Inhibitory and enhancing effects of NO on H2O2 toxicity: Dependence on the concentrations of NO and H2O2

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H₂O₂) toxicity and protect cells against H₂O₂ toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H₂O₂ toxicity in cultured liver endothelial cells over a wide range of NO and H₂O₂ concentrations. NO was generated by spermine NONOate (SpNO, 0.001–1 mM), H₂O₂ was generated continuously by glucose/glucose oxidase (GOD, 20–300 U/l), or added as a bolus (200 μM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H₂O₂-induced cell death. SpNO concentrations >0.1 mM were injurious with low H₂O₂ concentrations, but protective at high H₂O₂ concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H₂O₂ steady-state levels in line with inhibition of H₂O₂ degradation. Thus, the overall effect of NO on H₂O₂ toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H₂O₂ levels and enhancement being predominant with high NO and low H₂O₂ levels.     

In vitro cytotoxicity of heavy metals,acrylamide, and organotin salts to neural cells and fibroblasts

The cytotoxicity of neurotoxic agents was determined for a series of brain-derived cell types and compared with their toxic effects on BALB/c 3T3 fibroblasts, using the neutral red assay. Ranking of toxicants according to their potencies was the same for all cells tested and was in the order of methylmercury>cadmium> mercury>zinc>acrylamide. For a series of di- and triorganotins the ranking order was dibutyl>diphenyl>dibenzyl>diproyl> diethyl>dimethyltin and triphenyl>tribenzyl>trimethyltin, respectively. The test was sensitive enough to detect structure activity relationships between the degree of toxicity and the hydrophobic characteristics of the agents tested.Abbreviations Be₂Sn dibenzyltin dichloride - Be₃Sn triphenyltin dichloride - Bu₂Sn dibutyltin dichloride - CdCl₂ cadmium chloride - CH₃HgCl methylmercuric chloride - DMEM Dulbecco's Modified Eagle's Medium - Et₂Sn diethyltin dichloride - FBS fetal bovine serum - HgCl₂ mercuric chloride - Me₂Sn dimethyltin dichloride - Me₃Sn trimethyltin hydroxide - Phe₂Sn diphenyltin dichloride - Phe₃Sn triphenyltin hydroxide - Pr₂Sn dipropyltin dichloride - ZnCl₂ zinc chloride     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.     

Effects and mechanisms of H(2)O(2) on production of dicarboxylic acid.

The system of producing long chain dicarboxylic acid (DCA) by Candida tropicalis is an aerobic and viscous fermentation system. A method to overcome the gas-liquid transport resistance and to increase oxygen supply is by adding hydrogen peroxide (H(2)O(2)) to the fermentation system. Here we report that the H(2)O(2) not only can enhance the oxygen supply but also change the metabolism by inducing cytochrome P450, the key enzyme of a, o-oxidation. When C. tropicalis was cultivated in a 3-L bioreactor using the combination of aeration and H(2)O(2) feeding, DCA production rates increased by about 10% after a short period of decrease at the beginning. Furthermore, the experiments showed that the maximum activities of P450 could be induced at 2 mM H(2)O(2), and the inducible mechanisms are also discussed. Moreover, we suggest that alkane might be oxidized through the "peroxide shunt pathway" when H(2)O(2) is present. By adding H(2)O(2), the DCA yield in a 22-L bioreactor could increase by 25.3% and reach 153.9 g/L.     

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H₂O₂.Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H₂O₂ and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H₂O₂. Analysis of different hallmarks of programmed cell death was also carried out.Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H₂O₂, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H₂O₂ and nitric oxide contents and maintenance of the glutathione redox state were observed.Conclusions A decreased content of endogenous H₂O₂ may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H₂O₂, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.     

Growth of Rhodopseudomonas palustris under phototrophic and non-phototrophic conditions and its CO-dependent H2 production

A new hydrogen producing bacterium, Rhodopseudomonas palustris P4, originally isolated under an anaerobic/phototrophic condition, grew well under aerobic/chemoheterotrophic or anaerobic/chemoheterotrophic conditions and showed CO-dependent, H₂ production activity when transferred to anaerobic conditions. Cell growth was best under an aerobic/chemoheterotrophic condition as the doubling time of 1 h, while the H₂ production activity was highest in the cells grown under an aerobic/chemoheterotrophic condition at 20 mmol g^–1 cell^–1 h^–1.     

Activation of ERK signalling by Src family kinases (SFKs) in DRG neurons contributes to hydrogen peroxide (H2O2)-induced thermal hyperalgesia

Concomitant generation of reactive oxygen species during tissue inflammation has been recognised as a major factor for the development and the maintenance of hyperalgesia, out of which H₂O₂ is the major player. However, molecular mechanism of H₂O₂ induced hyperalgesia is still obscure. The aim of present study is to analyse the mechanism of H₂O₂-induced hyperalgesia in rats. Intraplantar injection of H₂O₂ (5, 10 and 20 µmoles/paw) induced a significant thermal hyperalgesia in the hind paw, confirmed by increased c-Fos activity in dorsal horn of spinal cord. Onset of hyperalgesia was prior to development of oxidative stress and inflammation. Rapid increase in phosphorylation of extracellular signal regulated kinase (ERK) was observed in neurons of dorsal root ganglia after 20?min of H₂O₂ (10 µmoles/paw) administration, which gradually returned towards normal level within 24?h, following the pattern of thermal hyperalgesia. The expression of TNFR1 followed the same pattern and colocalised with pERK. ERK phosphorylation was observed in NF-200-positive and -negative neurons, indicating the involvement of ERK in C-fibres as well as in A-fibres. Intrathecal preadministration of Src family kinases (SFKs) inhibitor (PP1) and MEK inhibitor (PD98059) prevented H₂O₂ induced augmentation of ERK phosphorylation and thermal hyperalgesia. Pretreatment of protein tyrosine phosphatases (PTPs) inhibitor (sodium orthovanadate) also diminished hyperalgesia, although it further increased ERK phosphorylation. Combination of orthovanadate with PP1 or PD98059 did not exhibit synergistic antihyperalgesic effect. The results demonstrate SFKs-mediated ERK activation and increased TNFR1 expression in nociceptive neurons during H₂O₂ induced hyperalgesia. However, the role of PTPs in hyperalgesic behaviour needs further molecular analysis.     

Rapid detection of cytotoxicity of food additives and contaminants by a novel cytotoxicity test, menadione-catalyzed H2O2 production assay

Menadione-catalyzed H₂O₂ production by viable animal cells was proportional to the viable cell number, and H₂O₂ production decreased with increasing cytotoxic effects after the incubation of cells with cytotoxic compounds. The cytotoxic effects of food additives, pesticides, antibiotics, heavy metals, phytotoxins, mycotoxins, and marine toxins were estimated using the above test employingNIH/3T3 and Neuro-2a cells. Synergistic effects of the toxin mixture were observed and acute cytotoxicity detected 1 h after the incubation of cells with toxins. This menadione-catalyzed H₂O₂production assay is rapid and simple compared to other popular cytotoxicity tests such as the MTT reduction assay and Neutral red inclusion test, requiring4 h. The menadione-catalyzed H₂O₂ production assay is expected to be a useful food safety test for rapidly detecting toxic compounds having a basic cytotoxic effect on common animal cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Platinum(II) and palladium(II) complexes with 2-Acetyl pyridine 4N-ethyl thiosemicarbazone able to overcome the cis-Platin resistance. Structure, antibacterial activity and DNA strand breakage

The reactions of Pd(II) and Pt(II) with 2-Acetyl Pyridine N(4)-Ethyl-Thiosemicarbazones, HAc4Et and 2-Acetyl Pyridine N(4)-1-(2-pyridyl)-piperazinyl Thiosemicarbazone, HAc4PiPiz and 2-Formyl Pyridine N(4)-1-(2-pyridyl)-piperazinyl Thiosemicarbazone, HFo4PiPiz afforded the complexes, [Pd(Ac4Et)], 1, [Pd(HAc4Et)₂]Cl₂, 2 and [Pd(Ac4Et)₂], 3[Pt(Ac4Et)], 4, [Pt(HAc4Et)₂]Cl₂, 5, [Pt(Ac4Et)₂], 6 and [Pd(Fo4PipePiz)Cl], 7, [Pd(Fo4PipePiz)₂], 8, [Pd(Ac4PipePiz)Cl], 9 and [Pd(Ac4PipePiz)₂], 10. The crystal structure of the complex [Pt(Ac4Et)₂], 6 has been solved. The platinum(II) atom is in a square planar environment surrounded by two cis nitrogen atoms and two cis sulfur atoms. The ligands are not equivalent, one being tridentate with (N,N,S) donation, the other being monodentate using only the sulfur atom to coordinate to the metal. The tridentate ligand shows a Z, E, Z configuration while the monodentate ligand shows an E, E, Z. Inter-molecular hydrogen bonds stabilize the structure, while the crystal packing is determined by –, and Pt – C interactions. The antibacterial effect of Pd(II) and Pt(II) complexes were studied in vitro. The complexes were found to have effect on Gram(+) bacteria, while the same complexes showed no bactericidal effect on Gram(–) bacteria. The effect of the Pd(II) and Pt(II) complexes on the in vitro DNA strand breakage was studied by agarose gel electrophoresis. The complexes 1-6 were found to exhibit a cytotoxic potency in a very low micromolar range and to be able to overcome the cisplatin resistance of A2780/Cp8 cells (Kovala-Demertzi et al. 2000).     

Eriodictyol protects against H(2)O(2)-induced neuron-like PC12 cell death through activation of Nrf2/ARE signaling pathway

Eriodictyol, a flavonoid isolated from the Chinese herb Dracocephalum rupestre has long been established as an antioxidant. The present study was designed to explore the protective effects of eriodictyol against hydrogen peroxide (H(2)O(2))-induced neurotoxicity with cultured rat pheochromocytoma cells (PC12 cells) and the possible mechanisms involved. For this purpose, differentiated PC12 cells were cultured and exposed to 200 μM H(2)O(2) in the absence or presence of eriodictyol (20, 40 and 80 μM). In addition, the potential contribution of the Nrf2/ARE neuroprotective pathway in eriodictyol-mediated protection against H(2)O(2)-induced neurotoxicity was also investigated. The results showed that H(2)O(2)-induced cell death can be inhibited in the presence of eriodictyol as measured by assays for MTT and apoptosis. Further study revealed that eriodictyol induced the nuclear translocation of Nrf2, enhanced the expression of heme oxygenase (HO-1) and γ-glutamylcysteine synthetase (γ-GCS), and increased the levels of intracellular glutathione. Treatment of PC12 cells with Nrf2 small interference RNA abolished eriodictyol-induced HO-1 and γ-GCS expression and its protective effects. In conclusion, these results suggest that eriodictyol upregulates HO-1 and γ-GCS expression through the activation of Nrf2/ARE pathway and protects PC12 cells against H(2)O(2)-induced oxidative stress.     

JNK1 activity lowers the cellular production of H2O2 and modulates the growth arrest response to scavenging of H2O2 by catalase

Hydrogen peroxide (H(2)O(2)) can interact with intracellular signaling pathways to regulate cell behavior. The c-Jun NH(2)-terminal kinase 1 (JNK1) signal, involved in diverse aspects of cellular functioning, is implicated as a cell sensor of redox stress. The growth-inhibitory effect of both high-level H(2)O(2) and H(2)O(2)-scavenging catalase treatments is accompanied by increased JNK1 activity. To investigate the role of this response in growth regulation, the JNK1 signal was increased by the introduction of ectopic HA-JNK1. HA-JNK1 expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase; however, with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H(2)O(2). Transient HA-JNK1 expression also reduced H(2)O(2) generation, and this effect was reversed by the JNK inhibitor SP600125. These results indicate that the JNK1 stress response contributes to growth inhibition by catalase treatment via inhibition of cellular H(2)O(2) production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon H(2)O(2) for efficient growth.     

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl^-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 M was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 m) using an IBA powder dip (3 g IBA kg^-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige and Skoog medium     

Changes in the content of glycogen and its metabolites during acute exposure ofAnabas testudineus (Bloch) to furadan

Significant differences were observed in glycogen metabolism ofAnabas testudineus exposed to an acute lethal (1.56 mg/litre) and a sublethal (0.56 mg/litre) concentration of furadan. At sublethal concentration, the muscle glycogen which was utilized during the early periods of exposure, was replenished in the later period of exposure and at 120 h, the muscle glycogen levels were higher than the control. At higher concentration, the liver glycogen levels showed an increase presumably at the expense of fuel reserves of the muscle.     

Effect of chronic exposure to cadmium and copper on Asellus aquaticus (L.) (Crustacea,Isopoda)

The effects of chronic exposure to 5 g·1^–1 cadmium or copper on the crustacean Isopod Asellus aquaticus (L.) were studied by analyzing survival and body growth in the first stages of the life-cycle and by determining fecundity and survival of embryo-bearing females. Juveniles survival is differently affected by the two metals in that embryonic development is more sensitive to cadmium while juvenile development is more sensitive to copper. Juvenile body growth is stimulated by cadmium and depressed by copper. Embryo-bearing female survival and fecundity are significantly reduced by cadmium but are not affected by copper. The consequence of environmental contamination by a sublethal cadmium or copper concentration is discussed.     

S. choloroleuca, S. mirzayanii and S. santolinifolia protect PC12 cells from H(2)O (2)-induced apoptosis by blocking the intrinsic pathway

Several studies have shown that neuronal cell death due to apoptosis is the major reason for cognitive decline in Alzheimer's disease. In this study, we report the anti-apoptotic effects of three Salvia species from Iran-S. choloroleuca, S. mirzayanii and S. santolinifolia-against H(2)O(2)-induced cytotoxicity in neuron-like PC12 cells. We showed that these antioxidant species could interfere with the intrinsic pathway of apoptosis by attenuating Bax/Bcl-2 ratio, decreasing outer mitochondrial membrane break and decreasing cytochrome c release to cytoplasm. Interestingly, we found that these species were able to replenish reduced glutathione level which affects cellular redox status and cytochrome c activity. Moreover, the decreased level of caspase-3, the executioner caspase, resulted in decrease of PARP-1 cleavage. Anti-apoptotic effects of these species along with their antioxidant effects, may represent a promising approach for treatment of neurodegenerative diseases.     

香蕉组织培养过程中内生菌污染的控制

研究了体外培养一种孟加拉传统香蕉（Musa spp. Cv. Kanthali）的茎尖组织。茎尖的原始细胞表面经无菌处理（0.1% HgCl₂处理12min）,接种6～15d后外植体地下茎部分仍有微生物污染（大部分是细菌）,杀死了85％的外植体。为确定无污染培养基,将等量外植体分别浸泡在含400mg/L氨苄青霉素和200mg/L庆大霉素（两种光谱抗生素）的培养基中1h。结果表明,经抗生素处理的外植体完全没有污染,但培养3周后不能再生。进行二次继代培养后,其中一部分外植体吸收了培养基并胀大,颜色由苍白转变成浅绿或深绿。三次继代培养后数天,不再观察到外植体的生长,所有经抗生素处理过的外植体都开始死亡。在未经抗生素处理的活外植体中,单个茎发育的最佳培养基是：MS+4.0mg/L BA+0.5mg/L KT+15% CW,平均生长时间为18～21d,但再生率很低,只有30％。茎细胞增殖的最佳培养基是：MS+4.0mg/L BA+2.0mg/L IAA+15% CW,每个茎平均只萌发3～4个芽。最后,在添加0.5mg/L IBA的一半浓度的MS培养基中,体外培养茎最大生根率达到90％。