• Clinical and Experimental Pediatrics
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• v.52 no.2
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• pp.213-219
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• 2009

Purpose:Iron is a critical nutritional element that is essential for a variety of important biological processes, including cell growth and differentiation, electron transfer reactions, and oxygen transport, activation, and detoxification. Iron is also required for neoplastic cell growth due to its catalytic effects on the formation of hydroxyl radicals, suppression of host defense cell activities, and promotion of cancer cell multiplication. Chronic transfusion-dependent patients receiving chemotherapy may have iron overload, which requires iron-chelating therapy. We performed this study to demonstrate whether the iron chelating agent deferoxamine induces apoptosis in Saos-2 osteosarcoma cells, and to investigate the underlying apoptotic mechanism. Methods:To analyze the apoptotic effects of an iron chelator, cultured Saos-2 cells were treated with deferoxamine. We analyzed cell survival by trypan blue and crystal violet analysis, apoptosis by nuclear condensation, DNA fragmentation, and cell cycle analysis, and the expression of apoptotic related proteins by Western immunoblot analysis. Results:Deferoxamine inhibited the growth of Saos-2 cell in a time- and dose-dependent manner. The major mechanism for growth inhibition with the deferoxamine treatment was by the induction of apoptosis, which was supported by nuclear staining, DNA fragmentation analysis, and flow cytometric analysis. Furthermore, bcl-2 expression decreased, while bax, caspase-3, caspase-9, and PARP expression increased in Saos-2 cells treated with deferoxamine. Conclusion:These results demonstrated that the iron chelating agent deferoxamine induced growth inhibition and mitochondrial-dependent apoptosis in osteosarcoma Saos-2 cells, suggesting that iron chelating agents used in controlling neoplastic cell fate can be potentially developed as an adjuvant agent enhancing the anti-tumor effect for the treatment of osteosarcoma.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.8
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• pp.979-984
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• 2006

Although iron is essential for many physiological processes, excess iron can lead to tissue damage by promoting the generation of reactive oxygen species (ROS). There is increasing evidence that ROS might play an important role in the pathogenesis of cardiovascular disease. However, the effects of iron excess on platelet function and the thrombotic response to vascular injury are not well understood. We examined the effects of iron excess-induced oxidative stress and the antioxidants on platelet aggregation. Oxidative stress was accessed by either free iron $(Fe^{+2})$ or hydrogen peroxide $(H_2O_2)$, as well as their combination on washed rabbit platelets (WPs) in vitro. When WPs were stimulated with either $Fe^{+2}$ alone or a subthreshold concentration of collagen, which gave an aggregatory curve with a little effect, and a dose dependent increase in platelet aggregation was observed by increasing concentrations of $Fe^{+2}$ with $H_2O_2$. This aggregation was associated with the iron-catalyzed formation of hydroxyl radicals from $H_2O_2$, and were inhibited by NAD/NADP (proton acceptor), catalase $(H_2O_2\;scavenger)$, tiron (iron chelator), mannitol (hydroxyl radical scavenger), and indomethacin (cyclooxygenase inhibitor), but not by NADH/NADPH (proton donor), superoxide mutase, and aspirin. However, NADH/NADPH, an essential cofactor for the antioxidant capacity by the supply of reducing potentials, showed the effect of an enhanced radical formation, suggesting a role for NADH/NADPH-dependent oxidase. These results suggest that iron $(Fe^{+2})$ can directly interact with washed rabbit platelets and this aggregation be mediated by OH formation as in the Fenton reaction, inhibited by radical scavengers.

• Journal of Soil and Groundwater Environment
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• v.12 no.2
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• pp.27-36
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• 2007

A recent study showed that MTBE can be degraded by Fenton's Reagent (FR). The treatment of MTBE with FR, however, has a definite limitation of extremely low pH requirement (optimum pH $3{\sim}4$) that makes the process impracticable under neutral pH condition on which the ferrous ion precipitate forming salt with hydroxyl anion, which result in the diminishment of the Fenton reaction and incompatible with biological treatment. Consequently, this process using only FR is not suitable for in-situ remediation of MTBE. In order to overcome this limitation, modified Fenton process using NTA, oxalate, and acetate as chelating reagents was introduced into this study. Modified Fenton reaction, available at near neutral pH, has been researched for the purpose of obtaining high performance of oxidation efficiency with stabilized ferrous or ferric ion by chelating agent. In the MTBE degradation experiment with modified Fenton reaction, it was observed that this reaction was influenced by some factors such as concentrations of ferric ion, hydrogen peroxide, and each chelating agent and pH. Six potential chelators including oxalate, succinate, acetate, citrate, NTA, and EDTA were tested to identify an appropriate chelator. Among them, oxalate, acetate, and NTA were selected based on their remediation efficiency and biodegradability of each chelator. Using NTA, the best result was obtained, showing more than 99.9% of MTBE degradation after 30 min at pH 7; the initial concentration of hydrogen peroxide, NTA, and ferric ion were 1470 mM, 6 mM, and 2 mM, respectively. Under the same experimental condition, the removal of MTBE using oxalate and acetate were 91.3% and 75.8%, respectively. Optimum concentration of iron ion were 3 mM using oxalate which showed the greatest removal efficiency. In case of acetate, $[MTBE]_0$ decreased gradually when concentration of iron ion increased above 5 mM. In this research, it was showed that modified Fenton reaction is proper for in-situ remediation of MTBE with great efficiency and the application of chelatimg agents, such as NTA, was able to make the ferric ion stable even at near neutral pH. In consequence, the outcomes of this study clearly showed that the modified Fenton process successfully coped with the limitation of the low pH requirement. Furthermore, the introduction of low molecular weight organic acids makes the process more available since these compounds have distinguishable biodegradability and it may be able to use natural iron mineral as catalyst for in situ remediation, so as to produce hydroxyl radical without the additional injection of ferric ion.

• The Journal of Korean Medicine
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• v.25 no.4
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• pp.61-78
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• 2004

Objective : This study was undertaken to determine whether Gamibaegi-eum (BGU) in vitro and in vivo exerts a beneficial effect against cell injury induced by reactive oxygen species (ROS) in the human intestine. Methods : Effects of BGU in vitro on cell injury were examined using Caco-2 cells, cultured human intestinal cell line. Exposure of cells to H₂O₂ induced increases in the loss of cell viability in a time and dose-dependent fashion. Results : BGU prevented H₂O₂-induced cell death and its effect was dose-dependent over a concentration range of 0.05­1%. H₂O₂-induced cell death was prevented by catalase, the hydrogen peroxide scavenger enzyme, and deferoxamine, the iron chelator. However, the potent antioxidant DPPD did not affect H₂O₂-induced cell death. H₂O₂ increased lipid peroxidation, which was inhibited by BGU and DPPD. H₂O₂ caused DNA damage in a dose-dependent manner, which was prevented by BGU, catalase, and deferoxamine, but not DPPD. BGU restored ATP depletion induced by H₂O₂. BGU inhibited generation of superoxide and H₂O₂ and scavenged directly H₂O₂. Oral administration of mepirizole in vivo at a dose of 200mg/kg resulted in ulcer lesions in the stomach and the proximal duodenum. Pretreatment of BGU(0.1%/kg, orally) and catalase (800Units/kg, i.v.) significantly decreased the size of ulcers. Mepirizole increased lipid peroxidation in the mucosa of the duodenum, suggesting an involvement of ROS. Pretreatment of BGU and catalase significantly inhibited lipid peroxidation induced by mepirizole. Morphological studies showed that mepirizole treatment causes duodenal injury and its effect is prevented by BGU. Conclusion : These results indicate that BGU exerts a protective effect against cell injury in vitro and in vivo through antioxidant action. The present study suggests that BGU may playa therapeutic role in the treatment of human gastrointestinal diseases mediated by ROS.

• Korean Journal of Clinical Laboratory Science
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• v.43 no.4
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• pp.161-170
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• 2011

Quercetin is one of the most distributed flavonoids in the plant kingdom and occurs naturally in a wide range of fruits and vegetables. This study was undertaken to determine whether quercetin exerts beneficial effect against necrotic and apoptotic cell death induced by hydrogen peroxide ($H_2O2$) in intestinal cells using the human-derived cultured T84 colonic epithelial cell line. Necrotic cell death was induced by exposing cells to 0.5 mM $H_2O_2$ for 2 h and apoptosis was induced by incubating cells in normal culture medium for 18 h following exposure of cells to 0.5 mM $H_2O2$ for 2 h. Cell viability was evaluated by the trypan blue exclusion assay and apoptosis was assessed by Hoechst 33258 staining and flow cytometry. $H_2O_2$ induced necrotic cell death in a time and dose-dependent fashion. Both necrotic and apoptotic cell deaths were not prevented by the antioxidants N,N'-diphenyl-p-phenylenediamine(DPPD) and Trolox, whereas both cell deaths induced by the organic hydroperoxide t-butylhydroperoxide (tBHP) were prevented by DPPD, suggesting that $H_2O_2$ induces cell death through a lipid peroxidation-independent mechanism. $H_2O2$-induced necrotic death was prevented by deferoxamine and 3-aminobenzamide, while the apoptotic cell death was not affected by these agents. Quercetin prevented both necrotic and apoptotic cell deaths induced by $H_2O_2$ in a dose-dependent manner. $H_2O_2$ caused activation of poly (ADP-ribose) polmerase (PARP), which was inhibited by deferoxamine, 3-aminobenzamide, and quercetin, but not DPPD. These results indicate that quercetin inhibits both necroticand apoptotic deaths of T84 cells. The anti-necrotic effect of quercetin may be attributed to its iron chelator activity rather than a direct $H_2O_2$ scavenging capacity and antioxidant. The present study suggests that quercetin may play a therapeutic role in the treatment of human gastrointestinal diseases mediated by oxidants.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.2
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• pp.173-183
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• 2020

An in vitro model for ischemia/reperfusion injury has not been well-established. We hypothesized that this failure may be caused by serum deprivation, the use of glutamine-containing media, and absence of acidosis. Cell viability of H9c2 cells was significantly decreased by serum deprivation. In this condition, reperfusion damage was not observed even after simulating severe ischemia. However, when cells were cultured under 10% dialyzed FBS, cell viability was less affected compared to cells cultured under serum deprivation and reperfusion damage was observed after hypoxia for 24 h. Reperfusion damage after glucose or glutamine deprivation under hypoxia was not significantly different from that after hypoxia only. However, with both glucose and glutamine deprivation, reperfusion damage was significantly increased. After hypoxia with lactic acidosis, reperfusion damage was comparable with that after hypoxia with glucose and glutamine deprivation. Although high-passage H9c2 cells were more resistant to reperfusion damage than low-passage cells, reperfusion damage was observed especially after hypoxia and acidosis with glucose and glutamine deprivation. Cell death induced by reperfusion after hypoxia with acidosis was not prevented by apoptosis, autophagy, or necroptosis inhibitors, but significantly decreased by ferrostatin-1, a ferroptosis inhibitor, and deferoxamine, an iron chelator. These data suggested that in our SIR model, cell death due to reperfusion injury is likely to occur via ferroptosis, which is related with ischemia/reperfusion-induced cell death in vivo. In conclusion, we established an optimal reperfusion injury model, in which ferroptotic cell death occurred by hypoxia and acidosis with or without glucose/glutamine deprivation under 10% dialyzed FBS.

• The Korean Journal of Pharmacology
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• v.27 no.2
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• pp.155-166
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• 1991

In order to evaluate a potential role of mitochondria in the mediation of toxicity of paraquat (PQ), submitochondrial particle and microsome of mouse liver were compared by oxygen radical generation and lipid peroxidation. With NADH in submitochondrial particle and NADPH in microsome as electron donors, PQ stimulated production of superoxide anion and $H_2O_2$ in both fractions. Under the same conditions, PQ enhanced the generation of ethylene from methional suggestiong stimulation of OH production by PQ. But these effects by PQ were somewhat lower in submitochondrial particle than in microsome. In addition, lipid peroxidation(measured as MDA production) was stimulated by PQ in both fractions. The stimulation of lipid peroxidation in both fractions seemed to occur by the same mechanism probably through perferryl ion. This was supported by the following findings: i) The lipid peroxidation in both fractions was partially inhibited by SOD and completely inhibited by DETAPAC(an iron chelator) but not by catalase or OH scavenger. ii) Addition of $ADP-Fe^{3+}$ further increased PQ-induced lipid peroxidation but decreased ethylene production from methional suggesting no correlation between OH production and lipid peroxidation. The redox-cycling of PQ in mitochondria appeared to be linked to NADH dehydrogenase, not to CoQ since all of the observed stimulations by PQ in submitochondrial particle were inhibited by p-hydroxymercuribenzoate(a NADH dehydrogenase inhibitor) but not affected by other respiratory chain blockers. The above results demonstrate that redox-cycling properties of PQ leading to oxygen radical generation and lipid peroxidation can also occur in mitochondria in the same manner as in microsome. Therefore, the observed actions of PQ in mitochondria suggest that mitochondria may also contribute to toxicity of this drug in vivo.

• Journal of Acupuncture Research
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• v.17 no.3
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• pp.208-218
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• 2000

This study was undertaken to determine if Salviae Radix (SR) exerts protective effect against oxidant-induced inhibition of phosphate uptake in renal proximal tubular cells. Membrane transport function and cell death were evaluated by measuring phosphate uptake and trypan blue exclusion, respectively, in opossum kidney (OK) cells, an established proximal tubular cell line. $H_2O_2$ was used as a model oxidant. $H_2O_2$ inhibited the phosphate uptake in a dose-dependent manner over the concentration range of 0.1-0.5 mM. Similar fashion was observed in cell death. However, the phosphate uptake was more vulnerable to $H_2O_2$ than cell death, suggesting that $H_2O_2$-induced inhibition of phosphate uptake is not totally attributed to cell death. Decreasedphosphate uptake was associated with ATP depletion and inhibition of $Na^+$-pump activity as determined by direct inhibition of $N^+-K^+$-ATPase activity. When cells were treated with $H_2O_2$ in the presence of 0.05% SR, the inhibition of phosphate uptake and cell death induced by $H_2O_2$ was significantly attenuated. SR restored ATP depletion and decreased $Na^+-K^+$-ATPase activity, and this is likely responsible for the protective effect of SR on decreased phosphate uptake. The protective effect of SR was similar to the $H_2O_2$ scavenger catalase. SR reacts directly with $H_2O_2$ to reduce the effective concentration of the oxidant. The iron chelator deferoxamine prevented the inhibition of phosphate uptake and cell death induced by $H_2O_2$, suggesting that $H_2O_2$-induced cell injury is resulted from an iron-dependent mechanism. These results indicate that SR exerts the protective effect against $H_2O_2$-induced inhibition of phosphate uptake by reacting directly with $H_2O_2$ like the $H_2O_2$scavenger enzyme catalase, in OK cells. However, the underlying mechanism remains to be explored.

• Tuberculosis and Respiratory Diseases
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• v.42 no.2
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• pp.184-205
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• 1995

Background: Pulmonary toxicity by bleomycin has multiple mechanisms including direct tissue toxicity due to oxygen-derived free radicals and indirect toxicity through amplification of pulmonary inflammation. To evaluate the effect of chelators or free radical scavenger to lung damage induced by bleomycin, penicillamine as a copper chelator, deferoxamine as an iron chelator and vitamin E as a free radical scavenger were administered. Methods: Two hundred Wistar rats were divided into five groups: Control, bleomycin treated, bleomycin-penicillamine treated, bleomycin-deferoxamine treated, and bleomycin-vitamin E treated groups. Rats sacrificed on day 1, day 3, day 4, day 7, day 14, and day 28 after treatment. Bronchoalveolar lavage, light microscopic and immunohistologic studies for type I, III, IV collagens, fibronectin, laminin and NBD phallicidin were evaluated. Results: There was a significant increase in the total cell counts of bronchoalveolar lavage on day 1 from all treated animals and vitamin treated group showed an abrupt decrease in total cell counts with decrease of neutrophils on day 3. Bleomycin-vitamin E treated group had the least histologic changes such as pulmonary fibrosis. The alveolar basement membranes were positive for type IV collegen and laminin. Basement membranes of bleomycin, bleomycin-penicillamine, or bleomycin-deferoxamine treated groups were disrupted and fragmented on day 4 or 7. The bleomycin-vitamin E treated group had intact basement membranes until day 28. Conclusion: Bleomycin-induced pulmonary fibrosis was related to the severity of acute injury to oxygen radicals or activation of neutrophils and disruption of basement membrane. Vitamin E seemed to be the most effective antioxidant in the inhibition of bleomycin-induced pulmonary injury and fibrosis.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.