Jeannette Vasquez-Vivar

Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: Potential implications in intracellular fluorescence detection of superoxide

Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500-530 nm; emission, 590-620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed.

Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine

Various detection methods of the specific product of reaction of superoxide (O2•−) with hydroethidine (HE), namely 2-hydroxyethidium (2-OH-E+), and with its mitochondria-targeted analog are described. The detailed protocol for quantification of 2-OH-E+, the unique product of HE/O2•− in cellular systems, is presented. The procedure includes cell lysis, protein precipitation using acidified methanol and HPLC analysis of the lysate. Using this protocol, we determined the intracellular levels of 2-OH-E+ and E+ in the range of 10 and 100 pmol per mg protein in unstimulated macrophage-like RAW 264.7 cells. In addition to HE, 2-OH-E+ and E+, we detected several dimeric products of HE oxidation in cell lysates. As several oxidation products of HE are formed, the superoxide-specific product, 2-OH-E+ needs to be separated from other HE-derived products for unequivocal quantification.

Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study.

©1997 Federation of European Biochemical Societies.

TETRAHYDROBIOPTERIN-DEPENDENT INHIBITION OF SUPEROXIDE GENERATION FROM NEURONAL NITRIC OXIDE SYNTHASE

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH4) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH4 analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields.l-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity forl-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with l-arginine. This effect is in accordance with the slow binding rates ofl-arginine to NOS in the absence of BH4.l-Arginine alone or in combination with BH4decreases the rates of NADPH consumption. The effect ofl-arginine on superoxide yields, however, was less dramatic than that caused by BH4 as much higher concentrations ofl-arginine are necessary to attain the same inhibition. In combination, l-arginine and BH4 inhibit the formation of superoxide generation and stimulate the formation ofl-citrulline. We conclude that, in contrast tol-arginine, BH4 does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.

Detection of superoxide anion using an isotopically labeled nitrone spin trap: potential biological applications

We describe the synthesis and biological applications of a novel nitrogen‐15‐labeled nitrone spin trap, 5‐ethoxycarbonyl‐5‐methyl‐1‐pyrroline N‐oxide ([15N]EMPO) for detecting superoxide anion. Superoxide anion generated in xanthine/xanthine oxidase (100 nM min−1) and NADPH/calcium‐calmodulin/nitric oxide synthase systems was readily detected using EMPO, a nitrone analog of 5,5′‐dimethyl‐1‐pyrroline N‐oxide (DMPO). Unlike DMPO‐superoxide adduct (DMPO–OOH), the superoxide adduct of EMPO (EMPO–OOH) does not spontaneously decay to the corresponding hydroxyl adduct, making spectral interpretation less confounding. Although the superoxide adduct of 5‐(diethoxyphosphoryl)‐5‐methyl‐pyrroline N‐oxide is more persistent than EMPO–OOH, the electron spin resonance spectra of [14N]EMPO–OOH and [15N]EMPO–OOH are less complex and easier to interpret. Potential uses of [15N]EMPO in elucidating the mechanism of superoxide formation from nitric oxide synthases, and in ischemia/reperfusion injury are discussed.

Altered Tetrahydrobiopterin Metabolism in Atherosclerosis. Implications for Use of Oxidized Tetrahydrobiopterin Analogues and Thiol Antioxidants

Objective—Tetrahydrobiopterin (BH4) is of fundamental importance for the normal function of endothelial NO synthase. The purpose of this study was to investigate the effects of hyperlipidemia on vascular BH4 levels and the effect of supplementation with sepiapterin in the presence and absence of N-acetylcysteine (NAC). Methods and Results—New Zealand White rabbits were fed normal chow (normocholesterolemic [NC] group) or hyperlipidemic chow (hyperlipidemic [HL] group) for 8 to 10 weeks. Mean cholesterol levels were 1465±333 and 53±17 mg/dL in the HL and NC group, respectively. Markedly diminished BH4 levels were found in the HL group compared with the NC group, but these levels could be restored after 6 hours of incubation with sepiapterin. Peak relaxations to acetylcholine and A23187 were impaired in the HL group. Supplementation with sepiapterin resulted in a further diminution of relaxation in the HL but not NC group. Incubation with NAC for 6 hours failed to raise BH4 levels, whereas NAC in conjunction with sepiapterin raised BH4 levels ≈221-fold. However, this increase did not improve relaxations to A23187 and acetylcholine. Conclusions—Prolonged exposure to sepiapterin impairs vasorelaxation in hyperlipidemia despite repletion of endogenous BH4. Antioxidant thiols do not correct this impairment. These studies have implications for the use of sepiapterin in the correction of vasomotor tone in atherosclerosis.

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

Human C-reactive protein induces endothelial dysfunction and uncoupling of eNOS in vivo.

BACKGROUND AND OBJECTIVE Elevated C-reactive protein (CRP) levels are associated with increased cardiovascular events and endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) activity in endothelial cells and inhibits endothelium-dependent nitric oxide (NO)-mediated vasodilation in vitro. Herein, we examined the effect of in vivo administration of CRP on endothelial function and underlying mechanisms in a valid animal model. METHODS Sprague-Dawley rats were injected intraperitoneally daily for 3 days with human CRP or human serum albumin (HuSA) at 20 mg/kg body weight. On day 4, mesenteric arterioles were isolated and pressurized for vasomotor study and aortic tissue was subjected to biochemical and molecular analysis. RESULTS Dilation of mesenteric arterioles to acetylcholine but not to sodium nitroprusside was significantly reduced following CRP treatment. The eNOS activity, eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and protein expression of GTPCH1 were significantly lower in aortic tissue homogenates from CRP-treated than HuSA-treated rats. CRP treatment also resulted in increased dihydroethidium staining for superoxide in aortic endothelium and membrane translocation of p47phox, a regulatory subunit of NADPH oxidase. CONCLUSION Our data provide novel evidence for the detrimental action of CRP in vivo by impairing eNOS-dependent vasodilation and uncoupling of eNOS.

4-Hydroxy-2-Nonenal Increases Superoxide Anion Radical in Endothelial Cells via Stimulated GTP Cyclohydrolase Proteasomal Degradation

Objective—4-Hydroxy-2-nonenal (4-HNE) is an abundant electrophilic lipid that mediates oxidative stress in endothelium by mechanisms that remain controversial. This study examines the effects of 4-HNE on nitric oxide (NO) and superoxide levels in bovine aorta endothelial cells (BAECs). Methods and Results—Exposure of BAECs to 4-HNE caused a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179 but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin and ascorbate supplemented cells suggesting that tetrahydrobiopterin (BH4) is a limiting factor in non supplemented cells. This was verified by quantification of BH4 by high-performance liquid chromatography analysis with electrochemical detection and by examining GTP cyclohydrolase I (GTPCH) protein levels and activity all of which were diminished by 4-HNE treatment. Analysis of 2-hydroxyethidium indicated that 4-HNE increased superoxide release in BAECs. The effects of 4-HNE on GTPCH and hsp90 were efficiently counteracted by proteasomal inhibition, indicating that depletion of BH4 by 4-HNE is attributable to specific mechanisms involving protein degradation. Conclusions—4-HNE by altering BH4 homeostasis mediates eNOS-uncoupling and superoxide generation in BAECs. By also decreasing phosphorylation of eNOS-serine 1179 4-HNE may specifically regulate NO/reactive oxygen species fluxes in the endothelium with important consequences to redox signaling.

Antiangiogenic action of redox-modulating Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP5+, via suppression of oxidative stress in a mouse model of breast tumor

MnTE-2-PyP(5+) is a potent catalytic scavenger of reactive oxygen and nitrogen species, primarily superoxide and peroxynitrite. It therefore not only attenuates primary oxidative damage, but was found to modulate redox-based signaling pathways (HIF-1alpha, NF-kappaB, SP-1, and AP-1) and thus, in turn, secondary oxidative injury also. Cancer has been widely considered an oxidative stress condition. The goal of this study was to prove if and why a catalytic SOD mimic/peroxynitrite scavenger would exert anti-cancer effects, i.e., to evaluate whether the attenuation of the oxidative stress by MnTE-2-PyP(5+) could suppress tumor growth in a 4T1 mouse breast tumor model. Tumor cells were implanted into Balb/C mouse flanks. Three groups of mice (n=25) were studied: control (PBS) and 2 and 15 mg/kg/day of MnTE-2-PyP(5+) given subcutaneously twice daily starting when the tumors averaged 200 mm(3) (until they reached approximately 5-fold the initial volume). Intratumoral hypoxia (pimonidazole, carbonic anhydrase), HIF-1alpha, VEGF, proliferating capillary index (CD105), microvessel density (CD31), protein nitration, DNA oxidation (8-OHdG), NADPH oxidase (Nox-4), apoptosis (CD31), macrophage infiltration (CD68), and tumor drug levels were assessed. With 2 mg/kg/day a trend toward tumor growth delay was observed, and a significant trend was observed with 15 mg/kg/day. The 7.5-fold increase in drug dose was accompanied by a similar (6-fold) increase in tumor drug levels. Oxidative stress was largely attenuated as observed through the decreased levels of DNA damage, protein 3-nitrotyrosine, macrophage infiltration, and NADPH oxidase. Further, hypoxia was significantly decreased as were the levels of HIF-1alpha and VEGF. Consequently, suppression of angiogenesis was observed; both the microvessel density and the endothelial cell proliferation were markedly decreased. Our study indicates for the first time that MnTE-2-PyP(5+) has anti-cancer activity in its own right. The anti-cancer activity via HIF/VEGF pathways probably arises from the impact of the drug on the oxidative stress. Therefore, the catalytic scavenging of ROS/RNS by antioxidants, which in turn suppresses cellular transcriptional activity, could be an appropriate strategy for anti-cancer therapy. Enhancement of the anti-cancer effects may be achieved by optimizing the dosing regime, utilizing more bioavailable Mn porphyrins (MnP), and combining MnP treatment with irradiation, hyperthermia, and chemotherapy. Mn porphyrins may be advantageous compared to other anti-cancer drugs, owing to their radioprotection of normal tissue and the ability to afford pain management in cancer patients via prevention of chronic morphine tolerance.