Praca oryginalna Original paper
The metabolism of normal and tumor cells varies so little that the use of this difference is very difficult. Therefore, in cancer therapy the differences in kinet-ics growth of normal and tumor tissues are used (16). Hence many researchers are interested in the biology of apoptosis – programmed cell death – and the pos-sibilities of controlling the process of stimulation and inhibition of apoptosis. Loss of the ability of apoptosis induces the process of carcinogenesis because it cre-ates the possibility of cell division with accumulated
mutations. It is also the same mechanism that becomes resistant to chemotherapy (27).
The same factor can induce apoptosis and/or necro-sis (23). Whether a cell starts a program of apoptonecro-sis or necrotic death is determined by the type and dose of the death-inducing agent and duration of exposure to this factor.
Development of new techniques for the analysis of cell death opens up the possibility of testing the effec-tiveness of selected doses of chemotherapeutic agents
Immunolocalization in the portal acinus of the liver
of different types of adriamycin induced cell death
depending on time
AGNIESZKA PEDRYCZ, ZBIGNIEW BORATYŃSKI*, JACEK MENDOCHA**
Department of Histology and Embryology with Laboratory of Experimental Cytology, Medical University of Lublin, Radziwiłłowska St., 20-080 Lublin, Poland
*Department of Animal Anatomy and Histology, Faculty of Veterinary Medicine University of Life Sciences in Lublin, Akademicka St., 20-950 Lublin, Poland
**Lublin’s Oncology Centre, Prusa St., 20-090 Lublin, Poland Pedrycz A., Boratyński Z., Mendocha J.
Immunolocalization in the portal acinus of the liver of different types of adriamycin induced cell death depending on time
Summary
The ability to control the phenomenon of apoptosis, its induction or inhibition, raises hopes for treating many diseases including cancer. Adriamycin, an antibiotic that is wildly used after treating cancer, induces apoptosis in liver cells in a certain and relatively quick way after its application.
The aim of the work was to obtain and examine the model of apoptosis and necrosis of hepatocytes with respect to their response to different damaging stimuli (adriamycin) depending on the time after the application in correlation with the ultrastructural construction, which is the result of the different location of hepatocytes within the portal acinus (of Rappaport).
There were 32 female white Wistar rats used in the study. They were divided into 4 groups (2 experimental and 2 control), 8 animals in each group. The adriamycin dose of 5 mg/kg was administered intraperitoneally to the rats in groups I and II and then the rats were decapitated after 4 weeks (group I) and after 8 weeks (group II). The rats in the control groups were given 0.5 ml 0.9% NaCl solution and then decapitated after 4 weeks (group III) and 8 weeks (group IV). In the research, preparations made from fragments of the right liver lobe were used for histological observations and immunohistochemical studies. In the immunohistochemical studies, a three-stage method was used. According to this method, hepatocytes were examined qualitatively and quantitatively for the presence of proteins involved in apoptosis, to which the death signals run: through mitochondrial pathways (caspase 3 and caspase 9), through intrinsic pathways by endoplasmic reticulum (caspase 3 and caspase 12), through extrincic pathways (caspase 3 and caspase 8) and one from inflammatory markers: caspase 1.
Histological images showed that the apoptosis phenomenon occurs after the administration of adriamycin in hepatocytes in a zonate way and is dependent on the time that has elapsed since its administration. Immunohistochemical studies showed, in both a qualitative and quantitative way, a phenomenon of apoptosis in hepatocytes (executive caspase 3) and necrosis (caspase 1). It was also proved that the signal for the induction of apoptosis showed zonation and mainly followed the mitochondrial pathway (caspase 9); the intrinsic pathway by endoplasmic reticulum was much less common (caspase 12); while even more rarely caspase 8 was identified as a marker of an extrinsic pathway to induce apoptosis.
by analyzing their effects in relation to apoptosis and necrosis (26). The expected result of this research is to determine the effective doses of chemotherapeutic agents for certain types of cancer and, moreover, to understand the factors that interact in the process of destroying the tumor tissue.
Animal tests showed that adriamycin is active against a broad spectrum of experimental tumors. In clinical oncology it is used to treat breast cancer, bronchus, thyroid, ovary, uterus chorioepithelioma, neuroblastoma, acute lymphoblastic and myeloblastic leukemia, Hodgkin’s disease and soft tissue and bone sarcomas (5).
In the nucleus of the cell adriamycin binds two places of the DNA double helix. This results in the inhibition of DNA-dependent DNA polymerase and RNA. It interferes with the process of replication, transcription and protein synthesis. DNA repair mecha-nisms are also effected (7).
Adriamycin inhibits the activity of succinate dehy-drogenase and NADH oxidase, important enzymes of the respiratory chain which causes oxygen shock in the cell (20). It was shown that adriamycin can also inhibit the enzyme system by removing free radicals, which increases the damage of cells (12).
Portal acinus is a liver functional unit created hy-pothetically by Rappaport. According to this concep-tion, portal acinus is created by a mass of liver cells arranged around the axis formed by the around portal artery and vein. The cells that lie in the neighborhood of classic hepatic lobule are part of the portal acinus. Portal acinus in cross section is square-shaped, with its two central veins and bramno-biliary space forming its summits (24).
The arrangement of hepatocytes in the liver remains constant regardless of its description. The concept of lobulus or acinus is only one of two different ways of looking at the same structure. Regardless of the chosen approach, they both show that hepatocytes are not all created equal. This fact is called heterogeneous hepatocytes. Thus there are some ultrastructural and functional liver cell differences depending on the dis-tance from the portal artery and underlying the division of portal acinus into three zones.
Firstly glycogen is deposited in the first zone (cells lying close to the portal artery), and only after some time it is deposited near the central vein (zone three). However, during starvation glycogenolysis begins in the third zone. Hepatocytes show greater activity of the enzymes responsible for the process of gluconeogen-esis while hepatocytes in zone three show a higher ac-tivity of enzymes responsible for the synthesis of fats. The present study is the result of the authors’ inter-ests in the phenomenon of apoptosis, which occurs after administration of adriamycin (1, 6).
Liver cells are exposed to free radicals formed dur-ing the process of biodegradation of adriamycin and they are responsible for the formation of oxygen shock,
leading to apoptosis. The severity of these reactions depends on the location of hepatocyte within one of three portal acinus (Rappaport) zones.
The aim of this study was to obtain and examine the model of apoptosis and necrosis of hepatocytes with respect to their response to different damaging stimuli (adriamycin) depending on the time after application in correlation with the ultrastructural construction, which is the result of the different location of hepatocytes within portal acinus (of Rappaport).
The authors wished to demonstrate the possibility of induction of apoptosis by modifying the time of action of adriamycin in order to achieve the desired therapeutic effect with the smallest possible side effects resulting from cell necrosis.
Material and methods
The study material consisted of 32 white Wistar female rats with an initial weight of 200-250 g aged 2.5-3 months.
The rats were randomly selected according to the simul-taneity principle of control and experimental groups.
The rats came from the inbreeding of the Department of Histology and Embryology, Medical University of Lublin and the Breeding of Laboratory Animals Jacek Kołacz, Warsaw.
The animals received standard feed and water ad libi-tum. They were kept in the Animal Quarters of the Depart-ment of Histology and Embryology, Medical University in Lublin, at the temperature of 20 ± 2°C and humidity of about 60%. The air temperature, lighting conditions and noise slightly changed during the day. The rats were kept in 0.2 m2 metal cages, 4 individuals each. The cage lining
was suitably changed.
The animals were divided into 4 equal groups, 8 females each. The groups of rats that received adriamycin at a dose of 5 mg/kg: (group I – were decapitated after 4 weeks and group II – were decapitated after 8 weeks).
The control groups of rats that received 0.5 ml. 0.9% NaCl (group III – were decapitated after 4 weeks and group IV – were decapitated after 8 weeks).
The studies on laboratory animals were approved by the Local Bioethics Committee at the Medical University of Lublin, no. 32/2000, 551/2005.
Hepatocytes death was induced by a single administration of adriamycin (Adriblastin FARMITALIA 10 mg dissolved in 5 ml of water for injection) at a dose of 5 mg/kg. The volume of the administered solution did not exceed 0.5 ml for a single specimen. In the control groups, 0.5 ml 0.9% NaCl was administered to every single female.
For immunohistochemical studies two liver samples of each rat were collected. The procedure dealing with the specimens was standard (fixed in 10% formalin, dehydrated in an alcohol series, X-rayed in xylene, embedded in paraffin blocks). Paraffin blocks were cut into slides of 5 µm thick-ness; preparations were placed on silanized glass.
There were two slides from each individual used in the study. Slides were de-waxed in xylene and decreasing con-centrations of ethyl alcohol. Slides were subjected to heat treatment under acidic conditions (10 M citrate buffer pH 6.0) in a microwave at 750 W and after a 5 min interval at
375 W. After cooling slides were rinsed with distilled water and then placed in a TBS buffer (Tris Buffered Saline). In the slides, endogenous peroxidase was blocked by incubat-ing them in a 0.3% H2O2 solution (99 ml TBS buffer + 0.1 g NaN3 + 1 ml 30% H2O2). Slides were washed in TBS buffer and incubated for 60 min at room temperature with rabbit primary antibodies at a dilution of 1/100 in buffer TBS/ BSA 1%: Caspase 1 (Lab Vision RB-9259-PO); Caspase 3 (Lab Vision AG-4 RB-1197-PO); Caspase 8 (Lab Vision Ab-4 RB-1200-PO); Caspase 9 (Lab Vision RB-1205-PO) and Caspase 12 (Alexis/Pro Scince PSC-2327). A set of ready-made staining reagents from DakoCytomation was used to obtain immunohistochemical reaction, which in-cluded: biotinylated secondary antibody against rabbit an-tibodies (Biotinylated Link Universal), Streptavidin merged with horseradish peroxidase (Streptavidin – HRP) and AEC – substrate – reaction’s pigment with horseradish peroxidase (AEC Substrate Chromogen). After staining by means of chromatogen, the slides were stained by Hematoxylin. For each tested product a negative control was made, which was a preparation to which no primary antibody was added. Photographic documentation was made by means of Colour Video Camera CCD-IRIS (Sony).
The results of immunohistochemical studies were as-sessed both qualitatively and quantitatively. In the quali-tative assessment, the intensity of color reaction in the areas of antigen-antibody reaction in the examined rat’s acinus of Rappaport (in different experimental and control groups) was taken into account. In order to better visualize changes in color, in the areas of the Rappaports’s aciunus a computer program was used to impose the image of the real mask color.
In the quantitative assessment, the number of slides was analyzed both in numbers and in percentage in which posi-tive, colorful, immunohistochemical reaction was observed with regard to individual zones of acinus of Rappaport.
The obtained results are collated as mean values and standard deviation, and analyzed statistically by means of the ONE WAY ANOVA test. There was an allowance for 5% risk of inference error and statistically significant dif-ferences at p ≤ 0.05.
Results and discussion
Two slides of 2 liver specimens from each of the 8 rats in a group were taken for qualitative and quan-titative evaluation of immunohistochemical studies. As a result 64 tests for each antibody were rated in the experimental and control groups.
Research results in the control groups showed no immunohistochemical staining (100%) for the tested antibodies in any of the evaluated attempts. Moreover, no immunohistochemical staining was noticeable in any of the negative control specimens (100%) (the preparation which had no primary antibody added).
The results from the experimental group are shown in the tables (Tab. 1-6) and photographs (Fig. 1-10).
The color immunohistochemical reaction in places of identified caspases was observed on slides and pres-ent on figures. Its discovery was evidence of apoptosis induction in different zones of the portal acinus and
allowed the identification of a signal path that was passed to apoptosis.
Caspase 3 – is the direct evidence for apoptosis induction, regardless of the pathway that has been ini-tialized. In experimental group I (5 mg/kg adriamycin – 4 weeks of the experiment) caspase 3 in zone 2 and 3 portal acinus was shown (Fig. 1). In the experimental group II (5 mg/kg adriamycin – 8 weeks of the experi-ment) caspase 3 were detected mainly in zone 1 and 2 of portal acinus, in part of the slides they were detected only in zone 1 or 2 (Fig. 2) (Tab. 2a, 2b).
Histological evaluation of the preparations of ex-perimental group II (5 mg/kg of adriamycin – 8 weeks of the experiment) showed characteristic changes of hepatocytes necrosis in portal acinus zone 3. In order to confirm the histological observations, an immuno-histochemical attempt of marking caspase 1 was made – one of the markers of inflammation associated with necrosis. The images confirm the histological observa-tions. In experimental group I there was no positive immunohistochemical staining confirming the presence of caspase-1 (Fig. 3). Only in experimental group II (5 mg/kg of adriamycin – 8 weeks of the experiment) did immunohistochemical reactions show the presence of the inflammation marker, caspase-1, and it was only in zone 3 of portal acinus (Fig. 4) (Tab. 3a, 3b).
Caspase 8 – an external pathway marker of the signal initiating apoptosis. In experimental group I (5 mg/kg of adriamycin – 4 weeks of the experiment) positive immunohistochemical staining was present in zone 3 portal acinus (Fig. 5). In experimental group II (5 mg/kg of adriamycin – 8 weeks of the experiment) in the substantial majority of preparations there was a positive immunohistochemical staining for caspase 8 (Fig. 6) (Tab. 4a, 4b).
Caspase 9 – an internal pathway marker – initiating the mitochondrial signal to apoptosis. In experimen-tal group I (5 mg/kg of adriamycin – 4 weeks of the experiment) positive immunohistochemical staining was found in portal acinus zone 2 and 3 (Fig. 7). In ex-perimental group II (5 mg/kg of adriamycin – 8 weeks of the experiment) positive immunohistochemical staining was observed in portal acinus zones 1 and 2 (Fig. 8) (Tab. 5a, 5b).
Immunohistochemical reaction for caspase 12 (an internal pathway marker by the endoplasmic reticulum, a course of inducing apoptosis) was marked. In experi-mental group I (5 mg/kg of adriamycin – 4 weeks of the experiment) positive immunohistochemical reaction was present mainly in portal acinus zone 3 (Fig. 9). In experimental group II (5 mg/kg of adriamycin Tab. 1. Identified caspases
Experimental group
Portal acinus zone
1 2 3
I – caspase 3, 9 caspase 3, 8, 9, 12 II caspase 3, 9, 12 caspase 3, 9, 12 caspase 1
– 8 weeks of the experiment) positive immunohisto-chemical staining was observed in portal acinus zones 1 and 2 (Fig. 10). Collective results for the designa-tion of caspase 12 – an internal path marker by the endoplasmic reticulum – were based on the analysis of immunohistochemical reaction in 32 preparations (Tab. 6a, 6b).
There are two main pathways leading to apoptosis described in the literature (2). The extrinsic pathway is induced by an external signal and is connected with the membranous death receptors possessing an intracel-lular domain called DD (death domain). In response to a binding death receptor, procaspase 8-initiating is
activated. The intrinsic pathway is activated by cel-lular stress. This stress occurs during cell exposure to radiation, chemicals, viral infections, damage of the growth factor or as a result of oxygen shock, and it initiates apoptosis via damage to the mitochondria (cytochrome c is released to the cytoplasm, which activates caspase 9) or damage to the endoplasmic reticulum, which leads to activation of caspase 12 and starts the caspase cascade (10-21). The cascade ends with the activation of effector caspases (3, 5, 6), which participate or initiate cellular DNA damage, inactivate DNA repairing enzymes and cut the protein cytoskel-eton (actin, spectrin) which leads to cell death (4). Tab. 2b. Caspase 3
Experimental groups
Number of preparations where the positive color of the immunohistochemical reaction as per details of every portal acinus zone
Zone Preparations without
a reaction
1 1 + 2 2 2 + 3 1 + 2 + 3 3
I (5 mg of adriamycin –
4 weeks of the experiment) 0%0 0%0 0%0 93.75%30 0%0 6.25%2 0%0
II (5 mg of adriamycin –
8 weeks of the experiment) 12.5%4 62.5%20 12.5%4 6.25%2 6.25%2 0%0 0%0
Fig. 1. Rat’s liver sample. Caspase 3 marked. Experimental Group I. AEC + H staining
Fig. 2. Rat’s liver sample. Caspase 3 marked. Experimental Group II. AEC + H staining
Tab. 2a. Caspase 3
Immunohistochemical reaction was present in portal acinus zone 2 and 3 in 94% of preparations
Immunohistochemical reaction in 63% of preparations was connected with portal acinus zone 1 and 2
0% 0% 0% 94% 0% 6% 0% Experimental group I
(5 mg of ADR – 4 weeks of experiment)
Zone 2 + 3 Zone 3 12% 63% 13% 6% 6% 0% 0% Experimental group II (5 mg of ADR – 8 weeks of experiment)
Zone 1
Both apoptotic and necrotic pathways of cell death remain in a relationship which is dependent on short-and long-term opening of the PTP, oxidative stress, ATP content in the cell and the high or low levels of Bcl-2. All of these conditions, depending on the availability of activated caspases, serve to influence whether the cell will go down the pathway to apoptosis or necrosis (9, 17).
In the literature many reports about inducing apopto-sis of various organs in both humans and experimental animals after adriamycin can be found (1, 6, 13).
In the present study after administration of adriamy-cin in white rats’ liver cells, apoptosis was observed,
differentiating cells in accordance with their location within one of the acinus hepaticus zones (Rappaport), and the track through which the signal to apoptosis follows was analyzed. The main transmission path to apoptosis after administration of adriamycin is via the mitochondrial path (internal), by releasing cytochrome c from mitochondria, formatting apoptosome from Apaf-1, activating caspase 9 and caspase 3 (executive). Similar observations were made by Serafino (25). The second transmission path signal to apoptosis in cells that were studied in this experiment started with the activation of caspase 12 (internal path – argyrophylic). Apoptosis activated by the destruction of endoplasmic Tab. 3b. Caspase 1
Experimental groups
Number of preparations where the positive color of the immunohistochemical reaction as per details of every portal acinus zone
Zone Preparations without
the reaction
1 1 + 2 2 2 + 3 1 + 2 + 3 3
I (5 mg of adriamycin –
4 weeks of the experiment) 0%0 0%0 0%0 0%0 0%0 6.25%2 93.75%30
II (5 mg of adriamycin –
8 weeks of the experiment) 0%0 0%0 0%0 25%8 0%0 75%24 0%0
Tab. 3a. Caspase 1
In the majority of the preparations (94%) there was no posi-tive immunohistochemical reactions for caspase 1
Immunohistochemical reaction in ¼ of the preparations is connected with zone 2 and 3 portal acinus. However, in the majority of the preparations (75%) it was observed only in zone 3
Fig. 3. Rat’s liver sample. No reaction to caspase 1. Experi-mental group I. AEC + H staining. Magn. 200 ×
Fig. 4. Rat’s liver sample. No reaction to caspase 1. Experi-mental group II. AEC + H staining. Magn. 280 ×
Experimental group I
(5 mg of ADR – 4 weeks of experiment)
0% 0% 0% 0% 0% 6% 94% Experimental group II (5 mg of ADR – 8 weeks of experiment)
0% 0% 0% 25% 0% 75% 0% Zone 2 + 3 Zone 3
reticulum was described by Yang (28). He noted that by the activation of caspase 12, the internal track is considerably higher than the other two (mitochondrial and receptor – external). In his studies Mehmet (19) noted that if during the initiation of apoptosis caspase activation occurs at the cell membrane (outer path) or mitochondrial (inner path – mitochondrial), caspase 12 is not activated. In this experiment it was not con-firmed, as both paths (mitochondrial and argyrophylic) were active in the study. The third path conveying the signal to apoptosis – the external path – was far less pronounced, although the marker in the form of cas-pase 8 was also determined in the study.
The data confirms the heterogeneity of hepatocytes that were observed in this experiment. The liver cells lying in zone 2 and especially 3 were proved to be the most sensitive to the adriamycin administration. The observed apoptosis was initiated mainly by the caspase 9 (mitochondrial path), and to a lesser extent by caspase 12 (endoplasmic reticulum path). The exter-nal path – by caspase 9 – was much less pronounced. Apoptosis in cells of zone 1 occurred only when there was a great period of time allowed to elapse between the administration of high doses (95 mg/kg of body weight) of adriamycin and the performance of the sample tests. It should finally be noted that the stud-Tab. 4a. Caspase 8
In 40% of preparations there was no immunohistochemical change. However, in 60% of preparations, the immunohi-stochemical reaction was positive and it was connected with zone 3
In the significant majority of preparations (90.6%) there was no immunohistochemical reaction
Tab. 4b. Caspase 8 Experimental groups
Number of preparations where the positive color of the immunohistochemical reaction as per details of every portal acinus zone
Zone Preparations without
the reaction
1 1 + 2 2 2 + 3 1 + 2 + 3 3
I (5 mg of adriamycin –
4 weeks of the experiment) 0%0 0%0 0%0 0%0 0%0 59.38%19 40.62%13
II (5 mg of adriamycin –
8 weeks of the experiment) 0%0 0%0 0%0 3.13%1 0%0 6.25%2 90.63%29
Fig. 5. Rat’s liver sample. Weak reaction to caspase 8 in 3 zone of portal acinus. Experimental group I. AEC + H staining. Magn. 140 ×
Fig. 6. Rat’s liver sample. No reaction to caspase 8. Experi-mental group II. AEC + H staining. Magn. 200 ×
Experimental group II (5 mg of ADR – 8 weeks of experiment)
0% 0% 0% 3% 0% 6% 91% Experimental group I
(5 mg of ADR – 4 weeks of experiment)
0% 0% 0% 0% 0% 59% 41% Zone 3
ies, after the dose of adriamycin, where examination of the samples obtained in zone 3 was carried out a long time after administration of the adriamycin, resulted in an image of necrosis whose marker was caspase 1.
The considered aspect of this study was the relation-ship between the apoptosis observed after the time elapsed since adriamycin administration. Literature confirms that the type of cell death after adriamycin depends on its dose (8). Eom noted that high doses of adriamycin induce apoptosis and a small death process starts with the inhibition of mitosis (8). Other authors
noted that the dose of adriamycin which does not ex-ceed 0.2 mg/ml affects apoptosis, whereas in higher doses – necrosis results (15).
In this experiment adriamycin was given at a dose of 5 mg/kg (experimental group I and II). This dose was reported as inducing apoptosis but not causing necrosis (22). In this experiment these observations were only partly confirmed, because it turned out that the dose of 5 mg/kg after 8 weeks of dosing caused necrosis in aciunus hepaticus zone 3. At all stages of this experiment caspase 1, which is involved in the inflammatory process causing necrosis, was marked Tab. 5a. Caspase 9
Positive immunohistochemical reaction in ¼ of the prepara-tions was present only in portal acinus zone 3. However, in the majority of preparations it was present in zones 2 and 3
In ¾ of the preparations positive immunohistochemical reac-tion was present in portal acinus zones 1 and 2
Fig. 7. Rat’s liver sample. Colour reaction on caspase 9. Expe-rimental Group I. AEC + H staining
Fig. 8. Rat’s liver sample. Staining reaction on caspase 9. Experimental Group II. AEC + H staining
Tab. 5b. Caspase 9 Experimental groups
Number of preparations where the positive color of the immunohistochemical reaction as per details of every portal acinus zone
Zone Preparations without
the reaction
1 1 + 2 2 2 + 3 1 + 2 + 3 3
I (5 mg of adriamycin –
4 weeks of the experiment) 0%0 0%0 0%0 68.75%22 6.25%2 25%8 0%0
II (5 mg of adriamycin –
8 weeks of the experiment) 0%0 75%24 0%0 12.5%4 12.5%4 0%0 0%0
Experimental group I
(5 mg of ADR – 4 weeks of experiment)
Zone 2 + 3 Zone 3 0% 0% 0% 69% 6% 25% 0% Experimental group II (5 mg of ADR – 8 weeks of experiment)
Zone 1 Zone 1 + 2 0% 75% 0% 12% 13% 0% 0%
immunohistochemicaly. In the entire experiment cas-pase 1 was at a comparable level with controls; only in the group of rats that received a dose of 5 mg/kg and were decapitated after 8 weeks was there noted significant growth in the hepatocytes located in the acinus hepaticus zone 3.
The results of this experiment confirm the reports that say that despite the fact that the greatest concentra-tion of adriamycin in the blood serum is obtained after 1 h following administration (3), its effect is often quite distant in time. It can be concluded that the observed long-term effect of adriamycin administration is
en-gendered by the apoptosis process of catalytic proteins, both those that are already in the cells and those which appear „de novo” in the apotesis process. Kaminski and Mazur believe that these may be proteins with short half-lives, which act as inducers or suppressors (14, 18). The observed disturbance in apoptosis process in the organization of chromatin, with some delays, is accompanied in the cytoplasm by changes induced by the activation of calcium-dependent transglutaminase. Their role is combined with the formation of perma-nent crosslinks „enveloping the cell”, which makes it resistant to attack by proteolytic enzymes (14). Tab. 6b. Caspase 12
Experimental groups
Number of preparations where the positive color of the immunohistochemical reaction as per details of every portal acinus zone
Zone Preparations without
the reaction
1 1 + 2 2 2 + 3 1 + 2 + 3 3
I (5 mg of adriamycin –
4 weeks of the experiment) 0%0 0%0 0%0 12.5%4 6.25%2 75%24 6.25%2
II (5 mg of adriamycin –
8 weeks of the experiment) 0%0 62.5%20 0%0 18.75%6 12.5%4 0%0 6.25%2 Tab. 6a. Caspase 12
Positive immunohistochemical reaction in ¾ of the prepara-tions was present in portal acinus zone 3
In 63% of the preparations positive immunohistochemical reaction was present in portal acinus zone 1 and 2
Fig. 9. Rat’s liver sample. Weak immunohistochemcal reaction to caspase 12 in zone 1, 2, 3 of portal acinus. Experimental group I. AEC + H staining . Magn. 280 ×
Fig. 10. Rat’s liver sample. Immunohistochemical reaction to caspase 12 in zone 1, 2 of portal acinus. Experimental group II. AEC + H staining. Magn. 200 ×
Experimental group I
(5 mg of ADR – 4 weeks of experiment)
Zone 3 0% 0% 0% 13% 6% 75% 6% Experimental group II (5 mg of ADR – 8 weeks of experiment)
Zone 2 + 3 Zone 1 + 2 0% 62% 0% 19% 13% 0% 6%
Administering 5 mg/kg m.c. induced apoptosis in hepatic cells of the rats. There was apoptosis after Adriamycin administration in the liver in the zonal way – depending on the location of the investigated cells in one zone of hepatic acinus (Rappaport). Apoptosis was more intensified if the explored cells were further from an artery but closer to the central vein of the hepatic lobule.
The zonal quality was indicated in the pathway of a signal to apoptosis, in more oxidated hepatocytes there was only an internal mitochondria pathway, and less oxidated hepatocytes were accompanied by the internal reticular pathway and the external path.
The period between adriamycin administration and hepatic cell investigation is a vital factor of the pro-cess, with the longer the period, the better the effect of apoptosis activation.
Investigation of apoptosis induction is worth per-forming in order to utilize this phenomenon in the therapy of neurodegenerative and neoplastic diseases.
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Author’s address: dr hab. Agnieszka Pedrycz, ul. Radziwiłłowska 11, 20-080 Lublin; e-mail: agnieszka.pedrycz-wieczorska@umlub.pl
