• Molecules and Cells
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• v.46 no.6
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• pp.329-336
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• 2023

Reactive oxygen species (ROS) serve as secondary messengers that regulate various developmental and signal transduction processes, with ROS primarily generated by NADPH OXIDASEs (referred to as RESPIRATORY BURST OXIDASE HOMOLOGs [RBOHs] in plants). However, the types and locations of ROS produced by RBOHs are different from those expected to mediate intracellular signaling. RBOHs produce O₂^•- rather than H₂O₂ which is relatively long-lived and able to diffuse through membranes, and this production occurs outside the cell instead of in the cytoplasm, where signaling cascades occur. A widely accepted model explaining this discrepancy proposes that RBOH-produced extracellular O₂^•- is converted to H₂O₂ by superoxide dismutase and then imported by aquaporins to reach its cytoplasmic targets. However, this model does not explain how the specificity of ROS targeting is ensured while minimizing unnecessary damage during the bulk translocation of extracellular ROS (eROS). An increasing number of studies have provided clues about eROS action mechanisms, revealing various mechanisms for eROS perception in the apoplast, crosstalk between eROS and reactive nitrogen species, and the contribution of intracellular organelles to cytoplasmic ROS bursts. In this review, we summarize these recent advances, highlight the mechanisms underlying eROS action, and provide an overview of the routes by which eROS-induced changes reach the intracellular space.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.4
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• pp.335-344
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• 2024

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca²⁺ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca²⁺ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC₅₀ of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca²⁺ transients by DPI. The L-type Ca²⁺ current (I_Ca) was decreased concentration-dependently by DPI (IC₅₀ of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca²⁺ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca²⁺ and Na⁺. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca²⁺ channel and RyR, thereby attenuating Ca²⁺-induced Ca²⁺ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

• Journal of Nutrition and Health
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• v.34 no.3
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• pp.299-305
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• 2001

The purpose of this study was to investigate the effects of catechin on the changes of microsomal mixed function oxidase(MFO) system and oxidative damage in rat liver exposed to microwave. Sprague-Dawley male rats weighing 200$\pm$10g body weight were randomly assigned to one normal and microwave exposed groups: microwave exposed groups were divided three groups: catechin free diet(MW) group, 0.25% catechin(MW+0.25C) group and 0.5% catechin(MW+0.5C) group to the levels of dietary catechin supplementation. The rats were irradiated with microwave at frequency 2.45GHz for 15 min and then the changes pattern of mixed function oxidase system and oxidative damage were investigated for 16 days. The activity of XOD in MW group was increased from 4 day to 8 days after irradiation, compared to normal group and decreased to the level of normal group 16 days. But catechin supplementation group were maintained to the normal level. The contents of cytochrome P(sub)450 and NADPH cytochrome P(sub)450 reductase activities in liver of MW group was increased from 4 day to 8 day after irradiation, compared to normal group and decreased to the level of normal group at 16 day. But catechin supplementation group were recovered to the normal level. The contents of superoxide radical in liver of MW group was increased 1.28, 1.25, 1.17 fold of normal group at 4,6 and 8 days days after irradiation. respectively, but catechin supplementation group were maintained the normal level. The contents of lipifuscin in liver have a same tendency in superoxide radical contents. These result suggested that the supplementation of catechin have control the mixed function oxidase system and oxidative damage and that may help to recover tissues from microwave damage. (Korean J Nutrition 34(3) : 299~305, 2001)

• The Korean Journal of Pharmacology
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• v.20 no.1 s.34
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• pp.47-54
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• 1984

Effect of ascorbic acid on various hepatic ethanol metabolizing enzymes including alcohol dehydrogenase(ADH), the microsomal . ethanol oxidizing system(MEOS), and catalase was quantitatively evaluated in liver microsomal and cytosolic preparation from Sprague-Dowley rats. In present study, ADH activities were no changed significantly by ascorbic acid. The MEOS activity, dependent on NADPH and $O_2$, was affected by azide (inhibitor of catalase) or exogenous catalase. In the presence of ascorbic acid, ethanol oxidation by rat liver microsomal preparation reacted with NADPH-generating system was increased by up to 22.5%, but decreased when liver microsome was reacted with $H_2O_2$ generated by xanthine and xanthine oxidase. Increase in the activity of the MEOS in the presence of ascorbic acid was greater in liver microsomal preparation pretreated with azide. Also ascorbic acid oxidized ethanol nonenzymatically. This ethanol oxidation induced by ascorbic acid was inhibited by OH radical scavengers (thiourea, sodium benzoate), but was not much affected by superoxide dismutase. From these results it was suggested that ascorbic acidcould interact directly with the MEOS, then promote the oxidation of ethanol. And, to some extent, ${\cdot}OH$-radicals or other radicals generated during the spontaneous autooxidation of ascorbic acid may be responsible for the production of acetaldehyde from ethanol.

• Parasites, Hosts and Diseases
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• v.61 no.4
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• pp.388-396
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• 2023

Entamoeba histolytica is an enteric tissue-invasive protozoan parasite causing amoebic colitis and liver abscesses in humans. Amoebic contact with host cells activates intracellular signaling pathways that lead to host cell death via generation of caspase-3, calpain, Ca²⁺ elevation, and reactive oxygen species (ROS). We previously reported that various NADPH oxidases (NOXs) are responsible for ROS-dependent death of various host cells induced by amoeba. In the present study, we investigated the specific NOX isoform involved in ROS-dependent death of hepatocytes induced by amoebas. Co-incubation of hepatoma HepG2 cells with live amoebic trophozoites resulted in remarkably increased DNA fragmentation compared to cells incubated with medium alone. HepG2 cells that adhered to amoebic trophozoites showed strong dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, suggesting intracellular ROS accumulation within host cells stimulated by amoebic trophozoites. Pretreatment of HepG2 cells with the general NOX inhibitor DPI or NOX2-specific inhibitor GSK 2795039 reduced Entamoeba-induced ROS generation. Similarly, Entamoeba-induced LDH release from HepG2 cells was effectively inhibited by pretreatment with DPI or GSK 2795039. In NOX2-silenced HepG2 cells, Entamoeba-induced LDH release was also significantly inhibited compared with controls. Taken together, the results support an important role of NOX2-derived ROS in hepatocyte death induced by E. histolytica.

• Journal of Nutrition and Health
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• v.33 no.6
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• pp.619-624
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• 2000

The purpose of this study was to investigate the effects of vitamin E on microsomal mixed function oxidase system of kidney in streptozotocin(STZ) induced diabetic rats. Sprague-Dawley male rats weighing 140$\pm$10g were randomly assigned to one control and three STZ-diabetic groups which were subdivided into vitamin E free diet(DM-0E group) 40mg vitamin E per kg diet(DM-40E group) and 400mg vitamin E per kg diet(DM-400E group). Vitamin E level of normal group was 40 mg per kg diet. Diabetes was experimentally induced by intravenous administration of 55 mg/kg B.W of STZ in citrate buffer(pH4.3) after 4 weeks feeding of experimental diets. Animals were sacrificed at the 6th day of diabetic state. The contents of cytochrome P450 in kidney were increased by 82, 54, 41% in DM-0E, DM-40E and DM-400E groups respectively when compared with normal group. The contents of cytochrome b5 in kidney were increased by 28% in DM-0E when compared with normal group but those of DM-40E and DM-400E groups were similar to that of normal group. The activities of NADPH-cytochrome P450 reductase in kidney that were increased by 35% in DM-0E group. Levels of TBARS(thiobarbituric acid reactive substance) in kidney were increased by 207, 129% and 72% in DM-0E and DM-400E groups respectively when compared with normal group but those of DM-40E and DM-400E groups were 26,44% lower than that of DM-0E groups. It is know that the activities of MFO system and lipid peroxidation were inhibited in kidney of STZ-induced diabetic rat by administeration of high doses of vitamin E.(Korean J Nutrition 33(6) : 619~624, 2000)

• Journal of Life Science
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• v.26 no.1
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• pp.8-16
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• 2016

Reactive oxygen species (ROS), at appropriate concentrations, mediate various normal cellular functions, including defense against pathogens, signal transduction, cellular growth, and gene expression. A recent study demonstrated that ROS and ROS-generating NADPH oxidase (Nox) are important in self-renewal and neuronal differentiation of subventricular zone (SVZ) neural stem cells in adult mouse brains. In this study, we found that endogenous ROS were detected in SVZ neural stem cells cultured from postnatal mouse brains. Nox4 was predominantly expressed in cultured cells, while the levels of the Nox1 and Nox2 transcripts were very low. In addition, the Nox4 gene was highly upregulated (by up to 10-fold) during neuronal differentiation. Immunocytochemical analysis detected the Nox4 protein mainly in neurons positive for the neuronal specific tubulin Tuj1. After differentiation, endogenous ROS were detected exclusively in neuron-like cells with processes. In addition, perturbation of the cellular redox state with N-acetyl cysteine, a ROS scavenger, during neuronal differentiation greatly inhibited neurogenesis. Lastly, knockdown of Nox4 using short hairpin RNA decreased neurogenesis. These findings suggest that Nox4 may be a major ROS-generating enzyme in postnatal SVZ neural stem cells, and Nox4-mediated ROS generation may be important in their neuronal differentiation.

• The Korea Journal of Herbology
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• v.36 no.3
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• pp.1-8
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• 2021

Objective : Reflux esophagitis (RE), one of gastroesophageal reflux disease (GERD), is a disease that causes inflammation due to reflux of stomach contents such as stomach acid and pepsin due to the unstable gastroesophageal sphincter, and is currently increasing worldwide. The currently used treatment for reflux esophagitis has various side effects. Therefore, in this study the effect of Toosendan Fructus extract on chronic acid reflux esophagitis in rats was evaluated in order to find a new treatment material for reflux treatment. Methods : After inducing reflux esophagitis through surgery, the group was separated and the drug was administered for 2 weeks; Normal rats (Normal, n=8), chronic acid reflux esophagitis rats (Control, n=8), Toosendan Fructus 200 mg/kg body weight/day-treated chronic acid reflux esophagitis rats (TF, n=8). After, we were taken esophageal tissue and esophageal mucosa damage was identified, and analyzed the expression of NADPH oxidase, AP-1/MAPK-related proteins, and tight junction proteins by western blot in esophageal tissue. Results : Toosendan Fructus administration significantly protected the esophageal mucosal damage of reflux esophagitis. Also, Toosendan Fructus significantly reduced the expression of NADPH oxidases (NOX2 and p22^phox) and AP-1/MAPK-related proteins (c-Fos, c-Jun, p-p38, p-ERK, and p-JNK). In addition, it significantly increased the expression of tight junction proteins (Occludin, Claudin-3, and Claudin-4). Conclusions : These results suggest that Toosendan Fructus reduced damage to the esophageal mucosa by protecting the esophageal mucosa by upregulating tight junctions proteins as well as inhibiting the AP-1/MAPK pathway through reducing NADPH oxidases expression.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.45 no.1
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• pp.57-67
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• 2019

Particulate matters with a diameter of < $10{\mu}m$ (PM10) exert oxidative stress and inflammatory events in various organs. The purpose of this study was to examine the molecular mechanism of reactive oxygen species (ROS) production induced by PM10 in the human epidermal keratinocytes (HEKs). When cultured HEKs were exposed to PM10, ROS production was induced and it was inhibited by apocynin, an antioxidant. The mRNA expression of NADPH oxidase (NOX) family was analyzed in order to examine their role in PM10-induced ROS production. PM10 increased the mRNA expression of NOX1, NOX2, dual oxidase (DUOX) 1 and DUOX2. HEKs expressed DUOX1 and DUOX2 at higher levels compared to other NOXs. The mRNA expression of dual oxidase maturation factors, DUOXA1 and DUOXA2, was also increased by PM10. We examined whether these calcium-dependent enzymes, DUOX1 and DUOX2, mediate the PM10-induced ROS production. A selective intracellular calcium chelator, BAPTA-AM, attenuated ROS production induced by PM10 or calcium ionophore A23187. The small intereference RNA (siRNA)-mediated down-regulation of DUOX2, but not DUOX1, attenuated the ROS production induced by PM10. PM10 increased the expression of inflammatory cytokines such as interleukin $(IL)-1{\beta}$, IL-6, IL-8 and interferon $(IFN)-{\gamma}$. SiRNA-mediated down-regulation of DUOX2 suppressed the PM10-induced expression of $IFN-{\gamma}$ but not other cytokines. This study suggests that DUOX2 plays a crucial role in ROS production and inflammatory response in PM10-exposed keratinocytes.

• The Korean Journal of Pesticide Science
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• v.2 no.2
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• pp.10-15
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• 1998

The bimolecular inhibition rate constants of carbofuran and N-dimethylphosphinothioyl carbofuran(PSC) to acetylcholinesterase(AChE) were $7.7{\times}10^{5}\;M^{-1}{\cdot}min^{-1}$ and $1.2{\times}10^{3}\;M^{-1}{\cdot}min^{-1}$, respectively. These results showed that PSC required a bioactivation process for its toxic action because it didn't inhibit the target enzyme effectively. The potency of PSC as an inhibitor of AChE increased when PSC and AChE were incubated with microsomes fortified with NADPH compared with microsome alone. Piperonyl butoxide(PBO) addition to these coupled systems greatly reduced the inhibition of the target enzyme by blocking the bioactivation process. In vivo inhibition study of mouse brain AChE, $I_{50}$ value for AChE was 28 mg/kg for PSC and the value increased to 57 mg/kg when PBO was pretreated. This result showed that cytochrome $P_{450}$ would also play a role in the bioactivation process of PSC in vivo. And conversioin of carbofuran from PSC was 55 % in a chemical oxidation system using meta-chloroperoxybenzoic acid. The oxidative activation of PSC to carbofuran was shown to be essential for showing its toxicological action and cytochrome $P_{450}$ was identified as an important enzyme which participated in this process.