Oxidative transformation of 2-acetylaminofluorene by a chemical model for cytochrome P450: A water-insoluble porphyrin and tert-butyl hydroperoxide

Article abstract of DOI:10.1016/j.bmc.2008.03.022

Oxidation of 2-acetylaminofluorene (AAF), a carcinogen, by a chemical model for cytochrome P450 was investigated to identify an active mutagen and elucidate the oxidation pathway. The oxidation system consisted of a water-insoluble tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide. The mutagen derived from AAF by the chemical model was 2-nitro-9-fluorenone (NO₂F{double bond, long}O), which was mutagenic in Salmonella typhimurium TA1538. AAF was oxidized initially at position 9 of the fluorene carbon by the chemical model forming 2-acetylamino-9-fluorenol (AAF-OH), and then oxidized further to 2-acetylamino-9-fluorenone (AAF{double bond, long}O) as a major product. Initial oxidation of the nitrogen formed 2-nitrofluorene (NO₂F), and further oxidation yielded 2-nitro-9-fluorenol (NO₂F-OH) as a minor product. These products, AAF-OH, AAF{double bond, long}O, NO₂F, and NO₂F-OH, and their presumable common intermediate, N-hydroxy-2-acetylaminofluorene, were oxidized by the chemical model, and the formation of NO₂F{double bond, long}O was determined. These results showed that NO₂F{double bond, long}O was the mutagen derived from AAF in the presence of the chemical model and was formed via oxidation of N-OH-AAF, NO₂F, and NO₂F-OH. These results may lead to a new metabolic pathway of AAF.

Full text of DOI:10.1016/j.bmc.2008.03.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 7070–7077
Oxidative transformation of 2-acetylaminofluorene
by a chemical model for cytochrome P450: A water-insoluble
porphyrin and tert-butyl hydroperoxide
Keiko Inami* and Masataka Mochizuki
Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo 105-8512, Japan
Received 9 January 2008; revised 5 March 2008; accepted 6 March 2008
Available online 10 March 2008
Abstract—Oxidation of 2-acetylaminofluorene (AAF), a carcinogen, by a chemical model for cytochrome P450 was investigated to
identify an active mutagen and elucidate the oxidation pathway. The oxidation system consisted of a water-insoluble tetrakis(pen-
tafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide. The mutagen derived from AAF by the chemical mod-
el was 2-nitro-9-fluorenone (NO2F@O), which was mutagenic in Salmonella typhimurium TA1538. AAF was oxidized initially at
position 9 of the fluorene carbon by the chemical model forming 2-acetylamino-9-fluorenol (AAF–OH), and then oxidized further
to 2-acetylamino-9-fluorenone (AAF@O) as a major product. Initial oxidation of the nitrogen formed 2-nitrofluorene (NO2F), and
further oxidation yielded 2-nitro-9-fluorenol (NO2F–OH) as a minor product. These products, AAF–OH, AAF@O, NO2F, and
NO2F–OH, and their presumable common intermediate, N-hydroxy-2-acetylaminofluorene, were oxidized by the chemical model,
and the formation of NO2F@O was determined. These results showed that NO2F@O was the mutagen derived from AAF in the
presence of the chemical model and was formed via oxidation of N-OH–AAF, NO2F, and NO2F–OH. These results may lead to
a new metabolic pathway of AAF.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
ogens in the mutation assay as a substitute for the meta-
bolic activation system. The metabolic activation
mechanism of 2-acetylaminofluorene (AAF) is well
known. The acetyl group of AAF is oxidized to N-hydro-
xy-AAF by cytochrome P450 in the metabolic activation,
followed by O-acetylation of N-hydroxy-2-acetylamino-
fluorene (N-OH–AAF) to N-acetoxy-2-aminofluorene.6,7
The ester contains an excellent leaving group and readily
breaks down to produce a nitrenium ion, and then in-
duces mutation by reaction with DNA bases. We have re-
ported that AAF is mutagenic in the presence of chemical
models.8 In this study, the oxidation mechanism of AAF
is elucidated by isolation and identification of the muta-
gen and the oxidation products derived from AAF in
the presence of the chemical model.
Environmental carcinogens are usually activated through
metabolic enzymes, such as cytochrome P450.1,2 In meta-
bolic studies of xenobiotics, medicine, or chemical carcin-
ogens, it is troublesome to handle enzymes because of
their restrictions in available reaction conditions (temper-
ature, solvent, and pH), and enzymes react with oxidized
products or reactive intermediates.3 Consequently, in-
stead of enzymes, biomimetic chemical models have been
helpful in metabolic studies.4 Okochi et al. have detected
mutagenicity of N-nitrosodialkylamines in Salmonella
typhimurium YG7108 and reproduced the enzymatic met-
abolic pathway of N-nitrosodialkylamines by isolating
the corresponding alcohols and aldehydes as their prod-
ucts, which suggest that N-nitrosodialkylamine under-
went oxidation through a-hydroxynitrosamine in
tetrakis(pentafluorophenyl)porphyrinatoiron(III) chlo-
ride (F5P) and tert-butyl hydroperoxide (t-BuOOH).5
Thus, the chemical model did oxidize and activate carcin-
2. Results
2.1. Mutagenicity of AAF by a chemical model for P450
in a mutation assay
Keywords: Chemical model; Cytochrome P450, 2-Acetylaminofluo-
rene; Oxidation.
A chemical model system for cytochrome P450, water-
insoluble F5P and t-BuOOH was used in the Ames assay
as a substitute for the S9 mix (Fig. 1).
*
Corresponding author. Tel./fax: +81 3 5400 2695; e-mail: inami-kk@
kyoritsu-ph.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.022

K. Inami, M. Mochizuki / Bioorg. Med. Chem. 16 (2008) 7070–7077
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Figure 1. Chemical structure of the metalloporphyrin (F5P), oxidant (t-BuOOH), and mutagen (AAF).
recrystallized from CCl4, yellow needles, mp 221.0–
1
222.0 ꢁC. H NMR, IR, and MS analyses showed that
oxidization of AAF in the presence of F5P/t-BuOOH
produced 2-nitro-9-fluorenone (NO2F@O). This finding
was confirmed by a comparison of spectra with those of
authentic NO2F@O. 1H NMR (500 MHz; CDCl3;
Me4Si) d: 7.43–7.49 (1H, m), 7.58–7.65 (1H, m), 7.68–
7.73 (2H, m), 7.76–7.79 (1H, m), 8.41–8.49 (2H, m);
m
max/cmÀ1 1714 (CO), 1520 (NO), 1338 (NO), EI-MS
m/z: 225 (M+, 100%), 195 (22), 151 (83). The mutagenic-
ity of the product was the same as that of the authentic
sample (data not shown).
2.3. Contribution of the direct mutagen derived from AAF
and the chemical models to the total mutagenicity of the
reaction solution
AAF decreased with an increase of NO2F@O in the
presence of the chemical model (Fig. 3A and B).
Figure 2. Mutagenicity of AAF in Salmonella typhimurium TA1538 in
the presence of F5P/t-BuOOH. F5P (250 nmol/plate) and t-BuOOH
(750 nmol/plate) in acetonitrile. The complete system contained AAF,
F5P, and t-BuOOH (d). Control systems lacked F5P (u) or t-BuOOH
(M).
AAF mutagenicity was assayed using S. typhimurium
YG7131, which is deficient in nitroreductase. The result
was compared to that of parent strain S. typhimurium
TA1538 (Fig. 4A). The nitro compound was activated
by reduction into hydroxylamine by nitroreductase in
S. typhimurim TA1538, and then the mutagenicity de-
rived from the hydroxylamine was detected, thus the
mutagenicity of the nitro compound decreased in nitro-
reductase-deficient strain. Mutagenicity of the mixture
of products derived from AAF oxidation decreased sig-
nificantly in S. typhimurium YG7131, and the mutage-
nicity of NO2F@O in the same strain also decreased
significantly. Furthermore, the amount of NO2F@O
formed, calculated by HPLC, accounted for the mutage-
nicity of the reaction mixture in S. typhimurium TA1538
(Fig. 4B).
AAF was mutagenic in a dose-dependent manner in the
presence of F5P/t-BuOOH in S. typhimurium TA1538
(Fig. 2). The activating system itself, F5P and t-BuOOH,
was not mutagenic in the absence of mutagens. In addi-
tion, the reaction mixture was not mutagenic in the ab-
sence of F5P or t-BuOOH.
2.2. Isolation and identification of AAF oxidation prod-
ucts from the reaction mixture
AAF and F5P were dissolved in acetonitrile, and
t-BuOOH was then added. The resulting solution was
stirred for 2 h at room temperature. The reaction mix-
ture was extracted with ethyl acetate, and was dried over
anhydrous sodium sulfate, and the solvent was subse-
quently evaporated. The residue was fractionated by sil-
ica gel column chromatography, and the fractions were
tested for mutagenicity. A fraction showing strong
mutagenicity contained a single compound, which was
A possible mechanism of AAF oxidation is shown in
Scheme 1. In pathway A of Scheme 1, the formation
of the nitro compound NO2F@O was initiated by the
oxidation of the methylene carbon of the fluorene ring.
While in pathway B of Scheme 1, the formation of
NO2F@O was initiated by the oxidation of nitrogen of

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K. Inami, M. Mochizuki / Bioorg. Med. Chem. 16 (2008) 7070–7077
Figure 3. Oxidation of AAF by F5P/t-BuOOH. (A) Decrease of AAF by F5P/t-BuOOH. AAF (500 nmol), F5P (250 nmol), and t-BuOOH (750 nmol)
were incubated in 3 mL of acetonitrile at 25 ꢁC. The complete system contained AAF, F5P, and t-BuOOH (d). Control systems lacked F5P (u) or
t-BuOOH (n). (B) Formation of NO2F@O by F5P/t-BuOOH. AAF (500 nmol), F5P (250 nmol), and t-BuOOH (750 nmol) were incubated in 3 mL
of acetonitrile at 25 ꢁC.
Figure 4. Contribution of NO2F@O to total mutagenicity in the reaction mixture. (A) Mutagenicity of AAF under F5P/t-BuOOH in Salmonella
typhimurium TA1538 (d) and Salmonella typhimurium YG7131 (m). (B) Correlation of mutagenicity of AAF by F5P/t-BuOOH with the calculated
mutagenicity based on NO2F@O formed. Mutagenicity of NO2F@O from AAF by F5P/t-BuOOH (d); mutagenicity of authentic sample (j).
the acetylamino group, followed by the oxidation of the
fluorene ring.
To examine the detailed oxidation pathway by the
chemical model, each of the four oxidation products,
AAF–OH, AAF@O, NO2F, and NO2F–OH, was oxi-
dized using the chemical model. AAF–OH was exclu-
sively oxidized to AAF@O by the chemical model
(Fig. 6A). AAF@O was barely oxidized by the chemical
model and NO2F@O was not significantly formed
(Fig. 6B). On the other hand, NO2F was oxidized to
NO2F–OH and NO2F@O (Fig. 7A), and NO2F–OH
was oxidized to NO2F@O (Fig. 7B).
2.4. Quantitative analysis of AAF oxidation products
For the purpose of elucidating the pathway in Scheme 1,
the oxidation products of AAF were detected using the
chemical model. The AAF oxidative products were ana-
lyzed by HPLC in the presence of the chemical models.
AAF–OH and AAF@O were detected in the reaction
mixture (Fig. 5A). Their retention times and UV spectra
were identical to those of the authentic samples. NO2F
and NO2F–OH were detected in the presence of the
chemical model using HPLC with a photodiode array
detector (Fig. 5B).
We proposed that NO2F@O was formed via N-OH–
AAF in the presence of the chemical model, as in the
S9 mix. N-OH–AAF could not be detected in the reac-
tion mixtures because of its high reactivity. N-OH–

K. Inami, M. Mochizuki / Bioorg. Med. Chem. 16 (2008) 7070–7077
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Scheme 1. Proposed mechanism of NO2F@O formation.
Figure 5. Formation of oxidation products from AAF and by F5P/t-BuOOH. (A) Formation of AAF–OH (d) and AAF@O (m). (B) Formation of
NO2F (d) and NO2F–OH (m).
Figure 6. Oxidation of AAF oxidation products by F5P/t-BuOOH. (A) Oxidation of AAF–OH (d) and formation of AAF@O (j) by F5P/t-BuOOH.
(B) Oxidation of AAF@O (d) and formation of NO2F@O (j) by F5P/t-BuOOH. AAF–OH or AAF@O (50 nmol), F5P (250 nmol), and t-BuOOH
(750 nmol) were incubated in 3 mL of acetonitrile at 25 ꢁC.

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K. Inami, M. Mochizuki / Bioorg. Med. Chem. 16 (2008) 7070–7077
Figure 7. Oxidation of AAF oxidative products by F5P/t-BuOOH. (A) Oxidation of NO2F (d) and formation of NO2F–OH (m) and NO2F@O (j)
by F5P/t-BuOOH. (B) Oxidation of NO2F–OH (d) and formation of NO2F@O (j) by F5P/t-BuOOH. NO2F or NO2F–OH oxidative product
(50 nmol), F5P (250 nmol), and t-BuOOH (750 nmol) were incubated in 3 mL of acetonitrile at 25 ꢁC.
in the environment,9 and also that 2-aminofluorene
is oxidized to NO2F by prostaglandin H synthase,10,11
and NO2F is metabolized to form NO2F@O by Cae-
norhabditis elegans.12,13 We have results that mutage-
nicity of AAF in the presence of S9 mix was not
derived from the nitro compound, since the mutage-
nicity under S9 mix was similar in S. typhimurium
TA1538 and YG7131 (data not shown). The result
suggested that reductases had high activities in the
S9 mix. We are speculating the formation of
NO2F@O in the absence of the reductases, and we
are working presently on detecting the nitro com-
pound in the reaction mixture containing AAF and
recombinant human CYP isoforms or prostaglandin
H synthase.
In order to estimate the contribution of NO2F@O to
the total mutagenicity in the reaction mixture, AAF
mutagenicity was assayed using S. typhimurium
YG7131, which is deficient in nitroreductase, and the
result was compared to that in a parent strain, S.
typhimurium TA1538 (Fig. 4A). The mutagenicity of
the mixture of products derived from AAF by the
chemical model decreased significantly in S. typhimuri-
um YG7131 similar to that of authentic NO2F@O.
The results suggested that the nitro compound formed
was responsible for the mutagenicity of AAF in the
presence of the chemical model. Furthermore, the
amount of NO2F@O formed was in accord with the
mutagenicity of the reaction mixture in S. typhimurium
TA1538. Those results showed that the mutagen de-
rived from AAF in the model was NO2F@O alone
(Fig. 4B).
Figure 8. Decrease of N-OH–AAF (d) and formation of NO2F (m),
NO2F–OH (j), and NO2F@O (r). N-OH–AAF (50 nmol), F5P
(250 nmol), and t-BuOOH (750 nmol) were incubated in 3 mL of
acetonitrile at 25 ꢁC.
AAF was oxidized in the presence of the chemical mod-
el, and NO2F, NO2F–OH, and NO2F@O were detected
(Fig. 8).
3. Discussion
A chemical model system for cytochrome P450, a por-
phyrin and an oxidant, was used in the Ames assay as
a biomimetic system of metabolic activation. In this
study, we elucidated the activation pathway of AAF
in the presence of water-insoluble F5P/t-BuOOH,
which activated AAF in a dose-dependent manner in
S. typhimurium TA1538 (Fig. 2). We isolated and
identified NO2F@O as a mutagen from a reaction
mixture. It has been reported that NO2F@O exists
As expected on the basis of the proposed oxidation
pathway outlined in Scheme 1, AAF was shown to be
metabolized to AAF–OH, AAF@O, NO2F, NO2F–
OH, and NO2F@O in the presence of the chemical mod-
el. The yield of AAF–OH, AAF@O, NO2F, NO2F–OH,
and NO2F@O was 68.2%, 22.2%, 0.4%, 0.6%, and 3.7%,

K. Inami, M. Mochizuki / Bioorg. Med. Chem. 16 (2008) 7070–7077
7075
Scheme 2. Pathway of formation of NO2F@O in the presence of the chemical model.
respectively. All formed oxidative products corre-
sponded to at least 95% of the theoretical based on the
observed AAF decomposition. AAF–OH and AAF@O
were reported to be formed from AAF by endogenous
enzymes.14,15 Furthermore, to examine the detailed oxi-
dation pathway by the chemical model, each of the four
oxidation products, namely, AAF–OH, AAF@O,
NO2F, and NO2F–OH, was oxidized using the chemical
model (Fig. 5). In pathway A in Scheme 1, AAF@O was
barely oxidized to form NO2F@O (Fig. 6). NO2F@O
was formed in the amount of 0.3 nmol (yield 0.6%) after
oxidation of AAF@O (50 nmol). On the other hand, in
pathway B in Scheme 2, NO2F@O was apparently
formed from NO2F and NO2F–OH in the presence of
the chemical model (Fig. 7). Thus, position 9 of fluorene
was oxidized as a major pathway; however, the pathway
to NO2F@O was not a major one.
4. Conclusion
The activation mechanism AAF has been studied in de-
tail. AAF was oxidized in vivo by cytochrome P450 to
N-OH–AAF, which is an active form with mutagenicity.
In this study, we showed that AAF was oxidized to
N-OH–AAF, followed by NO2F and NO2F–OH, and
then finally to NO2F@O, which was mutagenic in S.
typhimurium TA1538 in the presence of the chemical
model, as shown in Scheme 2. We demonstrated that the
use of a chemical model made it easy to identify the oxida-
tive products, and then elucidated the activation mecha-
nisms of AAF in the presence of chemical models. This
new pathway of AAF activation may be another pathway
in the metabolism of AAF, which has to be further exam-
ined for its demonstration. It is worth using a chemical
model for cytochrome P450 to predict possible oxidative
metabolites including mutagens.
We expected that NO2F@O was formed via N-OH–
AAF in the presence of the chemical model, as in
the S9 mix. Although N-OH–AAF could not be de-
tected in the reaction mixtures because of its high
reactivity, NO2F, NO2F–OH, and NO2F@O were
formed from N-OH–AAF in the presence of the chem-
ical model (Fig. 8). It has been reported that the N-
hydroxyacetylamino group was converted to a nitroso
group, which then was easily oxidized to a nitro
group.16,17 Floyd et al. have reported that N-OH–
AAF reacted with hydroperoxide to form 2-nitrosoflu-
orene via a nitroxyl radical.17 In addition, Nongkun-
sarn and Ramsden have reported that a nitroso
group was formed from imine via oxidation of the
oxaziridine ring.18,19
5. Experimental
5.1. Reagents
2-Amino-9-fluorenol (CAS No. 33417-27-5), 2-amino-9-
fluorenone (CAS No. 3096-57-9), 2-acetylaminofluorene
(AAF, CAS No. 53-96-3), tetrakis- (pentafluorophe-
nyl)porphyrinatoiron(III) chloride (F5P, CAS No.
36965-71-6) and tert-butyl hydroperoxide (t-BuOOH,
CAS No. 75-91-2) were purchased from Aldrich Chemical
Co., Inc. (Milwaukee, WI). Hydrazine hydrate (CAS No.
10217-52-4), 5% palladium/carbon, 2-nitrofluorene
(NO2F, CAS No. 607-57-8), and 2-nitro-9-fluorenone
(NO2F@O, CAS No. 3096-52-4) were obtained from
Wako Pure Chemical Industries, Ltd. (Okasa, Japan).
Scheme 2 summarizes the oxidative transformation of
AAF in the presence of F5P and t-BuOOH. We showed
a pathway in which the N-hydroxyacetylamino group
was oxidized to a nitro compound in the presence of
the chemical model, and that the nitro compound
formed was mutagenic in S. typhimurium TA1538.
5.2. Experimental
Melting points were measured on a Yanagimoto micro-
apparatus and were uncorrected. The EI-MS was ob-

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K. Inami, M. Mochizuki / Bioorg. Med. Chem. 16 (2008) 7070–7077
Table 1. HPLC analysis conditions
Compound
Conditions
AAF
NO2F@O
NO2F
Acetonitrile:20 mM sodium phosphate buffer (pH 7.4) 1:1, 0.5 mL/min, 7.5 min, 284 nm
Acetonitrile:20 mM sodium phosphate buffer (pH 7.4) 1:1, 0.5 mL/min, 13.0 min, 284 nm
Acetonitrile:20 mM sodium phosphate buffer (pH 7.4) 6:5, 0.5 mL/min, 23.4 min, 332 nm
Methanol:20 mM sodium phosphate buffer (pH 7.4) 3:2, 0.3 mL/min, 17.0 min, 332 nm
Methanol:20 mM sodium phosphate buffer (pH 7.4) 1:1, 0.5 mL/min, 3.6 min, 284 nm
Acetonitrile:methanol:H2O 7:3:3, 0.1 mL/min, 17.5 min, 284 nm
NO2F–OH
AAF–OH
AAF@O
Eluent, elution rate, retention time, and UV detector wavelength are shown for each compound.
tained with a Hitachi M-4100 mass spectrometer. The
NMR experiments were performed with JEOL JNM-
GX270, with Me4Si as an internal standard. HPLC
was performed using a Shimadzu LC-6A system [SPD-
6AV UV/vis spectrometric detector, Mightysil RP-18
column (5 lm, 250 · 3.0 mm)]. TLC was performed on
precoated Kieselgel 60F254 (Merck), and spots were
visualized under UV light.
for 2 h at room temperature, then added to water
(70 mL), and extracted three times with ethyl acetate
(40 mL). The organic layer was dried over anhydrous
sodium sulfate, and the solvent evaporated, leaving a
black solid (205 mg). The residue was fractionated using
silica gel column chromatography.
5.6. Quantitative analysis of the oxidation product
F5P (250 nmol), AAF (500 nmol), and t-BuOOH
(750 nmol) were incubated in acetonitrile (3 mL) at
25 ꢁC for various times (1, 3, 5, 10, and 20 min). The
products were analyzed by reverse-phase HPLC. Eluent,
elution rate, retention time, and UV detector wavelength
for each compound are listed in Table 1.
5.3. Chemical preparation
2-Acetylamino-9-fluorenol (AAF–OH) was prepared by
acetylation of 2-amino-9-fluorenol with acetic anhydride.
mp 248.0–249.0 ꢁC (lit., 249–250 ꢁC).20 2-Acetylamino-9-
fluorenone (AAF@O) was prepared by acetylation of 2-
amino-9-fluorenone with acetic anhydride and pyridine.
mp 231.0–232.0 ꢁC (lit., 233–236 ꢁC).20 N-Hydroxy-2-
acetylaminofluorene (N-OH–AAF)21 and 2-nitro-9-flu-
orenol (NO2F–OH)22 were synthesized by the method
reported.
Acknowledgments
This work was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Sci-
ence, Sports and Culture of Japan, and by a Grant-in-
Aid for Science Research Promotion Fund from the Ja-
pan Private School Promotion Foundation.
5.4. Bacterial mutation assay using the chemical model
A bacterial mutation assay was based on the Ames
test,23 and the chemical model, an iron(III) porphyrin
and an oxidant, was used as a substitute for the meta-
bolic activation system. The bacterial strains were
S. typhimurium TA1538, provided by Professor B.N.
Ames, the University of California, Berkeley, and
S. typhimurium YG7131, provided by Dr. T. Nohmi,
National Institute of Health Science, Japan.24
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An aliquot of F5P (250 nmol/20 lL) and AAF
(500 nmol/20 lL) in acetonitrile was mixed, and
t-BuOOH (750 nmol/20 lL) in 0.1 M sodium phos-
phate buffer (pH 7.4) was added. The mixture was then
incubated (25 ꢁC, 120 strokes/min) for 20 min, and a
0.1 M sodium phosphate buffer (pH 7.4, 0.5 mL), a cul-
ture of tester strains (0.1 mL), and top agar (2 mL)
were added. The mixture was then poured onto a min-
imal-glucose agar plate. After incubation for 44 h at
37 ꢁC, the colonies were counted. Duplicate plates were
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5.5. Reaction conditions with the model system
AAF (0.5 mmol) and F5P (0.06 mmol) were dissolved in
acetonitrile (30 mL), and t-BuOOH (15 mmol) was
added to the solution. The reaction mixture was stirred

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