Carcinogenesis vol.23 no.10 pp.1751–1757, 2002
Mutations induced by α-hydroxytamoxifen in the lacI and cII
genes of Big Blue transgenic rats
Tao Chen1,4, Gonçalo Gamboa da Costa2,
M.Matilde Marques2, Sharon D.Shelton1,
Frederick A.Beland3 and Mugimane G.Manjanatha1
1Division
of Genetic and Reproductive Toxicology, National Center for
Toxicological Research, Jefferson, AR 72079, USA, 2Centro de Quı́mica
Estrutural, Complexo I, Instituto Superior Técnico, Av. Rovisco Pais,
1049-001 Lisboa, Portugal, 3Division of Biochemical Toxicology, National
Center for Toxicological Research, Jefferson, AR 72079, USA
4To
whom correspondence should be addressed at HFT 130, NCTR, 3900
NCTR Road, Jefferson, AR 72079, USA
Email: [email protected]
The antiestrogen tamoxifen is widely used for the treatment
of breast cancer and more recently for the prevention of
breast cancer. A concern over the use of tamoxifen as a
chemopreventive agent is its carcinogenicity in rat liver,
through a genotoxic mechanism involving α-hydroxylation,
esterification, and DNA adduct formation, primarily by
reaction with dG. In a recent study [Gamboa da Costa
et al., Cancer Lett., 176, 37–45 (2002)], we demonstrated a
significant increase in the mutant frequency in the lacI
gene of Big Blue rats treated with tamoxifen, and a further
increase in rats administered α-hydroxytamoxifen. In the
present study, we have assessed mutation induction by
tamoxifen and α-hydroxytamoxifen in the liver cII gene of
Big Blue rats and have characterized the types of mutations
induced by α-hydroxytamoxifen in the liver lacI and cII
genes. The mutant frequencies in the liver cII gene were
80 ⍨ 13 ⍥ 10–6 in the control, 112 ⍨ 13 ⍥ 10–6 in
the tamoxifen-treated group (P < 0.01 vs. control), and
942 ⍨ 114 ⍥ 10–6 in the α-hydroxytamoxifen-treated
animals (P < 0.001 vs. control; P < 0.001 vs. tamoxifen).
Molecular analysis of the mutants indicated that the
α-hydroxytamoxifen-induced mutational spectrum differed
significantly from the control spectrum, but was very
similar to the spectrum induced by tamoxifen for both the
lacI and cII genes [Davies et al., Environ. Mol. Mutagen.,
28, 430–433 (1996); Davies et al., Carcinogenesis, 20, 1351–
1356 (1999)]. G:C → T:A transversion was the major type
of mutation induced by α-hydroxytamoxifen and tamoxifen,
while G:C → A:T transition was the main type of mutation
in the control. These results support the hypothesis that
α-hydroxytamoxifen is a major proximate tamoxifen metabolite causing the initiation of tumors in the liver of rats
treated with tamoxifen.
Introduction
Tamoxifen has been widely used for adjuvant therapy in the
treatment of patients with breast cancer for nearly 30 years,
and many millions of women have been treated with this drug
successfully. More recently the drug has been shown to reduce
the incidence of breast cancer in healthy women who are at
high risk of developing breast cancer (1,2). A concern over
the use of tamoxifen as a chemopreventive agent is the
© Oxford University Press
induction of endometrial cancer in women, and liver and
endometrial tumors in rats (3–7). The induction of liver tumors
by tamoxifen in rats has been associated with a genotoxic
mechanism involving DNA adduct formation and mutation
induction (8–11). The mechanism by which endometrial tumors
are induced by tamoxifen in women is not clear. Our recent
data suggest that induction of endometrial tumors in rats is
not due to the genotoxicity of tamoxifen (11,12); however, a
recent study with monkeys demonstrated that non-human
primates metabolize tamoxifen into genotoxic intermediates
and that tamoxifen–DNA adducts are detectable in various
tissues including the uterus (13,14).
The hepatic metabolism of tamoxifen gives rise to a variety
of metabolites. Although many putative reactive intermediates
of tamoxifen have been proposed (reviewed in ref. 15), it is
now generally believed that a substantial portion of the DNA
adducts arise from a minor metabolite, α-hydroxytamoxifen,
after conversion to a reactive sulfate ester by sulfotransferases
(16-19). The major DNA adduct resulting from this pathway is (E)-α-(deoxyguanosin-N2-yl)-tamoxifen, which is
accompanied by minor amounts of the Z diastereomer and
deoxyadenosine adducts (8,20).
Tamoxifen is a gene mutagen in rat liver, although it is not
mutagenic in several regulatory short-term tests. Tamoxifen
gave negative responses in the Salmonella typhimurium reversion assay and in the in vitro human lymphocyte chromosome
aberration assay. It was only weakly positive in a modified
in vivo/in vitro unscheduled DNA synthesis assay in rat
hepatocytes (21–23). In contrast to these short-term tests,
tamoxifen increased mutant frequencies ~3-fold over the background and induced mainly G:C → T:A transversion mutations
in the liver lacI and cII genes of Big Blue rats (9,24,25). Shortterm dosing of α-hydroxytamoxifen in a previous experiment,
however, resulted in only a 1.8-fold increase in mutant frequency compared with the controls, with no significant increase
in G:C → T:A transversion in the liver lacI gene of Big Blue
rats (10).
In a recent study (11), we used a subchronic dosing
regimen for treating Big Blue rats with tamoxifen or
α-hydroxytamoxifen. Intraperitoneal administration of 21 daily
doses of 54 µmol α-hydroxytamoxifen/kg body weight
increased the lacI mutant frequency in the liver to a value
77-fold higher than background and 24-fold greater than that
observed with tamoxifen. In the present study, we examined
the mutation induction by tamoxifen and α-hydroxytamoxifen
in the liver cII gene, and characterized the types of mutations
induced by α-hydroxytamoxifen in the lacI and cII genes of
rat livers.
Materials and methods
Treatment of animals and lacI mutation assay
Tamoxifen was purchased from Sigma (St Louis, MO). α-Hydroxytamoxifen
was prepared by the method of Foster et al. (26). Details of the treatment of
the rats and the lacI mutation assay are given elsewhere (11). Briefly, 8-weekold female Big Blue rats were treated intraperitoneally with 21 daily doses of
1751
T.Chen et al.
Table I. cII Mutant frequencies in the livers of Big Blue rats treated with tamoxifen or α-hydroxytamoxifen
Animal ID
Number of packaging
reactions
Control
C1
C2
C3
C4
C5
C6
2
2
2
2
2
2
41
25
18
36
23
30
458
295
241
508
239
503
000
000
000
000
000
000
90
85
75
71
96
60
2
2
2
2
2
1
40
62
33
53
36
25
312
516
326
453
320
271
000
000
000
000
000
000
128
120
101
117
113
92
196
178
140
127
184
256
238
183
145
161
170
251
000
000
000
000
000
000
824
973
966
789
1082
1020
Ave MF ⫾ SD: 80 ⫾ 13 ⫻ 10–6
Tamoxifen
T1
T2
T3
T4
T5
T6
Ave MF ⫾ SD: 112 ⫾ 13 ⫻ 10–6; P ⬍ 0.01 vs. control
α-Hydroxytamoxifen
A1
A2
A3
A4
A5
A6
Ave MF ⫾ SD: 942 ⫾ 114 ⫻ 10–6; P ⬍ 0.001 vs. control
1
1
1
2
2
2
and vs. tamoxifen
Number of
mutants
Total plaques
screened
MF (⫻10–6)
Treatment
MF: mutant frequency; SD: standard deviation.
Escherichia coli host strain G1250. To determine the total titer of packaged
phages, G1250 bacteria were mixed with 1:100 dilutions of phage, plated on
TB1 plates, and incubated overnight at 37°C (nonselective conditions). For
mutant selection, the packaged phages were mixed with G1250, plated on
TB1 plates and incubated at 24°C for 42 h (conditions for λ cII– selection).
After incubation at 24°C, λ phages with wild-type cII genes undergo
lysogenization and become part of the developing bacterial lawn, whereas
phages with mutated cII genes undergo lytic growth and give rise to plaques.
When incubated at 37°C, λ phages with wild-type cII genes also undergo a
lytic cycle, resulting in plaque formation. The cII mutant frequency was the
ratio of the number of mutant plaques (as determined at 24°C) to the total
number of plaques screened (as determined at 37°C).
Sequence analysis of lacI and cII mutants
Fig. 1. Mutant frequencies in the liver cII and lacI genes from control Big
Blue rats or Big Blue rats treated with tamoxifen or α-hydroxytamoxifen.
The data for the lacI gene are from ref. 11. a indicates a significant
difference between the treated group and the control; b indicates a
significant difference between rats treated with tamoxifen and
α-hydroxytamoxifen. The data are presented as the mean ⫾ SD from six
rats per group.
54 µmol tamoxifen/kg body wt, 54 µmol α-hydroxytamoxifen/kg body wt, or
the solvent (trioctanoin). Six rats from each treatment group were killed one
month after the last treatment. The livers were isolated, frozen, and stored
at –80°C. DNA extracted from the liver samples was packaged into λ vectors,
and the infectious phages with vectors were plated to assay lacI mutant
plaques as described previously (27,28).
cII Mutation assay
High-molecular-weight genomic DNA was extracted from the rat 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 λ phage,
plating the packaged DNA samples, and determination of mutant frequency
were carried out following the manual for λ select-cII mutation detection
system for Big Blue rodents (Stratagene). The λ shuttle vector containing the
cII target gene was rescued from total genomic DNA with λ phage packaging
extract (Transpack; Stratagene). The plating was performed with the
1752
The template DNA preparation from lacI mutants was performed as previously
described (27,29). Briefly, in vivo excision of LIZ was carried out by mixing
a verified mutant phage stock with XL1-Blue cells and ExAssist helper phages.
The pLIZ phagemids recovered from in vivo excision were selected by plating
out with SOLR cells on LB plates containing ampicillin. A single ampicillinresistant SOLR E.coli colony was selected and grown overnight in 3 ml of
medium. The phagemid DNA containing the entire lacI gene was extracted
using an ABI PRISM Miniprep Kit.
The cII target DNA for sequencing was amplified by PCR with two primers:
5⬘-AAAAAGGGCATCAAATTAACC-3⬘ and 5⬘-CCGTTGAGTATTTTTGCTG-3⬘. The cII mutant plaques were selected at random from different animals
and replated at low density to verify the mutant phenotype. Single wellisolated plaques were selected and transferred to a microcentrifuge tube
containing 25 µl of autoclaved distilled water. The tube was placed in boiling
water for 5 min and centrifuged at 12 000 g for 3 min. For PCR amplification,
10 µl of the supernatant was added to 40 µl of a PCR mastermix such that
the final amounts of the reagents were 1⫻ Taq polymerase reaction buffer,
10 pmol of each primer, 12.5 nmol of each dNTP, and 2.5 U of Taq polymerase.
The PCR reaction was performed with the following cycling parameters: a
3 min denaturation at 95°C, followed by 30 cycles of 30 s at 95°C, 1 min at
60°C, and 1 min at 72°C, with a final extension of 10 min at 72°C. The PCR
products were purified using PCR purification kits (Qiagen, Chatsworth, CA).
The lacI and cII mutant DNA was sequenced with an ABI Prism Big Dye
Terminator Cycle Sequencing Kit and a 377 DNA Sequencer (Applied
Biosystems, Foster City, CA). The primers for sequencing lacI mutations have
been described (27,29); the primers for cII mutation sequencing were the
same as those used for the PCR. The sequence data were analyzed with the
help of Sequence Navigator software (Applied Biosystems).
Mutations induced by α-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats
Table II. Mutations in the liver lacI gene of α-hydroxytamoxifen-treated Big Blue rats
Positiona
42
53
57
92
93
95
98
108
116
129
150
158
162
173
180
185
194
210
222
234
245
258
260
273
285
326
357
377/380
381
429/430
437
566
600
671
816
920
962
Total
Mutationb
Amino acid
change
Sequence context
5⬘→3⬘c
Number of
mutations
Number of
independent
mutations
C→T
G→T
C→T
C→T
G→A
G→T
G→A
-G
C→A
G→T
C→A
C→A
G→T
T→A
C→G
G→A
G→A
C→G
C→A
G→T
C→A
–C
C→A
C→A
C→A
G→C
G→T
C→G
⫹A
G→A
⫹T
G→T
C→A
C→T
G→T
–G
C→A
G→T
Thr→Met
Val→ Phe
Ala→Val
Arg→Cys
Arg→His
Arg→Leu
Val→Met
Frameshift
Ala→Asp
Val→Phe
Thr→Lys
Ala→Glu
Gly→Val
Leu→Gln
Pro→Ala
Arg→His
Gly→Arg
Thr→Ser
Ser→Stop
Gly→Val
Ser→Tyr
Frameshift
Ser→Stop
Gln→Lys
Ala→Glu
Arg→Pro
Glu→Stop
Ala→Gly
Frameshift
Arg→His
Frameshift
Gly→Val
Gln→Lys
Ser→Phe
Glu→Stop
Frameshift
Gln→Lys
Ala→Ser
gtaACGtta
gatGTCgca
gtcGCAgag
tccCGCgtg
tccCGCgtg
tccCGCgtg
cgcGTGgtg
gtgGTGaac
cagGCCagc
cacGTTtct
aaaACGcgg
gcgGCGatg
gcgGAGctg
gagCTGaat
attCCCaac
aacCGCgtg
gtgGCAcaa
caaCTGgcg
cagTCGttg
ctgAGTggc
accTCCagt
gccCTGcac
ccgTCGcaa
tcgCAAatt
gcgGCGatt
tctCGCgcc
gtaGAAcga
gcgGCGgtg
gcgCAACGCgtc
caaCGCgtc
gccATTgct
gtgGAAgct
cacCAGcaa
agtTCTgtc
gcgGAAcgg
ctgCGCgtt
gggCAAacc
cagGCGgtg
1
2
1
2
1
4
1
1
1
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
2
4
1
1
1
1
2
2
1
3
1
1
2
54
1
2
1
1
1
4
1
1
1
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
2
3
1
1
1
1
2
2
1
3
1
1
2
52
aThe position numbering is based on Farabaugh et
bPresented in terms of the sequence change on the
cUppercase indicates target codon and target bases
al. (32).
nontranscribed DNA strand. Abbreviations: –, deletion; ⫹, insertion.
are underlined.
Statistical analyses
One-way ANOVA followed by Student–Newman–Keuls test was used to
evaluate the differences in mutant frequencies among groups. Since the
variance increased with the magnitude of the mutant frequency, the data were
log-transformed before conducting the analysis. Mutational spectra were
compared using the computer program written by Cariello et al. (30) for the
Monte Carlo analysis developed by Adams and Skopek (31).
Results
cII Mutant frequency
Six female Big Blue rats per group were treated for 21
days by i.p. injection with 54 µmol tamoxifen, 54 µmol αhydroxytamoxifen, or the solvent only. One month after the
last treatment, the cII mutant frequency was assessed in the
liver (Table I). Compared with rats administered the solvent
alone (80 ⫾ 13 ⫻ 10–6), there was a significant increase in
the cII mutant frequency in the livers of Big Blue rats treated
with tamoxifen (112 ⫾ 13 ⫻ 10–6; P ⬍ 0.01) or
α-hydroxytamoxifen (942 ⫾ 114 ⫻ 10–6; P ⬍ 0.001). In
addition, the response observed with α-hydroxytamoxifen
was significantly greater than that induced by tamoxifen
(P ⬍ 0.001).
Mutations in the lacI gene induced by α-hydroxytamoxifen
The liver lacI mutant frequency from α-hydroxytamoxifentreated rats was 770 ⫾ 270 ⫻ 10–6, which is 77-fold
greater than the control frequency (Figure 1) (11). αHydroxytamoxifen-induced lacI mutations were evaluated by
DNA sequence analysis of 54 mutants isolated from the six
treated rats (Table II and summarized in Table IV). Mutations
that were found more than once in the mutants isolated
from a single animal were assumed to be siblings and were
considered to represent a single independent mutation.
Accordingly, a total of 52 independent mutations were
identified. Of the 52 mutations, 90% (47) were single base
pair substitutions and 10% (5) were frameshifts. Among the
single base pair substitutions, 98% (46/47) involved G:C
base pairs and 2% (1/47) occurred at A:T base pairs.
The most commonly occurring base pair substitution was
1753
T.Chen et al.
Table III. Mutations in the liver cII gene of α-hydroxytamoxifen-treated Big Blue rats
Positiona
Mutationb
Amino acid change
Sequence context 5⬘→3⬘c
Number of mutations
3
20
24
29
39–40
42
51
64
65
65
65/68
67–68
74
77
86
88
89
95
100
101
101
115
117d
123
123/125
125
126
135–136
141
145
155
157
160
163/166
178/185
179
179–184d
181
182
190
196
206
210
212
214
G→T
G→C
C→G
C→A
CG→AT
G→T
G→T
G→C
C→A
C→G
⫹A
AT→TA
G→T
C→A
C→A
G→T
C→A
C→A
G→T
G→A
G→T
C→T
G→A
C→A
⫹A
G→T
G→T
GG→TT
G→T
C→A
C→T
A→T
C→A
⫹T
⫹G
G→T
–G
G→T
G→T
G→A
G→T
G→T
G→T
C→T
C→G
C→T
–CG
C→A
CT→TG
C→G
G→T
C→A
–C
Met→Ile
Arg →Pro
Asn→Lys
Ala→Asp
Glu→STOP
Glu→Tyr
Leu→Phe
Ala→Pro
Ala→Glu
Ala→Gly
Frameshift
Met→STOP
Gly→Val
Thr→Asn
Thr→Lys
Ala→Ser
Ala→Glu
Ala→Asp
Gly→ Cys
Gly→Asp
Gly→Val
Gln→STOP
Gln→Gln
Ser→Arg
Frameshift
Arg→Met
Arg→Ser
Arg-Asp→Ser-Tyr
Trp→Cys
Pro→Thr
Ser→Leu
Met→Leu
Leu→Met
Frameshift
Frameshift
Trp→Leu
Frameshift
Gly→Cys
Gly→Val
Asp→Asn
Asp→Tyr
Arg→Leu
Leu→Phe
Ala→Val
Arg→Gly
Arg→STOP
Frameshift
Ala→Asp
Ala→Val
Leu→Val
Ala→Ser
Gln→Lys
Frameshift
catATGgtt
aaaCGCaac
cgcAACgag
gagGCTcta
cgaATCGAGagt
atcGAGagt
gcgTTGctt
atcGCAatg
atcGCAatg
atcGCAatg
atcGCAATGctt
gcaATGctt
cttGGAact
ggaACTgag
aagACAgcg
acaGCGgaa
acaGCGgaa
gaaGCTgtg
gtgGGCgtt
gtgGGCgtt
gtgGGCgtt
tcgCAGatc
tcgCAGatc
atcAGCagg
atcAGCAGGtgg
agcAGGtgg
agcAGGtgg
aagAGGGACtgg
gacTGGatt
attCCAaag
ttcTCAatg
tcaATGctg
atgCTGctt
ctgCTTGCTgtt
gaaTGGGGGGTCgtt
gaaTGGggg
gaaTGGGGGGTCgtt
tggGGGgtc
tggGGGgtc
gttGACgac
gacGACatg
gctCGAttg
cgaTTGgcg
ttgGCGcga
gcgCGAcaa
gcgCGAcaa
gcgCGAcaa
gttGCTgcg
gttGCTgcg
attCTCacc
ccgGCGgca
gaaCAAatc
gaaCAAatc
1
1
1
1
1
1
3
1
2
1
2
1
1
1
2
1
1
1
2
2
8
2
1
3
1
2
1
1
2
1
1
1
3
1
5
1
14
1
1
1
1
1
1
2
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
2
3
2
1
3
1
2
1
1
2
1
1
1
2
1
3
1
4
1
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
94
71
214–215
224
224–225
232
253
274
274
Total
aPosition
Number of
independent
mutations
1 is the first base of the start codon in the cII coding sequence.
nontranscribed DNA strand. Abbreviations: –, deletion; ⫹, insertion.
are underlined.
bPresented in terms of the sequence change on the
cUppercase indicates target codon and target bases
dPart of a double mutation.
G:C → T:A transversion (61%, 32/52), followed by
G:C → A:T transition (17%, 9/52).
Mutations in the cII gene induced by α-hydroxytamoxifen
A total of 94 cII mutants from six α-hydroxytamoxifen-treated
rats were successfully sequenced and 71 of them were found to
be independent (Table III and summarized in Table IV). Among
1754
the independent mutations, 76% (54/71) were base pair substitutions, 18% (13/71) were frameshift mutations and 6% (4/71)
were complex mutations (i.e. double base substitutions). Among
the base substitutions, 98% (53/54) were mutated at G:C base
pairs while only 2% (1/54) occurred at A:T base pairs. The
predominant base substitution was G:C → T:A transversion
(52%, 37/71), followed by G:C → A:T transition (14%, 10/71).
Mutations induced by α-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats
Table IV. Comparison of cII and lacI independent mutations in livers of Big Blue rats treated with solvent, tamoxifen or α-hydroxytamoxifen.
Type of mutation
Number (%) of mutations
Control
lacIa
Base substitutions
Transitions
G:C→A:T
A:T→G:C
Transversions
G:C→T:A
G:C→C:G
A:T→T:A
A:T→C:G
Frameshifts
Complex mutations
Total
54 (41%)
4 (3%)
23
15
9
4
17
5
131
(18%)
(11%)
(7%)
(3%)
(13%)
(4%)
(100%)
α-Hydroxytamoxifen
Tamoxifen
cIIb
27 (42%)
8 (12%)
10
7
0
5
8
0
65
(15%)
(11%)
(0%)
(8%)
(12%)
(0%)
(100%)
lacIc
2 (5%)
2 (5%)
16
1
0
2
9
5
37
(43%)
(3%)
(0%)
(5%)
(25%)
(14%)
(100%)
cIId
9 (23%)
3 (8%)
17
1
1
0
7
0
38
(45%)
(3%)
(3%)
(0%)
(18%)
(0%)
(100%)
lacI
9 (17%)
0 (0%)
32
5
1
0
5
0
52
(61%)
(10%)
(2%)
(0%)
(10%)
(0%)
(100%)
cII
10 (14%)
0 (0%)
37
6
1
0
13
4
71
(52%)
(9%)
(1%)
(0%)
(18%)
(6%)
(100%)
aData
from Chen et al. (33).
from Harbach et al. (34).
from Davies et al. (24).
dData from Davies et al. (25).
bData
cData
Comparison of mutational spectra
Table IV lists the independent cII and lacI mutations isolated
from livers of Big Blue rats treated with vehicle, tamoxifen
and α-hydroxytamoxifen. G:C → T:A transversion was the
major type of mutation induced by α-hydroxytamoxifen and
tamoxifen, while G:C → A:T transition was the main type of
mutation in the control. There was no significant difference
between the mutational spectra induced by α-hydroxytamoxifen in the liver lacI and cII genes (P ⫽ 0.3), while the
overall patterns of mutations in the control and α-hydroxytamoxifen-treated rats differed significantly (P ⬍ 0.0001 for both
the lacI and cII genes). Although the cII mutational spectrum
found for α-hydroxytamoxifen-treated rats was not significantly different from that of tamoxifen-treated rats
(P ⫽ 0.1), a significant difference was found between the
spectra of lacI mutation in the livers of tamoxifen-treated and
α-hydroxytamoxifen-treated rats (P ⬍ 0.0001). This was
mainly due to the higher induction of G:C → T:A transversion
in the α-hydroxytamoxifen-treated rats (61 vs. 43%).
Discussion
α-Hydroxytamoxifen is a much stronger mutagen than tamoxifen in rat liver
The mutant frequencies in the lacI and cII genes were
10 ⫾ 10 ⫻ 10–6 and 80 ⫾ 13 ⫻ 10–6 in the control,
32 ⫾ 18 ⫻ 10–6 and 112 ⫾ 13 ⫻ 10–6 in the tamoxifentreated rats and 770 ⫾ 270 ⫻ 10–6 and 942 ⫾ 114 ⫻ 10–6 in
the α-hydroxytamoxifen-treated animals (Figure 1). The net
increases of mutant frequency induced by tamoxifen over the
control were 22 ⫻ 10–6 in the lacI gene and 32 ⫻ 10–6 in the
cII gene, while the net increases by α-hydroxytamoxifen were
760 ⫻ 10–6 in the lacI gene and 862 ⫻ 10–6 in the cII gene.
Therefore, the net induction of mutant frequencies by αhydroxytamoxifen was 35-fold greater than tamoxifen in the
lacI gene and 27-fold greater in the cII gene. It has been
suggested that a dose of 103 µmol/kg tamoxifen would be
metabolized to the equivalent of 1 µmol/kg α-hydroxytamoxifen (10) or ~1% of the administered dose. Given the low rate
of conversion of tamoxifen to α-hydroxytamoxifen, it is not
unexpected that administration of tamoxifen and α-hydroxytamoxifen at equimolar doses will result in far more mutation
induction by α-hydroxytamoxifen.
In a previous study, oral treatment with 10 daily doses
of 103 mmol/kg α-hydroxytamoxifen resulted in ~1.8-fold
increase in mutation frequency but no significant increase in
G:C → T:A transversions (10). The lack of efficient induction
of mutations by oral administration of α-hydroxytamoxifen
may be due to inactivation of the chemical at the acidic pH
of the stomach. For example, in earlier studies we found that
with α-hydroxytamoxifen the binding to hepatic DNA was
four-fold higher when administered by i.p. injection compared
with gavage (12). Since the tamoxifen and α-hydroxytamoxifen
gave similar DNA adduct profiles when given by either route
(12), we elected to use i.p. dosing to maximize the chance for
mutant induction. Our results indicate that α-hydroxytamoxifen
induces mutations much more efficiently through i.p. injection
administration than oral treatment.
G:C → T:A transversion induced by α-hydroxytamoxifen
reflects the mutational specificity of tamoxifen–DNA adducts
The results from this study indicate that the mutation profiles
in the lacI and cII genes of liver from α-hydroxytamoxifentreated rats were significantly different from the corresponding
profiles in control rats (Table IV). Compared with the controls,
the mutations detected in α-hydroxytamoxifen-treated rats had
lower percentages of G:C → A:T transitions (17% in the lacI
and 14% in the cII for α-hydroxytamoxifen-treated rats vs.
41% in the lacI and 42% in the cII for the control rats) and
higher percentages of G:C → T:A transversions (61% in the
lacI and 52% in the cII for α-hydroxytamoxifen-treated rats
vs. 18% in the lacI and 15% in the cII for the control rats).
The types of mutations in the liver cII gene from
α-hydroxytamoxifen-treated rats were very similar to those
from tamoxifen-treated animals. With the lacI gene there
was a significant difference between the tamoxifen- and
α-hydroxytamoxifen-induced spectra; nonetheless, with both
compounds, the cII and lacI spectra shared common features:
1755
T.Chen et al.
most base substitutions occurred at G:C base pairs, with
G:C → T:A being the major type of mutation.
A mechanism by which tamoxifen induces G:C → T:A
transversions in rat liver has been suggested. Tamoxifen is
known to be metabolized to α-hydroxytamoxifen by
cytochrome P450 3A, and then converted into α-sulfoxytamoxifen by sulfotransferases. α-Sulfoxytamoxifen can bind
to DNA to form the major DNA adduct, (E)-α-(deoxyguanosinN2-yl)–tamoxifen (16–19). This is a bulky DNA adduct and
preferentially gives rise to G:C → T:A transversion mutation
like many other carcinogens, such as N-hydroxy-2acetylaminofluorene and benzo[a]pyrene, which also form
bulky DNA adducts (33,35). Our finding that the types of
mutations induced by α-hydroxytamoxifen are similar to those
induced by tamoxifen further supports the hypothesis that
α-hydroxytamoxifen is a major proximate tamoxifen metabolite causing mutations in rat liver.
The cII gene is an acceptable alternative to the lacI gene of
Big Blue rats in the determination of mutations
The cII assay is performed using a positive selection of
mutants. Compared with the lacI assay, it is faster and less
expensive to perform. In addition, the cII gene is 294 base
pairs in length (vs. 1080 base pairs for lacI), offering potentially
easier analysis of mutations (36). A primary purpose for using
the cII mutation assay and subsequent analysis of the cII
mutations was to confirm the results on mutant frequencies
and types of mutations induced by tamoxifen and α-hydroxytamoxifen in the lacI gene. An additional reason was to compare
the characteristics of the two systems by measuring mutant
frequencies and mutational spectra in the two genes from the
same DNA samples.
Our results indicate that the overall results provide some
interesting comparisons and contrasts between the two mutational systems. The net increase in mutation induction by
tamoxifen and α-hydroxytamoxifen was similar in the two
systems (22 ⫻ 10–6 in the lacI and 32 ⫻ 10–6 in the cII for
tamoxifen; 760 ⫻ 10–6 in the lacI and 862 ⫻ 10–6 in the cII
for α-hydroxytamoxifen). In addition, the mutational spectrum
in the liver cII gene from α-hydroxytamoxifen-treated rats was
not significantly different from that in the liver lacI gene
(P ⫽ 0.3). Our results confirm the finding of Harbach et al.
(34) that the smaller size of the cII coding region is a promising
alternative to the larger target of the lacI gene. Although
α-hydroxytamoxifen induced a higher mutant frequency in the
cII gene, there was a higher spontaneous background mutation
frequency in the liver cII gene (80 ⫻ 10–6) than that in the
liver lacI gene (10 ⫻ 10–6). The differences in the background
mutant frequencies resulted in a much greater fold- increase
in mutant frequency by α-hydroxytamoxifen in the lacI gene
as compared to the cII gene (77-fold vs. 12-fold). The packaging
efficiency was higher using the strain G1250 in the cII assay
than the strain SCS-8 in the lacI assay. The plaque-forming
units (PFU) per packaging reaction in the cII assay were
~180 000, whereas PFU per packaging reaction in the lacI
assay were ~100 000. This increase in PFU per packaging
reaction allowed more plaques to be screened in the cII assay
compared with the lacI assay, with a concomitant decrease in
the standard deviation. Consequently, although the increase in
the cII mutant frequency induced by tamoxifen was only 1.4fold, it was statistically significant (P ⬍ 0.01).
Conclusion
α-Hydroxytamoxifen induced a much greater mutant frequency
than tamoxifen in the liver lacI and cII genes of Big Blue rats.
1756
The spectra of mutations induced by α-hydroxytamoxifen in
the lacI and cII genes were similar to those induced by
tamoxifen but distinct from the control spectra. The main type
of mutations induced by α-hydroxytamoxifen was consistent
with the major tamoxifen-induced DNA adduct. These results
support the hypothesis that α-hydroxytamoxifen is a major
proximate tamoxifen metabolite causing induction of mutations
and the initiation of tumors in the liver of rats treated with
tamoxifen.
References
1. Early Breast Cancer Trialists’ Collaborative Group (1998) Tamoxifen for
early breast cancer: an overview of the randomised trials. Lancet, 351,
1451–1467.
2. Fisher,B., Costantino,J.P., Wickerham,D.L., et al. (1998) Tamoxifen for
prevention of breast cancer: report of the National Surgical Adjuvant
Breast and Bowel Project P-1 Study. J. Natl Cancer Inst., 90, 1371–1388.
3. Stearns,V. and Gelmann,E.P. (1998) Does tamoxifen cause cancer in
humans? J. Clin. Oncol., 16, 779–792.
4. Hard,G.C., Williams,G.M. and Iatropoulos,M.J. (1993) Tamoxifen and
liver cancer. Lancet, 342, 444–445.
5. Williams,G.M., Iatropoulos,M.J., Djordjevic,M.V. and Kaltenberg,O.P.
(1993) The triphenylethylene drug tamoxifen is a strong liver carcinogen
in the rat. Carcinogenesis, 14, 315–317.
6. Mäntylä,E., Nieminen,L. and Karlsson,S. (1995) Endometrial cancer
induction by tamoxifen in the rat. Eur. J. Cancer, 31A (Suppl. 5), S14.
7. Mäntylä,E.T.E., Karlsson,S.H. and Nieminen,L.S. (1996) Induction of
endometrial cancer by tamoxifen in the rat. In: Li,J.J., Li,S.A.,
Gustafsson,J.Å., Nandi S. and Sekely,L.I. (eds.) Hormonal Carcinogenesis.
II. Proceedings of the Second International Symposium, Springer, New
York, pp. 442–445.
8. Osborne,M.R., Hewer,A., Hardcastle,I.R., Carmichael,P.L. and
Phillips,D.H. (1996) Identification of the major tamoxifen-deoxyguanosine
adduct formed in the liver DNA of rats treated with tamoxifen. Cancer
Res., 56, 66–71.
9. Davies,R., Oreffo,V.I.C., Martin,E.A., Festing,M.F.W., White,I.N.H.,
Smith,L.L. and Styles,J.A. (1997) Tamoxifen causes gene mutations in the
livers of lambda/lacI transgenic rats. Cancer Res., 57, 1288–1293.
10. White,I.N.H., Carthew,P., Davies,R., Styles,J., Brown,K., Brown,J.E.,
Smith,L.L. and Martin,E.A. (2001) Short-term dosing of α-hydroxytamoxifen results in DNA damage but does not lead to liver tumours in
female Wistar/Han rats. Carcinogenesis, 22, 553–557.
11. Gamboa da Costa,G., Manjanatha,M.G., Marques,M.M. and Beland,F.A.
(2002) Induction of lacI mutations in Big Blue rats treated with tamoxifen
and α-hydroxytamoxifen. Cancer Lett., 176, 37–45.
12. Gamboa
da
Costa,G.,
McDaniel-Hamilton,L.P.,
Heflich,R.H.,
Marques,M.M. and Beland,F.A. (2001) DNA adduct formation and mutant
induction in Sprague-Dawley rats treated with tamoxifen and its derivatives.
Carcinogenesis, 22, 1307–1315.
13. Schild,L.J., Divi,R.L. and Poirier,M.C. (2002) Formation of tamoxifenDNA adducts in organs of monkeys dosed orally with tamoxifen for 30
days. Environ. Mol. Mutagen., 39 (Suppl. 33), 56.
14. Beland,F.A., Churchwell,M.I., Doerge,D.R., Malejka-Giganti,D., Zhang,X.,
Divi,R.L., Poirier,M.C., Gamboa da Costa,G. and Marques,M.M. (2002)
High performance liquid chromatography electrospray ionization mass
spectrometry analysis of tamoxifen DNA adducts in rats, monkeys and
humans. Proc. Am. Assoc. Cancer Res., 43, 343.
15. Phillips,D.H. (2001) Understanding the genotoxicity of tamoxifen?
Carcinogenesis, 22, 839–849.
16. Davis,W., Venitt,S. and Phillips,D.H. (1998) The metabolic activation
of tamoxifen and α-hydroxytamoxifen to DNA-binding species in rat
hepatocytes proceeds via sulphation. Carcinogenesis, 19, 861–866.
17. Shibutani,S., Dasaradhi,L., Terashima,I., Banoglu,E. and Duffel,M.W.
(1998) α-Hydroxytamoxifen is a substrate of hydroxysteroid (alcohol)
sulfotransferase, resulting in tamoxifen DNA adducts. Cancer Res., 58,
647–653.
18. Glatt,H., Bartsch,I., Christoph,S., Coughtrie,M.W.H., Falany,C.N.,
Hagen,M., Landsiedel,R., Pabel,U., Phillips,D.H., Seidel,A. and
Yamazoe,Y. (1998) Sulfotransferase-mediated activation of mutagens
studied using heterologous expression systems. Chem.-Biol. Interact., 109,
195–219.
19. White,I.N.H. (1999) The tamoxifen dilemma. Carcinogenesis, 20, 1153–
1160.
Mutations induced by α-hydroxytamoxifen in the lacI and cII genes of Big Blue transgenic rats
20. Osborne,M.R., Hardcastle,I.R. and Phillips,D.H. (1997) Minor products of
reaction of DNA with α-acetoxytamoxifen. Carcinogenesis, 18, 539–543.
21. Tucker,M..J., Adam,H.K. and Patterson,J.S. (1984) Tamoxifen. In:
Laurence,D.R., McLean,A.E.M. and Weatherall,M. (eds.) Safety Testing
of New Drugs. Academic Press, New York, pp. 125–161.
22. White,I.N.H., de Matteis,F., Davies,A., Smith,L.L., Crofton-Sleigh,C.,
Venitt,S., Hewer,A. and Phillips,D.H. (1992) Genotoxic potential of
tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and
C57BL/6 mice and in human MCL-5 cells. Carcinogenesis, 13, 2197–2203.
23. Styles,J.A., Davies,A., Lim,C.K., de Matteis,F., Stanley,L.A., White,I.N.H.,
Yuan,Z.-X. and Smith,L.L. (1994) Genotoxicity of tamoxifen, tamoxifen
epoxide and toremifene in human lymphoblastoid cells containing human
cytochrome P450s. Carcinogenesis, 15, 5–9.
24. Davies,R., Oreffo,V.I.C., Bayliss,S., Dinh,P.-A., Lilley,K.S., White,I.N.H.,
Smith,L.L. and Styles,J.A. (1996) Mutational spectra of tamoxifen-induced
mutations in the livers of lacI transgenic rats. Environ. Mol. Mutagen.,
28, 430–433.
25. Davies,R., Gant,T.W., Smith,L.L. and Styles,J.A. (1999) Tamoxifen induces
G:C → T:A mutations in the cII gene in the liver of lambda/lacI transgenic
rats but not at 5⬘-CpG-3⬘ dinucleotide sequences as found in the lacI
transgene. Carcinogenesis, 20, 1351–1356.
26. Foster,A.B., Jarman,M., Leung,O.-T., McCague,R., Leclercq,G. and
Devleeschouwer,N. (1985) Hydroxy derivatives of tamoxifen. J. Med.
Chem., 28, 1491–1497.
27. Chen,T., Aidoo,A., Manjanatha,M.G., Mittelstaedt,R.A., Shelton,S.D., LynCook,L.E., Casciano,D.A. and Heflich,R.H. (1998) Comparison of mutant
frequencies and types of mutations induced by thiotepa in the endogenous
Hprt gene and transgenic lacI gene of Big Blue® rats. Mutat. Res., 403,
199–214.
28. Manjanatha,M.G., Shelton,S.D., Culp,S.J., Blankenship,L.R. and
Casciano,D.A. (2000) DNA adduct formation and molecular analysis of
in vivo lacI mutations in the mammary tissue of Big Blue® rats treated
with 7,12-dimethylbenz[a]anthracene. Carcinogenesis, 21, 265–273.
29. Manjanatha,M.G., Chen,J.B., Shaddock,J.G., Jr., Harris,A.J., Shelton,S.D.
and Casciano,D.A. (1996) Molecular analysis of lacI mutations in Rat2TM
cells exposed to 7,12-dimethylbenz[a]anthracene: evidence for DNA
sequence and DNA strand biases for mutation. Mutat. Res., 372, 53–64.
30. Cariello,N.F. (1994) Software for the analysis of mutations at the human
hprt gene. Mutat. Res., 312, 173–185.
31. Adams,W.T. and Skopek,T.R. (1987) Statistical test for the comparison of
samples from mutational spectra. J. Mol. Biol., 194, 391–396.
32. Farabaugh,P.J., Schmeissner,U., Hofer,M. and Miller,J.H. (1978) Genetic
studies of the lac repressor. VII. On the molecular nature of spontaneous
hotspots in the lacI gene of Escherichia coli. J. Mol. Biol., 126, 847–863.
33. Chen,T., Mittelstaedt,R.A., Shelton,S.D., Dass,S.B., Manjanatha,M.G.,
Casciano,D.A. and Heflich,R.H. (2001) Gene- and tissue-specificity of
mutation in Big Blue® rats treated with the hepatocarcinogen N-hydroxy2-acetylaminofluorene. Environ. Mol. Mutagen., 37, 203–214.
34. Harbach,P.R., Zimmer,D.M., Filipunas,A.L., Mattes,W.B. and Aaron,C.S.
(1999) Spontaneous mutation spectrum at the lambda cII locus in liver,
lung, and spleen tissue of Big Blue® transgenic mice. Environ. Mol.
Mutagen., 33, 132–143.
35. Skopek,T.R., Kort,K.L., Marino,D.R., Mittal,L.V., Umbenhauer,D.R.,
Laws,G.M. and Adams,S.P. (1996) Mutagenic response of the endogenous
hprt gene and lacI transgene in benzo[a]pyrene-treated Big BlueTM
B6C3F1 mice. Environ. Mol. Mutagen., 28, 376–384.
36. Jakubczak,J.L., Merlino,G., French,J.E., Muller,W.J., Paul,B., Adhya,S.
and Garges,S. (1996) Analysis of genetic instability during mammary
tumor progression using a novel selection-based assay for in vivo mutations
in a bacteriophage λ transgene target. Proc. Natl Acad. Sci. USA, 93,
9073–9078.
Received May 2, 2002; revised June 27, 2002; accepted July 8, 2002
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