Diquat-induced oxidative damage in hepatic microsomes: effects of antioxidants.

The ability of the redox cycling compound, diquat, to induce lipid peroxidation and oxidative damage was investigated using hepatic microsomes. Antioxidants, with demonstrated efficacy in physical models of oxidative stress, were examined in a diquat model. Diquat (10 microM-3 mM) induced lipid peroxidation (TBARS) in hepatic microsomes prepared from Fischer 344 rats. Diquat (1 mM) also increased protein carbonyl formation, NADPH oxidation and superoxide anion radical production (acetylated cytochrome c reduction). The novel antioxidants U-74,006F, U-78,517G and the known antioxidant, DPPD, decreased diquat-induced lipid peroxidation to levels below that of the control. These antioxidants also decreased protein carbonyl formation caused by diquat. U-74,006F and U-78,517G reduced NADPH oxidation slightly; although this inhibition was statistically significant, the biological significance is questionable. DPPD had no effect on this parameter. U-78,517G inhibited the reduction of acetylated cytochrome c slightly, whereas the other antioxidants had little effect. Thus overall, the increase in NADPH oxidation and the production of superoxide anion by redox cycling of diquat were not substantially affected by antioxidants. Neither did the test compounds show evidence of activity as iron chelators. This leads to the suggestion that antioxidants are preventing diquat-induced oxidative damage by scavenging lipid peroxyl radicals and preventing the propagation of the lipid peroxidation process.     

Protective effect of L-theanine on carbon tetrachloride-induced acute liver injury in mice

We studied effects of L-theanine, a unique amino acid in tea, on carbon tetrachloride (CCl(4))-induced liver injury in mice. The mice were pre-treated orally with L-theanine (50, 100 or 200 mg/kg) once daily for seven days before CCl(4) (10 ml/kg of 0.2% CCl(4) solution in olive oil) injection. L-theanine dose-dependently suppressed the increase of serum activity of ALT and AST and bilirubin level as well as liver histopathological changes induced by CCl(4) in mice. L-theanine significantly prevented CCl(4)-induced production of lipid peroxidation and decrease of hepatic GSH content and antioxidant enzymes activities. Our further studies demonstrated that L-theanine inhibited metabolic activation of CCl(4) through down-regulating cytochrome P450 2E1 (CYP2E1). As a consequence, L-theanine inhibited oxidative stress-mediated inflammatory response which included the increase of TNF-α and IL-1β in sera, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in livers. CCl(4)-induced activation of apoptotic related proteins including caspase-3 and PARP in mouse livers was also prevented by L-theanine treatment. In summary, L-theanine protects mice against CCl(4)-induced acute liver injury through inhibiting metabolic activation of CCl(4) and preventing CCl(4)-induced reduction of anti-oxidant capacity in mouse livers to relieve inflammatory response and hepatocyte apoptosis.     

The antioxidant and antifibrogenic effects of the glycosaminoglycans hyaluronic acid and chondroitin-4-sulphate in a subchronic rat model of carbon tetrachloride-induced liver fibrogenesis

Hepatic fibrosis involves the interplay of many factors including reactive oxygen species. Recent reports described antioxidant properties of glycosaminoglycans (GAGs). Since several findings have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) may act as antioxidant molecules, the aim of this research was to evaluate the antioxidant effects of HYA and C4S treatment in a rat model of liver fibrosis. The effect on tissue inhibitors of metalloproteinases (TIMPs) was also studied. Liver fibrosis was induced in rats by eight intraperitoneal injections of CCl4, twice a week for 6 weeks. HYA or C4S alone (25 mg/kg) or HYA and C4S in combination (12.5 + 12.5 mg/kg) were administered daily by the same route during the 6 weeks. At the end of the 6-week treatment period (24 h after the last dose of GAGs), the following parameters were evaluated: (1) serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, as index of hepatic cell disruption; (2) hepatic conjugated dienes (CD), as index of lipid peroxidation; (3) hepatic TIMPs activity and expression; (4) hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, as index of endogenous defences; (5) hepatic hydroxyproline, as index of collagen deposition. CCl4-induced liver fibrosis enhanced lipid peroxidation and TIMPs activation, increased ALT and AST, depleted antioxidants SOD and GPx, and caused collagen deposition in liver tissue. Treatment with GAGs, especially when in combination, successfully reduced ALT and AST rise, lipid peroxidation by evaluating conjugated dienes, TIMPs activation and mRNA expression, partially restored SOD and GPx activities, and limited collagen deposition in the hepatic tissue. The data obtained showed that these molecules were able to limit hepatic injury induced by chronic CCl4 intoxication and especially limited liver fibrosis. They also confirm that HYA and C4S may exert antioxidant mechanism, while reduction of TIMPs expression suggests that GAGs may influence MMPs and TIMPs imbalance in liver fibrosis.     

Administration of the tris salt of alpha-tocopheryl hemisuccinate inactivates CYP2E1, enhances microsomal alpha-tocopherol levels and protects against carbon tetrachloride-induced hepatotoxicity

A series of tocopherol compounds were examined for their capacity to protect against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Of the tocopherol compounds tested in our study, only the tris salt of d-alpha-tocopheryl hemisuccinate (TS-tris) protected against CCl4-induced hepatotoxicity. The administration of d-alpha-tocopherol (alpha-T) and the nonhydrolyzable tocopherol ether, d-alpha-tocopheryloxybutyrate tris salt (TSE-tris), failed to protect against CCl4-induced hepatotoxicity. TS-tris was the only tocopherol which significantly decreased CYP2E1 activity after 18 h. This decrease in CYP2E1 activity is likely to limit the activation of CCl4 and protect against CCl4-induced hepatotoxicity. Our results also suggest that TS-tris protection against CCl4-induced hepatotoxicity correlates with the enhanced capacity of TS-tris to deliver alpha-T and increase the antioxidant status of hepatocytes. TSE-tris did not increase cellular alpha-T levels, while administration of TS-tris produced large increases in alpha-T levels in liver homogenates as well as in liver nuclei, microsomes, mitochondria and plasma membranes. This enhanced ability to deliver tocopherol equivalents to parenchymal liver cells may be related in part to the ability of TS-tris to form liposomes in aqueous solutions. TS-tris administration protected against CCl4-induced microsomal lipid peroxide formation and inactivation of the microsomal enzyme glucose-6-phosphatase (G6Pase). Supplementation of animals with alpha-T protected against microsomal lipid peroxide formation but not against the inactivation of G6Pase. Based on our findings, we propose that high cellular levels of alpha-T protect against CCl4-induced hepatotoxicity by scavenging CCl4 radicals as well as protecting against lipid peroxidation. Our results do not support the importance of microsomal lipid peroxidation as an early event in acute CCl4-induced hepatic necrosis.     

Hepatoprotective activity of Luffa acutangula against CCl4 and rifampicin induced liver toxicity in rats: a biochemical and histopathological evaluation

Hepatoprotective activity of hydroalcoholic extract of Luffa acutangula (HAELA) against carbon tetrachloride (CCl4) and rifampicin-induced hepatotoxicity in rats was evaluated and probable mechanism(s) of action has been suggested. Administration of standard drug- silymarin and HAELA showed significant hepatoprotection against CCl4 and rifampicin induced hepatotoxicity in rats. Hepatoprotective activity of HAELA was due to the decreased levels of serum marker enzymes viz., (AST, ALT, ALP and LDH) and increased total protein including the improvement in histoarchitecture of liver cells of the treated groups as compared to the control group. HAELA also showed significant decrease in malondialdehyde (MDA) formation, increased activity of non-enzymatic intracellular antioxidant, glutathione and enzymatic antioxidants, catalase and superoxide dismutase. Results of this study demonstrated that endogenous antioxidants and inhibition of lipid peroxidation of membrane contribute to hepatoprotective activity of HAELA.     

Protective effects of an extract of young radish (Raphanus sativus L) cultivated with sulfur (sulfur-radish extract) and of sulforaphane on carbon tetrachloride-induced hepatotoxicity

The protective effects of an extract of young radish (Raphanus sativus L) cultivated with sulfur (sulfur-radish extract) and of sulforaphane, an isothiocyanate, on carbon tetrachloride (CCl(4))-induced liver injury were observed in mice. CCl(4) produced a marked increase in the serum level of alanine aminotransferase (ALT), primed lipid peroxidation, and resulted in intense necrosis due to oxidative stress. Oral administration of the sulfur-radish extract and of sulforaphane after CCl(4)-induced liver injury both decreased the serum level of ALT, reduced the necrotic zones, inhibited lipid peroxidation, and induced phase 2 enzymes without affecting cytochrome P450-2E1 (CYP2E1). These results suggest that the administration of the sulfur-radish extract and of sulforaphane may partially prevent CCl(4)-induced hepatotoxicity, possibly by indirectly acting as an antioxidant by improving the detoxification system.     

The antioxidative activity of drugs in the liver microsomal system of rats]

The antioxidative properties of drugs--diethylcarbamazine citrate--DECC, dipyridamole-DP, levamisole and labinzarit--have been investigated in various microsomal lipid peroxidation (LPO) models: NADPH-, ascorbate- and CCl4-dependent. The most strong antioxidant of direct action turned out to be DP, DECC exhibited the antioxidative properties as a result of metabolic activity in monooxygenases system of rat liver microsomes. Levamisole and labinzarit turned out to be weak antioxidants. The control of microsomal membrane stability against Fe(2+)-ADP, NADPH-induced LPO, after being isolated from rat liver after the action of CCl4 without and with DECC, showed that DECC protected microsomal membranes from CCl4 in vivo and they remained stable against LPO in vitro.     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.     

Effects of melatonin on carbon tetrachloride-induced changes in rat serum

Carbon tetrachloride (CCl4) is a volatile organic chemical, which causes tissue damage, especially to the liver and kidney. In experimental animals it has been shown to be carcinogenic. This study was designed to evaluate the effects of exogenous melatonin administration on the CCl4-induced changes of some biochemical parameters in rat blood. Twenty-four male Wistar rats were randomly divided into three equal groups: Control, CCl4 and CCl4 plus melatonin (CCl4+MEL). Rats in CCl4 group were injected subcutaneously with CCl4 0.5 ml/kg in olive oil while rats in CCl4+MEL group were injected with CCl4 (0.5 ml/kg) plus melatonin (25 mg/kg in 10% ethanol) every other day for one month. Control rats were treated with olive oil. Serum urea, creatinine, total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total and conjugated bilirubin, alkaline phosphatase (ALP), gamma-glutamyl transferase (gamma-GT), total iron, and magnesium levels were determined. Serum AST, ALT, total and conjugated bilirubin, ALP, gamma-GT, and total iron levels were significantly higher in CCl4-treated rats than in the controls, while urea, total protein, and albumin levels were significantly lower. Melatonin treatment did not cause a significantly change in serum urea, total protein, and albumin levels. However, the elevations in AST, ALT, total and conjugated bilirubin, ALP, gamma-GT, and total iron levels induced by CCl4 injections were significantly reduced by melatonin. On the other hand, melatonin administration significantly decreased serum magnesium levels. These results indicate that melatonin could be a protective agent against the CCl4 toxicity in rats, most likely through its antioxidant and free radical scavenger effects.     

Inhibition of S-(1,2-dichlorovinyl)-L-cysteine-induced lipid peroxidation by antioxidants in rabbit renal cortical slices: Dissociation of lipid peroxidation and toxicity

Precision-cut, rabbit renal slices were used to examine the effects of three novel antioxidants (U-74006, U-74500, and U-78517) on S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced lipid peroxidation and toxicity. Slices exposed to DCVC showed a dose- and time-dependent increase in lipid peroxidation (TBARS) and a decrease in cellular viability, as evidenced by the loss of intracellular potassium, during the course of a 3 hour incubation. Subsequent studies employed DCVC concentrations of 100 μM. Microemulsion formulations of U-78517, U-74500, and U-74006 (100 μM) inhibited DCVC-induced lipid peroxidation by 100±, 50±, and <5% (not significant), respectively. However, none of these antioxidants had a significant effect on DCVC-dependent cytotoxicity, as indicated by intracellular potassium release. The effects of U-78517, the most potent of the three antioxidants, were similar to those observed with two model antioxidants, diphenyl-p-phenylenedi-amine (DPPD) and the iron chelator, deferoxamine. Aminooxyacetic (AOAA), an inhibitor of renal cysteine conjugate β-lyase, had only a minimal effect on DCVC-induced lipid peroxidation, and no effect on toxicity. These data represent the first report of DCVC-induced lipid peroxidation in rabbit renal cortical slices, a system which has been widely used to investigate mechanisms of nephrotoxicity, including that induced by DCVC. Our results demonstrate that DCVC-induced lipid peroxidation in renal slices can be inhibited by a variety of antioxidant compounds operating by different mechanisms. Because inhibition of lipid peroxidation had minimal effect on DCVC-dependent cytotoxicity, the data suggest that DCVC-induced lipid peroxidation is not a major mechanism in the cytotoxicity induced by this compound.     

In vitro and in vivo evaluation of antioxidant activity of Trichosanthes cucumerina aerial parts

The present study was conducted to determine whether aerial parts of Trichosanthes cucumerina extracts can exert significant antioxidant activity. The antioxidant activity of a hot water extract (HWE) and a cold ethanolic extract (CE) of T. cucumerina aerial parts was evaluated by assessing its (a) radical scavenging ability and prevention effect of lipid peroxidation in vitro, and (b) effects on lipid peroxidation and antioxidant enzyme activities, in vivo.In vitro antioxidant assays (DPPH, TBARS and carotene-linoleic acid assays) clearly demonstrated the antioxidant potential of HWE and CEE. Moreover, HWE increased SOD: by 91.2% and GPX by 104.4% while CEE increased SOD: by 115.5% and GPX by 96.4%) in CCl4-induced rats. Treatments with HWE and CE prevented the accumulation of lipid peroxidation products by 30.5% and 33.8%, respectively, in liver tissues compared to the rats exposed only to CCl4. In conclusion, the present investigation demonstrates for the first time that components in T. cucumerina aerial parts can exert significant antioxidant activity in vivo and in vitro.     

The critical steady-state hypoxic conditions in carbon tetrachloride-induced lipid peroxidation in rat liver microsomes

Defined steady-state oxygen partial pressures (PO2) were maintained constant with an oxystat system to study carbon tetrachloride (CCl4)-induced lipid peroxidation and oxygen uptake in rat liver microsomes. The initial rates of oxygen uptake and malondialdehyde formation indicated drastically increasing lipid peroxidation by decreasing PO2, attaining a maximum between 1-10 mmHg (0.1-1.3 kPa). Under these conditions, at the hypoxic end of the physiological PO2 in liver, CCl4 caused a 5-fold increase in the oxygen uptake rate and a 20-fold increase in the malondialdehyde formation rate while, at 80 mmHg (10.7 kPa) the haloalkane caused only an increase of 2- and 4-fold, respectively; in comparison, there was only a slight increase in NADPH-induced lipid peroxidation with increasing PO2. These data clearly demonstrate the critical role of low steady-state PO2 in CCl4-induced lipid peroxidation and support lipid peroxidation as a key factor in CCl4 hepatotoxicity.     

Protective effect of colchiceine against acute liver damage

Pretreatment of rats with colchiceine (10 micrograms/day/rat) for seven days protected against CCl4-induced liver damage. CCl4 intoxication was demonstrated histologically and by increased serum activities of alanine amino transferase (ALT), alkaline phosphatase (Alk. Phosph.) gamma glutamyl transpeptidase (GGTP), bilirubins and decreased activity of glucose-6-phosphatase (G-6Pase). Furthermore, an increase in liver lipid peroxidation and a decrease in plasma membrane GGTP and Alk. Phosph. activities were found. Colchiceine increased 1.5-fold the LD50 of CCl4 and prevented the release of intracellular enzymes as well as the decrease in GGTP and Alk. Phosph. activities in plasma membranes. It also completely prevented the lipid peroxidation induced by CCl4 and limited the extent of the histological changes.     

Promethazine inhibits the formation of aldehydic products of lipid peroxidation but not covalent binding resulting from the exposure of rat liver fractions to CCl4.

Promethazine is known to have protective activity in relation to CCl4-induced liver necrosis. This hepatoprotective property has been investigated with regard to the free radical scavenging and antioxidant properties of promethazine using isolated hepatocytes and microsomal suspensions. CCl4 is activated in both systems to free radical metabolites that bind covalently to lipid and protein, and initiate lipid peroxidation. A large number of carbonyl products is produced during CCl4-induced lipid peroxidation; promethazine strongly inhibits the production of all classes of carbonyl compounds in both microsomal suspensions and isolated hepatocytes. In contrast, promethazine is a very weak inhibitor of the covalent binding of metabolites of CCl4. We conclude that promethazine acts by scavenging the trichloromethylperoxyl radical and lipid peroxyl radicals, and is a weak scavenger of the trichloromethyl radical. These data, when considered together with the hepatoprotective effects of promethazine, suggest that lipid peroxidation is of relatively more importance than covalent binding in the pathogenesis of CCl4-induced liver necrosis.     

Protective effects of N-acetyl-L-cysteine against acute carbon tetrachloride hepatotoxicity in rats

In recent years, N-acetyl-L-cysteine (NAC) has been widely investigated as a potentially useful protective and antioxidative agent to be applied in many pathological states. The aim of the present work was further evaluation of the mechanisms of the NAC protective effect under carbon tetrachloride-induced acute liver injuries in rats. The rat treatment with CCl4 (4 g/kg, intragastrically) caused pronounced hepatolysis observed as an increase in blood plasma bilirubin levels and hepatic enzyme activities, which agreed with numerous previous observations. The rat intoxication was accompanied by an enhancement of membrane lipid peroxidation (1.4-fold) and protein oxidative damage (protein carbonyl group and mixed protein-glutathione disulphide formations) in the rat liver. The levels of nitric oxide in blood plasma and liver tissue significantly increased (5.3- and 1.5-fold, respectively) as blood plasma triacylglycerols decreased (1.6-fold). The NAC administration to control and intoxicated animals (three times at doses of 150 mg/kg) elevated low-molecular-weight thiols in the liver. The NAC administration under CCl4-induced intoxication prevented oxidative damage of liver cells, decreased membrane lipid peroxidation, protein carbonyls and mixed protein-glutathione disulphides formation, and partially normalized plasma triacylglycerols. At the same time the NAC treatment of intoxicated animals did not produce a marked decrease of the elevated levels of blood plasma ALT and AST activities and bilirubin. The in vitro exposure of human red blood cells to NAC increased the cellular low-molecular-weight thiol levels and retarded tert-butylhydroperoxide-induced cellular thiol depletion and membrane lipid peroxidation as well as effectively inhibited hypochlorous acid-induced erythrocyte lysis. Thus, NAC can replenish non-protein cellular thiols and protect membrane lipids and proteins due to its direct radical-scavenging properties, but it did not attenuate hepatotoxicity in the acute rat CCl4-intoxication model.     

The role of iron in the paracetamol- and CCl4-induced lipid peroxidation and hepatotoxicity

Treatment of non-induced or phenobarbital-induced, glutathione-depleted mice with 400 mg/kg paracetamol led to a marked ethane exhalation as an index of in vivo lipid peroxidation (LPO) and to a significant elevation of liver-specific serum enzyme activities. Similar effects were seen with rats treated with 0.5 ml/kg CCl4. Pretreatment with the iron-chelating agent desferrioxamine (DFO) clearly suppressed lipid peroxidation in all cases, but inhibited only the CCl4-induced hepatotoxicity. Treatment of mice with desferrioxamine alone showed no hepatotoxicity at all, nor did it influence liver GSH-levels. In addition, DFO had no effect on hepatic microsomal enzyme activities responsible for the bioactivation of both paracetamol and CCl4. These findings are consistent with the theories which indicate that lipid peroxidation requires the presence of Fe2+-ions, regardless of the initiating agent, and that LPO is involved in CCl4-toxicity, but most probably not in paracetamol-induced liver damage. Furthermore, Fe2+-ions might play a role as mediators of CCl4-hepatotoxicity.     

Antioxidative activity of flavonoids in various systems of lipid peroxidation

The antioxidant action of flavonols in different systems of lipid peroxidation (LPO) was studied. Quercetin and rutin were found to inhibit NADPH and CCl4-dependent LPO in rat liver microsomes, however, in the case of CCl4-dependent LPO, rutin had a very poor antioxidant effect. Study of flavonols oxidation by products of the cytochrome c catalyzed destruction of linoleic acid hydroperoxide demonstrated that the differences in the antioxidant offects of quercetin and rutin can be due to their different capability to terminate free radical chain reactions. The antioxidant effect of rutin was shown to be largely due to the chelating properties of this compound.     

C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro

C-Phycocyanin (from Spirulina platensis) effectively inhibited CCl(4)-induced lipid peroxidation in rat liver in vivo. Both native and reduced phycocyanin significantly inhibited peroxyl radical-induced lipid peroxidation in rat liver microsomes and the inhibition was concentration dependent with an IC(50) of 11.35 and 12.7 microM, respectively. The radical scavenging property of phycocyanin was established by studying its reactivity with peroxyl and hydroxyl radicals and also by competition kinetics of crocin bleaching. These studies have demonstrated that phycocyanin is a potent peroxyl radical scavenger with an IC(50) of 5.0 microM and the rate constant ratios obtained for phycocyanin and uric acid (a known peroxyl radical scavenger) were 1.54 and 3.5, respectively. These studies clearly suggest that the covalently linked chromophore, phycocyanobilin, is involved in the antioxidant and radical scavenging activity of phycocyanin.     

Protective effect of Aquilegia vulgaris (L.) on carbon tetrachloride-induced oxidative stress in rats

The ethyl ether extract of A. vulgaris inhibited in vitro microsomal lipid peroxidation (IC50 58.8 microg/ml) and showed moderate ability to scavenge superoxide radicals and to chelate iron ions. The extract (100 mg/kg body weight, po) decreased uninduced and enzymatic microsomal lipid peroxidation in the liver of male rats pretreated with CCl4 (1 ml/kg body weight) by 27 and 40%, respectively. Activity of antioxidant and related enzymes (catalase and glucose-6-phosphate dehydrogenase) inhibited by CCl4 was significantly restored after administration of the extract. The extract itself significantly enhanced superoxide dismutase activity. There was no effect of the extract on hepatic glutathione level and cytochrome P450 content, both were decreased by CCl4. Neither CCl4 nor the tested extract affected activities of NADPH-cytochrome P450 reductase and two monooxygenases, aniline hydroxylase and aminopyrine n-demethylase. It can be concluded that the protective effect of the A. vulgaris extract in CCl4-induced liver injury is mediated by inhibition of microsomal lipid peroxidation and restoring activity of some antioxidant and related enzymes.     

Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice

The present study was undertaken to evaluate the effect of aminoguanidine (AG) on carbon tetrachloride (CCl4)-induced hepatotoxicity. Treatment of mice with CCl4 (20 microl/kg, i.p.) resulted in damage to centrilobular regions of the liver, increase in serum aminotransferase and rise in lipid peroxides level 24 hours after CCl4 administration. Pretreatment of mice with AG (50 mg/kg, i.p.) 30 minutes before CCl4 was found to protect mice from the CCl4-induced hepatic toxicity. This protection was evident from the significant reduction in serum aminotransferase, inhibition of lipid peroxidation and prevention of CCl4-induced hepatic necrosis revealed by histopathology. Aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, did not inhibit the in vitro lipid peroxidation. Taken together, these data suggest a potential role of nitric oxide as an important mediator of CCl4-induced hepatotoxicity.