Reactive oxygen species are involved in regulation of pollen wall cytomechanics

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H₂O₂ and the hydroxyl radical (^?OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H₂O₂, ^?OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn‐5,10,15,20‐tetrakis(1‐methyl‐4‐pyridyl)21H, 23H‐porphin, or a spin‐trap α‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone, which eliminates ^?OH. The inhibiting concentrations of exogenous H₂O₂ and ^?OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo‐osmotic shock. ^?OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H₂O₂, as well as partial removal of endogenous ^?OH, over‐tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H₂O₂‐inducible inter‐polymer cross‐linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross‐links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. ^?OH is involved in local loosening of the intine in the germination pore region, while H₂O₂ is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.     

2D Amorphous V2O5/Graphene Heterostructures for High‐Safety Aqueous Zn‐Ion Batteries with Unprecedented Capacity and Ultrahigh Rate Capability

Rechargeable aqueous zinc‐ion batteries (ZIBs) are appealing due to their high safety, zinc abundance, and low cost. However, developing suitable cathode materials remains a great challenge. Herein, a novel 2D heterostructure of ultrathin amorphous vanadium pentoxide uniformly grown on graphene (A‐V₂O₅/G) with a very short ion diffusion pathway, abundant active sites, high electrical conductivity, and exceptional structural stability, is demonstrated for highly reversible aqueous ZIBs (A‐V₂O₅/G‐ZIBs), coupling with unprecedented high capacity, rate capability, long‐term cyclability, and excellent safety. As a result, 2D A‐V₂O₅/G heterostructures for stacked ZIBs at 0.1 A g^?1 display an ultrahigh capacity of 489 mAh g^?1, outperforming all reported ZIBs, with an admirable rate capability of 123 mAh g^?1 even at 70 A g^?1. Furthermore, the new‐concept prototype planar miniaturized zinc‐ion microbatteries (A‐V₂O₅/G‐ZIMBs), demonstrate a high volumetric capacity of 20 mAh cm^?3 at 1 mA cm^?2, long cyclability; holding high capacity retention of 80% after 3500 cycles, and in‐series integration, demonstrative of great potential for highly‐safe microsized power sources. Therefore, the exploration of such 2D heterostructure materials with strong synergy is a reliable strategy for developing safe and high‐performance energy storage devices.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^•−) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^•− and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^•− and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.     

A stopped‐flow study of the dual chemiluminescence for the luminol–KIO4–Mn2+ system in strong alkaline solutions

A novel phenomenon of dual chemiluminescence (CL) was observed for the KIO₄–luminol–Mn²⁺ system in strong alkaline solutions using the stopped‐flow technique. Scavenging study of the reactive oxygen species (ROS) suggested that the two CL peaks originated from different CL pathways precipated by distinct ROS (O₂^? and ^?OH for the first peak, mainly ¹O₂ for the second peak). Generation of these ROS at different time intervals from the reactions involving IO₄^?, O₂, and Mn²⁺ and their subsequent reactions with luminol induced the intense CL emission. The relative intensity of the two CL peaks can be tuned over a wide range by varying the concentrations of Mn^2?, luminol and KIO₄. Because of the involvement of different ROS in each pathway, the two CL peaks could respond quite differently to various substances. Moreover, variation of the intensity ratio of the two CL peaks altered the relative proportions of the corresponding ROS, thereby changing their responses to a given substance. The dual CL emission acts like a pair of tunable probes and it is believed that this CL system has great potential in analytical applications. Copyright © 2010 John Wiley & Sons, Ltd.     

Cross-talk between salicylic acid and NaCl-generated reactive oxygen species and nitric oxide in tomato during acclimation to high salinity

Hydrogen peroxide (H₂O₂) and nitric oxide (NO) generated by salicylic acid (SA) are considered to be functional links of cross‐tolerance to various stressors. SA‐stimulated pre‐adaptation state was beneficial in the acclimation to subsequent salt stress in tomato (Solanum lycopersicum cv. Rio Fuego). At the whole‐plant level, SA‐induced massive H₂O₂ accumulation only at high concentrations (10^?3–10^?2M), which later caused the death of plants. The excess accumulation of H₂O₂ as compared with plants exposed to 100 mM NaCl was not associated with salt stress response after SA pre‐treatments. In the root tips, 10^?3–10^?2M SA triggered the production of reactive oxygen species (ROS) and NO with a concomitant decline in the cell viability. Sublethal concentrations of SA, however, decreased the effect of salt stress on ROS and NO production in the root apex. The attenuation of oxidative stress because of high salinity occurred not only in pre‐adapted plants but also at cell level. When protoplasts prepared from control leaves were exposed to SA in the presence of 100 mM NaCl, the production of NO and ROS was much lower and the viability of the cells was higher than in salt‐treated samples. This suggests that, the cross‐talk of signalling pathways induced by SA and high salinity may occur at the level of ROS and NO production. Abscisic acid (ABA), polyamines and 1‐aminocyclopropane‐1‐carboxylic acid, the compounds accumulating in pre‐treated plants, enhanced the diphenylene iodonium‐sensitive ROS and NO levels, but, in contrast to others, ABA and putrescine preserved the viability of protoplasts.     

Rational design of an iminocoumarin-based fluorescence probe for peroxynitrite with high signal-to-noise ratio

As a high reactive oxygen species (ROS) and a reactive nitrogen species (RNS), peroxynitrite anion (ONOO⁻) is widely present in organisms and plays influential roles in physiological and pathological processes. It is of great significance to develop effective fluorescent probes for imaging peroxynitrite variation in living systems. Herein we present a novel fluorescent probe TQC0 for monitoring ONOO⁻ based on the iminocoumarin platform, and this probe was synthesized by the knoevenagel condensation between a dihydropyridine-salicylaldehyde derivative and 2-benzothiazole-acetonitrile, and subsequently masked with the boronate moiety. The obtained probe TQC0 exhibited a high signal-to-noise ratio (206-fold) and a quick ‘turn-on’ response (about 10 min) with great selectivity and sensitivity. Furthermore, the probe TQC0 was successfully applied for imaging ONOO⁻ in living cells with low cytotoxicity.     

Design and synthesis of a new fluorescent probe for cascade detection of Zn2+ and H2PO4− in water and targeted imaging of living cells

Design and synthesis of new fluorescence probes with good water‐solubility is of great importance to better understanding the significant role of ions which are related to biology and the environment. As important ions, zinc ion (Zn²⁺) and dihydrogen phosphate ion (H₂PO₄^?) display essential roles in living systems, and quantitative detection of these ions in water is still a challenge. In order to consider the significant role of the galactose moiety in the design of a water‐soluble fluorescence sensor, herein, we have developed a novel probe, Gal‐AQTF, for the cascade detection of Zn²⁺ and H₂PO₄^? with excellent selectivity in water. Through the introduction of the galactose moiety onto the sensor AQTF, which has been reported earlier by us, the water‐solubility, cell compatibility and targeting ability were enhanced. Gal‐AQTF has been successfully applied in the imaging of the living cells of HepG2 and A549, and illustrated good selectivity for the HepG2 cells which overly express the asialoglycoprotein (ASGP) receptor.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Hierarchical NiCo2S4@NiO Core–Shell Heterostructures as Catalytic Cathode for Long‐Life Li‐O2 Batteries

The critical challenges of Li‐O₂ batteries lie in sluggish oxygen redox kinetics and undesirable parasitic reactions during the oxygen reduction reaction and oxygen evolution reaction processes, inducing large overpotential and inferior cycle stability. Herein, an elaborately designed 3D hierarchical heterostructure comprising NiCo₂S₄@NiO core–shell arrays on conductive carbon paper is first reported as a freestanding cathode for Li‐O₂ batteries. The unique hierarchical array structures can build up multidimensional channels for oxygen diffusion and electrolyte impregnation. A built‐in interfacial potential between NiCo₂S₄ and NiO can drastically enhance interfacial charge transfer kinetics. According to density functional theory calculations, intrinsic LiO₂‐affinity characteristics of NiCo₂S₄ and NiO play an importantly synergistic role in promoting the formation of large peasecod‐like Li₂O₂, conducive to construct a low‐impedance Li₂O₂/cathode contact interface. As expected, Li‐O₂ cells based on NiCo₂S₄@NiO electrode exhibit an improved overpotential of 0.88 V, a high discharge capacity of 10 050 mAh g^?1 at 200 mA g^?1, an excellent rate capability of 6150 mAh g^?1 at 1.0 A g^?1, and a long‐term cycle stability under a restricted capacity of 1000 mAh g^?1 at 200 mA g^?1. Notably, the reported strategy about heterostructure accouplement may pave a new avenue for the effective electrocatalyst design for Li‐O₂ batteries.     

Non-invasive Analysis of Reactive Oxygen Species Generated in NH4OH-induced Gastric Lesions of Rats using a 300 MHz In Vivo ESR Technique

Free radicals are reportedly involved in mucosal injury, including NH⁴OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH⁴OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300?MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30?min after NH⁴OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to ?OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH⁴OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH⁴OH-treated living rats that ?OH produced from O₂^?- derived from neutrophils caused gastric lesion formation, while O₂^?- or H²O² derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration.     

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non‐phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H₂O₂ on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti‐angiogenesis. Our results showed that H₂O₂ dose‐dependently increased the generation of O₂ ^? (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H₂O₂ at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H₂O₂ at 4 nmol/cm² strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm²). Also, H₂O₂ (200～500 µM) could stimulate apoptosis in HUVECs. All the effects of H₂O₂ on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H₂O₂ to produce O₂ ^? and then to regulate angiogenesis. In summary, our results suggest that H₂O₂ as well as O₂ ^? mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.     

Synthesis and activity of a coumarin‐based fluorescent probe for hydroxyl radical detection

As a type of reactive oxygen species (ROS), hydroxyl radical (·OH) is closely associated with many kinds of diseases. The present study aimed to develo p a novel OH fluorescent probe based on coumarin, a new compound that has not been previously reported. This probe exhibited good linear range and selectivity for ·OHl, and is able to avoid interference from some metal ions and other kinds of ROS (H₂O₂, O₂^.‐, ¹O₂, and HClO). Meanwhile, this probe has been used to evaluate the ·OH‐scavenging efficiency of different compounds, such as isopropyl alcohol, cytosine, uracil, Tempo, Glutathione (GSH), and dimethyl sulfoxide (DMSO). Therefore, the present study shows that this probe not only can effectively measure the level of ·OH, but also can assess the ·OH‐scavenging efficiency of different compounds. Furthermore this current study suggested that following further optimization, this probe may be potentially applied in the diagnosis of oxidative stress in human body.     

Nitric oxide interferes with plant photo-oxidative stress by detoxifying reactive oxygen species

Oxidative stress within chloroplasts is originated due to light‐dependent O₂ reduction. This may be exacerbated by bipyridinium herbicides, which act at photosystem I as artificial electron acceptors. Their oxidation produces a superoxide anion that further dismutates to H₂O₂ and then, by the Fenton reaction, H₂O₂ may be reduced to the hydroxyl radical (OH^?). Reactive oxygen species (ROS), when produced in high amounts, provoke severe damage to the plant cell. Herein it is reported that two nitric oxide (NO) donors, sodium nitroprusside (100 µm ) and S‐nitroso‐N‐acetylpenicillamine (200 µm ), greatly reduced lipid peroxidation and the protein loss caused by the application of a high dose of the bipyridinium herbicide diquat to potato leaf pieces or isolated chloroplasts. Nitric oxide donors also protected the RNA against oxidative damage. Photo‐oxidative toxicity was correlated with an increase in photosynthetic electron transport and ROS production, but the rate of electron transport was restored and the ROS free amount was markedly reduced in the presence of NO. The specific activity of superoxide dismutase was not affected by diquat or NO donors, whereas just a small increase in catalase activity was observed after 24 h of treatment. These results provide strong evidence that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge ROS.     

An OFF–ON–OFF type fluorescent probe based on a naphthalene derivative for Al3+ and F− ions and its biological application

A novel fluorescent probe‐based naphthalene Schiff, 1‐(C2‐glucosyl‐ylimino‐methyl)‐naphthalene‐2‐ol (L) was synthesized by coupling d ‐glucosamine hydrochloride with 2‐hydroxy‐1‐naphthaldehyde. It exhibited excellent selectivity and highly sensitivity for Al³⁺ in ethanol with a strong fluorescence response, while other common metal ions such as Pb²⁺, Mg²⁺, Cu²⁺, Co²⁺, Ni²⁺, Cd²⁺, Fe²⁺, Mn²⁺, Hg²⁺, Li⁺, Na⁺, K⁺, Fe³⁺, Cr³⁺, Zn²⁺, Ag⁺, Ba²⁺ and Ca²⁺ did not cause the same fluorescence response. The probe selectively bound Al³⁺ with a binding constant (K_a) of 5.748 × 10³ M^?1 and a lowest detection limit (LOD) of 4.08 nM. Moreover, the study found that the fluorescence of the L ? Al³⁺ complex could be quenched after addition of F^? in the same medium, while other anions, including Cl^?, Br^?, I^?, NO₂^?, NO₃^?, ClO₄^?, CO₃^2?, HCO₃^?, SO₄^2?, HSO₄^?, CH₃COO^?, PO₄^3?, HPO₄^2?, S^2? and S₂O₃^2? had nearly no influence on probe behaviour. Binding of the [L ? Al³⁺] complex to a F^? anion was established by different fluorescence titration studies, with a detection limit of 3.2 nM in ethanol. The fluorescent probe was also successfully applied in the imaging detection of Al³⁺ and F^? in living cells.     

Co–B Nanoflakes as Multifunctional Bridges in ZnCo2O4 Micro‐/Nanospheres for Superior Lithium Storage with Boosted Kinetics and Stability

Transition metal oxides hold great promise as high‐energy anodes in next‐generation lithium‐ion batteries. However, owing to the inherent limitations of low electronic/ionic conductivities and dramatic volume change during charge/discharge, it is still challenging to fabricate practically viable compacted and thick TMO anodes with satisfactory electrochemical performance. Herein, with mesoporous cobalt–boride nanoflakes serving as multifunctional bridges in ZnCo₂O₄ micro‐/nanospheres, a compacted ZnCo₂O₄/Co–B hybrid structure is constructed. Co–B nanoflakes not only bridge ZnCo₂O₄ nanoparticles and function as anchors for ZnCo₂O₄ micro‐/nanospheres to suppress the severe volume fluctuation, they also work as effective electron conduction bridges to promote fast electron transportation. More importantly, they serve as Li⁺ transfer bridges to provide significantly boosted Li⁺ diffusivity, evidenced from both experimental kinetics analysis and density functional theory calculations. The mesopores within Co–B nanoflakes help overcome the large Li⁺ diffusion barriers across 2D interfaces. As a result, the ZnCo₂O₄/Co–B electrode delivers high gravimetric/volumetric/areal capacities of 995 mAh g^?1/1450 mAh cm^?3/5.10 mAh cm^?2, respectively, with robust rate capability and long‐term cyclability. The distinct interfacial design strategy provides a new direction for designing compacted conversion‐type anodes with superior lithium storage kinetics and stability for practical applications.     

Early burst of reactive oxygen species positively regulates resistance of eggplant against bacterial wilt

Reactive oxygen species (ROS) play a crucial role in the early response to plant biotic and abiotic stresses. In this study, bacterial wilt‐resistant and wilt‐susceptible eggplants were inoculated with Ralstonia solanacearum and the ROS content was analysed. The result revealed an increased accumulation of hydrogen peroxide (H₂O₂) and superoxide (O₂^?) in resistant and susceptible eggplant roots after R. solanacearum inoculation. H₂O₂ and O₂^? accumulation increased earlier in the inoculated resistant eggplant root than in the inoculated susceptible eggplant root. Real‐time polymerase chain reaction results revealed that respiratory burst oxidase homologue (Rboh) A, RbohB, RbohF and PR1 expression levels increased in inoculated resistant eggplant roots at an early stage (0–60 h postinoculation) and were at higher expression levels than those in susceptible eggplant roots. Ascorbate peroxidase, peroxidase and catalase activities were higher in inoculated resistant eggplant roots than in susceptible eggplant roots at the early stage. Hence, an early ROS burst positively regulates bacterial wilt resistance in eggplant.     

Pycnogenol enhances endothelial cell antioxidant defenses

Summary

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂^?), and hydroxyl radical (OH^?) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes—superoxide dismutase (SOD) and catalase (CAT)—play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H₂O₂ and O₂^? and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H₂O₂. Pycnogenol caused a concentration-dependent enhancement of H₂O₂ and O₂^? clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems.