Various substances are known to cause liver damage. One of these chemicals is carbon tetrachloride (CCl₄) which is a xenobiotic that induces hepatotoxicity in humans as well as in animals. The hepatotoxic effect of CCl₄ is considered to be a result from reductive dehalogenation by the P450 enzyme system to the highly reactive trichloromethyl radical, CCl₃. This free radical rapidly reacts with molecular oxygen to form a trichloromethyl peroxyl radical. Removal of hydrogen atoms from unsaturated fatty acids by such a radical creates carbon-centered lipid radicals. These lipid radicals also react rapidly with molecular oxygen to form lipid peroxyl radicals, thereby, initiating the process of lipid peroxidation. Unless scavenged by radical scavengers, these lipid peroxyl radicals in turn abstract hydrogen atoms from other lipid molecules, thus propagating the process of lipid peroxidation. In a recent study by, lipid peroxidation was considered to be the most important mechanism in the pathogenesis of liver damage induced by CCl₄. Similarly, it has previously been reported that levels of malondialdehyde (MDA), a marker of lipid peroxidation, are increased in CCl₄-toxicated liver. In addition, CCl₄ induces elevated levels of hepatic enzymes in serum that are markers of liver cell damage. Furthermore, histopathological changes occur in the liver after CCl₄ administration.

Caffeic acid phenethyl ester (CAPE) is an active component in honeybee propolis extracts and is considered to have medicinal properties. It has anti-inflammatory, immunomodulatory, anti-proliferative and anti-oxidant properties and has been shown to inhibit both lipooxygenase activity and suppress lipid peroxidation. However, as far as we know only few experimental studies on the protective effects of CAPE on CCl₄-induced hepatotoxicity have been performed yet. Therefore, we aimed to examine in the present study the effects of CAPE on CCl₄-induced hepatotoxicity in rats.

Adult male Wistar albino rats (weighing 170–220 g) supplied by the Firat (Euphrates) University Medical Faculty Experimental Research Unit were randomly divided into three groups with eight animals per group. The rats were kept in plexiglas cages (four animals per cage) and received standard chow and water ad libitum in an air-conditioned room with automatically regulated temperature (22±1°C) and light cycles (light: 07.00–19.00). All rats were allowed to acclimatize for 1 week prior to experimentation. Control rats (group I) received pure olive oil (1 ml subcutaneously (sc)) alone. Rats in group II were injected with CCl₄ (0.5 ml/kg body weight per 1 ml olive oil sc; EM Science, Cherry Hill, NJ, USA) every other day for 1 month. Rats in group III received CAPE (10 ?mol/kg body weight intraperitoneally) and an sc injection of CCl₄ every other day for 1 month. CAPE was synthesized in the Physico-Chemistry Laboratory using the technique described by.

All animals were killed by decapitation at the end of the experiment. Blood samples were collected in tubes, allowed to clot and the serum was removed by centrifugation at 2000g for 10 min. All serum samples were sterile, hemolysis-free, and were kept at 4°C before determination of the biochemical parameters.

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin and conjugated bilirubin levels were measured with an AU600 multiparameter analyzer (Olympus, Hamburg, Germany).

Livers of all rats were removed immediately after collection of the blood. Some of the liver tissue specimens were washed twice with a cold saline solution, placed into glass bottles, labelled, and stored at ?30°C for determination of MDA levels as an index of lipid peroxidation. The other liver tissue specimens were used for histopathological examination.

Tissues were homogenized after being cut into small pieces with scissors using ice-cold 150 mM KCl and were weighed. The MDA content of the homogenates was determined spectrophotometrically by measuring the presence of thiobarbituric acid-reactive substances. The results were expressed as nmol/g wet tissue.

Liver tissue specimens were fixed in neutral formalin solution (10%). Tissue specimens were embedded in paraffin wax and sectioned (thickness, 5 ?m). For histopathological evaluation, sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and examined with a BH2 light microscope (Olympus, Tokyo, Japan).

Quantitative data are expressed as mean±standard error of mean (SEM). Comparisons between groups were performed with the Kruskal–Wallis’s test for unpaired comparisons followed by the Mann–Whitney’s rank sum test. All analyses were performed with SPSS 11.0 for Windows software. P values of <0.05 were considered significant.

The biochemical parameters in serum and statistical comparisons of the groups are presented in Table 1. In the CCl₄-treated group, serum levels of AST, ALT, ALP, total bilirubin and conjugated bilirubin were significantly increased as compared with those in the control group (p<0.05). When rats were treated with CCl₄ and CAPE, significantly reduced elevations in serum AST, ALT, ALP, total bilirubin and conjugated bilirubin were found (p<0.05). Additionally, administration of CCl₄ to rats resulted in a significantly increased MDA production in livers (p=0.001), whereas in rats treated with CCl₄ and CAPE, levels of MDA were significantly reduced as compared with rats treated with CCl₄ only (p=0.001; Fig. 1).

The protective effects exerted by CAPE on CCl₄-induced liver hepatotoxicity as indicated by the biochemical data were confirmed by conventional histopathological examination. Livers of rats in the control group showed a normal histological appearance (Fig. 2), whereas livers of rats treated with CCl₄ showed classic histology of cirrhosis. Coagulative necrosis, massive fibrosis, mononuclear cell infiltration, hemorrhage, fatty degeneration and formation of regenerative nodules were observed. Additionally, apoptotic figures, microvesicular steatosis and hydropic degeneration in hepatocytes were found as well in this group. However, the histopathological changes observed after administration of CCl₄ were not found in rats treated with CCl₄ and CAPE, except for fatty degeneration.