Rat liver microsomal metabolism of o-aminophenol and N-(2-methoxyphenyl) hydroxylamine, two metabolites of the environmental pollutant and carcinogen o-anisidine in humans

Article abstract of DOI:10.1135/cccc2010077

o-Aminophenol and N-(2-methoxyphenyl)hydroxylamine are human metabolites of the industrial and environmental pollutant and bladder carcinogen 2-methoxyaniline (o-anisidine). The latter one is also a human metabolite of another pollutant and bladder carcinogen, 2-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of rat hepatic microsomes to metabolize these metabolites. N-(2-methoxyphenyl)hydroxylamine is metabolized by rat hepatic microsomes to o-aminophenol and predominantly o-anisidine, the parent carcinogen from which N-(2-methoxyphenyl)hydroxylamine is formed. In addition, two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, were generated. On the contrary, no metabolites were found to be formed from o-aminophenol by rat hepatic microsomes. Whereas N-(2-methoxyphenyl)hydroxylamine is responsible for formation of three deoxyguanosine adducts in DNA, o-aminophenol seems to be a detoxication metabolite of N-(2-methoxyphenyl)hydroxylamine and/or a parental carcinogen, o-anisidine; no o-aminophenol-derived DNA adducts were found after its reaction with microsomal cytochromes P450 and peroxidases.

Full text of DOI:10.1135/cccc2010077

Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1229
RAT LIVER MICROSOMAL METABOLISM OF o-AMINOPHENOL AND
N-(2-METHOXYPHENYL)HYDROXYLAMINE, TWO METABOLITES OF
THE ENVIRONMENTAL POLLUTANT AND CARCINOGEN o-ANISIDINE
IN HUMANS
a1
a2
a3
b
Karel NAIMAN , Petr HODEK , Jiří LIBERDA , Heinz H. SCHMEISER ,
c
a4,
Eva FREI and Marie STIBOROVÁ
*
a
Department of Biochemistry, Faculty of Natural Sciences, Charles University in Prague,
Albertov 2030, 128 43 Prague 2, Czech Republic; e-mail: naiman@email.cz,
hodek@natur.cuni.cz, jirik@natur.cuni.cz, stiborov@natur.cuni.cz
Research Group Genetic Alteration in Carcinogenesis, National Center for Tumour Diseases,
German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany;
e-mail: h.schmeiser@dkfz-heidelberg.de
Division of Preventive Oncology, National Center for Tumour Diseases,
German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany;
e-mail: e.frei@dkfz.de
1
2
3
4
b
c
Received Jume 24, 2010
Accepted August 17, 2010
Published online November 18, 2010
o-Aminophenol and N-(2-methoxyphenyl)hydroxylamine are human metabolites of the in-
dustrial and environmental pollutant and bladder carcinogen 2-methoxyaniline (o-anisidine).
The latter one is also a human metabolite of another pollutant and bladder carcinogen,
2
-methoxynitrobenzene (o-nitroanisole). Here, we investigated the ability of rat hepatic micro-
somes to metabolize these metabolites. N-(2-methoxyphenyl)hydroxylamine is metabolized
by rat hepatic microsomes to o-aminophenol and predominantly o-anisidine, the parent car-
cinogen from which N-(2-methoxyphenyl)hydroxylamine is formed. In addition, two
N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been
identified as yet, were generated. On the contrary, no metabolites were found to be formed
from o-aminophenol by rat hepatic microsomes. Whereas N-(2-methoxyphenyl)hydroxyl-
amine is responsible for formation of three deoxyguanosine adducts in DNA, o-aminophenol
seems to be a detoxication metabolite of N-(2-methoxyphenyl)hydroxylamine and/or a pa-
rental carcinogen, o-anisidine; no o-aminophenol-derived DNA adducts were found after its
reaction with microsomal cytochromes P450 and peroxidases.
Keywords: o-Anisidine; N-(2-Methoxyphenyl)hydroxylamine; o-Aminophenol; Metabolism;
Activation and detoxification.
2
-Methoxyaniline (o-anisidine) is a potent carcinogen, causing tumors
1
of the urinary bladder in rats and mice . The International Agency for
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1229–1247
©
2010 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2010077

1
230
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
Research on Cancer (IARC) has classified o-anisidine as a group 2B carcino-
1
gen , which is possibly carcinogenic to humans. o-Anisidine is used as an
intermediate in the manufacturing of a number of azo and naphthol
pigments and dyes, which are used for printing (90%) and for paper (3%)
2
and textile (7%) dyeing . This carcinogen is also a constituent of cigarette
smoke1 . Such a wide use of this aromatic amine could result in occupa-
–3
tional exposure. Hemoglobin adducts of o-anisidine were detected in blood
4
samples of persons living in urban or rural areas of Germany . The adducts
as well as o-anisidine found in urine might originate not only from the
sources mentioned above, but also from a possible o-anisidine precursor,
2
-methoxynitrobenzene (o-nitroanisole). This chemical was released into
the environment in the course of an accident in a German chemical plant,
5
causing subsequently local and regional contamination .
We have found that o-anisidine is oxidatively activated by peroxidase
and cytochrome P450 (CYP) to species binding to DNA in vitro6–10
.
o-Anisidine also forms DNA adducts in vivo. The same adducts as found
in DNA incubated with o-anisidine and human microsomes in vitro were
detected in urinary bladder, the target organ, and to a lesser extent, in liver,
8
kidney and spleen of rats treated with o-anisidine . The o-anisidine-derived
DNA adducts were identified as deoxyguanosine adducts formed from a me-
tabolite of o-anisidine, N-(2-methoxyphenyl)hydroxylamine8 . The same
deoxyguanosine adducts were also detected in DNA of the urinary bladder,
–10
1
1
kidney, liver and spleen of rats treated with o-nitroanisole , an oxidized
counterpart of o-anisidine. The data found previously8
,10–12
, indicate that
formation of N-(2-methoxyphenyl)hydroxylamine, the reactive metabolite
of both carcinogens, is critical for generation of DNA lesions in target or-
gans.
Recently, we have found that o-anisidine is oxidized by human, rat and
rabbit hepatic microsomes not only to N-(2-methoxyphenyl)hydroxyl-
amine, but that this compound is a subject of complex redox cycling reac-
tions, forming also o-aminophenol, o-nitrosoanisole and one additional
8
,10
metabolite, the structure of which has not been identified as yet
Scheme 1). N-(2-methoxyphenyl)hydroxylamine seems to also be a subject
(
of complex reactions, and its fate is dependent on the environment, in
which it occurs. The results of preliminary experiments utilizing rabbit
hepatic microsomes indicate that N-(2-methoxyphenyl)hydroxylamine can
be further metabolized forming three metabolites. Two of them were esti-
mated that might be o-aminophenol and a parent compound from which
1
0
N-(2-methoxyphenyl)hydroxylamine is generated, o-anisidine . When
nucleophiles such as DNA or proteins are present in the incubation mix-
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1231
8
ture, N-(2-methoxyphenyl)hydroxylamine forms the adducts . Neverthe-
less, metabolism of this compound by hepatic microsomes of rats, the
animal model mimicking the metabolism of a parent carcinogen,
o-anisidine, in human, has not been studied as yet. Therefore, metabolism
of N-(2-methoxyphenyl)hydroxylamine by rat hepatic microsomes was in-
vestigated in the present study.
While the formation of N-(2-methoxyphenyl)hydroxylamine was clearly
identified to be the activation pathway of o-anisidine and o-nitroanisole
metabolism8 , biological significance of formation of another major
o-anisidine metabolite, o-aminophenol, for detoxication/activation metabo-
,11
lism awaits further investigation. Even though o-aminophenol was found
to be oxidized by peroxidases1
3,14
, a metabolic conversion of this
SCHEME 1
Pathways of o-anisidine, N-(2-methoxyphenyl)hydroxylamine and o-aminophenol metabolism
showing the characterized metabolites and those proposed to form DNA adducts. The com-
pounds shown in brackets were not detected under the experimental conditions
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1
232
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
o-anisidine metabolite by microsomal CYP enzymes has not been investi-
gated as yet. In addition, significance of its metabolism by such enzymes
(
CYPs and peroxidases) for activation/detoxication of this compound has
not yet been examined. Therefore, investigation of potential of microsomal
CYP enzymes and peroxidases to activate o-aminophenol is another aim of
this study.
EXPERIMENTAL
Chemicals
Chemicals used were of analytical purity or better. 2-Methoxynitrosobenzene (o-nitroso-
8
,10
1
8,10
anisole) was synthesized as described
phenyl)hydroxylamine was synthesized by the procedure similar to that described earlier
and its identity was confirmed by electrospray mass and collision-induced dissociation spec-
and identified by H NMR
. N-(2-methoxy-
1
5
,
8
,10
tra and high field proton NMR spectroscopy
ESI mass spectra and H and C NMR spectroscopy
. o-Aminophenol identity was confirmed by
1
13
10
.
Preparation of Microsomes and Assays
The study was conducted in accordance with the Regulations for the Care and Use of Labo-
ratory Animals (311/1997, Ministry of Agriculture, Czech Republic), which complies with
the Declaration of Helsinki. Adult male Wistar rats (150–200 g) were fed ad libitum on pellet
chow and water. Microsomes from livers of ten male Wistar untreated rats or rats pre-treated
with β-naphthoflavone (β-NF), phenobarbital (PB), ethanol or pregnenolone-16α-carbonitrile
3β-hydroxy-20-oxopregn-5-ene-16α-carbonitrile) (PCN) were prepared as described ,16–18.
8
(
Hepatic microsomes of control (noninduced) rats and rats induced with β-NF, PB, ethanol
and PCN contained 0.6, 1.3, 2.7, 1.8 and 1.6 nmol CYP/mg protein, respectively. The activ-
ity of NADPH:CYP reductase in rat hepatic microsomes was measured using cytochrome c as
1
0
a substrate . NADPH:CYP reductase activities in hepatic microsomes of control (uninduced)
rats and rats induced with β-NF, PB, ethanol and PCN were 0.210, 0.199, 0.325, 0.201 and
0
.210 µmol/min/mg protein, respectively.
NADPH:CYP reductase was isolated from liver microsomes of rats pre-treated with PB as
1
7
described earlier
.
Incubations
Incubation mixtures used for study of the metabolism of N-(2-methoxyphenyl)hydroxyl-
amine and o-aminophenol contained the following concentrations in a final volume of 100 µl:
+
1
1
00 mM sodium phosphate buffer (pH 7.4), 1 mM NADP , 10 mM D-glucose 6-phosphate,
U/ml D-glucose 6-phosphate dehydrogenase (NADPH-generation system), a rat hepatic
microsomal fraction containing 0.7 nmol of CYP, and 1.0 mM N-(2-methoxyphenyl)hydroxyl-
amine or o-aminophenol dissolved in 10 µl of distilled water. The reaction was initiated by
adding the substrate. After incubation in open glass tubes (37 °C, 30 min), the reactions
were terminated by adding 100 µl of methanol and centrifuged at 5,000 g for 5 min. The
supernatants were collected and 30 µl of aliquots applied onto a high-performance liquid
chromatography (HPLC) column, where metabolites of N-(2-methoxyphenyl)hydroxylamine
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1229–1247

Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1233
were separated. Conversion of N-(2-methoxyphenyl)hydroxylamine with rat CYP enzymatic
systems to its metabolites was linear until 40 min. Compositions of incubation mixtures
used for study of the metabolism of N-(2-methoxyphenyl)hydroxylamine by rabbit
NADPH:P450 reductase were analogous to those described above, but 2 µM NADPH:CYP
reductase instead of microsomal enzymes was present. NADPH:CYP reductase was active
with its substrate, cytochrome c. The HPLC was performed on a C-18 high density reversed-
phase column (250 × 4.6 mm, 5 µm, Nucleosil 100-5HD, Macherey-Nagel, Duren, Germany).
Metabolites were eluted with 20% methanol, 80% 7.18 µM aqueous ammonia, pH 8.0 (v/v),
at a flow rate of 0.6 ml/min and monitored at 234 nm. N-(2-Methoxyphenyl)hydroxylamine
metabolites were analyzed by mass spectrometry and by comparing their chromatographic
properties on HPLC with those of synthetic standards. 2-Methoxynitrosobenzene, o-amino-
phenol, N-(2-methoxyphenyl)hydroxylamine and o-anisidine standards were eluted at reten-
tion times (r.t.) of 9.8, 10.8, 24.8 and 35.0 min, respectively.
Mass Spectrometry
Q
Positive- and negative-ion ESI mass spectra were recorded on a Finnigan LC -DECA
quadrupole ion trap mass spectrometer (ThermoFinnigan, San Jose (CA), USA). Metabolites
(
final concentration 1 pmol/µl) dissolved in methanol–water (1:1, v/v) were continuously
infused through a capillary held at 1.8 kV into the dynamic Finnigan nano-electrospray
ion source via a linear syringe pump (Harvard Apparatus Model 22) at a rate of 1 µl/min.
The ionizer and ion transfer optics parameters of the ion trap were as follows: spray voltage
1800 V, capillary temperature 150 °C, capillary voltage 14 V, tube lens offset –22 V, octapole 1
offset –7.4 V, lens voltage –16 V, octapole 2 offset –11.3 V, octapole r.f. amplitude 450 V
peak-to-peak (pp), and entrance lens voltage –66.9 V. Helium was introduced at a pressure of
0
.1 Pa to improve the trapping efficiency of the sample ions. The spectra were scanned in
7
the m/z range of 50–800 and the gating time was set to accumulate and trap 1 × 10 ions.
The mass isolation window for precursor ion selection was set to 2 amu and centered on the
1
2
C isotope of the pertinent ion. The background helium gas served as the collision gas for
the collision-induced dissociation (CID) experiment. The relative activation amplitude was
5% and the activation time was 30 ms. No broadband excitations were applied. Metabolites
were also characterized by the standard EI (electron impact) MS (70 eV) (FinniganMAT,
3
1
0
San Jose (CA), USA)
.
Covalent DNA Binding
Incubation mixtures used to study the binding of N-(2-methoxyphenyl)hydroxylamine or
o-aminophenol to DNA after its metabolism with hepatic microsomes of rats pre-treated
with ethanol contained in a final volume of 0.75 ml: 50 mM sodium phosphate (pH 7.4),
0
.5 mM N-(2-methoxyphenyl)hydroxylamine or o-aminophenol dissolved in distilled water
(
10 µl/0.75 ml incubation), 1 mM NADPH, hepatic microsomes containing 1 mg of protein
and 1 mg of calf thymus DNA. The reaction was initiated by adding N-(2-methoxy-
phenyl)hydroxylamine or o-aminophenol and reaction mixtures were incubated at 37 °C for
3
h. Control incubations were carried out either without microsomes or without DNA or
without N-(2-methoxyphenyl)hydroxylamine or without o-aminophenol. In order to evalu-
ate a spontaneous cleavage of N-(2-methoxyphenyl)hydroxylamine to nitrenium/carbenium
ions and their binding to DNA, 0.5 mM N-(2-methoxyphenyl)hydroxylamine was also incu-
bated only with DNA in the buffer mentioned above at 37 °C for 3 h. Incubation mixtures
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1229–1247

1
234
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
used to study the binding of o-aminophenol to DNA after its oxidation with peroxidases
contained in a final volume of 0.5 ml: 50 mM sodium phosphate (pH 7.4), 0.2 mM
o-aminophenol dissolved in distilled water (5 µl/0.5 ml incubation), 5 or 10 µg of HRP or
lactoperoxidase, respectively, 0.4 mM hydrogen peroxide and 1 mg of calf thymus DNA.
The reaction was initiated by adding hydrogen peroxide and reaction mixture was incubated
at 37 °C for 60 min. DNA was isolated from the water phase after extraction of incubations
1
9
with ethyl acetate by the phenol/chloroform extraction method as described
.
32P-Postlabeling Analysis
The standard procedure20, this procedure under the ATP-deficient conditions21, and the
2
2
32
nuclease P1 enrichment version
of the
P-postlabeling assay were performed as de-
8
,11,18,21
scribed
polyethylenimine (PEI) cellulose plates. (i) Essentially as described
vent was 3.5 M lithium formate, 8.5 M urea (pH 3.5); D4 solvent was 0.8 M lithium chloride,
.5 M Tris-HCl, 8.5 M urea (pH 8.0), followed by a final wash with 1.7 M sodium phosphate
. Labeled DNA digests were separated by two chromatographic methods on
6
,7,18
, except that D3 sol-
0
(
3
2
pH 6.0). D2 was omitted (method A). (ii) P-labeled adducts were also resolved by the
modification described by Reddy et al. . This procedure has been shown to be suitable for
2
2
resolution of DNA adducts formed by N-(2-methoxyphenyl)hydroxylamine or by o-anisidine,
8
,11
reductively activated in vitro and in vivo
sodium phosphate (pH 5.77); D2 was omitted; D3, 2.7 M lithium formate, 5.1 M urea (pH
.5); D4, 0.36 M sodium phosphate, 0.23 M Tris-HCl, 3.8 M urea (pH 8.0). After D4 develop-
. The solvents used in this case were: D1, 2.3 M
3
ment and brief water wash, the sheets were developed (along D4) in 1.7 M sodium phos-
phate (pH 6.0) (D5), to the top of the plate, followed by an additional 30–40 min
development with the thin layer chromatography (TLC) tank partially opened, to allow the
8
,11
radioactive impurities to concentrate in a band close to the top edge (method B)
. Adduct
levels were calculated in units of relative adduct labeling (RAL), which is the ratio of c.p.m.
of adducted nucleotides to c.p.m. of total nucleotides in the assay.
Preparation of Reference Compounds and 32P-Postlabeling Analysis of Adducts
An aliquot of 0.5 µmol of deoxyguanosine 3′-phosphate (dGp) was incubated in 50 mM
Tris-HCl buffer, pH 5.0, with 20 µmol of N-(2-methoxyphenyl)hydroxylamine without fur-
8
ther activation at 37 °C overnight in a total volume of 0.5 ml . After incubation and extrac-
tion with ethyl acetate, 20 µl aliquots were removed from the aqueous phase and directly
3
2
21
used for P-postlabeling analysis; the standard procedure and the nuclease P1 version of
the assay was utilized22. Resolution of the adducts on a PEI-cellulose TLC plate was carried
out by method B.
RESULTS
Metabolism of N-(2-Methoxyphenyl)hydroxylamine and o-Aminophenol
by Rat Hepatic Microsomes
When N-(2-methoxyphenyl)hydroxylamine was incubated with rat hepatic
microsomes in the presence of NADPH, four product peaks with r.t. of 8.2,
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1235
a
b
c
d
FIG. 1
HPLC elution profile of metabolites of 1 mM N-(2-methoxyphenyl)hydroxylamine formed dur-
ing incubation with hepatic microsomes of rats pre-treated with ethanol and NADPH for 30
min (a), of the same incubation mixture in time zero (b), of metabolites of 1 mM N-(2-
methoxyphenyl)hydroxylamine formed during incubation with NADPH:CYP reductase and
NADPH for 30 min (c), and of the same incubation mixture in time zero (d). Synthetic
o-aminophenol (e), N-(2-methoxyphenyl)hydroxylamine (f), and o-anisidine (g). For incuba-
tion conditions see Experimental. Peaks eluting between 2.0 and 5.5 min, solvent front,
NADPH and protein components of microsomes and NADPH-generation system
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236
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
1
0.8, 12.8 and 35.0 min, were separated by HPLC with UV monitoring
at 234 nm (see peaks in Fig. 1a for the profile obtained with hepatic micro-
somes of rats pre-treated with ethanol). The chromatographic properties
of an N-(2-methoxyphenyl)hydroxylamine metabolite with r.t. of 10.8 min
corresponded to those of o-aminophenol (Fig. 1). The mass spectrum
(
Fig. 2A) is consistent with the product being o-aminophenol. An addi-
tional product peak detected by HPLC, eluting at r.t. of 35.0 min, was
shown to be the parental compound, from which N-(2-methoxyphenyl)-
hydroxylamine is generated, o-anisidine (r.t. 35.0 min) (Fig. 1). The mass
spectra of this metabolite peak and o-anisidine were identical (not shown).
Chromatographic properties of an N-(2-methoxyphenyl)hydroxylamine
metabolite M1, eluting at r.t. of 8.2 min, did not correspond to any stan-
dard compound used for analysis. In the atmospheric pressure chemical
ionization mass spectrum, this metabolite showed the mass signal at m/z
1
22.8 (Fig. 2b), corresponding to that of the nitrenium/carbenium ion of
N-(2-methoxyphenyl)hydroxylamine. We have not ascertained whether
e
f
g
FIG. 1
(
Continued)
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1237
a
b
FIG. 2
a The positive- and negative-ion mass spectra of N-(2-methoxyphenyl)hydroxylamine metabo-
lite with r.t. 10.8 min. The protonated ion at m/z 110.4 and the negative ion at m/z 108.4 indi-
cate the molecular mass of o-aminophenol. b Atmospheric pressure chemical ionization mass
spectrum of N-(2-methoxyphenyl)hydroxylamine metabolite M1. The ion at m/z 122.8 indi-
cates the molecular mass of the nitrenium/carbenium ion of N-(2-methoxyphenyl)-
hydroxylamine
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1229–1247

1
238
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
this product is indeed the nitrenium/carbenium ion metabolite of N-(2-
methoxyphenyl)hydroxylamine, formed in the incubation, or product
formed in the mass spectrometer. Chromatographic properties of a product
peak M2, eluting at r.t. of 12.8 min, did not correspond to any standard
compound used for analysis. Its structure has not been characterized as yet.
Microsomes isolated from livers of uninduced rats and rats pre-treated
with β-NF (enriched with CYP1A1/2), PB (enriched with CYP2B1/2),
ethanol (enriched with CYP2E1) and PCN (enriched with CYP3A) were all
capable of metabolizing N-(2-methoxyphenyl)hydroxylamine, participating
differently in this reaction process (Table I). Among them, hepatic micro-
somes of rats pre-treated with ethanol were the most effective in N-(2-
methoxyphenyl)hydroxylamine metabolism, followed by microsomes of
rats pre-treated with PB, producing predominantly the reductive metabolite
of N-(2-methoxyphenyl)hydroxylamine, o-anisidine (Table I).
When N-(2-methoxyphenyl)hydroxylamine was incubated without hepa-
tic microsomal enzymes or without NADPH, metabolite M1, M2, o-amino-
phenol and o-anisidine peaks were also detectable by HPLC, but only under
acidic conditions (at pH 4.5 for 60 min). At pH 7.4, used for microsomal
incubations, their spontaneous formation was negligible; only a low but
detectable amount of o-anisidine was found (Table I). In the presence of
NADPH in the incubation mixture, the amount of this metabolite was
increased, by 2.4-fold (Table I). These findings indicate that conversion of
N-(2-methoxyphenyl)hydroxylamine in microsomes is mediated mainly
by enzymatic reactions, but participation of NADPH in formation of
o-anisidine cannot be excluded.
When N-(2-methoxyphenyl)hydroxylamine was incubated with NADPH
and NADPH:CYP reductase, the enzyme that is besides CYPs the prominent
component of the microsomal enzymatic system, formation of metabolite
M1 and o-anisidine was increased (Fig. 1, Table I). Our results suggest that
NADPH:CYP reductase plays a minor role in N-(2-methoxyphenyl)hydroxyl-
amine metabolism in microsomes, whereas CYP enzymes are more impor-
tant for this process.
In contrast to results found with N-(2-methoxyphenyl)hydroxylamine,
no metabolites were found to be formed from o-aminophenol when incu-
bated with rat hepatic microsomes and NADPH.
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
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240
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
DNA Adduct Formation by N-(2-Methoxyphenyl)hydroxylamine
and During Its Metabolism by Hepatic Microsomes
Using the 32P-postlabeling technique, we tested whether during the N-(2-
methoxyphenyl)hydroxylamine metabolism with hepatic microsomes of
rats pre-treated with ethanol, which were the most effective enzymatic
system metabolizing this compound, reactive species binding to DNA are
generated. DNA was isolated from incubation mixtures containing N-(2-
methoxyphenyl)hydroxylamine, microsomes and NADPH, and analyzed
for DNA adduct formation. The standard procedure and the nuclease P1
version of the 32P-postlabeling assay, suitable for detection of adducts medi-
ated in dGp by N-(2-methoxyphenyl)hydroxylamine (Fig. 3) or adducts in
8
DNA of the urinary bladder of rats pre-treated with o-anisidine were used.
Using these methods, up to three DNA adducts were found when hepatic
microsomes of rats treated with ethanol were incubated with DNA, N-(2-
methoxyphenyl)hydroxylamine and NADPH, having the same chromato-
graphic properties as adducts formed by N-(2-methoxyphenyl)hydroxyl-
FIG. 3
Autoradiographic profiles of 32P-labeled DNA adducts in calf thymus DNA formed by N-
(
2-methoxyphenyl)hydroxylamine incubated with hepatic microsomes of rats pre-treated with
ethanol (A, B), in dGp incubated with N-(2-methoxyphenyl)hydroxylamine (C), in DNA
formed by o-aminophenol incubated with hepatic microsomes of rats pre-treated with ethanol
(
D, E), and in DNA formed by o-aminophenol incubated with lactoperoxidase (F, G) (see Exper-
imental. Sample C was used as a positive control. The nuclease P1 version of the
3
2
P-postlabeling assay was used for analysis shown in panels (A), (D) and (F), the standard pro-
cedure for that in panels (B) and (C), and the standard procedure under ATP-deficient condi-
tions for that in panels (E) and (G). The method B (see Experimental) was utilized for
resolution of adducts
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1241
amine with dGp (Fig. 3, A–C). Similar to our previous work investigating
formation of DNA adducts by o-anisidine and N-(2-methoxyphenyl)-
8
hydroxylamine , only two DNA adducts (adduct spots 1 and 2) were, how-
ever, clearly detectable when the nuclease P1 version of the 32P-postlabeling
assay was used (Fig. 3, A). This finding indicates that the major dGp adduct
in DNA resulting from N-(2-methoxyphenyl)hydroxylamine (adduct spot 3
in Fig. 3) should to be the C8 adduct of the nitrenium ion with dGp.
Namely, the C8 adducts of dGp formed by nitrenium ions from N-hydroxy-
arylamines, including those from N-hydroxyanilines, are frequently de-
phosphorylated by nuclease P1 and, therefore, their detection by the
3
2
8
nuclease P1 version of the P-postlabeling assay is negligible . Indeed,
using the standard procedure of the 32P-postlabeling analysis, the adduct
spot 3 (Fig. 3), accounting more than 90% of the total RAL level and exhib-
iting sensitivity to dephosphorylation by nuclease P1 (Table II), was de-
TABLE II
Quantitative analysis of adducts formed by N-(2-methoxyphenyl)hydroxylamine with DNA
with or without rat hepatic microsomes
DNA adduct content determined by 2P-postlabelinga
3
6
RAL (adducts/10 nucleotides)
DNA adducts
Without microsomes
With microsomes
The standard procedure of the 32P-postlabeling assay
Spot 1b
Spot 2
Spot 3
Total
0.71 ± 0.04
0.03 ± 0.01
8.62 ± 0.52
9.36 ± 0.41
0.35 ± 0.03
0.01 ± 0.01
3.61 ± 0.22
3.97 ± 0.31
The nuclease P1 version of the 32P-postlabeling assay
Spot 1b
Spot 2
Spot 3
Total
2.27 ± 0.19
0.03 ± 0.01
0.04 ± 0.01
2.34 ± 0.23
1.18 ± 0.12
0.01 ± 0.01
0.01 ± 0.01
1.20 ± 0.12
a
The numbers represent mean ± SEM (n = 3) of triplicate in vitro incubations (two post-
labeling analyses of each sample). RAL, relative adduct labeling (the number of adducts per
normal nucleotides in modified DNA). The total adduct content is the sum of the RAL of in-
b
dividual adducts. see Fig. 3.
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1
242
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
tected. Levels of DNA adducts found in this experiment were lower than
those found in DNA incubated only with N-(2-methoxyphenyl)hydroxyl-
amine (Table II). This finding confirms the above results, showing that N-
(
2-methoxyphenyl)hydroxylamine is metabolized in microsomes to addi-
tional metabolites, whereas when this compound is incubated with DNA
it only decomposes to the nitrenium/carbenium ions that bind to DNA
(
Scheme 1). In addition, the nitrenium and/or carbenium ions may be scav-
enged by the microsomal proteins present in the incubation mixtures.
Metabolism of o-Aminophenol by Hepatic Microsomes and Peroxidases
Is a Detoxication Pathway for This Compound
In additional experiments, we also analyzed whether during metabolism of
o-aminophenol by hepatic microsomal systems (hepatic microsomes of rats
pre-treated with ethanol), reactive species forming DNA adducts are gener-
ated. The standard procedure under the ATP-deficient conditions, which is
more sensitive than the classical standard procedure, and the nuclease P1
version of the 32P-postlabeling assay and two chromatographic methods
(
see methods A and B in Experimental) were utilized in these experiments.
3
2
Using these P-postlabeling assays and separation methods A (not shown)
and B, no DNA adducts mediated by o-aminophenol metabolism in hepatic
microsomes were detectable (Fig. 3).
o-Aminophenol is oxidized with other oxidative enzymes, peroxidases,
forming the o-quinoimine metabolite1
3,14
(Scheme 1), the reactive com-
pound that might react with nucleophiles such as nucleophilic centers in
7
DNA . Therefore, we have analyzed whether during oxidation of o-amino-
phenol with peroxidases (horseradish peroxidase and lactoperoxidase were
used as models), DNA adducts are formed. The same 32P-postlabeling tech-
niques we employed for the analyses of DNA adduct formation by o-amino-
phenol incubated with microsomes (the standard procedure under the
ATP-deficient conditions and the nuclease P1 version of the 32P-postlabel-
ing assay and two chromatographic methods) were utilized for such a
study. Using these 32P-postlabeling assays and separation methods A (not
shown) and B, no DNA adducts mediated by o-aminophenol oxidation with
peroxidases were found (see Fig. 3, F and G for lactoperoxidase).
DISCUSSION
The results of this study show that rat hepatic microsomes can metabolize
N-(2-methoxyphenyl)hydroxylamine, a reactive metabolite of carcino-
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1243
genic o-anisidine and o-nitroanisole. This compound is responsible for
genotoxic effects of both carcinogens, because it is easily decomposed to
the nitrenium/carbenium ion forming DNA adducts (Scheme 1)8
,10,11
. The
results demonstrate that N-(2-methoxyphenyl)hydroxylamine is also fur-
ther metabolized by this subcellular enzymatic system to o-aminophenol
and the parent compound, o-anisidine. The structures of additional two
metabolites formed during N-(2-methoxyphenyl)hydroxylamine metabo-
lism have not been exactly characterized as yet. The formed o-anisidine
may be O-demethylated again to o-aminophenol (Scheme 1). The question
whether o-aminophenol is also formed from N-(2-methoxyphenyl)hydroxyl-
amine by its O-demethylation to N-(2-hydroxyphenyl)hydroxylamine,
which is subsequently reduced to o-aminophenol (Scheme 1), remains to be
answered. Because one of the N-(2-methoxyphenyl)hydroxylamine meta-
bolic products has not still been identified, we cannot exclude that this
metabolite might be N-(2-hydroxyphenyl)hydroxylamine. To confirm or
exclude this suggestion, this compound should be used as a standard.
Therefore, its synthesis is target of our future work. In contrast to rabbit
hepatic microsomes, which oxidize N-(2-methoxyphenyl)hydroxylamine
1
0
also to a 2-methoxynitrosobenzene , this nitrosoderivative was not found
to be generated by rat hepatic microsomes (present study).
Recently, redox cycling reactions, similar to those we found with
1
0
o-anisidine and N-(2-methoxyphenyl)hydroxylamine, were observed by
2
3
Kim et al. , who studied metabolism of several aromatic and heterocyclic
amines by a CYP1A2/NADPH:CYP reductase enzymatic system. They re-
ported that the CYP system catalyzes oxidation of the N-hydroxylated
intermediate formed from the carcinogenic heterocyclic amine 2-amino-
3
-methylimidazo[4,5-f]quinoline (IQ), to a nitrosoderivative. They demon-
strated that NADPH:CYP reductase can catalyze the reduction of the IQ
oxidation products, N-nitroso-IQ and N-hydroxyl-IQ, to N-hydroxyl-IQ
2
3
and the parent amine, IQ . N-hydroxylation products of two other aro-
2
3
matic amines investigated by Kim et al. , 2-aminofluorene and 4-amino-
biphenyl, are, however, reduced non-enzymatically, by NADPH. The results
of the present study show that reduction of N-(2-methoxyphenyl)hydroxyl-
amine to o-anisidine is essentially not mediated by NADPH or NADPH:CYP
reductase, but it is catalyzed by other enzymes present in hepatic micro-
somes used in this work. Because the efficiency of microsomes to reduce N-
(
2-methoxyphenyl)hydroxylamine to o-anisidine depends on levels of indi-
vidual CYP enzymes present in hepatic microsomes of rats pre-treated with
selective inducers of individual CYPs, the CYP enzymes should be more ef-
fective in this reduction than NADPH:CYP reductase. Nevertheless, identifi-
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1
244
Naiman, Hodek, Liberda, Schmeiser, Frei, Stiborová:
cation which of these CYP enzymes are responsible for this reaction awaits
further investigation.
Since N-(2-methoxyphenyl)hydroxylamine is responsible for formation
of DNA adducts, its formation is the activation pathway of o-anisidine
and/or o-nitroanisole metabolism8 . Because a role of o-aminophenol and
its metabolism in detoxication and/or activation of o-anisidine were not
known, these subjects were studied in this work. The results of this study
demonstrate that metabolism of o-aminophenol by hepatic microsomes or
peroxidases did not lead to formation of covalent DNA adducts. No metab-
olism of and DNA adduct formation by this compound were found in the
microsomal system. No DNA adducts were also generated during its oxida-
tion with peroxidases. This oxidation leads to formation of an o-quino-
imine metabolite1 , but this intermediate seems not to be reactive enough
to react with DNA. Formation of 2-aminophenoxazine-3-one that is a final
metabolite of the o-aminophenol oxidation by peroxidases seems to be
preferred (Scheme 1). This final 2-aminophenoxazine-3-one metabolite is
known to have significant biological activities such as anti-inflammatory
,11
3,14
2
4
25
and immunomodulatory properties and/or antimycobacterial and anti-
2
6
cancer activities . All these findings suggest that o-aminophenol and its
metabolism by peroxidases are not included into genotoxic processes occur-
ring during carcinogenesis caused by o-anisidine. Nevertheless, o-amino-
phenol might be considered to be mutagenic, because it induces sister
chromatid exchanges in a dose-dependent manner in cultured human lym-
phocytes in vitro and in Chinese hamster bone marrow cells in vivo . In
addition, Brennan and Schiestl28 reported that o-aminophenol is positive
in the deletion recombination assay in Sacchromyces cerevisiae. Furthermore,
even though o-aminophenol has not been found to form covalent DNA ad-
ducts, it was demonstrated in in-vitro experiments to cause DNA damage,
forming 8-oxy-7,8-dihydro-2′-deoxyguanosine in the presence of metal ions
2
7
2
9
such as Cu(II) . Hence, due to such processes, contribution of o-amino-
phenol to initiation of the o-anisidine-mediated carcinogenesis in the uri-
nary bladder, and in the development of tumors induced by other bladder
carcinogenic aromatic amines, which produce this compound as one of the
2
8
metabolites, cannot be excluded . Furthermore, O-demethylation reactions
produce formaldehyde (Scheme 1), which is known to modify DNA, gener-
3
0
ating several products including hydroxymethyl adducts and cross-links .
Formaldehyde is mutagenic in a variety of different test systems and carci-
nogenic in laboratory animals31 and has been described as “carcinogenic to
human” by the IARC and “reasonably anticipated to be a human carcino-
3
2
gen” by the U.S. Department of Health and Human Services (2004) .
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Aminophenol and N-(2-Methoxyphenyl)hydroxylamine Metabolism
1245
Therefore, on the one hand, it is plausible that formaldehyde-DNA adducts
could also play a role in carcinogenesis by o-anisidine. On the other hand,
however, formaldehyde produced in the cell is also detoxified by conjuga-
3
3
tion to glutathione and oxidized ; therefore, it is not likely a strong con-
tributor to carcinogenicity caused by o-anisidine.
It should be noted that tumor development in a specific organ is influ-
enced by promotional pressures on initiated cells in target organs and
not only by the levels of DNA adducts formed by the compounds like
o-anisidine. It is known that radicals formed from several carcinogens pro-
ducing oxidative DNA damage, such as 8-hydroxy-2′-deoxyguanosine,
are important not only in initiation, but also in promotion phases
3
4
of carcinogenesis . Therefore, their formation from o-aminophenol may be
one of the factors contributing to tumor promotion in o-anisidine- and/or
o-nitroanisole-mediated carcinogenesis. In addition, o-anisidine is oxidized
by several peroxidases6 , which are expressed in target organs (e.g. COX),
to form radicals besides DNA adducts. Hence, the production of such free
radicals in or near the target cells may be another factor important in the
promotional process in o-anisidine-mediated tumor development. How-
ever, the exact functions of such and/or other promotional pressures caused
both by o-anisidine and o-aminophenol in an o-anisidine-mediated tumori-
genesis remain to be resolved.
,7
Abbreviations: β-NF, β-naphthoflavone; CYP, cytochrome P450; EI, elec-
tron impact; dGp, deoxyguanosine 3′-phosphate; HPLC, high performance
+
liquid chromatography; HRP, horseradish peroxidase; NADP , nicotinamid-
adeninedinucleotide phosphate; NADPH, nicotinamidadeninedinucleotide
phosphate reduced; PB, phenobarbital (5-ethyl-5-phenylpyrimidine-
2
2
,4,6(1H,3H,5H)-trion); PCN, pregnenolone-16α-carbonitrile (3β-hydroxy-
0-oxopregn-5-ene-16α-carbonitrile); PEI-cellulose, polyethylenimine-
cellulose; RAL, relative adduct labeling; r.t., retention time; RPM, rotations
per minute; TLC, thin layer chromatography.
This research was supported by the Czech Science Foundation (Grant No. 303/09/0472) and
the Ministry of Education, Youth and Sports of the Czech Republic (MSM0021620808 and 1M0505).
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