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Peroxygenase Metabolism of N-Acetylbenzidine by Prostaglandin H Synthase

1999; Elsevier BV; Volume: 274; Issue: 21 Linguagem: Inglês

10.1074/jbc.274.21.14850

ISSN

1083-351X

Autores

Terry V. Zenser, Vijaya M. Lakshmi, Fong‐Fu Hsu, Bernard B. Davis,

Tópico(s)

Pharmacogenetics and Drug Metabolism

Resumo

Synthesis of prostaglandin H2by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mm ABZ to its final end product, 4′-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mm ascorbic acid was present. Similar results were observed whether 0.2 mm arachidonic acid or 0.5 mmH2O2 was used as the substrate. This metabolite co-eluted with syntheticN′-hydroxy-N-acetylbenzidine (N′HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N′HA by electrospray ionization/MS/MS. N′HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N′HA was substituted for ABZ, PHS metabolized N′HA to 4′-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4′-nitro-4-acetylaminobiphenyl, but not for N′HA. A 97% enrichment was observed for one atom of 18O, and a 17 ± 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N′HA with water was suggested to explain the lack of enrichment of N′HA and the low enrichment of two18O atoms into 4′-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N′HA by PHS and suggest a stepwise oxidation of ABZ to N′-hydroxy, 4′-nitroso, and 4′-nitro products. Synthesis of prostaglandin H2by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mm ABZ to its final end product, 4′-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mm ascorbic acid was present. Similar results were observed whether 0.2 mm arachidonic acid or 0.5 mmH2O2 was used as the substrate. This metabolite co-eluted with syntheticN′-hydroxy-N-acetylbenzidine (N′HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N′HA by electrospray ionization/MS/MS. N′HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N′HA was substituted for ABZ, PHS metabolized N′HA to 4′-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4′-nitro-4-acetylaminobiphenyl, but not for N′HA. A 97% enrichment was observed for one atom of 18O, and a 17 ± 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N′HA with water was suggested to explain the lack of enrichment of N′HA and the low enrichment of two18O atoms into 4′-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N′HA by PHS and suggest a stepwise oxidation of ABZ to N′-hydroxy, 4′-nitroso, and 4′-nitro products. Prostaglandin H synthase (PHS) 1The abbreviations used are: PHS, prostaglandin H synthase; ABZ, N-acetylbenzidine; PG, prostaglandin; N′HA, N′-hydroxy-N-acetylbenzidine; NHA, N-hydroxy-N-acetylbenzidine; dGp-ABZ, N′-(3′-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine; ESI, electrospray ionization; CAD, collisionally activated dissociation; HPLC, high pressure liquid chromatography; DPEA, 2,4-dichloro-6-phenylphenoxyethylamine. 1The abbreviations used are: PHS, prostaglandin H synthase; ABZ, N-acetylbenzidine; PG, prostaglandin; N′HA, N′-hydroxy-N-acetylbenzidine; NHA, N-hydroxy-N-acetylbenzidine; dGp-ABZ, N′-(3′-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine; ESI, electrospray ionization; CAD, collisionally activated dissociation; HPLC, high pressure liquid chromatography; DPEA, 2,4-dichloro-6-phenylphenoxyethylamine. catalyzes the metabolism of arachidonic acid in the presence of molecular oxygen to form prostaglandin (PG) H2 (1Ohki S. Ogino N. Yamamoto S. Hayaishi O. J. Biol. Chem. 1979; 254: 829-836Abstract Full Text PDF PubMed Google Scholar). PGH2 is the common substrate utilized to synthesize a family of biologically important compounds referred to as prostanoids. There are two PHS isozymes, COX-1 and COX-2, which are encoded by two different genes (2Kraemer S.A. Meade E.A. DeWitt D.L. Arch. Biochem. Biophys. 1992; 293: 391-400Crossref PubMed Scopus (203) Google Scholar,3Kujubu D.A. Herschman H.R. J. Biol. Chem. 1992; 267: 7991-7994Abstract Full Text PDF PubMed Google Scholar). COX-1 is the constitutive enzyme, whereas COX-2 is inducible. Whereas these isozymes differ substantially with respect to their expression and biology, they have a similar structure and express the same two catalytic activities (for a review, see Ref. 4Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar). Both isozymes catalyze a cyclooxygenase (bis-oxygenase) reaction in which arachidonic acid is converted to PGG2 and a peroxidase reaction in which PGG2 undergoes a two-electron reduction to PGH2. The two-electron oxidation of PHS yields the peroxidase spectral intermediate I that must be reduced to regenerate the resting enzyme (5Lambeir A.-M. Markey C.M. Dunford H.B. Marnett L.J. J. Biol. Chem. 1985; 260: 14894-14896Abstract Full Text PDF PubMed Google Scholar). A reducing cofactor(s) accomplishes this task and is required for the peroxidase reaction. By donating electrons to the oxidized PHS intermediate, reducing cofactors undergo co-oxidation. The peroxidase activity of both PHS isozymes is essential for biological activity. The biological reducing cofactor(s) for the peroxidatic activity of PHS is unknown. Many naturally occurring compounds have been tested for their efficiency in functioning as reducing cofactors (6Markey C.M. Alward A. Weller P.E. Marnett L.J. J. Biol. Chem. 1987; 262: 6266-6279Abstract Full Text PDF PubMed Google Scholar). These compounds include NADPH, NADH, glutathione, methionine, tryptophan, epinephrine, ascorbic acid, lipoic acid, and uric acid. Whereas the last four compounds were efficient cofactors, uric acid was considered the most likely endogenous candidate. A large number of synthetic chemicals were also found to exhibit significant reducing cofactor activity. During reduction of the 15-hydroperoxy group of PGG2 to a hydroxy group (PGH2), prostaglandin hydroperoxidase oxidizes, reducing cofactors by electron or oxygen transfer (for a review, see Ref. 7Eling T.E. Thompson D.C. Foureman G.L. Curtis J.F. Hughes M.F. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 1-45Crossref PubMed Scopus (291) Google Scholar). Most reducing cofactors donate an electron. Nitrogen-, carbon-, and sulfur-centered free radicals have been detected. For some cofactors, the radical product can undergo a second one-electron oxidation to form a two-electron oxidation product. In addition, the initial free radical product may disproportionate to give a two-electron oxidation product, i.e. an iminium cation, and the original substrate (8Lasker J.M. Sivarajah K. Mason R.P. Kalyanaraman B. Abou-Donia M.B. Eling T.E. J. Biol. Chem. 1981; 256: 7764-7767Abstract Full Text PDF PubMed Google Scholar). Other radical products may couple, forming dimers or trimers. With sulfide cofactors, the hydroperoxidase catalyzes peroxide reduction by the direct transfer of the peroxide oxygen to the acceptor molecule (9Egan R.W. Gale P.H. Van den Heuvel W.J.A. Baptista E.M. Keuhl Jr., F.A. J. Biol. Chem. 1980; 255: 323-326Abstract Full Text PDF PubMed Google Scholar, 10Ple P. Marnett L.J. J. Biol. Chem. 1989; 264: 13983-13993Abstract Full Text PDF PubMed Google Scholar). Except for sulfide reducing cofactors, no other peroxygenase reaction has been reported for PHS. Aromatic and heterocyclic amines represent an important group of PHS reducing cofactors. Exposure to these amines is common due to their occurrence in cooked foods, cigarette smoke, and pharmaceuticals and in the manufacture or use of dyes, chemicals, and antioxidants (11Schulte P.A. Ward E. Boeniger M. Hills B. King C.M. Romano L.J. Schuetzle D. Carcinogenic and Mutagenic Responses to Aromatic Amines and Nitroarenes. Elsevier Science, New York1988: 23-35Google Scholar). Benzidine, an aromatic amine reducing cofactor, is converted to a radical cation and subsequently converted to benzidinediimine, a two-electron oxidation product (12Wise R.W. Zenser T.V. Davis B.B. Carcinogenesis. 1983; 4: 285-289Crossref PubMed Scopus (38) Google Scholar, 13Zenser T.V. Mattammal M.B. Wise R.W. Rice J.R. Davis B.B. J. Pharmacol. Exp. Ther. 1983; 227: 545-550PubMed Google Scholar, 14Josephy P.D. Eling T.E. Mason R.P. Mol. Pharmacol. 1983; 23: 766-770PubMed Google Scholar). In contrast, monoacetylated benzidine, N-acetylbenzidine (ABZ), yields an oxygenated metabolite, 4′-nitro-4-acetylaminobiphenyl (15Smith B.J. DeBruin L. Josephy D. Eling T.E. Chem. Res. Toxicol. 1992; 5: 431-439Crossref PubMed Scopus (32) Google Scholar). Horseradish peroxidase does not form this metabolite. Metabolism of ABZ by PHS, but not by horseradish peroxidase, causes mutations that are attributed to 4′-nitro-4-acetylaminobiphenyl (15Smith B.J. DeBruin L. Josephy D. Eling T.E. Chem. Res. Toxicol. 1992; 5: 431-439Crossref PubMed Scopus (32) Google Scholar). The N′-hydroxy product of ABZ is not detected and is not thought to be an intermediate in 4′-nitro-4-acetylaminobiphenyl formation. A similar pattern of peroxidatic metabolism by PHS and horseradish peroxidase has also been reported for 2-aminofluorene (16Boyd J.A. Harvan D.J. Eling T.E. J. Biol. Chem. 1983; 258: 8246-8254Abstract Full Text PDF PubMed Google Scholar). N-Hydroxylation is an important step in aromatic amine activation. The reaction is catalyzed by cytochrome P-450s and, in some cases, flavin-containing monooxygenases. Rat liver microsomal cytochrome P-450 oxidizes ABZ toN′-hydroxy-N-acetylbenzidine (N′HA) andN-hydroxy-N-acetylbenzidine (NHA) (17Frederick C.B. Weis C.C. Flammang T.J. Martin C.N. Kadlubar F.F. Carcinogenesis. 1985; 6: 959-965Crossref PubMed Scopus (50) Google Scholar, 18Lakshmi V.M. Zenser T.V. Davis B.B. Drug Metab. Dispos. 1997; 25: 481-488PubMed Google Scholar). N′HA production was associated with N′-(3′-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation (dGp-ABZ) (18Lakshmi V.M. Zenser T.V. Davis B.B. Drug Metab. Dispos. 1997; 25: 481-488PubMed Google Scholar). This DNA adduct produces genotoxic lesions, causing mutations in various bacterial and mammalian test systemsin vitro (19Beland F.A. Beranek D.T. Dooley K.L. Heflich R.H. Kadlubar F.F. Environ. Health Perspect. 1983; 49: 125-134Crossref PubMed Scopus (205) Google Scholar, 20Melchior Jr., W.B. Marques M.M. Beland F.A. Carcinogenesis. 1994; 15: 889-899Crossref PubMed Scopus (73) Google Scholar, 21Heflich R.H. Morris S.M. Beranek D.T. McGarrity L.J. Chen J.J. Beland F.A. Mutagenesis. 1986; 1: 201-206Crossref PubMed Scopus (66) Google Scholar) and mutations in the oncogenes of tumors induced in vivo by benzidine (22Fox T.R. Schumann A.M. Watanabe P.G. Yano B.L. Maher V.M. McCormick J.J. Cancer Res. 1990; 50: 4014-4019PubMed Google Scholar). A recent study has demonstrated dGp-ABZ formation after PHS metabolism of ABZ (23Lakshmi V.M. Zenser T.V. Davis B.B. Carcinogenesis. 1998; 19: 911-917Crossref PubMed Scopus (19) Google Scholar). Thus, PHS metabolism of ABZ was reexamined to determine the mechanism of metabolism and the possible presence of an N′HA intermediate. [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 >98% (24Lakshmi V.M. Mattammal M.B. Spry L.A. Kadlubar F.F. Zenser T.V. Davis B.B. Carcinogenesis. 1990; 11: 139-144Crossref PubMed Scopus (24) Google Scholar). Benzidine-free base and hydrochloride salt, H2O2, glutathione, arachidonic acid, hematin, ascorbic acid, indomethacin, phenylbutazone, sodium cyanide, superoxide dismutase (bovine erythrocytes; 4.2 units/μg), mannitol, and diethylenetriaminepentaacetic acid were purchased from Sigma. 5,5-Dimethyl-1-pyrroline N-oxide was obtained from Aldrich. Ultima-Flo AP was purchased from Packard Instrument Co. 2,4-Dichloro-6-phenylphenoxyethylamine was a gift from Eli Lilly Laboratories (Indianapolis, IN). Furafylline was purchased from Gentest Corp. (Woburn, MA), whereas the other cytochrome P-450 inhibitors were purchased from Sigma. 4′-Nitro-4-acetylaminobiphenyl, N′HA, and NHA were synthesized by Dr. Shu Wen Li (Department of Biochemistry, St. Louis University Medical School, St. Louis, MO) using 4′-nitro-4-aminobiphenyl (TCI America, Portland, OR) as starting material (25Babu S.R. Lakshmi V.M. Hsu F.F. Zenser T.V. Davis B.B. Carcinogenesis. 1995; 16: 3069-3074Crossref PubMed Scopus (33) Google Scholar). The identity of these synthetic compounds was established by NMR and mass spectrometry. Seminal vesicle microsomes were prepared as described previously (26Zenser T.V. Mattammal M.B. Davis B.B. J. Pharmacol. Exp. Ther. 1978; 207: 719-725PubMed Google Scholar). [18O]H2O2 was purchased from Icon Isotope Services, Inc. (Summit, NJ). [3H]ABZ (0.06 mm) was added to 100 mm phosphate buffer, pH 7.4, containing 1 mg/ml ram seminal vesicle microsomes (PHS), 1 μg/ml hematin, and 1.0 mm diethylenetriaminepentaacetic acid in a total volume of 0.1 ml (27Lakshmi V.M. Mattammal M.B. Zenser T.V. Davis B.B. Carcinogenesis. 1990; 11: 1965-1970Crossref PubMed Scopus (11) Google Scholar). The reaction was started by the addition of 0.2 mm arachidonic acid or 0.5 mmH2O2 and incubated at 37 °C for 2 min. Blank values were obtained in the absence of either microsomes, H2O2, or arachidonic acid. The reaction was stopped by adding an equal volume of methanol, placed on ice, and centrifuged to obtain a clear supernatant. Metabolites in this supernatant were assessed using a Beckman HPLC with System Gold software that consisted of a 5-μm, 4.6 × 150-mm C-18 ultrasphere column attached to a guard column. The mobile phase contained 20% methanol in 20 mm phosphate buffer (pH 5.0), 0–2 min; 20–33% methanol, 2–8 min; 33–40% methanol, 8–15 min; 40–80% methanol, 15–22 min; and 80% to 20% methanol, 32–37 min (flow rate, 1 ml/min). Radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector. Data are 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 100%. A 10-ml reaction was stopped by three extractions with an equal volume of ethyl acetate. Extracts were pooled, concentrated to dryness under nitrogen, reconstituted with dimethylformamide, and purified using the HPLC solvent system described above. For N′HA purification, dimethylformamide contained 1 mm ascorbic acid, as did the tubes before HPLC collection. Fractions containing the metabolite were pooled, methanol was evaporated, and fractions were extracted three times with an equal volume of ethyl acetate. Organic fractions were pooled, back extracted with an equal volume of water, and evaporated to dryness, and the sample was kept at −70 °C for MS analysis. Oxygen uptake was measured using a Clark oxygen electrode and oxygen monitor (Model 53; Yellow Springs Instruments Co., Yellow Springs, OH). Experiments used 3.0 ml of air-saturated buffer at 37 °C. The complete reaction mixture contained the same reagents as described above. Reactions were started by the addition of H2O2 (28Lakshmi V.M. Zenser T.V. Sohani S. Davis B.B. Carcinogenesis. 1992; 13: 2087-2093Crossref PubMed Scopus (5) Google Scholar). To assess phenylbutazone metabolism, 0.25 mm phenylbutazone was added as described previously (29Marnett L.J. Bienkowski M.J. Pagels W.R. Reed G.A. Samuelsson B. Ramwell P.W. Paoletti R. Advances in Prostaglandin and Thromboxane Research. Raven Press, New York1980: 149-151Google Scholar, 30Lakshmi V.M. Zenser T.V. Mattammal M.B. Davis B.B. J. Pharmacol. Exp. Ther. 1993; 266: 81-88PubMed Google Scholar). ESI/MS analyses were performed on a Finnigan (San Jose, CA) TSQ-7000 triple-stage quadrupole spectrometer equipped with an ESI source and controlled by Finnigan ICIS software operated on a DECα workstation. Samples were flow-injected (5 μl/min) into the ESI source with a Harvard syringe pump. 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 220 °C. For collisionally activated dissociation (CAD) and tandem MS, the collision gas was argon (2.2–2.5 millitorr), and collision energies were varied between 24 and 26 eV. Product ion spectra were acquired in profile mode at a rate of 1 scan/3 s. MS analysis was used to determine oxygen incorporation into 4′-nitro-4-acetylaminobiphenyl using [18O]H2O2 as the substrate. To calculate the incorporation of one atom of 18O into 4′-nitro-4-acetylaminobiphenyl in the positive ion mode, peak heights at m/z 257, 259, and 261 were measured. The ratio of peak heights at 259 + 261/257 + 259 + 261 was divided by the fractional enrichment of [18O]H2O2. This result was multiplied by 100 to yield the percentage of 4′-nitro-4-acetylaminobiphenyl molecules with one atom of18O incorporated (16O-N-18O). A similar calculation was performed to determine the incorporation of two atoms of 18O into 4′-nitro-4-acetylaminobiphenyl. The ratio of peak heights at 261/257 + 259 + 261 was divided by the fractional enrichment of [18O]H2O2. This result was multiplied by 100 to yield the percentage of 4′-nitro-4-acetylaminobiphenyl molecules with two atoms of18O incorporated (18O-N-18O). The18O enrichment of H2O2 was 93% (ICON Isotope Services, Inc.). PHS metabolizes ABZ to 4′-nitro-4-acetylaminobiphenyl (Fig. 1). In control incubations, after the addition of 0.2 mm arachidonic acid, 4′-nitro-4-acetylaminobiphenyl represented 23% of the total radioactivity recovered by HPLC, with about 66% of the radioactivity represented by unmetabolized [3H]ABZ (Fig. 1A). After the addition of 1 mm ascorbic acid, the formation of 4′-nitro-4-acetylaminobiphenyl is significantly decreased to 5.1% of total radioactivity, and a new peak is observed eluting before ABZ and representing 3.3% of total radioactivity (Fig. 1B). With ascorbic acid, approximately 81% of the radioactivity was unmetabolized [3H]ABZ. The new peak co-eluted with synthetic N′HA but not with NHA. To examine the effect of ascorbic acid in more detail, a range of ascorbic acid concentrations was assessed (Fig. 2). The amount of [3H]ABZ metabolized represented 38% of the total radioactivity recovered by HPLC in the absence of ascorbic acid and decreased to 6% with 10 mm ascorbate. This decrease in ABZ metabolism was reflected in the corresponding decrease of 4′-nitro-4-acetylaminobiphenyl. The formation of the latter decreased from 25% of the total radioactivity in the absence of ascorbic acid to 1.2% with 10 mmascorbate. Ascorbic acid elicited a biphasic effect on the formation of the new metabolite (N′HA). At 3 mm, ascorbic acid elicited maximum N′HA formation (9.6% of the total radioactivity), whereas at 10 mm, the formation was reduced (4.7%). Although decreases in [3H]ABZ metabolism and 4′-nitro-4-acetylaminobiphenyl formation were observed with 0.03 mm ascorbic acid, increases in N′HA were not detected until 0.1 mm ascorbic acid was present. To identify the presumed N′HA metabolite (Fig. 1B), material was purified from a large scale incubation with 2 mmascorbic acid. The ESI mass spectra of both synthetic N′HA and NHA contain a MH+ ion at m/z 243. However, the identities of these molecular ion species can be distinguished by their daughter ion CAD tandem mass spectra. The CAD tandem mass spectrum of the MH+ ion derived from N′HA (Fig. 3A) contains an ion atm/z 201 due to ketene loss, which is seen for ABZ and N,N /-diacetylbenzidine. This ion is not observed in the CAD tandem mass spectrum of NHA, which instead contains an ion at m/z 200, representing acetyl loss due to C-N bond cleavage. The daughter ion CAD tandem mass spectrum obtained with the PHS-derived metabolite verified that this metabolite is N′HA (Fig. 3B). To further assess the presence of N′HA during the conversion of [3H]ABZ to [3H]4′-nitro-4-acetylaminobiphenyl, unlabeled N′HA was included in the reaction mixture (Fig. 1C). After the addition of 0.05 mm N′HA, a 3H peak was observed that co-eluted with the unlabeled N′HA standard (3.8% of the total radioactivity). With 0.05 mm N′HA, the formation of 4′-nitro-4-acetylaminobiphenyl was reduced from 23% of the total radioactivity to 2.8% of the total radioactivity, and the amount of unmetabolized [3H]ABZ increased from 66% to 86%. Solvent (dimethylformamide) in the absence of N′HA did not alter metabolism. The possible conversion of N′HA to 4′-nitro-4-acetylaminobiphenyl by PHS was assessed (Fig. 4). N′HA was substituted for ABZ in the reaction mixture. HPLC analysis indicated that a peak corresponding to 4′-nitro-4-acetylaminobiphenyl was observed. This product was not observed when 1 mm ascorbic acid was included in the incubation. The 4′-nitro-4-acetylaminobiphenyl metabolite was analyzed by ESI mass spectrometry and found to be identical to the authentic standard. The nitro metabolite was not converted to N′HA by incubation with 1 mm ascorbic acid. A variety of test agents were utilized to characterize ABZ metabolism by the ram seminal vesicle microsomal preparation. With arachidonic acid, indomethacin caused an almost complete inhibition of metabolism (Table I), whereas the cytochrome P-450 inhibitors DPEA, SKF-525A, α-naphthoflavone, and furafylline had no effect. With H2O2 as the substrate (TableII), indomethacin had no effect on metabolism, whereas sodium cyanide, a peroxidase inhibitor, reduced metabolism to 10% of the control incubation. Superoxide dismutase, mannitol, and 5,5-dimethyl-1-pyrroline N-oxide did not alter metabolism.Table IEffect of different test agents on arachidonic acid-dependent 4′-nitro-4-acetylaminobiphenyl formation by prostaglandin H synthaseCondition4′-Nitro-4-acetylaminobiphenyl% of controlaThe complete reaction produced 0.91 nmol of 4′-nitro-4-acetylaminobiphenyl.Complete100+ 0.01 mmindomethacin4+ 0.1 mm DPEAbDPEA, 2, 4-dichloro-6-phenylphenoxyethylamine.100+ 0.1 mm SKF-525A100+ 0.1 mmα-naphthoflavone92+ 0.1 mmfurafylline100a The complete reaction produced 0.91 nmol of 4′-nitro-4-acetylaminobiphenyl.b DPEA, 2, 4-dichloro-6-phenylphenoxyethylamine. Open table in a new tab Table IIEffect of different test agents on H2O2-dependent 4′-nitro-4-acetylaminobiphenyl formation by prostaglandin H synthaseCondition4′-Nitro-4-acetylaminobiphenyl% of ControlaThe complete reaction produced 1.04 nmole of 4′-nitro-4-acetylaminobiphenyl.Complete100+ 0.01 mmindomethacin100+ 50 mm NaCN10+ 50 mm DMPObDMPO, 5,5-dimethyl-1-pyrroline N-oxide.96+ 50 mmmannitol100+ 2 μg of superoxide dismutase100a The complete reaction produced 1.04 nmole of 4′-nitro-4-acetylaminobiphenyl.b DMPO, 5,5-dimethyl-1-pyrroline N-oxide. Open table in a new tab Oxygen incorporation into ABZ was assessed by oxygen uptake using H2O2 as the substrate. Using our assay conditions, 0.25 mm phenylbutazone was substituted for ABZ and used as a positive control to demonstrate oxygen uptake (29Marnett L.J. Bienkowski M.J. Pagels W.R. Reed G.A. Samuelsson B. Ramwell P.W. Paoletti R. Advances in Prostaglandin and Thromboxane Research. Raven Press, New York1980: 149-151Google Scholar, 30Lakshmi V.M. Zenser T.V. Mattammal M.B. Davis B.B. J. Pharmacol. Exp. Ther. 1993; 266: 81-88PubMed Google Scholar). Oxygen uptake was not detected during ABZ metabolism (data not shown). To assess the source of oxygen incorporated into ABZ, [18O]H2O2 was used as a substrate (Table III). 18O-N′HA enrichment was not detected. However, 18O enrichment of 4′-nitro-4-acetylaminobiphenyl was observed. In three separate experiments, 97% of the 4′-nitro-4-acetylaminobiphenyl molecules had one atom of 18O incorporated (16O-N-18O). A smaller percentage (17 ± 7%) of molecules contained 18O in both oxygen atoms of the nitro group (18O-N-18O).Table III18O-4′-Nitro-4-acetylaminobiphenyl enrichment after PHS-catalyzed N-acetylbenzidine metabolism with [ 18O]H2O2Experiment no.16O-N-18O18O-N-18O% of theoretical maximal18O incorporation197112971039731For MS 18O enrichment experiments, measurements in the positive mode determined the peak heights at m/z 257, 259, and 261. Calculations are described under “Experimental Procedures.” Open table in a new tab For MS 18O enrichment experiments, measurements in the positive mode determined the peak heights at m/z 257, 259, and 261. Calculations are described under “Experimental Procedures.” This report demonstrates that N′HA is formed during PHS metabolism of ABZ. This is the first time that PHS has been shown to produce anN-hydroxyarylamine. The presence of N′HA during ABZ peroxidatic metabolism was demonstrated by several methods using different experimental designs. [3H]N′HA was detected by HPLC after the addition of either ascorbic acid or unlabeled N′HA to the reaction mixture. The elution profile of N′HA was distinct from that of its structural isomer, NHA. In addition to the co-elution of [3H]N′HA with the synthetic N′HA standard, PHS-derived N′HA was identified by ESI/MS/MS. PHS was shown to metabolize N′HA to 4′-nitro-4-acetylaminobiphenyl. These results provide strong support for the conclusion that N′HA is formed during ABZ metabolism by PHS. The effects of ascorbic acid on ABZ metabolism are probably due to several factors (Figs. 1 and 2). Decreased ABZ metabolism could be explained by ascorbic acid acting as a reducing co-substrate for PHS (6Markey C.M. Alward A. Weller P.E. Marnett L.J. J. Biol. Chem. 1987; 262: 6266-6279Abstract Full Text PDF PubMed Google Scholar) and/or acting as a reducing agent, reducing an intermediate back to ABZ (13Zenser T.V. Mattammal M.B. Wise R.W. Rice J.R. Davis B.B. J. Pharmacol. Exp. Ther. 1983; 227: 545-550PubMed Google Scholar). Ascorbic acid is known to reduce nitroso compounds toN-hydroxyarylamines (31Mulder G.J. Unruh L.E. Evans F.E. Ketterer B. Kadlubar F.F. Chem. Biol. Interact. 1982; 39: 111-127Crossref PubMed Scopus (85) Google Scholar). Ascorbic acid also greatly increases the stability of N′HA in aqueous solutions (25Babu S.R. Lakshmi V.M. Hsu F.F. Zenser T.V. Davis B.B. Carcinogenesis. 1995; 16: 3069-3074Crossref PubMed Scopus (33) Google Scholar). The ability of unlabeled N′HA to elicit the appearance of [3H]N′HA is attributed to an isotope dilution effect (Fig. 1C). The small increase in [3H]N′HA compared with the large decrease in [3H]4′-nitro-4-acetylaminobiphenyl formation could be explained, in part, by decreased [3H]ABZ metabolism due to substrate competition from 0.05 mm unlabeled N′HA. Results suggest that the PHS conversion of ABZ to 4′-nitro-4-acetylaminobiphenyl occurs in several discrete steps with the oxidation of ABZ to N′HA, the oxidation of N′HA to 4′-nitroso, and the oxidation of the latter to 4′-nitro-4-acetylaminobiphenyl (Fig. 5). The accumulation of N′HA after the addition of ascorbic acid (Fig. 1B) is consistent with the reduction of 4′-nitroso-4-acetylaminobiphenyl, supporting the proposed reaction sequence. A transient N-hydroxyarylamine was detected during chloroperoxidase N-oxidation of 4-chloroaniline to its nitroso metabolite (32Corbett M.D. Chipko B.R. Batchelor A.O. Biochem. J. 1980; 187: 893-903Crossref PubMed Scopus (52) Google Scholar). Ring substituents that decrease electron density in the aromatic ring increased the ability to detectN-hydroxyarylamines (33Corbett M.D. Corbett B.R. Biochem. Arch. 1985; 1: 115-120Google Scholar). Reactions occurred in the absence of halide reducing cofactors. Oxidation of the nitroso to a nitro product was not detected. N-oxidation of amine to nitroso appeared to occur by two discrete two-electron transfers. Mass spectrometric analysis was used to directly establish the formation of N′HA by PHS and to determine the mechanism of metabolism. ESI/MS/MS demonstrated that the spectra of the new peak observed after the addition of ascorbic acid was identical to that of synthetic N′HA. The mass spectrum of PHS-derived N′HA was different from that observed with NHA. To assess the mechanism of ABZ metabolism, [18O]H2O2 was used as the substrate. 18O enrichment was observed in 4′-nitro-4-acetylaminobiphenyl, but not in N′HA. A subsequent study demonstrated a 97% 18O enrichment of 4′-nitro-4-acetylaminobiphenyl when N′HA was used as the reducing cofactor instead of ABZ. The inability to detect enriched N′HA may be attributed to a very rapid exchange of the [18O]N′HA with water. N-OH-arylamines undergo N-O bond heterolysis to form nitrenium ions, which react with an aqueous solvent (34Novak M. Kahley M.J. Eiger E. Helmick J.S. Peters H.E. J. Am. Chem. Soc. 1993; 115: 9453-9460Crossref Scopus (94) Google Scholar). This rapid exchange of [18O]N′HA would make it difficult to trap the first oxygen in 4′-nitro-4-acetylaminobiphenyl (Fig. 5) and would explain the relatively low enrichment observed for two 18O atoms into 4′-nitro-4-acetylaminobiphenyl. The results suggest that both ABZ and N′HA are accessible to the ferryl-bound oxygen from H2O2 and reduce PHS by oxygen transfer. Other results are also consistent with a peroxygenation mechanism. The lack of oxygen uptake during H2O2-mediated metabolism indicates that molecular oxygen is not required for the reaction. Hydroxyl radical or superoxide is not a source of oxygen or oxidants in the reaction because of the lack of effect of mannitol and superoxide dismutase. Oxidation by one-electron transfer would be expected to generate radical products, which should be inhibited by 5,5-dimethyl-1-pyrroline N-oxide (35Mottley C. Mason R.P. Biol. Magn. Reson. 1989; 8: 489-546Crossref Google Scholar). Radicals can also increase the oxygen uptake (28Lakshmi V.M. Zenser T.V. Sohani S. Davis B.B. Carcinogenesis. 1992; 13: 2087-2093Crossref PubMed Scopus (5) Google Scholar, 30Lakshmi V.M. Zenser T.V. Mattammal M.B. Davis B.B. J. Pharmacol. Exp. Ther. 1993; 266: 81-88PubMed Google Scholar) which was not observed. The lack of oxygen uptake and the lack of effect of specific inhibitors (Table II) provide strong support for metabolism of ABZ by the transfer of oxygen from H2O2. Sulfides, i.e. thioanisoles, are the only class of molecules previously reported to reduce PHS by oxygen transfer (9Egan R.W. Gale P.H. Van den Heuvel W.J.A. Baptista E.M. Keuhl Jr., F.A. J. Biol. Chem. 1980; 255: 323-326Abstract Full Text PDF PubMed Google Scholar, 10Ple P. Marnett L.J. J. Biol. Chem. 1989; 264: 13983-13993Abstract Full Text PDF PubMed Google Scholar). Several heme peroxidases catalyze peroxygenation reactions during organosulfur oxygenation and epoxidation of styrene (36Doerge D.R. Cooray N.M. Brewster M.E. Biochemistry. 1991; 30: 8960-8964Crossref PubMed Scopus (69) Google Scholar, 37Ortiz de Montellano P.R. Catalano C.E. J. Biol. Chem. 1985; 260: 9265-9271Abstract Full Text PDF PubMed Google Scholar). Chloroperoxidase and pea seed peroxygenase catalyze N-oxidation of aromatic amines to nitroso compounds by a peroxygenation mechanism (38Doerge D.R. Corbett M.D. Chem. Res. Toxicol. 1991; 4: 556-560Crossref PubMed Scopus (41) Google Scholar). Differences in reaction mechanisms among the various heme-dependent peroxidases and among substrates for the same peroxidase have been explained on the basis of accessibility of the heme moiety to substrates (39Ator M.A. Ortiz de Montellano P.R. J. Biol. Chem. 1987; 262: 1542-1551Abstract Full Text PDF PubMed Google Scholar, 40Harris R.Z. Newmyer S.L. Ortiz de Montellano P.R. J. Biol. Chem. 1993; 268: 1637-1645Abstract Full Text PDF PubMed Google Scholar). The peroxygenation mechanism for metabolism of ABZ is dramatically different from the electron transfer mechanism observed for PHS metabolism of benzidine, the diamine analogue of ABZ (12Wise R.W. Zenser T.V. Davis B.B. Carcinogenesis. 1983; 4: 285-289Crossref PubMed Scopus (38) Google Scholar, 13Zenser T.V. Mattammal M.B. Wise R.W. Rice J.R. Davis B.B. J. Pharmacol. Exp. Ther. 1983; 227: 545-550PubMed Google Scholar, 14Josephy P.D. Eling T.E. Mason R.P. Mol. Pharmacol. 1983; 23: 766-770PubMed Google Scholar, 41Oldfield L.F. Bockris J.O. J. Phys. Colloid Chem. 1951; 55: 1255-1274Crossref Scopus (26) Google Scholar, 42Rifi M.R. Tetrahedron Lett. 1969; 58: 5089-5092Crossref Scopus (7) Google Scholar, 43Wise R.W. Zenser T.V. Davis B.B. Carcinogenesis. 1985; 6: 579-583Crossref PubMed Scopus (30) Google Scholar, 44Josephy P.D. Eling T. Mason R.P. J. Biol. Chem. 1982; 257: 3669-3675Abstract Full Text PDF PubMed Google Scholar). Both chemical and enzymatic experiments indicate the oxidation of benzidine to one-electron (cation radical) and two-electron (benzidinediimine) oxidized products. A charge-transfer complex is also formed. The latter is a dimeric complex of benzidine and benzidinediimine, which is in equilibrium with the cation radical. Benzidinediimine is thought to be responsible for glutathione and DNA adduct formation (45Lakshmi V.M. Zenser T.V. Davis B.B. Toxicol. Appl. Pharmacol. 1994; 125: 256-263Crossref PubMed Scopus (19) Google Scholar). Formation of the dGp-ABZ adduct during PHS metabolism of ABZ (23Lakshmi V.M. Zenser T.V. Davis B.B. Carcinogenesis. 1998; 19: 911-917Crossref PubMed Scopus (19) Google Scholar) may be explained by the presence of N′HA (Fig. 5). N′HA generates nitrenium ions that react with DNA to produce dGp-ABZ (46Martin C.N. Beland F.A. Roth R.W. Kadlubar F.F. Cancer Res. 1982; 42: 2678-2686PubMed Google Scholar). It was proposed that PHS-derived 4′-nitro-4-acetylaminobiphenyl had to be reduced to N′HA before DNA adduct formation would occur (15Smith B.J. DeBruin L. Josephy D. Eling T.E. Chem. Res. Toxicol. 1992; 5: 431-439Crossref PubMed Scopus (32) Google Scholar). Whereas the latter may happen, this study demonstrates that N′HA is an intermediate available for immediate reaction with DNA. It is proposed that the reactions as illustrated in Fig. 5 occur in bladder epithelial cells and contribute to the initiation of bladder cancer, because: 1) bladder cells exhibit relatively high PHS but little cytochrome P-450 activity (47Danon A. Zenser T.V. Thomasson D.L. Davis B.B. Cancer Res. 1986; 46: 5676-5681PubMed Google Scholar, 48Wise R.W. Zenser T.V. Kadlubar F.F. Davis B.B. Cancer Res. 1984; 44: 1893-1897PubMed Google Scholar, 49Flammang T.J. Yamazoe Y. Benson R.W. Roberts D.W. Potter D.W. Chu D.Z.J. Lang N.P. Kadlubar F.F. Cancer Res. 1989; 49: 1977-1982PubMed Google Scholar), 2) ABZ is the major urinary metabolite observed in workers exposed to benzidine (50Rothman 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. Talaska G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5084-5089Crossref PubMed Scopus (96) Google Scholar, 51Hsu F. Lakshmi V. Rothman N. Bhatnager V.K. Hayes R.B. Kashyap R. Parikh D.J. Kashyap S.K. Turk J. Zenser T. Davis B. Anal. Biochem. 1996; 234: 183-189Crossref PubMed Scopus (30) Google Scholar), 3) bladder cells have been shown to peroxidatively activate an aromatic amine to form DNA adducts (52Hatcher J.F. Swaminathan S. Carcinogenesis. 1995; 16: 2149-2157Crossref PubMed Scopus (13) Google Scholar), 4) dGp-ABZ is detected in bladder cells from benzidine-exposed workers (50Rothman 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. Talaska G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5084-5089Crossref PubMed Scopus (96) Google Scholar), and 5) levels of dGp-ABZ correlate with urinary levels of ABZ (53Rothman 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. Zenser T. Cancer Epidemiol. Biomark. Prev. 1997; 6: 1039-1042PubMed Google Scholar). These experiments demonstrate that PHS is the seminal vesicle microsomal enzyme involved in ABZ metabolism. The effects of indomethacin are consistent with the distinct fatty acid cyclooxygenase (inhibition) and the peroxidase (absence of inhibition) activities of PHS (13Zenser T.V. Mattammal M.B. Wise R.W. Rice J.R. Davis B.B. J. Pharmacol. Exp. Ther. 1983; 227: 545-550PubMed Google Scholar). Cyanide, a peroxidase inhibitor, prevented ABZ metabolism and has been shown to inhibit PHS metabolism of benzidine (12Wise R.W. Zenser T.V. Davis B.B. Carcinogenesis. 1983; 4: 285-289Crossref PubMed Scopus (38) Google Scholar). 2,4-Dichloro-6-phenylphenoxyethylamine and SKF-525A prevented ABZ NADPH-dependent metabolism to N′HA by rat liver microsomes (18Lakshmi V.M. Zenser T.V. Davis B.B. Drug Metab. Dispos. 1997; 25: 481-488PubMed Google Scholar). The lack of involvement of cytochrome P-450s in ABZ seminal vesicle metabolism is suggested by the lack of inhibition observed with both general (2,4-dichloro-6-phenylphenoxyethylamine and SKF-525A) and specific 1A1/1A2 (α-naphthoflavone and furafylline) inhibitors (54Correia M.A. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York1995: 607-630Google Scholar,55Sesardic D. Boobis A. Murray B. Murray S. Segura J. De La Torre R. Davies D. Br. J. Clin. Pharmacol. 1990; 29: 651-663Crossref PubMed Scopus (286) Google Scholar). Previous studies have demonstrated that seminal vesicle microsomal cytochrome P-450 content and activity are very low (56Marnett L.J. Reed G.A. Johnson J.T. Biochem. Biophys. Res. Commun. 1977; 79: 569-576Crossref PubMed Scopus (86) Google Scholar, 57Marnett L.J. Johnson J.T. Bienkowski M.J. FEBS Lett. 1979; 106: 13-16Crossref PubMed Scopus (54) Google Scholar) and that PHS is the major peroxidase (58Pagels W.R. Sachs R.J. Marnett L.J. DeWitt D.L. Day J.S. J. Biol. Chem. 1983; 258: 6517-6523Abstract Full Text PDF PubMed Google Scholar). In summary, this study is the first to demonstrate that certain aromatic amines have access to the ferryl-bound oxygen of PHS and reduce this enzyme by oxygen transfer. N′HA was shown to be an intermediate in PHS metabolism of ABZ. N′HA is also an end product of ABZ metabolism by cytochrome P-450 (17Frederick C.B. Weis C.C. Flammang T.J. Martin C.N. Kadlubar F.F. Carcinogenesis. 1985; 6: 959-965Crossref PubMed Scopus (50) Google Scholar, 18Lakshmi V.M. Zenser T.V. Davis B.B. Drug Metab. Dispos. 1997; 25: 481-488PubMed Google Scholar). PHS peroxygenase metabolism of ABZ and N′HA produces 4′-nitro-4-acetylaminobiphenyl. N′HA could be responsible for dGp-ABZ formation during PHS metabolism of ABZ (23Lakshmi V.M. Zenser T.V. Davis B.B. Carcinogenesis. 1998; 19: 911-917Crossref PubMed Scopus (19) Google Scholar). Certain other aromatic and heterocyclic amines (16Boyd J.A. Harvan D.J. Eling T.E. J. Biol. Chem. 1983; 258: 8246-8254Abstract Full Text PDF PubMed Google Scholar, 59Morrison L.D. Eling T.E. Josephy P.D. Mutat. Res. 1993; 302: 45-52Crossref PubMed Scopus (23) Google Scholar) might also reduce PHS by oxygen transfer formingN-hydroxyarylamine metabolites, which might elicit toxic and carcinogenic effects. This manuscript is dedicated to Dr. Bernard B. Davis in recognition of 32 years of excellent scientific leadership. We thank Cindee Rettke and Priscilla DeHaven for excellent technical assistance.

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