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Does instant coffee prevent acute liver injury induced by carbon tetrachloride (CCl4)?
Hepatology Research 35 (2006) 163–168
Does instant coffee prevent acute liver injury induced by carbon tetrachloride (CCl4)? Ibrahim Hanifi Ozercan a,∗ , Adile Ferda Dagli a , Bilal Ustundag b , Mehmet Resat Ozercan a , Ibrahim Halil Bahcecioglu c , Hakim C ¸ elik d , Mehmet Yalniz c , c Orhan Kursat Poyrazoglu , Huseyin Ataseven c a
Fırat University, Faculty of Medicine, Department of Pathology, 23119 Elazig, Turkey Fırat University, Faculty of Medicine, Department of Biochemistry, 23119 Elazig, Turkey c Fırat University, Faculty of Medicine, Division of Gastroenterology, 23119 Elazig, Turkey d Harran University, Faculty of Medicine, Department of Biochemistry, Sanliurfa, Turkey b
Received 27 February 2006; received in revised form 6 April 2006; accepted 8 April 2006 Available online 5 June 2006
Abstract Objective: To investigate the protective effect of instant coffee (IC) on acute liver injury induced by CCl4 . Methods: The study included 32 rats which were allocated to four groups: control (n: 8), CCl4 (n: 8), CCl4 + IC (n: 8) and IC (n: 8). Malondialdehyde, which is a lipid peroxidation product, and levels of antioxidant capacity were measured and histopathological data were compared. Results: It was seen in the study that lipid peroxidation products that increased in the plasma and liver tissue of the CCl4 group decreased by IC administration. There was an increase in the measured antioxidant parameters, which were total antioxidant capacity (TAOC), sulphydryl (SH) and ceruloplasmin levels. Histopathologically, it was found that inflammation and necrosis which increased in the group administered CCl4 decreased significantly with IC administration, but there steatosis did not change. Conclusions: It was seen that IC had a protective role in acute liver injury induced by CCl4 , but did not affect steatosis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Instant coffee; Acute liver injury; Lipid peroxidation; Antioxidant
1. Introduction Drinking coffee is among the most common habits in the world [1]. Many studies have been conducted to determine the effect of widely consumed coffee on human health. The studies have demonstrated that although it causes hypercholesterolemia and coronary diseases, coffee prevents liver injury and the resulting cirrhosis, besides reducing hepatocellular carcinoma formation in the long run [2–6]. It has been reported that liver injury that results from an increase in free oxygen radicals due to various etiological ∗ Corresponding author. Tel.: +90 424 233 3555/2186; fax: +90 424 238 8096. E-mail address: [email protected] (I.H. Ozercan).
1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.04.004
causes leads to liver cirrhosis ([6–8]; Chen, 2005). There are many studies which have tried different substances as antioxidants for the purpose of protection in liver injury and the process leading to cirrhosis [9]. Studies on coffee have focused on its antioxidant activity. It has been reported that substances like cafestol and kahweol that are found in coffee can affect glutathione and sulphydryl groups through glutathione S-transferase enzyme [10]. Antioxidant molecules exercise their effect by inhibiting or preventing harmful reactions. Antioxidant defense system can be evaluated by measuring various antioxidant structures like antioxidant enzymes, vitamins, albumin, bilirubin, etc. in the serum or plasma. However, such measurements are not only time-consuming and expensive, but also require complicated techniques. Therefore, recent preference has been
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towards the measurement of the total antioxidant capacity, as it is more practical, less expensive and can demonstrate all the antioxidant structures in the organism [11,12]. Many studies about coffee have measured different antioxidants and their activities [8,10]. However, phenolic compounds that exercise the antioxidant effect of coffee have not been thoroughly clarified [13]. Besides, it has been reported that the agents in the coffee change according to the way coffee is prepared [14]. Therefore, the present study aims to investigate the effect of instant coffee on liver injury induced by CCl4 through the relation between TAOC, which is a good indicator of all the antioxidants in the organism, and histopathological changes.
2. Materials and methods 2.1. Experimental animals The study included 32 male Sprague–Dawley rats, which were divided into four equal groups. They were kept in specially manufactured cages in a room, which was sunny for 12 h. The study was carried out in strict conformance with the standard ethical rules about experimental animal studies. For feeding, a special rat food provided by the factory in Elazig was used. Water was supplied by special bottles with a drip pin placed inside the cages. Group 1, which is the control (C) group, was given tap water and Group 2 (CCl4 ) was administered CCl4 to induce acute intoxication, together with tap water. Group 3 (CCl4 + IC) was administered IC and CCl4 , and Group 4 (IC) was given only IC. Two grams of IC was dissolved in 170 mL of boiled water and left to cool in room temperature [14]. IC administration was started a day before CCl4 administration and the coffee was mixed into the water of rats every other day, gradually increasing the concentration from 25% at the beginning to 100% at the end. IC prepared specially was given at dose of 0.146 g/day for each rat in first 2 days of the experiment. The concentration of the IC was increased gradually as following: 0.234 g/day per rat in days 3 and 4, 0.263 g/day per rat in days 5 and 6 and 0.351 g/day per rat in days 7 and 8. CCl4 was injected through intraperitoneal route at a dose of 0.15 mL/100 g (suspended with olive oil at a rate of 1:1) every other day, three times starting from the second day until the end of the study. Rats which drank pure IC on the seventh day were decapitated after 24 h and blood and tissue samples were collected. 2.2. Biochemical analyses The plasma obtained by centrifuging the blood from rats was kept under −20◦ until the analysis. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (␥-GT), levels were determined by Olympus Au 600 autoanalyzer (Olympus Corp., Japan).
Liver tissue and plasma malondialdehyde levels: the plasma MDA (pMDA) levels were assessed by thiobarbituric acid method modified by Satoh [15] and Yagi [16]. The results were in nmol/mL. And the liver tissue MDA (tMDA) levels were assessed by Ohkawa method [17]. The results were in nmol/g tissue. 2.2.1. Measurement of the total antioxidant status of plasma The total antioxidant status of the plasma was measured using a novel automated colourimetric measurement method for the total antioxidant response developed by Erel [11,12]. In this method the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction, and reacts with the colourless substrate. O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in colour. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the colour change thereby providing an effective measure of the total antioxidant capacity of the plasma. The assay results are expressed as mmol Trolox eq./L, and the precision of this assay is excellent, being lower 3%. 2.2.2. Analyses of sulfhydryl groups Measurement of total free sulfhydryl groups of serum samples was assayed according to the method of Elman [18]. Briefly, 1 mL of buffer containing 0.1 M Tris, 10 mM EDTA, pH 8.2, and 50 all serum was added to cuvettes, followed by 50 all 10 mM dithiobisnitrobenzoicacid (DTNB) in methanol. Blanks were run for each sample as a test, but there was no DTNB in the methanol. Following incubation for 15 min at room temperature, sample absorbance was read 412 nm on a Cecil 3000 spectrophotometer. Sample and reagent blanks were subtracted. The concentration of sulfhydryl groups was calculated using reduced glutathion as free sulfhydryl group standard the result was expressed as millimolars. 2.2.3. Seruloplazmin analyses Serum ceruloplasmin levels were assessed by measuring its oxidase activity using p-phenylenediamine as substrate [19,20]. 2.3. Histopathological investigation The liver tissue specimens were buried into paraffin blocks undergoing a routine follow-up procedure after they were fixed at 10% formaldehyde. The 4 m sections were stained with hematoxylin–eosin (HE). The preparations were examined by Olympus BX-50 light microscopy. An experienced pathologist evaluated all liver biopsy specimens. The steatosis was rated as follows: lower than +: 25%, between ++: 25–50% and +++: 50–75% and higher than ++++: 75% depending on the ratio of liver cells consisting of fats to the normal liver cells. Necrosis was assessed by
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the number of focus in mm2 and inflammation by the number of inflammatory cells in mm2 [21]. 2.4. Statistical analysis Data were supplied in mean ± standard deviation. For comparisons between groups, Kruskal–Wallis single-way variance analysis test was used, and for dual-comparison Mann–Whitney U-test was used. For statistical evaluations, the SPSS package program, version 11.0 was used.
Graphic 1. The level of plasma total antioxidant capacity in study groups.
3. Results AST, ALT and GGT levels in the CCl4 group were significantly higher than those in the control group, while the levels in the CCl4 + IC group were significantly lower than those in the CCl4 group (p < 0.05). AST, ALT and GGT levels were higher in the group that was administered coffee only than in the control group, but not significantly (p > 0.05). Levels of plasma malondialdehyde, a lipid peroxidation product, were found to increase in the CCl4 group, relative to the control group, and decrease in the CCl4 + IC group (p < 0.05). Levels of MDA in liver tissue increased significantly in the CCl4 group, in comparison to the control group (p < 0.001). tMDA levels of the CCl4 + IC group were found lower than those in the CCl4 group, but the difference was not significant (p > 0.05). TAOC levels in the CCl4 group were significantly lower than those in the control group (p < 0.01). Administration of coffee to this group resulted in a significant increase in
Graphic 2. The level of plasma SH in study groups.
TAOC levels (p < 0.05) (Graphic 1). Plasma SH levels in the CCl4 group were significantly lower than those in the control group (p < 0.05), but not different from those in other groups (p > 0.05) (Graphic 2). Ceruloplasmin levels were similar to SH levels (Graphic 3; Table 1). Histological evaluation showed that the group that was administered IC was not significantly different from the control group in terms of steatosis, inflammation and necrosis (p > 0.05), but CCl4 and CCl4 + IC groups were (p < 0.001). When the group administered CCl4 and coffee was compared
Table 1 Results of biochemical parameters in study groups Control (Group 1)
CCl4 (Group 2)
CCl4 + IC (Group 3)
IC (Group 4)
p
AST (U/L)
163.70 ± 28.60a
1346.90 ± 301.01b
1039.30 ± 246.99
221.20 ± 67.77c
a: p < 0.001, Groups 1-3 and 1-2 b: p < 0.05, Group 2-3 c: p < 0.001, Groups 2-4 and 3-4
ALT (U/L)
63.90 ± 19.46a
1067.40 ± 184.07b
900.90 ± 161.59
91.70 ± 15.75c
a: p < 0.001, Groups 1-3 and 1-2 b: p < 0.05, Group 2-3 c: p < 0.001, Groups 2-4 and 3-4
GGT (U/L)
1.91 ± 0.51a
24.20 ± 15.06b
6.70 ± 3.15
3.24 ± 0.97c
a: p < 0.001, Groups 1-3 and 1-2 b: p < 0.05, Group 2-3 c: p < 0.001, Groups 2-4 and 3-4
Plasma MDA (nmol/mL)
1.49 ± 0.65a
3.70 ± 0.69c
2.37 ± 0.72b
1.57 ± 0.41d
a: p < 0.001, Group 1-2 b: p < 0.05, Group 1-3 c: p < 0.05, Groups 2-3 and 2-4 d: p < 0.01, Group 3-4
98.32 ± 11.50b
53.56 ± 5.86c
a: p < 0.001, Groups 1-2 and 1-3 b: p < 0.001, Group 2-4 c: p < 0.05, Groups 2-3 and 2-4
0.88 ± 0.11b
0.97 ± 0.10
a: p < 0.01, Group 1-2 b: p < 0.05, Group 1-3 c: p < 0.05, Groups 2-3 and 2-4
Liver tissue MDA (nmol/g tissue)
TAOC (mmol/eq. L)
SH (mmol/L) Ceruloplasmin (U/L)
50.17 ± 11.29a
1.02 ± 0.15a
0.31 ± 0.01 261.00 ± 45.86
110.18 ± 6.51
0.76 ± 0.06c
0.26 ± 0.02a 198.80 ± 20.62a
0.28 ± 0.01 228.60 ± 40.46
0.28 ± 0.01 230.40 ± 45.62
a: p < 0.05, Group 1-2 a: p < 0.05, Group 1-2
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Table 2 Results of histopathological evaluation in study groups Control (Group 1)
CCl4 (Group 2)
Steatosis (score: 0–4)
1.00 ± 00a
3.00 ± 47
Inflamation (cells/mm2 )
0.12 ± 09a
38.80 ± 16.98c
Necrosis (foci/mm2 )
0.07 ± 06a
1.07 ± 0.48c
CCl4 + IC (Group 3)
IC (Group 4)
p
1.10 ± 31b
a: p < 0.001, Groups 1-2 and 1-3 b: p < 0.001, Groups 4-2 and 4-3
26.10 ± 12.24
0.13 ± 05b
a: p < 0.001, Groups 1-2 and 1-3 b: p < 0.001, Groups 4-2 and 4-3 c: P < 0.05, Group 2-3
0.63 ± 0.24 b
0.09 ± 0.03
a: p < 0.001, Groups 2-1 and 2-4 b: p < 0.001, Groups 3-1 and 3-4 c: p < 0.05, Group 2-3
3.00 ± 47
Graphic 3. The level of plasma ceruloplasmin in study groups.
Fig. 2. Inflammation and steatosis in acute liver injury induced by CCl4 plus instant coffee (hematoxylin–eosine ×200).
Fig. 1. Inflammation and steatosis in acute liver injury induced by CCl4 (hematoxylin–eosine ×200).
with that was administered CCl4 only, a significant decrease was found in inflammation and necrosis values (p < 0.05). Instant coffee was not seen to have a steatosis-reducing effect (p > 0.05) (Figs. 1 and 2; Table 2).
4. Discussion CCl4 , a chemical with hepatotoxic effect, has been used as a model in many studies to induce liver injury [8,9,22]. Acute administration of this substance to experimental animals causes centrilobular necrosis and steatosis in the liver. Carbon tetrachloride (CCl4 ) is metabolized by mitochondrial
monooxigenase system (P450 2E1). During this process, an unstable trichloromethyl (CCl3 ) free radical is generated, which immediately transforms into trichloromethyl peroxide (Cl3 COO− ) [23,24]. These free radicals cause peroxidation in the fatty acids of the phospholipids constituting the cell membranes, which result in fragmentation of the lipid peroxide radicals, lipid hydroperoxides and other products, each acting like an active oxidizing agent. Consequently, the structure of the cell membrane and intracellular organelle membrane is completely deteriorated, and the structural damage expands. In chronic form, fibrosis and cirrhosis develop [25]. Lipid peroxidation has a vital role in the CCl4 -induced liver injury [26,27]. Although the effect of coffee on liver has been investigated in many studies, we have not found one which evaluates biochemical data together with liver histopathology. In our study it was observed that pMDA level and liver tMDA level in the CCl4 group increased significantly (p < 0.001) when compared to the levels in the control group and IC group, whereas pMDA levels significantly decreased in the group that was administered CCl4 and IC (p < 0.05). Instant coffee that is administered in addition to CCl4 was seen to reduce the inflammation caused by CCl4 . Inflammation plays an important role in liver injury associated with toxic substances and drugs. The increase observed in MDA levels, a lipid peroxidation marker, of rats administered CCl4 when membranes
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are affected by the events resulting from the activation of some mediators of pro-fibrogenic characteristics activated by hydrogen peroxide and similar structures that increase due to free oxygen radicals, decreased as a result of administration of coffee together with CCl4 [28,29]. There was no significant difference with regard to steatosis, inflammation and necrosis between the control group and the group that was administered IC only (p > 0.05). However, both CCl4 and CCl4 + IC groups were observed to have macro- and micro-vesicular intense steatosis. We think that hypercholesterolemia associated with cafestol in the coffee has a part in steatosis. There was not any significant difference between steatosis in the CCl4 + IC group and that in the CCl4 group. However, inflammation and necrosis in the CCl4 + IC group was less than those in the CCl4 group, and the difference between the values was significant (p < 0.05). The fact that instant coffee reduces inflammation and necrosis caused by CCl4 in the liver, while lessening the increase in liver enzymes indicates that it plays a role in the formation of hepatocyte injury. Many antioxidants have been reported to be effective in preventing the accumulation of transaminase and lipid peroxidation products in the plasma in case of liver injury induced by CCl4 [8,9,22,30]. Protective effects of various natural products in CCl4 hepatotoxicity have been reported [31]. Ustundag et al. [32] have reported that soy isoflavones are effective in liver injury caused by experimentally induced CCl4 , reduce the lipid peroxidation products and stimulate paraoxonase enzyme, which has an antioxidant characteristic. Abraham and Sing [33] have noted that coffee administration by gavage resulted in an increase in SH and glutathione S-transferase activity in the liver. In addition, Lam et al. [10] have stated in a study including rats and mice that kahweol increased the sulphydryl levels in the liver and that kahweol and cafestol together increased glutathione S-transferase activity. In our study, SH levels increased in the CCl4 + IC group, relative to the CCl4 group. This effect is associated with kahweol’s increasing glutathione S-transferase activity [10]. Ceruloplasmin levels also exhibited an increase similar to that of SH. When levels of TAOC, the best marker of the antioxidant state in the plasma, were considered besides such antioxidants as SH and ceruloplasmin, it was seen that the antioxidant capacity, which decreased with CCl4 administration, significantly increased in the group that was administered coffee. This increase indicates that coffee can be effective in activating antioxidant structures through mechanisms other than glutathione S-transferase, as it has been reported in several studies that coffee beans destroy free radicals by forming metal chelates [32,34]. Chelating capacity of highmolecular weight melanoidin and polyphenolic polymers is higher than their hydroxyl radical scavenging components. Besides, hydroxyl radical scavenging characteristics of highmolecular weight melanoidin-like compounds are stronger than those of low-molecular weight phenolic compounds [35].
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Boekschoten et al. [36] did not find a significant increase in the liver enzymes of 18 healthy volunteers who drank unfiltered coffee for 16 days. In our study, there was an insignificant increase in AST, ALT and GGT levels of the rats which were administered coffee only, relative to the control group. However, liver enzymes that showed a marked increase in the CCl4 group decreased significantly after coffee administration. It was seen in this study that besides lessening the increase of liver enzymes in acute liver injury induced by CCl4 , particularly long-term consumption of coffee prevented the process leading to liver injury by reducing AST, ALT and GGT levels in the liver [2,7,37,38]. Corrao et al. [6] have reported that drinking coffee reduces the risk of alcoholic and non-alcoholic liver cirrhosis. In addition, Gelatti et al. [3] have noted that long-term coffee consumption lessened the risk of hepatocellular carcinoma. The coffee dose that decreases the risk of development of liver cirrhosis and hepatocellular carcinoma has been reported as to be four or more cups/day. It has been shown that preventive role of coffee has been decreased in lower doses [3,39,40]. Coffee causes coronary heart diseases by increasing plasma total homocysteine and cholesterol levels [41–43]. Steatosis that increased in the CCl4 -administered group remained unchanged in the coffee added group. This effect of instant coffee was similar to the hypercholesterolemia causing effect of boiled coffee, as it has been shown in several studies that boiled coffee particularly triggered hypercholesterolemia [41–45]. Cholesterol-increasing effect of coffee has been attributed to cafestol, but instant coffee contains less cafestol than boiled coffee [14,46]. In conclusion, it was observed that instant coffee prevented lipid peroxidation and exercised a protective effect by increasing total antioxidant capacity, but did not have a significant influence on steatosis in acute liver injury induced by CCl4 . However, further studies including histopathological data are required to clarify the effects of various doses and durations of instant coffee consumption on liver injury.
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Does instant coffee prevent acute liver injury induced by carbon tetrachloride (CCl4)? Ibrahim Hanifi Ozercan a,∗ , Adile Ferda Dagli a , Bilal Ustundag b , Mehmet Resat Ozercan a , Ibrahim Halil Bahcecioglu c , Hakim C ¸ elik d , Mehmet Yalniz c , c Orhan Kursat Poyrazoglu , Huseyin Ataseven c a
Fırat University, Faculty of Medicine, Department of Pathology, 23119 Elazig, Turkey Fırat University, Faculty of Medicine, Department of Biochemistry, 23119 Elazig, Turkey c Fırat University, Faculty of Medicine, Division of Gastroenterology, 23119 Elazig, Turkey d Harran University, Faculty of Medicine, Department of Biochemistry, Sanliurfa, Turkey b
Received 27 February 2006; received in revised form 6 April 2006; accepted 8 April 2006 Available online 5 June 2006
Abstract Objective: To investigate the protective effect of instant coffee (IC) on acute liver injury induced by CCl4 . Methods: The study included 32 rats which were allocated to four groups: control (n: 8), CCl4 (n: 8), CCl4 + IC (n: 8) and IC (n: 8). Malondialdehyde, which is a lipid peroxidation product, and levels of antioxidant capacity were measured and histopathological data were compared. Results: It was seen in the study that lipid peroxidation products that increased in the plasma and liver tissue of the CCl4 group decreased by IC administration. There was an increase in the measured antioxidant parameters, which were total antioxidant capacity (TAOC), sulphydryl (SH) and ceruloplasmin levels. Histopathologically, it was found that inflammation and necrosis which increased in the group administered CCl4 decreased significantly with IC administration, but there steatosis did not change. Conclusions: It was seen that IC had a protective role in acute liver injury induced by CCl4 , but did not affect steatosis. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Instant coffee; Acute liver injury; Lipid peroxidation; Antioxidant
1. Introduction Drinking coffee is among the most common habits in the world [1]. Many studies have been conducted to determine the effect of widely consumed coffee on human health. The studies have demonstrated that although it causes hypercholesterolemia and coronary diseases, coffee prevents liver injury and the resulting cirrhosis, besides reducing hepatocellular carcinoma formation in the long run [2–6]. It has been reported that liver injury that results from an increase in free oxygen radicals due to various etiological ∗ Corresponding author. Tel.: +90 424 233 3555/2186; fax: +90 424 238 8096. E-mail address: [email protected] (I.H. Ozercan).
1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.04.004
causes leads to liver cirrhosis ([6–8]; Chen, 2005). There are many studies which have tried different substances as antioxidants for the purpose of protection in liver injury and the process leading to cirrhosis [9]. Studies on coffee have focused on its antioxidant activity. It has been reported that substances like cafestol and kahweol that are found in coffee can affect glutathione and sulphydryl groups through glutathione S-transferase enzyme [10]. Antioxidant molecules exercise their effect by inhibiting or preventing harmful reactions. Antioxidant defense system can be evaluated by measuring various antioxidant structures like antioxidant enzymes, vitamins, albumin, bilirubin, etc. in the serum or plasma. However, such measurements are not only time-consuming and expensive, but also require complicated techniques. Therefore, recent preference has been
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towards the measurement of the total antioxidant capacity, as it is more practical, less expensive and can demonstrate all the antioxidant structures in the organism [11,12]. Many studies about coffee have measured different antioxidants and their activities [8,10]. However, phenolic compounds that exercise the antioxidant effect of coffee have not been thoroughly clarified [13]. Besides, it has been reported that the agents in the coffee change according to the way coffee is prepared [14]. Therefore, the present study aims to investigate the effect of instant coffee on liver injury induced by CCl4 through the relation between TAOC, which is a good indicator of all the antioxidants in the organism, and histopathological changes.
2. Materials and methods 2.1. Experimental animals The study included 32 male Sprague–Dawley rats, which were divided into four equal groups. They were kept in specially manufactured cages in a room, which was sunny for 12 h. The study was carried out in strict conformance with the standard ethical rules about experimental animal studies. For feeding, a special rat food provided by the factory in Elazig was used. Water was supplied by special bottles with a drip pin placed inside the cages. Group 1, which is the control (C) group, was given tap water and Group 2 (CCl4 ) was administered CCl4 to induce acute intoxication, together with tap water. Group 3 (CCl4 + IC) was administered IC and CCl4 , and Group 4 (IC) was given only IC. Two grams of IC was dissolved in 170 mL of boiled water and left to cool in room temperature [14]. IC administration was started a day before CCl4 administration and the coffee was mixed into the water of rats every other day, gradually increasing the concentration from 25% at the beginning to 100% at the end. IC prepared specially was given at dose of 0.146 g/day for each rat in first 2 days of the experiment. The concentration of the IC was increased gradually as following: 0.234 g/day per rat in days 3 and 4, 0.263 g/day per rat in days 5 and 6 and 0.351 g/day per rat in days 7 and 8. CCl4 was injected through intraperitoneal route at a dose of 0.15 mL/100 g (suspended with olive oil at a rate of 1:1) every other day, three times starting from the second day until the end of the study. Rats which drank pure IC on the seventh day were decapitated after 24 h and blood and tissue samples were collected. 2.2. Biochemical analyses The plasma obtained by centrifuging the blood from rats was kept under −20◦ until the analysis. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (␥-GT), levels were determined by Olympus Au 600 autoanalyzer (Olympus Corp., Japan).
Liver tissue and plasma malondialdehyde levels: the plasma MDA (pMDA) levels were assessed by thiobarbituric acid method modified by Satoh [15] and Yagi [16]. The results were in nmol/mL. And the liver tissue MDA (tMDA) levels were assessed by Ohkawa method [17]. The results were in nmol/g tissue. 2.2.1. Measurement of the total antioxidant status of plasma The total antioxidant status of the plasma was measured using a novel automated colourimetric measurement method for the total antioxidant response developed by Erel [11,12]. In this method the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction, and reacts with the colourless substrate. O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in colour. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the colour change thereby providing an effective measure of the total antioxidant capacity of the plasma. The assay results are expressed as mmol Trolox eq./L, and the precision of this assay is excellent, being lower 3%. 2.2.2. Analyses of sulfhydryl groups Measurement of total free sulfhydryl groups of serum samples was assayed according to the method of Elman [18]. Briefly, 1 mL of buffer containing 0.1 M Tris, 10 mM EDTA, pH 8.2, and 50 all serum was added to cuvettes, followed by 50 all 10 mM dithiobisnitrobenzoicacid (DTNB) in methanol. Blanks were run for each sample as a test, but there was no DTNB in the methanol. Following incubation for 15 min at room temperature, sample absorbance was read 412 nm on a Cecil 3000 spectrophotometer. Sample and reagent blanks were subtracted. The concentration of sulfhydryl groups was calculated using reduced glutathion as free sulfhydryl group standard the result was expressed as millimolars. 2.2.3. Seruloplazmin analyses Serum ceruloplasmin levels were assessed by measuring its oxidase activity using p-phenylenediamine as substrate [19,20]. 2.3. Histopathological investigation The liver tissue specimens were buried into paraffin blocks undergoing a routine follow-up procedure after they were fixed at 10% formaldehyde. The 4 m sections were stained with hematoxylin–eosin (HE). The preparations were examined by Olympus BX-50 light microscopy. An experienced pathologist evaluated all liver biopsy specimens. The steatosis was rated as follows: lower than +: 25%, between ++: 25–50% and +++: 50–75% and higher than ++++: 75% depending on the ratio of liver cells consisting of fats to the normal liver cells. Necrosis was assessed by
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the number of focus in mm2 and inflammation by the number of inflammatory cells in mm2 [21]. 2.4. Statistical analysis Data were supplied in mean ± standard deviation. For comparisons between groups, Kruskal–Wallis single-way variance analysis test was used, and for dual-comparison Mann–Whitney U-test was used. For statistical evaluations, the SPSS package program, version 11.0 was used.
Graphic 1. The level of plasma total antioxidant capacity in study groups.
3. Results AST, ALT and GGT levels in the CCl4 group were significantly higher than those in the control group, while the levels in the CCl4 + IC group were significantly lower than those in the CCl4 group (p < 0.05). AST, ALT and GGT levels were higher in the group that was administered coffee only than in the control group, but not significantly (p > 0.05). Levels of plasma malondialdehyde, a lipid peroxidation product, were found to increase in the CCl4 group, relative to the control group, and decrease in the CCl4 + IC group (p < 0.05). Levels of MDA in liver tissue increased significantly in the CCl4 group, in comparison to the control group (p < 0.001). tMDA levels of the CCl4 + IC group were found lower than those in the CCl4 group, but the difference was not significant (p > 0.05). TAOC levels in the CCl4 group were significantly lower than those in the control group (p < 0.01). Administration of coffee to this group resulted in a significant increase in
Graphic 2. The level of plasma SH in study groups.
TAOC levels (p < 0.05) (Graphic 1). Plasma SH levels in the CCl4 group were significantly lower than those in the control group (p < 0.05), but not different from those in other groups (p > 0.05) (Graphic 2). Ceruloplasmin levels were similar to SH levels (Graphic 3; Table 1). Histological evaluation showed that the group that was administered IC was not significantly different from the control group in terms of steatosis, inflammation and necrosis (p > 0.05), but CCl4 and CCl4 + IC groups were (p < 0.001). When the group administered CCl4 and coffee was compared
Table 1 Results of biochemical parameters in study groups Control (Group 1)
CCl4 (Group 2)
CCl4 + IC (Group 3)
IC (Group 4)
p
AST (U/L)
163.70 ± 28.60a
1346.90 ± 301.01b
1039.30 ± 246.99
221.20 ± 67.77c
a: p < 0.001, Groups 1-3 and 1-2 b: p < 0.05, Group 2-3 c: p < 0.001, Groups 2-4 and 3-4
ALT (U/L)
63.90 ± 19.46a
1067.40 ± 184.07b
900.90 ± 161.59
91.70 ± 15.75c
a: p < 0.001, Groups 1-3 and 1-2 b: p < 0.05, Group 2-3 c: p < 0.001, Groups 2-4 and 3-4
GGT (U/L)
1.91 ± 0.51a
24.20 ± 15.06b
6.70 ± 3.15
3.24 ± 0.97c
a: p < 0.001, Groups 1-3 and 1-2 b: p < 0.05, Group 2-3 c: p < 0.001, Groups 2-4 and 3-4
Plasma MDA (nmol/mL)
1.49 ± 0.65a
3.70 ± 0.69c
2.37 ± 0.72b
1.57 ± 0.41d
a: p < 0.001, Group 1-2 b: p < 0.05, Group 1-3 c: p < 0.05, Groups 2-3 and 2-4 d: p < 0.01, Group 3-4
98.32 ± 11.50b
53.56 ± 5.86c
a: p < 0.001, Groups 1-2 and 1-3 b: p < 0.001, Group 2-4 c: p < 0.05, Groups 2-3 and 2-4
0.88 ± 0.11b
0.97 ± 0.10
a: p < 0.01, Group 1-2 b: p < 0.05, Group 1-3 c: p < 0.05, Groups 2-3 and 2-4
Liver tissue MDA (nmol/g tissue)
TAOC (mmol/eq. L)
SH (mmol/L) Ceruloplasmin (U/L)
50.17 ± 11.29a
1.02 ± 0.15a
0.31 ± 0.01 261.00 ± 45.86
110.18 ± 6.51
0.76 ± 0.06c
0.26 ± 0.02a 198.80 ± 20.62a
0.28 ± 0.01 228.60 ± 40.46
0.28 ± 0.01 230.40 ± 45.62
a: p < 0.05, Group 1-2 a: p < 0.05, Group 1-2
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Table 2 Results of histopathological evaluation in study groups Control (Group 1)
CCl4 (Group 2)
Steatosis (score: 0–4)
1.00 ± 00a
3.00 ± 47
Inflamation (cells/mm2 )
0.12 ± 09a
38.80 ± 16.98c
Necrosis (foci/mm2 )
0.07 ± 06a
1.07 ± 0.48c
CCl4 + IC (Group 3)
IC (Group 4)
p
1.10 ± 31b
a: p < 0.001, Groups 1-2 and 1-3 b: p < 0.001, Groups 4-2 and 4-3
26.10 ± 12.24
0.13 ± 05b
a: p < 0.001, Groups 1-2 and 1-3 b: p < 0.001, Groups 4-2 and 4-3 c: P < 0.05, Group 2-3
0.63 ± 0.24 b
0.09 ± 0.03
a: p < 0.001, Groups 2-1 and 2-4 b: p < 0.001, Groups 3-1 and 3-4 c: p < 0.05, Group 2-3
3.00 ± 47
Graphic 3. The level of plasma ceruloplasmin in study groups.
Fig. 2. Inflammation and steatosis in acute liver injury induced by CCl4 plus instant coffee (hematoxylin–eosine ×200).
Fig. 1. Inflammation and steatosis in acute liver injury induced by CCl4 (hematoxylin–eosine ×200).
with that was administered CCl4 only, a significant decrease was found in inflammation and necrosis values (p < 0.05). Instant coffee was not seen to have a steatosis-reducing effect (p > 0.05) (Figs. 1 and 2; Table 2).
4. Discussion CCl4 , a chemical with hepatotoxic effect, has been used as a model in many studies to induce liver injury [8,9,22]. Acute administration of this substance to experimental animals causes centrilobular necrosis and steatosis in the liver. Carbon tetrachloride (CCl4 ) is metabolized by mitochondrial
monooxigenase system (P450 2E1). During this process, an unstable trichloromethyl (CCl3 ) free radical is generated, which immediately transforms into trichloromethyl peroxide (Cl3 COO− ) [23,24]. These free radicals cause peroxidation in the fatty acids of the phospholipids constituting the cell membranes, which result in fragmentation of the lipid peroxide radicals, lipid hydroperoxides and other products, each acting like an active oxidizing agent. Consequently, the structure of the cell membrane and intracellular organelle membrane is completely deteriorated, and the structural damage expands. In chronic form, fibrosis and cirrhosis develop [25]. Lipid peroxidation has a vital role in the CCl4 -induced liver injury [26,27]. Although the effect of coffee on liver has been investigated in many studies, we have not found one which evaluates biochemical data together with liver histopathology. In our study it was observed that pMDA level and liver tMDA level in the CCl4 group increased significantly (p < 0.001) when compared to the levels in the control group and IC group, whereas pMDA levels significantly decreased in the group that was administered CCl4 and IC (p < 0.05). Instant coffee that is administered in addition to CCl4 was seen to reduce the inflammation caused by CCl4 . Inflammation plays an important role in liver injury associated with toxic substances and drugs. The increase observed in MDA levels, a lipid peroxidation marker, of rats administered CCl4 when membranes
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are affected by the events resulting from the activation of some mediators of pro-fibrogenic characteristics activated by hydrogen peroxide and similar structures that increase due to free oxygen radicals, decreased as a result of administration of coffee together with CCl4 [28,29]. There was no significant difference with regard to steatosis, inflammation and necrosis between the control group and the group that was administered IC only (p > 0.05). However, both CCl4 and CCl4 + IC groups were observed to have macro- and micro-vesicular intense steatosis. We think that hypercholesterolemia associated with cafestol in the coffee has a part in steatosis. There was not any significant difference between steatosis in the CCl4 + IC group and that in the CCl4 group. However, inflammation and necrosis in the CCl4 + IC group was less than those in the CCl4 group, and the difference between the values was significant (p < 0.05). The fact that instant coffee reduces inflammation and necrosis caused by CCl4 in the liver, while lessening the increase in liver enzymes indicates that it plays a role in the formation of hepatocyte injury. Many antioxidants have been reported to be effective in preventing the accumulation of transaminase and lipid peroxidation products in the plasma in case of liver injury induced by CCl4 [8,9,22,30]. Protective effects of various natural products in CCl4 hepatotoxicity have been reported [31]. Ustundag et al. [32] have reported that soy isoflavones are effective in liver injury caused by experimentally induced CCl4 , reduce the lipid peroxidation products and stimulate paraoxonase enzyme, which has an antioxidant characteristic. Abraham and Sing [33] have noted that coffee administration by gavage resulted in an increase in SH and glutathione S-transferase activity in the liver. In addition, Lam et al. [10] have stated in a study including rats and mice that kahweol increased the sulphydryl levels in the liver and that kahweol and cafestol together increased glutathione S-transferase activity. In our study, SH levels increased in the CCl4 + IC group, relative to the CCl4 group. This effect is associated with kahweol’s increasing glutathione S-transferase activity [10]. Ceruloplasmin levels also exhibited an increase similar to that of SH. When levels of TAOC, the best marker of the antioxidant state in the plasma, were considered besides such antioxidants as SH and ceruloplasmin, it was seen that the antioxidant capacity, which decreased with CCl4 administration, significantly increased in the group that was administered coffee. This increase indicates that coffee can be effective in activating antioxidant structures through mechanisms other than glutathione S-transferase, as it has been reported in several studies that coffee beans destroy free radicals by forming metal chelates [32,34]. Chelating capacity of highmolecular weight melanoidin and polyphenolic polymers is higher than their hydroxyl radical scavenging components. Besides, hydroxyl radical scavenging characteristics of highmolecular weight melanoidin-like compounds are stronger than those of low-molecular weight phenolic compounds [35].
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Boekschoten et al. [36] did not find a significant increase in the liver enzymes of 18 healthy volunteers who drank unfiltered coffee for 16 days. In our study, there was an insignificant increase in AST, ALT and GGT levels of the rats which were administered coffee only, relative to the control group. However, liver enzymes that showed a marked increase in the CCl4 group decreased significantly after coffee administration. It was seen in this study that besides lessening the increase of liver enzymes in acute liver injury induced by CCl4 , particularly long-term consumption of coffee prevented the process leading to liver injury by reducing AST, ALT and GGT levels in the liver [2,7,37,38]. Corrao et al. [6] have reported that drinking coffee reduces the risk of alcoholic and non-alcoholic liver cirrhosis. In addition, Gelatti et al. [3] have noted that long-term coffee consumption lessened the risk of hepatocellular carcinoma. The coffee dose that decreases the risk of development of liver cirrhosis and hepatocellular carcinoma has been reported as to be four or more cups/day. It has been shown that preventive role of coffee has been decreased in lower doses [3,39,40]. Coffee causes coronary heart diseases by increasing plasma total homocysteine and cholesterol levels [41–43]. Steatosis that increased in the CCl4 -administered group remained unchanged in the coffee added group. This effect of instant coffee was similar to the hypercholesterolemia causing effect of boiled coffee, as it has been shown in several studies that boiled coffee particularly triggered hypercholesterolemia [41–45]. Cholesterol-increasing effect of coffee has been attributed to cafestol, but instant coffee contains less cafestol than boiled coffee [14,46]. In conclusion, it was observed that instant coffee prevented lipid peroxidation and exercised a protective effect by increasing total antioxidant capacity, but did not have a significant influence on steatosis in acute liver injury induced by CCl4 . However, further studies including histopathological data are required to clarify the effects of various doses and durations of instant coffee consumption on liver injury.
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