• Toxicological Research
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• v.22 no.3
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• pp.301-306
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• 2006

Microcystin-LR(MC-LR), a cyanobacterial toxin produced by Microcystis aeruginosa, causes severe hepatotoxicity. Here we investigated the morphologic changes of rat hepatocyte spheroid induced by exposure of MC-LR($10^{-6}M$) in vitro. In addition, to determine the effects of such toxin in the process of hepatocyte spheroid formation, primarily isolated hepatocytes were incubated with MC-LR and the process of spheroid formation was observed. In both hepatocyte spheroid and suspension culture systems, the morphologic changes caused by MC-LR were noticible at 5 min post exposure and were characterized by the loss of microvilli, cytoplasmic vacuolation, the accumulation of lipid droplets, and blob formation. Especially, the size and numbers of blob on the cell surface were increased as the incubation time prolonged and the appearance of electron dense bodies were observed in the cytoplasm of hepatocyte at 20 min post exposure. Furthermore, bile canaliculi-like structures in the hepatocyte spheroids were slightly widened and the process of spheroids formation was inhibited in the isolated hepatocytes incubated with MC-LR. These results indicate that morphologic changes in. the hepatocyte membrane and organelles seem to be typical events in showing the MC-LR induced hepatotoxic effects and the spheroid culture method might be a useful experimental tool to evaluate hepatoxicity since it reflects the in vivo status of hepatocytes.

• YAKHAK HOEJI
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• v.37 no.2
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• pp.182-186
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• 1993

$\alpha$-Linolenic acid ethylester, $C_{19}$ spiroketalenolether polyyne, herniarin and steroid were isolated from the leaves of Artemisia selengensis (Compositae). The structures of the compounds were elucidated on the basis of spectroscopic evidence. Liver protective effects of these constituents were studied using galactosamine and CCI$_{4}$-induced cytotoxicity in primary cultured rat hepatocytes.

• Archives of Pharmacal Research
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• v.26 no.1
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• pp.64-69
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• 2003

To analyze vasopressin-induced $Ca^{2+}$ increase in liver cells, rat hepatocytes were isolated and attached to collagen-coated cover slips. Using fura-2, a $Ca^{2+}$-sensing dye, changes in intracellular $Ca^{2+}$ concentration by vasopressin were monitored. Results in this communication suggested that vasopressin-induced $Ca^{2+}$ increase were composed of both $Ca^{2+}$ release from internal $Ca^{2+}$ stores and influx from the plasma membrane. The $Ca^{2+}$ influx consisted of two distinguishable components. One was dependent on the presence of vasopressin and the other was not. SK＆F96365 blocked vasopressin-induced $Ca^{2+}$ influx in a dose-dependent manner. Vasopressin-induced $Ca^{2+}$ release from internal stores diminished in a primary culture of hepatocytes according to the culture time. However, changes in vasopressin-induced $Ca^{2+}$ influx across the plasma membrane differed from changes in the $Ca^{2+}$ release from internal stores, suggesting two separate signalings from receptor activation to internal stores and to the plasma membrane.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.423-427
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• 2001

Chitosan/alginate capsules were formed by electrostatic interactions and had appropriated mechanical strength, permeability to albumin, and stability to hepatocytes. Rat hepatocytes were isolated and immobilized in chitosan/alginate capsules. During the encapsulation process with hepatocyte, 10% of viability was decreased mainly due to the low pH of the chitosan solution. Among various capsule fabrication methods, the chitosan-alginate capsule showed the highest mechanical strength. Addition of collagen in the capsule with hepatocytes enhanced hepatic metabolism as well as the cell viability for 2 weeks of culture. The hepatocyte in the capsule without collagen decreased the viability to 10% for 2-week cultures.

• Toxicological Research
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• v.12 no.2
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• pp.289-293
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• 1996

Our previous studies demonstrated that quinone (menadione) is cytotoxic to rat platelets. In an attempt to assess the relative contributions of redox cycling and/or arylation in quinone-induced cytotoxicity, we have studied three quinones with different mechanisms: 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ; pure redox cycler), menadione (both redox cycler and arylator), and 1, 4-benzoquinone (pure arylator). The order of redox cycling capacity in platelet rich plasma (PRP) isolated from rats was menadione>DMNQ>1, 4-benzoquonone, which was consistent with the previous studies using isolated hepatocytes. 1, 4-Benzoquinone was more toxic to rat platelets than menadione, while DMNQ did not cause cell death at all. Lactate dehydrogenase inhibition studies revealed that 1, 4-benzoquinone inhibited significantly in a time-dependent manner, while menadione and DMNQ did not at all. These results suggested that arylation by quinone compounds might play a critical role in quinone-induced cytotoxicity in rat platelets.

• Toxicological Research
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• v.3 no.2
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• pp.129-141
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• 1987

Hepatocytes isolated from rats which have been pretreated with phenobarbital (80 mg/kg for 3 days), were able to take up N-acetylcysteine from surrounding medium and were able to synthesize the reduced glutathione ($GSH^{\ast}-3$) intracellularly. The N-acetylcysteine is quickly deacetylated after the uptake and increases the pool size of cysteine, which was very low initially (5 nmol/$10^6$ cells). From this increased intracellular cysteine pool, GSH was synthesized. Freshly isolated rat hepatocytes contained a high level of GSH (30 nmol/$10^6$ cells), but upon incubation with the diethylmaleate, it was markedly decreased (10 nmol/$10^6$ cells). The hepatocytes with depleted GSH have lost viability upon incubations with acetaminophen (5mM) and paraquat (2 mM). However, when the N-acetylcysteine (1 mM) was added to this incubation condition, these chemical induced hepatocellular necrosis were prevented for longer durations. This N-acetylcysteine dependent protective effect against the hepatotoxic chemicals was lost by adding methionine sulfoximine (10 mM), an inhibitor of GSH biosynthesis. Both the carbontetrachloride (5 mM) and chioroform (5 mM) added to the incubation medium caused rapid losses of GSH and cell viability, even without the prior depletion of cellular GSH. However, again, if the 1mM N-acetylcysteine was supplemented, the rates of losses of GSH and cell viability were retarded in both cases. Even though large amounts of the added N-acetylcysteine was present in the cell, N-acetylcysteine conjugate of acetaminophen was not formed. Instead, only large amounts of GSH conjugate of the drug was produced. Thus, it is concluded that the added N-acetylcysteine is taken up and utilized for resynthesis of GSH. In turn, this resynthesized GSH contributes to the protection against cytotoxicity inducible with hepatotoxic drugs.

• YAKHAK HOEJI
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• v.32 no.6
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• pp.428-434
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• 1988

The effects of calcium and calcium antagonist, nifedipine on the adrenergic receptor-stimulated glycogenolysis were investigated in isolated rat hepatocytes. The hepatic glycogenolysis induced by alpha-adrenergic receptor stimulation depended on calcium ions, and beta-adrenergic activation was unrelated to calcium ions. Nifedipine decreased the alpha-adrenergic agonist-induced glucose release significantly and the decrease was depended on calcium ions. The glucose release induced by beta-adrenergic agonist was not inhibited by nifedipine.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.288.1-288.1
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• 2002

Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. The aim of this study was to evaluate the ability of aqueous extract from the roots of Platycodon grandiflorum A. DC (Campanulaceae). Changkil (CK). to affect cellular response in primary cultures of rat hepatocytes to t-butyl hydroperoxide (t-BHP) induced oxidative stress and hepatotoxicity. CK-treated cells showed an increased resistance to oxidative challenge. as revealed by a higher percent of survival capacity in respect to control cells. CK added prior or simultaneously with I-BHP reduced enganced lipid peroxidation measured as production of malondialdehyde and enhnaced intracellular reduced glutathinoe depletion by t-BHP. Furhtermore. CK protected from the t-BHP-induced intracellular generation of reactive oxygen species assessde by montioting dichlorodihydrofluorescein fluorescence. it can be concluded that CK exerts an antioxidant action insice the cell. responsible for the abserved modulation of the cellular response to oxidative challenge. and CK have a marked anitioxdative and hepatoprotective potency.

• Journal of Pharmaceutical Investigation
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• v.26 no.1
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• pp.33-41
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• 1996

In order to study the effect of phenobarbital(PB) on the hepatic transport of diltiazem(DTZ), $Ca^{2+}$ channel blocker, we used isolated hepatocytes of rat which was intraperitoneally pretreated with phenobarbital sodium(75 mg/kg) for four days once a day. For the isolation of rat liver cells, a modification of the two step procedure of Seglen was used. DTZ was dissolved in incubation buffer to the final DTZ concentrations of 200, 400, 600, 800 and 1000 ng/ml in order to elucidate the uptake characteristics of DTZ by hepatocytes. Reactions were stopped at 10, 20, 30, 45, 60, 90, 120 and 300 sec. The initial velocity was determined by disappearance of diltiazem in the hepatocyte suspension. On the other hand, to determine the effect of PB on the in vitro hepatic intrinsic clearance of DTZ we obtained the metabolism rates of DTZ in the control and the PB-pretreated rat hepatocyte at various time intervals. According to pretreatment with PB, the size of hepatocyte and the amount of protein per $10^6$ cells were significantly (p<0.01) increased from $26.92{\pm}0.1364\;m$ to $35.31{\pm}1.00\;m$ and from $468{\pm}6.5\;{\mu}g/10^6$ cells to $628.8{\pm}12.1{\mu}g/10^6$ cells, respectively. In the case or hepatic uptake of diltiazem, $K_m$ was not different in the normalization by cell numbers and increased from $2.90\;{\mu}M\;to\;13.89\;{\mu}M$ in the normalization by protein amount. $V_max$ was increased regardless of normalization by protein amount and cell numbers, from $1.21\;{\mu}mole/min \;{\cdot}\;mg\;protein\;to\;3.96\;{\mu}mole/min\;{\cdot}\;mg\;protein\;and\;from\;2.38\;{\mu}mole/min\;{\cdot}\;10^6\;cells\;to\;2.83\;{\mu}mole/min\;{\cdot}\;10^6\;cells$, respectively. The in vitro hepatic intrinsic clearance of DTZ was significantly (p<0.01) increased from $0.640{\pm}0.038\;ml/mim\;{\cdot}\;10^6\;cells\;to\;2.385{\pm}0.212\;ml/min\;{\cdot}\;10^6\;cells$ due to PB-pretreatment. These results suggest that the uptake of DTZ by hepatocyte is extremely fast and PB enhances the hepatic intrinsic metabolic clearance of DTZ.

• YAKHAK HOEJI
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• v.46 no.6
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• pp.466-471
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• 2002

Oxidative stress is considered to be associated with many diseases, such as inflammatory and cardiovascular diseases, aging and cancer. An important etiological mechanism of these diseases may be a causal relationship between the presence of oxidants and the generation of lipid hydroperoxides derived from enzymatic reactions or xenobiotic metabolism. The hydroperoxides can be decomposed to alkoxy- (ROㆍ) and peroxy- (ROOㆍ) free radicals that can oxidize other cell components, resulting in changes in enzyme activity or the generation of mediators, which can cause further cell damage. The aim of this study was to evaluate the ability of aqueous extract from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil (CK), to affect cellular response in primary cultures of rat hepatocytes to t-butyl hydroperoxide (t-BHP) induced oxidative stress and hepatotoxicity. CK-treated cells showed an increased resistance to oxidative challenge, as revealed by a higher percent of survival capacity in respect to control cells. CK reduced t-BHP-enhanced lipid peroxidation measured as production of malondialdehyde and enhanced intracellular reduced glutathione depletion by t-BHP. Furthermore, CK protected from the t-BHP-induced intracellular generation of reactive oxygen species assessed by monitoring dichlorodihydrofluorescein fluorescence. It can be concluded that CK exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge, and CK have a marked antioxidative and hepatoprotective potency.