• Archives of Pharmacal Research
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• v.27 no.7
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• pp.757-762
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• 2004

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes including glutathione S-transferases (GSTs). NF-E2-related factor-2 (Nrf2) phosphorylation by protein kinase C (PKC) is a critical event for its nuclear translocation in response to oxidative stress. Previously, we have shown that peroxynitrite plays a role in activation of Nrf2 and Nrf2 binding to the antioxidant response element (ARE) via the pathway of phosphatidylinositol 3-kinase (PI3-kinase) and that nitric oxide synthase in hepatocytes is required for GSTA2 induction. In view of the importance of PKC and Pl3-kinase in Nrf2-mediated GST induction, we investigated the role of these kinases in peroxynitrite formation for GSTA2 induction by oxidative stress and determined the relationship between PKC and PI3-kinase. Although PKC activation by phorbol 12-myristate-13-acetate (PMA) did not increase the extents of constitutive and inducible GSTA2 expression, either PKC depletion by PMA or PKC inhibition by staurosporine significantly inhibited GSTA2 induction by tert-butylhydroquinone (t-SHa) a prooxidant chemical. Therefore, the basal PKC activity is req- uisite for GSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, induced GSTA2, which was not inhibited by PKC depletion, but slightly enhanced by PKC activation, suggesting that PKC promotes peroxynitrite formation for Nrf2-mediated GSTA2 induction. Treatment of cells with S-nitroso-N-acetyl-penicillamine (SNAP), an exogenous NO donor, in combination with t-BHQ may produce peroxynitrite. GSTA2 induction by SNAP ＋ t-BHQ was not decreased by PKC depletion, but rather enhanced by PKC activation, showing that the activity of PKC might be required for peroxynitrite formation. LY294002 a P13-kinase inhibitor blocked GSTA2 induction by t-BHQ, which was reversed by PMA-induced PKC activation. These results provide evidence that PKC may playa role in formation of peroxynitrite that activates Nrf2 for GSTA2 induction and that PKC may serve an activator for GSTA2 induction downstream of PI3-kinase.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.769-775
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• 2004

Hepatic ischemia and reperfusion (l/R) predisposes the liver to secondary stresses such as endotoxemia, possibly via dysregulation of the hepatic microcirculation secondary to an imbalanced regulation of the vascular stress genes. In this study, the effect of hepatic I/R on the hepatic vasoregulatory gene expression in response to endotoxin was determined. Rats were subjected to 90 min of hepatic ischemia and 6 h of reperfusion. Lipopolysaccharide (LPS, 1 mg/kg) was injected intraperitoneally after reperfusion. Plasma and liver samples were obtained 6 h after reperfusion for serum aminotransferase assays and RT-PCR analysis of the mRNA for the genes of interest： endothelin-1 (ET-1), its receptors $ET_A$ and $ET_B$, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), cyciooxygenase-2 (COX-2), and tumor necrosis factor-a (TNF-${\alpha}$). The activities of serum aminotransferases were significantly increased in the I/R group. This increase was markedly potentiated by LPS treatment. The ET-1 mRNA was increased by LPS alone, and this increase was significantly greater in both the I/R alone and I/R ＋ LPS groups compared to the sham. There were no significant differences in ETA receptor mRNA levels among any of the experimental groups. $ET_B$ mRNA was increased by both LPS alone and I/R alone, with no significant difference between the I/R alone and I/R ＋ LPS groups. The eN OS and HO-1 transcripts were increased by I/R alone and further increased by I/R ＋ LPS. The iNOS mRNA levels were increased by I/R alone, but increased significantly more by both LPS alone and I/R ＋ LPS compared to I/R alone. The TNF-${\alpha}$ mRNA levels showed no change with I/R alone, but were increased by both LPS alone and I/R ＋ LPS. The COX-2 expression was increased significantly by I/R alone and significantly more by I/R ＋ LPS. Taken collectively, significantly greater induction of the vasodilator genes over the constriction forces was observed with I/R ＋ LPS. These results may partly explain the increased susceptibility of ischemic livers to injury as a result of endotoxemia.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.781-789
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• 2004

The effect of 1,8-cineole on cytochrome P450 (CYP) expression was investigated in male Sprague Dawley rats and female BALB/c mice. When rats were treated orally with 200, 400 and 800 mg/kg of 1,8-cineole for 3 consecutive days, the liver microsomal activities of benzy-loxyresorufin- and pentoxyresorufin-D-dealkylases and erythromycin N-demethylase were dose-dependently induced. The Western immunoblotting analyses clearly indicated the induction of CYP 2B1/2 and CYP 3A1/2 proteins by 1,8-cineole. At the doses employed, 1,8-cineole did not cause toxicity, including hepatotoxicity. Subsequently, 1,8-cineole was applied to study the role of metabolic activation in thioacetamide-induced hepatotoxicity and/or immunotoxicity in animal models. To investigate a possible role of metabolic activation by CYP enzymes in thioacetamide-induced hepatotoxicity, rats were pre-treated with 800 mg/kg of 1,8-cineole for 3 days, followed by a single intraperitoneal treatment with 50 and 100 mg/kg of thioacetamide in saline. 24 h later, thioacetamide-induced hepatotoxicity was significantly potentiated by the pretreatment with 1,8-cineole. When female BALB/c mice were pretreated with 800 mg/kg of 1,8-cineole for 3 days, followed by a single intraperitoneal treatment with 100 mg/kg of thioace-tamide, the antibody response to sheep red blood cells was significantly potentiated. In addition, the liver microsomal activities of CYP 2B enzymes were significantly induced by 1,8-cineole as in rats. Taken together, our results indicated that 1,8-cineole might be a useful CYP modulator in investigating the possible role of metabolic activation in chemical-induced hepato-toxicity and immunotoxicity.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.806-810
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• 2004

Metformin is a biguanide antihyperglycemic agent often used for the treatment of non-insulin dependent diabetics (NIDDM). In this study, the pharmacokinetics and pharmacodynamics of metformin were investigated in Korean healthy volunteers during a fasting state for over 10 h. In order to evaluate the amount of glucose-lowering effect of metformin, the plasma concentrations of glucose were measured for a period of 10 h followed by the administration of metformin (oral 500 mg) or placebo. In addition, the concentration of metformin in blood samples was determined by HPLC assay for the drug. All volunteers were consumed with 12 g of white sugar 10 minutes after drug intake to maintain initial plasma glucose concentration. The time courses of the plasma concentration of metformin and the glucose-lowering effect were analyzed by nonlinear regression analysis. The estimated $C_{max}$, $T_{max}$, $CL_{t}$/F (apparent clearance), V/F(apparent volume of distribution), and half-life of metformin were 1.42$\{pm}$0.07 $\mu\textrm{g}$/mL, 2.59$\{pm}$0.18h, 66.12$\{pm}$4.6 L/h, 26.63 L, and 1.54 h respectively. Since a significant counterclock-wise hysteresis was found for the metformin concentration in the plasma-effect relationship, indirect response model was used to evaluate pharmacodynamic parameters for metformin. The mean concentration at half-maximum inhibition $IC_{50}$, $k_{in}$, $k_{out}$ were 2.26 $\mu\textrm{g}$/mL, 83.26 $H^{-1}$, and 0.68 $H^{-1}$, respectively. Therefore, the pharmacokinetic-pharmacodynamic model may be useful in the description for the relationship between plasma concentration of metformin and its glucose-lowering effect.

• Archives of Pharmacal Research
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• v.27 no.7
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• pp.683-692
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• 2004

Curcumin (diferuloylmethane) is a major naturally-occurring polyphenol of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. Curcumin has shown anti-carcinogenic activity in animal models. Curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase; and an effective inducer of heme oxygenase-1. Curcumin is also a potent inhibitor of protein kinase C(PKC), EGF(Epidermal growth factor)-receptor tyrosine kinase and LĸB kinase. Subsequently, curcumin inhibits the activation of NF(nucleor factor)KB and the expressions of oncogenes including c-jun, c-fos, c-myc, NIK, MAPKs, ERK, ELK, PI3K, Akt, CDKs and iNOS. It is proposed that curcumin may suppress tumor promotion through blocking signal transduction path-ways in the target cells. The oxidant tumor promoter TPA activates PKC by reacting with zinc thiolates present within the regulatory domain, while the oxidized form of cancer chemopreventive agent such as curcumin can inactivate PKC by oxidizing the vicinal thiols present within the catalytic domain. Recent studies indicated that proteasome-mediated degradation of cell proteins playa pivotal role in the regulation of several basic cellular processes including differentiation, proliferation, cell cycling, and apoptosis. It has been demonstrated that curcumin-induced apoptosis is mediated through the impairment of ubiquitin-proteasome pathway. Curcumin was first biotransformed to dihydrocurcumin and tetrahydrocurcumin and that these compounds subsequently were converted to monoglucuronide conjugates. These results suggest that curcumin-glucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are the major metabolites of curcumin in mice, rats and humans.

• Archives of Pharmacal Research
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• v.25 no.4
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• pp.511-521
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• 2002

The purpose of this study was to determine whether bromocriptine affects the catecholamines (CA) secretion evoked in isolated perfused rat adrenal glands, by cholinergic stimulation, membrane depolarization and calcium mobilization, and to establish the mechanism of its action. The perfusion of bromocriptine ($1~10{\;}{\mu}M$) into an adrenal vein, for 60 min, produced relatively dose-dependent inhibition in the secretion of catecholamines (CA) evoked by acetylcholine (ACh, 5.32 mM), DMPP ($100{\;}{\mu}M$ for 2 min), McN-A-343 ($100{\;}{\mu}M$ for 2 min), cyclopiazonic acid (CPA, $10{\;}{\mu}M$ for 4 min) and Bay-K-8644 ($10{\;}{\mu}M$ for 4 min). High $K^+$ (56 mM)-evoked CA release was also inhibited, although not in a dose-dependent fashion. Also, in the presence of apomorphine ($100{\;}{\mu}M$), which is also known to be a selective $D_2$-agonist, the CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with bromocriptine ($3{\;}{\mu}M$) in the presence of metoclopramide ($15{\;}{\mu}M$), a selective $D_2$-antagonist, the CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid considerably recovered as compared to that of bromocriptine only. Taken together, these results suggest that bromocriptine can inhibit the CA secretion evoked by stimulation of cholinergic receptors, as well as by membrane depolarization, in the perfused rat adrenal medulla. It is thought this inhibitory effect of bromocriptine may be mediated by inhibiting the influx of extracellular calcium and the release from intracellular calcium stores, through the activation of dopaminergic $D_2$-receptors located in the rat adrenomedullary chromaffin cells. Furthermore, these findings also suggest that the dopaminergic $D_2$-receptors may play an important role in regulating adrenomedullary CA secretion.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.68-76
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• 2004

Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.99-105
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• 2004

Isoflavones have been a central subject in research on the natural phytoestrogens found in Leguminosae. Their effects on bone formation and remodeling are important in that they can act like estrogen by binding on estrogen receptors on the target cell surface. We, therefore, believed that isoflavones may help in the treatment of patients with estrogen deficiency disease such as estrogen replacement therapy (ERT) for osteoporosis. As commonly known, osteoporosis is one of the hormonal deficiency diseases, especially in menopausal women. When estrogen is no longer produced in the body a remarkable bone remodeling process occurs, and the associated events are regulated by growth factors in the osteoblast lineage. In the present study, we investigated whether isoflavones (Isocal) extracted from Sophorae fructus affect the growth factors IGF-I and TGF-$\beta$ that have been known to be related with bone formation. In the study, we found that the active control (PIII) effectively enhanced the level of nitric oxide (NO) and growth factors, and thereby inhibited osteoclastogenesis. The most efficient concentration was $10^{-8}$% within five days, whereas the comparative control (soybean isoflavone) was not as effective even at a lower concentration. In conclusion, the products which contain enriched glucosidic isoflavone and nutrient supplements such as shark cartilage and calcium can be used for osteoporosis therapy by enhancing the production of IGF-I and TGF-$\beta$. Furthermore, the NO produced through endothelial constitutive NO synthase (ecNOS) may playa role in inhibiting bone reabsorption.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.106-110
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• 2004

The antioxidant activities of Acanthopanax senticosus stems were evaluated in $CCl_4$-intoxicated rats. The n-butanol fraction from the water extract of the stems, when pretreated orally at 200 mg/kg/day for 7 consecutive days in rats, was demonstrated to exhibit significant increases in antioxidant enzyme activities such as hepatic cytosolic superoxide dismutase, catalase and glutathione peroxidase by 30.31, 19.82 and 155%, respectively. The n-butanol fraction whereas showed a significant inhibition of serum GPT activity (65.79% inhibition) elevated with hepatic damage induced by $CCl_4$-intoxication. Eleutheroside B, a lignan component, isolated from the n-butanol fraction was found to cause a moderate free radical scavenging effection DPPH, its scavenging potency as indicated in $IC_{50}$ value, being 58.5$\mu$ M. These results suggested that the stems of A. senticosus possess not only antioxidant but also hepatoprotective activities.

• Archives of Pharmacal Research
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• v.27 no.1
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• pp.111-117
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• 2004

Hepatic microcirculatory failure is a major component of reperfusion injury in the liver. Recent data provided some evidence that endothelium-derived vasoconstrictors and vasodilators may be functionally important to the control of the total hepatic blood flow under these conditions of circulatory failure. Since Kupffer cells provide signals that regulate the hepatic response in ischemia/reperfusion (I/R), the aim of this study was to investigate the role of Kupffer cells in the I/R-induced imbalance of vasoregulatory gene expression. Rats were subjected to 60 min hepatic ischemia, followed by 5 h of reperfusion. The Kupffer cells were inactivated by gadolinium chloride ($GdCl_3$, 7.5 mg/kg body weight, intravenously) 1 day prior to ischemia. Liver samples were obtained 5 hrs after reperfusion for RT-PCR analysis of the mRNA for genes of interest: endothelin-1 (ET-1), its receptors $ET_A and ET_B$, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). ET-1 mRNA expression was increased by I/R. mRNA levels for $ET_A$ receptors showed no change, whereas $ET_B$ receptor transcripts increased in the I/R group. The increases in ET-1 and $ET_B$ mRNA were not prevented by the $GdCI_3$ pretreatment. The mRNA levels for iNOS and eNOS significantly increased within the I/R group with no significant difference between the I/R group and the $GdCl_3$-treated I/R group. HO-1 mRNA expression significantly increased in the I/R group and this increase was attenuated by $GdCI_3$. In conclusion, we have demonstrated that an imbalance in hepatic vasoregulatory gene expression occurs during I/R. Our findings suggest that the activation of Kupffer cells is not required for I/R-induced hepatic microvascular dysfunction.