DNA Alkylation by 4,5-Dioxovaleric Acid, the Final Oxidation Product

150

Chem. Res. Toxicol. 1998, 11, 150-157

DNA Alkylation by 4,5-Dioxovaleric Acid, the Final Oxidation Product of 5-Aminolevulinic Acid Thierry Douki,‡ Janice Onuki,† Marisa H. G. Medeiros,† Etelvino J. H. Bechara,† Jean Cadet,‡ and Paolo Di Mascio*,† De´ partement de Recherche Fondamentale sur la Matie` re Condense´ e, SCIB Laboratoire “Le´ sions des Acides Nucle´ iques”, CEA, Grenoble, 17, Avenue des Martyrs, F-38054 Grenoble Cedex 9, France, and Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜ o Paulo, CP 26077, CEP 05599-970 Sa˜ o Paulo, Brazil Received September 2, 1997

The heme precursor 5-aminolevulinic acid (ALA) accumulates under pathological conditions, namely, acute intermittent porphyria (AIP) and tyrosinosis, two diseases that are associated with increased liver cancer incidence. This has been previously linked to an enhanced production of reactive oxygen species generated by a metal-catalyzed ALA oxidation process, which was shown to cause DNA single-strand breaks and guanine oxidation within both isolated and cellular DNA. In the present work, we established that the final oxidation product of ALA, 4,5-dioxovaleric acid (DOVA), is an efficient alkylating agent of the guanine moieties within both nucleoside and isolated DNA. Adducts were produced through the formation of a Schiff base involving the N2-amino group of 2′-deoxyguanosine (dGuo) and the ketone function of DOVA, respectively. The modified dGuo nucleosides were characterized, following reduction into stable secondary amines, by extensive NMR, infrared, and mass spectrometry analyses. A method, based on the use of HPLC with electrochemical detection, was then developed for the sensitive measurement of the DOVA-dGuo adducts. Using this assay, we showed that the guanine moieties of isolated DNA can undergo the same reaction as the free nucleoside. The present data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that AIP may be involved in the induction of primary liver cell carcinoma.

Introduction 5-Aminolevulinic acid (ALA),1 a precursor of porphyrin IX in the biosynthesis of heme (Scheme 1), can accumulate in liver, brain, and other organs under pathologic conditions such as acute intermittent porphyria (AIP), tyrosinosis (1, 2), and lead poisoning (3). High incidence of primary liver cancer (PLC) has been reported in AIP and tyrosinosis and associated with the frequency of acute attacks when the ALA plasma level rises about 100-fold (4-6). This observation is an indication of possible mutagenic properties of ALA. Moreover, it has recently been observed that ALA is able to induce chromosomal aberrations in rat hepatocytes (7). The determination of the carcinogenic potential of 5-aminolevulinic acid has received additional attention with the recent use of ALA in cancer phototherapy (8-10). Nevertheless, little is known about the possible side effects of ALA and the chemical mechanisms associated with its DNA-damaging properties (Scheme 2). The involvement of reactive oxygen species produced during the metalcatalyzed oxidation of ALA by oxygen has been demon* To whom correspondence should be addressed. Fax: ++ (55) 11 8155579. E-mail: [email protected]. † Universidade de Sa ˜ o Paulo. ‡ SCIB Laboratoire “Le ´ sions des Acides Nucle´iques”. 1 Abbreviations: AIP, acute intermittent porphyria; ALA, 5-aminolevulinic acid; dGuo, 2′-deoxyguanosine; DOVA, 4,5-dioxovaleric acid; BSTFA, N-bis(trimethylsilyl)trifluoroacetamide; ESI-MS, electrospray ionization mass spectrometry; FT-IR, Fourier transform infrared spetroscopy; HPLC-EC, high-performance liquid chromatography coupled to electrochemical detection; GC/MS, gas chromatography associated with mass spectrometry; PLC, primary liver cancer.

Scheme 1. Enzyme Deficiencies in the Heme Biosynthetic Pathway Associated with ALA Overload

a Negative feedback inhibition by heme; in AIP, activated by certain drugs and metabolites. b Inhibited by lead ions in plumbism (lead poisoning). c Inhibited by succinylacetone in tyrosinosis. d Deficient biosynthesis in AIP.

strated by Bechara and co-workers (11). Support for the induction of DNA damage by ALA has been provided by the observation of increased levels of 8-oxo-7,8-dihydroguanine, an oxidation product of guanine, in both isolated DNA (12, 13) and liver DNA of rats treated with

S0893-228x(97)00157-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/31/1998

DNA Alkylation by the Oxidation Product of ALA Scheme 2. Possible Mechanisms of ALA DNA-Damaging Properties Associated with Its Mutagenicity

ALA (12). Noteworthy is the observed iron mobilization in rat brain and liver that is triggered by ALA chronic treatment (14). In addition, ALA has been found to be able to release iron from ferritin (15). 5-Aminolevulinic acid has also been shown to induce the formation of DNA strand breaks in plasmids in the presence of either ferrous ions (16) or ferritin (Di Mascio et al., unpublished results). Similar results have been recently obtained by using copper ion instead of iron (13). Less attention has been paid to the final oxidation product of ALA, 4,5dioxovaleric acid (DOVA). However, DOVA is expected to induce DNA base modifications as already shown for other reactive carbonyl derivatives. Indeed, malondialdehyde (17-19), acetaldehyde (20, 21), dicarbonyl compounds (22, 23), and a series of R,β-unsaturated aldehydes (24-28) have been identified as efficient DNAalkylating agents. In most cases, the major reactive site is the exocyclic amino group of guanine which is the most nucleophilic function of DNA. Therefore, we investigated the reactivity of DOVA with 2′-deoxyguanosine (dGuo) and identified two diastereoisomeric adducts. Their formation involves addition of the ketone function of DOVA to the N2-amino group of dGuo leading to a Schiff base formation. The reaction was shown to take place within isolated double-stranded DNA. These data may be relevant in symptomatic AIP carriers where prevalent PLC occurs.

Experimental Section Chemicals. 2′-Deoxyguanosine was purchased from PharmaWaldorf (Geneva, Switzerland). Sodium borohydride and calf thymus DNA were obtained from Sigma (St. Louis, MO). All other solvents and chemicals used were of analytical grade and purchased from Merck (Darmstadt, Germany). Water was purified on a Milli-Q system (Millipore, Bedford, MA). 4,5Dioxovaleric acid was a gift from Dr. Dieter Do¨rnemann (Philipps-Universita¨t, Marburg, Germany). HPLC Systems. The HPLC system consisted of a L7100 Merck pump connected to a 7125 Rheodhyne injector (Bellafonte, CA). For analytical purposes, the system was equipped with a 250- × 4.6-mm i.d. Hypersil C18 column (Interchim, Montluc¸ on, France) connected to either a Waters 990 diode array UV detector or a Waters 484 UV spectrometer set at 260 nm (Milford, MA). The isocratic eluent was a 25 mM solution of ammonium formate containing 5% methanol. The flow rate was 1 mL/min. When used for preparative separations, the system was equipped with a home-packed semipreparative (250- × 7-mm i.d.) Nucleosil 100-10 C18 octadecylsilyl silica gel (particle size 10 µm; Macherey-Nagel, Du¨ren, Germany) column. The

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 151 isocratic eluent was a 25 mM solution of ammonium formate containing 5% methanol. The flow rate was 3 mL/min. The HPLC-EC system consisted of a LC 10AD pump (Shimadzu, Tokyo, Japan) connected to a Spherex 5 C18 (Phenomenex, Torrance, CA) reverse-phase column (250- × 4.6-mm i.d., particle size 5 µm). The isocratic eluent was a 50 mM potassium phosphate buffer (pH 5.5). The detection was provided by a L-ECD 6A amperometric chemical detector (Shimadzu) and a 484 Waters UV spectrometer set at 285 nm. Data acquisition was performed using the software EZChrom Chromatography Data System, version 6.2 (Scientific Software, Inc.). Incubation of dGuo with DOVA and Reduction of the Adducts by Sodium Borohydride. 2′-Deoxyguanosine (10 mg) and DOVA (50 mg) were dissolved in 3 mL of water. The pH was then set at 7 by addition of 0.1 N NaOH. After 30 min of incubation, 50 µL was injected in the analytical HPLC system. The peaks corresponding to two major reaction products, namely, adducts 1 and 2, were observed on the chromatogram at retention times of 7.2 and 8.0 min, respectively. Then, NaBH4 (50 mg) was added to the bulk of the reaction mixture. The pH was kept below 10 by addition of 1 M HCl. A 50-µL aliquot fraction was collected, diluted with 200 µL of phosphate buffer (pH 7), and analyzed by HPLC. Two additional peaks, corresponding to compounds 3 and 4, were observed at retention times of 10.6 and 12.0 min, respectively. Addition of NaBH4 and HPLC analysis were repeated five times at 30-min intervals, until no change of the chromatogram was observed. The yield of the reduction was approximately 50%. The liquid phase was injected onto the semipreparative HPLC system after neutralization by addition of 1 M HCl. The fractions corresponding to the two peaks eluting at 12.7 and 13.4 min were collected separately. An aliquot of each of the collected fractions (100 µL out of 25 mL) was injected on the analytical HPLC. Each fraction was shown to contain a homogeneous compound corresponding to reduced adducts 3 and 4, respectively. Both fractions were freeze-dried overnight to remove ammonium formate. Mass Spectrometry Analyses of Reduced Adducts 3 and 4. The solution of sample to be analyzed by gas chromatography coupled to mass spectrometry (GC/MS) was freeze-dried overnight in silylation vials. Then, the resulting residue was derivatized for 20 min in 100 µL of a 1:1 (v/v) mixture of acetonitrile (silylation grade; Pierce, Rockford, IL) and N-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Fluka, Buchs, Switzerland) at 110 °C. GC/MS analyses were performed on a HP 5890 series II gas chromatograph (Hewlett-Packard, Les Ulis, France) equipped with a HP5-trace (Hewlett-Packard) capillary column (15 m, 0.25-mm i.d., 0.11-µm film thickness). The constant flow rate was 0.8 mL/min. The injection (1 µL) was performed in the splitless mode with the temperature of the injection port set at 250 °C. The temperature of the GC oven was maintained at 110 °C for 1 min and then raised to 280 °C at a rate of 15 °C/min. Detection in the positive mode was provided by a HP 5972 (Hewlett-Packard) mass spectrometer operating in the electron impact ionization mode. The mass spectrometer was used in the full scan mode with a 50-700 mass range. Only one peak was observed in the chromatogram of the respective silylated derivatives of compounds 3 and 4 (retention times of 10.26 and 10.39 min, respectively). The mass spectra of the two adducts were almost identical. Adduct 3: GC/MS EI, positive mode (m/z, relative abundance) 653, 6 [M]; 638, 3 [M - methyl]; 393, 50 [M + H dRTMS2]; 378, 35 [M + H - methyl - dRTMS2]; 303, 100 [M + H - TMSOH - dRTMS2]; 290, 88 [M + H - dRTMS2 TMSOCH2]. Adduct 4: GC/MS EI, positive mode (m/z, relative abundance) 653, 6 [M]; 638, 3 [M - methyl]; 393, 47 [M + H dRTMS2]; 378, 34 [M + H - methyl - dRTMS2]; 303, 100 [M + H - TMSOH - dRTMS2]; 290, 88 [M + H - dRTMS2 TMSOCH2].

152 Chem. Res. Toxicol., Vol. 11, No. 2, 1998 Electrospray ionization mass spectrometry analysis (ESI-MS) in the positive mode was performed on a Platform spectrometer (Fisons Instrument, Manchester, U.K.). The eluent (flow rate 5 µL/min) was a 1:1 mixture of water and acetonitrile. The sample (5 µg) was injected in 10 µL of a 0.25% aqueous solution of formic acid. Mixture of Adducts 3 and 4: ESI-MS, positive mode (m/z, relative abundance) 384, 15 [M + H+]; 268, 100 [M - dR + H + H+]; 250, 7 [M - H2O - dR + H + H+]. A mixture of 3 and 4 (100 µg) was freeze-dried and then acetylated overnight in 1 mL of a 1:1 mixture of pyridine and acetic anhydride. The sample was then evaporated under vacuum. Ethanol was added, and the resulting solution was transferred into a silylation vial. Ethanol was removed under vacuum, and then, the resulting residue was freeze-dried. It was then silylated by BSTFA and analyzed by GC/MS as described above. The major peak observed on the chromatogram corresponded to a disilylated and diacetylated derivative of the base moiety of 3 and 4. Acetylated and silylated adducts 3 and 4: GC/MS EI, positive mode (m/z, relative abundance) 495, 10 [M]; 480, 3 [M - methyl]; 452, 3 [M - CH3-CO-]; 436, 4 [M - CH3-CO-O-]; 295, 20; 280, 100. NMR Spectroscopy Studies. The products were dissolved in 1 mL of 99.5% D2O (Sigma) and dried under vacuum. The solid residues were dissolved in 500 µL of 99.99% D2O (Sigma). Both products 3 and 4 were first analyzed by 400-MHz 1H and 100-MHz 13C NMR spectroscopy using a AM 400 Bru¨cker spectrometer. 1H NMR spectra were calibrated by using the peak of HDO (4.9 ppm) as the reference. The 13C chemical shifts were calibrated with respect to methanol (49.9 ppm) used as internal reference. 2′-Deoxyguanosine was analyzed in a similar way. 1H and 13C NMR spectra of 4 were also obtained in DMSOd6. Chemical shifts were expressed with respect to tetramethylsilane (TMS) used as internal reference. Short- and longdistance 1H-13C heteronuclear coupling experiments were carried out with a 1:1 mixture of 3 and 4 in DMSO-d6 on a U 400 Varian spectrometer. 1H and 13C NMR features of 3 and 4 are listed in Tables 1-3. Infrared Spectroscopy Analysis. The infrared spectrum of a mixture of 3 and 4 was obtained in the solid state in potassium bromide (1 mg of a mixture of 3 and 4 and 100 mg of KBr) on a 1600 FT-IR Perkin-Elmer spectrometer (Foster City, CA). Two vibration bands were observed in the carbonyl region: ν (cm-1) 1682 (CdO of guanine), 1605 (-COO-). Hydrolysis of Reduced DOVA-dGuo Adducts. A mixture of the two diastereoisomeric reduced DOVA-dGuo adducts 3 and 4 (30 µg) was incubated in 1 mL of 0.1 M HCl at 50 °C. Aliquot fractions (50 µL) were collected after increasing periods of time (from 0 to 130 min) and analyzed by HPLC. The unique hydrolysis product exhibited a retention time of 14.2 min. The hydrolysis was repeated for 2 h in 1 mL of 0.1 M HCl with 3 mg of a 1:1 mixture of 3 and 4. The hydrolysis product was purified by HPLC and analyzed by 1H NMR (Table 1). A dynamic electrovoltamogram of the hydrolysis product of the reduced DOVA-dGuo adducts was determined on the HPLCEC system. A constant amount of product (0.3 µg) was injected. The ratio between the EC and UV response of the compound (retention time 13.8 min) was plotted against the potential applied to the EC detection cell (range 600-900 mV). Incubation of DNA with DOVA. A 1 mg/mL aqueous solution of calf thymus DNA (500 µL) was incubated for 2 h with 5 mg of DOVA at pH 7 in a water bath maintained at 37 °C. Then, 25 mg of NaBH4 was added to the solution. The pH was kept between 10 and 11 by addition of 1 M HCl. After 30 min, the sample was neutralized by addition of 1 M HCl. DNA was precipitated by addition of 1.5 mL of cold ethanol (-20 °C). The sample was then centrifuged. The solid fraction was dissolved in 250 µL of water and precipitated again by addition of ethanol. The resulting pellet was washed with 100 µL of 70% ethanol and centrifuged. The DNA sample was then dissolved in 500 µL of water. The pH was set at 4.8 by addition of 1 M

Douki et al. sodium acetate buffer. Nuclease P1 (25 units; Sigma) was added, and the sample was incubated overnight at 37 °C. The pH was then raised to 7.4 by addition of dephosphorylation buffer (500 mM TRIS-HCl, 1 mM EDTA, pH 8). Then, 50 units of bacterial alkaline phosphatase (GIBCO-Life Technologies, Gaithersburg, MD) was added. The resulting solution was incubated for 2 h at 37 °C. The enzymes were subsequently removed by addition of 250 µL of chloroform. The solution was centrifuged, and the aqueous fraction was collected and concentrated to 200 µL under vacuum. The resulting solution was split into two aliquot fractions which were subsequently injected on the analytical HPLC system. The fraction containing the adducts was collected and freeze-dried. The residue obtained was dissolved in 100 µL of 0.1 M HCl, and the resulting solution was heated at 50 °C for 2 h. The sample was then neutralized by addition of 1 M NaOH and analyzed by HPLC-EC with the potential of the electrode of the EC detector set at 850 mV. The injection volume was 50 µL out of 100 µL of solution. Purified hydrolyzed reduced DOVA-dGuo adduct (500 pmol) was added to the other half of the sample and the resulting solution analyzed by HPLC-EC.

Results Addition of DOVA to dGuo and Reduction of the Reaction Products. The reaction between DOVA and dGuo led to the formation of two major products (Scheme 3) which were separated by reverse-phase HPLC (Figure 1A). The UV absorption features of 1 and 2 were very similar to those of dGuo, with the exception of a slight red shift of approximately 5 nm. Attempts made to isolate compounds 1 and 2 failed because they rapidly decomposed with a half-life of 25 min in neutral aqueous solution at room temperature. Surprisingly, the HPLC chromatograms of solutions obtained after isolation and spontaneous decomposition of either 1 or 2 were identical, showing similarity to that of the crude reaction mixture. A major difference was a much higher relative amount of dGuo in the solution of decomposed adducts than in the first incubation mixture. This observation indicates that the decomposition of 1 and 2 leads to the release of both dGuo and DOVA which are then able to react with each other. Another interesting feature of the reaction is the dependence of the efficiency of the reaction of dGuo with DOVA on the concentration of the reagents. The yield of products 1 and 2 was near 90% at a high concentration of reagents (13 mM dGuo, 130 mM DOVA) but limited to 40% under more diluted conditions (9 mM dGuo, 20 mM DOVA). Altogether, these results strongly suggested that the formation of the DOVA-dGuo adducts was reversible in water. Adducts 1 and 2 are likely to be Schiff bases resulting from the initial addition of the exocyclic amino group of dGuo to a carbonyl group of DOVA. Identification of the reduction products of 1 and 2 (vide infra) showed that the Schiff base involves the C4* ketone of DOVA. The similarity in the yield of formation, chromatographic behavior, and UV spectrum suggests that 1 and 2 are diastereoisomers. This could be explained by the secondary attack of the N1 imine group of the guanine moiety on the aldehyde function of DOVA (Scheme 3). This would lead to the formation of a five-membered ring carrying an asymmetric carbon, namely, the former C5* atom of DOVA. Unfortunately, the instability of adducts 1 and 2 prevented any extensive spectroscopic studies allowing their more complete characterization. It was also not possible to determine whether the dehydration of the -NH-CHOH- link leading to the formation of a Schiff base occurred before

DNA Alkylation by the Oxidation Product of ALA

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 153

Scheme 3. Structure and Formation of DOVA-dGuo Adducts and Their Reduction Productsa

a The chemical structures of 3 and 4 were unambiguously determined by NMR, IR, UV, and mass spectroscopy. Since adducts 1 and 2 are unstable, their characterization was mainly inferred from UV spectroscopic analysis and identification of their reduction products. Therefore, the structures proposed for 1 and 2 are only tentative. The acidic hydrolysis of adducts 3 and 4 gives rise to 5.

Figure 1. HPLC elution profile of (A) the reaction mixture of dGuo and DOVA and (B) its NaBH4 reduction product.

or after cyclization of the adduct. In the latter case, a compound exhibiting a structure similar to that of the products obtained by addition of either glyoxal or its derivatives to 2′-deoxyguanosine (22, 23) could be an intermediate in the formation of 1 and 2. The reaction mixture of dGuo with DOVA was treated by NaBH4 in order to convert the likely labile Schiff base linkage of adducts 1 and 2 into a stable secondary amine function. Two additional peaks, corresponding to compounds 3 and 4, were observed on the HPLC chromatogram of the NaBH4-treated reaction mixtures (Figure 1B). It was clearly observed that products 3 and 4 were generated at the expense of adducts 1 and 2. When performed on small amounts of nucleoside, the reduction of 1 and 2 by a large excess of NaBH4 is quantitative (data not shown). However, when carried out with milligrams of DOVA-dGuo reaction mixture, the yield was limited to about 50%. This can be explained by the

high amount of salts produced by the successive addition of sodium borohydride and HCL. Compounds 3 and 4 exhibited UV absorption spectra similar to those of adducts 1 and 2. A major difference between the latter compounds and their reduction products was the high stability of adducts 3 and 4 (more than 3 days in aqueous solution at room temperature). Characterization of the Reduced DOVA-dGuo Adducts. Nucleosides 3 and 4 were characterized as the two diastereoisomers of the reduction products of the Schiff base generated by addition of DOVA to the N2amino group of the guanine moiety. The molecular weight of 3 and 4 was shown to be 383 as inferred from ESI-MS analysis. This can be rationalized in terms of a 1:1 DOVA adduct to dGuo which has lost one molecule of water and gained four hydrogen atoms. The latter gain is consistent with the reduction of both a Schiff base and the remaining carboxylic group of the DOVA moiety. The loss of one molecule of water is due to the formation of the Schiff base. Indeed, the infrared data unambiguously showed that a carboxylate group (ν ) 1604 cm-1) is present in the molecule. This rules out an intramolecular esterification or amidation of the latter group as an alternative explanation for the loss of a molecule of water in the formation of 3 and 4. Reduced adducts 3 and 4 were also analyzed separately by GC/MS following trimethylsilylation. The mass spectra (Figure 2) obtained for the TMS derivatives of both reduced DOVA-dGuo adducts were very similar, further suggesting that nucleosides 3 and 4 were diastereoisomers. The identification of the peak at m/z 653 as the molecular ion was confirmed by the observation of the loss of a methyl group (m/z 638) which is a characteristic fragmentation for trimethylsilylated compounds. In addition, an ion at m/z 393 (and its demethylated derivative) corresponding to

154 Chem. Res. Toxicol., Vol. 11, No. 2, 1998

Douki et al. Table 1. 1H NMR Chemical Shifts (in ppm with respect to HDO set at 4.9 ppm) of Reduced DOVA-dGuo Adducts and Their Hydrolysis Product (400 MHz, D2O) proton

3

4

H1′ H2′ H2′′ H3′ H4′ H5′ H5′′ H8 H4* H5*

6.45 (m) 3.07 (m) 2.45 (m) 4.77 (m) 4.17 (m) 3.93 (dd) 3.82 (dd) 8.04 (s) 4.21 (m) 3.85 (dd) 3.75 (dd) 2.45 (t) 2.04 (m) 1.93 (m)

6.45 (m) 3.02 (m) 2.63 (m) 4.74 (m) 4.18 (m) 3.92 (dd) 3.83 (dd) 8.07 (s) 4.25 (m) 3.85 (dd) 3.75 (dd) 2.46 (t) 2.09 (m) 1.93 (m)

H2* H3*

Figure 2. Electron impact mass spectrum of the tetrasilylated reduced DOVA-dGuo adduct 4 obtained by GC/MS analysis.

5

dGuo

7.97 (s) 4.13 (m) 3.84 (dd) 3.72 (dd) 2.45 (t) 2.05 (m) 1.95 (m)

6.40 (m) 2.89 (m) 2.62 (m) 4.74 (m) 4.24 (m) 3.92 (dd) 3.87 (dd) 8.09 (s)

a Protons labeled with an asterisk (*) are of the DOVA moiety, numbered like in the free acid.

Table 2. 13C NMR Chemical Shifts of Reduced DOVA-dGuo Adducts 3 and 4 and dGuoa

Figure 3. 400-MHz 1H NMR spectrum of reduced DOVAdGuo adduct 3 in D2O (HDO used as secondary reference set at 4.9 ppm).

the protonated base moiety of the nucleoside was also present among the main peaks of the mass spectrum. The molecular weight of 653 Da can be explained by the addition of four trimethylsilyl groups to 3 and 4 and an additional loss of a water molecule. This second dehydration can be explained by either an intramolecular esterification or an amidation of the carboxylic acid group within the DOVA moiety during the silylation step. It should be mentioned that GC/MS analysis following acetylation and BSTFA derivatization of 3 and 4 provided the mass spectrum of the diacetylated and disilylated base with an open chain for the DOVA moiety. This can be explained by the acetylation of both the hydroxyl and secondary amine groups of DOVA which makes impossible a further reaction of the latter functions with the carboxylic acid during the silylation. As observed for the mass spectrometry features, the 1 H NMR spectra (Figure 3) of reduced adducts 3 and 4 were almost identical (Table 1). The differences in the respective chemical shifts of the protons of 3 and 4 were lower than 0.05 ppm. In addition, the multiplicity of all the signals was identical for both adducts. The observation of signals corresponding to the nonexchangeable protons of a 2-deoxyribose moiety, inferred from 2DCOSY experiments, confirmed that 3 and 4 were nucleoside derivatives. The similarity between the 1H NMR features of the 2-deoxyribose ring of dGuo and adducts 3 and 4 also applied to their 13C NMR spectra (Table 2).

carbon

DEPT result

3b

4b

3+4c

dGuob

C1′ C2′ C3′ C4′ C5′ C2 C4 C5 C6 C8 C1* C2* C3* C4* C5*

CH CH2 CH CH CH2

85.4 39.2 72.2 87.9 62.8 153.7 nd 111.1 160.3 138.0 nd 34.2 28.2 53.7 64.6

85.0 39.2 72.2 87.9 62.7 nd nd nd nd nd nd 33.9 28.0 53.7 64.6

82.6 41.3 70.7 87.4 61.7 152.6 151.6 116.6 157.9 135.5 177.1 nd 26.8 51.7 62.2

83.2 40.0 71.2 88.0 62.1 154.1 151.4 117.0 157.5 136.1

CH2 CH2 CH CH2

a Carbons labeled with an asterisk (*) are of the DOVA moiety, numbered like in the free acid. nd, not detected. b At 100 MHz, in D2O (chemical shifts expressed with respect to methanol as the internal reference). c At 100 MHz, in DMSO-d6 (chemical shifts expressed with respect to TMS as the internal reference).

The chemical shifts of the H5′, H5′′, and H3′ protons on one hand and those of C5′ and C3′ carbons on the other hand were similar to those obtained for unmodified dGuo. This ruled out the addition of DOVA to one of the two hydroxyl groups of the 2-deoxyribose moiety of dGuo through an esterification of the carboxylic acid function. The signal of the H8 hydrogen atom of 3 and 4 exhibited a chemical shift similar to that of dGuo. In addition, the respective values of the chemical shift of the carbon atoms of the purine ring of 3 and 4, determined by 13C NMR and also by short- and long-distance heteronuclear 1 H-13C coupling analyses (Table 3), were similar to those observed for dGuo. These observations, together with the high similarity between the UV spectra of 3 and 4 on one hand and that of dGuo on the other hand, strongly suggest that the purine ring was not modified by the addition reaction. Evidence for the alkylation of the exocyclic amino group of the guanine moiety was provided by the observation of the loss of the corresponding signal (6.47 ppm for dGuo) in the 1H NMR spectrum obtained in DMSO-d6. However, an exchangeable proton was observed at δ ) 6.85 ppm. The relatively high value for the chemical shift of the latter resonance is more consistent with a NH than an OH proton. This may be rationalized in terms of the involvement of the exocyclic amino group of dGuo in the initial reaction with DOVA.

DNA Alkylation by the Oxidation Product of ALA Table 3. Correlations of Nonexchangeable Protons Observed in 2D Experiments Performed in DMSO-d6 on the Mixture of Adducts 3 and 4a proton

COSY 1H-1H

H1′ H2′ H2′′ H3′ H4′ H5′ H5′′ H8 H2* H3* H4* H5*

H2′, H2′′ H1′, H2′′, H3′ H1′, H2′, H3′ H3′, H4′ H3′, H5′, H5′′ H4′, H5′′ H4′ H3* H2*, H4* H3*, H5*, NH H4*

short-distance 1H-13C C1′ C2′ C2′ C3′ C4′ C5′ C5′ C8 C2* C3* C4* C5*

long-distance 1H-13C C8, C4, C3′ C1′, C3′, C4′ C3′, C4′ C1′, C4′ C1′, C3′ C3′, C4′ C4, C5, C6 C1*, C3*, C4* C1*, C2*, C4*, C5* C4*

a

Atoms labeled with an asterisk (*) are of the DOVA moiety, numbered like in the free acid.

Moreover, the amino proton exhibits a scalar coupling with the vicinal H-C4* methinic proton as inferred from 1 H NMR 2D-COSY analysis. It can thus be concluded that the initial addition of the amino group of guanine occurred on the ketone function of DOVA. Indeed, the NH would have been linked to a methylene group and resonate as a triplet in the case of addition on the aldehyde function. The latter function is then converted into a hydroxymethyl group upon reduction by NaBH4. The proposed structure also allows to account for the GC/ MS features of 3 and 4. Indeed, if a lactamization occurred within the latter compounds during the derivatization reaction, the resulting silylated derivatives would exhibit a trimethylsilylated hydroxymethyl group. The loss of this fragment would account for the predominant loss of 103 amu observed in the GC/MS spectrum. Moreover, evidence for the presence of a -CH2-O- group at the 5* position of the DOVA moiety was provided by the observation of two signals around 3.8 ppm in the 1H NMR spectrum of 3 and 4 (J5*a,5*b ) -20.8 Hz, J4*,5*a ) 4.4 Hz, J4*,5*b ) 5.9 Hz). The chemical shift of the H4* proton (4.21 ppm) is also in agreement with the presence of a vicinal secondary amine. The signals of the methylene groups 2* and 3* are located at 2.45 (J2*,3* ) 7.1 Hz) and around 2 ppm (J3*a,3*b ) -16.7 Hz, J3*b,4* ) 7.2 Hz), respectively. These attributions were confirmed by the correlations observed in a homonuclear 1H-1H COSY coupling experiment. Moreover, long- and short-range heteronuclear 1H-13C coupling experiments provided values for the chemical shifts of the carbon atoms of the DOVA moiety of 3 and 4 which were consistent with the proposed structure. The resonances of C2* and C3* in the low-field region are in agreement with their assignment as carbons of methylene groups linked to two other carbon atoms. The higher values of the chemical shifts of C5* and C4* can be accounted for by attachment of a heteroatom to both carbons. The presence of the C1* signal in the very low-field region (177.1 ppm) of the spectrum confirms that the NaBH4 reduction step has not modified the carboxylic acid function of the DOVA moiety. Detection of the DOVA-dGuo Adducts in DNA. The measurement of the DOVA-dGuo adducts in DNA required two preliminary steps. The first one was the optimization of the acidic hydrolysis of the alkylated nucleosides. The use of 0.1 N HCl at 50 °C allowed a quantitative release of the modified base moiety from 3 and 4. The UV absorption spectrum of the hydrolysis

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 155

product 5 was similar to those of the modified nucleosides 3 and 4. In addition, the 1H NMR spectrum of 5 was very similar to that of the base moiety of 3 and 4 (Table 1). These observations showed that the hydrolysis conditions were mild enough to allow the quantitative release of the base of the reduced dGuo-DOVA adducts without chemical modification of its structure. Interestingly, hydrolysis of either reduced adduct 3 or 4 led to the formation of the same compound. This provided an additional indication that the former nucleosides were diastereoisomers. The detection of the hydrolyzed reduced DOVA-dGuo adduct 5 was achieved by taking advantage of its electroactivity. Within the potential range tested for the determination of the dynamic electrovoltamogram of 5, the EC response did not reach a plateau. The detection potential was limited to 850 mV in order to avoid a too large increase of the background current. However, the detection limit was still close to 10 pmol, and the assay could be applied to the search of compound 5 within isolated DNA. It can be added that the nucleosides 3 and 4 are not electroactive at potentials below 950 mV. Therefore, the detection of the reduced dGuo-DOVA adducts has to be carried out at the base level. Commercially available calf thymus DNA was incubated with DOVA and subsequently reduced by NaBH4 in order to stabilize the DOVA-dGuo adducts. DNA was then hydrolyzed into nucleosides by successive incubation with nuclease P1 and alkaline phosphatase. The resulting mixture was injected on a first HPLC system, and the fractions corresponding to the retention time of the reduced DOVA-dGuo adducts were collected. This prepurification step was aimed at getting rid of the large amount of salts produced by the reduction step and at removing dGuo from the sample. Indeed, the latter nucleoside would then be hydrolyzed into guanine which is electroactive at the potential used for the detection of hydrolyzed reduced adduct 5. Because of the large excess of unreacted guanine in DNA, its presence would have interfered with the HPLC-EC analysis. Interestingly, two peaks, with retention times similar to those of adducts 3 and 4, were observed in the UV chromatogram of enzymatically digested DNA (data not shown). The purified fraction containing the mixture of adducts 3 and 4 was then hydrolyzed under acidic conditions and analyzed by HPLC with electrochemical and UV detection. Both HPLC elution profiles exhibited a peak corresponding to hydrolyzed reduced adduct 5 (Figure 4). Identification of the related electroactive product was also provided by the observation of a ratio between the EC and UV signals similar to that obtained with authentic 5. In addition, the compound observed on the HPLC chromatograms coeluted with authentic hydrolyzed reduced adduct 5. Altogether, these data demonstrate that the DOVA-dGuo adducts were produced within isolated DNA. The level of modification was determined to be 1300 adducts/106 normal nucleotides. However, this value might be underestimated since no evidence could be obtained for the completion of the NaBH4 reduction step.

Discussion 5-Aminolevulinic acid is a suspected endogenous mutagen associated with several porphyric disorders (4-6). The ability for ALA to damage both cellular and isolated

156 Chem. Res. Toxicol., Vol. 11, No. 2, 1998

Douki et al.

acterization of the DOVA-dGuo adducts by extensive NMR and MS studies required a preliminary reduction of the Schiff base into a more stable secondary amine group. The same approach was applied to their detection in isolated DNA. However, the relatively low stability of the DOVA-guanine adducts is compensated by the efficiency of the addition reaction of DOVA to dGuo. In addition, it is likely that the double-helix structure of DNA decreases the decomposition rate of the adducts compared to isolated dGuo, as observed for other hydrolytic processes such as deamination. It can be added that other aldehydes able to induce the formation of Schiff bases within DNA are known to be mutagenic (29, 30). In addition, glyoxal and other R-dicarbonyl compounds are both mutagens (31) and efficient alkylating agents of the N2 exocyclic amino group of guanine (22, 23). It should be emphasized that acetaldehyde adducts to guanine exhibiting a Schiff base structure have recently been detected in human white blood cells (21). Altogether, these data suggest that the formation of adducts between DOVA and guanine moieties of DNA might be biologically relevant in ALA mutagenicity.

Figure 4. HPLC UV and EC chromatograms of hydrolyzed DOVA-treated DNA (two upper panels). The lower panel shows the EC chromatogram of the same DNA sample spiked with 500 pmol of pure hydrolyzed reduced DOVA-dGuo adduct 5.

DNA through the production of reactive oxygen species during its iron-catalyzed oxidation (32) is now wellestablished (12, 16). ALA enoyl radical is an intermediate in the latter reaction leading to the release of the corresponding R-keto aldehyde, DOVA. Interestingly, ALA is able to promote its own oxidation since it releases iron, as observed in rat liver (15). A similar pathway is also likely to account for the iron deposits found in human liver biopsies of porphyric carriers (33). The species responsible for the release of iron from ferritin might be either the ALA enoyl radicals or the superoxide anions released during the oxidation of the former compound. Altogether, DOVA may accumulate in target organs upon ALA overload. Moreover, it is expected that DOVA, an R-keto aldehyde, can form adducts with the amino groups of DNA bases. The present work establishes a mechanism by which DOVA causes chemical damage to DNA. Using dGuo as a model compound, it was shown that DOVA is able to add to the exocyclic amino group of the guanine ring (Scheme 3). Two predominant diastereoisomers were generated as the result of the formation of a Schiff base between the exocyclic N2 nitrogen atom of guanine and the C4 carbon of DOVA. One intermediate in this reaction may exhibit a diol structure like the adducts generated by the reaction of glyoxal and its derivatives with guanine-containing compounds (22, 23). However, in the case of DOVA, a dehydration of the -NH-C-OH link takes place, leading to a Schiff base. Evidence was then provided for the formation of similar adducts within isolated DNA incubated with DOVA. This indicates that the involvement of the N2 exocyclic amino group of guanine in the base pairing with cytosine does not prevent its nucleophilic addition to the keto group of DOVA. However, the relative instability of the Schiff base group of the DOVA-guanine adducts has to be noted. The related dGuo adducts are efficiently reversed to dGuo and DOVA by hydrolysis. Therefore, the char-

Acknowledgment. This work was supported by the Fundac¸ a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo, FAPESP (Brazil), the Conselho Nacional para o Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq (Brazil), the Programa de Apoio aos Nu´cleos de Exceleˆncia, PRONEX/ FINEP (Brazil), and the Universidade de Sa˜o Paulo/ Commite´ Franc¸ ais d'Evaluation de la Coope´ration Universitaire avec le Bre´sil (USP-COFECUB/UC-23/96). J.O. is a recipient of a FAPESP fellowship. The authors thank Miriam Uemi (USP, Sa˜o Paulo), Franc¸ oise Sarazin, and Robert Nardin (CEA, Grenoble) for their assistance in the NMR analysis, Daniel Ruffieux (Biochimie C/CHRU, Grenoble) for the ESI-MS analyses, and Dr. Dieter Do¨rnemann (Philipps-Universita¨t, Marburg) for kindly providing 4,5-dioxovaleric acid.

References (1) Kappas, A., Sassa, S., and Anderson, K. E. (1983) In The Metabolic Basis of Inherited Diseases (Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S., Goldstein, J. L., and Brown, M. S., Eds.) pp 1301-1384, McGraw-Hill, New York. (2) Hindmarsh, J. T. (1986) The porphyrias: Recent advances. Clin. Chem. 32, 1255-1263. (3) Chisolm, J. J., Jr. (1971) Lead poisoning. Sci. Am. 224, 15-23. (4) Lithner, F., and Wetterberg, L. (1984) Hepatocellular carcinoma in patients with acute intermittent porphyria. Acta Med. Scand. 215, 271-274. (5) Gubler, J. G., Bergetzi, M. J., and Meyer, U. A. (1990) Primary liver carcinoma in two sisters with acute intermittent porphyria. Am. J. Med. 89, 540-541. (6) Thunnissen, P. L., Meyer, J., and de Koning, R. W. (1991) Acute intermittent porphyria and primary liver cancer. Neth. J. Med. 38, 171-174. (7) Fiedler, D. M., Eckl, P. M., and Krammer, B. (1996) Does δ-aminolaevulinic acid induce genotoxic effects? J. Photochem. Photobiol. B: Photobiol. 33, 39-44. (8) Kennedy, J. C., and Pottier, R. H. (1992) Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J. Photochem. Photobiol. B: Biol. 14, 275-292. (9) Ortel, B., Tanew, A., and Ho¨nigsmann, H. (1993) Lethal photosensitization by endogenous porphyrins of PAM cells modification by desferrioxamine. J. Photochem. Photobiol. B: Photobiol. 17, 273-278. (10) Peng, Q., Berg, K., Moan, J., Kongshaug, M., Giercksky, K. E., and Nesland, J. M. (1997) 5-Aminolevulinic acid-based photodynamic therapy: Principles and experimental research. Photochem. Photobiol. 65, 235-251. (11) Bechara, E. J. H., Medeiros, M. H. G., Monteiro, H. P., HermesLima, M., Pereira, B., Demasi, M., Costa, C. A., Abdalla, D. S. P., Onuki, J., Wendel, C. M. A., and Di Mascio, P. (1993) A free

DNA Alkylation by the Oxidation Product of ALA

(12)

(13) (14)

(15) (16)

(17)

(18)

(19) (20) (21) (22)

radical hypothesis of lead poisoning and inborn porphyrias associated with 5-aminolevulinic acid overload. Quim. Nova 16, 385-392. Fraga, C. G., Onuki, J., Lucesoli, F., Bechara, E. J. H., and Di Mascio, P. (1994) 5-Aminolevulinic acid mediates the in vivo and in vitro formation of 8-hydroxy-2′-deoxyguanosine in DNA. Carcinogenesis 15, 2241-2244. Hiraku, Y., and Kawanishi, S. (1996) Mechanism of oxidative DNA damage induced by δ-aminolevulinic acid in the presence of copper ion. Cancer Res. 56, 1786-1793. Demasi, M., Penatti, C. A. A., DeLucia, R., and Bechara, E. J. H. (1996) The prooxidant effect of 5-aminolevulinic in the brain tissue of rats: Implications in neuropsychiatric manifestations in porphyrias. Free Radical Biol. Med. 20, 291-299. Oteiza, P. I., Kleinman, C. G., Demasi, M., and Bechara, E. J. H. (1995) 5-Aminolevulinic acid induces iron release from ferritin. Arch. Biochem. Biophys. 316, 607-611. Onuki, J., Medeiros, M. H. G., Bechara, E. J. H., and Di Mascio, P. (1994) 5-Aminolevulinic acid induces single strand breaks in plasmid DNA in the presence of Fe2+ ions. Biochim. Biophys. Acta 1225, 259-263. Basu, A. K., O’Hara, S. M., Valladir, P., Stone, K., Mols, O., and Marnett, L. J. (1988) Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes. Chem. Res. Toxicol. 1, 53-59. Chaudhary, A. K., Nokubo, N., Ramachandra Reddy, G., Yeola, S. N., Morrow, J. D., Blair, I. A., and Marnett, L. J. (1994) Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science 265, 1580-1582. Vaca, C. E., Vodicka, P., and Hemminki, K. (1992) Determination of malondialdehyde modified 2′-deoxyguanosine-3′-monophosphate and DNA by 32P-postlabeling. Carcinogenesis 13, 593-599. Vaca, C. E., Fang, J.-L., and Schweda, E. K. H. (1995) Studies of the reaction of acetaldehyde with deoxynucleosides. Chem.-Biol. Interact. 98, 51-67. Fang, F. L., and Vaca, C. E. (1997) Detection of adducts of acetaldehyde in peritethial white blood cells of alcohol abusers. Carcinogenesis 18, 627-632. Shapiro, R., and Hachmann, J. (1966) The reaction of guanine derivatives with 1,2-dicarbonyl compounds. Biochemistry 5, 27992807.

Chem. Res. Toxicol., Vol. 11, No. 2, 1998 157 (23) Shapiro, R., Cohen, B. I., Shiuey, S.-J., and Mauer, H. (1968) On the reaction of guanine with glyoxal, pyruvaldehyde, and kethoxal, and the structure of the acylguanines. A new synthesis of N2alkylguanines. Biochemistry 8, 238-245. (24) 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. (25) Winter, C. K., Segall, H. J., and Haddon, W. F. (1986) Formation of cyclic adducts of deoxygunosine with the aldehydes trans-4hydroxy-2-hexenal and trans-4-hydroxy-2-nonenal in vitro. Cancer Res. 46, 5682-5686. (26) Eder, E., Scheckenbach, S., Deininger, C., and Hoffman, C. (1993) The possible role of R,β-unsaturated carbonyl compounds in mutagenesis and carcinogenesis. Toxicol. Lett. 67, 87-103. (27) Nath, R. G., and Chung, F.-L. (1994) Detection of exocyclic 1,N2propanodeoxyguanosine adducts as common DNA lesions in rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. (28) Douki, T., and Ames, B. N. (1994) An HPLC-EC assay for 1,N2propano adducts of 2′-deoxyguanosine with 4-hydroxynonenal and other R,β-unsaturated aldehydes. Chem. Res. Toxicol. 7, 511-518. (29) 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 in Salmonella tester strain TA104. Mutat. Res. 148, 25-34. (30) Dellarco, V. L. (1993) A mutagenicity assessment of acetaldehyde. Mutat. Res. 195, 1-20. (31) Bjeldanes, L. F., and Chew, H. (1979) Mutagenicity of 1,2dicarbonyl compounds: maltol, kojic acid, diacetyl and related substances. Mutat. Res. 67, 367-371. (32) Monteiro, H. P., Abdalla, D. S. P., Augusto, O., and Bechara, E. J. H. (1989) Free radical generation during δ-aminolevulinic acid autoxidation: Induction by hemoglobin and connections with porphyrinpathies. Arch. Biophys. Biochem. 271, 206-216. (33) Biempica, L., Kosower, N., Ma, M. H., and Goldfisher, S. (1974) Cytochemical and ultrastructural studies of liver in acute intermitent porphyria and porphyria cutanea tarda. Arch. Pathol. 98, 336-343.

TX970157D