Comparison of the effects of melatonin and pentoxifylline on carbon ...

Cell Biol Toxicol 2006; 22: 381–391. DOI: 10.1007/s10565-006-0019-y

C Springer 2006

Comparison of the effects of melatonin and pentoxifylline on carbon tetrachloride-induced liver toxicity in mice ˙ Bayram2 and M.R. S¸ekero˘glu1 T. Noyan1 , U. Kömüro˘glu1 , I. 1 Department of Biochemistry, 2 Department of Pathology, School of Medicine, Yuzuncu Yil University, Van, Türkiye Received 29 June 2005; accepted 8 March 2006; Published online 16 May 2006

Keywords: melatonin, pentoxifylline, carbon tetrachloride, liver damage, oxidative damage Abstract The purpose of the study was to determine whether along and in combination melatonin (MLT) and pentoxlfylline (PTX) exerted beneficial effects on histopathological changes and changes in oxidant and antioxidant systems in liver caused by CCl4 -induced liver toxicity in mice. Mice were randomly divided into six groups: control, olive oil, toxicity, MLT, PTX, PTX+MLT. MLT 10 mg/kg/day, PTX 50 mg/kg/day, and the same individual doses in MLT+PTX combination were given intraperitoneally to mice for 7 day. CCl4 0.8 mg/kg/day was administered on the 4th, 5th, and 6th days of therapy in all groups except the control and olive oil groups. In the toxicity group, increased concentrations of malondialdehyde (MDA) and lipid hydroperoxides (LOOH) and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) activities were found compared to the control and olive oil groups (p < 0.05). Compared to the toxicity group, both the PTX group and the PTX+MLT group had decreased MDA and LOOH levels, whereas MLT reduced only LOOH levels (p < 0.01). MLT, PTX and MLT+PTX increased the GSH-Px and CAT activities compared to the toxicity group (p < 0.05). MLT increased CAT activity compared to PTX and MLT+PTX (p < 0.05). Superoxide dismutase enzyme activity did not change in any group (p < 0.05). Histopatholically, ballooning, degeneration, apoptosis, and bridging necrosis were seen in the toxicity group. MLT, PTX and MLT+PTX decreased the apoptosis and bridging necrosis (p < 0.01), and PTX and MLT+PTX decreased balloon degeneration compared to the toxicity group (p < 0.01). These results indicate that administration of PTX and MLT alone and in combination before onset of liver toxicity might prevent the oxidative damage by reducing oxidative stress and increasing antioxidant enzyme levels. Abbreviations: ALT, alanine aminotransferase; AST, apartate aminotransferase; CCl4 , carbon tetrachloride; HE, hematoxylin–eosin; LDH, lactate dehydrogenase; LOOH, lipid hydroperoxides; MDA, malondialdehyde; MLT, melatonin; PTX, pentoxifylline; TBARS, thibarbituric acid-reactive substances Introduction Carbon tetrachloride (CCl4 ) has long been known as a hepatotoxin. It is widely used in ani-

mal liver injury models because damage by CCl4 is regarded as the analogue of liver damage caused by a variety of hepatotoxins in humans. The compound can induce both acute

382 and chronic liver injury. It is generally believed that CCl4 hepatotoxicity depends mainly on reductive bioactivation to trichloromethyl free radical (CCL3 . ) by cytochromes P450. The mitochondrial electron-transport chain is suggested to be responsible for activation of CCl4. The CCL3 . radical is highly toxic and may form many additional reactive intermediates in vivo (Castro et al., 1997; Stoyanovsky, and Caderbaum, 1999; Basu, 2003 ). Free radicals are formed in the energy/respiratory pathways of the body, as inflammatory mediators in the immune system, and in other biochemical pathways that are essential aspects of cellular metabolism (MacDonald-Wicks and Garg, 2003). Activated oxygen species, such as superoxide radical, hydrogen peroxide, and hydroxyl radical, produced by the partial reduction of oxygen, are highly unstable and extremely reactive. The short half-lives of many of these species make them highly toxic to tissues (Noyan et al., 2003). Rapid and extensive lipid peroxidation of the membrane lipids has been proposed as the basis of CCl4 hepatocellular toxicity (Basu, 2003). Pentoxifylline (PTX) (3,7-dimethyl-1-(5oxohexyl)xanthine) is a methylxanthine derivative that has been used for its regulatory effects on blood flow in the treatment of peripheral vascular disease, cerebrovascular disease, and a number of other conditions involving defective regional microcirculation (Ward and Clissold, 1987). PTX can enhance the chemotactic response of neutrophils but may inhibit phagocytosis and superoxide production by neutrophils and monocytes (Mandell, 1988). We have also demonstrated that PTX has antioxidant properties (Noyan et al., 2003). Melatonin (MLT) (N-acetyl-5-methoxytryptamine), is an indole amine synthesized during the night in the pineal gland (Pierrefiche et al., 1993). Its biological antioxidant activity is well known and it stands out as a powerful neutralizer of hydroxyl free radical (Tan et al., 1993). Because of its easy diffusion through membranes, and because it does not need specific receptors to carry out its

antioxidant activity. In addition, MLT regulates the activity and gene expression of antioxidant and pro-oxidant enzymes (Barlow-Walden et al., 1995; Reiter et al., 1997). Although MLT and PTX have been shown to exert a protective effect against acute liver injuries and ischemia–reperfusion injury in rat liver, their effects on liver toxicity have not been fully clarified. It has been reported that MLT exerts a protective effect against acute liver injuries induced by endotoxic shock and ischemia–reperfusion in rats through its antioxidant action (Ohta et al., 2000). In different studies, PTX showed variable effects on hepatocyte injury, from failure to alter indices of cell necrosis and cholestasis to prevention of liver cell damage (Abdel Salam et al., 2005). The aim of the present study was to examine the potential protective effects of PTX and MLT in a mouse model of acute intoxication with CCl4. We investigated changes in hepatic tissue morphology and function as well as the oxidative and antioxidative processes induced by CCl4 in hepatocytes.

Material and methods Sixty healthy male Mus musculus Swiss Albino mice weighing 38–52 g were used in this study. Mice were kept under standardized conditions for food, water, light and temperature. The approval of Yüzüncü Yıl University School of Medicine Animal Ethics Committee was obtained. The study took place over 7 days. To induce the hepatic toxicity, 0.08 mg/kg/day CCl4 diluted 1:3 in olive oil was injected daily via intraperitoneally (i.p.) on the 4th, 5th and 6th days of the therapy in all groups except the control and olive oil group. Animals were randomly separated into six groups, as shown below, that included 10 mice in each group. To avoid possible interactions of the compounds, all treatments were administrated at the same time and using different injectors. The experimental groups were as follows. Group 1 (control): animals were sham-treated with 0.8 ml/kg/day physiological serum i.p during

383 7 days. Group 2 (olive oil): animals were treated with 0.8 ml/kg/day olive oil i.p starting from the 4th day of the study for 3 days. Group 3 (CCl4 ): animals received only CCl4 . Group 4 (MLT): toxicity was induced as described and animals were treated with MLT 10 mg/kg/day i.p for 7 days. Group 5 (PTX): toxicity was induced as described and animals were treated with PTX 50 mg/kg/day i.p for 7 days. Group 6 (PTX+MLT): toxicity was induced as described and animals were treated with MLT 10 mg/kg/day plus PTX 50 mg/kg/day i.p for 7 days. Tissue sample preparation At the end of the study, animals were sacrificed at 09:00, after an overnight fast, by exsanguination under anesthesia. The liver tissues of each animal were removed, cleaned, dried, and processed for biochemical measurements. The homogenates were prepared on ice in homogenization buffer (0.1 mol/L phosphate, 0.1 mmol/L EDTA, pH 7.0) at a ratio of 1:4 (w/v). To each sample, 10 μl of 500 mmol/L BHT in acetonitrile was added to prevent formation of new peroxides during the assay. The homogenates were centrifuged at 20 000 g for 20 min at 4◦ C and frozen at −70◦ C. Analytic methods All chemicals were purchased from Sigma Chemical (St. Louis, MO, USA). The levels of thiobarbituric acid-reacting substances (TBARS), an end product of lipid peroxidation, in liver tissue was measured in tissue homogenates fluorimetrically at wavelengths of 525 nm for excitation and 547 nm for emission (Wasowicz et al., 1993). The concentration of lipid hydroperoxides (LOOH) was measured using the method described by Jiang et al. (1991). The absorbance was read at 560 nm after removal of any flocculated material by centrifugation. The apparent extinction coefficient (estimated by curve fitting the first-degree function) for H2 O2 , cumene hy-

droperoxide and butyl hydroperoxide at 560 nm was 4.3×104 mol/L/cm. Cu, Zn-SOD (superoxide dismutase) activities of liver tissue were determined by the method of Sun et al. (1988) based on the inhibition of nitroblue tetrazolium. The xanthine–xanthine oxidase system was used as a superoxide generator in the assay. The absorbance of the reduced product (formazone) was measured at 560 nm. Superoxide dismutase activity was measured as the degree of inhibition of this reaction. Catalase (CAT) activities of liver tissue were determined by Goth’s colorimetric method (Goth, 1991), in which the homogenate was incubated with H2 O2 substrate and the enzyme reaction was stopped by the addition of ammonium molybdate. The intensity of the yellow complex formed by molybdate and H2 O2 was measured at 405 nm. Glutathione peroxidase (GSH-Px) activities of liver tissues were determined by the modified method of Paglia and Valentine (1967). This method is based on the principle that GSH-Px catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted into the reduced form with concomitant oxidation of NADPH to NADP+ . The decrease in absorbance at 340 nm was measured. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lacate dehydrogenase (LDH) activities were measured by using a Roche-Hitachi PP Modular analyzer with Roche original reagents. Tissue protein concentration was determined using an Integra 800 analyser with Roche original reagents. Histopathological evaluation The tissue specimens were fixed in 10% formalin. Samples of liver were sectioned and stained with hematoxylin and eosin (HE) and subjected to histopathological evaluation by pathologists. Balloon degeneration, bridging necrosis, and apoptosis were measured using an ocular

384 micrometer, and total damage ratio was calculated as the sum of balloon degeneration, bridging necrosis and apoptosis. The following grading scheme was used to score the histological alterations: (−) absent; (+) mild; (++) moderate; (+++) severe. Statistical analysis The data are expressed as mean ± standard deviation (SD). Student’s t-test for first and last day body weight was used to determine differences among all groups. The Kolmogorov–Smirnov goodness-of-fit test was used to control whether the distribution of parameters was normal. Groups of data were compared by analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison tests. The Kruskal–Wallis test was also used to analyse the histopathological evaluation.

Results The final and initial body weights of mice are presented in Table 1. The final body weights of animals in groups 3, 4, 5 and 6 were significantly decreased compared with their initial values ( p < 0.05). The serum AST, ALT, and LDH activities are presented in Table 2 and Figure 6. AST, ALT, and LDH activities—indicators of liver function—were significantly increased in group 3 compared to all other groups (p < 0.05). The oxidant and antioxidant parameters are presented in Table 3 and Figures 7–9. The MDA and LOOH concentration in group 3 were significantly increased compared to groups 1 and 2 (p < 0.05). The MDA concentration in group 5 was significantly decreased compared to group 3 (p < 0.05). The MDA concentration in group 6 were also significantly decreased compared to groups 3 and 4 (p < 0.05). The LOOH concentration in group 4 were significantly increased compared to groups 1 and 2 (p < 0.05); however, there was a significant decrease in LOOH in group 4 com-

pared to group 3 (p < 0.05). The LOOH concentrations in group 5 and 6 were also significantly decreased compared to groups 3 and 4 (p < 0.05). The GSH-Px and CAT activities in group 3 were significantly decreased compared to groups 1 and 2 (p < 0.05). The CAT activity in group 4 was significantly increased compared to that in all other groups ( p < 0.05). The CAT activity in groups 5 and 6 was also significantly increased compared to group 3 (p < 0.05). The GSH-Px activities in groups 4 and 6 were significantly increased compared to groups 1 and 3 ( p < 0.05). The GSH-Px activity in group 5 was also significantly increased compared to that in groups 1, 2, and 3 (p < 0.05). The histopathological characteristics of all groups are given in Table 4 and Figures 1–4. Ballooning degeneration, apoptosis, and bridging necrosis were not seen in groups 1 and 2. The highest balloon degeneration, apoptosis, bridging necrosis, and total damage ratio were seen in group 3. Apoptosis, bridging necrosis, and total damage ratio decreased in groups 4, 5 and 6 compared to group 3 (p < 0.01), and there were no significant differences considering these parameters among 4, 5 and 6 groups. Balloon degeneration was decreased in groups 5 and 6 compared to group 3 (p < 0.01), but there was no significant difference between groups 3 and 4 (p > 0.05). Discussion Lipid peroxidation has been shown to be of great importance in mammalian physiology and pathophysiology in the last three decades. Increased lipid peroxidation is generally believed to be an important underlying cause of the initiation of oxidative stress related various tissue injuries, and cell death, and the progression of many acute and chronic diseases (Halliwell, 1997). In the present study CCl4 -induced toxicity caused an increase in the liver tissue MDA and LOOH levels as compared to values in the control and olive oil groups. These findings agree with previous studies (Recknagel et al., 1989;

385 Table 1. The comparison of mice’s body weights included the study dependent on time (mean±SD) Parameter

Days

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

Body weight. (g) Body weight. (g)

0 7

44.8 ± 4.8 45.7 ± 4.5

39.2 ± 2.8 39.9 ± 2.1

46.8 ± 4.1 44.4 ± 3.9∗∗

41.9 ± 3.4 39.7 ± 3.3∗

44.2 ± 2.2 40.2 ± 1.9∗∗

42.6 ± 2.8 41.4 ± 2.8∗∗

∗p

< 0.05. < 0.01.

∗∗ p

Table 2. Effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day) and MLT+PTX therapies on CCl4 -induced elevation of serum AST, ALT and LDH enzyme activities in mice Parameters Group 1

Group 2

Group 3

Group 4

1001.5 ± 192.0a∗∗.b∗∗

AST (U/L) 123.5 ± 35.4 390.4 ± 78.4 ALT (U/L) 31.7 ± 9.6 98.1 ± 29.2 1181.6 ± 401.9a∗∗.b∗∗ 383.8±92.4a∗.c∗∗ LDH (U/L) 762.3 ± 100.9 1703.2 ± 255.4 3816.6 ± 1309.3a∗∗.b∗∗ 1466.5±192.2c∗∗

547.1±103.0a∗∗.c∗∗

Group 5 561.6 ± 198.2a∗∗.c∗∗ 246.7 ± 125.1c∗∗

1986.5 ± 506.8c∗

Group 6 510.8 ± 223.4a∗.c∗∗ 411.7 ± 148.7a.∗.b∗.c∗∗ 2361.5 ± 867.3a.c∗

∗p

< 0.05. < 0.01. a Compared to group 1. b Compared to group 2. c Compared to group 3. ∗∗ p

Table 3. The effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day) and MLT plus PTX therapies on liver TBARS, LOOH contents and CAT, SOD and GSH-Px enzyme activities in mice (mean ± SD) Parameters

Group 1

Group 2

Group 3

Group 4

Group 5

Group 6

MDA (nmol/g pr) 250.9 ± 34.4 255.8 ± 44.4 443.1 ± 123.0a∗∗,b∗∗ 330.0 ± 50.5 209.7 ± 34.9c∗∗ 197.7 ± 65.9c∗∗, d∗ LOOH (nmol/g pr) 220.6 ± 18.9 228.6 ± 18.8 541.6 ± 66.8a∗∗,b∗∗ 329.6 ± 50.3a∗,b∗,c∗∗ 234.4 ± 16.9c∗∗,d∗ 191 ± 21.94c∗∗,d∗∗ CAT (kU/g pr) 121.7 ± 9.2 111.6 ± 9.7 80.5 ± 12.5a∗∗,b∗∗ 143.8 ± 6.9a∗,b∗∗,c∗∗ 118.4 ± 5.1c∗∗,d∗∗ 117.9 ± 8.6c∗∗,d∗∗ SOD (μg/g pr) 24.8 ± 5.1 23.2 ± 6.4 23.0 ± 4.9 22.9 ± 9.6 22.0 ± 3.7 25.9 ± 6.9 GSH-Px (U/g pr) 0.9 ± 0.0 1.02 ± 0.1 0.6 ± 0.2a∗,b∗ 1.2 ± 0.2a∗,c∗∗ 1.3 ± 0.1a∗∗,b∗c∗∗ 1.3 ± 0.1a∗,c∗∗ ∗p

< 0.05. < 0.01. a Compared to group 1. b Compared to group 2. c Compared to group 3. d Compared to group 4. ∗∗ p

Table 4. The effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day) and MLT+PTX therapies on ballooning degeneration, bridging necrosis, apoptosis and total damage ratio in the CCl4 -induced liver toxicity Median Group

Ballooning degeneration

Bridging necrosis

Apoptosis

Total damage ratio

1 2 3 4 5 6

0.00c

0.00c

0.00c

0.00c

0.00c

0.00c

2.00a 1.50ab 1.00b 1.00b

2.00a 1.00b 1.00b 1.00b

2.00a 1.00b 1.00b 1.00b

0.00c 0.00c 6.00a 3.50b 3.00b 3.00b

Different lower case represents significantly differences for each properties (p < 0.01).

386

Figure 1. Histopathological examination of liver tissue in control mice and mice treated with 0.8 mg/kg/day olive oil: normal histological characteristic are seen (CV; central vein) (HE × 20).

Figure 2. Histopathological changes of liver tissue in mice treated with 0.8 mg/kg/day CCl4 : centrilobular necrosis (CN) and diffuse numerous ballooning degenerations (BD) are seen in hepatocytes (HE × 20).

Zavodnik et al., 2005). During the initial phase of CCl4 toxicity following its administration, a large amount of CCl4 is converted to trichloromethyl radical or other radicals, which in turn accelerate several metabolic pathways (Stoyanovsky and Cederbaum, 1999; Basu, 2003). These radicals appear to affect the adjacent lipids in the tissues and induce lipid peroxidation. We think that in-

Figure 3. Histopathological changes of liver tissue in mice treated with 50 mg/kg/day PTX: mild pleomorphism and regeneration findings of hepatocytes are seen in the centrilobular area (HE × 20).

Figure 4. Histopathological changes of liver tissue in the mice treated with 10 mg/kg/day MLT: centrilobular necrosis and moderate ballooning degeneration are seen in the hepatocyte (HE ×20).

crease in the MDA and LOOH levels in the toxicity group are the result of increased lipid peroxidation associated with liver tissue damage by induced by CCl4 administration. In the present study, PTX administration caused a significant decrease in both MDA and LOOH levels, and MLT administration caused a decrease only in LOOH level as compared to the toxicity group.

387

Figure 5. Histopathological changes of liver tissue in mice treated with 10 mg/kg/day MLT plus 50 mg/kg/day PTX: mild regeneration findings of hepatocytes are seen in the centrilobular area (HE ×20).

The combined PTX+MLT treatment also caused a significant decrease in both MDA and LOOH levels compared to both toxicity and MLT groups. Protective effects of PTX against the free radical damage have been reported (Desmouliere et al., 1999; S¸ener et al., 2001; Noyan et al., 2003). S¸ener et al. (2001) reported that PTX caused decrease in MDA level and increase in GSH-Px activity in ischemia–reperfusion injury. We have also reported (Noyan et al., 2003) that PTX caused a decrease in lipid peroxidation and that it has an antioxidant effect. PTX has been shown to inhibit superoxide anion production by Kupffer cells in rat liver preservation and transplantation models (Kozaki et al., 1993; Britton and Bacon, 1994). Lee et al. (1997) have shown that PTX blocks hepatic stellate cell proliferation and activation induced by CCl4 treatment by interfering with oxidative stress. MLT is known to function as an antioxidant in vitro and in vivo (Daniels et al., 1995). Ohta et al. (2000) administered MLT at pharmacological doses and found that it prevented the progression of acute liver injury in rats intoxicated with CCl4 in a dose-dependent manner. In the present study, MLT did not show a decreasing effect on the MDA level in comparison with the toxicity group. This result may

indicate with dose-dependency. Thus, Daniels et al. (1995) reported that lipid peroxidation products accumulated when liver homogenates and microsomes were incubated in medium containing CCl4 , where co-incubation with MLT dosedependently inhibited the production of MDA. Daniels and co-workers (1995) also suggest that MLT may have failed to prevent the CCl4 -induced hepatic changes because the toxin is so heavily concentrated in the liver that additional MLT may have been required to abate the damage. Our data showed that PTX therapy may be more effective than MLT in reduction of liver lipid peroxidation. This result might be associated with inhibition of phosphodiesterase and increase of cAMP and cGMP levels by PTX (Ward et al., 1987). There is good evidence that cyclic nucleotides are able to prevent oxidative stress by reduction of lipid peroxidation (Abdollahi et al., 2003). Various previous studies have aimed to investigate the effects of various agents that protect against liver toxicity induced by CCl4 (Tan et al., 2000; Güven et al., 2003). We aimed to investigate the antioxidant and protective effects of PTX and MLT alone and in combination when administed before the formation of the liver toxicity. In the present study, CCl4 administration caused decreases in the activities of GSH-Px and CAT in liver. These findings agree with previous studies (Mandell, 1988; Güven et al., 2003). MLT and PTX administed alone and in combination also increased the CAT and GSH-Px activities compared to the toxicity group. However, SOD activity did not significantly change among the all groups. The histopathological findings of the present study are in agreement with the biochemical results. PTX, MLT, and PTX+MLT caused to decrease in apoptosis, bridging necrosis and total damage ratio. However, PTX and PTX + MLT decreased ballooning degeneration as compared to the toxicity group. There is a little information about pentoxifylline’s antioxidant activity. Reports relating to its antioxidant property are associated with leukocyte-driven radicals. PTX can enhance the chemotactic response

388

Figure 6. Effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day), and MLT+PTX on CCl4 -induced elevation of serum AST, ALT, and LDH activities in mice. a Compared to group 1; b compared to group 2; c compared to group 3; ∗ p < 0.05, ∗∗ p < 0.01.

of neutrophils, but may inhibit the phagocytosis and superoxide production by neutrophils and monocytes (Mandell, 1988; Demir and Erden, 1998). The reduction of neutrophils by PTX might be important because increased oxidative stress is

a feature of the CCl4 -induced liver injury and significant protection was obtained with the use of antioxidants (Jaeschke, 1990). PTX also exerts vasodilatory effects that depend in part on the release of nitric oxide (Kaye et al.,1996). This

Figure 7. Effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day), and MLT+PTX on CCl4 -induced elevation of tissue MDA and LOOH levels in mice. a Compared to group 1; b compared to group 2; c compared to group 3; d compared to group 4; ∗ p < 0.05, ∗∗ p < 0.01.

389

Figure 8. Effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day), and MLT+PTX on CCl4 -induced decrease of tissue CAT (kU/g protein) and SOD (μg/g protein) activities in mice. a Compared to group 1; b compared to group 2; c compared to group 3; d compared to group 4;∗ p < 0.05, ∗∗ p < 0.01.

is important because early vascular events have been suggested to have a role in liver damage caused by CCl4 , and increased hepatic arterial blood flow may act to lessen CCL4 -induced acute hepatic injury. PTX might protect the liver by alleviating early circulatory disturbance produced by CCL4 , thereby preventing acute liver damage (Abdel Salam et al., 2005). MLT was found to provide indirect as well as direct protection against free radical attack because it stimulates

antioxidative enzymes in addition to its direct scavenging ability. Melatonin as an antioxidant is effective in protecting nuclear DNA, membrane lipids, and cytosolic proteins from oxidative damage. Most studies have used pharmacological concentrations or doses of MLT to protect against free radical damage, in a few studies physiological levels of the indole have been shown to be beneficial against oxidative stress (Reiter et al., 1997). Information about the potential

Figure 9. Effect of MLT (10 mg/kg/day), PTX (50 mg/kg/day), and MLT+PTX on CCl4 -induced decrease of tissue GSH-Px activity in mice. a Compared to group 1; b compared to group 2; c compared to group 3; d compared to group 4;∗ p < 0.05, ∗∗ p < 0.01.

390 role of MLT in influencing the antioxidative defense system via enzymes involved in free radical metabolism is obviously incomplete. Pierrefiche and Laborit (1995) demonstrated that MLT stimulates the activity of glucose-6-phosphate dehydrogenase in liver. The importance of this lies in the fact that this enzyme resupplies the cell with reduced NADPH, which is required for regenerating reduced glutathione from oxidized glutathione via the enzyme GSH reductase. The present results may indicate that MLT and PTX have an antioxidant effect on the increase of the GSHPx and CAT activities. The combination of MLT with PTX might not provide additional benefits when compared to the use of MLT or PTX alone in the maintenance of antioxidant defense of liver tissues. In conclusion, these results indicate that administration PTX and MLT before liver toxicity occurs may prevent oxidative damage by reducing oxidative stress and increasing the antioxidant GSH-Px and CAT activities. Also, PTX alone and PTX+MLT have a stronger effect than MLT alone in reduction of lipid peroxidation. However, further studies are necessary to explain the preventive effects of MLT and PTX on CCl4 -induced hepatic toxicity.

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Address for correspondence: Tevfik Noyan, Yüzüncü Yıl ¨ Universitesi, Tıp Fakültesi, Biyokimya Bölümü Van, 65200, Türkiye. E-mail: [email protected]; [email protected]