Alanine Aminotransferase Leakage Research Articles

The antioxidant activity of polysaccharides from Armillaria gallica.

The purpose of this study was to investigate the antioxidant activity of Armillaria gallica polysaccharides. It explored whether Armillaria gallica polysaccharides (AgP) could prevent HepG2 cells from H2O2-induced oxidative damage. The results demonstrated that HepG2 cells were significantly protected by AgP, and efficiently suppressed the production of reactive oxygen species (ROS) in HepG2 cells. Additionally, AgP significantly decreased the abnormal leakage of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) caused by H2O2, protecting cell membrane integrity. It was discovered that AgP was also found to regulate the activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX), while reducing malondialdehyde (MDA), thus protecting cells from oxidative damage. According to the flow cytometry analysis and measurement of caspase-3, caspase-8, and caspase-9 activities, AgP could modulate apoptosis-related proteins and attenuate ROS-mediated cell apoptosis.

Efficacy of oltipraz in preventing acetaminophen-induced liver injury in mice.

Oltipraz (OPZ) is a synthetic dithiolethione with potential as a cancer chemopreventive agent, which can work by inducing detoxification enzymes. OPZ is an activator of nuclear factor erythroid 2-related factor 2 (Nrf2), suggesting its involvement in enzyme induction and possible protection against drug-induced liver injury. In this study, we present OPZ-mediated protection of mice against acetaminophen (APAP)-induced liver injury and discuss its possible contributing factors. Overnight-fasted male CD-1 mice were administered APAP intraperitoneally, and some mice were administered OPZ 16h before APAP. Hepatotoxicity was assessed by measuring serum alanine aminotransferase leakage and histopathological evaluation. The hepatic mRNA expressions of CYP2E1, glutamate cysteine ligase (GCL), and NAD(P)H:quinone oxidoreductase (NQO1) were measured by real-time reverse-transcription polymerase chain reaction. OPZ protected mice from APAP-induced liver injury in a dose-dependent manner, but did not alter hepatic glutathione (GSH) content or GCL expression in control mice, indicating that its hepatoprotective effect is not due to changes in basal GSH levels. OPZ did not affect CYP2E1 expression or APAP-induced early GSH depletion, suggesting it does not inhibit the metabolic activation of APAP to produce N-acetyl-p-benzoquinone imine. In contrast, after GSH depletion, OPZ accelerated hepatic GSH recovery. APAP significantly increased GCL expression during liver injury, but OPZ treatment only led to additional NQO1 expression. This suggests that NQO1 is responsible for the enhanced GSH recovery and protection against APAP-induced liver injury seen in OPZ-treated mice. In summary, OPZ protects against APAP-induced liver injury by inducing NQO1 expression and resulting in improved GSH recovery.

Comparative assessment of hepatoprotective properties of Artesunate and flavonoids from Artemisia annua on acetaminophen and carbon tetrachloride-induced cytotoxicity in primary mice hepatocytes

BackgroundArtesunate (ART) is a semi-synthetized molecule from Artemisinin, an active compound isolated from the medicinal plant Artemisia annua, widely used for the treatment of malaria. Previous studies reported that ART may exert a dual effect on the liver. Accordingly, this study investigated the potential protective action of ART against Acetaminophen (APAP) and Carbon tetrachloride (CCl4)-induced hepatotoxicity in primary mice hepatocytes, in comparison to that of flavonoid extracted from A. annua (FAA). In addition, the antioxidant properties of FAA were also assessed. MethodsThe antioxidant activities of FAA and Ascorbic acid (ASC) (0.01–100 μg/mL) were assessed through inhibition of lipid peroxidation, reduction of ferric and phosphomolydenum, and hydroxyl and DPPH radicals scavenging assays. The hepatoprotective effects of FAA and ART (0.1–100 μg/mL) were evaluated against APAP (11 mM) or CCl4 (4 mM) induced oxidative damage in primary mouse hepatocytes. Biochemical parameters associated with hepatotoxicity assessed include cell viability, cell membrane integrity, cellular glutathione, and antioxidant enzyme activities. ResultsThe obtained finding revealed FAA displayed a remarkable antioxidant activities as evidenced by the low IC50/EC50 values (3.85–19.32 μg/mL), comparable to that of ASC (3.26–18.04 μg/mL). When tested at 10 μg/mL, both FAA and ART significantly (p˂0.05) preserved cell viability, inhibited alanine aminotransferase leakage and lipid membrane peroxidation, and restored superoxide dismutase and catalase activities and glutathione content induced by APAP or CCl4 in a similar way as Silymarin. However, ART showed a significant (p˂0.05) cytotoxic effect on hepatocytes at 100 and 1000 μg/mL and did not confer obvious protection at 100 μg/mL. ConclusionOverall, our data demonstrated that ART harms mice hepatocytes at high concentration while conferring relative protection against APAP and CCl4-hepatotoxicity at low concentration. In contrast, FAA effectively protects liver cells without cytotoxicity effect, event at 100 μg/mL. Accordingly, ART should be given to the patient only under a medical prescription.

Thiacloprid-induced hepatotoxicity in zebrafish: Activation of the extrinsic and intrinsic apoptosis pathways regulated by p53 signaling pathway

Thiacloprid (THCP) is one of the major neonicotinoid insecticides, and its wide use has led to high detection in various media of aquatic environment, posing potential risks to aquatic organisms. This study was focused on the phenotypic responses and mechanisms of toxicity in zebrafish (Danio rerio) upon treatment with waterborne THCP (0.4, 4 and 40μM) for 21 days in vivo or 412.9μM for 24h in vitro. In vivo, we found that THCP induced severe oxidative stress, hepatic abnormalities, leakage of alanine aminotransferase and aspartate aminotransferase and apoptosis. The analysis of RNA-sequencing suggested the activation of the p53 signaling pathway under THCP exposure. The following in vitro study showed that THCP intoxication activated reactive oxygen species (ROS)-dependent p53 signaling pathway and induced hepatotoxicity in the zebrafish liver cells. The addition of p53 inhibitor pifithrin-α (10μM) exerted protection against of THCP-induced hepatotoxicity by reducing oxidative stress and inhibiting the p53 signaling pathway and apoptosis. Moreover, gene expression analyses indicated that both the extrinsic and intrinsic apoptosis pathways were involved in apoptosis induced by p53 activation. Overall, our results suggest that activation of the p53 signaling pathway is an important mechanism of THCP-induced hepatotoxicity.

Protection of mice against carbon tetrachloride-induced acute liver injury by endogenous and exogenous estrogens

Gender is a crucial factor determining susceptibility to drug-induced liver injury (DILI) in humans and experimental animals. However, no general concept of sex differences in DILI has been established, as metabolic events specific to one DILI model are difficult to apply to other DILI models. Herein, we examined sex differences in carbon tetrachloride (CCl4)-induced hepatotoxicity, a widely employed DILI model. Male and female CD-1 mice were intraperitoneally administered CCl4. Additionally, some male mice were administered genistein or another isoflavone to evaluate the effects of exogenous estrogens. Dose-dependent alanine aminotransferase leakage was observed at a CCl4 range of 0.5–10 mmol/kg, with male-dominant sex differences mainly observed at lower doses. No sex differences in hepatic glutathione levels or thiobarbituric acid-reactive substance formation were detected. CCl4 induced hepatic inflammatory genes, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, predominantly in female mice, which might be involved in DILI resistance, observed in female mice. Treatment of male mice with phytoestrogens, especially genistein, attenuated CCl4-induced hepatotoxicity. Moreover, genistein inhibited IL-6 and TNF-α expression, suggesting possible hepatoprotection via immunosuppression. In conclusion, female mice are resistant to CCl4-induced hepatotoxicity, and male mice were afforded protection by genistein, probably via mechanisms based on anti-estrogenic, antioxidant and/or anti-inflammatory effects.

A ROS-Sensitive Nanozyme-Augmented Photoacoustic Nanoprobe for Early Diagnosis and Therapy of Acute Liver Failure.

Early diagnosis of acute liver failure (ALF) is critical for curable treatment of patients, because most existing ALF therapies have narrow therapeutic time windows after disease onset. Reactive oxygen species (ROS), which lead to the sequential occurrences of hepatocyte necrosis and the leakage of alanine aminotransferase (ALT), represent early biomarkers of ALF. Photoacoustic imaging is emerging as a powerful tool for in vivo imaging of ROS. However, high-performance imaging probes that can boost the photoacoustic signals of the short-lived ROS of ALF are yet to be developed, and there remains a great challenge for ROS-based imaging of ALF. Herein, a ROS-sensitive nanozyme-augmented photoacoustic nanoprobe for successful in vivo imaging of ALF is presented. The deep-penetrating photoacoustic signals of the nanoprobe can be activated by the overexpressed ROS in ALF due to the synergy between nanocatalytic bubbles generation and thermoelastic expansion. Impressively, the nanozyme-augmented ROS imaging enables earlier diagnosis of ALF than the clinical ALT method, and the ROS-activated catalytic activity of nanoprobe permits timely nanocatalytic therapy of ALF.

Hepatoprotective effects of flavonoids from common buckwheat hulls in type 2 diabetic rats and HepG2 cells.

Flavonoids from common buckwheat hulls (BHFs) show significant antioxidant and antidiabetic potential. However, their hepatoprotective property is yet to be defined. This study aims to examine the hepatoprotective effect of BHFs in type 2 diabetes mellitus (T2DM) rats and chronic high glucose‐damaged HepG2 cells. Results showed that BHF treatment significantly relieves the state of insulin resistance, thereby reducing blood glucose and improving oxidative stress in T2DM rats. It is worth mentioning that BHF treatment improved diabetes‐induced liver damage disorders, manifested as the clearance of liver fat and the decline of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities. In vitro, HepG2 cells pretreated with BHFs maintained higher superoxide dismutase (SOD), glutathione peroxidase (GSH‐px), and catalase (CAT) activities than the unprotected group. In parallel, compared with the unprotected group, BHFs significantly reduced the leakage of ALT and AST in pre‐protected group dose‐dependently. These results indicated that BHFs had considerable antioxidant and hepatoprotective potential and could be promising to be used as nutraceuticals and dietary supplements to prevent and/or protect against liver disorders.

Effect of silibinin on ethanol- or acetaldehyde-induced damge of mouse primary hepatocytes in vitro

Silibinin, one of the flavonoids isolated from milk thistle seeds of Silybum marianum, has hepatoprotective properties against toxins in clinical. However, the detailed mechanisms have remained unclear. This study investigates the underlying mechanism of silibinin in the protection against ethanol- or acetaldehyde-induced damage of neonatal mouse primary hepatocytes in vitro. The results show that ethanol inhibited proliferation of hepatocytes in a time (12, 24, 36 h) and dose-dependent (0–800 mM) manner. However, silibinin did not show protective effect on ethanol (500 mM)-induced suppression of hepatocyte proliferation. Acetaldehyde, the toxic metabolite of ethanol, appearing immediately in individuals after drink also inhibited the proliferation of hepatocytes in a dose-dependent (0–12 mM) manner. Surprisingly, silibinin significantly increased the cell viability and reduced the leakage of alanine amino transferase (ALT) and aspartate amino transferase (AST) in acetaldehyde-treated hepatocytes, suggesting that silibinin protected cell injury caused by acetaldehyde treatment. The apoptosis-inducing effect of acetaldehyde was demonstrated by the increased number of cells in sub-G1 phase as well as caspase-3 activation. Further study shows that acetaldehyde induced autophagy in the hepatocytes. The autophagy inhibitors, 3-Methyladenine (3-MA) and chloroquine (CQ), further decreased the viability of cells treated with acetaldehyde, suggesting that autophagy plays a protective role against apoptosis. Consistently, silibinin (20 μM) significantly reduced the activation of caspase 3 or apoptosis and increased the conversion of LC3-I to LC3-II or autophagy. Taken together, it is concluded that silibinin does not repress the ethanol- induced hepatocyte injury, whereas silibinin reduces acetaldehyde-caused hepatocyte injury through down-regulation of apoptosis and up-regulation of autophagy.

Peculiarities of hemorrhagic vasculitis coexisting with liver steatosis

Our previous study revealed that 73.9 % of patients with adult-onset hemorrhagic vasculitis (HV) - IgA vasculitis have sonographic features of liver involvement and 61.3% of patients have gallbladder pathology, whereas liver functional tests were within normal limits. This study aimed to determine the clinical characteristics of adult-onset HV coexisting with liver steatosis (LS) as a first step of nonalcoholic non-alcoholic fatty liver disease. Methods. We retrospectively reviewed the medical records of 50 adult-onset HV patients, 20 were male and 30 female with a mean age of 46.0±2.0 years. Clinical, echocardiography, electrocardiography, abdominal sonogram and routine laboratory tests data (complete blood count, liver and renal function tests, C-reactive protein (CRP), glucose, uric acid, fibrinogen, serum protein electrophoresis) were analyzed. Patients were divided into two groups in order to evaluate the impact of LS on clinical course of adult-onset HV. In the main group with LS there were 15 male and 16 female with a mean age of 49.4±2.5 years, duration of treatment was 11.5±0.7 days). The control group without LS comprised five male and 14 female (39.0±3.0 years) with treatment duration 12.3±1.2 days. For normally distributed data, means for the two groups were compared using Student's t-test; Pearson's correlation was calculated to examine the association between two continuous variables. Results. In adult-onset HV patients with LS, inflammatory responses with elevated serum CRP level and leukocytosis have been observed. High functional activity of hepatocytes with increased acute-phase protein synthesis (CRP 2.2±0.37 vs 0.71±0.47, р<0.05), uric acid (0.35±0.02 vs 0.28±0.02 mcmol/l, р<0.05), and leakage of alanine aminotransferase (0.33±0.05 vs 0.21±0.02 mmol/g/l, р<0.05) from the mitichondrias of hepatocytes into the blood occurs following a hepatocellular injury. Adult-onset HV patients with LS had a higher frequency of gallbladder disorders (OR 3.43), and significantly larger sizes of left and right ventricles, left atrial and aortic root diameters (all р<0,05).

Structural characterization and in vitro hepatoprotective activity of polysaccharide from pine nut (Pinus koraiensis Sieb. et Zucc.)

A new purified polysaccharide (PNP40c-1) with a molecular weight of 2.06 × 105 Da was obtained from pine nuts (Pinus koraiensis Sieb. et Zucc.). Structural analysis indicated that PNP40c-1 is a homogeneous heteropolysaccharide composed of arabinose, rhamnose and glucose in a molar ratio of 2.98:1.00:0.52. The major backbone consisted of →3,4)-α-l-Arap(1→, →4)-α-l-Arap3Me(1→, →3)-α-l-Rhap(1→ and →6)-β-d-Glcp(1→, and the side chain is β-d-Glcp-(1→ linked at C4-position of →3,4)-α-l-Arap(1→. In addition, the hepatoprotective effect of PNP40c-1 was investigated by human hepatocyte cell line L02 treated with carbon tetrachloride (CCl4). Results suggested that PNP40c-1 could protect L02 cells from CCl4-induced damage by enhancing the activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), increasing the level of non-enzymatic antioxidant glutathione (GSH), suppressing lipid perioxidation and further reducing the leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Hence, PNP40c-1 could be a promising functional food to serve as hepatoprotective agent.

Cytotoxicity of 34 FDA approved small-molecule kinase inhibitors in primary rat and human hepatocytes

Of the 34 FDA approved oral small-molecule kinase inhibitors (KI), 23 (68%) have warnings for hepatotoxicity in product labeling. To better understand the mechanisms of KI hepatotoxicity and whether such effects can be predicted, we examined 34 KIs for cytotoxicity in primary rat and human hepatocytes. The hepatocytes were treated with KIs at ten concentrations normalized to maximal therapeutic blood levels (Cmax). At 5 and 24 h post treatment, lactate dehydrogenase or alanine aminotransferase leakage, caspase 3/7 activities and cellular adenosine triphosphate levels were measured. At 1 to 100-fold Cmax, while 5 KIs were neither toxic to human nor rat hepatocytes, 3 KIs showed similar cytotoxicity in both species and 26 KIs showed species-biased cytotoxicity, with 16 KIs being more toxic to human hepatocytes and 10 KIs being more toxic to rat hepatocytes. At concentrations of 1-, 2.5-, 5-, 10-, 100-fold Cmax, the number of cytotoxic KIs in human hepatocytes was 4, 8, 11, 14 and 27, respectively, and the corresponding number in rat hepatocytes was 1, 4, 9, 12 and 27, respectively. When hepatocyte cytotoxicity at 100-fold Cmax was used to predict KI clinical hepatotoxicity reflected in product labeling, the accuracy was 0.65 with human hepatocytes and 0.59 with rat cells. When the criterion of daily dose ≥100 mg or Cmax ≥1.1 μM was used to predict KI hepatotoxicity, the accuracy was 0.56 or 0.47, respectively. These results suggest both indirect and direct drug-induced hepatocyte toxicity may contribute to the mechanisms of KI-induced hepatotoxicity seen clinically and use of primary hepatocytes is a useful in vitro model to help predict such toxicity.

Active chemical fractions of stem bark extract of Khaya grandifoliola C.DC and Entada africana Guill. et Perr. synergistically protect primary rat hepatocytes against paracetamol-induced damage

BackgroundKhaya grandifoliola (Meliaceae) and Entada africana (Fabaceae) are traditionally used in Bamun (a western tribe of Cameroon) traditional medicine for the treatment of liver related diseases. In this study, the synergistic hepatoprotective effect of respective active fractions of the plants were investigated against paracetamol-induced toxicity in primary cultures of rat hepatocytes.MethodsParacetamol conferred hepatocyte toxicity, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, alanine aminotransferase (ALT), superoxide dismutase (SOD), catalase (CAT) activities, malondialdehyde (MDA) and glutathione (GSH) content assays. The crude extracts were fractionated by flash chromatography and fractions were tested for hepato-(protective and curative) activities. The most active fractions of both plants were tested individually, and in combination based on their respective half effective concentration (EC50).ResultsThe methylene chloride/methanol fractions of K. grandifoliola (75:25 v/v) (KgF25) and E. africana (90:10 v/v) (EaF10) were found to be the most hepato-protective with EC50 values of 10.30 ± 1.66 μg/ml and 13.47 ± 2.06 μg/ml respectively, comparable with that of silymarin (13.71 ± 3.87 μg/ml). These fractions and their combination significantly (P <0.05) improved cell viability, inhibited ALT leakage and MDA formation, and restored cellular CAT, SOD activities and GSH content. The combination was more effective in restoring biochemical parameters with coefficients of drugs interaction (CDI) less than 1.ConclusionThese findings demonstrate that the active fractions have synergistic action in the protection of rat hepatocytes against paracetamol-induced damage and suggest that their hepatoprotective properties may be maximized by using them in combination.

Standardized bioactive fraction of Phaleria macrocarpa (Proliverenol) prevents ethanol-induced hepatotoxicity via down-regulation of NF-κB-TNFα-caspase-8 pathway

ObjectiveTo verify that Proliverenol has a potential ability in protecting cells from ethanol-induced hepatotoxicity. MethodsActivity of Proliverenol against ethanol-induced apoptosis was evaluated at mRNA and protein levels in HepG2 cell exposed to Proliverenol for 1 and 3 h. ResultsProliverenol conferred hepatoprotective activity through increasing cell survival up to 53%–69% via up-regulation of APEX1 DNA repair enzyme for 3.0–4.7 fold and down-regulating of nuclear factor-κB, tumor necrosis factorα and caspase-8 expression, allowing them to prevent 4.5–6.9 fold of alanine aminotransferase (ALT) leakage in HepG2 cells. Our finding revealed that Proliverenol repressed expression of ALT, which is significantly important as possible alternative mechanism for increased blood transaminase activities. In addition, the result also showed that caspase-8 pathway seemed to be involved in the molecular pathway rather than directly inducing mitochondrial damage. ConclusionsThe data support our hypothesis that Proliverenol has a potential ability in protecting cells from ethanol-induced hepatotoxicity. We propose that Proliverenol provides hepatoprotective activity through up-regulating expression of APEX1 that repress DNA fragmentation, and down-regulating expression of nuclear factor-κB, tumor necrosis factorα and caspase-8, which therefore repress ALT leakage and its expression.

Exploration research on hepatotoxic constituents from Polygonum multiflorum root

By observing the cytotoxic effects of anthraquinones on HepG2 cell and using the precision-cut liver slices technique to authenticate the cytotoxic constituents, the paper aims to explore the material basis of Polygonum multiflorum root to cause liver toxicity. Firstly, MTT method was used to detect the effect of 11 anthraquinone derivatives on HepG2 cell. Then, the clear cytotoxic ingredients were co-cultured with rat liver slices for 6h respectively, and the liver tissue homogenate was prepared. BCA method was used to determine the content of protein in the homogenate and continuous monitoring method was used to monitor the leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamine amino transpeptidase (GGT) and lactate dehydrogenase (LDH). The toxic effect of these ingredients on liver tissue was tested by calculating the leakage rate of the monitored enzymes. As a result, rhein, emodin, physcion-8-O-β-D-glucopyranoside and physcion-8-O-(6'-O-acetyl)-β-D-glucopyranoside showed cytotoxic effects on HepG2 cell and their IC₅₀ values were 71.07, 125.62, 242.27, 402.32 μmol•L⁻¹ respectively, but the other 7 compounds are less toxic and their IC₅₀ values can not be calculated. The precision-cut liver slices tests showed that rhein group of 400 μmol•L⁻¹ concentration significantly increased the leakage rate of ALT, AST and LDH (P<0.01), and the rhein group of 100 μmol•L⁻¹ concentration only increased the leakage rate of LDH (P<0.05). With the increase of rhein concentration, the protein content in liver slices decreased significantly (P<0.05) with a certain range of does. Emodin group of 400 μmol•L⁻¹ concentration significantly increased the leakage rate of ALT, GGT and LDH (P<0.01). Physcion-8-O-β-D-glucopyranoside group of 800 μmol•L⁻¹ concentration also significantly increased the leakage rate of ALT, AST and LDH (P<0.01 or P<0.05), but the group of 200 μmol•L⁻¹ concentration only significantly increased the LDH leakage (P<0.05). Along with the increase of the concentration of physcion-8-O-β-D-glucopyranoside, the leakage rate of ALT, AST and LDH showed a trend of increase, but the protein content in liver slices was in decline. Furthermore, MTT reduction ability of liver slices significantly decreased (P<0.01) in the physcion-8-O-β-D-glucopyranoside group of 800 μmol•L⁻¹ concentration. The results suggested that rhein, emodin and physcion-8-O-β-D-glucopyranoside at high concentrations (≥400 μmol•L⁻¹) can produce some damage to the liver tissue. However, the exposure levels of these constituents are very low, so to reach the toxic concentration (400 μmol•L⁻¹ or 800 μmol•L⁻¹) an adult of 65 kg body weight will need at least a single oral 4 898 g, 339 g and 5 581 g of P.multiflorum root respectively, which is far from the statutory dose of crude P. multiflorum root (3-6 g) or its processed product (6-12 g). Therefore, the conclusion that anthraquinones are the prime constituents of the hepatotoxicity of P. multiflorum root are still not be proved.

Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2.

Hepatic stellate cells (HSCs), previously described for liver-specific mesenchymal stem cells (MSCs), appear to contribute to liver regeneration. Microvesicles (MVs) are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP) or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) in culture medium were also assessed. Results showed that (1) HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2) HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3) HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

Hepatoprotective Activity of Water Extracts from Chaga Medicinal Mushroom, Inonotus obliquus (Higher Basidiomycetes) Against Tert-Butyl Hydroperoxide-Induced Oxidative Liver Injury in Primary Cultured Rat Hepatocytes.

We examined the hepatoprotective activity of Inonotus obliquus water extract (IO-W) against tert-butyl hydroperoxide (t-BHP)-induced oxidative liver injury in the primary cultured rat hepatocyte. The 50% radical scavenging concentrations (SC50s) of IO-W for radical-scavenging activity against 2,2'-azino-bis-(3-ethylbenzothi- azoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) were 5.19 mg/mL and 0.39 mg/mL, respectively. IO-W pretreatment to the primary cultured hepatocytes significantly (p<0.05) protected the cells from t-BHP-induced cytotoxic injury even at a low concentration of IO-W (10 µg/mL). The cellular leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as malondialdehyde (MDA) formation caused by t-BHP were significantly (p<0.05) suppressed by IO-W pretreatment (>100 µg/ mL). In conclusion, this study demonstrates that IO-W exhibited hepatoprotective activity against t-BHP-induced oxidative liver injury in the primary cultured hepatocyte probably via its abilities of quenching free radicals, inhibiting the leakage of ALT, AST, and LDH, and decreasing MDA formation.

Role of store-operated Ca2+ channels in ethanol-induced intracellular Ca2+ increase in HepG2 cells

To investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs). HepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting. The ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment. Ethanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Efficacy of aqueous extract of Hippophae rhamnoides and its bio-active flavonoids against hypoxia-induced cell death

Objectives:To investigate the protective efficacy of aqueous extract of Hippophae rhamnoides against chronic hypoxic injury using primary rat hepatocytes.Materials and Methods:The extract was prepared using maceration method and characterized by its phenolic and flavonoid content and chemical antioxidant capacity using ferric reducing antioxidant power assay. Hepatocytes were maintained in hypoxia chamber (3% and 1% oxygen) for 72 h. The cells kept under normoxic condition served as control. The cells were treated with the extract and flavonoids; isorhamentin, kaempferol or qurecetin-3-galactoside. After the end of exposure period; cell survival, reactive oxygen species (ROS), leakage of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH), glutathione peroxidase (GPx), and superoxide dismutase (SOD) levels were measured.Results:The extract showed presence of high phenolic and flavonoid content with significant antioxidant activity in chemical assay. The cell exposed to hypoxia showed concentration dependent cell death and harbored higher reactive oxygen species. In addition, these cells showed significant leakage of intracellular LDH, ALT, and AST accompanied by the diminished levels/activities of GSH, GPx, and SOD. The treatment of cells with aqueous extract of H. rhamnoides reduced hypoxia-induced cell death and prevented increase in ROS levels and leakage of intracellular LDH, ALT, and AST from cells. Moreover, these cells maintained better levels/activities of GSH, GPx, and SOD in comparison to the respective controls. The major flavonoids present in aqueous extract of H. rhamnoides; quercetin-3-galactoside, kaempferol, and isorhamentin also prevented hypoxia induced cell injury individually or in combination, however, the protection offered by these compounds taken together could not match to that of the extract.Conclusions:Overall the findings reveal significance of aqueous extract of H. rhamnoides in controlling ROS-meditated hypoxic injury in cells and can be useful in many hepatic complications.

Short term effects of vanadium and nickel intoxication on rats liver antioxidant defence system

Hoda E. A. Farid, Medhat M. Abozid*, Kamal E. Mahmoud Department of Biochemistry, Faculty of Agricultural, Minufiya University (EGYPT) *Corresponding author: medh_latef@yahoo.com DOI: 10.7813/2075-4124.2012/4-5/A.2 ABSTRACT The present study was performed to investigate short term effects of vanadium and nickel intoxication on rats liver antioxidant defence system. After 4 weeks of oral treatment of 150 mg vanadium (V) /L or 180 mg nickel (Ni) /L and their combination. At the end of treatment, malondialehyde (MDA) level in liver tissues and serum alanine-(ALT) and aspartate-(AST) aminotransferase activities were increased but the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) activities were decreased significantly. Significant decrease was observed in the glutathione (GSH) content in all groups compared with control. Glutathione-S-transferase (GST) activity was showed the same behavior. Joint action analysis of vanadium, nickel and their mixture, depending upon biochemical data measurements showed antagonistic (An) effects in ALT, AST, GST, GSH, SOD and MDA while, additive effect (Ad) was found in CAT. In conclusion, vanadium and nickel treatments caused profound cell damage as indicated by increased MDA in liver and leakage of intracellular enzymes, ALT and AST to the blood, as well as decrease in antioxidant biomarkers. Key words: vanadium, nickel, antioxidant enzymes, malondialdehyde, rat. 1. INTRODUCTION There has been a growing interest for heavy metals, in recent years, studies on impact of trace heavy metals have continued to assume increasing relevance especially in the areas of petroleum chemistry and toxicology [1]. Metals play important roles in a wide variety of biological processes of living systems. Homeostasis of metal ions, maintained through tightly regulated mechanisms of uptake, storage and secretion is therefore critical for life and is maintained within strict limits [2]. Despite the fact that some studies showed its beneficial pharmaceutical effects, vanadium therapeutically exposure and the exposure associated with taking vanadium supplements, especially those which can provide more than 10 mg V/day [3], should be taken into consideration. Long-term intake of even small doses of vanadium could be toxic; no recommended dietary allowance has been clearly established [4]. The main target organ for nickel induced toxicity in animals is the respiratory tract [5]. The toxicity of nickel compounds appears to be related to the solubility of the compound. Several acute animal studies have reported soluble nickel sulfate as being the most toxic and insoluble nickel oxide the least toxic [6]. Vanadium and nickel metals not only are present in residual waters from the oil industry but also can be present as particulate matter in inhaled air [7] and as environmental heavy metals emitted from automotive materials [8]. Both metals are also present as aerosols or in the ashes of thermal power stations operating on oil fuel [9]. The recovery of heavy metals from industrial residues is an important task for environmental and economic reasons [10]. Although toxicity of individual heavy metals has been studied extensively, [11] the toxicological impact of a mixture is relatively less understood. Therefore, studies on mixture of heavy metals are necessary in order to understand their interactions on the living system and to evaluate their risk assessment [12]. Considering the above- stated, the toxic effect of vanadium and nickel in liver of rats after separate and combined exposures was planned to be carried in the present research by using antioxidant indices and marker enzymes of tissue damage (aminotransferases) as biochemical indicators. 2. MATERIALS AND METHODS 2.1. Chemicals Kits for glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were obtained from gamma trade company, Egypt. Ammonium metavanadate (NH

Hepatoprotective effect of lucidone against alcohol-induced oxidative stress in human hepatic HepG2 cells through the up-regulation of HO-1/Nrf-2 antioxidant genes

Lucidone was previously reported to exhibit anti-inflammatory activity in vitro and in vivo. In the present study, we characterized the mechanisms underlying the hepatoprotective effect of lucidone against alcohol-induced oxidative stress in vitro. Human hepatoma (HepG2) cells were pretreated with lucidone (1–10μg/mL) and then hepatotoxicity was stimulated by the addition ethanol (100mM). With response to ethanol-challenge, increased amount of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) release were observed, whereas lucidone pretreatment significantly inhibited the leakage of AST and ALT in HepG2 cells without appreciable cytotoxic effects. We also found that lucidone pretreatment significantly decreased ethanol-induced nitric oxide (NO), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), reactive oxygen species (ROS) and glutathione (GSH) depletion in HepG2 cells. Furthermore, Western blot and quantitative-PCR analyses showed that ethanol-exposure apparently down-regulated endogenous anti-oxidant hemoxygenase-1 (HO-1) expression, whereas pretreatment with lucidone significantly up-regulates HO-1 expression followed by the transcriptional activation of NF-E2 related factor-2 (Nrf-2). Interestingly, the profound up-regulation of HO-1 and Nrf-2 were observed in only ethanol-challenged cells, which evidenced that lucidone-induced induction of HO-/Nrf-2 were specific with oxidative stress. Thus, we concluded that lucidone-mediated up-regulation of phase-II enzymes and HO-1 via Nrf-2 signaling pathway may provide a pivotal mechanism for its hepatoprotective action.