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Hepatoprotective effect of l -carnitine against acute acetaminophen toxicity in mice
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Experimental and Toxicologic Pathology 59 (2007) 121–128 www.elsevier.de/etp
Hepatoprotective effect of L-carnitine against acute acetaminophen toxicity in mice Kursad Yapara, Asim Karta,, Mahmut Karapehlivanb, Onur Atakisib, Recai Tuncac, Serpil Erginsoyc, Mehmet Citild a
Department of Pharmacology and Toxicology, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey Department of Biochemistry, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey c Department of Pathology, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey d Department of Internal Medicine, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey b
Received 1 February 2007; accepted 26 February 2007
Abstract L-carnitine is a cofactor in the transfer of long-chain fatty acid allowing the b-oxidation of fatty acid in the mitochondria. It is also a known antioxidant with protective effects against lipid peroxidation. In this study, hepatoprotective effect of L-carnitine was investigated against acetaminophen (AA)-induced liver toxicity where mitochondrial dysfunction and oxidative stress are thought to be involved in AA hepatotoxicity. Sixty-four Balb/C mice were divided into eight groups. Mice were dosed with single-AA injection (500 mg/kg via the intra peritoneal route) with or without L-carnitine (500 mg/kg for 5 days starting 5 days before AA injection via intra peritoneal route) and sampled at 4, 8 and 24 h following AA injection. AA increased serum AST, ALT, total sialic acid (TSA) and MDA as well as tissue TSA and MDA levels significantly with the highest increase observed at 4 h, but there was a decrease in blood and tissue GSH level. Administration of L-carnitine significantly reduced AA-induced elevations in AST, ALT, TSA and MDA concentrations and increased GSH levels at all sampling points. AA also induced necrosis, hyperemia, sinusoidal congestion and hemorrhage with time-dependent increase in severity, but the degree of necrosis and histopathologic alterations were most severe at 24 h following AA administration. However, the degree of pathologic alterations was less severe with simultaneous L-carnitine application. These results suggest that AA results in oxidative damage in the liver with an acute effect. L-carnitine also has a prominent protective effect against AA toxicity and may be of therapeutic value in the treatment of AA-induced hepatotoxicity. r 2007 Elsevier GmbH. All rights reserved.
Keywords: L-carnitine; Acetaminophen; Sialic acid; Hepatoprotection; Oxidative stress
Introduction Corresponding author. Tel.: +90 474 242 6800; fax: +90 474 242 6853. E-mail address: [email protected] (A. Kart).
0940-2993/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2007.02.009
Acetaminophen (AA) is a widely used analgesic and antipyretic, which is safely employed in the therapeutic range in man and animals (Hjelle, 1986; Dargan and Jones, 2002). AA is readily absorbed by the
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gastrointestinal tract, and it is metabolized by the microsomal enzyme system in the liver. While 80–85% of AA’s biotransformation occurs via conjugation of glucuronide and sulfate by the transferase enzymes, 10–15% of AA is oxidized to the reactive oxygen species (ROS) through P-450-dependent mixed-function oxidases (Black, 1980; Dahlin et al., 1984; Brown et al., 1992; Vermeulen et al., 1992). The toxic metabolite produced at the therapeutic doses is detoxified by glutathione to cysteine and mercaptouric acid to be subsequently excreted from the body. However, AA taken in overdose or long-term use at the therapeutic level may lead to the saturation of conjugation pathway leading to the depletion of glutathione and to the increased formation of toxic reactive metabolites. Free toxic metabolites are then covalently bound to the macro molecules of cells leading to cellular necrosis (Mitchell et al., 1973; Jollow et al., 1973; Pumford et al., 1997). The toxic metabolite responsible for inducing liver damage is N-acetylp-benzoquinone imine (NAPQI), which could bind to cellular proteins leading to centrilobular necrosis in the liver (Dahlin, 1984). Previous reports indicate that mitochondrial dysfunction could play a role in the mechanism of AA hepatotoxicity. Reactive metabolites of AA, NAPQI, result in mitochondrial dysfunction and superoxide formation with the resultant effect of peroxynitrite and tyrosine nitration. The events in this mechanism affect mitochondrial permeability transition leading to disturbances in the permeability of the inner mitochondrial membrane (Masubuchi et al., 2005). L-carnitine is a g-three methyl amino-b-hydroxyl fatty acid, which is an essential cofactor in mitochondrial respiration playing an important role in the transfer of long-chain fatty acids from cytosol to mitochondria. By combination with carnitine to form acylcarnitine, acyl groups could be transferred from cytosolic coenzyme A on the outer surface of the mitochondrion membrane, then to the inner surface by exchange with free carnitine using an antiport mechanism. The acyl groups are then transferred from carnitine to coenzyme A within the mitochondrion (Kelly, 1998). Carnitine is also associated with buffering of excess acyl-Co A, which is potentially toxic to the cells, and it was reported that L-Carnitine has a protective effect on lipid peroxidation by reducing the formation of hydrogen peroxide (Brass, 2000; Rani and Panneerselvam, 2002). L-carnitine could also improve antioxidant status in rats and showed free radical scavenging activity as well (Kalaiselvi and Panneerselvam, 1998; Rani and Panneerselvam, 2001). Sialic acids are a family of nine-carbon neuraminic acid derivatives that are found in terminal residues of oligosaccharide chains of mucins, glycoproteins and glycolipids of cell membranes (Sillanaukee et al., 1999; Wang and Brand-Miller, 2003). It is reported that the level of sialic acids is increased following inflammation and several disease conditions including chronic liver
diseases (Sillanaukee et al., 1999). This might be an indicator of the disruption of cell membranes due to lipid peroxidation along with increased MDA concentration. In this study, it is aimed to investigate the protective effects of L-carnitine on acute AA toxicity at three different time points (at 4, 8 and 24 h following acute AA dosing) by evaluating histopathology and biochemical alterations including malondialdehyde (MDA), total sialic acid (TSA) and glutathione (GSH) concentrations in the blood and liver tissue.
Material and Methods Sixty-four male Balb/c mice (12–14-week old, 25–30 g bw) were obtained from University of Kafkas, Animal Research Farm. An approval from the Institutional Ethics Committee was obtained before the experiment. The mice were acclimatized 1 week prior to the planned experiments and fed with standard pelleted diet ad libitum. The mice were housed in stainless-steel cages (26 15 50 cm in size) in a controlled environment with temperature range at 2272 1C, humidity 5075% and a 12-h light/dark cycle. The mice were divided into eight groups each consisting of eight animals as follows: group 1 (control) was injected with single saline (0.02 ml/g body weight, 0.9% NaCl; Baxter, Mediflex, Eczacibasi, Istanbul, Turkey) via the intraperitoneal (i.p.) route. Group 2 received 500 mg/kg of L-carnitine daily (CARNITENEs, injectable solution in 5 ml sterile ampoule containing 200 mg/ml L-carnitine, 0.014 ml 10% hydrochloric acid and injectable water, Santa Farma Ilac Sanayii A.S., Istanbul, Turkey) with the injected volume of 62.5–75 ml for 25–30 g mice using a Hamilton microinjector applied for 5 days. Mice in groups 3, 4 and 5 were injected with a single i.p. dose of AA (Sigma Chemical Company, St. Louis, MO, AA was applied in saline solution at a concentration of 25 mg/ml with the injected volume of 0.5–0.6 ml where the mixture was prepared at 70 1C, then cooled to 37 1C for administration) at a dose of 500 mg/kg. The animals were then decapitated at 4, 8 and 24 following the AA application. Mice in groups 6, 7 and 8 were administered with 500 mg/kg of L-carnitine daily (starting 5 days before AA injection via the i.p. route) and a single dose of 500 mg/kg of AA and decapitated at 4, 8 and 24 h following the AA injection, respectively. Blood samples were collected from the heart via cardiac puncture under light ether anesthesia for GSH, MDA, TSA, AST and ALT measurements before killing the animals at determined time intervals. Blood GSH was measured from the whole blood. For MDA, TSA, AST and ALT measurements, all tubes were centrifuged
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(at 1200 g at 4 1C) for 10 min to obtain the serum. The serum samples were kept at 25 1C until they were analyzed. Serum AST and ALP concentrations were determined using a commercially available kit and using an autoanalyzer (Olympus Chemistry Analyzer AU 640, Type: 640-03, Japan). In addition, tissue samples from the liver were collected for tissue GSH, MDA and TSA concentrations. Liver tissues were immediately removed and washed with 0.15 M KCl (at 4 1C). The tissues were then homogenized in ice cold 0.15 M KCl by a homogenizator (Ultra Turrax Type T25-B, IKA Labartechnie, Germany) at 1600 rpm for 3 min. The homogenates were centrifuged at 5000g at 4 1C for 1 h. The supernatants were stored at 40 1C until they were analyzed. TSA was measured colorimetrically using a spectrophotometer (UV-1201, Shimadzu, Japan) by the method of Sydow (1985). For GSH and MDA concentrations, analyses were carried out by the method of Beutler et al. (1963) and Yoshoiko et al. (1979), respectively. For histopathological examinations, samples of liver tissue were taken from both anterior and posterior parts of the lobus intermedius at four different regions. The samples were then fixed in 10% neutral buffered formalin, embedded in paraffin wax and four sections at 4–6 mm were taken from each tissue sample and stained with haematoxylin–eosin (H&E). The percentage of the total necrotic area was assessed semiquantitatively under a light microscope with an ocular grid and a 40 objective. A total of 10 high-power fields were randomly chosen. For the statistical analysis, differences between the groups were tested by analysis of variance (ANOVA) and Tukey test using SPSS for Windows version 10.0. Data were presented as mean7standard errors, and p values less than 0.05 were considered significant.
Table 1.
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Results Serum and blood biochemical parameters in the treatment groups are presented in Table 1. The AST levels in the AA-treated groups were significantly increased compared to that of the control groups at 4, 8 and 24 h time points. The enzyme levels showed a gradual decrease towards 8 and 24 h. However, the difference from the control was still significantly higher. Pretreatment with the antioxidant compounds decreased AST levels significantly when compared with the corresponding group treated with AA only at 4, 8 and 24 h. Similar results were obtained for ALT levels. This enzyme was increased by AA treatment at 4 and 8 h but not at 24 h compared to that of the control. The ALT levels were decreased significantly with L-carnitine treatment at 4 and 8 h compared to the control and the respective AA-only groups. Serum TSA levels were found to be increased in the AA-treated groups at 4, 8 and 24 h compared to that of the control with a gradual decrease toward a 24 h time point. Mice receiving L-carnitine plus AA at 4, 8 and 24 h had significantly lower serum TSA levels compared to the control and the corresponding AA-only groups. Blood GSH levels of AA-treated mice at 4, 8 and 24 h were found to be significantly lower than that of the control with a gradual amelioration toward a 24 h time point. On the other hand, blood GSH levels were found to be similar in the L-carnitine plus AA-treated group at 4, 8 and 24 h compared to that of the control. Tissue biochemical parameters are presented in the treatment groups in Table 2. Hepatic TSA was increased following AA significantly at 4 h; however, the TSA concentrations at 8 and 24 h were not different from that of the control. L-carnitine treatment in AA groups prevented the increase significantly seen at 4 h. Liver MDA levels were increased after treatment with
Biochemical alterations in the serum of treatment groups
Groups
AST (U/L)
ALT (U/L)
TSA (U/L)
MDA (mmol/L)
GSH (blood) (mg/L)
1 2 3 4 5 6 7 8
125.3877.8f 119.7576.8f 742.50732.0a 513.25726.2bc 603.38724.7b 442.00719.1cd 390.25723.2d 284.63718.5e
42.3873.7cd 36.2573.0d 101.8878.5a 62.0074.3bc 86.1377.8ab 51.7574.8cd 56.5074.9cd 43.6373.8cd
608.86724.0d 532.67715.5e 773.91719.9a 632.15713.4cd 725.85717.7ab 613.18714.5d 693.15715.4bc 595.52710.5de
14.2570.6d 12.0370.4e 16.6270.4abc 15.9170.5bcd 16.9570.4ab 15.4070.3bcd 17.9470.4a 14.9570.3cd
79.0671.1bc 86.9371.0a 74.3871.1de 75.2670.7cde 71.1070.9ef 76.9370.9bcd 66.8171.0f 80.0370.9b
(control) (L-car) (AA-4 h) (L-car+AA-4 h) (AA-8 h) (L-car+AA-8 h) (AA-24 h) (L-car+AA-24 h)
Values presented as mean7SE (n ¼ 8). Means denoted with different superscripts are within the same column are statistically significant. AST: aspartate amino transferase, ALT: alanine aminotransferase, TSA: total sialic acid, MDA: malondialdehyde, GSH: glutathione. Group 1: (control) received 0.9% saline (i.p.); Group 2: was injected with L-carnitine (500 mg/kg, for 5 days); Group 3: acetaminophen (AA) only (500 mg/kg) injection (killed 4 h after AA treatment); Group 4: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 4 h after AA treatment); Group 5: AA only (500 mg/kg) injection (killed 8 h after AA treatment); Group 6: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 8 h after AA treatment); Group 7: AA only (500 mg/kg) injection (killed 24 h after AA treatment); Group 8: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 24 h after AA treatment).
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Table 2. groups
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Biochemical alterations in the liver of treatment
Groups
TSA (mg/g)
MDA (mmol/g)
GSH (mg/g)
1 2 3 4 5 6 7 8
0.52770.02b 0.44370.02c 0.71670.03a 0.53270.03bc 0.63170.03ab 0.52470.02bc 0.61370.04ab 0.52070.03bc
0.32170.03c 0.29570.02c 0.48670.01a 0.35170.03bc 0.46270.02a 0.33670.02bc 0.41870.02ab 0.33270.02bc
0.24270.02bc 0.37470.02a 0.15470.01d 0.24470.02bc 0.16270.01d 0.24870.02bc 0.18870.01cd 0.25870.01b
(control) (L-car) (AA-4 h) (L-car+AA-4 h) (AA-8 h) (L-car+AA-8 h) (AA-24 h) (L-car+AA-24 h)
Values presented as mean7SE (n ¼ 8). Means denoted with different superscripts are within the same column are statistically significant. TSA: total sialic acid, MDA: malondialdehyde, GSH: glutathione. Group 1: (control) received 0.9% saline (i.p.) Group 2: was injected with L-carnitine (500 mg/kg, for 5 days); Group 3: acetaminophen (AA) only (500 mg/kg) injection (killed 4 h after AA treatment); Group 4: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 4 h after AA treatment); Group 5: AA only (500 mg/kg) injection (killed 8 h after AA treatment); Group 6: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 8 h after AA treatment); Group 7: AA only (500 mg/kg) injection (killed 24 h after AA treatment); Group 8: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 24 h after AA treatment).
Fig. 2. The appearance of liver 4 h following L-carnitine plus acetaminophen treatment. The hepatocytes appear eosinophilic and enlarged. Caryopicnosis is present in some hepatocytes (H&E, X260).
Fig. 3. Histologic appearance of liver 8 h after acetaminophen administration at light microscopy. Widespread vacuolation and caryopicnosis are present, and the remaining hepatocytes are eosinophilic and necrotic (H&E, X90).
Fig. 1. Histologic appearance of the liver 4 h after acetaminophen administration at light microscopy. Extensive eosinophilia in the hepatocytes with some necrosis. Note the widespread vacuolation and caryopicnosis (H&E, X180).
AA at 4, 8 and 24 h. Although the levels were relatively decreased at 8 and 24 h compared to 4 h, they were still significantly higher than that of the control. L-carnitine prevented lipid peroxidation significantly, and MDA levels were reduced to the control levels at 4, 8 and 24 h. The AA treatment decreased liver GSH levels significantly compared to that of the control at 4 and 8 h after treatment. However, liver GSH level at 24 h was similar to that of the control. Pretreatment
with L-carnitine prevented the reduction in GSH significantly. Histopathological examinations revealed that livers were normal in appearance with no change in the lobular architecture in the control and L-carnitine groups. In the AA group (at 4th h), hepatocytes were swollen and eosinophilic in appearance particularly around the central veins. Hepatocytes in the centrilobular regions showed cytoplasmic vacuolization. In addition, majority of the hepatocytes showed picnosis and karyorhexis with the presence of necrotic areas. Sinusoids were focally congested with extensive hemorrhages (Fig. 1). In the AA plus L-carnitine group (at 4th h), hepatocytes surrounding the central veins were swollen and eosinophilic in appearance, and sinusoids
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Fig. 4. The appearance of liver 8 h following L-carnitine plus acetaminophen treatment. Hepatocytes appear eosinophilic with widespread caryopicnosis. There are some karyopicnosis and vacuolations (H&E, X180).
Fig. 5. Appearance of the liver 24 h following acetaminophen administration. Extensive necrosis with some vacuolations and karyopicnosis (H&E, X180).
were congested with no sign of hemorrhage (Fig. 2). In the AA group (at 8th h), confluent areas of necrosis and extensive hemorrhages were observed around all centrilobular regions. Nuclei of these hepatocytes were in the advanced stage of karyolysis or were completely absent (Fig. 3). In the AA plus L-carnitine group (at 8th h), scattered cells in the centrilobular regions had cytoplasmic vacuoles and increased eosinophilia. Nuclei of these hepatocytes were picnotic with very limited necrosis and hemorrhages (Fig. 4). In the AA group (at 24th h), massive confluent areas of necrosis were evident with considerable hemorrhage (Fig. 5). In the AA plus L-carnitine group (at 24th h), changes were similar to the AA plus L-carnitine group (at 8th h) (Fig. 6). Quantification of necrotic areas in the liver of AAtreated mice revealed that there was a progressive
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Fig. 6. Appearance of the liver 24 h following L-carnitine plus acetaminophen treatment. Hepatocytes appear eosinophilic, and there are widespread karyopicnosis (H&E, X180).
Fig. 7. Quantification of necrosis in the liver of the treatment groups with respect to time following last drug application. AA: acetaminophen, AA+L-car: acetaminophen plus L-carnitine.
increase from 4377% at 4 h and 62.75711 at 8 h up to 82711% at 24 h. However, the increase in necrotic areas in the mice applied AA and L-carnitine was from 8.2575% at 4 h and 2776 at 8 h up to 3278% at 24 h (Fig. 7). Statistical analysis revealed a statistically significant difference at the level of Po0.05 for necrotic areas between the only AA and AA plus L-carnitine applied groups at 4, 8 and 24 h time intervals.
Discussion Previous studies showed that several factors could be involved in the mechanism and pathophysiology of AA hepatotoxicty at the cellular level. Role of oxidative stress was reported to be one of the important factors in
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the development of hepatic cell injury (Knight et al., 2001). Lipid peroxidation was suggested to be closely related to AA-induced tissue damage, and MDA is a good indicator of the degree of lipid peroxidation (Sener et al., 2006). In this model of AA-induced hepatic injury, we observed a significant increase in the MDA content of the liver tissue at 4, 8 and 24 h. Although the levels were relatively decreased at 8 and 24 h compared to 4 h, they were still higher than that of the control. This observation is in agreement with the previous studies in which levels of lipid peroxidation products were increased following AA administration (Mitchell et al., 1985; Oak and Choi, 1998; Sener et al., 2006). Our results showed that L-carnitine causes a significant inhibition of MDA production. Thus, L-carnitine treatment effectively protected the liver tissue against oxidative damage. In addition to MDA, we observed an elevation of sialic acid concentration in the serum and liver tissue with the highest increase observed at 4 h. Release of sialic acids located on the terminal residues of glycolipids of cell membranes could ensue as a result of the breakdown of cell membranes and lipid peroxidation. The released sialic acid content resulting in the elevation of TSA concentration could indicate an altered structural integrity of glycolipids in the cellular membranes (O’Kennedy et al., 1991; Shutter et al., 1992). Previous reports showed that serum sialic acid concentrations are increased in several diseases including some forms of liver disease (Sillanaukee et al., 1999). Prevention of GSH depletion might be the most efficient way of direct protection against AA hepatotoxicity, since depletion of GSH in the liver tissue was shown to be an important factor in the pathophysiology of AA toxicity (Nagai et al., 2002; Lauterburg, 2002). Following AA toxicity, the liver tissue contains decreased GSH concentration and GSH peroxidase (GSH-Px) activities (Lores Arnaiz et al., 1995). In the current study, GSH depletion was prevented when L-carnitine was administered before AA. The increase in GSH level is also important for GSH-Px, which requires GSH as a cofactor, and the elevation in GSH level increases activity of GSH-Px (James et al., 2003). The toxic metabolite of AA, NAPQI, is detoxified by GSH to give rise to the AA-GSH conjugate. In the toxicity of AA, depletion of GSH following interaction with NAPQI is known to be an important event in the initiation of AA-induced cellular necrosis (Dahlin et al., 1984). The action of L-carnitine on the enhanced GSH level could support available GSH pool allowing the cell to detoxify more NAPQI and minimize the damage due to reduced levels of NAPQI available in the liver cells. Antioxidants like L-carnitine have been found to offer protection against AA-induced liver damage (Oz et al., 2004). The mechanism associated with the protective effect of L-carnitine could be also due to direct
antioxidant effect of L-carnitine. L-carnitine itself was also shown to be an antioxidant agent. It was reported that superoxide dismutase, which constitutes one of the major enzymatic antioxidant mechanisms against superoxide radical, prevented liver necrosis due to AA toxicity (Kyle et al., 1987; Nakae et al., 1990). Similarly, the antioxidant effect of L-carnitine was effectively utilized to prevent the toxic effect of several chemicals. For example, cisplatin-induced nephrotoxicity where oxidative stress and lipid peroxidation are thought to play a major role in the pathophysiology of nephrotoxicity, administration of L-carnitine in Sprague–Dawley rats normalized kidney function. In addition, L-carnitine attenuated the increased MDA and reduced GSH levels (Sayed-Ahmed et al., 2004). Other than nephrotoxicity, L-carnitine was shown to prevent ethanol-induced lesions in the gastric mucosa and protected against lipid peroxidation as well as normalized GSH of gastric mucosa in rats (Dokmeci et al., 2005). Methamphetamine neurotoxicity, mediated by peroxynitrite radicals, was protected by L-carnitine (Ashraf et al., 2002). In addition, it was reported that mitochondrial proteins could be the target for AA toxicity leading to the loss of energy production and cellular ion control (Masubuchi et al., 2005). The action of L-carnitine in mitochondrial energy production is to facilitate the transfer of long-chain fatty acids from cytosol to mitochondria, thereby playing an important role in the production of ATP (Kelly, 1998). Indeed, L-carnitine was shown to increase ATP production in the myocardium in cisplatin-induced cardiomyopathy (Al-Majed et al., 2006). In the present study, serum AST and ALT levels were increased following AA treatment indicating the cellular damage and decreased liver functions. The increases in lipid peroxides, serum and liver MDA and serum and liver TSA activity, which were accompanied by a significant reduction in GSH levels, indicate oxidative liver damage. L-carnitine pretreatment reduced AAinduced hepatotoxicity, as the biochemical parameters and the hepatic function tests were restored to control levels. Herein, hepatic toxicity was observed to be an acute effect, which is the most prominent damage appearing at 4 h following the administration of AA according to the biochemical parameters. However, the degree of histopathological alterations was observed more severely at the 24 h time point. The most plausible explanation for this pattern is that the biochemical alterations proceed before the histopathological changes, and the perturbations in the biochemical reactions could lead to the histopathological alterations later. In conclusion, findings of the present study showed once more that AA administered above the recommended therapeutic dose causes hepatic toxicity in mice,
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and that this is likely a result of reactive oxygen species. However, hepatic toxicity is an acute effect where the biochemical alterations are most prominent at 4 h after administration. In this study, it was demonstrated that L-carnitine also has a prominent protective effect against AA toxicity.
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Wang B, Brand-Miller J. The role and potential of sialic acid in human nutrition. Eur J Clin Nutr 2003;57:1351–69. Yoshoiko T, Kawada K, Shimada T. Lipid peroxidation in maternal and cord blood and protective mechanism against actived-oxygen toxicity in the blood. Am J Obstet Gynecol 1979;135:372–6.
Experimental and Toxicologic Pathology 59 (2007) 121–128 www.elsevier.de/etp
Hepatoprotective effect of L-carnitine against acute acetaminophen toxicity in mice Kursad Yapara, Asim Karta,, Mahmut Karapehlivanb, Onur Atakisib, Recai Tuncac, Serpil Erginsoyc, Mehmet Citild a
Department of Pharmacology and Toxicology, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey Department of Biochemistry, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey c Department of Pathology, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey d Department of Internal Medicine, College of Veterinary Medicine, University of Kafkas, 36040 Kars, Turkey b
Received 1 February 2007; accepted 26 February 2007
Abstract L-carnitine is a cofactor in the transfer of long-chain fatty acid allowing the b-oxidation of fatty acid in the mitochondria. It is also a known antioxidant with protective effects against lipid peroxidation. In this study, hepatoprotective effect of L-carnitine was investigated against acetaminophen (AA)-induced liver toxicity where mitochondrial dysfunction and oxidative stress are thought to be involved in AA hepatotoxicity. Sixty-four Balb/C mice were divided into eight groups. Mice were dosed with single-AA injection (500 mg/kg via the intra peritoneal route) with or without L-carnitine (500 mg/kg for 5 days starting 5 days before AA injection via intra peritoneal route) and sampled at 4, 8 and 24 h following AA injection. AA increased serum AST, ALT, total sialic acid (TSA) and MDA as well as tissue TSA and MDA levels significantly with the highest increase observed at 4 h, but there was a decrease in blood and tissue GSH level. Administration of L-carnitine significantly reduced AA-induced elevations in AST, ALT, TSA and MDA concentrations and increased GSH levels at all sampling points. AA also induced necrosis, hyperemia, sinusoidal congestion and hemorrhage with time-dependent increase in severity, but the degree of necrosis and histopathologic alterations were most severe at 24 h following AA administration. However, the degree of pathologic alterations was less severe with simultaneous L-carnitine application. These results suggest that AA results in oxidative damage in the liver with an acute effect. L-carnitine also has a prominent protective effect against AA toxicity and may be of therapeutic value in the treatment of AA-induced hepatotoxicity. r 2007 Elsevier GmbH. All rights reserved.
Keywords: L-carnitine; Acetaminophen; Sialic acid; Hepatoprotection; Oxidative stress
Introduction Corresponding author. Tel.: +90 474 242 6800; fax: +90 474 242 6853. E-mail address: [email protected] (A. Kart).
0940-2993/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2007.02.009
Acetaminophen (AA) is a widely used analgesic and antipyretic, which is safely employed in the therapeutic range in man and animals (Hjelle, 1986; Dargan and Jones, 2002). AA is readily absorbed by the
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gastrointestinal tract, and it is metabolized by the microsomal enzyme system in the liver. While 80–85% of AA’s biotransformation occurs via conjugation of glucuronide and sulfate by the transferase enzymes, 10–15% of AA is oxidized to the reactive oxygen species (ROS) through P-450-dependent mixed-function oxidases (Black, 1980; Dahlin et al., 1984; Brown et al., 1992; Vermeulen et al., 1992). The toxic metabolite produced at the therapeutic doses is detoxified by glutathione to cysteine and mercaptouric acid to be subsequently excreted from the body. However, AA taken in overdose or long-term use at the therapeutic level may lead to the saturation of conjugation pathway leading to the depletion of glutathione and to the increased formation of toxic reactive metabolites. Free toxic metabolites are then covalently bound to the macro molecules of cells leading to cellular necrosis (Mitchell et al., 1973; Jollow et al., 1973; Pumford et al., 1997). The toxic metabolite responsible for inducing liver damage is N-acetylp-benzoquinone imine (NAPQI), which could bind to cellular proteins leading to centrilobular necrosis in the liver (Dahlin, 1984). Previous reports indicate that mitochondrial dysfunction could play a role in the mechanism of AA hepatotoxicity. Reactive metabolites of AA, NAPQI, result in mitochondrial dysfunction and superoxide formation with the resultant effect of peroxynitrite and tyrosine nitration. The events in this mechanism affect mitochondrial permeability transition leading to disturbances in the permeability of the inner mitochondrial membrane (Masubuchi et al., 2005). L-carnitine is a g-three methyl amino-b-hydroxyl fatty acid, which is an essential cofactor in mitochondrial respiration playing an important role in the transfer of long-chain fatty acids from cytosol to mitochondria. By combination with carnitine to form acylcarnitine, acyl groups could be transferred from cytosolic coenzyme A on the outer surface of the mitochondrion membrane, then to the inner surface by exchange with free carnitine using an antiport mechanism. The acyl groups are then transferred from carnitine to coenzyme A within the mitochondrion (Kelly, 1998). Carnitine is also associated with buffering of excess acyl-Co A, which is potentially toxic to the cells, and it was reported that L-Carnitine has a protective effect on lipid peroxidation by reducing the formation of hydrogen peroxide (Brass, 2000; Rani and Panneerselvam, 2002). L-carnitine could also improve antioxidant status in rats and showed free radical scavenging activity as well (Kalaiselvi and Panneerselvam, 1998; Rani and Panneerselvam, 2001). Sialic acids are a family of nine-carbon neuraminic acid derivatives that are found in terminal residues of oligosaccharide chains of mucins, glycoproteins and glycolipids of cell membranes (Sillanaukee et al., 1999; Wang and Brand-Miller, 2003). It is reported that the level of sialic acids is increased following inflammation and several disease conditions including chronic liver
diseases (Sillanaukee et al., 1999). This might be an indicator of the disruption of cell membranes due to lipid peroxidation along with increased MDA concentration. In this study, it is aimed to investigate the protective effects of L-carnitine on acute AA toxicity at three different time points (at 4, 8 and 24 h following acute AA dosing) by evaluating histopathology and biochemical alterations including malondialdehyde (MDA), total sialic acid (TSA) and glutathione (GSH) concentrations in the blood and liver tissue.
Material and Methods Sixty-four male Balb/c mice (12–14-week old, 25–30 g bw) were obtained from University of Kafkas, Animal Research Farm. An approval from the Institutional Ethics Committee was obtained before the experiment. The mice were acclimatized 1 week prior to the planned experiments and fed with standard pelleted diet ad libitum. The mice were housed in stainless-steel cages (26 15 50 cm in size) in a controlled environment with temperature range at 2272 1C, humidity 5075% and a 12-h light/dark cycle. The mice were divided into eight groups each consisting of eight animals as follows: group 1 (control) was injected with single saline (0.02 ml/g body weight, 0.9% NaCl; Baxter, Mediflex, Eczacibasi, Istanbul, Turkey) via the intraperitoneal (i.p.) route. Group 2 received 500 mg/kg of L-carnitine daily (CARNITENEs, injectable solution in 5 ml sterile ampoule containing 200 mg/ml L-carnitine, 0.014 ml 10% hydrochloric acid and injectable water, Santa Farma Ilac Sanayii A.S., Istanbul, Turkey) with the injected volume of 62.5–75 ml for 25–30 g mice using a Hamilton microinjector applied for 5 days. Mice in groups 3, 4 and 5 were injected with a single i.p. dose of AA (Sigma Chemical Company, St. Louis, MO, AA was applied in saline solution at a concentration of 25 mg/ml with the injected volume of 0.5–0.6 ml where the mixture was prepared at 70 1C, then cooled to 37 1C for administration) at a dose of 500 mg/kg. The animals were then decapitated at 4, 8 and 24 following the AA application. Mice in groups 6, 7 and 8 were administered with 500 mg/kg of L-carnitine daily (starting 5 days before AA injection via the i.p. route) and a single dose of 500 mg/kg of AA and decapitated at 4, 8 and 24 h following the AA injection, respectively. Blood samples were collected from the heart via cardiac puncture under light ether anesthesia for GSH, MDA, TSA, AST and ALT measurements before killing the animals at determined time intervals. Blood GSH was measured from the whole blood. For MDA, TSA, AST and ALT measurements, all tubes were centrifuged
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(at 1200 g at 4 1C) for 10 min to obtain the serum. The serum samples were kept at 25 1C until they were analyzed. Serum AST and ALP concentrations were determined using a commercially available kit and using an autoanalyzer (Olympus Chemistry Analyzer AU 640, Type: 640-03, Japan). In addition, tissue samples from the liver were collected for tissue GSH, MDA and TSA concentrations. Liver tissues were immediately removed and washed with 0.15 M KCl (at 4 1C). The tissues were then homogenized in ice cold 0.15 M KCl by a homogenizator (Ultra Turrax Type T25-B, IKA Labartechnie, Germany) at 1600 rpm for 3 min. The homogenates were centrifuged at 5000g at 4 1C for 1 h. The supernatants were stored at 40 1C until they were analyzed. TSA was measured colorimetrically using a spectrophotometer (UV-1201, Shimadzu, Japan) by the method of Sydow (1985). For GSH and MDA concentrations, analyses were carried out by the method of Beutler et al. (1963) and Yoshoiko et al. (1979), respectively. For histopathological examinations, samples of liver tissue were taken from both anterior and posterior parts of the lobus intermedius at four different regions. The samples were then fixed in 10% neutral buffered formalin, embedded in paraffin wax and four sections at 4–6 mm were taken from each tissue sample and stained with haematoxylin–eosin (H&E). The percentage of the total necrotic area was assessed semiquantitatively under a light microscope with an ocular grid and a 40 objective. A total of 10 high-power fields were randomly chosen. For the statistical analysis, differences between the groups were tested by analysis of variance (ANOVA) and Tukey test using SPSS for Windows version 10.0. Data were presented as mean7standard errors, and p values less than 0.05 were considered significant.
Table 1.
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Results Serum and blood biochemical parameters in the treatment groups are presented in Table 1. The AST levels in the AA-treated groups were significantly increased compared to that of the control groups at 4, 8 and 24 h time points. The enzyme levels showed a gradual decrease towards 8 and 24 h. However, the difference from the control was still significantly higher. Pretreatment with the antioxidant compounds decreased AST levels significantly when compared with the corresponding group treated with AA only at 4, 8 and 24 h. Similar results were obtained for ALT levels. This enzyme was increased by AA treatment at 4 and 8 h but not at 24 h compared to that of the control. The ALT levels were decreased significantly with L-carnitine treatment at 4 and 8 h compared to the control and the respective AA-only groups. Serum TSA levels were found to be increased in the AA-treated groups at 4, 8 and 24 h compared to that of the control with a gradual decrease toward a 24 h time point. Mice receiving L-carnitine plus AA at 4, 8 and 24 h had significantly lower serum TSA levels compared to the control and the corresponding AA-only groups. Blood GSH levels of AA-treated mice at 4, 8 and 24 h were found to be significantly lower than that of the control with a gradual amelioration toward a 24 h time point. On the other hand, blood GSH levels were found to be similar in the L-carnitine plus AA-treated group at 4, 8 and 24 h compared to that of the control. Tissue biochemical parameters are presented in the treatment groups in Table 2. Hepatic TSA was increased following AA significantly at 4 h; however, the TSA concentrations at 8 and 24 h were not different from that of the control. L-carnitine treatment in AA groups prevented the increase significantly seen at 4 h. Liver MDA levels were increased after treatment with
Biochemical alterations in the serum of treatment groups
Groups
AST (U/L)
ALT (U/L)
TSA (U/L)
MDA (mmol/L)
GSH (blood) (mg/L)
1 2 3 4 5 6 7 8
125.3877.8f 119.7576.8f 742.50732.0a 513.25726.2bc 603.38724.7b 442.00719.1cd 390.25723.2d 284.63718.5e
42.3873.7cd 36.2573.0d 101.8878.5a 62.0074.3bc 86.1377.8ab 51.7574.8cd 56.5074.9cd 43.6373.8cd
608.86724.0d 532.67715.5e 773.91719.9a 632.15713.4cd 725.85717.7ab 613.18714.5d 693.15715.4bc 595.52710.5de
14.2570.6d 12.0370.4e 16.6270.4abc 15.9170.5bcd 16.9570.4ab 15.4070.3bcd 17.9470.4a 14.9570.3cd
79.0671.1bc 86.9371.0a 74.3871.1de 75.2670.7cde 71.1070.9ef 76.9370.9bcd 66.8171.0f 80.0370.9b
(control) (L-car) (AA-4 h) (L-car+AA-4 h) (AA-8 h) (L-car+AA-8 h) (AA-24 h) (L-car+AA-24 h)
Values presented as mean7SE (n ¼ 8). Means denoted with different superscripts are within the same column are statistically significant. AST: aspartate amino transferase, ALT: alanine aminotransferase, TSA: total sialic acid, MDA: malondialdehyde, GSH: glutathione. Group 1: (control) received 0.9% saline (i.p.); Group 2: was injected with L-carnitine (500 mg/kg, for 5 days); Group 3: acetaminophen (AA) only (500 mg/kg) injection (killed 4 h after AA treatment); Group 4: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 4 h after AA treatment); Group 5: AA only (500 mg/kg) injection (killed 8 h after AA treatment); Group 6: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 8 h after AA treatment); Group 7: AA only (500 mg/kg) injection (killed 24 h after AA treatment); Group 8: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 24 h after AA treatment).
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Table 2. groups
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Biochemical alterations in the liver of treatment
Groups
TSA (mg/g)
MDA (mmol/g)
GSH (mg/g)
1 2 3 4 5 6 7 8
0.52770.02b 0.44370.02c 0.71670.03a 0.53270.03bc 0.63170.03ab 0.52470.02bc 0.61370.04ab 0.52070.03bc
0.32170.03c 0.29570.02c 0.48670.01a 0.35170.03bc 0.46270.02a 0.33670.02bc 0.41870.02ab 0.33270.02bc
0.24270.02bc 0.37470.02a 0.15470.01d 0.24470.02bc 0.16270.01d 0.24870.02bc 0.18870.01cd 0.25870.01b
(control) (L-car) (AA-4 h) (L-car+AA-4 h) (AA-8 h) (L-car+AA-8 h) (AA-24 h) (L-car+AA-24 h)
Values presented as mean7SE (n ¼ 8). Means denoted with different superscripts are within the same column are statistically significant. TSA: total sialic acid, MDA: malondialdehyde, GSH: glutathione. Group 1: (control) received 0.9% saline (i.p.) Group 2: was injected with L-carnitine (500 mg/kg, for 5 days); Group 3: acetaminophen (AA) only (500 mg/kg) injection (killed 4 h after AA treatment); Group 4: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 4 h after AA treatment); Group 5: AA only (500 mg/kg) injection (killed 8 h after AA treatment); Group 6: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 8 h after AA treatment); Group 7: AA only (500 mg/kg) injection (killed 24 h after AA treatment); Group 8: L-carnitine (500 mg/kg, for 5 days) plus AA (killed 24 h after AA treatment).
Fig. 2. The appearance of liver 4 h following L-carnitine plus acetaminophen treatment. The hepatocytes appear eosinophilic and enlarged. Caryopicnosis is present in some hepatocytes (H&E, X260).
Fig. 3. Histologic appearance of liver 8 h after acetaminophen administration at light microscopy. Widespread vacuolation and caryopicnosis are present, and the remaining hepatocytes are eosinophilic and necrotic (H&E, X90).
Fig. 1. Histologic appearance of the liver 4 h after acetaminophen administration at light microscopy. Extensive eosinophilia in the hepatocytes with some necrosis. Note the widespread vacuolation and caryopicnosis (H&E, X180).
AA at 4, 8 and 24 h. Although the levels were relatively decreased at 8 and 24 h compared to 4 h, they were still significantly higher than that of the control. L-carnitine prevented lipid peroxidation significantly, and MDA levels were reduced to the control levels at 4, 8 and 24 h. The AA treatment decreased liver GSH levels significantly compared to that of the control at 4 and 8 h after treatment. However, liver GSH level at 24 h was similar to that of the control. Pretreatment
with L-carnitine prevented the reduction in GSH significantly. Histopathological examinations revealed that livers were normal in appearance with no change in the lobular architecture in the control and L-carnitine groups. In the AA group (at 4th h), hepatocytes were swollen and eosinophilic in appearance particularly around the central veins. Hepatocytes in the centrilobular regions showed cytoplasmic vacuolization. In addition, majority of the hepatocytes showed picnosis and karyorhexis with the presence of necrotic areas. Sinusoids were focally congested with extensive hemorrhages (Fig. 1). In the AA plus L-carnitine group (at 4th h), hepatocytes surrounding the central veins were swollen and eosinophilic in appearance, and sinusoids
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Fig. 4. The appearance of liver 8 h following L-carnitine plus acetaminophen treatment. Hepatocytes appear eosinophilic with widespread caryopicnosis. There are some karyopicnosis and vacuolations (H&E, X180).
Fig. 5. Appearance of the liver 24 h following acetaminophen administration. Extensive necrosis with some vacuolations and karyopicnosis (H&E, X180).
were congested with no sign of hemorrhage (Fig. 2). In the AA group (at 8th h), confluent areas of necrosis and extensive hemorrhages were observed around all centrilobular regions. Nuclei of these hepatocytes were in the advanced stage of karyolysis or were completely absent (Fig. 3). In the AA plus L-carnitine group (at 8th h), scattered cells in the centrilobular regions had cytoplasmic vacuoles and increased eosinophilia. Nuclei of these hepatocytes were picnotic with very limited necrosis and hemorrhages (Fig. 4). In the AA group (at 24th h), massive confluent areas of necrosis were evident with considerable hemorrhage (Fig. 5). In the AA plus L-carnitine group (at 24th h), changes were similar to the AA plus L-carnitine group (at 8th h) (Fig. 6). Quantification of necrotic areas in the liver of AAtreated mice revealed that there was a progressive
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Fig. 6. Appearance of the liver 24 h following L-carnitine plus acetaminophen treatment. Hepatocytes appear eosinophilic, and there are widespread karyopicnosis (H&E, X180).
Fig. 7. Quantification of necrosis in the liver of the treatment groups with respect to time following last drug application. AA: acetaminophen, AA+L-car: acetaminophen plus L-carnitine.
increase from 4377% at 4 h and 62.75711 at 8 h up to 82711% at 24 h. However, the increase in necrotic areas in the mice applied AA and L-carnitine was from 8.2575% at 4 h and 2776 at 8 h up to 3278% at 24 h (Fig. 7). Statistical analysis revealed a statistically significant difference at the level of Po0.05 for necrotic areas between the only AA and AA plus L-carnitine applied groups at 4, 8 and 24 h time intervals.
Discussion Previous studies showed that several factors could be involved in the mechanism and pathophysiology of AA hepatotoxicty at the cellular level. Role of oxidative stress was reported to be one of the important factors in
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the development of hepatic cell injury (Knight et al., 2001). Lipid peroxidation was suggested to be closely related to AA-induced tissue damage, and MDA is a good indicator of the degree of lipid peroxidation (Sener et al., 2006). In this model of AA-induced hepatic injury, we observed a significant increase in the MDA content of the liver tissue at 4, 8 and 24 h. Although the levels were relatively decreased at 8 and 24 h compared to 4 h, they were still higher than that of the control. This observation is in agreement with the previous studies in which levels of lipid peroxidation products were increased following AA administration (Mitchell et al., 1985; Oak and Choi, 1998; Sener et al., 2006). Our results showed that L-carnitine causes a significant inhibition of MDA production. Thus, L-carnitine treatment effectively protected the liver tissue against oxidative damage. In addition to MDA, we observed an elevation of sialic acid concentration in the serum and liver tissue with the highest increase observed at 4 h. Release of sialic acids located on the terminal residues of glycolipids of cell membranes could ensue as a result of the breakdown of cell membranes and lipid peroxidation. The released sialic acid content resulting in the elevation of TSA concentration could indicate an altered structural integrity of glycolipids in the cellular membranes (O’Kennedy et al., 1991; Shutter et al., 1992). Previous reports showed that serum sialic acid concentrations are increased in several diseases including some forms of liver disease (Sillanaukee et al., 1999). Prevention of GSH depletion might be the most efficient way of direct protection against AA hepatotoxicity, since depletion of GSH in the liver tissue was shown to be an important factor in the pathophysiology of AA toxicity (Nagai et al., 2002; Lauterburg, 2002). Following AA toxicity, the liver tissue contains decreased GSH concentration and GSH peroxidase (GSH-Px) activities (Lores Arnaiz et al., 1995). In the current study, GSH depletion was prevented when L-carnitine was administered before AA. The increase in GSH level is also important for GSH-Px, which requires GSH as a cofactor, and the elevation in GSH level increases activity of GSH-Px (James et al., 2003). The toxic metabolite of AA, NAPQI, is detoxified by GSH to give rise to the AA-GSH conjugate. In the toxicity of AA, depletion of GSH following interaction with NAPQI is known to be an important event in the initiation of AA-induced cellular necrosis (Dahlin et al., 1984). The action of L-carnitine on the enhanced GSH level could support available GSH pool allowing the cell to detoxify more NAPQI and minimize the damage due to reduced levels of NAPQI available in the liver cells. Antioxidants like L-carnitine have been found to offer protection against AA-induced liver damage (Oz et al., 2004). The mechanism associated with the protective effect of L-carnitine could be also due to direct
antioxidant effect of L-carnitine. L-carnitine itself was also shown to be an antioxidant agent. It was reported that superoxide dismutase, which constitutes one of the major enzymatic antioxidant mechanisms against superoxide radical, prevented liver necrosis due to AA toxicity (Kyle et al., 1987; Nakae et al., 1990). Similarly, the antioxidant effect of L-carnitine was effectively utilized to prevent the toxic effect of several chemicals. For example, cisplatin-induced nephrotoxicity where oxidative stress and lipid peroxidation are thought to play a major role in the pathophysiology of nephrotoxicity, administration of L-carnitine in Sprague–Dawley rats normalized kidney function. In addition, L-carnitine attenuated the increased MDA and reduced GSH levels (Sayed-Ahmed et al., 2004). Other than nephrotoxicity, L-carnitine was shown to prevent ethanol-induced lesions in the gastric mucosa and protected against lipid peroxidation as well as normalized GSH of gastric mucosa in rats (Dokmeci et al., 2005). Methamphetamine neurotoxicity, mediated by peroxynitrite radicals, was protected by L-carnitine (Ashraf et al., 2002). In addition, it was reported that mitochondrial proteins could be the target for AA toxicity leading to the loss of energy production and cellular ion control (Masubuchi et al., 2005). The action of L-carnitine in mitochondrial energy production is to facilitate the transfer of long-chain fatty acids from cytosol to mitochondria, thereby playing an important role in the production of ATP (Kelly, 1998). Indeed, L-carnitine was shown to increase ATP production in the myocardium in cisplatin-induced cardiomyopathy (Al-Majed et al., 2006). In the present study, serum AST and ALT levels were increased following AA treatment indicating the cellular damage and decreased liver functions. The increases in lipid peroxides, serum and liver MDA and serum and liver TSA activity, which were accompanied by a significant reduction in GSH levels, indicate oxidative liver damage. L-carnitine pretreatment reduced AAinduced hepatotoxicity, as the biochemical parameters and the hepatic function tests were restored to control levels. Herein, hepatic toxicity was observed to be an acute effect, which is the most prominent damage appearing at 4 h following the administration of AA according to the biochemical parameters. However, the degree of histopathological alterations was observed more severely at the 24 h time point. The most plausible explanation for this pattern is that the biochemical alterations proceed before the histopathological changes, and the perturbations in the biochemical reactions could lead to the histopathological alterations later. In conclusion, findings of the present study showed once more that AA administered above the recommended therapeutic dose causes hepatic toxicity in mice,
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and that this is likely a result of reactive oxygen species. However, hepatic toxicity is an acute effect where the biochemical alterations are most prominent at 4 h after administration. In this study, it was demonstrated that L-carnitine also has a prominent protective effect against AA toxicity.
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