Phenyl T-butyl Nitrone Research Articles

Isolation of mouse mesenchymal stem cells with normal ploidy from bone marrows by reducing oxidative stress in combination with extracellular matrix

BackgroundIsolation of mouse MSCs (mMSCs) with normal ploidy from bone marrow remains challenging. mMSCs isolated under 20% O2 are frequently contaminated by overgrown hematopoietic cells, and could also be especially vulnerable to oxidative damage, resulting in chromosomal instability. Culture under low oxygen or extracellular matrix (ECM) improves proliferation of MSCs in several species. We tested the hypothesis that culture under low oxygen in combination with ECM prepared from mouse embryonic fibroblast (MEF-ECM) could be used to purify proliferative mMSCs, and to reduce oxidative damage and maintain their chromosomal stability.ResultsOptimization of culture conditions under 20% O2 resulted in immortalization of mMSCs, showing extensive chromosome abnormalities, consistent with previous studies. In contrast, culture under low oxygen (2% O2) improved proliferation of mMSCs and reduced oxidative damage, such that mMSCs were purified simply by plating at low density under 2% O2. MEF-ECM reduced oxidative damage and enhanced proliferation of mMSCs. However, these isolated mMSCs still exhibited high frequency of chromosome abnormalities, suggesting that low oxygen or in combination with MEF-ECM was insufficient to fully protect mMSCs from oxidative damage. Notably, antioxidants (alpha -phenyl-t-butyl nitrone (PBN) and N-acetylcysteine (NAC)) further reduced DNA damage and chromosomal abnormalities, and increased proliferation of mMSCs. mMSCs isolated by the combination method were successfully used to generate induced pluripotent stem (iPS) cells by ectopic expression of Oct4, Sox2, Klf4 and c-Myc.ConclusionsWe have developed a technique that allows to reduce the number of karyotypic abnormalities for isolation of primary mMSCs and for limited culture period by combination of low oxygen, MEF-ECM, antioxidants and low density plating strategy. The effectiveness of the new combination method is demonstrated by successful generation of iPS cells from the isolated mMSCs. However, a culture system for mMSCs still is needed to prevent all the anomalies, especially after a long-term culture period.

An EPR study of free radical generation during maceration of uncooked vegetables

AbstractElectron paramagnetic resonance (EPR) spectroscopy has been used to study free radicals formed in a range of plant tissues as a result of physical damage, with the objective of gaining some insight into the free radical chemistry that is initiated when uncooked vegetable products (eg salads) are eaten. Chemical spin traps were used to aid the detection of unstable free radicals; more stable radicals were detected directly. Commonly observed ‘stable’ species were the monodehydroascorbate radical, which has a characteristic doublet spectrum, and a single‐peak resonance, which is presumed to come from free radical centres stabilised in macromolecules. In mushrooms (Agaricus spp), spin‐trapping experiments using either α‐(4‐pyridyl‐1‐oxide)‐N‐t‐butyl‐nitrone (4‐POBN) or phenyl‐t‐butyl‐nitrone (PBN) showed the formation of large quantities of adducts of the radical from 4‐hydroxymethylbenzene diazonium salts. Pleurotus species, in contrast, gave signals consistent with the formation of unidentified C‐centred radicals. With other foodstuffs, reaction with 4‐POBN was complex and signals from 4 − POBN· and ${\bf CO_{2}^{\cdot -}}$ adducts were observed along with the t‐butylhydronitroxide radical (an adduct breakdown product). Investigation of carrot hypocotyl rootstock in the presence of 5‐(diethoxyphosphoryl),5‐methyl‐1‐pyrroline‐N‐oxide (DEPMPO) revealed adducts of ·OH and unidentified C‐centred radicals. Free radical interactions between food components were demonstrated by the suppression of the signal from the 4‐POBN adducts of lettuce by onion, garlic, satsuma or vinaigrette, but not by olive oil. In addition, an appreciable decrease in spectral intensity of the 4‐POBN adduct from lettuce was observed in the presence of saliva, which suggests that saliva contains free radical scavengers which are able to compete successfully with the spin trap.© 2002 Society of Chemical Industry

Synthesis and radical scavenging activity of 3,3-dialkyl-3,4-dihydro-isoquinoline 2-oxides

The syntheses and antioxidant activities of several cyclic nitrones related to phenyl t-butyl nitrone (PBN) are described. These nitrones may act as radical scavengers and have potential uses in the treatment of stroke and septic shock.

Synthesis of benzazepine-based nitrones as radical traps

Benzazepine-based nitrones have been synthesized utilizing a modified Bischler-Napieralski reaction as the key step. These compounds are cyclic analogs of the radical trap phenyl t-butyl nitrone.

Microsurgical model of ischemia reperfusion in rat muscle: evidence of free radical formation by spin trapping.

The purpose of developing the experimental model described in this study was to verify the hypothesis that free radicals are formed during ischemia-reperfusion of skeletal muscle. Spin trapping technique, along with electron spin resonance spectroscopy (ESR), directly indicates the presence of reactive radicals, which are widely considered to be important in tissue injury. The experimental model was a rat pedicled rectus femoris muscle flap. The femoral artery and vein were cannulated to inject the "spin trap" and collect the effluent flow. The spin trap agent was phenyl-t-butyl nitrone (PBN) and Hank's balanced salt solution. Three injections and collections were made: a) before ischemia; b) after ischemia of 15, 30, 60, 120, and 180 minutes, but before blood flow had been restored; and c) after blood flow had been restored. No ESR signal was detected either before the ischemic period or after only 15 minutes of ischemia. PBN radical adducts were detected after 30, 60, 120, and 180 minutes of ischemia. A similar signal was detected when PBN was injected during reperfusion 10 minutes after the ischemic periods. The study demonstrated the presence of free radicals in an in vivo intact skeletal muscle ischemia-reperfusion model.

Neuroprotective effects of phenyl-t-butyl-nitrone in gerbil global brain ischemia and in cultured rat cerebellar neurons

We examined the ability of phenyl-t-butyl-nitrone (PBN), an electron spin trapper, to attenuate ischemia-induced forebrain edema and hippocampal CA1 neuronal loss in gerbils, and to protect rat cerebellar neurons in primary culture from glutamate-induced toxicity. PBN, given i.p. at 75 or 150 mg/kg 30 min before ischemia (5 min occlusion), inc reased survival (at 7 days) of CA1 nerons from 60 ± 14 (vehicle-treated, n =17) to 95 ± 15 (P<0.05, n = 15) and 145 ± 3 (P < 0.01, n = 15), respectively. When gerbis were treated with PBN (50 mg/kg, i.p.) immediately and 6 h after reperfusion, followed by b.i.d. for an additional 2 days, CA1 neurons survival improved from 35 ± 9 (vehicle, n = 20, 6 min occlusion) to 106 ± 17 (P < 0.01, n = 13). In gerbils exposed to a more severe ischemia (10 min), pretreatment with 150 mg/kg PBN increased the survival of CA1 neurons from 6 ± 6 (vehicle) to 27 ± 10 (P < 0.05, n = 11). Pretreatment with PBN, at 150 mg/kg, reduced forebrain edema (following 15 min eschemia) by 24.7% (P < 0.01, n = 16). PBBN at 50 mg/kg, i.p. had no hypothermic effect and at 75 or 150 mg/kg caused a transient hypothermia. The presence of PBN in the brain was confirmed in microdialysis samples and brain tissue extract using HPLC. In vitro, PBN prottected rat cerebellar neurons against 100 μM glutamate-induced toxicity with an EC 50 value of 2.7 mM. Our results further support the concept that free radicals contribute to brain injury following ischemia and suggest the potential therapeutic application of electron spin trappers in stroke.

On the hepatotoxicity of 1,1,2,2-tetrachloroethane

Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58–73% of the control) and NADPH-cytochrome ( P-450) c-reductase (to 29–35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20–64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (≈35% loss), UDP-glucuronosyl transferase (≈42% loss) and to a lesser extent glutathione S-transferase (≈17% loss) were all affected. Also, the activity of δ-aminolevulinic (ALA) synthetase was decreased (≈57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (≈33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl 2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.

Epr Spectroscopic Studies of Detection of a Carbon-Centered Free Radical During Acetylcholine-Induced and Endothelium-Dependent Relaxation of Guinea Pig Pulmonary Artery

Vascular smooth muscle relaxation by several vasodilators, including acetylcholine (Ach) and ATP, depends on the presence of intact endothelium. Ach is thought to activate muscarinic receptors on endothelium to release an endothelium-derived relaxing factor (EDRF) which brings about relaxation of smooth muscle. In order to assess the role of free radicals in the endothelium-dependent relaxation of blood vessel, we have studied the effect of a spin-trapping agent, phenyl t-butyl nitrone (PBN), on Ach-, ATP-, and sodium nitroprusside-induced relaxation of guinea pig pulmonary artery. Arterial strips were mounted in a 5-ml organ bath containing Krebs solution equilibrated with 95% O2 and 5% CO2 at 37 degrees C. After increasing vascular tone by a synthetic prostaglandin endoperoxide analog (50 ng/ml), the strips relaxed dose-dependently in response to Ach (5 x 10(-8) M), ATP (1.5 x 10(-6) M) or sodium nitroprusside (6 x 10(-9) M). Removal of the endothelium abolished the relaxation by Ach or ATP, but did not affect the relaxation by sodium nitroprusside. PBN inhibited Ach-induced relaxation of pulmonary artery dose-dependently, but had no effect on relaxations by ATP or sodium nitroprusside. PBN did not block radioligand binding to muscarinic cholinergic membrane receptors on both chick embryonic heart and guinea pig pulmonary artery endothelial cells indicating that it does not block the muscarinic receptors. Spin trapping in combination with electron paramagnetic resonance (EPR) spectral analysis revealed a carbon-centered radical with hyperfine splitting constants of aN = 16.0 G and aH beta = 3.85G in the lipid extracts of pulmonary artery (0.2-0.4 g) incubated with PBN (14 mM) and Ach (3 x 10(-6) M) for 20 min. No signal was detected when endothelium was removed. Our data suggest that the endothelium-dependent relaxation of pulmonary artery by Ach is associated with the generation of a free-radical and can be prevented by a spin-trapping agent. ATP, however, relaxes the arterial smooth muscle by a different mechanism.

Development of fluorinated, NMR-active spin traps for studies of free radical chemistry

Five fluorinated analogs of the spin trap phenyl-t-butyl nitrone (PBN) have been synthesized and evaluated for use as NMR-active traps. The introduction of the fluorine substituent allows selective observation of chemical reactions involving the spin traps. Although the paramagnetic adducts themselves are not directly observable by this approach as a consequence of extensive broadening, the reduced forms (hydroxylamines) can be readily observed. NMR studies of the trapping of the phenyl radical produced from the thermal decomposition of phenylazotriphenyl methane have been carried out. The observation of fluorinated benzaldehydes in these studies reflects the formation and subsequent degradation of oxygen-centered radicals under some conditions. Relative trapping efficiencies for the phenyl radical in the series 2-F, 4-F, 2,6-difluoro, 2-CF 3, and 4-CF 3 substituted PBN analogs have been determined based on an analysis of the 19F NMR resonance intensities of the reduced phenyl radical adducts. The relatively large proton hyperfine coupling constants observed for 2,6-difluoro and 2-CF 3 PBN analogs allow direct observation by ESR of adduct formation in solutions containing both PBN and either of these analogs. The introduction of fluorine substituents into the trap has only a small effect on trapping efficiency.

Hydralazine-dependent carbon dioxide free radical formation by metabolizing mitochondria.

The addition of hydralazine (1-hydrazinophthalazine) to rat liver mitochondria metabolizing malate/glutamate causes formation of a carbon-centered free radical which was spin-trapped with phenyl-t-butylnitrone (PBN) or dimethylpyrrolidine-N-oxide (DMPO). The coupling constants of the spin-trapped free radical were AN = 16.1, AH beta = 4.6 G for PBN and AN = 15.9, AH beta = 18.9 G for DMPO-trapped radical in aqueous solution. The spin-trapped free radical was shown to be the carbon dioxide anion free radical by independent synthesis, high pressure liquid chromatography separation, and electron paramagnetic resonance characterization. The amount of carbon dioxide anion free radical produced was absolutely dependent upon the presence of hydralazine and varied depending on mitochondrial substrate, with by far the highest amount produced by pyruvate. Studies with 13C-labeled pyruvate demonstrated that the carbon dioxide free radical came from C-1 of this compound.

Is gram-negative shock a free-radical-mediated condition?

The involvement of free radicals in endotoxic shock (a severe condition occurring in many Gram-negative septicaemias) has been investigated in a mouse model. Administration of endotoxin produced an increase in the e.s.r.-detectable signals due to ascorbyl radicals. In vivo spin-trapping experiments with the spin trap phenyl t-butyl nitrone resulted in the formation of poorly resolved adduct spectra. The results suggest that free radicals are produced during the endotoxic shock crisis and may play a significant role in the pathophysiology of this condition.

Spin trapping of free radical intermediates produced during the metabolism of isoniazid and iproniazid in isolated hepatocytes

By the use of spin trapping agents phenyl- t-butyl nitrone (PBN) and 4-pyridyl-1-oxide- t-butyl nitrone (4-POBN) free radical species were detected in isolated hepatocytes incubated with either isoniazid, iproniazid and their respective metabolites acetyl-hydrazine and isopropyl-hydrazine. The addition of bis-nitrophenyl phosphate, an inhibitor of the acylamidase enzymes, to isolated hepatocytes decreased the free radical activation of isoniazid and iproniazid, but not that of acetyl- and isopropylhydrazine, confirming that the radical species were originating from the biotransformation of these latter compounds. The ESR spectra were ascribed to the trapping of, respectively, acetyl and isopropyl free radicals on the basis of the analogies of the spectral feature with those of chemically-prepared spin adducts. Comparable ESR spectra were also observed during the metabolism of acetyl- and isopropylhydrazines by liver microsomes and their formation was inhibited by the omission of NADP +, anaerobic incubation and enzyme denaturation. In the microsomal preparations inhibitors of the mixed function oxidase system decreased to various extents the free radical formation and a similar effect was also observed following the destruction of cytochrome P-450 induced by pretreating the rats with CoCl 2. The addition of reduced glutathione also decreased the radical trapping indicating that GSH can effectively compete with the spin traps for the reaction with the free radicals. The incubation of isolated hepatocytes with isoniazid or acetyl-hydrazine reduced by 20–25% the intracellular GSH content, while a 50% decrease in GSH was present in the cells exposed to iproniazid and isopropyl-hydrazine. In the same hepatocyte preparations stimulation of lipid peroxidation and leakage of LDH were also observed during cell incubation with iproniazid and isopropyl-hydrazine, but not with isoniazid or acetyl-hydrazine and the extent of both phenomena correlated with the susceptibility of the above compounds to the free radical activation.

Spin-trapping study of the radiolysis of CCl4

This paper reports the spin trapping of radicals in liquid CCl4 irradiated with low-energy X-rays of different wavelengths, using phenyl-t-butylnitrone (PBN) as a spin trap. The unstable PBN-Cl adduct and the relatively stable PBN-CCl3 adduct have been detected. X-rays of energy above the K-edge for chlorine produce significantly more trapped radicals than those having energies below this K-edge value. This enhancement of radical formation is a consequence of the Auger effect, which follows the inner-shell photoabsorption. Experiments have also been made on trapping the radicals generated by the decay of tritium in a solution of tritiated methanol in CCl4.

Initiation and propagation of lipid peroxidation in cerebral infarction models. Experimental studies.

The possibility that cerebral ischaemia or cerebral hypoxia may initiate a series of free radical reactions in brain lipid constituents was explored by measuring sequential changes in chemiluminescence (CL) and electron spin resonance (ESR) during hypoxia or ischaemia load. Brain hypoxia was induced by means of arterial hypoxaemia (PaO2 17-22 mmHg), normocapnia (PaCO2 28-38 mmHg) and normotension (MABP 100-140 mmHg). To obtain lowered PaO2, 4% O2-96% N2 mixed gas was used for artificial ventilation. Spin trapping technique was used in ESR measurement and applied to the detection of free radicals generated in the ischaemic brain homogenate of three-vessel occlusion rat model (global highly ischaemic model with basilar artery coagulation and bilateral carotid artery clipping). Chemiluminescence (CL) began to rise in hypoxic or ischaemic loading and indicates high amounts at an early period of post-hypoxic or ischaemic state. The CL spectroanalysis by wavelength showed five peaks at 480 nm, 520-530 nm, 570 nm, 620-640 nm and 680-700 nm in both hypoxic and ischaemic brain. ESR measurement revealed the PBN (phenyl-t-butyl nitrone) trapped radical, which has hyperfine splitting constants of AN = 16.2-16.5 G and AH beta = 3.6-3.8 G in ischaemia model. An analysis of sequential change of PBN adduct intensity shows a peak at 30 min of ischaemic loading and a marked increase in the recirculation period. Preservation of ATP and marked lactic acidosis were seen in the 5 min hypoxic loading, elsewhere depletion of ATP and marked lactic acidosis were seen in the 5 min, 30 min ischaemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of spin traps in isolated rat hepatocytes and liver microsomes

Spin traps are increasingly employed in the detection of free radicals in biological systems, including liver microsomes and isolated hepatocytes. Two spin traps phenyl- t-butyl nitrone (PBN) and 4-pyridyl-1-oxide- t-butyl nitrone (4-POBN) have been tested for their effects on hepatocyte viability and mixed-function oxidase activity. High concentration of PBN but not of 4-POBN proved to moderately affect liver cell integrity, without interfering with intracellular ATP or cytochrome P-450 content. PBN also decreased hepatocyte GSH content, probably as the result of its metabolism to benzaldehyde. The two spin traps were found to inhibit aminopyrine demethylase and ethoxycoumarin deethylase activity in hepatocytes and microsomes. At low concentrations (1–5 mM) PBN enhanced aniline hydroxylase while high concentrations of the spin trap inhibited this activity. The inhibition of the monooxygenase system was not caused by damage of microsomal enzymes, but rather by competition with other substrates for the binding to the haemoprotein. The effects of spin traps on mixed function oxidase systems should be taken into account when evaluating the results of spin trapping experiments.

Phenyl-T-butyl-nitrone is active against traumatic shock in rats.

Oxygen free-radicals appear to be involved in the pathogenesis of shock; therefore trapping of these radicals would modify the evolution of experimental shock. Experiments were performed on rats submitted to 100% lethal whole body trauma (rotating drum) and their survival, pathology, acid-base status and hematocrit level observed. The spin trapping agent phenyl-t-butyl-nitrone (PBN) was administered before trauma (50, 100, 150 mg/kg i.p.) or at various intervals (30, 60 minutes) after establishment of a severe traumatic shock. It appeared that PBN administration was highly effective both in prevention and in reversion of traumatic shock in rats.

Mechanism of antihepatotoxic activity of atractylon, I: Effect on free radical generation and lipid peroxidation.

The mechanism of antihepatotoxic action of atractylon, a main sesquiterpenic constituent of Atractylodes rhizomes, was studied. Atractylon inhibited carbon tetrachloride (CCl4)-induced cytotoxicity in primary cultured rat hepatocytes and CCl4-induced lipid peroxidation by rat liver microsomes. However, atractylon increased the free radical generation by CCl4 with rat liver microsomes in the presence of a radical trapping agent, phenyl t-butyl nitrone (PBN). In addition, atractylon generated free radical per se. Experiments using 13CCl4 instead of CCl4 indicated that the increased free radicals consisted of those from 13CCl4 and from atractylon. Accumulated data support that although both CCl4 and atractylon generate free radicals respectively by rat liver microsomes, free radical from CCl4 conducts lipid peroxidation and produces liver lesion, while atractylon forms free radical which scavenges CCl3 radical in the absence of PBN, inhibits lipid peroxidation by CCl4 and suppresses CCl4-induced liver lesion.

Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique

When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal.

Mechanism of antihepatotoxic activity of glycyrrhizin. I: Effect on free radical generation and lipid peroxidation.

The mechanism of antihepatotoxic action of atractylon, a main sesquiterpenic constituent of ATRACTYLODES rhizomes, was studied. Atractylon inhibited carbon tetrachloride (CCl 4 )-induced cytotoxicity in primary cultured rat hepatocytes and CCl 4 -induced lipid peroxidation by rat liver microsomes. However, atractylon increased the free radical generation by CCl 4 with rat liver microsomes in the presence of a radical trapping agent, phenyl T-butyl nitrone (PBN). In addition, atractylon generated free radical PER SE. Experiments using 13 CCl 4 instead of CCl 4 indicated that the increased free radicals consisted of those from 13 CCl 4 and from atractylon. Accumulated data support that although both CCl 4 and atractylon generate free radicals respectively by rat liver microsomes, free radical from CCl 4 conducts lipid peroxidation and produces liver lesion, while atractylon forms free radical which scavenges CCl 3 radical in the absence of PBN, inhibits lipid peroxidation by CCl 4 and suppresses CCl 4 -induced liver lesion.

PROTON AND NITROGEN-14 ENDOR SPECTRA OF AMINOXYL SPIN ADDUCTS FROM PBN

Abstract Both N-14 and proton hyperfine splittings have been observed in the ENDOR spectra of spin adducts of phenyl t-butyl nitrone (PBN). From the differences in relative intensities of the ENDOR doublets the relative signs of the coupling constants can be ascertained.