• 한국환경과학회지
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• 제13권4호
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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.

• 한국양식학회지
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• 제11권2호
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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.

• 한국식품영양과학회지
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• 제27권2호
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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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• 제5권4호
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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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• 제13권4호
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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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• 제8권2호
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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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• 제5권1호
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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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• 제11권2호
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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.

• 한국어류학회지
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• 제12권3호
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• pp.173-179
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• 2000

Estradiol-$17{\beta}(E_2)$와 2,4-dichlorophenoxy acetic acid (2, 4- D)가 난황 전구물질의 합성에 미치는 영향을 넙치 Paralichthys olivaceus 간세포의 초대 배양을 통하여 조사하였다. 배양간세포의 생존율은 배양온도 $27^{\circ}C$에 서 가장 높게 나타났으며, $15^{\circ}C$에서는 생존율이 급격히 감소하여 약 50%의 생존율을 나타내었다. $E_2$에 의한 VTG 의 합성은 $10^{-6}M$에서 최대치를 나타내었다. 2, 4-D $10^{-7}\sim10^{-5}M$의 첨가에 의해 VTG의 합성은 이루어지지 않았다. 그러나, 저농도인 $10^{-8}M$에서는 VTG의 합성이 증가하였다. $E_2$ 및 2, 4-D에 의해 합성된 VTG는 $10^{-6}M$ tamoxifen의 첨가에 의해 유의하게 억제되었다 (P<0.01). 본 연구 결과에서 $E_2$와 2, 4-D의 동시 첨가는 VTG의 합성을 억제하지 않았다. 이러한 결과는 2, 4-D의 작용이 $E_2$와 유사한 작용을 가지지만, VTG의 합성에 있어서 $E_2$ 수용체에의 작용 양식은 서로 다른 것으로 추정된다.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.217-226
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• 2009

본 연구는 내분비 장애물질의 검출을 위하여 간세포의 단층 형성, 생존 및 기능에 미치는 어류 혈청의 영향에 대해 검토하였다. 한국산 메기의 간세포는 자신의 혈청 및 뱀장어, 틸라피아 등 타어종의 혈청에 의해 부착 및 단층이 형성되었으나, FBS는 메기 간세포의 단층을 형성시키지 못했다. 0.5에서 3%의 어류 혈청으로 메기 간세포의 단층을 형성 시킬 수 있는데, 이것은 FBS(5~20%) 사용의 1/10 이하로 적은 양이며, 어류 혈청이 FBS를 대체할 수 있고, FBS보다 간세포의 형태 및 기능 유지에 효과적인 것으로 나타났다. 어류 혈청이 첨가된 배양액에서 메기 간세포는 적어도 10일 이상 단층 형성을 유지할 수 있어, 내분비 장애물질 연구에 이용될 수 있을 것이다. 결론적으로 본 연구에서 개발된 어류 혈청을 사용한 메기 간세포 배양시스템과 효소면역측정법(ELISA)은 bisphenol A 등의 내분비 장애물질의 검출 및 연구를 위한 유용한 도구로서 이용될 수 있다고 생각된다.