• Development and Reproduction
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• v.13 no.4
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• pp.217-226
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• 2009

Effects of sera from several fish species on monolayer formation, viability and functions of catfish hepatocytes were investigated to establish a primary hepatocyte culture system for screening endocrine disruptors. Hepatocytes of Korean catfish (Silurus asotus) were attached and formed monolayer using the media supplemented with their own serum or sera from eel and tilapia, but not with fetal bovine serum (FBS). The amount of fish sera (0.5~3%) for monolayer culture of the catfish hepatocytes was less than 1/10 of FBS (5~20%) that is commonly used for primary culture of hepatocytes of other species. The results indicate that FBS can be replaced with sera from some fish species and the fish sera are more effective than FBS in maintaining the shape and functions of the hepatocytes. The primary culture of catfish hepatocytes was maintained monolayer with fish sera for at least 10 days, which makes possible to be used for screening the activities of endocrine disruptors. In conclusion, the primary culture system of hepatocytes with fish sera in the present study could be a useful tool for screening and studying endocrine disruptors.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.5
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• pp.496-501
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• 1992

The present work tested the inhibitory effects of bile acids on the cholesterol biosynthesis and the activity of HMG-CoA reductase in cultured rat hepatocytes. The uptake of bile acids in hepatocytes were increased in according to the different bile acid concentrations and culture times. The rate of cholesterol synthesis in cells were inversely decreased to the bile acid concentrations and culture times. As expected, insulin injection (4 units/100g body weight) showed an enhancing effect of the cholesterol synthesis and the HMG-CoA reductase activity. The addition of bile acids in medium of insulin-treated hepatocytes also showed the suppressing effect. This effect was directly confirmed in isolated hepatic icrosomes by the test of HMG-CoA reductase activity. In the test of $Na^+$,$K^+$-ATPase activity in the isolated hepatocyte membrane, only the cholic acid did not stimulate the enzyme system. The reason of such difference is not obvious, but this result indicates that the cholic acid could be absorbed by simple diffusion.

• Journal of Aquaculture
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• v.13 no.1
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• pp.79-85
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• 2000

The effects of estrogen and androgens on Vg gene expression were examined in primary hepatocyte culture and livers of the immature male trout. Specific primers of Vg cDNA were designed with already reported Vg gene nucleotide sequences. PCR product was sequenced and verified with Vg cDNA of rainbow trout. Total RNA was extracted from the cultured hepatocytes and livers of steroid-treated rainbow trout and then it was analyzed by reverse transcriptase- polymerase chain reaction (RT-PCR) analysis. The Vg mRNA and Vg protein synthesis were increased in rainbow trout in vivo and in vitro with E$_2$ and methyltestosterone (MT) There were dose and time-related effects of E$_2$ and MT on vitellogesis. Androgens such as progesterone androsterone and testosterone also stimulated Vg mRNA expression in vitro. The results show that androgens as well as E$_2$ can induce expression of Vg mRNA in trout in vivo and in vitro.

• Preventive Nutrition and Food Science
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• v.8 no.1
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• pp.61-65
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• 2003

This study was investigated to identify the regulatory mechanism of ACC gene expression by hormones and nutrition. The fragment of ACC promoter I (PI) -220 bp region was recombined to pGL3-Basic vector with luciferase as a reporter gene. The primary hepatocyte from the rat was used to investigate the regulation of ACC PI activity. ACC PI (-220 bp)/luciferase chimeric plasmid was transfected into primary rat hepatocyte by using lipofectin. ACC PI activity was shown by measuring luciferase activity. The addition of insulin, dexamethasone, and triiodothyronine to the culture medium increased the activity of ACC PI by 2.5-, 2.3- and 1.8-fold, respectively. In the presence of 1 $\mu$M dexamethasone, the effects of insulin was amplified about 1.2-fold showing the additional effects of dexamethasone. Moreover the activity of luciferase was increased by insulin, dexamethasone, and triiodothyronine treatment approximately 4-fold. These results indicated that insulin, dexamethasone and thyroid hormone coordinately regulate ACC gene expression via regulation of promoter I activity. On the -220 to ＋21 region of ACC PI, the addition of the glucose to the culture medium increased the activity of ACC PI. With 25 mM glucose, luciferase activity increased by 7-fold. On the other hand, on the -220 bp region, ACC PI activity was not changed by polyunsaturated fatty acids. Therefore, it can be postulated that there are response elements for insulin, triiodothyronine, dexamethasone, and glucose, but not PUFAs on the -220 bp region of ACC PI.

• Korean Journal of Environmental Agriculture
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• v.22 no.2
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• pp.94-99
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• 2003

The objective of present study was to investigate the antioxidative and hepatoprotective effects of selenium on cadmium-induced toxicity and lipid peroxidation in rat hepatocyte primary culture. To do this, two separate experiments were conducted. In Experiment 1, primary cultures of rat hepatocytes were incubated for 6 hr in the presence of various concentrations (1, 10, 50, 100, and $500\;{\mu}M$) of cadmium chloride. Cytotoxicity and lipid peroxidation were evaluated using the MTT assay and TBARS assay, respectively. Antioxidative and hepatoprotective effects were determined by measuring the activity of GOT and GSH-Px, respectively. Cell viability was reduced and lipid peroxidation was increased by cadmium in dose-dependent manners. There was significantly negative correlation (r=-0.943, p<0.01) between cell viability and lipid peroxidation GOT activity was increased and GSH-Px activity was decreased by cadmium at the concentration of $50\;{\mu}M$. In Experiment 2, primary cultures of rat hepatocytes were incubated for 6hr in the presence of 100\;{\mu}M$ of cadmium chloride and various concentrations (0.01, 0.1 and 1 ppm) of sodium selenite to assess the effect of selenium on cadmium-induced toxicity and lipid peroxidation. Cell viability and GSH-Px activity were increased by sodium selenite at the concentration of 1 ppm Whereas, lipid peroxidation and GOT activity were reduced by 0.1 ppm of sodium selenite. These results demonstrate that selenium has an antioxidative and hepatoprotective potentials against cadmium.

• Journal of Sericultural and Entomological Science
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• v.50 no.2
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• pp.93-100
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• 2012

To investigate the effects of Protaetia. brevitarsis extracts on the protection against liver damage by carbon tetrachloride($CCl_4$) in rat, two kinds of experiment were performed, firstly by the primary hepatocyte culture and secondly by the animal feeding. The primary hepatocyte culture with the extracts of P.brevitarsis showed significantly low activities of GPT, bile acid, and bilirubin, indicating an excellent protective effect against liver damage by $CCl_4$. Especially, below molecular weight 1,000 blew the water to have 32.1% recovery degree. In the seconde experiment, serum GPT activity was significantly decreased in water fraction of P. brevitarsis compared to $CCl_4$ treatment by 98.2%. Serum concentration of bile acid and bilirubin were tended to increased by $CCl_4$ treatment, but water fraction of P. brevitarsis and silymarin recovered the level. These consistent results in vitro and in vivo suggest that the extracts of P. brevitarsis may have strong protective effects against liver damage induced by the potential toxicants such as $CCl_4$.

• Toxicological Research
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• v.31 no.2
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• pp.137-149
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• 2015

Human hepatocytes, with complete hepatic metabolizing enzymes, transporters and cofactors, represent the gold standard for in vitro evaluation of drug metabolism, drug-drug interactions, and hepatotoxicity. Successful cryopreservation of human hepatocytes enables this experimental system to be used routinely. The use of human hepatocytes to evaluate two major adverse drug properties: drug-drug interactions and hepatotoxicity, are summarized in this review. The application of human hepatocytes in metabolism-based drug-drug interaction includes metabolite profiling, pathway identification, P450 inhibition, P450 induction, and uptake and efflux transporter inhibition. The application of human hepatocytes in toxicity evaluation includes in vitro hepatotoxicity and metabolism-based drug toxicity determination. A novel system, the Integrated Discrete Multiple Organ Co-culture (IdMOC) which allows the evaluation of nonhepatic toxicity in the presence of hepatic metabolism, is described.

• The Journal of Korean Medicine
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• v.19 no.1
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• pp.145-164
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• 1998

The purpose of this study is to evaluate the effect of Injinchunggantang-derivative on proliferation of hepatocyte in rats. Cell viability is studied by MTI assay. The gene related to cell replication such as p53, waf1, bcl-2 and $bcl-_{X_L}$ is quantitized by quantitative RT-PCR and the proteins coded by these genes are studied by Western blotting. The results are as follows. 1. The hepatocytes cultured in medium with lnjinchunggantang-derivative showed better viability compared with control grroup in MTI assay, and the hepatocytes cultured in medium with the Injinchunggantang-derivative-and-ethanol-mixed group showed better viability than the hepatocytes cultrued in 10% ethanol culture medium(control group), noting that Injinchunggantang-derivative has protective effect on hepatocyte injury. There was no dose- and time-dependence. 2. In quantitative RT-PCR, i) Bel-2 gene increased significantly both in Injinchunggantang-derivative group and in Injinchunggantang-derivative-and-ethanol-mixed group, while it showed no significant increase or decrease in other group. ii) $Bcl-_{X_L}$ gene increased significantly in Injinchunggantang-derivative group as well as in Injinchunggantang-deri vative-and-ethanol -mixed group. iii) P53 gene showed no significant increase or decrease in hepatocytes cultured in medium with 10% ethanol and in hepatocytes cultured in medium with Injinchunggantang-derivative-and-ethanol-mixed group, suggesting that 10% ethanol induced cell toxicity, thus increased p53 gene expression. iv) Wafl gene showed no significant increase or decrease in hepatocytes cutured in medium with Injinchtrnggantang-derivative, while increased in hepatocytes cultured in medium with 10% ethanol and in hepatocytes cultured in medium with Injinchtrnggantang-derivative-andethanol-mixed group, suggesting that 10% ethanol induced cell toxicity increased wafl gene expression. 3. In the study on protein by western blotting, the band of bcl-2 and $bcl-_{X_L}$ were widened in Injinchtrnggantang-derivative group. Especially the amount of $bcl-_{X_L}$ increased significantly compared with other groups. But in the study on p53 and wafl, there was no significant difference among those groups. Above study shows that Injinchunggantang-derivative has good effect on cell viability and that the genes resistant to cell death such as bcl-2 and $bcl-_{X_L}$ are induced by Injinchunggantang-derivative to resist to cell death by toxic agent And this is reconfirmed in protein study using' western blotting: These results suggest that Injinchunggantang-derivative has inhibitory effect on cell death as well as protective effect on hepatocyte. Therefore this prescription is recommended in various liver diseases such as chronic liver disease and-induced hepatic injury.

• Polymer(Korea)
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• v.32 no.2
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• pp.131-137
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• 2008

We have fabricated polystyrene (PS) nanofibrous scaffold for hepatocyte culture by electrospinning method and subsequently coated with specific ligand of Poly[N-p-vinylbenzyl-O-$\beta$-D-galactopyranocyl-($1{\rightarrow}4$)-D-gluconamide](PVLA) to enhance hepatocytes attachment. Rat hepatocytes behavior on the PVLA-coated and non-coated PS nanofibrous matrices have been investigated. Electrospun PS nanofiber structures revealed randomly aligned fibers with average diameter of 500 nm. It is observed that PS nanofibrous matrix could incorporate many cells into the interior of the matrix probably due to the suitable pore size. Cell viabilities cultured on PVLA-coated PS nanofibrous mats were maintained for 3 weeks, while it was decreased rapidly on PVLA-coated PS dishes. High hepatic functions especially for albumin secretion and ammonia removal were maintained at least for 2 weeks on nanofibrous mats but rapidly decreased on flat PS dishes. These results indicate that nanofibrous structure enabled 3-D culture with high level of cell-cell contact results in providing cell-cell communications and subsequent long-term maintenance of specific cell functions.

• Proceedings of the Korean Environmental Sciences Society Conference
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• 2003.11a
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• pp.159-164
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• 2003

Effects of Al on vitellogenin (VTG) and VTG mRNA induction by estradiol-17 $\beta$($E_2$) were examined in primary hepatocyte culture of rainbow trout. Hepatocytes were precultured for 2 days and then E2 ($2{\times}10^{-6}$M) and Al ($10^{-6}-10^{-4}$M) were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days. Media and hepatocytes were then analyzed by SDS-PAGE and Northern blotting for VTG and VTG mRNA, respectively. These metal had no appreciable effect on the viability of hepatocytes in culture. However, Al interfered with VTG production and VTG mRNA expression. Al reduced VTG production in a concentration-dependent way and a significant reduction accurred at Al concentrations greater than $5{\times}10^{-5}$M. VTG mRNA expression also decreased with a negative correlation with Al concentration (r＝-0.98). These results suggest that Al inhibit VTG production at the transcriptional level to reduce VTG mRNA expression.