Covalent Binding of Leukotriene A4 to DNA and RNA - Chemical

Chem. Res. Toxicol. 2003, 16, 551-561

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Covalent Binding of Leukotriene A4 to DNA and RNA Joseph A. Hankin,† David N. M. Jones,‡ and Robert C. Murphy*,†,‡ Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262, and Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206 Received February 7, 2003

Leukotriene A4 (LTA4) is a highly reactive electrophilic intermediate formed during the biosynthesis of the lipid mediators leukotriene B4 and leukotriene C4. Deoxynucleosides were found to react as nucleophiles with LTA4 in aqueous solutions as assessed by UV spectroscopy and electrospray ionization mass spectrometry. Aqueous solutions of native DNA and RNA were also found to react with LTA4 as assessed by mass spectrometric analysis of the constituent nucleosides derived from enzymatic hydrolysis of the nucleic acids. The most abundant adducts were observed for guanine- and adenine-containing deoxynucleosides and nucleosides. At neutral pH, these reactions led to an overall modification of deoxyguanosine/guanosine residues in DNA and RNA at 15 ( 1 adducts/107 bases and 230 ( 20 adducts/107 bases, respectively, determined by quantitative assay using stable isotope-labeled LTA4-nucleoside adduct. An estimation of the relative reactivity of LTA4 with each of the purine and pyrimidine bases in DNA and RNA was carried out by comparisons of the mass spectral ion abundance of the different adducts (LTA4-dAdo, LTA4-dCyd, LTA4-Thd, LTA4-Ado, LTA4-Cyd, and LTA4Urd) to the ion signal of known amounts of LTA4-dGuo and LTA4-Guo standards. The data were corrected for different mass spectrometric response factors that were experimentally determined for each adduct product. The structures of the two most abundant LTA4-Guo products were determined by NMR, UV spectroscopy, and mass spectrometry to be 5-hydroxy,12-[Guo-N2-yl]-6,8,11,14-eicosatetraenoic acid. Stimulation of human neutrophils with calcium ionophore led to the covalent modification of DNA within the cell as determined by mass spectrometric analysis of lipophilic nucleosides obtained after hydrolysis of extracted DNA. These observations, combined with the intracellular site of 5-lipoxygenase translocation and LTA4 biosynthesis at the nuclear envelope, suggest that LTA4 may have access to DNA and RNA within cells and furthermore modify nucleic acids in situ following the activation of 5-lipoxygenase and initiation of LTA4 biosynthesis.

Introduction The metabolism of arachidonic acid by 5-lipoxygenase leads to the production of leukotrienes, which are a class of biologically active eicosanoids thought to play a central role in the modulation of tissue inflammation (1, 2). Leukotriene B4 (LTB4) stimulates neutrophils through a specific G-protein-linked receptor to undergo chemotaxis and chemokinesis (3, 4). Leukotriene C4 (LTC4) was initially described as the slow-reacting substance of anaphylaxis (SRS-A), an agent responsible for bronchoconstriction and smooth muscle contraction (5, 6). Both of these leukotrienes are synthesized from a common intermediate, LTA41 (5,6-epoxy-7,9,11,14-eicosatetraenoic acid). Quite unexpectedly, the nuclear envelope has now * To whom coorespondence should be addressed. Tel: (303)398-1849. Fax: (303)398-1694. E-mail: [email protected]. † National Jewish Medical and Research Center. ‡ University of Colorado Health Sciences Center. 1 Abbreviations: BSA, bovine serum albumin; COSY, correlated NMR spectroscopy; LC/MS/MS, combined liquid chromatography and tandem mass spectrometry; LTA4, leukotriene A4, 5(S)-trans-5,6-oxido7,9-trans-11,14-cis-eicosatrienoic acid; LTA4-(d)Guo, covalent adduct of LTA4 with guanosine (adducts of LTA4 with other nucleosides or deoxynucleosides (d) are similarly noted using the standard three letter abbreviations for nucleosides); MRM, multiple reaction monitoring in a tandem quadrupole mass spectrometer where precursor ion and product ions are assigned to detect a specific compound; SPE, solid phase extraction; TOCSY, total correlated NMR spectroscopy.

been found to be the site where 5-lipoxygenase and 5-lipoxygenase-activating protein operate (7) to catalyze oxidation of arachidonic acid released by cPLA2 and carry out the initial addition of molecular oxygen to carbon-5 to form 5-hydroperoxyeicosatetraenoic acid and subsequently LTA4. LTA4 is a highly reactive electrophile that as a free acid has a half-life of approximately 1 s in aqueous solution at neutral pH (8). The mechanism of instability involves an acid-catalyzed opening of the epoxide ring to form a carbocation (9) with the subsequent nucleophilic addition of water to form 5,12- and 5,6-dihydroxyeicosatetraenoic acids. However, within specific cells, LTA4 undergoes enzymatically directed stereospecific addition of water to form LTB4 catalyzed by LTA4 hydrolase (10) or stereospecific addition of glutathione at carbon-6 to form LTC4 catalyzed by LTC4 synthase (11). Furthermore, evidence has accumulated that LTA4 can be stabilized and even transported out of the cell for subsequent conversion into the biologically active leukotrienes by acceptor cells in a process termed transcellular biosynthesis (12). Thus, within the cell, the halflife of LTA4 can be reasonably prolonged despite the electrophilic character of this epoxide. The nucleoside base subunits of DNA and RNA are known to have nucleophilic character and participate in

10.1021/tx034018+ CCC: $25.00 © 2003 American Chemical Society Published on Web 03/28/2003

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covalent binding reactions with various electrophilic agents, including reactive lipids (13) and drug metabolites (14). Such covalent binding can alter the properties of DNA in a manner that results in cytotoxic effects by interfering with DNA replication (15). Previously, isolated nucleosides in aqueous solution were found to have significant reactivity with LTA4 revealing that nucleosides were competitive with water in such reactions (16). Covalent adducts of LTA4 formed with various nucleosides were characterized as a mixture of 5,6- and 5,12-addition products, with several sites of the lipid component binding to the nucleophilic centers on the nucleoside base. These observations have now been extended to demonstrate the covalent binding of LTA4 with deoxynucleosides as well as to intact DNA and RNA with the observation that such reactions are facile and again competitive with the aqueous milieu. The two most abundant adducts of LTA4 with guanosine (LTA4Guo adducts) were studied in detail by mass spectrometry, UV, and NMR spectroscopy and found to be diastereoisomers linked at carbon-12 of LTA4 with covalent attachment of the guanosine at N2. Furthermore, endogenously generated LTA4 was found to bind to DNA within the human neutrophil upon stimulation of isolated cells with the calcium ionophore A23187.

Materials and Methods Materials. All solvents used in this work were of HPLC grade (Fisher Scientific) and were used without further purification. Water was purified to 18 MΩ resistance by flow through a Milli-Q Water System (Millipore, Danvers, MA). LTB4 and LTA4 methyl ester were purchased from Cayman Chemicals (Ann Arbor, MI). LTA4 ethyl ester was a kind gift of Dr. Joseph Mancini, (Merck Frost, Montreal, Quebec). Calf thymus DNA, calf liver RNA, proteinase K, DNase I, nuclease P1, alkaline phosphatase, snake venom phosphodiesterase, ATP, and BSA were purchased from Sigma (St. Louis, MO). Isotopically labeled deoxyguanosine triphosphate and [15N513C10] dGTP, with 15N and 13C greater than 98 atom % excess, and guanosine triphosphate ([15N]5 GTP), with 15N greater than 98 atom % excess, were purchased from Martek Biosciences Corporation (Columbia, MD). All HPLC columns were manufactured by Phenomenex (Torrence, CA), and SPE cartridges were obtained from Supelco (Bellefonte, PA). DNA isolation kits were purchased from Clontech Laboratories (Palo Alto, CA). NMR tubes (3 mm microtubes impedance matched to methanol and DMSO) were purchased from Shigemi Corp. (Allison Park, PA). NMR solvents (CD3OD, 100 atom % D4 and DMSO-d6, 100 atom % d6) were purchased from Cambridge Isotopes (Endover, MA). The hydrogen used for catalytic reduction was 99.99% pure (General Air, Denver, CO), and 1% Pd on alumina was purchased from Alpha Chemicals (Danvers, MA). Tritiated guanosine (4.0 Ci/ mmol) was purchased from Moravek (Brea, CA). Reactions of LTA4 and Deoxynucleosides, DNA, and RNA. The typical reaction carried out with LTA4 free acid and isolated nucleosides, DNA, or RNA followed a previously published protocol (16). LTA4 was prepared from its methyl or ethyl ester as previously described (17). Briefly, 6 nmol of LTA4 methyl ester, supplied in hexane with 1% triethylamine, was dried under a stream of nitrogen, dissolved in acetone (5 µL) mixed with 0.25 M NaOH (1 µL), and left at room temperature for 1 h. DNA and RNA were dissolved in water at 0.5 mg/mL, which corresponded to an approximate nucleoside concentration equivalent of 1.5 mM (based on an average deoxyribonucleotide weight of 325 g/mol for DNA and 341 g/mol for RNA). Aqueous solutions (25 µL) of the various nucleosides or nucleic acids (12.5 µg) were added to the LTA4 acetone solution. Reactions were left at room temperature for 1 h, and then, 5% formic acid solution (approximately 5 µL) was added to acidify the reaction

Hankin et al. solution and hydrolyze any remaining LTA4 free acid. Larger scale reactions of LTA4 with various nucleosides were carried out with aqueous solutions of nucleosides (26-100 mM) also containing 0.25 M NaOH and 5 mg/mL BSA. The concentrated nucleoside solution (100-200 µL) was added to the acetone solution of LTA4 (8-16 µg) for several hours at room temperature before it was acidified to pH 3 with formic acid. Control experiments were carried out at pH 10 by adding DNA and RNA to solutions of LTA4 that had been previously hydrolyzed in 0.1 N formic acid for 1 h at room temperature. The mixture was enzymatically digested and then worked up in the same fashion as the other experiments. DNA and RNA Digestions to Nucleosides and Adduct Isolation. DNA and RNA reaction solutions (25 µL) were brought to pH 7.5 by addition of HBSS (200 µL) and 1 M NH4HCO3 (20 µL). The DNA or RNA from reactions with LTA4 was enzymatically digested to individual nucleosides and LTA4nucleoside adducts using protocols developed by Blair et al. for DNA (18) and Crain for RNA (19). Briefly, for both DNA and RNA digestions, three enzymes were added sequentially to the nucleic acid solutions. Enzymes were made up freshly in the prescribed buffer solutions, and incubations were carried out for the designated times at 37 °C. For DNA hydrolysis, DNase was added (556 units/200 µg DNA) for 1.5 h followed by nuclease P1 (15.5 units/200 µg DNA) for 2 h followed by addition of alkaline phosphatase (30 units/200 µg DNA) for 1 h. For RNA conversion to nucleosides, nuclease P1 (20 units/200 µg RNA) was added and incubated for 2 h followed by addition of snake venom phosphodiesterase (0.02 units/200 µg RNA) for 2 h and then alkaline phosphatase (30 units/200 mg RNA) for 1 h. After the last digestion step, the pH was adjusted to 3-4 with formic acid. Internal standard (300 pg) was added after each digestion, and samples were purified using C-18 SPE cartridges that had been previously conditioned with three sequential washings of methanol and 3 vol of water. Each sample was added to the column followed by washing with 3 column vol of water and then eluting the lipophilic compounds with 3 vol of methanol. The eluant of each sample was taken to dryness using vacuum centrifugation and dissolved in 120 µL of water/ methanol (85/15). Reactions with isolated nucleosides required no enzymatic digestion and were simply adjusted to pH 3-4 with formic acid, purified using reversed phase SPE, dried, and dissolved as above. HPLC. RP-HPLC was carried out for both preparative purifications and analytical separations for this body of work. All RP-HPLC analyses utilized the same solvent system: solvent A, H2O with 8 mM ammonium acetate buffered to pH 5.7 with acetic acid and ammonium hydroxide; solvent B, 65:35 acetonitrile:methanol mixture. Analytical HPLC with mass spectrometric detection employed a 150 mm × 1 mm Columbus 5 µm, 110 Å, C-18 column with a flow rate of 50 µL/min and a linear gradient starting at 15% going to 40% B in 5 min, 60% B in 20 min, and 95% B in 5 min. Purification of synthetic LTA4nucleoside adducts involved a 150 mm × 2 mm LUNA 3 µm C-18(2) column and linear gradient described above with a flow rate of 200 µL/min. Elution of the adducts was detected by a UV monitor as well as by mass spectrometry with 10% of the sample split into the mass spectrometer. Purifications of isotopically labeled nucleosides enzymatically cleaved from isotopically labeled GTP and dGTP were carried out preparatively using a large 250 mm × 10 mm 10 µm LUNA C-18(2) column. A flow rate of 5 mL/min was used with a gradient of 0% B isocratic for 5 min, linear increase to 5% B over 5 min, and then to 25% B in 10 min (20). Normal phase HPLC used to separate nonenzymatic hydrolysis products of LTA4 from LTA4-nucleoside adducts employed a 150 mm × 2 mm Ultramex silica column with solvents C (75% hexane with 25% solvent D) and solvent system D (9:1 2-propanol:water, 2 mM ammonium acetate) and 0.3% acetic acid. The mobile phase flow rate was 200 µL/min using a gradient starting at 10% D, held isocratically for 5 min, and then a linear increase to 90% B over 5 min holding at 90% D for 3 min.

LTA4 Adducts of Nucleic Acids MS. The SPE-purified enzymatic digests from the reactions of LTA4 with DNA, RNA, or isolated nucleosides were analyzed by LC/MS and LC/MS/MS on a PE-SCIEX API 3000 tandem mass spectrometer (Toronto, Ontario). Instrumental conditions relevant to the analysis included electrospray voltage (negative ions) of -3800 V, declustering potential of -70 V, focusing potential of -220 V, collision gas thickness of 1.6 × 1015 molecules/cm2, and collision energy of -30 V. Positive ions were analyzed on this instrument with an electrospray voltage of +4500 V, declustering potential at +60 V, focusing potential of +200 V, and collision energy of 35 V with the same collision gas thickness as for negative ions. Medical grade air was used as the nebulizer gas for the electrospray ionization source, and ultrapure nitrogen (gas bleed off from a liquid nitrogen dewar) was used as the curtain and collision gases. Quantitation was carried out using MRM in negative ion mode, which involved precursor ion selection in Q1, collisional activation in Q2, and specific product ion selection in Q3. The dwell time was increased from 200 to 1500 ms for analyte and internal standard in quantitative analyses. A Thermoquest LCQ ion trap (San Jose, CA) was used for structural analysis and as an analytical detector in line with a UV detector during separation and purification procedures. The LCQ was operated in both positive and negative ion mode and was typically programmed to alternate between full scan mode and MS2 of the target adduct. MSn analysis was carried out by direct infusion of prepurified adduct compounds into the LCQ. Relevant product ions from MS2 analysis were used as precursor ions for MS3 analysis. Data were acquired and averaged over 1 min. The capillary temperature was set at 150 °C, the electrospray voltage was set at 3500 V (4000 V for positive ions), and the sheath gas flow was set at 70 units, auxiliary gas at 5 units. Quantitation. Internal standards for quantitative analysis were prepared from isotopically labeled [15N513C10]dGTP and [15N5]GTP, which were converted to monophosphorylated nucleosides by addition of 0.3 units ATPase/mg GTP for 2 h at 37 °C (21) and to nucleoside by addition of 150 units alkaline phosphatase/mg GTP (18). These labeled nucleosides (7-10 µmol) were purified by HPLC (20) and reacted with LTA4 free acid (25 nmol) as described above. After the reaction, the solution was adjusted to pH 3-5 with 5% formic acid and purified by RP-HPLC. Yields of the isolated adduct product in these reactions were in the range of 1-2% based on the amount of LTA4 added. Mass spectrometric determinations of the isotopic purity of the isolated internal standards showed an 87% atom excess for the LTA4-[15N513C10]dGuo adduct at m/z 599.3 with 0.5% LTA4-dGuo at m/z 584.3 as unlabeled species. The LTA4-[15N5]Guo adduct at m/z 605.3 showed an isotopic purity of 82% atom excess with 1.5% unlabeled LTA4-Guo at m/z 600.3. Quantitation of these internal standards was carried out by UV spectroscopy. The extinction coefficient for the adduct of LTA4-Guo was determined using radioactive guanosine adjusted with nonradioactive guanosine to a known specific activity (5.9 µCi/µmol). Products from the ensuing reaction were purified by HPLC, and isolated adducts were measured for radioactivity by scintillation counting. The number of moles of adduct were determined from the known specific activity of the added guanosine and the measured radioactivity as determined using an external standard and quench curve. This quantity and the UV absorbance were substituted into Beer’s law to calculate the ultraviolet extinction coefficient. Quantitation of LTA4-nucleoside adducts derived from guanosine and deoxyguanosine was carried out by stable isotope dilution mass spectrometry. The mass transitions used for MRM were based on the collision-induced decomposition of the molecular ions that lost the sugar residue as an unsaturated neutral species. A standard curve corresponding to the ratio of unlabeled to stable isotope-labeled internal standard expressed as a function of the quantity of known amounts of unlabeled adduct was found to be linear over the ranges of 1.6-100 pg and 100-5000 pg.

Chem. Res. Toxicol., Vol. 16, No. 4, 2003 553 The yields of other nucleoside adducts (deoxyadenosine, deoxycytidine, thymidine, adenosine, cytidine, uridine) formed in reactions of LTA4 with DNA or RNA were determined by monitoring the relative abundance of the specific mass transition ion pairs relative to LTA4-dGuo standard for DNA bases and LTA4-Guo standard for RNA bases. The variability of mass spectral response factors for the different adducts relative to the standards was assessed by simultaneously measuring the UV absorbance of each compound and mass chromatogram peak area acquired in MRM mode during RP-HPLC analyses. The synthetic LTA4 products of larger scale reactions were used for these determinations. For some LTA4-nucleoside adducts, it was necessary to carry out an additional normal phase HPLC separation to remove nonenzymatic hydrolysis products of LTA4 from the nucleoside adducts prior to quantitation by UV spectroscopy. Ultraviolet absorbance was used to quantitate the other adducts with the assumption of a similar extinction coefficient at a λmax between 270 and 272 nm for compounds similar in vibronic structure to the conjugated trienes of the LTA4-Guo adducts. The absolute quantities of other nucleoside adducts formed, therefore, are an approximation. Neutrophil Incubations. Human polymorphonuclear leukocytes (PMNs) were isolated from whole blood using the Percoll gradient centrifugation technique previously published (22). Separate experiments were carried out with different donors and total numbers of isolated cells between 1.5 and 4 × 108 neutrophils. Cells were suspended in HBSS to 2 × 107 neutrophils/mL, and two equal aliquots in each preparation were placed in 50 mL conical tubes at 37 °C. The calcium ionophore A23187 (2 µM) and arachidonic acid (2 µM) were added to the tubes followed by incubation with gentle shaking for 30 min in a water bath. Following this, cold ethanol (3 vol) was added to stop the reaction and cell suspensions were centrifuged at 450g to pellet precipitated proteins and cellular debris including the nuclei. The supernantant was carefully removed and stored for analysis of leukotriene production described below. The cell pellet was extracted using the protocols suggested in the commercial kit for DNA isolation (Clontec, Los Angeles, CA). The quantity of DNA extracted and precipitated by addition of 2-propanol was quantified in each experiment by UV absorbance (260 nm) after resolvation in buffer. The DNA was then enzymatically digested to individual nucleosides using the procedures described previously. NMR Spectroscopy. All NMR spectra were recorded at the UCHSC Biomolecular Facility using Varian Inova Spectrometers with a proton frequency of either 500 or 600 MHz. Samples (20-30 µg) were dissolved in CD3OD (120 µL) or DMSO-d6 (120 µL) for one-dimensional (1D) and two-dimensional (2D) experiments in 3 mm tubes impedance matched for either solvent. Each spectrometer is equipped with a Nalorac 3 mm IDTX probe with pulsed field gradients. All NMR spectra were recorded at 25 °C and referenced to an external standard of tetramethylsilane at 0.0 ppm. For the 1D spectrum of LTA4-Guo, a total of 64K data points were acquired over a spectral width of 12 ppm. A total of 872 transients were acquired with a 6 s recycle delay between each accumulation. The total acquisition time was approximately 2 h. The data were multiplied by a 0.4 Hz line broadening function and zero-filled to 128K points prior to Fourier transformation. The 2D gradient COSY spectrum was acquired with a spectral width of 10 ppm in both dimensions. Complex data points (660) were acquired in the indirect dimension with 32 transients per increment. Pulsed field gradients were used for echo type coherence selection during t1. Data were multiplied by an unshifted sinebell prior to Fourier transformation, and the absolute value of the data was calculated prior to analysis. A 2D Z-filtered TOCSY spectrum (23) was recorded using a DIPSI-2 mixing sequence (24) of 37 ms and a spin-lock field strength of 7.62 kHz. Complex data points (490) were acquired in t1, and frequency discrimination in the indirection dimension was achieved using the STATES-TPPI method (25). Data were multiplied by a mild Lorentz-Gaussian function in

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Figure 1. (A) RP-HPLC separation of a reaction of LTA4 (0.25 mM) with deoxyguanosine detected by negative ion electrospray ionization tandem mass spectrometry monitoring the mass transition m/z 584 f 468, corresponding to the adduct of LTA4 with deoxyguanosine. Inset: Collisionally induced dissociation mass spectrum of the precursor ion [M - H]- (m/z 584) of the adduct labeled LTA4-dGuoI corresponding to the adduct of LTA4 with deoxyguanosine using negative ion electrospray ionization. The characteristic ions correspond to the loss of neutral unsaturated deoxyribose fragment (m/z 468), loss of a neutral lipid fragment (m/z 266), and loss of a neutral nucleoside fragment (m/z 317). (B) RP-HPLC separation of a representative reaction of LTA4 (0.25 mM) with DNA (12 µg) at pH 10 following enzymatic digestion and purification of lipophilic products using SPE. The compounds identified as LTA4-dGuo adducts from the DNA reaction are indicated by specific mass transition for the precursor ion (m/z 584) to product ion (m/z 468) representing a loss of unsaturated deoxyribose product ion. both dimensions prior to Fourier transformation. All data were processed using Vnmr (Varian Inc.). Hydrogenation of the LTA4-Guo Adduct. Reduction of the LTA4-Guo adduct was carried out using Pd on Al2O3 (1 mg), which had been suspended in 100 µL of solvent (50:33:17; 0.8 mM ammonium acetate:CH3CN:MeOH, v/v/v) into the top portion of an aerosol barrier pipet tip (Fisher Scientific). Hydrogen gas was bubbled up through the bottom of the pipet at 3 psi so that the catalyst and liquid were mixed with welldispersed gas during the hydrogenation reaction. LTA4-Guo (300 pmol) was added in 100 µL of the above solvent, and hydrogen was bubbled into the mixture for 30 s. The pipet tip was then centrifuged at 200g, and the filtered sample was analyzed by HPLC coupled to a UV detector with the ion trap mass spectrometer set to acquire full scan and MS2 data for the reduced adduct expected at m/z 608.

Results The reaction of deoxyguanosine (1.5 mM) in solution with LTA4 (0.25 mM) yielded numerous adduct products (m/z 584.3) when analyzed by RP-HPLC/MS (Figure 1A). A similar clustered pattern of adduct products was evident when compared to the mass chromatogram of the previously described products of the reaction of guanosine with LTA4 (16). Collisional activation of each of the different products from Figure 1A yielded mass spectra that were similar in the major product ions produced but different in the relative abundances of these product ions. The mass spectrum of product LTA4-dGuoI derived from collisional activation of the precursor ion at m/z 584.3 (Figure 1A inset) showed ion fragments corresponding to the loss of the neutral unsaturated sugar (m/z 468), residual lipid fragment after loss of the neutral nucleo-

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Figure 2. (A) RP-HPLC separation of a representative reaction of LTA4 (0.25 mM) with RNA (13 µg) at pH 10 that had been enzymatically digested and lipophilic products purified by reversed phase separation. Compounds were detected by MRM of the precursor ion m/z 600, decomposed to the product ion m/z 468 following collisional activation. (B) Reversed phase separation of a reaction of LTA4 (0.25 mM) with guanosine as analyzed by MRM using the ion transition m/z 600 f 468 and the same HPLC separation conditions used to monitor the LTA4 with RNA.

side (m/z 317), and residual nucleoside after loss of the neutral lipid (m/z 266). Full collisional spectra acquired in positive ion mode yielded a similar mass chromatogram as in negative ion mode, and collisional activation of [M + H]+ at m/z 586 of LTA4-dGuoI yielded prominent ion fragments representing the loss of the unsaturated sugar (m/z 470), the nucleoside (m/z 319), and the protonated base (m/z 152) (data not shown). The proclivity of these reactions in aqueous solutions led to experiments with intact DNA and RNA. An analytical approach to assess covalent adduct formation was adopted where postreaction, DNA, and RNA were enzymatically digested to nucleoside components and analysis carried out on LTA4-nucleoside adducts using the established LC/MS/MS techniques, specifically MRM. The prominent loss of the neutral unsaturated sugar was used to screen DNA and RNA reactions for successful adduct formation. Analysis of the enzymatic digestion of DNA (12 µg) after reaction with LTA4 (Figure 1B) yielded a mass chromatographic trace (m/z 584 f 468) that was identical in distribution and pattern of products formed during the reaction of LTA4 with deoxyguanosine (Figure 1A) run under identical conditions. Similarly, RNA (13 µg) that was enzymatically digested to nucleosides after reaction with LTA4 (Figure 2A) gave a MRM chromatogram that was strikingly similar in mass transition, retention times, and pattern to the products obtained in the reaction of guanosine with LTA4 (Figure 2B). The UV absorbance extinction coefficient of the LTA4GuoI and LTA4-GuoII adducts was determined by synthesizing radioactive adduct of a known specific activity to enable simultaneously measurement of the number of moles by scintillation counting and UV absorbance signal by spectrophotometric measurement. The extinction coefficient for both adduct peaks, LTA4-GuoI and LTA4GuoII, was determined to be 54 000 at λmax 272 nm. This value served as the basis for subsequent mass spectrometric quantitative assays where the amounts of isotopically labeled and unlabeled internal standards used to

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Table 1. Adduct Yields from the Reaction of 250 µM LTA4 with Nucleosides and Nucleic Acids pH 7.4, no BSA

pH 7.4, BSAc

pH 10, no BSA

pH 10, BSAc

DNA (43 µg)b 15 ( 1d 23 ( 2 60 ( 7 119 ( 1 RNA (46 µg)b 230 ( 20 720 ( 50 300 ( 20 700 ( 30 deoxyguanosine (5 mM) 3600 ( 490 guanosine (5 mM) 2900 ( 650 a Yields are expressed as number of adducts/1 × 107 bases. b The amount of DNA and RNA was equivalent to the average nucleoside component concentration of 5 mM using an average molecular mass of 325 and 341 Da, respectively. c BSA added to a final concentration of 3 mg/mL. d SEM for three experiments.

Figure 3. Adduct formation following the reaction of LTA4 with RNA and DNA. The quantity of LTA4-dGuo and LTA4-Guo adducts formed in the reaction of 0.25 mM LTA4 with increasing amounts of DNA or RNA was analyzed in triplicate. Error bars indicate 1 SEM but were smaller than the symbol when not observed. Quantitation was carried out using mass spectrometry with isotopically labeled internal standard as described in the Materials and Methods.

establish a standard curve were determined by UV absorption. Basic conditions and the presence of BSA had previously been shown to stabilize LTA4 and increase its halflife substantially (8). The amount of adduct formed in reactions of LTA4 with DNA and RNA was studied in a quantitative assay as a function of various experimental conditions (Table 1). RNA was consistently more reactive than DNA, and reactions carried out under basic conditions or in the presence of BSA increased adduct yields with both nucleic acids. Reactions with isolated guanosine or deoxyguanosine yielded substantially larger amounts of adduct, especially in the case of DNA where the helical and supercoiled structure likely impeded contact between the nucleotide bases and the reactive electrophile. The yields of DNA and RNA adduct increased in a dose-dependent manner when nucleic acid amounts were increased from 25 to 400 µg (Figure 3). It should be stressed, however, that the conditions used to maximize yield of adducts were not physiologically relevant. There was evidence of adduct formation to other nucleosides in addition to deoxyguanosine and guanosine during reactions of LTA4 with DNA and RNA. Synthetic reactions carried out at larger scale (pH 10) using pure adenosine, deoxyadenosine, cytidine, deoxycytidine, thymidine, and uridine produced adducts with LTA4 in sufficient quantities for UV absorbance measurement and LC/MS/MS analysis. It was evident in these reactions that guanosine and deoxyguanosine were the most reac-

tive nucleosides with LTA4 and that cytidine and deoxycytidine were the least reactive. Tandem mass spectra were acquired on an ion trap mass spectrometer in negative ion mode for each of the different adducts (Figure 4). All of the adduct ions yielded similar fragments after collisional activation of the precursor ions including abundant product ions derived from the loss of the neutral unsaturated sugar group. An ion at m/z 317, representing the same residual lipid fragment after loss of the neutral nucleoside, was abundant in those adducts derived from the RNA nucleosides. The transitions from precursor ion to the fragment ions representing the loss of the neutral unsaturated sugar group were monitored for each nucleoside in LC/MRM mode on the triple quadrupole instrument when assessing adduct presence in the reactions of LTA4 with DNA and RNA (Table 2). The reaction of LTA4 with DNA (12 µg in the presence of 3 mg/mL BSA, pH 7.4) yielded observable adducts only with deoxypurines when measured by MRM. Reactions carried out with higher quantities of DNA (250 µg) yielded a large amount of adduct with deoxyadenosine and some evidence of adduct formation with deoxycytidine and thymidine (Figure 5). When this reaction was carried out at pH 10, a substantial increase in the thymidine adduct was observed, but no deoxycytidine adduct was observed (data not shown). The reactions of LTA4 with RNA (13 µg) in the presence of 3 mg/mL BSA, pH 7.4, yielded observable adducts with adenosine and uridine when compared to synthetic standards. When the reaction was carried out with larger amounts of RNA (250 µg), significant amounts of adduct with adenosine and uridine were found with additional formation of adducts with cytidine (Figure 6). In all cases, the most prevalent products of DNA and RNA remained the LTA4-dGuo and LTA4-Guo adducts, respectively. An assessment of the amounts of other adducts that formed in reaction of DNA and RNA with LTA4 was made by direct comparison of the mass spectral ion transition signals from LTA4-nucleoside adducts relative to quantified amounts of LTA4-dGuo and LTA4-Guo adducts produced in the same reactions. This approach required assessing the mass spectral response factors to determine if ionization efficiencies and collision energies were similar enough between the different compounds to make comparisons. Nanomole quantities of LTA4 adducts with each nucleoside were synthesized so that UV absorption spectra could be obtained and related to the signals acquired during mass spectrometric analysis. The amount of LTA4-nucleoside adducts was determined by selecting HPLC peaks from each reaction where the conjugated triene moiety was apparent in the UV spectra and using the assumption that the extinction coefficient of the UV absorbance at λmax 270-272 from the triene chromophore was identical to that determined for LTA4-GuoI and LTA4-GuoII. The ratio of integrated mass spectral peak area to the corresponding UV absorbance for the adducts derived in sequential experiments was calculated (Table 3). The peak area generated by the mass spectrometer appeared to estimate the true quantity of the other adducts within an order of magnitude. Therefore, in the reaction of LTA4 with DNA, the most reactive nucleoside base appeared to be deoxyguanosine although significant amounts of LTA4-dAdo formed. The reactions of LTA4 with RNA resulted in the largest amounts of addition to guanosine, although substantial amounts of addition to

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Figure 4. Tandem mass spectrometry (MS2) following collisional activation of the [M - H]- ion for the most abundant LTA4nucleoside adducts formed following the reaction of LTA4 (0.25 mM) with the corresponding nucleoside (26 mM) at pH 10. Precursor ions for the different adducts of LTA4-dAdo (m/z 568), LTA4-dCyd (m/z 544), LTA4-Thd (m/z 559), LTA4-Ado (m/z 584), LTA4Cyd (m/z 560), and LTA4-Urd (m/z 561) were selected and analyzed in an ion trap mass spectrometer using electrospray ionization.

Figure 5. MRM of LTA4-deoxynucleoside adducts following reaction with either DNA (upper panel) or isolated deoxynucleoside (lower panel). The analysis of DNA adducts was carried out after enzymatic hydrolysis and isolation of lipophilic DNA adducts. (A) MRM for the LTA4-dAdo adduct (m/z 568 f 452). (B) MRM for the LTA4-dCyd adduct (m/z 544 f 428). (C) MRM for the LTA4dThd adduct (m/z 559 f 443). Table 2. Mass and Mass Transitions for Adducts of LTA4 with Deoxynucleosides and Nucleosides nucleoside/ deoxynucleoside

molecular mass of adduct with LTA4 (Da)a

MRM transitionb [M - H]- f loss of sugar

deoxyguanosine deoxyadenosine deoxycytidine thymidine guanosine adenosine cytidine uridine

585.3 569.3 545.3 560.3 601.3 585.3 561.3 562.3

584.3 f 468.3 568.3 f 452.3 544.3 f 428.3 559.3 f 443.3 600.3 f 468.3 584.3 f 452.3 560.3 f 428.3 561.3 f 429.3

a Monoisotopic molecular mass. b Collision-induced decomposition of electrospray-generated molecular anion species. The loss of sugar ion corresponds to the mass of an unsaturated ribose or deoxyribose.

adenosine and uridine, and possibly significant amounts of LTA4-cytidine, formed. Structural Characterization of LTA4-Guo Adduct. Structural characterization of the two most abundant adducts following the reaction of LTA4 with RNA or LTA4 with guanosine was carried out using proton NMR, UV spectroscopy, and mass spectrometry. The

yield of the two target compounds (LTA4-GuoI and LTA4-GuoII) previously described (16) was increased by raising reacting guanosine concentration and the pH of the reaction system. Both adducts were found to be quite stable when stored dry for several months at -20 °C with no apparent degradation. Even at room temperature in methanol, the adduct was found to be quite stable as assessed both by HPLC and mass spectrometry. Both LTA4 adducts had strong UV absorption at λmax 272 nm and vibronic structures at 262 and 282 nm consistent with the conjugated triene (structure). Approximately 20-30 µg of both compounds of LTA4-GuoI and LTA4GuoII were accumulated in several large scale reactions and used for NMR analyses after purification by RPHPLC. Initial 1D and 2D NMR analyses were carried out in CD3OD, and chemical shift assignments of the LTA4GuoII adduct were established using 2D gradient COSY (26-28) and confirmed using 2D TOCSY (29). Each experiment typically required greater than 16 h to collect sufficient data at an appropriate signal-to-noise. The starting point for assignments was the H2-methylene protons of the eicosanoid backbone (Table 4), which had a characteristic triplet at 2.21 ppm. Almost the complete



Figure 6. MRM of LTA4-nucleoside adducts following reaction with either RNA (upper panel) or isolated nucleoside (lower panel). The analysis of RNA adducts was carried out after enzymatic hydrolysis and isolation of lipophilic nucleosides. (A) MRM for the LTA4-Ado adduct (m/z 584 f 452). (B) MRM for the LTA4-dCyd adduct (m/z 560 f 428). (C) MRM for the LTA4-Urd adduct (m/z 561 f 429). Table 3. Collision-Induced Decomposition Response Factors and Relative Yields of LTA4-Nucleoside Adducts Formed during the Reaction of LTA4 with DNA and RNA

Table 4. Table of Chemical Shifts from the 2D COSY NMR Experiments (500 MHz) for the Purified LTA4 Adduct with Guanosine, Designated LTA4-GuoII, in CD3OD

normalized mass relative spectral response abundances relative yield factor: MRM of LTA4 corrected for mass adducta

area/nmol adduct (UV)

nucleoside adducts

mass spectral response

LTA4-dGuo LTA4-dCyd LTA4-dAdo LTA4-dThd LTA4-Guo LTA4-Cyd LTA4-Ado LTA4-Urd

100b 45 55 240 100c 65 165 300

1d 0.007 0.305 0.009 1d 0.005 0.103 0.093

1 0.015 0.55 0.004 1 0.008 0.06 0.031

a Conditions of adduct formation include LTA (250 µM) with 4 each nucleoside (1 mg) at pH 10 containing BSA (3 mg/mL). b Deoxynucleotide adduct values are normalized to the ion yield following collision-induced decomposition (m/z 584 f 468) of a known quantity of LTA4-dGuo. c Nucleoside adduct values are normalized to the ion yield following collision-induced decomposition (m/z 600 f 468) of a known quantity of LTA4-Guo. d Conditions of reactions include LTA4 (250 µM) and DNA (130 µg) or RNA (130 µg) at pH 7.4 with 5 mg/mL BSA.

system for the eicosanoid backbone could be assigned unambiguously starting from this point. The exceptions were H8 and H9 of the conjugated triene and the methylene protons H18. It was most likely that H8 and H9 were overlapping with the signals from H7 and H10 in the region between 6.1 and 6.4 ppm. In the 1D spectrum, this region intergrates for four protons and was consistent with these four protons having very similar chemical shifts. The chemical shift assignments of H7 and H10 were made from correlations to H6 and H11, respectively, observed in the 2D TOCSY spectrum. The methylene protons of H18 were most likely overlapped with those of H17 and H19 in the region of 1.2-1.4 ppm. Again, this region integrated for six protons, consistent with this assignment. The spin system defined in the COSY spectrum clearly identified the major point of substitution of the guanine group at C12 of the eicosanoid backbone derived from LTA4. The peak from H12 (4.70 ppm) was coupled to a single vinyl proton at 5.75 ppm (H11), which was part of an extended set of vinyl protons, and to a methylene group (H13) with nondegenerate chemical shifts at 2.52 and 2.41 ppm. These methylene protons were both coupled to a single vinyl proton at 5.41 ppm (H14), which

H2 H3 H4 H5 H6 H7 H8/H9 H10 H11 H8′ H1′ H2′

LTA4 protons 2.21a 1.64a 1.54a 4.09 5.70 6.27 6.23, 6.33b 6.31 5.75 7.99 5.90 4.60

H12 H13 H14 H15 H16 H17 H18 H19 H20

guanosine protons H3′ H4′ H5′/H5′′

4.70 2.52, 2.41 5.41 5.54 2.06a 1.35a nac 1.27a 0.88a 4.32 4.06 3.83, 3.72

a Chemical shifts of these methylene protons are degenerate. The specific assignment of H8 and H9 could not be made as they are overlapped with signals from H7 and H10. See Discussion. c Not assigned (na), the signal for H18 could not be specifically assigned as it is overlapped with H17 and H19. See text for discussion. b

in turn was coupled to a single vinyl proton at 5.54 ppm (H15). H15 was further coupled to a methylene group (H16) at 2.06 ppm. This spin system could only arise if substitution occurred at the C12 of the eicosanoid backbone. The protons of the guanosine ribose ring formed an isolated spin system that was readily assigned in the COSY spectrum (Table 4). The H8 of the guanine ring appeared as an isolated singlet at 7.99 ppm. The 2D COSY and 2D TOCSY spectra of the compound identified as LTA4-GuoI confirmed its identity as a diastereoisomer of LTA4-GuoII and had very similar chemical shifts and an identical pattern of proton-proton connectivities in these spectra.

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Table 5. Tandem Mass Spectrometry (MS3) of Various Product Ions Obtained Following the Collisional Activation of [M-Sugar]- Fragment Ions from Stable Isotopically Labeled and Unlabeled Adducts of LTA4 with Deoxyguanosine (LTA4-dGuo and LTA4-Guo)

[M-sugar][M-sugar-H2O][M-sugar-43]other ions base a

LTA4-dGuo 584 f 468 f

LTA4-Guo 600 f 468 f

LTA4-15N13C-dGuo 599 f 478f

LTA4-15N-Guo 605 f 473 f

LTA4-Guo (reduced) 608 f 476 f

468 450 425 352 297 226 150

468 450 425 352 297 226 150

478 460 433 362 307 nda 160

473 455 429 357 302 231 155

476 458 433

Not detected.

Figure 7. (A) RP-HPLC chromatogram of LTA4-GuoII used as starting material for catalytic reduction experiments. The inset shows the UV absorption spectrum for the adduct with the λmax 272 nm. (B) RP-HPLC chromatogram monitored at 258 nm of LTA4-GuoII after catalytic reduction with Pd/Al2O3 and H2 gas. The inset shows the UV spectrum of the peak labeled R1 with the corresponding loss of the triene chromophore and new λmax at 258 nm. Chromatographic peaks labeled X were observed in the catalyst blank experiment. (C) RP-HPLC chromatogram monitored by the ion trap mass spectrometer; effluent split after the UV monitor. The peak labeled R1 was detected by monitoring m/z 608. The MS2 mass spectrum of R1 (inset) had prominent fragment ions corresponding to the loss of the unsaturated ribose (m/z 476) consistent with the conjugated triene reduced for this adduct.

Catalytic Reduction. The guanosyl atom attached at C-12 of the eicosanoid could not be deduced from the NMR data obtained. It was expected that catalytic reduction using a transition metal catalyst would selectively reduce the double bonds of the lipid (which dominate the UV absorption signal) allowing the heteroatom point of attachment to be discerned by characteristic UV absorption previously described for various alkyl guanosine adducts (30). Catalytic reduction of LTA4-GuoI and LTA4-GuoII adducts using palladium on alumina catalyst and hydrogen gas resulted in several products separated by RP-HPLC and detected by UV absorption and mass spectrometry (Figure 7). A single peak appeared on the mass chromatogram monitoring product ions derived from m/z 608, and this single peak corresponded to the adduct with four double bonds of the lipid reduced. This product was collisionally activated and found to lose an unsaturated ribose moiety (m/z 476) similar to the unreduced adduct (Figure 7C, insert). The UV absorption spectrum of the reduced adduct had a

maximum of λmax 258 nm and a shoulder at 285 nm consistent with guanosine adducts bonded to either N-1, N2, or N-3 nitrogen atom on the guanosine ring but not consistent with attachment of O6 of guanosine (30). To resolve the possible binding sites as N-1, N2, or N-3, further NMR and mass spectral analyses were carried out. Proton NMR experiments were performed in DMSOd6, which gave rise to a new peak at 10.1 ppm corresponding to the N-1 proton (31), a result inconsistent with linkage at either N-1 or N-3. Chemical shift values for protons on the N2-substituted nitrogen atom have been previously reported at 6.6 ppm. We did not observe any correlation from this region of the spectrum to protons at 4.7 ppm (H12) because of the poor signal-to-noise in this region. Using the tandem mass spectrometric technique MS3 in the negative ion mode (Table 5), collisional activation of m/z 468 derived from either LTA4-GuoI (m/z 600 f 468) or LTA4-dGuoI (m/z 584 f 468) resulted in the loss of a fragment of 43 Da (m/z 468 f 425), different from the neutral loss of 42 Da described in the collisional activation of positive ions of various substituted guanine adducts (32). The nitrogen-containing fragment, which was lost, could have corresponded to HNCO or H2NCHdCH2, both stable neutral molecules. This neutral loss was shifted to 44 Da for the LTA4-15N-GuoI adduct and a neutral loss of 45 Da for the doubly labeled LTA415 N and 13C-dGuoI adducts. These results were consistent only with the neutral species being lost as HNCO. This neutral loss was apparent also in the negative ion MS3 experiments with reduced LTA4-GuoI structure (m/z 608 f 476 f 433), which was obtained for the UV studies described above. This loss of HNCO was possible when considering the adduct structure having the alkyl group from the LTA4 linked at N2 of the guanosine and a charge remote fragmentation of the guanosine ring, likely leading to the formation of a conjugated linear molecule following extrusion of the stable neutral species HNCO. On the basis of NMR, UV spectroscopy, and mass spectrometry, the assignment of the chemical structure of diastereomers LTA4-GuoI and LTA4-GuoII was made as 5-hydroxy-12-[Guo-N2-yl]-6,8,10,14-eicosatetraenoic acid. On the basis of similar chemical behavior, similar chromatographic behavior, and tandem mass spectrometric product ions, the adducts LTA4-dGuoI and LTA4dGuoII were assigned as a diastereomeric pair 5-hydroxy12-[dGuo-N2-yl]-6,8,10,14-eicosatetraenoic acid. Neutrophil DNA Adducts. A set of experiments was carried out with PMNs (neutrophils), which were known to generate LTA4 upon stimulation with the calcium ionophore A23187 (33). Arachidonic acid (2 µM) was added to further increase the production of LTA4 in these cells. The DNA was extracted and quantified by UV spectroscopy and then digested and purified by SPE and



Figure 8. RP-HPLC and tandem mass spectrometric detection of LTA4 adducts formed with DNA following stimulation of PMN. (A) PMN cells (150 × 106) were incubated for 30 min in HBSS (control). DNA was extracted, enzymatically digested to individual nucleosides, and analyzed by LC/MRM (negative ions) for LTA4-dGuo adducts (m/z 584 f 468). The inset shows the RP-LC/MRM analysis of the supernatant of the experiments monitoring for LTB4 (I; m/z 335 f 195), 6-trans-LTB4 (I; m/z 335 f 195), 20-hydroxyLTB4 (II; m/z 351 f 195), and 20-carboxy-LTB4 (III; m/z 365 f 195). (B) PMN cells (150 × 106) were stimulated with A23187 (2 µM) and arachidonic acid (2 µM) for 30 min in HBSS. DNA was extracted, enzymatically digested to nucleosides, and analyzed by LC/ MRM (negative ions) for LTA4-dGuo adducts (m/z 584 f 468). The inset shows the LC/MS/MS analysis of the supernatant from the experiment monitoring for LTB4 (I; m/z 335 f 195), 6-trans-LTB4 (I; m/z 335 f 195), 20-hydroxy-LTB4 (II; m/z 351 195), and 20carboxy-LTB4 (III; m/z 365 f 195). (C) Synthetic LTA4-dGuo analyzed by reversed phase LC/MS/MS (negative ions, m/z 584 f 468) under the same HPLC conditions used for the PMN experiments above.

analyzed by LC/MS and LC/MS/MS. The extent of leukotriene production was assessed by LC/MS/MS analysis of the cell supernatant whereas the determination of LTA4-dGuo adducts was carried out following isolation of DNA from the cell pellets of the centrifuged cell suspension. In six of seven experiments, a measurable quantity of LTA4-dGuo adduct was observed (Figure 8) and in none of the control experiments (not stimulated with A23187) was any LTA4-dGuo adduct found. Analysis of the supernatant in each of the seven neutrophil experiments revealed a robust production of LTA4 as measured by LTB4, 6-trans-LTB4 isomers, 20-hydroxylLTB4, and 20-carboxy-LTB4 (34). There were no 5-lipoxygenase products observed in the control cell experiments. The DNA extracted from each of the cell incubations ranged from 120 to 250 µg; however, in the one experiment where no adduct was observed, only a yield of 40 µg total DNA was obtained. There was no evidence of adduct formation to nucleosides except for deoxyguanosine and no evidence for nucleoside or deribosylated adducts in the cytosol as assessed by LC/MS/MS.

Discussion The biosynthesis of leukotrienes is now recognized as a complex process often involving translocation of several critical proteins to the perinuclear membrane. These proteins include 5-lipoxygenase as well as the cytosolic phospholipase A2 (7). While in some cells 5-lipoxygenase itself is found in the cytosol, in many cells, it is typically found within the nucleus (34). Therefore, the site of leukotriene biosynthesis and the generation of the reactive electrophile, LTA4, likely take place in a region of the cell that is in close proximity to storage sites of DNA as well as RNA. While the chemical reactivity of LTA4

would suggest that it would exist within the cell for only a short period of time, it is clear that events such as binding to proteins to protect it from water as well as transport events into different cellular compartments can substantially prolong the half-life of LTA4. It is also clear that LTA4 can exit a cell and enter another cell where it can be enzymatically converted into biologically active LTB4 or LTC4 (12). It is entirely possible, therefore, that this electrophilic conjugated triene epoxide may find itself in an environment enriched in nucleophilic DNA and RNA. Previous work had established that isolated nucleosides and nucleotides of RNA had significant chemical reactivity with LTA4. The most prevalent products were formed in reactions of guanosine and GMP with LTA4, although reactivity was found for other bases as well (16). Characterization of these products by LC/MS and LC/ MS/MS revealed an array of products, which had different lipophilicity as demonstrated by RP-HPLC retention time. Nonetheless, these adducts generated the same precursor ions and had similar collision-induced decomposition mass spectra, although the relative abundances of products ions varied from one adduct to the next. These results suggested that not only could nucleosides such as guanosine attack carbon-12 or carbon-6 of the LTA4 structure but also multiple nucleophilic sites on guanosine itself participated in these reactions. The exact structural analysis of these products has not been carried out at this time because of the quantity of purified LTA4nucleoside adduct available for study. Nevertheless, the competitive nature of the reactions of LTA4 with nucleosides in an aqueous environment suggested that such reactions might occur in intact DNA or RNA or even within the nuclear environment of cells grown in culture.

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The present experiments revealed that LTA4 reacted with DNA as well as RNA in a fashion similar to those reactions with isolated deoxynucleosides and nucleosides. The profile of LTA4 adducts formed was very similar, although the yields were substantially less on a molar basis, for DNA relative to a pure nucleoside, suggesting that the double-stranded helical structure of DNA might physically hinder access of LTA4 to the more reactive sites of the nucleoside base. These electrophilic reactions were found most facile at conditions of basic pH because of the longer half-life of LTA4 under such conditions. Surprisingly, LTA4 reacted with DNA and RNA at neutral pH 7.4, even in the absence of a stabilizing protein such as albumin. This suggested further that the reaction of LTA4 with nucleic acids was rapid, facile, and highly competitive with water and furthermore suggested the possibility that this reaction might take place within the living cell where high concentrations of DNA were localized in the nucleus. The cell undoubtedly contained transport and lipid binding proteins that would further prolong the halflife of LTA4, possibly even facilitating an encounter with DNA or RNA. The number of adducts formed between DNA and LTA4 at neutral pH was on average found to be 16 adducts/107 bases. Under identical conditions, the number of adducts with RNA was found to be 15 times greater. Thus, it is possible that a more relevant target might be RNA for the electrophilic LTA4. To assess the covalent binding of LTA4 to DNA and RNA, a strategy was employed to enzymatically degrade isolated DNA/RNA to its component nucleosides followed by analysis of these unique lipophilic nucleoside adducts by RP-HPLC. The lipophilic LTA4 portion of the molecule permitted a simple and effective extraction and purification step using SPE to remove the unmodified nucleosides from the nucleoside-lipid adduct. The collision-induced decomposition of each of the nucleoside adducts of LTA4 was found to yield a characteristic fragment ion corresponding to the loss of an unsaturated neutral deoxyribose for DNA and ribose for RNA. This unique signature of the collisional activation of the target ions formed the basis for development of a MRM assay to specifically assess whether such adducts were formed under more physiologically relevant conditions. To assess the relative production of LTA4 adducts with each of the nucleoside bases, it was necessary to independently determine the relative ionization and fragmentation response factors for each of the adducts. The quantitation of the nucleoside adducts by ultraviolet spectroscopy as well as mass spectrometry revealed mass spectral response factors quite similar for all of the adducts and certainly within an order of magnitude. With this information, it was possible to assess that the most abundant nucleic acid adducts were found to occur between LTA4 and guanosine/deoxyguanosine as well as adenosine/deoxyadenosine. Adducts formed between LTA4 and uridine occurred between 5 and 10% as frequently as the adducts between LTA4 and deoxyguanosine. The adducts formed between LTA4 and cytidine or deoxycytidine were most difficult to assess, since they yielded the lowest amount of reaction product even when purified nucleosides were employed to generate standards for analysis. Structural analysis of the predominant diastereomeric peaks of LTA4 following the reaction with guanosine was carried out by various spectroscopic techniques. 2D COSY and TOCSY experiments revealed connectivity of guanosine to carbon-12 of the eicosanoid backbone and ruled

Hankin et al.

out connectivity to C-8 or N-7 of guanosine because of the prominent singlet at 8 ppm. This proton had been described to exchange rapidly in methanol-OD when guanosine was substituted at N-7 (35). Furthermore, catalytic reduction of the conjugated triene within the LTA4-Guo adduct yielded a product, which had a UV spectrum consistent with attachment at one of the nitrogen atoms. Identification of the specific nitrogen atom of attachment as N2 could be made using a combination of MS3 data with the specific loss of neutral HNCO and NMR analysis in DMSO-d6, which revealed a signal at 10.1 ppm that could be clearly defined as the N-1 proton. These data were only consistent with the structure of the LTA4-GuoII adduct as 5-hydroxy-12[Guo-N2-yl]-6,8,10,14-eicosatetraenoic acid. NMR analysis of the other LTA4 adduct eluting earlier on the HPLC (LTA4-GuoI) was virtually identical in terms of carbon bond connectivity establishing this as a diastereoisomer at carbon-12. The reaction of LTA4 generated endogenously within the human neutrophil was used to assess whether adducts of LTA4 with nuclear DNA could be formed under more physiological conditions. The method employed has been used extensively in studies of leukotriene biosynthesis, but previous investigations had not considered whether any LTA4 covalently bound to DNA residing in the nucleus. It was quite evident that adducts of deoxyguanosine (predominantly LTA4-dGuoII) were formed following stimulation of these cells. Abundant production of leukotrienes was clearly evident upon stimulation as measured by the production of LTB4, 6-trans-LTB4 isomers, and ω-oxidized LTB4. In summary, LTA4 has been found to covalently bind to nucleoside bases in both DNA and RNA with adducts of deoxguanosine and guanosine, respectively. More importantly, the formation of a DNA adduct with LTA4 was observed in human neutrophils stimulated to generate LTA4 ex vivo. Despite the complexity of these events, it is clear that electrophilic LTA4 can present itself to DNA, specifically guanosine residues within DNA that results in covalent binding.

Acknowledgment. This work was supported, in part, by grants from the National Institutes of Health, HL25785 and CA46934 (University of Colorado Cancer Center Core). We thank Professors Lawrence Marnett (Vanderbilt University) and James McCloskey (University of Utah) for helpful discussions.

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LTA4 Adducts of Nucleic Acids (9) Borgeat, P., and Samuelsson, B. (1979) Arachidonic acid metabolism in polymorphonuclear leukocytes: Unstable intermediate in formation of dihydroxy acids. Proc. Natl. Acad. Sci. U.S.A. 76, 3213-3217. (10) Haeggstrom, J. Z. (2000) Structure, function, and regulation of leukotriene A4 hydrolase. Am. J. Respir. Crit. Care Med. 161, S25-S31. (11) Lam, B. K., and Austen, F. K. (2000) Leukotriene C4 synthase. A pivotal enzyme in the biosynthesis of the cysteinyl leukotrienes. Am. J. Respir. Crit. Care Med. 161, S16-S19. (12) Sala, A., Bolla, M., Zarini, S., Muller-Peddinghaus, R., and Folco, G. (1996) Release of leukotriene A4 versus leukotriene B4 from human polymorphonuclear leukocytes. J. Biol. Chem. 271, 1794417948. (13) Blair, I. A. (2001) Lipid hydroperoxide-mediated DNA damage. Exp. Gerontol. 36, 1473-1481. (14) Pavanello, S., and Clonfero, E. (2000) Biological indicators of genotoxic risk and metabolic polymorphisms. Mutat. Res. 463, 285-308. (15) Friedberg, E. C. (1996) Relationships between DNA repair and transcription. Annu. Rev. Biochem. 65, 15-42. (16) Reiber, D. C., and Murphy, R. C. (2000) Covalent binding of LTA4 to nucleosides and nucleotides. Arch. Biochem. Biophys. 379, 119126. (17) Carrier, D. J., Bogri, T., Cosentino, G. P., Guse, I., Rakhit, S., and Singh, K. (1988) HPLC studies on leukotriene A4 obtained from the hydrolysis of its methyl ester. Prostaglandins, Leukotrienes Essent. Fatty Acids 34, 27-30. (18) Lee, S. H., Rindgen, D., Bible, R. H. J., Hajdu, E., and Blair, I. A. (2000) Characterization of 2′-deoxyadenosine adducts derived from 4-oxo-2-nonenal, a novel product of lipid peroxidation. Chem. Res. Toxicol. 13, 565-574. (19) Crain, P. F. (1990) Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol. 193, 782790. (20) Pomerantz, S. C., and McCloskey, J. A. (1990) Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry. Methods Enzymol. 193, 796-824. (21) Cantley, L. C. J., and Josephson, L. (1976) A slow interconversion between active and inactive states of the (Na-K)ATPase. Biochemistry 15, 5280-5287. (22) Haslett, C., Guthrie, L. A., Kopaniac, M. M., Johnson, R. B., and Henson, P. M. (1985) Modulation of multiple neutrophil functions


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by preparative methods or trace contamination of bacterial lipopolysaccharide. Am. J. Pathol. 119, 101-110. Rance, M. (1987) Improved techniques for homonuclear rotatingframe and isotropic mixing experiments. J. Magn. Reson. 74, 557-564. Shaka, A. J., Lee, C. J., and et al. (1988) Iterative schemes for bilinear operators-application to spin decoupling. J. Magn. Reson. 77, 274-293. Marion, D., Ikura, M., and et al. (1989) Rapid recording of 2D NMR-spectra without phase cyclingsapplication to the study of hydrogen-exchange in proteins. J. Magn. Reson. 85, 393-399. Aue, W. P., Bartholdi, E., and et al. (1976) 2-Dimensional spectroscopy - application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229-2246. Nagayama, K., Kumar, A., and et al. (1980) Experimentaltechniques of two-dimensional correlated spectroscopy. J. Magn. Reson. 40, 321-334. Bax, A., and Freeman, R. (1981) Investigation of complex networks of spin-spin coupling by two-dimensional NMR. J. Magn. Reson. 44, 542-561. Braunschweiler, L., and Ernst, R. R. (1983) Coherence transfer by isotropic mixing-application to proton correlation spectroscopy. J. Magn. Reson. 53, 521-528. Singer, B., and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York. Selzer, R R., and Elfarra, A. A. (1996) Synthesis and biochemical characterization of N1-, N2-, and N7-guanosine adducts of butadiene monoxide. Chem. Res. Toxicol. 9, 126-132. Gregson, J. M., and McCloskey, J. A. (1997) Collision-induced dissociation of protonated guanine. Int. J. Mass Spectrom. Ion Processes 165/166, 475-485. Borgeat, P., and Samuelsson, B. (1979) Arachidonic acid metabolism in polymorphonuclear leukocyte effects of ionophore A23187. Proc. Natl. Acad. Sci. U.S.A. 76, 2148-2152. Peters-Golden, M., and Brock, T. G. (2000) Intracellular compartmentalization of leukotriene biosynthesis. Am. J. Respir. Crit. Care Med. 161, S36-S40. Tomasz, M. (1970) Extreme lability of the C-8 proton: A consequence of 7-methylation of guanine residues in model compounds and in DNA and its analytical application. Biochim. Biophys. Acta 199, 18-28.

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