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Effect of carnosine against thioacetamide-induced liver cirrhosis in rat
Peptides 31 (2010) 67–71
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Effect of carnosine against thioacetamide-induced liver cirrhosis in rat A. Fatih Aydın a, Zeynep Ku¨sku¨-Kiraz a, Semra Dog˘ru-Abbasog˘lu a, Mine Gu¨llu¨og˘lu b, Mu¨jdat Uysal a, Necla Koc¸ak-Toker a,* a b
Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey Department of Pathology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2009 Received in revised form 25 November 2009 Accepted 25 November 2009 Available online 1 December 2009
Carnosine (b-alanyl-L-histidine) is a dipeptide with antioxidant properties. Oxidative stress has been proposed to be involved in thioacetamide (TAA)-induced liver cirrhosis in rats, that is similar to human disease. In this study we aimed to investigate the role of carnosine on the development of TAA-induced cirrhosis. 200 mg TAA/kg body weight has been given i.p. twice a week for three months to female wistar rats. Another group received same dose of TAA in the same pattern plus 2 g carnosine/L of drinking water for three months. TAA administration resulted in hepatic fibrosis, significant increases in plasma transaminase activities as well as hepatic hydroxyproline and lipid peroxide levels, while liver glutathione (GSH) and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) protein expressions and activities decreased. Carnosine was found to behave as an antioxidant reducing malondialdehyde (MDA) and diene conjugate (DC) levels although it was not effective on increased transaminase activities and decreased antioxidants. It also did not affect the histopathological changes observed in TAA group. Thus our findings indicate that carnosine appears to attenuate peroxidation as an antioxidant itself but does not seem to prevent the development of TAA-induced cirrhotic process. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Thioacetamide Carnosine Cirrhosis Oxidative stress SOD GSH-Px
1. Introduction Liver cirrhosis associated with various pathological processes, is characterized by progressive fibrosis producing liver injury, portal hypertension and carcinoma [12]. Fibrosis resulting from activation of stellate cells is considered to be provoked by oxidative stress and cytokines [11,28]. Indeed induction of free radical generation, mitochondrial dysfunction and depletion of antioxidants are effective in the progression of fibrosis and cirrhosis [25]. In several experimental models for the induction of cirrhosis, hepatotoxic agents such as thioacetamide (TAA), carbon tetrachloride and ethionine have been used [22,25,37]. TAA is a widely used agent to develop liver cirrhosis in rats that is similar to human cirrhosis. TAA is metabolically activated to thioacetamide sulfoxide and further to thioacetamide-S,S-dioxide. In fact toxic effects of TAA are attributed to those reactive metabolites [9,10]. TAAinduced liver cirrhosis is observed to be associated with lipid peroxidation and depletion of antioxidants [1,21,32,35]. Accordingly reduction of oxidative stress appears to be helpful for the regression of fibrosis and cirrhosis. Thus use of radical scavengers and antioxidants to prevent fibrosis have been suggested benefi-
* Corresponding author. Tel.: +90 2124142188; fax: +90 2126215642. E-mail address: [email protected] (s.$. Koc¸ak-Toker). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.11.028
cial. For this purpose hydroxyl radical scavengers like dimethylsulfoxide and dimethylthiourea [6] extract of solanum nigrum [17], melatonin [11], selenium [30], curcumin [7] and taurine [4] have been applied in TAA-induced liver cirrhosis and reduction of radical mediated hepatic damage has been reported to inhibit the development of cirrhosis. In recent years carnosine has provoked interest as an antioxidant although its discovery has been documented almost a century before [15]. Carnosine (b-alanyl-L-histidine) is a dipeptide which is found at relatively high concentrations in mammalian tissues [2]. It has several functions such as membrane protecting activity, pH buffering capacity and metal chelating ability [2,15]. Carnosine is also a potent scavenger of reactive oxygen species and aldehydes. It inhibits lipid peroxidation and protein oxidation and prevents advanced glycation product formation [2,15]. Therefore, it has been proposed that carnosine may be an effective agent to prevent oxidative stress-induced pathologies such as ischemia-reperfusion [13], TAA-induced [23] and alcohol-induced liver damage [20], atherosclerosis [31], diabetic complications [19], aging [3,16] and Alzheimer’s disease [14]. In a previous study [23] carnosine acutely co-administered with TAA exhibited a preventive effect on oxidative stress and hepatotoxicity by acting as a non-enzymatic antioxidant. Therefore in the present study we aimed to investigate the effect of carnosine in TAA-induced cirrhosis.
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2. Materials and methods 2.1. Animals Female Wistar rats (body weight 160–200 g) were obtained from Center for Experimental Medical Research Institute of Istanbul University. The animals were allowed free access to food and water and were kept in wire-bottomed stainless steel cages. Food and water consumption were measured periodically. The experimental procedures used in this study met the guidelines of the Animal Care and Use Committee of the University of Istanbul. 2.2. Treatments Animals were divided into four groups. Control group received standard laboratory chow. Carnosine group received 2 g carnosine/L of drinking water for three months. The water supply was refreshed daily. TAA group received 200 mg TAA/kg body weight i.p. twice a week for three months. TAA plus carnosine group received 200 mg TAA/kg body weight i.p. twice a week plus 2 g carnosine/L of drinking water for three months. At the end of three months, rats were fasted overnight and killed by collecting blood into heparinized tubes by cardiac puncture. Carnosine and other chemicals were of highest purity and purchased from Sigma–Aldrich (USA). 2.3. Determinations Livers were quickly removed and washed in ice-cold saline and kept at 70 8C until they were analyzed. Plasma alanine transaminase (ALT) and aspartate transaminase (AST) activities were determined by using Roche autoanalyzer. Liver hydroxyproline content was determined by using liver specimens (100–250 mg) which were homogenized in buffer and hydrolyzed in 2 mL 6 N HCl at 110 8C for 24 h. After that the hydrolysis samples were treated and quantified spectrophotometrically at 565 nm [36]. Tissues were homogenized in ice-cold 0.15 M KCl (10%, w/v). Lipid peroxidation was assessed by two different methods in the tissue homogenates. First, the levels of malondialdehyde (MDA) were measured by thiobarbituric acid test [26]. The breakdown product of 1,1,3,3-tetraethoxypropane was used as standard. Second, diene conjugate (DC) levels were determined in tissue lipid extracts at 233 nm spectrophotometrically and calculated using a molar extinction coefficient of 2.52 104 M1 cm1 [8]. Liver glutathione (GSH) levels were measured with 5,50 dithiobis-(2-nitrobenzoate) at 412 nm [5]. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined in postmitochondrial fraction of the liver, which was separated by sequential centrifugation. In brief, liver homogenates were centrifuged at 600 g for 10 min at 4 8C to remove crude fractions. Then, supernatants were centrifuged at 10,000 g for 20 min to obtain the postmitochondrial fraction. SOD activity was assayed by its ability to increase the effect of riboflavinsensitized photooxidation of o-dianisidine [24]. GSH-Px activity was measured using cumene hydroperoxide as substrate [27]. Protein levels were determined using bicinchoninic acid [33]. Western blotting was performed to study the protein expression of CuZnSOD and GSH-Px proteins. Liver samples (10%, w/v) were homogenized in a solution containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Ipegal 630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin and 10 mg/mL soy bean trypsin inhibitor at 0–4 8C using a homogenizer (Tissue ruptor, Qiagen Sample and Assay Technologies). Homogenates were centrifuged at 13,000 g at 4 8C for 20 min. The supernatant was processed for determination of total protein concentration by using bicinchoninic acid [33].
SDS-polyacrylamide gel electrophoresis (PAGE) was performed using the Bio-Rad Mini Protean III gel system [18]. Equal amounts of protein (50 mg/well) were loaded onto 12% SDS-PAGE for each sample and proteins were transferred to PVDF membranes [38]. After blocking, membranes were incubated for 16 h at 4 8C with rabbit polyclonal antibody against CuZnSOD (sc-11407, Santa Cruz Biotechnology) or GSH-Px protein (sc-30147, Santa Cruz Biotechnology). They were washed and further incubated for 1.5 h at room temperature with secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase, sc-2004, Santa Cruz Biotechnology). The chemiluminescence signals were visualized by exposing the membranes to Kodak Biomax X-ray film for 1–5 min and then the bands were quantitated using the image analysis software. Values of western blot (SOD and GSH-Px) were normalized with an internal standard (actin). Accordingly the membranes were stripped of primary and secondary antibodies by incubation in a solution of 0.2 M glycine, 0.05% Tween 20 at pH 2.5 for 15 min at 80 8C. Membranes were reprobed using monoclonal anti-actin antibody as the primary antibody (sc-1616R, Santa Cruz Biotechnology). 2.4. Histopathological analysis The liver tissue samples for the histopathological examination were washed with 0.9% NaCl solution and fixed in 10% formalin solution. After standard tissue processing procedures, the tissues were embedded in paraffin. The 0.3 mm-thick sections were prepared and stained with hematoxylin–eosin (HE) and Masson Trichrome. They were examined under light microscopy by an expert who was blinded to the treatments. Cirrhosis was scored semi-quantitatively as described in Zhang et al. [39]. According to this; 0 was accepted as normal liver; 1, as thickened perivenular collagen and thin collagen septa; 2, as thin septa with incomplete bridging between portal regions; 3, as thin septa and extensive bridging; and 4, as thickened septa with complete bridging of portal regions and nodular appearance. 2.5. Statistical analysis The results were expressed as mean SD. Experimental groups were compared using Kruskal–Wallis variance analysis test. Where significant effects were found, pos hoc analysis using Mann– Whitney U-test was performed, and p < 0.05 was considered to be significant. 3. Results Daily access to food and water was observed to be same during the period of three months and no difference in body weight gain was observed among all groups at the end of three months (data not shown). Results are shown in Figs. 1–4 and Table 1. According to this: (a) Carnosine treatment alone did not alter plasma ALT and AST activities as well as hepatic oxidative stress parameters and GSH content and SOD and GSH-Px activities in rats. (b) TAA treatment caused significant increases in plasma ALT (71.5%) and AST (97.4%) activities. Liver protein levels were found reduced and hydroxyproline content increased significantly. Hepatic MDA and DC levels were elevated, while GSH content decreased. Lower protein levels and activities of SOD and GSH-Px were also observed. (c) Plasma ALT and AST activities, liver protein and hydroxyproline levels did not change in TAA plus carnosine group. Hepatic MDA and DC levels were found to return to the values of control group. On the other hand, carnosine treatment did not affect
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Fig. 1. Malondialdehyde (MDA) and diene conjugate (DC) levels in livers of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car treated rats (means SD). Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney U-test; p < 0.05.
Fig. 3. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) expressions as relative densitometric units (rdu) in livers of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car treated rats (means SD). Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney U-test; p < 0.05.
TAA-induced changes in liver GSH content, SOD and GSH-Px protein levels and activities. (d) Normal histology was observed in the liver tissues of the carnosine group. Cirrhosis was observed in the liver tissues from the groups which had TAA and TAA plus carnosine. The tissues displayed thin fibrous bands separating the parenchyma into nodules. The portal tracts and the fibrous septa contained inflammatory infiltration and ductular reaction. Scoring of cirrhosis was determined to be 3.41 0.51 in TAA group and 3.42 0.53 in TAA plus carnosine group. The hepatocytes in the nodules displayed regenerative atypia and large cell displasia, as well as small cell dysplasia. Foci of well differentiated hepatocellular carcinoma measuring 0.4 cm in largest diameter were detected in the two rats of the group which had TAA plus carnosine. 4. Discussion
Fig. 2. Glutathione (GSH) levels and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in livers of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car treated rats (means SD). Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney Utest; p < 0.05.
TAA is a commonly used chemical compound to induce experimental liver fibrosis that mimics human liver cirrhosis. In the present study TAA administration to rats for three months has been observed to cause micro- and macronodular hepatic fibrosis as assessed histopathologically. Liver injury was also determined by biochemical parameters (plasma ALT and AST and liver hydroxyproline levels). Plasma AST and ALT activities were found to increase 97.4% and 71.5%, respectively. Liver hydroxyproline levels elevated 294.3%. Free radicals are believed to play a major role in the development of TAA-induced liver cirrhosis [1,4,22,35]. Exacerbation of lipid peroxidation and depletion of antioxidants have been reported by various investigators [4,11,34]. Indeed in the present
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Fig. 4. The representative hepatic histopathology: (control): normal portal tracts (PT), central hepatic venules (CHV) and lobular areas in normal liver tissue (H&E, 40); (carnosine): noncirrhotic liver tissue displaying minimal mononuclear cell infiltration in portal tracts (H&E, 40); (TAA): cirrhotic liver tissue displaying fibrous bands (B) and nodular liver parenchyma (N) among them (H&E, 100); (TAA plus carnosine): fibrous bands (B), parenchymal nodules (N) and a macronodule (MN) larger than the other nodules in the cirrhotic liver tissue (H&E, 100).
study the prooxidant–antioxidant balance has been observed to change on the behalf of oxidation, as also found in the previous study [4]. TAA administration resulted in 43.2% increase in liver MDA levels and 51.7% increase in DC content. On the other hand liver GSH levels, and protein levels and activities of SOD and GSHPx significantly decreased. The reduction of the number and function of mitochondria in hepatocytes of cirrhotic rats have been considered to cause uncoupling in oxidative phosphorylation leading to accumulation of NADH and lactate and diminished energy synthesis rate. This is also suggested to decrease hepatic protein synthesis in cirrhosis, since most of the cell energy is used by the process [29]. In fact, decrease in liver protein levels of SOD and GSH-Px and in total liver protein content observed in the present study appear to confirm this suggestion. Accordingly, use of antioxidants for amelioration has been suggested in experimental cirrhosis models. Different antioxidants have been shown to prevent liver fibrosis and cirrhosis [4,6,7,11,17,30]. Although the literature on carnosine is diverse and many vital functions are attributed to the dipeptide, its use as an antioxidant has been recently searched [3,16,19,20]. In a previous study we have shown that carnosine in vivo prevented acute TAA-induced hepatic oxidative stress by acting as an antioxidant itself without mingling with other elements of the antioxidant system [23]. Depending on that issue, we aimed to investigate the effect of carnosine as an antioxidant in chronic TAA administration for the first time in literature. In fact there are few papers in literature reporting the effect of carnosine on chronic conditions. Carnosine has been given as 0.5, 1 and 2 g/L of drinking water to mice after being treated with ethanol to induce chronic liver injury [20].
Application of 2 g/L carnosine decreased MDA levels and increased GSH content and catalase and GSH-Px activities. On the other hand, when diabetic mice were given 0.5 and 1 g carnosine/L of drinking water, MDA levels reduced dose dependently and catalase activity increased, whereas only 1 g/L of carnosine increased GSH-Px activity [19]. In both studies carnosine is found protective through exhibiting antioxidative and anti-inflammatory effects. In the present study, however, carnosine and TAA have been applied simultaneously. Carnosine dosage (2 g/L of drinking water) was similar to the preferred dose in literature. Considering the daily water consumption of rats, carnosine intake was calculated to be approximately 150 mg/kg body weight/day. Carnosine was observed to significantly lower the increased MDA and DC levels. No effect on decreased GSH content and SOD and GSH-Px activities and protein expressions were determined. In various studies carnosine is reported to react with a variety of deleterious aldehydes to form carnosine–aldehyde adducts and to have a metal chelating effect [2,15]. In this study carnosine appears to attenuate peroxidation as an antioxidant itself in cirrhosis. The paper about the effect of polaprezinc, a zinc–carnosine chelate, on liver fibrosis in dietary methionine and choline deficient mice is the only recent research about carnosine and liver fibrosis. Methionine and choline deficient diet is described to develop steatohepatitis with mild fibrosis, increased lipid peroxidation, activated hepatic stellate cells. Polaprezinc application is shown to attenuate fibrosis turning the balance between collagen synthesis and degradation towards degradation, and to reduce lipid peroxidation, suppress hepatic stellate cell activation and inhibit mRNA expression of pro-inflammatory cytokines. [34].
Table 1 Plasma ALT, AST activities, liver protein and hydroxyproline content of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car-treated rats (means SD). Groups
n
ALT (U/L)
AST (U/L)
Liver protein (mg/g)
Liver hydroxyproline (mg/g wet liver)
Control Car TAA TAA + Car
8 6 12 7
42.4 7.27a 39.2 7.63a 72.7 14.9b 67.6 17.4b
81.7 12.0a 77.0 11.5a 161.3 27.2b 181.3 40.0b
198.4 18.6a 181.4 11.8a 157.7 22.6b 160.0 19.0b
0.35 0.03a 0.38 0.06a 1.03 0.2b 1.01 0.09b
Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney U-test; p < 0.05.
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Although both zinc and carnosine are known for radical scavenging and antioxidant activities, the researchers consider zinc as the main contributor for the action of polaprezinc on development of steatohepatitis. However in this study carnosine is not found to attenuate fibrosis as shown by unchanged liver hydroxyproline levels and plasma ALT and AST activities and histopathological examination. Since fibrosis present in cirrhotic liver is a consequence of massive accumulation of extracellular matrix proteins, especially collagen, hydroxyproline levels may as well implicate the present amount of collagen. As it is suggested they may be used to determine the extent of fibrosis [17]. Thus in the present study carnosine alone does not seem to prevent the development of TAAinduced cirrhotic process. Acknowledgment This work was supported by the Research Fund of University of Istanbul (Project No. 2779). References [1] Abul H, Mathew TC, Dashti HM, Al-Bader A. Level of superoxide dismutase, glutathione peroxidase and uric acid in thioacetamide-induced cirrhotic rats. Anat Histol Embryol 2002;31:66–71. [2] Aldini G, Facino RF, Beretta G, Carini M. Carnosine and related dipeptides as quenchers of reactive carbonyl species: from structural studies to therapeutic perspectives. Biofactors 2005;24:77–87. ¨ zdemirler-Erta G, Koc¸ak-Toker N, Uysal M. The [3] Aydın AF, Ku¨c¸u¨kgergin C, O effect of carnosine treatment on prooxidant-antioxidant balance in liver, heart and brain tissues of male aged rats. Biogerontology; in press. ¨ , C¸evikbas¸ U, Aykac¸-Toker G, Uysal M. [4] Balkan J, Dog˘ru-Abbasog˘lu S, Kanbag˘lı O Taurine has protective effect against thioacetamide-induced liver cirrhosis by decreasing oxidative stress. Hum Exp Toxicol 2001;20:251–4. [5] Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1979;61:882–8. [6] Bruck R, Shirin H, Aeed H, Matas Z, Hochman A, Pines M, et al. Prevention of hepatic cirrhosis in rats by hydroxyl radical scavengers. J Hepatol 2001; 35:457–64. [7] Bruck R, Ashkenazi M, Weiss S, Goldiner I, Shapiro H, Aeed H, et al. Prevention of liver cirrhosis in rats by curcumin. Liver Int 2007;27:373–83. [8] Buege JA, Aust JD. Microsomal lipid peroxidation. Method Enzymol 1978; 52:302–10. [9] Chilakapati J, Shankar K, Korrapati MC, Hill RA, Mehendale HM. Saturation toxicokinetics of thioacetamide: role in initiation of liver injury. Drug Metab Dispos 2005;33:1877–85. [10] Chilakapati J, Korrapati MC, Hill RA, Warbritton A, Latendresse JR, Mehendale HM. Toxicokinetics and toxicology of thioacetamide sulfoxide: a metabolite of thioacetamide. Toxicology 2007;230:105–16. [11] Cruz A, Padillo FJ, Torres E, Navarrete CM, Munoz-Castaneda JR, Caballero FJ, et al. Melatonin prevents experimental liver cirrhosis induced by thioacetamide in rats. J Pineal Res 2005;39:143–50. [12] Friedman SL. Liver fibrosis—from bench to bedside. J Hepatol 2003;38:S38–53. [13] Fouad AA, El-Rehany MAA, Maghraby HK. The hepatoprotective effect of carnosine against ischemia/reperfusion liver injury in rats. Eur J Pharmacol 2007;572:61–8. [14] Hipkiss AR. Could carnosine or related structures suppress Alzheimer’s disease? J Alzheimers Dis 2007;11:229–40. [15] Hipkiss AR, Brownson C. Carnosine reacts with protein carbonyl groups: another possible role for the anti-ageing peptide? Biogerontology 2000;1:217–23. [16] Hipkiss AR, Brownson C, Carrier MJ. Carnosine, the anti-ageing, anti-oxidant dipeptide, may react with protein carbonyl groups. Mech Aging Dev 2001;122:1431–45.
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[17] Hsieh CC, Fang HL, Lina WC. Inhibitory effect of solanum nigrum on thioacetamide-induced liver fibrosis in mice. J Ethnopharm 2008;119:117–21. [18] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [19] Lee Y, Hsu C, Lin M, Liu K, Yin M. Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation. Eur J Pharmacol 2005;513:145–50. [20] Liu W, Liu T, Yin M. Beneficial effects of histidine and carnosine on ethanolinduced chronic liver injury. Food Chem Toxicol 2008;46:1503–9. [21] Low Ty, Leow Ck, Salto-Tellez M, Chung MCM. A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. Proteomics 2004;4:3960–74. [22] Lukivskaya O, Patsenker E, Lis R, Buko VU. Inhibition of inducible nitric oxide synthase activity prevents liver recovery in rat thioacetamide-induced fibrosis reversal. Eur J Clin Invest 2008;38:317–25. ¨ zdemirler G, Koc¸ak-Toker N, C¸evikbas¸ U, Uysal M. Role of [23] Mehmetc¸ik G, O carnosine in preventing thioacetamide-induced liver injury in the rat. Peptides 2008;29:425–9. [24] Mylroie AA, Collins H, Umbles C, Kyle J. Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol Appl Pharmacol 1986;82:512–20. [25] Natarajan SK, Thomas S, Ramamoorthy P, Basivireddy J, Pulimood AB, Ramachandran A, et al. Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models. J Gastroenterol Hepatol 2006;21:947–57. [26] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. [27] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70: 158–69. [28] Palacios RS, Roderfeld M, Hemmann S, Rath T, Atanasova S, Tschuschner A, et al. Activation of hepatic stellate cells is associated with cytokine expression in thioacetamide-induced hepatic fibrosis in mice. Lab Invest 2008;88:1192– 203. [29] Perez MJ, Sanchez-Medina F, Torres M, Gil A, Suarez A. Dietary nucleotides enhance the liver redox state and protein synthesis in cirrhotic rats. J Nutr 2004;134:2504–8. [30] Rai M, Pai MRSM, Sabitha P, Pai P. Hepatoprotective influence of selenium in experimental liver cirrhosis. Kuwait Med J 2001;33:333–6. [31] Rashid I, Van Reyk DM, Davies MJ. Carnosine and its constituents inhibit glycation of low-density lipoproteins that promotes foam cell formation in vitro. FEBS Lett 2007;581:1067–70. [32] Sanz N, Diez-Fernandez C, Andres D, Cascales M. Hepatotoxicity and aging: endogenous antioxidant systems in hepatocytes from 2-, 6-, 12-, 18- and 30month-old rats following a necrogenic dose of thioacetamide. Biochim Biophys Acta 2002;1587:12–20. [33] Smith PK, Krohn RI, Hermanson GT, Mallia AK, Garner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76–85. [34] Sugino H, Kumagai N, Watanabe S, Toda K, Takeuchi O, Tsunematsu S, et al. Polaprezinc attenuates liver fibrosis in a mouse model of non-alcoholic steatohepatitis. J Gastroenterol Hepatol 2008;23:1909–16. [35] Sun F, Hayami S, Ogiri Y, Haruna S, Tanaka K, Yamada Y, et al. Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver. Biochim Biophys Acta 2000;1500:181–5. [36] Switzer BR. Determination of hydroxyproline in tissue. J Nutr Biochem 1991;2:229–31. [37] Torres EM, Bouza JIP, Bravo AL, Hernandez MMA, Marino EC. Experimental thioacetamide-induced cirrhosis of the liver. Histol Histopathol 1991;6: 95–100. [38] Towbin H, Stashelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 1979;76:4350–4. [39] Zhang J, Wang H, Yu H. Thioacetamide-induced cirrhosis in selenium-adequate mice displays rapid and persistent abnormity of hepatic selenoenzymes which are mute to selenium supplementation. Toxicol Appl Pharmacol 2007;224:81–8.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Effect of carnosine against thioacetamide-induced liver cirrhosis in rat A. Fatih Aydın a, Zeynep Ku¨sku¨-Kiraz a, Semra Dog˘ru-Abbasog˘lu a, Mine Gu¨llu¨og˘lu b, Mu¨jdat Uysal a, Necla Koc¸ak-Toker a,* a b
Department of Biochemistry, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey Department of Pathology, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2009 Received in revised form 25 November 2009 Accepted 25 November 2009 Available online 1 December 2009
Carnosine (b-alanyl-L-histidine) is a dipeptide with antioxidant properties. Oxidative stress has been proposed to be involved in thioacetamide (TAA)-induced liver cirrhosis in rats, that is similar to human disease. In this study we aimed to investigate the role of carnosine on the development of TAA-induced cirrhosis. 200 mg TAA/kg body weight has been given i.p. twice a week for three months to female wistar rats. Another group received same dose of TAA in the same pattern plus 2 g carnosine/L of drinking water for three months. TAA administration resulted in hepatic fibrosis, significant increases in plasma transaminase activities as well as hepatic hydroxyproline and lipid peroxide levels, while liver glutathione (GSH) and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) protein expressions and activities decreased. Carnosine was found to behave as an antioxidant reducing malondialdehyde (MDA) and diene conjugate (DC) levels although it was not effective on increased transaminase activities and decreased antioxidants. It also did not affect the histopathological changes observed in TAA group. Thus our findings indicate that carnosine appears to attenuate peroxidation as an antioxidant itself but does not seem to prevent the development of TAA-induced cirrhotic process. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Thioacetamide Carnosine Cirrhosis Oxidative stress SOD GSH-Px
1. Introduction Liver cirrhosis associated with various pathological processes, is characterized by progressive fibrosis producing liver injury, portal hypertension and carcinoma [12]. Fibrosis resulting from activation of stellate cells is considered to be provoked by oxidative stress and cytokines [11,28]. Indeed induction of free radical generation, mitochondrial dysfunction and depletion of antioxidants are effective in the progression of fibrosis and cirrhosis [25]. In several experimental models for the induction of cirrhosis, hepatotoxic agents such as thioacetamide (TAA), carbon tetrachloride and ethionine have been used [22,25,37]. TAA is a widely used agent to develop liver cirrhosis in rats that is similar to human cirrhosis. TAA is metabolically activated to thioacetamide sulfoxide and further to thioacetamide-S,S-dioxide. In fact toxic effects of TAA are attributed to those reactive metabolites [9,10]. TAAinduced liver cirrhosis is observed to be associated with lipid peroxidation and depletion of antioxidants [1,21,32,35]. Accordingly reduction of oxidative stress appears to be helpful for the regression of fibrosis and cirrhosis. Thus use of radical scavengers and antioxidants to prevent fibrosis have been suggested benefi-
* Corresponding author. Tel.: +90 2124142188; fax: +90 2126215642. E-mail address: [email protected] (s.$. Koc¸ak-Toker). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.11.028
cial. For this purpose hydroxyl radical scavengers like dimethylsulfoxide and dimethylthiourea [6] extract of solanum nigrum [17], melatonin [11], selenium [30], curcumin [7] and taurine [4] have been applied in TAA-induced liver cirrhosis and reduction of radical mediated hepatic damage has been reported to inhibit the development of cirrhosis. In recent years carnosine has provoked interest as an antioxidant although its discovery has been documented almost a century before [15]. Carnosine (b-alanyl-L-histidine) is a dipeptide which is found at relatively high concentrations in mammalian tissues [2]. It has several functions such as membrane protecting activity, pH buffering capacity and metal chelating ability [2,15]. Carnosine is also a potent scavenger of reactive oxygen species and aldehydes. It inhibits lipid peroxidation and protein oxidation and prevents advanced glycation product formation [2,15]. Therefore, it has been proposed that carnosine may be an effective agent to prevent oxidative stress-induced pathologies such as ischemia-reperfusion [13], TAA-induced [23] and alcohol-induced liver damage [20], atherosclerosis [31], diabetic complications [19], aging [3,16] and Alzheimer’s disease [14]. In a previous study [23] carnosine acutely co-administered with TAA exhibited a preventive effect on oxidative stress and hepatotoxicity by acting as a non-enzymatic antioxidant. Therefore in the present study we aimed to investigate the effect of carnosine in TAA-induced cirrhosis.
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2. Materials and methods 2.1. Animals Female Wistar rats (body weight 160–200 g) were obtained from Center for Experimental Medical Research Institute of Istanbul University. The animals were allowed free access to food and water and were kept in wire-bottomed stainless steel cages. Food and water consumption were measured periodically. The experimental procedures used in this study met the guidelines of the Animal Care and Use Committee of the University of Istanbul. 2.2. Treatments Animals were divided into four groups. Control group received standard laboratory chow. Carnosine group received 2 g carnosine/L of drinking water for three months. The water supply was refreshed daily. TAA group received 200 mg TAA/kg body weight i.p. twice a week for three months. TAA plus carnosine group received 200 mg TAA/kg body weight i.p. twice a week plus 2 g carnosine/L of drinking water for three months. At the end of three months, rats were fasted overnight and killed by collecting blood into heparinized tubes by cardiac puncture. Carnosine and other chemicals were of highest purity and purchased from Sigma–Aldrich (USA). 2.3. Determinations Livers were quickly removed and washed in ice-cold saline and kept at 70 8C until they were analyzed. Plasma alanine transaminase (ALT) and aspartate transaminase (AST) activities were determined by using Roche autoanalyzer. Liver hydroxyproline content was determined by using liver specimens (100–250 mg) which were homogenized in buffer and hydrolyzed in 2 mL 6 N HCl at 110 8C for 24 h. After that the hydrolysis samples were treated and quantified spectrophotometrically at 565 nm [36]. Tissues were homogenized in ice-cold 0.15 M KCl (10%, w/v). Lipid peroxidation was assessed by two different methods in the tissue homogenates. First, the levels of malondialdehyde (MDA) were measured by thiobarbituric acid test [26]. The breakdown product of 1,1,3,3-tetraethoxypropane was used as standard. Second, diene conjugate (DC) levels were determined in tissue lipid extracts at 233 nm spectrophotometrically and calculated using a molar extinction coefficient of 2.52 104 M1 cm1 [8]. Liver glutathione (GSH) levels were measured with 5,50 dithiobis-(2-nitrobenzoate) at 412 nm [5]. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined in postmitochondrial fraction of the liver, which was separated by sequential centrifugation. In brief, liver homogenates were centrifuged at 600 g for 10 min at 4 8C to remove crude fractions. Then, supernatants were centrifuged at 10,000 g for 20 min to obtain the postmitochondrial fraction. SOD activity was assayed by its ability to increase the effect of riboflavinsensitized photooxidation of o-dianisidine [24]. GSH-Px activity was measured using cumene hydroperoxide as substrate [27]. Protein levels were determined using bicinchoninic acid [33]. Western blotting was performed to study the protein expression of CuZnSOD and GSH-Px proteins. Liver samples (10%, w/v) were homogenized in a solution containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Ipegal 630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin and 10 mg/mL soy bean trypsin inhibitor at 0–4 8C using a homogenizer (Tissue ruptor, Qiagen Sample and Assay Technologies). Homogenates were centrifuged at 13,000 g at 4 8C for 20 min. The supernatant was processed for determination of total protein concentration by using bicinchoninic acid [33].
SDS-polyacrylamide gel electrophoresis (PAGE) was performed using the Bio-Rad Mini Protean III gel system [18]. Equal amounts of protein (50 mg/well) were loaded onto 12% SDS-PAGE for each sample and proteins were transferred to PVDF membranes [38]. After blocking, membranes were incubated for 16 h at 4 8C with rabbit polyclonal antibody against CuZnSOD (sc-11407, Santa Cruz Biotechnology) or GSH-Px protein (sc-30147, Santa Cruz Biotechnology). They were washed and further incubated for 1.5 h at room temperature with secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase, sc-2004, Santa Cruz Biotechnology). The chemiluminescence signals were visualized by exposing the membranes to Kodak Biomax X-ray film for 1–5 min and then the bands were quantitated using the image analysis software. Values of western blot (SOD and GSH-Px) were normalized with an internal standard (actin). Accordingly the membranes were stripped of primary and secondary antibodies by incubation in a solution of 0.2 M glycine, 0.05% Tween 20 at pH 2.5 for 15 min at 80 8C. Membranes were reprobed using monoclonal anti-actin antibody as the primary antibody (sc-1616R, Santa Cruz Biotechnology). 2.4. Histopathological analysis The liver tissue samples for the histopathological examination were washed with 0.9% NaCl solution and fixed in 10% formalin solution. After standard tissue processing procedures, the tissues were embedded in paraffin. The 0.3 mm-thick sections were prepared and stained with hematoxylin–eosin (HE) and Masson Trichrome. They were examined under light microscopy by an expert who was blinded to the treatments. Cirrhosis was scored semi-quantitatively as described in Zhang et al. [39]. According to this; 0 was accepted as normal liver; 1, as thickened perivenular collagen and thin collagen septa; 2, as thin septa with incomplete bridging between portal regions; 3, as thin septa and extensive bridging; and 4, as thickened septa with complete bridging of portal regions and nodular appearance. 2.5. Statistical analysis The results were expressed as mean SD. Experimental groups were compared using Kruskal–Wallis variance analysis test. Where significant effects were found, pos hoc analysis using Mann– Whitney U-test was performed, and p < 0.05 was considered to be significant. 3. Results Daily access to food and water was observed to be same during the period of three months and no difference in body weight gain was observed among all groups at the end of three months (data not shown). Results are shown in Figs. 1–4 and Table 1. According to this: (a) Carnosine treatment alone did not alter plasma ALT and AST activities as well as hepatic oxidative stress parameters and GSH content and SOD and GSH-Px activities in rats. (b) TAA treatment caused significant increases in plasma ALT (71.5%) and AST (97.4%) activities. Liver protein levels were found reduced and hydroxyproline content increased significantly. Hepatic MDA and DC levels were elevated, while GSH content decreased. Lower protein levels and activities of SOD and GSH-Px were also observed. (c) Plasma ALT and AST activities, liver protein and hydroxyproline levels did not change in TAA plus carnosine group. Hepatic MDA and DC levels were found to return to the values of control group. On the other hand, carnosine treatment did not affect
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Fig. 1. Malondialdehyde (MDA) and diene conjugate (DC) levels in livers of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car treated rats (means SD). Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney U-test; p < 0.05.
Fig. 3. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) expressions as relative densitometric units (rdu) in livers of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car treated rats (means SD). Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney U-test; p < 0.05.
TAA-induced changes in liver GSH content, SOD and GSH-Px protein levels and activities. (d) Normal histology was observed in the liver tissues of the carnosine group. Cirrhosis was observed in the liver tissues from the groups which had TAA and TAA plus carnosine. The tissues displayed thin fibrous bands separating the parenchyma into nodules. The portal tracts and the fibrous septa contained inflammatory infiltration and ductular reaction. Scoring of cirrhosis was determined to be 3.41 0.51 in TAA group and 3.42 0.53 in TAA plus carnosine group. The hepatocytes in the nodules displayed regenerative atypia and large cell displasia, as well as small cell dysplasia. Foci of well differentiated hepatocellular carcinoma measuring 0.4 cm in largest diameter were detected in the two rats of the group which had TAA plus carnosine. 4. Discussion
Fig. 2. Glutathione (GSH) levels and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in livers of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car treated rats (means SD). Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney Utest; p < 0.05.
TAA is a commonly used chemical compound to induce experimental liver fibrosis that mimics human liver cirrhosis. In the present study TAA administration to rats for three months has been observed to cause micro- and macronodular hepatic fibrosis as assessed histopathologically. Liver injury was also determined by biochemical parameters (plasma ALT and AST and liver hydroxyproline levels). Plasma AST and ALT activities were found to increase 97.4% and 71.5%, respectively. Liver hydroxyproline levels elevated 294.3%. Free radicals are believed to play a major role in the development of TAA-induced liver cirrhosis [1,4,22,35]. Exacerbation of lipid peroxidation and depletion of antioxidants have been reported by various investigators [4,11,34]. Indeed in the present
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Fig. 4. The representative hepatic histopathology: (control): normal portal tracts (PT), central hepatic venules (CHV) and lobular areas in normal liver tissue (H&E, 40); (carnosine): noncirrhotic liver tissue displaying minimal mononuclear cell infiltration in portal tracts (H&E, 40); (TAA): cirrhotic liver tissue displaying fibrous bands (B) and nodular liver parenchyma (N) among them (H&E, 100); (TAA plus carnosine): fibrous bands (B), parenchymal nodules (N) and a macronodule (MN) larger than the other nodules in the cirrhotic liver tissue (H&E, 100).
study the prooxidant–antioxidant balance has been observed to change on the behalf of oxidation, as also found in the previous study [4]. TAA administration resulted in 43.2% increase in liver MDA levels and 51.7% increase in DC content. On the other hand liver GSH levels, and protein levels and activities of SOD and GSHPx significantly decreased. The reduction of the number and function of mitochondria in hepatocytes of cirrhotic rats have been considered to cause uncoupling in oxidative phosphorylation leading to accumulation of NADH and lactate and diminished energy synthesis rate. This is also suggested to decrease hepatic protein synthesis in cirrhosis, since most of the cell energy is used by the process [29]. In fact, decrease in liver protein levels of SOD and GSH-Px and in total liver protein content observed in the present study appear to confirm this suggestion. Accordingly, use of antioxidants for amelioration has been suggested in experimental cirrhosis models. Different antioxidants have been shown to prevent liver fibrosis and cirrhosis [4,6,7,11,17,30]. Although the literature on carnosine is diverse and many vital functions are attributed to the dipeptide, its use as an antioxidant has been recently searched [3,16,19,20]. In a previous study we have shown that carnosine in vivo prevented acute TAA-induced hepatic oxidative stress by acting as an antioxidant itself without mingling with other elements of the antioxidant system [23]. Depending on that issue, we aimed to investigate the effect of carnosine as an antioxidant in chronic TAA administration for the first time in literature. In fact there are few papers in literature reporting the effect of carnosine on chronic conditions. Carnosine has been given as 0.5, 1 and 2 g/L of drinking water to mice after being treated with ethanol to induce chronic liver injury [20].
Application of 2 g/L carnosine decreased MDA levels and increased GSH content and catalase and GSH-Px activities. On the other hand, when diabetic mice were given 0.5 and 1 g carnosine/L of drinking water, MDA levels reduced dose dependently and catalase activity increased, whereas only 1 g/L of carnosine increased GSH-Px activity [19]. In both studies carnosine is found protective through exhibiting antioxidative and anti-inflammatory effects. In the present study, however, carnosine and TAA have been applied simultaneously. Carnosine dosage (2 g/L of drinking water) was similar to the preferred dose in literature. Considering the daily water consumption of rats, carnosine intake was calculated to be approximately 150 mg/kg body weight/day. Carnosine was observed to significantly lower the increased MDA and DC levels. No effect on decreased GSH content and SOD and GSH-Px activities and protein expressions were determined. In various studies carnosine is reported to react with a variety of deleterious aldehydes to form carnosine–aldehyde adducts and to have a metal chelating effect [2,15]. In this study carnosine appears to attenuate peroxidation as an antioxidant itself in cirrhosis. The paper about the effect of polaprezinc, a zinc–carnosine chelate, on liver fibrosis in dietary methionine and choline deficient mice is the only recent research about carnosine and liver fibrosis. Methionine and choline deficient diet is described to develop steatohepatitis with mild fibrosis, increased lipid peroxidation, activated hepatic stellate cells. Polaprezinc application is shown to attenuate fibrosis turning the balance between collagen synthesis and degradation towards degradation, and to reduce lipid peroxidation, suppress hepatic stellate cell activation and inhibit mRNA expression of pro-inflammatory cytokines. [34].
Table 1 Plasma ALT, AST activities, liver protein and hydroxyproline content of control, carnosine (Car), thioacetamide (TAA) and TAA plus Car-treated rats (means SD). Groups
n
ALT (U/L)
AST (U/L)
Liver protein (mg/g)
Liver hydroxyproline (mg/g wet liver)
Control Car TAA TAA + Car
8 6 12 7
42.4 7.27a 39.2 7.63a 72.7 14.9b 67.6 17.4b
81.7 12.0a 77.0 11.5a 161.3 27.2b 181.3 40.0b
198.4 18.6a 181.4 11.8a 157.7 22.6b 160.0 19.0b
0.35 0.03a 0.38 0.06a 1.03 0.2b 1.01 0.09b
Values not sharing a common letter are significantly different by Kruskal–Wallis test followed by Mann–Whitney U-test; p < 0.05.
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Although both zinc and carnosine are known for radical scavenging and antioxidant activities, the researchers consider zinc as the main contributor for the action of polaprezinc on development of steatohepatitis. However in this study carnosine is not found to attenuate fibrosis as shown by unchanged liver hydroxyproline levels and plasma ALT and AST activities and histopathological examination. Since fibrosis present in cirrhotic liver is a consequence of massive accumulation of extracellular matrix proteins, especially collagen, hydroxyproline levels may as well implicate the present amount of collagen. As it is suggested they may be used to determine the extent of fibrosis [17]. Thus in the present study carnosine alone does not seem to prevent the development of TAAinduced cirrhotic process. Acknowledgment This work was supported by the Research Fund of University of Istanbul (Project No. 2779). References [1] Abul H, Mathew TC, Dashti HM, Al-Bader A. Level of superoxide dismutase, glutathione peroxidase and uric acid in thioacetamide-induced cirrhotic rats. Anat Histol Embryol 2002;31:66–71. [2] Aldini G, Facino RF, Beretta G, Carini M. Carnosine and related dipeptides as quenchers of reactive carbonyl species: from structural studies to therapeutic perspectives. Biofactors 2005;24:77–87. ¨ zdemirler-Erta G, Koc¸ak-Toker N, Uysal M. The [3] Aydın AF, Ku¨c¸u¨kgergin C, O effect of carnosine treatment on prooxidant-antioxidant balance in liver, heart and brain tissues of male aged rats. Biogerontology; in press. ¨ , C¸evikbas¸ U, Aykac¸-Toker G, Uysal M. [4] Balkan J, Dog˘ru-Abbasog˘lu S, Kanbag˘lı O Taurine has protective effect against thioacetamide-induced liver cirrhosis by decreasing oxidative stress. Hum Exp Toxicol 2001;20:251–4. [5] Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1979;61:882–8. [6] Bruck R, Shirin H, Aeed H, Matas Z, Hochman A, Pines M, et al. Prevention of hepatic cirrhosis in rats by hydroxyl radical scavengers. J Hepatol 2001; 35:457–64. [7] Bruck R, Ashkenazi M, Weiss S, Goldiner I, Shapiro H, Aeed H, et al. Prevention of liver cirrhosis in rats by curcumin. Liver Int 2007;27:373–83. [8] Buege JA, Aust JD. Microsomal lipid peroxidation. Method Enzymol 1978; 52:302–10. [9] Chilakapati J, Shankar K, Korrapati MC, Hill RA, Mehendale HM. Saturation toxicokinetics of thioacetamide: role in initiation of liver injury. Drug Metab Dispos 2005;33:1877–85. [10] Chilakapati J, Korrapati MC, Hill RA, Warbritton A, Latendresse JR, Mehendale HM. Toxicokinetics and toxicology of thioacetamide sulfoxide: a metabolite of thioacetamide. Toxicology 2007;230:105–16. [11] Cruz A, Padillo FJ, Torres E, Navarrete CM, Munoz-Castaneda JR, Caballero FJ, et al. Melatonin prevents experimental liver cirrhosis induced by thioacetamide in rats. J Pineal Res 2005;39:143–50. [12] Friedman SL. Liver fibrosis—from bench to bedside. J Hepatol 2003;38:S38–53. [13] Fouad AA, El-Rehany MAA, Maghraby HK. The hepatoprotective effect of carnosine against ischemia/reperfusion liver injury in rats. Eur J Pharmacol 2007;572:61–8. [14] Hipkiss AR. Could carnosine or related structures suppress Alzheimer’s disease? J Alzheimers Dis 2007;11:229–40. [15] Hipkiss AR, Brownson C. Carnosine reacts with protein carbonyl groups: another possible role for the anti-ageing peptide? Biogerontology 2000;1:217–23. [16] Hipkiss AR, Brownson C, Carrier MJ. Carnosine, the anti-ageing, anti-oxidant dipeptide, may react with protein carbonyl groups. Mech Aging Dev 2001;122:1431–45.
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