Sulfinamide Formation following Peroxidatic Metabolism of - American

Washington University, St. Louis, Missouri 63121. Received February 26, 1999. Arylaminerhemoglobin conjugates identified as sulfinamides are considere...
0 downloads 0 Views 152KB Size
Download PDF
96

Chem. Res. Toxicol. 2000, 13, 96-102

Sulfinamide Formation following Peroxidatic Metabolism of N-Acetylbenzidine 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 63121 Received February 26, 1999

Arylamine-hemoglobin conjugates identified as sulfinamides are considered dosimeters for the bioavailability of metabolically formed N-oxidation products. This report considers peroxidation as an alternative pathway for aromatic amine metabolism and examines horseradish peroxidase metabolism of N-acetylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [3H]ABZ was incubated with 1 mM glutathione, a decrease in the total extent of metabolism was observed along with detection of a new metabolite (ABZ-SG), representing 12% of the total radioactivity. Optimum ABZ-SG formation occurred at 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. In the absence of glutathione, a molar ratio of H2O2 to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N′-hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to γ-glutamyltranspeptidase, and completely hydrolyzed by 0.1 N HC1 or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N′-(glutathion-S-yl)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolism, was prevented by 0.3 mM NaN3, 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM histidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited ABZ metabolism. The lack of effect of 50 mM mannitol and 2 µg of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Studies also indicated that molecular oxygen is not a source of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The proposed mechanism for sulfinamide formation, involving two consecutive one-electron oxidations with subsequent rearrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemoglobin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N′-(glutathion-S-yl)-N-acetylbenzidine S-oxide.

Introduction N-Acetylbenzidine (ABZ)1 is an important metabolite in individuals exposed to benzidine. ABZ is the major metabolite observed in urine (1) and plasma of workers exposed to benzidine, and the major media metabolite observed following incubation of human liver slices with benzidine (2). In exfoliated bladder cells from workers exposed to benzidine, dGp-ABZ was the main DNA adduct that was detected (3). The urinary levels of free ABZ correlate with urinary levels of dGp-ABZ (4). This DNA adduct produces genotoxic lesions, causing mutations in various bacterial and mammalian test systems in vitro (5-7) and mutations in oncogenes of tumors * To whom correspondence should be addressed: VA Medical Center (GRECC/11 G-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; dGp-ABZ, N′-(3′-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine; DMPO, 5,5-dimethyl1-pyrroline N-oxide; DETAPAC, diethylenetriaminepentaacetic acid; HPLC, high-pressure liquid chromatography; DMSO, dimethyl sulfoxide; ESI/MS, electrospray ionization mass spectrometry; CAD, collisionally activated dissociation; ABZ-SG, N′-(glutathion-S-yl)-Nacetylbenzidine S-oxide.

induced in vivo by benzidine (8). dGp-ABZ may initiate benzidine-induced bladder cancer. Thus, N-acetyltransferase metabolism of benzidine to ABZ appears to be a necessary step in DNA adduct formation and has been demonstrated in human liver with the reaction favoring NAT1, rather than NAT2 (2, 9). Peroxidase metabolism of ABZ results in dGp-ABZ formation (10). Peroxidatic activation of ABZ may be responsible for bladder cell dGp-ABZ formation (3) because these cells contain high levels of prostaglandin H synthase, an enzyme with peroxidatic activity, and low levels of cytochrome P450 (11-13). In addition, bladder cells have been shown to peroxidatically activate an aromatic amine to form a DNA adduct (14). dGp-ABZ formation was prevented by glutathione, which formed a conjugate with ABZ (10). Adducts and conjugates of carcinogens are biomarkers of exposure and can be mechanistically relevant to carcinogenesis. Arylamines can undergo sequential cytochrome P450-mediated N-oxidation to N-hydroxyl, nitroso, and nitro metabolites. Nitroso metabolites react with thiols to form sulfinic acid amides (15). Hydrolytic cleavage of the hemoglobin sulfinamide yields the pri-

10.1021/tx990031b CCC: $19.00 © 2000 American Chemical Society Published on Web 01/22/2000

Sulfinamide Formation

mary amine (16). Analysis of the latter provides a method for biomonitoring exposure, and data about metabolism and disposition of the primary aromatic amine. For example, 4-aminobiphenyl is a constituent of cigarette smoke (17), and forms a sulfinamide conjugate with hemoglobin, and its level is elevated in smokers compared to nonsmokers (18, 19). More hemoglobin conjugate is observed with the rapid cytochrome P450 1A2 oxidizer and slow N-acetylation phenotype (20). Thus, knowledge of the mechanism of carcinogen metabolism allows evaluation of susceptibility, an important factor in risk assessment, and the development of prevention strategies. An ABZ sulfinamide was detected as the main hemoglobin conjugate in rats treated with benzidine (21, 22) and is expected to be present in hemoglobin from benzidine-exposed workers (3). Detection of this sulfinamide was taken to indicate the involvement of N-oxidation products of cytochrome P450 ABZ metabolism. While we have detected rat liver cytochrome P450-mediated formation of N′-hydroxy-N-acetylbenzidine and N-hydroxy-Nacetylbenzidine (23), peroxidation is an alternative pathway for aromatic amine metabolism which should be considered. To gain insight into the role peroxidation might play in ABZ metabolism, horseradish peroxidase metabolism was assessed in the presence of glutathione and the resulting conjugate identified.

Experimental Procedures Caution: N-Acetylbenzidine is hazardous and should be handled carefully. ABZ and [3H]ABZ were synthesized by acetylation of benzidine using glacial acetic acid with a final product purity of >98% (24). [3H]Benzidine (180 mCi/mmol) and [3H]glutathione (44.8 Ci/mmol) were purchased from Chemsyn (Lenexa, KS) and NEN Life Science Products, Inc. (Boston, MA), respectively. Horseradish peroxidase (type VI), benzidine-free base and hydrochloride salt, H2O2, glutathione, ascorbic acid, sodium cyanide, superoxide dismutase (bovine erythrocytes, 4.2 units/µg), mannitol, methionine, thiourea, histidine, sodium azide, 2-methyl- 2-nitrosopropane, γ-glutamyltranspeptidase (type 1 from bovine kidney), and DETAPAC were purchased from Sigma Chemical Co. (St. Louis, MO). DMPO was obtained from Aldrich Chemical Co. (Milwaukee, WI). Ultima-Flo AP was purchased from Packard Instruments (Meriden, CT). N′-Hydroxy-N-acetylbenzidine and 4′-nitro-4-acetylaminobiphenyl were synthesized by S. Wen, using 4′-nitro-4-aminobiphenyl (TCI America, Portland, OR) as the starting material (25). The identity of these oxidative metabolite standards was established by mass spectrometry. Metabolism of ABZ by Horseradish Peroxidase. The reaction mixture (0.1 mL) contained 0.06 mM [3H]ABZ, 10 µg/ mL horseradish peroxidase, and the indicated concentrations of glutathione in phosphate buffer (pH 5.5) and 0.1 mM DETAPAC (26). H2O2 (0.05 mM) was added to start the reaction, and the incubation was continued at 37 °C for 3 min. Blank values were obtained in the absence of either peroxidase or H2O2. The reaction was stopped by adding 0.01 mL of 10 mM ascorbic acid and 0.2 mL of dimethylformamide, and the mixture was placed on ice. Metabolism was 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. For solvent system 1, the mobile phase contained 20% methanol in 20 mM phosphate buffer (pH 5.0) 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 5% acetonitrile in 20 mM ammonium acetate buffer (pH 7.0) from 0 to 2 min, 5 to 10% acetonitrile from 2 to 10 min, 10 to 50% acetonitrile from 20 to 25 min, and 50 to 5% acetonitrile

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 97 from 30 to 35 min, at a flow rate of 1 mL/min. Radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector and expressed as a percentage of total radioactivity recovered by HPLC. The amount of ABZ metabolized was determined by subtracting the percentage of ABZ recovered (unmetabolized) from 98% (purity of ABZ). Formation of an ABZ sulfinamide conjugate with hemoglobin was also assessed. To the reaction mixture described above was added 800 µg/mL cyanohemoglobin. The reaction was stopped by addition of catalase (10 µg/mL), and extracted four times with ethyl acetate (2:1, v/v). An equal volume of 10% TCA was added at 4 °C. The precipitate was washed with an ethyl acetate/ ether mixture (1:1, v/v), and resuspended in 0.1 N NaOH. Following a 60 min incubation at 37 °C, an equal volume of 10% TCA was added at 4 °C, and the neutralized supernatant was extracted three times with ethyl acetate (2:1, v/v). Radioactivity in the organic extract was assessed, and further analyzed for ABZ by HPLC. Data are expressed as picomoles of ABZ sulfinamide-hemoglobin conjugate formed. Metabolite Purification. For ABZ-SG purification, a 10 mL reaction mixture was extracted with 2 volumes of ethyl acetate. This organic extraction was repeated three times and the residual solvent evaporated from the aqueous phase with nitrogen. The latter was applied to a 500 mg C-18 Bakerbond spe column. After an 8 mL water wash, ABZ-SG was eluted with 3 mL of 100% methanol. The methanol eluent was concentrated to dryness under nitrogen, reconstituted with methanol, and purified using HPLC solvent system 2 described above. Fractions containing the conjugate were pooled and evaporated, and the spe protocol described above was repeated. The organic phase was evaporated to dryness and the sample kept at -70 °C for MS or NMR analysis. Oxygen Uptake Studies. Oxygen uptake was assessed using a Clark oxygen electrode and oxygen monitor (model 53 from Yellow Springs Instruments Co., Yellow Springs, OH). In the experiments, we used 3.0 mL of air-saturated buffer at 37 °C. The complete reaction mixture contained the same reagents as described above. Using these assay conditions, 0.25 mM phenylbutazone was substituted for ABZ and used as a positive control to demonstrate oxygen uptake (27, 28). Reactions were started by adding H2O2 (29). Mass Spectral Identification of Metabolites. ESI/MS analyses were conducted on a Finnigan (San Jose, CA) TSQ7000 triple-quadrupole mass spectrometer equipped with Finnigan ICIS software operated on a DEC alpha station. The glass capillary was maintained at 220 °C, and the electrospray needle was operated at 4.5 kV. The collision energy for CAD tandem mass spectrometry was performed at 25 eV. The collision gas (argon) pressure was set at 2.2 mTorr. All samples were dissolved in methanol and flow-injected into the ESI chamber using a Harvard (South Natick, MA) syringe pump, which was operated at a flow rate of 5 µL/min. For source CAD MS/MS, the skimmer voltage (40 V) was optimized to maximize the intensity of the ion used for tandem mass spectrometry. NMR Analysis. Samples were prepared in 100% d6-DMSO (Aldrich gold label) under a dry nitrogen purge. About 30 µg of the sample was dissolved in 1 mL of DMSO and transferred into high-quality 5 mm tubes and capped under nitrogen. NMR spectra were acquired on a Varian Inova-500 instrument with the proton resonance frequency at 499.97 MHz with an INVERSE probe (1H 90° pulse of 9 µs). One 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 the VNMR software. A line-broadening parameter of 0.5 Hz was used for the Fourier transformation. Chemical shifts were referenced to TMS at 0.0 ppm.

Results Illustrated in Figure 1 is the metabolism of [3H]ABZ in the absence and presence of 1 mM glutathione. In the absence of glutathione, all metabolites elute after ABZ

98

Chem. Res. Toxicol., Vol. 13, No. 2, 2000

Lakshmi et al.

Figure 2. Dose-response effect of glutathione on the horseradish peroxidase metabolism of N-acetylbenzidine. The elution time and distribution of radioactive metabolites were assessed by HPLC as indicated in Figure 1.

Figure 1. HPLC analysis of horseradish peroxidase metabolism of N-acetylbenzidine in the absence and presence of 1 mM glutathione.

with the major metabolite peak eluting at 32 min. This peak represented 52% of the total radioactivity in the chromatogram and nearly 70% of the total metabolism of ABZ that was observed. The 32 min peak is labile and proved to be difficult to purify and identify. In the presence of 1 mM glutathione, a new peak (ABZ-SG) is observed which eluted before ABZ and represented 12% of the total radioactivity and 23% of the total metabolism of ABZ. With glutathione, the amount of ABZ remaining increased from 22 to 48% of total radioactivity, while the total radioactivity in the 32 min peak decreased from 52 to 19%. A recent study has demonstrated a novel peroxidatic oxidation of ABZ by prostaglandin H synthase to form N′-hydroxy-N-acetylbenzidine and 4′-nitro-4acetylaminobiphenyl (30). Neither of these compounds was detected in the incubation mixtures illustrated in Figure 1. In addition, conditions which favored N′hydroxy-N-acetylbenzidine formation (1 mM ascorbic acid) also did not result in the detection of this compound. To examine the effect of glutathione in more detail, a range of glutathione concentrations was investigated (Figure 2). As the concentration of glutathione was increased from 0 to 10 mM, the amount of ABZ metabolized and the amount of the 32 min peak that formed decreased. The formation of ABZ-SG was biphasic with

Figure 3. Effect of H2O2 on horseradish peroxidase-mediated ABZ metabolism in the presence and absence of 0.3 mM glutathione. The elution time and distribution of radioactive metabolites were assessed by HPLC as indicated in Figure 1.

formation increasing from 0 to 0.3 mM glutathione and then decreasing after 1 mM. To further assess conditions for ABZ metabolism by horseradish peroxidase, a range of H2O2 concentrations was examined with 0.05 mM ABZ (Figure 3). Differences in metabolism were noted in the absence and presence of glutathione. In the absence of glutathione, maximum formation of the 32 min peak and complete metabolism of ABZ occurred at 50 µM H2O2. In the presence of 0.3 mM glutathione, the level of metabolism of ABZ increased in a linear manner up to 100 µM H2O2. The level of the 32 min peak increased linearly from 6 to 100 µM H2O2. ABZ-SG formation was observed at all concentrations of H2O2 that were tested, and the level of formation increased linearly with H2O2 concentration.

Sulfinamide Formation

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 99

Table 1. Effect of Different Test Agents on ABZ-SG Formation and N-Acetylbenzidine Metabolism by Horseradish Peroxidasea % of control condition

ABZ metabolized

ABZ-SG formed

complete with 50 mM NaCN with 0.3 mM NaN3 with 50 mM DMPO with 4 mM 2-methyl-2-nitrosopropane with 50 mM mannitol with 2 µg of superoxide dismutase with 1 mM thiourea with 0.1 mM ascorbic acid with 1 mM methionine with 1 mM histidine

100 0 100 78 57 100 100 100 0 95 95

100 0 0 0 32 100 100 0 0 90 11

a The complete reaction, with 0.3 mM glutathione, metabolized 2.4 nmol of N-acetylbenzidine and produced 0.64 nmol of ABZSG. Values represent the average of at least duplicate determinations.

A variety of test agents previously shown to inhibit peroxidation, quench radicals, and react with reactive oxygen species were examined for their effect on ABZ metabolism and ABZ-SG formation (Table 1). At the concentration of agents that was tested, only 50 mM NaCN and 0.1 mM ascorbic acid completely inhibited both ABZ metabolism and ABZ-SG formation. Several agents were found to completely inhibit ABZ-SG formation, but only moderately inhibit ABZ metabolism. These agents include 0.3 mM NaN3, 50 mM DMPO, 1.0 mM

thiourea, and 1.0 mM histidine. Inhibition by these agents did not result in the synthesis of additional metabolites. 2-Methyl-2-nitrosopropane (4 mM) also inhibited both ABZ metabolism and ABZ-SG formation. Other agents that were tested, but were not effective, include 50 mM mannitol, 2 µg of superoxide dismutase, and 1 mM methionine. The new product observed in the presence of glutathione, ABZ-SG, was purified and further characterized. ABZ-SG was susceptible to γ-glutamyltranspeptidase treatment. It was completely hydrolyzed after 10 min at room temperature in the presence of 0.1 N HC1 or NaOH with ABZ being the main product. The negative ion ESI mass spectra of the metabolite gave ions at m/z 546 and 568, representing (M - H)- and (M - 2H + Na)ions, respectively. The isotopic abundance of the molecular ion indicates that the compound contains one sulfur [abundance of the (M - H + 2)- ion, observed, 10%; calculated, 10.4%]. In addition, the molecular ion is 16 m/z higher than that expected for a thioether conjugate, indicating the addition of an oxygen atom. These data are consistent with ABZ-SG being N′-(glutathion-S-yl)N-acetylbenzidine S-oxide. 1

H NMR analysis was used to further determine the structure of the ABZ-SG conjugate (Table 2). Spectral parameters for ABZ and glutathione have been previously published (31, 32). All of the glutathione protons were accounted for in the conjugate spectrum (positions 1-11). For ABZ, the amide NH proton at position 16 was

Table 2. 1H NMR Spectral Parameters of N′-(Glutathion-S-yl)-N-acetylbenzidine S-Oxide

100

Chem. Res. Toxicol., Vol. 13, No. 2, 2000

Lakshmi et al. Scheme 1

detected in the conjugate (9.970 ppm) along with the CH3 protons (2.054 ppm) at position 17. All eight aromatic protons of ABZ were detected in the conjugate, which suggests that substitution occurred through the nitrogen of the amino group and not through the aromatic carbon atoms. Furthermore, the NH2 signal observed at 5.138 ppm with ABZ was not detected in the conjugate spectrum. A new resonance, which integrated to an exchangeable proton, was detected at 4.525 ppm and tentatively assigned to NH at position 12 in the conjugate. A noticeable feature of the NMR spectra was the 0.588 ppm high field shift of the proton at position 11 compared to that at position 1. This is consistent with proton shielding from the sulfoxide. Thus, the 1H NMR spectrum of the conjugate is consistent with that expected for N′-(glutathion-S-yl)-N-acetylbenzidine S-oxide. To determine if molecular oxygen was incorporated into the sulfinamide, oxygen uptake studies were performed. Results suggest that oxygen incorporated into ABZ-SG is not derived from molecular oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was also assessed. Addition of hemoglobin to the incubation mixture resulted in the detection of

radioactivity bound to protein. The extent of binding in incubations containing hemoglobin was reduced to values observed with the blank (minus either H2O2 or horseradish peroxidase) by 10 mM NaCN. Approximately 3.5 ( 0.1 pmol of ABZ sulfinamide-hemoglobin conjugate was formed.

Discussion The study is the first to demonstrate the formation of a sulfinamide following peroxidatic activation of an aromatic amine in the presence of glutathione. ABZ-SG formation required only low concentrations of glutathione for maximum conjugate formation. In the absence of glutathione, peroxidatic metabolism with equal molar amounts of H2O2 and ABZ resulted in complete metabolism of ABZ and maximum formation of the 32 min peak. In the presence of 0.3 mM glutathione, nearly a 50% reduction of ABZ metabolism was observed along with equal amounts of ABZ-SG and the 32 min peak (Figures 2 and 3). Glutathione appears to function as both a nucleophile and a reducing agent. This inhibition observed with glutathione has also been reported with

Sulfinamide Formation

peroxidatic metabolism of benzidine and was attributed to reduction of a radical intermediate and/or diimine back to the parent compound (33). Results with ABZ are consistent with those conclusions. Both ESI/MS and NMR analyses indicated that ABZ-SG was N′-(glutathion-S-yl)-N-acetylbenzidine S-oxide. The peroxidaseactivated ABZ intermediate also formed a sulfinamide adduct with hemoglobin. A variety of test agents were used to further evaluate ABZ-SG formation. Several of the agents (sodium azide, DMPO, thiourea, and histidine) inhibited conjugate formation, but had little effect on the total metabolism of ABZ. These agents may be interacting with and/or reducing an ABZ intermediate(s) back to ABZ. None of these agents elicited the formation of additional metabolites. Complete inhibition by 50 mM sodium cyanide and 0.1 mM ascorbic acid is consistent with cyanide inhibition of peroxidases (34), and with ascorbate being a substrate for peroxidases (35) and/or reducing the radical or diimine back to ABZ (36-38). The lack of an effect of mannitol and superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Although methionine can function as a nucleophile and react with certain electrophilic aromatic amines (39), it did not prevent ABZ-SG formation. Although thiyl radicals are most likely present during the reaction, they are probably not involved in ABZ-SG formation. The observed inhibition of ABZ metabolism by glutathione may involve reduction of an ABZ radical with concomitant formation of a thiyl radical, as has been demonstrated for other aromatic amines (40). Thiyl radicals have been shown to react with the heterocyclic amine 2-amino-4-(5-nitro-2-furyl)thiazole and form a thioether, but not a sulfinamide conjugate (29). Thiyl radicals react with molecular oxygen to form peroxyl radicals, resulting in oxygen uptake (41, 42). Alternatively, glutathione disulfide anion free radicals may also be formed which can react with oxygen to form superoxide. These glutathione reaction products could be responsible, in part, for the small amount of oxygen uptake that is observed. Horseradish peroxidase may oxidize ABZ to a twoelectron oxidation product. The molar ratio of H2O2 to ABZ of 1:1 (Figure 3) in the absence of glutathione is consistent with a two-electron oxidation of ABZ. Horseradish peroxidase and prostaglandin H synthase peroxidatically metabolize benzidine to its two-electron oxidized product benzidinediimine (33, 38, 43). The latter reacts with glutathione to form the thioether conjugate 3-(glutathion-S-yl)benzidine (38). DMPO did not alter benzidinediimine formation of DNA adducts or 3-(glutathionS-yl)benzidine (26, 38), but did inhibit horseradish peroxidase ABZ-SG formation (Table 1). This suggests that ABZ oxidation may involve two consecutive oneelectron oxidations in the formation of a two-electron product. Attempts to prepare the two-electron oxidation product of ABZ have been unsuccessful. The source of the sulfinamide oxygen was not determined. Oxygen uptake studies demonstrated that a small amount of oxygen was consumed during the reaction. However, selective inhibitors of ABZ-SG formation did not inhibit oxygen uptake. The oxygen uptake observed may be due, in part, to thiyl radicals reacting with molecular oxygen (41, 42). Recent studies of ABZ metabolism by prostaglandin H synthase demonstrated conversion to N′-hydroxy-N-acetylbenzidine and 4′-nitro-

Chem. Res. Toxicol., Vol. 13, No. 2, 2000 101

4-acetylaminobiphenyl by a peroxygenation reaction which was not inhibited by 100 mM DMPO (30). Since neither of these ABZ oxidized metabolites was detected during horseradish peroxidase metabolism of ABZ and DMPO was inhibitory, formation of these oxygenated metabolites in the present report is unlikely. It is difficult at this time to identify the source for the oxygen atom in ABZ-SG. Perhaps an intermediate in ABZ-SG formation reacts with the oxygen atom of water (see Scheme 1 below). The mechanism for ABZ activation by horseradish peroxidase to bind glutathione and form a sulfinamide conjugate is depicted in Scheme 1. According to this scheme, ABZ is oxidized by two consecutive one-electron oxidations to a radical cation and diimine monocation. A similar scheme has been described for benzidine (37, 38). The diimine monocation is a resonance structure of the ABZ nitrenium ion. This intermediate has been proposed recently as the reactive intermediate responsible for ABZ forming a DNA adduct (44). Glutathione acts as a nucleophilic trap for this reactive intermediate forming a sulfenamide. This labile conjugate loses a proton, forming a resonance-stabilized cationic intermediate, ArN+SG, that can be trapped by reaction with a water molecule at the sulfur atom. This sulfur-stabilized nitrenium ion has been previously proposed to be responsible for sulfinamide formation in the reaction of glutathione with nitrosoarenes (15). ABZ plays an important role in the metabolism and disposition of benzidine, and may be involved in toxic and carcinogenic processes. ABZ is the major metabolite observed in urine (1) and plasma of workers exposed to benzidine, and is represented in DNA (3) and hemoglobin adducts (21, 22). The novel peroxidatic activation of ABZ in forming N′-(glutathion-S-yl)-N-acetylbenzidine S-oxide was demonstrated. Thus, peroxidation could be involved in the formation of ABZ sulfinamide conjugates of hemoglobin and may play a greater role in ABZ metabolism than previously reported.

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. 1H NMR analysis was performed by Dr. Narayana Mysore, Shell Chemicals, a subsidiary of Shell Oil Co., Houston, TX. We thank Cindee Rettke and Priscilla DeHaven for excellent technical assistance.

References (1) Hsu, F.-F., Lakshmi, V., Rothman, N., Bhatnager, V. K., Hayes, R. B., Kashyap, R., Parikh, D. J., Kashyap, S. K., Turk, J., Zenser, T., and Davis, B. (1996) Determination of benzidine, N-acetylbenzidine and N,N′-diacetylbenzidine in human urine by capillary gas chromatography/negative ion chemical ionization mass spectrometry. Anal. Biochem. 234, 183-189. (2) Lakshmi, V. M., Bell, D. A., Watson, M. A., Zenser, T. V., and Davis, B. B. (1995) N-Acetylbenzidine and N,N′-diacetylbenzidine formation by rat and human liver slices exposed to benzidine. Carcinogenesis 16, 1565-1571. (3) Rothman, N., Bhatnagar, V. K., Hayes, R. B., Zenser, T. V., Kashyap, S. K., Butler, M. A., Bell, D. A., Lakshmi, V., Jaeger, M., Kashyap, R., Hirvonen, A., Schulte, P. A., Dosemeci, M., Hsu, F., Parikh, D. J., Davis, B. B., and Talaska, G. (1996) The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers. Proc. Natl. Acad. Sci. U.S.A. 93, 5084-5089.

102

Chem. Res. Toxicol., Vol. 13, No. 2, 2000

(4) Rothman, N., Talaska, G., Hayes, R., Bhatnagar, V., Bell, D., Lakshmi, V., Kashyap, S., Dosemeci, M., Kashyap, R., Hsu, F., Jaeger, M., Hirvonen, A., Parikh, D., Davis, B., and Zenser, T. (1997) Acidic urine pH is associated with elevated levels of free urinary benzidine and N-acetylbenzidine and urothelial cell DNA adducts in exposed workers. Cancer Epidemiol., Biomarkers Prev. 6, 1039-1042. (5) Beland, F. A., Beranek, D. T., Dooley, K. L., Heflich, R. H., and Kadlubar, F. F. (1983) Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary bladder carcinogenesis. Environ. Health Perspect. 49, 125-134. (6) Melchior, W. B., Jr., Marques, M. M., and Beland, F. A. (1994) Mutations induced by aromatic amine DNA adducts in pBR322. Carcinogenesis 15, 889-899. (7) Heflich, R. H., Morris, S. M., Beranek, D. T., McGarrity, L. J., Chen, J. J., and Beland, F. A. (1986) Relationships between the DNA adducts and the mutations and sister-chromatid exchanges produced in Chinese hamster ovary cells by N-hydroxy-2-aminofluorene, N-hydroxy-N′-acetylbenzidine and 1-nitrosopyrene. Mutagenesis 1, 201-206. (8) Fox, T. R., Schumann, A. M., Watanabe, P. G., Yano, B. L., Maher, V. M., and McCormick, J. J. (1990) Mutational analysis of the H-ras oncogene in spontaneous C57BL/6 x C3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens. Cancer Res. 50, 4014-4019. (9) Zenser, T. V., Lakshmi, V. M., Rustan, T. D., Doll, M. A., Deitz, A. C., Davis, B. B., and Hein, D. W. (1996) Human N-acetylation of benzidine: Role of NAT1 and NAT2. Cancer Res. 56, 39413947. (10) Lakshmi, V. M., Zenser, T. V., and Davis, B. B. (1998) N′-(3′Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism. Carcinogenesis 19, 911-917. (11) Danon, A., Zenser, T. V., Thomasson, D. L., and Davis, B. B. (1986) Eicosanoid synthesis by cultured human urothelial cells: Potential role in bladder cancer. Cancer Res. 46, 5676-5681. (12) Wise, R. W., Zenser, T. V., Kadlubar, F. F., and Davis, B. B. (1984) Metabolic activation of carcinogenic aromatic amines by dog bladder and kidney prostaglandin H synthase. Cancer Res. 44, 1893-1897. (13) Flammang, T. J., Yamazoe, Y., Benson, R. W., Roberts, D. W., Potter, D. W., Chu, D. Z. J., Lang, N. P., and Kadlubar, F. F. (1989) Arachidonic acid-dependent peroxidative activation of carcinogenic arylamines by extrahepatic human tissue microsomes. Cancer Res. 49, 1977-1982. (14) Hatcher, J. F., and Swaminathan, S. (1995) 32P-Postlabeling analysis of adducts generated by peroxidase-mediated binding of N-hydroxy-4-acetylaminobiphenyl to DNA. Carcinogenesis 16, 2149-2157. (15) Kazanis, S., and McClelland, R. A. (1992) Electrophilic intermediate in the reaction of glutathione and nitrosoarenes. J. Am. Chem. Soc. 114, 3052-3059. (16) Green, L. C., Skipper, P. L., Turesky, R. J., Bryant, M. S., and Tannenbaum, S. R. (1984) In vivo dosimetry of 4-aminobiphenyl in rats via a cysteine adduct in hemoglobin. Cancer Res. 44, 42544259. (17) Patrianakos, C., and Hoffmann, D. (1979) Chemical studies of tobacco smoke. LXIV. On the analysis of aromatic amines in cigarette smoke. J. Anal. Chem. 3, 150-154. (18) Vineis, P., Bartsch, H., Caporaso, N., Harrington, A. M., Kadlubar, F. F., Landi, M. T., Malaveille, C., Shields, P. G., Skipper, P., Talaska, G., and Tannenbaum, S. R. (1994) Genetic ally based N-acetyltransferase metabolic polymorphism and low-level environmental exposure to carcinogens. Nature 369, 154-156. (19) Bryant, M. S., Vineis, P., Skipper, P. L., and Tannenbaum, S. R. (1988) Hemoglobin adducts of aromatic amines: Associations with smoking status and type of tobacco. Proc. Natl. Acad. Sci. U.S.A. 85, 9788-9791. (20) Landi, M. T., Zocchetti, C., Bernucci, I., Kadlubar, F. F., Tannenbaum, S., Skipper, P., Bartsch, H., Malaveille, C., Shields, P., Caporaso, N. E., and Vineis, P. (1996) Cytochrome P4501A2: Enzyme induction and genetic control in determining 4-aminobiphenyl-hemoglobin adduct levels. Cancer Epidemiol., Biomarkers Prev. 5, 693-698. (21) Birner, G., Albrecht, W., and Neumann, H.-G. (1990) Biomonitoring of aromatic amines III: hemoglobin binding of benzidine and some benzidine congeners. Arch. Toxicol. 64, 97-102. (22) Zwirner-Baier, I., and Neumann, H.-G. (1998) Biomonitoring of aromatic amines V: acetylation and deacetylation in the metabolic activation of aromatic amines as determined by haemoglobin binding. Arch. Toxicol. 72, 499-504.

Lakshmi et al. (23) Lakshmi, V. M., Zenser, T. V., and Davis, B. B. (1997) Rat liver cytochrome P450 metabolism of N-acetylbenzidine and N,N′diacetylbenzidine. Drug Metab. Dispos. 25, 481-488. (24) Lakshmi, V. M., Mattammal, M. B., Spry, L. A., Kadlubar, F. F., Zenser, T. V., and Davis, B. B. (1990) Metabolism and disposition of benzidine in the dog. Carcinogenesis 11, 139-144. (25) Babu, S. R., Lakshmi, V. M., Hsu, F. F., Zenser, T. V., and Davis, B. B. (1995) Glucuronidation of N-hydroxy metabolites of Nacetylbenzidine. Carcinogenesis 16, 3069-3074. (26) Lakshmi, V. M., Mattammal, M. B., Zenser, T. V., and Davis, B. B. (1990) Mechanism of peroxidative activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT): comparison with benzidine. Carcinogenesis 11, 1965-1970. (27) Marnett, L. J., Bienkowski, M. J., Pagels, W. R., and Reed, G. A. (1980) Mechanism of Xenobiotic Cooxygenation Coupled to Prostaglandin H2 Biosynthesis. In Advances in Prostaglandin and Thromboxane Research (Samuelsson, B., Ramwell, P. W., and Paoletti, R., Eds.) pp 149-151, Raven Press, New York. (28) Lakshmi, V. M., Zenser, T. V., Mattammal, M. B., and Davis, B. B. (1993) Phenylbutazone peroxidatic metabolism and conjugation. J. Pharmacol. Exp. Ther. 266, 81-88. (29) Lakshmi, V. M., Zenser, T. V., Sohani, S., and Davis, B. B. (1992) Mechanism of formation of the thioether conjugate of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). Carcinogenesis 13, 2087-2093. (30) Zenser, T. V., Lakshmi, V. M., Hsu, F. F., and Davis, B. B. (1999) Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. J. Biol. Chem. 274, 14850-14856. (31) Lakshmi, V. M., Hsu, F. F., McGarry, A. E., Davis, B. B., and Zenser, T. V. (2000) Hypochlorous acid-mediated activation of N-acetylbenzidine to form N′-(3′-monophosphodeoxyguanosin-8-yl)N-acetylbenzidine. Toxicol. Sci. 53, 202-212. (32) Mulder, G. J., Unruh, L. E., Evans, F. E., Ketterer, B., and Kadlubar, F. F. (1982) Formation and identification of glutathione conjugates from 2-nitrosofluorene and N-hydroxy-2-aminofluorene. Chem.-Biol. Interact. 39, 111-127. (33) Wise, R. W., Zenser, T. V., and Davis, B. B. (1985) Prostaglandin H synthase oxidation of benzidine and o-dianisidine: Reduction and conjugation of activated amines by thiols. Carcinogenesis 6, 579-583. (34) Saunders, B. C., Holmes-Siedle, A. G., and Stark, B. P. (1964) Peroxidase, Butterworths, Washington, DC. (35) Markey, C. M., Alward, A., Weller, P. E., and Marnett, L. J. (1987) Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. J. Biol. Chem. 262, 6266-6279. (36) Wise, R. W., Zenser, T. V., and Davis, B. B. (1983) Prostaglandin H synthase metabolism of the urinary bladder carcinogens benzidine and ANFT. Carcinogenesis 4, 285-289. (37) Zenser, T. V., Mattammal, M. B., Wise, R. W., Rice, J. R., and Davis, B. B. (1983) Prostaglandin H synthase-catalyzed activation of benzidine: A model to assess pharmacologic intervention of the initiation of chemical carcinogenesis. J. Pharmacol. Exp. Ther. 227, 545-550. (38) Lakshmi, V. M., Zenser, T. V., and Davis, B. B. (1994) Mechanism of 3-(glutathion-S-yl)-benzidine formation. Toxicol. Appl. Pharmacol. 125, 256-263. (39) Bartsch, H., Dworkin, M., Miller, J. A., and Miller, E. C. (1972) Electrophilic N-acetoxyaminoarenes derived from carcinogenic N-hydroxy-N-acetylaminoarenes by enzymatic deacetylation and transacetylation in liver. Biochim. Biophys. Acta 286, 272-298. (40) Eling, T. E., Mason, R. P., and Sivarajah, K. (1985) The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical. J. Biol. Chem. 260, 1601-1607. (41) Ross, D., Albano, E., Nilsson, U., and Moldeus, P. (1984) Thiyl radicalssFormation during peroxidase-catalyzed metabolism of acetaminophen in the presence of thiols. Biochem. Biophys. Res. Commun. 125, 109-115. (42) Harman, L. S., Carver, D. K., Schreiber, J., and Mason, R. P. (1986) One- and two-electron oxidation of reduced glutathione by peroxidases. J. Biol. Chem. 261, 1642-1648. (43) Josephy, D. P., Eling, T. E., and Mason, R. P. (1983) Co-oxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase. J. Biol. Chem. 258, 5561-5569. (44) Dicks, A. P., Ahmad, A. R., D’Sa, R., and McClelland, R. A. (1999) Tautomers and conjugate base of the nitrenium ion derived from N-acetylbenzidine. J. Chem. Soc., Perkin Trans. 2, 1-3.

TX990031B