Khaled A. Bayoumi, Alaa M. Shehab, Iman F. Gaballah and Ehab R. Ibrahim*

THE POTENTIAL PROTECTIVE ROLE OF AMINOGUANIDINE AND α-LIPOIC ACID IN AMELIORATION OF CARBON TETRACHLORIDE-INDUCED HEPATOXICITY IN ALBINO RATS

Khaled A. Bayoumi, Alaa M. Shehab, Iman F. Gaballah and Ehab R. Ibrahim*

Departments of Forensic Medicine & Clinical Toxicology and Histology *, Faculty of Medicine, Cairo University

ABSTRACT

Background: Carbon tetrachloride (CCl4) is a potent environmental hepatotoxin which is a well-known model compound for producing toxicity by generation of free radicals. Hypothesis: It has been shown that aminoguanidine (AG) is a nitric oxide synthase inhibitor and α-lipoic acid (ALA) is a potent scavenger of a variety of free radicals. AIM: To determine the protective roles of aminoguanidine and lipoic acid against CCl4 hepatotoxicity in albino rats. MATERIALS AND METHODS: Male albino rats were randomly divided into five groups (8 rats/group). Groups I and II included normal control and CCl4 groups respectively; group III was given AG (100 mg/kg 2% solution in saline); group IV was given ALA (100mg/kg 1% solution in saline); group V was given AG and ALA in the same previously mentioned doses. Each of the groups III, IV and V received single oral daily dose of drugs for 2 weeks followed by concurrent administration of subcutaneous CCl4 (1 ml/kg) on alternate days for a week. Liver damage was depicted biochemically and histologically. RESULTS: Rats insulted with CCl4 showed significant reduction of hepatic function and marked alterations in the morphological changes of the liver. In addition, CCl4 violated the antioxidant status as evidenced by increase in lipid peroxidation in liver tissue as well as decrease reduced glutathione (GSH) and activities of superoxide dismutase (SOD) and catalase (CAT). Whereas pretreatment with either AG or ALA alone reduced these changes and attenuated the pathological effects of CCL4 induced liver injury, the combination of both drugs was better than each drug alone. CONCLUSION: These results clearly show that AG and / or ALA treatment significantly antagonize the effects of CCl4 on the liver. Therefore, these two components could be potential hepatoprotective agents.

KEY WORDS: Carbon tetrachloride; Aminoguanidine; Lipoic acid; Liver; Albino rats.

INTRODUCTION

Liver diseases are one of the most serious health problems in the world today but, despite great advances in modern medicine, their prevention and treatment lines still remain limited (Hussain et al., 2012). The liver is particularly susceptible to chemically induced injury due to its extensive metabolic capacity and cellular heterogeneity. Oxidative stress occurs due to imbalance between reactive oxygen species (ROS) formation and scavenging by antioxidants. Excess generation of ROS can cause oxidative damage to biomolecules resulting in lipid peroxidation and carcinogenesis (Khan and Sultana, 2009). Till date available modern drugs have not been able to come up with a satisfactory answer for liver disorders because of high cost and additional adverse effects. It is therefore necessary to search for alternative drugs for the treatment of liver diseases to replace the currently used drugs of doubtful efficacy and safety (Kumar et al., 2013).

Carbon tetrachloride (CCl4) is a colorless, volatile, heavy and nonflammable liquid well known for hepatic and renal toxic actions. The metabolism of CCl4 into trichloromethyl and peroxy trichloromethyl free radicals has been reported to cause liver cirrhosis, steatosis and necrosis (Weber et al., 2003). Furthermore, CCl4 treatment has been used as a model to induce fatty liver for studying possible interacting effects of a compound or a treatment (Recknagel, 1967). It should be noted that CC14 produces an experimental damage that histologically resembles viral hepatitis (James and Pickering, 1976).

Aminoguanidine (AG) is an antioxidant substance that acts as a competitive and selective inhibitor for inducible nitric oxide synthase (iNOS) (Ara et al., 2006). Nitric oxide (NO) is an important mediator of hepatotoxicity (Hortelano et al., 1999). It is derived from two sources in liver. Hepatocytes and Kupffer cells contain (iNOS), the activity of which is markedly increased in inflammation. Endothelial cells contain constitutive NO synthase (eNOS). Nitric oxide is known to react with superoxide radical, forming the more potent oxidizing agent peroxynitrite (ONOO-) (Ischiropoulos et al., 1992). The latter can react directly with sulfhydryl residues in the cell membranes leading to lipid peroxidation as well as with DNA resulting in cytotoxicity (Radi et al., 1991). The enzyme iNOS is expressed during the development of cirrhosis. Therefore, the prevention of iNOS expression may have an important role in both cirrhosis and endotoxaemia (Heller et al., 2000). The action of AG has been known to be associated with reduction of peroxinitrite, which has deleterious roles in inductions of inflammation, lipid peroxidation and DNA fragmentation (Potenza et al., 2009). Previous investigations have also demonstrated that AG reduced hydrogen peroxide (H2O2) induced intracellular hydroxyl radical formation and apoptosis, further demonstrating a potential antioxidant activity (Ihm et al., 1999). AG protection could be through decreasing the metabolic activation of CC14 by directly inhibiting P4502E1, the isoenzyme most effective in the activation of CC14 (Doglru-Abbasogllu et al., 2002).

There are conflicting data in the literature to support both a protective and a cytotoxic role of NO in liver and kidney (Gardner et al., 2002; Li et al., 2003 and Liu et al., 2003). Furthermore, pretreatment with the selective iNOS inhibitor, AG, protects against hepatotoxicity induced by endotoxin (Shiomi et al., 1998) or hepatotoxic agents as paracetamol (Gardner et al., 2002) and results in renal failure improvement in cirrhotic rats (Islas-Carbajal et al., 2005).

Alpha lipoic acid (ALA) has become one of the most recognized and in-demand health supplements. It is a naturally occurring, short-chain fatty acid containing two sulfur molecules. ALA is both water and fat soluble and, therefore, is distributed in both the cellular membranes and the cytosol in plants and animals (Wollin & Jones, 2003). ALA plays an essential role in mitochondrial dehydrogenase reactions (Packer et al., 1997) and has found considerable attention as a very potent antioxidant that fights free radicals and improves recovery (Moini et al., 2002). Due to its readily absorption throughout the body, it may protect the integrity of cells ranging from the brain to the liver (Keith et al., 1997). It shows beneficial effects in oxidative stress conditions because of its synergistic action with other antioxidants (Suzuki et al., 1993). ALA is taken up and reduced by cells to dihydrolipoic acid (DHLA), which can be released into the extracellular medium (Arner et al., 1996). ALA and DHLA can serve as powerful antioxidants through several mechanisms, including scavenging of free radicals, chelation of metal ions, and regeneration of endogenous and exogenous antioxidants, such as ubiquinon, vitamins C and E, and glutathione (Kozlov et al., 1999). There is growing evidence that orally delivered ALA may not be used as a metabolic cofactor but, instead, has a unique property of neutralizing free radicals without itself being consumed in the process (Shay et al., 2009).

Given that oxidative stress plays a basic role in CCl4 hepatotoxicity, the present study was undertaken to explore the influence of AG and/or ALA on CCl4 induced hepatic toxicity. To this end, the antioxidant and the radical scavenging activities of AG and ALA were evaluated by estimating the activities of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), as well as the extent of lipid peroxidation in the liver tissue. In addition, the study also examined the protective effects of AG and/or ALA on liver functions in CCl4 intoxicated rats. To our knowledge no report is available until now, for the protective action of combined aminoguanidine and lipoic acid on CCl4-induced toxicity in vivo.

MATERIALS AND METHODS

Experimental Animals:

Random – bred, adult male albino rats (Wistar strain) weighing approximately 125 – 150 gm were obtained from the Animal House of National Research Centre, Dokki, Cairo, Egypt. Animals were evaluated prior to initiation of the study to ensure a healthy condition and acclimation to the study environment. Clinically acceptable animals were randomly assigned into 5 groups (8 animals / group), so that there were no statistically significant differences among group body weight means. Four animals were housed in each stainless steel wire mesh cage on a bedding of wood chips at constant temperature of 25 ± 2o C, illumination (12 h light/dark) and had free access to commercial food and tap water, ad libitum and left for an initial adaptation period of 1 week before any experimental manipulation.

All animals received human care according to the criteria outlined in “Guide for the care and use of laboratory animals” prepared by the National Academy of Sciences and published by the National Institute of Health.

Test Materials:

Aminoguanidine, alpha-lipoic acid were purchased from Sigma, St. Louis, MO, USA. Carbon tetrachloride (CCl4) was obtained from Egyptian company for chemicals and pharmaceuticals (ADWIA). Biochemical kits were purchased from the Gamma Trade Company for Pharmaceutical and Chemicals, Dokki, Egypt.

Experimental groups:

Rats were randomly divided into:

Group I rats served as normal (vehicle) control and received oral saline at equivalent volume of that of the test drugs in the presence of 0.05 mol/l NaOH for 2 weeks followed by concurrent subcutaneous (sc) administration of liquid Paraffin (LP) in the lower abdomen 1 ml/ kg on alternate day for one week; group II rats were given oral saline at equivalent volume of that of the test drugs in the presence of 0.05 mol/l NaOH for 2 weeks followed by concurrent administration of carbon tetrachloride (CCl4) subcutaneously (sc) in the lower abdomen, in a suspension of liquid paraffin (1: 2 v/v) at the dose of 1 ml/kg BW on alternate days for a week for induction of hepatotoxicity (Achilya et al., 2003); group III was given aminoguanidine (AG) (100 mg/kg 2% solution in saline) (Ahmed et al., 2011) ; group IV was given alpha lipoic acid (ALA) (100mg/kg 1% solution in saline in the presence of 0.05 mol/l NaOH) (Pari and Murugavel, 2004); group V was given AG and ALA in the same previously mentioned doses.

Groups (III, IV and V) received single oral daily dose of drugs through a gastric tube for 2 weeks followed by concurrent administration of CCl4 on alternate days for a week.

Different doses of above mentioned drugs, LP and CCl4 were administered to rats daily in the morning according to the ethical guidelines for the care of the laboratory animals (Zimmerman, 1983). Two hours after the last administration, blood samples were collected by cardiac puncture under chloral hydrate (6 ml. of 7 % chloral hydrate / kg, Sigma, St. Louis, USA) anesthesia. Blood samples were placed in heparinized tubes, allowed to clot and centrifuged at 3000×g for 10 min to obtain sera which were used to determine SGOT, SGPT, SALP and total bilirubin. Immediately after blood collection, the animals were scarified by cervical dislocation and liver specimens were promptly removed and perfused with phosphate buffer solution (PBS) [PH = 7.4] containing 0.16 mg/ml heparin. Then livers were isolated and dissected into 2 parts. The first part was taken and placed in 10% neutral formalin, dehydrated in ascending series of ethanol, cleared in xylol and then embedded in paraffin for histopathological examination.

The second part was immersed immediately in liquid nitrogen and kept at -80o C for determination of malondialdehyde (MDA), reduced glutathione (GSH), contents, superoxide dismutase (SOD) and catalase (CAT).

Clinical chemistry:

Serum biochemical estimations:

Estimation of total bilirubin “mg/dl” (Perry et al., 1983); glutamic oxalacetic transaminase (SGOT) “unit/dl” and glutamic pyruvic transaminase (SGPT) “unit/dl” (Reitman and Frankel, 1975) and alkaline phophatase (SALP) “unit/l” (Tietz and Rinker, 1983).

Liver biochemical estimations:

The levels of lipid peroxidation i.e. thiobarbituric acid reactive substances (TBARS) in the liver tissue were measured according to Okhawa et al., (1979). The levels of lipid peroxides were expressed as μmoles of malondialdehyde (MDA)/g of liver tissue. The reduced glutathione (GSH) content of liver tissue was determined as per reported method of Ellman, (1959) and expressed as μg/g of liver tissue. The superoxide dismutase (SOD) and catalase (CAT) activities in liver tissue were assayed as per the methods of Kakkar et al., (1984) and Aebi, (1974) respectively. The SOD activity was expressed as unit/mg of liver tissue and CAT was expressed in terms of μmol of hydrogen peroxide decomposed/min/mg of liver tissue.

Histopathology:

Liver sections of 6 microns thick were prepared and stained with haematoxylin and eosin (Drury and Wallington, 1980). The extent of liver damage was assessed under light Olympus microscope.

Statistical analysis:

The data were analyzed using IBM computer and SPSS- P.C 4.1 statistical package. All results were expressed as the mean ± standard error of mean (SEM). They were analyzed for statistical significance by one-way ANOVA followed by Dunnett's post hoc test of significance. P < 0.05 was statistically significant.

RESULTS

Number of deaths of animals:

We started the study with 8 rats/normal control group and 14 rats/CCl4 group. Groups III, IV and V were 10 rats/group. In CCl4 group, 6 rats died while in the treatment groups 2 rats died and the extreme values were excluded giving 8 rats/group used for statistical analysis.

Serum biochemical parameters: there were significant elevations (p < 0.001) of SGOT, SGPT, SALP and serum bilirubin in the CCl4 group as compared to the normal control group. Treatment with AG, ALA or both significantly (p < 0.001) reduced the SGOT, SGPT, SALP and serum bilirubin levels towards the normal values (Table 1). Rats of Group-V showed comparable SGOT, SGPT, SALP and serum bilirubin levels to that of normal control rats.

Liver biochemical parameters: Treatment of rats with CCl4 increased lipid peroxidation as evident by significant rise (p < 0.001) in the levels of MDA in CCl4 animals as compared to normal control group. Treatment with AG, ALA or both significantly (p < 0.001) reduced the MDA levels when compared with CCl4 animals (Table 2). Significant depletion in the level of GSH was noticed in CCl4 group (p < 0.001) as compared with normal control group. Significant elevation in GSH level towards normal level occurred on administration of AG (p < 0.05) and ALA or combination of AG & ALA (p < 0.001) as compared with CCl4 group (Table 2). There were significant (p < 0.001) decrease in SOD and CAT activities in CCl4 group compared with normal control group. Administration of AG (p < 0.05) and ALA or combination of AG & ALA (p < 0.001) significantly restored SOD and CAT activities towards normal values when compared with CCl4 animals (Table 3).

Histopathological Results:

Liver sections of control rats showed the normal histological structure of hepatic lobules (Figure 1). Livers challenged with CCl4 showed centrilobular hepatocellular necrosis, marked fatty and cellular infiltration (Figure 2). However, pretreatment with either AG or ALA resulted in partial improvement of hepatocytes and preserved hepatic architecture (Figures 3 & 4), while pretreatment with both drugs together resulted in marked improvement in the hepatic architecture nearly to the control groups (Figure 5).

DISCUSSION

Deaths due to hepatic disorders are increasing in number and there is only limited number of drugs available for the treatment. Currently the researchers across the world are focusing their attention to develop an ideal hepatoprotective agent. Since reactive oxygen species are very important causes of liver damage, an antioxidant may be a useful tool for protecting the liver cells (Jain et al., 2008).

In the present study, CCl4 caused a significant increase in the mean values of SGPT, SGOT, SALP and serum bilirubin. Plasma membrane, endoplasmic reticulum, mitochondria, and Golgi apparatus are the main subcellular structures affected by CCl4 exposure. Damage to plasma membrane of hepatocytes results in release of enzymes in circulation (Reynolds, 1963). On the other hand, serum bilirubin levels reflect the functional state of hepatic cells (Gupta et al., 2004). These results are in accordance with previous studies by Drotman & Lawhorn (1978); Mukherjee (2003); Weber et al., (2003); Soni et al., (2008); Karwani & Sisodia (2011) and Feroz et al., (2013). Kumar et al., (2013) reported similar results but the rise in bilirubin level was not to the same extent as SGPT, SGOT and SALP. This could be explained by the fact that blood collection in Kumar et al., (2013) study was 24 hours after CCl4 administration while bilirubin reaches peak serum level in the second hour after CCl4 administration and probably declines afterwards (Gumucio, 1989).

The findings of the present study indicated that hepatic injury was reduced by aminoguanidine (AG) or alpha lipoic acid (ALA) drugs with a reduction in plasma levels of SGOT and SGPT as well as the cell membrane enzyme SALP and serum total bilirubin. The study also indicated the usefulness of combining both AG and ALA. The protective effect of AG against the enzyme leakage seems to be through the liver cell membrane integrity restoration and is independent of any effects on liver GSH contents (Raza et al., 2003). It is noteworthy that ALA protects the liver against CCl4 toxicity by regenerating endogenous antioxidants and inhibiting the elevation of liver aminotransferases (Morakinyo et al., 2012).

These results are in agreement with the results obtained by Raza et al., (2003) and Ahmed et al., (2011), who illustrated the hepatoprotective effect of AG and Sivaprasad et al., (2004); Abdel-Zaher et al., (2008); Foo et al., (2011) and Morakinyo et al., (2012) who demonstrated the ALA hepatoprotective effects. In contrast, AG treatment against CCl4 induced hepatoxicity was ineffective in reducing the leakage of hepatic enzymes according to reports of some other investigators (Skamarauskas et al., 1996 and Kamel et al., 2011). This opposition may be explained on the basis of using different dose regimens.

The present study revealed increased lipid peroxidation as evidenced by a significant rise in liver MDA and significant decrease in superoxide dismutase (SOD) and catalase (CAT) activities in CCl4 treated rats. This enhanced lipid peroxidation results in tissue damage and failure of antioxidant defense mechanisms (Ashok et al., 2001). SOD and CAT are endogenous enzymatic antioxidants present in all oxygen metabolizing mammalian cells involved in the clearance of superoxide and hydrogen peroxide (H2O2) , so each of them is an index of increased H2O2 production (Meneghini, 1997). GSH, one of the major tripeptide non-enzymatic biological hepatic antioxidants, is involved in removal of free radicals and a substrate for glutathione peroxidase. Deficiency of GSH within the living organisms can lead to tissue damage and injury (Leeuwenburgh and Ji, 1995). A significant decrease in liver GSH was observed in CCl4 treated rats in the present study. This may be due to enhanced substrate utilization by glutathione peroxidase, as there is a direct correlation between GSH depletion and enhanced lipid peroxidation (Al-Shabanah et al., 2000).

Numerous studies reported similar rise in MDA (Pramod et al., 2008; Soni et al., 2008; Neetu & Sangeeta, 2010 and Taye & Abdel-Raheem, 2012) and decrease in SOD, CAT and GSH (Lin et al., 2008 and Dikshit et al., 2011) on CCl4 intoxication.