OBJECTIVETo investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs).

METHODSHepG2 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.

RESULTSThe 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.

CONCLUSIONEthanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Calcium ; metabolism ; Calcium Channels ; metabolism ; Ethanol ; adverse effects ; Hep G2 Cells ; Hepatocytes ; drug effects ; metabolism ; Humans

OBJECTIVETo investigate the effect of N,N-dimethylformamide (DMF) on calcium homeostasis and calpain I and II gene expression in human hepatocytes (HL-7702).

METHODSHL-7702 cells were exposed to different concentrations of DMF (10, 25, 50, 100, or 200 mmol/L); other HL-7702 cells, which were used as a control group, were exposed to the equal volume of DMEM; the intracellular Ca(2+) concentration was monitored using the calcium fluorescent probe (fluo-3/AM). After 24-h exposure to DMF (10, 25, 50, 100, 150, or 200 mmol/L), the morphology of hepatocytes was observed under an inverted phase contrast microscope, and the cell viability was measured by MTT assay. After 24-h exposure to DMF (10, 25, 50, 100, or 150 mmol/L), the mRNA expression levels of calpain I and II in hepatocytes were measured by real-time quantitative PCR.

RESULTSThere were significant differences in cell viability among different exposure groups (P < 0.01); the 50, 100, 150, and 200 mmol/L DMF exposure groups had a significantly lower cell viability than the control group (P < 0.05). Under the inverted phase contrast microscope, HL-7702 cells gradually lost the original shape, with swelling and shrinking, as the dose of DMF increased, and those treated with 150 mmol/L DMF even became round and floated. The fluorescence density of fluo-3 in hepatocytes increased as the dose of DMF rose, demonstrating a dose-response relationship, and there were significant differences among these exposure groups (P < 0.05). There were significant differences in mRNA expression levels of calpain I and II among these exposure groups (P < 0.01), and the expression increased as the dose of DMF rose; but DMF did not promote the mRNA expression of calpain I at a concentration of 150 mmol/L.

CONCLUSIONDMF can cause damage to hepatocytes, which is related to intracellular calcium increase and calpain mRNA increase.

Calcium ; metabolism ; Calpain ; metabolism ; Cell Line ; Dimethylformamide ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Humans

Cell Line ; Cytochrome P-450 CYP1A1 ; drug effects ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Microsomes, Liver ; drug effects ; metabolism ; Quercetin ; pharmacology

OBJECTIVETo investigate the effect of ginsenoside (GSS) in regulating level of sex hormone receptors in human liver cell line HL-7702.

METHODSThe growth of HL-7702 were detected by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay for choosing the available concentration of GSS; and the effect of GSS on sex hormone receptors in HL-7702 cells were detected by immuno-histochemistry, Western blot and RT-PCR.

RESULTSGSS significantly enhance the protein and mRNA expressions of estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) in HL7702 cell in a dose-dependent manner, the levels of expressions in the GSS treated group were higher than those in the solvent control group respectively (P < 0.05).

CONCLUSIONGSS can up-regulate the protein and mRNA expressions of sex hormone receptors in HL-7702.

Cell Line ; Ginsenosides ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Receptors, Estrogen ; metabolism

Acetyl-CoA Carboxylase ; metabolism ; Cell Line ; Curcumin ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Humans

Cell Line ; Cholesterol ; biosynthesis ; Hepatocytes ; drug effects ; metabolism ; Humans ; Hydroxymethylglutaryl-CoA Synthase ; metabolism ; Lipogenesis ; Quercetin ; pharmacology

Calcium ; metabolism ; Calcium Signaling ; Epinephrine ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Humans

OBJECTIVETo investigate the cytotoxic effect of multi-walled carbon nanotubes (MWCNTs) on human liver L02 cells and its relevant mechanism.

METHODSMWCNTs, carboxyl modification MWCNTs (MWCNTs-COOH), and hydroxyl modification MWCNTs (MWCNTs-OH) were characterized by transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The carbon nanotubes at concentrations of 12.5, 25, 50, 100, and 200 μg/ml were incubated with human liver L02 cells for 24, 48 and 72 hours, respectively. The cell viability was evaluated by water soluble tetrazolium salts assay and the intercellular reactive oxygen species induced by the carbon nanotubes were detected by 2', 7'-dichlorodihydrofluorescein diacetate method.

RESULTSTransmission electron microscope showed that the average outside diameters (10 to 20 nm) and the average length (10 to 30 μm) of the three MWCNTs were similar. Scanning electron microscope indicated that the three MWCNTs had a similar surface topography. X-ray photoelectron spectroscopy demonstrated that the MWCNTs-COOH and MWCNTs-OH had relatively high peak areas at 289 and 286ev, respectively,indicating that they have been modified by carboxyl and hydroxyl groups,respectively. Water soluble tetrazolium salts assay showed that the MWCNTs-COOH was less cytotoxic when compared to MWCNTs which demonstrated to be slightly more cytotoxic than MWCNTs-OH. The capability to induce increase in intracellular reactive oxygen species was in the following order: MWCNTs > MWCNTs-COOH > MWCNTs-OH.

CONCLUSIONSModification of MWCNTs with carboxyl group and hydroxyl group improves the biocompatibility of MWCNTs to some extents. MWCNTs-COOH has better compatibility than MWCNTs at the low concentration,and MWCNTs-OH showed better compatibility than MWCNTs after 48 hours. Different mechanisms may be involved in the interaction between cells and the MWCNTs with different chemical surfaces.

Cell Survival ; drug effects ; Cells, Cultured ; Hepatocytes ; drug effects ; metabolism ; Humans ; Nanotubes, Carbon ; chemistry ; toxicity ; Reactive Oxygen Species ; metabolism

OBJECTIVE: To investigate the effect and possible mechanism of lidocaine precondition on calcium overload and apoptosis of the hepatocytes induced by cell hypoxia-reoxygenation. METHODS: The cultured L02 hepatocytes were randomly divided into 3 groups: a hypoxia-reoxygenation group (Group I), a lidocaine precondition group (Group II), and a normal control group (Group III). After 4 hours of cell hypoxia and 10 hours of reoxygenation, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations in the nutritive medium were detected. The cytoplasm ionic calcium concentration was measured by fluoro spectrophotometer. The apoptosis was measured by flow cytometry and fluorescent microscope. The appearance and ultra-microstructure changes of hepatocytes were observed by inverted microscope and electronic microscope. RESULTS: Cytoplasm ionic calcium concentration and apoptosis was positively correlated (r=0.7652, R(2)=0.5855, P< 0.05). The ALT concentration in the nutritive medium, AST concentration in the nutritive medium, cytoplasm ionic calcium concentration and the ratio of apoptosis of Group I and II were significantly higher than those of Group III(P< 0.05).The appearance and ultra-microstructure changes of Group I and II were worse than those of Group III. The ALT concentration in the nutritive medium, AST concentration in the nutritive medium, cytoplasm ionic calcium concentration and the ratio of apoptosis of Group II were significantly lower than those of Group I (P< 0.05). The ultra-microstructure injury of hepatocytes of Group II were less serious than those of Group I. CONCLUSION Precondition with lidocaine can attenuate calcium overload of hepatocytes induced by hypoxia-reoxygenation in vitro,and decrease the ratio of apoptosis.
Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Lidocaine ; pharmacology ; Oxygen ; metabolism

OBJECTIVETo investigate the effects of hydroquinone (HQ) on expression of Polymerase eta (Pol eta) and DNA damage in human hepatic cells (L-02), and to explore the role and possible mechanism of Pol eta involved in the process of DNA damage-tolerance.

METHODSAfter L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was detected by MTT assay; DNA impairment was detected by single cell gel electrophoresis (SCGE); Real-time fluorescent quantitative PCR and Western blotting methods were used to measure the expression of Pol eta at the mRNA and protein level in L-02 hepatic cells exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L).

RESULTSMTT assay showed that HQ with concentrations from 0 to 80 micromol/L had little effect on the survival rate of L-02 (P>0.05); whereas the survival rate of the group of 160 micromol/Lwas significantly higher than that of the control (P<0.01) after being treated with HQ for 24 h; the higher dose of HQ presented, the more degrees of DNA damage were produced. It was found that HQ in a low concentration (1-80 micromol/L) could induce the expression of Pol eta which was in proportion to the increasements of HQ concentration; the expression levels of mRNA and protein were reached to the maximum when treated with 80 micromol/L; the expression of Pol eta decreased (the relative quantity values were 2.32 +/- 0.16 and 1.20 respectively) once the concentration of HQ exceeded 160 micromol/L as compared with the group of 80 micromol/L, but it was higher than that of the control.

CONCLUSIONThis study suggested that Pol eta might involve in the process of DNA damage-tolerance induced by HQ in the hepatic cells.

Cell Survival ; drug effects ; Cells, Cultured ; DNA Damage ; drug effects ; DNA Repair ; DNA-Directed DNA Polymerase ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Hydroquinones ; adverse effects ; Mutagens