Chem. Res. Toxicol. 1994, 7, 857-860
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Formation of Etheno Adducts in Reactions of Enals via Autoxidation Hauh-Jyun Candy Chen and Fung-Lung Chung* Section of Nucleic Acid Chemistry, Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received June 21, 1994@ 4-Hydroxy-2-nonenal (HNE) a n d crotonaldehyde (CAI are a,P-unsaturated aldehydes (enals) produced by lipid peroxidation. Previous studies have shown that enals form tricyclic propano adducts with purine nucleosides. When epoxidized by tert-butyl hydroperoxide, enals yield t h e more reactive epoxy aldehydes which are capable of modifying adenine a n d guanine in DNA by forming etheno adducts. The epoxides are considerably more mutagenic and tumorigenic t h a n the parent aldehydes. In this study, we found that, in addition to the propano adducts, etheno adducts are detected upon incubation of enals with deoxyadenosine (dAdo) or deoxyguanosine (dGuo) at 37 “C (pH 7.4). When carried out under oxygen-enriched atmosphere, the reaction of HNE with dAdo yielded 1,Wethenodeoxyadenosine (1,W-EdAdo) a n d 7-(1’,2’dihydroxyheptyl)-lJVj-EdAin 0.03% a n d 0.48% yield. The reaction with dG gave 0.19% 1,Wethenodeoxyguanosine (1,W-EdGuo) and 0.79% 7 4 1’,2’-dihydroxyheptyl)-l,W-EdGuo. I n t h e case of CA, t h e reaction with dAdo produced 7-(l‘-hydroxyethyl)-lfl-EdAdoin 0.67% yield, while t h e reaction with dGuo yielded 0.34% 1,W-EdGuo a n d 0.97% 7-(l’-hydroxyethyl)-l,WEdGuo, respectively. When the above reactions were carried out under nitrogen, yields of etheno adducts were significantly reduced. These results provide evidence for a pathway by which the etheno adducts may be formed by enals via autoxidation.
Introduction It is widely recognized t h a t endogenous compounds may cause genetic damage which result in mutations (I). For example, oxygen free radicals are known to react with DNA bases, yielding a variety of oxidized lesions such as 8-hydroxyguanine (2,3). Recently, exocyclic adducts including 1P-propanodeoxyguanosine, 1 P - e t h e n o d e oxyadenosine (l,iP-EdAdo),l 3,N4-ethenodeoxycytidine (3,N4-EdCyd), and iP,3-ethenodeoxyguanosine ( i P , 3 EdGuo) have been detected as background adducts in tissue DNA of untreated rodents, suggesting that these are endogenous adducts (4-6). Although the exact mechanism by which the exocyclic DNA adducts are formed in vivo is not yet clear, the preliminary evidence suggests t h a t lipid peroxidation may be involved (5, 7). Like malondialdehyde, a&unsaturated aldehdyes (enals) are products of lipid peroxidation. Among these aldehydes, trans-4-hydroxy-2-nonenal (HNE) and crotonaldehyde (CA) are of particular interest, because of their known mutagenic and tumorigenic activities (8-10). Enals readily react with cellular sulfhydryl groups and primary amines of proteins (12,121. More importantly, these bifunctional aldehdyes modify guanine and adenine by forming exocyclic adducts either via direct Michael addition or through the formation of epoxides (13-1 7). The enals yield the cyclic propano adducts, whereas the epoxy aldehydes form the etheno adducts (Scheme 1). Epoxides of enals can be produced by organic hydroperoxides, such as tert-butyl hydroperoxide (16). Epoxide of acrolein (glycialdehyde) was detected upon incubation
* Corresponding author.
Abstract published in Advance ACS Abstracts, October 15, 1994. Abbreviations: HNE, trans-4-hydroxy-2-nonenal;CA, crotonaldehyde; a d o , deoxyadenosine; dGuo, deoxyguanosine; dCyd, deoxycytidine;lP-EdAdo, 1P-ethenodeoxyadenosine; lJP-EdGuo, I F ethenodeoxyguanosine; 3,N4-EdCyd,3,N4-ethenodeoxycytidine. @
of acrolein with rat liver or lung microsomes (18). To the best of our knowledge, this is the only study in which an epoxide of enal was detected upon microsomal incubation. The scarcity is possibly due to low level of formation as a result of other metabolic pathways (29,20) or the intrinsic chemical instability of epoxides. There is a marked difference in the reactivities of enals and their epoxides with proteins and DNA. While enals bind readily to proteins through free thiol groups by Michael addition, the epoxy aldehydes are considerably more reactive toward nucleic acids (7). These differences may explain why epoxy aldehydes are more mutagenic and tumorigenic, and their parent aldehdyes are more toxic (21). Previously, we have characterized the etheno adducts from reaction of the epoxide of HNE with nucleic acids (7, 16). The structures of etheno adducts formed in the reactions of the epoxides of HNE and CA with deoxyadenosine (dAdo) or deoxyguanosine (dGuo) are shown in Scheme 1. In this study, we found t h a t the etheno adducts are products of reaction of HNE or CA incubated with dAdo or dGuo in the presence of oxygen. Their yields were considerably decreased when the reactions were carried out under nitrogen. These observations provide evidence that etheno adducts are produced by autoxidation of CA and HNE, a plausible pathway by which etheno adducts are formed endogenously.
Experimental Procedures Chemicals. dAdo, dGuo, and 1,W-EdAdo were purchased from Sigma (St. Louis, MO). CA (99%+) was purchased from Aldrich Chemical Co. (Milwaukee, WI). HNE was synthesized by a modified procedure (22). The epoxide of HNE, 2,3-epoxy4-hydroxynonanal, was synthesized by reaction with tert-butyl hydroperoxide (16).2,3-Epoxybutanal was prepared from CA upon reaction with sodium hypochlorite (23). 1,P-Ethenodeoxyguanosine (l,W-EdGuo, 4) was prepared from chloroacetal-
0893-228x/94/2707-085~~04.50/00 1994 American Chemical Society
Chen and Chung
858 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
Scheme 1. (A) Propano Adducts Derived from Reactions of dAdo or dGuo with HNE or CA; (B)Etheno Adducts Derived from Reactions of dAdo or dGuo with the Epoxides of HNE or CA R
dR 1 .N6-propano dA R=CSH11CH(OH) or CH3 4
H
dR
1,N2-propano dG
1
dR 1
n
2: R=C#11CH(OH) 3: R=CH3 R
n
dGuo
4
4
dehyde (24). 7-(l', 2'-Dihydroxyhepty1)-1,iV-EdAdo(2) and 741'hydroxyethyl)-l,iV-EdAdo (3)were synthesized by reacting dAdo with 2,3-epoxy-4-hydroxynonanaland 2,3-epoxybutanal, respectively (7,17). 7-(1',2'-Dihydroxyheptyl)-l,W-EdGuo (5) was synthesized from reactions of dGuo with 2,3-epoxy-4hydroxynonanal and 7-(l'-hydroxyethyl)-l$P-EdGuo (6)from 2,3-epoxybutanal(16,17).The structures of all these adducts were characterized by their proton NMR, mass, and U V spectra. Since the proton NMR of 3 and 6 have not been reported, these data are presented here (the primed numbers refer to the carbons in the sugar moiety). 3 (MeOH-dd: 6 1.80 (d, J = 6.5 Hz, 3H, CH3), 2.56 (m, lH, CY-H), 2.90 (m, lH, CY-H), 3.85 (m, 2H, CU-H), 4.12 (m, lH, C4'-H), 4.66 (m, lH, C3'-H), 5.36 (q, J = 6.5 Hz, lH, CHsCH), 6.65 (t, J = 6.7 Hz, lH, Cl'-H), 7.51 (9, lH, C8-H), 8.54 (9, lH, C2-H), 9.26 (9, lH, C5-H); 6 (MeOH-dr): 6 1.65 (d, J = 6.5 Hz, 3H, CH3), 2.44 (m, lH, C2'H), 2.83 (m, lH, CY-H), 3.82 (m, 2H, C5'-H), 4.07 (m, lH, C4'H), 4.61 (m, lH, CY-H), 5.48 (4,J = 6.5 Hz, lH, CH&H), 6.43 (dd, J = 7.6, 6.4 Hz, lH, C1'-H), 7.22 (s,lH, C6-H), 8.18 (9, lH, C2-H). Instrumentation. Proton NMR spectra were obtained on a Bruker AM 360 WB instrument (Billereca, MA). The low resolution mass spectra were obtained on a Hewlett Packard Model 5988A spectrometer (Wilmington, DE). Chromatography. The HPLC was performed on a Waters System (Waters Associates, Milford, MA) equipped with two Model 510 pumps, a Model 660 solvent programmer, a Reodyne injector, and a Waters 994 programmable photodiode array detector. A reversed phase B & J octadecylsilane (ODs) 250 x 4.6 mm column (Baxter, Edison, NJ) was eluted with a linear gradient of HzO and CH3CN at a flow rate of 1.0 mumin with UV detection at 210 nm. System 1: 0-5 min, 5% CH3CN;5-60 min, 5-40% CHsCN; System 2: 0-5 min, 5% CH3CN; 5-60 min, 5-35% CH3CN; System 3: 0-5 min, 5% CH3CN; 5-60 min, 5-30% CH3CN, System 4: 0-5 min, 5% CHzCN, 5-60 min, 5-20% CH3CN.
Reactions of HNE and CA with dAdo and dGuo under Aerobic Conditions. Typically,a 20 mL vial containing a 1.0 mL solution of dAdo or dGuo (10 mM) in 0.1 M sodium phosphate buffer (pH 7.4) was bubbled with oxygen for 2 min. It was then sealed with a rubber septum and tightened with a copper wire. The atmosphere inside the reaction vial was purged for 1 min with a stream of oxygen through the inlet needle while another needle was used as an outlet. HNE (10 pL, 62 pmol) or CA (5 pL, 62 pmol) was added through a syringe. The top of the septum was then sealed with silicon glue, and the reaction mixture was incubated at 37 "C for 72 h. The reaction mixture was directly analyzed by HPLC using the systems described above. Reactions were also carried out under identical conditions but in ambient air. Reactions of HNE and CA with dAdo and dGuo under Anaerobic Conditions. Procedures were identical to those described above except that nitrogen was used instead of oxygen.
Results and Discussion Previous studies have shown that the cyclic propano adducts are products from reactions of e n d s with deoxyribonucleosides (Scheme 1)(13,14).The formation of the propano adducts with enals is initiated by Michael addition to the olefinic carbon followed by ring closure with the aldehyde. The detection of etheno adducts in the present study upon reaction of HNE or CA with dAdo or dGuo, even though they formed only in small quantities, was unexpected. Since we had previously shown that both the substituted and unsubstituted EdAdo and EdGuo adducts are products of reactions of epoxy aldehydes (Scheme 1)(2,161, the formation of etheno adducts from enals can be best rationalized by the presence of epoxides in these reactions, although a direct evidence
Chem. Res. Toxicol., Vol. 7, No.6, 1994 859
Etheno Adducts /?om Enals via Autoxidation
0.5 r
0.51-
retention time (min) retention time (min) Figure 1. HPLC chromatograms of reaction mixtures of (a) HNE or (b) CA with &do under aerobic (top) and anaerobic (bottom) conditions. In (a),the peak at 21.4min is adduct 1.The peaks at 42.1 and 43.0min are isomers of adduct 2. In (b),peaks at 25.8and 26.2 min are isomers of adduct 3.HPLC System 1 was used for (a), and System 3 was used for (b). Table 1. Retention Times of Etheno Adducts in Reversed Phase HPLC compd retention timeQ(min) HPLC System 1 21.4 1 2a 42.1and 43.0 1 Sa 25.8and 26.2 3 4 16.3 2 17.4 4 56 46.6 2 6a 24.5and 26.0 4 The isomers separated into two peaks. The isomers showed Q
as one peak.
Table 2. Percent (%) Yields of Etheno dAdo and dGuo Formed from Reactions of HNE or CA under Aerobic and Anaerobic Conditionsa CA
HNE
EdAdo EdGuo EdAdo 1
2
4
6
1
3
EdGuo 4
6
aerobic
under 02 0.03 0.48 0.19 0.79 NDb 0.67 0.34 0.97 under ambient 02 ND 0.36 0.04 0.58 ND 0.43 0.23 0.87 anaerobic under Nz ND ND ND ND ND 0.16 ND 0.05 Percentage yields were calculated based on dAdo or dGuo.
of epoxide formation is still lacking. From a mechanistic point of view, the etheno adducts could also be formed from 2-haloacetaldehydes (25)or 1-halooxiranes (26).The lack of a halogen source in these reactions excludes these possibilities. In order to provide evidence t h a t the etheno adducts are indeed formed via autoxidation of enals during the reaction, we studied their formation under conditions in which the ambient air was replaced by either oxygen or nitrogen. Figure 1shows typical HPLC chromatograms of reaction mixtures obtained from dAdo in the presence and absence of oxygen. In the HNE reaction, peaks eluting a t 42.1 and 43.0 min were assigned as the two isomeric 7-(1’,2’-dihydroxyheptyl)-l,IP-EdAdo (2) (7). Similarly, reactions with CA yielded peaks at 25.8 and 26.2 min, which were assigned as the isomeric 7-(1’hydroxyethy1)-lP-EdAdo (3) ( I 7). These assignments were based on comigration with the synthetic standards and on their characteristic UV spectra. The retention times of the etheno dAdo and dGuo adducts in various HPLC conditions are summarized in Table 1. Etheno dAdo and dGuo adducts have distinct UV spectra with absorption maxima a t 231, 259 (sh), 268, 279, and 300 (sh) nm (pH 7.0) and at 228 and 283 nm (pH 7.01, respectively (7, 16). Nair and Offerman had reported t h a t 7-(l’-hydroxyethyl)-l,IP-ethenoadenosineand 1,Wethenoguanosine were the main products of reactions of 2,3-epoxybutanal with guanosine and adenosine at pH 5 and 10, respectively (17). At pH 7.4, we found that a major etheno product was the substituted etheno adduct 3 or adduct 6 in the reaction with dAdo or dGuo. We also observed t h a t adduct 3 or 6 was a pair of diastereomers because of the chirality on the alkyl substituent. As summarized in Table 2, yields of etheno adducts were increased when the reactions were performed in a n
Not detectable. oxygen-enriched atmosphere, and their yields were diminished under anaerobic conditions. Analogous results were observed in the reactions with dGuo or dAdo. Thus, these results support t h a t autoxidation of enals is responsible for the formation of etheno adducts. The small amount of the etheno adducts still detected in the CA reactions under anaerobic conditions is likely due to traces of the oxidation product, since CA had not been stored under nitrogen. It is also possible that CA may undergo autoxidation with residual oxygen that remained in the incubation mixture. We did not, however, detect etheno adducts under anaerobic conditions with the freshly prepared HNE. As expected, reactions of CA or HNE with dGuo gave the 1,W-propano-dGuo adducts as the major products with 7.4% or 6.4% yield, respectively. These adducts were identified by comparing their UV and retention times with those of the synthetic standards. Unlike the etheno adducts, yields of the propano adducts were virtually unchanged (2~0.1%)between reactions carried out under aerobic and anaerobic conditions. The epoxidation of alkenes by co-oxidation of aldehydes, presumably via a peroxycarboxylic acid intermediate formed by addition of oxygen to the aldehydes, has been demonstrated (27). Enals are bifunctional molecules possessing both alkene and aldehyde groups. Therefore, they are prone to epoxidation by the peroxycarboxylic acids derived from oxidation. It is tempting to speculate that the mechanism involves the addition of oxygen to the aldehyde, yielding a peroxycarboxylic acid which transfers an oxygen atom to the olefinic group, thus producing the epoxide and the corresponding carboxylic acid. However, evidence for the epoxy aldehyde formation under the conditions used in this study has
860 Chem. Res. Toxicol., Vol. 7, No. 6, 1994
not yet been obtained. Previously, we observed that HNE was epoxidized in the light-exposed tetrahydrofuran, possibly by a tetrahydrofuran hydroperoxide (28). Regardless of the identity of the intermediate, the present study demonstrates t h a t enals can undergo autoxidation in a pH 7.4 aqueous buffer, resulting in the formation of etheno adducts in the presence of dAdo or dGuo. These findings suggest a biologically relevant pathway for endogenous formation of etheno adducts. Acknowledgment. This work was supported by Grant CA 43159 from the National Cancer Institute. The authors thank Dr. Mingyao Wang for helpful discussion. References (1) Marnett, L. J., and Burcham, P. C. (1993) Endogenous DNA
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