Chem. Res. Toxicol. 1990, 3, 102-110
102
32P-Postlabeling Measurement of Adenine N-l-Oxide in Cellular DNA Exposed to Hydrogen Peroxide J. F. Mouret, F. Odin, M. Polverelli, and J. Cadet* Laboratoires de Chimie, Dgpartement de Recherche Fondamentale, Centre d’Etudes Nucleaires de Grenoble, 85 X,F 38041 Grenoble Cedex, France Received October 31, 1989
A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-l-oxide, the specific H202-mediatedoxidation product under nonradical conditions. This has required the chemical synthesis of both 2‘-deoxyadenosine N-l-oxide 3’-monophosphate and 2’-deoxyadenosine N-l-oxide 5‘-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order t o improve the rate of phosphorylation and t o prevent self-radiolysis processes. [32P]-2’-deoxyadenosine N-l-oxide 5’-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close t o 1 modified adenine N-l-oxide/106 bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H202.
Introduction It is now generally accepted that oxidative DNA’ damage involving both base modification as well as oligonucleotide strand breaks is implicated in various deleterious biological effects including mutagenesis, carcinogenesis, and lethality (1-4). However, the determination of radical oxidative pyrimidine and purine lesions within cellular DNA remains a challenging problem. Various approaches have been used for monitoring these types of lesions. In particular, attempts were made to develop radioimmunological assays (RIA), which are in most cases applied to whole DNA. Polyclonal antibodies have been raised against thymine glycol (5-7), 5-(hydroxymethy1)uracil (8),7,8-dihydro-8-oxoadenine (9), and 8,5’-cycloadenosine 5’-monophosphate (10). However, the main limitation of these serological assays is their relatively low sensitivity. One major exception deals with a monoclonal antibody that was reported to be able to detect 1thymine glycol/106 thymine residues (11, 12). The use of endonucleases may represent an alternative when considering whole DNA (13). However, this approach suffers from a lack of specificity. For example, endonuclease I11 has been found to recognize a wide variety of nucleobase lesions, including thymine glycol, 5-hydroxy-5-methylhydantoin, and urea (14-16) as well as unidentified oxidized purine nucleobases ( I 7). A more general approach is based on chromatographic analysis of modified DNA components following digestion of the DNA by either acidic hydrolysis of the N-glycosidic bond of the modified bases or enzymatic cleavage of phosphodiester bonds with a cocktail of exonucleases and phosphatase. Several improvements were made in both the specificity and the sensitivity of detection techniques using high-performance liquid chromatography (HPLC) analysis. Electrochemical detection was shown to be a sensitive and specific tool for monitoring the formation of 8-hydroxy-2’-deoxyguanosine in cellular DNA exposed to various oxidants (18-22). The limit for detection of this compound was found to be less than 0.1 ng. Another
* To whom correspondence should be addressed. 0893-228~/90/2703-0102$02.50/0
interesting method is capillary gas chromatography-mass spectrometry, which was successfully applied for the study of various radiation-induced DNA base damages (23-25). However, the GC-MS method, which requires derivatization of the modified bases at high temperatures, may not be able to detect thermolabile products. Radioactivity detection requires the incorporation of 3H- or 14C-radiolabeled nucleobase or nucleoside in the DNA of cultured cells. However, one main limitation of this approach when applied for the detection of oxidative type damage deals with self-radiolysis processes, which are expected to generate the same DNA degradation products as those resulting from exposure to reactive oxygen species. This was illustrated by the presence of a significant amount of thymine glycol in [3H]thymineDNA of nonirradiated living cells (26). The yield of thymine glycol, as measured by the acetol assay ( 2 3 , was found to represent 0.37 and 0.05% of the overall radioactivity of [3H]thyminein the DNA of Micrococcus radioduram and Chinese hamster V-79cells, respectively. As an alternative to all these methods, the 32P-postlabeling assay reported a few years ago by Randerath et al. (28,29) appears to be a promising technique for monitoring DNA base lesions in cellular DNA. This assay consists of an enzymatic digestion of the modified DNA, leading to the formation of nucleoside 3’-monophosphates. In a Abbreviations: DNA, deoxyribonucleic acid; RNA, ribonucleic acid; HPLC, high-performanceliquid chromatography; TLC, thin-layer chromatography; PEI, poly(ethy1enimine); GC-MS, capillary gas chromatography-mass spectrometry; NMR, nuclear magnetic resonance; TSP, 3-(trimethylsilyl)[ 2,2,3,3-*H4]propionate;FAB, fast atom bombardment; ATP, adenosine 5’-triphosphate;bicine, N,iV-bis(2-hydroxyethyl)glycine; dANOX, 2’-deoxyadenosineN - I-oxide; DMT,, 4,4‘-dimethoxytriphenylmethyl; DMTdANOX, 5‘-0-(4,4’-dimethoxytriphenylmethyl)-2’-deoxyadenosine N-l-oxide; 5‘-dAMP-NOX, 2’-deoxyadenosine N-l-oxide 5’monophosphate; 5’-dTMP, thymidine 5’-monophosphate;5’-dGMP, 2‘deoxyguanosine 5’-monophosphate;5‘-dCMP,2’-deoxycytidine5’-mOnOphosphate; 5’-dUMP, 2’-deoxyuridine 5’-monophosphate;5‘-dAMP, 2’deoxyadenosine 5’-monophosphate;5-Me-5’-dCMP, 5-methyl-2’-deoxycytidine 5’-monophosphate; 3‘-dNps, thymidine 3’-monophosphate (3’dTMP), 2’-deoxyguanosine 3’-monophosphate (3’-dGMP), 2’-deoxycytidine 3’-monophosphate (3’-dCMP),and 2’-deoxyadenosine 3’-moncphosphate (3’-dAMP); 3’-dAMP-NOX, 2’-deoxyadenosine N-l-oxide 3’monophosphate; EDTA, ethylenediaminetetraacetic acid disodium salt.
0 1990 American Chemical Society
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 103
Formation of Adenine N-1-Oxide in DNA Scheme I. Experimental Procedure for Standard (37) and Adapted 32P-PostlabelingAssaya
Microccocal nuclease Phosphodiesterase I1
R
I
R
Figure 1. Formation of adenine N-1-oxide by oxidation of adenine with hydrogen peroxide.
1
Nucleoside-3‘-monophosphates (dNP + dXP)
.
and on the u n a m b i g u o u s characterization of the a d e n i n e N-1-oxide. It should be noted that this compound is specifically produced b y oxidation of DNA with hydrogen peroxide under nonradical conditions ( 4 1 , 42).
Materials and Methods nucleoside-3’,5’-biphosphates 32pdNp + 32pdXp
dxp small amOunt of normal nucleotides
I
32P ATP
T4 kinase
nuclease
Pi
32pdX
1 +
32pdN
final purification steps * Partisil SAX 10 WLC C18 reverse phase WLC
1
32pdX
2D
1
separation
dNP indicates normal nucleotides (3’-dTMP, 3’-dCMP, 3’dGMP, 3’-dAMP), and dXp denotes modified 3’-nucleotides.
subsequent step, t h e resulting nucleotides a r e phosphor y l a t e d b y T 4 polynucleotide kinase i n the presence of [32P]ATP of high specific activity (Scheme I). T h i s method was initially applied to the detection of base damage i n d u c e d b y various polycyclic a r o m a t i c h y d r o c a r b o n s (29-32) and b y alkylating a g e n t s (33, 34). In addition, 5-methylcytosine, a relatively m i n o r nucleobase of DNA, and 5-bromouracil, an analogue of thymine, have also been q u a n t i t a t e d b y using the 32P-postlabeling assay (35-38). It should also be noted that t w o a t t e m p t s based on t h i s a p p r o a c h were recently made for the m e a s u r e m e n t of oxidized nucleobases. T h y m i d i n e glycol, m e a s u r e d as its 5’-monophosphate derivative, w a s shown to be f o r m e d upon exposure of thymidine 3’-monophosphate or isolated calf t h y m u s DNA to ionizing radiation (39). The second o n e d e a l t w i t h the d e t e r m i n a t i o n of the reactivity of t h e four DNA nucleoside 3’-monophosphates toward L-ascorbic a c i d i n t h e presence of hydrogen peroxide. It was f o u n d that under these conditions only 2’-deoxyguanosine 3‘m o n o p h o s p h a t e was significantly oxidized. The m a i n decomposition p r o d u c t following phosphorylation b y T 4 p o l y n u c l e o t i d e k i n a s e w a s t e n t a t i v e l y i d e n t i f i e d as [32P]-8-hydr~~y-2’-deoxyguanosine 3’,5‘-bisphosphate (40). In t h i s paper we r e p o r t an a d a p t a t i o n of t h e 32P-postlabeling assay (Scheme I) which allows the q u a n t i t a t i v e m e a s u r e m e n t of a d e n i n e N-1-oxide (Figure 1) within cellular DNA exposed to hydrogen peroxide. E m p h a s i s was placed on the quantitative aspect of the measurement
Chemicals. The 2’-deoxynucleoside 5’-monophosphates were purchased from Sigma (St. Louis, MO), and the related 3’monophosphates were obtained from Pharmacia (Uppsala, Sweden). 2’-Deoxyadenosine was from Genofit (Geneva, Switzerland). 4,4’-Dimethoxytriphenylmethylchloride was obtained from Aldrich Chemie (Milwaukee, WI). 2-Mesitylenesulfonyl chloride and triethylamine were purchased from Fluka Chemie AG (Buchs, Switzerland). Anhydrous pyridine and 2-cyanoethyl phosphate (barium salt dihydrate) were from Merck (Darmstadt, West Germany). Radiolabeled [ Y - ~ ~ P ] A(3000 T P Ci/mmol) and 2’-deoxy[1’,2’,2,8-3H]adenosine 5’-triphosphate (69 Ci/mmol) were purchased from Amersham (Amersham, U.K.). Enzymes. Micrococcal nuclease was obtained from Sigma whereas proteinase K, RNase, lysozyme, and calf spleen phosphodiesterase were purchased from Boehringer (Mannheim, West Germany). Nuclease P1 was obtained from BRL (Bethesda, MD), and T 4 polynucleotide kinase was from New England Biolabs (Beverly, MA). HPLC Analysis. Several HPLC apparatus were used for both preparative and analytical separations: The first system consisted of a Model M6000A dual-piston pump and a R401 refractive index detector (Waters Associates, Milford, MA). Sample injection was carried out by using a Model 7125 Rheodyne valve (Berkeley, CA). The second system consisted of M6000A and M501 pumps, a system controller 721, a data module, Model 730 (Waters Associates), a variable UV wavelength spectrophotometer (Cecil Instrument, Cambridge, U.K.), and a fraction collector (Gilson, Middleton, WI). The third system consisted of 2 Model 201 pumps, a Model l l l B UV detector, a Model 811 dynamic mixer, a Model 702 gradient manager interfaced with an Apple IIe microcomputer, and a Model 201-102 fraction collector (Gilson, Middleton, WI). Partisil SAX 10 analytical columns, 10 pm (250/4.6 mm i.d.), were obtained from Whatman (Clifton, NJ). Semipreparative (300/7.5 mm i.d.) and analytical reverse-phase columns (250/4.5 mm id.) were home-packed with 10-pm Nucleosil octadecylsilyl silica gel (Macherey Nagel, Duren, West Germany). Two-dimensional thin-layer chromatography separations were carried out on silica DC-plastik folien Kieselgel60F254 (Merck, Darmstadt, West Germany). The solvents consisted of propan1-01, methanol, 20% ammonia, and water (45:15:30:10 v/v) for the first direction and 95% ethanol and 1 M ammonium acetate, pH = 7, (70:30 v/v) for the second development. Radioactivity was quantitated by using Beckman Ready-Solv scintillator fluid and a Beckman LS 2800 counter (Fullerton, CA). Localization of the radioactive spots were made by autoradiography using X-ray-sensitive films (Kodak, Rochester, NY). Spectrometer Analysis. ‘HNMR spectra were recorded a t 200.13 MHz (Bruker AM 200 spectrometer) and a t 400.13 MHz (Bruker AM 400 spectrometer) in deuterium oxide a t 20 “C. 3-Trimethylsilyl[2,2,3,3-2H4]pr~pionate was used as internal standard (TSP = 0.00 ppm). Assignments were checked by homonuclear double- or triple-resonance experiments. H(2’) and H(2”) were further assigned on the basis of coupling constant arguments (43). Spectral analysis (LAOCOON 111) and computersimulated spectra were the final test of the chemical shifts and coupling constants. Positive and negative ion fast atom bombardment (FAB) mass spectra (glycerol matrix) were obtained on a Kratos spectrometer,
104 Chem. Res. Toxicol., Vol. 3, No. 2, 1990 Model MS 50, equipped with a FAB gun 8-keV xenon atom. Preparation of 3‘- and 5‘-Monophosphates of %’-Deoxyadenosine N-1-Oxide. (A) 2’-Deoxyadenosine N-1-Oxide 3’-Monophosphate. ( 1 ) 5’-0-(4,4’-Dimethoxytriphenylmethyl)-2’-deoxyadenosineN-1-Oxide (DMTdANOX). 2’Deoxyadenosine N-1-oxide (500 mg) was dissolved twice in 20 mL of anhydrous pyridine. 4,4’-Dimethoxytriphenylmethylchloride (1.2 equiv, 760 mg) was added and dissolved in 20 mL of anhydrous pyridine. The reaction was held at 4 OC for 15 h and then stopped by addition of 50 mL of cold water. The reaction products were extracted twice with 150 mL of ethyl acetate, and the resulting solution was then dried by adding 1 g of sodium sulfate. The solution was evaporated to dryness, and the resulting products were separated on a preparative column (150/45 mm i.d.) filled with silica gel Bio-Si1 A, 100-200 mesh (Bio-Rad, Richmond, VA). The compounds were eluted with a step gradient of chloroform and methanol. Fractions of 200 mL were collected and evaporated to dryness. Fraction 6 (15% methanol) contained 745 mg of 5’-0-(4,4‘-dimethoxytriphenylmethyl)-2‘-deoxyaden~ine N-1-oxide (yield 70%): ‘H NMR (200.13-MHz, D20,TSP) 6 2.47 (m, 1 H, H-2’9, 2.94 (m, 1 H, H-29, 3.69 (m, 2 H, H-5’,5”), 3.72 (s, OCH3),3.74 (s, OCH3),4.13 (m, 1 H, H-4’),4.63 (m, 1 H, H-3’), 6.40 (t, 1 H, H-l’), 6.73 (m, trityl), 7.21 (m, trityl), 8.30 (s, 1 H, H-8), 8.44 (m, 1 H, H-2); FAB-MS m/z (relative intensity) (positive mode) 570 (20, [(M + H)’]), 303 (100, [(DMT, + H)+]),268 (7, [(dANOX + HI+]), 152 (70, [ ( B + H)+]). (2) 2’-Deoxyadenosine N-1-Oxide 3’-(Cyanoethyl monophosphate). DMTdANOX (580 mg) was dissolved twice in 5 mL of anhydrous pyridine. Cyanoethyl phosphate (960 mg) as the pyridinium salt and 1.04 g of 2-mesitylenesulfonyl chloride were added and subsequently dissolved in 5 mL of anhydrous pyridine. The reaction was held for 6 h at room temperature and then stopped by 500 pL of water. The dedimethoxytritylation was achieved by treating the residue with 3 mL of 80% acetic acid for 10 min, and then the acidic solution was evaporated to dryness. The resulting residue was dissolved in 20 mL of chloroform, and the phosphorylated compound was extracted by 20 mL of water (2X). HPLC purification, of the compounds subsequent to concentration was carried out by using an octadecylsilyl silica gel reverse-phase semipreparative column (300/7.5 mm) with water-methanol (97:3 v/v) as the eluent system at a flow rate of 3 mL/min. Evaporation to dryness of the combined fractions ( k ’ = 3.5) gave 200 mg of a homogeneous compound, as shown by TLC analysis, which was assigned as 2’-deoxyadenosine N-1oxide 3’-(cyanoethyl monophosphate) (yield 50%): ‘H NMR (200.13 MHz, D,O, TSP) d 2.92 (m, 2 H, H-2’,2”), 2.94 (m, CH,CH,O), 3.92 (m, 2 H, H-5’,5”), 4.17 (m, CH,CH,O), 4.43 (m, 1 H, H-4’), 5.07 (m, 1 H, H-39, 6.59 (t, 1 H, H-l’), 8.50 (s, 1 H, H-8), 8.66 (s, 1H, H-2); FAB-MS m/z (relative intensity) (negative mode) 399 (60, [(M - H)-]), 383 (20, [(M - H - O)-]), 150 (60, [(B - H)-I), 134 (25, [(B - H - O)-]), 97 (75, phosphate), 79 (100, phosphate). (3) 2’-Deoxyadenosine N-1-Oxide 3’-Monophosphate. 2’Deoxyadenosine N-1-oxide 3’-(cyanoethyl monophosphate) (100 mg) was dissolved in 10 mL of methanol saturated with ammonia, and the resulting solution was held a t room temperature for 15 h and then evaporated to dryness. The resulting compound was purified by using an octadecylsilyl silica gel reverse-phase HPLC semipreparative column (300/7.5 mm) with water as eluent at a flow rate of 3 mL/min. Evaporation to dryness of the combined fractions (k’ = 1) gave 65 mg of 2’-deoxyadenosine N-1-oxide 3’-monophosphate (yield 70%): ‘HNMR (400.13 MHz, D,O, TSP) 6 2.86 (m, 1 H, H-2”, J2’,3,= 6.2 Hz, J,,,,,, = -14.1 Hz), 3.04 (m, 1 H, H-2’, Jy,3, = 3.2 Hz), 3.92 (m, H-5”, J5,,5,r= -12.6 Hz), = 3.3 Hz, J4,,5c, = 4.8 3.95 (m, 1 H, H-5’), 4.43 (m, 1 H, H-4’, J48,5, Hz), 5.06 (m, 1 H, H-3’, J3’,48 = 3.1 Hz), 6.63 (t, 1 H, H-1’, J1c,2’ = 7.5 Hz, J,,,2,, = 6.2 Hz), 8.56 (s, 1 H, H-8), 8.70 (s, 1 H, H-2); FAB-MS m/z (relative intensity) (negative mode) 368 (4, [(M H + Na)-]), 241 (20),150 (24, [(B - H)-]), 134 (IO, [(B - H - O)-]), 97 (30, phosphate), 91 (18), 79 (100, phosphate), 63 (24), 59 (22). (B) 2’-Deoxyadenosine N-1-Oxide 5’-Monophosphate. 2’-Deoxyadenosine 5’-monophosphate (500 mg) was dissolved in 135 mL of 10 mM phosphate buffer (Tritisol, Merck) (pH = 7). An ethanolic solution of m-chloroperbenzoic acid (0.1 M; 135 mL) was added, and the solution was left a t 37 O C for 2 days. Subsequently to evaporation, the m-chloroperbenzoic acid was elim-
Mouret et al. inated by two extractions with 200 mL of diethyl ether. The reaction mixture was applied to an octadecylsilyl silica gel reverse-phase HPLC semipreparative column and subsequently eluted with water a t a flow rate of 2 mL/min. Evaporation to dryness of the fraction ( k ’ = 0.75) gave 330 mg of 2’-deoxyadenosine N-1-oxide 5’-monophosphate (yield 70%): ‘H NMR (200.13 MHz, DzO, TSP) 6 2.67 (m, 1 H, H-2’, JrS, = 6.2 Hz, J2‘,rj = -14.0 Hz), 2.90 (m, 1 H, H-2”, J2,,,3,= 3.6 Hz), 4.08 (m, 1 H, H-5’, Js,y= -11.0 Hz), 4.08 (m, 1 H, H-5”), 4.34 (m, 1 H, H-4’, J4t,5r = J4,,5r! = 3.8 Hz), 4.79 (m, 1 H, H-3’, J3,,4‘= 2.9 Hz), 6.57 (t, 1 H, H-i’, J1,,23 = 7.0 Hz, Jit,p = 6.4 Hz), 8.62 (s, 1 H, H-8), 8.64 (s, 1 H, H-2); FAB-MS m/z (relative intensity) (positive mode) 392 (2.5, [M + H + 2Na)+]), 370 (1.5, [(M + H + Na)+]), 354 (0.5, [(M + H - 0 + Na)’]), 207 (16, [(SGLY + Na)’]), 115 (loo), 93 (32, [(GLY)+]). Photolysis of 2’-Deoxyadenosine N-1-Oxide 5’-MOI10phosphate. A solution of 2’-deoxyadenosine N-1-oxide 5’monophosphate (35 mg/lO mL) in water was exposed to a UV source (254 nm) for 3 h. The resulting products were separated on an octyadecylsilyl silica gel reverse-phase HPLC semipreparative column in the ion-suppression mode with 0.05 M triethylamine acetate (pH = 7) and methanol (95:5 v/v) a t a flow rate of 3 mL/min. Evaporation to dryness of the fastest eluting compound (k’= 1.29) gave 7 mg of a homogeneous product which was assigned as 3-(5-monophospho-2-~-erythro-pentofuranosyl)-4-ureido-5cyanoimidazole (yield 20%): ‘H NMR (200.13 MHz, DzO, T S P b 2.61 (2 H, H-2’,2”), 4.11 (2 H, H-5’, H-5”), 4.25 (1H, H-4’), 4.72 (1 H, H-3’), 6.18 (1 H, H-l’), 8.10 (1 H, H-8); FAB-MS m/z (relative intensity) (negative mode) 346 (24, [(M - HI-]), 329 (16), 183 (40), 128 (loo), 97 (70, phosphate), 91 (98), 79 (54,phosphate), 59 (74). Evaporation to dryness of the combined fractions ( k ’ = 2.35) gave 10 mg of 2’-deoxyisoguanosine 5’-monophosphate (yield 28.5%): ‘H NMR (200.13 MHz, DZO, TSP) 6 2.66 (1H, H-29, 2.88 (1 H, H-2”), 4.11 (2 H, H-5’, H-5”), 4.32 (1H, H-4’), 4.77 (1 H, H-3’),6.54 (1H, H-l’), 8.57 (1 H, H-8); FAB-MS m/z (relative intensity) (negative mode) 346 (34, [(M - H)-]), 275 (14), 183 (loo), 97 (48, phosphate), 91 (98), 79 (34, phosphate), 71 (42), 59 (84). The slowest eluting compound ( k ’ = 6.59) was identified as 2‘-deoxyadenosine 5’-monophosphate (2.4 mg, yield 6.7%) by comparison of the ‘H NMR and FAB spectroscopic features with those of the authentic sample. Enzymatic Digestion of Highly Modified DNA. Nicktranslated [ 1’,2’,2,&3H]-2’-deoxyadenosineDNA (BRL) was mixed with 15 pg of unlabeled DNA in 180 pL of phosphate buffer (Tritisol) (pH = 7). This DNA (5 pg, 60 pL) was oxidized for 10 min or 1 h with 60 pL of a 0.1 M ethanolic solution of mchloroperbenzoic acid. The reaction was stopped by precipitating the DNA with cold ethanol. The resulting precipitate was dissolved in 20 pL of water and dialyzed against water for 1 h by using a VM 0.05-pm filter (Millipore). Subsequently, 2.5 pg of DNA was enzymatically digested with 1 pL (15 units) of micrococcal nuclease and 2 pL (0.4 units) of speen phosphodiesterase in the presence of 20 mM sodium succinate (pH = 6) and 10 mM CaCl, (final volume 20 pL). The reaction was carried out a t 37 “C for 2 h and was then stopped by ethanolic precipitation. The supernatant was evaporated to dryness and analyzed by using a reverse-phase analytical column (250/4.5 mm) with an analytical gradient of 0.05 M triethylamine acetate (pH = 7) and methanol (vide infra). In Vivo Modification of DNA. Proteus mirabilis (wild-type) cells were cultivated overnight (44) in the presence of 0.08% of caseine hydrolysate. The medium was eliminated by centrifugation at lOOOOg, and the pellet was washed twice with 50 mL of 0.1 M phosphate buffer (pH = 7.5). The bacterial cells (1 OD at X = 600 nm) were then resuspended in 150 mL of 0.1 M phosphate buffer, pH = 7.5. The cells were exposed to 10 mM hydrogen peroxide at 4 “C for 30 min. The reaction was stopped by centrifugation at loooOg for 15 min, and the pellet was washed twice with 50 mL of phosphate buffer. Isolation of Cellular DNA. DNA was isolated according to the procedure described by Marmur (45) with the following modifications. The bacterial cells were killed by osmotic shock using 3 mL of 0.5 M saccharose, 0.3 M Tris-HC1 (pH = 7.5), and 6 mL of cold water; 0.2 mL of 0.25 M EDTA and 20 mg of
Formation of Adenine N-1-Oxide in DNA lysozyme were then added, and the reaction was held at 37 “C for 1h. The proteins were denatured (30 min at 37 “C) by using sodium dodecyl sulfate (final concentration 1%) in standard sodium citrate buffer and then digested with 25 pL of proteinase K (200 mg/mL). RNA was digested with 25 p L of RNAse (10 mg/mL). DNA was extracted twice with a mixture of isoamyl alcohol and chloroform (19:l v/v). DNA recovery from 1OD of bacterial cells averaged 0.1 mg. DNA concentrationwas estimated spectrophotometrically in water by using 20 A260 units/mg for native DNA as the standard (Azso/A2m =. 1.7). 32P-Postlabeling Analysis of Adenine N-Oxide. The modified DNA (1pg) was digested as described below, and then the 2-deoxyadenosine N-1-oxide 3‘-monophosphate was separated from the other 3’-dNps using a C18reverse-phase HPLC analytical column with a methanolic gradient in 0.05 M triethylamineacetate (pH = 7) (0-5 min 0%, 5-25 min l o % , 25-35 min 10%). Appropriate fractions containing the 2’-deoxyadenosine N-1-oxide 3’-monophosphate ( k ’ = 5,04) were pooled and lyophilized t o eliminate the buffer. To 10 pL of 2‘-deoxyadenosine N-1-oxide S‘-monophosphate were added 5 p L of [32P]ATP(50 pCi), 2 p L of lox kinase buffer (0.4 M bicine, 0.1 M MgC12, 1 mM spermidine, 0.1 mM dithiothreitol), and 1 pL (10 units) of T4 polynucleotide kinase. The reaction was held at 37 “C for 30 min, and then 6 p L of 1.7 M acetic acid, 84 p L of l x nuclease P1 buffer [30 mM sodium acetate (pH = 5.31,O.l mM ZnSO,], and 10 p L (10 units) of nuclease p l were added (1h at 37 “C). As a control, a known amount (3 pg) of thymidine 3’,5’-bisphosphatewas digested by the nuclease P1 in order to follow t,he quantitative liberation of thymidine 5’monophosphate. Purification of the [32P]-2’-deoxyadenosineN-1-oxide 5’monophosphate from [32P]ATPwas performed by using a Partisil SAX 10 analytical column, with a gradient of 0.6 M NaHzP04 (pH = 4) in water (0 min 2 % , 0-5 min 2 % , 5-30 min lo%, 30 min 100%). Thanks to the presence of UV-detectable levels of unlabeled 2‘-deoxyadenosine N-1-oxide 5’-monophosphatethe appropriate fraction was collected (k’= 5.93) and evaporated to dryness. The resulting compound was purified on a octadecylsilyl silica gel reverse-phase HPLC analytical column. The analysis was performed by using a methanolic gradient in 0.05 M triethylamine acetate (pH = 7) (0-25 min 10% methanol, 30-40 min 20% methanol). The 32P-radiolabeledproduct (k’ = 3.08) was finally lyophilized. In order to assign the structure of the purified compound, a known amount of 2‘-deoxyadenosine N-1-oxide 5‘-monophosphate (50 pg) was added and exposed for 1 h to a 254-nm UV source (final volume 3 mL). Subsequent to evaporation the reaction products were analyzed by two-dimensionalthin-layer chromatography using propan-1-01,methanol, 20% ammonia, and water (45:15:30:10v/v) in the first dimension and 95% ethanol and 1 M ammonium acetate (pH = 7) (7030v/v) in the second direction.
Results and Discussion The syntheses of 2’-deoxyadenosine N-1-oxide 3’monophosphate and of the corresponding 5’-monoester derivative were carried out in order to facilitate the search and quantitative analysis of adenine N-1-oxide in cellular DNA. Quantitative Measurement of Adenine N-1-Oxide. Synthesis of Modified Nucleoside 3’- or 5’-Monophosphates. 2’-Deoxyadenosine N-1-oxide 3’-monophosphate was obtained by using the cyanoethyl triester method starting from the corresponding modified nucleoside (46). The compound, which was purified on a octadecylsilyl silica gel reverse-phase HPLC column, was unambiguously assigned on the basis of FAB mass spectrometry analysis as well as by ‘H and 31PNMR measurements. The FAB-MS spectrum in the negative mode shows a notable pseudomolecular ion at m/z 368 [(M - H + Na)-]. This is indicative of the gain in mass of 102 amu corresponding to the incorporation of a phosphate group. In addition, the presence of peaks at m / z 79 and 97 is
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 105 characteristic of a phosphate group. 2’-Deoxyadenosine N-1-oxide 5’-monophosphate was prepared by specific oxidation of 2’-deoxyadenosine 5’monophosphate with m-chloroperbenzoic acid (47). The structure of the 2’-deoxyadenosine N-1-oxide 5’-monophosphate was assigned on the basis of FAB mass spectrometry and ‘H NMR. The FAB mass spectrum in the positive mode shows a pseudomolecular ion at m/z 370 [ (M + H + Na)+]. The gain of 16 amu in the molecular weight of the starting nucleotide is indicative of the presence of an additional atom of oxygen. In addition, the ion at m/z 354 [(M H - 0 Na)+] corresponding to the loss of an oxygen atom from the molecular ion is diagnostic for a N-oxide structure (48). The main difference in the NMR spectrum with the 2‘-deoxyadenosine N-1-oxide 3’-mOnOphosphate consists of a downfield shift for H-5’ and H-5” (0.22 ppm) and an upfield effect for H-3’. In addition, the presence of an additional scalar coupling (J= 7 Hz) in the H-5’,5’’ pattern provides further support for the presence of a phosphate group. Enzymatic Digestion of a Highly Modified DNA. The first requirement for the quantitative measurement of the adenine N-1-oxide was the complete enzymatic digestion of the modified biopolymer. For this purpose [ 1’,2’,2,8-3H]-2’-deoxyadenosine-labeled DNA was oxidized by m-chloroperbenzoic acid. The extent of the micrococcal nuclease and spleen phosphodiesterase digestion was based on the quantitation of the resulting 3’-nucleotide and nondigested DNA contained in the supernatant and ethanol precipitate, respectively. The 3’-nucleotide was found to represent 96.9% of the total radioactivity in the control experiment and 96.6% for the DNA exposed 10 min to m-chloroperbenzoic acid. It should be noted that under these oxidation conditions 3.5% of [ 1’,2’,2,8-3H]-2’deoxyadenosine N-1-oxide 3’-monophosphate was generated. These data strongly suggested that the enzymatic digestion of an exposed DNA containing a very low amount of adenine N-1-oxide (less than 1 X should be complete. Elimination of the 3’-Phosphate by the P1 Nuclease. The oxidized and normal nucleotides were found to be better resolved as nucleoside 5’-monophosphates rather than as the 3‘,5‘-bisphosphate derivatives. The 5‘-nucleotides were obtained after elimination of the 3‘-phosphate group from the 3’,5‘-bisphosphate derivatives using nuclease P1. The reaction was shown to be quantitative when synthesized [ 1’,2’,2,8-3H]-2’-deoxyadenosine N-loxide 3‘,5‘-bisphosphate was used as the substrate. This labeled compound was obtained by first synthesizing [1’,2’,2,8-3H]-2’-deoxyadenosine N-1-oxide 3’-monophosphate. This nucleotide was diluted with known 1 pg of the corresponding unlabeled nucleotide and phosphorylated in the presence of a large amount of nonradioactive ATP. C,, RP-HPLC of the products of the reaction between nuclease P1 and [ 1’,2’,2,8-3H]-2’-deoxyadenosine N-1-oxide 3‘,5’-bisphosphate revealed a single product, namely, [ 1’,2’,2,8-3H]-2’-deoxyadenosine N-1-oxide 5’monophosphate. Quantitative Phosphorylation by T4 Polynucleotide Kinase. (A) Choice of the Phosphorylation Conditions. Another important prerequisite for the quantitative determination of adenine N-1-oxide is the enzymatic phosphorylation mediated by T4 polynucleotide kinase. Various approaches have been previously used to enhance the rate of phosphorylation. The addition of an excess of nonradioactive ATP (29) was found to lead to a quantitative phosphorylation. However, the decrease of the specific activity of the [ T - ~ ~ P ] A T reduces P the sen-
+
+
106 Chem. Res. Toxicol., Vol. 3, No. 2, 1990
Mouret et al.
Table I. Phosphorylation Yield of Various %’-Deoxynucleoside3’-Monophosphates
3:dTMP
% of phosphorylation” nucleoside 3’-monophosphate 1 ng 1OOpg 2’-deoxycytidine 3’-monophosphate 3.16 29.7 thymidine 3’-monophosphate 0.90 21.7 2’-deoxyadenosine 3’-monophosphate 1.78 13.5 2‘-deoxyguanosine 3’-monophosphate 1.19 3.4 2’-deoxyadenosineN-1-oxide 3’-monophosphate 1.74 7.5
3‘-dGMP
1
/
/
a Theoretical cpm/experimental cpm. Theoretical cpm was obtained for 1 ng of 2’-deoxyadenosineN-l-oxide 3’-monophosphate as follows: 1 ng = 2.88 X M = 8.64 X 10“ Ci (ATP: 3000 Ci/mmol) = 19.18 X loe cpm (1 Ci = 2.22 X 10l2cpm).
-6
-5
Figure 3. HPLC elution profile of the separation of 3‘-dAMPNOX from a mixture of normal nucleotides (3’-dTMP,3’-dCMP, 3’-dGMP, 3’-dAMP). The separation was achieved by using on an octadecylsilylsilica gel reverse-phase HPLC analytical column and an appropriate methanolic gradient in 0.05 M triethylamine acetate (pH = 7 ) (0 min 0% MeOH, 5-25 min 10%).
-4
-3
-2
3
4
5
6
7 log (cpml
Figure 2. 32P-postlabelingdetermination of a known amount of
3’-dAMP-NOX: standard calibration curve (-); determination
of adenine N-l-oxide standards in the presence of cellular DNA (---). Each bar represents the range of three independent de-
terminations.
sitivity of the assay. Another possibility is to use radioactive ATP of higher specific activity (9000 Ci/mmol instead of 3000 Ci/mmol) (30). Under this “deficient” condition the DNA-carcinogen adducts were found to be labeled to a greater extent than the normal nucleotides. In the present work, in order to improve the phosphorylation efficiency, the phosphorylation of 2’-deoxyadenosine N-l-oxide 3’-monophosphate by the T4 polynucleotide kinase was investigated. (B) Phosphorylation of Nucleoside 3’-MOnOphosphates. 2’-Deoxyadenosine N-l-oxide 3’-mOnOphosphate (1 ng or 100 pg) was enzymatically phosphorylated, and the yields were compared with those obtained following similar reaction using the four normal nucleoside 3’-monophosphates (Table I). These results show a notable difference in the phosphorylation rate, consistent with previous observations (35). It is interesting to note that, for 1 ng, the 2’-deoxyadenosine N-l-oxide 3’-monophosphate was phosphorylated with similar efficiency to all the normal 3’-nucleotides. In addition, the phosphorylation yield was found to be increased by reducing the amount of substrate. However, the phosphorylation efficiency was higher for cytosine and thymine nucleosides than for the three purine nucleotides. Therefore, the phosphorylation of the 2’deoxyadenosine N-l-oxide 3’-monophosphate was studied. Figure 2 demonstrates that, for less than 1 ng, the phosphorylation was linear over 3 orders of magnitude of substrate concentration. The slope of the curve can be considered as a characteristic of the reactivity of the T4 polynucleotide kinase for the substrate and should be different for each damaged or normal nucleotide.
(C) Quantitation of Adenine N-l-Oxide Isolated from DNA. To quantitatively determine the formation of adenine N-l-oxide within DNA, it was necessary to eliminate nucleoside 3‘-monophosphates without decreasing the amount of 2‘-deoxyadenosine N-l-oxide 3’monophosphate. To remove the normal nucleotides from the DNA-carcinogen adducts, different approaches have been previously used. One was based on the observed resistance of the DNA-carcinogen adducts to nuclease P1 (49) or to the 3’-phosphatase activity of the T4 polynucleotide kinase (32). Another took advantage of the lipophilic nature of these adducts (50). Neither of these assays can be applied to oxidized nucleotides (vide supra). Instead, we adapted a chromatographic technique that had been successfully used to separate methylated bases (33) and hydrophobic DNA-carcinogen adducts (51) from other 3’-nucleotides. Thus a chromatographic system using a volatile solvent was developed to allow the HPLC separation of the 2’deoxyadenosine N-l-oxide 3’-monophosphate from the other nucleoside 3’-monophosphates (Figure 3). The modified 3’-nucleotide was then phosphorylated by T4 polynucleotide kinase after complete elimination of the volatile solvent. In order to assist in the quantitative determination of adenine N-l-oxide within DNA, known amounts of 2‘deoxyadenosine N-l-oxide 3’-monophosphate standard (10, 100, or 1000 pg) were mixed with nonoxidized DNA. The radioactivity recovered after the 32P-postlabelingassay was used to plot the curve shown in Figure 2. Finding the same slope value indicated that this experimental protocol can be applied to the quantitative determination of adenine N-l-oxide within cellular DNA. However, the quantitative determination of high levels of DNA damage still remains a problem because of the low phosphorylation yield. In order to increase the amount of phosphorylated lesions, a dilution of the modified 3’-nucleotide should be achieved. Under these conditions the determination of a high level of DNA damage was expected to be quantitative. On the other hand, adenine N-l-oxide (3.2/106 bases) was found to be present in the DNA of untreated cells. This level of adenine N-l-oxide cannot be accounted for by self-
Formation of Adenine N-1-Oxide in DNA
Chem. Res. Toxicol., Vol. 3, No. 2, 1990 107 Column: Partisill0 SAX Eluent :NaH2P04 pH=4
0.2,
5:dCMP
‘
’1 /5’dUMP I
5’dAMP.NOX
8
0.1
Figure 4. Autoradiogram of the two-dimensional TLC separation of the reaction products of the 32p polynucleotide kinase mediated phosphorylation of the HPLC fraction containing 3’-dAMP-NOX.
radiolysis during sample processing due to the elimination of 2’-deoxyadenosine 3’-monophosphate prior to 32P phosphorylation of 2’-deoxyadenosine N-1-oxide 3‘monophosphate. It is more likely to result from autoxidative processes within the cell and/or during DNA extraction. Purification and Characterization of the Lesion. (A) Purification of 2’-Deoxyadenosine N-1-Oxide 5’Monophosphate. Two-dimensional thin-layer chromatography analysis of 32Ppolynucleotide kinase mediated phosphorylation of the HPLC fraction containing 2’deoxyadenosine N- 1-oxide 3’-monophosphate (Figure 4) shows the presence of several radioactive compounds. In order to remove the contaminating products, two successive purification steps were found to be necessary. These contaminants were identified as [ T - ~ ~ P I A T 2’-deoxyP, uridine 5’-monophosphate, 5-methyl-2’-deoxycytidine5’monophosphate, and 2’-deoxyguanosine 5’-monophosphate. The presence of these nucleotides is probably due to contamination of the initial HPLC fraction by the corresponding 3’-nucleotides. (1) Elimination of [32P]ATPUsing an Anion-Exchange HPLC Column. Two methods have been previously used to remove [32p]ATP. The method developed by Randerath (29) was based on the use of potato apyrase for destroying ATP. Unfortunately, this enzyme has a contaminating phosphatase activity which may lower the effective yield of 5’-nucleotides. The method developed by Bodell is based on the use of an anion-exchange column which retains ATP when 0.1 M NaH2P04is used as the solvent (38). Under these conditions the 5’-nucleotides are eluted but the ionic strength of the eluent is too high to be used for thin-layer chromatography, and the chromatographic resolution was poor. The separation of [32P]2’-deoxyadenosine N-1-oxide 5’-monophosphate from A T P and the self-radiolysis products of ATP was achieved by an anion-exchange HPLC analysis (Figure 5) using a known amount of 2’-deoxyadenosine N-1-oxide 5’-monophosphate as an internal standard. (2) Final Mapping of the DNA Damage. ?l?-Labeled DNA adducts have previously been separated from 32Plabeled normal bases by reverse-phase TLC (34) followed by PEI-cellulose TLC mapping (29). However, this analytical method was not effective for resolving oxidized [32p]nucleotides. HPLC separation, which has been shown to be suitable for analyzing [3P]DNA-carcinogen adducts (52,53), alkylated nucleotides (33,52), or base analogues (38),was used in the present work. The complete sepa-
0
20
10
30
50
6bmin.
Figure 5. Elimination of ATP and purification of 5’-dAMP-N0X using anion-exchange column (Partisil S A X 10). The compounds were eluted by using a gradient of 0.6 M NaH2P04(pH = 4) in water (0 min 2% NaH2P04,0-30 min lo%, 30 min 100%). Column :Cia Eluent :TEAA 0.05M p k 7 + Me0H
0.2
r ,5-dTMP
\
5’FHMdUMP dUMP C
\ aAMP-NOX
I
k
5‘dGMP
/ 5’-dAMP
0.1
I
0.
io
20
30
iomin.
Figure 6. Final HPLC purification of 5’-dAMP-NOX on an octadecylsilyl silica gel reverse-phase analytical column. T h e compounds were eluted by using an appropriate methanolic gradient of methanol in 0.05 M triethylamine acetate (pH = 7) (0-25 min 10% methanol, 30-40 min 20% methanol).
ration of 2’-deoxyadenosine N-1-oxide 5’-monophosphate was achieved by using reverse-phase HPLC in the ionsuppression mode (Figure 6). The final two-dimensional TLC separation (Figure 7) shows the presence of one predominant radiolabeled compound. (B) Characterization of 2’-Deoxyadenosine N-lOxide 5’-Monophosphate. A microreaction was used to assign the structure of the expected compound obtained after phosphorylation. This microreaction is based on the photolability of 2’-deoxyadenosine N- 1-oxide 5’-monophosphate, which leads to the formation of three main decomposition products (Figure 8). The three main photoproducts were characterized as 2’-deoxyisoguanosine 5’-monophosphate, 3-(5-monophospho-2-~-eryythro-pentofuranosyl)-4-ureido-5-cyanoimidazole,and 2’-deoxyadenosine 5’-monophosphate (Figure 9) on the basis of
Mouret et al.
108 Chem. Res. Toxicol., VoZ. 3, No. 2, 1990
Table 11. Adenine N-1-Oxide Induced by H202 in P. mira bilis control DNA exposed DNA 24 360 ppm 102 436 cpm radioactivity recovered” 3.2 26.6 adenine N-1-oxideb “Variations in the quantitative measurement of the adenine N1-oxide were found not to exceed 5-10% in the range of 1-10 fmol. Higher variations were observed when using different batches of polynucleotide kinase and [32P]ATP. However, a similar value for the formation of adenine N-1-oxide was obtained after considering the related calibration curve. Adenine N-1-oxide per lo6 base pairs.
Figure 7. Final two-dimensional TLC of 5’-dAMP-NOX after two HPLC purification steps.
5:dAMP
ureidonuclmtid@
0
5!d AMP-NOX
dR@
5-isodGMP
1
Figure 8. Two-dimensional TLC separation of the photolysis products of 5’-dAMP-NOX. S’-dAMP -NOX
IhS
I
I
Acknowledgment. We gratefully acknowledge the financial support of l’Or6al and Centre National d’Etudes Spatiales (Grant 89/1305). We thank Dr. Dan Levy for valuable discussions and critical reading of the manuscript.
1
in
N
“H2
1
2
superior to deproteinization using phenol, which increased the background level of adenine N-1-oxide by a factor of 5. This 32P-postlabeling assay can be applied to as little as 1pg of DNA for which the limit of detection was about 5 pg. The radioactivity recovered after the 32P-postlabeling assay and the corresponding amount of adenine N-1-oxide are listed in Table 11. The extent of DNA damage was calculated by using the DNA calibration curves shown in Figure 2. The amount of adenine N-1-oxide was found to be 8.3 times higher in the exposed DNA than in the control DNA. This large difference indicates that H202is able to induce the formation of a nonradical lesion within bacteria a t nonlethal doses. Thus the formation of adenine N-loxide can be used as a convenient way to monitor the action of hydrogen peroxide within cellular DNA. In addition, the high sensitivity and the low amount of DNA needed for the assay suggest that this procedure is available for monitoring human exposure to oxidative stress. Thus the present 32P-postlabelingassay should be adaptable to a wide variety of oxidized nucleobases. Work is in progress to study the formation of 5-(hydroxymethy1)uracil and 8-hydroxyguaninewithin cellular DNA exposed to various oxidizing agents.
3
Figure 9. Structure of the main W photodecomposition products of 2’-deoxyadenosine N-1-oxide 5’-monophosphate: (1) 2’Deoxyadenosine 5’-monophosphate, (2) 3-(2-D-erythrO-pentOfuranosy1)-4ureid&kyanoimidazole,and (3) 2’-deoxyisoguanosine 5’-monophosphate.
extensive FAB mass spectrometry and ‘HNMR measurements (data not shown). In addition, the corresponding nucleosides obtained by dephosphorylation with alkaline phosphatase showed identical FAB-MS and lH NMR features to those of the authentic compounds obtained by UV photolysis of the 2’-deoxyadenosine N-loxide (54-56). Application of the Postlabeling Assay to Cellular DNA. Wild-type P. mirtzbilis cells (44) were exposed to the action of 10 mM hydrogen peroxide under conditions previously described (21). The survival of the exposed cells to hydrogen peroxide was found to be 90%. To minimize the background (vide supra) occurring during extraction and purification of DNA, efforts were made to search for less oxidizing experimental conditions. The isoamyl alcohol-chloroform extraction procedure (45) proved to be
Registry No. 3’-dCMP, 6220-63-9; 3’-dAMP, 15731-72-3; 3’-dAMP-NOX, 125927-62-0; 3’-dTMP, 2642-43-5; 3’-dGMP, 6220-62-8; 5’-dAMP, 653-63-4; 5’-dAMP-NOX, 51785-75-2; DMTdANOX, 125950-27-8; dANOX, 3506-01-2; DMT,-C1, 40615-36-9; H202, 7722-84-1; adenine N-1-oxide, 700-02-7; 3-(2~erythro-pentofuranosyl)-4ureid~5-cyanoimidmle, 125927-63-1; 2’-deoxyisoguanosine 5’-monophosphate, 125972-63-6;2’-deoxyadenosine N-1-oxide 3’-(cyanoethyl monophosphate), 125950-143; cyanoethyl phosphate, 2212-88-6; polynucleotide kinase, 3721165-7.
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