Human Liver-derived HepG2 Cells Research Articles

Hepatoprotective and free radical scavenging activities of prenylflavonoids, coumarin, and stilbene from Morus alba.

Chemical investigation of the EtOH extract of Morus alba L. (Moraceae), as guided by free radical scavenging activity, furnished 5,7-dihydroxycoumarin 7-methyl ether (1), two prenylflavones, cudraflavone B (2) and cudraflavone C (3), and oxyresveratrol (4). Compounds 1 and 4 showed superoxide scavenging effects with the IC(50) values of 19.1 +/- 3.6 and 3.81 +/- 0.5 microM, respectively. Compound 4 exhibited a DPPH free radical scavenging effect (IC(50) = 23.4 +/- 1.5 microM). Compounds 2 and 4 showed hepatoprotective effects with EC(50) values of 10.3 +/- 0.42 and 32.3 +/- 2.62 micro, respectively, on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells.

Sesquiterpenes with hepatoprotective activity from Cnidium monnieri on tacrine-induced cytotoxicity in Hep G2 cells.

Bioassay-guided fractionation of the EtOH extract of Cnidium monnieri (Apiaceae) furnished two hepatoprotective sesquiterpenes, torilin (1) and torilolone (2), together with a new derivative, 1-hydroxytorilin (3). Compounds 1 and 2 showed hepatoprotective effects on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. The EC50 values of compounds 1 and 2 were 20.6 +/- 1.86 (P < 0.01) and 3.6 +/- 0.1 (P < 0.01) microM, respectively. Silybin as a positive control showed an EC50 value of 69.0 +/- 3.4 microM.

Differences in the Regulation of the Classical and the Alternative Pathway for Bile Acid Synthesis in Human Liver: NO COORDINATE REGULATION OF CYP7A1 AND CYP27A1

It has been reported that there is a coordinate regulation of sterol 27-hydroxylase (CYP27A1) and cholesterol 7alpha-hydroxylase (CYP7A1) in rats. Thus, the levels of the mRNA corresponding to these two enzymes were found to change in the same direction in rat liver and in isolated rat hepatocytes. In contrast, other groups have not seen such regulation of CYP27A1 in rabbit liver or in rat liver when using an activity assay. In the present work, the effect of bile acid treatment on human CYP27A1/luciferase reporter activity was studied in a transient transfection assay in human liver-derived HepG2 cells. Neither the endogenous 27-hydroxylase activity nor the CYP27A1/luciferase reporter activity were down-regulated by treatment of HepG2 cells with chenodeoxycholic acid or taurochenodeoxycholic acid. We also measured CYP27A1 mRNA and CYP7A1 mRNA in liver of humans subjected to treatment with chenodeoxycholic acid, ursodeoxycholic acid, hydroxymethylglutaryl (HMG)-CoA reductase inhibitor and a combination of HMG-CoA reductase inhibitor and cholestyramine. There was a 60-fold variation in the levels of CYP7A1 mRNA but only a 5-fold variation in the levels of CYP27A1 mRNA. There was no correlation between the two mRNA species. It is concluded that, in humans, there is little or no coordinate regulation of CYP7A1 and CYP27A1 at the transcriptional level, and that CYP27A1 is not subject to a negative feedback control by bile acids. The results underline that marked species differences may exist in mechanisms for control of synthesis of bile acids and cholesterol homeostasis.

Bakuchiol: a hepatoprotective compound of Psoralea corylifolia on tacrine-induced cytotoxicity in Hep G2 cells.

Bioassay-guided fractionation of the H(2)O extract of the seeds of Psoralea corylifolia furnished one hepatoprotective compound, bakuchiol (1), together with two moderately active compounds, bakuchicin (2) and psoralen (3), on tacrine-induced cytotoxicity in human liver-derived Hep G2 cells. The EC(50) values of compounds 1 - 3 are 1.0, 47.0, 50.0 microg/ml, respectively. Silymarin as a positive control showed the EC(50) value with 5.0 microg/ml.

Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids

Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7α-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species. —Chen, W., E. Owsley, Y. Yang, D. Stroup, and J. Y. L. Chiang. Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids.

Inhibition of aflatoxin B1 genotoxicity in human liver-derived HepG2 cells by kolaviron biflavonoids and molecular mechanisms of action.

Kolaviron biflavonoids have demonstrated antihepatotoxic activity in animal studies. The present study investigated the possible chemopreventive potential of kolaviron in inhibiting aflatoxin B1 (AFB1) genotoxicity in HepG2 cells. Kolaviron inhibition of AFB1-induced cytotoxicity by clonogenic assay and genotoxicity by [3H]thymidine incorporation in unscheduled DNA synthesis were evaluated, including antioxidant potential of kolaviron determined by its reduction in the intracellular reactive oxygen species level induced by hydrogen peroxide. Induction of AFB1-detoxicating enzymes such as cytochrome P450 3A4 (3A4) and glutathione S-transferases (GSTs) A1-1/ A2-2 (alpha) and M1B (mu) was determined by reverse transcription polymerase chain reaction (RT-PCR) and northern blotting for the messages and western immunoblot analysis for protein. Kolaviron significantly (P < 0.01) and dose-dependently inhibited the cytotoxicity (by 71.6%) and genotoxicity (47.1%) of AFB1 in HepG2 cells. The antioxidant potential of kolaviron compared favourably with values for the standard antioxidant trolox C (53.8% at only 4.5 x 10(-2)-fold kolaviron concentration) but was below that of butylated hydroxyanisole (58.1% at a ninefold kolaviron concentration). It induced about threefold increases in the messages for 3A4 and GSTs alpha and mu, including a twofold increase in GSTalpha protein. Kolaviron may have chemopreventive potential in inhibition of human AFB1 genotoxicity and possibly hepatocarcinogenesis.

Assay to Detect Inhibitory Substances in Serum of Patients with Acute Liver Failure

Patients with acute liver failure accumulate toxic substances in the circulation which may impair recovery of hepatic function. The aim of this study was to test an in vitro assay to detect inhibitory substances in the serum of patients with acute liver failure. Human liver-derived HepG2 cells were incubated for 24h in 96 well plates (30,000 cells/well) with sera (10%) from 24 patients with acute liver failure due to paracetamol overdose or NANB hepatitis and 11 normal controls. DNA synthesis was determined from the incorporation of 3H-thymidine and cell viability by the metabolism of the tetrazolium dye MTS. HepG2 cells exposed to acute liver failure sera incorporated significantly less 3H-thymidine (median 30% of control, range 0.2-169%) than normal sera (100%, 76-133%, p=0.002). Cell viability was also reduced (75%, 33-112% vs 100%, 96-105%, p<0.001). There was no correlation between these values and patient outcome or levels of plasma TNF-alpha or serum interferon-gamma. The assay detected inhibitory substances in sera of patients with acute liver failure and could be used to monitor the use of liver support systems.

2-Methyl-thiazolidine-2,4-dicarboxylic acid protects against paracetamol induced toxicity in human liver derived HepG2 cells.

The effects of 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP) on paracetamol-induced toxicity were investigated and evaluated in a human liver derived HepG2 cell line. Incubation of the cells with CP (2 mM and 10 mM) drastically attenuated the GSH and cysteine depletion caused by toxic concentrations of paracetamol (1 mM and 5 mM). When CP (10 mM) was introduced alone into the medium, the level of malondialdehyde and the reactive oxygen species were maintained at the control levels with a simultaneous increase of non-protein sulfhydryl in the cells. Thus, the results of our work prove that CP is a non-toxic precursor of cysteine and GSH, and successfully prevents paracetamol toxicity in HepG2 cells.

Sphingomyelin synthase is absent from endosomes.

Both the Golgi and the endosomes have recently been proposed as the main site of SM-synthase, the enzyme responsible for sphingomyelin (SM) biosynthesis. To settle this confusion, we studied the subcellular distribution of SM-synthase in human liver-derived HepG2 and baby hamster kidney BHK-21 cells. To discriminate between Golgi and endosomes we made use of 3,3-diaminobenzidine (DAB) cytochemistry. Cells were incubated with a conjugate of transferrin (Tf) and horseradish peroxidase (HRP), or with unconjugated HRP, to label the recycling pathway and the complete endocytic pathway (including lysosomes) with peroxidase activity, respectively. After cell homogenization, the peroxidase activity was used to induce a local deposition of DAB-polymer. The total SM-synthase activity was not affected by this procedure, and, in contrast to endosomes labeled with (125)I-Tf, organelles containing SM-synthase did not increase in buoyant density as determined by Percoll density gradient fractionation. Thus, little, if any, SM-synthase localizes to the endocytic pathway of HepG2 and BHK-21 cells. In experiments performed at low temperature to inhibit vesicular transport, we found less than 10% of newly synthesized short-chain SM at the cell surface. We conclude that most SM-synthase activity is present in the Golgi, and to a small extent at the cell surface.

Uptake and metabolism of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine by human liver-derived cells: HepG2 cells as a model for thyroid hormone handling by human liver.

The uptake and metabolism of T3 and rT3 was studied in human liver-derived HepG2 cells. The results showed a saturable, time-dependent, and ouabain-sensitive increase in nuclear bound T3. The effects of ouabain (0.5 mmol/L) and unlabeled T3 (10 nmol/L and 10 mumol/L) were much more pronounced at the nuclear level, suggesting the presence of a nonspecific component in total cellular binding. Nuclear binding of rT3 remained below the detection limit in all experiments. Comparison of rT3 metabolism in HepG2 cells and primary cultures of rat hepatocytes showed an approximately 10-fold lower iodide production in HepG2 cells. Iodide production was decreased in the presence of ouabain and almost absent in the presence of propylthiouracil (100 mumol/L). Our data confirmed the presence of a carrier-mediated uptake system for both T3 and rT3. Metabolism data indicated functional type I deiodinase activity in HepG2 cells, the presence of glucuronidating enzymes, and the absence of thyroid hormone sulfotransferase activity. Based on these data, we propose that HepG2 cells provide an appropriate model for thyroid hormone handling by human liver. In addition, we suggest that in human liver sulfation of thyroid hormone, and therefore deiodination of T3 is of only minor importance.

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.