Increased Reactive Oxygen Species Formation Research Articles

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) induces oxidative stress, DNA strand breaks, and poly(ADP-ribose) polymerase-1 activation in human breast carcinoma cell lines

The formation of reactive oxygen species (ROS) plays a critical role in 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced toxicities in mammalian cells since it promotes cell proliferation, growth arrest, and apoptosis. In this study, we investigated whether TCDD induces oxidative stress and DNA damage in human ERα(+)/MCF-7 and ERα(−)/MDA-MB-231 breast cancer cells and whether this is accompanied by the initiation of DNA repair events. Results indicated that viability of MCF-7 and MDA-MB-231 cells was concentration- and time-dependently reduced by TCDD. Further, we observed significant increases in ROS formation and decreases in intracellular glutathione (GSH) in these two cell lines after TCDD treatment. Overall, the extent of cell death was greater in MCF-7 cells than in MDA-MB-231 cells whereas the magnitude of ROS formation and GSH depletion was greater in MDA-MB-231 cells than in MCF-7 cells. In addition, we observed that at non-cytotoxic concentration (1 nM for 5 h), TCDD induced decreases in intracellular NAD(P)H and NAD + in MCF-7 and MDA-MB-231 cells. These decreases were completely blocked by three types of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The catalytic activation of PARP-1 in cells treated with TCDD was confirmed by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 and MDA-MB-231 cells exposed to TCDD as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that TCDD induces decreases in intracellular NAD(P)H and NAD + through PARP-1 activation mediated by formation of DNA strand breaks. In addition, we demonstrated that the extent of oxidative stress and DNA damage was greater in MDA-MB-231 cells than in MCF-7 cells, with a strong correlation to estrogen receptor (ER) status. In conclusions, our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to TCDD and that the TCDD-induced oxidative stress and DNA damage may, in part, contribute to TCDD-induced carcinogenesis.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin–proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A 2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg − 1 ) attenuated protein degradation and increased protein synthesis in skeletal muscle.

Neuroprotective effect of a ginseng ( Panax ginseng) root extract on astrocytes primary culture

A standardized aqueous extract of Panax ginseng radix was tested for its antioxidant effect on primary astrocytes culture on an oxidant stress model generated by H 2O 2. The results indicated that this extract had a significant effect on the reduction of astrocytic death induced by H 2O 2. Dose–response experiments revealed that this ginseng extract increased cell viability at a wide range of concentrations. Therefore, we investigated the effects of this extract on antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidases (GPx) and glutathione reductase (GR), on glutathione content (reduced and oxidized forms and red/ox index) and on the intracellular reactive oxygen species (ROS) formation. Exposure of astrocytes to H 2O 2 decreased the activities of antioxidant enzymes, and increased ROS formation. Ginseng root extract reversed the effect of almost all of these parameters in H 2O 2-injured primary cultures of rat astrocytes.

NADPH oxidase modulates myocardial Akt, ERK1/2 activation, and angiogenesis after hypoxia-reoxygenation

Recent studies have demonstrated that reactive oxygen species (ROS) mediate myocardial ischemia-reperfusion (I/R) and angiogenesis via the mitogen-activated protein kinases and the serine-threonine kinase Akt/protein kinase B pathways. NADPH oxidases are major sources of ROS in endothelial cells and cardiomyocytes. In the present study, we investigated the role of NADPH oxidase-derived ROS in hypoxia-reoxygenation (H/R)-induced Akt and ERK1/2 activation and angiogenesis using porcine coronary artery endothelial cells (PCAECs) and a mouse myocardial I/R model. Our data demonstrate that exposure of PCAECs to hypoxia for 2 h followed by 1 h of reoxygenation significantly increased ROS formation. Pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI, 10 microM) and apocynin (Apo, 200 and 600 microM), significantly attenuated H/R-induced ROS formation. Furthermore, exposure of PCAECs to H/R caused a significant increase in Akt and ERK1/2 activation. Exposure of PCAEC spheroids and mouse aortic rings to H/R significantly increased endothelial spheroid sprouting and vessel outgrowth, whereas pharmacological inhibition of NADPH oxidase or genetic deletion of the NADPH oxidase subunit, p47(phox) (p47(phox-/-)), significantly suppressed these changes. With the use of a mouse I/R model, our data further show that the increases in myocardial Akt and ERK1/2 activation and vascular endothelial growth factor (VEGF) expression were markedly blunted in the p47(phox-/-) mouse subjected to myocardial I/R compared with the wild-type mouse. Our findings underscore the important role of NADPH oxidase and its subunit p47(phox) in modulating Akt and ERK1/2 activation, angiogenic growth factor expression, and angiogenesis in myocardium undergoing I/R.

Role of Oxidative Stress in Parity‐Induced Endothelial Dysfunction

Oxidative stress has been implicated in a wide variety of degenerative states including cardiovascular disease. There is now considerable evidence to suggest that there is increased oxidative burden during pregnancy. But it is not clear whether, in the long-term, this oxidative burden is a significant contributor to cardiovascular disease in older women. We examined the effect of parity on coronary artery function and on vascular reactive oxygen species (ROS). The experiments were done on multiparous and age-matched virgin rats. Vascular reactivity to acetylcholine (ACh) was measured in small coronary arteries using wire myography in the presence and absence of superoxide dismutase (SOD, 100 U/ml). ROS formation was measured using the chemoluminescent luminol derivative L-012. There was reduced ACh-mediated maximal relaxation in coronary arteries from multiparous rats (Parous: 49.2%±2.9; Virgins: 94.6%±3.1; P<0.05). This effect was associated with oxidative stress as indicated by increased superoxide anion formation (Parous: 3.9±0.6 URL; Virgins: 1.4±0.5 URL). Incubation with 100U/ml SOD for 30 min improved maximal vascular relaxation to ACh (Parous: 76.6%±3.1). Coronary arteries from both groups relaxed similarly to sodium nitroprusside. These data suggest that parity induces coronary endothelial dysfunction through decreased nitric oxide availability and increased ROS formation.

Thalidomide Induces gamma-Globin Gene Expression in Adult Primary Erythroid Cells through Increased Intracellular Reactive Oxygen Species Mediated Activation of p38 MAPK Signal Pathway.

Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide's therapeutic effect are still being defined. We hypothesize that thalidomide induce the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin gene expression in a dose-dependent manner. The averaged gamma/gamma+beta percentage ratio was 12.89% ± 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% ± 0.07% in Epo alone (P<0.01). Interestingly, we found that intracellular ROS level was significantly increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner. Thalidomide also increased the histone H3 phospho-acetylation and histone H4 acetylation. In contrast, treatment with the anti-oxidant N-acetylcycteine (NAC) inhibited the thalidomide induced p38 activity and histone H3, H4 acetylation. These data indicate that thalidomide induced the gamma-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.

Anxiety-Induced Plasma Norepinephrine Augmentation Increases Reactive Oxygen Species Formation by Monocytes in Essential Hypertension

An association between anxiety and depression and increased blood pressure (BP) and cardiovascular disease risk has not been firmly established. We examined the hypothesis that anxiety and depression lead to increased plasma catecholamines and to production of reactive oxygen species (ROS) by mononuclear cells (MNC) in hypertensive individuals. We also studied the role of BP in this effect. In Protocol 1, a cross-sectional study was performed in 146 hypertensive patients to evaluate whether anxiety and depression affect BP and ROS formation by MNC through increasing plasma catecholamines. In Protocol 2, a 6-month randomized controlled trial using a subtherapeutic dose of the alpha(1)-adrenergic receptor antagonist doxazosin (1 mg/day) versus placebo in 86 patients with essential hypertension was performed to determine whether the increase in ROS formation by MNC was independent of BP. In Protocol 1, a significant relationship was observed between the following: trait anxiety and plasma norepinephrine (r = 0.32, P < .01); plasma norepinephrine and ROS formation by MNC (r = 0.36, P < .01); and plasma norepinephrine and systolic, diastolic, and mean BP (r = 0.17, P = .04; r = 0.26, P = .02; r = 0.23, P < .01, respectively). In Protocol 2, subtherapeutic doxazosin treatment (1 mg/day) had no significant effect on BP. However doxazosin significantly decreased ROS formation by MNC compared with placebo (P < .01). Trait anxiety may increase plasma norepinephrine and increase ROS formation by MNC independent of BP in hypertensive patients.

Protective effect of a synthetic anti‐oxidant on neuronal cell apoptosis resulting from experimental hypoxia re‐oxygenation injury

Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured neuronal cells exposed to hypoxia-reoxygenation (H/R) injury, as a model for stroke, yield a burst of reactive oxygen species (ROS) as measured with electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping. Added superoxide dismutase inhibited spin-adduct formation verifying that superoxide radical anion was formed in neuronal cells following H/R injury. The intracellular ADP/ATP ratio increased rapidly over the first 5 h following injury and this was due primarily to the decreased cellular pools of ATP, consistent with the notion that H/R promotes mitochondrial dysfunction leading to decreased ATP reserve and increased ROS formation. As an early response to the enhanced oxidative stress, genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), and the oxygen-sensor neuroglobin increased significantly. Up-regulation of the HO-1 gene was paralleled by increased HO protein expression and activity. Despite this cellular response, apoptosis increased significantly following H/R injury indicating that the endogenous anti-oxidant defenses were unable to protect the cells. In contrast, addition of a phenolic anti-oxidant, bisphenol (BP), prior to H/R injury, inhibited ROS production and gene regulation and significantly decreased neuronal cell apoptosis. Added BP was converted stoichiometrically to the corresponding diphenoquinone indicating the synthetic anti-oxidant effectively decreased oxidative stress through a radical scavenging mechanism. Together, these data indicate that BP has the potential to act as a neuro-protective drug.

Intra‐ and extracellular reactive oxygen species generated by blue light

Blue light from dental photopolymerization devices has significant biological effects on cells. These effects may alter normal cell function of tissues exposed during placement of oral restorations, but recent data suggest that some light-induced effects may also be therapeutically useful, for example in the treatment of epithelial cancers. Reactive oxygen species (ROS) appear to mediate blue light effects in cells, but the sources of ROS (intra- versus extracellular) and their respective roles in the cellular response to blue light are not known. In the current study, we tested the hypothesis that intra- and extracellular sources of blue light-generated ROS synergize to depress mitochondrial function. Normal human epidermal keratinocytes (NHEK) and oral squamous cell carcinoma (OSC2) cells were exposed to blue light (380-500 nm; 5-60 J/cm(2)) from a dental photopolymerization source (quartz-tungsten-halogen, 550 mW/cm(2)). Light was applied in cell-culture media or balanced salt solutions with or without cells present. Intracellular ROS levels were estimated using the dihydrofluorescein diacetate (DFDA) assay; extracellular ROS levels were estimated using the leucocrystal violet assay. Cell response was estimated using the MTT mitochondrial activity assay. Blue light increased intracellular ROS equally in both NHEK and OSC2. Blue light also increased ROS levels in cell-free MEM or salt solutions, and riboflavin supplements increased ROS formation. Extracellularly applied ROS rapidly (50-400 muM, <1 min) increased intracellular ROS levels, which were higher and longer-lived in NHEK than OSC2. The type of cell-culture medium significantly affected the ability of blue light to suppress cellular mitochondrial activity; the greatest suppression was observed in DMEM-containing or NHEK media. Collectively, the data support our hypothesis that intra- and extracellularly generated ROS synergize to affect cellular mitochondrial suppression of tumor cells in response to blue light. However, the identity of blue light targets that mediate these changes remain unclear. These data support additional investigations into the risks of coincident exposure of tissues to blue light during material polymerization of restorative materials, and possible therapeutic benefits.

Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology.

Increased production of mitochondrial reactive oxygen species (ROS) by hyperglycemia is recognized as a major cause of the clinical complications associated with diabetes and obesity [Brownlee, M. (2001) Nature 414, 813-820]. We observed that dynamic changes in mitochondrial morphology are associated with high glucose-induced overproduction of ROS. Mitochondria undergo rapid fragmentation with a concomitant increase in ROS formation after exposure to high glucose concentrations. Neither ROS increase nor mitochondrial fragmentation was observed after incubation of cells with the nonmetabolizable stereoisomer L-glucose. However, inhibition of mitochondrial pyruvate uptake that blocked ROS increase did not prevent mitochondrial fragmentation in high glucose conditions. Importantly, we found that mitochondrial fragmentation mediated by the fission process is a necessary component for high glucose-induced respiration increase and ROS overproduction. Extended exposure to high glucose conditions, which may mimic untreated diabetic conditions, provoked a periodic and prolonged increase in ROS production concomitant with mitochondrial morphology change. Inhibition of mitochondrial fission prevented periodic fluctuation of ROS production during high glucose exposure. These results indicate that the dynamic change of mitochondrial morphology in high glucose conditions contributes to ROS overproduction and that mitochondrial fission/fusion machinery can be a previously unrecognized target to control acute and chronic production of ROS in hyperglycemia-associated disorders.

Carotid artery intima-media thickness and reactive oxygen species formation by monocytes in hypertensive patients

Carotid artery intima-media thickness (IMT) is a widely accepted index for assessing atherosclerosis, and is known to be a risk indicator for cardiovascular and cerebrovascular events. Oxidative stress and inflammation are also known to play critical roles in the pathogenesis of vascular events. We studied the association between IMT and inflammatory markers, such as oxidative stress in polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNCs) in 156 patients with essential hypertension. Reactive oxygen species (ROS) formation by PMNs and MNCs was measured by gated flow cytometry. CRP and traditional risk factors, such as age, gender, body mass index, hemoglobin A(1c), and total cholesterol were also measured. The subjects were divided into a plaque group (max-IMT>or=1.1 mm, n=40), and a nonplaque group (max-IMT<1.1 mm, n=116). ROS formation by MNCs was significantly increased in the plaque group when compared with the nonplaque group (P<0.0001). Multiple regression analysis revealed a significant correlation between IMT and ROS formation by MNCs (r=0.407, P<0.0001), or CRP (r=0.216, P=0.0029) or hemoglobinA1c (r=0.158, P=0.0270) or age (r=0.157, P=0.0447). No significant correlation was observed between IMT and ROS formation by PMNs. These results suggest that carotid artery IMT may be affected by increased ROS formation by MNCs, and that increased ROS formation by MNCs may be related to the development of atherosclerosis. We propose that ROS formation by MNCs is a marker for prediction of carotid atherosclerosis.

Ferric iron increases ROS formation, modulates cell growth and enhances genotoxic damage by 4-hydroxynonenal in human colon tumor cells

Iron is a relevant risk factor for colorectal cancer due to its genotoxic properties. Here we hypothesised that iron-overload causes other toxic effects, which contribute to carcinogenesis. For this, we investigated formation of reactive oxygen species (ROS), DNA repair, cell growth and glutathione (GSH) in human colon tumor cells (HT29 clone 19A) treated with ferric nitrilotriacetate (Fe-NTA, 0–2000 μM). Intracellular formation of ROS was analysed with the peroxide-labile fluorescent dye carboxy-dichlorodihydrofluorescine-diacetate. DNA repair, reflected as the persistency of DNA damage induced by selected genotoxins, was determined with the Comet assay. Cell growth and GSH were measured by fluorimetrical analysis. Key findings were that ROS formation increased with time (1000 μM Fe-NTA, p < 0.001). DNA damage was largely repaired after 120 min, but was not affected by 10 μM Fe-NTA. In contrast, 10 μM Fe-NTA significantly increased DNA damage induced by 4-hydroxynonenal. Doses of 25 μM Fe-NTA increased cell growth ( p < 0.05), whereas high concentrations (2000 μM) resulted in growth arrest ( p < 0.05), that was accompanied by increased GSH levels ( p < 0.01). In conclusion, high concentrations of Fe-NTA caused cellular effects, which reflect a stress response, and resulted in formation of ROS. Carcinogenic risks from ferric iron could be derived also from lower concentrations, which enhance tumor cell growth and cause progenotoxic effects.

Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species

Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-Delta12,14 -prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.

Nox1 Overexpression Potentiates Angiotensin II-Induced Hypertension and Vascular Smooth Muscle Hypertrophy in Transgenic Mice

Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vessel wall, but the importance of individual Nox homologues in specific layers of the vascular wall is unclear. Nox1 upregulation has been implicated in cardiovascular pathologies such as hypertension and restenosis. To investigate the pathological role of Nox1 upregulation in vascular smooth muscle, transgenic mice overexpressing Nox1 in smooth muscle cells (TgSMCnox1) were created, and the impact of Nox1 upregulation on the medial hypertrophic response during angiotensin II (Ang II)-induced hypertension was studied. These mice have increased expression of Nox1 protein in the vasculature, which is accompanied by increased superoxide production. Infusion of Ang II (0.7 mg/kg per day) into these mice for 2 weeks led to a potentiation of superoxide production compared with similarly treated negative littermate controls. Systolic blood pressure and aortic hypertrophy were also markedly greater in TgSMCnox1 mice than in their littermate controls. To confirm that this potentiation of vascular hypertrophy and hypertension was due to increased ROS formation, additional groups of mice were coinfused with the antioxidant Tempol. Tempol decreased the level of Ang II-induced aortic superoxide production and partially reversed the hypertrophic and hypertensive responses in these animals. These data indicate that smooth muscle-specific Nox1 overexpression augments the oxidative, pressor, and hypertrophic responses to Ang II, supporting the concept that medial Nox1 participates in the development of cardiovascular pathologies.

Matrix metalloproteinase-2 and -9 are induced differently by doxorubicin in H9c2 cells: The role of MAP kinases and NAD(P)H oxidase

Dysregulation of myocardial metalloproteinases (MMPs) is now regarded as an early contributory mechanism for the initiation and progression of heart failure. Doxorubicin is a strongly cardiotoxic anticancer drug. This study investigates the effects of doxorubicin on myocardial MMP-2 and MMP-9 activation. After pre-treatment with or without carvedilol or dexrazoxane, we exposed H9c2 cardiomyocytes to doxorubicin to evaluate reactive oxygen species (ROS) formation and MMP-2 and MMP-9 expression and activation. To investigate the signaling pathways leading to doxorubicin-induced MMP activation, we also examined the phosphorylation of three members of the MAPK family (ERK1/2, p38, and JNK), the effects of selective inhibitors of ERK1/2, p38, and JNK on MMP transcription and activity, the transcription of the NAD(P)H oxidase subunit Nox1, and the effects of the NAD(P)H oxidase inhibitor DPI on MMP activation. Doxorubicin induces a significant increase in ROS formation and a rapid increase of MMP expression and activation. Pre-treatment with carvedilol or dexrazoxane prevented these effects. We also found that p38 is the MAPK that is mainly responsible for MMP-9 activation through an NAD(P)H-independent mechanism. ERK and JNK modulate the transcription of the NAD(P)H oxidase subunit Nox1, while the JNK/ERK NAD(P)H oxidase cascade is an important pathway that mediates doxorubicin signaling to MMP-2. Inhibition of NAD(P)H oxidase attenuates the increase in MMP-2, but augments the doxorubicin-induced increase in MMP-9. Enhancement of MMP-2 and MMP-9 in cardiac myocytes in response to doxorubicin is mediated by the cooperation of ERK, JNK, and p38 kinase pathways, most of which are redox dependent.

NO restores HIF-1α hydroxylation during hypoxia: Role of reactive oxygen species

The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its α subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1α by prolyl hydroxylases (PHDs) recruits the von Hippel–Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the α subunit. Hypoxic stabilization of HIF-1α has been reported to be antagonized by nitric oxide (NO). By using a HIF-1α–pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1α by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1α stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1α protein stabilization. In vitro HIF-1α–pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1α destabilization by NO donors under hypoxia via modulation of PHD activity.

Impairment of Diazoxide-Induced Formation of Reactive Oxygen Species and Loss of Cardioprotection in Connexin 43 Deficient Mice

Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient (Cx43+/-) mice. Because connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/- mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 micromol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/- cardiomyocytes. In contrast, diazoxide (200 micromol/L) increased ROS formation by 43+/-10% versus vehicle in WT, but only by 18+/-4% in Cx43+/- cardiomyoctes (P<0.05). Two hour-simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7+/-1% versus 39+/-2%, (Cx43+/-): 8+/-1% versus 40+/-3%, P<0.01). Although menadione protected WT and Cx43+/- cardiomyocytes, diazoxide increased viability (17+/-2%, P<0.01) in WT, but not in Cx43+/- (9+/-1%). Menadione (37 microg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/- mice (24+/-4% versus 24+/-5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35+/-4% versus 55+/-3% of area at risk, P<0.01), but not in Cx43+/- mice (56+/-2% versus 54+/-3%). Cardiomyocytes of Cx43+/- mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.

ROS formation and glutathione levels in human oral fibroblasts exposed to TEGDMA and camphorquinone

Glutathione (GSH) is important for the self-protection of cells against oxidative stress and toxic xenobiotics, whereas reactive oxygen species (ROS) at elevated concentrations may cause detrimental alterations of cell membranes, DNA, and other cellular structures. The present investigation addressed the effects of triethylene-glycoldimethacrylate (TEGDMA) and camphorquinone (CQ) on glutathione metabolism and the formation of ROS in oral cells. Primary human pulp fibroblasts were exposed to various concentrations of TEGDMA and CQ (0.1-5 mM). Subsequently, GSH concentration and ROS formation were analyzed with the use of the monobromobimane assay (GSH) and 2',7'-dichlorofluorescein diacetate (DCFH-DA) (ROS). The endogenous ROS hydrogen peroxide (H2O2) was used as a positive control (0.02-2 mM). TEGDMA significantly decreased GSH at concentrations between 0.5 and 5 mM (p<0.05), but did not elevate ROS levels. Contrary, CQ increased ROS formation at concentrations>or=1 mM, but had only a moderate effect on GSH at the highest test concentration. Hydrogen peroxide increased ROS and simultaneously decreased GSH at concentrations of >or=0.2 mM. These data show that the investigated substances may cause cell damage due to various mechanisms, GSH decrease and/or ROS increase. As a consequence, TEGDMA and CQ released into an aqueous environment from resinous materials might interact, thus generating significant cytotoxic effects even at low concentrations.

Effects of Naphthalene Quinonoids on the Induction of Oxidative DNA Damage and Cytotoxicity in Calf Thymus DNA and in Human Cultured Cells

The purpose of this study was to examine the differences in the induction of DNA damage and cytotoxic effects by quinonoid derivatives of naphthalene in calf thymus DNA (ct-DNA) and in human T47D breast cancer cells. Results indicated that copper(II) and NADPH were essential for causing oxidant-mediated aldehydic DNA lesions (ADLs), including abasic sites and aldehydic base/sugar lesions, in ct-DNA exposed to 1,2-naphthalenediol (NCAT), 1,4-naphthalenediol (NHQ), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1,4-NQ). The ADLs induced by naphthalene quinonoids in ct-DNA decrease in the rank order NCAT congruent with 1,2-NQ > NHQ >> 1,4-NQ. Results from the analyses in cells indicated that after 1.5-5 h of exposure all naphthalene quinonoids induced a cytotoxic response in T47D cells at concentrations 10-100 microM or above, where NHQ and 1,4-NQ were approximately 5-10 times more efficient than NCAT and 1,2-NQ in the induction of cell death. In addition, NHQ, 1,2-NQ, and 1,4-NQ were not able to produce measurable levels of ADLs in cells at concentrations up to 1.25 mM, whereas NCAT (0.75-1.25 mM) induced a significant increase in the number of ADLs in T47D cells after 1.5 h of exposure when compared to control. The specific type of ADLs induced by NCAT is resistant to cellular excision repair pathway. Results from the measurements of reactive oxygen species (ROS) indicated that all naphthalene quinonoids induced increases in ROS formation in T47D cells. The induction of ROS formation in cells by naphthalene quinonoids decreases in the rank order 1,4-NQ congruent with 1,2-NQ > NHQ > NCAT. Overall, results from our investigation suggest that naphthalene quinonoids cause cell death at concentrations well below the concentrations at which they induce the formation of ADLs, perhaps by altering intracellular redox status.

Xanthine oxidase, nitric oxide synthase and phospholipase A 2 produce reactive oxygen species via mitochondria

The formation of reactive oxygen species (ROS) has been suggested to be associated with excitotoxicity but the involvement of cytoplasmic enzymes in ROS formation is not clearly known. In the present study, we examined the role of xanthine oxidase (XO), nitric oxide synthase (NOS) and phospholipase A 2 (PLA 2) in glutamate-induced oxidative stress in rat cortical slices. Glutamate-induced ROS formation and mitochondrial depolarization were measured in rat cortical slices in presence of allopurinol, L-NAME and 4-bromophenacylbromide, the specific inhibitors of XO, NOS and PLA 2, respectively. Upon stimulation of slices with glutamate, a significant increase in ROS formation and mitochondrial depolarization was observed. However, pretreatment of slices with allopurinol, L-NAME and 4-bromophenacylbromide inhibited the glutamate-induced ROS formation and mitochondrial depolarization. The glutamate-induced ROS formation was dependent on the concentration of these inhibitors and also on the duration of the treatment. Allopurinol was found to be less effective as compared to L-NAME and 4-bromophenacylbromide. The combined treatment of slices with these enzyme inhibitors showed further inhibition in ROS formation and mitochondrial depolarization. The inhibition in ROS formation as well as mitochondrial depolarization by allopurinol, L-NAME and 4-bromophenacylbromide clearly suggests that the activation of XO, NOS and PLA 2 by calcium during glutamate receptor stimulation may release some chemicals which depolarize mitochondria resulting in ROS formation.