• 미생물학회지
• /
• 제52권2호
• /
• pp.236-241
• /
• 2016

탈유비퀴틴 효소 중 PPPDE 상과에 속하는 Schizosaccharomyces pombe의 $sdu1^+$ 유전자가 유비퀴틴 C-말단 가수분해 효소 활성을 갖는 단백질을 인코딩하고, 산화적 및 일산화질소 스트레스 방어에 관여함이 이전에 밝혀진 바 있다. 예비적인 본 연구는 정상적인 및 과잉발현의 조건에서 S. pombe 유비퀴틴 C-말단 가수분해 효소 활성의 활성산소종 의존성 조절에 초점을 맞추었다. 과산화수소, 수퍼옥사이드 라디칼 생성하는 메나디온 및 일산화질소 생성하는 sodium nitroprusside (SNP)에 노출시킨 S. pombe 세포에서 유비퀴틴 C-말단 가수분해 효소 활성이 감소되었다. 환원형 글루타치온과 그 전구체인 N-acetylcysteine은 과산화수소의 존재 유무에 상관없이 유비퀴틴 C-말단 가수분해 효소 활성을 현저하게 증강시켰다. 그러나, 과산화수소의 부재 시 혹은 존재 시 활성산소종에 미치는 글루타치온과 N-acetylcysteine의 영향은 같은 조건 하에서의 유비퀴틴 C-말단 가수분해 효소 활성 패턴과 상반되었다. 과잉발현의 유비퀴틴 C-말단 가수분해 효소 활성을 보이는 재조합 플라즈미드 pYSTP를 보유하는 S. pombe 세포에서 유비퀴틴 C-말단 가수분해 효소 활성도 과산화수소, 메나디온 및 SNP에의 노출되는 조건에서 감소되었지만, 벡터 대조 세포에서 보다는 높게 유지되었다. 요약하면, S. pombe 유비퀴틴 C-말단 가수분해 효소 활성은 활성산소종에 의하여 하향조절 되지만, 그 의의는 현재로썬 알려지고 있지 않은 상태이다.

• Toxicological Research
• /
• 제27권3호
• /
• pp.161-166
• /
• 2011

Carbon black, a particulate form of pure elemental carbon, is an industrial chemical with the high potential of occupational exposure. Although the relationship between exposure to particulate matters (PM) and cardiovascular diseases is well established, the cardiovascular risk of carbon black has not been characterized clearly. In this study, the cytotoxicity of carbon black to vascular smooth muscle and endothelial cells were examined to investigate the potential vascular toxicity of carbon black. Carbon black with distinct particle size, N330 (primary size, 28~36 nm) and N990 (250~350 nm) were treated to A-10, rat aortic smooth muscle cells and human umbilical vein endothelial cell line, ECV304, and cell viability was assessed by lactate dehydrogenase (LDH) leakage assay. Treatment of carbon black N990 resulted in the significant reduction of viability in A-10 cells at 100 ${\mu}g$/ml, the highest concentration tested, while N330 failed to cause cell death. Cytotoxicity to ECV304 cells was induced only by N330 at higher concentration, 200 ${\mu}g$/ml, suggesting that ECV304 cells were relatively resistant to carbon black. Treatment of 100 ${\mu}g$/ml N990 led to the elevation of reactive oxygen species (ROS) detected by dichlorodihydrofluorescein (DCF) in A-10 cells. Pretreatment of antioxidants, N-acetylcysteine (NAC) and sulforaphane restored decreased viability of N990-treated A-10 cells, and N-acetylcysteine, but not sulforaphane, attenuated N990-induced ROS generation in A-10 cells. Taken together, present study shows that carbon black is cytotoxic to vascular cells, and the generation of reactive oxygen contributes to the development of cytotoxicity. ROS scavenging antioxidant could be a potential strategy to attenuate the toxicity induced by carbon black exposure.

• 한국환경농학회지
• /
• 제28권4호
• /
• pp.462-467
• /
• 2009

Exposure of formaldehyde (FA), one of the major compounds in pesticides and in the onset of sick house syndrome, has been implicated in the development of diverse diseases. Liver is a very important organ to body metabolism and drug detoxification. Apotosis of hepatocytes is associated with the onset of liver diseases such as hepatitis. However, the apoptotic effect of FA in hepatocytes is not clear. Therefore, this study was conducted to investigate the effect of FA on the apoptosis in HepG2 cells, a human hepatocyte cell line. As a result, FA (> $500\;{\mu}M$) decreased cell viability and increased lactate dehydrogenase activity in HepG2 cells, which was blocked by the treatment of vitamin E and N-acetylcysteine (NAC). In addition, FA decreased glutathione (GSH) contents and Bcl-2 levels, while increasing lipid peroxide formation and Bax levels. It also cleaved caspase-3 form, which was blocked by the treatment of vitamin E and NAC. It is insisted that FA induced apoptosis via oxidative stress in human hepatocytes.

• 한국식품과학회지
• /
• 제43권4호
• /
• pp.483-489
• /
• 2011

본 연구에서는 다양한 생리활성이 보고된 폴리페놀 화합물인 EGCG의 화학안정성, H2O2 생성능 및 세포독성에 대하여 다양한 항산화제와의 조합에 의한 변화를 분석하였다. EGCG는 생리적 조건에서 갈색화합물로 산화되면서 불안정화되는데, catalase를 제외한 각종 항산화제제 SOD, ascorbic acid, NAC 및 GSH는 EGCG 갈색산화물의 생성을 유의적으로 저해하였다. EGCG에 의해 생성되는 $H_2O_2$는 catalase에 의해 거의 완벽하게 제거되었으며, SOD와 NAC에 의해서도 유의적으로 감소하였다. 하지만 GSH 및 고농도의 ascorbic acid의 존재 시 오히려 $H_2O_2$ 수준이 증가하는 현상을 나타내었다. EGCG의 HeLa 및 HT-29 세포에 대한 독성은 catalase, SOD 및 NAC 등과 같은 항산화제 존재 하에 유의적으로 감소하였고 NAC에 의한 EGCG 세포독성의 감소는 첨가된 NAC의 농도 증가에 따라 더욱 두드러졌다. 그러나 GSH 존재하에 EGCG의 독성은 GSH와 EGCG농도에 따라 다른 조절 양상을 나타내었으며, ascorbic acid에 의해서 EGCG의 세포독성이 약간 증가하는 현상을 나타내었다. 본 결과는 EGCG와 함께 처리된 다양한 항산화제들이 ROS의 소거 뿐만 아니라 EGCG 화학안정성 등 다른 요인에 영향을 미칠 수 있으며, 항산화제의 존재 하에 변형된 EGCG활성에 대해 ROS관련 기작 외에 다양한 요인들에 대한 고려가 함께 이루어져야 함을 시사한다.

• Toxicological Research
• /
• 제16권2호
• /
• pp.133-139
• /
• 2000

This study was designed to evaluate the mechanism by which reactive nitrogen intermediates (RNI) induced the cytotoxicity of human doparminergic neuroblastoma SH-SY5Y cells. 3-Morpholino-sydnonimine (SIN-l), a donor of peroxynitrite (ONOO) and sodium nitroprusside (SNP), a donor of nitric oxide (NO) induced cell detachment and apoptotic death, as characterized by chromatin condensation, the ladder pattern fragmentation of genomic DNA and morphological nuclear changes. SIN-l also induced the activation of caspase 3-like protease in a time-dependent manner. Exogenous antioxidants, such as reduced glutathione (GSH), N-acetylcysteine (NAC), and selenium protected the cells from apoptotic death and reduced the activation of caspase 3-like protease by SIN-1. Furthermore, SIN-l directly reduced the intracellular levels of glutathione. Taken together, these data suggested that RNI including NO and peroxynitrite decrease the concentration of intracellular antioxidant such as GSH, which lead to the apoptotic death of human neuroblastoma SH-SY5Y cells.

• The Korean Journal of Physiology and Pharmacology
• /
• 제24권3호
• /
• pp.267-276
• /
• 2020

T In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-Ras^V12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-Ras^V12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-Ras^V12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-Ras^V12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-Ras^V12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that Ras^V12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

• Journal of Microbiology and Biotechnology
• /
• 제23권5호
• /
• pp.699-706
• /
• 2013

We have previously reported that N-acetylcysteine (NAC) not only delayed apoptosis but also enhanced the production of recombinant erythropoietin (EPO) in Chinese hamster ovary (CHO) cell culture. To investigate the production enhancement mechanism, the effects of similar thiol-reducing agents were studied. Intriguingly, all mild reducing agents examined including mercaptoethanesulfonic acid (MESNA), thiolactic acid (TLA), and thioglycolate (TG) were shown to block apoptosis and increase EPO production. A pulse-chase study of EPO secretion revealed that all four thiol-reducing agents increased the EPO secretion rate; among them TLA showed the highest rate. In terms of product quality, the sialic acid content of the glycoprotein is one of the most important factors. It was reported that a number of glycoproteins produced by CHO cells often have incomplete sialylation, particularly under high-producing conditions. Human ${\alpha}2,3$-sialyltransferase (${\alpha}2,3$-ST) was introduced into EPO-producing CHO cells in order to compensate for the reduced sialylation during supplementation with NAC. When ${\alpha}2,3$-ST was expressed in the presence of NAC, reduced sialylation was restored and an even more sialylated EPO was produced. Thus, our study is significant in that it offers increased EPO production while still allowing the prevention of decreased sialylation of EPO.

• 동의생리병리학회지
• /
• 제22권5호
• /
• pp.1322-1329
• /
• 2008

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruits on cell viability in human glioma cells. The present study was undertaken to examine the effect of mulberry fruit (Mar; Fructus) on cell viability and to determine its underlying mechanism in human glioma cells. Cell viability and cell death were estimated by MTT assay and trypanblue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with $DiOC_6$(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Mori Fructus resulted in apoptotic cell death in a dose- and time-dependent manner. Mori Fructus increased ROS generation and the Mori Fructus-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in Mari Fructus-induced cell death. Western blot analysis showed that Mori Fructus treatment caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). Mori Fructus induced depolarization of mitochondrial membrane potential and its effect was inhibited by the antioxidants NAC and catalase. Mori Fructus induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by Mori Fructus and caspase inhibitors prevented the Mori Fructus-induced cell death. These findings suggest that Mori Fructus results in human glioma cell death through ROS-dependent mitochondrial pathway in human glioma cells.

• The Korean Journal of Physiology and Pharmacology
• /
• 제3권5호
• /
• pp.521-528
• /
• 1999

Reactive oxygen species (ROS), generated by infiltrating neutrophils, are considered as an important regulator in the pathogenesis and deveolpment of pancreatitis. The present study aims to investigate whether neutrophils primed by $4{\beta}-phorbol\;12{\beta}-myristate\;13{\alpha}-acetate$ (PMA) affect the productions $H_2O_2$ and lipid peroxide (LPO), $NF-_{\kappa}B$ activation and cytokine production in pancreatic acinar cells, and whether these alterations were inhibited by an antioxidant, N-acetylcysteine (NAC) and superoxide dismutase (SOD). $H_2O_2$ (ferrithiocyanate method), LPO (as thiobarbiturate reactive substances), and cytokines $(IL-l{\bata},\; IL-6,\;TNF-{\alpha};\;enzyme-linked\;immunosorbent\;assay)$ and $NF-_{\kappa}B$ activation (electrophoretic mobility shift assay) were analyzed in acinar cells treated with or without PMA-primed neutrophils in the absence or presence of NAC (10 mM) or SOD (300 U/ml). As a result, the productions of H2O2, LPO and $TNF-{\alpha}$ were increased with the ratio of PMA-primed neutrophils to acinar cells while the productions of LPO, $IL-l{\beta},\;IL-6\;and\;TNF-{\alpha}$ were increased with time. PMA-primed neutrophils resulted in the activation of $NF-_{\kappa}B.$ Both NAC and SOD inhibited neutrophil-induced alterations in acinar cells. In conclusion, ROS, generated by neutrophils, activates $NF-_{\kappa}B,$ resulting in upregulation of inflammatary cytokines in acinar cells. Antioxidants might be clinically useful antiinflammatory agents by inhibiting oxidant-mediated activation of $NF-_{\kappa}B$ and decreasing cytokine production.

• Archives of Pharmacal Research
• /
• 제28권3호
• /
• pp.311-318
• /
• 2005

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC­PK1 cells were treated with H$_2$O$_2$ in the absence of serum to induce cell death. Subsequent to exposure to H$_2$O$_2$, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H202-treated control cells, it was observed that 0.5 $\mu$M of desipramine and 25 $\mu$M of NAC exhibited about 90 and $95\%$ of cytoprotection, respectively, against H$_2$O$_2$-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and $3\%$ respectively, when compared to $71\%$ in H$_2$O$_2$-exposed cells. Cellular glutathione level in 500 $\mu$M H202-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H$_2$O$_2$-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H$_2$O$_2$-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.