Thiols and polyamines in the cytoprotective effect of taurine on carbon tetrachloride‐induced hepatotoxicity

The mechanism by which taurine (2‐aminoethanesulfonic acid) protects hepatocytes injury induced by carbon tetrachloride (CCl₄) is not fully understood. In a previous study, we reported that cellular polyamines play an important role in this mechanism. The relationship between cellular glutathione (GSH), protein‐SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamine on the cytoprotective ability of taurine in CCl₄‐induced toxicity in isolated rat hepatocytes, was examined. CCl₄ induced a LDH release and decreased cellular thiols and polyamine levels. Treating with taurine reversed these depletions. The effect of CCl₄ was also reversed by the addition of exogenous polyamines. Pretreating with α‐difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, which is a key enzyme in polyamine biosynthesis and therefore used to deplete cellular polyamine, prevented the protective effect of taurine. Adding diethyl maleate, a cellular glutathione‐depleting agent, reduced the effect of exogenous polyamines. The role of polyamine in the cytoprotective effect of taurine in CCl₄‐induced toxicity may therefore be by preventing, among others, GSH and protein‐SH depletions. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 71–76, 1999     

Effect of β-alanine administration on carbon tetrachloride-induced acute hepatotoxicity

Summary. Mice were supplemented with β-alanine (3%) in drinking water for one week. β-Alanine intake reduced hepatic taurine levels, but elevated cysteine levels significantly. Hepatotoxicity of CCl₄ in mice fed with β-alanine was decreased as determined by changes in serum enzyme activities. Hepatic glutathione and taurine concentrations after CCl₄ challenge were increased markedly by β-alanine intake. The enhanced availability of cysteine for synthesis of glutathione and/or taurine appears to account for the hepatoprotective effects of β-alanine against CCl₄-induced liver injury.     

Dexamethasone protects cultured rat hepatocytes against cadmium toxicity: involvement of cellular thiols

In the present study, the effects of dexamethasone on cadmium-induced toxicity were evaluated in isolated rat hepatocytes. Hepatocytes were cultured for 24 h in William’s E medium containing fetal calf serum (10%), insulin (0.1 IU/ml), and glucagon (0.01 μM) in the absence or presence of 0.1 μM dexamethasone. Cadmium chloride, 5 or 10 μM, was added to the medium and the toxicity was evaluated for up to 48 h after treatment. Lactate dehydrogenase (LDH) release, the reduced and oxidized glutathione ratio (GSH/GSSG), protein-SH groups, and lipid peroxidation levels were evaluated. Cadmium induced a dose- and time-dependent LDH release in control hepatocytes at 24 h (Cd 10 μM 42%) while hepatocytes pretreated with dexamethasone showed lower necrosis (Cd 10 μM 12% at 24 h). GSH/GSSH ratio and protein-SH groups were higher while lipid peroxidation was lower in dexamethasone-treated hepatocytes as compared with untreated cells. In conclusion, cadmium toxicity was associated with an increase in intracellular oxidative stress responsible for accelerated cell death. The use of dexamethasone prevented cadmium damage, suggesting that the cytoprotective action of this hormone is related to its effect in preventing changes in thiols such as glutathione and protein-SH groups.     

Protective effects of the apigenin-O/C-diglucoside saponarin from Gypsophila trichotoma on carbone tetrachloride-induced hepatotoxicity in vitro/in vivo in rats

This study investigated the hepatoprotective activity of saponarin, isolated from Gypsophila trichotoma Wend., using in vitro/in vivo hepatotoxicity model based on carbone tetrachloride (CCl₄)-induced liver damage in male Wistar rats. The effect of saponarin was compared with those of silymarin. In vitro experiments were carried out in primary isolated rat hepatocytes. Cell incubation with CCl₄ (86 μmol l⁻¹) led to a significant decrease in cell viability, increased LDH leakage, decreased levels of cellular GSH and elevation in MDA quantity. Cell pre-incubation with saponarin (60–0.006 μg/ml) significantly ameliorated CCl₄-induced hepatic damage in a concentration-dependent manner. These results were supported by the following in vivo study. Along with decreased MDA quantity and increased level of cell protector GSH, seven day pre-treatment of rats with saponarin (80 mg/kg bw; p.o.) also prevented CCl₄ (10%, p.o.)-caused oxidative damage by increasing antioxidant enzyme activities (CAT, SOD, GST, GPx, GR). Biotransformation phase I enzymes were also assessed. Administered alone, saponarin decreased EMND and AH activities but not at the same extent as CCl₄ did. However, pre-treatment with saponarin significantly increased enzyme activities in comparison to CCl₄ only group. The observed biochemical changes were consistent with histopathological observations where the hepatoprotective effect of saponarin was comparative to the effects of the known hepatoprotecor silymarin. Our results suggest that saponarin, isolated from Gypsophila trichotoma Wend., showed in vitro and in vivo hepatoprotective and antioxidant activity against CCl₄-induced liver damage.     

Vitamin D and melatonin protect the cell’s viability and ameliorate the CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines

Carbon tetrachloride (CCl₄) is widely used to induce liver toxicity in in vitro/in vivo models. Lipid peroxidation (LPO) begins with toxicity and affects cell viability. Recently, the beneficial effects of melatonin and Vitamin D on cell proliferation in human normal and cancer cells were found. This study was planned to evaluate antioxidant and cytoprotective activity of melatonin and Vitamin D in CCl₄ induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines. Based on the cytotoxicity assay, melatonin and Vitamin D were evaluated for cytotoprotective potential against CCl₄ induced toxicity in HepG2 and Hep3B liver cell lines by monitoring cell viability, LPO and glutathione (GSH) level. Different dosages of CCl₄ (0.1, 0.2, 0.3 and 0.4 % v/v) were applied to HepG2 and Hep3B cells in order to determine the most toxic dosage of it in a time dependent manner. The same experiments were repeated with exogenously applied melatonin (MEL) and Vitamin D to groups treated with/without CCL₄. Cell viability was determined with MTT measurements at the 2nd, 24th and 48th h. GSH content and Malondialdehyde levels were measured from the cell lysates. As a result, both melatonin and Vitamin D administration during CCl₄ exposure protected liver cells from CCl₄ induced cell damage. Increase in LPO and decrease in GSH were found in the CCl₄ groups of both cells. Contrary to these results administration of MEL and Vitamin D on cells exhibited results similar to the control groups. Therefore, melatonin and Vitamin D might be a promising therapeutic agent in several toxic hepatic diseases.     

Isolation, characterization and antioxidative effect of phyllanthin against CCl4-induced toxicity in HepG2 cell line

The present study was an attempt to investigate the hepatoprotective and antioxidative property of Phyllanthus amarus (P. amarus) extract and phyllanthin. Phyllanthin, one of the active lignin present in this plant species was isolated from the aerial parts, by silica gel column chromatography employing gradient elution with hexane-ethyl acetate solvent mixture. It was obtained in high yields (1.23%), compared to reported procedures and the purity was ascertained by HPTLC and reversed-phase HPLC analysis. Characterization of phyllanthin was done by mp, UV-Visible spectrophotometry, elemental analysis, FT-IR, ¹H NMR, ¹³C NMR and mass spectral analysis. Free radical scavenging activity of P. amarus extract and phyllanthin was also examined using DPPH assay. The protective effect of P. amarus extract and phyllanthin was studied on CCl₄-induced toxicity in human hepatoma HepG2 cell line. The results indicated that CCl₄ treatment caused a significant decrease in cell viability. In addition, the toxin treatment initiated lipid peroxidation (LPO), caused leakage of enzymes like alanine transaminase (ALT) and lactate dehydrogenase (LDH) with a significant decrease in glutathione (GSH) levels. It was observed that phyllanthin effectively alleviated the changes induced by CCl₄ in a concentration-dependent manner, with much smaller strengths as compared to P. amarus extract.     

Protective effects of hepatocyte-specific glycyrrhetic derivatives against carbon tetrachloride-induced liver damage in mice

Glycyrrhetic acid (GA), the main hydrolysate of glycyrrhizic acid extracted from the roots of the Chinese herb Glycyrrhiza glabra, was reported to be accumulated in hepatocytes due to the extensive distribution of GA receptors in liver. A series of hepatocyte-specific derivatives on the basis of anetholtrithione and glycyrrhizic were designed and synthesized. The potential beneficial effect was evaluated in carbon tetrachloride (CCl₄)-induced liver injury model. In addition, the hepatoprotective activity of these derivatives was assessed by measuring levels of serum marker enzymes, including serum glutamate oxaloacetate transaminase (GOT), serum glutamate pyruvate transaminase (GPT), alkaline phosphatase (AKP), lactate dehydrogenase (LDH) and the ratio of GSH to GSSG. Gratifyingly, compounds 5a–c (100 mg/kg, p.o.) markedly prevented CCl₄-induced elevation of levels of serum GPT, GOT. A comparative histopathological study of liver exhibited almost a normal liver lobular architecture and cell structure of the livers, as compared to CCl₄-treated group. These findings were confirmed with the histopathological observations, where hepatocyte-specific glycyrrhetic acid derivatives 5a–c were capable of reversing the toxic effects of CCl₄ on hepatocytes.     

Hepatoprotective effect of Origanum vulgare in Wistar rats against carbon tetrachloride-induced hepatotoxicity

The effect of an aqueous extract of Origanum vulgare (OV) leaves extract on CCl₄-induced hepatotoxicity was investigated in normal and hepatotoxic rats. To evaluate the hepatoprotective activity of OV, rats were divided into six groups: control group, O. vulgare group, carbon tetrachloride (CCl₄; 2 ml/kg body weight) group, and three treatment groups that received CCl₄ and OV at doses of 50, 100, 150 mg/kg body weight orally for 15 days. Alanine amino transferase (ALT), alkaline phosphatase (ALP), and aspartate amino transferase (AST) in serum, lipid peroxide (LPO), GST, CAT, SOD, GPx, GR, and GSH in liver tissue were estimated to assess liver function. CCl₄ administration led to pathological and biochemical evidence of liver injury as compared to controls. OV administration led to significant protection against CCl₄-induced hepatotoxicity in dose-dependent manner, maximum activity was found in CCl₄?+?OV3 (150 mg/kg body weight) groups and changes in the hepatocytes were confirmed through histopathological analysis of liver tissues. It was also associated with significantly lower serum ALT, ALP, and AST levels, higher GST, CAT, SOD, GPx, GR, and GSH level in liver tissue. The level of LPO also decreases significantly after the administration of OV leaves extract. The biochemical observations were supplemented with histopathological examination of rat liver sections. Thus, the study suggests O. vulgare showed protective activity against CCl₄-induced hepatotoxicity in Wistar rats and might be beneficial for the liver toxicity.     

Protective effect of gamma-aminobutyric acid (GABA) against cytotoxicity of ethanol in isolated rat hepatocytes involves modulations in cellular polyamine levels

Summary. Gamma-aminobutyric acid (GABA) is considered to be a multifunctional molecule with various physiological effects throughout the body. It is also evident that the liver contains GABA and its transporter. However, the functions of GABA in liver have not been well documented. In this study, the cytoprotective effect of GABA against ethanol-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. Addition of ethanol induced decrease of cell viability in a dose-dependent manner. However, treatment with GABA resulted in a dose-dependent recovery from ethanol (150 mM)-induced cytotoxicity. GABA reversed the ethanol-induced decrease in intracellular polyamine levels. Furthermore, the addition of polyamines also reversed the ethanol-induced decrease of cell viability. These results suggest that GABA is protective against the cytotoxicity of ethanol in isolated rat hepatocytes and this effect may be modulated by the maintenance of intracellular polyamine levels.     

Effects of testosterone and 17, β– estradiol on the polyamine metabolism in cultivated normal rat kidney epithelial cells

Summary. Ornithine decarboxylase (ODC) and diamine oxidase (DAO) are important enzymes involved in the metabolism of polyamines (putrescine, spermidine and spermine). The influence of testosterone (T) and 17, β– estradiol (E₂) on the activity of ODC and DAO was examined in cultivated normal rat kidney (NRK) epithelial cells. The results showed an increase in enzyme activities 4 hours or 12 hours after hormonal treatment. Both T and E₂ led to a significant increase (1.6-fold) in ODC protein level as compared to the controls. Cellular concentration of spermidine and spermine increased (2.2- and 2.6-fold respectively) 4 hours after T addition. A higher levels in concentrations of putrescine (1.4-fold) and spermine (1.5-fold) 12 hours after E₂ treatment were observed. These results suggest that the biosynthesis and terminal oxidation of the polyamines in NRK epithelial cells are androgen- and estrogen-mediated and depend on the hormonal sensitivity of the cells. Received April 5, 1999, Accepted December 20, 1999     

The hepatoprotective effect of coumarin and coumarin derivates on carbon tetrachloride-induced hepatic injury by antioxidative activities in rats

Coumarins are a vast group of natural compounds and some of them possess antioxidant activities. The comparison of the antioxidant activity of some coumarins with various chemical molecular structure has not been investigated in previous studies. Therefore, this study was aimed to investigate the hepatoprotective effect against carbon tetrachloride (CCl₄) -induced hepatic injury by coumarin (1,2-benzopyrone) and coumarin derivatives, esculetin (6,7-dihydroxycoumarin), scoparone (6,7-dimethoxycoumarin), and 4-methylumbelliferone (7-hyroxy-4-methyl) in male Sprague–Dawley rats. Product of lipid peroxidation, malondialdehyde (MDA), activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) were evaluated for oxidative stress in hepatic injury. Gamma glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH) were detected in plasma as a biomarker of hepatic injury. Significantly elevated levels of MDA and lowered levels of SOD and CAT activities were observed in liver of rats exposed to CCl₄, when compared to control values. Similarly, administration of CCl₄ increased LDH and GGT levels in serum. Pre-treatment of rats with esculetin (35 mg kg⁻¹, orally) and scoparone (35 mg kg⁻¹, orally) significantly prevented CCl₄-induced decrease in MDA levels and increase in SOD and CAT, whereas 4-methylumbelliferone (35 mg kg⁻¹) and coumarin (30 mg kg⁻¹) had no effect against CCl₄-induced rise in serum enzymes. Esculetin and scoparone also showed protective properties as was evidenced in reduced LDH and GGT levels in serum. The results of this study indicate that the chemical structures of coumarins play an important role in the prevention of oxidative stress.     

<Emphasis Type="Italic">Undaria pinnatifida</Emphasis> fucoidan extract protects against CCl<Subscript>4</Subscript>-induced oxidative stress

This study was performed to elucidate the effects of Undaria pinnatifida fucoidan extract (UPFE) in preventing CCl₄-induced oxidative stress. UPFE (100 mg/kg) was intraperitoneally administered to rats for 14 days. On day 15, CCl₄ dissolved in olive oil (50% CCl₄) was injected 12 h before they were anesthetized and dissected. To measure UPFE-mediated antioxidation, we examined the levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in serum, as well as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in liver homogenates. CCl₄ treatment markedly increased the levels of GOT, GPT, ALP, LDH, and MDA and significantly decreased levels of SOD, CAT, and GPx. UPFE pretreatment decreased levels of GOT, GPT, ALP, LDH, and MDA, by 62.8, 68.5, 41.9, 72.7, and 122%, respectively and increased those of SOD, CAT, and GPx by 111.1, 15.9, and 52.6%, respectively. These results showed that UPFE has antioxidant effects against CCl₄-induced oxidative stress.     

Index

Hydrogen peroxide (H₂O₂) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3′,4′,-tetrahydroxy flavone) against H₂O₂-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H₂O₂. Moreover, fisetin protected against H₂O₂-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H₂O₂ treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H₂O₂. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H₂O₂-induced cells damage. Taken together, our results suggest that fisetin protects against H₂O₂-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.     

Antioxidant and hepatoprotective potential of Aegle marmelos Correa. against CCl4-induced oxidative stress and early tumor events

The antioxidant properties and inhibitory effect on early tumor promoter markers of A. marmelos (25 and 50 mg/Kg b. wt. orally) have been evaluated. Male Wistar rats were pre-treated for seven consecutive days with A. marmelos prior to CCl₄ (1 mL Kg^{? 1} body weight p. o., in corn oil [1:1 v/v]) treatment. Pre-treatment with A. marmelos suppressed lipid peroxidation (LPO), xanthine oxidase (XO) and release of serum toxicity marker enzymes viz, SGOT, LDH, SGPT dose-dependently and significantly (p < 0.001). Hepatic antioxidant status viz, reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), quinone reductase (QR), catalase (CAT) were concomitantly restored in A. marmelos-treated groups (p < 0.001). In addition, A. marmelos pretreatment also prevented the CCl₄-enhanced ornithine decarboxylase (ODC) and hepatic DNA synthesis significantly (p < 0.001). In conclusion, carbon tetrachloride-induced liver toxicity was strikingly attenuated by A. marmelos treatment and the study gives some insight into the mechanisms involved in diminution of free radical generating toxicants and enhancement of the antioxidant armory, hence preventing further tissue damage, injury and hyperproliferation.

Thus, these findings indicate that A. marmelos attenuates CCl₄-mediated hepatic oxidative stress, toxicity, tumor promotion and subsequent cell proliferation response in Wistar rats.     

Protective effect of C-phycocyanin against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo

This study focused on the hepatoprotective activity of C-phycocyanin (C-PC) against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo. In in vitro study, human hepatocyte cell line L02 was used. C-PC showed its capability to reverse CCl₄-induced L02 cells viability loss, alanine transaminase (ALT) leakage and morphological changes. C-PC also showed the following positive effects: prevent the CCl₄-induced overproduction of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA); prevent changes in superoxide dismutase (SOD) activity; and reduce glutathione (GSH) level. In vivo, C-PC showed its capability to decrease serum ALT and aspartate transaminase (AST) levels in CCl₄-induced liver damage in mice. The histological observations supported the results obtained from serum enzymes assays. C-PC also showed the following effects in mice liver: prevent the CCl₄-induced MDA formation and GSH depletion; prevent SOD and glutathione peroxidase (GSH-Px) activity; and prevent the elevation of transforming growth factor-beta1 (TGF-β1) and hepatocyte growth factor (HGF) mRNAs. Both the in vitro and in vivo results suggested that C-PC was useful in protecting against CCl₄-induced hepatocyte damage. One of the mechanisms is believed to be through C-PCs scavenging ability to protect the hepatocytes from free radicals damage induced by CCl₄. In addition, C-PC may be able to block inflammatory infiltration through its anti-inflammatory activities by inhibiting TGF-β1 and HGF expression.     

S-allyl cysteine prevents CCl4-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50–200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

The physiological role of biogenic amines redox reactions in mitochondria. New perspectives in cancer therapy

Summary. In tumours, polyamines and amine oxidases increase as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H₂O₂ and aldehydes produced by the reaction. Increasing the incubation temperature from 37 to 42 °C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. Since the tumour cells release endogenous substrates of BSAO, the administration of spermine is not required. Combination with hyperthermia improves the cytocidal effect of polyamines oxidation products. Our findings show that multidrug resistant (MDR) cells are more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity which induces the generation of intracellular ROS prior to the onset of mitochondrial permeability transition (MPT). It makes this new approach attractive, since the development of MDR is one of the major problems of conventional cancer therapy.     

Alterations in susceptibility to carbon tetrachloride toxicity and hepatic antioxidant/detoxification system in streptozetocin-induced short-term diabetic rats: Effects of insulin and Schisandrin B treatment

The streptozotocin-induced short-term (2 week) diabetic rats showed an increase in susceptibility to carbon tetrachloride (CCl4)-induced hepatocellular damage. This diabetes-induced change was associated with a marked impairment in the hepatic glutathione antioxidant/detoxification response to CCl₄ challenge, as indicated by the abrogation of the increases in hepatic reduced glutathione (GSH) level, glucose-6-phosphate dehydrogenase and microsomal glutathione S-transferases (GST) activities upon challenge with increasing doses of CCl₄. While the hepatic GSH level was increased in diabetic rats, the hepatic mitochondrial GSH level and Se-glutathione peroxidase activity were significantly reduced. Insulin treatment could reverse most of the biochemical alterations induced by diabetes. Both insulin and schisandrin B (Sch B) pretreatments protected against the CCl₄ hepatotoxicity in diabetic rats. The hepatoprotection was associated with improvement in hepatic glutathione redox status in both cytosolic and mitochondrial compartments, as well as the increases in hepatic ascorbic acid level and microsomal GST activity. The ensemble of results suggests that the diabetes-induced impairment in hepatic mitochondrial glutathione redox status may at least in part be attributed to the enhanced susceptibility to CCl4 hepatotoxicity. Sch B may be a useful hepatoprotective agent against xenobiotics-induced toxicity under the diabetic conditions. (Mol Cell Biochem 175: 225–232, 1997)     

Protective Effects of Garlic and Silymarin on NDEA-Induced Rats Hepatotoxicity

Background ­— The present study was conducted to investigate the chemopreventive effects of garlic extract and silymarin on N-nitrosodiethylamine (NDEA) and carbon tetrachloride (CCl₄)-induced hepatotoxicity in male albino rats. Methods and Results — Animals were pretreated with garlic, silymarin or both for one week prior to the injection of NDEA. Then animals received a single injection of NDEA followed by weekly subcutaneous injections of CCl₄ for 6 weeks. Oral administration was then continued along with the injection of CCl₄ for the duration of the experiment. Serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), hepatic lipid peroxidation (LPO), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione-S-transferase (GST) and glutathione reductase (GSR) were measured. Injection of NDEA induced a significant elevation in serum AST, ALT and ALP. In the liver, NDEA increased oxidative stress through the increase in LPO and decrease in SOD, and GSH-dependent enzymes. Although administration of garlic or silymarin significantly reduced the liver toxicity, combined administration was more effective in preventing the development of hepatotoxicity. Conclusion — These novel findings suggest that silymarin and garlic have a synergistic effect, and could be used as hepatoprotective agents against hepatotoxicity.