Comparison of hprt and lacl mutant frequency with DNA adduct formation in N-Hydroxy-2-acetylaminofluorene-treated big blue rats

Article abstract of DOI:10.1002/em.1028

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF)is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacl mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 × 10^-6 compared with 3.2 × 10^-6 in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacl mutant frequencies in the liver of 97.6, 155.6, and 406.8 × 10^-6, respectively, compared with a control frequency of 25.7 × 10^-6; rats given four doses had lacl mutant frequencies in spleen lymphocytes of 55.8 × 10^-6 compared with a control frequency of 20.4 × 10^-6. Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by ³²P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/μg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Published 2001 Wiley-Liss, Inc.

Full text of DOI:10.1002/em.1028

Environmental and Molecular Mutagenesis 37:195–202 (2001)
Comparison of hprt and lacI Mutant Frequency With
DNA Adduct Formation in N-Hydroxy-2-
acetylaminofluorene–Treated Big Blueா Rats
Tao Chen,1* Roberta A. Mittelstaedt,1 Anane Aidoo,1 L. Patrice Hamilton,2
Frederick A. Beland,2 Daniel A. Casciano,3 and Robert H. Heflich1
1Division of Genetic and Reproductive Toxicology, National Center for Toxicological
Research, Jefferson, Arkansas
2Division of Biochemical Toxicology, National Center for Toxicological Research,
Jefferson, Arkansas
3Office of the Director, National Center for Toxicological Research,
Jefferson, Arkansas
N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is doses had lacI mutant frequencies in spleen lym-
the proximate carcinogenic metabolite of the pow- phocytes of 55.8 ϫ 10Ϫ6 compared with a control
erful rat liver carcinogen 2-acetylaminofluorene. In frequency of 20.4 ϫ 10Ϫ6. Additional rats were
this study, transgenic Big Blueா rats were used to evaluated for DNA adduct formation in the liver,
examine the relationship between in vivo mutage- spleen lymphocytes, and bone marrow by 32P-
nicity and DNA adduct formation by N-OH-AAF in postlabeling. Adduct analysis was conducted 1
the target liver compared with that in nontarget day after one, two, and four treatments with N-OH-
tissues. Male rats were given one, two, or four AAF, 5 days after one treatment, and 9 days after
doses of 25 mg N-OH-AAF/kg body weight by i.p. two treatments. N-(Deoxyguanosin-8-yl)-2-aminoflu-
injection at 4-day intervals, and groups of treated orene was the major DNA adduct identified in all
and control rats were euthanized up to 10 weeks the tissues examined. Adduct concentrations in-
after beginning the dosing. Mutant frequencies creased with total dose to maximum values in sam-
were measured in the spleen lymphocyte hprt ples taken 1 day after two doses, and remained
gene, and lacI mutant frequencies were deter- essentially the same after four doses. In samples
mined in the liver and spleen lymphocytes. At 6 taken after four doses, adduct levels were 103, 28,
weeks after beginning the dosing, the hprt mutant and 7 fmol/␮g of DNA in liver, spleen lympho-
frequency in spleen lymphocytes from the four-dose cytes, and bone marrow, respectively. The results
group was 16.5 ϫ 10Ϫ6 compared with 3.2 ϫ indicate that the extent of both DNA adduct forma-
10Ϫ6 in control animals. Also at 6 weeks, rats tion and mutant induction correlates with the organ
given one, two, or four doses of N-OH-AAF had specificity for N-OH-AAF carcinogenesis in the rat.
lacI mutant frequencies in the liver of 97.6, 155.6, Environ. Mol. Mutagen. 37:195–202, 2001.
and 406.8 ϫ 10Ϫ6, respectively, compared with a
control frequency of 25.7 ϫ 10Ϫ6; rats given four
Published 2001 Wiley-Liss, Inc.†
Key words: 32P-postlabeling; liver; spleen lymphocytes; bone marrow; transgene
INTRODUCTION
the male rat liver, which contains high concentrations of
enzymes (CYP1A2, SULT1C1, N,O-acyltransferase) im-
portant to the activation of 2-AAF to DNA-reactive elect-
rophiles (N-sulfooxy 2-AAF and N-acetoxy-2-aminoflu-
orene). This metabolism and DNA adduct formation are
strongly related to the induction of tumors [DeBaun et al.,
1968, 1970a,b; Miller et al., 1968; Miller, 1970; Heflich and
Neft, 1994].
2-Acetylaminofluorene (2-AAF) is a powerful carcinogen
in several animal species, with hepatocarcinogenesis in the
male rat being a particularly potent tumorigenic response.
Because of their use as models for studying the roles of
metabolic activation and DNA damage in chemical carci-
nogenesis and mutagenesis, 2-AAF and its metabolites are
among the most intensively studied of all chemical carcin-
ogens [Heflich and Neft, 1994]. The N-hydroxylation of
2-AAF to the proximate carcinogen N-hydroxy-2-acetylamin-
*Correspondence to: Tao Chen, National Center for Toxicological Re-
ofluorene (N-OH-AAF) was the first direct demonstration of search, HFT-120, Division of Genetic and Reproductive Toxicology, 3900
NCTR Road, Jefferson, AR 72079. E-mail: TChen@NCTR.FDA.GOV
the metabolism of a parent chemical carcinogen to a more
active form [Cramer et al., 1960; Miller et al., 1961]. Received 28 August 2000; provisionally accepted 17 October 2000; and in
final form 5 January 2001
Studies of 2-AAF metabolism and subsequent DNA adduct
formation indicate that extensive adduct formation occurs in
Published 2001 Wiley-Liss, Inc. †This article is a US Gov-
ernment work and, as such, is in the public domain in the
United States of America.

196
Chen et al.
TABLE I. Treatment and Sacrifice Protocol for Measuring
DNA Adducts in Male Big Blue Rats Treated
With N-OH-AAF
The carcinogenicity of chemicals like 2-AAF is thought
to depend on their ability to damage DNA and induce
mutations in critical oncogenes and tumor-suppressor genes
[Knudson, 1986, 1993]. The mutagenicity of 2-AAF and its
metabolites has been studied extensively in a variety of in
vitro systems [Heflich and Neft, 1994] and in the in vivo rat
lymphocyte hprt assay [Beland et al., 1999; Mittelstaedt et
al., 1999]. Investigations of 2-AAF mutagenesis in target
tissues for carcinogenesis, however, were limited until re-
cently to the analysis of tumor oncogenes. The development
of animal models having transgenic shuttle vectors has
provided a valuable tool for analyzing in vivo somatic cell
mutation in any tissue from which sufficient DNA can be
recovered. These assays permit the direct comparison of
cancer incidence with the frequency and types of mutations
induced by an agent [Gossen et al., 1989; Kohler et al.,
1991; Provost et al., 1993].
Treatment
groupa
Day 1 Day 2 Day 5 Day 6 Day 9 Day 13 Day 14
1
2
3
4
5
6
Kb
K
K
K
Tb
T
T
T
T
K
K
T
T
T
K
K
T
T
aEach group consisted of four Big Blue rats with the exception of group 1,
which included three transgenic rats, one each killed on days 2, 6, and 14
as controls.
bK, time killed; T, time of treatment.
Animal Treatments
Several studies have reported the mutagenicity of 2-AAF
or N-OH-AAF in the liver of transgenic mice [Gunz et al.,
1993; Shephard et al., 1993; Brooks et al., 1995; Frijhoff et
al., 1998; Ross and Leavitt, 1998]; however, no investiga-
tion to date has taken advantage of transgenic rats. In the
present study, we used the Big Blue௡ rat [Dycaico et al.,
1994, 1996] to evaluate the mutagenicity and DNA adduct
formation by N-OH-AAF in the liver and nontarget tissues
of rats. Mutations were measured in the transgenic lacI gene
from the liver and spleen lymphocytes and in the hprt gene
of spleen lymphocytes. We sought to address the following
issues: (1) the tissue specificity of N-OH-AAF mutagenicity
in the rat; (2) the relationship between DNA adduct forma-
tion and the mutant frequency induced by N-OH-AAF in the
rat; and (3) the mutant frequency induced by N-OH-AAF in
the lacI transgene in comparison with that in the endoge-
nous hprt gene.
Male Fischer 344 rats were obtained from our breeding colony. Male Big
Blue௡ rats, homozygous for the lacI transgene with 30–40 copies per rat
genome, were purchased from Taconic Farms (Germantown, NY). We
followed the recommendations set forth by our Institutional Animal Care
and Use Committee for the handling, maintenance, treatment, and termi-
nation of the animals.
Fischer 344 rats were used to establish the doses of N-OH-AAF and the
approximate expression time for N-OH-AAF mutation induction used in
experiments conducted with transgenic animals. Four groups of 6-week-old
male rats received 0, 12.5, 25, or 50 mg N-OH-AAF/kg body weight (b.w.).
The N-OH-AAF was dissolved in propane-1,2-diol and administered in
single intraperitoneal (i.p.) injections of 0.8 ml/kg b.w. At 2, 8, 14, and 20
weeks after the exposure, three rats per group were killed by CO2 anes-
thesia, and the hprt mutant assay was performed on spleen lymphocytes
isolated from each animal.
For experiments with Big Blue௡ rats, 6-week-old male animals were
given either one, two, or four doses of the propane-1,2-diol vehicle or one,
two, or four doses of 25 mg N-OH-AAF/kg b.w. delivered in propane-1,2-
diol. The doses were administered i.p. in a volume of 0.8 ml/kg and given
at 4-day intervals, with the first treatment on day 1 and the fourth on day
13. Five rats from each treatment group were killed by CO2 anesthesia at
2, 4, 6, 8, and 10 weeks after beginning the treatment (2 weeks was day 14
of the treatment schedule). The liver and spleen lymphocytes were isolated
from each animal and used for the mutant frequency assays.
MATERIALS AND METHODS
Separate groups of treated and control Big Blue௡ rats were used to
measure DNA adducts in the liver, spleen lymphocytes, and bone marrow.
As outlined in Table I, the N-OH-AAF treatment scheme was identical to
that used for the Big Blue௡ mutagenicity assays. The termination schedule
was more complex, however: rats were euthanized 1 day after the first,
second, and fourth doses; 4 days after one treatment; and 9 days after two
treatments.
Preparation of N-OH-AAF
N-Hydroxy-2-aminofluorene (N-OH-AF) was prepared in our laboratory
from 2-nitrofluorene (Aldrich, Milwaukee, WI) by the method of Lotlikar
et al. [1965]. The N-OH-AF (11.3 g) was suspended in 400 ml of argon-
purged ethyl acetate and reacted with 1.1 equivalents (6.0 ml) of acetic
anhydride in the presence of 600 ␮l of triethylamine. After stirring for 30
min at room temperature, 200 ml of 7.5 M ammonium hydroxide was
added and the mixture was stirred for an additional 30 min. The ethyl
acetate was evaporated under reduced pressure, and the residue was sus-
pended in 400 ml of diethyl ether, which was extracted three times with 1
N potassium hydroxide. The potassium hydroxide solution was washed
repeatedly with diethyl ether until the organic layer was clear. The aqueous
layer was then acidified with concentrated HCl and the precipitated N-OH-
AAF was collected by filtration, washed with water, and dried under
Lymphocyte hprt Assay
The lymphocyte hprt mutant assay was performed as described previ-
ously [Chen et al., 1998, 1999]. Briefly, spleens were removed aseptically
from the rats, teased apart with 25- to 26-gauge needles, and washed with
cold, supplemented phosphate-buffered saline to release the cells. The
lymphocytes were isolated by Accu-Paque™ (Accurate Chemical, West-
bury, NY) density-gradient centrifugation, as described previously [Aidoo
et al., 1991, 1993]. For each sample, two sets of lymphocyte cultures were
reduced pressure. The N-OH-AAF gave a single spot by thin-layer chro- established in three 96-well microtiter plates. One set of plates was used for
matography, had a melting point of 149–151°C (literature 145–147°C
[Lotliker et al., 1965]), and the appropriate UV spectrum.
determining cloning efficiency (CE) under nonselective conditions, and the
other was the selection set with culture medium supplemented with 2.5 ␮g

N-OH-AAF Mutation in Big Blueா Rats
197
6-thioguanine/ml. Both sets of plates were incubated in a humidified
atmosphere of 5% CO2 in air. After 12–14 days, the plates were scored for
clone formation by microscopic examination. The CE for each set of
cultures and the frequency of 6-thioguanine-resistant (TGr) lymphocytes
were calculated as previously reported [Aidoo et al., 1991].
TABLE II. Mutant Frequencies in the hprt Gene of Spleen
Lymphocytes From Male Fischer 344 Rats Treated With
Single Intraperitoneal Doses of N-OH-AAF
Time
TGr mutant
(weeks)a
Dose (mg/kg)
CE (%)b
frequency (ϫ10Ϫ6 b
)
2
0
4.9 Ϯ 0.2
5.5 Ϯ 0.2
6.5 Ϯ 0.4
6.5 Ϯ 0.5
10.5 Ϯ 1.7
9.9 Ϯ 1.6
11.2 Ϯ 0.5
10.4 Ϯ 0.9
11.6 Ϯ 0.3
14.0 Ϯ 0.9
15.3 Ϯ 1.7
10.4 Ϯ 0.9
7.7 Ϯ 1.2
5.6 Ϯ 0.7
NDc
0.5 Ϯ 0.5
2.1 Ϯ 0.5
2.8 Ϯ 1.2
3.2 Ϯ 1.2
0.7 Ϯ 0.4
6.0 Ϯ 0.9**
5.4 Ϯ 0.4*
4.7 Ϯ 1.5*
1.2 Ϯ 0.4
2.5 Ϯ 0.8
2.5 Ϯ 0.4
5.0 Ϯ 2.7
3.4 Ϯ 1.2
2.3 Ϯ 1.2
ND
lacI Mutant Assays
12.5
25.0
50.0
0
12.5
25.0
50.0
0
12.5
25.0
50.0
0
12.5
25.0
50.0
High-molecular-weight genomic DNA was extracted from the lympho-
cytes and livers using the RecoverEase™ DNA Isolation Kit (Stratagene,
La Jolla, CA) and stored at 4°C until DNA packaging was performed. The
packaging of the ␭ phage, plating for lacI mutant plaques, and determina-
tion of mutant frequency were carried out following recommended Big
Blue௡ protocols [Rogers et al., 1995 and the 1992 manual for the Big
Blue௡ assay system (Stratagene)]. Briefly, the ␭ shuttle vectors containing
the target lacI gene were packaged using Transpack௡ Packaging Extract
(Stratagene). The resulting phages were preadsorbed to SCS-8 E. coli cells
and plated on 25 ϫ 25-cm2 NZY agar trays. The plates were incubated
overnight at 37°C and counted to determine the number of mutant (blue)
and nonmutant (clear) plaques. The lacI mutant frequency was calculated
by dividing the number of mutant plaque-forming units (PFUs) by the total
number of PFUs analyzed.
8
14
20
6.3 Ϯ 0.6
3.0 Ϯ 0.6
aWeeks after the exposure to N-OH-AAF.
bData are means Ϯ SEM (n ϭ 3).
cND, not determined.
DNA Adduct Analysis
*P Ͻ 0.05 vs. control.
**P Ͻ 0.01 vs. control.
Hepatic nuclei were isolated by the method of Basler et al. [1981], and
DNA was prepared from liver nuclei, spleen lymphocytes, and bone
marrow by enzymatic digestion, solvent extractions, and precipitations
using slight modifications of the method described in Beland et al. [1984].
DNA adducts were analyzed by 32P-postlabeling using an n-butanol en-
hancement procedure [Gupta, 1985; Poirier et al., 1991], with the adducts
being resolved by thin-layer chromatography, as described in Beland et al.
[1999]. The adducts were identified through comparison to a synthetic
standard, N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), that was
prepared by reacting N-OH-AF with DNA [Beland et al., 1980]. Adduct
levels were quantified through comparison to a standard modified at a level
of 66 fmol/␮g of DNA and obtained from treating a mouse with [ring-
3H]2-AAF [Poirier et al., 1991].
hprt and lacI Mutant Frequencies in Big Blueா Rats
Considering the weak induction of hprt mutants by single
doses of N-OH-AAF and the time for mutation induction,
male Big Blue௡ rats were treated with one, two, or four
doses of 25 mg N-OH-AAF/kg b.w. at 4-day intervals, and
the rats were euthanized for mutant assays at 2, 4, 6, 8, and
10 weeks after the initial treatment. Mutant frequencies
were measured in the spleen lymphocyte hprt gene from
these rats (Table III). There were significant dose-related
increases in hprt lymphocyte mutant frequency at all assay
times (P Ͻ 0.05 to P Ͻ 0.001). Also, the hprt lymphocyte
mutant frequencies in rats treated with four doses of N-OH-
AAF were significantly higher than the concurrent control
frequencies 4 and 6 weeks after beginning the dosing (Table
III).
Statistical Analysis
The significance of DNA adduct levels and mutant frequencies was
tested by analysis of variance, with comparisons between pairs of groups
made with Bonferroni and Student–Newman–Keuls tests. Because the
magnitude of the variance of adduct values increased with the mean of the
adduct levels, these data were transformed to either their square roots (liver
and bone marrow) or natural logarithms (spleen lymphocytes) before
conducting the analyses. Linear regression analysis was used to test for
dose response.
Mutant frequencies were measured in the liver and spleen
lacI gene of rats killed 6 weeks after beginning the treat-
ment, which was within the optimum expression period for
hprt mutant induction. Linear regression analysis indicated
a significant dose response in liver lacI mutant frequency
among the different dose groups (P Ͻ 0.001; Table IV).
Rats given one, two, or four doses of N-OH-AAF had lacI
RESULTS
hprt Lymphocyte Mutant Frequency Assays in Fischer
344 Rats
Male Fischer 344 rats were injected with single doses of mutant frequencies of 97.6, 155.6, and 406.8 ϫ 10Ϫ6
,
N-OH-AAF, and the lymphocyte hprt assay was performed respectively, with the four-dose response being a 16-fold
on these animals 2, 8, 14, and 20 weeks later (Table II). The increase over the control frequency of 25.7 ϫ 10Ϫ6. In
only significant differences between the mutant frequencies contrast, the lacI mutant frequency in spleen lymphocytes
in control and treated animals were found at week 8. None from rats given four doses of N-OH-AAF was only 55.8 ϫ
of the data sets from the different sampling times showed a 10Ϫ6 compared with a frequency of 20.4 ϫ 10Ϫ6 in control
significant dose response (P Ͼ 0.05).
rats (Table V), a 2.7-fold increase. Although the mutant

198
Chen et al.
TABLE III. Mutant Frequencies in the hprt Gene of Spleen
Lymphocytes From Big Blue௡ Rats Treated With One or
More Doses of 25 mg N-OH-AAF/kg b.w.
TABLE IV. Mutant Frequencies in the Liver lacI Gene from
N-OH-AAF–Treated Big Blue௡ Rats 6 Weeks After
Beginning Treatment
Time
Number of Total dose
TGr mutant
Total dose
(mg/kg)a
Animal
code
Total
plaques
Mutant
plaques
Mutant frequency
(ϫ10Ϫ6 b
(weeks)a
doses
(mg/kg)
CE (%)b
frequency (ϫ10Ϫ6 b
)
)
2
0
1
2
4
0
1
2
4
0
1
2
4
0
1
2
4
0
1
2
4
0
25
50
100
0
25
50
100
0
25
50
100
0
25
50
100
0
4.6 Ϯ 0.4
5.5 Ϯ 0.4
5.0 Ϯ 0.6
5.0 Ϯ 0.4
5.3 Ϯ 0.5
5.5 Ϯ 0.2
6.2 Ϯ 0.9
7.3 Ϯ 0.8
6.1 Ϯ 0.9
5.9 Ϯ 0.7
5.2 Ϯ 0.6
6.4 Ϯ 0.7
7.8 Ϯ 0.9
9.7 Ϯ 1.6
9.4 Ϯ 1.9
9.1 Ϯ 2.1
9.3 Ϯ 1.2
6.2 Ϯ 0.4
5.9 Ϯ 1.1
5.2 Ϯ 1.1
1.2 Ϯ 0.5
3.6 Ϯ 0.9
6.1 Ϯ 1.7
9.5 Ϯ 3.5
2.8 Ϯ 0.6
4.9 Ϯ 1.0
11.0 Ϯ 2.3
15.1 Ϯ 3.6**
3.2 Ϯ 0.7
8.6 Ϯ 2.1*
6.8 Ϯ 1.4
16.5 Ϯ 1.7***
3.1 Ϯ 0.9
6.6 Ϯ 0.9
6.8 Ϯ 0.9
9.8 Ϯ 2.1
3.3 Ϯ 0.5
14.1 Ϯ 4.2
13.7 Ϯ 3.5
12.4 Ϯ 5.4
0
BFC6W1
BFC6W2
BFC6W3
BFC6W4
BFC6W5
Average
247,980
249,740
277,854
262,904
252,734
225,842
4
8
3
12
6
6.6
16.1
32.0
10.8
45.6
23.7
4
25.7 Ϯ 6.1
25
BFL6W1
BFL6W2
BFL6W3
BFL6W4
BFL6W5
Average
161,000
242,580
157,133
142,093
234,260
187,413
18
23
9
7
41
19.6
111.8
94.8
57.3
49.3
175.0
6
97.6 Ϯ 22.5
8
50
BFM6W1
BFM6W2
BFM6W3
BFM6W4
BFM6W5
Average
160,360
247,820
188,296
142,720
247,558
197,351
35
33
16
28
36
29.6
218.3
133.1
85.0
196.2
145.4
10
25
50
100
155.6 Ϯ 23.7**
100
BFH6W1
BFH6W2
BFH6W3
BFH6W4
BFH6W5
Average
158,660
115,788
151,373
140,158
221,413
157,478
48
51
62
64
94
63.8
302.5
440.5
409.6
456.6
424.5
aWeeks after beginning N-OH-AAF treatment.
bData are means Ϯ SEM (n ϭ 5).
*P Ͻ 0.05 vs. control.
**P Ͻ 0.01 vs. control.
***P Ͻ 0.001 vs. control. Data sets from all time points have significant
dose responses (P Ͻ 0.05–P Ͻ 0.001).
406.8 Ϯ 27.2***
aDelivered as 0–4 doses of 25 mg N-OH-AAF/kg b.w. at 4-day intervals.
bData are means Ϯ SEM (n ϭ 5).
**P Ͻ 0.01 vs. control.
frequencies from the livers and spleen lymphocytes of con-
trol animals were not significantly different, the difference
in mutant frequencies for the same tissues from treated
animals was highly significant (P Ͻ 0.001). A lacI mutant
assay performed with lymphocytes taken from rats at week
10 after starting the four-dose treatment produced a mutant
***P Ͻ 0.001 vs. control. Dose response, calculated by linear regression
analysis, significant at P Ͻ 0.001.
TABLE V. Mutant Frequencies in the Spleen Lymphocyte
lacI Gene from Big Blue௡ Rats 6 Weeks After Beginning
Treatment With Four Doses of N-OH-AAF
frequency of 58.0 ϫ 10Ϫ6, indicating that lacI mutants were Total dose
Animal
code
Total
plaques
Mutant
plaques
Mutant frequency
(ϫ10Ϫ6 a
(mg/kg)
)
fully expressed in the spleen by the 6-week time point.
Because of the relatively weak mutant induction by the
0
BFC6W1
BFC6W2
BFC6W3
BFC6W4
BFC6W5
Average
310,000
348,000
310,000
303,000
292,000
312,600
6
7
5
11
3
6.4
19.4
20.1
16.1
36.3
10.3
four-dose treatment, spleen lacI mutant frequencies were
not determined for rats given one or two doses of N-OH-
AAF.
20.4 Ϯ 4.3
N-OH-AAF-DNA Adducts in Big Blueா Rats
100
BFH6W1
BFH6W2
BFH6W3
BFH6W4
BFH6W5
Average
359,000
329,000
320,000
302,000
303,000
322,600
11
35
17
12
15
18
30.6
106.4
53.1
39.7
49.5
Male Big Blue௡ rats were treated either one, two, or four
times with 25 mg N-OH-AAF/kg b.w., as for the mutage-
nicity assays, and euthanized according to the schedule
outlined in Table I. DNA adduct levels were determined by
55.8 Ϯ 13.2*
32P-postlabeling in liver, spleen lymphocytes, and bone
marrow from samples obtained 1 day after one, two, and
four doses, 5 days after one dose, and 9 days after two
aData are means Ϯ SEM (n ϭ 5).
*P ϭ 0.034 vs. 0 mg/kg data.
doses. In each tissue from N-OH-AAF–treated rats, a major The DNA adduct levels were highest in liver (103 fmol/␮g
DNA adduct was detected that had chromatographic prop- of DNA after four doses), followed by spleen lymphocytes
erties identical to those of a dG-C8-AF standard (Fig. 1). (28 fmol/␮g of DNA) and then bone marrow (7 fmol/␮g of

N-OH-AAF Mutation in Big Blueா Rats
199
Fig. 1. Phosphorimages from 32P-postlabeling analyses of a: DNA stan- marrow DNA). The 32P-postlabeling and elution conditions are outlined in
dard containing dG-C8-AF; b: liver DNA from a control male Big Blue௡ Materials and Methods. The location of the origin of chromatography is
rat, and c–e: DNA from male Big Blue௡ rats treated intraperitoneally with indicated in (a).
N-OH-AAF (c: liver DNA; d: spleen lymphocyte DNA; and e: bone
DNA) (Fig. 2). The levels of adducts measured 1 day after a maximum TGr mutant frequency of 9 ϫ 10Ϫ6 compared
completing treatments with two or four doses did not differ with 1 ϫ 10Ϫ6 in control rats.
from one another. These adduct concentrations, however,
The relatively weak responses produced by single doses
were significantly greater than the levels obtained after a of N-OH-AAF in the lymphocyte hprt assay prompted us to
single dose, which did not differ from the background use multiple doses of the carcinogen in assays conducted
radioactivity observed with the control rats. The adduct with transgenic rats. Multiple doses did result in higher
levels in liver and bone marrow decreased significantly mutant frequencies than achieved with single doses (com-
when assessed 9 days after the second dose. This trend did pare hprt mutant frequencies in Tables II and III, and the
not occur in spleen lymphocytes (Fig. 2).
liver lacI responses produced by one and four doses in
Table IV). DNA adduct analysis, however, indicated that
doses 3 and 4 did not produce increases in DNA binding
relative to the levels of binding achieved by two doses of 25
DISCUSSION
Treatment of Big Blue௡ rats with the hepatocarcinogen mg N-OH-AAF/kg b.w. (Fig. 2). This was true for measure-
N-OH-AAF resulted in the induction of mutations and/or ments made with all three tissues analyzed: liver, spleen,
DNA adducts in spleen lymphocytes and bone marrow, as and bone marrow cells. The leveling out of binding values
well as the liver. Although our data confirm the utility of with repeated treatment is typical for animals being fed
using lymphoid cells as surrogates for detecting the geno- 2-AAF or given multiple doses of N-OH-AAF [e.g., Beland
toxicity of tissue-specific carcinogens, the responses were et al., 1982; Poirier et al., 1991] and may be a result of the
clearly strongest in the liver. DNA adduct concentrations processes acting to reduce DNA damage being approxi-
were 15-fold higher in the liver than in bone marrow, and mately compensated for by the additional DNA binding
the induced lacI mutant frequency was 11-fold greater in the occurring with subsequent treatments. A reduction in adduct
liver than in spleen lymphocytes. The responses in the hprt levels after the second N-OH-AAF dose can be seen for the
lymphocyte assay were positive, but reached no more than liver and bone marrow DNA-binding data in Figure 2 (2
16.5 ϫ 10Ϫ6 vs. a control value of 3.2 ϫ 10Ϫ6. The hprt doses ϩ repair values). This reduction was most likely
response was comparable to what we previously found for caused by DNA repair, but it could be a reflection of the
the induction of hprt lymphocyte mutants in Fischer 344 elimination of damaged cells by apoptosis, adduct dilution
rats fed the parent compound 2-AAF [Beland et al., 1999]. resulting from cell division, or some combination of these
In that study, 1 month of feeding 0.02% 2-AAF resulted in processes. Although the concentration of liver DNA adducts

200
Chen et al.
study. In male rat liver, 2-AAF and N-OH-AAF produce
two additional adducts: N-(deoxyguanosin-8-yl)-2-acetyl-
aminofluorene (dG-C8-AAF) and 3-(deoxyguanosin-N2-yl)-
2-acetylaminofluorene (dG-N2-AAF) [Beland and Kadlu-
bar, 1985, 1990]. Although both of these adducts can be
detected by 32P-postlabeling analyses, dG-C8-AAF tends to
label very poorly [Culp et al., 1993]. Furthermore, after
multiple doses, these adducts are relatively minor (ϳ10%)
compared to dG-C8-AF [Beland et al., 1982; Culp et al.,
1993], which may explain our failure to observe them in the
current study. Several investigators have postulated that,
rather than acting as mutagenic lesions, these adducts pro-
duce toxicity that is important to the promotion of 2-AAF
carcinogenesis [Kroese et al., 1988; Heflich and Neft,
1994].
Comparing the lacI mutant frequencies with the adduct
concentrations indicates that four doses of N-OH-AAF in-
duced four mutants per fmol adduct per ␮g DNA in the liver
(381 induced lacI mutants/103 fmol dG-C8-AF/␮g DNA),
but only one mutant per fmol adduct per ␮g DNA in spleen
lymphocytes (35.4 induced lacI mutants/28 fmol dG-C8-
AF/␮g DNA). There is evidence, however, that many of the
mutants in spleen lymphocytes originate from mutations
induced in the bone marrow [Jansen et al., 1996; Walker et
al., 1999b]. This is because the majority of spleen lympho-
cytes in adult rodents originate in the bone marrow, and the
bone marrow cells divide at a much higher rate than do
spleen lymphocytes. Calculating the efficiency of inducing
spleen lymphocyte lacl mutants using bone marrow DNA
adduct binding yields a value of five induced mutants/fmol
adduct/␮g DNA (35.4 induced lacI mutants/7 fmol dG-C8-
AF/␮g DNA), which is consistent with the value of four,
calculated for liver lacI mutants. If the assumption is correct
that most spleen lacI mutants originate in the bone marrow,
these data can be interpreted as indicating that N-OH-AAF
adducts produce mutations with almost equal efficiency in
the liver and spleen lymphocytes and that any dG-C8-AAF
or dG-N2-AAF adducts formed in the liver had little effect
on the lacI mutant frequency.
One of the concerns about using lacI as a reporter of in
vivo mutation has been the extent to which mutations in the
transgene mirror the mutational events occurring in endog-
enous genes. To examine this question, several studies have
compared lymphocyte mutant frequencies in the lacI trans-
gene with those of the endogenous hprt gene [Skopek et al.,
1995, 1996; Walker et al., 1996, 1997, 1999a; Sisk et al.,
1997; Chen et al., 1998; Manjanatha et al., 1998]. The
results of these studies indicate that the frequency of hprt
and lacI mutants induced by various mutagenic carcinogens
are rarely the same, but depend on both the nature of the
damage induced by the mutagen and the nature of the target
gene. For instance, agents that are mainly point mutagens
generally produce higher mutant frequencies in the lacI
Fig. 2. DNA adduct levels in liver, spleen lymphocytes, and bone marrow
from N-OH-AAF–treated Big Blue௡ rats. Six-week-old male rats were
treated intraperitoneally at 4-day intervals with 25 mg N-OH-AAF/kg b.w.
or the solvent alone. Adduct levels were assessed by 32P-postlabeling 1 day
after one, two, or four doses, and 4 days after one dose and 9 days after two
doses (ϩ repair). Data are presented as the means Ϯ SEM. Bars with
different letters differ significantly from each other (P Ͻ 0.05). Note
different scales used for y-axes.
after doses 2 and 4 was the same, the induced lacI mutant
frequency in liver after dose 4 was nearly threefold higher
than that after dose 2 (Table IV). This observation indicates
that the mutagenic response is a function of the length of
time a given adduct level persists in DNA, as well as the
absolute concentration of the adducts.
The major DNA adduct produced in vivo by 2-AAF and
N-OH-AAF is dG-C8-AF [Beland and Kadlubar, 1985,
1990; Heflich and Neft, 1994], the adduct found in this gene than in the hprt gene. This is because the lacI gene has

N-OH-AAF Mutation in Big Blueா Rats
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a larger target for point mutation, especially for point mu-
tation at G:C basepairs [discussed in Chen et al., 1998].
As demonstrated in this study, the major adduct produced
by N-OH-AAF is dG-C8-AF, and a large number of in vitro
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carcinogens in the Big Blue௡ rat. 7,12-Dimethylbenz[a]an-
thracene, which forms somewhat more bulky adducts with
dA than with dG, induced 1.7-fold more lacI mutants than
hprt mutants [Manjanatha et al., 1998], whereas thiotepa,
which like N-OH-AAF produces mainly dG adducts, in-
duced 2.8-fold more lacI than hprt mutants [Chen et al.,
1998].
A strength of transgenic shuttle vector systems for de-
tecting in vivo mutation is that mutations can be measured
in any tissue from which sufficient DNA can be recovered.
Thus, the transgenic assays may be useful for demonstrating
the tissue-specificity of in vivo mutation, which could be
extremely useful in evaluating the tissue-specific carcino-
genicity of chemical agents. The results of this study are
consistent with the expectation that the hepatocarcinogen
N-OH-AAF should produce a higher mutant frequency in
the target liver than the nontarget tissues like spleen lym-
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with published liver lacI mutant frequencies for mice
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specificity to the response. Whereas the N-OH-AAF mutant
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less than twofold background to about fivefold background
[Gunz et al., 1993; Shephard et al., 1993; Brooks et al.,
1995; Frijhoff et al., 1998; Ross and Leavitt, 1998]. Al-
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comparable to our rat treatment, these mutagenicity results
are generally consistent with the greater carcinogenic po-
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and suggest the potential of the Big Blue௡ system for
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Accepted by—
G. Hoffmann