The protection by heme oxygenase‐1 induction against thioacetamide‐induced liver toxicity is associated with changes in arginine and asymmetric dimethylarginine

This study was designed to investigate the role of HO‐1 induction in prevention of thioacetamide (TAA)‐induced oxidative stress, inflammation and liver damage. The changes in hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity as well as plasma arginine and asymmetric dimethylarginine (ADMA) levels were also measured to evaluate nitric oxide (NO) bioavailability. Rats were divided into four groups as control, hemin, TAA and hemin + TAA groups. Hemin (50 mg kg^?1, i.p.) was injected to rats 18 h before TAA treatment to induce HO‐1 enzyme expression. Rats were given TAA (300 mg kg^?1, i.p.) and killed 24 h after treatment. Although TAA treatment produced severe hepatic injury, upregulation of HO‐1 ameliorated TAA‐induced liver damage up to some extent as evidence by decreased serum alanine transaminase, aspartate transaminase and arginase activities and histopathological findings. Induction of HO‐1 stimulated antioxidant system and decreased lipid peroxidation in TAA‐treated rats. Myeloperoxidase activity and inducible NO synthase protein expression were decreased, whereas DDAH activity was increased by hemin injection in TAA‐treated rats. Induction of HO‐1 was associated with increased arginine levels and decreased ADMA levels, being the main determinants of NO production, in plasma of TAA‐treated rats. In conclusion, our results indicate that HO‐1 induction alleviated increased oxidative stress and inflammatory reactions together with deterioration in NO production in TAA‐induced liver damage in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Upregulation of heme oxygenase‐1 inhibits the maturation and mineralization of osteoblasts

Heme‐oxygenase‐1 (HO‐1), an important enzyme involved in vascular disease, transplantation, and inflammation, catalyzes the degradation of heme into carbon monoxide and biliverdin. It has been reported that overexpression of HO‐1 inhibits osteoclastogenesis. However, the effect of HO‐1 on osteoblast differentiation is still not clear. We here used adenoviral vector expressing recombinant human HO‐1 and HO‐1 inducer hemin to study the effects of HO‐1 in primary cultured osteoblasts. The results showed that induction of HO‐1 inhibited the maturation of osteoblasts including mineralized bone nodule formation, alkaline phosphatase activity and decreased mRNA expression of several differentiation markers such as alkaline phosphatase, osteocalcin, and RUNX2. Furthermore, downstream products of HO‐1, bilirubin, carbon monoxide, and iron, are involved in the inhibitory action of HO‐1. HO‐1 can be induced by H₂O₂, lipopolysaccharide and inflammatory cytokines such as TNF‐α and IL‐1β in osteoblasts and also in STZ‐induced diabetic mice. In addition, endogenous PPARγ ligand, 15‐deoxy‐Δ^12,14‐prostaglandin‐J2 (15d‐PGJ2) markedly increased both mRNA and protein levels of HO‐1 in osteoblasts via PI3K‐Akt and MAPK pathways. Blockade of HO activity by ZnPP IX antagonized the inhibitory action on osteocalcin expression by hemin and 15d‐PGJ2. Our results indicate that upregulation of HO‐1 inhibits the maturation of osteoblasts and HO‐1 may be involved in oxidative‐ or inflammation‐induced bone loss. J. Cell. Physiol. 222: 757–768, 2010. © 2009 Wiley‐Liss, Inc.     

Implications of paraquat and hydrogen peroxide-induced oxidative stress treatments on the GABA shunt pathway in Arabidopsis thaliana calmodulin mutants

Arabidopsis mutants with T-DNA insertion in seven calmodulin genes (CAM) were used to determine the specific role of CAM in the tolerance of plants to oxidative stress induced by paraquat and hydrogen peroxide (H₂O₂) treatments. Arabidopsis calmodulin mutants (cam) were screened for seedling growth, seed germination, induced oxidative damage, and levels of γ-aminobutyric acid (GABA) shunt metabolites. Only the cam5-4 and cam6-1 mutants exhibited an increased sensitivity to paraquat and H₂O₂ during seed germination and seedling growth. In response to treatments with 3 μM paraquat and 1 mM H₂O₂, only the cam5-4, cam6-1 mutants showed significant changes in malonaldehyde (MDA) levels in root and shoot tissues, with highly increased levels of MDA. In terms of the GABA shunt metabolites, GABA was significantly elevated in root and shoot tissues in response to the paraquat treatments in comparison to alanine and glutamate, while the levels of all shunt metabolites increased in root tissue but not in the shoot tissue following the H₂O₂ treatments. GABA, alanine and glutamate levels were significantly increased in root and shoot of the cam1, cam4, cam5-4, and cam6-1 mutants in response to paraquat (0.5, 1 and 3 μM), while they were increased only in the root tissue of the cam1, cam4, cam5-4, and cam6-1 mutants in response to H₂O₂ (200 and 500 μM, 1 mM). These data show that the cam5-4 and cam6-1 mutants were sensitive to the induced oxidative stress treatments in terms of seed germination, seedling growth, and oxidative damage. The accumulation of GABA shunt metabolites as a consequence of the induced oxidative stress treatments (paraquat and H₂O₂ treatments) suggests that the GABA shunt pathway and the accumulation of GABA metabolites may contribute in antioxidant machinery associated with reactive oxygen species and in the acquisition of tolerance in response to induced oxidative stress in Arabidopsis seedlings.     

Terrein reduces age‐related inflammation induced by oxidative stress through Nrf2/ERK1/2/HO‐1 signalling in aged HDF cells

This study investigated whether multiple bioactivity of terrein such as anti‐inflammatory and anti‐oxidant inhibits age‐related inflammation by promoting an antioxidant response in aged human diploid fibroblast (HDF) cells. HDF cells were cultured serially for in vitro replicative senescence. To create the ageing cell phenotype, intermediate stage (PD31) HDF cells were brought to stress‐induced premature senescence (SIPS) using hydrogen peroxide (H₂O₂). Terrein increased cell viability even with H₂O₂ stress and reduced inflammatory molecules such as intracellular adhesion molecule‐1 (ICAM‐1), cyclooxygenase‐2 (COX‐2), interleukin‐1beta (IL‐1β) and tumour necrosis factor‐alpha (TNF‐α). Terrein reduced also phospho‐extracellular kinase receptor1/2 (p‐EKR1/2) signalling in aged HDF cells. SIPS cells were attenuated for age‐related biological markers including reactive oxygen species (ROS), senescence associated beta‐galactosidase (SA β‐gal.) and the aforementioned inflammatory molecules. Terrein induced the induction of anti‐oxidant molecules, copper/zinc‐superoxide defence (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and heme oxygenase‐1 (HO‐1) in SIPS cells. Terrein also alleviated reactive oxygen species formation through the Nrf2/HO‐1/p‐ERK1/2 pathway in aged cells. The results indicate that terrein has an alleviative function of age‐related inflammation characterized as an anti‐oxidant. Terrein might be a useful nutraceutical compound for anti‐ageing. Copyright © 2015 John Wiley & Sons, Ltd.     

l‐cysteine desulfhydrase‐related H2S production is involved in OsSE5‐promoted ammonium tolerance in roots of Oryza sativa

Previous studies revealed that rice heme oxygenase PHOTOPERIOD SENSITIVITY 5 (OsSE5) is involved in the regulation of tolerance to excess ammonium by enhancing antioxidant defence. In this study, the relationship between OsSE5 and hydrogen sulfide (H₂S), a well‐known signalling molecule, was investigated. Results showed that NH₄Cl triggered the induction of l ‐cysteine desulfhydrase (l ‐DES)‐related H₂S production in rice seedling roots. A H₂S donor not only alleviated the excess ammonium‐triggered inhibition of root growth but also reduced endogenous ammonium, both of which were aggravated by hypotaurine (HT, a H₂S scavenger) or dl ‐propargylglycine (PAG, a l ‐DES inhibitor). Nitrogen metabolism‐related enzymes were activated by H₂S, thus resulting in the induction of amino acid synthesis and total nitrogen content. Interestingly, the activity of l ‐DES, as well as the enzymes involved in nitrogen metabolism, was significantly increased in the OsSE5‐overexpression line (35S:OsSE5), whereas it impaired in the OsSE5‐knockdown mutant (OsSE5‐RNAi). The application of the HT/PAG or H₂S donor could differentially block or rescue NH₄Cl‐hyposensitivity or hypersensitivity phenotypes in 35S:OsSE5‐1 or OsSE5‐RNAi‐1 plants, with a concomitant modulation of nitrogen assimilation. Taken together, these results illustrated that H₂S function as an indispensable positive regulator participated in OsSE5‐promoted ammonium tolerance, in which nitrogen metabolism was facilitated.     

Effects of salicylic acid on paraquat tolerance in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Foliar spraying ofArabidopsis thaliana (Columbia ecotype) plants with a 1.0-mM salicylic acid (SA) solution significantly improved their tolerance to subsequent paraquat (PQ)-induced oxidative damage. Leaf injuries, including losses of chlorophyll, protein, and fresh weight, were reduced. Our analysis of antioxidant enzymes in the leaves showed that SA pre-treatment effectively retarded rapid decreases in the activities of Superoxide dismutase (SOD), catalase, and ascorbate peroxidase that are normally associated with PQ exposure. In addition, guaiacol peroxidase activity was remarkably increased. In a native gel assay of peroxidase (POD) isozymes, staining activity of the POD1 isozyme, which disappeared in plants exposed only to 10 μM PQ, was significantly recovered by the 1.0-mM SA pre-treatment POD2 isozyme activity was also pronounced in all SA-treated plants compared with the control. A 12-h SA pre-treatment, without subsequent PQ stress, also caused a small increase in the endogenous H₂O₂ content that accompanies the symptoms of mild leaf injuries. This enhanced level occurred in parallel with a slight SOD increase and a catalase decrease. From our results, it can be assumed that, due to the small increase in SOD as well as catalase inactivation via SA pre-treatment, a moderate increase in H₂O₂ levels may occur. In turn, a large induction of guaiacol peroxidase leads to enhanced PQ tolerance inA. thaliana plants.     

Tat‐antioxidant 1 protects against stress‐induced hippocampal HT‐22 cells death and attenuate ischaemic insult in animal model

Oxidative stress‐induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat‐Atox1 and examined the roles of Tat‐Atox1 in oxidative stress‐induced hippocampal HT‐22 cell death and an ischaemic injury animal model. Tat‐Atox1 effectively transduced into HT‐22 cells and it protected cells against the effects of hydrogen peroxide (H₂O₂)‐induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat‐Atox1 regulated cellular survival signalling such as p53, Bad/Bcl‐2, Akt and mitogen‐activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat‐Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat‐Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat‐Atox1 protects against oxidative stress‐induced HT‐22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat‐Atox1 has potential as a therapeutic agent for the treatment of oxidative stress‐induced ischaemic damage.     

Hydrogen peroxide produced by NADPH oxidase: a novel downstream signaling pathway in melatonin‐induced stress tolerance in Solanum lycopersicum

The beneficial effects of melatonin on abiotic stress have been demonstrated in several plants. However, little is known about the signal transduction pathway of melatonin involved in the plant stress response. Here, we manipulated the melatonin levels in tomato plants through a chemical approach. The roles of melatonin in stress tolerance were studied by assessing the symptoms, chlorophyll fluorescence and stress‐responsive gene expression. Moreover, both chemical and genetic approaches were used to study the roles of hydrogen peroxide (H₂O₂) in melatonin‐induced signal transduction in tomato plants. We found that melatonin activates NADPH oxidase (RBOH) to enhance H₂O₂ levels by reducing its S‐nitrosylation activity. Furthermore, melatonin‐induced H₂O₂ accumulation was accompanied by obtainable stress tolerance. Inhibition of RBOH or chemical scavenging of H₂O₂ significantly reduced the melatonin‐induced defense response, including reduced expression of several stress‐related genes (CDPK1, MAPK1, TSPMS, ERF4, HSP80 and ERD15) and reduced antioxidative enzyme activity (SOD, CAT and APX), which were responsible for the stress tolerance. Collectively, these results revealed a novel mechanism in which RBOH activity and H₂O₂ signaling are important components of the melatonin‐induced stress tolerance in tomato plants.     

Overexpression of a peroxiredoxin gene from <Emphasis Type="Italic">Tamarix hispida</Emphasis>, <Emphasis Type="Italic">ThPrx1</Emphasis>, confers tolerance to oxidative stress in yeast and Arabidopsis

Peroxiredoxins (Prxs) are ubiquitous thiol-specific antioxidant enzymes that are critically involved in cell defense and protect cells from oxidative damage. In this study, a putative Type II Prx (ThPrx1) was identified and characterized from Tamarix hispida. The expression of ThPrx1 is highly induced in response to hydrogen peroxide (H₂O₂) and methyl viologen (MV) stresses. When expressed ectopically, ThPrx1 showed enhanced tolerance against oxidative stress in yeast and Arabidopsis. In addition, transgenic Arabidopsis plants overexpressing ThPrx1 displayed improved seedling survival rates and increased root growth and fresh weight gain under H₂O₂ and MV treatments. Moreover, transgenic Arabidopsis plants showed decreased accumulation of H₂O₂, superoxide (O₂^??) and malondialdehyde (MDA), increased superoxide dismutase (SOD) activity compared to wild-type (WT) plants under oxidative stress. Moreover, transgenic plants maintained higher photosynthesis efficiency and lower electrolyte leakage rates than that of WT plants under stress conditions. These results clearly indicated that ThPrx1 plays an important role in cellular redox homeostasis under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress.     

Effect of elevated CO2, temperature and limited water supply on antioxidant status during regrowth of nodulated alfalfa

Atmospheric CO₂ is a major contributor to the greenhouse effect and is one of the main inducers of climate change. Previous studies with nodulated alfalfa plants have shown that elevated CO₂ increased the growth of plants grown under well‐watered or limited water supply conditions. The beneficial effects of atmospheric CO₂ enrichment included higher photosynthetic rates, growth and water‐use efficiency and an increase in the root/shoot ratio. However, at the moment, we do not have information on the possible implications of the beneficial effect of elevated CO₂ as it may relate to a higher capacity of the violaxanthin–antheraxanthin–zeaxanthin (VAZ) cycle, the dissipation of excess radiation as heat and the effect on photooxidation, and to an improved leaf antioxidant system (Halliwell–Asada cycle). The aim of the present study was to determine the effects of the interaction between CO₂ (ambient, around 350 vs 700 μmol mol⁻¹), temperature (ambient vs ambient + 4°C) and water availability (well irrigated vs partially irrigated) on the leaf antioxidant status of nodulated alfalfa during regrowth. Parameters measured in this study included relative growth rate (RGR), H₂O₂ content, oxidative damage [measured as thiobarbituric acid‐reacting substances (TBARS)], leaf pigment composition (chlorophylls and xanthophylls), ascorbate (ASA) and glutathione pool levels and antioxidant enzymes. Our results revealed that during alfalfa regrowth, the effects of elevated CO₂, limited water supply, temperature and their interactions on growth were not related to significant or general changes in leaf antioxidant capacity, H₂O₂ accumulation or oxidative stress (TBARS concentrations). The beneficial effects of CO₂ enrichment in well‐watered and limited water‐subjected plants were not associated with an increase in the capacity of alfalfa leaves to dissipate excess radiation as heat through the VAZ cycle or with an increase in the antioxidant capacity, measured in terms of Halliwell–Asada cycle enzymes and antioxidant compounds. Furthermore, elevated CO₂ did not affect RGRs during the last 15 days of regrowth and reduced the activity of several antioxidant enzymes (catalase, superoxide dismutase and glutathione reductase and ASA peroxidase in limited water‐subjected plants), suggesting a lower basal rate of oxygen activation and H₂O₂ formation, leading to a relaxation of the antioxidant system.     

Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance

The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens‐mediated transformation. Catalase activity in progeny from transgenic plants was approximately three‐fold higher than that in wild‐type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µm paraquat under moderate light intensity, whereas leaf discs from wild‐type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild‐type leaf discs at 24 h after treatment with 1 µm paraquat and 10 mm H₂O₂, respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wild‐type plants were sprayed with 100 µm paraquat and exposed to high light illumination (800 µmol m^?2 s^?1). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild‐type plants. The catalase activity and the photosynthesis activity (indicated by the F_v/F_m ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO₂ fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 °C) under high light intensity (1000 µmol m^?2 s^?1). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photo‐oxidative stress invoked by paraquat treatment, drought stress and chilling stress.     

Auxin and its transport play a role in plant tolerance to arsenite‐induced oxidative stress in Arabidopsis thaliana

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)‐containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole‐3‐acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H³‐IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H₂O₂ in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)‐mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high‐temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high‐temperature stress.