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Dose- and time-dependent effects of luteolin on carbon tetrachloride-induced hepatotoxicity in mice
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Experimental and Toxicologic Pathology 61 (2009) 581–589 www.elsevier.de/etp
Dose- and time-dependent effects of luteolin on carbon tetrachloride-induced hepatotoxicity in mice Robert Domitrovic´a,, Hrvoje Jakovacb, Cˇedomila Milina, Biserka Radosˇevic´-Stasˇic´b a
Department of Chemistry and Biochemistry, Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia Department of Physiology and Immunology, Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia
b
Received 28 August 2008; accepted 17 December 2008
Abstract Carbon tetrachloride (CCl4) is a well-known model compound for producing chemical hepatic injury. This study investigated the protective effects of the flavonoid luteolin on the CCl4-induced hepatotoxicity in mice. Luteolin dissolved in dimethyl sulfoxide (DMSO) was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose, and once daily for 2 consecutive days. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). CCl4-induced hepatotoxicity was reduced in a dose- and time-dependent manner, as determined by decreased serum aminotransferase activities and liver histopathology. CCl4 intoxication resulted in an overexpression of heat shock protein gp96 in the mice liver, which was strongly attenuated by luteolin pretreatment. Luteolin has also decreased oxidative stress produced by CCl4, as suggested by improvement in the Cu/Zn superoxide dismutase activity. The effect of luteolin on myeloperoxidase, an indicator of inflammatory cell infiltration, was also investigated. Treatment of the mice with luteolin resulted in a significant decrease in the myeloperoxidase activity. The hepatoprotective effect of luteolin against CCl4 hepatotoxicity was higher in animals pretreated with luteolin for 2 consecutive days. This suggests that the protection might be due to induction of some adaptive mechanisms. The data indicate that luteolin could be effective in protecting mice from the hepatotoxicity produced by CCl4. r 2008 Elsevier GmbH. All rights reserved. Keywords: Carbon tetrachloride hepatotoxicity; Luteolin; Cu/Zn superoxide dismutase; Myeloperoxidase; Histopathology; Timeand dose-dependent; Heat shock protein; gp96
Introduction The liver plays a central role in transforming and clearing metabolites and xenobiotics, and is susceptible to the toxicity from these agents. Carbon tetrachloride (CCl4) is a well-known model compound for inducing liver injury. Its biotransformation by the hepatic microsomal cytochrome P450 produces hepatotoxic Corresponding author. Tel./fax: +385 51 651 135.
E-mail address: [email protected] (R. Domitrovic´). 0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.12.005
metabolites, namely trichloromethyl free radicals. The covalent binding of the trichloromethyl free radicals to the cell proteins is considered to be the initial step in a chain of events that eventually lead to cell necrosis (Recknagel et al., 1989; Williams and Burk, 1990; Brautbar and Williams, 2002). During liver damage, inflammatory cell infiltration may occur, which can be quantified by measuring the activity of myeloperoxidase (MPO), an enzyme found within the azurophilic granules of polymorphonuclears such as neutrophils and monocyte/macrophage mononuclears, including Kupffer cells (Reynolds et al., 2002).
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Flavonoids, a large class of polyphenolic compounds present in fruits and fruit products, vegetables, and plant-derived beverages such as tea and wine, are associated with a variety of beneficial properties (Middleton et al., 2000; Havsteen, 2002). Due to their antioxidant activity (Rice-Evans et al., 1996), they are presumed to protect tissues against oxidative stress and associated pathologies such as cancer, coronary artery disease and inflammation. Polyphenols act as antioxidants by scavenging reactive oxygen and nitrogen species, chelating redox-active transition metal ions preventing them from catalyzing free radical formation, but also several other ways (Frei and Higdon, 2003). Luteolin (30 ,40 ,5,7-tetrahydroxyflavone) is an important member of the flavonoid family, present in glycosylated forms and as aglycone in various plants. Luteolin is reported to have antiinflammatory (Ziyan et al., 2007; Ueda et al., 2002), antioxidant (Perez-Garcia et al., 2000), antiallergic (Veda et al., 2002), antitumorigenic (Ju et al., 2007), anxiolytic-like (Coleta et al., 2007), and vasorelaxative properties (Woodman and Chan, 2004). Heat shock proteins (HSPs) are induced in response to various stresses and to protect cells from such stresses (Lee et al., 2004). The 90 kDa HSP family, one of the most abundant proteins in eukaryotic cells, plays an important role in the folding of newly synthesized proteins and stabilization and refolding of denatured proteins in stress conditions (Sreedhar et al., 2004). The progressive cellular damage caused by reactive oxygen species contributes to protein misfolding and accumulation of HSPs. HSP gp96 (Grp94, glucose regulated protein 94) is a constitutively expressed endoplasmic reticulum lumenal protein that is upregulated in response to cellular stress such as heat shock, oxidative stress or glucose depletion (Ruddon and Bedows, 1997). HSP gp96 has not been previously studied in toxic liver damage. The main purpose of this study was to investigate the potential effects of luteolin in reducing oxidative stress and inflammation in the liver of mice caused by CCl4, as well as enhancement of hepatic proliferative capability, which could provide helpful information for the prevention of liver damage. The present investigation focuses on evaluation of the efficacy of different dose regimes of luteolin for their protection against CCl4-induced hepatotoxicity. The parameters analysed were serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, histopathology, HSP gp96 protein expression, hepatic Cu/Zn superoxide dismutase (Cu/Zn SOD) and myeloperoxidase activities.
alanine aminotransferase and aspartate aminotransferase, dimethyl sulfoxide (DMSO), hexadecyltrimethylammonium bromide (HTMABr), o-dianisidine dihydrochloride, hydrogen peroxide (H2O2) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Superoxide Dismutase Assay Kit was obtained from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals and solvents were of the highest grade commercially available.
Animals Male Balb/c mice from our breeding colony, 2–3 month old, were divided into 8 groups with 6 animals per group. Mice were fed a standard rodent diet (pellet, type 4RF21 GLP, Mucedola, Italy), and water ad libitum. The animals were maintained at 12 h light/dark cycle, at constant temperature (2071 1C) and humidity (5075%). All experimental procedures were approved by the Ethical Committee of the Medical Faculty, University of Rijeka.
Experimental design The control group animals were given DMSO as a single dose (group I) or once daily for 2 consecutive days (group II). Luteolin dissolved in DMSO was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose (groups III and IV), and once daily for 2 consecutive days (groups V and VI). Groups VII and VIII received vehicle (DMSO) as a single dose and once daily for 2 consecutive days, respectively. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p., dissolved in olive oil), except the control groups. Twenty-four hours after administrating CCl4, the mice were killed by cervical dislocation, blood was collected from the orbital sinus and the serum was separated to determine ALT and AST activities. The abdomen was cut open quickly and the liver was perfused thoroughly with isotonic saline, excised, blotted dry and divided into multiple samples. Liver samples were used to assess the Cu/Zn SOD and MPO activities, and protein content. A portion of the livers were preserved in a buffered formalin solution to obtain the histological sections.
Hepatotoxicity studies
Materials and methods Olive oil was provided by Zvijezda, Croatia. Luteolin, carbon tetrachloride, diagnostic kits for the serum
Serum levels of ALT and AST, as markers of hepatic function, were measured by using a Bio-Tek EL808 Ultra Microplate Reader (BioTek Instruments, Winooski, VT, USA) according to manufacturer’s instructions.
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Superoxide dismutase activity assay Livers were homogenized in 50 mM phosphate buffer saline (PBS), pH 7.4, using a Polytron homogenizer (Kinematica, Lucerne, Switzerland) for 1 min at 4 1C. The homogenate was subjected to centrifugation using Beckman L7-65 Ultracentrifuge (Beckman, Fullerton, USA) at 12,000g for 15 min, at 4 1C. The supernatants and homogenates were used for superoxide dismutase activity assay. Cu/Zn SOD activity in homogenate supernatants was assessed by the degree of suppression of xanthine reduction in the presence of NADH and xanthine oxidase (Superoxide Dismutase Assay Kit, Cayman Chemical). The increase of absorbance was monitored spectrophotometrically at 450 nm (Bio-Tek EL808 Ultra Microplate Reader). The assay kit utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of Cu/Zn SOD was defined as the amount of enzyme required to inhibit the reduction of xanthine by 50%. Protein content in liver homogenates was estimated by Bradford’s method (Bradford, 1976).
Myeloperoxidase activity assay Liver homogenates were prepared in 50 mM PBS, pH 6.0 (containing 5 mM DTT). The homogenate was centrifuged at 12,000g for 20 min, at 4 1C. For the biochemical determination of MPO activity, the supernatant was discarded and the pellet was washed again. After decanting the supernatant, the pellet was extracted by suspending the material in 0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer (pH 6.0, 4 1C) for 2 min. The samples were immediately frozen and subjected to three freeze–thaw cycles, with sonication between cycles. Supernatant MPO activity was assayed as described previously (Bradley et al., 1982). Briefly, 0.1 ml of supernatant was mixed with 2.9 ml of 50 mM PBS, pH 6.0, containing 0.167 mg of o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The rate at which a colored product formed during the MPO-dependent reaction of o-dianisidine dihydrochloride was measured. The change in absorbance at 460 nm was recorded at 15-s intervals over 2 min using a spectrophotometer (Cary 100, Varian, Mulgrave, Australia). One unit of MPO activity is defined as that which degrades 1 mmol of peroxide per minute at 25 1C (Barone et al., 1991). Enzyme activity was expressed as mmol/mg protein using the molar absorbency index (1.13 104) of oxidized o-dianisidine.
Histological examinations Liver tissues were placed in plastic cassettes and immersed in neutral buffered formalin for 24 h. The
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fixed tissues were processed routinely, embedded in paraffin, sectioned, deparaffinized, and rehydrated using the standard techniques. The extent of CCl4-induced necrosis was evaluated by assessing the morphological changes in the liver sections stained with hematoxylin and eosin (H&E), using standard techniques.
Immunohistochemistry Tissue expression of gp96 protein in the liver was analysed by immunohistochemistry. Immunohistochemical studies were performed on paraffin embedded liver tissue using DAKO EnVision+System, Peroxidase (DAB) kit according to the manufacturer’s instructions (DAKO Corporation, USA). Briefly, slides were incubated with peroxidase block to eliminate endogenous peroxidase activity. After washing, monoclonal anti-Grp94 antibody (Monoclonal rat Anti-Grp94 antibody, Clone 9G10, Stressgen, Canada), diluted 1:30 in phosphate-buffered saline supplemented with bovine serum albumin was added to tissue samples and incubated overnight at 4 1C in a humid environment, followed by 45 min incubation with peroxidase labeled polymer conjugated to goat anti-mouse immunoglobulins containing carrier protein linked to Fc fragments to prevent nonspecific binding. The immunoreaction product was visualized by adding substrate-chromogen (DAB) solution. Tissues were counterstained with hematoxylin and 37 mM ammonia water, dehydrated in a gradient of alcohol and mounted with mounting medium. The specificity of the reaction was confirmed by substitution of anti-Grp94 antibody with mouse irrelevant IgG2a immunoglobulin (clone DAK-G05; Dako, USA), used in the same conditions and dilutions as a primary antibody.
Statistical analysis Data were analysed using StatSoft STATISTICA version 7.1 software. Differences between the groups were assessed by a nonparametric Man–Whitney and Kruskal–Wallis tests. Values in the text are mean7 standard deviation (SD). Differences with po0.05 were considered to be statistically significant.
Results We did not observe a significant difference between control groups I and II or between CCl4-treated groups VII and VIII. Therefore, the data from groups I and II or from groups VII and VIII were combined and then evaluated as a unique control and CCl4 groups, respectively.
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Hepatotoxicity studies Serum ALT and AST levels were highly elevated at 24 h after CCl4 treatment. Pretreatment with luteolin decreased ALT levels in CCl4-treated mice when administered once daily for 2 consecutive days, 50 mg/kg. AST levels were reduced in all groups receiving luteolin except for the lowest dose, with the highest reduction in mice pretreated with 50 mg/kg of luteolin for 2 consecutive days (Table 1).
Superoxide dismutase activity assay CCl4 administration significantly decreased the Cu/Zn SOD activity, however, pretreatment with luteolin significantly ameliorated the lost of Cu/Zn SOD activity, in a dose-dependent manner (Fig. 1).
Myeloperoxidase activity assay MPO activity, an indicator of neutrophil and monocyte/macrophages infiltration, was significantly higher in the liver tissue of CCl4-treated mice than in that of the control group, which indicates that a severe inflammatory response had taken place in the development of liver damage. In contrast, the data showed that pretreatment with luteolin resulted in a remarkable inhibition of MPO activities in CCl4-treated mice (Fig. 2).
Histological examinations Liver tissue from control mice (Fig. 3A) showed normal liver histology and hepatocyte structure. In the group treated with CCl4, we have found large areas of extensive, mainly centrilobular necrosis with loss of hepatocyte structure, vacuolar fatty change and mild inflammatory cell infiltration, comprised predominantly of mononuclear cells with occasional polymorphonuclears (Fig. 3B). In the liver of mice pretreated with 5 mg/kg of Table 1. The effect of luteolin pretreatment on the serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in experimental groups.
Control (vehicle) Luteolin 5 mg/kg Luteolin 50 mg/kg Luteolin 5 mg/kg, 2 days Luteolin 50 mg/kg, 2 days CCl4
AST (U/L)
ALT (U/L)
41.377.8a 28497193b 21627119c 2110736c 1225769d 29957248b
44.775.5a 35007212b 36437115b 3615754b 1411731c 34357131b
Each value represents the mean7SD for 6 mice, and that for vehicle control or CCl4 group represents the mean7SD for 12 mice. Values not sharing a common letter are significantly different (po0.05).
Fig. 1. The effect of luteolin on the CCl4-induced oxidative stress. Mice were pretreated with luteolin as a single dose (5 or 50 mg/kg, i.p.) or once daily for 2 consecutive days (2 5 or 2 50 mg/kg, i.p.). The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). Mice were sacrificed 24 h after the CCl4 administration. The hepatic Cu/Zn SOD activity was measured as described in Materials and methods. Each bar represents the mean7SD for 6 mice, and that for vehicle control or CCl4 group represents the mean7SD for 12 mice. Values not sharing a common letter are significantly different (po0.05).
luteolin large necrosis with mild inflammatory cell infiltration was also observed (Fig. 3C). The liver of mice pretreated with 50 mg/kg (Fig. 3D) and 5 mg/kg of luteolin for 2 days (Fig. 3E) showed moderate necrosis with a low presence of inflammatory cells. The liver of mice pretreated with 50 mg/kg of luteolin for 2 days showed minimal hepatocellular necrosis, maintained lobular architecture and hepatocyte structure, with no evidence for inflammatory cell infiltration and fatty changes (Fig. 3F).
Immunohistochemistry The liver of control mice showed normal liver histology without significant gp96 immunopositivity (Fig. 4A). The data revealed that CCl4 intoxication is followed by a high upregulation of gp96 expression in viable hepatocytes (Fig. 4B). In the group treated with CCl4 intense cytoplasmic gp96 immunopositivity of viable liver parenchymal cells restricted in periportal areas was found, while gp96 expression was barely observed in pericentral areas affected by a massive necrosis. A markedly immunopositive single hepatocytes at the edge of necrotic areas could be found. In the liver of mice pretreated with 5 mg/kg of luteolin moderate
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Fig. 2. The effect of luteolin on the CCl4-induced oxidative stress. Mice were pretreated luteolin as a single dose (5 or 50 mg/kg, i.p.) or once daily for 2 consecutive days (2 5 or 2 50 mg/kg, i.p.). The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). Mice were sacrificed 24 h after the CCl4 administration. The hepatic myeloperoxidase activity was measured as described in Materials and methods. Each bar represents the mean7SD for 6 mice, and that for vehicle control or CCl4 group represents the mean7SD for 12 mice. Values not sharing a common letter are significantly different (po0.05).
gp96 immunopositive periportal areas were found, with a weak immunoreactivity in necrotic areas (Fig. 4C). The liver of mice pretreated with 50 mg/kg of luteolin showed mixed areas of normal hepatocytes with both intense and weak immunopositivity (Fig. 4D). In the group pretreated with 5 mg/kg of luteolin for 2 days weak cytoplasmic immunoreactivity of both viable hepatocytes and necrotic areas were observed (Fig. 4E). Pericentral immunopositivity was the least prominent in the mice pretreated with 50 mg/kg of luteolin for 2 days (Fig. 4F). In this group we found a weak immunostaining of hepatocytes located in necrotic areas, encompassed by normal hepatocytes without notable gp96 immunopositivity.
Discussion The liver is the major organ responsible for the metabolism of drugs and toxic chemicals, and therefore is the primary target organ for nearly all toxic chemicals (Bissel et al., 2001; Kaplowitz, 2000). Liver injury induced by CCl4 is the best-characterized system of the xenobiotic-induced hepatotoxicity and is a commonly used model for the screening the anti-hepatotoxic and
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hepatoprotective activity of drugs (Recknagel et al., 1989; Williams and Burk, 1990; Brautbar and Williams, 2002). Literature overview shows that most of the studies on luteolin actions under oxidative stress conditions have been performed in vitro (Lima et al., 2006; Oh et al., 2004). With regard to well-known antioxidant and antiinflammatory activities of luteolin, in this study we attempt to examine its potential beneficial effects during in vivo toxicological model of liver injury. Luteolin might be a rational candidate drug for the prevention of liver injury among individuals exposed to hepatotoxic agents. In the present study, we used the murine model of CCl4-induced liver injury to investigate the hepatoprotective effect of luteolin in a time- and dose-dependent manner. Since many cases of acute toxic liver damages are triggered by free radical formation and further driven by local inflammatory response, potent antioxidant and inflammatory properties of luteolin seems to be protective during these conditions. In addition, considering specific functional pool and regenerative capacity of the liver, permissive effect of luteolin to allow a sufficient regenerative response by reducing intensity of liver damage could be also expected. The results of the present study show that CCl4 administration causes severe acute liver damage in mice, demonstrated by remarkable elevation of serum AST and ALT levels. The increased serum levels of AST and ALT have been attributed to the damaged structural integrity of the liver. This is because they are intracellular enzymes, released into circulation after hepatocyte damage or necrosis (Sallie et al., 1991). These findings were also confirmed by histological observation. The changes mostly included hepatocellular necrosis or apoptosis, fatty accumulation, inflammatory cells infiltration and other histological manifestations, which were also consistent with the findings of other authors (Brattin et al., 1985; Sun et al., 2001). CCl4 injection induced oxidative stress, as evidenced by decreased Cu/Zn SOD activity and gp96 overexpression, and increased intrahepatic myeloperoxidase activity, suggesting accumulation of immune cells. Invading neutrophils or monocyte/macrophages could release MPO at inflammatory sites within the liver, and MPO generated hypochlorous acid (HClO) could damage bystander hepatic cells (Reynolds et al., 2002). Pretreatment with luteolin could ameliorate CCl4induced hepatotoxicity in mice, as demonstrated by the lower serum aminotransferase activities. This effect is in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and regeneration of hepatocytes (Thabrew et al., 1987). A microscopic examination showed that the severe hepatic lesions and necrosis induced by CCl4 were ameliorated by the administration of luteolin, with the most significant
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Fig. 3. The effect of luteolin pretreatment on the CCl4-induced liver damage in mice. Mice were pretreated with luteolin (5 or 50 mg/kg, i.p.) once or once daily for 2 consecutive days. The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.), except the control groups. Mice were sacrificed 24 h after the CCl4 administration. Liver sections from mice treated with: (A) vehicle, (B) CCl4, (C) luteolin 5 mg/kg plus CCl4, (D) luteolin 50 mg/kg plus CCl4, (E) luteolin 5 mg/kg for 2 days plus CCl4, (F) luteolin 50 mg/kg for 2 day plus CCl4. Insets show inflammatory cell infiltration. Original magnification 400. Insets: original magnification 1000. H&E stain.
effect in mice pretreated with 50 mg/kg luteolin for 2 consecutive days. Moreover, luteolin showed a remarkable antiinflammatory activity. Luteolin reduced MPO activity in the liver of all treated groups, indicating the protection of hepatocytes from excessive inflammatory response and MPO generating HClO. Inflammation itself could inhibit CCl4-induced hepatotoxicity by the suppression of cytochrome P-450 activities and metallothionein (MT) induction (DiSilvestro and Carlson, 1992), although the induction of MT synthesis seems to be independent of free radical production in the liver (Min et al., 1992). Recently, we have shown MT induction in periportal and pericentral hepatocytes in CCl4-intoxicated mice pretreated with luteolin
(Domitrovic´ et al., 2008). However, the detailed mechanisms of luteolin hepatoprotective action with a multiple dose pretreatment need to be clarified. Previous studies have shown the inhibitory effect of luteolin on CCl4-induced liver microsomal lipid peroxidation (Cholbi et al., 1991). In this study, luteolin significantly reduced oxidative stress in the liver of CCl4treated mice in time- and dose-dependent manner, as suggested by a significant restoration of hepatic Cu/Zn SOD activity, which was also confirmed by histological observations. Oxidative stress can trigger a cellular stress response characterized by induction of antioxidants, acute phase reactants, and HSPs, which are presumed to play a role in limiting tissue damage.
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Fig. 4. The effect of luteolin pretreatment on the gp96 expression in mice liver. Mice were pretreated with luteolin (5 or 50 mg/kg, i.p.) once or once daily for 2 consecutive days. The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.), except the control groups. Mice were sacrificed 24 h after the CCl4 administration. Liver sections from mice treated with: (A) vehicle, (B) CCl4, (C) luteolin 5 mg/kg plus CCl4, (D) luteolin 50 mg/kg plus CCl4, (E) luteolin 5 mg/kg for 2 days plus CCl4, (F) luteolin 50 mg/kg for 2 day plus CCl4. Original magnification 400. Immunohistochemical stain for gp96 protein.
Impaired hepatic antioxidant defence could led to protein denaturation and/or misfolding. HSP90 family is not only involved in protein folding, but also plays key roles in the activation and assembly of a range of specific proteins involved in signal transduction, cell cycle control, or transcriptional regulation (Pratt, 1998). HSP gp96, a member of the HSP90 family, is expressed in a gradient from chronic hepatitis through liver cirrhosis and hepatocellular carcinoma (Hartl and Hayer-Hartl, 2002; Schueller et al., 2004). Under normal physiological conditions, the tissue-resident protein gp96 occurs in the endoplasmic reticulum. Recently, involvement of gp96 in liver regeneration as an innate sensor of damage has been suggested (Mrakovcˇic´-Sˇutic´
et al., 2008). Liver damage induced by partial hepatectomy was followed by a high upregulation of gp96 expression in regenerating hepatocytes. In this study, intense gp96 immunopositivity of viable liver parenchymal cells suggest the protection from necrosis induced by a high dose of CCl4, particularly in hepatocytes found at the edge of necrotic areas. Positive staining in the disintegrated area of liver parenchyma indicates necrosis, with HSPs released from cells as a result of necrotic death (Binder et al., 2000). The immunohistochemical study showed a decreasing tendency of cytoplasmic gp96 protein level from the CCl4-intoxicated group to the groups pretreated with luteolin, in a time- and dose-dependent manner. The data suggest
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that the overexpression of gp96 in liver tissue is an early molecular response in acute, chemically induced liver damage in mice, which correlates with the degree of tissue damage. The results of this study suggest that the pro tective effect of luteolin might be due to induction of adaptive mechanisms, which could ameliorate hepatocyte damage. A single dose pretreatment affected gp96 expression and the activity of Cu/Zn SOD and myeloperoxidase much less than pretreatment for 2 consecutive days. As suggested by Lin et al. (1999), a prolonged pretreatment can either induce or inhibit enzyme activities via alteration of enzyme biosynthesis. The levels of antioxidant enzymes are regulated by gene expressions as well as by post-translational modifications. Although their functions are to scavenge reactive oxygen and nitrogen species, they may also be targets of various oxidants (Fujii and Taniguchi, 1999). Kang and Kim (1997) suggested that Cu/Zn SOD could be fragmentated by the product of catalysis, H2O2, due to the peroxidative reaction of superoxide dismutase. Hepatoprotective effects of luteolin, at least in part, could originate from its inhibitory ability on free radical formation or due to its free radical scavenging activity. Additionally, the inhibitory effect of luteolin on inflammatory cell infiltration might also contribute to its hepatoprotective properties. In conclusion, luteolin has shown a significant timeand dose-dependent hepatoprotective effect on acute liver toxicity induced by CCl4. Luteolin seems to be a beneficial drug for the prevention of acute liver toxicity, although further studies are necessary.
Acknowledgements This work was supported by the grants from Ministry of Science, Education and Sport, Republic of Croatia (Projects nos. 062-0000000-3554 and 062-0621341-0061). The authors wish to thank Mihaela Staver for her assistance in the early trials of this study.
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Experimental and Toxicologic Pathology 61 (2009) 581–589 www.elsevier.de/etp
Dose- and time-dependent effects of luteolin on carbon tetrachloride-induced hepatotoxicity in mice Robert Domitrovic´a,, Hrvoje Jakovacb, Cˇedomila Milina, Biserka Radosˇevic´-Stasˇic´b a
Department of Chemistry and Biochemistry, Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia Department of Physiology and Immunology, Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia
b
Received 28 August 2008; accepted 17 December 2008
Abstract Carbon tetrachloride (CCl4) is a well-known model compound for producing chemical hepatic injury. This study investigated the protective effects of the flavonoid luteolin on the CCl4-induced hepatotoxicity in mice. Luteolin dissolved in dimethyl sulfoxide (DMSO) was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose, and once daily for 2 consecutive days. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). CCl4-induced hepatotoxicity was reduced in a dose- and time-dependent manner, as determined by decreased serum aminotransferase activities and liver histopathology. CCl4 intoxication resulted in an overexpression of heat shock protein gp96 in the mice liver, which was strongly attenuated by luteolin pretreatment. Luteolin has also decreased oxidative stress produced by CCl4, as suggested by improvement in the Cu/Zn superoxide dismutase activity. The effect of luteolin on myeloperoxidase, an indicator of inflammatory cell infiltration, was also investigated. Treatment of the mice with luteolin resulted in a significant decrease in the myeloperoxidase activity. The hepatoprotective effect of luteolin against CCl4 hepatotoxicity was higher in animals pretreated with luteolin for 2 consecutive days. This suggests that the protection might be due to induction of some adaptive mechanisms. The data indicate that luteolin could be effective in protecting mice from the hepatotoxicity produced by CCl4. r 2008 Elsevier GmbH. All rights reserved. Keywords: Carbon tetrachloride hepatotoxicity; Luteolin; Cu/Zn superoxide dismutase; Myeloperoxidase; Histopathology; Timeand dose-dependent; Heat shock protein; gp96
Introduction The liver plays a central role in transforming and clearing metabolites and xenobiotics, and is susceptible to the toxicity from these agents. Carbon tetrachloride (CCl4) is a well-known model compound for inducing liver injury. Its biotransformation by the hepatic microsomal cytochrome P450 produces hepatotoxic Corresponding author. Tel./fax: +385 51 651 135.
E-mail address: [email protected] (R. Domitrovic´). 0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.12.005
metabolites, namely trichloromethyl free radicals. The covalent binding of the trichloromethyl free radicals to the cell proteins is considered to be the initial step in a chain of events that eventually lead to cell necrosis (Recknagel et al., 1989; Williams and Burk, 1990; Brautbar and Williams, 2002). During liver damage, inflammatory cell infiltration may occur, which can be quantified by measuring the activity of myeloperoxidase (MPO), an enzyme found within the azurophilic granules of polymorphonuclears such as neutrophils and monocyte/macrophage mononuclears, including Kupffer cells (Reynolds et al., 2002).
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Flavonoids, a large class of polyphenolic compounds present in fruits and fruit products, vegetables, and plant-derived beverages such as tea and wine, are associated with a variety of beneficial properties (Middleton et al., 2000; Havsteen, 2002). Due to their antioxidant activity (Rice-Evans et al., 1996), they are presumed to protect tissues against oxidative stress and associated pathologies such as cancer, coronary artery disease and inflammation. Polyphenols act as antioxidants by scavenging reactive oxygen and nitrogen species, chelating redox-active transition metal ions preventing them from catalyzing free radical formation, but also several other ways (Frei and Higdon, 2003). Luteolin (30 ,40 ,5,7-tetrahydroxyflavone) is an important member of the flavonoid family, present in glycosylated forms and as aglycone in various plants. Luteolin is reported to have antiinflammatory (Ziyan et al., 2007; Ueda et al., 2002), antioxidant (Perez-Garcia et al., 2000), antiallergic (Veda et al., 2002), antitumorigenic (Ju et al., 2007), anxiolytic-like (Coleta et al., 2007), and vasorelaxative properties (Woodman and Chan, 2004). Heat shock proteins (HSPs) are induced in response to various stresses and to protect cells from such stresses (Lee et al., 2004). The 90 kDa HSP family, one of the most abundant proteins in eukaryotic cells, plays an important role in the folding of newly synthesized proteins and stabilization and refolding of denatured proteins in stress conditions (Sreedhar et al., 2004). The progressive cellular damage caused by reactive oxygen species contributes to protein misfolding and accumulation of HSPs. HSP gp96 (Grp94, glucose regulated protein 94) is a constitutively expressed endoplasmic reticulum lumenal protein that is upregulated in response to cellular stress such as heat shock, oxidative stress or glucose depletion (Ruddon and Bedows, 1997). HSP gp96 has not been previously studied in toxic liver damage. The main purpose of this study was to investigate the potential effects of luteolin in reducing oxidative stress and inflammation in the liver of mice caused by CCl4, as well as enhancement of hepatic proliferative capability, which could provide helpful information for the prevention of liver damage. The present investigation focuses on evaluation of the efficacy of different dose regimes of luteolin for their protection against CCl4-induced hepatotoxicity. The parameters analysed were serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, histopathology, HSP gp96 protein expression, hepatic Cu/Zn superoxide dismutase (Cu/Zn SOD) and myeloperoxidase activities.
alanine aminotransferase and aspartate aminotransferase, dimethyl sulfoxide (DMSO), hexadecyltrimethylammonium bromide (HTMABr), o-dianisidine dihydrochloride, hydrogen peroxide (H2O2) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Superoxide Dismutase Assay Kit was obtained from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals and solvents were of the highest grade commercially available.
Animals Male Balb/c mice from our breeding colony, 2–3 month old, were divided into 8 groups with 6 animals per group. Mice were fed a standard rodent diet (pellet, type 4RF21 GLP, Mucedola, Italy), and water ad libitum. The animals were maintained at 12 h light/dark cycle, at constant temperature (2071 1C) and humidity (5075%). All experimental procedures were approved by the Ethical Committee of the Medical Faculty, University of Rijeka.
Experimental design The control group animals were given DMSO as a single dose (group I) or once daily for 2 consecutive days (group II). Luteolin dissolved in DMSO was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose (groups III and IV), and once daily for 2 consecutive days (groups V and VI). Groups VII and VIII received vehicle (DMSO) as a single dose and once daily for 2 consecutive days, respectively. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p., dissolved in olive oil), except the control groups. Twenty-four hours after administrating CCl4, the mice were killed by cervical dislocation, blood was collected from the orbital sinus and the serum was separated to determine ALT and AST activities. The abdomen was cut open quickly and the liver was perfused thoroughly with isotonic saline, excised, blotted dry and divided into multiple samples. Liver samples were used to assess the Cu/Zn SOD and MPO activities, and protein content. A portion of the livers were preserved in a buffered formalin solution to obtain the histological sections.
Hepatotoxicity studies
Materials and methods Olive oil was provided by Zvijezda, Croatia. Luteolin, carbon tetrachloride, diagnostic kits for the serum
Serum levels of ALT and AST, as markers of hepatic function, were measured by using a Bio-Tek EL808 Ultra Microplate Reader (BioTek Instruments, Winooski, VT, USA) according to manufacturer’s instructions.
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Superoxide dismutase activity assay Livers were homogenized in 50 mM phosphate buffer saline (PBS), pH 7.4, using a Polytron homogenizer (Kinematica, Lucerne, Switzerland) for 1 min at 4 1C. The homogenate was subjected to centrifugation using Beckman L7-65 Ultracentrifuge (Beckman, Fullerton, USA) at 12,000g for 15 min, at 4 1C. The supernatants and homogenates were used for superoxide dismutase activity assay. Cu/Zn SOD activity in homogenate supernatants was assessed by the degree of suppression of xanthine reduction in the presence of NADH and xanthine oxidase (Superoxide Dismutase Assay Kit, Cayman Chemical). The increase of absorbance was monitored spectrophotometrically at 450 nm (Bio-Tek EL808 Ultra Microplate Reader). The assay kit utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of Cu/Zn SOD was defined as the amount of enzyme required to inhibit the reduction of xanthine by 50%. Protein content in liver homogenates was estimated by Bradford’s method (Bradford, 1976).
Myeloperoxidase activity assay Liver homogenates were prepared in 50 mM PBS, pH 6.0 (containing 5 mM DTT). The homogenate was centrifuged at 12,000g for 20 min, at 4 1C. For the biochemical determination of MPO activity, the supernatant was discarded and the pellet was washed again. After decanting the supernatant, the pellet was extracted by suspending the material in 0.5% hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer (pH 6.0, 4 1C) for 2 min. The samples were immediately frozen and subjected to three freeze–thaw cycles, with sonication between cycles. Supernatant MPO activity was assayed as described previously (Bradley et al., 1982). Briefly, 0.1 ml of supernatant was mixed with 2.9 ml of 50 mM PBS, pH 6.0, containing 0.167 mg of o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The rate at which a colored product formed during the MPO-dependent reaction of o-dianisidine dihydrochloride was measured. The change in absorbance at 460 nm was recorded at 15-s intervals over 2 min using a spectrophotometer (Cary 100, Varian, Mulgrave, Australia). One unit of MPO activity is defined as that which degrades 1 mmol of peroxide per minute at 25 1C (Barone et al., 1991). Enzyme activity was expressed as mmol/mg protein using the molar absorbency index (1.13 104) of oxidized o-dianisidine.
Histological examinations Liver tissues were placed in plastic cassettes and immersed in neutral buffered formalin for 24 h. The
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fixed tissues were processed routinely, embedded in paraffin, sectioned, deparaffinized, and rehydrated using the standard techniques. The extent of CCl4-induced necrosis was evaluated by assessing the morphological changes in the liver sections stained with hematoxylin and eosin (H&E), using standard techniques.
Immunohistochemistry Tissue expression of gp96 protein in the liver was analysed by immunohistochemistry. Immunohistochemical studies were performed on paraffin embedded liver tissue using DAKO EnVision+System, Peroxidase (DAB) kit according to the manufacturer’s instructions (DAKO Corporation, USA). Briefly, slides were incubated with peroxidase block to eliminate endogenous peroxidase activity. After washing, monoclonal anti-Grp94 antibody (Monoclonal rat Anti-Grp94 antibody, Clone 9G10, Stressgen, Canada), diluted 1:30 in phosphate-buffered saline supplemented with bovine serum albumin was added to tissue samples and incubated overnight at 4 1C in a humid environment, followed by 45 min incubation with peroxidase labeled polymer conjugated to goat anti-mouse immunoglobulins containing carrier protein linked to Fc fragments to prevent nonspecific binding. The immunoreaction product was visualized by adding substrate-chromogen (DAB) solution. Tissues were counterstained with hematoxylin and 37 mM ammonia water, dehydrated in a gradient of alcohol and mounted with mounting medium. The specificity of the reaction was confirmed by substitution of anti-Grp94 antibody with mouse irrelevant IgG2a immunoglobulin (clone DAK-G05; Dako, USA), used in the same conditions and dilutions as a primary antibody.
Statistical analysis Data were analysed using StatSoft STATISTICA version 7.1 software. Differences between the groups were assessed by a nonparametric Man–Whitney and Kruskal–Wallis tests. Values in the text are mean7 standard deviation (SD). Differences with po0.05 were considered to be statistically significant.
Results We did not observe a significant difference between control groups I and II or between CCl4-treated groups VII and VIII. Therefore, the data from groups I and II or from groups VII and VIII were combined and then evaluated as a unique control and CCl4 groups, respectively.
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Hepatotoxicity studies Serum ALT and AST levels were highly elevated at 24 h after CCl4 treatment. Pretreatment with luteolin decreased ALT levels in CCl4-treated mice when administered once daily for 2 consecutive days, 50 mg/kg. AST levels were reduced in all groups receiving luteolin except for the lowest dose, with the highest reduction in mice pretreated with 50 mg/kg of luteolin for 2 consecutive days (Table 1).
Superoxide dismutase activity assay CCl4 administration significantly decreased the Cu/Zn SOD activity, however, pretreatment with luteolin significantly ameliorated the lost of Cu/Zn SOD activity, in a dose-dependent manner (Fig. 1).
Myeloperoxidase activity assay MPO activity, an indicator of neutrophil and monocyte/macrophages infiltration, was significantly higher in the liver tissue of CCl4-treated mice than in that of the control group, which indicates that a severe inflammatory response had taken place in the development of liver damage. In contrast, the data showed that pretreatment with luteolin resulted in a remarkable inhibition of MPO activities in CCl4-treated mice (Fig. 2).
Histological examinations Liver tissue from control mice (Fig. 3A) showed normal liver histology and hepatocyte structure. In the group treated with CCl4, we have found large areas of extensive, mainly centrilobular necrosis with loss of hepatocyte structure, vacuolar fatty change and mild inflammatory cell infiltration, comprised predominantly of mononuclear cells with occasional polymorphonuclears (Fig. 3B). In the liver of mice pretreated with 5 mg/kg of Table 1. The effect of luteolin pretreatment on the serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in experimental groups.
Control (vehicle) Luteolin 5 mg/kg Luteolin 50 mg/kg Luteolin 5 mg/kg, 2 days Luteolin 50 mg/kg, 2 days CCl4
AST (U/L)
ALT (U/L)
41.377.8a 28497193b 21627119c 2110736c 1225769d 29957248b
44.775.5a 35007212b 36437115b 3615754b 1411731c 34357131b
Each value represents the mean7SD for 6 mice, and that for vehicle control or CCl4 group represents the mean7SD for 12 mice. Values not sharing a common letter are significantly different (po0.05).
Fig. 1. The effect of luteolin on the CCl4-induced oxidative stress. Mice were pretreated with luteolin as a single dose (5 or 50 mg/kg, i.p.) or once daily for 2 consecutive days (2 5 or 2 50 mg/kg, i.p.). The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). Mice were sacrificed 24 h after the CCl4 administration. The hepatic Cu/Zn SOD activity was measured as described in Materials and methods. Each bar represents the mean7SD for 6 mice, and that for vehicle control or CCl4 group represents the mean7SD for 12 mice. Values not sharing a common letter are significantly different (po0.05).
luteolin large necrosis with mild inflammatory cell infiltration was also observed (Fig. 3C). The liver of mice pretreated with 50 mg/kg (Fig. 3D) and 5 mg/kg of luteolin for 2 days (Fig. 3E) showed moderate necrosis with a low presence of inflammatory cells. The liver of mice pretreated with 50 mg/kg of luteolin for 2 days showed minimal hepatocellular necrosis, maintained lobular architecture and hepatocyte structure, with no evidence for inflammatory cell infiltration and fatty changes (Fig. 3F).
Immunohistochemistry The liver of control mice showed normal liver histology without significant gp96 immunopositivity (Fig. 4A). The data revealed that CCl4 intoxication is followed by a high upregulation of gp96 expression in viable hepatocytes (Fig. 4B). In the group treated with CCl4 intense cytoplasmic gp96 immunopositivity of viable liver parenchymal cells restricted in periportal areas was found, while gp96 expression was barely observed in pericentral areas affected by a massive necrosis. A markedly immunopositive single hepatocytes at the edge of necrotic areas could be found. In the liver of mice pretreated with 5 mg/kg of luteolin moderate
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Fig. 2. The effect of luteolin on the CCl4-induced oxidative stress. Mice were pretreated luteolin as a single dose (5 or 50 mg/kg, i.p.) or once daily for 2 consecutive days (2 5 or 2 50 mg/kg, i.p.). The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). Mice were sacrificed 24 h after the CCl4 administration. The hepatic myeloperoxidase activity was measured as described in Materials and methods. Each bar represents the mean7SD for 6 mice, and that for vehicle control or CCl4 group represents the mean7SD for 12 mice. Values not sharing a common letter are significantly different (po0.05).
gp96 immunopositive periportal areas were found, with a weak immunoreactivity in necrotic areas (Fig. 4C). The liver of mice pretreated with 50 mg/kg of luteolin showed mixed areas of normal hepatocytes with both intense and weak immunopositivity (Fig. 4D). In the group pretreated with 5 mg/kg of luteolin for 2 days weak cytoplasmic immunoreactivity of both viable hepatocytes and necrotic areas were observed (Fig. 4E). Pericentral immunopositivity was the least prominent in the mice pretreated with 50 mg/kg of luteolin for 2 days (Fig. 4F). In this group we found a weak immunostaining of hepatocytes located in necrotic areas, encompassed by normal hepatocytes without notable gp96 immunopositivity.
Discussion The liver is the major organ responsible for the metabolism of drugs and toxic chemicals, and therefore is the primary target organ for nearly all toxic chemicals (Bissel et al., 2001; Kaplowitz, 2000). Liver injury induced by CCl4 is the best-characterized system of the xenobiotic-induced hepatotoxicity and is a commonly used model for the screening the anti-hepatotoxic and
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hepatoprotective activity of drugs (Recknagel et al., 1989; Williams and Burk, 1990; Brautbar and Williams, 2002). Literature overview shows that most of the studies on luteolin actions under oxidative stress conditions have been performed in vitro (Lima et al., 2006; Oh et al., 2004). With regard to well-known antioxidant and antiinflammatory activities of luteolin, in this study we attempt to examine its potential beneficial effects during in vivo toxicological model of liver injury. Luteolin might be a rational candidate drug for the prevention of liver injury among individuals exposed to hepatotoxic agents. In the present study, we used the murine model of CCl4-induced liver injury to investigate the hepatoprotective effect of luteolin in a time- and dose-dependent manner. Since many cases of acute toxic liver damages are triggered by free radical formation and further driven by local inflammatory response, potent antioxidant and inflammatory properties of luteolin seems to be protective during these conditions. In addition, considering specific functional pool and regenerative capacity of the liver, permissive effect of luteolin to allow a sufficient regenerative response by reducing intensity of liver damage could be also expected. The results of the present study show that CCl4 administration causes severe acute liver damage in mice, demonstrated by remarkable elevation of serum AST and ALT levels. The increased serum levels of AST and ALT have been attributed to the damaged structural integrity of the liver. This is because they are intracellular enzymes, released into circulation after hepatocyte damage or necrosis (Sallie et al., 1991). These findings were also confirmed by histological observation. The changes mostly included hepatocellular necrosis or apoptosis, fatty accumulation, inflammatory cells infiltration and other histological manifestations, which were also consistent with the findings of other authors (Brattin et al., 1985; Sun et al., 2001). CCl4 injection induced oxidative stress, as evidenced by decreased Cu/Zn SOD activity and gp96 overexpression, and increased intrahepatic myeloperoxidase activity, suggesting accumulation of immune cells. Invading neutrophils or monocyte/macrophages could release MPO at inflammatory sites within the liver, and MPO generated hypochlorous acid (HClO) could damage bystander hepatic cells (Reynolds et al., 2002). Pretreatment with luteolin could ameliorate CCl4induced hepatotoxicity in mice, as demonstrated by the lower serum aminotransferase activities. This effect is in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and regeneration of hepatocytes (Thabrew et al., 1987). A microscopic examination showed that the severe hepatic lesions and necrosis induced by CCl4 were ameliorated by the administration of luteolin, with the most significant
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Fig. 3. The effect of luteolin pretreatment on the CCl4-induced liver damage in mice. Mice were pretreated with luteolin (5 or 50 mg/kg, i.p.) once or once daily for 2 consecutive days. The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.), except the control groups. Mice were sacrificed 24 h after the CCl4 administration. Liver sections from mice treated with: (A) vehicle, (B) CCl4, (C) luteolin 5 mg/kg plus CCl4, (D) luteolin 50 mg/kg plus CCl4, (E) luteolin 5 mg/kg for 2 days plus CCl4, (F) luteolin 50 mg/kg for 2 day plus CCl4. Insets show inflammatory cell infiltration. Original magnification 400. Insets: original magnification 1000. H&E stain.
effect in mice pretreated with 50 mg/kg luteolin for 2 consecutive days. Moreover, luteolin showed a remarkable antiinflammatory activity. Luteolin reduced MPO activity in the liver of all treated groups, indicating the protection of hepatocytes from excessive inflammatory response and MPO generating HClO. Inflammation itself could inhibit CCl4-induced hepatotoxicity by the suppression of cytochrome P-450 activities and metallothionein (MT) induction (DiSilvestro and Carlson, 1992), although the induction of MT synthesis seems to be independent of free radical production in the liver (Min et al., 1992). Recently, we have shown MT induction in periportal and pericentral hepatocytes in CCl4-intoxicated mice pretreated with luteolin
(Domitrovic´ et al., 2008). However, the detailed mechanisms of luteolin hepatoprotective action with a multiple dose pretreatment need to be clarified. Previous studies have shown the inhibitory effect of luteolin on CCl4-induced liver microsomal lipid peroxidation (Cholbi et al., 1991). In this study, luteolin significantly reduced oxidative stress in the liver of CCl4treated mice in time- and dose-dependent manner, as suggested by a significant restoration of hepatic Cu/Zn SOD activity, which was also confirmed by histological observations. Oxidative stress can trigger a cellular stress response characterized by induction of antioxidants, acute phase reactants, and HSPs, which are presumed to play a role in limiting tissue damage.
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Fig. 4. The effect of luteolin pretreatment on the gp96 expression in mice liver. Mice were pretreated with luteolin (5 or 50 mg/kg, i.p.) once or once daily for 2 consecutive days. The control mice were given DMSO. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.), except the control groups. Mice were sacrificed 24 h after the CCl4 administration. Liver sections from mice treated with: (A) vehicle, (B) CCl4, (C) luteolin 5 mg/kg plus CCl4, (D) luteolin 50 mg/kg plus CCl4, (E) luteolin 5 mg/kg for 2 days plus CCl4, (F) luteolin 50 mg/kg for 2 day plus CCl4. Original magnification 400. Immunohistochemical stain for gp96 protein.
Impaired hepatic antioxidant defence could led to protein denaturation and/or misfolding. HSP90 family is not only involved in protein folding, but also plays key roles in the activation and assembly of a range of specific proteins involved in signal transduction, cell cycle control, or transcriptional regulation (Pratt, 1998). HSP gp96, a member of the HSP90 family, is expressed in a gradient from chronic hepatitis through liver cirrhosis and hepatocellular carcinoma (Hartl and Hayer-Hartl, 2002; Schueller et al., 2004). Under normal physiological conditions, the tissue-resident protein gp96 occurs in the endoplasmic reticulum. Recently, involvement of gp96 in liver regeneration as an innate sensor of damage has been suggested (Mrakovcˇic´-Sˇutic´
et al., 2008). Liver damage induced by partial hepatectomy was followed by a high upregulation of gp96 expression in regenerating hepatocytes. In this study, intense gp96 immunopositivity of viable liver parenchymal cells suggest the protection from necrosis induced by a high dose of CCl4, particularly in hepatocytes found at the edge of necrotic areas. Positive staining in the disintegrated area of liver parenchyma indicates necrosis, with HSPs released from cells as a result of necrotic death (Binder et al., 2000). The immunohistochemical study showed a decreasing tendency of cytoplasmic gp96 protein level from the CCl4-intoxicated group to the groups pretreated with luteolin, in a time- and dose-dependent manner. The data suggest
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that the overexpression of gp96 in liver tissue is an early molecular response in acute, chemically induced liver damage in mice, which correlates with the degree of tissue damage. The results of this study suggest that the pro tective effect of luteolin might be due to induction of adaptive mechanisms, which could ameliorate hepatocyte damage. A single dose pretreatment affected gp96 expression and the activity of Cu/Zn SOD and myeloperoxidase much less than pretreatment for 2 consecutive days. As suggested by Lin et al. (1999), a prolonged pretreatment can either induce or inhibit enzyme activities via alteration of enzyme biosynthesis. The levels of antioxidant enzymes are regulated by gene expressions as well as by post-translational modifications. Although their functions are to scavenge reactive oxygen and nitrogen species, they may also be targets of various oxidants (Fujii and Taniguchi, 1999). Kang and Kim (1997) suggested that Cu/Zn SOD could be fragmentated by the product of catalysis, H2O2, due to the peroxidative reaction of superoxide dismutase. Hepatoprotective effects of luteolin, at least in part, could originate from its inhibitory ability on free radical formation or due to its free radical scavenging activity. Additionally, the inhibitory effect of luteolin on inflammatory cell infiltration might also contribute to its hepatoprotective properties. In conclusion, luteolin has shown a significant timeand dose-dependent hepatoprotective effect on acute liver toxicity induced by CCl4. Luteolin seems to be a beneficial drug for the prevention of acute liver toxicity, although further studies are necessary.
Acknowledgements This work was supported by the grants from Ministry of Science, Education and Sport, Republic of Croatia (Projects nos. 062-0000000-3554 and 062-0621341-0061). The authors wish to thank Mihaela Staver for her assistance in the early trials of this study.
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