Earticle

We investigated the antioxidative effect of black garlic extract (BGE) on paraquat (PQ)- induced oxidative stress in mice. A DPPH radical scavenging activity assay showed that BGE had potent free radical scavenging activity, comparable to that exhibited by vitamin C. Mice were administered with either vitamin C or two levels of BGE by oral gavage for 10 days, with PQ being injected intraperitoneally on day five. It was found that BGE reduced the liver enzyme levels induced by PQ injection in mice. The concentrations of plasma and hepatic malonedialdehyde were both significantly reduced in the BGE groups compared with the levels in the PQ group, whereas the hepatic superoxide dismutase and catalase activities were significantly increased in the BGE groups compared with the PQ group. These findings suggest that BGE has potent antioxidative activities in vivo and thus could prevent the oxidative stress induced by PQ injection in mice by two mechanisms: the induction of antixoxidative enzyme systems and direct scavenging of reactive oxygen species.

흔하게 사용되어온 제초제인 paraquat는 파킨슨병의 원인이 될 수 있는 유력한 위험 요소이다. 헴산화효소-1(HO-1)은 산화적 스트레스와 소포체 스트레스의 marker인데, 여러 가지 자극에 의해 heme을 분해하여 biliverdin, 일산화탄소, 철 성분으로 전환시킨다. 본 연구에서는 뇌의 흑색질 유래의 도파민 세포주 SN4741에서 paraquat가 시간별, 농도별로 HO-1을 활성화시키는 기작을 조사하였다. HO-1이 paraquat에 의해 활성화되는 것은 주로 유전자 전사 수준에서 조절되었다. HO-1 유전자의 promoter와 5' enhancer인 E1, E2를 결실시킨 실험에서, E2 enhancer가 도파민 세포에서 paraquat에 의한 HO-1 유전자 발현을 유도하는 핵심 부위로 판명되었다. E2 enhancer 부위를 돌연변이 시킨 실험 결과는 전사인자 활성 단백질-1 (AP-1) 결합부위를 통해 HO-1 발현이 유도됨을 밝히게 되었다. 또한, 도파민 세포에서 HO-1 유전자 발현의 조절과 신호전달 과정의 관계를 조사하기 위해 MAP kinase들의 특이적 저해제를 처리하고 paraquat로 자극을 준 결과, JNK 저해제인 SP600125가 가장 현저하게 paraquat에 의한 HO-1 발현을 억제하였다. 결론적으로, 도파민 세포에서 paraquat가 HO-1을 유도하는 데는 E2 enhancer가 중요하게 작용하고, AP-1과 JNK 경로를 통해 HO-1 발현이 조절된다는 사실을 처음으로 밝히게 되었다.

Paraquat, a widely used herbicide, has been suggested as a potential risk factor for Parkinson's disease. Heme oxygenase-1(HO-1), a marker for oxidative stress and endoplasmic reticulum (ER) stress, is known to catalyze heme to biliverdin, carbon monoxide and free iron in response to various stimuli. Here we show that paraquat activates HO-1 expression in a timeand dose-dependent manner in substantia nigra (SN) dopaminergic neuronal cells. Activation of HO-1 by paraquat was regulated primarily at the level of gene transcription. Deletion analysis of the promoter and the 5' distal enhancers, E1 and E2, of the HO-1 gene revealed that the E2 enhancer is a potent inducer of the paraquat-dependent HO-1 gene expression in dopamninergic neuronal cells. Mutational analysis of the E2 enhancer further demonstrated that the transcription factor activator protein-1 (AP-1) plays an important role in mediating paraquat-induced HO-1 gene transcription. Moreover, using specific inhibitors of the mitogen-activated protein kinases (MAPKs), we investigated the role of paraquat and MAPKs for HO-1 gene regulation in dopaminergic cells. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 significantly suppressed the expression of HO-1 by paraquat. All these results demonstrate that induction of HO-1 by paraquat requires the activation of the AP-1 and JNK pathway.

본 연구에서는 절대적 호기성인 Vitreoscilla가 지닌 FeSOD의 특성을 밝히고자 paraquat와 iron을 사용하여 실험하였다. Vitreoscilla에서 FeSOD의 활성도는 초산화 음이온을 생성하는 paraquat에 대해 커다란 영향 없이 일정하게 발현되었다. 이 결과는 Vitreoscilla는 호기적인 물질대사 동안 생성되어지는 초산화 음이온에 대해 방어할 수 있을 만큼 충분한 FeSOD를 평상시 지니고 있음을 의미한다. 또한 다른 요소에 의한 FeSOD의 발현 가능성이 고려되어졌다. 보조 인자인 Fe 를 처리한 결과 활성도가 증가됨을 알 수 있었다. 이는 CN-resistant respiration을 증가시키는 초산화 음이온에 대한 조절 기작과는 달리 apoenzyme 상태가 보조 인자인 금속 이온 (Fe)에 의해 holoenzyme 상태로 전환되었음을 의미한다. PQ와 Fe를 함께 처리하였을 경우, FeSOD의 증가는 PQ에 의해서 생성된 초산화 음이온에 의해 apo 상태에서 hole 상태로 FeSOD의 활성이 증진되는 synergism effect로 설명되어진다. 결론적으로 metalloenzyme인 SOD의 경우는 이런 전환이 활성 속도를 제한하거나 조절할 수도 있는 posttranslational 수준의 조절 기작이 존재함을 알 수 있었다.

Superoxide dismutase which is metalloenzyme that decomposes superoxide radicals into hydrogen peroxide and molecular oxygen. Vitreoscilla has FeSOD. Expression of FeSOD to paraquat was largely constitutive. This suggests that the basal level of FeSOD is sufficient to provide protection against superoxide generated during normal aerobic metabolism. Induction of SOD by iron supports that insertion of the active site metal into the corresponding apoprotein. The effect of paraquat on induction by iron seemed that iron brought the synergism effect in SOD activity with paraquat. It suggests that the relief of growth inhibition is due to protection against the lethality of O2 afforded by the elevated SOD. There may be control of FeSOD activity posttranslationally. Posttranslation control of enzyme function is particularly feasible for a metalloenzyme, for which conversion of apo- to holoenzyme may be the rate-limiting or regulatory step.

The tannins represent a highly heterogeneous group of water-soluble plant polyphenols that may play an important role in antimutagenic and antioxidant properties. We investigated the antioxidant function of tannic acid in comparison to other phenolic compounds including catechin, chlorogenic acid, cinnamic acid, ellagic acid, and gallic acid for their ability to scavenge several stable radicals and reactive oxygen species (ROS) such as ●DPPH+,●ABTS+ , hydrogen peroxide, hydroxyl radical, and superoxide radical. The ability of tannic acid to decrease paraquat-induced lipid oxidation in mouse liver and lung through its antioxidant properties was also assessed. The results showed that almost all the tested compounds have stable radical scavenging activity except cinnamic acid. Tannic acid, gallic acid, and ellagic acid demonstrated remarkable ROS scavenging properties toward H2O2, ●OH2-, ●O2- and especially only tannic acid could inhibit paraquat-induced lipid peroxidation effectively in mouse liver and lung. Based on these results, it appears that increased number of galloyl and ortho-hydroxyl groups enhances the antioxidant activity of phenolic compounds and tannic acid is evaluated as the most effective antioxidant among all the tested compounds. These results suggest that the tannins, especially tannic acid, can be used as therapeutic agent for various diseases caused by ROS.

활성산소 소거 효소인 superoxide dismutase (SOD)가 결핍된 효소 변이주들을 대상으로 하여, 활성산소 유발물질인 paraquat을 배지에 첨가하여 배양하면서 산소 독성을 관찰하였다. 호기 상태에서 는 MnSOD (mitochondria SOD) 결핍 효모는 CuZnSOD (세포질 SOD) 결핍 효모보다 성장이 많 이 저하되었지만, 혐기 상태에서는 이들 SOD 결핍 효모 모두 성장 속도에서 야생 효모와 큰 차이를 보이지 않았다. Paraquat으로 처 리한 결과, 호기 배양 에서 CuZnSOD 결핍 효모는 O.OlmM 이상에서 성 장하지 않음을 알 수 있었다. 따라서 O.OOlmM paraquat을 배지에 첨가한 후 호기적으로 배양하면서 세포내 성분의 변화를 관찰하였더니, CuZnSOD 결핍 효모에셔는 catalase 역가가 떨어진 반면 glutathion peroxidase 역가와 세포막 지질의 과산 화물이 증가하였으며, MnSOD 결핍 효모에서는 catalase 역가와 glutathion peroxide 역가가 모두 조금씩 증가하면서 세포막 지질의 과산화물은 그다지 변화하지 않았다. 이러한 사실로부터 CuZnSOD 가 없는 경우 활성산소 소거계로써 catalase 보다 glutathion peroxidase가 훨씬 활성화되어지지만, 이렇게 활성화된 glutathion peroxidase로는 세포질내 산소 radical을 완벽히 제거하는데에는 다소 불충분 하다는 사실을 알 수 았었다. 한편, 혐기적 배양에서 는 SOD 결핍 효모들의 catalase 역가는 모두 감소 한 반면, glutathion peroxidase 역가는 다소 증가하였고, 또한 세포막 지질의 과산화물은 다소 감소하는 추세를 보였다. 이는 혐기 상태에서 산소 radical 이 소량밖에 생기지 않으므로, 활성화된 glutathion p peroxidase에 의해 어느 정도 극복되어지기 때문인 것으로 사료된다.

Using superoxide dismutase (SOD)-deficient mutants of Saccharomyces cerevisiae, the oxygen toxicity induced by paraquat was studied. In aerobic culture condition, yeasts lacking MnSOD (milochondrial SOD) showed more significant growth retardation than CuZnSOD (cytoplasmic SOD)-deficient yeasts. However, not so big differences in growth pattern of those mutants compared with wild type were observed under anaerobic condition. When exposed to paraquat, the growth of yeasts lacking CuZnSOD was severely affected by higher than 0.01mM of paraquat in culture medium. By the analysis of several cellular components ivolved in free radical generating and scavenging system, it was found that, under aerobic condition, the content of lipid peroxides in cell membrane as well as cellular activity of glutathion peroxidase of CuZnSOD-deficient mutants was increased in the presence of paraquat, although significant decrease of catalase activity was observed in those stratns. In MnSOD-deficient yeast, however, increment in cellular activity of glutathion peroxldase and catalase by paraquat was observed without any deterioration of membrane lipid. It implies that the lack of mitochondrial SOD could be compensated by both of glutathion peroxldase and catalase, but that only glutathion peroxidase might act for CuZnSOD in cytoplasm. In contrast, all of SOD-deficient mutants showed a significant decrease in catalase activity, but slight increase in the activities of glutathion peroxidase, when cultivated anaerobically in the medium containing paraquat. Nevertheless, any significant changes of lipid peroxides in cell membranes were not observed during anaerobic cultivation of SOD-deficient mutants. It suggests that a little amount of free radicals generated by paraquat under anaerobic condition could be sufficiently overcome by glutathion peroxidase but not by catalase.

Minocycline is a semisynthetic second-generation tetracycline that can easily penetrate the blood-brain barrier. Its neuroprotective properties were demonstrated in various disease models such as cerebral ischemia, amyotrophic lateral sclerosis, and Huntington's disease. In addition, minocycline was found to be able to block 6-OHDA- and MPTP-induced dopaminergic neurodegeneration associated with Parkinson's disease (PD). The present study investigated neuroprotective properties of minocycline in a paraquat-induced mouse model of PD. Paraquat, a quaternary nitrogen herbicide, induced the c-Jun N-terminal kinase (JNK) and caspase-3 activation in Substantia Nigra pars compacta (SNpc) dopaminergic neurons in vivo. These effects were attenuated by minocycline (40mg/kg, i.p.) pretreatment. Results in this study support a role of minocycline against toxic insults and suggest that its therapeutic potential for PD

산화질소(nitric oxide: NO) 공여체인 $100{\mu}M$ sodium nitroprusside (SNP)를 배추 잎에 전처리한 후 이어서 $2{\mu}M$ paraquat (PQ)처리 시, PQ에 의해 유도된 산화적 손상에 대한 잎의 내성이 효과적으로 증진되었다. 24 시간 광 배양기간 동안 PQ 단독 처리구 잎에서는 생체량, 엽록소 및 단백질 함량이 현저하게 감소하였으나 PQ 노출 전에 3시간 SNP 전처리로 이들 잎 손상이 의미 있게 완화되었다. 게다가 PQ 처리에 기인된 malondialdehyde (MDA)와 $H_2O_2$ 함량 증가도 SNP 전처리에 의해 유의하게 억제되었다. 잎에서 이들 PQ 독성에 대한 SNP의 방어효과와 관련하여 ascorbate-glutathione 회로 구성 효소의 활성도 변화를 조사하였다. PQ 단독 처리구에서 APX, DHAR 및 GR 효소 활성도는 배양 6시간후에 급격히 감소되어 대조구 잎과 비교 시 각각 대조구의 19%, 50%, 39% 수준의 활성도 값을 보였다. 그러나, 이들 효소 활성도 값 감소는 SNP 전처리에 의해 현저하게 억제되어 6시간 배양 후에 PQ 단독처리구 보다 각각 5배, 2배, 1.5배 높은 값을 나타내었다. 또한, 그 이후 24시간 배양 때까지 PQ 단독 처리구보다 계속 높은 활성도를 보이면서 점차로 감소하였다. 이들 결과로부터, PQ에 노출된 배추 잎에서 SNP 전처리에 의한 ascorbate-glutathione 회로의 활성화가 $H_2O_2$의 축적을 억제하며 그로인해 PQ에 의해 유도된 산화스트레스로부터 잎을 방어하는 것으로 생각되었다. 동시에 이 들 결과는 산화질소가 배추 잎에서 PQ 스트레스에 대한 항산화 방어자로서의 역할을 하는 것을 의미한다.

Pretreatment of chinese cabbage leaves with $100{\mu}M$ sodium nitroprusside (SNP), a nitric oxide (NO) donor, effectively improved their tolerance to subsequent $2{\mu}M$ paraquat (PQ)-induced oxidative damage. The fresh weight, and chlorophyll and protein contents in primary leaves treated with PQ alone were noticeably reduced over 24 h light incubation. However, these leaf injury symptoms were significantly alleviated with $100{\mu}M$ SNP pretreatment for 3 h prior to PQ exposure. In additions, the increase of the contents of malondialdehyde (MDA) and $H_2O_2$ due to PQ exposure were significantly inhibited by SNP pretreatment. Together with the protective effects of SNP against PQ toxicity in leaves, the changes of ascorbate-glutathione cycle enzymes activities were examined. In the PQ alone treatment, the activities of APX, DHAR, and GR after 6 h incubation were rapidly reduced and showed 19%, 50% and 39% respectively, compared with those of the control. However, the decreases in these enzyme activities were significantly inhibited by SNP pretreatment. As a result, their activities were higher than those of PQ alone treatment by 5 times, 2 times, and 1.5 times, respectively, at 6 h incubation. Thereafter, these enzymes decrease their activities gradually showing high levels than those of PQ alone. Based on the above results, it can be assumed that the activation of ascorbate-glutathione cycle by SNP pretreatment in chinese cabbage leaves exposed to PQ can prevent $H_2O_2$ accumulation, thereby leading to protection against PQ-induced oxidative stress. Also, these results indicate that NO acts as an protectant against PQ stress in the leaves of chinese cabbage.

Purpose: Paraquat, a globally used herbicide, is highly toxic to human beings. Hence, we reviewed some cases of paraquat poisoning in Korea. Methods: We analyzed the clinical and laboratory findings of 50 patients poisoned with paraquat retrospectively. The patients were admitted to the department of internal medicine in the Eumseong KeumWang hospital from January 2008 to December 2008. Results: Among 50 cases of paraquat poisoning, 28 cases were male. Twenty-four cases (48%) were over 60 years old. Fourty-nine patients ingested paraquat on purpose as suicidal attempts, while 1 patient underwent accidental ingestion. Seven patients swallowed less than one mouthful of paraquat, of which 4 patients survived. Eleven patients swallowed two mouthfuls of paraquat, of which 8 patients survived. Thirty-two patients swallowed over three mouthfuls of paraquat and they all died. Thirty-one patients with leukocytosis died. Twenty-one patients with metabolic acidosis died. Increased levels of blood amylase and glucose were related to high mortality, and increased level of blood creatinine was related to severe mortality. Hemoperfusions were accomplished in 27 patients of paraquat poisoning, of which 12 patients survived. Conclusion: Paraquat is a highly toxic herbicide. When patients arrive at the hospital, laboratory findings, urine paraquat concentrations, arrival time, and the amount of paraquat consumed must be considered for treatment plan.

연구배경 : Paraquat는 P-450 reductase에 의해 반응성 산소유리기(ROS)를 발생시켜 세포막, 단백질, 핵산 등과 반응함으로써 세포손상을 유도하며 급성 폐 손상을 일으킨다. 최근 급성 폐 손상 및 급성 호흡곤란 증후군에 있어서 폐상피세포의 아포프토시스가 중요한 역할을 한다고 알려지기 시작하였다. 이에 반응성 산소유리기에 의한 폐 손상의 대표적 물질인 paraquat로 인한 폐상피세포의 세포죽음이 아포프토시스인지 확인하고 dexamethasone, N-acetylcysteine, 그리고 bcl-2가 paraquat로 인한 폐상피 세포죽음에 어떠한 영향을 미치는지 등을 연구하였다. 방법 : 폐상피세포주인 A549와 BEAS-2B 세포주, 그리고 bcl-2 construct를 유전자 주입한 A549 pcDNA3-bcl-2 세포주를 이용하였다. 아포프토시스는 Annexin V assay를 이용하여서 판정하였으며 세포독성 검사는 MTT assay를 이용하였다. Paraquat는 0, $1{\mu}M$, $10{\mu}M$, $100{\mu}$M, 1 mM, 10 mM의 농도로 사용하였다. Dexamethasone은 $1{\mu}M$의 농도로 paraquat 투여 12시간 전에 전처치하였고, N-acetylcysteine은 1 mM의 농도로 paraquat 투여 1시간 전에 전처치하였다. 결과 : 양 세포주 모두에서 paraquat는 농도와 시간 경과에 따라서 세포죽음을 증가시켰고, 이러한 세포죽음은 아포프토시스였다. N-acetylcysteine과 dexamethasone은 시간과 농도에 따라 약간의 차이가 있으나 전반적으로 10~30%의 방어효과가 있었다. Bcl-2를 과발현시킨 A549-bcl-2 세포주에서 A549-neo 세포주에 비해 paraquat에 의한 세포독성이 약 20~30% 정도 차단되었다. 결론 : Paraquat는 폐상피세포에서 아포프토시스를 유도하며, paraquat에 의한 아포프토시스는 마이토콘드리아 경로에 의해 일어날 것으로 추정된다.

Background: Paraquat is extremely toxic chemical material, which generates reactive oxygen species (ROS), causing multiple organ failure. In particular, paraquat leads to irreversible progressive pulmonary fibrosis. Exaggerated cell deaths exceeding the normal repair of type II pneumocytes leads to mesenchymal cells proliferation and fibrosis. This study examined the followings; i) whether or not paraquat induces cell death in lung epithelial cells; ii) whether or not paraquat-induced cell deaths are apoptosis or necrosis; and iii) the effects of N-acetylcysteine, dexamethasone, and bcl-2 on paraquat-induced cell deaths. Methods: A549 and BEAS-2B lung epithelial cell lines were used. The cell viability and apoptosis were evalluated using a MTT assay, Annexin V staining was monitored by fluorescence microscopy, The level of bcl-2 inhibition was examined by establishing stable A549 pcDNA3-bcl-2 cell lines throung the transfection of pcDNA3-bcl-2 with the mock. Results: Paraquat decreased the cell viability in A549 and BEAS-2B cells in a dose and time dependent manner. The Annexin V assay showed that apoptosis was the type of paraquat-induced cell death. Paraquat-induced cell deaths was significantly inhibited by N-acetylcysteine, dexamethasone, and bcl-2 overexpression. The cell viability of A549 cells treated with N-acetylcysteine, and dexamethasone on the paraquat-induced cell deaths were increased significantly by 10 ~ 20%, particularly at high doses. In addition, the cell viability of A549 pcDNA3-bcl-2 cells overexpressing bcl-2 was significantly higher than the untransfected A549 cells. Conclusion: Paraquat induces apoptotic cell deaths in lung epithelial cells in a dose and time dependent manner. The paraquat-induced apoptosis of lung epithelial cells might occur through the mitochondrial pathway.

목적: Paraquat에 의한 폐 손상의 장기간 추적 CT소견을 알아보고자 하였다. 대상과 방법: Paraquat 중독 환자 중에서 6개월 이상의 추적 CT사진을 얻은 6명의 CT를 분석하였다. 추적 CT는 7-84개월(평균 25.7개월)에 걸쳐 1-6번(평균: 3.3번)을 시행하였다. 1-2개월, 3-12개월, 1-2년, 2-3년, 7년이상의 CT소견들을 분석하였다. 결과: Paraquat에 의한 폐 손상의 HRCT에서 1-2개월에는 기관지 확장증을 동반한 불규칙한 모양의 경화를 보였고(5/5), 3-12개월에는 불규칙한 모양의 경화와 마쇄유리상음영을 보였고(5/5), 1년이후에는 불규칙한 모양의 경화와 마쇄유리상음영(4/5), 혹은 국소적인 벌집모양(1/5)을 보였다. 같은 환자의 추적 CT에서 병변의 방사선음영이 줄었고 국소적인 경화의 일부는 소실되었다. 결론: Paraquat에 의한 폐 손상 환자의 추적 HRCT의 소견은 중독후 1-2개월에는 불규칙한 모양의 경화를, 3-12개월이후에는 불규칙한 모양의 경화와 마쇄유리상음영, 혹은 벌집모양을 보였다. 중독후 3-12개월이후부터 Paraquat에 의한 폐 손상은 호전되었다.

Purpose: To determine the long-term follow-up CT findings of paraquat-induced lung injury. Materials and Methods: Six patients who ingested paraquat underwent sequential follow-up CT scanning during a period of at least six months, and the results were analysed. Scans were obtained 1-6 (mean, 3.3) time during a 7-84 (mean, 25.7) months period, and the findings at 1-2 months, 3-12 months, 1-2 years, 2-3 years, and more than above 7 years after poisoning were analyzed. Results: We observed irregular-shaped areas of consolidation with traction bronchiectasis at 1-2 months (5/5), irregular-shaped consolidation and ground-glass opacity (5/5) at 3-12 months, and irregular-shaped consolidations/ground-glass opacity (4/5) and focal honeycombing (1/5) one year later. In the same patients, follow-up CT scans showed that some areas of focal consolidation could not be visualized and the radio-opacity of the lesions had decreased. Conclusion: The HRCT findings of paraquat-induced lung injury were irregular shaped areas of consolidation 1-2 months after ingestion, and irregular-shaped consolidation and ground-glass opacity or focal honeycombing 3-12 months later. At this thim slight improvement was observed.