evaluation of the changes resulting … of the changes... · evaluation of the changes resulting...

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.