanced chemiluminescence signal was captured using a Fluor-S MAX Multimager (Bio-Rad, Hercules, CA) in ultrasen-
Oxopropenylation of DNA sitive mode (f-stop ) 1.4) for 5 min. The band intensities were quantified using Quantity One software (Bio-Rad). To quantify the amount of DNA that was actually immobilized, the following procedure was added to the immunoslot blot assay described by Leuratti et al. (34). After the chemiluminescence assay, membranes were washed in PBS overnight and then stained with 5 µg/mL PI in PBS for 3 h at room temperature. Membranes were then washed with PBS for 30 min and sealed in plastic wrap. Staining was quantified with a Fluor-S MAX Multimager in fluorescence mode (f-stop ) 8) and Quantity One software. Standard curves were produced by plotting the adduct level against the quotient of the enhanced chemiluminescence signal divided by the PI staining intensity. The M1G levels of unknown samples were based on standard curves analyzed in parallel on the same blot. Chemical Modification of DNA. Calf thymus DNA was dissolved in 10 mM potassium phosphate buffer (pH 7.0), and aliquots were stored at -20 °C until they were used. For bleomycin modifications, a stock solution of the Fe-bleomycin complex was prepared by mixing a 5:4 molar ratio of ferrous ammonium sulfate with bleomycin sulfate in water just prior to use. DNA (0.3 mg/mL) in 100 mM potassium phosphate buffer (pH 7.4) was treated for 30 min with varying concentrations of the Fe-bleomycin complex at room temperature and then frozen at -80 °C until analysis was carried out. Stocks of base propenals and N-oxopropenyllysine were prepared in DMSO and stored at -20 °C. Sodium-MDA stocks were prepared in water just prior to use. Concentrations of stock solutions were determined by UV absorbance using the following extinction coefficients (M-1 cm-1): thymine propenal (304 ) 26 900), cytosine propenal (314 ) 26 800), adenine propenal (258 ) 34 000), N-oxopropenyllysine (280 ) 46 000) (33), and MDA (268 ) 34 200). DNA (0.3 mg/mL) was modified in 100 mM potassium phosphate buffer with varying concentrations of the modifying agents at 37 °C for 24 h (or less for time course experiments). DMSO was added to a total concentration of 6.25% in all reaction mixtures. The pH of all reaction mixtures was 7.4 except in the pH study. Reactions were stopped by ethanol precipitation as described above. The DNA pellet was washed twice with cold 70% ethanol and redissolved to a concentration of 0.1 mg/mL in 10 mM potassium phosphate buffer (pH 7.0) for 1 h at room temperature by vibrational agitation. γ-Irradiation of DNA. Calf thymus DNA in 10 mM potassium phosphate buffer (pH 7.0) (0.1 mg/mL) was subjected to varying amounts of radiation from a 137Cs source emitting 4100 rad/min at room temperature. Mutagenicity Assay. All mutagenicity assays were carried out as previously described using S. typhimurium strain hisD3052 (36). This strain was kindly provided by B. Ames (University of California, Berkeley, CA). Briefly, mutagens dissolved in 0.1 mL of water (sodium-MDA) or DMSO (base propenals and Noxopropenyllysine) were added to 0.1 mL of an overnight culture and 2 mL of top agar enriched with minimal histidine/biotin warmed to 45 °C. Control experiments were performed under the same conditions, with the vehicle added instead of the mutagen solution. The components were mixed by gentle vortexing and immediately poured onto minimal glucose agar plates. The inverted plates were grown in the dark with ventilation at 37 °C for 48 h. Plates were scored for colony growth, and revertant frequencies were calculated by linear regression. Data Analysis. Linear regression analyses were performed with Prism 2.0b software (GraphPad Software Inc., San Diego, CA). Statistical significance was tested with an unpaired Student’s t test assuming equal variance using Microsoft Excel (Office 98).
Results M1G Adduct Analysis. Our laboratory has used an immunoaffinity chromatography/gas chromatography/
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1237
Figure 1. Immunoslot blot standard curve. Varying amounts of MDA-modified calf thymus DNA containing 2.03 M1G adducts/104 bases were diluted with unmodified calf thymus DNA to the indicated levels of M1G. DNA (1 µg) was added to each well and vacuum-filtered onto the membrane. The quotient of the ECL signal divided by the PI fluorescence is plotted against the adduct level. Points are means of three determinations ( the standard error of the mean (SEM). The solid line represents the best fit by linear regression analysis (r2 ) 0.996).
mass spectrometry method to quantify M1G adducts in DNA treated with modifying agents in vitro and in DNA from bacteria, rodents, and humans. Unfortunately, the assay required a large quantity of DNA (∼1 mg) and was not a high-throughput method ( β-ethoxyacrolein . β-isobutoxyacrolein), whereas increasing the leaving group ability increases mutagenicity [β-chloroacrolein . β-hydroxyacrolein (sodium-MDA) . NR-oxopropenyltryptophan ∼ 0]. Scheme 4A depicts a proposed mechanism for M1G formation from thymine propenal that begins with 1,2-addition to the aldehyde, consistent with previous reports from our laboratory (39). Decomposition of the carbinolamine produces N2-oxopropenylguanine and the conjugate base of thymine as the formal leaving group. The leaving group ability is directly related to the stability of the developing negative charge produced by bond breakage. The ability to stabilize a negative charge is correlated to the pKa of the conjugate acid of the leaving group. The relevant pKa values for the released leaving groups are listed in Scheme 4B. The pKa values predict, on the basis of stabilization of leaving groups, that the order of reactivity is as follows: adenine propenal > cytosine propenal > thymine propenal > β-hydroxyacrolein (MDA) > β-alkylaminoacrolein (e.g., N-oxopropenyllysine). The difference in reactivity between thymine propenal and MDA is greater than the disparity in leaving-group pKa. In the case of sodium-
a (A) Reaction of thymine propenal with guanine by initial 1,2addition followed by release of the conjugate base of thymine and (B) pKa values of modifying agent leaving groups.
MDA, which exists as the negatively charged enolate at physiological pH, protonation is doubly critical. Charge repulsion between the enolate and negatively charged DNA would hamper initial collision of the reactants. Addition of one proton to the enolate produces neutral β-hydroxyacrolein, and protonation is required to stabilize the hydroxide leaving group (i.e., pKa ) 15.7). Cytotoxicity. Base propenals are toxic to cells and inhibit cell growth, DNA synthesis, RNA synthesis, and protein synthesis (29, 30). These findings suggest that base propenals react with cellular proteins to elicit toxicity, but DNA is also a potential target. Thymine and adenine propenal inhibit DNA synthesis in HeLa cells with IC50 values of 23 and 30 µM, respectively (29). The inhibition of DNA synthesis by thymine propenal precedes the inhibition of protein and RNA synthesis, suggesting a role for DNA damage or inhibition of the replication machinery (29). Our results, which show that base propenals induce DNA adducts and cause mutations in bacteria, support the hypothesis that DNA is a target of base propenal toxicity. The contribution of DNA lesions to base propenal cytotoxicity may be explained by recent results that demonstrate the ability of M1G to inhibit DNA replication in vitro.2 Our laboratory has shown that MDA induces cell cycle arrest in human tumor cells in a p53-independent manner with a parallel increase in M1G levels (40). Cells treated with 1 mM MDA irreversibly arrest at the G2/M checkpoint and show increases in the level of p53 in p53 wild-type cells and increases in the level of p21 in both p53 wild-type and p53 null cells. This increase in tumor suppressor proteins can be cautiously interpreted as a protective reaction to DNA damage to 2
Unpublished data.
Oxopropenylation of DNA
allow time for DNA repair. Because the same DNA lesions are produced by MDA and base propenals, one might predict a similar induction of tumor suppressor proteins and cell cycle arrest by base propenals. Location of Oxopropenylating Agents. Voitkun et al. (41) have shown that treatment of histones with acidhydrolyzed tetramethoxypropane (MDA equivalents) enhances formation of covalent protein-DNA cross-links. The authors found that cross-linking with DNA did not occur with MDA-modified bovine serum albumin, a protein that has no intrinsic affinity for DNA, but abundant lysine residues. Furthermore, DNA-protein cross-links formed more readily with histone H1 than with the histone fraction IIS, which was attributed to the greater abundance of lysine residues in histone H1. These studies indicate that oxopropenyl units can be delivered from lysine residues, but the reaction depends on protein context. Although we show that NR-acetyl-N-oxopropenyllysine methyl ester is only weakly reactive toward DNA, this reactivity might be enhanced if the oxopropenyl group is adventitiously bound to an appropriate lysine residue of a DNA-binding protein. A similar argument can be made for base propenals. Unlike MDA, which is generated in lipid membranes, base propenals are generated directly in chromatin. Whereas MDA must diffuse through a cellular milieu laden with nucleophiles (e.g., glutathione) before it can react with DNA, DNA is likely the first nucleophile encountered by newly formed base propenals. Conclusion. We have shown that the endogenously produced electrophiles, MDA, base propenals, and Noxopropenyllysine, can react with DNA to form M1G. Of these, the base propenals are the most potent oxopropenylating agents, and therefore may contribute significantly to the MDA-DNA adduct burden in tissues. Furthermore, we have identified base propenals as a new group of endogenously produced mutagens.
Acknowledgment. This work was supported by a research grant and center grants from the National Institutes of Health (CA47479, CA87819, ES00267, and CA68485). We thank Peter Dedon, Chiara Leuratti, and J. Scott Daniels for helpful advice.
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