Tert-butyl Nitrone Research Articles

ChemInform Abstract: 2,4,6-Trimethoxyphenyl tert-Butyl Nitrone (MO)3PBN, as a Detector of Hydroxyl Radicals in the Presence of Peroxides, Superoxide, and Peroxyl Radicals.

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Calicheamicin γ1I and phenyl tert-butyl nitrone (PBN): observation of a kinetic isotope effect by an ESR study

ESR study of enediyne calicheamicin gamma(1)(I) with phenyl tert-butyl nitrone (PBN) gave a significant kinetic isotope effect (k(H)/k(D) = 1.8) for the formation of the phenyl radical PBN monoadducts.

Solvent-free synthesis of nitrones in a ball-mill

Various C-aryl and C-alkyl-nitrones were synthesized within 0.5–2 h via condensation of an equimolar amount of aldehydes and N-substituted-hydroxylamines under solvent-free conditions in a ball-mill apparatus. Reactions can be performed without the need of excluding air and moisture and yields the expected products with no need for further purification. The study has been complemented by Differential Scanning Calorimetry (DSC) and solid-state 13C MAS nuclear magnetic resonance experiments. We have also studied the temperature profile during the reaction. A comparative study with the corresponding solvent-free microwave activated reaction showed the superiority of the ball-milling method; 31 examples are described, including the synthesis of the anti-aging agent C-phenyl- N- tert-butyl nitrone (PBN) and one of its analogues C-2-pyridyl- N- tert-butylnitrone (2-PyBN).

α-Phenyl- N- tert-butyl nitrone (PBN) derivatives: Synthesis and protective action against microvascular damages induced by ischemia/reperfusion

Nitrones 4– 7, structurally related to PBN ( 1), were prepared by reaction of the corresponding aromatic aldehydes with N- tert-butyl hydroxylamine. The protective effects of these nitrones against microvascular damages in ischemia/reperfusion in the ‘hamster cheek pouch’ assay were studied and 1, as well as 4a, 4b, and 7 (derived from piperonal, O-benzyl vanillin, and furfural, respectively), showed to be more active than shark cartilage or α-tocopherol. No correlation was found between the protective effect of these nitrones and their log P (partition coefficient) or their capacity to trap ·OH and ·CH 3 radicals.

Spin-Trapping of the p-Benzyne Intermediates from Ten-Membered Enediyne Calicheamicin γ1I

In the presence of thiols, the ten-membered-ring enediyne calicheamicin gamma1I generates a p-benzyne biradical that initiates oxidative cleavage of double-stranded DNA. Application of spin-trapping has successfully provided ESR and mass spectroscopic evidence for the formation of the monoadducts with phenyl tert-butyl nitrone (PBN). [reaction: see text].

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H 2O 2 with concurrent evolution of O 2. This pseudocatalatic degradation of H 2O 2 to O 2 is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n- tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H 2O with subsequent regeneration of ascorbate by the reduction of MDA with H 2O 2 evolving O 2 through the intermediate formation of O 2 −. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN − in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN −. Further kinetic studies indicate that SCN − itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN − blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H 2O 2 to O 2. Binding studies by optical difference spectroscopy indicate that SCN − binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN − acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.

Electron paramagnetic resonance evidence of hydroxyl radical generation and oxidative damage induced by tetrabromobisphenol A in Carassius auratus

Tetrabromobisphenol A (TBBPA) is one of the most widely used brominated flame retardants (BFRs). To confirm its putative oxidative stress-inducing activity, freshwater fish Carassius auratus were injected intraperitoneally with TBBPA. One experiment lasted 3 h to 28 days after a single injection of 100 mg/kg TBBPA, and the other lasted 24 h after a single injection of 0–300 mg/kg TBBPA. Reactive oxygen species (ROS) were trapped by phenyl- tert-butyl nitrone (PBN) and detected by electron paramagnetic resonance (EPR). Protein carbonyl (PCO) and lipid peroxidation product (LPO) content were also determined. A six-line EPR spectrum was detected in the sample prepared in air, and a multiple one was obtained in nitrogen. The observed spectrum in nitrogen fits the simulation one with PBN/ OCH 3 and PBN/ CH 3 quite well. As compared to the control group, TBBPA significantly induced ROS production marked by the intensity of the prominent spectra in liver and bile. TBBPA (100 mg/kg) also significantly increased PCO content in liver starting 24 h and LPO content 3 days after injection. Either PCO or LPO content showed significant relation with ROS production. Based on the hyperfine constants and shape of the spectrum, ROS induced by TBBPA was determined as OH. The results clearly indicated that TBBPA could induce OH generation and result in oxidative damage in liver of C. auratus.

Comparison of Neuroprotective Effects Induced by α-Phenyl- N- tert-butyl nitrone (PBN) and N- tert-Butyl-α-(2 sulfophenyl) nitrone (S-PBN) in Lithium-Pilocarpine Status Epilepticus

The status epilepticus (SE) induced in rats by lithium-pilocarpine (Li-pilo) shares many common features with soman-induced SE including extensive limbic neuropathology. Reactive oxygen species are hypothesized to play a role in the SE induced neuropathology and we propose that the free radical scavengers α-phenyl- N- tert-butyl nitrone (PBN) and N- tert-butyl-α-(2 sulfophenyl) nitrone (S-PBN) may be neuroprotective. PBN or S-PBN were administered either immediately following pilocarpine (exposure treatment) or 5 min after the onset of SE as determined by ECoG activity. SE was allowed to continue for 3 h before termination with propofol. The rats were sacrified 24 h following pilocarpine administration. S-PBN induced minor effects to reduce SE duration and improve neurological deficit 24 h following pilocarpine administration. One hundred and fifty milligrams per kilograms PBN administered 5 min after SE onset produced significant neuroprotection in the parietal, occipital, perirhinal and piriform cortices as well as the lateral amygdala. One hundred and fifty milligrams per kilograms S-PBN was neuroprotective only in the occipital and perirhinal cortex while 300 mg/kg S-PBN exacerbated cortical neuropathology. S-PBN administered 5 min after SE onset exacerbated neuropathology in thalamic regions. In contrast, PBN and S-PBN administered as exposure treatment exacerbated neuropathology in thalamic and CA3 regions. The differential neuroprotective effects of PBN and S-PBN may be the result of the poor brain penetration by S-PBN. The results suggest that free radical scavenger activity is neuroprotective in cortical regions during cholinergic convulsions. Regional variations in drug-induced neuroprotectant activity in Li-pilo SE are common and suggest multiple mechanisms of neuropathology.

Hydroxyl radical production and oxidative damage induced by cadmium and naphthalene in liver of Carassius auratus

Freshwater goldfish ( Carassius auratus) were exposed to cadmium (Cd) from 0 to 5 mg/L, and naphthalene (NAP) from 0 to 50 mg/L. Twenty-four hours after the exposure, reactive oxygen species (ROS) was trapped by phenyl- tert-butyl nitrone and detected by electron paramagnetic resonance (EPR). Protein carbonyl (PCO) and lipid peroxidation (LPO) content were determined. The activities of superoxide dismutase (SOD) and catalase (CAT) were also measured. The EPR spectra signals were characterized by prominent six-line spectra, which were defined as hydroxyl radical ( OH). As compared to the control group, Cd and NAP significantly induced OH production marked by the intensity of the prominent spectra at higher concentrations. Both xenobiotics also increased LPO content and PCO content, depending on the concentrations. Either LPO or PCO content showed significant relation with OH production. Cd increased the activity of SOD and decreased that of CAT at 5 mg/L, and NAP increased the activities of SOD and CAT at 5 mg/L. The results clearly indicated that these two structurally different non-redox cycling xenobiotics could induce OH generation and result in oxidative damage in liver of C. auratus, and these effects were concentration-dependent.

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO [rad] and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO ), it forms methyl radicals ( CH 3) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO formation. However, we recently reported that CH 3 might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated α-[4-pyridyl 1]- N- tert-butyl nitrone, d 0/d 9-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO and DMSO. In addition to identifying the two well-known free radicals, CH 3 and OCH 3, from this interaction, we also characterized two additional free radicals, CH 2OH and CH 2S(O)CH 3. Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/ CH 3 was not detected. However, with the addition of the LC/MS technique, we found ∼0.75 μM POBN/ CH 3 hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.

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.

Generation of superoxide anion and most likely singlet oxygen in irradiated TiO 2 nanoparticles modified by carotenoids

Photosensitization of the TiO 2 nanoparticles with carotenoids leads to the formation of superoxide anion and singlet oxygen on red light irradiation. It has been shown that carotenoids facilitate generation of superoxide radical anion O 2 − in irradiated TiO 2 colloids. At low carotenoid concentrations (<3×10 −5 M) the rate constant of O 2 − production exceeds that obtained in the absence of carotenoid. EPR experiments with 2,2,6,6-tetramethyl-4-piperidone and spin trapping with α-phenyl- N- tert-butyl nitrone and 5,5-dimethyl-pyrroline- N-oxide showed that both superoxide anion and singlet oxygen can be produced in irradiated TiO 2 suspensions in toluene and CH 2Cl 2. The PBN–O 2 − and DMPO–O 2 − spin adducts were detected by the 9 GHz EPR/ENDOR spin trapping technique. The dismutation of superoxide radical anion was proposed to generate singlet oxygen through intermediate formation of HO 2 radicals. The PBN–HO 2 spin adduct was identified by the 9 GHz EPR/ENDOR and 95 GHz EPR spin trapping technique.

ESR study of camphorquinone/amine photoinitiator systems using blue light-emitting diodes

New light-activation units equipped with high-illuminant blue light-emitting diodes (LEDs) have recently been proposed as a replacement for the halogen units that are widely used in dentistry to polymerize light-cured resins. The photoinitiators in light-cured dental resins, typified by the camphorquinone (CQ)/amine photoinitiator system, generate primary radicals with light irradiation that attack the double bonds of resin monomers. The physical properties of the cured resins are affected by the generation of primary radicals during the initial stage of polymerization. This study examined two types of photoinitiator systems, CQ/DMPT and CQ/DMAEMA, and three types of curing units, a new LED unit and two conventional halogen units. The primary radicals generated by irradiation were quantified using electron spin resonance (ESR) spectroscopy with a trapping method, using phenyl- tert-butyl nitrone as the trapping agent. The energy efficiencies of the LED and halogen units were compared by quantifying the generated radicals and emitted light energy (J/cm 2). The energy required to generate a given amount of radicals using the LED unit was smaller than that using the halogen units ( p<0.05). These results suggest that the new LED unit performs better than conventional halogen units with respect to light energy.

Synthesis of a new family of glycolipidic nitrones as potential antioxidant drugs for neurodegenerative disorders

This paper deals with the synthesis of a novel series of amphiphilic glycosylated spin-traps derived from α-Phenyl- N- tert-butyl nitrone (PBN) and an initial characterization of their anti-caspase-3 activity. Preliminary investigation of their anti-apoptosis effect showed they dramatically inhibit the activity of caspase-3 in cultured neuronal cells following induction of apoptosis by hydrogen peroxide.

Methylmercury-induced reactive oxygen species formation in neonatal cerebral astrocytic cultures is attenuated by antioxidants

Excessive generation of reactive oxygen species (ROS) has been suggested as a causal factor in various neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease [Brain Res. 830 (1999) 10–15; Biochem. J. 310 (1995) 83–90; Free Radic. Biol. Med. 27 (1999) 612–616]. The present work examined the role of ROS in the neurotoxicity of methylmercury (MeHg). ROS formation in primary astrocytic cultures of neonatal rat cerebral cortex was monitored by 2′,7′-dichlorodihydrofluorescein diacetate (H 2DCF-DA) fluorescence. MeHg, at 10 and 20 μM caused a significant increase in ROS formation (10 μM, P<0.01; 20 μM, P<0.001). Additional studies established the effectiveness of antioxidants/free radical scavengers in attenuating the MeHg-stimulated ROS formation in the following rank-order: (1) Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a non-thiol containing antioxidant, (2) n-propyl gallate (PG), a free radical scavenger, (3) superoxide dismutase (SOD), an antioxidant enzyme that dismutates superoxide anion radical, (4) α-phenyl- tert-butyl nitrone (PBN), a lipophilic hydroxyl radical spin trapping agent. A significant inhibition of MeHg-induced ROS generation was also noted in astrocytes preincubated (3 h) with arachidonyl trifluoromethyl ketone (AACOCF 3, 20 μM, P<0.05), a specific inhibitor of cytosolic phospholipase A 2 (cPLA 2). Conversely, pretreatment (24 h) with 100 μM buthionine- l-sulfoxamine [BSO, a glutathione (GSH) synthesis inhibitor], significantly increased ( P<0.05) ROS formation in MeHg treated astrocytes compared to controls. Combined, these studies invoke ROS as potent mediators of MeHg cytotoxicity and support the hypothesis that excessive ROS generation, at least in part, plays an important role in MeHg-induced neurotoxicity.

Deferoxamine, allopurinol and oxypurinol are not neuroprotective after oxygen/glucose deprivation in an organotypic hippocampal model, lacking functional endothelial cells

Reactive oxygen species-induced reperfusion injury of the brain is an important cause of neonatal morbidity and mortality following perinatal hypoxia–ischemia. Deferoxamine, allopurinol and oxypurinol have previously been shown to be neuroprotective in vivo during or directly after hypoxia–ischemia. To further characterize and more precisely elucidate whether the neuroprotective properties of these agents are mediated via neuronal and glial cells, or whether endothelial cells contribute to this effect, we tested their ability to protect CA1 neurons in organotypic hippocampal slices. Hippocampal slices obtained from 8-day-old rats were cultured for 7 days and exposed to oxygen/glucose deprivation for 50 min, or used as control slices. Cell damage was assessed at 48 h after oxygen/glucose deprivation using propidium iodide staining. At different time points following oxygen/glucose deprivation we administered dizocilpine, 6-cyano-7-nitroquinoxaline-2,3-dione, and α-phenyl- N- tert-butyl nitrone for validation purposes. Deferoxamine, allopurinol or oxypurinol were used as test substances. As expected, 89% and 98% protection was demonstrated with dizocilpine present during or during/after oxygen/glucose deprivation resp. α-Phenyl- N- tert-butyl nitrone administered during/after oxygen/glucose deprivation provided 44% protection. However, iron chelation with deferoxamine and inhibition of xanthine oxidase by allopurinol or oxypurinol did not confer neuroprotection. The neuroprotective effect of deferoxamine, allopurinol or oxypurinol, as seen in vivo, may be obtained via inhibition of the production of damaging factors by blood born substances or endothelial cells.

Antioxidant flavonoids from leaves of Polygonum hydropiper L.

Ten flavonoid compounds were isolated from the dried leaves of Polygonum hydropiper L. (Laksa leaves), and identified as 3- O-α- l-rhamnopyranosyloxy-3′,4′,5,7-tetrahydroxyflavone; 3- O-β- d-glucopyranosyloxy-4′,5,7-trihydroxyflavone; 6-hydroxyapigenin; 6″- O-(3,4,5-trihydroxybenzoyl) 3- O-β- d-glucopyranosyloxy-3′, 4′, 5, 7-tetrahydroxyflavone; scutillarein; 6-hydroxyluteolin; 3′,4′,5,6,7-pentahydroxyflavone; 6-hydroxyluteolin-7- O-β- d-glucopyranoside; quercetin 3- O-β- d-glucuronide; 2″- O-(3,4,5-trihydroxybenzoyl) quercitrin; quercetin. Evaluation of the antioxidative activity, conducted in vitro, by using electron spin resonance (ESR) and ultraviolet visible (UV–vis) spectrophotometric assays, showed that these isolated flavonoids possess strong antioxidative capabilities. Measurement of the Trolox equivalent antioxidant capacity (TEAC) values, against ABTS (2,2′-azinobis(3-ethyl-benzo-thiazoline-6-sulphonic acid) radicals and phenyl- tert-butyl nitrone (PBN) azo initiator (AI) also showed strong anti-oxidative activity. The most powerful of the antioxidants was 2″- O-(3,4,5-trihydroxybenzoyl) quercitrin (galloyl quercitrin). A combination of two flavonoid compounds was tested for synergistic anti-oxidative capacity, but no significant improvement was observed.

In vitro blood–brain barrier permeability and cerebral endothelial cell uptake of the neuroprotective nitrone compound NXY-059 in normoxic, hypoxic and ischemic conditions

The free radical trapping nitrone compounds α-phenyl- N- tert-butylnitrone (PBN), 2-sulfophenyl- N- tert-butylnitrone (S-PBN) and disodium 2,4-disulfophenyl- N- tert-butyl nitrone (NXY-059) are effective neuroprotective agents in experimental models of both transient and permanent focal ischemia. A recent in vivo study suggested that NXY-059 had poor brain uptake in a transient ischemia model. We have now examined its blood–brain barrier permeability and cerebral endothelial uptake during hypoxic and ischemic conditions using an in vitro model of the blood–brain barrier. The in vitro blood–brain barrier permeability and cerebral endothelial uptake of NXY-059 and S-PBN were low during normoxic conditions. In contrast, PBN had very high blood–brain barrier penetration in vitro which confirmed earlier in vivo results. The permeability of [ 14C]NXY-059 increased 3.5 times after 9 h of hypoxia or 3 h of ischemia. There was, respectively, a 5-fold and more than 10-fold increase, after 6 and 9 h of ischemia. The control molecule [ 3H]inulin ( M r≈5000) showed a similar increase in permeability under the same experimental conditions indicating a major change in the transport properties of the endothelium. There was a 60% reduction in the ATP levels of astrocytes after 3 h of ischemia and a 90% reduction after 9 h. The reduction in ATP levels in endothelial cells was somewhat lower. The uptake of NXY-059 in cerebral endothelial cells under normoxic, hypoxic or 9 h of ischemic conditions was negligible. NXY-059, S-PBN and PBN showed no effects on vesicular transport or the integrity of the blood–brain barrier in normoxic or ischemic conditions, nor did the compounds induce any change in the ATP levels of the cells. In conclusion, it is possible that the increase in blood–brain barrier permeability of [ 14C]NXY-059 which occurs during prolonged ischemia in vitro reflects a change which may be of importance to the neuroprotective effects of this nitrone free radical trapping agent.

In vivo detection of aflatoxin-induced lipid free radicals in rat bile

Aflatoxin B1 (AFB1), a potent hepatotoxin and hepatocarcinogen, is metabolized in the liver via cytochrome P-450 to an AFB1-8,9-epoxide intermediate. The formation of the AFB1-8,9-epoxide correlates with the pathological changes observed in numerous mammalian species. Oxidative damage has been postulated to play a major role in the mechanisms associated with AFB1-induced cytotoxicity and carcinogenecity in mammalian species. The aim of this study was to detect and identify free radical intermediates from the hepatic metabolism of AFB1 in vivo. Rat bile ducts were cannulated and rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (α-(4-pyridyl-1-oxide)- N- tert-butyl nitrone) (1 g/kg i.p.), and bile was collected over a period of 2 h at 20-min intervals. ESR spectroscopy was used to detect a carbon-centered radical adduct of 4-POBN in rat bile. The effect of metabolic inhibitors, such as deferoxamine mesylate (DFO), an iron chelator, and SKF 525A, a cytochrome P-450 inhibitor, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in free radical formation by pre-treatment with both DFO and SKF 525A. This indicates that oxidation of AFB1 generates free radical species via CYP metabolism and an iron-mediated redox mechanism.

Analysis of tert-butyl hydroperoxide induced constrictions of perfused vascular beds in vitro

tert-Butyl hydroperoxide ( t-BOOH), an inducer of oxidative stress in vitro, elicits constrictor responses of the isolated, rat kidney and mesenteric arteries perfused (5 ml/min) with physiological salt solutions (PSS) at 37°C gassed with carbogen. We hypothesized that generation of superoxide anions (O 2 −) accounts for these responses. We assessed responses to t-BOOH in preparations with/without endothelium, and in the absence/presence of antioxidant compounds, catalase and tempol, scavengers of hydroxyl (OH −) radical and O 2 −, respectively. t-BOOH (0.01–50 μmol) induced (expressed as % of 50 μmol KCl vasoconstriction) were abolished by endothelium denudation, perfusion with Ca 2+-free PSS and by nifedipine, (1 nM). Infusion of t-BOOH (0.1 μM) did not significantly (P > 0.05) affect phenylepherine E max in the mesenteric arteries, however it reduced phenylepherine E max responses in the kidney from 94.9 ± 3.9 % to 64.7 ± 4.7 %. Nitroblue tetrazolium, as well as α-phenyl N- tert-butyl nitrone, at 100 μM, but not catalase (500 IU) significantly attenuated t-BOOH (10 μmol) vasoconstrictor responses. Tempol (100 μM), a membrane permeable antioxidant, also significantly reduced t-BOOH (10 μmol) responses from 17.0 ± 1.9 % (control) to 9.6 ± 0.5 % (+tempol) in the mesenteric arteries and from 40.4 ± 4.2 % (control) to 20.7 ± 1.5 % (+tempol) in the kidney. Our data suggest that t-BOOH elicits vasoconstriction via two distinct mechanisms: (i) increased influx of extracellular Ca 2+, and (ii) generation of free radicals including O 2 − anions.