Production of reactive oxygen species (ROS) by devil stinger (Inimicus japonicus) during embryogenesis

In aqua-cultural industry, the seed production of devil stinger, a valuable fish in Japan, has not succeeded yet due to the cryptogenic mass mortality. We found that survival rate of the larvae of devil stinger increased by the addition of green tea extract rich in catechin into rearing tank. Generation of reactive oxygen species (ROS) was detected in the embryo of devil stinger by chemiluminescence analysis under the normal growth conditions without addition of specific stimulants. Even in the unfertilized egg, certain level of ROS was detected. ROS were continuously detected during the development from fertilized egg to larva and tended to increase gradually. Observation of embryos and post-hatching larvae with hypersensitive photon-counting microscopy indicated that ROS were produced on the surface of embryo and the head region of larva especially peripheries of eyes. When the embryo proteins were analyzed by immunoblotting using antibody against the human neutrophil cytochrome b558 large subunit (gp91 phox), a main band of approximately 91 kDa was detected, suggesting the presence of NADPH oxidase-like ROS generating system in the embryo of devil stinger. After treatment with streptomycin and penicillin G for 1 day, the level of ROS production in larvae decreased with increase in the survival rate of larvae. Our results suggest that devil stinger has ROS generation system that is already activated at fairly early stage of development before the maturation of usual immune system.     

Production of reactive oxygen species (ROS) by various marine fish species during the larval stage

Previous studies have indicated that the devil stinger produces reactive oxygen species (ROS) during early development from fertilized egg to larva. To determine whether ROS generation is a common feature in marine fish species, we conducted chemiluminescence analysis using ROS specific probe (L012) on larvae of six marine fish species. Marbled rockfish, black rockfish, and devil stinger showed higher levels of chemiluminescence response (CR), whereas the levels of CR of sevenband grouper, tiger puffer, and red seabream were fairly lower. These CRs were inhibited by the addition of superoxide dismutase. Hypersensitive photon-counting microscopic observation of black rockfish suggested that ROS production was concentrated in the head area. Our results suggest that the larvae of these six marine fishes produce ROS to considerably different extents depending on species, and that rockfish species, belonging to ovoviviparous fish, tend to produce much higher levels of ROS especially at the later larval stage.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Induction of Reactive Oxygen Species in Neurons by Haloperidol

Abstract: Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT-22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT-22 cells are equally sensitive to HP with an IC₅₀ of 35 µ M in the primary neurons and 45 µ M in HT-22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as β-estradiol and vitamin E, lowers the level of ROS and protects diol and vitamin E, lowers the level of ROS and protects HT-22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca²⁺, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.     

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.     

Reactive oxygen species‐induced activation of ERK and p38 MAPK mediates PMA‐induced NETs release from human neutrophils

Neutrophils/polymorphonuclear leukocytes (PMNs), an important component of innate immune system, release extracellular traps (NETs) to eliminate invaded pathogens; however understanding of the role of signaling molecules/proteins need to be elucidated. In the present study role of p38 MAPK and extracellular signal regulated kinase (ERK) against phorbol 12‐myristate 13‐acetate (PMA) induced reactive oxygen species (ROS) generation and NETs formation has been investigated. Human neutrophils were treated with PMA to induce free radical generation and NETs release, which were monitored by NBT reduction and elastase/DNA release, respectively. PMA treatment led to the time dependent phosphorylation of p38 MAPK and ERK in PMNs. Pretreatment of PMNs with SB202190 or U0126 did not significantly reduce PMA induce free radical generation, but prevented NETs release. Pretreatment of PMNs with NADPH oxidase inhibitor (diphenyleneiodonium chloride) significantly reduced free radical generation, p38 MAPK and ERK phosphorylation as well as NETs release, suggesting that p38 MAPK and ERK activation was downstream to free radical generation. The present study thus demonstrates ROS dependent activation of ERK and p38 MAPK, which mediated PMA induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540, 2013. © 2012 Wiley Periodicals, Inc.     

活性氧诱发人类11号染色体基因突变

对体外产生的和内源性刺激产生的活性氧 (ROS)诱发人类 11号染色体 (Hchr 11)基因突变规律及其突变谱进行研究 .体外羟自由基 (·OH)用过氧化氢 (H2 O2 )与Fe2 + 反应产生 ,并用化学发光(CL)进行相对定量分析 ;内源性ROS用佛波醇酯 (PMA)刺激人外周血白细胞产生 ,并用CL和特异性抗氧化物检测和鉴定 ;用包含单条Hchr 11的人 中国仓鼠卵巢细胞 (AL)为靶 ,经CD59表面抗原抗体筛选突变细胞克隆 ,研究ROS诱发的Hchr 11基因突变 ;突变克隆细胞DNA用Hchr 11上 5种标志基因引物进行多重PCR分析 ,结合琼脂糖凝胶电泳绘制基因突变谱 .结果表明 ,体外ROS可诱发Hchr 11基因突变 ,且·OH诱发基因突变的能力明显强于H2 O2 ,两者的突变谱也存在明显差异 ;PMA可刺激人外周血白细胞产生大量的多种ROS ,并诱发Hchr 11基因突变 ,突变谱综合了H2 O2 和·OH的所有特征 ;一些抗氧化物对内源性产生的ROS诱发Hchr 11基因突变有明显抑制作用 .提示体外和内源性ROS可诱发Hchr 11基因突变 ,不同的活性氧分子诱发的基因突变可能具有特异性     

Survival,Growth and Reproduction of Cryopreserved Larvae from a Marine Invertebrate,the Pacific Oyster (Crassostrea gigas)

This study is the first demonstration of successful post-thawing development to reproduction stage of diploid cryopreserved larvae in an aquatic invertebrate. Survival, growth and reproductive performances were studied in juvenile and adult Pacific oysters grown from cryopreserved embryos. Cryopreservation was performed at three early stages: trochophore (13±2 hours post fertilization: hpf), early D-larvae (24±2 hpf) and late D-larvae (43±2 hpf). From the beginning (88 days) at the end of the ongrowing phase (195 days), no mortality was recorded and mean body weights did not differ between the thawed oysters and the control. At the end of the growing-out phase (982 days), survival of the oysters cryopreserved at 13±2 hpf and at 43±2 hpf was significantly higher (P<0.001) than those of the control (non cryopreserved larvae). Only the batches cryopreserved at 24±2 hpf showed lower survival than the control. Reproductive integrity of the mature oysters, formely cryopreserved at 13±2 hpf and 24±2 hpf, was estimated by the sperm movement and the larval development of their offspring in 13 crosses gamete pools (five males and five females in each pool). In all but two crosses out of 13 tested (P<0.001), development rates of the offspring were not significantly different between frozen and unfrozen parents. In all, the growth and reproductive performances of oysters formerly cryopreserved at larval stages are close to those of controls. Furthermore, these performances did not differ between the three initial larval stages of cryopreservation. The utility of larvae cryopreservation is discussed and compared with the cryopreservation of gametes as a technique for selection programs and shellfish cryobanking.     

Expression of mt2 and smt-B upon cadmium exposure and cold shock in zebrafish (Danio rerio)

Metallothionein-2 (mt2) and similar to metallothionein-B (smt-B) are included in the MT gene family. The objective of this study was to compare mt2 and smt-B messenger (m)RNA expressions after cadmium exposure and cold shock with whole-mount in situ hybridization in immature zebrafish (Danio rerio) and with a semi-quantitative RT-PCR in mature zebrafish. Three-day post-fertilization (dpf) larvae were treated with 0, 0.08, 0.26, and 0.89 microM cadmium for 24 and 48 h, and some larvae were challenged with a normal (28.5 degrees C) or low temperature (12 degrees C) for 12, 24, and 48 h. Results were obtained. (1) During embryonic and larval development, mt2 mRNA existed at 6 h post-fertilization (hpf), and the level rapidly increased to 24 hpf, then it gradually increased with further larval growth. smt-B was found at 12 hpf, and it also rapidly increased to 24 hpf, but remained constant during further larval development. (2) The mt2 mRNA signals and whole-body Cd contents displayed dose- and time-dependent responses after Cd exposure. After cold shock, mt2 mRNA signals also showed time-dependent expression. But smt-B mRNA signals were not appeared by either challenge. Besides, mature zebrafish were treated with 1.78 microM Cd and found that the highest levels of smt-B mRNA (smt-B/beta-actin) appeared in brain, and seems a reverse expression between smt-B mRNA and mt2 in brain after Cd exposure. Apparently, mt2 is possibly more relevant to Cd detoxification and cold shock adaptation in zebrafish larvae compared to smt-B, but smt-B might be related to certain physiological functions in neural (or brain) of mature zebrafish.     

Effect of sanazole and metrinidazole on production of reactive oxygen species by macrophages]

The in vitro effect of sanazole (AK-2123; N-(2'-methoxyethyl)-2-[3"-nitro-1"-triazolyl]acetamide) and metronidazole (1-beta-hydroxyethyl-2-methyl-5-nitroimidazole) on phorbol-12-myristate-13-acetate (PMA)-stimulated and spontaneous (without stimulation by PMA) production of reactive oxygen species (ROS) by peritoneal and splenic murine macrophages was studied. ROS production was analyzed using fluorescent probe 2',7'-dichlorofluoresceine diacetate (DCFH-DA). An increase in the spontaneous production of ROS by macrophagal cells with therapeutic concentration of sanazole (0.6-1.25 mM) in the incubation medium was observed. At these concentrations metronidazole had no effect on spontaneous production of ROS by macrophagal cells. PMA-stimulated ROS production was inhibited by high concentrations (2.5-10 mM) of sanazole and metronidazole. The spontaneous generation of ROS by peritoneal macrophages was stimulated by sanazole at all tested concentrations (0.6-10 mM).     

表没食子儿茶素没食子酸酯对活性氧的抑制作用

生物体内的活性氧(Reactive oxygen species,ROS)过量引起氧化应激将导致脂质、DNA和蛋白质氧化损伤,从而引发一系列生理和病理反应。绿茶中茶多酚的主要成分表没食子儿茶素没食子酸酯((-)-Epigallocatechin-3-gallate,EGCG)具有强抗氧化性,能有效抑制ROS。本文简要介绍了生物体内ROS的来源和EGCG的特性及其对ROS的抑制作用。通过检测玫瑰红水溶液在光敏化时所产生~1O_2的1 270 nm近红外发光,分析比较了EGCG和迭代钠(NaN_3)对~1O_2发光的淬灭过程,发现EGCG对~1O_2的淬灭效果比NaN_3更好,为EGCG淬灭~1O_2的定量研究提供理论依据。     

Reactive Oxygen Species and Relative Enzyme Activities in the Development of Aerial Roots of Chinese Banyan (Ficus microcarpa)

Although previous research has indicated that reactive oxygen species (ROS) regulate cell extension and tissue ontogenesis, the functions of ROS in aerial roots have not been previously studied. This research evaluated ROS production and dissipation in aerial roots of Chinese banyan (Ficus microcarpa). Aerial root segments (4 cm long) were cut from trees and divided into developmental zones 1, 2, and 3 (0–5, 5–15, and 15–25 mm from root tip, respectively). According to histochemical and biochemical determinations, production of the superoxide radical (O ₂ ^·– ), hydrogen peroxide (H₂O₂), and the hydroxyl radical (^·OH) decreased from zone 1 to zone 3. The detected ROS increased with the application of exogenous stimulators of ROS generation and decreased with the application of exogenous inhibitors of ROS generation. Based on protein content, superoxide dismutase (SOD) activity increased but peroxidase (POD) and catalase (CAT) activities decreased from zone 1 to zone 3, whereas based on root segment fresh weight, SOD and CAT activities did not differ among the zones but POD activity decreased from zone 1 to 3. We conclude that ROS are generated mainly in the rapidly developing zones of aerial roots and suggest that NADPH oxidase, POD, and SOD control ROS generation. POD activity and the hydroxyl cycle seem particularly important in ROS generation in aerial roots.     

Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of γ-glutamyltransferase expression

Abstract

γ-Glutamyltransferase (GGT) plays a significant role in antioxidant defence and participates in the metabolism of glutathione (GSH). The enzyme is up-regulated after acute oxidative stress and during pro-oxidant periods, but the underlying regulatory mechanisms are not well known. The present investigation studied whether the endogenous reactive oxygen species (ROS) level was a determinant for GGT expression. A substantial amount of ROS is produced through the NADPH oxidase (NOX) system and knockdown of p22phox, a sub-unit of NOX1-4, resulted not only in reduced ROS levels but also in reduced GGT expression in human endometrial carcinoma cells. Phorbol-12-myristate-13-acetate (PMA) is an activator of NOX and it was found that PMA treatment of human colon carcinoma cells both increased cellular ROS levels and subsequently up-regulated GGT expression. On the other hand, the NOX inhibitor apocynin reduced ROS levels as well as GGT expression. The GGT mRNA sub-type A was increased after PMA-induced NOX activation. These results demonstrate that ROS generated from NOX enzymes are a significant determinant for GGT expression and activity.     

Syndecan-4 regulates platelet-derived growth factor-mediated MAP kinase activation by altering intracellular reactive oxygen species

The cell adhesion receptor, syndecan-4, regulates cellular interactions with both the extracellular matrix and soluble ligands. Accumulating evidence also suggests that cell adhesion is involved in generating reactive oxygen species (ROS). Here, we investigated the role of syndecan-4 in regulating growth factor-induced ROS generation. Rat embryo fibroblasts (REFs) overexpressing syndecan-4 exhibited increased ROS levels compared to control cells. Expression of the non-phagocytic NADH oxidase component Nox1 was increased in syndecan-4-overexpressing REFs and syndecan-4-mediated ROS generation was diminished when levels of Nox1 were knocked-down with small inhibitory RNAs. In addition, syndecan-4 enhanced platelet-derived growth factor (PDGF)-induced MAP kinase activity in parallel with ROS generation. Collectively, these data suggest that syndecan-4 regulates PDGF-induced MAP kinase activation by altering ROS generation.     

Endogenous production of reactive oxygen species by the NADPH oxidase complexes is a determinant of γ-glutamyltransferase expression

γ-Glutamyltransferase (GGT) plays a significant role in antioxidant defence and participates in the metabolism of glutathione (GSH). The enzyme is up-regulated after acute oxidative stress and during pro-oxidant periods, but the underlying regulatory mechanisms are not well known. The present investigation studied whether the endogenous reactive oxygen species (ROS) level was a determinant for GGT expression. A substantial amount of ROS is produced through the NADPH oxidase (NOX) system and knockdown of p22phox, a sub-unit of NOX1-4, resulted not only in reduced ROS levels but also in reduced GGT expression in human endometrial carcinoma cells. Phorbol-12-myristate-13-acetate (PMA) is an activator of NOX and it was found that PMA treatment of human colon carcinoma cells both increased cellular ROS levels and subsequently up-regulated GGT expression. On the other hand, the NOX inhibitor apocynin reduced ROS levels as well as GGT expression. The GGT mRNA sub-type A was increased after PMA-induced NOX activation. These results demonstrate that ROS generated from NOX enzymes are a significant determinant for GGT expression and activity.     

Combined incubation of colon carcinoma cells with phorbol ester and mitochondrial uncoupling agents results in synergic elevated reactive oxygen species levels and increased γ-glutamyltransferase expression

The NADPH oxidase (NOX) is a significant determinant for the expression and activity of γ-glutamyltransferase (GGT), which is frequently upregulated after increased levels of reactive oxygen species (ROS) and oxidative stress. Earlier studies on human colon carcinoma HT-29 cells have shown that treatment with phorbol 12-myristate 13-acetate (PMA) activates NOX thus increasing the intracellular level of ROS and upregulating GGT. Another important source of cellular ROS is the mitochondria, and treatment with the mitochondria uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) results in increased ROS levels. The present study shows that when HT-29 cells were simultaneously treated with both agents, a significant and synergic increase in intracellular ROS was detected. NOX activity contributed at least 50 % of this increase as inhibiting NOX activity with apocynin or downregulating the NOX activity using siRNA against p22 phox reduced the synergic ROS production. The combined FCCP and PMA treatment also provoked highly increased GGT mRNA levels after 24 h whereas only minor and delayed increases in GGT protein and enzyme activity levels were detected. The results strongly indicate that ROS production by both mitochondria and NOX is involved in the regulation of GGT expression in colon carcinoma cells.     

Reactive Oxygen Species Scavenging Enzymes and Down-Adjustment of Metabolism Level in Mitochondria Associated with Desiccation-Tolerance Acquisition of Maize Embryo

It is a well-known fact that a mature seed can survive losing most of its water, yet how seeds acquire desiccation-tolerance is not well understood. Through sampling maize embryos of different developmental stages and comparatively studying the integrity, oxygen consumption rate and activities of antioxidant enzymes in the mitochondria, the main origin site of reactive oxygen species (ROS) production in seed cells, we found that before an embryo achieves desiccation-tolerance, its mitochondria shows a more active metabolism, and might produce more ROS and therefore need a more effective ROS scavenging system. However, embryo dehydration in this developmental stage declined the activities of most main antioxidant enzymes and accumulated thiobarbituric acid-reactive products in mitochondria, and then destroyed the structure and functional integrity of mitochondria. In physiologically-matured embryos (dehydration-tolerant), mitochondria showed lower metabolism levels, and no decline in ROS scavenging enzyme activities and less accumulation of thiobarbituric acid-reactive products after embryo dehydration. These data indicate that seed desiccation-tolerance acquisition might be associated with down-adjustment of the metabolism level in the late development stage, resulting in less ROS production, and ROS scavenging enzymes becoming desiccation-tolerant and then ensuring the structure and functional integrity of mitochondria.