• Toxicological Research
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• 제9권2호
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• pp.217-224
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• 1993

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a well-known dopamine neuron-specific toxin. But the involvement of oxidative damage in the pathogenesis of MPTP-induced parkinsonism is still uncertain. In this study, by using 2',7',-dichlorofluorescin diacetate(DCFH-DA) that detects intracellular oxidative processes, the effect of MPTP on dichlorofluorescein fluorescence in dissociated cells from fetal rat mesencephalon in culture was investigated. At 7th day in culture, cells were loaded with DCFH-DA, and exposed to 1 mM MPTP or MPP+. MPTP induced dichlorofluorescein-fluorescence which was peaked at 3 min and mostly faded away 30 min after MPTP treatment.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권6호
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• pp.461-467
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• 2005

Background. 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. Materials and methods. The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ using ${\alpha}$-minimal essential medium (${alpha}$-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 ${{\mu}M$. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. Results. For concentration between 5 and 10 ${\mu}M$, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 ${\mu}M$. In the concentration of 15 ${\mu}M$, the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 ${\mu}M$ was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. Conclusion. These results suggests an incubation period of 15 minutes at 15 ${\mu}M$ of CFSE provides best labelling of MC3T3 in vitro.

• Environmental Analysis Health and Toxicology
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• 제11권1_2호
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• pp.49-57
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• 1996

The production of reactive oxygen species on addition of hexavalent chromium (potassium dichromate, $K_2Cr_2O_7$ ) to lung cells in culture was studied using flow cytometer analysis. A Coulter Epics Profile flow cytometer was used to detect the formation of reactive oxygen species after $K_2Cr_2O_7$ was added to A549 cells grown to confluence. The cells were loaded with the dye, 2',7'-dichlorofluorescein diacetate, after which cellular esterases removed the acetate groups and the dye was trapped intracellularly. Reactive oxygen species oxidized the dye, with resultant fluorescence. Increased doses of Cr(VI) caused increasing fluorescence (10-fold higher than background at 200 gM). Addition of Cr(III) compounds, as the picolinate or chloride, caused no increased fluorescence. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that three (as yet unidentified) spectral "signals" of the free radical type were formed on addition of 20, 50, 100 and 200 gM Cr(VI) to the A549 cells in suspension. Two other EPR 'signals" with the characteristics of Cr(V) entities were seen at field values lower than the standard free radical value. radical value.

• Fisheries and Aquatic Sciences
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• 제14권3호
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• pp.179-185
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• 2011

The antioxidant activities of a methanolic extract of Eisenia bicyclis and its organic solvent fractions, including dichloromethane ($CH_2Cl_2$), ethyl acetate (EtOAc), n-butanol (n-BuOH), and water ($H_2O$) fractions, were investigated. Scavenging activities against DPPH, hydroxyl, superoxide anion, and peroxynitrite radicals were evaluated using electron spin resonance spectrometry; intracellular reactive oxygen species (ROS) were evaluated by a 2',7'-dichlorofluorescein diacetate assay using RAW264.7 mouse macrophages. The antioxidant activities of the individual fractions were: EtOAc>n-BuOH>$CH_2Cl_2$ >$H_2O$. The EtOAc fraction exhibited strong radical scavenging activity and a significantly reduced ROS level in RAW264.7 cells. Moreover, the phenolic contents of the extract and fractions followed the same order as their radical scavenging activities. Our results indicate that E. bicyclis is a valuable natural source of antioxidants that may be applicable to the functional food industry.

• Archives of Pharmacal Research
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• 제27권2호
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• pp.194-198
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• 2004

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human degenerative diseases such as cancer, aging, arteriosclerosis, and rheumatism. Much attention has been focused on the development of safe and effective antioxidants. To discover sources of antioxidative activity in marine algae, extracts from 17 kinds of seaweed were screened for their inhibitory effect on total ROS generation in kidney homogenate using 2',7'-dichlorofluorescein diacetate (DCFH-DA). ROS inhibition was seen in three species: UIva pertusa, Symphyocladia latiuscula, and Ecklonia stolonifera. At a final concentration of 25 $\mu\textrm{g}$/mL, U. pertusa inhibited 85.65$\pm$20.28％ of total ROS generation, S. latiscula caused 50.63$\pm$0.09％ inhibitory, and the Ecklonia species was 44.30$\pm$7.33％ inhibition. E. stolonifera OKAMURA (Lam-inariaceae), which belongs to the brown algae, has been further investigated because it is commonly used as a foodstuff in Korea. Five compounds, phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5), isolated from the ethyl acetate soluble fraction of the methanolic extrclct of E. stolonifera inhibited total ROS generation.

• Preventive Nutrition and Food Science
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• 제19권4호
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• pp.261-267
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• 2014

As a part of ongoing research to elucidate and characterize antioxidant and anti-inflammatory nutraceuticals, solvent-partitioned fractions from Spergularia marina were tested for their ability to scavenge radicals and suppress inflammation. The results of the 2',7'-dichlorofluorescein diacetate assay indicate that solvent-partitioned fractions from S. marina scavenged intracellular radicals in $H_2O_2$-stimulated mouse macrophages. The tested fractions decreased the generation of nitric oxide (NO) and the expression of inflammation mediators, namely, inducible nitric oxide synthase (iNOS) and interleukin (IL)-6, by lipopolysaccharide (LPS)-induced mouse macrophages, indicating that S. marina decreases inflammation. Among all tested fractions [i.e., $H_2O$, n-buthanol (n-BuOH), 85% aqueous methanol (aq. MeOH), and n-hexane], the 85% aq. MeOH fraction showed the strongest antioxidant and anti-inflammatory response. The 85% aq. MeOH fraction scavenged 80% of the free radicals produced by $H_2O_2$-induced control cells. In addition, NO production was 98% lower in 85% aq. MeOH fraction-treated cells compared to LPS-induced control cells. The mRNA expression of iNOS and IL-6 was also suppressed in 85% aq. MeOH fraction-treated cells. The results of the current study suggest that the phenolic compound components of S. marina are responsible for its antioxidant and anti-inflammatory effects.

• 대한수의학회지
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• 제62권1호
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• pp.3.1-3.7
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• 2022

Disulfiram (DSF) is a marketed drug to treat patients with alcohol dependence by inhibiting aldehyde dehydrogenase. Over the last few decades, DSF has been shown to have anticancer effects through different mechanisms. Moreover, this effect can be elevated when used with copper (Cu). Subsequent studies have been conducted on various cancers, but few on lymphoma. This study investigated the anticancer effects of DSF on lymphoma and how this effect changed when treated with Cu. DSF synergistically decreased the metabolic activity of EL4 lymphoma cells when combined with Cu. At 1 µM of DSF alone, the metabolic activity of EL4 cells decreased by 49% compared to the control, whereas it decreased by 87% with a DSF + CuCl₂ treatment. Rhodamine 123 and 2',7'-dichlorofluorescein diacetate staining showed that DSF induced the reduction of the mitochondrial membrane potential and promoted the production of reactive oxygen species. In particular, the combined treatment of DSF + Cu induced cell death based on multiple assays, including annexin V-fluorescein isothiocyanate/propidium iodide staining. Overall, DSF has anticancer effects on lymphoma cells and exhibits synergistic effects when combined with Cu. This study provides some valuable information to broaden the use of DSF in clinics and basic research.

• 대한의생명과학회지
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• 제23권2호
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• pp.128-132
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• 2017

Streptozotocin (STZ), bisphenol A (BPA), and diethylstilbestrol (DES) are known as endocrine disruptors, occurs oxidative stress in animal cells. Generally, oxidative stress induces reactive oxygen species (ROS) and lipid peroxidation of sperm and lead to decreased viability and fertility in pigs. Therefore, we investigated the influence of STZ, BPA and DES on ROS production and lipid peroxidation on boar sperm. Collected sperm were incubated with semen extender containing $10{\mu}M\;STZ$, $10{\mu}M\;BPA$ and $20{\mu}M\;DES$ for 3, 6 and 9 hours. Intracellular ROS and lipid peroxidation of sperm were analyzed by 2', 7'-dichlorofluorescein diacetate and malondialdehyde methods. The results show that, intracellular ROS was not significantly different among the all treatments, but lipid peroxidation was significantly increased in STZ group at 3 hour after incubation with boar sperm (P<0.05). These results suggest that STZ stimulates lipid peroxidation more than ROS production and may exert a negative effect on sperm fertility.

• 대한화장품학회지
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• 제32권4호
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• pp.263-267
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• 2006

태양광선과 산소는 피부세포에 자유라디칼(free radical)과 활성산소종(reactive oxygen species, ROS)을 생성시켜 세포막 지질의 과산화와 염증을 유발한다. 본 연구는 자외선에 의한 피부손상에 대한 머위추물물의 피부 보호효과에 대한 것으로 UVB가 조사된 각질형성세포에서이 항산화 및 염증관련 사이토카인의 억제효과를 측정하였다. 라디칼에 의한 지질과산화에 대한 억제효과는 $100{\mu}g/mL$에서 70.1%로 우수하게 나타났으며, 세포내에서 ROS에 의해 형광을 띠는 물질로 전환되는 $CM-H_2DCFDA$를 이용하여 ROS의 양을 측정한 결과 자외선에 의해 증가된 세포내 ROS의 양이 머위추출물을 처리함으로써 $500{\mu}g/mL$ 농도에서 45% 이상의 우수한 소거효과를 나타냈다. 또한 각질형성세포에서 UVB에 의해 생합성이 증가되는 $IL-1{\alpha}$ 및 $PGE_2$의 생합성 억제효과는 $100{\mu}g/mL$에서 각각 25.7%, 59.3%를 저해하는 것으로 우수한 효과를 나타내었다. 따라서 머위추출물은 각질형성세포에서 자외선 조사에 의한 피부손상에 대한 보호효과를 가진 화장품에서 우수한 소재로 적용될 수 있다.

• 한국약용작물학회지
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• 제27권6호
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• pp.365-375
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• 2019

Background: Oxidative stress plays an important role in neuro-degenerative disorders such as Alzheimer's disease. Oxidative stress is mediated by reactive oxygen species (ROS), which are implicated in the pathogenesis of numerous diseases, and account for the toxicity of a wide range of compounds. Methods and Results: In order to study the neuro-protective effect of the complex extracts of Aster scaber Thunberg (AS) and Chaenoleles sinensis Koehne (CSK) against hydrogen peroxide in PC12 cells, cell viability was evaluated by the MTT assay using tetrazole, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the intracellular ROS levels were determined the by 2',7'-dichlorofluorescein diacetate (DCF-DA) assay. In order to examine the anti-amnesic effects of the complex extracts of AS and CSK, behavioral tests were performed on male ICR mice. The ameliorating effect of the complex extracts against Aβ_1-42-induced learning and memory impairment was analyzed by y-maze and passive avoidance tests. The AS and CSK extracts showed neuro-protective activity both in vitro and in vivo, and the neuro-protective effect of their 60 : 40 (AS : CSK) mixture was better than that of the other mixtures. Moreover, the complex extracts synergistically inhibited acetylcholinesterase activity and rapid peroxidation. Conclusions: A mixture of the AS and CSK extracts could be used to develop functional foods and serve as raw materials for the development of therapeutics against Alzheimer's disease.