• 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.

• 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.

• 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.

• 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.

• Archives of Pharmacal Research
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• v.13 no.4
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• pp.355-358
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• 1990

In order to investigate the cellular mechanisms of hypoglycemic of brazilin, hepatocyte monolayer culture was introduced and, glycogen synthesis rate and insulin binding were measured as parameters. Glycogen synthesis and insulin sensitivity were remarkably augmented by the treatment of brazilin. Brazilin slightely increased insulin binding. Scatchard analysis revealed that this increase in insulin binding was not due to increase in the binding capacity but in binding affinity. These results suggest that the augmentation of hepatic glycogenesis and insulin sensitivity by brazilin may play an important role in the improvement of hyperglycemia.

• Toxicological Research
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• v.8 no.2
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• pp.285-302
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• 1992

Human and rat hepatocytes were isolated by nonperfusion method and cultured for longer than 5 days. Human liver biopsy sample and rat liver were used as hepatocyte source. Several physical and chemical factors which were influencing on hepatocyte isolation procedure were examined and a batch isolation procedure was established for small size sample of rat liver. Isolated hepatocytes showed normal morphlologica characteristics in microscopy and electron microscopical examinations and a morphologica response to phalloidin. Isolated cells were cultured as a monolayer and proven to have intact morphological characteristics for longer than 15 days. Because human liver sample is harder and tighter compared with rat liver, a standard procedure for rat hepatocytes was slightly modified to reduce mechanical damage. Similarly with rat hepatocytes, isolated human hepatocytes showed a normal morphological characteristics and could be cultured for longer than 15days. Human and rat hepatocytes were examined on their functional integrities including cytochrome-P450 related enzyme activity and it's inducibility, hormonal inducibility of AIB uptake and TAT activity, albumin synthesis, DNA synthesis, cellular protein maintenance. In all parameters used in the present study, human and rat hepatocytes showed normal functional characteristics.

• Toxicological Research
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• v.5 no.1
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• pp.9-15
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• 1989

The effect of butylated hydroxytoluene (BHT) and its major metabolite, 3, 5-di-tert-butyl-4-hydroxybenzoic acid (BHT-acid) on the uptake of taurocholate into hepatocytes was studied using the primary culture of rat hepatocytes. Hepatocyte were isolated by an in situ collagenase perfusion technique and maintained as a monolayer in serum-free meadia for 24 hours before use. The uptake of taurocholate was saturable with an apparent Km of 12.8+2.8 MuM and Vmax of 0.18+0.01 nmol/mg/min. Both BHT and BHT-acid inhibited the hepatocellular uptake of taurocholate when they were added to the culture.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.186-192
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• 2001

A primary culture of human fetal hepatocytes was obtained through a therapeutic abortion process at 26 weeks of gestation period. More than $10^8$ cells were seeded on a plastic plate. These hepatocytes were infected with hepatitis B virus (HBV). The HBV was purified from serum of one chronic HBV carrier. Transformed hepatocytes were subcultured in a 10% FBS-supplemented medium. The morphology of the transformed cell was epithelial-like. The cells from the first pass showed signs of early proliferation and had a latent period of more than 3 months after 6-7 passages. After the rest period, the transformed cell proliferated actively and they were subcultured every three days. Transformed hepatocytes were characterized by detection of the HBV transcript by RT-PCR. The secretion of virions from transformed cells was investigated by PCR with the cell medium. Two types of virions secreted into the culture medium were examined by using the transmission electron microscope. Another approach to study the secretion of virions in to culture medium was carried out with HBV antibody. HBsAg was detected in the culture medium of transformed cells using ELISA and Western blot analyses. These data suggested that the human fetal hepatocyte cell line has been established by infection of HBV, in which this cell line secreted viral particles into the culture medium.

• Korean Journal of Ichthyology
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• v.12 no.3
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• pp.173-179
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• 2000

Effects of Estradiol-$17\beta(E_2)$ and 2, 4-dichlorophenoxy acetic acid (2, 4-D) on vitellogenin(VTG) production were investigated in primary hepatocyte culture of olive flounder, Paralichthys olivaceus. Highest survival rate of hepatocyte were observed at $27^{\circ}C$, which markedly declined equal to 50% of those of $15^{\circ}C$. Vitellogenin production peaked at the concentration of $10^{-6}M\;E_2$. No effect was observed on VTG production at various concentrations of 2, 4-D. However, a low concentration of 2, 4-D (ie, $10^{-8}M$) only appeared increased VTG production. $E_2$ or $10^{-8}M$ 2, 4-D-primed VTG production was markedly inhibited by the addition of $10^{-6}M$ tamoxifen to the culture medium(P<0.01). Inhibition was not affected by combinational treatment with $10^{-6}M$ $E_2$ and $10^{-6}M$ 2, 4-D. These results from the current investigation suggest that 2, 4-D mimics $E_2$, but the mechanism of reaction in inducing the $E_2$ receptor are different in VTG production in oliver flounder hepatocytes.

• 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.