Chem. Res. Toxicol. 1989, 2, 267-271
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Identification of 3,N4-Propanodeoxycytidine 5’-Monophosphate Formed by the Reaction of Acrolein with Deoxycytidine 5‘-Monop hosphate Raymond A. Smith,? Daniel S. Williamson,+and Samuel M. Cohen*it$* Department of Pathology and Microbiology and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68105 Received December 5, 1988
Acrolein reacts with deoxycytidine 5’-monophosphate a t physiological p H to form one major adduct. A second minor adduct can be detected when a 3-fold excess of acrolein is present in the reaction mixture. T h e products were separated by ion-pair HPLC on two reverse-phase columns connected in series using triethylammonium formate as ion-pair reagent. T h e major adduct was characterized as 3-(5‘-monophospho-2’-deoxyribosyl)-7,8,9-trihydro-7-hydroxypyrimido[3,4-c]pyrimidin-2-one(3,N4-propanodeoxcytidine5’-monophosphate). This mixture of diastereomers was formed by addition of C1 of acrolein t o the exocyclic amino group a t the 4-position of cytidine, followed by ring closure between C3 of acrolein and N3 of the heterocyclic ring. In order t o address the utility of 32Ppostlabeling for the detection of this exocyclic adduct in acrolein-modified nucleic acids, an acrolein-deoxycytidine 3’-monophosphate reaction mixture was subjected to 32Ppostlabeling. 3’-Dephosphorylation with nuclease PI and the 3’-phosphatase activity of T 4 polynucleotide kinase yields a nucleotide 5’- [32P]monophosphatewhich cochromatographs with 3,N4-propanodeoxycytidine 5’-monophosphate. These data indicate that 32P postlabeling and 3’-dephosphorylation can be used in conjunction with ion-pair HPLC for the detection and quantitation of acrolein-modified nucleotides.
Introduction Acrolein is structurally the simplest member of a group of a,@-unsaturated aldehydes and may be important in terms of potential human exposure and subsequent adverse effects on human health. It is a ubiquitous air pollutant arising from many sources including automotive exhausts and fires ( I ) . In addition, it is of economic importance, finding widespread use as an intermediate in the chemical industry (2). Of particular concern for human health, acrolein is present a t high concentrations in cigarette smoke (3) and is also a metabolite of the widely used chemotherapeutic and immunosuppressive drug cyclophosphamide (4). Smoking and cyclophosphamide therapy have both been implicated as causative agents in the induction of human bladder cancer (2, 4 ) . Although the carcinogenicity of acrolein has not been demonstrated, it causes base pair substitution mutations in Salmonella typhimurium in the absence of an activating system ( 5 ) and is, therefore, capable of direct reaction with DNA. The product of the reaction of acrolein with deoxyguanosine or guanine residues in DNA has been characterized as 1,W-propanodeoxyguanosine(6). Oxidation of the acrolein adducts of 1-methylcytosineand 9-methyladeninehas been used to deduce the orientation of addition and infer the structures of other cytosine and adenosine derivatives (7, 8).
32P postlabeling is being applied to the detection of adducted nucleotides (as 5’-[32P]monophosphates) in acrolein-modified homopolynucleotides and DNA (9, IO). This approach requires the synthesis and characterization *Towhom correspondence should be addressed at the Department of Pathology and Microbiology, University of Nebraska Medical Center, 42nd and Dewey Ave., Omaha, NE 68105. Department of Pathology and Microbiology. Eppley Institute for Research in Cancer and Allied Diseases.
*
0893-228~/89/2702-0267$01.50/0
of acrolein-modified nucleoside 5’-monophosphates. The synthesis, purification, and structural characterization of an acrolein adduct of deoxycytidine 5’-monophosphate is the subject of this paper.
Experimental Procedures Deoxycytidine monophosphates were purchased from Sigma Chemical Co. (St. Louis, MO). Acrolein (gold label reagent, 99.9% pure, containing 200 ppm hydroquinone) was from Aldrich Chemical Co. (Milwaukee, WI). Micrococcal nuclease (Staphylococcus aureus) and phosphodiesterase I1 (bovine spleen) were obtained from Cooper Biomedical (Malvern, PA). Nuclease PI (Penicillium citrinium) and T4 polynucleotide kinase were purchased from Pharmacia-LKB Biotechnology (Piscataway, NJ). Nonradioactive ATP and [y-32P]ATP (>5000 Ci/mmol) were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN) and Amersham International (Arlington, IL), respectively. Triethylamine was purchased from Eastman Kodak (Rochester, NY), and all other materials were obtained from commercial sources. Deoxycytidine 3’-monophosphate was dissolved in PBS (1 mmol/mL) and incubated (37 “C, 16 h) with a 3-fold excess of acrolein. Aliquots containing 125 nmol of nucleotide were mixed with an equal volume of succinate buffer (40 mM sodium succinate, 20 mM CaCl,, p H 6.0) prior to transfer of 32Pfrom [y32P]ATP(50 pM, 10-30 Ci/mmol) to the 5’-hydroxyl groups of the 3’-monophosphatesby T4 polynucleotide kinase (0.25 unit/pL) in kinase buffer (10 mM Bicine-NaOH, 10 mM MgCl,, 10 mM dithiothreitol, 1.0 mM spermidine, p H 9.3). After 3 h a t 37 “C, the products were 3’-dephosphorylated by addition of nuclease P,(5 units) and T4 polynucleotide kinase (14 units) in 0.1 volume of phosphatase buffer (500 mM sodium acetate, 100 mM MgCl,, p H 4.5), deoxycytidine 5’-monophosphate (50 nmol) was added, and the incubation was continued (1 h, 37 “C). The products of this 32Ppostlabeling procedure were analyzed by ion-pair HPLC on two 3.9 X 300 mm 10-pm Bondapak CI8 columns connected in series, eluted (1 mL/min, 70 OC) with 10 mM triethylammonium phosphate (TEAP) in water, p H 5.0 (solvent A). Two minutes after injection, the system was programmed from 0 to 10% solvent B (10 mM TEAP in methanol, p H 6.0) in 10 0 1989 American Chemical Society
268 Chem. Res. Toxicol., Vol. 2, No. 4 , 1989 min by using gradient profile 2 on a Waters Model 680 gradient controller. The chromatography was completed by linear elution (profile 6) to 30% B in 50 min and 100% B at 53 min from the start of the gradient. After a 3-min isocratic plateau, the system was returned to 100% solvent A by 60 min. UV and 32P-labeled peaks were detected with a Waters 481 UV/visible spectrophotometer (254 nm) and a Ramona 5 LS flow-through radioactivity monitor, respectively. The 5 LS was operated in the stream split mode, and saturation of the radioactivity monitor by excess [y-32P]ATPwas prevented by purging (30 min, 5 mL/min) the flow cell with 1.0 M phosphoric acid. A timed events program activated the pump on the 5 LS a t 40 min after injection, and simultaneously the stream split (which was set at 100%) diverted the HPLC eluate to waste. Ten minutes after the next injection the timed events program shut the 5 LS pump off and the HPLC eluate was automatically diverted to the Ramona flow cell. This sequence of events also eliminates the peak of inorganic 32Pfrom the beginning of the chromatogram and accounts for the changes in the 32Pbase line seen a t 10 and 40 min in Figure 1. To characterize the products of the postlabeling analysis, deoxycytidine 5'-monophosphate (20 mg/mL in PBS) was incubated with an equimolar or 3-fold molar excess of acrolein. Adducts were purified by ion-pair HPLC as described above by using formic acid instead of phosphoric acid. In addition, the internal diameter of the columns and the flow rate were increased to 7.8 mm and 1.5 mL/min, respectively. UV-absorbing materials were collected by a FRAC-100 fraction collector operated in the peak cutting mode, pooled, and adjusted to pH 10.0 by the addition of 0.1 M NaOH. The ion-pair reagent was extracted by shaking with an equal volume of ether and the pH returned to 10.0 before two further extractions with ether. The solution was then adjusted to pH 3.0 by addition of 0.1 M HCl and extracted three times with an equal volume of ether. The 'H NMR spectrum of the material did not change following the pH adjustments and extractions which were performed in as short a period of time as possible, typically 7-10 min at room temperature. The nucleotide was concentrated by rotary evaporation (at 35 "C) and desalted by chromatography on a column (2.5 X 40 cm) of Sephadex G-25 eluted with distilled water. All NMR experiments were performed on a Varian XL-300 spectrometer at 27.0 "C using the standard 5-mm broad-band switchable probe. The two-dimensional (2-D) experiments were performed nonspinning. Slight modification of the radio frequency transmitter and receiver gating was required for performance of the indirect-detection experiments.
Smith et al.
Figure 1. Ion-pair HPLC analysis of 32P-labeled nucleoside 5'-monophosphates on two 10-pm Bondapak C18columns (3.9 X 300 mm i.d.) a t 1 mL/min and 70 "C. Panel A: Elution profile of acrolein-modified deoxycytidine 5'-monophosphate (UV markers). Panel B: 32Ppostlabeling analysis of deoxycytidine 3'-monophosphate. Panel C: 32Ppostlabeling analysis of acrolein-deoxycytidine 3'-monophosphate reaction mixture. Peak identification: (a) deoxycytidine 5'-monophosphate; (b) adduct 1 (3,N4-propanodeoxycytidine 5'-monophosphate); (c) adduct 2.
A a
B
b
Results Ion-pair HPLC of deoxycytidine 3'-monophosphate that had been incubated with acrolein prior to 32Ppostlabeling provides evidence for t h e f o r m a t i o n of one n e w p e a k of radioactivity. T h i s material is more highly retained than the p a r e n t nucleotide and cochromatographs w i t h t h e major a d d u c t f o r m e d b y reaction of acrolein w i t h deoxycytidine 5'-monophosphate (Figure 1). Increasing t h e amount of acrolein b y a factor of 3 results i n increased conversion of the s t a r t i n g m a t e r i a l (Figure 2, p a n e l A versus B) as well as t h e f o r m a t i o n of a second (minor) a d d u c t (Figure 2, panel B, p e a k c). T h e s e materials were purified b y HPLC and the ion-pair reagent was removed b y solvent extraction prior t o structural determination b y NMR spectroscopy. The general s c h e m e used for s t r u c t u r a l elucidation of the adduct utilized classic as well as recently developed 2-D NMR experiments. T h e approach relies upon u n a m biguous a s s i g n m e n t of t h e sugar and exocyclic p r o p a n o p r o t o n resonances via phase-sensitive d o u b l e q u a n t u m filtered correlation spectroscopy (DQF-COSY) (11). Figure 4 shows the DQF-COSY s p e c t r u m of a d d u c t 1. The sugar p r o t o n s a r e assigned in a stepwise fashion s t a r t i n g w i t h the easily recognized H1' p r o t o n . The resonance of 6.3 p p m integrates to o n e p r o t o n and is assigned as t h e terminal m e t h i n e p r o t o n of t h e exocyclic function, H 7 . The H 4 a n d H5 p r o t o n s m a y b e assigned provisionally o n t h e
I 10
20 MINUTES
30
Figure 2. Ion-pair HPLC analysis of acrolein-deoxycytidine 5'-monophosphate reaction mixture on two 10-pm Bondapak C18 columns (7.8 X 300 mm id.) a t 1.5 mL/min and 70 "C. Panel A: Equimolar amounts of each reactant. Panel B: Threefold excess of acrolein. In each case an aliquot of the reaction mixture corresponding to 3 pmol of deoxycytidine 5'-monophosphate was analyzed. Peak identification as in Figure 1. basis of their characteristic relative chemical shifts. Unambiguous confirmation is provided b y subsequent experiments; therefore, t h e correlation of H 4 and H5 is not included in Figure 4. Acquisition parameters are given in t h e legend. The second step i n a s s i g n m e n t requires correlation of each proton with its directly bound carbon. To accomplish this, t h e phase-sensitive 'H-detected heteronuclear multiple q u a n t u m coherence (HMQC) e x p e r i m e n t (12) w a s applied. T h i s e x p e r i m e n t allows one-bond correlation of
Chem. Res. Toxicol., Vol. 2, No. 4, 1989 269
Acrolein-Deoxycytidine 5'-Monophosphate Adduct
c7, c3' c4
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Figure 4. DQCOSY spectrum of adduct 1. The spectral width in both dimensions is 237.4 Hz, 512 increments in tl were used, and the spectrum results from a phase-sensitive Fourier transformation of a 2 X 2048 X 2048 matrix of complex oints. The experiment required 5.8 h to acquire. The standard H spectrum is shown across the top. The residual HOD peak is labeled as
P
such. protons and carbons with excellent resolution and sensitivity. Indeed, a 5-7-fold increase in sensitivity is observed in our hands compared to the conventional 13C-detected experiment, even with use of the decoupler coil of our
130
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4 Figure 5. HMQC spectrum of adduct 1. The 'H spectral width was 2505.6 Hz and the 13C spectral width was 11800 Hz; 256 increments in tl were acquired. The spectrum results from phasesensitive Fourier transformation of a 2 X 1024 X 1024 matrix of complex points. The experiment required 6.5 h to acquire. The 13C spectrum across the top required 15.1 h to acquire. broad-band 5-mm switchable probe for lH observation. Figure 5 shows the HMQC spectrum of adduct 1. The conventional I3C spectrum is shown across the top, and the 'H spectrum is along the left vertical axis. The correlations appear as doublets in the proton dimension because we are detecting only protons directly bound to a 13Cnucleus. Hence, this large (140-200-Hz) coupling appears in the detected signals. (These are the 13C satellites which can be observed in normal 'H spectra.) Note also that the proton homonuclear I3C couplings show up as small splittings in the satellite signals. 13C decoupling during acquisition was not used since the small amount of material made accurate pulse width calibration difficult for the required WALTZ decoupling scheme. The assignment of all protonated carbons is straightforward and is shown in the 13Cspectrum. All acquisition parameters are given in the legend. The final step in the assignment procedure entails use of long-range carbon-proton correlation. For this purpose, 'H-detected heteronuclear multiple-bond correlation (HMBC) experiment was employed (14). Figure 6 shows the results of a HMBC experiment performed on adduct 1. Two types of correlations arise in this experiment, one-bond and multiple-bond correlation. The former are equivalent to the satellites seen in the HMQC experiment. They are attenuated by application of a low-pass J-filter (14), but some of this signal does leak through since an average l J H C is used for the filter, leading to nonoptimal suppression for some spin systems. These satellites are connected with vertical lines in the figure. The other type of correlations arise from scalar coupling of protons to carbons two or three bonds away. These appear also as doublets in the proton dimension if the long-range coupling constant is large enough, but generally this fine structure is not visible. The acquisition parameters are given in the legend. As can be seen, the H1' proton has a long-range correlation to C4, confirming H4 is correlated t o C5, C4 (1-bond satellites), and C2 and Cl'. The H5 shows correlations to C4 and C5a but not to C2 (4JHcis too small),
270 Chem. Res. Toxicol., Vol. 2, No. 4 , 1989 C4
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Figure 6. HMBC spectrum of adduct 1. The ‘H and 13Cspectral widths are the same as in Figure 5, and 512 increments in t , were acquired. The total experiment time was 16.5 h. The spectrum results from Fourier transformation of 2048 X 2048 complex points, and an absolute value mode display is used. Vertical lines connect the satellites representing one-bond correlations identical with those seen in the HMQC data. All proton resonances are labeled within the contour plot. The small offset in F1in the low-intensity correlations is due to proton homonuclear coupling evolution during t P The upper portion of the spectrum is plotted at a 50% higher vertical scale than the lower portion. confirming the C5a and C2 assignments. Finally, the downfield H9 methylene proton of the exocyclic propano function shows a correlation to C5a and C2 while the exocyclic methine proton only shows correlation to C5a. This confirms the cyclic adduction and orientation of the propano function, since the H9 protons occupy the only exocyclic position which will allow correlation to two quaternary carbons. These data are consistent with the structural assignment of the acrolein-deoxycytidine adduct shown in Figure 3. In addition to the NMR data presented, a UV spectrum revealed a, A a t 280 nm. Fast atom bombardment mass spectrometry (FAB MS) of adduct 1did not reveal an M + 1 peak; however, a 96% intensity 171 m f z peak was observed which corresponds to base plus 5 H+ (B + 5). This can be rationalized in light of the number of sites available for reduction in the base moiety. Also, a 46% peak a t 157 m / z was seen which is consistent with loss of nitrogen from the B + 5 fragment. Negative FAB MS added no additional information. In addition to the major adduct, there was a second minor adduct in the deoxycytidine 5’-monophosphate acrolein reaction mixtures (Figure 2). However, there was no radioactivity associated with the minor adduct (Figure 1) when 32P-labeledsamples of acrolein-modified deoxycytidine 3’-monophosphate was cochromatographed with UV marker compounds. Attempts to characterize this minor adduct were unsuccessful since it converts into the major adduct upon isolation. The available NMR data (not shown) suggests a disubstituted moiety which is stabilized by release of one acrolein molecule to form the major adduct.
Discussion In a previous report, we were unable to detect cytosine adducts in poly(dC) that had been incubated with acrolein
and analyzed by 32Ppostlabeling (10). This was not due to lack of reactivity between acrolein and cytosine residues since chromatography of the reaction mixture under conditions which bound polymeric material provided an eluate with increased UV absorbance compared to that of the control. Reaction of acrolein with deoxycytidine 3’monophosphate provided a substrate for 32Ppostlabeling. After 3’-dephosphorylation, this radioactive material cochromatographed in ion-pair HPLC with a marker compound (Figure 1)synthesized by reaction of acrolein with deoxycytidine 5’-monophosphate (10). The kinase reaction was performed for 3 h since this has been shown to be the optimum incubation period for labeling nucleotides with 32P from [ T - ~ ~ P I A T( 1P5 ) . Many adducted 3’,5’-bisphosphates are reported to be resistant to 3’-dephosphorylation by nuclease Pl (16), and this problem was circumvented by the use of nuclease P1 and T4 polynucleotide kinase. This latter enzyme contains a pH-dependent 3’-phosphatase which is only active a t neutral pH’s, and this activity has been used for the 3‘-dephosphorylation of 32P-labeledvinyl chloride adducts (17,18). The structure of this adduct has been determined by nuclear magnetic resonance spectroscopy. Ion-pair HPLC was used since chromatography of the reaction mixture on silica or polymeric anion-exchange columns was unsuccessful due to inadequate resolution and/or tailing of peaks. Ion-pair HPLC has been used in the resolution of nucleoside 5’-monophosphates (19) as well as trycyclic nucleoside 5’-monophosphates (20). The tetraalkylamines used in these systems cannot be removed by rotary evaporation or freeze-drying; the more volatile triethylamine was used as the ion-pair reagent in this work (21). Solvent extraction was used for removal of the triethylammonium formate since rotary evaporation or freeze-drying resulted in the formation of a yellow product which was insoluble in organic or aqueous solvents. Phosphoric acid was used as the counterion to triethylamine in analysis of 32P-labeled digests in order to inhibit accumulation of inorganic 32P on the HPLC columns. For adduct purification, the phosphoric acid was replaced by formic acid since this latter agent is readily removed during ether extraction. The present study indicates that acrolein reacts with deoxycytidine 5’-monophosphate to form a major and minor adduct. This latter material appears to be diadducted and is only detectable when a 3-fold molar excess of acrolein is present in the reaction mixture, conditions that also result in increased conversion of dC5’P to the major product, adduct 1 (Figure 2). Multiple adduction to purine nucleosides has been reported for malondialdehyde (22, 23) and acrolein (8). Adduct 1 has been characterized by NMR spectroscopy as an exocyclic 3,N4-propanodeoxycytidine5’-monophosphate which is formed by addition of C1 of acrolein to the exocyclic amino group of cytosine followed by ring closure between C3 of acrolein and N3 of cytosine. This orientation for the addition of acrolein is the reverse of that proposed (7, 8) following reaction with cytosine derivatives under nonphysiological conditions (1.0 M sodium acetate, p H 4.37, 25 OC). The product of the reaction between acrolein and 1-methylcytosine was oxidized by heating in alkaline silver oxide. Spectral and chromatographic properties (in one TLC system) of the derivative (37-66% yield) and synthetic N4-(2-carboxyethyl)-l-methylcytosinewere identical. This apparent homology was, therefore, used to infer the structure of acrolein cytidine or deoxycytidine adducts and deduce the orientation of acrolein addition to these nucleosides. This derivatization procedure does not allow for possible intramolecular rearrangements (24), and this may
Acrolein-Deoxycytidine 5’-Monophosphate Adduct contribute to the discrepancy in assignment of the orientation of acrolein adduction. Addition of acrolein to deoxycytidine 5’-monophosphate results in the formation of a new chiral center at C7 (Figure 3) which is derived from the C1 of acrolein. This chiral center is in addition to those of deoxyribose, and adduct 1 should exist as a mixture of diastereomers. Indeed, a small splitting is observed in the H4 peak in the ‘H spectrum which does not represent a scalar coupling, as this small in-phase splitting does not evolve to antiphase in a simple spin-echo experiment (not shown). We take this as evidence for an equimolar mixture of diastereomers. The reaction of acrolein with other DNA constituents results in the formation of molecules with additional chiral centers (6, 8). The techniques described in this report for the synthesis and characterization of adducted deoxynucleoside 5’monophosphates are also applicable to deoxynucleoside 3’,5’-bisphosphatesl and may be of considerable value in the characterization of carcinogen-modified nucleotides that have been detected by 32Ppostlabeling analysis. In addition, the indirect detected long-range proton-carbon correlation technique allows complete assignment of the synthetic adducts without recourse to chemical derivatization for proof of structure.
Acknowledgment. This work was supported by grants from Nebraska State Department of Health (87-01R), CA44886 and CA36727 from the National Cancer Institute, and RR01968 from the NIH. We thank Dr. Phillip Issenberg for performing mass spectral analysis and Dr. Donald L. Nagel for assistance with NMR studies. The technical assistance of Ilene Pinnt and T. Scott Tibbels, as well as the secretarial support of Patricia Smith and Deboraha Coleman, is gratefully acknowledged. Supplementary Material Available: Table giving 13C and
‘HNMR chemical shifts (1 page). Ordering information is given on any current masthead page.
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