- Email: [email protected]
Caffeic acid phenethyl ester protects kidneys against carbon tetrachloride toxicity in rats
Journal of Ethnopharmacology 97 (2005) 273–280
Caffeic acid phenethyl ester protects kidneys against carbon tetrachloride toxicity in rats M. Ogeturka , I. Kusa , N. Colakoglub , I. Zararsiza , N. Ilhanc , M. Sarsilmaza,∗ b
a Department of Anatomy, Faculty of Medicine, Firat University, 23119 Elazig, Turkey Department of Histology and Embryology, Faculty of Medicine, 23119 Firat University, Elazig, Turkey c Department of Biochemistry, Faculty of Medicine, Firat University, 23119 Elazig, Turkey
Received 31 March 2004; accepted 10 November 2004 Available online 12 January 2005
Abstract Caffeic acid phenethyl ester (CAPE), an active component of propolis produced by honeybees, exhibits antioxidant and anti-inflammatory properties. The aim of this study was to investigate possible protective effects of CAPE on carbon tetrachloride (CCl4 )-induced renal damage in rats. A total of 24 animals were divided into three equal groups: the control rats received pure olive oil subcutaneously, rats in the second group were injected with CCl4 (0.5 ml/kg, s.c. in olive oil) and rats in the third group were injected with CCl4 (0.5 ml/kg) plus CAPE (10 mol/kg, i.p.) every other day for one month. At the end of the experimental period, the animals were sacrificed and blood samples were collected. Serum urea and creatinine levels and renal malondialdehyde (MDA) contents were determined. Histopathological examination of the kidney was also performed using light microscopic methods. It was found that kidney MDA levels were increased significantly following CCl4 exposure and this increase was significantly inhibited by CAPE treatment, while no significant changes were observed in serum urea and creatinine levels. CCl4 administration alone also caused histopathologically prominent damage in the kidney compared to the control group. Glomerular and tubular degeneration, interstitial mononuclear cell infiltration and fibrosis, and vascular congestion in the peritubular blood vessels were observed in the renal cortex. With exception of rare vascular congestions, these histopathological changes were disappeared in rats treated with CCl4 plus CAPE. In view of the present findings, it is suggested that CAPE protects kidneys against CCl4 toxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Caffeic acid phenethyl ester; Carbon tetrachloride; Nephrotoxicity; Histopathology; Light microscopy
1. Introduction Carbon tetrachloride (CCl4 ), a clear, heavy, and nonflammable liquid is most widely used for experimental induction of hepatic cirrhosis (Sundari et al., 1997; Bahcecioglu et al., 1999). It is known to be nephrotoxic as well as hepatotoxic to humans (Abraham et al., 1999). Furthermore, it has been identified as a probable human carcinogen based on evidence of tumors in animals (Thrall et al., 2000) In years past, carbon tetrachloride was widely used as a dry ∗ Corresponding author. Tel.: +90 424 2370000x6035; fax: +90 424 2379138. E-mail addresses: [email protected], [email protected] (M. Sarsilmaz).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.019
cleaning solvent until it was recognized as a potent hepatotoxin, nephrotoxin and carcinogen. Today, it is primarily used as an organic solvent (Kovacic et al., 2002). Administration of CCl4 causes an increase in lipid peroxidation products (Daniels et al., 1995; Abraham et al., 1999; Donder et al., 1999) and a decrease in the activity of enzymes protecting lipid peroxidation in the kidney (Dogukan et al., 2003). These detrimental effects of CCl4 have been attributed to conversion of CCl4 to highly toxic trichloromethyl and trichloromethyl peroxyl free radicals by cytochrome P450 enzyme, resulting in cell injury (Sundari et al., 1997; Abraham et al., 1999; Bahcecioglu et al., 1999). Several antioxidant agents, including ginkgo biloba and black tea extracts, antioxidant vitamins (C and E), and melatonin have been reported to reduce CCl4 -induced nephrotoxicity (Bahcecioglu
274
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
et al., 1999; Donder et al., 1999; Turkdogan et al., 2001; Fadhel and Amran, 2002). The caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is know to have antioxidant, immunomodulatory, anti-inflammatory and anticarcinogenic properties and has been used as a folk medicine for many years (Banskota et al., 2001; Ozen et al., 2004). It has been shown to be a pharmacologically safe compound (Zhao et al., 2003). It has also been shown to suppress lipid peroxidation (Ilhan et al., 1999; Irmak et al., 2001) and to stimulate the activity of antioxidant enzymes (Fadillioglu et al., 2004; Ozen et al., 2004). Although there is considerable interest in the role of CAPE as a dietary antioxidant, its effects on CCl4 induced renal damage have not been investigated to date. The aim of the present study was therefore to investigate whether CAPE treatment prevents CCl4 -induced nephrotoxicity. For this purpose, we have examined histopathological effects of CCl4 and possible protective effect of CAPE on tissue damage of rat kidney. We have also examined tissue malondialdehyde (MDA) levels in order to evaluate lipid peroxidation and serum urea and creatinine levels in order to evaluate renal function. 2. Materials and methods 2.1. Preparation of CAPE Caffeic acid phenethyl ester applied in this study was synthesized in the Physico-Chemistry Laboratory according to standard method described by Grunberger et al. (1988). 2.2. Animals and treatments Adult male Wistar albino rats (weighing 170–220 g) obtained from Firat University Medical Faculty Experimental Research Unit were randomly divided into three groups with eight animals per group. All animals received humane care in compliance with the European Community Guidelines on the care and use of laboratory animals (86/609/EEC). The rats were kept in plexiglas cages (4 rats/cage) where they received standard chow (supplied from Elazig Feed Plant, Elazig, Turkey) and water ad libitum in an air-conditioned room with automatically regulated temperature (22 ± 1 ◦ C) and lighting (07.00–19.00 h). All rats were allowed to acclimate for a week prior to experimentation. Control rats subcutaneously received pure olive oil (1 ml). In the second group, rats were injected with CCl4 (0.5 ml/kg body weight per 1 ml olive oil s.c.; EM Science, Cherry Hill, NJ, USA) every other day for one month. Rats in the third group concomitantly received CAPE (10 mol/kg body weight intraperitoneally) and CCl4 for the same duration and at the same injection interval. 2.3. Determination of serum urea and creatinine levels All animals were sacrificed by decapitation 24 h after the final injections. Blood samples were collected in routine bio-
chemical test tubes and centrifuged at 2000 × g for 10 min at 4 ◦ C. Serum urea and creatinine levels were measured by an AU600 multiparametric autoanalyzer (Olympus, Hamburg, Germany). 2.4. Preparation of tissues At the end of the collection of the blood samples, the kidneys of all rats were excised immediately, decapsulated and divided longitudinally into two equal sections. Ones were placed in formaldehyde solution for routine histopathological examination by light microscopy. The other sections were washed in ice-cold saline, placed into glass bottles, labeled and stored at −30 ◦ C until processed. 2.5. Determination of MDA levels Tissues (after cutting into small pieces with scissors) were homogenized in ice-cold Tris–HCl buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Tempest Virtishear, Model 278069; The Virtis Company, Gardiner, NY) for 2 min at 5000 rpm after weighed. The MDA content of the homogenates was determined by Wasowicz’s method (Wasowicz et al., 1993) based on the reaction of MDA with thiobarbituric acid at 95–100 ◦ C. Fluorescence intensity was measured in the upper nbutanol phase by a fluorescence spectrophotometer (Hitachi, Model F-4010) with adjusted excitation at 525 nm and emission at 547 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3tetramethoxypropane). The results were expressed as nmol/g wet tissue. 2.6. Histopathological analysis of kidney The kidney tissue specimens separated for histopathological examination were fixed in neutral formalin (10%). Following routine tissue procedures of light microscopy, kidneys were paraffin-sectioned (5 m-thick), stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and examined with an Olympus BH2 light microscope. All sections were evaluated for the degree of tubular and glomerular injury, vascular congestion, interstitial mononuclear inflammatory cell infiltration and fibrosis by an experienced observer unaware of the treatment. The following grading scheme was used to score the histological alterations: (−) absent; (+) mild; (++) moderate; (+++) severe. 2.7. Statistical analysis 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 soft-
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Fig. 1. Serum urea and creatinine levels in the control, CCl4 and CCl4 plus CAPE-treated rats. There are no statistically significant differences between the groups.
275
Fig. 2. Malondialdehyde (MDA) levels in kidneys of rats divided into control, CCl4 and CCl4 +CAPE subgroups.
3.2. Tissue MDA levels ware (SPSS Inc., Chicago, IL, USA). Differences were considered to be statistically significant at p < 0.05.
3. Results All animals survived to scheduled sacrifice. 3.1. Serum urea and creatinine levels Serum urea and creatinine levels are shown in Fig. 1. CCl4 administration at doses of 0.5 ml/kg every other day for one month (15 injections) decreased both serum urea and creatinine levels relative to control values which was more prominent in the urea level; however, these changes were not statistically significant. CAPE treatment, further, decreased serum urea and creatinine levels, but also not significantly.
As shown in Fig. 2, there were marked increases in kidney MDA levels in CCl4 -treated rats (p < 0.001 versus controls). CAPE treatment significantly reduced the elevations in MDA levels (p < 0.001 versus treated with CCl4 only), and it provided statistically equal levels with controls (p = 0.114). 3.3. Renal histopathology The histopathologically observed changes ranged in severity from none (control group; Fig. 3) to mild (CCl4 +CAPE group; Fig. 8) to severe (CCl4 group; Figs. 4–7). The evaluation of the kidney sections of control rats showed normal structural and architectural integrity (Fig. 3). Chronic administration of CCl4 caused significant morphological damage to the kidneys, especially to the renal cortex. The affect of CCl4 on the medulla, however, was limited. In CCl4 -treated kidneys, the affected glomeruli which were observed in addition to the renal corpuscles with normal appearance exhib-
Fig. 3. Kidney section from control rats treated with olive oil. Glomeruli and tubules appear normal. Masson’s trichrome, ×100.
276
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Fig. 4. Section of a kidney from a CCl4 -treated rat showing mild dilatation of Bowman’s space with glomerular atrophy (arrows) in addition to the renal corpuscles with normal appearance (*), H&E, ×100.
ited different forms of degeneration. Some glomeruli showed mild dilatation of Bowman’s space with glomerular atrophy (Fig. 4), whereas a small number of others, however, exhibited congestion in the capillary loops with an adhesion between visceral and parietal layers of Bowman’s capsule (Fig. 5). Most of renal tubules were dilated (Fig. 5) and their epithelial cells tended to be vacuolated with a foamy appearance (Fig. 6). In the renal cortex, interstitial inflammatory cell infiltrations were clearly apparent after CCl4 treatment. In addition, mild mononuclear inflammatory cell infiltrations were observed in cortico-medullary border similar to those in the cortex. There was an evident increase in the connec-
tive tissue cells in these regions of infiltration as an indicator of fibrosis. Regarding blood vessels, there was considerable congestion in the peritubular vessels (Fig. 7). These findings observed in the renal cortices of CCl4 -administrated rats were spread throughout the subcortical areas as well. In the CCl4 +CAPE group, interstitial inflammatory cell infiltrations were absent, and histological appearances of the glomeruli and tubules were normal. An increase in the connective tissue cells was also not observed after CAPE treatment, but rare vascular congestions were present in both the cortical and cortico-medullar regions (Fig. 8). Histopathological findings are summarized in Table 1.
Fig. 5. Section of a kidney from a CCl4 -intoxicated rat exhibiting congestion in the capillary loops (c) with disappearance of Bowman’s space (arrows). Tubular dilatations are evident (*), H&E, ×200.
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
277
Fig. 6. Foamy appearance in the tubular epithelial cells in a histopathologic section of a rat given only CCl4 (arrows), H&E, ×200.
4. Discussion In this study, we hypothesized that CAPE would effectively protect kidneys by its antioxidant and antiinflammatory effects on CCl4 -induced injury. To the best of our knowledge, this is the first study to evaluate these effects of CAPE in an attempt to prevent kidney damage from CCl4 . Our results demonstrate that CAPE will be able to reduce the damage to the rat kidney induced by chronic CCl4 poisoning. This was verified by both biochemical (MDA levels) and histopathological observations. CCl4 , a hepatotoxin and nephrotoxin, was used for the purpose of inducing renal damage in this study since it has
previously been shown to exert its toxic effects on the kidney besides well-known liver (Dogukan et al., 2003; Ozturk et al., 2003). The mechanism of CCl4 hepatotoxicity is well documented in the rat model (Sharma et al., 1997; Sundari et al., 1997; Sotelo-Felix et al., 2002) According to these reports, CCl4 -induced liver injury is due to the conversion of CCl4 to CCl3 and CCl3 O2 by the cytochrome P450 enzyme. These highly reactive free radicals cause cell damage. However, the pathogenesis of CCl4 -induced renal injury has not been clearly clarified. While Rincon et al. (1999) showed that effects of CCl4 on kidney structure and function depended on the functional state of the liver, Ogawa et al. (1992) suggested etiologic independence of the renal
Fig. 7. Section of a kidney from a CCl4 -intoxicated rat exhibiting an evident increase in the connective tissue with mononuclear inflammatory cell infiltration (*) and considerable vascular congestions around the tubules (arrows). Masson’s trichrome, ×100.
278
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Fig. 8. Renal section from a rat treated with both CCl4 and CAPE showing a markedly similar histological structure to that of control rats with exception of rare vascular congestions (arrows). Masson’s trichrome, ×100.
and hepatic events. The oxidative damage to the lipids and proteins of the rat kidney resulting from chronic toxicity of CCl4 inhalation was reported by Abraham et al. (1999). It has also been reported that systemically administered CCl4 in rats was distributed at higher concentrations in the kidney than in the liver (Sanzgiri et al., 1997). Since the kidney has an affinity for CCl4 (Abraham et al., 1999) and contains cytochrome P450 predominantly in the cortex (Rush et al., 1984; Ronis et al., 1998), we think that the mechanism of CCl4 nephrotoxicity is probably the same as that of the liver and also independent from the diminished functionality of the liver. Numerous experimental studies have demonstrated the beneficial effects of antioxidant treatment on CCl4 -induced tissue injury. Propolis, a honeybee product, mostly contains flavonoids and phenolic compounds, which have been reported to have antioxidative properties (Banskota et al., 2001). Gonzalez et al. (1995) and Merino et al. (1996) showed that Cuban red propolis extract exerts a protective effect on CCl4 -induced liver injury in rats. In these studies, propolis extract was found to significantly reduce the elevations in serum ALT and MDA levels, and liver triglycerides induced by CCl4 . Similarly, Sharma et al. (1997) also found that propolis decreased the levels of liver MDA, and serum Table 1 Grading of the histopathological changes in the kidney sections. Scoring was done as follows: (−) absent; (+) mild; (++) moderate; (+++) severe Parameter
Control
CCl4
CCl4 +CAPE
Interstitial fibrosis Interstitial cell infiltration Glomerular changes Tubular dilatation Vascular congestion
− − − − −
++ +++ ++ ++ +++
− − − − +
ALT and AST which were elevated by alcohol and CCl4 . Moreover, Basnet et al. (1996) examined the hepatoprotective activity of Brazilian propolis against CCl4 -toxicity in rats and found that CCl4 -induced elevations in serum ALT, AST and LDH levels were significantly decreased by propolis pretreatment. On histopathological evaluation of propolis in CCl4 induced liver injury, Merino et al. (1996) found a significant reduction of ballooned cells in the liver of propolistreated rats compared with the CCl4 -treated group. Sharma et al. (1997) also found that multifocal necrotic changes of liver induced by CCl4 and alcohol administration minimized or erased completely by propolis treatment. Furthermore, a dose-dependent hepatoprotective effect of propolis on isolated rat hepatocytes against CCl4 toxicity has been observed in vitro (Mahran et al., 1996), besides the above-mentioned in vivo models. Thus, the current findings are generally consistent with a protective effect of propolis extract at the level of the kidney against CCl4 toxicity. Recently, a few studies have shown the nephroprotective properties of an active component of propolis extract, CAPE. Ozen et al. (2004) reported the protective effects of CAPE against cisplatin-induced nephrotoxicity in rats. It was also demonstrated that acute administration of CAPE suppressed ischemia-reperfusion-induced renal lipid peroxidation and tissue injury (Irmak et al., 2001; Ozyurt et al., 2001). Furthermore, Fadillioglu et al. (2004) reported, although not specific only to the nephroprotective properties of CAPE, that the activities of antioxidant enzymes were increased with CAPE treatment. We revealed that CAPE inhibited CCl4 induced lipid peroxidation in kidney tissue. Significant increases in MDA content in CCl4 -treated rat kidneys were returned to control values with CAPE administration. Thus, it is assumed that CAPE exerts its protective effects in CCl4
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
toxicity through its stimulating effects on the antioxidative defense system as well as its scavenging and/or antioxidant properties. Although there are numerous studies demonstrating that CCl4 causes significantly increases in MDA levels in various tissues including the kidney (Daniels et al., 1995; Donder et al., 1999), however, a limited number of studies have investigated the effects of CAPE on these CCl4 -induced alterations. In addition, no published data has ever demonstrated the influence of CAPE on CCl4 -induced elevation in renal lipid peroxide levels. Regarding to other biochemical parameters which were intended to evaluate renal function, we found no statistically significant changes in serum urea and creatinine concentrations in CCl4 -treated rats in harmony with previous studies (Wirth et al., 1997; Palaparthy et al., 2001; Croquet et al., 2002; Ozdogan et al., 2002). In the present study, we also found that chronic administration of CCl4 damaged the renal cortical and subcortical areas, but had limited effects in the medulla. Based on our light microscopic observations which were in keeping with MDA changes, we detected glomerular and tubular lesions, interstitial inflammatory cell infiltration, interstitial fibrosis and vasocongestion in the kidney sections of CCl4 -treated rats. CAPE substantially reduced CCl4 -induced kidney damage in rats. Presence of a cytochrome P450 system in the kidney cortex and a prostaglandin endoperoxidase in the medulla (Rush et al., 1984) may account for the difference in the level of damage in different regions of the kidney. Similarly, Ozturk et al. (2003) found that subcutaneous injections of CCl4 to rats at daily doses of 1 ml/kg for seven-day extensively caused glomerular and tubular alterations in the renal cortex, and these effects were prevented by betaine pretreatment. Dogukan et al. (2003) also reported that chronic administration of CCl4 (0.15 ml/kg, sc, in olive oil) three times a week for seven weeks in rats caused various degrees of tubular and glomerular changes, interstitial mononuclear cell proliferation and fibrosis in the kidney. In conclusion, the results obtained herein indicate that CAPE exerts a protective effect on CCl4 -induced kidney damage in rats, possibly through its antioxidant properties. We therefore propose that CAPE may be beneficial in reducing tissue damage in patients exposed to toxic doses of CCl4 , but further studies are required to determine the optimal doses of this compound.
References Abraham, P., Wilfred, G., Cathrine, S.P., 1999. Oxidative damage to the lipids and proteins of the lungs, testis and kidney of rats during carbon tetrachloride intoxication. Clinica Chimica Acta 289, 177–179. Bahcecioglu, I.H., Ustundag, B., Ozercan, I., Ercel, E., Baydas, G., Akdere, T., Demir, A., 1999. Protective effect of Ginkgo biloba extract on CCI4 -induced liver damage. Hepatology Research 15, 215–224. Banskota, A.H., Tezuka, Y., Kadota, S., 2001. Recent progress in pharmacological research of propolis. Phytotherapy Research 15, 561–571. Basnet, P., Matsushige, K., Hase, K., Kadota, S., Namba, T., 1996. Four di-O-caffeoyl quinic acid derivatives from propolis. Potent hepato-
279
protective activity in experimental liver injury models. Biological & Pharmaceutical Bulletin 19, 1479–1484. Croquet, V., Moal, F., Veal, N., Wang, J., Oberti, F., Roux, J., Vuillemin, E., Gallois, Y., Douay, O., Chappard, D., Cales, P., 2002. Hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension. Journal of Hepatology 37, 773–780. Daniels, W.M., Reiter, R.J., Melchiorri, D., Sewerynek, E., Pablos, M.I., Ortiz, G.G., 1995. Melatonin counteracts lipid peroxidation induced by carbon tetrachloride but does not restore glucose-6 phosphatase activity. Journal of Pineal Research 19, 1–6. Dogukan, A., Akpolat, N., Celiker, H., Ilhan, N., Bahcecioglu, I.H., Gunal, A.I., 2003. Protective effect of interferon-␣ on carbon tetrachloride-induced nephrotoxicity. Journal of Nephrology 16, 81–84. Donder, E., Baydas, G., Ozkan, Y., Ercel, E., Yalniz, M., Dogan, H., 1999. Investigation of antioxidant effect of melatonin against carbon tetrachloride toxicity in various tissues. Biomedical Research 10, 141–145. Fadhel, Z.A., Amran, S., 2002. Effects of black tea extract on carbon tetrachloride-induced lipid peroxidation in liver, kidneys, and testes of rats. Phytotherapy Research 16, 28–32. Fadillioglu, E., Oztas, E., Erdogan, H., Yagmurca, M., Sogut, S., Ucar, M., Irmak, M.K., 2004. Protective effects of caffeic acid phenethyl ester on doxorubicin-induced cardiotoxicity in rats. Journal of Applied Toxicology 24, 47–52. Gonzalez, R., Corcho, I., Remirez, D., Rodriguez, S., Ancheta, O., Merino, N., Gonzalez, A., Pascual, C., 1995. Hepatoprotective effects of propolis extract on carbon tetrachloride-induced liver injury in rats. Phytotherapy Research 9, 114–117. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E.M., Efros, L., Caldwell, M., Estevez, V., Nakanishi, K., 1988. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 44, 230–232. Ilhan, A., Koltuksuz, U., Ozen, S., Uz, E., Ciralik, H., Akyol, O., 1999. The effects of caffeic acid phenethyl ester (CAPE) on spinal cord ischemia/reperfusion injury in rabbits. European Journal of Cardiothoracic Surgery 16, 458–463. Irmak, M.K., Koltuksuz, U., Kutlu, N.O., Yagmurca, M., Ozyurt, H., Karaman, A., Akyol, O., 2001. The effect of caffeic acid phenethyl ester on ischemia-reperfusion injury in comparison with ␣-tocopherol in rat kidneys. Urological Research 29, 190–193. Kovacic, P., Sacman, A., Wu-Weis, M., 2002. Nephrotoxins: widespread role of oxidative stress and electron transfer. Current Medicinal Chemistry 9, 823–847. Mahran, L.G., el-Khatib, A.S., Agha, A.M., Khayyal, M.T., 1996. The protective effect of aqueous propolis extract on isolated rat hepatocytes against carbon tetrachloride toxicity. Drugs under Experimental and Clinical Research 22, 309–316. Merino, N., Gonzalez, R., Gonzalez, A., Remirez, D., 1996. Histopathological evaluation on the effect of red propolis on liver damage induced by CCl4 in rats. Archives of Medical Research 27, 285–289. Ogawa, M., Mori, T., Mori, Y., Ueda, S., Azemoto, R., Makino, Y., Wakashin, Y., Ohto, M., Wakashin, M., Yoshida, H., Iesato, K., 1992. Study on chronic renal injuries induced by carbon tetrachloride: selective inhibition of the nephrotoxicity by irradiation. Nephron 60, 68–73. Ozdogan, O., Goren, M.Z., Ratip, S., Giral, A., Moini, H., Enc, F., Birsel, S., Berkman, K., Tozun, N., 2002. Role of endothelin-1 in a cirrhotic rat model with endotoxin induced acute renal failure. Hepatology Research 24, 114–124. Ozen, S., Akyol, O., Iraz, M., Sogut, S., Ozugurlu, F., Ozyurt, H., Odaci, E., Yildirim, Z., 2004. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. Journal of Applied Toxicology 24, 27–35. Ozturk, F., Ucar, M., Ozturk, I.C., Vardi, N., Batcioglu, K., 2003. Carbon tetrachloride-induced nephrotoxicity and protective effect of betaine in Sprague-Dawley rats. Urology 62, 353–356.
280
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Ozyurt, H., Irmak, M.K., Akyol, O., Sogut, S., 2001. Caffeic acid phenethyl ester changes the indices of oxidative stress in serum of rats with renal ischaemia-reperfusion injury. Cell Biochemistry and Function 19, 259–263. Palaparthy, R., Kastrissios, H., Gulati, A., 2001. Pharmacokinetics of diaspirin cross-linked haemoglobin in a rat model of hepatic cirrhosis. Journal of Pharmacy and Pharmacology 53, 179–185. Rincon, A.R., Covarrubias, A., Pedraza-Chaverri, J., Poo, J.L., Armendariz-Borunda, J., Panduro, A., 1999. Differential effect of CCl4 on renal function in cirrhotic and non-cirrhotic rats. Experimental and Toxicologic Pathology 51, 199–205. Ronis, M.J., Huang, J., Longo, V., Tindberg, N., Ingelman-Sundberg, M., Badger, T.M., 1998. Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary conditions. Biochemical Pharmacology 55, 123–129. Rush, G.F., Smith, J.H., Newton, J.F., Hook, J.B., 1984. Chemically induced nephrotoxicity: role of metabolic activation. Critical Reviews in Toxicology 13, 99–160. Sanzgiri, U.Y., Srivatsan, V., Muralidhara, S., Dallas, C.E., Bruckner, J.V., 1997. Uptake, distribution, and elimination of carbon tetrachloride in rat tissues following inhalation and ingestion exposures. Toxicology and Applied Pharmacology 143, 120–129. Sharma, M., Pillai, K.K., Husain, S.Z., Giri, D.K., 1997. Protective role of propolis against alcohol–carbon tetrachloride-induced hepatotoxicity in rats. Indian Journal of Pharmacology 29, 76– 81. Sotelo-Felix, J.I., Martinez-Fong, D., Muriel, P., Santillan, R.L., Castillo, D., Yahuaca, P., 2002. Evaluation of the effectiveness of Rosmarinus officinalis (Lamiaceae) in the alleviation of carbon tetrachloride-
induced acute hepatotoxicity in the rat. Journal of Ethnopharmacology 81, 145–154. Sundari, P.N., Wilfred, G., Ramakrishna, B., 1997. Does oxidative protein damage play a role in the pathogenesis of carbon tetrachloride-induced liver injury in the rat? Biochimica et Biophysica Acta 1362, 169– 176. Thrall, K.D., Vucelick, M.E., Gies, R.A., Zangar, R.C., Weitz, K.K., Poet, T.S., Springer, D.L., Grant, D.M., Benson, J.M., 2000. Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling. Journal of Toxicology and Environmental Health Part A 60, 531–548. Turkdogan, M.K., Agaoglu, Z., Yener, Z., Sekeroglu, R., Akkan, H.A., Avci, M.E., 2001. The role of antioxidant vitamins (C and E), selenium and Nigella sativa in the prevention of liver fibrosis and cirrhosis in rabbits: new hopes. Deutsche tierarztliche Wochenschrift 108, 71–73. Wasowicz, W., Neve, J., Peretz, A., 1993. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clinical Chemistry 39, 2522–2526. Wirth, K.J., Bickel, M., Hropot, M., Gunzler, V., Heitsch, H., Ruppert, D., Scholkens, B.A., 1997. The bradykinin B2 receptor antagonist Icatibant (HOE 140) corrects avid Na+ retention in rats with CCl4 -induced liver cirrhosis: possible role of enhanced microvascular leakage. European Journal of Pharmacology 337, 45–53. Zhao, W.X., Zhao, J., Liang, C.L., Zhao, B., Pang, R.Q., Pan, X.H., 2003. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro. World Journal of Gastroenterology 9, 1278–1281.
Caffeic acid phenethyl ester protects kidneys against carbon tetrachloride toxicity in rats M. Ogeturka , I. Kusa , N. Colakoglub , I. Zararsiza , N. Ilhanc , M. Sarsilmaza,∗ b
a Department of Anatomy, Faculty of Medicine, Firat University, 23119 Elazig, Turkey Department of Histology and Embryology, Faculty of Medicine, 23119 Firat University, Elazig, Turkey c Department of Biochemistry, Faculty of Medicine, Firat University, 23119 Elazig, Turkey
Received 31 March 2004; accepted 10 November 2004 Available online 12 January 2005
Abstract Caffeic acid phenethyl ester (CAPE), an active component of propolis produced by honeybees, exhibits antioxidant and anti-inflammatory properties. The aim of this study was to investigate possible protective effects of CAPE on carbon tetrachloride (CCl4 )-induced renal damage in rats. A total of 24 animals were divided into three equal groups: the control rats received pure olive oil subcutaneously, rats in the second group were injected with CCl4 (0.5 ml/kg, s.c. in olive oil) and rats in the third group were injected with CCl4 (0.5 ml/kg) plus CAPE (10 mol/kg, i.p.) every other day for one month. At the end of the experimental period, the animals were sacrificed and blood samples were collected. Serum urea and creatinine levels and renal malondialdehyde (MDA) contents were determined. Histopathological examination of the kidney was also performed using light microscopic methods. It was found that kidney MDA levels were increased significantly following CCl4 exposure and this increase was significantly inhibited by CAPE treatment, while no significant changes were observed in serum urea and creatinine levels. CCl4 administration alone also caused histopathologically prominent damage in the kidney compared to the control group. Glomerular and tubular degeneration, interstitial mononuclear cell infiltration and fibrosis, and vascular congestion in the peritubular blood vessels were observed in the renal cortex. With exception of rare vascular congestions, these histopathological changes were disappeared in rats treated with CCl4 plus CAPE. In view of the present findings, it is suggested that CAPE protects kidneys against CCl4 toxicity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Caffeic acid phenethyl ester; Carbon tetrachloride; Nephrotoxicity; Histopathology; Light microscopy
1. Introduction Carbon tetrachloride (CCl4 ), a clear, heavy, and nonflammable liquid is most widely used for experimental induction of hepatic cirrhosis (Sundari et al., 1997; Bahcecioglu et al., 1999). It is known to be nephrotoxic as well as hepatotoxic to humans (Abraham et al., 1999). Furthermore, it has been identified as a probable human carcinogen based on evidence of tumors in animals (Thrall et al., 2000) In years past, carbon tetrachloride was widely used as a dry ∗ Corresponding author. Tel.: +90 424 2370000x6035; fax: +90 424 2379138. E-mail addresses: [email protected], [email protected] (M. Sarsilmaz).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.019
cleaning solvent until it was recognized as a potent hepatotoxin, nephrotoxin and carcinogen. Today, it is primarily used as an organic solvent (Kovacic et al., 2002). Administration of CCl4 causes an increase in lipid peroxidation products (Daniels et al., 1995; Abraham et al., 1999; Donder et al., 1999) and a decrease in the activity of enzymes protecting lipid peroxidation in the kidney (Dogukan et al., 2003). These detrimental effects of CCl4 have been attributed to conversion of CCl4 to highly toxic trichloromethyl and trichloromethyl peroxyl free radicals by cytochrome P450 enzyme, resulting in cell injury (Sundari et al., 1997; Abraham et al., 1999; Bahcecioglu et al., 1999). Several antioxidant agents, including ginkgo biloba and black tea extracts, antioxidant vitamins (C and E), and melatonin have been reported to reduce CCl4 -induced nephrotoxicity (Bahcecioglu
274
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
et al., 1999; Donder et al., 1999; Turkdogan et al., 2001; Fadhel and Amran, 2002). The caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is know to have antioxidant, immunomodulatory, anti-inflammatory and anticarcinogenic properties and has been used as a folk medicine for many years (Banskota et al., 2001; Ozen et al., 2004). It has been shown to be a pharmacologically safe compound (Zhao et al., 2003). It has also been shown to suppress lipid peroxidation (Ilhan et al., 1999; Irmak et al., 2001) and to stimulate the activity of antioxidant enzymes (Fadillioglu et al., 2004; Ozen et al., 2004). Although there is considerable interest in the role of CAPE as a dietary antioxidant, its effects on CCl4 induced renal damage have not been investigated to date. The aim of the present study was therefore to investigate whether CAPE treatment prevents CCl4 -induced nephrotoxicity. For this purpose, we have examined histopathological effects of CCl4 and possible protective effect of CAPE on tissue damage of rat kidney. We have also examined tissue malondialdehyde (MDA) levels in order to evaluate lipid peroxidation and serum urea and creatinine levels in order to evaluate renal function. 2. Materials and methods 2.1. Preparation of CAPE Caffeic acid phenethyl ester applied in this study was synthesized in the Physico-Chemistry Laboratory according to standard method described by Grunberger et al. (1988). 2.2. Animals and treatments Adult male Wistar albino rats (weighing 170–220 g) obtained from Firat University Medical Faculty Experimental Research Unit were randomly divided into three groups with eight animals per group. All animals received humane care in compliance with the European Community Guidelines on the care and use of laboratory animals (86/609/EEC). The rats were kept in plexiglas cages (4 rats/cage) where they received standard chow (supplied from Elazig Feed Plant, Elazig, Turkey) and water ad libitum in an air-conditioned room with automatically regulated temperature (22 ± 1 ◦ C) and lighting (07.00–19.00 h). All rats were allowed to acclimate for a week prior to experimentation. Control rats subcutaneously received pure olive oil (1 ml). In the second group, rats were injected with CCl4 (0.5 ml/kg body weight per 1 ml olive oil s.c.; EM Science, Cherry Hill, NJ, USA) every other day for one month. Rats in the third group concomitantly received CAPE (10 mol/kg body weight intraperitoneally) and CCl4 for the same duration and at the same injection interval. 2.3. Determination of serum urea and creatinine levels All animals were sacrificed by decapitation 24 h after the final injections. Blood samples were collected in routine bio-
chemical test tubes and centrifuged at 2000 × g for 10 min at 4 ◦ C. Serum urea and creatinine levels were measured by an AU600 multiparametric autoanalyzer (Olympus, Hamburg, Germany). 2.4. Preparation of tissues At the end of the collection of the blood samples, the kidneys of all rats were excised immediately, decapsulated and divided longitudinally into two equal sections. Ones were placed in formaldehyde solution for routine histopathological examination by light microscopy. The other sections were washed in ice-cold saline, placed into glass bottles, labeled and stored at −30 ◦ C until processed. 2.5. Determination of MDA levels Tissues (after cutting into small pieces with scissors) were homogenized in ice-cold Tris–HCl buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Tempest Virtishear, Model 278069; The Virtis Company, Gardiner, NY) for 2 min at 5000 rpm after weighed. The MDA content of the homogenates was determined by Wasowicz’s method (Wasowicz et al., 1993) based on the reaction of MDA with thiobarbituric acid at 95–100 ◦ C. Fluorescence intensity was measured in the upper nbutanol phase by a fluorescence spectrophotometer (Hitachi, Model F-4010) with adjusted excitation at 525 nm and emission at 547 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3tetramethoxypropane). The results were expressed as nmol/g wet tissue. 2.6. Histopathological analysis of kidney The kidney tissue specimens separated for histopathological examination were fixed in neutral formalin (10%). Following routine tissue procedures of light microscopy, kidneys were paraffin-sectioned (5 m-thick), stained with hematoxylin and eosin (H&E) and Masson’s trichrome, and examined with an Olympus BH2 light microscope. All sections were evaluated for the degree of tubular and glomerular injury, vascular congestion, interstitial mononuclear inflammatory cell infiltration and fibrosis by an experienced observer unaware of the treatment. The following grading scheme was used to score the histological alterations: (−) absent; (+) mild; (++) moderate; (+++) severe. 2.7. Statistical analysis 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 soft-
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Fig. 1. Serum urea and creatinine levels in the control, CCl4 and CCl4 plus CAPE-treated rats. There are no statistically significant differences between the groups.
275
Fig. 2. Malondialdehyde (MDA) levels in kidneys of rats divided into control, CCl4 and CCl4 +CAPE subgroups.
3.2. Tissue MDA levels ware (SPSS Inc., Chicago, IL, USA). Differences were considered to be statistically significant at p < 0.05.
3. Results All animals survived to scheduled sacrifice. 3.1. Serum urea and creatinine levels Serum urea and creatinine levels are shown in Fig. 1. CCl4 administration at doses of 0.5 ml/kg every other day for one month (15 injections) decreased both serum urea and creatinine levels relative to control values which was more prominent in the urea level; however, these changes were not statistically significant. CAPE treatment, further, decreased serum urea and creatinine levels, but also not significantly.
As shown in Fig. 2, there were marked increases in kidney MDA levels in CCl4 -treated rats (p < 0.001 versus controls). CAPE treatment significantly reduced the elevations in MDA levels (p < 0.001 versus treated with CCl4 only), and it provided statistically equal levels with controls (p = 0.114). 3.3. Renal histopathology The histopathologically observed changes ranged in severity from none (control group; Fig. 3) to mild (CCl4 +CAPE group; Fig. 8) to severe (CCl4 group; Figs. 4–7). The evaluation of the kidney sections of control rats showed normal structural and architectural integrity (Fig. 3). Chronic administration of CCl4 caused significant morphological damage to the kidneys, especially to the renal cortex. The affect of CCl4 on the medulla, however, was limited. In CCl4 -treated kidneys, the affected glomeruli which were observed in addition to the renal corpuscles with normal appearance exhib-
Fig. 3. Kidney section from control rats treated with olive oil. Glomeruli and tubules appear normal. Masson’s trichrome, ×100.
276
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Fig. 4. Section of a kidney from a CCl4 -treated rat showing mild dilatation of Bowman’s space with glomerular atrophy (arrows) in addition to the renal corpuscles with normal appearance (*), H&E, ×100.
ited different forms of degeneration. Some glomeruli showed mild dilatation of Bowman’s space with glomerular atrophy (Fig. 4), whereas a small number of others, however, exhibited congestion in the capillary loops with an adhesion between visceral and parietal layers of Bowman’s capsule (Fig. 5). Most of renal tubules were dilated (Fig. 5) and their epithelial cells tended to be vacuolated with a foamy appearance (Fig. 6). In the renal cortex, interstitial inflammatory cell infiltrations were clearly apparent after CCl4 treatment. In addition, mild mononuclear inflammatory cell infiltrations were observed in cortico-medullary border similar to those in the cortex. There was an evident increase in the connec-
tive tissue cells in these regions of infiltration as an indicator of fibrosis. Regarding blood vessels, there was considerable congestion in the peritubular vessels (Fig. 7). These findings observed in the renal cortices of CCl4 -administrated rats were spread throughout the subcortical areas as well. In the CCl4 +CAPE group, interstitial inflammatory cell infiltrations were absent, and histological appearances of the glomeruli and tubules were normal. An increase in the connective tissue cells was also not observed after CAPE treatment, but rare vascular congestions were present in both the cortical and cortico-medullar regions (Fig. 8). Histopathological findings are summarized in Table 1.
Fig. 5. Section of a kidney from a CCl4 -intoxicated rat exhibiting congestion in the capillary loops (c) with disappearance of Bowman’s space (arrows). Tubular dilatations are evident (*), H&E, ×200.
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
277
Fig. 6. Foamy appearance in the tubular epithelial cells in a histopathologic section of a rat given only CCl4 (arrows), H&E, ×200.
4. Discussion In this study, we hypothesized that CAPE would effectively protect kidneys by its antioxidant and antiinflammatory effects on CCl4 -induced injury. To the best of our knowledge, this is the first study to evaluate these effects of CAPE in an attempt to prevent kidney damage from CCl4 . Our results demonstrate that CAPE will be able to reduce the damage to the rat kidney induced by chronic CCl4 poisoning. This was verified by both biochemical (MDA levels) and histopathological observations. CCl4 , a hepatotoxin and nephrotoxin, was used for the purpose of inducing renal damage in this study since it has
previously been shown to exert its toxic effects on the kidney besides well-known liver (Dogukan et al., 2003; Ozturk et al., 2003). The mechanism of CCl4 hepatotoxicity is well documented in the rat model (Sharma et al., 1997; Sundari et al., 1997; Sotelo-Felix et al., 2002) According to these reports, CCl4 -induced liver injury is due to the conversion of CCl4 to CCl3 and CCl3 O2 by the cytochrome P450 enzyme. These highly reactive free radicals cause cell damage. However, the pathogenesis of CCl4 -induced renal injury has not been clearly clarified. While Rincon et al. (1999) showed that effects of CCl4 on kidney structure and function depended on the functional state of the liver, Ogawa et al. (1992) suggested etiologic independence of the renal
Fig. 7. Section of a kidney from a CCl4 -intoxicated rat exhibiting an evident increase in the connective tissue with mononuclear inflammatory cell infiltration (*) and considerable vascular congestions around the tubules (arrows). Masson’s trichrome, ×100.
278
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Fig. 8. Renal section from a rat treated with both CCl4 and CAPE showing a markedly similar histological structure to that of control rats with exception of rare vascular congestions (arrows). Masson’s trichrome, ×100.
and hepatic events. The oxidative damage to the lipids and proteins of the rat kidney resulting from chronic toxicity of CCl4 inhalation was reported by Abraham et al. (1999). It has also been reported that systemically administered CCl4 in rats was distributed at higher concentrations in the kidney than in the liver (Sanzgiri et al., 1997). Since the kidney has an affinity for CCl4 (Abraham et al., 1999) and contains cytochrome P450 predominantly in the cortex (Rush et al., 1984; Ronis et al., 1998), we think that the mechanism of CCl4 nephrotoxicity is probably the same as that of the liver and also independent from the diminished functionality of the liver. Numerous experimental studies have demonstrated the beneficial effects of antioxidant treatment on CCl4 -induced tissue injury. Propolis, a honeybee product, mostly contains flavonoids and phenolic compounds, which have been reported to have antioxidative properties (Banskota et al., 2001). Gonzalez et al. (1995) and Merino et al. (1996) showed that Cuban red propolis extract exerts a protective effect on CCl4 -induced liver injury in rats. In these studies, propolis extract was found to significantly reduce the elevations in serum ALT and MDA levels, and liver triglycerides induced by CCl4 . Similarly, Sharma et al. (1997) also found that propolis decreased the levels of liver MDA, and serum Table 1 Grading of the histopathological changes in the kidney sections. Scoring was done as follows: (−) absent; (+) mild; (++) moderate; (+++) severe Parameter
Control
CCl4
CCl4 +CAPE
Interstitial fibrosis Interstitial cell infiltration Glomerular changes Tubular dilatation Vascular congestion
− − − − −
++ +++ ++ ++ +++
− − − − +
ALT and AST which were elevated by alcohol and CCl4 . Moreover, Basnet et al. (1996) examined the hepatoprotective activity of Brazilian propolis against CCl4 -toxicity in rats and found that CCl4 -induced elevations in serum ALT, AST and LDH levels were significantly decreased by propolis pretreatment. On histopathological evaluation of propolis in CCl4 induced liver injury, Merino et al. (1996) found a significant reduction of ballooned cells in the liver of propolistreated rats compared with the CCl4 -treated group. Sharma et al. (1997) also found that multifocal necrotic changes of liver induced by CCl4 and alcohol administration minimized or erased completely by propolis treatment. Furthermore, a dose-dependent hepatoprotective effect of propolis on isolated rat hepatocytes against CCl4 toxicity has been observed in vitro (Mahran et al., 1996), besides the above-mentioned in vivo models. Thus, the current findings are generally consistent with a protective effect of propolis extract at the level of the kidney against CCl4 toxicity. Recently, a few studies have shown the nephroprotective properties of an active component of propolis extract, CAPE. Ozen et al. (2004) reported the protective effects of CAPE against cisplatin-induced nephrotoxicity in rats. It was also demonstrated that acute administration of CAPE suppressed ischemia-reperfusion-induced renal lipid peroxidation and tissue injury (Irmak et al., 2001; Ozyurt et al., 2001). Furthermore, Fadillioglu et al. (2004) reported, although not specific only to the nephroprotective properties of CAPE, that the activities of antioxidant enzymes were increased with CAPE treatment. We revealed that CAPE inhibited CCl4 induced lipid peroxidation in kidney tissue. Significant increases in MDA content in CCl4 -treated rat kidneys were returned to control values with CAPE administration. Thus, it is assumed that CAPE exerts its protective effects in CCl4
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
toxicity through its stimulating effects on the antioxidative defense system as well as its scavenging and/or antioxidant properties. Although there are numerous studies demonstrating that CCl4 causes significantly increases in MDA levels in various tissues including the kidney (Daniels et al., 1995; Donder et al., 1999), however, a limited number of studies have investigated the effects of CAPE on these CCl4 -induced alterations. In addition, no published data has ever demonstrated the influence of CAPE on CCl4 -induced elevation in renal lipid peroxide levels. Regarding to other biochemical parameters which were intended to evaluate renal function, we found no statistically significant changes in serum urea and creatinine concentrations in CCl4 -treated rats in harmony with previous studies (Wirth et al., 1997; Palaparthy et al., 2001; Croquet et al., 2002; Ozdogan et al., 2002). In the present study, we also found that chronic administration of CCl4 damaged the renal cortical and subcortical areas, but had limited effects in the medulla. Based on our light microscopic observations which were in keeping with MDA changes, we detected glomerular and tubular lesions, interstitial inflammatory cell infiltration, interstitial fibrosis and vasocongestion in the kidney sections of CCl4 -treated rats. CAPE substantially reduced CCl4 -induced kidney damage in rats. Presence of a cytochrome P450 system in the kidney cortex and a prostaglandin endoperoxidase in the medulla (Rush et al., 1984) may account for the difference in the level of damage in different regions of the kidney. Similarly, Ozturk et al. (2003) found that subcutaneous injections of CCl4 to rats at daily doses of 1 ml/kg for seven-day extensively caused glomerular and tubular alterations in the renal cortex, and these effects were prevented by betaine pretreatment. Dogukan et al. (2003) also reported that chronic administration of CCl4 (0.15 ml/kg, sc, in olive oil) three times a week for seven weeks in rats caused various degrees of tubular and glomerular changes, interstitial mononuclear cell proliferation and fibrosis in the kidney. In conclusion, the results obtained herein indicate that CAPE exerts a protective effect on CCl4 -induced kidney damage in rats, possibly through its antioxidant properties. We therefore propose that CAPE may be beneficial in reducing tissue damage in patients exposed to toxic doses of CCl4 , but further studies are required to determine the optimal doses of this compound.
References Abraham, P., Wilfred, G., Cathrine, S.P., 1999. Oxidative damage to the lipids and proteins of the lungs, testis and kidney of rats during carbon tetrachloride intoxication. Clinica Chimica Acta 289, 177–179. Bahcecioglu, I.H., Ustundag, B., Ozercan, I., Ercel, E., Baydas, G., Akdere, T., Demir, A., 1999. Protective effect of Ginkgo biloba extract on CCI4 -induced liver damage. Hepatology Research 15, 215–224. Banskota, A.H., Tezuka, Y., Kadota, S., 2001. Recent progress in pharmacological research of propolis. Phytotherapy Research 15, 561–571. Basnet, P., Matsushige, K., Hase, K., Kadota, S., Namba, T., 1996. Four di-O-caffeoyl quinic acid derivatives from propolis. Potent hepato-
279
protective activity in experimental liver injury models. Biological & Pharmaceutical Bulletin 19, 1479–1484. Croquet, V., Moal, F., Veal, N., Wang, J., Oberti, F., Roux, J., Vuillemin, E., Gallois, Y., Douay, O., Chappard, D., Cales, P., 2002. Hemodynamic and antifibrotic effects of losartan in rats with liver fibrosis and/or portal hypertension. Journal of Hepatology 37, 773–780. Daniels, W.M., Reiter, R.J., Melchiorri, D., Sewerynek, E., Pablos, M.I., Ortiz, G.G., 1995. Melatonin counteracts lipid peroxidation induced by carbon tetrachloride but does not restore glucose-6 phosphatase activity. Journal of Pineal Research 19, 1–6. Dogukan, A., Akpolat, N., Celiker, H., Ilhan, N., Bahcecioglu, I.H., Gunal, A.I., 2003. Protective effect of interferon-␣ on carbon tetrachloride-induced nephrotoxicity. Journal of Nephrology 16, 81–84. Donder, E., Baydas, G., Ozkan, Y., Ercel, E., Yalniz, M., Dogan, H., 1999. Investigation of antioxidant effect of melatonin against carbon tetrachloride toxicity in various tissues. Biomedical Research 10, 141–145. Fadhel, Z.A., Amran, S., 2002. Effects of black tea extract on carbon tetrachloride-induced lipid peroxidation in liver, kidneys, and testes of rats. Phytotherapy Research 16, 28–32. Fadillioglu, E., Oztas, E., Erdogan, H., Yagmurca, M., Sogut, S., Ucar, M., Irmak, M.K., 2004. Protective effects of caffeic acid phenethyl ester on doxorubicin-induced cardiotoxicity in rats. Journal of Applied Toxicology 24, 47–52. Gonzalez, R., Corcho, I., Remirez, D., Rodriguez, S., Ancheta, O., Merino, N., Gonzalez, A., Pascual, C., 1995. Hepatoprotective effects of propolis extract on carbon tetrachloride-induced liver injury in rats. Phytotherapy Research 9, 114–117. Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E.M., Efros, L., Caldwell, M., Estevez, V., Nakanishi, K., 1988. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 44, 230–232. Ilhan, A., Koltuksuz, U., Ozen, S., Uz, E., Ciralik, H., Akyol, O., 1999. The effects of caffeic acid phenethyl ester (CAPE) on spinal cord ischemia/reperfusion injury in rabbits. European Journal of Cardiothoracic Surgery 16, 458–463. Irmak, M.K., Koltuksuz, U., Kutlu, N.O., Yagmurca, M., Ozyurt, H., Karaman, A., Akyol, O., 2001. The effect of caffeic acid phenethyl ester on ischemia-reperfusion injury in comparison with ␣-tocopherol in rat kidneys. Urological Research 29, 190–193. Kovacic, P., Sacman, A., Wu-Weis, M., 2002. Nephrotoxins: widespread role of oxidative stress and electron transfer. Current Medicinal Chemistry 9, 823–847. Mahran, L.G., el-Khatib, A.S., Agha, A.M., Khayyal, M.T., 1996. The protective effect of aqueous propolis extract on isolated rat hepatocytes against carbon tetrachloride toxicity. Drugs under Experimental and Clinical Research 22, 309–316. Merino, N., Gonzalez, R., Gonzalez, A., Remirez, D., 1996. Histopathological evaluation on the effect of red propolis on liver damage induced by CCl4 in rats. Archives of Medical Research 27, 285–289. Ogawa, M., Mori, T., Mori, Y., Ueda, S., Azemoto, R., Makino, Y., Wakashin, Y., Ohto, M., Wakashin, M., Yoshida, H., Iesato, K., 1992. Study on chronic renal injuries induced by carbon tetrachloride: selective inhibition of the nephrotoxicity by irradiation. Nephron 60, 68–73. Ozdogan, O., Goren, M.Z., Ratip, S., Giral, A., Moini, H., Enc, F., Birsel, S., Berkman, K., Tozun, N., 2002. Role of endothelin-1 in a cirrhotic rat model with endotoxin induced acute renal failure. Hepatology Research 24, 114–124. Ozen, S., Akyol, O., Iraz, M., Sogut, S., Ozugurlu, F., Ozyurt, H., Odaci, E., Yildirim, Z., 2004. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. Journal of Applied Toxicology 24, 27–35. Ozturk, F., Ucar, M., Ozturk, I.C., Vardi, N., Batcioglu, K., 2003. Carbon tetrachloride-induced nephrotoxicity and protective effect of betaine in Sprague-Dawley rats. Urology 62, 353–356.
280
M. Ogeturk et al. / Journal of Ethnopharmacology 97 (2005) 273–280
Ozyurt, H., Irmak, M.K., Akyol, O., Sogut, S., 2001. Caffeic acid phenethyl ester changes the indices of oxidative stress in serum of rats with renal ischaemia-reperfusion injury. Cell Biochemistry and Function 19, 259–263. Palaparthy, R., Kastrissios, H., Gulati, A., 2001. Pharmacokinetics of diaspirin cross-linked haemoglobin in a rat model of hepatic cirrhosis. Journal of Pharmacy and Pharmacology 53, 179–185. Rincon, A.R., Covarrubias, A., Pedraza-Chaverri, J., Poo, J.L., Armendariz-Borunda, J., Panduro, A., 1999. Differential effect of CCl4 on renal function in cirrhotic and non-cirrhotic rats. Experimental and Toxicologic Pathology 51, 199–205. Ronis, M.J., Huang, J., Longo, V., Tindberg, N., Ingelman-Sundberg, M., Badger, T.M., 1998. Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary conditions. Biochemical Pharmacology 55, 123–129. Rush, G.F., Smith, J.H., Newton, J.F., Hook, J.B., 1984. Chemically induced nephrotoxicity: role of metabolic activation. Critical Reviews in Toxicology 13, 99–160. Sanzgiri, U.Y., Srivatsan, V., Muralidhara, S., Dallas, C.E., Bruckner, J.V., 1997. Uptake, distribution, and elimination of carbon tetrachloride in rat tissues following inhalation and ingestion exposures. Toxicology and Applied Pharmacology 143, 120–129. Sharma, M., Pillai, K.K., Husain, S.Z., Giri, D.K., 1997. Protective role of propolis against alcohol–carbon tetrachloride-induced hepatotoxicity in rats. Indian Journal of Pharmacology 29, 76– 81. Sotelo-Felix, J.I., Martinez-Fong, D., Muriel, P., Santillan, R.L., Castillo, D., Yahuaca, P., 2002. Evaluation of the effectiveness of Rosmarinus officinalis (Lamiaceae) in the alleviation of carbon tetrachloride-
induced acute hepatotoxicity in the rat. Journal of Ethnopharmacology 81, 145–154. Sundari, P.N., Wilfred, G., Ramakrishna, B., 1997. Does oxidative protein damage play a role in the pathogenesis of carbon tetrachloride-induced liver injury in the rat? Biochimica et Biophysica Acta 1362, 169– 176. Thrall, K.D., Vucelick, M.E., Gies, R.A., Zangar, R.C., Weitz, K.K., Poet, T.S., Springer, D.L., Grant, D.M., Benson, J.M., 2000. Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling. Journal of Toxicology and Environmental Health Part A 60, 531–548. Turkdogan, M.K., Agaoglu, Z., Yener, Z., Sekeroglu, R., Akkan, H.A., Avci, M.E., 2001. The role of antioxidant vitamins (C and E), selenium and Nigella sativa in the prevention of liver fibrosis and cirrhosis in rabbits: new hopes. Deutsche tierarztliche Wochenschrift 108, 71–73. Wasowicz, W., Neve, J., Peretz, A., 1993. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clinical Chemistry 39, 2522–2526. Wirth, K.J., Bickel, M., Hropot, M., Gunzler, V., Heitsch, H., Ruppert, D., Scholkens, B.A., 1997. The bradykinin B2 receptor antagonist Icatibant (HOE 140) corrects avid Na+ retention in rats with CCl4 -induced liver cirrhosis: possible role of enhanced microvascular leakage. European Journal of Pharmacology 337, 45–53. Zhao, W.X., Zhao, J., Liang, C.L., Zhao, B., Pang, R.Q., Pan, X.H., 2003. Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro. World Journal of Gastroenterology 9, 1278–1281.