Iranian Journal of Pharmacology and Therapeutics Razi Institute for Drug Research (RIDR) of Iran University of Medical Sciences and Health Services (IUMS) ISSN: 1735-2657 Vol. 4, Num. 2, 2005, pp. 118-123

Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 2, 2005, pp. 118-123

Protective Effect of N-Acetyl Cysteine in Carbon Tetrachloride-Induced Hepatotoxicity in Rats

NARASIMHANAIDU KAMALAKKANNAN, RAJAGOPALAN RUKKUMANI, KODE ARUNA, PENUMATHSA SURESH VARMA, PERIYASAMY VISWANATHAN and VENUGOPAL PADMANABHAN MENON

Department of Biochemistry (N.K, R.R, K.A., P.S.V., V.P.M.); Department of Pathology (P.V.); Rajah Muthiah Medical College and Hospital, Annamalai University, Annamalainagar, Tamil Nadu, India.

Address correspondence to: Dr. Venugopal P. Menon, Pro-fessor and Chairman, Department of Biochemistry & Center for Micronutrient Research, Annamalai University, Anna-malainagar, Tamil Nadu, INDIA. E-mail: biocmr@sify.com

Received August 12, 2005; Revised October 29, 2005; Accepted November 5, 2005

Code Number: pt05025

ABSTRACT

The present study determines the efficacy of N-acetyl cysteine (NAC) on marker enzymes, lipid peroxida-tion and antioxidants in carbon tetrachloride induced hepatotoxicity in rats. Carbon tetrachloride (CCl₄) (3 mL/kg/week) administered subcutaneously to albino Wistar rats for a period of three months significantly increased the activities of marker enzymes in plasma such as aspartate transaminase, γ-glutamyl trans-ferase and alkaline phosphatase and increased the levels of thiobarbituric acid reactive substances and hydroperoxides in plasma and tissues (liver and kidney). A significant decrease in the levels of plasma antioxidants (glutathione, vitamin C and vitamin E) was also noted. Further, a decrease in the concentra-tion of glutathione and the activities of superoxide dismutase, catalase and glutathione peroxidase in the tissues were observed. N-acetyl cysteine (150 mg/kg) was orally administered to normal and carbon tet-rachloride-treated rats for a period of three months. N-acetyl cysteine decreased the activities of marker enzymes, lipid peroxidation and improved the antioxidant status in carbon tetrachloride-treated rats. But there were no significant alterations in these parameters in normal rats treated with N-acetyl cysteine. Histopathological observations of the liver also showed the protective effect of N-acetyl cysteine in carbon tetrachloride-induced hepatotoxicity in rats. The results of this study show the protective action of N-acetyl cysteine in carbon tetrachloride-induced hepatotoxicity in rats. This is mainly due to the effective antioxi-dant potential of N-acetyl cysteine.

Keywords: N-acetyl cysteine, Hepatotoxicity, Carbon tetrachloride

Carbon tetrachloride is commonly used as a model to evaluate hepatotoxicity [ 1 ]. Carbon tetrachloride me-tabolism begins with the formation of the trichloro-methyl free radical, CCl₃˙ through the action of the mixed function cytochrome P450 oxygenase system of the endoplasmic reticulum [ 2 ]. The CCl₃ radical reacts with various biologically important substances such as amino acids, nucleotides and fatty acids, as well as pro-teins, nucleic acids and lipids. In the presence of oxy-gen, the CCl₃ radical is converted to the trichloromethyl peroxy radical (CCl₃OO˙). This radical is more reactive and is capable of abstracting hydrogen from polyunsatu-rated fatty acids (PUFA) to initiate the process of lipid peroxidation [ 3 ].

Modulation of cellular thiols has been used to pro-tect the hepatocytes against attack by reactive oxygen intermediates and is currently being investigated as a novel therapeutic strategy in different liver pathologies. One of the most extensively studied agents is N-acetyl-L-cysteine, a sulfur-containing amino acid that pos-sesses many biological properties. It is credited as a drug with multiple therapeutic applications [ 4 ]. NAC could significantly interfere with the pathophysiology of free radicals producing drug induced oxidative stress [ 5 ].

Reports have shown that NAC treatment protects against acetaminophen hepatotoxicity in patients [ 6 ] and in rats [ 7 , 8 ]. Also, there are few reports on the protec-tive role of NAC in CCl₄-induced toxicity in patients [ 9 - 11 ] and in rats [ 12 - 15 ]. But there are no detailed reports on the antioxidant defense of NAC in CCl₄-induced hepatotoxicity in a long run. Hence we considered it worthwhile and carried out this investigation to assess the effect of NAC on marker enzymes, nonenzymic and enzymic antioxidants in CCl₄-induced hepatotoxicity in rats.

MATERIALS AND METHODS

Animals

Male albino Wistar rats of body weight 150-180 g were obtained from the Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University and were maintained there. The rats were housed in polypropylene cages lined with husk. They were fed on a standard pellet diet (Agro Corporation Private Ltd., Bangalore, India) and water ad libitum.

Materials

N-acetyl-L-cysteine was obtained from Sigma Chemical Company, St. Louis, MO, USA. CCl₄ was purchased from Merck Ltd., Mumbai, India. All other chemicals and biochemicals used in our study were of high analytical grade.

Experimental Design

In our study, a total of 24 rats were used. The rats were divided into 4 groups of 6 rats each.

• Group I. Normal control rats.
• Group II. Normal rats orally administered with NAC (150 mg/kg body weight) [ 16 ].
• Group III. Rats subcutaneously injected with CCl₄ (3 mL/kg body weight/week) [ 17 ].
• Group IV. Rats orally administered with NAC (150 mg/kg body weight) along with subcutane-ous injection of CCl₄ (3 mL/kg body weight/week).

The experiment was carried out for a period of three months. All the experimental protocols were approved by the Ethical Committee of Annamalai University. After the last treatment, the animals were fasted over-night and killed by cervical dislocation. Blood was col-lected in heparinised tubes. Plasma was separated and used for various biochemical estimations. Liver and kidney were collected in ice-cold containers, washed with saline, homogenised with appropriate buffer and used for various estimations.

In plasma, the levels of marker enzymes such as AST [ 18 ], ALP [ 19 ], GGT [ 20 ] and the levels of TBARS [ 21 ], hydroperoxides [ 22 ], GSH [ 23 ], vitamin C [ 24 ] and vitamin E [ 25 ] were estimated by standard pro-cedures.

In liver and kidney, the concentration of TBARS [ 21 ], HP [ 22 ], GSH [ 23 ], superoxide dismutase [ 26 ], catalase [ 27 ] and glutathione peroxidase [ 28 ] were also estimated.

For histopathological studies, livers from animals of different groups were perfused with 10% neutral forma-lin solution. Paraffin sections were made and stained using hematoxylin-eosin (H&E) stain. After staining, the sections were observed under light microscope and photographs were taken.

Statistical Analysis

Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The values are mean ± S.D. for 6 rats in each group. p-Values < 0.05 were con-sidered as significant.

RESULTS

Effect of NAC on Marker Enzymes

The effect of oral administration of NAC on plasma AST (aspartate transaminase), GGT (γ-glutamyl trans-ferase) and ALP (alkaline phosphatase) activities in normal and CCl₄-induced rats is presented in Table 1 . A significant increase in the activities of these marker en-zymes were observed in CCl₄-treated rats. On treatment with NAC, the activities of these enzymes were found to be significantly decreased.

Effect of NAC on Lipid Peroxidative Products and Nonenzymic Antioxidants in Plasma

Table 2 shows the changes in the levels of plasma TBARS (thiobarbituric acid reactive substances), HP (hydroperoxides), vitamin C, vitamin E and GSH (glutathione) in normal and CCl₄-treated rats. There was a significant increase in the levels of TBARS and hydrop-eroxides and a decrease in vitamin C, vitamin E and GSH in CCl₄-treated rats. Treatment with NAC signifi-cantly decreased the elevated levels of TBARS and hy-droperoxides and increased the levels of vitamin C, vi-tamin E and GSH in plasma.

Effect of NAC on Lipid Peroxidative Products in the Tissues

There was a significant increase in the concentration of TBARS and hydroperoxides in the tissues (liver and kidney) of CCl₄-administered rats ( Table 3 ). Oral administration of NAC significantly decreased the concen-tration of TBARS and hydroperoxides in all the tissues.

Effect of NAC on Tissue Enzymic Antioxidants

Table 4 presents the activities of antioxidant enzymes (superoxide dismutase [SOD] and catalase) in normal and CCl₄-treated rats. The activities of these enzymes were significantly decreased in the tissues of CCl₄-administered rats. On treatment with NAC, the decreased activities of these enzymes were brought back to near normal.

Table 5 shows the concentration of GSH and the activity of glutathione peroxidase in the tissues of CCl₄-administered rats. The concentration of GSH and the glutathione peroxidase (GPx) activity were found to be decreased upon CCl₄ administration. Oral administration of NAC restored the changes brought about by the administration of CCl₄.

Histological Examination of the Liver

Histopathological examination of the liver sections from normal rats showed normal parenchymal architec-ture (Fig 1-A). The liver of rats treated with NAC alone did not show any noticeable alterations (Fig 1-B). In rats treated with CCl₄ alone, the liver sections showed thick-ening of blood vessels (Fig 1-C) and microvesicular fatty changes around portal triad (Fig 1-D). In rats treated with CCl₄ + NAC, only mild sinusoidal dilata-tion was observed (Fig 1-E).

Oral administration of NAC to normal rats did not show significant effect in any of the parameters studied.

DISCUSSION

Carbon tetrachloride induced lipid peroxidation results in changes of structures of the endoplasmic reticu-lum and other membranes, loss of metabolic enzyme activation and reduction of protein synthesis leading to liver damage [ 29 ]. In this study, CCl₄ administration to rats lead to a marked elevation in the levels of plasma AST, GGT and ALP. This might be due to the release of these enzymes from the cytoplasm, into the blood circu-lation rapidly after rupture of the plasma membrane and cellular damage [ 30 ]. Treatment with NAC significantly reduced the levels of these marker enzymes in CCl₄ treated rats. This implies that NAC tends to prevent liver damage, suppresses the leakage of enzymes through cellular membranes, preserves the integrity of the plasma membranes and hence restores these enzymes levels.

Lipid peroxidation as well as altered levels of some endogenous scavengers are taken as indirect in vivo reli-able indices for oxidative stress [ 31 ]. Increased levels of TBARS and hydroperoxides were observed in plasma and tissues of CCl₄-treated rats. Lowered levels of TBARS and hydroperoxides by oral administration of NAC could be related to its antioxidant capacity to scavenge reactive oxygen species. NAC contains free sulfhydryl groups and it may directly react with electro-philic compounds such as free radicals [ 32 ].

Excessive liver damage and oxidative stress caused by CCl₄ depleted the levels of GSH, vitamin C and vi-tamin E in our study. Oxidative stress induced by CCl₄ results in the increased utilisation of GSH and subse-quently the levels of GSH is decreased in plasma and tissues. Utilisation of vitamin E is increased when oxidative stress is induced by CCl₄ and this shows the pro-tective role of vitamin E in mitigating the elevated oxi-dative stress. Vitamin C scavenges and destroys free radicals in combination with vitamin E and glutathione [ 33 ]. It also functions cooperatively with vitamin E by regenerating tocopherol from the tocopheroxyl radical [ 34 ]. A decrease in the levels of vitamin C may indicate increased oxidative stress and free radical formation in CCl₄-induced liver injury.

N-acetyl cysteine treatment effectively restored the depleted levels of these nonenzymic antioxidants. NAC could significantly interfere with the pathophysiology of free radical producing drug induced oxidative stress [ 5 ]. Wong et al. have reported the ability of NAC in regulat-ing GSH concentration and thus protect liver damage from reactive metabolites formed from CCl₄ [ 15 ]. In-crease in GSH levels could also contribute to the recy-cling of other antioxidants such as vitamin E and vita-min C [ 35 ].

A major defense mechanism involves the antioxi-dant enzymes including SOD, catalase and GPx which convert active oxygen molecules into non-toxic compounds.CCl₄-administration decreased the activities of these antioxidant enzymes and GSH concentration in the tissues. Oral administration of NAC restored the activities of these enzymes and glutathione in rats treated with CCl₄. NAC contributes significantly to the intracellular antioxidant defense system by acting as a powerful consumer of superoxide, singlet oxygen and hydroxyl radicals [ 36 ].

NAC induces its beneficial effect mainly through maintaining –SH groups of enzymes and membrane proteins in the reduced state [ 37 ]. NAC can prevent the hepatic GSH depletion as well it can slow the decrease of hepatic GSH. The hepatoprotective effects of NAC may also be due to its ability to enhance glutathione production by providing more substrate for reactive intermediates that promote detoxification mechanisms [ 38 ]. This also may be the reason for the restoration of other antioxidant enzymes such as SOD and catalase.

Histopathological examination of the liver also pro-vided supporting evidence for our study. Rats treated with NAC reduced the damage caused by CCl₄ admini-stration. This clearly indicates the membrane stabilizing effect of NAC by scavenging free radicals and preserv-ing the integrity of the membranes. The overall results of our study confirm the protective effect of N-acetyl cysteine in CCl₄-induced hepatotoxic-ity in rats by its ability to stabilize cell membranes, scavenge free radicals and antioxidant properties. The present investigation has also confirmed the usefulness of NAC as an effective hepatoprotectant.

ACKNOWLEDGEMENT

We thank UGC for sanctioning a project. The first author is a Junior Research Fellow in the project.

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