ultrastructural and immunohistochemical evaluation of apoptosis in

Bull Vet Inst Pulawy 49, 475-478, 2005
ULTRASTRUCTURAL AND IMMUNOHISTOCHEMICAL
EVALUATION OF APOPTOSIS IN FOETAL RAT LIVER
AFTER ADRIAMYCIN ADMINISTRATION
AGNIESZKA PEDRYCZ, ZBIGNIEW BORATYŃSKI1,
MARCIN WIECZORSKI AND JUSTYNIAN VISCONTI
Department of Histology and Embryology,
Medical University of Lublin, 20-080 Lublin, Poland
1
Department of Animal Anatomy and Histology, Faculty of Veterinary Medicine,
Agricultural University of Lublin, 20-950 Lublin, Poland
e-mail: [email protected]
Received for publication June 30, 2005.
Abstract
The purpose of this study was to show apoptosis in
foetal hepatocytes as a late effect of adriamycin given to
female rats in a single dose ( 5 mg/kg of b.w.,
intraperitoneally) a long time before planned pregnancy. Rats
were decapitated on 20th d of pregnancy and for further
investigation the foetal liver was taken. Apoptosis was
evaluated on the basis of the analysis of electron microscope
picture of hepatocytes and the immunohistochemical
identification of BCL-2 and BAX proteins. The BAX
expression was significantly increased in the experimental
group compared to the control group, whereas BCL-2 was not
expressed (the same as in the control group). The high level of
apoptotic index in the experimental group was caused by both
the lack of BCL-2 protein expression and the intensive
expression of the BAX protein. In the group of control foetuses
apoptosis was not intensive, but was present as physiological
natural cell death during embryogenesis process. In
hepatocytes of foetuses whose mothers were treated with
adriamycin in single dose a long time before pregnancy, there
was observed increased apoptosis.
Key words: rat foetus, adriamycin, liver,
apoptosis, electron microscopy, immunohistochemistry.
It is known that adriamycin (ADR),
antineoplastic antibiotic cause apoptotic death in
hepatocytes of adult rats (10, 16). Apoptosis is actually,
without a doubt, one of the most often described
phenomenon. Natural, physiological cell death programmed death - happens as a result of organism
aging, but not only because of this. Altruist cells commit
suicide for the sake of the whole organism. Apoptotic
physiological cell death is often determined as a result of
the organizational comfort of cell structure
disintegration, that is mitochondria, endoplasmic
reticulum with a lack of typical necrotic signs.
Physiological apoptosis appears, among others, in
embryogenesis. During foetal life, part of new-arised
cells decrease to leave the best cells in new organism.
The present study evaluated foetal liver cells of rats
whose mothers 4 weeks before the planned pregnancy
were treated with adriamycin.
A dose of 5 mg/kg of body weight used in the
present study is known as one which in experimental
animals, including rats, allows for full-term pregnancy
(11). It is also known that adriamycin administered to
pregnant female rats causes tracheo-oesophageal fistulas
with oesophageus atresia in foetuses, which could be
used in a foetal rat model of oesophageal atresia (3, 12,
13). Osteotic changes in foetal rats exposed to
adriamycin are also relatively more frequent (5).
Because of numerous metabolic pathways, the
liver is especially exposed to the harmful effect of
exogenous substances, including drugs. This study
shows a typical physiological death of hepatic cells of
healthy rat foetuses and apoptosis of rat foetal
hepatocytes after the damaging effect of exogenous
factors. In apoptosis study, the evaluation of a picture
from electron microscope was used and BCL-2 and
BAX
proteins
were
identified
using
immunohistochemistry method. BAX protein is one of
the main factors of apoptosis. BCL-2 is anti-apoptotic
protein. Immunohistochemical detection of places of
BAX protein expression could assess the localization
and intensity of physiological and pathological
apoptosis, which could help in distinguishing these two
types of cell death.
Material and Methods
Sixteen foetuses were used in the experimental
group, chosen randomly two from each of 8 female rats
which were treated with adriamycin administered
intraperitoneally 4 weeks before fertilization in a single
476
dose of 5 mg/kg of body weight. The control group
consisted of 16 foetuses chosen randomly two from each
of 8 female rats which were treated with 0.5 ml of 0.9%
NaCl administered intraperitoneally in a single dose 4
weeks before fertilization.
After the decapitation of the pregnant females
(on 20th d of pregnancy) the chosen foetuses were also
decapitated and liver samples were taken from them.
The samples taken for electron microscopy were fixed in
fluid containing 2% paraformaldehyde and 2.5%
glutaraldehyde in 0.1 M Sørensen phosphate buffer.
Then the preparations were treated with osmium
tetraoxide, stained in uranyl acetate, dehydrated in
increasing concentrations of ethanol and embedded in
Aralchit ACM Fliska resins. The preparations were cut
into 0.5-0.7 µm and 60 nm thick sections with Reichert
Ultracut S ultramicrotom. The ultra-thin sections were
stained with 8% solution of uranyl acetate in 0.5% acetic
acid and lead citrate. The photographic documentation
was performed with Tesla BS-500 electron microscope.
Samples taken for immunohistochemical
studies were fixed in 10% formalin, and then after
dehydratation and embedding in paraffin cut into 7 µm
sections. To identify BAX and BCL-2 proteins,
preparations from both groups were used. For each
preparation a negative control was performed (a slide
without primary antibody).
The proteins expression level was evaluated
with a standard three-step immunohistochemical
procedure. Rabbit BCL-2 and BAX antibodies were
used as a primary antibody. Then biotinylated secondary
antibody was added, and then horse-radish peroxidase
conjugated with streptavidin. Since streptavidin has a
great affinity to biotin, it binds to the place where
primary antibody coated the background, and after
adding a chromatogen (DAB or AED) a reddish colour
appears.
Hepatocyte nuclei showed different shapes and
size. In some cells pyknotic nuclei with chromatin
condensed circumpherentially were found (Fig. 4). In
some cases an evident vacuolization of the nuclei was
observed. In some cells “rinsed” cytoplasm, without
organelles - cells “empty inside” was observed (Fig. 3),
while in other cells, the destruction of cell organelles
was found. Mitochondria were often swollen, with
bright matrix and cristal destruction, some of them were
damaged, with ruptured mitochondrial membrane.
Rough endoplasmic reticulum was quite often
defragmented (Fig. 3). An increased amount of
lysosomes was also found in hepatocyte cytoplasm.
Numerous autophagosomes were observed close to
cholangial tubules (Fig. 3). Perisinusal spaces were often
dilated and swollen with the content of damaged
hepatocyte cytoplasm. Connective tissue proliferation
was observed, which was the evidence of cell membrane
damage, which was also shown by the presence of
erythrocytes in hepatocyte cytoplasm (Figs 3 and 4).
Changes were also observed in blood stem cells
- deformed cells, “naked nuclei”. These changes as a
result led to atrophia of haematopoetic focuses (Figs 3
and 4). Numerous cell divisions were the evidence of
attempts at liver regeneration.
The expression of BAX protein was observed
in the experimental (Fig. 8) and control groups (Fig. 6),
but in the experimental group BAX was expressed more
strongly (Fig. 8). In the control group apoptotic changed
cells were disposed rather steadily. The most intensive
expression in the experimental group was observed in
periportal regions of hepatic lobes.
BCL-2 protein was not expressed in rat foetal
liver preparations either in the experimental or in the
control groups (Figs 5, 6, 7 and 8).
Discussion
Results
Foetal hepatocytes from the control group
showed some difference as compared to the hepatocytes
from adult individuals known from manual descriptions,
which was due to the developmental stadium, intensity
of proliferation, and liver embryogenesis. Foetal
hepatocytes were much smaller with a smaller amount
of the cytoplasm in relation to the nucleus as compared
to mature hepatocytes (Fig. 1). The lack of
specialization was manifested with a smaller amount of
cell organelles in the cytoplasm (Fig. 1).
Haematopoetic cell focuses in different stages
of the development, as well as erythroblasts in blood
vessel lumen were visible in the foetal liver (Fig. 2). The
observed divisions of hepatocytes and blood cells were
the evidence of intensive proliferation (Fig. 1). There
were no signs of cell damage. (Fig. 2)
Some of the foetal hepatocytes from the
experimental group were characterized by steatosis and
necrosis. But the most frequent changes were apoptotic
features.
The digestive system, together with the liver
and pancreas, derives from the primary gut. The gut
develops during the formation of the head and caudal
and lateral folds of embryo. After the fold formation is
finished, the gut can be divided into anterior, medium,
and posterior gut. The anterior gut could be divided into
the head part from the oral-throat membrane to the place
of forming lung bud and the caudal part - from lung bud
to the place where a liver bud is formed. The liver bud
appears, as a epithelium thickening in the most caudal
part of the primary anterior gut.
Adriamycin toxicity is known and proved. The
histological ultrastructural changes present in this study
(cell lysis, effacement of cell structures, swelling of
mitochondria with brightening of the matrix and partial
crest destruction, some mitochondria damage,
defragmentation of rough endoplasmatic reticulum) fully
confirm earlier reports, describing damage of adult rat
liver after adriamycin treatment (10). These changes are
typical for apoptosis. A few stages of apoptotic cell
degeneration could be distinguished.
477
Fig. 1. Electronogram of part foetal liver of rat from
control group. Visible cell in division (arrow) and nuclei
(n). 3000x.
Fig. 2. Electronogram of part foetal liver of rat from
control group. Hepatocytes and blood cells present.
Erythroblast in blood vessel (arrows). 3000x.
Fig. 3. Electronogram of foetal hepatocytes of rat from
experimental group. Significant cell damage, formation of
autophagosomes (AF). 3000x.
Fig. 4. Electronogram of foetal hepatocytes of rat from
experimental group. Visible pyknotic nuclei in
hepatocytes. 3000x.
478
Fig. 5. Section of foetal liver from control group.
BCL-2 expression. 360x.
Fig. 6. Section of foetal liver from control group.
BAX expression. 360x.
Fig. 7. Section of foetal liver from experimental group.
BCL-2 expression. 360x.
Fig. 8. Section of foetal liver from experimental group.
BAX expression. 400x.
The early morphologic changes can include the damage
of plasmatic membranes, decrease of cell volume, and
nuclear pyknosis. More advanced changes are complete
destruction and fragmentation of nuclei with forming of
apoptotic bodies. The final stage is phagocythosis of
apoptotic bodies and the remains of the destroyed cell by
other cell with phagocytic capacities.
Asakura et al. (2) proved that adriamycin
conjugated with glutathione via glutaraldehyde inhibited
the activity of glutathione S-transferase, playing an
important part in apoptosis induction, in rat hepatoma
cells.
It was proved that therapeutical doses of
adriamycin increase lipid peroxidation in microsomes
and in mitochondria of the liver, especially in the
presence of Fe+3 ions (8, 14).
Ganey et al. (7) showed that the toxicity of that
drug is oxygen-dependent. In periportal regions of
hepatic lobes, which are rich in oxygen, an increase in
oxygen intake was proportionally dependent on
adriamycin dose, whereas the compound had no
influence on oxygen intake in hepatocytes in region of
central vein, which is poor in oxygen (7). Perhaps that is
why in the present experiment BAX protein expression
present in experimental liver sections was located
mainly in periportal regions of hepatic lobes in contrast
to control group where BAX protein expression were
disposed rather steadily in all preparations.
BCL-2 oncoprotein evaluated in the present
experiments regulates programmed cell death (15) by
providing a survival advantage to rapidly proliferating
cells, while BAX protein promotes apoptosis by
enhancing cell susceptibility to apoptotic stimuli. The
multidominian proapoptotic molecule BAX is required
to initiate the mitochondrial pathway of apoptosis (4).
Increased expression of BAX protein could be the
evidence that the mitochondrial pathway of apoptosis
started. The presence of performed apoptotic process
was confirmed by microscopical view.
Apoptosis is an important mechanism
regulating cell number and their development in
different organs and tissues, which is essential in
embryogenesis and aging processes, as well as in
removing harmful and useless cells from the body (6).
Apoptosis of cells in the healthy human embryo was
observed by Lichnowsky et al. (9). They noticed an
increase in BCL-2 and BAX protein expression using
the immunohistochemical method. They assessed
BAX/BCL-2 apoptotic index. The low value of
apoptotic index was mostly accompanied by high
479
expression of BCL-2 – antiapoptotic protein. It suggests
the presence of apoptosis in embryogenesis, which is
regulated by high level of antiapoptotic protein.
In the present study, increased expression of
BCL-2 protein in the investigated rat foetal liver was not
observed, which suggests the lack of proliferation and
regeneration processes. BAX protein found in the
control rat foetal liver shows the presence of the
apoptosis process. Apoptosis in the control group
dominated the proliferation processes; however, it was
significantly smaller than in the experimental group. The
high level of apoptotic index in the experimental group
was caused both by lack of the BCL-2 protein
expression and intensive expression of the BAX protein.
The increase in BCL-2 expression and decrease
in BAX level in the liver was observed by Akcali et al.
(1) during hepatocyte regeneration after hepatectomy.
Opposite relations were observed in the experimental
group of the present study. Increase in the BAX
expression and the lack of changes in the BCL-2
expression as compared to the control group suggests
increased hepatocyte apoptosis.
In mammal cells two main ways of apoptotic
signal transmission were described: external and internal
(mitochondrial) way. The internal way is a response to
factors causing DNA damage, and the process takes
place mostly in mitochondria, most often in connection
with proapoptotic proteins from BCL-2 group, including
BAX protein.
The increase in the BAX protein activity
observed in the present study and visible mainly thanks
to immunohistochemical methods could suggest that the
apoptotic signal was transmitted in mitochondrial way.
A possibility is not ruled out that apoptosis could be
initiated also in the ways not investigated in the present
study.
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