Medycyna Wet. 2011, 67 (7) 467
Praca oryginalna Original paper
Anthracyclines, which include adriamycin, were discovered in the 1960s. Adriamycin is the 14-hydro-xydaunomycin produced by Streptomyces peuceticus s. Caesius mutant (5, 7). It is an antibiotic used in oncology and haematology (15). The highest serum level of the drug is observed 1 h after its intravenous administration. Adriamycin is relatively quickly eli-minated from the plasma (8). It diffuses passively to cells, where it accumulates in nuclei and lysosomes. In the nucleus, adriamycin binds DNA, which blocks the synthesis of DNA and RNA, as well as impairs the processes of replication, transcription, and synthesis of proteins. Moreover, it impairs the mechanisms of DNA repair. It is most effective in the phase S of the cell cycle (3).
In the process of adriamycin transformation in microsomes mediated by NADPH, p-450 cytochrome reductase, semiquinone is formed (9). It is a free radi-cal form responsible for cytotoxic effects of the drug (17, 19). Adriamycin inhibits the activity of respirato-ry chain enzymes (succinic dehydrogenase, NADH oxidase) (1-3). Moreover, it suppresses the enzymatic system, which removes free radicals (16). In our pre-vious studies, we described the apoptotic index and histological assessment of renal tubular epithelial cells of rats during antracyclines therapy. We also observed the ultrastructure of rat hepatocytes after adriamycin treatment (13, 14).
In the present study, the apoptotic index and histo-logical structure of apoptotic cells in the rat liver after
Apoptotic index and histological assessment
of hepatocytes after adriamycin treatment
ZBIGNIEW BORATYÑSKI, AGNIESZKA PEDRYCZ*, PIOTR SIERMONTOWSKI**, JANUSZ KOCKI***, AGATA ZAJ¥C****
Department of Animal Anatomy and Histology, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-950 Lublin, Poland
*Department of Histology and Embryology with Laboratory of Experimental Cytology, Medical University, 20-080 Lublin, Poland
**Maritime and Hyperbaric Medicine Department, Military Institute of Medicine, 81-519 Gdynia, Poland ***Laboratory of Clinical Genetics, Medical University, 20-080 Lublin, Poland
****Department of Anaesthesiology and Intensive Care, Provincial Specialist Hospital, 22-100 Che³m, Poland
Boratyñski Z., Pedrycz A., Siermontowski P., Kocki J., Zaj¹c A.
Apoptotic index and histological assessment of hepatocytes after adriamycin treatment Summary
Adriamycin is an antineoplastic antibiotic which is biodegragated mainly in the liver. The purpose of this study was to analyse the apoptotic index and the histological structure of apoptotic cells in the rat liver. Rats were examined 4 and 7 weeks after adriamycin treatment. The animals were divided into four equal groups: groups I and III, consisting of rats treated with a single intraperitoneal dose of adriamycin (5 mg/kg b.w) and decapitated after 4 or 7 weeks; groups II and IV comprising control rats treated with a single intraperitoneal dose of 0.9% NaCl (0.5 ml) and decapitated after 4 or 7 weeks. After decapitation, liver specimens were collected and embedded in paraffin. Slides were stained with haematoxylin and eosin. The degree of apoptosis was determined quantitatively using the apoptotic index. Statistical analysis was performed using the one-way ANOVA test and Students t-test.
A single dose of adriamycin induced apoptosis of hepatocytes, which increased with time The apoptotic index was significantly higher in the group examined 7 weeks after adriamycin administration. The histologi-cal structure of the liver in the experimental groups was similar. The lesions observed were fohistologi-cal. The blurring of the hepatic cell membrane and a focal disintegration of architectonics concerning the shape and size of hepatocytes were visible. Chromatin in the nuclei was dispersed. Some nuclei showed peripheral chromatin condensation. The cytoplasm of some cells showed numerous vacuoles. Naked nuclei and congestion resulting from vessel damage were visible.
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adriamycin administration were examined. Addition-ally, the influence of time after adriamycin treatment on the observed changes was investigated.
Material and methods
The study material consisted of 32 white female Wistar rats aged 2.5-3 months whose baseline body weight was 200-250 g. The animals received standard feed and water ad libitum.
The animals were divided into four equal groups: group I rats treated with a single intraperitoneal dose of adria-mycin (5 mg/kg b.w). and decapitated after 4 weeks, group II control rats treated with a single intraperitoneal dose of 0.9% NaCl (0.5 ml) and decapitated after 4 weeks, group III rats treated with a single intraperitoneal dose of adria-mycin (5 mg/kg b.w.) and decapitated after 7 weeks, and group IV control rats treated with a single intraperitoneal dose of 0.9% NaCl (0.5 ml) and decapitated after 7 weeks. The experiments on animals were approved by the Local Ethics Committee.
After decapitation, the specimens from the right hepatic lobe were collected, fixed in 10% buffered formalin, dehy-drated in graded ethanol, cleared in xylene, and embedded in paraffin. The 5 µm sections were stained with haemato-xylin and eosin (HE).
During the microscopic analysis of the hepatocytes, spe-cial attention was paid to features suggesting apoptosis. Three specimens from each animal were examined. The degree of apoptosis was determined quantitatively using the apoptotic index. Nuclei stained dark with haematoxy-lin with a reduced perimeter and diameter were accepted as pyknotic, that is, as one of possible manifestations of apoptosis. In the specimens (magnified about 1,000 ×), the number of pyknotic nuclei in 100 liver cells was counted and compared between an experimental group and the ap-propriate control group. The results were presented as per-centages. The photographic documentation was prepared using the Jenaval Contrast camera (Carl Zeiss, Germany). The results were presented as means and the standard deviation (± SD) of the mean. Statistical analysis was per-formed using the one-way ANOVA test and Students t-test. The significance level of p £ 0.05 was accepted.
Results and discussion
The microscopic morphology of rat livers from control groups II and IV was characterised by normal architectonics with clearly visible and defined lobu-les. The cytoplasm of hepatocytes was stained pink with visible basophilic, evenly distributed granules. One or two bright violet and distinct nuclei with one or two nucleoli and regular nuclear membranes and chromatin stroma were visible in the cell centre. The lumen of sinuses showed hepatic macrophages and a low number of erythrocytes.
The microscopic picture of the liver in experimen-tal groups (I and III) (fig. 1, 2) was similar. The obser-ved changes were focal. Hepatocytes had blurred cel-lular membranes. Focal changes in the shape and size of hepatocytes were visible. Hepatic trabeculae were
focally damaged. Nuclei of hepatocytes were different in size, shape, and colour. Some nuclei revealed peri-pheral chromatin condensation.
The cytoplasm of hepatocytes was markedly brigh-ter than in control groups. The cytoplasm of some cells showed numerous vacuoles (fig. 1). A characteristic lesion resulting from the adriamycin treatment was an increased number of peroxysomes in the cytoplasm of some cells seen under a light microscope. The lumen of sinuses was focally reduced (fig. 1) or markedly widened (fig. 2). The hepatic macrophages were significantly larger and more numerous than those in controls (fig. 1). Naked nuclei or erythrocytes causing congestion resulting from vessel damage were visi-ble. The examination of the apoptotic index was per-formed on 100 cells from each of the three specimens
Fig. 1. Group I. The liver section of the rat treated with ad-riamycin and decapitated 4 weeks later. The photomicrograph shows focal abnormalities in architectonics, irregular hepa-tocytes, hepatocytes with brightened cytoplasm, nuclei of various shape, colour, and size, numerous erythrocytes in sinuses. H + E, 400 ×
Fig. 2. Group III. The liver section of the rat treated with adriamycin and decapitated 7 weeks later. The photomicro-graph reveals disintegrated architectonics, hepatocytes with brightened cytoplasm, few nuclei, widened sinuses with nu-merous hepatic macrophages and erythrocytes. H + E, 400 ×
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collected from every animal. No statistically signifi-cant differences in the number of apoptotic cells were observed between control groups (p = 0.42). How-ever, statistically significant differences were found between the experimental groups (p = 0.008) and be-tween the experimental and control groups (p £ 0.001) (tab. 1). A statistically significant increase in the num-ber of apoptotic cells was observed in groups I and III compared to controls. The highest percentage of apop-totic cells was found in group III seven weeks after adriamycin administration (32.50 ± 4.64). This per-centage was statistically significantly higher than that in the group examined 4 weeks after adriamycin treat-ment (tab. 1). Adriamycin induced hepatocyte apopto-sis, which increased with time of the experiment.
Apoptotic cells die within several hours. Apoptotic bodies undergo quick phagocytosis. The apoptotic cells in this study shrank and reduced in volume. Under a light microscope the specimens stained with haema-toxylin and eosin showed pyknotic nuclei, nuclei with peripherally condensed chromatin. On this basis, the apoptotic index was calculated, and it was markedly increased in cells exposed to adriamycin. The apopto-tic index also increased with the time of this experi-ment.
Under a light microscope, parenchymal dimness was observed, which resulted from the oedema of many mitochondria. Such changes in hepatocytes were also described in hepatocytes exposed to other toxic substances. Moreover, they were observed in oxygen deficiency, fasting or shock (6).
Another characteristic lesion noted under a light microscope was an increased number of peroxysomes in the cytoplasm of dying cells, which shows their significant involvement in processes of detoxification. Peroxysomes play an important role in the detoxifica-tion of many metabolites and xenobiotics by their oxidation. They are formed by blastogenesis from the smooth endoplasmic reticulum, and their enzymes are synthesised in the rough endoplasmic reticulum. Eom et al. (4) and Park et al. (12) demonstrated that the kind of death that cells undergo following the admini-stration of adriamycin was dose-dependent. High doses of the compound induced apoptosis and low ones caused death initiated by mitosis abnormalities.
Adriamycin induces apoptosis mainly in hepatic cells, through which it is excreted, in kidneys, where it simultaneously causes the experimental nephrotic
syndrome (11), and in the heart, where its action is highly cardiotoxic (10, 18). The apoptotic index of hepatocytes observed in this study increased with time as a result of adriamycin action.
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Authors address: Prof. dr hab. Zbigniew Boratyñski, ul. Akademicka 12, 20-950 Lublin; e-mail: zbyszek.boratynski@up.lublin.pl
Tab. 1. Mean number of apoptotic cells in the rat liver (x ± sd)
Explanation: * significant difference at p £ 0.001
s l a m i n A Group Meannumberofapopitccells s l o rt n o C II _4,50 ±1.76 V I _4,30 ±2.80 l a t n e m ir e p x E s p u o r g I 22,66*±5.57 II I 32,50 ±4.64
