Reductions of nitro and 9-oxo groups of environmental nitrofluorenes by the rat mammary gland in vitro

Article abstract of DOI:10.1021/tx0000567

Nitrofluorenes and C-9-oxidized nitrofluorenes are widespread environmental genotoxins which may be relevant for breast cancer on the basis of their carcinogenicities, particularly of 2,7-dinitrofluorene (2,7-diNF), for the rat mammary gland. Since their metabolism to active carcinogens may involve nitroreduction, this study examined the reduction of 2-nitrofluorene (2-NF) and 2,7-diNF and their 9-oxo- and 9-hydroxy (OH) derivatives by the rat mammary gland. Cytosolic fractions catalyze NADH- and NADPH-dependent reductions of the 2-nitro and 9-oxo to the respective 2-amino and 9-OH compounds at rates 4- and ≥10-fold greater than those with microsomes. Rates of amine formation catalyzed by cytosol from 2,7-diNF are greater than the rate from 2-NF and increase for C-9-oxidized derivatives: 9-oxo-2-NF >> 9-OH-2-NF > 2-NF and 9-OH-2,7-diNF 9-oxo-2,7-diNF >> 2,7-diNF. Nitroreduction is inhibited by O₂ or allopurinol (20 μM), dicoumarol (100 μM), and rutin (50 μM). 9-Oxoreduction is inhibited by rutin, dicoumarol, and indomethacin (100 μM), but not by O₂ or allopurinol. Pyrazole or menadione does not inhibit nitro or 9-oxoreduction. Xanthine, hypoxanthine, 2-hydroxypyrimidine, and N'-methylnicotinamide support cytosol-catalyzed nitro, but not 9-oxo, reduction. The data suggest that the nitroreduction is catalyzed largely by a xanthine oxidase and partially by a diaphorase and 9-oxoreduction by a carbonyl reductase. The extents of the nitro and carbonyl reductions of the nitrofluorenes may determine their reactivities with DNA, and thus genotoxicities for the mammary gland.

Full text of DOI:10.1021/tx0000567

Chem. Res. Toxicol. 2000, 13, 793-800
793
Red u ction s of Nitr o a n d 9-Oxo Gr ou p s of En vir on m en ta l
Nitr oflu or en es by th e Ra t Ma m m a r y Gla n d in Vitr o
Clare L. Ritter,† Richard W. Decker,† 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 March 7, 2000
Nitrofluorenes and C-9-oxidized nitrofluorenes are widespread environmental genotoxins
which may be relevant for breast cancer on the basis of their carcinogenicities, particularly of
2,7-dinitrofluorene (2,7-diNF), for the rat mammary gland. Since their metabolism to active
carcinogens may involve nitroreduction, this study examined the reduction of 2-nitrofluorene
(2-NF) and 2,7-diNF and their 9-oxo- and 9-hydroxy (OH) derivatives by the rat mammary
gland. Cytosolic fractions catalyze NADH- and NADPH-dependent reductions of the 2-nitro
and 9-oxo to the respective 2-amino and 9-OH compounds at rates 4- and g10-fold greater
than those with microsomes. Rates of amine formation catalyzed by cytosol from 2,7-diNF
are greater than the rate from 2-NF and increase for C-9-oxidized derivatives: 9-oxo-2-NF >
9-OH-2-NF > 2-NF and 9-OH-2,7-diNF . 9-oxo-2,7-diNF > 2,7-diNF. Nitroreduction is
inhibited by O2 or allopurinol (20 µM), dicoumarol (100 µM), and rutin (50 µM). 9-Oxoreduction
is inhibited by rutin, dicoumarol, and indomethacin (100 µM), but not by O2 or allopurinol.
Pyrazole or menadione does not inhibit nitro or 9-oxoreduction. Xanthine, hypoxanthine,
2-hydroxypyrimidine, and N′-methylnicotinamide support cytosol-catalyzed nitro, but not 9-oxo,
reduction. The data suggest that the nitroreduction is catalyzed largely by a xanthine oxidase
and partially by a diaphorase and 9-oxoreduction by a carbonyl reductase. The extents of the
nitro and carbonyl reductions of the nitrofluorenes may determine their reactivities with DNA,
and thus genotoxicities for the mammary gland.
oxidation of 2-aminofluorene (2-AF), which is used in syn-
thetic petroleums and dye and rubber industries (6, 7).
In addition, 9-oxo-2,4,7-triNF is a photoconducting agent,
a fungicide, and an industrial chemical reagent (4).
In tr od u ction
The nitrated polycyclic aromatic hydrocarbons (nitro-
PAHs),1 including nitrofluorenes, originate from incom-
plete combustion of fossil fuels and atmospheric nitration
and are present in exhausts of coal, kerosene, diesel, and
gasoline heaters and engines (1-3). Environmental
exposure to genotoxic and carcinogenic nitro-PAHs through
inhalation, ingestion, and skin presents a health risk to
humans (3). Among the nitro-PAHs listed as “possibly
carcinogenic to humans” by the International Agency for
Research on Cancer (3), 2-nitrofluorene (2-NF) is formed
at high levels in petroleum exhausts and is found in both
the particulate and gas phase with especially high levels
in air in winter (4, 5). In the air of Tokyo, the level of
2,7-dinitrofluorene (2,7-diNF) is the highest among those
of the characterized nitro-PAHs and is ∼30-fold greater
than that of 2-NF and 10-fold greater than that of
1-nitropyrene (4). Other nitrofluorenes in diesel exhaust
include 2,5-dinitrofluorene (2,5-diNF) and 9-oxo deriva-
tives of 2-NF, 2,7-diNF, and 2,4,7-trinitrofluorene (2,4,7-
triNF). 2-NF and 9-oxo-2-NF are also formed by photo-
The mammary gland of female Sprague-Dawley (SD)
rats is the site most susceptible to tumor induction with
incidences of 100, 44, and 12.5% after ingestion of 2,7-
diNF, 2-NF, and 2,5-diNF, respectively, at 1.62 mmol/
kg of diet for 8 months (8, 9). 2,7-DiNF induced mammary
tumor incidence and multiplicity equal to that of 2-acetyl-
aminofluorene and also yielded mammary tumors (12.5%)
in male rats (9). One oral dose of 0.32 mmol of 9-oxo-
2,4,7-triNF induced a 35% mammary tumor incidence in
female SD rats (10). In our recent study involving
intramammary injections of 2.04 µmol of a nitrofluorene
in 50 µL of dimethyl sulfoxide (DMSO) into each of eight
mammary glands per rat, 2,7-diNF exhibited the greatest
carcinogenic potency, yielding a 100% mammary tumor
incidence with a latent period of 18-48 weeks compared
to the 33-87% incidences and latent periods of 22-82
weeks for 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 9-oxo-2,7-diNF,
2,5-diNF, 9-oxo-2,4,7-triNF, or DMSO (11). Both 2,7-diNF
and 9-oxo-2,7-diNF yielded a greater tumor multiplicity
than did DMSO. The carcinogenicity of 2,7-diNF was very
similar to that of 6-nitrochrysene and greater than that
of 4-nitropyrene tested in DMSO at the same dose level
per gland (12, 13). The data suggest that contamination
of the environment with certain nitro-PAHs, even at low
levels, poses substantial carcinogenic risk which may be
relevant for breast cancer (14).
* To whom correspondence should be addressed: VA Medical
Center, One Veterans Drive (151), Minneapolis, MN 55417. E-mail:
malej001@maroon.tc.umn.edu.
† University of Minnesota.
‡ Veterans Affairs Medical Center.
1
Abbreviations: nitro-PAHs, nitrated polycyclic aromatic hydro-
carbons; 2-NF, 2-nitrofluorene; 2,7-diNF, 2,7-dinitrofluorene; 2,5-diNF,
2,5-dinitrofluorene; 2,4,7-triNF, 2,4,7-trinitrofluorene; 2-AF, 2-amino-
fluorene; 2,7-diAF, 2,7-diaminofluorene; SD, Sprague-Dawley; DMSO,
dimethyl sulfoxide; OH, hydroxy; NH2, amino; IS, internal standard;
XO, xanthine oxidase.
10.1021/tx0000567 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/27/2000

794 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Ritter et al.
272.0433 (calcd); 1H NMR (500 MHz, DMSO-d6) δ 8.41-8.24
(m, 6H, aromatic H), 6.47 (d, H, OH, J ) 6.5 Hz), 5.76 (d, H,
CH, J ) 6.5 Hz). Exchangeable protons were identified by
addition of D2O; the resonance at 6.47 ppm disappeared, and
the resonance at 5.76 ppm collapsed to a singlet: 13C NMR (125
MHz, DMSO-d6) δ 149.7 (C), 148.0 (C), 143.5 (C), 124.9 (CH),
122.8 (CH), 120.3 (CH), 73.0 (CH-OH).
The carcinogenicity of 2,7-diNF by the intramammary
route (11) indicated that the mammary gland is capable
of metabolic activation of this compound and possibly
other nitrofluorenes to DNA-reactive species involved in
the initiation of tumorigenesis. Nitroreduction appears
to play a key role in the activation of nitrofluorenes to
mutagens (15, 16) and of 2-NF to an ultimate carcinogen
(4, 5, 17, 18). In our previous studies, the rates of
nitroreduction of nitrofluorenes and C-9-oxidized nitro-
fluorenes catalyzed by bovine milk xanthine oxidase (XO)
and bacterial diaphorase paralleled their reported muta-
genicities in Salmonella typhimurium (19), and nitro-
reductions of nitrofluorenes catalyzed by mammary gland
cytosol suggested one- and/or two-electron reactions (20).
Preliminary HPLC analyses of the products of nitro-
reduction revealed that mammary gland cytosol, unlike
XO or diaphorase, catalyzes a carbonyl reduction of the
9-oxo group to a 9-OH group as well as nitroreduction.
The tumorigenicity or metabolism of 9-OH-2,7-diNF (a
new compound synthesized for this study) has not been
examined. However, the carcinogenic potential of 9-OH
nitrofluorenes is strongly suggested by the findings that
9-OH-2-NF is the most mutagenic metabolite excreted
in urine and feces of rats treated with 2-NF (21) and
treatment of rats with 9-OH-2-NF yields substantial
levels of DNA adducts and focal lesions in the liver (22).
Thus, the aim of the study presented here was to examine
the reductive metabolism of 2-NF and 2,7-diNF and their
C-9-oxidized, i.e., 9-oxo and 9-OH, derivatives by the rat
mammary gland. The effects of nitro and carbonyl
reductions on the product formation were determined by
HPLC. The cofactor dependence and effects of known
inhibitors of reduction were also examined.
9-Oxo-2-NH2-7-NF (40 mg, 0.167 mmol) (19) was dissolved
in 22 mL of toluene under reflux and cleared by centrifugation.
The supernatant was mixed with up to 2 portions of aluminum
isopropoxide (56 mg, 0.275 mmol) under reflux for 1-2 h to
complete reduction, which was monitored by a change of color
from red to yellow and TLC on Analtech Uniplate Silica Gel G
(Analtech Inc., Newark, DE) developed in an ethyl acetate/tert-
butyl methyl ether mixture (98:2) with Rf values of 0.64 and
0.72 for 9-OH-2-NH2-7-NF and 9-oxo-2-NH2-7-NF, respectively.
The mixture was taken to dryness under reduced pressure in a
rotary evaporator and extracted in acetone (4.4 mg/mL) under
reflux. The solution was evaporated to dryness and the residue
redissolved in acetone and subjected to preparative TLC in the
system described above. The band with an Rf of 0.64 was
extracted in acetone under reflux and the extract evaporated
to dryness with N2 and dried over P2O5. 9-OH-2-NH2-7-NF (16%
yield, mp >300 °C dec) was 97% pure as determined by HPLC:
UV/vis λmax 400, 257 nm [ꢀ(methanol) ) 13 250 and 8503 M-1
1
cm-1, respectively]; FAB-MS 242.1 (M+), 241.1 [(M - H)-]; H
NMR (500 MHz, DMSO-d6) δ 8.19 (d, H, C-8, J ) 2.0 Hz), 8.17
(dd, H, C-6, J ) 8.5, 2.0 Hz), 7.69 (d, H, C-5, J ) 8.5 Hz), 7.57
(d, H, C-4, J ) 8.5 Hz), 6.82 (d, H, C-1, J ) 2.0 Hz), 6.07 (dd, H,
C-3, J ) 8.5, 2.0 Hz), 5.96 (d, H, OH, J ) 7.5 Hz), 5.74 (s, 2H,
NH2), 5.41 (d, H, CH, J ) 7.5 Hz). Exchangeable protons were
identified by addition of D2O; resonances at 5.96 and 5.74 ppm
disappeared, and the resonance at 5.41 ppm collapsed to a
singlet.
Ma m m a r y Gla n d . Female SD rats (Specific Pathogen Free;
Harlan Sprague Dawley, Madison, WI) were acclimated for 1
week in a controlled environment with a 12 h light/dark cycle
and maintained on a Teklad Certified Rodent Diet (Harlan
Teklad, Madison, WI) and water ad libitum. When they were
50-60 days old, rats were decapitated after CO2 asphyxiation.
Mammary glands from individual rats were trimmed of extra-
neous tissue on an ice block, minced, and rinsed with cold 50
mM Tris-HCl buffer (pH 7.4) containing 0.15 M KCl. The
mammary tissue was frozen in liquid N2 and stored at -80 °C
for up to 6 months. For tissue fractionation, all buffers were
ice-cold and all procedures carried out at 4 °C unless specified
otherwise. Frozen mammary gland was pulverized with a
Bessman pulverizer, and homogenized in 2 volumes (w/w) of
50 mM Tris-HCl buffer (pH 7.4) containing 0.15 M KCl and 1
mM dithiothreitol (DTT) with a Polytron homogenizer type
PT10/35 (Kinematica, Gmbh, Lucerne, Switzerland). The ho-
mogenate was centrifuged at 14000g for 20 min at 4 °C and the
solidified fat removed. Cytosolic and microsomal fractions were
prepared from the supernatant as described (24) immediately
before use. Microsomes were stored overnight on ice in homog-
enization buffer. The amount of protein was determined with
Coomassie Plus Protein assay reagent (Pierce, Rockford, IL).
Exp er im en ta l P r oced u r es
Ca u t ion : The nitrofluorenes should be handled according
to the NIH Guidelines for the Laboratory Use of Chemical
Carcinogens (Publication 81-2385, U.S. Government Printing
Office, Washington, DC).
Rea gen ts. Glass-distilled water was used throughout. Sol-
vents and reagents were of the highest available purity.
Aluminum isopropoxide, 2-NF, 2-AF, 2,7-diNF, 2-NH2-7-NF,
9-oxo-2-NF, 9-oxo-2-AF, 2,7-diaminofluorene (2,7-diAF), and
4-phenylphenol were from Aldrich (Milwaukee, WI), and 9-oxo-
2,7-diNF was from Pfaltz and Bauer, Inc. (Waterbury, CT).
9-OH-2-NF, 9-OH-2-AF, and 9-oxo-2-NH2-7-NF were synthe-
sized as described previously (19). All fluorenyl compounds and
4-phenylphenol were recrystallized until pure by HPLC.
Syn th eses of 9-OH-2,7-d iNF a n d 9-OH-2-NH2-7-NF . The
9-oxo compounds were reduced to 9-OH compounds with alu-
minum isopropoxide (22, 23). All solvents were flushed with N2
and reduction procedures conducted under N2. 9-Oxo-2,7-diNF
(250 mg, 0.92 mmol) in 90 mL of toluene was refluxed for 2 h
with aluminum isopropoxide (550 mg, 2.7 mmol) to give ∼90%
reduction. The mixture was taken to dryness under reduced
pressure in a rotary evaporator. The residue was stirred for 10
min in 20 mL of 1 N H2SO4 in 20% 2-propanol. The insoluble
material (195 mg) was collected, washed with cold water, and
dried over P2O5. It was dissolved (1.8 mg/mL) in a benzene/
toluene mixture (8:2) by refluxing and cleared by centrifugation.
The supernatant was refluxed for ∼3 min with a small amount
of charcoal, filtered, condensed, and cooled to 4 °C and then to
-20 °C. The precipitate was collected and purified by repeated
recrystallizations. 9-OH-2,7-diNF (14% yield, mp >300 °C dec)
was 98% pure as determined by HPLC: UV/vis λmax 335, 318
(shoulder), 254 nm [ꢀ(ethanol) ) 23 554, 20 294, and 8247 M-1
cm-1, respectively]; FAB-MS 272.1 (M-), 425.1 [(M + matrix )
3-nitrobenzyl alcohol, 153)-]; HR FAB m/z 272.0432 (observed),
Deter m in a tion by UV/Vis Sp ectr om etr y of th e Effect
of Mod ifier s on Red u ction of 9-Oxo-2-AF . Cytosolic protein
(0.2 mg/mL) was added to cuvettes containing 50 mM Tris-HCl
buffer (pH 7.4) containing 1 mM DTT at 37 °C (final volume of
1 mL). To specified incubations was added pyrazole (1 mM),
rutin (20 µM), indomethacin (100 µM), or menadione (10 µM)
in methanol (final concentration of 1.8%). 5â-Androstan-17â-
ol-3-one or 5R-androstane-3,17-dione (100 µM) was added in
ethanol (final concentration of 1%). Dicoumarol (100 µM) was
added in 0.025 N NaOH (100 µL/mL), and allopurinol (20 µM)
was added in buffer. NADH (0.5 mM) was added and the UV/
vis absorbance baseline recorded from 260 to 600 nm. 9-Oxo-
2-AF (28 µM in 20 µL of DMSO) was added and the absorbance
spectrum recorded every 20 s for up to 10 min. The loss of A283
of 9-oxo-2-AF and the increase in A302 due to 9-OH-2-AF gave

Reductions of Nitro and C-9-Oxidized Fluorenes
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 795
isosbestic points at 290 and 316 nm. The initial rates of increase
in A302 relative to A316 were linear with time and protein
concentration and were corrected for changes in the controls
which contained inactive cytosol. The rates of change were
compared in incubations containing modifier or its solvent.
Product formation was confirmed by HPLC analysis of extracts
of selected incubations.
Deter m in a tion of P r od u cts fr om Nitr oflu or en es In cu -
ba ted w ith Ma m m a r y Gla n d F r a ction s. Mixtures (1 mL) of
0.2-0.5 mg/mL cytosolic protein or 0.5 mg/mL microsomal
protein, 28 µM nitrofluorene compound in 40 µL of DMSO, and
0.5 mM cofactor in 50 mM Tris-HCl buffer (pH 7.4) containing
1 mM DTT were incubated at 37 °C in 2 mL amber vials.
Anaerobic incubations contained an O2-scavenging system
consisting of 19 µg/mL catalase, 16 µg/mL glucose oxidase, and
75 µM glucose and were subjected to a gentle stream of argon
for 10 min and then 5 min pulses every 15 min (19). Controls
either contained inactive protein or lacked the cofactor or nitro
compound. At the end of timed intervals, 10 nmol of 4-phenyl-
phenol was added as an internal standard (IS) in 10 µL of a
2-propanol/methanol mixture (2:1), and the mixture was im-
mediately applied to a Baker C18 extraction column precondi-
tioned with argon-flushed solvent and buffer. After washing with
buffer was carried out, the nitrofluorenes and their metabolites
were eluted with 5% DMSO in CH2Cl2. The eluates were dried
over Na2SO4, and the CH2Cl2 was evaporated at 25 °C under a
stream of N2 passed through an Oxy-Trap (Alltech Associates,
Deerfield, IL). Recoveries of all available standard compounds
were g95% except for 2,7-diNF (∼87%). Whereas 9-OH-2,7-diNF
was susceptible to oxidation (e3.5% was detected by HPLC as
9-oxo-2,7-diNF after extraction), 9-OH-2-NF and 9-OH-2-NH2-
7-NF were not recovered as such. Mixtures of mononitrofluo-
renes and their metabolites were separated on modified HPLC
system A (22) containing methanol, 2-propanol, and 0.2 M acetic
acid (pH 3.8) with gradients of 20 to 40% methanol and 80 to
60% acid within 5 min, 40 to 66% methanol, 0 to 4% 2-propanol,
and 60 to 30% acid within 2 min, 66 to 75% methanol, 4 to 5%
2-propanol, and 30 to 20% acid within 3 min, and 75 to 65%
methanol, 5 to 10% 2-propanol, and 20 to 25% acid within 6
min. Mixtures of dinitrofluorenes and their metabolites were
separated on HPLC system C (22) containing methanol, 2-pro-
panol, and 0.2 M acetic acid (pH 4.2) with gradients of 30 to
70% methanol and 70 to 30% acid within 10 min, 70 to 74%
methanol, 0 to 6% 2-propanol, and 30 to 20% acid within 3 min,
and 74 to 80% methanol, 6 to 10% 2-propanol, and 20 to 10%
acid within 3 min. Identification of compounds was based on tR
and UV/vis spectra superimposed with those of their standards
and was verified in select incubations by LC/MS or by MS with
direct infusion of compounds collected after HPLC separation.
The peak areas were integrated in the modified gradient A at
F igu r e 1. HPLC (system C) profile of the standard dinitro-
fluorene compounds and their amino derivatives superimposed
on the profile (filled peaks) of the extract of an incubation of
9-OH-2,7-diNF with mammary gland cytosol and NADH. Desig-
nations of the peaks (UKN is unkown) are at the wavelengths
used for integration of the areas.
F igu r e 2. UV/vis absorbance spectra of 9-OH-2,7-diNF and
9-oxo-2,7-diNF, their respective 2-amino derivatives, and un-
knowns UKN I-III recorded with the diode array detector
during HPLC elution.
260 nm for IS (tR ) 10.83 min), 280 nm for 9-oxo-2-NF (tR
11.66 min), 9-oxo-2-AF (tR ) 9.94 min), 2-AF (tR ) 10.50 min),
300 nm for 9-OH-2-AF (tR ) 9.11 min), 330 nm for 2-NF (tR
)
temperature on a 500 MHz Varian Inova spectrometer, and
FAB-MS analyses were performed with a VG 7070E-HF mass
spectrometer at the Department of Chemistry, University of
Minnesota. LC/MS analyses were performed at the University
of Minnesota Cancer Center with a Finnigan TSQ 7000 triple-
stage quadropole mass spectrometer.
Sta tistica l An a lysis. For comparison of two groups or no
fewer than three groups, a two-tailed t test or one way ANOVA
with Neuman-Keuls post test, respectively, was performed with
GraphPad Prism version 2.0 for Macintosh (GraphPad Software,
San Diego, CA).
)
13.59 min), 9-OH-2-NF (tR ) 10.44 min), and 360 nm for
2-nitrosofluorene (tR ) 13.86 min). In gradient C, peak integra-
tion was at 260 nm for 9-oxo-2-NH2-7-NF (tR ) 12.01 min) and
IS (tR ) 12.86 min), 280 nm for 2,7-diAF (tR ) 4.62 min) and
9-oxo-2,7-diNF (tR ) 14.1 min), 340 nm for 9-OH-2,7-diNF (tR
) 11.80 min) (m/z 271) and 2,7-diNF (tR ) 14.80 min), and 390
nm for 9-OH-2-NH2-7-NF (tR ) 9.62 min) (m/z 243) and 2-NH2-
7-NF (tR ) 12.48 min). Compounds were quantitated from the
peak area relative to standard curves and corrected for recovery
of the IS. The HPLC profiles of dinitrofluorenes and their amino
derivatives (Figure 1) and the UV/vis spectra recorded during
the chromatography (Figure 2) are shown. Initial rates were
calculated from time intervals in which product detection was
linear with time and protein concentration.
Resu lts
Nitr o a n d 9-Oxor ed u ction s of Nitr oflu or en es a n d
9-Oxon itr oflu or en es Ca ta lyzed by Ma m m a r y Gla n d
Cytosol. The products of the reduction of nitrofluorenes
catalyzed by mammary gland cytosol or microsomes were
determined by HPLC analyses. The products from the
incubation of 2-NF, 2,7-diNF, 9-oxo-2-NF, 9-OH-2-NF,
9-OH-2,7-diNF, 9-oxo-2,7-diNF, or 9-oxo-2-NH2-7-NF with
In str u m en ta tion . HPLC data were obtained with a Hewlett-
Packard 1090 liquid chromatograph equipped as described
previously (25). UV/vis data were obtained with Hewlett-
Packard 8452A diode array and Beckman UV-70 spectropho-
tometers. 1H and 13C NMR spectra were obtained at ambient

796 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Ritter et al.
Ta ble 2. Cofa ctor s Utilized by Ma m m a r y Gla n d Cytosol
or Micr osom es for Red u ction s of 9-Oxon itr oflu or en esa
reduction of
NO2 to NH2
reduction of
9-oxo to 9-OH
9-oxo-
9-oxo-
9-oxo-
9-oxo-
cofactor (0.5 mM)
cytosol
NADH
NADPH
hypoxanthine
2-hydroxypyrimidine
xanthine
N′-methylnicotinamide
microsomes
NADH
2-NF 2,7-diNF 2-NF 2,7-diNF
1
1
1
0.55
0
-
-
-
1b
0.62c
0.74
0.54
0.53
0.35
0.25
1.11b
1.76
0
0
0
0
d
-
-
-
1
0.56
0.1
-
-
-
1
4.8
0
-
-
-
NADPH
hypoxanthine
a
Mammary gland cytosol (0.2 mg/mL) or microsomes (0.5 mg/
mL) from at least two separate rats were incubated in duplicate
under argon for 30 or 60 min, respectively, and products were
identified and quantitated as described in Experimental Proce-
F igu r e 3. Fate of 9-oxo-2-NF or 9-oxo-2,7-diNF incubated with
mammary gland cytosol. Pathway A, carbonyl reductase-
dependent, catalyzed by cytosol with NADH or NADPH, but
not hypoxanthine. Pathway B, nonenzymic. Pathway C, nitro-
reductase-dependent, catalyzed by cytosol with NADH, NADPH,
or hypoxanthine.
b
dures. 9-OH-2,7-diNF may be underestimated due to oxidation.
c Values represent the ratios of the amounts of total (9-OH + 9-oxo)
amine or total (NO2 + NH2) 9-OH products relative to those with
d
NADH. Not determined.
rate of NADH-dependent reduction of 9-oxo greatly
exceeds that of the nitro group of the mononitro com-
pound, i.e., the formation of 9-OH-2-NF is ∼68-fold faster
than that of 9-oxo-2-AF from 9-oxo-2-NF. The effects of
oxidation state at C-9 of mono- and dinitrofluorenes differ
since total amine formation from 9-oxo-2-NF > 9-OH-2-
NF > 2-NF and from 9-OH-2,7-diNF . 9-oxo-2,7-diNF
> 2,7-diNF. The addition of a second electron-withdraw-
ing nitro group at the C-7 of 2-NF increases the rate of
reduction of the 2-nitro to the 2-amino with the rates of
amine formation from 2,7-diNF, 9-OH-2,7-diNF, and
9-oxo-2,7-diNF being greater than those from 2-NF,
9-OH-2-NF, and 9-oxo-2-NF, respectively. The rate of
9-oxoreduction of the 9-oxo-2-AF is similar to that of
9-oxo-2-NF. Although 9-OH-2,7-diNF formation may be
underestimated due to its nonenzymatic oxidation and
enzymatic conversion to unknowns, the level of 9-OH-2-
NH2-7-NF determined from 9-oxo-2,7-diNF relative to
those from 9-OH-2,7-diNF and 9-oxo-2-NH2-7-NF sug-
gests that the 9-oxoreduction is enhanced by the presence
of the 2-amino group. 2-NF or 2-AF is not detected in
incubations of 9-oxo-2-NF or 9-OH-2-NF with mammary
gland cytosol, nor is 2,7-diNF or 2-NH2-7-NF detected
from 9-oxo-2,7-diNF, 9-OH-2,7-diNF, or 9-oxo-2-NH2-7-
NF. No 2,7-diAF is detected from the dinitro compounds.
Ta ble 1. Effects of Nitr o Gr ou p s a n d th e Sta te of
Oxid a tion a t C-9 on Red u ction of Nitr oflu or en es a n d
C-9-Oxid ized Nitr oflu or en es by Ma m m a r y Gla n d Cytosol
apparent rate of
formation
a
nitrofluorene
2-NF
9-OH-2-NF
9-oxo-2-NF
product
(nmol min-1 mg-1
)
2-AF
0.04 ( 0.01
0.09 ( 0.02
14.37 ( 4.53
0.21 ( 0.07
0.12 ( 0.06
12.83 ( 4.10
0.12 ( 0.04
2.99 ( 0.59c
0.15 ( 0.25d
0.15 ( 0.09
0.62 ( 0.26e
g60
9-OH-2-AF
9-OH-2-NF
9-oxo-2-AF
9-OH-2-AF
9-OH-2-AF
2-NH2-7-NF
9-OH-2-NH2-7-NF
9-OH-2,7-diNF
9-oxo-2-NH2-7-NF
9-OH-2-NH2-7-NF
9-OH-2-NH2-7-NF
9-oxo-2-AFb
2,7-diNF
9-OH-2,7-diNF
9-oxo-2,7-diNF
9-oxo-2-NH2-7-NF
a
Reductions were determined from incubations of mammary
gland cytosol with 28 µM nitrofluorene (4% DMSO) and 0.5 mM
NADH at timed intervals as described in Experimental Proce-
dures. Values are means ( SD from duplicate incubations with
b
at least three different cytosols. The product of nitroreduction
of 9-oxo-2-NF. c Nitroreduction significantly (P < 0.001) greater
d
than that of other nitrofluorenes. May be underestimated due
to product oxidation. e Significantly (P ) 0.0015) greater than the
rate of formation of 9-OH-2-AF from 9-oxo-2-NF.
Cofa ct or R eq u ir em en t s for Nit r o a n d 9-Oxo-
r ed u ction s of 9-Oxon itr oflu or en es Ca ta lyzed by
Ma m m a r y Gla n d Cytosol a n d Micr osom es. Like
NADH, NADPH supports cytosol-catalyzed reductions of
nitro to amino and 9-oxo to 9-OH groups (Table 2).
Compared to NADH, NADPH is a poorer electron donor
for both nitro and 9-oxoreductions with 9-oxo-2-NF.
Hypoxanthine, xanthine, N′-methylnicotinamide, and
2-hydroxypyrimidine support cytosol-catalyzed nitro but
not 9-oxoreductions. The microsomal fraction of mam-
mary gland contains relatively low levels of reductase
activity, i.e., NADH- and NADPH-supported rates of
conversion of 9-oxo-2-NF to amine and 9-OH products are
∼24 and 1%, respectively, of those of cytosol (data not
shown). Hypoxanthine supports only minimal microsome-
catalyzed amine formation (∼10% of the NADH-
supported rate) from 9-oxo-2-NF.
mammary gland cytosol and NADH indicate reduction
of the 9-oxo to 9-OH as well as the reduction of the nitro
group to the amine (the structures of the C-9-oxidized
substrates and products are shown in Figure 3). Both
reductions are dependent on the active enzyme. The
nitroreductase activity is partially unstable with losses
of g30% in cytosols stored overnight in ice or frozen at
-80 °C, whereas the 9-oxo reducing activity appears to
be stable at -80 °C in the presence of 1 mM DTT. Thus,
cytosols were prepared in 1 mM DTT immediately before
use. The XO and diaphorase activities in mammary gland
cytosols from no fewer than four rats each assayed in
duplicate with xanthine (26) and menadione (27) are 4.98
( 1.42 and 88.3 ( 15.5 nmol min-1 mg-1, respectively.
The rates of both the 9-oxo and nitro reductions are
dependent on the state of oxidation at C-9 and the
number of nitro groups of the nitrofluorene (Table 1). The

Reductions of Nitro and C-9-Oxidized Fluorenes
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 797
Ta ble 3. Cofa ctor Dep en d en ce of Red u ction P r od u cts F or m ed in In cu ba tion s of C-9-Oxid ized Nitr oflu or en es w ith
Ma m m a r y Gla n d Cytosola
nmol of productb
substrate
9-oxo-2-NF
time (min)
30
product
NADH
NADPH
hypoxanthine
9-OH-2-NF
9-oxo-2-AF
9-OH-2-AF
9-oxo-2-NH2-7-NF
9-OH-2-NH2-7-NF
9-OH-2,7-diNF
9-oxo-2-NH2-7-NF
9-OH-2-NH2-7-NF
9-OH-2-NH2-7-NF
25.08 ( 4.02
0.145 ( 0.095
0.88 ( 0.473
0.223 ( 0.310
12.65 ( 1.52
0.127 ( 0.284g
0.377 ( 0.300
6.60 ( 3.68
14.02 ( 1.01
0.37 ( 0.13
0
1.80 ( 0.94c
0.263 ( 0.018d
0
e
9-OH-2,7-diNF
9-oxo-2,7-diNF
30
30
-
1.58 ( 0.72f
-
8.20 ( 3.29d
2.32 ( 0.69g
0
0
3.77 ( 2.46d
5.18 ( 0.91
0
0
9-oxo-2-NH2-7-NF
10
17.75 ( 2.60
-
a
Mammary gland cytosol (0.2 mg/mL) was incubated under anaerobic conditions with 28 µM substrate (4% DMSO) and 0.5 mM cofactor;
b
the products were analyzed by HPLC as described in Experimental Procedures. Values are means ( SD with cytosols from at least two
separate rats, each incubated in duplicate. c Statistically different (P < 0.01) from the product with NADH or NADPH. Statistically
d
different (P e 0.025) from the product with NADH. e Not determined. f Statistically different (P e 0.005) from the product with NADH.
g
May be underestimated due to product oxidation.
Ta ble 4. Effects of Mod ifier s on Red u ction s of Nitr o a n d 9-Oxo Gr ou p s of Nitr o a n d Am in o F lu or en es by
Ma m m a r y Gla n d Cytosola
reduction of NO2 to NH2
reduction of 9-oxo to 9-OH
substrate
modifier (µM)
substrate
inibition (%)b
inhibition (%)b
O2
9-OH-2-NF, 9-oxo-2-NF,
9-OH-2-NF, 9-oxo-2-NF,
2,7-diNF, 9-OH-2,7-diNF
9-oxo-2,7-diNFc
2,7-diNF
9-OH-2-NF, 2,7-diNF
93 ( 14
64 ( 17
9-oxo-2-AF, 9-oxo-2-NF
0
dicoumarol (100)
9-oxo-2-AF, 9-oxo-2-NF,
9-oxo-2-NH2-7-NF
9-oxo-2-AF
39 ( 26
menadione (10)
allopurinol (20)
rutin (50)
0
0
0
88 ( 3
27 ( 6
9-oxo-2-AF
9-oxo-2-AF, 9-oxo-2-NF,
9-oxo-2-NH2-7-NF, 9-oxo-2,7-diNF
9-oxo-2-AF, 9-oxo-2-NF
9-oxo-2-AF
9-oxo-2-AF
9-oxo-2-AF
68 ( 17
pyrazole (1000)
indomethacin (100)
5â-androstan-17â-ol-3-one (100)
5R-androstane-3,17-dione (100)
9-oxo-2-NF, 2,7-diNF
0
0
d
-
24 ( 11
15 ( 14
7 ( 7
d
-
d
-
a
Reductions of nitro and 9-oxo groups were assessed by UV/vis and/or HPLC analyses in incubations containing 28 µM substrate and
b
0.5 mM NADH, unless specified otherwise, with mammary gland cytosol as described in Experimental Procedures. Inhibition of initial
rates of cytosol-dependent changes in reduction with two to seven cytosols, each incubated in duplicate. c Determined with 0.5 mM
d
N′-methylnicotinamide as a cofactor. Not determined.
The effect of the electron donor on the amounts of
products depends on the substrate as shown with 9-oxo-
2-NF, 9-OH-2,7-diNF, 9-oxo-2,7-diNF, and 9-oxo-2-NH2-
7-NF with NADH, NADPH, or hypoxanthine as a cofactor
for the cytosol-catalyzed reductions (Table 3). After 30
min, the total amount of amine from 9-oxo-2-NF with
hypoxanthine is ∼1.8- or 2.8-fold greater than with
NADH or NADPH, respectively, and the amount of 9-OH
products is greater with NADH than with NADPH. From
9-OH-2,7-diNF, the amounts of amine are slightly greater
with NADH than with hypoxanthine (12.87 vs 9.78 nmol).
The extent of reduction of 9-oxo-2,7-diNF to amine is
2-fold greater with NADH than with hypoxanthine (6.98
vs 3.77 nmol) and similar to that with NADPH. Hypo-
xanthine does not support any 9-oxoreduction. Levels of
9-oxo-2-NH2-7-NF from 9-OH-2,7-diNF are higher with
hypoxanthine as a cofactor than with NADH and do
not correlate with the levels of 9-OH-2-NH2-7-NF. At
shorter incubation times, no 9-oxo-2-NH2-7-NF is de-
tected from 9-OH-2,7-diNF even though large amounts
of the 9-OH-2-NH2-7-NF are formed, suggesting that
little or no oxidation of the latter occurs during incubation
or extraction (Tables 1 and 3). Thus, it appears that the
9-oxo-2-NH2-7-NF detected in incubations of cytosol with
9-OH-2,7-diNF is derived from 9-oxo-2,7-diNF formed
during the incubation.
(tR ) 9.8 min; λmax ) 345 nm), and UKN III (tR )
12.1 min; λmax ) 370 nm) (Figures 1 and 2). The peak
areas of UKN I-III in the fresh extract from a 30 min
anaerobic incubation of 9-OH-2,7-diNF with 0.2 mg/mL
cytosolic protein and 0.5 mM NADH are ∼4, 15, and
15%, respectively, of that of 9-OH-2-NH2-7-NF. With
hypoxanthine as a cofactor, the respective areas are
18-, 0.67-, and 3.5-fold as great as with NADH. The
same unidentified peaks are also found from incubations
of cytosol with 9-oxo-2,7-diNF and NADH, but not
hypoxanthine. However, they are not found in incuba-
tions of 9-oxo-2-NH2-7-NF with cytosol and either cofactor
or in controls with inactive cytosol. The data suggest
that these unknowns represent or are derived from
intermediates of nitroreduction of 9-OH-2,7-diNF, e.g.,
9-OH-2-nitroso-7-NF and/or 9-OH-2-NHOH-7-NF. Be-
cause of the small amounts, the parent ions could not
be unequivocally identified from the LC/MS spectra, and
the peaks from HPLC were unstable to collection and
lyophilization.
Effects of Mod ifier s on Ma m m a r y Gla n d Cytosol-
Ca ta lyzed Nitr o a n d 9-Oxor ed u ction s of F lu or en yl
Com p ou n d s. Since the use of different cofactors in the
reductions of nitrofluorenes and C-9 oxidized nitrofluo-
renes suggests involvement of several enzymes in the
reductions of the nitro and 9-oxo groups, the effects of
known modifiers of enzymic nitro and carbonyl reductions
on the initial rates of these reductions catalyzed by
mammary gland cytosol were examined with NADH as
a cofactor (Table 4). Nitroreduction is inhibited by O2,
In addition to the amine, 9-OH-2,7-diNF incubated
with active cytosol and NADH or hypoxanthine yields
unknown products that can be detected with HPLC
system C: UKN I (tR ) 8.8 min; λmax ) 375 nm), UKN II

798 Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Ritter et al.
dicoumarol (100 µM), allopurinol (20 µM), and rutin (50
µM) by 93, 64, 88, and 27%, respectively. Menadione (10
µM) has no effect on nitroreduction when N′-methylnico-
tinamide replaces NADH. Reduction of the 9-oxo group
is most sensitive to rutin (50 µM) (68% inhibition) and
is also inhibited 39% by dicoumarol (100 µM) and 24%
by indomethacin (100 µM). In contrast to nitroreduction,
reduction of 9-oxo is insensitive to O2 and allopurinol (20
µM). Reduction of the 9-oxo group is also unaffected by
menadione (10 µM). 5R-Androstane-3,17-dione (100 µM)
and 5â-androstan-17â-ol-3-one (100 µM), substrates for
carbonyl reducing steroid dehydrogenases, did not act as
competitive substrates for the reduction of 9-oxo-2-AF.
Pyrazole (1000 µM) has no effect on reduction of the nitro
or 9-oxo group.
activity may be due to a diaphorase isozyme since a
purified DT diaphorase [NAD(P)H:quinone oxidoreduc-
tase] from rat tumor was found to be nonspecific in its
requirement of reduced pyridinium electron donors uti-
lizing 1-methylnicotinamide as efficiently as NADH (N′-
methylnicotinamide was not evaluated) (30). The nitro-
reduction of the nitrofluorene compounds by bacterial
diaphorase (19), the demonstration herein of diaphorase
activity in the mammary gland cytosols, and the dicou-
marol inhibition of cytosol-catalyzed amine formation
(Table 4) and of the nitrofluorene-dependent reduction
of acetylated cytochrome c (20) suggest that diaphorase
may contribute to the cytosol-catalyzed nitroreduction of
the nitrofluorenes. However, dicoumarol also inhibits
other enzymes, including XO (ref 31 and our unpublished
data). Whether the weak inhibition of nitroreduction by
the antioxidant rutin (50 µM), a flavone glucuronide, is
a function of enzyme inhibition is unclear. The lack of
inhibiton by pyrazole (Table 4) indicates that alcohol
dehydrogenase is not involved in nitroreduction (32). On
the basis of O2 uptake during NADH-dependent 9-oxo-
2,4,7-triNF reduction catalyzed by mammary gland
cytosol and microsomes (20) and on the basis of the
inhibition by O2 of amine formation from nitrofluorenes
catalyzed by diaphorase and XO (19), the inhibition by
O2 herein (Table 4) is attributed to its interaction with
nitro anion radical and other O2-sensitive intermediates.
Microsomal nitroreduction may be linked to the NADPH
cytochrome P450 and NADH cytochrome c reductases
found in rat mammary gland (33). Although microsomal
nitroreduction of 9-oxo-2-NF seems to prefer NADH, it
may be underestimated with NADPH because of the
rapid carbonyl reduction.
Reduction of the 9-oxo group differs from that of the
nitro group in the effects of electron donors, O2, and
inhibitors. Only NADH and NADPH support 9-oxo-
reduction (Table 3), indicating that neither XO nor
aldehyde oxidase is a catalyst (34). The lack of involve-
ment of XO in 9-oxoreduction is also supported by the
insensitivity to allopurinol of cytosol-catalyzed reduction
and the lack of 9-oxoreduction of the nitrofluorenes by
milk XO (19). Likewise, the lack of inhibition by pyrazole
indicates that alcohol dehydrogenase is not involved in
9-oxoreduction. Among the many enzymes associated
with carbonyl reduction in human and rat liver (35),
dicoumarol-sensitive DT diaphorase and flavone-sensitive
carbonyl reductase have received special attention
because of their potential involvement in xenobiotic
metabolism. DT diaphorase has been identified in rat and
human mammary tissue (36, 37) and carbonyl reductase
in human breast tissue (37). The mammary gland cyto-
solic carbonyl reducing activity with the 9-oxonitrofluo-
renes is partially inhibited by 100 µM dicoumarol and
strongly inhibited by flavones (Table 4). Recently, two
enzymes were purified from rat liver cytosol that cata-
lyzed reduction of quinones, aldehydes, and ketones,
including stereoselective carbonyl reduction (38). One
enzyme utilized NADH and NADPH, while the other
preferred NADPH. Both enzymes were inhibited by
quercitrin; neither was inhibited by pyrazole and only
the former by dicoumarol. The dicoumarol-insensitive,
NADPH-specific enzyme catalyzed reduction of 5R- and
5â-3-keto steroids and resembled 3R-hydroxysteroid
dehydrogenase in catalysis and inhibition. Although
3R-hydroxysteroid dehydrogenase is present in the rat
mammary gland (39), the low level of inhibition by
Discu ssion
This study shows that the cytosolic and microsomal
fractions of the rat mammary gland catalyze NADH- and
NADPH-dependent reductions of the nitrofluorenes to
aminofluorenes and 9-oxonitrofluorenes to the 9-OH-
nitro- and/or 9-OH-aminofluorenes. The cytosolic fraction
contains greater carbonyl and nitro reducing activities
for the C-9-oxidized nitrofluorenes than the microsomal
fraction. The effects of the structure of the nitrofluorenes
are similar for nitroreduction catalyzed by mammary
gland cytosol (Table 1), XO, or diaphorase (19) and for
the nitrofluorene-stimulated reduction of acetylated cyto-
chrome c catalyzed by mammary gland cytosol (20). Thus,
the rate of reduction of nitrofluorene to amine catalyzed
by the cytosol of the mammary gland is greater for the
2,7-diNF than for the 2-NF compounds and is greater
with increasing oxidation state at C-9. However, 9-OH-
2,7-diNF (not available for the previous studies) is
converted to amine faster than is 9-oxo-2,7-diNF, even
though their rates of one-electron nitroreduction cata-
lyzed by XO or cytosol and monitored with acetylated
cytochrome c are similar (data not shown), suggesting
that the one-electron nitroreduction is not rate-limiting
for amine formation. No evidence for reduction of both
nitro groups of 2,7-diNF was found. Several mammalian
enzymes catalyze nitroreductions, including aldehyde
oxidase, cytochrome c reductase, NADPH cytochrome
P450 reductase, XO, and diaphorase (28). The effects of
electron donors and reduction modifiers on product
profiles catalyzed by the mammary gland cytosol suggest
that more than one enzyme contributes to the nitro-
reduction of the nitrofluorenes. The mammary gland
cytosol-catalyzed nitroreduction of 9-oxo-2,7-diNF is
supported with NADH, NADPH, N′-methylnicotinamide,
2-hydroxypyrimidine, xanthine, or hypoxanthine as the
electron donor (Table 2). XO also catalyzes 9-oxo-2,7-diNF
reduction with NADH, NADPH, hypoxanthine, and 2-
hydroxypyrimidine, but not with N′-methylnicotinamide
(our unpublished data). The extents of utilization of
xanthine, hypoxanthine, and 2-hydroxypyrimidine (Table
2) and inhibition by allopurinol (Table 4) (29) as well as
the demonstration herein of XO activity in the mammary
gland cytosol suggest its substantial contribution to
the cytosolic NADH-dependent nitroreduction. Although
aldehyde oxidase, unlike XO, utilizes N′-methylnicotin-
amide, the lack of inhibiton by menadione of the N′-
methylnicotinamide-dependent reduction (29) contra-
indicates its involvement in the cytosol-catalyzed nitro-
reduction of the nitrofluorenes (Table 4). This latter

Reductions of Nitro and C-9-Oxidized Fluorenes
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 799
indomethacin and the ineffectiveness of the 3-keto
androstanes as competitive substrates of 9-oxo-2-AF
(Table 4) (38, 40) suggest little contribution from this
reductase to the NADH-dependent cytosol-catalyzed
carbonyl reduction of 9-oxo-2-AF. Thus, the activity of
the mammary gland catalyzing the NADH-dependent
reduction of the 9-oxo group resembles the liver activity
that is partially sensitive to dicoumarol, is sensitive to
flavone, and utilizes both NADH and NADPH. This
activity appears to be different from the quinone reducing
diaphorase activity purified from rat liver, which is
inhibited by 1 µM dicoumarol, but not by 10 µM rutin
(41). An exception to the cytosolic location of most
carbonyl reducing activities is 11â-hydroxysteroid de-
hydrogenase-1, which is located in the endoplasmic
reticulum and catalyzes carbonyl reduction of steroids
and nonsteroids (42). Like 11â-hydroxysteroid dehydro-
genase, the microsomal carbonyl reductase activity from
mammary gland utilizes NADPH and NADH with a
preference for NADPH (Table 2). Thus, the data indicate
that the mammary gland of the rat has a powerful
capacity to catalyze the carbonyl- and nitroreduction of
9-oxonitrofluorenes via different reductases. The relative
extents of the reductions may depend on tissue O2 levels,
cofactor availability, and the substrate with possible
consequences for carcinogenicity.
Activation of nitrofluorenes to DNA-reactive species
may be determined by both carbonyl- and nitroreduction.
DNA adducts consistent with nitroreduction were iso-
lated from the mammary gland of rats treated with
1-nitropyrene, 4-nitropyrene, or 1,6-dinitropyrene (18,
43-47). However, specific hydroxylations of 1-nitropyrene
(48) increased or decreased the extent of reactivity with
DNA, and nitroreduction of ring-oxidized 1-nitropyrene
was suggested to account for the mixture of adducts found
in the mammary gland of 1-nitropyrene-treated rats (44,
45). Oxidation of nitrofluorenes at C-9 could also alter
the levels and characteristics of the DNA adducts. Oral
administration of 9-OH-2-NF yielded a unique, as yet
unidentified, hepatic DNA adduct (22). In addition,
adducts which appeared similar on HPLC and TLC
separation to those from liver of rats treated with 2-NF,
including the dG-C8-AF, were found, suggesting reduc-
tion of 9-oxo to -CH2- before or after adduct formation.
Although reduction of 9-oxo or 9-OH to -CH2- is not
observed under the conditions of the in vitro incubations
with mammary gland herein, such reduction may occur
in vivo and be revealed by analyses of the metabolites
and/or DNA adducts formed in the rat. Upon intra-
mammary application, 2,7-diNF and 9-oxo-2,7-diNF were
carcinogenic at the site of application (11). Although two,
as yet unidentified, DNA adducts were detected in liver
of male rats treated with 2,7-diNF (49), adducts in
mammary gland from nitrofluorenes have not been
analyzed. Determination of DNA adducts of 2,7-diNF,
9-OH-2,7-diNF, and 9-oxo-2,7-diNF formed in the
mammary gland is needed to elucidate the roles of C-9
oxidation and carbonyl and nitro reductions in activation
of the nitrofluorenes.
synthetic 9-hydroxy-2,7-dinitrofluorene and 9-hydroxy-
2-amino-7-nitrofluorene, and interpretation of spectra.
We also thank Ms. Nancy Raha (University of Minnesota
Cancer Center) for verification of HPLC peak identities
by MS analyses of the compounds described above as
metabolites.
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Ack n ow led gm en t. This study 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. We
thank Dr. Letitia Yao (NMR Laboratory, Department of
Chemistry, University of Minnesota) for NMR spectra of
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