Differential effects of dietary flavonoids on reactive oxygen and nitrogen species generation and changes in antioxidant enzyme expression induced by proinflammatory cytokines in Chang Liver cells.

This study was aimed to investigate the differential protective effect of dietary flavonoids against oxidative stress induced by proinflammatory stimuli in parenchymal liver cells. Chang Liver cells were incubated with a cytokine mixture (CM) supplemented with the flavonols quercetin and kaempferol, the flavanone taxifolin and the flavone apigenin (5-50 microM). Concentrations of oxidised and reduced glutathione, generation of different ROS/RNS, and expression of antioxidant enzymes were measured. Oxidised glutathione concentration and the oxidised/reduced glutathione ratio were increased by the CM. These effects were significantly prevented by quercetin, kaempferol and taxifolin at all tested concentrations. Effects of apigenin reached a lesser extent and were not significant at 25 microM. Treatment with quercetin and kaempferol prevented the production of peroxides, superoxide anion and nitric oxide induced by CM. Taxifolin 50 microM and apigenin 25-50 microM caused a significant increase in peroxides and nitric oxide generation. Protein concentration of the different antioxidant enzymes was generally reduced by kaempferol and quercetin in comparison to CM, although quercetin 25 and 50 microM increased Mn SOD protein concentration. GPx protein level was significantly increased by apigenin 25 and 50 microM. Changes in mRNA tended to be parallel to those in protein concentration. Our study reveals that important differences exist between flavonoids with different structural features in their capacity to abrogate the generation of different ROS/RNS, and suggests that the modulation of antioxidant enzymes by flavonoids may be also important in their antioxidant effects in liver cells.     

The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 μM) esculetin for 8 h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8 h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 μM esculetin for 8 h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.     

Dihydroisotanshinone I protects against menadione-induced toxicity in a primary culture of rat hepatocytes

Dihydroisotanshinone I is a phenanthrenequinone derivative isolated from the roots of Salvia trijuga Diels. The present study demonstrated the hepatoprotective effect of dihydroisotanshinone I against menadione-induced cytotoxicity in a primary culture of rat hepatocytes. Pretreating the cells with dihydroisotanshinone I at concentrations ranging from 2.5 microM to 20 microM for 24 hours caused dose-dependent protection against hepatotoxicity induced by menadione. Intracellular glutathione level and activity of DT-diaphorase have been suggested to play important roles in menadione-induced cytotoxicity. However, treating the hepatocytes with 20 microM dihydroisotanshinone I for 24 hours did not cause a significant change in glutathione level and DT-diaphorase activity. On the contrary, adding dihydroisotanshinone I to freshly isolated hepatocytes at concentrations between 50 nM to 200 nM inhibited NADH-induced superoxide production dose-dependently as indicated by the decrease of lucigenin-amplified chemiluminescence. In addition, dihydroisotanshinone I at concentrations ranging from 5 microM to 20 microM inhibited tert-butyl hydroperoxide-induced lipid peroxidation dose-dependently in isolated hepatocytes as indicated by the level of malondialdehyde. These results suggest that the protective action of dihydroisotanshinone I against menadione-induced hepatotoxicity is attributed to its antioxidant properties including the free radical scavenging activity and inhibition of lipid peroxidation. Abbreviations. DTD:DT-diaphorase GSH:glutathione LDH:lactate dehydrogenase MDA:malondialdehyde MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TBHP: tert-butyl hydroperoxide     

The effect of quercetin on the mRNA expression of different antioxidant enzymes in hepatoma cells

The flavonol quercetin shows a wide range of effects in biological systems. We investigated whether quercetin exerts its proposed antioxidant properties via the antioxidant enzyme system. Quercetin in a concentration range from 5 to 100 microM decreased manganese superoxide dismutase, glutathione peroxidase, and copper zinc superoxide dismutase mRNA expression levels each by 30-40% in rat hepatoma H4IIE cells. Catalase mRNA expression levels increased about 30% but only with the cytotoxic concentration of 100 microM. Despite the down-regulation of antioxidant enzyme mRNA expression quercetin treatment of cells induced only a mild oxidative stress. Pretreatment of H4IIE cells with quercetin even protected against an oxidative stress resulting from hydrogen peroxide exposure. In conclusion, the antioxidant capacity of quercetin was shown not to be due to the antioxidant enzyme system.     

Protective mechanisms of Aralia continentalis extract against tert-butyl hydroperoxide-induced hepatotoxicity: in vivo and in vitro studies

In the present work, we investigated the protective effects of the ethanol extract of Aralia continentalis roots (AC) on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in a cultured Hepa1c1c7 cell line and in mouse liver. Pretreatment with AC prior to the administration of t-BHP significantly prevented the increase in serum levels of hepatic enzyme markers (ALT, AST) and lipid peroxidation and reduced oxidative stress, as measured by glutathione content, in the liver. Histopathological evaluation of the livers also revealed that AC reduced the incidence of liver lesions. The in vitro study showed that AC significantly reduced t-BHP-induced oxidative injury in Hepa1c1c7 cells, as determined by cell cytotoxicity, intracellular glutathione content, lipid peroxidation, reactive oxygen species (ROS) levels, and caspase-3 activation. Also, AC up-regulated phase II genes including heme oxygenase-1 (HO-1), NAD(P)H:quinone reductase, and glutathione S-transferase. Moreover, AC induced Nrf2 nuclear translocation and ERK1/2 and p38 activation, pathways that are involved in inducing Nrf2 nuclear translocation. Taken together, these results suggest that the protective effects of AC against t-BHP-induced hepatotoxicity may, at least in part, be due to its ability to scavenge ROS and to regulate the antioxidant enzyme HO-1 via the ERK1/2 and p38/Nrf2 signaling pathways.     

The ameliorative effect of 23-hydroxytormentic acid isolated from Rubus coreanus on cisplatin-induced nephrotoxicity in rats

Previously, the authors demonstrated that the triterpenoid glycoside niga-ichigoside F? (NIF?) and its aglycone 23-hydroxytormentic acid (23-HTA) isolated from the unripe fruits of Rubus coreanus (Rosaceae) ameliorate cisplatin-induced toxicity in renal epithelial LLC-PK? cells. In the present study, the nephroprotective effects of NIF? and 23-HTA were investigated in Sprague-Dawley rats with acute renal injury induced by a single intraperitoneal (i.p.) injection of cisplatin (7 mg/kg). Pretreatment with 23-HTA (10 mg/kg/d, per os (p.o.)) significantly reduced cisplatin-induced elevations in blood urea nitrogen (BUN) and serum creatinine level, whereas NIF? (10 mg/kg, p.o.) slightly reduced these levels. In addition, pretreatment with 23-HTA prevented cisplatin-induced hydroxyl radical generation, malondialdehyde (MDA) production, glutathione (GSH) depletion, and cisplatin-induced changes in the activities of oxidant and antioxidant enzymes in rat renal tissues. In addition, histopathological examinations showed that 23-HTA pretreatment reduced cisplatin-induced acute tubular necrosis and histological changes. In contrast, NIF? was found to have a slight or no influence on cisplatin-induced oxidative enzymes and acute tubular necrosis. Taken together, these results suggest that protective effect of 23-HTA pretreatment on cisplatin-induced renal damage is associated with the attenuation of oxidative stress and the preservation of antioxidant enzymes.     

Differential effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rat

1. Gavage administration of the natural flavonoids tangeretin, chrysin, apigenin, naringenin, genistein and quercetin for 2 consecutive weeks to the female rat resulted in differential effects on selected phase 1 and 2 enzymes in liver, colon and heart as well as antioxidant enzymes in red blood cells (RBC). 2. Glutathione transferase (GST) activity assayed by use of the substrate 1-chloro-2,4-dinitrobenzene was significantly induced by apigenin, genistein and tangeretin in the heart but not in colon or liver. 3. In RBC chrysin, quercetin and genistein significantly decreased the activity of glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx), whereas superoxide dismutase (SOD) was only significantly decreased by genistein. 4. The oxidative status of the animal, measured as plasma malondialdehyde, revealed that chrysin, quercetin, genistein, and beta-naphthoflavone (BNF) significantly protected against, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP)-induced oxidative stress. Hepatic PhIP-DNA adduct formation was not affected by any of the administered flavonoids, whereas PhIP-DNA adduct formation in colon was slightly, but significantly, inhibited by quercetin, genistein, tangeretin and BNF. 5. The observed effects of chrysin, quercetin and genistein on antioxidant enzymes, concurrently with a protection against oxidative stress, suggest a feedback mechanism on the antioxidant enzymes triggered by the flavonoid antioxidants. 6. Despite the use of high flavonoid doses, which by far exceed the human exposure levels, the effect on drug metabolizing and antioxidant enzymes was still very minor. The role of singly administered flavonoids in the protection against cancer and heart disease is thus expected to be limited.     

Alpha-lipoic acid regulates heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells

Oxidative stress is a known mechanism induced, among other things, by arsenic toxicity. As a response, the cell triggers the synthesis of antioxidant and stress response elements like glutathione and heme oxygenase. Alpha-lipoic acid (ALA) is a well-known antioxidant that confers protection to oxidative stress conditions. We analyzed the effect of ALA pretreatment on Nrf2-responsive gene expression of HepG2 cells exposed to As(3+). Cells were treated with 5mM ALA and 8h later exposed to 50μM As(3+) for 24h, analyzing MTT-activity, glutathione content, Nrf2 induction and antioxidant gene expression. As(3+) increased glutathione (154%), heme oxygenase, glutamate cystein ligase, modifier subunit and metallothionein (35-fold, 10-fold and 9-fold, respectively). ALA prevented the strong expression of heme oxygenase by As(3+) exposure (from 35- to 5-times of control cells), which correlated with the reduction of Nrf2 observed in As(3+) group. ALA pretreatment can down-modulate the response mediated by Nrf2 and provide protection to As(3+) exposed HepG2 cells.     

Cytotoxicity of reactive oxygen species and related agents toward undifferentiated and differentiated rat phenochromocytoma PC12 cells

The cytotoxicity of reactive oxygen species and related agents toward cultured rat adrenal medullary phenochromocytoma PC12 cells was examined. These species and agents include hydrogen peroxide, linoleic acid hydroperoxide (LOOH), tert-butyl hydroperoxide, paraquat, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN), and a hypoxanthine-xanthine oxidase system. The respective 50% lethal concentrations (LC50) for undifferentiated and differentiated PC12 cells were found to be 275 and 165 microM of hydrogen peroxide, 58.3 and 35.3 microM of LOOH, 536 and 212 microM of tert-butyl hydroperoxide, 42.5 and 26.5 mM of paraquat, 79.5 and 74.5 mM of AAPH, 412 and 300 microM of AMVN, and 37.2 and 16.6mU x ml(-1) xanthine oxidase activity of the hypoxanthine-xanthine oxidase system. These results show that the differentiated cells were more susceptible to these oxidative agents than the undifferentiated cells. The glutathione peroxidase activity level of the undifferentiated cells was 2-3 times higher than the differentiated cells, the catalase activity level also tended to be higher, the superoxide dismutase activity level was higher on a per-protein-quantity basis but lower on a per-cell-number basis, and the total and reduced glutathione concentration levels were considerably higher. The enhanced susceptibility of the differentiated cells may result from decreases in the activity of glutathione peroxidase and the concentration of its substrate, reduced glutathione (GSH). Further, the preincubation of PC12 cells with alpha-tocopherol or L-buthionine-(R,S)-sulfoximine (BSO) lowered or enhanced their cytotoxicities, respectively.     

Potential in vitro Protective Effect of Quercetin,Catechin, Caffeic Acid and Phytic Acid against Ethanol-Induced Oxidative Stress in SK-Hep-1 Cells

Phytochemicals have been known to exhibit potent antioxidant activity. This study examined cytoprotective effects of phytochemicals including quercetin, catechin, caffeic acid, and phytic acid against oxidative damage in SK-Hep-1 cells induced by the oxidative and non-oxidative metabolism of ethanol. Exposure of the cells to excess ethanol resulted in a significant increase in cytotoxicity, reactive oxygen species (ROS) production, lipid hydroperoxide (LPO), and antioxidant enzyme activity. Excess ethanol also caused a reduction in mitochondrial membrane potential (MMP) and the quantity of reduced glutathione (GSH). Co-treatment of cells with ethanol and quercetin, catechin, caffeic acid and phytic acid significantly inhibited oxidative ethanol metabolism-induced cytotoxicity by blocking ROS production. When the cells were treated with ethanol after pretreatment of 4-methylpyrazole (4-MP), increased cytotoxicity, ROS production, antioxidant enzyme activity, and loss of MMP were observed. The addition of quercetin, catechin, caffeic acid and phytic acid to these cells showed suppression of non-oxidative ethanol metabolism-induced cytotoxicity, similar to oxidative ethanol metabolism. These results suggest that quercetin, catechin, caffeic acid and phytic acid have protective effects against ethanol metabolism-induced oxidative insult in SK-Hep-1 cells by blocking ROS production and elevating antioxidant potentials.     

Antigenotoxic effect of Xanthohumol in rat liver slices.

Xanthohumol (XN), the principal prenylated flavonoid in the hop plant, Humulus lupulus L., is suggested to have cancer chemo-preventive activities. Its mechanisms of protection have been proposed to be inhibition of metabolic activation, induction of detoxifying enzymes and antioxidant activity. Our previous study showed that XN efficiently protected human hepatoma HepG2 cells against the genotoxic effects of two pro-carcinogens (2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo(a)pyrene (BaP)) that are dependent on cytochrome P450 (CYP) mediated metabolic activation, and against genotoxic effects of the oxidative damage inducing tert-butyl hydroperoxide (tBOOH). In the present study, we investigated the antigenotoxic effects of XN in precision-cut rat liver slices. Using the comet assay, we detected that at non-cytotoxic concentrations (0.01-10 microM) XN completely prevented IQ and BaP-induced DNA damage. The protective effects of XN against tBOOH-induced DNA damage was less efficient; the maximal 50% reduction of DNA damage was observed at 0.1 microM XN. In rat microsomes, XN (0.001-10 microM) inhibited CYP1A activity (7-ethoxycoumarin (7EC) de-ethylation) in a concentration-dependent manner. Surprisingly, no inhibition of 7EC metabolism by XN was observed in rat liver slices. XN also did not have any influence on mRNA expression of the enzymes CYP1A2 and quinone reductase (QR). These results indicate that inhibition of metabolic activation of pro-carcinogens by CYP1A is not likely to be the mechanism of its antigenotoxic action. In conclusion, XN efficiently protects DNA against genotoxicity of IQ and BaP and against oxidative DNA damage. Although the mechanism of the protective effect of XN is unclear, our results indicate that XN exhibits antigenotoxic effects in fresh liver tissue and provide additional evidence for the cancer preventive potential of XN.     

Standardized Hypericum perforatum reduces oxidative stress and increases gene expression of antioxidant enzymes on rotenone-exposed rats

Since oxidative stress is implicated in the pathophysiology of dementia and depression, this study was designed to investigate the pro-oxidant activity of rotenone, the protective role of standardized extract of Hypericum perforatum (SHP), as well as the mRNA levels of antioxidant enzymes, in brain homogenates of rats following exposure to rotenone and SHP extract. Quercetin in liposomes, one active constituent, was tested in the same experimental conditions to serve as a positive control. The animals received pretreatment with SHP (4 mg/kg) or quercetin liposomes (25 and 100 mg/kg) 60 min before of rotenone injection (2 mg/kg). All treatments were given intraperitoneally in a volume of 0.5 ml/kg body weight, for 45 days. Rotenone treatment increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and levels of malondialdehyde (MDA). The content of reduced glutathione (GSH) was decreased due to chronic rotenone treatment. Rotenone significantly induced the gene expression of CuZnSOD, MnSOD; CAT and GPx in brain. In contrast, SHP extract exerted an antioxidant action which was related with a decreased of MnSOD activity and mRNA levels of some antioxidant enzymes evaluated. Liposomal quercetin treatment resulted in a significant preservation of the activities of antioxidant enzymes and a decreased in the mRNA levels of these antioxidant enzymes. One possible mechanism of action of SHP extract may be related to quercetin in protecting neurons from oxidative damage. Therefore standardized extract of H. perforatum could be a better alternative for depressed elderly patients with degenerative disorder exhibiting elevated oxidative stress status.     

Quercetin, a bioflavonoid, attenuates ferric nitrilotriacetate-induced oxidative renal injury in rats

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces acute proximal tubular necrosis as a consequence of lipid peroxidation and oxidative tissue damage, that eventually leads to high incidence of renal adenocarcinomas in rodents. This study was designed to investigate the effect of quercetin, a bioflavonoid with antioxidant potential, on Fe-NTA-induced nephrotoxicity in rats. One hour after a single intraperitoneal (i.p.) injection of Fe-NTA (8 mg iron/kg), a marked deterioration of renal architecture and renal function was observed. Fe-NTA induced a significant renal oxidative stress demonstrated by elevated thiobarbituric acid reacting substances (TBARS) and reduction in activities of renal catalase, superoxide dismutase and glutathione reductase. Pretreatment of animals with quercetin (2 mg/kg, i.p.) 30 minutes before Fe-NTA administration markedly attenuated renal dysfunction, morphological alterations, reduced elevated TBARS and restored the depleted renal antioxidant enzymes. These results clearly demonstrate the role of oxidative stress and its relation to renal dysfunction, and suggest a protective effect of quercetin on Fe-NTA-induced nephrotoxicity in rats.     

Protective mechanisms of anthocyanins from purple sweet potato against tert-butyl hydroperoxide-induced hepatotoxicity

Anthocyanins have been shown to exert anti-proliferative, anti-inflammatory effects and anti-carcinogenic activity. In the present work, we investigated the protective effects of anthocyanin fraction (AF) from purple sweet potato on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in HepG2 cell line and in rat liver. The result showed that the oral pretreatment of AF before t-BHP treatment significantly lowered the serum levels of the hepatic enzyme markers (ALT and AST) and reduced oxidative stress of the liver by evaluation of malondialdehyde and glutathione. Histopathological evaluation of the livers also revealed that AF reduced the incidence of liver lesions. The in vitro result showed that AF significantly reduced t-BHP-induced oxidative injury, as determined by cell cytotoxicity, intracellular glutathione content, lipid peroxidation, reactive oxygen species (ROS) levels, and caspases activation. Also, AF up-regulated antioxidant enzymes including heme oxygenase-1 (HO-1), NAD(P)H:quinone reductase, and glutathione S-transferase. Moreover, AF induced Nrf2 nuclear translocation and Akt and ERK1/2 activation, pathways that are involved in inducing Nrf2 nuclear translocation. Taken together, these results suggest that the protective effects of AF against t-BHP-induced hepatotoxicity may, at least in part, be due to its ability to scavenge ROS and to regulate the antioxidant enzyme HO-1 via the Akt and ERK1/2/Nrf2 signaling pathways.     

Quercetin attenuates cadmium-induced oxidative damage and apoptosis in granulosa cells from chicken ovarian follicles

The attenuating effect of quercetin on cadmium-induced oxidative damage and apoptosis was investigated in cultured granulosa cells from chicken ovarian follicles. Results showed that exposure to 5 μM CdCl(2) induced a decrease in granulosa cell number and viability, caused chromatin condensation and DNA fragmentation. Moreover, cadmium treatment markedly increased malondialdehyde level and decreased glutathione peroxidase and superoxide dismutase activities. Furthermore, cadmium provoked higher BAX expression, inhibited expression of BCL2 and X-linked inhibitor of apoptosis protein (XIAP) and activated caspase-3. However, simultaneous supplementation with 1 μg/ml quercetin protected granulosa cells against cadmium-induced cytotoxicity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and alleviating apoptosis by modulating XIAP, BAX and BCL2 expression, and inhibiting caspase-3 activity. Therefore, these results suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in granulosa cells through attenuating lipid peroxidation, elevating intracellular antioxidant status and inhibiting apoptosis to ensure reproductive health.     

Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture

The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 microg/mL), followed by T. testudinum and Erythroxylum extracts, whereas no significant protective effects was produced by Dictyota extract treatment. Antioxidant properties of the natural products against lipid peroxidation and GSH depletion induced by tert-butyl hydroperoxide were then investigated. The results show that at 36 h pre-treatment of cells with mangiferin or MSBE, concentrations of T. testudinum and Erythroxylum extracts ranging from 25 to 100 microg/mL significantly inhibited lipid peroxidation induced by tert-butyl hydroperoxide (100 and 250 microM) and increased the GSH levels reduced by the toxicant. D. pinnatifida inhibited lipid peroxidation, but did not preserve GSH levels. In conclusion, MSBE, E. minutifolium, E. confusum and T. testudinum extracts and mangiferin showed hepatoprotective activity against induced damage in all the experimental series, where mangiferin and the extracts of MSBE and T. testudinum were the best candidates to inhibit "in vitro" damage to rat hepatocytes. This hepatoprotective effect found could be associated with the antioxidant properties observed for the products.     

Cytotoxicity of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on HepG2 cells in relation to glutathione perturbation.

Tanshinones are abietane type-diterpene quinones isolated from the roots of Radix Salvia miltiorrhiza (Danshen), a well-known traditional Chinese medicine in the treatment of cardiovascular diseases. Among the major diterpenes isolated, including cryptotanshinone, tanshinone I, tanshinone IIA and dihydrotanshinone, tanshinone IIA had been shown to posses various pharmacological activities including antioxidant, protection/prevention from angina pectoris and myocardial infarction, and anticancer properties. Tanshinone IIA, usually the most abundant tanshinone present in the herb, has been the focus of studies in its clinical potential, among which its ability to inhibit the proliferation of cancer cell lines. The aim of this study was to study the cytotoxicity of the tanshinones on human HepG2 cells in vitro in relation to intracellular glutathione perturbation (reduced glutathione, GSH and oxidized glutathione, GSSG). Studies using MTT assay showed that all tanshinones decreased cell viability of HepG2 cells in a concentration-dependent manner, with the cell viability decreased to 60% and 35% after 24 h and 48 h treatment, respectively. Assessment of apoptotic cells with fragmented DNA by flow cytometry indicated that only tanshinone IIA (12.5 and 25 microM) induced apoptosis in the cancer cells. Tanshinone IIA and cryptotanshinone caused significant decreases in G(1) cells by 23% and 13%, respectively, after 24 h treatment. The declines in G(1) cells were compensated by increases in G(2)/M (15% for tanshinone IIA) and S cells (8% and 13% for tanshinone IIA and cryptotanshinone, respectively). All the tanshinones studied, except tanshinone IIA, elevated GSH/GSSG ratio at low concentrations (1.56 and 3.13 microM), but the ratio decreased, indicating oxidative stress at high concentrations (6.25-25 microM). Taken together, tanshinone IIA caused HepG2 cytotoxicity through apoptosis without influencing oxidative stress, while the other tanshinones showed lower efficacy in inducing apoptosis in the HepG2 cells.     

Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells: evaluation by the comet assay

In the present study, the chemoprotective effects of quercetin, rutin and ursolic acid on tert-butyl hydroperoxide (t-BHP)-induced DNA damage in a human hepatoma cell line (HepG2) were investigated by the comet assay. To determine whether protection was due to direct chemical interactions alone or to cellular-mediated responses three different types of treatments were used: simultaneous incubation of cells with individual test compounds and the toxicant; pre-treatment with test compound before addition of the toxicant followed or not by a recovery period. The expression of Hsp70 was quantified by Western blotting to test the involvement of heat shock proteins in the cellular responses to the test compounds. In addition, effects on proliferation were evaluated by the MTT assay. The results show that quercetin and ursolic acid prevented DNA damage and had antiproliferative properties in HepG2 cells suggesting an anticarcinogenic potential for these compounds. The protective effects of quercetin against t-BHP-induced DNA damage seem to be due to both direct effects on t-BHP toxicity and to cellularly mediated indirect effects which reflect the potentiation of the cellular antioxidant defenses. Ursolic acid seems to exert effects only through cellularly mediated mechanisms since it was not protective in simultaneous incubation. Quercetin and ursolic acid also showed to increase the rate of DNA repair. Rutin did not have effects at any level. These results, obtained with liver cells, emphasize and confirm the chemopreventive potential of quercetin and ursolic acid, which may help explain the lower cancer incidence in human population with high dietary intakes of fruits and vegetables. These results also demonstrate that Hsp70 is not involved in the observed effects in HepG2.     

Changes in antioxidant defense systems induced by thiram in V79 Chinese hamster fibroblasts.

The role of antioxidant defence systems in protection against oxidative damage of lipids and proteins induced by fungicide thiram during in vitro exposure was investigated in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. We analyzed the catalytic activities of superoxide dismutases (SOD1 and SOD2), Se-dependent and Se-independent glutathione peroxidases (GSH-Px), glutathione reductase (GR), and catalase (CAT), as well as total glutathione/glutathione disulfide ratio (GSH(total)/GSSG). Thiram treatment resulted in an increase in activities of SOD1, Se-dependent GSH-Px, and GR at the highest tested dose (150 microM). On the contrary, inhibition of CAT and Se-independent GSH-Px activities, and no significant changes in the level of SOD2 activity was observed at any tested doses (100-150 microM). GSH(total)/GSSG ratio in the 100 microM thiram treated cells was not significantly changed comparing to the control, despite significant decrease of GSH total (50%). In 150 microM thiram treated cells the ratio falls to 43% of control value. Pretreatment with l-buthionine sulfoximine (L-BSO), an inhibitor of GSH synthesis, significantly enhanced decrease in CAT and Se-independent GSH-Px activities, as well as GSH(total)/GSSG ratio, and reduced Se-dependent GSH-Px activity, following exposure to thiram. Simultaneously, L-BSO pretreatment enhanced increase in SOD1 activity, and had no effect on SOD2, following thiram exposure. Pretreatment with N-acetyl cysteine (NAC), a GSH precursor, prevented enzymatic changes in CAT, Se-dependent GSH-Px, GR, SOD1 activities, and significantly decreased SOD2 activity following exposure to thiram. GSH(total)/GSSG ratio was restored to the control value. This study suggests that following the changes in antioxidant defense systems thiram can act through the production of free radicals.     

α‐Lipoic acid protects against microcystin‐LR induced hepatotoxicity through regeneration of glutathione via activation of Nrf2

Microcystins (MCs), as the most dominant bloom‐forming strains in eutrophic surface water, can induce hepatotoxicity by oxidative stress. Alpha‐lipoic acid (α‐LA) is a super antioxidant that can induce the synthesis of antioxidants, such as glutathione (GSH), by nuclear factor erythroid 2‐related factor 2 (Nrf2). However, the potential molecular mechanism of α‐LA regeneration of GSH remains unclear. The present study aimed to investigate whether α‐LA could reduce the toxicity of MCs induced in human hepatoma (HepG2), Bel7420 cells, and BALB/c mice by activating Nrf2 to regenerate GSH. Results showed that exposure to 10 μM microcystin‐leucine arginine (MC‐LR) reduced viability of HepG2 and Bel7402 cells and promoted the formation of reactive oxygen species (ROS) compared with untreated cells. Moreover, the protection of α‐LA included reducing the level of ROS, increasing superoxide dismutase activity, and decreasing malondialdehyde. Levels of reduced glutathione (rGSH) and rGSH/oxidized glutathione were significantly increased in cells cotreated with α‐LA and MC‐LR compared to those treated with MC‐LR alone, indicating an ability of α‐LA to attenuate oxidative stress and MC‐LR‐induced cytotoxicity by increasing the amount of rGSH. α‐LA can mediate GSH regeneration through the Nrf2 pathway under the action of glutathione reductase in MC‐LR cell lines. Furthermore, the data also showed that α‐LA‐induced cytoprotection against MC‐LR is associated with Nrf2 mediate pathway in vivo. These findings demonstrated the potential of α‐LA to resist MC‐LR‐induced oxidative damage of liver.