Dichlorofluorescein Oxidation Research Articles

Calcium-independent phospholipase A2modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.

Correlation Analyses of Phytochemical Composition, Chemical, and Cellular Measures of Antioxidant Activity of Broccoli (Brassica oleraceaL. Var. italica)

Chemical measures of antioxidant activity within the plant, such as the oxygen radical absorbance capacity (ORAC) assay, have been reported for many plant-based foods. However, the extent to which chemical measures relate to cellular measures of oxidative stress is unclear. The natural variation in the phytochemical content of 22 broccoli genotypes was used to determine correlations among chemical composition (carotenoids, tocopherols and polyphenolics), chemical antioxidant activity (ORAC), and measures of cellular antioxidation [prevention of DNA oxidative damage and of oxidation of the biomarker dichlorofluorescein (DCFH) in HepG2 cells] using hydrophilic and lipophilic extracts of broccoli. For lipophilic extracts, ORAC (ORAC-L) correlated with inhibition of cellular oxidation of DCFH (DCFH-L, r = 0.596, p = 0.006). Also, DNA damage in the presence of the lipophilic extract was negatively correlated with both chemical and cellular measures of antioxidant activity as measured by ORAC-L (r = -0.705, p = 0.015) and DCFH-L (r = -0.671, p = 0.048), respectively. However, no correlations were observed for hydrophilic (-H) extracts, except between polyphenol content and ORAC (ORAC-H; r = 0.778, p < 0.001). Inhibition of cellular oxidation by hydrophilic extracts (DCFH-H) and ORAC-H were approximately 8- and 4-fold greater than DCFH-L and ORAC-L, respectively. Whether ORAC-H has more biological relevance than ORAC-L because of its magnitude or whether ORAC-L bears more biological relevance because it relates to cellular estimates of antioxidant activity remains to be determined. Chemical estimates of antioxidant capacity within the plant may not accurately reflect the complex nature of the full antioxidant activity of broccoli extracts within cells.

Spectrofluorometric determination of intracellular levels of reactive oxygen species in drug-sensitive and drug-resistant cancer cells using the 2′,7′-dichlorofluorescein diacetate assay

This article examines a non-invasive spectrofluorometric method using the 2′,7′-dichlorofluorescein diacetate (DCHF-DA) assay for quantifying the intracellular reactive oxygen species (ROS i) produced in four cultured cancer cell lines: drug-sensitive (K562) and drug-resistant (K562/ adr) human erythromyelogenous leukemia cell lines, and drug-sensitive (GLC4) and drug-resistant (GLC4/ adr) human small cell lung carcinoma cell lines. The oxidation of the probe to the fluorescent dichlorofluorescein (DCF) was continuously monitored by following the DCF fluorescence intensity as a function of time using a standard spectrofluorometer in the presence of an extracellular DCF fluorescence quencher (Co 2+). By fitting the spectrofluorometric data to a kinetic model based on the following two reactions: (i) deacetylation of DCHF-DA to the oxidant-sensitive compound 2′,7′-dichlorofluorescein (DCHF) by cellular esterase enzymes (pseudo-first-order rate constant: k e) and (ii) oxidation of DCHF by ROS i (second-order rate constant: k 2), the parameters intervening in DCF formation, k e and the product of k 2 by the ROS i concentration, were quantitatively determined for the different cell lines studied. The results revealed that the intracellular esterase content or activity is similar in K562, K562/ adr, and GLC4 cells, but 5-fold higher in GLC4/ adr cells. The product k 2[ROS i] was found to be similar in the four cell lines considered, with a mean value of (5.3±0.9)×10 −7 cell −1 s −1. Assuming that H 2O 2 (in combination with peroxidases) is the primary responsible species for DCHF oxidation in intact cells, and using the rate constant value k 2 = 790 ± 62 M - 1 s - 1 established in our laboratory for the reaction of DCHF with H 2O 2 in the presence of horseradish peroxidase, the mean value of the intracellular levels of ROS i in those cells was estimated to be 0.67±0.16 nM per cell. Such a value compares favorably to H 2O 2 intracellular steady-state concentrations that have been estimated in the literature for a few other cell types.

Environmental oxygen tension affects phenotype in cultured bone marrow-derived macrophages.

This study tested the hypothesis that the unique phenotype of alveolar macrophages (AM) is maintained through adaptation to the relatively high oxygen partial pressure (P(O2)) of the lung, through modification of redox-sensitive transcription factors. BALB/c mouse bone marrow-derived macrophages (BMC) were differentiated under different P(O2) and compared functionally to AM and peritoneal macrophages (PM). BMC differentiated in normoxia (P(O2) 140 Torr, BMC(high)) were similar to AM in having low phagocytic and antigen presenting cell (APC) activities. However, BMC grown in low oxygen tension as found in other tissues (<40 Torr, BMC(low)) were better phagocytes and APCs, similar to PM. BMC(high) were more oxidative intracellularly than BMC(low), based on oxidation of dichlorofluorescein and higher glutathione disulfide/glutathione (GSH) ratios, despite having more GSH. Finally, lipopolysaccharide-induced nuclear factor-kappaB translocation, measured by laser scanning cytometry, was reduced in BMC(high) and AM, compared with BMC(low) and PM, respectively. These data suggest that regulation of the AM phenotype may occur, at least in part, via inhibition of NF-kappaB by the unique redox environment.

REACTIVE SPECIES MEDIATED INJURY OF HUMAN LUNG EPITHELIAL CELLS AFTER HYPOXIA-REOXYGENATION

This study tested the hypothesis that hypoxia exposure predisposed lung epithelial cells to reactive oxygen species-(ROS) mediated cellular injury. Human lung carcinoma cells (ATCC line H441) having epithelial characteristics (including lamellar bodies, surfactant protein [SP]-A, and SP-B) were cultured in air (air/5% CO 2) or hypoxia (< 1% O 2 /5% CO 2) for 0 to 24 hours before imposition of oxidant stress. Cellular manganese superoxide dismutase (MnSOD) activity (units/mg protein) decreased significantly after 24 hours of hypoxia. In normoxic culture after hypoxia, the cells produced increased ROS, detected as dichlorofluorescein (DCF) fluorescence and H 2 O 2 accumulation in medium. Antioxidants N-acetylcysteine (N-Ac) and ebselen inhibited increased DCF fluorescence after hypoxia. To test their ability to tolerate oxidant stress, some cells were incubated with antimycin A (100 μ M) and trifluorocarbonylcyanide phenylhydrazone (10 μ M) (anti A + FCCP), a mitochondrial complex III inhibitor and respiratory chain uncoupler, which together increase mitochondrial superoxide (O 2 -) and H 2 O 2 production. Lung epithelial cells preexposed to hypoxia released more lactate dehydrogenase (LDH) than normoxic controls in response to increased O 2 - production. Increased LDH release from hypoxia-preexposed cells treated with anti A + FCCP was inhibited by 1 mM N-Ac. Rotenone and myxothiazole increased DCF oxidation more in hypoxic than in normoxic cells, suggesting that mitochondrial electron transport complex I may have been altered by hypoxia preexposure.

Alterations in cellular Ca 2+ and free iron pool by sulfur amino acid deprivation: the role of ferritin light chain down-regulation in prooxidant production

Deficiency of sulfur amino acids occurs in certain pathophysiological states such as protein-calorie malnutrition. Sulfur amino acid deprivation (SAAD) increases oxidative stress through a decrease in GSH. Ferritin expression is induced by oxidative stress, which confers resistance to oxidative insults. The effects of SAAD on the changes in cellular Ca 2+ and free iron pool, prooxidant production and the ferritin light chain (FLC) expression were comparatively evaluated in Hepa1c1c7 and Raw264.7 cells. [Ca 2+] i was rapidly increased by SAAD. Sulfhydryl-containing compounds prevented the increase in [Ca 2+] i in cells under SAAD, supporting the role of redox-state in the regulation of [Ca 2+] i. Thapsigargin or Ca 2+-free medium inhibited the increase in [Ca 2+] i, showing that Ca 2+ originated from endoplasmic reticulum as well as from extracellular source. Inhibition of Ca 2+ mobilization decreased the fluorescence of Phen Green SK inside cells, representing the inhibition of free iron release. Both inhibition of Ca 2+ mobilization and iron chelation decreased dichlorofluorescein oxidation, indicating the possibility that the increase in [Ca 2+] i affected that in cellular free iron and prooxidant production. FLC protein level was immunochemically detectable in Raw264.7 cells, but not in Hepa1c1c7 cells. SAAD alone (or in combination with FeSO 4) down-regulated FLC protein expression, while SAAD increased the FLC mRNA level in both Hepa1c1c7 and Raw264.7 cells. Calcium or iron chelators prevented increases in the FLC mRNA. These results provided evidence that changes in cellular Ca 2+ and iron pool by SAAD increased cellular oxidative stress and that the down-regulation of FLC protein by SAAD would further enhance prooxidant production in spite of the increase in FLC mRNA.

Analysis of agonist-evoked nitric oxide release from human endothelial cells: role of superoxide anion.

1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production. 2. The duration of *NO release was increased only in the simultaneous presence of extracellular L-arginine and exogenous superoxide dismutase. In contrast, the inhibition of membrane reduced nicotinamide adenine dinucleotide (phosphate) oxidases, the major source of *O2- in endothelial cells, did not prolong *NO release, although extracellular L-arginine was also present. Comparison of these two experimental conditions suggested that H2O2 was involved in the extension of the *NO signal. 3. The present study demonstrates that, in the absence of external L-arginine, *O2- production does not constitute the major pathway controlling the duration of agonist-induced *NO signal. These results suggest that L-arginine and H2O2 act jointly to maintain nitric oxide synthase in an activated form.

Effect of sodium butyrate on reactive oxygen species generation by human neutrophils.

Short-chain fatty acids enema has been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms that lead to this response have not been well characterized. The aims of this study were to investigate the effect sodium butyrate has on reactive oxygen species (ROS) generation by human neutrophils, which are responsible for mucosal injury. Human neutrophils incubated with or without sodium butyrate were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). ROS generation was largely differentiated with flow cytometry assays of hydroethidine oxidation and dichlorofluorescein oxidation for superoxide anion and hydrogen peroxide respectively, and luminol-dependent chemiluminescence for myeloperoxidase-mediated oxidants. Sodium butyrate (up to 50 mM) did not alter hydroethidine oxidation upon stimulation of the OZ or PMA. However, sodium butyrate at a concentration of 25 mM elevated dichlorofluorescein oxidation to 125 + 8% (P = 0.028) of control upon stimulation of OZ and to 191 +/- 30% (P = 0.0016) upon stimulation of PMA. Contrary to these results, sodium butyrate greatly inhibited chemiluminescence responses in a dose-dependent manner. The inhibition by 50 mM sodium butyrate was 61 +/- 6% upon OZ and 71 +/- 9% upon PMA, respectively. These data indicate that sodium butyrate up-regulates hydrogen peroxide generation but down-regulates generation of myeloperoxidase-mediated oxidants, the latter being more potent in killing microorganisms and in inducing tissue injury. A possible mechanism is suggested whereby sodium butyrate may inhibit myeloperoxidase activity and hence attenuate the destructive activities of neutrophils in UC.

Effect of Sodium Butyrate on Reactive Oxygen Species Generation by Human Neutrophils

Background: Short-chain fatty acids enema has been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms that lead to this response have not been well characterized. The aims of this study were to investigate the effect sodium butyrate has on reactive oxygen species (ROS) generation by human neutrophils, which are responsible for mucosal injury. Methods: Human neutrophils incubated with or without sodium butyrate were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). ROS generation was largely differentiated with flow cytometry assays of hydroethidine oxidation and dichlorofluorescein oxidation for superoxide anion and hydrogen peroxide respectively, and luminol-dependent chemiluminescence for myeloperoxidase-mediated oxidants. Results:

Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-1 and reactive oxygen species.

This study evaluated the changes in the biomechanical properties of endothelial cells (ECs) induced by neutrophil adhesion and the roles of ICAM-1 and reactive oxygen species (ROS) in modulating these changes. Neutrophil adherence to 24-h TNF-alpha-activated pulmonary microvascular ECs induced an increase in the apparent stiffness of ECs within 2 min, measured with magnetic twisting cytometry. An anti-ICAM-1 Ab blocked the EC stiffening response without inhibiting neutrophil adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening response induced by neutrophils. The neutrophil-induced increase in the apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl radical scavenger), allopurinol (a xanthine oxidase inhibitor), or deferoxamine (an iron chelator), suggesting that ROS may be involved in mediating the EC stiffening response. The cellular sources of ROS were determined by measuring the oxidation of dichlorofluorescein. Neutrophil adherence to TNF-alpha-activated ECs induced ROS production only in ECs, and not in neutrophils. This ROS production in ECs was completely prevented by the anti-ICAM-1 Ab and partially inhibited by allopurinol. These results suggest that ICAM-1-mediated signaling events during neutrophil adherence may activate xanthine oxidase, which in turn mediates the ROS production in ECs that leads to stiffening. ROS generated in ECs on neutrophil adherence appear to mediate cytoskeletal remodeling, which may modulate subsequent inflammatory responses.

Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion.

Cardiomyocyte death after ischemia/reperfusion correlates with oxidant stress, and antioxidants confer protection in that model. Preconditioning (PC) with hypoxia or adenosine also confers protection, leading us to hypothesize that PC protects by attenuating oxidant generation during subsequent ischemia/reperfusion. Chick cardiomyocytes were preconditioned with 10 minutes of hypoxia or adenosine (100 micromol/L), followed by 1 hour of simulated ischemia and 3 hours of reperfusion. Adenosine PC decreased cell death from 50+/-3% to 18+/-4% and enhanced the return of contractions during reperfusion, as observed previously with hypoxic PC. A transient burst of dichlorofluorescein (sensitive to H2O2 oxidation that was significantly attenuated by PC initiated by hypoxia or adenosine was seen at reperfusion. The protein kinase C (PKC) inhibitor Go-6976 and the mitochondrial ATP-sensitive K(+) (K(ATP)) channel inhibitor 5-hydroxydecanoate each abolished protection and abrogated the PC-induced attenuation of reperfusion oxidant stress. By contrast, when given only at reperfusion, the K(+) channel opener pinacidil or the antioxidants 2-mercaptopropionylglycine and 1,10-phenanthroline decreased oxidant stress at reperfusion and improved survival and return of contractions. Thus, PC protection is associated with an attenuation of the oxidant burst at reperfusion, regardless of the method by which PC is triggered. Loss of PC protection associated with PKC inhibition or K(ATP) channel inhibitors is associated with a restoration of that oxidant stress. These results suggest a mechanism for PC protection and reveal a functional link between PKC activation and K(ATP) channel activation in that pathway.

Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase:: Possible implications for oxidative stress measurements

The oxidation of 2′-7′-dichlorofluorescin (DCFH) to the fluorescent 2′-7′-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H 2O 2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H 2O 2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H 2O 2, was still strongly inhibited by catalase, demonstrating that H 2O 2 was present and necessary for DCF formation. H 2O 2was apparently formed during either chemical or enzymatic deacetylation of 2′-7′-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF •−). Oxidation of DCF •− to DCF by oxygen would yield superoxide (O 2 •−). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H 2O 2/HRP system. Both radicals were also detected in the absence of added H 2O 2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O 2 •− in cells because O 2 •− itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H 2O 2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H 2O 2, the use of this probe to measure H 2O 2 production in cells is problematic.

Enalaprilat inhibits hydrogen peroxide production by murine mesangial cells exposed to high glucose concentrations.

Oxidative stress is considered to play a role in the pathogenesis of diabetic nephropathy. Angiotensin-converting enzyme (ACE) inhibitors are hypotensive drugs with a well-known effect in preventing the progression of chronic renal failure. Their mechanism of action is not clearly established. The effect of enalaprilat on hydrogen peroxide (H2O2) production by cultured murine mesangial cells exposed to 5.5 (basal condition), 30 and 50 mM glucose concentrations was examined over 8 h. A fluorimetric method quantifying, in arbitrary units, the intracellular dichlorofluorescein (DCFH) oxidation to the highly fluorescent compound 2'7'dichlorofluorescein (DCF) from the non-fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) was employed (a method not previously reported for cultured mesangial cells). Experiments were repeated three times in quadruplicate wells. H2O2 production by mesangial cells exposed to 50 mM glucose was significantly increased after 1 h, compared to cells exposed to 5.5 and 30 mM glucose. This observation was not reproduced with 50 mM mannitol. Addition of 100 ng/ml enalaprilat to cells with 50 mM glucose significantly inhibited H2O2 production during the 8 h of the assay. This response was similar to that obtained with 100 ng/ml catalase. Increasing enalaprilat concentrations (10, 50 and 100 ng/ml) also significantly decreased the constitutive H2O2 generation in the presence of 5.5 mM glucose. Angiotensin II and saralasin, both at 1 microM, did not modify H2O2 production by cells exposed to 5.5 mM glucose. In contrast, 1 microM staurosporine, a protein kinase C (PKC) antagonist, significantly decreased H2O2 generation in the presence of 50 mM glucose. Enalaprilat has an antioxidant effect in cultured mesangial cells. This action is not linked to ACE inhibition, but may be related to an inhibition of the PKC system.

Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion

We have examined H2O2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H2O2 content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-fold). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhancement of H2O2 production by IL-beta-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentration of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 beta-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesion to HSE there was significantly (P < 0.05) enhancement of H2O2 content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1 Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 micrograms/ml anti-mouse IL-1 beta neutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1 beta (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H2O2. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells.

Alterations in reactive oxygen, pH, and calcium in astrocytoma cells during lethal injury.

Exposure of cultured human astrocytoma cells to iodoacetic acid results in rapid depletion of cellular ATP and cell death. Pathophysiological changes in the injured cells, including formation of reactive oxygen species (ROS), cell viability, glutathione, pH, and cytosolic calcium, were characterized at the cellular level via fluorescence microscopy. After iodoacetic acid treatment, cellular ATP and intracellular glutathione fell sharply to undetectable levels within 2 h. ROS, as detected by the oxidation of dichlorofluorescein, appeared in 20 min and reached a maximum before the loss of membrane integrity. Cells became acidotic within 10 min. Cytosolic free calcium concentration exhibited a slow increase and then a sharp influx shortly before the rupture of the cell membrane. The addition of lipophilic antioxidants, nordihydroguaiaretic acid or the troloxamine U-78517F, eliminated the accumulation of ROS and delayed the onset of cell death without affecting other parameters observed in the early phase of the injury. We conclude that ROS is formed and may play important roles during lethal cell injury caused by energy depletion.

Oxidative stress, antioxidant capacity and ethylene production during ageing of cut carnation (Dianthus caryophyllus) petals

The objective of this work was to study the role of free radicals in relation to ethylene production during ageing of cut carnation petals. Ethylene production by freshly cut flowers was negligible, but 8 d after cutting ethylene production began to increase and reached a peak by day 9, before beginning to decline again. The efflux of electrolytes (membrane damage index) increased 101% and 2 ,7-dichlorofluorescein oxidation rate (oxidative stress index) increased 53% from day 8 to day 11 after detachment. Ethylene peak was either (a) not affected significantly by the supplementation of exogenous ethylene on the day of cutting, or (b) expanded between days 7-9 after ethylene supplementation on day 6 of cutting, or (c) was inhibited by amino-oxyacetic acid and paraquat treatments. After ethylene supplementation, conductivity and 2,7-dichlorofluorescein oxidation increased significantly as compared to control petals, and the activity of antioxidant enzymes was not affected. However, both w-tocopherol and glutathione content decreased significantly after ethylene supplementation on day 6 after detachment. Amino-oxyacetic acid treatment prevented the increases in conductivity and 2,7dichlorofluorescein oxidation, did not alter the activities of antioxidant enzymes and significantly increased the content of a-tocopherol and glutathione as compared to control carnation petals. Paraquat treatment paralleled qualitatively ethylene supplementation after 6 d of cutting. Taken as a whole, the data presented here may be understood as experimental evidence of a close association between ethylene production and oxidative stress in ageing of cut carnations.

Syndrome de Pickwick et syndrome d'apnées du sommeil: Étude de 21 patients

The leaves of Syzygium cumini (L.) or Skeels (Myrtaceae) are widely used in Brazilian folk medicine to treat diabetes.The present study evaluated the functional capacity, biochemical parameters, oxidative stress and DNA damage from eight weeks of intervention with a crude hydroalcoholic extract of S. cumini leaves (EBH) and continuous aerobic training (TAC) in diabetic (D) rats.A hydroalcoholic (50%) extract was prepared by ultrasound and phytochemical parameters (total phenols, total tannins and myricetin content) were analyzed. Thirty-seven male Wistar rats were divided into five groups: normoglycemic controls (CONT), diabetic controls (D-CONT), diabetics treated with extract (D+EBH), trained diabetic (D+TAC) and diabetics treated with extract and trained (D+EBH+TAC). Functional capacity was assessed with a maximum exercise capacity test; biochemical parameters with enzymatic kits; oxidative stress by superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and oxidized dichlorofluorescein (DCF), and the DNA damage by the comet assay.The D+TAC and D+EBH+TAC groups showed better functional capacity at the end of interventions. The D+EBH group showed glucose and triglyceride reduction, lowest DNA damage index in the blood, liver, kidney, heart, lung and gastrocnemius muscle, improved SOD levels in the liver, kidney and lung, improved CAT levels in the kidney and lower lipid peroxidation in all tissues studied, compared to the D-CONT group. The exercise (D+TAC) was effective in reducing triglycerides, improving SOD levels in the lung, reducing lipid peroxidation in all tissues studied and reducing the DCF oxidation in the kidney, in addition to protecting against DNA damage in the blood and heart. However, the additive effect of the intervention protocols when combined (EBH+TAC) was observed only in improving the gastrocnemius SOD levels. The phytochemical analyses showed a high content of phenols and the presence of myricetin glycosides.The findings in this study suggest a crude hydroalcoholic extract of S. cumini leaves has potential hypoglycemic, hypolipidemic and protective properties acting against oxidative stress and against DNA damage, probably due to its phenols and myricetin glycoside content and the antioxidant properties of these constituents. Moreover, exercise was suggested to have beneficial effects on diabetes, improving functional capacity, ameliorating blood triglyceride control and decreasing lipid peroxidation, but with no effects on ameliorating blood glucose levels. The association of intervention protocols presented an additive effect on the antioxidant SOD activity in the muscle cells of diabetic rats.

Effect of ageing on oxygen radical generation by soybean seeds

SynopsisRates of leakage of protein and electrolytes were measured in soybean embryonic axes from soybean seeds stored for 8–42 months. A significant increase in the content of proteins and electrolytes was determined in the external medium where aged embryonic axes were incubated. Lipid peroxidation was evaluated by the measurement of thiobarbituric acid-reactive substances (TBARS) in isolated axes from aged seeds. No significant differences were detected either in the contentin vivonor in the rate of generationin vitro. To assessin vivooxidative condition of the axes, an assay employing 2′,7′-dichlorofluorescein (DCFH-DA) oxidation was used. Embryonic axes from seeds stored for 8 and 42 months showed 1.1±0.1 and 4.1±0.1 fluorescence units per min per axes, respectively, after 24 h of imbibition. This significant increase in fluorescence was measured at all the tested incubation times. Antioxidant defences, evaluated as chemiluminescence induced by t-butyl hydroperoxide and α-tocopherol content, showed a significant increase with storage time. These data suggest that the damage due to enhanced peroxidative events are limited by the increased amount of potent lipid soluble antioxidant.

C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils.

The acute phase protein, C-reactive protein (CRP), when heat-aggregated (Agg-CRP), potentiates immunoglobulin G (IgG) Fc receptor-mediated luminol-enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of monocytes and neutrophils to Agg-CRP, superoxide anion release was measured by cytochrome c reduction. In addition, the extracellular release of hydrogen peroxide was distinguished from hydrogen peroxide generation using a phenol red oxidation assay. Finally, a flow cytometric determination of dichlorofluorescein (DCFH) oxidation was employed as an index of intracellular peroxide production. Although Agg-CRP alone did not stimulate hydrogen peroxide generation by either monocytes or neutrophils, it significantly enhanced hydrogen peroxide generation in response to heat-aggregated IgG (Agg-IgG). In contrast, Agg-CRP did not enhance the extracellular release of either hydrogen peroxide or superoxide anion from Agg-IgG-stimulated cells. The capacity of Agg-CRP to enhance selectively intracellular oxidative product generation was confirmed when measuring DCFH oxidation in Agg-IgG-stimulated cells. To evaluate whether this selective enhancement of intracellular oxidative events could be attributed, at least in part, to a scavenging effect of Agg-CRP, a cell-free oxygen radical-generating system was employed. Agg-CRP did not significantly diminish the lucigenin-amplified CL response induced by the xanthine/xanthine oxidase reaction. These results indicate that although Agg-CRP enhances the intracellular generation of reactive oxygen intermediates by monocytes and neutrophils, extracellular release of those products is not influenced by cell interaction with Agg-CRP. It is tempting to speculate that CRP can selectively boost the microbicidal activities of monocytes and neutrophils within an inflammatory site by amplifying the intracellular generation of reactive oxygen products without increasing damage to surrounding normal tissues.

Generation of hydrogen peroxide in resting and activated platelets.

The production of hydrogen peroxide was measured by following the oxidation of dichlorofluorescein (DCFH) entrapped into platelets. Resting platelets produced nanomolar quantities of DCF, which was proportional to the concentration of platelets and was steady during 1 h of incubation. A significant increase of basal DCF fluorescence was induced by stimuli namely thrombin, arachidonic acid, the Ca2+ ionophore A23187 and PMA. The effect of agonists has been also measured in the presence of 3-amino-1,2,4-triazole (AT) or N-ethylmaleimide (NEM), inhibitors of catalase and glutathione peroxidase, respectively. A further significant enhancement of DCF produced in stimulated platelets was detected only in the presence of NEM. A correlation was found between the increase in DCF and externally added hydrogen peroxide or the oxidizing species formed by xanthine oxidase plus acetaldehyde. The yield was not affected by superoxide dismutase and was higher in the presence of AT or NEM. A cooperative effect in the presence of both inhibitors was shown. Glutathione peroxidase plus glutathione diminished the level of DCF to basal levels.