Hydrogen Peroxide-Mediated Growth of the Root System Occurs via Auxin Signaling Modification and Variations in the Expression of Cell-Cycle Genes in Rice Seedlings Exposed to Cadmium Stress

The link between root growth, H₂O₂, auxin signaling, and the cell cycle in cadmium (Cd)‐stressed rice (Oryza sativa L. cv. Zhonghua No. 11) was analyzed in this study. Exposure to Cd induced a significant accumulation of Cd, but caused a decrease in zinc (Zn) content which resulted from the decreased expression of OsHMA9 and OsZIP. Analysis using a Cd‐specific probe showed that Cd was mainly localized in the meristematic zone and vascular tissues. Formation and elongation of the root system were significantly promoted by 3‐amino‐1,2,4‐triazole (AT), but were markedly inhibited by N,N’‐dimethylthiourea (DMTU) under Cd stress. The effect of H₂O₂ on Cd‐stressed root growth was further confirmed by examining a gain‐of‐function rice mutant (carrying catalase1 and glutathione‐S‐transferase) in the presence or absence of diphenylene iodonium. DR5‐GUS staining revealed close associations between H₂O₂ and the concentration and distribution of auxin. H₂O₂ affected the expression of key genes, including OsYUCCA, OsPIN, OsARF, and OsIAA, in the auxin signaling pathway in Cd‐treated plants. These results suggest that H₂O₂ functions upstream of the auxin signaling pathway. Furthermore, H₂O₂ modified the expression of cell‐cycle genes in Cd‐treated roots. The effects of H₂O₂ on root system growth are therefore linked to auxin signal modification and to variations in the expression of cell‐cycle genes in Cd‐stressed rice. A working model for the effects of H₂O₂ on Cd‐stressed root system growth is thus proposed and discussed in this paper.     

Circadian expression of DNA methylation and demethylation genes in zebrafish gonads

This research aimed at investigating the light synchronization and endogenous origin of daily expression rhythms of eight key genes involved in epigenetic mechanisms (DNA methylation and demethylation) in zebrafish gonads. To this end, 84 zebrafish were distributed into six tanks, each one containing 14 fish (7 males and 7 females). Animals were subjected to 12 h light:12 h dark cycles (LD, lights on at ZT0 h) and fed randomly three times a day during the light phase. Locomotor activity rhythms were recorded in each tank for 20 days to test their synchronization to light. Then, zebrafish were fasted for one day and gonad samples were collected every 4 h during a 24 h cycle (ZT2, 6, 10, 14, 18, and 22 h). The results revealed that most of the epigenetic genes investigated exhibited a significant daily rhythm. DNA methylation genes (dnmt4, dnmt5, dnmt7) exhibited a daily rhythm of expression with a nocturnal acrophase (ZT14:01~ZT22:17 h), except for dnmt7 in males (ZT2:25 h). Similarly, all DNA demethylation genes (tet2, tdg, mb4, gadd45aa, and apobec2b) revealed the existence of statistically significant daily rhythms, except for gadd45aa in females. In females, tdg, mb4, and apobec2b presented a nocturnal peak (ZT14:20 ~ ZT22:04 h), whereas the tet2 acrophase was diurnal (ZT4:02 h). In males, tet2, tdg, and gadd45aa had nocturnal acrophases (ZT18:26~ZT21:31 h), whereas mb4 and apobec2b displayed diurnal acrophases (ZT5:28 and ZT4:02 h, respectively). To determine the endogenous nature of gene expression rhythms, another experiment was performed: 12 groups of 14 fish (7 males and 7 females) were kept in complete darkness (DD) and sampled every 4 h during a 48 h cycle (CT2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 h). Under DD, most of the genes (7 out of 8) presented circadian rhythmicity with different endogenous periodicities (tau), suggesting that the epigenetic mechanisms of DNA methylation and demethylation in the gonads follow an internal control, functioning as part of the translation network linking the environment into somatic signals in fish reproduction.     

Cu/Zn superoxide dismutase is differentially regulated in period gene-mutant mice

The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9 h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.     

The limitations of heart rate as a predictor of metabolic rate in fish

Although telemetered heart rate (f_H) has been used as a physiological correlate to predict the metabolic rate (as oxygen consumption, V?O₂) of fish in the field, it is our contention that the method has not been validated adequately for fish. If f_H in fish is to be used to estimate V?O₂, a single linear (or log-linear) relationship must be established for each species between the two variables which allows V?O₂ to be predicted accurately under all environmentally relevant conditions. Our analyses of existing data indicate that while a good linear (or log-linear) relationship can be established between f_H and V?O₂, the conditions under which the relationship applies may be quite restricted. Physiological states and environmental factors affect the relationship between f_H and V?O₂ significantly such that several curves can exist for a single species. In addition, there are situations in which f_H and V?O₂ do not covary in a significant manner. In some situations f_H can vary over much of its physiological range while V?O₂ remains constant; in others V?O₂ may vary while f_H is invariate. The theoretical basis for this variability is examined to explain why the use of telemetered f_H in predicting V?O₂ of fish may be limited to certain specified applications.     

Metabolism of reactive oxygen species in cotton cytoplasmic male sterility and its restoration

To elucidate reactive oxygen species (ROS) metabolism of cotton cytoplasmic male sterility and the effects of restorer gene on the metabolism of ROS, the metabolism changes in the production and scavenging of ROS and gene expression related to ROS-scavenging enzymes were investigated in the anther mitochondria of CMS line, maintainer line and hybrid F₁. During the abortion preliminary stage (sporogenous cell division stage), anthers of CMS line had a little higher superoxide (O₂ ⁻) production rate and hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents than those of maintainer or hybrid F_1. Simultaneously, a little higher ROS contents might serve as a signal to increase the activity of superoxide dismutase (SOD) in anthers of CMS line to reduce the ROS damage to the anther development. But at the abortion peak (pollen mother cell meiosis stage), anthers of CMS line had extraordinarily higher ROS contents and lower ROS-scavenging enzymic activities compared with the hybrid F₁, during which the ROS contents and ROS-scavenging enzymic activities in hybrid F₁ were approximate to those of maintainer line. The expression of Mn-sod and apx mRNA in anther of CMS line was obviously inhibited when ROS produced with a great deal during anther abortion, however the gene expression in hybrid F₁ kept normal with the maintainer. Excessive accumulation of O₂^·−, H₂O₂ and MDA, significant reduction of ROS-scavenging enzymic activities and lower gene expression level of ROS-scavenging enzyme were coinstantaneous with male cells death in anthers of CMS line. But when the restorer gene was transferred into CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F₁. The restorer gene likely plays an important role in keeping the dynamic balance between the production and elimination of ROS.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Importance of sandy bottoms in coral reefs to the oscillation of daily rhythms in the tropical wrasse Halichoeres trimaculatus

Most wrasse species swim during the day and bury themselves in the sandy bottoms of shallow reefs at night. This study aimed to evaluate the importance of sandy bottoms to the day-active/night-inactive rhythmicity of the tropical wrasse Halichoeres trimaculatus. Actogram analysis revealed that fish were active during the photophase and inactive during the scotophase in aquariums with both sandy and bare bottoms. When fish were kept in aquariums with bare bottoms, rhythmicity was maintained under constant dark conditions (DD) but became obscured under constant light conditions (LL), suggesting that a day-active/night-inactive rhythmicity is regulated by the circadian system. Robust fluctuations in Period1 (wPer1) and Period2 (wPer2) expression were observed in the pectoral fin tissue under light–dark conditions (LD). Similar fluctuations in wPer1 expression persisted under DD. When fish were kept under LD conditions for 7 days and then DD for 20 days, the emergence of fish from the sandy bottom was delayed gradually. At the same time, the peak time of wPer1 expression under DD was retarded from 06:00 to 10:00. Although wPer2 expression was dampened under DD, it increased after exposing fish to light. These results suggest that wPer1 and wPer2 are differentially involved in the day-active/night-inactive rhythmicity, and that blocking light with a sandy bed at night and exposing fish to light during emergence in the morning play important roles in maintaining consistent activities in wrasse species.     

水稻磷脂氢谷胱甘肽过氧化物酶在蛋白质水平上的组织和诱导表达特征

蛋白质是生命活动的主要承担分子,了解蛋白质在有机体中的时空分布对于正确解析蛋白质的功能十分重要．磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是目前发现的唯一能够直接还原膜上脂类过氧化物的抗氧化酶,在保护生物膜免受过氧化损伤方面有着重要作用．采用Western blot技术,分析了水稻PHGPx (OsPHGPx) 在水稻不同组织以及多种胁迫条件下的蛋白质表达特征．结果表明,OsPHGPx在成熟水稻植株内主要分布于叶组织中,以旗叶中含量最高,而在水稻幼苗中则在茎及叶组织中均检测到较强的杂交信号．OsPHGPx在幼苗中的表达受到H₂O₂和NaCl的强烈诱导,但植物激素对其表达的影响较弱．H₂O₂和NaCl的诱导效果呈现出时间及剂量的相关性,当用0.5 mmol/L H₂O₂处理12 h或用500 mmol/L NaCl处理24 h,此时OsPHGPx表达量达到最大值．对H₂O₂清除剂二甲基硫脲处理的水稻幼苗,外源H₂O₂的再处理并不能诱导OsPHGPx的表达,而NaCl的诱导效果并不受影响,说明H₂O₂可能并不介导NaCl诱导OsPHGPx的表达．这些结果为进一步研究OsPHGPx在水稻中生物学功能奠定了基础．     

Protection against oxidative stress by vitamin D in cone cells

Photoreceptor degeneration (PD) refers to a group of heterogeneous outer retinal dystrophies characterized by the death of photoreceptors. Both oxidative stress and inflammation are involved in the pathogenesis of PD. We investigate whether vitamin D has a potential for the treatment of PD by evaluating the anti‐oxidative stress and anti‐inflammatory properties of the active form of vitamin D₃, 1,α, 25‐dihydroxyvitamin D₃, in a mouse cone cell line, 661W. Mouse cone cells were treated with H₂O₂ or a mixture of H₂O₂ and vitamin D; cell viability was determined. The production of reactive oxygen species (ROS) in treated and untreated cells was measured. The expression of key anti‐oxidative stress and inflammatory genes in treated and untreated cells was determined. Treatment with vitamin D significantly increased cell viability and decreased ROS production in 661W cells under oxidative stress induced by H₂O₂. H₂O₂ treatment in 661W cells can significantly down‐regulate the expression of antioxidant genes and up‐regulate the expression of neurotoxic cytokines. Vitamin D treatment significantly reversed these effects and restored the expression of antioxidant genes. Vitamin D treatment also can block H₂O₂ induced oxidative damages. The data suggested that vitamin D may offer a therapeutic potential for patients with PD.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

Gpx1 is a stationary phase-specific thioredoxin peroxidase in fission yeast

The genome sequence of Schizosaccharomyces pombe reveals only one gene for a putative glutathione peroxidase (gpx1⁺). The Gpx1 protein has a peroxidase activity but preferred thioredoxin to glutathione as an electron donor when examined in vitro and in vivo, and therefore is a thioredoxin peroxidase. Besides H₂O₂, it can reduce alkyl and phospholipid hydroperoxides. Expression of the gpx1 gene was elevated at the stationary phase, and we found that it supported long-term survival of S. pombe. The mutant also exhibited some defect in the activity of aconitase, an oxidation-labile Fe-S enzyme in mitochondria. Activity of sulfite reductase, a labile Fe-S enzyme in the cytosol, was also dramatically lowered in the mutant in the stationary phase. The Gpx1 protein, without any obvious targeting sequence, was localized in mitochondria as well as in the cytosol. Therefore, Gpx1 must serve to ensure optimal mitochondrial function and cytosolic environment, especially in the stationary phase.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Facing warm temperatures during migration: cardiac mRNA responses of two adult Oncorhynchus nerka populations to warming and swimming challenges

The main findings of the current study were that exposing adult sockeye salmon Onchorhynchus nerka to a warm temperature that they regularly encounter during their river migration induced a heat shock response at an mRNA level, and this response was exacerbated with forced swimming. Similar to the heat shock response, increased immune defence‐related responses were also observed after warm temperature treatment and with a swimming challenge in two different populations (Chilko and Nechako), but with some important differences. Microarray analyses revealed that 347 genes were differentially expressed between the cold (12–13° C) and warm (18–19° C) treated fish, with stress response (GO:0006950) and response to fungus (GO:0009620) elevated with warm treatment, while expression for genes involved in oxidative phosphorylation (GO:0006119) and electron transport chain (GO:0022900) elevated for cold‐treated fish. Analysis of single genes with real‐time quantitative PCR revealed that temperature had the most significant effect on mRNA expression levels, with swimming and population having secondary influences. Warm temperature treatment for the Chilko population induced expression of heat shock protein (hsp) 90α, hsp90β and hsp30 as well as interferon‐inducible protein. The Nechako population, which is known to have a narrower thermal tolerance window than the Chilko population, showed even more pronounced stress responses to the warm treatment and there was significant interaction between population and temperature treatment for hsp90β expression. Moreover, significant interactions were noted between temperature treatment and swimming challenge for hsp90α and hsp30, and while swimming challenge alone increased expression of these hsps, the expression levels were significantly elevated in warm‐treated fish swum to exhaustion. In conclusion, it seems that adult O. nerka currently encounter conditions that induce several cellular defence mechanisms during their once‐in‐the‐lifetime migration. As river temperatures continue to increase, it remains to be seen whether or not these cellular defences provide sufficient protection for all O. nerka populations.     

Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide

We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H₂O₂). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H₂O₂. We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H₂O₂ induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H₂O₂ induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.     

MicroRNA-210 alleviates oxidative stress-associated cardiomyocyte apoptosis by regulating BNIP3

Oxidative stress-induced myocardial apoptosis and necrosis are involved in ischemia/reperfusion (I/R) injury. This study was performed to investigate microRNA (miR)-210’s role in oxidative stress-related myocardial damage. The expression of miR-210 was upregulated in myocardial tissues of I/R rats, while that of Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3) was downregulated. To simulate in vivo oxidative stress, H9c2 cells were treated with H₂O₂ for 48 h. MiR-210 level was increased upon H₂O₂ stimulation, peaked at 8 h, and then decreased. An opposite expression pattern of BNIP3 was observed. BNIP3 was demonstrated as a direct target of miR-210 via luciferase reporter assay. H₂O₂-induced cell apoptosis was attenuated by miR-210 mimics, whereas aggravated by miR-210 inhibitor. MiR-210 knockdown-induced cell apoptosis in presence of H₂O₂ was attenuated by BNIP3 siRNA. Our work demonstrates that miR-210 plays a protective role in H₂O₂-induced cardiomyocyte apoptosis at least by regulating the pro-apoptotic BNIP3.     

Protective effect of vanillic acid in hydrogen peroxide-induced oxidative stress in D.Mel-2 cell line

The overproduction of reactive oxygen species (ROS) causes oxidative stress, such as Hydrogen peroxide (H₂O₂). Acute oxidative stress is one of the main reasons for cell death. In this study, the antioxidant properties of vanillic acid- a polyphenolic compound was evaluated. Therefore, this study aims to check the effectiveness of vanillic acid in H₂O₂-induced oxidative stress in D. Mel-2 cell line. The efficacy was determined by biochemical tests to check the ROS production. The cytotoxicity of H₂O₂ and vanillic acid was checked by MTT assay. The DNA fragmentation was visualized by gel electrophoresis. Protein biomarkers of oxidative stress were analyzed by western blotting. The results depict a promising antioxidant effect of vanillic acid. The IC₅₀ value of vanillic acid and H₂O₂ was found 250 μg/ml and 125 μg/ml, respectively. The catalase activity, SOF, GPx, and PC was seen less in H₂O₂ treated group compared with the control and vanillic acid treated group. However, the TBRAS activity was hight in H₂O₂ treated group. The effect of H₂O₂ on DNA fragmentation was high as compared with vanillic acid-treated cells. The protein expression of Hsp70, IL-6 and iNOS was seen significant in a vanillic acid-treated group as compared with H₂O₂ treated group. These results reinforce that at low concentration, vanillic acid could be used as an antioxidant agent in the food and pharmaceutical industries.