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EVALUATION OF THE CHANGES RESULTING FROM TAMOXIFEN
EVALUATION OF THE CHANGES RESULTING FROM TAMOXIFEN RESULTING FROM TAMOXIFEN
ADMINISTRATION. A COMBINED DNA
RESULTING FROM TAMOXIFEN ADMINISTRATION. A COMBINED DNA A COMBINED DNA
FLOWCYTOMETRIC AND HISTOPATHOLOGICAL STUDY
A COMBINED DNA FLOWCYTOMETRIC AND
HISTOPATHOLOGICAL STUDY
TamoxifenTamoxifen
Tamoxifen is a non steroidal tiphenylethylene first synthesized in p y y y1966. The drug was initially developed as an The drug was initially developed as an oral contraceptive, but instead of blocking ovarian function tamoxifen blocking ovarian function, tamoxifen was found to induce ovulation.
TamoxifenTamoxifenTamoxifenTamoxifen
T if is id l s d s ti st iTamoxifen is widely used as an anti estroginadjuvant therapy for breast cancerp ti ts d s h m p ti tpatients and as a chemopreventive agentfor healthy women at high risk for breast
ncancer.
TamoxifenTamoxifenTamoxifenTamoxifen
Since 1971 it has been usedsuccessfully to treat many millions ofwomen.
Treatment results showed anincrease in disease free survival andd i f bdecrease in recurrence rate of breastcancer.
TamoxifenOn October 29,1998, the Food and Drug Administration (FDA) approved Nolvadex Adm n strat on (FDA) approved Nolvadex (tamoxifen citrate) for reducing the incidence of breast cancer in women at high risk for developing the disease.
TamoxifenTamoxifenTamoxifenTamoxifen
With the widespread therapeutic andWith the widespread therapeutic andemerging prophylactic use oftamoxifen there has been muchtamoxifen, there has been muchdiscussion about side-effects of thedrug particularly its carcinogenicitydrug, particularly its carcinogenicity
TamoxifenTamoxifenTamoxifenTamoxifen
Numerous studies have established anincreased incidence of endometrialf mcancer among women taking tamoxifen.
Recent results confirm not only anincreased incidence of endometrialfcancer but also increased mortality fromthe disease
TamoxifenTamoxifenTamoxifenTamoxifen
A number of studies are compatiblewith genotoxic activity.g y
Tamoxifen induces micronuclei inTamoxifen induces micronuclei inmetabolically competent human cellscauses aneuploidy and chromosomalcauses aneuploidy and chromosomalaberrations
TamoxifenTamoxifen
I t t if i t tIn rats, tamoxifen is a potenthepatocarcinogen in bothp gmales and females.
TamoxifenTamoxifen
It has been characterized as ahuman carcinogen by thehuman carcinogen by theInternational Agency forResearch on CancerResearch on Cancer.
AimAimAimAim
The aim of the present study is toevaluate the effect of tamoxifenadministration on uterus, kidney,spleen and liver tissues in mice byp ydetecting the histopathologic changesas well as the DNA ploidy and cellp ycycle phase analysis.
Material and methodsMaterial and methodsMaterial and methodsMaterial and methodsA total of 40 female Swiss Albino mice wereA total of 40 female Swiss Albino mice wereinvolved in the study.
The animals were divided at random into 2 groups:Mice of the first group were served as controlf f g pgroup, were orally administered with normal saline3 times /week for 6 weeks.
Mice of the second group were orally administeredwith 20mg/kg tamoxifen 3 times /week for 6 weeks.
Material and methodsMaterial and methodsMaterial and methodsMaterial and methods
Animals were sacrificed and the uterusspleen liver and kidneys were removed andspleen liver and kidneys were removed andeach organ was divided into 2 pieces.
one piece was fixed in 10% formaldehydesolution dehydrated, embedded in wax,
d d f d d d hsectioned, deparafinised and stained withH&E for histopathological examination.
Material and methodsMaterial and methodsMaterial and methodsMaterial and methods
The other parts of the previouslymentioned organs were directlyg ytransferred to the flow cytometrylaboratory where single cell suspensionsy g pwere prepared and stained withpropidium iodide (PI) and subjected top p ( ) jDNA flow cytometric analysis.
RESULTSRESULTSRESULTSRESULTS
Uterine tissue:The results of the histopathologicalThe results of the histopathologicalexamination indicated that there is apathological change in the form ofpathological change in the form ofsquamous metaplasia and dysplasia ofthe glandular epithelial cellsthe glandular epithelial cells.
Dysplastic Squamous cells, lining endometrum (following drug application) are large in size, with vesicular nuclei (1) and prominent nuclei (2), kerato hyaline granules (3) are abundant in upper layers.
The effect of TAM treated on DNA ploidy in th t i ti the uterine tissue.
1 0 01 0 0
5 1 3 38 0
1 0 0
ploi
d ce
lls
4 8 . 6 65 1 . 3 3
4 0
6 0
id a
nd a
neu
02 0
% o
f dip
oi
0D i p l o i d A n e u pl o i d
C o n tr o l T A MC o n tr o l T A M
Percent of diploid and aneuploid cells in the uterus of the normal control and 6 weeks
treated animals
Percent of cell cycle phases of diploid y p pand aneuploid cells in the uterus
80%
100%
hase
s S
G2-M
60%
80%
l cyc
le p
h G0-G1
40%
nt o
f cel
l
0%
20%
Per
cen
c ontr ol TA M contr ol TAM
D iploid ce lls Ane uploid c ells
ResultsResults
Histopathological examination of KidneyHistopathological examination of Kidneytissues showed a minimal change inthe form of focal renal lesion in thethe form of focal renal lesion in therenal tubular epithelium.
renal tubular epithelial cells with higher nuclear cytoplasmic ratio, hyper chromaticnuclei (»), density esinophilic cytoplasm
The effect of TAM treatment on DNA ploidy in the kidney tissues.
1 0 0 9 7 .51 0 0
ells
6 0
8 0
neup
loid
ce
4 0
6 0
ipoi
d an
d an
0 2 .5
0
2 0
% o
f di
0D i pl oi d A n e u pl o i d
C o n tro l T A M
Percent of diploid and aneuploid cells in the kidney of the normal control
and 6 weeks treated animals.
Percent of cell cycle phases of diploid and aneuploid cells in The kidney
80%
100%
phas
es S
G2-M
G0-G1
60%
ll cy
cle
p G0 G1
20%
40%
ent o
f cel
0%
20%
Perc
e
control TAM control TAM
Diploid cells Aneuploid cells
ResultsResults
No histopathologicl changes wasNo histopathologicl changes wasdetected in the spleen.
spleen of mice treated with tamoxifen for 6 weeks revealing no pathological changes
The effect of TAM treatment on DNA ploidy in the spleen tissues
1 0 0 1 0 01 0 0
lls
6 0
8 0
neup
loid
ce
4 0
6 0
poid
and
an
0 02 0
% o
f dip
0D i pl oi d A n eu pl o i d
C o n tro l T A MC o n tro l T A M
P e r c e n t o f d ip lo id a n d a n e u p lo id c e l ls in th e s p le e n o f th e n o r m a l c o n tr o l a n d T A M
tr e a te d a n im a ls .
Percent of cell cycle phases of diploid and aneuploid cells in The spleenp p
100%s S
80%
e ph
ases
G2-M
G0-G1
%
60%
cell
cycl
e
20%
40%
cent
of c
0%
Per
c
control TAM control TAM
Diploid cells Aneuploid cells
ResultsResultsResultsResults
No histopathological changes wereNo histopathological changes weredetected in the liver tissues oftamoxifen treated animalstamoxifen treated animals.
section from liver in tamoxifen treated mice, showing no pathological changes
The effect of TAM treated on DNA ploidy in the liver tissuesp y
1 0 0 1 0 01 0 0
ells
6 0
8 0
neup
loid
ce
4 0
6 0
dipo
id a
nd a
0 0
0
2 0
% o
f d
D i p l o i d A n e u pl o i d
C o n tr o l T A M
Percent of diploid and aneuploid cells in the liver of the normal control and TAM treated
animals
Percent of cell cycle phases of diploid and aneuploid cells in The liveraneuploid cells in The liver.
100% S
80%
G2-M
G0-G1
40%
60%
20%
0%
Control TAM Control TAM
Diploid cells Aneuploid cells
ConclusionConclusionFrom the study we can conclude that tamoxifenmay induce ploidy changes and disturbance in thecell cycle in the uterus kidney and spleen tissuescell cycle in the uterus, kidney and spleen tissuesof female mice.
Tamoxifen may induce cell proliferation in the cellsof the uterus and spleen and this is clear from theincreased percentage of cells in the S phase inincreased percentage of cells in the S phase inthese two tissues.
Beside the histopathological investigation we mayneed to use the DNA flow cytometric analysis todetect the early changes resulting from tamoxifent ct th ar y chang s r su t ng from tamo f nadministration that may results into malignanttransformantion.
