Chem. Res. Toxicol. 2006, 19, 571-576
571
Formation of Acrolein Adducts with 2′-Deoxyadenosine in Calf Thymus DNA Agnieszka J. Pawłowicz, Tony Munter, Yan Zhao, and Leif Kronberg* Department of Organic Chemistry, A° bo Akademi UniVersity, Biskopsgatan 8, FIN-20500 Turku/A° bo, Finland ReceiVed December 13, 2005
Acrolein is a ubiquitous environmental contaminant that has been found to be mutagenic in prokaryotic and eukaryotic cells. In the present study, we examined the reactions of acrolein with 2′-deoxyadenosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions. The deoxynucleoside adducts were isolated by reversed-phase liquid chromatography, and their structures were determined by their UV absorbance, mass spectrometry, and 1H and 13C NMR spectroscopy. The reaction of 2′-deoxyadenosine with acrolein resulted in the formation of four structurally different adducts (dAI, dAII, dAIII, dAIV). The structures of the novel acrolein adducts, dAIII and dAIV, were assigned as 3-[N6-(2′-deoxyadenosinyl)]propanal (dAIII) and 9-(2′-deoxyribosyl-6-(3-formyl-1,2,5,6-tetrahydropyridyl)purine (dAIV), respectively. The adduct dAIII was found to arise via a Dimroth rearrangement of adduct dAI, while the adduct dAIV was shown to be formed upon further reaction of acrolein with dAIII. In the reaction of acrolein with calf thymus DNA, all studied 2′-deoxyadenosine-acrolein adducts were observed. For the first time, it is shown that the N6-adduct and the adducts which are derived from two acrolein units are formed in calf thymus DNA. Introduction Acrolein, a reactive R,β-unsaturated aldehyde, is a ubiquitous environmental contaminant. It is formed exogenously during the incomplete combustion of fossil fuels, including engine exhausts, wood, and overheating frying oils (1) and endogenously as a product of lipid peroxidation (2). It occurs also in relatively high concentration in cigarette smoke (3). Acrolein is formed during the metabolism of the anticancer drug cyclophosphamide and appears to be involved in the bladder toxicity of this drug (4-6). Moreover, acrolein is an initiator of tumor growth in the rat urinary bladder (7) and is mutagenic in a variety of short-term assays, including Salmonella (8, 9) and Chinese hamster V79 cells (10). Acrolein is strongly mutagenic to DNA-repair-deficient xeroderma pigmentosum fibroblasts but not to normal human fibroblasts (11). Previously, it has been shown that acrolein reacts with calf thymus DNA bases to form DNA adducts. Adducts of 2′deoxyguanosine (12), 2′-deoxycytidine, 2′-deoxyuridine (13, 14), and thymidine (15) have been identified. Chung et al. (16) detected acrolein-derived guanosine adducts as a prevalent lesion in human and rodent tissues. The hydroxypropano-deoxyguanosine adduct was used as a model to study the mutagenicity of the acrolein-derived deoxyguanosine adducts (17). Although the mutational specificity of the hydroxypropano-deoxyguanosine adduct is known, the spectrum of mutations by acrolein is still unknown. In previous work on the structural characterization of adducts formed in reaction of acrolein with 2′-deoxyadenosine, the 9-hydroxy-1,N6-propano-2′-deoxyadenosine adduct was identified (18). This adduct was later found to be accumulated in the nuclei of rat liver cells exposed to acrolein (19). In a very recent work, we reported on the structural and conformational determination of a 2′-deoxyadenosine adduct that was comprised of * To whom correspondence should be addressed. E-mail: leif.kronberg@ abo.fi. Phone: +358-2-21 54 183. Fax: +358-2-21 54 866.
Scheme 1. Structures of Adducts Formed in the Reaction of Acrolein with 2′-Deoxyadenosine
two units of acrolein, that is, compound dAII, Scheme 1 (20). However, LC-DAD1 analyses of the reaction mixture of 2′-deoxyadenosine with acrolein showed the occurrence of two additional peaks that represented adducts of unknown structures. The aims of this study were to structurally characterize these adducts and to determine the formation of 2′-deoxyadenosine adducts in calf thymus DNA incubated with acrolein.
Materials and Methods Caution: Acrolein has been found to be mutagenic in bacteria and mammalian cells. Therefore, caution should be exercised in the handling and disposal of the chemical. Chemicals. 2′-Deoxyadenosine and acrolein were purchased from Fluka Chemie AG (Buchs, Switzerland). Calf thymus DNA (Type I: sodium salt, highly polymerized), nuclease P1 from Penicillium citrinum, alkaline phosphatase (Escherichia coli Type III), acid phosphatase (wheat germ Type I), and Bis-tris buffer were obtained from Sigma-Aldrich (St. Louis, MO). Acetonitrile was gradient grade for chromatography from Merck (Darmstadt, Germany). 1 Abbreviations: LC-DAD, liquid chromatography with diode array detection; LC-ESI-MS/MS, liquid chromatography/electrospray ionization tandem mass spectrometry; MRM, multiple reaction monitoring; COSY, correlation spectroscopy (1H-1H); HMBC, heteronuclear multiple bond connectivity NMR spectroscopy (long-range 1H-13C COSY); Bis-tris, bis[2-hydroxyethyl]iminotris[hydroxymethyl]-methane hydrochloride; dsDNA, double-stranded deoxyribonucleic acid; ssDNA, single-stranded deoxyribonucleic acid.
10.1021/tx0503496 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/10/2006
572 Chem. Res. Toxicol., Vol. 19, No. 4, 2006 LC-DAD Methods. LC-DAD analyses were carried out on an Agilent 1100 Series liquid chromatographic system consisting of a quaternary pump, a vacuum degasser, an autosampler, a thermostated column compartment, a diode-array detector (UV), and Agilent ChemStation data system (Agilent Technologies, Espoo/ Esbo, Finland) using reversed-phase C18 analytical column (5 µm, 4 mm × 125 mm, Hypersil BDS-C18, Agilent Technologies). The column was eluted with a gradient from 1% acetonitrile in 0.01 M ammonium acetate (pH 7.0) to 30% acetonitrile over the course of 20 min at a flow rate of 1 mL/min. Preparative isolation of the products was conducted on a semipreparative reversed-phase C18 column (5 µm, 10 mm × 250 mm, Hypersil BDS-C18, Krotek, Tampere/Tammerfors, Finland). The column was coupled to an Agilent HPLC system equipped with an Agilent 1100 Series G1364C (analytical scale) fraction collector (Agilent Technologies). LC-ESI-MS/MS Methods. The LC-ESI-MS/MS analyses were performed on an Agilent 1100 Series LC/MSD Trap SL instrument (Agilent Technologies) equipped with an electrospray source and operated in the positive ion mode. Ionization was carried out using nitrogen as both nebulizer gas (40 psi) and drying gas (12 L/min) heated to 350 °C. The capillary exit offset had a value of 115.3 V, and skim 1 voltage was set at 40 V. The maximum ion accumulation time was 2.00 ms, and the target value was 20 000. Collisioninduced dissociation experiments coupled with multiple tandem mass spectrometry (MSn) employed helium as collision gas. The fragmentation amplitude was varied between 0.7 and 1.0 V. The compounds were introduced through the LC system using the same chromatographic conditions as previously described for LC-DAD analyses, with the exception of the flow rate which was adjusted to 0.5 mL/min and the use of an Agilent binary LC pump. Pure compounds were introduced directly into the MS source by a syringe pump at a flow rate of 5 µL/min using as solvent a 1:1 (v/v) mixture of 0.01 M aqueous ammonium acetate/acetonitrile. The drying gas temperature was 325 °C, and the flow rate was 5 L/min with the nebulizer gas pressure set at 15 psi. Analyses of DNA Adducts by LC-ESI-MS/MS. A Micro LC triple-quadrupole mass spectrometer (Micromass, Manchester, U.K.) equipped with an electrospray interface was used with a source block temperature of 120 °C and a desolvation temperature of 325 °C. Nitrogen was used as the desolvation gas (616 L/h) and the cone gas (33 L/h), while argon was used as the collision gas, at a collision cell pressure of 5.8 × 10-3 mbar. Positive ions were acquired in multiple reaction monitoring (MRM) mode with a dwell time of 0.25 s and interchannel delay of 0.05 s. The HPLC separations were performed on an Agilent 1100 system consisting of a binary pump, a vacuum degasser, an autosampler, and a thermostated column oven. The DNA samples were chromatographed on a reversed-phase C18 analytical column (5 µm, 4 mm × 125 mm, Hypersil BDS-C18). The column was eluted with a gradient consisting of 0.01 M aqueous ammonium acetate and acetonitrile starting from 1% acetonitrile and ending after 20 min at 30% acetonitrile at the flow rate of 0.5 mL/min and with the injection volume of 20 µL. NMR and UV Spectroscopy. The 1H NMR and 13C NMR spectra of adducts were recorded on a Bruker Avance 600 NMR spectrometer at 600 and 150 MHz, respectively (Bruker, Germany). Spectra of the adduct dAIII and dAIV were recorded at 25 °C in Me2SO-d6, and the solvent was used as the internal standard. The 1H NMR signal assignments were based on chemical shifts and 1H-1H and 13C-1H correlation data. The assignment of carbon signals was based on chemical shifts and 13C-1H correlations. The UV spectra of the isolated compounds were recorded by the diode array detector as the peaks eluted from the LC column. Preparation of 3-[N6-(2′-Deoxyadenosinyl)]propanal (dAIII) and 9-(2′-Deoxyribosyl)-6-(3-formyl-1,2,5,6-tetrahydropyridyl)purine (dAIV). Acrolein (558 mg, 10.0 mmol) was allowed to react with 2′-deoxyadenosine (250 mg, 1.0 mmol) in 100 mL of a 0.5 M phosphate buffer solution at pH 7.4. The reaction was performed at 37 °C. The progress of the reaction was followed by LC-DAD and LC-ESI-MS/MS analyses on the C18 analytical column. After 5 days, the reaction was stopped and the reaction mixture was
Pawłowicz et al. concentrated by rotary evaporation to about 10 mL. Separation and purification of the compounds were carried out using the semipreparative column and the fraction collector. The column was eluted isocratically with 5% acetonitrile in 0.01 M ammonium bicarbonate (pH 6.4) for 15 min and then with a gradient from 5 to 40% acetonitrile over the course of 11 min at a flow rate of 3 mL/ min. The solutions containing the pure compounds were rotaryevaporated to dryness and dried under vacuum. The residues were subjected to spectroscopic and spectrometric studies. dAIII had the following spectral characteristics: UVmax 268 nm, UVmin 236 nm (HPLC eluent, approximately 13% acetonitrile in ammonium acetate buffer, pH 7). The positive ion electrospray mass spectra showed the following ions (m/z, relative abundance, formation): 308 (100, MH+); MS2 of 308, 192 (100, MH+ - deoxyribosyl + H); MS3 of 308, 136 (100, MH+ - deoxyribosyl - C3H5O + H). dAIV had the following spectral characteristics: UVmax 279 nm, UVmin 250 nm (HPLC eluent, approximately 23% acetonitrile in ammonium acetate buffer, pH 7) (Figure 3). The positive ion electrospray mass spectra showed the following ions (m/z, relative abundance, formation): 346 (100, MH+); MS2 of 346, 230 (100, MH+ - deoxyribosyl + H); MS3 of 230, 202 (34, MH+ - deoxyribosyl - CHO + H), 148 (100, MH+ deoxyribosyl - C5H6O + H). The 1H and 13C NMR spectroscopic data of dAIII and dAIV are presented in Tables 1 and 2, respectively. Small-Scale Reactions of Acrolein with 2′-Deoxyadenosine. Acrolein (22 mg, 0.38 mmol) was allowed to react with 2′deoxyadenosine (10 mg, 0.038 mmol) in 4 mL of 0.5 M phosphate buffer solutions at pH 4.6, 7.4, and 8.8 for 7 days. The reaction mixtures were stirred at 37 °C, and aliquots were analyzed by LCDAD and LC-MS. Rearrangement of dAI to dAIII. A sample of dAI in 0.01 M phosphate buffer (pH 7.0) was incubated at 37 °C, and aliquots were analyzed at various times by analytical LC-DAD. Reaction of dAIII with Acrolein. Acrolein (12.2 mg, 0.22 mmol) was allowed to react with compound dAIII (6.7 mg, 0.022 mmol) in 4 mL of 0.5 M phosphate buffer at pH 7.4 and 37 °C. The progress of the reaction was followed by LC-DAD analyses for 7 days. Determination of Deoxynucleoside Adduct Yields. Quantitative 1H NMR analyses, using 1,1,1-trichloroethane for dAIII and dAIV as an internal standard, were performed on aliquots of the adducts. Standard solutions were prepared for LC-DAD analyses by taking an exact volume of the NMR sample and diluting it with an appropriate volume of water. The quantitative determination of adducts in the reaction mixtures was made by comparing the peak area of the adducts in the standard solutions with the area of the adduct peaks in the reaction mixtures. Adducts were quantified using UV detection at 254 nm. The molar yields of the products were calculated from the original amount of 2′-deoxyadenosine in the reaction mixture. Reaction of Acrolein with Single- and Double-Stranded Calf Thymus DNA. Single-stranded (ss) DNA was made from the double-stranded (ds) DNA by heating the DNA to 100 °C for 5 min and rapidly cooling the solution on ice (21). Acrolein (40 mg, 0.71 mmol) was allowed to react with ssDNA and dsDNA (5 mg) in 5 mL of 0.1 M phosphate buffer (pH 7.4) at 37 °C for 3 days. The DNA was precipitated by addition of 5 M NaCl (1 mL) and cold ethanol (15 mL) and cooling the solution at -20 °C. The mixture was centrifuged at 3000 rpm, and the supernatant was collected. The recovered DNA was first washed with cold 70% ethanol (5 mL), then with cold ethanol (5 mL), and after that dissolved in water (5 mL). The DNA was reprecipitated from the solution by addition of cold ethanol (15 mL) and cooling at -20 °C. The DNA was recovered by centrifugation and dissolved in 4 mL of 100 mM Bis-tris buffer (pH 6.5) containing 2 mM MgCl2. The DNA was enzymatically hydrolyzed by adding Nuclease P1 (dissolved at a concentration of 1 mg/mL in 1 mM ZnCl2) to obtain a concentration of 50 units/mL. The mixture was stirred and incubated at 37 °C for 4 h. Then, bacterial alkaline phosphatase
Acrolein Adducts with 2′-Deoxyadenosine Table 1. 1H and
13C
Chem. Res. Toxicol., Vol. 19, No. 4, 2006 573
Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH-H of Protons in dAIII
proton
δ (ppm)
multiplicity
H-8 (1H) H-2 (1H) NH
8.35 8.22 7.91
s s, br s, br
JH,H (Hz)
carbon
δ (ppm)
C-8 C-2
139.98 152.73
H-1′
C-4 C-5 C-6
148.47 120.16 154.82
H-1′
CHO C-a C-b
203.07 34.42 43.63
H-b CHO; H-b CHO
C-1′ C-2′ a
84.42
H-2′, H-3′, H-4′
C-3′ C-4′ C-5′
71.44 88.47 62.33
H-2′, H-4′, H-5′ H-5′
HMBC
Purine Unit
a
CHO (1H) H-a (1H) H-b (2H)
9.72 3.76 2.76
t br dt
H-1′ (1H) H-2′ (1H) H-2′′ (1H) H-3′ (1H) H-4′ (1H) H-5′(1H) H-5′′(1H)
6.35 2.72 2.26 4.41 3.88 3.62 3.52
dd ddd ddd dt dd dd dd
Acrolein Unit 1.7 6.6; 1.5 Sugar Unit 7.8; 6.1 13.4; 7.8; 5.6 13.1; 6.1;2.9 5.6; 2.7 6.8; 4.3 11.6; 4.3 11.6; 4.1
Signal overlapped by solvent. Table 2. 1H and proton
δ (ppm)
13C
Chemical Shifts (δ) and Spin-Spin Coupling Constants, JH,H of Protons in dAIV multiplicity
JH,H (Hz)
carbon
δ (ppm)
HMBC
Purine Unit H-8 (1H) H-2 (1H)
8.44 8.27
s s
C-8 C-2 C-4 C-5 C-6
139.26 152.23 150.57 120.06 153.87
H-1′ H-2, H-8, H-1′ H-8, H-2 H-2, H-8
CHO C-aa C-b C-c C-d C-ea
193.22
H-c
26.38 149.89 139.05
H-c H-b CHO, H-b
C-1′ C-2′a
84.15
H-2′, H-3′ H-1′, H-4′
C-3′ C-4′ C-5′
71.24 88.41 62.16
H-4′, H-5′, H-2′ H-1′, H-2′, H-5′
Acrolein Units
a
CHO H-a (2H) H-b (2H) H-c (1H)
9.48 4.39 2.55 7.19
s br ddd ddd
H-e (2H)
4.78
br
H-1′ (1H) H-2′ (1H) H-2′′ (1H) H-3′ (1H) H-4′ (1H) H-5′ (1H) H-5′′(1H) OH-5′ (1H) OH-3′ (1H)
6.37 2.67 2.28 4.40 3.87 3.60 3.51 5.19 5.40
dd ddd ddd td td dt dt t d
12.2; 5.9; 2.7 5.7; 3.9; 1.8
Sugar Unit 7.4; 6.3 13.3; 7.4; 5.8 13.3; 6.2; 3.2 6.1; 3.2 4.3; 2.7 11.8; 4.4 11.8; 5.0 5.6 4.1
Signal overlapped by solvent.
was added to give a final concentration of 6 units/mL, and wheat germ acid phosphatase (dissolved in Bis-tris-MgCl2 buffer at a concentration of 10 mg/mL) was added to obtain a final concentration of 0.4 units/mL. The mixture was incubated at 37 °C for 18 h. The enzyme digest mixture was loaded onto a Centricon YM-3 filter that had been prerinsed with water and centrifuged at 4000 rpm. The ultrafiltrate was recovered and concentrated by rotary evaporation to near dryness and reconstituted in 300 µL of a mixture of 20% methanol in water.
Results and Discussion Reaction of Acrolein with 2′-Deoxyadenosine. LC-DAD and LC-ESI-MS/MS analyses of the small-scale reactions of acrolein with 2′-deoxyadenosine showed the formation of four major product peaks, dAI-dAIV (Figure 1). The compounds dAI and dAII were obtained at higher yields under acidic conditions (pH 4.6). Work on their structural determination has been previously published (18, 20). The most favorable condition for the formation of compounds dAIII and dAIV was pH 7.4 and 5 days of reaction at 37 °C. The maximum yields for
dAIII and dAIV were 3.0% and 17.9%, respectively (Figure 2). The conditions giving the highest yields of the adducts were applied in large-scale reactions to isolate sufficient amounts of the products and determine their structures. On the basis of UV, NMR spectroscopy, and electrospray mass spectrometry, the structures of the adducts were assigned as 3-[N6-(2′-deoxyadenosinyl)]propanal (dAIII) and 9-(2′-deoxyribosyl)-6-(3-formyl1,2,5,6-tetrahydropyridyl)purine (dAIV) (Scheme 1). The UV spectrum of dAIII showed an intense absorption maximum at 268 nm and absorption minimum at 236 nm and indicated substitution at the N6-position (22-24) (Figure 3). The positive ion electrospray mass spectrum of the adduct showed a protonated molecular ion peak at m/z ) 308. The ion peak recorded at m/z ) 192 was attributed to the loss of the deoxyribosyl unit from MH+. The fragmentation pattern differs from that of 9-hydroxy-1,N6-propano-2′-deoxyadenosine (dAI) for which a fragment ion was observed at m/z ) 174 that corresponds to the loss of water.
574 Chem. Res. Toxicol., Vol. 19, No. 4, 2006
Figure 1. C18 analytical column LC-DAD chromatogram at 215 nm of the reaction mixture of acrolein with 2′-deoxyadenosine in 0.5 M phosphate buffer (pH 7.4) held at 37 °C for 5 days. For analysis conditions, see Materials and Methods.
Figure 2. Formation of dAIII and dAIV during a reaction taking place at pH 7.4 and 37 °C.
Figure 3. UV absorbance spectra of dAIII and dAIV. The UV spectra were recorded with the diode array detector as the compounds eluted from the LC column.
The 1H NMR spectrum of dAIII displayed six signals, besides the signals from the protons of the deoxyribose moiety (Table 1). The singlet signal at δ ) 8.35 ppm was assigned to the purine H-8 proton and showed a long-range H-H correlation to the anomeric sugar proton H-1′ at δ ) 6.35 ppm. The H-2 proton in the adenine moiety gave a signal at δ ) 8.22 ppm.
Pawłowicz et al.
The aldehyde proton appeared at δ ) 9.72 ppm as a triplet (J ) 1.7 Hz) due to two-bond coupling to the H-b protons at δ ) 2.76 ppm. A 1H-1H correlation (COSY) was observed between the H-b and the formyl protons. The signal of H-b was split additionally into a triplet (J ) 6.6 Hz) because of coupling to the methylene protons H-a at δ ) 3.76 ppm. The signal from the proton bound to the exocyclic nitrogen appeared at δ ) 7.91 ppm. In the 13C NMR spectrum, three carbon signals at δ ) 203.07, 43.63, and 34.42 ppm were found in addition to the 10 carbon signals from the purine and deoxyribose moiety (Table 1). The signal observed at δ ) 203.07 ppm was assigned to the formyl carbon, and the signals at δ ) 43.63 and 34.42 ppm were assigned to C-a and C-b, respectively. The NMR, UV, and the mass spectrometric data were consistent with the structure of dAIII presented in Scheme 1. A ring-chain tautomerism of dAIII could not be observed. Upon storage of dAI in an aqueous solution at neutral pH, dAIII was formed. This transformation can be explained by a ring opening of dAI to yield an N1-propanal adduct, which in turn undergoes the Dimroth rearrangement to the N6-propanal adduct (dAIII) (25-29). Also the finding that dAIII could not be observed prior to the formation of dAI indicates that dAIII is not formed through a Michael reaction at the exocyclic amino group. The UV of dAIV exhibited an absorption maximum at 279 nm and absorption minimum at 250 nm (Figure 3). In the mass spectrum, the ion peak at m/z ) 346 corresponded to the protonated molecular ion. The ion peak at m/z ) 230 corresponded to the cleavage of the deoxyribosyl unit and the ion peak at m/z ) 202 to the loss of the deoxyribosyl moiety and the carbonyl group. The 1H NMR spectrum of dAIV exhibited seven proton signals, in addition to the signals from the protons of the deoxyribose moiety (Table 2). The one-proton singlets at δ ) 8.44 and δ ) 8.27 ppm were assigned to the purine protons H-8 and H-2, respectively. H-8 was assigned on the basis of the observed 1H-1H correlation with H-1′ signal in the deoxyribosyl unit at δ ) 6.37 ppm. The one-proton singlet at δ ) 9.48 ppm was assigned to the formyl proton based on a one-bond 13C-1H correlation to the carbon signal at δ ) 193.22 ppm. The methylene protons H-a and H-e in the tetrahydropyridine ring appeared as broad singlets at δ ) 4.39 and 4.78 ppm, respectively. The signal at δ ) 2.55 ppm was assigned to H-b. It was split into double double doublet and exhibited 1H1H correlation to the H-a protons and the H-c proton. The methine proton H-c appeared at δ ) 7.19, and it exhibited 1H1H correlations with the H-b protons and the formyl proton. The 13C NMR spectrum of dAIV displayed six carbon signals in addition to the 10 signals from 2′-deoxyadenosine (Table 2). The formyl carbon was observed at δ ) 193.22 ppm. The signals at δ ) 149.89 and δ ) 139.05 ppm were assigned to the doublebonded carbons C-c and C-d, respectively. In the HMBC spectrum, the C-d carbon showed correlations to the H-b and the formyl proton. The signals of methylene carbons, C-e and C-a, bonded to the exocyclic nitrogen atom of 2′-deoxyadenosine, were overlapped by solvent, and it was not possible to determine the chemical shifts from the 13C-1H correlation spectra because of the broadening of the proton signals. The final signal, C-b, was observed at δ ) 26.38 ppm. Collectively, the spectral data of the compound are consistent with the structure of dAIV (Scheme 1). Further proof of the structure of dAIV was obtained from the experiment where pure dAIII was reacted with acrolein. In
Acrolein Adducts with 2′-Deoxyadenosine
Chem. Res. Toxicol., Vol. 19, No. 4, 2006 575
Scheme 2. Mechanism of the Formation of dAIV from dAIII
approximately three times faster than the reaction of acrolein with 2′-deoxyadenosine yielding dAI. We are currently exploring these reaction kinetics further.
Conclusions
Table 3. Retention Times and Recorded Transitions of Nucleoside Adducts Analyzed in the DNA Hydrolysate nucleoside retention adduct time (min) dAI dAII dAIII dAIV
7.1 9.0 10.9 18.5
cone collision voltage energy (V) (eV)
transition
mode of formation
308 > 192 364 > 248 308 > 192 346 > 230
MH+ - dR + H MH+ - dR + H MH+ - dR + H MH+ - dR + H
10 10 10 19
13 13 13 14
Table 4. Yields of the Adducts Found in the DNA Hydrolysate adducts mol/106 mol of normal nucleotides
dAI
dAII
dAIII
dAIV
dsDNA ssDNA
42 142
126 1405
2 21
2 57
this reaction, dAIV was formed as the main product. dAIV is thus likely to be obtained by intramolecular aldol condensation of the two acrolein-derived propanal tails bound to N6 resulting in the tetrahydropyridine ring (Scheme 2). Very recently, the cyclic adduct consisting of a tetrahydropyridine unit was found in “lysine-containing model peptides” (30). Reaction of Acrolein with DNA. The reactions of acrolein with single- and double-stranded calf thymus DNA were performed at pH 7.4 for 3 days, and the modified DNA was enzymatically hydrolyzed to the deoxynucleosides. The adducts were identified in the hydrolysate by positive ion electrospray MS/MS spectra and coelution with the 2′-deoxyadenosine standards. The ion peaks monitored were the protonated molecular ions and the fragment peaks obtained by cleavage of the deoxyribosyl moiety (m/z ) 116) from the parent ions (Table 3). The major adduct was found to be dAII (Table 4). The level of dAII corresponded to 162 pmol/mg DNA (190 adducts mol/ 106 mol of normal nucleotides) in dsDNA and 1200 pmol/mg DNA (1405 adducts/106 nucleotides) in ssDNA. The adducts dAI, dAIII, and dAIV were also detected in the hydrolysate. The levels of dAI, dAIII, and dAIV in dsDNA corresponded to 42 pmol/mg DNA (49 adducts/106 nucleotides), 3 pmol/mg DNA (4 adducts/106 nucleotides), and 3 pmol/mg DNA (4 adducts/106 nucleotides), respectively. In ssDNA, dAI was detected at the level of 150 pmol/mg DNA (176 adducts/106 nucleotides) and dAIII at the level of 24 pmol/mg DNA (28 adducts/106 nucleotides). The corresponding level for dAIV was 54 pmol/mg DNA (63 adducts/106 nucleotides). The formation of the adducts comprising of two acrolein units (dAII and dAIV) cannot be described by an attack of a preformed acrolein condensation product, but rather through a sequential attack of two units of acrolein (20). Since it is rather unlikely that two molecules of acrolein would attack the same adenine moiety in DNA, the formation and the high yields of dAII and dAIV may be due to higher reactivity toward acrolein of the adducts dAI and dAIII than of 2′-deoxyadenosine. In other words, the former exocyclic amino nitrogen (N6) in dAI and dAIII will readily undergo a Michael reaction with a second unit of acrolein. Preliminary experiments in our laboratory have shown that the reaction of acrolein with dAI yielding dAII is
Structural characterization of deoxynucleoside adducts of acrolein is important for studies on the possible interactions of acrolein and DNA and in clarifying the mechanism of the genotoxicity of acrolein. The results of this work demonstrate that acrolein reacts with 2′-deoxyadenosine, and the reaction results in the formation of four adducts. In this work, two novel adducts were fully characterized by spectroscopic and spectrometric methods. The compounds were identified as 3-[N6-(2′deoxyadenosinyl)]propanal (dAIII) and 9-(2′-deoxyribosyl-6(3-formyl-1,2,5,6-tetrahydropyridyl)purine (dAIV). Furthermore, it was shown that, besides dAI, the N6 and the adducts consisting of two units of acrolein (dAII and dAIV) were formed in doubleand single-stranded calf thymus DNA and that the major adducts were those consisting of two units of acrolein. dAII, dAIII, and dAIV have not been previously detected in DNA. Acrolein appears to induce base pair substitution mutations in Salmonella TA 104 (8), and it has been reported previously that the 9-hydroxy-1,N6-propanodeoxyadenosine adduct may contribute to the biological effects of acrolein in the mutation assay (18). Our studies show that other 2′-deoxyadenosineacrolein adducts can be involved in the mechanisms of mutagenic activity of acrolein. Of special interest is the question concerning the biological significance of the adducts comprising two acrolein units (dAII and dAIV). Further studies are needed to show whether these adducts are formed in vivo or not. Acknowledgment. This work was supported by the Graduate School of Bioorganic and Medicinal Chemistry, A° bo Akademi University.
References (1) Izard, C., and Libermann, C. (1978) Acrolein. Mutat. Res. 47, 115138. (2) Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins. J. Biol. Chem. 273, 16058-16066. (3) International Agency for Research on Cancer (1986) IARC Monograph on the EValuation of the Carcinogenic Risk of Chemicals to Humans 38, pp 133-161, IARC, Lyon, France. (4) International Agency for Research on Cancer (1981) IARC Monograph on the EValuation of the Carcinogenic Risk of Chemicals to Humans 26, pp 165-202, IARC, Lyon, France. (5) Crook, T. R., Souhami, R. L., and McLean, A. E. (1986) Cytotoxicity, DNA cross-linking, and single strand breaks induced by activated cyclophosphamide and acrolein in human leukemia cells. Cancer Res. 46, 5029-5034. (6) Ramu, K., Fraiser, L. H., Mamiya, B., Ahmed, T., and Kehrer, J. P. (1995) Acrolein mercapturates: Synthesis, characterization, and assessment of their role in the bladder toxicity of cyclophosphamide. Chem. Res. Toxicol. 8, 515-524. (7) Cohen, S. M., Garland, E. M., St. John, M., Okamura, T., and Smith, R. A. (1992) Acrolein initiates rat urinary bladder carcinogenesis. Cancer Res. 52, 3577-3581. (8) Marnett, L. J., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Naturally occurring carbonyl compounds are mutagens Salmonella tester strain TA104. Mutat. Res. 148, 2534. (9) Parent, R. A., Caravello, H. E., and San, R. H. C. (1996) Mutagenic activity of acrolein in S. typhimurium and E. coli. J. Appl. Toxicol. 16, 103-108. (10) Smith, R. A., Cohen, S. M., and Lawson, T. A. (1990) Acrolein mutagenicity in the V79 assay. Carcinogenesis 11, 497-498. (11) Curren, R. D., Yang, L. L., Conklin, P. M., Grafstrom, R. C., and Harris, C. C. (1988) Mutagenesis of xeroderma pigmentosum fibroblasts by acrolein. Mutat. Res. 209, 17-22.
576 Chem. Res. Toxicol., Vol. 19, No. 4, 2006 (12) Chung, F.-L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (13) Smith, R. A., Williamson, D. S., and Cohen, S. M. (1989) Identification of 3,N4- propanodeoxycytidine 5′-monophospate formed by the reaction of acrolein with deoxycytidine 5′-monophospate. Chem. Res. Toxicol. 2, 267-271. (14) Chenna, A., and Iden, C. R. (1993) Characterization of 2′-deoxycytidine and 2′-deoxyuridine adducts formed in reactions with acrolein and 2-bromoacrolein. Chem. Res. Toxicol. 6, 261-268. (15) Chenna, A., Rieger, R. A., and Iden, C. R. (1992) Characterization of thymidine adducts formed by acrolein and 2-bromoacrolein. Carcinogenesis 13, 2361-2365. (16) Chung, F.-L., Nath, R. G., Nagao, M., Nishikawa, A., Zhou, G.-D., and Randerath, K. (1999) Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts. Mutat. Res. 424, 71-81. (17) Khullar, S., Varaprasad, C. V., and Johnson, F. (1999) Postsynthetic generation of a major acrolein adduct of 2′-deoxyguanosine in oligomeric DNA. J. Med. Chem. 42, 947-950. (18) Smith, R. A., Williamson, D. S., Cerny, R. L., and Cohen, S. M. (1990) Detection of 1,N6-propanodeoxyadenosine in acrolein-modified polydeoxyadenylic acid and DNA by 32P postlabeling. Cancer Res. 50, 3005-3012. (19) Kawai, Y., Furuhata, A., Toyokuni, S., Aratani, Y., and Uchida, K. (2003) Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model. J. Biol. Chem. 278, 50346-50354. (20) Pawłowicz, A. J., Munter, T., Klika, K. D., and Kronberg, L. (2006) Reaction of acrolein with 2′-deoxyadenosine and 9-ethyladenines Formation of cyclic adducts. Bioorg. Chem. 34, 39-48. (21) Selzer, R. R., and Elfarra, A. A. (1999) In vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts. Carcinogenesis 20, 285-292.
Pawłowicz et al. (22) Selzer, R. R., and Elfarra, A. A. (1996) Characterization of N1- and N6-adenosine adducts and N1-inosine adducts formed by the reaction of butadiene monoxide with adenosine: Evidence for the N1-adenosine adducts as major initial products. Chem. Res. Toxicol. 9, 875-881. (23) Munter, T., Cottrell, L., Hill, S., Kronberg, L., Watson, W. P., and Golding, B. T. (2002) Identification of adducts derived from reactions of (1-chloroethenyl)oxirane with nucleosides and calf thymus DNA. Chem. Res. Toxicol. 15, 1549-1560. (24) Qian, C., and Dipple, A. (1995) Different mechanisms of aralkylation of adenosine at the 1- and N6-positions. Chem. Res. Toxicol. 8, 389395. (25) Plna, K., Nilsson, R., Koskinen, M., and Segerba¨ck, D. (1999) 32Ppostlabeling of propylene oxide 1- and N6-substituted adenine and 3-substituted cytosine/uracil: formation and persistence in vitro and in vivo. Carcinogenesis 20, 2025-2032. (26) Solomon, J. J., Mukai, F., Fedyk, J., and Segal, A. (1988) Reactions of propylene oxide with 2′-deoxynucleosides and in vitro with calf thymus DNA. Chem.-Biol. Interact. 67, 275-294. (27) Barlow, T., Takeshita, J., and Dipple, A. (1998) Deamination and Dimroth rearrangement of deoxyadenosine-styrene oxide adducts in DNA. Chem. Res. Toxicol. 11, 838-845. (28) Macon, J. B., and Wolfenden, R. (1968) 1-Methyladenosine. Dimroth rearrangement and reversible reduction. Biochemistry 7, 3453-3458. (29) Wilson, M. H., and McCloskey, J. A. (1973) Isotopic labeling studies of the base-catalyzed conversion of 1-methyladenosine to N6-methyladenosine. J. Org. Chem. 38, 2247-2249. (30) Kaminskas, L. M., Pyke, S. M., and Burcham, P. C. (2005) Differences in lysine adduction by acrolein and methyl vinyl ketone: Implications for cytotoxicity in cultured hepatocytes. Chem. Res. Toxicol. 18, 1627-1633.
TX0503496
