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Communications Interchain Cross-Linking of DNA Mediated by the Principal Adduct of Acrolein Ivan D. Kozekov,† Lubomir V. Nechev,† Ana Sanchez,‡ Constance M. Harris,† R. Stephen Lloyd,‡ and Thomas M. Harris*,† Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 and Sealy Center for Molecular Science, The University of Texas Medical Branch, Galveston, Texas 77555 Received July 30, 2001
A DNA duplex containing the primary acrolein adduct, 3-(2-deoxy-β-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin-10(3H)-one (2), of deoxyguanosine in a 5′-CpG sequence context spontaneously but reversibly formed an interchain cross-link with the exocyclic amino group of deoxyguanosine in the opposing chain. The linkage was sufficiently stable that the cross-linked duplex could be isolated by HPLC and characterized by MALDITOF mass spectrometry. Enzymatic degradation gave bis-nucleoside 6, which was independently prepared by direct reaction of 2 with dGuo.
Introduction
Scheme 1
Bis-electrophiles have the potential to form simple and cyclic adducts with the individual nucleobases in DNA or cross-links between the bases. In general, the adducts of single bases are much better characterized than the cross-links and there is a paucity of methods for synthesis of DNA containing site-specific cross-links. Acrolein typifies this situation. Acrolein (1, Scheme 1) is a mutagen and tumor initiator formed in cells via lipid peroxidation and also present in tobacco smoke (1, 2). The 1,N 2-hydroxypropano adduct (2) of deoxyguanosine has been identified in the DNA of rodents and humans exposed to acrolein (3, 4). Synthetic methods have been developed for site-specific preparation of oligodeoxynucleotides containing 2 (5, 6), which now make it possible to study the effects of this acrolein adduct on DNA structure and biological properties. Very low mutagenicity of 2 relative to the unhydroxylated 1,N 2-propano analogue has led to the proposal that 2 opens to form the N 2-(3-oxopropyl) adduct 3 in duplex DNA (7, 8). A recent two-dimensional NMR study by de los Santos et al. has provided support for this proposal (9). Some evidence exists for acrolein forming interchain cross-links (10), but they have never been structurally characterized. Herein we describe experiments establishing that 2 is capable of forming an interchain cross-link to the N 2 position of the opposing guanine in a 5′-CpG sequence context.
Experimental Section 1H
NMR spectra were recorded at 400.13 MHz on a Bruker AM400 NMR spectrometer in DMSO-d6. Nucleosides and oligo* To whom inquiries should be addressed. Phone and Fax: (615) 322-2649. E-mail
[email protected]. † Vanderbilt University. ‡ UTMB-Galveston.
nucleotides were purified and the reactions were followed on a Beckman HPLC system (System Gold software, pump module 125) with a diode array UV detector (module 168) monitoring at 260 nm. For purification and monitoring reactions of nucleosides, a YMC ODS-AQ column (250 × 4.6 mm) was used with water-acetonitrile mixtures (1.5 mL/min). For oligonucleotides, a YMC ODS-AQ column (250 × 10 mm) with 0.1 M ammonium
10.1021/tx010127h CCC: $20.00 © 2001 American Chemical Society Published on Web 10/18/2001
Communications formate-acetonitrile mixtures was used with the following gradient: 1 to 10% acetonitrile over 15 min, 10 to 20% acetonitrile over 5 min, hold for 5 min, 100% acetonitrile over 3 min, hold for 2 min and then return to 1% acetonitrile over 3 min (5.0 mL/min). Oligonucleotide containing 1,N 2-hydroxypropanodeoxyguanosine (2) was synthesized as described previously (6). Unmodified oligonucleotides were purchased from Midland Certified Reagent Company, Midland, TX. Mass Spectrometry. Low- and high-resolution FAB mass spectra were obtained at the Mass Spectrometry Facility at the University of Notre Dame, Notre Dame, IN. Negative ion MALDI-TOF mass spectra of modified oligonucleotides were obtained on a Voyager Elite DE instrument (Perseptive Biosystems) using a 3-hydroxypicolinic acid (3-HPA) matrix containing ammonium hydrogen citrate (7 mg/mL) to suppress multiple sodium and potassium adducts. Enzymatic Hydrolysis. Oligonucleotide (0.2-0.5 A260 units) was dissolved in 30 µL of buffer (pH 7.0, 10 mM Tris-HCl, 10 mM MgCl2) and incubated with DNase I, phosphodiesterase I and alkaline phosphatase at 37 °C for 90 min (11). The mixture was analyzed by HPLC. 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-(N 2-deoxyguanosinyl)-pyrimido[1,2-a]purin-10(3H)one (6). 1,N 2-Hydroxypropanodeoxyguanosine (2) (3.5 mg, 0.011 mmol) and deoxyguanosine (6 mg, 0.022 mmol) were dissolved in 200 µL of DMSO. The reaction mixture was stirred at 100 °C for 5 days. The solvent was evaporated under vacuum and the residue was dissolved in 500 µL of pH 7.0, 0.05 M sodium phosphate buffer. HPLC purification of the reaction mixture gave 4.0 mg (65%) of 6. 1H NMR (DMSO-d6): δ (ppm) 10.14 (bs, 1H, dG-N1H), 8.07 (bs, 1H, NH-5), 7.91 (s, 1H, H-2), 7.85 (s, 1H, dG-H-8), 7.22 (m, 1H, dG-N 2H), 6.57 (s, 1H, H-8), 6.15 (t, 1H, H-1′), 6.05 (t, 1H, H-1′), 5.24 (m, 1H, 3′-OH), 5.21 (m, 1H, 3′-OH), 4.85 (m, 2H, 5′-OH), 4.30 (m, 2H, H-3′), 3.75 (m, 2H, H-4′), 3.48 (m, 4H, H-5′, H-5′′), 3.27 (2H, H-6 buried under the water peak, identified by COSY), 2.63 (m, 1H, H-2′), 2.53 (m, 1H, H-2′), 2.22 (m, 2H, H-7 and H-2′′), 2.14 (m, 1H, H-2′′), 1.85 (m, 1H, H-7). HRMS (FAB+) m/z calcd for C23H28N10O8 [M + H]+ 573.2170, found 573.2175. 1,3-Bis(2′-deoxyguanosin-N 2-yl)propane (7). Compound 6 (2.0 mg, 0.0034 mmol) in 200 µL of H2O/methanol (1:1) was treated with sodium borohydride (1.0 mg, 0.027 mmol). The mixture was stirred at room temperature for 8 h and then quenched with 1 mL of 5% acetic acid. HPLC purification gave 1.5 mg (75%) of 7. This compound was identical with an authentic sample synthesized as described previously (12). 1H NMR (DMSO-d6): δ (ppm) 10.48 (bs, 2H, N1H), 7.84 (s, 2H, H-8), 6.42 (bs, 2H, N 2H), 6.09 (t, 2H, H-1′), 5.23 (s, 2H, 3′-OH), 4.83 (m, 2H, 5′-OH), 4.29 (m, 2H, H-3′), 3.74 (m, 2H, 4′-H), 3.45 (m, 4H, H-5′, H-5′′), 3.26 (4H, N-CH2 buried under the water peak, identified by COSY), 2.53 (m, 2H, H-2′), 2.13 (m, 2H, H-2′′), 1.74 (m, 2H, C-CH2-C). Synthesis of Cross-Linked Oligonucleotide Duplex. A mixture of 5′-d(GCTAGC-2-AGTCC) (10.0 A260 units) and 5′d(GGACTCGCTAGC) (10.0 A260 units) in phosphate buffer (100 µL, 0.05 M, pH 7.0) containing 1 M KCl was incubated at 37 °C. The reaction was followed by HPLC. After 7 days a steady state was reached, showing ∼50% conversion. The peak for the crosslinked oligonucleotide (16.53 min) was isolated and lyophilized. An aliquot (0.2 A260 units) of the product was hydrolyzed enzymatically and analyzed by HPLC. The peak for bisnucleoside 6 was identified by coelution with an authentic sample, synthesized as described above. MALDI TOF [M - H]- found: 7327.8 and 7345.2. Calcd for a cross-linked duplex containing 5 or 6: 7327.3. Calcd for a cross-linked duplex containing 4: 7345.3. Reduction of the Cross-Linked Duplex. An aliquot of the cross-linked oligonucleotide (∼1.0 A260 units) was dissolved in phosphate buffer (30 µL of pH 7.0, 0.05 M) and treated with NaCNBH3 (1 mg) at room temperature for 24 h. The reaction was quenched with 5% acetic acid (100 µL) and purified by HPLC. MALDI-TOF [M - H]- calcd: 7329.3. Found: 7330.2.
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Figure 1. (A) HPLC analysis of the cross-linking reaction. Elution sequence: cross-linked duplex, adducted oligonucleotide, complementary strand. (B) Yield of cross-link as a function of time. HPLC analysis of an enzymatic hydrolysate showed a peak for bisnucleoside 7, identified by coelution with an authentic sample.
Results and Discussion In preliminary experiments to search for acroleininduced interchain cross-links, the duplex formed from 5′-d(GCTAGC-2-AGTCC) labeled with 32P phosphate at the 5′ terminus was incubated with the complement sequence 5′-d(GGACTCGCTAGC) (0.1 M sodium phosphate, 5 mM MgCl2, pH 7.0, 25 °C). After various time intervals, the reaction was terminated by treatment with NaBH4 for 10 min. Electrophoresis was carried out under denaturing conditions. Autoradiography revealed the appearance of a new species of lower mobility appropriate for a cross-linked duplex which reached a steady state after ∼3 days. To establish the structure of the cross-link, the experiment was repeated on a larger scale with UV detection rather than 32P labeling to monitor cross-linking. Similar reaction conditions were used (0.05 M sodium phosphate, 1.0 M KCl, pH 7.0, 37 °C). The reaction was monitored directly, i.e., without prior reduction. HPLC (Figure 1A) showed gradual formation of the cross-linked duplex. After 7 days, the reaction had reached steady state and product represented ∼50% of the mixture (Figure 1B). Capillary gel electrophoresis carried out under denaturing conditions confirmed this result (Figure 2). The crosslinked oligonucleotide, isolated by preparative HPLC, was relatively stable under conditions that maintained duplex structure, undergoing no more than 20% reversion to the starting oligonucleotides in 16 h at room temperature in pH 7.0, 0.05 M phosphate buffer. However, it reverted completely to the starting oligonucleotides within 1 h in unbuffered H2O. Treatment of the cross-linked oligonucleotide duplex with NaCNBH3 (pH 7.0, 24 h, room temperature) followed by enzymatic degradation led to near quantitative formation of reduced bis-nucleoside 7 in which there is a three-carbon tether between the N 2 positions of the deoxyguanosines. The structure was established by comparison with an authentic sample we had previously prepared by reaction of 1,3-diaminopropane with a 2halopurine equivalent of dGuo (12). This result demonstrates that the cross-link is formed by reaction of the aldehyde moiety of 3 with the exocyclic amino group of dGuo and is consistent with the unreduced bis-nucleoside being imine 5 (or carbinolamine 4 and/or cyclic bisnucleoside 6 in equilibrium with 5).
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Figure 2. Bottom trace: Capillary gel electrophoresis of the cross-linking experiment after 7 days. Top traces: Starting oligodeoxynucleotides.
MALDI-TOF negative ion mass spectra of the unreduced, cross-linked duplex showed signals at m/z 7345.2 and 7327.8, which are consistent with (M - H)- ions for the cross-linked species containing carbinolamine 4 plus 5 and/or 6. In addition, they showed much stronger signals for the two starting oligonucleotides, which we attribute to reversal of the cross-link during preparation of the MALDI sample. The 7345.2 ion cannot be assigned unambiguously to 4 because it might also arise from the un-cross-linked duplex that had failed to dissociate in the MALDI matrix.. As an additional approach for probing the structure of the cross-link, enzymatic hydrolysis was carried out on unreduced, cross-linked duplex. Nuclease digestion gave bis-nucleoside 6 in 25% yield plus nucleoside 2. An authentic sample of 6 was prepared in 65% yield by reaction of 2 with deoxyguanosine (DMSO, 6 days, 100 °C). The existence of the propano ring in 6 was established via NMR spectroscopy, in particular a cross-peak between the carbonyl carbon of the right-hand ring and the methine proton of the tether was observed in the HMBC spectrum. The stability of 6 in aqueous solution is much greater than that of the cross-linked oligonucleotide duplex. At pH 7.0 and ambient temperature, the halflife of 6 is 64 h. Bis-nucleoside 6 exists as a mixture of two diastereomers. They are separable by HPLC but reequilibrate (via 5) with a half-life of ∼1.5 h in pH 7.0 buffer. Treatment of 6 with NaBH4 (H2O/MeOH, 8 h, room temperature) gave bis-nucleoside 7 in 75% yield after HPLC purification. A crotonaldehyde analogue of 6, arising from treatment of DNA with acetaldehyde, was recently reported (11). The 1H NMR spectrum of 6 and our observations concerning its stability are consistent with data reported for this analogue. Overall, the combined evidence suggests that the acrolein cross-link is imine 5 or carbinolamine 4 in equilibrium with 5. Cyclic bis-nucleoside 6 would appear to be excluded by the difference in observed stability. However, detailed NMR studies of the cross-linked duplex will be required to establish unambiguously the structure(s) of the cross-link. We have reported structural studies of a DNA duplex containing 7, also in a CpG context (12). The trimethylene
Communications
cross-link is accommodated without degradation of duplex stability. NMR spectroscopy showed the two guanines and the trimethylene tether lie approximately in a single plane. Both carbinolamine 4 and imine 5 could readily be accommodated by this structure. It is noteworthy that the previously reported structural and mutagenesis studies on 2 were carried out with the adduct in 5′-CpG sequence contexts. In view of our result, further structural and biochemical studies need to be carried out. In particular, the effect of cross-linking on biological processing needs to be addressed. Acrolein may also generate several other types of cross-links. There has been a report of intrachain crosslinks between guanines being produced by acrolein (10). Adduct 2 could conceivably form an interchain cross-link in a 5′-GpC sequence. It is not out of the question that cross-linking of 2 to the exocyclic amino groups of adenine and cytosine could occur since NMR structural studies of DNA duplexes containing unfunctionalized 1,N 2propano deoxyguanosine have shown the nucleoside to be rotated into a syn conformation, placing the trimethylene moiety in the major groove (13). The adducts of higher homologues of acrolein including crotonaldehyde (4) and 4-hydroxynonenal (14) may also form cross-links. Close parallels exist between the DNA adducts of acrolein and adducts arising from the epoxides of vinyl monomers, which act as two-carbon aldehydic bis-electrophiles. For example, the epoxide of vinyl chloride yields the hydroxyethano analogue of 2 (15). The chemistry and biology of the two-carbon adducts have been studied in greater detail than those of acrolein and homologues. Interestingly, there is genetic (16) and biophysical (17) evidence for the formation of cross-links by these twocarbon bis-electrophiles, but the cross-links have never been structurally characterized. It seems likely that DNA cross-links mediated by aldehydic bis-electrophiles are widespread in nature.
Acknowledgment. We gratefully acknowledge financial support from the U.S. Public Health Service (Grants ES00267, ES05355, and ES07781).
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acrolein-derived deoxyguanosine adduct in Escherichia coli. J. Biol. Chem. 276, 9071-9076. de los Santos, C., Zaliznyak, T., and Johnson, F. (2001) NMR characterization of a DNA duplex containing the major acroleinderived deoxyguanosine adduct γ-OH-1,N 2-propano-2′-deoxyguanosine. J. Biol. Chem. 276, 9077-9082. Kawanishi, M., Matsuda, T., Nakayama, A., Takebe, H., Matsui, S., and Yagi, T. (1998) Molecular analysis of mutations induced by acrolein in human fibroblast cells using supF shuttle vector plasmids. Mutat. Res. 417, 65-73. Wang, M., McIntee, E. J., Cheng, G., Shi, Y., Villalta, P. W., and Hecht, S. S. (2000) Identification of DNA adducts of acetaldehyde. Chem. Res. Toxicol. 13, 1149-1157. Dooley, P. A., Tsarouhtsis, D., Korbel, G. A., Nechev, L. V., Shearer, J., Zegar, I. S., Harris, C. M., Stone, M. P., and Harris, T. M. (2001) Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5′-(CpG) motif: Model of a malondialdehyde cross-link. J. Am. Chem. Soc. 123, 1730-1739. Singh, U. S., Moe, J. G., Reddy, G. R., Weisenseel, J. P., Marnett, L. J., and Stone, M. P. (1993) 1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)3 frameshift hotspot of Salmonella typhimurium hisD3052:
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Hoogsteen base-pairing at pH 5.8. Chem. Res. Toxicol. 1993, 6, 825-836. Chung, F.-L., Nath, R. G., Ocando, J., Nishikawa, A., and Zhang, L. (2000) Deoxyguanosine adducts of t-4-hydroxy-2-nonenal are endogenous DNA lesions in rodents and humans: detection and potential sources. Cancer Res. 60, 1507-1511. Guengerich, F. P., Persmark, M., and Humphreys, W. G. (1993) Formation of 1,N 2- and N 2,3-ethenoguanine from 2-halooxiranes: isotopic labeling studies and isolation of a hemiaminal derivative of N 2-(2-oxoethyl)guanine. Chem. Res. Toxicol. 6, 635-648. Nivard, M. J. M., and Vogel, E. W. (1999) Genetic effects of exocyclic DNA adducts in vivo: heritable genetic damage in comparison with loss of heterozygosity in somatic cells. In DNA Adducts: Identification and Biological Significance (Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, D., and Bartsch, H., Eds.) pp 335-349 International Agency for Research on Cancer, Lyon, France. Spengler, S. J., and Singer, B. (1988) Formation of interstrand cross-links in chloroacetaldehyde-treated DNA demonstrated by ethidium bromide fluorescence. Cancer Res. 48, 4804-4806.
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