Chem. Res. Toxicol. 1994, 7, 511-518
511
An HPLC-EC Assay for 1,M-PropanoAdducts of %Deoxyguanosine with 4-Hydroxynonenal and Other a,@-UnsaturatedAldehydes Thierry Doukit and Bruce N. Ames*J CEAIDbpartement de Recherche Fondamentale sur la M a t i b e Condensbe SESAMILAN, 38054 Grenoble Cedex 9, France, and Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, California 94720-3202 Received January 31,1994"
An assay was developed for the 1,W-propanoguanine adducts generated in DNA by reaction with biologically relevant a,@-unsaturated aldehydes. The analysis takes advantage of the electrochemical activity of the adducts released as modified bases by quantitative acidic hydrolysis of the DNA. The detection limit of the method is around 1pmol in DNA. Emphasis was placed on the detection of the derivatives of 4-hydroxynonenal (a final product of lipid peroxidation) and hexenal. The adducts were detected in calf thymus DNA incubated with these two unsaturated aldehydes. The 4-hydroxynonenal-l~-propan0guanine derivatives were not observed in DNA extracted from young rat kidneys or liver. The technique was shown to be also applicable t o a series of a,@-unsaturatedaldehydes-1,W-propanoguanineadducts which were found to be electrochemically active at relatively low potential and efficiently separated by reverse-phase HPLC.
Introduction a,@-Unsaturatedaldehydes such as acrolein are known to be mutagenic compounds (1, 2,3). Acrolein is generated by combustion of organic matter, is present in cigarette smoke, and is widely used industrially (annual production: 30000 tons). Aldehydic compounds can also be produced by biological processes. For instance, malondialdehyde (MDA)' and 4-hydroxynonenal (HNE), together withsimilar compoundsgenerated in lower amounts such as 4-hydroxyhexenal (HHE),are associated with lipid peroxidation ( 4 ) . HNE, which is thought to be generated by decomposition of w-&polyunsaturated fatty acids as shown by its presence in autooxidized linolenic and arachidonic acid (5), was reported to be one of the major final products of lipid peroxidation in isolated rat hepatocytes (6) and was detected in most rat organs (7, 8). HNE has a conjugated aldehyde structure, which enables it to react readily with biomolecules, such as proteins (9, 10). Moreover, it is mutagenic in rodent cells (11),which indicates that it is able to induce DNA modifications. In addition, HNE was found to be 4000 times more efficient than MDA in inducing SOS response in Salmonella
* To whom correspondence should be addressed.
CEAIDBpartement de Recherche Fondamentale sur la Matiete Condensee SESAM/LAN. Tel: 33-76-88-31-91;Fax: 33-76-88-50-90. t University of California-Berkeley. Tel: 510-642-5165; Fax: 610643-7935; e-mail:
[email protected]. @Abstractpublished in Advance ACS Abstracts, May 15, 1994. 1 Abbreviations: dG-Hex, hexenal-lJv2-propano-2'-deoxyguanosine adduct; dG-HNE-H+, hydrolyzed 4-hydroxynonenal-1J-propano-2'deoxyguanosineadduct; dG-Hex-H+,hydrolyzed hexenal-1 Jv2-propano2'-deoxyguanosine adduct; dG-HNE, 4-hydroxynonenal-1J-propano2'-deoxyguanosine addud; EC, electmchemical detection;EDTA, disodium ethylenediaminetetraacetate;GC-MS, gas chromatographycoupled with mass spectrometry; Gua-HNE, guanine-4-hydroxynonenaladduct; GuaHex, guanine-hexenal adduct; Hex, hexenal; HF/pyridine, 70% (w/w) solutionof hydrogen fluoride in pyridine;HHE, 4-hydroxyhexenal;HNE, 4-hydroxynonenal; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; RP, reverse phase; NMR, nuclear magnetic resonance; tR, retention time; SDS, sodium dodecyl sulfate; TMS, trimethylsilyl group; Tris, tris(hydroxymethy1)aminomethane; TSP, potassium (trimethylsily1)propionate;UV, ultraviolet. t
typhimurium (12). Consequently, HNE, together with MDA and other aldehydic products, was proposed to be partly responsible for the mutagenic properties of lipid peroxidation (13, 14). The mutagenicity of a,@-unsaturatedaldehydes was shown to be related to their ability to undergo 1,4conjugated addition on nucleobases such as guanine, which was found to be the most reactive (15,161. The N-2 amino group of the guanine ring reacts first with the C-3 atom of the aldehyde, and the transient species generated undergoes a further cyclization by reaction of the aldehyde function on the N-1 amine group of the nucleobase (Figure 1). l p - p r o p a n o cyclic guanine adducts were isolated i n vitro and characterized for acrolein and crotonaldehyde (1, 17). With acrolein, an additional isomeric adduct is produced by the addition of the N-1 amino group on the C-3 atom of the unsaturated aldehyde (18) (Figure 1, structure 5). MDA, reacting under its 8-hydroxyacrolein tautomeric form, also undergoes a conjugated addition on guanine, followed by a double dehydration, giving rise to a pyrimidopurinone adduct (19) (Figure 1, structure 6). The biological relevance of the 1p-propanoguanine adducts was established by their detection in the DNA of bacteria incubated with acrolein (1)and of rodents treated with N-nitrosopyrrolidine and crotonaldehyde (20). The major guanine-malondialdehyde adduct was reported to be present in rat and human urine (21). However, results based on the use of liquid chromatography coupled with mass spectrometry are in disagreement with this observation (22). In addition, 1 p - p r o p a n o cyclic adducts of 2'deoxyguanosine were isolated i n vitro, by incubation of the nucleoside with HNE and HHE (23). Therefore, the determination of the amount of 1JVzpropanoguanine adducts in DNA appears to be a major tool for the study of the mutagenicity of a,@-unsaturated aldehydes. We present here a sensitive assay for this class of DNA lesions, based on an acidic hydrolysis followed by a high-performance liquid chromatography separation coupled with an electrochemical detection (HPLC-EC).
0893-228~19412707-0511~04.50f 0 0 1994 American Chemical Society
512 Chem. Res. Toxicol., Vol. 7, No. 4, 1994
Douki and Ames
L 1
2
3
4
CG) 644
5
6
Figure 1. Formation and structure of the unsaturated aldehydes adducts: (1)2‘-deoxyguanosine; (2) a,&unsaturated aldehyde (R = H, acrolein; R = methyl, crotonaldehyde; R = ethyl, pentenal; R = propyl, hexenal; R = butyl, heptenal, R = CHsCHzCHOH,HHE; R = CH&H&CHOH, HNE); (3) 1,W-propano-2’-deoxyguanosine;(4) 1,W-propanoguanine;(5) isomeric 1,W-propano adduct obtained with acrolein; (6) MDA-pyrimidopurinone adduct.
It was mainly developed with the HNE and hexenal derivatives, but the extension of the method to other unsaturated aldehydes was also investigated.
Materials and Methods Chemicals and Enzymes. 2’-Deoxyguanosine,guanine, tris(hydroxymethy1)aminomethane (Tris), pronase (Streptomyces griseus), and calf thymus DNA were purchased from Sigma (St. Louis, MO). Acrolein, crotonaldehyde, truns-2-pentenal, trans2-hexenal, 2-methylacrolein,trans 2-heptenal, and 70% hydrogen fluoride stabilized in pyridine were Aldrich products (Milwaukee, WI).HPLC-grade methanol and acetonitrile, ACS concentrated hydrochloric acid, sodium hydroxide, and disodium ethylenediaminetetraacetate (EDTA) were purchased from Fisher Scientific (Fair Lawn, NJ). Sodium dodecyl sulfate (SDS) was obtained from Bio-Rad (Hercules, CA). HNE and HHE were synthesized following the procedure reported by Esterbauer et al. (24). RNase T1 (Aspergillus oryzae) and RNase A (bovine pancreas) were from Boehringer-Mannheim (Mannheim, Germany). High-Performance Liquid Chromatography. The chromatographic system consisted of an HP 1090diode array detector HPLC system (Hewlett-Packard, Kennett Square, PA) equipped with a 5-pm particle size 4.6 X 25 mm Supercosil LC18 DB octadecylsilylsilicagel column (Supelco,Bellefonte,PA). Except where noted, the isocratic eluent was a 8020 0.025 M ammonium formate/acetonitrile mixture. The elution was monitored at 260 nm. Electrochemical detection was performed with a 5100 A Coulochem detector (ESA, Bedford, MA) connected to a Model 5010 two-electrodeelectrochemicalcell (ESA,Bedford, MA). The isocratic eluent was a mixture of acetonitrile with a 50 mM concentration of pH 5.5 phosphate buffer. The dynamic electrovoltamograms were determined by plotting the ratio between the EC and the UV signal of the HPLC peaks corresponding to the adducts for decreasing detection potentials. Gas Chromatography-Mass Spectrometry Measurements. GC-MS analysis were performed on an HP 5890 GC interfaced to a 5989 MS engine (Hewlett-Packard, Palo Alto, CA). The DB5 column (12-mlength,0.25-mm internal diameter, 0.2-pm film thickness, J&W Scientifics, Folsom, CA) was used with helium as a carrier gas at a linear velocity of 35 cmes-1. The ionization was provided by electron impact (energy 70 eV). A splitter ratio of 1/20 was used. The column temperature was maintained at 100 “C for 1min, raised to 280 “C with a 10 “C min-1 rate, and maintained at this temperature for 5 min. Before
injection, the samples were dried by lyophilization and silylated for 2 h at 110 “C in sealed vials with 50 pL of silylation-grade pyridine and 50 pL of silylation-grade bis(trimethylsily1)trifluoroacetamide containing 1% trimethylsilyl chloride (Pierce, Rockford, IL). A 4-pl aliquot was injected for each analysis. Nuclear Magnetic Resonance Experiments. The ‘H NMR spectra were recorded on a Brucker AM 400 spectrometer. The exchangeable protons of the samples were first removed by evaporation of a solution in 1 mL of 99.8% deuterium oxide (Eurisotop, France). The residue obtained was dissolved in 0.5 mL of 99.96% deuterium oxide prior to the analysis. The chemical shifts were obtained from first-order analysis and expressed with respect to potassium (trimethylsily1)propionate (TSP) used as internal reference. Synthesis of the 2’-Deoxyguanosine and Guanine Adducts. 2’-Deoxyguanosine (50 mg) was incubated under stirring for 6 days with 20 mg of HNE in 10 mL of pH 9.4 carbonate buffer at 50 “C. The solution was lyophilized and the sample dissolved in 1mL of 8020 water/acetonitrile mixture and injected on the HPLC system. Under the conditions used, 2’-deoxyguanosine was not retained and eluted in the void volume. The fraction eluted between 10 and 25 min was collected, freezedried, and injected on the same HPLC system. Four peaks (dGHNE 1, 2, 3, and 4) were observed, with the two first poorly resolved. Their respective retention times were 16.5, 17, 19.6, and 21.5 min. The overall amount of adduct was 7 mg (yield 9%). dG-HNE 1 + 2: 1H NMR features (400.15 MHz, DzO, TSP) 6 (ppm) 8.19 (lH, s, H-8 dG), 6.69 (lH, s, H-1 HNE), 6.55 (lH, m, H-1’ dG), 4.90 (lH, m, H-3‘ dG), 4.36 (lH, m, H-4’ dG), 4.20 (lH, m, H-3 HNE), 4.01 (2H, m, H-5’,5’’ dG), 3.61 (lH, m, H-4 HNE), 2.50 (lH, m, H-P”dG), 2.08 (lH, m, H-2’ dG), 1.86/1.71 (2H, m, H-2 HNE), 1.65 (2H, m, H-5 HNE), 1.59 (6H, t, H-6,7,8 HNE), 1.16 (3H, H-9 HNE). dG-HNE 3 + 4 lH NMR features (400.15 MHz, DzO, TSP) 6 (ppm) 8.10 (lH, s, H-8 dG), 6.66 (lH, s, H-1 HNE), 6.52 (lH, m, H-1’ dG), 4.90 (lH, m, H-3’ dG), 4.34 (lH, m, H-4’ dG), 4.02 (2H, m, H-5’,5’‘dG), 3.89 (lH, m, H-3 HNE), 3.43 (lH, m, H-4 HNE), 2.58 (lH, m, H-2”dG), 2.04 (lH, m, H-2’ dG), 1.85 (2H, m, H-2 HNE), 1.55 (8H, m, H-5,6,7,8 HNE), 1.13 (3H, t, H-9 HNE). The hexenal-1,W-propano-2‘-deoxyguanosine adducts were prepared by incubation of 10 mg of 2’-deoxyguanosine with 100 pL of hexenal for 5 days at 50 “C in 2 mL of pH 9.4 carbonate buffer. The reaction mixture was lyophilized,dissolved in water,
Chem. Res. Toxicol., Vol. 7, No. 4, 1994 513
HPLC-EC Aldehydes and injected on RP-HPLC. Two peaks were collected (dG-Hex 1 and 2; t~ 7.5 and 8.1 min.). dG-Hex 1 2: 1H NMR features (400.15 MHz, DzO, TSP) 6 (ppm) 8.36 (lH, s,H-8dG),6.65 (lH,H-1Hex), 6.53 (lH,m,H-1' dG), 4.90 (lH, m, H-3' dG), 4.36 (lH, m, H-4' dG), 4.02 (2H, m, H-5',5"dG), 2.99 (lH, m, H-2" dG), 2.73 (lH, m, H-2' dG), 2.53 (lH, m, H-3 Hex), 1.98 (2H, m, H-2 Hex), 1.45 (4H, m, H-4,5 Hex), 1.24 (3H, t, H-6 Hex). The other 1,Wpropano adducts were prepared by incubation of 10 mg of 2'-deoxyguanosine in 2 mL of pH 9.4 carbonate buffer with 100 pg of unsaturated aldehyde for 2 days at room temperature. The samples were thenlyophilized and the adducts isolated by HPLC. A 5% acetonitrile in 25 mM ammonium formate mixture was used for the isolation of the acrolein adduct ( t R 9 min), 10% for the derivatives of 2-methylacrolein ( t 7.9 ~ min), HHE ( t R 8.7,9.2, 11.5, and 12.1 min), crotonaldehyde ( t ~ 8.3 and 9.0 min), and pentenal ( t 18.7 ~ and 20.0 min), and 20% for the sample treated with heptenal (15.7 and 16.1 min). Guanine (5 mg) was incubated for 5 days in 2 mL of 50 mM pH 9.4 carbonate buffer with 5 mg of HNE. After lyophilization and dissolution in water, the reaction mixture was injected on the HPLC system. Guanine was not retained, and two adducts (Gua-HNE) with respective retention times of 7.8 and 9.4 min were collected. The chromatogram of the GC-MS analysis followingsilylation of adduct 1exhibited a peak at 17.8min with major ions at m/z = 523 (guanine HNE 3 trimethylsilyl groups (TMS)) and 508 (guanine + HNE + 3 TMS - 1methyl). 5 mg of guanine were incubated for 5 days with 20 pL of hexenal in 1mL of pH 9.4 50 mM carbonate buffer at 50 OC. The sample was then lyophilized, dissolved in water, and injected on HPLC. One peak was observed in addition to guanine with a 5.2-min retention time, corresponding to the hexenal/guanine adduct (Gua-Hex). Acidic Hydrolysis of the 2'-Deoxyguanosine Adducts. The mixture of dG-HNE was lyophilized in polypropylene tubes. Part of the sample was then dissolved in 100 pL of HF/pyridine and left at room temperature for 2 h. The reaction mixture was diluted in 1mL of water, and the pH was raised to 7 by addition of sodium hydroxide. The sample was lyophilized,and the residue obtained was dissolved in water. An equal amount of dG-HNE was hydrolyzed in 1mL of 0.1 N hydrochloric acid at 90 "C for 2 h. The sample was cooled to room temperature and neutralized by addition of sodium hydroxide. The two hydrolyzed samples were injected on the HPLC system, and the two corresponding chromatograms were found to exhibit the same two major peaks at 13.5and 15.3 min, with a similar UV signal intensity. The two hydrolyzed adducts (dG-HNE-H+ 1 and 2) were collected together. Upon hydrolysis, the mixture of the dG-HNE adducts 1and 2 gave rise to a compound eluting at 13.5 min, whereas the mixture of 3 and 4 was converted into a compound eluting at 15.4 min. The chromatogram of the GC-MS analysis of the silylation product of the mixture exhibited two major couples of peaks. At 21.9 and 22.3 min, the molecular ions were at m/z = 523 (guanine HNE + 3 TMS) whereas at 23.7 and 23.9 min, they were at m/z = 595 (guanine + HNE 4 TMS). The corresponding radiolabeled dG-HNE-H+ were prepared by incubation of 1.5 mg of dG with 5 mg of HNE and 250 pCi of [3H-C8]dG (Amersham,U.K.). After 7 days, the sample was lyophilized and dissolved in 1mL of 0.1 N HC1. After 2 h at 90 "C, the solution was neutralized by addition of sodium hydroxide, and the adducts were isolated by HPLC. dG-HNE-H+ 1 + 2: lH NMR features (400.15 MHz, D20, TSP) 6 (ppm) 7.89 (lH, s, H-8 Gua), 6.53 (lH, s, H-1 HNE), 3.86 (lH, m, H-3 HNE), 3.72 (lH, m, H-4 HNE), 1.60 (2H, m, H-2 HNE), 1.35 (8H, m, H-5,6,7,8 HNE), 0.97 (3H, t, H-9 HNE). The two dG-Hex adducts were hydrolyzed by treatment with 1 mL of 0.1 N HC1 for 2 h at 90 "C. After neutralization, the sample was injected on the HPLC system. A product (dG-HexH+) with a 7.2-min retention time was collected. An aliquot fraction was lyophilized,silylated, and analyzed by GC-MS. The chromatogram exhibited a major peak at 20.5 min with a molecular ion at m/z = 465 (guanine + Hex + 3 TMS). A 10
+
+
+
+
+
times smaller peak ( t 19 ~ min) with molecular ion at m/z = 393 (guanine + Hex + 2 TMS) was also observed. dG-Hex-H+: lH NMR features (400.15 MHz, DzO, TSP) 6 (ppm) 8.06 (lH, H-8 Gua), 6.59 (lH, H-1 Hex), 2.54 (lH, H-3 Hex), 1.89 (2H, m, H-2 Hex), 1.68 (2H, m, H-4), 1.55 (2H, m, H-5 Hex), 1.25 (3H, t, H-6 Hex). Similarly,a 0.1 N HC1treatment followed by HPLC separation, using the eluents described for the 2'-deoxyguanosine adducts, ~ min), provided the hydrolyzed adducts of acrolein ( t 5.1 ~ min), 2-methylacrolein ( t R 5.3 min), crotonaldehyde ( t 5.8 ~ min), HHE ( t 6.2 ~ and 7.1 min), and heptenal pentenal ( t 11.0 ( t R 13.5 min). Treatment of Calf Thymus DNA with 4-Hydroxynonenal and Hexenal. Calf thymus DNA (1mg) was treated for 3 days with 5 mg of HNE in 1mL of pH 9.4 carbonate buffer at 45 OC. The sample was lyophilized and the residue dissolved in 500 pL of water. The DNA was precipitated by addition of 50 pL of 3 M pH 5 sodium acetate and 1mL of ethanol. The suspension was cooled to -20 "C and centrifuged at 20000g. A total of 960 pg was recovered, as inferred from the 260-nm UV absorption measured on an Ultrospec I1 spectrophotometer (LKB-Pharmacia, Uppsala, Sweden). The DNA was hydrolyzed by treatment with 1 mL of 0.1 N HC1 at 90 OC for 2 h. After neutralization by addition of sodium hydroxide, the sample was prepurified by using 1-g phase 6-mL C18 Supelclean cartridges (Supelco, Bellefonte, PA). It was washed by 2 fractions of 3 mL of water and 2 mL of 9010 watedmethanol, and the fraction containing dG-HNE-H+was eluted by 3 mL of methanol. After evaporation and dissolution in 500 pL of water, the sample was analyzed by HPLC-EC, with a 600-mV detection potential. Fractions corresponding to 50 pg of hydrolyzed DNA were used for each injection. Calf thymus DNA (4 mg) was incubated for 5 days with 20 pL of hexenal in 1 mL of pH 9.4 carbonate buffer at 50 OC. The sample was lyophilized and the DNA purified by precipitation. The sample was dissolved in water, split into two equal aliquot fractions, and lyophilized. One fraction was hydrolyzed by 100 pL of HF/pyridine at 25 "C for 2 h and the other by 1mL of 0.1 N HC1 at 90 OC for 2 h. After neutralization, each of these two hydrolysis products were purified by elution on a Supelclean C18 silica cartridge. The samples were washed with 3 mL of water and 2 mL of 95:5 water/methanol, and the adducts were collected by elution of 3 mL of methanol. For each hydrolysis technique, one half (800 pg of hydrolyzed DNA) was lyophilized and silylated prior to GC-MS analysis performed in single-ion monitoring (m/z = 393 and 378). The other half was dissolved in 1mL of water and usedfor the electrochemicalanalysis (potentials: channel 1,300 mV; channel 2,550 mV). Extraction of Rat Liver and Kidney DNA. Six animals were used in this experiment, using the following procedure. A 3-month-old male Fischer 344 rat (Simonsen, Gilroy, CA) was decapitated following light COz anesthesia. The liver and the kidneys were quickly excised and immediately placed in ice-cold buffer (100 mM Tris, 10 mM EDTA, pH 7.4). The envelope and the medulla of the kidneys were removed. The tissues were finely cut with scissors and homogenized in 9 times the tissue weight of buffer, using a Potter-Elvehjem glass-Teflon homogenizer. The sample was lysed with a 1% final concentration of SDS. The homogenates were centrifuged at lOOOg for 20 min to spin down the nuclei, and the pellet was resuspended in once the original tissue weight of buffer. A 1-mL aliquot of each organ nuclei fraction was incubated with RNase T1 (5 units/mL final concentration) and RNase A (2.5 units/mL final concentration) at 50 "C for 1h, and thereafter with Pronase (0.5 mg/mL final concentration) at 50 "C for 1 h. The samples were extracted once with an equal volume of phenol, once with 2 volumes of phenol/chloroform/isoamylalcohol (25:24:1), and once with an equal volume of chloroform/isoamyl alcohol (24:l). The DNA was precipitated by addition of 10% (v/v) of pH 5 3 M sodium acetate and 2 volumes of cold ethanol. The amount of DNA isolated after centrifugation (20000g) was 1mg and 900 pg for the liver and the kidneys, respectively, as inferred from the 260-
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Chem. Res. Toxicol., Vol. 7, No. 4, 1994
dG-HNF! 1.2
Figure 2. HPLC profile of (1)the dG-HNE adducts and of (2) the previous sample after 0.1 N HC1 treatment at 90 "C for 2 h. HPLC conditions are described in the experimental part. The amount injected is around 100 pmols.
nm UV absorption. The absence of significantamount of RNA in the isolated DNA was establishedby HPLC analysis following digestion into nucleosides by nuclease P1 and alkaline phosphatase (data not shown). The DNA samples were suspended in 1 mL of 0.1 N HCl and heated at 90 O C for 2 h. They were then neutralized by additionof sodium hydroxide and prepurified on a C18 cartridge as described above. The methanol fraction was evaporated, and the residue was dissolved in 400 pL of a 8020 water/acetonitrile mixture and analyzed by HPLC-EC (electrode potentials 350 and 530 mV for channels 1 and 2, respectively).
Results Formation and Hydrolysis of the Modified Nucleosides. Treatment of 2/-deoxyguanosine with HNE and hexenal was found to provide the 1JP-propano adducts as the major products, as reported by Winter et al. (23). Four diastereoisomers of dG-HNE and two of dG-Hex were obtained, and all compounds were efficiently isolated by RP-HPLC (Figure 2.1). Their UV absorption spectra, exhibiting the characteristic red shift of N-2-substituted guanine, and chromatographic and 1H NMR features were in agreement with those reported. Like hexenal, crotonaldehyde, pentenal, and heptenal generated two diastereoisomeric adducts, whereas HHE behaved like HNE with formation of four compounds. Treatment of 2'deoxyguanosine with acrolein yielded three main compounds eluting on reverse-phase high-performance liquid chromatography (RP-HPLC). Only the product corresponding to third peak, characterized by Chung e t al. (18)as the 1,W-propano adduct, was collected. It is worth mentioning that 2-methylacroleingenerates only one major product with UV features similar to the other 1 F propano-2/-deoxyguanosineadducts. These modified 2/-deoxyguanosinewere converted into their base derivatives by acidic treatment. The four dGHNE and dG-HHE adducts were hydrolyzed into I F -
propanoguanine adducts by hydrochloric acid (Figure 2.2). The use of hydrogen fluoride stabilized in pyridine, tested with the HNE derivatives, led to the same result. The acidic hydrolysis of the dG-HNE adducts also showed that the four dG-HNE diastereoisomers were eluted in two couples of enantiomeric base moieties since dG-HNE 1 and 2 were hydrolyzed into dG-HNE-H+1, and dG-HNE 3 and 4 into dG-HNE-H+ 2. Similarly, the two diastereoisomeric adducts of 2/-deoxyguanosine with crotonaldehyde, pentenal, hexenal, and heptenal were hydrolyzed into one mixture of enantiomeric compounds, eluted in one single peak on RP-HPLC. In addition, the hydrolysis of the acrolein and 2-methylacrolein adducts generated a single product. The structure of dG-HNE-H+ and dG-Hex-H+ was confirmed by GC-MS analysis following silylation. Derivatization of dG-HNE-H+ provided the tri- and tetrasilylated compounds, which were both eluted as two peaks because of the separation of the diastereoisomers. The hydrolyzed dG-Hex was converted into its di- and trisilylated derivatives. The mass spectra of the tetrasilylated hydrolyzed dG-HNE (Figure 3.1) had a molecular ion at mlz = 595. Fragments corresponding to the loss of a methyl (M - 15) and a trimethylsilylated hexanol (M 173) group due to the fragmentation of the aldehyde side chain were observed. Similarly, the loss of a methyl (M - 151, an ethyl (M - 29), and a propyl (M - 43) group was observed for the trisilylated dG-Hex-H+ (Figure 3.2). For dG-HNE-H+, the fragment corresponding to the loss of a TMS-OH group (M- 901, characteristic of derivatized hydroxyl group, was followed by a further fragmentation with loss of a methyl (mlz = 490), a butyl (mlz = 4481, and a pentyl group (mlz = 434). No such result was observed with dG-Hex-H+,thus providing evidence that the TMSOH group lost in dG-HNE-H+ comes off the C-4 atom of the aldehyde. Another major ion for the dG-HNE-H+ adduct corresponds to the loss of 72 mass units, due to a rearrangement involving N-3 and the N-2 TMS group. It should be added that the resulting fragment underwent a further loss of a methyl group since a peak a t mlz = 436 (M - 87) was also observed. This rearrangement, also observed to a smaller extent for the disilylated hydrolyzed dG-Hex, does not occur in unmodified guanine because the N-2 TMS group can rotate and is thus unlikely to react with N-3. In contrast, the cyclic structure of the 1JP-propano adduct induces a stable conformation favorable to the reaction. The two compounds obtained by treatment of guanine with HNE were also found to be additional products of the unsaturated aldehyde. Their mass after silylation was 523 for the major derivatives, which corresponds to the addition of 3 TMS groups on a guanine/HNE adduct. However, the Gua-HNE adducts are different from the two dG-HNE-H+ diastereoisomers, as shown by different retention times on RP-HPLC and electrochemical properties (vide infra). Similar observations were made for Gua-Hex compared with dG-Hex-H+. Electrochemical Detection. The hydrodynamic electrovoltamograms of the modified 2'-deoxyguanosine and their hydrolyzed derivatives were determined for acrolein, 2-methylacrolein, crotonaldehyde, pentenal, hexenal, heptenal, HHE, and HNE. Figure 4 shows the dynamic electrovoltamogramsfor the HNE and hexenal derivatives. For a given type of adduct, the half-potential was found to be independent of the nature of the a,b-unsaturated
Chem. Res. Toxicol., Vol. 7,No. 4, 1994 515
HPLC-EC Aldehydes 1
PndaMWi850000
1
1.
450000
OTMS 0
1
I
M-Wl
800000 750000 700000 650000 600000
Mac3 450
550000
500000 250000.
450000 400000 200000~ 350000
300000 250000
hi+ 465
200000 150000 100000
50000
50000
0
pit--'
0
4:
It-->
260
280
300
320
340
360
380
400
420
440
460
Figure 3. Mass spectra of (1)the tetraderivatized (TMS)hydrolyzed dG-HNE; (2) the triderivatized hydrolyzed dG-Hex. GC-MS conditions are described in the experimental part.
300
400
500
600
700
800
500
600
700
800
potential (mV)
25
-. 300
400
potential (mV) Figure 4. Electrovoltamograms of the (1)HNE and (2) hexenal derivatives: (a) guanine adducts; (b) dG adducts; (c) hydrolyzed dG adducts. HPLC conditions are described in the experimental part.
aldehyde, and no difference was found for the different diastereoisomers of a same adduct. The half-wave potential of the 2/-deoxyguanosineadducts was found to be in the 730-750-mV range. Hydrolysis was found to decrease the oxidation potential of modified 2j-deoxynucleosides to between 440 and 470 mV. It should be noted that two HPLC systems were used for the study of the electrochemical properties of dG-HNE-H+ and dGhex-H+, showing a drastic solvent effect on the electrochemical properties. When 35% methanol was used in the HPLC eluent ,the half-oxidation wave potential was found to be 80 mV higher than with 20% acetonitrile. It is also worth mentioning that the half-oxidation wave potentials of the two adducts obtained by direct reaction of HNE and hexenal with guanine were found to be around 900 mV.
Figure 5. HPLC-EC chromatograms of hydrolyzed calf thymus DNA (1)treated with hexenal(20 pg); (2) treated with HNE (50 pg). HPLC conditions are described in the experimental part.
Measurements in Calf Thymus DNA. Samples of calf thymus DNA were incubated for several days with HNE and hexenal. The excess aldehyde was efficiently removed by lyophilization. The DNA was recovered by precipitation with an excellent yield (>95%)and hydrolyzed into bases by using 0.1 N HC1 for 2 h at 90 "C. The hexenal-treated DNA was also hydrolyzed by using HF/ pyridine. The following prepurification step took advantage of the high hydrophobicity of the 1P-propanoguanine adducts. They were retained on a CIS silica gel cartridge that was washed with water and a mixture of water and methanol to remove the more polar compounds. The adducts were quantitatively recovered by fractions of pure methanol, as shown by the use of radiolabeled dG-HNE-H+ standards. The prepurified samples were analyzed by HPLC-EC (Figure 5). An identical procedure was used for the DNA extracted from rat kidney and liver.
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The amount of material in each injection corresponds to 500 pg of hydrolyzed DNA. The amount of hexenal-treated DNA in each injection was around 20 pg, and each measurement was duplicated for each of the acidic treatments. It is worth mentioning that injection of the equivalent of 2 pg of hydrolyzed DNA still allowedan easy detection of the adducts. The amounts of the hexenal adducts, determined by comparison with a known amount of standard (200 pmol) in each of the HC1 (results of two injections: 138 and 132 pmol) and HF/pyridine (160 and 148pmol) hydrolyzed samples, were very close, corresponding to an average value of 1 modification for 95 guanine residues. The measurement of the adducts was also made by GC-MS. A calibration curve in the 5-50 fmol of detected product range (r2 = 0.945, n = 4) was determined. The measurement of hydrolyzed hexenal-treated calf thymus DNA (4 GC-MS determinations, varying between 12 and 26 fmol detected) corresponded to a modification rate of 1/110 guanine residues (10 pmol for 400 pg of DNA), consistent with the result obtained by HPLC-EC. The HPLC-EC analysis of the HNE-treated DNA hydrolyzed by HC1 was made by two injections of 50 pg of hydrolyzed DNA. The amounts of each of the two couples of adducts were determined by using a calibration curve in the 5-150-pmol range (r2= 0.998 for both couple of adducts, n = 6) to be 173 and 112 pmol and 179 and 119 pmol, respectively. This corresponds to one modification for 125 guanine residues.
Discussion Formation and Hydrolysis of the Nucleoside Adducts. The observation of the formation of the 1,Wpropano adducts of 2‘-deoxyguanosine with acrolein, crotonaldehyde, pentenal, hexenal, HNE, and HHE was in agreement with published data (17,18, 23, 25, 26). 2-Methylacrolein was recently reported to generate the two types of 1,W-propano adducts similar to those of acrolein (25). However, only one major product was isolated in this work, likely because the second type of adduct is produced in a much lower amount because of the steric hindrance of the methyl group. No data were available about the heptenal 1,W-propanoguanine adduct, and its similar behavior to that of the lower analogs (acrolein, crotonaldehyde, pentenal, and hexenal) provides further evidence that the ability of an a,p-unsaturated aldehyde to generate 1,W-propanoguanine adducts is not dependent on the length of its side chain. The modified base moieties were released from the nucleoside or the DNA by acidic treatment, using either hot HC1 or HF/pyridine. The latter system was recently reported to allow the hydrolysis of modified DNA components under milder conditions than the classical hot acidic treatment (27,28). The observation of similar results for the hydrolysis of the dG-HNE products and the hexenal-treated DNA by using either HCl or HF/ pyridine strongly indicates that no degradation of the adducts occurred even under high-temperature acidic conditions. This allows one to use the HC1 treatment, which is easier and safer than HF/pyridine hydrolysis, for quantitative release of the 1,W-propanoguanine adducts from DNA. The incubation of guanine with the two unsaturated aldehydes generated compounds different from that obtained by hydrolysis of the 2’-deoxyguanosine derivative,
Douki and Ames
as shown by a difference of chromatographic behavior and of electrochemical properties. No such result was found in the literature since the 1,W-propanoguanine adducts are prepared by hydrolysis of the nucleoside derivatives (17,18,26).No further characterization of Gua-HNE and Gua-Hex was made because they are much less biologically relevant than the 2’-deoxyguanosine derivatives. Indeed, the reactivity of the guanine moiety is very similar in the nucleoside and in DNA because the N-9 position is substituted by a deoxyribose ring in both molecules. However, it should be mentioned that 7,8-cyclic adducts were described as minor products of the reaction of 2ldeoxyguanosinewith some unsaturated aldehydes (25,26). Guanine may preferentially react with HNE and Hex through this mechanism in contrast with 2‘-deoxyguanosine because of the difference of subsitution of the N-9 position that dramatically modifies the reactivity of the purine ring. HPLC-EC.The use of high-performance liquid chromatography coupled with an electrochemical detection in the oxidation mode was successfully applied to the detection of modified nucleosides (29-31). This technique is sensitive and can be highly specific for compounds with low oxidation potentials. HPLC-EC detection was recently applied to the measurement of the malondialdehyde pyrimidopurinone adduct following partial reduction of the adduct to generate an N-2 amino group (32). In a 1,W-propanoguanine adduct, the N-2 position is already a secondary ene-amine, which makes the compound electroactive in a range compatible with an electrochemical detection without required preliminary modification. This is shown by the relatively low oxidation potential of 730 mV observed for all the unsaturated aldehyde studied in this work. Hydrolysis of the modified nucleoside induced a decrease of this half-oxidation wave potential to approximately 450 mV. This property of bases compared to nucleoside, also reported for the MDA adduct (321,can be explained by the formation of an extra oxidizable NHgroup on position N-9. It is likely that all 1,W-propano adducts generated by reaction of an a,p-unsaturated aldehyde with 2’-deoxyguanosine could be detected by this HPLC-EC assay. Indeed, the 1,W-propanoguanine structure seems to be the main requirement for an electroactivity compatible with an HPLC-EC detection, as shown by the identical electrochemical properties of the series of adducts studied in this work. A substitution of either the C-4 (HNE,HHE), C-2 (2-methylacrolein), or C-3 (acrolein compared to the higher analogs) atoms of the a,@-unsaturated aldehyde has no effect on the electrochemical properties. Because of the higher electroactivity of the base derivatives versus their 2‘-deoxyguanosine precursors, the acidic hydrolysis releasing the base was preferred to an enzymatic digestion providing the nucleosides. Moreover, for HNE and HHE, the isomers of the bases are eluted in two peaks instead of four for the nucleoside. Similarly, the two diastereoisomeric 2’-deoxyguanosine adducts obtained with crotonaldehyde, pentenal, hexenal, and heptenal were separated on RP-HPLC whereas only one peak was obtained with the corresponding bases. Consequently, for the same amount of lesion, the detection of the base is twice as sensitive as the nucleoside. I t should also be noted that the use of two electrodes in the electrochemical cell allowed a preliminary oxidation of the eluted hydrolysis mixture at 350 mV that considerably decreased the signal noise before the 530-mV detection. The high hydropho-
Chem. Res. Toxicol., Vol. 7, No. 4, 1994 517
HPLC-EC Aldehydes
]I
ll
I
3
4
Figure 6. HPLC separation of (1) 1,W-propano-dG adducts and (2) their hydrolyzed products for 1: acrolein, 2: crotonaldehyde, 3: pentenal, 4 hexenal, and 5: heptenal,; RP-HPLC, gradient from 5% to 20% acetonitrile in 25 mM ammonium formate; UV detection 260 nm.
bicity of the 1,W-propanoguanine adducts is another advantage since it allows an efficient separation of the adducts from the polar hydrolysis byproducts and unmodified bases, both in the prepurification step and on the RP-HPLC system. Altogether, these features allowed a very low background signal in the part of the HPLC-EC elution profile corresponding to the hexenal- and HNE1,W-propanoguanine adducts, even when amounts as high as 500 pg of hydrolyzed DNA extracted from tissues were injected. The detection limit was estimated to be around 1pmol, corresponding to the smallest integratable peak. The calibration curve for the two couples of HNE adducts was linear in the 1-150-pmol range, which is indicative of a high reliability of the technique. These results should make possible the measurement of a modification rate of 1/105guanine residues in 150-200 pg of DNA. The HPLCEC assay could thus be a powerful tool for the study of the mutagenicity of this class of highly reactive compounds since the homologous series of the a,@-unsaturatedaldehydes were efficiently separated by RP-HPLC (Figure 6). Measurements in DNA. Incubation of isolated calf thymus DNA with both hexenal and HNE resulted in the formation of the corresponding 1,W-propanoguanine adducts. Their detection using the HPLC-EC assay represents the first identification of these adducts in DNA. The extensive study of the formation of the 1,Wpropanoguanine adducts of hexenal in calf thymus DNA i n vitro showed the reliability of the method, using either HC1 or HF/pyridine DNA hydrolysis. Both GC-MS and HPLC-EC techniques were used and provided results in the same range (1.05% against 0.9% of modified guanine for the HPLC-EC and GC-MS, respectively). No attempt to further improve the GC-MS assay (use of an internal standard, optimization of the parameters) was done since the HPLC-EC technique appeared to be sensitive without requiring any derivatization step and is thus simpler to
use and more reliable. In addition, a very high amount of DNA could be injected, in contrast with the limited injection volume in the GC-MS technique. The HNE-1,W-propanoguanine adducts were not detected in rat liver or kidney DNA, in spite of a high amount of nucleic acid analyzed (500 pg per injection). An upper limit for the modification rate of 2 fmol/pg of young rats DNA (1 modified guanine for lo6 bases) can thus be determined. For comparison, the amount of 8-oxo-7,8dihydroguanine, a major product of oxidative stress, in rat liver DNA is approximately 10fmol/pg (33). However, the detection limit of the assay should be at least 1order of magnitude more sensitive to allow us to rule out the biological relevance of the 1,W-propanoguanine-HNE adducts. It would also be interesting to study the involvement in HNE mutagenicity of other DNA lesions, such as 2,3-ethenoguanine generated in vitro by incubation of isolated DNA with 2,3-epoxy-4-hydroxynonenal,a possible HNE metabolite (34, 35). Other mutagenic mechanisms such as the formation of cross-links with proteins through a HNE molecule could also be involved, as suggested by the observation of a higher yield of DNAprotein cross-links with unsaturated aldehydes by comparison with their saturated analogs (36).
Acknowledgment. This work was supported by National Cancer Institute Outstanding Investigator Grant CA39910 and by the National Institute of Environmental Health Sciences Center Grant ES01896. We thank Dr. Helen Yeo for her critical comments on the manuscript. References (1) Foiles, P. G., Akerkar, S. A., and Chung, F.-L. (1989) Application
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