DNA adducts from nitroreduction of 2,7-dinitrofluorene, a mammary gland carcinogen, catalyzed by rat liver or mammary gland cytosol

Article abstract of DOI:10.1021/tx010172p

Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from combustion of fossil fuels. Nitro aromatic compounds undergo nitroreduction to N-hydroxy arylamines that bind to DNA directly or after O-esterification. This study analyzes the DNA binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene (2,7-diNF), a potent mammary carcinogen in the rat. Potential adduct(s) of 2,7-diNF was (were) generated by reduction of 2-nitroso-7-NF with ascorbate/H⁺ in the presence of calf thymus DNA. The major adduct was characterized by HPLC/ESI/MS and ¹H NMR spectrometry as N-(deoxyguanosin-8-yl)-2-amino-7-NF, and a minor one was determined by HPLC/ESI/MS to be a deoxyadenosine adduct of 2-amino-7-NF. Products from enzymatic nitroreduction were monitored by HPLC and DNA adduct formation by ³²P-postlabeling. Xanthine oxidase/hypoxanthine-catalyzed nitroreduction of 2,7-diNF, 2-nitrofluorene (2-NF), and 1-nitropyrene (1-NP) yielded the respective amines to similar extents (30-50%). However, the level of the major adducts (～0.15/10⁶ nucleotides) from 2-NF [N-(deoxyguanosin-8-yl)-2-aminofluorene] and 2,7-diNF [N-(deoxyguanosin-8-yl)-2-amino-7-NF] was ≤2% that from 1-NP. In the presence of acetyl CoA, nitroreduction of 2-NF catalyzed by rat liver cytosol/NADH yielded the same adduct at a level of 2.2/10⁶ nucleotides. Liver or mammary gland cytosol with acetyl CoA yielded mainly N-(deoxyguanosin-8-yl)-2-amino-7-NF from 2,7-diNF at >30 adducts/10⁶ nucleotides, levels comparable to those from 1,6-dinitropyrene and 4- or 49-fold greater than the respective levels without acetyl CoA. Recovery of 2-nitroso-7-NF and 2-amino-7-NF from cytosol-catalyzed reduction of 2,7-diNF indicated nitroreduction and an N-hydroxy arylamine intermediate. Likewise, the presence of 2-acetylamino-7-NF indicated that reactivity with acyltransferase(s) was not prevented by the nitro group at C7. These data are consistent with activation of 2,7-diNF via nitroreduction to the N-hydroxy arylamine and acetyl CoA-dependent O-acetylation of the latter to bind to DNA. Enzymatic nitroreduction of 2,7-diNF was greatly enhanced by 9-oxidation. The nitroreduction of either 9-oxo-2,7-diNF or 9-hydroxy-2,7-diNF catalyzed by liver cytosol with acetyl CoA yielded two adducts (>2/10⁶ nucleotides). Differences in the TLC migration of these adducts, compared to those from 2,7-diNF, and the lack of 2,7-diNF formation in the incubations suggested retention of the C9-oxidized groups. The relative ratios of the amine to amide from nitroreductions of 9-oxo-2,7-diNF and 2,7-diNF catalyzed by liver cytosol suggested that the 9-oxo group decreased reactivity with acyltransferase and, thus, the amount of N-acetoxy arylamine that binds to DNA. The mammary gland tumorigenicity of 2,7-diNF and the extent of its activation by the tumor target tissue shown herein suggest relevance of this environmental pollutant for breast cancer.

Full text of DOI:10.1021/tx010172p

536
Chem. Res. Toxicol. 2002, 15, 536-544
DNA Ad d u cts fr om Nitr or ed u ction of 2,7-Din itr oflu or en e,
a Ma m m a r y Gla n d Ca r cin ogen , Ca ta lyzed by Ra t Liver
or Ma m m a r y Gla n d Cytosol
Clare L. Ritter,†,‡ Sandra J . Culp,§ J ames P. Freeman,§ M. Matilde Marques,|
Frederick A. Beland,§ and Danuta Malejka-Giganti*,†,‡
Veterans Affairs Medical Center, Minneapolis, Minnesota 55417, Department of Laboratory
Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455, National Center
for Toxicological Research, J efferson, Arkansas 72079, and Centro de Qu´ımica Estrutural,
Instituto Superior Te´cnico, 1049-001 Lisboa, Portugal
Received November 9, 2001
Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from
combustion of fossil fuels. Nitro aromatic compounds undergo nitroreduction to N-hydroxy
arylamines that bind to DNA directly or after O-esterification. This study analyzes the DNA
binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene (2,7-diNF), a potent
mammary carcinogen in the rat. Potential adduct(s) of 2,7-diNF was (were) generated by
reduction of 2-nitroso-7-NF with ascorbate/H+ in the presence of calf thymus DNA. The major
1
adduct was characterized by HPLC/ESI/MS and H NMR spectrometry as N-(deoxyguanosin-
8-yl)-2-amino-7-NF, and a minor one was determined by HPLC/ESI/MS to be a deoxyadenosine
adduct of 2-amino-7-NF. Products from enzymatic nitroreduction were monitored by HPLC
and DNA adduct formation by 32P-postlabeling. Xanthine oxidase/hypoxanthine-catalyzed
nitroreduction of 2,7-diNF, 2-nitrofluorene (2-NF), and 1-nitropyrene (1-NP) yielded the
respective amines to similar extents (30-50%). However, the level of the major adducts (∼0.15/
106 nucleotides) from 2-NF [N-(deoxyguanosin-8-yl)-2-aminofluorene] and 2,7-diNF [N-(deoxy-
guanosin-8-yl)-2-amino-7-NF] was e2% that from 1-NP. In the presence of acetyl CoA,
nitroreduction of 2-NF catalyzed by rat liver cytosol/NADH yielded the same adduct at a level
of 2.2/106 nucleotides. Liver or mammary gland cytosol with acetyl CoA yielded mainly
N-(deoxyguanosin-8-yl)-2-amino-7-NF from 2,7-diNF at >30 adducts/106 nucleotides, levels
comparable to those from 1,6-dinitropyrene and 4- or 49-fold greater than the respective levels
without acetyl CoA. Recovery of 2-nitroso-7-NF and 2-amino-7-NF from cytosol-catalyzed
reduction of 2,7-diNF indicated nitroreduction and an N-hydroxy arylamine intermediate.
Likewise, the presence of 2-acetylamino-7-NF indicated that reactivity with acyltransferase(s)
was not prevented by the nitro group at C7. These data are consistent with activation of 2,7-
diNF via nitroreduction to the N-hydroxy arylamine and acetyl CoA-dependent O-acetylation
of the latter to bind to DNA. Enzymatic nitroreduction of 2,7-diNF was greatly enhanced by
9-oxidation. The nitroreduction of either 9-oxo-2,7-diNF or 9-hydroxy-2,7-diNF catalyzed by
liver cytosol with acetyl CoA yielded two adducts (>2/106 nucleotides). Differences in the TLC
migration of these adducts, compared to those from 2,7-diNF, and the lack of 2,7-diNF formation
in the incubations suggested retention of the C9-oxidized groups. The relative ratios of the
amine to amide from nitroreductions of 9-oxo-2,7-diNF and 2,7-diNF catalyzed by liver cytosol
suggested that the 9-oxo group decreased reactivity with acyltransferase and, thus, the amount
of N-acetoxy arylamine that binds to DNA. The mammary gland tumorigenicity of 2,7-diNF
and the extent of its activation by the tumor target tissue shown herein suggest relevance of
this environmental pollutant for breast cancer.
N-hydroxy arylamines that, per se and/or upon esterifi-
cation of the N-hydroxy function, react with DNA to form
covalent adducts (4). Hence, nitroreduction is considered
a relevant pathway in the metabolic activation of nitro-
PAHs to mutagenic and carcinogenic species. The carci-
nogenicity for the mammary gland of female Sprague-
Dawley rats upon systemic and local administration of
In tr od u ction
Nitrated polycyclic aromatic hydrocarbons (nitro-
PAHs)1 are widespread environmental pollutants from
the incomplete combustion of fossil fuels and atmospheric
nitration (1-3). Nitroreduction of nitro-PAHs yields
* To whom correspondence should be addressed at the VA Medical
Center (151), One Veterans Dr., Minneapolis, MN 55417. Phone: 612-
725-2000, ext. 4583. Fax: 612-725-2255. E-Mail: malej001@tc.umn.edu.
† Veterans Affairs Medical Center.
1 Abbreviations: AAF, acetylaminofluorene; AcCoA, acetyl CoA; AF,
aminofluorene; AP, aminopyrene; BisTris, bis(2-hydroxyethyl)amino-
tris(hydroxymethyl)methane; ESI-MS, electrospray ionization mass
spectrometry; IS, internal standard; NF, nitrofluorene; NP, nitro-
pyrene; PAHs, polycyclic aromatic hydrocarbons; XO, xanthine oxidase.
‡ University of Minnesota.
§ National Center for Toxicological Research.
| Instituto Superior Te´cnico.
10.1021/tx010172p CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/08/2002

DNA Adducts from Nitroreduction of Nitrofluorenes
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 537
nitro-PAHs suggests the relevance of exposure to envi-
ronmental nitro-PAHs for human breast cancer (5).
Nitrofluorenes and their C9-oxidized derivatives con-
stitute a significant fraction of the nitro-PAHs present
in the environment (3, 6). The carcinogenicity of nitro-
fluorenes for rat mammary gland has been reported (7-
9). The mammary gland tumorigenicity of 2,7-dinitro-
fluorene (2,7-diNF) administered in diet equaled that of
2-acetylaminofluorene (2-AAF) (8), and intramammary
injections of 2,7-diNF induced a carcinogenic response
similar to or greater than that of 6-nitrochrysene and
4-nitropyrene (9). 9-Oxo-2,7-diNF was less tumorigenic
than 2,7-diNF upon intramammary injection (9), but has
not been tested via systemic exposure.
Our earlier study established that nitroreduction of
2-nitrofluorene (2-NF), 2,7-diNF, and their C9-oxidized
derivatives to amino compounds is catalyzed by xanthine
oxidase (XO) and diaphorase (10). The rates of amine
formation increased with the state of oxidation at C9 (9-
oxo-2,7-diNF > 2,7-diNF; and 9-oxo-2-NF > 9-hydroxy-
2-NF > 2-NF) and the number of nitro groups (2,7-diNF
> 2-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF) of the sub-
strate. Our subsequent study showed that rat mammary
gland cytosol could also catalyze the nitroreduction of the
nitrofluorenes (11). The rate of amine formation catalyzed
by mammary cytosol from 2,7-diNF was greater than that
from 2-NF, and the rate from C9-oxidized nitrofluorenes
was greater than from nitrofluorenes: 9-oxo-2-NF >
9-hydroxy-2-NF > 2-NF; and 9-hydroxy-2,7-diNF .
9-oxo-2,7-diNF > 2,7-diNF. In addition, an active car-
bonyl reductase in the rat mammary gland catalyzed
reduction of the 9-oxo to 9-hydroxy nitrofluorenes. These
studies suggested that the facile nitro and carbonyl
reductions of 2,7-diNF and C9-oxidized nitrofluorenes
would yield appreciable levels of the N-hydroxy aryl-
amine intermediates that are involved in the binding to
DNA.
The DNA adducts formed from exposure of rats to 2-NF
have been investigated (12); however, little is known
about adduct formation from the more carcinogenic 2,7-
diNF or the C9-oxidized nitrofluorenes. The major in vivo
adducts, N-(deoxyguanosin-8-yl)-2-aminofluorene (2-AF)
and N-(deoxyguanosin-8-yl)-2-AAF, from rats treated
with 2-NF, were consistent with nitroreduction to N-hy-
droxy-2-AF followed by activation via O-acetylation. In
one study, hepatic adducts determined by 32P-TLC auto-
radiography indicated a 4.9-fold increase in their levels
after oral compared to ip administration of 2-NF to male
Wistar rats (13). In the same study, administration of
2,7-diNF yielded two hepatic adducts at levels largely
independent of the mode of administration and compa-
rable to those after ip administration of 2-NF. The
adducts from 2,7-diNF were not identified. Another study
from the same laboratory showed that ip administration
of 9-hydroxy-2-NF to male Wistar rats yielded levels of
hepatic adducts similar to those from 2-NF (14). Among
the adducts from 9-hydroxy-2-NF, N-(deoxyguanosin-8-
yl)-2-AF indicated reduction of 9-hydroxyfluorene to
fluorene, while another adduct suggested retention of the
C9-oxidized group.
termine the effect of C9-oxidation on adduct formation.
To accomplish these goals, adducts from the nitroreduc-
tion of 2,7-diNF were generated by reducing 2-nitroso-
7-NF with ascorbate in the presence of DNA, isolated
from the DNA hydrolysate, and purified by HPLC. DNA
adduct formation from 2-NF, 2,7-diNF, 9-hydroxy-2,7-
diNF, and 9-oxo-2,7-diNF, as a consequence of anaerobic
enzymatic nitroreduction, was determined using 32P-
postlabeling assays. Nitroreduction, which was monitored
by HPLC, was catalyzed by XO/hypoxanthine or liver and
mammary gland cytosols in the presence of NADH. To
examine the effect of O-acetylation of the nitroreduction
intermediates, acetyl CoA (AcCoA) was added to the
cytosolic incubations. 1-Nitropyrene (1-NP) and 1,6-
dinitropyrene (1,6-diNP), for which binding to DNA is
unaffected or enhanced, respectively, by AcCoA (15),
served as controls.
Exp er im en ta l P r oced u r es
Rea gen ts. Glass-distilled water was used throughout. Sol-
vents and reagents were of the highest purity available. Calf
thymus DNA (type 1), XO (milk), hypoxanthine, NADH, and
catalase were from Sigma Chemical Co. (St. Louis, MO), and
ascorbic acid was from ACROS Organics (Fair Lawn, NJ ).
Phenol, bis(2-hydroxyethyl)aminotris(hydroxymethyl)methane
(BisTris), and EDTA were from Fisher BioTech (Pittsburgh, PA),
and AcCoA was from Calbiochem (LaJ olla, CA). 2-NF, 2-AF, 2,7-
diNF, 2-amino-7-NF, 2,7-diAF, 1-NP, 1,6-diNP, 1-aminopyrene
(1-AP), 4-phenylphenol, 3-chloroperoxybenzoic acid, and silica
gel were from Aldrich Chemical Co. (Milwaukee, WI), and 9-oxo-
2,7-diNF was from Pfaltz and Bauer (Waterbury, CT). All
fluorenyl compounds and 4-phenylphenol were recrystallized
until determined to be pure by HPLC. 9-Hydroxy-2,7-diNF and
9-hydroxy-2-amino-7-NF (11), 9-oxo-2-amino-7-NF (10), 2-acetyl-
amino-7-NF (16), 1-amino-6-NP, and 1-acetylamino-6-NP (17)
were synthesized as described previously.
In str u m en ta tion . HPLC was conducted with a Hewlett-
Packard system consisting of a 1090 liquid chromatograph, with
a diode array UV/vis detector, a Rheodyne model 7125 injector,
and ChemStation software, or a Waters Associates system
consisting of two model 510 pumps and a model 660 automated
gradient controller, equipped with a Rheodyne model 7125
injector and a Hewlett-Packard 1050 diode array spectrophoto-
metric detector. UV/vis spectra were obtained with a Beckman
DU-70 or DU-40 spectrophotometer. Mass spectra were obtained
on a VG 7070E-HF mass spectrometer or a Finnigan MAT 95
mass spectrometer operated in the chemical ionization mode or
a Finnigan TSQ-7000 LC/MS system operated in the electro-
spray ionization mode. For the latter spectral measurements,
40% acetonitrile containing 0.1% formic acid was used at a flow
rate of 0.2 mL/min. 1H NMR spectra were obtained on a Varian
Inova spectrometer, operated at 500 MHz, or a Varian Unity
300 spectrometer operated at 300 MHz. Chemical shifts are
reported in ppm downfield from tetramethylsilane, and coupling
constants are reported in hertz.
Syn th esis of 9-Oxo-2-a cetyla m in o-7-NF . 9-Oxo-2-amino-
7-NF (8.3 mg; 0.032 mmol) was dissolved in 10 mL of freshly
redistilled pyridine at 95 °C with stirring. After cooling the
mixture to 80 °C, acetic anhydride (12 µL, 0.13 mmol) was
added. The mixture was stirred at 80 °C, and the reaction was
monitored by TLC on Uniplate silica gel plates (Analtech,
Newark, DE) developed in chloroform/ethyl acetate/glacial acetic
acid (20:79:1). Three additional 10-15 µL aliquots of acetic
anhydride were added once every 4 h. The solvent was evapo-
rated under reduced pressure and the residue heated in 10 mL
of 70% ethanol. Insoluble material was removed by centrifuga-
tion and the solution concentrated under reduced pressure and
then chilled on ice. The precipitate was removed by centrifuga-
tion and dried over P2O5 at room temperature. The HPLC
analysis of the product indicated that g98% of the UV/vis
The aims of the present in vitro study were to (i)
characterize DNA adduct formation from the nitroreduc-
tion of 2,7-diNF, (ii) investigate the relationship between
levels of nitroreduction and adduct formation, (iii) de-
termine whether O-acetylation enhanced the binding to
DNA of 2,7-diNF following nitroreduction, and (iv) de-

538 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Ritter et al.
absorbance monitored at 260, 280, 340, and 390 nm was due to
a single peak. 9-Oxo-2-acetylamino-7-NF (5.2 mg, 58% yield, mp
272 °C) had UV/vis λmax 353, 295, and 261 nm [ꢀ(ethanol/H2O,
1:1) ) 9130, 16 400, and 20 500 M-1 cm-1, respectively]; CI-MS
283 (MH+), 241 (MH+-CH2CO), 195 (MH+-CH2CO-NO2);
FAB-high-resolution MS, m/z 283.0734 (observed), 283.0719
(calcd).
septum while mixing to give a final volume of 5 mL. The mixture
was flushed with argon for 15 min and incubated for 18 h. In
experiment 2, conditions were slightly modified. Ascorbic acid
(5.44 µmol in 0.51 mL of water) was added to each of three vials
containing 17 mg of DNA and 2.3 µmol of 2-nitroso-7-NF (added
in 0.68 mL of DMSO) in the buffer to give a final volume of 17
mL. After 4.5 and 17 h, additional aliquots of ascorbic acid (5.44
µmol in 0.51 mL of water) were added. After 24 h, the contents
of the vials were combined for product and DNA adduct
analyses.
Syn th esis of 2-Nitr oso-7-NF . The synthesis of 2-nitroso-7-
NF was based on the procedure of Vance et al. (18) and was
carried out under nitrogen. 2-Amino-7-NF (25 mg, 0.11 mmol)
was dissolved in 25 mL of dichloromethane by stirring at room
temperature. The solution was cooled to 4 °C, and 3-chloro-
peroxybenzoic acid (38 mg, ∼0.17 mmol) was added. The mixture
was stirred for 1 h at 4 °C, and then washed sequentially with
1 volume of 1 N NaOH, H2O (twice), 6 N HCl, and H2O (twice).
The dichloromethane solution was dried over anhydrous sodium
sulfate and evaporated under reduced pressure. The residue was
dissolved in 2 mL of benzene and applied to a column containing
10 g of silica gel (70-230 mesh, 60 Å) in hexane/benzene (1:1).
The column was eluted with hexane/benzene (3:7) and the eluate
monitored by HPLC with online UV/vis detection. Fractions with
In cu ba tion of Nitr o Com p ou n d s w ith DNA d u r in g
En zym a tic Red u ction . The nitro compounds (28 nmol in 40
µL of DMSO) were added to a mixture of 2 mg of DNA, 10 µg of
catalase, 1 mg of cytosolic protein, and 1 µmol of AcCoA (in
selected incubations) in 50 mM sodium pyrophosphate, pH 7.0.
Reduction was initiated by addition through the septum of
NADH (2 µmol in 50 µL of water) followed by flushing with
argon for 15 min. The total volume was 1 mL. After 3 h,
additional NADH (2 µmol in 50 µL) was added to the mixture
followed by flushing with argon and incubation for 18 h.
Similarly, reduction was initiated by the addition of XO (0.54
Sigma unit) to the nitro compounds (28 nmol in 40 µL of DMSO),
2 mg of DNA, 10 µg of catalase, and 3.76 µmol of hypoxanthine
in 0.5 mM sodium citrate buffer, pH 5.8, in a total volume of 1
mL. After 3 h, additional hypoxanthine (3.76 µmol in 100 µL)
and XO (0.0108 Sigma units in 20 µL) were added, and
incubation was continued for 18 h. Controls were incubated with
heat-inactivated enzymes or DMSO in place of the nitro
compounds.
a
single peak containing >99% of the UV/vis absorbance
monitored at 260, 280, 340, and 390 nm were collected and
evaporated to dryness at 40 °C under reduced pressure. 2-Ni-
troso-7-NF (3.4 mg, 13% yield, mp 195 °C dec) had UV/vis λmax
359 and 343 nm [ꢀ(dichloromethane/ethanol/cyclohexane, 0.5:
0.5:99) of 30 900 and 28 400 M-1 cm-1, respectively], which
agreed with the published values (18). CI-high-resolution MS
(MH+) 241.0620 (observed), 241.0613 (calcd); 1H NMR (500
MHz, CD2Cl2): d 8.48 (br s, H8), 8.36 (br d, J ) 8.5 Hz, H6),
8.21 (br d, J ) 8 Hz, H3), 8.16 (d, J ) 8 Hz, H4), 8.07 (d, J )
8.5 Hz, H5), 7.96 (br s, H1), 4.21 (s, H9).
HP LC An a lysis of P r od u cts fr om Red u ction s of Nitr oso
or Nitr o Com p ou n d s. Aliquots from incubation mixtures were
diluted to 1 mL with the incubation buffers, and, in most cases,
5 µL of 4-phenylphenol was added as an internal standard (IS).
The extraction procedure and HPLC analysis were as described
previously (11). Briefly, the mixtures were extracted with Baker
C18 solid-phase extraction columns and eluted with dichloro-
methane containing 5% DMSO. The eluate was dried over
sodium sulfate, concentrated under a gentle stream of argon at
38 °C, and analyzed using the HPLC system with a gradient of
methanol, 2-propanol, and 0.2 M acetic acid, pH 4.2, at: 30-
70% methanol and 70-30% acid within 10 min; 70-74%
methanol, 0-6% 2-propanol, and 30-20% acid within 3 min;
and 74-80% methanol, 6-10% 2-propanol, and 20-10% acid
within 3 min (11). Identification was based on tR (min) and UV/
vis spectra superimposed with those of the standards. Peak
areas were integrated at 260 nm for the IS (tR, 12.8), 9-oxo-2-
amino-7-NF (tR, 11.8), 9-oxo-2-acetylamino-7-NF (tR, 12.4),
1-acetylamino-6-NP (tR, 13.6), and 1-NP (tR, 17.6); 280 nm for
2-AF (tR, 12.3), 2,7-diAF (tR, 4.5), 9-oxo-2,7-diNF (tR,13.9),
1-amino-6-NP (tR, 14.7), and 1-AP (tR, 14.4); 340 nm for 2-NF
(tR, 16.0), 2,7-diNF (tR, 14.7), 2-acetylamino-7-NF (tR, 13.0),
2-nitroso-7-NF (tR, 15.2), and 9-hydroxy-2,7-diNF (tR, 11.9); and
390 nm for 2-amino-7-NF (tR, 12.4), 9-hydroxy-2-amino-7-NF (tR,
9.6), and 1,6-diNP (tR, 16.9). Compounds were quantified from
peak areas relative to those of standards and corrected for
recovery of the IS, unless otherwise noted.
An im a ls a n d P r ep a r a tion of Tissu e Cytosols. Female
Sprague-Dawley rats (Specific Pathogen Free from Harlan
Sprague Dawley, Indianapolis, IN) were housed in a controlled
environment with a 12 h light/12 h dark cycle and main-
tained on Teklad Certified Rodent Diet (Harlan Teklad, Madi-
son, WI) and water ad libitum for 1 week. At 60 days of age,
they were asphyxiated with CO2 and decapitated. Mammary
glands and livers (perfused with saline) were removed, cleaned
of extraneous tissue, rinsed, blotted, weighed, frozen in liquid
nitrogen, and stored at -80 °C. For preparation of cytosolic
fractions, frozen mammary gland was pulverized with a Bess-
man pulverizer. Powdered mammary gland and frozen liver
pieces were homogenized with a Polytron homogenizer type
PT10/35 (Kinematica, Gmbh, Lucerne, Switzerland) in 2 and 4
volumes (w/w), respectively, of 50 mM sodium pyrophosphate
buffer, pH 7.0, containing 1 mM dithiothreitol. The homogenate
was centrifuged at 14000g for 20 min at 4 °C, and the solidified
fat was removed. The cytosolic supernatant fraction was ob-
tained from centrifugation at 100000g for 60 min at 4 °C. Protein
was determined with the Coomassie Plus Protein assay kit
(Pierce, Rockford, IL).
Gen er a l P r oced u r e for Rea ction s of Nitr o or Nitr oso
Com p ou n d s w ith DNA u n d er Red u cin g Con d ition s. All
solvents were flushed with argon before use. Reaction mixtures,
protected from light, were kept under argon in amber vials with
septa seals and were incubated in a shaking water bath at 37
°C. At termination, 0.1-0.25 mL aliquots were removed for
HPLC analyses of reduction products. The remaining material
was mixed with 1 volume of phenol saturated with 50 mM
BisTris-HCl buffer, pH 7.1, containing 1 mM EDTA and stored
at -80 °C until being analyzed for DNA adducts.
In cu ba tion of 2-Nitr oso-7-NF w ith Ascor bic Acid a n d
DNA. The reducing equivalents of the freshly prepared ascorbic
acid solutions were confirmed to be 2 mequiv/mmol (19).
2-Nitroso-7-NF was dissolved in DMSO within 1 min of being
added to the incubation mixture to give final concentrations of
134 µM compound and 4% DMSO. In experiment 1, an aqueous
solution of 3.2 µmol of ascorbic acid was added to 5 mg of calf
thymus DNA in 10 mM sodium citrate buffer, pH 5.0. 2-Nitroso-
7-NF (0.67 µmol in 0.2 mL of DMSO) was added through the
Isola tion a n d Ch a r a cter iza tion of th e DNA Ad d u ct
fr om 2-Nitr oso-7-NF . Reactions with DNA, 2-nitroso-7-NF,
and ascorbate were extracted 3 times with phenol saturated
with 50 mM BisTris-HCl, 1 mM EDTA, pH 7.1, once with
phenol/chloroform/isoamyl alcohol (50:48:2), and once with
chloroform/isoamyl alcohol (48:2). The DNA was precipitated
with ethanol and sodium chloride, washed with 70% ethanol,
redissolved in 5 mM BisTris-HCl, 0.1 mM EDTA, pH 7.1, and
hydrolyzed to nucleosides (20). The adducts were enriched by
extraction with 1-butanol, the 1-butanol was evaporated, and
the residue was dissolved in 20% aqueous methanol. Initial
adduct purification was accomplished by elution from a Sepha-
dex LH20 column with 70% methanol. The adduct was further
purified by HPLC using a C18 µBondapak reversed phase
column that was eluted with a linear gradient of 80-50% of
100 mM ammonium acetate, pH 5.3, and 20-50% of acetonitrile

DNA Adducts from Nitroreduction of Nitrofluorenes
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 539
Ta ble 1. P r od u cts fr om Rea ction of 2-Nitr oso-7-NF w ith
Ascor bic Acid a n d DNAa
incubation
conditions
products
unreacted
ascorbic time 2-nitroso-7-NF 2-amino-7-NF adducts/103
expt acid (mM) (h)
(µM)
(µM)
nucleotides
2.4
1
2
0.64
0.32b
0.31
0.30
22
4.5
8.6
28.2
17
6
1.8
26.0
4.2
a
Incubations containing 134 µM 2-nitroso-7-NF are described
b
under Experimental Procedures. Ascorbate added in three ali-
quots during incubation.
F igu r e 1. HPLC elution profile of adducts from interaction of
DNA with ascorbate-reduced 2-nitroso-7-NF. The inset shows
the UV spectra of the 16.1 (s) and 20.2 (- - -) min peaks.
in 30 min. The flow rate was 2 mL/min, and the peaks were
monitored at 254 and 280 nm. Adduct levels were quantified
from the absorbance at λmax of 393 nm of the UV/vis spectrum
using the ꢀ396 nm(methanol) of 18 900 M-1 cm-1 determined for
2-amino-7-NF.
After enzymatic hydrolysis to nucleosides, the modified
DNA was analyzed by HPLC, which indicated two peaks,
in a 14:1 ratio, that eluted at 16.1 and 20.2 min (Figure
1). The UV absorbance maxima were at 287 and 397 nm
for the major peak, and 293 and 387 nm for the minor
peak, respectively (Figure 1). The peaks were isolated for
further characterization. Electrospray ionization mass
spectrometry (ESI-MS) of the product with tR 16.1 min
in the positive ion mode (Figure 2A) yielded an ion at
m/z 492, which corresponded to a protonated derivative
of 2-amino-7-NF and deoxyguanosine. A prominent frag-
ment observed at m/z 376 was consistent with loss of
deoxyribose from the protonated molecule. Additional
characterization of the structure of the adduct was
obtained by 1H NMR (Figure 3), the proton chemical
shifts for which are shown in Table 2. Assignments are
based upon comparisons to the spectra of deoxyguanosine
and model fluorene compounds, combined with homo-
nuclear decoupling experiments and chemical exchange
of labile protons with D2O. All the protons of the 2-amino-
7-NF and deoxyguanosine moieties were detected, al-
though the H2′ of deoxyguanosine was partially obscured
by the residual DMSO resonance. Six aromatic protons
were present, representing H1, H3, H4, H5, H6, and H8
of 2-amino-7-NF. Also present was a two-proton reso-
nance for H9 (δ 4.06). These data indicated that linkage
occurred through the NH of 2-amino-7-NF, an interpre-
tation supported by the presence of a one-proton NH
resonance at δ 8.92. All of the deoxyribose protons for
deoxyguanosine could be assigned, as well as a two-
proton resonance for the exocylic NH2 at δ 6.69 and a
one-proton imino resonance at δ 11.26. Notably absent
was a resonance that could be assigned to H8 of deoxy-
guanosine (δ ∼8.0). Based upon the combined mass
DNA Isola tion fr om Liver a n d Ma m m a r y Gla n d Cytosol
In cu ba tion s. Cytosolic incubations (1 mL) were extracted
sequentially with an equal volume of phenol saturated with 50
mM BisTris-HCl, 1 mM EDTA, pH 7.1, phenol/chloroform/
isoamyl alcohol (50:48:2), and chloroform/isoamyl alcohol (48:
2). The DNA was precipitated with ethanol and sodium chloride,
collected by centrifugation, and dissolved in 700 µL of 5 mM
BisTris-HCl, 0.1 mM EDTA, pH 7.1. RNase A (30 µg) and RNase
T1 (23 units) were added, and the samples were incubated at
37 °C for 1 h. The samples were then treated with 28 µL of 25%
sodium dodecyl sulfate and 50 µg of proteinase K, incubated
for an additional hour at 37 °C, and sequentially extracted with
phenol, phenol/chloroform/isoamyl alcohol, and chloroform/
isoamyl alcohol. The DNA was precipitated with ethanol and
sodium chloride, collected by centrifugation, dissolved in 5 mM
BisTris-HCl, 0.1 mM EDTA, pH 7.1, and quantified by UV
spectroscopy.
32P -P ostla belin g An a lyses. Approximately 10 µg of DNA
was 32P-postlabeled using 1-butanol enrichment (21). Adducts
were separated by TLC and visualized as described (22). Adducts
were identified and quantified through comparisons to DNA
samples modified with N-(deoxyguanosin-8-yl)-2-AF at 4.1 ad-
ducts/105 nucleotides (for 2-NF adducts) (23), N-(deoxyguanosin-
8-yl)-1-amino-6-NP at 3.8 adducts/107 nucleotides (for 1,6-diNP
adducts) (24), and N-(deoxyguanosin-8-yl)-2-amino-7-NF at 2.4
adducts/103 nucleotides (for 2,7-diNF adducts).
A modified DNA standard was not available for the 1-NP
adduct; thus, N-(deoxyguanosin-8-yl)-1-amino-6-NP was used
for quantitation. Likewise, DNA standards were not available
for 9-hydroxy-2,7-diNF and 9-oxo-2,7-diNF adducts. For these
samples, N-(deoxyguanosin-8-yl)-2-AF was used for quantita-
tion.
Sta tistica l An a lyses. Data were evaluated by ANOVA
followed by a Student-Newman Keuls test. Values are reported
as the mean ( SD. When necessary, the data were transformed
before the analyses to attain equal variance, normal distribu-
tion, or both.
1
spectral and H NMR data, the structure of the adduct
eluting at 16.1 min was assigned as N-(deoxyguanosin-
8-yl)-2-amino-7-NF (Figure 3).
Resu lts
ESI-MS (Figure 2B) of the 20.2 min peak (Figure 1)
gave a protonated molecule at m/z 476 and a BH2 ion at
m/z 360, both being consistent with a deoxyadenosine
adduct of 2-amino-7-NF. The amount of material was
insufficient for further analysis.
DNA Ad d u cts fr om En zym a tic Nitr or ed u ction s of
2-NF a n d 2,7-d iNF . To initiate nitroreduction, the
nitrofluorenes were incubated with XO/hypoxanthine or
cytosol of liver or mammary gland and NADH under
argon. DNA was added to the incubations to bind the
presumed N-hydroxy arylamine intermediates. Since the
reactivity of the latter with DNA may increase via
Ch a r a cter iza tion of th e DNA Ad d u ct fr om Re-
d u ction of 2-Nitr oso-7-NF by Ascor ba te. 2-Nitroso-
7-NF was reduced by ascorbate in the presence of calf
thymus DNA at pH 5.0 using two protocols (Table 1).
Addition of ascorbate in three aliquots, each at a ∼2.4
molar ratio vs a single aliquot at a 4.8 molar ratio,
resulted in 1.3 vs 6.4% residual 2-nitroso-7-NF with
approximately 20% of the substrate being recovered as
2-amino-7-NF in both cases. Multiple vs single additions
of ascorbate yielded 4.2 vs 2.4 adducts/103 nucleotides,
respectively.

540 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Ritter et al.
F igu r e 3. 1H NMR spectrum, recorded in DMSO-d6, of the
major adduct, N-(deoxyguanosin-8-yl)-2-amino-7-NF, obtained
from the reaction of DNA with ascorbate-reduced 2-nitroso-7-
NF.
Ta ble 2. 1H NMR Sp ectr a l P a r a m eter s for
N-(Deoxygu a n osin -8-yl)-2-a m in o-7-NF
δ (ppm)a
assignment
2.04 (1H, m)b
dG, H2′′
dG, H2′
2.49 (1H, m)c
3.78 (2H, s)
3.93 (1H, s)
dG, H5′, H5′′
dG, H4′
4.06 (2H, s)
fluorene, H9
dG, H3′
dG, OH
4.43 (1H, d, J ) 4.8)
5.39 (1H, bs)d
6.11 (1H, bs)d
dG, OH
6.34 (1H, dd, J ) 9.5, 5.9)
dG, H1′
dG, NH2
6.69 (2H, s)d
7.77 (1H, d, J ) 8.4)
7.95 (1H, d, J ) 8.4)
8.00 (1H, d, J ) 8.7)
8.17 (1H, s)
fluorene, H3
fluorene, H4
fluorene, H5
fluorene, H1
fluorene, H6
fluorene, H8
fluorene, NH
dG, NH
8.26 (1H, d, J ) 8.7)
8.38 (1H, s)
F igu r e 2. ESI mass spectra obtained in the positive ion mode
for the major (A) and minor (B) DNA adducts arising from the
reaction with ascorbate-reduced 2-nitroso-7-NF. ACN, aceto-
nitrile.
8.92 (1H, s)d
11.26 (1H, bs)d
a
b
Recorded in DMSO-d6. Abbreviations: dG, deoxyguanosine;
b, broad; d, doublet; m, multiplet; s, singlet. c Partially obscured
by the DMSO-d5 resonance. Exchanged with D2O.
d
acyltransferase-catalyzed O-acylation of the N-hydroxy
function (4), the effect of AcCoA on DNA adduct forma-
tion from 2,7-diNF during nitroreduction catalyzed by
liver or mammary gland cytosol was determined. In
addition, 1-NP and 1,6-diNP were included as positive
controls with the XO and tissue cytosol systems, respec-
tively, since binding to DNA in vitro of 1,6-diNP, but not
1-NP, is enhanced by AcCoA under nitroreductive condi-
tions (15).
Ta ble 3. P r od u cts fr om Red u ction of 2-NF , 2,7-DiNF , a n d
1-NP Ca ta lyzed by Xa n th in e Oxid a se (XO)a
substrate
(28 µM)
DNA adducts/
catalyst
XO
amine (µM)b
106 nucleotidesc
2-NF
14.2 ( 0.7
0.2 ( 0.1
8.4 ( 0.1
NDe
8.8 ( 2.9
0.3 ( 0.4
0.2 ( 0.2
0.03 ( 0.00
0.1 ( 0.05
0.1 ( 0.04
11.1 ( 1.6f
0.1 ( 0.1
d
-
2,7-diNF
1-NP
XO
d
-
XO
XO-catalyzed nitroreduction was confirmed by the
demonstration of the amines 2-AF, 2-amino-7-NF, and
1-AP as the major products detected from 2-NF, 2,7-diNF,
and 1-NP, respectively (Table 3). The extent of amine
formation from all three nitro compounds was similar.
32P-Postlabeling of DNA indicated one major DNA adduct
from the XO-catalyzed reduction of 1-NP (Figure 4A).
This adduct has been previously characterized as N-(de-
oxyguanosin-8-yl)-1-AP (25). Its level was significantly
higher than that of the adducts detected from reduction
of 2-NF and 2,7-diNF with XO (Table 3). The latter
adducts (Figure 4B,C) coeluted with N-(deoxyguanosin-
8-yl)-2-AF and N-(deoxyguanosin-8-yl)-2-amino-7-NF, re-
spectively (not shown).
d
-
a
Incubation conditions and assays are described under Experi-
b
mental Procedures. Values refer to 2-AF from 2-NF, 2-amino-7-
NF from 2,7-diNF, and 1-AP from 1-NP. Values (mean ( SD) are
from duplicate incubations and are corrected for recovery of IS.
c Values (mean ( SD) are derived from 32P-postlabeling analyses
d
of duplicate incubations and represent the major adduct. XO and
hypoxanthine were omitted. e ND, not detected; value assumed to
be 0 (i.e., ebackground). f Significantly greater (p < 0.05) than
adduct level with 2-NF or 2,7-diNF.
Formation of nitroreduction products from nitro-
fluorenes was dependent on active tissue cytosol (Table
4). Detection of arylamides among the nitroreduction
products in the presence of AcCoA indicated enzymatic

DNA Adducts from Nitroreduction of Nitrofluorenes
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 541
Ta ble 4. P r od u cts fr om Red u ction of 2-NF , 2,7-DiNF , 9-Oxo-2,7-d iNF , 9-Hyd r oxy-2,7-d iNF , or 1,6-DiNP Ca ta lyzed by Liver
or Ma m m a r y Gla n d Cytosol in th e P r esen ce of DNA, w ith (+) or w ith ou t (-) AcCoAa
nitroreduction products (µM)b
DNA adducts/
substrate (28 µM)
tissue
AcCoA
aminec
amided
other
NDf
106 nucleotidese
2-NF
liver
+
+
+
+
-
-
+
+
-
-
+
+
+
+
+
+
+
0.25 ( 0.23
0.45 ( 0.05
2.1 ( 1.5
ND
8.7 ( 1.5
ND
2.2 ( 0.1
0.03 ( 0.00
31.4 ( 13.0j,k
0.2 ( 0.2
liverg
liver
ND
2,7-diNF
13.3 ( 10.3h
ND
0.42 ( 0.33i
ND
liverg
liver
2,7-diNF
16.5 ( 9.0
ND
ND
0.23 ( 0.32i
ND
7.1 ( 3.7
liverg
ND
0.1 ( 0.0
2,7-diNF
mammary gland
mammary glandg
mammary gland
mammary glandg
liver
8.2 ( 0.02l
ND
6.9 ( 0.7h
ND
0.60 ( 0.00i
ND
34.6 ( 3.5k
0.05 ( 0.02
0.7 ( 0.02
0.02 ( 0.02
2.2 ( 2.1
2,7-diNF
13.1 ( 7.1
ND
ND
ND
ND
ND
9-hydroxy-2,7-diNF
9-oxo-2,7-diNF
1,6-diNP
0.43 ( 0.04m
ND
0.97 ( 0.01m
ND
3 UKNn
ND
liverg
0.1 ( 0.0
liver
7.7 ( 10.2m
0.08 ( 0.11m
3.0 ( 2.8
ND
1.3 ( 1.7m
ND
2 UKNo
ND
2.3 ( 2.6
liverg
0.1 ( 0.0
liver
0.3 ( 0.2
ND
0.9 ( 1.3
2 UKNp
2 UKNp
2 UKNp
37.0 ( 31.2j
0.2 ( 0.1
liverg
1,6-diNP
mammary gland
5.3 ( 5.7
40.6 ( 5.9
a
b
Incubation conditions and assays are described under Experimental Procedures. Values (means ( SD) are from 2-4 incubations
and are corrected for recovery of IS, except where noted. c Values refer to 2-AF from 2-NF, 2-amino-7-NF from 2,7-diNF, 9-oxo-2-amino-
7-NF from 9-hydroxy-2,7-diNF, the sum of 9-oxo-2-amino-7-NF and 9-hydroxy-2-amino-7-NF from 9-oxo-2,7-diNF, and 1-amino-6-NP
d
from 1,6-diNP. Values refer to 2-AAF from 2-NF, 2-acetylamino-7-NF from 2,7-diNF, 9-oxo-2-acetylamino-7-NF from 9-hydroxy-2,7-
diNF or 9-oxo-2,7-diNF, and 1-acetylamino-6-NP from 1,6-diNP. e Values (mean ( SD) are derived from 32P-postlabeling analyses of 2-6
incubations and represent the major adduct. f ND, not detected; value assumed to be 0 (i.e., ebackground). Inactivated by heating at
g
h
i
100 °C for 5 min. Significantly greater (p < 0.05) than the respective tissue levels of amide from 1,6-diNP. Value refers to 2-nitroso-
j
k
7-NF. Significantly greater (p < 0.05) than the adduct level from 2-NF, 9-hydroxy-2,7-diNF, or 9-oxo-2,7-diNF. Significantly greater (p
l
< 0.05) than the respective adduct level in the absence of AcCoA. Significantly greater (p < 0.05) than the amine level detected in the
m
n
liver. Values are not corrected for recovery of IS (not added). Unknown products at tR 8.02, 9.52, and 10.4 min detected by HPLC.
o Unknown products at tR 8.02 and 10.4 min detected by HPLC. Unknown products at tR 16.5 and 16.7 min detected by HPLC.
p
F igu r e 4. 32P-Postlabeling images of DNA obtained from
incubations with (A) 1-NP, (B) 2-NF, and (C) 2,7-diNF in the
presence of XO/hypoxanthine.
F igu r e 5. 32P-Postlabeling images of DNA obtained from (A)
the reaction with 2-nitroso-7-NF in the presence of ascorbate;
(B) an incubation of 2,7-diNF with mammary gland cytosol in
the absence of AcCoA; and (C) an incubation of 2,7-diNF with
mammary gland cytosol in the presence of AcCoA.
N-acetylation of the amines. In the presence of AcCoA,
the level of amine from 2,7-diNF was significantly greater
with mammary gland than liver cytosol although the
levels of total nitroreduction products were similar. The
level of amide was significantly greater from 2,7-diNF
than from 1,6-diNP with either cytosol. Small amounts
of the intermediate reduction product 2-nitroso-7-NF
were recovered from incubations of 2,7-diNF with cytosols
of liver and, with added AcCoA, of mammary gland.
There was no evidence for reduction of both nitro groups
of 2,7-diNF to yield 2,7-diAF.
fold greater with 2,7-diNF (Table 4). Incubations of 2,7-
diNF with the liver or mammary gland cytosol and
AcCoA resulted in nearly identical levels of N-(deoxy-
guanosin-8-yl)-2-amino-7-NF, and these were similar to
those of N-(deoxyguanosin-8-yl)-1-amino-6-NP from 1,6-
diNP (Table 4). Levels of N-(deoxyguanosin-8-yl)-2-amino-
7-NF were 4.4- and 49-fold greater with liver and
mammary gland, respectively, in the presence than in
the absence of AcCoA.
32P-Postlabeling analyses of DNA from the cytosolic
incubations with 2-NF showed one major adduct with the
chromatographic characteristics of N-(deoxyguanosin-8-
yl)-2-AF (not shown). The chromatographic characteris-
tics of the major adducts from ascorbate-reduced 2-nitroso-
7-NF (Figure 5A) and 2,7-diNF incubated with mammary
gland cytosol (Figure 5B) or mammary gland cytosol in
the presence of AcCoA (Figure 5C) were comparable to
those of the standard N-(deoxyguanosin-8-yl)-2-amino-
7-NF (not shown). Adduct patterns from incubations of
2,7-diNF with liver cytosol were similar to those with
mammary gland. Although the extent of nitroreduction
was similar with 2,7-diNF and 2-NF in the presence of
liver cytosol and AcCoA, DNA adduct formation was 14-
DNA Ad d u ct F or m a tion fr om Nitr or ed u ction of
9-Hyd r oxy-2,7-d iNF a n d 9-Oxo-2,7-d iNF . Liver cytosol
was incubated with AcCoA, NADH, and 9-hydroxy-2,7-
diNF or 9-oxo-2,7-diNF overnight under argon. The
detection of 9-oxo-2-amino-7-NF, 9-hydroxy-2-amino-7-
NF, and 9-oxo-2-acetylamino-7-NF from 9-oxo-2,7-diNF
by HPLC analyses (Table 4) indicated the nitroreduction
followed by N-acetylation of the amine and also carbonyl
reduction. The presence of 9-oxo-2-amino-7-NF and 9-oxo-
2-acetylamino-7-NF among the products from 9-hydroxy-
2,7-diNF indicated nitroreduction and N-acetylation of
the amine either before or after oxidation of the carbonyl.
These data indicate that 9-oxo-2-amino-7-NF is a sub-
strate for the N-acyltransferase of liver cytosol. Three

542 Chem. Res. Toxicol., Vol. 15, No. 4, 2002
Ritter et al.
Sch em e 1. Activa tion of 2,7-DiNF in Vitr oa
F igu r e 6. 32P-Postlabeling images of DNA obtained from the
incubation of (A) 9-hydroxy-2,7-diNF and (B) 9-oxo-2,7-diNF
with liver cytosol in the presence of AcCoA.
unidentified compounds (tR 8.02, 9.52, and 10.4 min) were
recovered from incubations with 9-hydroxy-2,7-diNF, two
of which (tR 8.02 and 10.4 min) were also recovered from
incubations with 9-oxo-2,7-diNF.
32P-Postlabeling analyses of DNA indicated a major
and a minor adduct from 9-hydroxy-2,7-diNF (Figure 6A),
which appeared to comigrate with a minor and the major
adduct, respectively, from 9-oxo-2,7-diNF (Figure 6B).
The migration patterns of these adducts were different
from that of the major adduct from 2,7-diNF (Figures 4C
and 5). The levels of DNA adduct formation from 9-hy-
droxy-2,7-diNF and 9-oxo-2,7-diNF were similar to that
found with 2-NF and significantly lower than those
observed with 2,7-diNF and 1,6-diNP (Table 4). Incuba-
tion of 9-hydroxy-2,7-diNF or 9-oxo-2,7-diNF with XO/
hypoxanthine and DNA (1 mg/mL) under argon for 2 h
yielded ∼0.1 adduct/106 nucleotides (data not shown), a
level similar to that observed with 2-NF and 2,7-diNF
under similar conditions (Table 3).
a
Cytosol is from rat liver or mammary gland. Compounds in
brackets are the presumed intermediates.
the N-hydroxy arylamines of 2-NF and 2,7-diNF were less
reactive with DNA than that of 1-NP.
32P-Postlabeling analyses of DNA from incubations of
2-NF and 2,7-diNF with liver or mammary gland cytosol,
with or without AcCoA, indicated that the major adducts
were the same as with XO, i.e., N-(deoxyguanosin-8-yl)-
2-AF and N-(deoxyguanosin-8-yl)-2-amino-7-NF, respec-
tively. 2-NF incubated with liver cytosol in the presence
of AcCoA yielded 2-AAF, a product of nitroreduction and
acyl transfer (Table 4). The greater levels of DNA adduct
compared to those obtained with XO catalysis are con-
sistent with nitroreduction to N-hydroxy-2-AF, which is
further activated via O-acylation as previously reported
(12). In incubations of 2,7-diNF with liver or mammary
gland cytosol, similar levels of nitroreduction products
were found in the presence or absence of AcCoA. N-Acetyl-
2-amino-7-NF was formed in the presence of AcCoA and
indicated that 2-amino-7-NF and, presumably, N-hy-
droxy-2-amino-7-NF were substrates for acyltransferase
(Scheme 1). Addition of AcCoA increased formation of
N-(deoxyguanosin-8-yl)-2-amino-7-NF from 2,7-diNF cata-
lyzed by the cytosols, consistent with an acyltransferase-
catalyzed activation of an N-hydroxy arylamine via
O-acetylation. Liver or mammary gland cytosol with
added AcCoA yielded similar levels of adducts from 2,7-
diNF. Thus, the relative levels of adducts were not
affected by the apparently greater acyltransferase activ-
ity in liver indicated by the ratios of arylamide to
arylamine (Table 4) and a 2.5-fold greater rate of AcCoA-
dependent binding of N-hydroxy-2-AAF to DNA by liver
than mammary gland cytosol observed in our earlier
study (26). In the present study, the levels of deoxy-
guanosine adducts from 2,7-diNF were comparable to
those from 1,6-diNP despite the greater level of reduction
products from 2,7-diNF. The data indicate that factors
other than the extent of nitroreduction and acetylation
determine the level of adduct formation. The level of
N-(deoxyguanosin-8-yl)-2-amino-7-NF formation in incu-
bations of 2,7-diNF with liver cytosol in the absence of
AcCoA was high relative to that obtained with mammary
gland cytosol. The lack of 2-acetylamino-7-NF among the
Discu ssion
Nitroreduction of nitro-PAHs is considered to be a
principal route to species yielding DNA adducts critical
for tumorigenesis (4, 12). N-Hydroxy arylamines, inter-
mediate products of the nitroreduction, may bind to DNA
directly or be activated via esterification, particularly
O-acetylation. DNA adducts from nitroreduction of 2,7-
diNF, a potent mammary gland carcinogen (8, 9), had
not been characterized previously. In this study, we
demonstrated the enzyme-catalyzed formation of 2-ni-
troso-7-NF and 2-amino-7-NF from 2,7-diNF. These
products indicated that nitroreduction had occurred and
suggested an N-hydroxy-2-amino-7-NF intermediate
(Scheme 1). N-Hydroxy-2-amino-7-NF was then gener-
ated in situ from reduction of 2-nitroso-7-NF by ascorbate
in the presence of calf thymus DNA. Two adducts were
isolated from this reaction, and based on ESI-MS and
1H NMR analyses, the major adduct was identified as
N-(deoxyguanosin-8-yl)-2-amino-7-NF. The second ad-
duct, which was formed in lower amounts, was tenta-
tively identified as a derivative of 2-amino-7-NF and
deoxyadenosine.
Adduct formation from the enzyme-catalyzed nitro-
reductions of 2-NF and 2,7-diNF was compared by 32P-
postlabeling assays using N-(deoxyguanosin-8-yl)-2-AF
and N-(deoxyguanosin-8-yl)-2-amino-7-NF as the respec-
tive standards. The major DNA adducts from nitro-
reduction of 2-NF and 2,7-diNF catalyzed by XO (Table
3) were identical to the above standards and, hence, likely
derived from their respective N-hydroxy arylamines.
Although levels of amine formation from 2-NF, 2,7-diNF,
and also 1-NP were similar, adduct levels from 2-NF and
2,7-diNF were e2% of those from 1-NP, suggesting that

DNA Adducts from Nitroreduction of Nitrofluorenes
Chem. Res. Toxicol., Vol. 15, No. 4, 2002 543
reduction products suggests that formation of the adduct
in this instance was AcCoA-independent and involved
another activity and/or donor. The electron-withdrawing
nitro group at C7 of 2,7-diNF may increase the stability
of its N-hydroxy arylamine (16) and, thus, increase its
availability to other enzymes. Likewise, the 7-nitro group
increases the density of the positive charge on the
nitrogen to “harden” the nitrenium ion (18), resulting in
greater probability of reaction with deoxyguanosine.
Thus, a “hardened” nitrenium ion and/or stabilized
hydroxylamine may contribute to the greater adduct level
from 2,7-diNF than from 2-NF with liver cytosol and
AcCoA.
oxyguanosin-8-yl)-2-amino-7-NF should facilitate bio-
monitoring of populations exposed to 2,7-diNF. Genotoxic
activation of nitro-PAHs may also be catalyzed by human
P450 1B1 (27), which is expressed in breast tissue (28),
and is also inducible in the liver and lung of rats exposed
to diesel exhaust (29). Further studies are needed to
establish the role of nitroreduction and/or P450 oxidation
in activation of 2,7-diNF in vivo.
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. Keith Fagerquist of the Department of Chem-
istry, University of Minnesota, for the mass spectra of
9-oxo-2-acetylamino-7-NF and 2-nitroso-7-NF, and Dr.
Letitia Yao of the same department for the NMR spec-
trum of 2-nitroso-7-NF. We also thank Lonnie R. Blan-
kenship of the National Center for Toxicological Research
and Richard W. Decker of the VA Medical Center for
technical assistance.
In our previous studies, rates of nitroreduction by XO
or mammary gland cytosol were greater for C9-oxidized
derivatives of 2-NF and 2,7-diNF than for the parent
compounds (10, 11). In the present study, liver cytosol
with added AcCoA catalyzed conversion of 9-oxo-2,7-diNF
and 9-hydroxy-2,7-diNF to the respective amines and to
9-oxo-2-acetylamino-7-NF as well as two or three un-
identified products (Table 4). The formation of 9-oxo-2-
acetylamino-7-NF indicates that the 9-oxo-2-amino-7-NF
is a substrate for acyltransferase. However, the ratio of
amide to amine from the 9-oxidized 2,7-diNF compounds
is low relative to that from 2,7-diNF, suggesting lower
activities of the 9-oxidized aminonitrofluorenes with
acyltransferase of liver cytosol. Thus, 9-oxidation of 2,7-
diNF may decrease formation of the presumed N-acetoxy
amine intermediate and lead to the lower levels of adduct
from 9-oxo-2,7-diNF and 9-hydroxy-2,7-diNF relative to
those from 2,7-diNF. A decreased affinity of transacylase
for the 9-oxidized compounds may contribute to the lower
carcinogenicity of 9-oxo-2,7-diNF, compared to 2,7-diNF,
upon intramammary injection observed in our previous
study (9). 9-Hydroxy-2,7-diNF and 9-oxo-2,7-diNF, which
are interconverted by carbonyl reductase and nonenzymic
oxidation (11), yielded the same two adducts in inverse
proportions, which may be adducts retaining the 9-oxo
and 9-hydroxy groups. In this study, N-(deoxyguanosin-
8-yl)-2-amino-7-NF was not among the adducts from
9-oxo-2,7-diNF or 9-hydroxy-2,7-diNF, consistent with the
lack of 2,7-diNF among the metabolites from cytosol-
catalyzed reduction of the 9-oxidized 2,7-diNF com-
pounds. Oral administration of 9-hydroxy-2-NF to rats
yielded an adduct that was similar on TLC and HPLC
to N-(deoxyguanosin-8-yl)-2-AF (14), suggesting reduction
of the C9-oxidized group to a methylene group. The
relatively low level of adducts from 9-oxo-2,7-diNF we
observed in the present study suggests that its tumori-
genicity upon intrammammary injection arose from a low
level of in vivo conversion to 2,7-diNF or from adducts
unique to the C9-oxidized compounds. Examination of
metabolites and DNA adducts formed in vivo from the
C9-oxidized dinitrofluorenes is needed to determine
whether reduction of the C9-oxidized group contributes
to the biological activity.
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