The Effects of Pumpkin Seed Oil on Carbon Tetrachloride Induced Chronic Hepatic Damage in Rats and Detection of Hepatic Apoptosis by Caspase Activity

Liver is a well-recognized target organ that is most exposed to toxic substances due to its anatomical localization and importance of function (Kumar et al., 2017). Acute and chronic exposure of carbon tetrachloride results in hepatotoxicity which shows itself at the level of cell organelles and biochemical alterations in liver tissue (Recknagel et al., 1989). It is well known that this damage is caused by oxidative stress and subsequent free radicals. The free radical derivatives act on unsaturated fatty acids in the cell membrane, producing lipid peroxidation and acting on the liver by disrupting cell membranes of hepatocytes (Basu, 2003; Manibusan et al., 2007). However, it is suggested that liver damage may also be due to the activation of other cells in the liver (endothelial-satellite cells and hepatocytes) by the action of pro-inflammatory mediators released from activated Kupffer cells (Weber et al., 2003). 

The pumpkin (Cucurbita pepo L.), which has grown in the temperate and subtropical regions of the world and has been used in Europe since the 16th century, is quite rich in unsaturated fatty acids and fibres (Eraslan et al., 2013; Andjelkovic et al., 2010). Pumpkin seed is also rich in unsaturated fatty acids, antioxidants, proteins and phytosterols known to have anti-atherogenic and hepatoprotective effects (Makni et al., 2008; Ryan et al., 2007). Studies have shown that pumpkin show estrogenic, antiviral, antibacterial, antifungal, anthelminthic, anticarcinogenic and most importantly antioxidant properties due to these contents (Caili et al., 2006).

This study aimed to determine the effects of pumpkin seed oil (PSO) known to have various biological activities, on CCl4 induced hepatic damage by assaying plasma vitamin E, 8-OHDG, serum SDH (sorbitol dehydrogenase), ALT (alanine aminotransferase) and liver MDA (malondialdehyde), SOD (superoxide dismutase), GPx (glutathione peroxidase) levels and immunohistochemical analyses of apoptosis by caspase-3,-4, -8 and -9 activity of liver tissues in rats.

The rats were maintained in accordance with the Guidelines for Animal Experimentation approved by Erciyes University, Experimental Animal Ethics Committee (permit no: 16/052). CCl4 was obtained from Merck Ltd. and PSO was purchased from Bukas Inc. Co., Izmir, Turkey and content of PSO is given in Table 1.



A total of 80 Wistar rats were divided into eight groups of ten rats each: Group I served as control, receiving vehicle 0.9% NaCl (1.0 mL/kg); Group II was given intraperitoneally CCl4 at a dose of 0.2 mL/kg, 1:1 mixture with corn oil, twice a week for 8 weeks. Groups III, IV and V were daily treated with PSO through gavage for 8 weeks (1, 2 and 3 mL/kg, respectively). Groups VI, VII and VIII were administered with intraperitoneally CCl4 (0.2 mL/kg) twice a week and simultaneously PSO by gavage for 8 weeks (1, 2 and 3 mL/kg, respectively).

All treated animals were anesthetized by ketamine and xylazine (Green et al., 1981) injection and then sacrificed by cervical dislocation 24h after the last administration of CCl4. Systemic necropsy was performed and blood samples were taken. The samples were centrifuged at 3000 rpm for 10 min and the sera were stored in the 20°C before they were analysed. A portion of liver was stored at -80°C for MDA, SOD and GPx analysis until needed. All tissue samples were fixed in 10% neutral buffered formalin solution for light microscopic examinations (Luna, 1968). To demonstrate caspase activity in tissues, the Avidin Biotin Peroxidase Complex (ABC) technique was performed according to the standard procedure provided in the commercial kit (Zymed, Histostain-Plus Kit). Anti-caspase-3 (active) (Novus NB100-56113) (1/2000), anti-caspase-8 (Abcam ab25901) (1/100), anti-caspase-4 (Novus NBP1- 51267) (1/2000) and anti-caspase-9 (Abcam ab25758) (1/100) were used as primary antibodies. As a negative control PBS was applied to liver tissues and as a positive control, primary antibodies were applied to the control tissues recommended by the primary antibody manufacturers.

Serum ALT activity was determined with spectrophotometer by using commercial kits (Biolabo). Serum vitamin E levels were determined spectrophotometrically. Plasma 8-OHdG levels were determined with ELISA by using commercial kits (Northwest Life NWK-8OHDG02). Liver tissue samples were homogenized according to MDA, SOD and GPx measurement procedures and separated into supernatants. Levels of MDA (Cayman), activities of SOD (Cayman) and GPx (Cayman) in liver were determined with ELISA by using commercial kits.

No macroscopic-microscopic lesions were found in the livers from animals of Group I and Groups III and IV (Fig 1). The liver of the Group II showed colour changes varying from dark-red to grey-white. Histopathological examination of the Group II displayed steatosis in the cytoplasm of hepatocytes (Fig 2a). Necrotic changes in the hepatocytes around central vein resulted in pink homogenous mass formation. Inflammatory cell infiltration close to the portal area and Kuppfer cell hyperplasia in parenchyma were observed (Fig 2b). In Group VI, necrosis in hepatocytes, fatty changes in cytoplasm and cell infiltrations were also similar with Group II (Fig 2c, 2d). Severity of cell infiltration and steatosis were lesser in Group II than Group VII (Fig 2e, 2f) and Group VIII (Fig 2g, 2h).



​

While the sections immunostained with caspase-4 and caspase-8 showed negative reaction, caspase-3 and caspase-9 showed mild positive immune reaction within normal apoptotic process in the livers from animals from Group I and Groups III and V (Fig 3). Immuno-histochemically stained sections revealed multiple apoptotic hepatocytes with an intense immune reaction for caspase-3, -8 and -9 in the perivascular cytoplasmic hepatocytes with lipid vacuoles and caspase-4 negative reaction in Group II (Fig 4a, 4b, 4c), Group VI (Fig. 4d, 4e, 4f), Group VII (Fig 4g, 4h, 4i) and Group VIII (Fig 4j, 4k, 4l).

​

​

Effects of PSO application on serum ALT activity, vitamin E, plasma 8-OHdG, liver tissue MDA levels and SOD and GSH-Px activities in the liver treated with CCl4 were given in Table 2. Serum ALT activity was not significantly different between the Group I and the Groups III and IV. The highest serum ALT activity was determined in the Group II and Group VI. In this case, the ALT enzyme activity, which was increased by CCl4 administration and evaluated as indicative of liver function, was not affected by the 1 mL PSO administration. However, the administration of 2 and 3 mL PSO to the CCl4-treated groups significantly reduced ALT activity. Even in the Group VIII, there was no significant difference in the statistical significance of ALT activity when compared with control group values (Table 2).



Vitamin E levels were increased according to the level of pumpkin given to rats. The highest level of vitamin E was detected in the Group V. There was no significant difference in vitamin E levels among the other groups. Plasma 8 OHdG levels were not affected by either CCl4 or PSO administrations. Treatment of CCl4 caused a significant increase in MDA levels in Group II while PSO administration had a non-significant decrease in CCl4+PSO treated groups. The lowest level of SOD and GPx activity were determined in the CCl4-treated group and addition of PSO caused significant increases in liver tissue in a dose dependent way (Table 2).

Chronic administration of CCl4 has been reported to cause liver toxicity resulting in hepatitis, fibrosis and cirrhosis in rats (Basu, 2003; Yehia et al., 2013; Khan et al., 2009; Gutierrez et al., 2010). Free radical metabolites caused by CCl4 toxicity, which mediated by the cytochrome P450 enzyme system, react with unsaturated fatty acids in the cell membrane to initiate lipid peroxidation or break down cell membranes by binding to proteins (Basu, 2003; Manibusan et al., 2007).

It has determined that chronic administration of CCl4 in different doses cause severe necrosis surrounded by a fibrous tissue composed of fibroblasts and collagen bundles, especially in the liver portal area that forms pseudolobulation and inflammatory cell infiltrations, as well as micro- and macrovesicular lipid vacuoles in hepatocytes with ballooning or vacuolar degeneration in rats (Yehia et al., 2013; Khan et al., 2009, Gutierrez et al., 2010). Similarly, in the present study, CCl4 administration at a dose of 0.2 mL/kg was resulting in inflammatory cell infiltration ranged from mild to severe and fibrosis in the hepatic parenchyma. With this, it can be said that CCl4 dependent hepatotoxicity and consequently histopathological changes may be due to lipid peroxidation resulting from the toxic metabolites of CCl4 Manibusan et al. (2007) which are depending on the passage of ions through the cell membrane due to lipid peroxidation, the activation of membrane enzymes and intracellular signal transduction, which is important for the continuity of normal cell physiology. It can also be suggested that increased oxidative stress causes mitochondrial damage in hepatocytes, which can result from reduced mitochondrial oxidation of fatty acids and consequent fatty acid esterification resulting in triglyceride accumulation in hepatocytes (Aranda et al., 2010).

The number of studies using PSO for the therapeutic effect on liver damage induced by CCl4 in rats is limited (Nkosi et al., 2006; Mohamed et al., 2009). In these studies, it has been reported that PSO has been partially normalized histopathological changes in liver damage from CCl4 or other toxic substances. In the present study, parallel to the findings of above researchers showing that liver damage was inhibited with by PSO and the severity of fat vacuoles in the hepatocytes and the inflammatory cells in the portal area decreased depending on the amount of PSO given, which might be due to the hepatoprotective property of PSO.

Hepatocyte apoptosis is an important factor in the development of liver injury in various liver diseases (Canbay et al., 2004). According to studies conducted over the last decade, hepatocyte apoptosis is thought to be the basis of cell death and the first cellular response to many toxic liver diseases (Bilodeau, 2003; Canbay et al., 2004).

Mechanisms that stimulate apoptosis are involved extrinsic and intrinsic signalling pathways (Leach, 1998). The extrinsic apoptotic pathway involves both caspase-8 and caspase-9, whereas intrinsic pathways only involve caspase-9 apoptosis. In the present study, as some researchers have reported (Cao et al., 2014; Karakus et al., 2011; Eckle et al., 2004; Liu et al., 2014) there was an increase in caspase-3 activity in hepatocytes after CCl4 administration. Contrary to the findings of Liu et al. (2014) who suggested an activation of caspase-9 but not caspase-8 in CCl4-treated rats, in the present study CCl4 treatment resulted in the activation of caspase-8 and caspase-9 in the liver of rats. This demonstrates that, CCl4 induces apoptosis by using both the intrinsic and extrinsic pathways. In the present study, decreased activity of caspase-3, caspase-8 and caspase-9 in PSO treated groups may be related to the hepatoprotective effect of PSO in a dose dependent way.

Alanine aminotransferase enzyme activity, a liver-specific enzyme, increases in blood due to loss of cell integrity in hepatic diseases with degenerative, necrotic and parenchymal tissue loss (Evans, 2009). Several studies have showed that significant reductions in ALT levels due to liver damage by administration of PSO (Nkosi et al., 2006; Mohamed et al., 2009). In the present study, administration of 2 and 3 mL/kg PSO to CCl4 treated groups caused a decline in the activities of ALT enzymes in serum compared to the Group II and this may be due to the result of hepatoprotective effects of polyphenol and unsaturated fatty acid contents in PSO (Makni et al., 2008; Ryan et al., 2007; Mohamed et al., 2009). However, administration of 1 mL/kg PSO did not affect ALT activity.

Malondialdehyde, as the end product of lipid peroxidation, leads to the formation of hydrogen peroxide and reactive oxygen species (Kelly et al., 1998). On the other hand, nitric oxide released from Kupffer cells, hepatocytes and endothelial cells was reported to be important mediator against inflammation and tissue damage caused by CCl4 (Breikaa et al., 2013). Consistent with earlier researchers (Yehia et al., 2013; Gutierrez et al., 2010) who reported elevated liver MDA levels in CCl4-induced liver damage at different doses, the MDA levels was also increased in the CCl4-treated groups in the present study indicating to CCl4 triggers lipid peroxidation and leads finally to hepatotoxicity. Nkosi et al., (2006) and Xu (2000) have reported that PSO decreased MDA levels to normal values and these reductions could be attributed to antiperoxidative components of PSO. Similarly, increased MDA levels were numerable decreased in groups treated with CCl4+PSO may be due to the antiperoxidative effect of PSO.

Superoxide dismutase enzyme converts the highly reactive superoxide radical to H2O2; GSH-Px and CAT break down the H2O2 and protects the body by minimizing the potential damage of hydroxyl radicals. The inhibition of these protective mechanisms leads to increased susceptibility to cellular damage induced by free radicals (Valko et al., 2007). In this study, the decrease in liver SOD and GPx activities in the CCl4 group increased with PSO treatment were similar with some researchers (Mohamed et al., 2009; Zaib and Khan, 2014). This is due to the ability of vitamin E, β-Carotene and antioxidant polyphenol components of PSO to counteract the deleterious effects of toxic metabolites of CCl4 (Abdel-Rahman, 2006; Mohamed et al., 2009).

As a result, it is seen that PSO may have beneficial effect on some biochemical and oxidative stress parameter alterations induced by CCl4. Using of PSO at a dose of 3 mL/kg is more effective than the doses of 1 and 2 mL/kg on the histopathological changes of CCl4 induced liver damage. It is thought that PSO may have a therapeutic effect on hepatotoxicity, but this effect is partly reflected to liver tissue. In order to define the full effect of PSO on the hepatotoxicity without creating any adverse effects, further studies investigating different dosages and times are needed.

This study was supported by the Fund of Erciyes University Scientific Research Projects (Project no: TCD-2016-6791).