Protective effect of aqueous extract of Perilla frutescens on tert-butyl hydroperoxide-induced oxidative hepatotoxicity in rats.

The leaves of perilla [Perilla frutescens (L.) Britt. var. japonica (Hassk.) Hara] are often used in Asian gourmet food. The object of this study was to evaluate the protective effects of an aqueous extract of perilla leaves on the tert-butyl hydroperoxide (t-BHP)-induced oxidative injury observed in rat livers. The treatment of the hepatocytes with the perilla leaf extract (PLE) significantly reversed the t-BHP-induced cell cytotoxicity and lipid peroxidation. In addition, PLE exhibited ferric-reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activities. The in vivo study showed that the pretreatment with PLE (1000 or 3000 mg/kg) for 5 days before a single dose of t-BHP (i.p.; 0.2 mmol/kg) significantly lowered the serum levels of aspartate aminotransferase and alanine aminotransferase, reduced the indicators of oxidative stress in the liver, such as the glutathione disulfide content and lipid peroxidation level in a dose-dependent manner, and remarkably increased the activity of hepatic gamma-glutamylcysteine synthetase. Histopathological examination of the rat livers showed that PLE reduced the incidence of liver lesions induced by t-BHP. Based on the results described above, it is suggested that PLE has the potential to protect liver against t-BHP-induced hepatic damage in rats.     

Non-toxic concentrations of peroxynitrite commit U937 cells to mitochondrial permeability transition-dependent necrosis that is however prevented by endogenous arachidonic acid

The present study was aimed at examining the mechanism whereby an otherwise non-toxic concentration of peroxynitrite promotes a rapid necrotic response in U937 cells in which cytosolic phospholipase A(2) is pharmacologically inhibited or genetically depleted. We found that loss of viable cells is appreciable 15min after addition of peroxynitrite, does not further increase at 30min and is mediated by mitochondrial permeability transition (MPT). Both MPT and toxicity were prevented by exogenous arachidonic acid (AA). Various experimental approaches produced results consistent with the notion that the AA-dependent protective mechanism takes place 10-15min after addition of peroxynitrite. The observation that the extent of DNA strand scission induced by peroxynitrite did not vary under conditions of different AA availability suggests that this event is either upstream to mitochondrial dysfunction or irrelevant for cytotoxicity. Collectively, these data indicate that a non-toxic concentration of peroxynitrite commits U937 cells to MTP-dependent necrosis that is however prevented by endogenous AA. Thus, mitochondria are a likely target of the cytoprotective signalling triggered by AA.     

Protective effect of linalool,myrcene and eucalyptol against t-butyl hydroperoxide induced genotoxicity in bacteria and cultured human cells

We studied the protective effect of monoterpenes myrcene, eucalyptol and linalool against t-butyl hydroperoxide (t-BOOH) induced genotoxicity in reverse mutation assay with Escherichia coli WP2 IC185 strain and its oxyR mutant IC202, and with the comet assay in human hepatoma HepG2 and human B lymphoid NC–NC cells. The monoterpenes were tested in concentration ranges 0.05–1.5 mg/plate and 0.01–1.0 μg/ml in bacteria and mammalian cells, respectively. Suppression of t-BOOH induced mutagenesis was detected only in IC202 strain, and correlated with the observed inhibition of lipid peroxidation by the three monoterpenes. Linalool and myrcene strongly suppressed t-BOOH induced mutagenesis. Eucalyptol, in addition to moderate suppression of t-BOOH induced mutagenesis, suppressed also spontaneous mutagenesis. In NC–NC cells linalool and myrcene reduced t-BOOH induced DNA damage by about 50% at 0.01 μg/ml, while eucalyptol was less efficient (about 50% reduction at 1.0 μg/ml). In HepG2 cells linalool and eucalyptol reduced DNA damage by 30% and 40%, respectively, while myrcene was ineffective. The repair of t-BOOH induced DNA damage, studied in HepG2 cells, was not affected by monoterpenes. The results indicate that linalool, eucalyptol and myrcene have substantial protective effect against oxidant induced genotoxicity, which is predominately mediated by their radical scavenging activity.     

Antioxidant effects of the chestnut (Castanea crenata) inner shell extract in t-BHP-treated HepG2 cells,and CCl4- and high-fat diet-treated mice

The antioxidant effects of chestnut inner shell extract (CISE) were investigated in a tert-butylhydroperoxide (t-BHP)-treated HepG2 cells, and in mice that were administered carbon tetrachloride (CCl₄) and fed a high-fat diet (HFD). Pre-incubation with CISE significantly blocked the oxidative stress induced by t-BHP treatment in HepG2 cells (P < 0.05) and preserved the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase compared to group treated with t-BHP only. Similarly, the CCl₄- and HFD-induced reduction of antioxidant enzymes activities in liver was prevented by CISE treatment compared to control groups. Furthermore, hepatic lipid peroxidation were remarkably lower (P < 0.05) in the CISE-treated groups with t-BHP or HFD. To determine the active compound of CISE, the fractionation of CISE has been conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the t-BHP-induced ROS generation and reduction in antioxidant enzyme activity in an in vitro model system. From these results, it was demonstrated that CISE has the ability to protect against damage from oxidative stressors such as t-BHP, CCl₄ and HFD in in vitro and in vivo models. The CISE might be useful for the prevention of oxidative damage in liver cells and tissues.     

Size-dependent cytotoxicity of amorphous silica nanoparticles in human hepatoma HepG2 cells

The purpose of this study is to compare the potential cytotoxicity induced by amorphous silica particles with different sizes. The effects of one fine particle (498 nm) and three nanoparticles (68, 43, and 19 nm) on cultured human hepatoma (HepG2) cells were investigated by detecting morphological changes, cell viability, cytomembrane integrity, DNA damage, cell cycle distribution, and apoptosis after the cells were treated with 100 μg/mL of four silica particles for 24 h. The results indicated that in HepG2 cells, the cytotoxicity generated by silica particles strongly depended on the particle size, and smaller silica particle possessed higher toxic effect. In order to further elucidate the possible mechanisms of cell injuries, intracellular reactive oxygen species (ROS) was measured. Increased ROS level was also observed in a size dependent way. However, the result showed the fine particle did not promote intracellular ROS level significantly, while cell injuries were detected in this treated group. Thus, our data demonstrated that exposure to different sizes of silica particles resulted in a size dependent cytotoxicity in cultured HepG2 cells, and ROS generation should be one possible damage pathway but might not be completely responsible for the toxic effect produced by silica particles.     

A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation

Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timing at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.     

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H₂O₂ across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H₂O₂ release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H₂O₂ release, assessed by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H₂O₂ release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells

In the present study the chemopreventive effects of water soluble AquaROX^® 15 and oil soluble VivOX^® 40 rosemary extracts against 4-nitroquinoline-N-oxide (NQNO) and 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ) induced mutagenicity in the reverse mutation assays with Salmonella typhimurium TA98 and against t-butyl hydroperoxide (t-BOOH), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced DNA damage in HepG2 cells were studied, applying the comet assay. The results showed comparable protective effect of AquaROX and VivOX against oxidative DNA damage, whereas protection against indirect active genotoxic carcinogens was more efficient by VivOX.     

Reactive oxygen species-mediated induction of apoptosis by a plant alkaloid 6-methoxydihydrosanguinarine in HepG2 cells

We have found in the previous study that 6-methoxydihydrosanguinarine (6ME), a benzophenanthridine alkaloid isolated from Hylomecon species, may have potential as a chemotherapeutic agent. However, the mechanisms of 6ME-induced cell death have not been investigated. The purpose of the present study was to determine the apoptosis-inducing potential of 6ME in human hepatocarcinoma HepG2 cells and the role of reactive oxygen species in 6ME-induced apoptosis. It can be concluded from the results that 6ME inhibits the growth of HepG2 cells in a concentration- and time-dependent manner (IC50=3.8+/-0.2 microM following 6 h incubation). Treatment of HepG2 cells with 6ME resulted in the release of mitochondrial cytochrome c followed by the activation of caspase proteases, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. 6ME increased the expression of p53 and bax and decreased the expression of bcl-2. The cytotoxic effect of 6ME is mediated by the time-dependent generation of reactive oxygen species. Our results also show that preincubation of HepG2 cells with vitamin C decreased the expression of p53 and bax and inhibited the release of cytochrome c, activation of downstream caspase and the cleavage of poly(ADP-ribose) polymerase, thus inhibiting the apoptosis inducing effect of 6ME.     

Protective effects of dried flower extracts of Hibiscus sabdariffa L. against oxidative stress in rat primary hepatocytes

Dried flower extracts of Hibiscus sabdarrifa L., a local soft drink material and medical herb, was found to possess antioxidant activity in the present study. In the preliminary studies, antioxidant potential of three fractions of the ethanol crude extract (HS-C: chloroform-soluble fraction; HS-E: ethyl acetate soluble fraction; HS-R: residual fraction) obtained from the dried flowers of Hibiscus sabdarrifa L. were evaluated by their capacity of quenching 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and inhibiting xanthine oxidase (XO) activity. HS-E showed the greatest capacity of scavenging free radical (EC₅₀ = 0.017 mg/ml), and HS-C showed the strongest inhibitory effect on XO activity (EC₅₀ = 0.742 mg/ml). Furthermore, antioxidant bioactivities of these crude extracts were investigated using a model of tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat primary hepatocytes. All fractions were found to inhibit significantly the unscheduled DNA synthesis (UDS) induced by t-BHP at a concentration of 0.20 mg/ml. HS-C and HS-E also decreased the leakage of lactate dehydrogenase (LDH) and the formation of malondialdehyde (MDA) induced by t-BHP (1.5 m ) considerably at a concentration of 0.10 and 0.20 mg/ml in the rat primary hepatocyte cultures. These results indicated that the dried flower extracts (HS-C and HS-E) of H. sabdarrifa L. protect rat hepatocytes from t-BHP-induced cytotoxicity and genotoxicity by different mechanisms.     

Differential sensitivity to resveratrol-induced apoptosis of human chronic myeloid (K562) and acute lymphoblastic (HSB-2) leukemia cells

The in vitro effects of resveratrol (RES) on apoptotic pathway in human chronic myeloid (K562) and acute lymphoblastic (HSB-2) leukemia cells were investigated. RES treatment of both cell types significantly and irreversibly inhibited their growth, associated with extensive apoptosis and increase in hypodiploid cells. Cell cycle analysis showed accumulation in G(1) phase in HSB-2 drug exposed cells, while only K562-treated cells exhibited a marked accumulation in S phase with a concomitant decrease in G(1) and G(2)/M at 24 h. Moreover, RES caused internucleosomal DNA fragmentation, even if K562 cells were found less sensitive to the drug, as compared to HSB-2 cells, which also reacted earlier to the treatment. RES-induced apoptosis was associated with an increase of Bax expression and a marked release of cytochrome c from mitochondria. Interestingly, K562 cells exhibited a basal content of glutathione 10-fold that of HSB-2 cells, which increased after 24-48 h RES exposure, together with increment of glutathione reductase and peroxidase activities. However, the major resistance to apoptosis of K562 cells cannot be attributed to their higher pool of reducing power, since neither the inhibition of glutathione synthesis by buthionine sulphoximine nor glutathione depletion by diethylmaleate, sensitized these cells. In addition, glutathione enrichment of HSB-2 cells by N-acetylcysteine did not prevent the apoptotic effects of RES. Our data indicate that RES commitment to apoptosis in both cell lines is independent from the intracellular content of glutathione, while it is associated with either the enhanced expression of Bax and cytochrome c release.     

Phloroglucinol: Antioxidant properties and effects on cellular oxidative markers in human HepG2 cell line

Phloroglucinol is an ubiquitous secondary metabolite encountered in a free state or polymerised as phlorotannins in brown macroalgae, and present in higher plants. FRAP and TEAC assays measured the antioxidant properties of phloroglucinol in non-biological conditions. Additionally, the biological effects of phloroglucinol (4–400 μM) were scrutinised using cellular oxidative stress markers, such as the generation of ROS, antioxidant defences (concentration of GSH and activities of GPx, GR and GST), and levels of MDA as a marker for lipid peroxidation. The direct effect was assessed immediately after an incubation period, whereas for the protective effect, the incubation period was followed by 3-h treatment with the pro-oxidant t-BOOH. The results indicated that despite having a higher radical scavenging capacity than Trolox after 30 min, phloroglucinol was not a suitable antioxidant standard for phlorotannins. Regarding the biological effects, phloroglucinol had no impact on cell viability, reduced levels of ROS and increased antioxidant defences in the direct treatment for most concentrations. The results of the protective effect were mitigated as phloroglucinol failed to protect from ROS generation but evoked a significant recovery of the stress-altered cellular antioxidant defences to restful conditions. Additionally, MDA levels were greatly reduced, preventing a radical chain oxidation.     

Cooperation between Apo2L/TRAIL and bortezomib in multiple myeloma apoptosis

The proteasome inhibitor bortezomib is currently an important drug for treatment of relapsed and refractory multiple myeloma (MM) and for elderly patients. However, cells from some patients show resistance to bortezomib. We have evaluated the possibility of improving bortezomib therapy with Apo2L/TRAIL, a death ligand that induces apoptosis in MM but not in normal cells. Results indicate that cotreatment with low doses of bortezomib significantly increased apoptosis of MM cells showing partial sensitivity to Apo2L/TRAIL. Bortezomib treatment did not significantly alter plasma membrane amount of DR4 and DR5 but increased Apo2L/TRAIL-induced caspase-8 and caspase-3 activation. Apo2L/TRAIL reverted bortezomib-induced up-regulation of β-catenin, Mcl-1 and FLIP, associated with the enhanced cytotoxicity of combined treatment. More important, some cell lines displaying resistance to bortezomib were sensitive to Apo2L/TRAIL-induced apoptosis. A cell line made resistant by continuous culture of RPMI 8226 cells in the presence of bortezomib (8226/7B) was highly sensitive to Apo2L/TRAIL-induced apoptosis. Moreover, RPMI 8226 cells overexpressing Mcl-1 (8226/Mcl-1) or Bcl-x_L (8226/Bcl-x_L) also showed enhanced resistance to bortezomib, but co-treatment with Apo2L/TRAIL reverted this resistance. These results indicate that Apo2L/TRAIL can cooperate with bortezomib to induce apoptosis in myeloma cells and can be an useful adjunct for MM therapy.     

Induction apoptosis of luteolin in human hepatoma HepG2 cells involving mitochondria translocation of Bax/Bak and activation of JNK

Since hepatocellular carcinoma remains a major challenging clinical problem in many parts of the world including Eastern Asia and Southern Africa, it is imperative to develop more effective chemopreventive and chemotherapy agents. Herein, we present an investigation regarding the anticancer potential of luteolin, a natural flavonoid, and the mechanism of its action in human hepatoma HepG2 cells. Using DNA fragmentation assay and nuclear staining assay, it showed that luteolin induced apoptosis of HepG2 cells. Luteolin induced the cytosolic release of cytochrome c and activated CPP32. We found that Bax and Bak translocated to mitochondria apparently, whereas Fas ligand (FasL) was unchanged after a treatment with luteolin for 3 h. In addition, it showed that c-Jun NH2-terminal kinase (JNK) was activated after the treatment of luteolin for 3-12 h. Further investigation showed that a specific JNK inhibitor, SP600125, reduced the activation of CPP 32, the mitochondrial translocation of Bax, as well as the cytosolic release of cytochrome c that induced by luteolin. Finally, the apoptosis induced by luteolin was suppressed by a pretreatment with SP600125 via evaluating annexin V-FITC binding assay. These data suggest that luteolin induced apoptosis via mechanisms involving mitochondria translocation of Bax/Bak and activation of JNK.     

Hepatoprotective and antioxidative effects of total phenolics from Laggera pterodonta on chemical-induced injury in primary cultured neonatal rat hepatocytes

Although Laggera pterodonta as a folk medicine has been widely used for several centuries to ameliorate some inflammatory ailments as hepatitis in China, there have been no studies of the hepatoprotective and antioxidative effects of this plant. In this paper, the hepatoprotective effect of total phenolics from L. pterodonta (TPLP) against CCI₄-, d-GalN-, TAA-, and t-BHP-induced injury was examined in primary cultured neonatal rat hepatocytes. TPLP inhibited the cellular leakage of two enzymes, hepatocyte ASAT and ALAT, caused by these chemicals and improved cell viability. Moreover, TPLP afforded much stronger protection than the reference drug silibinin. Meanwhile, DPPH and superoxide radicals scavenging activities of TPLP were also determined. The present investigation is the first to report chemical-induced injury model in primary cultured neonatal rat hepatocytes and provide evidence for the hepatoprotective and antioxidative effects of L. pterodonta. Neutralizing reactive oxygen species by nonenzymatic mechanisms may be one of main mechanisms of TPLP against chemical-induced hepatocyte injury. Furthermore, The total phenolic content of L. pterodonta and its main component type were quantified, and its principle components isochlorogenic acids were isolated and authenticated. These data support the folkloric uses of L. pterodonta in the treatment of hepatitis.     

Implication of caspases and subcellular compartments in tert-butylhydroperoxide induced apoptosis

Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.     

Protective effects of hesperidin against oxidative stress of tert-butyl hydroperoxide in human hepatocytes

Increasing evidence regarding free radical generating agents and the inflammatory process suggest that accumulation of reactive oxygen species (ROS) could involve hepatotoxicity. Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables, has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, antihypercholesterolemic, and anticarcinogenic actions. However, the cytoprotection and mechanism of hesperidin to neutralize oxidative stress in human hepatic L02 cells remain unclear. In this work, we assessed the capability of hesperidin to prevent tert-butyl hydroperoxide (t-BuOOH)-induced cell damage by augmenting cellular antioxidant defense. Hesperidin significantly protected hepatocytes against t-BuOOH-induced cell cytotoxicity, such as mitochondrial membrane potential (MMP) deplete and lactate dehydrogenase (LDH) release. Hesperidin also remarkably prevented indicators of oxidative stress, such as the ROS and lipid peroxidation level in a dose-dependent manner. Western blot showed that hesperidin facilitated ERK/MAPK phosphorylation which appeared to be responsible for nuclear translocation of Nrf2, thereby inducing cytoprotective heme oxygenase-1 (HO-1) expression. Based on the results described above, it suggested that hesperidin has potential as a therapeutic agent in the treatment of oxidative stress-related hepatocytes injury and liver dysfunctions.     

Drinking of Salvia officinalis tea increases CCl(4)-induced hepatotoxicity in mice.

In a previous study, the drinking of a Salvia officinalis tea (prepared as an infusion) for 14 days improved liver antioxidant status in mice and rats where, among other factors, an enhancement of glutathione-S-transferase (GST) activity was observed. Taking in consideration these effects, in the present study the potential protective effects of sage tea drinking against a situation of hepatotoxicity due to free radical formation, such as that caused by carbon tetrachloride (CCl(4)), were evaluated in mice of both genders. Contrary to what was expected, sage tea drinking significantly increased the CCl(4)-induced liver injury, as seen by increased plasma transaminase levels and histology liver damage. In accordance with the previous study, sage tea drinking enhanced significantly GST activity. Additionally, glutathione peroxidase was also significantly increased by sage tea drinking. Since CCl(4) toxicity results from its bioactivation mainly by cytochrome P450 (CYP) 2E1, the expression level of this protein was measured by Western Blot. An increase in CYP 2E1 protein was observed which may explain, at least in part, the potentiation of CCl(4)-induced hepatotoxicity conferred by sage tea drinking. The CCl(4)-induced hepatotoxicity was higher in females than males. In conclusion, our results indicate that, although sage tea did not have toxic effects of its own, herb-drug interactions are possible and may affect the efficacy and safety of concurrent medical therapy with drugs that are metabolized by phase I enzymes.     

Contribution of the Ah receptor to the phenolic antioxidant-mediated expression of human and rat UDP-glucuronosyltransferase UGT1A6 in Caco-2 and rat hepatoma 5L cells

UDP-glucuronosyltransferases (UGTs) represent major phase II enzymes of drug metabolism which are regulated in a tissue-specific manner by endogenous and environmental factors. Among the latter, aryl hydrocarbon receptor (AhR) agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and phenolic antioxidants such as tert-butylhydroquinone (tBHQ) are known to induce the expression of human UGT1A6 in Caco-2 cells. While binding of the TCDD-activated AhR to one xenobiotic response element (XRE) in the 5'-flanking regulatory region of UGT1A6 was characterised previously, the mechanism responsible for tBHQ induction is unknown. Therefore, it was investigated whether antioxidant response elements (AREs) are involved in tBHQ induction of UGT1A6. Transfectants of 3 kb of its regulatory region and its deletion mutants were treated with tBHQ. These studies suggested a region with approximately 2-fold induction, including an ARE-like motif, 15 bp downstream of the previously characterised XRE. Transfectants of the point-mutated ARE-like motif showed marginally reduced response to tBHQ, but surprisingly, loss of response to TCDD, suggesting interference of flanking proteins with the AhR/Arnt complex. Coordinate responses of UGT activity after treatment with TCDD or tBHQ were also observed in rat hepatoma 5L cells, mutants without the AhR and with recomplemented AhR. The results suggest a contribution of the AhR pathway and of proteins binding to the XRE flanking region to the induction of human UGT1A6 by both AhR agonists and phenolic antioxidants.