467
Chem. Res. Toxicol. 1990, 3, 467-472
Studies of the Reaction of Malondialdehyde with Cytosine Nucleosides Koni Stone, Adam Uzieblo,+and Lawrence J. Marnett* A. B. Hancock Memorial Laboratory for Cancer Research, Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received M a y 22, 1990 The reaction of malondialdehyde (MDA) with cytosine nucleosides was investigated, and the structures of 1:l and 3:l MDA-cytosine adducts were identified. Ultraviolet and NMR spec(M$) troscopy indicates the structure of the 1:l adduct is N4-(3-~~~-1-truns-propenyl)cytosine and the structure of the 3:l adduct is 6-(5*,7*-diformyl-2*H-3*,6*-dihydro-2*,6*-methano1*,3*-oxazocin-3*-yl)-2-oxopyrimidine (M3C). Both adducts are analogous to previously identified MDA-adenine adducts, The time courses of adduct formation in the reaction of MDA with deoxycytidine a t different pH's and using different sources of MDA were determined. M3Cdeoxyribose is the major product a t acidic pH whereas M,C-deoxyribose appears to be the sole adduct formed a t neutral pH. These results suggest that adducts formed between nucleic acid bases and oligomers of MDA may not play a major role in MDA mutagenesis.
Introduction Malondialdehyde (MDA)' is a product of lipid peroxidation and is generated during prostaglandin and thromboxane biosynthesis (1-4). It is a mutagen in bacterial and mammalian cells and a carcinogen in rodents (5-8). Its widespread occurrence in the animal kingdom and its genotoxic activity make it a potential endogenous carcinogen in humans. MDA reacts with nucleic acids to form adducts that may be responsible for its genotoxic activity (9-13). Adducts result from the addition of MDA or its oligomers to the deoxynucleoside bases (11-14). Scheme I summarizes the reactions of MDA with guanine and adenine nucleosides (9, 12, 13). Golding et al. have isolated and identified the dimer and trimer of MDA shown in Scheme I (15). MDA also reacts with cytosine nucleosides and forms an adduct that contains one molecule of nucleoside and three molecules of MDA (11). The spectral properties of the 3:l MDA-cytidine adduct are quite similar to the 3:l MDA-adenosine adduct, the structure of which was recently revised to M3A-R (13). We have reinvestigated the reaction of MDA with cytosine nucleosides and have identified two adducts that are structurally analogous to MIA and M3A. In addition, we have conducted studies to compare the relative amounts of adduct formed under different conditions. The major reaction products at acidic pH are the oligomeric adducts. However, the extent of their formation at neutral pH is unknown. Also, MDA is generated at low concentrations in vivo; thus, it is important to determine the amounts of the adducts formed under conditions where an excess of MDA is not present. Our results indicate that the product profile is significantly altered by pH and the conditions used to generate MDA. Materials and Methods 2'-Deoxycytidine was purchased from Cruachem (Herndon, VA). Cytidine was from Aldrich (Milwaukee, WI). Tetraethoxypropane (TEP)was from Fluka Chemical Co. (Ronkonkoma, NY).These compounds were determined to be greater than 95% pure by NMR analysis. Deuterated solvents for NMR were from Aldrich Chemical (Milwaukee, WI). High-purity solvents for 'Present address: Proteins International, 1858 Starbat Dr., Rochester, MI 48093.
0893-228x/90/2703-0467$02.50/0
Scheme I. Reactions of MDA and MDA Oligomers with Guanine and Adenine Nucleosides
H\N.H
0
Ribose
M,GR
MDA
MIA-R
"DA?i no' Dimer of MDA
'""7 Hb 0
Adenosine
-
H
r?
'N Ribose
Trimer of MDA
M+R
HPLC were from Burdick and Jackson (Muskegon, MI). Synthesis of NaMDA. MDA was synthesized by hydrolyzing 2 g (TEP)in 5 mL of aqueous HC1 (pH 1.3) for 2 h. The solution was neutralized to pH 7.0 with 1.0 N NaOH and then extracted Abbreviations: MDA, malondialdehyde; TEP,tetraethoxypropane; MIG, pyrimido[1,2-a]purin-l0(3H)-one;M2G,6-cis-3,5,6,12-tetrahydro12-oxo-6,10-methano-lOIf-[ 1,3,5]oxadiazocino[5,4-a]purine-9-carbox-
aldehyde; MIA, NB-(3-0xo-l-tra~-propenyl)adenine; MsA, 6-(5*,7*-di-
formyl-2*~-3*,6*-dihydro-2*,6*-methano-l*,3*-oxazocin-3*-yl)purine; MIC, N4-(3-oxo-l-trans-propenyl)cytosine; M3C, 6-(5*,7*-diformyL2*H3*,6*-dihydro-2*,6*-methano-l*,3*-oxazocin-3*-yl)-2-oxopyrimidine; dR, 2-deoxyribofuranose; R, ribofuranose; COSY, correlation spectroscopy; lH-13C COSY, heteronuclear correlated spectroscopy; DEFT, distor-
tionless enhancement by polarization transfer.
0 1990 American Chemical Society
Stone et al.
468 Chem. Res. Toxicol., Vol. 3, No. 5, 1990 with diethyl ether to remove any remaining TEP. The aqueous layer was concentrated by rotary evaporation and divided into four fractions, and each fraction was applied to two ClS solid-phase extraction columns connected in series (J.T. Baker Chemical Co., Phillipsburg, NJ). MDA polymers adsorbed to the columns, and the NaMDA was eluted with water. The filtrates were treated with activated charcoal to remove traces of colored impurities and then lyophilized. The white solid was 98% pure of organic impurities by NMR analysis [D20: 8.7 ppm (d, 2 H), 5.35 ppm (t, 1 H)]. The product contained 40% NaCl, as determined by UV = 34 mM-'). absorbance of the NaMDA a t 267 nm Synthesis of M3C-R. The M3C-R adduct was prepared by dissolving 500 mg of cytidine in 20 mL of water and adding it to a solution of 918 mg of T E P in 20 mL of 0.05 N HCl. The pH of the reaction mixture was adjusted to 4.0 and stirred a t 37 O C . During the first hour of the reaction, the pH was maintained a t 4.0 with 0.05 N HCl. After 48 h, the reaction mixture was neutralized to 7.0 with 0.05 N NaOH. Its volume was reduced under vacuum (37 "C) to 3 mL and injected onto an MPLC column (2.5 X 50 cm, C1840-wm packing, Baker Chemical Co.). The column was equilibrated and washed with 20% methanol in water. The green fraction was collected and injected onto a reversed-phase semipreparative column (10 X 250 mm, C18Ultrasphere ODS-3 DP-5, Beckman) and eluted with 15% acetonitrile with a flow rate of 2.0 mL/min. A single peak eluted a t 13 min which was determined to be the M3C adduct by UV and NMR spectroscopic analysis. The isolated yield was 1.4%. Synthesis of MIC-dR a n d M3C-dR. To prepare these adducts, 110 mg of 2'-deoxycytidine hydrochloride was reacted with 500 mg of TEP in water at pH 4.2 for 48 h at 37 O c a 2 The reaction mixture was extracted with 3 volumes of diethyl ether to remove any unreacted TEP, and the adducts were purified by HPLC on a semipreparative C18column (10 X 250 mm, Ultrasphere ODS, Beckman). The following aqueous solvent system was employed: 5% acetonitrile isocratic for 15 min, 5-10% linear gradient of acetonitrile over 10 min, 10% acetonitrile for 15 min, and 10-50% linear gradient of acetonitrile over 5 min. Under these chromatographic conditions, the MIC-dR adduct eluted at 27.9 min and the diastereomers of M3C-dR eluted at 36.8 and 39.5 min. The purified compounds were prepared for NMR spectroscopy by lyophilizing from D 2 0 three times. Time Course of Reaction of TEP w i t h %'-Deoxycytidine at pH 4.2 o r 7.2. For these experiments, 62 mg of 2/-deoxycytidine hydrochloride was dissolved in water, and the pH was adjusted to 7.2 or 4.2 with 0.1 N NaOH. The aqueous volume was adjusted to 800 wL, 500 wL of T E P was added to the reaction vial, and the reaction vial was placed in a shaker a t 37 OC. At various times, aliquots were removed and diluted for HPLC analysis on a C18 analytical HPLC column (4.6 X 250 mm, 5 pm, Ultrasphere ODS, Beckman). MIC-dR (10 min) and M3C-dR (14.4 and 15.2 min) were eluted with a 5-20% acetonitrile linear gradient over 20 min. The UV absorbance a t 326 nm was integrated and the concentrations of the adducts in the reaction mixture were quantitated by using standard curves. Each data point was the average of a t least three separate determinations. Time Course of Reaction of NaMDA w i t h %'-Deoxycytidine at pH 4.2 o r 7.2. NaMDA (21.6 mg) was combined with 62 mg of 2'-deoxycytidine in water. The pH of this solution was 1.2. To avoid excess polymerization of the MDA a t low pH, the solution was neutralized with 1 N sodium hydroxide prior to adding the NaMDA. Then, the pH was adjusted to either 4.2 or 7.2 with 1 N HCI. Water was added to give a total volume of 800 pL. The reaction vial was placed in a 37 OC shaker, and aliquots were periodically removed and analyzed as described above. Instrumentation. UV spectra were recorded on a HewlettPackard 8452A diode array spectrophotometer. UV spectra of peaks eluting from HPLC columns were recorded with a Hewlett-Packard Model 1040A diode array detector. HPLC was performed with a Varian Model 9010 HPLC. For the pH time course studies, either a Varian 2050 UV detector or a HewlettPackard Model 1040A diode array detector was used to monitor absorbance at 326 nm. Peaks were integrated and quantitated
* The yield of MIC-dR could be increased by performing the reaction
at pH 5.4.
Table I. Seectral Data and Assignments chemical shifts, PPm carbon 'H 13C multiplicity 1.96, 2.18 23.6 CH2 3.95 60.7 CH2 2.32, 2.58 39.6 CH2 4.04 15.8 CH 4.13 87.0 CH 4.35 69.9 CH 6.23 87.3 CH 6.76 77.9 CH 6.89 94.4 CH 7.69 164.7 CH 144.2 CH 8.43 8.49 145.3 CH 9.17 185.5 CH 9.32 184.9 CH 126.2 C 123.7 C 156.8 C 161.9 C "UV max (H,O):
6323
for M.C-dR" proton assignment 9*a,9*b 5',5" 2',2/' 6* 4'
3' 1' 2*
5 8* 4* 6 11*
10* 5* 7* 2 4
= 21 300; e238 = 15006.
by using the Rainin Dynamax program on a Macintosh desktop computer or the Hewlett-Packard HPLC software on a Hewlett-Packard Chemstation. NMR Spectroscopy. NMR spectra were recorded on Bruker instruments. The 13C broad-band decoupled and DEPT spectra were recorded by using an AC200 instrument. The DEPT (distortionless enhancement by polarization transfer) (16)experiments were acquired with 0 = 135O. An AC300 instrument was employed for the homonuclear correlated (COSY) 2D experiments. The experimental details for these experiments are described in the legend of Figure 1. Chemical shifts are reported in ppm and are referenced to HOD as the internal standard. A heteronuclear correlated 2D experiment was done on an AM400 instrument, using an inverse detection probe to observe protons coupled to 13C. The pulse sequence is described by Bax et al. (17). A sample of M3C-R was prepared by dissolving 3.8 mg of 0.5 mL of D20 in a 5-mm Wilmad 528PP NMR tube. The spinner was turned off for the data acquisition to minimize noise in Fl. A total acquisition time of 12 h was required to obtain 128 scans for each of 308 t l valus. The data were zero filled to 1K data points in Fl and sine bell shifted in the F2 domain. The data were then Fourier transformed in a magnitude calculation. This resulted in a 1K by 1K data matrix.
Results Structure of M3C-dR. T h e major MDA-deoxycytidine a d d u c t was synthesized b y reacting TEP with deoxycytidine at p H 4.2 and 37 "C for 48 h. B y use of t h e chromatographic system described u n d e r Materials and Methods, two diastereomers of t h e 3:l deoxycytidine add u c t were separated. T h e compounds from these two HPLC peaks had identical NMR and W spectra and were subsequently combined t o increase t h e mass for 13C and 2D COSY NMR experiments. T h e 1D 'H and 13Cspectra of t h e 3:l cytidine a d d u c t were similar to the spectra for t h e M3A adduct. Therefore, i t was expected that t h e former adduct would have a structure related t o M3A, and assignments were m a d e o n t h e basis of this analogy a n d t o assignments for cytidine (13,18). A 2D COSY experi m e n t with M3C-dR was performed t o verify the scalar couplings. The expected couplings between the protons and H6.)were present, in the propano bridge (H2., HSOa,-, as shown in Figure 1. T h e long-range coupling between H2.a n d H,, was also detected. T h e UV absorption maxi m a a n d N M R chemical shifts a r e summarized in T a b l e I, a n d t h e structure is shown i n Figure 4. Proton-CarbonConnectivities for M3C-R. I n order to verify t h e proton-carbon connectivities for t h e 3:l
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 469
Malondialdehyde-Cytosine Adducts 'H
.........____...___._._.
..........________.____. (
80
90
j
50
60
70
20
30
40
10
' t
0
20 30
2*-6'
~ . . . . . . ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ 40
50
3'-4'
~
60 70
80
9.0
# 100
90
80
70
60
50
40
30
20
10
PM
Figure 1. Two-dimensional COSY spectrum of M C dR. The sample was prepared by dissolving 4.8 mg of the addict in 500 pL of D,O. The plot results from 96 scans for each of 256 tl values. The matrix was zero filled in the Fl domain and then Fourier transformed to yield a matrix that contained 512 by 512 data points, and this was symmetrized. The water peak was suppressed by selective irradiation for 1.3 s. Two dummy scans were taken before each FID was acquired to allow for presaturation of the water peak. The solid lines show the vicinal and geminal couplings, and the dashed lines show the long-range couplings.
MDA-cytosine adduct, a lH-13C COSY experiment with inverse detection was performed (Figure 2). The ribose adduct (M3C-R)was used to avoid congestion in the 2.0 ppm region of the proton spectrum; the 2',"' and 9*a,b protons have chemical shifts from 6 1.96 to 2.58 ppm. This is a coupled spectrum, so every proton signal is split into a doublet by the 13C to which it is attached. The center of the doublet corresponds to the chemical shift of the proton, and horizontal lines have been drawn to depict this. The most upfield carbon (6 17.6 ppm, C6.) is attached to the proton at 6 4.09 ppm in the M3C-R adduct. This is analogous to the M,A-R spectrum, and this 13Cchemical shift is 15-20 ppm upfield from where the methine carbon in a bicyclononane ring would be expected (13). The long-range couplings between the carbons at 6 125 (C7*) ppm and the aldehyde protons at 6 9.2 and 6 129 (Hll,) and 6 9.3 (Hlo,) ppm, respectively, were also observed. These couplings were used to assign the quaternary carbons in the adduct. The UV absorbance maxima and NMR chemical shifts for M3C-R are summarized in Table 11, and the structure is shown in Figure 4. Identification of MIC-dR. A 2'-deoxycytidine adduct corresponding to MIA-dR was not detected in the early attempts at adduct production. This was due to poor separation of MIC-dR and 2'-deoxycytidine on reversedphase HPLC with an isocratic solvent system of 10% acetonitrile/ water. Separation was eventually achieved by first washing the deoxycytidine off the column with 5% isocratic acetonitrile, and then employing a gradient of 5-10'70 acetonitrile to elute the MIC-dR. The UV spectrum was very similar to the spectrum of MIA-R, with an absorbance maximum at 324 nm. The NMR spectrum displayed three resonances in addition to the signals for 2'-deoxycytidine. These signals were assigned to a prop-
,
180
lQlO*
8'
-I
1
140 jjl20
160
4'
5'7'
,
100
J,
8
--,
6o
,
4o
Z T P M l3C
9a,b' 6'
Figure 2. Two-dimensional plot of the inverse detected lH-13C COSY of M3C-R. The vertical axis re resents a proton spectrum where protons that are coupled to C are observed. This is a coupled spectrum, so the contour peaks are doublets that are a t 6, f (1I2)JH.C. The horizontal axis represents the l3C chemical shifts. The solid lines show the direct proton-carbon connectivities in the adduct moiety. The dashed lines show the long-range coupling between the aldehyde protons and the neighboring quaternary carbons in the adduct. The expected cytidine proton-carbon connectivities are all present, but these lines have been omitted for the sake of clarity.
P
Table 11. Summary of Spectral Data from UV, I3C DEPT, and HeteroCOSY Experiments on M,C-R" correlated chemical shifts, ppm carbon proton 'H 13C multiplicity assignment 9*a,9*b 1.99. 2.22 25.4 CHI 5',"' 3.95 61.7 CH; 17.6 CH 6* 4.09 4' 4.21 85.4 CH 3' 4.35 70.2 CH 2' 75.9 CH 4.80 1' CH 5.96 92.8 2* 6.83 79.5 CH 5 6.89 96.1 CH 8* CH 7.69 166.1 4* 8.43 145.5 CH 6 147.2 CH 8.49 11* CH 9.17 193.0 10* 192.4 CH 9.32 5* 128.1 C 7* 125.5 C 2 163.5 C C 4 170.5 "UV max (H20): e323 = 21 300; €238 = 15006.
enal group attached to N4 of the cytosine base. A 2D COSY NMR spectrum was obtained to show the scalar couplings (Figure 3). Off-diagonal elements were obvious between the propenal protons at 6 5.95,9.34, and 8.34 ppm. Proton assignments for this adduct are summarized in Table 111, and the structure is shown in Figure 4. Time Course Experiments. A study of the time course of adduct formation was performed to determine the
Stone et al.
470 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
0 2 0 4 0 6 0 8 0 1 0 0 1 2 0
Time (hr) 1
. 9 5
Y O
7 0
80
h 0
5 0
4 0
1 I,
2 5
pp''
Figure 3. Two-dimensional COSY spectrum of MIC-dR. The sample was prepared by dissolving 800 fig of M,C-dR in 500 p L of D20. The experimental details are given in the legend of Figure 1. The solid lines show the vicinal couplings, and the dotted lines show the coupling between H8 and H7. The H, proton is not observed in the 1 D spectrum due to exchange; the 1 D proton spectrum is plotted above the 2 D contour plot.
*
H I* 0
N
OAN
k
H
I
M,C-dR
B
5 H6
M&R
M&dR
Figure 4. Structures of MIC-dR and M,C-R(dR). Table 111. Chemical Shifts and Assignments for M,C-dR 'H chemical multiproton J, Hz assignments shifts, ppm plicity 2' 2.40 mult 2" mult 2.56 mult 51,511 3.82 4' mult 4.09 mult 3' 4.45 8.6, 13.7 dofd 9 5.95 1' t 6.3 6.25 d 7.4 5 6.31 d 7.4 6 8.21 8.34 d 13.7 a 9.34 d 8.6 10
relative amounts of the adducts formed under various reaction conditions. Previous studies of the reaction of TEP and deoxyadenosine indicated that the oligomeric adducts were not formed above pH 6.0. However, quantitation of the deoxyadenosine adducts was difficult at lower pH as depurination began to occur below pH 5.4. 2'-Deoxycytidine was used as the nucleoside, since it does not undergo deribosylation. This simplified the product profile; therefore, it was anticipated that quantitation of the deoxycytidine adducts would more accurately reflect the kinetics of the reaction. The MIC-dR and M,C-dR adducts were used as chromatographic standards. We were interested in the relative amount of the adducts formed at pH 7.2 compared to pH 4.2 (the pH used to synthesize the 3:l adduct standards). Also, two different sources of MDA were compared. The MDA was generated in situ by hydrolysis of TEP, or it was
.
.
.
. I
-64 . I I , I 9 I 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0
Time (hr)
Figure 5. Amount of adducts produced from the reaction of MDA or T E P with 2'-deoxycytidine at pH 4.2. (A) Amount of M3dC formed from reaction with MDA (8)or with T E P (+). (B) Comparison of the amounts of MldC (e)and M3dC (a) produced by reaction of T E P with dC a t pH 4.2.
added directly to the reaction vial as the sodium salt. Figure 5A compares the time course of formation of M,C-dR a t pH 4.2 in reactions of 2'-deoxycytidine with NaMDA added directly or generated in situ from TEP. When NaMDA was added directly, the M3C-dR adduct was detected at 2 h. However, when MDA was generated from TEP, M3C-dR was not detected until 8.5 h. The MDA reaction reached a maximum at 20 h and produced an amount of M3C-dR 2 orders of magnitude greater than the TEP reaction. After 120 h, the TEP reaction produced approximately the same amount as the NaMDA reaction at 20 h. This corresponded to a yield of 10%. The amounts of the MIC-dR and M3C-dR formed at pH 4.2, with hydrolysis of TEP as the source of MDA, are compared in Figure 5B. The MIC-dR adduct was detected at the first time point (2 h) and reached a maximum at 30 h. After 60 h, the amount of the 1:l adduct decreased. As stated above, the 3:l adduct was not detected until 8.5 h after the start of the reaction. The maximum amount of the 3:l adduct was 300 times larger than the maximum amount of the 1:l adduct. The amount of MIC-dR formed from hydrolysis of TEP a t pH 4.2 is compared to the same reaction at pH 7.2 in Figure 6A. The reaction at pH 4.2 produced a maximum after 60 h, and then the amount decreased. The pH 7.2 reaction produced more of the 1:l adduct than the pH 4.2 reaction, and the amount of the 1:l adduct did not diminish; rather, it remained constant after 37 hr. The maximum amount of the MIC-dR produced corresponds to a yield of 0.03%. The time course of MIC-dR from NaMDA at pH 7.2 is similar to that of the reaction with TEP at the same pH (Figure 6B). The reaction a t pH 4.2 with NaMDA produced detectable amounts of MIC-dR up until the 24-h time point. This is illustrated by the chromatograms in Figure 7A,B. The MIC-dR peak is shown in the chromatogram from the 4-h time point (Figure 7A); after 24 h (Figure 7B) there is no M,C-dR. In the same chromatograms, the amount
Chem. Res. Toxicol., Vol. 3, No. 5, 1990 471
Malondialdehyde-Cytosine Adducts
action as shown in the graph of the entire data set in Figure 6.
Discussion
B
0'30] -
The two cytidine adducts identified in this report increase the number of adducts produced from the reaction of MDA with nucleosides to six (Scheme I; Figure 4). Other adducts may be formed, but the yields are too low for identification of their structures with the techniques presently a~ailable.~ The known adducts contain one or more molecules of MDA. Theoretically, they could arise from sequential addition of MDA molecules to the nucleoside base or by addition of preformed oligomers of MDA to the base (11, 14). There is evidence that the oligomeric adducts me not formed by sequential addition, and this implicates reaction with MDA oligomers (13). Indeed, Golding et al. have isolated and identified a dimer and a trimer of MDA that could serve as precursors to the M,G, M3A, and M3C adducts (Scheme I) (15). Treatment of the trimer with acid converts it to a bicyclic derivative that is obviously homologous to the structures of M3A and M3C (eq 1).
I
Ei 0.20
83
g 0.10 0.ooY. 0
I
2
.
0
'
I
4
0
I
e
J
'
8
I
0
I
'
1
0
0
Time (hr)
Figure 6. Amount of MIC-dR produced from the reaction of MDA or TEP with 2'-deoxycytidine. (A) Comparison of the reactions of deoxycytidinewith TEP at pH 4.2 (D) and 7.2 (+). (B)Comparison of the reactions of deoxycytidinewith MDA (e) or TEP (EI) at pH 7.2.
(I l
1 11 M.C.dR
I " '
I '
0
50
MC-dR
I
" l " " I " ' IO u
IS0
2uu
Time ( m i d
Figure 7. HPLC chromatogramsof reactions of 2'-deoxycytidine with MDA or TEP. Samples were analyzed with a reversed-phase C18 analytical column, using an aqueous gradient of 5 % to 20% acetonitrile over 20 min and a flow rate of 1.0 mL/min. (A and B) Reaction of MDA with deoxycytidine at pH 4.2 after 2 h (A) or 20 h (B). (C and D) Reaction of TEP with deoxycytidine at pH 7.2 after 4 h (C) or 46 h (D).
of M3C-dR increased with time, as shown in the graph of the entire data set in Figure 5. Regardless of the source of MDA, the reactions at pH 7.2 did not produce detectable amounts of M3C-dR. This is illustrated by the chromatograms in Figure 7C,D, which show the product profiles at 4 and 46 h, respectively. The M3C-dR adduct is not present in either chromatogram and was not detected throughout the course of the reaction. The MIC-dR adduct increased with time during this re-
The importance of individual adducts to MDA mutagenesis is uncertain. MDA induces frameshift and basepair substitution mutations in some strains of Salmonella typhimurium (5,19).Previous investigations indicate that both carbonyl equivalents must react for MDA to induce frameshift mutations in strain hisD3052 (20). Whether similar molecular requirements exist for mutagenesis of other bacterial strains is unknown. MIG and the oligomeric adducts are products of reaction of both carbonyl equivalents, but MIA and MIC are not. However, MIA and MIC are possible precursors of cross-links between complementary strands of DNA molecules or between DNA and protein molecules. Thus, derivatives of MIA and MIC may lead to formation of lesions that revert hisD3052. Using reaction conditions analogous to those described in the present report, we have been unable to detect crosslinks beteen MDA and two molecules of guanine, adenine, or cytosine nucleosides. However, cross-links between MDA and a-amino acids are produced under highly acidic conditions, and our laboratory previously reported evidence for generation of MDA cross-links between complementary strands of DNA molecules at low pH and high MDA concentrations (19). In, addition, the MDA dimer reacts with n-propylamine to form an amine adduct analogous to a cross-link (15). Thus, the role of cross-links in MDA mutagenesis is uncertain. It was hoped that by studying the reaction of MDA with cytosine nucleosides, we could develop conditions that would enable us to convert cytosine residues in oligonucleotides (e.g., T,CT,) to M,C derivatives. Such adducted oligonucleotides could then be hybridized to their complement and the duplex analyzed for the presence of cross-links. Unfortunately, the MIC-dR adduct is produced in very low yield. The maximum isolated yield of MIC-dR from a preparative-scale reaction was 0.3% at pH 5.4, and the major product at this pH was M3C-dR. Although MIC-dR is the major adduct at pH 7.2, the estiK. Stone, unpublished results.
472 Chem. Res. Toxicol., Vol. 3, No. 5, 1990
mated yield was 0.03% from an analytical-scale reaction. In addition, we found that MIC-dR is unstable. Upon decomposition,it regenerates deoxycytidine and produces unknown compounds that elute soon after the solvent front on HPLC. The combination of low yield and instability eliminates the possibility of introducing MIC into oligonucleotides by direct modification with MDA. There is no analytical methodology for simultaneous quantitation of all MDA-derived adducts in DNA. Thus, a comprehensive picture of the levels of individual adducts formed following incubations with MDA is not available. In particular, the extent to which oligomeric MDA adducts form is unknown. The procedures for synthesis of M2G, M3A, and M3C adducts employ low pH and high MDA concentrations. We performed experiments to determine the effect of pH on the relative amounts of MIC and M3C adducts in which we either added MDA directly or generated it in situ from TEP. The latter source was designed to control the rate of MDA production and minimize oligomer formation. With either source of MDA, M3C predominated over M,C by nearly 2 orders of magnitude at pH 4.2. However, at pH 7.2 with pure N W A or TEP, the only detectable adduct was MIC. These findings are consistent with previous observations that MDA is stable to oligomerization at neutral pH (22). The fact that MIC formed at approximately the same rate at pH 7.2 from MDA or TEP is consistent with the low reactivity of MDA at neutral pH. MDA exhibits a pK, of 4.46, so the ratio of conjugate base to free acid is 550:l. Only the free acid is reactive to nucleophiles, so apparently the rate of its generation from TEP is greater than the rate of ita reaction with cytosine nucleosides. Otherwise, the rate of formation of MIC would have been much lower in reactions with TEP that in reactions with MDA. Oligomeric MDA-deoxycytidine adducts are the major products of reaction of MDA and deoxycytidine at acidic pH but are undetectable at neutral pH. The inability to detect M3C adducts at neutral pH suggests they and other oligomeric adducts do not play an important role in MDA mutagenesis. The possibility exists that oligomeric adduds are formed in low yield on intact DNA but represent very efficient premutagenic lesions that could account for the mutagenicity of MDA. However, Basu et al. reported that a polymerized solution of MDA is less mutagenic than MDA toward S. typhinurium hisD3052 (19). Unfortunately, the exact composition of MDA oligomers in that experiment was not determined, so it is not certain if MDA dimers and trimers were present. Because of the recent demonstration that the MDA oligomers, which are precursors to M2G, M3A, and M3C, can be isolated and purified, it is now possible to directly test these oligomers for mutagenic activity and thereby evaluate the importance of the MDA dimer and trimer in mutagenesis.
Acknowledgment. This work was supported by a grant from the National Cancer Institute (CA47479). We are
grateful to Michael P. Stone for helpful discussions and assistance with the 2D NMR experiments.
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