Hydroxyl Free Radical Formation Research Articles

Eradication of Pseudomonas aeruginosa biofilms and persister cells using an electrochemical scaffold and enhanced antibiotic susceptibility.

Biofilms in chronic wounds are known to contain a persister subpopulation that exhibits enhanced multidrug tolerance and can quickly rebound after therapeutic treatment. The presence of these “persister cells” is partly responsible for the failure of antibiotic therapies and incomplete elimination of biofilms. Electrochemical methods combined with antibiotics have been suggested as an effective alternative for biofilm and persister cell elimination, yet the mechanism of action for improved antibiotic efficacy remains unclear. In this work, an electrochemical scaffold (e-scaffold) that electrochemically generates a constant concentration of H2O2 was investigated as a means of enhancing tobramycin susceptibility in pre-grown Pseudomonas aeruginosa PAO1 biofilms and attacking persister cells. Results showed that the e-scaffold enhanced tobramycin susceptibility in P. aeruginosa PAO1 biofilms, which reached a maximum susceptibility at 40 µg/ml tobramycin, with complete elimination (7.8-log reduction vs control biofilm cells, P ≤ 0.001). Moreover, the e-scaffold eradicated persister cells in biofilms, leaving no viable cells (5-log reduction vs control persister cells, P ≤ 0.001). It was observed that the e-scaffold induced the intracellular formation of hydroxyl free radicals and improved membrane permeability in e-scaffold treated biofilm cells, which possibly enhanced antibiotic susceptibility and eradicated persister cells. These results demonstrate a promising advantage of the e-scaffold in the treatment of persistent biofilm infections.

Corneal Endothelium and Phacoemulsification.

Phacoemulsification has become the most commonly used procedure in cataract surgery owing to the development of new devices and surgical techniques; however, corneal endothelial damage still represents a serious complication. Although several factors are known to cause corneal endothelial damage, effects of irrigating pressure and temperature rise in the anterior chamber should also be considered. Furthermore, free radical formation is another important harmful factor. Ultrasound in aqueous solution induces cavitation, which results in the formation of hydroxyl free radicals that damage the corneal endothelium. The best protection is the use of ophthalmic viscosurgical devices. In addition, the use of H2 gas as an anti-free radical agent in the irrigating solution is discussed. At present, corneal endothelial injury is usually assessed using the limited range of specular microscopy. Corneal volume might be a new parameter with which to assess total corneal endothelial function.

Antioxidant Activity of Caffeic Acid against Iron-Induced Free Radical Generation--A Chemical Approach.

Caffeic acid (CA) is a phenolic compound widely found in coffee beans with known beneficial effects in vivo. Many studies showed that CA has anti-inflammatory, anti-mutagenic, antibacterial and anti-carcinogenic properties, which could be linked to its antioxidant activity. Taking in consideration the reported in vitro antioxidant mechanism of other polyphenols, our working hypothesis was that the CA antioxidant activity could be related to its metal-chelating property. With that in mind, we sought to investigate the chemical antioxidant mechanism of CA against in vitro iron-induced oxidative damage under different assay conditions. CA was able to prevent hydroxyl radical formation promoted by the classical Fenton reaction, as determined by 2-deoxyribose (2-DR) oxidative degradation and DMPO hydroxylation. In addition to its ability to prevent hydroxyl radical formation, CA had a great inhibition of membrane lipid peroxidation. In the lipid peroxidation assays CA acted as both metal-chelator and as hydrogen donor, preventing the deleterious action promoted by lipid-derived peroxyl and alkoxyl radicals. Our results indicate that the observed antioxidant effects were mostly due to the formation of iron-CA complexes, which are able to prevent 2-DR oxidation and DMPO hydroxylation. Noteworthy, the formation of iron-CA complexes and prevention of oxidative damage was directly related to the pH of the medium, showing better antioxidant activity at higher pH values. Moreover, in the presence of lipid membranes the antioxidant potency of CA was much higher, indicating its enhanced effectiveness in a hydrophobic environment. Overall, our results show that CA acts as an antioxidant through an iron chelating mechanism, preventing the formation of free hydroxyl radicals and, therefore, inhibiting Fenton-induced oxidative damage. The chemical properties of CA described here—in association with its reported signaling effects—could be an explanation to its beneficial effects observed in vivo.

Cu(II)-vitamin D interaction leads to free radical-mediated cellular DNA damage: a novel putative mechanism for its selective cytotoxic action against malignant cells.

Vitamin D (vit D) is a known anticancer molecule, and cancer cells are reported to have elevated levels of Cu(II) ions. In this study, we show that interaction of vit D and Cu(II) leads to the formation of hydroxyl free radicals, superoxide anion and hydrogen peroxide, which causes severe oxidative stress, selectively in malignant cells. We show that the production of these reactive oxygen species causes cellular DNA fragmentation which may cause cell death. A novel putative chemical mechanism explaining how vit D causes cell death by DNA damage, selectively in malignant cells, is proposed.

IS-039 Antioxidant Strategies: Where Are We Now?

<h3></h3> Part of asphyxia-related brain damage occurs <b><i>upon </i></b>reoxygenation. Renewed availability of oxygen activate biochemical pathways and neuronal cell death. Important pathways are: 1) Calcium-induced formation of neurotransmitters; 2) formation of (pro-) radicals; 3) activation of inflammation; 4) induction of apoptosis; 5) depletion of growth factors. Four important sources of free radicals are: 1) Nitric oxide (NO)-related formation of peroxynitrite. It is reported that selective iNOS/eNOS inhibitor 2-iminobiotin induced neuroprotection after asfyxia in animal models. 2) Pro-radicals, such as non proteinbound-iron (NPBI), lead to formation of hydroxyl free radicals. NPBI chelation with deferoxamine, which has also a stabilising effect on HIF1-alpha and stimulates trophic factors, showed encouraging results in experimental models. 3) Formation of superoxide radical by metabolisation by xanthine-oxidase (XO) can be blocked by XO-inhibitors such as allopurinol. 4) Metabolisation of arachidonic acid to prostaglandin leading to superoxide can be blocked by cyclo-oxygenase inhibitors. Since XO-derived superoxide occurs upon reoxygenation after asphyxia, a trial with allopurinol to the mother with signs of perinatal fetal hypoxia has been started. Activation of inflammatory factors after asphyxia is recognised to be related to post-aphyxial brain damage. Rather than monotherapy directed to one pathway, a combination of drugs intervening in various pathways in relation with the time-profile of these pathways, might achieve optimal reduction of reperfusion injury.

Studies on SnCl2-doped TiO2 photocatalyst for Pyrocatechol Photodegradation

SnCl2-doped TiO2 photocatalyst for pyrocatechol photodegradation aimed to examine the effect of Sn/Ti ratio and H2O2+UVC irradiation. The preparation was carried out by the impregnation and the characterization consisted of WAXD, SEM, and laser particle size. Using 1 mM pyrocatechol in the CSTR with various TiO2 photocatalysts, the remaining pyrocatechol was determined by UV-vis spectrophotometry to calculate % photodegradation. From WAXD results, it was found that SnO2 incorporation to the TiO2 bulk phase but the morphologies did not change. And the particle size changed due to the attraction force of the surface charging. Without H2O2 addition, it showed 15.7% photodegradation by TiSn10UVC compared to 11.37% of TiDark. Thus, UVC was the important factor for photodegradation. With H2O2+UVC irradiation, the maximum photodegradation reached 34.6% by TiSn10HOUVC, but those of HOUVC and TiHOUVC showed 25.0% and 26.5%, respectively. The Sn-doping affected the formation of more hydroxyl free radical from H2O2+UVC irradiation and charge separation. But higher Sn/Ti ration, it gave segregate SnO2 which increased recombination center with the adverse effect on the lower photodegradation. The conclusion revealed that SnCl2-doping with the ratio of Sn/Ti =10 onto TiO2 photocatalyst showed the highest photoactivity with H2O2+UVC irradiation. Moreover, the chemical kinetics is also discussed.

Advanced Treatment of Sodium Acetate in Water by Ozone Oxidation

Ozone oxidation is an advanced oxidation process for treatment of organic and inorganic wastewater. In this paper, sodium acetate (according to chemical oxygen demand [COD]) was selected as the model pollutant in water, and the degradation efficiencies and mechanism of sodium acetate in water by ozone oxidation were investigated. The results showed that the ozone oxidation was an effective treatment technology for advanced treatment of sodium acetate in water; the COD removal rate obtained the maximum value of 45.89% from sodium acetate solution when the pH value was 10.82, ozone concentration was 100 mg/L, reaction time was 30 minutes, and reaction temperature was 25 degrees C. The COD removal rate increased first and decreased subsequently with the bicarbonate (HCO3-) concentration from 0 to 200 mg/L, the largest decline being 20.35%. The COD removal rate declined by 25.38% with the carbonate (CO3(2-)) concentration from 0 to 200 mg/L; CO3(2-) has a more obvious scavenging effect to inhibit the formation of hydroxyl free radicals than HCO3-. Calcium chloride (CaCl2) and calcium hydroxide (Ca(OH)2) could enhance the COD removal rate greatly; they could reach 77.35 and 96.53%, respectively, after a reaction time of 30 minutes, which was increased by 31.46 and 50.64%, respectively, compared with only ozone oxidation. It was proved that the main ozone oxidation product of sodium acetate was carbon dioxide (CO2), and the degradation of sodium acetate in the ozone oxidation process followed the mechanism of hydroxyl free radicals.

Overview and Prospect of Strengthen Ozone Oxidation Technology in Water Treatment

Based on the enhanced formation of hydroxyl free radical (OH), Strengthen ozone oxidation can intensify the oxidizing ability and lower reaction selectivity as a kind of the Advanced Oxidation Technologies (AOTs). This paper introduces the research progress of strengthen ozone oxidation technology such as H2O2 strengthen, catalytic strengthen, UV strengthen, ultrasound strengthen, hydrodynamic cavitations strengthen et al, and points out the research focus and application prospects of this technology.

SU-E-I-78: Neuromelanin in the Subthalamic Nucleus of Patients with Parkinson's Disease: An Electron Spin Resonance Spectroscopy Study.

Parkinson disease and related syndromes are associated directly with the concentrations of neuromelanin, iron and other heavy metals, and nowadays it is discussed the possible protective role of neuromelanin by the sequester redox active iron ions, reducing the formation of free hydroxyl radicals and therefore inactivating the iron ions that induce oxidative stress. The aim of this work is to study the concentration ratios between iron ions and neuromelanin in subthalamic nucleus of patients with Parkinson's disease (PD) using Electron Spin Resonance (ESR). Necropsy samples of subthalamic nucleus from eight human brains were studied: three non-affected by any neurodegenerative disease and five with Parkinson's disease. The samples were stored in formaldehyde and washed with a solution of 0.01 molar of ethylenediaminetetraacetic acid. ESR experiments were development in a JEOL FA-200 X-Band spectrometer at different temperatures between -170° C to room temperature. The relative concentrations of each species were estimated from the double integral values of the fitted spectra. For all samples, ESR spectra showed to be composed of three different signals following the Curie's law. One signal was attributed to high-spin ferric ions (g∼ 4.3) in rhomboedric symmetry, Cu(II) ions (close to g=2.0) and neuromelanin (g∼ 2.01). The ferric ions concentration ratio between patients and controls was 3.0±0.2. The same ratio for neuromelanine was 0.24±0.06. Our preliminary results indicated a significant increment of iron concentration in PD samples which agrees with previous histochemical and biochemical reports. This finding and the clear reduction of neuromelanin concentration in PD samples suggest the possible role of neuromelanin as iron ions storage.

Can the soil bacterium Cupriavidus necator sense ZnO nanomaterials and aqueous Zn2+ differentially?

Synthetic metal oxide nanomaterials exert toxicity via two general mechanisms: Release of free ions at concentrations which exert toxic effects upon the target cell, or specific surface-mediated physicochemical processes leading to the formation of hydroxyl free radicals and other reactive oxygen species which act to disrupt cell membranes and organelles. From a regulatory standpoint this presents a potential problem since it is not trivial to detect free metal ions in the presence of nanoparticles in biological or natural media. This makes efforts to identify the route of uptake difficult. Although in vitro studies of zinc oxide nanoparticles suggest that toxicity to the soil bacterium Cupriavidus necator is exerted in a similar manner to zinc acetate, we found no free Zn ion is associated with nanoparticle suspensions. The proteome of cells subjected to equal concentrations of either the nanoparticle or zinc acetate suggest that the mode of toxicity is quite different for the two forms of Zn, with a number of membrane-associated proteins up-expressed in response to nanoparticle exposure. Our data suggests that nanoparticles act to interrupt cell membranes thereby causing cell death rather than exerting a strictly toxic effect. We also identify potentially useful genes to serve as biomarkers of membrane disruption in toxicogenomic studies with nanoparticles or to engineer biosensor organisms.

Evidence of hydroxyl free radical generation by calcium overload in rat myocardium.

Although calcium (Ca2+) is important in cardiac dysfunction and has also been reported as a source of oxidative toxicity, the connection between Ca2+ overload and oxygen free radicals in the myocardium is not clear. We have investigated whether Ca2+ overload generates hydroxyl free radicals in rat ventricle. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl free radical reacts with salicylate. Ringer's solution containing salicylic acid (0.5 nmol microL-1 min-1) was infused through a microdialysis probe in the region of the left anterior descending coronary artery of the rat ventricle. A positive linear correlation was obtained between Ca2+ and hydroxyl free radical formation trapped as 2,3-DHBA (r2 = 0.976) and 2,5-DHBA (r2 = 0.982) in the myocardial dialysate. The administration of ouabain (1 mg kg-1, i.v.), a Ca2+ elevator, into the femoral vein significantly increased the level of 2,3- and 2,5-DHBA. These results indicate that Ca2+ overload generates hydroxyl free radicals in rat heart.

Aniracetam, a pyrrolidinone-type cognition enhancer, attenuates the hydroxyl free radical formation in the brain of mice with brain ischaemia.

We demonstrate here that aniracetam has the ability to block the formation of cytotoxic hydroxyl radicals (.OH) during ischaemia-reperfusion of mouse brain. The fact that brain ischeamia for 40 min followed by reperfusion increased .OH was evidenced by detection of a peaked increase at 20 min after an ischaemic insult in the formation of 2,3-dihydroxybenzoate (DHBA) from salicylate in cerebroventricular perfusate, a means of monitoring .OH formation. A clearcut increase in dopamine was also observed during and after brain ischaemia. The ischaemia-reperfusion mice given aniracetam at an intraperitoneal dose of 30 or 100 mg kg-1 showed a smaller increase in the formation of DHBA than those given the vehicle only. Aniracetam at 100 mg kg-1 significantly suppressed the formation of DHBA by approximately 80%, becoming evident at 20 min after reperfusion and thereafter. Protection against death in mice insulted with a 40-min brain ischaemia (3/13 vs 13/25) was observed following 100 mg kg-1 aniracetam. The increase in the dopamine levels was substantially reduced following aniracetam treatment and the reduction became significant at 20 min after reperfusion and thereafter in parallel with attenuation by aniracetam of DHBA formation. This finding suggests that the inhibitory activity of aniracetam in attenuating the hydroxyl free-radical formation in ischaemic mice is probably due, at least in part, to its palliative action on the dopaminergic neurons.

The exploration of the antibacterial mechanism of fe3+ against bacteria

This study demonstrated that the bacteria could adsorb Fe3+ and reduce Fe3+ to Fe2+. Iron had significant bacteriostatic effects, which were directly proportional to the iron concentration and under the influence of pH and chelator. It presumed that the inhibition of Fe3+ acts through the formation of hydroxyl free radicals.

Hyperbaric oxygenation alleviates MCAO-induced brain injury and reduces hydroxyl radical formation and glutamate release

The present study examined the effect of hyperbaric oxygen (HBO) on the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), the products of salicylate trapping of hydroxyl free radicals, and glutamate release in the striatum during acute ischemia and reperfusion. Non-HBO rats (n = 8) were subjected to 1-h ischemia. Study rats (n = 8) were treated with HBO at 2.8 ATA for 1 h during ischemia. Artificial CSF solution containing 5 mM sodium salicylate was perfused at 1 microl/min. Samples were continuously collected at 15 min intervals and the levels of 2,3-DHBA, 2,5-DHBA, and glutamate were analyzed. The lesion volume was determined by TTC stain. Occlusion of the middle cerebral artery induced a significant increase in the levels of 2,3-DHBA and 2,5-DHBA. A peak of approximately two and fourfold of baseline levels was reached at 45 min and was maintained at elevated levels during reperfusion. The level of glutamate increased approximately two times at 30 min during ischemia, continued to increase, and reached approximately three times baseline level during reperfusion. HBO significantly alleviated brain injury associated with decreased levels of 2,3-DHBA, 2,5-DHBA and glutamate. This study suggests that the decreased glutamate release and the reduced formation of hydroxyl free radicals might contribute to the neuroprotective effect of HBO.

Promotion of oxidative stress in kidney of rats loaded with cystine dimethyl ester

Cystinosis is a systemic genetic disease caused by a lysosomal transport deficiency accumulating cystine in most tissues. Although tissue damage might depend on cystine accumulation, the mechanisms of tissue damage are not fully understood. Studies performed in fibroblasts of cystinotic patients and in kidney cells loaded with cystine dimethyl ester (CDME) suggest that apoptosis is enhanced in this disease. Considering that oxidative stress is a known apoptosis inducer, our main objective was to investigate the effects of CDME loading on several parameters of oxidative stress in the kidney of young rats. Animals were injected twice a day with 1.6 micromol/g body weight CDME and/or 0.26 micromol/g body weight cysteamine (CSH) from the 16th to the 20th postpartum day and killed after 1 or 12 h. CDME induced lipoperoxidation and protein carbonylation and stimulated superoxide dismutase, glutathione peroxidase (GPx), and catalase activities, probably through the formation of superoxide anions, hydrogen peroxide, and hydroxyl free radicals. Coadministration of CSH, the drug used to treat cystinotic patients, prevented, at least in part, those effects, possibly acting as a scavenger of free radicals. These results suggest that the induction of oxidative stress might be one of the mechanisms leading to tissue damage in cystinotic patients.

Reactive oxidative and nitrogen species in the nigrostriatal system following striatal 6-hydroxydopamine lesion in rats

Oxidative stress is a major contributing factor in the pathogenesis of Parkinson's disease. We therefore investigated the effect of the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on hydroxyl-free radical and peroxynitrite formation in the intrastriatal 6-OHDA rat model of Parkinson's disease. The hydroxylation product of salicylate (2,3-dihydroxy-benzoic acid) as well as the hydroxylation and nitration products of d-phenylalanine (2- and 3-hydroxyl-phenylalanine, nitrotyrosine and nitrophenylalanine) were assessed in tissue samples of the striatum and, for the first time, the substantia nigra of adult rats at four different time points (25 min, 2 h, 4 h and 7 days) after unilateral stereotaxic intrastriatal injection of 6-OHDA. In the striatum, maxima of hydroxylating and nitrating markers were found at early time points after 6-OHDA lesion. These results suggest a direct interrelation between 6-OHDA-autoxidation and/or the increased dopamine turnover and hydroxyl-free radical and peroxynitrite formation. In the substantia nigra, i.e., at a distance from the injection site of the neurotoxin, an increase in hydroxyl-free radical formation was observed at 7 days after 6-OHDA lesion, with this modification possibly being independent of 6-OHDA autoxidation and rather representing a long-term effect of the toxin. Furthermore, we conclude that apart from the formation of reactive oxygen species, the production of reactive nitrogen species occurs in this experimental Parkinson's disease model. Finally, the similarity between the 6-OHDA model and Parkinson's disease supports the notion that reactive oxygen species as well as reactive nitrogen species may play an important role in the pathogenesis of this neurodegenerative disorder.

Study on production of free hydroxyl radical and its reaction with salicylic acid at lead dioxide electrode

The formation of free hydroxyl radical ( OH) in oxygen transfer reactions at PbO 2 electrodes and the reaction between the electrochemically produced OH and salicylic acid (SA) were studied by cyclic voltammetry and capillary electrophoresis-amperometric detection (CZE-AD) in this paper. Experiments showed that OH was formed in the potential range of 1.0–1.4 V corresponding to the anodic discharge of H 2O at the PbO 2 electrode, while this phenomenon was not obvious at the Pt electrode. Optimum CZE-AD conditions for indirect determination of OH were explored. It was also found that when salicylic acid was used to trap OH produced on the PbO 2 electrode surface, the amount of 2,5-dihydroxybenzoic acid produced is much more than that of 2,3-dihydroxybenzoic acid. This phenomenon is explained by the proposal that SA first forms an active aromatic radical on the PbO 2 electrode surface before trapping OH, where OH performs a stronger preference in attacking at the 5-position than for attacking at the 3-position of the aromatic radical.

The biogenic trace amine tyramine induces a pronounced hydroxyl radical production via a monoamine oxidase dependent mechanism: an in vivo microdialysis study in mouse striatum

Tyramine is a biogenic trace amine that releases monoamines and is a good substrate for monoamine oxidase (MAO)-A/B. Here we investigated whether tyramine affects hydroxyl radical formation in the intact and lesioned dopaminergic system. Male C57bl/6 mice received systemic and local tyramine administrations. Hydroxyl radical formation and dopamine (DA) overflow were determined in the striatum using in vivo microdialysis in combination with the salicylate hydroxylation assay. Systemic injection of tyramine neither enhanced extracellular dopamine nor induced hydroxyl radical formation. In contrast, when tyramine was incorporated into the dialysate fluid, hydroxyl radical formation and extracellular dopamine levels were significantly enhanced. Systemic pretreatment with the MAO-A/B inhibitor tranylcypromine or with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly diminished the tyramine-induced hydroxyl radical formation by 73.1% and 80.6%, respectively. We conclude that the mechanism of tyramine-induced hydroxyl free radical formation involves MAO metabolism and requires an intact dopaminergic system. Pharmacological intervention on the MAO-mediated formation of hydroxyl free radicals seems to be a promising strategy to prevent oxidative damage in the nigrostriatal dopaminergic system.

The biogenic trace amine tyramine induces a pronounced hydroxyl radical production via a monoamine oxidase dependent mechanism: an in vivo microdialysis study in mouse striatum

Tyramine is a biogenic trace amine that releases monoamines and is a good substrate for monoamine oxidase (MAO)-A/B. Here we investigated whether tyramine affects hydroxyl radical formation in the intact and lesioned dopaminergic system. Male C57bl/6 mice received systemic and local tyramine administrations. Hydroxyl radical formation and dopamine (DA) overflow were determined in the striatum using in vivo microdialysis in combination with the salicylate hydroxylation assay. Systemic injection of tyramine neither enhanced extracellular dopamine nor induced hydroxyl radical formation. In contrast, when tyramine was incorporated into the dialysate fluid, hydroxyl radical formation and extracellular dopamine levels were significantly enhanced. Systemic pretreatment with the MAO-A/B inhibitor tranylcypromine or with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) significantly diminished the tyramine-induced hydroxyl radical formation by 73.1% and 80.6%, respectively. We conclude that the mechanism of tyramine-induced hydroxyl free radical formation involves MAO metabolism and requires an intact dopaminergic system. Pharmacological intervention on the MAO-mediated formation of hydroxyl free radicals seems to be a promising strategy to prevent oxidative damage in the nigrostriatal dopaminergic system.

The role of superoxide dismutase and α-tocopherol in the development of seizures and kindling induced by pentylenetetrazol - influence of the radical scavenger α-phenyl- N- tert-butyl nitrone

Previous experiments have shown that the generation of free hydroxyl radicals in rat brain homogenates is increased following pentylenetetrazol (PTZ) kindling. The present study was performed in order to evaluate the involvement of endogeneous radical defence systems as the superoxide dismutase (SOD) and the level of α-tocopherol, an important lipid-soluble and membrane-bound antioxidant in brain homogenate of rats after acute seizure and kindling induced by PTZ. The activities of the total SOD were significantly reduced after acute seizure and tend towards an enhancement in kindled animals. Western blot analysis shows an upregulation of Mn-SOD in rat brain homogenates after kindling. The level of the chain-breaking antioxidant α-tocopherol was reduced in acutely convulsing rats and was not modified in kindled rats. Second, we studied the influence of exogeneously supplied radical scavenger α-phenyl- N- tert-butyl-nitrone (PBN) on seizure and kindling following PTZ treatment. After a single injection of PTZ at a dose evoking clonic–tonic seizures, PBN did not modify either the formation of free hydroxyl radicals measured by the levels of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA or the susceptibility to PTZ. In the kindling group, subchronic treatment with PBN (over a period of 4 weeks) prevented the increase in the formation of free hydroxyl radicals, and the susceptibility to PTZ was transiently decreased during the development of kindling, but PBN did not influence the susceptibility to PTZ in fully kindled rats. Pretreatment with PBN increased the activities of total SOD and the protein content of Mn-SOD and decreased the level of α-tocopherol in comparison to saline controls. The results suggest that the formation of free hydroxyl radicals is not reflected by an enhanced susceptibility to PTZ classified according to the modified RACINE scale. Additionally, it may be assumed that the increased generation of hydroxyl radicals in kindled animals is not primary caused by an exhaustion of both the defence systems measured. Adaptive mechanisms, as the induction of Mn-SOD, may be taken into consideration to counteract oxidative stress-mediated free radical formation.