• 약학회지
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• 제53권6호
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• pp.314-320
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• 2009

We examined metabolic conversion of cysteine into glutathione (GSH) and taurine in rat liver under oxidative stress. Administration of tert-butylhydroperoxide (t-BHP) into the portal vein of male rats resulted in a rapid elevation of serum sorbitol dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities, which decreased gradually in 24 hr. Hepatic cysteine concentration was reduced in 3 hr, and recovered progressively, reaching a level greater than 200% of the normal value in 24 hr. GSH was increased both in liver and blood at 9 hr after t-BHP challenge, whereas hypotaurine or taurine was not altered. $\gamma$-Glutamylcysteine synthetase (GCS) activity was increased from 9 hr after t-BHP treatment, but protein expression of the GCS-heavy subunit was not changed in liver. Activity or expression of cysteine dioxygenase was not affected by t-BHP treatment. Taken together, these data show that an acute oxidant challenge to the rats may induce upregulation of cysteine availability and GCS activity, resulting in an enhancement of hepatic GSH synthesis, but the increased cysteine level does not stimulate taurine synthesis via cysteine sulfinate pathway. It is indicated that the regulation of GSH and taurine biosynthesis from cysteine is not solely dependent on the cysteine concentration in rat liver under oxidative stress.

• 한국환경보건학회지
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• 제27권2호
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• pp.82-91
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• 2001

This study was carried out to elucidate the protective effect of zinc chloride(ZnCl$_2$) and its mechanism against the immuno-cytotoxicity of methylmercury chloide($CH_3$HgCl). This study was observed in the culture of EMT-6 cells which are originated from mammary adenocarcinoma of Balb/c mouse. Cytotoxicity of metals was measured by cell viability and NO$_2$$^{[-10]}$ , and mitochondrial function was evaluated by adenosine triphosohate (ATP) production. $CH_3$HgCl significantly decreased the sythesis of nitric oxide(NO), ATP and glutathione(GSH) in a dose-dependent manner. ZnCl$_2$ significantly increased the synthesis of GSH in a dose-dependent manner, but synthesis of NO and ATP were not changed. The immuno-cytotoxicity of $CH_3$HgCl was not fully protected when combined addition of ZnCl$_2$, whereas ZnCl$_2$ prior to addition of $CH_3$HgCl completly protected the Hg-induced immuno-cytotoxicity. Similarly, intracellular accumulation of mercury significantly decreased by ZnCl$_2$. Degree of diminution of intracellular mercury was larger in ZnCl$_2$ prior to addition of $CH_3$HgCl than in combined addition of ZnCl$_2$ and $CH_3$HgCl.. Dithiothreitol(DTT) or buthionine sulfoximine(BSO) addition at 50$\mu$M or less, which was not toxic to the cells, did not affect synthesis of NO and ATP. DTT increased intracellular GSH level and DTT pretreatment protected toxicity induced by $CH_3$HgCl as shown complete recover in the NO and ATP values. BSO decreased intracellular GSH level and BSO pretreatment exaggerated toxicity induced by $CH_3$HgCl as shown synergistic reduction in the NO and ATP values. These results indicated that the protective effects of zinc against immuno-cytotoxicity of methylmercury associated with increasing cellular level of GSH. Increased intracellular GSH transports methylmercury to out of cells. In accordance with intracellular level of mercury decreased, immuno-cytotoxicity of methylmercury decreased. These result also suggest that the protective mechanism of zinc against the mercury toxicity would be exerted in the immune system in vivo.

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2001년도 Korean Environmental Mutagen Society
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• pp.166-166
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• 2001

• Journal of Preventive Medicine and Public Health
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• 제32권2호
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• pp.170-176
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• 1999

본 연구는 EMT-6 세포를 이용하여 무기수은, 유기수은 및 카드뮴의 세포독성에 대한 glutathione(GSH)의 방어효과를 알아보고자 하였다. 무기수은, 유기수은 및 카드뮴을 첨가한 배양조건에서 EMT-6 세포의 세포생존율, ${NO_2}^-$ 및 ATP 생성량은 첨가한 중금속의 농도가 증가할수록 용량의존적으로 감소하였다. GSH, OTC 및 BSO를 단독 첨가한 배양조건은 세포의 세포생존율과 NO2- 및 ${NO_2}^-$ 생성량에 영향을 주지 않았다. 수은화합물 및 카드뮴과 GSH를 동시 첨가한 배양조건에서는 세포생존율이 90% 이상 유지되었고, ${NO_2}^-$ 및 ATP 생성량은 기본배양조건과 비슷한 수준으로 나타났다. $16{\mu}M$의 무기 및 유기수은과 $160{\mu}M$의 카드뮴을 첨가한 실험조건에 GSH를 동시 첨가했을 경우 방어효과는 GSH의 농도에 따라 용량의존적으로 증가하였다. 세포내에서 수은 및 카드뮴의 세포독성에 대한 GSH역할을 알아보고자 GSH, OTC, BSO 전처리 실험을 한 결과, GSH의 전처리는 이들이 세포막을 통과하지 못하기 때문에 대조군과 비슷한 양상으로 나타난 반면에 BSO를 전처리한 군에서는 세포내 GSH 농도의 감소로 수은의 세포 독성이 증가하여 대조군에 비하여 ${NO_2}^-$와 ATP 생성량이 현저히 감소하였다. 또한 세포내 GSH의 농도를 증가시키는 OTC를 전처리한 결과 수은의 독성에 대한 방어효과가 시간 및 용량 의존적으로 현저하게 증가하였다. 이러한 실험결과는 수은의 세포독성에 대한 GSH의 방어효과가 GSH 세포내 농도와 밀접한 관련이 있음을 간접적으로 보여주고 있다. 본 연구의 결과는 수은 및 카드뮴의 독성에 대한 GSH의 방어작용이 단순히 -SH기와 중금속의 결합에 의한 결과가 아니라 세포내에서 GSH 분자가 갖는 고유의 기능으로 판단되며, 특히 중금속에 의한 에너지대사의 장애를 GSH가 회복시킬 수 있음을 보여준다.

• BMB Reports
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• 제31권3호
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• pp.254-257
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• 1998

The gshI gene from the Escherichia coli K-12 strain codes for ${\gamma}-glutamylcysteine$ synthetase which mediates the rate-limiting step of glutathione biosynthesis. The isolated gshI gene from E. coli K-12 has an unusual translation initiation codon, UUG. The 494th amino acid is Ala rather than Gly which was found in a mutant strain E. coli B. In order to improve the translational rate of the gshI gene of E. coli K-12, the initiation codon, UUG, was changed to the usual AUG codon by the site-specific mutagenesis. This change has resulted in a 53% increase of ${\gamma}-glutamylcysteine$ synthetase activity. The enzyme activity was also improved by replacing $Ala^{494}$ with Val (A494V) or Leu (A494L). The replacement of $Ser^{495}$ with Thr (S495T) also resulted in a 62% increase of the enzyme activity. Therefore, the specific activity of ${\gamma}-glutamylcysteine$ synthetase was increased with the increasing chain length of the aliphathic amino acid at the site of the 494th amino acid (Ala<$Val{\leq}Leu$).

• Biomolecules & Therapeutics
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• 제13권4호
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• pp.256-262
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• 2005

We examined the signaling molecules involved in the 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and increase in cellular glutathione (GSH) level in SK-N-SH cells. The 6-OH-DA-induced cell death was significantly prevented by the pretreatment with N-acetylcysteine (NAC), a thiol antioxidant, and BAPTA, an intracellular $Ca^{2+}$ chelator. Although 6-OHDA induced ERK phosphorylation, the pretreatment with PD98059, an ERK inhibitor, did not block 6-OHDA-induced cell death. In addition, the 6-OHDA-induced activation of caspase-3, a key signal for apoptosis, was blocked by the pretreatment with NAC and BAPTA. While the level of reactive oxygen species (ROS) was significantly increased in the 6-OHDA-treated cells, the cellular GSH level was not altered for the first 6-hr exposure to 6-OHDA, but after then, the level was significantly increased, which was also blocked by the pretreatment with NAC and BAPTA, but not by PD98059. Depletion of GSH by pretreating the cells with DL-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor, rather significantly potentiated the 6-OHDA-induced death. In contrast to the pretreatment with NAC, 6-OHDA-induced cell death was not prevented by the post-treatment with NAC 30 min after 6-OHDA treatment. The results indicate that the GSH level which is increased adaptively by the 6-OHDA-induced ROS and intracellular $Ca^{2+}$ is not enough to overcome the death signal mediated through ROS-$Ca^{2+}$ -caspase pathway.

• 한국어병학회지
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• 제32권1호
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• pp.21-28
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• 2019

It has been reported that various anti-oxidant substances stimulate non-specific immune responses in fishes. In this study it was examined whether N-acetylcysteine (NAC), a precusor for anti-oxidant glutathione (GSH) synthesis, can modulate non-specific immune function in Far Eastern catfish Silurus asotus. Immune functions were assessed using the respiratory burst activity monitored by chemiluminescence (CL) responses in isolated leucocyte. NAC stimulated CL responses with doses of 10 or 100 mg/kg, but not with 1 mg/kg after 48 hr injection. It was observed with 10 mg/kg NAC that CL activity continued to elevate from 24 hr through 96 hr post-dosing, and returned to the near preinjection level by 10 days. To understand whether NAC can also activate CL activity in vitro, NAC was directly added to isolated catfish leucocytes. It was observed, however, that NAC can not stimulate CL at reasonable concentration ranges in vitro. As NAC is a precursor for the strong anti-oxidant glutathione (GSH), a putative immune stimulator, it was assessed whether GSH can also stimulate CL responses. Observed results show that GSH activated CL both in vivo and in vitro. The data obtained collectively support the proposition that NAC indirectly stimulates non-specific immune functions in catfish by enhancing GSH biosynthesis, but not by direct action of NAC. Such effects may have beneficial significance in aquaculture for practical utilization.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1997년도 춘계학술대회
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• pp.109-109
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• 1997

Inhibitory effect on DNA topoisomerase-I, rate of glutathione conjugation and cytotoxicity of naphthoquinone derivatives were correlated. During 5 min exposure of the derivatives to glutathione (GSH), it was found that 14% of 5,8-dimethoxy-1,4-naphthoquinone(DMNQ) was converted into a GSH-conjugate, whereas 5,8-dihydroxy-1,4-naphthoquinone(DHNQ) did not interact with GSH, implying that DMNQ exerted higher electrophilicity than DHNQ. However, DHNQ (IC$\_$50/, 0.15 ${\mu}$M) showed stronger cytotoxicity in L1210 cells than DMNQ(IC$\_$50/, 0.45 ${\mu}$M). The stronger cytotoxicity of DHNQ, compared to DMNQ, could be ascribed to more rapid redox cycling. Both naphthoquinones (IC$\_$50/, 60-65 ${\mu}$M) exhibiting about the same inhibitory effect on DNA topoisomerase-I were more potent than 1,4-naphthoquinone(1,4-NQ, IC$\_$50/, 134 ${\mu}$M). Thus, 5,8-oxy groups in the structure seem to be important for the inhibition of the enzyme. DMNQ showed a broader dose range while maintaining a good antitumor activity against S-180 fluid tumor. For these reasons, DMNQ was taken as useful pharmacophore for structural modification. Introduction of 1-hydroxyalkyl groups at C-2 of DMNQ lowered all of the activities mentioned above, while acetylation of 1-hydroxyalkyl moiety enhanced the activities by 4-5 times. Introduction of the same side chains at C-6 exhibited stronger activities than 2-substituted ones. Based on these results it was suggested that the quinonoid moiety in 6-substituted DMNQ was more exposed to cellular nucleophiles such as DNA, thiols of enzymes and so on. The synthesis of DHNQ or DMNQ derivatives are going on, and the corelationship between structure-activity will be discussed.

• 한국동물생명공학회지
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• 제34권2호
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• pp.86-92
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• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• Tuberculosis and Respiratory Diseases
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• 제41권5호
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• pp.475-483
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• 1994

연구배경 : N-acetylcysteine(ACE)은 임상에서는 객담용해제로 널리 쓰이고 있으나 시험관내 실험 또는 동물실험에서 ACE는 염증세포에서의 산소유리기를 감소시키고, 세포내 강력한 항산화기능을 갖고있는 glutathione(GSH)의 합성을 촉진한다고 알려졌다. 저자들은 만성폐쇄성폐질환 환자에게 통상 투여용량인 매일 600mg씩 1주일간 ACE를 투여했을때 말초혈액내 호중구의 superoxide 분비, chemotaxis 등의 기능에 변화가 오는지 또는 혈장의 GSH 농도가 증가되는지를 살펴보고 동시에 시험관내 실험으로 정상인의 호중구에 ACE를 가했을때 ACE의 농도에 따라 superoxide나 GSH의 양에 변화가 오는지를 관찰하였다. 방법 : ACE 투여 전, 후에 만성폐쇄성폐질환 환자의 말초혈액에서 호중구를 분리하여 PMA로 자극하거나 안했을때의 superoxide 분비를 분광광도계로, luminol-enhanced chemiluminescence를 luminometer로, 혈장의 GSH 농도를 분광광도계로 각각 측정하였다. 한편 정상인의 호중구를 분리하여 $10^{-2}-10^{-5}$ mole의 ACE와 혼합배양시의 superoxide, chemiluminescence 및 GSH를 각각 같은 방법으로 측정하였다. 결과 : ACE를 투여하기 전, 후의 말초혈액내 호중구의 superoxide 분비는 $1.97{\pm}1.75nM/1.4{\times}10^6\;cells/15min$, $2.75{\pm}2.14nM$이었고 PMA로 자극하였을때는 $67.52{\pm}13.41nM$, $67.99{\pm}13.54\;nM$로서 각각 유의한 차이가 없었다(p>0.05). 호중구의 chemiluminescence도 ACE 투여전, 후에 $2.64{\pm}2.29mV$, $2.91{\pm}3.58mV$이었고 PMA로 자극하였을때는 $40.76{\pm}30.09mV$, $37.33{\pm}30.03mV$로서 유의한 차이가 없었다(p>0.05). 호중구의 chemotaxis를 chemotactic index로 비교하였을때 ACE투여전, 후에 $55.71{\pm}16.34$, $59.68{\pm}11.57$이었고 혈장의 GSH는 $0.37{\pm}0.17nM/4{\times}10^5cells$, $0.39{\pm}0.18nM$로서 역시 유의한 차이가 없었다(p>0.05). 정상인의 호중구를 분리하여 $10^{-5}-10^{-2}$ mole의 ACE와 동시배양하며 PMA로 자극하였을때 superoxide는 ACE를 가하지 않은 대조군에서의 $56.54nM/1.4{\times}10^6\;cells/15min$에 비해 ACE의 $10^{-2}$ mole에서 37.0nM로서 유의하게 감소하였고 ACE의 농도에 따라 감소하는 양상을 보였다(r=-0.269, p<0.05). 호중구의 GSH 농도는 대조군의 $0.27nM/4{\times}10^5\;cells$에 비하여 ACE의 $10^{-3}$ mole에서 0.35nM, $10^{-2}$ mole 에서 1.15 nM로서 유의하게 증가하여 ACE의 농도에 따라 증가하였다(r=0.72, p<0.01). 결론 : ACE를 만성폐쇄성폐질환 환자에게 매일 600mg씩 1주일간 투여하였을때 말초혈액내 호중구의 superoxide 분비, chemotaxis에 영향을 주지 않았고 혈장의 glutathione 농도에도 변화가 없었다. 그러나 정상인의 호중구를 분리하여 ACE와 같이 배양한 실험에서 ACE는 농도에 비례하여 호중구의 superoxide 분비를 억제하였고 GSH를 증가시켰다. 이상으로 미루어 ACE는 염증세포에서의 superoxide 분비를 억제하고 GSH를 증가시킴으로서 oxidant-antioxidant 간의 불균형으로 야기되는 ARDS, 폐기종, 간질성폐질환등의 치료에 이용될수 있을것으로 생각되나 폐기종을 포함한 만성폐쇄성폐질환의 치료에 이용되기 위해서는 ACE의 혈중농도 또는 폐포세척액내의 농도를 충분히 증가시키는 방법이 강구되어야 할 것으로 사료된다.