To the mechanism of 2-nitroanisole carcinogenicity: In vitro metabolism of 2-nitroanisole mediated by peroxidases and xanthine oxidase

Article abstract of DOI:10.1135/cccc19980857

The in vitro enzymatic metabolism of carcinogenic 2-nitroanisole was investigated using peroxidases (horseradish peroxidase and prostaglandin H synthase) and xanthine oxidase catalyzing oxidative and reductive reactions, respectively. The oxidation of 2-nitroanisole catalyzed by horseradish peroxidase exhibits the Michaelis-Menten kinetics. The Michaelis constant (K_m) and the maximal velocity (V_max) values for this substrate were determined at pH 5.0, 7.0, 7.6 and 8.0. At optimal pH (7.6), the K_m and V_max values are 0.219 μmol/1 and 34.45 pmol/min per nmol peroxidase, respectively. The oxidation of 2-nitroanisole is inhibited by radical trapping agents (NADH, ascorbate, glutathione and nitrosobenzene). This indicates that the peroxidase-mediated oxidation of 2-nitroanisole proceeds via a radical mechanism. Active oxygen species are formed during the horseradish peroxidase-catalyzed reactions in the presence of NADH, hydrogen peroxide and 2-nitroanisole. 2-Nitroanisole is also oxidized by mammalian prostaglandin H synthase. Using the nuclease P1-enhanced variation of the ³²P-postlabelling assay, the formation of DNA adducts was detected in DNA treated with 2-nitroanisole and xanthine oxidase. No DNA binding was detected after oxidation of 2-nitroanisole with horseradish peroxidase and prostaglandin H synthase. The results presented (the formation of DNA adducts after 2-nitroanisole activation by xanthine oxidase and that of radicals and/or superoxide radicals during the reactions with peroxidases) strongly suggest the participation of 2-nitroanisole both in the initiation and in the promotion phases of carcinogenesis.

Full text of DOI:10.1135/cccc19980857

To the Mechanism of 2-Nitroanisole Carcinogenicity
857
TO THE MECHANISM OF 2-NITROANISOLE CARCINOGENICITY: in vitro
METABOLISM OF 2-NITROANISOLE MEDIATED BY PEROXIDASES
AND XANTHINE OXIDASE
a
b
b
Marie STIBOROVA , Heinz H. SCHMEISER and Eva FREI
a Department of Biochemistry, Charles University, 128 40 Prague 2, Czech Republic;
e-mail:stiborov@prfdec.natur.cuni.cz
b Department of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280,
69120 Heidelberg, Germany
Received February 25, 1998
Accepted May 1, 1998
The in vitro enzymatic metabolism of carcinogenic 2-nitroanisole was investigated using peroxidases
(horseradish peroxidase and prostaglandin H synthase) and xanthine oxidase catalyzing oxidative and
reductive reactions, respectively. The oxidation of 2-nitroanisole catalyzed by horseradish peroxidase
exhibits the Michaelis–Menten kinetics. The Michaelis constant (Km) and the maximal velocity (Vmax
)
values for this substrate were determined at pH 5.0, 7.0, 7.6 and 8.0. At optimal pH (7.6), the Km and
Vmax values are 0.219 µmol/l and 34.45 pmol/min per nmol peroxidase, respectively. The oxidation
of 2-nitroanisole is inhibited by radical trapping agents (NADH, ascorbate, glutathione and nitroso-
benzene). This indicates that the peroxidase-mediated oxidation of 2-nitroanisole proceeds via a radi-
cal mechanism. Active oxygen species are formed during the horseradish peroxidase-catalyzed
reactions in the presence of NADH, hydrogen peroxide and 2-nitroanisole. 2-Nitroanisole is also oxidized by
mammalian prostaglandin H synthase. Using the nuclease P1-enhanced variation of the 32P-postlabel-
ling assay, the formation of DNA adducts was detected in DNA treated with 2-nitroanisole and xan-
thine oxidase. No DNA binding was detected after oxidation of 2-nitroanisole with horseradish
peroxidase and prostaglandin H synthase. The results presented (the formation of DNA adducts after
2-nitroanisole activation by xanthine oxidase and that of radicals and/or superoxide radicals during
the reactions with peroxidases) strongly suggest the participation of 2-nitroanisole both in the initia-
tion and in the promotion phases of carcinogenesis.
Key words: Carcinogens; Nitro compounds; Oxidation; Reduction; Radicals; DNA adducts; Initia-
tion and promotion phases of carcinogenesis.
Aromatic amines and nitro compounds rank among a group of carcinogenic or poten-
tially carcinogenic compounds, presenting a considerable danger for human popula-
tion1,2. These compounds are often found in a workplace (e.g. in chemical industry) and
in the environment, where they add to local and regional pollution (car exhausts, tech-
nological spills). The compounds of this group are thus intensively studied in many
laboratories and are also in the center of interest of many public institutions.
This is also the case for 2-nitroanisole (2-methoxynitrobenzene). 2-Nitroanisole is
used primarily as a precursor in the synthesis of 2-anisidine, an intermediate in the
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

858
Stiborova, Schmeiser, Frei:
manufacture of many azo dyes3. An industrial accident at Hoechst in Germany caused
a large-scale leakage of this compound in 1993 and subsequent local and regional con-
tamination. As a result, researchers in Germany and other countries of the European
Union started to address in particular the following research problems: (i) the toxicity
and carcinogenicity of this compound, (ii) what are the dangerous concentrations of
2-nitroanisole, (iii) the metabolic pathway of 2-nitroanisole and (iv) the persistence of
residues of this compound and/or its metabolites in environment and organisms after
the accident.
2-Nitroanisole is a toxic compound, causing anaemia, characterized by increased le-
vels of methemoglobin and accelerated destruction of erythrocytes3. 2-Nitroanisole ex-
hibits carcinogenic activity, causing neoplastic transformation in urinary bladder and,
to a lesser extent, in spleen, liver and kidneys3. The compound is weakly mutagenic in
the Ames test with the Salmonella typhimurium TA100 strains3. However, its mut-
agenic activity could not be proved with other strains3 (e.g. TA97, TA98, TA1535,
TA1537). This chemical also exhibits a low activity in cytogenetic tests. It induces a
slight increase in chromosomal aberrations and of sister chromatid exchanges, but only
at high concentration3. However, these weak mutagenic effects sharply constrast with
its strong carcinogenicity. The data mentioned above imply a rather enigmatic mecha-
nism of the 2-nitroanisole carcinogenicity. Indeed, up to the present time, it has not
been determined exactly, if this compound is a genotoxic or epigenetic carcinogen and
in which phase of carcinogenesis it is involved. But these data are necessary to resolve
the mechanism of 2-nitroanisole carcinogenicity.
The major route of metabolism of 2-nitroanisole in vivo is oxidative demethylation to
2-nitrophenol, which appears in urine predominantly as the sulfate conjugate4. A second
pathway involves reduction to 2-anisidine (2-methoxyaniline); at blood concentrations
at which the metabolism and elimination of 2-nitroanisole are linear, 2-anisidine is a
minor metabolite formed in liver4. However, at higher doses the 2-nitrophenyl sulfate
pathway may reach saturation, leading to the formation of proportionally more 2-ani-
sidine3.
The efficiences and balance of oxidative and/or reductive enzyme systems catalyzing
the 2-nitroanisole biotransformation (detoxication and/or activation) are unknown.
These are, however, the first essential data needed to explain the mechanisms, by which
2-nitroanisole is implicated in carcinogenesis. Therefore, to answer this question, the
complex study carried out in our laboratories is aimed at finding which enzymes are
able to convert 2-nitroanisole. In the present study, we used peroxidases (abundant
enzymes catalyzing oxidative reactions of several xenobiotics in urinary bladder)5–8 to
determine their abilities to oxidize 2-nitroanisole in vitro. Horseradish peroxidase and
mammalian prostaglandin H synthase (PHS) are used as model peroxidases. We report
here their kinetic and catalytic characteristics with respect to 2-nitroanisole as the sub-
strate. Moreover, the participation of oxidative and reductive reactions catalyzed by
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To the Mechanism of 2-Nitroanisole Carcinogenicity
859
peroxidases and xanthine oxidase, respectively, in the formation of DNA adducts from
2-nitroanisole, are reported.
EXPERIMENTAL
Chemicals
Chemicals were obtained from the following sources: arachidonic acid, indomethacin and horseradish
peroxidase from Sigma; L-ascorbic acid from Merck; NADH, yeast alcohol dehydrogenase, nitroso-
benzene, DNA (from calf thymus), ferricytochrome c, superoxide dismutase and glutathione (re-
duced) from Boehringer; 2-nitroanisole from Fluka; xanthine oxidase and hypoxanthine from Serva.
All chemicals were reagent grade or better. Enzymes and chemicals for the 32P-postlabelling assay
were obtained commercially from sources described previously9.
Analytical Method
The assay mixture for the demethylation of 2-nitroanisole contained, in 1 ml, 200 µg horseradish
peroxidase, 0.01–1.0 µmol 2-nitroanisole dissolved in ethanol, 1 µmol H2O2 and 50 µmol sodium
acetate (pH 5.0) or 50 µmol potassium phosphate (pH 7.0, 7.6 or 8.0). The mixtures were incubated
for 10 min at 37 °C. The reaction was terminated by addition of 500 µl of 20% trichloroacetic acid.
The amount of formaldehyde formed was measured as described by Nash10. Inhibition of 2-nitroani-
sole oxidation by radical scavengers was performed in the same reaction mixture, but 0.5 or 1 µmol
NADH, or ascorbate, or glutathione dissolved in 50 µmol of potassium phosphate buffer (pH 7.6), or
1 µmol of nitrosobenzene dissolved in ethanol was added.
Kinetic analyses were carried out using the non-linear least-square method described by Cleland11.
NADH oxidation was measured in a final volume of 1 ml containing: 50 µmol potassium phos-
phate (pH 7.0, 7.6 or 8.0) or 50 µmol sodium acetate (pH 5.0), 0.1 µmol NADH, 0–0.220 µmol
H2O2, 0–15 µg horseradish peroxidase and 0–1.5 µmol 2-nitroanisole. The reaction was started by
addition of 2-nitroanisole, and the disappearance of NADH with time was followed at 340 nm using
a Beckman D-62 spectrophotometer at 20 °C.
The same reaction mixture was used for estimation of ferricytochrome c reduction except that 20 nmol
ferricytochrome c was added. Reduction of ferricytochrome c was monitored by the increase in ab-
sorbance at 550 nm (ref.12) at 20 °C. The molar extinction coefficient of cytochrome c at 550 nm
(ref.12) was 2.9 . 104 mol/cm.
Oxygen consumption was measured with a Clark-type electrode at 20 °C.
Fresh ram seminal glands were obtained from a local slaughterhouse, trimmed of excess fat and
tissue, and stored at –70 °C until use. Microsomes were prepared as described previously13 and used
as a source for PHS. PHS-Cyclooxygenase activity was determined by measuring the arachidonic
acid-dependent oxygen uptake in a 2.0-ml chamber equipped with a Clark-type oxygen electrode. The
incubation mixture contained (in 1 ml) 1.5 mg microsomal protein, 50 µmol potassium phosphate
(pH 7.6) and 10 µl ethanol (2-nitroanisole solvent), with different concentration of 2-nitroanisole,
preincubated for 1 min at 37 °C. The reaction was initiated by addition of arachidonic acid (0.1 µmol)
dissolved in 2 µl of ethanol. Some reaction mixtures also contained 100 nmol of indomethacin, which
is known as an inhibitor of the PHS-cyclooxygenase activity7. The initial rate of arachidonic acid
oxidation was determined from the slope of the linear portion of the oxygen uptake curve.
Protein concentrations were estimated according to Lowry et al.14 with bovine serum albumin as a
standard.
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860
Stiborova, Schmeiser, Frei:
Incubation mixtures used for the modification of DNA by 2-nitroanisole oxidized with peroxidases
contained in final volume of 1.0 ml: 50 µmol potassium phosphate (pH 7.6), 200 µg horseradish
peroxidase and 1.0 µmol H2O2, 1.5 mg microsomal protein and 0.1 µmol arachidonic acid, 1.0 µmol
2-nitroanisole and 1 mg calf thymus DNA. Control incubations were carried out either without per-
oxidases or without 2-nitroanisole. Incubations were performed at 37 °C for 60 min.
The incubations with xanthine oxidase were carried out according to Howard and Beland15. 2-Nitro-
anisole (1.0 or 10.0 µmol) was added to a deaerated and argon-purged solution of 1.0 mg of calf
thymus DNA containing 1 µmol hypoxanthine and xanthine oxidase (1 unit) in 50 µmol potassium
phosphate (pH 6.0) (the total volume was 1.0 ml). Mixtures were incubated 4 h at 37 °C under hy-
poxic conditions in the dark. Both control incubations without xanthine oxidase and without 2-nitro-
anisole were performed. In both enzymatic systems, reactions were stopped by extraction with 2
volumes of ethyl acetate (twice). DNA was isolated by the phenol/chloroform procedure (twice) as
described by Kirby16, modified by Schoepe et al.17 and precipitated with ethanol18,19. The DNA con-
tent was quantified spectrophotometrically at 260 nm.
32P-Postlabelling and Recovery of Individual Nucleotide Adducts
For DNA modified by 2-nitroanisole after reaction with peroxidases or xanthine oxidase, the nuclease
P1 version of the 32P-postlabelling assay17 was used. DNA was hydrolyzed to deoxyribonucleoside
3′-monophosphates using micrococcal nuclease and spleen phosphodiesterase. Nuclease P1-treated
samples were labelled using T4 polynucleotide kinase nad [γ-32P]ATP under conditions described
previously20,21. The same 32P-postlabelling procedure was used for control incubations (either with-
out enzymes or without 2-nitroanisole). Labelled digests were chromatographed on thin layer plates
of poly(ethylenimine)–cellulose (PEI–cellulose), with modifications described by Reddy et al.22. This
procedure is suitable for the resolution of benzoquinone-, phenol-, or hydroquinone derived ad-
ducts22. The solvents used were as follows: D1, 2.3 M sodium phosphate (pH 5.77); D2 was omitted;
D3, 2.7 M lithium formate, 5.1 M urea (pH 3.5); D4, 0.36 M sodium phosphate, 0.23 M Tris-HCl, 3.8 M
urea (pH 8.0). After D4 development and a brief water wash, the sheets were developed (along D4)
in 1.7 M sodium phosphate (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 radioac-
tive impurities to concentrate in a band close to the top edge. The specific activity of [γ-32P]ATP and
efficiency of the kinase reaction were assayed as described23. Typical values for specific activity
were 2 000 ± 300 Ci/mmol. Autoradiography and evaluation of relative adduct labelling (RAL)
values were performed as described previously using Cerenkov counting23,24
.
RESULTS
Oxidation of 2-Nitroanisole by Peroxidases
Horseradish peroxidase in the presence of hydrogen peroxide oxidizes carcinogenic
2-nitroanisole. The formation of formaldehyde illustrating demethylation of this carci-
nogen was proved. No activity was observed when any of the component was omitted
from the reaction mixture. The rates of demethylation of 2-nitroanisole by peroxidase
were measured at pH 5.0, 7.0, 7.6 and 8.0 and were 15.0, 23.8, 28.0 and 26.0 pmol
formaldehyde/min per nmol peroxidase, respectively. The highest rate of demethylation
was found at pH 7.6.
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To the Mechanism of 2-Nitroanisole Carcinogenicity
861
Formaldehyde formation was measured in the reaction medium which contained per-
oxidase, hydrogen peroxide and various 2-nitroanisole concentrations. The reactions
followed the Michaelis–Menten kinetics (Fig. 1). Under the conditions used, the maxi-
mal velocity (Vmax) and the apparent Michaelis constant (Km) values were the highest
and lowest, respectively, at pH 7.6 (Table I).
The conversion of 2-nitroanisole is strongly inhibited by NADH, glutathione, ascor-
bate and nitrosobenzene, which are known to be radical scavengers25 (Table II). These
findings indicate that radicals are involved in the reaction mechanism.
We studied the mechanism of inhibition of the 2-nitroanisole oxidation by ascorbate
and NADH. Low formaldehyde production was detected when 1 mmol/l ascorbate was
present in the incubation mixture. Two possibilities of the mechanism of the ascorbate
inhibition of the 2-nitroanisole oxidation could be suggested. Ascorbate may be the
substrate of peroxidase, and it may compete with 2-nitroanisole, or it may react with
reactive metabolites of 2-nitroanisole. Ascorbate as a subtrate is practically not con-
TABLE I
Kinetic parameters of 2-nitroanisole oxidation with horseradish peroxidase
a
a
pH
Km
Vmax
mmol/l
pmol HCHO/min nmmol peroxidase
5.0
7.0
7.6
8.0
0.345 ± 0.020
0.308 ± 0.025
0.219 ± 0.017
0.386 ± 0.021
21.50 ± 1.81
32.25 ± 1.99
34.45 ± 2.01
33.00 ± 1.70
a Means and standard deviations of three experiments.
30
25
V
3
4
2
20
15
10
1
FIG. 1
5
Dependence of the rate of oxidation of 2-nitro-
anisole with horseradish peroxidase on the 2-nitro-
anisole concentration. V (pmol/min per nmol
peroxidase) at pH: 1 5.0, 2 7.0, 3 7.6 and 4 8.0
0
0.0
0.2
0.4
0.6
0.8
1.0
2-Nitroanisole, mM
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862
Stiborova, Schmeiser, Frei:
verted by peroxidase (measured at 265 nm) under the conditions used in the experi-
ments with the peroxidase system alone (∆A/min = 0.03), but a rapid oxidation oc-
curred in the presence of 1 mmol/l 2-nitroanisole (∆A/min = 0.75). Ascorbate is
therefore not a peroxidase substrate but reduces the radicals formed from 2-nitroanisole
back to 2-nitroanisole.
Likewise, free radical(s) from 2-nitroanisole act as catalyst in the oxidation of
NADH by hydrogen peroxide and peroxidase. In the absence of 2-nitroanisole in the
reaction mixture, the oxidation of NADH by the peroxidase system was negligible
(0.002 NADH/min per nmol peroxidase); the same was true for the system with this
compound but without hydrogen peroxide (0.003 nmol NADH/min per nmol peroxi-
dase). However, in the complete system (peroxidase, H2O2, NADH, 2-nitroanisole),
NADH was oxidized (Table III). An effective oxygen uptake during the reaction in the
above system was also observed (Table III). We found that NAD+ is formed during the
reaction. When most of the NADH (in the reaction mixture) was oxidized, the reaction
adjusted to pH 8.8, and an NADH-generating system (alcohol dehydrogenase and ethyl
alcohol) was added, NADH was fully recovered, indicating that the product was NAD+.
As shown in Table IV, 2-nitroanisole enhanced the reduction of ferricytochrome c in
the peroxidase–H2O2–NADH system. No reduction of ferricytochrome c occurred with
2-nitroanisole alone in the absence of hydrogen peroxide. The enhancement by 2-nitro-
anisole of the reduction rate was completely prevented by superoxide dismutase (Table IV)
suggesting that the superoxide radical was formed, being responsible for the reduction
of ferricytochrome c. Ferricytochrome c decreased the oxygen consumption by the per-
oxidase–H2O2–NADH–2-nitroanisole system. Addition of 0.05, 0.1, 0.15 and 0.2
TABLE II
The effect of radical scavengers on the 2-nitroanisole demethylation catalyzed by horseradish peroxidase
Scavenger
Concentration
mmol/l
Ratea of 2-nitroanisole demethylation
pmol HCHO/min nmol peroxidase
None
28.00 ± 1.21
Ascorbate
0.5
1.0
0.5
1.0
0.5
1.0
1.0
14.42 ± 1.12 (51.5)b
1.51 ± 0.12 (5.4)
12.21 ± 1.15 (43.6)
2.04 ± 0.18 (7.3)
20.36 ± 1.76 (72.7)
2.88 ± 0.21 (10.3)
22.40 ± 2.10 (80.0)
Glutathione
NADH
Nitrosobenzene
a The values are averages and standard deviations of three parallel experiments. b The figures in the
parentheses are percentage values relative to controls.
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To the Mechanism of 2-Nitroanisole Carcinogenicity
863
mmol/l ferricytochrome c resulted in 51, 75, 89 and 100% inhibition of oxygen con-
sumption, respectively. However, no effect of ferricytochrome c on NADH oxidation
by the system was observed. This indicates that superoxide radicals are not responsible
for NADH oxidation and that the oxidation of NADH is mediated by the reaction of
peroxidase with 2-nitroanisole (see above). These findings confirm that radicals are
formed by a one-electron oxidation of 2-nitroanisole catalyzed by peroxidase, which is
effective in the oxidation of NADH.
Although horseradish peroxidase has served as a suitable model peroxidase for mam-
malian peroxidases in the present work and in other studies25,26, it was of interest to
investigate the metabolism of 2-nitroanisole by PHS, another enzyme with peroxidative
activity present in several mammalian tissues, including the tissues of lower urinary
TABLE III
NADH oxidation and oxygen uptake ratesa in the peroxidase–H2O2–2-nitroanisole system
System
Conversion rate
Oxygen uptake rate
nmol NADH/min nmol peroxidase
pmol O2/min nmol peroxidase
Complete
0.232 ± 0.0091
0.002 ± 0.0003
0.003 ± 0.0003
0
0.211 ± 0.012
0.040 ± 0.003
0.045 ± 0.004
0
– 2-nitroanisole
– H2O2
– peroxidase
a Values are averages and standard deviations of three parallel experiments.
TABLE IV
Ferricytochrome c reduction ratea (ν) in the 2-nitroanisole-mediated NADH oxidation with the per-
oxidase–H2O2 system at various 2-nitroanisole concentrations (c)
c, mmol/l
v, nmol/min
0
0.033 ± 0.010
0.083 ± 0.050
0.150 ± 0.013
0.206 ± 0.018
0.230 ± 0.021
0.255 ± 0.022
0
0.1
0.2
0.3
0.4
0.5
0.5b
a Values are averages and standard deviations of three parallel experiments; b 0.2 nmol of superoxide
dismutase added.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

864
Stiborova, Schmeiser, Frei:
tract5–7,27. These tissues are the target organs for 2-nitroanisole. Conversion of arachi-
donic acid requires the incorporation of molecular oxygen catalyzed by PHS cyclo-
oxygenase and the presence of a reducing cofactor for the reduction of endogeneous
substrates (prostaglandins) catalyzed by the hydroperoxidase activity of the enzyme7,13
.
Oxygen uptake serves as a means of measuring cyclooxygenase activity of PHS
(refs7,13). We investigate if 2-nitroanisole serves as a cofactor for PHS by measuring
oxygen incorporation into arachidonic acid catalyzed by ram seminal microsomes con-
taining PHS. Table V shows the concentration-dependent stimulation of oxygen uptake
produced by 2-nitroanisole, which is completely inhibited by indomethacin, a well-
known PHS cyclooxygenase inhibitor. At high concentrations of 2-nitroanisole, the
stimulating effect of this compound was decreased (Table V). These findings indicated
that 2-nitroanisole serves as a cosubstrate for PHS, radicals being formed during the
reaction7.
The Binding of 2-Nitroanisole to DNA
Using the nuclease P1 version of the 32P-postlabelling assay, we did not detect DNA
binding of 2-nitroanisole metabolite(s) after oxidation by horseradish peroxidase of
PHS (Fig. 2). Although the reactive intermediates (radicals) are formed in this system,
the data mentioned above imply that available amounts of these radicals should be very
low or that the radicals might be quenched before the DNA is reached.
Because reduction can be an alternative metabolic pathway for 2-nitroanisole in
vivo4, we studied the possible DNA binding of the metabolites, formed in such reduc-
TABLE V
Stimulation of PHS-cyclooxygenase activity (measured as oxygen uptake rate per protein contenta, ν)
by various concentrations of 2-nitroanisole
2-Nitroanisole concentration
ν
µmol/l
mmol/min kg
0
10
106.0 ± 9.7
165.1 ± 12.1
196.8 ± 17.2
259.8 ± 21.1
278.7 ± 24.3
157.2 ± 16.4
9.1 ± 0.9
50
100
150
200
50b
a
b
The numbers are averages and standard deviations of triplicate analyses; 100 nmol of indometh-
acin was added.
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To the Mechanism of 2-Nitroanisole Carcinogenicity
865
tive reactions. Out of the several mammalian reductases (microsomal NADPH : cyto-
chrome P450 oxidoreductase, xanthine oxidase, aldehyde oxidase, lipoyl dehydroge-
nase, DT-diaphorase) that are known to catalyze reduction of nitro compounds in
mammalian tissues28, xanthine oxidase was chosen for our experiments. Using the same
method for the covalent DNA binding determination (the above-mentioned nuclease P1
version of the 32P-postlabelling assay), we investigated formation of DNA adducts by
2-nitroanisole in the system containing this mammalian reductase. After inspection of
autoradiographs, five major and several minor adduct spots were detected in DNA
treated with 2-nitroanisole and the xanthine oxidase system (Fig. 2). In contrast, control
incubations carried out in parallel either without xanthine oxidase, or without 2-nitro-
anisole were free of adduct spots even after prolonged exposure times. Quantitative
analysis of the adducts formed is shown in Table VI.
a
b
c
d
FIG. 2
Autoradiographs of PEI–cellulose TLC maps of 32P-labelled digest of DNA, which was treated with
2-nitroanisole (1 µmol) activated with horseradish peroxidase (a), activated with PHS (b), activated
with xanthine oxidase (c) and of that which was treated with 2-nitroanisole (10 µmol) activated with
xanthine oxidase (d). Analysis was performed by the nuclease P1 version of the assay. Autoradio-
graphy was done at –70 °C for 16 h (a, b) and for 2 h (c, d). The origins are located at the bottom
left corner, D3 direction from bottom to top and D4 direction on four directional TLC on PEI–cellu-
lose from left to right (see Experimental or refs20–24
)
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

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DISCUSSION
It is well established that the development of cancer (carcinogenesis) requires multiple
steps. In several model systems, the stages of initiation, promotion and progression can
be operationally defined through the use of discrete chemical agents29,30
.
Although the mechanism, by which 2-nitroanisole is involved in carcinogenic pro-
cesses remains to be explained, one could speculate about a possible pathway. A pre-
requisite for 2-nitroanisole participation in the initiation stage of carcinogenesis is, that
this compound forms adducts with DNA. In the present work we observed that activa-
tion of 2-nitroanisole leading to formation of DNA adducts in vitro is caused by the
xanthine oxidase system, which is known to catalyze reductive reactions15,28. The ulti-
mate carcinogens formed by the reductive enzyme systems may hence be involved in
the initiation of carcinogenesis induced by 2-nitroanisole. These results also strongly
suggest that 2-nitroanisole acts as a genotoxic carcinogen.
Here, 32P-postlabelled adducts of 2-nitroanisole–DNA were resolved by the four-di-
rectional PEI–cellulose TLC technique modified by Reddy et al.22 for analysis of polar
adducts (Fig. 2). In contrast to the original Randerath method24, this modification
makes visible also adducts which contain only one benzene ring18,22. Therefore, the
ultimate carcinogen formed from 2-nitroanisole probably contains only one benzene
ring, too. The nitro group in 2-nitroanisole molecule is in all probability responsible for
2-nitroanisole activation, since it was postulated by Howard and Beland15 and Kedderis and
Miwa28 that reduction of nitro compounds, such as carcinogenic or mutagenic poly-
cyclic nitroaromatic hydrocarbons30–32 and nitroheterocyclic compounds used as thera-
peutic, antimicrobial or antiparasitic agents, is important for their biological activity. At
present, we can not determine the exact mechanism of reductive activation of 2-nitro-
anisole. Nevertheless, we can speculate that similarly to other nitroaromatic com-
TABLE VI
Quantitative analysis of adducts formed from calf thymus DNA reacting with 2-nitroanisole and xanthine
oxidase at various 2-nitroanisole concentrations (c)
Total DNA adduct contenta
c, mmol/l
RAL . 107
mmol/g
0
0
0
1.0
5.70 ± 0.62
1.71 ± 0.19
10.0
13.80 ± 1.23
4.14 ± 0.37
a
The numbers are averages and standard deviations of triplicate analyses. Relative adduct labelling
(RAL) represents the number of adducts per normal nucleotides in modified DNA (see refs20–24).
Total adduct content was determined by summing up of RAL of individual adducts.
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

To the Mechanism of 2-Nitroanisole Carcinogenicity
867
pounds, the nitro group of 2-nitroanisole should undergo one-electron reduction to form
the nitro anion radical, which is than converted by another one-electron reduction for-
ming the nitroso group. This compound can be further reduced by two electrons to
N-(2-methoxyphenyl)hydroxylamine. Further studies with that compound, aimed at re-
solving the mechanism of this reductive activation, are already in progress. Proposed
metabolic pathway of 2-nitroanisole is shown in Scheme 1.
The reactions catalyzed by peroxidases were studied in detail in our laboratories. At
present, 2-nitroanisole has been found to be oxidized by horseradish peroxidase to re-
active metabolites with NADH and/or ascorbate oxidation activity. In addition, it medi-
ates an effective oxygen uptake producing active forms of oxygen (superoxide radicals)
under the conditions used. These findings could imply that the reactions are one-elec-
tron redox processes having free radicals as primary products, similarly to those ob-
served with several other xenobiotics oxidized by peroxidase to radicals7,8,20,25–27. The
radicals of 2-nitroanisole are also formed in its oxidation reactions catalyzed by PHS.
OCH
3
NO
2
peroxidative
oxidation
reduction
OCH
OCH
3
NHOH
DNA-adducts
radicals
OH
3
NO
NH
2
2
SCHEME 1
Several lines of evidence suggest a role of free radicals not only in the initiation of
carcinogenesis (formation of adducts) but particularly also in processes of tumor pro-
motion33,34 and/or even progression35. We did not prove the participation of radicals
derived from 2-nitroanisole by the action of peroxidases in the adduct formation under
the conditions used. However, in view of the role played by radicals and superoxide
radicals in carcinogenesis, formation of these species during the peroxidase-catalyzed
oxidation of 2-nitroanisole can have biological consequences in the second (promo-
tional) phase of carcinogenesis. In addition to peroxidases, other oxidative enzymes
could also be effective in 2-nitroanisole oxidation and they might even be responsible
for the DNA adduct formation. Therefore, the studies with major oxidative enzymes
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

868
Stiborova, Schmeiser, Frei:
metabolizing xenobiotics in organisms (cytochromes P450) in biotransformation of
2-nitroanisole are under way in our laboratories.
The analysis of induction type of bladder cancer caused by 2-nitroanisole, in depend-
ence on different concentrations used, has shown a striking similarity to the induction
type previously found for a well-characterized bladder carcinogen, N-[4-(5-nitro-2-
furyl)thiazol-2-yl]formamide (FAFNT) (ref.36). For this compound, a two-stage model
of carcinogenesis has been postulated. According to the model, FAFNT is involved
both in the initial phase of carcinogenesis, modifying DNA of the target cells, and in
the promotion phase, stimulating the target cell proliferation. However, it was not
proved experimentally, whether the model is also valid for 2-nitroanisole. The data
described in this paper (the formation of DNA adducts after 2-nitroanisole activation by
xanthine oxidase and that of radicals or superoxide radicals during the reactions with
peroxidases) strongly imply the participation of 2-nitroanisole both in the initiation and
promotion phases of carcinogenesis. The model of induction type of bladder cancer
caused by FAFNT is therefore probably applicable to 2-nitroanisole. The importance of
the data presented here in an in vivo system has yet to be determined.
This study was supported by German Cancer Research Center and by the grant of the Ministry of
Education of the Czech Republic (VS96141).
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