N-Acetylbenzidine− DNA Adduct Formation by Phorbol 12-Myristate

in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pa...
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Chem. Res. Toxicol. 2000, 13, 785-792

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N-Acetylbenzidine-DNA Adduct Formation by Phorbol 12-Myristate-Stimulated Human Polymorphonuclear Neutrophils 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 February 16, 2000

N′-(3′-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pathways leading to dGp-ABZ formation in human peripheral white blood cells. [3H]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism required H2O2, but not NaCl. While transformation by HOCl was completely inhibited by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduced 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product with MPO, but not with HOCl. Previously identified oxidation products of ABZ, N′-hydroxy-N-acetylbenzidine or 4′-nitro-4-acetylaminobiphenyl, were not detected. With DNA or dGp present, a new product was observed that corresponded to synthetic dGpABZ in its HPLC elution profile, in nuclease P1 hydrolysis to dG-ABZ, and in 32P-postlabeling analysis. The HOCl-derived adduct was identified by electrospray ionization mass spectrometry, with collision-activated dissociation, as dGp-ABZ. Metabolism of [3H]ABZ by peripheral blood cells was stimulated about 3-fold with 30 ng/mL β-phorbol 12-myristate 13-acetate (PMA). Using 32P-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if either 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct formation. With dGp, dGp-ABZ formation could be detected with as little as 0.12 × 106 neutrophils. Using specific chromatographic and enzymatic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.

Introduction N′-(3′-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ)1 is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine (1, 2). Moreover, adduct levels in human peripheral white blood cells correlate with levels in exfoliated bladder cells. The sum of the amounts of urinary benzidine metabolites also correlates with the level of dGp-ABZ in both peripheral white blood cells and exfoliated bladder cells. This is the only adduct detected in rats, mice, or hamsters treated with either benzidine or N-acetylbenzidine (ABZ) and the major adduct detected in rat liver following administration of * 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]. † St. Louis University School of Medicine. ‡ Washington University. 1 Abbreviations: ABZ, N-acetylbenzidine; dG, 2′-deoxyguanosine; dGp, 2′-deoxyguanosine 3′-monophosphate; dpG, 2′-deoxyguanosine 5′monophosphate; dGp-ABZ, N′-(3′-monophosphodeoxyguanosin-8-yl)-Nacetylbenzidine; DETAPAC, diethylenetriaminepentaacetic acid; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; DMSO, dimethyl sulfoxide; ESI/MS, electrospray ionization mass spectrometry; CAD, collision-activated dissociation; HOCl, hypochlorous acid; MPO, myeloperoxidase; PMA, β-phorbol 12-myristate 13-acetate.

N,N′-diacetylbenzidine (3, 4). dGp-ABZ causes frameshift mutations, point mutations, and sister chromatid exchanges in in vitro systems, and mutations in ras protooncogenes in tumors (5-8). Thus, dGp-ABZ may play an important role in the initiation of bladder cancer. Determining the metabolic pathways leading to dGp-ABZ formation will contribute to a better understanding of aromatic amine carcinogenesis, and provide strategies for bladder cancer prevention, biomarkers, and risk assessment. Polymorphonuclear neutrophils express peroxidatic activity (9). During phagocytosis or following addition of β-phorbol 12-myristate 13-acetate (PMA) to neutrophils, a respiratory burst is triggered in which membraneassociated NADPH oxidase reduces molecular oxygen to superoxide anion which dismutates to form H2O2. Myeloperoxidase (MPO) present in neutrophils uses H2O2 to produce cytotoxic oxidants, such as hypochlorous acid (HOCl) in the presence of Cl-. Following addition of phorbol myristate acetate, benzidine and other arylamines are activated to bind neutrophil DNA (10). Although specific adducts were never identified, subsequent studies demonstrated that binding could be initiated by incubating aromatic amines with MPO and H2O2.

10.1021/tx0000320 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/20/2000

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HOCl can also activate arylamines to bind DNA (11). Thus, arylamine oxidation by MPO may be direct and/or indirect involving hypochlorous acid. ABZ is the major metabolite observed in urine (12) and plasma of workers exposed to benzidine. ABZ is a substrate for prostaglandin H synthase, and peroxidatic metabolism of ABZ elicits mutations in Salmonella typhimurium (13, 14). Prostaglandin H synthase is expressed at high levels in bladder cells (15, 16) and can activate ABZ to form dGp-ABZ (17). Because the same adduct is formed in bladder cells and in white blood cells, similar mechanisms of adduct formation might exist in both cell types, with white blood cells as a surrogate biomarker. This study was designed to determine whether in vitro activation of ABZ by human polymorphonuclear neutrophils can lead to the dGp-ABZ reported in human peripheral white blood cells.

Experimental Procedures Materials. [3H]Benzidine (180 mCi/mmol) was purchased from Chemsyn (Lenexa, KS). ABZ and [3H]ABZ were synthesized by acetylation of benzidine using glacial acetic acid with a final product purity of greater than 98% (18). Carrier-free [γ-32P]ATP (7000 Ci/mmol) was purchased from ICN (Irvine, CA). Benzidine-free base and hydrochloride salt, taurine, sodium hypochlorite (NaOCl), H2O2, cytochrome c, superoxide dismutase, glutathione, ascorbic acid, diethylenetriaminepentaacetic acid (DETAPAC), proteinase K, DNA (calf thymus, type I), 2′deoxyguanosine 3′-monophosphate (dGp), 2′-deoxyguanosine 5′monophosphate (dpG), 2′-deoxyguanosine (dG), micrococcal nuclease (EC 3.1.31.1), potato apyrase (grade I, EC 3.6.1.5), and PMA were purchased from Sigma Chemical Co. (St. Louis, MO). Nuclease P1 (EC 3.1.30.1), spleen exonuclease (EC 3.1.16.1), and T4 polynucleotide kinase (EC 2.7.1.78) were from Boehringer Mannheim (Indianapolis, IN). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Aldrich Chemical Co. (Milwaukee, WI). The dGp-ABZ standard was prepared as previously described (19). MPO from human polymorphonuclear leukocytes (180-220 units/mg of protein) was purchased from Calbiochem (La Jolla, CA). Ultima-Flo AP was purchased from Packard Instruments (Meriden, CT). Caution: N-Acetylbenzidine is carcinogenic and should be handled in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (20). Metabolism of ABZ by MPO. [3H]ABZ (0.06 mM, 0.2 µCi, 1% ethanol) was added to 100 mM phosphate buffer (pH 5.5) containing 1 µg/mL MPO, 0.1 mM DETAPAC, and 100 mM NaCl in a total volume of 0.1 mL. The reaction was started by the addition of H2O2 to a final concentration of 0.1 mM and the mixture incubated at 37 °C for 5 min. Blank values were obtained in the absence of either MPO or H2O2. The reaction was stopped by adding 2 volumes of dimethylformamide containing 2 mM ascorbic acid and the mixture placed on ice and centrifuged to obtain a clear supernatant. Metabolism was assessed in this supernatant by HPLC as described below. Reaction mixtures containing 1 mg/mL DNA or dGp were extracted twice with 2 volumes of ethyl acetate, and the aqueous fraction was processed as described below. The ethyl acetate extracts were pooled, evaporated to dryness, redissolved in 0.1 mL of an 80:20 methanol/dimethylformamide mixture, and analyzed by HPLC. In experiments where buffer was passed over Chelex 100 resin to remove transition metal ions, no effect on metabolism was observed. Reaction of ABZ with HOCl. [3H]ABZ (0.06 mM, 0.2 µCi, 1% ethanol) was added to 100 mM phosphate buffer (pH 5.5) containing 0.1 mM DETAPAC in a total volume of 0.1 mL. The reaction was started by the addition of NaOCl to a final concentration of 0.1 mM and the mixture incubated at 37 °C for 10 min. A 1 mM stock solution of NaOCl was made in 0.1 N NaOH. Blank values were obtained in the absence of NaOCl.

Lakshmi et al. Incubations were stopped and samples processed as described above for MPO. Preparation of Human Polymorphonuclear Neutrophils. Human blood was mixed with EDTA (0.2% final concentration) and immediately layered over an equal volume of PMN 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 use of human polymorphonuclear neutrophils was approved by the Human Studies Committee, and informed consent was obtained from participants. To qualitatively assess the PMA responsiveness of different preparations of cells, superoxide production was assessed (21). 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 blanks. Incubation of Neutrophils with ABZ. 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 [3H]-N-acetylbenzidine at the indicated PMA concentrations. To evaluate metabolism, some incubations contained DNA (0.7 mg/mL) or dGp (0.7 mg/mL). Cells were not included in blank incubations. The reaction was stopped by placing on ice, and the mixture was extracted twice with 2 volumes of ethyl acetate. The aqueous fraction, including the neutrophils, was processed as described below for adduct analysis. The ethyl acetate extracts were pooled, evaporated to dryness, redissolved in 0.1 mL of an 80:20 methanol/dimethyl sulfoxide (DMSO) mixture, and analyzed by HPLC. HPLC Analysis of Metabolites. Metabolites were assessed using a Beckman HPLC with System Gold software, which consisted of a 5 µm, 4.6 mm × 150 mm C-18 ultrasphere column attached to a guard column. For solvent system 1, the mobile phase contained 20 mM phosphate buffer (pH 5.0) in 20% methanol from 0 to 2 min, 20 to 33% methanol from 2 to 8 min, 33 to 40% methanol from 8 to 15 min, 40 to 80% methanol from 15 to 22 min, and 80 to 20% methanol from 32 to 37 min, at a flow rate of 1 mL/min. For solvent system 2, the mobile phase contained 20 mM phosphate buffer (pH 5.0) in 50% methanol from 0 to 2 min, 50 to 60% methanol from 2 to 7 min, 68 to 90% methanol from 7 to 27 min, and 90 to 50% methanol from 35 to 40 min, at a flow rate of 1 mL/min. Radioactivity in HPLC eluents was assessed using a FLO-ONE radioactive flow detector. Data are expressed as the percentage of total radioactivity recovered by HPLC. The amount of ABZ metabolized was determined by subtracting the percentage of ABZ recovered (unmetabolized) from 98% pure ABZ. Some fractions were also collected for 32P-postlabeling. Preparation of DNA. DNA was precipitated following addition of 2 volumes of ethanol, adjusting the concentration of NaCl to 0.25 M, and left overnight at -20 °C (19). DNA was purified using a proteinase K (1 mg/mL)/detergent (1% SDS) digestion followed by extraction with phenol and a 24:1 chloroform/isoamyl alcohol mixture. The resulting DNA was treated with ribonuclease A and T1, and phenol extracted. NaCl and ethanol were added to precipitate DNA overnight at -20 °C. The purity and quantitation of DNA were determined by absorbance at 260 and 280 nm. A A260/A280 ratio of approximately 1.7 was achieved for each sample. DNA samples were enzymatically hydrolyzed to dGp adducts by digestion with micrococcal nuclease and spleen phosphodiesterase (22). Preparation of the dGp Adduct. Enrichment of the adduct from unmodified nucleotides was achieved with n-butanol extraction (23). The aqueous fraction was made 1 mM with respect to tetrabutylammonium chloride and extracted with an equal volume of water-saturated n-butanol. Three n-butanol extractions were pooled, back extracted with water twice, evaporated using a speed vac, and dissolved in 0.05 mL of distilled water.

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Table 1. Effect of Various Test Agents on MPO and HOCl Oxidation of N-Acetylbenzidinea test agent (concentrated) complete without 100 mM NaCl with 10 mM taurine with 100 mM DMPO with 1 mM glutathione with 1 mM ascorbic acid

MPO (nmol of HOCl (nmol of ABZ metabolized) ABZ metabolized) 2.7 ( 0.2 1.7 ( 0.2b 1.4 ( 0.3 0.9 ( 0.5 0.9 ( 0.3c 0.5 ( 0.1

3.0 ( 0.2 0.1 ( 0.03 0.8 ( 0.2 0.2 ( 0.1 0.1 ( 0.1

a Samples were analyzed by HPLC using solvent system 1. Test agents were included in incubations that contained 0.06 mM [3H]ABZ and either 0.1 µg (20 milliunits) of MPO and 0.1 mM H2O2 or 0.1 mM HOCl. Values represented are the means ( SEM of nanomoles of ABZ metabolized. b The final NaCl concentration of 0.1 mM is attributed to MPO preparation. c New observed metabolite.

Nuclease P1 Sensitivity of the dGp Adduct. The enriched adduct sample was added to a reaction mixture containing 0.1 M sodium phosphate (pH 5.5) and 0.15 mM ZnCl2 at 37 °C (24). One aliquot was incubated with 2 µg of nuclease P1, while another aliquot was incubated without nuclease for the indicated time. The sensitivity is indicated by cleavage of the 3′phosphate of dGp-ABZ to dG-ABZ or dpGp-ABZ to dpG-ABZ. Samples were analyzed by HPLC using solvent system 1. Mass Spectral Analysis. Electrospray ionization mass spectrometry (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 collisionactivated dissociation (CAD) tandem mass spectra, the collision gas was argon (2.2-2.5 mTorr) and the collision energy was set at 50 eV. Product ion spectra were acquired in the profile mode at a scan rate of one scan per 3 s. Adduct Analysis by 32P-Postlabeling. The dGp adducts were analyzed by 32P-postlabeling as previously described (19, 22). Labeled adducts were separated on PEI-cellulose sheets using the following multicomponent solvent systems: D-1, 1.7 M sodium phosphate (pH 5.5); D-3, 4 M ammonium hydroxide; and D-4, 0.6 M lithium formate/0.5 M Tris-HCl/7 M urea (pH 8.0). 32P-labeled adducts were observed by autoradiography. Adducts were extracted from sheets with a 1:1 mixture of 14 M ammonium hydroxide and methanol. Adduct content was calculated on the basis of recovery of radioactivity from TLC spots and the specific activity of ATP, and expressed as femtomoles per milligram of DNA or dGp. Authentic dGp-ABZ was prepared as previously described (19). A corresponding standard was prepared by incubation of N′-hydroxy-N-acetylbenzidine with either DNA or dGp.

Results [3H]ABZ

Transformation of was observed with MPO and HOCl. HPLC analysis indicated that similar metabolite peaks were observed with each oxidant condition. Metabolites were labile with several attempts at purification and identification being unsuccessful. Previously identified oxidation products of ABZ, N′-hydroxy-Nacetylbenzidine or 4′-nitro-4-acetylaminobiphenyl (25, 26), were not observed. The characteristics of metabolism are indicated in Table 1. When the concentration of NaCl was reduced from 100 to 0.1 mM, the level of MPO metabolism of ABZ was reduced 37%. Taurine (10 mM), a HOCl substrate, caused nearly complete inhibition of HOCl transformation, but only inhibited MPO metabo-

Figure 1. Illustrated are HPLC chromatographs of [3H]ABZ reactions with either HOCl or MPO in the presence of dGp or DNA. Panel A represents the reaction of HOCl and ABZ with dGp present. This reaction mixture was extracted with ethyl acetate and then with n-butanol. The n-butanol extract is depicted in panel B. Panel C represents the n-butanol extract from the reaction of MPO and ABZ with DNA present. Samples were analyzed using solvent system 1.

lism by 48%. DMPO (100 mM), a radical trapper, inhibited transformation of ABZ by about 70% for both conditions. Ascorbic acid and glutathione were effective inhibitors of both reactions. With MPO, glutathione caused the formation of a new peak, presumably a

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Figure 3. (A) ESI CAD tandem mass spectra of the HOClderived adduct in the positive ion mode and (B) positive ion MS3 mass spectrum of the ion at m/z 376 arising from source CAD. Scheme 1 illustrates the fragmentation pattern of the adduct.

Figure 2. Illustrated are HPLC chromatographs of nuclease P1-treated [3H]dGp-ABZ adduct samples. dGp-ABZ was derived from the n-butanol fraction following ethyl acetate extraction of reaction mixtures (Figure 1B). Panel A depicts partial nuclease P1 hydrolysis of the adduct derived from HOCl and ABZ with dGp. Panel B depicts the adduct derived from DNA incubations with MPO and ABZ in the absence of nuclease P1. Panel C depicts the same adduct as panel B, but with nuclease P1.

conjugate. Thus, MPO and HOCl exhibit unique characteristics for transforming ABZ. Activation of [3H]ABZ by MPO and HOCl to form DNA adducts was also assessed. Using incubation conditions

described in Table 1 for HOCl and ABZ, the inclusion of 1 mg/mL 2′-deoxyguanosine 3′-monophosphate (dGp) resulted in a new peak of radioactivity that corresponded to synthetic dGp-ABZ (Figure 1A). This adduct peak corresponded to 31% of the total radioactivity recovered by HPLC and could be partially purified into n-butanol following ethyl acetate extraction (Figure 1B). Subsequent experiments demonstrated dGp-ABZ formation during incubation of [3H]ABZ and 1 mg/mL DNA with either HOCl or MPO. DNA was isolated from the MPO reaction mixture and hydrolyzed to 3′-monophosphates by digestion with micrococcal nuclease and spleen phosphodiesterase (Figure 1C). The dGp-ABZ metabolite has an identical elution time in all three panels of Figure 1. Thus, the same adduct was formed during ABZ transformation by either MPO or HOCl. To further characterize dGp-ABZ formation by MPO and HOCl, the n-butanol-purified 3H-labeled adduct was subjected to nuclease P1 hydrolysis (Figure 2). Nuclease P1 treatment of the HOCl-derived dGp-ABZ resulted in the formation of a new peak labeled dG-ABZ (Figure 2A), which comigrated with the product of the reaction of HOCl and ABZ in the presence of 1 mg/mL dG. While a similar peak was present in a MPO-derived DNA preparation (Figure 2B), the magnitude of this peak increased dramatically after nuclease P1 treatment (Figure 2C). Identical elution profiles were observed with the synthetic dGp-ABZ standard (not shown). In separate experiments utilizing 32P-postlabeling analysis, dGp-ABZ derived from HOCl was also demonstrated to correspond to the synthetic standard (not shown). Thus, dGp-ABZ prepared with either MPO or HOCl was sensitive to nuclease P1, yielded an identical hydrolysis product, and is identical to our standard. The HOCl-derived adduct was further identified by mass spectrometry. The ESI mass spectrum gives an [M + H]+ ion at m/z 572 in the positive ion mode. The product ion spectrum resulting from CAD of the [M + H]+ ion at m/z 572 (Figure 3A) yields a major fragment ion at m/z 376, representing protonated N′-(guanin-8-yl)acetylbenzidine. The structural information about the m/z 376 ion was further confirmed by the tandem mass

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Scheme 1. Fragmentation Pathway of dGp-ABZ by ESI CAD Tandem Mass Spectrometry in the Positive Mode

spectrum obtained by source CAD/MS/MS (Figure 3B), which contains ions equivalent to the tandem mass spectrum arising from protonated N′-(3′-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (m/z 572, Figure 3A). The fragmentation pathway to ion formation is shown in Scheme 1 and is consistent with the structure of the HOCl-derived adduct being dGp-ABZ. Metabolism of 0.02 mM [3H]ABZ by human polymorphonuclear neutrophils was assessed. In incubations containing 2 × 106 cells/0.3 mL, about 12% of the ABZ was metabolized. Following addition of 30 ng/mL PMA, 33% of the ABZ was metabolized. HPLC analysis indicated that metabolites similar to those observed with the MPO and HOCl reactions were detected. As with the latter reactions, the metabolites were labile and were not identified. Using conditions similar to those described above, cells were incubated in the presence of either 0.7 mg/mL DNA or dGp, and analyzed by 32P-postlabeling. Results observed with 32P-postlabeling experiments are illustrated in Figure 4. In human polymorphonuclear neutrophils incubated with 0.02 mM ABZ for 30 min in the presence of DNA (Figure 4A), d32PGp-ABZ was not observed, using TLC conditions previously optimized for detecting this adduct (17, 19). d32PGp-ABZ was observed in cells stimulated with 30 ng/mL PMA (Figure 4B). The adduct observed with PMA-stimulated cells corresponded to the d32PGp-ABZ standard (Figure 4C), as indicated by comigration (Figure 4D). The amount of dGp-ABZ formed was quantitated following 32P-postlabeling (Table 2). d32PGp-ABZ was detected only in cells incubated with PMA. More adduct was detected with incubations containing DNA or dGp. The level of adduct formation in cells exposed to PMA

Figure 4. 32P-postlabeled material separated on PEI-cellulose. The following postlabeled samples are represented: (A) human polymorphonuclear neutrophils incubated with 0.02 mM ABZ in the presence of 0.7 mg/mL DNA, (B) PMA-stimulated neutrophils incubated with 0.02 mM ABZ in the presence of 0.7 mg/mL DNA, (C) dGp-ABZ standard, and (D) samples B and C combined.

and DNA (3.6 ( 0.7 fmol/106 cells) was not altered by the presence of 0.1 mM indomethacin (3.5 ( 1.0 fmol/ 106 cells). With dGp, d32PGp-ABZ formation could be detected with as few as 0.12 × 106 neutrophils. To further evaluate the dGp-ABZ formed by neutrophils, the 32P-postlabeled cell sample containing DNA and incubated with PMA was eluted from PEI-cellulose sheets (Figure 4) and analyzed by HPLC (Figure 5). The sample

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Table 2. Human Peripheral White Blood Cell Formation of dGp-ABZa conditions control without PMA with PMA DNA without PMA with PMA dGp without PMA with PMA

dGp-ABZ (fmols/106 cells) 0 0.024 ( 0.002 0 3.6 ( 0.7 0 12.5 ( 1.5

a Neutrophils were incubated with 0.02 mM ABZ and 30 ng/ mL PMA in the absence or presence of either 0.7 mg/mL DNA or dGp. dGp-ABZ formation was quantitated as d32PGp-ABZ following 32P-postlabeling. Values represent the means ( SEM of femtomoles of dGp-ABZ per 106 cells.

Figure 5. HPLC profile of the 32P-labeled bisphosphate adduct (d32PGp-ABZ) recovered from PEI-cellulose sheets. Cells were incubated with 30 ng/mL PMA and 0.7 mg/mL DNA. In Panel A, samples were incubated in the absence of nuclease (control), while panel B represents the same sample after nuclease P1 treatment to form d32PG-ABZ.

was investigated with and without nuclease P1 treatment which converts the bisphosphate adduct (d32PGp-ABZ) to its 5′-monophosphate analogue (d32PG-ABZ). The single peak observed was converted to an additional peak with nuclease P1 treatment. With increasing time of nuclease incubation, all the radioactivity was converted to the new peak. Identical elution profiles were observed with the synthetic dGp-ABZ standard analyzed by 32Ppostlabeling and subjected to nuclease P1 treatment (not shown). Analogous results were observed with cell samples containing dGp instead of DNA. In addition, the product of the reaction of HOCl and ABZ in the presence of 1 mg/ mL dpG comigrated with d32PG-ABZ. Thus, neutrophilderived dGp-ABZ is identical to our standard.

Discussion This study demonstrates for the first time transformation of ABZ by HOCl, MPO, and human peripheral white blood cells and provides a metabolic basis for the reported presence of dGp-ABZ in peripheral white blood cells (2).

Lakshmi et al.

While products of HOCl and MPO transformation were not identified due to their lability, they appeared to have similar HPLC elution profiles. N′-Hydroxy-N-acetylbenzidine and 4′-nitro-4-acetylaminobiphenyl have been reported as prostaglandin H synthase peroxygenase products of ABZ (25, 26) and were not detected. MPO metabolism of ABZ probably does not require NaCl, because metabolism of ABZ was only inhibited 37% when the concentration of NaCl was reduced from 100 to 0.1 mM. A similar reduction in chloride concentration results in a more than 100-fold decrease in the rate of chloridedependent oxidation of 5,5′-dithiobis(2-nitrobenzoic acid) (27). ABZ, like other aromatic amines (28), may be a reducing cosubstrate for MPO and not require in situ HOCl formation for initiation of transformation. Taurine appears to compete very effectively with ABZ for transformation by HOCl with little ABZ transformation being observed. The partial inhibition of MPO metabolism by taurine suggests that HOCl formed in situ is responsible for at least some MPO metabolism. Inhibition of both HOCl and MPO transformation by DMPO is consistent with a radical-mediated process (29). Ascorbic acid and glutathione inhibition of both reactions is compatible with their antioxidant properties. In addition, glutathione appeared to form a new product with MPO, which is probably a conjugate (30). The mechanism of ABZ activation in forming dGp-ABZ is not completely understood. Benzidine has been shown to undergo two sequential one-electron oxidations to a radical cation and then a diimine before binding macromolecules (31-33). In contrast to benzidine, the ABZ twoelectron oxidation product has not been successfully prepared for experimental use. Peroxidatic oxidation of ABZ may result in the formation of a less ring-activated intermediate, such as a diimine monocation, which is a resonance structure of the ABZ nitrenium ion. The latter may be responsible for MPO-catalyzed dGp-ABZ formation. MPO oxidizes 3,5,3′,5′-tetramethylbenzidine by two sequential one-electron oxidations (28). The ABZ diimine monocation intermediate has been proposed as the reactive intermediate responsible for dGp-ABZ formation (34). HOCl oxidizes a variety of biological compounds (35, 36), including aromatic amines (11, 37-39), and is likely to react with ABZ forming a radical cation that is then oxidized to the diimine monocation, as proposed above for MPO. DMPO could inhibit oxidation by either directly scavenging HOCl or removing radical cations, which being in equilibrium with the diimine would, in turn, remove diimine. An alternative mechanism for HOCl oxidation is the formation of a chloramine, as has been reported for other amines (40). HOCl oxidation of nitrogencontaining aromatic compounds, such as diclofenac (41), aminopyrine (37), and clozapine (38), results in Nchlorination products which have been proposed to lose HCl or chlorine anion, forming iminoquinone, dication, or nitrenium ion intermediates responsible for covalent binding. In contrast, HOCl oxidation of 4-aminobiphenyl, a primary amine, has been reported not to result in the formation of an N-chloro intermediate, but forms deeply colored products, probably dimers or polymers (42). Thus, a common mechanism for MPO and HOCl activation of ABZ may involve a diimine monocation intermediate in equilibrium with the diimine. Incubation of ABZ with either the MPO or HOCl oxidizing system and DNA or dGp resulted in the formation of dGp-ABZ. This product was identified by

Neutrophil N-Acetylbenzidine-DNA Adduct Formation

comparison to the synthetic dGp-ABZ standard by HPLC elution profile, nuclease P1 hydrolysis to dG-ABZ, 32Ppostlabeling, and ESI mass spectrum analysis. In addition, the DNA-derived adduct was isolated by techniques which yield 3′-monophosphate (dGp) adducts (22). Combination of the unique methods of sample treatment described above with selective methods for identification of the product derived from each treatment confirms the formation of dGp-ABZ. Incubation of human polymorphonuclear neutrophils with ABZ resulted in dGp-ABZ formation as determined by 32P-postlabeling. Adduct formation was dependent upon addition of PMA. The latter is known to stimulate an oxidant burst in these cells, which results in H2O2 production and release of MPO. In the presence of NaCl, neutrophils form HOCl (9). A 300-fold increase in the extent of adduct formation was observed by inclusion of DNA in the incubation mixtures. Even larger increases in adduct formation were observed when dGp was included in incubation mixtures. This suggests that neutrophil-generated reactive metabolites are available for eliciting adduct formation in adjacent cells and tissues. Neutrophil-derived dGp-ABZ was identified by comparison to the synthetic adduct with 32P-postlabeling, HPLC of the postlabeled adduct, and nuclease P1 hydrolysis of the postlabeled adduct (d32PGp-ABZ) to d32PGABZ. Indomethacin, an inhibitor of prostaglandin H synthase, did not alter adduct formation. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO. To assess the relevance of our results to those from peripheral white blood cells of workers exposed to benzidine, adduct values in Table 2 were recalculated as relative adduct labeling (RAL) and then adjusted for an incubation time of 24 h from 30 min. With this adjustment, the values for PMA-stimulated cells in the absence and presence of DNA are 48 and 740 RAL × 109, respectively. These values correspond to that reported in peripheral white blood cells from workers manufacturing benzidine dye (median, 26.3; range, 3.3-107.4) and benzidine (median, 415.2; range, 67.6-975) (2). Thus, the results from Table 2 appear to be comparable to what was observed in workers exposed to benzidine over a long period of time. Human polymorphonuclear neutrophils may serve as surrogates for monitoring exposure to aromatic amines. These results clearly demonstrate that neutrophils produce dGp-ABZ by a peroxidatic mechanism involving MPO. While bladder cell dGp-ABZ may also be formed by a peroxidatic mechanism, the enzyme involved may be prostaglandin H synthase which can be expressed at high levels in bladder (15, 16, 43) and can also activate ABZ to form dGp-ABZ (17). High levels of ABZ in the urine and plasma of workers exposed to benzidine correspond to dGp-ABZ detection in bladder cells and peripheral white blood cells (1, 2). Peripheral blood cell DNA adducts have been proposed as a surrogate source for assessing exposure to a variety of carcinogens, including those in cigarette smoke (44, 45). There is a strong link between chronic infection/ inflammation and cancer (for a review, see ref 46). A recent study demonstrating MPO-mediated activation of a variety of aromatic amines, including 4-aminobiphenyl, 4,4′-methylenebis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine, in binding DNA suggests that the data

Chem. Res. Toxicol., Vol. 13, No. 8, 2000 791

reported in the current study might be relevant to other aromatic amines in other tissues (47). The previous study considered the role of polymorphonuclear neutrophilderived MPO as the source of this enzyme in lung and in the formation of human lung DNA adducts by aromatic amines in cigarette smoke. These cells were thought to accumulate as a result of particulate matter in the lungs of smokers. Individuals who inherit two copies of an allele that reduces transcription of the MPO gene may be at decreased risk of lung cancer (48). Schistosomiasis, a chronic parasitic urinary tract infection endemic to Egypt, elicits a chronic inflammatory reaction with associated pathology (granuloma formation) and a high incidence of bladder cancer (49). It has been estimated that chronic inflammation caused by chronic infections causes about 21% of new cancer cases in developing countries compared with 9% in developed countries (50). Lower-income individuals are more likely to be employed in jobs involving direct contact with hazardous materials and to have poorer health. These factors increase the risk of DNA adduct formation and cancer. Further study is needed to determine whether the migration of human polymorphonuclear leukocytes into the urothelium in response to infections, like Schistosoma haematobium, contributes to MPO-derived DNA adducts playing a role in the development of bladder cancer.

Acknowledgment. This work was supported by the Department of Veterans Affairs (T.V.Z.) and National Cancer Institute Grant CA72613 (T.V.Z.). Mass spectrometry was performed at the Mass Spectrometry Resource Center, Washington University School of Medicine, through NIH Grants RR-00954 and AM-20579. We thank Cindee Rettke and Priscilla DeHaven for excellent technical assistance.

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