Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Evidence for the presence and activity of a complete antioxidant defence system in mature sieve tubes

The phloem is the major route for the transport of solutes and nutrients from source to sink organs in plants. The functional transport phloem consists of parenchymal tissue, enucleate sieve elements, and the intimately connected companion cells. The general absence of a nucleus and functional ribosomes in sieve tubes poses problems especially for damage avoidance and repair of sieve element components. To examine how sieve tubes can remain functional during oxidative stress, we analysed phloem sap of cucumber and pumpkin plants with respect to the presence of antioxidant defence enzymes, their enzymatic activity, and activity changes after exposure to drought stress. Using 1D SDS-PAGE and nano ESI MS/MS, the presence of proteins such as cytosolic Cu/Zn superoxide dismutase, monodehydroascorbate reductase, and peroxidase could be shown. Moreover, activities for several antioxidant enzymes (superoxide dismutase, dehydroascorbate reductase, peroxidase) in phloem exudate could be demonstrated. The activity of these enzymes in phloem sap from cucumber and pumpkin plants increased in response to drought stress. The presented results together with earlier findings provide evidence supporting the presence of a complete machinery of antioxidant defence enzymes and detoxifying metabolites important for avoiding damage to essential components of the sieve elements due to oxidative stress.     

RAS and connective tissue in the heart

Circulating angiotensin (Ang) II has well-known endocrine properties in the cardiovasculature. AngII, produced de novo within the heart, has various autocrine and paracrine properties on resident cells expressed via AT(1) receptor-ligand binding. Herein, we review the heart's renin-angiotensin system and its role in connective tissue turnover involving heart valve leaflets and fibrous tissue that appears at sites of injury, such as following myocardial infarction.     

Effects of angiotensin II type 1 receptor blockade on the oxidative stress in spontaneously hypertensive rat tissues

The aim of this work was to investigate the production of oxidative damage in homogenized kidney, liver and brain of spontaneously hypertensive rats (SHR), as well as the involvement of angiotensin (Ang) II in this process. Groups of 12-week-old SHR and Wistar Kyoto rats (WKY) were given 10 mg/kg/day losartan in the drinking water during 14 days. Other groups of WKY and SHR without treatment were used as controls. The production of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (Gpx) were determined. No significant difference in TBARS was observed between untreated SHR or WKY rats; GSH content was lower in the liver but higher in the brain of SHR compared to WKY rats. In tissues from the SHR group, SOD and Gpx activities were reduced, whereas CAT activity was slightly increased in kidney. TBARS levels did not change in WKY rats after losartan administration, but were reduced in SHR liver and brain. Losartan treatment decreased GSH content in WKY kidney, but increased GSH in SHR liver. The activity of the antioxidant enzymes was not modified by losartan in WKY rats; however, their activities increased in tissues from treated SHR. The lower activity of antioxidant enzymes in tissues from hypertensive rats compared to those detected in normotensive controls, indicates oxidative stress production. Ang II seems to play no role in this process in normotensive animals, although AT1 receptor blockade in SHR enhances the enzymatic activity indicating that Ang II is implicated in oxidative stress generation in the hypertensive animals.     

Functional relevance of Gedunin as a bona fide ligand of NADPH oxidase 5 and ROS scavenger: An in silico and in vitro assessment in a hyperglycemic RBC model

Clinical evidence suggests that type 2 diabetes therapy can greatly benefit from the suppression of reactive oxygen species generation and the activation or restoration of cellular antioxidant mechanisms. In human, NADPH oxidase (NOX) is the main producer of reactive oxygen species (ROS) that supress the activity of endogenous antioxidant enzymes. In the present study, the antioxidant potential of Gedunin was studied. In silico findings reveal its strong binding affinity with NOX5 C terminal HSP90 binding site that disrupts NOX5 stability and its ability to generate ROS, leading to restoration antioxidant enzymes activities. It was found that Gedunin suppressed hyperglycaemia induced oxidative stress in an in vitro RBC model and markedly reversed glucose induced changes including haemoglobin glycosylation and lipid peroxidation. A significant restoration of activities of cellular antioxidant enzymes; superoxide dismutase, catalase and glutathione peroxidase in the presence of Gedunin revealed its ability to reduce oxidative stress. These results substantiated Gedunin as a bona fide inhibitor of human NOX5 and a ROS scavenging antioxidant with promising therapeutic attributes including its natural origin and inhibition of multiple diabetic targets.     

Angiotensin II downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway

Angiotensin II (Ang II) plays a profound regulatory effect on NADPH oxidase and the functional features of vascular adventitial fibroblasts, but its role in antioxidant enzyme defense remains unclear. This study investigated the effect of Ang II on expressions and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in adventitial fibroblasts and the possible mechanism involved. Ang II decreased the expression and activity of CAT in a dose- and time-dependent manner, but not that of SOD and GPx. The effects were abolished by the angiotensin II type 1 receptor (AT1R) blocker losartan and AT1R small-interfering RNA (siRNA). Incubation with polyethylene glycol-CAT prevented the Ang II-induced effects on reactive oxygen species (ROS) generation and myofibroblast differentiation. Moreover, Ang II rapidly induced phosphorylation of ERK1/2, which was reversed by losartan and AT1R siRNA. Pharmacological blockade of ERK1/2 improved Ang II-induced decrease in CAT protein expression. These in vitro results indicate that Ang II induces ERK1/2 activation, contributing to the downregulation of CAT as well as promoting oxidative stress and adventitial fibroblast phenotypic differentiation in an AT1R-mediated manner.     

Angiotensin II and extracellular matrix homeostasis

As a circulating hormone, endocrine properties of angiotensin (Ang) II are integral to circulatory homeostasis. Produced de novo its autocrine/paracrine properties contribute to biologic responses involving various connective tissues (e.g. extracellular matrix, adipose tissue, bone and its marrow). In this brief review, we develop the concept of extracellular matrix homeostasis, a self regulation of cellular composition and structure, wherein fibroblast-derived AngII regulates elaboration of TGF-beta 1, a fibrogenic cytokine responsible for connective tissue formation at normal and pathologic sites of collagen turnover.     

Heme oxygenase-1 expression levels are cell cycle dependent

Heme oxygenase-1 (HO-1) is a stress protein, which has been suggested to participate in defense mechanisms against agents that may induce oxidative injury, such as angiotensin II (Ang II). The purpose of the present study was to examine the role of human HO-1 in cell-cycle progression. We investigated the effect of Ang II on HO-1 gene expression in serum-deprived media to drive human endothelial cells into G(0)/G(1) (1% FBS) compared to exponentially grown cells (10% FBS). The addition of Ang II (100 ng/ml) to endothelial cells increased HO-1 protein and activity in G(0)/G(1) in a time-dependent manner, reaching a maximum HO-1 level at 16 h. Real-time RT-PCR demonstrated that Ang II increased the levels of HO-1 mRNA in G(0)/G(1) as early as 1 h. The rate of HO-1 induction in response to Ang II was several-fold higher in serum-starved cells compared to cells cultured in continuous 10% FBS. The addition of Ang II increased the generation of 8-epi-isoprostane PGF(2 alpha). Inhibition of HO-1, by Stannis mesoporphyrin (SnMP), potentiated Ang II-mediated DNA damage and generation of 8-epi-isoprostane PGF(2 alpha). These results imply that expression of HO-1 in G(0)/G(1), in the presence of Ang II, may be a key player in attenuating DNA damage during cell-cycle progression. Thus, exposure of endothelial cells to Ang II causes a complex response involving generation of superoxide anion, which may be involved in DNA damage. Upregulation of HO-1 ensures the generation of bilirubin and carbon monoxide (CO) in G(0)/G(1) phase to counteract Ang II-mediated oxidative DNA damage. Inducibility of HO-1 in G(0)/G(1) phase is essential and probably regulated by a complex system involving oxygen species to assure controlled cell growth.     

Mas receptor mediates cardioprotection of angiotensin‐(1‐7) against Angiotensin II‐induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress

Angiotensin II (Ang II) plays an important role in the onset and development of cardiac remodelling associated with changes of autophagy. Angiotensin1‐7 [Ang‐(1‐7)] is a newly established bioactive peptide of renin–angiotensin system, which has been shown to counteract the deleterious effects of Ang II. However, the precise impact of Ang‐(1‐7) on Ang II‐induced cardiomyocyte autophagy remained essentially elusive. The aim of the present study was to examine if Ang‐(1‐7) inhibits Ang II‐induced autophagy and the underlying mechanism involved. Cultured neonatal rat cardiomyocytes were exposed to Ang II for 48 hrs while mice were infused with Ang II for 4 weeks to induce models of cardiac hypertrophy in vitro and in vivo. LC3b‐II and p62, markers of autophagy, expression were significantly elevated in cardiomyocytes, suggesting the presence of autophagy accompanying cardiac hypertrophy in response to Ang II treatment. Besides, Ang II induced oxidative stress, manifesting as an increase in malondialdehyde production and a decrease in superoxide dismutase activity. Ang‐(1‐7) significantly retarded hypertrophy, autophagy and oxidative stress in the heart. Furthermore, a role of Mas receptor in Ang‐(1‐7)‐mediated action was assessed using A779 peptide, a selective Mas receptor antagonist. The beneficial responses of Ang‐(1‐7) on cardiac remodelling, autophagy and oxidative stress were mitigated by A779. Taken together, these result indicated that Mas receptor mediates cardioprotection of angiotensin‐(1‐7) against Ang II‐induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress.     

Development of oxidative stress and cell damage in the liver of rats with water-immersion restraint stress

We examined how oxidative stress and cell damage develop in the liver of rats subjected to water-immersion stress (WIRS). In rats subjected to WIRS for 1.5, 3 or 6 h, serum alanine aminotransferase and aspartate aminotransferase activities increased time-dependently. In the liver tissue, vacuolization and apoptosis occurred at 1.5 h of WIRS and vacuolization further developed without further appearance of apoptosis at 3 h or 6 h. Serum lipid peroxide (LPO) and NOx (nitrite/nitrate) concentrations increased at 3 h of WIRS and these increases were enhanced at 6 h. In liver tissue, increases in LPO and NOx concentrations and myeloperoxidase activity and decreases in ascorbic acid and reduced glutathione concentrations and superoxide dismutase activity occurred at 3 h of WIRS and these changes were enhanced at 6 h, although vitamin E concentration and xanthine oxidase activity were unchanged. These results indicate that oxidative stress in the liver of rats with WIRS develops after the appearance of cell damage in the tissue, and suggests that oxidative stress is caused through disruption of the antioxidant defense system and increases in NO generation and neutrophil infiltration in the liver, which may contribute to the progression of cell damage in the tissue.     

Estrogen-dependent inhibition of dextrose-induced endoplasmic reticulum stress and superoxide generation in endothelial cells

Increased oxidative stress and endoplasmic reticulum stress (ER stress) have been implicated in atherosclerosis. Estrogens have potent antioxidant activity but their effects on ER stress have not been well studied. Therefore, we studied the effects of estradiol and related sex steroids on dextrose-induced ER stress and superoxide (SO) generation in human umbilical vein endothelial cells (HUVECs). Oxidative stress was measured using hydroethidine fluorescence and MCLA chemiluminescence. ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (ES-TRAP) assay and by Western blot analysis of the expression of GRP78, JNK1, and phosphorylated JNK1, markers for ER stress. A supraphysiological dextrose concentration (27.5mM) increased ER stress and SO generation compared to treatment with a physiological concentration (5.5mM) of dextrose. In the presence of estradiol or testosterone (T), ER stress and SO generation were significantly reduced. In contrast to T-treated cells, dihydrotestosterone and 5-methyltestosterone were ineffective at alleviating ER stress or SO generation. When HUVECs were treated with T and the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione, T was no longer effective at suppressing ER stress or inhibiting SO generation. Changes in GRP78 expression and JNK activity in HUVECs support the results obtained in the ES-TRAP assay. These results indicate that dextrose-induced endoplasmic reticulum stress and superoxide generation are reversed by estradiol and testosterone; however, the latter requires aromatase-dependent conversion to estradiol.     

ACE2-mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function

Oxidative stress in the central nervous system mediates the increase in sympathetic tone that precedes the development of hypertension. We hypothesized that by transforming Angiotensin-II (AngII) into Ang-(1-7), ACE2 might reduce AngII-mediated oxidative stress in the brain and prevent autonomic dysfunction. To test this hypothesis, a relationship between ACE2 and oxidative stress was first confirmed in a mouse neuroblastoma cell line (Neuro2A cells) treated with AngII and infected with Ad-hACE2. ACE2 overexpression resulted in a reduction of reactive oxygen species (ROS) formation. In vivo, ACE2 knockout (ACE2(-/y)) mice and non-transgenic (NT) littermates were infused with AngII (10 days) and infected with Ad-hACE2 in the paraventricular nucleus (PVN). Baseline blood pressure (BP), AngII and brain ROS levels were not different between young mice (12 weeks). However, cardiac sympathetic tone, brain NADPH oxidase and SOD activities were significantly increased in ACE2(-/y). Post infusion, plasma and brain AngII levels were also significantly higher in ACE2(-/y), although BP was similarly increased in both genotypes. ROS formation in the PVN and RVLM was significantly higher in ACE2(-/y) mice following AngII infusion. Similar phenotypes, i.e. increased oxidative stress, exacerbated dysautonomia and hypertension, were also observed on baseline in mature ACE2(-/y) mice (48 weeks). ACE2 gene therapy to the PVN reduced AngII-mediated increase in NADPH oxidase activity and normalized cardiac dysautonomia in ACE2(-/y) mice. Altogether, these data indicate that ACE2 gene deletion promotes age-dependent oxidative stress, autonomic dysfunction and hypertension, while PVN-targeted ACE2 gene therapy decreases ROS formation via NADPH oxidase inhibition and improves autonomic function. Accordingly, ACE2 could represent a new target for the treatment of hypertension-associated dysautonomia and oxidative stress.     

Lipid peroxides induce expression of catalase in cultured vascular cells

Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H(2)O(2)). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H(2)O(2) after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress.     

Long-term effects of intrauterine malnutrition on vascular function in female offspring: implications of oxidative stress

Maternal malnutrition is known to impair fetal growth and predispose to the development of hypertension and type 2 diabetes. Recently, studies have demonstrated that intrauterine malnutrition is followed later in male offspring by oxidative stress characterized by increased superoxide generation due to activation of NADPH oxidase and reduced antioxidant defenses. However, few studies have investigated the mechanisms involved in endothelial dysfunction in female offspring. We evaluated the effects of the exogenous application of superoxide scavengers on the endothelium-dependent vasorelaxation in the mesenteric microvessels of female offspring. In addition, we examined indicative parameters of oxidative stress by measuring superoxide anion concentration and the activity of superoxide dismutase (SOD) as a marker of antioxidant defenses. Pregnant female Wistar rats were fed either a normal diet or 50% of this, throughout gestation. Intrauterine malnutrition induced hypertension and increased superoxide production without affecting SOD activity. Topical application of MnTMPyP (SOD mimetic) and apocynin (NADPH oxidase inhibitor) significantly improved the altered arteriolar responses to acetylcholine and bradykinin. In addition, incubation with apocynin reduced superoxide generation in these female offspring. The data suggest that after exposure to intrauterine malnutrition, female offspring present an increased superoxide production that is, at least in part, responsible for an endothelial dysfunction observed in these animals. These effects may be mediated via modulation of enzyme systems that generate superoxide.     

Ceramide response post-photodamage is absent after treatment with HA14-1

The oxidative stress induced by photodynamic therapy using the phthalocyanine Pc 4 (PDT) can lead to apoptosis, and is accompanied by photodamage to Bcl-2 and accumulation of de novo ceramide. Similar to PDT, the oxidative stress inducer and Bcl-2 inhibitor HA14-1 triggers apoptosis. To test the specificity of the ceramide response, Jurkat cells were exposed to an equitoxic dose of HA14-1. Unlike PDT, HA14-1 did not induce accumulation of de novo ceramide, although levels of sphingomyelin, phosphatidylserine and phosphatidylethanolamine were below control values after either treatment. In contrast to PDT, (i) the transient inhibition of serine palmitoyltransferase induced by HA14-1 was associated with the initial decrease in de novo ceramide, and (ii) HA14-1-initiated inhibition of sphingomyelin synthase and glucosylceramide synthase did not result in accumulation of de novo ceramide. These results show that the ceramide response to PDT is not induced by another pro-apoptotic stimulus, and may be unique to PDT as described here.     

Hypercholesterolemia increases mitochondrial oxidative stress and enhances the MPT response in the porcine myocardium: beneficial effects of chronic exercise

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We tested the hypothesis that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response and that exercise training could reverse this phenotype. MPT was assessed by mitochondrial swelling in response to 10-100 μM Ca(2+). FH pigs did show an increased MPT response to Ca(2+) that was associated with decreases in the expression of the putative MPT pore components mitochondrial phosphate carrier (PiC) and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation, as well as decreased levels of reduced GSH in cardiac mitochondria. Expression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin-2 (Trx2), and peroxiredoxin-3 (Prx3) was greatly reduced in the FH pigs. In contrast, cytosolic catalase expression and activity were increased. However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce mitochondrial oxidative stress, and return MnSOD, Trx2, Prx3, and catalase expression/activities to normal. We conclude that FH reduces mitochondrial antioxidants, increases mitochondrial oxidative stress, and enhances the MPT response in the porcine myocardium, and that exercise training can reverse these detrimental alterations.     

Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

Altered arachidonic acid biosynthesis and antioxidant protection mechanisms in Schwann cells grown in elevated glucose

In cultured Schwann cells, elevated glucose induces alterations in arachidonic acid metabolism that cause a decrease in the content of glycerophospholipid arachidonoyl-containing molecular species (ACMS). This could result from decreased de novo arachidonic acid biosynthesis, or increased arachidonic acid release from phospholipids. Incorporation of radioactive 8,11,14-eicosatrienoic acid into ACMS was lower for cells grown in 30 mm versus 5 mm glucose, consistent with a decrease in delta5 desaturase activity. However, neither basal arachidonic acid release from prelabeled cells nor stimulated generation of arachidonic acid in the presence of the reacylation inhibitor, thimerosal, the phosphotyrosine phosphatase inhibitor, bipyridyl peroxovanadium, or both together, were altered by varying the glucose concentrations, indicating that arachidonic acid turnover did not contribute to ACMS depletion. Free cytosolic NAD+ /NADH decreased, whereas NADP+ /NADPH remained unchanged for cells grown in elevated glucose, implying that decreased desaturase activity is a result of metabolic changes other than cofactor availability. Schwann cells in elevated glucose were susceptible to oxidative stress, as shown by increased malondialdehyde, depleted glutathione levels, and reduced cytosolic superoxide dismutase activity. Glutathione-altering compounds had no effect on ACMS levels, in contrast to N -acetylcysteine and alpha-lipoic acid, which partly corrected ACMS depletion in phosphatidylcholine. These findings suggest that in the Schwann cell cultures, a high glucose level elicits oxidative stress and weakens antioxidant protection mechanisms which could decrease arachidonic acid biosynthesis and that this deficit can be partly corrected by treatment with exogenous antioxidants.