Lactols in Hydrolysates of DNA Treated with α-Acetoxy-N

Crotonaldehyde (0.76 mmol) was similarly reacted with DNA (100 mg).b Neutral .... Tricker, A. R., Stickler, D. J., Chawla, J. C., and Preussmann, R. (...
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Chem. Res. Toxicol. 1998, 11, 1567-1573

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Lactols in Hydrolysates of DNA Treated with r-Acetoxy-N-nitrosopyrrolidine or Crotonaldehyde Mingyao Wang, Pramod Upadhyaya, Trieu T. Dinh, Leo E. Bonilla, and Stephen S. Hecht* University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware Street SE, Minneapolis, Minnesota 55455 Received July 13, 1998

R-Acetoxy-N-nitrosopyrrolidine (R-acetoxyNPYR) is a stable precursor to R-hydroxyNPYR, the initial product of metabolism and proposed proximate carcinogen of N-nitrosopyrrolidine (NPYR). Crotonaldehyde (2-butenal) is a metabolite of NPYR and also a mutagen and carcinogen. Both R-acetoxyNPYR and crotonaldehyde form DNA adducts, but these reactions have not been completely characterized. In previous studies, we detected substantial amounts of unidentified radioactivity in hydrolysates of DNA that had been treated with radiolabeled R-acetoxyNPYR. In this study, we have characterized these products as 2-hydroxytetrahydrofuran, the cyclic form of 4-hydroxybutanal, and paraldol, the dimer of 3-hydroxybutanal. These products were identified by comparison to standards and by conversion to 2,4dinitrophenylhydrazones. 2-Hydroxytetrahydrofuran is the major product in neutral thermal hydrolysates of R-acetoxyNPYR-treated DNA and is derived predominantly from N2-(tetrahydrofuran-2-yl)deoxyguanosine 8. Paraldol is present to a lesser extent than 2-hydroxytetrahydrofuran in these reactions and is formed from paraldol-releasing adducts, which in turn are produced in the reaction of crotonaldehyde, a solvolysis product of R-acetoxyNPYR, with DNA. Other products in hydrolysates of R-acetoxyNPYR-treated DNA are N7-substituted guanines 5 and 6, cyclic N7-C8 guanines 4, 11, and 12, and 1,N2-propanodeoxyguanosines 9 and 10. Paraldol is a major product in hydrolysates of crotonaldehyde-treated DNA, being present in amounts 100 times greater than those of previously identified adducts 9 and 10. The results of this study provide a more complete picture of the reactions of R-acetoxyNPYR with DNA and yield some new insights about possible endogenous DNA adducts formed from crotonaldehyde.

Introduction N-Nitrosopyrrolidine (NPYR)1 is a representative carcinogenic cyclic nitrosamine. R-AcetoxyNPYR (Figure 1) is a stable precursor to R-hydroxyNPYR (1), the major initial metabolite and proximate carcinogen of NPYR (1, 2). NPYR is a well-established hepatocarcinogen in the rat and a respiratory carcinogen in the Syrian golden hamster (3). NPYR occurs in the human diet and in tobacco smoke (4, 5). It is a likely product of nitric oxidemediated endogenous nitrosation in humans and has been detected in human urine (6-9). Crotonaldehyde (2butenal) is a mutagen and a hepatocarinogen in rats (10, 11). It occurs in the human diet, tobacco smoke, and mobile source emissions as well as being a product of lipid peroxidation (11, 12). It is also a solvolysis product of R-acetoxyNPYR and a metabolite of NPYR (Figure 1) (13). In this study, we identified lactols as significant products in hydrolysates of DNA treated with R-acetoxyNPYR or crotonaldehyde. Our goal is to understand the metabolism and DNA adduct formation by NPYR. DNA adducts are important in carcinogenesis. While adduct formation from simple Abbreviations: R-acetoxyNPYR, R-acetoxy-N-nitrosopyrrolidine; 2,4-DNP, 2,4-dinitrophenylhydrazone; 2,4-DNP reagent, 2,4-dinitrophenylhydrazine reagent; 3-HOBA, 3-hydroxybutanal; 4-HOBA, 4-hydroxybutanal; R-hydroxyNPYR, R-hydroxy-N-nitrosopyrrolidine; 2-hydroxyTHF, 2-hydroxytetrahydrofuran; NPYR, N-nitrosopyrrolidine; NTH, neutral thermal hydrolysis; PCI, positive ion chemical ionization. 1

acyclic nitrosamines such as N-nitrosodimethylamine and N-nitrosodiethylamine is well-characterized and results from predictable reactions of alkanediazonium ions with DNA bases (3), adduct formation from cyclic nitrosamines such as NPYR is more complex, presumably due to the intermediacy of aldehydic diazonium ions and related intermediates such as 2, 3, and 7 (Figure 1), which are formed by spontaneous decomposition of R-hydroxyNPYR (1). An understanding of DNA adduct formation from NPYR will provide insights about these pathways for other carcinogenic cyclic nitrosamines such as N-nitrosopiperidine, N-nitrosomorpholine, and N′-nitrosonornicotine, for which the spectrum of DNA adducts is largely uncharacterized (14-18). Moreover, DNA adducts and related products of DNA hydrolysis can be used as biomarkers for the uptake and metabolic activation of carcinogens. This is particularly important in the case of nitrosamines such as NPYR which are extensively metabolized in vivo. Thus, only small amounts of NPYR and 2-hydroxyTHF, a major metabolite resulting from R-hydroxylation in vitro, are detected in the urine of NPYR-treated animals (2, 6). In previous studies, we have characterized several DNA adducts as products of the reaction of R-acetoxyNPYR with DNA. These adducts are illustrated in Figure 1. The N7-C8 guanine adduct 4, the open chain N7substituted adduct 5, and the N2-tetrahydrofuranyl adduct 8 are the most prevalent (15, 19). Adducts 9-12

10.1021/tx980165+ CCC: $15.00 © 1998 American Chemical Society Published on Web 11/12/1998

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Figure 1. Products formed in the solvolysis of R-acetoxyNPYR and in its reactions with DNA. See the text for details. NTH is neutral thermal hydrolysis.

are formed via crotonaldehyde, a product of the solvolysis of R-acetoxyNPYR (13, 20-22). Adducts 4, 5, 9, and 10 have been detected in rat liver DNA following treatment with NPYR (20, 23, 24). Despite what appears to be a relatively complete characterization of DNA adduct formation from R-acetoxyNPYR, chromatograms of neutral thermal hydrolysates of DNA that had been treated with radiolabeled R-acetoxyNPYR showed substantial amounts of unidentified material. One of these chromatograms is illustrated in Figure 2. This chromatogram had large unidentified early eluting peaks, labeled P9 and P12. Characterization of this early eluting radioactivity was the focus of this study.

Experimental Section Caution: R-AcetoxyNPYR is a mutagen and chemically activated form of the carcinogen NPYR. Crotonaldehyde is a mutagen and carcinogen. These compounds should be handled with extreme care, using appropriate safety wear and ventilation at all times. Apparatus. HPLC analyses were carried out with Waters Associates (Waters Division, Millipore, Milford, MA) systems equipped with a model 991 or 996 photodiode array detector, a

Gilson model 116 UV detector (Gilson Medical Electronics, Middletown, WI), an RF-10AXL spectrofluorometric detector (Shimadzu Scientific Instruments, Columbia, MA), or a β-RAM radioactivity HPLC flow-through detector (IN/US Systems, Tampa, FL). The following systems were used: (1) two 4.6 mm × 25 cm Partisil-10 SCX strong cation exchange columns (Whatman, Clifton, NJ) and an elution medium of 40 mM ammonium phosphate buffer (pH 2.0) at a flow rate of 1 mL/ min with fluorescence (excitation at 290 nm and emission at 380 nm) or radio-flow detection; (2) two 4.6 mm × 25 cm Supelcosil LC 18-DB columns (Supelco, Bellefonte, PA), the eluant being a gradient from 0 to 20% CH3CN in H2O over the course of 20 min at a flow rate of 1 mL/min with UV detection (280 nm); (3) the same columns as in system 2 with isocratic elution by 5% CH3CN in 10 mM sodium phosphate buffer (pH 7.0) for 10 min and then a gradient from 5 to 25% CH3CN over the course of 60 min at a flow rate of 1 mL/min with UV detection (254 nm); (4) a 9.4 mm × 50 cm Partisil 10 ODS-3 Magnum 9 column (Whatman) with elution by a gradient from 0 to 80% CH3CN in H2O over the course of 20 min at a flow rate of 4 mL/min with UV detection (280 nm); (5) a 4.6 mm × 25 cm 5µ OD5 octadecyl column (Burdick and Jackson, Baxter, McGaw Park, IL) with elution by a gradient from 40 to 60% CH3CN in H2O over the course of 40 min at a flow rate of 1 mL/min with UV detection (365 nm); and (6) the same column as in system 5, the eluant in this case being a gradient of 0 to

Lactols in DNA Hydrolysates

Figure 2. HPLC analysis of a neutral thermal hydrolysate of [2,3,4,5-3H]-R-acetoxyNPYR-treated DNA. The analysis was performed in a previous study (15). The traces were obtained using HPLC system 1. The upper trace illustrates the retention times of standard adducts 4, 5, 11, and 12 (Figure 1) and guanine (G), as detected by fluorescence. The lower panel illustrates radio-flow detection of products in the neutral thermal hydrolysate. 30% CH3CN in H2O over the course of 30 min at a flow rate of 1 mL/min with UV (254 nm) or radio-flow detection. GC/MS was carried out on a Finnigan MAT 90 doublefocusing mass spectrometer (Finnigan MAT, Bremen, Germany) interfaced with a Hewlett-Packard model 5890 gas chromatograph equipped with split-splitless injection and a 0.25 mm × 60 m DB-5 column (J & W Scientific, Folsom, CA). Samples were introduced at 50 °C, with no oven temperature ramp. Mass spectra were obtained using PCI with CH4 or NH3 as the reagent gas. Electron ionization mass spectra were obtained by direct insertion. The probe was heated at 25 °C/min to a final temperature of 150 °C. 1H NMR data were obtained on a GE Omega 300 spectrometer. Chemicals and Enzymes. R-AcetoxyNPYR and 2-hydroxyTHF were synthesized (25, 26). 2,4-DNP reagent was prepared by dissolving 250 mg of 2,4-dinitrophenylhydrazine (Eastman Organic Chemicals, Rochester, NY) in 100 mL of 6 N HCI. Crotonaldehyde-2,4-DNP and 4-HOBA-2,4-DNP were prepared from crotonaldehyde and 2-hydroxyTHF (13). Paraldol was allowed to react with 2,4-DNP reagent, and the products were separated by HPLC system 5. Their electron ionization mass spectra were determined. [3H]H2O was purchased from Amersham Life Sciences, Inc. (Arlington Heights, IL). All other reagents and enzymes were obtained from Aldrich Chemical Co. (Milwaukee, WI) and Sigma Chemical Co. (St. Louis, MO). 2-(2-Hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol) was prepared essentially as described previously (27). To 50 g of cold acetaldehyde was added 1.25 mL of 10% aqueous NaOH dropwise while the mixture was being stirred at 5 °C. The mixture was kept at 4-5 °C for 30 min, then stirred for 1 h at 5 °C, and then adjusted to pH 3-4 with tartaric acid. After filtration, the product was distilled in vacuo at an oil bath temperature of 140 °C. The distillate [bp 57 °C (1 mmHg), 5.4 g] was further purified using HPLC system 5, with a retention time of 10 min: 1H NMR for the major conformer of paraldol (Figure 1) (D2O) δ 5.03 (m, 1H, ring 4-CHOH), 4.83 (m, 1H, ring 2-CH), 3.92 (m, ring 6-CHCH3 + 2-CHOHCH3), 1.69 (m, 4H, ring 5-H2 + CH2CHOH), 1.2 (m, ring 6-CH3 + CHOHCH3), essentially as described in ref 28; MS PCI (CH4) m/z (relative intensity) 161 (M - 15, 2), 89 (CHOHCH2CHOHCH3, 9), 73 (22), 71 (OdCHCH2CHCH3, 100). Reactions. (1) r-AcetoxyNPYR and DNA. R-AcetoxyNPYR (6.0 g, 38 mmol) was added to a solution of 5 g of calf

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1569 thymus DNA in 300 mL of 0.1 M phosphate buffer (pH 7). The mixture was incubated at 37 °C for 20 h while it was being shaken. The reaction mixture was diluted with 300 mL of H2O and then extracted three times with 300 mL of CHCl3/isoamyl alcohol (24:1). After each extraction, the mixture was centrifuged at 10000g for 20 min. The supernatants were removed, combined, and mixed with 30 mL of 5 M NaCl and 450 mL of ethanol to precipitate the DNA. The DNA was washed with 450 mL of 70% ethanol and then twice with 100 mL of ethanol. It was dried under a stream of N2. The DNA was hydrolyzed and analyzed as described below. (2) Crotonaldehyde and DNA. Crotonaldehyde (56 mg, 0.76 mmol) was added to a solution of 100 mg of calf thymus DNA in 6 mL of 0.1 M phosphate buffer (pH 7). The mixture was incubated at 37 °C for 20 h while it was being shaken. The reaction mixture was diluted with 22 mL of H2O and then extracted with 12 mL of CHCl3/isoamyl alcohol (24:1). The mixture was centrifuged at 10000g for 20 min. The supernatant was removed and mixed with 2.8 mL of 5 M NaCl and 30 mL of ethanol to precipitate the DNA. The DNA was washed with 5 mL of 70% ethanol and then twice with 10 mL of ethanol. It was dried under a stream of N2. For neutral thermal and acid hydrolysis, DNA (10 mg) was dissolved in 1 mL of 10 mM sodium cacodylate buffer (pH 7.0) and the mixture was heated at 100 °C for 1 h. To the resulting mixture was added 0.1 mL of 1 N HCl to precipitate the partially apurinic DNA. An aliquot of the supernatant was analyzed for adducts 4, 11, and 12 using HPLC system 1. Another aliquot (0.8 mL) was mixed with 50 µL of 2,4-DNP reagent and the mixture allowed to stand overnight at room temperature. The mixture was extracted twice with 3 mL of CH2Cl2. The extracts were concentrated to dryness, redissolved in methanol, and analyzed by HPLC system 5. The precipitated, partially apurinic DNA was dissolved in 1 mL of 0.1 N HCl and the mixture heated at 85 °C for 1 h to release adducts 9 and 10, as the guanine base, which were analyzed using HPLC system 1. For enzyme hydrolysis, DNA (15 mg) was dissolved in 1.5 mL of 10 mM Tris-HCl/5 mM MgCl2 buffer (pH 7.0). The mixture was incubated with 7500 units of DNaseI at 37 °C for 10 min and then with 0.38 unit of phophodiesterase I and 2300 units of alkaline phosphatase for an additional 60 min. Enzymes were removed by centrifugation using a centrifree MPS device (MW cutoff of 30 000; Amicon, Inc., Beverly, MA). The hydrolysate (0.4 mL) was analyzed for adducts 8-10 by HPLC system 3. Another portion of the hydrolysate (0.8 mL) was treated with 50 µL of 2,4-DNP reagent, extracted with CH2Cl2, and analyzed by HPLC system 5. Collection and Analysis of P9 and P12. The retention times of paraldol and 2-hydroxyTHF were established by HPLC system 1. Neutral thermal hydrolysates of DNA reacted with R-acetoxyNPYR were analyzed in this system. Material eluting at the retention times of 2-hydroxyTHF and paraldol (1.5-1.7 mL) was collected and then concentrated to 1 mL. 2,4-DNP reagent (50 µL) was added to each concentrated solution. After standing overnight at room temperature, the reaction mixture was extracted twice with 3 mL of CH2Cl2. The extracts were combined and concentrated in vacuo to dryness. The residue was dissolved in methanol and analyzed by HPLC system 5. The retention times of paraldol and 2-hydroxyTHF were also established by HPLC system 2. Neutral thermal hydrolysates of DNA reacted with R-acetoxyNPYR were analyzed, and the material eluting at the correct retention times was collected as described above. The collected material was concentrated and repurified in the same HPLC system. This material was concentrated and analyzed by GC/MS.

Results Preliminary data indicated that P9 and P12 had the same retention times and formed the same 2,4-DNPs as two products of the solvolysis of R-acetoxyNPYR-paraldol and 2-hydroxyTHF, respectively (Figure 1). Paraldol

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Table 1. HPLC Retention Times of Paraldol, 2-HydroxyTHF, and Derived 2,4-DNPs retention time (min)

paraldol 2-hydroxyTHF 3-HOBA-2,4-DNP crotonaldehyde-2,4-DNP 4-HOBA-2,4-DNP

HPLC system 1

HPLC system 2

HPLC system 5

8.39 9.56 -

12.6 23.8 -

11.7 35.9 11.2

is the dimer of 3-HOBA, while 2-hydroxyTHF is the cyclic form of 4-HOBA. Therefore, these standards were prepared and were used to establish the retention times of P9 and P12 with the strong cation exchange and reversephase HPLC systems employed in this study (Table 1). In previous studies, we have found that 2,4-DNP formation is an effective way to isolate and quantify lactols (2, 13, 29). Published data on 3-HOBA-2,4-DNP, the expected product of derivatization of paraldol, are ambiguous with respect to the possible formation of crotonaldehyde-2,4-DNP (30). Therefore, we reacted paraldol with 2,4-DNP reagent. The products were separated by HPLC (Table 1) and identified by their electron ionization mass and UV spectra. The electron ionization mass spectrum of the product which was eluted at 11.7 min had a molecular ion at m/z 268 and a base peak at m/z 45 ([CH3CHOH]+). Its UV spectrum was similar to that of 4-HOBA-2,4-DNP (Figure 3). These results demonstrate that the 11.7 min peak is 3-HOBA2,4-DNP. The product which was eluted at 35.9 min had a molecular ion and base peak of m/z 250, expected for crotonaldehyde-2,4-DNP; its retention time and UV spectrum were identical to those of crotonaldehyde-2,4DNP. Crotonaldehyde-2,4-DNP presumably formed from dehydration of either 3-HOBA or 3-HOBA-2,4-DNP under the reaction conditions. Thus, reaction of paraldol with 2,4-DNP reagent gives a mixture of 3-HOBA-2,4DNP and crotonaldehyde-2,4-DNP. DNA that had been treated with R-acetoxyNPYR was precipitated, washed once with 70% ethanol and twice with ethanol, dried, and subjected to neutral thermal

hydrolysis. The hydrolysates were analyzed using strong cation exchange HPLC. Material which eluted at the same retention times as those of paraldol and 2-hydroxyTHF was collected, derivatized with 2,4-DNP reagent, and analyzed by HPLC. Panels A and B of Figure 4 are the chromatograms obtained upon derivatization of the material which coeluted with paraldol and 2-hydroxyTHF, respectively. The two peaks indicated by arrows in Figure 4A had the same retention times as 3-HOBA2,4-DNP and crotonaldehyde-2,4-DNP, the products of derivatization of paraldol with 2,4-DNP reagent. Similarly, the indicated peak in Figure 4B had the same retention time as that of 4-HOBA-2,4-DNP, which is formed upon derivatization of 2-hydroxyTHF. The UV spectra of the standard and isolated 2,4-DNPs were identical (Figure 3A-F). The same DNA hydrolysates were analyzed by reversephase HPLC. The material which was eluted at the retention times of paraldol and 2-hydroxyTHF was collected and analyzed by GC/MS. The mass spectra of standard paraldol and 2-hydroxyTHF and the corresponding isolated products were essentially identical (Figure 5A,B). These results demonstrate that paraldol and 2-hydroxyTHF are products of the neutral thermal hydrolysis of R-acetoxyNPYR-treated DNA. DNA treated with R-acetoxyNPYR was also hydrolyzed enzymatically, and these hydrolysates were treated with 2,4-DNP reagent. The results of this analysis demonstrated the presence of 3-HOBA-2,4-DNP, crotonaldehyde-2,4-DNP, and 4-HOBA-2,4-DNP, as in the neutral thermal hydrolysates. It seemed likely that the source of the paraldolreleasing adducts in R-acetoxyNPYR-treated DNA was crotonaldehyde, which is known to be a product of the solvolysis of R-acetoxyNPYR and is known to react with DNA. Therefore, we investigated the reaction of crotonaldehyde with DNA. This DNA was extensively purified and then subjected to either neutral thermal or enzyme hydrolysis, followed by derivatization with 2,4DNP reagent. The results of this analysis demonstrated the presence of 3-HOBA-2,4-DNP and crotonaldehyde-

Figure 3. UV spectra of standard 2,4-DNPs (A, C, and E) and 2,4-DNPs isolated from the traces shown in Figure 4 (B, D, and F).

Lactols in DNA Hydrolysates

Figure 4. HPLC trace obtained upon analysis (system 5) of (A) P9 and (B) P12 of Figure 2 after treatment with 2,4-DNP reagent. The indicated peaks were identified by their retention times and UV spectra (Figure 3).

2,4-DNP, as in the studies described above. Products of the neutral thermal or enzymatic hydrolysis of DNA treated with either R-acetoxyNPYR or crotonaldehyde are summarized in Table 2. 2-HydroxyTHF is the major product of both neutral thermal and enzyme hydrolysis of DNA treated with R-acetoxyNPYR. The total amount of 2-hydroxyTHF and the N2-tetrahydrofuranyl adduct 8 in the enzyme hydrolysates is similar to the amount of 2-hydroxyTHF in the neutral thermal hydrolysate, consistent with the known conversion of 8 to 2-hydroxyTHF under neutral thermal hydrolysis conditions (14). Levels of paraldol are significantly lower than those of 2-hydroxyTHF in hydrolysates of R-acetoxyNPYR-treated DNA. In hydrolysates of DNA treated with crotonaldehyde, paraldol is present in amounts more than 100 times greater than the amounts of each of the adducts 9-12, previously characterized in these reactions (Table 2). Paraldol levels are greater in neutral thermal hydrolysates than in enzyme hydrolysates of both types of DNA, suggesting the presence of a thermally unstable paraldolreleasing adducts.

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Figure 2 is a chromatogram of a neutral thermal hydrolysate of [2,3,4,5-3H]-R-acetoxyNPYR-treated DNA from an earlier study (15). P9 and P12 in this chromatogram are about the same size. But the data in Table 2 demonstrate that levels of paraldol are lower than those of 2-hydroxyTHF in neutral thermal hydrolysates of R-acetoxyNPYR-treated DNA. Therefore, P9 of Figure 2 must contain something in addition to paraldol. Our data indicate that this is [3H]H2O. Thus, when hydrolysates of [2,3,4,5-3H]-R-acetoxyNPYR-treated DNA were stored at -20 °C for 1 year, the P9 level increased at the expense of the P12 level ([3H]-2-hydroxyTHF). The newly formed material did not form a 2,4-DNP derivative and had the same retention time as [3H]H2O in HPLC systems 1 (retention time of 7.11 min) and 6 (retention time of 2.55 min). However, in samples of neutral thermal hydrolysates of [U-14C]-R-acetoxyNPYR-treated DNA, the ratios of P9 and P12 were similar to those shown in Table 2, and did not change after storage for 1 year. These data indicate that P9 of Figure 2 is a mixture of [3H]H2O and paraldol.

Discussion In an earlier study, we noted the presence of substantial amounts of unidentified early eluted radioactivity in neutral thermal hydrolysates of [2,3,4,5-3H]-R-acetoxyNPYR-treated DNA (Figure 2) (15). This was in contrast to chromatograms obtained using fluorescence detection in which adduct 4 predominated (20). In this paper, our major objective was to characterize this unknown radioactivity. This goal was accomplished. The main early eluted products derived from R-acetoxyNPYR-treated DNA are paraldol, the dimer of 3-HOBA, and 2-hydroxyTHF, the cyclic and predominant form of 4-HOBA. [3H]H2O was also identified. Thus, P9 of Figure 2 is comprised of paraldol and [3H]H2O, while P12 is 2-hydroxyTHF. The N2-tetrahydrofuranyl adduct 8 is the source of most of the 2-hydroxyTHF observed in these experiments.

Figure 5. PCI mass spectra of standard and isolated (HPLC system 2) paraldol (A) and 2-hydroxyTHF (B).

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Table 2. Quantitation of Product Formation in the Reactions of r-AcetoxyNPYR or Crotonaldehyde with DNAa product (mmol/mol of G or dG)b reactant R-acetoxyNPYR

crotonaldehyde

2-hydroxyTHFc

4d

neutral thermal or acidf

9.9 (

2.1f

53.6 ( 3.5

24.3 ( 4.4

NDg

enzyme neutral thermal or acidf

4.1 ( 0.8 9.6 ( 3.0

32.7 ( 0.6 ND

NQ ND

27.3 ( 2.3 NQ NQh NQ ND 0.07 ( 0.04 0.033 ( 0.006 0.043 ( 0.006

enzyme

4.3 ( 0.5

ND

ND

ND

hydrolysis conditions

paraldolc

8

9 and 10e 1.8 ( 0.2

NQ

11 0.07 ( 0.01

NQ

12 0.08 ( 0.01

NQ

R-AcetoxyNPYR (38 mmol) was allowed to react with calf thymus DNA (5.0 g) for 20 h at 37 °C in 0.1 M phosphate buffer (pH 7.0). Crotonaldehyde (0.76 mmol) was similarly reacted with DNA (100 mg). b Neutral thermal and acid hydrolysis products expressed per mole of G and enzyme hydrolysis products per mole of dG. Values are mean ( SD (n ) 3). c Quantified as their 2,4-DNPs: 3-HOBA2,4-DNP and crotonaldehyde-2,4-DNP (6:1 ratio) from paraldol and 4-HOBA-2,4-DNP from 2-hydroxyTHF. d Numbers refer to structures in Figure 1. Adducts 5 and 6 were not quantified. e Quantified as guanine base. f Acid hydrolysis for adducts 9 and 10 only. g ND means not detected. h NQ means not quantified. a

We have previously shown that this adduct is unstable at the nucleoside level, decomposing to 2-hydroxyTHF and deoxyguanosine with a t1/2 of 26 min at pH 7 and 37 °C (6, 14). Thus, the formation of 2-hydroxyTHF upon neutral thermal and enzyme hydrolysis of R-acetoxyNPYR-treated DNA is completely consistent with previous data. Comparison of the amounts of 2-hydroxyTHF present in neutral thermal and enzyme hydrolysates indicates that adduct 8 is the main source of 2-hydroxyTHF. There may also be some minor 2-hydroxyTHFreleasing adducts in this DNA, since in some experiments we analyzed each portion of the HPLC chromatogram of the enzyme hydrolysate for material which gives 4-HOBA-2,4-DNP upon derivatization and obtained evidence for an additional 2-hydroxyTHF-releasing adduct (data not shown). The most likely source of the paraldol released upon hydrolysis of R-acetoxyNPYR-treated DNA is an adduct or adducts formed in the reaction of crotonaldehyde with DNA. Crotonaldehyde is known to be a solvolysis product of R-acetoxyNPYR (13). In 1983, our group demonstrated that both crotonaldehyde and R-acetoxyNPYR react with deoxyguanosine, producing the diastereomeric 1,N2-propanodeoxyguanosine adducts 9 and 10 (21). We subsequently demonstrated that crotonaldehyde and acrolein produce 1,N2-propanodeoxyguanosine adducts in DNA (22). Others have confirmed these results (31). In several recent studies, Chung and Nath have demonstrated that crotonaldehyde adducts 9 and 10 are present in human and laboratory animal DNA, apparently arising partly from lipid peroxidation and perhaps also from exogenous sources (32-35). These studies suggest that crotonaldehyde may be an endogenous carcinogen. The results of the present study suggest that DNA damage by crotonaldehyde may be more extensive than previously realized, since the levels of paraldol-releasing adducts are about 100 times greater than those of the 1,N2-propanodeoxyguanosine adducts 9 and 10. It will be important to determine whether similar ratios of paraldol-releasing adducts to 1,N2-propanodeoxyguanosine adducts are found in human DNA. Chung and Nath have shown that levels of adducts 9 and 10 range from approximately 0.003 to 1 µmol/mol of guanine in human DNA. If the ratio of paraldol-releasing adducts to 9 and 10 is the same as observed here, one would expect levels of paraldolreleasing adducts in human DNA to range from 0.3 to 100 µmol/mol of guanine. For comparison, levels in human lung DNA of 8-oxodeoxyguanosine, a common indicator of oxidative DNA damage, are reported to range approximately from 2 to 12 µmol/mol of guanine (36). Although our results support the concept that crotonaldehyde is the main source of the paraldol-releasing

adducts in R-acetoxyNPYR-treated DNA, they could also be formed by reaction of a 3-oxobutyl carbocation with DNA. This would be produced by rearrangement of a carbocation derived from intermediate 3 of Figure 1. In a previous study, we did not find evidence for the production of 3-HOBA or paraldol in the solvolysis of R-acetoxyNPYR or in incubations of NPYR with rat liver microsomes and cofactors (13). However, in this study, we did identify paraldol as a solvolysis product of R-acetoxyNPYR (data not shown), supporting the intermediacy of a 3-oxobutyl carbocation. In this study, our detection limits were better and the reactions were run on a larger scale. We do not know the structures of the paraldol-releasing adducts in crotonaldehyde- or R-acetoxyNPYR-treated DNA. Crotonaldehyde is known to undergo Michael additions with deoxyguanosine at both the N2- and 7-positions (20, 21, 37). This is followed by ring closure to give adducts 9-12. Bis-adducts have also been observed (37). The paraldol-releasing adducts may be adducts in which Michael addition, but no ring closure, has occurred. Cross-links could also be formed. These adducts could produce 3-HOBA upon hydrolysis, and 3-HOBA is known to dimerize to paraldol. Alternatively, crotonaldehyde could react with H2O, producing paradol, which could then react with DNA. We are currently exploring these hypotheses by developing methods for stabilizing these adducts in DNA. In Figure 1, our current understanding of DNA adduct formation from R-acetoxyNPYR is summarized. Ring opening of R-hydroxyNPYR (1) gives 4-oxobutanediazohydroxide (2) which most likely is converted to diazonium ion 3 (2). This may react directly with DNA to produce adducts 4 and 5, or adduct 6 after oxidation (20). We refer to these as type 1 adducts (6). They are reasonably stable in DNA, with half-lives of 33-54 h at 37 °C, but depurinate upon neutral thermal hydrolysis (19, 20, 23). Diazonium ion 3 or a derived carbocation can cyclize to give cyclic oxonium ion 7 which either may react directly with DNA to produce 8 or may give 8 by way of a reactive 2-tetrahydrofuranyl derivative (14). Adducts formed by this pathway are called type 2 adducts. Intermediates 2, 3, and 7 react with H2O to produce three solvolysis products: 4-HOBA, which exists mainly as the lactol 2-hydroxyTHF; 3-HOBA, which exists mainly as the dimer paraldol; and crotonaldehyde (2, 13, 28). Crotonaldehyde reacts with DNA to produce adducts 9-12 as well as the paraldol-releasing adducts (20, 21, 37). Adducts 9 and 10 may also be produced from intermediate 3 or its decomposition products since the ratio of 9 and 10 to 11 and 12 is substantially greater in R-acetoxyNPYRtreated DNA than in DNA reacted with crotonaldehyde

Lactols in DNA Hydrolysates

(Table 2). Adducts formed by reaction with DNA of stable solvolysis products of R-acetoxyNPYR such as crotonaldehyde are referred to as type 3 adducts. Overall, the major DNA adducts produced from R-acetoxyNPYR are the N7-C8 cyclic adduct 4 and the open chain N7 adduct 5 (type 1 adducts), the N2-tetrahydrofuranyl adduct 8 (a type 2 adduct), and the paraldol-releasing adducts (type 3 adducts). In summary, 2-hydroxyTHF and paraldol have been identified as substantial products in neutral thermal and enzyme hydrolysates of DNA treated with R-acetoxyNPYR. They are formed from the N2-tetrahydrofuranyl adduct 8 and crotonaldehyde-derived paraldol-releasing adducts, respectively. Paraldol-releasing adducts appear to be major products in the reaction of crotonaldehyde with DNA. These results define more completely the reactions of R-acetoxyNPYR and crotonaldehyde with DNA. Moreover, 2-hydroxyTHF and paraldol may be useful as biomarkers of DNA modification by NPYR and crotonaldehyde.

Acknowledgment. This study was supported by Grant CA-44377 from the National Cancer Institute. We thank Dr. Edward J. McIntee for obtaining 1H NMR spectra.

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