Spin trapping and cytoprotective properties of fluorinated amphiphilic carrier conjugates of cyclic versus linear nitrones.
ABSTRACT: Nitrones have been employed as spin trapping reagent as well as pharmacological agent against neurodegenerative diseases and ischemia-reperfusion induced injury. The structure-activity relationship was explored for the two types of nitrones, i.e., cyclic (DMPO) and linear (PBN), which are conjugated to a fluorinated amphiphilic carrier (FAC) for their cytoprotective properties against hydrogen peroxide (H(2)O(2)), 3-morpholinosynonimine hydrochloride (SIN-1), and 4-hydroxynonenal (HNE) induced cell death on bovine aortic endothelial cells. The compound FAMPO was synthesized and characterized, and its physical-chemical and spin trapping properties were explored. Cytotoxicity and cytoprotective properties of various nitrones either conjugated and nonconjugated to FAC (i.e., AMPO, FAMPO, PBN, and FAPBN) were assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) reduction assay. Results show that of all the nitrones tested, FAPBN is the most protective against H(2)O(2), but FAMPO and to a lesser extent its unconjugated form, AMPO, are more protective against SIN-1 induced cytotoxicity. However, none of the nitrones used protect the cells from HNE-induced cell death. The difference in the cytoprotective properties observed between the cyclic and linear nitrones may arise from the differences in their intrinsic antioxidant properties and localization in the cell.
Project description:Reactive nitrogen species (RNS) such as nitrogen dioxide ((•)NO(2)), peroxynitrite (ONOO(-)), and nitrosoperoxycarbonate (ONOOCO(2)(-)) are among the most damaging species present in biological systems due to their ability to cause modification of key biomolecular systems through oxidation, nitrosylation, and nitration. Nitrone spin traps are known to react with free radicals and nonradicals via electrophilic and nucleophilic addition reactions and have been employed as reagents to detect radicals using electron paramagnetic resonance (EPR) spectroscopy and as pharmacological agents against oxidative stress-mediated injury. This study examines the reactivity of cyclic nitrones such as 5,5-dimethylpyrroline N-oxide (DMPO) with (•)NO(2), ONOO(-), ONOOCO(2)(-), SNAP, and SIN-1 using EPR. The thermochemistries of nitrone reactivity with RNS and isotropic hfsc's of the addition products were also calculated at the PCM(water)/B3LYP/6-31+G**//B3LYP/6-31G* level of theory with and without explicit water molecules to rationalize the nature of the observed EPR spectra. Spin trapping of other RNS such as azide ((•)N(3)), nitrogen trioxide ((•)NO(3)), amino ((•)NH(2)) radicals and nitroxyl (HNO) were also theoretically and experimentally investigated by EPR spin trapping and mass spectrometry. This study also shows that other spin traps such as 5-carbamoyl-5-methyl-pyrroline N-oxide, 5-ethoxycarbonyl-5-methyl-pyrroline N-oxide, and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide can react with radical and nonradical RNS, thus making spin traps suitable probes as well as antioxidants against RNS-mediated oxidative damage.
Project description:Nitrones (e.g. ?-phenyl-N-tert-butyl nitrone; PBN) are cerebroprotective in experimental stroke. Free radical trapping is their proposed mechanism. As PBN has low radical trapping potency, we tested Sgk1 induction as another possible mechanism. PBN was injected (100?mg/kg, i.p.) into adult male rats and mice. Sgk1 was quantified in cerebral tissue by microarray, quantitative RT-PCR and western analyses. Sgk1+/+ and Sgk1-/- mice were randomized to receive PBN or saline immediately following transient (60?min) occlusion of the middle cerebral artery. Neurological deficit was measured at 24?h and 48?h and infarct volume at 48?h post-occlusion. Following systemic PBN administration, rapid induction of Sgk1 was detected by microarray (at 4?h) and confirmed by RT-PCR and phosphorylation of the Sgk1-specific substrate NDRG1 (at 6?h). PBN-treated Sgk1+/+ mice had lower neurological deficit ( p?<?0.01) and infarct volume ( p?<?0.01) than saline-treated Sgk1+/+ mice. PBN-treated Sgk1-/- mice did not differ from saline-treated Sgk1-/- mice. Saline-treated Sgk1-/- and Sgk1+/+ mice did not differ. Brain Sgk3:Sgk1 mRNA ratio was 1.0:10.6 in Sgk1+/+ mice. Sgk3 was not augmented in Sgk1-/- mice. We conclude that acute systemic treatment with PBN induces Sgk1 in brain tissue. Sgk1 may play a part in PBN-dependent actions in acute brain ischemia.
Project description:Cyclic nitrones have been employed for decades as spin trapping reagents for the detection and identification of transient radicals, and have been employed as pharmacological agent against ROS-mediated toxicity. The short half-life of the nitrone-superoxide adducts limits the application of nitrones in biological millieu, and therefore investigaton of the redox properties of the superoxide adducts is important. Moreover, computational investigation of the redox properties of the nitrones and their corresponding spin adducts may provide new insights into the nature of their pharmacological activity against ROS-induced toxicity. In general, electron-withdrawing group substitution at the C-5 position results in higher EAs and IPs making these substituted nitrones more susceptible to reduction but more difficult to oxidize compared to DMPO. One-electron reduction and oxidation of nitrones both resulted in elongated N-C(2) bonds indicating the tendency of radical anion and cation forms of nitrone to undergo ring-opening. The EAs and IPs of various O(2)(*-) adducts indicate that DEPMPO-O(2)H is the most difficult to reduce and oxidize compared to the O(2)(*-) adducts of DMPO, EMPO, and AMPO. In general, nitroxides gave higher EAs compared to nitrones making them more suceptible to reduction. One-electron oxidation of nitroxides leads to elongation of the N-C(2) bond but not for their reduction. The energetics of redox reaction of O(2)(*-) adducts was also explored. Results indicate that the reduction of O(2)(*-) adducts with O(2)(*-) is preferred followed by their oxidation by O(2) and then by O(2)(*-), but the maximum difference between these free energies of redox reactions in aqueous solution is only 0.21 kcal/mol. The preferred decomposition pathways for the one-electron oxidation and reduction of nitroxides was also explored, and formation of potentially biologically active products such as NO, H(2)O(2), and hydroxamic acid was predicted.
Project description:1. The metabolic activation of carbon tetrachloride to free-radical intermediates is an important step in the sequence of disturbances leading to the acute liver injury produced by this toxic agent. Electron-spin-resonance (e.s.r.) spin-trapping techniques were used to characterize the free-radical species involved. 2. Spin trapping was applied to the activation of carbon tetrachloride by liver microsomal fractions in the presence of NADPH, and by isolated intact rat hepatocytes. The results obtained with the spin trap N-benzylidene-2-methylpropylamine N-oxide ('phenyl t-butyl nitrone') (PBN) and [13C]carbon tetrachloride provide unequivocal evidence for the formation and trapping of the trichloromethyl free radical in these systems. 3. With the spin trap 2-methyl-2-nitrosopropane, however, the major free-radical species trapped are unsaturated lipid radicals produced by the initiating reaction of lipid peroxidation. 4. Although pulse radiolysis and other evidence support the very rapid formation of the trichloromethyl peroxy radical from the trichloromethyl radical and oxygen, no clear evidence for the trapping of the peroxy radical was obtainable. 5. The effects of a number of free-radical scavengers and metabolic inhibitors on the formation of the PBN-trichloromethyl radical adduct were studied, as were the influences of changing the concentration of PBN and incubation time. 6. High concentrations of the spin traps used were found to have significant effects on cytochrome P-450-mediated reactions; this requires caution in interpreting results of experiments done in the presence of PBN at concentrations greater than 50 mM.
Project description:Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)? levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 ?M) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.
Project description:Nitrones are potential synthetic antioxidants against the reduction of radical-mediated oxidative damage in cells and as analytical reagents for the identification of HO2* and other such transient species. In this work, the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) and PCM/mPW1K/6-31+G(d,p) density functional theory (DFT) methods were employed to predict the reactivity of HO2* with various functionalized nitrones as spin traps. The calculated second-order rate constants and free energies of reaction at both levels of theory were in the range of 100-103 M-1 s-1 and 1 to -12 kcal mol-1, respectively, and the rate constants for some nitrones are on the same order of magnitude as those observed experimentally. The trend in HO2* reactivity to nitrones could not be explained solely on the basis of the relationship of the theoretical positive charge densities on the nitronyl-C, with their respective ionization potentials, electron affinities, rate constants, or free energies of reaction. However, various modes of intramolecular H-bonding interaction were observed at the transition state (TS) structures of HO2* addition to nitrones. The presence of intramolecular H-bonding interactions in the transition states were predicted and may play a significant role toward a facile addition of HO2* to nitrones. In general, HO2* addition to ethoxycarbonyl- and spirolactam-substituted nitrones, as well as those nitrones without electron-withdrawing substituents, such as 5,5-dimethyl-pyrroline N-oxide (DMPO) and 5-spirocyclopentyl-pyrroline N-oxide (CPPO), are most preferred compared to the methylcarbamoyl-substituted nitrones. This study suggests that the use of specific spin traps for efficient trapping of HO2* could pave the way toward improved radical detection and antioxidant protection.
Project description:Peroxynitrite is a reactive oxidant produced in vivo in response to oxidative and other stress by the diffusion-limited reaction of nitric oxide and superoxide. This article is focused on the identification of free radical intermediates of uric acid formed during its reaction with peroxynitrite. The experimental approach included the ESR spin trapping of the radical generated from the reaction between uric acid and peroxynitrite at pH 7.4 and mass spectrometry studies of the trapped radicals. Using PBN (N-tert-butyl-alpha-phenylnitrone) as the spin trapping agent, a six-line ESR spectrum was obtained and its hyperfine coupling constants, a(N)=15.6 G and a(H)=4.4 G, revealed the presence of carbon-based radicals. Further structural identification of the PBN-radical adducts was carried out using liquid chromatography-mass spectrometry. After comparison with the control reactions, two species were identified that correspond to the protonated molecules (M+1) at m/z 352 and 223, respectively. The ions of m/z 352 were characterized as the PBN-triuretcarbonyl radical adduct and the m/z 223 ion was identified as the PBN-aminocarbonyl radical adduct. Their mechanism of formation is discussed.
Project description:We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1'Z)-1,1'-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50?=?1.24?±?0.39 ?M) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity?=?95.8?±?3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.
Project description:Herein we report the synthesis, antioxidant and neuroprotective power of homo-tris-nitrones (HTN) 1-3, designed on the hypothesis that the incorporation of a third nitrone motif into our previously identified homo-bis-nitrone 6 (HBN6) would result in an improved and stronger neuroprotection. The neuroprotection of HTNs1-3, measured against oligomycin A/rotenone, showed that HTN2 was the best neuroprotective agent at a lower dose (EC50 = 51.63 ± 4.32 ?M), being similar in EC50 and maximal activity to ?-phenyl-N-tert-butylnitrone (PBN) and less potent than any of HBNs 4-6. The results of neuroprotection in an in vitro oxygen glucose deprivation model showed that HTN2 was the most powerful (EC50 = 87.57 ± 3.87 ?M), at lower dose, but 50-fold higher than its analogous HBN5, and ?1.7-fold less potent than PBN. HTN3 had a very good antinecrotic (IC50 = 3.47 ± 0.57 ?M), antiapoptotic, and antioxidant (EC50 = 6.77 ± 1.35 ?M) profile, very similar to that of its analogous HBN6. In spite of these results, and still being attractive neuroprotective agents, HTNs 2 and 3 do not have better neuroprotective properties than HBN6, but clearly exceed that of PBN.
Project description:?-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 ?m. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-?-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.