A Schiff Base Is a Major DNA Adduct of ... - ACS Publications

Chem. Res. Toxicol. 2001, 14, 423-430

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A Schiff Base Is a Major DNA Adduct of Crotonaldehyde Mingyao Wang, Edward J. McIntee, Guang Cheng, Yongli Shi, Peter W. Villalta, and Stephen S. Hecht* University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Received November 9, 2000

Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in human tissues as well as tissues of untreated laboratory animals. A second class of crotonaldehyde-DNA adducts releases 2-(2hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these adducts are quantitatively more significant than the Michael addition adducts in vitro. In this study, we demonstrate that the major source of the paraldol-releasing DNA adducts of crotonaldehyde is a Schiff base. Reaction of crotonaldehyde with DNA, followed by treatment with NaBH3CN and enzyme hydrolysis, resulted in the formation of N2-(3-hydroxybutyl)dG (10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with dG demonstrated that the Schiff base precursor to N2-(3-hydroxybutyl)dG is N2-(3-hydroxybutylidene)dG (7), identified by UV, LC-APCI-MS, and MS/MS. Four isomers of N2-(3hydroxybutylidene)dG were observed. The (R)- and (S)-isomers were identified by reactions of chiral paraldol with dG; each existed as a pair of interconverting (E)- and (Z)-isomers. These data indicate that the structure of the major Schiff base DNA adduct in crotonaldehyde-treated DNA is N2-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and accounts for more than 90% of the paraldol released from crotonaldehyde-treated DNA. However, the adduct is stable in DNA and therefore is a likely companion to the Michael addition adducts in human DNA.

Introduction Crotonaldehyde (1, Scheme 1) is a mutagen and carcinogen (1, 2). This simple R,β-unsaturated aldehyde is a common environmental contaminant, found in mobile source emissions, the atmosphere, tobacco smoke, and other thermal degradation mixtures (1). Crotonaldehyde is also a product of lipid peroxidation and a metabolite of the hepatocarcinogen, N-nitrosopyrrolidine (3, 4). In a shuttle vector system, crotonaldehyde induces a variety of mutations, including dG-dThd transversions, dGdAdo transitions, and tandem base substitutions (5). These mutations result from crotonaldehyde-DNA adducts. Initial investigations of crotonaldehyde’s reactions with dG and DNA led to the discovery of the Michael addition pathway, resulting in 1,N2-propano-dG adducts 2 as well as adducts 3-5 (Scheme 1) (6-9). Diastereomers of each of these products have been characterized. Chung and co-workers have detected two diastereomers of 2 (with methyl and hydroxy trans to each other) in DNA of various tissues from untreated laboratory animals as well as humans (8, 10). They propose that these adducts arise from endogenous lipid peroxidation as well as exogenous exposures such as cigarette smoke. The presence of DNA adducts of crotonaldehyde in human tissues suggests that they may play some role in cancer development. We have recently discovered a second pathway of crotonaldehyde-DNA adduct formation (11). When cro* To whom correspondence should be addressed: University of Minnesota Cancer Center, Mayo Mail Code 806, 420 Delaware St. SE, Minneapolis, MN 55455. Telephone: (612) 624-7604. Fax: (612) 6265135. E-mail: [email protected].

tonaldehyde-treated DNA is subjected to enzymatic hydrolysis (EH), neutral thermal hydrolysis (NTH),1 or acid hydrolysis, significant amounts of 2-(2-hydroxypropyl)4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12, Scheme 1) are released. Paraldol is the dimer of 3-hydroxybutanal (6). Paraldol-releasing DNA adducts were 13-75 times more prevalent than Michael addition adduct 2 in crotonaldehyde-treated DNA (11). This suggests that these adducts may also be found in significant quantities in human DNA. Therefore, we investigated the structures of the paraldol-releasing DNA adducts. We identified several diastereomers of N2-[2-(2-hydroxypropyl)-6-methyl-1,3-dioxan-4-yl]deoxyguanosine (N2-paraldol-dG, 11) and some related adducts resulting from incomplete hydrolysis of the adducted DNA (11). However, N2paraldol-dG and related adducts accounted for less than 10% of the paraldol released from EH or NTH of crotonaldehyde-treated DNA. At present, we have characterized the major paraldol-releasing DNA adduct of crotonaldehyde as N2-(3-hydroxybutylidene)dG (7, Scheme 1).

Experimental Section Apparatus and Assay Conditions. HPLC analyses were carried out with Waters Associates (Waters Division, Millipore, Milford, MA) systems equipped with a model 991 or 996 photodiode array detector. Flow rates were 1 mL/min unless noted otherwise. The following solvent elution systems were 1 Abbreviations: 2,4-DNP, 2,4-dinitrophenylhydrazine; N2-paraldoldG, N2-[2-(2-hydroxypropyl)-6-methyl-1,3-dioxan-4-yl]deoxyguanosine; NTH, neutral thermal hydrolysis; paraldol, 2-(2-hydroxypropyl)-4hydroxy-6-methyl-1,3-dioxane.

10.1021/tx000234w CCC: $20.00 © 2001 American Chemical Society Published on Web 03/17/2001

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Scheme 1. Adduct Formation in the Reaction of Crotonaldehyde (1) with dG or DNAa

a Several stereoisomers of Michael addition products 2-5 as well as N2-paraldol-dG (11) have been characterized (4, 7-9, 11). EH, enzyme hydrolysis; NTH, neutral thermal hydrolysis.

used: (1) two 4.6 mm × 25 cm, 5 µm Supelcosil LC 18-BD columns (Supelco, Bellefonte, PA) with isocratic elution by 5% CH3CN in 10 mM sodium phosphate buffer (pH 7) for 10 min and then a gradient from 5 to 25% CH3CN over the course of 60 min and detection by UV (254 nm), for analysis of reactions of crotonaldehyde or racemic paraldol with dG, and hydrolysates of crotonaldehyde-treated DNA; (2) one of the columns used in system 1 with isocratic elution by 5% CH3OH in 40 mM ammonium acetate buffer (pH 6.6) for 10 min and then a gradient from 5 to 35% CH3OH over the course of 60 min at a flow rate of 0.5 mL/min, with detection by UV (254 nm), for collection of Schiff bases for LC-APCI-MS analysis and analysis of reactions of chiral paraldol with dG; and (3) a 4.6 mm × 25 cm, 5 µm OD5 octadecyl column (Burdick & Jackson, Baxter, McGaw Park, IL) with elution by a gradient from 40 to 60% CH3CN in H2O over the course of 40 min with UV detection (365 nm), for analysis of 2,4-dinitrophenylhydrazones of crotonaldehyde and 3-hydroxybutanal. LC-MS and LC-MS/MS analyses were carried out on a Finnigan MAT LCQ Deca instrument (Thermoquest LC/MS Division, San Jose, CA) interfaced with a Waters Alliance 2690 HPLC multisolvent delivery system and equipped with an SPD10A UV-vis detector (Shimadzu Scientific Instruments, Columbia, MD). For LC-APCI-MS, the same column as in HPLC system 3 was used, with elution by a gradient from 0 to 60% CH3CN in H2O over the course of 60 min at a flow rate of 0.5 mL/min. The APCI source was set as follows: voltage, 3.5 kV; current, 4.8 µA; capillary temperature, 200 °C; and vaporizer temperature, 400 °C. For LC-ESI-MS, the same column as in HPLC system 3 was used, with elution by a gradient from 0 to 30% CH3CN in 1% acetic acid over the course of 60 min at a flow rate of 0.5 mL/min. The ESI source was set as follows: voltage, 2.0 kV; current, 11 µA; and capillary temperature, 275 °C. MS/MS data were acquired with the following parameters: parent mass, 338 amu; isolation width, 1.5 amu; activation amplitude, 25%; activation Q, 0.25; and activation time, 30 ms.

NMR data were obtained on an 800 MHz instrument (Varian, Inc., Palo Alto, CA) using Shigemi 3 mm tubes (Shigemi, Inc., Allison Park, PA). Chemicals and Enzymes. 2,4-Dinitrophenylhydrazine (2,4DNP) reagent was purchased from Eastman Organic Chemicals (Rochester, NY). Crotonaldehyde and NaBH3CN were obtained from Aldrich Chemical Co. (Milwaukee, WI). Freshly opened bottles of crotonaldehyde had no detectable impurities, as determined by GC analysis (11). Alkaline phosphatase was purchased from Boehringer Mannheim Co. (Indianapolis, IN). Paraldol and chiral paraldol were prepared as described previously (11, 12). All other chemicals and enzymes were obtained from Sigma Chemical Co. (St. Louis, MO). Reactions. (1) Crotonaldehyde and DNA. Crotonaldehyde (0.4 mmol) from a freshly opened bottle was allowed to react with calf thymus DNA (20 mg) in 2 mL of 0.1 M phosphate buffer (pH 7) at 37 °C for 96 h. The modified DNA was precipitated by addition of ethanol and then washed with 70% ethanol and ethanol. This DNA sample was used for the analyses presented in Figures 1 and 2. (2) Crotonaldehyde or Paraldol and dG. Crotonaldehyde (0.18 mmol) was allowed to react with dG (20 mg) in 10 mL of 0.1 M phosphate buffer (pH 7) at 37 °C for 22 h. Paraldol or chiral paraldol (0.13 mmol) was allowed to react with dG (6.7 mg) in 2.5 mL of 0.1 M phosphate buffer at 37 °C for 22 (racemic) or 96 h (chiral). Hydrolysis of DNA. EH and NTH were carried out as previously described (11, 13). Treatment of DNA with NaBH3CN followed by enzymatic hydrolysis was also performed as described previously (9). Paraldol was also treated in this way. Stability of the Schiff Base Adduct in DNA. Aliquots of DNA (2.5 mg) that had been allowed to react with crotonaldehyde for 24 h were washed with ethanol, then were dissolved in 1 mL of Tris-HCl buffer (pH 7), and allowed to stand at 37 °C for 4, 8, 24, or 70 h. The DNA was then treated with

Crotonaldehyde-DNA Adduct

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Figure 1. DNA was allowed to react with crotonaldehyde and then subjected to NTH (A) or treated with NaBH3CN prior to NTH (B). The resulting mixtures were treated with 2,4-DNP reagent and then analyzed by HPLC (system 3). Chromatograms are illustrated. The arrows in panel B represent the retention times of the 2,4-dinitrophenylhydrazones of 3-hydroxybutanal and crotonaldehyde, which were detected only in panel A.

Figure 2. DNA was allowed to react with crotonaldehyde and then subjected to enzyme hydrolysis (A) or treated with NaBH3CN prior to enzyme hydrolysis (B). The resulting mixtures were analyzed by HPLC (system 1), and the chromatograms are illustrated. Numbers above the peaks refer to the structures in Scheme 1. NaBH3CN and enzymatically hydrolyzed. The hydrolysates were analyzed for 10 by HPLC system 2.

Results In a previous study, we carried out HPLC analysis of enzymatic hydrolysates of crotonaldehyde-treated DNA (11). Fractions were collected from each portion of the chromatogram, and each fraction was assayed for paraldol (12) by reaction with 2,4-DNP reagent. When paraldol itself or a paraldol-releasing DNA adduct is present, the 2,4-dinitrophenylhydrazones of 3-hydroxybutanal and crotonaldehyde are detected. The results of this study demonstrated that, during enzymatic hydrolysis of this crotonaldehyde-treated DNA, large amounts of paraldol itself are released, presumably from an unstable DNA adduct. This elutes in the 5-15 min retention time fraction and accounts for at least 90% of the paraldol detected at various points in the chromatogram by the 2,4-DNP reagent method. The remaining 10% is produced

from DNA adducts such as N2-paraldol-dG which release paraldol when treated with the acidic 2,4-DNP reagent. On the basis of these results, we speculated that the major unstable paraldol-releasing DNA adduct of crotonaldehyde was a Schiff base such as 7 or 8. In our study of acetaldehyde-DNA reactions, we developed conditions for analyzing the Schiff base N2ethylidene-dG, which is the major DNA adduct of acetaldehyde (9). Reaction of acetaldehyde-treated DNA with NaBH3CN results in near-quantitative conversion of N2ethylidene-dG to N2-ethyl-dG. If a Schiff base were responsible for release of paraldol during hydrolysis of crotonaldehyde-treated DNA, then reaction of this DNA with NaBH3CN prior to hydrolysis should suppress release of paraldol. As shown in Figure 1A, NTH of crotonaldehyde-treated DNA followed by treatment with 2,4-DNP reagent resulted in formation of the 2,4-dinitrophenylhydrazones of 3-hydroxybutanal and crotonaldehyde, as expected. However, these products were not detected when the DNA was treated with NaBH3CN prior

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Figure 3. UV spectra of (A) N2-ethylidene-dG, (B) N2-ethyl-dG, (C) peak C of Figure 5A, and (D) peak A of Figure 2B. All spectra were obtained in 0.1 M phosphate buffer (pH 7.0).

to NTH (Figure 1B). These results suggested the presence of a Schiff base. In the next step, crotonaldehyde-treated DNA was either hydrolyzed enzymatically or allowed to react with NaBH3CN before enzyme hydrolysis. The hydrolysates were analyzed by HPLC. As shown in Figure 2A, the 1,N2-propano-dG adducts 2 as well as N2-paraldol-dG adducts 11 were detected in hydrolysates of DNA that had not been treated with NaBH3CN, consistent with previous results. However, hydrolysates of the NaBH3CN-treated DNA showed two major new peaks (A and B) eluting at 37.8 min, just prior to the 1,N2-propano-dG diastereomers 2 (Figure 2B). It is evident from Figure 2B that the levels of these peaks far exceed those of 1,N2propano-dG adducts 2 or N2-paraldol-dG (11). Peaks A and B were also formed upon NaBH3CN treatment of reaction mixtures of crotonaldehyde with dG, or paraldol with dG. The UV spectra of peaks A and B were identical; that of peak A is shown in Figure 3D. It is identical to that of N2-ethyl-dG (Figure 3B). ESI-MS of peaks A and B each give a base peak at m/z 340, which is M + 1 of a hydroxybutyl-substituted dG. 1H NMR data for peaks A and B, collected together from HPLC chromatograms of the NaBH3CN-treated paraldol-dG reaction mixture, are summarized in Table 1. All assignments were confirmed by COSY. The 1H NMR data are similar to those previously reported for N2-(4-hydroxybutyl)dG (14). Collectively, these spectral data conclusively establish the structure of peaks A and B as two diastereomers of N2(3-hydroxybutyl)dG (10). Our next goal was to isolate and characterize the postulated Schiff base precursor to 10. As the Schiff base would be unstable to conditions of DNA hydrolysis, we

Figure 4. Chromatogram obtained upon HPLC analysis (system 1) of products formed in the reaction of crotonaldehyde with dG. Numbers by the peaks refer to the structures in Scheme 1. Peaks C-F are isomers of N2-(3-hydroxybutylidene)dG (see the text).

examined the reaction of crotonaldehyde with dG. This produced the chromatogram illustrated in Figure 4. Peaks C-F are the hypothesized Schiff base precursors to 10. Peaks C-F were also formed but in greater relative yield in the reaction of paraldol (12) with dG as illustrated in Figure 5A. The UV spectra and retention times of peaks C-F in Figures 4 and 5A are identical. When the paraldol-dG reaction mixture was treated with NaBH3CN, peaks C-F disappeared with appear-



Table 1. 1H NMR Data (800 MHz) for N2-(3-Hydroxybutyl)dGa

deoxyguanosyl protons C-8H

N2-H

1′-H

2′-Ha,b

3′-H

4′-H

5′-Ha,b

3′-OH

5′-OH

7.79 (s)

7.60 (bs)

6.13 (dd) J ) 7.2, 7.2 Hz

2.17 (m) 2.62 (m)

4.34 (m)

3.79 (m)

3.49 (m) 3.55 (m)

5.25 (bs)

4.91 (bs)

butyl protons 1-Ha,b (t)b

3.29 J ) 6.8 Hz a

2-Ha,b

3-H

4-CH3

1.53 (m) 1.59 (m)

3.69 (m)

1.07 (d) J ) 6.4 Hz

Collected via HPLC; see Figure 5B. b Obscured in the DMSO spectrum. Measured with DMSO and D2O.

Figure 5. Chromatogram obtained upon HPLC analysis (system 1) of products formed in the reaction of paraldol with dG (A) or of paraldol with dG followed by treatment with NaBH3CN (B). Numbers above the peaks refer to the structures in Scheme 1. Peaks C-F are isomers of N2-(3-hydroxybutylidene)dG (see the text).

ance of N2-(3-hydroxybutyl)dG (Figure 5B). Treatment with NaBH3CN of each of peaks C-F of Figure 4A gave N2-(3-hydroxybutyl)dG, identified by retention time, UV, and MS. Furthermore, treatment of each of peaks C-F of Figure 5A with 2,4-DNP reagent gave a mixture of the 2,4-dinitrophenylhydrazones of 3-hydroxybutanal and crotonaldehyde. These data demonstrate that peaks C-F in Figures 4 and 5A are the Schiff base precursors to N2(3-hydroxybutyl)dG. We sought further information about the structures of Schiff base peaks C-F. The UV spectra of peaks C-F were identical; that of peak C of Figure 5A is shown in Figure 3C. A shoulder appears at 275 nm compared to 280 nm in the spectrum of N2-(3-hydroxybutyl)dG (Figure 3D). An identical relationship exists between N2-ethylidene-dG, the Schiff base of acetaldehyde and dG (Figure 3A), and N2-ethyl-dG [Figure 3B (9)]. These UV data, together with the chemical studies described above, provide convincing evidence that peaks C-F of Figures 4 and 5A are Schiff bases. MS data on peaks C-F were obtained by collecting them in HPLC system 2, cooling immediately to -80 °C,

and then analyzing by LC-APCI-MS and MS/MS. These data are summarized in Table 2. MS data for peaks C and D (together) and for each of peaks E and F were similar. Each exhibited an M + 1 ion at m/z 338, as well as ions corresponding to loss of H2O, deoxyribose, deoxyribose with H2O, and deoxyribose with CH3CHO. MS/MS analysis of m/z 338 gave similar fragments for each peak, C-F. These results are consistent with N2-(3-hydroxybutylidene)dG (7), Scheme 1, as the structure of the Schiff base. When peaks E and F were collected without cooling, and then immediately re-injected under the same conditions, the chromatogram illustrated in Figure 6 was obtained. These results demonstrate that peaks E and F are unstable, giving dG as well as smaller amounts of peaks C and D. After the mixture stood for 3 h at room temperature, peaks C-F had completely reverted to dG plus paraldol, as determined by HPLC. Due to the instability of peaks C-F, we were not able to obtain interpretable NMR data. Further information about the structures of peaks C-F was obtained by examining the reactions with dG of

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Table 2. APCI-MS and MS/MS Data for Peaks C-F of Figures 4 and 5Aa (A) APCI-MS

peak

m/z 338 (M + 1)

m/z 320 (M + 1 - H2O)

m/z 294 (M + 1 - CH3CHO)

m/z 222 (M deoxyribose + 1)

m/z 204 (M deoxyribose H2O + 1)

m/z 178 (M deoxyribose CH3CHO + 1)

C + Db E F

10 33 36

8 18 25

NDc ND 3

27 100 100

20 50 62

9 22 28

(B) MS/MS of m/z 338 peak

m/z 294

m/z 222

m/z 204

C D E F

26 24 16 22

100 100 100 100

13 11 7 12

a Values are relative intensities. b Peaks for dG + 1 (m/z 268, relative intensity of 35%) and G + 1 (m/z 152, relative intensity of 100%) were also observed, presumably due to dG in the sample. c ND, not detected.

Figure 6. Chromatogram obtained upon HPLC analysis (system 1) of peaks E and F, which had been collected from the mixture shown in Figure 5A, and re-injected using the same HPLC system.

chiral 3-hydroxybutanal. (S)- and (R)-3-hydroxybutanal (as the corresponding paraldol dimers 12) were prepared as described previously by reduction of either ethyl (S)or (R)-3-hydroxybutyrate with diisobutylaluminum hydride. Reaction of (S)-3-hydroxybutanal (as paraldol dimer 12) with dG gave the chromatogram illustrated in Figure 7A in which peaks D and F prevailed, while the corresponding (R)-isomer gave the chromatogram shown in Figure 7B in which peaks C and E prevailed. Co-injection of these two mixtures of products gave four peaks, C-F. These results demonstrate that peaks D and F have an (S)-3-hydroxybutylidene group, whereas the corresponding configuration in peaks C and E is (R). Therefore, peaks D and F must be (E)- or (Z)-isomers of N2-(S)-(3-hydroxybutylidene)dG, whereas peaks C and E are (E)- or (Z)-isomers of N2-(R)-(3-hydroxybutylidene)dG. These data are all consistent with our assigned structure, N2-(3-hydroxybutylidene)dG (7). However, we could not fully exclude Schiff base 8 in the absence of NMR data. It was possible that treatment of 8 with NaBH3CN could yield N2-(3-hydroxybutyl)dG (10), if the hemiacetal linkage in 8 dissociated under the reaction conditions. To test this possibility, we treated paraldol (12) with NaBH3CN under the same conditions used for reduction of paraldol-dG reaction mixtures. If the hemiacetal linkage of 9 were stable under these conditions, we should obtain products with a molecular weight of 178 resulting from reduction of the aldehyde carbonyl group in 9, whereas if it were unstable, the major product would be 1,3-butanediol, with a molecular weight of 90. LCAPCI-MS analysis of the reaction mixture showed the presence of several peaks having ions at m/z 179, which is consistent with M + 1 of a reduction product in which

the hemiacetal linkage of 9 was stable. Although these products were not identified, the results indicate that the hemiacetal linkage of 8 would be stable during NaBH3CN reduction. Therefore, 10 would not be the expected major product of NaBH3CN treatment of 8. The LCAPCI-MS data which we obtained on the Schiff base products C-F are also not consistent with 8, as we could not detect any ions at m/z 426, which is M + 1 of 8. Furthermore, the presence of two pairs of interconverting isomers (C-F), as discussed above, is consistent with 7, while there are 16 stereoisomers of 8. We investigated the stability of the Schiff base adduct in DNA by incubating crotonaldehyde-treated DNA at pH 7.0 and 37 °C, treating with NaBH3CN, hydrolyzing enzymatically, and analyzing for N2-(3-hydroxybutyl)dG (10). The Schiff base adduct was stable in DNA for at least 70 h under these conditions.

Discussion The results of this study clearly demonstrate that a Schiff base is a major DNA adduct of crotonaldehyde. All of our data are consistent with N2-(3-hydroxybutylidene)dG (7, Scheme 1) as the structure of this Schiff base. Thus, reaction of crotonaldehyde with DNA followed by treatment with NaBH3CN produces N2-(3-hydroxybutyl)dG. UV and MS data obtained from reactions of crotonaldehyde or paraldol with dG also support 7 as the structure of the Schiff base. However, the instability of 7 at the nucleoside level, leading to its decomposition during enzyme hydrolysis, prevented us from obtaining spectral data directly on the DNA adduct. Therefore, it is possible that the Schiff base in DNA is not identical to that formed in the dG reactions. We do not consider this likely on the basis of previous studies of crotonaldehyde-DNA reactions which indicate that adducts in DNA are structurally the same as those formed in reactions with dG (6, 9, 11, 15, 16). In contrast to the instability of 7 at the nucleoside level, our data show that the Schiff base is stable in DNA, where it is presumably protected from hydrolysis. The concentration of crotonaldehyde used in the DNA reactions in this study was high, 200 mM, to facilitate adduct identification. However, we have previously detected substantial amounts of paraldol-releasing adducts in DNA reacted with 0.2 mM crotonaldehyde, and it is very likely that these adducts would also be detected at lower concentrations (11). As shown in Figure 2B, levels of the Schiff base adduct far exceed those of Michael



Figure 7. Chromatograms obtained upon HPLC analysis (system 2) of products formed in the reaction of paraldol with dG: (A) paraldol from (S)-3-hydroxybutanal and (B) paraldol from (R)-3-hydroxybutanal. Peaks D and F are (E)- or (Z)-isomers of N2-(S)(3-hydroxybutylidene)dG, and peaks C and E are (E)- or (Z)-isomers of N2-(R)-(3-hydroxybutylidene)dG. Numbers above the peaks refer to the structures in Scheme 1. The major isomer of 11 formed from (S)-3-hydroxybutanal under these conditions is the first eluting of the four peaks observed with racemic paraldol, and the major isomer from (R)-3-hydroxybutanal is the third eluting peak. [note that in Figure 7 and in the corresponding text of ref 11 describing the formation of 11, the (R)- and (S)-labels were inadvertently switched, and in Figure 8 and the corresponding text of ref 11, the A and B peak labels were switched].

addition adducts 2 in DNA reacted with crotonaldehyde, consistent with our previous finding that paraldolreleasing DNA adducts were 13-75 times more prevalent than 2. The relatively high concentration of the Schiff base compared to that of 2 in crotonaldehyde-treated DNA could have important implications. Chung and co-workers have shown that adducts 2 are present in human tissues, ranging in concentration from 0.003 to 6 µmol/mol of G (8, 10, 17-19). While the highest levels were found in the oral mucosa of smokers, considerable amounts of these adducts have also been detected in various tissues of apparently unexposed individuals. These adducts, which have also been detected in tissues of laboratory animals, most likely arise from endogenous lipid peroxidation reactions. Since the Schiff base adduct is stable in DNA and is formed in vitro in amounts considerably greater than the amount of adducts 2, it is likely that significant amounts of this adduct are present in human DNA. This could have important implications with respect to cancer etiology. Therefore, we are developing MS methods for quantifying this adduct in human DNA. While the concentration of Schiff base adduct 7 in DNA reacted with crotonaldehyde exceeds those of adducts 2, the opposite is seen in reactions with dG. Inspection of Figure 4 indicates that levels of adducts 2 are greater than those of 7 in dG reacted with crotonaldehyde. Formation of the Schiff base can occur by addition of H2O to crotonaldehyde, producing 3-hydroxybutanal (6). The amount of 6 in these reaction mixtures will be far smaller than that of crotonaldehyde. This indicates that 6 reacts considerably faster with DNA than does crotonaldehyde. Apparently, Schiff base formation occurs more rapidly than Michael addition in the DNA reactions, but not in the dG reaction. Alternatively, hydrolysis of the Schiff base back to dG and 6 may compete significantly with the forward reaction, whereas this is not the case in DNA. An alternate pathway leading to Schiff base adduct 7 is initial formation of a crotonaldehyde Schiff base with

dG or DNA followed by Michael addition of H2O, and tautomerization. We consider this less likely than the mechanism shown in Scheme 1 because NaBH3CN treatment did not appear to produce major products resulting from reduction of a crotonaldehyde Schiff base. However, further studies would be necessary to exclude this possibility. As shown in Figure 6, collection of Schiff base peaks E and F, followed by re-injection in the same system, results in appearance of peaks C and D as well as dG. This indicates that peaks E and F, and C and D, interconvert and are therefore pairs of (E)- and (Z)isomers. We could not assign the stereochemistry. However, facile (E)-(Z) interconversion of Schiff bases has been previously demonstrated, particularly in cases where the group attached to nitrogen is aromatic (20, 21). Our data are consistent with these studies. In summary, this study demonstrates that a Schiff base is a major DNA adduct of crotonaldehyde, being responsible for most of the paraldol released upon hydrolysis of crotonaldehyde-treated DNA. This Schiff base is stable in DNA and is present in substantially greater quantities than the Michael adducts 2, suggesting that the Schiff base adduct may be present in significant quantities in human DNA.

Acknowledgment. This study was supported by Grant CA-85702 from the National Cancer Institute. S.S.H. is an American Cancer Society Research Professor, supported by ACS grant RP-00-138.

References (1) International Agency for Research on Cancer (1995) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Dry Cleaning, Some Chlorinated Solvents and Other Industrial Chemicals, Vol. 63, pp 272-391, Lyon, France. (2) Chung, F. L., Tanaka, T., and Hecht, S. S. (1986) Induction of liver tumors in F344 rats by crotonaldehyde. Cancer Res. 46, 1285-1289.

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