Identification of DNA Adducts of Methylglyoxal - Chemical Research

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Identification of DNA Adducts of Methylglyoxal Matthias Frischmann,† Clemens Bidmon,† Ju¨rgen Angerer,‡ and Monika Pischetsrieder*,† Institute of Pharmacy and Food Chemistry, University of Erlangen-Nuremberg, Schuhstrasse 19, D-91052 Erlangen, and Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine, University of Erlangen-Nuremberg, Schillerstrasse 25, D-91054 Erlangen, Germany Received May 11, 2005

Methylglyoxal (MG) is a sugar degradation product, which is endogenously formed by fragmentation of triose phosphates during glycolysis, ketone body metabolism of acetone, and catabolism of threonine. Food, beverages, and medical products are important exogenous sources with concentrations of up to 100 µM MG. MG is a reactive dicarbonyl compound, which easily modifies amino groups of proteins (glycation reaction) and thereby induces proinflammatory responses. Moreover, increased mutation frequencies in mammalian cells after treatment with MG have been reported, which are caused by stable modifications of DNA bases. Thus far, two types of adducts have been identified, which are formed during the reaction of free guanine or 2′-deoxyguanosine with high MG concentrations. In this study, we investigated the prolonged exposure of DNA to physiological MG concentrations. DNA was incubated with MG, enzymatically hydrolyzed to release the free nucleosides, and then analyzed by LC-MS/MS. We detected four products, which were derived from the reaction of 2′-deoxyguanosine and 2′-deoxyadenosine with 1 and 2 equiv of MG each. The adducts with 1 equiv of MG were identified as N2-(1-carboxyethyl)-2′-deoxyguanosine (CEdG) and N6-(1-carboxyethyl)-2′-deoxyadenosine. LC-MS/MS was optimized for these compounds, and incubation of DNA was repeated using physiological concentrations of 10 µM MG. Thereby, CEdG proved to be the most sensitive and suitable marker for the reaction of DNA with MG (negative MRM mode, three mass transitions [M - 1]- 338f178, 338f106, and 338f149).

Introduction 1

Methylglyoxal (MG) is a sugar degradation product, which is found in tobacco, food, and beverages such as toast, soy sauce, coffee, and whiskey as well as in medical products such as conventional peritoneal dialysis fluids (1, 2). Moreover, MG is endogenously formed by fragmentation of triose phosphates during glycolysis, keton body metabolism of acetone, and catabolism of threonine (3-5). Depending on these various sources, physiological concentrations of up to 100 µM MG have been reported. MG is a reactive dicarbonyl compound, which reversibly binds to proteins at arginine, lysine, and cysteine residues. Furthermore, irreversible binding easily leads to advanced glycation end products, which are stable modifications of amino acids via the so-called glycation reaction (6, 7). Similar to glyoxal, MG is detoxified by the glyoxalase system, a glutathione (GSH)-dependent metabolic pathway for the conversion of R-oxoaldehyds RCOCHO to the corresponding R-hydroxyacids RCH(OH)COOH (8). Because of the pivotal role of GSH in the glyoxalase system, its depletion by oxidative stress may * To whom correspondence should be addressed. Tel: ++49-91318524102. Fax: ++49-9131-8522587. E-mail: pischetsrieder@ lmchemie.uni-erlangen.de. † Institute of Pharmacy and Food Chemistry. ‡ Institute and Outpatient Clinic of Occupational, Social and Environmental Medicine. 1 Abbreviations: MG, methylglyoxal; cMG-dGuo, 1,N2-(1,2-dihydroxy-2-methyl)ethano-2′-deoxyguanosine; dGuo, 2′-deoxyguanosine; dAdo, 2′-deoxyadenosine; OPD, o-phenylendiamine; MRM, multiple reaction monitoring; CEdG, N2-(1-carboxyethyl)-2′-deoxyguanosine; CEdA, N2-(1-carboxyethyl)-2′-deoxyadenosine; MG2-dGuo, bis adduct of methylglyoxal and 2′-deoxyguanosine; MG2-dAdo, bis adduct of methylglyoxal and 2′-deoxyadenosine.

severely reduce the protecting capabilities of the cell and lead to an accumulation of MG, which subsequently might evoke cellular reactions. Several cellular effects of MG have been reported. Recent publications showed that MG induces a proinflammatory response in mesothelial cells and apoptosis in rat Schwann cells (9, 10). Furthermore, MG is a known mutagen: It causes DNA and DNA-protein cross-links in human keratinocytes, increased point mutations in Salmonella typhimurium, and increased mutation frequencies in mammalian cells (11-13). The occurrence of point mutations correlated with the glycation rate of DNA (14). Several adducts of free nucleosides and nucleotides have already been described. The first studies were carried out by Shapiro et al., who reacted guanine with more than 30-fold molar excess of MG (0.7 M) for 18 h at 65 °C. Under these conditions, the guanine was completely converted into the cyclic adduct 1,N2-(1,2-dihydroxy-2-methyl)ethano-2′-deoxyguanosine (cMG-dGuo; Scheme 1; 15). Vaca et al. used a 32P-postlabeling method and detected cMG-dGuo in DNA that was treated for 25 h at 37 °C with 40 mM MG, in lymphocytes that were incubated with 3 mM MG overnight (16), and in human bucal epithelial cells that were exposed for 1 h to 30 mM MG (17). Schneider et al. identified a bis adduct of dGuo and MG, N2,7-bis(1-hydroxy-2-oxopropyl)-dGuo, as the main product, when dGuo was incubated with a 20-fold excess of MG (40 mM) for 48 h at 37 °C (18). In this study, we developed a highly sensitive LC-MS/MS method to measure MG-derived DNA AGEs. N2-(1-Carboxyethyl)-2′-deoxyguanosine (CEdG), a novel

10.1021/tx0501278 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/27/2005

Methylglyoxal DNA Adducts Scheme 1. Putative Reaction Pathway of the Formation of CEdG

adduct of MG and dGuo, was identified as the major detectable product, when DNA was exposed for a prolonged time to physiological MG concentrations.

Experimental Procedures Caution: MG has been found to be mutagenic in bacteria and mammalian cells. Caution should therefore be exercised in the handling of the compound. Materials and Methods. dAdo monohydrate, dGuo monohydrate, ammonium formate, 2,3-butanedione, sodium phosphate, salmon sperm DNA, and alkaline phosphatase (EC 3.1.3.1) were purchased from Fluka (Buchs, Switzerland). A MG 40% aqueous solution, o-phenylendiamine (OPD), ammoniumbicarbonate, ammoniumacetate, and phosphodiesterase (EC 3.1.4.1) were purchased from Sigma-Aldrich (Munich, Germany). Pyruvaldehyde 1,1-dimethyl acetal was obtained from Merck (Darmstadt, Germany), nuclease S1 (EC 3.1.30.1) was purchased from Fermentas (St. Leon-Rot, Germany), HPLC grade acetonitrile was purchased from Fisher Scientific (Loughborough, United Kingdom), dialysis membranes were purchased from Visking (London, United Kingdom), a cutoff filter (Nanosep spin columns) was purchased from Pall Life Science (Dreieich, Germany), and PBS solution, pH 7.4, was purchased from Biochrom (Berlin, Germany). Synthesis, Distillation, and Quantification of MG. The synthesis of MG by acid hydrolysis of pyruvaldehyde 1,1dimethyl acetal and subsequent distillation was carried out as described in the literature (19). For quantification, 500 µL of the collected fractions of freshly distilled MG and 2,3-butanedione as calibration standard (10, 5, 2, 1, and 0.2 mM in H2O) was diluted with 400 µL of PBS and derivatized overnight with 40 µL of OPD (0.2 M in methanol) at room temperature. The resulting 2-methylquinoxaline (2-MQ) and 2,3-dimethylquinoxaline (2,3-DMQ) were diluted 1:10 in PBS and analyzed with Agilent 1100 series HPLC/DAD at 316 nm [injection volume, 50 µL; column, Agilent Zorbax Eclipse XDB-C8, 4.6 mm × 150

Chem. Res. Toxicol., Vol. 18, No. 10, 2005 1587 mm with 5 µm particle size; eluent A, 5 mM aqueous ammonium formate buffer; eluent B, acetonitrile; gradient elution (time/ %A), 0 min/95, 5 min/55, 10 min/10, 17 min/10, 19 min/95, and 25 min/95; flow rate, 300 µL/min; tR(2-MQ) ) 14.6 min; and tR(2,3-DMQ) ) 15.1 min]. The concentration of 2-MQ was calculated by using the calibration curve of 2,3-DMQ under the assumption that the compounds have the same response factor. Distillation yielded a solution of 74.5 mM freshly synthesized MG. Synthesis of CEdG. The synthesis of CEdG was carried out as described in the literature (20). Briefly, 50 mg of dGuo suspended in 1 mL of 1 M phosphate buffer (pH 7.4) was incubated with 100 mg of dihydroxyacetone at 70 °C in a shaking water bath. dGuo was dissolved at 70 °C in the course of the reaction. After 24 h, both diastereomers of CEdG were isolated by preparative HPLC using 50 mM ammonium formate buffer solution and methanol as eluents. Synthesis of cMG-dGuo. The cyclic adduct of dGuo and MG was synthesized analogously to the synthesis of the cyclic adduct of guanine and MG as reported by Shapiro et al. (15). A 467 mg amount of dGuo monohydrate (1.65 mmol) and 8.5 mL of purchased MG 40% aqueous solution (about 52 mmol) were added to 75 mL of 0.043 M sodium phosphate buffer (pH 7.0). The reaction mixture was heated for 18 h at 65 °C. HPLC-MS/MS Instrument Setup. An Agilent 1100 series HPLC system (Palo Alto, CA) with degasser, binary pump, column compartment, and DAD, a Perkin-Elmer PE200 autosampler (Boston, MA), and an Applied Biosystems API 2000 electrospray-MS/MS (Foster City, CA) were used. General chromatographic conditions were as follows: Agilent Zorbax Eclipse XDB-C8 column, 4.6 mm × 150 mm with 5 µm particle size; eluent A, 5 mM aqueous ammonium formate buffer, pH 6.2; eluent B, acetonitrile; gradient elution (time/%A): 0 min/ 95, 6 min/40, 8 min/10, 17 min/10, 20 min/95, and 25 min/95; and flow rate, 300 µL/min. DNA bases were detected by DAD at their absorption maximum of 254 nm. General MS parameters were as follows: negative ionization; ion spray voltage, -4500 V; nebulizer gas, 30 psi; heater gas, 75 psi; heater gas temperature, 420 °C; declustering potential, -21 V; focusing potential, -340 V; and entrance potential, -10.5 V. In the full scan mode, negative ionization and a mass range of 300-450 amu were applied. Transition Ions of CEdG. A product ion scan of [M - H]at a m/z value of 338 in the negative mode at a collision energy of -20 V leads to fragment ions with m/z values of 294 (- CO2), 222 (- deoxyribose), and 178 (- CO2, - deoxyribose). Transition Ions of Bis Adduct of MG and dGuo (MG2-dGuo). A product ion scan of [M - H]- at a m/z value of 410 in the negative mode at a collision energy of -36 V leads to fragment ions with m/z values of 338 (- MG), 294 (- CO2, MG), and 178 (- CO2, - deoxyribose, - MG). Transition Ions of N2-(1-Carboxyethyl)-2′-deoxyadenosine (CEdA). A product ion scan of [M - H]- at a m/z value of 322 in the negative mode at a collision energy of -20 V leads to fragment ions with m/z values of 278 (- CO2), 206 (- deoxyribose), and 162 (- CO2, - deoxyribose). Transition Ions of Bis Adduct of MG and dAdo (MG2-dAdo). A product ion scan of [M - H]- at a m/z value of 394 in the negative mode at a collision energy of -30 V leads to fragment ions with m/z values of 322 (- MG), 278 (- CO2, MG), and 162 (- CO2, - deoxyribose, - MG). The loss of CO2 indicating the presence of a carboxylic group in all four compounds was affirmed by a neutral loss scan of 44 amu (collision energy, -15 V). Product ion scans were also repeated in the positive mode. Because of an approximately 100fold higher sensitivity with negative than with positive polarity, a negative multiple reaction monitoring (MRM) method was developed using the three most intensive mass transitions for each of the four compounds. LC-MS/MS (negative MRM mode): collision gas, N2; collision gas setting, 9; transitions, 338/178, 338/106, 338/149 (CEdG), 322/162, 322/118, 322/146 (CEdA), 410/178, 410/149, 410/133

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(MG2-dGuo), and 394/162, 394/133, 394/174 (MG2-dAdo); and scan time, 150 ms per mass transition. For the MRM method, 12 different collision energies between -20 and -50 V were used, so that for each fragment the ion maximum intensity was achieved. The limit of detection of CEdGA/B using the MRM method was determined as 0.5 ng/mL for each diastereomer by injection of a purified CEdGA/B standard (resolution settings: Q1 unit resolution, Q3 low resolution). Incubation of dAdo and dGuo with MG. Fifty micromoles of dAdo monohydrate and 50 µmol of dGuo monohydrate were dissolved in 10 mL of 10 mM purchased MG in PBS and incubated at 37 °C for 2 weeks. Samples of 300 µL were taken immediately after mixing and after 0.5, 2, 3, 5, 7, and 14 days of incubation. The excess of MG was immediately inactivated by addition of 200 µmol of OPD giving the 2-methylquinoxaline. Then, the samples were stored at -20 °C until analysis. The incubation was carried out in triplicate. This experiment was also repeated with 100 µM MG, whereby two additional samples were taken after 4 and 6 days. In some cases, the reaction was carried out in the same way but was not stopped by OPD. After the incubation time, these samples were immediately injected into the LC-MS/MS. Furthermore, dGuo was reacted with MG according to Schneider et al. (18). Briefly, dGuo (20 µmol) and an aqueous solution of purchased MG 40% (400 µmol) were dissolved in 10 mL of 100 mM sodium phosphate buffer (pH 7.4). The reaction mixture was incubated for 18 h at 37 °C. Incubation of DNA with 100 mM, 10 mM, and 10 µM MG. Purchased MG (final concentrations, 100 and 10 mM, respectively) and 10 mg of salmon sperm DNA were dissolved in 10 mL of PBS and incubated for 1 week at 37 °C. The incubation was also repeated with 10 µM of freshly synthesized MG. One milliliter of the incubated DNA solutions was extensively dialyzed (MWCO 14000) against distilled water, lyophillized, and resolved in 100 µL of water for DNA hydrolysis. DNA Hydrolysis. Ten microliters of 0.1 M ammonium acetate buffer (pH 5.3) and 10 µL of nuclease S1 (10 U) were added to the resolved DNA samples and incubated for 2 h at 45 °C. Then, 10 µL of 1 M ammonium bicarbonate buffer (pH 8.0) and 10 µL of snake venom phosphodiesterase (0.008 U) were added and incubated for another 2 h at 37 °C. An aliquot of 8 µL of alkaline phosphatase (1 U) was added and incubated for 1 h at 37 °C. The samples were centrifuged through a 10 kDa cutoff filter for 10 min at 14000g to remove enzymes. Isolation of CEdA. A 0.5 mmol amount of dAdo monohydrate and 2 mmol of MG were dissolved in 10 mL of PBS and incubated for 16 h at 50 °C. A peak pair with the same UV spectrum, mass spectrum, and fragmentation pattern was detected. The second peak was isolated for 1H NMR analysis using a similar chromatographic system as described above for HPLC-MS/MS instrument setup. 1H NMR was carried out on a Bruker Avance 600 system (Karlsruhe, Germany). The 1H NMR spectrum and the mass spectrum of the isolated adduct are consistent with the structure of CEdA. 1H NMR (D2O): δ 8.12 (s, 1H, H-2), 8.09 (s, 1H, H-8), 6.32 (t, 1H, H-1′), 4.62 (m, 1H, H-3′ obscured by H2O), 4.49 (m, 1H, H-4′), 4.02 (q, 1H, H-a), 3.65 (m, 2H, H-5′), 2.70 (m, 1H, H-2′), 2.40 (m, 1H, H-2′), 1.37 (d, 3H, H-b).

Results Formation and Detection of DNA Adducts by MG. DNA was incubated with 100 and 10 mM MG for 1 week at 37 °C and then enzymatically hydrolyzed. A HPLC method was developed to separate the resulting nucleosides using diode array detection. The samples were then analyzed adding the mass spectrometer for full scan in negative mode, giving evidence for four adducts with [M - H]- at m/z values of 322, 338, 394, and 410 (Figure 1). Further investigation using precursor and product ion scan confirmed those adducts to be stable reaction products of MG with DNA nucleosides (data not shown).

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Figure 1. LC-MS mass spectra (Q1 fullscan) of hydrolyzed DNA after incubation with 100 mM MG; time window, 7.9-9.6 (top) and 10.6-11.1 (bottom) min, respectively.

The adducts with m/z values of 322 and 338 appeared as pairs of peaks, and the two other adducts appeared as peak quartets, indicating the presence of diastereomers. A new LC-MS/MS method for the sensitive detection of these four products was developed as follows: The three most intensive transitions obtained from the previous product ion scans of each compound were used for MRM. Figure 2 shows four chromatograms monitoring the most intensive transition for each reaction product. The peak pairs for the compounds with m/z values of 338 and 322 as well as the peak quartets for the compound with a m/z value of 410 are visible. Because of the gradient, the four peaks of the product with a m/z value of 394 are not resolved by this method. Identification of the Adducts. The UV spectra of the four adducts showed the same maximum at 254 nm as the unmodified dAdo or dGuo, respectively, indicating that they are modifications of these nucleosides. In addition, the observed masses corresponded with the calculated masses for single charged ions [M - 1]- of the nucleoside plus 1 or 2 equiv of MG (m/z 338 and 410 for dGuo and 322 and 394 for dAdo). The compound with a m/z value of 338 appeared as a pair of peaks in the chromatogram, and in the mass spectrum, the loss of deoxyribose and CO2 was observed as main fragmentation (338f178). The same properties have been described for the two diastereomers of CEdG (CEdGA,B; Figure 3). CEdGA,B are glycation products of DNA, resulting from the reaction of dGuo with various carbonyl compounds, such as dihydroxyacetone or glucose. Therefore, we synthesized and purified CEdGA,B as described in the literature (20) as a reference compound.

Methylglyoxal DNA Adducts

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Figure 4. Time course of the adduct formation from equimolar concentrations of nucleosides and 10 mM MG. Results given as a ratio of the peak areas of modified nucleosides to unmodified dGuo; 2, MG2-dGuoA-D/dGuo; [, CEdGA,B/dGuo; and 9, CEdAA,B/ dGuo.

Figure 2. LC-MS/MS chromatograms (MRM) for the detection of modified nucleosides formed by the reaction of DNA with 10 mM MG. Chromatograms show the four most intensive transitions 338/178 (a), 410/178 (b), 322/162 (c), and 394/162 (d).

Figure 3. Structures of CEdGA,B (left molecule) and CEdAA,B (right molecule). Asterisks indicate the new chiral centers leading to two diastereomers for each compound.

The peak pair with m/z 338 showed a retention time, UV spectra, and fragmentation identical to those of the standard. Thus, the adducts of dGuo with 1 equiv of MG could unequivocally be identified as the two diastereomers of CEdG. For structural assignment of the compound with a m/z value of 322, one peak of the pair was isolated by HPLC. 1 H NMR analysis indicated a -CHX-CH3 group bound to the purine (δ 4.02 q/1H H-a, 1.37 d/3H H-b). The chemical shift of the substituent is almost identical to that of the carboxyethyl group in CEdG (δ 4.18 q/1H, 1.39 d/3H). The presence of a carboxyethyl group is also confirmed by the loss of CO2 and deoxyribose (322f162) as main fragmentation in the MS/MS spectra, which is analogous to the fragmentation observed for CEdG. In conclusion, these facts indicate the formation of the two diastereomers of CEdA (CEdAA,B; Figure 3). The isolation of the two adducts formed by the reaction of 2 equiv of MG with dGuo (MG2-dGuo) and dAdo (MG2-dAdo), respectively, turned out to be difficult due to the instability of these compounds during the isolation process, so that their structures could not be determined unequivocally. Time Course of Adduct Formation during the Reaction of dGuo and dAdo with MG. To determine

the formation rate of the four new MG adducts, equimolar concentrations of dGuo and dAdo (5 mM each) were incubated with 10 mM MG. Several samples were taken within a period of 2 weeks to monitor the formation rate of the glycated nucleosides. Excess MG was removed by the immediate addition of OPD, which leads to the formation of 2-methylquinoxaline. The incubation of dGuo and dAdo was also repeated with a physiological concentration of 100 µM MG. The formation of the modified nucleosides was investigated by summing the peak areas of all diastereomers for each adduct. Results are given as the ratio of the areas of the modified nucleoside and dGuo. In the presence of 10 mM MG, MG2-dGuoA-D were the main products with the highest formation rate. MG2-dAdoA-D could not be detected. Furthermore, the amount of CEdGA,B was up to 42-fold higher than the amount of CEdAA,B over the whole incubation time and was still increasing after 2 weeks (Figure 4). In the reaction mixtures containing 100 µM MG, only CEdGA,B were detectable (Figure 5). The reaction was also repeated with 10 mM dGuo and equimolar concentrations of MG without stopping the reaction by OPD. We monitored the formation of adducts by product ion scan of the m/z value 338. The LC-MS/ MS runs were carried out immediately after each sampling. Interestingly, after short-term incubation, a peak pair was detected, which did not correspond to CEdGA,B (Figure 6a). We identified this product as the cyclic adduct of MG and dGuo, cMG-dGuo (Scheme 1), by synthesis of the reference compounds according to Shapiro et al. (15). Then, the main mass transition of cMGdGuo, 338f265, was added to our MRM method and its formation rate was compared to those of CEdGA,B (Figure 7). Within the incubation time of 1 day, the amount of cMG-dGuo distinctly decreased, whereas the areas of CEdG steadily increased. After 1 week, 97% of the peak areas of the modified nucleosides could be assigned to CEdGA,B (Figure 7). Reaction of DNA with Freshly Synthesized MG. Purchased MG can contain minor impurities (21), which might influence the results, as the formation of CEdGA,B cannot unequivocally be assigned to the reaction of MG

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Figure 5. Time course of the adduct formation from 100 µM MG, dGuo, and dAdo. Results given as a ratio of the peak areas of CEdGA,B to unmodified dGuo; [, CEdGA,B/dGuo.

Figure 6. Incubation of dGuo with 10 mM MG without removing the excess of MG by the addition of OPD. Mass transitions for cMG-dGuoA,B (338/265, tR ) 10.7 and 10.9 min) and CEdGA,B (338/178, tR ) 8.10 and 9.30 min) at the beginning (a) and after 7 days (b) of incubation.

itself. To exclude any reaction of minor byproducts, we synthesized MG by acid hydrolysis of pyruvaldehyde 1,1-dimethyl acetal and subsequent distillation of the released MG. The MG concentrations of the collected fractions were determined by HPLC. Incubation of DNA with a final dilution of 10 µM MG was carried out for 1 week at 37 °C. In accordance with the results obtained with the free nucleosides, CEdGA,B once again proved to be the main products of the reaction of DNA with low MG concentrations under physiological conditions (Figure 8). None of the other reaction products could be detected. Furthermore, it was shown that CEdGA,B is also formed at very low MG concentrations as present in vivo.

Figure 7. Time course of the product formation from dGuo with 10 mM MG without removing the excess of MG by the addition of OPD. The formation rate is expressed by the ratio of the peak area of the product to the sum of the peak areas of all modified nucleosides detected in the reaction mixture; [, cMG-dGuoA/B; and 9, CEdGA,B.

Discussion

Figure 8. Incubation of DNA with 10 µM freshly synthesized MG for 1 week at 37 °C leads to the formation of CEdG (tR(CEdG-A) ) 8.20 min, tR(CEdG-B) ) 9.40 min). The chromatogram shows mass transitions for CEdG (338/178, 338/106, and 338/149).

In this study, we reinvestigated the reaction of MG with nucleosides and DNA using LC-MS/MS techniques. Two peak pairs and two peak quartets were detected as the main products when DNA was reacted with an excess of MG at 37 °C. The first peak pair was assigned to a mono adduct of MG with dGuo due to its molecular mass. Its structure was then unequivocally identified as the two

diastereomers of CEdG (CEdGA,B) by synthesis of the reference compounds and comparison of retention time, UV spectra, and fragmentation pattern in the mass spectrum. Thus, we identified for the first time CEdGA,B as DNA adducts, which are formed by MG at physiological temperatures.

Methylglyoxal DNA Adducts

Figure 9. Possible structures of MG2-dGuo. Left molecule: a bis adduct reported in the literature (18). Right molecule: putative structure of an adduct, which could explain the loss of CO2 in the mass spectrum. A second equivalent of MG is weakly bound to one of the endocyclic nitrogens of CEdG.

CEdGA,B were first described by Ochs and Severin as a reaction product of dGuo and glyceraldehyde (22). Later, various other carbonyl compounds, such as dihydroxyacetone, glucose, or dehydroascorbic acid, were identified as precursors of CEdGA,B or the analogous guanosine derivative carboxyethyl-2′-guanosine (CEGA,B) (23-25). Furthermore, N2-carboxyethyl-9-methylguanine was obtained by heating methylguanine with a 2-fold excess of MG so that it was postulated that CEdG is also formed during the reaction of DNA with methylgloxal (26). Scheme 1 shows the postulated reaction pathway for the formation of CEdGA,B, involving two tautomerization steps, similar to the Amadori rearrangement of sugars (27) and other R-hydroxyaldehydes (28). N2Carboxymethyl-guanosine (CMG), the glyoxal derived analogue to CEdG, has been described before. However, preliminary studies on the reaction conditions indicate that CMG is formed from glyoxal rather at elevated temperatures, whereas the cyclic adduct predominates at 37 °C, even during long-term incubation (29). The mass spectra of the second peak pair indicate the presence of two diastereomers of a reaction product of dAdo and MG. For identification, one peak of the pair was isolated from a reaction mixture of dAdo and MG. To increase the product yield, the mixture was heated at 50 °C for 16 h. NMR and MS data strongly suggest the presence of the two diastereomers of CEdA (CEdAA,B), a so far unknown DNA adduct. In contrast to the known glycation products of DNA, which are all derivates of dGuo, CEdAA,B are the first modifications of dAdo. The formation of CEdAA,B could explain earlier results from Krymkiewicz, who found that 14C-labeled MG binds to polyadenilic acid, however, with a much lower efficiency as compared to the reaction with polyguanylic acid (30). Isolation of the two peak quartets was not possible, due to their instability and degradation during the isolation process. However, the mass spectrum of the peak quartets indicates the presence of diastereomeric adducts of dGuo and dAdo with two molecules of MG. Schneider et al. described the formation of a bis adduct of dGuo with a 20-fold excess of MG (Figure 9; 18). Therefore, we also reacted MG with dGuo as described by Schneider et al. and analyzed the reaction mixture by LC-MS/MS in the positive mode. In this experiment, a bis adduct was detected with the same retention time as MG2-dGuo. Both products showed also the same fragmentation pattern, similar to this one reported by Schneider et al. Interestingly, in the negative mode, a different fragmentation pattern was detected. The three main fragments appeared at 338, 294, and 178, indicating the loss of weakly bound MG, of CO2 and MG, as well as the loss of CO2, deoxyribose, and MG. The latter fragment was also observed for CEdG. MG2-dAdo gave the analo-

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gous fragmentation pattern. Furthermore, a neutral loss scan was performed, which showed a mass loss of 44 in the negative mode for MG2-dGuo indicating the presence of a carboxylic group and the separation of CO2 during fragmentation. The neutral loss scan was also repeated in positive mode at different collision energies, but no loss of CO2 was detected. Therefore, the purified standard of CEdG was injected for neutral loss scan at different collision energies. In accordance with the results for the bis adduct, the loss of CO2 was only detected in negative mode and not in positive mode, although the presence of a carboxylic group in the structure of CEdG is unequivocal, due to the NMR data. The MS data strongly suggest, therefore, that the bis adducts are derivatives of CEdGA,B and CEdAA,B, to which a second molecule of MG is reversibly bound (Figure 9). The binding of the second MG to N7 is in accordance with findings of Schneider et al. (18) and would explain degradation due to depurination during purification (31). Despite sterical hindrance, the MG residue could also be linked, in principle, to N1 or N3 positions of the nucleobases. The postulated structures can be present in four diastereomeric forms, which would explain the appearance of a peak quartet in the HPLC chromatogram. Thus, further experiments are required to elucidate if MG2-dGuo and the previously identified bis adduct are identical or coelute under the conditions applied. However, because both bis adducts are only formed at high, unphysiological MG concentrations, no further attempts were undertaken to characterize the binding site and structure of the second MG residue. Both possible structures are shown in Figure 9. Shapiro et al. first investigated the reaction of guanosine with more than 12-fold excess of MG. After 5 min of incubation, the guanosine was completely converted into the cyclic adduct cMG-Guo (15). Using 32P-postlabling, Vaca et al. detected cMG-dGuo, when DNA was incubated with 40 mM MG for 25 h or in the DNA of cells, which were treated with 3 mM MG overnight or with 30 mM MG for 1 h (16, 17). In contrast, in none of our reaction mixtures of MG and DNA, peaks were recorded with masses corresponding to the cyclic adducts. Likewise, the cyclic adducts were not detectable, when the free nucleotide was incubated with MG as long as the reaction was stopped with OPD to trap unreacted MG. Therefore, we prepared cMG-dGuo according to Shapiro et al. (15). The diastereomers of the cyclic adduct appeared in the HPLC-MS/MS as a novel peak pair with m/z of 338, which differed in retention time and fragmentation pattern from CEdGA,B. Then, we incubated dGuo with an excess of MG without trapping unreacted MG by OPD and recorded a time course of product formation between 1 and 7 days (Figure 7). Immediately after the addition of MG, cMG-dGuo was detected in the highest concentrations, whereas CEdGA,B was not present. During further incubation, the concentration of cMGdGuo decreased during the first 24 h rapidly and then more slowly until it was hardly detectable after 1 week. In contrast, the concentration of CEdGA,B increased accordingly and the two diastereomers were the main products after 7 days of reaction time. This time course indicates a very rapid and reversible formation of cMGdGuo and a slower formation of the stable CEdGA,B. Alternatively, cMG-dGuo could be an intermediate in the formation of CEdGA,B (Scheme 1). The low stability of the cyclic adducts at neutral or slightly alkaline pH, par-

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ticularly in the absence of the dicarbonyl compound, was reported before (15, 32). Because cMG-dGuo is formed reversibly, the dicarbonyl trapping reagent OPD most likely shifts the reaction equilibrium toward the educts. Additionally, the nucleophil OPD may also react directly with cMG-dGuo leading to degradation (33). Furthermore, the cyclic adduct could be partially degraded during enzymatic DNA hydrolysis (34). Thus, it can be concluded that cMG-dGuo is formed after short-term reaction of DNA with MG, whereas CEdGA,B predominate during long-term exposure of DNA to MG or when DNA analysis is performed several days after short-term exposure. This conclusion is supported by the previous studies where cMG-dGuo was detected, when the reaction was carried out for e25 h. In contrast, in our studies, DNA was incubated for 1 week. When the reaction of DNA or nucleosides was carried out at physiological temperatures with various concentrations of MG, it turned out that the formation of the bis adducts and of CEdAA,B is only favored in the presence of a large excess of MG. With physiological concentrations of MG (10-100 µM), only CEdGA,B were detected. Therefore, we conclude that CEdGA,B are the products, which are most likely detected when DNA modifications by MG in vitro or in vivo are monitored. CEdGA,B are stable end products, which accumulate during prolonged lifetime of the DNA and which can be reliably analyzed by LC-MS/MS after enzymatic hydrolysis. Among the four MG-DNA modifications that are detected under the applied conditions, CEdGA,B are formed in the highest concentrations.

References (1) Nagao, M., Fujita, Y., Sugimura, T., and Kosuge, T. (1986) Methylglyoxal in beverages and foods: its mutagenicity and carcinogenicity. IARC Sci. Publ. 70, 283-291. (2) Tauer, A., Knerr, T., Niwa, T., Schaub, T. P., Lage, C., PasslickDeetjen, J., and Pischetsrieder, M. (2001) In vitro formation of N(epsilon)-(carboxymethyl)lysine and imidazolone under conditions similar to continuous ambulatory peritoneal dialysis. Biochem. Biophys. Res. Commun. 280 (5), 1408-1414. (3) Richard, J. P. (1993) Mechanism for the formation of methylglyoxal from triosephosphates. Biochem. Soc. Trans. 21 (2), 549-553. (4) Kalapos, M. P. (1999) Possible physiological roles of acetone metabolism in humans. Med. Hypotheses 53 (3), 236-242. (5) Lyles, G. A., and Chalmers, J. (1992) The metabolism of aminoacetone to methylglyoxal by semicarbazide-sensitive amine oxidase in human umbilical artery. Biochem. Pharmacol. 43 (7), 1409-1414. (6) Lo, T. W., Westwood, M. E., McLellan, A. C., Selwood, T., and Thornalley, P. J. (1994) Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin. J. Biol. Chem. 269 (51), 32299-32305. (7) Ahmed, M. U., Brinkmann Frye, E., Degenhardt, T. P., Thorpe, S. R., and Baynes, J. W. (1997) N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem. J. 324 (Part 2), 565-570. (8) Thornalley, P. J. (1998) Glutathione-dependent detoxification of alpha-oxoaldehydes by the glyoxalase system: Involvement in disease mechanisms and antiproliferative activity of glyoxalase I inhibitors. Chem.-Biol. Interact. 111-112, 137-151. (9) Welten, A. G., Schalkwijk, C. G., ter Wee, P. M., Meijer, S., van den Born, J., and Beelen, R. J. (2003) Single exposure of mesothelial cells to glucose degradation products (GDPs) yields early advanced glycation end-products (AGEs) and a proinflammatory response. Peritoneal Dial. Int. 23 (3), 213-221. (10) Fukunaga, M., Miyata, S., Liu, B. F., Miyazaki, H., Hirota, Y., Higo, S., Hamada, Y., Ueyama, S., and Kasuga, M. (2004) Methylglyoxal induces apoptosis through activation of p38 MAPK in rat Schwann cells. Biochem. Biophys. Res. Commun. 320 (3), 689-695.

Frischmann et al. (11) Roberts, M. J., Wondrak, G. T., Laurean, D. C., Jacobson, M. K., and Jacobson, E. L. (2003) DNA damage by carbonyl stress in human skin cells. Mutat. Res. 522 (1-2), 45-56. (12) Murata-Kamiya, N., Kamiya, H., Kaji, H., and Kasai, H. (2000) Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells. Mutat. Res. 468 (2), 173-182. (13) Migliore, L., Barale, R., Bosco, E., Giorgelli, F., Minunni, M., Scarpato, R., and Loprieno, N. (1990) Genotoxicity of methylglyoxal: Cytogenetic damage in human lymphocytes in vitro and in intestinal cells of mice. Carcinogenesis 11 (9), 1503-1507. (14) Pischetsrieder, M., Seidel, W., Munch, G., and Schinzel, R. (1999) N(2)-(1-Carboxyethyl)deoxyguanosine, a nonenzymatic glycation adduct of DNA, induces single-strand breaks and increases mutation frequencies. Biochem. Biophys. Res. Commun. 264 (2), 544-549. (15) Shapiro, R., Cohen, B. L., Shiuey, S., and Maurer, H. (1969) On the reaction of guanine with glyoxal, pyruvaldehyde and kethoxal, and the structure of the acylguanines. A new synthesis of N2alkylguanines. Biochemistry 8, 238-244. (16) Vaca, C. E., Fang, J. L., Conradi, M., and Hou, S. M. (1994) Development of a 32P-postlabeling method for the analysis of 2′deoxyguanosine-3′-monophosphate and DNA adducts of methylglyoxal. Carcinogenesis 15 (9), 1887-1894. (17) Vaca, C. E., Nilson, J. A., Fang, J. L., and Grafstro¨m, R. C. (1998) Formation of DNA adducts in human buccal epithelial cells exposed to acetaldehyde and methylglyoxal in vitro. Chem.-Biol. Interact. 108, 197-208. (18) Schneider, M., Quistad, G. B., and Casida, J. E. (1998) N2,7-bis(1-hydroxy-2-oxopropyl)-2′-deoxyguanosine: Identical noncyclic adducts with 1,3-dichloropropene epoxides and methylglyoxal. Chem. Res. Toxicol. 11 (12), 1536-1542. (19) McLellan, A. C., and Thornalley, P. J. (1992) Synthesis and chromatography of 1,2-diamino-4,5-dimethoxybenzene, 6,7-dimethoxy2-methylquinoxaline and 6,7-dimethoxy-2,3-dimethylquinoxaline for use in a liquid chromatographic fluorimetric assay of methylglyoxal. Anal. Chim. Acta 263, 137-142. (20) Seidel, W., and Pischetsrieder, M. (1998) DNA-glycation leads to depurination by the loss of N2-carboxyethylguanine in vitro. Cell. Mol. Biol. 44 (7), 1165-1170. (21) Kalapos, M. P. (1999) Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. Toxicol. Lett. 110 (3), 145-175. (22) Ochs, S., and Severin, T. (1994) Reaction of 2′-deoxyguanosine with glyceraldehyde. Liebigs Ann. Chem. 851-853. (23) Seidel, W., and Pischetsrieder, M. (1998) Immunochemical detection of N2-[1-(1-carboxy)ethyl]guanosine, an advanced glycation end product formed by the reaction of DNA and reducing sugars or L-ascorbic acid in vitro. Biochim. Biophys. Acta 1425 (3), 478484. (24) Nissl, J., Ochs, S., and Severin, T. (1996) Reaction of guanosine with glucose, ribose, and glucose 6-phosphate. Carbohydr. Res. 289, 55-65. (25) Larisch, B., Pischetsrieder, M., and Severin, T. (1997) Formation of guanosine adducts form L-ascorbic acid under oxidative conditions. Bioorg. Med. Chem. Lett. 7, 2681-2686. (26) Papoulis, A., al-Abed, Y., and Bucala, R. (1995) Identification of N2-(1-carboxyethyl)guanine (CEG) as a guanine advanced glycosylation end product. Biochemistry 34 (2), 648-655. (27) Micheel, F., and Frowein, A. (1957) Die Amadori-Umlagerung aliphatischer N-Glykoside. Chem. Ber. 89, 1599-1605. (28) Liu, Z., and Sayre, L. M. (2003) Model studies on the modification of proteins by lipoxidation-derived 2-hydroxyaldehydes. Chem. Res. Toxicol. 16, 232-241. (29) Seidel, W., and Pischetsrieder, M. (1998) Reaction of guanosine with glucose under oxidative conditions. Bioorg. Med. Chem. Lett. 8, 2017-2022. (30) Krymkiewicz, N. (1973) Reactions of methylglyoxal with nucleic acids. FEBS Lett. 29, 51-54. (31) Shabarova, Z., and Bogdanov, A. (1994) Advanced Organic Chemistry of Nucleic Acids, VCH, Verlag, Weinheim. (32) Shapiro, R., and Hachman, J. (1966) The reaction of guanine derivatives with 1,2-dicarbonyl compounds. Biochemistry 5, 27992807. (33) Brock, A. K., Kozekov, I. D., Rizzo, C. J., and Harris, T. M. (2004) Coupling products of nucleosides with the glyoxal adduct of deoxyguanosine. Chem. Res. Toxicol. 17, 1047-1056. (34) Dennehy, M. K., and Loeppky, R. N. (2005) Mass spectrometric methodology for the determination of glyoxaldeoxyguanosine and O6-hydroxyethyldeoxyguanosine DNA adducts produced by nitrosamine bident carcinogens. Chem. Res. Toxicol. 18 (3), 556-565.

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