Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro

Article abstract of DOI:10.1021/tx980152w

Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes contributes to human health risks. Since nitroreduction leads to activation of many nitro compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron reductants was examined. Rates of nitroreduction catalyzed by xanthine oxidase (XO)/hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased with the number of nitro groups and oxidation at C-9: 9-oxo-2,4,7-triNF > 9-oxo-2,7- diNF > 2,7-diNF > 9-oxo-2-NF = 2,5-diNF > 9-hydroxy-2-NF > 2-NF. Ascorbate catalyzed one-electron reduction to nitro anion radicals which reacted with molecular O₂ to yield superoxide. Rates of O₂ uptake with 9-oxo-2,4,7- triNF and 9-oxo-2,7-diNF were 63 and 0.17 times those, respectively, with equivalent concentrations of nitrofurazone, a classical substrate. Superoxide formation was indicated by the ~75% regeneration of O₂ upon addition of superoxide dismutase and catalase. 9-Oxo-2,4,7-triNF stimulated O₂ uptake in the presence of XO/NADH with typical Michaelis-Menten kinetics with an apparent K(m) of 0.476 ± 0.054 μM versus a K(m) of 6.18 ± 0.719 μM for nitrofurazone. HPLC analyses of products from reduction catalyzed by XO or diaphorase of Clostridium with NADH showed the following trends for the rates of amine formation from 9-oxo-2,7-diNF > 2,7-diNF; 9-oxo-2-NF > 9-hydroxy-2- NF > 2-NF; 2,7-diNF > 2-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF. Little or no amine was formed in 95% O², suggesting O²-labile intermediates. The data herein suggest that oxidation at C-9 and multiple nitro groups increase the potential for nitroreduction of the nitrofluorenes in vivo which may lead to genotoxic effects.

Full text of DOI:10.1021/tx980152w

Chem. Res. Toxicol. 1998, 11, 1361-1367
1361
Nitr or ed u ction of Nitr a ted a n d C-9 Oxid ized F lu or en es
in Vitr o
Clare L. Ritter and Danuta Malejka-Giganti*
Department of Laboratory Medicine and Pathology, University of Minnesota,
Minneapolis, Minnesota 55455, and Veterans Affairs Medical Center,
Minneapolis, Minnesota 55417
Received J une 22, 1998
Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes
contributes to human health risks. Since nitroreduction leads to activation of many nitro
compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron
reductants was examined. Rates of nitroreduction catalyzed by xanthine oxidase (XO)/
hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased
with the number of nitro groups and oxidation at C-9: 9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF >
2
,7-diNF > 9-oxo-2-NF ) 2,5-diNF > 9-hydroxy-2-NF > 2-NF. Ascorbate catalyzed one-electron
reduction to nitro anion radicals which reacted with molecular O to yield superoxide. Rates
of O uptake with 9-oxo-2,4,7-triNF and 9-oxo-2,7-diNF were 63 and 0.17 times those,
respectively, with equivalent concentrations of nitrofurazone, a classical substrate. Superoxide
formation was indicated by the ∼75% regeneration of O upon addition of superoxide dismutase
and catalase. 9-Oxo-2,4,7-triNF stimulated O uptake in the presence of XO/NADH with typical
Michaelis-Menten kinetics with an apparent K of 0.476 ( 0.054 µM versus a K of 6.18 (
.719 µM for nitrofurazone. HPLC analyses of products from reduction catalyzed by XO or
2
2
2
2
m
m
0
diaphorase of Clostridium with NADH showed the following trends for the rates of amine
formation from 9-oxo-2,7-diNF > 2,7-diNF; 9-oxo-2-NF > 9-hydroxy-2-NF > 2-NF; 2,7-diNF >
2
2
-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF. Little or no amine was formed in 95% O , suggesting
2
O -labile intermediates. The data herein suggest that oxidation at C-9 and multiple nitro
groups increase the potential for nitroreduction of the nitrofluorenes in vivo which may lead
to genotoxic effects.
In tr od u ction
Nitrated polycyclic aromatic hydrocarbons contribute
to environmental pollution since they are found in
emissions from diesel, airplane, and other fossil fuel
combustions as well as in cigarette smoke (1-6). Their
genotoxicity upon metabolic activation suggests a health
F igu r e 1. 2-Nitrofluorene.
risk to humans. Among the more than 200 environmen-
tal nitro polycyclic aromatic hydrocarbons, 1-nitropyrene
and 2-nitrofluorene (2-NF)1 (Figure 1) predominate.
Other nitrofluorenes identified in environments, includ-
ing workplaces, are 2,5-diNF, 2,7-diNF, and 9-oxo deriva-
tives of 2-NF, 2,7-diNF, and 2,4,7-triNF (1). 2-NF was
mutagenic in bacterial and mammalian cells and carci-
nogenic in rodents, and it is considered “possibly carci-
nogenic to humans” by the International Agency for
Research on Cancer (2). A major metabolite of 2-NF in
the rat and human lung in vitro was 9-hydroxy-2-NF (7,
decrease in mutagenicity in the nitroreductase deficient
TA98NR strain indicated that the activity of nitrofluo-
renes was a result of nitroreduction. In rats adminis-
tered 2-NF or 9-OH-2-NF, the hepatic DNA adducts
included 8-[N-(2-aminofluorenyl)]-2′-deoxyguanosine (11,
1
3), an adduct derived from interaction with the reduced
nitro compound. The levels of this adduct from 2-NF
correlated with levels of preneoplastic liver foci, suggest-
ing significance of nitroreduction in the initiation of liver
tumorigenesis.
Reduction of the nitro group can proceed via one- or
8
). Additional nitro groups and oxidation at C-9 of 2-NF
-
-
two-electron (1e or 2e ) mechanisms as shown in
led to the increased mutagenicity of nitrofluorenes in
Salmonella typhimurium TA98 (Table 1) (9-12). The
-
Scheme 1. Reduction via 1e produces a nitro anion
2
radical that reacts with molecular O to regenerate the
parent nitro compound with concomitant superoxide
anion radical production. Mason and Holtzman first
demonstrated nitro anion radical formation by ESR
spectroscopy and showed the futile redox cycling of the
*
Address correspondence to this author: Veterans Affairs Medical
Center (151), One Veterans Drive, Minneapolis, MN 55417. Tele-
phone: (612) 725-2000, ext. 4583. Fax: (612) 725-2255. E-mail:
malej001@maroon.tc.umn.edu.
1
Abbreviations: AF, aminofluorene; 1e- or 2e , one- or two-electron;
-
radical with O
and catalase-sensitive O
tion is often described as “O
reduction is often referred to as “O
2
by means of superoxide dismutase (SOD)
SOD, superoxide dismutase; XO, xanthine oxidase; DMSO, dimethyl
sulfoxide; DTPAC, diethylenetriaminepentaacetic acid; IS, internal
standard. The abbreviations for 2-NF and related compounds are listed
in Table 1.
-
2
uptake (14). Thus, 1e reduc-
-
2
-sensitive”, whereas 2e
-insensitive” since no
2
1
0.1021/tx980152w CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/10/1998

1
362 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Ritter and Malejka-Giganti
Ta ble 1. Mu ta gen icity a n d Red u ction P oten tia ls of Nitr oflu or en esa
mutagenicity
compound
-nitrofluorene
-hydroxy-2-nitrofluorene
-oxo-2-nitrofluorene
,5-dinitrofluorene
,7-dinitrofluorene
-oxo-2,7-dinitrofluorene
-oxo-2,4,7-trinitrofluorene
abbreviation
2-NF
9-OH-2-NF
9-oxo-2-NF
2,5-diNF
2,7-diNF
9-oxo-2,7-diNF
9-oxo-2,4,7-triNF
E1/2 (V)
-1.07d
TA98b
TA98/TA98NRc
e
f
2
9
9
2
2
9
9
1
8.1 to 17
g
h
-
0.21
1.89
- f
-
-
i
j
-0.90
-0.94d
- e
d
j
f
-0.93
-0.64
34 to 222
105 to 211
7.1 to 14
i
f
j
j
f
7.9
i
f
2.9f
-0.27
153 to 194
a
The values are derived from published data (9-12, 15, 16). b Values are relative to 2-NF ) 1; revertants per nanomole of compound
c
were determined in S. typhimurium TA98. Ratio of revertants per nanomole of compound in nitroreductase proficient TA98 to
nitroreductase deficient TA98NR strains. Reference 15. e Reference 9. f Reference 10. g Data not available. h Reference 11. Reference
d
i
j
1
6. Reference 12.
Sch em e 1. Red u ction s of Nitr o Gr ou p
Exp er im en ta l P r oced u r es
Ca u tion : The nitrofluorenes should be handled according to
the NIH Guidlines for the Laboratory Use of Chemical Carcino-
gens Publication No. 81-2385 (U.S. Government Printing Office,
Washington, DC).
Ma ter ia ls. Glass-distilled water was used throughout. All
solvents were HPLC grade. Dimethyl sulfoxide (DMSO) was
from Fisher Scientific (Pittsburgh, PA). Acetylated cytochrome
c, Chelex 100, hypoxanthine, XO from milk, diaphorase from
Clostridium kluyverii, allopurinol, dicoumarol, SOD, NADH,
ascorbate, and diethylenetriaminepentaacetic acid (DTPAC)
were from Sigma Chemical Co. (St. Louis, MO). 2-NF, 2-AF,
2
,7-diNF, 2-amino-7-NF, 9-oxo-2-NF, 9-oxo-2-aminofluorene (9-
oxo-2-AF), and 4-phenylphenol were from Aldrich Chemical Co,
Inc. (Milwaukee, WI). 2,5-DiNF and 9-oxo-2,7-diNF were from
Pfaltz and Bauer, Inc. (Waterbury, CT). 9-Oxo-2,4,7-triNF was
from MacKenzie Corp. (Bush, LA). 9-OH-2-NF and 9-OH-2-AF
were prepared by the published method (22). All fluorenyl
compounds and 4-phenylphenol were recrystallized until pure
by HPLC. DMSO was preflushed with argon.
radical is involved. However, the highly reactive nitroso
and hydroxylamine intermediates on the pathway to
amine are difficult to detect and are likely O -labile.
2
The ease of reduction of the nitro group, which may
be characterized by the reduction potential, depends on
the structure of the compound and is important in
mediating its biological effects. Reduction potentials
reported for nitrofluorenes (15, 16) are shown in Table
In str u m en ta tion . The rate of O
a YSI 5331 O probe (Yellow Springs Instrument Co., Yellow
Springs, OH) in a 1.7 mL vessel maintained at 37 °C with a
YSI model 53 O monitor connected to a recorder. The UV λmax
2
uptake was measured with
2
2
values were determined with a Beckman DU-70 UV/vis spec-
trophotometer (Beckman Instruments, Fullerton, CA). HPLC
data were obtained with a Hewlett-Packard 1090 liquid chro-
1
. A relationship between the mutagenicity in S. typh-
imurium and first half-wave reduction potential of 2-NF,
,7-diNF, and 9-oxo-2,7-diNF was reported (17). Even
1
13
matograph equipped as previously described (23). H and
C
2
NMR spectra were obtained at ambient temperature on a 500
MHz Varian Inova spectrometer, and FAB-MS analysis was
performed with a Finnigan Mat 95 mass spectrometer at the
Department of Chemistry, University of Minnesota, Minneapo-
lis, MN. LC/MS analyses were performed at the University of
Minnesota Cancer Center with a Finnigan TSQ 7000 triple-
stage quadropole mass spectrometer in the positive or negative
ion mode.
though mutagenic potencies of nitrofluorenes appear to
be dependent on ease of their nitroreduction (Table 1),
to our knowledge no study has attempted to correlate
their reduction potential with nitroreductase-catalyzed
reductions. To gain insight into nitroreduction of nitro-
fluorenes possibly occurring in vivo, we examined the
enzymatic and nonenzymatic nitroreduction in vitro of a
series of nitrofluorenes and compared these reductions
to those of the classical nitroreductase substrates, nitro-
furazone and nitrofurantoin. Thus, we examined the
effect of structure of nitrofluorenes on reduction by
xanthine oxidase (XO), which is found in most mam-
Syn th esis of 9-Oxo-2-a m in o-7-NF . Sodium borohydride
(31 mg, 0.82 mmol) was added slowly to a solution of 9-oxo-2,7-
diNF (243 mg, 0.9 mmol) in polyethylene glycol 400 (24.3 mL)
and absolute ethanol (4.9 mL), and the mixture was kept under
reflux (∼95 °C) for 15 min. After the mixture cooled to ambient
temperature and addition of another volume of absolute ethanol
(4.9 mL), the reaction mixture was mixed with an equal volume
of cold water and chilled at 4 °C. The precipitate was collected,
-
malian tissues and has been shown to catalyze 1e
nitroreductions (18, 19). The nitrofluorenes found to be
most susceptible to nitroreduction catalyzed by XO were
selected for further assays which included monitoring of
O uptake catalyzed by ascorbate or XO. Use of ascor-
2
bate, which can reach a level of 1 mM in vivo, was based
2 5
washed with water, and dried over P O . The crude product
(
47% yield, ∼82% pure by HPLC) was recrystallized from
toluene (with charcoal) to yield the compound: mp ∼290 °C dec;
97.6% pure by HPLC; UV λmax 260 and 386 nm [ꢀ(methanol) )
26 900 and 13 650 M cm , respectively]; H NMR (500 MHz,
DMSO-d
NMR (125 MHz, DMSO-d
-
1
-1
1
1
3
6
) δ 8.4-6.6 (m, 6H, aromatic H), 6.1 (s, 2H, NH
2
);
C
on the work of Rao et al. (20) who showed by ESR
6
) δ 152.3-109.3 (12 aromatic C),
92.1 (CdO); FAB-MS m/z (relative intensity) 240.054 (M , 100),
10.060 [(M - NO) , 16], 194.061 [(M - NO ) , 14], 182.063
spectroscopy and O
2
uptake that ascorbate was a catalyst
+
1
2
-
of 1e nitroreduction of nitrofurantoin and other nitro
compounds. In addition, the extent of amine formation
from nitrofluorenes catalyzed by XO or diaphorase, a
+
+
2
+
+
[
(M - NO
Nitr o Com p ou n d -Stim u la ted Red u ction of Acetyla ted
Cytoch r om e c. Buffers were saturated with argon or 95% O
2
- CO) , 4], 166.065 [(M - NO
2
- CO) , 9].
-
catalyst of 2e reduction (21), was measured by HPLC.
2

Nitroreduction of Nitrofluorenes
5% CO ). Unless specified otherwise, incubation mixtures (1
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1363
(
2
mL) in 50 mM Tris-HCl (pH 7.5) at 37 °C contained 30 µg of
XO, 50 µM acetylated cytochrome c, and 14 µM nitro compound
added in 20 µL of DMSO, and 10 or 20 µM allopurinol in select
incubation mixtures. Anaerobic incubation mixtures were
2
saturated with argon and contained an O -scavenging system
of 16 µg of glucose oxidase, 135 µg of glucose, and 0.285 unit of
catalase. The buffer in septum-covered cuvettes was flushed
with gas for 5 min. Additions were made by syringe through
the septum. XO was added and the mixture gently purged with
gas for 2 min and again for 20 s after each addition of
cytochrome c, nitro compound, and cofactor. Reactions were
started with the admixing of the electron donor, NADH or
hypoxanthine. The rate of reduction was calculated from the
-
1
-1
initial increase in A550nm (ꢀ ) 18 500 M cm ). No reduction
of acetylated cytochrome c was detected under any conditions
with hypoxanthine and nitro compound without active enzyme.
Corrections for the nitro compound-independent anaerobic
reduction varied from ∼30% of the 2-NF-dependent rate to 0.2%
of the 9-oxo-2,4,7-triNF-dependent rate.
F igu r e 2. Nitro compound-stimulated reduction of acetylated
cytochrome c (50 µM) catalyzed by XO (0.03 mg) in 50 mM Tris-
HCl buffer (pH 7.5) with 0.5 M hypoxanthine with or without
10 µM allopurinol under anaerobic conditions at 37 °C. Nitro
compound (14 µM) was added in DMSO to give a final concen-
tration of 2%. Experiments were conducted as described in
Experimental Procedures. Values are the means ( SD from two
separate experiments, each with triplicate incubations. The
effect of allopurinol was significant at p e 0.05.
Nitr o Com p ou n d -Stim u la ted
concentration of O at saturation was determined with a XO/
hypoxanthine/catalase system (24). O uptake measurements
2
O Con su m p tion . The
2
2
were based on published procedures (20, 25). Incubation
mixtures (1.7 mL) in the above buffer, treated with Chelex 100
and 1 mM DTPAC to remove metal ions, contained 2.5 mM
ascorbate or 230 µg/mL XO as the catalyst. The reaction was
started by the addition of nitro compound in argon-flushed
DMSO (final concentration of 1-8%) or 1 mM NADH for the
determinations with XO. Formation of superoxide anion radical
was demonstrated by the effect of SOD (160 units/mL) and
7
5% methanol and 0 to 5% 2-propanol in acid followed by a 6
min linear gradient of 75 to 65% methanol and 5 to 10%
-propanol in acid. A DuPont Zorbax C18 analytical column
2
2
catalase (0.005 unit/mL) (23). The rate of O consumption was
(
Mac-Mod Analytical, Inc., Chadds Ford, PA) (150 mm × 4.6
calculated from the initial decrease in saturation and was
corrected for nitro compound-independent and nonenzymic
changes.
mm i.d.) with a 0.5 µM prefilter (Rheodyne, Inc., Cotati, CA)
and 10 µL sample loop was used. The operating temperature
was 30 °C, the flow rate 0.8 mL/min, and the operating pressure
Deter m in a tion of Am in e P r od u cts fr om Nitr oflu or en es.
1
25 bar. Compound identification was based on UV spectra and
Buffers were saturated with argon or 95% O
2 2
(5% CO ).
t
R
values superimposed with those of standard compounds
Incubation mixtures (1 mL) in 2 mL amber vials with septa caps
fitted with inlet and outlet ports for continuous gas exchange
contained diaphorase (67 units in 0.39 mg) or XO (0.16 unit in
chromatographed under identical conditions and verified in
select incubation mixtures by LC/MS analyses. The areas of
peaks separated with gradient A were integrated at 260 nm for
1
0
.33 mg), 28 µM nitro compound added in 40 µL of DMSO, and
.5 mM NADH or allopurinol in 50 mM Tris-HCl (pH 7.5) at 37
-
IS (t
R
) 9.46 min), 280 nm for 9-oxo-2-NF (t
R
-
) 10.41 min) (m /
z 225) and 9-oxo-2-AF (t
R
) 7.43 min) (m / z 195), 300 nm for
) 5.66 min) (m / z 180), and 330 nm for 9-OH-
°C. Anaerobic incubation mixtures contained the O
2
-scavenging
-
9
2
-OH-2-AF (t
-NF (t
R
system described above. Controls contained inactive enzyme
or lacked NADH or nitro compound. Incubations were termi-
nated at intervals of 5-60 min by heating in a boiling water
bath for 2 min. Mixtures were chilled on ice, except those
containing 2-NF and 2,7-diNF which were cooled to 37 °C to
prevent precipitation of the nitro compound. 4-Phenylphenol
-
R
) 8.49 min) (m / z 227); with gradient B at 260 nm for
IS (t
R
) 11.14 min) and 9-oxo-2-amino-7-NF (t
R
) 11.9 min)
+
+
(
m / z 240) and 280 nm for 9-oxo-2,7-diNF (t
R
) 13.4 min) (m / z
2
2
1
2
70); with gradient C at 260 nm for IS (t
-amino-7-NF (t ) 12.0 min), 280 nm for 9-oxo-2,7-diNF (t
4.1 min), 340 nm for 2,7-diNF (t ) 14.8 min), and 390 nm for
-amino-7-NF (t ) 12.5 min); and with gradient D at 260 nm
) 9.53 min), 280 nm for 2-AF (t ) 8.7 min), and 9-oxo-
) 10.7 min), and 330 nm for 2-NF (t ) 13.1 min). The
R
) 12.9 min) and 9-oxo-
R
R
)
R
(10 nmol in 10 µL of 2:1 2-propanol/methanol) was added as an
R
internal standard (IS). The mixtures were centrifuged at 2000g
for 5 min and the supernatants applied to a Baker C18 extraction
column activated with 1 mL each of acetonitrile and buffer. After
washing with 1 mL of buffer, the organic compounds were eluted
for IS (t
-NF (t
R
R
2
R
R
compounds were quantified from peak areas relative to standard
curves and corrected for the extraction of the IS. Rates of amine
formation were calculated within the times of linear formation.
Sta tistica l An a lysis. The Newman-Keuls test was used;
a p value of e0.05 was considered statistically significant. The
correlation coefficient was obtained by regression analysis using
GraphPad Prism version 2.00 for MacIntosh (GraphPad Soft-
ware, San Diego, CA).
with 1 mL of acetonitrile. The eluate was dried over Na
2 4
SO
and evaporated with a stream of N passed through an Oxy-
2
Trap (Alltech Associates, Deerfield, IL). Extracts from incuba-
tion mixtures with 9-OH-2-NF or 9-oxo-2-NF were dissolved in
2
9
-propanol/methanol (2:1) and those with 2-NF, 2,7-diNF, or
-oxo-2,7-diNF in DMSO. Solutions were analyzed as follows
by HPLC with solvent systems containing methanol, 2-propanol,
and 0.2 M acetic acid (pH 4.2): (A) a 10 min linear gradient of
Resu lts
4
5 to 55% methanol and 0 to 20% 2-propanol in acid, followed
by a 6 min linear gradient of 55 to 85% methanol and 20 to
Nitr o Com p ou n d -Stim u la ted Red u ction of Acety-
la ted Cytoch r om e c. Since nitrofurazone-stimulated
reduction of acetylated cytochrome c was virtually abol-
ished by air (data not shown), the extent of stimulation
of XO-dependent reduction of acetylated cytochrome c by
the nitrofluorenes and nitrofurazone was determined
under an argon atmosphere with hypoxanthine as a
cofactor (Figure 2). The rates of reduction (9-oxo-2,4,7-
1
7
0% 2-propanol in acid; (B) a 10 min linear gradient of 30 to
0% methanol in acid followed by a 2 min linear gradient of 70
to 80% methanol and 0 to 10% 2-propanol in acid, then
isocratically for 4 min; (C) a 10 min linear gradient of 30 to 70%
methanol in acid followed by a 3 min linear gradient of 70 to
7
4% methanol and 0 to 6% 2-propanol in acid and then by a 3
min linear gradient of 74 to 80% methanol and 6 to 10%
-propanol in acid; and (D) a 10 min linear gradient of 45 to
2

1
364 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Ritter and Malejka-Giganti
Ta ble 2. Effect of SOD a n d Ca ta la se on th e Nitr o Com p ou n d -Stim u la ted O2 Up ta k e Ca ta lyzed by Ascor ba te
O2 (µM/min)a
concentration
(mM)
without SOD
and catalase
with SOD and
catalase
compound
% decrease
nitrofurantoin
1.0
0.2
0.2
0.025
0.025
13.4 ( 1.45
0.553 ( 0.043
3.19 ( 0.46
20.2 ( 1.87
0.321 ( 0.082
4.54 ( 0.12b
66
88
75
69
c
0.066 ( 0.062b
9
-oxo-2,7-diNF
nitrofurazone
-oxo-2,4,7-triNF
nitrofurazone
c
b
0.782 ( 0.129
b
9
6.21 ( 0.62
NDd
a
Incubations are described in Experimental Procedures. Values are the means ( SD of three to six determinations and are corrected
for nitro compound-independent O2 uptake. The effect of SOD and catalase was significant at p e 0.05. c Added in DMSO to give a final
concentration of 8%. Not determined.
b
d
Ta ble 3. Kin etic P a r a m eter s for Nitr o
Com p ou n d -Stim u la ted O2 Up ta k e Ca ta lyzed by XO
triNF . 9-oxo-2,7-diNF > 2,7-diNF ≈ 9-oxo-2-NF > 2,5-
a
diNF > 9-OH-2-NF > 2-NF) increased with the number
Vmax
of nitro groups and the extent of oxidation at C-9. The
compound
Kmb (µM)
(µM/min) log(Vmax/Km)
rate of nitrofurazone reduction was similar to that of
-oxo-2,7-diNF. The XO inhibitor, allopurinol, at 10 µM
9
9
-oxo-2,4,7-triNF
-oxo-2,4,7-triNF,
0.476 ( 0.054 36.1 ( 4.12
1.88
9
almost completely inhibited reduction with all nitro
compounds except for 9-oxo-2,4,7-triNF. With the latter
compound, increasing the concentration of allopurinol to
SOD, and catalase 2.87 ( 0.112 23.6 ( 2.55
0.905
0.699
nitrofurazone
6.18 ( 0.719 30.9 ( 3.58
a
Reactions in mixtures containing nitro compound added in
DMSO at a final concentration of 1% to 50 mM Tris-HCl (pH 7.5)
and 1 mM DTPAC were started by the addition of 1 mM NADH
as described in Experimental Procedures. b Values ( SE were
derived from the double-reciprocal plots of data from three
independent experiments with triplicate incubations at each
substrate concentration.
2
0 µM did not increase the extent of inhibition.
Under aerobic conditions, the reduction of a nitro
compound to a nitro anion radical generates superoxide
Scheme 1). Because XO catalyzes oxidation of hypox-
anthine without a nitro compound (24), NADH was used
as a cofactor to determine the effect of O on the nitro
(
2
compound-dependent reduction of acetylated cytochrome
c (50 µM) catalyzed by XO (0.007 unit/mL) and stimulated
by 14 µM 9-oxo-2,7-diNF. The rate was 1.44 nmol/min
under aerobic conditions compared to 3.84 nmol/min
under argon. Addition of SOD decreased the rate of
reduction under aerobic conditions by 38%.
to nitrofurazone (Table 3). Because of the rapid rate of
nitro-independent O consumption by XO/hypoxanthine,
2
NADH was used as a cofactor. The lower Km and the
log Vmax/Km for 9-oxo-2,4,7-triNF indicated its facile
enzymatic reduction. Vmax was similar for both com-
pounds. Addition of 0.1 mM allopurinol to mixtures of
XO, NADH, and 9-oxo-2,4,7-triNF led to stimulation
instead of inhibition of O2 uptake (data not shown),
suggesting that allopurinol was an electron donor. Thus,
Nitr o Com p ou n d -Stim u la ted O
2
Con su m p tion .
Nitro anion radicals undergo redox cycling with O
2
,
resulting in regeneration of parent nitro compound and
production of superoxide anion radical and hydrogen
the initial rate of O uptake was compared in incubation
2
peroxide (Scheme 1). For every mole of superoxide acted
mixtures with 0.1 mM NADH or allopurinol. The rate
3
on by SOD and catalase, /
decreasing the observed rate of uptake of O
4
mol of O
2
is regenerated,
by a
of O uptake was significantly (p e 0.05) greater with
2
2
allopurinol than with NADH (8.74 ( 1.51 vs 4.28 ( 0.91
µM/min). No O2 was consumed in the absence of an
electron donor or with inactive XO.
theoretical 75% (25). To generate nitro anion radicals,
ascorbate was used since it catalyzes nitroreduction via
-
1
e (20) without the influence of protein. Thus, stimula-
En zym e-Ca ta lyzed Am in e F or m a tion fr om Ni-
-
-
tion of ascorbate-catalyzed O
2
uptake with and without
tr oflu or en es. Nitroreduction via 1e and 2e ultimately
leads to amine formation (Scheme 1). The extent of
amine formation in extracts of incubation mixtures of
2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, and 9-oxo-2,7-
diNF with XO or diaphorase was monitored by HPLC
(Table 4). Since NADH was a cofactor for both enzymes,
it was used in these studies. The rates of amine forma-
tion in anaerobic incubation mixtures increased with the
state of oxidation at C-9 (9-oxo-2-,7-diNF > 2,7-diNF and
9-oxo-2-NF > 9-OH-2-NF > 2-NF) and the number of
nitro groups (2,7-diNF > 2-NF and 9-oxo-2,7-diNF >
SOD and catalase by nitrofluorenes was compared to that
by nitrofurantoin and nitrofurazone (Table 2). Among
the nitrofluorenes, only 9-oxo-2,7-diNF and 9-oxo-2,4,7-
triNF were sufficiently soluble to yield measurable O
uptake. The rate of nitrofurantoin-stimulated O uptake
was decreased by 66% in the presence of SOD and
catalase. 9-Oxo-2,7-diNF at 0.2 mM stimulated O
2
2
2
uptake at a rate that was ∼20% of that of nitrofurazone.
These rates were decreased by 88 and 75%, respectively,
in the presence of SOD and catalase. The highly reactive
-
9
-oxo-2,4,7-triNF was compared to nitrofurazone at 0.025
9-oxo-2-NF) of the substrate. Since diaphorase is a 2e
mM. The rate of O consumption with 9-oxo-2,4,7-triNF
2
reductase (21), formation of an O -reactive nitro anion
2
was ∼63-fold greater than that in the presence of
nitrofurazone and was decreased 69% by SOD and
catalase.
radical would not be expected. O2 prevented amine
formation from the compounds tested, suggesting that
O -labile intermediates were formed. With XO, a catalyst
2
-
Flavin enzymes such as XO also catalyze nitroreduc-
of 1e nitroreduction (18, 26), the effect of O
2
was similar,
-
tion of many nitro compounds via 1e (18). The kinetic
except that a small amount of 9-oxo-2-amino-7-NF was
detected from 9-oxo-2,7-diNF. Since allopurinol sup-
parameter log Vmax/K
m
of nitroreductase enzymes mea-
sured by O uptake was shown to correlate with the
2
2
ported O uptake with XO-catalyzed reduction of 9-oxo-
reduction potentials of a series of nitro compounds (25).
Poor solubility of nitrofluorenes limited kinetic analysis
by this method to 9-oxo-2,4,7-triNF which was compared
2,4,7-triNF, it was added as an electron donor for selected
compounds. Only 9-oxo-2,7-diNF yielded an appreciable
amount of amine, ∼7% of the amount with NADH. No

Nitroreduction of Nitrofluorenes
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1365
Ta ble 4. En zym e-Ca ta lyzed Am in e F or m a tion fr om Nitr oflu or en es
amine formation (nmol min- mg-1)a
1
diaphorase/NADH
argon
XO/NADH
XO/allopurinol
argon
nitrofluorene
amine
O2
argon
O2
b
2
-NF
2-AF
9-OH-2-AF
9-oxo-2-AF
2-amino-7-NF
9-oxo-2-amino-7-NF
0.034 ( 0.008
0.128 ( 0.024
0.484 ( 0.007
0.108 ( 0.046
0.821 ( 0.044
-
0
0
0.004 ( 0.002
0.056 ( 0.003
0.519 ( 0.098
0.381 ( 0.012
0.693 ( 0.156
-
-
9
9
2
9
-OH-2-NF
-oxo-2-NF
,7-diNF
0
0
0.002 ( 0
0.002 ( 0.001
-
-
-
-oxo-2,7-diNF
0.001 ( 0.001
0.015 ( 0.01
0.049 ( 0.003
a
The incubations and determinations of amines are described in Experimental Procedures. Values are the means ( SD of at least
b
three determinations. Not determined.
amine was detected without cofactor or with inactive XO
from any of the nitro compounds examined.
the first wave reduction potentials of the nitrofluorenes
(15, 16) (Table 1) was 0.957.
Nitroreduction of 9-oxo-2,7-diNF and 9-oxo-2,4,7-triNF
-
via 1e was also demonstrated by O
2
uptake due to the
Discu ssion
futile cycling with O
2
of the respective nitro anion radicals
The data herein indicate that nitrofluorenes undergo
nitroreduction catalyzed by ascorbate or flavin enzymes.
Nitroreduction via 1e catalyzed by XO and measured
generated by ascorbate or XO (Tables 2 and 3). Nitrore-
duction via 1e of compounds, including nitrofurantoin,
by ascorbate was confirmed by O2 uptake and ESR
detection of nitro anion radical (20). The rate of O2
uptake (13.4 nmol/min) reported herein with 1 mM
nitrofurantoin at 37 °C was comparable to the rate
-
-
with acetylated cytochrome c revealed that the extent of
9
-oxo-2,7-diNF-stimulated reduction of acetylated cyto-
chrome c was decreased by air. The partial inhibition of
this reaction upon addition of SOD indicated some
intermediary superoxide anion radical, presumably formed
by futile cycling of the nitro anion radical (Scheme 1).
Thus, the decrease by air could reflect the competition
calculated from a trace of O
solution at 30 °C (20). An almost 75% decrease in the
observed rate of O uptake effected by addition of SOD
and catalase is consistent with the theoretical regenera-
2
consumption of a similar
2
3
between O
2
and cytochrome c for the nitro anion radical
tion of /
during the futile recycling of nitro anion radical with O
(25). The relative rates of stimulation of O uptake by
4 2 2 2
mol of O from the superoxide and H O formed
since the rate constants for reaction with acetylated
cytochrome c were 10-100 times greater for radicals of
substrates (including nitro compounds) than for super-
oxide radical (27). It is also possible that other reduced
nitro intermediates, e.g., hydroxylamines, are reactive
2
2
nitro compounds (9-oxo-2,4,7-triNF . nitrofurazone >
9-oxo-2,7-diNF) differ from those for XO-catalyzed cyto-
chrome c reduction (9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF
> nitrofurazone), suggesting that the latter rates may
be affected by enzyme affinity or reactivity of the reduced
nitro intermediates with cytochrome c.
2
with both O and cytochrome c as has been reported for
nitropyrenes (28, 29). Hence, the data strongly suggest
that aerobic 9-oxo-2,7-diNF-stimulated reduction of acety-
lated cytochrome c catalyzed by XO involves both super-
oxide and reduced nitro compound(s). Likewise, both
these species were postulated for nitronaphthalene- and
nitrofurazone-stimulated cytochrome c reduction (30, 31)-
uptake studies also showed a catalysis of 1e-
O
2
nitroreduction of 9-oxo-2,4,7-triNF by XO/NADH (Table
). Herein, an apparent K for XO/NADH-catalyzed
reduction of nitrofurazone was 6.18 ( 0.72 µM. This was
considerably lower than the K
3
m
.
The anaerobic conditions used herein to compare
reduction of nitrofluorenes show the rate of nitro anion
radical formation and its reactivity in the absence of O
The rate of reduction of 14 µM nitrofurazone by XO/
m
of 100 µM for NADPH-
cytochrome P450 reductase-catalyzed reduction of nitro-
2
.
furazone reported by Orna and Mason (25), whose data
showed that K
son of the K
m
varied with the flavoenzyme. Compari-
and log(K /Vmax) values indicated that
-
1
-1
hypoxanthine (∼100 nmol mg min ), reported herein
m
m
-
1
(
Figure 2), was comparable to the rate of 270 nmol mg
9-oxo-2,4,7-triNF had a much greater apparent affinity
for XO than did nitrofurazone which has a similar
reduction potential. Given the broad substrate specificity
of XO and the ease with which 9-oxo-2,4,7-triNF forms
charge transfer complexes, a high level of substrate
enzyme interaction is not surprising. Similarly, the effect
of binding parameters unrelated to electron potential
-
1
min derived from uric acid formation and reported for
anaerobic XO/xanthine-catalyzed reduction of a 30 µM
solution (18).
The effects of the nitrofluorene structures on the rates
of anaerobic nitroreduction (Figure 2) and mutagenicities
(
Table 1) were similar in that both were enhanced by
yielded a 40-fold range in K
closely related nitroacridines, catalyzed by XO/xanthine
19). In our study with XO, the extent of regeneration
of O effected by SOD and catalase was comparable to
m
values for reduction of
oxidation at C-9 and an increased number of nitro groups.
However, the mutagenicity of 9-OH-2-NF was lower than
that of 2-NF, which may be due to differences in nitrore-
ductase specificities or assay conditions. The 2.9-17-fold
greater mutagenicities of the nitrofluorenes in nitrore-
ductase-proficient TA98 than in nitroreductase deficient
TA98NR strains indicate activation by nitroreduction.
The relationship of reduction potential or electron affinity
to mutagenicity of nitrofluorenes has been explored (9,
(
2
that with other flavin enzyme systems (14, 25).
Nitroreduction to amine is a multistep process and,
thus, depends on chemical and enzymic reactivity of the
intermediates as well as the nitro compound. XO/NADH
catalyzed amine formation from 2-NF, 9-OH-2-NF, 9-oxo-
2-NF, 2,7-diNF, and 9-oxo-2,7-diNF (Table 4) with an
order of reactivity similar to that of XO/hypoxanthine-
catalyzed reduction of acetylated cytochrome c (Figure
1
7, 32), whereas its relationship to the rate of enzyme-
-
catalyzed 1e nitroreduction of nitrofluorenes has not.
The correlation coefficient calculated between the initial
rates of acetylated cytochrome c reduction (Figure 2) and
2
2). Even an atmosphere of 95% O did not completely

1
366 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Ritter and Malejka-Giganti
prevent amine formation from 9-oxo-2,7-diNF under 1e-
reducing conditions, suggesting rapid dismutation of
radical. The mutagenicity of the nitrofluorenes deter-
mined in S. typhimurium under aerobic conditions (Table
(2) IARC (1989) Diesel and Gasoline Engine Exhausts and Some
Nitroarenes, Monographs on the Evaluation of the Carcinogenic
Risks to Humans. Vol. 46, IARC, Lyon, France.
(3) Fu, P. P. (1990) Metabolism of nitro-polycyclic aromatic hydro-
carbons. Drug Metab. Rev. 22, 209-268.
1
) suggests that these compounds are susceptible to O
2
-
(4) M o¨ ller, L., Lax, I., and Eriksson, L. C. (1993) Nitrated polycyclic
aromatic hydrocarbons: a risk assessment for the urban citizen.
Environ. Health Perspect. 101 (Suppl. 3), 309-315.
-
insensitive 2e nitroreduction. The mammalian diapho-
rase, NAD(P)H quinone reductase, is considered to
catalyze obligatory 2e quinone reduction (21). Since
nitro compounds are usually poor substrates, their reduc-
tion by diaphorase has received less attention than
quinone reduction but evidence indicates that nitroaro-
matics and quinones may bind to different sites (33).
Because of its stability, availability, and bacterial origin
(
5) M o¨ ller, L. (1994) In vivo metabolism and genotoxic effects of
nitrated polycyclic aromatic hydrocarbons. Environ. Health Per-
spect. 102 (Suppl. 4), 139-146.
6) Beland, F. A., and Marques, M. M. (1994) DNA adducts of
nitropolycyclic aromatic hydrocarbons. In DNA Adducts: Iden-
tification and Biological Significance (Hemminki, K., Dipple, A.,
Shuker, D. E. G., Kadlubar, F. F., Segerback, D., and Bartsch,
H., Eds.) pp 229-244, IARC Scientific Publications No. 125, IARC,
Lyon, France.
-
(
(like that of Salmonella nitroreductase), diaphorase from
(
7) M o¨ ller, L., T o¨ rnquist, S., Beije, B., Rafter, J ., Toftgård, R., and
Gustafsson, J .-Å. (1987) Metabolism of the carcinogenic air
pollutant 2-nitrofluorene in the isolated perfused rat lung and
liver. Carcinogenesis 8, 1847-1852.
8) Gøtze, J .-P., Lindeskog, P., and T o¨ rnquist, C. S. I. (1994) Effects
of induction and age-dependent enzyme expression on lung
bioavailability, metabolism, and DNA binding of urban air
particulate-absorbed benzo[a]pyrene, 2-nitrofluorene, and 3-amino-
-
Clostridium was used herein to demonstrate 2e nitrore-
duction. Diaphorase catalyzed rapid reduction of 9-oxo-
2
to their respective amines. The amine formation, like
the 1e oxidation catalyzed by XO, was O
which was probably due to O -labile intermediate(s) as
suggested above. Other possibilities are that the nitro,
unlike the quinone, reductase activity of the Clostridium
diaphorase is O
tion of these compounds. J arabak found differences in
quinone reductions by diaphorases from rat liver and C.
kluyverii and suggested that results with the latter were
,7-diNF > 9-oxo-2-NF > 9-OH-2-NF g 2,7-diNF > 2-NF
(
-
2
-sensitive
2
1
1
,4-dimethyl-5H-pyridol-(4,3)-indole. Environ. Health Perspect.
02 (Suppl. 4), 147-156.
(
9) Vance, W. A., Wang, Y. Y., and Okamoto, H. S. (1987) Disubsti-
tuted amino-, nitroso-, and nitrofluorenes: a physicochemical
basis for structure-activity relationships in Salmonella typh-
imurium. Environ. Mutagen. 9, 123-141.
-
2
-sensitive or it catalyzed 1e nitroreduc-
(
10) McCoy, E. C., Rosenkranz, E. J ., Rosenkranz, H. S., and Mer-
melstein, R. (1981) Nitrated fluorene derivatives are potent
frameshift mutagens. Mutat. Res. 90, 11-20.
-
consistent with 1e reduction (34). However, the differ-
ences in the relative rates of amine formation, especially
between 9-oxo-2-NF and 2,7-diNF, catalyzed by diapho-
rase and XO could result from differences in mechanisms
of reduction.
Allopurinol was an electron donor for XO-catalyzed
nitroreduction of nitrofluorenes that could be detected
(11) Cui, X.-S., Bergman, J ., and M o¨ ller, L. (1996) Preneoplastic
lesions, DNA adduct formation and mutagenicity of 5-, 7-, and
9
-hydroxy-2-nitrofluorene, metabolites of the air pollutant 2-ni-
trofluorene. Mutat. Res. 369, 147-155.
(12) Kitchin, R. M., Bechtold, W. E., and Brooks, A. L. (1988) The
structure-function relationships of nitrofluorenes and nitrofluo-
renones in the Salmonella mutagenicity and CHO sister-
chromatid exchange assays. Mutat. Res. 206, 367-377.
via O
2
consumption (Table 2) or amine formation (Table
uptake
(
13) M o¨ ller, L., Cui, X.-S., Torndal, U.-B., and Eriksson, L. C. (1993)
Preneoplastic lesions and DNA adduct formation of the airborne
genotoxic agents 2-nitrofluorene and 2,7-dinitrofluorene. Car-
cinogenesis 14, 2627-2632.
4
). Although the rate of allopurinol-supported O
2
with 9-oxo-2,7-diNF was relatively rapid, the further
reduction to amine was slow, suggesting that electrons
were provided during the conversion of allopurinol to
oxipurinol, the actual inhibitor of XO (35).
In summary, nitroreduction of the nitrofluorenes is
structure-dependent with a wide range in rates which
correlate with their reduction potentials. The environ-
mental prevalence of the C-9 oxidized nitrofluorenes and
their enhanced rates of nitroreduction under anaerobic
conditions suggest that nitrofluorenes are a health risk
(14) Mason, R. P., and Holtzman, J . L. (1975) The role of catalytic
2
superoxide formation in the O inhibition of nitroreductase.
Biochem. Biophys. Res. Commun. 67, 1267-1274.
15) Bock, H., and Lechner-Knoblauch, U. (1985) The electrochemical
reduction of aromatic nitro compounds in aprotic solution. Z.
Naturforsch. 40b, 1463-1475.
(
(16) Empis, J . M. A., and Herold, B. J . (1986) Substituent effects in
fluoren-9-one ketyls. Part 2. The electrolytic reduction of fluoren-
9
-ones studied by cyclic voltammetry and electron spin resonance
spectroscopy. J . Chem. Soc., Perkin Trans. 2, 425-430.
17) Klopman, G., Tonucci, D. A., Holloway, M., and Rosenkranz, H.
S. (1984) Relationship between polarographic reduction potential
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nitroimadazoles by reduced flavin mononucleotide and by xan-
thine oxidase. Biochem. Pharmacol. 29, 2684-2687.
(
(
(
to humans. In vivo where O
2
levels are e5% (36),
nitroreduction could yield DNA-reactive species leading
to aminofluorenyl adducts and oxidative DNA damage.
19) O’Connor, C. J ., McLennan, D. J ., Sutton, B. M., Denny, W. A.,
and Wilson, W. R. (1991) Effect of reduction potential on the rate
of reduction of nitroacridines by xanthine oxidase and by dihydro-
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R. P. (1987) Generation of radical anions of nitrofurantoin,
misonidazole, and metronidazole by ascorbate. Arch. Biochem.
Biophys. 255, 419-427.
Ack n ow led gm en t. We thank Mr. Richard Decker
and Ms. Kristen Bennett for technical assistance. We also
thank Dr. Leo Bonilla (University of Minnesota Cancer
Center) for verification of HPLC peak identities by MS
analyses and Dr. Letitia Yao (NMR Laboratory, Depart-
ment of Chemistry, University of Minnesota) for NMR
spectra of a synthetic 9-oxo-2-amino-7-nitrofluorene. This
work was supported by a grant from the National Cancer
Institute (CA-28000), the U.S. Public Health Service, and
Biomedical Research Funds, the U.S. Department of
Veterans Affairs.
(
(
21) Li, R., Bianchet, M. A., Talalay, P., and Amzel, L. M. (1995) The
three-dimensional structure of NAD(P)H:quinone reductase, a
flavoprotein involved in cancer chemoprotection and chemo-
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(
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