[CANCER RESEARCH54, 3357—3360,
July 1, 19941
Advances in Brief
Tamoxifen Induces Hepatic Aneuploidy and Mitotic Spindle Disruption after a
Single in Vivo Administration to Female Sprague-Dawley Rats'
Linda M. Sargent, Yvonne P. Dragan, Nicole Bahnub, John E. Wiley, Carol A. Sattler, Pauline Schroeder,
Gerald L SaWer, V. Craig Jordan, and Henry C. Pitot@
McArdle Laboratory for Cancer Research, The Medical Schoo4 University of Wisconsin@ Madison@ Wisconsin 53706 [L. M. S., Y. P. D., N. B., C. A. S., P. S., G. L. S., H. C. P.J;
Medical Genetics@ East Carolina University Medical Schoo4 Greenville, North Carolina 27858 [J. E. W.J; and Lurie Cancer Center, Northwestern University, Chicago, Illinois
60611 [V. C. J.J
Abstract
says in rats have demonstrated
Tamoxifen has found extensive use in the treatment of all stages of
human breast cancer. The efficacy of tamoxifen treatment for the pre
vention
of second
primary
tumors
and
Its chemosuppressive
action
prevention
of this disease
In women.
Recently,
tamoxifen
has been shown
to induce hepatocellular carcinomas in rats. For determination of the
mechanism
of Induction
of these tumors and assessment
of the possibility
of risk of human cancer development from tamoxifen treatment, female
Sprague-Dawley
rats (five rats per treatment)
were administered
tamox
Ifen at doses ranging from 03 to 35 mgi'kg.One day after treatment, the
rats were sacrificed, sad the hepatocytes were isolated and cultured for
50 h. Colcemid
was added 3 h prior to harvest, and the hepatocytes
were
then prepared for karyotypic evaluation. One hundred metaphase spreads
were examined
per animaL Tamoxifen
treatment
resulted in the induction
of aneuploldy In approximately 70% of the examined hepatocytes at the
doses used. In addition,
premature
condensation
(2—10%)and endoredu
plication (5-10%) were observed in hepatocytes of rats treated with
tamoxifen.
Furthermore,
exchanges
between
chromosomes
as well as
chromosomebreakage were observed. Examinationof the cultured hepa
both unipolar
spindles and incompletely
elongated
splndles
Ex
posure of rats to a single U? V1VO
dOSeOf tamoxifen produced multiple
changes In rat hepatocytesIncludingclastogenicdamage at dosescompa
rable to that administered
to humans. The occurrence of aneuploldy
induction, premature condensation, chromosome breakage, and Improper
mitotic spindle formation IndIcates that risk versus benefit of tamoxifen
ifen is an effective promoting agent in hepatocarcinogenesis in the rat
a reactive intermediate capable of binding to protein in a cell-free
system. In addition, 32P-postlabeled DNA adducts can be detected
after repeated doses of tamoxifen (10—12).Further studies have dem
onstrated that tamoxifen can induce a G@block in MCF-7 cells (13).
Francavila et a!. (14) have demonstrated that tamoxifen can inhibit
hepatic proliferation after a partial hepatectomy, suggesting that this
agent also induces a G@block in hepatocytes. Tamoxifen induces the
formation of micronuclei in a metabolism-competent lymphoblastoid
cell line (1 1, 12). Because of the striking carcinogenic effect of
tamoxifen on rat liver in vivo, its structural similarity to DES,3 and its
ability to induce micronuclei, the effects of tamoxifen on hepatic
ploidy and chromosomal integrity were investigated to determine
whether either of these types of genetic damage might be integral to
the carcinogenic action of tamoxifen.
In order to determine whether tamoxifen induced aneuploidy or chromo
some damage in rat liver in vivo, we administered tamoxifen citrate (Sigma
Chemical Co., St. Louis, MO) to groups containing five female Sprague
Dawley rats (Harlan Sprague-Dawley, Madison, WI) in doses of 0.3, 3.0, and
35.0 mg/kg by gavage. Alternatively,
the solvent, trioctanoin
(Pfaltz and
treatment should be carefully evaluated.
Bauer, Waterbury, CT), or the positive control for induction of aneuploidy
(15), 35 mg TCPOBOP, synthesized and provided as a gift by Dr. James
Introduction
Miller, McArdle Laboratory, was administered. Twenty-four hours after dos
ing, the rat livers were perfused with a collagenase solution as described
The antiestrogen, tamoxifen, has been used extensively to prevent
@
of malignant
Materials and Methods
tocytes from rats treated with tamoxifen by electron microscopy demon
strated
incidence
(8). Mani and Kupfer (9) have demonstrated that tamoxifen can form
In
animal models have led to Initiation ofclinical trials to test its emeacy for
an increased
liver neoplasms compared with that in untreated animals (7). Tamox
the recurrence of human breast neoplasms, as well as the appearance
of further primary lesions (1). The efficacy of tamoxifen treatment for
all stages of breast cancer (1, 2) and the low incidence of acute side
previously (16). Hepatocytes were separated from littoral cells by a Percoll
(Pharmacia,
LKB Biotechnology,
Piscataway,
NJ) isodensity centrifugation
and immediately plated in 75-cm collagen-coated flasks (16) at a density of
2.6 X
cells in 15 ml of serum-free “supplemented―
Dulbecco's modified
Eagle's medium/Ham's
F-12 medium (17). The medium was supplemented
effects associated with its use (3) have led to the institution of
with bovine serum albumin (0.2%), 4-(2-hydroxyethyl)-1-piperazineethanesul
large-scale clinical trials using tamoxifen as a chemopreventive agent
fonic acid (18 mM), and sodium pyruvate (5 mM). The medium was changed
in individuals with an increased risk of breast cancer (4, 5). This use
after 3 h, and 10 ng epidermal growth factor (Collaborative Biomedical
of tamoxifen for nonmalignant indications has prompted a reconsid
eration of the toxicology of tamoxifen. While studies on the toxicity
of tamoxifen have not shown significant mutagenicity in microbial
Products, Bedford, MA) was added per ml of medium to stimulate cell
division. After 47 h ofculture, 40 ng/ml Colcemid was added to each flask, and
the cells were incubated for an additional 3 h. Parallel cultures were treated
with 25 g.LM5-bromo-2'-deoxyuridine
(Sigma) for the last 6 h of culture to
permit analysis of late replication banding (18). Cultures were randomly
systems (6), several experimental results suggest that tamoxifen may
possess a carcinogenic potential. Recent results from chronic bioas
selected, and the cells were preparedfor examinationby electronmicroscopy
by methods described previously (19) or harvested for karyotypic analysis. The
Received 3/9/94; accepted 5/19/94.
Thecostsof publication
of thisarticleweredefrayedinpartbythepaymentof page
charges.Thisarticlemustthereforebe herebymarkedadvertisementInaccordancewith
18U.S.C.Section1734solelyto Indicatethisfact.
1This work was supportedin part by National Cancer Institute Grants CA-07175,
hepatocytes were removed from the dish with 0.01% collagenase and harvested
by hypotonictreatment(0.075MKC1)for 8.5 mm. Once removedfrom the
flask, the hepatocytcs
were fixed with mcthanol:acctic
acid (3:1, v/v), and
slides were prepared as described by Sargent et a!. (20). One hundred meta
CA-22484,andCA-57245,andby SpecialInstitutionalGrantSIO-15fromtheAmerican phases of good morphology were randomly selected from cultures of each rat.
CancerSociety.
2 To whom requestsfor reprints should be addressed,at McArdle Laboratory for
CancerResearch,
University
ofWisconsln,
1400UnIversity
Avenue,Madison,
Wisconsin
53706.
3 The abbreviationsused are: DES, diethylstilbestrol;TCPOBOP, 1,4-bis[2-(3,5-dl
ch1omnvridvlnxv@1benzene.
3357
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TAMOXIFEN INDUCES HEPA11C ANEUPLOIDY IN VIVO
Table 1 Effect of a single oral dose of tamoxifen on rat hepatic chromosomal number and integrity
Average percentage of hepatocytes in cultures from five rats at each dose ±SD demonstrating chromosome breakage and other aberrations after treatment in vivo with tamoxifen
in trioctanoin at 0.3—35mg/kg administered in vivo by gavage. Values for hepatocyte cultures from five rats receiving the solvent only or TCPOBOP are also given. TCPOBOP was
administered at a dose of 35 mg/kg by gavage (23).
PrematureTreatmentAneuploidyBreakageCondensationExchangesEndoreduplicationSolvent
2.0<1.0<1.0<1.035
control3
±3.05.0
±
5.0―<1.0<1.0<1.00.3
mg TCPOBOP/kg65
±6.5a10.0
±
±8.0°
70 ±5.0―8.0
mg Tam/kg
2.0―35
3 mg Tam/kg71
2.0―a mg Tam/kg85
Significant
difference
±2.0
±7.0―23.0
from
thecontrol
±1.0
9.4 ±3.02.0
±4.0―10.0
±1.0―
±3.0―
5.0 ±2.0―5.0
±2.0―10.0
6.0 ±
±
treatment (P < 0.05); Tam, tamoxifen.
In the animals examined, approximately 70% of the hepatocytes were tet
raploid (chromosome number is 84), and 30% were diploid (chromosome
than 0.3 mg/kg may be necessary to demonstrate a dose-dependent
effect for induction of aneuploidy by tamoxifen. Tamoxifen treatment
number is 42). A spread was considered
induced a dose-related increase in premature condensation and chro
aneuploid if it deviated from the
established diploid or tetraploid number of chromosomes. The metaphases
were analyzed blindly for ploidy, breakage, exchanges, premature condense
tion, and endoreduplication.
@
4.0 ±2.5―8.0
±5.0―10.0
The significance
of differences between groups
was determined by nonparametric
statistics; evidence of a dose-response
relationship was tested by regression analysis. The alpha level was adjusted to
take into account the number of comparisons.
Results
Karyotypic analysis of metaphase spreads from Sprague-Dawley
female rats was performed to determine the effect of a single tamox
lien treatment on the number and integrity of hepatic chromosomes.
Only a low level of breakage and aneuploidy similar to that reported
previously for control untreated rats was observed in the solvent
treated rats (Table 1). TCPOBOP, which has previously been shown
to induce aneuploidy in the rat liver in vivo (15), was used as a
positive control to demonstrate that aneuploidy could be detected with
this model. The present study showed that 35 mg TCPOBOP/kg body
weight resulted in substantial aneuploidy (Table 1). Tamoxifen also
mosome breakage (Table 1) that was significantly greater than that
observed in solvent-treated rats (P < 0.05). Additionally, a dose
dependent induction of endoreduplication was observed with tamox
ifen treatment. Furthermore, a significant level (P < 0.05) of aneu
ploidy, chromosomal
exchanges, and endoreduplication
were
observed for all doses of tamoxifen administered (Table 1).
Fig. 1 is a photomicrograph of a metaphase of a type typically
observed in rats treated with 35 mg tamoxifen citrate/kg. The spread
is aneuploid. The presence of diplochromosomes (paired homologous
chromosomes) indicates that the spread has undergone endoredupli
cation. In addition, chromosomes without a homologue can be ob
served (Fig. 1, arrow). Also, two exchanges are shown in this mets
phase spread (Fig. 1, double arrows). All of the observed exchanges
were between homologous chromosomes, indicating that the cx
all doses of tamoxifen tested (P < 0.05). Since the level of aneuploidy
changes occurred during S synthesis (21).
Since the mechanisms of induction of aneuploidy and endoredupli
cation can be quite varied (22), two factors potentially responsible for
these effects were examined. In order to determine whether the
tamoxifen-induced aneuploidy and endoreduplication are associated
with an inhibition of chromosome synthesis, we examined metaphase
spreads by late replication banding (18). In these studies, all meta
phase spreads investigated showed a normal banding pattern with a
noted was independent of the dose of tamoxifen used, doses lower
light-staining, late-replicating X chromosome (data not shown), mdi
induced a marked level of aneuploidy compared with the solvent
control (P < 0.05). The level of aneuploidy was extremely high
compared with the control. Aneuploidy was significantly increased for
&
S
a
Fig. 1. A hepatic metaphase spread prepared from
a female Sprague-Dawley rat administered 35 mg/kg
tamoxifen citrate and sacrificed 24 h later is shown.
1'•
This metaphase spread demonstrates aneuploidy, cx
changes, and endoreduplication. The two exchanges
are indicated by double arrows, while the three chro
mosomes missing a homologue are indicated by sin
gle arrows. Hepatocytes were isolated by perfusion
and Percoll isodensity centrifugation before culture
and preparation for cytogenetic analysis. Further de
tails on the methods are described in Table 1 and in
“Materials
and Methods.―
4:
F
3358
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TAMOXIFEN
INDUCES
HEPATIC
eating that the observed aneuploidy and endoreduplication were not
due to a block in S synthesis.
Examination of hepatic metaphase spreads from solvent- and ta
moxifen-treated rats by electron microscopy shows some interesting
differences in mitotic spindle integrity (Fig. 2). Bipolar spindles were
noted in control cultures (Fig. 2A), while in a significant number of
cultures from tamoxifen-treated rats, monopolar spindles (Fig. 2B)
were observed. Of the 36 informative spindles examined, 9 were
monopolar. In addition, the organelles were not excluded from the
spindle in a number of these spreads. In some tamoxifen-treated
hepatocytes (data not shown), a normal bipolar spindle appeared to
have formed, but during anaphase the spindle elongation was much
less than in untreated hepatocytes. Thus, while tamoxifen treatment
does not appear to affect S phase DNA synthesis, there is clearly an
ANEUPLOIDY
IN VIVO
effect of the in vivo administration of this agent on the structure of the
mitotic apparatus that could lead to aneuploidy (22).
Discussion
The administration of tamoxifen induces premature condensation of
the hepatic chromosomes, chromosome exchanges, and chromosome
breaks. Compounds structurally related to tamoxifen can block cell
cycle progression (23). In addition, another structural analogue of
tamoxifen, DES, induces mitotic arrest (24). Tamoxifen induces a G@
block in MCF-7 cells (13) and possibly in hepatocytes in vivo (14).
Since the blockage of cell cycle progression can result in premature
condensation, this action of tamoxifen should be explored to uncover
the mechanism by which tamoxifen causes premature condensation.
The presence of tamoxifen-induced
“@
p.@.
@
.
chromosomal breakage. Since the exchanges were observed in this
_n.••.p
.
DNA adducts (10—12)may result
in premature condensation, exchanges between chromosomes, and
.@
study only between homologous chromosomes, the exchanges cc
curred during S (21). Thus, tamoxifen-induced
DNA damage results
in changes in cell cycle progression and further karyotypic instability.
Tamoxifen administration
in vivo results in aneuploidy, an alter
ation in the number of chromosomes, and endoreduplication, which is
the result of two or more synthesis cycles in the absence of an
intervening mitosis. A block in DNA synthesis or the inhibition or
delay of spindle formation can result in endocycles and in aneuploidy.
The presence of a light-staining, late-replicating X chromosome in
dicates that the endoreduplication and aneuploidy that occurred after
tamoxifen treatment were not due to a block in S synthesis (18). The
presence of monopolar spindles and incompletely elongated spindles
from which the organelles were not excluded suggests that the aneu
ploidy and endoreduplication observed with tamoxifen treatment may
be due to the induction of spindle aberrations. A structural analogue
of tamoxifen, DES, has also been shown to induce micronuclei (25),
aneuploidy (26), and cellular transformation (27). In addition, DES
treated cells have been shown to undergo mitotic arrest (24) and to
contain monopolar spindles (28). Therefore, this class of agents ap
pears to have multiple effects on the cell that result in the disruption
of the normally tightly regulated coordination of spindle formation
and chromosomal integrity.
Multiple factors are involved in the maintenance and proper for
mation of the mitotic spindle apparatus. Although the mechanism by
which tamoxifen induces aneuploidy and karyotypic instability is
unknown, tamoxifen is known to alter cellular calcium levels (29) and
acts as a calmodulin antagonist (30). Although the role of calmodulin
in spindle assembly is not known, calmodulin is associated with the
spindle pole body and is required for proper formation of the spindle
apparatus (31). Calmodulin-defective mutants in yeast contain a single
spindle pole body, monopolar spindles, and shortened spindles that
appear not to have elongated (31). The phenotypes associated with
these calmodulin mutants include aneuploidy in some of the cells, as
well as a low percentage of cells with endoreduplication (32). Since
these phenotypes are similar to the observed effects of tamoxifen, the
aneuploidy and endoreduplication observed in the rat may be due to
inhibition of the action of calmodulin. Furthermore, yeast mutants that
have improper spindle pole body formation also contain monopolar
spindles (31, 32), as was observed in the present study in rat hepato
Fig. 2. Electron micrograph ofa dividing hepatocyte prepared from a Sprague-Dawley
rat followingtrioctanoinintubation(A)or treatmentwith3 mg/kgtamoxifenCitrate(B).
The metaphasespreadin (A) hasa spindleapparatusof normallengthandcontainstwo
poles. The metaphase cell in (B) contains a monopolar spindle. In addition, the organelles
arc not excluded from the metaphase plate in the spread provided in (B). B, arrow, the
single pair of centrioles in this spindle apparatus.
cytes, further supporting the possibility that tamoxifen may alter both
the number and integrity of chromosomes through its inhibitory action
on calmodulin or one of the spindle pole body proteins.
The effectiveness of tamoxifen as a clastogenic agent indicates
that tamoxifen induces changes in both the number and structural
integrity of the chromosomes
and may increase the progression
of
cells toward frank neoplasia. Aneuploidy and endoreduplication
3359
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TAMOXIFEN INDUCES HEPATIC ANEUPLOIDY IN VJVO
tamoxifen on hepatocyte proliferation in vivo and in vitro. Hepatology, 9: 614—620,
1989.
are often associated with malignant conversion (33). In addition,
the hallmark of the stage of progression is one of an evolving
karyotypic instability, including an increase in aneuploidy and
chromosome breakage with gene deletions, rearrangements, and
duplications (34). Since tamoxifen possesses clastogenic action as
evidenced by the induction of micronuclei, aneuploidy, and chro
mosomal breakage, its carcinogenic action may include effective
ness during the stage of progression. This supposition is supported
by the induction of transformation of Syrian hamster embryo cells
15. Rossi, A. M., Zaccaro, L, Rosselli, F., and Quatirone, C. Clastogenic effects induced
in mice and rats by 1,4-bis[2-(3,5-dichloropyridyloxy)Jbenzene,a phenobarbital-like
enzyme inducer and liver tumour promoter. Carcinogenesis (Lond.), 9: 1147—1151,
1988.
16. Xu, Y-H., Sattler, G. L., and Pitot, H. C. A method for the comparative study of
replicative DNA synthesis in GOT-positive and GOT-negative hepatocytes in primary
culture isolated from carcinogen-treated rats. In Vitro Cell. Dcv. Biol., 24: 995—1000,
1988.
17. Li, Y., Sattler, 0., and Pitot, H. Oxaloacetate induces DNA synthesis and mitosis in
primary cultured rat hepatocytes in the absence of EGF. Biochem. Biophys. Res.
Commun., 193: 1339—1346, 1993.
by tamoxifen (35). Retrospective studies have found an increased
incidence of endometrial cancers in tamoxifen-treated women (36).
The present study performed in rat hepatocytes indicates that
tamoxifen alters the number of chromosomes, increases the mci
dence of chromosomal aberrations, and may disrupt mitotic spindle
integrity. The carcinogenic action of tamoxifen for the rat liver,
coupled with the occurrence of aggressive breast and uterine
18. Huret, J., Delabar, J., Marlens, F., Aurias, A., Tanger, J., and Sinet, D. Downs
syndrome with duplication of a region of chromosome 21 containing the CuZn
superoxide dismutase gene without detectable karyotypic abnormalities. Hum. Genet.,
75: 251—257,1987.
19. Sattler, C. A., Sawada, N., Sattler, G. L, and Pitot, H. C. Electron microscopic and
time lapse studies of mitosis in cultured rat hepatocytes. Hepatology, 8: 1540—1549,
1988.
20. Sargent, L, Xu, Y-h., Sattler, 0. L, Meisner, L, and Pitot, H. C. Ploidy and
karyotype of hepatocytes isolated from enzyme-altered foci in two different protocols
tumors, albeit at a low incidence in women treated with tamoxifen,
of multistage hepatocarcinogenesis in the rat. Carcinogenesis (Lond.), 10: 387—391,
1989.
suggests that a careful assessment of the risk as well as the benefit
of chronic tamoxifen administration should be considered in the
nonmalignant
21. DiPaolo, J., and Popescu, N. Banding pattern analysis of initial structural chromo
some alterations induced by N-methyl-N-nitroso-N-nitmsoquinone in Syrian hamster
cells. Mutat. Res., 44: 359-368, 1977.
use of this drug.
22. Paweletz,
N., Ghosh,
S., and Vig, B. K. Mitosis
and the induction
of aneuploidy.
In:
Mechanisms of Chromosome Distribution and Aneuploidy, pp. 217—229.Alan R.
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Tamoxifen Induces Hepatic Aneuploidy and Mitotic Spindle
Disruption after a Single in Vivo Administration to Female
Sprague-Dawley Rats
Linda M. Sargent, Yvonne P. Dragan, Nicole Bahnub, et al.
Cancer Res 1994;54:3357-3360.
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Department at [email protected].
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