From the Synthesis of prostaglandin H2
by 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 mM
H2O2 was used as the substrate. This metabolite
co-eluted with synthetic
N'-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 two
18O 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)1 catalyzes the
metabolism of arachidonic acid in the presence of molecular oxygen to
form prostaglandin (PG) H2 (1). 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 (2,
3). 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. 4). 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 (5). 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 (6). 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. 7). 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 (8). 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 (9, 10). 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 (11).
Benzidine, an aromatic amine reducing cofactor, is converted to a
radical cation and subsequently converted to benzidinediimine, a
two-electron oxidation product (12-14). In contrast, monoacetylated
benzidine, N-acetylbenzidine (ABZ), yields an oxygenated
metabolite, 4'-nitro-4-acetylaminobiphenyl (15). 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 (15). 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 (16).
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 to
N'-hydroxy-N-acetylbenzidine (N'HA) and
N-hydroxy-N-acetylbenzidine (NHA)
(17, 18). N'HA production was associated with
N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation (dGp-ABZ) (18). This DNA adduct produces genotoxic lesions,
causing mutations in various bacterial and mammalian test systems
in vitro (19-21) and mutations in the oncogenes of tumors
induced in vivo by benzidine (22). A recent study has demonstrated dGp-ABZ formation after PHS metabolism of ABZ (23). Thus,
PHS metabolism of ABZ was reexamined to determine the mechanism of
metabolism and the possible presence of an N'HA intermediate.
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 >98% (24). 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 (25). The identity of these synthetic compounds was
established by NMR and mass spectrometry. Seminal vesicle microsomes
were prepared as described previously (26).
[18O]H2O2 was purchased from Icon
Isotope Services, Inc. (Summit, NJ).
Metabolism of ABZ by PHS--
[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 (27). The reaction was started by the addition
of 0.2 mM arachidonic acid or 0.5 mM
H2O2 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%.
Metabolite Purification--
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 Oxygen Uptake Studies--
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 (28). To assess
phenylbutazone metabolism, 0.25 mM phenylbutazone was added
as described previously (29, 30).
Mass Spectral Identification of Metabolites--
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 DEC
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 of
18O 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 of 18O incorporated (18O-N-18O). The
18O 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 mM
ascorbate. 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.
VA Medical Center,
Department of Medicine,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C for MS analysis.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
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Fig. 1.
HPLC analysis of PHS metabolism of
N-acetylbenzidine. Ram seminal vesicle microsomes
were incubated with 0.06 mM
[3H]N-acetylbenzidine, and 0.2 mM
arachidonic acid was added to start the reaction. Where
indicated, 1 mM ascorbic acid or 0.05 mM
unlabeled N'HA was included in the incubation.
4'-Nitro-ABZ, 4'-nitro-4-acetylaminobiphenyl.
View larger version (19K):
[in a new window]
Fig. 2.
The dose-response effect of ascorbic acid on
arachidonic acid-dependent PHS metabolism of
N-acetylbenzidine. Samples were analyzed by HPLC
as illustrated in Fig. 1. 4'-Nitro-ABZ,
4'-nitro-4-acetylaminobiphenyl.
To identify the presumed N'HA metabolite (Fig. 1B), material
was purified from a large scale incubation with 2 mM
ascorbic 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 at
m/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).
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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.
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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, -naphthoflavone, and furafylline had no
effect. With H2O2 as the substrate (Table
II), 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.
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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 (29, 30). 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).
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DISCUSSION |
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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 an N-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 (6) and/or acting as a reducing agent, reducing an intermediate back to ABZ (13). Ascorbic acid is known to reduce nitroso compounds to N-hydroxyarylamines (31). Ascorbic acid also greatly increases the stability of N'HA in aqueous solutions (25).
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.
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A transient N-hydroxyarylamine was detected during chloroperoxidase N-oxidation of 4-chloroaniline to its nitroso metabolite (32). Ring substituents that decrease electron density in the aromatic ring increased the ability to detect N-hydroxyarylamines (33). 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 (34). 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 (35). Radicals can also increase the oxygen uptake (28, 30) 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 (9, 10). Several heme peroxidases catalyze peroxygenation reactions during organosulfur oxygenation and epoxidation of styrene (36, 37). Chloroperoxidase and pea seed peroxygenase catalyze N-oxidation of aromatic amines to nitroso compounds by a peroxygenation mechanism (38). 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 (39, 40).
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 (12-14, 41-44). 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 (45).
Formation of the dGp-ABZ adduct during PHS metabolism of ABZ (23) may be explained by the presence of N'HA (Fig. 5). N'HA generates nitrenium ions that react with DNA to produce dGp-ABZ (46). It was proposed that PHS-derived 4'-nitro-4-acetylaminobiphenyl had to be reduced to N'HA before DNA adduct formation would occur (15). 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 (47-49), 2) ABZ is the major urinary metabolite observed in workers exposed to benzidine (50, 51), 3) bladder cells have been shown to peroxidatively activate an aromatic amine to form DNA adducts (52), 4) dGp-ABZ is detected in bladder cells from benzidine-exposed workers (50), and 5) levels of dGp-ABZ correlate with urinary levels of ABZ (53).
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 (13). Cyanide, a peroxidase inhibitor, prevented ABZ metabolism and
has been shown to inhibit PHS metabolism of benzidine (12).
2,4-Dichloro-6-phenylphenoxyethylamine and SKF-525A prevented ABZ
NADPH-dependent metabolism to N'HA by rat liver microsomes (18). 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 (-naphthoflavone and furafylline) inhibitors (54,
55). Previous studies have demonstrated that seminal vesicle microsomal
cytochrome P-450 content and activity are very low (56, 57) and that
PHS is the major peroxidase (58).
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 (17, 18). 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 (23). Certain other aromatic and heterocyclic amines (16, 59)
might also reduce PHS by oxygen transfer forming N-hydroxyarylamine metabolites, which might elicit toxic and
carcinogenic effects.
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ACKNOWLEDGEMENTS |
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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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FOOTNOTES |
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* This work was supported by the Department of Veterans Affairs (T. V. Z. and B. B. D.) and National Cancer Institute Grant CA72613 (to T. V. Z.). Mass spectrometry was performed at the Mass Spectrometry Resource Center, Washington University School of Medicine, through National Institutes of Health Grants RR-00954 and AM-20579.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: VA Medical Center (GRECC/11G-JB), St. Louis, MO 63125-4199. Tel.: 314-894-6510; Fax: 314-894-6614; E-mail: zensertv{at}slu.edu.
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ABBREVIATIONS |
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The 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.
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REFERENCES |
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