• Archives of Pharmacal Research
• /
• 제27권7호
• /
• pp.757-762
• /
• 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.

• 한국환경보건학회:학술대회논문집
• /
• 한국환경보건학회 2005년도 국제학술대회
• /
• pp.341-344
• /
• 2005

The expression of the glutathione S-transferase (GST), whose induction accounts for antioxidant defense system, is regulated by activation of CCAAT/enhancer binding protein ${\beta}$ ($C/EBP{\beta}$), Sick house syndrome (SHS) presents healthy damage owing to the indoor environment of a building. Toluene has been implicated in one of the important causes of SHS. The present study investigated the effects of toluene treatment on the induction of GSTA2 gene and its mechanism. H411E cells treated with toluene, and GSTA2 expression was determined by immunoblot analysis. The translocation of $C/EBP{\beta}$ was assessed by immunocytochemical assays. $C/EBP{\beta}$ DNA binding activity was determined by electrophoretic mobility shift assays. The role of the C/EBP binding site in the induction of the GSTA2 gene was assessed by luciferase reporter-gene activity. Toluene induced GSTA2 protein expression. In toluene-treated cells, $C/EBP{\beta}$ translocated to the nucleus and bound to the consensus sequence of C/EBP (TTGCGCAA). Toluene treatment increased luciferase reporter-gene activity in cells transfected with the C/EBP-containing regulatory region of the GSTA2 gene. Oxidative stress is believed to play an important role in the induction of GSTA2 gene by toluene This study shows that toluene-induced GSTA2 gene expression is dependent upon nuclear translocation and binding of $C/EBP{\beta}$ to the C/EBP response element in the GSTA2 gene promoter.

• 한국독성학회:학술대회논문집
• /
• 한국독성학회 2003년도 춘계학술대회 논문집
• /
• pp.72-72
• /
• 2003

Induction of glutathione S-transferases is associated with cancer chemoprevention. We reported that PD98059, an MKK1 inhibitor, induces glutathione Stransferase A2 (rGSTA2). This report comparatively examines the role of CCAAT/enhancer binding protein (C/EBP) and Nrf-2 in the induction of rGSTA2 by PD98059. PD98059 at the concentrations effective for the inhibition of MKKI increased the rGSTA2 protein and mRNA levels in H4IIE cells. (omitted)

• Toxicological Research
• /
• 제17권
• /
• pp.251-256
• /
• 2001

The expression of phase II detoxifying enzymes is affected by a variety of compounds and the induction of the enzymes plays an essential role in chemoprevention. A variety of phytochemicals such as sulfur-containing chemoprotective agents (SCC) may trigger cellular signals and activate phase II gene expression through ARE activation. see induces glutathione S-transferases. Studies were conducted to investigate the role of mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase) in the induction of GST (e.g. rGSTA2) by sec. We also studied the MAP kinase pathway responsible for the GST expression by see and compared that with the pathway activated by oxidative stress as a result of sulfur amino acids deprivation (SAAD). see inhibited phosphorylation of ERK1/2 although the effect of see on JNK and p38 MAP kinase was minimal. Wortmannin and LY294002. PI3-kinase inhibitors. abolished the increases in rGSTA2 mRNA and protein levels by SCC. Deprivation of cystine and methionine caused oxidative stress in H4IIE cells. as evidenced by a decrease in the reduced glutathione and an increase in prooxidant production. Electrophoretic mobility shift assay revealed that the ARE complex consisting of Nrf-1/2 and Maf proteins was activated 12~48 h. The rGSTA2 mRNA and protein levels were increased by SAAD. Activation of ARE and induction of rGSTA2 were both completely inhibited by PI3-kinase inhibitors. Inhibition of p38 MAP kinase by SB203580 prevented the ARE-mediated rGSTA2 induction. The results of this study showed that PI3-kinase might play an essential role in the ARE-mediated rGSTA2 induction by see or SAAD and that the dual MAP kinase pathways were responsible for the enzyme induction.

• Toxicological Research
• /
• 제13권4호
• /
• pp.339-347
• /
• 1997

Disulfiram (DSF) and diethyldithiocarbamate (DDC), a reduced form of DSF, protect the liver against toxicant-induced injury through inhibition of cytochrome P450 2E1. The effect of DSF and DDC on the levels of major hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) expression was comparatively studied, given the view that these enzymes are involved in terminal detoxification events for high energy intermediates of xenobiotics. Treatment of rats with a single dose of DSF (20-200 mg/kg, po) resulted in 2- to 15-fold increases in the mEH mRNA level at 24 hr with the ED$_{50}$ value being noted as 60 mg/kg. The mEH mRNA level was elevated ~15-fold at 24 hr after treatment at the dose of 100 mg/kg, whereas the hepatic mRNA level was rather decreased from the maximum at the dose of 200 mg/kg, indicating that DSF might cause cytotoxicity at the dose. In contrast to the effect of DSF, DDC only minimally elevated the mEH mRNA level at the doses employed. DSF moderately increased the major GST mRNA levels in the liver as a function of dose, resulting in rGSTA2, rGSTA3/5 or rGSTM1 mRNA levels being elevated 3- to 4-fold at 24 hr post-treatment, whereas the rGSTM2 mRNA level was not altered. DDC, however, failed to stimulate the mRNA levels for major GST subunits, indicating that the reduced form of DSF was ineffective in stimulating the GST the expression. The effect of other organosulfides including aldrithiol, 2, 2'-dithiobis(benzothiazole) (DTB), tetramethylthiouram disulfide (TMTD) and allyl disulfide (ADS) on the hepatic mEH and GST mRNA expression was assessed in rats in order to further confirm the increase in the gene expression by other disulfides. Treatment of rats with aldrithiol (100 mg/kg, po) resulted in a 16-fold increase in the mEH mRNA level at 24 hr post-treatment. DTB, TMTD and ADS also caused 5-, 9- and 12-fold increases in the rnRNA level, respectively, as compared to control. Thus, all of the disulfides examined were active in stimulating the mEH gene in the liver. The organosulfides significantly increased the rGSTA2, rGSTA3, rGSTA5 and rGSTM1 mRNA levels at 24 hr after administration. In particular, aldrithiol was very efficient in stimulating the rGSTA and rGSTM genes among the disulfides examined. These results provide evidence that DSF and other sulfides effectively stimulate the mEH and major GST gene expression at early times in the liver and that DDC, a reduced form of DSF, was ineffective in stimulating the expression of the genes, supporting the conclusion that reduced form(s) of organosulfur compound(s) might be less effective in inducing the mEH and GST genes through the antioxidant responsive element(s).

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1997년도 춘계학술대회
• /
• pp.93-93
• /
• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
• /
• pp.82.1-82.1
• /
• 2003

The protective adaptive response to electrophiles and reactive oxygen species is mediated by enhanced expression of phase II detoxifying genes including glutathione S-transferases. ${\alpha}$-Lipoic acid, which exerts prooxidant or antioxidant activities, has been shown to activate the insulin signaling pathway and thus to induce insulin-like actions via PI3-kinase and Akt. Our previous studies have shown that PI3-kinase plays an essential role in Nrf2-or C/EBP${\beta}$-mediated glutathione S-transferase A2 (GSTA2) induction. (omitted)

• Toxicological Research
• /
• 제14권3호
• /
• pp.293-300
• /
• 1998

Previous studies have shown that the heterocycles including thiazoles are efficacious in inducing phase phase II metabolizing enzyme as well as certain cytochrome P450s and that the inductin of these matabolizing enzymes by the heterocyclic agents is highly associated with their hepatotoxicity. In the present study, the effects of benzylisothiazole (BIT), which has a isothiazole moiety, on the expression of microsomal epoxide hydrolase (mEH), major glutathione S-transerases and cytochrome P450s were studied in the rat liver in association with its hepatotoxicity. Treatment of rats with BIT(1.17 mmol/kg, 1~3d) resulted in substantial increases in the mEH. rGSTA2, rGSTA2, rGSTM1 and rGSTM2 mRNA levels, whereas rGSTA3 and rGSTA5 mRNA levels were increased to much lesser extents. A time-course study showed that the mRNA levels of mEH and rGSTs were greater at 24hr after treatment than those after 3 days of consecutive treatment. Relative changes in mEH and rGST mRNA levels were consistent with those in the proteins, as assessed by Western immunoblot analysis. Hepatic cytochrom P450 levels were monitored after BIT treatment under the assumption that metabolic activation of BIT may affect expression of the enzymes in conjunction with hepatotoxicity. Immunoblot analysis revealed that cytochrome P450 2B1/2 were 3-to 4-fold induced in rats teatd with BIT(1.17 mmol/kg/day.3days), whereas P450 1A2, 2C11 and 3A1/2 levels were decreased to 20~30% of those in unteatd rats. P450 2E1 was only slightly decreased by BIT. Thus, the levels of several cytochrome P450s were suppressed by BIT treatment. Rats treated with BIT at the dose of 1.17mmol/kg for 3 days exhibited extensive multifocal nodular necrosis with moderate to extensive diffuse liver cell degeneration. No notable toxicity was observed in the kidney. These results showed that BIT induces mEH and rGSTs in the liver with increases in the mRNA levels, whereas the agent significantly decreased major cytochrome P450s. The changes in the detoxifying enzymes might be associated with the necrotic liver after consecutive treatment.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2000년도 춘계총회 및 학술대회
• /
• pp.101.1-101.1
• /
• 2000

• 한국식품과학회지
• /
• 제39권6호
• /
• pp.688-693
• /
• 2007

아세트아미노펜(APAP)으로 유도된 간독성 모델에 미치는 잔대를 주재료로 하는 추출물(ATJH)의 간보호 효능을 관찰하기 위하여 동물모델에서 혈청 간기능 지표효소의 활성도를 측정하고 조직학적인 변화를 관찰하였다. APAP를 투여한 동물군은 ALT와 AST활성을 현저하게 증가시켰다. ATJH를 1000 mg/kg의 용량으로 3일간 전투여한 후 APAP로 간 독성을 유도한 동물은 증가된 AST, ALT활성을 약 60-80% 감소시켰으며, 500 mg/kg의 용량으로 장기간(7일간) 전투여한 동물에서도 APAP독성방어효과가 현저하였다. APAP를 고농도(450 mg/kg)로 투여하여 간손상에 의한 사망을 유도한 동물군에서 ATJH는 생존율을 대조군에 비하여 130% 증가시켰다. APAP 단독 투여한 군에서 80-90% 정도 간세포 괴사가 관찰되었으나 ATJH(500, 1000 mg/kg)를 3일간 전투여 한 군에서는 간조직 손상이 억제되었다. 동물모델에서 ATJH에 의한 간독성 방어효능의 기전을 연구하기 위하여 간세포에서 제2상 해독화 효소인 GST의 단백 발현 변화를 면역화학적으로 관찰하였다. ATJH는 농도의존적으로 GSTA2, GSTA3/5의 단백 발현을 유의성있게 유도하였다. 본 연구에서는 잔대를 주원료로 한 추출물이 간세포의 해독화효소의 발현을 증가시킴으로서 APAP에 의해 유발된 간손상을 억제함을 처음으로 증명하였고, 간조직 보호를 위한 화학적 예방 효능을 갖는 활성물질로서 ATJH의 가능성을 제시한다.