• Fisheries and Aquatic Sciences
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• v.1 no.1
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• pp.24-29
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• 1998

Effect of extracellular calcium in vitellogenin (VTG) production in response to estradiol-17 $\beta$ $(E_2,\;2\times10^{-6}M)$ was examined in primary hepatocyte culture of rainbow trout, Onchorhynchus mykiss. Total calcium in estrogenized sera significantly increased, compared with the control, while diffusible calcium was insignificant. However, diffusible calcium in the incubation medium with $E_2$ was significantly reduced, compared with the control. The uptake of extracellular calcium by cultured hepatocytes signifIcantly increased 90 min after $E_2$ addition. Moreover, the accumulation of intracellular calcium increased in the cultures with $E_2$, regardless of the calcium concentrations in the incubation media. In addition, $E_2-primed $ VTG production was significantly decreased by withdrawal of E_2$ from the incubation medium. Moreover, VTG production by $E_2-primed$ hepatocytes was reduced by removing calcium from the incubation medium with or without $E_2$. These results suggest that the entry of extracellular calcium into the cytoplasm is an important step for VTG production in primary hepatocyte cultures in rainbow trout.

• Fisheries and Aquatic Sciences
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• v.5 no.4
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• pp.251-257
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• 2002

Effects of bisphenol-A (BPA) on vitellogenin (VTG) synthesis and estrogen-estrogen receptor $(E_2- ER)$ binding activity were examined in primary hepatocyte cultures of rainbow trout, Oncorhynchus mykiss. Hepatocytes were precultured for 2 days and then $estradiol-17\beta\;(E_2,\;2\times10^{-6}M)\;BPA\;(10^{-5}-10^{-8}M)$ and/or 4-hydroxy-tamoxifen $(4-OHT,\;10^{-6} M)$ were simultaneously added to the incubation medium. Hepatocytes were cultured for 5 more days and then spent medium was analyzed by SDS-PAGE for VTG production. The addition of BPA to the incubation medium had no effect on the viability of hepatocytes in the culture. On the other hand, BPA increased VTG production in a concentration-dependent way and a significant increment occurred at BPA concentrations greater than $10^{-6}$M. Although VTG was increased by the addition of $E_2\;(2\times10^{-6}\;M)\;or\;BPA\;(10^{-5}M)$, its were reduced by a simultaneous 4-OHT $(10^{-6}\;M)$ addition. BPA inhibited $E_2-human$ ER binding activity by $72\%$ at $10^{-5}$ M of BPA. These results suggested that BPA induced VTG synthesis by BPA-ER binding activity in the hepatocyte of rainbow trout.

• YAKHAK HOEJI
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• v.54 no.5
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• pp.377-386
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• 2010

Single-dimensional (1-D) and two-dimensional (2-D) LC methods were utilized to separate peptides from various sources followed by MS/MS analysis. Two-dimensional ultra-high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than 1-D LC. The most popular 2-D LC approach used today for proteomic research combines strong cation exchange and reversed-phase LC. We have evaluated an alternative mode for 2-D LC of peptides using 2-D RP-RP nano UPLC Q-TOF Mass Spectrometry, employing reversed-phase columns in both separation dimensions. As control experiments, we identified 129 proteins in 1-D LC and 322 proteins in 2-D LC from E. coli extract peptides. Furthermore, we applied this method to rat primary hepatocyte and a total of 170 proteins were identified from 1-D LC, and 527 proteins were identified from all 2-D LC system. The in-depth protein profiling established by this 2-D LC MS/MS from rat primary hepatocyte could be a very useful reference for future applications in regards to drug induced liver toxicity.

• Journal of Aquaculture
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• v.11 no.2
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• pp.279-282
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• 1998

Effects of high concentration of intracellular calcium on estradiol-induced vitellogenin(VTG) induction were examined using ouabain in Primary hepatocyte culture in the rainbow trout Oncorhynchus mykiss. Ouabain increases cytosolic free calcium as a result of inhibition of $Na^+ - Ca^{2+}$ exchanger. Ouabain markedly reduced VTG production to the control level, despite of calcium concentrations in the incubatin medium. Therefore, ouabain would reduce VTG production not by increasing intracellular calcium bt directly by inhibiting $Na^+ - K^+$ ATPase.

• Fisheries and Aquatic Sciences
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• v.1 no.2
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• pp.153-158
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• 1998

Effects of cyclic (c) AMP and G-protein related reagents (3-isobutyl-l-methyxanthine (IBMX), Forskolin (FSK), cholera toxin (CTX), and pertussis toxin (PTX≫ on estradiol-17$\beta$ induced vitellogenin (VTG) induction were examined in primary hepatocyte cultures in rainbow trout Oncorhynchus mykiss. The addition of IBMX, FSK, or CTX to the incubation medium markedly increased VTG production, while PTX was not effective in stimulating the production. It is well known that cAMP regulates phosphorylation and dephosphorylation through mediation of protein kinase A. These results suggest that VTG production is highly dependent on cAMP state in hepatocytes because of its highly phosphorylated nature.

• Journal of the Korean Society of Food Science and Nutrition
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• v.27 no.2
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• pp.344-349
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• 1998

The purpose of this study was carried out to investigate the effect of scoparone(6, 7-dimethoxyco-umarin) on liver function. Sprague-Dawley rats were treated with scoparone at a dose of 20mg/kg body weight for 5 days. Hepatic bile flow, liver weight, BSP(bromosulfophthalein) biliary excretion, alanine aminotransferase(ALT) and aspartate aminotransferase(AST) activities, malondialdehyde production and lactate dehydrogenase(LDH) release were assayed. Among them, ALT and AST activities, malondialdehyde production and LDH release were assayed by using primary hepatocyte cultures at a concentration of 0.1mg/ml. Scoparone treatment had no effect on liver weight and hepatic bile flow. Scoparone treatment not only increased BSP biliary excretion, but also recovered the decreased BSP biliary excretion by CCl4, Also scoparone significantly decreased with the increases of ALT and AST activities, malondialdehyde production and LDH release induced by CCl4. These results suggested that scoparone could protect the liver damage by chemicals via promoting the liver excretory function.

• Toxicological Research
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• v.3 no.1
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• pp.1-8
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• 1987

In vitro induction of cytochrome 450 associated monooxygenase activities by phenobarbital (PB) and 3-methylcholanthrene (MC) was investigated in primary cultures of adult rat hepatocytes. PB and MC were added to the culture 24 hr after the initial plating of hepatocytes. A signiftcant increase of the activities of 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase were observed in MC and PB treated culture. MC caused about 500% induction of the initial oxidation rates of both enzymes in 48 hr. However the PB maintained both enzyme activities close to the level of freshly isolated hepatocytes. Biphenyl 4-hydroxylase and aminopyrine N-demethylase activities were also induced by MC and PB. But the level of induction was less than that occuring with 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase. When aflatoxin $B_1$ was added to the hepatocyte cultures which have been treated with MC or PB, it caused a significant increase of the unscheduled DNA synthesis at higher dose of aflatoxin $B_1$ as compared to those of untreated control hepatocyte cultures. The results suggest that microsomal enzyme activities can be selectively controlled preferably in hepatocyte cultures by the in vitro induction method. This principle may be useful for studying the metabolism and other toxicological studies.

• Journal of Biomedical Engineering Research
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• v.19 no.3
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• pp.215-224
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• 1998

This paper is fundamental study to develope the extracorporeal liver support system for patient with fulminant hepatic failure(FHF) or being expected for orthotopic liver transplantation. The polyurethane foam, which is composed of the density of 33kg/m3, the average pore diameter of 500${\mu}{\textrm}{m}$, the closed window of 60-70%, was manufactured with the prepolymer of 15% NCO-, Hepatocytes were inoculated to form spheroids in polyurethane foam. The time of spheroid formation in BSA(Bovine Serum Albumin) coated polyurethane foam was shorter than that in raw polyurethane foam. To verify the function of hepatocyte spheroids, we measured ammonia removal rate, urea and albumin secretion rate. Polyurethane foam was suitable for culture of hepatocyte spheroids. And culture of hepatocyte spheroids in polyurethane foam has high possibility in using as an extracorporeal liver support system.

• Journal of Environmental Science International
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• v.13 no.4
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• pp.413-420
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• 2004

The objectives of present study were to investigate the effects of benzo[a]pyrene(BaP) on cytotoxicity, lipid peroxidation and antioxidant enzymes in rat hepatocyte primary culture. Primary cultures of rat hepatocytes were incubated for 24 hr, 48 hr or 72 hr in the presence of various concentrations (0, 10, 20, 30, 50 or 100 $\mu.$ M) of BaP. Cytotoxicity and cell viability were determined by measuring glutamic oxaloacetic transaminase(GOT) activity, lactate dehydrogenase(LDH) activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MIT) value. Lipid peroxidation was evaluated using thiobarbituric acid reactive substances(TBARS) assay. Effects on antioxidant system were determined by measuring glutathione peroxidase(GPx) activity, glutathione reductase(GR) activity and glutathione concentration. Activities of GOT and LDH, MTT value as well as TBARS concentration were not affected by up to 100 $\muM$ of BaP for 24 hr incubation. However, BaP at the concentration of 50 $\muM$ for 48 hr incubation or at the concentration of 30 $\muM$ for 72 hr incubation began to increase LDH activity and TBARS concentration but decrease MTT value, representing that BaP caused cytotoxicity and decreased cell viability in dose- and time-dependent manners. GPx activity began to be decreased by BaP at the concentration of 50 $\muM$ for 72 hr incubation. Whereas, GR activity began to be decreased by BaP at the concentration of 20 $\muM$ for 72 hr incubation. Glutathione concentration began to be decreased by BaP at the concentration of 20 $\muM$ for 72 hr incubation and was further reduced to 90% by 100 $\muM$ of BaP. These results demonstrate that BaP caused cytoctoxicity and decreased cell viability by increasing lipid peroxidation and decreasing glutathione concentration as well as activities of GPx and GR.

• YAKHAK HOEJI
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• v.50 no.6
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• pp.358-366
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• 2006

The aim of this study was to investigate the protective effects of glycynhizin, active glycosides of Glycyrrhizae Radix, and baicalin, bioactive flavonoid isolated from Scutellariae Radix, on hepatocyte injury induced by carbon tetrachloride(CCl$_4$, 10 mM), tert-butyl hydroperoxide (TBH, 0.5 mM), and D-galactosamine (GaIN, 30 mM). Primary cultures of rat hepatocyte (18 hr cultured) were treated with CCl$_4$, TBH, or GaIN and various concentrations (0.1, 1, 10, and 100 ${\mu}$M) of glycyrrhizin or baicalin. Activity was accessed by determining the release of lactate dehydrogenase (LDH) and aminotransferses. CCl$_4$ significantly increased the levels of LDH, alanine aminotransferase (ALT), and aspartate aminotransferase(AST) and these increases were prevented by baicalin concentrations of 0.1,1, and 100 ${\mu}$M. The increases in ALT and AST levels were reduced by glycyrrhizin concentration of 100 ${\mu}$M. The level of LDH was markedly increased by TBH, and this increase was reduced by both glycyrrhizin and baicalin. ALT and AST levels were increased by TBH, which were prevented by glycynhizin and bacalin, respectively: GaIN markedly increased the levels of LDH, ALT and AST These increases was significantly reduced by both glycyrrhizin and baicalin. These results suggest that glycynhizin and baicalin possess the hepatoprotective activity.