Nitrating Reactive Nitric Oxygen Species Transform Acetaminophen to

Laubach, V. E., Shesely, E. G., Smithies, O., and Sherman, P. A. (1995) Mice lacking inducible nitric oxide synthase are not resistant to ...
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Chem. Res. Toxicol. 2000, 13, 891-899

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Nitrating Reactive Nitric Oxygen Species Transform Acetaminophen to 3-Nitroacetaminophen Vijaya M. Lakshmi,† Fong Fu Hsu,‡ Bernard B. Davis,† and Terry V. Zenser*,†,§ VA Medical Center, Division of Geriatric Medicine, and Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63125-4199, and Department of Medicine, Washington University, St. Louis, Missouri 63130 Received May 24, 2000

Nitrating reactive nitric oxygen species (RNOS) elicit many of the deleterious effects of the inflammatory response. Their high reactivity and short half-life make RNOS analysis difficult. Reaction of acetaminophen (APAP) with RNOS generated by various conditions was evaluated by HPLC. When [14C]APAP was incubated at pH 7.4, the same new product (3NAP) was produced by at least three separate pathways represented by the following conditions: myeloperoxidase oxidation of NO2-, NO2Cl, and ONOO- or Sin-1. Diethylamine NONO and spermine NONO did not convert APAP to 3NAP. 3NAP was stable at pH 5, 7.4, or 9, and at pH 7.4 with ONOO-, spermine NONO, Sin-1, or H2O2. HOCl transformed 3NAP, which was prevented by APAP, ascorbic acid, taurine, or NO2-. ONOO--derived 3NAP was identified by 1H NMR as 3-nitroacetaminophen or 3-nitro-N-acetyl-p-aminophenol, and the product mass was verified by EI/ESI mass spectrometry. Human polymorphonuclear neutrophils incubated with [14C]APAP and stimulated with β-phorbol 12-myristate 13-acetate produced 3NAP in the presence of NO2-. Neutrophil 3NAP formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO2-. Spermine NONO supported 3NAP formation by stimulated cells in the absence of NO2-. Results demonstrate that 3NAP is a product of nitrating RNOS generated by at least three separate pathways and may be a biomarker for nitrating mediators of inflammation.

Introduction Nitrating reactive nitric oxygen species (RNOS)1 are thought to contribute to the deleterious effects of inflammation in a variety of disease states (1-5). Upregulation of the inducible isoform of nitric oxide synthase (iNOS) during inflammation can produce high levels of nitric oxide (NO) (6). RNOS can be derived from a series of complex reactions involving NO with either oxygen or superoxide (7). RNOS include NO, peroxynitrite anion (ONOO-), dinitrogen trioxide (N2O3), and nitrogen dioxide radical (NO2•). Nitrite (NO2-) is a relatively stable NO product and a major end product and is used in combination with nitrate (NO3-) as a measure of the extent of iNOS upregulation. Levels of NO2- in healthy humans range from 0.5-3.6 µM in plasma (8) to 30-210 µM in saliva (9). Concentrations of NO2- as high as 0.1 mM are found in respiratory fluid and gastric juice as well as plasma during inflammation (9). * To whom correspondence should be addressed: VA Medical Center (GRECC/11G-JB), St. Louis, MO 63125-4199. Phone: (314) 894-6510. Fax: (314) 894-6614. E-mail: [email protected]. † VA Medical Center and Division of Geriatric Medicine, St. Louis University School of Medicine. ‡ Washington University. § Department of Biochemistry, St. Louis University School of Medicine. 1 Abbreviations: RNOS, reactive nitric oxygen species; iNOS, inducible nitric oxide synthase; NO, nitric oxide; ONOO-, peroxynitrite anion; NO2•, nitrogen dioxide radical; APAP, acetaminophen; DETAPAC, diethylenetriaminepentaacetic acid; PMA, β-phorbol 12-myristate 13-acetate; DEA-NONO, diethylamine NONOate; Sin-1, 3-morpholinosydnonimine; ESI, electrospray ionization; CAD, collisionally activated dissociation; EI, electron impact.

Effects of RNOS can include nitrosation, nitration, and oxidation (7). Nitrosation of primary amines results in the reaction of diazonium ions with water and subsequent deamination. Deamination of DNA by RNOS can lead to mispairing, depurination, and strand breakage, contributing to mutagenesis (5, 10). ONOO- nitrates certain phenolic compounds such as tyrosine to yield 3-nitrotyrosine. A considerable amount of evidence has accumulated suggesting that nitration of LDL, formation of 3-nitrotyrosine, is an important event in atherogenesis (11, 12). Nitration of certain tyrosine residues may exert dramatic physiologic effects by altering kinase reactions and subsequent signaling pathways (7). At least three pathways generate RNOS capable of nitrating tyrosine. As mentioned above, intermediates derived from ONOO- produce 3-nitrotyrosine (13). The same product can be derived from human polymorphonuclear neutrophils by a myeloperoxidase-catalyzed H2O2dependent oxidation of NO2- to NO2•-like radicals (14). Nitryl chloride (NO2Cl) also reacts with tyrosine to form 3-nitrotyrosine. NO2Cl is formed by the reaction of hypochlorous acid (HOCl) with NO2- (15). HOCl is formed by myeloperoxidase-mediated oxidation of chloride anion. 3-Nitrotyrosine is used as a marker for RNOS. Acetaminophen (APAP), a phenolic compound, is a popular analgesic antipyretic drug tolerated well at the recommended oral adult dose of 300-1000 mg for adults and children 12 years of age and older (16). Following a therapeutic dose, peak plasma concentrations occur at 0.5-2 h and range up to 0.2 mM APAP. APAP is rapidly and completely absorbed from the gastrointestinal tract

10.1021/tx000115g CCC: $19.00 © 2000 American Chemical Society Published on Web 08/10/2000

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with 90-100% of it recovered in urine within 24 h. A small amount of APAP is excreted unchanged in urine, and about 90% is excreted after conjugation in liver as an O-glucuronide or sulfate. Bioactivation by cytochrome P450 contributes to hepatotoxicity. APAP is also a substrate for peroxidases, which catalyze one- and/or twoelectron oxidation (17-19). Determination of the role of RNOS in disease processes depends on their identification and measurement in lesions of tissues or in biological fluids from individuals affected by or at risk for a specific disease. The high reactivity and short half-life of RNOS make their analysis difficult. An indirect measure of RNOS can be achieved by assessing products formed by their reaction with a particular target molecule. 3-Nitrotyrosine and NO2-/ NO3- have been used as biomarkers to assess increased levels of RNOS. However, NO2-/NO3- determinations reveal little about the RNOS and can be influenced by diet (9). While NO has a very short half-life, 3-nitrotyrosine levels in tissue likely reflect an estimate of steady-state values over time and protein turnover. NO2-/ NO3- determinations utilize body fluids, while 3-nitrotyrosine measurements employ protein hydrolysates from tissue or serum. Biomarkers are useful in interpreting the effects of treatment variables such as pharmacological intervention, lifestyle habits, nutritional deficits, and nutritional supplements. This study was designed to assess the in vitro effects of different RNOS on APAP, a popular over-the-counter drug, and apply these results to intact cells with the ultimate goal of finding biomarkers for nitrating RNOS that can be utilized in humans. The concentrations of NO2- and APAP that are used are those expected during an inflammatory response or after ingestion of the recommended dose of APAP (9, 16).

Experimental Procedures Materials. [ring-U-14C]APAP (46 mCi/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). APAP, NaNO2, NaOCl, H2O2, cytochrome c (horse heart), superoxide dismutase (bovine erythrocytes), catalase (bovine liver), ascorbic acid, diethylenetriaminepentaacetic acid (DETAPAC), NaCN, and β-phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Co. (St. Louis, MO). Diethylamine NONOate (DEA-NONO), spermine NONOate, 3-morpholinosydnonimine (Sin-1), and myeloperoxidase from human polymorphonuclear leukocytes (180-220 units/mg of protein) were purchased from Calbiochem (San Diego, CA). Alkaline solutions of ONOO- were prepared from acidified NO2and H2O2, and quantitated spectrophotometrically (302 ) 1.67 mM-1 cm-1) as described previously (13). Stock solutions were kept at -70 °C. The 3NAP standard was chemically synthesized from p-aminophenol. The nitrated product was prepared by mixing p-aminophenol with concentrated HNO3 and H2SO4 at 0-5 °C for 1 h. The purified product was added to acetic anhydride and heated under reflux for 2 h. Silica gel columnpurified 3NAP was identified by NMR/MS analysis. Ultima-Flo AP was purchased from Packard Instruments (Meriden, CT). Reaction of APAP with RNOS. [14C]APAP (0.06 mM) was incubated in 100 mM potassium phosphate buffer (pH 7.4), containing 0.1 mM DETAPAC in a total volume of 0.1 mL at 37 °C. For incubation with myeloperoxidase, 1 µg/mL peroxidase was added in the presence or absence of the indicated concentration of NO2-, and the reaction was started by addition of 0.05 mM H2O2. Incubations with DEA-NONO, spermine-NONO, ONOO-, Sin-1, or HOCl were started immediately following their addition. The pH was checked at the conclusion of these incubations and did not change by more than ( 0.1 pH unit. Incubation times were 10 min for myeloperoxidase, 1 min for

Lakshmi et al. ONOO-, and 30 min for other conditions. Blank values were obtained in the absence of either RNOS generating agent or H2O2. The reaction was stopped by adding 0.1 mL of 10 mM ascorbic acid and/or 10 µg/mL catalase, and the mixture was placed on ice. Formation of 3NAP was assessed by HPLC as described below. Preparation of Human Polymorphonuclear Neutrophils. Human blood was mixed with EDTA (0.2% final concentration) and immediately layered over an equal volume of neutrophil isolation media from Robins Scientific Corp. (Sunnyvale, CA). Neutrophils were isolated by centrifugation using the manufacturer’s specifications. Red blood cell contamination was eliminated by hypotonic lysis at 4 °C. Cells were resuspended in Hank’s balanced salt solution at a density of 10 × 106 cells/ mL. The Human Studies Committee approved the use of human polymorphonuclear neutrophils, and informed consent was obtained from participants. To assess PMA responsiveness of different preparations of cells, superoxide production was assessed (20). Superoxide-specific reduction of cytochrome c was assessed spectrophotometrically (550 ) 21.1 mM-1 cm-1) and was inhibited by superoxide dismutase (10 µg/mL). Values observed with cells in the absence of PMA were considered as blanks. Metabolism of APAP by Neutrophils. Neutrophils (2 × 106 cells in 0.3 mL) were incubated in 12 mm × 75 mm polypropylene tubes at 37 °C for 30 min. Cells were incubated with 0.02 mM [14C]APAP in the presence or absence of 30 ng/ mL PMA. Blank values were obtained in the absence of cells. The reaction was stopped by placing the mixture on ice, the mixture sonicated three times for 15 s, and 0.3 mL of methanol added. The supernatant was evaporated, and the residue was dissolved in 0.1 mL of methanol and analyzed by HPLC. HPLC Analysis of Metabolites. Metabolites were assessed using a Beckman HPLC system with System Gold software, which consisted of a 5 µm, 4.6 mm × 150 mm C-18 ultrasphere column attached to a guard column. The mobile phase contained 20 mM ammonium acetate buffer (pH 7.0) in 5% acetonitrile from 0 to 2 min, 5 to 10% from 2 to 10 min, 10 to 50% from 20 to 25 min, and 50 to 5% from 35 to 45 min, at a flow rate of 1 mL/min. Using these solvent conditions, the 3NAP standard eluted at 20.1 min. The radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector. Data are expressed as a percentage of total radioactivity or picomoles recovered by HPLC. The amount of APAP that was metabolized was determined by subtracting the percentage of APAP recovered (unmetabolized) from 98% (purity of APAP). Mass Spectral Analysis. A Finnigan SSQ-7000 GC/MS system (interfaced with a Varian 3300 gas chromatograph), controlled by Finnigan ICIS software operated on a DEC alpha station, was used for the analysis. The machine was tuned by auto-tune mode and further tuned manually with PFTFA as the calibrant. The TMS derivatives of both standard and biological samples of 3NAP were separated by a J & W DB-1 capillary column (15 m × 0.25 mm, 0.25 µm film thickness) operated in a splitless injection mode. The injector, transfer line, and source temperatures were set at 250, 250, and 200 °C, respectively. The GC oven was programmed at 100 °C for 1 min, increased to 220 °C at 12 °C/min, and kept at 220 °C for 1 min. A standard electron energy at 70 eV (300 µA) was used. The multiplier voltage was set at 2 kV (the detector is operated with a conversion dynode at 15 kV). Electrospray ionization (ESI) MS analyses were performed on a Finnigan (San Jose, CA) TSQ-7000 triple-stage quadrupole spectrometer equipped with a Finnigan ESI source and controlled by Finnigan ICIS software operated on a DEC alpha workstation. Samples were loop injected onto the ESI source with a Harvard syringe pump at a flow rate of 5 µL/min. The electrospray needle and the skimmer were at ground potential, and the electrospray chamber and the entrance of the glass capillary were at 4.4 kV. The heated capillary temperature was 200 °C. For collisionally activated dissociation (CAD) tandem mass spectra, the collision gas was argon (2.2-2.5 mTorr), and

3-Nitroacetaminophen Formation

Chem. Res. Toxicol., Vol. 13, No. 9, 2000 893 hundred twenty-eight transients were signal averaged with a recycle delay of 5 s and a 45° tip. The time domain data were processed on a SUN workstation using VNMR software. A linebroadening parameter of 0.5 Hz was used for the Fourier transformation. Chemical shifts were referenced to TMS at 0.0 ppm.

Results

Figure 1. HPLC analysis of myeloperoxidase metabolism of 0.06 mM [14C]APAP in the absence and presence of 0.1 mM NaNO2. Reactions were started by addition of 0.05 mM H2O2. A2 and A3 refer to metabolites with masses corresponding to an APAP dimer and trimer, respectively. the collision energy was set at 22 eV. Product ion spectra were acquired in the profile mode at a scan rate of one scan per 3 s. NMR Analysis. Samples were prepared in 100% DMSO-d6 (Aldrich gold label) under a dry nitrogen purge. About 30 µg of the sample was dissolved in 1 mL of DMSO and the mixture transferred into high-quality 5 mm tubes which were capped under nitrogen. NMR spectra were acquired on a Varian Inova500 instrument with a proton resonance frequency of 499.97 MHz with an INVERSE probe (1H 90 pulse of 9 µs). One

Metabolism of 0.06 mM [14C]APAP by myeloperoxidase was assessed in the presence of 0.05 mM H2O2. Under the conditions of this incubation, approximately 63% of APAP was metabolized (Figure 1, top panel). Previous studies have identified polymers as the major products of APAP peroxidatic metabolism and have characterized these products in detail (21, 22). The products eluting at 15 (dimer) and 24.8 min (trimer) represented 14 and 19% of the total radioactivity recovered by HPLC, respectively. The masses of these myeloperoxidase products were determined by ESI/MS and the products not characterized further. Addition of 0.1 mM NO2- resulted in 73% metabolism of APAP and the formation of a new peak at 20.1 min (Figure 1, bottom panel). The new peak was labeled 3NAP and represented 7% of the total radioactivity recovered by HPLC. Dimer and trimer products were also observed in the presence of NO2-. The results suggest formation of the new product may involve RNOS. A variety of conditions, which generate RNOS, were evaluated for conversion of APAP to 3NAP (Table 1). When 0.06 mM APAP was incubated with myeloperoxidase in the presence of 0.05 mM H2O2 and 0.1 mM NO2-, 245 ( 21 pmol of 3NAP was formed. This reaction is thought to generate NO2• as a RNOS (14). Myeloperoxidase-mediated metabolism was prevented by 1 mM NaCN, a peroxidase inhibitor. In the presence of physiologic concentrations of chloride (100 mM), a myeloperoxidase substrate, 166 ( 23 pmol of 3NAP was observed. Taurine (0.5 mM), an HOCl scavenger, did not alter the metabolism in the absence or presence of chloride. DEANONO (0.2 mM) and spermine-NONO (1 mM) are both NO-donor molecules and are thought to generate a variety of RNOS, including N2O3 (23). Neither compound was effective in producing detectable levels of 3NAP. NO2Cl, generated by incubation of NO2- with HOCl (15), elicited a small amount of 3NAP (43 ( 4 pmol). A bolus addition of ONOO- was effective in eliciting formation of 3NAP. The amount of product observed with 0.1 mM ONOO- (467 ( 73 pmol) was more than that observed with myeloperoxidase (245 ( 21 pmol). Preincubation of ONOO- in buffer for 10 min prior to APAP addition failed

Table 1. Formation of 3NAP by Different Reactive Nitric Oxygen Speciesa conditions

3NAP formed (pmol)

myeloperoxidase, 0.05 mM H2O2, and 0.1 mM NaNO2 with 1 mM NaCN with 0.5 mM taurine with 100 mM NaCl with 100 mM NaCl and 0.5 mM taurine 0.2 mM DEA-NONO 1 mM spermine-NONO 0.1 mM NaNO2 and 0.1 mM HOCl 0.1 mM ONOOwith a 10 min preincubation before APAP addition 2 mM Sin-1

245 ( 21 0 208 ( 22 166 ( 23 146 ( 19 0 0 43 ( 4 469 ( 73 0 59 ( 7

RNOS NO2

NO, N2O3 NO, N2O3 NO2Cl ONOOONOO-

a Incubations contained 0.06 mM [14C]APAP, 100 mM potassium phosphate buffer (pH 7.4), and 0.1 mM DETAPAC in 0.1 mL and were incubated at 37 °C. Incubation times were 10 min for myeloperoxidase, 1 min for ONOO-, and 30 min for other conditions. 3NAP formation was analyzed by HPLC, and values represent means ( SEM. Some RNOS thought to be generated by each condition are indicated.

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Table 2. Stability of [14C]3NAPa conditions

[14C]3NAP remaining (nmol)

phosphate buffer pH 5.0 pH 7.4 pH 9.0 0.1 mM HOCl with 0.5 mM APAP with 0.5 mM ascorbic acid with 0.25 mM taurine with 0.1 mM NaNO20.1 mM ONOO1 mM spermine-NONO 2 mM Sin-1 0.1 mM H2O2

10 ( 0 10 ( 0 10 ( 0 6.3 ( 0.1 9.6 ( 0.1 10 ( 0 10 ( 0 9.6 ( 0 9.8 ( 0.1 9.7 ( 0.1 9 ( 0.1 10 ( 0

a [14C]3NAP (0.1 mM, 10 nmol) was incubated in 100 mM phosphate buffer at the indicated pHs for 18 h (0.1 mL). Additional incubations were carried out in 100 mM phosphate buffer (pH 7.4) containing 0.1 mM DETAPAC for 5 min at 37 °C. Reaction mixtures containing HOCl were stopped by addition of 1 mM methionine. Samples were analyzed by HPLC, and values represent means ( SEM.

Table 3. 1H NMR Spectral Parameters for Acetaminophen and 3-Nitroacetaminophen

Figure 2. EI mass spectra of the 3-nitroacetaminophen-TMS2 standard (A) and the biological product isolated from PMAstimulated human polymorphonuclear neutrophil incubations (B). ESI tandem mass spectrum of the 3-nitroacetaminophen standard (C). Scheme 1 illustrates the fragmentation pattern of the corresponding EI or ESI mass spectra.

to demonstrate product formation. Sin-1 elicits a sustained release of ONOO- and was also evaluated (24). At 2 mM Sin-1, a small amount of 3NAP was produced (59 ( 7 pmol). The stability of 3NAP was assessed to determine if this could explain its low yield under certain conditions used above. An equal molar concentration of HOCl (0.1 mM) resulted in an ∼40% loss of [14C]3NAP (Table 2). A new product, which eluted in front of 3NAP, was observed, but not characterized further. Loss of 3NAP was prevented by 0.5 mM APAP, 0.5 mM ascorbic acid, or 0.25 mM taurine. Inclusion of an equal molar concentration of NO2- to generate NO2Cl also prevented HOCl loss of 3NAP. ONOO- (0.1 mM) did not cause depletion of [14C]3NAP. [14C]3NAP was also stable in the presence of 2 mM Sin-1, 1 mM spermine-NONO, and 0.1 mM H2O2. To determine its pH stability in 100 mM potassium phosphate buffer, 3NAP was incubated at pH 5, 7.4, and 9 for 18 h. No loss of 3NAP was observed at any pH. Except for HOCl, 3NAP was stable under the conditions that were tested. Thus, the stability of 3NAP did not influence its synthesis reported in Table 1. 1H NMR analyses were used to determine the structure of the new product derived from the reaction of ONOOwith APAP. 1H NMR spectral parameters for APAP and

the new product are given in Table 3. The D2O-exchangeable amide NH proton at position 1 of APAP (9.133 ppm) was detected in the new product (10.065 ppm). The CH3 protons of APAP (2.000 ppm) at position 7 were also detected in the new product (2.030 ppm). A single D2Oexchangeable phenolic proton at position 4 was present in both APAP (9.650 ppm) and the 20.1 min peak (10.656 ppm). While both protons meta to OH in position 4 are present along with the ortho proton in position 5, the new product is missing the ortho proton in position 3. Thus, the 1H NMR spectrum of the new product is consistent with that expected for 3-nitroacetaminophen or 3-nitroN-acetyl-p-aminophenol (3NAP). The ONOO--derived product was analyzed further by MS (Figure 2). Using electron impact (EI) MS, the trimethylsilyl (TMS) derivative of the synthetic 3NAP yields the M+• ion at m/z 340, corresponding to a di-TMS molecule (3-nitroacetaminophen-TMS2) (Figure 2A). The derivative gives a prominent ion at m/z 325 by loss of CH3-. Further losses of TMSOH and TMSH from m/z 325 give rise to m/z 235 and 251 ions, respectively. The ion corresponding to an NO2- loss is observed at m/z 294. The postulated fragmentation pathways for ion formation are illustrated in Scheme 1. These results are consistent with the synthetic product derived from the reaction of ONOO- with APAP being 3-nitroacetaminophen. This structural assignment is further supported by the ESI mass spectrum (Figure 2C). In negative ion mode, the synthetic 3NAP yields a prominent deprotonated molecular ion (M - H)- at m/z 195, which gives abundant

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Scheme 1. Fragmentation Pathways Observed for (A) Negative Ion ESI Tandem Mass Spectrometry of 3-Nitroacetaminophen and (B) the EI Mass Spectrum of the 3-Nitroacetaminophen-TMS2 Derivative

fragment ions upon CAD. The tandem mass spectrum yields fragment ions at m/z 150 and 165 corresponding to (M - NO2)- and (M - H - NO)-, respectively. Ions at m/z 178 and 152 arise from loss of OH- and CH3CO-, respectively. Further loss of NO from m/z 152 gives an ion at m/z 122. The ion at m/z 46, corresponding to an NO2- ion, was also observed. These analyses further confirmed that the structure of the new product is 3-nitroacetaminophen (3NAP). To assess the effect of RNOS generated by intact cells on 3NAP formation, human polymorphonuclear neutro-

phil metabolism of [14C]APAP was assessed with 30 ng/ mL PMA in the presence and absence of NO2- (Figure 3). Metabolism of APAP in the absence of NO2- was similar to that observed by myeloperoxidase in Figure 1A with metabolite peaks at 15.1, 24.6, and 25.8 min (Figure 3, top panel). In the presence of 1 mM NO2-, 3NAP formation was observed (Figure 3, bottom panel). This metabolite represented about 13% of the total radioactivity recovered by HPLC. In separate experiments, the material eluting at 19-21 min was collected, a TMS derivative prepared, and analysis performed by

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Lakshmi et al. Table 4. Formation of 3NAP by Human Polymorphonuclear Neutrophilsa

conditions

3NAP formed (pmol)

PMNs and APAP with PMA with 1 mM NO2with 1 mM NO2- and PMA with 1 mM NO2-, PMA, and NaCN with 1 mM NO2-, PMA, and catalase with 1 mM NO2-, PMA, and taurine with 1 mM NO2-, PMA, and superoxide dismutase with 0.1 mM NO2- and PMA with 0.1 mM NO2-, PMA, and taurine with PMA and spermine-NONO with PMA, spermine-NONO, and catalase with PMA, spermine-NONO, and taurine

0 0 520 ( 31 14 ( 14 89 ( 27 480 ( 23 558 ( 40 89 ( 20 104 ( 6 63 ( 6 8(5 77 ( 17

a Human polymorphonuclear neutrophils (PMNs; 2 × 106 cells) were incubated with 0.02 mM [14C]APAP in 0.3 mL of HBSS at 37 °C for 30 min. The following test agents were present at the indicated concentrations: 30 ng/mL PMA, 0.1 or 1 mM NO2-, 1 mM NaCN, 1 mM taurine, 330 munits/mL superoxide dismutase, 10 µg/mL catalase, and 0.25 mM spermine-NONO. When present, PMA and spermine-NONO were added 5 and 6 min, respectively, after the start of the 37 °C incubation. Formation of 3NAP was assessed by HPLC, and values represent means ( SEM.

Scheme 2. Three Pathways for 3NAP Formationa

Figure 3. Illustrated is the HPLC elution profile of incubations of human polymorphonuclear neutrophils with 0.02 mM [14C]APAP in the absence and presence of 1 mM NaNO2. PMA (30 ng/mL) was added 5 min after the incubations were started.

EI/MS. The mass spectrum of the metabolite from PMAstimulated neutrophils (Figure 2B) is identical to that of ONOO--derived 3NAP (Figure 2A). Thus, the metabolite isolated from neutrophils is 3-nitroacetaminophen. In addition, the material eluting at 19-21 min was collected and rechromatographed on the same gradient with HPLC buffer at pH 5.5, instead of pH 7.0. The rechromatographed sample eluted as a single peak at 26.2 min with a purity of >95%. This decrease in the pH of the HPLC buffer increased the elution time of 3NAP by 6 min, consistent with the lower pKa of phenols with an ortho-substituted nitro group (25). Further assessment of the formation of 3NAP by neutrophils indicated that it was not detected in the absence of either PMA or NO2- (Table 4). 3NAP formation in the presence of both PMA and 1 mM NO2- (520 ( 31 pmol) was completely inhibited by 1 mM NaCN and reduced by more than 80% with catalase. However, neither taurine (1 mM) nor superoxide dismutase altered 3NAP formation. Formation of 3NAP could also be detected with 0.1 mM NO2- (89 ( 20 pmol) which was also not altered by taurine. Spermine-NONO (0.25 mM) initiated 3NAP formation (63 ( 6 pmol) by PMAstimulated neutrophils in the absence of NO2- which was prevented by catalase, but not taurine.

a Mediators of inflammation (endotoxin and cytokines) induce iNOS, increasing the extent of NO synthesis from L-arginine (L-Arg). NO reacts with superoxide (O2•-) to form peroxynitrite anion (ONOO-) which can form several RNOS intermediates (i.e., NO2•), some of which can nitrate acetaminophen (APAP) to form 3NAP (pathway 1). NO2- is the major end product of NO and ONOO- metabolism. NADPH oxidase reduces molecular oxygen to O2•-, which dismutates to H2O2. Myeloperoxidase (MPO) oxidizes NO2- to a RNOS, NO2•-like radicals, that nitrates APAP (pathway 2). HOCl oxidizes NO2- to NO2-Cl (nitryl chloride) which reacts with APAP in forming 3NAP (pathway 3). [NO2•] denotes known and unknown intermediates. Inhibitors are available to prevent 3NAP formation by each pathway.

Discussion This is the first paper to demonstrate nitration of APAP to form 3NAP by RNOS associated with the inflammatory response (Table 1). 3NAP was formed by at least three separate pathways (Scheme 2) represented by the following conditions: myeloperoxidase oxidation of NO2-, NO2Cl, and ONOO-. With myeloperoxidase, a chloride concentration (100 mM) 1000-fold higher than that of NO2- only reduced the extent of 3NAP formation by 30%. Previous studies have demonstrated that oxidation of NO2- is preferred over chloride by myeloperoxidase (14). ONOO- was much more effective than either myeloperoxidase/NO2- or NO2Cl in producing 3NAP. Sin-1 produces a sustained release of NO and superoxide, causing continuous formation of ONOO- (24). The amount

3-Nitroacetaminophen Formation

of ONOO- produced by 2 mM Sin-1 should exceed the 0.1 mM ONOO- bolus addition (Table 1). However, 3NAP produced by Sin-1 was only about 13% of that observed with 0.1 mM ONOO-. As demonstrated with Sin-1 formation of 3-nitrotyrosine (26), secondary effects of Sin-1 byproducts (i.e., NO and superoxide) with APAP may cause a decreased level of 3NAP formation. NO donors, DEA-NONO and spermine-NONO, elicit controlled release of NO and did not produce 3NAP. Using very different reaction conditions (ambient temperature for a 12 h incubation), 1 mM spermine-NONO reacted with 1 mM tyrosine to produce a 0.3% yield of 3-nitrotyrosine (27). A proportional yield of 3NAP during our 30 min incubation would not be detectable using our HPLC method. The formation of 3NAP may occur by the same mechanism as formation of 3-nitrotyrosine. Peroxynitrite nitrates tyrosine solely through a radical mechanism, which involves reaction of tyrosine phenoxyl radical with NO2• (28). Oxidation of NO2- by myeloperoxidase also yields a NO2•-like radical (14), and a similar radical could be derived from NO2Cl (15). Dityrosine, a product of myeloperoxidase tyrosine metabolism (29), is indicative of a free radical-coupling reaction of two tyrosine phenoxyl radicals. Peroxidatic metabolism of APAP to phenoxyl radicals has been demonstrated (18), and the identification of polymeric products in the myeloperoxidase reaction mixtures is consistent with this conclusion. APAP reactions with ONOO- or NO2Cl also involve a dimer and trimer of APAP (not shown). The NO donorcontrolled release of NO results in the generation of nitrosation species such as N2O3, not NO2• (23). Thus, APAP phenoxyl radicals would be expected to react with NO2•, forming 3NAP by a radical mechanism. Human polymorphonuclear neutrophils were used as a representative component of the inflammatory response. These cells exhibit an oxidant burst following stimulation with PMA. Membrane-associated NADPH oxidase reduces molecular oxygen to superoxide anion which dismutates to form H2O2. Myeloperoxidase catalyzes the metabolism of H2O2 in the presence of reducing cosubstrates, such as NO2- (14). In the presence of NO2-, PMA-stimulated neutrophils metabolized APAP to 3NAP. This product was not observed in the absence of either PMA or NO2-. The lack of inhibition by taurine indicates that NO2Cl is not mediating 3NAP formation (Scheme 2, pathway 3). In addition, the lack of superoxide dismutase inhibition suggests ONOO--mediated APAP metabolism is also not contributing to 3NAP formation (pathway 1). Inhibition of 3NAP formation by catalase and NaCN is consistent with myeloperoxidase-mediated formation by pathway 2. Spermine-NONO, which did not directly elicit 3NAP formation (Table 1), supported 3NAP formation by PMA-stimulated cells in the absence of NO2-. The extent of 3NAP formation with spermineNONO was reduced by catalase, but not taurine. This is consistent with one-electron oxidation of NO to NO+ with immediate hydration to NO2- (30), which is oxidized by neutrophil myeloperoxidase (Scheme 2, pathway 2). Results suggest 3NAP formation during an inflammatory response in vivo. Direct chemical synthesis of this biological product was achieved by nitration and acetylation of p-aminophenol. A characteristic property of 3NAP shared by other phenols with an ortho-substituted nitro group is intramo-

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lecular hydrogen bonding between these two functional groups. Ionization of the hydroxyl serves to extend the conjugation of the aromatic ring and shifts the secondary UV maximum of 3NAP from 370 nm at pH 3.5 to 430 nm at pH 9.5. This characteristic is shared with 3-nitrotyrosine in which the pKa of the phenol for tyrosine is about 10, while that of 3-nitrotyrosine is shifted to about 6.8. 3-Nitrotyrosine exhibits UV absorption maxima at pH 3.5 and 10 of 357 and 430 nm, respectively (25). 3NAP, formed by reaction of APAP with NO2- at acidic pH, is a clinical assay for diagnosing APAP overdose in serum samples (31). 3NAP has been termed 2-nitro-4acetamidophenol and characterized by its absorption spectra (32, 33). Its first reported synthesis may have been in 1891 (34). We can find no prior evidence for structural identification of 3NAP, using techniques similar to those in this report. The lack or slight formation of 3NAP observed with some reagents is not due to their reaction with 3NAP (Table 2). A variety of biological antioxidants, such as ascorbic acid, taurine, and glutathione, are oxidized by HOCl (35). Thus, the inhibition of 3NAP transformation by APAP, ascorbic acid, taurine, and NO2- is consistent with these reagents competing with 3NAP for reaction with HOCl. It is likely that APAP and biological antioxidants would protect 3NAP from loss by HOCl in vivo, and that 3NAP would be recovered relatively unaltered in biological samples. Recent studies have demonstrated a similar stability profile for 3-nitrotyrosine (36). With the recognition that 3-nitrotyrosine represents an in vivo biomarker of nitrating RNOS-associated inflammatory conditions (1-5), a number of methods were developed for its measurement (37). Protein-associated 3-nitrotyrosine can be analyzed by either immunohistochemical or enzyme-linked immunosorbent assays. These measurements allow tissue localization and, while only semiquantitative, have demonstrated a role of RNOS in tissue injury associated with many diseases. HPLC methods can be more sensitive and specific. However, HPLC analysis provides no structural information about the analyte and can be confounded by coeluting but structurally different molecules. A quantitative, selective, sensitive stable isotope dilution GC/MS method has been developed to measure the level of 3-nitrotyrosine (38). The primary source of artifactual nitration with these methods is due to tissue NO2-/NO3- (the level of which is expected to be elevated in inflammation). This is a special problem with methods requiring acid hydrolysis of proteins to amino acids, because of the high efficiency of acid-catalyzed NO2-/NO3- nitration. Antibody detection techniques using peroxidases or acidic conditions also generate artifactual increases in the level of 3-nitrotyrosine. While 3-nitrotyrosine measurements are taken with serum proteins, the use of atherosclerotic and other tissues requires invasive techniques. 3-Nitrotyrosine levels in protein are likely to reflect its average accumulation over time and protein turnover rate. The current method for assessing 3NAP has been modified, using a stable isotope (3-nitro-N-acetyl-paminophenol-d3) to increase precision. The pharmacokinetics of APAP specify a relatively narrow window during which it is available to react with nitrating RNOS before being excreted in urine (16). 3NAP measurements are thought to complement and extend current measurements of RNOS, using 3-nitrotyrosine. 3-Nitrotyrosine

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analysis can require invasive techniques, and is sensitive to artifactual influence, and its MS determination (38) is much more technically difficult than the method for 3NAP. 3NAP can serve as a quantifiable, real-time biomarker for nitrating RNOS associated with inflammation. 3NAP is formed by nitrating RNOS generated by at least three separate pathways (Scheme 2). PMA-stimulated neutrophils metabolized APAP to form 3NAP. Results are consistent with this reaction involving myeloperoxidase (Scheme 2, pathway 2). 3NAP could be useful in identifying different nitrating RNOS (i.e., ONOO-) and their pathways for generation in experiments utilizing knockout mice (i.e., iNOS or myeloperoxidase) or animals treated with selective antiinflammatory drugs (i.e., peroxynitrite decomposition catalysts or superoxide dismutase mimics) (39-41).

Acknowledgment. We thank Cindee Rettke and Priscilla DeHaven for excellent technical assistance. This work was supported by the Department of Veterans Affairs (T.V.Z.) and National Cancer Institute Grant CA72613 (T.V.Z.). MS was performed at the Mass Spectrometry Resource Center, Washington University School of Medicine, through NIH Grants RR-00954 and AM20579. 1H NMR analysis was performed by Dr. Narayana Mysore, Shell Chemicals, subsidiary of Shell Oil Co., Houston, TX.

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